INTRODUCTION

Training/Internship is an integral part of the program in Laboratory Medicine and is designed to

provide interns with an opportunity to integrate and apply previously acquired knowledge and

technical skills in actual clinical settings. Under the guidance of experienced Medical Laboratory

Professionals and other qualified laboratory personnel and health professionals, interns learn more

about diagnostic test procedures, quality control methods and programs, and instrumentation in the

clinical laboratory. They also gain an understanding of the roles and functions of the Medical

Laboratory Professionals.

The training/internship provides applied learning experiences during which the intern should:

1. Practice and acquire clinical laboratory skills

2. Practice skills in problem-solving

3. Perform quality control procedures

4. Learn and adapt new procedures

5. Operate and maintain various laboratory machines and instruments

6. Understand the responsibilities, roles, and functions of the Medical Laboratory Professionals

7. Report accurate and precise results to supervisors8. Learn how to correlate tests results to patient

clinical diagnosis.

The internship program is conducted in the affiliated hospital laboratories of the program, where

interns learn by participating in the workload of a supervising technologist/specialist/consultant.

Emphasis in each internship discipline is given on: a) organization of work, b) use of automated

instrumentation, c) the relation of laboratory results to patient diagnosis, and d) the establishment and

use of programs for quality control and preventive maintenance of laboratory instruments.

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 1st Semester: Fun. English, Sc & Society, Env. Health, Comm. Healthcare, HumanAnatomy.

2nd Semester: HumanPhysiology,Biochemistry&BioPhysics,MedicalEntomology&

Parasitology.

3rd Semester: Hematology, Clinical Immunology & Serology.

4th Semester: Clinical Pathology, Clinical Biochemistry, Histotechnology &cytotechnology..

5thSemester:ClinicalEndocrinology&Andrology,ClinicalMicrobiology,Bloodtransfusion

& Blood Bank.

6th Semester: Modern Biomedical Instrumentation, Computer Application including Ms

Office, Dissertation Project.

With the introduction I now proceed to state briefly work done by me during 6 months internship

training at .........................................................................................................................

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 AIMS & OBJECTIVES

The basic purpose of internship training are expending our knowledge about the subject along with
other ancillary matters like.
 To gain knowledge about the patient handling and how to give instructions to the patient
before the collection of sample.
 To gain knowledge about collection, preservation, testing process and handling individual
sample according to the disease condition of the patient.
 To learn the advance techniques of independent approach, manners as well as
development of personal skill.
 To learn the principle operation techniques to use different modern and sophisticated
instruments used for human health and health related disorders.
To learn at the time of test how to handle different hazardous and non-hazardous sample and after
how to dispose such type of samples.

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 Department of Microbiology

 Sterilization and disinfection

 Media preparation

 Collection of specimen

 Bacterial identification (Staining technique)

 Bio-chemical tests

 Features of pathogenic bacteria

 Antibiotic sensitivity test

 Serological tests

 Parasitological test ( Stool examination )

 Mycological test (Fungal examination )

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  STERILISATION AND DISINFECTION

Sterilizationis defined as the process where all the living microorganisms, including bacterial
spores are killed. Sterilization can be achieved by physical, chemical and physiochemical means.

Disinfection is the process of elimination of most pathogenic microorganisms (excluding bacterial

spores) on inanimate objects. Disinfection can be achieved by physical or chemical methods.
Chemicals used in disinfection are called disinfectants. Different disinfectants have different target
ranges, not all disinfectants can kill all microorganisms. Some methods of disinfection such as
filtration do not kill bacteria, they separate them out. Sterilization is an absolute condition while
disinfection is not. The two are not synonymous.

Decontaminationis the process of removal of contaminating pathogenic microorganisms from the

articles by a process of sterilization or disinfection. It is the use of physical or chemical means to
remove, inactivate, or destroy living organisms on a surface so that the organisms are no longer
infectious.

Sanitization is the process of chemical or mechanical cleansing, applicable in public health

systems. Usually used by the food industry. It reduces microbes on eating utensils to safe, acceptable
levels for public health.

Asepsisis the employment of techniques (such as usage of gloves, air filters, UV rays ETC) to
achieve microbe-free environment.

Bactericidal is that chemical that can kill or inactivate bacteria. Such chemicals may be called
variously depending on the spectrum of activity, such as bactericidal, virucidal, fungicidal,
microbicidal, sporicidal, tuberculocidal or germicidal.

Antibiotics are substances produced by one microbe that inhibits or kills another microbe. Often
the term is used more generally to include synthetic and semi-synthetic antimicrobial agents.

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Flow chart sterilization and disinfection

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  MEDIA PREPARATION

 MEDIA PREPARATION: Media are important ingredients for a laboratory to

initiate growth of micro-organism for the purpose of diagnosis, research etc. So
preparation of media is a vital part of laboratory activities.
 DIFFERENT TYPES OF MEDIA AND THEIRPREPARATION:
 MacConkey’s Agar Media:
 Distilled water ------ 100 ml
 Peptone ------ 2 gm
 Sodium chloride ------ 0.5 gm
 SodiumTaurochoate ------ 0.5%
 Bile salt ------ 0.5 gm
 Agar- Agar powder ------ 2.5gm
Heat he mixture in a water bath to make a homogeneous solution and then check the pH
of the solution. Then autoclave at 15 lbs pressure for 30 minutes.
Add 1% Lactose and 1% Neutral red solution (indicator) to the 1 st part of MacConkey’s
Agar and then steam for 30 minutes in an autoclave. Finally pour the solution in sterile
petridis and after 15 minutes keep them in a refrigerator.

 Nutrient Agar Media :

 Distilledwater ------ 100 ml
 Peptone ------ 1 gm (%)
 Sodiumchloride ------ 0.5 gm (%)
 Beef heartextract ------ 1gm (%)
 Agar-Agar ------ 2.5gm (%)
Heat is done to make a homogeneous solution. Check the pH of the solution 7.6 and then
autoclave it at 15 lb pressure for 30 minutes. Then pour it in a sterile petri-dish.

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  Nutrient broth Media:
It is a basic transport culture medium used in the preparation of blood agar and other
media.It is used to maintain cultures of control organism semi solid form and in the
solidform.

Composition :
 Distilledwater ------ 100 ml
 Peptone ------ 1 gm (%)
 Sodium Chloride ------ 0.5 gm (%)
 Beefextract ------ 1 gm
Heat to make a homogeneous solution. Check the pH of the solution 7.6. Then make the
tubes of 3 to 4 ml and then plug the tubes with cotton. Then autoclave at 15 lbs pressurefor
30 minutes.
 Peptone water:
 ThisisacommonliquidmediumwhichisusedasabaseinsugarfermentationsandfortheIndoletest.Itc
anbeusedforseedcultureforantibioticsensitivitytestingbytheKirby-Bauermethod.

 Distilled water ------ 100 ml

 Peptone ------ 1 gm (%)
 Sodium Chloride ------ 0.5 gm (%)
Use water bath to make a homogeneous solution. Check the pH of the solution 7.6. Then
make the tubes of 3 to 4 ml and then plug the tubes with cotton. Then autoclave at 15 lbs
pressure for 30 minutes.
 Blood agar media:
It is ageneral purpose enriched and solid medium,which supports the growth of most
ordinary bacteria.Blood supplies a numberof substances for the growthof fastidious
organisms.
Composition :
 Sterile nutrientagar ------ 200 ml
 Steriledefibrinated sheep ------ 25 ml
blood

oProcedure: 1) The temperature of the nutrient agar should be 54 to 56 degree C.

2)Then add to it the sheep blood, which should be at room temperature. So the final
temperatureisshallnotbelow45degreeC.and the final maximum temperature48to52
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degree C. Then stir well. Heat the solution to make a homogeneous.3)Then pour it inapetri-
dish.Thenputtheplateinsideanincubatorfor24hrstochecktheagarplate for
growth (i.e. whether the plate contaminated ornot.)
 Muller Agar media:
Thismediumisusedfordiffusionmethodofanti-
microbialsusceptibilitytestingofbacteria(Kirby-BauerMethod).
composition
 Beef extract: 2.0g
 Acid Hydrolysate of casein: 17.5g
 Starch: 1.5g
 Agar: 17.0g
o Preparation:
1. Suspend 38 g of medium (or the components listed above) in 1 liter of purified water.

2. Mix thoroughly.

3. Heat with frequent agitation and boil for 1 minute to completely dissolve the
components.
4. Autoclave at 121°C for 15minutes.

5. Cool to 45°C.

6. Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface
to give uniform depth.
7. Allow to solidify at room temperature.

8. Check prepared Mueller Hinton Agar to ensure the final pH is 7.3 ±1 at25 degree c
9. Prepared media can be stored at 4 to 8°C. Mueller-Hinton agar is stable for
approximately 70 days from the date of preparation.

Sabouraud Dextrose agar :

Sabouraud Dextrose Agar (SDA) is used for the isolation, cultivation, and maintenance of
non-pathogenic and pathogenic species of fungi and yeasts .

Composition of SDA Agar:

Ingredients gm/liter

Dextrose 40.0
Peptone 10.0
Agar 15.0
Final pH ( at 25°C) (5.6±0.2)

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Preparation of SDA agar :

1. Suspend 65 g of the medium in one liter of distilled water.

2. Heat with frequent agitation and boil for one minute to completely dissolve the
medium.
3. Autoclave at 121° C for 15 minutes.
4. Cool to 45 to 50°C and pour into petri dishes or tubes for slants.

Sabouraud Dextrose chloramphenicol agar:

Sabouraud Dextrose chloramphenicol Agar (SDCA) is recommended for the isolation and the
enumeration of yeasts and molds, especially when the samples are highly contaminated with
bacteria. It is also used as selective isolation media for Candida albicans in cosmetic products.

COMPOSITION OF SDCA AGAR:

The composition can be adjusted in order to obtain optimal performance.
For 1 liter of media : -
Dextrose ................................................................................................................ 40,0 g
Pancreatic digest of animal tissues .......................................................................... 5,0 g
Pancreatic digest of casein ...................................................................................... 5,0 g
Chloramphenicol .................................................................................................... 50 mg
Bacteriological agar. ............................................................................................... 15,0 g

pH of the read-to-use media at 25 °C : 5,6 ± 0,2.

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  COLLECTION OF SPECIMEN
COLLECTION OFSPECIMEN:
 Sputum:
1. Before collect the sample the mouth should berinsed.
2. Always collect the first morning sample, it represents the pulmonary secretion
accumulated overnight.
3. In case of children nasopharyngeal swab may be taken which can be represents the
bronchialpathogens.
4. The sample must be taken in a cotton plug sterile test tube with cotton plug broom steak.
 Throat swab:

1. The sample is collect in the morning beforebrush.

2. Throat swab should be collected with a sterile swab from the tonsils tonsilar fossa,and

posterior pharyngealwall.

3. The sample must be taken in a cotton plug sterile test tube with cotton plug broom steak.

 Urine:

1. First morning urine should becollected.

2. Mid stream urine sample must becollected.

3. The sample should be collected ¾th of a sterile testtube.

 Pus:

1. Pus sample should be collected from the wounds of the patient, in a sterile cotton plug
test tube with a cotton plug broom steak.
 Stool:

1. First morning stool sample are collected in a sterile container.

N.B. – Beside this all fluid sample (ascetic fluid etc.) are collected in a sterile test tube.

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  INOCULATION OF THESAMPLE:
1. Pus, throat swab, sputum sample are must be inoculated in blood agar and MaC-
Conkey agar media both for confirmation of the growth of the bacteria (Streptococus
or staphylococcus).
2. The other sample like urine, ascetic fluid is inoculated in Mac-Conkey media (For
identify the growth of E. coli, klebsiella orProteus).
3. Stool sample must be inoculated in TCBS media for identify the growth of Vibrio
cholerae.
The method of the culture used in the laboratory are-

1. Streakmethod

2. Lawnmethod

3. Strokemethod

4. Stab method

Mainly streaking method are used in the laboratory.

Streakmethod:
Lift the bottom of the petri-dish containing the medium with the lef than and hold it round
the sidewith the thumb and middle finger. In most cases the method of plating inuse.
The inoculumissmeared over area Atogive well in oculumsor well.The loopisre sterilized
andthendrawnfromthewellintwoorthreeparallellinesontothefreshsurfaceofthemedium and then
continued. At each step the inoculums is derived from the most distal part of the strokes
which are precedingit.
When the inoculum is small or the medium is selective it can be more heavily inoculated.
Several loop full of the specimen are used to spread the wall (A). The loop is then resterilized
in a flame, recharged by rubbing it over area A and then used to seed the remainder the plate
by successive parallel strokes. Our main aim is to get discrete colonies. Sometimes when we

have to put more than one culture in a plate we the following procedure i. e. we divided the plate
into quadrants and use one quadrant for one sample.
 Incubation :
After inoculation the sample in media it is incubating in the incubator for 24 hrs at 37° C.

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Lawn plates Technique :

A lawn plate has the microbes spread on top of the agar with a sterile spreader so colonies will grow
only on the surface. Lawn plates are best made individually in class and can be used to show
microbial growth or testing anti-microbial chemicals.

lawn plate procedure :

1. Using aseptic techniques suck approximately 0.3ml of a liquid microbial culture into a sterile glass
or plastic pipette.
2. Re-flame the mouth of the bottle taking care not to squeeze the teat of the pipette and recap
3. Using an upright sterile plate, lift one side of the lid towards the Bunsen burner to reduce
contamination and squeeze in the liquid. Discard the pipette into the Virkon beaker and squeeze the
pipette to suck Virkon in and out.
4. Use a sterile spreader to spread the liquid culture over the surface of the agar. Alternatively a sterile
cotton wool bud can be dipped in liquid culture and swabbed over the surface of the agar.

Flow chart of lawn plate technique

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  BACTERIOLOGY
BACTERIALIDENTIFICATION:
After isolation of bacteria in pure culture from a specimen, it has to be identified. the
following studies are necessary to characteristic a bacterial strain.
A. Morphology of bacteria colony
B. Growth in liquid media
C. Staining
D. Hanging drop preparation
E. Biochemical test
F. Antigenic structure
G. Antibiotic sensitivity test.
Morphology of bacterial colony:
 Size… In mm (ex- pin head size is characteristic of staphylococci)
 Shape…. Circular, dome, irregular etc. (Ex. E. coli have dome shapedcolony)
 Surface…… Smooth, rough, granularetc.
 Elevation …. Colony may be flat , raised , low, convex ETC .
 Edge …..Entire, crenated, lobate, undulate etc.
 Colour… ......White, Golden, Pink etc. (Ex. E. coli have pink colour colony &Strepto
or Staphylococcus have white colour colony).
 Hemolysis …. Hemolysin produced in some bacteria leads to hemolysis .
 Consistensy …. Mucoid , friable , firm , butyroius .

GROWTH IN LIQUID MEDIA ;

Commonly used liquid media are peptone water and nutrient broth. Bacterial growth appears in
following forms:
(i) Uniform turbidity- Most of the Gram negative bacteria grow in this form.
(ii) Deposit at bottom- This occurs in growth of streptococci, chains of this bacteria being heavier
may settle down as deposit.
(iii) Surface pellicle formation- All aerobes have tendency to grow on surface of media due to more
content of oxygen present on the surface e.g. Pseudomonas sp.

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GRAM’SSTAINING:
 Principle:
When bacteria are stained some bacteria are got the primary stainand when they treated
with decolorizing agent they do not lose their original color. They are called the gram
positive bacteria and they are appeared in violet color because of Crystal Violet dye. Other
bacteria lose their primary color during decolourisation and got the counter stain at last time,
they arecalled the Gram Negative bacteria. They are appeared in pink color for the
counter stainSafranin.
 Reagentrequire:
 Methyl violet… 0.5% in 10cc distilled water.
 Gram’s iodine 1gm.
 Absolute alcohol…… Fo rdecolorizing
 Safranin… 5% in 100 cc distilled water.
 Procedure:

1. Fix the sample on the slide by heating the slide by passing over the flame for (2 to 3
times). The heating should not be over.
2. Methylvioletsolutionispouredovertheslideanditskeepfor1minute.Thenwashby
tapwater.
3. After that Gram’s iodine solution is poured over the slide. Then it is allow acting for 2
minutes.
4. Then add drop by drop absolute alcohol over the smear for decolorized the smear until
faint yellow.
5. Then counter stain by Safranin for 1minute.
6. Then again wash by tapwater.
7. Ten kept the slide into the slanting position for drying theslide.
8. After that the slide is observe under the microscope by using oil immersion objective.
If the violet color rods or cocci appear then it is the Gram Positivebacteria(e.g.
Staphylococcus, Streptococcus, Mycobacterium tuberculosis).
If the pink color rods or cocci appear then it is the Gram negative bacteria (e.g. E. coli,
Klebsiella, Neisseria).
 ACID FAST BACILLI STAIN OR THE ZIEHL-NEELSENSTAIN:

 Principle:Mycobacterium tuberculosis is very difficult to identify and staining the

organism as organism is coated with lipid containing cell wall. They bind carbolfuchsin
tightlyandresistrestainingwithstrongdecolorizingagentsuchasalcohol&strongacid. Acid
fast negative bacteria readily loss the stain when they treated with alcohol and strong
acid. Heat is applied in Ziehl Neelsen or stain method for the detection of
mycobacterium tuberculosis. Cold method detect mycobacterium leprae , Nocardia
Asteroids etc. All these methods used carbolfuchsin as the primary stain and phenol as
mordant. Following the counter stain by methylene blue or malachite green. De-
colorized acid fast, Negative bacilli & other cells kept blue colour in contrast with the
red colour acid fast bacilli.

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  ReagentRequired: 
1. (a) Carbolfuchsin
i) Basic fuchsin powder – 3gm
ii) Ethyl alcohol – (95%) – 100 ml
Mix it inmorter.

(b) 50% Phenol solution

Then mix the (a) solution 1 ml with 90 ml of (b) solution.

2. 25% sulphuricacid.
3. Counter stain by Methyleneblue.
i) Methylene blue – 0.3 gm
ii) Distilled water – 100ml

Procedure:
1. Collect the sample in a white sterile container. Then fix the sample on the slide by
heatingmethod.
2. Cover the slide with carbol fuchsin and heat until steama rises. Discontinue heating
and then re continue when steam stops arising. Discontinue heating and then re
continue when steam stops raising. Continue this process for a period of 5 minutes.
Stain must not be allowed to evaporate and dry. If necessary pour more carbol fucsin
to keep the whole slide covered withliquid.
3. Wash withwater.
4. Covertheslidewith25%SULPHURICACID.Keepforaminute.Washtheslidewith water
and pour or more acid. The dye is basic and its combination with acid produces a
compound which is yellowish brown. The aim of washing to remove the compound of
acid and stain and allow it to fresh acid to react with the preparation. Decolorisation
takes a total time of 1- minutes and it is said to be over when after washing, the film is
faintlypink.
5. Wash slide well withwater.
6. Treat slide with 95% alcohol for two minutes. Then wash withwater.
7. Counter stain with Loeffler’s Methylene Blue for 15 to 20seconds.
8. Wash dry. See in 10X (Oil immersion).
If the acid fast bacilli is seen then Mycobacterium Tuberculosis is present in the sample.

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 BIO- CHEMICALTESTS:
 INDOLETEST:
 Reagent :Kovacreagent
i) Para di methyle aminobenzaldehyde
ii) Amylalcohol
iii) Cons.HCL
 Principle:This is a test her bacteria breakdown protein or not.

 Procedure:Some bacteria are able to convert tryptophantoindole in presence of Kovas

reagent. If the test is positive a pink color ring is formed at the junction. E.g – E.coli.
 CITRATE TEST:This is a biochemical test to find out the bacteria which utilizes
citrate as sole of carbon source.
 Procedure:A positive test is indicated by the growth of bacteria and a deep color due to
the presence of indicator Bromothymol blue. E.g – Citobactor,Klebsiella.

 TRIPLE SUGAR IRON TEST(TSI):

 Principle:This is a biochemical test to know whether the bacterium utilizes both
lactose and glucose or only glucose.
 Procedure:In a test tube containing both lactose and glucose a loop of bacteria is
inserted. Then it is kept overnight incubation. The result may be of two types-
a) If only glucose is fermented yellow color is showed along with a black ring if H 2S
is present. This is to be represented as A/A +_H2S.
b) If only glucose is fermented pink color occurs in slant and yellow in bolt with a
black ring if H2S is present. This is to be represent as K/A +_H2S.
E.g. – Salmonella typhi, Proteus vulgaris, Proteus mirabilis.

 UREASETEST:
To find out presence of enzyme (Urease) in the bacteria. Urea convert to Ammonia and
CO2 in presence of indicator Phenol red turns into deep pink color. This is biochemical
test for Identifying bacteria. This test indicates wheter a bacterium contain enzyme Urease
or not. This is a positive urease test. E.g. – Klebsiella sp. Proteus sp.

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 OXIDASETEST:

Tetramethyl Paraphenyllene Diamine Di Hydrocholoride is the reagent of this test. This is

a test to find out whether a bacterium contain cytochrome oxidase. 1.1% solution of fresh
solutionofthisreagentismade.Takefilterpaperandpourthereagent,with a glass rod and
platinumloop.Wetakealoopfullofbacteriaandbreakitonfilterpaper.Apositivereaction
indicates a deep purple color in 10 seconds. E.g. – Pseudomonas, Vibrio,Neisseria.
 CATALASE TEST:

We take 3% of H2O2 in a test tube and mix bacteria full loop in it. The development of
bubbles on the slide indicates the positive catalase test. E.g. – Staphylococcus aureus.
 COAGULASE TEST: Pick up a few colonies of the bacteria from an agar culture and
emulsify in two drops of saline placed on a slide/ tube. If the coagulation appear then it
indicates the positive coagulase test. E.g. – Staphylococcusaureus.

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  FEATURE OF SOME COMMON PATHOGENICBACTERIA:
 Escherechiacoli.:This genus was named after Escherichia who was the first to describe
the colon bacilli. E. coli lives in the human or animal intestine. When it is voided in faeces
it remain in the environment for some days. Detection of E coli in drinking water is taken
as evidence of recent pollution with human or animal faeces.
 Morphology:E.coli is a Gram negative,straightrod.It is motile by peritrichate flagella
through.Somestrainmaybenon-motile.Capsuleandfimbriaearefoundinsomestrain. Spores
are not formed. Many strains have a combination of character. This is because of
conjugation and transduction between bacterial strains.
 Pathogenicity: Escherichia coli is associated with four disease-
 Urinary tract infection.
 Diarrhea/Gastroenteritis.
 Pyogenic infection
 Septicemia.
Klebsiella sp.:The genus Klebsiella consist of no motile, capsulated rods which grow very
well on ordinary media forming large dome shaped mucoid colonies which are sticky.
Klebsiellaarewidelydistributedinnatureoccurringbothascommensalsin intestineandas saprophytes
in soil and water. They are classified into threespecies.
 Klebsiella pneumoniae.
 Klebsiella ozaenae
 Klebsiella rhinoscleromatis.
 Diseaseproduce: 
Pneumonia – Klebsiella pneumonia is a rare disease and occur with some predisposing such
as-
 Middle ofage
 Diabetes
 Alcoholism
 Chronic bronchopulmonary disease.
Theoutcomeispoorcasefatalityis80%.Thereis involvement of one or more lobes of the lung
and massive mucoid inflammatory exudates. Necrosis and abscess formation is more
common than pneumococcal pneumonia.

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 Urinary tract infection: Klebsiella is a common cause of UTI and they a red resistant
to most antibiotics. It also causes pyogenic infection such as abscess, meningitis and
septicemia. Rarely it can cause be o fdiarrhea.

 Proteus:
Proteus is Gram negative bacilli. They are called proteus after the greek God Proteus who
could assume any phase. Proteus is characterized by spreading growth on agar.
 Morphology : Pleomorphism exist with long filaments and granular forms. They
have petricheat flagella and exhibit swimming motility, best seen at 20 degree C. They
are non-capsulated andnon sporing.
 Pathogenicity:Proteus species are saprophytes and are found everywhere. They occur
as commensals in the intestine and sometimes on the skin over the moist parts of the
body. They are opportunist pathogens and may cause many types of infection. They are
known to cause hospital acquired infection especially in burn. Urinary tract infection is
the commonest disease caused by proteus. It also produce pyogenic lesion of variety
types like abscess and infection of wounds, ear or respiratorytract.
 Staphylococcusaureus:
 Morphology: Staphylcoccus aureus are Gram positive spherical cocci, 1 micrometer in
the diameter, arranged in grape like clustur due to cell division occurring in three planes
with daughter cell tending to remain in close contact after cell division. They may be
found singly, in pair or in short chains or 3 or 4 cells. They are non-motile, nonsporing
andnon capsulated.
 Pathogenicity:Staphylococcus aureus causes manyinfections-
 Cutaneousinfection
 Deepinfection
 Staphylococcus enterocolitis
 Staphylococcal food poisoning
 Various exfoliative syndrome
 Toxic Shock Syndrome

 Staphylococcus Pyogens:
 Morphology:They are spherical or oval cells,0.5to1.0micron in diameter,arranged in
chain. The chain formation is due to the cocci dividing in one plane and the daughter
cells failing to separate completely. They are non motile and nonsporing. Some strains
haveacapsulemadecapofhyaluronicacid.Capsulesarebestseeninveryyoungculture.
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Culture: Streptococcus pyogenes is an aerobe and a facultative anaerobe. Grows best at
temperatures of 37 degree C. It is fastidious.
 THE CULTURE MEDIA USED FOR THE GROWTH OF STREPTOCOCCUS ARE:

1. Blood agar:After incubation of 24 hours the colonies are small (0.5 to 1 mm), circular,
semitransparent, low convex discs with an area of clear hemolysis are promoted by 10%
CO2.
2. Selective medium like crystal violet blood agar and PNF horse blood agar are used when
we wanted to inhibit many throat commensallyorganism.
3. Crystal violet blood agar:The addition of low concentration of crystal violet (1 in 50000
or 0.002%) to blood agar inhibits the growth of some bacteria e. g.staphylococci.
 Pathogenicity: Streptococcus pyogenes produces pyogenic infection with tendency
to spread locally along lymphatics and through bloodstream.
 Respiratoryinfection.
 Skin infection
 Genital infection
 Other superlative infection
 Non superlative infection
 Glomerulonephritis.
 SENSITIVITY TEST (ANTIBIOTICSTEST):

After identifying the bacteria we must have done the antibiotic test. It is very important thing
for the treatment. Antibiotic discs are used to detect the bacterial activity of the sample by
using Muller Hinton Agar.

Procedure:

1. At first the prepared Muller Hilton agar in a petri dish is taken.

2. Then the antibiotics disc are taken and bring it to the room temperature.
3. Thenpickupacolonyfromthegrowthbyaloopandputitinapreparednutrientbroth,then keep in
an incubator for 1 hour.
4. ThenbacteriamixednutrientbrothpouredontheMolarHiltonagarmediaandallowdrying.
5. After drying the media the antibiotics discs are put on the media one by one linewise Then
the resistant area is observed for thereport.

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The list of the antibiotics for different types of the sample purpose----
Gram Negative Bacteria:- Gram Positive Bacteria:-

Nitrofurantoin(NIT)- For Urine Vancomycin(VA)

Amikacin(AK) Linezolid(LZ)

Piperacillin- Tazobactam(PIT) Teicoplanin(TEI)

Cefepime(CPM) Levofloxacin(LE)

Gentamycin(GEN)(10) Clindamycin()

Levofloxacin(LE) Erythromycin(E)- Without Urine

Imipenem(IPM) Azithromycin(AZM)- Without Urine

Cefotaxime(CTX) Amikacin(AK)

Polymyxin –B(PB) Gentamycin(GEN)(10)

Enterococcus:- Pseudomonas:-

Vancomycin(VA) Ceftazidime(CAZ)- Incase of positive

Linezolid(LZ) Aztreonam(AO/AT)

Teicoplanin(TEI) Imipenem(IMP)

Levofloxacin(LE) Polymyxin- B(PB)

Gentamycin (HLG)(120) Piperacillin- Tazobactam(PIT)

Chloramphenicol(C) Levofloxacilin(LE)

Tetracycline(T) Amikacin(AK)

Ampicillin(AMP) Gentamycin(GEM)(10)

Nitrofurantoin(NIT)- For Urine

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 SEROLOGY

Serology definition :
the scientific study or diagnostic examination of blood serum, especially with regard to the response
of the immune system pathogen or introduced substances.

Antigen Antibody reaction :Antigen-antibody reaction or antigen-antibody interaction is a

particular chemical interaction between antibodies generated by B cells of the white blood cells and
antigens during the immune reaction. The process of agglutination combines antigens and antibodies.

Stages of Antigen-Antibody Reaction

There are three stages to the interactions between Ag and Ab.

 The first stage of the reaction entails the formation of the Ag-Ab complex.
 The second stage results in visible phenomena like agglutination, precipitation, etc.
 The third stage involves the destruction of Ag or neutralization of Ag.

Types of Antigen-Antibody Reaction

The types of antigen-antibody reactions are as follows:

 Precipitation Reaction
 Agglutination Reaction
 Complement Fixation
 Immunofluorescence
 ELISA – Enzyme-Linked Immuno Sorbent Assay

Precipitation Reaction
An insoluble precipitate of Ag-Ab complex is produced when a soluble Ag and its Ab combine in the
presence of an electrolyte (NaCl) at a specific pH and temperature. Precipitin is the Ab that causes
precipitation, and the reaction is termed a precipitation reaction.

The precipitation reaction occurs in both liquid and gel media.

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Agglutination Reaction
The particles are clustered or agglutinated when a certain Ag is combined with its Ab in the presence
of electrolytes at an appropriate temperature and pH. The clumps of cellular Ag formed by the
serum’s Ab are known as agglutinins.

Agglutinogens are the name for the aggregated particulate antigens.

 Slide Agglutination: This is a fast and convenient way to identify the presence of
agglutinating antibodies.
 Tube Agglutination: This is a common technique for estimating the quantity of Ab. A
constant volume of the Ag suspension is introduced after serially diluting the Ab-containing
serum with saline in multiple small test tubes.
A control tube is retained that contains no antiserum. The tubes are incubated up until observable
agglutination. The tube demonstrating the highest agglutination is known as the titre.

 Passive Agglutination Test: This test is equivalent to the hemagglutination test, but the
physical characteristics of the reaction are different .

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Dengue NS1 antigen Microlisa:

Principle :DENGUE NS1 Ag MICROLISA is a solid phase enzyme linked immunosorbent assay
(ELISA) based on the “Direct Sandwich” principle. The microwells are coated with Anti-dengue
NS1antibodies with high reactivity for Dengue NS1 Ag. The samples are added in the wells followed
by addition of enzyme conjugate (monoclonal anti-dengue NS1 antibodies linked to Horseradish
Peroxidase (HRPO)). A sandwich complex is formed in the well wherein dengue NS1 (from serum
sample) is “trapped” or “sandwiched” between the antibody and antibody HRPO conjugate. Unbound
conjugate is then washed off with wash buffer. The amount of bound peroxidase is proportional to the
concentration of dengue NS1 antigen present in the sample. Upon addition of the substrate buffer and
chromogen, a blue color develops. The intensity of developed blue color is proportional to the
concentration of dengue NS1 antigen in sample. To limit the enzyme-substrate reaction, stop solution
is added and a yellow color develops which is finally read at 450nm spectrophotometrically.

Procedure : Once the assay has started, complete the procedure without interruption. All the
reagents should be dispensed in the centre of the well and the tip of the pipette should not touch the
wall of the microwell. Fit the strip holder with the required number of Anti-Dengue NS1 antibody
coated strips. The sequence of the procedure must be carefully followed. Arrange the assay control
wells in a horizontal or vertical configuration. Configuration is dependent upon reader software.
1. Add 50 µl Diluent in all the wells.
2. Add 50 µl Negative Control in A-1well.
3. Add 50 µl Calibrator in B-1, C-1 & D-1 well.
4. Add 50 µl Positive Control in E-1 well.
5. Add 50 µl sample in F-1 well onwards.
6. Add 100 µl of working Conjugate Solution in each well.
7. Ensure thorough mixing of controls, samples to be tested & working conjugate to get reproducible
results.
8. Apply cover seal.
9. Incubate at 37ºC + 1ºC for 90 min. + 1min.
10. While the samples and working Conjugate are incubating, prepare working Wash Solution as
specified in preparation of reagents.
11. Take out the plate from the incubator after the incubation time is over and, wash the wells 6 times
with working Wash Solution.
12. Add 150 µl of working substrate solution in each well.
13. Incubate at room temperature (20-30ºC) for 30 min. in dark.
14. Add 100 µl of stop solution.
15. Read absorbance at 450 nm. within 30 minutes in ELISA READER. (Bi chromatic absorbance
measurement with a reference wavelength 600-650 nm is recommended when available).

. CALCULATION OF RESULTS TEST VALIDITY: Ensure the following is within specified

acceptance criteria

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 i) NC O.D. must be < 0.3. If it is not so, the run is invalid and must be repeated.
ii) PC O.D. must be > 1.0. If it is not so, the run is invalid and must be repeated.
iii) Mean Calibrator O.D. must be > 0.35. If it is not so, the run is invalid and must be repeated.
iv) Cut off value must be >1.5 x NC O.D. If it is not so, the run is invalid and must be repeated.
v) Ratio of PC O.D. / cut off must be > 1.1. If it is not so, the run is invalid and must be repeated.

a. Cut off value = mean O.D. of calibrator x calibration factor .

b. Calculation of sample O.D. ratio :Calculate sample O.D. ratio as follows:

sample O.D
Sample O.D Ratio =
Cut of value

INTERPRETATION OF RESULTS :
a. If the Dengue NS1 Ag Units is < 9 then interpret the sample as Negative for Dengue NS1 Antigen.
b. If the Dengue NS1 Ag Units is between 9 - 11 then interpret the sample as Equivocal for Dengue
NS1 Antigen.
c. If the Dengue NS1 Ag Units is > 11 then interpret the sample as Positive for Dengue NS1 Antigen.

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 Dengue IgM Microlisa
PRINCIPLE :
Dengue IgM Microlisa test is an enzyme immunoassay based on “MAC Capture ELISA”. Anti
human IgM antibodies are coated onto microtiter wells. Specimens and controls are added to the
microtiter wells and incubated. Antibodies to Dengue if present in the specimen, will bind to the Anti
human IgM antibodies absorbed onto the surface of the wells. The plate is then washed to remove
unbound material. Horseradish peroxidase (HRPO) conjugated Dengue antigen (DEN 1-4) is added to
each well. This dengue antigen conjugate will bind to dengue specific IgM antibodies which is
complex with anti human IgM antibodies. Finally substrate solution containing chromogen and
hydrogen peroxide is added to the wells and incubated. A blue colour will develop in proportion to
the amount of Dengue antibodies present in the specimen. The colour reaction is stopped by a stop
solution. The enzyme substrate reaction is read by EIA reader for absorbance at a wavelength of 450
nm. If the sample does not contain Dengue IgM antibodies then enzyme conjugate will not bind and
the solution in the wells will be either colourless or only a faint background colour develops.
TEST PROCEDURE :
The sequence of the procedure must be carefully followed. Arrange the assay control wells in a
horizontal or vertical configuration. Configuration is dependent upon reader software.
1. Add 100 µl Negative Control in A-1well.
2. Add 100 µl calibrator in B-1, C-1 & D-1 wells.
3. Add 100 µl Positive Control in E-1 well.
4. Add 100 µl of each sample diluted in sample diluent (1:100), in each well starting from F-1 well.
(Refer TUBE DILUTION).
5. Apply cover seal.
6. Incubate at 37ºC + 1ºC for 60 min. + 1min.
7. While the samples are incubating, prepare working Wash Solution as specified in preparation of
reagents.
8. Take out the plate from the incubator after the incubation time is over and, wash the wells 5 times
with working Wash Solution.
9. Add 100 µl of Enzyme Conjugate in each well.
10. Apply cover seal.
11. Incubate at 37ºC + 1ºC for 60 min + 1min.
12. Aspirate and wash as described in step no. 8.
13. Add 100 µl of working substrate solution in each well.
14. Incubate at room temperature (20-30ºC) for 30 min. in dark.
15. Add 50 µl of stop solution.

16. Read absorbance at 450 nm and 630 nm (reference filter) within 30 minutes in ELISA READER.

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CALCULATION OF RESULTS

a. Cut off value = mean O.D. of calibrator x calibration factor

b. Calculation of sample O.D. ratio : Calculate sample O.D. ratio as follows:
Sample O.D
sample O.D Ratio =
Cut off value

INTERPRETATION OF RESULTS:
a. If the Dengue IgM Units is < 9 then interpret the sample as Negative for Dengue IgM antibodies.
b. If the Dengue IgM Units is between 9 - 11 then interpret the sample as Equivocal for Dengue IgM
antibodies. c. If the Dengue IgM Units is > 11 then interpret the sample as Positive for Dengue IgM
antibodies .

WIDAL TEST

 WIDALTESTFORSALMONELLAINFECTION: Thisisappliedforthediagnosis of
enteric fever that include typhoid (Salmonella typhi) and paratyphoid (Salmonella
paratyphi).Specificantibodies(agglutinin)areusuallydetectableinthepatient’sblood after
6 days of entericfever.
 Principle:Antibodies found in the serum are produced in response to exposure to
Salmonella organisms and agglutinates a bacterial suspension of Salmonella (non-
infective), which carries homologous antigens. Two types of agglutinins are found in
patient, each of which reacts with ‘O’ and ‘H’ antigens. Thus in the test, four specific
antigenic suspensions areused-
Salmonella typhi O(somatic)
Salmonella typhi H (flagella, non-specific)
Salmonella paratyphi AH
Salmonella paratyphi BH
 Slide Agglutination Method:
 Specimen:A fresh serum sample.
 Reagents: commercial kits available for performing the test.

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Equipment:
1. Glass plate with outline circle.
2. Disposable Pasteu rpipette.
3. Rotatormachine.
4. Timer.
5. Applicatorstick.Testtube.

 Procedure:
1. Preparea1:20dilutionofeachserumspecimenbynormalsaline(NS-1.9ml+Serum-
0.1 ml)
2. The glass plates (at least 6 circle) for the febrile agglutination test are often provided
in the kit. Mark them from 1 to6.
3. Place one drop of diluted serum in each of the first four circle: 1 2 3 and 4. Place one
drop of positive control serum in each of the last two circle: 5 and6.
4. Place one drop of antigen suspension, provided in the kit, in its correspondingcircle.

5. Preparea1:20dilutionofeachserumspecimenbynormalsaline(NS-1.9ml+Serum-
0.1 ml)
6. The glass plates (atleast 6 circle) for the febrile agglutination test are often provided in
the kit. Mark them from 1 to6.
7. Place one drop of diluted serum in each of the first four circle: 1 2 3 and 4. Place one
drop of positive control serum in each of the last two circle: 5 and6.
8. Place one drop of antigen suspension, provided in the kit, in its correspondingcircle-

30 | P a g e
 Number Circle Serum specimen Antigen added

1 1: 20 diluted test serum O

2 H

3 A(H)

4 B(H)

5 Positive control O

6 Any of the (H, AH or BH)

9. Mixtheantigensuspensionandthedilutedserumwiththehelpofseparateanapplicator stick
for eachcircle.
10. Holdtheglassplatenearanadequatelightsource.Rotatetheslideintherotatorslowly or
manually for 3minutes.
11. Record the observed degree of agglutination asfollows-

Complete agglutination 4+

75% agglutination 3+

50% agglutination 2+

25% agglutination 1+

NO agglutination Negative (-)

12. Specimenswithpositivereactionarefurthersubjectedtotitredeterminationbythetube
method, other are reported as‘Negative”.

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 Quantitative:
1. Prepare a glass plate with four rows of five circleseach.
2. With the help of a 0.2 ml pipette (0.01 ml graduation), deliver the following amounts
of each serum specimen in the five circles in the row. Use four rows for four types of
antigens to be tested (O, H, AH, BH). Following is an example with the firstrow-
Row 1 Antigen ‘O’

Circle NO. Volume of serum Corresponding Agglutination

(ml) titre reaction

1 0.08 1:20 4+
2 0.04 1:40 3+
3 0.02 1:80 2+
4 0.01 1:160 1+(Titre)
5 0.005 1:320 0

3. Choose the first row of 5 circles and add the first antigen suspension (O antigens) in
equal quantities (0.03 ml) in each of the circles contains the serum specimens in
different ratio. Mix each circle with a different applicatorstick.
4. Rock the slide by manually and read theagglutination.
5. Read and record the degree of agglutination and report the titrevalue.

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 HEPATITIS B SURFACE ANTIGEN DETECTION(HBSAG):
 Dipstick test for HepatitisB:
 Principle:The dip stick test for the detection of hepatitis B surface
antigen(HBsAg)takes advantages of the formation of a visible spot by precipitating
immune-complexes. The nitrocellulose strips has zones of chemicals and antibodies
against hepatitis B. With theapplication of test sample, a positively reacted specimen
appears as redbands.
 Materialprovided: 
1. Test cards/ test strips individually foilpouched.
2. Sample dispensing plasticdropper.

 Materialrequired: 
1. Lancet.
2. Pipette.
3. Heparinized capillary tubes and rubberbulb.
4. Positive and Negativecontrol.
 Sample:Whole blood, plasma orserum.
 Assayprocedure: 
1. Bring all reagents and specimens to roomtemperature.
2. Remove the test card from the foil pouch and place on a clean drysurface.
3. Identify the test card for each specimen orcontrol.
4. Dispense100µl(3drops)ofthespecimenorcontrolintothesamplewellonthecard.
5. Interpret test results at 15minutes.

 Reading: Result should be read within 15 minutes. Do not interpret test results after 20
minutes.
 Interpretation:

Result Interpretation

A purple red band appear at test region. Positive

The absence of the purple red test band in the Negative

test region.

If the control band is not appear. Invalid

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  HEPATITIS–C-VIRUS (HCV)DETECTION:
 Dipstick test for HepatitisC:
 Principle:The HCV rapid test is a chromatographic immune assay (CIA) for the
detection of antibodies to HCV in human serum/plasma and whole blood. HCV
recombinant antigens are precoated onto membrane as a capture reagent on the test
region. During the test, specimen is allowed to react with the colloidal gold particles,
whichhavebeenlabeledwithHCVrecombinantantigens.IfantibodiestoHCVpresent,
apinkcoloredbandwilldevelopedonthemembraneinproportiontotheamountof

HCV antibodies present in the specimen. Absence of this pink colored band in the test region
suggests a negative result. To serve as a procedural control, a purple colored band in the control
region will always appear regardless the presence of antibodies to HCV.
 Accessories reagents:
1. HCV test device with adescant.
2. Capillarypipette.
3. Assaydiluents.
 Specimen:Human serum orplasma.
 Assayprocedure: 
1. Remove the test device from foil pouch; place it on a flat, drysurface.
2. Using a capillary pipette add 10 µl of plasma (up to black line) into the sample
wells (S).

3. Add 4 drops (About 120µl) of assay diluents into sample wells(S).

4. Interpret test result in 5- 20minutes.
Interpretation of the result

Interpretation Result

The presents of only one color band (Control band “C” ) Negative

The present of two color bands (“T” and “C”) Positive

If the control band “C” is not visible Invalid

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 STOOL EXAMINATION
General Stool Examination (GSE) is carried out in laboratories for various diagnostic purposes.
Examination of stool is very helpful in the diagnosis of disease of the gastrointestinal tract. Mostly a
clean container which does not contain any detergent or disinfectant is sufficient for all types of stool
examinations including stool culture. It consist the following tests:-
PHYSICAL EXAMINATION OF STOOL: Sample should be examined immediately after
collection. Samples left standing prolonged will deteriorate helminths, Ovum, other parasites and
increase the numbers of monilia and bacteria which gives wrong results, however the following
aspects of stool should be examined:
(1) Quantity: the adult person excretions about 150-250 gm. /day of feces, about (1/3- 1/2) of feces
dry weight is bacteria.
(2) Consistency and form: Normal stool is well formed. But in constipation (Dehydration) the stool
is solid (Hard) and the semi-solid (soft or loose) seen when taking certain medications and laxatives.
In abnormal cases such as diarrhea and dysentery the stool appear liquid, or watery in nature. In
cholera the stools have a rice water appearance. In cases of malabsorption of fats the stools are pale
bulky and semi-solid.
(3) Colour: 1- Normal colours of stools are light to dark brown due to the Presence of bile
pigments.
2- Dark black: In cases with bleeding into the intestinal tract the stools become dark tarry in nature
due to the formation of acid hematin ,if the bleeding is in the small intestines. In case of bleeding in
large intestines or rectum stool color may be bright red due to fresh blood.
3- Red color: Resulted from eating certain colorful foods such as red beets.
4- Clay colour: The stool may be clay coloured due to absence of stercobilinogen in biliary tract
obstruction.
(4) Odor: The normal fecal odor of stool resulted from Indole and Skatol. Odor of stools may become
offensive in conditions like, Intestinal amoebiasis. In cases of bacillary dysentery and cholera the
stools are not foul smelling due to the absence of fecal matter.
(5) Blood: 1- The blood is present on the outer surface of the feces and this caused either by
contamination from menstrual cycle blood in women or bleeding hemorrhoids from the blood vessels.
2- Blood should be noted in stools if present as it is indicative of Ulceration or presence of any other
pathology like malignancy.
(6) Mucus: Is present in certain conditions like amoebic or bacillary dysentery.
(7) Parasite: Stools may contain adult helminths. Nematodes like Ascaris are easily visible as their
size is large. Hook worms and Proglottids of cetodes may also present. These may be visible to the
naked eye.

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 MICROSCOPIC EXAMINATION OF STOOL:
The laboratory diagnosis of most parasitic infections is by the demonstration of ova of the parasite in the
stools of the infected person. The stool is collected in a clean container. The stool can be examined by
the following techniques.
(a)Wet mounts examination.
(b)Iodine examination.
(a) Saline wet mount examination: The stool is emulsified in normal saline and a large drop is
placed on a glass slide and is then covered with a cover slide. Then examined under a light
microscope, it is important to examine specimen under 10X objective lens at first to observe large
molecules, cells, ova and helminths, then to the 40X objective to complete the test. It is preferable to
keep the condenser down and the intensity of the light low for proper visualization of the ova and
cysts. The thickness of the film should be such that one is able to see the printed letters of the
newspaper through it.
(b) Iodine examination: Iodine preparation leads to better visualization of morphological details of
ova and cysts as it stains the glycogen in them. However it has the disadvantage that the live
trophozoites of Entamoeba histolytica and other live parasites cannot be seen as the iodine kills them.
The examination instructions in normal saline must be followed the same in iodine test.
Microscopic examination include the following:
(1) Pus cells: Observed in stool the same procedure as in urine.
(2) RBCs: Observed in stool the same procedure as in urine.
(4) Protozoa:
(a) Entamoeba histolytica: To investigate the vegetative phase (trophozoite) and cyst, causing
amoebic dysentery disease.
(b) Entamoeba coli: trophozoite + cyst Note: - most of children diarrhea less than 2 years cause by
Entamoeba coli.
(c) Giardia lamblia, trophozoite + cyst, Cause watery diarrhea disease in children, especially.
(d) Balantidium coli, trophozoite + cyst, causing Balantidiasis in colon.
(5) Worms : (a) Enterobius vermicularis (pinworm): investigating the eggs that are of convex and
flat surface and a pointed end.
(b) Ascaris lumbricoides: investigating for eggs which characterized by the content of granular
yellow to Brown irregular albumin membrane.
(c) Hookworm (Ancylostoma duodenale): investigating the eggs where the egg yolk is divided and
surrounded by a thin membrane.

(d) Tapeworms, (Taenia solium): investigating the worm pieces called (gravid segments or
Proglottids) that comes out with the feces.

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(e) Schistosoma mansoni: Investigating the eggs distinct by lateral spin.
(f) Tapeworms, (Taenia solium): investigating the worm pieces called (gravid segments or
Proglottids) that comes out with the feces.
(g) Schistosoma mansoni: Investigating the eggs distinct by lateral spin.

CHEMICAL EXAMINATION OF STOOL:

(a) pH: The pH of stools is acidic in amoebic dysentery and is alkaline in bacillary dysentery.

(b) Occult blood: Presence of blood in feces which is not apparent on gross inspection and which can
be detected only by chemical tests is called as occult blood. Causes of occult blood present in a
number of diseases including malignancy of the gastrointestinal tract. The reagents used are:
1- Benzidine reagent: - Development of blue color is indicative of presence of occult blood in the
stool specimen.
2- Orthotolidine: Development of green color Benzidine test is also highly sensitive and false-positive
reactions are common. Since bleeding from the lesion may be intermittent, repeated testing may be
required.

STOOL CULTURE:
1. Specimen of stool (at least 4 ml or 4 cm³) is collected in a clean, dry, container with a tightly
fitting lid.
2. Do not let sample stand for a long period of time (so as not to die eccentric parasitic and inspection
preferably within an hour of taking the sample).
3. Early morning is the best sample because the stool here all night and the chances of the emergence
of complex parasites and eggs are the largest.
4. For children prefer to urinate first before taking a stool sample so as not to mix urine with stool
sample.
5. Stool should not be contaminated with urine, water, soil, or menstrual blood. Urine and water
destroy trophozoites; soil will introduce extraneous organisms and also hinder proper examination.
6. Patient's name, date, time & number must be labeled on the sample container.
7. Stools should be examined as early as possible after receipt in the laboratory (preferably within 1
hour of collection). If delay in examination is anticipated, sample may be refrigerated because
Parasites are best detected in warm, freshly passed stools.
8. Patient must not use laxatives prior collecting stool sample.
9. Patient must refrain from taking certain medicines before the test such as: pH medications, diarrhea
medications, anti - parasite drugs, antibiotics.
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 10. The patient must wear gloves before handling a stool sample in order to avoid transmission or use
the tool to move the sample in the container. And don't take a stool sample base of the bathroom
(toilet) floor.
11. The patient must wash his hands thoroughly after taking the sample. And the sample water or
soap does not mix.

Procedure:
Stool is cultured by taking a sample by loop and cultured on different types of culture media
according to the type of bacteria or diagnosis of case investigated as follows:
1- It is cultured on Thiosulfate citrate bile salts sucrose agar media if the patient is suspected of
cholera infection.
2- It is cultured on Lowenstein–Jensen medium for Mycobacterium tuberculosis if the person
suspected of gastrointestinal tuberculosis.
3- It is cultured on blood agar if suspected of infection with Staphylococcus aureus which it is blood
hemolytic.
4- It is cultured on MacConkey agar medium to detect lactose fermentation bacteria in pink color
colonies include (E .coli, Klebsiella and Enterobacter). But if it's not lactose fermenter, it is either
Proteus which is identified by (diffusion phenomenon), or pseudomonas which is identified by
(pyocynin test). And to distinguish between Shigella and Salmonella, by using serological and
biochemical.

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Mycology Examination
Medical mycology is the branch of medical science that deals with the study of medically important
fungi. The name ‘fungus’ is derived from Greek ‘mykes’ meaning mushroom (a type of edible fungus).
Fungi differ from bacteria and other eukaryotes in many ways.
Morphological Classification of fungi

Based on the morphological appearance, there are four main groups of fungi given as follows :
1. Yeast: They grow as round to oval cells that reproduce by an asexual process called budding in which
cells form protuberances which enlarge and eventually separate from the parent cells. Examples include:
Cryptococcus neoformans (pathogenic) , Saccharomyces cerevisiae (non-pathogenic).
2. Yeast-like: In some yeasts (e.g. Candida), the bud remains attached to the mother cell, elongates and
undergoes repeated budding to form chains of elongated cells known as pseudohyphae. They can be
differentiated from true hyphae as they have constrictions at the septa
3. Molds: They grow as long branching filaments of 2–10 µm width called hyphae Hyphae are either
septate (i.e. form transverse walls) or nonseptate (there are no transverse walls and they are
multinucleated, i.e. coenocytic. Examples of true molds include—Dermatophytes, Aspergillus,
Penicillium, Rhizopus, Mucor, etc.
4. Dimorphic fungi: They exist as molds (hyphal form) in the environment at ambient temperature
(25°C) and as yeasts in human tissues at body temperature (37°C). Several medically important fungi are
thermally dimorphic such as: Histoplasma capsulatum.

Specimen collection
Specimen Collection It depends on the site of infection such as skin scraping, hair, nail, sputum, etc. For
systemic mycoses, blood sample may also be collected. Cerebrospinal fluid (CSF) is collected for
cryptococcal meningitis
Microscopy:
Fungal elements can be detected in the clinical specimens by direct microscopic examination of material
from the lesion.
Potassium hydroxide (KOH) preparation: Keratinized tissue specimens such as skin scrapings
and plucked hair samples are treated with 10% KOH which digests the keratin material so that the fungal
hyphae will be clearly seen under the microscope. Heat the slide gently over the flame and leave it aside
for 5–10 minutes before examination.
„ About 10% is the usual concentration of KOH used
„ About 20–40% KOH is needed for the specimens such as nail that otherwise takes a longer time to
dissolve
„ Biopsy specimens as they take longer time to dissolve, are usually dissolved in 10% KOH in a test
tube and examined after overnight incubation
„ Glycerol (10%) can be added to prevent drying
„ DMSO (dimethyl sulfoxide) can be added to help in tissue digestion.
Gram stain: It is useful in identifying the yeasts (e.g. Cryptococcus) and yeast like fungi (e.g.
Candida). They appear as gram-positive budding yeast cells.
39 | P a g e
India ink and nigrosin stains:They are used as negative stains for demonstration of capsule of
Cryptococcus neoformans.
Lactophenol cotton blue (LPCB):It is used to study the microscopic appearance of the fungal
isolates grown in culture. It contains:
„ Phenol acts as disinfectant
„ Lactic acid preserves the morphology of fungi
„ Glycerol prevents drying
„ Cotton blue stains the fungal elements blue.
Culture Condition
™ Temperature: Most of the fungi grow well at 25–30°C except the dimorphic fungi that grow at both
25°C and 37°C
™ BOD incubators (biological oxygen demand): It is a special incubator used in diagnostic mycology,
which is capable of maintaining low temperature
™ Incubation time: Culture plates should be incubated for 2–3 weeks
™ Antibiotics such as cycloheximide (actidione), chloramphenicol and gentamicin can be added to the
culture media to inhibit bacterial growth

Microscopic Appearance of Fungi

A. Teased mount: A bit of fungal colony is teased out from the culture tube and the LPCB mount is
made on a slide and viewed under microscope. If proper teasing is not done, then the intact morphology
may not be identified properly. Identification is based on the following:
„ Nature of hyphae (such as septate or aseptate, hyaline or phaeoid, narrow or wide) and
„ Type of sporulation (conidia or sporangiospores).
B. Slide culture: Though this is a tedious procedure, it gives the most accurate in situ microscopic
appearance of the fungal colony. A sterile slide is placed on a bent glass rod in a sterile petri dish. Two
square agar blocks measuring around 1 cm2 (smaller than the coverslip) are placed on the slide. Bit of the
fungal colony is inoculated onto the margins (at the center) of the agar block. Then the coverslip is
placed on the agar block and the petri dish is incubated at 25°C. After sufficient growth occurs, LPCB
mounts are made both from the coverslip and the underneath slide
C. Cellophane tape mount: The impressions are taken by placing the cellophane tape on the colonies
present on the surface of SDA plate, then LPCB mount is made from the cellophane tape. This is easy to
perform than slide culture and in-situ fungal morphology is also maintained.

40 | P a g e
 REPORT FINDING
A.BACTRIOLOGY-
Urineculture:
Clinicalsignificance:
Aurinecultureisalabtesttocheckforbacteriaorothergermsinaurinesample. Itcanbe used to
check for a urinary tract infection in adults and children.

TABLENO:1
AnalysisofUrineculturereport.

NO. Results(Norm
OF Testname- Totalnum alvalue-<10^4)
DAYS URINECULT beroftest
URE Growth percentage No Percentage
Growth
1 40 30 75% 10 25%
2 44 28 64% 16 36%
3 30 19 63% 11 37%
4 42 34 81% 8 19%
5 36 25 69% 11 31%
6 46 36 78% 10 22%
7 URINE 40 22 55% 18 45%
8 CULTURE 50 36 72% 14 28%
9 45 29 65% 16 35%
10 56 30 54% 26 46%
11 60 45 75% 15 25%
12 40 25 62% 15 38%
13 36 25 69% 11 31%
14 44 31 70% 13 30%

41 | P a g e
TABLENO:2

Analysis of infective bacterial infection.

NO. OF INFECTIVEBACTERIALNAME
DAYS E.coli Klebbseilla Proteus Pseudomonas Staph. CONS Enterococcus
areus
1 10 9 0 4 4 1 2
2 9 8 2 4 2 0 3
3 8 5 2 2 1 1 0
4 12 9 4 4 3 0 2
5 7 8 0 2 4 1 3
6 11 15 0 4 2 0 4
7 9 7 2 2 2 0 0
8 12 14 3 3 3 1 1
9 9 10 3 2 0 1 2
10 10 14 1 2 0 0 3
11 14 16 0 4 6 1 4
12 8 7 2 2 2 0 4
13 9 10 2 0 2 0 2
14 6 12 0 3 6 1 3

42 | P a g e
 Miscellaneousculture:
Clinicalsignificance:
Miscellaneous culture includes Bacterial culture and gram stain. This culture type is
appropriate for culture of superficial body sites , and it meant for isolation of
Aerobic bacteriaonly. If sample type requires culture for both Aerobes and
Anarobes, such as deep wounds, a wound culture should be ordered instead.

TABLENO:3

Analysis of miscellaneous culture report.

Testname- Totalnum Results(Norma

MISCELLANE beroftest lvalue-<10^4)
NO. OF OUSCULTURE
DAYS
Growth percentage No Percentage
Growth
1 30 20 67% 10 33%
2 25 18 72% 7 28%
3 30 18 60% 12 40%
4 35 26 74% 9 26%
5 36 26 72% 10 28%
6 MISCELLANEO 26 20 76% 6 24%
7 CULTURE 34 22 64% 12 36%
8 35 26 74% 9 26%
9 27 15 55% 12 45%
10 40 25 62% 15 38%
11 36 26 72% 10 28%
12 28 16 57% 12 43%
13 36 28 78% 8 22%
14
14.02.23 40 26 65% 14 35%
15.02.23
15 30 20 67% 10 33%

43 | P a g e
TABLENO:4

Analysis of infective bacterial infection.

INFECTIVEBACTERIALNAME
NO. OF E.coli Klebseilla Proteus Pseudomonas Staph. CONS enterococcus streptococcus
DAYS aerus aerus
1 2 2 2 4 6 2 1 1
2 1 1 1 4 8 1 1 1

3 2 2 0 5 7 0 1 0

4 2 4 2 4 8 2 1 2

5 1 2 2 6 6 1 1 1
6 2 2 3 7 8 0 0 0

7 2 3 2 5 10 2 1 1

8 1 1 0 4 5 0 0 1
9 2 4 2 4 8 2 1 2
10 1 1 1 3 6 1 0 0
11 2 2 2 5 7 1 1 0
12 2 4 2 4 8 1 1 2

13 0 2 2 3 5 0 2 1

45 | P a g e
 Fluidculture:
Clinicalsignificance:
A sensitivity test checks to see what kind of medicine ,such as an antibiotic, will
work best to treat the illness or infection. For a culture, a sample of body fluid or
tissueisaddedtoasubstancethatpromotesthegrowthofgerms.Ifnogermsgrow, the culture
is negative.

TABLENO:5
Analysis of fluid culture report.

NO. OF Testname- Totalnum Results(Norm

DAYS FLUIDCULT beroftest alvalue-<10^4)
URE
Growth percentage No Percentage
Growth
1 10 04 40% 06 60%
2 14 03 22% 11 78%
3 08 02 25% 06 75%
4 FLUID 11 04 37% 07 63%
CULTURE
5 12 02 17% 10 83%
6 08 01 13% 07 87%
7 12 03 25% 09 75%
8 07 02 29% 05 69%
9 05 00 00% 00 00%
10 09 02 23% 07 77%
11 10 04 40% 06 60%
12 12 03 25% 09 75%
13 08 02 25% 06 75%
14 09 01 12% 08 88%
15 10 03 30% 07 70%

46 | P a g e
TABLENO:6

Analysis of infective bacterial infection.

NO. INFECTIVEBACTERIALNAME
OF E.coli Klebseilla Proteus Pseudomonas Staph. CONS enterococcus streptococcus
DAYS- aerus aerus
1 1 0 0 1 1 0 1 0
2 0 0 0 1 1 1 0 0

3 0 0 0 0 2 0 0 0

4 1 0 0 1 2 0 0 0

5 0 0 0 1 1 0 0 0
6 0 0 0 0 0 0 1 0

7 0 0 0 1 1 1 0 0

8 0 0 0 1 1 0 0 0

9 0 0 0 0 0 0 0 0

10 1 0 0 1 0 0 0 0
11 0 0 0 1 2 1 0 0

12 1 0 0 0 1 0 1 0

13 0 0 0 0 1 1 0 0
14 1 0 0 0 0 0 0 0
15 1 0 0 1 1 0 0 0

47 | P a g e
 1. Bloodculture:
Clinica lsignificance:
A blood culture test helps doctor figure out if the patient have a kind of infection
thatisinhis/herbloodstreamandcanaffectentirebody.Doctorscallthisasystemic infection.
The test checks a sample of blood for bacteria that might be causing the infection.

TABLENO:7
Analysisofbloodculturereport.

No. of DAYS Testname– Results(Norm

TotalBLO alvalue-<10^4)
ODCULTURE number
Growth percentage No percentage
Growth
1 14 02 14% 12 86%
2 18 02 12% 16 88%
3 09 01 11% 08 89%
4 BLOOD 12 03 25% 09 75%
5 CULTURE 15 02 14% 12 86%
(BD
6 10 01 10% 09 90%
BACTEC^tm
7 10 02 20% 08 80%
Automated
8 13 02 15% 11 85%
bloodculture
9 system) 08 03 38% 05 62%
10 09 01 11% 08 89%
111 12 03 25% 09 75%
12 10 02 20% 08 80%
13 07 01 14% 06 86%
14 09 01 11% 08 89%
15 15 02 14% 13 86%

48 | P a g e
TABLENO:8

Analysis of infective bacterial infection.

NO. OF INFECTIVEBACTERIALNAME
DAYS E.coli Klebseilla Proteus Pseudomonas Staph CONS enterococcus streptoco
spp. aeruginosa aerus ccus
1 0 0 0 1 0 1 0 0
2 0 0 0 1 0 0 0 1

3 0 0 0 1 0 0 0 0

4 1 0 0 0 1 0 0 1
5 0 0 0 1 1 0 0 0

6 0 0 0 0 0 0 0 1

7 0 0 0 1 0 1 0 0

8 0 0 0 1 1 0 0 0
9 0 0 0 2 0 1 0 0

10 1 0 0 0 0 0 0 0
11 0 0 0 1 0 1 0 1
12 0 0 0 1 0 0 0 1
13 0 0 0 0 0 1 0 0

14 1 0 0 0 0 0 0 0
15 0 0 0 2 0 0 0 0

49 | P a g e
TABLENO:9
Analysis of anaerobic culture report.
TEST NAME-
ANAEROBIC TOTAL RESULTS- Clostridum species pesent.
CULTURE(RC TEST Proteolyticanaerobes(blackingofmeat)
M- NUMBE
RobertCooked R
Son Meat
media) GROWTH PERCENTAG NOG PERCENTAG
E ROWTH E

1 OPERATION 105 02 2% 103 88%

THEATOR
SAMPLES 85 00 00% 85 100%
(ANAEROBIC
2 CULTURE)

50 | P a g e
 B.MYCOLOGY-
Fungalculture:

Clinicalsignificance:
Afungalculturetestisusedtofindoutwhetheryouhaveafungalinfection.Thetest
mayhelpidentifythetypeoffungasthatyouhave.Thetestisalsousedtohelpguide treatment
and to see if treatment is working.

TABLENO:10
Analysis of total no of sample of mycology.

Total Differenttypeofsample
No. of numbe
days r Urine Miscellaneous Pus Fluid Blood
oftest
1 6 2 3 0 1 0
2 5 1 3 0 1 0
3 3 0 1 1 0 1
4 4 1 2 1 0 0
5 5 1 1 0 1 2
6 4 0 0 0 3 1
7 6 1 2 3 0 0
8 5 1 1 2 1 0
9 4 3 0 0 1 0
10 3 1 2 0 0 0

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TABLENO:12
Analysis of various type of fungal growth of mycology sample.

Results
Candida Aspergillus penicilliu Mucor Rhizop
NO.
m us
Of
C. C. C. C. A. A. A. P.
Days albican tropic krusei glabrat flavous fumigato niger marneffei
s alis a us
1 (1) 0 0 0 (1) 0 (3) (1) (1) 0
5.5% 5.5% 16.67% 5.5% 5.5%

2 0 (2) 0 0 (2) (1) (2) 0 0 (1)

10% 10% 5% 10% 5%

3 0 0 (1) 0 (1) 0 (2) 0 0 0

5.8% 5.8% 11.76%

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C.SEROLOGY-
HepatitisB:
Clinicalsignificance:

Hapatitis B is a potentially life- threatenting liver infection caused by the hepatitis B virus
(HBV).Itisamajorglobalhealthproblem.Itcancausechronicinfectionandputspeopleat high risk
of death from cirrhosis and liver cancer.

i. Rapidcardtest:

TABLENO:13
AnalysisofHepatitisBcardtest.

TestName:Hepat Tota Result

itisB lnoof Positive Percentage Negative Percentage
(HBsAg) test
1 105 4 3.81% 101 96.19%
2 112 5 4.46% 107 95.53%
3 99 2 2.02% 97 97.97%
4 125 6 4.8% 119 95.2%
5
113 2 1.76% 111 98.23%
6 RapidTestof 89 1 1.12% 88 98.87%
7 Hepatitis B 108 2 1.85% 106 98.14%
8 (HBsAg) 116 3 2.58% 113 97.41%
9 120 2 1.66% 118 98.33%
10 113 3 2.65% 110 97.34%
11 109 1 0.94% 108 99.05%
12 105 2 1.96% 103 98.03%
13 101 3 2.97% 99 97.02%

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ELISAProcedure:TA
BLENO:14
Analysis of Hepatitis BELISA test.

N0. Of Testname Totalno Result

Days oftest
Positive Percentage Negative Percentage
1 87 2 2.29 85 97.70%
2 85 1 1.17% 84 98.82%
3 87 4 4.59% 83 95.40%
4 87 0 0 87 100%
5 84 5 5.95% 79 94.04%
6 82 2 2.43% 80 97.56%
7 HepatitisB 87 4 4.59% 83 95.40%
(HBsAG)
8 87 1 1.14% 86 98.85%
ELISA
9 87 0 0 87 100%
10 85 0 0 85 100%
11 87 6 6.89% 81 93.10%
84 2 2.38% 82 97.61%
12 87 4 4.59% 83 95.40%

54 | P a g e
 HepatitisC:
Clinicalsignificance:

HepatitisCinfectionthatcontinusesovermanyyearscancausesignificantovermanyyears can
cause significant complications, such as:Scarring oftheliver(cirrhosis). After decades of
hepatitis C infection ,cirrhosis may occur. Scarring in liver makes it difficult for liver to
function.

ii. Rapidcardtest:
TABLENO:15
AnalysisofHepatitisCrapidcardtest.

NO Of TestName:Hep Tota Result

Days atitisC lnoof Positive Percentage Negative Percentage
(AntiHCV) test
1 105 1 0.95% 104 99.04%
2 112 3 2.67% 109 97.32%
3 99 1 1.01% 98 98.98
4 125 2 1.6% 123 98.4%
5 113 5 4.42% 108 95.57%
RapidTestof
6 89 1 1.12% 88 98.87%
7 HepatitisC 108 4 3.70% 104 96.29%
(AntiHCV)
8 116 7 6.03% 109 93.96%
9 120 5 4.16% 115 95.83%
10 113 3 2.65% 110 97.34%
11 109 1 0.91% 108 99.08%
12 105 2 1.90% 103 98.09%
13 101 3 2.97% 102 97.02%

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` `

WIDAL TEST
Clinical significance:
The Widal test is an advanced way to check for antibodies that body makes against the
salmonellabacteriathatcausestyphoidfever.ItlooksforOANDHantibodiesinapatient’s sample
blood (serum). This test helps detect life threatening illness like typhoid fever.

TABLENO:17
Analysis of WIDAL test.

NO Of Testname Totalno Result

Days oftest
Positive Percentage Negative Percentage
1 4 2 50% 2 50%
2 3 0 0 3 100%
3 1 0 0 1 100%
4 2 0 0 2 100%
5 Widal 5 1 20% 4 80%
Test
6 4 0 0 4 100%
7 6 3 50% 3 50%
8 2 1 50% 1 50%
9 1 0 0 1 100%
10 4 2 50% 2 50%
11 3 0 0 3 100%
12 5 0 0 5 100%
13 6 2 33.33% 4 66.67%

56 | P a g e
 Cartridge based nucleic acid amplication test(CB-NAATGene Xpert):
Clinica significance:
CBNAATprovidesarobustandapromisingroleinearlydiagnosisofTBinheadandneckas
wellasothercasesofsmearnegativeTBsuchasTB.Itshighspecificityandlesstimetaking procedure
makes it an excellent tool for timely diagnosis of such cases.

TABLENO:18
AnalysisofCB-NAATtest.

NO Of Testname Totalno Result

Days oftest
Detected Percentage Non Percentage
detected
1 8 0 0 8 100%
2 4 0 0 4 100%
3 12 1 8.33% 11 91.66%
4 12 0 0 12 100%
5 8 1 12.5% 7 87.5%
6 4 2 50% 2 25%
7 CB-NAAT 12 0 0 12 100%
GeneXpert
8 8 1 12.5% 7 87.5%
9 8 1 12.5% 7 87.5%
10 8 2 25% 6 75%
11 12 3 25% 9 75%
12 4 0 0 4 100%
13 12 0 08.33 12 100%
.

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 WORKINGPICTURESATMICROBIOLOGYANDSEROLOGY
DEPARTME
ISOLATEDORGANISMESCHERICHIACOLIINMACONKEYAGA ENTEROCOCCUSspp
R

VITEK^R2COMPACTFORACCURATEMICROBIALIDENTIFICATIO
NANDSENSITIVITY
ENTEROCOCCUSspp.
58 | P a g e
114
BIOCHEMISTRY

59 | P a g e
 Dept. of Biochemistry
 Quality control and calibration in biochemical laboratory

 Determination of plasma glucose

 Determination of Liver function test

 Determination of lipid profile test

 Determination of kidney function test

 Determination of Thyroid function test

 Determination of pancreas function test

 Determination of cardiac enzyme test

 Determination of electrolytes

60 | P a g e
Quality control in biochemistry laboratory

Quality control definition :Quality control in the medical laboratory is a statistical process used
to monitor and evaluate the analytical process that produces patient results and establishing conditions
such that the quality of all tests performed in the medical lab assists clinicians in practicing good
medicine. When a diagnostic test is performed in the medical laboratory, the outcome of the test is a
result. The result may be a patient result or it may be a quality control (QC) result. The result may be
quantitative (a number) or qualitative (positive or negative) or semi-quantitative (limited to a few
different values).
Total laboratory automation has replaced manual testing of parameters but still laboratory errors like
pre examination /pre analytical, examination/ analytical and post examination/ post analytical errors
are in the rise. The only way to keep a check on laboratory errors is by quality control at all phases of
testing.
Definition of calibration :Calibrators are used to measure the accuracy of test results and
often referred to as 'standardized samples'.
In an analytical chemistry , a calibration curve, also known as a standard curve, is a general
method for determining the concentration of a substance in an unknown sample by comparing the
unknown to a set of standard samples of known concentration. A calibration curve is one approach to
the problem of instrument calibration; other standard approaches may mix the standard into the
unknown, giving an internal standard . The calibration curve is a plot of how the instrumental
response, the so-called analytical signal, changes with the concentration of the analyte (the substance
to be measured).

Westgurd rules :The following control rules are used to interpret the control data:
1. 12s – One control observation exceeding the mean ±2SD used only as a “warning” rule that
initiates testing of the control data by the other control rules
2. 13s- One control observation exceeding the mean ±3SD primarily sensitive to random error.
3. 22s – Two consecutive control observations exceeding the same mean +2SD or mean -2SD
primarily sensitive to systematic error.
4. R4s – One observation exceeding the mean +2SD and another exceeding the mean -2SD is
primarily sensitive to random error.
5. 41s– Four consecutive observations exceeding the mean +1SD or the mean -1SD primarily
sensitive to systematic error.
6. 10x– 10 consecutive control observations falling on one side of the mean(above or below, with no
other requirement on size of the deviations) - sensitive to systematic error.
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Errors in biochemistry laboratory :
Every quantitative measurement has some uncertainty associated with it. This uncertainty is referred
to as the errors, which is a measure of the difference between the true value (derived from standard
samples of known compositions) and the analytical value.

(1) Pre-analytical errors

(2) Analytical errors

(3) Post analytical errors

1. Pre-analytical errors

(a) Wrong technique during collection of sample: Example: Blood collected for calcium estimation
after applying tourniquet.

(b) Choosing wrong sample carrying media: Example: Blood sample for glucose estimation is
collected and carried in EDTA vial.

(c) Error in drawing amount of sample: Example: Excess amount of blood is mixed with citrate for P-
time determination.

(d) Sample collected from a wrong area of the body: Venous blood is collected for doing ABG
analysis.

(e) Temperature for keeping a sample for some days before analysis is not properly maintained.
(f) Failed to give proper instruction to a patient: Example: Fasting blood for "lipid profile" is collected

at 8:00 am, when patient has his last meat at 11:00 pm (12 hours strict fasting mandatory).

(g) Improper documentation: Example: Patient details are not properly noted on the sample vial

or wrong bar-code for patient identification is attached.

(h) Fact suppression by the patient: Example: Patient suppressed that he was on fast day before doing
OGTT).

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 2. Analytical errors :
This may be of two kinds:

a) Systematic error

(b) Random error

(a) Systematic errors: These are consistent errors which can be identified and either eliminated or
reduced. They are most commonly caused by a fault or inherent limitations in the apparatus being
used but may also be influenced by poor analytical design. These errors may be either Constant or
Proportional. Again both of these systematic errors have three common causes.

Analyst error: (i) Misuse of manual or automatic pipette, (ii) Incorrect preparation of stock solutions,
(iii) Incorrect calibration, (iv) Incorrect use of pH meter.

(2) Instrument error: (i) Electronic, (ii) Defective matrix of the sample.

(3) Method error: Error in the procedure applied.

3. Post analytical error :

Common post-analytical include failure to report test results, delay in report , incorrect calculation
critical results not reported and result sent to wrong.

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  DETERMINATIONOFGLUCOSEINPLASMA/SERUM:
 Clinicalsignificance:Glucose is the main source of energy for the human body.Glucose
is converted either into glycogen to be stocked in the liver or into triglyceride to be
stocked in the fatty tissue. Glucose concentration in blood is regulated by several
hormones, including two antagonists: insulin & glucagon. Quantification of glucose in
blood is used to diagnose metabolic carbohydrates disorders such as diabetes, neonatal
glycaemia, idiopathic hypo glycaemia and pancreaticdisease.

The main physiological troubles are linked to hyper glycaemia(Type1Diabetes

mellitus and Type 2 Diabetes mellitus). Type 1 Diabetes mellitus is insulin dependent
and appears mainly before 30 years old. Type 2 Diabetes mellitus is non insulin
dependentandusuallyappearsafter40yearsold,but can occure earlier for obese people.
Otherdiabetes has secondary origin and appears after endocrine or hepatic diseases.
 Method: Enzymatic GOD- POD method(Colorimetric)

 Principle: Enzymatic colorimetric determination of glucose according to the

followingreaction-
Glucose oxidase
Glucose+O2 ---------------------------------------- Glucuronic acid+H2O2
Peroxidase
2H2O2 +Phenol+4-aminoantipyrine ----------- Quinonemine dye (Red) +4H2O

 Reagentcomposition:
1. R1 – Bufferreagent.
2. R2 – Glucosereagent.
Working reagent:- Add R1(1 bottle) and R2(1 bottle) reagent for the preparation of he
working reagent.
3. R3 – Glucosestandard.
 Sample: Fasting serums are required in case of fasting glucose assay and free of
hemolysis. Plasma collected on sodium fluoride containing vial or any inhibitor of
glucosevial.
 Reference value: Serum/plasma- Fasting – 70- 110mg/dl.

Post parental-Up to 140 mg/dl.

 Procedure:
o Wavelength: 505 nm (492-550)
o Temperature:37°C.

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 Reading is taken after the following distribution of reagent and sample against the Blank and
standard.
Dispense Blank(B Standard(S Test(T
) ) )
Working 1ml 1ml 1ml
reagent
Distilled water 10μl - -
Standard - 10μl -
Test sample - - 10μl

Afterdispensingthereagentandthesamplecontainingtubeisthoroughlymixedandincubates at 37C
temperature in theincubator.

 Calculation: Glucose concentration in plasma/serumconcentration:

O.D of the test

= -------------------------- Standard concentration
O.D of the standard
(Here generally used standard concentration is 100 mg/dl).

DETERMINATION OF SERUM/ PLASMAUREA:

Urea is the end product of protein metabolism. It is synthesized in the liver from ammonia
product by the catabolism of amino acids. It is transported by the blood to the kidney from
where it is excreted. Increased levels are found in renal diseases, urinary tract obstruction,
shock and congested heart failure.
Method : Berthelotmethod.
Principle: Urea is hydrolyzed in presence of water and urease to produce Ammonia and CO2.
Under alkaline condition, Ammonia so formed, react with hypochlorite and phenolic
chromogento form coloure dIndophenol,which is measured at 578nm(570-
600nm).Sodiumnitroprussideactasacatalyst.Theintensityofthecolourisproportional of urea in
the sample.

Urease
Urea+H2O ----------------- Ammonia +CO2.
Sodium Nitroprusside
Ammonia + Phenol+ Hypochlorite -------------------------------------- Indophenol

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  Reagent Composition:
1. R1 – Ureasereagent.
Working reagent :- 1 ml of urease reagent add to 50 ml of distilled water.
2. R2 –Chromogenreagent.
Working chromogen reagent :- 40 ml of chromogen reagent is mixed with 160 ml of
distilled water.
3. R3 – Ureastandard.
 Otherrequirements:
1. Clear and dry small test tube with test tuberack.
2. Micropipette &tips.
3. Incubator.
4. Spectrophotometriccolorimeter.
 Procedure:
1. All the reagent and sample are brought to the room temperature before thetest.
2. For one sample three test tube is taken in a rack and marked them as Blank (B),
Standard(S)andTest(T).Afterthatthereagentandsampleisdispensedasfollowing way.

Dispense Blank Standard Test

Solution 1 1.5ml 1.5ml 1.5ml

Serum/plasma - - 10μl
Reagent 3
(Standard) - 10μl -

Mixing is done and incubates the tube at 37°C temperature for 3 minutes.


Solution 2 1.5ml 1.5ml 1.5ml


3. After that the content of the three tubes is mixed well and incubates at 37°C for 5
minutes.
4. Blank is set with the reagent blank in the spectrophotometric colorimeter.
5. Theabsorbanceofthecolourproducedinthetesttubeismeasuredagainstblankand
standard at 570- 600 nm wavelength.

6. Then the result iscalculated.

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  Calculation:

Absorbance of test
Serum urea(mg/dl) = -------------------------------------(Concentration ofStandard)
Absorbance of Standard
Blood Urea Nitrogen (BUN) = Urea in mg/dl  0.467

 Normal range:Serum/plasma urea concentration = 12.8- 42.8mg/dl

Urine urea concentration = 15.6 – 42 mg/dl.

 DETERMINATION OF SERUM/URINECREATININE:
Creatinine is synthesized in the liver and passes in the circulation and is taken up
almost entirely by the skeletal muscle for conversion to creatine phosphate which serves as a
storage form of energy in skeletal muscle. About 2% of total creatine is converted directly to
the Creatinine. So the production of Creatinine is related to the total muscle mass, remain
approximatelythesameinplasmaandurinefromdaytodayunlessmusclemasschange.The
elevated level of Creatinine is found in renal dysfunction, reduced renal blood flow (Shock,
Cardiacfailure).

 Method:Alkaline picratemethod.
 Principle:Picric acid in an alkaline medium reacts with Creatinine to form a orange colour
compound. The intensity of the colour produced directly proportional to the amount of
Creatinine present in the sample (Serum/Urine).
Alkaline medium
Creatinine+Picricacid -------------------------- Alkaline picrate (Orange).
 Referencevalue: 
Serum Creatinine-
 Adult male- 0.6- 1.2mg/dl
 Adult female- 0.5- 1.1 mg/dl
UrineCreatinine-
 Adult male- 1.1- 3.0gm/dl
 Adult female- 1 – 1.8 gm/dl

 Reagent:
1. Picricacid
2. Bufferreagent
3. Creatinine standard (2 mg/dl)

67 | P a g e
 Sample:Serum or urine. Fasting blood serum is required. 24 hour collected urine is
preferred. Dilute the urine specimen with distilled water in the ratio of 1:50 before the assay.
 Anotherrequirements:
1. Test tube &rack
2. Micropipette
3. Incubator
4. Centrifuge
5. Pasteur pipette
6. Spectrophotometric colorimeter.
 Procedure:
A. Deproteinization of specimen:-
1. All the sample and specimen are brought to the room temperature before thetest.
2. Clean dry test tube is taken in a rack. For each test one tube isrequired.
3. Then 3 ml of picric acid and 0.2 ml of serum is added and is mixedproperly.
4. Then the tube is centrifuged at 2000- 3000 rpm for 10 minutes to obtain clear
supernatant.
B. Colour development:-
1. Thre clean dry test tubes(foronetest)is taken and marked for Blank(B), Standard
(S), and Test (T). Dispense the reagent and produced supernatant as following way.
Dispense Blank (B) Standard(S) Test(T)
Supernatant
- - 1.1ml
sample
Picric acid 1.0ml 1.0ml 1.0ml
Distilled water 0.1ml - -
Creatinie
- 0.1ml -
standard
Buffer reagent 0.1ml 0.1ml 0.1ml

2. After that the tubes are mixed well and tubes are kept at room temperature for 20
minutes.
3. Then the orange colour solution is produced in the test tube and standard tube.
4. The intensity of colour is measured of the test (sample) and standard against blank.
The result is then calculated.

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 Calculation:

Absorbance of test
Serum Creatinine (mg/dl) = ------------------------------------  2 mg/dl (Concentration of
Standard)
Absorbance of Standard

Absorbance of test
UrineCreatinine =  1 gm/dl
Absorbance of Standard

Urine Creatinine gm/24 hours= X gm/dl Total volume.

 DETERMINATION OF URIC ACID INPLASMA/SERUM:

Uric acid is a nonprotein nitrogenous waste product of the body derived from the purine
which scame from dietary urine which is endproduct of nucleo protein digestion (richinred
meat).SerumuricacidlevelisoftenrisinginGOUTandincreasemetabolismofnucleoprotein of our
body (i.e. Leukemia, Polycythemia). The determination has diagnostic value as
differentiatebetweenGOUTandnon-GOUTarthritis.Italsoelevatedrenalfailureanduremia.

 Method: Enzymatic (Uricase) method- End point reaction.

 Principle:The enzyme Uricase in the reagent acts on uric acid to catalyzed following
oxidationreaction.
Uricase
Uric acid + 2H2O+O2 ------------------------------------------- Allantoin+ H2O2 +CO2
Oxidation

Peroxidase
H2O2 ------------------------------------  H2O +[O]
Splitting
Then the phenolic chromogen present in the reagent 4- dichlorophenolsulphonate (DCFS)
and4-aminoantipyrinegetoxidizedtoformaredpurplecolourcompoundIntensityofwhich can be
measured at 546 nm (530- 550 nm) of green filter. The absorbance of the red colour
compound is directly proportional to the amount of Uric acid present in the serum or plasma.

 Reagents:
1. R1 – Buffer enzyme (Uric acidreagent).
2. R2 – Chromogenreagent
Working reagents:- R1 and R2 are mixed properly for the preparation of the working
reagents.
3. R3 – Uric acidstandard.

 Sample:Fasting serum or heparinized plasma is required for thetest.

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  Reference value:Serum uric acid concentration-
 Male- 3.4- 7.0mg/dl
 Female- 2.4- 5.7mg/dl
 Assayprocedure: 

Reaction type- End point

Reagent volume- 1.0 ml
Sample volume – 0.02 ml
Incubation time- 20 minutes
Incubation temperature – Room temperature
Zero setting with: Distilled water
Wavelength: 546 nm

Sta All the reagent and samples are brought to room temperature before start the assay.

All the reagent and sample are dispense in three test tubes (for one sample) marked as Blank
(B), Standard (S) and Test (T) as following way-

Dispense Blank Standard Test

Uric acid working

1 ml 1 ml 1 ml
reagent

Distilled water 20 μl - -

Uric acid standard - 20 μl -

Test sample - - 20 μl

All the test tube is mixed and incubates at 20- 25°C for 20 minutes.

After incubation optical density is measured at 546 nm against blank within 60 minutes.
 Calculation:

Absorbance of test
Serum uric acid conc.(mg/dl)= -------------------------------------  Concentration ofStandard
Absorbance of Standard

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  DETERMINATION OF CHOLESTEROL IN PLASMA/SERUM:
 Principle: Cholesterol and its esters are released from lipoprotein by detergents.
Cholesterol esterase hydrolyses the esters. In the subsequent oxidation by cholesterol
oxidase ,H2O2 is liberated. The colorimetric indicator is quinoneimine is generated
from 4- amino antipyrine and phenol by H2O2 under the catalytic action of peroxidase
(Trinder’sreaction).
Cholesterol esterase
Cholesterolester+H2O2 -----------------------------------------------------------------  Cholesterol + Fattyacid

Cholesterol oxidase
Cholesterol+ O2 ------------------------------------------- Cholesterol- 3 – one + H2O2
Standard concentration: 8.08 mg/dl

Peroxidase
2H2O2 + 4- aminoantipyrine+Phenol ----------------------------- Quinoneimine + 4H2O

 Method:CHOD- PAP method (Enzymatic and colorimetricmethod).

 Reagentcomposition:
1. R1 – Cholesterol enzyme reagent.
2. R2 – Cholesterolstandard.
 Sample:Fasting serum, heparin plasma or EDTA plasma is the sample ofchoice.
 Referencevalue: 
Desirable :<= 200 mg/dl
Borderline high risk : 200- 240 mg/dl
High risk: >= 240 mg/dl

 Assay procedure: 
Wavelength:- 500 - 546nm
Light path:-1 Cm
Temperature:- 37°C
Measurement:- Against reagent blank.

71 | P a g e
 1. All tge reagent and sample are brought to the room temperature before start theassay.
2. Then reagent and sample are dispensed in three test tubes marked as for blank(B), for
standard (S) and for test(T).

Dispense Blank Standard Test

Reagent 1 1000 μl 1000 μl 1000 μl

Distilled water 10 μl - -

Choesterol standard - 10 μl -

Test sample - - 10 μl

3. After proper distribution of the sample and reagent the three test tubes is mixed and
incubate at 20- 25C for 10 minutes or 5 minutes at 37°C. After incubation the optical
density or absorbance is read within 60 minutes against standard and reagent blank at
500- 546nm.
 Calculation:
Absorbance of test
Serum cholesterol conc.(mg/dl) = ------------------------------------ × Concentration ofStandard
Absorbance of Standard

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  DETERMINATION OF TRIGLYCERIDES INSERUM:
 Principle: Determination of triglycerides involves enzymatic splitting with lipoprotein
lipase. Indicator is quinoneimine , which is generated from 4 aminoantipyrine and 4-
chlorophenol by hydrogen peroxide under the catalytic action ofperoxide.

Glycerol+ ATP ----------------------------  Glycerol- 3- phosphate + ADP

 Reagentscomposition:
1. R1 - Triglyceride enzymereagent.
2. R2 – Triglyceride standardsolution.
 Sample:Fasting serum, heparin plasma or EDTA plasma.
 Reference value:
Desirable :<= 200 mg/dl
Borderline high risk : 200- 400 mg/dl
High risk: >= 400 mg/dl
 Assay procedure:
Wavelength:- 500 - 546nm
Light path:-1 Cm
Temperature:- 37°C
Measurement:- Against reagent blank.
1. All the reagent and sample are brought to the room temperature before start the assay.
2. Then reagent and sample are dispensed in three test tubes marked as for blank (B), for
standard (S) and for test(T).
Dispense Blank Standard Test
Reagent 1 1000μl 1000 μl 1000 μl

Distilled water 10 μl - -

TG standard - 10μl -

Test sample - - 10 μl

3. After proper distribution of the sample and reagent the three test tubes is mixed and
incubate at 20- 25C for 10 minutes or 5 minutes at 37°C. After incubation the optical
density or absorbance is read within 60 minutes against standard and reagent blank at
500- 546nm.
 Calculation:

Absorbance of test
Serum Triglyceride conc. (mg/dl) = -------------------------------------  Concentration of
Standard
Absorbance of Standard
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  DETERMINATION OF HDL- CHOLESTEROL IN/SERUM:
 Clinical significance: HDL- cholesterol and coronary heart diseases are inversely
related. Low concentration of HDL- cholesterol are associated with higher risk of
coronary heart disease. Thus HDL- cholesterol in combination with total cholesterol
determination is a good index of the risk of coronary heartdisease.
 Principle:Chylomicrons, VLDL (very low density lipoprotein) and LDL fractions in
serumorplasmaareseparatedfromHDLbyprecipitatingwithPhosphotungsticacidand
Magnesium chloride. After centrifugation, the cholesterol in the HDL fraction, which
remains in the supernatant, is assayed with enzymatic cholesterol method, using
cholesterol Esterase, cholesterol oxidase, Peroxidase and the chromogen 4 –
aminoantipyrine.
 Method: Pohosphotungstedmethod.
 Sample:Fasting serum is preferred for the test. EDTA or heparinised plasma can also
beuse.

 Reference value:HDL- cholesterol in plasma/serum 30- 70mg/dl.

 Reagents:
1. R1 - HDL enzymatic/chromogenicreagent.
2. R2 - Bufferreagent.
Working reagent:- one bottle of R1 is mixed with the one bottle of R2.
3. R3 – HDLstandard.
4. R4 – Precipitating reagent.
 Assayprocedure: 
The sample, the precipitating reagent and the working reagent are brought to the room
temperature before the use for the assay.
1. Precipitation:Atirst, sample and precipitating reagents are dispenses into a centrifuge
tubeaccording to following table-
Dispense Test

Serum/plasma 0.20 ml/ 200 μl

Precipitating reagent 0.20 ml/ 200 μl

Then the content of the tube is mixed and centrifuge at 3500- 4000 rpm for 10 minutes. Then
clear supernatant is separated immediately and the cholesterol is determined as following
formula in section 2.
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 2. Cholesterol
Assay:Wavelength:- 492-
550 nm Light path:-1 Cm
Temperature:-37°C
Zero setting: - Against reagent blank.
1. All the reagent and sample are brought to the room temperature before start theassay.
2. Then reagent and sample are dispensed in three test tubes marked as for blank (B), for
standard (S) and for test(T).
0 Blank Standard Test

Working reagent 1000 μl 1000 μl 1000 μl

HDL- standard - 20 μl -

Supernatant from section 1 - - 20 μl

3. After proper distribution of the sample and reagent the three test tubes is mixed and
incubate 5 minutes at 37°C. After incubation the optical density or absorbance is read
against standard and reagent blank.

 Calculation:
Absorbance of Test
Serum HDL- cholesterol conc. (mg/dl) = ---------------------------------  Conc. of Std. (50
mg/dl)
Absorbance of Standard

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  DETERMINATION OF SERUMBILIRUBIN:

Bilirubin is mainly formed from the haem portion of aged or damaged RBCs. It then
combined with albumin to form a complex which is not water soluble. This is referred to as
indirectorun-conjugatedbiliruin.Intheliverthisbilirubincomplexiscombinedwithgluconic acid
into a water soluble conjugate. This is referred to as conjugated or direct bilirubin. Elevated
levels of bilirubin are found in liver disease, excessive haemolysis (Haemolytic jaundice) and
obstruction of the biliary tract (Obstruct jaundice) and in drug induced reaction. The
differentiation between the direct and indirect bilirubin is important in diagnosing the cause
ofhyper-bilirubinaemia.

 Method:Malloy and Evelynmethod.

 Principle:The method is based on Van den Berg reaction. When bilirubin reacts with
diazo reagent, purple colored azobiliubin is formed. Method is used as reaction
accelerator, since total bilirubin is soluble in it. By using only distilled water, direct
bilirubin is determine. The difference between total bilirubin & direct bilirubin gives
measureofindirectbilirubin.Theopticaldensitiesoftotaltest&directtestaremeasured against
respective blanks at 540 nm (Green filter, 510-560).
 Normalrange:
Total bilirubin:- 0.25 – 1.00mg/dl
Direct bilirubin:- 0.00- 0.25mg/dl
Indirect bilirubin:- 0.25- 0.75 mg/dl

 Specimen:Fasting serum or heparinizedplasma.

 Requirements:
i) Test tube with test tuberack.
ii) Serologicalpipette.
iii) Stopwatch.
iv) Photometer.
 Reagents:
1. R1 DiazoA
2. R2 DiazoB
Diazo mixture:- add 1 bottle of Diazo A with 1 bottle of Diazo B.
3. Diazo blank
4. Methanol
5. Bilirubinstandard.

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  Procedure:
1. All the reagent sand sample are brought to room temperature before performing the test.
2. Four test tubes are taken and labeled.
3. Fresh diazo mixture is prepared by mixing 5.0 ml of diazo A and 0.15 ml of DiazoB.
4. Pipette all the reagents and sample are as follows-

Dispense Total test Total Blank Direct test Direct blank

Distilled water (ml) 1.8 1.8 1.8 1.8

Serum (ml) 0.2 0.2 0.2 0.2

Diazo mixture (ml) 0.5 - 0.5 -

Diazo blank reagent

- 0.5 - 0.5
(ml)

Methanol (ml) 2.5 2.5 - -

Distilled water (ml) - - 2.5 2.5

5. Keep it in dark place for 30minutes.

6. Intensity is read at 540 nm (Green filter).

7. Opticaldensityoftheartificialbilirubinstandardistobereadat540nmbytransferring the
standard solution in a drycuvette.
 Calculation:
O.D of total Bilirubin = O.D of total test – O.D of total blank.
O.D of direct Bilirubin = O.D of direct test – O.D of direct blank.
O.D of total Bilirubin
Total Bilirubin mg/dl = ----------------------------------  standard concentration (10
mg/dl)

O.D of standard
Direct Bilirubinmg/dl= ----------------------------------  Standard concentration (10 mg/dl)
O.D of standard
Indirect Bilirubin mg/dl = (Total bilirubin – Direct bilirubin) mg/dl.
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  DETERMINATION OF TOTALPROTEIN:
Proteins are constituents of muscle, enzymes, hormones and several other key functional and
structuralunitsinthebody.Theyareinvolvedinthemaintenanceofthenormaldistributionof water
between blood and the tissues. Consisting mainly of albumin and globulin the functions vary
independently and widely in disease. Increase levels are found mainly in dehydration.
Decrease levels are found mainly in malnutrition, impaired synthesis and protein losses as in
hemorrhage or excessive proteincatabolism.
 Method:Modified Biurate method.
 Principle:The peptic bond of protein reacts with the cupric ion in alkaline solution to
form a colour chelate compound, the absorbance of which measured in 578 nm. The
biurate reagent contains sodium tartarate,potassium tartarate,which help sinmaintaining
solubility of this complex in alkaline media. The absorbance of the final colour is
proportional to the concentration of the total protein in thesample.
 Normalvalue:
Adult: - 6.4- 7.8 gm/dl
Children:- 6.0- 7.5 gm/dl
Specimen:- 10- 12 hours fasting blood serum
Reagents:
1. R1 – Biuretreagent.
2. R2 – Proteinstandard.
 Otherrequirements:
i) Test tube with test tuberack
ii) Serologicalpipette
iii) Colorimeter.
 Procedure:
1. At first all the reagent and sample should be brought to room temperature before
performing thetest.
2. Three test tube is to be taken and leveled Blank(B), Standard (S) and Test (T)
respectively.
3. Pipettes all the reagents and sample are asfollows-
Dispense Blank Standard Test
Biurate reagent 1 ml 1 ml 1ml
Serum - - 0.01 ml
Protein standard (6.5 gm/l) - 0.01 ml -
4. Mix and incubate at 37C for 5minutes.
5. After5minutesmeasuredtheabsorbanceofstandardandtestagainstblankin578nm,
calorimetrically. :
6. Calculation
O.D of the test
Total serumprotein(gm/dl)= --------------------------------------- Standard concentration (8gm/dl)
O.D of the standard

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  DETERMINATION OF SERUMALBUMIN:

Albuminconsistsofapproximately60%oftetotalproteininthebody,the major part consist of

globulin. It is synthesized in the liver and maintains the osmotic pressure in body. Albumin
also helps in the transportation of drugs, hormones and enzymes. Elevated levels are rarely
seen and are usually associated with dehydration. Decreased levels are seen in liver diseases
(Hepatitis,Cirrhosis).Malnutrition,kidneydisorders,increasefluidlossduringextensiveburns and
decreased absorption in gastro- intestinal disease.

 Method:BCGmethod.

 Principle:Albumin binds with dye Bromocresol green in a buffered medium to form a

green coloured complex. The intensity of the colour formed is directly proportional to
the amount of the albumin present in thesample.

 Normalvalue:

Albumin- 3.5 – 5.0gm/dl.

Globulin- 2.4 – 3.2gm/dl.

 Reagents:
1. BCGreagent.
2. Albuminstandard.
3. Distilledwater.

 Requirements:
i) Test tubes with test tuberack.
ii) Serologicalpipettes.

 Sample:Fastingserum.

 Procedure:
1. At first all the reagent and sample should be brought to room temperature before
performing thetest.
2. Three test tubes are to be taken and leveled Blank (B), Standard (S) and Test (T)
respectively.
3. Pipettes all the reagents and sample are asfollows-

79 | P a g e
 Dispense Blank Standard Test

BCG reagent 1 ml 1 ml 1ml

Distilled water 10μl - -

Albumin standard - 10μl -

Sample - - 10μl

4. Mix and incubate at room temperature for 5minutes.

5. After 5 minutes measured the absorbance of standard and test against blank in 630
nm,calorimetrically.

 Calculation:
O.D of the test
Serum Albumin (gm/dl) = -------------------------------  standard concentration (4gm/dl)
O.D of the standard

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 SERUM ALKALINE PHOSPHATASE TEST :

CLINICAL SIGNIFICANCE :
Human ALP consists of a group of enzymes which hydrolyse phosphates at an alkaline pH. ALP is
found in practically all tissues of the body but in high concentrations in the osteoblasts of bone, liver,
placenta, kidney, intestinal wall and lactating mammary glands. In adults the ALP normally found
circulating in the serum is largely derived from the liver. In children or in adolescents going through
pubertal growth spurts, there is an additional contribution from bone and this accounts for the higher
reference interval for these groups. Pregnancy also raises the normal values of ALP. Raised ALP
levels are often observed in bone disease or liver disease involving the biliary tract. If the source of
the isoenzyme is not apparent then estimation of GGT may help differentiate between the two. A
raised GGT in the presence of a raised ALP would suggest the liver is the primary source. Increased
ALP (usually normal GGT) is seen in Osteomalacia and Rickets, primary hyperparathyroidism with
bone involvement, Pagets disease, secondary carcinoma in bone and some cases of osteogenic
sarcoma. Increased levels of ALP (usually with a raised GGT) is seen in cholestasis, hepatitis,
cirrhosis, space occupying lesions and malignancy with bone or liver involvement or direct
production. Low levels of ALP may be observed in conditions which cause arrested bone growth or in
hypophosphatasia.

PRINCIPLE :
The method according to IFCC recommendation. This method utilises 4-nitrophenyl phosphate as the
substrate. Under optimised conditions ALP present in the sample catalyses the following reaction.
ALP
AMP + 4-NPP +H2O 4- nitrophenol + phosphate .
Mg2+/alkaline Ph

At the pH of the reaction, 4-nitrophenol has an intense yellow colour. The reagent also contains a
metal ion buffer system to ensure that optimal concentrations of Zinc and Magnesium are maintained.
The metal ion buffer can also chelate other potentially inhibitory ions which may be present. The
reaction is monitored by measuring the rate of increase in absorbance at 405 or 415 nm which is
proportional to the activity of ALP in the serum.
PROCEDURE :

Wavelength: 420 (405 – 430) nm

Cuvette: 1 cm

Two reagents method - substrate start

Reagent blank Calibrator Sample

Reagent 1 0.800 ml 0.800 ml 0.800 ml
Sample - - 0.020 ml
Calibrator - 0.020 ml -
Distilled water 0.020 ml - -

81 | P a g e
Mix and after 5 min .incubate 37 degree C .

Reagent 2 0.200 ml 0.200 ml 0.200 ml

Mix, incubate 1 min. at 37°C and then measure the initial absorbance of calibrator and sample against
reagent blank. Measure the absorbance change exactly after 1, 2 and 3 min. Calculate 1 minute
absorbance change (ΔA/min)
CALCULATION :
Δ Asam /min
. 1. ALP (U/l) × Ccal
Δ Acal /min
Ccal = calibrator concentration
2. Using factor: ALP (U/l) = f x ΔA/min
f = factor
f = 2764 (at 405 nm)

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 DETERMINATION OF TSH :
Principle :
The TSH is a solid phase sandwich ELISA method. The samples, and anti-TSHHRP/Biotin conjugate
are added to the wells coated with Streptavidin. TSH in the patient’s sample forms a sandwich
between two specific antibodies to TSH. Unbound protein and HRP conjugate are washed off by
wash buffer. Upon the addition of the substrate, the intensity of color is proportional to the
concentration of TSH in the samples. A standard curve is prepared relating color intensity to the
concentration of the TSH.
Material required:

1. Precision pipettes: 10-100µl, 20-200µl, 100-1000µl

2. Disposable pipette tips

3. Disposable Gloves
4. ELISA reader
5. ELISA washer STORAGE AND STAB
6. D.W

TEST PROCEDURE
1. Secure the desired number of coated wells in the holder. Dispense 25 µl of Standards and Serums
into the appropriate wells.
2. Dispense 50 µl of Enzyme Conjugate reagent into each well. Incubate at room temperature (18-
25°C), for 45 minutes.
3. Remove the incubation mixture by emptying the plate content into a waste container. Rinse and
empty the microtiter plate 5 times with Wash Buffer (1X). Strike the microtiter plate sharply onto the
absorbent paper or paper towels to remove all residual water droplets.
4. Dispense 100 µl of TMB Substrate into each well. Incubate at room temperature(18-25°C) in the
dark, for 20 minutes.
5. Stop the reaction by adding 100 µl of Stop Solution to each well. Gently mix for 10 seconds until
the blue color completely changes to yellow.
6. Read the optical density at 450/630 nm with a microtiter plate reader within 15 minutes.

CALCULATION OF RESULTS:Construct a standard curve by plotting the absorbance obtained

from each reference standards against its concentration in µIU/ml on the graph paper, with
absorbance values on the vertical or Y axis and concentrations on the horizontal or X axis. Use the
absorbance values for each specimen to determine the corresponding concentration of TSH in µIU/ml
from the standard curve. Any diluted specimens must be corrected by the appropriate dilution factor.

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 DETERMINATION SERUM AMYLASE :

CLINICAL SIGNIFICANCE :
α-Amylase is derived mainly from the salivary glands and the exocrine pancreas. α-Amylase catalyses
the hydrolysis of α-1-4 glucosidic linkages of starch and other related polysaccharides to produce
maltose and other oligosaccharides. The enzyme is a relatively small molecule which is rapidly
cleared by the kidneys and excreted in the urine. α-Amylase is most frequently measured in the
diagnosis of acute pancreatitis when serum levels may be grossly elevated. In acute pancreatitis α-
amylase starts to rise approximately 4 hours after the onset of pain, reaches a peak at 24 hours and
remains elevated for 3-7 days. Hyperamylasamia is also associated with other acute abdominal
disorders, biliary dysfunction, salivary gland disorders, ruptured ectopic pregnancy and
macroamylasamia.

PRINCIPLE:
2-Chloro-4-nitrophenol-β -1- 4 galactopyranosylmaltotrioside (CNP-G) is a direct substrate for
determination of α-amylase activity, which does not require the presence of ancillary enzymes. The
rate of 2-chloro-4-nitrophenol formation can be monitored at (400-420) nm and is proportional to the
α-amylase activity.
Amylase
Gal – G2 - α-CNP Gal – G2 + CNP
PROCEDURE:
Wavelength: 405 (400 – 420) nm
Cuvette: 1 cm
Working solution 1000 µ
Sample 20 µ

Mix, incubate 1 min. at 37°C and then measure the initial absorbance of calibrator and sample against reagent
blank. Measure the absorbance change exactly after 1, 2 and 3 min. Calculate 1 minute absorbance change
(ΔA/min).
CALCULATION:
Δ Asam /min
1. Amylase activity (U/l) × Ccal
Δ Acal /min.
Using factor: Amylase activity (U/l) = f x ΔA/min f = factor f = 3128 (at 405 nm)

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DETERMINATION OF CK MB:
Clinical significance : Creatine kinase-MB (CK-MB) is a form of an enzyme found primarily in
heart muscle cells. This test measures CK-MB in the blood. CK-MB is one of three forms (isoenzymes) of the
enzyme creatine kinase (CK). Test results may vary depending on your age, gender, health history, the method
used for the test, and other things. Your test results may not mean you have a problem. Ask your healthcare
provider what your test results mean for you. Levels of CK-MB do not rise in your blood within the first 4 to 6
hours after a heart attack. You may need to have repeated tests to see if you've had a heart attack. Higher levels
of CK-MB may mean that you have had a heart attack or have other heart problems. These include:
Myocarditis, an infection and inflammation of the heart muscle Pericarditis, an infection and inflammation of
the thin sac that surrounds the heart Cardiac defibrillation, when an electric shock is used to fix the heart
rhythm. Higher levels of CK-MB may also mean more of the heart was damaged in the attack. Higher levels
may also be caused by muscle damage elsewhere in your body, by diseases that affect your muscles, and by
trauma to your chest.
PRINCIPLE:An antibody to the anti CK-M inhibits completely CK-MM and subunit (M) of the CK-
MB. The activity of the non inhibited CK-B subunit is then assayed by the following series of
reactions:

CK Phosphocreatine +ADP creatine +ATP

ATP + Glucose ADP +Glucose-6-phosphate.

G6P + NADP 6-Phosphogluconate + NADPH + H+ .

The rate of NADPH formation, measured photometrically, is proportional to the catalytic

concentration of CK-B present in the sample.
Working reagent (WR):
 Dissolve one tablet of R 2 in one vial of R 1. Cap vial and mix gently to dissolve contents. Stability:
8 days at 2-8°C or 24 hours at 15- 25°C.
PROCEDURE :
1. Assay conditions: Wavelength: ……………………………………..……340 nm
Cuvette......................................................... 1 cm light path
Constant temperature.................. 25°C / 30°C / 37°C
2. Adjust the instrument to zero with distilled water or air.

3. Pipette into a cuvette:

Working reagent 10ml
Sample 40 µL

4. Mix then Incubate for 10 minute.

5. Read initial absorbance (A) of the sample, start the stopwatch and read again after 5 minutes (A2).
6. Calculate the difference between absorbances : ΔA= A2 – A1.
CALCULATIONS:
l . ΔA x 825 = U/L de CK-B ΔA x 1651 = U/L de CK-MB
Units: One international unit (IU) is the amount of enzyme that transforms 1 μmol of substrate per
minute, in standard conditions. The concentration is expressed in units per liter of sample (U/L).
85 | P a g e
 REPORTANDFINDING
Biochemistry (FBS,PPBS,NA,K)

SL PATIENT ID A SEX DATE GLUCO GLUCOS NA+ K+

NO G SEFAST EP.P. (mg/dl) (mg/dl)
E ING (mg/dl)
(Y) (mg/dl)

1. MCHK/84956 41 M 28/10/23 95 121 138.8 4.03

2. MCHK/84959 20 F 29/10/23 83 130 142.4 3.89

3. MCHK/84989 25 F 07/11/23 220 580 138.2 4.74

4. MCHK/84990 28 F 07/11/23 122 216 144 4.92

5. MCHK/84991 48 M 07/11/23 84 134 140.9 4.14

6. MCHK/84992 36 M 07/11/23 88 149 135.5 4.87

7. MCHK/84998 46 M 08/11/23 110 155 137.5 4.73

8. MCHK/84899 62 M 09/11/23 96 130 139.8 4.52

9. MCHK/84960 55 F 12/11/23 80 133 141.5 4.43

10. MCHK/84985 56 M 14/11/23 111 250 144.8 4.60

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 REPORT
Biochemistry (LFT, CRP)

S NA M E AG SE DATE TOTAL DIREC SGO SG ALKA TOT ALBU CRP

LN E( X BILIRU T T PT PHOS AL MIN
O. Y) BIN BILIRU PRO
BIN TEIN

11 MCHK/8 48 M 04/11/23 0.5 0.3 115 85 75 7.7 3.5 -

5123
12 MCHK/85 39 F 04/11/23 2.7 1.5 47 53 451 7.7 4.4 -
124
13 MCHK/85 25 F 04/11/23 0.1 0.6 127 86 72 5.2 1.8 6.8
125
14 MCHK/85 28 F 04/11/23 2.7 0.8 58 59 117 7.2 7.5 -
126
15 MCHK/85 48 M 04/11/23 0.9 0.3 27 18 144 7.4 2.2 1.9
127
16 MCHK/85 28 M 04/11/23 1.7 0.5 35 34 332 7.1 2.9 -
128
17 MCHK/85 46 M 04/11/23 0.3 0.8 38 24 180 6.0 3.4 -
129
18 MCHK/85 62 M 04/11/23 0.9 0.1 20 26 107 7.2 1.0 4.5
130
19 MCHK/85 55 F 04/11/23 3.5 1.8 41 27 78 7.1 3.9 2.9
131
20 MCHK/85 56 M 04/11/23 1.0 0.9 44 37 98 6.8 3.2 4.8
132

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CLINICAL PATHOLOGY &
HEMATOLOGY

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DEPARTMENT OF CLINICAL PATHOLOGY &HEMATOLOGY (CENTRAL LABORATORY)

 CLINICALPATHOLOGY
 Routine examination of Urine.
 Examination of various body fluids by Neubauer chamber.
 Examination of OPD blood samples by using Hematological analyzer.
 Differential count of OPD blood samples by leishman staining method.

 HAEMATOLOGY
 Collection of blood samples for Prothrombin time andAPTT.
 Counting of P. time and APTT byanalyzer.
 Preparation of peripheral blood smears of IPD bloodsamples.
 Staining of blood smears by using leishmanstain.

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 CLINICAL PATHOLOGY

1. Urine test (Physical, Chemical &Microscopical).

2. Stool test (Physical, Chemical &Microscopical).
3. Occult blood test in urine andstool.

Working in clinical pathology laboratory are required to examine various body fluids, like
blood,urine,spinalfluid,Pleuralfluid,serousfluidandsynovialfluid.Laboratoryinvestigation in
clinical pathology primarily focuses on physical examination of fluid, clinical microscopy
and simple chemicalscreening.

COLLECTION OF URINE: There are various methods for collection of urine. Method of
collection to be used depends on the nature of investigation .
Time of Collection
1. A single specimen: This may be a first morning voiding, a random specimen, or a post-
prandial specimen. The first voided specimen in the morning is the most concentrated and has
acidic pH in which formed elements (cells and casts) are well preserved. This specimen is
used for routine examination, fasting glucose, proteins, nitrite, microscopic analysis for
cellular elements, pregnancy test, orthostatic proteinuria, and bacteriological analysis.
The random specimen is a single specimen collected at any time of day. It is used for
routine urine examination. Post-prandial specimen (collected 2 hours after a meal in the
afternoon) is sometimes requested for estimation of glucose (to monitor insulin therapy in
diabetes mellitus) or of urobilinogen.
2. 24-hour specimen: After getting up in the morning, the first urine is discarded. All the
urine voided subsequently during the rest of the day and the night is collected in a large bottle
(clean bottle of 2 liter capacity with a cap). The first urine after getting up in the morning on
the next day is also collected. The urine should be preserved at 4-6°C during the period of
collection. The container is then immediately transported to the laboratory. The urine is
thoroughly mixed and an aliquot is used for testing. This method is used for quantitative
estimation of proteins and hormones.
Preservation of Urine Sample The urine sample should ideally be examined within 1-2 hours
of voiding.
• Hydrochloric acid: It is used for preservation of a 24- hour urine sample for adrenaline,
noradrenaline, vanillylmandelic acid, and steroids.
• Toluene: It forms a thin layer over the surface and acts as a physical barrier for bacteria and
air. It is used for measurement of chemicals.
• Boric acid: A general preservative.
• Thymol: It inhibits bacteria and fungi.
• Formalin: It is an excellent chemical for preservation of formed elements.

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 EXAMINATION OFURINE:
 PHYSICAL EXAMINATION OFURINE:
 Volume :. The average 24-hr urinary output in adults is 600-2000 ml. The volume varies
according to fluid intake, diet, and climate.

 Colour:Normal urine color nearly colorless to dark yellow or strawcolor:

 Appearance:Normal, freshly voided urine is clear in appearance. Causes of cloudy or turbid
urine. Foamy urine occurs in the presence of excess proteins or bilirubin.

 Odor:Freshly voided urine has a typical aromatic odor due to volatile organic acids. After
standing, urine develops ammoniacal odor (formation of ammonia occurs when urea is
decomposed by bacteria).

 Sp.Gravity: To measure the Sp. Gravity of urine we normally use Refractometer

method.
 Materials:
I. Refractometer scale.
II. Urinesample.

 Refractometermethod:
1. Clean the glass surface of the instrument with distilled water and soft cloth.
2. Close the cover over the glass surface and insert a drop of urine sample with the help
of Pasteurpipette.
3. Hold the instrument up to a lightsource.
4. Read the Sp. Gravity from the scale at the point where the dividing line between the
bright and dark fieldsmeet.

 CHEMICAL EXAMINATION OFURINE:

 pH TEST:pH normal urine varies from 6 to 6.8 which is acidic. A rough estimated of
pH is made by indicator paper, blue litmus or wide range indicatorpaper.
 TEST:To estimate the albumin of urine “heat and acetic acid test” s is require.
 Materials:
1. Urinesample.

2. 3% glacial aceticacid.

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  HEAT AND ACETIC ACIDTEST:
 Procedure:

a. The urine should be acidic, a few drops of 3% acetic acid is added to an alkaline urine
to make it slightlyacidic.
b. Urine should be clear,turbid.

c. At first centrifuge the turbid urine in a testtube.

d. Taken out the supernatant portion forexamination.

e. Take urine in a clean test tube filling 2/3portion.

f. Boil upper half inch of thetube.

g. A white cloud appears in a heated portion, due to presence of protein orphosphate.

h. Add 2 to 3 drops of 3% glacial aceticacid.

i. Ifcloudinessdisappears -------- Phosphate.

Ifcloudinesspersists ------------ Albumin.

 Observation:

Negative --- Nocloudiness.

Trace --- Barely visiblecloudiness.

1+ --- Definite cloud without granular flocculation.

2+ --- Heavy and granular cloud without flocculation.
3+ --- Dense cloud without markedflocculation.
4+ --- Thick, cloudy precipitate andcoagulation.

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 SUGAR TEST:
To sugar test of the urine “Benedict’s Test” isessential.
 Materials:
I. Benedict’s qualitativereagent.
II. Urinesample.
 BENEDICT’STEST:
a. 5ml Benedict’s qualitative reagent taken in a testtube.
b. Boil for 1minute.
c. Add 8 drops urine drop bydrop.
d. Boil for 3 to 5 minutes, then cool themixture.

Observation:

Negative --- No change incolor.

Trace --- Pale green with slide cloudiness.

1+ --- Greendeposit.

2+ --- Yellowdeposit.

3+ --- Orange to redprecipitate.

4+ --- Brick red precipitate.

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  MICROSCOPIC EXAMINATION OFURINE:
 Materials:
I. Urinesample,
II. Centrifugetube,
III. Glass slide,
IV. Cover slip,
V. Microscope.
 Method:
1. Mix the urine very well and pour into a 10 ml centrifuge tube.
2. Centrifuge with another balanced test tube for 3-5 minutes at 2500RPM.
3. Pour off the supernatant quickly and completely into another test tube.
4. Re-suspended the deposit by shaking thetube.
5. Place 1 drop of the deposit on a glassslide.
6. Cover it with a coverslip.
7. Observe it first under low power objective in a subdued light.
 Observation:
1. Red cells, pus cells, renal epithelial cell are identified under high powerobjective.
2. Casts are identified under low power objective, but finer structures are identified
under HP [High power] objective.
3. Crystyals are examined under low powerobjective.
4. Bacteria, yeast cells, trichomonas are examined under HP objective.

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  OCCULT BLOOD TEST INURINE:
 Method: Benzidinemethod.
 Principle:The peroxides activity of hemoglobin decompose hydrogen peroxide and the
liberate active oxygen oxidases the compou ndbenzidine.
 Reagents:
1. Saturated solution of benzidine in glacial acetic acid.
2. Hydrogen peroxide (3% v/v in distilled water).
 Procedure:
1. Mix equal parts of tworeagents.
2. Add equal volume ofurine.
3. The appearance of a green or blue colour within 5 min indicates presence of blood.
Report asfollows.
Trace --- Faint green
1+ --- Green
2+ --- Greenish blue
3+ --- Blue
4+ --- Deep blue

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 HEMATOLOGY

I. Blood Sample Collection by Venipuncture

II. Routine haematological test

 Heamoglobin estimation
 Total count of RBC
 Total count of WBC
 Differential count of WBC in peripheral blood smear
 Total platelet count
 Erythrocyte sedimentation rate(ESR)
 Packed cell volume
 Red cell indices (MCV, MCH, &MCHC)
Reticulocyte count
III. Coagulation profil etest:
 ClottingTime.
 BleedingTime.
 ProthrombinTime.

IV. Special HeamatolgicalTest:

 Absolute EosinophilCount.
 HLA Typing (Human Lymphocyte Analysis).

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 BLOOD SAMPLE COLLECTION BYVENIPUNCTURE:

The Volume of blood obtained by venipuncture is sufficient to carry out multiple

tests. Venipuncture can be done either by the syringe method or vacuum tube method. The
latter is disposable and is not very popular in developing countries because of the high coast.

 Procedure:
 Allthethingsareassembledrequiredduringbloodcollection.Thethingsaretourniquet,
cotton, alcohol, syringe, container, anticoagulantetc.
 Patient is identified and decided the total amount of blood needed for all the tests.
 The containers are selected and labelled them with the patient’sidentification.
 Then the patient is asked to sit alongside the table used for blood collection, Patients
arm is laid on table and palm upwards. For indoor patients are said to lay arm in an
outstretchedposition.
 The puncture is selected carefully after toeing up thetourniquet.
 Afterfeelingtheveininlefthandtheskinisdisinfectedwithaswabdippedinmethanol or
70%alcohol.
 The syringe is then checked it the needle is fixedtightly.
 After holding the syringe in right hand and bevel with uppermost it is pushed firmly
and steadily into the centre of vein at 30-40°angle.
 Then the blood is appeared in barrel and the tourniquet is released without disbursing
the needle.
 A Swales of cotton over the hidden point of the needle is placed.
 The needle is removed from vein and the patient is asked to press the cotton swabs for
fewminutes.
 The needle is removed from syringe and gently expelled the blood into appropriate
container.

 The blood is mixed immediately and thoroughly but gently with anticoagulant (it
required).
 Immediately the syringe and needle is rinsed without cold water to preventclotting.
 If bleeding is topped, an adhesive tape is applied on the puncturesite.

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  ROUTINE HEMATOLOGICALTEST:

 DETERMINATION OF HAEMOGLOBIN CONCENTRATION(HB%):

Hemoglobin is a conjugated protein present in RBCs. It carries oxygen from the lungs to
the tissue cells and carbon dioxide- The gaseous waste from the cells to the lungs. Hemoglobin
consists of two components-haem (Iron+Proteoporhyrin) and globin (amino acid chains).There
are three types of hemoglobin present in adultperson

Structure Normal%

a. HbA (adult HB) --------- α2β2 97-98%

b. HbA2 --------- α2δ2 1.5-3%
c. HbF (foetal Hb) --------- α2γ2 Absent
 Clinicalsignificance:
A decrease in haemoglobin conc in blood below normal is a sign of anaemia and this
leadstoreduceoxygencarryingcapacityleadingtoanoxemlaischemicchangesandultimately
tonecrosis.
Elevated haemoglobin levels can be observed in Ploycythemia congenital heart diseases.
Normal Values:
Male-15-18 gm%
Female-11-16gm%
Children (at1yr)-10-14 gm%
Infants-14-20gm%
 Cyanmethemoglibin Method:
 Principle:In the presence of potassium Ferricyanide at alkaline pH, hemoglobin and its
derivatives are oxidized to methemoglobin. Methemoglobin, so formed reacts with
potassium cyanide to form cyanmethemoglobin, a red coloured complex, which is
measured colorimetri 540 nm (green filter). The color intensity is proportional to the Hb
concentration of the bloodsample.
Haemoglobin + Potassium ferricyanide-Methemoglobin.

Mehemoglobin + Potassium cyanide- Cyanmethemoglobin.

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  Reagents:
Reagent-1: Drabkin’s Solution (contain Potassium cyanide-50mg & Potassium
ferricyanide 200mg & Distilled water-1000ml)
Reagent-2: Cyanmethemoglobin std.
Sample: Whole blood sample (0.02 ml) is used.
 Procedure:
1. Take test tubes for three test tubes for blank rendered and test. Level them and
proceed as followingsteps.
Pipette into test tubes Test Blank Standard

Drabkin solution 1.0ml --- 5.0ml

Cyanmethemoglobin std --- 1ml ---

Sample 20μl --- 20µl

It is mixed thoroughly and after 5minutes the test sample is measured against blank at 540nm.
Standard: The O.D. of Reagent 2 (Cyanmethemoglobin standard) is directly measured either
on the spectrophotometer on the colorimeter against Drabkin’s solution.

 Calculations:
Blood Hb in gm %= OD of test/OD of std ×concentration of std. Mg% × Dilution factor
= OD of test/OD of std × 60 × 0.250
= OD of test/OD of std × 15
 Precaution:Store Drabkin’s reagent in brown bottle, as it decomposes on light
exposure. Once hehemoglobin cyanide solution has been prepared, Hb estimation must
be carried out with in 6 hours.
Drabkin’s solution should be clear and pale yellow. If it is turbid, it should be
discarded. It can be stored at cool temperature, 4-6°C in the refrigerator.
 Acid Hematin Method (Sahli’s Method):
It is recommended for place where colorimeter is available. It is not recommended
because all forms of Hb are not measured by this method.

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 Principle:Hb is converted to acid hematin by reaction with HCL. The acid hematin
solution is further diluted with HCL acid until its colour matches exactly that of
permanent standard of comparator box. He Hb concentration is read directly from the
hemoglbinometertube.
 Reagent andEquipments:
1. 0.1 N Hydro Choric Acid,
2. Sahli Haemoglobinometer,
3. Haemoglobinometer pipette.
4. Haemoglobinometer tube.
 Procedure:
1. Fill the haemoglobinometer tube up to 20 mark with 0.1 N Hydro ChloricAcid.
2. Draw the blood onto haemoglobinometer pipette (20µl) from anticoagulated test
bloodsample.
3. Wipe out surface of the pipette with the wetcotton.
4. Blowthebloodintoacidsolutionofhaemoglobinometertubebyadding0.1NHCL, until
the colourmatch.
5. ReadtheHbconcentrationdirectlyfromthemarkreached.Thereadingmaybein% or
ingm/dl.

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 DETERMINATION OF TOTAL COUNT OFRBC:
 Introduction:The Human red cell is normally a circular ,non nucleated,biconcavedisc.
The red blood cell contain hemoglobin.The surface area of red cell is much greater than
that ofthe sphere of the same size. There are exchange of oxygen &carbon dioxide is
maximalwith the biconcave configuration.
 Clinical significance:The total RBC count is performed to assess the red cell mass in
the blood. The change in erythrocyte number is frequently detected in clinical practice
by estimation of hemoglobin rather than total RBC count, as estimation of hemoglobin
easy and less expensive more over total RBC count is still performed in some
conditions to detect he red cell population, especially if the count is expectedto be a
very high as in polycythemia
 Principle:The blood specimen is diluted with RBC diluting fluid which does not
remove the WBC but allows the RBC to be counted under 400X magnification in a
known volume of fluid. Finally, the number of cells in undiluted blood is calculated.

 Specimen:Well mixed anticoagulated (EDTA) venous blood is mostlyused.

 Requirements:
i. RBC dilutingfluid:
 Trisodiumcitrate 3.2 gm.
 Formalin 1.0gm.
 Distilledwater 100ml
ii. Micro-pipette
iii. Graduatedpipette
iv. Improved neubauer chamber with coverslip

 Method:Hemocytometric method [include improved hemocytometerchamber]

Normal Range :

Male : 4.5-6.0×106 Cell/µl of Blood

Female: 4.0-4.8×106 Cell/µl ofBlood

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 Procedure:

1). Collect all the equipment which needed for practical, & clean all the equipment
properly.

2). Take adequate RBC diluting fluid in a watch glass.

3) Make a finger prick under aseptic conditions & such blood in to the pipette & dilute
the blood with RBC diluting fluid.

4). After proper mixing take some amount of mixed diluting fluid through pipette.
5). Discard first two drops of fluid from the pipette.
6). Charge the neubauer’s chamber and allow two to five minutes for proper charge by
which the cells settle downeasily.

7). Place the counting chamber.

8). Switch to low power objective, adjust light and locate the large square in the
centre

9. Now switch to high power objective.

10). The red blood cells are counted in the four squares including centre of neubauer’s
chamber.

 Calculation:

TotalNOofRBC×Dilutingfactor
Total RBCCount =
Total no of area count × area of each squre × depth of fluid

If Total NO of RBC=N
Diluting factor=200
Total NO of area=5
Total NO of each area square=1/5
Depth of fluid=1/10-0.1
So, Total RBC Count=N × 200/5 × 1/5 × 1/5 × 1/10= N × 200 × 50=N × 10,000

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  DETERMINATION OF TOTAL COUNT OF WBC[LEUKOCYTE]:
 Introduction:Leukocyte [White Blood Cell] are nucleated cells that are involved inthe
defense mechanism of the body, Unlike red cells, White cells use the blood stream
primarily for transportation to their place of function in the body tissue. Leukocytes are
classified as granulocytes, & agranulocytes. Granulocytes are neutrophils, eosinophils,
basophils & agranulocytes are lymphocytes &monocytes.
 Clinical Significance:Total leukocyte counter is a part of the routine heamotologic
investigation to assesss the nature & severity of an infection in some diseases,alteration
inleukocytecountalonemaybediagnostic,butfrequentlytheleucocytecountisordered with
other investigations especially with the differential leukocyte count to aid in diagnostic.

When the total leukocyte count increase above the normal the condition is called
leucocytosis and when the count decrease below normal range then the condition is
called leucocytopenia.

 Specimen:EDTA mixed venous blood or fresh capillaryblood.

 Principle:Bloodisdilutedwithacidsolution,whichremovestheredcellsbyheamolysis
&accentuatesthenucleiofthewhitecells.Thecountingofthewhitecellsthenbecomes
easycountingisdoneusingamicroscopeunderlowpowerobjectiveandwithknowledge

of the volume of fluid examined & the dilution of the blood obtained. The number of white
cells/mm2 of undiluted whole blood is calculated.
 Method:Hemocytometric method [include improved hemocytometerchamber]
 Requirement:

i) Microscope

ii) Improved neubauer chamber

iii) WBC pipette

iv) WBC diluting fluid [Turk’sfluid]

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 oComposition of WBC diluting fluid:

i). 1% glacial acetic acidsolution.

ii). Creation violet stain or aqueous methylene blue [0.3%]

iii). Distilledwater.

 NormalRange:
Adults : 4,000-11,000/mm3 of blood

Newborns : 10,000-25,000/mm3 of blood

Infants : 6,000-18,000/mm3 of blood

Children : 5,000-15,000/mm3 ofblood

 Procedure:

1). Assemble al equipment needed for practicals clean it properly.

2). Take adequate WBC diluting fluid in a watch glass.

3). Make a finger prick under aseptic conditions & such blood into the pipette &dilute
the blood with WBC dilutingfluid.

4). After proper mixing take some amount of mixing diluting fluid through pipette.

5). Discard the first two drops of fluid from thepipette.

6. Charge the neubauer’s chamber allow two-five minutes for proper charge bywhich
the cells settle downeasily.

7. Place the countingchamber.

8. Switch to low power objective, adjust light & located the large square in thecentre.

9). Now switch to high powerobjective.

10 .The white blood cells in the four corner squares arecounted.

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  Calculation:

White cell total count = Total NO of WBC × diluting factor/Total number of area count × area
of each square × depth of fluid

Where,

i) Dilution =1:20,

ii) Area counted =4

iii) Each square =1mm

iv) Depth of fluid = 1/10 =(0.1)

v) No of white cells counted =N

Now Total white blood cells = N × 20/4 × 1 × 1 ×1/10 = N × 20 × 10/4 = N × 50

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  DETERMINATION OF DIFFERENTIAL COUNT OF WBC:
 Introduction: Differential count is the percent (%) distribution of various white cells in
the peripheral blood as determined from a blood smear stained with a polychromatic
stain.
 Clinical Significance: Differential count is useful to identify change in the distribution
of white cells which may be related to specific type of disorders. It also gives idea
regarding the severity of the diseases and the degree of response of thebody.
 Principle:The polychromic staining solution contain methylene glue and cassin. The
basic and acidic dyes induced multiple colors when applied to cell. Methanol acts as
fixative and also a solvent the natural components of the cell are stained by bothdyes.
 Speciman:EDTA-Anti coagulated blood or fresh capillaryblood.

 NormalRange:

i) Neutrophils -40%-75%

ii) Eosinophils -1%-6%

iii) Basophils -0%-1%

iv) Lymphocytes -20%-45%

v) Monocytes -2%-10%

 Requirements:

a) Microscope slider & A glassspreader

b) Cederwoodoil

 Reagent:

a) Leishmanstain:

i) Leishmanpowder -0.5gm

ii) Acetone freemethylalcohol -100ml

iii) Eosin -7gm

b) Buffer (pH7):

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  Procedure:Thin smear is prepared by spreading a small drop of blood evenly aslide.
o Blood FilmPreparation:
1. Take a clean dry grease freeslide.
2. Transfer a small drop of blood near the edge of theslide.
3. Place the spreader slide at an angel of 30°degree. Pull back the spreader until it
touches the drop of blood. Let the blood run among the edge of the spreader.
4. Push the spreader forward to the end of the slide with a smoothmovement.
5. Dry the blood smear at roomtemperature.
o Staining TheFilm:
1. Coverthesmearwiththestainingsolutionbyadding10-15dropsonthesmear.Wait for
2minutes.

2. Add equal number of the drops of buffer solutions. Mix the reaction mixture
adequately by blowing on it through a pipette. Wait for 10minutes.
3. Wash the smear by using tapwater.
4. Stand the slide in a drainingrack.
o Examination Of Film:
1. First examine the stained smear under the low power (40 X) and adjust thefilm.
2. Then examine the film under high power (60 X).
3. Examine the film by moving from one field to the next field. Record the type of
Leukocytes seen in eachfield.
4. Count at least a total of 100 Leucocytes.
5. Add equal number of the drops of buffer solutions. Mix the reaction mixture
adequately by blowing on it through a pipette. Wait for 10minutes.
6. Wash the smear by using tapwater.
7. Stand the slide in a drainingrack.
o Examination Of Film:
 First examine the stained smear under the low power (40 X) and adjust thefilm.
 Then examine the film under high power (60 X).
 Examine the film by moving from one field to the next field. Record the type of
Leukocytes seen in eachfield.
Count at least a total of 100 Leucocytes

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  DETERMINATION OF PLATELETCOUNT:
 Introduction:Platelets are smallest cells in blood circulation, they participate in blood
clotting process. They are membrane encapculated fragments of megacarioocytes.
Although they are encecleated and have a diameter of 1-4 (µ) microne. The life span of
platelets is 9-10 days. Its determination is requested in investigation of bleeding
disorders.
 ClinicalSignificance:Decrease of platelet count is known as Thrombocytopenia is
often associated with pro longed bleeding and poor clot retraction. It also
occursin-

i) Aplasticanemia.

ii) Megeloblasticanemia.

iii) Hypersplenism.

iv) Acuteleukaemia.

v) Cytotoxicchemotherapy.

vi) HIVinfection.

vii) Septicaemia.
Increase in platelet count is known as Thrombocytosis, Improtant a high platelet count may
also place a risk for a bleeding. It occurs in conditions like.
 Polycythemiavera.
 Chronic myelogonousleukaemia.
 Spleenctomy.
 Renalfailure.
 Normal Range:1.5-4.5lakhs/mm3 
 Speciman:

1) EDTA anti=coagulated blood is the recommended specimen for plateletcount.

2) Capillary blood can be used but venous blood generally for satisfactory results as
capillary blood gives lower values than venousblood.

 Principle:The diluents prevents coagulation as it fixes the platelets and prevents them
from clumping. No attempt is made to lyses the RBC’s. Platelets are identified by their
size, shape and darker. The dye provides the background during cell counting. This dye
does not stain the platelet and it is essential for counting procedure.

108 | P a g e
  Reagents:
Diluting fluid:

1) Tri Sodium Citrate(.106M) -3.8gm

2) Neutralformaldehyde (40%) -0.2ml

3) Brilliantcresolblue -0.1gm

4) De-ionisedwater -100ml

 Equipments:

1) Hemocytometer.

2) Sahli’spipette.

3) Microscope.

4) Testtube.

5) Petri dish with filterpaper.

 Procedure:

 Transferred 3.98ml. of diluents into a test tube.

 The blood specimen is gently mixed for about2minute.

20/µl of fresh non-coagulated blood is added into the test tube and the contents of the pipette is
rinsed with the diluting fluid for 3-4minutes.

 Immediately the diluents is mixed with the specimen for at least 5minutes.

 The hemocytometer chamber is then placed on the stage of the micro scope. By using
Sahli’s pipette a drop of specimen is transferred on each side of the counting chamber.

 Place the mounted hemocytometer into a moist surface and let it stay undisturbed for 15
minutes. The cover slip is placed on the countingchamber.

 The red cell counting is focused under low magnification and then under high
magnification ofmicroscope.

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 Observation:Platelets are bluish and must be distinguished from debris. They are oval,
round or shaped with varies size from1-5µm.
 Calculation:

Platelet count/µm or mm3 = Number of platelets × dilution/ volume of fluid

= (N × 200)/0.1

Where, volume of fluid = 1 × 0.1 = 0.1 µm

Dilution = 400/.02 =200

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  DETERMINATION OF ERYTHROCYTE SEDIMENTATION RATE(ESR):
 Introduction:The whole blood is allowed to settle ,sedimentation of the erythrocyte
will occurs the rate at which the red cells fall is known as the erythrocyte
sedimentationrate.
 ClinicalSignificance: Erythrocytese dimentation rate or ESR is an one specific test that
reflects changes in plasma protein which accompany most of the acute & chronic
infectionssomeofthesepathologiccondition.ItsmeansifthereishighESR,wecansay that
there is a disease but we cannot diagnosis of the disease ESR does not change in
functionaldisease.
Principle:The erythrocyte sedimentation rate of ESR of blood may determined by the
heightofmmofcolumnofclearplasmalyingaboutaventralcolumnofbloodattheend of 1hour where a sample of
blood treated with an anti coagulant is left standing in an long tube place vertically.
 Specimen:Citrated ant coagulated blood determine ESR within 2 hours after blood
collection.
 Method: WESTERGREN Method, wintrobe method
 NormalRange:
1) Westergern method-Male-5-15 mm/hr

-Female-5-20 mm/hr
2) Wintro bemethod -Male-0-9mm/hr

-Female-0-20mm/hr
 Reagent:

1) Anticoagulant -1mm of 3.8% Na citratesol

2) Patient blood -4ml

 WestergrenMethod:

1) Westergren’s pipette (open at both ends) is about 30 cm long a bore diameter of

about 2.5mm.
2) The lower 20 cm are marked from 0 (top) to 200(bottom).

3) Anticoagulant used is 3.8% trisodium citrate solution. One part of anticoagulant is

added of 4 parts ofblood.

4) Thepipetteacceptsabout1mlofblood.Fillthepipettebysuckintillthe0markand clamp it
vertically in the Westergre’srack.
5) Read the upper level of red cells exactly after 1hour.

This is a better method than windtrobe’s since the reading obtained is magnified as column is
lengthier.
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Calculation:Mean ESR = 1st hour reading + 2nd hourreading/2/2.

HEMATOCRIT [ PACKED CELL VOLUME(PCV)]:

 Introduction:Hematocrit means “Blood separation” the hematocrit measures the

percentage of volume of the packed red cells. Therefore, hematocrit also known as
packed cell volume [PCV].
 ClinicalSignificance:

i) The value of hematocrit is used in determination of blood indices, especially MCV

& MCHC indices help in the diagnosis’s classification of varies types ofanaemia.

ii) Hematocrit is one of the important factors that determines viscosity ofblood.

 Principle:Anti coagulated blood is taken in a wintrobe tube filled to the graduation

mark & then centrifugal for the prescribed length of time the volume of packed cells is
read directly from the graduations mark on the wintrobe.
 Method: Wintrobe method
 NormalRange:

AdultMale - 46%[42%-52%]

Adult Female - 42%[38%-48%]

Reagent: A sample of venous blood [fresh or EDTAblood]
 Procedure:

i) Fill the wintrobe tube with blood with the help of the Pasteur pipette to the 10cm
mark

ii) Place the wintrobe tube in one of the cup of the centrifuge.

iii) Centrifuge for 30 minutes at 3000 rpm[Rapid/minute]

iv) After 30 minutes, stop the centrifuge stake out the tube & read the packed cell
volume.

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  DETERMINATION OF ERYTHROCYTEINDICES:
 Determination Of Mean Corpuscular Volume(MCV):

MCV is derived from PCV and total erythrocyte count. This is the average volume of red
cells. The volume is expressed in femtometer (fl). MCV is calculated by following
formula.

MCV=PCV × 10/RBC count in million

Normal Values:MCV : 86±10fl
 Determination Of Mean Corpuscular Hemoglobin(MCH):

The MCH is the average haemoglobin content of a red cell. The weight is expressed in
picogram (pg). It is calculated as follows:

MCH = Hb × 10/RBC Count in millions

 Normal Values:MCH : 29.5±2.5pg

 Mean Corpuscular Hemoglobin Concentration(MCHC):

It is expressed of the average haemoglobin concentration per unit volume (100) of

packed red cells. It is expressed in g/dl. MCHC is calculated by following two fannulas.
MCHC= (MCH/MCV) × 100
MCHC= Hb (g/dl) × 100/PCV (%)
 Normal Values:MCHC : 32.5±2 g/dl or(%)
 DETERMINATION OF RETICULOCYTECOUNT:

 Introduction:Reticulocytes are Juvenile red cells that pass into the blood stream from
the bone marrow. Roticulocytes stay in circulation for about 24 hours and mature in to
erythrocytes.
 Clinical Significance:The number of Reticulocytes in the blood circulations indicates
the degree of activity of bone marrow, and when the marrow is very active theirnumber
increase this known as Reticulocytosis, in case of aplastic anemia the reticulocyte count
isdecreased.
 Principle:Supra vital staining method is used for reticulocyte count. Blood is mixed
with the stain and the stain enters the cells in living condition. The RNA in the cell is
precipitated by staining as dark blue network or reticulum. Blood smear is made
afterwards, since a direct count is not possible, a relative count is taken against the
number of red blood cells and expressed as a percentage of bloodcells.
 Specimen:EDTA-Anti coagulated blood is commonly used or fresh capillaryblood.
 Normal Range:0.2-2% ofRBC

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  Requirements:

i) Grease free glassslides

ii) Testtube.

iii) Pasteur pipette with rubberteats.

iv) Capillarytube.

v) Test tuberack.

vi) Microscope.

vii) Reagent- Staining solution: It is prepared asfollow:

 Brilliant cresyl blue 1.0gm or New methylene bluepowder

 Sodium citrate0.4gm
 Sodium chloride0.85gm
 Distilled water 100ml

Dissolve first sodium citrate in normal saline and then the dye. Filter it and store in a plastic
container. It is stable at 2-8°C.

 Procedure:

1) Take 2-3 drops of blood and 1 drop of methylene blue powder and mix itproperly.

2) After gently mixing stay it for 20 Minutes at room temperature .

3) Now take this mixture through Pasteurpipette

4) Now drop one drop of this mixture onslide.

5) Prepare a thin smear of the stained blood specimen with the help of a spreader slide
and air dryit.

6) Examine the smear first under the low power objective for scanning, and locate a
thin portion of the smear when red cells are evenlydristributed.

7) Changetotheoilimmersionobjective.Reticulocyteareidentifiedbyfinedeepviolet
filament and granules arranged in anetwork.
 Calculation:

Reticulocyte percentage (%) = Number of reticulocyte count × 100

Total number of RBC Count

114 | P a g e
  COAGULATION PROFILETEST:
 DETERMINATION OF COAGULATION TIME(CT):
 Introduction:This is also known as Lee & White clotting time, this method seems for
coagulation factor.
 Clinical Significance:Only severe clotting factor deficiency is recognized by this
method prolonged clotting time. This is indicting the missing coagulation factor.
 Normal Range:4-9minutes
 Method:Lee & White method-
1. Make a clean vein puncture with as little trauma as possible to the connective tissue
between skin andvein.
2. Withdraw 4-5 ml blood by dry glass syringe. Delivered 1ml of blood in each of the
three 8 × 75 mm testtubes.
3. Thethreetubeswithbloodareallowedtostandinuprightpositionatroomtemperature.
4. Coagulation in the tube can be ascertained by tilting the tube or by gentle tipping. The
firsttubeisgentlytiltedeveryminuteandtheremainingtubesareexaminedeveryhalf minute.
The average of the clotting time in three tubes gives theresult.
Since the speed of coagulation increased with temperature, the test should be done at
the particular temperature (37°C).
 DETERMINATION BLEEDING TIME(BT):
 Introduction:Determination of bleeding time recognizes vascular defect and platelet
disorder.
 Clinical Significance:Prolonged bleeding time is generally found with
thrombocytopenia (Platelet count < 50,000 cells/µl) and where there is a dysfunction,
bleeding time is high with a normal plateletcount.
 NormalRange:

1) Duke method: 2-5minute

2) IVY method: 5-11minute

 Method:IVY’s method
1. A sphygmomanometer cuff is wrapped around the patient’s arm above the elbow and
inflated to 40mm hg and the same pressure is maintained throughout thetest.
2. Volar surface of forearm is cleansed with rectified spirit and an area of skin devoid of
superficialveinsisselected.Theskinisstretchedlaterallybythumbbetweenthumb and fore
finger of left hand, and two separate punctures, 5-10 cm apart, are made in quick
succession by free hand, using a disposable lancet or No.11 surgical blade. Any
microlancet with about one mm width a cutting depth of 2.5 mm is suitable.
3. Timing is begun as soon as the punctures are made and bleedingstarts.
4. Blood extruding from the cut is blotted of gently but completely with filter paper at
15/30 seconds interval till the bleeding spots. The time isnoted.
When the bleeding has ceased, a sterile adhesive strip is place on the wounds.

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  DETERMINATION OF PROTHROMBIN TIME (QUICK’SMETHOD):
 Introduction:Prothrombin or factor II is synthesized in the liver under the influence of
fat solublevit-E.
 Clinical Significance:Prothrombin (factor II) is synthesized in the liver in the presence
ofvitaminK.FactorVIIisallsosynthesizedintheliver,whichisrelatedtoprothrombin. In
clotting mechanism in stage 2, prothrombin is converted to thrombin, which transforms
soluble fibrinogen into insoluble fibrin clot. Abnormal prothrombin time suggests stage
2 defect. Prolonged prothrombin time is related to the deficiencies of
factorsII,V,VII, and X.Since excess of coumarin groupd rugsmaylead to hemorrhagic
conditions, prothrombin time determination is also used to monitor the drugtherapy.
 Normal Range:14± 2seconds
 Specimen:Citratedplasma.
 Name of the method:Quick’smethod
 Principle:Thrombokinase preparation (Thromboplastin preparation of human or rabbit
brain)containingcalciumionsisaddedtocitratedplasma.INthepresenceoffactorVII, stage 2
of coagulation mechanism is triggered and the clotting time is recorded after the
additionofthrombokinaseinthepresenceofcalciumions.SincefactorsXII,Xi,VIIIand
platelets are bypassed, the test depends upon the activity of factors VII, V, X, II and I.
Deficiency of any of these factors may cause prolongation of clot formation in thistest.
 Requirements:

1) Water bath(37°C)
2) Stop watch.
3) Test tubes (10 × 100mm)
4) Brain thromboplastin (or commercially available thrombokinasetablets)
5) 0.15 g/dl calciumchloride
 Procedure:

1) Place a test tube containing about 2 ml of calcium chloride at37°C

2) Pipette 0.1 ml of plasma in a small test tube (10 × 100mm).

3) Add0.1ml of brain thromboplastin and mix (oruse thrombokinase tablets

according to manufacturer’sdirections).

4) Wait for 2minutes.

5) Add 0.1 ml of pre-warned calcium chloride solution, mix and start the stopwatch.
6) Holdthetubeinfrontofasourceoflightandkeeptiltingthetubegently.Atthefirst
appearance of fibrin clot, stop the watchimmediately.

7) Record thetime.
8) Repeat the steps 1 to 7 twice to check the reliability ofresults.

9) Repeat the procedure by using normalplasma.

10) Report prothrombin time inseconds.

116 | P a g e
 
SPECIAL HAEMOTOLOGICALTEST:
 DETERMINATION OF ABSOLUTE EOSINOPHILSCOUNT:
 Clinical Significance:Increased eosinophils count is often associated with allergic
reactions.Parasiticinfections,brucellosisandincertainleukemia.Increaseintheadrenal
function. (Hyperadrenalism or Cushing’s syndrome) is associated with a fall in
eosinophilscount.
 Normal Range:40-440/cu mm(µl)
 Specimen:EDTA or heparinized blood
 Principle:Blood is diluted with a special diluting fluid, which removes red cells and
stains the eosinophils red. These cells are then counted under low powder (10X) in a
known volume of fluid by using Neubauer countingchamber.
 Requirements:

1) Microscope
2 . Improved Neubauer chamber of Fuch Rosenthal countingchamber.

3. Diluting fluid: (Hingleman’ssolution)-

a) Yelloweosin,

b) 95%phenol,

c) Formalin,Distilledwater
 Procedure:

1) Pipette 0.36 ml of diluting fluid in a testtube.

2) Add 0.04 ml of blood (use Hb pipette,twice).

3) Mix and keep for 10minutes.

4) Mix the diluents and charge the counting chamber.

5) Let it stand under a moist Petridis for about 2 to 3minutes.

6) Count the cells under low power objective with reducedlight.

if improved Neubauer counting chamber is used, count cells in all nine squares.

 Calculations:

Total number of eosinophils, cu mm(µl) = Numberofcellscounted×10

0.9 × volume of fluid ×area counted ×depth

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  REPORTANDFINDING

Patients Report
Hematology
(RBC, TC, DC, Platelets Count)

Lab Age Sex Date RBC TC DC (%) Plet.

No. (Y) (M/ (mill (/cum Count
F) ion) m) (lacks/m
N L M E B m3)

Rx- 34 M 12.12.23 3.3 5100 57 30 05 03 00 2.5

35

Rx- 37 F 12.12.23 7.7 22300 90 05 02 01 00 1.0

36

Rx- 29 M 12.12.23 2.4 15400 30 43 03 13 00 6.9

37

Rx- 22 M 12.12.23 3.7 2800 62 68 05 01 00 1.8

38

Rx- 37 F 12.12.23 2.5 8200 86 20 02 01 00 46,000

39

Rx- 18 M 12.12.23 3.3 16000 68 20 05 01 00 80,000

40

Rx- 48 M 12.12.23 2.5 3300 38 55 03 03 00 2.3

41

Rx- 35 M 12.12.23 2.8 18700 97 02 02 00 00 4.5

42

Rx- 32 F 12.12.23 4.5 4500 59 33 06 07 00 1.7

43
Rx- 26 M 12.12.23 1.6 2700 25 68 04 00 00 12000
44

118 | P a g e
Figure 13:Hematological Analyzer

119 | P a g e
 Patient’s Report
Clinical Pathology (Urine RE/ME)

Lab. Age/S Physical Chemical Microscopical

No. ex
Volu Color S. G. Sug Prote P. E. R.B
me pH ar in Cell Cell .C
I-78 45/F 30 ml Straw 1.001 6.7 Nill Nill 0-5 1-2 1+

I-79 36/M 30 ml Pale 1.009 6.4 Nill Nill 1-2 1-7 Nil
yellow

I-83 62/F 30 ml yellow 1.004 6.6 Colo Trace 0-2 1-2 Nil
r (3+)
redu
ced
I-69 28/F 30 ml Straw 1.003 6.0 Nil Trace 0-1 1-2 Nil
(1+)

OPD- 33/F 30 ml yellow 1.002 6.8 Nill Trace 5-6 0-2 Nil
36 (2+)

I-70 45/M 30 ml Light 1.009 7.0 Nill Nil 6-8 2-3 1+

yellow

OPD- 60/M 30 ml Straw 1.005 7.0 Nill Nill 5-6 0-1 2+

50

OPD- 7/F 30 ml Straw 1.006 4.5 Nill Nill 3-4 0-1 Nill
80

OPD- 23/F 30 ml Pale 1.005 6.7 Nill Nill 0-2 0-3 1+

81 yellow

120 | P a g e
121 | P a g e
 DEPARTMENT OF PATHOLOGY
 HISTOPATHOLOGY
 Processing of Histologytissues.
 Tissue processing by automated tissueprocessor.
 Blockpreparation.
 Section cutting byMicrotome.
 Staining of Tissue Section byusingHaematoxylin & Eosin Staining
Method.
 Per-iodic acid SchiffReaction.
 Mounting & slidepresentation.
 MuseumTechnique
.
 CYTOLOGYAND CYTOPATHOLOGY
 Collection of body fluids by FNACtechnique.
 Leishman-Giemsa stainingmethod.
 Papanicolaou stainingmethod.
 Ziehl-neelsen stainingmethod.
 Mounting & slidepresentation.

122 | P a g e
HISTOLOGY AND HISTOPATHOLOGY

 Processing of histologytissues:

The specimen comes to the histology laboratory from the operation theatre or morgue
and it is collected by surgical biopsy, major resection, autopsy, cytological smear, fine needle
aspiration biopsy etc.

Te basic steps of histology are-

 Receiving.
 Registration.
 Labeling ofspecimen.
 Grossing.
 Fixation
 Dehydration
 Clearing
 Impregnation
 Embedding and blockmaking
 Sectioncutting
 Staining.
 Receiving:When a biopsy or autopsy material sent to a laboratory, the technician
must check thefollowing:

a. Whether it is in a fixative or not (15 to 20 times of atissue).

b. Presence of tissue is there ornot.
c. Clinical short notes of thepatient.
d. Whether it is referred by a physician orsurgeon.
e. Where from the tissue istaken.
f. Physical exam of the big tissue (length, breadth, weight,colour).

123 | P a g e
  Registration:

Afterreceivingthetissuemaintainingoflogbookorregisterisavitalstepfortheother steps. I
will be maintain by registration of date of tissue receiving, patient’s identity, name of the
organ from where the tissue collected, nature of the tissue,size and weight of the tissue etc.
Details of clinical biopsies are entered in the appropriate book, include a record of the number
of pieces taken and each specimen is numbered.

 Labeling ofspecimen:

Correct labeling and identification of specimen are essentials of any technique of

processing. If any mistake in labeling, may lead to incorrect diagnosis and also cause the
death of the patient.

 Grossing of the specimen:

After proper labeling the tissues are send for grossing. This process is two types; the
first one is Pre-grossing which is done in case of large solid tissues. Due to its large volume
and solidity the internal portion of the tissue is not fixed well. So, for better fixation these
tissues are cut vertically or horizontally and filled with cotton in the dissected portion and put
into the fixative for 12 hours at least. After 12 hour fixation next day the tissues are again
grossed in the 3-5-2 mm length, breadth and thickness and put in to the tissue container or
cassette with proper label.

 Fixation:

During fixation, tissues are fixed in complete physical and partly chemical state. Most
fixatives act by denaturation or precipitation of the cell protein or by making soluble
componentsanytissueremovedfromthebodystartsdecomposingimmediatelybecauseofloss of
blood supply and oxygen, accumulation of product of metabolism, action of autolytic
enzymes and putrefaction by bacteria. This process of decomposition is prevented byfixation.
Fixation is the method of preserving cells and tissues in life like conditions as per aspossible.

Types of fixative:-

Fixatives may be simple or complicated.

 Simple fixative consists of one substance (e.g.formalin)

 Compound fixative has two or moresubstances.

124 | P a g e
 Fixatives can also be divided into following three groups-
 Micro anatomical fixatives, which preserve the anatomy of thetissue.
 Cytological fixatives, which may be cytoplasmic or nuclear and preserve respective
intracellularconstituents.
 Histochemical fixatives, employed for demonstration of histochemical
constituents and enzymes.

Commonly used fixatives are – formalin, Glutaraldehyde, Picric acid (Bouin’s fluid),
Alcohol (e.g. Carnoy’s fixative), Osmium tetraoxide etc.

 Dehydration:
The term dehydration means “removal of water form”, in this process water from cells and
tissues is removed so that this space is subsequently taken up by wax. Dehydration is carried out
by passing the tissue after washing the fixative direct in to the 70% alcohol andkeep it over
night,next day it will be transfer to 90% alcohol and keep it for over night,next day it will be
transfer to absolute alcohol and keep thre successive changes of alcohole achforone hour. That
will generally complete the dehydration in case of thintissue.

 Clearing:

This is the process in which alcohol from tissues and cells is removed and is replaced
by a fluid in which wax is soluble and it also makes the tissue transparent. Xylene is the most
commonly used clearing agent.

 Impregnation:
This is the process in which empty space in the tissues and cells after removal ofwater are taken
up by the paraffin wax. This hardens the tissue which helps in section cutting. Impregnation is
done in molten paraffin wax which has the molten point of 56°C (54°- 60°C) generally the
temperature of the paraffin is maintained just 2°C higher than the melting point of the paraffin
waxused.

 Embedding & Blockmaking:

Embedding of tissue is done in molten wax. Wax blocks are conventionally prepared using
metallic L (Leukart’sMould). The moulds are paced over a smooth surfaced glass file.
Moltenwaxispouredinthecavityofthemoulds.Theprocessedtissuepiecesareputintowax with
number tag and examine surface spacing downward. Wax is allowed to solidify. After
solidification the L moulds are removed.

 Sectioncutting:
Put the paraffin block having tissue in the block holder of the rotary microtome. Cut
the section by operating the microtome manually after adjusting the thickness at 5- 6
micrometer. Section are picked from the knife with the help of a forceps or camel hair brush
these are made to float in a water bath which is kept at a temperature of 40- 45C i.e. slightly
below the melting point of wax. This removes folds in the section. From water bath sections
are picked on a clean glass slid the glass slide is placed in an hotplate mentioned at a
temperature of 56C for 20- 30 minutes for proper dry and better adhesion coating adhesivefor
section can be used before picking up sections, these includes egg albumin, glycerin (Mayer’s
albumin mixture). This section is now ready forstaining.
125 | P a g e
 Staining:
Various dyes (Either the natural or synthetic) are used to prepared the staining
solution. For routine staining purpose mainly hematoxylin and eosin stains are used.
The important steps involved in routine staining procedure are as follows-
 Driving:The slides are completely dried in hot air oven before de-paraffinization.

 Deparaffinization:To remove the paraffin from the slide, after cooling it to room
temperature, it is placed in xylene for 3- 5 minutes and again for additional 3- 5
minutes in another xylem ebath.
 Hydration:The slides are passed through decreasing concentration on (Absolute,
90%, 70%, 50%). Finally these slides are washed in distilled water. This ensures
complete hydration of the sections.

 Staining:It is carried out by the prescribed procedure.

 Dehydration:After staining the sections are rapidly agitated through 70% and 90%
alcoholsolution,Followedbytwochangesinabsolutealcoholandfinallytwochanges in
absolute alcohol and finally two changes in xylene (Clearingagent).

 Mounting:This is carried out by placing a coverslip on the mounting media (DPX)

laid over thespecimen.

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  STAINING OF TISSUE SECTION BY USING HEMATOXYLIN AND EOSIN
 Principle:
Stains the nucleus light blue which turns red in the presence of acid. The cell differentiation
Hematoxylin and eosin are the principle stains used for the demonstration of nucleus and the
cytoplasmic inclusion. Alum acts as the mordant and hematoxylin containing
alumisachievedbytreatingthetissuewithacidsolution.Thecounterstainingisperformedby using
eosin solution which imparts pink colour tocytoplasm.

 Requirements:
1. Couplin’s jar.
2. Dropping bottles (50ml).
3. Coverslips.
4. Dissection needles, forceps, scalpelsetc.
5. Canada Balsam.
6. Slide washingtray.

 Reagents:
 Harris’s HaematoxylinStain
 1% Eosin solution.
 1% acid alcoholsolution.
 Staining solution:
1. Bring the section to water, after drying, deparaffinization andhydration.
2. Stain by Harris’s hematoxylin for 3- 5minutes.
3. Wash of the stain by running tap water for fewseconds.
4. Decolorized by 1% hydrochloric acid for fewseconds.
5. Blue in running tap water for 20- 30 minutes. (Examine under microscope for proper
nuclear staining, if it under stained the nagainstainit and if it is overstained
decolorized again).
6. Counter stain b watery yellow eosin for 1- 5minutes.
7. Wash the reverse side of the slide under running tapwater.
8. Dehydrate the slide.
9. Clearing in Xylene.
10. Mount in DPX or Canada balsam with a coverslip.
 Result:
Nucleus:- Blue
Cytoplasm:- Red
Collagen:- Pink
Red blood cells:- Break red.
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  Per- iodic acid Schiff reaction (PAS):
 Purpose:This staining is done for the detection of carbohydrate present intissue.
 Procedure:
1. Bring section down towater.
2. Oxidizes for 5- 10 minutes in 1% aquous periodicacid.
3. Wash in running tap water for 5 minute and rinse in distilledwater.
4. Treat with Schiff reagent in dark place for 10- 30minutes.
5. Transfer directly to first sulphite and rinse for 1minute.
6. Transfer directly to second sulphite and rinse for 2minute.
7. Transfer directly to third sulphite and rinse for 2minutes.
8. Wash for 10 minute for running tapwater.
9. Counter stain is done with ½ diluted haematoxylin for 30 seconds and blue in running
tap water for 5minutes.
10. Dehydrate inalcohol.
11. Clear inxylene.
12. Mount in DPX.]

 Result:
PASpositivesubstances : Bright red (Magenta)
Nucleus :Blue
Othertissueconstituents : Yellow.

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 CYTOLOGY AND CYTOPATHOLOGY
Cytology more commonly known as cell biology; study of cell substance.Cell composition and
the interaction of cells with other cells and the larger environment in which they exist. Cytology
can also refer to cytopathology, which analyzes cell structure to diagnosis the disease.
Microscopic and molecular studies of cells can focus on either multi celled or single celled
organism.
Currently cytology has following branches:
A. Exfoliativecytology
B. Aspirationcytology
C. Imprintcytology.

Exfoliative cytology:
This is the study of cells which are spontaneously shed off from epithelial surfaces into body
cavities or fluid. The cells can also be obtained by scarping, brushing or wash of the body
surface; the principle of the technique is that in diseased states rate of exfoliation of cells is
increased.
Exfoliative cytology is applied in diagnosing diseases of the following:-
a. Female genital tract
b. Respiratorytract
c. Gastro intestinaltract
d. Urinarytract
e. Body fluid (Pleural, Peritoneal, CSF, Semenetc)
f. Buccal smear for sex chromatin.
Aspiration Cytology:
In this study, samples are obtained from diseased tissue by fine needle aspiration (FNA) or aspiration
biopsy cytology (ABC).

Aspiration cytology is applied for diagnosis of palpable or non- palpable lesion.

1. Palpable mass lesionin-
a. Lymphnodes.
b. Breast.
c. Thyroid.
d. Salivarygland
e. Soft tissuemasses.
f. Bones.
g. Non – palpable mass lesionin-
h. Abdominalcavity.
i. Thorasiccavity.
j. Retroperitoneum
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Imprint cytology:

In imprint cytology touch preparation from cut surfaces of fresh unfixed surgically excised
tissue are prepared on clean glass slides. These are fixed, stained and examined immediately.
It is considered complementary to frozen section.

 LEISHMAN – GEIMSASTAIN:
 Reagents:
1. Leishmanstain-
a. Leishman powder- 0.15 gm.
b. Methanol- 100ml.
2. Giemsa stain-
a. Giemsa powder- 0.75 gm
b. Methanol- 65ml.
c. Glycerol- 35ml.

 Procedure:
1. Fix the dry smear with leishman’s stain for 2minutes.
2. Then add double volume of buffer solution and mixwell.
3. Wash the slide under tap water after 10minutes.
4. Then flood the slide with Giemsa stain, which is prepared by diluting the stain with
distilled water in 1:10dilution.
5. It is left for another 25minutes.
6. Wash the slide in ta pwater.
7. Air dry and mount the slide
 Result:
1. Nuclei- Bluish red
2. Acidophilic granules- Pink to red.
3. Basophilic granules-Blue.

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  Papanicolaou Stain(PAP):
 Reagents:
1. Alcohol ether mixture.
2. Harrish hematoxylin stain
3. 1% acid alcohol.
4. 70%, 90% alcohol, absolute alcohol.
5. Orange green- 6 (OG-6).
6. Eosin azide – 36 (EA-36)
7. Xylene.
8. DPX.

 Procedure:
1. Fix the smear in alcohol ether mixture for 20 – 30 minutes.
2. Rinse in distilledwater.
3. Stain in Harris’s hematoxylin for 4minutes.
4. Wash in tap water for 1- 2minutes.
5. Differentiate in 1% acid alcohol.
6. Blue in running tapwater.
7. Rinse in tapwater.
8. Transfer the smear in 70% alcohol and then 95% alcohol for fewseconds.
9. Stain in Orange green 6 for 1- 2minutes.
10. Rinse in 3 changes of 95% alcohol for few seconds ineach.
11. Stain in eosin azide – 36 for 1- 2minutes.
12. Rinse in 3 changes of 95% alcohol for few second in each.
13. Dehydrate in absolutealcohol.
14. Clear inXylene.
15. Mount in DPX or Canadabalsam.
 Result:
Nucleus :Blue
Acidophiliccells : Red to orange
Basophiliccells : Green to bluish green
Ceratenised cell or penetratedbyblood : Variation ofred.

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 PICTURESOFHISTOPATHOLOGICALTISSUESAMPLE

FETUS LEFTLEG OVERIANCYST

INTESTINE CHOCOLATECYST KIDNEY

BONE TUMOR PROSTATECHIPS

133 | P a g e
BLOOD BANK

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 DEPARTMENT OF BLOOD BANK

 BLOODBANK

 ABOGROUPING

 THE COMPATIBILITY TST (CROSSMATCHING)

 TTITESTING

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 BLOOD BANK

 Blood bank & blood transfusion unit:

a. In-house blood collection:Collection of in bloodbank.

b. Urgen tlaboratory:
1. Blood grouping & Rhfactor
2. Compatibility testing or crossmatching
3. Direct & indirect Coombstest
4. Dutest
c. Component separation:
1. R.B.C. or Packed cellseparation
2. Plateletseparation
3. F.P.P. or plasmaseparation
4. Wash R.B.Cpreparation
5. Cryoprecipitate
d. TTI testing laboratory:
1. HIV test(ELISA)
2. HCV test
3. VDRL or RPRtest
4. HBs Agtest

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  Clinical significance of blood transfusion:

Blood transfusion today is a major medical service that is rendered to patients needing
replacement of whole blood or blood components. Since the beginning to the Second World
War, transfusion of blood & blood products has become accepted as a routine & relatively
safe procedure in the management of patients. The blood bank tries to select a donor’s blood
that willbecompatiblewithrecipient’sblood.Therecipient’splasmashouldnothaveanyreactive
antibody towards the red cell or donor which may lead to haem agglutination or hemolysis of
donor’s redcells.

 ABO bloodgroups:

During blood transfusion, an identical ABO blood group of the donor is ideal. Persons
belonging to blood group “O” are considered to be “universal donors”, i.e. their blood can be
giventoindividualsofanyotherbloodgroups.TheredcellsdonotcarryeitherAorBantigen & hence
they do not react with their corresponding antibodies. On the other hand, those with blood
group AB are “universal recipients”. They will accept blood from any of the blood groups-A,
B, AB or O. this is because they neither have anti-A oranti-B.

Transfusion of specific component blood, as needed by the patient ,is preferred over
transfusion ofwhole blood. Whole blood is needed in case of large scale blood less. The
whole blood arrengements the recipient’s total blood volume which may lead to congestive
heart failure.
Anemicpatientswithadequatebloodvolumebutwhoaredeficientinredbloodcellpopulation
require packed red cells. Transfusion of plasma, plasma components & platelet concentrates
are the therapeutic measures recommended for bleedingpatients.

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 IN-HOUSE BLOODCOLLECTION:
 Blood collectionprocedure:
 Preparation:Before starting to draw blood, sauce that the following materials are
available & within reachable distance properly labeled blood collection bottle with
donors identification member; bleeding set & air way attached; pilot tubes;
tourniquet/pacer cuff; forceps; stripper; adhesive tape; rubber bond; savlon; alcohol;
iodine swab; local anesthetic & syringe & needle forinjection.
 Procedure:

1. The donor is advised to lie on the bleeding table & made sure he/she is relaxed &
comfortable. The bottle is placed approximately 30 – 40 on below the level oftable.
2. The tourniquet/pacer cuff is applied to the upper arm & select a prominent antecubital
vein.
3. The tourniquet is released & disinfected venipuncture site using savlon, iodine &
alcohol swab in that order. The skin is allowed to dry before inserting theneedle.
4. The tourniquet is re-applied & injected local anesthetic subcutaneously at the site of
venipuncture using aseptictechnique.
5. Thevenipunctureisperformedwitha15or16gaugeneedle.Thebottleorbagisgently agitated
to mix the blood with anticoagulant. For this purpose agitating machine may beused.

6. Whentherequiredamountofbloodhasbeencollected,tubeisclamped;thetourniquet is
released & removed the object from the donor’shand.
7. When the required amount of blood is collected before the needle has been removed
fromvein,theneedleisdrawnfromthebottle&putted5–10ml.Sampleinto2sterile, dry pilot
test tubes, each labeled with the same number as that or the main blood
collectionbottle.
8. An alcohol swab is placed over the needle & then removed it. Pressure is applied over
the puncture site with alcohol swab. The donor is asked to press swales firmly overthe
area for 3 – 5 minutes & preferably with arm hold straight up in an extendedposition.
9. The air way needle from the blood collecting bottles is promptly removed & replaced
the cays after applying 70% alcohol swab an openareas.
10. Finally, tally the members on the bottle with the donor slip, record thebook.
11. A rubber band is putted around the pilot tubes & collectingbottles.
12. Did not let the donor’s stand up immediately after blood donation. Make sure that any
bleeding from the venipuncture had stopped with a Band-Aid wound iscovered.
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13. The bottle is stored at 2 –4⁰C.

Blood collection procedure

 BLOOD BANKLABORATORY:
 Determination of bloodgroup
 Principle:The house of blood transfusion has become steadily more frequent now a
139 | P a g e
 days. To detect or to decide whether the donor’s blood matches the recipient’s blood,
the blood group test isdone.
 Requirements:
1. Glassslide.
2. Pasteurpipette.
3. Applicatorsticks.
 Reagents:
1. Anti-Asera.
2. Anti-Bsera.
3. Anti-Dsera.
4. Normalsaline.
 Procedure:
1. A suspension of RBC’s in normal saline isprepared.
2. On 1 half of glass slide is placed 1 drop of Anti-A blood groupingsera.

3. On the other half of the glass slide placed 1 drop Anti-B blood grouping sera &
Anti-Dsera.
4. Using a Pasteur pipette added 1 drop of cell suspension to each halfslide.
5. With separator applicator sticks mixed each cells sera mixturewell.
6. Tilt the slide back & forth & observed foragglutination.

ANTI-A ANTI-B ANTI-D PROBABLE BLOOD GROUP

+ - + A(+)
- + + B(+)
- - + O(+)
+ + + AB(+)
- - - O(-)

Agglutination: +
Not agglutination: -
 Significance:The use of blood groupsare:-
1. To ensure compatible bloodtransfusion,
2. To eliminate haemolytic disease of the newborns due to Rhincompatibility,
3. To detect susceptibility to variousdiseases

4. COMPATIBILITY TESTING OR CROSSMATCHING :

Before the recipient receives blood transfusion, a compatibility test must be run within the
laboratory with the donor’s red cells & the recipient’s serum. This is called major cross
matching. The primary purpose of major cross match is to find out any incompatibility of
donor’scellswithpatient’sserumisordertoavoidtransfusionreactions.Theminorcrossmatch is rarely
140 | P a g e
requested when the compatibility of the recipient’s red cells is tested against donor’s
serum.Compatibility test or cross matching is performed subsequent to the ABO grouping & Rh
typing of the recipient’s & donor’s blood. It is the final criterion as to the suitability of a
particular donor blood for a particularrecipient.
Therecipient’sbloodisobtainedfreshwhilethedonor’sbloodisobtainedfromthepilottube. ACD anti
coagulated donor’s blood should not be more than 21 days & constantly stored at 4⁰C.
 Principle:Serum of the recipient is tested against the red cells of the donor under
different conditions in order to establish their compatibility or non- agglutination.
Agglutination in any of the conditions indicates the presence of incompatible antibody
in patient, natural orimmune. The three phases of compatibility testing are listed below &
illustrated in saline phases, where the immunologic reaction between red cells suspended in
saline medium & the antibody occurs at room temperature.

1. Thermophase with protein: Where the red cells are suspended in the antibody (serum)
with 22% albumin (protein) & incubated for 30 minutes at37⁰C.
2. Antihuman globulin(AHG) phase: Where the incubated cells are washed ( to remove
freeglobulin)&reactedwithantihumanglobulinserum(Coombsreagentorantihuman
globulin).
 CROSS MATCHING BY SLIDEMETHOD:
The above slide method is commonly done in blood bank for quick cross matching.
 Requirements:
1. Glass slide,marker
2. Disposable plasticsticks
3. Microscope
4. 4% red cell suspension of donor’s &recipient’s
5. Donor’s & recipient’sserum
 Procedure:
1. Take a slide & draw a line centrally to divide into 2parts.
2. Marked 1 part ‘P’ for major cross matching & part ‘D’ for minor cross matching.
3. On the ‘P’ slide add 1 drop of patient’s serum & 1 drop of donor’s 4% cell
suspension.
4. Onthepartof‘D’add1dropofdonor’sserum&1dropofpatient’scellsuspension.
5. Mix the content of each slide by gently rotating theslide.
6. Incubate at room temperature (21-25⁰C)for10minutes.
7. Examine both macroscopically & microscopically for agglutination.
 Result:There is no agglutination in both sides of the side that indicates donor’s blood
compatible with recipient’sblood.
 COOMBS TEST:Antihuman globulin technique is very useful in recognizing weak
immunologic reactions. It is widely used in identification of Du compatibility testing,
antibody screening & identification of sensitized redcells.
 DIRECT COOMBS TEST:This recognizes sensitized red cells when the sensitizing
141 | P a g e
 occurs within the body, i.e, in haemolytic disease of new born (HDN) & autoimmune
hemolytic anemia. This test is performed to detect anti-D antibody or other antibodies
attached to the red cell surface within the bloodstream.
 Principle:Antihuman globulin or Coombs test detect sensitized re cells where the
redcellgetcoatedwithIgGantibodyorglobulinbutdonotagglutinatewhensensitize red
cells come in contact with anti human globulin reagent they agglutinate.
 Reagents:
1. Anti human globulin or Coombs reagent.
2. Pre sensitized red cells or Coombs sensitive cells.
3. Saline(0.85%).
 Specimen:Collected blood is preferred over whole blood (citrate) is required for
direct Coombs test. In case of indirect Coombs test for antibody screening serum
specimen will beneeded.
 Procedure:
1. Prepare 4% cell suspension in isotonic saline of the red blood cell to betested.
2. With a clean pasture pipette add 1 drop of the prepared red cell suspension to a
small testtube.
3. Wash 3 times with normal saline to remove all trace of serum or freeglobulin.
4. Decant completely after lastwashing.
5. Add 2 drops of AHG serum to the sedimentedcells.
6. Mix well & centrifuge for 1 minute at 1500r.p.m.
7. Re- suspend the cells by gentle agitation & examine macroscopically for
agglutination.
8. Examine for agglutination by holding the tube against a lighted background &
tapping the bottom of thetube.

 Observation:
Ifagglutinationoccurs ----------------------- Positive Coombs test

Ifnoagglutination ---------------------------- Negative Coombstest

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  INDIRECT COOMBS TEST:
 Principle:Here the sensitization of red cells is done in the laboratory by incubating
theredcellswiththecorrespondingantibodyat37⁰Cfor30minutes.Thetestisapplied in
detecting the presence of unexpected antibody in the serum which will react with
corresponding antigen on the red cells.
 Specimen:In case of indirect Coombs test for antibody screening, is needed it need
not to be a fasting sample.
 Procedure:
1. Label there test tubes as ‘T’ (test serum), ‘PC’ (positive control), ‘NC’ (negative
control).
2. In the ‘T’ marked tube add 1 drop of test serum.
3. In ‘PC’ marked tube add 1 drop of Anti-Dserum.
4. In ‘NC’ marked tube add 1 drop of saline or bovine albumin(22%).
5. In each test tube add 1 drop of 4% saline suspension of the pooled Rh +ve (‘O’
group) cells or Coombs sensitive cells.
6. Incubate all the three tubes at 37⁰C for 30minutes.
7. Wash the cells 3 – 4 times with normal saline to remove excess serum or free
globulin.
8. Add 2 drops Coombs serum (AHG serum) to each tube & mixwell.
9. Keep for 5 minutes & centrifuge for 1 minute, at 1500r.p.m.
10. Resuspend the cells & examine macroscopically or microscopically for
haemagglutination.
 Observation:

Ifagglutinationoccurs -------------- Positive Coombstest

Ifnoagglutination ------------------ Negative Coombstest

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 Du TEST:

 Requirements:
1. Testtube
2. Micro-pipette

3. Pasteurpipette
4. Centrifugemachine
5. Waterbath
6. Microscope
 Reagents:
1. Anti-human serum (Coombs serum/Anti-IgG)
2. Bovinealbumin.

 Procedure:
1. Prepare 5% red blood cell suspension in isotonicsaline.
2. Level one tube as ‘T’ & add 1 drop of anti-D serum init.
3. Level second tube as ‘C’ & add 22% Bovine albumin toit.
4. By a Pasteur pipette add 1 drop of cell suspension to each & mixwell.
5. Incubate at 37⁰C for 15minutes.
6. After incubation wash the cell with normal saline for threetimes.
7. Decant the supernatant afterwashing.
8. Add 2 drops of anti-human serum to eachtube.
9. Mix & centrifuge at 15,000 RPM for 1minute.
10. Resuspend the cells by gentle agitation & examine macroscopically & confirm the
resultmicroscopically.
 Interpretation:

OBSERVATION ANTI-D (ANTI- BOVINE INTERPRETATION

NO. RhoSERUM) ALBUMIN
CONTROL
1 0 0 D(Rho) –
NEGATIVE
2 + - D(Rho) – POSITIVE
3 + + TEST INVALID

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  TTI TESTINGLABORATORY
 HCVMICROELISA:
 Principle of the assay:The 3rd generation HCV Micro ELISA is based on a highly
sensitive technique, Enzyme linked immuno sorbent assay which detects antibodies
againstHCVinhumanserum&plasma.TheHCVproteinsarepresentinserumatlevels well
below the limits of detection. Thus, immuno diagnosis of HCV infection is based on
detection of host generated antibodies ( anti-HCVs) to viralproteins.

The 3rd generation HCV Micro ELISA utilizes a combination of antigen with the sequence
of both HCV structural & nonstructural antigen i.e. CORE, E1, E2, NS3, NS4, & NS5. It
has an obvious advantage over the available 2nd generation & 1st generation ELISA with
improved sensitivity & specificity.

The combination of antigens for the structural & non-structural HCV proteins are coated onto
the micro wells diluted samples & controls are then incubated. Antibodies to HCV, if present,
bind to the immobilized HCV antigens on the micro well during this incubation. The micro
wells are then thoroughly washed with the diluted wash buffer to remove excess of unbound
anti-HCV or other human IgGs which may interfere with the test. An enzyme conjugate, anti-
human IgG conjugated with HRPO is added. The excess of enzyme conjugates againremoved
with diluted wash buffer .At this stage the micro well shold only the bound antigen-antiHCV-
enzyme conjugate complex in the next step,the freshly prepared substrate solutionisincubated
with the complex in the micro wells. The enzyme substrate reaction leads to development of a
blue color which is indicative of the Ag-Ab reaction which has occurred in the micro well. In
the final step the stop solution is added & the optical density of the developed color is red
photometrically.

 Kit & its components:

1. Micro wells: 12 strips (12X8 wells). Break way micro well coated with HCV antigens
packed in a sealed pouch withdessicant.
2. Sample diluents: 1 bottle (20 ml). Buffer containing protein & anti-microbial agentsas
preservative.
3. Enzyme Conjugate Concentrate (100x): 1 vial (0.25 ml), Anti-human IgGs conjugated
with Horseradish peroxidase with proteinstabilizers.
4. Conjugate Diluent: 1 bottle (15 ml) buffer containing proteinstabilizers.
5. Wash buffer concentrate (25X): 1 bottle (50 ml) PBS with surfactant. Dilute 1.25 with
distilled water beforeuse.
6. TMB Concentrate (100X): 1 vial (0.25 ml) to be diluted in TMB substrate diluents
beforeuse.
7. Substrate (TMB diluents): 1 bottle (20 ml) buffer containingsubstrate.
8. Control: 1 vial (2.0 ml) ready to use normal human serum negative for antibodies
against HCV, HIV-1 & HIV-2 & HBs Ag. Containing sodium azide aspreservative.
9. Control: 1 vial (2.0 ml) ready to use inactivated & diluted human serum reactive for
HCV antibodies, non reactive for HIV-1, HIV-2 & HBsAg containing sodium azideas
preservative.
10. Stop solution: 1 vial (6 ml) ready to use, 2N Sulfuricacid.
11. Plate sealers: Adhesive backed sheets for sealing micro wellplate/strips.

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  Additional material & instrumentsrequire:
1. Micro pipette
2. Micro tips
3. Timer
4. ELISA reader
5. ELISA washer
6. Distilled or deionizedwater
7. Incubator37⁰C
8. Graduated cylinders for reagent dilution
9. Vortex Mixer
10. Sodium hypochloridesolution
11. Disposablegloves
12. Paper towels or absorbent tissue glassware
 Specimen collection &preparation:
1. Only human serum or plasma samples should be used for the test. While preparing
serum samples, remove the serum from the clot as soon as possible to avoid
hemolysis. Fresh serum/plasma samples arepreferred.
2. Specimensshouldbefreeofmicrobialcontamination&maybestoredat2–8⁰Cfor
oneweekorfrozenat-20⁰Corlower.Avoidrepeatedfreezing&thawing.
 Preparation of reagents:
Prepare the following reagents just before or during assay procedure. Reagents & samples
should be at room temperature(20–30⁰C)beforebeginningtheassay.Allcontainersusedfor
preparationofreagentsmustbecleanedthoroughly&rinsedwithdistilledordeionizedwater. Pre-
warm the incubator to37⁰C.

 Samplepreparation:

Tube dilution: Mark the tubes carefully for the proper identification of the samples. Dilute the
serum samples to be tested with sample diluents (1:1 dilution) in separate tubes (200 µl diluent
+ 20 µl sample) use a separate tip for each sample & then discard as biohazardous waste.
 Micro welldilution:
a. Pipette 100µl of sample diluents into the microwell.
b. Add 10µl of serum sample to betested.
c. Ensure thorough mixing of the samplediluents.

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 Preparation of washbuffer:
a. Check the buffer concentrate for the presence of salt crystals. If crystals are present in
thesolutionresoluillizebywarmingat37⁰Cuntilallcrystalsdissolve.
b. Prepareatleast50ml(2mlconcentratedbufferwith48mldistilledordeionizedwater) of
buffer for each HCV Micro ELISA strip used. Mix will before use.
 Washprocedure:
1. Compete washing will adversely affected the testoutcome.
2. Aspirate the well contents into waste container. Then till the wells completely with
wash buffer avoiding overflow or buffer from one well to another & allow to soak
(approx 30 seconds). Aspirate completely & repeat the wash & soak procedure for
additional times for a total of washes.
 Testprocedure:
1. Add 100 µl of the sample diluent to A-1 well as blank.
2. Add 100 µl negative control in each well no. B-1 & C-1 respectively. Negative
control is ready to use & hence no dilution is required.
3. Add100µlpositivecontrolinD-1,E-1,&F-1wells,positivecontrolisreadytouse& hence no
dilution isrequired.
4. Add 100 µl sample diluents in each well starting from G-1 well followed by addition
of10µl
sample(referMicrowelldilution)alternativelytransfer100µlofeachsample, dilutedin
sample diluents (1-11), in each well starting from G-1 well (refer tube dilution).
5. Applycoverseal&incubateat37⁰C+2⁰Cfor30minutes+2minutes.
6. While the samples are incubating prepare working wash solution & working
conjugateas specified in ‘Preparation ofreagents’.
7. After the incubation is over, wash the wells 5 times with working wash solution
according to the wash procedure given in the previoussection.
8. Add 100 µl of working conjugate solution in each well includingA-1.
9. Applycoverseal&incubateat37⁰C+2⁰Cfor30minutes+2minutes.
10. While conjugate is incubating ,prepare substrate solution in last 5 minutes of
incubation asspecified in ‘ preparation of reagents’ protect from light.
11. Aspirate & wash the wells after incubation as described in step no.7.
12. Add 100 µl working substrate solution in each well includingA-1.
13. Incubate at room temperature (20 – 30⁰C) in dark for 30minutes.
14. Add 50 µl of stop solution.
15. Read absorbance at 450 nm in ELISA READER after blanking A-1 well.
Bichromatic absorbance measurement with a reference wavelength 600 – 650 nm is
recommended.

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  Positive control acceptance criteria:

NC or NC x must be <0.150 if it is not so, the run is invalid & must be repeated.

NC 0.042 B1 well

0.036 C1 well

Total 0.078/2 = 0.039

Cut – off value:

The cut-off value is calculated as below:

Cut off value = 0.1 X PC X + 0.1

e.g. PC X = 1.27 then

Cut off value = 0.1 X 1.27 + 0.1 = 0.227

 Interpretation of result:
1. Test specimens with absorbance value less than the cut off value are non reactive for
Anti-HCV.
2. Test specimens with absorbance value greater than or equal to the cut off value are
reactive forAnti-HCV.
3. Test specimens with absorbance value within 10% below the cut off should be
considered suspect for the presence of antibodies & should be retested induplicate.
4. Specimens with absorbance value equal to or greater than the cut off value are
considered initially reactive. Original specimen should be retested induplicate.

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  HIV TEST:
 Summary & explanation of the test:

The available research data indicates that Acquire immunodeficiency Syndrome (AIDS) is
caused by HIV virus transmitted by sexual contact,exposure to blood or certain blood
products, by aninfected mother to her child during pre-natal & post natal period. The
two type of HIVviruses(HIV-1&HIV-
2)havebeenisolatedfrompatientswithAIDS&AIDSrelatedcomplex (ACR). These two virus
belong to the retrovirus group & are slow viruses.

 Principle:
Specimens & controls are added to the micro titer wells & incubated. Antibodies to HIV-1 &
HIV-2 if present in the specimen, will bind to the specific antigens absorbed on to the surface of
the wells. The plate is then washed to removed unbound material. Horse radish peroxidase
(HRP) conjugated anti human IgG is added to each well. This conjugate will bind to HIV
antigen-antibody complex present. Finally substrate solution containing chromogen & hydrogen
peroxide is added to the wells & incubated. A blue color will develop in proportion
totheamountofHIV-1 &/orHIV-2antibodies presentinthe specimen.Thecolorreactionis
stoppedbyastopsolution.TheenzymesubstratereactionisreadbyEIAreaderforabsorbance at a
wavelength of 450 nm. If the sample does not contain HIV-1 or HIV-2 antibodies then enzyme
conjugate will not bind & the solution in the wells will be either colouries or only a faint
background color develops.

Micro-wells HIV strip plates: 12 strips ( 12 X 8 wells). Break way micro well coated with
HCV antigens packed in a sealed pouch with dessicant.
Sample diluents: 1 bottle ( 20 ml) buffer containing protein & anti microbial agents as
preservative.

Enzyme conjugate concentrate (100 X) : 1 vial ( 0.25 ml), Anti-human IgGs conjugated with
horse radish peroxidase with protein stabilizers.
Conjugate diluents: 1 bottle ( 15 ml) buffer containing protein stabilizers.

Wash Buffer concentration ( 25 X ) : 1 bottle PBS with surfactant. Dilute 1.25 with distilled
water before use.

TMB concentrate (100X) : 1 vial (0.25 ml) to be diluted in TMB substrate diluents before use.
Substrate ( TMB diluents) : 1 bottle (20 ml) buffer containing substrate.
Control:1vial(2.0ml)readytousenormalhumanserumnegativeforantibodiesagainstHCV, HIV-1
& HIV-2 & HBs Ag. Containing sodium azide as preservative.
Control: 1 vial (2.0 ml) ready to use inactivated & diluted human serum reactive for HCV
antibodies, non reactive for HIV-1, HIV-2 & HBs Ag containing sodium azide as preservative.

Stop solution: 1 vial (6ml) ready to use, 2N sulfuric acid.

Plate sealer: Adhesive backed sheets for sealing micro well/ strips.

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  Additional material & instruments required:
1. Micro pipettes
2. Micro tips
3. Timer
4. ELISA reader
5. ELISA washer
6. Distilled / de ionizedwater
7. Incubator37⁰C
8. Graduated cylinder for reagent dilution
9. Vortex mixer sodium hypochloridesolution
10. Disposablegloves
11. Paper towels/ absorbent tissueglassware

 Specimen collection &preparation:

1. Only human serum or plasma samples should be used for the test. While preparing
serum samples,remove the serum from the clot as soon as possible to avoid hemolysis.
Fresh serum/plasma samples arepreferred.
2. Specimens should be free of microbial contamination & may be stored at 2 – 8⁰C for
oneweekorfrozenat-20⁰Corlower.Avoidrepeatedfreezing&thawing.
 Preparation of reagent:

Prepare the following reagents or during procedures. Reagents & samples should be at room
temperature (20–30⁰C)before beginning the assay & can remain at room temperature during
testing. Return reagents to 2 – 8⁰C after use. All containers used for preparation of reagents
mustbecleanedthoroughly&rinsedwithdistilledordeionizedwater.Pre-warmtheincubator
at37⁰C.

 Sample preparation:

Tube dilution: Mark the tubes carefully for the proper identification of the samples. Dilute the
serum samples to be tested with sample diluents(1:1dilution)in separate tubes(200µldiluents
+ 20 µl sample) use a separate tip for each sample & then discard as biohazardous waste.

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 Micro well dilution:
1. Pipette 100 µl of sample diluent into the microwell
2. Add 10µl of serum sample to betested.
3. Ensure thorough mixing of the sample to betested.
 Preparation of wash buffer:
1. Check the buffer concentrate for the presence of salt crystals. If crystals are present in
thesolutionresoluillizebywarmingat37⁰Cuntilallcrystalsdissolve.
2. Prepare at least 50 ml ( 2 ml concentrated buffer with 48 ml. distilled or de ionized water)
of buffer for each HCV Micro ELISA strip used. Mix will beforeuse.
3. Mix 20 ml. 25 X wash buffer concentrate with 480 ml. of distilled or de ionizedwater. Wash
buffer is stable for 2 months when stored at 2 –8⁰C.
 Wash procedure:
1. Complete washing will adversely affect the test outcome.
2. Aspirate the well contents into waste container. Then till the wells completely with
washbufferavoidingoverfloworbufferfromonewelltoanother&allowsoaking (approx 30
seconds). Aspirate completely & repeat the wash & soak procedure 4 additional times for a
total of washes.
 Tes tprocedure:
1. Add 100 µl of the sample diluent to A-1 well asblank.
2. Add 100 µl negative control in each well no. B-1 & C-1 respectively. Negative
control is ready to use & hence no dilution isrequired.
3. Add100µlpositivecontrolinD-1,E-1,&F-1wells,positivecontrolisreadytouse& hence no
dilution isrequired.
4. Add 100 µl sample diluents in each well starting from G-1 well followed by addition
of10µlsample(referMicrowelldilution)alternativelytransfer100µlofeachsample, diluted
in sample diluents (1-11), in each well starting from G-1 well (refer tube dilution).
5. Applycoverseal&incubateat37⁰C+2⁰Cfor30minutes+2minutes.
6. While the samples are incubating prepare working wash solution & working
conjugate as specified in ‘Preparation ofreagents’.
7. After the incubation is over, wash the wells 5 times with working wash solution
according to the wash procedure given in the previoussection.
8. Add 100 µl of working conjugate solution in each well includingA-1.
9. Applycoverseal&incubateat37⁰C+2⁰Cfor30minutes+2minutes.
10. While conjugate is incubating, prepare substrate solution in last 5minutes of incubation as
specified in ‘ preparation of reagents’ protect fromlight.
11. Aspirate & wash the wells after incubation as described in step no.7.
12. Add 100 µl working substrate solution in each well includingA-1.
13. Incubate at room temperature (20 – 30⁰C) in dark for 30minutes.
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 14. Add 50 µl of stopsolution.

15. Read absorbance at 450 nm in ELISA READER after blanking A-1 well. Bichromatic
absorbance measurement with a reference wavelength 600 – 650 nm isrecommended.

 Negative control acceptance criteria:

NC or NC X must be < 0.150 if it is not so, the run is invalid & must berepeated.
 Positive control acceptancecriteria:
1. Positive control must be ≥0.50
2. Determine the mean (PCX) value if one of three positive control values is outside of
these limits, recalculate PCX based upon the two acceptable positive controlvalues.

3. If two of the three positive control values are outside the limits the assay is invalid &
the test must berepeated.
 Absorbance:NC – 0.042 B1 well PC- 1,412 D1well

0.040 C1 well - 1,392 E1 well

Total 0.822 wells - 1,407 F1 well

Total 4,2112

 Interpretation of result:
1. Test specimen with absorbance value less than the cut off value are non reactive for
Anti-HCV.
2. Test specimens with absorbance value greater than or equal to the cut off value are
reactive forAnti-HCV.
3. Test specimens with absorbance value within 10% below the cut off value should be
considered suspect for the presence of antibodies & should be retested induplicate.
Specimens with absorbance value equal to or greater than the cut off value are considered initially
reactive. Original specimen shouldbe retested induplicate.

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 REPORTANDFINDING
URGENTLABORATORY:

ClinicalSignificance:
Before the recipient receives blood transfusion , acompatibility test must be run within
the laboratory with the donor’s red cells & the recipient’s serum. This is called major
cross matching.The primary purpose of major cross match is to find out any
incompatibility ofdonor’s cells with patient’s serum is order to avoid transfusion
reactions. The minor crossmatch is rarely requested when the compatibility of the
recipient’s red cells is tested against donor’s serum.

CompatibilitytestorcrossmatchingisperformedsubsequenttotheABOgrouping&Rh
typingoftherecipient’s&donor’sblood.Itisthefinalcriterionastothesuitabilityofa particular
donor blood for a particular recipient.

Therecipient’sbloodisobtainedfreshwhilethedonor’sbloodisobtainedfromthepilot tube.
ACD anti coagulated donor’sblood should not be more than 21 days& constantly stored at
4⁰C.

TABLENO:1
Analysis of total no of CRBC blood bag in a week.

Date Blood Bags of CRBC

A+ A- B+ B- AB+ AB- O+ O-
53 15 95 11 42 7 87 8

51 12 99 8 39 10 92 5
56 17 105 16 48 14 98 9
64 21 116 14 45 11 102 14

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TABLENO:2
Analysis of total FreshFrozenPlasma(FFP)bag in a week.

Date FFP
A+ A- B+ B- AB+ AB- O+ O-
61 24 74 21 69 22 73 15
53 22 71 18 77 16 69 12
65 28 84 29 84 25 88 17
68 31 89 33 81 22 96 21

TABLENO:3
Analysis of total Randam Doner Platelet(RDP)bag in a week.

Date BloodBagsofRDP
A+ A- B+ B- AB+ AB- O+ O-
36 21 43 23 27 20 39 26
31 25 39 25 31 28 30 29
32 21 48 39 26 33 44 33
26 29 57 33 38 28 49 31

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