Processing of Blood

[[p.1]]
Processing of Blood
8.1 DONOR'S BLOOD
Each unit of blood collected by way of donation from a healthy donor has to be processed first and
preserved for transfusion to a compatible recipient. Unlike all other laboratory the produce of blood
bank is issued for human consumption. And as such, it must be processed to ensure the suitabilityfor
consumption.Aunit of unprocessed bloodis not onlypotentiallydangerous, but alsocould be a causeof
fatality in a recipientconsumer. Any unit of blood,thus collected,is labelledand numberedas per serial
number (annual)on the date of collection. So date of collection·and a numberis must, following which
grouping bothABO and Rh is done and this too is mentionedon the label.The label also containsname
of certain diseases clearance for which is mandatory. In India, they are malaria, syphiIlis, hepatitis B
surface antigens.HepatitisC virushas recentlybeenaddedto the mandatory list and has to beclearedby
test before any blood bag is transfused, and AIDS (acquiredimmuno deficiency syndromecaused by
HIVI and HIV2). CMV is not mandatoryin India. In USA the list includesanother two diseasesin the
mandatory list and they are CMV and HTLV. Physicalcondition 'of the container must be inspected.
Content must be clot free and no haemolysisetc. Containermust not bear any weak or leaky area and it
has not crossed the limitof expiryusuallytwo yearsafter the date of manufacture. The blood ha~ thus to
be tested and cleared for the diseases,named in the mandatory list beforeits final preservation till then '
the blood is kept under observation in the recommended range of temperature. Any blood unit not
clearedha~ tobediscardedbyincineration. Thoseunitsclearedarepreservedforfutureuseviz.transfusion:
Syphilis
This is a very highly contagious disease that can be transmittedthrough transfusion of infected blood.
So any blood showingweakreactivityhas to be retestedfor clearance,and if stillreactive, is issuedafter'
72 hours of preservationat 4°C. A wider margin of 96 hours is preferable. In syphilis the blood is not
discarded, after 72 hoursat 4°C, the germsbecomesineffective and annihilated. For detectionof syphilis
VDRL test is routinelyemployed.
Material required
VDRL slide, VDRL antigen, Buffered saline, inactivated serum, slide rotation shaker, known positive
and negative sera.
151

[[p.2]]
152
Blood Banking and Transfusion
VDRL slide are simple glass slides with concavities arranged in order.
VDRL antigen is cardiolipin Lecithin.
Method
Emulsion of VDRL antigen is first prepared with buffered saline.
l'he inactivated serum of donor is mixed with emulsion of VDRL antigen (according to instruction
in the accompanying insert) in the concavity of VDRL slide.
Keep the slide over a rotator-shaker for 4 minutes minimum at 180 rpm speed.
Ensure that it rotates through 3/4th ofcircle, observe for agglutination-naked eye examination doubtful
cases confirmed' under microscope compare with positive and negative control run along with test.
Gradation though not very clear cut but can be arbitrarily divided in three for interpretation of result.
If no agglutination observed result can be interpreted as negative of course after confirmation under
microscope (Table 8.1).
Tllble···.~~.tll.
VDRL·;tes~
Type of test
Nameof
Antig.en
Specificity
Gradation of
Inference
test
used
agglutination
Flocculation VORL
Cardiolipin
Non-specific
(a) Well definedlarge clump
Definitely
Lecithin
positive
(b) A mediumsize clump
Positive
(c) Not well defined but fine clump
Weak
.reactive
Other quick tests are also available viz. RPR (Rapid Plasma Reagin).
Specific test or confirmatory test is TPHA (Trepanoma Pallidum Haemagglutination Test)
Malaria
Malaria can also be transmitted through transfusion. It is parlicularly relevant to transfusion practice in
India; because of endemic nature of the disease. So each and every blood unit' has to be tested and
cleared for malaria.
Routinely test for malaria is done from smears: (i) Thin sm.earand (ii) Thick smear.
(i) Thin smear is prepared over a glass slide by putting a drop ofuncoagulated fresh blood and then
on that drop put another slide so that blood spreads uniformly at the edge of the second slide
and now draw the second slide uniformly over the first slide In a way that the drop of blood
spreads uniformly over the first slide, smear becoming gradually thinner at the end where we
stop. Both the slide must be clear.. dry, dust and grease free.
(ii) Thick smear : This has the advantage of having increased volume. of blood on the slide and
probability ofpositive diagnosis increases. However, it is difficult to identify the infecting species
in thick smears
Thick smears are prepared by putting a drop of blood on a clear, dry, dust and grease free all
glass slide and spread it with a glass rod until it covers an area of approximately 2 em in
diameter. Ideal thick smears are those through which newspaper print can be read.

[[p.3]]
Processing of Blood
153
For stainingof smear various methods are used. Some of these are :
(a) Giemsa stain
(b) Leishman stain
(c) Field's stain
(d) J.S.B. stain (Jaswant Singh, Bhattacharya stain) commonly used in Blood Bank Laboratory is
the former two.
Glemsa
Fix smear by dipping it in acetone free methyl alcohol for a few seconds.Air, dryand cover it for 10-20
minutes with 5% Giemsastain, Washwith distilled water and air dry.Used both for thick and thin films.
Leishiam's stain
Fix smear as above.With slide on a level bench, add 7-8 drops of Leishman'sstain leave for 20 seconds.
Add 12 to 15 drops (double the amount) distilled water (pH 7.2). Leave for twenty minutes.Wash with
distilled water and dry. Used both for thick and thin films. Basic differenceis that thin films has to be
fixed before staining whereas thick films need not be fixed before staining.
Slides,arenow examinedunder microscopefor detectionof malarial parasitepresent in smear. The
smear may show presenceof parasite in different forms,
But if the number of parasite are very few, it becomes difficult to detect them, say in case of one
parasite, present in per millilitreof blood at least half a millionof parasitewill be transfused,undetected
by smear staining method. So some of laboratory now prefers IHA-Indirecthaemaglutination test for
detection of an..timalarial antibody.
Indirect
haem~gglutinatlon
test
The principle is that since the particulate antigen can be demonstrated by their visible agglutination,
presence of specific antibody (ies) and the reaction between soluble antigen and antibody in solution
could not be detected by naked eye. Hence soluble antigens are coated over inert particle like sheep
erythrocytelatex,bentoniteparticlesetc.,thusthereactionbetweenantigencoatedparticleand antibodies
are made visible to,naked eye.
Material
Sheep erythrocyte, tannic acid, tubes all glass, centrifuge machine, U-bottomed microtitre plate,
micropipettestipsfordroppinganddilution,PBSpH7.2BSA(Bovineserumalbumin);standardreagents
given malarial antigen and antiserum.
Preparation of sheep erythrocyteantigen is the first step in the method.Polysaccharide antigengets
adsorbed to tannic acid pretreated sheep erythrocyte easily and the erythrocytes gets uniformly and
effectively coated.
(a) .The sheep erythrocytesare washed 3/4 times with PBS pH 7.2 by centrifugingat 1500rpm for
ten minutes after each wash supernatantis decanted as completely as possible.
(b) No,w make a 3% suspensionof sheep erythrocytein PBS ph 7.2.
(c) 1/20,000 dilution of tannic acid is now made from stock solution.

[[p.4]]
154
Blood Banking and Transfusion
(d) Equal quantity of 3% R.B.C. suspension
of sheep and 1120.000 dilution of tannic
acid are mixed well in a conical flask
taking care ro add RBC to tannic acid
solution during preparation of mixture so
'that an even coating of RBC takes place.
(e) Flask is now incubated at 4°C for twenty
minutes. Shake well after 15 minutes to
avoid settling of cells.
(f) Contents are transferred to tubes and
centrifuged at 1200 rpm for ten minutes
and tannic acid removed later with pipette.
(g) Cells are now washed twice with PBS pH
7.2.
(h) Resuspend the cells to make a 3%
suspension.
(i) Now make a 5 ml of the optimal dilution
of the antigen (the highest dilution of
antigen which gives maximum antibody
titre with standard antisera is taken as
optimal dilution) and mix with 5 ml of3%
tanned RBC.
Rg. 8.1. Single channel pipette.
G) Incubate at 37°C for thirty minutes with
intermittent shaking.
(k) Centrifuge at 1200 rpm for ten minutes and antigen is removed. Washed with PBS-BSA and
1.5% cell suspension made.
(I) 0.025 ml of PBS-BSA washed red cell is added to each cavity of first row (test row) of a U-
bottomed microtitre plate.
(m) 0.025 ml of sample is now added to the first cavity offirst row (test row) and doubling dilutions
are made upto the last cavity.
(n) Run control along with test.
(0) Plate is shaken gently for two minutes and incubated at RT for 60 minutes, under seal and cover,
then keep overnight at 4°C.
(p) Readings are now taken and interpreted as given below:
(i) Compact button with a regular margin is taken as negative reaction.
(ii) Particulate agglutination is graded as 4+, 3+, 2+, 1+ depending on the intensity of reaction
exhibited.
(iii) Titre for the sample can also be recorded.
Hepatitis
This is another disease transmitted by blood transfusion and it could be due to transfusion of hepatitis 'B'
virus or non-A, non-B hepatitis virus. The later two is rarely encountered and carrier state is also very

[[p.5]]
rare and in comparison hepatitis 'B' virus is more significant clinically for
the harm it causes in form ofpost-transfusion hepatitis and other sequelles.
So in blood transfusion practice, more importance is given to detection of
hepatitis 'B' virus. Hepatitis 'B' was discovered by Blumberg et al. in 1965.
Hepatitis 'B' virus infection is associated with certain clinical,
serological and biochemical findings which are supportive to the diagnosis .
as well as gives clue to the suspicion for such infection.
Incubation period is long and varies between 60 to 180 days with past
history of blood transfusion or transfusion of components or history of
handling blood and its products viz. lab technicians, Surgeons, anaesthetist
may also acquire the disease after handling of infectious cases.
Raised SGPT level along with jaundice and elevation ·of serum
bilirubin level.
Most cases-will show a positive serology for HBV.
And as such screening for HBSAG has gained importance and made
mandatory in processing each and every blood unit.
Processing"of Blood
155
HEPATITIS 'B'
Fig. 8.2. Virus surface
antigen.
Diagnostic methods
(a) Counterimmuno electrophoresis (CIEP)
(b) Second generation tests RPHA (Reverse Passive Haemagglutination tests).
(c) Third generation test RIA and ELISA-commonly practiced.
(d) Other tests viz. latex agglutination for quick estimation, passive haemagglutinatioi. test and
complement fixation tests are also employed in certain laboratory for screening purpose.
The counter immunoelectrophoresis method was earlier practiced but discontinued now-a-days in
favour of better result oriented third generation tests.
CIEP is based on the principle that proteins are amino acids and they can be electrically positive,
negative ",nd neutral. And in an electrical field like charges repel and opposite charges attract. The
HbSAg in an electrical field moves towards anode and gammaglobulin moves towards cathode. This
property of opposite mobility is utilised to our advantage in immunoelectrophoresis. And under an
appropriate physico chemical environment, the two, i.e., antigen and antibody, when meets produces a
sharp line of white precipitation. In a three hole system HbSAg +ve sample will produce a line of
precipitation between the test serum and antibody wells. The sample which contains anti-IfbS identifies
itself by producing a precipitation between test serum and well containing the Australia antigen.
This is a second generation test and sensitivity approximately ranges between 800 to 1000 ng/mI.
The most commonly practiced method in India is RPHA.
RPHA
Principle
It is based on principle that RBCs coated with anti-HBS will agglutinate in the presence of Hepatitis B
surface antigen. The reactive specimen 'show agglutination which is exhibited bya diffuse.cell pattern
upon settling. A non-reactive specimen does not cause agglutination .and settles into a compact cell
button.

[[p.6]]
-' .
..
~ ",.\
"
..--:--
156
Blood Banking and Transfusion
Procedure (Fig. 8.3)
(i) Put 25 J.l litre specimen diluent in each
U-bottomed well of a microtitre plate
marked for test and also in wells for
positive and negative control.
(ii) Add 7 J.ll of test serum in the wells
marked for test.
(iii) To positive control well add 7 ~ of
positive control serum.
(iv) To negative control well add 7 J.lI of
negative control serum.
(v) Mix.
(vi) Add 25 J.ll ofcells to each well (antibody
to HbSAg) chimpanzee coated human red
blood cells (lyophilized).
(vii) Mix again.
(viii) Cover the plate and incubate at R.T. for
two hours.
(ix) Read the result using a top reflecting
mirror.
Nonreactive
Test Result
WeakReactive
Test Result
Fig. 8.3. RPHA result Interpretation.
StrongReaetlve
Test Result
ModerateReactive
Test R suIt
Interpretation
The cells settling pattern of a strongly reactive
specimen will appear as a large diffused pattern
of cells with irregular periphery.
The cell settling pattern of a weakly reactive specimen will appear as a smaller, more dense pattern
of cells with slight irregular periphery and larger than negative control. Moderate reactive specimen
show cell settling pattern intermediate to the above two.
Non-reactive cell settling pattern is distinct, compact button ofcell with a smooth regular periphery
(no agglutination).
But since the result varies with different rmke and batch, it is always recommended that any positive
reaction is conftrmed before declaring the result. Confirmation can be done by the following method or
Elisa.
Conventional method is to take two tubes for confirmatory test:
(a) Take one volume of test sample and one volume of test serum to each of the tube mark them A
andB.
(b) To A add equal volume, i.e.• one volume of known HbSAg negative serum.
(c) To B add equal volume, i.e., one volume of known anti-HbSAg.
(d) Incubate both tubes for fifteen .to twenty minutes at RT.
(e) Set two rows of tubes corresponding to A & B.
(f) Serially dilute the contents of tube respectively in corresponding rows of A and B.
(g) To each dilution in two rows A and B, add one volume of antibody coated red cells.

[[p.7]]
Processing of Blood
157
(h) Mix the contents,
(i) Incubate at RT for two hours.
(j) Read the result.
Interpretation
If the testsampleshows the fall in titre by at least two tubes,the test is confirmed positive. It can be more
than two but not less than two. But, if both rows show same titre the original test is not confirmed as
positive.
In somecentres to economisethe expenditurethe RPHA is. done as routine test and positivesamples
are repeated in Elisa because Elisa considered more sensitive than RPHA but it should not preclude the
use of confirmatory test described above.
'ELISA (Enzyme linked immunosorbent Assay or Enzyme Immune assay) : This is also a third
generation test and considered more sensitive and specific in comparison to RPHA.
Material required
1. Microwell plate - Monoclonal anti-HbS coated on to solid phase
2. Wash fluid
3. Mechanical washer
4. Reagents and controls
5. Elisa reader,
Read the instroction carefully in the insert because it varies according to make or manufacturer and
follow it scrupulously while performing the test. The generally followed procedure is described below.
Principle
In Elisa the antibody is tagged with enzyme. Addition of enzyme substrate will lead to development of
colour which can be read macroscopically (naked eye) or spectroscopically(ELISA reader),
Procedure
1. Take correct volume of serum to be tested as per instruction in the insert and put them in the
respective wells.
2. Similarly positive and negative control equal to the volume of test serum is taken and put into
the respective wells,
.
3. The wells in the microwell plate containing monoclonal anti-HbS coated on to solid phase
along with test serum and positive and negativecontrol is now covered and incubate at 37°C for
the recommended period.
4. Washingstage: In this stage wells are washedthoroughly4 to 5 times with wash fluidprovided.
Washingis done either manually or mechanicallydepending on facility available.This removes
unbound extraneous protein and fluid. Antigen present in test serum and anti-HbS remains
firmly bound.
5. Addition of conjugate: In this stage the enzyme linked with nonclonal anti-HbS is added to
each well of microplate, volume as per instruction in insert.
6. Cover the plate and incubate at 37°C for recommended period.
7. Washingstage II: Washfouror fivetimesto removetheunboundedenzyme.labelledmonoclonal
antibody.

[[p.8]]
158
Blood.Banking and Transfusion
8. Add substrateinto wells as per direction and incubate at RTwhile protectingfrom light.Incubate
for recommended period.
9. Add blocking reagent at the end of incubation period. Blocking reagents are added to each well
as per instruction. Blocking reagents are added to stop the reaction.
10. Read the result either macroscopically (naked eye) and compare with control, this will give
onlyqualitativeestimation.Forquantitativeestimationrecordobservance valuespectroscopically
(ELISA reader) at 450 om.
11. Calculation to be done as per instruction in the insert.
The method described above is known as sandwiteh ELISA technique.
Radioirnmune assay
This also follows the principles of sandwitch technique as described above, but it will require an
uninterrupted supply of 1125• Radioiodine and special storage system recommended by BARC. Source
of supply of radioiodine is BARC and needs monitoring of working system by BARe. So for this
purpose, it is not commonly" practiced and is not described.
Detection of HCV antibody in human plasma/serum by ELISA technique
The ELISA technique described is a third generation test, and it utilises a combination of stroctural and
non-structural antigen, namely Core, NS3, NS4 and NS5. This combination of antigens are"coated onto
the microwells. If antibodies are present in the sample of serum/plasma to betested will get bind to the
coated antigen present in the microwell (ready to use) to which enzyme conjugate is added followed by
addition of fresh enzyme substrate and stop solution in the last stage. Blue colour is indicativeof positive
reaction and presence of antibody in the test sample.
Sun1mary of procedure
(a) First remove strips after the pack attains the room temperature (20° to 30°C).. Required number
of strips to be removed, balance kept intact for further test.
(b) Serum samples to be tested to be diluted (1 : 1 dilution) 200 ml diluent is added to 20 J.ll of
sample serum, if tube dilution is preferred.
Sample can be diluted in microwell also add 10 J.11 of sample serum to ]00 J.1l of sample diluent.
(c) Now assay procedure can be started:
.(i) 100 ul of sample diluent in AI microwell at blank,
(ii)
]00 J.lI of negative control in Bland C.I .
(iii) 100 J11 of positive control in DI' E1 and Fl.
(iv) Add 100 J11 of sample diluent in each well starting from O}well then add 10 JlI of sample
microwell dilution (b above).
(v) Apply cover and seal the well incubate at 37°C for 30 minutes.
(vi) After the Step V is over, wash the well 5 times with working wash solution.
(vii) Add 100 f..11 of working conjugate solution in each well starting from AI.
(viii) Apply cover and seal incubate at 37°C for 30 minutes in dark environment.
(ix) Washthe wells by aspiration at least 5 times.
(x) Add 100 ul of working substrate solu~on
in each well starting from At.
(xi) Incubate at room temperature (20 to30°C) for 30 minutes, protect from light.
(xii)" 'Add 50 JlIof stop solution after incubation is over.
(xiii) Read absorbance at 450 om in Elisa reader, A) well to beblanked.

[[p.9]]
Processing of Blood
159
Calculationof result and validityof test to be done as per literatureprovidedby manufacturerof kit.
Interpretation of result
Specimens withabsorbancelessthancutoff value- non-reactive toanti-HCVspecimens,withabsorbance
value greater or equal are considered reactive confirmationof reactive sample done by RffiA.
Detection of HIV (Human Immuno Deficiency Virus)
This is a dreaded virus whichcauses the disease knownasAcquiredImmunoDeficiencySyndrome.The
first case was discovered in later part of 1981,the discoveryof virus and its discovereris controversial.
However, theAIDS is responsiblefor manya silentdeaths in manyof the countriesprior to its discovery,
It was in early part of 1984 a research group in National Cancer Institute, USA, discovered the virus
which they prefer to name as T-lymphocyte retrovirustype-Ill Human1LV III or HTLVIII and the one
isolated in Pasteur Institute, Paris by another group almost at same time named it as lymphadenopathy
relatedvirusorLAV. ThisLAVIH1LVIII hasbeendetectedin blood,semenandsalivaaverageincubation
period is 29 months in adults and 12months in children,but it can even'lie dormantfor four to five years
in vivo before producing any symptom.Monthly update on HIV infectionsof AIDS in India by NACO
for period upto June 1993, states that seropositivecases were 12244 by Western blot and out of which
1841were"biood donors, i.e., 15.04%of total.That the diseasecan spreadthroughbloodtransfusioncan
be confirmedby anotherstudyconductedby Vaidya et al. in Mumbaishowsthat of 130patientreceiving
transfusionof blood/products 14 were detected seropositiveby ELISAlWB: There were tl% patient of
thalassamia, 15.38% haernophilliaand 7.6% of idiopathicthrombocytopenic purpuraand remainingof 7
cases seropositivity were attributed as others. In June 1993,break up by NACO also shows 260 cases
received bloodlbloodproducts,i.e., a 2.12% of total are transfusionrelatedAIDS.The range of duration
between the first blood transfusionand detection of seropositivityin HIV-I could be as wide asI to 22
years with a mean of 4.5 years of course, it depends on various other factors that may influence the
appearance of clinical symptoms.This emphasisesthe essentialityof screeningfor detection of HIV in
blood meant for transfusion purpose. High risk group among the donors are homosexual/heterosexual
Fig. 8.4. 1. GP 120, 2. GP 41, 3. P 10, 4". Viral membrane, 5. P 24, 6. RNA,
7. Reverse transcriptase physical structure of HIV.

[[p.10]]
160
Blood Banking and Transfusion
sexually promiscus persons, patients suffering from sexually transmitted diseases, intravenous, drug
users, professional blood donors, prostitutes, health care persons exposed to blood and blood products
in course of the professional discharge of their duty. These persons could be screened at donation stage
itself by cross-questioning. This will economise the expenditure greatly, that we spend for testing the
blood serologically.
Serological test commonly practiced are ELISA for general screening of all the blood/as the
preliminary investigating procedure, followed by western Blot as a confirmatory procedure for sample'
detected reactive by ELISA. The other serological procedure are also there but has not been accepted as '
standard, e.g., latex agglutination, particle agglutination and immunoblot technique.
ELISA can be done by three techniques :
(a) Sandwitch technique (already described in HbsAg detection).
(b) Competitive ELISA: Basic principle involved is competition between anti-HIV in test sample
or control sera and that in conjugate to get attached to solid phase HIV antigen prepared by
recombinant DNA method.
(c) Indirect ELISA: In this technique bound anti-HIV from sample/control attached to solid phase
is detected indirectly by addition of enzyme labeUed anti-human globulin, i.e., conjugate. All
these techniques can be employed and different manufacturers produces ELISA kit that employs
different technology. At present the strategy is to discard any blood that is found reactive by
first screening test. But for confirmation, two tests employing different technique of different
manufacturer can bedone in second screening, if both becomes positive, it can be confirmed as
cent percent positive. In doubtful cases, one may go for confirmation by western blot, This
confirmation is necessary to identify the AIDS cases and notify the person suffering from AIDS.
It is essential because the AIDS infectedperson may become source of further spread. But so
far unit of blood or blood product is concerned, it should be discarded if it is reactive in first
screening itself, irrespective of screening procedure adopted.
Procedure
Since the procedure vary from batch to batch produced by different manufacturers, it is always
recommended to follow the instructions in the insert. A general outline ofcompetitive ELISA and indirect
ELISA is viven below. Sandwitch elisa has already been discussed in section of HbsAg detection.
Material and reagents required
Microtitre plate with solid phase to which peptides or recombinant protein, mv virions are attached.
These plates with Ll-shapco wells are commonly used but in all cases supplied along with kit. Required
reagents most of them are included in kit supplied. Wash fluid are also supplied with the kit.
The materials not supplied with kit are micropipettes or disposable tips, multichannel dispenser,
incubator or water bath 37°C or 45°C, 'incubator box, reagent trough. Microwellplate washing system,
plate reader. Reagents not supplied is sodium hypochlorite solution. Software package, if required may
be arranged but this is not essential for the test procedure.
Specimen for test
The test sample may be serum or plasma and again serum is commonly preferred and due care should be
taken that serum samples are fully clotted and should not contain any contaminant or visible particle,
viz., clot etc. If it is contaminated, it is discarded and if clot is present, it is removed by centrifugation.

[[p.11]]
Processing of Blood
181
Sampleof serum is preserved at 2°C to goC before testing. If serum preservedfor72 hours, then store it
at 15°Cor at a temperaturein which it freezes. Defreezingof such sample is to be done in a water bath
at 56°C for 2 to 3 hours as the case may be. Temperature should not cross more than 56°C in any case.
Essentialprecautions to be undertaken are :
(a) Allow the 'wholekit to attain room temperature before use.
(b) Ensure that manufacturers has certified that all controls are inactivated and does not contain
any HbsAg.
(c) Kit has been stored at recommendedtemperatureof 2°C to goC and is withinthe limit of expiry.
(d) Handle the acid supplied and acid used for glassware washing with due care.
(e) Use gloves while doing tests to ensure personal safety andalso use protectiveglass over eyes to
protect them from splashing that may occur during manual washing of plate.
(f) Do not pipette by mouth.
(g) Washing is an essential step. Aspirationofliquid and washing the well by filling the wells upto
the brim is to be done with due care to avoid erroneous result,
(h) Conjugate reconstitutionis to be done as per instruction.
(i) Always u~e
disposable tips and make it a habbit to discard the tips after every single use. It
preventscross contamination, very essential to prevent contaminationbetween control, reagent
etc.
G) Do not allow the well to remain dry during procedureand also ensure that outerside of bottom
of wells does not have any water moleculeattached to it and it is dry before the plate is placed
in the reader.
(k) All disposables to bediscarded after use and non-disposables are sterilised before reuse.
(1) Washhands, thoroughly after the procedure is completed.
(m) Always follow the instruction given in the insert and never try to modify at any stage.
Procedure
Competitive ELISA
(a) Reconstitute the reagents as per instructionafter it has attained room temperature.
(b) Solid phase microplate is also allowed to.attain room temperature by exposing it to room
temperature.
(c) The test serum or plasma specimen and control sera 50 J.1l of each are placed into wells as
follows. Use the number of required strips only,rest can be removed and stored at 2°C to goC.
(i) Test serum/plasma50 J.1l into each well. Leave six well for control (read instructionsfor
how much control to be used).
(ii) 50 J.lI of negative control in each of two wells.
(iii) 50 J.lI of cut off control in each of three wells.
.(iv) 50 J.1l of positive control in one well.
(d) Reconstitute conjugate and add appropriate amount as per instruction in each well with a
multichannel dispenser.
(e) Incubate at 37°C for one hour (read instruction)after covering the wells.
(f) Reconstitutionof substratehas to bedone in the meantimeand it shouldbereadyfor use before
the incubation period is over.
(g) Washthoroughly the whole plate manually or mechanicallyby the wash fluid provided.

[[p.12]]
162
Blood Banking and Transfusion
SerumSample~
,
Added to Microwell
~
~
+
I
Co~ugme
•
I
(Incubate)
/ "
(A) ShowingCompetitionin HIV
PositiveTest Samples
,i,
/.)~
'---'
Serum Sample
Add to Microwefl
.- Conjugate
Incubate)
(B) No competitionin HIV
NegativeTest Samples
Serum Sample with HIV Antibody
,i.
SerumSamplewith No HIV Antibody
•
Solidphaseattached Antigenat Bottom of Well
A
Enzyme Labelled HIV Specific Antibody
I
J'l~
HIV Specific Antibody
Fig. 8.5. Competitive Elisa.
(h) Add substrate (read instruction) in appropriate amount to all the wells.
(i) Incubate as .per instruction.
Add stop solution to each well with multi-channel dispenser in the same sequence that was
followed during addition of reconstituted substrate.
Read result : Absorbance of each well at 450 nm by using a microwell plate reader. Calculate
cut off value as per instruction in the insert and compare the absorbances of test samples to the
cut off value and interpret result as per instruction.
Principle : In Enzyme linked .immunosorbant Assay sheet anchor for the test is solid phase
attached protein material from virions or synthetic peptide or recombinant protein.
In the particular type of .Elisa·described above, competitive Elisa, Recombinant protein (prepared
by DNA technique) using HIV-l core (P24) and"envelope (GP41) antigen are captured onto mouse
monoclonal antibody attached to a solid phase (plastic bead or microtitre plate well). This is then treated
with test specimen (serum or plasma) or control sera and the conjugate followed by a period ofincubation
during which competitv-: takes place between antibody to HIV-l in specimen or sera and the conjugate.
If antibody present in specimen or control sera, it will block the binding of conjugate or otherwise
binding with conjugate will occur. Washing done and substrate added incubated in the dark. Enzyme
reaction is now terminated by adding stop solution usually sulphuric acid. Anintense colour develops in
non-reactive. specimen while no development colour occurs in reactive specimens.
The method described shows how to detect HIV-l by competitive ELISA in a reactive specimen. To
be confirmed by retesting in duplicate, and if both retest by alternative immunoassay are non-reactive,
then the result can be interpreted as non-reactive, but if both reactive or one reactive and other nOI)-
reactive, then it .has to be confirmed further by western blot, IFA (Immunofluoroscent Assay), Ripa
(Radioimmunoprecipitation assay) in a reference laboratory.

[[p.13]]
Processing of Blood
163
Any detection of HIV-2, it became necessary, to develop technology/procedure that can detect both
HIV-l and mv-2, so that any blood' or blood product containing anyone or either is not transfused'
ina.dvertantly. So now-a-days procedure that can detect both HIV-l and HIV-2 arc now recommended.
They areELISA, Immunocomb (B1 - Spot) Irnmunoblot (HIVIcheck) etc. are practiced.
ELISA for detection of HIV·1 and HIV·2
This is also a blood screening procedure and confirmatory diagnostic procedure and is used for donor
screening purpose to detect both HIV-l.and HIV-2.
Principle: Asolid phase enzyme immunoassay and the technique described here is known as indirect
Elisa. We detect HIV-l and HIV-2 antibody not directly but by use of an enzyme labelled
antihumanglobulin antibody. On incubation at a recommended temperature for a recommended period,
this enzyme labelled anti-human globulin antibody binds to anti-HIV-l and HIV-2, which are human
immunoglobulins.
Synthetic peptides are coated on to solid phase (Plastic beads or microtitre plate wells).This peptide
antigens specially displays the immunodominant antigenic determinant of HIV-l and HIV-2 to which
when a positive specimen is added forms a stable Ag-Ab complex. This is washed and enzyme labelled
AHG antibody is again added which in turn binds with complex, remains in the well. Enzyme substrate
added, colour development proportionate to anti-HIV antibody takes place. Negative sample does not
show any colour.
Precaution : Same as described for competitive ELISA.
Material supplied: Variablefrom kit to kit.manufactured by different manufacturers.
Material required: Depends on materials supplied.
Procedure
Reagent preparation
It is done strictly as per instruction in the insert and no modification by the operator is to beundertaken
to avoid the possibility of erroneous result.
I. Remove microplate and reagent etc., at least 20 to 30 minutes before the test so that at the time
of test the whole kit reached room temperature stage.
2. Appropriate amount of test specimen is separately taken in clean tubes and diluted with sample
diluent as per instructions given.
.
3. Controls: Positive, negative and cut off prepared as per instruction to run alongwith each kit or
set of test.
4. Appropriate amount of diluted sample of patients are dispensed in wells.
,5. Incubate atRT for half an hour or as per instruction.
6. Aspirate and wash at least 5 times with wash solution.
7. Appropriate amount of conjugate - Enzyme labelled antihuman globulin antibody is added to
each of the wells including controls.
8. Incubate for half an hour at RT or as per instructions.
9. Aspirate and wash as described above.
10. Add freshly prepared substrate solution to each well.

[[p.14]]
164
Blood Banking and Transfusion
abe
Microwellmagnified
Solid phaseantigen showing
immunodominant epitope for 1 and 2
Anti HIV 1 and 2 (igG)
Ag-Ab complex
Enzymelabelled anti human IgG
Bindingof Ag-Ab Complexwith AHG
~
Substrate
,
Test Specimen
liJlWlW~
d
e
9
SEQUENCEOF EVENT
(a) Microwellcoated with peptideantigen
(b) Add test specimencontainingHIV antibody
(c) Ag..Ab complex formed after incubation
(d) Wash. add enzymelabelledantihuman(conjugate)
(e) Bindingof AHG with Ag·Ab complexafter incubation
(1) Wash add substrateand incubate
(9) Add stop solutioncolour develops(Positive)
Fig. 8.6. Indirect Elisa.
11. Incubate at RT for half an hour or as per instruction.
12. Add stop solution as per instruction in the insert, development colour takes place in proportion
to anti-HIV-l and HIV-2 in test specimen that is specimen showing colour development are
reactive and no colour develops in non-reactive specimen, i.e., negative samples.
13. Read result by recording absorbance in respect of each well in a plate reader usually at an
wavelength of 450 mm or as per instructions given by manufacturer.
Calculation of mean of positive control, negative control and cut off determination is done and
results interpreted as per instruction.
The indirect ELISA described as above is also a screening test for blood and is not a confmnatory
diagnostic test. Any positive test (or reactive specimen) has to be confirmed as described before in
competitive ELISA.
Apart from ELISA there are other less time consuming tests for detection of HIV-l and HIV-2in test
specimens method recommended are immunocomb and immunoblot described as under.
Immunocomb
Principle: This is also a solid phase enzyme immunoassay that detects both HIV-l and HIV-2 separately.
HIV-l and HIV-2 are fixed separately on plastic solid phase. This when treated with serum plasma
specimen which contains antibody gets attached to respective spot on plastic solid phase and are then
treated with enzyme labelled AHG. AHG gets bound to Ag-Ab complex. This when treated with enzyme
substrate develops colour in presence of antibody, Therefore, the antibody use is again indirectly detected
by use of AHG Since this bi-spot solid phase can detect both HIV-l and HIV-2, so this is a differential
screening for HIV-l and HIV-2 and definitely an improvisation over Elisa where technique described
cannot differentiate between HIV-I and HIV-2.

[[p.15]]
Processing of Blood
165
Material
(a) Basic materials required are a plastic card, the lower part of which is arranged in a form of
comb and each segmentof comb has three spots arranged in pattern shown below in Fig. 8.7.
(b) Developing plate : Each developingplate consistsof compartmentA to F,each compartmentIS
dividedin 12wells. Andallthewellsofeachcompartmentcontainsa reagent. Sosixcomparement
contains six solution reagent details (Fig. 8.7).
ProceduJ'e
Given as compartmentwise :
A. Containsspecimendiluent,appropriateamountof test specimenis addedtoeach wellseparately
with a micropipette. Insert the immunocomb card. Now incubate at room temperature for a
period of ten minutes.
B. Contains wash solution, insert immunocomb card after removing front compartment A, then
agitate twice or thrice incubate at room temperaturefor two minutes.
C. Contains diluted enzyme labelled AHG After two minutes of incubation, the immunocard is
removed from B, and placed in 'C' compartmentand again incubated for ten minutes at room
temperature.
D. Contains wash solution. After removing from C after ten minutes incubation, insert in
compartment'0', wash the segment with agitationand incubateat Rf'for two minutes.
E. Contains wash solution, wash with agitation and incubate for two minutes.
F. Contains chromogen substrate. Inserttheimmunocomb cordafterremovingfrom'E'compartment
and in F compartment, it is again incubatedfor ten minutesat RT.
Stop reactionis done by re-inserting again at 'E' compartmentand incubatingfor one minute. Bring
out thecard and makeit air dry.Run positiveand negativecontrolseparatelyalong witheach (estassay.
A f-.
·8 f-.-
C ~
0 r-.
E ~
F r-.
1
2
3
4
5
6
(
8
9
10 11 12
DEVELOPINGPLATE AS VIEWED FROMTOP
Specimendiluent
Wash solution
Diluted Enzyme labelled AHG
Wash solution
Wash solution
Chromogensubstrate solution
INDIVIDUALSEGMENTOF COOMB
Plastic solid phase arranged
like Comb with twelve
segment in Lower part
10to12mm
6 to 8 mm
2to4mmlt
Fig. 8.7. Immunocomb.
•
•
•
Internalcontrol goat AHG
Synthetic HIV-2 Peptide GP 36
Synthetic
H1V-1 peptide (GP41 and GP120)

[[p.16]]
166
Blood Banking and Transfusion
Read results : Results are read from development of colour on the spots.
Interpretation : A non-reactive specimen will show no colour development on the .lowertwo spot
and presence of an upper internal control at the top.
Areactivespecimenwillshowcolourdevelopmentdependingon presenceofrespectiveHlvantibody,
i.e., for HIV-l at lower spot and HIV-2 at middle spot Equivocal tests are to be confirmed by western
blot similarly reactive tests are confirmed by western blot.
'
Immunoblot (quick HIV)
Principle : Synthetic peptide antigen for HIV-I and HIV-2 are dispersed and held immobilised in flow
through membrane. Test specimen containing antibodies to HIV-l and HIV-2, when allowed to pass
throughthe membranegets bound to the antigen and formsAg-Ab complex.Anindicatorsuch ascolloidal
gold conjugated anti-human immunoglobulin is added and allowed to be soaked in. Results are ready
within minutes.Test serum positive gives a red colour with non-reactive specimen does not develop any
colour.
Materials required: Synthetic peptide for HIV-I and HIV-2 captured in a flow through membrane
support, set in a plastic box (supplied in the kit) and buffers and wash solution along with colloidal gold
conjugate.
Procedure
1. Open up the pouch that contains the small box, remove the lid and bring the box to room
temperature along with the whole kit.
2. Add appropriate amount of buffer on the membrane in inner circle. This wets the membrane.
3. Add one drop of test specimen (serum/plasma).
4. Add buffer again in appropriate amount arid allowed to be soaked in.
5. Now wash with wash solution as per instruction in insert.·
6. Add appropriate amount of colloidal gold conjugate allow to soak and again wash with wash
solution.
Results ready within 5 minutes shows red colour spot on the membrane indicating reactive sample;
~r otherwise membrane remains clear, i.e., non-reactive specimen. This has to be confirmed further by
western blot test, other test such as latex agglutination and particle agglutination test are also described,
Particle
agglutinatl~n
test
This test is based on the principle of immune-agglutination, test serum if contains antibody to HIV will
react with particles coated with purified antigen resulting in agglutination.
To eliminate non-specific reaction, detergentdisrupted HIV particle or artificially synthesised HN
particles are attached to gelatin particles.
Gelatin particles.are biologically inert or inactive and does not contain any leukocyte antigen and
causes minimal adsorptionof serum constituents possibilityof non-specificagglutinationis thus virtually
eliminated.' .
Serodia HIV is the kit used for particle agglutination in a microtitre plate. This is also accepted
standard procedure for test HIV"-l and where ELISA is not available or difficult to perform particle
agglutination test is the recommended procedure.

[[p.17]]
Processing of Blood
167
Test procedure
1. Use the microtitre plate supplied with kit.
2. The tes,tserum is diluted with serum diluent provided (usually PBS). After serial dilution 1 : 4 :
1 : 8 and 1 : 16 of the serum proceed to the next step.
3. Add appropriate amount of uncensitised particles respectively in secondtube and sensitised
particles in thirdtube, thus making final dilution of 1 : 16 and 1 : 32 in second and third tube.
4. Shake the plate gently with automatic vibrator for thorough mixing and cover the plate in
incubator at RT for two hours.
5. Read the result and interpret by observing the pattern of agglutination. A negative result is
indicated by compact button'at the bottom of the well. A positive result is indicated by scattered
cell pattern at the bottom of well of microplate visually. Fully automatic fastec 901 provided
with kit can also interpret the result.
Advantages : Simple, no washing, less time consuming results are comparable to ELISA and
sometimes better.
Latex agglutination test: The principle is that HIV glycoprotein antigen are produced by recombinant
DNA method. This when treated with test specimen (serum) produces agglutination, if the specimen'
contains anti-HIVe
Procedure
1. All materials required for the test are allowed to obtain.
2. Take the slide coated with HIV latex particle.
3. 'Appropriate amount of test specimen added.
4. Slide is rocked gently for 5 minutes.
5. Agglutination pattern will appear if HIV antibodies are present in test specimen.
Read results: Appearance of agglutination indicates reactive sample, i.e., positive for HIV. This has
to be confirmed by western blot.
Western blot
This procedure is adopted as a confirmatory diagnostic test for the cases found reactive by screen test
and also repeatedly reactive by retest procedure.
But now as per policy, the test detected reactive by screening only is discarded and further use is not
risked for transfusion purpose. So the procedure of confirmatory diagnosis has lost its importance for
blood transfusion services. Nevertheless, a transfusionist ought to know the procedural outline oftechnique
as they are to identify the donors (HIV positive) and inform the central monitoring organisation
accordingly.
Principle
The western blot is also an immuno blot basically but here, we take help of an electrophoresis to separate
viral antigen according to their relative molecular weight. The separated HIV antigens are transferred to
nitrocellulose membrane and strips of nitrocellulose membrane impregnated with viral antigen are
incubated with test specimen.These strips are now treated with enzyme labelled AHa antibody and

[[p.18]]
168
Blood Banking and Transfusion
substrate is added, colour develops and forms a distinct pattern. In short the technique is thus an
immunoelectro-phoretically transferred blot detected by enzyme linked AHG antibody.
Procedure (basic steps only)
1. Immunoelectrophoresis is conducted to purified HIV virions over a polyacrylamide slab gel in
presence of sodium dodecyl sulphate. Viral antigens gets separated according to their relative
molecular weight
2. These are now transferred to a membrane of nitrocellulose.
3. Membranes having the complete spectrum representing all the viral antigens are cut into strips.
4. Strips are incubated withtest specimen as per instruction.
5. HIV antibody present will get bound to the viral antigen impregnated in thenitrocellulose strips.
6. These Ag-Ab complex are now treated with biotinylated AHG and later detected by avidin
conjugate.
.
7. Substrate when added develops colour. Interpretation of result done by comparing with bands
of a strip of strongly reactive control. Basic minimum criteria for a positive diagnosis is the
presence of antibody to at least one antigen respectively in Pol, gag and env.
The procedure described is a very brief outline of actual technique and may give the impression that
test is very simple. But actually the test is very 'difficult and complicated and need to practiced only by
skilled, expert technologist.
The test though said to be a confirmatory diagnostic test, but it is not full proof, and it fails to
diagnose the HIV infection during the window phase. There is always a gap between the entry of HIV
virus and appearance of a detectable amount of HIV .antibodies in the serum of an infected person and
this is defined as window' phase. The antibody though remain absent during window phase, the P24
antigen is found in serum and CSF of HIV infected person. 'The western blot testis basedonprinciple of
antibody detection. As such, it fails to detect the P24 antigenemia during window phase approximately
2 to 3 months. Following this period seroconversion occurs, antigenis lost and antibody appears.P24
antigen may reappear again at a very late stage terminal stage when anti-HIV antibodies concentration
usually drops down below the detectable label in the senlm. In such cases WB test is inconclusive.
However, peR (Polymerase chain reaction) is able to detect the P24 antigen in such cases. The chance
of a blood donor being in window phase may range somewhere between 1 : 44,000. to 1 : 3,50,000. A
study conducted by Nuchapraynon C. et al. on Thai voluntary donors 1,25472 cases for P24 in addition
to Elisa and WB screening concluded th~t
P24 test-might prevent 60 post-transfusion HIV annually. No
such study has been conducted in India. But if an analogy is drawn, we cannot afford. to neglect and
overlook the possibility of transmission of infection albeit in decimal incidence per 100 in a year, and if
we consider the total number of voluntary donors or replacement donors in India, the figure of transmission
during window phase may well cross beyond 100 annually. As such the importance of screening for P24
or search for antibodies in early phase of disease deserves due consideration. Again since HIV-l and
HIV-2 are known to share core antigen; so the following tests may serve useful purpose.
(a) peR based confirmatory assay.
(b)
Virus isolation.
(c) Flowcytomeric IRS (IlnITIUnO Reactive Bead) assay for quantitative and early detection of HIV
antibodies-detection by fluorescent antiglobulin test. A close view on serological profile ofmv
infection will reveal that anti-GP 41' appears quiet early (Fig. 8.8).

[[p.19]]
Processing of Blood
169
/
./
;---
/
.
... _
/L..·
.
/
/
/
/
/
..
, ..-' .......
,
.,
I
•
,
:
I
I
I,
I
Oa....---'-----..&..----:--
--'-
~
_
1
2
3
4
Month
Month
Month
Month
Years
(
(
(
(
- - - - -
P24 Antigen
............ Anti GP41
>(
)(
Anti P24
ShowingDecline
Of Anti P24 and Risein P24 Antigen
Note GP 41 Antibody is Steadythroughout
Fig. 8.8. Serological profile of HIV infection.
In IRB assay polystyrene beads of four different diameters each coated with different HIV recombinant
DNA manufactured, protein (P24, P31, GP41, GP120) bound anti-HIV antibodies were detected by
fluorescent antiglobulin. Here we have the added advantage of using IgM and IgG specific fluorescent
antibody probes. Anti GP41 is an IgM that appears in serum before the anti-P24.This procedure test is
practiced for simultaneous detection of two or more specific antibody; this procedure can also be adapted
to detect HN antigen, HIV specific immune complex and other blood borne infectious agent. The test
has been specifically developed for HIV-l. Since in USA (the test has been developed by collaborative
effort between university of California and Transmed Biotech
and National Heart Lung and Blood
Institute) the HIV-2 is uncommon. In India test is not yet been marketed. This test can be called as an
improvement on present generation of test with added advantage for early detection at window phase.
It has been discussed earlier that even WB considered and recommended as a confirmatory diagnostic
test is not a full proof method for HIV detection. Similarly Elisa that is commonly used for screening
purpose is not fullproof and fails to detect all pure positive and false negative cases. A transfusionist is
more concerned with false negative cases. The clinocoepidemological impact and bioburden on the
health status of the community, the medicologal implication of certifying a false negative case should
make us cautious. In this context, it will ~e
worthwhile to refer to a comparative study of particle
agglutination PAA (serodia) and ELISA of 1130 serum conducted at I1H Mumbai and quoted by Dr.
Z.S. Bharucha in "Strategies for ensuring safe blood and blood products" reveal the deficiency in highly
claimed perfection of ELISA (Table 8.2) comparison of ELISA and Serodia (PAA).
Table 8.2.· Reporting by ELISA and Serodia
Total no. of sera tested 1130
Pos
WB (Ind)
NeG
False positive
False negative
ELISA
60
8
8
14
2
Serodia
64
II
9
20
0

[[p.20]]
170
Blood Banking and Transfusion
Above observation though in a small number of cases is quite significant and therefore, it is
recommended is that each batch of kit supplies must be tested for validity before being employed for use'
irrespective of the recommendation claim by manufacturer's validity testing.
The objective of HIV antibody testing can be considered under four broad categories:
1. Screening of blood and blood products includes safe donation and transfusion
2. Surveillance
3. Diagnostic
4. Academical
We are more concerned with No.1. The sensitivity and specificity are the two major parameters that
determines the validity/accuracy of any laboratory test procedure (Table 8.3) and its performance in
distinguishing between adiseased and healthy person. Sensitivity by definition is the ability to detect a
high proportion of true positives and as such there will be fewer number of false negative in a test with
high sensitivity. More important for transfusion point of view. Specificity by definition is the ability to
detect the number of true negative cases as such there will be fewer false positive results in a test with
high specificity. Specificity is more important from diagnostic point of 'view.
Predictive value ofpositive result: Higher the prevalence of infection in a community,greater is the
probability of cases being detected as truly positive. So with prevalence (total number of cases old and
new at a given point of time) increased greater will be the positive predictive value, i.e., it reflects the
diagnostic power of the test "in other words sample testing false positive should decline.
Predictive value ofnegative results: Here the the prevalence is inversly proportional to true negative
results, i.e., as the prevalence increases, the negative predictive value decreases. It can thus be inferred
that with increase in prevalence a proportionate increase may occur in sample testing false negative.
Since it is of prime importance for a transfusionist to take care of transfusion/donation safety so a
test procedure giving minimal or nil false negative and markedly sensitive should be choosen for such
type of screening test.
While evaluating it has to be compared with claim of manufacturer. It has been observed that when
non-identical reference tests are used test kit under evaluation may yield disparate sensitivity and broad
outline is that less sensitive the reference test, the more sensitive the test under evaluation will appear.
The predictive valuedata are again influencedby point prevalence ofAIDS or HIV virusin thecommunity
under study.
Table 8.3.
Mea~uremen,t
of
val~dity
Screening test results
True diagnosis
Total
Diseased
Not diseased
a
b
a+b
Positive
(True positive)
(False positive)
c
d
e+d
Negative
(False negative)
(True negative)
Total
a+c
b+d
a+ b + e + d
Sensitivity: ala + c; Specificity: dIb + d; PPV ala + b; NPV die + d

[[p.21]]
Processing of Blood
171
In evaluation of blood donor screening test, take into consideration the :
(a) Characteristics of assay kit
(b) Evaluate the manufacturer's claim.
Characteristic of assay kit includes :
(i) Sensitivity and specificity; index specified.
(ii) Format of the particular test.
(iii) Technology employed conform to standard prescribed.
(iv) Cut off calculation - cut off may be fixed or floating.
(v) Time required.
(vi) Reagent requirements, dilution, stability and expiry date, storage instruction etc.
(vii) Sample specimen collection, dilution and other requirements.
(viii) Control with special instruction if any.
(ix) Accessories and equipment normally required to run the test.
(x) Whether procedure.is cost-effective and small test runs are possible or not.
Evaluation of manufacturer's claim : A limited laboratory trial may be conducted to evaluate the
manufacturer's claim.
Protocol
(a) Normal donor samples 1000 to 3000.
(b) More than one.kit should be used.
(c) Analysis of result.
Results are analysed in terms of following and interpretation done with reference to "reference
reactive" samples already placed in each evaluating plate.
Analysis in terms of :
(i) IR (initial reactive sample) versus RR (Repeat Reactive, sample) and correlation between these
two "results.
(ii) Repeat duplicate and their correlation.
(iii) Numbers of positive, negative and borderline (equivocal).
(iv) Tests confirmed and indeterminate by recommended confirmatory test.
(v) Variability in supplied hems controls, cutoff, reagent.
(vi) Consistency between different kit supplied in a particular lot.
(vii) Quality of software, equipment and accessories.
(viii) Operator's training and skill.
(ix) While confirming any test result by a particular methodology at least 100 to 200 specimens
should be subjected to trial test viz. 100 to 200 known positive specimen is minimal requirement
to confirm sensitivity.
(x) Qualification panel (minimum eight with different levels of reactivity), seroconverter panel (for
assessment of sensitivity), dilution panel (information on linearity and clue to sensitivity index).

[[p.22]]
172
Blood Banking and Transfusion
Selection of test kits for serological investigation, are therefore, done on the basis sensitivity,
specificity and predictive value. For transfusion/donation safety, a kit with high sensitive index is always
preferredto avoid false negative as much as possible. Similarly low specificity is also avoided because
it leads to discard of blood unit for its association with high rate of initial positive result. The present
policy of GO.l. is to discard any unit showing positive result for mv in the first test. However, a kit with
low specificity may show positivity even in duplicate retest needing further confirmation by a confirmatory
test. So to avoid all this, it is better not to accept a kit with low specificity.
Accepted kit, therefore, should be highly sensitive and also not of very low specificity. Such
acceptance should be supported by minimal acceptable statistical standard, i.e. at least 200 specimen
confirmsthe reference test result and limited laboratory trial (for 1000 to 3000) conducted on study
population similar to test targeted population while giving due precaution in :
(a)
Specimen collection, and
(b)
Blinding the operators from the reference test value.
Strategy I : All samples are tested with one ELISA or rapid/simple test, thereafter, referred to as test
(Table 8.4).
Strategy II : All samples are first tested with one test. Any reactive samples are subjected to a
second test based on different principle and or a different antigen preparation.
Strategy III : All samples are first tested with one test. Any reactive samples are retested with
different test. Samples reactive by the second test based on different principle are subjected to a third
and different test.
However, what is the strategy to be adopted in a particular region/country is decided by concerned
authority of that country and so far screening of blood donor is concerned the present policy is to be
discard any initial HIV reactive unit without waiting for further confirmation.
N·AT (Nucleic acid testing)
Nucleic acids are:
(a) RNA: Ribonucleic acid
(b) DNA: Deoxyribonucleic acid
Through NAT technology minutest or minimally present virus in human plasma could be detected
through geometric amplification of nucleic acids viz. RNA, DNA.
Table
8~4.
WHO recommendation for HIV testing strategy
Objective of te~ting
Prevalence of infection
Testing 'strategy
(a)
Transfusion/donation safet.y
All prevalences
I
(b)
Surveillance
More than 10%
I
Less than or equal to 10%
IT
(c)
Diagnosis
All prevalences
IT
Clinical symptoms of HIV
More than 10%
II
Infection/AIDS Asymptomatic'
Less than or equal to 10%
ill

[[p.23]]
Processing of Blood
173
Polymerase chain reaction (peR) and transcription mediated amplification (TMA) are the two
procedure. In the former procedure steps arenucleic acid extraction, purification followed by
ultracentrifugation finally is amplification. Amplified nucleic acid are detected by using thermocycler.
In TMA procedure, three virus nucleic acid, viz. HIV-l, RNA, HBV DNA and HeV RNA are
simultaneously screened from human plasma sample in one tube. Steps involved are target capture and
amplification of nucleic acid. Amplified nucleic acids are identified and detected. Readers might have
noted that in this procedure no ultracentrifugation and thermocycler are employed.
NATis very useful in narrowing the window period in HIV, HBV and HeV respectively to 8 to 10
days, 25 to 28 days and 20 to 24 days approximately.
TMA, a three in one procedure may prove more beneficial and useful for blood donor screening
with sole aim of safe transfusion of blood.
8.2 RECIPIENT BLOOD
Pretransfusion test on recipient sample
The recipient/patient sample is received in blood bank has also to be processed before transfusion so
that a compatible unit of blood component is transfused to the patient. This is to ensure safe transfusion
of blood
Specimen or sample collection : TQe blood may be collected directly from the patient or from an
infusion line :
1. Direct collection is usually done from the veins of anticubital fossa commonly. if the veins are
damaged in this place for repeated transfusion, then veins from any other place is choosen. A
tourniquet may be tied proximal to venepuncture site and the blood is drawn by a disposable
needle and syringe slowly (force is avoided to prevent haemolysis) and blood amount 2 to 5 ml.
The needle withdrawn local pressure with spirit swab applied and tourniquet untied. The amount
of blood so withdrawn is transferred to all glass plain vial directly or it may be collected in
EDTA vial. Common practice is plain vial for sample required. for grouping for transfusion
purpose.
2. From infusion line, if the blood is collected one is to ensure that the infusion line does not
contain dextran type of fluid and in that case the sample collection is avoided. In such cases
sample collection to be done before dextran is started. In other cases where blood is collected
from an infusion line first few ml of blood is discarded.
In case of infants sample is collected by heel prick method if veins are not available and upto the age
of at least 3 months, the infants sample to be accompanied by mother's sample. Mother's sample is
required to know the antibody present.
Precautions
1. Milking of collection site or squeezing of the site is to be avoided.
2. If blood is to be collected for platelet transfusion, the donor in such cases must not have taken
aspirin group of drug in last 72 hours.
3. 'After collection of sample the vial is to,be labelled immediately.

[[p.24]]
174
Blood Banking and Transfusion
Label to contain Name, Age, Sex of the patient/recipient statistical number of hospital and ward,
bed number ofhospital. It is preferable, if diagnosis could be given. For cord blood, it must be mentioned
as cord blood and post-transfusion sample also to be mentioned over the label. Vial is then stoppered
with rubber stopper and despatch immediately to blood bank.
The sample of blood must be accompanied by a form in which the purpose of sending the blood to
blood bank is clearly mentioned and signed. If the sample is meant for blood transfusion, it is then
accompanied by a duly filled up requisition form, sanction slip and a presigned receipt form without
which samples are not accepted in registration counter of blood bank.
1. After the sample is received in the laboratory, the particulars in the requisition form are cross-
checked with that over the label on the sample vial.
2. Contents of the vials are checked, it should not contain any extraneous particle, any contaminant
or the blood sample should not be haemolysed. ,
3. History of previous transfusion record should be checked in the column meant for this purpose
in the requisition form. Also see the history of previous pregnancy etc.
4. The sample received should be stored at 2° to 8°C after the necessary laboratory procedure is
over for a minimum period of seven days, it can be discarded after seven days.
5. The sample received has to be tested for ABO and Rh grouping. ABO grouping by both cell and
serum grouping,and also Rh grouping is done.
6. Antibody detection to be done in the way as described. Refer to chapter on antibody identification
etc.' The detection test, if done in different media at different temperature definitely gives clue
to such antibody.
Once these procedure are over we can then proceed for choice and selection of blood and 4 or 5
units are choiced or selected from the blood available in stock and which are certified free of Syphilis,
Malaria, Hb.Ag, HIV, HeV etc., and these units are cross-matched with patient/recipient blood and
compatible unit issued.
8.3 CHOICE AND SELECTION OF BLOOD
Normally the first choice goes for the same ABO group and sameRH group and 4 or 5 units are selected
according to seniority because the policy of storage in blood banking is "first in first out" and is strictly
observed unless specified otherwise viz. demand for fresh blood. This is observed to prevent expiry of
blood. Blood choosed must be within the limit of expiry period and must not contain any clot, no
haemolysis or contaminant observed, supernatant must be as clear as possible. Unit must not be a leaking
one, i.e., container intact along with tubing and stored in a recommended temperature. It is certified free
for malaria, syphillis, HbSAg, HIV and HeV. Any deviation from the guidelines described should not
be risked because it may lead to fatality for patient and medicolegal involvement for technologist.
Now these selected unit of blood are rendered to cross-match with recipient sample and the compatible
unit is issued.
The best policy for safe transfusion is to choice and transfuse blood of same ABO and Rh group.
But in some emergency when the particular group is not available, a choice of alternative blood group
has to made by transfusionist. In such cases transfusionist makes a suggestion to clinician and obtain his
written approval for transfusion of an alternative group of blood. Only on obtaining such approval, the
transfusionist selects the alternative group of blood, general guideline being thesame as described earlier.
4 to 5 units are taken outof stock and crossmatchedwith recipients sample and compatible unit is issued.

[[p.25]]
· Processing of Blood
175
A choice of alternative blood group that can be used in emergency life saving cases is given in the Table
8.5. It is emphasised again to the readers that blood of same ABO and same Rh group is the best and
should be the first and last choice unless the compelling circumstances warrants use of alternative group
of blood.
Table 8.5. Alternate choice for ABO group
ABO group
Alternate choice
First choice
Second choice
1.
A
0
X
2.
A]
0
X
3.
A2 with anti-At
0
X
4.
At with anti-HI
None
None
5.
B
·0
X.
6.
AlB
AorB
0
7.
A2 B with anti-AI
A2 0r B
0
In cases where A subtyping could not be done, it is better to transfuse B as an alternate first choice. 0 group
though accepted as an universal donor, but there are some '0' group which are known as "Dangerous universal
donor". In this type of '0' group, there is presence of high titre anti-A and anti-B, these 'O' group persons with high
titred agglutininsand heomolysinare known' as "Dangerousuniversaldonor" and if blood from such individualsare
transfused ~s an alternate choice, must be titred for antibody and a titre below 1 : 100 is considered safe for
transfusion purpose.
Emergency method for low titre '0'
The serum is diluted 1 : 100. Now take one volume of 1 : 100 serumand to it, add one volume of 20/0 cells
suspension. Cell serum mixture is now incubated at room temperature (20
0e to 25°C) for 30 minutes. At
the end of incubation period. The mixture is centrifuged at 1000 r.p.m. for one minute. Read the result.
If there is agglutination, titre is more than 100 andif no agglutination, the titre is less than 100. The low
titred '0
1 group can be considered safe for transfusion whereas high titred group '0' is unsafe for transfusion.
Haemolysin activity can be detected by putting "A" and "B" cells in 101serum and incubate at 37°C
for at least two hours, after two hours, i.e. at the end of incubation period the supernatant in the tube is
carefully observed and if it is reddish in colour, it exhibits the haemolysin activity and considered unsafe
for transfusion. If no colour change of supernatant, the cells are not destroyed' and as such no haemolysis,
the haemolysin is thus not present. Such blood is considered safe for transfusion.
But since alternate choice for '0' is made only in emergency cases when same blood group is not
available and if the transfusion saves the life of the patient, it is better to transfuse '0' packed cell from
where plasma is very carefully separated. Such '0' packed cell are to be prepared by refrigerated centrifuge
and not by sedimentation method.
Note of caution
It is always advisable that if a patienthas already received more than two units of blood from alternate
choice, continue the same instead of switching over to the original ABO group of the patient unless it is

[[p.26]]
176
Blood 'Banking and Transfusion
warranted by non-availability of alternate choice again. In those cases freshly drawn sample from the
patient is cross-matched with group specific blood and after thorough satisfaction regarding the
compatibility, with group specific blood unit, one can transfuse the group specific compatible unit. This
is particularly to be followed strictly in case of 'AB' group. In this type of circumstances as described
above and the patient is "AB" group, it is better to continue with the second choice, i.e, '0' group
provided it is low titred and haemolysin free. Alternate choice in Neonates/infants is very difficult because
of their weak constitutio, they cannot withstand blood loss and has to be replenished as a life saving
measure. We must thus be very much careful in selecting the alternate choice ABO group so that it does
not lead to haemolytic reaction and ultimate fatal risk to the recipient.
In case there is incompatibility between mother and infant, the best is to transfuse packed cell that is
compatible with both serum, of mother and infant. In case of exchange transfusion where the demand
isfor whole blood such packed cell can be reconstitutedwith FFP same as that of baby blood group. The
other alternate choice for whole blood is '0' packed cell reconstituted in "AB" neutral FFP.
The Table 8.6 may be useful in taking decision on alternate choice in infants.
Table 8.6. Altemate choice of ABO group in infants
Child
A
B
AB
0
Mother
A
* 'A' or '0'
0
* 'A' or'O'
0
B
0
B orO
B orO
0
AB
* 'A' or '0'
'B'or'O'
* A, se-o
None
0
0
0
None
0
* Subtypes of A group has to be done and A subtype has to be transfused though they are weakly developed and
difficult to detect in infants. It is always better to transfuse the exact subtype if it can be detected.
Alternate choice for Rh group
The policy should be, always stick to the original group unless compelling circumstances warrants the
use of alternate choice.
Rh is highly immunogenic and careful alternate choice is to be done particularly in female of child
bearing age.
While doing Rh grouping, if it turns out to be negative, then make yourself sure that you are not
dealing with Du'
A definite case of Rh negative patient can be transfused with Rh +ve blood provided the patient
does not contain Rh antibodies and to detect this one has to undertake antibody screening for Rh antibody.
Rh antibody is better detected by enzyme viz. papain. If the patient is a female of child bearing age and
life saving transfusion is required, it is preferable to change the ABO group instead of Rh group because
of inherent danger of developing into HDN at the time of pregnancy.
A DU +ve individual is considered as Rh +ve as donor and Rh -ve as a recipient. This is of relevance
particularly with "high Du". High Du is more prevalent in black population, "Negros". Infants suffering
from HDN are transfused with Rh" (negative) blood only. If it becomes necessary to change the Rh
system, we may do Rh grouping in fresh sample obtained from the recipient and if it does not show

[[p.27]]
Processing of Blood
177
antibody (can be detected by papain). We may switch over to Rh system of the recipient viz. recipient is
AlB Rh +ve and 'Nedo not have AlB Rh +ve donor, then we can either use AI Rh +ve blood or B Rh +ve
blood and likewise. It has been also observed that in very few r r and r"r individual mayproduce antibody
when transfused with Rh +ve blood and are considered as Rh +ve since they stimulate antibody when
such bloods' are transfused to IT individuals. Again for Rh null blood group, there is no alternate choice
and same is true for Bombay group of blood.
Basically whether it involves, i.e., alternate choice involves change in Rh or ABO group is not the
main issue but it is the compatibility of alternatively selected blood. For this purpose, cross-matching is
essential which gives the transfusionist an idea regarding the antibody and helps him in choice of blood,
lacking in the corresponding antigen for safe transfusion practice, without any future sequele, i.e., no
immediate, delayed or anamnestic response.
8.4 CROSS MATCHING
Cross matching is one of the most important serological procedure pertaining to blood group serology
and is the fundamental procedure responsible for safe transfusion of blood.
Basically an antigen-antibody reaction, a correct interpretation of which is the most essential
preliminary step in the practice of safe transfusion of blood. By cross matching we are.able to detect the
atypical and clinically significant antibody mostly IgM and IgG present in the recipient serum or in
Donors serum, also by autocontrol, We are able to detect auto-antibody in patient himself. Normally
group specific donor units are selected for cross matching with recipients serum (major 'cross matching)
or with donors serum and recipient cell (minor cross matching) and in case of non-availability of group
specific blood selection of units are done from alternate choice (ref. immediate previous section)
compatibility test is then undertaken and a unit compatible with recipient's blood is issued for transfusion
purpose. An incompatible unit is potentially dangerous and not beissued for transfusion. If all the group
specific blood unit are found incompatible presence of a typical or clinically significant antibody is
suspected in recipients serum and a screening test for detection of antibody and identification of antibody
is undertaken. Once antibody is identified, then the particular antigen negative group specific donor unit
is selected and again a compatibility test is done to find out the compatible donor unit for transfusion
purpose with the aim of achieving the normal cell survival rate in the post-transfusion period.
Cross matching is thus. an absolute essential procedure for safe blood transfusion. Therefore, the
sample received from the patient are tested for ABO and Rh grouping and group specific donor unite; are
choosen from the units preserved in store and in case of non-availability, units are selected from alternate
choice after obtaining approval from clinician for use <?f alternate choice. Cross matching of every unit
of blood is carried out as follows. The. blood group antibodies mainly belongs to IgM, IgG and IgA
occasional, and they can be detected by saline, albumin, enzyme and antiglobulin reagent and thus, we
can perform the compatibility test in three gradual phases according to following scheme as under in the
Table 8.7.
The two other types of cross matching usually employed are: (a) Minor crossmatching and (b) auto
control. Minor crossmatching is the anti-thesis of major crossmatching test and detects the antibodies
present in the donor serum. Unlike the major crossmatch test, reactant used in this test are Donors
serum/plasma is treated with Recipient's/patient's cell. If incompatibility is detected, a transfusion of
such blood has of little consequence because. the antibodies contained in donor's plasma entering the
recipient's body drop by drop through venous circulation encounters large and excess amount ofantigen

[[p.28]]
178
Blood Banking and Transfusion .
Table 8.7. Detection of common antibody by major crossmatch
Typeof
Reactant
Medium and
Thermal status
Antibodies usually detected
crossmatch
type of test
.Major
Patient's serum
Saline
At RT 20° to 25°
MostlyIgm -A, -B, a-Al'-M,
containing antibody +
centigrade
-N,-Lewis a, b, -Lu
b
Donor'scell
4°C
-H, -I, -i, -PI, -Lea, ~Le
mostly
containing antigen
IgM
Alubmin
37°C
MostlyIgG -D, -C, -E, -e, -f, -c
24° to 37°C
PPl' PK, IgM usually
~ Papain cystein
a
b
37°C
-C, -D, -E, -JK , -e, -JK , -c
mostlyIgG
Enzyme
24° to 37°C
PP1 PK IgM usually
AHG
37°C
MqstlyIgG, bDt --<;, -f, liE, -c, -
Lu , -et -Lu b, -Jk , -I~
,1)5s, -
K,-Fy,-Fy,-K,-Js -Js
and very little antigen-antibody binding can take place under the prevailing circumstances. Finally
when the whole process is completed, the antibodies present in donor's plasma gets diluted in recipient's
blood volume and concentration of antibody is effectively reduced through dilution and other process as
under:
1. Volume dilution 250 ml : 6000 ml, i.e, 1 : 24 is the titre of antibody and is not an effective titre
for naturally occurring antibody.
2. Antibodies during course of circulation get absorbed by antigens present in vascular endothelium.
3. In secretors individual neutralisation may occur by soluble blood group substances present in
plasma. For the reason described above, most of the blood bank laboratory does not give much
importance to minor crossmatching while doing the compatibility test. But a fair opinion will be
to do the minor crossmatch test simultaneously along with.major crossmatch.
The reasons being
(a) All the blood bank laboratory does not do screening of donor's plasma.
(b) The factors descnoed above hold true, if donor's plasma contains low titre of antibody and in
present day, where immunisation is one of the means for effective disease preventive mechanisms
and because of these prophylactic vaccination (bacterial antigen etc.) the .level of naturally
occurring anti-A and anti-B level may rise as a result of immunisation. These donor antibody
may rarely produce any classical haemolytic transfusion reaction but may be responsible for
secondary or anamnestic response during subsequent transfusion which is more dangerous.
(c) Antibodies to other blood group antigen apart from A, B, 0 viz. produced during pregnancy are
usually high titred, and since they remain attached to red cell membrane and does not occur in
soluble form, the reduction of antibody through absorption and neutralisation cannot take place
effectively. Such donor antibody may not produce the book picture of haemolytic transfusion
reaction but may cause decreased cell survival. The transfusion thus becomescounter productive.

[[p.29]]
Processing of Blood
179
(d) Incubator effect: In multiple transfusion cases, different donor's blood are transfused frequently.
And during such transfusions a donor blood may be transfused which is compatible with recipient
serum. But during subsequent transfusion, a donor blood may be transfused that may contain an
antigen incompatible to antibody present in the first donor leads to an antigen antibody reaction
and destruction of red cells of second transfusion. This is called incubator effect. .The recipient
here experiences the reaction not for own fault but as a media that contains the reacting element
namely antibody and antigen from different donors.
Few examples described thus emphasises the need for simultaneous minor cross matching before
issuing a blood as .a compatible unit,
Autocontrol is also run along with each cross matching test, separately for recipient and donor to
detect any auto-agglutinin present or not.
Aims and objects of cross matching
(i) To ensure that patient/recipient is supplied with a compatible unit of antigen negative blood.
(ii) To prevent haemolytic transfusion reaction overt or covert.
(iii) To detect immunogenic auto antigen and auto antibody, and blood to be issued has to be processed
accordingly. All these are done with sole aim for safe transfusion of blood.
Cross matching is thus a mandatory procedure and in no case uncrossmatched blood to be issued
even if there is a written request, and how to deal with such emergency situation, will be discussed later.
The following para contains the normal steps and procedure for cross matching.
Procedure
Take four tubes and add two drops of recipients serum to all the tubes + two drops 2%.to 5% of donor
cell suspension in saline. Number all the tubes as I, II, III, IV. Remove tube No.1, put it at 4°C to 12°C
to detect cold agglutinin if any; use sedimentation method.
Room temperature phase
Take tube II, III and IV. To tube III, 22%. Bovine serum Albumin is added in equal volume and similarly
to tube IV, add papain cystine solution in equal volume that is two drops of the papain cystin solution,
Mix and incubate all the tubes at room temperature (20°C to 25°C) for 45 minutes to 60 minutes
(sedimentation method) and for 5 to 10 minutes (spin method). In spin method centrifuge at 1000 r.p.m,
for 1 min. The spin can also be done after sedimentation but it is optional. Read the result and observe
for both haemolysis and agglutination, if non-reactive proceed to next phase.
Warm phase or thermo-phase
If no agglutination or haemolyses is observed then take all the three tubes and incubate them in a water
bath gradually raising the temperature to 37°C (body temperature) and incubate for twenty minutes. All
warm reacting .antibodies indifferent media is thus detected in this stage. In saline some examples of
anti-Lu" is detected in warm phase and rest are mostly detected in RT phase. Read result and observe for
both agglutination and haemolysis. If no reaction is observed, discard tube II and proceed to antiglobulin
phase with tube III and IV.

[[p.30]]
180
Blood Banking and Transfusion
3
4
Incubate at 4° to 12
QC
~~~~.~~~~-~~~~~~~~.~~~~
•• foreold agglutinin
(Sedimentation
method)
A.S. -2 drops
R.S. -2 drops
+
+
Incubate at Room
D.C.S. -2 drops
D.C.S. -2 drops
temperature Sedimentation
+
P.C. -2 drops
BSA -2 dro s (220/0)
Method 45 to 6~
min spin
p
method -5-10 min
____-'l__~
Incubate at 37°C for 20 mins
----------~---i...
1. Incubate at 37°C for
another 10 mins
2. Centrifuge
3. Wash (3 to times) and
decant
4. Add AHG 1 to 2 drops'
5. Look for reaction
p.e.
Papain Cystein
R.S.
Recipients serum, D.C.S. -Donor' Cell suspension 2 to 50/0
8.S.A. Bovine Serum albumin, AHG-Antihuman Globulin
Anti-
[
globulin
Phase
Fig. 8.9. Cross matching (major) different stages.
Antiglobulin phase
The tubes III and IV respectively contains BSA and enzyme; since both tubes are already incubated for
twenty minutes at 37°C, another ten minutes of incubation done at 37°C. This is followed by centrifugation
of both tubes at 1000 f.p.m. for one minute. Observe for agglutination and haemolysis and abscence
indicates presence of some undetected incomplete antibodies. Thorough washing of contents of both
tube with physiological normal saline. Decant supernatant after each wash as completely as possible.
Repeat decant and wash cycle for 3 or 4 times. Add one to two drops of AHG to contents of both tubes
after the last wash. Read the result immediately, if no reaction, wait for five minutes at RT and again read
for result. If no reaction test the validity by adding IgG coated red cell.
If test is valid and no reaction observed after 5 minutes at RT, the blood samples are compatible.
Similar procedure is observed for minor crossmatch and if both procedure and autocontrol does not
reveal any agglutination or haemolysis blood is considered safe for transfusion.
Compatibility test by gel technology
Recent advancement in gel technology can also be applied for doing the compatibility test.

[[p.31]]
Processing of Blood
181
A card with six microtubules fixed and each of these containing polyspecific and monoclonal anti-
C3d within gel matrix are supplied by manufacturer with following additional reagents :
(a) Diluent: A modified LISS for red cell suspension.
(b) Test cell reagents.
Procedure requires following essentials to be observed :
(i) Identify the card with patients or donors name and registration number.
(ii) Cards damaged, seals broken, dried cards or airbubbles inside tubules avoided.
(iii) Dispenser, pipettor, tips, suspension tubes, incubators, card centrifuge.
(iv) Donor red cell suspension prepared by putting 10 ul of packed red cell in 1 ml of diluent
containing modified LISS, then mix gently, and 0.8% of red cell suspension is ready for use.
(v) Plasma or serum of the recipient.
(vi) All material required are allowed to attain room temperature before use.
Procedure for compatibility test is described below :
A. Identify the microtubules with patients name and registration number.
B. Use the required number of tubules by removing aluminium foil.
c. 50 J..lI of donor's red cell suspension in appropriate tubule.
D. 50 JlI of patient's own red cell suspension.
E. 25 fl.l patient's plasma or serum in all microtubules.
F. Incubate at 37°C for 15 minutes.
G. Centrifuge for ten minutes in the card c~ntrifuge.
H. Read and record result.
Negative reaction that includes autocontrol also indicates compatibility.
Positive reaction indicates incompatibility. Further test needs to be performed for detecting antibody
in recipient's serum or plasma.
8.5 SPECIAL TYPE OF eROS: MATCHING
A. Cross matching ifdone normally takes minimum two hours to give compatibility result and that
too without antibody screening and identification etc. But certain emergent conditions has to be
issued urgently. In such cases LISS solution is used because it reduces the incubation period.
ABO and Rh grouping of recipient is done first. From the stock obtain 2/3 donor units. Wash
the cells 2/3 times and after last wash, make cell suspension in Liss. Now take equal volume of
recipients serum and LISS suspended cell 2 to 5% and incubate for ten minutes at RT look for
results, if non-reactive proceed to antiglobulin phase look for the result and if no agglutination
or haemolysis is observed; units are compatible. oth at RT and AHG at 37°C.
Issue blood with remark "Emergency crossmatching" set up full crossmatching later, and if any
incompatibility detected,"then the concerned clinicianimmediately informed so that blood can
be discontinued.
In absence of LISS solution we can adopt the following procedure:
1. Take 2 drops of serum and 2 drops of group specific donor cell suspension 10%.

[[p.32]]
182
Blood Banking and Transfusion
2. Mix andincubate at 37°C for 15 minutes.
3. Spin the tube at 1000 rpm for one minute.
4. Dislodge the cell button by gentle shaking.
5. Put a little amount from the tube on a slide observe for results macroscopically and with
optical aid.
6. If non-reactive, then wash the test cells 3 to 4 times in saline.
7. Proceed to antiglobulin phase.
8. If no agglutination and haemolysis is observed after antiglobulin, then the unit is compatible.
Issue the unit with remark "Emergency crossmatching",
Follow the next steps as described earlier in LISS method.
B. Massive transfusion and cross matching of blood: If ten or more. than ten units are transfused
within a period of 24 hours, then it is very difficult to detect antibodies in serum even by
Indirect AHG test and in such cases a short cut procedure is adopted.
(a) 2 drops of recipients serum + one drop of 50/0 saline suspension of cells are taken.
(b) Cell and sera mixture is mixed thoroughly.
(c) Spin at 3400 rpm for 15 sees.
(d) Gently dislodge the cell button with gentle shaking.
(e) Put the content on slide and observe for agglutination and haemolysis macroscopically or
with optical aid.
(f) A non-reactive unit is deemed compatible with serum of the massively transfused patient.
This non-reactive unit of group specific human blood IS released for further transfusion.
C.
Cross matching of blood in °AIHA : Though best but not always practicable is to withheld the
transfusion as long as the condition ·of the patient permit and transfuse when it is absolutely
indicated.
In autoimmune disorder our aim is not to aggravate the .condition further, but to find a suitable,
compatible antigen negative blood is a herculean task and met with failure in majority of cases.
And in view of emergent condition of patient, we can't wait for long time doing all the detailed
investigation. So a mid-level solution ofthe problem can be obtained by issuing least incompatible
blood. Two procedures are as followed :
(a)
Individual in whom autoantibody detected in serum:
(i) Procedure :
(i) A set of ten tubes in a row horizontally.
(ii) Vertical row is put according to number of donors selected from group specific donors
say seven.
(iii) In the horizontal row select the second tube and from second to tenth tube put 8 drops
of saline in each tube.
(iv) In second tube only put eight drops of serum of the patient and do serial dilution up to
tenth tube horizontally.
(v) Now make vertical transfer of serially diluted solution in the tubes arranged vertically
for 7 donors in a row..See the arrangement as given in Table 8.8.

[[p.33]]
Processing of Blood
183
~
Table 8.8. Diluted patients serum
S + Saline 8 + 8
sL dilution
1 : 1
1 : 2
1 : 4
1: 8
1 : 16
1 : 32
1 : 64
I : 128
1 : 256
1 : 5]2
Donor 1
!
1 drop
J,
J;
t J,
J,
!
J,
J,
!
Donor2
J,
Donor 3
J,
Donor4
!
Donor 5
!
Donor6
J,
Donor7
!
1 drop
(vi) Vertical transfer of one drop in each of donor tube seven in number from second tube.
(viii) First tube also put 8 drops of serum and make vertical transfer of one drop to each
tube of first vertical row.
(viii) Now to all the tubes set horizontally for donor No.1 put one drop C?f 2 to 5% donor's
cell suspension
up~o
tube 10 and likewise for the respective donors viz. donor 2, 3
and 4.
(ix) Incubate at room temperature for 45 minutes to 60 minutes.
(x) Observe for agglutination and/or haemolysis.
(xi) Donor showing least reaction is the least incompatible blood and may be considered
for transfusion (Table 8.9).
Table 8.9.
1 : 1
1 : 2
1 : 4
1:8
1 : 16
1: 32
.l : 64
1 : 128
1 : 256
1 : 512
Donor I
+++
++
++
+
W+
W
-
-
-
-
Donor2
W+
+
W
-
W+
-
-
-
-
-
Donor3
++++
+++
+++
++
+
w+
w
-
-
-
Donor·No. 2 is showing least reaction and may be considered for transfusion since this is
the least incompatible unit, while issuing the blood remark should be labelled "Least
incompatible blood to stop immediately on suspicion ofslightest reaction". Some serologist
also suggest that the reaction shown by least incompatible should be less than reaction
shown by patient's serum against his own red cell. And such blood unit is more safer for
transfusion.
(ii) If antibody remained attached to red cell membrane only in such cases antibody has to be
eluted by heat eluation, either eluation etc. Once the antibody eluate is made, then compatible
blood unit against this antibody eluate has to be searched for. The unit which is suitable
has to be transfused. For elution refer to Chapter 14. DAT positive AIHA cases usually

[[p.34]]
184
Blood Banking and Transfusion
show abscence of antibody in serum and in such type of cases antibody eluate are tested.
Both IAT and DAT are important in cases of AIHA. In comparison, if IATis stronger, than
the reaction exhibited by DAT, it signifies the most likely presence of aIle antibody which'
may be confirmed by testing the serum with panel of cells.
D. New born infants and crossmatching : The new born infants do not have isoagglutinin of their
own, but possess the antibody transferred from the mother during foetus stage in utero. So in
such cases, it is better to transfuse a blood which is compatible to mother. Refer to choice of
blood groups in the previous section. If mother is 'A' and child is 'B', then choice is '0'. Serum
grouping is not advocated in ABO typing of infants. So for infants under 4 months, ABO and D
typing has to be done and maternal serum tested for agglutinin and group specific blood
crossmatched with maternal serum. The compatible unit is then transfused.
E. Compatibility for intrauterine transfusion : Here also blood is tested for compatibility by cross
matching with mother's serum. Usual choice is orr blood except in those cases where'D' positive
mother have developed anti-D mixtures. Kell being next in order of immunogenicity so blood
should be preferably kell negative too.A chance leakage has to be taken into count and it is thus
advocated that in subsequent transfusion mother's serum to betested against a panel of cells and
antigen negative unit to be transfused. Blood meant for intrauterine transfusion should be
rigorously tested and blood has to be free from significant transfusion transmitted disease
(Syphillis, HIV, Hev, CMV, HbSAg, Toxoplasma, Malaria), not more than 24 hours old and
preferably irradiated saline washed packed red cell.
F. Cross matching in leukaemia: In leukaemia, the antigen A is very weakly reactive and few
cases of B also weakly reactive. As such special attention has to begiven during ABO typing of
recipient who may happen to be leukaemic, correct ABO typing will allow to the technologist to
pick up the group specific blood and do the crossmatching properly. Once the crossmatching is
over,. compatible unit can be issued.
G. Cross matching in cases with acquir~d
'B': It is usually associated with gram negative scepticimia,
malignancy of colon and rectum and in some cases of intestinal obstruction. It is very difficult
to identify acquired 'B' antigen by routine ABO typing, but the reactions exhibited by cell (B
acquired) and anti-B sera mixture are never stronger than with actual 'B' and B sera. This
discrepancy may give rise to suspicion or clue to presence of acquired 'B'. In such cases when
'B' cells are acetylated, they no longer react with anti-B', And since most of the cases acquired,
'B' is associated with AI' they show reactivity with AI lectin or Dolichos biflorus extract. Also,
they react negatively with recipient's own anti-'B'. In such cases wrong ABO typing may give
rise to crossmatching problem and a wrong unit of blood may be issued. So basically, it is the
proper ABO typing and Rh of the recipients is the preliminary important and essential step for
crossmatching and any incorrect interpretation mayinduce disastrous consequences.
H. Cold antibody : In case where cold antibody is detected, crossmatch is performed at 1DOC and
37°C and a unit compatible at both is issued. In case of non-availability surgeon advised to
avoid hypothermia.
Specimen collection
For cross matching purpose at least 2 ml to 5 ml of blood has to .be sent along with 'requisition form. The
specimen collected is from veins in a plain vial container which is well stoppered. The specimen should

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Processing of Blood
185
be labelled properly with Name, Age, Ward No. and bed number withstatistical number. Haemolyse and
contaminated sample is not accepted. If the requisition form mentions the age below 4 months ask for
both mother and babies blood sample separately and also confirm whether the blood of baby is cord
blood or not.
For AIHA cases samples to be collected in EDTA vial and not in plain vial:
Drawback in crossmatching
(a) Rh typing errors cannot be detected by crossmatching.
(b) It can detect only antibodies specific for red cell antigen of the donor. A donor unit without
appropriate antigen or with very weak antigen may thus fail to detect the corresponding antibody
even if it is present in serum of the patient.
(c) Present method of compatibility testing cannot detect any antibodies to either leucocyte antigen
or platelet antigen.
8.6 ISSUE OF BLOOD IN EMERGENCY LIFE SAVING SITUATION
This is influenced by three conditions mainly :
(i) Availability of particular group of blood
(ii) Time available at hand
(iii) Availability of blood sample
In such situations when partially or uncrossmatched blood has to be issued, a written request from
the physician is a must where he requests for release ofsuch blood and clearly takes responsibility of any
eventuality that may arise out of such transfusion including transfusion transmitted disease.
(a) Firstly the ABO and Rh grouping of patient is quickly done, if required slide technique may be
employed (it is quicker than tube technique). See that recipient sample is properly labelled and
tallies with. requisition form. If time permits spin tube technique may be employed.
Selected blood from the stock, do a saline crossmatching at R.T. again over a slide (both major
and minor) proceed for antiglobulin test with liss suspended cell, conduct the test at 37~C.
If
compatible release blood with remark on label "Emergency cross match".
Start full cross match immediately. after issue of blood and if incompatibility detected inform
physician immediately for discontinuation of transfusion.
(b) If less time is available, antiglobulin phase may be omitted and blood released with remark
"Partially crossmatched blood emergency release".. Follow the procedure same as above after
the blood is issued.
(c) If no time is available, release group specific blood after ABO and Rh grouping without any
crossmatch with remark "Uncross matched blood - Emergency release". Follow procedure
described as above after Issue of the blood.
(d) If no blood sample of patient is available viz. in certain burn .cases or infants, where blood is
urgently required, release '0' Rh negative packed cell and not whole blood. A female infant or a
female ·of child bearing age of course is an exception and in these recipients should not be
transfused with '0' Rh negative and instead '0' Rh +ve packed red cell may be transfused.
Remarks on the label "Alternate choice uncrossmatched, untitred blood". Follow procedure as
before and in'the meantime wait for samples to arrive.

[[p.36]]
186
Blood Banking and Transfusion
(e) Inaccidental cases SFH (Stromafree haemoglobin)if available is the best choice also, it is the
alternatechoice in situation as described above in (d) till the time we establish properABO and
Rh grouping.SFH is not a substituteof blood but an emergencyarrangementto carryoxygen to
vital parts of the body. So ultimately blood has to betransfused.
(f) In case of non-availabilityof a particular group alternate choice blood may be issued (Ref. the
section choice of blood) with remarks "Alternatechoice-uncrossmatchedlpartial crossmatched,
Emergencyrelease". Follow procedure as described after the issue of blood.
In ~ach
and every case of such emergencyrelease physician must be contactedimmediately or later
depending on the outcome of full set crossmatchingand serological tests and inform him of the actual
status of bloodunit released for transfusion.
'8~7
:ISSUEOF BLOOD TONE.ONATE .AND INFANTS
It has already been discussedthat the neonates and infants below 4 months does not have their antibody
developed by that time and the antibody present in circulation is the antibody transfused from
mother during foetus stage in utero as the IgG can cross placental barrier, this IgG is destroyed very
slowly in circulation of a new born and infant. And both humoral and immune system are very poorly
developed.
Infants below4 monthsABO typingis not advocatedby reverse method(serumgrouping)andABO
typing is done by forward (cell grouping) method. After establishment of ABO and D, type material
serum is used for cross match and issue of group specific blood. Alternate choice of blood has already
been dealt with in section concerned in this chapter.
Neonates and infants should always be. issued with fully investigated, safe and fresh blood. Fresh
blood providesenough2-3 D.P.G essentialfor oxygenpreservationduring storageand helps in boosting
up of oxygen content in the infants.
The blood should be fully investigated in infants because of immature immune system, they are
more disease prone and susceptibleto infection.In infants to avoid transfusionTransmitted disease, we
should take special precaution for C.M.V. apart from other investigation, because it is one of lTD that
can be transmittedthroughblood and bloodproducts and has special relevancein infants withimpaired
immunity. Though less common in India, but it is better not to overlook and a screening of blood and
components for transfusion, particularlyin infants is always advisable,or a CMV negativeblood donor
panel may be maintained.
For CMV screening immunoassays are available, also efT is available, but for mass and quick
screening, these methodsare not very useful.Presentlyavailablelatex iagglutinationtest are suitablefor
screening of blood because of simplicity and quickness of the procedure.
In .case of infants, a blood issued should be as fresh as possible in any case never more than 7
(seven).days and fully investigated, compatible, warm blood. If alternate choice blood is released in
emergency,it should. bear the same characteristicsas described above. And in case uncrossmatched or
partiallycrossmatched, itshouldbementionedoverthelabel.In infant,'itisadvisable toissuecrossmatched
blood as far as practicablebecausechildrensand infantsdue to immaturityin differentsystemarealways
susceptibleand maynot be able to bearthe bruntor the impact.An utmostcautionis therefore, advocated
for issue of blood in infants.
.

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187
Recommended for further reading
1. A Handbook ofMedical LaboratoryTechnology, Dr; V.H. Talib, 1990, Sponsored by WHO.
2. BloodTransfusion inClinicalMedicine, P.L. Mollison, Seventhedition, 1983, BlackwellScientific Publication.
3. Introduction to Transfusion Medicine, Barucha and Chauhan, First edition, 1990,D.K. Publishers.
4. Procedure in Immunohaematology and Blood Banking, H.M. Bhatia, 1977, 11B, ICMR, Seth,as. Medical
College, Mumbai.
5. Techniques in BloodBanking, H.M. Bhatia, 1985, Technical ReportSeries3, lIB, lCMR, Seth,as. Medical
College, Mumbai.
6. Transfusion MedicineTechnical Manual, SaranR.K. andMakroo, R.N.,secondedition, 1991, DGHS, Nirman
Bhawan, New Delhi in collaboration with WHO.
.
7. Textbook of Preventive and Social Medicine, Park and Park, 10th edition, 1985, Banarasidas Bhanot,
Jabalpur.
Journal.s" and ArtiCles·
1. Bloodlines 1: OrthoDiagnosticSystem: Evaluation ofBloodDonorScreening Test, CelsoBianco, pp. 3 to
10, Selectionoftest kits/or use in clinicalserologylaboratory - Joseph, M. Campos, pp. 11 to 16.
2. Care(Centre for AIDS Research and Control), Callingpublishedby ICMR, New Delhi, Vol. No.4, Oct. to
Dec., 1991, Acute HIV illness, MalviyaA and KumarAshok; pp. 33 to 35.
3. Care Calling, ICMR,New Delhi, Vol. 5, No.1, January-March, ·1992, Strategies for Ensuringlife Blood
and Blood Products, Dr. ZS. Bharucha, pp. 40 to 42.
4. CareCalling, ICMR, New Delhi, Vol. 6, No.1, January-March. 1993, Kalidoscope, Dr. R.R. Gangakhedkan
pp.41.
5. lmmunohaematology Bulletin, VoL 23,No.2, March-April 1992,ICMR, KEMHospital, Mumbai. Molecular
Approachesto the DetectionofAgents in PersistentViral Infections, lYas, GN., pp. 1 to 7.
6. Transfusion Bulletin, Vol. 5, No.3, Oct.-Dec. 1992, Editor ISBTI,.Pune. Cost Effective Strategies for
Serological Testing ofAIDS,pp. 6 to 8.