THE UNITED REPUBLIC OF

TANZANIA

MINISTRY OF HEALTH

NATIONAL MEDICAL
STANDARD OPERATING
PROCEDURES FOR HEALTH
CENTRE LEVEL
LABORATORY
Version 01:2024
i

JANUARY 2024
NATIONAL MEDICAL STANDARD OPERATING PROCEDURES FOR HEALTH CENTRE LEVEL
LABORATORY

PUBLISHED IN 2024

©MINISTRY OF HEALTH,

MAGUFULI GOVERNMENT CITY, AFYA ROAD/STREET, MTUMBA,

PO BOX 743,

40478 DODOMA, TANZANIA.

LANDLINE: +255 (0)26 232 3267

EMAIL: [email protected]

WEBSITE: www.moh.go.tz

ISBN: 978-9912-9833-0-4

Any section(s) or clause(s) within this document, can be used by the Government of
Tanzania Departments, implementers and stakeholders, provided that, the Ministry of
Health is acknowledged

ii
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 Table of Contents
ABBREVIATIONS AND ACRONYMS ................................................................................................. I

TERMS AND DEFINITIONS .............................................................................................................V

FOREWORD ................................................................................................................................VI

ACKNOWLEDGEMENTS ..............................................................................................................VII

EXECUTIVE SUMMARY ................................................................................................................. 8

SCOPE ........................................................................................................................................... 9
PRIMARY BENEFICIARIES .................................................................................................................... 9
SECONDARY BENEFICIARIES ................................................................................................................ 9

CHAPTER 1: SAMPLE COLLECTION ........................................................................................... 10

1.0 GENERAL CONSIDERATIONS .............................................................................................. 10

1.2 COLLECTION OF BLOOD SAMPLES ...................................................................................... 11
1.3 COLLECTION OF URINE SAMPLES ....................................................................................... 14
1.4 FAECAL SAMPLES COLLECTION PROCEDURE ...................................................................... 17
1.5 COLLECTION OF BODY FLUIDS............................................................................................ 17
1.6 WOUND SAMPLES TO INCLUDE PUS, ABSCESS, TISSUE, ETC. ................................................ 22
1.8 COLLECTION OF THROAT AND NASAL SWABS ..................................................................... 23
COLLECTION OF OROPHRYANGEAL SWABS.................................................................................. 25
1.7 SPUTUM SAMPLES ............................................................................................................ 26

CHAPTER TWO: PARASITOLOGY .............................................................................................. 28

2.1. PROCEDURE FOR MALARIA RAPID TEST (MRDT) ................................................................ 28

2.1.1 PURPOSE ..................................................................................................................................... 28
2.2.2 SCOPE ......................................................................................................................................... 28
2.2.3 PRINCIPLE .................................................................................................................................... 28
2.2.4 SAMPLE REQUIREMENTS ................................................................................................................ 28
2.2.5 EQUIPMENT ................................................................................................................................. 28
2.2.6 MATERIALS .................................................................................................................................. 28
2.2.7 STORAGE AND STABILITY ................................................................................................................ 29
2.2.8 SAFETY ........................................................................................................................................ 29
2.2.9 CALIBRATION ................................................................................................................................ 29
2.2.10 QUALITY CONTROL ...................................................................................................................... 29
2.2.11 PROCEDURE STEPS ...................................................................................................................... 29
2.2.12 BIOLOGICAL REFERENCE INTERVALS ................................................................................................ 29
2.2.13 INTERPRETATION AND REPORTING OF RESULTS ................................................................................ 29
2.2.14 LIMITATIONS OF THE PROCEDURE AND SOURCES OF ERROR ................................................................. 30
2.2.15 PERFORMANCE CHARACTERISTICS .................................................................................................. 30
2.2.16 SUPPORTING DOCUMENTS ........................................................................................................... 30
2.2.17 REFERENCES ............................................................................................................................... 30
2.2. PROCEDURE FOR MALARIA MICROSCOPY.......................................................................... 30
2.2.1 PURPOSE ..................................................................................................................................... 30
2.2.2 SCOPE ......................................................................................................................................... 30
2.2.3 RESPONSIBLE ................................................................................................................................... 30
2.2.4 PRINCIPLE .................................................................................................................................... 31
2.2.5 SAMPLE REQUIREMENTS ................................................................................................................ 31
2.2.6 EQUIPMENT ................................................................................................................................. 31
2.2.7 MAINTENANCE ............................................................................................................................. 31
2.2.8 MATERIALS .................................................................................................................................. 31
2.2.9 STORAGE AND STABILITY ................................................................................................................ 32
2.2.10 SAFETY ...................................................................................................................................... 32
2.2.10 CALIBRATION .............................................................................................................................. 32
2.2.11 QUALITY CONTROL ...................................................................................................................... 32
2.2.12 PROCEDURE STEPS ...................................................................................................................... 32
2.2.13 EXAMINATION OF THE BLOOD FILMS ............................................................................................... 33
2.2.14 BIOLOGICAL REFERENCE INTERVALS NOT APPLICABLE........................................................................ 34
2.2.15 REPORTING AND INTERPRETATION OF RESULTS MALARIA PARASITES.................................................... 34
2.2.16 CRITICAL VALUES ......................................................................................................................... 34
2.2.17 LIMITATION OF THE PROCEDURE AND SOURCES OF ERRORS................................................................ 34
2.2.18 PERFOMANCE CHARACTERISTICS .................................................................................................... 34
2.2.19 SUPPORTING DOCUMENTS NOT APPLICABLE ................................................................................... 35
2.2.20 REFERENCES ............................................................................................................................... 35
2.3 PROCEDURE FOR URINE MICROSCOPY ............................................................................... 35
2.4 PROCEDURE FOR STOOL ROUTINE EXAMINATION............................................................... 39
2.4.1 PURPOSE ..................................................................................................................................... 39
2.4.2 SCOPE ......................................................................................................................................... 39
2.4.2.1 RESPONSIBLE ............................................................................................................................. 39
2.4.3 PRINCIPLE .................................................................................................................................... 39
2.4.4 SAMPLE REQUIREMENTS ................................................................................................................ 39
2.4.5 EQUIPMENT ................................................................................................................................. 39
2.4.6 SAFETY ........................................................................................................................................ 40
2.4.7 CALIBRATION ................................................................................................................................ 40
2.4.8 QUALITY CONTROL ........................................................................................................................ 40
2.4.9 BIOLOGICAL REFERENCE INTERVALS .................................................................................................. 41
2.4.10 INTERPRETATION AND REPORTING OF RESULTS ................................................................................ 41
2.5 PROCEDURE FOR EXAMINATION OF BLOOD FOR MICROFILARIAE ........................................ 42
2.5.1 PURPOSE ..................................................................................................................................... 42
2.5.2 SCOPE ......................................................................................................................................... 43
2.5.3 RESPONSIBLE ................................................................................................................................ 43
2.5.4 PRINCIPLE .................................................................................................................................... 43
2.5.5 SAMPLE REQUIREMENTS ................................................................................................................ 43
2.5.6 EQUIPMENT ................................................................................................................................. 43
2.5.7 MATERIALS .................................................................................................................................. 43
2.5.8 STORAGE AND STABILITY ................................................................................................................ 44
2.5.9 SAFETY ........................................................................................................................................ 44
2.5.10 CALIBRATION .............................................................................................................................. 44
2.5.11 QUALITY CONTROL ...................................................................................................................... 44
2.5.12 PROCEDURE STEPS ...................................................................................................................... 44
2.5.13 BIOLOGICAL REFERENCE INTERVALS - NOT APPLICABLE ...................................................................... 45
2.5.14 REPORTING AND INTERPRETATION OF RESULTS ................................................................................ 45
2.5.15 LIMITATION OF THE PROCEDURE AND SOURCES OF ERRORS................................................................ 46
2.5.16 PERFORMANCE CHARACTERISTICS .................................................................................................. 46
2.5.17 SUPPORTING DOCUMENTS SAMPLE COLLECTION MANUAL ................................................................. 46
2.5.18 REFERENCES ............................................................................................................................... 46
CHAPTER THREE: BLOOD TRANSFUSION .................................................................................. 47

3.1 PROCEDURE FOR ABO AND RHESUS BLOOD GROUPING ...................................................... 47

3.1.1 PURPOSE ..................................................................................................................................... 47
3.1.2 SCOPE ......................................................................................................................................... 47
3.1.3 RESPONSIBLE ................................................................................................................................ 47
3.1.4 PRINCIPLE .................................................................................................................................... 47
3.1.5 SAMPLE REQUIREMENTS ................................................................................................................ 47
3.1.6 EQUIPMENT ................................................................................................................................. 47
3.1.7 MATERIALS .................................................................................................................................. 48
3.1.8 STORAGE AND STABILITY ................................................................................................................ 48
3.1.9 SAFETY ........................................................................................................................................ 48
3.1.10 CALIBRATION .............................................................................................................................. 48
3.1.11 QUALITY CONTROLS .................................................................................................................... 48
3.1.12 PROCEDURE STEPS ...................................................................................................................... 50
3.1.13 BLOOD GROUPING PROCEDURE...................................................................................................... 50
3.1.14 BIOLOGICAL REFERENCE INTERVALS ................................................................................................ 51
3.1.15 INTERPRETATION AND REPORTING OF RESULTS INTERPRETATION OF RESULTS ....................................... 51
3.1.16 LIMITATIONS OF THE PROCEDURE AND SOURCE OF ERROR ................................................................. 52
3.1.16 PERFORMANCE CHARACTERISTICS .................................................................................................. 52
3.1.17 SUPPORTING DOCUMENTS ........................................................................................................... 52
3.1.18 REFERENCES ............................................................................................................................... 52
3.2 PROCEDURE FOR ESTIMATION OF HAEMOGLOBIN BY USING COPPER SULPHATE SOLUTION. 52
3.2.1 PURPOSE ..................................................................................................................................... 52
3.2.2 SCOPE ......................................................................................................................................... 52
3.2.3 RESPONSIBLE ................................................................................................................................ 52
3.2.4 PRINCIPLE .................................................................................................................................... 52
3.2.5 SAMPLE REQUIREMENTS ................................................................................................................ 53
3.2.6 EQUIPMENT ................................................................................................................................. 53
3.2.7 MATERIALS .................................................................................................................................. 53
3.2.8 PREPARATION OF COPPER II SULPHATE SOLUTION .............................................................................. 53
3.2.9 PREPARE WORKING SOLUTION ........................................................................................................ 53
3.2.10 STORAGE AND STABILITY .............................................................................................................. 53
3.2.11 SAFETY ...................................................................................................................................... 54
3.2.12 CALIBRATION .............................................................................................................................. 54
3.2.13 QUALITY CONTROL ...................................................................................................................... 54
3.2.14 PROCEDURE STEPS ...................................................................................................................... 54
3.2.15 BIOLOGICAL REFERENCE INTERVALS ................................................................................................ 55
3.2.16 INTERPRETATION AND REPORTING OF RESULTS ................................................................................ 55
3.2.17 LIMITATIONS OF THE PROCEDURE AND SOURCES OF ERROR ............................................................... 55
3.2.18 PERFORMANCE CHARACTERISTICS .................................................................................................. 55
3.2.19 SUPPORTING DOCUMENTS ........................................................................................................... 55
3.2.20 REFERENCES ............................................................................................................................... 55
3.3 PROCEDURE FOR COMPATIBILITY TESTING ......................................................................... 56
3.3.1 PURPOSE ..................................................................................................................................... 56
3.3.2 SCOPE ......................................................................................................................................... 56
3.3.3 RESPONSIBLE ................................................................................................................................ 56
3.3.4 PRINCIPLE .................................................................................................................................... 56
3.3.5 SAMPLE REQUIREMENTS ................................................................................................................ 56
3.3.6 EQUIPMENT ................................................................................................................................. 56
3.3.7 MATERIALS .................................................................................................................................. 56
3.3.8 STORAGE AND STABILITY ................................................................................................................ 57
3.3.9 SAFETY ........................................................................................................................................ 57
3.3.10 CALIBRATION .............................................................................................................................. 57
3.3.11 QUALITY CONTROL ...................................................................................................................... 57
3.3.12 PROCEDURE STEPS ...................................................................................................................... 57
3.3.13 BIOLOGICAL REFERENCES INTERVALS .............................................................................................. 58
3.3.14 REPORTING AND INTERPRETATION OF RESULTS ................................................................................. 58
3.3.15 LIMITATION OF THE PROCEDURE AND SOURCES OF ERRORS................................................................ 58
3.3.16 PERFORMANCE CHARACTERISTICS .................................................................................................. 58
3.3.17 SUPPORTING DOCUMENTS ........................................................................................................... 58
3.3.18 REFERENCES ............................................................................................................................... 59

CHAPTER FOUR: HAEMATOLOGY............................................................................................. 60

4.1 PROCEDURE FOR SICKLING SCREENING TEST....................................................................... 60

4.2 PROCEDURE FOR URIT-12 HEMOGLOBIN METER ................................................................. 62
4.3 PROCEDURE FOR DETERMINATION OF HAEMOGLOBIN LEVEL ............................................. 64
4.4 PROCEDURE FOR DETREMANATION OF HEMOGLOBIN LEVEL .............................................. 66
4.5 PROCEDURE FOR CD 4 COUNT TEST BY USING BD FACS PRESTO ........................................... 68
4.6 PROCEDURE FOR DETERMINATION OF CLOTTING TIME ....................................................... 71
4.7 PROCEDURE FOR DETERMINATION OF THE BLEEDING TIME ................................................ 73
4.8 PROCEDUIRE FOR FULL BLOOD COUNT BY USING URIT BH – 40P HAEMATOLOGY ANALYSER. 75
4.9 PROCEDURE FOR FULL BLOOD COUNT USING OF ABX PENTRA 80 HAEMATOLOGY ANALYSER
78
4.10 PROCEDURE FOR FULL BLLOD COUNT USING SINNOWA HB - 7021 ..................................... 81
4.11 PROCEDURE FOR FULL BLOOD COUNT USING BHA 3000 ................................................... 83
4.12 PROCEDURE FOR PERFORMING FULL BLOOD COUNT BY USING MS4 HAEMATOLOGY
ANALYSER.................................................................................................................................. 86

CHAPTER 5: CLINICAL CHEMISTRY AND IMMUNOLOGY ............................................................ 91

5.1 PROCEDURE FOR BLOOD GLUCOSE BY USING ACCU-CHECK GLUOCOMETER......................... 91

5.2 PROCEDURE FOR TESTING BLOOD GLUCOSE BY GLUCO PLUS ............................................... 94
5.3 PROCEDURE FOR URIT 50 (URINE CHEMISTRY ANALYZER) ................................................... 97
5.4 PROCEDURE FOR (URIT-560) URINE ANALYZER ................................................................... 99
5.5 ROCEDURE FOR PERFORMING URINE BIOCHEMISTRY BY USING CYBOW READER 300 ........ 101
5.6 PROCEDURE FOR DETERMINATION OF ALT BY USING DIRUI-DR 7000 CHEMISTRY ANALYZER
105
5.7 PROCEDURE FOR DETERMINATION OF AST BY USING DIRUI-DR7000 CHEMISTRY ANALYZER
107
5.8 PROCEDURE FOR SA-30 SEMI AUTOMATED CHEMISTRY ANALYZER ................................... 109
5.9 PROCEDURE FOR OPERATING CLINDIAG FA 200 CHEMISTRY TEST ..................................... 112
5.10 PROCEDURE FOR DETERMINATION OF IMMUNOASSAYS BY USING GETEIN 1100
IMMUNOANALYSER ................................................................................................................. 115
5.11 PROCEDURE FOR OPERATING FIA 8000 ANALYSER .......................................................... 117
5.12 PROCEDURE FOR OPERATING ALERE AFFINION AS100ANALYSER ..................................... 120

CHAPTER SIX: SEROLOGY....................................................................................................... 124

6.1 PROCEDURE FOR SYPHILIS ANTIBODIES RAPID TEST .......................................................... 124

6.2 PROCEDURE FOR (HIV) TESTING BY USING BIOLINETM HIV 1/2 3.0 TEST ............................ 126
6.3 PROCEDURE FOR PERFORMING (HIV) BY USING UNIGOLD TEST ......................................... 129
6.4 PROCEDURE FOR URINE PREGNANCY TEST ....................................................................... 132
6.5 PROCEDURE FOR HEPATITIS C ANTIBODY RAPID TEST ....................................................... 135
6.6 PROCEDURE FOR CRYPTOCOCCAL ANTIGEN RAPID TEST PROCEDURE ................................ 138
6.7 PROCEDURE FOR PERFORMING (HBSAG) RAPID TEST......................................................... 141
6.8 PROCEDURE FOR SARS-COV-2 ANTIGEN RAPID DIAGNOSTIC TEST ..................................... 144
6.9 PROCEDURE FOR DENGUE VIRUS ANTIBODY DETECTION RAPID TEST ................................ 147
6.10 PROCEDURE FOR PLAGUE RAPID TEST ............................................................................ 150
6.11 PROCEDURE FOR HELICOBACTER PYLORI ANTIGEN TEST.................................................. 152
6.12 PROCEDURE FOR HELICOBACTER PYLORI ANTIBODY RAPID TEST ..................................... 154
6.13 PROCEDURE FOR BRUCELLA ANTIBODY DETECTION ........................................................ 157
6.14 PROCEDURE FOR SALMONELLA TYPHI ANTIBODIES QUANTIFICATION METHOD ............... 159
6.15 PROCEDURE FOR CHORELA RAPID DIAGNOSTIC TEST ...................................................... 163

CHAPTER 7: BACTERIOLOGY AND MYCOLOGY ........................................................................ 167

7.1 POTASSIUM HYDROXIDE WET MOUNT PREPARATION ...................................................... 167

7.2 PROCEDURE FOR ZIEHL NIELSEN STAIN ............................................................................. 170
7.3 PROCEDURE FOR AURAMINE O PHENOL STAINING ........................................................... 173
7.4 PROCEDURE FOR GRAMS STAINING ................................................................................. 177

CHAPTER EIGHT: MOLECULAR BIOLOGY ................................................................................ 180

8.1 DIAGNOSIS OF MTB/RIF TESTING USING A TRUENAT MACHINE ......................................... 180

8.2 DIAGNOSIS OF MTB/RIF BY USING GENEXPERT SYSTEM .................................................... 185
8.3 DETERMINATION OF HIV -1 VIRAL LOAD BY USING GENEXPERT SYSTEM ............................ 190
8.4 DETERMINATION OF HIV EARLY INFANT DIAGNOSIS BY USING GENEXPERT SYSTEM........... 200

CHAPTER NINE: ANATOMICAL PATHOLOGY ........................................................................... 211

9.1 PROCEDURE FOR MORTUARY SERVICES ........................................................................... 211

NOTE PAD ................................................................................................................................ 231

NOTE PAD ................................................................................................................................ 232

NOTE PAD ................................................................................................................................ 233

Annex 1: List of Subject Matter Experts who developed these SOPs .................. 218
Annex 2: Biological Reference Intervals for Full Blood Count.............................. 221
Annex 3: Biological Reference Intervals for Coagulation Profile .......................... 222
Annex 4: Biological Reference Intervals for Urine Biochemistry .......................... 223
Annex 5: Biological Reference Intervals for Clinical Chemistry and Immunoassays
224
Annex 6: Critical or Panic Values that call for Immediate Actions ........................ 228
Annex 7: Charts for Biochemical Identifications of Common Enterobacteriaceae and
other Enteric Organisms......................................................................................... 229
ABBREVIATIONS AND ACRONYMS
For the purposes of this NMLSOP document, these abbreviations and acronyms will
apply:

Abbreviations Acronyms
AIDS Acquired Immunodeficiency Syndrome
AJLM African Journal for Laboratory Medicine
AMMP-1 Adult Morbidity and Mortality Project Phase 1
AMR Antimicrobial Resistance
ANC Antenatal Care
APECSA Association of Pathologists of East, Central and Southern
Africa
APHL Association of Public Health Laboratories
APT Association of Pathologists of Tanzania
ASCP American Society for Clinical Pathology
ASLM African Society for Laboratory Medicine
ASM American Society for Microbiology
BEmONC Basic Emergency Obstetric and Neonatal Care
BMC Bugando Medical Centre
BRM Biorisk Management
BSC Biological Safety Cabinet
BSc Bachelor of Science
BSL Biosafety Level
BUQ Bottom-Up Quantification
CCM Chama Cha Mapinduzi (Ruling Party in the United
Republic of Tanzania)
CD4 Cluster of Differentiation 4
CDC Centers for Disease Control and Prevention
CDs Cluster of Differentiations
CEmONC Comprehensive Emergency Obstetric and Neonatal Care
CEPD Continuing Education and Professional Development
CHF Community Health Financing
CHMT Council Health Management Team
CHSB Council Health Service Board
CHWs Community Health Workers
CLS Community Laboratory Services
CLSI Clinical and Laboratory Standards Institute
COPECSA College of Pathologists, East, Central and Southern Africa
CPD Continuing Professional Development
CPL Central Pathology Laboratory
CTRL Central Tuberculosis Reference Laboratories
DDHCTSU Director, Diagnostic and Health Care Technical Services
Unit
DED District Executive Director
DHs District Hospitals
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Abbreviations Acronyms
DLTs District Laboratory Technologists
DMO District Medical Officer
DO Data Officer
DP Development Partner
DQA Data Quality Assurance
DTS Dried Tube Specimen
EAC East African Community
EAPHLN East African Public Health Laboratory Network
ECSA East, Central, and Southern African
eLIS electronic Laboratory Information System
eLMIS Electronic Logistics Management Information System
EOC Emergency Operations Centre
EQA External Quality Assessment
FBO Faith Based Organisations
FYDP Five Years’ Development Plan
GCLA Government Chemistry Laboratory Agency
GHSA Global Health Security Agenda
GOT Government of Tanzania
HCRF Health Commodity Revolving Fund
HCTS Health Care Technical Services
HIV Human Immunodeficiency Virus
HLI Health Links Initiative
HLPC Health Laboratory Practitioners’ Council
HMIS Health Management Information Systems
HMTs Health Management Teams
HSSP IV Health Sector Strategic Plan IV
HSSP V Health Sector Strategic Plan V
I-TECH International Training and Education Centre for Health
IATA International Air Transport Association
ICAP International Center for AIDS Care and Treatment
Programs
IDSR Integrated Disease Surveillance and Response
IHR International Health Regulations
IMPACT Information Mobilise for Performance Analysis and
Continuous Transformation
IMTU International Medical and Technological University
ISBN International Standard Book Number
ISO International Organization for Standardization
IT Information Technology
KCMC Kilimanjaro Christian Medical Centre
KIU Kampala International University
KPI Key Performance Indicator
LEMM Laboratory Equipment Management Module
LIO Laboratory Information Officer
LIS Laboratory Information System
LMIS Logistic Management Information System

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Abbreviations Acronyms
LO Logistic Officer
LQA Laboratory Quality Assurance
MDG Millennium Development Goals
MeLSAT Medical Laboratory Scientists Association of Tanzania
MMAM Mpango wa Maendeleo ya Afya ya Msingi
MNCH Mother and Neonatal Child Health
MoH Ministry of Health
MOU Memorandum of Understanding
MS Marketing Surveillance
MSc Master of Science
MSD Medical Stores Department
MSD Medical Stores Department
MTBDR Mycobacterium Tuberculosis Drug Resistance
MTEF Medium Term Expenditure Framework
MUHAS Muhimbili University of Health and Allied Sciences
NACP National AIDS Control Programme
NACTE National Council for Technical Education
NBTS National Blood Transfusion Services
NCDs N0n-Communicable Diseases
NGO Non-Governmental Organisation
NHLS National Health Laboratory Services
NHLSP National Health Laboratory Strategic Plan
NIMR National Institute for Medical Research
NMCP National Malaria Control Programme
NMLSSP III National Medical Laboratory Services Strategic Plan III
NPHL National Public Health Laboratory
NPHLA National Public Health Laboratory Agency
NRL National Reference Laboratory
NSCLQS National Sub-Committee on Laboratory Quality System
NSGHLS National Standard Guidelines for Health Laboratory
Services
NSGRP National Strategy for Growth and Reduction of Poverty
NSLQMS National Sub-committee for Laboratory Quality
Management Systems
NTDs Neglected Tropical Diseases
NTLP National Tuberculosis and Leprosy Programme
PEPFAR President’s Emergency Plan for AIDS Relief
PHDR Poverty and Human Development Report
PHIA Population-based HIV Impact Assessment
PHLB Private Health Laboratory Board
PHLS Public Health Laboratory Services
PLHIV People Living with HIV and AIDS
PO-RALG President’s Office-Regional Administration and Local
Government
POC Point of Care
PPM Planned Preventive Maintenance

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Abbreviations Acronyms
PPP Public Private Partnership
PT Proficiency Testing
QA Quality Assurance
QC Quality Control
QMS Quality Management System
QO Quality Officer
RAS Regional Administrative Secretary
RBF Results-based Financing
RFM Result Framework Matrix
RHMT Regional Health Management Team
RLQA Regional Laboratory Quality Assurance
RLTs Regional Laboratory Technologists
RMO Regional Medical Officer
RRH Region Referral Hospital
RTQII Rapid Testing Quality Improvement Initiative
SADC Southern African Development Community
SADCAS Southern African Development Community Accreditation
System
SCM Supply Chain Management
SD Standard Deviation
SDG Sustainable Development Goals
SLIPTA Stepwise Laboratory Improvement Process Towards
Accreditation
SLMTA Strengthening Laboratory Management Toward
Accreditation
SO Safety Officer
SWOC Strength Weakness Opportunities and Challenges
TA Technical Assistance
TB Tuberculosis
TCU Tanzania Commission for Universities
THPS Tanzania Health Promotion Support
TIKA Tiba kwa Kadi
TMDA Tanzania Medicine and Medical Devices Authority
TOR Terms of Reference
TOT Trainer Of Trainee
TSPAS Tanzania Service Provision Assessment Survey
URT United Republic of Tanzania
WHO World Health Organization
ZACDS Zonal Advisory Committee on Diagnostic Services

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TERMS AND DEFINITIONS
For the purposes of this NMLSOP document, these terms and definitions will apply:

Terms Applicable Definitions

Demand Refers to consumers' desire to acquire the services, and
the willingness to pay for it.
Access Refers to the ability of people to reach places and services
and the ability of places to be reached by people and
goods
Quality Refers to the degree to which a set of inherent
characteristics of products or service fulfils requirements
Resilience Refers to the process of adapting well in the face of
adversity, trauma, tragedy, threats, or significant sources
of stress - such as relationship problems, serious health
problems, or workplace and financial stressors
Accountability Refers to responsibility of an individual to complete
assigned tasks and to perform the duties required by their
job
Learning Refers to the process of acquiring new understanding,
knowledge, behaviours, skills, values, attitudes and
preferences
Operational plan Refers to a comprehensive and actionable plan that
defines how team’s functions and activities contribute to an
organisations overall business goal

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FOREWORD
This is the first National Medical Laboratory Standard Operating Procedure
(NMLSOP).

The document outlines the Standard Operating Procedures that the laboratory tests
are to be performed in various facilities during the period of one year’s towards
strengthening the provision of quality medical laboratory services. The plan was
developed through consultative process involving various stakeholders within the
Ministry of Health and other Ministry of the government of Tanzania.

This plan describes the background of medical laboratory sciences, as well as the
context in which it was established in the country. It also features the current roles,
functions and structure of the organisation. Furthermore, contents of the plan include;
stakeholders’ analysis, strengths, weaknesses, opportunities, and challenges that face
medical laboratory services. In identifying these elements, SWOC and Political,
Economic, Social, Technological, Environmental and Legal (PESTEL) analysis were
used.

The NMLSOP have been developed in line with the other medical laboratory’s technical
procedures. This NMLSOP is presented in seven (7) chapters set in a path to
overcome the weaknesses and challenges as well as proving a tool to take advantage
of organisation’s strength in exploring the existing opportunities. The SOPs include
purpose, scope, responsibilities, principle and many steps. This NMLSOP is expected
to be reviewed annually from January 2024 to January 2025.

Successful implementation of this NMLSOP depends on the availability of technical

and financial resources to coordinate its execution. Hence, all stakeholders should be
aware of their roles and responsibilities at the national, regional, and council levels
outlined in this strategy. It is expected that, MoH through the Director, Diagnostic and
Health Care Technical Services Unit (DDS) will provide the required leadership and
guidance.

Dr. John A. K. Jingu

PERMANENT SECRETARY

vi
ACKNOWLEDGEMENTS
The National Medical Laboratory Standard Operating Procedure (NMLSOP) is a
product of dedicated efforts and contributions of various stakeholders. The MoH
acknowledges the contribution of PO-RALG, Development and Implementing Partners,
Non-Government Organisations (NGOs), Institutions or facilities (National Hospitals,
Zonal Hospitals, Specialised Hospitals, Regional Referral Hospitals, District Hospitals,
Health Centers, Dispensaries), Regulatory Agencies (Health Laboratory Practitioner’s
Council and Private Health Laboratories Board), Programmes (NASHCoP, NTLP,
NMCP, NBTS) and Individuals towards improving the quality of health care service
delivery through improved medical laboratory services.

In particular, the MoH would like to thank the Global Fund (GF) and Centres for
Disease Control and Prevention (CDC) for their financial and technical support through
the consultancy in development of this strategic plan. Special appreciations are
extended to technical experts and individuals for their active participation and
constructive inputs and comments provided in reviewing this guideline (ANNEX 01).

Our appreciations go to the Director of diagnostic and Health Care Technical Services
Unit (DDS) Dr Alex S. Magesa for steering up the whole process; Acting Head of
Laboratory Services (Ag. HLS) Mr. Reuben S. Mkala for field coordination; and to all
laboratory technical team or individuals who played a pivotal role in developing this
NMLSOP by participating in the consultation workshops, meetings, providing relevant
information and offering their expert opinion when consulted. Last but not the least;
appreciations go to David Ocheng, who facilitated the whole

Prof.Tumaini J. Nagu
CHIEF MEDICAL OFFICER

vii
EXECUTIVE SUMMARY
In developing this National Medical Laboratory Standard Operating Procedure
(NMLSOP) for the period 2024-2025 consideration has been made on a number of
National and International guiding and operational guidelines. National Standard for
Medical Laboratories (2017); Health Care Technology Policy Guideline (2004);
Operational Plan for the National Laboratory System to Support HIV and AIDS Care
and Treatment (2005); National Laboratory Quality Assurance Framework to Support
Health Care Interventions (2007); Standard Guidelines for the Facilities and
Operations of Forensic Bureau Laboratory (2008); International Health Regulations
(IHR) of 2005; Global Health Security Agenda (GHSA) 2024 Framework; and the
Ruling Party Election Manifesto 2020.

The main objective of this SOPs is to enable the laboratories at all levels to effectively
and efficiently carry out their core functions of laboratory tests as stipulated by their
mandate, strategically coordinate and allocate the available resources by prioritising
functions with effective impact in line with the National Health Policy (2007) and the
Sustainable Development Goals (SDGs).

There are four chapters in this document. Chapter one provides; a brief introduction
and background information, justification and objectives for the NMLSOP.

This National Medical Laboratory Standard Operating Procedure has four strategic
directions:

1. Enhanced conducive Political, Social and Economic environment for Medical

Laboratory services (Resilience);
2. Improved and strengthened Medical Laboratory Diagnostic services (Access);
3. Heightened effective collaboration and partnerships for coordinated action
(Demand);
4. Strengthened Quality Management systems, Surveillance and Monitoring and
Evaluation (Quality).

For each objective above, strategies, targets, activities and performance indicators
were derived. The NMLSOP Matrix specifically describes the sequence of objectives,
strategies, targets, activities and performance indicators are considered the guiding
framework for the development and implementation of annual operational plans.

Page 8 of 245
Scope
The scope of this National Medical Laboratory Standard Operating Procedure
(NMLSOP) is to provide a step-by-step instruction for performing laboratory tests.
Primary Beneficiaries
The primary beneficiaries are Laboratory Practitioners’ who perform examination of
biological samples in the laboratories and non-laboratory testers performed specialised
laboratory tests at point-of-care testing sites.

Secondary Beneficiaries
The Secondary users of this document will include but not be limited to:

Policy makers, medical care managers and administrators, medical devices regulatory
authorities, medical laboratories, intervention programmes, Regional Health
Management Teams and Council Health Management Teams, zonal medical
equipment workshops, biomedical engineers, healthcare technical services and end-
users engaged in strengthening the quality of medical diagnostic services in the
country.

Additionally, other beneficiaries include: Development and Implementing Partners who

provide technical assistance to support the Government of Tanzania (GOT) to
implement the medical laboratory agenda, public and private medical institutions and
laboratories, and higher learning medical laboratory training institutions, prospective
accrediting agencies, medical professional bodies and clinical laboratory medicine.

The objective of NMLSOP is to ensure that Laboratory personnel implement National

Medical Laboratory Standard Operating Procedure for continuity of quality medical
diagnostic laboratory services as needed by the clients.

Page 9 of 245
CHAPTER 1: SAMPLE COLLECTION
1.0 GENERAL CONSIDERATIONS
The collection of samples for laboratory tests from patients consists of following steps:

i. Documentation/Registration of the patient

ii. Collection of sample
iii. Dispatch of sample to respective department
1.1.1 Handling of Biological Samples
Laboratory staffs are often confronted with the problem of handling of biological
samples from patients. The following must be observed for personal protection;

i. All biological samples must be considered hazardous and infected.

ii. Wearing of personal protective equipment (PPEs),
iii. Exercising due care to prevent spillage/splashes while transferring blood to
containers from syringe,
iv. The sample containers be labelled with adequate information.
1.1.2 Samples for Culture
i. As far as possible samples for culture should be obtained before
administration of antimicrobial agents.
ii. If it is not possible, then the laboratory should be informed about the
therapeutic agent(s) so that this fact is considered before issuing laboratory
report.
iii. Material should be collected from the appropriate site where the likelihood
and possibility of isolation of suspected organisms is high.
iv. Sometimes patient’s active participation is necessary for sample collection
v. (sputum or urine), so he/she should be instructed properly and accordingly.
vi. Sufficient quantity of samples is to be collected to permit complete
examination.
vii. Samples are to be placed in sterile containers.
viii. Some samples are directly collected in culture media. Contact laboratory if
such collection is required.
ix. Proper labelling of samples should always be done with patient’s name, test
type, date and site of collection etc.
x. The relevant clinical information is to be recorded on the request form.
xi. Any condition, circumstances or situation that will require special procedures
should also be noted on the request from.
xii. Samples should be collected during working hours except in emergency, so
that the services of qualified microbiologist will be available to directly
supervise processing of the sample.

10
 xiii. The most appropriate samples for isolation of viral, chlamydial or rickettsial
agents depend on the nature of the illness.
xiv.The material should be collected as early as possible in the acute phase of
the disease, because these agents tend to disappear relatively rapidly after
the onset of the symptoms.
xv. Vesicle fluid is preferably collected in a syringe or capillary pipette and
immediately diluted in an equal volume of skimmed milk or tissue culture
medium.
xvi.All samples for viral culture should be frozen and stored at -70°C until culture
is initiated.
1.1.3 Dispatch of Samples from Reception to the Laboratory Sections
i. Match the containers and respective request forms, number them and enter in the
dispatch register/computer.
ii. Verify while handing over/taking away to respective department of the laboratory.
iii. Notify the concerned department about urgent and special tests.
1.1.4 Sample Transportation
i. Exterior of the container should not be soiled/contaminated with the samples.
ii. Sufficient absorbent materials must be used to pack the sample, so that it absorbs
the spilled liquid in case of leakage/breakage during transit to reference/referral
laboratory.
iii. Sample containers must be leak proof and unbreakable. Plastic containers are
preferred.
iv. Samples must be promptly delivered to the laboratory for valid results.
v. If applicable, appropriate transport media should be used.
vi. Samples are to be refrigerated or incubated at 37°C, as the case may be, if there is
a delay in transport of samples to laboratory.
vii. An appropriately filled request form should always accompany all samples to guide
the pathologist/ health laboratory practitioner in selection of suitable media or
appropriate technique.
1.2 COLLECTION OF BLOOD SAMPLES
Consider the following recommended order of veins during blood drawing;

• Median cubital vein (first choice),

• Cephalic vein (second choice),
• Basilic vein (third choice).
1.2.1 Blood sample for serology
i. Serological tests are required in most of the bacterial, viral and parasitic
diseases.
ii. A clotted blood sample is preferred.
iii. A vacuum collection system is both convenient as well as reliable.

11
 iv. Wherever applicable, paired samples are to be collected during acute and
convalescent phases of illness in certain viral and other infections to
document a diagnostic rise in antibody titre.
v. Protect blood samples from extremes of heat and cold during transport.
vi. Whole blood is to be stored at 2-8°C.
vii. Serum can be frozen at -20°C or lower temperature for long term storage.
1.2.2 Blood sample for culture
i. Make sure you have the appropriate media for blood culture, as the media
may vary depending upon the type of pathogen suspected.
ii. Wash the hands with soap and water and wear sterile gloves.
iii. Withdraw the blood following the procedure described on the procedure for
collection of venous blood. Change needle before injecting the blood into the
culture bottle.
iv. Thoroughly clean the rubber bung of the culture bottle with iodine solution
and inject an amount of blood equal to 10% of the volume of medium (for 30
ml medium 3 ml blood and for 50 ml medium, 5 ml blood is needed).
v. After the needle has been removed, the site should be cleaned with 70%
alcohol/spirit swab again.
vi. Don’t store the containers and caps separately.
vii. Blood obtained for culture of suspected anaerobes should not be exposed
to air in any way.
1.2.3 Venous blood
i. Welcome and greet the patient, introduce
yourself.
ii. Make the patient sit comfortably on the
phlebotomy chair. iii. Identify the patient by
asking his/her particulars and compare them
with the request form.
iii. Check the request form for the requested investigations and Inform the
patient about the samples to be collected.
iv. Where possible, ask the patient to remove any tight - fitting sleeved clothing,
or to roll up loose sleeves.
v. Lable the containers before obtaining sample.
vi. Select syringe of appropriate size so that the quantity of blood required can
be obtained in single prick. If multiple or high volume of samples is required,
use a butterfly needle or a canula.
vii. Select appropriate vein (preferably antecubital) from forearm. Cleanse the
skin over the venepuncture site in a circle approximately 5 cm in diameter
with 70% alcohol/spirit swab and allow to air dry, do not blow.
viii. If the sample is to be collected for blood culture then skin is to be thoroughly
sterilised, following the procedure as follows:
a. Starting in the centre of a circle

12
 b. apply 2% iodine (or povidone-iodine) in everwidening circles until the
entire chosen area has been saturated with iodine.
c. Allow the iodine to dry on the skin for at least 1 min.
d. Completely remove the iodine with 70% alcohol/spirit swab following the
pattern of application.
viii. Apply a tourniquet tight enough to obstruct venous flow only and relocate
the vein to be punctured but don’t touch the proposed site of needle entry
or the needle itself. Ask the patient to clench the fist to make the veins
prominent. If the vein is not visible, palpate it with fingers. In case the veins
of forearm are not visible/palpable, other sites such as dorsum of the hand
may be selected.
ix. With bevel up, insert the correct needle size on the veins at an angle
between 15 - 30o then draw the blood into the appropriate collection tubes,
(make sure the patient arm in a downward position to prevent reflux).
x. Mix immediately after drawing each tube that contain an additive by gently
inverting the tube 8 - 10 times. Do not mix vigorously in order to avoid
haemolysis.
xi. Release the torniquet from the patient, Withdraw the needle and apply
pressure to the puncture site using dry cotton balls/gauze pad. Do not
withdraw the piston too forcefully as it can collapse the vein and it may cause
frothing/ haemolysis of the sample.
xii. Apply pressure with thumb on antiseptic swab at puncture site for 2 - 4 min
till the blood ooze stops.
xiii. If syringe was used, safely remove the needle from the syringe before
distribution.
xiv.The blood from syringe is distributed to appropriate, labelled containers.
NOTE:
In case of multiple blood sample collection, consider the following recommended order
of draw;

i. First tube blood culture,

ii. Second tube: non-additive tube (e.g. red stopper),
iii. Third tube: coagulation tube (e.g. blue stopper),
iv. Last tube: additive tube (e.g. lavender or green tube).
1.2.4 Capillary Blood Collection
i. Wear sterile gloves
ii. Assemble All Collection Tools
iii. Clean the ring fingertip with 70% isopropyl alcohol swab or 70% spirit starting
the middle and leave outward to prevent contaminating the area,
iv. Leave the site to air-dry,
v. Hold the finger firmly place the new sterile lancet device at the site on the
finger,

13
 vi. Wipe first drop of blood with a clean dry gauze or cotton wool,
vii. Collect the sample with the second drop of blood using appropriate sample
collection devices such as blood capillary tube,
viii. Apply pressure with a clean dry gauze pad or cotton wool until bleeding stop,
ix. Transfer the collected blood sample into appropriate sample container or
testing devices.
1.2.5 Procedure for Performing Neonate Capillary Blood Collection
i. Use the most medial or lateral portions of the planter surface of the heel
Limit the depth of the puncture wound by using an automated lancet.
ii. Only consider using the whole plantar surface of the foot (using automated
lancets of 2.2mm in length or less) for neonates over 33 weeks’ gestation if
they are having multiple/frequent heel pricks
iii. Position the neonate: ensure the foot is lower than the body.
iv. Choose a puncture site – do not use a previous puncture site.
a. Clean the heel site (i.e. gauze and water) if the foot appears unclean (e.g.
faecal material).
b. Encircle the foot with the palm of the hand and the index finger.
c. Make a quick puncture with the automated lancet device
d. Wipe off the first drop of blood with a gauze swab
e. Collect the sample with the second drop of blood using any of the
collection devices such as slides or rapid test
f. Apply pressure with a clean dry gauze pad until bleeding stop
1.3 COLLECTION OF URINE SAMPLES

Urine samples are collected for routine and culture examinations to diagnose urinary
tract infections (UTIs), both lower UTIs (cystitis-infection of the bladder) and upper UTIs
(pyelonephritis-infection of the kidney). Unlike most other cultures, colony counts are
done on urine samples to determine the number of organisms present in the sample.
Generally, >100,000 organisms/ml of a single isolate indicate an individual has a UTI.
However, mixed UTIs do occur and some individuals with UTIs will have counts lower
than 100,000 organisms/ml.

Most organisms that cause UTIs are normal enteric flora, including E. coli, Proteus,
Klebsiella, Enterobacter, and Enterococcus species. In young, otherwise healthy,
sexually active females, Staphylococcus saprophyticus can be found to be the cause
of UTI.

UTIs caused by Proteus species can be complicated by the formation of urinary calculi
or stones. The large amount of urease that Proteus produces can alkalinize the urine.

14
If there are minerals such as phosphates or carbonates in the urine, the alkaline pH
can cause them to precipitate out and form stones.

1.3.1 TYPES OF URINE SAMPLE

A. First morning urine sample
It provides concentrated urine as the bladder incubated it the whole night. It is best for
nitrite, protein, good for microscopic examination and culture and sensitivity. The casts
may have deteriorated and bacteria may affect true glucose reading.

B. Random (routine) urine sample

It is the most common type and most convenient sample. It is good for observing
physical characteristics, chemical analysis and identification of casts, crystals and
cells.

C. Second-voided urine sample

The first morning sample is discarded and second sample is collected. Formed
elements remain intact.

D. Mid stream (clean catch) urine sample

The portion of urine that does not contain the first and last portions of the sample.

E. Post-prandial
It is collected after meal (usually after 2 hours). It is good for glucose and protein
estimation. Urine sugar testing now has limited diagnostic or prognostic value.

F. Timed sample
It is a combination of all voiding over a length of time. Two-hour sample is good for
urobilinogen and 24-hour sample is good for quantitative urinary components
estimation. Timed urine samples are collected in dynamic function tests.

G. Foley catheter
Disinfect a portion of the catheter with alcohol, puncturing the tubing directly with a
sterile syringe and needle and aspirate the urine. Place urine in a sterile container, it
should never be collected from drainage bag.

H. Suprapubic urine
Urine sample collected by suprapubic aspiration and cystoscopy.

1.3.1.1 Procedures for Collection of Urine Sample

15
Urine sample is often collected by patient him/herself. Therefore, the patient needs to
be properly instructed to have correct sample collection. An uncontaminated
midstream urine (MSU) sample is the best and following methods are to be used for
its collection:

A. Females
i. Wash the genital area thoroughly with - Clean water (may be omitted for
urine Routine Examination).
ii. With two fingers of one hand, hold the outer folds of vagina (labia) apart.
With the other hand, rinse the area from the front to the back with running
tap water.
iii. Start urination so that the stream of urine should flow without touching the
skin. After a few moments, place a sterile container under the stream of
urine. Remove it from the urine stream the moment required amount of urine
is collected.
iv. Secure and tighten the cap on the container.
B. Males
i. Wash the genital, area thoroughly with Clean water (may be omitted for urine
Routine Examination).
ii. Start urination and after a few moments, place a sterile container under the
stream of urine. Collect the required amount of urine and remove the
container from urine stream.
iii. Secure and tighten the cap.
C. Infants, uncooperative and debilitated patients
i. Plastic bags may be attached after careful and thorough washing of genital
area.
ii. The bags should be watched so that they can be removed immediately after
patient has passed the urine.
iii. If the patient has not voided urine within 30 min the collecting bag is
removed.
iv. Patient needs to be re-scrubbed and a new collection device is to be
attached.
D. Urine collection for Mycobacterium tuberculosis
i. Three consecutive early morning samples (>90 ml each) collected in sterile
container are superior to 24h collection.
ii. Boric acid (1.6%) is used as preservative in case of 24h urine collection in
exceptional situations e.g., when patient cannot report daily for sampling.
iii. Suprapubic aspiration in ward by a doctor is preferred in catheterised
patients.

16
1.4 FAECAL SAMPLES COLLECTION PROCEDURE
Faecal samples are collected for routine and culture examinations to find the causative
agent of infectious diarrhoea. Rectal swabs are often helpful in identifying the cause of
acute bacterial diarrhoea when stool sample cannot be collected readily.
It is important to remember that there are many causes of diarrhoea other than
infectious agents such as metabolic disorders, certain drugs, and food intolerances
(allergies). In a routine stool culture, the following organisms are often isolated:
Salmonella species, Shigella species and Vibrio cholera. Viruses and parasites are
also common causes of diarrheal disease.
1.4.1 Procedure for Collection of faecal samples
i. Faeces should be passed directly into a clean, waxed cardboard container
that is fitted with a tight cover.
ii. Avoid contact with residual soap/detergent, disinfectant or urine in the
bedpan.
iii. Faeces obtained are transferred to another clean, wide mouthed and screw
capped container. The sample should include any pus, blood, mucus or
formed elements that may have passed with stool.
iv. Sample (~1 ml) is added to 10 ml sterile alkaline peptone water in suspected
cholera cases.
v. If viral infection is suspected, faeces are extracted with sterile buffered
saline. Faeces (~1 ml) are mixed with 9 ml sterile buffered saline, allowed to
sediment for 30 min (or centrifuged). The supernatant is transferred to a
sterile container, frozen and kept below -40°C until processed.
1.5 COLLECTION OF BODY FLUIDS
The primary body fluid that are collected for routine and culture examinations includes;
cerebrospinal fluid (CSF), joint fluid, pleural fluid, and peritoneal (ascites) fluid.

1.5.1 Cerebrospinal fluid (CSF)

i. CSF routine and culture examinations are performed to diagnose
meningitis due to Viruses, fungi, and bacteria. Acute bacterial meningitis
(ABM) is a medical emergency.
ii. The age and immune status of the patient influence the type of bacterial
pathogen most likely to cause ABM:
iii. Neonates 0 – 2 months; E. coli, Streptococcus agalactiae - Group B
streptococci, Listeria monocytogenes
iv. months - 2 years; Haemophilus influenza and Neisseria meningitidis
v. Older than 2 years; Neisseria meningitides – most common in children and
young adults, Streptococcus pneumoniae – most common in older adults
1.5.1.1 Procedures for Collection of CSF samples

17
 CSF is normally collected from sub-arachnoid space of spinal cord at lumber level by
puncture with a long needle. A physician in the ward under strict aseptic conditions
performs the procedure.

i. Sample shall be collected in 2-4 ml quantities in 3-4 sterile screw capped

bottles that are serially numbered and must be sent to the laboratory
immediately.
ii. In case CSF is to be cultured for M. tuberculosis then at least 5 ml sample
is needed. CSF shall be tested as soon as it arrives in the laboratory.
iii. CSF in the first bottle is sometimes contaminated with blood and should
be kept aside.
iv. Fluid from second bottle is used for routine tests while fluid from third
bottle is used for bacterial culture etc.
v. If tuberculous meningitis is suspected, 4th bottle is kept in refrigerator
undisturbed to see whether a pellicle or coagulum forms.
NB: CSF must never be refrigerated (if for bacterial culture as it kills H.
Influenzae) and should be kept at 37°C.
1.5.1.2 Body Effusions (Exudates and Transudates)
An effusion is fluid which collects in a body cavity or joint. Fluid which collects due to
an inflammatory process is referred to as an exudate (needs investigations) and that
which forms due to a non-inflammatory condition is referred to as a transudate (needs
no microbiological investigations). Effusions include; pleural, pericardial, synovial,
peritoneal, and hydrocele fluids.

1.5.1.3 Pleural and Pericardial Fluids

Main purpose of testing is to ascertain their transudative or exudative nature and to
find a causative organism if an infective process is indicated. See sputum sample for
list of organisms that can be isolated from pleural samples.

1.5.1.4 Peritoneal Fluid - Ascites

The common indications for paracentesis are ascites of unknown origin, suspected
intestinal perforation, haemorrhage or infarct, infections like tuberculosis,
complications of cirrhosis (spontaneous bacterial peritonitis) and suspected
intraabdominal malignant disorders.

1.5.1.5 Joint fluid (synovial fluid)

Joint fluid cultures are performed to diagnose septic arthritis (most cases of arthritis
are NOT infectious; they are due to strain on a joint or immunological diseases).

18
The three most common causes of septic arthritis are: Staphylococcus aureus,
Neisseria gonorrhoea, and Coagulase-negative staphylococci in patients with joint
replacement prosthetics

1.5.1.6 Hydrocele Fluid

Usually from the sac surrounding the testes. Occasionally Wuchereria bancrofti
microfilariae and rarely Brugia species can be found in hydrocele fluid.
1.5.1.6.1 Collection of aspiration fluids (effusions)
i. Collection of synovial, pleural, pericardial, peritoneal, or hydrocele fluid is
carried out by a medical officer or competent nurse.
ii. Label each container with the date and the patient’s identifiers
iii. After aspiration, aseptically dispense the fluid into 3 tubes as follows:
• 5 to 10 ml is in a sterile tube for microbiological examination.
• 5 ml in anticoagulant (heparin, trisodium citrate or EDTA) for
estimation of cell count and protein concentration.
• 2-3 ml in a plain tube and allowed to clot (normal fluid does not clot).
iv. If the sample cannot be examined immediately, fluid should be frozen and
stored at -70°C until examined.
COLLECTION OF GENITAL SAMPLES
i. Indicated for the diagnosis of bacterial sexually transmitted diseases,
primarily gonorrhea (GC) or non-gonococcal cervicitis or urethritis (NGU).
The most common cause of NGU is Chlamydia trachomatis.
ii. immunological and molecular tests for the diagnosis of chlamydial
infections includes (PCR, DNA probes, etc.) .
iii. Vaginal secretions are also sent to the laboratory for the diagnosis of
vaginitis.
iv. The diagnosis of vaginitis can be made with a wet mount – yeast,
trichomonas, bacterial vaginosis (BV), culture – yeast, or Gram stain –
yeast and BV.
v. Urethritis, cervicitis: Neisseria gonorrhoeae, Chlamydia trachomatis, Other
agents of NGU
vi. Vaginitis: Candida albicans, Trichomonas vaginalis and BV
1.5.2 Urethral swabs
Possible pathogens; Neisseria gonorrhoea, Chlamydia trachomatis, and Trichomonas
vaginalis.
1.5.3 Collection of urethral discharge from male patients
i. Cleanse around the urethral opening using a swab moistened with sterile
physiological saline.
ii. Gently massage the urethra from above downwards.
iii. Using a swab, collect a sample of discharge.

19
 iv. Make a smear of the discharge on a microscope slide by gently rolling the swab
on the slide. This will avoid damaging pus cells which contain the bacteria.
Note: Very few pus cells may be present if the patient has recently passed urine.
Allow 2–4 hours after urination before collecting a samples.
i. When culture is indicated (see previous test),
ii. Collect a sample of pus on a sterile cotton-wool swab.
iii. If possible, before inserting the swab in a container of Amies transport medium,
inoculate a plate of culture medium.
iv. Label the samples and deliver them to the laboratory as soon as possible.
v. Isolation of N. gonorrhoeae from urine
Note:
• A rectal swab is also required from homosexual patients. A selective medium is
required to isolate N. gonorrhoea from a rectal sample.
• In acute urethritis, it is often possible to detect N. gonorrhoea in pus cells passed in
urine, especially the first voided urine of the day (centrifuged to sediment the pus
cells).
1.5.4 Cervical swabs and possible pathogens
A. From non-puerperal women:
Neisseria gonorrhoea, Chlamydia trachomatis (serovars D-K), Streptococcus
pyogenes, herpes simplex virus.
B. From women with puerperal sepsis or septic abortion:
Streptococcus pyogenes, other beta-haemolytic streptococci, Staphylococcus
aureus, Enterococcus species, anaerobic cocci, Clostridium perfringens,
Bacteroides, Proteus, Escherichia coli and other coliforms, Listeria
monocytogenes.
1.5.5 Collection of cervical samples from female patients
i. A samples collected from the endocervical canal is recommended for the
isolation of N. gonorrhoeae by culture. Use a sterile vaginal speculum to
examine the cervix and collect the samples.
ii. Moisten the speculum with sterile warm water, and insert it into the vagina.
iii. Cleanse the cervix using a swab moistened with sterile physiological
saline.
iv. Pass a sterile cotton-wool swab 20–30 mm into the endocervical canal and
gently rotate the swab against the endocervical wall to obtain a samples.
v. When gonorrhoea is suspected, before inserting the swab in Amies
transport medium, if possible inoculate a plate of culture medium. Label

20
 the sampless and deliver to the laboratory as soon as possible. Inoculated
culture plates must be incubated within 30 minutes.
1.5.6 Vaginal Swabs
Vaginal discharge may be due to infection of the vagina or infection of the cervix or
uterus. Pathogens causing vaginal infections include Trichomonas vaginalis, Candida
species, and Gardnerella vaginalis with anaerobes.
1.5.7 Collection of vaginal discharge
To detect T. vaginalis, C. albicans and G. vaginalis
Two preparations are required:

A. Wet preparation to detect motile T. vaginalis

i. Use a sterile swab to collect a samples from the vagina.
ii. Transfer a sample of the exudate to a microscope slide.
iii. Add a drop of physiological saline and mix.
iv. Cover with a cover glass.
v. Label and deliver to the laboratory for immediate examination
B. Dry smear for Gram staining to detect Candida and examine for clue cells
Although yeast cells can be seen in an unstained wet preparation, the Gram positive
cells and pseudohyphae of C. albicans are more easily seen in a Gram stained smear.
i. Use a sterile swab to collect a samples from the vagina.
ii. Transfer a sample of the exudate to a microscope slide and spread it to make a thin
smear.
iii. Allow the smear to air-dry, protected from insects and dust.
iv. Label and deliver to the laboratory with the wet preparation.
1.5.8 Collection of samples to detect T. pallidum
To detect motile T. pallidum spirochetes, a samples must be collected before antibiotic
treatment.

i. Wearing protective rubber gloves, cleanse around the ulcer (chancre) using
a swab moistened with physiological saline. Remove any scab which may
be present.
ii. Gently squeeze the lesion to obtain serous fluid. Collect a drop on a clean
cover glass and invert it on a microscope slide.
iii. Immediately deliver the preparation to the laboratory for examination by
darkfield microscopy

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1.6 WOUND SAMPLES TO INCLUDE PUS, ABSCESS, TISSUE, ETC.

Indicated for primarily to diagnose skin and soft tissue infections (SSTIs). SSTIs may
be caused by a variety of organisms; different organisms depending on how the wound
or injury occurred. Fungi, parasites, and viruses are also important causes of certain
types of SSTIs.

Community-acquired: Staphylococcus aureus, Streptococcus pyogenes, Clostridium

perfringens and other anaerobic bacteria).

Hospital-acquired: Staphylococcus aureus, Enteric Gram-negative rods – E. coli,

Pseudomonas aeruginosa, Acinetobacter species, and other non-fermenting
Gramnegative rods, Streptococcus pyogenes, Clostridium species and other
anaerobic bacteria

1.5.9 Collection of Wound samples - General considerations

i. Samples should be collected by a medical officer or an experienced nurse.
ii. Pus from an abscess is best collected at the time the abscess is incised and
drained, or after it has ruptured naturally.
iii. When collecting pus from abscesses, wounds, or other sites, avoid
contaminating the sample with commensal organisms from the skin.
iv. As far as possible, a samples from a wound should be collected before an
antiseptic dressing is applied.
v. When pus is not being discharged, use a sterile cotton-wool swab to collect a
sample from the infected site.
vi. Immediately after collection, immerse the swab in Amies transport container.
vii. Label the samples and as soon as possible deliver it with a completed request
form to the laboratory.
viii. When myeloma is suspected: Obtain a samples from a draining sinus tract using a
sterile hypodermic needle to lift up the crusty surface over the sinus opening. This
method of samples collection has the advantages that the pus obtained is usually free
from secondary organisms and the draining granules can usually be seen clearly and
removed for microscopic examination. Transfer the pus to a sterile container.

22
 ix. When tuberculosis is suspected: Aspirate a sample of the pus and transfer it to a sterile
container.
x. When the tissue is deeply ulcerated and necrotic (full of dead cells): Aspirate a sample
of infected material from the side wall of the ulcer using a sterile needle and syringe.
Transfer to a sterile container.
xi. Fluid from pustules, buboes, and blisters: Aspirate a samples using a sterile needle and
syringe. Transfer to a sterile container.
xii. Serous fluid from skin ulcers, papilloma, or papules, that may contain Treponema:
Collect a drop of the exudate directly on a clean cover glass and invert it on a clean
slide. Immediately deliver the samples to the laboratory for examination by dark-field
microscopy.
Caution: Samples from patients with suspected plague or anthrax are highly
infectious. Label such samples HIGH RISK and handle them with care. In a health
centre for dispatch to a microbiology laboratory Collect the samples using a sterile
cotton-wool swab.
Insert it in a container of Amies transport medium, breaking off the swab stick to allow
the bottle top to be replaced tightly.
In a hospital with a microbiology laboratory
• Using a sterile technique, aspirate or collect from a drainage tube up to 5 ml of
pus.
• Transfer to a leak-proof sterile container. When the material is aspirated fluid
from a pustule, transfer the fluid to a sterile, leak-proof container. Stopper, and
seal in a leak-proof plastic or metal container.
Note: It is not possible to transport exudate from a suspected treponemal ulcer
because the Treponema remain motile for only a short time.
Make a smear of the material on a clean slide (for Gram staining) and allow to air-dry
in a safe place. Heat-fix the smear.
Caution: Do not make a smear for transporting when the samples is from a
patient with suspected anthrax.
Send the samples with a completed request form to reach the microbiology laboratory
within 6 hours.
1.8 COLLECTION OF THROAT AND NASAL SWABS
Throat cultures are performed to diagnose streptococcal pharyngitis (infection with
Streptococcus pyogenes (Group A streptococci). The most common causes of
pharyngitis (sore throat) are viruses, which cause over 75- 80% of all cases. Of the

23
bacteria that cause pharyngitis, Group A streptococci is the major cause and therefore,
with few exceptions the only bacteria that is reported from a throat culture is
Streptococcus pyogenes.
Exceptions include:
Corynebacterium diphtheria which in areas of the world where vaccination is prevalent,
is a rare cause of pharyngitis
Neisseria gonorrhoea can cause pharyngitis, however many pharyngeal infections with
N. gonorrhoea are mild or asymptomatic.
If the physician suspects either of these two organisms, he/she must let the lab know
because the isolation of either requires special culture techniques.
1.5.10 Throat Swabs
Throat swab cultures are to be taken under direct vision with good light.
Areas of exudation, membrane formation, any inflammation or if not seen then tonsillar
crypts are the sites of choice.
1.5.11 Nasal swabs
Nasopharyngeal swabs are better taken by treating physician/surgeon himself. For
recovery of viral agents, washings are collected after gargles with nutrient broth by the
patient.
Nasal Sample for Mycobacterium leprae
The nasal sample for M. leprae can be taken as follows:

Nasal swab
i. Make the patient sit with his head bent backwards but facing the light.
ii. Insert and repeatedly rotate the swab into one of the nasal cavities, against
upper part of the nasal septum.
iii. Make 2-3 evenly spread smears.
iv. Air dry the slides, wrap in a paper and send to the laboratory.
Nasal washings and nasal blow
i. Make the patient sit.
ii. Place a few drops of sterile saline in the nose.
iii. After 3 min, ask the patient to blow hard his nose on a small sheet of plastic or
cellophane. (This plastic or cellophane can be given to the patient to take it
home and ask him to blow hard onto the sheet, the following morning, soon after
waking and before washing.
iv. The patient can bring it directly to the laboratory).
v. Transfer some of the mucus pieces from the washing to a slide with a clean
wooden stick and make thin smear.

24
 vi. Air dry slide and send it to the testing area
Collection of Nasopharyngeal Swabs
Assemble equipment for Nasopharyngeal swab collection and PPEs for prevention of
infections.

i. Lable the VTM with required information and fill the register and all necessary
forms with all necessary information.
ii. Remove the swab from the package. Do not touch the soft end with your hand
or anything else.
iii. Insert the nasopharyngeal swab into the nasopharnx region.
iv. Leave in place for a few seconds.
v. Slowly remove swab while slightly rotating it.
vi. Break the applicator’s stick end and put tip of swab into ATM containing vial
VTM
COLLECTION OF OROPHRYANGEAL SWABS
Assemble equipment for orophryangeal throat swab collection and prevention of
infections.

i. Label the VTM with required information and fill the register and all necessary
forms with all necessary and correct information.
ii. Remove the swab from the package. Do not touch the soft end with your hand
or anything else.
iii. Have the patient open his/her mouth wide.
iv. Insert swab in the area of tonsils.
v. Use tongue depressor if the patient is not able to resist gagging and closing the
mouth while the swab touches the back of the throat near the tonsils.
vi. Rotate swab to obtain adequate sample
vii. Break the applicator’s stick end and put tip of swab into vial containing VTM

1.6 COLLECTION OF SKIN SMEARS

i. Ensure all required material and supplier are in place.
ii. Ask the patient to sit on the prepared chair at phlebotomy.
iii. Confirm the identity of the patient by asking his/her name and compare with
name written on the request form.
iv. Clearly explain to patient what you want to do and ask for verbal consent
v. Select the site, you should take a smear from two sites only;

vi. One ear lobe and One active lesion

1.6.1 Sample from ear lobe

25
 i. Clean the skin at the smear site with swab and let it air dry. ii. Pinch the skin
firmly between your thumb and forefinger
ii. Make an incision in the skin about 5mm long and 2mm deep. Keep on pinching
to make sure the cut remains blood less iv. Turn the scalpel 90o and hold it at
right angle to the cut
iii. Scrap inside the cut once or twice with side of the scalpel to collect tissue fluid
and pulp (there should be no blood in the sample as this may interfere with
staining and reading of the slide
iv. Stop pinching the skin and absorb any bleed with dry cotton swab
v. Spread the material scrapped from the incision on to the slide, ensure you
spread it evenly with the flat of the scalpel making 8mm diameter
vi. Clearly label your slide with patient ID similar to that appearing on patient
request form
1.6.2 Sample from active lesion
i. Rub the scalpel with cotton wool drenched with alcohol.
ii. Pass the blade through the flame of the spirit burner for 3-4 seconds and let it
to cool without touching anything
iii. Select the most active looking lesion (active means lesion that are raised and
reddish in colour)
1.7 SPUTUM SAMPLES
Sputum cultures are performed to diagnose infections such as pneumonia and
pulmonary tuberculosis that is caused by Mycobacterium tuberculosis. Bacteria
associated with Community-Acquired Pneumonia (CAP):

i. Streptococcus pneumoniae – most common bacterial cause of CAP

ii. Haemophilus influenzae
iii. Moraxella catarrhalis
iv. Staphylococcus aureus - particularly following a viral infection such as influenza
v. Klebsiella pneumoniae – particularly in individuals with chronic conditions such
as alcoholism
vi. Mycoplasma pneumonia* – particularly in young individuals in closed quarters
vii. Bacteria associated with Hospital-Acquired Pneumonia:
a. Streptococcus pneumoniae
b. Enteric Gram-negative rods such as E. coli, Klebsiella, Enterobacter,
Citrobacter, and Serratia
c. Staphylococcus aureus
d. Pseudomonas aeruginosa
e. Acinetobacter species
f. Haemophilus influenzae

26
1.6.3 Sputum Collection Procedure
i. Label the sample container on the body of the container, not on the lid and fill
out the sputum examination request form.
ii. Instruct the patient and demonstrate how she or he can produce and collect
good sputum;
a. Access to a well ventilated place (outside the laboratory working area),
b. Mouth wash (rinsing with water),
c. Breathe in deeply 2-3 times, and breathe out hard each time.
d. Cough deeply from the chest and collect the sputum into the container.
e. Opening and closing the sputum container so as there are no leaks or
smearing on the exterior of the container.
f. Hand wash steps
iii. Emphasize the need for the patient to supply the most useful sample, the
normally thick, yellowish (sometimes blood-streaked), purulent material brought
up from the lungs after a deep, productive cough.
iv. Emphasize that saliva produced by spitting is not sputum. However, if the only
sample the patient can produce is salivary, do submit it to the laboratory as it
can still yield useful information.
v. Encourage the patient to bring the collected sample back to the unit as quickly
as possible.
vi. For M. tuberculosis culture, a series of three fresh, early morning samples (5-
10 ml) are collected and kept in the refrigerator. If amount is less, the patient is
advised to collect 24 h sputum or until 50 ml is obtained.
vii. M.tuberculosis can be recovered from the gastric contents in infants, debilitated
patients and those who are unable to cooperate in the collection of sputum. This
can be obtained by gastric aspiration performed as an indoor procedure.
viii. Gastric washings are better collected early in the morning, in fasting state.
These are neutralised soon after collection by N/10 NaOH.

27
CHAPTER TWO: PARASITOLOGY
2.1. PROCEDURE FOR MALARIA RAPID TEST (MRDT)

2.1.1 Purpose

This procedure provides instructions for In vitro qualitative screening test for detection
of malaria parasites (P. falciparum, P. vivax, P. ovale and P. malariae) in whole blood

2.2.2 Scope

This procedure applies to all Health Laboratory Practitioners who works in parasitology
section on performing MRDT test.

2.2.2.1 Responsible

Head of section is responsible to ensure implementation and competence assessment

for all staff that will perform this test

2.2.3 Principle

Test is based on principle of immune-chromatography in which nitrocellulose

membrane is pre-coated with two monoclonal antibodies as two separates lines. One
monoclonal antibody (test line PAN), is PAN specific to lactose Dehydrogenase
(phLDH) of the plasmodium species (plasmodium falciparum, P. vivax, P. ovale and P.
malariae). And the other line (test line pf) consist of monoclonal antibody specific to
histidine rich protein 2 (HRP2) of plasmodium falciparum. When the test sample along
with assay diluent flows through intercellular membrane, monoclonal antibody
conjugated with colloidal gold which are PAN specific to plDH and falciparum specific
for HRP2 binds to plasmodium antigen released from lysed blood sample. These
antigen –conjugate complex moves through the nitrocellulose membrane and binds to
corresponding immobilised antibody at test lines, which leads to the formation of colour
band/bands indicating reactive results. The control band will appear irrespective of
reactive or non-reactive sample.

2.2.4 Sample Requirements

Whole blood from EDTA/Finger prick

2.2.5 Equipment

Timer

2.2.6 Materials

Gloves, Test kit, Lab Coat, and

28
2.2.7 Storage and Stability

MRDT sample should be tested within 1hour after collection if not possible stored at 2-
8oc for 7 days.

2.2.8 Safety

i. All personal protective equipment (PPE) must be worn when performing this
procedure.
ii. All samples must be regarded as potentially infectious.
iii. Refer to National infection prevention and control Guidelines (IPC)

2.2.9 Calibration

Not Applicable

2.2.10 Quality Control

Process known positive and negative blood sample daily before performing patient
samples.

2.2.11 Procedure Steps

Follow the actions described below for specimen collection step-by-step.

i. Allow all kit components and specimen to room temperature prior to testing
ii. Remove the test device from the foil pouch; place it on a flat, dry surface.
iii.Label the test device with the patient identification number/name
iv. Transfer 5µl of whole blood collected in an inverted cup/special capillary
provided into sample well by touching sample pad. (see manufacture
instruction)
v. Add 4 drops of assay diluents into the squire assay diluents well. (See
manufacture instruction)
vi. Read the result within 15 min. (Do not interpret after 30 min) and read result.
(See manufacture instruction).

2.2.12 Biological Reference Intervals

Not Applicable

2.2.13 Interpretation and Reporting of Results

Negative

i. The presence of one colour band (“C” Control line) within the result window
indicates a negative result. Positive
ii. “C” and “P. falciparum the presence of two coloured bands (“Pf” test line and
“C” control line) within the result window no matter which band appears first,
indicate P.F positive result. Positive

29
 iii. “C”, “P.f” and “Pan” the presence of three colored band (“Pf”, Pan “Test line and
“C” Control line) within the result window no matter which band appears first,
indicate P.F positive or mixed infection of P.F and P.V or P.M or P.O Invalid
result
iv. If no coloured band appear, at control line “c” within stipulated time then the
result is invalid.

2.2.14 Limitations of the procedure and sources of error

Test kit cannot detect malaria antigen if parasites are less than 100. The test is limited
to detect HRP2, an antigen to Malaria Plasmodium species that may persist after
treatment or passed P. falciparum infection.

2.2.15 Performance Characteristics

Refer to package insert

2.2.16 Supporting Documents

Sample collection manual

2.2.17 References

Manufacturer inserts

2.2. PROCEDURE FOR MALARIA MICROSCOPY

2.2.1 Purpose

This procedure provides instructions for the examination of malaria parasite to

diagnose and monitor treatment outcome of malarial infection.

2.2.2 Scope

This procedure is used during examination of malaria parasites at the Laboratory

using Microscopy.

2.2.3 Responsible

• Qualified and trained health Laboratory Practitioners are responsible for

performing this test procedure
• The Head of Parasitology unit is responsible for ensuring the effective
implementation and maintenance of this procedure.

30
2.2.4 Principle

2.2.4.1 Giemsa stain

Giemsa stain is a Romanowsky stain contains methylene blue which is basic stain and
eosin is acidic stain, the malaria parasites has DNA in the nucleus which is basic in
nature and RNA in the cytoplasm is acidic in nature, during staining the reactions takes
place whereby acidic part of a parasite will pick-up basic part of the stain and the basic
part of the parasite will pick-up the acidic part of the stain, that’s why the nucleus of the
parasite will show red color and the cytoplasm will stain bluish. Malaria parasites are
identified by microscopic examination of thick or thin blood films stained with Giemsa.
Thick blood films are used for detecting parasites, thin blood films are used for more
detailed morphological examination and for determining parasite species. Thick blood
films consist of several layers of blood cells, so that a large volume of blood is
examined. The thick film staining technique ruptures red cells, leaving white cells and
parasites intact. The thin film staining technique preserves the morphology of blood
cells and malaria parasites.

2.2.5 Sample Requirements

Whole blood collected in EDTA tube is required Blood slides

2.2.6 Equipment

Hot plate, Tally counters or differential counter, Microscope, Timer, Weighing scale,
and PH Meter

2.2.7 Maintenance

To increase the life-span of microscopes, preventive maintenance, including cleaning

the objectives and replacing parts as necessary. should be part of routine internal QC
and must be properly done and documented. Microscopes should be covered when
not in use to avoid exposure to dust, and proper precautions must be taken in humid
areas to avoid fungal growth on the lenses and in the microscope. Also Hot plate, Tally
counter, Timer, weighing scale and PH meter should be maintained as per
manufacture instructions.

2.2.8 Materials

• High-quality immersion oil should be used according to the manufacturer’s

recommendations.
• High-quality microscope slides, free of surface abrasions, preferably have a
frosted end for labeling and purchased from a reputable supplier should be
used.
• 10% alcohol-based Giemsa stain,
• markers, lancets, syringes, needles, Vacutainer-type needles, alcohol swabs,
lens-cleaning solution, lens-cleaning tissues, buffer tablets, pH calibration
solutions, cotton-wool, gloves, safety glasses (including the over-spectacle
type), filter paper and glycerol. gloves, sharps, boxes, gowns and detergents,
and slide boxes.

31
2.2.9 Storage and Stability

• Unstained blood slides should be stored at 2-8 c for 3 months

• Stained blood slides should be stored at room temperature for 3 months on slide
boxes.

2.2.10 Safety

• Adhere to safety precaution as stated in safety manual/IPC guideline

• All personal protective equipment(PPE) should be worn when performing
procedure All samples should be regarded as potential infectious

2.2.10 Calibration

Calibration of PH meter and weighing scale should be done once per year.
Maintenance of microscopes should be done as planned.

2.2.11 Quality Control

QC slides should be used to check the quality and performance of the Giemsa stain,
Microscope and Laboratory personnel. Malaria-positive and negative blood should be
used to prepare QC thick and thin films. Before examining patient slides, the QC slides
should be checked first, If the QC slides are satisfactory, the patient slides can be
examined. Slides must be selected regularly for cross-checking, either by sending
them to a crosschecking center or during routine supportive supervisory visits.

2.2.12 Procedure Steps

A. Preparation of thick and thin blood film

i. Prepare both thick and thin film on the same microscope glass slide.
ii. Label the slides with the unique patient ID including the date of examination.
iii. Put Slide card for making thin and thick blood films, showing size of blood drops
and area of slide to cover for a thin film and thick film
iv. Put the microscope glass slide on the slide card for thick and thin film
v. Pipette 2µl of blood and pour it on the smallest circle on the slide card
vi. Place the spreader in front of the 2µl drop of blood at a 30 o- 45o angle. Use a
clean microscope slide with a smooth edge as a spreader.
vii. Pull back the spreader and hold until the blood evenly spreads along the width
viii. Push the slide forward in a smooth continuous motion
ix. Avoid hesitation or a jerky motion when spreading the blood
x. Pipette 6µl of blood and pour on the large circle on the slide card.
xi. Using another slide, spread the drop of blood within the confined area to make
a thick film.
xii. Put the slide films on the slide rack and allow it to dry.
xiii. Fix the thin smear for one second by either spraying or dipping into absolute
methanol.
xiv.Air dry the slides on a slide rack with the fixed thin film facing down

B. Preparation of Giemsa Stock Solution (500ml) from Giemsa powder

32
 i. Measure and dissolve 3.8g of Giemsa powder in 250ml of methanol
ii. Measure 250ml of glycerol and add to the solution above
iii. Ripen the stock solution by placing in direct sunlight for about 1 week or place
in water bath of temperature 56oC and shake at interval.
iv. Filter the stock solution before storing in a cool dry place with label and date of
preparation

C. Preparation of 10% Giemsa stain from Giemsa Stock solution

i. Add 1 part of stock solution to the 9 parts of 7.2 buffer solution

ii. Prepare 30ml of 10% working giemsa solution as follows:
iii. 3mls of Giemsa +25ml of buffered water PH of 7.2, mix and transfer to a clean
caped leakproof bottle
iv. Lable and keep in a dry place

D. Staining of blood films

i. Arrange the slides on a staining rack with the sample side facing up
ii. With an aid of a disposable pipette, flood the films with 10% Giemsa solution
and leave to stain for 15 minutes.
iii. Decant the Giemsa and wash in buffered water at pH 7.2
iv. Clean the back of each slide with cotton wool or gauze
v. Air dry the slides

2.2.13 Examination of the blood films

i. Place a drop of immersion on the thick film

ii. Place the slide on the microscope stage
iii. Swing the X10 objective into position and bring the film into focus using the
course adjustment.
iv. Use the X10 and X40 objectives to check quality of the slide(s) before reading
and reporting;
v. Swing the X100 oil immersion objective into position and focus using the fine
adjustment
vi. Choose the correct smear reading pattern either Horizontal, start from up right
to left or Vertical, start from upright down.
vii. Systematically, Examine the thick film.
viii. If you do not see any parasites, continue examining the whole film.
ix. If parasites are seen, start counting number of white blood cells and parasites
simultaneously up to a WBC of 200.
x. If the parasitemia level is less than 10/200WBC, continue to count up 500 WBC
xi. Report the number of parasites count per 200 or 500 white blood cells in the
Blood parasites worksheet.
xii. Retain the read slides for 3 Months

33
2.2.14 Biological Reference Intervals Not Applicable

2.2.15 Reporting and Interpretation of Results Malaria parasites

• Typical Malaria parasites have the following features on the Giemsa stained
films;
• Purple red chromatin dot, Blue cytoplasm, Brown-black/yellowish green pigment
and Distinct morphology

2.2.15.1 Interpretation

In stained blood films, trophozoites appear as red stained chromatin dots with blue
staining cytoplasm. If doubtful on parasites seen, search for definite ones. Do not make
a diagnosis on the basis of structures that resemble rings or chromatin dots alone.
Structures that may be confused with malaria parasites are platelets, portions or other
red cell inclusion bodies

2.2.15.2 Reporting

If no parasites are seen report as “No parasites seen”

If malaria parasites are seen, report species identified and count e.g. “Plasmodium
species seen 50/200WBC”

2.2.16 Critical values

The following are the Panic/Critical values for Malaria microscope test results. In case
of the patient result falling inside the indicated values, call the Doctor or relevant ward
and record the details of the conversation on the Panic Result Book

Test Critical value

Malaria for Under 5 years >100 P. falciparum
Asexual/200wbc’s
Malaria for >5 years& adult >1000 P. falciparum
Asexual/200wbc’s
None Tropical people ANY POSITIVE

2.2.17 Limitation of the Procedure and Sources of Errors

• Poor storage of reagents or using the reagents after expiry date may cause false
results.
• Difficulty in distinguishing young ring-stage parasites

2.2.18 Perfomance characteristics

Refer to the method verification report of this procedure.

34
2.2.19 Supporting Documents Not Applicable

2.2.20 References

• Practical Laboratory Manual-Jane Carter and Orgenes Lema

• Standard Operating Procedure Essential Laboratory Tests {AMREF-2008} EXT
120
• Basic Malaria Microscopy Part I. Learner’s Guide, Second edition, WHO.

2.3 PROCEDURE FOR URINE MICROSCOPY

2.3.1 Purpose

The purpose of this procedure is to provide step by step instructions for performing
macroscopic and microscopic examination of urine sediment samples

2.3.2 Scope

This procedure is used during examination of urine samples in Parasitology section in

medical laboratory.

2.3.3 Responsible

The Registered medical laboratory personnel is responsible for effective

implementation and maintenance of this procedure.

2.3.4 Principle

2.3.4.1 Macroscopic Examination

The urine is visualized with naked eyes to determine its appearance (turbidity and
colour).

2.3.4.2 Microscopic Examination

The urine sediment is analyzed by a microscope to observe the presence of white

blood cells, red blood cells, parasites and other abnormalities in urine sample.
Identification of cells (WBC (pus cells), RBGs and Epithelial), casts, crystals,
amorphous phosphates, bacteria and parasites in urine is based on their different
cellular and intra-cellular morphology under light microscopy.

2.3.5 Sample Requirements

20 mL, minimum 1 mL Fresh, cleanly voided urine collected in a clean container

Early morning voided mid-stream urine. Other samples include;

Random urine (collected at any time of the day)

35
Terminal urine sample collected at any time of the day for demonstrating ova of
Schistosoma haematobium

First voided urine sample in the morning is used to demonstrate Trichomonas vaginalis
in males

In infants and babies, a random urine sample collected as early as possible is used for
all types of urine investigation.

2.3.6 Equipment

Microscope, Centrifuge machine, Refrigerator, Timer, and Forceps

2.3.7 Materials

Gloves, Cover slips, Glass slides, Test tubes, Centrifuge tubes, Gauze, Grease pencil,
Lens paper, Waste containers, Urine container, Laboratory coat, Marker pen

2.3.8 Storage and stability

Process urine sample within 1hour of collection, if not possible refrigerate at 2-8oC
immediately and test within 12 hours.

2.3.9 Safety

i. Adhere to safety precautions as stated in the Safety Manual

ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
iv. Refer to National infection prevention and control Guidelines for health waste
management and safety practice.

2.3.10 Calibration

Urine analyzers, timer and centrifuge should be calibrated as per schedule

Maintenance of all equipments should be done as planed

2.3.11 Quality Control

Quality control should be performed daily before processing patient samples by

analysing known positive and negative samples.

2.3.12 Procedure

2.3.12.1 Macroscopic examination

Observe the appearance of the urine for color and clarity/turbidity

36
2.3.12.2 Microscopic examination

i. Label the centrifuge tubes with the laboratory unit number using a grease pencil
and arrange into a rack.
ii. If the urine sample is to be tested both for urinalysis and culture, mix well by
inverting three times and aliquot the sample into a centrifuge tube and label
exactly as the original sample
iii. Transfer the centrifuge tubes containing urine sample into the centrifuge.
iv. Make sure the centrifuge tubes are balanced well.
ii. Centrifuge the urines at 3000rpm for 5 minutes.
iii. Remove the centrifuge tubes from the centrifuge
iv. Pour off the supernatant into the sink with running water and leave the urine
deposit
v. Transfer the rack containing arranged centrifuge test tubes to the working area
vi. Arrange the marked slides according to the number of urine sample
vii. Re-suspend the urine deposit by tapping the bottom of the centrifuge tube.
viii. Transfer a drop of the deposit onto a clean glass slide.
ix. Put on a cover slip
x. Place the slide on the microscope stage and reduce the iris diaphragm
xi. Examine the slide systematically using x 10 objective and count the cells, casts,
crystals, amorphous phosphates and parasites (these structures are more likely
to be seen around the edges of the cover slip.
xii. Swing the x40 objective into position
xiii. Open the iris diaphragm very slightly to allow just enough light to provide a
contrast of cells, casts, crystal and amorphous phosphates against the bright
back ground (these structures are more likely to be seen around the edges of
the cover slip).
xiv.structures cannot be seen if a very bright light is used
xv. Put the used glass slide and cover slip in a container with disinfectant.

2.3.13 Biological Reference interval

Macroscopy
Color Straw - Dark yellow
Appearance Clear - Hazy

Microscopy
Red blood cells: 0–2/hpf.

WBC (PUS CELL)/Pus cells: 0–5/hpf

Casts 0-4/hpf

Bacteria Negative

2.3.14 Interpretation and Reporting of Results

A. Macroscopic

37
Report the Turbidity and color of the urine e.g. Clear yellowish, Blood stained etc.

B. Microscopic

• Report a range of the actual highest number of WBC (pus cells), RBC and
Parasites per high power field as counted under x40 objective (e.g. 0 – 2
RBC/HPF, 3 – 5 WBC/HPF, 10 – 12 S. haematobium ova/HPF) etc.
• For crystals, Epithelial cells, yeasts or casts, report as +, ++ or +++.
o Where + indicates 1 to 9 observed findings per field;
o ++ indicates 10 to 100 observed findings per field;
o +++ indicates above 100 observed findings per field.
• If no cells or parasites are seen report as “No parasites or cells seen.”

C. Results interpretation

• Schistosomes Haematobium ova in urine indicates Schistosomiasis disease

• Presences of more than 3 Red blood cells for males, more than 8 red blood cells
for females and any RBC or WBC (pus cell) for children and notification of more
than 8 WBC (Pus Cell) in either male or female is pathological significant.
• White blood cells may indicate infection or inflammation.
• Red blood cells may indicate kidney disease, a blood disorder, or bladder
cancer.
• Bacteria can indicate infection.
• Skin cells can indicate infection or kidney disease.
• Crystals may be a sign of kidney stones.
• Casts, or tube-shaped proteins, may be a sign of a kidney disorder.
• Parasites can indicate parasitic disease in various parts of the body.

2.3.15 Limitations of the Procedure and Sources of Errors

• Inadequate centrifugation of sample

• Poor collection of urine sample
• Prolonged storage of urine sample at wrong temperature Expired reagents
• Technical competency level
• Presence of artifacts such as plant cells

2.3.16 Performance Characteristics Not Applicable

2.3.17 Supporting document Sample Collection Manual
2.3.18 References

• Monica Cheesbrough: District Laboratory Practice in Tropical Countries, Vol 1,

Tropical Health Technology, 1998.
• Practical Laboratory Manual-Jane Carter and Orgenes Lema

38
2.4 PROCEDURE FOR STOOL ROUTINE EXAMINATION

2.4.1 Purpose

This procedure provides instructions for examination of stool macroscopic and

microscopic to detect abnomalities/Parasites. The most common parasites include the
roundworms Ascaris lumbricoides and Necator americanus (commonly called
hookworm); the tapeworms D. latum, Taenia saginata, and, rarely, T. solium; the
amoeba E. histolytica; and the flagellate G. lamblia.

2.4.2 Scope

This procedure is used during examination of stool in Parasitology Section.

2.4.2.1 Responsible

• The Registered medical laboratory personnel are responsible for implementing

this test procedure.
• The Head of Parasitology section is responsible for ensuring the effective
implementation and maintenance of this procedure

2.4.3 Principle

Normal saline retains the morphology of the organism in its natural shape and color,
free helminthes eggs from debris.

Iodine - stains the internal structure of cyst to brown/yellow color so to allow the study
of cyst morphology. It also kills the organisms to allow internal structure easily seen.

2.4.4 Sample Requirements

Stool collected in clean plastic screw capped container which has spoon like. Include
macroscopic worms or worm segments as well as bloody and mucoid portions of the
sample.

About 150mg stool should be collected.

2.4.5 Equipment

Microscope, Tally counters or differential counter, Centrifuge and Fume hood

2.4.5.1 Maintenance

Maintenance of microscopes should be done as planned

39
 2.4.5.2 Materials

Normal saline, 1% Iodine, 10% formalin, Wooden applicator stick, Grease pencil,
Gloves, Microscopic slides, Cover slip, Marker and Stool container

2.4.5.3 Storage and stability

Stool sample should be at the laboratory within two hours after collection. If a liquid or
soft stool sample can’t be examined within 30 minutes of passage, place it in a
preservative; if a formed stool sample can’t be examined immediately, refrigerate it or
place it in preservative.

2.4.6 Safety

• All samples must be considered as potentially infectious and must be handled

and examined with care.
• All personal protective equipments (PPE) should be worn when performing
procedure
• Adhere to safety precautions as stated in the Safety manual
• Refer to National infection prevention and control Guidelines for health waste
management and safety practice.

2.4.7 Calibration

Calibration of centrifuge should be done as per schedule

2.4.8 Quality Control

Quality control should be performed daily before processing patient samples by using
known positive and negative samples

2.4.8.1 Procedure

A.Macroscopic Observations

Observe the stool sample for the following; Color of the samples, Consistency,
Presence of blood, mucus, and, or, pus. Whether the samples contain worms.

B.Wet Preparation by Saline and 1% Iodine

i. Using marker label a microscope slide with the laboratory number

ii. Place a drop of fresh physiological saline at one end of a slide and a drop of 1%
Iodine on the other end.
iii. Using applicator stick, pick up a size of match head stool (~2 mg) and mix with
a drop of saline and a similar amount with Iodine to make a smooth thin preparation.

40
 iv. If stool is formed, the portion should include the inside and outside parts of the
sample
iv. For mucoid or watery stool, mix the entire contents before picking a portion to
mix with saline.
v. Cover the preparations with a cover slip.
vi. Start with 10x objective to systematically, examine the entire saline preparation.
vii. Use the 40x objective to assist in the detection and identification of parasitic
elements (eggs, ova, cyst etc). Always examine several microscope fields with this
objective before reporting ‘No parasites seen’.
viii. Use the iodine preparation to assist in the identification of cysts.
ix. Report the findings on Stool analysis worksheet.
x. Discard the used microscope slide on the container with disinfectant.

2.4.9 Biological Reference Intervals

Not Applicable

2.4.10 Interpretation and Reporting of Results

2.4.10.1 Interpretation

i. Adult helminths or portions of helminths may be recovered and seen with a

naked eye. Examples include E. vermucularis adult worms, Ascaris lumbricoides adult
worms, and tapeworm proglottids.
ii. Occasionally, other helminths may be recovered (hookworm, Strongyloides
stercoralis), but identification requires the use of the microscope.
iii. The appearance of stool will yield diferrent interpretation such as; blood and
mucus in faeces: might be suggestive of amoebic dysentery, intestinal
schistosomiasis, invasive balantidiasis (rare infection), and severe T. trichiura
infections. Other non-parasitic conditions in which blood and mucus may be found
include bacillary dysentery, Campylobacter enteritis, ulcerative colitis, intestinal
tumour, and haemorrhoids.
iv. Presence of pus: This can be found when there is inflammation of the intestinal
tract. Many pus cells can be found in faecal sampless from patients with bacillary
dysentery. They can also be found in amoebic dysentery but are less numerous.
v. Pale coloured and frothy (containing fat) samples: might be suggestive of
giardiasis and other infections as- sociated with intestinal malabsorption.
vi. Pale coloured faeces: (lacking stercobilinogen) might be suggestive of an
obstructive jaundice.
vii. Mucoid and blood diarrhea might be suggestive to presence of E.
histolytica

2.4.10.1 Reporting

A.Macroscopic Findings - Report the following:

41
 i. Colour of the samples.
ii. Consistency, i.e. whether formed, semiformed, unformed, watery.
iii. Presence of blood, mucus, and, or, pus. If blood is present note whether this is mixed
in the faeces. If only on the surface this indicates rectal or anal bleeding.
iv. Presence of worms, e.g. A. lumbricoides (large roundworm), E. vermicularis
(threadworm) or tapeworm segments, e.g. T. solium, T. saginata.
B.Microscopic Findings – Report the Following

• Report presence of any ova, trophozoites or cysts seen, specifying the species,
e.g. “Entamoeba histolytica trophozoites seen”.
• If no parasites are seen report as “ No ova or cysts seen”.

2.4.10.2 Limitation of the Procedure and Sources of Error

• Delay in examination of stool sample may cause missing of some parasites in

wet prepation which are dectected still alive. Examples of such organisms are
Strongloides stercolaris, Giardia lamblia E. Histolytica trophozoites, etc.
• Presence of urine kills trophozoites (false-negative results). Excessive heat or
cold.

2.4.10.3 Performance Characteristics

Refer to the method verification of this procedure.

2.4.10.4 Supporting Documents Not Applicable

2.4.10.5 References

• Practical Laboratory Manual-Jane Carter and Orgenes Lema

• Monica Cheesbrough: District Laboratory Practice in Tropical Countries,Vol 1,
• Tropical Health Technology, 1998.
• Brunner & Suddarth’s Handbook of Laboratory and Diagnostic Tests, 2010.

2.5 PROCEDURE FOR EXAMINATION OF BLOOD FOR MICROFILARIAE

2.5.1 Purpose

This procedure provides instructions for processing of blood, lymphatic, and

cerebrospinal fluid for the recovery of lymphatic filariasis. Diagnosis of filarial infections
is often confirmed by demonstration of the parasite.

42
2.5.2 Scope

This procedure is used during examination procedures to diagnose filarial infections in

the Laboratory using Microscopy.

2.5.3 Responsible

• Qualified and trained Medical Laboratory Technician, s are responsible for

performing this test procedure.
• The Head of Parasitology unit is responsible for ensuring the effective
implementation and maintenance of this procedure.

2.5.4 Principle

A. Giemsa stain

Giemsa stain is a Romanowsky stain contains methylene blue which is basic stain and
eosin is acidic stain, the parasites has DNA in the nucleus which is basic in nature and
RNA in the cytoplasm is acidic in nature, during staining the reactions takes place
whereby acidic part of a parasite will pick-up basic part of the stain and the basic part
of the parasite will pick-up the acid part of the stain, that’s why the nucleus of the
parasite will show red color and the cytoplasm will stain bluish.

B. Wet preparation

Microfilariae are seen in wet preparations of blood on direct microscopic examination

or in the deposit of a blood sample after lysis with formalin and centrifugation.

2.5.5 Sample Requirements

Whole blood collected in EDTA tube should be collected around midnight (22:00 –
04:00 for W. Bancrofti and 10:00 – 15:00 for L. Loa), as this is the time when parasite
is present in the blood for microfilaria worms.

2.5.6 Equipment

Hot plate, Tally counters or differential counter, Microscope, Centrifuge, Timer,

Weighing scale and pH Meter

2.5.7 Materials

• 10 % Giemsa stain working solution,

• 70% Methylated spirit,
• Distilled water,
• Immersion oil,
• Tap water,

43
 • buffer solution,
• Cover slips,
• Glass slide,
• Rack,
• Slide drying rack,

2.5.8 Storage and Stability

The Giemsa stock stain should be stored in a dark bottle and take precautions to avoid
moisture entering the stain.

2.5.9 Safety

• All sampless must be considered as potentially infectious and must be handled

and examined with care.
• All personal protective equipments (PPE) should be worn when performing
procedure
• Adhere to safety precautions as stated in the Safety manual
• Refer to National infection prevention and control Guidelines for health waste
management and safety practice.

2.5.10 Calibration

Calibration of centrifuge, pH meter and weighing scale should be performed as

planned. maintenance of microscopes should be done as planned

2.5.11 Quality Control

Positive and negative control samples are processed once a day in the morning before
patient samples.

2.5.12 Procedure Steps

Step 1: Procedural Steps - Wet Preparation

i. Collect 10 ml of venous blood and dispense it into 10 ml of water.

ii. Mix the blood gently in water and leave for 10 minutes to give time for all the
red cells to lyze.
iii. Centrifuge the haemolyzed sample for 10 minutes at slow to medium speed,
i.e. RCF 300–500 g.
iv. Using a Pasteur pipette, immediately remove and discard the supernatant fluid.
v. Transfer the sediment to a slide, add a small drop of methylene blue and cover
with a cover glass. The stain will be taken up by the nuclei and show whether
the microfilariae are sheathed.

44
 vi. Examine the entire preparation microscopically for motile microfilariae using the
10× objective with the condenser iris closed sufficiently to give good contrast.
vii. Count the number of microfilariae in the entire preparation. Divide the number
counted by 10 to give the approximate number of microfilariae per ml of blood
(mf/ml).
viii. If unable to identify the species with certainty, continue with step 2 (examination
with 10% Giemsa stain under oil immersion objective).

Step 2: Procedural Steps – 10 % Giemsa Staining Procedure

i. Remove the cover glass and add a small drop of plasma, serum, or albumin
solution.
ii. Mix and spread thinly. Allow the preparation to dry completely.
iii. The addition of albumin, plasma, or serum (known to be microfilaria-free) will
help to prevent the preparation from being washed from the slide during
staining.
iv. Fix with absolute methanol or ethanol for 2–3 minutes.
v. Flood the films with 10% Giemsa solution and leave to stain for 15 minutes.
vi. Decant the Giemsa and wash in buffered water at pH 7.2 or distilled water
vii. Clean the back of each slide with cotton wool or gauze
viii. Place a drop of immersion on the thick film and place the slide on the
microscope stage
ix. Swing the x10 objective into position and bring the film into focus.
x. Swing the x100 oil immersion objective into position and focus.
xi. Examine the thick film systematically starting from the top left hand corner and
move from field to field.

2.5.13 Biological Reference Intervals - Not Applicable

2.5.14 Reporting and Interpretation of Results

A. Microfilaria

Species are identified by noting the arrangement of the nuclei towards the end of the
tail and the presence of sheath. W. bancrofti and Loa loa have a sheath; M. perstans
does not have a sheath. In Loa loa and M. perstans, the nuclei reach the tail tip and
the tail tip is rounded. In W. bancrofti, the tail tip is pointed.

B. Trypanosomes

Trypanosomes have elongated, flat, narrow bodies, often curved. In wet preparations,
they move rapidly by means of an undulating membrane and flagellum. In stained
preparations, the kinetoplast, a dark staining round body from which the flagellum
originates, is seen.

45
2.5.15 Limitation of the Procedure and Sources of Errors

• Incorrectly timed samples for microfilaria or trypanosomes

• Poor storage of reagents or using the reagents after expiry date

2.5.16 Performance Characteristics

Refer to the method verification report of this procedure.

2.5.17 Supporting Documents Sample collection manual

2.5.18 References

• Practical Laboratory Manual-Jane Carter and Orgenes Lema

• Standard Operating Procedure Essential Laboratory Tests {AMREF-2008} EXT
120

46
CHAPTER THREE: BLOOD TRANSFUSION
3.1 PROCEDURE FOR ABO AND RHESUS BLOOD GROUPING

3.1.1 Purpose

This procedure provides instructions for performing ABO and Rhesus (D) blood
grouping using tube method.

3.1.2 Scope

This procedure is used in Blood Transfusion unit when performing ABO & Rhesus (D)
blood group typing for donors and patients.

3.1.3 Responsible

The head of Blood Transfusion and competent medical laboratory personnel are
responsible for ensuring this procedure is effectively implemented and maintained.

3.1.4 Principle

This method is based on immunophenotyping principle. The known antibodies A&B

(antisera) react with unknown antigens on the red cells surface to form agglutination
or haemolysis; this is known as forward or cells grouping. Also known antigens (A
and B) react with unknown antibodies in the patient or donor serum/plasma to form
agglutination or haemolysis; this is known as backward or serum blood grouping.

The D antigen on the red cells surface reacts which known D antibodies (anti-D) to
form agglutination which determines the Rhesus group of an individual, either as Rh
(D) Positive (agglutination) or Rh (D) Negative (no agglutination).

3.1.5 Sample Requirements

2-3mls of EDTA sample, 2-3mls of clotted sample from plain tube centrifuge the sample
at 3500rpm (RCF) for 5 minutes.

3.1.6 Equipment

Centrifuge, Microscope, Refrigerator, Timers, 37˚C water bath and Thermometer

47
3.1.7 Materials

Reagent Consumables
Anti –A Gloves

Anti-B Laboratory coat

Anti-D (saline, IgM) Test tubes, Test tube rack,

slides
Incomplete anti-D (IgG)
Marker pens,
Anti-Human Globulin Serum (AGS)
Beakers,
0.85% Physiological Saline
Pasteur pipettes,
Low ionic strength solution (LISS)

3.1.8 Storage and Stability

• Store serum or plasma at 20C -80C for 7 days.

• Whole blood is stored at 20C - 80C for 3 days
• Anti-sera should be kept at 20C – 80C or as per manufacturer instructions

3.1.9 Safety

• Adhere to safety precautions as stated in the Safety manual/ IPC guideline

• All personnel protective equipment (PPE) must be worn when performing
this procedure.
• All samples must be regarded as potentially infections.

3.1.10 Calibration

Calibration of auxiliary equipment should be done as per calibration schedule

3.1.11 Quality Controls

Perform Quality Controls once in a week, when new antisera received and when new
control cells are prepared.

3.1.11.1 Controlling of anti-sera

• Commercial anti-sera are quality controlled by reacting them with known cell
Suspension of A, B, O, Rh (D) Positive and Rh (D) Negative.
• Arrange 6 tubes labelled A, B, AB, O and D positive and D negative.
• Put one drop of 2-5% cell suspension of A, B, AB, O Rh (D) Positive and O
Rh (D) Negative to the corresponding tubes above
• Add one drop Anti-A, Anti-B, Anti-AB and Anti-D into corresponding tubes.

48
3.1.11.2 Controlling of O sensitized cells

• Label two tubes as O sensitized cells and O un sensitised cells

• Put one drop of corresponding of 3-5% cell suspension in the two tubes
above.
• In the two tubes, add one drop of AHG each.

3.1.11.3 Controlling of physiological saline

• Label two tubes as saline and distilled water

• Put one drop of saline and one drop of distilled water to the respective tubes
above
• Add one drop of O Rh (D) Positive cells to the two tubes

3.1.11.4 Common step

• Centrifuge all tubes above at light speed (1000rpm for 1 minute).

• Agglutination or haemolysis indicates positive reactions.
• Expected reactions are shown in table below
• Record results on ABO & Rh blood.

3.1.11.5 Interpretation of IQC results

Commercial Cell suspensions

A B AB Rh D Rh D O- Unsensitised
Antisera cells cell s cells sensitized cells
pos Neg cells

cells Cells
Anti A + - + -
Anti B - + + -
Anti AB + + + -
Anti D +
LISS +++
AHG + -
Saline + to ++
Distilled Haemolysis
water

Key:

+ Agglutination

- No agglutination

Note: Strength of agglutination is graded from 1+ (separate agglutination) to 3+

(one solid agglutination)

49
3.1.12 Procedure Steps

Prepare of 2- 5% cell suspension

i. Place 2-3 drops of donor red blood cells into a tube

ii. Fill the tube (3/4) with 0.85% normal saline
iii. Centrifuge the tube at 3400rpm for 2 to 3 minutes. Decant supernatant fluid.
(Repeat 3 times)
iv. Transfer a drop of packed red cells from the above tubes and add 19 drops of
saline to make 2% to 5% donor red cell suspension

3.1.13 Blood grouping procedure

A. Forward/Cell Grouping

i. For each patient/donor label 3 test tubes as Anti-A tube (A), Anti-B tube (B),
and anti-D tube (D).
ii. Arrange the labelled test tubes in the test tube rack
iii. Add one volume of anti A into tube A
iv. Add one volume of anti-B into tube B
v. Add one volume of anti-D into tube D
vi. To each of the above tubes add one drop of 2 - 5% cell suspension and mix
well.
vii. Centrifuge the three tubes at 1000rpm for one minute
viii. Examine the contents of the tubes for the evidence of agglutination.
ix. Read, interpret, and record the test results.

B. Backward/Serum Grouping

i. For each patient/donor, label 2 test tubes as A cells and B cells.

ii. Add two drops of serum to each tube. iii. Add one drop of A reagent cells
into tube labelled A cells,
iii. Add one drop of B reagent cells into tube labelled B cells
iv. Mix the contents of the tubes gently
v. Centrifuge the tubes at 1000 rpm for one minute.
vi. Examine the serum for evidence of haemolysis, gently suspend the cell
bottoms and examine them for agglutination macroscopically and
microscopically.
vii. Read, interpret and record test results.

3.1.13.1 Procedure for Weak Rh (D)

i. If the above anti-D reaction is negative, confirm weak Rh (D) as follows:

ii. Add two drops of LISS
iii. Incubate at 37˚C for 15 minutes in water bath. In absence of low ionic
strength solution (LISS) incubate at room temperature for 30 minutes
iv. Spin for 1000rpm for 1 minute
v. Observe for agglutination macroscopically and microscopically or for
haemolysis

50
 vi. If agglutination or haemolysis is observed at this stage, report result as Rh
(D) Positive and the procedure ends here.
vii. If there is still no agglutination, proceed as follows:

a. Wash contents of the tube 3 times with physiological saline

b. Discard supernatant after third wash
c. Add one drop Anti-Human Globulin Serum (AGS)
d. Spin for 1000rpm for one minute
e. Observe for agglutination (macroscopically and microscopically) or
haemolysis
f. If agglutination is observed at this stage, report results as Rh (Du)
Positive
g. If reaction is still negative, add one drop of O sensitised cells to the tube
h. Spin for 1000rpm for 1minute
i. Observe for agglutination (macroscopically and microscopically) or
haemolysis
j. Presence of agglutination or haemolysis indicates a valid negative result.
k. Absence of agglutination or haemolysis means the test is invalid;
therefore, it has to be repeated.

Note: For purposes of transfusion, patients with Rh(Du) Positive should be

given rhesus negative blood.

3.1.14 Biological Reference Intervals

Not Applicable

3.1.15 Interpretation and Reporting of Results Interpretation of results

Patient/Donor Cell Grouping Patient/ Donor Serum

Grouping
Anti-A Anti-B Ant-AB Anti-D Blood group & A-cells B- Blood
Rhesus factor group
cells
+ - + + A Rh (D) Pos - + A
- + + + B Rh (D) Pos + - B
+ + + + AB Rh (D) Pos - - AB
- - - + O Rh (D) Pos + + O

KEY: + Means Agglutination

- Means No Agglutination

3.1.15.1 Reporting of results

Results report should include the ABO Type and Rhesus D reaction results, e.g. Blood
group A Rh (D) Positive, or Blood Group A Rh (D) Negative

51
3.1.16 Limitations of the Procedure and Source of Error

• Avoid haemolysed samples as this may lead to false negative results.

• Patients who have had recent multiple transfusions may develop
alloantibodies that can interfere with antigen – antibody reactions

3.1.16 Performance Characteristics

Refer to the method verification report

3.1.17 Supporting Documents

Sample collection manual, safety manual, and quality manual.

3.1.18 References

i. Technical manual of the American Association of Blood Banks

ii. Mollison P.L., Blood Transfusion in Clinical Medicine 8th Ed. Oxford.
Blackwell Scientific, Practical haematology by Decie iii. Guidance manual on
“ABO and Rh blood grouping” (Institute of Biologicals-India)
iii. Anti-sera insert kit (Anti-A, Anti- B, Anti-AB, Anti- D Monoclonal blood
grouping antibodies for tube and slide test) T Tulip diagnostics (P) LTD.

3.2 PROCEDURE FOR ESTIMATION OF HAEMOGLOBIN BY USING COPPER

SULPHATE SOLUTION

3.2.1 Purpose

To provide guidance on the procedure of Haemoglobin estimation level to blood donors

using the Copper Sulphate solution technique

3.2.2 Scope

This procedure provides guidance on haemoglobin estimation of blood donors using

copper sulphate (CUSO4) method in NBTS Blood collection teams and its satelites.

3.2.3 Responsible

Trained qualified and competent certified registered medical personnel and other
authorised medical personnel.

3.2.4 Principle

A blood droplet is allowed to fall into copper sulphate solution of a specific gravity 1.053
and the movement of droplet is observed, If the specific gravity is higher than solution,
the drop will sink within 15 sec or else it will remain suspended for some time.

52
3.2.5 Sample Requirements

Capillary Blood

3.2.6 Equipment

Not applicable

3.2.7 Materials

• Reagents: 70% Ethyl alcohol, Copper Sulphate solution with specific gravity
of 1.053
• Consumables: Sterile swabs, Picker, Sharp container, Capillary tubes,
Gloves, Universal bottle, Waste containers and Timer

3.2.8 Preparation of Copper II Sulphate solution

i. Weigh 170 gms of hydrous Copper Sulphate Powder/Crystals

ii. Put into volumetric flask
iii. Dissolve 170gms of hydrous Copper Sulphate Powder/Crystals with 1 litre
of distilled water.
iv. Mix well until all Crystals dissolves
v. Label the solution as STOCK SOLUTION with Preparation date, Batch
number and expiry date

NB: Calculate expiry date six months from date of preparation

vi. Store and keep stock solution at room temperature in a tightly capped
brown glass bottle.

3.2.9 Prepare Working Solution

To prepare 1 Litre of working solution;

• Dispense 480mls of distilled water using measuring cylinder into a volumetric

flask.
• Add 520mls of prepared stock solution using measuring cylinder - Mix
thoroughly

3.2.10 Storage and Stability

Copper Sulphate working solution stored at room temperature in brown bottle in three
months

53
3.2.11 Safety

i. Decontaminate working surfaces twice daily, in the morning and

afternoon and when needed, all generated records are kept.
ii. Adhere to safety precautions as stated in the Safety manual
iii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iv. All samples must be regarded as potentially infections.
v. Refer to National infection prevention and control Guidelines for health
waste management and safety practice.
vi. All spills should be wiped thoroughly using 0.5% sodium hypochlorite
solution.

3.2.12 Calibration

Not applicable

3.2.13 Quality Control

Reagent: Test specific gravity of copper sulphate solution using Hydrometer

i. Accept the copper sulphate solution if the specific gravity is 1.053

ii.Reject copper sulphate if specific gravity is not equal to 1.053
iii.
Record findings of the copper sulphate inspection and testing report form.
iv.Then select sample with slightly bellow and slightly high with a cut off value
of High (12.6 -13.0) g/dl and Low (12.0 – 12.4) g/dl.
v. Accept the copper sulphate solution, if drop of blood with haemoglobin level
less than 12.5 g/dl floats and blood with haemoglobin level higher than
12.5g/dl sinks. Perform corrective action if results are outside an acceptable
limits

3.2.14 Procedure Steps

Follow the actions described below step-by-step:

i. Explain the Procedure to the Blood Donor

ii. Welcome and greet the donor, offer a chair to seat
iii. Explain the procedure and reassure the donor.
iv. Select and clean Site
v. Select donors' middle finger or the finger medial to the middle finger
vi. Clean the upper top of right-side area with cotton wool swab soaked in 70%
ethyl alcohol in a spiral movement starting at the intended site out ward
vii. Leave it until dried do not blow the site
viii. Remove the lancets cover

54
 ix. Using the index finger and thumb, squeeze and hold the donor's·fingertip at
the upper joint tightly and prick
x. Wipe off the first drop of blood once with a dry swab
xi. Draw blood into the Capillary Tube
xii. Gently press the pricked fingertip to draw blood Note; Do not Squeeze the
pricked finger because it may introduce tissue fluid, dilute blood and give
false low Haemoglobin
xiii. Hold Capillary tube at approximately 60 degrees and let the blood flow into
the capillary tube up to not less than 3/4 capacity
xiv.Avoid air entering the capillary tube by ensuring smooth flow and
undisrupted flow of blood into the capillary tube
xv. Close the upper tip of capillary tube by placing your finger tip
xvi.Give clean dry swab to donor and instruct them to press finger with thumb
till bleeding stops.
xvii. Release a drop of Blood to Copper Sulphate Solution
xviii. Hold capillary tube at least 1 cm above the surface of copper sulphate
solution
xix.Release your finger tip to allow one drop of blood freely into labeled
container of fresh daily prepared and quality control copper sulphate
solution.

3.2.15 Biological Reference Intervals

Above or equal 12.5 g/dl

3.2.16 Interpretation and Reporting of Results

• Accept if HB is greater/equal than 12.5g/dl

• Reject if HB is less than 12.5 g/dl

3.2.17 Limitations of The Procedure and Sources of Error

Not applicable

3.2.18 Performance Characteristics

Not applicable

3.2.19 Supporting Documents

Sample collection manual, Quality manual, Safety manual

3.2.20 References

AAB technical manual 12th edition, AfSBTC 4th Edition.

55
3.3 PROCEDURE FOR COMPATIBILITY TESTING

3.3.1 Purpose

This procedure provides instructions for performing compatibility testing, which is used
to select blood and blood components that will not cause harm to the recipient (patient)

3.3.2 Scope

This procedure is used in Blood Transfusion section when performing compatibility

testing prior to issue of a unit of blood to recipient.

3.3.3 Responsible

The head of Blood Transfusion and competent medical laboratory personnel are
responsible for ensuring this procedure is effectively implemented and maintained.

3.3.4 Principle

This method is based on immunophenotyping principle. The known red cell antigens
from donor are mixed with unknown antibodies from the recipient (patient) to detect if
there is any incompatibility caused by ABO, Rhesus and/or other blood groups
antibodies.

3.3.5 Sample Requirements

• Patient/ recipient serum from a clotted blood sample in plain tube

• Donor cells from a segment tubing of the blood unit

3.3.6 Equipment

Centrifuge, Water bath, timer, refrigerator

3.3.7 Materials

Reagent Consumables
Incomplete anti-D (IgG) Test tubes, Test tube rack, grease pencil,

Anti-Human Globulin Beakers,

Serum (AGS) Physiological Saline,

Sensitized cell Pasteur pipettes

56
3.3.8 Storage and Stability

Venous blood must be used within 3 hours at room temperature then after should be
refrigerated at 2-8˚C. Blood Donor units should be stored at 1-6˚C

3.3.9 Safety

• Adhere to safety precautions as stated in the Safety manual

• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections. iv. Refer to National
infection prevention and control Guidelines for health waste management and
safety practice.

3.3.10 Calibration

Calibration should be done as per schedule.

3.3.11 Quality control

Quality Controls for Antihuman globulin should be done on a daily basis before
performing patient samples..Internal quality control should be prepared from patient
samples or known organisation.

3.3.12 Procedure Steps

3.3.12.1 Immediate Spin Saline Technique

i. Select a unit of blood from the blood bank storage with the same group as the
recipient. If there is no unit with the same blood group as the recipient, blood
group O packed red cells can be used as universal donor. Also recipients with
AB blood group are considered as universal recipients of packed red cells.
ii. Label a tube for each donor red cell suspension being tested with the patient’s
serum.
iii. Add two drops of patient’s serum or plasma to each tube.
iv. Add one drop of donorcells suspension into appropriate test tubes
v. Rinse the Pasteur pipette 5 times during transferring of cells and serum to avoid
contamination
vi. Mix the contents of the tube(s) and centrifuge at 1000rpm for 60 seconds.
vii. Gently re-suspend the cell buttons and observe for haemolysis or agglutination.
viii. Read, interpret, and record test results. ix. If compatible, proceed with Indirect
Antiglobulin Technique

57
3.3.12.2 Interpretation and reporting:

• Agglutination or haemolysis means a positive (incompatible) test results

• A smooth suspension of red cells after resuspension of the red cells button
means negative results and indicates a compatible immediate spin cross match.

Note: In emergency cases where the blood unit is required immediately, perform
Immediate Spin Saline Technique. Issue the blood unit if it is compatible then
proceed with Indirect Antiglobulin test. If there is any incompatibility,
immediately call the ward to stop the transfusion and re-call the blood unit.

3.3.13 Biological References Intervals

Not applicable

3.3.14 Reporting and Interpretation of results

3.3.14.1 Interpretation of results

Results should be reported in such a way that will indicate the recipient’s blood group
and the donor’s blood unit number to which the donor’s bloods is compatible or not
compatible.

3.3.14.2 Reporting of results

Report as compatible when there is no agglutination or incompatible when there is no

agglutination.

3.3.15 Limitation of the Procedure and Sources of Errors

• Haemolysis samples may lead to false negative results.

• Patients who have had recent multiple transfusions may develop allo-antibodies
that can interfere with antigen – antibody reactions

3.3.16 Performance Characteristics

Refer to the method verification report.

3.3.17 Supporting Documents

Quality manual, sample collection manual and safety manual

58
3.3.18 References

Pam S. Helekar, D.P. Blackall et.al. American Association of Blood Bank 15 Edition,
1985. (method 3.1) and (method 3.2.1)

59
CHAPTER FOUR: HAEMATOLOGY
4.1 PROCEDURE FOR SICKLING SCREENING TEST
4.1.1 Purpose
The purpose of this procedure is to provide instruction for performing a screening test
to determine abnormal type of Haemoglobin called Haemoglobin S in blood.

4.1.2 Scope
This procedure is used by all trained laboratory staff while performing sickling test.in
the Laboratory

4.1.3 Responsible
Qualified, trained and competent Medical Laboratory Scientist, Technologist and
Technician are responsible for doing this test procedure.
Section heads are responsible for ensuring the effective implementation and
competency assessment for this procedure

4.1.4 Principle
When a drop of blood is sealed between a cover slip and a slide, the decline in oxygen
tension due to oxidative processes in the blood cells leads to sickling. In this method
added with blood drop chemical reducing agents such as sodium met bisulphite. This
rapidly reduces oxyhemoglobin to reduced haemoglobin, and then this will be
accelerating sickling.

4.1.5 Sample Requirements

3 - 4 ml of venous whole blood collected in EDTA tube (purple top vacuum). Sample
must be free from haemolysis, lipemia and icterus.

4.1.6 Equipment
Light Microscope, and Hot plate

4.1.7 Materials
Freshly prepared 2% Sodium Metabisulphite (Diluting 0.2gm in 10ml of distilled water).
Vaseline/paraffin wax, Pipette, Cover glass, Glass slide and Applicator stick

4.1.8 Storage and Stability

Processed whole blood is stable at 2oC to 8oC for 3 days.
Reagents are freshly prepared and stored at room temperature on daily basis. Do not
use reagents that is more than 24 - hour post preparation.

60
4.1.9 Safety
Adhere to safety precautions as stated in the Safety manual/IPC guideline All personal
protective equipment (PPE) must be worn when performing this procedure.
All samples must be regarded as potentially infections.

4.1.10 Calibration
Not Applicable

4.1.11 Quality Control

Known positive control samples and negative control samples should be tested the
same way as patient sample.

4.1.12 Procedural Steps

i. Place one drop of the blood to be tested in a glass slide. ii. Add 1- 2 drops of freshly
prepared 2% sodium met bisulphite to the drop of blood and mix well with an applicator
stick.
iii. Place a cover glass on top of the sample and press down lightly on it to remove
any air bubbles and to form a thin layer of the mixture. Wipe of the excess sample.
iv. Carefully rim the cover glass with molten paraffin wax or Vaseline, completely
sealing the mixture under the cover slip.
v. Incubate for 24 hours at room temperature, or for one hour at 370C. vi. Examine
the prepared glass slide for the present of sickle cells after one hour using 40 X
objective.
vii. If there is negative findings within one hour, allow the prepared slide to stand at
room temperature for 24 hours, and examined under microscopy

4.1.13 Biological Reference Interval Not applicable.

4.1.14 Interpretation and Reporting Of Results
• Report Positive when the presence RBCs appear as moon shaped or shaped
like a “C” showing they are sickle cells.
• Report Negative when the presence RBCs appear round Normal looking red
cells
• The results should be interpreted along with other clinical features. Further tests
might be necessary to confirm the disease condition.
4.1.15 Limitation of the Procedure and Sources of Errors
Haemolysed samples. Iron deficiency or blood transfusions within the past 3 months
can cause a false negative result

4.1.16 Performance Characteristics

Refers to method verification report

61
4.1.17 Supporting Documents
Sample collection manual

4.1.18 References
Monica Cheesbrough Handwrite
4.2 PROCEDURE FOR URIT-12 HEMOGLOBIN METER
4.2.1 Purpose
This procedure is used to describe step by step on how to operate the URIT-12
Haemoglobin Meter using human whole blood sample in the laboratory.

4.2.2 Scope
This procedure is applied in testing hemoglobin parameter using human whole blood
sample in the haematology department/section in the laboratory

4.2.3 Responsible
A trained and competent laboratory scientist, laboratory technologists and assistant
laboratory technologists are responsible for performing this procedure.
The head of section or assigned personnel will be responsible for ensuring that this
procedure is effectively implemented.

4.2.4 Principle
The URIT-12 Hemoglobin Meter utilizes optical reflectance for determination of the
total hemoglobin. A drop of whole blood is applied to the test spot on the strip, blood
immediately disperses within the membrane contacting the reagent, then reaction
product could absorb spectrum in the range of 500nm – 600nm. The meter’s optical
detector automatically measures the change in membrane reflectance. The intensity
of reflectance is inversely proportional to the hemoglobin concentration. The meter
calculates and displays the total hemoglobin concentration in gram/decilitre (g/dL) in
12 seconds based on mathematical conversion.

4.2.5 Sample Requirements

Fresh capillary or EDTA-anticoagulated venous whole blood

4.2.6 Equipment
URIT-12 Hemoglobin Meter

4.2.7 Materials
The materials required in this procedure are Clean gloves, Laboratory coats,
Micropipettes, Cuvetes,Sharp container, Pricker and Alcohol swab

62
4.2.8 Storage and Stability
Anticoagulated blood is stable up to 72 hours at 2-8 ⸰c

4.2.9 Safety
• Personnel Protective Equipment must be worn at all times
• All samples must be treated as potentially infectious.
• Adhere to safety precautions as stated in the Safety manual/IPC guidline
4.2.10 Calibration
Not Applicable

4.2.11 Quality Control

Use Hemoglobin HQ-A Control Solution or known higher and low concentration made
in-houseto run on weekly basis to determine the accuracy of the patient results.
4.2.12 Procedural Steps
i. Massage the patient’s middle or ring finger from knuckle up to the tip to stimulate
blood flow
ii. Insert the test strip into the strip holder with the notched end in first and the hole
facing up. The notched end on the top of strip should no longer be visible when test
strip is inserted correctly and fully
iii. Perform finger prick. Avoid “Milking” Apply light pressure to obtain one drop of
blood.
iv. Take 13-15µl of whole blood with capillary tube or transfer pipette
v. Rapidly drip the blood into the sample spot on the strip when the meter shows
blood symbol and ensure the test strip is covered by blood sample completely. vi.
During the test do not disturb or move the meter or strip, even press any key of meter
vii. The test results will be displayed in less than 30 seconds
viii. Record the test results displayed on the machine. ix. Remove the test strip and
immediately dispose off into highly infectious waste container.
4.2.13 Biological Reference Intervals
Infant 14.0 - 22.0g/dl, Children 11.1 - 14.1g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl

4.2.14 Interpretation and Reporting Of The Results

Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician

63
4.2.15 Limitation of the Procedure and Sources of Errors
Only whole blood or EDTA anticoagulated blood should be used.

4.2.16 Performance Characteristics

Refer into method verification report

4.2.17 References
URIT – 12 Hemoglobin meter Operation Manual
4.3 PROCEDURE FOR DETERMINATION OF HAEMOGLOBIN LEVEL
USING HEMOCHROMAX PLUS
4.3.1 Purpose
The procedure provides instructions to laboratory staff on operation of Hemochroma
Plus Machine.

4.3.2 Scope
This procedure is applicable when performing quantification of Haemoglobin (Hb)
concentration in Hospital Laboratory.

4.3.3 Responsible
Qualified, trained and competent Medical Laboratory Scientist, Technologist and
Technician are responsible for doing this test procedure. Section heads are
responsible for ensuring the effective implementation of this procedure.

4.3.4 Principle
The hemochroma PLUS analyser utilizes a dual wavelength LED light sources by
which the haemoglobin absorbance is detected and converted into an electrical signal.
The signal is direct proportional to the amount of haemoglobin present in the blood
sample.

4.3.5 Sample Requirements

Whole blood, capillary or venous ant coagulated collected blood into EDTA tube.

4.3.6 Equipment
Hemochroma PLUS machine

4.3.7 Materials
Hemochromax Plus Micro calibrator cuvette, Calibrator ID chip cuvettes, , Gauze, 70%
methylated spirit and Blood lancet or prickers

4.3.8 Storage and Stability

64
Anticoagulated blood is stable up to 3 days at 2-8 ⸰c

4.3.9 Safety.
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personnel must be worn protective equipment (PPE) when performing this
procedure.
• All samples must be regarded as potentially infections.
4.3.10 Calibration
It should be done when the machine is not working properly or when provides a doubt
result.

4.3.11 Quality Control

Use commercial IQC materials or known higher and low concentration made inhouse
to run on daily basis before performing the patient sample

4.3.12 Procedure
i. Establish good relationship with the patient
ii. Make sure the patient is sitting comfortably
iii. Lightly massage to stimulate circulation. Only use the middle or ring finger.
The patient should not wear the ring on that finger iv. Pres lightly and draw finger-prick
blood into a micro cuvette by bringing the cuvette in contact with the blood drop on the
fingertip and puncture the side to a depth of cuvette.
v. Remove any excessive blood from the outside of the cuvette. vi. Insert the cuvette
containing blood sample to the Hb machine
vii. Wait until displaying of the test results and record the findings into the system
and register book
viii. Pull the cuvette holder out to its loading position and discard the used
microcuvette in sharp box
4.3.13 Biological Reference Interval
Infant 14.0 - 22.0g/dl, Children 11.1 - 14.1g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl

4.3.14 Interpretation and Reporting of Results

Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician

65
4.3.15 Limitation of the Procedure and Sources of Errors
i. Only whole blood should be used
ii. Air bubbles in the optical eye caused by inadequate filling of the cuvettes may
lead into false results
4.3.16 Performance Characteristics
Refer to method verification report

4.3.17 Supporting Documents

Sample Collection Manual, Safety Manual,

4.3.18 References
• Monica Cheesbrough. District laboratory practice in tropical countries, Part 2.
2000.
• Hemochromax PLUS package insert
• Hemochromax PLUS user manual
4.4 PROCEDURE FOR DETREMANATION OF HEMOGLOBIN LEVEL
USING HEMOCUE 201+ MACHINE
4.4.1 Purpose
This procedure provides instructions for the performance of Haemoglobin Estimation
using Hemocue 201+ machine.

4.4.2 Scope
This procedure applies to all competent laboratory staffs during determination of
haemoglobin level by using Hemocue 201+ machine.

4.4.3 Responsible
Qualified, trained and competent health laboratory practitioners in the laboratory are
responsible for implementation of this procedure.

4.4.4 Principle
The reaction in the microcuvette is a modified azidemethemoglobin reaction. The
erythrocytes are haemolysis to release the haemoglobin. The haemoglobin is
converted to methoglobin and the combined with azide to form azide methoglobin. The
measurement takes place in the analyser in which the transmittance is measured the
absorbance and haemoglobin level is calculated. The absorbance is directly
proportional the haemoglobin concentration

4.4.5 Sample Requirements

66
Capillary whole blood sample or Anti-Coagulated Whole blood (collected in EDTA
anticoagulant)

4.4.6 Equipment
Hemocue 201+ machine

4.4.7 Materials
Hemocue Hb 201+ microcuvette, Lancet/Pricker for capillary sample, Pipette or any
other transfer device for venous sample or control materials

4.4.8 Storage and Stability

Ant-Coagulated whole blood is stable up to 4 hours at room temperature and up to 24
hours at 4°C– 8°C.

4.4.9 Safety
• Personnel Protective Equipment must be worn at all times
• All samples must be treated as potentially infectious.
4.4.10 Calibration
Not Applicable

4.4.11 Quality Control

The Hemocue Hb 201+ has an electronic self-test daily IQC or the use of known higher
and low concentration made in-house can also be applied.

4.4.12 Procedural Steps

i. Establish good relationship with the patient
ii. Make sure the patient is sitting comfortably
iii. Lightly massage to stimulate circulation. Only use the middle or ring finger.
The patient should not wear the ring on that finger iv. Pres lightly on the fingertip and
puncture the side to a depth of cuvette.
v. Remove any excessive blood from the outside of the cuvette. vi. Insert the
cuvette containing blood sample to the Hb machine
vii. Wait until displaying of the test results and record the findings into the system
and register book
viii. Pull the cuvette holder out to its loading position and discard the used
microcuvette in sharp box

Note: The microcuvette should be filled within 3 minutes after it has been taken out of its
package.

4.4.13 Biological reference Intervals

67
Infant 17.0-22.0g/dl, Children 11.0-13.0g/dl, Adult male 13.0 -17.0g/dl, Adult female
12.0-15.0g/dl

4.4.14 Interpretation and Reporting of Results

Interpretation of results
Interpretation of the results is based on the biological reference intervals.
Reporting of results
The obtained results will be reported in g/dl.
Critical results: HB ≤ (5.07) g/dl is considered as critical and communicate with
clinician

4.4.15 Limitation of the Procedure and Sources of Error

• Only whole blood should be used
• Air bubbles in the optical eye caused by inadequate filling of the cuvettes
• Delayement in transfer of filled cuvettes with blood into HB machine
4.4.16 Performance Characteristics
Refers into method verification report

4.4.17 Supporting Documents

Waste Management Procedure /IPC guideline Quality Control Result Procedure

4.4.18 References:
Monica Cheesbrough. District laboratory practice in tropical countries, Part 2. 2000.
Hemocue Hb 201+ cuvette kits insert and use manual.
4.5 PROCEDURE FOR CD 4 COUNT TEST BY USING BD FACS PRESTO
4.5.1 Purpose
The purpose of this procedure is to provide detailed information on how to analyse and
detect CD4 T Cell enumeration on blood sample by using BD FACS presto.

4.5.2 Scope
This procedure applicable in haematology to analyse and detect CD4 T Cell by using
BD FACS presto.

4.5.3 Responsible
Qualified, trained and competent Medical Laboratory Technician and Technologist are
responsible for doing this test procedure. Section heads are responsible for ensuring
the effective implementation and competency assessment for this procedure.

4.5.4 Principle

68
The BD FACS Presto™ cartridge the CD4/%CD4/Hb cartridge contains dried
fluorochrome-conjugated antibody reagents. When blood reacts with the reagents, the
antibodies in the reagent bind to the surface antigens on the BD FACS Presto
Cartridge: lymphocytes and monocytes. After the incubation period, the cells are
analysed on the BD FACS Presto Near-Patient CD4 Counter (the instrument). The
software identifies the cell populations of interest and calculates CD4 absolute counts,
CD4 percentages of lymphocytes, and haemoglobin concentration. The system
measures total haemoglobin by spectrophotometric method, using absorbance at an
isobestic point for oxyhemoglobin and deoxy-hemoglobin, with Correction for scatter.

4.5.5 Sample Requirements

Blood sample on K2EDTA vacuum tubes. All K2EDTA samples must be received and
set up within 24 hours from collection time.

4.5.6 Equipment
BD FACS Presto

4.5.7 Materials
Reagent Consumables
BD FACS presto Blue and yellow tips
cartridge BD FACS presto print out paper
BD disposable 100 µl Pipette
4.5.8 Storage and Stability
• Do not refrigerate whole blood SAMPLE before sample preparation.
• Do not use previously fixed and stored samples.
4.5.9 Safety
• Blood samples may contain infectious agents that are hazardous to your health.
Observe Standard Universal precautions.
• Ensure the instrument and environment you working are kept clean and free
from infectious substance such as human blood to avoid contamination.
• Spills should be immediately disinfected with 0.5% Sodium Hypochlorite
Solution.
4.5.10 Calibration Not Applicable.
4.5.11 Quality Control
BD Facs presto has internal electronic self-test for IQC or the use of known higher and
low CD4 counts made in-house can also be applied

4.5.12 Procedural Steps

i. Open the cartridge package and label the patient ID on to the cartridge.

69
ii. Face the inlet port up. iii. Invert the tube 10 times to mix the contents well. iv.
Use the pipette to obtain the sample.
v. Gently squeeze the bulb on the pipette to form a drop of blood on the tip of the
pipette. vi. Carefully dispense the sample into the inlet port. Hold the cartridge by its
ridges only.
vii. Make sure the blood reaches the top of the inlet port. If necessary, gently
squeeze the bulb on the pipette to dispense more blood.
viii. Make sure the cartridge is level, with the barcode side up, at all times. Make
sure that blood appears in the part of the channel not covered by the channel protector,
next to the containment zone.
ix. Discard the pipette into a biohazard us waste container.
x. Close the cartridge cap securely and Set the on-board timer. xi. Place the
cartridge, barcode side up, on the work station
xii. Press the Run Test tab. xiii. Press Patient ID.
xiv. Enter the patient’s ID and press Accept. xv. The Confirmed Patient ID
screen opens. xvi. Press Accept and Insert the cartridge:
xvii. Select your Operator ID and press Accept. Then cartridge door on the
instrument opens will open.
xviii. Note: If possible, complete the following two steps within 30 seconds. xix.
Remove the channel protector from the cartridge. xx. Hold the cap with the
channel facing upwards.
xxi. Insert the prepared cartridge into the cartridge door. The cartridge door closes.
xxii. Press Accept to eject the cartridge within 30 seconds.
4.5.13 Biological Reference Interval
Analyte Gender Reference interval SI UNIT
Absolute CD 4 count Male 462 – 1306 cells/uL
Female 440 – 1602 cells/uL
%CD4 of lymphocytes Male 29 – 54 %
Female 32 – 55 %
HAEMAGLOBIN Male 13.5 – 18.0 g/dL
Female 12.0 – 16.0 g/dL
4.5.14 Interpretation and Reporting of Results
Interpretation of results
• The results are displayed on the screen and print automatically.
Reporting of Results
• Report the obtained results as displayed on the screen.
Critical values
• If the CD4 count is low (below 200cells/ l for adult and below 450 cells/ l for
children) the result will be regarded as critical and communicate with clinician
4.5.15 Limitations of the procedure and sources of error

70
• Use the cartridge only with the BD FACS Presto instrument.
• The use of expired cartilage may result into false results
• Improper filling of the test device may not give the proper results
4.5.16 Performance Characteristics
Refer to Method verification report

4.5.17 Supporting Documents

Quality Manual, Sample Collection Manual, Safety Manual.

4.5.18 References
Becton Dickinson BD FACS presto Operating Manual

4.6 PROCEDURE FOR DETERMINATION OF CLOTTING TIME

4.6.1 Purpose
The purpose of this standard operating procedure (SOP) is to provide guidelines to be
followed for performing clotting time

4.6.2 Scope
This procedure is to be performed at point of care or any health facility to detect the
clotting time

4.6.3 Responsible
The Section heads are responsible section is responsible for ensuring the effective
implementation and maintenance of this procedure. Qualified, competent and
registered Medical Laboratory practitioners are responsible for implementing this test
procedure.

4.6.4 Principle
The presence of activator augments the contact activation phase of coagulation, which
stimulates the intrinsic coagulation pathway. Clotting time can be performed manually,
whereby the operator measures the time interval from when blood is injected into the
test tube to when clot is seen along the sides of the tube.

4.6.5 Sample Requirements

Whole blood

4.6.6 Equipment
Stop watch, water bath and thermometer

4.6.7 Materials

71
Disposable gloves, Laboratory coat, 70% alcohol, Masks, and Sterile lancets

4.6.8 Storage and Stability

Not applicable

4.6.9 Safety
• Adhere to safety precautions as stated in the facility Safety manual
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
• Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
4.6.10 Calibration
• Perform equipment calibration of the Stop watch, water bath and thermometer
as per auxiliary equipment calibration schedule

4.6.11 Quality Control

Not Applicable

4.6.12 Procedural Steps

Two methods can estimate clotting time:
❑ Capillary method of bleeding time.
i. Prick the finger with the lancet. ii. Hold the capillary over the blood, and the
capillary will fill automatically.
iii. Now, after regular intervals, break the capillary. iv. When a clot starts
forming, that is the endpoint and clotting time. ❑ Test tube method of clotting time.
i. Perform this test at 37 ° C.
ii. Take 4 ml of blood for the tube method and start the time.
iii. Note the time when there is the first appearance of the clot formation.
iv. This test can be done in multiple tubes to be more accurate.
4.6.13 Biological Reference Interval
Not Applicable

4.6.14 Interpretation and Reporting of Results

i. The expected range is 4 to 10 minutes.
ii. The glass tube method clotting time is 5 to 15 minutes. iii. Results are given
in amount of minutes takes for bleeding time to stop
4.6.15 Limitation of the Procedure and Sources of Error
i. This test is only prolonged in severe deficiency.
ii. Normal clotting time is despite prolonged bleeding time seen in
thrombocytopenia.

72
iii. This may be normal in patients taking anticoagulant therapy.
iv. This is usually normal when the intrinsic and common pathways are present in
an amount not exceeding 1% of the normal plasma level.
4.6.16 Performance Characteristics
Refer the method verification report of the procedure.

4.6.17 Supporting Documents

Sample collection manual

4.6.18 References
A manual of laboratory diagnostic tests. Edition 7, Lipipicontt William and Wilkins, by
Frances Talaska Fishbach, RN, BSN, MS, and MarshallaBrnet 11, RN, BSN, MS,
Ph.D.

4.7 PROCEDURE FOR DETERMINATION OF THE BLEEDING TIME

4.7.1 Purpose
The purpose of this procedure is to provide instructions for investigation of bleeding
time.

4.7.2 Scope
This procedure is used in Haematology section when performing bleeding time

4.7.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head Haematology is responsible for ensuring the effective implementation and
maintenance of this procedure.

4.7.4 Principle
Bleeding time is a medical test done to assess platelet function of a patient. It involves
cutting the underside of the subject's forearm, in an area where there is no hair or
visible veins. The cut is of a standardized width and depth, and is done quickly by an
automatic device. A blood pressure cuff is used above the wound, to maintain venous
pressure at a special value. The time it takes for bleeding to stop (i.e. the time it takes
for a platelet plug to form) is measured. Cessation of bleeding can be determined by
blotting away the blood every several seconds until the site looks 'glassy'.

4.7.5 Sample Requirements

Plasma/whole blood, Serum (2-5ml)

73
4.7.6 Equipment
Timer, Thermometer, Light Microscope, Sphygmomanometer

4.7.7 Materials
Marker pen, Examination Gloves, Filter paper, 70% alcohol swabs and Sterile lancets

4.7.8 Storage and Stability

Not Applicable

4.7.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.7.10 Calibration
Perform calibration of equipment (Timer and Thermometer) as per calibration schedule

4.7.11 Quality Control

Not Applicable

4.7.12 Procedural Steps

i. Apply the blood pressure cuff to the arm just above the elbow. ii. Inflate the
device to 40 mm of mercury and maintain at this level. iii. Clean the anterior
surface of the fore arm with 70% alcohol swabs.
iv. Make two clean punctures about 2 mm long and 2 mm deep being careful to
avoid underlying veins.
v. Blot the blood with the filter paper every 15 seconds but be careful and make
sure you only touch the top of the drop.
vi. start the stop watch as the first drop of the blood appears, immediately when
the blood ceases stop the timer and record the time.
vii. Calculate the average of the 2 punctures and record bleeding time.
viii. If it’s a prolonged bleeding time then perform Prothrombin Time (PT), Activated
Partial Thromboplastin Time (APTT) and platelets count.
4.7.13 Biological Reference Interval
Not Applicable

4.7.14 Interpretation and Reporting of Results

Interpretation of results
i. Interpret results in terms of minutes taken for bleeding to stop. ii. Normal
ranges are around 1½ - 5 minutes.

74
NOTE; If the bleeding time is greater than 10 minutes, stop the test and apply pressure to the
wound. Report the results as greater than 10 minutes.

Reporting of results
Report results as: Bleeding time (in minutes)
Critical value
Findings greater than 10 minutes.

4.7.15 Limitation of the Procedure and Sources of Error

Anything that alters platelet function can interfere with the bleeding time. Some
examples include aspirin, thrombocytopenia, and uremia

4.7.16 Performance Characteristics

Not Applicable

4.7.17 Supporting Document

Sample collection manual

4.7.18 References
4.8 PROCEDUIRE FOR FULL BLOOD COUNT BY USING URIT BH – 40P
HAEMATOLOGY ANALYSER.
4.8.1 Purpose
This procedure provides instruction on how to operate urit BH-40P haematology
analyser in determining full blood count

4.8.2 Scope
This procedure applies in the Haematology section when performing FBC (Full blood
count) analysis using URIT BH - 40P Haematology Analyser.

4.8.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this procedure. The Head Haematology is responsible for ensuring the
effective implementation and maintenance of this procedure.

4.8.4 Principle
The URIT BH – 40P Automated Haematology Analyser is a multi-parameter, it can
display 21 parameters and 3 histograms. Analyser adopts electrical impedance
method for WBC, RBC and PLT test and colorimetric method for HGB test. The
electrical impedance method is based on non-conductivity of blood cells. When the
blood cells in diluents pass through the ruby aperture, resistance will change, based

75
on that we can get the counting and volume of blood cells. The colorimetric methods
to measure and calculate HGB. Add lyse into the diluents sample, and then RBC will
be dissolve and release haemoglobin. Then the haemoglobin combines with lyse to
form cyanohemoglobin. Measure the transmission light intensity of this compound in a
sample cup through the monochromatic light of 540 nm wavelength and then compare
it with the result in blank state to get the haemoglobin concentration (blank state refers
to the state that only has diluents in sample cup).

4.8.5 Sample Requirements

Whole blood sample in EDTA-K2.2H2O tube.

4.8.6 Equipment
• URIT BH-40 Automated Haematology Analyser
• Perform start up, maintenance, troubleshooting and shut down the URIT BH-
40 Automated Haematology analyser as per manufacturer’s instrument instructions

4.8.7 Materials
Diluent, Lyse, Probe detergent, Set of Controls kit, Marker pen, Examination Gloves
Capillary tube, Plane test tube, Thermal paper, and Gloves

4.8.8 Storage and Stability

• Keep the Set of Controls at 2oC – 8oC
• Never use reagent and control beyond its expiration date
• Blood sample be kept at temperature between 2oC- 8oC for 7 days.
4.8.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.8.10 Calibration
• URIT calibrates the analyser in factory before shipment.
• Use the user URIT BH-40P to recalibrate the analyser when there is shifts or
trends in some parameters
4.8.11 Quality Control
Run all quality controls; QC1 (Low), QC 2 (Normal) and QC 3 (High) before
examination of patient samples

4.8.12 Procedural Steps Running Patient Samples

i. Pre- diluent peripheral blood mode
• Present the empty sample tube under aspiration probe.

76
• At main menu screen, click “Drain”; the diluents will be dispensed into the tube.
• Remove the tube, add 20µl of the blood sample to the tube, and gently shake
the tube to make them well mixed.
• After that present the well mixed sample under the aspiration probe; make sure
the probe touches bottom slightly.
• Press RUN key on the front panel and remove the sample after hearing beep
sound. The result will be available after analysis is performed. ii. Whole Blood
Mode
• Gently shake the tube to well mix the blood sample, then present the sample
tube beneath the probe, make sure the probe touches tube bottom slightly.
• Press RUN key and remove the sample after hearing beep sound. The results
will be available after analysis is performed.
iii. Ant coagulated Peripheral Blood Mode
• Gently shake the tube to well mix the blood sample, then present the sample
tube beneath the probe, make sure the probe touches tube bottom slightly.
• Press RUN key and remove the sample after hearing beep sound. The results
will be available after analysis is performed.
4.8.13 Biological Reference Interval
See annex 1.

4.8.14 Interpretation and Reporting of Results

Interpretation of results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
Reporting of results
Results are automatically printed from the URIT BH-40P and then review by section
head. Critical value

WBC > 20 x109/L, HGB <5g/dL, PLT < 50 or 1000 x109/L

4.8.15 Limitation of the Procedure and Sources of Error

• The test will be affected by hemolysed blood and coagulated blood.
• Samples with extreme lipemic, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated haemoglobin values.
4.8.16 Performance Characteristics
Refer to the method verification report of this procedure.

4.8.17 Supporting Document

Sample collection manual

4.8.18 References

77
URIT BH- 40P Operation manual
4.9 PROCEDURE FOR FULL BLOOD COUNT USING OF ABX PENTRA 80
HAEMATOLOGY ANALYSER
4.9.1 Purpose
This procedure provides instructions for operation and maintenance of ABX Pentra 80
analyzer for Full blood picture

4.9.2 Scope
This procedure applies to all Full Blood Count tests done on ABX Pentra 80 analyser
in the haematology section

4.9.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head Haematology is responsible for ensuring the effective implementation and
maintenance of this procedure.

4.9.4 Principle

The ABX Pentra 80 is an automated haematology analyser used for counting and
differentiating the cellular components in whole blood using electrical impedance,
cytochemical staining, light scatter and spectrophotometer.

The principle behind cell counting is based on disruption of electric current as particles
pass through an orifice. An electric current applies on both sides of this orifice. Cells
do not conduct electric current, therefore their passage through the orifice leads to a
change of the electric current established between both electrodes. These electric
current differences are registered and increment a counter at every cell passage.

A chemical agent is used to separate erythrocyte and leukocyte populations, because

of size overlapping and quantities discrepancies. This chemical agent contained in
Lysis (ACTI-DIFF) pops the cytoplasmic membrane of the red cells. Erythrocyte
population disappears leaving the leukocytes.

A haemoglobin preservative is added in lysing agent to measure haemoglobin scaled

down in a 540nm photometric tank at the end of the counting. The haemoglobin
measurement is made from the first dilution. The lysing agent has a powerful

78
haemoglobin reducer (potassium cyanide) and then the haemoglobin measurement
follows Drabkin method with a 540nm reading. The integration of luminous intensity
transmitted is evaluated according to the BEER-LAMBERT formula. An enzymatic
liquid (ABX CLEANER) ensures the system cleanliness between every analysis and
prevents carryover between samples.

4.9.5 Sample Requirements

2 to 4mls of whole blood collected in K3 EDTA tube
4.9.6 Equipment
ABX Pentra 80 Analyser and Perform start up, maintenance, troubleshooting and shut
down the ABX Pentra 80 Haematology analyser as per manufacturer’s instrument
instruction.
4.9.7 Materials
ABX Pentra 80 analyser reagent pack, Laboratory coat, Biohazard waste container.
0.5% Sodium hypochlorite solution, Distilled water, Protective gloves, Methanol, A4
paper

4.9.8 Storage and Stability

Control kit is stable until expiry date and should be kept at 2oC – 8oC. Blood sample be
kept at temperature between 2oC- 8oC for 7 days.

4.9.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.9.10 Calibration
Calibrate the ABX Pentra 80 analyser under the following conditions:

• Change of software
• Major component replacement
4.9.11 Quality Control
Run control QC materials (Low, Normal and High) before patient samples

4.9.12 Procedural Steps

i. Click the STAT MODE key.
ii. Then write the patient ID, Age, Gender.
iii. Click the VALIDATE key.
iv. Mix well the patient sample.

79
 v. Place the sample on the tube holder of analyser.
vi. Press the door of analyser inside to run the sample.
vii. The patient results will be printed automatically.
4.9.13 Biological Reference Interval See annex 1.
4.9.14 Interpretation And Reporting Of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Results are automatically printed from the ABX Pentra 80 and then review by section
head.
• Communicate the Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.9.15 Limitation of the Procedure and Sources of Error

i. Sickled red blood cells may not be accurately recognised and may give
erroneous results
ii. Samples with cold agglutinins may falsely decrease the red cell count. The
indices will indicate that the haemoglobin and haematocrit values do not agree.
Thin film is recommended. These samples should be incubated for 30 minutes
at 37 oC and reanalysed.
iii. Platelet clumps and neonatal samples may interfere with Drabkins method of
haemoglobin determination.
iv. Samples with extreme lipaemia, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated haemoglobin values.
v. Samples with nucleated red blood cells may falsely elevate white cell count.
Additionally, presence of nucleated red cells may interfere with white cell
differential count. Samples from patients with elevated chylomicrons ad those
receiving total parenteral nutrition (TPN) including a high lipid concentration may
falsely elevate the platelet count.
vi. Aggregated platelets may falsely elevate the white blood cell count and
percentage of lymphocytes.
vii. The presence of immature white blood cells, including blasts, may affect the
accuracy of the differential. The instrument will give an << I >> or ‘M’ error code
if the blasts are suspected. A thin film is recommended.
viii. Clinical studies have shown that the Full Blood Count is not affected by
presence of malaria parasites, Howell-Jolly bodies, cryogoblins and red cell
fragments.
4.9.16 Performance Characteristics

80
Refer to the method verification report of this procedure.

4.9.17 Supporting Document

Sample collection manual

4.9.18 References
ABX PENTRA 80 operator’s manual
4.10 PROCEDURE FOR FULL BLLOD COUNT USING SINNOWA HB - 7021
4.10.1 Purpose
This procedure provides instructions for operation and maintenance of SINNOWA HB
– 7021 Hematology Analyser.

4.10.2 Scope
This procedure applies to all Full Blood Count tests done on SINNOWA HB – 7021
analyser in the haematology section

4.10.3 Responsible
• Qualified, trained and competent health laboratory practitioners are responsible
for implementing this test procedure.
• The Haematology section head is responsible for ensuring the effective
implementation and maintenance of this procedure.

4.10.4 Principle
The instrument adopts the method of impedance to measure and count cells. The
conductivity liquid (mainly diluents) provides constant current source to the electrode
thus they circuit can form a steady impedance circulation. When cells pass through the
aperture, the conductive liquid is replaced by cells. Change of circuit resistance
produces electrical pulse. The amplitude varies when the cells of different size pass
through the aperture. Consequently the number and volume of cells pass through the
aperture can be calculated based on the amplitude.
4.10.5 Sample Requirements
2 to 4mls of whole blood collected in K3 EDTA tube

4.10.6 Equipment
• SINNOWA HB – 7021 Haematology Analyzer, Refrigerator
• Perform start up, maintenance, troubleshooting and shut down the SINNOWA HB –
7021 Haematology analyser as per manufacturer’s instrument instruction
4.10.7 Materials
Reagents consumables

81
 SINNOWA Diluent Reagent, Laboratory coat, Biohazard waste container.
SINNOWA Lyse Reagent, 0.5% Sodium hypochlorite solution, Distilled
SINNOWA Detergent, SINNOWA water, Protective gloves, Methanol, Printing
Probe Detergent, paper
4.10.8 Storage and Stability
• Control kit is stable until expiry date and should be kept at 2 oC – 8oC
• Blood sample be kept at temperature between 2oC- 8oC for 7 days.
• Store reagents as per manufacturer instructions
4.10.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.10.10 Calibration
The SINNOWA HB – 7021Hematology Analyser System requires commercial
calibrator material or assayed whole blood for calibration.
Calibrate the SINNOWA HB – 7021Hematology Analyser under the following
conditions:

• Change of software
• Major component replacement
4.10.11 Quality Control • Run the three levels of control LOW, NORMAL and
HIGH” for “SINNOWA HB – 7021 Hematology Analyzer to ensure quality of results
Perform Quality Control:-
i. Before analyzing the samples
ii. After replacement of the reagents
iii. After maintenance component replacement, or a field service action
iv. If there is any doubt in accuracy of the test results
v. After a reagent lot number change
vi. After a software change
vii. Following calibration
viii. According to your laboratory’s quality control program
ix. According to manufacturer requirements
4.10.12 Procedural Steps
i. Turn on the power on rear panel and indicator shows red light
ii. The instrument initializes test program
iii. Diluents, lyses and detergent will be sucked and tubing system cleaned
iv. If initialization is finished the display shows all parameters WBC, RBC and PLT

82
 v. Shift to select ID and then press OK
vi. Select the test selection from the drop down menu
vii. Gently mix the sample
viii. Open the sample tube and place it under the Open Mode Probe
ix. Raise the tube until the end of the probe is deeply immersed in the sample.
x. Press the Touch Plate to activate aspiration
xi. Remove the tube when the beep sounds and replace the cap.
xii. When the cycle is finished, the results post to the Data log and are displayed in
the Run View
xiii. Print the results
4.10.13 Biological Reference Interval See annex 1.
4.10.14 Interpretation and Reporting of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Results are automatically printed from the SINNOWA HB – 7021 and then review by
section head.
• Communicate the Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.10.15 Limitation of the Procedure and Sources of Error

• Keep reagent away from direct sunlight and protect them from evaporation.
• Use reagent container cap attached to each inlet tube, the cap will minimize
evaporation and contamination.
• Never use reagent, control and calibrators beyond their expiration date.
4.10.16 Performance Characteristics
Refer to the method verification report of this procedure.

4.10.17 Supporting Document

Sample collection manual

4.10.18 References
• SINNOWA HB – 7021 Hematology Analyser user manual.

4.11 PROCEDURE FOR FULL BLOOD COUNT USING BHA 3000

HAEMATOLOGY ANALYSER
4.11.1 Purpose

83
This procedure provides instructions for operation and maintenance of BHA 3000
Haematology Analyser.

4.11.2 Scope
This procedure applies in haematology section for performing Full Blood Count tests
done on BHA 3000 analyser

4.11.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure. The Haematology section head is responsible for
ensuring the effective implementation and maintenance of this procedure.

4.11.4 Principle
BHA -3000 Automatic Hematology Analyser provides a 3 part differential blood count
uses an electrical impedance to count red blood cells, platelets and volume
distributions, and uses calorimetry to measure the haemoglobin and relevant
parameters will be enumerated.
This system uses electrical impedance method to count red blood cells, platelets and
white blood cells. When the absorbed quantitative sample is diluted by a quantitative
conductive solution, it is sent to the detection unit of the instrument. The detection unit
has a detection aperture, with a pair of positive and negative electrodes on both sides
of the aperture, which is connected with a constant current power supply. Due to the
bad conductor characteristic of these cells, when the cells in the diluted sample pass
through the detection aperture under a constant negative pressure, the DC resistance
between the electrodes will change, thus forming a pulse change at both ends of the
electrode in portions to the size of the cell volume. When cells continuously pass
through the aperture, a series of electrical pulses are generated at both ends of the
electrodes. The number of pulse is equal to the number of cells passing through the
aperture. And the pulse amplitude is proportion to the cell volume. Amplify the collected
electrical pulses, and calculates the number of electric pulse in the red blood/platelets
channel and WBC channel. The pulses are then classified according to different
channel voltage threshold, hence determines the cell volume distribution

4.11.5 Sample Requirements

2 to 4mls of whole blood collected in K3 EDTA tube

4.11.6 Equipment

84
BHA 3000 Haematology Analyzer, Refrigerator
4.11.7 Materials
DIL-3 Diluent and HR-1 Lyse, Controls (Normal level (N), Low- level (L) and High- level
(H) Laboratory coat, Biohazard waste container. 0.5% Sodium hypochlorite solution,
Distilled water, Protective gloves, Printing paper

4.11.8 Storage and Stability

• Control kit is stable until expiry date and should be kept at 2 oC – 8oC
• Blood sample be kept at temperature between 2oC- 8oC for 7 days.
• Store reagents as per manufacturer instructions
4.11.9 Safety
• Adhere to safety precautions as stated in the Safety manual/IPC guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
4.11.10 Calibration
Calibrate the BHA 3000 Haematology Analyzer Analyser under the following
conditions:

• Change of software
• Major component replacement
4.11.11 Quality Control
Run the three levels of control LOW, NORMAL and HIGH” for BHA 3000 Haematology
Analyzer to ensure quality of results
Perform Quality Control:

i. Before analyzing the samples

ii. After replacement of the reagents
iii. After maintenance component replacement, or a field service action
iv. If there is any doubt in accuracy of the test results
v. After a reagent lot number change
vi. After a software change
vii. Following calibration
viii. According to your laboratory’s quality control program
ix. According to manufacturer requirements
4.11.12 Procedural Steps
i. Turn on the power on rear panel and indicator shows red light
ii. The instrument initializes test programme
iii. Diluents, lyses and detergent will be sucked and tubing system cleaned

85
 iv. If initialization is finished the display shows all parameters WBC,RBC and PLT
v. Shift to select ID and then press OK
vi. Select the test selection from the drop down menu
vii. Gently mix the sample
viii. Open the sample tube and place it under the Open Mode Probe
ix. Raise the tube until the end of the probe is deeply immersed in the sample.
x. Press the press Plate to activate aspiration
xi. Remove the tube when the beep sounds and replace the cap.
xii. When the cycle is finished, the results post to the Data log and are displayed in the Run
View
xiii. Print the results
4.11.13 Biological Reference Interval See annex 1.
4.11.14 Interpretation and Reporting of Results
• Normal: if the results are within acceptable range.
• Abnormal (pathological): if the results are out of acceptable range
• Communicate the following Critical value with clinicians
Test Critical Value
Haemoglobin Less than 5 g/dl (50 g/L)
White Blood Count Less than 2.0 or greater than 18.0 cells/mm3
Platelet Count Less than 50,000/ mm3 or greater than
800,000/mm 3

4.11.15 Limitation of the Procedure and Sources of Error

• Keep reagent away from direct sunlight and protect them from evaporation.
• Use reagent container cap attached to each inlet tube, the cap will minimize
evaporation and contamination.
• Never use reagent, control and calibrators beyond their expiration date.
4.11.16 Performance Characteristics
Refer to the method verification report of this procedure.

4.11.17 Supporting Document

Sample collection manual

4.11.18 References
BHA 3000 Haematology Analyser user manual.
4.12 PROCEDURE FOR PERFORMING FULL BLOOD COUNT BY USING MS4
HAEMATOLOGY ANALYSER
4.12.1 Purpose

86
This procedure provides instructions for operation and maintenance of MS4 analyser
for Full blood count

4.12.2 Scope
This procedure applies to all Full blood count tests done on MS4 analyser in the
Laboratory hematology section

4.12.3 Responsible
• Qualified and trained Health Laboratory Practitioners are responsible for
implementing this test procedure.
• Section head is responsible for ensuring the effective implementation and
maintenance of this procedure.

4.12.4 Principle
The MS4-S is an automated haematology analyser used for counting and
differentiating the cellular components in whole blood using electrical impedance,
cytochemical staining, light scatter and spectrophotometer.
The principle behind cell counting is based on disruption of electric current as particles
pass through an orifice. An electric current applies on both sides of this orifice. Cells
do not conduct electric current, therefore their passage through the orifice leads to a
change of the electric current established between both electrodes. This electric
current difference is registered and increment a counter at every cell passage.
A chemical agent is used to separate erythrocyte and leukocyte populations, because
of size overlapping and quantities discrepancies. This chemical agent contained in
Lysis (ACTI-DIFF) pops the cytoplasmic membrane of the red cells. Erythrocyte
population disappears leaving the leukocytes. A Haemoglobin preservative is added in
lysing agent to measure Haemoglobin scaled down in a 540nm photometric tank at the
end of the counting. The Haemoglobin measurement is made from the first dilution.
The lysing agent has a powerful Haemoglobin reducer (potassium cyanide) and then
the Haemoglobin measurement follows Drabkin method with a 540nm reading. The
integration of luminous intensity transmitted is evaluated according to the BEER-
LAMBERT formula.
An enzymatic liquid (TRANSFLUX) ensures the system cleanliness between every
analysis and prevents carryover between samples.

4.12.5 Sample Requirements

87
2 to 4mls of whole blood collected in K3 EDTA purple top color tube

4.12.6 Equipment
MS4-S Hematology Analyser, and roller mixer
4.12.7 Materials
MS4-S analyser reagent pack, Lab coat, Biohazard waste container, 5-6% Sodium
hypochlorite, Distilled water, Protective gloves and Methanol

4.12.8 Storage and Stability

Blood is stable for about 4 hours at room temperature or 24hours at 2- 6oC

4.12.9 Safety
i. Treat all samples as potentially infectious.
ii. Keep hands away from the sample carrier when the analysis begins
iii. Some components inside the MS4s have sharp edges or angular corners, therefore
operate with caution to avoid cuts to the hands
iv. When performing maintenance procedure, take similar precautions as you would take
when handling patient samples
4.12.10 Calibration
All auxiliary equipment should be calibrated annually

4.12.11 Quality Control

Use commercially quality control or in house prepared quality control samples

Running of quality control

i. Take the QC material from the refrigerator and let them stay at room temperature
for 15 minutes.
ii. After 15 minutes’ mix well the controls one by one by inverting the tube gently at
least (x7) seven times without creating bubbles.
iii. Press ANALYSIS key (Tube like symbol) then DOWN arrow (↓).
iv. Select Quality CT (to run controls) then use RIGHT arrow ( ) to select control
levels.
v. Select LOW if you start with low control up to High control using UP and DOWN
arrows on key board or on machine. (↑) or (↓)
vi. Place the control tube on the tube holder
vii. Press ENTER key on keyboard or ( ) on the machine to validate
viii. Repeat the procedure to the Normal and High controls

88
 ix. The QC results will be printed automatically
x. Plot the results of the QC on the Levy-Jennings chart and verify that the QC
has passed before running patients’ samples.
4.12.12 Procedural Steps
Running of Patient Samples
i. Press Analysis key in the machine
ii. Press RIGHT arrow
iii. Select the gender female or male by using UP and DOWN arrows (↑) or (↓)
iv. Press the ENTER key (using key board) or ( ) on the machine to validate
v. Write the name of the patient (Full Name)
vi. Mix well the sample
vii. Place the sample on the tube holder of the machine.
viii. Press ENTER on keyboard or ( ) on the machine to start running the sample.
ix. The patient results will be printed automatically.
4.12.13 Biological Reference Intervals
See annex 1.
Critical value/results should be recorded and immediately communicated to the
requesting clinician.

4.12.14 Interpretation and Reporting of Results

Results are reported and communicated through the appropriate locally established
procedure

4.12.15 Limitation of the Procedure and Sources of Errors

i. Exposing reagents to direct sunlight may cause them to deteriorate
ii. Sickled red blood cells may not be accurately recognised
iii. Samples with cold agglutinins may falsely decrease the red cell count.
iv. Platelet clumps and neonatal samples.
v. Samples with extreme lipaemia, chylomicrons or extremely high bilirubin
concentrations might produce falsely elevated Haemoglobin values.
vi. Samples with nucleated red blood cells may falsely elevate white cell count.
vii. Samples from patients with elevated chylomicrons and those receiving total
parenteral nutrition (TPN) including a high lipid concentration may falsely
elevate the platelet count.
viii. Circulating micro megakaryocytes may be counted as white cells
ix. Aggregated platelets may falsely elevate the white blood cell count and
percentage of lymphocytes.
4.12.16 Performance Characteristics

89
Method verification should be carried out and its report will be referred to fulfil this
requirement.

4.12.17 Supporting Documents

Laboratory quality policy manual, Laboratory safety policy manual and Laboratory
sample collection manual

4.12.18 References

MS4S operator’s manual, 2013-07

90
CHAPTER 5: CLINICAL CHEMISTRY AND IMMUNOLOGY
5.1 PROCEDURE FOR BLOOD GLUCOSE BY USING ACCU-CHECK
GLUOCOMETER
5.1.1 Purpose
This procedure provides instructions for use of blood glucose test strips for
determination of Blood Glucose level. Blood glucose is measured mainly in the
diagnosis and maNot applicablegement of diabetes mellitus.

5.1.2 Scope
This procedure will be used for Blood Glucose testing in the laboratory and at point
of care testing sites (POCT).

5.1.3 Responsible
Qualified and competent registered and licensed Health laboratory practititioners and
Trained health care providers respectively are responsible for doing this test
procedure.
The head of clinical chemistry section is responsible for ensuring the effective
implementation of this procedure.

5.1.4 Principle
The test strips contain a capillary that sucks up a reproducible amount of blood.
Glucose in the blood reacts with an enzyme electrode containing Glucose oxidase
(or Glucose dehydrogeNot applicablese).The enzyme is reoxidized with an excess of
a mediator ferricyanide ion, a ferrocene derivative or osmium bipyridyl complex. The
mediator in turn is reoxidised by reaction at the electrode, which generates an
electrical current. The total charge passing through the electrode is proportioNot
applicablel to the amount of glucose in the blood that has reacted with enzyme.

5.1.5 Sample requirements

i. Capillary or 1ml Fluoride-oxalate venous anticoagulated blood (fasting,
post-prandial, or random samples). Do not collect blood from an arm
receiving an I.V. infusion. Fasting samples: This refers to blood collected
after a period of no food intake. For adults the fasting time is usually 10 to
16 hours. For children the fasting time is 6 hours unless a longer time is
indicated, e.g. when investigating hypoglycaemia. The drinking of plain
water is permitted.

91
 ii. Post-prandial samples: This describes blood collected after a meal has
been taken. The sample is usually taken as a 2 hour post - prandial
samples.
iii. Random samples: This refers to a blood sample collected at any time,
regardless of food intake.
5.1.6 Equipment Glucometer Maintenance
Conduct maintenance as required by manufacturer instructions

5.1.7 Materials
Glucose test strips, Lancets, Cotton wool or gauze or alcohol swab, Sharp box or
Container, waste bin, Disposable gloves, Laboratory coat

5.1.8 Storage and stability

All related materials should be stored as the per manufactures instructions. Sample
should be processed within 1hour after collection

5.1.9 Safety
i. All samples must be considered as potentially infectious and must be handled
and examined with care. ii. All person applicable protective equipment (PPE) should
be worn when performing procedure
iii. Adhere to safety precautions as stated in the Safety manual

Refer to National infection prevention and control Guidelines for health waste
management and safety practice.

5.1.10 Calibration
Equipment should be calibrated as per schedule.

5.1.11 Quality control

Process internal quality control before examing the patient samples on daily base

5.1.12 Procedure Steps

i. Compare the code number on the chip with the corresponding code number on
the label of the test strip container where applicable. ii. The three-digit number on
the code chip (e.g.689) must match the three-digit number on the label. (Leave the
meter turned off).
iii. Gently slide the code chip into the slot on the side of the meter. (You must
feel the code lock into place)
iv. To turn on the Glucose meter, press the S button and hold it down for more
than 3 seconds until the depicted display appears.

92
 v. Wear gloves clean the patient’s finger using the alcohol swab and allows it to
dry.
vi. Take one strip from the container. Close cap tightly and make sure the yellow
color in the round window on the back of the test strip matches the yellow
color above 0 mg/dL on the container. If it looks green do not use it.
vii. Insert the test strip, with the orange pad facing up, until it will go no further
into the meter. Do not bend the test strip. (The arrow heads are almost no
longer visible when the test strip is inserted correctly.)
viii. Make sure the code on the meter matches the code on the test strip container.
ix. When you see the flashing blood drop, hold the lancet device against the side
of patient fingertip and press the release button.
x. Gently squeeze patient fingertip to get a drop of blood. xi. Once the the blood
drop appears on the screen, you have 2 minutes to apply the drop of blood.
xii. Touch the blood drop to the center of the square orange pad. Do not bend the
test strip.
xiii. An hourglass symbol appears on the screen, and then the test result appears.
xiv. To remove the lancet, take off the lancet device cap and point the lancet end
away.
xv. Slide out the rejector to discharge the lancet into an appropriate container for
sharp objects.
xvi. Applying Blood with Test Strip outside of the Meter. xvii. Take the test strip
out of the meter
xviii. Touch the center of the square orange pad to the drop of blood. Do not bend the
test strip. xix. Within 20 seconds, insert the test strip, with the orange pad to the drop
of blood.

5.1.13 Biological Reference Intervals

Fasting Blood glucose (mmol/L) Random Blood Glucose
Blood/Plasma: 3.9 – 5.6 mmol/L (70 - 100 mg/dl) ≤6.9 (125 mg/dl)

5.1.14 Interpretation and reporting of results

i. Results are displayed in either mg/dl or mmol/liter depending on which unit of
measurement is selected. Report the value in the agreed SI unit.
ii. If the result is lower than 10mg/dL (0.6mm/L) “Lo” is displayed instead of a
result.
iii. “Lo” may indicate that your blood is very low. iv. If the result is higher than
600mg/dL (33.3mmol/L), “Hi” is displayed instead of a result.
v. Fasting blood glucose between 5.6 – 6.9 mmol/L (100 – 125 mg/dl) indicates high
risk to diabtes. vi. Two separate test results of 7.0 mmol/L (126 mg/dl) or higher
indicate diagnosis of diabetes.

Critical value

93
 Fasting blood glucose <2mmol/L
>20mmol/L

5.1.15 Limitation of the Procedure and Sources of Error

i. Falsely elevated glucose results may be obtained when a person’s blood
contains bilirubin (unconjugated) >340 µmol/l (>20 mg/dl), triglycerides >57
mmol/l
ii. Abnormal uric acid levels may also interfere with test results. Caution is needd
in the interpretation of neoNot applicablete blood glucose values <2.8 mmol/l
(<50 mg/dl).
iii. Abnormal haematocrit values may affect test results. iv. Haematocrit levels
below 0.20 may cause falsely low glucose values when the glucose
concentration is less than or equal to 11.1 mmol/L. Values above 0.55 may
cause falsely low glucose values when the glucose is above 11.1 mmol/l.
5.1.16 Perfomance Characteristics Refer to the verification report
5.1.17 Supporting Document Not applicable
5.1.18 References
ACCU-CHEK Active user’s manual.
5.2 PROCEDURE FOR TESTING BLOOD GLUCOSE BY GLUCO PLUS
5.2.1 Purpose
This Standard Operating Procedure (SOP) is aimed to describe step by step on how
to operate Gluco plus device

5.2.2 Scope
This procedure for Gluco-plus device will be used for blood glucose chemistry testing
in health facility in Tanzania

5.2.3 Responsible
Trained, qualified and competent laboratory registered practitioners are responsible
for performing this procedure.
The head of section for chemistry is responsible for ensuring the effective
implementation and competency assessment for this procedure .

5.2.4 Principle Not applicable

5.2.5 Sample Requirements Whole blood
5.2.6 Equipment
Perform the procedure for start-up, maintenance, troubleshooting and shut down the
Gluco-plus as per manufacturer’s instrument instructions

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5.2.7 Materials
Test strips and Lancing device

5.2.8 Storage and Stability

• Gluco-plus strips should be stored at room temperature
• All reagents should be protected from direct sunlight, extreme heat, and
freezing during shipment and storage.
• Temperatures below 32° F (0°C) may cause reagent layering that changes the
tonicity and conductivity of the reagents.
• Sample stability after collection of venous whole blood: ✓ Run Samples within
one hour of collection.
5.2.9 Safety
• Decontaminate working surfaces twice daily, in the morning and afternoon
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
• Refer to Manufacturer instruction, National infection prevention and control
• Guidelines for health waste management and safety practice.
5.2.10 Calibration Not Applicable
5.2.11 Quality Control
Control solution
Test prepared GLUCOPLUS control Solution;
i. Once per week
ii. When opening new strips kit
iii. When you suspect the meter or test strips are not working properly. iv. If
you drop/damage the meter.
v. Record results on QC Form.

5.2.12 Procedural Steps

Step Action
Changing strip code
1 Check the code on the test strip vial before inserting the test strip.
2 Insert the test strip to turn ON the meter and match the code on the meter
with the code on the strips vial.
3 If the code is already matched press OK to go to APPLY SAMPLE screen.
4 If the code in Meter does not match the code on the test strip vial, Press
S button until you hear a beep sound. Press S or M to match the code
number on the test strips vial.
Setting time
1 Switch on the analyser

95
 2 Press S button until you hear a beep sound. Press off button to save the
strip code.
3 Month number will blink use S and M button to select the required month
4 Press the off button to save month and date number will blink and use S
and M button to select the required date.
5 The time hours will blink use S and M to set time .
Performing a Quality Control test
1 Perform weekly and as per meter (machine) protocol.
2 Control used is GLUCOPLUS Selected Control solution.
3 Prepare and Apply Control solution.
4 Touch the sample tip of the test strip to the control drop. Verify check
window filled.
5 Meter display will count down from 5 to 1 and then display result.
6 Compare the control solution results with correct Control range printed on
test strips vial. (Example; 6.2 – 8.2 mmol/L). If the result are not within
control range repeat the control solution test.
Testing Blood
1 Use lancing device to puncture site, normally fingertip.
2 Insert a test strip into the strip port to turn on meter.
3 Touch the blood sample to the sample tip at the end of the test strip.
4 The meter display will count down from 5 to 1 and then display result.
5 If the “HI” or “LO” message appears on the meter, the result is above 33.3
or below 1.1. Repeat test to verify.
Error Codes
E-1 Problem with Meter – Do not use the meter
E-2 Meter or strip Problem – Repeat the test with New strip.
E-3 Meter was not ready – Repeat the test with a new strip. Apply SAMPLE
or CONTROL appears on the display.
E-4 Strip Problem – Repeat test with a new strip.
E-5 Strip problem or Sample too small – Repeat the test with a new strip
and new sample.
HI.E Temperature too high—repeat test in a cooler area.
LO.E Temperature too low—repeat test in a warmer area.
E-6 Battery LOW — replace battery soon.
5.2.13 Biological Reference Interval
Fasting Blood glucose (mmol/L) Random Blood Glucose
Blood/Plasma: 3.9 – 5.6 mmol/L (70 - 100 mg/dl) ≤6.9 (125 mg/dl)

5.2.14 Interpretation and Reporting of Results vii. Results are displayed in either
mg/dl or mmol/liter depending on which unit of measurement is selected.
Report the value in the agreed SI unit.
viii. If the result is lower than 10mg/dL (0.6mm/L) “Lo” is displayed instead of a
result.

96
 ix. “Lo” may indicate that your blood is very low.
x. If the result is higher than 600mg/dL (33.3mmol/L), “Hi” is displayed instead
of a result.
xi. Fasting blood glucose between 5.6 – 6.9 mmol/L (100 – 125 mg/dl) indicates
high risk to diabtes. xii. Two separate test results of 7.0 mmol/L (126 mg/dl)
or higher indicate diagnosis of diabetes.
Critical value
Fasting blood glucose <2mmol/L
>20mmol/L

5.2.15 Limitation of the Procedure and Sources of Errors

If the “HI” or “LO” message appears on the meter, the result is above 33.3 or below

5.2.16 Perfomance Characteristics

Refer the method verification reports from for this procedure and equipment
manufacturer user manual

5.2.17 Supporting document

Sample Collection Manual, Safety Manual, Quality Manual

5.2.18 References
1. User manual for Gluco-plus
2. Manufacturers package insert
5.3 PROCEDURE FOR URIT 50 (URINE CHEMISTRY ANALYZER)
5.3.1 Purpose
This procedure is provides description for performing urine biochemistry by using
URIT 50 semi-automated Urine analyser.

5.3.2 Scope
This procedure is applied for testing Urine sample at the health facilities in Tanzania.

5.3.3 Responsible
A trained and competent health laboratory practitioners are responsible for
performing this procedure. The head of section of biochemistry and parasitology is
responsible of ensuring the implementation of this procedure.

5.3.4 Principle
The analyser measures change of the reflectance of reagent strips pads, A detector
integrated in the system is composed of light source and a light receiver, the light
from which goes through in the spherical integrator and reflect to the reagent pads

97
on strip. The absorbance (reflectance) varies according to the color of reagent pads,
the darker of the reagent pads higher the absorbance is and less light is reflected.
Conversely the lighter the reagent pad is the lower the absorbance is and more light
is reflected degree of color developed is direct proportion to the concentration of
analyte in urine” .

5.3.5 Sample Requirements

4mls of uncentrifuged mild stream urine sample is used.

5.3.6 Equipment
Urit-50

5.3.7 Materials
Disposable gloves, Laboratory coats, Urine container, Waste container, Marker pen,
Urine strips from urit G10, G11 or G14, Gauze

5.3.8 Storage and Stability

i. Store urine sample at room temperature for 30 minutes to 2 hours or 24 hours
in refrigerator.
ii. Reagent strips and calibrator should be stored to free and clean area at
37°C iii. Control materials should be stored as per the manufacturers
instructions

5.3.9 Safety
Samples and control materials at this section should be treated as infectious material
and should be handled careful.

5.3.10 10.0. Calibration Not applicable

5.3.11 Quality Control
i. Put on PPE
ii. Install the strip holder into machine
iii. Switch on the machine and wait the machine for initialization
iv. Put the dry or calibrator strip on the strip holder till D sound
v. Wait the machine to scan and print result
vi. Record the QC result.

Note: Return calibrator strip into its container and discard other used materials according to
standard operating procedures

5.3.12 Testing Procedures

98
 i. Deep the reagent strips of G series into urine sample and put it on the dry gauze
to remove excess urine on the back of the strip.
ii. Put the sample strip on the strip holder till D sound
iii. Read the patient result on the machine
iv. Record result to the register
5.3.13 Biological Reference Intervals See annex 3.
5.3.14 Interpretation And Reporting Of Results
Refer to the insert which present on the reagents strips of G series G10, G11, G14.
Report result according to insert present on the reagent strip bottle

5.3.15 Limitation of the Procedure and Sources of Errors

The test relies on correct collection of sample by the patient, and if this is not done
properly the results may not be accurate

5.3.16 Performance Characteristics Refer to method verification

5.3.17 Supporting Documents URIT 50 user amnual
5.3.18 References
URIT 50 urine user manual.
5.4 PROCEDURE FOR (URIT-560) URINE ANALYZER
5.4.1 Purpose
This procedure provides instructions for determining urine biochemical test using the
URIT-560 analyzer

5.4.2 Scope
This procedure is used in Clinical chemistry section of the user when performing urine
using the URIT-560 analyzer

5.4.3 Responsible
The section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained

5.4.4 Principle
The analyzer measures changes in reflectance of the reagent strips pads. A detector
integrated in the system is composed of a light source and a light receiver, the light
from which goes through spherical integrator and reflect at the reagent pads on the
strips. The absorbance varies according to the color of the reagent pads. The darker
the reagent pads is the higher the absorbance is and less light is reflected.
Conversely, the lighter the reagent pad is the lower the absorbance is, and more light

99
 is reflected; ie. The degree of color development is proportion to the concentration of
analyte in urine.
The reflected light goes in to the optical-electronic detector system, which transforms
the optical into electrical. The strength of the electricity correlates which reflectance.
Then the electrical cables will be processed by CPU after being transformed by I/V
converter, and the test results can be printed out by printer.

5.4.5 Sample Requirements

Dry, wide-necked, leak proof container 10-20 ml of urine specimen.

5.4.6 Equipment
URIT-560, Printer

5.4.7 Materials
Urine strips (URIT G Series), Printer thermal paper, Urine container, Gloves

5.4.8 Storage and Stability

Sample is stable for 2hrs at room temperature or 24hrs at 2-80C

5.4.9 Safety
i. Personnel Protective Equipment must be worn at all times
ii. Samples must be treated as potentially infectious.
5.4.10 Calibration
Perform calibration as per manufacturer instructions

5.4.11 Quality Control

Currently no control

5.4.12 Procedural Steps.

i. Give the patient a sterile, dry, wide-necked, leak proof container and request10-
20 ml of urine specimen. ii. Explain to the patient the need to collect the urine with
as little contamination as possible i.e. a clean-catch specimen.
iii. On the instrument, press (image test) to enter into the image test interface.
iv. Insert or Pour urine on the test strip, put test strip into test strip holder until the
sound of alarm raised.
v. Then wait the printer out results.
5.4.13 Biological Reference Intervals.
Parameter Abbreviation Biological Reference Intervals
Urobilinogen URO Normal
Glucose GLU Negative

100
 Bilirubin BIL Negative
Ketones KET Negative
Specific gravity S.G 1.003-1.029
Occult blood BLD Negative
Ph Ph 4.5 - 7.8
Protein PRO Negative
Nitrite NIT Negative
Leukocytes LEU Negative
5.4.14 Interpretation And Reporting Of Results
Results are automatically printed from the machine. Attach results printout with report
form

5.4.15 Limitation of the Procedure and Sources of Errors.

• Urine must be processed within 2hr to avoid growth of bacteria which
consuming glucose and developing ammonia in urine, loose of ketone bodies,
Increase of PH
• Urine if not processed on time store in refrigerator 2oC – 8oC for 24
5.4.16 Performance Characteristics
Refer into method verification report

5.4.17 Supporting Documents

Equipment maintenance form, sample collection manual, quality manual

5.4.18 References
• URIT-560 operator’s manual
• Standard guard line for Health Laboratory 2007 Edition.
5.5 ROCEDURE FOR PERFORMING URINE BIOCHEMISTRY BY USING
CYBOW READER 300
5.5.1 Purpose
This Standard Operating Procedure (SOP) is aimed to describe step by step on how
to operate the CYBOW™ READER 300 semi-automated Urine analyser using Urine
sample at the health facilities in Tanzania

5.5.2 Scope
This procedure applies to all staff who works in parasitology section on performing
urinalysis test.

5.5.3 Responsible

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A trained, qualified and competent laboratory registered practitioner are responsible
for performing this procedure. The head of sections is responsible for ensuring the
implementation of this procedure.

5.5.4 Principle
The CYBOW reader 300 are reflectance photometer. The strip is illuminated by white
light, and the reflected light from the strip is detected by the sensor. The RGB signal
is digitized, and this digitized image is interpreted by the processor. The intelligent
image analyser SW locates the strip and the pads, and based on this colour data the
parameter values are determined. The results including the date and the time of
measurement, sequence number and the ID are stored printed out by the internal
printer.

5.5.5 Sample Requirements

10-20mls of mild stream urine sample collected in sterile wide mouth container is
required for performing this test. Do not centrifuge urine sample before bio chemical
test.

5.5.6 Equipment
Perfom the CYBOWTM READER 300 procedure for start-up, maintenance,
troubleshooting and shut down the urine analyser as per manufacturer’s instrument
instruction.

5.5.7 Materials/REAGENTS
Gloves, Marker pen, Laboratory coat, Waste container, Gauze, Urine container and
CYBOW strip.

5.5.8 Storage and Stability

Sample, reagents, calibrators and control materials should be stored as per the
manufacturer instructions.

5.5.9 Safety
• Adhere to safety precautions as stated in the Safety manual
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
• Front cover of machine should be covered during operation to avoid sample
contamination.
• Used only power cord specified for CYBOWTM READER 300
• Avoid excessive dust, wet/damp condition and provide proper ventilation.

102
 • Do not wipe the body clean with benzene, thinner, gasoline. This may
discolour
5.5.10 Calibration
The calibration of instrument should be done prior to first time use and then the 2 nd
calibration process is recommended in every 4 weeks with calibration strip provided
in the package.

5.5.11 Quality Control

Run internal Quality Control samples daily before examing patient samples to ensure
quality of examination results. Other conditions that drive controls include:

After a reagent lot number change

After maintenance, component replacement, or a field service action ✓
After a software change ✓ Following calibration.
According to regulatory requirements
5.5.12 Procedural Steps
• Check if the urine was received within 1 hour of collection and in a sterile
universal container
• Mix the urine by swirling the container and dip a CYBOW strip into urine,
making sure that the entire measuring region of the strip is immersed then
running strip on instrument.
Mode of running strip on CYBOWTM READER 300
1. General mode • Select the general mode by pressing direction key(◄) and
press Enter button to return to the standby mode.
• After the 1st strip dipped in urine and placed on a plate, press start key(►).
• Put 1st-10th (max.) strip on the plate one by one after dipping each in selected
urine.
• Once last reagent strip on batch is placed, press Enter button
• After incubation time of the 1st strip, it will start to loading the results of the strip
on by one.

2. One by one mode

• Select one by one mode by pressing Direction key (◄) and press
Enter button.
• After the 1st strip is placed on the loading plate press start key (►).
• Press direction key whenever each of the next strip is placed on the
plate one by one.
• Once last strip is place on strip loading plate, press Enter button.
• After incubation time of the 1ststrip, it start to read and print result of
the strip one by one.
3. Quick mode

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 • Select the quick mode by press direction key(◄) and press Enter
button
• Put the strips (incubation is done) once strip loading plate
continuously.
• Once the last strip is placed on the loading plate, press Enter button.
• Test result is shown on the LCD and automatically saved in the
memory
5.5.13 Biological Reference Intervals See annex 3.
5.5.14 Interpretation And Reporting Of Results
Chemical urinalysis
Report the reading when the immersed strip is compared to the colours on the strips
container
Macroscopic examination
Report whether the urine is Clear, Slightly cloudy, Cloudy or turbid. Report the colour
of the urine which will range from Light yellow, Yellow, Amber, Red to Brown
Microscopic examination of urine sediment
• White Blood Cells/Pus Cells/ leucocytes.
Report the average number of cells per High Power Field, example 2-5
WBCs/HPF
• Red Blood Cells
Report the average number of cells per High Power Field, example 2-5
RBCs/HPF
• Casts
Identify the type of cast and report as number per High Power Field
• Crystals
Identify the type of crystals and report their presence • Epithelial cells
Identify the type whether squamous, transitional or renal epithelial cells,
quantify them and report per high power field. Otherwise report the presence
of epithelial cells if can’t be identified.
• Trichomonas Vaginalis
Report as “seen” or “not seen”
• Yeast
Report as “seen” or “not seen”

• Spermatozoa
Report for males and not for females

5.5.15 Limitation of the Procedure and Sources of Errors

Urine samples should be tested within one hour of collection

104
 If any delay happen put the urine sample in the refrigerator to avoid bacterial growth.
Do not use urine dipsticks beyond expiry date

5.5.16 Performance Characteristics

Refer to the method verification report of this procedure

5.5.17 Supporting Documents

Safety manual and Sample collection manual

5.5.18 References
Manufacturer’s Package Insert in multistrips kit
Cheesbrough, Monica Health Laboratory Manual for Tropical Countries
Graff’s Text Book for Urinalysis and body fluids, Second edition, Lillian A. Mundt and
Kristy Shanahan
5.6 PROCEDURE FOR DETERMINATION OF ALT BY USING DIRUI-DR 7000
CHEMISTRY ANALYZER
5.6.1 Purpose
This procedure provides instructions for determining Alanine Aminotransferase
(ALT) using the DIRUI-DR7000 Analyzer

5.6.2 Scope
This procedure is used in Clinical chemistry section for analysing ALT using the
DIRUI-DR7000 clinical chemistry Analyzer.

5.6.3 Responsible personnel

Qualified, trained and competent Health Laboratory Assistant technologists,
Technologists, and Scientists are responsible for performing this procedure.. The
section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained.
5.6.4 Principle
Kinetic method for the determination of ALT activity according to the
recommendations of the Expert Panel of the International Federation of Clinical
Chemistry (IFCC). Without pyridoxalphosphate activation. ALT is measured by the
reagent rate analysis by the coupled reaction with lactate dehydrogenase (LDH) to
reduce NADH (measured at a wavelength of 340nm) to NAD+. The rate of decrease
in absorbance at 340 nm due to NADH depletion is proportional to the ALT activity in
the sample.
5.6.5 Sample requirement
30µl serum or plasma, sample free from hemolysis.

105
5.6.6 Equipment
DIRUI DR7000 Semi Automated Chemistry Analyzer, Centrifuge machine

5.6.7 Materials
Reagent Kits, Calibrator/Stsndard, Control Kits (LEVEL I and II), Disposable gloves
Laboratory coat, Sample Cups, Reaction Wells, Transfer Pipettes

5.6.8 Storage and stability

Refer to the facility laboratory sample collection manual

5.6.9 Safety
i. Adhere to safety precautions as stated in the facility laboratory Safety
manual/IPC guidline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
5.6.10 Calibration
i. Perfom equipment calibration when; - ii. There is a change in the reagent lot
number. iii. If the QC result falls outside the acceptable ranges. iv. The machine
blinks on the QC-calibration indicating that the calibration is expired.
v. There is a change in the system software
vi. System maintenance/ component replacement procedure is performed
5.6.11 Quality control
QC shluld be performed before patient samples, after calibration of reagent

5.6.12 Procedure Steps

Running the Calibrator, controls or samples;
i. Label the tubes for blank, calibrator, Controls or samples
ii. Prepare the working reagents as indicated by the industrial manufacturer
iii. Pipette the working reagents R1 480µl into the labelled tubes
iv. Pipette 30µl of distilled water into the tube labelled blank.
v. Incubate for 300s
vi. Add 120µl of R2 reagent
vii. Incubate for 60s
viii. Read the result
5.6.13 Biological reference intervals See annex 4.
5.6.14 Interpretation and Reporting of Results
Interpretation of results
Perfomed results will be displayed on the machine screen

106
 Result reporting
Once all of the results are accepted or validated, a final report will automatically be
printed out.

5.6.15 Limitation of the Procedure and Sources of Error

Hemolyed, lipemic samples, icterus and anticoagulants such as citrate, oxalate and
fluoride (for other tests except Glucose) and drugs such as hydroxocobalamin and
Cephalosporin antibiotics.

5.6.16 Performance Characteristics Refer to the verification report

5.6.17 Supporting document
Sample collection manual, quality manual

5.6.18 References
DIRUI-DR7000 Chemistry analyser user manual
5.7 PROCEDURE FOR DETERMINATION OF AST BY USING DIRUI-DR7000
CHEMISTRY ANALYZER
5.7.1 Purpose
This procedure provides instructions for determining ASAT using the DIRUI-DR7000
Analyzer

5.7.2 Scope
This procedure is used in Clinical chemistry section of the user when performing
blood analysis using the DIRUI-DR7000 clinical chemistry Analyzer

5.7.3 Responsible
The section head of Clinical Chemistry is responsible for ensuring this procedure is
effectively implemented and maintained.

5.7.4 Principle
Kinetic method for the determination of Aspartat-Aminotransferase (AST) activity
according to the recommendations of the Expert Panel of the International Federation
of Clinical Chemistry (IFCC). Without pyridoxalphosphate activation. AST is
measured by the reagent rate analysis by the coupled reaction with Malate
dehydrogenase (MDH) to reduce NADH (measured at a wavelength of 340nm) to
NAD+. The rate of decrease in absorbance at 340 nm due to NADH depletion is
proportional to the AST activity in the sample.

5.7.5 Sample Requirements

107
 Serum or plasma, sample free from hemolysis and not contaminated

5.7.6 Equipment
DIRUI DR7000 Semi Automated Chemisry Analyzer•
Cleaning and Maintenance
i. Use a large amount of distilled water to rinse the tubing by click the rinse
interface.
ii. And drain the liquid from the tubing if necessary. iii. Remove the waste liquid
bottle from the back of the analyzer.
iv. Keep the instrument vertical during move and transport.
v. Try best to avoid vibration. vi. And check and debug the instrument before use.
5.7.7 Materials
Reagent Kits, Calibrator/Standard,Control Kits (LEVEL I and II), Supplies, Disposable
gloves, Laboratory coat, Sample Cups, Reaction Wells and Transfer Pipettes

5.7.8 Storage and Stability

• Reagent Should be kept at temperature of 2-8°C and sealed in dry place
without sunshine. The shelf life is 18 months .
• Under condition of 2-8°C, the open vial stability is 30 days
5.7.9 Safety
Personnel Protective Equipment must be worn at all times and samples must be
treated as potentially infectious.

5.7.10 Calibration
It is suggested to use supplementary calibrator as instructed. When lot number is
changed or QC is invalid, calibration shall be conducted again. Procedures for
reagent, Calibration, QC and Sample preparation

5.7.11 Quality Control

It is suggested to use QC products produced by DIRUI.

5.7.12 Procedural steps

i. Label the tubes for blank, calibrator, Controls or samples
ii. Prepare the working reagents as indicated by the industrial manufacturer
iii. Pipette the working reagents R1 480µl into the labelled tubes
iv. Pipette 30µl of distilled water into the tube labelled blank.
v. Incubate for 300s
vi. Add 120µl of R2 reagent
vii. Incubate for 60s
viii. Read the results

108
 5.7.13 Biological Reference Intervals.
See annex 4.

5.7.14 Interpretation and Reporting of Results

Reporting of results
Results are automatically printed from the machine Attach results printout with report
form.

5.7.15 Limitations of the procedure and sources of error. Gross

hemolysis, Lipemic AND Icterus specimen
5.7.16 Performance Characteristics
Refer verification report

5.7.17 Supporting Documents

Equipment Maintenance Form.
18.0 References
DIRUI DR7000 Analyser operator’s manual
National standard guard line for Health Laboratory 2007 Edition
5.8 PROCEDURE FOR SA-30 SEMI AUTOMATED CHEMISTRY ANALYZER
5.8.1 Purpose
This Procedure describes step by step on how to operate the SA-30 a semiautomated
chemistry analyser to perform basic chemistry tests using human serum, plasma or
cerebral spinal fluid (CSF) sample.

5.8.2 Scope it is applied in testing biochemistry parameters using human serum or

plasma sample in the biochemistry department/section.

5.8.3 Responsible
All qualified, trained and competent laboratory scientist, laboratory technologists and
assistant laboratory technologists are responsible for performing this procedure. The
head of section of biochemistry is responsible of ensuring the implementation of this
procedure.

5.8.4 Principle
The principle of the instrument is based on the phenomenon of different wave band
absorbance from substance, which is in line with Lambert-Bill Law.
(The greater the concentration of the sample, the more light is absorbed, the less
light is transmitted, and the darker of the color)

109
5.8.5 Sample Requirements
The 2 - 4ml of whole blood collected in plain tube (red top) for serum or EDTA (purple
top) for plasma or in heparinized tube free from hemolysis. Cerebral spinal fluid (CSF)
when required.

5.8.6 Equipment
SA-30 semi- automated chemistry analyser, Centrifuge

5.8.7 Materials
Micropipettes and tips, Marker pen, Thermal paper and PPE

5.8.8 Storage and Stability

i. Store serum/plasma at room temperature 25 – 35 °C for 8 hours
ii. Tested sample stored at 2 -8 °C to 7 days. iii. Store reagent,calibrator and
controls per manufacture recommendation
5.8.9 Safety
Treat all Samples and control materials as infectious material and should be handled
careful as per IPC guidelines

5.8.10 Calibration
Quality Control procedure is the same as analytic procedure of unknown sample
(Use Randox Calibrator (pipette 500µl of reagent to tube and add 25µl calibrator mix
gently and incubate at 370C for 8-10 minutes for end point test method, no need of
extra incubation for kinetics method)

5.8.11 Quality Control

Quality Control procedure is the same as analytic procedure of unknown sample (Use
Randox QC ,pipette 500µl of reagent to tube and add 25µl control mix gently and
incubate at 37C for 8- 10 minutes end point test method, no need of extra incubation
for kinetics test method)

5.8.12 Procedural Steps

i. Prepare for sample and reagent. ii. For End point test method, Add
R1 + R2 + Sample in ratio and mix thoroughly and incubate at 37℃ for 8 – 10
Min (refer to reagent user manual)
iii. For kinetics test method, Add R1 + R2 + Sample in ratio and mix thoroughly,
no need of extra incubation.
iv. For HDL, LDL add R1 + Sample in in ratio, mix them thoroughly incubate at
37℃ in 2 min then add R2 and incubated them in 5-7 Min,
v. Click “Test” on main menu to enters next page to start testing.

110
 vi. Press PUSH button to aspirate distilled water, to calibrate AD value. The
AD value should be 45000 to 60000.
vii. Click “Continue” to test reagent blank. viii. Select “YES” to aspirate
reagent blank to test reagent blank absorbance.
ix. Press PUSH button to aspirate reagent blank to test reagent blank
absorbance.
x. Click “Continue” to test STD
Select “NO”, device will use last factor and perform the sample test directly.
xi.
Select “YES”, device will aspirate standard to test STD xii. Press
PUSH button to aspirate standard, then device will test standard
absorbance and calculate factor automatically
xiii. Click “Continue” to next to test the sample directly or perform control test.
xiv. Press PUSH button to aspirate sample or control, then device will test
the sample or control and display test result automatically
5.8.13 Biological Reference Intervals See annex 4.
5.8.14 Interpretation And Reporting Of Results
Interpretation
i. If the result of the particular parameter lies within the established
reference range, it means that the patient has normal particular
parameter.
ii. If the result of the particular parameter lies below or above the
established reference range, it means that the patient has abnormal
particular parameter and requires intervention as per the clinical history
and the laboratory findings. Reporting results
Report the results as they are displayed on the screen of the machine

5.8.15 Limitation of the Procedure and Sources of Errors

i. Avoid using the haemolysed and lipemic sample as this will cause falsely
elevated values. In this case inform the requesting physician and ask for
another specimen.
ii. Avoid exposure of the freshly dissolved substrate to strong sunlight, since the
reagent is light sensitive. The change in absorbance will increase with an
increase in temperature, since the pH of the reagent will be different at
different temperatures
iii. Serum must be separated by centrifugation as soon as possible after
collection of the patient's blood sample, preferably <2 hours, otherwise,
phosphate present in erythrocytes will be released into the serum causing
falsely elevated values
iv. Grossly bloody CSF may give spuriously elevated values. Undue delay in
analysis may give low values. The report to the requesting physician should
include the appearance of the CSF before and after centrifugation.

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 5.8.16 Performance Characteristics Refer to method verification report
5.8.17 Supporting Documents
Sample collection manual, Safety manual, Quality manual

5.8.18 References
SA-30 semi automated chemistry analyser user manual
5.9 PROCEDURE FOR OPERATING CLINDIAG FA 200 CHEMISTRY TEST
5.9.1 Purpose
This procedure provides instructions for determining basic Chemistry tests using
Clindiag FA 200 analyzer.

5.9.2 Scope
This procedure is used at Clinical chemistry section for processing basic chemistry
tests using Clindiag FA 200 analyzer.

5.9.3 Responsible
Qualified and trained Health Laboratory Personnel are responsible for doing this test
procedure.

5.9.4 Principle
The test principle of the biochemistry analyser is mainly based on the lambert Beer
law.
The reagents and the samples to be tested are mixed at a certain proportion. The
mixture is placed in a calorimetric dish at a certain temperature for incubation, its
absorption of light of specific wavelength is continuously measured, and finally the
concentration of the measured substance is automatically calculated according to the
value of absorbance (change).The procedure uses Lambert Beers Law which state
that the absorptive capacity of a dissolved substance is direct proportional to its
concentration to the solution.

5.9.5 Sample Requirements

Serum is most preferred sample
Refer to the facility Laboratory sample collection manual.

5.9.6 Equipment
Clindiag FA200 Chemistry analyser.

5.9.7 Materials
Reagent kits, cleaning solution, HD- high efficiency cleaning agent, Sample cups,

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Calibrators and controls, Gloves, Laboratory coat, A4 paper, Micropipette,
Micropipette tips

5.9.8 Storage and Stability

Samples are stored at 2-8oC after testing for 3days.

5.9.9 Safety
i. Adhere to safety precautions as stated in the Safety manual. ii. All personal
protective equipment (PPE) must be worn when performing this procedure.
iii. All samples must be regarded as potentially infections. iv. Refer to National
infection prevention and control Guidelines for health care.
v. Avoid any contact between hands and eyes and nose during sample collection
and testing.
vi. Do not use kit beyond the expiration date. vii. Do not reuse the test device.
viii. All spills should be wiped thoroughly using 1% sodium hypochlorite
solution.
5.9.10 Calibration
Machine should be calibrated when

• There is a change in the reagent lot number.

• If the QC result falls outside the acceptable ranges.
• There is a change in the system software
• System maintenance/ component replacement procedure is performed
5.9.11 Quality Control
Follow the following steps to run internal Quality Control.sample i. Click on
task.
ii. Click on add Quality control(QC.)
iii. Click on ADD
iv. Select QC batch number.
v. Select a QC item in QC list
vi. Select type of container
vii. Input the position of QC material on the sample plate
viii. Click on OK
ix. Allow the amchine to perfom test till final results
5.9.12 Procedural Steps
i. Click Task
ii. Click Add sample
iii. Click ADD
iv. Enter patient information
v. Select the test item

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 vi. Select the sample cup number
vii. Click OK
viii. Click Test on left side ix. Select test sample
x. Then click start test
xi. When sample processing is complete, Select the results from the menu bar,
patient result review will display, select name of patient on the left side.
xii. All the undone tests will be shown on the results as NA with a reason on the
right side. xiii. Re-running patient sample, repeat the patient order with no
change in a sample cup number

5.9.13 Biological Reference Intervals See annex 4.

5.9.14 Interpretation And Reporting Of Results
Results interpretation
Click on <Results> then <sample Results>. The results appear on the computer
screen, from here you can select the desired results and release them.
Reporting of the results
To print the results, select the sample interface in browse result and click print the
test results. Results. Critical results
Critical results should be immediately communicated to the clinicians requested the
examination. Refer to the annex …. For more details on critical results for clinical
chemistry assays.

5.9.15 Limitation of the Procedure and Sources of Errors

Do not proces hemolysed samples as they might lead to falsely high results of
potassium and low results for glucose.
Samples for glucose investigation should be processed within 2 hours of collection;
any delay would cause falsely low results.
Potential operator errors and clindiag FA 200 system technology limitations.
Communicate the following Panic/Critical values to the clinicians

5.9.16 Performance Characteristics

Method verification of this procedure should be done and that the report should be
referred to verify compliance to this requirement.

5.9.17 Supporting Documents

Sample collection manual

5.9.18 References
Refer to equipment instruction manual Clindiag FA 200

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5.10 PROCEDURE FOR DETERMINATION OF IMMUNOASSAYS BY USING
GETEIN 1100 IMMUNOANALYSER
5.10.1 Purpose
This procedure provides description on determination of immunoassays by using
Getein 1100 Immunofluorescence Quantitative analyser.

5.10.2 Scope
This procedure is used for processing and analysis of immunol assay tests in the
biochemistry department/section at the hospital laboratory.

5.10.3 Responsible
A qualified,trained and competent laboratory scientist, laboratory technologists and
assistant laboratory technologists are responsible for performing this procedure. The
head of section of biochemistry is responsible of ensuring the implementation of this
procedure.

5.10.4 Principle
The detection element scans the binding area and converts the optical signal to
electrical signal. The voltage variation between the test line and background has a
linear relationship with the antigen concentration which can be used to calculate the
concentration. In conclusion the antigen concentration in whole blood, plasma,
serum, urine can be calculated quantitatively according to optical signal of the test
line.

5.10.5 Sample Requirements

The 2 - 4ml of whole blood collected in plain tube (red top) for serum or EDTA (purple
top) for plasma or in heparinized tube free from hemolysis. urine sample will be
collected in urine container if required.

5.10.6 Equipment
Getein1100 Immunofluorescence Analyzer, stop watch

5.10.7 Materials
Disposable gloves, Micropipettes and its tips, Containers for waste segregation,
Marker pen, Getein test card

5.10.8 Storage and Stability

Store unproceesed samples at room temperature for 12 hours. Store performed
samples at 2 - 8°c up to 7 days.

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 Calibrator, controls and test kit devices should be stored as per manufactures
instruction

5.10.9 Safety
i. Samples and control materials should be treated as infectious material ii. Worn
PPE all of the time while working
5.10.10 Calibration
Calibration of the assay should be peformed as per manufacturer instruction.

5.10.11 Quality Control

Use commercial available controls or inhouse controls to run QC as per schedule

5.10.12 Procedural Steps

i. Refer to the user manual or material data sheet to specific items including
reaction time and sample volume carefully for accurate information
ii. Add patient information including ID, name, age, gender, types of sample
and test mode to be used
iii. Click start after inserting the card, test item will be auto-recognized and the
result will be shown on the screen. User can also see the voltage waveform
by slide to the left side
iv. Normally, the test card will auto-quit after testing if not please click on “Quit”
icon to quit manually
5.10.13 Biological Reference Intervals See annex 4.
5.10.14 Interpretation And Reporting Of Results
Interpretation of results
If the result of the particular parameter lie within established reference range, it
means that the patient has normal particular parameter
If the results of the particular parameter lie below or above established reference
range, it means that the patient has abnormal particular parameter and requires
intervention as per the clinical history and the laboratory finding.
Reporting of results
Report the result as they are displayed on the screen of the machine.

5.10.15 Limitation of the Procedure and Sources of Errors

Only used for in vitro analysis of human whole blood, serum, plasma, urine or stool
freezed samples can not be used for testing due to loss of enzyme or hormone
activities

5.10.16 Performance Characteristics

Reffer to verification report.

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5.10.17 Supporting Documents
• Sample collection manual
• Material data sheet or reagent manual
• Safety manual
5.10.18 References
Getein 1100 user manual

5.11 PROCEDURE FOR OPERATING FIA 8000 ANALYSER

5.11.1 Purpose
This procedure provides instructions for operating FIA 8000 Quantitative
Immunoassay Analyzer for biomarkers.

5.11.2 Scope
This FIA8000 is an analyzer that used to measure biomarkers in human whole blood,
serum, plasma or urine samples.

5.11.3 Responsible
Qualified and competent registered Health Laboratory practititioners are responsible
for doing this test procedure. The head of section of chemistry is responsible for
ensuring the effective implementation and competency assessment for this
procedure.

5.11.4 Principle
The combination of the antigensin the sample, the gold-labelantibodyin the colloidal
gold pad or nitrocellulose membrane, and theantibody pre-coated on the test linecan
form a purplish red streak on the test line. The colour intensity of the test lineis
proportionate to the quantity of antigens detectedin the sample. The analysersystem
can obtain the photo-electric signal intensity of the complexby scanning the test line
with a photo-electric component. Then the voltage difference between the voltage of
the test line and the background is obtained. The voltagedifference has a linear
relationship with the antigen concentrationwhichcan be used to calculate the antigen
concentration. The relationship has been established and varying from the measured
parameter. In conclusion, the antigen concentration in whole blood, plasma, serum,
urine can be calculated quantitatively in one-step according to the colour intensity of
the test line.

5.11.5 Sample Requirements Centrifuged Serum and plasma

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 5.11.6 Equipment
FIA 8000 Quantitative Immunoassay Analyzer

5.11.7 Materials
Test kit, Power source, Printing paper, QC Card, QC SD, Gloves

5.11.8 Storage and Stability

Fresh sample is preferred however if can not be done sample can be stored at 2-8⁰C
not more than 3days.

5.11.9 Safety
i. Decontaminate working surfaces twice daily, in the morning and afternoon ii.
Adhere to safety precautions as stated in the Safety manual
iii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iv. All samples must be regarded as potentially infections.
v. Avoid any contact between hands and eyes and nose during sample collection
and testing.
vi. All spills should be wiped thoroughly using 1% sodium hypochlorite solution
vii. Decontaminate the biohazpus waste before disposal.
5.11.10 Calibration
Perfom equipment calibration when; -
i. There is a change in the reagent lot number.
ii. If the QC result falls outside the acceptable ranges.
iii. The machine blinks on the QC-calibration indicating that the calibration is
expired.
iv. There is a change in the system software
v. System maintenance/ component replacement procedure is performed
5.11.11 Quality Control
Quality Control (QC) card should be run before processing patient sample for each
day

5.11.12 Procedural Steps

i. Centrifuge collected whole blood samples to obtain serum or plasma.
ii. Mix urine samples thoroughly before testing.
iii. Allow samples to reach room temperature before testing.
iv. Turn on the analyser and select the test.
v. Touch the screen to turn on the analyzer.
vi. Select the test you want to perform from the list of available tests.
vii. Insert the test card.

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 viii. Open the test card slot.
ix. Carefully insert the test card into the slot, making sure that the arrows on the
test card are pointing in the same direction as the arrows on the analyzer.
x. Close the test card slot.
xi. Add the sample.
xii. Follow the on-screen instructions to add the sample to the test card. Be
careful not to overfill the sample well.
xiii. Touch the screen to start the test.
xiv. The analyzer will automatically begin to process the sample.
xv. Read the results.
xvi. The analyzer will automatically read the results and display them on the
screen.
xvii. The results may be displayed in a variety of formats, such as quantitative
results, qualitative results, or graphs.

5.11.13 Biological Reference Intervals See annex 4.

5.11.14 Interpretation And Reporting Of Results
Interpretation of results
Interpretate results based on the Biological reference interval.
Normal results are patient results which fall within the reference range for the
particular test. Abnormal results are those that fall below or above the reference
range. The test report is labeled H: High and L: Low to show the abnormality obtained.
Reporting of results
Report the obtained and displayed results in request form/register
Critical results
Analyte Less Than Greater Than
Amylase 25U/L 150U/L
Chloride 85 mmol/L 115mmol/L
CK 30U/L 200U/L
Creatinine 26umol/L 120umol/L
Glucose(fasting) 2.5mmol/L 20.0mmol/L
Potassium 2.5mmol/L 6.0mmol/L
Sodium 120mmol/L 160mmol/L
Bilirubin Total 3.4umol/L 20.5umol/L
Biliribun Total for new Born Newborn
24hours ≥ 1374umol/L
48hours ≥ 2224umol/L
84hours ≥2904umol/L
One week to one month ≥3424umol/L
Urea (BUN) ≤1.0mmol/L ≥ 54mmol/L

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5.11.15 Limitation of the Procedure and Sources of Errors
Haemolyzed sample should not be used since the color changes caused by
haemolysis may result to wrong results. Refer to package insert for interfering
substances for specific test

5.11.16 Performance Characteristics Refer to the method verification report.

5.11.17 Supporting Documents Sample collection manual
5.11.18 References
User manual for FIA 8000 Analyser.
5.12 PROCEDURE FOR OPERATING ALERE AFFINION AS100ANALYSER
5.12.1 Purpose
The purpose of this procedure is to provide instructions on how to operate ALERE
AFFINION AS100 analyser for HbA1c, Lipid panel and C - reactive protein.

5.12.2 Scope
This procedure is used in Clinical chemistry section for processing HbA1c,Lipid panel
and C-Reactive Protein.

5.12.3 Responsible
A trained, qualified and competent laboratory registered practitioners are responsible
for performing this procedure. The head of section for chemistry is responsible for
ensuring the effective implementation and competency assessment for this
procedure .

5.12.4 Principle
A Test Cartridge with patient sample or control is placed in the cartridge chamber of
the Analyzer. By manually closing the lid, the Test Cartridge is transported into the
analysis compartment of the Analyzer. Test and lot-specific information is obtained
from the barcode label (Figure 2). When the Test Cartridge enters the Analyzer, the
integrated camera reads the barcode. The calibration data for the actual lot are read,
which then initiates the processing of the Test Cartridge. The sample and reagents
are automatically transferred between the wells. An integrated camera monitors the
entire process. Light-emitting diodes (LEDs) illuminate the reaction area, which can
be either a colored membrane or a reaction well. The camera detects the reflected or
transmitted light, which is converted to a test result and displayed on the touch

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 screen. When the user accepts the result, the lid covering the cartridge chamber
opens automatically and the used Test Cartridge can be removed and discarded. The
Analyzer is then ready for the next run.

5.12.5 Sample Requirements

Whole blood/Serum / plasma as specified in the reagents insert or as stated in the
sample collection manual.

5.12.6 Equipment
Alere Affinion AS100

5.12.7 Materials
Test cartridge, Calibrators, Controls, PPEs, Pipette tips 100 -200ul, Pipette tips 100,
1000ul, Sample container rack, waste bin

5.12.8 Storage and Stability

• Test cartridge should be stored in refrigerator at 2-8oC in sealed foil pouches
and only stable until expiration date. If not refrigerated they can be stored
at room temperature(15-25oC) for four weeks.
• Test cartridges should not be exposed to direct sunlight at relative humidity
below 90%.
• Whole blood samples can be stored refrigerate at 2-8o C for 3 days. Plasma
and serum samples can be refrigerated for 10 days or frozen up 1 year if
the tubes are properly sealed.
5.12.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC guidline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
5.12.10 Calibration
Perform calibration as per Alere Afinion™ AS100 user manual.

5.12.11 Quality Control

Quality control should be done as prescribed in the quality management procedure.
Use commercial or in-house made quality control materials to perform on daily basis
before testing patient samples. Commercially available quality materials should be
used to verify performance of the procedure at least after 100 patient samples have
been tested. Lot to lot verification should also be used to check performance
acceptability of reagents.

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5.12.12 Procedural Steps Analyzing a patient/control sample
1 Touch to enter the patient sample mode.
Touch to enter the control mode. A “C” in the upper left corner
indicates that the Analyzer is in the control mode. The lid opens
automatically.
If the lid is left open from the previous run and “Insert Cartridge” is
displayed, this step is omitted and you can start with step 2.

2 Insert the Test Cartridge with the barcode label facing left.
Be sure that the Test Cartridge is correctly placed in the cartridge
chamber.

3 Close the lid manually. The Analyzer will start processing the Test
Cartridge.
The processing time depends on the test in use.

4 Touch and enter the patient ID.

Touch to confirm.
Touch and enter the control ID or Alere Afinion™ Control Data.
Touch to confirm.
Entering the patient ID, control ID or Alere Afinion™ Control Data will
not interrupt the processing.
5 Record the result, then touch to accept.
If a printer is connected, touch to print the result.
The lid opens automatically.
The result will be saved in the result records.

6 Remove the used Test Cartridge from the cartridge chamber and
discard it in a suitable waste container. Insert a new Test Cartridge or
close the lid manually.
Keep the lid closed to protect the cartridge chamber when the
Analyzer is not in use.

5.12.13 Biological Refences See annex 4.

5.12.14 Interpretation And Reporting Of Results Interpretation of results
• Interpretation of results is based on the Biological reference interval;
• Normal results are patient results which fall within the reference range for the
particular test.
• Abnormal results are those that fall below or above the reference range.
Reporting of Results
Report the displayed/printed results into register/request form

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5.12.15 Limitation of the Procedure and Sources of Error
i. Icteric samples that appears with yellow colour of the serum or plasma due to
bilirubin accumulation.
ii. Samples tested after 24 hours may give unreliable results Avoid using lipemic
samples
5.12.16 Performance Characteristics Refer the method verification reports .
5.12.17 Supporting Document
i. Sample Collection Manual, Safety Manual, Quality Manual

5.12.18 References
ALERE AFFINION AS100analyzer user manual
Manufacturer package insert

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CHAPTER SIX: SEROLOGY
6.1 PROCEDURE FOR SYPHILIS ANTIBODIES RAPID TEST
6.1.1 Purpose
This procedure provides instructions for Qualitative detection of antibodies of all
isotopes against Treponema pallidum

6.1.2 Scope
The procedure is used in all Laboratory areas for screening syphilis infection

6.1.3 Responsible
Qualified and trained Medical Laboratory Technicians, Technologists and Scientist
are responsible for implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.1.4 Principle
The syphilis Ab Rapid test strip (Serum/plasma) is a lateral flow chromatographic
immunoassay based on the Principle of the double antigens–sandwich technique, In
this test syphilis recombinant antigen is immobilized in the test line region of the strip
test device, After sample is added to the sample well of the device it react with syphilis
recombinant antigen coated particles in the test. This mixture migrates
chromatographically along the length of the test strip and interacts with immobilized
syphilis antigens.
If the sample contains syphilis antibodies a coloured line will appear in the test line
region indicating positive results. If the sample does not contain syphilis antibodies a
coloured line will not appear in the region, indicating a negative result

6.1.5 Sample Requirements

Whole blood/plasma sample in purple tube (EDTA) Serum from clotted blood sample
in plain tube.

6.1.6 Equipment
Centrifuge, Timer, Micropipette
Maintenance
Maintenance of the equipment should be performed as per schedule

6.1.7 Materials

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Reagents Consumables
Syphilis Ab Rapid Test Strips kit Marker pen
Known Positive control, Examination Gloves
Known Negative control

6.1.8 Storage and Stability

• The kit should be stored at 2-30 oC until the expiry date printed on the sealed
pouch or as instructed by the manufacturer.
• Do not freeze the kit or exposing it over 300C.
• Store Serum and plasma sample at 2-80C for up to 3 days.
• For long term storage, serum/plasma should be kept below -20oC
6.1.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC
guideline
ii. All personnel protective equipment (PPE) must be worn when
performing this procedure.
iii. All samples must be regarded as potentially infections.
6.1.10 Calibration
Perform calibration of equipment as per calibration schedule

6.1.11 Quality Control

A known Negative and Positive in-house controls once every week and whenever a
new kit is opened.

6.1.12 Procedural Steps

i. Bring the test kit and sample to room temperate before use.
ii. Remove the test from its sealed pouch, and use it as soon as possible.
iii. Place the test strip on a clean, dry flat surface
iv. Label the test strip with the Patient ID
v. For serum or plasma specimen;
vi. Hold the dropper vertically and transfer 2 drops of serum or plasma
(approximately 60µl) onto the specimen pad of the test strip.
vii. Read test results in 15 minutes. Do not interpret results after 15 minutes.
viii. For whole blood specimen; ix. Hold the dropper vertically and
transfer 2 drops of whole blood (approximately 50ul) onto the specimen
pad of the test strip.
x. Then add 1 drop of buffer (approximately 30ul) and start the timer.
xi. Read test results in 15 minutes. xii. Do not interpret test results
after 15 minutes
6.1.13 Biological Reference Interval Not applicable
6.1.14 Interpretation and Reporting of Results Interpretation of results

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 Negative - Only one coloured band appears on the control(C) region. No
apparent band on the test (T) region
Positive - In addition to a pink coloured control (C) band, a distinct pink coloured
band will also appear in the tests (T) region
Invalid – a total absence of colour in both regions or no coloured line appears
on the control (C) region is an indication of procedure error or the tests
deterioration. Repeat the test with a new kit. Reporting of results

Report results as: Syphilis - Negative or syphilis – Positive Critical value

Not applicable

6.1.15 Limitation of the Procedure and Sources of Error

i. The syphilis Ab rapid test strip should be stored at room temperature
(15-30°c) ii. Humidity and temperature can adversely affect results. iii.
Do not use test if pouch is damaged or broken
iv. Do not use it beyond expiration date.
v. Do not perform the test in a room with strong air flow. i.e. an electric fan
strong air-condition
vi. Test is for single use only. Do not re use test.
6.1.16 Performance Characteristics
Refer manufacture kit insert for specificity and sensitivity.

6.1.17 Supporting Document

Sample collection manual, Safety manual

6.1.18 References
Manufacturer Kit insert for syphilis

6.2 PROCEDURE FOR (HIV) TESTING BY USING BIOLINETM HIV 1/2 3.0 TEST
6.2.1 Purpose
The purpose of this procedure is to describe the method of performing Bio line
HIV1/HIV-2 rapid test assay.
6.2.2 Scope
This procedure is applicable to all HIV-1/HIV-2 rapid test using Bio line HIV-1/HIV-2

6.2.3 Responsible
Qualified, registered, licenced and trained Medical personnel are responsible for
implementing this test procedure.

126
The Head of Serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.2.4 Principle
SD Bioline HIV-1/HIV-2 is a rapid HIV qualitative immune-chromatographic assay
used to detect antibodies to HIV in human blood, serum or plasma as the sample had
been added to sample pad. As the sample migrates through the conjugate pad, it
reconstitutes and mixes with the selenium colloid-antigen conjugate. This mixture
continues to migrate through the solid phase to the immobilized recombinant antigens
and synthetic peptides at the sample window site. If antibodies to HIV-1 and/or HIV-
2 are present in the sample, the antibodies bind to the antigen-selenium colloid and
to the antigen at the client window, forming a red line at the client window site. If
antibodies to HIV-1 and/or HIV-2 are absent, the antigen selenium colloid flow past
the client window and no red line is formed at the client window site.

6.2.5 Sample Requirements

2-3mls Whole blood/plasma/ serum

6.2.6 Equipment
Timer, Centrifuge, Micropipette, Refrigerator.
Maintenance
Maintenance of equipment should be performed as per schedule

6.2.7 Materials
Abbott Bioline TM HIV 1/2 3.0 Test/kit, Assay diluent, Disposable gloves, Laboratory
coat

6.2.8 Storage and Stability

The test kit should be stored at a temperature between 1 oC and 30oC or as per
manufacturer claims
Whole blood; If the blood sample is not immediately tested, it should be refrigerated
at 2-80C for 3days
Plasma or serum; If plasma or serum sample is not tested immediately, it should be
refrigerated at 2-80C for 7 days
For storage period longer than 2week, freezing below -200C is required. They should
be brought to room temperature 15-300C prior to use.

6.2.9 Safety

127
Adhere to safety precautions as stated in the facility Safety manual /IPC guideline
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infectious.

6.2.10 Calibration
Perform calibration of equipment as per calibration schedule

6.2.11 Quality Control

Run known Negative and Positive in-house controls daily before performing patient
samples or when new test kit is opened.

6.2.12 Procedure Steps

i. Bring reagents and samples to room temperature before use. ii. Tear off the
desired number of test strips from the 10-test card by bending and tearing off
along the perforated line.
iii. Label the strips with sample identification number or patient/client
identification number.
iv. Peel the foil cover from the reagent area of the test strips.
For serum or plasma samples;
v. Apply 10 µl of sample using a precision pipette to the sample pad (marked
by the arrow symbol).
vi. In the absence of precision pipette apply 1 drop of sample using plastic
Pasteur pipette provided by manufacture in the kit.
vii. Then apply 4 drop of buffer to the sample pad.
viii. Wait for a 10 to 20 minutes and read results.
For whole blood collected by finger prick method; ix. Apply 20 µl of sample
(collected by EDTA capillary tube) to the sample pad (marked by the arrow
symbol).
x. In the absence of precision pipette or EDTA capillary tube, apply 1 drop of
sample using plastic Pasteur pipette provided by manufacture in the kit.
xi. then apply 4 drops of buffer to the sample pad. Wait for 10 to 20 minutes
and read results.
For whole blood collected by venepuncture method; xii. Apply 20 µl of sample
using a precision pipette to the sample pad (marked by the arrow symbol).
xiii. Then apply four (4) drops of buffer to the sample pad.
xiv. Wait for 10 minutes (up to 20 minutes) and read results.
6.2.13 Biological Reference Interval

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 Not applicable

6.2.14 Interpretation and Reporting of Results

Result interpretation
Negative Result
The presence of only control line(C) within the result window indicate a negative result
Positive Result
The presence of two lines as C and T -1(1) within the window indicates positive
results for HIV-1
The presence of two lines as C and T -2 (2) within the window indicates positive
results for HIV-2
The presence of three lines as C, T-1(1) and T-2(2) within the result window indicates
a positive result for HIV-1 and/or HIV -2
Invalid results
No presence of control line (C) or/and pink/purple band observed in the result window
Indicate an invalid result. The direction may not have been followed correctly or the
test may have deteriorated. It is recommended that the specimen be retested.
Reporting of results
i. Reactive test Results will be reported as POSITIVE
ii. Non-reactive test results will be reported as NEGATIVE
Critical value
Not applicable

6.2.15 Limitation of the Procedure and Sources of Error

• Avoid haemolysed sample and beware of lipemic samples
• Samples other than blood have not been validated to give accurate results.
• Intensity of the patient bar does not necessarily correlate to the titre of antibody
• A negative result with BIOLINE HIV-1/2 does not exclude the possibility of an
infection with HIV.
6.2.16 Performance Characteristics
Refer to the method verification report of this procedure.

6.2.17 Supporting Documents

Sample collection Manual, HIV rapid testing algorithm

6.2.18 References
Package insert Abbott Bioline TM HIV-1/2 3.0

6.3 PROCEDURE FOR PERFORMING (HIV) BY USING UNIGOLD TEST

6.3.1 Purpose

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The purpose of this procedure is to describe the method of testing HIV-1and HIV-2
using Trinity Biotech Uni-Gold test assay.

6.3.2 Scope
This procedure is applicable in all sites that perform Trinity Biotech Uni-Gold HIV test

6.3.3 Responsible
Qualified, registered, licenced and trained Medical personnel are responsible for
implementing this test procedure.

The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.3.4 Principle
Recombinant proteins representing the immune-dominant regions of the envelope
proteins of HIV-1 and HIV-2, glycoprotein gp-41, gp120 (HIV-1) and glycoprotein
gp36 (HIV-2) respectively are immobilized at the test region of the nitrocellulose strip.
These proteins are also linked to colloidal gold and impregnated below the test region
of the device. A narrow band of the nitrocellulose membrane is also sensitized as a
control region. Antibodies to HIV-1 and HIV-2 react with the colloidal gold linked
antigens. The antibody protein-colloidal gold complex moves chromatographically
along the membrane to the test and control regions of the test device.

6.3.5 Sample Requirements

Whole blood/plasma sample collected in purple tube (EDTA)
Serum from clotted blood sample in plain tube
Centrifuge sample at 3000rpm for 5 minutes to obtain serum of plasma.

6.3.6 Equipment
Stop watch, Micropipette, Centrifuge, refrigerator

6.3.7 Materials
Uni-GoldTM HIV test-kit, Disposable gloves, Laboratory coat, 70% alcohol

6.3.8 Storage and stability

Uni-GoldTM HIV test device and wash solution should be stored between 2-270C or
as per manufacturer instructions
Whole blood specimen should be stores at 2-80C for up to 3 days at -200C or below

6.3.9 Safety

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 • Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
• All personal protective equipment (PPE) must be worn when performing this
procedure.
• All samples must be regarded as potentially infections.
6.3.10 Calibration
Perform equipment calibration as per Schedule

6.3.11 Quality Control

Run known Negative and Positive in-house controls once a week.
The test strips contain a control line, which turns colored if the run is valid.

6.3.12 Procedure Steps

i. Bring reagents and samples to room temperature at least 20 minutes before
use.
ii. Remove the test device from its protective wrapper.
iii. Label the device with sample identification number or patient/client
identification number.
iv. Peel the foil cover from the reagent area of the test strips.
v. For serum or plasma or whole blood collected by finger prick or
venipuncture samples;
vi. Using one of the disposable pipettes supplied with the kit, fill it with the
sample.
vii. Holding the pipette over the sample port, add two drops of sample
(approximately 60 µl) carefully to the sample port of the test device.
viii. Add two drops (approximately 60 µl) of wash reagent to sample port and
start the timer.
ix. Wait for a minimum of 10 minutes (up to 12 minutes) and read results
6.3.13 Biological Reference Intervals
Not Applicable

6.3.14 Interpretation and Reporting of Results

Results interpretation Reactive test results
Two pink/red lines of the intensity in the device window, the first adjacent to
letter ‘T’ (test) and the second adjacent to ‘C’ (control). Non – reactive test
results

A pink/red line of the intensity adjacent to the letter ‘C’ (control). But no
pink/red line adjacent to ‘T’ (test) this indicates a Non-Reactive result. Invalid
results

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 No pink/red line appears in the device window adjacent to the letter “C”
(control) irrespective of weather or not a pink /red line appears in the device
window adjacent to “T” (test). This is an INVALID result that cannot be
interpreted. An invalid result must be repeated
Results reporting
• Reactive test results: Report as HIV TEST POSITIVE
• Non-reactive test results-repeat 1st and 2nd tests following the national
HIV testing algorithm
• If the test results are still discordant report - INCONCLUSIVE then
inform the patient for retesting after 14 days
• If after 14 days, the test is still discordant report - INCONCLUSIVE
Collect fresh venous blood sample, refer the sample for ELISA testing
Critical Values Not Applicable
6.3.15 Limitation of the Procedure and Sources of Error
i. Avoid hemolyzed sample and beware of lipemic samples when
interpreting results.
ii. The BIOLINE HIV-1/2 test is designed to detect antibodies to HIV-1 and
HIV-2 in human serum, plasma and whole blood. Other body fluids or
pooled samples may not give accurate results.
iii. Intensity of the patient bar does not necessarily correlate to the titer of
antibody in the sample. iv. A negative result with BIOLINE HIV-1/2 does
not exclude the possibility of an infection with HIV.
6.3.16 Performance Characteristics
Refer to the method verification report

6.3.17 Supporting Documents

Sample collection manual
HIV rapid testing algorithm

6.3.18 References
Manufacture package insert (Trinity Biotech Uni-Gold HIV)

6.4 PROCEDURE FOR URINE PREGNANCY TEST

6.4.1 Purpose
This procedure provides instructions for Qualitative detection of HCG in urine

6.4.2 Scope
The procedure is used in the serology section in detection of pregNot applicablency

6.4.3 Responsible

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Qualified,trained and competent Medical Laboratory Technicians, Technologists and
scientist are responsible for implementing this test procedure.
The Head Microbiology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.4.4 Principle
The Human Chorionic GoNot applicabledotropin One Step PregNot applicablency
Test Strip (urine) is rapid chromatographic immunoassay for the qualitative detection
of Human Chorionic GoNot applicabledotropin in urine to aid in early detection of
pregNot applicablency. The test uses two lines to indicate results. The test line is pre
coated with a monocloNot applicablel Human Chorionic GoNot applicabledotropin
antibody to selectively detect elevated level of Human Chorionic GoNot
applicabledotropin. The control line is pre coated with goat anti-mouse IgG antibody.
The test also includes a burgundy coloured conjugate paid containing another
monocloNot applicablel HCG antibody conjugated with colloidal gold. The assay is
conducted by immersing the test strip in a urine specimen and observing the
formation of coloured lines .The specimen migrate via capillary action along the
membrane to reach with coloured conjugate. Positive specimen reacts with the
specific antibody HCG coloured conjugate to form a coloured line at the test line
region of the membrane. Absence of this coloured line suggest a negative results

6.4.5 Sample requirements

Fresh Urine collected from either morning,evining or any other time

6.4.6 Equipment
Stop watch
Refrigerator

6.4.7 Materials
Reagents Consumables
HCG test kit Disposable gloves, Laboratory coat

6.4.8 Storage and stability

Test strips reagent are stable at 2 to 30o C up to expiration date or as per
manufacturer instruction
If sample canot be tested within 1 hour of collection, it should be stored at 2 - 8oC for
24hrs

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6.4.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline. ii. All personal protective equipment (PPE) must be worn when
performing this procedure.
iii. All samples must be regarded as potentially infections.

6.4.10 Calibration
Perform equipment calibration as per schedule

6.4.11 Quality control

Run known Negative and Positive in-house controls (known patient or EQA sample)
once a week or when the new kit is opened

6.4.12 Procedure Steps

i. Remove the test strip from the sealed pouch and use it as soon as
possible.
ii. With arrow pointing towards the urine specimen immerse the test strip
vertically in the urine specimen for at least 10 to 15 seconds. Do not pass
the maximum line (MAX) on the test strip when immersing the strip.
iii. Place the test strip(s) on the non absorbent flat surface, start the timer and
wait for the colored line(s) to appear. The results should be ready in 5
minutes.
6.4.13 Biological reference intervals
Not applicable

6.4.14 Interpretation and reporting of results

Results interpretation
POSITIVE: two distinct colored lines appear. One line should be the control line
region (C) and another line should be on test line region (T).
NEGATIVE: one colored line appears in control line region (C) no apparent colored
line appears in the first line (T).
INVALID: control line fails to appear in both the control region. Reporting of results
Report results as PregNot applicablency test Negative or PregNot applicablency test
Positive.

6.4.15 Limitations of the Procedure and Sources of Error

i. The hCG one step pregnancy test strip (urine) is preliminary qualitative
test therefore neither the quantitative nor the rate of increase of hCG can
be determined by this test.

134
 ii. Very dilute urine specimen as indicated by low specific gravity may not
contain representative level of hCG. If pregnancy is still suspected the first
morning urine specimen should be collect 48 hours later and tested.
iii. first trimester pregnancies terminate for natural reasons, a test result that
is weakly positive should be confirmed by retesting with first morning urine
specimen collected 48 hours later.
iv. This test may produce false positive results. A number of conditions other
than pregnancy including trophoblastic disease and certain non
trophoblastic neoplasms including testicular tumours, prostate cancer,
breast cancer and lung cancer, causes elevated level of hCG. Therefore
the presence of hCG in the urine should not be used for the diagnosis
pregnancy unless this condition has been ruled out.
v. This test may produce false negative results. False negative results may
occur when the levels of hCG are below the levels of sensitivity level of
the test. When pregnancy is still suspected a first morning urine specimen
should be collected 48 hours later and tested. In case pregnancy is
suspected and the test continue to produce negative result see a physician
for further diagnosis.
6.4.16 Performance Characteristics
Refer to the method verification report of this procedure

6.4.17 Supporting documents

Sample collection manual

6.4.18 References
HCG package insert

6.5 PROCEDURE FOR HEPATITIS C ANTIBODY RAPID TEST

6.5.1 Purpose
The purpose of this procedure is to give instructions on how to perform Hepatitis C
Virus Antibody (HCV Ab) rapid test.

6.5.2 Scope
This procedure is applicable to all site perform hepatitis C Virus Antibody rapid tests

6.5.3 Responsible
It is the responsibility of the Head of serology Section to ensure effectively
implemented by all personnel working in the serology section.

6.5.4 Principle
The HCV Ab Rapid test Strip is a lateral flow chromatographic immunoassay based
on the Principle of the double antigen- sandwich technique. The test strip consists of

135
1) a burgundy colored conjugate pad containing HCV antigens conjugated with
colloidal gold (HCV Ag conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose
membrane strip contain a test band (T band) and a control band (C band). The T
band is pre-coated with non- conjugated HCV antigens, and the C band is pre-coated
with goat anti-rabbit IgG. When an adequate of test specimen is dispensed into the
sample well of the strip, the specimen migrates by capillary action across the strip.
The antibodies: either the IgG, the IgM, or the IgA, to HCV if present in the specimen
will bind to the HCV Ag conjugates. The immunocomplex is then captured on the
membrane by the pre coated HCV antigens, forming a burgundy colored T band,
indicating a HCV Ab positive test result. Absence of the T band suggests a negative
result. The test contains an internal control (C band) which should exhibit a burgundy
colored band of the immunocomplex of goat anti rabbit IgG / rabbit IgG-gold
conjugates regardless the presence of any antibodies to HCV. Otherwise, the test
result is invalid and the specimen must be retested with another device.

6.5.5 Sample requirement

2-3mls whole blood/serum/plasma. To obtain serum, Centrifuge blood collected in
plain red top tube at 3000rpm per 3 minutes. To obtain Plasma, Centrifuge blood
collected in EDTA tube at 3000rpm per 3 minutes

6.5.6 Equipment
Timer, Centrifuge and Refrigerator

6.5.7 Materials
Reagents Consumables
HCV Ab test kit (Test strips, disposable Disposable gloves,
dropper and HCV Ab buffer) Laboratory coat
Micropipette
6.5.8 Storage and stability
a. The test device in the sealed pouch can be stored at 2-400C or as instructed
by manufacturer up to the expiration date. The test device must remain in the
sealed pouch until use. DO NOT FREEZE
b. Store whole blood at 2-80c for up 3 days
c. Serum and plasma maybe stored at 2-80c for up 7 days, for long term storage,
serum and plasma specimens should be kept at-200c or below.
6.5.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline

136
 ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
6.5.10 Calibration
Listed equipment will be calibrated as per calibration schedule.

6.5.11 Quality control

Run known Negative and Positive in-house controls once a week or when new test
kit is opened

6.5.12 Procedure Steps

Allow test strip, specimen, buffer and/or controls to equilibrate to room temperature
prior to testing.
i. Remove the test device from the foil pouch and use it as soon as possible.
best results will be obtained if the assay is performed within one hour.
ii. Place the test device on a clean and level surface.
iii. Label the test device with sample ID
iv. For venepuncture whole blood samples; Hold the dropper vertically and
transfer 2 drops of venipuncture whole blood (approximately 50ul) to the
sample pad of the strip, then add 1 drop of buffer (approximately30ul) and
start the timer.
For finger stick whole blood sample; allow 2 hanging drops of fingerstick
whole blood (approximately 50ul) to fall into the center of the sample pad on
the test strip, then add 1 drop of buffer(approximately 30ul) and start the timer.
For serum or plasma sample; Hold the dropper vertically and transfer
1 drop of serum or plasma(approximately30ul) to the sample pad of the
test strip, then add 1 drop of buffer (approximately 30ul) and start the
timer.
v. Wait for the red line(s)to appear. the result should be read in 15 minutes.do
not interpret the result after 15 minutes

6.5.13 Biological Reference Intervals

Not Applicable

6.5.14 Interpretation and Reporting of Results Interpretation of results

Positive; two colored lines should be observed. The line in the test region (T) is the
prone line; The line in the control region (C) is the control line, which is used to
indicate proper performance of the device.
Negative; The control line appears in the test, but the test line is not visible.

137
 Invalid; No line appears in the control region. Under no circumstances should a
positive sample be identified until the control line forms in the viewing area if the
control line does not form, the test result is inconclusive and the assay should be
repeated
Reporting of results
Reactive test result - HCV Ab rapid test positive
Non - reactive test results - HCV Ab rapid test Negative
6.5.15 Limitations Of The Procedure And Source Of Error
i. The HCV Ab Rapid Test cassette (whole blood, serum, plasma) is for in
vitro diagnostic use only. This test should be used for the detection of
antibodies to HCV in whole blood, serum or plasma sample.
ii. The HCV Ab Rapid Test cassette (whole blood, serum, plasma) will only
indicate the presence of antibodies to HCV in the sample and should not
be used as the sole criteria for the diagnosis of hepatitis C viral infection.
iii. A negative result can occur if the quantity of the antibodies to HCV
present in the specimen is below the detection limits of the assay, or the
antibodies that are detected are not present during the stage of disease in
which a sample is collected.
6.5.16 Performance Characteristics
Refer to method verification report of this procedure

6.5.17 Supporting Document

Sample collection manual

6.5.18 References
HCV Ab Package inserts
Kit manufacturer paper insert

6.6 PROCEDURE FOR CRYPTOCOCCAL ANTIGEN RAPID TEST

PROCEDURE
6.6.1 Purpose
The purpose of this procedure is to give instructions on how to perform cryptococcal
antigen rapid test.

6.6.2 Scope
This procedure will be used by all staff and students perform CrAg test

6.6.3 Responsible

138
It is the responsibility of the Head of serology Section to ensure effectively
implemented and maintained.

6.6.4 Principle
The CrAg Lateral Flow Assay is a dipstick sandwich immune-chromatographic assay.
Specimens and specimen diluent are added into an appropriate reservoir, such as a
test tube, and lateral flow device is placed into the reservoir. The test uses specimen
wicking to capture gold- conjugated, anti-Crag monocloNot applicablel antibodies and
gold conjugated control antibodies deposited on the test membrane. If Crag is
present in the specimen, then it binds to the gold conjugated, anti-CrAg. The gold
labelled antibody antigen complex continues to pick up the membrane where it will
interact with the test line, which has immobilized ant Crag monocloNot applicablel
antibodies. The gold labelled antibody-antigen complex forms a sandwich at the test
line causing a visible line o form. With proper flow and reagent reactivity, the wicking
of any specimen, positive or negative, will cause the gold- conjugated control
antibody to move to the control line. Immobilized antibodies at the control line will
bind to the gold conjugated control antibody and form a visible control line. Positive
test results create two lines (test and control). Negative test results from only one line
(control). If control line fails to develop then the test is invalid.

6.6.5 Sample Requirement

2-3mls serum, plasma, whole blood (venous and finger prick) and cerebral spinal
fluid;
NOTE 1: To obtain serum, Centrifuge blood collected in plain red top tube at 3000rpm
per 3 minutes
NOTE 2: To obtain Plasma, Centrifuge blood collected in EDTA tube at 3000rpm per
3 minutes

6.6.6 Equipment
Timer, Centrifuge and Refrigerator

6.6.7 Materials
Reagents Consumables
CrAg test kit (CrAg LF Test strip, LF Disposable gloves,
Specimen diluents, LF titration diluents, Laboratory coat
CrAg positive control) Micropipette
6.6.8 Storage And Stability

139
 i. If a delay is encountered in sample processing, store at 2-8ºc for up to
72 hours is permissible.
ii. CSF, plasma, serum may be stored for longer period at -20ºc provided
they are not repeatedly thawed and refrozen
iii. Whole blood in transit should be maintained at 2-8ºc not -20ºc.
6.6.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
6.6.10 Calibration
Perform Equipment calibration as scheduled.

6.6.11 Quality Control

i. To ensure assay validity, a procedural control bar is incorporated in the
assay device.
ii. Run known positive and negative in-house controls weekly and when the
new kit of test strips is opened to verify kit.
6.6.12 Procedure Steps
i. Add one drop /40µ pipette LF Sample diluents to an appropriate labeled
reservoir. (Test tube) it is also a good practice to label the strip. ii. Add 40µ of
sample to the reservoir and mix. iii. Submerge the white end of a CrAg LF test
strip into the sample.
iv. Wait 10 minutes after inserting the strip
v. Read and record the results
6.6.13 Biological Reference Intervals
Not applicable

6.6.14 Interpretation And Reporting Of Results

Interpretation of results
Positive Results
Two red bands appear on the membrane. One band appear on the control region (C)
and another band appears on the test region (T) Negative Results
Only one red band appears on the control region C. No apparent red band appears
in the test region T.
Invalid Results

140
 No visible band at all or there is visible band only in the test region and not in the
control region, Repeat the procedure.
Reporting of results
For the positive result report as Cryptococcal antigen rapid test Positive
For the Negative result report as Cryptococcal antigen rapid test Negative
6.6.15 Limitations Of The Procedure And Source Of Error
i. The assay Performance Characteristics have not been established for
matrices other than serum, plasma, whole blood and CSF
ii. Depending on the disease and organism prevalence, testing should not be
performed as screening procedure for general population. The predictive
value of a positive or negative serologic result depends on the pre-test
likelihood of cryptococcal disease being present. Testing should only be done
when clinical evidence suggests the diagnosis of cryptococcal disease
iii. Testing hemolyzed serum samples could lead false negatives due to the high
background color on the strip
iv. This assay was not evaluated for potential interference related to specimen
pre-treatment with 2-mercatoethanol or with specimens including the
following substances: Vaginal cream, caffeine, ascorbic acid, intraconazole,
amphotericin B, acetaminophen, or acetylsalicylic acid
6.6.16 Performance Characteristics
Refer to method verification report of this procedure

6.6.17 Supporting Document

Sample collection manual

6.6.18 References

6.7 PROCEDURE FOR PERFORMING (HBsAg) RAPID TEST

6.7.1 Purpose
The purpose of this procedure is to give instructions on how to perform Hepatitis B
Virus Surface Antigen Rapid test in human whole blood, plasma or serum.

6.7.2 Scope
This procedure is applicable in all sites that perform Hepatitis B Virus surface antigen
in whole blood ,serum or plasma sample qualitatively.

6.7.3 Responsible
It is the responsibility of the Head of serology section and all laboratory personnel to
ensure effective implementation of this procedure.

6.7.4 Principle

141
 HBsAg is an antibody sandwich immunoassay. Colloidal gold conjugated
monoclonal antibody reactive to HBsAg is dry-immobilized onto a nitrocellulose
membrane strip. When the sample is added, it migrates by capillary diffusion through
the strip rehydrating the gold conjugate. If present, HBsAg will bind with the gold
conjugate antibody to form particles. These particles will continue to migrate along
the strip until the test zone (T) where they are captured by ant-HBs antibody
immobilized there and a visible red line appears. If there is no HBsAg in sample, no
red line will appear in the T zone. The gold conjugate will continue to migrate alone
until is captured in the control zone (C) by immobilized goat, ant-mouse IgG antibody
aggregating a red line, to serve as an internal process control, a control band should
always be seen after test is completed. Absence of a colored control line in the control
region is an indication of an invalid result.

6.7.5 Sample requirements

2-3mls Whole blood, centrifuged Serum or plasma samples

6.7.6 Equipment
i. Timer
ii. Refrigerator
iii. Centrifuge
6.7.7 Materials
Reagents Consumables

HBsAg test kits (HBsAg Device, Disposable gloves,

disposable specimen droppers) Laboratory coat
Assay diluent Micropipette
6.7.8 Storage And Stability
a. The test device in the sealed pouch can be stored at 2-400C or as
instructed by manufacturer up to the expiration date. The test device
must remain in the sealed pouch until use.DO NOT FREEZE.
b. Store whole blood at 2-80c for up 3 days
c. Serum and plasma maybe stored at 2-80c for up 7 days, for long term
storage, serum and plasma specimens should be kept at-200c or
below.
6.7.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.

142
 iii. All samples must be regarded as potentially infections.
6.7.10 Calibration
Perform Equipment calibration as scheduled.

6.7.11 Quality Control

Run known Negative and Positive in-house controls once a week or when new test
kit is opened .

6.7.12 Procedure Steps

i. Remove the test strip from foil pouch and use as soon as possible. Note:
Check and verify the device’s integrity before and after opening the foil
pouch
ii. Label the test device with patient ID Whole blood specimen:
iii. Hold the dropper vertically and transfer 2 drops of whole blood (approximately
50-60 ul) to the specimen area, then add one drop of buffer
Serum/ plasma;
i. Immerse the strip into the sample tube with the arrow end pointing towards
the sample.
ii. Let it stay immersed until you see liquid traveling up past the MAX word.
iii. Lay the strip (MAX side facing up) flat on a clean, dry, non-absorbent surface
iv. If cassette; Add 60µl (2 drop) of serum or plasma in to the sample window and
allow to soak in.
v. Read the results at 15-20 minutes. Ensure that the background of the test
area is white before interpreting the results
6.7.13 Biological Reference Intervals
Not Applicable

6.7.14 Interpretation And Reporting Of Results

Interpretation Of Results
NEGATIVE: if only one line (control line) appears in result line area, interpret
the result as negative. This shows that the concentration of HBsAg in the
sample is under the detection limit.
POSITIVE: if only two line (control line and test) appears in result line area,
interpret the result as positive.
INVALID: Control line fails to appear. Insufficient sample volume or incorrect
procedural technique is the most likely reasons for control line failure. Review
the procedure and repeat the test with a new test strip.
Reporting of results
Reactive test Results. Report as HBsAg rapid test positive
Non-reactive test results: Report as HBsAg rapid test negative

143
 6.7.15 Limitations Of Procedure And Source Of Error
i. HBsAg Rapid Test Kit detects HBsAg in human serum or plasma and
is only screening test. All reactive samples should be confirmed by
supplemental assays like PCR OR ELISA.
ii. A non -reactive result does not exclude the possibility of exposure or
infection with Hepatitis B virus.
iii. Patients with auto-immune liver diseases may show falsely reactive
results.
iv. This test is standardized to work best when the test procedure
mentioned in the package insert is strictly followed. Any deviation from
the test procedure may lead to erroneous results.
6.7.16 Performance Characteristics
Refer to method verification report of this procedure

6.7.17 Supporting Documents

Sample collection manual

6.7.18 References
HBsAg Package insert kit

6.8 PROCEDURE FOR SARS-COV-2 ANTIGEN RAPID DIAGNOSTIC TEST

6.8.1 Purpose
The purpose of this standard operating procedure (SOP) is to provide guidelines to
be followed for performing Rapid Antigen Detection Test for COVID-19 using the
Standard COVID-19 Ag detection assay kit

6.8.2 Scope
This procedure is to be performed at point of care or any health facility

6.8.3 Responsible
The Head of Serology is responsible for ensuring the effective implementation and
maintenance of this procedure
Qualified, competent and registered Medical Laboratory practitioners are responsible
for implementing this test procedure.

6.8.4 Principle
It is a rapid chromatographic immunoassay for qualitative detection of specific
antigens to SARS-CoV-2. When the liquid sample is dropped on the sample pad, the
antigen in the sample forms an immunocomplex with the antibody labelled with
colloidal gold. Its complex moves along with the liquid sample, and makes a contact

144
with the antibody immobilized on the membrane, followed by forming an
immunocomplex with the immobilized antibody, resulting in generation of a coloured
red purple line. Appearance of red purple line on the membrane indicates the
presence of antigen in the sample. Since the liquid of the sample migrates through
the membrane very fast, it makes it possible to detect the presence or absence of
antigen within 15 minutes.

6.8.5 Sample Requirements

Nasopharyngeal swab sample collected from nostril of the suspect individual.
Oropharyngeal swab sample collected from the posterior pharynx and tonsillar area
of the suspect individual.

6.8.6 Equipment
Stop watch, Micropipette/Supplied capillary

6.8.7 Materials
Reagent Consumables
Test devices Buffer Disposable gloves
Laboratory coat
70% alcohol
Mask
6.8.8 Storage And Stability
Store Covid 19 rapid kit devoice 2-30OC Protected from sunlight and should not be
frozen
6.8.9 Safety
i. Adhere to safety precautions as stated in the facility Safety manual
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections. iv. Refer to
National infection prevention and control Guidelines for health waste
management and safety practice.
6.8.10 Calibration
Perform equipment calibration as per schedule

6.8.11 Quality Control

Run known Negative and Positive in-house controls once a week.
The test strips contain a control line, which turns coloured if the run is valid.

6.8.12 Procedural Steps

145
 i. Peel off the aluminium foil seal from the sample processing tube containing
the extraction buffer.
ii. After the sample collection, plunge the swab up and down in the sample
processing tube for at least 15 seconds, taking care not to spill the contents
out of the tube.
iii. Remove the swab while pinching wall of the tube with the swab and rotating
the swab, to extract the liquid from the swab. iv. Firmly attach the dropper lid
to the top of the sample processing tube.
v. Remove the test cassette from the sealed pouch. vi. Sample adding: Reverse the
sample processing tube, holding the tube upright, and slowly add 3-4 drops to the
sample ole (S) of the test cassette then start the timer.
vii. Timing observation: judge the result 15 minutes after sample adding; do not
observe the results after 30 minutes later.
viii. After the test, put the medical wastes into the biosafety bag.
6.8.13 Biological Reference Interval
Not Applicable

6.8.14 Interpretation and Reporting of Results

Results interpretation
A Positive: Two distinct coloured bands appear on the strip.
Negative: Only one distinct coloured band on the strip.
Invalid: If no control band is seen.
Reporting of results
Report negative results as SARS-COV-2 – Negative.
Report positive result as SARS-COV-2 – Positive
Critical value
Any positive results
6.8.15 Limitation of the Procedure and Sources of Error
i. The kit is not intended for testing liquid sample such as wash or aspirate
sample or swab in transport media as a result can be compromised by
over dilution.
ii. Insufficient sample volume or incorrect procedural techniques are the
most likely reason for control line failure
6.8.16 Performance Characteristics
Refer manufacturer reagent kit insert for sensitivity and specificity

6.8.17 Supporting DocumentsS

Sample collection manual

6.8.18 References

146
Manufacture package insert kit for SARS COV-2

6.9 PROCEDURE FOR DENGUE VIRUS ANTIBODY DETECTION RAPID TEST

6.9.1 Purpose
This procedure provides details instruction for screening of dengue IgG, IgM antibody
by using rapid test strip as an aid in the diagnosis of infection with Dengue virus

6.9.2 Scope
This procedure is used in serology section when performing rapid Dengue antibody
rapid tests.

6.9.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.9.4 Principle
Dengue test utilizes immunochromatography whereby mouse anti- human IgM and
human IgG antibodies are immobilized on the nitrocellulose membrane respectively,
as two individual test lines (IgM line and IgG line) in the test window of the test device.
The IgG line in the test window is closer to the sample well and followed by IgM line.
As the test sample flows through the membrane within the test device, the coloured-
dengue specific recombinant antigen-colloidal gold conjugate complexes with
specific antibodies (IgM and or IgG) of dengue virus if present in the sample. This
complex moves further on the membrane to the test region where it is captured by
the anti-human IgM and or human IgG antibodies coated on the membrane leading
to formation of a coated band, which indicates a positive test results. Absence of the
coloured band in the test window indicates a negative test result. A built in control
line will always appear in the test window when the test has performed properly
regardless of the presence or absence of anti-Dengue virus antibodies in the sample.
Dengue NSI antigen test is a solid phase immunochromatographic assay. As the test
sample flows through the membrane within the test device and mobilize the gold anti-
NSI conjugate that it is coated on the conjugate pad if NSI it is present then the result
it is the formation of coloured band of the test (T)line region

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6.9.5 Sample Requirements
Plasma/whole blood, Serum (2-5ml)
NOTE 1: To obtain serum, Centrifuge blood collected in plain red top tube at
3000rpm/RCF per 3 minutes
NOTE 2: To obtain Plasma, Centrifuge blood collected in EDTA tube at
3000rpm/RCF per 3 minutes

6.9.6 Equipment
Timer ,Centrifuge ,refrigerator and Thermometer

6.9.7 Materials
Reagents consumables
• Dengue IgG/IgM antibody and NSI • Marker pen
antigen Cassette • Examination Gloves
• Buffer • Gauze
• Transfer pipette for dengue NSI
• Capillary pipette for
Dengue IgG/IgM
• Known Positive control,
• Known Negative control
6.9.8 Storage and Stability
i. The test kit should be stored at 15-25ºC in the sealed pouch for the
duration of the shelf-life (refer to manufacturer instruction)
ii. If the samples are not to be tested they should be refrigerated
immediately at 4 - 8 ºC
iii. If storage periods > 5 days the sample should be frozen at -200C
6.9.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
6.9.10 Calibration
Perform calibration of equipment (Timer ,Centrifuge
,refrigerator and Thermometer)as per calibration schedule

6.9.11 Quality Control

i. Control samples weather commercial or in-house made are run the same way
as patient sample on weekly bases and whenever a new kit is opened.
ii. Serology Section head should review Quality control records

148
6.9.12 Procedural Steps
i. Bring the samples and the test components to room temperature if
refrigerated or frozen. Mix the sample well prior to assay
ii. Remove the test strip from the foil pouch and use it as soon as possible
iii. Use transfer pipette to transfer sample by depressing the bulb of the pipette
iv. If capillary used withdraw 5ul of sample, the black bar near the opening end
of pipette indicates the required 5ul of sample.
v. Drop the sample in the corner pointed by S1»
vi. Hold the pipette in a vertical position over the left “S” sample well on the
device
vii. Transfer 2 drops of sample into well
viii. Dispense 2 drops of sample buffer to the right ”S” sample well
ix. Read result at the end of 20 minutes
6.9.13 Biological Reference Interval
Not Applicable
6.9.14 Interpretation and Reporting of Results
Interpretation of results
NEGATIVE
• If only the “C” line is developed, the test indicates that no detectable
antibodies to dengue are present in the specimen
POSITIVE
• Lines showed to control and NSI –NS1 POSITIVE during window period
• Lines showed to control and IGM-IgM Positive Chronic dengue
• Lines Showed to control and IgG- IgG Positive during early infections BUT
has been treated
INVALID
If NO line is developed at “C”, the assay is invalid regardless of colour development
on the “T” line. Repeat the test Reporting of results
Report results as: Dengue - Negative or Dengue – Positive

Critical value
Positive findings

6.9.15 Limitation of the Procedure and Sources of Error

This kit is intended ONLY for testing of individual samples. Don’t use it for testing of
cadaver samples, saliva, urine or other blood samples or pooled (mixed) blood

6.9.16 Performance Characteristics

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 Refer manufacture kit insert for specificity and sensitivity. Also Method verification of
this procedure should be done and that the report should be referred to verify
compliance to this requirement. for Dengue rapid test procedure

6.9.17 Supporting Documents

Sample collection manual
6.9.18 References
Dengue IgG/IgM antibody + NSI antigen Cassette Test Rapid Test Strip Package
insert.

6.10 PROCEDURE FOR PLAGUE RAPID TEST

6.10.1 Purpose
This procedure provides details instruction of detecting acute bacterial infection
caused by Yersinia pestis

6.10.2 Scope
This procedure is used in serology section when performing rapid Plague rapid
tests(F1RDT)

6.10.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.10.4 Principle
F1RDT detect pathogen‐specific antigens (the F1 capsular antigen, which is part of
the outer surface of Yersinia pestis, the bacteria causing plague) in a small quantity
of different body fluids through lateral flow immunochromatography. The test is
simple to perform and provides a result within 15 minutes. It can be performed in the
pus contained in the buboes (swellings), or in the sputum (mucous coughed up from
the respiratory tract) of people with suspected pneumonic plague.

6.10.5 Sample Requirements

Bubo aspirate, urine, and sputum,serum

6.10.6 Equipment
Centrifuge, timer, thermometer

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6.10.7 Materials
Reagents Consumables
F1RDT test kit • Marker pen
• Examination Gloves
• Gauze

6.10.8 Storage and Stability

Refer to manufacturer storage instructions of test devices and samples

6.10.9 Safety
i. Adhere to safety precautions as stated in the Safety manual/IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iii. All samples must be regarded as potentially infections.
6.10.10 Calibration
Perform calibration of equipment (Timer ,Centrifuge and Thermometer)as per
calibration schedule

6.10.11 Quality Control

Refer to manufacturer insert package

6.10.12 Procedural Steps

Refer to manufacturer insert package

6.10.13 Biological Reference Interval

Not Applicable

6.10.14 Interpretation and Reporting of Results

Interpretation of results
Refer to manufacturer insert package Reporting of results
Report results as: Plague - Negative or Plague – Positive Critical value
Positive findings

6.10.15 Limitation of the Procedure and Sources of Error

F1RTD test needs to be combined with other laboratory evaluations to confirm the
diagnosis

6.10.16 Performance Characteristics

Method verification of this procedure should be done and that the report should be
referred to verify compliance to this requirement. of this procedure

151
 6.10.17 Supporting Documents
Sample collection manual

6.10.18 References
Chanteau S, Rahalison L, Ratsitorahina M, Mahafaly, Rasolomaharo M, Boisier P, et
al. Early diagnosis of bubonic plague using F1 antigen capture ELISA assay and
rapid immunogold dipstick. International Journal of Medical
Microbiology 2000;290(3):279-83.

6.11 PROCEDURE FOR HELICOBACTER PYLORI ANTIGEN TEST

6.11.1 Purpose
This procedure provides instructions for the rapid detection of Helicobacter pylori
antigen in human stool sample.

6.11.2 Scope
This procedure is used in serology section to all rapid H. pylori antigen tests.

6.11.3 Responsible
Qualified, trained, and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.11.4 Principle
This is a ready to use test that is based on the homogeneous membrane system
technology with latex microspores to perform the test, an aliquot of diluent sample is
added to sample well of the test cassette. The sample flows through a label pad
containing Pylori antibody coupled to red –colored colloidal gold. In the presence of
antigens, they bind to the antibody coated on the colloidal gold particles to form
antigen-antibody-gold complexes. These complex moves on the nitrocellulose
membrane by capillary action towards the test line region on which Pylori specific to
the antibody on the membrane in the form of a line. A second red control line will
always appear in the results windows to indicate that the test has been correctly
performed and the test device functions properly. If pylori is not present or lower than
the detection limit of the test, only the control line will be visible. If the control line
does not develop, the test in invalid.

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6.11.5 Sample Requirements Stool sample.
6.11.6 Equipment
Timer

6.11.7 Materials
Reagents consumables
Test cassette dilution buffer Marker pen
-Known Positive control, Examination Gloves
-Known Negative control

6.11.8 Storage and Stability

I. Store un opened test device at 2- 30 ̊C.if stored at 2-8 C
̊ , ensure that
the test device is brought to room temperature before opening. Do not
freeze the kit or expose the kit over 30 ̊C. (refer to manufacturer
instruction)
II. Store sample at 2-8 ̊C for up to 72 hours.
6.11.9 Safety iv. Adhere to safety precautions as stated in the Safety manual/IPC
guideline
v. All personal protective equipment (PPE) must be worn when performing
this procedure.
vi. All samples must be regarded as potentially infections.
6.11.10 Calibration
Perform calibration of equipment as per calibration schedule

6.11.11 Quality Control

Analyse known Negative and Positive in-house controls once every week and
whenever a new kit is opened.

6.11.12 Procedural Steps

i. Remove the test device from its foil pouch by tearing along the notch and use
as soon as possible
ii. Unscrew the cap of the sample collection tube. iii. Randomly stab the sample
collection stick into the fecal sample at list 6
different sites
iv. Return the sample collection stick into the sample tube and tighten the cap
v. Shake the sample collection tube vigorously to mix the sample and the
extraction buffer
vi. Hold the sample collection tube upright and break off the tip of the sample
collection tube, invert the sample collection tube and transfer two drops of the
mixture into the sample pad of the test strip then start timer
vii. Wait 10-15 minutes and read the results for the colored lines to appear

153
 viii. Do not read result after 15 minutes
6.11.13 Biological Reference Interval
Not Applicable

6.11.14 Interpretation and Reporting of Results

Interpretation of results
Negative - Only one coloured band appears on the control(C) region. No apparent
band on the test(T) region
Positive - In addition to a pink coloured control (C) band, a distinct pink coloured band
will also appear in the tests(T) region
Invalid – a total absence of colour in both regions or no coloured line appears on the
control (C) region is an indication of procedure error or the tests deterioration. Repeat
the test with a new kit.
Reporting of results
Report results as: H pylori - Negative or H pylori – Positive

6.11.15 Limitation of the Procedure and Sources of Error

1.1. The test is a qualitative assay and is not for quantitative determination of
antibodies concentration levels in human stool only
1.2. The results obtained should only be interpreted in conjunction with other
diagnostic results and clinical information.
1.3. A negative result can occur if the quantity of the. Pylori antigen presence
in the sample below the detection limits of the assay, or the antigen that
are detected are not present during the stage of diseases in which a
sample is collection.

6.11.16 Performance Characteristics

Refer manufacture kit insert for specificity and sensitivity. Also Method verification of
this procedure should be done and that the report should be referred to verify
compliance to this requirement. for H pylori procedure

6.11.17 Supporting Documents

Sample collection manual

6.11.18 References
Manufacturer Kit insert for H pylori
6.12 PROCEDURE FOR HELICOBACTER PYLORI ANTIBODY RAPID TEST
6.12.1 Purpose

154
Rapid chromatographic immunoassay for the qualitative detection of antibodies (IgG)
anti-Helicobacter pylori (H. pylori) in human serum or plasma. It is used as an aid in
the diagnosis of infection with H. pylori

6.12.2 Scope
This procedure is used in serology section when performing rapid H. pylori antibody
tests.

6.12.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
implementing this test procedure.
The Head serology is responsible for ensuring the effective implementation and
maintenance of this procedure.

6.12.4 Principle
The H. Pylori rapid Test device is a lateral flow chromatographic assay based on the
Principle of the antibody – sandwich technique. The membrane is pre coated with
H.pylori Antigen on the test line region of the test. During testing H. pylori Antibodies
in the serum or plasma sample reacts with coated antigen and migrates upward on
the membrane chromatography by capillary action to the membrane and generate a
colored line. The presence of this colored line in the test region indicates a positive
result, while its absence indicates a negative result

6.12.5 Sample Requirements

Plasma/whole blood, Serum (2-5ml)
NOTE 1: To obtain serum, Centrifuge blood collected in plain red top tube at 3000rpm
per 3 minutes
NOTE 2: To obtain Plasma, Centrifuge blood collected in EDTA tube at 3000rpm per
3 minutes

6.12.6 Equipment
Timer, Centrifuge, refrigerator and Thermometer

6.12.7 Materials
Reagents consumables
Test cassette/strips buffer Marker pen
-Known Positive control, Examination Gloves
-Known Negative control

155
 6.12.8 Storage and Stability
III. Store unopened test device at 2- 30 ̊C. If stored at 2-8 C̊ , ensure that
the test device is brought to room temperature before opening. Do not
freeze the kit or expose the kit over 30 C ̊ . (refer to manufacturer
instruction)
IV. Store sample at 2-8 C ̊ for up to 72 hours.
6.12.9 Safety vii. Adhere to safety precautions as stated in the Safety manual/IPC
guideline
viii. All personal protective equipment (PPE) must be worn when performing
this procedure.
ix. All samples must be regarded as potentially infections.
6.12.10 Calibration
Perform calibration of equipment (Timer, Centrifuge,
refrigerator and Thermometer)as per calibration schedule

6.12.11 Quality Control

i. Analyse known Negative and Positive in-house controls the same way as
sample testing procedure once every week and whenever a new kit is opened.
ii. Serology Section head should review Quality control records
6.12.12 Procedural Steps
i. Remove the test device from its foil pouch by tearing along the notch and use
as soon as possible
ii. Place the test strip on a clean and level surface, hold the dropper vertically
and transfer 1 drop of plasma/serum to the sample pad.
iii. Add 1 drop of buffer, then start the timer
iv. Read the results in 10-15 minutes
6.12.13 Biological Reference Interval
Not Applicable

6.12.14 Interpretation and Reporting of Results

14.1 Interpretation of results
Negative - Only one coloured band appears on the control(C) region. No apparent
band on the test(T) region
Positive - In addition to a pink coloured control (C) band, a distinct pink coloured band
will also appear in the tests(T) region
Invalid – a total absence of colour in both regions or no coloured line appears on the
control (C) region is an indication of procedure error or the tests deterioration.
Repeat the test with a new kit.

156
 14.2 Reporting of results
Report results as: H pylori - Negative or H pylori – Positive
14.3 Critical value Not applicable
6.12.15 Limitation of the Procedure and Sources of Error
• The test is for in vitro diagnostic use only
• The test should not be used as the sole criteria for the diagnosis of pylori
infection since it only indicates the presence of antibodies in the sample
6.12.16 Performance Characteristics
Refer manufacture kit insert for specificity and sensitivity. Also Method verification of
this procedure should be done and that the report should be referred to verify
compliance to this requirement. for H. pylori procedure

6.12.17 Supporting Documents

Sample collection manual

6.12.18 References
Manufacturer Kit insert for H pylori
6.13 PROCEDURE FOR BRUCELLA ANTIBODY DETECTION
6.13.1 Purpose
This procedure provides instructions for Qualitative detection of antibodies of all
isotopes against Brucella species.

6.13.2 Scope
The procedure is used in the serology section in the diagnosis of brucellosis.

6.13.3 Responsible
Qualified, registered and competent health laboratory practitioners are responsible
for implementing this test procedure. Section heads are responsible for ensuring the
effective implementation and maintenance of this procedure.

6.13.4 Principle
This test is based on antigen /antibody reaction. The smooth, attenuated stained
Eurocell antigen suspensions are mixed with the patient’s serum. Specific antibodies
to Brucella antigens if present in the patient serum will react with the antigen
suspensions to produce an agglutination reaction. No agglutination indicates the
absence of specific antibodies to Brucella antigens.

6.13.5 Sample Requirement

157
 Serum sample is prefarable for this procedure. Allow blood to clot and Centrifuge the
sample at 3000rpm for 5 minutes

6.13.6 Equipment
Centrifuge, Pipettes, Shaker and Stop watch

6.13.7 Materials
Stained Eurocell-A/ Eurocell -M Antigen suspensions, Slide Test, 70%
alcohol, Known Positive control, Known Negative control, Marker pen,
Examination Gloves.

6.13.8 Storage and Stability

Reagent should be stored at 2-8°C. Sample can be stored at room temperature for
4hrs then can be stored at 2-8°C If serum separated can be stored at -20°C for 1year.

6.13.9 Safety
i. Adhere to safety precautions as stated in the facility Safety manual
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections. iv. Refer to National
infection prevention and control Guidelines for health care services in
Tanzania
6.13.10 Calibration
Calibration of Centrifuge should be done as per schedule.
Maintenance
Maintenance of Centrifuge should be done as planned.

6.13.11 Quality Control

Run the positive and negative controls sample daily before performing patient
samples or run QC Parallel with patient sample.

6.13.12 Procedural Steps

i. Prior to the start bring all reagents to room temperature (20 to 25˚C). ii.
Shake and mix the Eurocell antigen suspension well before dispensing.
iii. Place one drop of positive and negative control onto the reaction circle of glass
slide.
iv. Place 80ul of saline onto the next reaction circle of the glass slide.
v. Place 80ul of patient serum to be tested onto the next reaction circle. vi. Add
one drop of the appropriate Eurocell antigen suspensions in each of the above
circles. vii. Mix contents of each circle uniformly over the entire circle with

158
 separate mixing sticks. viii. Gently rock the slide back and forth/observe for
agglutination macroscopically for one minute
6.13.13 Biological Reference Interval
Not applicable

6.13.14 Interpretation and Reporting of Results

Interpretation of results
Agglutination is a POSITIVE test results and indicates the presence of specific
antibodies to Brucella in the patient’s serum. No agglutination is a NEGATIVE test
results
Reporting of results
Report results as Brucella antibody – NEGATIVE or Brucella antibody – POSITIVE

6.13.15 Limitation of the Procedure and Sources of Error

The test cannot distinguish between past infection and current infection

6.13.16 Performance Characteristics

Refer to the verification report of this procedure

6.13.17 Supporting Document

Result management procedure

6.13.18 References
Monica Cheesbrough: District Laboratory Practice in Tropical Countries, Vol 1,
Tropical Health Technology, 1998.
URIT 560 Urine Analyzer– User Manual for Urine Chemistry Analyzer
Practical Laboratory Manual-Jane Carter and Orgenes Lema
6.14 PROCEDURE FOR SALMONELLA TYPHI ANTIBODIES QUANTIFICATION
METHOD
6.14.1 Purpose
This procedure provides instructions for in vitro detection and quantitative estimation
of specific antibodies to salmonella present in Human serum

6.14.2 Scope
The procedure is used in serology section when performing widal test by slide
method.

6.14.3 Responsible

159
Qualified full registered and competent laboratory practitioners are responsible for
implementing this test procedure.
The Head of serology section is responsible for ensuring the effective implementation
and maintenance of this procedure.

6.14.4 Principle
This test is based on the Principle of direct agglutination reaction. The smooth
suspension of killed salmonella bacilli carries homologous O and H antigens. When
patient serum (containing antibodies to S typhi and S paratyphi) is incubated with
respective antigens, visible agglutination occurs. Arising titre of antibodies is
indicative of Enteric fever

6.14.5 Sample Requirements

Serum sample is preferable for this procedure. Allow blood to clot
Centrifuge the sample at 3000rpm for 5 minutes

6.14.6 Equipment
Timer, Centrifuge, Pipettes, Water bath

6.14.7 Materials
Stained salmonella antigen set, Stained salmonella antigen S typhi “O”, Stained
salmonella antigen S typhi “H”, Disposable gloves, pipettes, White Tile or slide,
Sample rack, Test tube rack, 5%Sodium hypochlorite to wipe and disinfect the spills
and Marker Pen

6.14.8 Storage and Stability

Reagent should be stored at 2-8°C
Sample can be stored at room temperature for 4hrs then can be stored at 2-8°C if
serum separated can be stored at -20°C for 1year.

6.14.9 Safety
i. Decontaminate working surfaces twice daily, in the morning and
afternoon
ii.
iii. Adhere to safety precautions as stated in the facility Safety manual
iv. All personal protective equipment (PPE) must be worn when
performing this procedure.

160
 v. All samples must be regarded as potentially infections. vi. Refer to
National infection prevention and control Guidelines for health care
services in Tanzania,
6.14.10 Calibration
Centrifuge should be calibrated as per schedule.
1.1. Maintenance
Maintenance of Centrifuge should be done as planed

6.14.11 Quality Control

Analyze the positive and negative controls sample daily before performing patient
samples or run QC Parallel with patient sample.

6.14.12 Procedural Steps

Rapid slide test (Widal screening test)
i. Clean the glass slide or white tile provided in the kit and wipe
ii. Place 1 drop of undiluted serum to be tested in each of the first two
circle (1-2)
iii. Add one drop of antigen “O” and “H” in circles 1,2 respectively
iv. Mix the contents of each circle with separate stick and spread to fill
the entire circle area
v. Rock the slide for one minute and observe for agglutination
vi. If agglutination is visible within one minute then proceed for
quantitative estimation Quantitative slide/white tile test
Circle Serum Volume Appropriate Antigen Titre
Drop
01 0.08ml 1 drop Mix and Rotate for 1:20
one minute and
02 0.04ml 1 drop observed 1:40

03 0.02ml 1 drop agglutination 1:80

04 0.01ml 1 drop 1:160

05 0.005ml 1 drop 1:320

6.14.13 Biological Reference Interval
 When value of O and H antigen are less than 1:160 NEGATIVE
 When value of O and H antigen are greater than 1:160 POSITIVE
6.14.14 Interpretation And Reporting Of Results
i. Salmonella typhi O: 1:(Respective titre)
ii. Salmonella typhi H: 1 :(Respective titre)

Rapid/ white tile widal test

161
 Granular agglutination in case of “O” and flocculating agglutination in case of “H”
indicate positive reaction Quantitative slide/white tile test
A diagnostic titre of 1:80 suggest positive results

6.14.15 Limitation of the Procedure and Sources of Error

Rapid slide tests or quantitative slide tests are non-specific type of test. The positive
results should be further confirmed by tube test and other microbiological
investigations

6.14.16 Performance Characteristics

Refer data for verification report
6.14.17 Supporting Documents
Not applicable

6.14.18 References
Reagent package insert for widal test

162
6.15 PROCEDURE FOR CHORELA RAPID DIAGNOSTIC TEST
6.15.1 Purpose
This procedure provides instructions for performing rapid Bioline Cholera Ag O1/
0139 test

6.15.2 Scope
The procedure is used for performing rapid Bioline Cholera Ag O1/ 0139 test in
Microbiology section.

6.15.3 Responsible
Qualified, trained and competent health laboratory practitioners are responsible for
performing this test

6.15.4 Principle
Cholera antigen test contains a membrane strip which is precoated with mouse
monoclonal anti- vibrio cholera O1 antibody on test line O1 region and with the
device. These line in result window are not visible before applying any specimen. The
control line is used for procedural control. Control line should always appear if the
test procedure is performed properly and the test reagents of control line are working.
A purple test line will be visible in the result window if vibrio cholera O1 and/or O139
antigens are present in the specimen.
When a specimen is added to the test, vibrio cholera O1 and O139 antigens in the
specimen react with colloidal gold-labelled V. Cholerae O1 and V. Cholerae O139
specific antibodies and form a complex of antigen-antibody colloidal gold conjugates.
As this complex migrates along the length of the result window by capillary action,
the complex is captured by the mouse monoclonal V. cholerae 01 antibody on test
line 01 (O1) and mouse monoclonal V. cholerae 0139 antibody in the test line 0139
(0139) across the result window and generates a colored line. In the absence of V.
cholerae 01 and 0139 antigen in specimens, a complex is not formed and no colored
test line appears in the result window of test device.

6.15.5 Sample Requirement

Human stool sample

6.15.6 Equipment
Refrigerator

6.15.7 Materials

163
 Disposable gloves, Timer, Biohazard bag, Test device, Sample collection tube with
extraction buffer, Sample collection swab for solid stool samples and Sample
collection dropper for liquid stool samples

6.15.8 Storage and Stability

a) For best result stool sample should be tested as soon as possible after
collection.
b) Extracted stool sample is stable for 72 hours when store in 2-8⁰C
c) Do not use stool sample in transport media or preservatives
6.15.9 Safety
a) Wear protective gloves while handling sample and wash hands after testing
b) Do not mix or interchange different specimens
c) Decontaminate and dispose all sample test kits and potentially
contaminated materials in a biohazard container as if they were infectious.
d) Do not mix reagents of different lots or those of other products.
6.15.10 Calibration
Not applicable

6.15.11 Quality Control

a) the cholera antigen rapid test has three test lines O1, O139 and control line.
The control line is used for procedural control and shows that the diluent
has been applied successfully and that the active ingredients of main
components on the strip are functional.
b) Use samples with known results (positive and negative) to test each test kit
batch before use and ensure the results correlate with the respective control
samples.
6.15.12 Procedural Steps
Specimen Collection
In case of solid stool sample.
a) Loosen the filter cap of the specimen collection tube.
b) Collect the sufficient stool sample (about 50mg) from different 4-5 sites of
stool specimens using the specimen collection swab.
c) Immediately place the swab into the specimen collection tube.
d) Vigorously mix the solution by rotating the swab at least 10 times against
the side of the specimen collection tube.
e) Release as much liquid as possible from the swab by squeezing the sides
of the swab as the swab is withdrawn.
f) Discard the swab and then assemble filter cap on the specimen collection
tube.
In case of liquid stool sample.
a) Loosen the filter cap of the specimen collection tube.

164
 b) Draw liquid specimens up to the fill line (about 300ul) using disposable
dropper.
c) Immediately transfer liquid specimen into the specimen collection tube.
d) Discard the dropper and then assemble filter cap on the specimen collection
tube.
6.15.13 Testing Procedure.
a) Bring the test device and sample to reach a temperature between 15-30⁰C
for at least 30 minutes in case they were refrigerated.
b) Remove the test device form the foil pouch and place it on a flat, dry surface.
c) Shake the collection tube thoroughly to ensure proper mixing of the sample
with extraction buffer.
d) Loosen the nozzle cap of the specimen collection tube.
e) Hold the collection tube vertically and dispense 3 drops(70ul) into the
specimen well of test device.
f) Wait a minimum of 15 minutes then read results. Do not read test results
after 15 minutes; reading results after 15 minutes can yield false results.
6.15.14 Biological Reference Intervals
Not applicable

6.15.15 Interpretation And Reporting Of Results

A coloured control line will appear in the left section of the result window to show that
the test is working properly.
Coloured lines will appear in the middle and right section of the result window. These
lines are test line 0139 and test line 01 (0139, 01).
Negative Result: The presence of only control line (C) within the result window
indicates a negative result.
Positive Result: The presence of two lines as control line (C) and test line 01 (O1)
within the result window indicates a positive result for V. cholerae 01 antigen. The
presence of two lines as control line (C) and test line 0139 (0139) within the result
window indicates a positive result for V. cholerae 0139 antigen.
The presence of three lines as control line (C) test line 01 (O1) and test line 0139
(0139) within the result window indicates a positive result for V. cholerae 01 and 0139
antigen.
Caution: The presence of any line, no matter how faint, the result is considered
positive.
Invalid Result: If the control line (C) is not visible within the result window after
performing the test, the result is considered invalid.

165
6.15.16 Limitations Of The Procedure And Sources Of Error
a) A negative result does not exclude the possibility of V. cholerae 01 and/or
0139 infection in a patient. Failure to detect V. cholerae 01 and/or 0139 may
be a result of factors such as collection of specimens at an improper time in
the disease when few bacteria are present and improper sampling or handling
of the specimen.
b) A positive result does not preclude the presence of other enteric pathogens.
While the relationship between cholera and gastroenteritis is well established,
concurrent infection with other microbial pathogens is possible. Additional
microbiological tests should be performed in parallel with Bioline Cholera Ag
O1/ 0139 test kit in order to exclude other possible causes of the illness.
c) Test results should be interpreted in conjunction with information available
from epidemiological studies, clinical symptoms of the patient and other
diagnostic procedures.
6.15.17 Performance Characteristics
Refer to manufacture user manual

6.15.18 Supporting Documents

Sample collection manual

6.15.19 References
Refer to manufacture user manual

166
CHAPTER 7: BACTERIOLOGY AND MYCOLOGY
7.1 POTASSIUM HYDROXIDE WET MOUNT PREPARATION
7.1.1 Purpose
This procedure provides instructions for examination of wet preparations that is
mainly used to examine samples and cultures for motile bacteria, C.S.F for
capsulated yeast cells and for fungi.

7.1.2 Scope
This procedure applies to the microbiology section and health laboratory practitioners
in the laboratory settings.

7.1.3 Responsible
Competent Health Laboratory Practitionersare responsible for implementing this test
procedure.
The Head of Microbiology/who asned is responsible for ensuring the effective
implementation and maintenance of this procedure.

7.1.4 Principle
A KOH preparation is used for Samples such as skin scrapings, nail, infected hairs,
or for other Samples such as sputum to clear out background debris that may be
confused with fungal elements. KOH dissolves proteinaceous tissues, including
keratin, and renders them transparent. This enables fungi to be visualized more
easily. The use of KOH is not necessary for Samples such as CSF where background
debris is minimal.

7.1.5 Sample Requirements

KOH preparation is used ideally in suspected cases of dermatophytosis, i.e., fungal
infection of skin, hair, or nails. Also used for specimen such as sputum, pus, and
urine sediment.

7.1.6 Equipment Microscope

7.1.7 Materials
Potassium hydroxide 10 or 20%, Potassium hydroxide 10 g, Glycerol 10ml, Deionized
water 80ml, (optional) helps prevent the KOH mount from drying. Dispense working
solution in a dropper bottle. Microscope slides, 24 x 50 mm cover slips, sterile
forceps, mounting needle

167
7.1.8 Storage and Stability
Potassium hydroxide, 10 or 20% solution is stable for one year at room temperature

7.1.9 Safety
Observe standard safety precautions when handling Samples. Refer to Safety
Manual
Discard all used materials (e.g., sticks, pipettes, disposable forceps) in a bucket
containing bleach.

7.1.10 Calibration Not applicable

7.1.11 Quality Control
Fungal spores may contaminate the KOH solution, and may give false positive
results. Solution must be examined for signs of contamination (e.g., turbidity) before
each use.

7.1.12 Procedure Steps

i. Place the material to be examined on a glass slide, add a drop of 10% KOH
ii. Place a cover slip over the preparation. iii. Let stand for five to ten
minutes. iv. If clearing is not complete, an additional ten minutes is necessary.

Note: Keratinous (skin, nails) Samples should be left at room temperature for 20 to 30 minutes
to allow digestion and “clearing” of the keratin, after clearing, press coverslip gently to make
a thin mount.

v. Examine the slide microscopically on low power and confirm observations with
high power. Observe for hyphae, conidia, budding yeasts, spherules or
sclerotic bodies, etc. Consult photomicrographs in appropriate references
when necessary, to identify fungal structure.

7.1.13 Biological Reference Intervals Not applicalbe

7.1.14 Interpretation and Reporting of Results
Interpretation
Dermatophytes in skin or nail are seen as branching hyphae or arthrospores and
often appear slightly greenish in color, with hyphae running across the colorless host
cells. Most hyphae will be parallel-sided and around 2 µm in width. Yeasts are
present as budding cells, pseudohyphae, or yeast mycelium.
In infections caused by dematiaceous fungi, the hyphae are often brown
(dematiaceous septate hyphae).

168
 Artifacts, such as fibers, may be distinguished from hyphae from the lack of septa,
tapering ends, and size differences.
Reporting of Results
Report the type of fungal structure seen. Do not report quantity.
Examples: “Fungal elements seen (septate hyphae)”
“Fungal elements seen (yeast cells and pseudohyphae)”
Report negative results as: “No fungal elements seen”
Report as: “Fungal elements seen (Malassezia spp.)”

7.1.15 Limitations of the Procedure and Sources of Errors

i. Cotton swabs should not be used in preparing these slides as the cotton strands
may resemble hyphae. ii. The contrast between unstained fungal elements that
may be present and the background mounting fluid can be accentuated by
narrowing the iris diaphragm to reduce the amount of incident light; or use of
phase-contrast microscopy that can greatly enhance visualization of organisms.
iii. KOH preparations are presumptive and should not be substituted for culture
and further identification.
iv. KOH should not be stored in a glass bottle as it will leach minerals from the
glass. The minerals will result in a cloudy, flocculant solution. This can be
avoided by storing the KOH in plastic, polystyrene or other non-glass container.
v. Gentle heating may speed the activity of the KOH, but it may be harmful to the
specimen if overdone. vi. KOH preparations are not permanent; the reagent will
eventually destroy the fungi. The addition of small amount of glycerol to the
preparation will preserve it for several days.
vii. KOH combined with calcofluor white is a more sensitive method, but a
fluorescent microscope with appropriate filter is required.
viii. India ink, added to the KOH, stains fungal elements and helps them stand out
against the background. Reagent is prepared as follows: Make a 10% KOH
solution in Parker Super Quink Ink, permanent blue black (50 ml of ink + 5 g of
KOH pellets). Centrifuge KOH-ink solution at 2,000 x g for ten minutes. Pour
supernatant into plastic (not glass) sterile tube. Store at room temperature.
7.1.16 Performance Characteristics Not applicable
7.1.17 Supporting Documents Not applicable
7.1.18 References
Lynne S. Garcia, Henry D. Isenberg. Clinical Microbiology Procedures
Handbook. 3rd edition.
American Society for Microbiology. Washington DC, USA. 2010.

169
 Guidelines on Standard Operating Procedures for Laboratory Diagnosis of
HIV/AIDS Opportunistic Infections in Patients. WHO Regional Office for
Southeast Asia. New Delhi. June 2001
Bailey and Scott’s Diagnostic Microbiology. 13th edition. Mosby, Inc. St. Louis,
Missouri, USA. 2013.

7.2 PROCEDURE FOR ZIEHL NIELSEN STAIN

7.2.1 Purpose
This procedure provides instructions for performing Ziehl-Nielsen stain of sputum or
other body fluid samples.

7.2.2 Scope
This procedure is to be used for detection of Acid Fast Bacilli in sputum or other body
fluid samples in the Laboratory

7.2.3 Responsible
The section heads and technical staffs are responsible for implementing this
procedure.

7.2.4 Principle
This procedure is used to stain mycobacterium tuberculosis and mycobacterium
leprae. These bacteria are also called acid fast bacilli. They stain with carbolfuschin,
which is a red dye. They retain the dye when treated with acid, which is because of
the presence of mycolic acid in their cell wall.

7.2.5 Sample Requirements

The Sputum and body fluids such as CSF, synovial, pericardial, synovial, ascitic,
blood, pus, bone marrow, tissue biopsies or pleural fluid samples.

7.2.6 Equipment
Centrifuge (when necessary), Bunsen burner/spirit lamp, Light Microscope and
Biosafety cabinet

7.2.7 Materials
ZN stains reagents: 1% Carbol Fuchsin stain, 20% Sulphuric acid and 0.125%
Methylene blue. Acid Fast Bacilli Positive and negative Control slides for zn.
Personal protective gears, Gauze, Glass slides, Staining racks, Drying rack, Clean
Glass slides, Applicator stick, Micropipette or Pasture
pipettes,sputum container,timer

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7.2.8 Storage and Stability
Sputum samples should be proccesed within 3days if stored at room temperature
and if not possible store it at 2 to 8°C for 10 days.

7.2.9 Safety
Decontaminate working surfaces as recommended by IPC Guidelines.
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infections.
Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
7.2.10 Calibration
Centrifuge and safety cabinety should be calibrated as per schedule

7.2.11 Quality Control

Perform IQuality control by Smearing and staining known Negative and positive
sample /EQA before examining any patient sample every day. Perform lot to lot for
every new batch of reagent received

7.2.12 Procedure Steps

i. Place the fixed slides with smear upwards on a staining rack over a sink about
1 cm apart.
ii. Flood the smear with filtered carbol fuchsin staining solution.
iii. Prepare a torch by dipping its cotton wool end in burning spirit and light it.
Use only a few drops on acid alcohol or 70% v/v ethanol or methanol.
iv. Heat the slide keeping the torch a little below the slide and moving it
continuously forth and back along the line until steam arises, repeat twice at
intervals of 3-5 minutes. Do not overheat. Allow slides to stand for at least
30 minutes.
v. Tilt the slide using forceps to drain off the staining solution.
vi. Rinse the slide well with clean water from a beaker (or running tap water).
vii. Pour the 20% Sulphuric acid or 3%Hcl over the smears, covering them
completely. Allow to act for 3 minutes.
viii. Tilt the slide with forceps to drain off the acid solution; then gently rinse the
slide again with clean water.
ix. Repeat covering by sulphuric acid or Hcl solution and rinsing once for smears
that are still red.
x. Flood smear with methylene blue solution for 1 minute.
xi. Tilt the slide with forceps draining off the Methylene blue solution. xii.
Wash with clean water.

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xiii. Using forceps take the slide from rack, drain off water and stand the slide on
the edge to air dry on the drying rack.
xiv. Examine the smear microscopically first with the 40x objective to see the
distribution of material, then with the oil immersion objective to look for Acid
Fast Bacilli . Open fully the condenser iris when using the oil immersion lens.
After examining a positive smear, the oil immersion objective must be wiped
clean.

Note: The stained smear should show a light blue colour from methylene blue. If the smear is
dark blue, it usually indicates that the smear is too thick.

7.2.13 Biological Reference interval

Not applicable

7.2.14 Interpretation and Reporting of Results

Acid Fast Bacilli appears as Red, straight or slightly curved rods, occurring singly or
in small.
If any definite red bacilli are seen, report the smear as Negative If no red bacilli are
seen in at least 100 fields and ‘Acid Fast Bacilli positive’ if give an indication of the
number of bacteria present as follows:
More than 10 AFB/HPF field Report + + + OR +3
1 – 10 AFB/HPF field Report + + OR +2
10 - 99 AFB/100 fields Report + OR +1
-9 AFB/100 fields Report the exact number/100
If no red bacilli are seen in at least 100 Report as negative
fields
7.2.15 Limitation of the Procedure and Sources of Error
i. Re use of containers or positive slides.
ii. Contaminated stain prepared with water containing
environmental mycobacteria.
iii. Use of scratched slides.
iv. Acid Fast Bacilli floated off one slide and became attached to another during
staining procedure because of no distance between each slide.
v. Poor quality of staining solutions.
vi. Taking improper portion of sample for smear preparation. vii. Improper focal
distance for examination.
viii. Use of poorly prepared staining solution. ix. Overheating during fixing.
x. Too long interval between staining and reading, especially when slides not kept
in dark or poorly stained.

7.2.16 Performance Characteristics Not applicable

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 7.2.17 Supporting Document
Patients Results Register - TB 05
Internal Quality Control review form

7.2.18 References
Clinical Microbiology Procedures Handbook. American Society for Microbiology.
Washington D.C., USA, 2nd edition, 2007.
Monica cheesbrough (2005). District Laboratory Practice in Tropical countries.
Cambridge University Press, New York, USA, 2nd edition, 2005.
WHO, (2003). Mannual of basic techniques for a health laboratory. Geneva. 2 nd
edition, 2003.
Manual of Clinical Microbiology. American Society for Microbiology (ASM),
Washington D.C., USA. 9th edition, 2007.
7.3 PROCEDURE FOR AURAMINE O PHENOL STAINING
7.3.1 Purpose
The Auramine O staining technique applies to identification of Acid Fast Bacilli in
patient sample using fluorescence Microscopy which increase the rate of detection
compared to the light microscopy.

7.3.2 Scope
This procedure is to be used for detection of Acid Fast Bacilli in sputum or other body
fluid samples in the Laboratory by using Auramine O reagents.

7.3.3 Responsible
Competent Health Laboratory Practitioners are responsible for implementing this test
procedure.
The Head Microbiology is responsible for ensuring the effective implementation and
maintenance of this procedure

7.3.4 Principle
The fluorochrome dye, Auramine-Rhodamine, forms a complex with mycolic acids
contained in the acid fast cell wall of organisms which resist decolorization by
acidalcohol. The counterstain, Methylene blue 0.3%, renders tissue and its debris
non fluorescent, thus reducing the possibility of artifacts. The cells visualized under
ultraviolet light appear bright yellow or reddish orange.

7.3.5 Sample Requirements

Mucoid, purulent or blood stained samples.

7.3.6 Equipment

173
Biosafety cabinet, Fluorescent microscopes, Timer, Bunsen burner/ Spirit lamp

7.3.7 Materials
Auramine –O phenol 0.1%, Hydrochloric acid in Alcohol 0.5%, Methylene blue 0.3%,
Microscopic slides, Slide holding box, Gloves, Gauze/Cotton wool, Beaker, Forceps
Applicator sticks/Disposable, Pencil, Dry rack, Slide racks, wide mouth sputum
container for routine samples or Falcon tube 50ml for referral samples.

7.3.8 Storage and Stability

Sputum samples should be proccesed within 3 days if stored at room temperature
and if not possible store it at 2 to 8°C for 10 days.

7.3.9 Safety
Decontaminate working surfaces as recommended by IPC Guidelines.
All personal protective equipment (PPE) must be worn when performing this
procedure.
All samples must be regarded as potentially infections.
Refer to National infection prevention and control Guidelines for health waste
management and safety practice.
7.3.10 Calibration
Centrifuge and safety cabinety should be calibrated as per schedule

7.3.11 Quality Control

Internal Quality Control should be done by smearing and staining known Negative
and positive sample before examining any patient sample every day. Perform lot to
lot for every new batch of reagent received/prepared.

7.3.12 Procedure
Film Preparation
i. Label the slides properly using a unique laboratory number
ii. Place the labeled slides, the samples and the applicator stick /Pasteur pipette
in the Biological safety cabinet
iii. Match each slide with the corresponding sputum or sample container. iv.
Proceed to smearing, taking the labeled slides and opening containers one by
one, do the smearing from the center of the slide, outwards making small coil
– like movements (A thin smear ,allow to air dry).
v. Select a small portion of purulent or muco-purulent material with the
applicator stick for a direct sputum smear and then transfer it to the slide, if a
stick is used, break it in two pieces and used ragged ends for dissecting
sputum and for smearing.

174
 vi. Spread the material carefully over the area equal to about 2×1 cm using
repeated coil like movements, without touching the margins of the slides and
should be in the middle.
vii. Make the smear as even as possible by continuing this process until no thick
parts remain. Remove excess material with the second stick and discard in
the biohazard bag.
viii. The thickness of the smear should be such that a newspaper can bared bye
read through the dried smear held about 10cm above it (Translucent).
ix. Warm the slides on the slide warmer in the biological safety cabinet to dry
and fix the smear for at least 30minutes.
x. Re-fix the smears by passing a flame under the slides before staining.
xi. If the slide warmer is not functioning, air dry the smear then fix them by
passing a flame under the smear ,the smear should face upward ,do not over
heat ,or else Acid Fast Bacilli staining will be poor.
Fluorescent Microscopy Staining Procedure
Place the slides on staining rack. IT IS A MUST to keep distance of at least 1cm
between every slide. Otherwise there is a possibility that acid fast bacilli may cross
contaminate the negative smears due to over flooding or splashes from positive
smear to a negative smear

i. Cover the smear completely with filtered 0.1% auramine solution Do not
heat
ii. Leave for 15 minutes iii. Wash the slides well with distilled water or
running water iv. Pour the acid alcohol solution over the slides.
v. Allow to act for 2- 3 minutes.
vi. Gently rinse each slide with distilled or running water. vii. Repeat
decolourization if macroscopically visible stains are still visible.
viii. Flood smear with 0.5% methylene blue counter solution for 1 minute .Time is
critical because counterstaining for longer time may quench the acid fast
bacilli
ix. Gently wash off counterstain with distilled or running water.
x. Stand the slides on edge to drain and air dry on the slide rack away from
direct sun light.
Examination
i. Keep stained smears in the dark (box or folder) till reading, and read as soon
as possible (within 24hours) since fluorescence fades quickly out of the box.
ii. Use 20 x objectives for scanning and 40x confirmation; scan the stained smear
systematically from one side to another side
iii. One length has to be scanned before reporting a Negative. iv. Acid-fast
bacilli appear bright yellow against the dark background material.
v. Store the slides in a slide box according to the study, following the laboratory
Number as they will be needed for EQA.

175
Note: Acid fast bacilli appear bright yellow against dark background, report as possible for
Acid Fast Bacilli if at least one acid-fast bacillus was seen in a well stained smear, even if you
think they might be other mycobacterium other than tubercle bacilli. Tubercle bacilli are quite
variable in shapes from very short fragments to elongated types. They may be uniformly
stained or with one or many gaps, or even granular. They occur singly or in small groups
(coded), and rarely in large clumps. The typical appearance of bacillli are usually rather long
and slender, straight or slightly curved rods.

7.3.13 Biological Reference interval Not applicable

7.3.14 Interpretation and Reporting of Results
If fluorescent Acid Fast Bacilli are seen, report the smear as Acid Fast Bacilli
positive, and give an indication of the number of bacilli present in plus signs (+ to
+++).
The results have to be reported on the working sheet and on patients result register
(MTB Register 05 if available).
If no fluorescent rods are seen, report the smear as (NO AFB SEEN).
Report Fluorescence (200magnigication, Fluorescence
one length 20x=field 200=HPF (400magnigication,
one length 40x =field 200=HPF
Negative Zero AFB/1 length Zero AFB/1 length
Scanty 1-29 AFB/1 Length 1-19 AFB/1 Length
1+ 30-299 AFB/1 Length 20-199 AFB/1 Length on average
2+ 10-100 AFB/ 1field on average 5-50 AFB/1 field on average
3+ >100 AFB/ Field on average >50 AFB/ Field on average

Critical values
Presence of Acid Fast Bacilli

7.3.15 Limitation of the Procedure and Sources of Error

i. Direct reagents to Sun light
ii. Poor reagent quality
iii. Using wrong reagent Concentration
iv. Using unfiltered reagent
v. Using expired reagent
vi. Poor sample quality
7.3.16 Perfomance characteristics Not applicable
7.3.17 Supporting documents
Patients Results Register - TB 05 Internal Quality Control

7.3.18 References

176
 International union against tubercle and lung diseases. The public health service
national tuberculosis Reference laboratory and the national laboratory Network
,Paris 1998
Smithwick R.W laboratory Manual for acid fast microscopy .US Department of
Health ,Education and Welfare,CDC,1979
Angra P, Becx-Bleumink M,Glipin C,et al,Ziehl Neelsen staining ,strong red on
week blue, or weak red under strong blue Int J Tuberic Lung Dis 2007:11:1160-
1

7.4 PROCEDURE FOR GRAMS STAINING

7.4.1 Purpose
This procedure provides instructions on the steps to be followed when performing
Gram staining

7.4.2 Scope
This procedure will be used by all laboratory personnel perform gram staining to
identify bacteria

7.4.3 Responsible
It is the responsibility of the Head of Microbiology Section to ensure effectively
implemented and maintained .

7.4.4 Principle
Gram stain based on the ability of bacteria cell wall. When the bacteria are stained
with primary stain (crystal violet) and fixed by the mordant (Iodine), Gram Positive
bacteria retain the primary stain when decolorized by ethanol (alcohol) because the
cell walls of gram positive bacteria have THICK layer of protein-sugar components
called peptidoglycan and low lipid contents. Upon decolourization Gram positive
bacteria causes thick cell wall to dehydrate and shrink, which closes the pores in the
cell wall and prevent the stain from existing the cell, therefore ethanol cannot remove
crystal Violet-Iodine complex that is bound to the thick layer which peptidoglycan and
appears BLUE. While Gram Negative bacteria are decolorized by ethanol. For the
gram negative bacteria cell wall takes up the crystal violet-iodine complex but due to
the thin layer of peptidoglycan and thick outer layer with form of lipids, crystal violate-
iodine complex gets washed off, when they are exposed to decolorizer dissolves the
lipids in the cell walls which allow the crystal violet-iodine complex to lead out of the
cells then when again stained with counterstain (safranin) they pick up the safranin
and appears red in color.

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7.4.5 Sample Requirement
Fresh collected Pus, urine sediment, CSF, sputum, other body fluids

7.4.6 Equipment
Microscope, Timer, Bunsen burner/hot plate

7.4.7 Materials
Reagents Consumables
• Crystal violet as primary stain
• Lugol’s iodine as mordant • Gloves
• 10% Acid/Acetone as • Gauze
decolourizer • Waste bins
• Grass slides
• Neutral red/safranin as counter
• Applicator stick
stain
7.4.8 Storage and Stability
i. Samples are stable at 2-80C for 7 daysdays
ii. store reagentas instructed by manufacturer
7.4.9 Safety
i. Adhere to safety precautions as stated in the Facility Safety manual/ IPC
guideline
ii. All personal protective equipment (PPE) must be worn when performing this
procedure.
iii. All samples must be regarded as potentially infections.
7.4.10 Calibration
Perform Equipment calibration as scheduled.

7.4.11 Quality Control

Quality control is done daily when receiving/preparing a new batch of reagents. A
known sample ATCC for gram positive Staphylococcus aureus and for gram negative
Escherichia coli bacteria respectively are used.
7.4.12 Procedural Steps
Smear preparation and staining
i. Slide with one end frosted should be used for making smears so that a lead
pencil can be used to label the slide clearly.
ii. Use a sterile applicator stick to add one drop of normal saline on the slide.
iii. Use a sterile applicator stick to transfer one pure colony on the slide and make
a smear iv. Allow smear to air dry on a flat safe place.
v. Flood the fixed smear with crystal violet for 30 seconds
vi. Decant crystal violet and rinse gently slide with running tap water
vii. Flood the slide with Lugol’s iodine for 30 seconds

178
 viii. Rinse off iodine gently with flowing tap water ix. Decolorize by letting the
reagent flow over the smear while the slide is held at an angle or tilt slide.
x. Adjust decolourisations time to thickness of smear
xi. Remove excess decolorizer with gentle flow of tap water
xii. Flood with neutral red for 30 seconds
xiii. Remove excess neutral red with gentle flow of tap water
xiv. Drain slide and air dry in an upright position
Reading smears
Place the slide on the microscope and ensure that the smear is facing upward

7.4.13 Biological Reference Intervals

Not Applicable

7.4.14 Interpretation And Reporting Of Results Interpretation of results

• If Bacteria pick the color of the counter stain (Neutral red/dilute
Carbolfuschin/safranin) THEN report as Gram Negative (Cocci, Bacilli/Rod
depending on morphological observed/identified cells)
• IF stained bacteria pick the color of the primary stain (crystal violet) THEN
report as Gram Positive (Bacilli/Rods or cocci depending on observed
identified cells) Reporting of results
• Results should be reported as Gram Positive Rods or Cocci according to the
morphology of the bacteria or
• Gram NEGATIVE Rods or Bacilli according to the shape of bacteria
7.4.15 Limitations Of The Procedure And Source Of Error
Avoid over staining, Avoid over decolourization Do not use expired reagent.
7.4.16 Performance Characteristics
Refer to method verification report of this procedure

7.4.17 Supporting Document Sample collection manual

7.4.18 References
Districtlaboratory practice in tropical countries. Part 2: AuthorMonica Cherbourg 6th
edition

179
CHAPTER EIGHT: MOLECULAR BIOLOGY
8.1 DIAGNOSIS OF MTB/RIF TESTING USING A TRUENAT MACHINE
8.1.1 Purpose
The purpose of SOP is to describe the stepwise procedure for the rapid detection of
Mycobacteria tuberculosis (MTB) and detection of rifampicin resistance using
Truenat MTB/RIF test for use with the Truenat Dx system is a semi-quantitative
realtime PCR in-vitro diagnostic test for: The detection of MTB DNA in sputum
samples, detection of rifampicin resistance-associated mutations of the rpoB gene.
The purpose of SOP is to describe the stepwise procedure for the rapid detection of
Mycobacteria tuberculosis (MTB) and detection of rifampicin resistance using
Truenat. MTB/RIF test for use with the TrueNat Dx system is a semi-quantitative real-
time PCR in-vitro diagnostic test for; detection of MTB DNA in sputum samples and
detection of rifampicin resistance-associated mutations of the rpoB gene.

8.1.2 Scope
This SOP describes the use of the Truenat™ MTB Plus assay, a chip-based
RealTime Polymerase Chain Reaction (PCR) test, for the semi-quantitative, detection
and diagnosis of Mycobacterium tuberculosis complex bacteria (MTBC) in human
sputum samples

8.1.3 Responsible
Qualified and competent health laboratory practitioners are responsible for
implementing this test procedure.
The section head is responsible for ensuring the effective implementation and
maintenance of this procedure.

8.1.4 Principle
The TrueNat assays utilize chip-based real-time micro-polymerase chain reaction
(PCR) for detection of TB and RIF-resistance from Deoxyribonucleic Acid (DNA) that
is extracted from sputum sample within an hour.
Truenat™ MTB Plus works on the principle of Real-Time Polymerase Chain Reaction
where the sputum sample is first liquefied and lysed thereafter the extracted DNA
from the sample is then amplified by the Truelab Real-Time microPCR analyser. The
purified DNA is then dispensed into the reaction well of the Truenat™ MTB Plus chip
and the test is started. Description of the apparatus

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Trueprep AUTO Sample Prep device Truelab™ Real Time micro PCR Analyser
Figure1. Truenat Dx System hardware components
8.1.5 Sample Requirements
Sputum sample type, collected as either Spot or morning sputum sample

8.1.6 Equipment
True prep AUTO Sample Prep device, Truelab Real Time micro PCR Analyser and
Refrigerator

8.1.7 Materials
Extraction of Amplification of Materials required but Reagents and
DNA purified not provided Solutions
DNA
• Trueprep • Truenat™ • Liquefaction
AUTO v2 • Truelab Duo Positive buffer
Universal Real Time Control • Lysis buffer
Cartridge Quantitative Kit - Panel I • Conc. bleach
Based micro PCR • Powder free and 70%
Sample Analyzer disposable alcohol
Prep • Truenat™ MTB gloves
Device Plus micro PCR • Two waste
• Trueprep chip disposal
AUTO • Truelab™ micro containers, with
MTB PCR printer lids, containing
Sample • Truepet SPA bleach
Pre- fixed volume (6 solutions
treatment µl) Precision • Timer
Pack micropipette • Two waste
• Trueprep bags
• • DNase and
AUTO v2 • Micro tube
RNase-free
Universal Stand
pipette tips with
Cartridge • . Cartridge
filter barrier
Based Holder
Sample • Two cryovials
Prep Kit racks

Note: Connect a new reagent pack to the Trueprep Auto v2 device by inserting the Plug-in
Connector into the slot provided.

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8.1.8 Storage and Stability store at room temperature for 3 days
Store at 2°C -8°C

8.1.9 Safety
• Treat all biological samples, including used cartridges, as if capable
of transmitting infectious agents. Because it is often impossible to
know which might be infectious, all biological samples should be
treated with universal precautions.
• Wear protective disposable gloves, laboratory coats and eye
protection when handling samples and reagents.
• Wash hands thoroughly after handling samples and test reagents.
Follow safety procedures for working with chemicals and handling
biological samples, (See safety manual)
• Dispose used cartridges according to infectious waste material
disposal guidelines.
8.1.10 Calibration
Calibration is perfumed as per schedule

8.1.11 Quality Control

• Use Truenat Positive Control Kit- Panel containing Positive Control
and Negative Control or use PBS as a negative control and a known
positive culture sample.
• Run positive and negative controls at least one time per month or ➢
When opening a new o test kit lot.
 If the temperature of the storage area falls outside of 2-30°C.
 New user prior to performing testing on the clinical sample.
 Accept patient results if the positive controls give positive results
while negative controls give negative results. Corrective action
should be taken in case of QC failure either by repeating the control
and/or informing the supervisor.
 Whenever a new shipment of test kits is received.
• Records QC results in the TB register.
8.1.12 Procedural Steps
A. Equipment start-up procedure Press the “Power” button to switch on the
Truenat device.
Press ‘start’ and ‘eject’ simultaneously to reset when prompted to change the
Reagent Pack and reset
B. Sample Processing procedure
Prepare Sample and Extract DNA
i. Wear gloves for sample handling.
ii. Label Sputum sample with patient details or lab ID

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 iii. Add 2 drops of liquefaction buffer to the sputum sample
iv. Close the cap and swirl gently to mix
v. Incubate for 10 minutes at room temperature. If sample is not pipetteable after
10 minutes, incubate for another 5 minutes with swirling at 2-minute intervals
vi. Transfer 0.5 ml of liquefied sputum sample into the lysis buffer bottle using a 1 ml
transfer pipette
vii. Add 2 drops of liquefaction buffer into the lysis buffer bottle, swirl gently to
mix and incubate for 3-5 minutes
viii. Remove the cartridge from the pouch, label it and place it on the cartridge
stand. Take out the elute collection tube (ECT) and label it appropriately.
Keep it aside for later use. Keep the elute transfer pipette in the pouch for
later use.
ix. Transfer the entire contents of the lysis buffer tube to the sample chamber
(black cap) of the cartridge using 3 ml transfer pipette
x. Switch “on” the Trueprep® AUTO v2 device. Press “eject” button to open and
gently pull out the cartridge holder
xi. Place the cartridge in the tray, and gently push to close the cartridge holder.
Press “start.”
The device will beep at the end of the DNA extraction process (20 minutes),
and the cartridge holder will eject automatically.
xiii. Gently pull out the cartridge holder, remove the cartridge, and place it on the
cartridge stand.
xiv. Carefully pierce the elute chamber with the provided elute transfer pipette, and
transfer the entire elute into the ECT. Discard the transfer pipette and cartridge
C. Running a PCR TB Test
i. Switch “on” the Truelab microPCR analyser by pressing the red button in the
back right corner for 2 seconds. LED will glow in Green. Wait for 30-50
seconds for “boot-up screen” to appear followed by “home screen.”
ii. Select USER ID, enter password. Press “Sign in” to Log in
iii. Select test profile “MTB” or “MTB Plus”. To confirm selection tap “PROCEED”
and enter patient details (referred by, patient ID, gender, patient name & age)
iv. Select sample type (sputum).
v. Open a TRUENAT™ MTB Plus chip pouch *Pull out the orange desiccant
pouch and confirm that it is orange in colour.
vi. Gently take out the chip without touching white well portion and place it on the
chip tray by aligning it in the slot provided
vii. Press “START TEST” on the screen. Chip tray opens. “Please Load Sample” will
appear. (Don’t press “YES” until chip loading is complete.
viii. Open the master mix tube, discard the stopper and place the tube in the micro
tube stand. *Check for white cake at the bottom of the micro tube.

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 ix. Attach the 6ul micro tip provided in the pouch to the single push pipette.
x. Transfer 6ul of the elute from ECT into the master mix tube
xi. Allow the master mix to stand for 30 SECONDS to get a clear solution. *Do
not mix by tapping, shaking or reverse pipette. *Do not discard the pipette tip.
xii. Transfer the elute from the master mix tube to the white reaction well of the
chip (Figure 16). *Avoid spillage of the clear solution outside the white reaction
well. *Discard the pipette tip and master mix tube.
xiii. Click “YES” on the device screen to start the test. The PCR will be completed
in 35 minutes.
xiv. Tap the “Open/Close Tray” button to eject the chip tray and discard the used
chip immediately after the reaction.
xv. If MTB is detected test the same elute for RIF resistance using the Truenat
MTB RIF Dx chip as a follow-on test. The test takes about 55 minutes.
D. Running a RIF-Resistance Test
i. If MTB is detected in a sample, Run a RIF resistance test.
iii. Use a portion of the same DNA eluate to test for RIF resistance using a
Truenat MTB-RIF Dx chip.
iv. Start by returning to Step 3 in the PCR TB test process and repeat for
RIFresistance by Selecting “MTB RIF” as the test type in the Truelab micro–
PCR Analyser.
v. RIF-resistance testing takes an additional 60 minute.
8.1.13 Biological Reference Interval Not applicable
8.1.14 Interpretation and Reporting of Results
Interpretation of results
At the end of the test run, the result screen will display;

i. “DETECTED” for Positive result.

ii. “NOT DETECTED” for Negative results. iii. MTB load as “HIGH”,
“MEDIUM”, “LOW” or “VERY LOW” for positive.
iv. The result screen also displays the validity of the test run as “VALID” or
“INVALID”.

NOTE: Invalid samples have to be repeated with fresh samples from the sample
preparation stage.
IPC will co-amplify in most positive cases also, in some samples having a high
target load, the IPC may not amplify, however, the test run is still considered valid
Sample with MTB DETECTED should be tested for MTB RIF
Reporting of results
Click “VIEW RESULTS” on the menu bar. The View Results window appears.

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 Optional: Press “Print” to print the result page using Truelab® microPCR printer.

Critical value
MTB Detected RIF Resistance Detected

8.1.15 Limitation of the Procedure and Sources of Error

i. Optimal performance of this test requires appropriate sample collection,
handling, storage and transport to the test site.
ii. Though very rare, mutations within the highly conserved regions of the target
genome where the Truenat™ assay primers and/or probe bind may result in
the under-quantitation of or a failure to detect the presence of the concerned
pathogen.
iii. The instruments and assay procedures are designed to minimize the risk of
contamination by PCR amplification products. However, it is essential to follow
good laboratory practices and ensure careful adherence to the procedures
specified in this package insert for avoiding nucleic acid contamination from
previous amplifications, positive controls or samples. iv. A sample for
which the Truenat™ assay reports “Not Detected” cannot be concluded to be
negative for the concerned pathogen. As with any diagnostic test, results from
the Truenat™ assay should be interpreted in the context of other clinical and
laboratory findings.
v. The performance of the test has not been evaluated with samples processed
by methods other than those described in the package insert.
vi. Do not open the cartridge lid except when adding sample.
vii. Do not use a cartridge if it appears wet or if the lid seal appears to have been
broken.
viii. Do not use a cartridge that has a damaged reaction tube. ix. Each single-use
cartridge is used to process one test. Do not reuse spent cartridges.
8.1.16 Performance Characteristics
Refer to the method verification report of this procedure.

8.1.17 Supporting Document Sample collection manual

8.1.18 References
• Truenat MTB Plus package insert version 5.
• The Trueprep™ AUTO v2 Universal Cartridge Based Sample Prep Device
user manual.
• TBRL Bamenda Biosafety manual, Version 4.0, section 10.
• Truenat™-A Point-of-care Real Time PCR Test for Tuberculosis, video by
Molbio available at https://youtu.be/ydR2I5S2v3

8.2 DIAGNOSIS OF MTB/RIF BY USING GENEXPERT SYSTEM

8.2.1 Purpose

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This procedure provides instructions for performing sample which is suspect with
MTB/Rif

8.2.2 Scope
This procedure is used for detection of the Mycobacterium tuberculosis complex
bacteria and their rifampicin susceptibility using the Gene Xpert MTB/Rif system in
microbiology section in the Laboratory.

8.2.3 Responsible
Qualified, trained, Competent and Registered health laboratory personnel is
responsible for ensuring the effective implementation for this procedure.

8.2.4 Principle
The Gene Xpert MTB/RIF system is a fully automated nested real-time PCR system,
which detects MTB complex DNA in smear positive and negative sputum samples
and other body fluid i.e. pleural fluid, ascetic fluid CSF and Pus. It simultaneously
identifies mutations in the rpoB gene, which are associated with rifampicin

resistance.

8.2.5 Sample Requirements

• Collect minimum 1ml and maximum 4ml of sputum or other body fluid
• Do not accept samples with obvious food particles or other solid particulatesor
blood stained (for this do Auramine O or ZN Stain)
8.2.6 6.0 Materials
• MTB/RIF cartridges, Sample Reagent, Disinfectant solution (0.5% Jik and 70%
alcohol), Sterile disposable transfer pipettes, Sterile screw-capped sample
collection containers, Disposable gloves, Plastic bag for waste disposal,
Labels and/or indelible labeling marker, Sterile pipettes for sample processing.

8.2.7 Equipment
• Gene Xpert machine, Microscope, Personal Protective Equipment such as N95
respirator and Timer

8.2.8 Storage and stability

• Sputum sample may be stored t 2-8 oC before examinations.
• Protect the Sputumu samples from dierect sunlight.
• Store the Gene xpert catradges at 2-8oC
8.2.9 Safety

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 i. Decontaminate working surfaces twice daily, in the morning and afternoon
ii. Adhere to safety precautions as stated in the Safety manual
iii. All personal protective equipment (PPE) must be worn when performing
this procedure.
iv. All samples must be regarded as potentially infections.
v. Avoid any contact between hands and eyes and nose during sample
collection and testing.
vi. Do not use kit beyond the expiration date which appears on the package
label or device in a damaged pouch.
vii. All spills should be wiped thoroughly using 1% sodium hypochlorite
solution.
8.2.10 Calibration
Calibarate auxillary equipment used in this operating this procedure once a year and
kep record available is performed once per year.

8.2.11 Quality Control

• The quality control will be performed on weekly bases (every week).
• Positive and negative known sample will be used.
• Ensure that all MTB/RIF cartridges and sample reagents used have passed
the required are used within their expiry date
8.2.12 Procedure Steps
Start-up the Gene Xpert instrument and Preparation of sample
i.Disinfect the working area by 0.5% jik.
ii.
Label each Xpert MTB/RIF cartridge with the sample ID (e.g. NEW
X_2016, HIV X_2016 or KID X_2016). Do not put the label on the lid
of the cartridge or obstruct the existing 2D barcode on the cartridge.
Write on the sides of the cartridge or affix ID label.
iii. Leave sample in leak-proof sputum collection container.
iv. Unscrew lid of sputum collection container, add Sample Reagent 2:1
(v/v) to sample and close the lid again.
v. Shake vigorously 10 - 20 times.
vi. Incubate for 5 minutes at room temperature.
vii. Shake the sample again vigorously 10 – 20 times.
viii. Continue incubation for another 10 minutes.
Note: Samples should be liquefied with no visible clumps of sputum. If there
are still clumps of sputum, shake again vigorously and incubate for another 35
min.

8.2.13 Preparing the Cartridge

• Start the test within 30 minutes of adding the sample to the cartridge

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 • Using the sterile transfer pipette provided, aspirate the liquefied
sample into the transfer pipette until the meniscus is above the
minimum mark (= 2ml). Open the cartridge lid. Transfer sample into
the open port of the Xpert MTB/RIF cartridge
NOTE: It is crucial that no bubbles are created when transferring the sample
into the cartridge as this can lead to an error (no result).
Dispense slowly to minimize the risk of aerosol formation.
Close the cartridge lid. Make sure the lid snaps firmly into place.
Note: Remaining liquefied sample may be kept for up to 12 hrs at 2-8oC
should repeat testing be required.
8.2.14 Start the test on the GeneXpert instrument
Note: Before start processing the sample, check that the Gene Xpert instrument is functioning
and the modules are available.

I. Turn on the computer, and then turn on the Gene Xpert instrument.
II. On the Windows desktop, double-click the Gene Xpert shortcut icon.
III. Log on to the Gene Xpert System software using your user name and
password.
IV. Click on “CHECK STATUS” and check if modules are available. If not
proceed to “Troubleshooting” in User’s manual.
V. In the GeneXpertDx System window, click “CREATE TEST”. The Scan
Cartridge Barcode dialog box appears.
VI. Scan the barcode on the Xpert MTB/RIF cartridge.
VII. The Create Test window appears.
VIII. Using the barcode information, the software automatically fills the boxes for
the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and
Expiration Date.
IX. In the Sample ID box (ID=Patient names), scan or type the sample ID (e.g.
NEW X_2016, HIV X_2016 or KID X_2016). Make sure you type the correct
sample ID. The sample ID is associated with the test results and is shown in
the “View Results” window and all the reports X. Choose module.
XI. Click “Start Test”.
XII. In the dialog box that appears, type your password.
XIII. Open the instrument module door with the blinking green light and load the
cartridge.
XIV. Close the door.
XV. The test starts and the green light stops blinking.
XVI. Wait until the system releases the door lock at the end of the run, then open
the module door and remove the cartridge.
XVII. Dispose of used cartridges in the appropriate sample waste containers
according to your institution’s standard practices
8.2.15 Biological Reference interval Not Applicable

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Interpretation and Reporting of Results
• MTB DETECTED, Rif Resistance DETECTED –RR
• MTB DETECTED, Rif Resistnce INDETEMINATE –TI
• MTB DETECTED Rif Not DETECTED – T
• MTB NOT DETECTED – N
• This is DNA based test, meant for New TB suspect, ensure don’t enrol
‘follow-up’ patient.
The results are interpreted by the GeneXpert Dx system from measured
fluorescent signals and embedded calculation algorithms and will be
displayed in the “View Results” window. Lower Ct values represent a
higher starting concentration of DNA template; higher Ct values
represent a lower concentration of DNA template.
Critical values MTB DETECTED, Rif Resistance DETECTED –RR
8.2.16 Limitation of the Procedure and Sources of Error
• Perform the test and validate results as per this SOP and details of test package
insert. Reliable results depend on proper sample collection, handling, and
storage. A positive test result does not necessarily indicate the presence of
viable organisms. It is however, presumptive for the presence of MTB and
rifampicin resistance. The results might be affected by antecedent or
concurrent anti-TB drug therapy.

8.2.17 Performance Characteristics

Refer to the verification report of Gene Xpert.

8.2.18 Supporting Document

Sample collection manual and safety manual

8.2.19 References
1. Clinical Microbiology Procedures Handbook. American Society for
Microbiology. Washington D.C., USA, 2nd edition, 2007.
2. Monica cheesbrough (2005). District Laboratory Practice in Tropical countries.
Cambridge University Press, New York, USA, 2nd edition, 2005.
3. WHO, (2003). Mannual of basic techniques for a health laboratory. Geneva.
2nd edition, 2003.
4. Manual of Clinical Microbiology. American Society for Microbiology (ASM),
Washington D.C., USA. 9th edition, 2007.
5. Manual for the Laboratory Identification and Antimicrobial Susceptibility
Testing of Bacterial Pathogens of Public Health Importance in the Developing
World. U.S. Centers for Disease Control and Prevention (CDC), Atlanta,
Georgia, U.S.A, and World Health Organization (WHO) Geneva Switzerland.
2003.

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 6. International Union against Tuberculosis and Lung Disease. The Public Health
Service National Tuberculosis Reference Laboratory and the National
Laboratory Network. Paris; 1998.
8.3 DETERMINATION OF HIV -1 VIRAL LOAD BY USING GENEXPERT
SYSTEM
8.3.1 Purpose
This SOP outlines the steps for qualitative in vitro diagnostic HVL test by using
automated GeneXpert system.

8.3.2 Scope
The Xpert HIV-1 VL assay is an in vitro reverse transcriptase polymerase chain
reaction (RT-PCR) assay for the detection and quantification of Human
Immunodeficiency Virus type 1 (HIV-1) RNA in human plasma from HIV-1 infected
individuals, using the automated GeneXpert Instrument Systems. The assay can
quantify HIV-1 RNA over the range of 40 to 10,000,000copies/mL. The Xpert HIV-1
VL assay is validated for quantification of RNA from HIV-1 Group M (subtypes A, B,
C, D, F, G, H, J, K, CRF01_AE, CRF02_AG, and CRF03_AB), Group N, and Group
O.
The Xpert HIV-1 VL assay is intended for use in conjunction with clinical presentation
and other laboratory markers for disease prognosis and for use as an aid in assessing
viral response to antiretroviral treatment as measured by changes in plasma HIV-1
RNA levels.
The Xpert HIV-1 VL assay is not intended to be used as a donor screening test for
HIV-1 or as a diagnostic test to confirm the presence of HIV-1 infection

8.3.3 Responsible
The Qualified, competent and registered health laboratory practitioners are
responsible to carry out this procedure.
The head of section is responsible for ensuring the effective implementation and
competency assessment for this procedure

8.3.4 Principles Principle of the Procedure

GeneXpert Instrument Systems automate and integrate sample preparation, nucleic
acid extraction and amplification, and detection of the target sequence in simple or
complex specimens using real-time reverse transcriptase PCR (RT-PCR). The
systems consist of an instrument, personal computer, and preloaded software for

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running tests and viewing the results. The systems require single-use disposable
GeneXpert cartridges that contain the RT-PCR reagents and carry out the sample
extraction and RT-PCR processes. Because the cartridges are self-contained,
crosscontamination between samples is minimized. For a full description of the
systems, refer to the appropriate GeneXpert Dx Operator Manual or GeneXpert
Infinity Operator Manual.
The HIV-1 VL assay includes reagents for the detection of HIV-1 RNA in specimens
and two internal controls used for quantitation of HIV-1 RNA. The internal controls
are also used to monitor the presence of inhibitor(s) in the RT and PCR reactions.
The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the
cartridge, probe integrity, and dye stability

Principle of operation
Each GeneXpert Dx module processes one sample. You insert the sample and
applicable reagents into a GeneXpert cartridge, create a test, load the cartridge into
an available instrument module, and then start the test. During the test, the system
performs the following steps:
i. Moves the sample and reagents into different chambers in the
cartridge for sample preparation.
ii. Hydrates the reagent beads. iii. Performs probe checks to
ensure that the sample preparation is successful (only if the assay
definition requires this step).
iv. Moves the sample and reagent mixture into the reaction tube.
v. Starts the PCR cycles and real-time detection.
8.3.5 Sample Requirement.
• DBS collected as per SOP for collection of DBS.
• Anticoagulated whole blood(WB) B in sterile tubes using EDTA (lavender
top) as the anticoagulant as per the manufacturer’s intructions for use.
• A minimum of 100 μL of WB is required for the HIV-1 Qualitative assay.
8.3.6 Equipment
Perform equipment Start up, Maintenance, trouble shoot and shut down refer
manufacturer instructions.
Biosafety cabinet, Data computer connected to LIS (Optional), Printer (optional),
GeneXpert instrument and GeneXpert Software should be available.

8.3.7 Materials
Reagent kit content Extra supplies

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The HIV-1 Qual assay kit contains sufficient reagents to • Serviette/Wipe
process 10 specimens or quality control samples. • Bleach
The kit contains the following: • 70 % alcohol or
HIV-1 Qual assay Cartridges with Integrated Reaction Tubes methylated
10 spirit
• Bead 1, Bead 2, and Bead 3 (freeze-dried) 1 of each • Labelling
per cartridge marker
• Lysis Reagent (Guanidinium Thiocyanate) 1.4 mL per • Optional:
cartridge sterile pipettes
• Rinse Reagent 0.5 mL per cartridge for sample
• Elution Reagent 2.5 mL per cartridge processing
• Lab coat,
• Binding Reagent 2.4 mL per cartridge
• Non -
• Proteinase K Reagent 0.48 mL per cartridge
powdered
gloves

HIV-1 Qual assay Sample Reagent Set (Sample Reagent) 10

• Lysis Reagent (Guanidinium Thiocyanate) 1.0 mL per
vial Disposable 1 mL Transfer Pipettes 1 bag of 10 per kit
Disposable 100 μL Transfer Micropipettes 1 bag of 10 per kit
CD 1 per kit
• Assay Definition Files (ADF)
• Instructions to import ADF into GeneXpert software
• Instructions for Use (Package Insert)
8.3.8 Storage and stability
Reagents
• Store the HIV-1 Qualitative assay cartridges and reagents at 2–28 °C.
• Do not use any reagents that have become cloudy or discoloured.
• Do not use a cartridge that has leaked.
• Use cartridge within 30 minutes after adding the sample
• Reagents are stable until their expiration dates when stored and handled
as per instruction for use.
Samples
• DBS cards may be stored at 18–30 °C for 30 days or – 15°C - 20 °C or
colder for up to 4 months, or -70 °C for longer storage.
• EDTA-anticoagulated WB may be stored at 31–35 °C for up to 8 hours,
15–30 °C for up to 24 hours or at 2–8 °C for up to 72 hours, prior to sample
preparing and testing
8.3.9 Safety
i. Treat all biological specimens, including used cartridges, as if capable of
transmitting infectious agents.

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 ii. Wear protective disposable gloves, laboratory coats, and eye protection
when handling specimens and reagents. Wash hands thoroughly after
handling specimens and test reagents.
iii. Follow safety procedures for working with chemicals and handling
biological samples.
iv. When processing more than one sample at a time, open only one
cartridge; add sample and close the cartridge before processing the next
sample. Change gloves between samples.
v. Do not substitute HIV-1 Qual assay reagents with other reagents.
vi. Do not open the HIV-1 Qual assay cartridge lid except when adding the
Sample Reagent and WB or the Sample Reagent treated DBS sample.
vii. Do not use a cartridge if it appears wet or if the lid seal appears to have
been broken.
viii. Do not shake the cartridge. Shaking or dropping the cartridge after
opening the cartridge lid may yield invalid results.
ix. Do not use a cartridge that has a damaged reaction tube.
x. Each single-use HIV-1 Qual assay cartridge is used to process one
specimen. Do not reuse spent cartridges. xi. The single-use
disposable pipette is used to transfer one specimen. Do not reuse spent
disposable pipettes.
xii. In the event of contamination of the work area or equipment with specimen
or control materials, disinfect the area with a 1:10 bleach solution and then
70% ethanol. Wipe work surfaces dry completely before proceeding.
8.3.10 Calibration
There is no need to calibrate the GeneXpert Dx instrument. Cepheid performs all of
the necessary calibrations before you receive the system. However, Cepheid
recommends that the instrument be recalibrated after 1 year of use, based on the
initial installation date (or based on the previous calibration for subsequent years) or
at 2000 tests per instrument module, whichever comes first.

8.3.11 Quality control

The GeneXpert Dx System automatically performs internal quality control for each
sample. During each test, the system uses one or more of the following controls:
Internal control (IC)—Verifies the performance of the PCR reagents and prevents a
false negative result. The internal control PCR assay assesses if there is any
inhibition, possibly by components, in the test sample. The internal control is provided
in the cartridge and should be positive in a negative sample.
Endogenous control (EC)—Normalizes targets and ensures sufficient sample is
used in the test. Because of its low variability, the endogenous control can also be

193
used to indicate sample-inhibitor contamination. The endogenous control is taken
from the specimen sample.
Each test includes a Sample Volume Adequacy (SVA), a Sample Processing Control
(SPC) and Probe Check Control (PCC).
Sample Volume Adequacy (SVA): Ensures that the sample was correctly added to
the cartridge. The SVA verifies that the correct volume of sample has been added in
the sample chamber. The SVA passes if it meets the validated acceptance criteria. If
the SVA does not pass, an ERROR 2096 will display if there is no sample or an
ERROR 2097 if there is not enough sample. The system will prevent the user from
resuming the test.
Sample Processing Control (SPC): Ensures that the sample was correctly
processed. The SPC is an Armoured RNA in the form of a dry bead that is included
in each cartridge to verify adequate processing of the sample virus. The SPC verifies
that lysis of HIV-1 has occurred if the organism is present and verifies that the
specimen processing is adequate. Additionally, this control detects
specimenassociated inhibition of the RT-PCR reaction. The SPC should be positive
in a negative sample and can be negative or positive in a positive sample. The SPC
passes if it meets the validated acceptance criteria.
In addition to the controls, the GeneXpert Dx instrument performs a probe check
during the first stage of the test.
Probe Check Control (PCC): Before the start of the PCR reaction, the GeneXpert
Instrument System measures the fluorescence signal from the probes to monitor
bead rehydration, reaction tube filling, probe integrity, and dye stability. The PCC
passes if it meets the validated acceptance criteria.
External Controls: Internal quality controls should be done weekly by using known
HIV-1 DETECTED and HIV -1 NOT DETECTED as the same as the routine EID/DBS
samples.
8.3.12 Procedural steps
Follow the actions described step by step to do each specific task

Start-up the GeneXpert instrument

- Turn on the GeneXpert Dx instrument, and then turn on the computer.
- On the Windows desktop, double-click the GeneXpert Dx shortcut icon.
- Log on to the GeneXpert Dx System software using your user name and
password.
- Click on “CHECK STATUS” and check if modules are available. If not proceed
to “Troubleshooting” in User manual.
! Note: Before start processing the specimen, check that the GeneXpert instrument
is functioning and the modules are available.

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Preparing of cartridge(s)
EDTA anticoagulated Whole Blood
i. Wear protective disposable gloves. ii. Disinfect the work area by 0.5
% bleach solution followed by 70 % alcohol
iii. Wear protective disposable powder free gloves.
iv. Label the Sample Reagent vial with the specimen identification.
v. Inspect the test cartridge for damage. If damaged, do not use. vi.
Open the cartridge lid.
vii. Use the 1 mL transfer pipette provided to transfer 750 μL of the sample
reagent into the sample chamber of the cartridge.
viii. Allow the Sample Reagent to adjust to room temperature and mix the
bottle by inverting before transferring to the cartridge. Transfer exactly
750 μL into the sample chamber of the cartridge.
ix. Mix the Whole Blood sample by inverting the vial (EDTA or lavendertop
tube) at least seven times. Immediately transfer 100 μL using the
micropipette provided by squeezing the upper bulb and then releasing
to aspirate the blood.
x. Squeeze again to dispense the blood into the sample chamber of the
cartridge where it will mix with the Sample Reagent already in the
sample chamber. Alternatively, use an automatic pipette to dispense
the blood into the sample chamber of the cartridge. Do NOT pour the
specimen into the chamber!

Figure 1. HIV-1 Qual Assay 1 mL Transfer Pipette

195
 Figure 2. HIV-1 Qual Assay 100 μL Transfer Micropipette

Figure 3. HIV-1 Qual Assay Cartridge (Top View)

❑ DBS sample
i. Wear protective disposable gloves.
ii. Disinfect the working area. iii. Wear protective disposable
powder free gloves. iv. Before starting, remove the vial containing
the Sample Reagent from the kit and, if it was refrigerated, allow to adjust
to room temperature. If the vial has not been stored in an upright position,
make sure the buffer is settled in the bottom by giving the vial a firm
shake.
v. Turn on Thermo Mixer to heat to 56 °C.
vi. Label the Sample Reagent vial with the specimen identification.
vii. Using sterilized scissors, excise one entire DBS from the filter
paper card for each specimen. Follow the delineated lines when
excising the DBS. If perforated circles are used, use clean and
sterile pipette tips to detach the DBS.
viii. Unscrew the lid on the vial containing the Sample Reagent and
place one DBS in the vial. Ensure that the DBS is fully submerged
in the Sample Reagent buffer.

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 ix.
Place the vial with the DBS in a Thermo Mixer and incubate for 15
minutes at 56 °C while rotating at 500 rpm.
x. Inspect the test cartridge for damage. If damaged, do not use. xi.
Open the cartridge lid
xii. Use the 1 mL transfer pipette provided to transfer all the liquid from
the lysed DBS specimen into the sample chamber of the cartridge.
Ensure the pipette is filled above the third mark on the transfer
pipette. Avoid suction of the DBS with the pipette. Do NOT pour the
specimen into the chamber!
xiii. Close the cartridge lid, ready to start the test.
Notes! Change gloves between specimen, and each new procedure.
Starting the Test
i. In the GeneXpert System window, click Create Test. The scan
Cartridge Barcode dialog box appears.
ii. Scan the barcode on the HIV-1 Qual assay cartridge. iii. Using the
barcode information, the software automatically fills the boxes for the
following fields: Select Assay, Reagent Lot ID, Cartridge SN, and
Expiration Date.
iv. Type the Patient ID, make sure the Patient ID is typed correctly.
v. Type in the Sample ID. Make sure the Sample ID is typed correctly.
vi. On the Notes field, enter the words KATAVI RRHL to indicate a
testing laboratory name on the patient report.
vii. Open the instrument module door with the blinking green light and load
the cartridge.
viii. Click Start Test (Gene Xpert Dx). Enter you’re your user name and
password, if requested.
ix. Close the door.
x. The test starts and the green light stops blinking. When the test is
finished, the light turns off. xi. Wait until the system releases the
door lock before opening the module door and removing the cartridge.
Result viewing, and printing
i. In the Gene Xpert Dx System window, Click the View Results icon to view
results. This view result window appears.
ii. If the software reports “Error”, Invalid, or No result, repeat the test using
new DBS circle.
iii. Should the test again show Error, Invalid, or No result, proceed to
troubleshooting manual to exclude technical problems before requesting a
new specimen.
iv. Upon completion of the test, click the Report button of the View Results
window to view and/or generate a PDF report file.

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 v. Report should be done in the Laboratory HEID register and a tick on the
respective result blank space on the HEID request form. Report as; HIV -
1 DETECTED*.
HIV -1 NOT DETECTED
vi. Report “please submit a new sample” if the system repeatedly did not
produce a result and you have excluded and/or fixed a technical problem.
vii. HIV -1 DETECTED is a critical result that needs immediate action
including but not limited to; retesting using new DBS circle with a new
cartridge, and result notification to the respective referring health facility.
8.3.13 Biological Reference Intervals Not Applicable
8.3.14 15. Result interpretation and Reporting of Results.
• The results are interpreted automatically by the GeneXpert Instrument System
from measured fluorescent signals and embedded calculation algorithms and
are clearly shown in the View Results window. Possible results are shown in
Table below:
Result Interpretation
HIV-1 The HIV-1 target nucleic acids are detected.
DETECTED • The HIV-1 target nucleic acids have a Ct within the
See Figure 1. valid range.
• SPC: NA (not applicable); SPC is ignored because
the HIV-1 target amplification occurred.
• Probe Check: PASS; all probe check results pass.
HIV-1 NOT The HIV-1 target nucleic acids are not detected. SPC meets
DETECTED acceptance criteria.
See Figure 2. • SPC: PASS; SPC has a Ct within the valid range.
• Probe Check: PASS; all probe check results pass.
INVALID Presence or absence of the HIV-1 target nucleic acids
cannot be determined. Repeat test with new sample and
cartridge.
• SPC: FAIL; SPC Ct is not within valid range.
• Probe Check: PASS; all probe check results pass
ERROR Presence or absence of HIV-1 target nucleic acids cannot be
determined. Repeat test with new sample and cartridge.
• HIV-1: NO RESULT
• SPC: NO RESULT
• Probe Check: FAIL*; all or one of the probe check
results fail.
*If the probe check passed, the error is caused by the
maximum pressure limit exceeding the acceptable range or
by a system component failure.

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 NO RESULT Presence or absence of HIV-1 target nucleic acids cannot be
determined. Repeat test with new sample and cartridge.
A NO RESULT indicates that insufficient data were collected.
For example, the operator stopped a test that was in
progress.
• HIV-1: NO RESULT
• SPC: NO RESULT
• Probe Check: NA (not applicable).

Figure 4 HIV -1 DETECTED

Figure 5. HIV -1 NOT DETECTED

8.3.15 Limitation of the Procedure and Sources of Error
Good laboratory practices and changing gloves between handling specimens are
recommended to avoid contamination of reagents.

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 Rare mutations within the target region of the HIV-1 Qual assay may affect primer
and/or probe binding resulting in failure to detect the virus.
A negative test result does not preclude HIV-1 infection. Results from the HIV-1 Qual
assay should be interpreted in conjunction with clinical presentation and other
laboratory markers.
8.3.16 Performance Characteristics
Refer to the manufacture package insert and verification report for detailed
information on Performance Characteristics of the testing procedure

8.3.17 Supporting Documents

• Sample collection manual and safety manual
• Quality manual
8.3.18 References
• Ministry of Health, Community Development, Gender, Elderly and Children,
Standard Operating Procedures for qualitative HIV-1 HEID testing using
GeneXpert
• Xpert HIV-1 Qual -1 Assay Package Insert 308-3048 Rev J
• GeneXpert Dx System. Operator Manual
8.4 DETERMINATION OF HIV EARLY INFANT DIAGNOSIS BY USING
GENEXPERT SYSTEM
8.4.1 Purpose
This SOP outlines the steps for qualitative in vitro diagnostic HIV-1 test by using
automated GeneXpert system.

8.4.2 Scope
The HIV-1 Qual assay, is a qualitative in vitro diagnostic test designed to detect
Human Immunodeficiency Virus Type 1 (HIV-1) total nucleic acids on the automated
Gene Xpert Systems using human whole blood (WB) and dried blood spot (DBS)
specimens from individuals suspected of HIV-1 infection. The HIV-1 Qualitative assay
is intended to aid in the diagnosis of HIV-1 infection in conjunction with clinical
presentation and other laboratory markers. The assay is intended to be used by
laboratory professionals or specifically-trained healthcare workers. The assay is not
intended to be used as a blood donor screening test for HIV-1.

8.4.3 Responsible

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Section head is responsible effective implementation of this procedure. Only
competent laboratory staffs should carry out this procedure. It is the responsibility of
each staff to read, understand and implement this procedure.

8.4.4 Principle
Principle of the Procedure
The GeneXpert (GX) Instrument Systems automate and integrate sample
preparation, nucleic acid extraction and amplification, and detection of the target
sequence in simple or complex samples using real time reverse transcription PCR
(RT-PCR). The systems consist of an instrument, personal computer, and preloaded
software for performing tests and viewing the results. The systems require the use of
single-use disposable GeneXpert cartridges that hold the RT-PCR reagents and host
the RT-PCR processes. Because the cartridges are self-contained,
crosscontamination between samples is minimized. The HIV-1 Qual assay includes
reagents for the detection of HIV-1 total nucleic acids in specimens as well as an
internal control to ensure adequate processing of the target and to monitor the
presence of inhibitor(s) in the RT and PCR reactions. The Probe Check Control
(PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity,
and dye stability.

8.4.5 Principle of operation

Each GeneXpert Dx module processes one sample. You insert the sample and
applicable reagents into a GeneXpert cartridge, create a test, load the cartridge into
an available instrument module, and then start the test. During the test, the system
performs the following steps:
i. Moves the sample and reagents into different chambers in the cartridge
for sample preparation.
ii. Hydrates the reagent beads. iii. Performs probe checks to ensure that
the sample preparation is successful (only if the assay definition
requires this step).
iv. Moves the sample and reagent mixture into the reaction tube.
v. Starts the PCR cycles and real-time detection.
8.4.6 Sample requirements
DBS collected as per SOP for collection of DBS.

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EDTA Anticoagulated WB in sterile tubes using EDTA (lavender top) as the
anticoagulant as per the manufacturer’s instructions for use. A minimum of 100 μL of
WB is required for the HIV-1 Qualitative assay.

8.4.7 Equipment
Start up, Maintainance, trouble shoot and shud down refer manaufacturer instructions
Thermo Mixer C for incubation with smart block, Biosafety cabinet, Data computer
connected to LIS (Optional) and Printer (optional)

8.4.8 Materials
Materials (Reagents and consumables) used to perform the test.
Reagent kit content Extra consumables
The HIV-1 Qual assay kit contains sufficient reagents to • DBS Collection
process 10 specimens or quality control samples. Kit (Filter paper
The kit contains the following: cards, e.g.,
HIV-1 Qual assay Cartridges with Integrated Reaction Whatman 903,
Tubes 10 Munktell or equivalent,
• Bead 1, Bead 2, and Bead 3 (freeze-dried) 1 of each lancets, desiccants,
per cartridge plastic
• Lysis Reagent (Guanidinium Thiocyanate) 1.4 mL per • sealable bags,
cartridge and swabs)
• Rinse Reagent 0.5 mL per cartridge • Scissors,
• Elution Reagent 2.5 mL per cartridge sterile
(recommended for
• Binding Reagent 2.4 mL per cartridge
excising DBS from filter
• Proteinase K Reagent 0.48 mL per cartridge
paper if not using a
HIV-1 Qual assay Sample Reagent Set (Sample Reagent)
perforated DBS card)
10
• Sterile pipette
• Lysis Reagent (Guanidinium Thiocyanate) 1.0 mL per
tips
vial Disposable 1 mL Transfer Pipettes 1 bag of 10 per kit
• Serviette/Wipe
Disposable 100 μL Transfer Micropipettes 1 bag of 10 per • Bleach
kit CD 1 per kit
• 70 % alcohol or
• Assay Definition Files (ADF) methylated
• Instructions to import ADF into GeneXpert software spirit
• Instructions for Use (Package Insert) • Distilled water
• Labelling
marker
• Optional:
sterile pipettes
for sample
processing

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 • Lab coat,
• Non - powdered
gloves

8.4.9 Storage and Stability.

DBS cards may be stored at 18–30 °C for 30 days or – 15°C - 20 °C or colder for up
to 4 months, or -70 °C for longer storage.
EDTA-anticoagulated WB may be stored at 31–35 °C for up to 8 hours, 15–30 °C for
up to 24 hours or at 2–8 °C for up to 72 hours, prior to preparing and testing the
specimen.

8.4.10 Safety
i. Treat all biological specimens, including used cartridges, as if capable of
transmitting infectious agents.
ii. Wear protective disposable gloves, laboratory coats, and eye protection
when handling specimens and reagents. Wash hands thoroughly after
handling specimens and test reagents.
iii. Follow safety procedures for working with chemicals and handling biological
samples.
iv. When processing more than one sample at a time, open only one cartridge;
add sample and close the cartridge before processing the next sample.
Change gloves between samples.
v. Do not substitute HIV-1 Qual assay reagents with other reagents.
vi. Do not open the HIV-1 Qual assay cartridge lid except when adding the
Sample Reagent and WB or the Sample Reagent treated DBS sample.
vii. Do not use a cartridge if it appears wet or if the lid seal appears to have been
broken.
viii. Do not shake the cartridge. Shaking or dropping the cartridge after opening
the cartridge lid may yield invalid results.
ix. Do not use a cartridge that has a damaged reaction tube.
x. Each single-use HIV-1 Qual assay cartridge is used to process one
specimen. Do not reuse spent cartridges. xi. The single-use disposable
pipette is used to transfer one specimen. Do not reuse spent disposable
pipettes.
xii. In the event of contamination of the work area or equipment with specimen or
control materials, disinfect the area with a 1:10 bleach solution and then 70%
ethanol. Wipe work surfaces dry completely before proceeding.
8.4.11 Calibration
You do not need to calibrate the GeneXpert Dx instrument. Cepheid performs all of
the necessary calibrations before you receive the system. However, Cepheid

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recommends that the instrument be recalibrated after 1 year of use, based on the
initial installation date (or based on the previous calibration for subsequent years) or
at 2000 tests per instrument module, whichever comes first.

8.4.12 Quality control

Quality control is an important part of in vitro diagnostic testing because it ensures
you are performing the tests correctly and that your GeneXpert Dx System is working
properly. The GeneXpert Dx System automatically performs internal quality control
for each sample. During each test, the system uses one or more of the following
controls:
o Internal control (IC)—Verifies the performance of the PCR reagents and
prevents a false negative result. The internal control PCR assay assesses if
there is any inhibition, possibly by components, in the test sample. The internal
control is provided in the cartridge and should be positive in a negative sample.
o Endogenous control (EC)—Normalizes targets and ensures sufficient
sample is used in the test. Because of its low variability, the endogenous
control can also be used to indicate sample-inhibitor contamination. The
endogenous control is taken from the specimen sample.
Each test includes a Sample Volume Adequacy (SVA), a Sample Processing Control
(SPC) and Probe Check Control (PCC).
Sample Volume Adequacy (SVA): Ensures that the sample was correctly added to
the cartridge. The SVA verifies that the correct volume of sample has been added in
the sample chamber. The SVA passes if it meets the validated acceptance criteria. If
the SVA does not pass, an ERROR 2096 will display if there is no sample or an
ERROR 2097 if there is not enough sample. The system will prevent the user from
resuming the test.
Sample Processing Control (SPC): Ensures that the sample was correctly
processed. The SPC is an Armoured RNA in the form of a dry bead that is included
in each cartridge to verify adequate processing of the sample virus. The SPC verifies
that lysis of HIV-1 has occurred if the organism is present and verifies that the
specimen processing is adequate. Additionally, this control detects
specimenassociated inhibition of the RT-PCR reaction. The SPC should be positive
in a negative sample and can be negative or positive in a positive sample. The SPC
passes if it meets the validated acceptance criteria.
In addition to the controls, the GeneXpert Dx instrument performs a probe check
during the first stage of the test.
Probe Check Control (PCC): Before the start of the PCR reaction, the GeneXpert
Instrument System measures the fluorescence signal from the probes to monitor

204
bead rehydration, reaction tube filling, probe integrity, and dye stability. The PCC
passes if it meets the validated acceptance criteria.
External Controls: Internal quality controls should be done weekly by using known
HIV-1 DETECTED and HIV -1 NOT DETECTED as the same as the routine EID/DBS
samples.
8.4.13 Procedural steps
Follow the actions described step by step to do each specific task

Start-up the GeneXpert instrument

- Turn on the GeneXpert Dx instrument, and then turn on the computer.
- On the Windows desktop, double-click the GeneXpert Dx shortcut icon.
- Log on to the GeneXpert Dx System software using your user name and
password.
- Click on “CHECK STATUS” and check if modules are available. If not proceed
to “Troubleshooting” in User manual.

Note: Before start processing the specimen, check that the GeneXpert instrument is
functioning and the modules are available.

Preparing of cartridge(s) for EDTA anticoagulated Whole Blood

i. Wear protective disposable gloves.
ii. Disinfect the work area by 0.5 % bleach solution followed by 70 % alcohol
iii. Wear protective disposable powder free gloves. iv. Label the Sample
Reagent vial with the specimen identification.
v. Inspect the test cartridge for damage. If damaged, do not use. vi. Open the
cartridge lid.
vii. Use the 1 mL transfer pipette provided to transfer 750 μL of the sample
reagent into the sample chamber of the cartridge.
viii. Allow the Sample Reagent to adjust to room temperature and mix the bottle
by inverting before transferring to the cartridge. Transfer exactly 750 μL into
the sample chamber of the cartridge. ix. Mix the Whole Blood sample by
inverting the vial (EDTA or lavender-top tube) at least seven times.
Immediately transfer 100 μL using the micropipette provided by squeezing
the upper bulb and then releasing to aspirate the blood.
x. Squeeze again to dispense the blood into the sample chamber of the cartridge
where it will mix with the Sample Reagent already in the sample chamber.
Alternatively, use an automatic pipette to dispense the blood into the sample
chamber of the cartridge. Do NOT pour the specimen into the chamber!

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 Figure 6. HIV-1 Qual Assay 1 mL Transfer Pipette

Figure 7. HIV-1 Qual Assay 100 μL Transfer Micropipette

Figure 8. HIV-1 Qual Assay Cartridge (Top View)

DBS sample
i. Wear protective disposable gloves.
ii. Disinfect the working area. iii. Wear protective disposable powder free
gloves. iv. Before starting, remove the vial containing the Sample Reagent from
the kit and, if it was refrigerated, allow to adjust to room temperature. If the vial
has not been stored in an upright position, make sure the buffer is settled in the
bottom by giving the vial a firm shake.

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 v. Turn on Thermo Mixer to heat to 56 °C.
vi. Label the Sample Reagent vial with the specimen identification. vii. Using
sterilized scissors, excise one entire DBS from the filter paper card for each
specimen. Follow the delineated lines when excising the DBS. If perforated circles
are used, use clean and sterile pipette tips to detach the DBS.
viii. Unscrew the lid on the vial containing the Sample Reagent and place one DBS
in the vial. Ensure that the DBS is fully submerged in the Sample Reagent buffer.
ix. Place the vial with the DBS in a Thermo Mixer and incubate for 15 minutes at
56 °C while rotating at 500 rpm.
x. Inspect the test cartridge for damage. If damaged, do not use.
xi. Open the cartridge lid
xii. Use the 1 mL transfer pipette provided to transfer all the liquid from the lysed
DBS specimen into the sample chamber of the cartridge. Ensure the pipette
is filled above the third mark on the transfer pipette. Avoid suction of the DBS
with the pipette. Do NOT pour the specimen into the chamber!
xiii. Close the cartridge lid, ready to start the test.

Notes Change gloves between specimen, and each new procedure.

Starting the Test

i. In the GeneXpert System window, click Create Test. The scan Cartridge
Barcode dialog box appears.
ii. Scan the barcode on the HIV-1 Qual assay cartridge.
iii. Using the barcode information, the software automatically fills the boxes for
the following fields: Select Assay, Reagent Lot ID, Cartridge SN, and
Expiration Date.
iv. Type the Patient ID, make sure the Patient ID is typed correctly.
v. Type in the Sample ID. Make sure the Sample ID is typed correctly.
vi. Open the instrument module door with the blinking green light and load the
cartridge.
vii. Click Start Test (Gene Xpert Dx). Enter you’re your user name and
password, if requested.
viii. Close the door. ix. The test starts and the green light stops blinking.
When the test is finished, the light turns off.
x. Wait until the system releases the door lock before opening the module door
and removing the cartridge.

8.4.14 Biological Reference Intervals.

Not Applicable.

8.4.15 Interpretation and reporting of Results

207
The results are interpreted automatically by the GeneXpert Instrument System from
measured fluorescent signals and embedded calculation algorithms and are clearly
shown in the View Results window. Possible results are shown in Table below:
Result Interpretation
HIV-1 DETECTED The HIV-1 target nucleic acids are detected.
See Figure 1. • The HIV-1 target nucleic acids have a Ct within the valid
range.
• SPC: NA (not applicable); SPC is ignored because the
HIV-1 target amplification occurred.
• Probe Check: PASS; all probe check results pass.
HIV-1 NOT The HIV-1 target nucleic acids are not detected. SPC meets
DETECTED acceptance criteria.
See Figure 2. • SPC: PASS; SPC has a Ct within the valid range.
• Probe Check: PASS; all probe check results pass.
INVALID Presence or absence of the HIV-1 target nucleic acids cannot be
determined. Repeat test with new sample and cartridge.
• SPC: FAIL; SPC Ct is not within valid range.
• Probe Check: PASS; all probe check results pass
ERROR Presence or absence of HIV-1 target nucleic acids cannot be
determined. Repeat test with new sample and cartridge.
• HIV-1: NO RESULT
• SPC: NO RESULT
• Probe Check: FAIL*; all or one of the probe check results
fail. *If the probe check passed, the error is caused by the
maximum pressure limit exceeding the acceptable range or by a
system component failure.
NO RESULT Presence or absence of HIV-1 target nucleic acids cannot be
determined. Repeat test with new sample and cartridge.
A NO RESULT indicates that insufficient data were collected. For
example, the operator stopped a test that was in progress.
• HIV-1: NO RESULT
• SPC: NO RESULT
• Probe Check: NA (not applicable).

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8.4.16 Limitation of the Procedure and Sources of Error.
Good laboratory practices and changing gloves between handling specimens are
recommended to avoid contamination of reagents.
Rare mutations within the target region of the HIV-1 Qual assay may affect primer
and/or probe binding resulting in failure to detect the virus.
A negative test result does not preclude HIV-1 infection. Results from the HIV-1 Qual
assay should be interpreted in conjunction with clinical presentation and other
laboratory markers

8.4.17 Performance Characteristics

Refer to the manufacture package insert for detailed information on Performance
Characteristics of the testing procedure.

8.4.18 Supporting Documents

Sample collection manual, Quality manual

209
8.4.19 References
Ministry of Health, Community Development, Gender, Elderly and Children, Standard
Operating Procedures for qualitative HIV-1 HEID testing using GeneXpert
Xpert HIV-1 Qual -1 Assay Package Insert 308-3048 Rev J
GeneXpert Dx System. Operator Manual

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CHAPTER NINE: ANATOMICAL PATHOLOGY
9.1 PROCEDURE FOR MORTUARY SERVICES
9.1.1 Purpose
This procedure provides instructions for providing mortuary services including
autopsy practice, embalming as well as safety of personnel, visitors and community.

9.1.2 Scope
This document provides guidelines for the mortuary staff and administration
recommended standards for mortuary facilities in settings and for communication
between staff involved in autopsy procedures or autopsy related processes.
Body storage
A body cold store having a capacity appropriate for the mortuary workload should be
maintained at a temperature of about 2- 6 o C.
If long term storage is required, the body should have maintained at approximately
20 o C.
Labelling procedures should be established so that body identification is made easy.

9.1.3 Responsible
Qualified and trained mortuary personnel are responsible for implementing this
procedure.
The Head of Laboratory is responsible for ensuring the effective implementation and
maintenance of this procedure.

9.1.4 Principle Not applicable

9.1.5 Sample requirements Not applicable
9.1.6 Equipment
Refrigerator, Trolley, Post-mortem kit

9.1.7 Materials
10% formalin, Gloves, Leak proof bag, gloves, water resistant gown/plastic apron
over water repellent gown, and surgical masks, goggles or face shield, shoe covers

9.1.8 Storage and stability

Dead body: For short term store at 2-6 ͦ C, For long term store -20 ͦ C
10% formalin: Store at room temperature

9.1.9 Safety

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 - PPE shall be used to prevent skin and mucous membrane contact with blood
and other body fluid. These may include the use of gloves, N95 masks,
protective eye wear, face shields, shoe covers, plastic aprons/gowns hair
bornets, cut resistant gloves and laboratory coats.
Surgical or post mortem gloves must be worn by all personnel involved in the
autopsy procedure Hand washing:
- Hand and other skin surface should be washed with soaps and water
immediately after contact with blood or other body fluid. Hand shall be washed
each time with running water and soap.
- Sufficient and appropriate disinfectant should be 0.5% chlorine solution for
routine mortuary work, embalming and post-mortem, then removed and rinsed
with distilled water before being dried and stored.
- Appropriate vaccination and follow up of immunity status should be offered to
all mortuary staff and record should be maintained including any refusal of an
offer of immunization.
- The Mortuary personnel is responsible to ensuring that he/she complies with
policies guarding personal safety, handling of bodies and bodily fluids as well
as the safe operation of Mortuary equipment.
- Put bio hazardous waste in a red biohazard bag
- Put the red bag in the bio hazardous waste box
- Tape box closed
- Put taped box in bio hazardous waste pick up location
- The Medical Attendants must wear complete PPE when handling hazardous
waste.
Autopsy Instruments:
- All instruments are to be cleaned and disinfected between examinations,
instruments should be dried and laid out on a non-metal surface
- Autopsy Tables and Garbage Disposals: Autopsy tables should be cleaned
and disinfected between autopsies or at the end of the day
- Following the autopsy, the disposals and drain on each autopsy table should
be cleaned.
9.1.10 Calibration
Calibration for refrigerators should be done as per schedule

9.1.11 Quality control Not applicable

9.1.12 Procedure for receiving died body from inside the facility.
After receiving a call from the ward there is a dead body the following procedure
should be followed.
- Prepare the trolley by making sure it is well covered.
- Make sure you have put on proper personal protective equipment such as
apron, face mask, boot and gloves.

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9.1.13 In the ward you should observe the following before:
If the dead body is well labelled
> If the mortuary forms have been filled in and signed by the nurse
>Make sure you sign the death book before you take the body

9.1.14 Once you reach in the mortuary do the documentation first

- inform the relatives about the process that they should follow including how to
do the payments and how to get the discharge summary.
- Dead body should be kept in a cooling unit (with tags as above)
- Make sure to label on the form the number of the fridge (unit) and the fridge
should have a tag number which should match with that attached to the body
inside.

Note: Tags have to be attached to the big toes /or around the circumference of the
ankle

Receiving dead body from outside the facility brought by police, relatives or
good Samaritans.
After receiving a dead body from the police or relatives from home the following
procedures should be followed
i. First get the information, document the police/transport ID/telephone number
ii. Prepare the trolley to receive the dead body
iii. Check the body appearance and attached properties, if it’s a police case after
documentations put the dead body directly to the cooling unit and wait for
post-mortem exam to be done.
iv. Provide the mortuary identity card to the police, if will be later on provided to
the relative
v. If it’s a home cases after documentation inform the relatives about the other
process of payments.
vi. After payments request the nearby relative to attend the embalming
procedure then keep the dead body in the cooling unit after following the
identification procedure and make sure it is well labelled.
vii. Make sure you label the form to indicate which unit the dead body has been
put in.
9.1.15 Releasing the died body from mortuary to relatives
Before giving out dead bodies following procedure should be followed;
i. For the dead body from the facility make sure the relative comes with the
discharge summary, burial certificate and the proper receipt.
ii. Check if the information has been filled well in the mortuary register book.
iii. When relative have accomplished all the process and have all the form
needed, assist them to prepare the dead body and keep it in a good condition

213
 iv. Allow them to leave after finishing all the process.
v. For out - patients make sure the relatives come with the burial certificate/or
letter from local government if they have it and proper receipts.
vi. When they have accomplished all process, assist them to prepare the body in
the washer rooms and make sure equipment, water and sanitation is well
controlled.
9.1.16 Handling personal property and clothing
Personal property that arrives with decedent remains shall be processed as follows;
i. Decedent’s personnel property will be entered into register book upon
admission.
ii. Clothing should be described by colours and items
iii. Personal effect such as jewellery, watch as well as money should only
be removed in the presence of relatives/police. iv. Personal effects
such as jewellery should be described in non-valuable terms e.g. yellow
metal or coloured stone.
9.1.17 Embalming procedure
It consists of arterial infusion of embalming fluid (10% Formalin). Since most bodies
are not disposed within 72hrs after death, hence the importance of a mortuary
establishing this service.
i. Arrange all your equipment that are needed for embalming. ii.
Prepare the formalin solution 10% concentration
iii. Put on the proper protective equipment
iv. Prepare the body to be embalmed
v. Start the embalming procedure
vi. Make sure that body has a label and tag
vii. Clean the area and the equipment that have been used.
9.1.18 Autopsy procedure
i. Autopsy has to be complete routinely.
ii. A complete autopsy is defined to include a detailed external examination as
entire body and an internal examination to include the removal and dissection
of all thoraco –abdominal and neck organs, opening the head with the removal
and examination of the brain
iii. A complete autopsy does not require histological examination
iv. A patient autopsy is defined as an examination that surface any part of the
defined complete autopsy e.g. not opening any of the body cavities or not
examining organs.
v. An external examination is defined as a detailed description of the decedents
remains including scars, surgical incisions, medical devices and tattoos.
9.1.19 Pre-autopsy procedures

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Prior to autopsy the medical attendant will set up the autopsy work room according
to the case examination status including preparing tables for body dissection,
preparing instruments, preparing sample containers and collection tubes, preparing
paper work for daily case load and taking radiographs.
Autopsy work room should be set up with the following instruments supplies, in
certain cases it will be necessary to equip the autopsy work room with specialized
instruments or additional supplies.

9.1.20 The standard autopsy precautions including using:

- A surgical scrub or suits
- Surgical cap
- Impervious gown of apron with full sleeves coverage
- A form of eye protection e.g. goggles or face shield
- Shoe covers
- Double surgical gloves with interposed layer of cut proof synthetic mesh.
- Surgical masks that may protect nose and mouth from splashes of body fluids but
do not always offer protection from airborne pathogens
- Use of respiration in adequate resources
- Safety practices to prevent injury from sharp items including hand washing as
necessary after glove removal
9.1.21 Peri autopsy procedure
i. Remove bodies from body storage colder in the stage in autopsy suits
ii. Identification by the relatives and authorised photographer can take photos
iii. Undress and transfer remaining to autopsy table
iv. Remove medical intervention devices and wash remains
v. Assist photographer in taking autopsy photographs and ID photos
vi. Perform initial Y or median incision
vii. Remove chest plate
viii. Open thoracic and abdominal cavities
ix. Medical attendant has to assist the pathologist in obtaining toxicology samples
(blood, bile, urine, vitreous, gastric, liver, brain)
x. Remove organs
xi. Weigh and record organs weight
xii. Open the entire length of the gastro intestinal tract
xiii. Elevate head
xiv. Incise and reflect scalp
xv. Remove brain
xvi. Remove duct
xvii. Obtain decedents fingerprints
xviii. Perform other autopsy procedures as directed (removing spinal cord, opening
inner extremities to exam pulmonary thrombo –emboli, stripping pavietal

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 pleural, incising the psoas muscles, assisting with preparation of sex kits and
DNA cards. Measure the length of a died.
9.1.22 Post Autopsy Procedures
i. Put organs in viscera bag ii. Replace body organs
iii. Close the thoracic, abdominal and cranial cavities with sutures
iv. Clean the body and replace in body bag
v. Indicate completion of examination by inciting a ONE on body bag
vi. Return body to refrigerated storage
vii. Put specimen in designated area depending on processing instructions;
a) Toxicology samples are put in toxicology refrigerator in the yellow
tray labelled” toxicology”
b) Histology sections are put in the yellow tray on top of toxicology
refrigerator labelled “histology”
c) Microbiology sample are put in the yellow tray on top of toxicology
refrigerator labelled “micro”
viii. Thoroughly clean and disinfect autopsy and dissection tables, sinks, drains,
instruments, dry erase boards floor area
ix. Between examinations all instruments surfaces should be cleaned with a 10%
bleach solution.
9.1.23 Body storage and organization
i. Body should be stored in clean, closed body bags with no leakage of fluids
on rack or tray
ii. Place body in empty compartment head toward the wall
iii. Tag body storage compartment with decedent name and case number
iv. Log body on body part(s) into box and cart inventory sheet
v. Maintain an accurate box or cart inventory sheet. The inventory sheet should
be updated appropriate daily
vi. Empty trays to the cleaned and disinfected and kept in box
vii. All trays should be cleaned and disinfected following a release or transfer
9.1.24 Receiving and releasing remains
Receiving Remains;
i. Identify ID bracelet with Police to tag
ii. Obtain appropriate signatures on transport notification form
iii. Take photography using the camera
iv. Obtain decedent height and weight
v. Record personal property (clothing)
vi. Complete intake of body into the logbook
vii. Complete intake of body in FACTS
viii. Log body on box and cart sheet ix. Place body in refrigerated
storage
Releasing Remains:

216
 i. Confirm in FACTS that the body is ready to be released and that the receipt
of remain seen has been properly completed by communications unit.
ii. Print and sign copy of the Receipt of remains form.
iii. Sign body out of release logbook
iv. Obtain funeral home representatives signatures, number and initials where
appropriate
v. Have to tag and receipt of remains form witnesses
vi. Clean and disinfect tray before retaining it to cold box
9.1.25 Sample Storage and Retrieval
All stock specimens retained in clean and dry bottle in 10% buffered formalin label
write the Post Mortem Examination Number, name, age, sex, address and
anatomical site

9.1.26 Transportation of remains

If a body is to be shipped out of the country, a letter stating that the autopsy showed
no evidence of any infections or communicable diseases is required.
In the contents of such a letter must include Identification details such as name and
date of death that match the details in the transit/burial permit or death certificate.

9.1.27 Supporting documents

National guideline for operating of Mortuary services 2020, National guideline for
establishment of Mortuary services 2020, IPC Guideline 2018

9.1.28 References
1. Standard guidelines for the facilities and operation of mortuaries in Tanzania
2008
2. Hutchins GM. Practice guidelines for autopsy performance. Archives of
pathology and laboratory medicine, 1994, 118(1):19-25
3. Advices ED, Sims KL. Enhancing autopsy performance and reporting. A
system for a 5-day completion time. Archives of pathology and laboratory
medicine,1996,120(3):249-53.

217
Annex 1: List of Subject Matter Experts who developed these SOPs

No NAME TITILE FACILITY

1. Adam Mwakyoma Laboratory Scientist Kilimanjaro Christian
Medical Centre (KCMC),
Moshi
2. Amedeus Mushi Laboratory Scientist & HLPC, MoH, Dodoma
Epidemiologist
3. Anjela Maufi Laboratory Scientist Kitete RRH, Tabora
4. Antusa Massawe Laboratory Scientist Dunga H/C, Tanga
5. Baraka Mathias Laboratory Scientist Maranatha Hospital,
Mbeya
6. Betrand Msemwa Microbiology & Manyara Catholic University of
Health and Alliened
Sciences (CUHAS),
Mwanza
7. Bezard Ngumbuchi Quality Officer PHLB, Dodoma
8. Ceif Abdul Quality Officer NPHL, Dar es Salaam
9. David Ocheng Laboratory Expert/ Ilala, Dar es Salaam
Consultant
10. Desmond Kileo ICTO DHCTSU, MoH, Dodoma
11. Dickson Charles Laboratory Scientist Cardinal Rugambwa
Kakuntebe Hospital, Dar es Salaam
12. Dominic Fwiling’afu Registrar PHLB, MoH, Dodoma
13. Dr. Alex Magesa DDS DHCTSU, MoH, Dodoma
14. Dr. Goodluck Tesha Epidemiologist Jhpiego, Dar es Salaam
15. Dunstan Haule Laboratory Quality Officer NBTS-HQ, Dar es Salaam
16. Edward Lushishi Laboratory Technologist Singida RRH, Singida
17. Emmanuel Ndaki Laboratory Scientist NBTS Lake zone,
Mwanza
18. Eng. Gervas H. Swai Biomedical Engineer DHCTSU, MoH, Dodoma
19. Eng. Suniva S. C. Head Health Care and DHCTSU, MoH, Dodoma
Haule Technical Services
20. Ezekiel Mng’ong’o Laboratory Scientist Maranatha Hospital,
Mbeya
21. Farhiya Mohamed Laboratory Quality Officer NBTS Eastern Zone, Dar
es Salaam
22. Ferdinand Matata Head of Diagnostic PO-RALG, Dodoma
Services
23. Frank Changala Laboratory Scientist NPHL, Dar es Salaam
24. Dr Haji Hussein Technical officer ICAP, Dar es Salaam
25. Frank Mushi Laboratory Scientist Manyara RRH, Manyara
26. Frank Shemhande Laboratory Quality Officer SBNRH-Ndanda, Mtwara
27. Godfrey Mahundi Laboratory Manager Dodoma MC Hospital
28. Helman Mhangala Deputy Laboratory NBTS Central Zone,
Manager Dodoma
29. Ibrahim Mauki Laboratory Scientist NPHL, Dar es Salaam

218
30. Idrissa Hassan Laboratory Service PORALG, Dodoma
Coordinator
31. Innocent A. Chinguile Laboratory Scientist & Training, Dodoma MoH,
Epidemiologist
32. Jackson Luvamunda Quality Officer Sinza HC, Dar es Salaam
33. Jackson Zengo Deputy National DHCTSU, Dodoma MoH,
Laboratory Quality Officer
34. Jacob Lusekelo National Laboratory DHCTSU, Dodoma MoH,
Quality Officer
35. James Mziray Laboratory Service PO-RALG, Dodoma
Coordinator
36. Joakim Chacha LS-HSEEP DHCTSU, MoH, Dodoma
37. Joyce Samson Laboratory Quality Officer Sekou-Toure RRH,
Mwanza
38. Kadogosa Saimon Laboratory Technologist Singida RRH, Singida
39. Lanja Kahemela Laboratory Quality Officer MNH, Mloganzila, Dar es
Salaam
40. Maige Ngeleja DSCMC MoH, Dodoma
41. Mary F. Mtui Registrar HLPC, MoH, Dodoma
42. Mayala Lushina Laboratory Quality Officer MNH, Upanga, Dar es
Salaam
43. Michael Mazoya Laboratory Scientist NBTS, Dar es Salaam
44. Mzelifa Daudi Microbiologist University of Dodoma
(UDOM), Dodoma
45. Pius R. Tarimo Laboratory Scientist & Kilimanjaro Christian
Epidemiologist Medical University
College (KCMUCo),
Moshi
46. Rajabu Muninge Laboratory Scientist Maranatha Hospital,
Mbeya
47. Rashid Nassoro Laboratory Quality Officer Morogoro RRH, Morogoro
48. Reuben Abednego Laboratory Scientist NPHL, Dar es Salaam
49. Reuben Lema Deputy Laboratory Morogoro Morogoro RRH,
Manager
50. Reuben S. Mkala Ag. Head of Laboratory DHCTSU, Dodoma MoH,
Service
51. Richard Kinyaha Laboratory Scientist Kibong’oto IDH,
Kilimanjaro
52. Robert Makala Regional Laboratory RAS - Manyara
Technologist (RLT)
53. Sabra Rashid Laboratory Scientist MoH, Unguja-Zanzibar
54. Said Lunemhya Laboratory Scientist Benjamin Mkapa ZRH,
Dodoma
55. Scolastica S. Laboratory Quality Officer Mirembe Hospital,
Manyama Dodoma
56. Susu Jeremiah Susu Laboratory Quality Officer Benjamin Mkapa ZRH,
Dodoma
57. Tamaly Lutufyo Laboratory Manager CCBRT, Dar es Salaam

219
58. Ulfat Rahim Laboratory Technologist TMJ, Dar es Salaam
59. William S Mollel Laboratory Technologist MOLLEL LABORATORY,
Dodoma
60. Yahya Mnung’a MeLSAT-President MeLSAT, Dar es Salaam
61. Yohana Shirima Laboratory Technologist NBTS Northern zone,
Moshi
62. Yulitha Barnabas Laboratory Manager Dodoma RRH, Dodoma
63. Zacharia Omary Laboratory Manager Sekou-Toure RRH,
Mwanza

220
Annex 2: Biological Reference Intervals for Full Blood Count

Parameter Reference Range

Adult Infants Children

Males Females Birth 1 month 1 year SI Unit
WBC 4.00 - 10.0 4.0 - 10 10 - 26 5.0 - 9.0 6.0 - 16.0 x103 /µL
NEU 40 - 80 40 - 80 40 - 80 40 - 80 40 - 80 %
LYM 20 - 40 20 - 40 20 - 40 20 - 40 20 - 40 %
MON 2 - 10 2 - 10 2 - 10 2 - 10 2 - 10 %
EOS 1–6 1-6 1-6 1-6 1-6 %
BASO <1 - 2 <1 - 2 <1 - 2 <1 - 2 <1 - 2 %
RBC 4.5 - 5.5 3.8 - 4.8 5.0 - 7.0 3.0 - 5.4 3.9 - 5.1 x106 /µL
HGB 13.0 - 17.0 12.0 -15.0 14.0 - 22.0 11.5 - 16.5 11.1 - 14.1 g/dL
MCV 83.0 - 99.0 83.0 - 99.0 100 -120 92 - 116 72 - 84 Fl
MCH 27.0 - 32.0 27.0 - 32.0 31.0 - 37.0 30.0 - 36 25.0 - 29.0 Pg
MCHC 31.5 - 34.5 31.5 - 34.5 30.0 - 36.0 29.0 - 37.0 32.0 - 36.0 g/dl
Hct 40 – 50 36 - 46 45 - 75 33 - 53 30 - 38 %
RDW
PLT 150 - 410 150 - 410 100 - 450 200 - 500 200 - 550 x103 /µL
MPV

221
Annex 3: Biological Reference Intervals for Coagulation Profile

Test Range SI Unit

Prothrombin Time 9.40 - 12.50 Sec
Activated Prothrombin Time 25.40 – 36.90 Sec
Fibrinogen 2.20 – 2.80 g/l
Factor V 0.62 -1.39 IU
Factor VII 0.50 -1.29 IU
Factor VIII 0.50 -1.50 IU
Factor IX 0.65 -1.50 IU
Free protein S (male) 74.10 - 145.10 %
Free protein S (female) 54.70 - 123.70 %
Protein S activity 63.50 - 149.00 %
Protein C may be less in neonates, infants and 70.00 – 140.00 %
increase in adolescence
Plaminogen (activity) 80.20 - 132.50 %
Plamin inhibitor 98.00 – 122.00 %
Homocysteine 4.30 - 11.10 μmol/L
D-dimmer ≤ 232 ng/ml
Von Will brand factor ristocetin cofactor activity 480 - 201.90 %
(blood group O)
Von Will brand Factor ricostein factor activity 60.80 - 239.80 %
(blood A+B+AB)

222
Annex 4: Biological Reference Intervals for Urine Biochemistry

Parameter Abbreviation Biological Reference Intervals

Urobilinogen URO Normal
Glucose GLU Negative
Bilirubin BIL Negative
Ketones KET Negative
Specific gravity S.G 1.003-1.029
Occult blood BLD Negative
Ph Ph 4.5 - 7.8
Protein PRO Negative
Nitrite NIT Negative
Leukocytes LEU Negative

223
Annex 5: Biological Reference Intervals for Clinical Chemistry and
Immunoassays

Test Name Normal range SI Unit

Parameter/Analyte Sub category
Alanine aminotransferase
0 - 55 U/L
(ALT)
Albumin 35 - 50 g/l
Male 15 -125 U/L
Female 15 -125 U/L
Male child 0 - 500 U/L
Female Child 0 - 500 U/L
Children U/L
Aged 1 day <250 U/L
Aged 2 - 5 days <231 U/L
Alkaline Phosphate
Aged 6 days - 6 months <449 U/L
Aged 7months - 1 year <462 U/L
Aged 1 - 3years <281 U/L
Aged 4 - 6years <269 U/L
Aged 7 - 12 years <300 U/L
Aged 13 - 17 years (M) <390 U/L
Aged 13 - 17 years (F) <187 U/L
Aspatate Aminotransferase 5 - 34 U/L
Bilirubin – Direct 0 – 8.6 µmol/L
Bilirubin – Total 3.4 - 20.5 General µmol/l
Total Protein 64 - 83 g/l
Male 12- 64 U/L
Female 9 - 36 U/L
Gamma Glutamyl Transferase
Male child 9 - 36 U/L
Female child 9 - 36 U/L
CSF protein 0-4.3 lumbar fluid g/L
CSF glucose One third of Glucose g/L
Asciti Protein 60 - 80 g/L
Ascitic Glucose 70 - 100 mg/dL
Adult Male 63.6 - 110.5 µmol/L
Adult Female 50.4 - 98.1 µmol/L
Creatinine
Male child 27 - 88 µmol/L
Female child 27 - 88 µmol/L
Male 3.2 - 7.4 µmol/L
Female 2.5 - 6.7 µmol/L
Blood Urea Nitrogen (BUN)
Male child 3.2 - 7.4 µmol/L
Female Child 2.5 - 6.7 µmol/L
Cholesterol Total <5.2 mmol/L
HDL - Cholesterol 1.04 - 1.55 mmol/L
LDL – Cholesterol 0 - 3.34 mmolL
Triglycerides 0 - 1.69 mmol/L

224
Test Name Normal range SI Unit
Sodium ( Na ) 136 - 145 mmol/L
Potassium ( K ) 3.5 – 5.1 mmol/L
Chloride (Cl ) 98 – 107 mmol/L
Amylase Total 25- 125 U/L
Male 30 – 200 U/L
Female 29- 168 U/L
Creatine Kinase ( CK )
Male child 30 – 200 U/L
Female child 29-168 U/L
Lactate dehydrogenase 125 – 220 IU/L
(LDH)
Lipase 13- 60 U/L
Male 2.1 – 2.55 mmol/L
Famale 2.1-2.55 mmol/L
Calcium
Male child 2.2 – 2.7 mmol/L
Female child 2.2 – 2.7 mmol/L
Glucose 3.3 - 6.1 mmol/L
Male 5.5 – 25.8 µmol/L
Female 4.5 - 25.8 µmol/L
Iron
Male child 5.5 – 25.8 µmol/L
Female child 4.5 – 25.8 µmol/L
% Saturation (Iron 20 - 50 %
saturation)
Male 0.21 - 0.42 mmol/L
Female 0.15 - 0.35 mmol/L
Uric Acid
Male child 0.21 - 0.42 mmol/L
Female child 0.15 - 0.35 mmol/L
Phosphorus 0.74 - 1.52 mmol/L
Sodium 24hrs Urine 27 – 287 mmol/24 hours
Potassium 24hrs Urine 25 – 125 mmol/24hrs
Male 1.74 - 3.64 g/L
Female 1.8 - 3.82 g/L
Transferrin
Male child 1.86 – 3.88 g/L
Female child 1.86 - 3.88 g/L
Alpha Feto Protein 0.0 - 1.09 ng/ml
13.8-17.5 Female pg/ml
High Sensitive Troponin
28.9-39.2 Male pg/ml
Vitamin B12 187-883 pg/ml
Ferritin 10 - 250 ng/ml
Folate 3.72 - 50.4 ng/ml
PSA 0.0 - 4.0 ng/ml
TSH 0.49 - 4.67 IU/ml
T4 0.47 - 4.67 ng/L
T3 1.45 - 3.48 pg/ml
CK-MB 0.0 - 6 %

225
Test Name Normal range SI Unit
Tacrolimus 3 – 20 ng/ml
BNP 0-142 pg/ml
Cyclosporine 30.0-1500 ng/ml
CEA 0-5 ng/ml
CA-125 0-35 IU/mL
Less than 5 for non pregnant mIu/mL
B-HCG
25 for early pregnancy mIu/mL
Vitamin D 0 - 160 ng/ml
Immunoglobulin G 5.40-18-22 Male g/l
5.52-16.31 Female g/l
1-12 months <15 IU/mL
1-5 years <60 IU/mL
Immunoglobulin E 6-9 years <90 IU/mL
10-15 years <200 IU/mL
Adults <100 IU/mL
Male 63 – 645 mg/dl
Female 65 – 517 mg/dl
Immunoglobulin A
Male child 21- 291 mg/dl
Female child 21 - 281 mg/dl
Male 0.22-2.40 g/l
Female g/l
Immunoglobulin M
0.33-2.93 g/l
Either 0.22-2.93 g/l
D - Dimer 0 .0 – 198 ng/L
CRP 0.0-5.0 mg/L
Follicular phase 21-251 pg/ML
Midcycle phase 38-649 pg/ML
Estradiol Lueal phase 21-312 pg/ML
Postmenopausal female 1028 pg/ML
Male 11-44 pg/ML
Male ng/ml
Prolactin 3.46-19.40 ng/ml
Female5.18-26.53 ng/ml
Male 4.94-32.01 nmol/L
Testosterone
Female 0.38-1.97 nmol/L
Follicular phase 0.1-0.3 ng/ml
Luteal phase 1.2-15.9 ng/ml
Postmenopausal 0.1-0.2 ng/ml
Progesteron First trimester 2.8-147.3 ng/ml
Second trimester 22.5-95.3 ng/ml
Third trimester 27.9-242.5 ng/ml
Male 0.1-0.2 ng/ml
Male 1.14-8.75 ng/ml
LH Follicular phase 2.39-6.60 ng/ml
Midcycle peak-9.06-74.24 ng/ml

226
Test Name Normal range SI Unit
Luteal phase 0.909.33 ng/ml
Postmenopausal ng/ml
10.39 - 64.57 ng/ml
Male 0.95 - 11.95 mIu/mL
Follicular phase 3.03 - 8.08 mIu/mL
Midcycle peak 2.55 - 16.69 mIu/mL
FSH
Luteal phase 1.38 - 5.47 mIu/mL
Postmenopausal mIu/mL
26.72 - 133.41 mIu/mL
Male 0.66 - 1.07 mmol/L
Female 0.66 - 1.07 mmol/L
Magnesium
Male child 0.70 - 0.86 mmol/L
Female 0.70 - 0.86 mmol/L
ADA 0 - 15 U/L
Glycated haemoglobin
4-6 %
(HBA1C)

227
Annex 6: Critical or Panic Values that call for Immediate Actions

Analyte Less Than Greater Than

Amylase 25 U/L 150 U/L
Chloride 85 mmol/L 115 mmol/L
CK 30 U/L 200 U/L
Creatinine 26 umol/L 120 umol/L
Glucose(fasting) 2.5 mmol/L 20.0 mmol/L
Potassium 2.5 mmol/L 6.0 mmol/L
Sodium 120 mmol/L 160 mmol/L
Bilirubin Total 3.4 umol/L 20.5 umol/L
Biliribun Total for new Born Newborn
24hours ≥ 1374 umol/L
48hours ≥ 2224 umol/L
84hours ≥2904 umol/L
One week to one month ≥3424 umol/L
Urea (BUN) ≤1.0mmol/L ≥ 54 mmol/L
HGB < 5 mg/dl > 20 g/dl
CD4 200 cells/ l

228
Annex 7: Charts for Biochemical Identifications of Common Enterobacteriaceae and other Enteric Organisms

MAC
Organism TSI Oxidase H2S Gas Motility Indole Urea Citrate Haemolysis Comment
Reaction
Serratia Red pigment at
NLF K/A or A/A - - + + - - + -
mercesens room temp on MHA
Grow with
Proteus mirabilis NLF K/A - + + + - + +(weak) - swarming xters on
BA
A/A or Grow with
Proteus vulgaris NLF K/A - + + + + + +/- - swarming xters on
BA
Citrate pos (non-
Salmonella sp NLF K/A - + + + - - + -
typhoid salmonella)
Salmonella typhi NLF K/A - Wk+ + + - - - - Black ppt on
SSA&XLD
Shigella sonnei NLF K/A - - - - - - - - No black ppt on
SSA/XLD
Other Shigella sp NLF K/A - - - - - - -
Vibrio cholerae NLF A/A - + + - - - - + String test-positive
Vibrio NLF K/A + - - + + - + +
parahaemolyticus
Green pigmentation
P.aeruginosa NLF K/NC + - - + - + + +/-
on MHA
Acinetobacter spp NLF NC - - - - - - - - Coccoide rods
Morganella NLF K/A - - + + + + - -
morganii
Providencia spp NLF K/A - - + + + -
Yersinia NLF K/A - - - +(25/-35°C) +/- +/- - -
enterocolitica
Edwardsiella NLF K/A - + + + + - - -
tarda
Grows with
E.coli LF A/A - - + + + - - +/- precipitate bile salt
on MCA
229
 MAC
Organism TSI Oxidase H2S Gas Motility Indole Urea Citrate Haemolysis Comment
Reaction
Enterobacter Often resistant to
aerogenes LF A/A - - + + - - + - Ampicillin and
cephalosporin
Grow with very
Klebsiella
LF A/A - - + - - + + - mucoid
pneumoniae
colonies
Klebsiella oxytoca LF A/A - - + - + + + -
Citrobacter Late LF A/A or K/A - + + + - +/- + -
freundii

230
NOTE PAD

231
NOTE PAD

232
NOTE PAD

233
 FOR FURTHER INFORMATION CONTACT:

PERMANENT SECRETARY
MINISTRY OF HEALTH,
MAGUFULI GOVERNMENT CITY, AFYA ROAD/STREET, MTUMBA,
PO BOX 743,
40478 DODOMA, TANZANIA.
LANDLINE: +255 (0)26 232 3267
EMAIL: [email protected]
WEBSITE: www.moh.go.tz