Practical Manual

BSc (Hons) in Biomedical Science

Department of Biomedical Science
Faculty of Health Sciences
KIU
About this resource
Clinical Biochemistry is central to all areas of biological life sciences. It provides theoretical
and practical aspects of biochemistry and its applications. This manual, explains the basic
techniques necessary to carry out biochemical investigations safely and effectively, is intended
as a guide for undergraduates and the technicians in the laboratory.
As a fulfilment of the degree, the practicals listed in the manual has to be studied and acquire
necessary skills. KIU, is a non-state degree awarding Higher Education Institute which awards
the Bachelor of Science (Honours) in Biomedical Science. For further details, contact
Department of Biomedical Science.
Email: [email protected]
Institution address: Department of Biomedical Science, Faculty of Health Sciences, KIU,
249/1, Malabe Rd, Koswatta, Battaramulla, Sri Lanka

Copyright
Clinical Biochemistry – Practical Manual is copyright.
Educational use: Electronic or paper copies of the resource or individual pages from it may be
made for classroom and educational uses while the KIU is credited and identified as copyright
holder.
© 2021 KIU
 Clinical Biochemsitry Practical Manual 2

Table of contents
1 PRACTICAL 01: SPECIMEN COLLECTION, LABELLING, REJECTION CRITERIA 3
1.1 REQUEST FORMS ........................................................................................................................ 3
1.2 INAPPROPRIATE SUBMISSIONS / REJECTION CRITERIA ............................................................... 4
1.3 SPECIMEN LABELLING ................................................................................................................ 4
1.4 SPECIMEN COLLECTION.............................................................................................................. 5
2 PRACTICAL 02: URINE FULL REPORT 1 ............................................................................ 9
2.1 PHYSICAL EXAMINATION........................................................................................................... 9
2.2 CHEMICAL EXAMINATION PH AND SPECIFIC GRAVITY ............................................................ 10
3 PRACTICAL 03: URINE FULL REPORT 2 .......................................................................... 12
3.1 PREPARATION OF WET SMEAR.................................................................................................. 12
4 PRACTICAL 04: URINE FULL REPORT 3 .......................................................................... 19
4.1 DETERMINATION OF REDUCING SUBSTANCE IN URINE ............................................................ 19
4.2 DETERMINATION OF KETONE BODIES IN URINE ...................................................................... 21
4.3 DETECTERMINATION OF PROTEIN IN URINE ............................................................................. 23
5 PRACTICAL 05: URINE FULL REPORT 4 .......................................................................... 26
5.1 DETERMINATION OF BILIRUBIN (BILE PIGMENTS) IN URINE ..................................................... 26
5.2 DETECTION OF UROBILINOGEN IN URINE ................................................................................. 27
5.3 DETECTION OF BILE SALTS IN URINE ........................................................................................ 27
5.4 URINE STRIP METHOD .............................................................................................................. 28
6 PRACTICAL 06: STANDARD CURVE PREPARATION ................................................... 30
6.1 PREPARATION OF DILUTION SERIES ......................................................................................... 30
6.2 PREPARATION OF STANDARD CURVE ....................................................................................... 31
7 PRACTICAL 07: PLASMA GLUCOSE TEST ...................................................................... 33
8 PRACTICAL 08: ORAL GLUCOSE TOLERANCE TEST ................................................. 35
9 PRACTICAL 09: LIPID PROFILE ......................................................................................... 37
9.1 TOTAL CHOLESTEROL .............................................................................................................. 37
9.2 TRIGLYCERIDES ....................................................................................................................... 38
9.3 HDL – C ................................................................................................................................... 39
9.4 LDL – C ................................................................................................................................... 41
10 PRACTICAL 10: INTERNAL QUALITY CONTROL IN CHEMICAL PATHOLOGY
LABORATORY .................................................................................................................................. 42
11 PRACTICAL 11: SERUM ENZYMOLOGY.......................................................................... 45
11.1 ESTIMATION OF SERUM TRANSAMINASES ACTIVITY ........................................................... 45
12 PRACTICAL 12: URINE PREGNANCY TEST .................................................................... 47

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Practical 01
Specimen collection, Labelling, rejection criteria
1 Practical 01: Specimen collection, Labelling, rejection criteria
The lab shall conduct tests for the hospital in-house, OPD, walking-in patients, corporate-sector
clients, the government sector and other private sector clients (other labs), maintaining
confidentiality and cordiality in order to meet the needs of the patients and all clinical personnel
responsible for patients care.

Quality laboratory results begin with proper collection and handling of the specimen delivered
for analysis. Correct patient preparation, specimen collection, specimen packing, storage and
processing of vital importance and proper guidelines regarding the above should be followed
to maintain the quality of a given laboratory. All specimens should be handled with universal
precautions, as they may well be hazardous and infectious.

1.1 Request forms

Introduction

Assay request forms are available from the laboratory. Complete the request form for each
patients with all required patient information.

These include,

• Patient name, sex, age and medical record or lab reference number
• Collection date and time
• Type and amount of specimen submitted
• Condition of the specimen
• Volume collected
• Source of specimen
• Presumptive clinical diagnosis and any patient history
• Complete billing information
• Mark boxes indicating the test (s) requested.

Procedure

1) Check the given request forms and comment.

2) Prepare a request form for given tests – UER, FBS, lipid profile

Discussion

1. Explain the importance of request form

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2. Discuss the changes should be included in a STAT samples

3. Mentioned the factors you should be considered, if submitting more than one
specimen per patient and specimens to be stored and transport at different
temperature.

1.2 Inappropriate submissions / Rejection criteria

Introduction

It is the policy of the laboratory to reject specimens when there is a failure to follow these
guidelines.

Procedure

You have provided 13 request forms with specimens.

Mentioned the accepting and rejection criteria for each

Discussion

Explain the accepting and rejecting criteria for cerebrospinal fluid.

1.3 Specimen labelling

Introduction

Label all specimen containers with the following:

Patient name

Patient ID number

Specimen type(s)

Date collected.

Identifying information can be provided by writing directly onto the vials in indelible ink. If
labels are used, they should be secured to insure retention during freezing.

Procedure

1) Place label directly under the cap

2) Name at the top of the tube
3) Label with BHT number, Age, sex, Date of collection
4) Leave visible window to see blood
5) Place only one label on each tube

Discussion

List down the incorrect labelling methods

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Discuss the importance of following correct labelling method.

1.4 Specimen collection

Introduction

• Biological specimens that are analysed in clinical laboratories include,

• Whole blood
• Serum
• Plasma
• Stool
• Saliva
• Synovial, cerebrospinal, amniotic, pleural, pericardial, ascitic fluid and other
body fluids
• Solid tissues

1.4.1 Blood

The most common samples of laboratory testing are whole blood, serum or plasma. Blood for
analysis may be obtained from veins, arteries or capillaries. Venous blood is usually the
specimen of choice and venepuncture is the method for obtaining this specimen. In young
children, Skin puncture is frequently used to obtain what is predominantly capillary blood.
Arterial puncture is used mainly for blood gas analyses. A syringe or an evacuated blood tube
is used to obtained blood specimens.

If whole blood or plasma is desired for testing, an anticoagulant must be added to the
specimen during the collection procedure.

Procedure

1) Identify the blood collection containers

01.
Volume of the blood - …………………………………………………..
Type of anticoagulant - ……………………………………………
……………………………………………
……………………………………………
Test performed - ……………………………………………….
02.
Volume of the blood - …………………………………………………..
Type of anticoagulant - ……………………………………………
……………………………………………
……………………………………………

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Test performed - ……………………………………………….

03.
Volume of the blood - …………………………………………………..
Type of anticoagulant - ……………………………………………
……………………………………………
……………………………………………
Test performed - ……………………………………………….
04.
Volume of the blood - …………………………………………………..
Type of anticoagulant - ……………………………………………
……………………………………………
……………………………………………
Test performed - ……………………………………………….

2) Identify the blood collection procedures

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1.4.1.1 Separate plasma and serum from bllod

Introduction

Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot.
The clot is removed by centrifugation and the resulting supernatant, designated serum, is
carefully removed using a Pasteur pipette.

Plasma is produced when whole blood is collected in tubes that are treated with an
anticoagulant. The blood does not clot in the plasma tube. The cells are removed by
centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet
using a Pasteur pipette.

Procedure: Plasma separation

1. Draw blood from patient

2. Collect the blood into an anticoagulant blood collection tube
3. Mix well with anticoagulant
4. Allow to stand for 10 minutes
5. Centrifuged at 2500 – 3000 rpm for 5 - 10 minutes
6. Observe the separation
7. Remove the supernatant using a Pasteur pipette

Procedure: Serum separation

1. Draw blood from patient to a plain tube (tube without anticoagulant)

2. Allow the blood to clot at room temperature for 10 to 15 minutes
3. After blood has clotted completely and then centrifuged at 2500 – 3000rpm for 5 – 10
minutes.
4. Observe the seperation
5. Remove the supernatant using a Pasteur pipette

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Discussion

Discuss the difference between serum and plasma

Explain the pre analytical errors in each step in analytical phase.

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Practical 02
Urine Full Report 1
2 Practical 02: Urine Full Report 1
Introduction

Urine is an excretory product of our body. It is formed in the kidney. Urine examination helps
in the diagnosis of various renal as well as systemic diseases. Urine is readily available and
easily collected specimen. Urine contain information, which can be obtained by inexpensive
laboratory tests, about many of the body’s major metabolic functions

Urine analysis performed under 03 categories,

Physical examination
Chemical examination
Microscopic examination

2.1 Physical Examination

Introduction

Physical examination includes,

Color observation

Clarity/ Appearance

Color
Freshly excreted normal color of the urine is pale yellow/ yellow/ dark yellow
Color of the urine is from urochrome
Clarity
Clarity is transperancy/ torbidity of a urine samples.
Normal urine specimen clarity is clear
Procedure
• Mix urine samples well.
• View through a clear container.
• View against a white background.
• Maintain adequate room lighting.
• Determine color and clarity.

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2.2 Chemical examination pH and specific gravity

2.2.1 pH

Introduction

Along with the lungs, the kidneys are the major regulators of the acid-base content in the body.
They do this through the secretion of hydrogen in the form of ammonium ions, hydrogen
phosphate, and weak organic acids, and by the reabsorption of bicarbonate from the filtrate in
the convoluted tubules.
Healthy individual usually produces a first morning specimen with a slightly acidic pH of 5.0
to 6.0. More alkaline pH is found following meals. pH of normal random samples can range
from 4.5 to 8.0.
Use pH meter/ pH paper to measure the pH of the specimen
Procedure

1. Observe the urine specimens and comment on the color, clarity and pH, specific gravity

2.2.2 Specific Gravity

Specific gravity is defined as the density of a solution compared with the density of a similar
volume of distilled water at a similar temperature.
Urine is actually water that contains dissolved chemicals, the specific gravity of urine is a
measure of the density of the dissolved chemicals in the specimen.
Measure of specimen density, specific gravity is influenced not only by the number of particles
present but also by their size.
Specific gravity provides valuable preliminary information and can be easily performed by
Direct methods
urinometer (hydrometer)
harmonic oscillation densitometry (HOD)
Indirect methods
refractometer
chemical reagent strip.
Specific gravity of a normal healthy individual – 1.015 – 1.025
Urinometer
• Consists of a weighted float attached to a scale that has been calibrated in terms of
urine specific gravity

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• The weighted float displaces a volume of liquid equal to its weight and has been
designed to sink to a level of 1.000 in distilled water.
• The additional mass provided by the dissolved substances in urine causes the float to
displace a volume of urine smaller than that of distilled water
• The urinometer reading may also need to be corrected for temperature, inasmuch as
urinometers are calibrated to read 1.000 in distilled water at a particular temperature
• The calibration temperature is printed on the instrument.

• If the specimen is cold, 0.001 must be subtracted from the reading for every 3oC that
the specimen temperature is below the urinometer calibration temperature.
• Conversely, 0.001 must be added to the reading for every 3oC that the specimen
measures above the calibration temperature.
Procedure
1. An adequate amount of urine is poured into a proper-size container.
2. Urinometer is added with a spinning motion.
3. The scale reading is then taken at the bottom of the urine meniscus.
Discussion
Differentiation of haematuria from haemoglobinuria from their physical properties
Identification of the presence of protein in urine by physical properties
Identification of the presence of bilirubin in urine by physical properties
Tabulate advantage and disadvantages using urinometer

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Practical 03
Urine full Report 2
3 Practical 03: Urine full Report 2
Introduction
The second part of routine urinalysis is the microscopic examination of the urinary sediment.

Its purpose is to detect and to identify insoluble materials present in the urine.

The blood, kidney, lower genitourinary tract, and external contamination all contribute formed
elements to the urine.

These include

RBCs WBCs epithelial cells

Casts bacteria yeast
Parasites mucus spermatozoa
Crystals artifacts

Some of these components are of no clinical significance and others are considered normal
unless they are present in increased amounts, examination of the urinary sediment must include
both identification and quantitation of the elements present.

most time-consuming part of the routine urinalysis

3.1 Preparation of wet smear
Procedure

1. Preparation and Examination of the Urine Sediment

2. Before analysis

Well mixed specimen

Centrifuged and take the deposit

Procedure
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Procedure
Preparation of wet smear
1. Take fresh or well-preserved urine samples (Standard amount of urine, usually
between 10 and 15 mL)
2. Mix urine samples
3. Transfer urine into a conical centrifuged tube.
Centrifugation

1. Centrifuge for 5 minutes at a relative centrifugal force (RCF) of 400.or 1500 rpm for
5 minutes
Sediment Preparation

1. Remove the supernatant using pasture pipette.

2. Do not remove the sediment.
3. uniform amount of urine (1 ml) and sediment should remain.
4. Resuspend the sediment in the last drop of urine by tapping the bottom of the tube
until well mixed.
5. Place a drop (10µl) of suspend into a clean glass slide.
6. Put the cover slip
Examination of the Sediment

1. Observe under both low (X10) and high (X40) power

2. first examined under low power
3. Then examined under high power (X40)
4. Observe minimum of 10 fields in each power
5. Record the results
Complete the practical document

Structure: …………………………………..

Appearance: ………………………………….

Observed under : ……………….....................

Reporting method : ………………………......

Normal level : ………………………………..

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Structure: …………………………………..

Appearance: ………………………………….

Observed under : ……………….....................

Reporting method : ………………………......

Normal level : ………………………………..

Structure: …………………………………..

Appearance: ………………………………….

Observed under : ……………….....................

Reporting method : ………………………......

Normal level : ………………………………..

Structure: …………………………………..

Appearance: ………………………………….

Observed under : ……………….....................

Reporting method : ………………………......

Normal level : ………………………………..

Draw three fields you observed under high power field

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Crystals

frequently found in the urine are rarely of clinical significance.

They may appear as true geometrically formed structures or as amorphous material

The primary reason for the identification of urinary crystals is to detect the presence of the
relatively few abnormal types

liver disease

inborn errors of metabolism

renal damage caused by crystallization of iatrogenic compounds within the

tubules

Crystals are usually reported as rare, few, moderate, or many per hpf. Crystals are formed by
the precipitation of urine solutes, including inorganic salts, organic compounds, and
medications.

Crystal pH Appearence

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Practical 04
Urine full Report 3
4 Practical 04: Urine full Report 3
Changes that can occur in the volume, appearance, constituents and specific gravity of urine
are commonly used to diagnose a wide range of disease.

Abnormal chemical constituents in urine include,

Reducing substances (glucose, fructose, galactose, vitamin C)

Proteins (albumin, globulin, haemoglobin, Bence Jones protein)

Ketone bodies (Acetone, acetoacetic acid)

Bile salts and bilirubin (Bile pigments)

Urobilinogen (in increased amount)

4.1 Determination of reducing substance in urine

Objectives
• To describe the copper reduction method for detection of urinary reducing
substances
• To list down the possible causes of interference factors
Introduction
In healthy individuals, glucose filtered by the glomerulus is reabsorbed in the proximal
convoluted tubule

Therefore, glucose is not present in the urine

Clinical significance,

Hyperglycaemia associated

Renal associated

4.1.1 Benedict test

Introduction

Banedict’s reagent contains CuSo4. In hot alkaline solutions reducing substances reduce blue
Cu2+ ions to brick red (Cu+) Cu2O precipitate.

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As the reaction proceeds, the colour of the reaction mixture changes progressively from blue
to green, yellow, orange and brick red depending on the concentration of the reducing
substance. A negative reaction indicates the original blue color of the Cu2+.

Procedure

1. Pipette 5 ml of Benedict’s reagent into a test tube.

2. Add 8 drops of urine to the Benedict’s reagent.
3. Keep for 5 mintes in a boiling water bath..
4. Cool under running tap water
5. Observe the color change with precipitation
6. Interpret the test result using following table.
Color of precipitate Color of the Approximate Reporting method
solution strength of
reducing sugars
(%)
No precipitate Blue with green 0.1
opalescence

Slight yellow Blue on standing 0.2

Slight orange Green (Blue on 0.3

standing)

Definite orange Green (Blue on 0.5

standing)

Deep orange Green (Blue on 1.0

standing)

Brick red Blue colour 2.0

almost disappears

Discussion

List down the factors that are causing false positive results in the above test?
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List down the factors that are causing false negative results in the above test?
Other methods available to measure glycosuria?
Diseases associated with hyperglyceamia which cause glucosuria

Disease associated with renal glycosuria which cause glucosuria

4.2 Determination of Ketone bodies in Urine

Objectives
• To describe the principle of the Rothera’s test
• To list down the possible causes of interference factors

Ketone bodie sare “three intermediate products of fat metabolism”. Under normal conditions,
metabolized fats are completely broken down to water and carbon dioxide. Only negligible
amount (1mg/24 hrs) of ketone bodies are excreted in urine

Types of ketone bodies:

Β hydroxybutyric acid 78 %,

Acetoacetic acid 20 %,

Acetone 2%

Both acetone and beta hydroxybutyric acid are produced from acetoacetic acid

4.2.1 Rothera’s test

Principle

Acetoacetic acid or acetone reacts with nitroprusside in alkaline solution to form a purple-
colored complex”

Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25 mg/dl of acetone

Procedure

Take 5 ml of urine in a test tube and saturate it with ammonium sulphate.

Add a small crystal of sodium nitroprusside. Mix well.

Slowly run along the side of the test tube liquor ammonia to form a layer.

Observe the pink-purple ring at the interface

Interpretation

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No clor change / Brown ring Nil

Slight purple ring +

Moderate purple ring ++

Dark purple ring +++

4.2.2 Gerhardt’s test (Confirmatory test for acetoacetic acid)

Principle

Ferric chloride solution with acetoacetic acid to form a characteristic brown red precipitate.

Procedure

Take 3 ml urine into a test tube.

Add 3 % FeCl3 solution drop by drop. Mix while adding.

Filter the brown- red precipitate formed.

Add 3 % FeCl3 solution continuously to the filtrate until a purple color appear

Heat and see whether the color disappear or not

Interpretation

Positive – Purple color in 2nd step but on heating purple color disappear – Acetoacetic acid

Negative – Color less or purple color in 2nd step and color persis on heating - Salicylates

Discussion

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Discuss the principle of the sodium nitroprusside reaction, including sensitivity and possible
causes of interference.

Discuss the clinical significance of ketonuria.

4.3 Detectermination of protein in urine

Objectives

• To perform different type of tests to identify proteinuria

• 3% sulfosalicylic acid method

• Acid heat coagulation

• Strip method

• To understand the clinical significance of proteinuria

• To understand the false negative and false positive test reactions

4.3.1 3% sulfosalicylic acid method (SSA) – Confirmatory test for Protein

Principle

Also called as cold precipitation test. Negatively charged sulfosalicylic acid neutralizes the
positive charge on proteins causing denaturation. Due to less solubility protein precipitation
will occur.

Procedure

1. Add 3 ml of urine into a two-test tube

2. Label test tube as “test” and “negative control”

3. Add 3 ml of 3% sulfosalicylic acid to the test tube

4. Add 3 ml of water for control tube

5. Mix well

6. Immediately compare the test and Control by observation for turbidity

Turbidity indicates the presence of proteins. According to the protein concentration turbidity
will gradually change. Results can be interpret using a control chart.

Interpretation

No Cloudiness / Clear Negative

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Slight Cloudiness +

Moderate cloudiness ++

Marked cloudiness +++

Cloudiness with precipitate ++++

Discussion

Discuss the sulfosalicylic acid (SSA) test for urine protein, including interpretation and sources
of interference.

Acid Heat coagulation test (Preliminary test for proteins)

Principle

Albumin is coagulated when heated. Precipitate at the iso-electric point

Procedure

1. Fill 2/3rd of the test tube with urine

2. Add few drops of dilute acetic acid

3. Heat the top half of it

4. Lower part serve as control

5. Note the appearance of the turbidity

Note

Without adding acetic acid

Heat the top 1/3 of the urine in the test tube.

Observe the increase in turbidity in the upper portion to the lower unheated part. This indicates
the presence of proteins and phosphates. Turbidity due to phosphate dissolves with the addition
of about three drops of acetic acid. Turbidity remaining indicates the presence of proteins.

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Discussion

Explain the reason for adding few drops of acetic acid to the urine sample

This test is used as a screening test to identify Bence Jones Protein. Explain the method of
identification and clinical significance of the test.

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Practical 05
Urine Full Report 4
5 Practical 05: Urine Full Report 4
5.1 Determination of bilirubin (bile pigments) in urine
Introduction

Bilirubin in not normally decreased in the urine. When it is found, the condition is referred to
as “bilirubinuria”. Urine containing 8.4 µmol/L or more of bilirubin has a characteristic yellow
brown color.

5.1.1 Fouchet’s test

Principle

Barium chloride is used to precipitate the sulphates in the urine. Any bilirubin present becomes
attached to the precipitated barium chloride. When fouchet’s reagents is added to the
precipitate, the iron III (ferric) chloride oxidizes the bilirubin to green bilirubin.

Procedure

1. Check the pH of urine. If alkaline, acidify with diluted acetic acid.

2. Add 5 ml of 10% BaCl2 solution to 10 ml urine in a test tube.
3. Mix and observe for the presence of a precipitate
4. Fliter through a whatman No:1 filter paper.
5. Dry filter paper over placing it on a second filter paper
6. Add a drop of fouchet’s reagents to the filtered precipitate
7. Observe the color

Interpretation

Green color – Positive

Discussion

What are the precautions should be followed during fouchet’s test?

Discuss the clinical significance of bilirubinuria

Discuss the principle of the fouchet’s test for urinary bilirubin, including possible sources of
error.

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5.2 Detection of urobilinogen in urine

Introduction

Urobilinogen which is a reduced form of bilirubin is normally present only in trace amounts in
the urine of a healthy person. The measurement of the amount of urobilinogen in urine is a
good index for assessing the stages of Jaundice and monitoring impairment of liver function.

5.2.1 Ehrlich’s test

Principle

Urobilinogen reacts with p-dimethylaminobenzaldehyde to form a red condensation product.

Procedure

Add 0.5 ml Ehrlich’s reagent to freshly voided 2 ml urine.

Mix the contents and leave for 3-5 min at room temperature.

If no red color is produced warm the contents to 50oC

5.3 Detection of bile salts in urine

Introduction

The hepatocytes synthesis bile salts from cholesterol which is either newly synthesized in the
liver or derived from plasma lipids. Two primary bile acids are formed, cholic and
chemodeoxycholic, which are the conjugated with glycine or taurine to for corresponding bile
salts.

5.3.1 Hay’s test

Principe

Bile salts posses the unusual property of lowering the surface tension of a solution markedly
even when present in small concentrations.

Procedure

1. Sprinkle small amount of sulphur powder lightly on the surface of urine in a test tube.

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Interpretation

Sulphur powder sinks to the bottom – Positive

Sulphur powder keeps floating - Negative

5.4 Urine strip method

Objectives

▪ State the proper care and storage of commercial reagent strip

▪ Summarize the clinical significance of the following substances and describe the
chemical principles

pH Specific gravity

Protein Glucose

Ketones Blood

Bilirubin Urobilinogen

nitrite leukocytes

▪ Compare and contrast the sensitivity, specificity, and potential interferences of

commercial reagent strip and manual method

Introduction

Reagent strips consist of chemical-impregnated absorbent pads attached to a plastic strip.

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Absorbent pad comes in contact with urine – color producing chemical reaction takes place.
Interpretation will be done by comparing the color produced on the pad with a chart supplied
by the manufacturer

Procedure

1. Dip the reagent strip completely, briefly, into a well-mixed specimen

2. Remove excess urine from the strip by running the edge of the strip on the container
when withdrawing it from the specimen

3. Blot the edge of the strip on a disposable absorbent pad

4. Wait specified length of time for reactions to take place

5. Comparing the colored reactions against the manufacturer’s chart using a good light
source

Discussion

State the proper care and storage of commercial reagent strip

Compare and contrast the sensitivity, specificity, and potential interferences of commercial
reagent strip and manual method

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Practical 06
Standard curve preparation
6 Practical 06: Standard Curve Preparation
Objectives,

• Prepare a dilution series by using given methods

• Measure absorbance using the spectrophotometer

• Draw a standard curve

Principle

Principles of spectrophotometer is Beer Lambert’s low

Beer’s law

Amount of transmitted light decreases exponentially with an increase in the concentration of

the absorbing molecules

Lambert’s law

Transmitted light decreases exponentially with the thickness of the absorbing molecule
(Thickness of the medium)

It states that the absorbance of light by a material in a solution is directly proportional to

its concentration in that solution.

A = ϵlc

Where, A – absorbance, ϵ - molar absorptivity, l – length of solution, c – concentration

6.1 Preparation of dilution series

Preparation of dilution series using double dilution method (dependent method)

1. Five glass tubes were taken and they were labelled as S1, S2, S3, S4 and S5
2. 1 ml of distilled water was added to each tube expect S1 tube
3. 2 ml of 200 mg/dl standard glucose solution was transferred to S1 tube
4. 1 ml of 200 mg/dl standard transferred to S2 tube from S1. It was mixed well
5. 1 ml of diluted standard transferred to S3 tube from S2. It was mixed well
6. 1 ml of diluted standard transferred to S4 tube from S3. It was mixed well
7. Measure the concentration in each tube

Preparation of dilution series using simple dilution method (independent method)

1. Six glass tubes were taken and they were labelled as S1, S2, S3, S4, S5 and S6
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 Clinical Biochemsitry Practical Manual 31

2. Mix the solution according to the following table

S1 S2 S3 S4 S5 S6

Working standard 50 100 200 300 400 500

concentration
5.55mmol/l (µl)

Distilled water (µl) 1950 1900 1800 1700 1600 1500

Final concentration
mmol/l

6.2 Preparation of standard curve

1. Six glass tubes were taken and they were labelled as S1, S2, S3, S4, S5 and S6
2. Mix the solution according to the following table

Blank S1 S2 S3 S4 S5 S6

Working reagent (µl) 1000 1000 1000 1000 1000 1000 1000

Distilled water (µl) 10

S1 (µl) 0 10

S2 (µl) 4 10

S3 (µl) 10

S4 (µl) 10

S5 (µl) 10

S6 (µl) 10

3. Plugged the Cotton plugs for all 6 tubes and incubate for 10 minutes at 37oC.
4. Measure the absorbance of each samples against 500 nm using reagent blank using a
spectrophotometer

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 Clinical Biochemsitry Practical Manual 32

5. Plot a graph, absorbance Vs Concentration of the samples.

Discussion

Compare and contrast the two-dilution series method.

Discuss the importance of standard curve.

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 Clinical Biochemsitry Practical Manual 33

Practical 07
Plasma glucose test
7 Practical 07: Plasma glucose test
Introduction

The glucose level in blood is maintained within a narrow range (60-100 mg/dl) under diverse
conditions (feeding, fasting, or sever exercise) by the regulatory hormones (insulin, glucagon,
epinephrine).

The most frequently encounter disorder of carbohydrate metabolism in blood is Diabetes

Mellitus (Hyperglycemia). Ketoacidosis and hyperosmolar coma may be caused if plasma
sugar increased more than 300 mg/dl.

If plasma glucose level lower than 50 mg/dl consider as hypoglycemia. If lower than 30 mg/dl
plasma glucose level persist for long period, it may cause irreversible encephalic damage.

Principle

Glucose oxidases catalyze the oxidation of glucose to gluconic acid. The formed hydrogen
peroxide is detected by a chromogenic oxygen acceptor, phenol-aminophenazone in the
presence of peroxidase.

Procedure

1. Let stand reagents and specimens at room temperature

2. Mix the reagents as given

Pipette into well identified test tubes Blank Standard Assay

Reagent 1 mL 1 mL 1 mL

Demineralized water 10µ

Standard 10µ

Specimen 10µ

3. Mix well. Let standard for 10 minutes at 37oC or 20 minutes at room temperature
4. Read absorbance at 510 nm against reagent blank

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 Clinical Biochemsitry Practical Manual 34

5. Calculate the glucose concentration by using beer and lambert law.

Discussion

State the interpretation procedure in fasting blood glucose test, random glucose test and 2-hour
postprandial glucose tolerance test.

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 Clinical Biochemsitry Practical Manual 35

Practical 08
Oral Glucose tolerance test
8 Practical 08: Oral Glucose Tolerance test
Principle:

This test is used to measure the maximum amount of glucose which can be tolerate by the cells.
This is also used to identify the efficiency of insulin and the activity of β cells of pancreas as
well as glucose absorption of the gut.

Procedure:

1. Add reagents and samples as follows,

QC QC
fasting 1h 2h Blank
(normal) (path)

Glucose reagent
1000 1000 1000 1000 1000 1000
(μl) 100 mg/dl

Samples (μl) 10 (distilled

10 10 10 10 10
water)

2. Incubate all tubes at 37oC for 10 minutes. Perform intermittent mixing.

3. Measure the absorbance against 510 nm wave length in spectrophotometer.

Calculation

Beer Lambert’s law,

Absorbance α concentration
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡𝑒𝑠𝑡 = × 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑

Interpretation

Condition Fasting 1st hour 2nd hour

Less than renal

Normal <100mg/dl <140 mg/dl
threshold level

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 Clinical Biochemsitry Practical Manual 36

Impaired 100 -125 mg/dl indefinitive 140-200 mg/dl

Exceed renal
Diabetes >126 mg/dl >200mg/dl
threshold level

More than renal

Gut abnormality <100 mg/dl <140 mg/dl
threshold level

No significance No significance
Hypoglycemia <100mg/dl
increase increase

Normal pregnancy <150 mg/dl <190mg/dl <165 mg/dl

Test sample

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 Clinical Biochemsitry Practical Manual 37

Practical 09
Lipid Profile
9 Practical 09: Lipid Profile
Introduction
A group of tests that measure the levels of different types of lipids in the blood.

Lipid profile is done to,

• To screen for suspected heart Disease

• To monitor the efficacy

• Patients with risk factors

• To assess the risk of heart disease

• Patients whose plasma is seen to be lipaemic

9.1 Total Cholesterol

• The reagents typically use a bacterial cholesteroll ester hydrolase to cleave cholesterol
esters

𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 𝑒𝑠𝑡𝑟𝑒𝑟𝑠 + 𝐻2𝑂 →→→ 𝑐ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 + 𝐹𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑𝑠

• The 3-OH group of cholesterol is then oxidized to a ketone in an oxygen – requiring

reaction catalyzed by cholesterol oxidase.

𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙 + 𝑂2 →→→ 𝑐ℎ𝑜𝑙𝑒𝑠 − 4 − 𝑒𝑛 − 3 − 𝑜𝑛𝑒 + 𝐻2𝑂2

Procedure

Mix the solution

Blank S1 S2 QCN QCN QCP QCP T1 T2

1 2 1 2

Working 1000 1000 1000 1000 1000 1000 1000 1000 1000
Solution (µl)

Distilled 10
water(µl)

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 Clinical Biochemsitry Practical Manual 38

Standard 10 10
(TC) (µl) –
200 mg/dl

QC normal 10 10
(µl)

QC path (µl) 10 10

Sample (µl) 10 10

Absorbance

9.2 Triglycerides
First step is the lipase – catalysed hydrolysis of triglycerides to glycerol and fatty acids

𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠 + 𝐻2𝑂 →→→→→ 𝐺𝑙𝑦𝑐𝑒𝑟𝑜𝑙 + 3 𝑓𝑎𝑡𝑡𝑦 𝑎𝑐𝑖𝑑𝑠

Glycerol is phosphorylated in an ATP acquiring reaction catalyzed by glycerokinase

𝐺𝑙𝑦𝑐𝑒𝑟𝑜𝑙 + 𝐴𝑇𝑃 →→→→→ 𝐺𝑙𝑦𝑐𝑒𝑟𝑜𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐴𝐷𝑃

Glycerophosphate is oxidized to dihydroxyacetone and H2O2 in a glycerophosphate oxidase-

catalyzed reaction

𝐺𝑙𝑦𝑐𝑒𝑟𝑜𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝑂2 →→→→→ 𝐷𝑖ℎ𝑦𝑑𝑟𝑜𝑥𝑦𝑎𝑐𝑒𝑡𝑜𝑛𝑒 + 𝐻2𝑂2

Specific

Do not detect glucose or phospholipids

Linea in the concentration range up to 700 mg/dl

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 Clinical Biochemsitry Practical Manual 39

Blank S1 S2 QCN QCN QCP QCP T1 T2

1 2 1 2

Working 1000 1000 1000 1000 1000 1000 1000 1000 1000
Solution
(µl)

Distilled 10
water(µl)

Standard 10 10
(TG) (µl) –
200 mg/dl

QC normal 10 10
(µl)

QC path 10 10
(µl)

Sample (µl) 10 10

Absorbance 0 0.352 0.249 0.120 0.114 0.181 0.171 0.132 0.125

9.3 HDL – C
Concentration of cholesterol associated with HDL is determined after non – HDL lipoproteins.

Physically removed. Effectively masked – Automated

Non HDL, - Precipitate with polyanions

• VLDL

• IDL

• Lipoprotein (a)

• LDL

• Chylomicrons

Treat serum sample with polyanion – divalent cation combinations

Centrifuge the sample
Remove the precipitate
Measure the cholesterol present in supernatant

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 Clinical Biochemsitry Practical Manual 40

HDL assays are considered inaccurate in samples contain triglycerides concentrations above
400 mg/dl
To minimize this error, samples should be pretreated to remove or reduce interference by
triglyceride-rich lipoproteins

• Centrifugation

• Filtration

• Dilution

Procedure

QCN 1 QCN 2 QCP 1 QCP 2 T1 T2

HCL 1000 1000 1000 1000 1000 1000

precipitant
(µl)

QC normal 500 500

(µl)

QC path (µl) 500 500

Sample (µl) 500 500

1. Reagents and samples were added as above table and mixed vigorously.
2. All tubes were kept in room temperature for 10 minutes
3. Centrifuge all tubes at 4000 rpm for 10 minutes.
4. Supernatant were separated

Blank S1 S2 QCN QCN QCP QCP T1 T2

1 2 1 2

Working 1000 1000 1000 1000 1000 1000 1000 1000 1000
Solution
(µl)

Distilled 100
water(µl)

Standard 100 100

(HDL) (µl)
– 50 mg/dl

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 Clinical Biochemsitry Practical Manual 41

QC normal 100 100

(µl)

QC path 100 100

(µl)

Sample (µl) 100 100

Absorbance 0 1.779 1.697 0.517 0.503 0.742 0.794 0.539 0.537

9.4 LDL – C
Manual – Determined indirectly

Indirect method

“ Friedewald equation

mg/dl
[𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠]
𝐿𝐷𝐿 − 𝐶 = [𝑇𝑜𝑡𝑎𝑙 𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙] − [𝐻𝐷𝐿 − 𝐶] −
5
[𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠]
[𝑉𝐿𝐷𝐿] =
5
mmol/L
[𝑇𝑟𝑖𝑔𝑙𝑦𝑐𝑒𝑟𝑖𝑑𝑒𝑠]
𝐿𝐷𝐿 − 𝐶 = [𝑇𝑜𝑡𝑎𝑙 𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙] − [𝐻𝐷𝐿 − 𝐶] −
2.22
𝐿𝐷𝐿 − 𝐶 = [𝑇𝑜𝑡𝑎𝑙 𝐶ℎ𝑜𝑙𝑒𝑠𝑡𝑒𝑟𝑜𝑙] − [𝐻𝐷𝐿 − 𝐶] − [𝑉𝐿𝐷𝐿 − 𝐶]

Discussion

Prepare a lipid profile.

Interpret the test results.

Discuss the limitation is lipid profile

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 Clinical Biochemsitry Practical Manual 42

Practical 10
Internal quality control in chemical pathology laboratory
10 Practical 10: Internal quality control in chemical pathology laboratory
Under optimal conditions, perform 20 measurements on the same control serum. Follow the
method as carefully as possible using the same reagents and equipment for each of the 20
measurements.
Calculate the mean (average value) by adding up all the results and dividing the total by 20.
For each result, work out the difference in value from the mean.
Multiply each difference value by itself to give the squared difference values
Add squared difference, to give the sum of the squared differences
Calculate what is called the standard deviation (SD) using the formula

Calculate the optimal CV using the formula

Check whether the optimal CV is below the maximum allowed for the particular test

Using above data draw levy Jennings chart.

Question 01
Measured glucose concentration (mmol/L) for normal QC data in Laboratory A
1 6.5 11 6.8

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 Clinical Biochemsitry Practical Manual 43

2 6.7 12 6.5

3 6.5 13 6.7

4 6.6 14 6.9

5 6.8 15 6.6

6 6.9 16 6.8

7 6.5 17 6.7

8 6.7 18 6.5

9 6.8 19 6.7

10 6.4 20 6.8

Question 02
Measured glucose concentration (mg/dl) for normal QC data in Laboratory B
1 96.27 1’ 96.27

2 93.12 2’ 92.49

3 99.01 3’ 92.17

4 102.50 4’ 101.32

5 98.48 5’ 100.37

6 93.43 6’ 101.32

7 92.80 7’ 99.01

8 95.33 8’ 99.43

9 97.85 9’ 104.16

10 106.03 10’ 107.95

Measured glucose concentration (mg/dl) for pathological QC data in Laboratory B

1 290.72 1’ 258.20

2 267.04 2’ 274.62

3 289.45 3’ 255.05

4 268.30 4’ 271.77

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 Clinical Biochemsitry Practical Manual 44

5 268.90 5’ 266.09

6 250.94 6’ 260.09

7 258.52 7’ 258.84

8 254.44 8’ 268.62

9 257.6 9’ 243.0

10 262.9 10’ 270.8

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 Clinical Biochemsitry Practical Manual 45

Practical 11
Serum enzymology
11 Practical 11: Serum enzymology
Introduction

Enzymes are the protein catalysts that increase the rate of specific chemical reactions in the
body. Enzymes are found within cells. Injury or death of tissues can cause the release of tissue
specific enzymes into the blood stream. Elevated enzymes levels/ enzyme activity are often
indicators of tissue problems and are used in the diagnosis of disease.

The selection of which enzyme to measure in serum for diagnostic or prognostic purposes
depends on a number of factors. One of the properties of enzymes that make them so important
as diagnostic tool is the distribution of enzymes among various tissues and the specificity, they
exhibit relative to the reactions they catalyse. Greater specificity is achieved in three ways,

Interpreting investigations of clinical features

Test pattern recognition

Isoenzyme determination

Enzymes of clinical interest

• Alanine transaminase
• Alkaline phosphatase
• Amylase
• Aspartate transaminase
• Creatinine kinase
• Gamma- glutamyl transferase
• Lactase dehydrogenase
• Lipase
• 5’- Nucleotidase
• Trypsin
11.1 Estimation of serum transaminases activity
Introduction

Transaminases are widely distributed throughout the body. Aspartate transaminase (AST) is
found primary in the heart, liver, skeletal muscle and kidney. Alanine transaminase (ALT) is
found primarily in the liver and kidney.

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 Clinical Biochemsitry Practical Manual 46

Liver disease is the most important cause of increased transaminase activity in serum. ALT is
the more liver-specific enzyme. N most types of liver disease, ALT activity is higher than that
of AST.

11.1.1 Estimation of serum ALT/ GPT) activity

Principle

𝐿 𝐴𝑙𝑎𝑛𝑖𝑛𝑒 + 2 − 𝑜𝑥𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 ↔ 𝐿 − 𝐺𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 + 𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒

𝑃𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 + ↔ 𝐷 − 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷+

Procedure

1. Add 100 µL of serum into 1000 µL of Reagent 1 (Tris, L-Alanine, LDH).

2. Mix and incubate for 5 min at 37oC.
3. Add 250 µLof Reagent 2 (Oxoglutarate + NADH) into the sample mixture and mix
4. Read absorbance at 340 m, after 1 min and start stopwatch.
5. Read absorbance again 1, 2 and 3 min thereafter.

Calculation

From absorbance readings calculate ∆A/min and multiply by 2143.

ALT activity (U/L) = ∆A/min X Factor

Discussion

Explain the importance of measuring ALT in disease diagnosis

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 Clinical Biochemsitry Practical Manual 47

Practical 12
Urine pregnancy test
12 Practical 12: Urine pregnancy test
Introduction

One – step pregnancy test

Human chorionic gonadotropin (hCG) is a glycoprotein hormone produced by the developing

placenta shortly after fertilization. In normal pregnancy, hCG can be detected in both urine
and serum as early as 7 to 10 days after conception. The appearance of hCG in both urine and
serum soon after conception, and its subsequent rapid rise in concentration during early
gestational growth, make it an excellent marker for the early detection of pregnancy. hCG can
be used as a tumor marker as its beta subunit is secreted by some cancer including hydatidiform
mole formation, testicular cancer.

Principle

Rapid chromatographic immunoassay - Two-site sandwich immunoassay

Qualitative detection of human chorionic gonadotropin (hCG) in urine

Three zones in the reagent strip,

Reaction zone –soluble anti hCG antibody-enzyme

–Mouse monoclonal antibodies linked to an enzyme

Test Zone –immobilized polyclonal mixture of hCG antibody-dye substrate

Control Zone –the dry substrate + anti-(anti hCG Ab Enzyme conjugates) Ab

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 Clinical Biochemsitry Practical Manual 48

Procedure

1. Allow the test strip, urine specimen to RT prior to testing

2. Bring the pouch to RT before opening it
3. Remove the test strip from the sealed pouch and use it as soon as possible
4. With arrows pointing toward the urine specimen
5. Immerse the test strip vertically in the urine specimen for at least 5 seconds

Do not pass the maximum line on the test strip when immersing the strip. Place the strip on a
non-absorbed flat surface, start the timer and wait for the red lines to appear. The results should
be read at 3 minutes.

Interpretation

Positive –
–Two distinct red lines appear
–One line should be in control and another should be in the test region
Negative
–One redlines appears in the control region
–No apparent red or pink line appears in the test region
Invalid
–Control lines fails to appear
–Insufficient specimen volume or incorrect procedural techniques
Discussion

Discuss the precautions should follow during the testing procedure

Explain the principle behind the chCG pregnancy strip test

Explain the pre-analytical errors occur in this test method

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