Some Related Topics

[[p.1]]
Some Related Topics
9.1 GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PD) DEFIC~ENCY
It is not an uncommon condition and falls under the group of genetically transmitted error of metabolism
that exhibits deficiency of G6PD in red cells and since it is 'X' - linked it is more common in males and
fully expressed in homozygous person. In females particularly heterozygotes present a group of cells
which are normal and another group which is deficient, thus it is not fully expressed in heterozygotes. It
has a worldwide distribution found amongst black population ofAfrica and America, then mediterranean
consisting of Sardinians, Sicilians and Greek. Also in Kurdish Jews, Iranians and Arabs. In India, it was
first described by Buxi et a1. (1964) in neonatal jaundice cases due to deficiency of G6PD. In India
(amongst the investigated races) :
Parsees
15.7%
Cutchi Bhanusali
13%
Punjabi Khatris
14%
Sindhis
3%
Bengali
5%
CIUfi1ica~
features
Mild haemolytic anemia, of course, not in all cases, suffering from G6PD deficiency, but in a small
proportion ofcases. The condition has gained importance because it predisposes to some cases ofneonatal
jaundice and drug induced haemolytic anemia, drugs commonly responsible are antimalarials viz.
primaquine, sulpha group of drugs, sulphanilamide and sulphacetamide, sulfamethoxole and
sulphapyridine, analgesics and antipyretics, water soluble vitamin, mostly vitamin K, nitrofuration, fava
beans etc. India being. endemic for malaria, we must be cautious while treating malaria or any fever
cases where tests to detect G6PD deficiency should be made mandatory.
laboratory finde91lgs
Red cell morphology is usually normal but the survival time for RBC is shortened, osmotic fragility is
also normal. In acute haemolytic anemia induced by drug administration spherocyte and poikilocytes
may be found with appearance of Heinz bodies - stains deep purple with supravital stain viz. methyl
188

[[p.2]]
Some Related Topics
189
violet, very minute bodies found attached to erythrocyte membrane. Urine 'may bedark coloured due to
haemolytic.episode induced by drug administration; it cures spontaneously alongwith drug withdrawal.
Pathophysiology of the disease reveals. that haemolytic susceptibility of the deficient person can
occur with exposure to drugs as stated above and intercurrent infections and bad obstetric history in
immature/premature child giving rise to severe hemolytic anemia with associated hyperbilirubinimia
(neonatal jaundice). The haemolytic susceptibility is more with aged RBCs, since these cells have,
decreased contents of G6PD. Physiologically the cell content of G6PD gradually reduces as the RBC
gets aged.
In a G6PD deficient person the predisposing conditions stated above increases, the oxygen demand
(00) of red cells, thereby, activating the aerobic glycolysis (PPP) (Fig. 9.1). In G6PD deficient person,
there is limited regeneration of NADPH (an essential intermediary for cell metabolism) which ultimately
leads to oxidative denaturation of haemoglobin. This denatured, oxidised haemoglobin precipitates as
Heinz body gets attached to the membrane of RBC.
.
Pentose Phosphate pathway (Aerobic Glycolysis)
.---
NADP
NADP H
---.--.
Glucose 6 Phosphate
dehydrogenase
---------------.. 6 phospho gluconate
1
Glucose
1
G6P
Pentose
Fig. 9.1.
The condition can be detected by dye reduction test or easier screening test like Fluorescent spot test
commonly adopted dye decolourisation test at present is DPIP. Rate of decolorisation, therefore, gives
measure of G6PD activity.
Dichlorophenol - Indophenol test (DPIP)
Principle : It has been observed that G6PD helps conversion of G6P to 6PG in presence of NADP. In
this process NADP gets reduced to NADPH. In DPIP test phenothiazine methosulphate (PMS) is employed
as an electron, carrier between NADPH and DPIP; as shown by following reaction (Fig. 9.2).
Reagents required : ..
1. 0.74 M Trisbuffer pH 8.0 (8.94 gm/loo ml) containing 0.5 mM DPIP (14.5 mg/lOO ml).
2. 3.0 mM NADP (2.3 mg/ml) stored at 20°C.
3. 50.0 mM G6P (15.2 mg/ml) stored at 20°C.
4. 2 mg/IOO ml phenothiazine methosulphate (PMS) freshly prepared and stored at 4°C in dark,
Reagents 1 to 4 are mixed in proportion 8.05 : 05 : 1 volume before use.
Procedure: 0.02 ml (20 J.1l) anticoagulated blood is added in 1.0 ml distilled water and the red cells
are haemolysed by mixing. Add 0.5 ml of reagent mixture mix and overlaid with paraffin oil. The tubes
are incubated in a water bath at 37°C and the time taken for decolourisation from blue to red is noted.
This is due to presence of hae~oglobin
in blood samples. The reduced DPIP is actually colourless.

[[p.3]]
190
Blood Banking and Transfusion
NADP + NADPH
G6PD
~
NADP
NADP H
~
Meth Hb Reductase
Fig. 9.2. Dye decolourisation test. PMS acting as electron carrier.
Interpretation
1. Normal sample decolourises within 5 to 20 minutes.
2. Delayed decolourisation occurs within 20 minutes to 80 minutes - Female heterozygotes.
3. No change in colour within 80 minutes and decolourisation occurring beyond 80 minutes, the
sample is deficient in G6PD. Homozygous and Hemizygous suffering from disease are usually
completely deficient and decolourisation occurs after 80 minutes.
Fluorescent spot test
The fluorescence ofNADPH under long wave UV light is used asan indicator ofG6PD'activity. Glucose
6 phosphate G6P is converted into 6PG in the following reaction:
G6P + NADP
G6PD) 6PG + NADPH + H+
As 6PGD is present in all haemolysate NADP is"reduced further as follows :
6PG + NADP
G6PD) Ribulose - 5-P + NADPH + H+
Oxidised glutathione (GSSG) is added to the reaction mixture to reconvert the small amount of
NADPH formed in mildly G6PD deficient haemolysate in following reaction :
ossa+ NADPH + H+
OR) 2GSH + NADP+
The screening test, therefore, measures the difference between the activity of G6PD and the
glutathione reductase activity.
Reagent mixture
20 ml of screening mixture contains the following :
G6P 0.01 M (6 mg/2 ml)
2.0ml

[[p.4]]
NADP 7.5 mM (12 mg/2 ml)
1% saponin
Tris HCl buffer 0.75 m (0.908 gIlD ml) pH 7.8
GSSG 8 mM (10 mg/2 ml)
Distilled water
2.0·ml
4.0ml
6.0ml
2.0ml
4.0 ml
Some Related Topics
191
Procedure : 10 Jll of whole blood are added to 100 ul of screening mixture and a spot made on
whatman filter paper no. 1 at zero time. The mixture is allowed to incubate at room temperature an.d
spots are made at 5 minutes intervals upto 30 minutes. Results are read under long wave UV light after
drying.
Interpretation
Normal sample ': Slight fluorescence at zero time with bright fluorescence in subsequent spot.
Deficient sample : Donot show fluorescence in any of the spot.
It is sometimes necessary to add less quantity of blood in samples with high haematocrit to avoid
quenching of fluorescence by increased haemoglobin content.
Its relevance with blood transfusion involves a definite though minor risk. The minor red cell
abnormality in donors particularly with sickle cell trait, Thalassemia trait and G6PD deficiency cannot
be overlooked.
The G6PD deficient donor RBC has a reduced life span of 80 to 100 days and any person who has
been transfused with G6PD deficient blood inadvertantly ingests the drugs that aggravates the condition
will suffer from rapid haemolysis of donors blood in his circulation, thus failing the purpose for which
the'blood was transfused. The haemolytic process is more severe in white population than in black, in
whom the haemolytic process is limited to older red cells only.
Since the condition is likely to get aggravated with hepatitis, malaria and other intercurrent infection,
we ate to ensure that blood transfused is of good quality and properly checked, so that any recipient
suffering from G6PD deficiency does not acquire any transfusion transmitted disease.
9.2 'HUMAN LEUCOCYTE ANTIGEN (HLA) SYSTE.M, OTHE.R LEUCOCYTE ANTIGEN AND
PLATELET ANTIGEN
The alloantigen found on erythrocytes, except A, Band H, Lewis PI and Ii are mostly confined to
erythrocyte membrane. But the HLA antigen are found on :
1. All nucleated cells.
2. Platelets.
3. Rarely on RBC - Bg, antigen; A28 and B7, B8 and B17 etc. The above described expression of
HLA antigen are determined by A, Band C loci.
There are also certain antigens determined by 'D' loci and are expressed on ~ lymphocytes, epidermal
cells and nionocytes.
Because of their wide distribution throughout body tissues and solid organ, they have become
increasingly relevant to present day clinical practice - Transfusion - Blood, Transplant - organ and Bone
marrow, Test for Paternity, Traits in anthropology and investigation thereof Tendencies - Familial and
disease association, investigation thereof.

[[p.5]]
Glycoxytase I
192
Blood Banking and Transfusion
In this communication we have restricted
ourselves mainly to basics of HLA system and
clinico-laboratory aspect relevant to transfusion
and transplant. To understand the extreme
polymorphism of HLA comparable only to Rh
system and the inherent complicacy of the
system, one requires a clear basic knowledge of
structure and genetics ofHLA and biochemistry.
Ge~etics,
structure
Class I
Class II
Class II
Centromere
HLALOCUS
Fig. 9.3.
Humanchromosome 6 dtagramatic representation
mainlydepictingHLA locusat distal third awayfrom
centromere, nearerto Glo 1 (Notasper scaJe)
The HLA complex is also referred to as human
MHC (Major histocompatibility complex) is
positioned a little away from centromere in the
distal third of chromosome 6 in human being
(Fig. 9.3)" Chromosome 6 is a human counterpart
of murine chromosome 17 (most widely studied chromosome for HLA). The MHC locus is a closely
linked locus and during meioses, it follows the law of segregation and law of independent assortment.
Accordingly the closely linked loci of multiple allelomorphic genes segregates enbloc and in a family at
genetic equilibrium, one would expect to find four possible genotype combination. Offspring will inherit
one haplotype from mother and another from father (Table 9.1).
Crossing over in closely linked loci (loci that are closely placed that-recombination occurs less than
50%, of the time are said to be linked) of HLA series A, B, C and D is not uncommon and observed in
member of families. The frequency of crossing over between closely linked loci are said to be dependent
on the distance separating them in other words, it is proportional to separating distance between two
loci.
Table 9.1·. Heplotype. HLA
Parents
Possible garnets
Offspring
Probability of occurrence
Patemal- ab
a'b}-E=
1 : 4
Maternal-cd
C, d
be
bd
Linkage disequilibrium is another important genetic phenomenon observed in HLA system. Normal
combination results in genetic equilibrium in a population. But it has been observed that certain allelic
combination are encountered predominantly whereas some combinations are rarely met. This tendency
of certain allele to occur frequently than would be expected by normal combination or on the basis of
chance alone is known PS linkage disequilibrium, i.e., two genetically linked loci has failed to reach a
equilibrium state in a population. The extent of. disequilibrium can be measured and is expressed as
difference between the observed and expected and is called delta (a) value in other words, it is the
measure or degree of gametic association. Polymorphism is another feature in genetics of H~A
system

[[p.6]]
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193
and IS oecause of multiple of alleles encoded in each locus the HLA system has extreme polymorphism
even more than Rh system and is unparalleled. This different antigenic combination numbering millions
can be expected.
The diagrammatic representation (not as per scale) showing the location that MHC locus occupies
at distal third of chromozome 6, a little away from centromere. The three molecules of HLA region are
shown diagrammatically on right hand side where class 1 molecule include HLA A, Band C antigens
'and class II include HLA-D, DR, DQ, DP whereas class ill molecule includes complement component
·C2, C4, A, Band Bf-Properdin factor B. In addition, it also include gene encoding for adrenal cytochrme
P-450 enzymes and specific steroid for 21 hydroxylase. This genetic mapping showing HLA complex
that measures approximately 2.0 CM (centimorgan - the frequency ofrecombination between two linked
loci expressed as a per cent and reflects the distance between the two loci) and occupies a 1000th part of
total human geno~e.
There is no silent or amorphs in HLA system because HLA antigen are usually
expressed codominantly.
Class 1 molecule : The antigens are HLA A, Band C found on all nucleated cells and platelets.
Structurally consists of a heavy chain mol wt 43 KD (kilodalton) and a light chain mol. wt. being 12 KD.
Class 1molecule has one extracellular one transmembrane and one intracellular portion. The extracellular
portion has an aminoterminal while intracellular portion has a carboxy terminal. Extra cellular portion
consists of heavy chain that carries the serological determinant in (Xl' ~ and'Cl:J and each one consists of
90 aminoacid approximately. a2 and ~
has disulphide bonds. The a l and (12 is responsible for
polymorphism in HLA system. a l has aminoterminal and a 3 has the carboxy terminal having a
transmembrane portion has 24 hydrophobic aminoacids and intracellular portion having hydrophilic
aminoacid residue adjacent to the surface of cell membrane. The
~2 microglobulin 'consists of light
chai~.
The ~ and ~2 microglobulin are non-polymorphic and like other immunoglobulins.
Class II molecule referred to as HLAD region and has three subtypes DR, DP and DQ. This HLA D
region was originally detected by mixed lymphocyte culture using homologus typing cells from persons
of known D locus. This region is thus also named as Mixed lymphocyte reaction genes or MLRregion
and is human counterpart of murine IA· antigens that has relevancy with immune responsiveness in that
animal. Since the distribution pattern of murine and human antigen is somewhat similar, an analogy can
Class II
Class III·
Class I
I
I
I
O.Bem
B
C
A
...-----------tl
I
I
0.2 em ,I-----.of
0.7 em
D
(MLR)
(Ir?)
DR
DO
DP
I
GL01
I
!
CENTROMERE
•
3 Centimorgan (Approx)
Fig. 9.4. Genemapping (approx.) Human Chromosome 6, showing major histocompatibility (MHC) locus.

[[p.7]]
194
Blood Banking and Transfusion
thus be drawn with the humanimmune response and it has also been proved beyond doubt that human
chromosome 6 exerts considerable influenceon :
(i) Self recognition and surveillance that constitutedefence mechanismof the body.
(ii) Immunological behaviour, butthe exact identification of Ir genesin MHC is controversial and
arbitrarilybeen assignedthe MLR region.
TheclassIIantigenscaneitherbeselectedserologically or byMLC,hasarestrictedtissuedistribution
and has been observedon ~ lymphocytes, macrophages, skin epidermalcells and sperm,Their presence
on fibroblasts, granulocytes, platelet and T (resting)cells could not be detected.
Structurally consistsof ex. chain mol. wt. 33 to 35 KD and ~ chain mol. wt. 26 to 29 KD and bothof
themoccuras glycoprotein over cell surface;encodedby geneson humanchromosome6. They together
consists of 4 domains in extracellular, transmembrane and intracellular portion of the molecule. The
extracellular portionconsistsof aminotenninaldomain(Xl and ~J domain,themembraneproximal domain
~ and ~2; connectingpolypeptide and transmembranous region,intracellularregion.The polymorphism
is related to ~l' domain only the rest are non-polymorphic. The connecting polypeptideis hydrophillic
whereas trans-membranous part is hydrophobic.
The intracellularportionis associatedwith anotherchain, invariantchain or gamma chain encoded
by genes on human chromosome 5..It is positioned invertedly with aminoterminal consisting of 216
aminoacidis intracellularand carboxyterminalis extracellular. At cell surfacethe invariantchain is not
associated with class II molecule.
The ex chainand ~ chainof classII moleculerespectively contain232and236aminoacids(Fig.9.5).
Class III moleculeincludesthe MHC linkedcomplementcomponentviz. C2, C4 A and B and also
BF (properdinfactor B). It also includes steroid specific21 hydroxylase.
Functionally all three molecules are important and takes'active part in immunological reaction in
our body and are associatedwith pathophysiology in MHC linked deficiencystate.
Class I moleculeconsistingof HLAA, B and C antigensstimulatesproductionof alloantibody and
the specificity of cytotoxic T cells are controlled by them as it has been observed that viral antigen
occurring on cell surfacewithclass I antigenare destroyedby cytotoxicT cells directedagainstthem so
.
.
Cell
membrane
ONH2
E
IX
NH 13<§g)
x
(Invariant chain)
COOH ~
2
CHO
NH2
T
Y
NH2 C
R
11, <§g)
~
Il.
CAl
A
C
E
l
<®><®>
C
~132M
l
®>
s
u
CHO
S
~2
l
S
A
R
a 2
C
COOH
C
Class I Molecule
Class I Molecule ~
~
COOH coos
.-J L Intracellular-.J
cooa
Cell----J----
membmnoo
~T~~memb~~~~~~~~~~~~~~~~~
Fig. 9.5. Structure and biochemistry of HLA molecule.

[[p.8]]
Some Related Topics
195
it can also recognise (self non-self)and thereby selectivelydestroys the virus.Class II moleculescontrols
the interaction between T and T cells (suppressor and helper). T cell and macrophages and between T
cells and B cells. Class III moleculesthat includecomplementcomponentsetc. takes part in complement
activation, production of 21 hydroxylase (deficiency produces virlism), opsonisation.
Since HLA system has important bearing with blood transfusionand organ tissue transplant, it will
be worthwhile to study the laboratory aspects of HLA system viz. typing and antibody screening, being
dealt later in this section.
·HLA and blood transfusion
(a) Multitransfusedrecipients of whole blood and packed cells is reported to have developed high
incidenceof HLAantibodiesof course,it does not mean thatrecipientof singleblood transfusion
does not develop antibody at all.
(b) Recipients of massive transfusion of whole blood and packed cells, e.g., in open heart surgery
cases also develop antibody. These antibodies are detected a week after transfusion and more
significantlyin second week and its presence may continue even beyond two months.They are
inconspicuous by their absence, if the blood is allowed through a cotton wool filter during
process of transfusion.
(c) From above observation we can logically deduce that a blood unit which is leucocyte depleted
as compared to whole blood or packed cells has less chance of developing HLA antibodies and
experimentally confirmed by Miller et al., 1975.Also blood unit stored in frozen condition at
20°C or below and is thawed and separately washed before transfusion develops Jessantibody.
(d) The positiveeffectof-previous transfusionfrom a potential.donorin renal transplantand negative
effect of previous transfusionon bone marrow transplant requires further explorationin view of
the contradictory effects observed.
Pregnancy and HLA
The HLA antibodies were detected in 24th week of pregnancy and significantpresence were reported in
10% by Overweg andEngelfriet, 1969, in primipara.
Since multiparasare more exposed to pregnancyand those who has conceived more than four times
are exposed to both haplotypes of their husband,
Specificity HLAantibody- HLAantibodiesare IgG type of antibody,i.e., immune antibody. Natural
antibody are yet to be reported. The antigen antibody reaction thus exhibits both agglutination and
complement mediated effect of cytotoxic killer cells.
HLA antibodiesrelevant to pregnancyand blood transfusionrespectively are HLAB and HLA-A,B
and C. It willbe worthwhileto mentionin this connectionthat HLA antibodiesformed during pregnancy
could be inhibited by plasma from husband of the patient. The liLA antibodies of A, B, C series can be
detected serologicallyin some cases after blood transfusion (multiple and massive). The importance of
HLAD antigen and/or MLR antigen in blood transfusion, is controversial and is of little significance
details of which is beyond the scope of this book.
Laboratory aspects of HLA system
There are different aspects of laboratory investigation which varies purpose viz. population genetics,
anthropology, paternity testing, transplant etc.

[[p.9]]
196
Blood Banking and Transfusion
Objectives
(a) Tissue typing
(b) Antibody screening
Indications
Tissue typing in blood transfusion is employed to r.
1. .Maintain donor panel
2. Source for compatible platelet
Antibody screening
1. It is done to find out-anyHLA specific antibody particularly in recipients with history of multiple
transfusion and massive transfusion to ensure safe transfusion in future.
2. Source for antibody.
Typing procedures
1. Lymphocytotoxicity test
2. Mixed lymphocyte culture for HLA-D region
3. Platelet complement fixation test
4. Leucocyte micro agglutination test
The No.1 and 2 are in common practice and is discussed in detail.
Lymphocyto toxicity test
It serves double purpose, i.e., HLA typing and eFTA (Complement Fixation Test Assay).
Principle : Basically, it is a serological test including cell lysis by antigen antibody complex in
presence of complement, i.e., complement mediated antigen antibody reaction.
The HLA antisera binds to the target antigens 011 the cell surface of lymphocyte forming Ag-Ab
complex. This activates the rabbit complement leading to cell lysis causing alteration in appearance of
cells which can be detected under phase contrast microscope by entry of certain dyes (eosin, trypsin
blue) in dead cells and not in the living cells. Formalin is added to stop the reaction and entry of dye into
the cells is thus inhibited. Dead cells look larger and dark depending on type of dye used whereas living
cell will appear as small refractile body. Test is performed in a microtitre .plate so very often called
microlymphocyto toxicity test.
Reagents and equipments
(a) Monospecific cytotoxic anti-HLA sera.
(b) Paraffin oil.
(c) Rabbit complement : Pooled sera from a minimum six young rabbit (less than 6 months old)
.tested for high complement activity and abscence .oflymphocytotoxic antibody should be used
undiluted and unabsorbed. It must give at least 20 to 30% reaction when tested with positive
HLA typing sera of 1 : 2 dilution usually the rabbit complement is stored at -70°C to -80°C or
in liquid nitrogen.

[[p.10]]
Some Related Topics
197
It is also available commercially in case commercial preparation is used follow instructions in
the insert.
(d) Positive control: Antilymphocytic serum.
(e) Negative control: Heat inactivated foetal calf serum.
(f) 5% eosin dye: Stored at 4°C to 6°C. If of non-commercial grade, filter daily before use.
(g) 40% w/v formaldehyde : Commercially available usually stored at room temperature, buffered
to neutral pH before use.
Equipments commonly used
(a) Polystyrene microtitre plate with sixty wells preferably teransaki plate.
(b) Pispenser :
(i) Hamilton syringe 1 JLl and 4 fll.
(ii) Multichannel dispenser.
(c) Phase contrast microscope.
(d) Incubator with arrangement for temperature adjustment.
Preparation oftyping tray are now done as follows :
1. Bring the microtitreplate usually 60 tests are done at a time to prevent extra cost of course
in urgency removable strips can be used.
2. Wells are layered with one drop of liquid paraffm each.
3. One III of HLA antisera is injected, under the oil, into appropriate well with Hamilton
syringe - seras if not clear should be high speed centrifuged at 10°C and then used. The
seras of at least three well defined specificity are used. It is better to use sera of asmany
specificity as possible.
(d) The known positive and negative control are used.
(e) Trays are now stored usually at temperature of -20°C or it can also be stored frozen between -
40°C to -80°C.
Testing procedure stepwise
(a) Bring out the prepared plates and thawed outside to bring it at R.T.
(b) One JlI purified lymphocyte suspension, cell count 2000 celVfJl is dispensed into each well by
disposable syringe, tip touching the bottom of the well.
. Avoid carryover and ensure p~oper
mixing of cell and sera peripheral blood lymphocytes or T
lymphocytes are used for class I antigen typing, whereas b lymphocytes are only used for class
.II antigen typing.
.
(c) Plates are now incubated at R.T. (20°C to 25°C) for 30 minutes for class I antigen (-A, -B and
-C series) and one hour at 37°C for HLA (D~).
(d) 5 JlI of Rabbit complement (preferably fresh and pooled) to each well with a multichannel
dispenser and incubate at RT for 60 minutes for HLA class I antigen and 90 to 120'minutes for
HLA class II antigen.

[[p.11]]
198
Blood Banking and Transfusion
(e) Add three Jll of 5% eosin dye to each well and allowed to react at R.T. for 2 to 5 minutes, the
non-viable cells takes up the dye whereas viable cells remains unstained.
(0 At end of incubation at room temperature 6 JlI to 40% buffered formaldehyde pH adjusted to
7.2 to 7.4 (salmon coloured) by adding KOH,is now added to each well which stops the above
reaction.
(g) Seal the test plate with its lid or any glass, lid to flatten the drops. Lids are placed tightly taking
care for any air bubble.
(h) Keep the whole plate covered with lid at 4°C overnight (allowing for settling ofcells to bottom).
If quick result required, reading can be taken after 15 minutes or the plates can be briefly centrifuged
for 1 to 2 minutes at 120 x g.
Reading of result is done by an inverted phase contrast microscope using a lOx objective and
interpretation of reactions are done in terms of the percentage of dead cells. The percentage of dead cells
(larger and stained dark red) are assessed with reference to positive and negative control.
Living lymphocytes are small and refractible.
Scoring and interpretation are shown in Table 9.2.
Peripheral blood lymphocyte isolation
Principle: Defibrinated and diluted blood samples are layered on a density gradient (Ficoll Matrizoite)
and centrifuged, lighter' mononuclear cells forms a band at interface of plasma and density gradient,
heavier blood cells settles at the bottom. Lighter cells can thus be separated..
Materials and reagents
(a) Preservative free sodium heparin 500 iulml.
(b) 6% dextran in saline (mol. wt. 150,000 to 170,000).
(c) Carbonyl iron powder.
(d) Lymphoprep : Mixture of sodium metrizoate and Ficoll with 1.077 density-commercially
available.
Table 9.2. Interpretation and scoring
Score
Percentage of cell lysis
. Interpretation
0
oto 15%
Negative
1
15 to 20%
Doubtful negative
2
21 to 40%
+
3
41 to 60%
++
4
61 to 80%
+++
5
81 to 100%
++++
Note : The above procedure described are the general principles of NIH technique adjustmentsare done, depending
on type of reagents (commercial/prepared in own laboratory) and other prevailing working conditions in different
laboratory.

[[p.12]]
Some Related Topics
199
(e) Ammonium chloride - ammonium bicarbonate solution:
1. NH4CI-7 gm
2. NH4HC03-72 ml
Distilled water-l litre
(0 Neutral AB serum or Bovine Albumin.
(g) Tissue culture medium - RPMI 1640 supplemented with Benzyl penicillin (50,000 unit),
streptomycinsulphate (50 mg), 4.4% NaHC03 (25 ml) and 3% glutemine (5 ml) per 500 ml, pH
7.0 to 7.2 (commercially available).
(h) Refrigerated centrifuge.
Stepwise procedure
1. Specimen: 10 ml of blood is collected in 0.1 to 0.2 ml of preservative free sodium heparin.
Fresh sample is preferred, and if at all necessary sample may be stored at 10°C to 20°C for
about 6 hours.
2. Heparinised blood is now mixed with dextran in a proportion of 4 : 1, allowed to stand at 30°C
for 30 min. Majority of red cells sediment and supernatant plasma is collected, which contains
leucocyte.
3. The plasma is centrifuged at 3000 rpm for 10 minutes to sediment leucocyte and the remaining
red cells. Supernatant is discarded leaving about 1 ml of sediment in which the button is
resuspended.
4. A pinch of carbonyl iron powder is added to 1 ml suspension and mixed continuously for 15
min. in 37°C in a water bath carbonyl iron is engulfed by granulocytes and monocyte which are
then brought down to the bottom of the tube with help of a magnet. Supernatant is collected.
5. The supernatant is carefully layered on 1ml of lymphoprep and centrifuged ten minutes at 2000
rpm (400 G) at 20°C.
6. The lymphocytesform a ring betweenplasma on the top and lymphoprepbelow.3/4th of plasma
is discarded.The remainingplasmaandlymphocyteare collected.Ifred cellsare furtherobserved
at the time of first wash, they are lysed withNH4CI- NH4HC03 solution; about 1ml of chilled
lysate is used for 5 min. at 22°C to 24°C to. lyse red cells.
7. Lymphocytesare washed twicein normal saline and finally suspendedin RPMI 1640containing
1% neutralAB serum or 5% bovine albumin. The count-oflymphocyte is adjusted to 2000 JlI to
2400lJll.
Cell counting is done in Neubauer chamber. Take any comer square of a neubauer chamber, it
contains 16 small square. Count the cells in any 4 smaller squares of 16 small squares. One smallest
square 1/16 of big, comer square =1/160JlI =.00625 ul,
If, now one smallest square contains 10 cells in 1/160JlI, then the amount of cells present in 0.1 JlI
will be thus :
=160 cells/O.l JlI =1600 cells/ul
x cells =
1
10 in
= x cell in 0.1 JlI
160
10x160
10
So

[[p.13]]
= 10 ml
200
Blood Banking and Transfusion
Then put it in the following formula to know the dilution required
Counted cell number (in 4 smallest square) x ml suspension
= ml final cone.
Desired cell number*
40 x 5 illl of suspension
200
50
= 50 =4 ml
So, in this case, cells centrifuged again supernatant discarded and resuspended in 4 ml to make a
count of 2000/JlI.
Conversely, if the cell count is ITIOre, say 100
100 x 5 nll
50
So the suspension has to be diluted upto 10 ml to obtain a cell count of 2000/J.11.
In this case :
50x160
IOx4
800
= 4
=200 in 0.1 JlI =2000/JlI
i.e., the desired cell count
Separated lymphocytes showing the above concentration of cells per microlitre should show at least
90% viable cells.
The above method does simply isolate the peripheral blood lymphocyte but to have pure lymphocyte
suspension of HLA-A, B, C, platelet elimination is required to be done and this is done by giving a low
speed spin after step 6 of lymphocyte separation
Separation of T-Iymphocyte and B-Iymphocyte - BPL solution obtained as above can be further
separated into T-Iymphocyte (A, B, C typing) and B lymphocyte (MLR typing) and the usual method
practiced are :
1. Nylon wool procedure
2. Separation by rosetting from sheep red blood cells
3. Lymphokwik method
1. NYLON WOOL PROCEDURE
Principle: B lymphocyte due to their cell surface properties gets adhered to nylon wool and this property
of B cell is exploited to get B· cell and T cell separated from one another. Once the B cells gets adhered,
T cells can be ·washed off and both collected in separate containers and centrifuged later to get enriched
content of B cells andT cells available.
Procedure: Two procedures are available:
(i) Nylon wool - drinking straw
(ii) Nylon woo] - syringe
* Desired number is the number of cells in one bigger corner square of neuber chamber so that the total no. of cells
becomes 2000/J.il.
.

[[p.14]]
Some Related Topics
201
Materials and reagent
(a) Nylon wool - drinking straw (plastic) or nylon wool - 2 ml disposable syringe depending on
procedure.
(b) Scrubbed nylon wool (3 dernier, type 200).
(c) RPMI - 1640 to pH 7.2.
(d) Inactivated Fcs or McCoy's 5A medium MOD, with 30% Fcs.
(e) Long and short pasteur pipette (15 ern, 25 em),
(0 37°C incubator.
(g) Heater.
(h) Petridish.
(i) P.P.B.L. 'suspension having minimum concentration of 4000 cells/ul,
Nylon wool-drinking straw
(i) Plastic straws of 0.5 em diameter are taken and length is shortened by cutting at 14 em level,
mark 6 cm from one end. Now heat seal the bottom at 45 degree angle with ,tip of serrated
forceps.
(ii) Soak 0.1 gm of nylon wool per straw in medium of 1:1 RPMI 1640and McCoys 5A in petridish.
(iii) Gently tease apart the scrubbed nylon wool and loosely pack it into drinking straw upto the
mark previously made, Again', add0.2 ml of above medium in the straw to prevent drying.
Ensure that there is no gap left in loosely packed column of nylon wool. Now seal the other end
of.tube and store at -20oe to -30°C.
Separation of T and B cells
(i) Nylon wool columns arethawed before use and brought to room temperature. Nowmake very
small opening approx. 2 mm at the tip of sealed end.
(ii) The nylon wool columns are washed.in the medium (RPMI-McCoy 5A).
(iii) Now the columns are filled up with the medium, upto 1 em above the nylon wool level that is 6
em + 1 cm = 7 cm.
(iv) Place it horizontally in a 37°C incubator and warm it for 30 minutes.
(v) Take 0.5 ml of P.P.B.L. suspension in the medium and top the prewarmed nylon wool cohimn,
i.e., 7 em (N.W. column) is topped with PPBL suspension again and 0.2 ml of medium on the
top of PPBL suspension to prevent drying.
(vi) Columnis now laid flat and incubated at 37°C for 30 minutes.
(vii) Now make a rent atthe bottom' ofstrawand held itvertically over a 10 ml plastic tube marked
'T'-wash the column with 10 to 15 ml of warm medium to elute out the non-adherent T-
lymphocytes and process continued till the medium is no longer cloudy.
(viii) After this hold the straw perfectly vertically and add 1 to 2 mlof medium. Hold the end of the
.straw closed and roll the strain between thumb and finger and now press out the content in
another tube marked 'B'. The process is repeated till the nylon wool becomes dry.
(ix) The contents of both tubes 'T' and 'B' are washed thrice in the medium.
(x) The cell count of each tube is adjusted 2 x 106 cells/ml. Also check the purity and viability.

[[p.15]]
202
Blood Banking and Transfusion
Nylon wool syringe
(a) Take a 2 ml disposable syringe.
(b) Fill it up loosely ensuring that no gap is left in between with 0.15 g scrubbed nylon wool
washed in Hank's PBS.
(c) Now add 10 ml of medium (1:1 RPMI and McCoys) prewarmed at 37°C.
(d) The lymphocyte pellets are resuspended in 1 ml of warm medium.
(e) Resuspended pellets are now poured into the column and allowed to be soaked in.
(f) Seal both end of syringe and incubate at 37°C for 30 min.
(g) Wash out the nonadherent T cells with warm medium and collect in tube marked 'T'.
(h) B cells are eluted by putting 2 ml of medium each time while pushing the plunger of the syringe
and collect it into a plastic tube marked IB' which contains 1 ml of warm medium.
(i) Centrifuge both the tubes at lOOO rpmfor ten minutes. Repeat the wash for three times. Adjust
cell count 2000-3000/~1.
G) Check for purity and viability.
Other method for T and B cell separation are :
2. SEPARATION BY ROSETTING FROM SHEEP RBe (SRBe)
Principle: A further separation methodis based on rosetting with sheep red blood cell. The strength of
binding (rosetting) can be increased by pretreatment of SRBC with neuroaminodase or amino
ethylisothioceramin bromide hydrobrontide (AET). Subsequently the SRBC's are incubated with PBL
suspension. Lymphocytes possess cell surface receptors which have affinity for SRBC and thus SRBC's
clusters around the T lymphocytes which being heavier can be separated from 'B' lymphocytes by density
gradient centrifugation followed by separation by lysis of SRBC and 'T' cells'are thus freed.
Nellroaminodase procedure
(a) Make a working solution of Neuroaminodase type VI with distilled water (sterile and double
distilled).
(b) Dispense in 150 ml aliquots and kept in frozen state.
(c) SRBC's are washed 2 to 3 times in phosphate buffer solution.
(d) Centrifuge washed SRBC at 2000 rpm for 10 minutes cell button is now resuspended in 5 ml of
PBS.
(e) Before starting with actual procedure neuroaminodase aliquots are thawed and brought to RT.
(f) SRBC suspension is now mixed with aliquots of neuroaminodase and incubated at 37°C for 30
minutes.
(g) Wash the mixture thrice in PBS and again put to centrifuge at 2000 rpm for ten minutes.
(h) Decant the clear supernatant and resuspend the cell button (of 1% concentration) in 10% FCS-
RPMII640.
(i) Stored at 4°C, limit for storage period, 7 days.
U) Separation of lymphocytes can be done by mixing equal volume of PBL (2000 cells/ul) with
neuro - SRBC mixture (less than one week old) after bringing it to RT. Mix thoroughly.

[[p.16]]
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203
(k) Now make a 5% solution of polybrene.
(1) Take 100ml of above (1 : 30 dilution in PBS) and add to neuro SRBC mixture,put to centrifuge
at 200 g for 5 minutes.
(m) Incubate for 15 minutes at 4°C.
(n) Resuspend the cell button and underlayerit with density gradient mixture put to centrifugefor
minimum twenty minutes at 400 g at RT.
(0) White ring forms at interphaseconsist ofB lymphocyte,is pipettedout and resuspendedin 3 ml
of RPMI 1640.
(p) Supernatant is discarded.
(q) Rosetted 'T' cell that comprises the cell button is dislodged by shaking.
(r) Add 3 ml of 0.85% Tris-ammonium chloride to the cell button and incubate 37°C for three
minutes lysis of SRBC's occur and 'T' lymphocytesare now freed.
(s) Wash the 'B' and 'T' lymphocyteseparately at least for threetimes.
(t) .Adjustcell count 2 X106 cells/ml. Purity and viability checked.
Lympho Kwik method
Principle : In this method monoclonal antibody with preadded complementare used. The different cell
population in a mixed state are separatedby using the above reagent in a viscous mediumand unwanted
cells arelysed specificmonoclonal antibodywith complement'T'cells than B cells are made to lyse and
vice versa and purified cell suspension of particular cell is made.
Antibody. screening
Principle : The lymphocyte toxic antibody are detected by testing them as usually against a panel of
lymphocyteswhich must include as maximum as possible representation of HLA specificity. The test is
done in a complementmediatedcytotoxictechnique,The test may be done with freshlycollectedPBLor
a stored frozen and thawedlymphocytes. T cells may be used to identifythe class I antibodyand 'B' cells
to identify the class II antibody.
Run controls with every series of test and incubations done both at RT and 37°C and incubation
time increased respectively to one hour at RT before and after adding complement and two hours at
37°~
before and after adding of complement.
Indicationsfor antibodyscreeningarealreadygivenat thebeginningof thischapteranotherindication
that may be added is pre-transplantwork up.
I. Antibody reagent source: Sera should be strongly reactive and monospecific. Sera for anti-DR
activity should be adsorbed with platelet to make it free from class I antibody. All the prepared
sera should be retested for specificity, titre and avidity, and an optimal dilution to be prepared
on the basis of three described criteria.
2. Multitransfused sera showing positive result acts as a guidance for future transfusion purpose
so compatible unit of blood may be transfused.
3. In pretransplant work up : If the transplant recipient shows a positive sera, than it has to be
reported in terms of strength of reaction and the percentageof panelreactivity so that a suitable
donor can be choosen by avoiding the HLA specificities.

[[p.17]]
204
Blood Banking. and Transfusion
Mixed lymphocyte culture test
Principle : Lymphocyte population from two different individuals are mixed and incubated with each
other in tissue culture. Now on the property of recognising the foreign elements, the responders will
recognise the antigenic disparity for HLA 0 on the stimulator population of cells. These simulatorswere
pretreated and made inactive with anti-proliferating agent viz. Radiation or Mitomycin C. Responders
for recognition of HLA 0 - undergoes blastogenic transformation which can be measured by adding
tritiated thymidene to maximally stimulated mixed lymphocyte culture after 5 to 6 days and estimating
the amount of thymidine incorporated by a beta scintillation counter.
Procedure
Step A :
(i) Collection of samples, 20 ml of blood is collected in heparin from :
(a) Recipient (Leukaemia patient in remission)
(b) Living related donors
(c) Unrelated control.
(ii) PBL separated from heparinised blood by density gradient centrifugation procedure and
suspended in sterile medium RPMI 1640-.with 1% antibiotic.
(iii) Half the amount of each PB;L suspension is either treated with mitomycin C (50.ug/ml) and
incubated at 37°C for 30 minutes or are irradiated by 2000 rads and cells are thus rendered
inactive.
(iv) Wash both treated and untreated cells at least" thrice with RPMI-1640 with 1% antibiotic
(penicillin, streptomycin and amphoteriein B) mixture.
(v) Adjust the concentration of both treated and untreated cells at 2000 cells/ul in ten per cent FCS-
RPMI with 1% antibiotic.
(vi) Set up culture in sterile tissue culture plate.
(vii) Tissue culture plate is incubated at 37°C in 5% CO2 atmosphere for about 5~ days. Asepsis and
sterility is must.
.Step B :
(i) After 5Y2 days the plate is brought out.
(ii) One uci of 3H Thymidine is added to each well avoid carry over - use Hamilton syringe,
(iii) Incubate for 18hours at 37°C in 5% CO2 humidified atmosphere.
(iv) Use automated cell harvester to harvest the cell culture on the fibre glas~
filter.
(v) Dry the filter discs at RT and place in scintillation vials each containing the scintillation fluid.
(vi) Thymidine incorporation is measured in Beta'scintillation counter and expressed in counts per
minim per culture.
Interpretation ofresults: Each laboratory has its own standard set for normal and positive reactivity.
A value of S.l. above 2 is considered significant.
Other white cell antigens
They are mainly granulocyte and platelet antigen.

[[p.18]]
So.me Related Topics
205
A. Granulocyte antigen : Granulocytes also expresses their own antigens and has been observed
on mature neutrophil granulocyte and on monocytes. Granulocyte related antigens which were
discovered first, were NAp N~
and NB 1 who have independent control from two loci NA and
NB. Later on another antigen Nel was also detected.
'Unlike HLA these NA, NB and NC series of antibody attacks foetal neutrophil during pregnancy
and may cause destruction of neutrophil leading to granulocytopenia in new born with its
subsequent sequale. And 'in transfusion related reaction, it may cause febrile episode or febrile
transfusion reaction.
NO] and NE) were later on discovered in a case of autoimmune neutrophil granulocytopenia
and chronic benign neutropenia respectively. Another series ofantigen HGA comprising HGA 1,
HGA2, HGA3 defined with antibodies were observed in cases of granulocyte transfusion and
cases ~f multiple' transfusion, 'ntis series of ~ntibodies
are shared with monocytes.
Antibodies : Immunisation by pregnancy and transfusion can thus give rise to antibody to these
granulocyte antigen, Sincethese antibodies destroys the granulocytes in vivo and in tum releases
toxic 'material which may lead to febrile transfusion reaction, In massive transfusion of
granulocyte the reactions may besevere withfatal outcome. They can be detected by agglutination
reaction in most of the cases exceptin (i) ND) - detected by immunofluorescent test, (ii) HGA2
by colour fluorochromatic microgranulocyto toxicity assay.
B. Platelet antigen: Originally described platelet antigen is PLAI and has' a frequency of 97% in
population. TIle next major platelet antigen PLA2• Both of them are associated with platelet
'membrane glycoprotein, Antibodies are formed in persons lacking the platelet antigens.
Immunisation occurs due to transfusion or by pregnancy. Antibodies can cause rapid destruction
of transfused platelet resulting in thrombocytopenic purpura. in recipient. during post-transfusion
period or thrombocytopenia in new born in cases of pregnancy. But since the antigens are
widely distributed so the immunisation occurs very infrequently. In some cases cross-reaction
between anti-PL~1
and recipients PLAt negative platelets are also reported and these have a
very fatal outcome' due to rapid destruction ofrecipient's own platelet, this'can be inhibited only
by stopping transfusion of antigen incompatible platelet.
Other platelet antigens of less significance are Baka, Ko", Kob, Pleb and Duzo.
. .
.
..
They can be detected by platelet agglutination test.
(i) Direct
(ii) Fluoroscin labelled anti.globulin serum.
To ensure the transfer of compatible platelet the platelet immunofluorescence test is employed'in
crossmatching, Like other major crossmatching, here also serum from recipient is tested against donor's
platelet. They are incubated together after which platelets are washed and tested with antiglobulin serum
labelled with fluoroscein. Incompatible platelet will exhibit strong fluoroscence.
Ge~eral
guideline for platelet transfusion
Preferably ABC compatible, HLA matched platelet are to be transfused to avoid any future complication.
Regarding ABO compatibility :
(a) A and AB platelets are to be transfused to A and AB recipient.
(b) Any group platelets can be given to '0' group recipient.

[[p.19]]
206
Blood Banking and Transfusion
(c) IB' group platelet are preferred for 'BIgroup recipient, alternatively plateletephorosis of'B' group
donor.
(d) For Rh" recipient particularly women, preference is given to Rh" donor platelet, alternatively
plateletephorosis of Rh" donor or if Rh +ve platelet are given, it is to be followed by Rho
immunoglobulin injection.
However, it has been observed that HLA unmatched platelet or ABO incompatible platelet has the
same effect but to avoid any future complication, a plasma compatibility to detect the presence of strong
anti-A and anti-'B' is recommended.
9.3 ORGAN TRANSPLANT AND BLOOD TRANSFUSION
History of organ transplantation actually started with tumour transplantation in mice and later on around
1916, the normal tissue graft were experimented where it was observed that autografts (skin graft within
same strain) were successful but not the allografts (between different strains of same species). And a
search for the cause lead to the discovery of genetic base - it was found by Snell around 1948 that the
"Major Histocompatibility locus" in mouse is responsible for allograft rejection. MHC in mouse is
identified as H2 locus in chromosome 17. Human counterpart is located in short ann of chromosome
number 6 nearer to GLO and PGM3 and is known as HLA locus or MHC.
Organ transplantation is a relatively recent development in medical science which plays a major
role in improving the quality of life and its span, but the graft rejection is a stumbling block in its
persuasion and implementation. This Immunologically mediated rejection depends on individuals ability
to self and nonself recognition. This is known as immune response regulated through an integration of
two opposing function namely "help and suppression". The immunological balance in a normal human
being is controlled at different genetic levels but the MHC locus plays the most determinant role through
antigenic recognition, production of antibody, suppression, lymphocytic proliferation and cytotoxicity
etc.
Since immune mediated graft rejection is the major obstacle in the transplantation procedure, so it
was rightly conceived by experimental scientist that by circumventing the immune reaction through
induction of immunosuppression can help in acceptance of graft. Thus the cytotoxic and
immunosuppressive agents like azathioprine, prednisolone and more recently cyclosporin were introduced
to suppress the' immune mediated graft rejection, but met .with only moderate success. So further
experiments on this line lead to the observation that blood transfusion plays a significant role particularly
in renal and bone marrow transplant and in the following para we will make a brief discussion on the
role of blood,transfusion in renal transplant and BMT.
Monozygotic twin: Twins produced from one zygote (a fertilised egg cell).
.RIA identifical : Each offspring of family inherits one HLA haplotype each from parent and four
genotypes are thus possible and probability of occurrence is 1 : 4 so 25% of sibling will inherit same
parental haplotype and are called as "HLA identifical".
Haplo identical: The 50% of 'siblings will inherit and share one haplotype and are known as
haploidentical.
Haplodistinct :The 25% of siblings will not inherit any of the haplotype and are called as haplodistinct.
Order of histocompatible donor are as follows:
(i) Monozygotic twins

[[p.20]]
Some Related Topics
207
(ii) HLA identical
(iii) Haploidentical sibling
(iv) Haploidentical child or parent
(v) First order relative - grand parents, cousins etc.
While selecting donors preference is given in the order as stated above because survival rate of
transplant in sl. 1 and 2 is definitely better than the rest.
Similarly while transfusing the blood in a transplant case in pre-, post- or.peri-transplant period, we
better observe the order of HLA identical or haploidentical blood for transfusion to avoid any haemolytic
transfusion reaction thatjeopardises the whole procedure. This is particularly relevant to peri-transplant
or post-transplant transfusion.
Renal transplant and blood transfuslon
It has been observed that a peri-transplant transfusion of HLA uncross matched blood in some cases of
renal transplant evoked HLA antibody production resulting in rejection of the graft particularly in cases
where renal graft too is not from a histocompatible donor. On the other hand transfusion of blood prior
to transplant has been shown to have better outcome by way of improving the survival rate. This effect
could be due to immunologicalresponse in the following terms: (a) Enhancementof antibodyproduction,
(b) Cellular immunity.
A~
Transfusion of random donor blood in case of non-identical donor or identical donor blood
from histocompatible donor evokes production of antibody to all possible antigen in a recipient
who is to undergorenal transplant at a later date. A selection of suitable kidney will thus include
the following criteria.
(i) Histocompatibility
(ii) Kidney that is antigen negative to other antibodies - developed.
Selection of kidney will be thus against a wider range of antibodies thereby reducing the chance
of immunisation and subsequent graft rejection.
B. Antibodiesin the serum have been observed to interfere with expressionof cellular immunity in
experiments conducted in vitro. They do so by blocking the MLR response through killing of
responder cells in MLR by cytotoxic antibodies but on the other hand, B lymphocyte antibody
effects the block through reacting with responder cells. The role of B lymphocytes in kidney
transplant is yet to be understood clearly since there are two types of B lymphocytes - warm
reacting type, reacting at 37°C, has antibodies directed against DRW antigens and the other is
cold reacting type said to possess autoantibody. The former is reported to have impaired graft
survival whereas the later has a better graft survival.
How much blood has to be transfused pre-transplant and at what intervai between two transfusion
and between last transfusion and transplant has been determined through trial and error method.
It was observed that only transfusion is sufficient in some cases and some required more than one.
So, it was decided that minimum two to three transfusion of frozen red blood cells or lucocyte depleted
red cells (minus buffycoat) because they have lesser tendency to develop cytotoxic antibody.
Time period is 1110re than two three weeks but less than 6 months pretransplant,the interval between
two successive transfusion is not critical but could be done at anytime within six months but the gap
between last transfusion and transplant should not be more or less than 2 to 3 weeks, ths gap is necessary

[[p.21]]
208
Blood Banking and Transfusion
for formation of antibodies and also antibody level, most cases starts falling after three weeks. The exact
mechanism, how the prior transfusion exerts beneficial effect on graft survival or acceptance is not yet
clearly understood and so instead of confining our views to present state of knowledge, we should have
an open mind for forthcoming informations that will help in better understanding or may require to
change the present view.
Rarely practiced now-a-days, due to advent in immunosuppresive drug therapy.
Bone marrow transplant and blood transfusion
The relation between the bone marrow transplant and blood transfusion has been widely studied because,
the BMT has revolutionised the treatment and improved the survival rate in haematological conditions
viz. Aplastic anemia, PNH and Fanconis anemia (non-malignant condition); acute leukaemia,
lymphoblastic leukaemia, acute myelogenons leukaemia (malignant condition). Success of BMT is
dependent on supportive blood transfusion therapy. There is no denial of the beneficial effect of blood
transfusion on BMT, but the exact mechanism as to how blood transfusion helps in improved survival of
BMT is' yet to be clearly understood. In view of the' havoc caused by blood transfusion during pre-
transplantperiod which is in contradiction to the' experience of kidney transplant, our views needs to be
redefined in respect of blood transfusion in BMT.
-
BMT is always done in fILA identical cases though there were some reported .success in HLA non-
identical cases but the majority trend is in favour of HLA ~ identical donor. The ABO compatibility is
considered less critical for BMT so in cases of non-availability of ABO compatible donor a donor who
is HLA identical but ABO incompatible can be tried.'So in BMT,there will be two types of donors and
the role of blood transfusion vis a vis these donors will be discussed :
A. HLA identical and ABO compatible donor
B. HLA identical but ABO incompatible donor
To start with, we must have some idea regarding other general principles followed in bone marrow
transplant :
1. Since the donor and recipient are not cent per cent genetically identical so a pre-transplant
conditioning of the patient with an immunosuppressive regimen is necessary to· avoid future
.graft rejection.
2. Togive supportive post-transplant measure to improve the survival rate and reduce graft rejection
rate. This could be in. form of blood and blood component, antibiotics, stem cell transfusion
.parenteral hyperalimentation for negative caloric and nitrogen balance, protective environment.
3. Selection of proper donor who should be essentially HLA identical preferably siblings.
4. To prevent the occurrance of GVRD (graft vs host disease) the most common fatal complication
ofBMT.
The most common application of BMT is,done in case ofAplastic onemia and Acute leukaemia and
blood transfusion in relations to this will be discussed here.
In case of HLA identical ABO compatible donors the recommendations are as follows:
Pre-transplant period
(a) In aplastic. anemia cases where pretransplant conditioning is done with cytoreducive and
antimitotic drugs with a sole aim to blunt sensitisation of the patient. In such situation blood
transfusion, even if from a HLA identical sibling is not recommended because by blood
transfusion we can sensitise the :

[[p.22]]
,some. Related Topics
209.
(i) Non-HLA antigen, and
(ii) Tissue antigen and thereby jeopardising the acceptance of graft by the recipient.
Andthis negative outcome of blood transfusion has also been confirmed by statistical evidence.
If at all blood transfusion becomes a necessity, blood transfused should be minimal and from a
random donor, preferably leucocyte depleted RBC or mechanically washed RBC and should be
thoroughly crossmatched before transfusion. Platelet-transfusion may be necessary only in case
of profuse bleeding usually observed with count below 5000/lll..Thoroughcrossmatching is a
must before transfusion. But for all practical purpose,' it is better to avoid any sort of transfusion
(blood or component) during pre-transplant period in Aplastic anemia cases.
(b) Acute leukaemia: The situation in acute leukaemia is again different. In leukaemia, patient
undergoes intensive immunosuppressive therapy including radiation. Blood transfusion thus
becomes essential and due intense immunosuppression, the sensitisation is less likely to occur.
So blood transfusion, if necessary is carried out during remission under cover of antirniotic
drugs. But the transfusion should preferably be restricted to random donor andleukocyte depleted
preparation to have a better graft survival and the units transfused also to be restricted to one or
two.
Post transplant period
The intense haematopoitic suppression as a result of precondition regimens, the time required for
engraftment and the engrafted graft to take. up the function' varies between 4 to' 6 weeks. Here the'
transfusion need is inevitable and has to be followed vigorously irrespective of the type of haematologic
disorders treated. This improves the prospect of graft acceptance.
Following blood or blood products may be considered it is ofcourse influenced by two conditions :
I. Assessed requirement of patient.
II. Physician's choice :
(a) Irradiated blood, where blood or blood products are irradiated by 1500 rads to check
proliferation of lymphocyte' and possibility of GVHD.
(b) Leukocyte depleted red blood cells.
(c) Platelet transfusion· is usually taken up in cases where platelet count goes below 20,OOO/fl).
(d) Granulocyte transfusion.
(e) GM-CSF intravenous infusion is a newer approach to reduce the period of post-transplant
neutropenia. In cases of autologus transplant of bone marrow a dose of 2 to 32 mgmlkg/
day is recommended for intravenous infusion 3 days after BMT for a period of 14 days.
General principles in respect of post-transplant blood or blood products transfusion are:
(i) Random donor HLA identical blood is preferred in first instance.
(ii) In case of non-availability of such blood family donor and sibling donor may be
considered, thus first preference to HLA identical, then to HLA haploidentical and
then to non-identical.
(iii) Refractory platelet. from random donor are treated in same line and if patients are
refractory to both random donor and related donor platelet, the choice is random
donor platelet along with prednisolone therapy.

[[p.23]]
210
Blood Banking and Transfusion
(iv) Blood or blood products transfused should be as fresh as possible, contain no
contamination and properly crossmatched to ensure compatibility.
(v) Duration and interval between successive transfusion varies from patient to patient
and entirely dependent on physicians assessment of patient.
(vi) Granulocyte transfusion usually given for intercurrent infection should be supported
by appropriate antibiotic.
(vii) Haematocrit to be maintained between 25 to 30%.
HLA identical ABO incompatible donor
The incompatibility may be of two types mainly major and minor; third is a combination of two - a rare
event.
(a) The major incompatibility of ABO group are those where donor is either group 'A' or 'B' and
recipient are blood group '0'.
(b) The minor incompatibility includes the cases where donor is '0' blood group and the recipient is
either 'A' or 'B' group.
(c) Major - minor incompatibility where donor is 'A' and recipient is blood group 'B', it may even be
vice versa.
The marrow transplantation and subsequent follow up in major incompatibility is a difficult task
with formidable problem like acute haemolytic transfusion reaction and persistent post-transplant
haemolysis, Experimentally, it has been observed that if antibody titre can be significantly reduced the
problem can be successfullytackled. This is possible either by whole blood exchange or plasmaexchange
during pretransplant period. The former procedure can be executed through CFC machine. PPP or FFP
are commonlyusedinplasmaexchangeandisexchangeddailyfor threedays immediatepriortotransplant.
Post-transplant haemopoitic suppression and haematocrit value could be maintained by group '0' RBe
(leukocyte depleted) and is continued till the circulating antibody disappears, nCT becomes normal
usually occurs within three months post-transplant period.
In minor incompatibilitymarrow transplantation does not pose much problem. However,the plasma
from bone marrow is removed by the process of centrifugation, thereby, the antibodies are reduced. The
red cells transfused should be '0' type that is donor.type, leukocyte depleted RBe is preferred. The
plasma transfusion, if at all becomes necessary, should be of recipient type. This type of blood or plasma
transfusion is to be continued with frequent checking for recipient's cells in circulation.
In major minor incompatibility, the accepted recommendation for transfusion is for '0' group RBC
and/or plasma of 'AB' group (neutral plasma) to be followed strictly to prevent any haemolysis of red
cells.
9.4 GRAFT VERSUS HOST DISEASE (GVHD)
This can be described as a syndrome, where foreign cells mount an assault on the tissues ofthe host after
successful transplantation or engraftment, a very common complication observed in BMT.
Pathophysiology
Transfusion oflymphocytes or its precursor (alive) in animmunosuppressed recipient or patient induces
a graft vs host reaction. Mitotically active lymphocytes present in stored blood when transfused in an

[[p.24]]
Some Related Topics
211
'Table,'9.3. Immunologically incompetent 'patients
I
Congenital
I
. I
Acquired
I
Immunodeficiency
states
I
Immature immunological
systemviz. premature
babyor intrauterine
foetus
I
Immunosuppression
by externalagentviz.
radiation, automitotic
drugs
I
Diseasestatesviz.
acuteleukaemia and
other haemotological
malignancy
immunologically incompetent patient vide supra (Table 9.3) leads to an engraftment of bone marrow by
immunologically competent lymphocyte or its precursor, subsequently mounts an assault on the host
tissue resulting in full blown graft vs host reaction of GVHD..
"An alternative hypothesis states that itmay be due to aberration in suppressor 'T' cells of the host
that may allow development of autocytotoxic cells "Reinhertzetal" quoted by Mollison, P.L.
Clinical feature
It varies from mild to severe depending on the type of graft vs host reaction, most of them terminates
with a fatal result.
Haematological picture : Pancytopenia, haemolytic anemia.
Peripheral : Lymphadenopathy, enlargement of liver and spleen, loose motion and frequent skin
infection..
Any sort of transfusion ofstored blood containing active lymphocyte and its precursors in the immuno-
incompetent patients described above may lead to development of GVHD subsequently. These active
lymphocyte may contaminate the platelet or granulocyte which are usually separated from whole blood.
So transfusion of granulocyte or platelet may also cause GVHD in immunosuppressed patient.
Prevention and treatment
To prevent occurrance of.GVHD, it has ~een
recommended to use irradiated blood which restricts the
mitotically active lymphocyte and its precursors. Once the disease has occurred, it is to be treated with
fresh blood transfusion preferably leukocyte depleted and irradiated the other supportive measures viz.
parenteral hyperalimentation under mild antibiotic (having less toxic side effect) cover. Blood is irradiated
by a radiation dose of 1500 cads.
Books recommended for further reading
1. Blood Transfusion in Clinical Medicine, P.L. Mollison, Seventh edition, 1981, BlackwellScientificpublication.
2. Clinical Practice of Blood Transfusion, L.D. Petz and Scott, N. Swisher; First edition, 1981, Churchill
Livingstone Limited.
3. Clinical Diagnosisof Management, Laboratory Method, Todd, Sanford, Davidson, Second edition (Indian),
1989, ArrBS.
4. Introduction to Transfusion Medicine, Bhurucha, 2.S. and Chauhan, D.M., First edition, 1990, D.K.
Publishers.
5. Procedures inBloodBanking and Immunohaematology, H.M. Bhatia, 11H (ICMR), Seth as. Medical College,
Mumbai,

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Blood Banking and Transfusion
6. Techniques in Blood Banking, H.M. Bhatia, 11H.(Technical Report), Series 3, 1985, 11H (ICMR), Seth GS.
Medical College, Mumbai.
7. Transfusion Medicine Technical Manual, Saran, R.K. and Makroo, R.N., 1991, Sponsored by .WHO and
Published by DGHS, New Delhi.
Journal and articles
1. Impact ofthe HLA System on ImmunogeneticResearch, Vol. 5, No.5, Sept.-Oct., 1984,lmmunohaematology
Bulletin.
2. Structure and Genetic Aspect of RIA System - Nafisa, M., Contractor, Vol. 10, No.4, July-August, 1988,
Immunohaematology Bulletin.
3. Haematopoiotic Growth Factor; Colony Stimulating Factors, S.H. Advani, lmmunohaematology Bulltein,
Vol. 23, No.6, Nov.-Dec., 1992.