Addly's Guide of Standard Operation Procedure in Kota Belud Hospital

INDUSTRIAL TRAINING 11
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S.O.P REPORT OF INDUSTRIAL TRAINING

WRITTEN BY:
ADDLY MADI
1102008100129
DIPLOMA TECHNOLGY SCIENCE MEDICAL LABORATORY

Diploma Technology Science Medical Laboratory
School oI Health Science
Advanced Management and Technology Centre


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ACKNOWLEDGMENT

SpeciaI thanks to:

1. Ms. Marliana Muhammad, Mr Marcel Kuiming who gave us guidance and
support about this ndustrial Training and settle up our problems.

2. Mdm. Clorina Jesson, Head of Pathology Department Hospital Kota Belud
who gave us opportunity to learn a lot in this department.

3. Mr. Jefri Md Yassin, Mr Daman Trip, Mr. Jaafar Linggam, Mr.Saidin
Sulukan, who always there to give advice and help us when we need it.

4. Our highest appreciation to all the MLTs in Pathology Department of
Hospital Kota Belud because they have been teaching us the wonders of
laboratory's world using their most affectionate methods and without feeling
bored.

5. Lots of thanks to all the trainees from Advanced Management and
Technology Centre (PTPL) and Masterskill University College of Health
Sciences for all the guidance, cooperation and teamwork either directly or
indirectly.


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Mission & Vision of Sabah HeaIth Department
(JKNS)

'ISION
Make the individual, Iamily and community in the State concerned and involved in health
care towards the creation oI a healthy community.

MISION
To realize the vision oI the department through increased health promotion and more
eIIective prevention, treatment and rehabilitation services quality by members oI the
practice oI proIessionalism, teamwork, caring and smart partnerships with all sectors in
order to better quality oI liIe.


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HAEMATOLOGY LABORATORY

Introduction

Haematology laboratory is a laboratory that deals mainly with blood samples. The
bloods taken here are mostly stained and are mainly examined Ior cell abnormalities,
either in the morphology or the number. As we know, in a normal blood sample, cells like
erythrocytes, leukocytes and so on present in a speciIic quantity. A decrease and increase
in the number oI certain speciIic blood cells indicates the condition oI the patient.
Furthermore, the presence oI abnormal morphology helps in the diagnosis oI the disease.
Bone marrow aspiration samples are also taken to this laboratory where it is stained and
examined.

A) FULL BLOOD COUNT, PERIPHERAL BLOOD FILM AND FULL BLOOD
PICTURE

OB1ECTI'E
To determined the parameter oI white blood cell (WBC), red blood cell (RBC),
Hemoglobin (Hb), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume
(MCV), ), Mean Corpuscular Hemoglobin Concentration (MCHC) and platelet in blood
by using CELL DYNN 3200 Open Tube (OT), automated hematology system. Each
results and reading obtained can help doctor to interpret the pathological condition oI the
patient such as anemia and leukemia. Other test also needed Ior conIirmation oI the
disease.

SPECIMEN
O Whole blood in EDTA test tube

Cell Dyn 32000

Purpose: The CELL DYN 3200 is a multi parameter with Iive parts diIIerential count,
automated hematology analyzer Ior Iull blood count on EDTA anticoagulant whole blood.


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Reagent: 1. Dilvent sheath
2. Hb lysed
3. WBC lysed
4. QC material and calibrator
5. Enzymatic Cleaner

Daily Start up Procedure
1. Check reagents Iirst number, expiring date, levels and replace reagent containers iI
necessary.
2. Check printer paper, add more paper as necessary.
3. Check tubing in the normally closed valves and sample transIer peristaltic pump Ior
breaks, crimps or other obstruction.
4. Run break ground counts until acceptable results.

Procedures
1. AIter control and calibration checked and veriIied ready Ior use, analyzer in REAMP
mode.
2. Press MAIN.
3. Operator ID enter ID.
4. Run sample.
5. Check and veriIy patient ID name, lab number. Mix well sample. Put under probe
and press START.
6. The FBC determination oI the whole blood to be tested is automatically big the
analyzer as soon as the samples have been aspirated.
O Every 30 sample run precision / replicate
7. Result automatically prints out.
O Check report, veriIy and validate by perIormer.
8. End oI the day.
O Wash analyzer go to special protocol auto cleaner PRESS
9. Completed auto cleaner.
10. Stand by shut down.


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B) Prothrombin Time and Activated Partial Thromboplastin Time (PT/APTT):

OBJECTIVE
To determine the PT and APTT by using the STA-Compact

PRINCIPLES
The STA-Compact detects clot Iormation using a turbo-densitometric measuring
principle. A light beam passes through the cuvette containing the test plasma on to a
photo-detector. The photo-detector measures the intensity oI the transmitted light and
relays any changes to the electronic circuitry oI the instrument that processes the
inIormation continuously. The time required Ior the clot Iormation is indicated on the
digital display.

Prothrombin Time (PT):
O For deIects oI deIicits in Factors II, V, VII, and X and severe dysIunction or
hypoIibrinogenemia; includes monitoring oI long term anticoagulant therapy
aIIecting these Iactors, such as coumarin.

Activated Partial Thromboplastin Time (APTT):
O Primarily Ior deIects or deIicits in Factors VIII, IX, XI, and contact activation
components, but also sensitive to all other clotting Iactors except VII; Irequently
used to monitor heparin therapy.

INTERPRETATION
1. Normal PT is 11-16 s, prolonged iI abnormalities oI Iactors, such as liver disease,
warIarin, DIC or vitamin K deIiciency.
2. Normal APTT is less than 30-40s.Prolonged time showed deIiciencies or
inhibitors to Iactors. Common causes oI a prolonged aPTT are DIC, liver diseases,
massive transIusion with stored blood, administration oI heparin or contamination
with heparin, circulating anticoagulant or deIiciency oI a coagulation Iactor other
than Iactor VII.


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D) ERYTHROCYTE SEDIMENTATION RATE (ESR)

OB1ECTI'E
To measure the presence and intensity oI morbid processes within the body. This test is a
useIul supplement to clinical method that accelerated particularly in chronic disease and
in inIlammatory disease.

PRINCIPLE
Erythrocytes sediments at a speciIic rate in blood which has rendered in coagulable, this
rate is determined primarily by the plasma proteins and the number, shape, and surIace
properties oI the erythrocytes.

PROCEDURE
1. Venous blood is diluted with 31.3 g/l trisodium citrate in the proportion one part
oI citrate to Iour part oI blood.
2. The sample was mix well.
3. The blood was draw up into the micropipette 300 mm.
4. The tube was placed exactly vertical and leave undisturbed Ior 60 minutes Iree
Irom vibrations, draughts, and not exposed to direct sunlight.
5. The height oI clear plasma above the upper limit oI the column oI sedimenting red
cells was then read to nearest mm.

E) RETICULOCYTE COUNT
Reticulocyte is a prematured red blood cell containing basophilic rebonucleoprotein`
residue in the nucleus. The basophilic component will combine with new methylene blue
Ior granule-like or Iilament-like Iormation.

F) CD4/CD8 count
PRINCIPLE
O The FACSCount instrument (Becton Dickinson Immunocytometry Systems) is a
compact cell counter with a built-in computer designed to use unlysed whole
blood, collected in liquid EDTA. When whole blood is added to the tubes oI
sample reagent pair, the Iluorochrome-labeled antibodies bind speciIically to

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antigens on the surIace oI lymphocytes. The FACSCount instrument detects 2
colors and measures relative size. The CD3 cells will Iluoresce red and the CD4
and CD8 cells will Iluoresce yellow when analyzed on FACSCount instrument. A
known umber oI reIerence beads is contained in each reagent tube and Iunctions
as a Iluorescence and quantitation standard Ior calculating the absolute counts Ior
the CD4, CD8 and CD3 T lymphocytes. From CD4/CD8 count, we can
interpret the pathological condition Ior an example, in HIV patient, the CD4 and
CD8 count is very low.

G) TEST: D-DIMER

Test principle: The latex particles provided in the D-Dimer Test are coated with mouse
antihuman D-Dimer monoclonal antibodies. Test samples containing D-dimers when
mixed with the latex particle suspension make the particles agglutinate. At a
predetermined concentration oI D-dimers that the D-dimers Test is designated Ior, the
agglutination oI the particles procedures macrospic dumps that can be visualized by the
naked eye.

Reagent : D-DI TEST (Stage)
Reagent 1 : 1.5 ml ready-Ior-use latex particles coated with mouse monoclonal anti
human D-dimer antibodies (2F7).
Reagent 2 : 20 ml ready-Ior-use glycine buIIer
Reagent 3&4 : Lyophilized human negative and positive plasmas Ior use as controls.

Preparation of Reagent and Storage:
1. Allow the reagent 1 and 2 to stand at room temperature (18`C-25`C) Ior 30 minutes.
Resuspend the reagent 1 by vial inversions several times beIore use.
2. Reconstitute each vial (reagent 3 and 4 with 1ml oI distilled water). AIter the solution
stand at room temperature (18`C-25`C) 3o minutes. Swirl the vial gently beIore use.
Normal Range: 0.5 (ug/ml FEU)


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1. SCREENING TEST

Procedure:
1. Patients plasmas are tested undiluted. All the reagents must be at room
temperature.
2. In each appropriately identiIied article on the test card place 20 ul oI the sample
(patient undiluted, reagent 3 and 4).
3. Shake the reagent 1 vial several times. Place 20 ul oI reagent 1 next to the test
sample in each circle. Use separate stirring rods, combine and mix the two drops
in each circle.
4. Manually rockle the test in such a manner that the liquid swirls around in each
circle. Continue rocking the card Ior 2-3 minutes.
5. Compare the agglutination pattern oI each circle with those oI the negative and
positive controls (Reagent 3 and 4).

2. SEMI-QUANTITATI'E DETERMINATION

Procedure:
To semi-quantitative the positive D-Dimer level oI sample, we reagent 2 to serially dilute.
Follow the same assay procedure described above Ior the screening test.

Results:
Plasma D-Dimer levels are obtained by multiplying the detection limit oI D-Di Test by
the dilution number. For example, iI the 1:g dilutation is highest dilatation that procedures
agglutination, then the plasma D-Dimer level is equal to or greater than 4 ug/ml (FEU)
(0.5 ugl/ml x g).

Appropriate concentrations are given in the Iollowing table Ior test samples assayed
undilutated and at dilatation 1:2 through 1:16.


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Test Sample Approximate D-Dimer
Level (ug/ml - FEU)
Undiluted 1:2 1:4 1:8 1:16
Negative 0.5~
Positive Negative ~0.5 0.1
Positive Positive Negative ~1.0 2.0
Positive Positive Positive Negative ~2.0 4.0
Positive Positive Positive Positive Negative ~4.0 8.0
Positive Positive Positive Positive Positive ~8.0

Test Report : D-Dimer
Normal Range 0.5 ug/ml (FEU)


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BIOCHEMISTRY LABORATORY

Biochemistry laboratory is divided into 3 sections. There are coordination zone,
miscellaneous zone, diagnosis zone and elecsys zone.

Coordination zone
-receive sample
-labeling sample
-recording sample

Routine section
-diagnosis done Ior several test by using HITACHI 912
-serum bilirubin test Ior newborn
-arterial blood gas
-electrolyte

Non-routine section
-diagnosis Ior special test such as identiIication oI hormone level
-HbA1C test


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A) ROUTINE SECTION USING HITACHI 912

GENERAL
HITACHI 912 is a model analyzer that consist oI sample carousel, 2 reagent
carousels which is holding 32 reagent containers, and a reaction disk with 120 reusable
cuvettes. Samples can be pipetted every 10 seconds Irom cups or primary tubes.
InsuIIicient sample volume is detected by a liquid level sensor. Results Ior most test is
available within 5 minutes. Reagents are barcoded and placed in a reIrigerated
compartment oI the analyzer.A unique Ieature oI the 911 is the ability to add multiple
reagent per method. It has autodilution capabilities Ior serum and urine samples,autorun
and autocalibration Ieatures.
This system is also capable oI analyzing the ISE test (Na,K,Cl) through the ion
selective electrode (ISE) that senses a speciIic ion selectivity, and the reIerence (REF)
electrode.It is used to measure the potential diIIerence between the 2 electrodes, which is
generated iI the sample pass through electrodes.

LIST OF TESTS
List oI tests that run using this machine.
O Urea
O Sodium
O Potassium
O Chloride
O Creatinine
O Uric acid
O Total protein
O Albumin
O Total bilirubin
O ALP
O ALT
O Ca
2

O Phosphate
O Mg
O AST

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O LDH
O Glucose(FBS/2HPP/RBS)
O Iron
O UIBC

B) BLOOD GAS ANALYZER

PURPOSE
To analyse pH and blood gases Ior acid base status in patient using a Iully automated
blood gas system.This blood gas test in chemical pathology unit using radiometer ABL5
and Bayer Rapidleb 248 blood Gas Analyser.

PRINCIPLE OF TEST
The blood gas are measured using a Iully automated system. The measured parameters
are pH, pCO
2
and
,
pO
2.
whereas the calculated parameters are standed and actual
bicarbonate(HCO
3-
),total CO
2,
in vivo and in vitro base excess.The Iull results oIcthe
measured and calculated parameter will give a picture oI acid base status oI a patients
having problem oI either metabolic alkalosis or acidosid and respiratory alkalosis which
arises Irom many pathological condition.

C) NON-ROUTINE SECTION USING ARCHITECT i200SR

PRINCIPLE OF TEST
The principle oI test is speciIic to each test. For example TSH test. TSH test is based on
sandwich principles. The total duration oI assay is 18 minutes.

O 1st incubation: 50 l oI sample, biotinylated monoclonal TSH-speciIic antibody
and a monoclonal TSH-speciIic antibody labeled with a ruthenium complex react
to Iorm a sandwich complex.
O 2
nd
incubation : AIter addition oI streptavidin-coated micro particles, the complex
becomes bound to the solid phase via interaction oI biotin and streptavidin.
O The reaction mixture is aspirated into the measuring cell where the micro particles
are magnetically captured onto the surIace oI the electrode. Unbound substances

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are then removed with ProCell.application oI a voltage to the electrode then
induces chemiluminescent emission which is measured by a photomultiplier.
O Results are determined via calibration curve which is instrument-speciIically
generated by 2-point calibration and a master curve provided via the reagent
barcode.

Thyroid-stimulating hormone (TSH)
Thyroid-stimulating hormone (TSH,thyrotropin) is a glycoprotein having a molecular
weight oI approximately 30,000 Dalton and consisting oI two subunits. The B-subunit
carries the TSH-speciIic immunological and biological inIormation, whereas the u-chain
carries species-speciIic inIormation and has an identical amino acid sequence to the u-
chains oI LH,FSH and HCG.
TSH is Iormed in speciIic basophile cells oI the anterior pituitary and is subject to a
circadian secretion sequence. The hypophyseal release oI TSH (thyrotrophic hormone) is
the central regulating mechanism Ior the biological action oI thyroid hormones. TSH has
stimulating action in all stages oI thyroid hormones Iormation secretion; it also has a
proliIerative eIIect. The determination oI TSH serves as the initial test in thyroid
diagnostics. Even very slight changes in the concentrations oI the Iree thyroid hormones
bring about much greater opposite changes in the TSH level. Accordingly, TSH is a very
sensitive and speciIic parameter Ior assessing thyroid Iunction and is particularly suitable
Ior early detection Ior exclusion oI disorders in the central regulating circuit between the
hypothalamus, pituitary and thyroid. Elecsys TSH employs monoclonal antibodies
speciIically directed against human TSH.The antibodies labeled with ruthenium complex
consist oI a chimeric construct Irom human and mouse-speciIic components. As a result,
interIering eIIects due to HAMA (human anti- mouse antibodies) are largely eliminated.

LIST OF TESTS
Below is the list oI tests that run using Architect i200SR
O TSH
O FT4
O T4
O FT3
O T3

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O -HCG
O Ierritin
O Troponin T
O Ca 125
O Prolactin
O Carcinoembryonic enzyme (CEA)
O LH
O FSH
O Progesterone
O Estradiol
O Testosterone
O Cortisol

Carcinoembryonic enzyme (CEA)
CEA is mainly Iound in the Ietal GIT and Ietal serum. It also occurs in slight
quantities in intestinal, pancreatic and hepatic tissue oI healthy adults. The Iormation
oI CEA is repressed aIter birth and accordingly serum CEA values are hardly
measurable in healthy adults. Elevated CEA concentrations are Irequently Iound in
cases oI colorectal adenocarcinoma. Slight to moderate CEA elevations occurs in 20-
50 oI benign diseases oI the intestine, pancreas, liver and the lungs. Smokers also
have elevated CEA values. The main indication Ior CEA determinations is the Iollow-
up and therapy management oI colorectal carcinoma.
-HCG
HCG is produced in the placenta during pregnancy. In non-pregnant women, it also
can be produced by tumors oI the trophoblast, germ cell tumors with trophoblastic
components and some non-trophoblastic tumors. The elevated values here serve as an
indication oI chorionic carcinoma, hydatiIorm mole or multiple pregnancies.
Depressed values indicate threatening or missed abortion, ectopic pregnancy, gestosis
or intra uterine death. Measurement oI HCG makes also a contribution to the testing
Ior Down`s syndrome.
Elevated HCG concentrations not associated with pregnancy are Iound in patients
with other diseases such as tumors oI the germ cells, ovaries, bladder, pancreas,
stomach, lung and liver.

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Troponin T
The Elecsys Troponin T assay can be used as an aid in the diIIerential diagnosis oI
acute coronary syndrome to identiIy necrosis e.g. acute myocardial inIarction. The
test is Iurther indicated Ior the risk stratiIication oI patients presenting with acute
coronary syndrome and Ior cardiac risk in patients with chronic renal Iailure. The test
may be also useIul Ior the selection oI more intestine therapy and intervention in
patients with elevated levels oI cardiac troponin T.

Prolactin
During pregnancy the concentration oI prolactin rises under the inIluence oI elevated
progesteron and estrogen production. The stimulating action oI prolactin on the
mammary gland leads post partum to lactation. Hyperprolactinemia (in men and
women) is the main cause oI Iertility disorders. The determination oI prolactin is also
determined when breast cancer and pituitary tumors are suspected.

URINE PARAQUAT
1) Add 1ml alkaline dithionite reagent to 1ml urine.
2) A blue colour indicates the presence oI paraquat, diquat or both. Reaction may not
be immediate observe again aIter 20 minutes.
3) Writing the result.
Note:-
II paraquat is present a blue colour develops immediately, the depth oI colour
increasing with the concentration oI paraquat. A pale green colour may be obtained
with low concentration oI paraquat.

MICROALBUMIN
1) Remove one strip Irom bottle and replace the cap tightly. Dip the test pads into
the urine, making sure both pads are wetted.
2) Immediately remove the strip, dragging the edge oI the strip against the rim oI
the urine container to remove excess urine.
3) Press START button on CLINTEK 50 analyzer at the same time as the strip is
removed Irom the urine.
4) Blot the strip by touching the edge only to a paper towel.

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5) Place the reagent strip, with the reagent pads Iacing up, onto the instrument`s
test/Ieed table. Slide the strip along the table until it touches the end oI the
table.
6) The table is automatically pulled into the instrument, where the strip is
identiIied and read. Results a displayed or printed as soon as they are
available.

PLASMA AMMONIA
1) Pipette specimen into test tubes accordingly.

Reagent blank Standard Sample
Sample - - 0.2ml
Distilled water 0.2ml - -
Standard - 0.2ml -
Reagent 1 3.0ml 3.0ml 3.0ml
2) Mix and allow to stand Ior 5 minutes and read initial absorbance oI sample and
blank (A1) using Beckman Spektrophotometer.
3) Add 0.02 ml GLDH solution into each oI the test tubes. Mix and incubate Ior 5
minutes and read Iinal absorbance oI sample and blank (A2).
4) The plasma ammonia concentration is calculated as the Iormula below:-
Ammonia (Umol/L) A sample A blank X 294
-------------------------
A standard A blank

ALANINE TRANSFERASE TEST
1) Received and check specimens.
2) CentriIuge specimen at 1500rpm Ior 5 minutes and separate serum Irom specimen.
3) Run specimen Ior 20 minutes in Modular P800:-
i) Touch workplace/Test selection
ii) Enter the sample location
iii) Enter the sample id
iv) Touch sample type, sample cup, sample vol.
v) Touch the desired test key or proIile key.

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vi) Load patient sample.
4) writing the results.

ALBUMIN TEST
a. Touch workplace/Test selection
b. Enter the sample location
c. Enter the sample id
d. Touch sample type, sample cup, sample vol.
e. Touch the desired test key or proIile key.
I. Load patient sample.

ALKALINE PHOSPHATASE TEST

AMYLASE TEST
3) Run specimen Ior 20 minutes in Hitachi 912 Analyzer:-

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ASPARTATE AMINO TRANSFERASE TEST

CALCIUM TEST


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TOTAL CHOLESTEROL TEST

CORTISOL TEST

CREATINE TEST

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CREATINE KINASE TEST

DIRECT BILIRUBIN TEST

ELECTROLYTES TEST

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FERRITIN TEST

IRON TEST

GAMMA GLUTAMYL TRASPEPTIDASE TEST

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GLUCOSE TEST

HIGH DENSITY LIPOPROTEIN CHOLESTEROL TEST


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MAGNESIUM TEST

PHOSPHATE INORGANIC TEST

TOTAL BILIRUBIN TEST

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TOTAL PROTEIN TEST

TRIGLYCERIDES TEST

PROGESTERONE TEST
3) Run specimen Ior 20 minutes in CentriIuge 5810:-

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PROLACTIN TEST

CA 125 TEST

CA 199 TEST

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THYROID STIMULATING TEST

FREE THYROXINE (FT4) TEST


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TRIIODOTHYRONINE TEST

TOTAL T4 TEST

LACTATE DEHYDROGENASE TEST

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LITHIUM TEST

LUTEINISING HORMONE TEST

LOW DENSITY LIPOPROTEIN CHOLESTEROL TEST

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ESTRADIOL TEST

FOLLICLE STIMULATING HORMONE TEST


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CORTISOL TEST


D) URINE DRUG TEST
Drug screening test is done via enzyme immunoassay (EIA).There are three types
oI drugs screened here; canabinoids, amphetamine and opiates. The screening test is done
by using a bottle oI urine Irom the patient. II the result is positive, conIirmation test
should be done .The conIirmatory test is done via thin layer chromatography .But iI the
result is negative, the conIirmatory test should not be done by using the patient urine. II
the conIirmatory test is positive, the patient is declared as positive and iI negative the
patient is declared as negative. The conIirmatory test is done to make sure that iI the
patient is really positive in drug test or not.
The conIirmatory test can be divided into 2 steps. First step is detection oI
cannabinoids in urine by thin layer chromatography and the second step is detection oI
morphine in urine by thin layer chromatography. Both conIirmatory tests should be done
to know the type oI drug the patient takes iI the patient is positive in conIirmatory test.


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RECEIVAL OF URINE SAMPLES FOR ABUSED DRUG TESTING

1) All urine specimens Ior abused drug testing are checked on receival and a special
receival Iorm is duly completed by the staII on duty. A proper chain oI custody
documentation shall be properly maintained throughout the series oI events involved
in the testing processes.
2) Incomplete drug test reguisition Iorms and/or urine specimens that Iail to comply with
the receival criteria shall be rejected and the Iorwarding client or agent inIormed
promptly either verbally, by phone or by correspondence. Rejection Ior any urine
specimen shall be endorsed by an authorized staII.
3) The urine specimens are promptly assigned a lab reIerence number. This number shall
be properly labeled to the urine container Ior a speciIic urine samples and also stated
(or printed) on each copy oI the request Iorms Ior particular urine specimens. Details
oI the drug test requisition shall be entered into the computer.
4) When analysis cannot be perIormed immediately, then all the urine specimens are
kept under lock and key in a dedicated Ireezer (at -20c).
5) BeIore any analytical procedure is perIormed, ensure that all the urine specimens
being tested are Iully thawed and homogenously mixed beIore aliquoting Ior the
respective test.

SCREENING TEST FOR ABUSED DRUG
1) Allow all the urine specimens being tested to thaw completely aIter taking out Irom
the Ireezer. The urine specimens are then sorted or arranged careIully and correctly
identiIied against the respective test requisition Iorms Ior the particular urine
specimen and Iinally a batch test worklist is made.

- Multi-drug one step multi-line screen test device (urine): Only Ior medical check
up cases (reIer to respective package insert in conducting the test procedure).
- Employing an automated chemistry/immunoassay analyzer applying EMIT
technology-Ior all cases. All batch test run shall be perIormed together with
quality control and calibrators according to the recommendation oI the reagents
manuIacturers.
- For control cut-oIIs, the NIDA recommendations shall be complied:-
ATS 1000 ng/ml

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Opiates 300 ng/ml
Cannabinoids 100 ng/ml
2) ReIer to reagent package insert instructions and/or instrument operation and
instruction manual when running the test on the particular technology preIerred.
3) The analytical data generated Irom the batch test run Ior each urine specimen, control
and calibrators shall then be correctly entered/recorded in the worklist. The
interpretation oI the analytical data shall then made into a test report Ior each
particular urine specimens and veriIied and endorsed by the authorized staII. The
Results are then chopped unto each copy oI the drug test requisition Iorms Ior
respective urine specimens.
4) All urine specimens that yielded negative during the screening test shall be reported
as negative.
5) Update the test results into the computer Ior all the urine specimens tested.
6) All the urine specimens screened and tested negative` are disposed aIter 2 days Irom
the date oI screening test.
7) The 'Non-Negative urine specimens identiIied during the screening test are then
subjected to a comIirmatory test procedure cannot be perIormed immediately, then
restore all the urine specimens into the Ireezer as number (4).
8) LTAT Ior drug oI abuse screening test shall be not more than 3 working days.

CONFIRMATION TEST FOR ABUSED DRUG

1) Allow all the urine specimens being analysed to thaw completely aIter taking out
Irom the Ireezer. The urine specimens are then sorted or arranged careIully and
correctly identiIied against the respective test requisition Iorms Ior the particular urine
specimen and Iinally a batch test worklist is made.
2) PerIorm conIirmatory test Ior screened positive urine specimens by employing thin
layer chromatography (TLC) methods or by gas Chromatography mass spectrometry
(GCMS) techniques. ReIer to 'A Compilation oI the Test Procedures in The
Detection oI Drug Presence in Urine Specimen at HQE.
3) For all batch run, known controls or panel oI standard values are perIorm together.
QC controls are also perIormed in the same batch run.

36
4) Urine specimens are discarded only aIter Iinal analytical report is veriIied, validated
and endorsed. Intention to dispose any urine drug specimens shall be approved and
conIirmed by the scientiIic oIIicer in charge.
5) Under any circumstances where the analytical process or outcome are inconclusive
and doubtIul, the urine specimens shall be sent to reIerence lab Iurther analysis.
6) Final drug analysis reports are veriIied, validated, endorsend and updated into the
computer/Iile.
7) A copy oI the original drug request Iorm and report containing details oI analysis data
are Iiled accordingly Ior each case analyzed.
8) LTAT Ior conIirmation oI ATS, Opiates and Cannabinoids is not more than 7
working days.

REPORTING FOR ABUSED DRUG

1) Drug analysis reports are sorted, whole work processes involved reviewed,
counterchecked, veriIied and Iinally endorsed by an authorized scientiIic oIIicer.
2) A copy oI the original drug request Iorm and report containing printed/written details
oI analysis data and inIormation are archived accordingly Ior each case analyzed.
3) Drug analysis report copies are disseminated/extended to:-
- Original requesting station/department/organization (attentioned to
requesting oIIicer as stated in the drug request Iorm)
- HQE drug unit record Iile.
4) All drug analysis Iinal report are documented and recorded into dispatch register
book. Representative oI respective stations/department/organization who personally
comes to collect these reports shall be acknowledged receival oI reports by signing
and writing their identity as well as stating the time and date oI report collection.
Reports dispatched by post shall be documented in a special register book kept at the
pathology general oIIice.
5) Retrieval oI drug analysis reports shall be carried out only aIter prior approval given
by the scientiIic oIIicer in charge being the custodian oI these conIidential.


37


38

MODIFIED ORAL GLUCOSE TOLERANCE TEST (MOGTT)
1) The patient should be identiIied.
2) The patient must Iast overnight (10-16 hrs) water, but no other beverage is permitted.
3) Fasting blood glucose specimen is withdrawn and a specimen oI urine obtained.
4) 75g oI glucose is given, dissolved in 250-350 ml oI water. The patient should drink
this within a period oI 5 minutes.
5) Further sample oI blood and urine are obtained at 60 mins and 120 mins aIter the
glucose load.
6) IdentiIy sample and place in biohazard plastic bag and send to laboratory Ior analysis.
7) Analysis the urine and writing report.

PREGNANCY TEST
2) Removed the test strip Irom its Ioil wrapper by tearing along the splice`.
3) Fill a test tube/container with urine sample and hold the strip in a vertical position.
The urine level should not be higher than the 'max level indicated on the strip.
4) Place the test strip into the tube or container containing the sample. The strip must be
placed in the tube or container so that the words 'dip and read are in an upright
position.
5) Read results aIter 5 minutes.
6) Inter results in the test request Iorm and record book.
7) The test results is checked and veriIied.
8) Dissemination oI results to the requesting party.

URINE FEME

OB1ECTI'E
To detect abnormalities in urine by using Multistix reagent strip Ior urinalysis and
microscopic examination

PRINCIPLE
The test is based on visual evaluation oI the color Irom the urine sample. This test will
determine the urine substances by urinalysis strips. Each element oI the urine will be

39
absorbed into each test portion oI the strip and the subsequent chemical and enzymatic
reactions will change the color oI the test paper accordingly. The degree oI coloring will
be proportional to the concentration oI each element in the urine. For the reconIirmation
oI the substances microscopic examination was done.

INTERPRETATION

Full examination:
O By using urinalysis strip, we can detect presence oI chemical in urine such as
protein, glucose, acetone, urobilinogen and bile. These chemical will indicate
physiological changes and pathological condition in the body Ior example:
Protein renal Iailure
Glucose diabetes mellitus
Acetone diabetic ketoasidosis, starvation
Urobilinogen liver Iailure, hepatitis
Bile bile duct obstruction
O Appearance :
Turbid
Clear
colour
O SpeciIic gravity

Microscopic examination:
O Red blood cell usually indicate bleeding oI urinary tract
O White blood cell/ pus usually indicate inIection or inIlammation oI urinary tract
O Cast usually indicate renal impairment
O Epithelial cell the presence oI epithelial cell in small amount in pregnant woman
considered normal. However the presence oI epithelial cell in male is an
indication oI prostate cancer
O Crystal renal calculi


40

PROCEDURE
2) Mix the specimen well.
3) Pour 10ml oI urine in to a graduated centriIuge tube.
4) CentriIuge at 1500 rpm Ior 5 minutes.
5) Remove the supernatant Iluid by the careIul decantation or aspiration.
6) Resuspend the sediment by gently tapping the bottom oI the tube.
7) Place a drop oI resuspended sediment on one area oI a slide and cover with cover slip.
8) Examine with both low (100x) and high (400x) powers Iields.
9) Enter results in the request Iorm and record book.
10) The test results is checked and veriIied.
11) Dissemination oI results to the requesting party.

STOOL/URINE REDUCING SUGAR
1) Add 0.4ml (8 drops) oI urine or stool (pea size) to 5 ml oI qualitative Benedict`s
reagent in a test tube.
2) Place the test tube in a boiling water bath Ior 5 minutes, then examine. When the
test is negative, the solution remains a clear blue and shows at most a white
turbidity due to phosphates. As the amount oI reduction increases, a precipitate
appears which progress Irom green to yellow and Iinally to orange and brick red
with a proportionate discharge oI the original blue colour. The approximate
amount oI glucose can be estimated Irom the appearance.
3) Lastly, writing the result.

NEONATE BILIRUBIN
1) CentriIuge specimen and separate serum Irom specimen.
2) Arrange serum specimen and get a suitable amount oI aliguot and put it in a
vial/cuvette using a pipette where the position has been speciIied.
3) PerIorm calibration on bilirubinometer machine.
4) Run test using bilirubinometer.
5) Lastly, writing the result.


41

URINE HAEMOGLOBIN/RBC
1) Dip the test strip brieIly (max 1 second) into the urine.
2) When withdrawing the strip, wipe its edge along the rim oI the vessel to remove
excess urine.
3) Insert test strip to the analyzer. For visual reading, compare the colors aIter 6o
seconds.
4) Writing the result.


42


43

BLOOD BANK

A) ABO and Rhesus grouping by tile method

OB1ECTI'E
To determine the ABO and Rh grouping oI the blood sample

PRINCIPLE
Agglutination oI the red blood cells with a given antisera is a positive test result which
indicates the presence oI the corresponding antigen on the red cell. Absence oI the
agglutination is the absence oI the particular antigen on the red cell.

PROCEDURE
1. Use pre labeled tile identiIying sections Ior antisera and cell to be used as shown
below.
ANTI-A ANTI-B ANTI-AB CELL A CELL B CELL O RHCTRL
ANTI- D
2. Drop 1 drop oI antisera to ABO cell in appropriate sections.
3. Drop 1 drop oI sample serum Ior O cell, A cell and B cell Ior ABO reverse
grouping.
4. Drop 1 drop oI blood sample Ior Anti-A, Anti-B and Anti-AB Ior ABO Iorward
grouping and Anti-D and Rh Ctrl Ior Rhesus (D) Typing.
5. Mix cell and antisera using clean applicator stick thoroughly over an area about
2cm diameter.
6. Keep cell and antisera mixture in a continuous gentle motion and observe Ior
agglutination. Grade the agglutination and record.
7. Care must be taken not to allow drying up at the edges oI cell suspension to
prevent Ialse positive result.

INTERPRETATION
1. Agglutination oI red blood cells constitute a positive result.
2. A smooth suspension oI red blood cells at the end is a negative result.


44

B) Hemoglobin estimation by Copper Sulphate method
O To estimate hemoglobin (Hb) level Ior blood donors semiquantitatively ie
~12.5g5 or 12.5g
O Interpretation (aIter 15seconds drop oI blood):
- Sinking : ~12.5g
- Floating: 12.5g.

C) Coomb`s test by tube method

OB1ECTI'E
To detect human immunoglobulin and/or complement components bound to human
erythrocytes.

PRINCIPLE
The direct Anti-Human Globulin (AHG) test is used to demonstrate whether or not red
cells have been coated (sensitized) with antibody and/or complement in vivo. The test is
useIul in the diagnosis oI hemolytic disease oI new born (HDN), autoimmune hereditary
(AIHA) and in investigation oI red blood cells sensitized by drugs and delayed
transIusion reactions. The indirect AHG test is used to demonstrate the presence oI allo
antibodies and/or complement in the serum (in vivo).

PROCEDURE
O Direct AHG test
1. Prepare a 3-5 red cells suspension Irom patient blood in saline (one drop oI red
blood cell saline).
2. Add one oI above red blood cell suspension to an appropriately labeled test tube
and wash red cells in saline a minimum oI three times with saline.
3. Following the Iinal wash, completely decant the supernatant saline and blot the
tubes edges on the clean absorbent tissue to ensure the removal oI all residual
saline and obtain the resultant dry` red cells button.
4. Add two drops oI AHG reagent to each tube containing a dry` button oI washed
red cells. Mix tube gently but thoroughly to suspend/separate recipient red cells.

45
5. CentriIuge immediately Ior 15s at 3000rpm.
6. Gently dislodge recipient red cells button and examine Ior agglutination.
7. Grade and record result.
8. Negative or weak positive AHG test result should be appropriately controlled by
Coomb`s control cells.

O Indirect Coomb`s test
1. Label three test tube I, II and III.
2. Add three drops oI serum to be tested in each tube.
3. Add one drop oI screening cells I,II and III to each tube respectively.
4. Mix the tube well and incubate at room temperature Ior 1-5 min.
5. Record result.
6. CentriIuge both tubes Ior 15 s at 3000 rpm.
7. Add two drops oI Albumin or LISS to each tube.
8. Mix the tube well and incubate at 37C Ior 30 min.
9. a`1CentriIuge both tubes Ior 15 s at 3000 rpm.
10. Record the result.
11. Wash the cells Ior three times with 60 s Ior centriIugation.
12. Add two drops AHG reagent to the cell sediment.
13. Mix well and centriIuge Ior 15 s at 3000 rpm.
14. Re-suspend the cells by gentle agitation and examine tubes macroscopically Ior
agglutination.
15. It`s recommended all positive result be graded Irom 4 to - at each stage oI
reading.
16. To all negative and weak positive test, add Coomb`s control cell to validate test
result.


46

INTERPRETATION
Positive: hemagglutination oI tests cells in the antiglobulin test phase oI a direct or
indirect antiglobulin test procedure constitute a positive test results and within the
accepted limitations oI test procedure, indicate the presence oI IgG and/or complement
(C3) on the red cells.
Recommended grading oI reactions strengths

Score Grade Macroscopic Microscopic
12 C Complete agglutination
- a single agglutinate oI
strongly reactive red cells
No Iree cells detected
10 Strong reaction, one or two
large agglutinates
A Iew detached masses oI
agglutinated cells
8 Strong reaction, a number oI
large agglutinates
Large agglutinates in a sea
oI smaller clumps, and a
scattering oI Iree cells
5 Many small agglutinates Many agglutinates oI up to
20 cells in background oI
small clump and Iree cells
3 /- Weak granularity in cell
suspension
Scattered agglutinates oI 6-
8 cells, with mostly Iree
cells
1 W An even cell suspension A Iew small clumps oI 3-4
cells in a sea oI Iree cells
0 0 An even cell suspension No agglutinates detected


47

D) Cross-matching by tube method
OB1ECTI'E
To determine compatibility testing in crossmatching oI donor cell and patient serum.

PRINCIPLE
This method used to determine the agglutination Ior ABO blood group and to detect
antibody or complement bound to red cell. The agglutination mean that the donor cell and
patient serum have the speciIic complementary.

PROCEDURE
Room temperature phase
1. Label the three tubes S` (Saline test), A` (Albumin test) and C` (Auto control).
2. Do the ABO grouping Ior the patient.
3. CentriIuge the patient blood to get the serum.
4. Add two drops oI patient serum into each tube.
5. Add two drops oI 22 albumin into tube A`.
6. Add one drop oI 3-5 red cell suspension donor into tubes S` and A`.
7. Add one drop oI 3-5 red cell suspension patient into tube C`.
8. Mix well and centriIuge the three tubes at 3000 rpm Ior 15 s.
37C phase
1. The three tubes are incubated at 37C Ior 30 min.
2. CentriIuge the tubes at 3000 rpm Ior 15 s.
AHG phase
1. Wash the cell oI tube A` and C` Ior three times with saline.
2. Decant the all saline.
3. Add two drops oI AHG into the two tubes.
4. Mix well and centriIuge the tubes at 3000 rpm Ior 15 s.
6. The all negative test should be conIirmed by the AHG Coomb`s Control Cell.


48


49

SEROLOGY LABORATORY
Most oI the tests done in the serology lab used kits that contain speciIic antigens. There
are more than 5 tests done in this laboratory. The most common tests done are ANTI-HIV
I & II, HBsAg, Anti-HCV and VDRL screening oI blood and blood products using the
EVOLIS (BIO-RAD) and MUREX machine.

` Blood screening by using E'OLIS (BIO-RAD) machine are stated below:
A) HI'

PRINCIPLE OF THE GENSCREEN PLUS HI' ANTIGEN-ANTIBODY (HI' Ag-
Ab) KIT

GENSCREEN PLUS HIV Ag-Ab kit is an enzyme immunoassay based on the principle
oI the sandwich technique Ior the detection oI HIV antigen and oI the various antibodies
associated with HIV 1 and / or HIV 2 virus in human serum/plasma.

The GENSCREEN PLUS HIV Ag-Ab solid phase is coated with:
O Monoclonal antibodies against p24 HIV 1 antigen
O PuriIied antigens: gp 160 recombinant proteins with an artiIicial Iunctional
consensus polypeptide which is composed oI variable sequences oI the virus
inserted into well-preserved sequences oI HIV-1 group O strains and a peptide
mimicking the immunodominant epitope oI the HIV2 envelope protein.

THE GENSCREEN PLUS HIV Ag-Ab conjugates are based upon the use oI:
O Biotinylated polyclonal antibodies to HIV Ag (conjugate 1)
O Avidin and HIV antigens peroxidase conjugate (gp41 and gp36 peptides
mimicking the immunodominant epitopes oI the HIV 1 and HIV 2 envelope
glycoproteins, and the same Iunctional consensus polypeptide oI HIV1 used Ior
the solid phase) (conjugate)


50

The assay procedure includes the Iollowing reaction steps :
O Conjugates 1 (biotinylated polyclonal antibody to p24 HIV 1 Ag ) is added into
the microplate wells.
O Serum samples to be assayed and controls are pipetted into the wells
1. HIV antigens, iI present, will bind with the monoclonal antibody
coated to the solid phase and the conjugate 1.
2. HIV 1 and/ or HIV 2 antibodies, iI any, will bind to the antigens
immobilized on the solid phase.
3. Deposition oI conjugate 1 and sample is validated through a colour
change, Irom yellow-green to blue.

O AIter incubation at 37C Iollowed by the washing step, conjugate 2 is added :
1. Avidin reacts with biotinylated Ab-Ag-Ab complexes.
2. Peroxidase labelled, puriIied HIV 1 and HIV 2 antigens bind in
turn to the antibodies captured on the solid phase.

O AIter incubation at 18.30 C the unbound conjugate 2 Iraction is removed by
washing. AIter incubation in presence oI the substrate at room temperature, the
presence oI the complexed conjugate is shown by a change oI colour.
O The reaction is stopped and absorbance are read using a spectrophotometer at
450/620-700 nm. The absorbance measured on a sample determines the presence
or absence oI HIV Ag or HIV 1 and/ or HIV 2 antibodies.

INTERPRETATION OF THE RESULTS OF GENSCREEN

PLUS HIV ANTIGEN-
ANTIBODY (HIV Ag-Ab) KIT

Samples with absorbance value less than the cut-oII value are considered to be negative
by the GENSCREEN

PLUS HIV Ag-Ab test.
Result just below the cut-oII value (CO -10 OD CO ) should however, be
interpreted with caution ( it is advisable to retest in duplicate the corresponding samples
when the systems and laboratory procedures permit).

51
Samples with absorbance values equal to or greater than the cut-oII value are initially
considered to be positive by the GENSCREEN

PLUS HIV Ag-AB test. They should be
retested in duplicate beIore Iinal interpretation.
II aIter retesting oI a sample, the absorbance values oI the 2 duplicates are less than the
cut-oII value, the initial result is non repeatable and the sample is declared to be negative
with the GENSCREEN

PLUS HIV Ag-AB test.
Non repeatable reactions are oIten caused by :
O Inadequate microplate washing
O Contamination oI negative samples by serum or plasma with a high antibody titre
O Contamination oI the substrate solution by oxidizing agents
O Contaminations oI the stopping solution

II aIter retesting the absorbance oI one oI the duplicates is equal to or greater than the
cut-oII value, the initial results is repeatable and the sample is declared to be positive
with GENSCREEN

PLUS HIV Ag-Ab test.

B) HB' DETECTION
MONOLISA
HBs Ag ULTRA

PRINCIPLE
MONOLISA
HBsAg ULTRA assay is a one step enzyme immunoassay based on the

principle oI the 'sandwich type using monoclonal antibodies and polyclonal antibodies
selected Ior their ability to bind themselves to the various subtypes oI HBs Ag now
recognized by the WHO and the most part oI variant HBV strains.

The MONOLISA
HBsAg ULTRA solid phase is coated with monoclonal antibodies.

The MONOISA
HBsAg ULTRA conjugates are based on the use oI monoclonal

antibodies Irom mouse and polyclonal antibody Irom goat against the HBsAg. These
antibodies are bound to the peroxidase.

The assay procedure includes the Iollowing reaction steps:
1. Distribution oI control sera and samples into the wells oI the microplate. This
distribution can be visually controlled: there is a clear diIIerence oI colouration

52
between empty well and well with samples. This distribution can also be
controlled automatically by reading at 490/620-700 nm (optional).
2. Distribution oI the red coloured conjugate into wells. This distribution can also be
visually controlled: AIter the conjugate solution addition, the colour oI the well
becomes red. It is possible to control automatically this distribution by the
spectrophotometric reading at 490/620-700 nm (optional). The sample deposition
can also be controlled at this step oI the manipulation by automatic reading at
490/620-700 nm.
3. AIter incubation at 37C during one hour and halI the unbound conjugate is
removed by washing.
4. Distribution oI coloured substrate. This distribution can be visually controlled :
there is a clear diIIerence oI colouration between empty well and well with the
pink solution. This distribution can also be controlled automatically by reading at
490 nm (optional).
5. AIter 30 minutes incubation in presence oI the substrate in dark and at room
temperature (18-30C), the presence oI the complexed conjugate is shown by a
change oI colour.
6. Distribution oI the stopping solution. This distribution can be visually controlled:
The substrate solution which initially pink becomes uncoloured Ior the non
reactive samples well and turn blue to yellow Ior the positive sample wells.
7. Reading oI the optical densities at 490/620-700 nm and interpretation oI the
results.
Interpretation oI the results
Samples with ratio values lower than 1 are considered to be negative by the
MONOLISA
HBsAg ULTRA. Results just below the cut-oII value (sample ratio
between 0.9 and 1) should however, be interpreted with caution. It is advisable to retest in
duplicate the corresponding samples when the systems and laboratory procedures permit.
Samples with ratio values equal to or greater than 1 are considered to be initially positive
by the MONOLISA
HBsAg ULTRA. They should be retested in duplicate beIore Iinal

interpretation.
II aIter retesting oI a sample, the ratio values oI the 2 duplicates are less 1, the initial
result is non repeatable and the sample is declared to be negative with the MONOLISA

HBsAg ULTRA.

53
II aIter retesting oI a sample, the ratio values oI the 2 duplicates are greater than 1, the
initial result is repeatable and the sample is declared to be positive, subject to the
limitations oI the procedure, described below.
The samples which have retested twice and Iound negative but with one value near the
cut-oII value (ratio between 0.9 and 1) should be considered with care. It is advised to
retest the patient with another method or another sample.
To veriIy the speciIicity oI the reaction, every positive result in accordance with the
interpretation criterias oI the MONOLISA
HBs Ag ULTRA should be conIirmed by a

neutralization method oI the HBs Ag.
Non repeatable reactions are oIten caused by :
O Inadequate microplate washing,
O Contamination oI negative samples by serum or plasma with a high HBs Ag
concentration,
O Contamination oI the substrate solution by oxidizing agents,
O Contamination oI the stopping solution.

C) HC' DETECTION
MONOLISA
Anti-HC' PLUS 'ersion 2

PRINCIPLE OF THE TEST
MONOLISA
Anti-HCV PLUS Version 2 is based upon the oI a solid phase prepared

with puriIied antigens :3 recombinant proteins produced by E.coli Irom clones selected in
the non structural area (NS3 and NS4) and in the structural area oI the hepatitis C virus
genome. Detection is with goat anti-human IgG antibody puriIied by aIIinity
chromatography and coupled to peroxidase.
The perIormance oI the test includes the Iollowing reaction steps :
1) The samples to be tested and the control sera are added to the wells. II antibodies
to HCV are present, they will bind to the antigens Iixed on the solid phase.
2) The peroxidase-labelled antibodies to human IgG are added aIter a washing step.
They in turn bind to the speciIic antibodies captured on the solid phase.
3) AIter removal oI the unbound enzymatic conjugate, the antigen antibody complex
is revealed by addition oI substrate.

54
4) AIter the reaction has been stopped, the absorbance values are read using a
spectrophotometer at 450/620-700 nm. The absorbance measured Ior a sample
allows the presence or absence oI antibody to HCV to be determined. The colour
intensity is proportional to the quantity oI antibody to HCV bound on the solid
phase.

INTERPRETATION OF THE RESULTS
Samples with an optical density less than the cut-oII value are considered to be negative
with the MONOLISA
Anti-HCV PLUS Version 2 test.

However, results just below the cut-oII value (OD sample ~ OD Cut-oII 10) should be
interpreted with care and the corresponding samples retested in duplicate. Samples with
an optical density higher than, or equal to the cut-oII value are considered to be positive
and must be retested in duplicate beIore the Iinal interpretation.
AIter retesting, the sample is considered to be positive iI the second or third measurement
is positive, i.e. higher than, or equal to, the cut-oII value. The sample is considered to be
negative iI broth values are less than the cut-oII value.

` Blood screening by using MUREX machine are stated below:

A) Detection of Hepatitis B Surface Antigen

PURPOSE
O To detect the presence oI hepatitis B surIace antigen in human serum or plasma

PRINCIPLE
In Murex HBsAg Version 3, the sample is preincubated in microwells coated with
mixture oI mouse monoclonals speciIic Ior diIIerent epitopes on the determinant oI
HBsAg. AIIinity puriIied goat antibody to HBsAg conjugated to horseradish peroxsidase
is then added to the sample in the well. During the two incubation steps any HBsAg
present in the sample is bound to the well in an antibody-antigen-antibody-enzyme
complex. In the absence oI HBsAg no conjugate will be bound. AIter washing to remove
sample and unbound Conjugate, a solution containing 3,3`,5,5`-tetramethybenzidine

55

(TMB) and hydrogen peroxide is added to the wells. Wells which contain HBsAg and
hence bound Conjugate will develop a purple colour which is converted to orange when
the enzyme reaction is determined spectrophotometrically and is directly proportional to
the amount oI Conjugate bound and hence the concentration oI HBsAg in the sample.

METHOD
1. Pre test preparation by placing reagent and specimens at room temperature 30
minutes beIore use.
2. Arrangement oI Specimens
For each test batch, arrange specimens as per appendix 1. Start with specimen
no.1 in well F1. The wells Ior controls are as Iollows
A1 negative control
B1 negative control
C1 positive control
D1 blank E1 blank

3. Allow another MLT to sample 5 specimens at random per rack and veriIy that
these specimens tally with records in request Iorm/record book. The MLT who
veriIies shall endorse on Iorm/book samples veriIied. II there is any
discrepancy(ies), stop test and investigate to resolve discrepancy(ies).II there are
no discrepancy(ies) continue test.
4. Label rack and Miniprep rack with date and test batch example, 010801-0101.
5. Label Microtitre plate with test, date and test batch example, HBsAg-010801-
0101
6. Use two or more microtitre plate iI specimens tested above 91 specimens and
label microtitre plate with number, test and test batch example,
1-HBsAg-0101


56

TEST METHOD
1. Dispensing control, specimen and specimen diluent into microtitre plate using
miniprep or manual method
2. Incubation
3. Do not wash aIter incubation. Add conjugate to each well oI microtitre plate using
miniprep or manual method as prescription below
4. AIter add conjugate, shake the plate using plate shaker (as prescription 2.2) Ior 10
second or manually agitate by tapping the sides Ior 10 second
5. AIter add conjugate, incubate microtitre plate in Stuart ScientiIic Incubator` Ior
30 to 33 minutes (37 C).
6. Washing
7. AIter wash, add Substrate to each well oI microtitre plate using miniprep or
manual method
8. AIter add substrate, incubate microtitre plate in Stuart ScientiIic Incubator` Ior 30
to 33 minutes (37 C)
9. AIter incubate, add Stop reaction (0.5M to 2M Sulphuric acid) to each well oI
microtitre plate using miniprep or manual method
10. Reading

B) Detection of antibodies to Hepatitis C 'irus (HC')

PURPOSE
O To detect the presence oI anti-HCV in human serum or plasma

PRINCIPLE
In murex anti-HCV (version 4.0) diluted sample is incubated in microwells coated with
high puriIied antigens with contain sequences Irom the core, NS3, NS4 and NS5 regions
oI HCV. During the course oI the Iirst incubation any anti-HCV antibodies in the sample
will bind to the immobilized antigens. Following washing to remove unbound material,
the captured anti-HCV antibodies are incubated with peroxsidase conjugated monoclonal
anti human IgG. During the course oI the second incubation the conjugate will bind to
antibody immobilized in the Iirst step. AIter removal oI excess conjugate, bound enzyme

57
is detected by addition oI the solution containing 3,3`,5,5`-tetramethylbenzidine (TMB)
and hydrogen peroxide. A purple colour will develop in the wells which contained anti-
HCV positive samples. The enzyme reaction is terminated with sulphuric acid to give an
orange colour which is read photometrically. The amount oI Conjugate bound, and hence
colour, in the wells, is directly related to the concentration oI antibody in the sample.

C) Detection Of Antibodies To Work Human Immunodeficiency 'irustypes 1(Hiv-1,
Hiv-1 Group O) And 2(Hiv-2) In Human Serum Or Plasma

PURPOSE
O To detect the presence oI anti-HIV in human serum or plasma

PRINCIPLE
Murex HIV-1.2.0 is based on microwells coated with a synthetic peptide representing an
immunodominant region oI HIV-1 (O), recombinant protein derived Irom the envelope
proteins oI HIV-1 and HIV-2 and an HIV core protein. The conjugate is a mixture oI the
same epitopes all labeled with horseradish peroxidase.
Test specimens and control sera are incubated in the wells and antibodies to HIV in the
sample or control sera bind to the antigens on the microwell; sample and any excess
antibodies are then washed away. In a subsequent step, conjugate is added which in turn
binds to any speciIic antibody already bound to the antigens on the wells. Sample not
containing speciIic antibody will not cause the conjugate to bind to the well. Unbound
conjugate is washed away and a solution containing 3.3`,5,5`-tetramethylbenzidine
(TMB) and hydrogen peroxide is added to the wells. Wells with bound Conjugate develop
a purple colour which is converted to an orange colour when the reaction is stopped with
sulphuric acid. AIter incubation the enzymic reactions are terminated with sulphuric acid
and the colour is read spectrophotometrically at 450 nm. The amount oI Conjugate, and
hence colour, in the wells is directly related to the concentration oI antibody to HIV in the
sample.


58

METHODS
1. Pre test preparation.
2. Arrangement oI Specimens. For each test batch, arrange specimens as per appendix 1.
Start with specimen no.1 in well F1. The wells Ior controls are as Iollows
A1 negative control D1 positive control
B1 negative control E1 positive control
C1 negative control
3. Dispensing control, specimen and specimen diluent into microtitre plate using
miniprep
4. Incubation
5. Washing
6. AIter wash, add conjugate to each well oI microtitre plate using miniprep or manual
method.
7. AIter add conjugate, incubate microtitre plate in Stuart ScientiIic Incubator` Ior 30 to
33 minutes (37 C).
8. AIter incubation, wash microtitre plate.
9. AIter wash, add Substrate to each well oI microtitre plate using miniprep or manual.
10. AIter add substrate, incubate microtitre plate in Stuart ScientiIic Incubator` Ior 30 to
33 minutes (37 C).
11. AIter incubate, add Stop reaction (0.5M to 2M Sulphuric acid) to each well oI
microtitre plate using miniprep or manual method.
12. Reading
13. Read within 15 minutes at absorbance 450 nm using 620nm to 690 nm as the
reIerence.
INTERPRETATION OF RESULT
Negative Result - Samples giving an absorbance less than the Cut-oII value are
considered negative in the assay.
Reactive Result
Samples giving an absorbance equal to or greater than the Cut-oII value are considered
initially reactive in the assay. Such samples should be retested in duplicate using the
original source. Sample that are reactive in at least one oI the duplicated retested are

59
considered repeatedly reactive murex HIV-1.2.O and are presumed to contain antibodies
to HIV-1 or HIV-2. Such samples should be Iurther investigated and the presence oI
antibodies against HIV conIirmed by other tests. Samples that are non-reactive i.e with an
absorbance less than that oI the Cut-oII value, should be considered non-reactive Ior HIV
antibodies.

OTHER TEST A'AILABLE IN SEROLOGY LABORATORY
SERODIA

HI'-1/2-confirmation test for HI'

PRINCIPLE OF THE PROCEDURE
SERODIA

HIV 1/2 is manuIactured using gelatin particle carriers sensitized with
inactivated HIV 1 and HIV 2 antigens separately. The test is based on the principle that
sensitized particles are agglutinated by the presence oI antibodies to HIV 1 and HIV 2 in
human serum/plasma.

INTENDED USE
This SERODIA

HIV 1/2 kit is used to detect antibodies to HIV 1 and HIV 2 in serum or
plasma specimens. It detects anti-HIV antibodies but it does not detect the virus antigen.
As with any antibody detection assay, SERODIA HIV 1/2 may not be capable oI
detecting very small amounts oI antibody associated with early inIection or the very early
seroconversion period.

IMMUNOTREP
CARBON ANTIGEN


IMMUNOTREP
CARBON ANTIGEN is a modiIied Iorm oI IMMUNOTREP VDRL

ANTIGEN which contains carbon particles to improve the visual reading oI the result.
When binding occurs between cholesterol/ cardiolipin / lecithinin the reagent and the
reagin antibodies in the samples, the results can be seen macroscopically in the Iorm oI
black clumps. No visual Ilocculation indicates a negative result.


60

RESULTS AND INTERPRETATION
Known level value samples should be tested with each test run. II users known samples
do not give expected results, test results must be considered invalid.

Qualitative method
Medium and large aggregates reactive
Finely dispersed aggregates weak reactive
No aggregates visible, smooth grey appearance non reactive

Semi-quantitative method
The titre is the last dilution that produces a reactive result.
Titres oI 1/128 has been detected with IMMUNOTREP
CARBON ANTIGEN with no

prozone (Hook) eIIect.

SYPHILIS TPPA LIQUID (HUMAN GmbH) - CONFIRMATION TEST FOR
'DRL POSITI'E SPECIMEN

Principle of the Test:
O Test is based on indirect haemagglutination test Ior detection oI speciIic
antibodies against T. pallidum.
O Uses avian erythrocyte coated with T. pallidum antigen
O Serum containing speciIic antibodies will react with the sensitized cells to
Iorm characteristics patterns in microtitration plates
O Antibody to non-pathogenic Treponemas are absorbed by an extract oI
ReiIer`s Treponemas included in suspensions

Test procedure:
1. Dropping oI serum diluent : 100 3l l ( well 1) and 25 3l (well 2 & 3)
2. Dropping oI test serum/ PC/NC to well 1 mix well
3. TransIer 25 3l oI mixture (well 1) to well 2 mix well
4. TransIer 25 3l oI mixture Irom the last well (well 3)

61
5. Add 75 3l oI suspended SCC ( Control cells - blue cap) well 2
6. Add 75 3l oI suspended STC ( Test cells - white cap) well 2
7. Shake plate gently to ensure thorough mixing
8. Place plate on white level surIace, away Irom vibration and direct sunlight
9. Leave Ior 45-60 minutes read plates (plate interpretation)

IM QUICK TEST

Up to 90 oI patients with InIectious Mononucleosis show high titres oI heterophilic
antibodies. The cause oI I.M. has not yet been isolated but it has been reported that there
is a relationship between the disease and the Epstein-Barr Virus.

The IM QUICK TEST uses especially treated with horse erythrocytes with high
speciIicity Ior the heterophilic antibodies associated with I.M.
The stabilized reagent does not react with normal Frossman antibodies.
Latex particles have been added to the reagent to assist in the resuspension oI the
erythrocytes.

Contents
ER - Erythrocyte Reagent (white dropper)
PC - Positive Control serum (red dropper)
NC - Negative Control serum (green dropper)

INTERPRETATION, QUALITY CONTROL
Positive Control serum (PC) and Negative Control serum (NC) should be run with each
test and compared with the unknown specimen to distinguish possible granularity Irom
agglutination. The test is considered negative when there is no diIIerence in agglutination
is observed between the unknown specimen and Negative Control serum (NC). Positive
Control serum (PC) and positive sera must show distinct agglutination within 2 minutes.


62

RHEUMATOID FACTOR

The HUMATEX RF Test is based upon the agglutination reaction between Rheumatoid
Factor (RF) oI a patient specimen or control serum and human IgG coated onto
polystyrene latex particles. A positive reaction is indicated by a distinctly visible
agglutination oI the latex particles in the test cells oI the slide.

CONTENTS
LR - RF Latex Reagent
PC - Positive Control serum (red cap)
NC - Negative Control serum (green cap)
GBS - Glycine-NaCl BuIIer

QUALITATIVE DETERMINATION (screening test)
Pipetting Scheme
Bring LR, PC, NC and serum samples to room temperature.
Mix LR careIully prior to use to suspend the latex particles completely
Pipette/ drop onto separate cells oI the slides :
Serum sample
PC, red cap
NC, green cap
40 3l
1 drop
1 drop
LR, white cap, onto all sample and control
cells
1 drop each
Mix with separate sticks and spread the Iluid over the entire area oI the particular cell
Tilt the slide back and Iorth Ior 2 minutes so that the mixture rotates slowly inside the
cells or place the slide on an automated rotator at 100 r.p.m.
At the end oI the 2 minutes read the results under bright artiIicial light.

Interpretation oI Results
Distinct agglutination indicates a RF content oI more than 20 IU/ml in the non-diluted
serum specimen.
Sera with positive results in the screening test should be re-tested in the titration test.

63
Diagnostic value
The clinical signiIicance oI RF determinations consists oI diIIerentiating between
rheumatoid arthritis and rheumatoid Iever. It was Iound that in rheumatoid arthritis
approximately 80 oI the cases examined showed the presence oI rheumatoid Iactor.
Whereas Ior rheumatoid Iever, the rheumatoid Iactor is almost always absent. The RF test
is more Irequently positive in long term active process than in diseases which are less
active or are still in early stages. RF is occasionally Iound in the serum oI patients with
polyarthritis nodosa, SLE, hepatitis and certain other diseases.

Rheumatoid Factor Agglutination Test
O For the detection oI Rheumatoid Iactor(s) in serum as an aid in the diagnosis oI
Rheumatoid Arthritis

TYPHI DOT (WIDAL TEST)

PURPOSE
O To detect and qualitative sample type

PRINCIPLE
In Widal test, antibodies produced in patient`s serum in response to bacterial antigens
agglutinate with homologous bacterial antigens in the stained bacterial suspensions.
Somatic constituents oI some strains oI Proteus are shared in some species oI Rickettsia.
ThereIore in the Weil-Felix test, sera Irom people with certain rickettsial inIections
produce antibodies which agglutinate with the somatic antigens oI the Proteus strains in
the suspensions. The bacterial cells which are standardized, killed suspensions oI smooth
organisms have been stained to Iacilitate the reading oI reactions.

INTERPRETATION
Positive test: Agglutination reaction observed
Negative test: No Agglutination observed


64

DENGUE 'IRUS RAPID TEST STRIP

EXPLANATION OF THE TEST

Dengue viruses, transmitted by the mosquito, 0d08 albopictu8 and 0d08 a0pti are
widely distributed throughout the tropical and subtropical areas oI the world. There
are Iour known distinct serotypes (dengue virus 1, 2, 3 and 4 ). In children, inIection
is oIten subclinical or causes a selI-limited Iebrile disease. However, iI the patient is
inIected a second time with a diIIerent serotype, a more severe disease, dengue
hemorrhagic Iever or dengue shock syndrome, is more likely to occur. Dengue is
considered to be the most important arthropod borne viral disease due to the human
morbidity and mortality it causes.

Traditionally, the serological diagnosis oI an acute dengue virus inIection has relied
on showing a 4-Iold or greater rise in anti-dengue virus antibody between paired
acute- and convalescent phase sera Irom a patient. The haemagglutination-inhibition
test has been the most commonly used serological assay Ior dengue diagnosis.

Rapid and reliable tests Ior primary and secondary inIections oI dengue are essential
Ior patient management. Primary Dengue inIection is associated with mild to high
Iever, headache, muscle, pain and skin rash. Immune response includes lgM
antibodies produced by the 5
th
day oI symptoms and persist Ior 30-60 days. lgG
appear the 14
th
day and persist Ior liIe. Secondary inIections oIten result in high Iever
and in many cases with haemorrhagic events and circulatory Iailure. Secondary
inIections show that lgG titre rise within 1-2 days aIter the onset oI symptoms and
induce IgM response aIter 20 days oI inIection.

AccuSens Dengue lgG / lgM Rapid Test is a solid phase immunochromatographics
assay Ior the rapid, qualitative and diIIerential detection oI IgG and IgM antibodies to
dengue virus in human serum, plasma or whole blood. This test is intended Ior
proIessional use as aid in the presumptive diagnosis between primary and secondary
dengue inIection.


65
This test provides only a preliminary test result. ThereIore, isolation oI the virus,
antigen detection in Iixed tissues, RT-PCR and serological test like
haemagglutination-inhibition test, more speciIic alternative diagnosis method must be
used in order to obtain a conIirmation oI dengue virus inIection.

AccuSens Dengue lgG / lgM Rapid Test is designed to simultaneously detect and
diIIerentiate IgG and IgM antibodies to Dengue virus in human serum, plasma or
whole blood. This test also can detect all 4 dengue serotypes by using a mixture oI
recombinant dengue envelope proteins. AccuSens Dengue lgG / lgM Rapid Test strip
has 3 pre-coated line, 'G ( Dengue IgG Test Line ), 'M ( Dengue IgM Test Line ),
and 'C ( control line ) on the surIace oI the strip. A purple 'G and 'M lines will be
visible in the result window iI there are enough IgG and/or IgM antibodies to Dengue
virus in the sample.

When a specimen is added to the test, anti-Dengue IgG and IgM in the specimen
sample react with recombinant Dengue virus envelope proteins oI colloidal gold
conjugates and Iorms a complex oI antibodies and colloidal gold conjugates. As the
mixture migrates along the test strip by capillary action, the anti-Dengue IgG or IgM
complex is captured by the relevant anti-human IgG or anti-human IgM immobilized
in two lines across the test strip and generate a coloured line.

Total PSA- total (free+ complexed) Prostate-Specific Antigen

PSA is a glycoprotein having which has the Iunction oI a serine proteinase. The
proteolytic activity oI PSA in blood is inhibited by the irreversible Iormation oI
complexes with protease inhibitors such as alpha 1-antichymotrypsin, alpha 2-
microglobulin and other acute phase proteins. Besides these complexes, about 30 oI the
PSA present in blood occurs in the Iree Iorm, but is proteolytically inactive.

Elevated concentrations oI PSA in serum are generally indicative oI pathologic condition
oI prostate (i.e. prostatitis, benign hyperplasia or carcinoma). As PSA is also present in
para-urethral and anal glands, as well as in breast tissue or with breast cancer, low levels

66
oI PSA can also be detected in sera Irom woman. PSA may still be detectable even aIter
radical prostatectomy.

The main areas in which PSA determinations are employed are the monitoring oI
progress and eIIiciency oI therapy in patients with prostate carcinoma or receiving
hormonal therapy. The steepness oI the rate oI Iall in PSA down to no-longer detectable
levels Iollowing radiotherapy, hormonal therapy or radical surgical removal oI the
prostate provides inIormation on the success oI therapy. An inIlammation or trauma oI
the prostate can lead to PSA elevations oI varying duration and magnitude.

Intended uses
The Elecsys total PSA immunoassay, is a quantitative in vitro diagnostic test Ior total
(Ireecomplexed) Prostate-speciIic antigen in human serum and plasma. It is indicated Ior
the measurement oI total PSA in conjunction with digital rectal examination as an aid in
the detection oI prostate cancer in men aged 50 years or older. Prostate biopsy is required
Ior diagnosis oI prostate cancer. The test is Iurther indicated Ior serial measurement oI
tPSA to aid in the management oI cancer patients.

Immuno Concepts HEp-2000
Colorzyme

ANA -Ro Test

SUMMARY AND EXPLANATION OF THE TEST

The Immuno Concepts HEp-2000
Colorzyme

ANA Ro Test System uses the indirect
enzyme antibody technique. Diluted patient samples are incubated with antigen substrate
to allow speciIic binding oI autoantibodies to cell nuclei. II anti-nuclear antibodies are
present, a stable antigen-antibody complex is Iormed. AIter washing to remove non-
speciIically bound antibodies, the substrate is incubated with an anti-human antibody
conjugated to horseradish peroxidase (HRP). When results are positive, there is the
Iormation oI a stable three-part complex consisting oI HRP-conjugated anti-human
antibody bound to human antinuclear antibody which is bound to nuclear antigen. This
complex is visualized by incubating the slide in Colorzyme

color reagent which contains

67
an enzyme speciIic substrate. The reaction between the enzyme-labeled antibody and
enzyme speciIic substrate results in a color reaction visible by standard light microscopy
oI the slide. In positive samples, the cell nuclei will show a blue-purple staining pattern
characteristic oI the particular nuclear antigen distribution within the cells. II the sample
is negative Ior ANA, the nucleus will show no clearly discernible pattern oI nuclear
staining. The cytoplasm may demonstrate weak staining while the non-chromosome
region oI the mitotic cells may demonstrate a darker staining.

Anti-HA' IgM
The hepatitis A virus is a RNA-containing virus that lacks an envelope. It belongs to the
Iamily oI picornaviruses. To date, just one human serotype and 7 genotypes have been
described. The viral capsid consists oI 3 proteins (VP1-VP3) that Iorm an
immunodominant structure on the surIace oI the viral particle that is highly conserved
between all genotypes. AIter vaccination or natural inIection, the immune response is
directed against this structure. Hepatitis A is the most common Iorm oI acute viral
hepatitis. It is transmitted by the Iecal-oral route. The disease has not been known to take
a chronic course, nor does the virus persist in the organism.

An acute hepatitis A inIection can be assumed iI anti-HAV IgM antibodies are detected.
Anti-HAV IgM antibodies can always be detected at the onset oI the diseases, and usually
disappear 3 to 4 months later. Anti-HAV IgM can also be detected in some patients Ior a
longer period oI time, however HAV IgM antibodies develop only very rarely aIter
vaccination.

Assays to detect anti-HAV IgM antibodies are used in the diIIerential diagnosis oI acute
hepatitis to determine a hepatitis A inIection.

Test principle
-capture test principle. Total duration oI assays: 18 minutes.
O 1
st
incubation: Pretreatment oI 10 L oI the automatically 1: 400 diluted sample (
using Elecsys Diluent Universal ) with anti-Fdy reagent to block speciIic lgG in

68
the presence oI monoclonal anti-HAV antibodies labeled with ruthenium
complex
a
.
O 2
nd
incubation: AIter addition oI biotinylated monoclonal h-lgM-speciIic
antibodies, HAV antigen, and streptavidin-coated microparticles, the anti-HAV
lgM antibodies present in the sample Iorm a sandwich complex with the HAV
antigen and the ruthenium-labeled anti-HAV antibody which becomes bound to
the solid phase via interaction oI biotin and streptavidin.
O The reaction mixture is aspirated into the measuring cell where the microparticles
are magnetically captured onto the surIace oI the electrode. Unbound substances
are then removed with ProCell. Application oI a voltage to the electrode then
induces chemiluminescent emission which is measured by a photomultiplier.
O Results are determined automatically by the Elecsys soItware by comparing the
electrochemiluminescence signal obtained Irom the reaction product oI the sample
with the signal oI the cutoII value previously obtained by anti-HAV lgM
calibration.

Expected values
The cutoII is selected such that the anti-HAV lgM concentration is above the cutoII index
when acute HAV inIection is present. In case oI a past hepatitis A inIection, the anti-
HAV lgM concentration is usually below the cutoII index oI 1.0. In the course oI most
acute hepatitis A inIections, the anti-HAV lgM concentration decreases within 3-4
months aIter onset oI the Iirst symptoms and can then no longer be detected. Anti-HAV
lgM antibodies are persistent only in exceptions and can then be detected beyond this
period.

Intended use
Immunoassay Ior the in vitro qualitative determination oI lgM antibodies to the hepatitis
A virus in human serum and plasma. The assay is used as an aid to detect an acute or
recently acquired hepatitis A virus inIection. The electrochemiluminescence
immunoassay 'ECLIA' is intended Ior use on the Roche Elecsys 2010 and MODULAR
ANALYTICS E170 ( Elecsys module ) immunoassay analyzers.


69


70
MICROBIOLOGY LABORATORY

There are Iour sections in Microbiology Laboratory. There are recording, culturing,
diagnosis and media preparation section. Mostly, in this laboratory, we learn to Iind out
or detect the kind oI microorganism.

RECORDING SECTION
In this section, all clinical sample and patient record will be recorded according to each
classiIication such as stool and cholera sample, blood and routine sample. Routine
samples are including sputum, csI, and urine.

CULTURING SECTION
AIter collecting clinical specimens, all specimens will be cultured and incubated.
Bacterial colonies Iormed are used Ior Iurther test and identiIication.

DIAGNOSIS SECTION
To identiIy diseases, several test are used to conIirm and detect pathogen that
causes diseases.

MEDIA PREPARATION SECTION
To cover all types oI culture media prepared in tubes, bottles and plastic Petri dishes used
in bacteriological work.


71
Inoculation procedures for routine specimens

Urine specimens

1. Prepare blood agar and MacConkey agar on sterile petri dish.
2. Label number oI each specimen on the petri dish.
3. Inoculate specimen on blood agar and MacConkey agar.
4. Incubate both plates at 37
o
C Ior 18-24 hours.

URINE CULTURE
PURPOSE
For the diagnosis oI acute or chronic urinary tract inIections (UTI) caused by bacteria or
Iungi.

PRINCIPLE
Most urinary tract inIections are caused by single isolates oI bacteria or Iungi. In about
10 oI the patients who have chronic UTI or have had surgical manipulations oI the
urinary tract, two organisms may be present and both may contribute to the disease
process. The isolation oI the same 2 organisms in repeated urine samples is likely to be
signiIicant oI inIection. In patients with indwelling urinary catheters, urine samples
should be collected aseptically aIter the urinary catheters have been changed. The
presence oI 3 or more organisms in the urine sample is strong presumptive evidence oI
improper collection or transportation oI the urine specimen. In most UTI, enteric bacteria
are the most common etiologic agent with E. coli isolated Iar more Irequently than any
other organisms. Common pathogens oI UTI include:
. E.coli
2. Coagulase-Negative $taphlococcu8
3. l0b8i0lla
4. $. aur0u8 .
5. Ent0robact0r
6. $. 8aprophticu8
7. Prot0u8
8. $tr0ptococcu8 Group D

72
. P80udomona8
.Moran0lla
.$0rratia
SAMPLE TYPE
O Clean-voided midstream urine.
O Catheter urine (indwelling/ non-indwelling).
O Suprapubic needle aspiration oI bladder.

Table V: Growth Characteristics oI Colonies on Hectoin Agar (18 Hours Incubation)

Colonies

Size

Appearance

E. coli

1-1.5 mm

O yellow, opaque with a slightly deeper
coloured centre.
O Blue colonies - non-lactose stains

l0b8i0lla spp.

-

mucoid, yellow to whitish blue

Pseudomonas

-

green, with matt surIace & rough edges

Salmonella

-

Ilat, blue colonies

E. fa0cali8

0.5 mm

yellow colonies

$. aur0u8

0.75 mm

deep yellow, uniIorm in colour

Corynebacteria

Tiny

grey colonies

Lactobacilli

Tiny

grey with a rough surIace


73
Pus or wound specimens

4. Incubate both plates at 37
o
C Ior 18-24 h.

PUS CULTURE

PURPOSE
For the detection oI microorganisms which cause skin & soIt tissues inIections.

PRINCIPLE
The skin is subject to minor traumas which may destroy its integrity and allow
microorganisms to enter into the deeper layers Irom the external environment. Wounds
can be classiIied into surgical, traumatic and physiological wounds.

Purulent exudates are produced as a result oI bacterial invasion oI the skin & the
wound. Pus exudates are taken Irom the inIected site to determine the type oI organism
causing the inIection & are cultured on microbiological media to isolate& identiIy the
pathogen and to test it Ior antibiotic susceptibility.

Whenever possible it is more satisIactory to aspirate the pus using a sterile syringe
& needle. Pus is sampled with a swab only when there is little inIectious material. Swabs
should be placed in Amies transport medium to preserve the anaerobes and the organisms
which are sensitive to drying.

SAMPLE TYPE
Pus or exudates.


74
IdentiIy pathogen accordingly by using biochemical tests. (ReIer to Table IV)
Carry out sensitivity test on the pathogens isolated.

Table IV: IdentiIication oI Pathogens Isolated Irom Pus Specimens

Pathogen

Biochemical Tests Ior IdentiIication

$. aur0u8

1. Coagulase
2. Catalase positive
3. II results indeterminate, conIirm by Staph.
agglutination test.

B-haemolytic Streptococci
on Blood agar

Gram stain (iI need to rule out Irom corynebacteria)

Alpha-haemolytic Streptococci
on Blood agar
-$. pn0umonia ( green pigment
appear)

1. Optochin
2. Catalase negative

cin0tobact0r spp.

1. Oxidase
2. KIA
O-F
Motility

Pasteurella & Other Unusual
Gram-Negative Organisms

1. Oxidase
2. KIA & Motility

Anaerobes

Gram Stain


75
CSF

4. Incubate both plate at 37
o
C Ior 18-24 hours.

Inoculation procedures stool C&S specimens

1. Prepare Hectoin agar, Sorbitol MacConkey agar and Selenite F. broth.
2. Label number oI each specimen on Hectoin agar, Sorbitol MacConkey agar and
Selenite F. broth.
3. Inoculate specimens on Hectoin media and Sorbitol MacConkey agar.
4. Put stool swab into Selenite F. broth (On the second day, inoculate specimen into
Selenite F. broth then, inoculate Ior the second time on Hectoin agar. Incubate
broth and media at 37
o
C Ior 18-24 h).

STOOL CULTURE

PURPOSE
To detect causative pathogens oI watery diarrhoea, dysentery & enteric Iever that
involves the gastrointestinal tract.

PRINCIPLE
Watery stool is one oI the signs & symptoms oI gastrointestinal inIections. In
enteric inIection, the organism is usually present in the blood in the early stage oI
inIection & diarrhoea may appear later. However blood is always negative Ior watery
diarrhoea & dysenteric inIections & thereIore stool culture must be relied upon Ior
diagnosis. Stool is collected & cultured on media to isolate, identiIy & test the pathogen
Ior antibiotic susceptibility.

Human gastrointestinal tract is populated by a myriad oI microorganisms
including many bacteria and viruses. Stool cultures can yield numerous bacteria species

76
that may be considered as commensals or potential pathogens. The plating media used to
isolate causative agents oI gastroenteritis have been designed to suppress or inhibit the
growth oI common bowel Ilora and provide distinguishing characteristics to the
pathogenic microorganisms on these media.

SAMPLE TYPE
1. Stool
2. Rectal swab

Table III: Colonial Morphology oI Enteric Pathogens in DiIIerent Types oI Selective
Media

Organism

Macconkey Agar

TCBS
Salmonella Convex, colourless ,2-3 mm.
-
Shigella Non-lactose Iermenter
-
$. tphi Non-lactose Iermenter
-
J. chol0ra0 Non-lactose Iermenter Ilat,
yellow,
2-3 mm.

J. paraha0molticu8
Non-lactose Iermenter
Ilat, green,
3-5 mm.


77
Inoculation procedures for blood for C&S specimens

4. Incubate - Blood agar & Sabouraud`s agar at 37
o
C Ior 18-24 hours.
- Blood agar anaerobic at 37
o
C Ior 48 hours.
5. II there is no growth aIter 48 hour incubation, reincubate until the seventh
day
6. IdentiIy the pathogen isolated by biochemical tests.
7. Carry out antibiotic sensitivity testing on the pathogen isolated.

Inoculation procedures for inoculation of RSJC specimens

1. Prepare TCBS agar on sterile petri dish.
2. Label number oI each specimens on the petri dish.
3. Inoculate specimen on TCBS agar.
4. Incubate broth and media at 37
o
C Ior 18-24 h).

Biochemical test:
- antiserum test to distinguish between Jibrio chol0ra0 Ogawa, Jibrio chol0ra0
Inaba and Jibrio chol0ra0 Hikojima


78
HIGH 'AGINAL SWAB (H'S)

PURPOSE
High vaginal swab culture is carried out to detect the microbial agents which cause
puerperal sepsis, septic abortion, vaginitis, cervicitis, bacterial vaginosis & pelvic
inIlammatory diseases in women & vulvo-vaginitis in children.

PRINCIPLE
Bacteriological examination oI genital specimens Irom patients with acute
venereal disease is relatively straight Iorward. However, examination oI genital
specimens Irom women with various non-speciIic acute genital inIections is usually
diIIicult as they are always contaminated by the normal vaginal Ilora. In general, the
bacteriological diagnosis oI genital inIections in Iemale patients involves the collection oI
a high vaginal swab or an endocervical swab which is examined under the microscope by
Gram-stained smear & cultured on culture media to isolate & identiIy the established
pathogens. The pathogens are then tested Ior antibiotic susceptibility.
Bacterial vaginosis is characterized by decrease in numbers oI lactobacilli &
increase in numbers oI anaerobes, Gardn0lla vainali8 & Mcopla8ma homini8. However,
true causative agent(s) are not known at this time. The condition is best diagnosed by the
presence oI `Clue cells" in Gram stain in conjunction with physical Iindings such as pH,
odour oI the vaginal discharge. Cultures Ior organisms associated with bacterial
vaginosis, including Gardn0r0lla vainali8 are not recommended.

SAMPLE TYPES
Vaginal swab.


79
Table 1.Biochemical Tests Ior the IdentiIication oI Pathogens in Genital InIections

Pathogen

Biochemical Tests Ior IdentiIication

N. gonorrhoeae

1. Gram stain
2. Oxidase

Candida

1. Germ Tube
2. Chlamydospore Test

Staphylococci

1. Catalase
2 Coagulae

ColiIorms: E.coli

1. KIA
2. LIA
3. Citrate
4. Motility


80
SENSITI'ITY TEST
PURPOSE
O To determine the inhibitory activity oI an

PRINCIPLE
antimicrobial agent against a particular strain oI bacteria.The disk diIIusion susceptibility
test is used. A standard inoculum oI the organism to be tested is seeded onto the surIace
oI an agar plate, & Iilter paper discs containing deIined amounts oI antibiotics are
applied. The plate is then incubated overnight. The antibiotic diIIuses Irom the disc into
the medium, dependent on its physical & chemical characteristics. Zones oI inhibition oI
growth oI the organism develop & the diameters are determined by the susceptibility oI
the organism, its growth rate & the diIIusibility oI the antibiotics. The diameter oI the
zones oI inhibition are measured & converted to sensitive', intermediate' & `resistant'
categories by reIerence to a table.

PROCEDURE
Disk Susceptibility Testing: Step by Step

Prepared inoculum; incubate broth culture Ior 2 hours

Dip a sterile cotton swab into broth culture; raise swab above liquid and rotate against
wall oI tube to remove excess liquid.

Apply inoculum to plate, covering entire surIace; rotate 60 degrees and swab; rotate 60
degrees and swab again.

Place not more than 6 disks on an agar plate.

Invert plate (agar side up) and incubate at 35
o
C Ior 16 to 18 hours

AIter 24 hours, check agar Ior conIluent lawn oI growth and circular zones oI inhibition.

Measure the diameter oI zone oI inhibition to nearest mm.

81
SPUTUM CULTURE

PURPOSE
For the detection oI microorganisms which cause middle & lower respiratory tract
inIections.

PRINCIPLE
Cough is one oI the signs & symptoms oI the respiratory tract inIections. Cough
usually becomes productive oI sputum which is a purulent discharge generated in the
alveoli & small air passages. Examination oI expectorated sputum has been the primary
means oI diagnosing the causes oI bacterial respiratory inIections. Sputum is collected &
cultured on microbiological media to isolate & identiIy the pathogens. For the diagnosis
oI pertussis in children, nasopharyngeal aspirate is collected & cultured Ior ord0t0lla
p0rtu88i8 & ord0t0lla parap0rtu88i8.
The accuracy in establishing an etiological diagnosis Ior a lower respiratory tract
inIection depends on the number oI organisms produced in respiratory secretions & on
whether the causative species is normally Iound in the oropharyngeal Ilora. Sputum is
usually contaminated with normal Ilora oI the respiratory tract. It may yield a Iew
colonies oI Streptococcus pneumonia, H. inIluenza, yeast, coliIorms, $. aur0u8 and
haemolytic streptococci which are also known to cause pneumonia. Hence, no
bacteriological report Irom expectorated sputum can be judged alone as being normal or
abnormal but must be taken together with the direct smear and clinical Iindings.

SAMPLE TYPE
Sputum


82
TEST METHOD
Table II: Biochemical Tests Ior The IdentiIication oI Respiratory Pathogens

Organism

Smear

Growth Quantity

. influ0n:a0

pus cell~25
pleomorphic Gram-ve rods

~ Iew colonies

$. aur0u8

pus cells ~25
many Gram positive cocci in clusters

2 or more

$. pn0umonia0

pus cells ~25
Gram positive cocci with capsules

~ Iew colonies

l0b8i0lla
pn0umonia0

pus cells ~25
numerous gram negative rods

almost pure 2 to
3

Candida

Gram positive yeast cells

2 to 3


83
GRAM STAIN
Gram stain is used to stain microorganisms Irom specimens or cultures to
diIIerentiate the diIIerent species oI organisms. It indicates the proportion oI diIIerent
species in a specimen & it is also a guide to Iurther procedure in the analysis oI a
specimen.
Gram stain involves staining a Iixed smear with crystal violet, applying iodine as
mordant, decolouring the primary stain with iodine/acetone and counterstain with
saIranine. As a result, Gram-positive cells will be purple black to purple in colour and
gram-negative cells will be red pink in colour.

OXIDASE
O To aid in the identiIication oI genera such as
Pseudomonas (usually ) Enterobacteriaceae (-)
Neisseria () Acinetobacter (-)
Vibrio () Yersinia (-)
O To aid in species identiIication such as
-Pseudomonas maltophilia (-) Irom other Pseudomonas spp. (usually )

Reading oI Results
Positive test: Development oI a dark purple colour on the inoculum
streak within 10 sec.
Delayed Positive test: Development oI a dark purple colour within 10-60 sec.
Negative test: No colour development on the inoculums streak or the
colour develops aIter 60 sec.

Interpretation
Positive: Pseudomonas aeruginosa
Negative : Escherichia coli.


84


85
CYTOLOGY LABORATORY

To establish the anatomical source oI cytological specimen, the specimens are classiIied
into 3 categories.
SPECIMEN DESCRIPTION
1. Gynae All pap smear received Irom Klinik
Kesihatan` and other hospital.
2. Non-Gynae (QNG) Sputum, body Iluid, brushing and imprint
smear
3. Fine needle aspiration cytology (FNAC) Body Iluid

A) FINE NEEDLE ASPIRATION (FNAC)

There are two types oI sample involved:
I) Dry aspiration
Numerous cells suspended in small amount oI tissue Iluid and have creamy consistency.
II) Wet aspiration
Small number oI cells suspended in Iluid/blood.

PROCEDURE
1) Direct
smearing
- label slides, expelled the specimen in the needle onto the
labeled slide.
- Use another needle aspirate the specimen in the plastic hub oI
the Iirst needle.
a) Dry aspiration: smeared with the Ilat oI a slide exert a light
pressure to achieve a thin and even spread. Do not press too
Iirm as this may cause crush artiIact.
b) Wet aspiration: quickly move the smearing slide Irom one end
oI the specimen slide, then smearing with the Ilat oI the slide.
2) Indirect
smearing
- Iluid specimen are processed by centriIugation
3) Alcohol
Iixed smear
- Wet smear are immersed into 95 ethyl alcohol
- stain with Pap stain

86
PROCESS
1. Process specimen in bio-hazard cabinet.
2. Slides were labeled with laboratory number and name as on specimen container.
3. The plain tubes were labeled according to the specimen laboratory number.
4. For air drying smear
-The wet smear are Iast drying using hair dryer
-These smears are stain with MGG stain

B) PAPANICOLAOU STAIN

OB1ECTI'E
O To demonstrate nuclear details.
O Preservation oI keratin

PRINCIPLE
O Used in conjunction with wet Iixation in 95 ethanol and consists oI Harris`s
Haemotoxylin, Orange-G (OG)-6 and Eosin Alcohol (EA)50.
O Harr`s Haemotoxylin has strong aIIinity Ior nuclei (nuclear staining), OG-6 was used to
stain keratinized cytoplasm, EA50 has polychrome properties and was used to stain
cytoplasm according to cell maturation.


87
PROCEDURE
wet smears Iixed in 95 ethanol

75 ethanol (30 dips)
Rinse in running water (11 dips)
Harr`s Haemotoxylin (10 minutes)
Rinse in running water
DiIIerence in 0.5 acid alcohol (7dips)

Blueing in running water (10 minutes)
95 ethanol (2 - 3 minutes)
OG-6 (1-2min)
Rinse in 95 ethanol (5 dips)
E.A 50 (5 minutes)
Rinse in 95 ethanol (5 dips)
Absolute ethanol I (1 minute)
Absolute ethanol II (2 minutes)
Absolute ethanol III (3 minutes)
Clearance 1 (1 minute)
Clearance 2 (2 minutes)
Clearance 3 (3 minutes)

88
C) GIEMSA STAINING

PRINCIPLE
Giemsa's stain is a member oI the Romanowski group oI stains, which are deIined as being
the black precipitate Iormed Irom the addition oI aqueous solutions oI methylene blue and
eosin, dissolved in methanol. The variants oI the Romanowski group diIIer in the degree oI
oxidation (polychroming) oI the methylene blue stain prior to the precipitation.

The stain class was originally designed to incorporate cytoplasmic (pink) staining with
nuclear (blue) staining and Iixation as a single step Ior smears and thin Iilms oI tissue
(spread preparations oI omentum). Minor modiIications oI working stain concentration and
staining time have been made over the years Ior Iixed tissue sections.

The Romanowski stains are extremely tedious to prepare, and are best purchased as the
commercially available pre-made stock stain.

Technical Points

1. (step 2) - Usually the staining is perIormed at room temperature overnight, however,
increasing the stain temperature shortens staining time. Sections stained at 37C Ior
several hours, (staining time assessed by microscopical examination), produce better
results than sections stained at 60C Ior a shorter period. The higher the staining
temperature, the greater the intensity oI blue staining, but without the equivalently
increased red staining - see technical point 2 below.

2. DiIIerentiation with acetic acid will vary according to the staining time and temperature,
but it is generally achieved within 30 secs. The diIIerentiating agent removes only the
blue dye component, thus increasing the apparent intensity oI the red component.

Method

1. Bring sections to distilled water
2. Stain with diluted Giemsa's stain made up Iresh (see technical point 1)
3. Rinse in distilled water
4. DiIIerentiate with 0.5 aqueous acetic acid (see technical point 2)

89
5. Dehydrate rapidly
6. Clear and mount

Results

Bile pigments........................................................................green
Collagen, muscle, bone..............................................................pale pink
Micro-organisms, Iungi, parasites.............................................purplish-blue
Starch granules, cellulose...........................................................sky blue
Pigments (native colour is yellow/brown, or iI Iixed in
dichromate containing Iixative)............ ...green
Nuclei.........................................................................................dark blue to violet
Erythrocytes................................................................. .............salmon pink
Cytoplasm............................................................varying light blue shades

C) DIFF QUICK (MODIFIED GIEMSA)

OB1ECTI'E
O To demonstrate Helicobacter like organism.

PRINCIPLE
O The DiII Quick method provides a rapid alternative to conventional-staining
procedure. It can be used on blood Iilms, urinary deposits and semen examination.
The method can be used Ior routine screening but is most useIul Ior urgent
diIIerential counts. The stain has a shelI liIe oI approximately 2 years.

PROCEDURE

Dip slide into the Iixative reagent Ior 7 times

Dip slide into Quick Dip I Ior 5-7 times

Dip slide into Quick Dip II Ior 7-10 times

90

Rinse under tap water until all excess blue is removed

INTERPRETATION
O The Helicobacter like organism is stain with dark blue color.

Reagent Formulae

1. Giemsa's stain, stock solution obtain Irom commercial sources.
Giemsa reagent improves with age, expiry is unimportant.
Giemsa stain, working solution
Giemsa stock solution ------ 40 drops
Distilled water --------------- 40 ml
The diluted stain keeps well, but is best made up Iresh each time.

2. Acetic acid 0.5

Giemsa solution with addition oI BuIIer solution will produce clear cell structure staining.
The nucleas will be stained with reddish or purple stain while the cytoplasm will be
stained with basophilic and kreatinising stain. AIter staining, the slide appear as bluish-
purple slide.

D) LABORATORY REPORT OF PAP SMEAR SPECIMEN
There are several criteria evaluated Ior pap smear specimen:
O Adequacy oI specimen
O General categorization
i. Within normal limits
ii. Benign cellular changes due to :
a. Reactive changes associated with:
-inIlammation
-atrophy with inIlammation
-radiation

91
-intra uterine contraceptive device
b. InIection :
-Trichomona8 vainali8
-ctinomc08 8pp.
-0rp08 8impl0 viru8
-candida
O Epithelial cells abnormality
i. Squamous cell - atypical squamous cells oI undetermined signiIicance
- Low grade squamous intraepithelial lesion
- Human papilloma virus
- High grade squamous intraepithelial lesion
- squamous cell carcinoma

ii. Glandular cells - endometrial cells, benign in post menopausal
- atypical glandular cells oI undetermined signiIicance
- endocervical adenocarcinoma
- endometrial adenocarcinoma
- extrauterine adenocarcinoma
-adenocarcinoma


92


93
HISTOLOGY LABORATORY

There are Iour sections in histopathology laboratory:

1. Receive specimen
The category oI specimen that received here include internal (Irom this hospital),
external (Irom other hospital), autopsy and Irozen section. The common specimens
are small biopsy, appendix, Fallopian tubes, product oI conception (POC) and small
cyst / lump. All the specimen should be together with Iorm that have been approve by
medical oIIicer. II not, the specimen will be rejecting.

2. Grossing and embedding of histopathology specimen
The objective oI grossing is to select the appropriate tissue samples Ior histopathology
examination and to ensure proper Iixation the specimens beIore processing by slicing
the relevant specimen. For small specimen, this process is done by lab technician but
Ior big specimen, the specimen will be sent to Queen Elizabeth Hospital and
identiIied by pathologist. AIter grossing, the tissue block is prepared by embedded the
tissue using a wax.

3. Sectioning and fishing
These processes are to prepare the tissue sample onto the slide so the lab technician or
pathologist could see the specimen under the light microscope. Microtome is used to
cut the tissue block to become a slice and then the section is brings into the water bath
(54
0
C). Then, the best thin layer oI the section is picking up using a slide. This is the
process that we called Iishing. The slide then has been incubate Ior one hour in the
incubator (58
0
C) to discard the wax.

4. Staining and Mounting
There are many types oI staining that have been used in histopathology laboratory:


94
A) HAEMATOXYLIN AND EOSIN

OB1ECTI'E
O To demonstrate clearly tissue structure (nuclear and cytoplasm)

PRINCIPLE
O Haematoxylin itselI is not a stain, it is its oxidation product, haematin, which is
the natural dye.
O Haematin on its own has poor aIIinity Ior tissue and is inadequate as a nuclear
stain without the presence oI mordant.
O Harris`s Haematoxylin is an alum haematoxylin which is chemically ripened with
mercuric oxide and useIul particularly to give clear nuclear staining.
O Eosin is the most suitable stain to combine with an alum haematoxylin to
demonstrate the general histological architecture oI a tissue particularly to
distinguish between the cytoplasm oI diIIerent types oI cells and between the
diIIerent type oI connective tissue Iibers and matrices by staining them diIIering
shades oI red and pink.

PROCDURE
1. Histoclear I 3 minutes
2. Histoclear II 3 minutes
3. Absolute alcohol I 3 minutes
4. Absolute alcohol II 3 minutes
5. 90 alcohol 3 minutes
6. Wash well in running water 3 minutes
7. Haematoxylin 40 minutes
9. Decolorized in acid alcohol 1 dip
11. Ammonia solution 2 dips
12. Wash well in running water 3-4 minutes
13. Counter stain in 0.5 Eosin 5 minutes
14. Wash well in running water 1 dip

95
15. 80 alcohol 1 dip
16. 95 alcohol I 1 dip
17. 95 alcohol II 1 dip
18. Absolute alcohol I 1 dip
19. Absolute alcohol II 1 dip
20. Absolute alcohol III 1 dip
21. Histoclear 1 dip

INTERPRETATION
O Blue colour - nucleus
O Red colour - muscle Iibers
O Pink colour - collagen
O Bright red colour - red blood cell

B) PERIODIC ACID SCHIFF`S (PAS) FOR NEUTRAL
MUCOPOLYSACCHARIDE

OB1ECTI'E
O To demonstrate polysaccharide, mucopolysaccharide and mucoprotein.

PRINCIPLE
The substances containing vicinal glycol groups or their amino or alkylamino derivatives
are oxidized by periodic acid to Iorm dialdehydes which is combining with SchiII`s
reagent to Iorm an insoluble magenta compound. The PAS stain is a histochemical
reaction in that the periodic acid oxidizes the carbon to carbon bond Iorming aldehydes
which react to the Iuchsin-sulIurous acid which Iorm the magenta color.

PROCEDURE:
1. DeparaIIinize and hydrate to distilled water.
2. Place slides into 0.5 Periodic acid Ior 5 minutes.
3. Rinse in distilled water.
4. *SchiII's Reagent, microwave HIGH power, Ior 45 - 60 seconds, until
deep magenta.

96
5. Wash in running tap water Ior 5 minutes.
6. Counterstain in hematoxylin Ior 3 minutes.
7. Wash in tap water, blue hematoxylin, rinse in distilled water.
8. Dehydrate in alcohol, clear, and coverslip.
* Conventional method: SchiII's Reagent, room temperature Ior 30 minutes.
RESULTS:
Glycogen, Iungus: magenta
Nuclei blue
NOTES:
1. To check stain, pour 10 ml oI Iormaldehyde in cylinder, add a Iew
drops oI SchiII's, it should turn red-purple immediately.
2. Discard SchiII's iI it turns pink while sitting in the reIrigerator.
3. The white precipitate at the bottom oI the SchiII's can be redissolved
iI desired by gently warming and stirring the solution.

PROCEDURE

Wash the slide

The slide is covered with 0.5 periodic acid Ior 10 minutes.

Place the slide in coplin jar that contained distilled water Ior a Iew seconds

Cover the slide with SchiII reagent Ior 15 minutes

Rinse under running tap water Ior 5 minutes.

The slide then is counter stained with Heamatoxylin Ior 1.5 minutes to stain the
nucleus

Rinse under running water Ior 5 minutes.

Mount


97
INTERPRETATION
O Acid mucosubstances blue in colour
O Neutral polysaccharides magenta (bright pink in color)

C) ZEIHL NEELSEN STAIN

PRINCIPLE:
O As the causative agents Ior Leprosy (Mcobact0rium l0pra0) and Nocardiasis
(ocardia a8t0roid08) are much less acid and alcohol Iast than Mcobact0rium
tub0rculo8i8 bacilli, a more gentle dewaxing and minimal exposure to organic
solvents is required Ior adequate staining. The turberculle bacilli which is acid
Iast, when heated with Carbol Fuchsin will be stained Red and the other
microorganisms will be decolorised by the 3 Acid Alcohol and later will be
stained Blue by the Methelene blue.

CAUTION: HOT SOLUTIONS, POTENTIALLY TOXIC METHOD:
(1) Heat slides in slide dryer to Iacilitate dewaxing.
(2) Take sections to water.
(3) Drain slide and place in a slide mailer with 15 ml oI SoIt Carbol Fuchsin.
(4). Fill Slide Mailer (~ 15 ml volume), so that Iluid comes up to slide Irosting.
Place Mailer into microwave in a beaker, in case oI spillage, leave cap oI mailer
open.. Microwave on FULL POWER, First time 10 seconds FULL POWER, Second
Time 7 seconds FULL POWER
(Check slides each time. CAUTION halt iI solution boils.)
(5) Allow to stand Ior 2 - 5 minutes.
(6) Wash excess stain Irom slide.
(7) Wash well in running water, wipe away excess stain 5 min
(8) DiIIerentiate individually with 0.5 Acid Alcohol, control microscopically 1 - 3 min
(Use 5" Sulphuric acid for Atypical AFB)
(9) Wash in running tap water. 5 min
(10) Filter on Working Methylene Blue 20 secs
Avoid overstaining as some weak staining bacterial staining can be replaced by
methylene blue.
(11) Wash with water.

98
(12) Drain and air dry thoroughly.
(13) Dip slides in xylene and mount in DPX.

RESULTS:
Acid Fast Bacilli RED (Leprae bacilli are short rods)
Nocardia RED (Nocardia organisms are long, thin, and Iilamentous)
Erythrocytes PALE PINK
Background BLUE OBJECTIVE

PROCEDURE
1. DeparaIIinize in Xylol-oil (I) 15 minutes
2. DeparaIIinize in Xylol-oil (II) 15 minutes
3. Drain and blot dry
4. Carbol Fuschin Stain (boiled-oven) 30 minutes
5. Rinse under running water 3 minutes
6. Decolorized with 3 acid alcohol
7. Leave Ior 3 minutes
8. Rinse with running tap water
9. Counter stain with Methelene Blue 2 minutes
10. Leave Ior 1 minute
11. Rinse with running tap water
12. Take out the slide Irom the staining rod and leave to dry
13. Look Ior rod-shaped bacilli in red under x100 power microscopy

INTERPRETATION
O Acid Iast bacilli - red
O Others (nuclei and other tissue constituent) - blue

E) IMMUNOFLUORESCENT STAIN

PURPOSE
The specimen are Iirst treated with 3 hydrogen peroxide to suppressed
endogenous peroxidase activity. They are than incubatedwith an approximately

99
characterized and diluted rabbit, mouse or goat primary antibody and Iollow by sequential
15 minutes incubation with the provided biotinylated link antibody and peroxidase-
labeled sterptavidin. Staining is completedaIter incubation with the provided substrate
chromogen solution, resulting in a brown colored precipitate at the antigen site.

PROCEDURE
Slide (test control positive)

Oven

Bring section to water

Target retrieval solution (1/10) 800ml

Microwave 12 minutes (observe every 5 minutes)
(3 -4 bubbles)
Incubate at room temperature (20 minutes)

Put the slide into sequenza

Wash 3 times (3min, 3min, 5min) with distilled water

Peroxidase (20 minutes)

Wash 3 times (3min, 3min, 5min) with Tris BuIIer

Add primary antibody and leave it Ior 1 hour


Add secondary antibody (mouse / rat) and leave it Ior 1 our



100
Remove the slide Irom sequenza and put onto the rod

Chromogen (1ml DAB Subs. BuIIer 1 drop chromogen)

Look under light microscope until brown color appear at slide control

Wash with tap water

Counter stain with haematoxylin (20 -30 seconds)
Depends on the darker oI brown color
Brown Haematoxylin ~ length
Brown Haematoxylin length

Wash with tap water

Oven


101


102
PARASITOLOGY LABORATORY

Blood Smear for Malarial Parasite (BSMP)

Procedure

1. Receive and check specimen
2. Allow the blood Iilm to dry thoroughly
3. Fix the thin Iilm by dipping it in a container oI methanol Ior a Iew seconds
4. Stain Ior 5-15 min with 10 Giemsa (10ml Giemsa, Azur-Eosin 90ml 7.2 BuIIer)
5. Wash slide
6. Place the slide in the rack to drain and dry
7. Using a 10x ocular and 100x oil immersion objective Ior screening
8. Enter BSMP result in the test request Iorm and record it in malaria record book.
(II ve reIer to method oI counting malaria parasite
9. The BSMP test result is checked and veriIied
10. Dissemination oI BSMP result to the requesting party.

Positive Result :
Finding Code Interpretation
1 10 parasite/ 100 TBF Moderate
11 100 parasite/ 100 TBF Badly moderate
1 10 parasite/ 1 TBF Severe
~ 10 parasite/ 1 TBF Badly severe

TBF : Thick blood Iilm


103
Faecal concentration procedure-formalin-ether / ethyl acetate / gasoline

Procedure

1. With an applicator stick add 1.0-1.5 g oI Iaeces to 10 ml oI Iormalin in a
centriIuge tube and stir to Iorm a suspension.
2. Strain the suspension through the 400m mesh sieve or 2 layer oI wet surgical
gauze directly into a diIIerent centriIuge tube or into a small beaker. Discard the
gauze.
3. Add more 10 Iormalin to the suspension in the tube to bring the total volume to
10 ml.
4. Add 3.0 ml oI ether ( or ethyl acetate or gasoline ) to the suspension in the tube
and mix well by putting a rubber stopper in the tube and shaking vigorously Ior 10
seconds.
5. Remove the stopper and place the tube in the centriIuge; balance the tubes and
centriIuge at 400-500g Ior 2-3 minutes.
6. Remove the tube Irom the centriIuge; the contents consist oI 4 layers: (a) top layer
oI ether ( or ethyl acetate or gasoline ), (b) a plug oI Iatty debris that is adherent to
the wall oI the tube, (c) a layer oI Iormalin, and (d) sediment.
7. Gently loosen the plug oI debris with an applicator stick by a spiral movement and
pour oII the top 3 layer in a single movement, allowing the tube to drain inverted
Ior at least Iive seconds. When done properly a small amount oI residual Iluid
Irom the walls oI the tube will Ilow back onto the sediment.
8. Mix the Iluid with the sediment ( sometimes it is necessary to add a drop oI saline
to have suIIicient Iluid to suspend the sediment ) with a disposable glass pipette.
TransIer a drop oI the suspension to a slide Ior examination under a coverslip; an
iodine-stained preparation can also be made.
9. Examine the preparations with the 10X objective or, iI needed Ior identiIication,
higher power objectives oI the microscope in a systemic manner so that the entire
coverslip area is observed. When organism or suspicious objects are seen, switch
to higher magniIication to see more detailed morphology oI the object in question.


104
Kato-Katz technique - cellophane faecal thick smear

Procedure

1. Place a small mound oI Ieacal material on newspaper or scrap paper and press the
small screen on top so that some oI the Ieaces are sieved through the screen and
accumulate on top.
2. Scrape the Ilat-sided spatula across the upper surIace oI the screen to collect the
sieved Iaeces.
3. Place template with hole on the centre oI a microscope slide and add Ieaces Irom
the spatula so that the hole is completely Iilled. Using the side oI the spatula pass
over the template to remove excess Iaeces Irom edge oI the hole ( the spatula and
screen may be discarded or, iI careIully washed, may be reused ).
4. Remove the template careIully so that the cylinder oI Iaeces is leIt on the slide.
5. Cover the Iaecal material with the pre-soaked cellophane strip. The strip must be
very wet iI the Iaeces are dry and less so iI the Iaeces are soIt ( iI excess glycerol
solution is present on upper surIace oI cellophane wipe with toilet paper). In dry
climates excess glycerol will retard but not prevent drying.
6. Invert the microscope slide and Iirmly press the Iaecal sample against the
hydrophilic cellophane strip on another microscope slide or on a smooth hard
surIace such as a piece oI tile or a Ilat stone. The Iaecal material will be spread
evenly between the microscope slide and the cellophane strip. It should be
possible to read newspaper print through the smear aIter clariIication.
7. CareIully remove slide by gently sliding it sideways to avoid separating the
cellophane strip or liIting it oII. Place the slide on the bench with the cellophane
upwards. Water evaporates while glycerol clears the Iaeces.
8. For all except hookworm eggs, keep slide Ior one or more hours at ambient
temperature to clear the Iaecal material prior to examination under the
microscope. To speed up clearing and examination, the slide can be placed in a
40C incubator or kept in direct sunlight Ior several minutes.
9. 8cari8 and Trichuri8 eggs will remain visible and recognizable Ior many months
in these preparations. Hookworms eggs clear rapidly and will no longer be visible
aIter 30-60 minutes. $chi8to8om0 eggs may be recognizable Ior up to several

105
months but it is preIerable in a schistosomiasis endemic area to examine the slide
preparations within 24 hours.
10. The smear should be examined in a systemic manner and the number oI eggs oI
each species reported. Later multiply by the appropriate number to give the
number oI eggs per gram oI Iaeces ( by 20 using a 50 mg template; by 50 Ior a 20
mg template; and by 24 Ior a 41.7 mg template ). With high egg counts, to
maintain a rigorous approach while reducing reading time, the Stoll quantitative
dilution technique with 0.1 mol/liter NaOH may be recommended .


106
Direct Faecal Smears -Saline and Iodine wet mount preparations

1. With a wax pencil or other marker, write the patient`s name or identiIication
number and the date at the leIt-hand end oI the slide.
2. Place a drop oI saline in the centre oI the leIt halI oI the slide and place a drop oI
iodine solution in the centre oI the right halI oI the slide. ( Note : iodine wet mount
preparations are most useIul Ior protozoa, less so Ior helminthes )
3. With an applicator stick or match, pick up a small portion oI Iaeces ( approximately
2mg which is about the size oI a match head ) and add it to the drop oI saline : add a
similar portion to the drop oI iodine. Mix the Iaeces with the drops to Iorm
suspensions.
4. Cover each drop with a cover slip by holding the cover slip at an angle, touching the
edge oI the drop, and gently lowering the cover slip onto the slide so that air bubbles
are not produced. ( Note : ideal preparations containing 2mg oI Iaeces are uniIorm
not so thick that Iaecal debris can obscure organisms, nor so thin that blank spaces
are present )
5. Examine the preparations with the 10X objective or, iI needed Ior identiIication,
higher power objectives oI the microscope in a systematic manner ( either up and
down or laterally ) so that the entire cover slip area is observed. When organisms or
suspicious objects are seen, switch to higher magniIication to see the more detailed
morphology oI the object in question.


107
Staining Procedures for Protozoa in Faeces

The use oI Lugol`s iodine Ior staining wet mount preparations Irom Iresh or Iormalin-
preserved Iaecal specimens is described on Plate 1. Here are presented some procedures
Ior permanent staining oI smears prepared Irom Iresh, PVA or SAF preserved Iaecal
material. Many details oI preparation oI Iaecal smears and the application oI various
staining procedures are also presented in the reIerences listed in the introduction.

Permanent stains Ior Iaecal smears

A. Trichrome stain

Use : Very good stain Ior Iresh and PVA preserved Iaecal smears : does not give good
staining results with SAF preservation.

Preparation : Add 10ml oI glacial acetic acid to 6g oI chromotrope 2R, 3g oI light
greenSF and 7g oI phosphotungstic acid in a clean Ilask. Swirl to mix and let stand Ior
30 min. Add 100ml oI distilled water and mix thoroughly; the stain should be a deep
purple. Store in a glass-stoppered bottle; the stain is stable and is used undiluted.

1. Place slides, Iixed in either Schaudinn`s Iixative or PVA, into 70 alcohol Ior 2
min.
2. Add Lugol`s diluted iodine solution to 70 ethanol to produce a colour oI strong
tea: place slides in the solution Ior 5 min.
3. Place slides in two changes oI 70 alcohol.
4. Stain slides in undiluted trichrome stain Ior 10 min.
5. Remove slides, drain thoroughly and place them in 90 acidiIied alcohol
( prepared by adding 4.5ml oI glacial acetic acid to 1 litre oI 90 ethanol ) Ior 2
3 seconds.
6. Dip slides in 95 alcohol to rinse and then dehydrate through 100 ethanol and
xylene or through carbol-xylene mixture.
7. Using resinous mounting medium, place a coverslip on the smear


108
B. Iron Haematoxylin stain

Use : Very good stain Ior Iresh, PVA or SAF preserved Iaecal smears.

Preparation :
Stock solution A : dissolve 1g oI haematoxylin crystals in 100ml oI 95 alcohol;
allow solution to stand in light Ior 1 week and then Iilter.

Stock solution B: mix 1g oI Ierrous ammonium sulIate,1g oI Ierric ammonium sulIate
and 1ml oI hydrochloric acid in 97ml oI distilled water.

Prepare a working solution by combining 25ml each oI stock solutions A and B;
prepare at least 3-4 h prior to staining. Prepare picric acid solution Ior destaining by
adding 25ml oI saturated aqueous picric acid to 25ml oI distilled water.

1. Place slides into 70 alcohol Ior 5 min;into 50 alcohol Ior 2 min;into tapwater
Ior 5 min; into working haematoxylin stain solution Ior 10 min; into distilled
water alcohol containing 1 drop oI ammonia Ior 5 min; and into 95 alcohol Ior 5
min.
2. Dehydrate through 100 ethanol and xylene or through carbol-xylene mixture.
Using resinous mounting medium, place a coverslip on the smear.

C. ModiIied Ziehl-Neelsen technique ( acid-Iast stain )

Use : For detection oI Cryptosporidium, Cyclospora and other coccidian inIections.
1. Prepare a thin smear oI Iaeces
2. Air-dry and Iix in methanol Ior 2-3min
3. Stain with cold carbol-Iuchsin Ior 5-10min
4. DiIIerentiate in 1 HCL-ethanol until colour ceases to Ilow out oI smear.
5. Rinse in tapwater.
6. Counterstain with 0.25 malachite green ( or methylene blue ) Ior 30 sec
7. Rinse in tapwater
8. Blot or drain dry.

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