Common Blood Bank Reagent Solution Buffer and Special Methods

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Common Blood Bank
Reagent, Solution Buffer
and Special Methods
14.1 SOLur'Ot.Js,·
(A) Standar'd solution is one in which exact concentration of the solute is known
Buffer solution are those which are capable of maintaining their, hydrogen .ion concentrations or pH
under all circumstances and situation.
Molar solution is that solution which contains one mol of a solute in one litre of water. One mol of
solute is equal to the gram molecular weight i.e., molecular weight expressed in grams.
Normal solution is a solution which contains one gram equivalent weight of the solute in one litre
where equivalent weight is :
Molecular weight
Valency
MonovaJentatoms (Sodium, Hydrogenetc.) has molecularweight = equivalentweight(an equivalent
weight of a substance is that weight which will combine or replace one gm of hydrogen).
(B) Weter
All these solutions are preparedin water unless stated otherwise and waterfor laboratory use are prepared
by distillation or deionisation.
According to college of American PathologistGlamlin, 1978 quoted by Henry and Macpherson in
the clinical diagnosis and management by laboratory method, Ioc cit) the reagent water is graded as
below:
Type I reagent water
Procedures which requiremaximumwaterpurityviz. preparationof standardsolution,ultramicrochemical
analyses, measurements at nanogram or subnanogram concentration, tissue and/or cell culture.
Type II reagent wafer
For most laboratory testing in chemistry; hematology, microbiology and immunology etc.
381-

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382
Blood Banking and Transfusion
Type III reagent water
For most qualitative testing viz. urine analysis, parasitological and histological works, washing of
instruments, glasswares, general purpose use in laboratory.
Specific resistance is the resistance in ohms of a column of solution 1 em long and 1 cm2 in cross
sectionalarea. The gradingof reagent water as above is done on the basis of specific resistanceat 25°C.
(C) 'Methods of expressing concentration
Concentration is defined as weight per unit volume.
It is expressed as (1) Percent, (2) Molar, (3) Normal.
1.
Per cent : Gerraway.has expressed per cent in three ways:
(a) Weight unit weight (w/w) : A 10% solution contains 10 gm of solute in 100 gm of solution
or (90 gm of solvent).
(b) Weight unit volume (w/v). : A 10% solution contains 10 gm of solute dissolved in final
volume of 100 ml of solution.
(c) Volume unit volume (v/v) : A 10% solution contains 10 ml of concentrate (solute) per 100
ml of the solvent.
2.: Molarity : 1 mole of a substance = molecular weight of compound expressed in grams. If 1
mole of a substance is dissolved in a final solution of one. litre,the solution is called one molar
or simply molar.
For example 1 M solution of Sodium chloride or common salt (NaCl).
Molecular weight of NaCl is ~3 + 35.5 1 M solution of NaCIwill contain 58.5 gm of NaCIin 1
litre of solution. Molarity of solution is expressed as :
I
x concentrationin thesolutionin gms/ltr.
Molecularweight of substance
'
3. Normal: Normality of a solution is expressed as :
Normality=(Molarity) x (Valency)
Example
Normality of a 1M solution of FeS04 (Ferrous sulphate), valency of iron in the compound is 2.
Normality=M x 2 =2.
In case of a normal solution, concentrationis expressedin terms of their combiningweights.Agram
equivalent contains same number of chemically active particles. 1 meq(milliequivalent) is 1/1000of an
equivalent.
Mg
Meq= gm. eq. wt.
To obtain meqllitre
(Mg/IOOml)xIO
meqlltr.= Molecular weight

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·Common
BI~oct
Bank Reagent, Solution Buffer and Special Methods
383
(Mg/IOOml)xIOx2
=
Molecular weight
Or conversly
(meq/L) x (Molecularwt.)
Mg/IOO ml =
20
.(D) 'Speciflc gravity
Knowledgeabout specificgravityis usefulin preparingnormalsolutionof a liquid.
(s.p. gravityof solution)x (Volume) x (% of materialin solution) =Wt. of solutein solution,
Topreparea normalsolutionof a liquidof known specificgravityand per cent cone, in solution:
(Eq. wt.) x 100
ml of liquidrequiredper litre= (sp. gr.) x cone.
(mol. wt.) x 100
=--------~--
(sp.gr.) x (conc.) x (valency)
In blood bankingthe commonly used solutionis Normalsalineor Physiologic saline.This is a type
of Physiologic solutionand a Physiologic solutionhavenearlysimilarcomposition as that-of bodyfluid
The physiologic saline (Na:CI 0.85 gm to 0.9 gm per 100 00) also known as normal saline equals the
osmotic pressure as that of contents of RBC in human blood. The pH of this isotonic saline varies
between6.7 to 7.4, pH can beadjustedby using phosphate buffer.
14.2 'BUFFERS
There aredifferent type of buffersolutionavailableand those whichare commonly used ill blood bam
are described. Buffersalreadydefinedearlier usuallyconsistsof salts of a weak acid or salt of a weal
base.These buffershave a rangeof pH varyingbetween5 to 8. Before discussing the buffers, we mus
have a clear conceptionof osmolarity, osmolality. Osmolarity : A solutioncontaining 1 osmol (gmw,
number of particles of ions into which the substance dissociates into solution) of solute per litre 01
solution has 1 osmolar concentration.
Osmolality : 1.osmol of soluteper kg of solventhas a concentration of osmolal.
pH : It gives th~
hydrogenion concentration in moles, litre.
pKa : log Ka (acid dissociation constant)
In buffersolution,the pH is relatedto the undissociated acidconcentration andto thecorresponding
anion:
pH =pK + Jog
(A)
a
(HA)
where. (A) is the molar concentration. of anion. (HA) is the molarconcentration of acid.

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384
Blood Banking and Transfusion
ISO..Osmotic phosphate buffer
For an iso-osmotic buffer, the recommended osmolality of stock solution are 25.05 gIlitre for N~PO
4'
2H20 (acid solution and 17.92 g/litre for N~HP04
(alkaline solution).
(a) NaH2P04, 2H20 (150 mmol/litre) 23.4 gllitre
(b)
N~HP04
(150 mmol/litre) 21.3 g/litre.
The working solution of desired value (pH is made by mixing solution of (a) and (b) as mentioned
in few of the examples as given in Table 14.1.
Table 14.1. pH values
Desired pH
Acid stock solution (a) in ml
Alkaline stock solution ())) in ml
6.0
83 ml
17 ml
7.0
32ml
68 ml
7.2
24011
76-ml
The maximum buffering capacity is at the pK value of weak acid or a weak base. For example, if we
desire to have apH value of 5.8, more acid will be required to bring down the pH from 6 to 5.8. It has
been observed that efficient buffering covers a pH range of approximately 1 unit on either side of pK
value ofthe acid or base.
Sorensen's phosphate buffer
This is a mixture of working buffer solution containing fifteenth molar monobasic potassium phosphate
and fifteenth molar dibasic sodium phosphate solution.
(a) Monobasic potassium phosphate : 1/15M KH2P04 having a molecular weight of 9.1 gm is
dissolved in redistilled water to make up to 1 litre. .
(b) Dibasic sodium phosphate: 1/15M Na2HP0 4 (disodium hydrogen orthophosphate).
1/15M Na2HPO4.2H20 : Disodium hydrogen orthophosphate containing two moles of water is
u.ed for preparing stock solution. (b) 11.857 or 11.9 gm disodium phosphate duohydrate
(Na2HP0 4.2H20 ) is dissolved in redistilled water to makeup to 1 litre.
Prepare working buffer solution of desired pH by mixing stock·solutions a and b as mentioned
below in the few examples (Table 14.2).
Table 14.2.
Desired pH
5.2
6.8
7..1
8.0
Stock solution a (in ml)
9.75 ml
5.0ml
3.0 ml
0.5 ml
Stock solution b (in ml)
0.25 ml
5.0ml
7.0ml
9.5 ml

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Oornmon Blood Bank Reagent, Solution Buffer and Special Methods
38(i
While preparing Sorensen's buffer mixture (not iso-osmotic) ensure that stock solutions are crystal
clear, test for chloride and sulphate should be negative.
Phosphate buffered saline
This is prepared by mixing equal volumes of normal saline (sodium chloride solution 0.9%) and iso-
osmotic phosphate buffer pH 7.0. The phosphate buffered saline being iso-osmotic can be a better
suspending medium for RBC and in this suspending medium RBC can be kept as long as 24 hours.
Tris He, buffer
This buffer is prepared by mixing the desired weight ofTris (hydroxymethyl) aminomethane buffer with
redistilled water and then Hel is added, to (Table 14.3) adjust pH up to the desired level. So to make a
Tris Hel buffer solution take the required amount Tris (hydroxymethyl) aminomethane into a volumetric
flask ofdesired capacity and then add 50 ml ofdistilled water to dissolve the content. Now add appropriate
volume of I moJ/HCI, mix it thoroughly and make up the volume with water up to the desired level (100
mI, 15000,300 ml or 100 mmolll, 150 mmolll, 300 mmolll). This buffer is recommended to be used for
a pH range between 7.0 and 9.0 and it has an added advantage of being one of stable buffer.
'Table 14.3. pH Tris-HCI.buffer
Desired pH
Volume of HCI
7.20
44.5 ml
7.0
33.5 ml
8.0
28.0 ml
8.50
15.0ml
9.00
-5.000
14.3 COMMONLY USED REAGENT IN :BLOOD BANK·
A. Bovine serum albumin (BSA)
Prepared from bovine blood and is used mostly in blood group serology as a high' protein medium that
enhances the agglutination reaction probably acts by reducing the zeta potential or repulsionbetween
RBC. Commercially available as 22% (commonly used) and 30%.
The other variety commercially available is of 1 to 7% concentration used as reagent stabiliser for
other reagents.
BSA is prepared by removing the globulin fraction. This can-be done by cold ethanol fractionation.
Use of BSA
It can be used alone or in combination AHG respectively for detection of IgG and antibody detection
test-I - i (IgM) antibody are potentiated by BSA. As stabiliser in other reagent.
Specification
Free ofcontamination, protein concentration optimum (between 20 to 30%), pH and conductivity optimum,
Quality control check is done with anti-D.

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388
Blood Banking and Transfusion
Technique
One stage method :
(i) Add two to three drops of serum in a labelled test tube.·
(ii) Add one to two drops of 2 to 3% RBC suspension in saline.
(iii) Add 22% BSA, two drops in the same labelled tube.
(iv) Mix thoroughly and then incubation is done at 37°C for fifteen to twenty minutes.
(v) Centrifuge for 2-3 minutes at 1000 rpm.
(vi) Resuspend the cell button by gentle tapping.
(vii) See for agglutination and/or haemolysis.
(viii) Negative or weak agglutination to be confirmed with optical aid/microscope.
Albumin layering teehnlque
(i) First two steps same as before.
(ii) Mix and incubate for 30 minutes at 37°C.
(iii) Centrifuge for two to three minutes at 1000 rpm.
(iv) Add two drops of 22% BSA in such a manner that it trickles down by the side of the test tube
and does not disturb the cell button. The albumin forms a layer on the top of cell which .is
allowed to be settled at the top. Mixing is not done.
(v) Incubate for 15 to 20 minutes at 37°C.
(vi) Resuspend the cell button by gentle tapping.
(vii) Negative and weak results are confirmed by optical aid and/or microscope.
B•.Enzymes
Enzymesare proteins having molecular weight ranging between 24,000 to 30,000 daltons. They potentia.es
the agglutination through different ways :
(a) f\"odifying red cell membrane:
(i) By exposing the latent antigen (Rh, Jk etc.) by removing the sialic acid or sialoglycoprotein
from red cell surface.
(ii) Enhancing the antibody reactivity : By removing structures that interferes with access to
antibody.
(iii) Denaturation of antigen.
(iv) Change in shape and surface of RBC.
(b) Potentiating the stage of agglutination reaction viz. sensitisation and agglutination of sensitised
cells probably through exposure of latent antigen, reducing the zeta potential and increasing the
association constant, antigen clustering.
The enzymes commonly used for blood bank laboratory are proteolytic enzymes viz. papain,
trypsin, ficin and bromelin. 'Out of all these enzymes named, papainis preferred for its easy
availability and cheaper price, next in order of preference is Ficin.

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One stage technique
The one stage technique is less time consuming because it does not require pretreatment of red cells.
One stage method of cystein activated papain reported by Low in 1955and slightly modified by Hekkar
et aI., 1957, further modified by Bhatia, ]964 (Procedures in BloodBanking and Immunohaematology
loe cit.)
(a) Ten grams of papain powder is dissolved in PBS 2~O ml, the pH has to be 6.2.
(b) Allow to settle for 15 minutes, then shake vigorouslyto mix.
(c) Centrifuge, then filter through Whatman filter No.1, discard the deposit.
(d) 4.85 gm of cystein hydrochloride is dissolved in 25 ml of PBS (pH 6.2) mix well.
(e) Add this cystein solution to the filtered papain.
(f) Make up to 1000 ml by adding buffer.
(g) Incubate at 37°C for 1 to 1~ hour.
(h) Filter again through Whatman filter paper No.1.
(i) Incubate over night at 4°C.
G) Next day, adjust pH,to 6.2 with 1 m NaOH.'
(k) Store in deep freezer at 20°C in small aliquots. Bhatia in 1964 observed that addition of 1 ml of
3.8% sodium citrate in 10 rnl of cystein papain gives better result.
Preparation of phosphate b~r
(pH 6.2)
MIlS KH2P04 : 9.078 gms in 1000 ml of redistilled water.
(acid)
MJl~
N~HP04:
11.876 gms in 1000 ml of redistilled water.
(base)
To prepare 100 ml papain cystein, we require 80 ml (acid) and 20 ml (base) mixed well, will give
100 ml of PBS (pH 6.2). The amount of acid and base, thus required for 1000 ml PBS can thus be
calculated.

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388
Blood Banking and Transfusion
Test
(i) Take one volume of PC from the stock solution.
(ii) Take one volume of antiserum.
(iii) Take one volume of 2% saline suspension of red cell.
(iv) The above three are mixed strictly adhering to the order as follows:
(a) One volume anti-serum.
(b) One volume P.C. (this mixture should not be allowed to' settle more than 5 minutes).
(c) Layer 2% red cell suspension one volume through the side of test tube within 5 minutes of
mixing serum and P.C. The red cell sinks first through papain and then through anti-serum
during the incubation period of 30 minutes at 37°C or at RTfor 1 hour but not more than
90 minutes in any case.
(v) Results are observed after mild centrifugation at end of incubation.
(vi) Run positive and negative control with the test.
Indication
To detect anti-D antibody in particular, it can also detect anti-PI' anti-Lewis and anti-H etc., but the
sensitivity of the test may get much reduced, if serum, cell and papain are mixed together and the
procedure as described are not followed.
FICIN
Stock solution is prepared by adding 250 mg of dry powder to 25 ml of Hendry's buffer (iso-osmotic
phosphate buffer pH 7.4).
Buffer solution
NaH2 P04 2H20 : 2.34 gm/IOO ml
(acid)
NaZHP04 : 1.63 gm/IOO ml
(alkali)
Mix 19 ml of (acid) solution to 81 ml of (alkali) solution to make 100ml of iso-osmotic phosphate
buffer.
Ficin treatment of red cells
(a) Ficin 1 volume from stock solution is mixed with 39 volumes of iso-osmotic PB.
(b) Warm the 40 volumes (39 + 1) to. 37°C.
(c) To this warm mixture of (b) above add 1'volume of washed packed RBC.
(d) Incubate for 15 to 20 minutes at 37°C.
(e) After incubation, wash the RBC three to four times with normal saline at 37"C till it is required
foruse.
(f) Store the ficin treated RBC at 37°C till it is required. for use..

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Common Btood Bank Reagent, Solution Buffer and Special Methods
"389
Test
For test take equal volume of test serum and 2% of Ficin treated RBe and incubate at 37°C for one hour
and then read the result by observing for agglutination. It is mandatory that test serum be kept"at 37°C
for ten minutes before 2% ficin treated red cell suspension is added and then incubated for one hour at
37 CC.
Use
(i) Antibody detection and identification: 0.1% ficin solution in buffered saline at pH 7.4.
(ii). Auto absorption of cold agglutinin: "0.1% ficin is used..
Disadvantage
Ficin is irritant and contact with skin may cause sloughening so necessary protection may be taken when
preparing ficin solution.
ZZAP REAGENT
Composition
(a) 0.2 M dithiotheritol (DTI): 2.5 ml
(b) 1% papain : 0.5 ml
(c) 0.15 PBS (pH 7.4) : 2.0 ml
Mix thoroughly and adjust pH between 6.0 to 6.5 storage is done at 4°C. Maximum storage period
5 days.
Use
Detection of al1oantibody : It can be done by absorbing the autoantibody (warm), patient's own red cells
are treated with Z~AP
and employed for absorption of autoantibody. Autoantibody once absorbed, co-
existing alloantibody is set free for detection. 'ZZAP reagent is known for its destructive effect on kell,
puffy and S antigen.
AB serum (neutral)
The serum is collected from voluntary AB donor who does not possess either anti-A or anti-B. The
senlffi from such donors after collection are processed under all asceptic condition and then kept overnight
at 4°C. At the end of incubation observe the serum carefully, remove any clot, if there is any. Add
preservative (sodium azide), filter the serum in seitz filter and store frozened in deep freeze at -20°C.
Standardisation is done by checking with ABO groups in saline, enzyme, albumin and Coomb's
method. Tests are done at 4°C,-RT and 37°C. At least 200 samples are tested. A standard serum must not
show rouleaux formation.
Indication for uses
(a) Negative control
(b) Diluent

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390
Blood Banking and Transfusion
Low ionic strength solution (LISS)
The solution was described by Low and Messeter
0.3 M Sodium glycinate (pH 6.7)'
(a)
0.17 M Sodium chloride
(b)
0.15 M Phosphate buffer (ph 6.7)
(c)
In a litre of LISS solution the ingredients (a), -(b) and (c) are in proportion of 80 :' 18 : 2.
(i) Since sodium glycinate is not available in market, glycine is used in its place, 18 gm of glycine
is dissolved in 500 ml of redistilled water.
(ii) Sodium chloride 1.79 gm is dissolved in lOQ ml of water redistilled.
(iii) For both (i) and (ii) above, pH is adjusted to 6.7 by adding IN NaOH.
(iv) 0.15 M phosphate buffer (pH 6.7) is made.
Add 0.15 M NaH2 P04, '2H20 (23.4 gmllitre) to 25 ml of alkaline solution of 0.15 M Na2HP04
(21.3 gm/litre), drop by drop addition is continued and pH is intennittently checked. Addition
and mixing is stopped as soon as pH is 6.7.
(v) 0.15 M Phosphate buffer (pH 6.7) is now taken and 20 ml is added to the solution no. (i).
(vi) Now add solution no. (ii) full amount to solution no. (v).
(vii) Make up,to 1000 ml by adding redistilled water to solution no. (vi) with continuous mixing,
adjust pH to 6.7.
(viii) Sterilise by autoclaving after dispensing the stock solutions in 50 to 100 ml vials store at 4°C. A
low concentration of sodium azide (0.1 gmllitre) helps as preservative and keeps the stock
solution free of bacterial contamination.
Standardisation and quality control :
1. pH 6.7 (range 6.6 to 6.8). '
2. osmolarity of the solution ranges between, 270 to 320 mmollkg.
3. Conductivity: 3.4 to 3.7 mohmlcm at 23°C.
4. A titration is carried out weak anti-D solution and CcDee RBC in two sets, one for new liss
prepared and compare it with old liss placed in the second set, Results of both sets should be
comparable to each other.
Use
In cross matching : Antibody quantification, screening and identification, indirect Coomb's test.
Standard cells for screening
For screening ofunknown serum for theantibodies red cells are employed. To get standard red cells, two
donors are selected from a panel of donors. The two donors selected are supposed to possess as many as
red cell antigen between them.
Essential antigens required for the purpose of screening are D, C, E, c, e, M, N, S, S, PI' JKa, JKb,
Fya, Fyb, K, k and Kp8 , Lua•Cw are optional.
Weak:antigens like Duffy and Kidd may not react with single dose of antigen and to have dosage
effect requires homozygous red cells.

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20.5 gm
4.2gm
8.0gm
1000 ml
Common Blood Bank Reagent. Solution Buffer and Special Methods
391
The panel of cells thus made can be stored for 4 weeks in Alsevar's solution.
Modified Alsevar's solution consists of :
Dextrose (114 mmollL)
Sodium chloride (72 mmollL)
Trisodium citrate (dihydrate) (27 mmollL)
Water (redistilled)
pH adjusted to 6.1 by adding citric acid (0.5 g). Solution is then sterilised by autoclaving.
Recommended proportion 4 : 1 (Blood: Solution).
The cell panel can be preserved in the above solution. A simplified cell panel that serves as nearly all
purpose is described below. All the cells meant for panel purpose should be clear negative for Direct
Coomb's test and W~.
Recommended panel of cells of ten should be .'0' group as follows :
Donor genotype
1. R1R1
2. ~R2
3. R1R2
4.
IT
5.
IT
6. rr
7. r"r
8.
IT
9. other
10. Rh type to include one CW cell.
All these panel of cells contains 2 or 3 cell types that contain essential antigen described above, e.g.
2 to 3 MM cells',2 or 3 S+ cells (Ss, SS one each), 2 or 3 S-cells, 1 to 2 Lu (a+), 2 or 3 Fy'" (Fya+b+ one
and Fya + b - one).
Preparation of panel of cell
(a) According to panei of cells as described collect ten CPD samples of group '0'.
(b) Make 2% cell suspension of each sample.
(c) Take one volume of antisera in different tubes, e.g. anti-S, anti-Jk", anti-JKb, then anti-C, D, E,
e, c etc. as per essential antigen list mentioned earlier.
(d) To the antisera containing tubes add 2 volumes of 2% cell suspension to each.
(e) Incubate at 37°C for 1 hour see for agglutination.
(f) Wash thrice with normal saline, decant completely.
(g) Now add two volume of AHG to the cell button and mix slowly, allow to settle for five minutes
at RT, see for agglutination.
(h) If no agglutination mild centrifugation at low speed - look for agglutination again.

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392
Blood Banking and Transfusion
Agglutination indicates presence of-particular antigen against which antisera was taken in that tube.
Once antigen is known, the cell can be preserved as Alsever's solution for 4 weeks and can be used for
detection of specific antibody.
·Pooled .cells
(a) Take fresh CPD sample of known A, Band 0 cells, three each.
(b) 0.5 ml of cells from each sample groupwise.
(c) Wash three to four times with normal saline.
(d) 5% cell suspension of each sample.
(e) Do the pooling of cells A with A, B with B and 0 with O.
(0 Pooled cells are now tested anti-A sera, anti-B sera and anti-AB sera respectively for A pooled
cells, B pooled cells and 0 pooled cells.
(g) If agglutination shown with respective antisera then use the.pooled cells.
Caution
Pooled cells at RT can be.kept for six hours only so if not in use it can be stored at 4°C. Every morning
prepare fresh pooled cell.
Lectins
Lectins are saline extracts of seeds and can agglutinate specific antigen viz. Dolichos bifloros extract
can agglutinate AI' ulex europeus reacts with 'H' antigen.
(a) Anti-AI can be prepared from Dolichos biflorus. Dolichos biflorus seeds are soaked overnight
in 25 ml of normal saline. Ground the seeds meticulously in a grinder, the extract is now
transferred to a separate container. Add 25 ml of normal saline again to the seeds while pouring
slowly shake the container, reground the content, repeat the procedure again. The extract of
which are collected separately each time in a separate container is pooled in one container.
Test checking done with A] and ~ cells and firmness to agglutination as well the grading is the
determining criteria for the dilution to be used for the purpose in future. Dilution is done with
normal saline pH adjusted to 7.0 preserved at 20°C.Recommended titre for anti-AI lectin is 1 :
.16 with AI cells, firmness to agglutination is +4.
(b) Ulex Europeaus seed extracts are used as anti-H for detecting 'H' antigen. The seeds are first
grounded by means of a grinder and to each one gram add 10 ml of saline. The whole mixture
is now agitated continuously for one hour in a mechanical device. After one hour centrifuge at
3000 rpm for 8 to 10 minutes, supernatant is collected and sediment discarded. Ifthe supernatant
is cloudy recentrifuge is advised. After recentrifuge, seitz filter. Collect the filtered fluid in a
separate container, procedure is repeated with fresh seeds and collection 2 or 3 are pooled.
Pooled extract are tested and checked for activity particularly with Oh cells which must give negative
reaction. It can also be checked with secretor and non-secretor saliva.
A. CQPper sulphate solution
Screening of donors haemoglobin particularly in outdoor donation.
Preparation of stock solution 170 to g of crystalline CuSO4' 5H20 is dissolved in l1itre ofredistilled
water working solution can be prepared by adding 52 ml of stock solution to 48 ml of redistilled water.

[[p.13]]
C~m01Ort;
ardod 'Bal'lk'Re~gent
..,Solution, BU'ffer and Special Methods
393
The specific gravity of working solution should be 1.053. This specific gravity is equal to 12.5 g/dl of
haemoglobin. Thus, it is an indirect measure of haemoglobin. After making the working solution, check
the specific gravity which must be within ± 0.003 of required value.
Specific gravity can be calculated as follows:
weight of copper sulphate solution
S
f C 804
1 .
----~~-~----x
p. gr. 0
u
so ution
weight of water
Technique
(a) Make a skin puncture (finger prick) under proper asepsis, let the blood flow freely from puncture
site which is held approximately 1 em above the tube containing CuSO4 solution.
(b) CuSO4 solution, 30 ml of specific gravity 1.053 is kept in a clean test tube. 30 ml of solution can
be used for 25 tests only. After which fresh solutions are to be taken.
(c) One drop of blood is collected from the fingerprics. Weight for twelve seconds, if the drops
floats on the surface or rises from bottom, interpretation is that the drop is lighter than CuSO4
solution' and the haemoglobin content of blood is less than 12.5 gm/dl. But, if it sinks within 12
to 15 seconds, then the 'haemoglobin is equal to or higher than 12.5 gmldl of blood,
B.. Cyanmethaemoglobin method
This is also known as HICN method is based on the principle that solution'containing potassium cyanide
or potassium ferricyanide, when treated with blood, the haemoglobin and allied compositions are converted
into cyanmethaemoglobin or HeN. Absorbance of the solution is measured at 540 nm ± 10 nm with a
light green filter.
TechnIque
(a) Capillary or venous blood sample obtained respectively by skin puncture or by venepuncture
(collected in EDTA) can be used. Fresh blood to be used immediately.
(b) 20 J.lI of blood is added to 4 ml or 5 ml of diluent reagent modified drabkins reagent containing
potassium Ferricyanide, potassium cyanide and other ingredients,
(c) Allow to settle for 5 to 10 minutes or as per manufacturers instruction. HICN is fanned by this
time and this has to be compared with the standard.
(d) Filter recommended for 540 nm usuallylight green filter is selected.
(e) Photoelectric colorimeter is set at zero against blank ortransmission at 100%.
(0 Pour the diluted Haemoglobin standard into 'cuvette and record the optical density or per cent
transmission (cyanmeth-HB standard).
(g) Pour the undiluted haemoglobin standard into cuvette and record the 00 or per cent transmission.
(h) Plot a graph Hb in mg/dl on horizontal axis and 00 on vertical axis.
(i) The value of cyanmeth Hb in mg/dl is given on the vial, half of this value will give the
concentration of cyanmeth-Hb in tube containing 1 : 1 dilution of Hb standard.
(j) The equivalent g/dl Hb concentration in the diluted and undiluted standard to be calculated as
per instruction in the leaflet.
(k) Draw a line connecting the two plotted points on the graph which should pass through origin.

[[p.14]]
394
Blood Banking and Transfusion
Alternatively
Directly calculated instead of putting into graph :
OD of unknown (test)
(Cone. of Std. in mg/dl x dilution factor)
Hb =
x
...:-
o ---~-----
00 ofstandard
1000
For example
O.385x60x251 =13.8g/dI
0.418xlOOO
(c) Acid Haematin method of Sahli
Principle
Hb is converted into acid haematin by hydrochloric acid. The brown colour of the compound is matched
against brown glass standard in a comparator.
Apparatus
Sahli's haemoglobinometer consisting of the comparator with glass standard, a square Hb tube marked
both in grams and percentage figures and Hb pipette marked at 20 cumm O.lN Hel and distilled water.
Technique
(a) Fill the Hb calibrated tube upto the mark 20 (not less) with O.IN Hel by means of a dropper.
o (b) Fill the Hb pipette exactly upto 20 cumm mark by gentle controlled sucking, the pipette is held
horizontally while.pipetting, the blood is drawn from oxalated vial or directly from skin puncture
(finger prick) site. Slight excess can be removed by touching the mouth of pipette with gauze or
gloved fingertip of technician/operator. Great excess will interfere with result. In such case
pipette cleaned, dried and refilled withfresh sample. Wipe off the outside blood at the mouth of
pipette with dry gauge.
(c) Empty the pipette into acid in the tube, keeping the point of pipette touched to the bottom of
tube and gently blowing off the blood without causing bubbles. Rinse the pipette at least three
times by drawing in and discharging the blood acid mixture, Now withdraw the pipette halfway
up the tube and rinse the outside of pipette with a few drops of acid.
(d) Mix the acid haematin solution in the tube with the glass rod and 'allow the tube to stand for ten
minutes, In this interval at least 95% colour of acid haematin is developed.
(e) Now dilute the solution of acid haematin by adding distilled water drop by drop, stirring the
mixture all the time with glass rod. The comparator is held against good daylight and the addition
of water continued till the colour of solution matches perfectly with that of standards. Take the
reading in grams per cent. The bottom of meniscus is read.
EDTA (Ethyline Diamine Tetra. Acetic acid)
Composition
1. Ethyline diamine tetracetic acid dipotassium or disodium salt - 100 g.

[[p.15]]
Common Blood Bank Reegent, Solution Buffer and Special Methods
395
44.5 gm or 41.0 gm
75 ml
1000ml
2. Water (redistilled) : 1000ml, allow appropriate volumeto dry in bottles/vials at 20°Cso as to
give a concentration of 1.5 to 0.25 mgmlml of blood.
Neutral EDTA (pH 7.0) 110mmolll
EDTA dipotassium or disodium salt
1 mmol/l NaOH
Redistilled water
Uses
1. Collection of bloodsamples.
2. EDTA is anti-complementary, s<? not recommended fo~
samples required complement assay.
3. EDTAprevents plateletclumping sochoiceanticoagulant forsamples meantforplatelet counting.
.
.
.
Fig. 14.1. Binocular microscope.
(i) For bloodcoagulation study.
(ii) Platelet function studies.
(iii) Buffered citratehelps in stabilising plasmapH.
Use
Trl Sodium Citrate
(i).N~ C6Hs0 7, 2HzO 109mmol/l- 32 gm
(ii) Redistilled water- 1000mI.
Dispense in smallvialsof 10ml,steriliseby autoclaving 3.2%to 3.8%of aqueous solution for 9 ml
of wholeblood (1 : 9)..
14.4 TECHNIQUES IN BLOOD BANKING
(A) Microscopy
Magnification by a microscope (Fig. 14.1).
Magnification at 105 mm. Initial magnification of the
objectivemultiplied by the magnification of the eye piece.
.
.
.. .
.
.
;
'tabte
:14
~4
. Magrilflc;atlon
.
.
-
. ' -
- -
.-
.
..
. .
Objective
Magnification
the objective
Total
Focal length
Eye piece
Magnification
2J3 or 16 mm
10
lOx
100
1/6or4mm
45
lOx
450
1/12 or 1.8 mm
100
lOx
1000

[[p.16]]
396
Blood Banking and Transfusion
Use of microscope
(a) Put the slide on the stage and focus under low power,
(b) Next under high power or oil immersion.
(c) Coarse adjustment and then fine adjustment.
(d) In low power, lower the condenser and use concave mirror, similarly with high power.
(e) With unstained specimen close the diaphragm for better definition of the object.
Working with oil immersion lens
(a) First define field under low power.
(b) Place a drop of oil and move the 'slide so that oil drop is just below the objective, lower the
objective so that it is in contact with the oil and almost touches the slide.
(c) Look through the microscope, raise the objective through coarse adjustment until the object is
in focus, final focussing is done by fine adjustment.
(d) After work is over the oil immersion is cleansed with xylol.
Dark ground illumination
Light illuminating the object must not enter the objective directly. Light admitted must be that which has
been reflected by object itself. A special condenser is used which allows a wide cone of light and then
stops that portion which normally enters the front of the objective. The oblique rays which normally
miss objective, when it is scattered by the object, enter the objective to illuminate the image of the
specimen against an entirely dark background.It is used to examine organisms in living state without
staining. It increases visibility without increase in resolving power.
Specia~
type of microscope
(i) Phase contrast microscope: It is method of illumination which enables observation of entirely
transparent·objects without staining.
Principle: Ordinary specimens are visible because they have an amplitude or intensitycontrast
many specimen do not change the amplitude of light but change the phase of light wave. These
phase changes are converted into amplitude changes which are visible to the eye. Special phase
contrast apparatus in place of·condenser and phase contrast objectives are required.
(ii) Electron microscope : In principle it works by using light of shorter wavelength to increase
resolving power. A powerful electron beam is passed in vacuum through magnetic fields which
,replaces the usual optical system of microscope and image of the material under examination is
photographed. The lenses are circular electromagnets whose focus varies as strength of applied
magnetic field. A magnification of 10,000 to 50,000 can be obtained in practice and objects as
small as 1-2 micron photographed.
(iii) Fluorescent microscope : Some substances emit light when stimulated. The source of light is
UV lamp or a carbon arc. The filter used in the system permits only short wave blue rays to fall
on the object, if the object is autofluorescent, it emits colour. Alternatively the object can be
stained by fluorescent dyes, rhodamine, auramine or fluorocin isothiocyanate making them
visible under microscope.

[[p.17]]
Cotnmot:\,Blood,'Bani< R9agent, Solution Buffer and Special Methods
397
B. Centrifugation
This is a common procedure used in blood bank routine working laboratory and also in component
separation room for separating the component.
Principle
Centrifuge machineusesthecentrifugal forceas a meansto separatephasesof differing density(quantity
of particlespresentper unit).The principleof balancein regardto symmetry andgeometryon eitherside
of rotatinghead is to be observed. Effectivecentrifugation thereforedependson two main criteria: (a)
time, (b) centrifugal force. The relative-centrifugal force can be calculatedfrom a nomogram whenthe
radius'r' is knownor it can be calculatedfrom the formulae as given below :
ReF =1.118 x 10-5 x r x N2
where
ReF
Relativecentrifugal force in unit of Ig' i.e. multipleof gravitational force
r
Radiusin eM
N
Speed of rotation (revolution per minute)
1.118 x 10-5
0.00001118, a constant
Typesof centrifuge.
Table 14.5 centrifuges and portable centrifuges are usually non-refrigerated units mainly used for
laboratorypurposewhereas heavydutycentrifugeare refrigerated unit fitted with cups to holdthe blood
bags, main use for componentseparation.
In laboratory centrifuge tubes (glasses'or plastic) are used. Plastic tubes are supplied alongwith
centrifuge machine.
Glass tubes of differentsizes are availablein blood bank laboratory:
(a) 75 mm x 10mm
(b) 100mm x 12mm
(c) 150nun x 12 mm
,(d) 75 mm x 12mm
(e) 150mm x 19mm
(t) 38 mm x 6.4 rnm (precipitin tube) mainly Use for groupingpurpose.
I
Table top
centrifuge
(for lab use)
I
Centrifuge'
I
Portable
(floor'tYpe)
Non refrigerated
I
I
Heavy duty centrifuge
Non-portable refrigerated
(Component separation)
G force upto 50,000. Cups
are fitted to hold blood bags
Fixed
angle
G force
upto
.5000
Swing
.head
G force
upto
3000
Microhaematocrit
centrifuge G force into
12000, tiny capillary
tube are fed into fixed
rotating head
Fixed
arrangement
For'36 tubes
G force upto
5000'
Swing
4 to 16tubes
G force
upto
3000

[[p.18]]
398
Blood Banking and Transfusion
Tubes of appropriate size are fed into the tube holder. Tubescontains the substance to be centrifuged.
For balance on either side of head tubes of equal size, shape and weight are to be placed in equal length
tube holder, same principle is followed in case of heavy dutycentrifuge. Manual of instruction contains
the operating variables which has to be consulted before the machine is put into use. Time given in
manual usually indicates the acceleration time unless stated otherwise.
(C) Plpettlng
Pipetting is one of the basic procedure in blood bank or any serological laboratory. Pipettes used for
clinical laboratory can be manual type, semi-automatic and automatic -type. Most laboratory use the
manual type of pipette.
Manually operated pipettes can be of: (a) Volumetric type, (b) Graduated type.
(a) The volumetric type : Either delivers or contains a specific volume for transfer. The delivery-
type are mostly used in blood bank or serological laboratory is identified by an opaque ring near
the top. They work on blowout principle is designed to deliver the required volume only. The
container type pipette are designed in such a way that it measures the total volume of liquid held
and work on washout principle to deliver the exact volume,
(b) Graduated type : They are designed and calibrated for measuring uniform fractional volume
and its delivery these-types are also used for serological work.
The semi-automatic and automatic type are
respectively for sampling and sample diluting purpose.
The former is operated manually and the later electrically
manually operated semi-automatic pipettes works on
"air-displacement" principle mostly whereas the
automatic or electrically operated type works mostly on
principle of "Peristalsis". These pipettes are mostly used
in cases where accuracy and precision are required.They
can be -of different variety viz. single channel,
multichannel (Fig. 14.2), then tip ejector type and many
others.These pipettes are now-a-days most preferred type
of pipettes because no risk of mouth pipetting is involved.
This is one of the greatest risk to which an operatorl
technician is exposed in dealing with an unknown sample
in blood bank laboratory.
Fig. 14.2. Multichannel pipette.
(0) Thawing
The word thawing literally means "to become so warm as to men ice or snow". In blood banking by
thawing the frozen blood or components are melted first and then used. The frozen blood or component
could not be immediately melted and the procedure followed for thawing/melting are as follows :
The frozen red cells are thawed-in a water bath at 37°e for about ten minutes followed by
deglycerolisation prior to use.
-Both FFP and epPT are stored in a frozen state and has to be thawed before use. The former is
thawed at 30
0e to 37°e in a water bath with continuous gentle agitation/massaging of bag for a period

[[p.19]]
Go.mmon·BJood :Bank Reagent, Solution Buffer and Spe¢lal Methods
399
of ten to fifteen minutes. This is to prevent formation of fibrin flake, this reduces the expected clotting
property in FFP. The common mistake of thawing FFPat room temperature helps precipitationoffibrin
to occur at 12° to 15°C.
Again thawing of FFP is done at 4°C to manufacture CPPT. This thawing can be done at 4°C in a
walk-in-cooleror a cold room at 4t) to 6°C for 18 hours continuously and is known as "air-thaw" or at
4°C in cold circulation water bath. Now-a-days rapid thawing in water bath is more preferred than slow
thawing in air at. 4°C to 6°C.
.(E) Titration
This is one of the common procedure followed in blood bank laboratory to find out/quantifythe amount
and/or concentration of antibody. Titration is also used in other serological procedure.
A volume of 2% red cell suspensionin saline is added to one volumeof serum and diluted seriallyin
two fold dilution, the mixture is mixed well with repeated pipetting twice or thrice and then incubated at
RT for one hour : 10M antibody detection.
Operative variables are pipetting, incubation temperature, time and media depends on -type of
investigation to be carried out. Described above is the procedure for detection of IgM antibody but in
case of IgG, the media could be either albumin or enzyme, or AHG may be added, temperaturepreferred
is 37°C and incubation time recommended is 1 to 1Y2 hour. Pipetting has to be. skilled and meticulous
now-a-days the semiautomatic tip ejector variety is preferred. Care is taken that the seal mouth is not
broken.
The titration be of single'serial dilution technique as described above.starting from 1 : 1 to 1 : 512
commonly employed procedure for antibody detection and quantification:
Procedure
(a) Deliver 0.1 ml of saline in all the test tubes arranged ina raw except the first one.
(b) Now add 0.1 ml of serum to be tested in tubes land 2.
(c) With a clean pipette, mix the contents in tube 2 by pipetting twice/thrice.
(d) Transfer one volume from tube 2 to tube 3 (use new pipette preferably),so the dilutions are 1 :
1, 1 : 2, 1 : 4 respectively in tubes 1, 2 and 3.
(e) Same procedure of transferring,of 0.1 ml fromtube (3) to (4) is continued through up to tube
(10) (dil 1 : 512). After final dilution, the last drop of 0.1 ml transferred. from tube (10) is either
discarded or retained. Mix the contents of all the tubes well by two or three repeated pipetting.
(t) Now add 0.1 ml of 2-5% red cell suspension in saline to all the tubes, mix well by shaking.
(g) Incubate at recommended temperature for IgG or IgM for 1Y2 hour and 1 hour respectively.
(h) Read the result for saline antibody (IgM) after and of incubation.. Reciprocal of the highest
serum dilution that shows detectable agglutination is the titre of antibody present.
(i) If the antibody suspectedis of IgG type or warm, then wash all the tubes with saline for three to
four times.
G) Add one volume of AHG sera to all the tubes.
(k) Centrifuge all tubes at 1000rpm for one minute or 3400 rpm for 15 seconds.
(1) Read results and record as AHG titre.

[[p.20]]
400
Blood Banking and Transfusion'
Master dilution technique
Commonly employed in cases of :
(i) AIliA to find out less incompatible blood that can be recommended for transfusion.
(ii) Serum is studied for reactivity by titrating with different red blood cells.
In master dilution technique, the dilution is kept-constant. Here instead one volume to start with, a
larger volumes of serum and diluent are used so that dilutions are kept constant (as recommended in
single dilution) in tubes 1 to 10 of different rows. This is done by transferring diluted.serum for each
tube of the row from the corresponding master tube.
(F) Elution
It is the process by which antibody taken up by the red cells from its own serum, are removed and
subsequently identified. The commonly employed method for elution are :
(a) Heat elution
(b) Ether elution
(c) Chemical elution.
(a) Heat elution
Originally described by Landsteiner and Miller, materials required :
(i) Prepare 6% bovine serum albumin from the available BSA i.e. 22% alburnin commercially
available.
(ii) Two to three ml of packed red cell washed in normal saline at least 5 (five) times.
(iii) Collect supernatant saline after fifth wash or final wash i.e. on sixth wash.
(iv) Neutral AB serum may be prepared if 6% BSA is not available.
Test procedure
(i) Washthe red cells supposed to be eluted for six times with normal saline of appreciablevolume.
Final wash is done by equal volume of normal saline. The supernatantis collected and is used as
negative control that run parallel with test.
(ii) Nowadd afurthervolumeof normalsalineor 6% BSAor neutralAB serumat anequalproportion
to the washed packed'RBC.
(iii) Put the above couection in a tube.orany suitable glass container which is now to be heated at
56°C in a water bath under constant agitation for te~
minutes,
(iv) After ten minutes of heating put the test tubes for centrifugation in a prewarmed centrifuge, the
best way to do it is to put the cups of centrifuge in the same water bath or dry heat applicationin
hot air oven up to 56°C.
The test tubes containing eluates are now put into ·the prewarmed cups of centrifuge, then
centrifuge at 1000 rpm for two minutes or 3400 rpm for 30 to 50 seconds. Light spin for long
duration is better than the later.
(v) Supernatant is now collected immediately without any delay.
(vi) Run the test with the supernatant so collected, identify the antibody.
(vii). Negative control (step .1) is run parallel with the test.

[[p.21]]
'Gom.m()n' Blood., Bank,,Reagent, ,So~utipn'
,Buffer and Special Methods
401
Indication for uses
ABO-HDN : To elute saline antibody in particuiar.
Sometimes used for dilution of weak antigens on RBCs.
(b) Ether elution
Originally described by Rubin in 1963 materials required :
1. Six times washed packed RBC : 2 ml at least.
2. The supernatant fluid from the sixth wash.
3. Diethyl ether: Fresh, discoloured appearance indicates that oxidation has taken place and is not
acceptable for the test.
Test procedure
(i) Eluate is made by washing of red cells with saline.
.(ii) Supernatant from final wash is collected separately.
(iii) Now add saline equal to the volume of washed RBe.
(iv) Add fresh ether equal to the total volume of saline and cell mixture.
(v) Cap the mouth of test tube, mix the contents well by shaking slowly for 1 minute or by repeated
inversion for one minute.
(vi) After the end of one minute allow the tube to settle and then very carefully remove the cap,
volatile ether is now released slowly.
(vii) Incubate for 30 minutes at 37°C.
(viii) Spun the tube at 3400 rpm, for minimum of ten minutes.
(ix) After ten minutes remove the test tube, observe that there is three layers present in the tube ether
at top, haemoglobin stained pinkish red layer at the bottom - eluate, middle layer of red cell
stroma.
(x) Top layer is discarded by aspirating.
(xi) Drive the pipette through the middle layer and carefully aspirate eluate from the bottom layer.
(xii)
~e
eluate is incubated for 15 minutes at 37°C.
(xiii) While incubating push air bubble by a pipette into the content so that residual ether is driven out.
The eluate is now subjected for antibody test. The test is ron parallel with the recovered supernatant
from final wash step (ii) negative control.
Indication and uses
To detect wann incomplete antibody in haemolytic disease ofnewborn, AllIA (autoantibody), haemolytic
transfusion reaction (alloantibody).
IgG antibody in DCT positive cases.
(c) Chemical elution
In chemical elution the antibody are eluted by causing split in the immune complex without harming the
RBCs and cell membrane in the process. The chemical reagents used are (i) Chloroquin diphosphate,

[[p.22]]
402
Blood Banking and Transfusion··
(ii) ZZAPreagent(described earlier). Describedbelowisthecommonlypracticedchloroquine diphosphate
method.
Materials reqUired
(i) Test cells
(ii) Anti-humanglobulin reagent IgG component
(iii) Chloroquine diphosphate
Test
(i) Take20mgofchloroquinediphosphate in 100mlof0.1mPBS(7.2pH)anddissolve completely.
AdjustpH to 5.0 using5N NaOH. Storagetemperaturerecommended'for stock solutionis 4°C.
(ii) Take one volume of packed RBC washed well in saline and to this one volume, add 4 (four)
volumes of chloroquinediphosphatefrom stock solution.
(iii) Mix well by shaking or repeated inversion and incubate at RT for 120 minutes minimum.
Intermittentmixing is done at intervals during the period of incubation.
(iv) After theend of incubation,wash 4/5 times with saline.
(v) Resuspendedthe cellbutton in saline and make a 5% saline suspension.
(vi) One volumeof 5% saline suspensionis now taken to itis added one volume ofAHG followed
by DCT.
(vii) Observe for reaction macroscopically, if positive, the steps (i) to (iv) as described above are
repeated and continued till no reaction is observed in DCT.
(viii) If non-activity is observed, a cell suspensionof 2.5% in saline is made.
Indication and uses
The antibody eluted red cells can be used for antigentyping and/or auto absorptionprocedurefor warm
auto-antibody.
(G) Absorption
This is the process by which antibody are removed from the serum by getting them absorbed by red
blood cells. The RBCs employedcontainsthe correspondingantigenby this methodwe can get the aIlo-
antibody absorbed or auto-antibody (cold, warm) absorbed.
(8) Absorption of allo-antlbody
By this method the allo-antibody presentin the serumis absorbedby the red cell, thusthe serumis made
free of particular antibody, of coursethe unabsorbedantibodywillremain as residualantibodyin serum.
Materials required
(i) Selection of red cells: It dependson type of antibodyto beabsorbedand those whichare not to
be absorbedor retained. Thus the red cells selected will have corresponding antigenor lacking
in' antigenrespectively or as desired.
(ii) Large volumesof normal saline.

[[p.23]]
::·gqmJ~bp;:-;atQoci:.~ank':-Reage·nt;_SolutiQn
.Buffer and Special Methods
403
.. ",
~.:."
. :;: .:",; ..;' '.-
~
~
-' ...
.
'. .'
':"
'.'..~
"
..
",
~ .
'.'
'.
Procedure
(i) Wash the required or approximate, large volume of red cells twice or thrice with normal saline.
(ii) Centrifuge at high speed for about 5 minutes to tight pack the cells at the bottom of the tube
.supernatant fluid is discarded as completely as possible.
(iii) Divide cell button equally in two test tubes.
(iv) Equal volume of test serum is added to one of the test tubes as in above.
(v) Incubation is done at required temperature for about 30 to 6O·minutes. It is ensured that antibody
reacts at that temperature. Shake the tube intermittently during incubation.
(vi) After incubation heavy spin centrifugation is done and serum recovered.
(vii) Test the serum and see that antibody intended to be removed and or retained are absent or
present respectively. If not repeat the procedure with the second test tubes containing the red
cells.
(b) Absorption of auto--antlbody
1. Warm auto-antibody absorption
This procedure is intended to absorb the antibody reacting at 37°C.
Materials required
(i) Sample in anti-coagulant from the patient.
(ii) Sample in plain vial prewarmed to 37°C, serum separation is also done at 37°C.
(iii) Warm (37°C) normal saline.
Procedure
(i) Wash patents RBC with normal saline warmed upto 37°C twice or thrice. Recover the supernatant
saline from final wash.
(ii) Washed red cells-are resuspended in normal saline 1 : 1.
(iii) The above cell saline mixture is heated in a water bath at 56°C for five minutes with continuous
agitation of the contents in the tube. This causes heat elution of auto-antibody.
(iv) A light spin is done and supernatant removed.
(v) Saline wash with warm normalsaline of the cell button two or three times. Centrifuge (heavy
spin to tight pack the cells) and discard supernatant as completely as possible.
(vi) Take one-volume of red cell as in above and add one volume of 1% papain, mix well and
incubate at 37°C for 15 to 20 minutes.
(vii) Saline wash the cells 2/3 times, discard the supernatant.
(viii) Divide the red cells in cell button in two equal volume.
(ix) Take one tube containing-the-red-cells, add equal volume of serum to the washed packed RBC.
(x) Mix well and incubate at 37°C for half to one hour with continuous agitation.
(xi) Do a heavy spin and 'supemarant recovered.
(xii) Supernatant tested against the cells (autologus). If still reactive repeat steps' (ix) to (xi) with
second tube as in step (viii) warm auto-antibody absorption.

[[p.24]]
404
Blood Banking and Transfusion
This procedure is intended to absorb the antibody reacting at room temperature and with auto-
control obtained from the patient.
2. Cold auto-antibody absorption
Materials required
(i) Fresh blood samples in anti-coagulant.
(ii) Fresh blood samples in plain vial,
(iii) Serum for absorption is first clotted in ice and preserved at cold temperature in refrigerator.
(iv) Anti-coagulated sample placed at 37°C and allowed to settle at 37°C over table top. Plasma is
separated out, the cells are saline washed twice or thrice by prewarmed normal saline (37°C).
Procedure
(i) Take one volume of 1% papain solution and is added to one volume of washed packed autologus
RBC (1 : 1).
(ii) Mix well by shaking and the enzyme cell mixture is incubated at 3~oC
for 12 to 15 minutes.
(iii) Now wash the cells thrice with normal saline. Supernatant saline from final wash is discarded.
(iv) Divide the washed cells in two equal volume in two test tubes.
(v) Add serum for absorption equal volume to the cells in one of the test tube as in step (iv) above.
(vi) Mix well and put inside the refrigerator for half to one hour. The tube agitated or shaken
intermittently will help better absorption of antibody.
(vii) Serum is separated by a light spin, this serum is now tested for reactivity with autologus RBCs
to know the status of the serum, if serum is still reactive, it indicates serum is not completely
free and the procedure from (v) to (vii) is repeated with the second tube (in step (iv) to make
the serum free of auto-antibody.
To both wann and cold auto-antibody absorption history of transfusion in patient in recent past is of
immense importance. There can be chance removal of aHo-antibody in addition to auto-antibody in
recently transfused cases.
Indication and uses of absorption
(i) Auto-antibody once removed from the serum allo-antibody could be detected.
(ii) Indirectly the corresponding antigen present in RBC could be confirmed,
(iii) Multiple antibody, if suspected in sera of multi-transfused cases could be detected by exclusion.
(iv) Reagent source for rare antibody, Rare antibody required could be separated by removing the
other antibodies.
(H) haemolysln test
Some antibody has the ability to haemolyse red cells in presence of complement. The test which can
detect this ability is known as haemolysin test, materials required.
(a)
Fresh serum less than 24 hours old.
(b) Neutral AB serum free from lysin - an alternative source of complement.

[[p.25]]
~·CQrTunon·:~lood..·,Bank·
"Reagent, .Solution. Buffer and Special Methods
405
(c) 2 to 5% saline suspension of red cells.
Procedure
(a) 2 drops of serum : Fresh and preferably less than 24 hours old. In case of non-availability of
required fresh serum, the old serum may be reinforced with equal volume of neutralAB serum
that will act as source·of complement.
(b) Two drops fresh serum or reinforced serum are taken in two tubes. Each tube containing two
drops of serum are marked 1 and 2.
(c) Add two drops of 2 to 5% saline suspension of washed red cells A to tube 1 and B to tube 2.
(d). Mix well and incubate for at least 110to 120 minutes at 37°C.
(e) Centrifuge at 3000 rpm for 5 minutes, the supernatant is examined against white background
properly and adequately lighted to detect haemolysis. Presence of haemolysis is indicated by
pink to red colouration of the supernatant which is infered as positive test.
(f) Rough scoring can be done fro-n cell button size and thecolour of supernatant pink to red.
(g) For quantitativeanalysis the titre is determined by seriallydiluting the serum and then red cells
and, complement is added to each dilution. This method is not completely satisfactory as the
recognition of 100% haemolysis by abscence of cell button and determination of endpoint by
no haemolysis or trace haemolysis are subject to argument.
(h) The other method'for quantitative analysis is to find out 00 of the supernatant fluid at 541 om
and percentageof lysis can thus be obtained by comparing with control. This percentagelysis is
plottedin a graphpaperagainstthelogof anti-serumdilution.Minimumhaemolyticdose (MHD)
is indicated at a point from where the plateau starts when all the points are joined together.
Both the procedurefor quantitativetests are not beyond criticismand are not completelysatisfactory
and since so much of precision is not required for clinical work, it is better to restrict to qualitative
analysis only.
Indication end use.s
(i) Detection of lytic anti-A and anti-B in dangerous universal donor.
(ii) Incompleteantibody (IgG) in AB and 0 group, their capabilityto cause haemolysisin a mixture
of cell, serum and complement.
(I) .Adsorptionand Elution
Materials required
(i) Respective antisera.
(ii) '0' group cells and'A' group, 'B' group cells.
(iii) Anti-human globulin serum (AHG).
(iv) Normal saline.
Procedure
(i) Salinewashingtwice/thriceof RBC to be tested,collect at least 1ml of washedred cellspacked.

[[p.26]]
406
Blood Banking and Transfusion
(ii) Equal volume of corresponding antisera is then added to washed packed red cells. The antisera
added is of the group for which weak variant is suspected viz., anti-A, if weak variant of 'A' is
suspected.
(iii) Mix well the cell sera mixture and incubate for one to one and half hour at room temperature.
(iv) Centrifuge the mixture 1000 rpm for one minute. and discard the supernatant fluid.
(v) Saline wash red cells in cell button above' with large volumes of normal saline for at Ieast six
times. Recover the supernatant after final wash and kept in separate tube.
(vi) Equal volume of normal saline is now added to the washed packed red cells as in above and
heat at 56°C for ten minutes, tube is intermittently shaken. The adsorbed antibody is thus heat
eluted.
(vii) Centrifuge at 1000 rpm for one minute, recover the supernatant and discard the cells.
(viii) Cherry brown coloured supernatant contains the heat eluted antibody that has to be tested.
The eluate is subjected to testing take three different samples of '0' group cel.ls and three different
samples/group of cells of corresponding antisera used viz. 'A' cells, if antisera A is used. Test the eluate
with all three '0' cells and corresponding group of cells three samples - B, (in case of antisera 'B' has
been used) at RT, at 37°C and with AHG.
Supernatant recovered and kept separately as in step (v) above.
Interpretation of test result
(i) If reactive with corresponding cells in AHG and non-reactive with '0' cells, it indicates that
tested cells have active antigen that can bind the group specific antibody.
(ii) If eluate reacts also with '0' cells the result is indicative of non-specific reactivity.
(iii) .Supernatant collected in step (v). if shows reactivity with the corresponding cells, i.e., 'A' cells
or 'B' cells Indicates presence of free antibody and the test with eluate· are not considered valid.
Indication and uses
To detect weak variants or subgroups of 'A' and 'B'.
(J) Anti-human globulin consumption test
The test is based on the principle that human globulin will inhibit the activity of AHG Failure to
demonstrate the reactivity when sensitised red cells are treated with AHG is indicative of inhibition.
Material required
(i) AHG
(ii) Strongly sensitised 'D' positive RBC
(iii) Test fluid containing cell (RBC, leucocyte, platelet) suspension in saline.
Procedure
The RH sensitised cells are used as indicator in the test. The sensitised cells viz., RBC, leucocyte,
platelet are treated with AHG reagent. The fall in the titre of AHG or antiglobulin using Rh sensitised
cells suggests consumption of antiglobulin by sensitised (antibody coated) RBC, leucocyte and platelet.

[[p.27]]
7.5 mg/litre
100ml
24gms
20ml
1000ml
2.5gm
1000ml
Common Blood ·Bank Reagent. Solution Buffer and Special Methods
407
This is shown by difference in titre value of the two separately set raws, a difference of two tubes or
more is significant.
Indication and uses
To detect incomplete antibody to red cells, leucocyte and platelet.
To detect presence of contaminating protein viz., IgG, if mixed with IgM etc.
(K) Acid elution slide test for HbF
This test is originally described by Kleihaur and Betke. This test is based on the principle adult
haemoglobin (HbA) and other haemoglobins are soluble at acid pH and gets eluted from the cells whereas
HbF is not soluble in acid pH and is retained within the cells. On staining the foetal cells containing HbF
appears as pink coloured cell in a field of adult ghost (denatured) cells.
Materials required
Equipment: Microscope, slide, staining tray, reagents :
(i) Fixative: 80% ethanol, once stock prepared can be preserved for 4 weeks. If precipitate forms
the solution can be used after filtering.
(ii) Elution solution.
Solution:
(a) Haematoxylin
90% Ethanol
(b) Ferric chloride
2.5 mollHCI
Redistilled water
Foruse the mixture of elution solution is made in following proportion a : b=5 : 1.
Counterstain :
Eosin
Redistilled water
Technique
Make a fresh film and dry it in air. After drying is over, fix the films with 80% ethanol for five minutes
in a Koplin's jar, Rapid rinsing of slide is done in water, the slides are made to stand vertically over
blotting paper and dried at.RT in air for 15 to 20 minutes. Slides are now immersed in elution solution
mixture for a period of twenty seconds. After 20 seconds slides are withdrawn and immediately washed
in slow running tap water. The slides are then air-dried at RT. The. slides are now placed in counterstain
for a period of two minutes and allowed to dry. Cells containing HbF stains red and adult denatured cells.
stain pink or pale pink. Under microscope the slide is examined foetal ceils stand out as well defined red
coloured/deep pink.coloured cells while the adult cells are lightly coloured ghost cells mostly pale pink.
The slide is counted for 100,000 adult cells and the number of foetal cell present is counted. Amount of
foetal blood in maternal circulation is calculated on the basis of Table i 4.6.

[[p.28]]
408
Blood Banking and Transfusion
'Table 14.6. Foetar ;;~nd'
rriat~ma'"
bloOd. ,.,r.eia~iortship
~
No. of foetalcell present
Amountof foetal blood in
per 100,000adult cells
maternalcirculation (in 00)
1 to 4
0.15 ml
3 to 8
0.25 mI
8to 16
0.50 mi
14 to 24
0.75 ml
19 to 3i
1.00.00
41 to 64
2.00 ml
63 to 96
3.00ml
105to 157
5.00ml
Calculation of anti-D doses to be given by injection as immunoprophylaxis in RH HON.
Volume of FMH = percentage of foetal cells in adult maternal cell x 50
.
Volume of Foeto maternal haemorrhage
No. of doses required =
30
1 dose =300 ~g anti-D covers for 30 ml of foetomaternal haemorrhage.
Maximum single dose recommended is 1500 ug and given by deep intramuscular injection in the
buttock.
A dose more than this is to be given in divided from at an interval of 48 to 72 hours.
(L) $illgerls method for estlmatlon of HbF
This method is also known as alkali denaturation method and is based on the principle that foetal
haemoglobin is resistant to denaturation by alkali in comparison to other haemoglobin. So a strong
alkali is added to haemolysate that causes denaturation ofother haemoglobin and HbF is left undamaged,
Ammonium sulphate (half saturated) is added to stop denaturation by lowering pH of mixture this
precipitates denatured haemoglobin which is now filtered out. Filtrate contains undamagedlundenatured
haemoglobin which is measured and expressed in percentage.
.
M~tf#(ial
requlred
Equipment: Stopwatch, balance, pH meter, pipette and micropipette, 5 ml and 20 01, 100 ul, Whatman
filter paper, spectrophotometer.
.
R~ag~nt
(i) 0.083 molllitre
NaOH
NaOH
3.32.g
Redistilled water up to
100 ml
(ii) Half saturated Ammonium sulphate with Hel

[[p.29]]
CQ~mQt)
BI~
,Bank ,Ae~gant.
'SblutionBuffer and Special Methods
409
Supernatant of saturated ammonium sulphate
Redistilled water
400 ml
10 mollL nci
2 ml
pH adjusted
2.0 to 3.0
Saturated solution ofAmmonium sulphate could beprepared by addition of (NH4)2 SO4 to distilled
water kept cold at 4°C to ~oC
in ratio of 1 gm : 1 ml. Allow the mixture to settle after thorough mixing.
The supernatant.is collected after the mixture is settled.
Technique
Place two tubes and mark them (aj.and (b) :
(i) In the tube marked (a), place 1.6 ml of 0.983 M NaOH solution.
(ii) In tube (b), put 5 m1 of redistilled water.
(iii) AddO.l ml of haemolysate to the tube marked (a), the pipette rinsed several times before
withdrawal, the content mixed well by shaking of tube,
(iv) Start a stopwatch and exactly after one minute, add 3.4 ml of half saturated ammonium sulphate
solution.
(v) Mix by repeated inversion and then filter mixturethrough Whatman filter paper.
(vi) In tube marked (b), add 0.1 mlof haemolysate (Preparation of haemolysate).
(vii) Determine OD.;.Qf tubes (a) and (b) with contents at 420 nm wavelength.
Calculation
OD of tube (a)
Percentage haemoglobin F = OD of tube (b)
OD of test
Where (a) is test and (b) is control = OD
f
I xlOO
o
contro
Normal value in adult - HbF less than 2%
Normal value in new born - HbF 60% to 70%
False low value is obtained in cases of samples obtained from' cord where HbF is more than 40%
since Singer's method is reported to give accurate result between 10 to 40%.
Prepara.tion of haemolysate
(i) Blood Sample is collected in EDTA.
(ii) Wash the cells 2/3 times in normal saline, remove the supernatant after centrifugation.
(iii) To the cell button add redistilled water equal volume to cell button, this will cause lysis of red
cell.
(iv) To the lysed cell add 0.5 ml of CCL4 (carbon tetrachloride).
(v) Now the mixture consisting of packed cell button with equal volume of water and carbon
tetrachloride in proportion of 1 : 1 : 0.5 is centrifuged for 1/2 (half) an hour with light spin.

[[p.30]]
410
Blood Banking and Transfusion
(vi) After end 'of half hour, the tube is brought out of centrifuge. The content will be divided in three
layer Lysate (top layer), stroma (middle layer), carbon tetrachloride (bottom layer).
(vii) Remove lysate from the top without disturbing stroma,
Lysate can be preserved by adding one drop of pot cyanate to the amount of lysate obtained and
stored at (-) 20°C. But use of fresh sample is always recommended for better and result oriented test.
(M)HbA2 estimation by electrophoresis
This is based on the principle of movement of charged particles in an electrical field. At pH· 8.4
haemoglobin is a negatively charged protein and as such migrate towards anode in an electric field with
cellulose acetate as supporting media various type of haemoglobin are separated because of structural
and electrical variation and identification can thus be made easily.
Materials required
Apparatus : Standard electrophoresis apparatus, cellulose acetate strips, Whatman No.3 chromatography
paper to be used as bridging strips.
Beeqents
Tris buffer pH 8.4
Tris (hydroxyethyl aminomethane)
EDTA
Boric acid
Redistilled water
10.2 g
0.6 g
3.2 g
1000 ml
Procedure
1. Tank is prepared first with above buffer and Whatman paper as wicks.
2. Cut cellulose acetate strips 5 x 10 em.
3. Soak strips with glazy size down in buffer for about 4 to 5 minutes then blot gently.
4. Place two of the strips in tank and secure it well.250 volts applied and current to 3 milliampere
stabilise for 15 to 20 minutes.
5. Tum off voltage and apply approximately 0.01 ml ofhaemolysate to each of the strip preferably
towards cathodeside. Wait for 2 to 3 minutes.
6. Reset the voltage and current at 250 volts and 3 milliampere respectively. Wait for 45 minutes,
maximum separation is reported to have occurred by 45 minutes.
7. Now once separation is over turn off the power supply.
8. Carefully cut the strips to separate HbA and ~ and strips for HbA is put into the container with
16 ml of distilled water and strips for HbA2 is put into container with 4 ml of distilled water,
9. Put the elution solution on roller mixer for 30 minutes minimum,
10. Read the OD in photoelectric colorimeter at 413 om against blank (clean piece of cellulose
acetate).

[[p.31]]
,.Common Blood Bank Reagent, Solution Buffer and Special Methods
411
Calculation
ODA2xl00
4F
% A2= (ODA)+ODA2 x
Normal range of A2 is 1.8 to 3.8%.
(.N) SickUng test
This is based on the principle that deoxygenation produces sickling of red cell, i.e. distortion of cell
membrane and shape into a form akin to the sickle.This is due to polymerisationof HbS which aligns
into some elongated shape objects like fibre thought to be responsiblefor distortion of cell size, shape
and membrane.
Material required
Equipment : Microscope, slide and cover slip.
Reagent
Stock solution A :
Disodium hydrogen phosphate
Redistilled water
Stock solution B :
Sodium dithionite
Redistilled water
1.62 gms
100mI
1.985 gms
100 ml
Working solutionis preparedby mixingA andB freshly in proportion 3 : 2 (A: B)
Method
(i) Take 5 drops of working solution and add to one drop of anticoagulated whole blood 011 slide.
(ii) Cover immediatelywith cover slide and seal with petroleumjelly mixed with'paraffin.
(iii) Observe under microscopeat end of 10 minutes and one hour.
(Ol Nestroft
This is naked eye single tube recordingofosmotic fragility test. This test is undertakenfor field study of
thalassemia cases.
Materials· required
Glass equipment : Test tube, disposable syringe and needle.
Reagents
(i) EDTAvial for collection of blood
(ii)
10% phosphate buffered saline
NaCI
9gm

[[p.32]]
412
Blood Banking and Transfusion
Na2HP0 4
NaH2 P0 4, 2H20
Redistilled water
J..36 gm
0.243 gm
100 ml
Test
(a) Take one tenth of 10% buffered saline i.e., 1 ml of PBS (10%) in 9 ml of redistilled water.
(b) Take 3.6 ml of 1% PBS (step a) in 6.4 ml of distilled water, the concentration thus, arrived at is
0.36%.
(c) 2 ml of 0.36% PBS is added to 20 JlI of blood in EDTA, mix well and wait for 30 minutes.
(d) At the end of half an hour take the reading as follows.
(e) Place a newspaper behind the tube containing test mixture. Test is negative if the writing on
paper is visible when seen through the contents of the glass tube containing test mixture and if
the letters are invisible test is positive and if indistinct the inference is doubtful.
Fallacy of test: Iron deficiency anemia gives the same picture so any positive case in the field has
to be confirmed by electrophoresis HbA 2 is raised in p thalassemia or thalassemia major of Indian
variety.
(Pi Dcnath-Landstelner test
For diagnosis of paroxysmal cold haemoglobinurea (PCH) this testis employed. The quantitative test is
described below.
Materials reouired
Equipment : Test tubes, tube holding racks, water bath working at 37°C. Freeze that can maintain
temperature at O°C alternatively ice may be procured, centrifuge.
Reagent: Neutral AB serum, group '0' RBC normal saline.
Procedure
(i) Serial double dilution of test serum in fresh neutral AB serum.
(ii) Equal volume of 20/0 suspension of '0' RBC is added to each tube.
(iii) Dip the tubes in crushed ice (maintained at DOC) for a minimum period of half an hour.
(iv) After the end of half hour transfer all the tubes to water bath at 37°C and incubates for one hour
in the water bath at 37°C~.
(v) After one hour take out all the tubes and centrifuge them at 1000 rpm for one to two minutes
and observe the haemolysis.
Read results as follows :
Macroscopic observation
Complete haemolysis with no cell button =+4
Supernatant if deep red
= +3
Supernatant when only red
=+2
Supernatant pale pink
+1
Trace or weak haemolysis
+ ±

[[p.33]]
Common Blood Bank Reagent, Solution Buffer and Special Methods
413
This titre is given by reciprocal of highest dilution showing weak haemolysis or trace haemolysis
graded ±.
(Q) Detection of IG status of autoantibody
Autoantibody are mostly IgM and IgG and to know the exact status of auto-antibody one of the
immunoglobulin has to be excluded to establish the presence.
.
The test is based on this principle, it has been observed the 2 mercapto ethanol (2ME)· and
Dithiothreitol (OTT)has unique property ofsplitting the interchain sulphahydril bonds ofgaII:UDa globulin
thus rendering them ineffective for the purpose of agglutination. This holds true particularly with IgM
antibodies but not with IgG in any case, the IgA are in intermediate zone (some IgA are inhibited and
some are not).
Materials required
Equipment: Water bath 37°C, glass tube, tube racks, pipette.
Reagents' and chemiCals:
2 mercaptoethanol or Dithiothritol (OTT), iso-osmotic phosphte buffer (pH 7.2) described in section of
buffers, serum and appropriate red cells.
Procedum
(i) Take one volume of serum (undiluted).
(ii) 2 ME 0.1 molllitre in isoosmotic phosphate buffer or DTT0.1 molllitre in isoosmotic phosphate
buffer.
(iii) Appropriate red cell.
Steps·
(a) Mix one volume of serum with one volume of 2 ME.- test, mark the tube T.;
(b) Add one volume of serum to one volume of phosphate buffer alone - control, mark the tube 'C'.
(c) Incubate both test and control at 37°C for 120 to 130 minutes.
(d) After the end of incubation titrate both the serum T and control C with appropriate red cells,
Inference : If IgG is present, the titration value of treated serum T and control serum C will be the
same.
If IgM antibody is present, the treated serum T cannot'agglutinate the red cell or ifat all, it will show
a very much lower titre than that is obtained with control serum C (untreated with chemicals).
D17 can be used in place of 2 ME, if test is to be conducted with OTI.
(.Rl. Complement· system assay·
The C 3 and C4 are the complements commonly determined complements in the laboratory, the other
components are usually determined in some special cases like genetic deficiency etc. and is beyond the
perview of this book. A brief discussion of C3 and C4 assay is given below.

[[p.34]]
75 ml
1881nl
338 ml
700 ml
414
Blood Banking and Transfusion
The C3 and C4 component can be estimated by immunodiffusion technique of Mancini and is based
on the principle that the antiserum to C3 and C4 is incorporated in agarose and then poured on a glass
slide, multiple wells are cut into the agarose slide and antigen standards of known amoiunt are placed
into some well and the unknown test sera in others. An adequate time for diffusion is allowed, the
antigen diffuses into antisera containing ag~rose
and forms a ring of precipitation around the wells
where antigen and antibody are in optimal concentration. A standard curve is drawn by measuring the
area/diameter 'of ring aroundthe standard. The concentration of antigen in test sera can be determined by
direct inspection since it is proportional to the area/diameter of the ring and can be derived from the
graph (Fig. 14.3).
Malerials required
(i) Equipment : Glass slide, refrigerator for keeping the moist chamber, moist chamber, water
bath, hot plate, Hamilton syringe, gel punch, measuring lens.
(ii) Reagents :
(a) Barbitone buffer (pH 8.4)
Sodium dithyl barbiturate
10.28 gms
Barbituric acid
1.85 gms
Redistilled water
1000ml
(b) Specific anti-sera against C3 and C4•
(c) Standard for C3 and C4•
(d) Stain (amido black stain) :
Amido black powder
500 gm
Methanol
250 ml
Glacial acetic acid
50ml
Redistilled water
200 ml
Stock solution is made for 500 ml, and the stain is filtered first before adding redistilled
water.
(e) Destain :
Glacial acetic acid
Methanol
Redistilled water
Total volume required
(f) Agarose gel for full slide
Agarose
25 mg
Berbitone buffer
2.5 ml
Polyethylene glycol
80 mgm
Boil the agar in a beaker when the agar is dissolved, add polyethylene glycol 80 mg to the agar at 45
to 80aC, while adding PEG frequent stirring is to be done. A mixture of antisera (say anti-C3), 20 J.lI is
diluted in barbitone buffer 2.5 ml is made and the mixture contained in the tube is also heated up to 48°C
by keeping in water bath. The mixture (antisera buffer) at 48°C is now slowly added to agar - PEG at
48°C in water bath. The temperature is critical.

[[p.35]]
Common 'Blood. Bank Reagent, 'Solution Buffer and Special Methods
415
..-
--4W--
....Standard 3
Q)
60
~
"0
50
c
::Je
~
40
0)
c
'C
'0
30
ES
20 1----oofJA--oIf-------... Standard 2
.5
Unknown
m 10 t---e~.--.--+----I.-------
..Standard 1
«
B
10
20
30
40
50
60
(Not as per Scale)
(mgldl) concentration of Antigen
Well punched
garget plate
Outer diameter of
precipitation ring
Std. 1 +---+---<
Std.24----+---<
®
Std. 3 4---_+_-<G
@)
Unknown .--.......---<
Fig. 14.3.
Method
(a) Take a clean microslide, place it on an evenly topped uniformly levelled table.
(b) Slowly pour the agargel on the microslide and allow it to settle. Semisolidified agargel on
microslide may be transferred into a refrigerator kept on levelled even surface within the freeze
for approximately 15 minutes which helps quick solidification.
(c) Solidified agar gel on_ microslide is now brought out of the freeze. Wells of2-3 mm size are now
punched in the solidified agar gel at an evenly spaced and predetermined distance.
(d) With the help of hamilton syringe put respective standards 1 Ill, 2 JlI and 3 fll in three punched
out wells respectively in the same line. A mental record of the wells for standards 1, 2, 3 and of
the particular line of well is made.
(e) Rest of punched..out well filled with 2 Jll of unknown test sera.
(1) Keep the microslide on a moist part of cotton evenly surfaced or moist filter paper in a petridish
moist chamber. Transfer the moist.chamber into a refrigerator (domestic) and keep it overnight
(18 to 24 hours). The preservation period should not be more than 24 hours. Complete diffusion
occurs by this time.
(g) After the slide is brought out, it is washed several times in normal saline 5/6 times and finally in
distilled water 5/6 times.
(h) Slide is now allowed to airdry at RT. When it is dry, the top of slide is lightly blotted with
ordinary filter paper to ensure complete dryness.
(i) Immerse in Amido black stain solution for 5 to 10 minutes, rinse in water and destained with
destain solution, till a clear background is seen.
G) Measure the diameter of precipitation ring around the standard and put them in a graph against
their respective antigen concentration (in mgldl).

[[p.36]]
416
Blood Banking and Transfusion
(k) The diameter of precipitation ring around unknown test sera plotted in the graph and the value
of antigen concentration can be deducted from the standard graph.
Precautionary measure for better result are recommended
(a) Antisera and standard must be very good quality, antisera lacking in sharp specificity may give
rise to illdefined ring or multiple ring. Similarly standard must be of good quality. Deterioration
of unknown test sera may give rise to false ring formation double/triple,
(b) Temperature is very critical, denaturation may occur above 56°C.
(c) Due care for carryover has to be taken.
(d) Evaporation of buffer has to be adjusted for.
(5) Plasma viscosity
Plasma viscosity is mainly dependent on the concentration of plasma protein. It has aJ;1 association with
ESR (closely linked with an increased production of protein). But then the change in viscosity of plasma
is observed to be more closely linked with clinical severity of disease than the ESR, plasma viscosity can
be measured by viscometer and has different type, viz., capillary viscometer, rotational viscometer and
rolling ball type. Different viscometer acts on different principle. More commonly used is capillary
viscometer.
In capillary viscometer, a comparative study is made between the flow rate of plasma and distilled
water under similar conditions such as pressure, temperature, bore and length of capillary tube.
Observations are recorded as viscosity of plasma in relation to distilled water. The sample of blood 'is
obtained from vein (0.5 ml to 1 ml) in·EDTA,(1.2 mg/ml). Test is conducted usually at 25°C and it is
maintained throughout the test variation of 0.5 may be allowed. It is always recommended that the test
be performed as per instruction manual supplied by manufacturer.
Normal plasma viscosity varies between 1.15 to 1.34 with an observed mean at 1.25. The measurement
is expressed in mPNS.
Whole blood viscosity can also be measured by capillary viscometer, it is mainly influenced by
packed cell volume, others that can influence the resistance flow of blood are plasma viscosity, red cell
deformation and aggregation. The clinical application being limited, the detail discussion is not within
the scope of this book.
(T) Hereditary .erythrobl8stlc multinuclearity v.,!!. positive acidified serum fest (HEM-PAS)
In HEMPAS, dyserythropoiesis (ineffective and/or abnormal erythropoiesis) is the main feature. It may
be acquired or congenital. Acquired type is found in different haematological condition (consultTextbook
of Haematology), The congenital type, also associated with splenomegaly, is an autosomal recessive
disorder. The congenital type has been graded into three on the basis of marrow changes, serological
findings and haematological changes. The HEM-PAS'belongs to grade Il 'of congenital dyserythropoietic
anemia showing :
(i)
Binuclearity or multinuclearity of erythroid precursor.
(ii) Electron microscopy reveals karryorhexis and excess endoplasmic reticulum giving the
appearance of a double cell membrane.

[[p.37]]
Com~ori.Blood.
Bank Reagent. ,Solution Buffer and, Special Methods
417
(iii) HEMPAS cells are agglutinated or lysed by an IgM antibody (anti HEMPAS) at 15°Cto 22°C.
.
These IgM antibody is found in good numberofnormal persons, it is believed that these antibody
reacts with, ~n antigen found on the surface of HEMPAS cells.
(iv)
I~. has an increased sensitivity to complement and can fix C3 component of complement in an
increasing amount.
(v) High agglutination score anti-i.
(vi) Lysis of both anti-I and anti-i.
(vii) Polyagglutinability are observed with some of inherited antigen and HEMPAS is one of them.
The reason could be due to no. (iii) stated above.
(viii) Normocytic anemia.
(ix) Positive acidified serum test.
The' acidified serum test is found positive in both HEMPAS and PNH (Paroxysmal nocturnal
haem~globinurea).
The paroxysmal nocturnal haemoglobinurea is a sleep related condition more common in yO':lng
adults, manifested as haemosiderinurea arid haemoglobinurea due to intravascular haemolysis.
Haematologically, one can find aneniia normocytic or hypochromic microcytic anemia with reticulocytosis.
Marrow may show hypercellularity with normoblastic hyperplasia; but hypocellularity may also be
observed. Auto haemolysis test may show increased lysis at 48 hours. TIlls is ~ acquired defect and
typical picture is observed in around 20% of patient. This is an intrinsic defect of red cells and it has
been observed that these cells binds more C3 as compared to normal cells. The basic membrane defect
leading to ~ 'increased sensitivity to complement in PNH cells is still not clear.
Acidifiedserum test: Originally described by Ham-in 1939and thus also known as Hamtestmaterials
,and equipment. Tubes, racks, water bath at 56°C,incubator.
Reagents and chemical: AB neutral serum, heat inactivated serumand 0.25 N Hel, patients red cell'
50% suspension and 50% normal red cell suspension; 'patients serum.
Procedure
(i) Take 4 (four) tubes and mark them A, B, C, D.
(ii) In A put equal volume ofAB neutral serum fresh and 50% suspension of patients red cell 0.5 ml
each.
(iii) In B put equal volume (0.5 ml) of fresh AB neutral serum, 0.25 N Hel, and 50% of patient red
cell.
'(iv) In C put patients own serum, 0.25 N HCI and 50% suspension of patients red cell in equal
volume (0.5 ml).
(v) In D put heat inactivated serum (serum is inactivated at 56°C for 30 minutes in water bath), 0.25
N Hel and 50% suspension of patients red cell equal volume of 0.5 ml.
(vi) Incubate all the tubes at 37°C for a period of 60 minutes.
(vii) After incubation period is over, read the result, the tube will either show lysis of different
grades or no lysis in all (Table 14.7).

[[p.38]]
418
Blood Banking and Transfusion
fable 14.7
Disease
Acidifiedserum test
Remarks
Sucrose'haemolysis
A
B
C
D
HEMPAS
Trace
Lysis
Lysisnot
Not
In A 30% of cases
Not positive
lysis
++to +++
observed
observed (-)
usually shows lysis
PNH
Trace
Lysis++to
+
Not
If D shows'lysis then
Positive
+++
observed (-)
the test is notpositive
for PNH*
* Positivityi.e. lysisin D maybedue to presenceof spherocytesor cells sensitisedby antibodymayberesponsible.
Spherocytescouldbeexcludedby auto-haemolysis testandUS (hereditary spherocytes) canbeexcludedby osmotic
fragility test.
Sucrose haemolysis test
The test is based on the principle that sucrose by providing alow ionic strength medium to the cell,
serum mixture helpsbinding ofcomplement.In paroxysmalnocturnal haemoglobinurea a good member
of cell populationhas increasedsensitivityto complement. The sucrosehaemolysistest has a diagnostic
value in PNH..
Material and equipment
Tubes, racks, centrifuge, photocolorimeterlspectrophotometer.
Reagents and chemicals
(i) Test cells: Blood sample collected in EDTA, centrifuged, supernatant discarded, washed at
least thrice with 0.15 M NaCl and then make 50% suspension with 0.15 M NaCl.
(ii) Serum: AB neutral serum.
(iii) Stocksolution ofsucrose:486 gm.ofsucroseand5.1gm ofsodiumbarbitaldissolvedinredistilled
water of 500 ml volume. pH is adjusted between 7.2 to 7.4 and make up to 1000ml,
(v) Working solution is made by taking 20 ml from the stock solution and to it, add redistilled water
8000.
Test
(a) Take four tubes A, B, C; D.
(b) In A, sucrose 0.90 ml and cells and serum equal voluu«. (0.05 ml) is taken and mixed properly.
(c)
In B, working solution of sucrose 0.95 ml and test celt. 0.05 mI is taken and mixed properly.
(d) In tube C, working solution of sucrose 0.05 ml and A.B neutral serum 0.05 ml is taken.
(e) In tube D, 0.05 ml of test cell is mixed with 0.01 M !'!H40 H volume O.~5
mI.
(f) All the tubes are now incubated at room temperature for at least one hour.
(g)
4 ml of saline (0.15 M NaCl) is then added to the tubes and cells are separated out by
centrifugation.

[[p.39]]
··Common- Blood.' Bank: Reagent" Solution Buffer and Special Methods
419
(h) Takethe supernatant fluidfromeach tubeseparately and recordthe OD at 540 om againstwater
blank.
(i)· Percentage of haemolysis can be-calculated from the formulae below:
.
ODA-(ODB-ODc)xlOO
% of haemolysis =
(ODD- OOB)
Interpretation
Cell lysis below5% is insignificant, cell lysis5 to 10%borderlinecases and needsfurtherconfirmation,
cell lysis above 10%is significantfor PNH.
Faist positive
AIHA and some forms of megaloblastic anemia.
False negative
Faulty serumor serumlackingin complement.
(U) Autohaemolysis
It has been observedsome red cells when incubatedin their own plasma or serum for a period of 48
hours at 37°C showsincreasedhaemolysis as comparedto DOnna! in vitro.
This phenomenon as described above is commonly observed in hereditary spherocytosis (HS)
. clinicallymanifests as malaise, discomfort.in abdomen maybeduetosplenomegaly, anemiaandjaundice
areassociated manifestation. Ahereditary condition ofautosomal dominanttypecharacterisedbyincreased
osmoticfragility andautohaemolysis. Otherlaboratory findingincludespherocytic redcellwithdefective
membranepermeable to sodiumin excess amountthat may ultimately lead to cell rupturedue to inflow
of water to maintainthe balance. RBC count moderately decreasedspherocytes of varyingnumbermay
be observed (smallhyperchromic red cell with decreaseor absenceof centralpallor).WBCand platelet
count may beslightlyincreased. On veryrare occasion aplasticanemiawithhypoactive marrow maybe
observed. Transfusion of blood from a HS donor will cause destruction of RBC in spleen and reduced
red cell survivalin the recipient,
The auto haemolysis test is one of the test that hasdiagnosticvaluefor HS cases, exact mechanism
of lysis is not clearly understood but defect in membrane and inability to maintain cation gradient
influences the process of lysis. Autohaemolysis test is not in much practice now-a-days due to advent in
specificenzymetest,however, interestedreadersmayconsultpracticalhaematology byDacieandLewis"
for details of procedure.
Normalbloodsamplesshowslysisless than3.5%at theend of 48 hoursincubation at recommended
temperature withoutglucoseand less than 0.9% with added glucose.
In HS autohaemolysis withoutglucoseis alwaysincreasedbut withadditionof glucose,thisincrease
maybeless.It isalsoincreasedincasesofhereditary nonspherocytic anemiaandshowsslighttomoderate
autohaemolysis.

[[p.40]]
420
Blood Banking and Transfusion
(V) Osmotic fragility test
It is based on the principle that surface membrane of RBC has a semipermeable characteristic.
Assumingthat 0.85% to 9.0% provides a physiological or isotonic environment and that the distilled
water provides most unfavourable ionic environment, then the salt concentrations of varying strength
within the intermediate range will cause lysis ofRBC. The mechanism oflysis though obscure is believed
to be caused by inflow of water into RB.C that results in spherical swelling of RBC and once the critical
volume is reached, cell leaks at first and then ruptures releasing haemoglobin" which is measured by a
photocolorimeter from supernatant fluid.
Hypotonic saline buffer pH 7.4 is used and blood added in proportion of 1 : 100 and incubated at RT
for the test.
Procedure
Materials and equipment : Tubes, rack, centrifuge, photocolorimeter, graph paper.
Reagents and chemicals: Freshly drawn blood collected in heparinised vial or defibrinated sample
is always to be preferred.
Buffer pH 7.4 (described below) preparation of stock solution of buffer.
Buffer saline osmotically equivalent to 100 gmllitre, pH 7.4.
NaCI
90gm
N~
HP04
13.62 gms
NaH'2 PO4' 2~O
2.46 gms
Water (redistilled) up to 1000 ml (pH is adjusted to 7.4) stock solution prepared can be stored for a
long time.
Preparation of hypotonic saline
Take 100 ml volumetric flask and mark them distinctly at 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50,
0.55, 0.60, 0.65, 0.70, 0.75, 0.90.
Now take stock solutions and add to each flask ten times the volume marked.
For example, marking 0.30, stock solution to be added is 0.30 x 10 = 3 ml to the flask marked 0.30.
Now add redistilled water 97 ml to make to 100 ml. This makes a hypotonic saline of 0.30%. After
adding the required quantity of stock solution in each of labelled volumetric flask, add redistilled water
to make it upto 100 ml.
(i) 10 ml of above solutions are now put into tubes marked 'T' and concentration of hypotonic
saline, viz., 0.l5% etc. are serial-wise placed in rack to make a set.
(ii) Another set of tube of equal numbers are also taken and marked 'D.' to this tube add 10 ml "of
distilled water in each.
(iii) 0.1 ml of blood (test cells) is added to each tube in both the sets.
(iv) Cap tubes by paraffin and mix by repeated inversion.
(v) Incubate at RT for sixty minutes.
(vi) After end of incubation centrifuge at 1200 g for 5 minutes.
(vii) Unlysed cells and stroma settles at bottom oftubes, now all the tubes are brought out ofcentrifuge.

[[p.41]]
Common Blood Bank Reagent. Solution Buffer and Special Methods
,421
(viii) Observe for lysis. The lysis in tubes '0' should be complete preferably with no residue at the
bottom.
(ix) Reading is taken from supernatant fluid separated from each tube blanked against 0.90 T sample.
(x) Reading taken at 540 nm and reading for '0' should bebetween 0.4 to 0.8 (acceptable range). If
this is not achieved, the supernatants to be equally and suitably diluted to achieve the range.
Calculation :
ODof T
% haemolysis = OD of D xlOO
(xi) The reading, i.e., % of haemolysis for each
concentration of hypotonic saline is now
plotted in a graph paper with % of
haemolysis
placed
vertically
and
concentration of saline in horizontal axis.
The normal fragility curve
(i·J.~
i4.4)
appears as a symmetrical sigmoid curve,
median corpuscularfragility ofnormal range
is 0.44 ± 0.02 at room temperature (20° to
25°C) in pH 7.4 in a fresh saline sample.
Increase in osmotic fragility is depicted by
a shift to the right of normal· and decrease
in osmotic fragility by a shift to the left of
nonnaL
100
80
.!l 60
rn
~
~ 40
20
O~ 1
0.2"
0.3
0.4
0.5
0.6
0.7
0.8
0/0 NaCI
~ .-Ig. 14.4.
Interpretation
Increased osmotic fragility is a feature usually observed with spherocytes - hereditary spherocytosis"and
.insome cases ofAIHA,and occasionallyin ABOHDN.In HS, test done with defibrinated blood incubated
at 37°C for 24 hours shows greater increase in osmotic fragility. Decreased osmotic fragility is a feature
usually observed in flatter cells that can expand more, and therefore, it gets lysed at a lesser concentration.
(hypotonic solution) thando normal cells sickle anemia, T.major, T.minor, HbE disease, Hbe thalassemia
etc.
In PNH, osmotic fragility is not increased and similarly in some of symptomatic haemolytic anemia.
(W) Microplates
Now-a-days microplates are being increasingly used in serological and other laboratory discipline, it has
a fruitfulapplication in blood bank too and days are not far" when it will occupy a right place in routine
work of blood bank laboratory viz., ABC and Rh typing, Coomb's test and other serological works.
Microplates are injection moulded plates made of polystyrene material. The plate has wells either
V-shaped or V-shaped, total of 96 wells in a plate of size 12 em x 8 em. The wells are arranged in letters
A to H, 8 rows of number 1 to 12. These polystyrene plates have electrostatic property that interferes
with the outcome of the result and as such plates with low static property are recommended for use. The .
plates used are better disposed off for safety purpose a reuse of plates are not recommended and strip
system ofeight strips is more economical so that only required amount ofstrips are used and rest preserved,

[[p.42]]
422
Blood Banking and Transfusion
it has been observed that albumin and tween 20" diluent has a limiting influence on electrostatic property
ofpolystyrene plates and therefore, anyone of the above two are used as red cell diluent, the later is also
used as primer for the plates in some of the tests. Recommended strength for wash solution and redcell
diluent is 0.02%. Albumin, and can be prepared as follows from the commercial albumin available:
(i) Albumin 30% - 6 mI.
(ii) Sodium chloride (N/tO) to make upto 1000 ml pH adjusted 6 to 8.
Prerequisites
Wash red cells with 0.02% albumin saline at least twice. It can be done in the 'U' wells by putting 25 J.11
of blood in each well and then the wash solutions are added. The plate is now centrifuged at 1000 rpm
for 3 to 4 minutes. Remove the supernatant and resuspend in diluting agent albumin-saline or LISS so as
to make a final concentration of 2 to 5% red cell suspension 3%, suspension is recommended for better
result. In 'V' wells further lower concentration is required and range is 0.1% to 0.2% ofred cell suspension.
If desired, enzyme enhancement effect may be achieved by pre-treatment of cells with 1% papain or
0.5% bromelin.
Someof operators prefer priming of plates intended to be used. This is done by rinsing the plates "in
Tween 20, shaken and then dried overnight at 37°C.
(i) Antisera (anti-A, anti-B, anti-AB, anti-D)
(ii) Pooled cells (A, B,.0)
(iii) P.C. solutions.
Above mentioned reagents and microplate preparation are intended for ABO and Rh grouping.
Materials and equipment
(i) Microplates either 'U' or 'V' wells as per requirement, plates with low static property is
recommended.
(ii) Centrifuge machine for plates: Fitted with four plate carriers (single or double).
(iii) A shaker for shaking the plate with variable speed controller and timer.
(iv) Semiautomated cell washer.
(v) Magnifying glass for reading the result.
(vi) Automated readers.
Saline method for ABO and Rh typing
(i) Blood sample to be tested is taken, cell and serum separated and cell suspension of required
strength is made in saline, all samples to be tested arranged serially and numbered 1 to 12
(microplate used is 8 x 12) serial arrangement done as follows la - (2 to 5% cell suspension). lb
- separated serum from sample No.1. Arrange them in a rack in two rows of tubes cell and
serum.
(ii) Dispense one volume of antisera A, B, AB, anti-D respectively in rows A, B, C, D of microtitre
plate.
(iii) Then put one volume of A, Band 0 pooled cell respectively in rows E, F, G.
(iv) One volume of '0' pooled cell in row 'H'.

[[p.43]]
,Common Blood,Bank Reagent, Solution Buffer and Special Methods
423
(v) Put one volume of 2% to 5% cell suspension in saline from the test sample in rows A, B, C, 0,
from tube la, 2a, 3a and like wise upto numbers 12 of microplate well.
(vi) Put one volume of serum in wells of microplate in E, F, G, H respectively from-testsample lb,
2b, 3b and likewiseupto 12,e.g., one volume of lb, sample in number 1 well of each E, F,G,H
rows then from 2b in No.2 well of E, F, G,H.
(vii) Add one volume of papaincystein to each well of row H.
(ix) Keep the plates on a shaker for 30 to 35 seconds for proper mixing of the contents of the well.
(x) Incubate at room temperature(20°C-25°C) for 60 minutes.
(xi) At the end of incubation period, the plates are centrifuged at 500 rpm for at least 40 seconds.
(xii) Read the results after shaking again or the plates can be kept tilted at an angle of 60° to 710° till
the negative control begins to trail (usually 5 minutes).
Inference
(a) Negative reaction, a compact button of cell trailing/streamingdown in tilted positionof 60°Cto
70°C.
(b) Weak positive reaction: Thecell button is not compact, rather loose cell button which fails to
stream down in tilted position of 60°C to 70°C.
(c) Positive reaction: Cell fails to trail and remain in the bottom of the well usually at the centre
forming a carpet pattern.
(d) H thereis positivereactionwith'0' papaincystein,l.e.,in H row producedfor antibody. Screening
Oh and other antibody that you expect in serum of that particular serum number.
Antiglobulin test
Antiglobulin test in microplate may beconducted either in saline media or in LISS. The materials and
equipments, reagents and chemicals, same as described before.
Procedure for Indirect Coomb's test (leT)
(i) Dispense serum (test) and washed cells in saline orLISS 5% suspension in anequal volume of
25 J..L1 each in the appropriate wells of microplate,
(ii) Incubate at 37°Cfor thirty to fortyfive minutes..
(iii) At the end of incubation periodthe wells are filled with normal saline.
(iv) The plates are centrifugedat 1000'~rpm
for 35 to 40 seconds.
(v) The plates are slowly tapped and supernatant saline decanted out of the well.
(vi) Repeat procedure/steps(iv) and (v) for three times more,
(vii) ,Now take 50 JlI of standardised Coomb's serum andadd to all the wells under test.
(viii) Shake and mix well for half to one minute.
(ix) Allow to stand at RT for about ten minutes.
(x) Centrifuge at. low spin 300 rpm for 40 seconds,
(xi) Read the result after gentle shaking or keep microplate tilted at 60° to 700 till negative control
begins to stream down.

[[p.44]]
424
Blood Banking and Transfusion
(xii) Validity of test is confirmed by adding one volume of IgG coated red cells to any test well that
is non-reactive centrifuge at 1000 rpm for one minute, look for agglutination a negative result
invalidates the whole test and full procedure stepwise to be repeated.
NB
(a) Positive and negative control must accompany all test.
(b) AHG serum or Coomb's serum (polyspecific) has to be standardised for microplate testing.
Direct Coomb's test
(i) Take 25 III of 2 to 5% red cell suspension in saline or in LISS.
(ii) Wash the cells with normal saline as described in ICT, at least for four times.
(iii) Add one volume of standardised polyspecific Coomb's serum.
(iv) Follow all the steps as described in ICT steps IX to XIII.
(v) Read and record result as in step XII of ICT.
Books and journals recommended for further reading
1. A Handbook ofMedical Laboratory Technology, Y.R. Talib, 1988, sponsored by WHO.
2. Blood Transfusion in ClinicalMedicine, P. L.Mollison, Seventhedition,1983,BlackwellScientificPublication.
3. Blood Group Serology, Boomian, Dodd, Lincoln, Sixth edition, 1988, Churchill Livingstone.
4. CurrentMedicalDiagnosisand Treatment. marcusA, KniPP. Milton,J. Chatton,1977edition, LangeMedical
Publication.
5. Clinical Diagnosis and Management by Laboratory methods, Todd, Sanford, Davidshon, edited by John
Bernard Henry, seventb Asian edition, ArrBS.
6. Hematology, Willial1i, E. Beutlet; A.l. Erslev; Marshall A.,. Lichtman, 3rd edition, 1983,McGraw Hill Book
Company.
7. Introduction to Transfusion Medicine, Bharuchaand Chouhan, First edition, 1990,D.K. Publisher, Mumbai.
8. Laboratory Medicine Haematology, John B. Miale, 1977, C.~ Mosby Company.
9. Practical Haematology. Dacie and Lewis, fifth edition, Chu'rchill Livingstone.
10. Procedures in Blood Banking and lmmuno Haematology, H.M..Bhatia. 1977, ICMR, Mumbai.
11. Techniques in Blood Banking, H.lv/. Bhatia, 1985, llH, ICMR~
Mumbai.
12. Transfusion Medicine TechnicalManual, Saran and Makroo, 199/, DGHS, Nirman Bhawan, New Delhi.
Journals, articles
J. Apte, s. v Preventive Haemaiology; Immunohaematology Bulletin, 1992, July-August. Vol. 23, No.4, p. 5.
2. Highlights ofthe XVI Annual Conference of' Mumbai Haematology Group. 1992, Ian-Feb; Vol. 23, No.1,
p. 7 (Immunohaematology Bulletin).
-3. Various Relevant Appendices described ill Indian Pharmacopia, Transfusion Bulletin, 1993, April, Vol. 6,
No. J, Transfusion Bulletin.