Grouping and compatibility issues and
their resolution
3rd Basic Haematopathology Course
TMH
Mumbai
ABO system
 First human blood group system to be discovered
 Only blood group system in which individuals have
predictably antibodies in their serum to antigens that are
absent from their RBCs
 4 major ABO phenotypes : A, B, O and AB - determined by
the presence or absence of two antigens (A and B) on red
cells
•Presence or absence of naturally occurring antibodies termed isohemaagglutinins, directed against missing A and B antigens.
•Immunizing source- is gut and environmental bacteria which have been
shown to possess ABO like structures on their lipopolysaccharide coats.
• Predominantly IgM ,activate complement and react at room
temperature or colder
•Produce strong direct agglutination reactions during ABO testing
 ABO antibodies – Initiated at birth but detected only by 3 to
6 months of age ,peaks between 5-10 years and declines later
in life
 ABO antigens – develop by 37th day of fetal life and fully
developed by 2-4 years of age and remain constant for life
 Forward grouping or front typing:
Detection of antigens on an individual’s RBCs using known sources
of commercial antisera
 Reverse grouping or back typing:
Detection of ABO antibodies in the patient’s serum using known
reagent RBCs namely A1 and B cells
Discrepancy – A conflict or variation …between
things that ought to be the same
Collins
dictionary
In the blood bank
 Reverse typing discrepant with forward grouping
 Patients phenotype inconsistent with antibody profile
 Group/type inconsistent with previously assigned group/type
 Crossmatch results discrepant with prior or subsequent
compatibility testing
Classification of grouping discrepancies
 Group I – Unexpected reactions in serum grouping due to weakly
reacting or missing antibodies
 Group II – Unexpected reaction in cell grouping due to weakly
reacting or missing antigens
 Group III – Unexpected reactions in cell and serum grouping caused
by protein or plasma abnormalities
 Group IV – Miscellaneous problems
Common sources of technical errors
 Inadequate identification of blood specimens, test tubes or
•
•
•
•
•
slides
A mix- up in samples
Clerical error
Cell suspension either too heavy or too light
Failure to add reagent
Failure to follow manufacturer’s instructions
 Contaminated poor quality reagents
• Missed observation of hemolysis
• Uncalibrated centrifuge
• Unclean/ contaminated glassware
General principles for resolution
 Eliminate the chance of clerical error or misidentification by retesting
where possible on a fresh sample.
 Test with alternate platforms and reagents
 Use washed cells and saline replacement/dilution to eliminate type III
reactions
 Use enhancement techniques to bring out weak or missing reactions
 Review and analyze the patient’s history for a possible role
Lab tests that aid in resolution
The tools to him that has the ability to handle them.
French proverb
 Multiple platforms and reagents
 Antibody screening and identification
 Non-routine serological tests (enhancement techniques,
secretor test, adsorption and elution, etc.)
 Extended phenotyping
 Genotyping
Case 1 - Where a discrepancy aided
diagnosis!
 A 55 year old lady slated to undergo an orthopaedic surgery
elsewhere, was referred to our blood bank. Results of grouping done
elsewhere were:Anti A Anti B Anti
AB
A cells B cells O
cells
4+
2+
4+
4+
Forward group:AB
2+
2+
Reverse group:???
• Microscopy: Rouleaux
•Rouleaux – Stacking of erythrocytes in a coin like fashion
(right) can be mistaken for agglutination (left)
•Saline dilution/replacement will neutralise rouleaux
formation
 With saline dilution reverse group consistent with AB.
 In view of the clinical history of fracture, patient’s clinician
informed of need to exclude plasma cell dyscrasia.
 On further investigation, patient diagnosed to have myeloma
 The presence of a discrepancy may point to a
significant clinical condition
Case 2 – Clinical history to the rescue!
 Sample from a 1 year old child was sent for grouping
 Grouping results
Anti A
Anti B
Anti
AB
A cells B cells O cells
Neg
Neg
Neg
Neg
Forward group:O
Neg
Neg
Reverse group: AB?
 Sample retested after incubation at 4 degrees Celsius showed
similar results.
 Clinical history enquired into:- history of recurrent infections;
suspected case of severe combined immunodeficiency syndrome
 Inference: Serum grouping negative because of
hypogammaglobulinemia.
 Clinical history can play an important role in resolving a
discrepancy
Case 3 – A blood group makeover!
 59 year old gentleman posted for surgery for carcinoma colon.
 Previously documented A group
 Repeat grouping:-
Anti A
Anti B
4+
2+
Anti
AB
4+
Forward group: AB
A cells B cells O cells
Neg
4+
Neg
Reverse group: A?
 Patient’s serum tested against patient’s own cells gave a
negative reaction
 Acquired B suspected in view of the above, and clinical
history
 Confirmed by secretor test: Showed only A substance
 Inference: A group with acquired B
CH2OH
CH2OH
O
OH
OH
Bacterial deacetylating
enzymes
O
OH
O
OH
OH
NHCOA immunodminant sugar
(N- acetyl D galactosamine)
O
OOH
o
NH2
Acquired B
(D galactosamine)
Anti B
CH2OH
OH
O
OH
OH
OH
B immunodominant sugar
(D galactose)
Acquired B phenotype
 Patients with gastrointestinal lesions, particularly carcinoma colon.
 Always occur in group A patients, nearly always A1
 Acquired B cells do not agglutinate with patient’s own anti B
 Secretors do not secrete B substance
 Reversible on treatment with acetic anhydride
 Ph sensitive:- Anti B reagents with PH beyond the range of 6.0-8.5 do
not agglutinate acquired B cells
Case 4 – An argument in favour of different
phases of compatibility testing!
 A 19 year old youth posted for an elective surgery was grouped in
our blood bank.The patient had received 16 uneventful transfusion
prior to this
 Grouping results
Anti A
Anti B
Anti AB A cells B cells O cells
4+
4+
4+
Forward group:AB
Neg
Neg
Neg
Reverse group:AB
•Antibody screen - Negative
Crossmatch results
Phase of
testing
Donor 1
Group AB
Donor 2
Group A
Donor3
Group O
saline
Neg
Neg
Neg
37o C
3+
3+
Neg
Coombs
4+
4+
Neg
Patients cells tested with A1 lectin – Negative reaction
signifying A2B group
Incompatible units tested with lectin and found to be
positive signifying A1 phenotype.
Crossmatch performed with A2B unit was compatible
Inference: A2 with anti A1
 Significance:- Anti A1 reactive at 37o C – clinically
significant!!
 Would have been missed if immediate spin
crossmatch alone was done eg. type and
screen/electronic crossmatch scenario
Case – 5 Bombay group
Anti A
Anti B
A1 cells
B cells
O cells
Auto
control
Results
0
0
4+
4+
4+
0
Oh
Bombay
•Reported by Bhende in 1952 in Bombay
•Inheritance of double dose of h gene producing genotype hh
•No expression of ABO genes and no H antigen and hence ABH antigens not
formed
•Serum contains anti A, Anti B, Anti A,B and Anti H
•Anti H – potent and clinically significant at 370 C
•IgM antibody-activates complement and RBC lysis
•Transfuse blood only from another Bombay individual
Case 6 – Dilemma after resolution!
 55 year old lady slated for elective surgery referred from
elsewhere. She had received 2 uneventful transfusions of O
positive blood elsewhere one year previously.
 Grouping performed elsewhere.
Anti A Anti B Anti
AB
Neg Neg Neg
Forward group: O
A
B
O
cells cells cells
4+
4+
Neg
Reverse group: O
Case 6 – Dilemma after resolution!
 55 year old lady slated for elective surgery referred from
elsewhere. She had received 2 uneventful transfusions of O
positive blood elsewhere one year previously.
 Grouping performed elsewhere.
Anti A Anti B Anti
AB
Neg Neg Neg
Anti
H
Neg
A
cells
4+
B
cells
4+
O
cells
Neg
Our grouping
Phas Anti
e
A
Anti
B
Anti
AB
Anti
H
H
A
lectin cells
B
cells
O
cells
poole
d
O
cells
dono
r1
O
cells
dono
r2
O
cells
dono
r3
IS/
LISS
Neg
Neg
Neg
Neg
Neg
4+
4+
Neg
Neg
Neg
Neg
4O
inc
Neg
Neg
Neg
Neg
-
4+
4+
Neg
Neg
Neg
Neg
37O
inc
Neg
Neg
Neg
Neg
-
4+
4+
Neg
Neg
Neg
Neg
Coo
mbs
Neg
Neg
Neg
Neg
-
4+
4+
Neg
Neg
Neg
2+
Lewis typing: Le(a-b+) suggesting secretor positive status
Inference: Parabombay (Oh secretor)
Para-bombay (Oh secretor)
 H deficient secretor
 Little or no A,B and H antigens
Ulex europaeus (common gorse)
 RBCs may be agglutinated by strong anti-H.
 Usually not agglutinated by anti-A and anti-B but may occasionally be
agglutinated by potent antisera or anti-AB
 Weak H like antibody (HI) reactive at low temperature almost always
present in sera. This is not inhibited by secretor saliva and does not
agglutinate cord cells.
 Normal H levels in saliva because of secretor status
Bombay categories
Classificatio
n
Gene
Bombay
(Red cell H
deficient,
non
secretor)
Glycosyltransfe
rase
Antigens
Secreti
on
Antibodies
hh sese
None or A
A and/or B and/or B
(depending on
ABO genotype)
in RBC or
serum
None
None
Anti-A, antiB, anti-H
Parabombay
(Red cell H
partially
deficient,
non
secretor)
hh (weak
variant)
sese
A and/or B
(depending on
genotype)
Weak A and/or B
depending on
genotype. Weak
H in Oh.Residual
H if A or B
enzymatically
removed in others
None
Anti H, anti A
and/or anti B
(depending
on ABO
genotype)
Parabombay
(Red cell H
deficient
secretor)
hh
Se
A and/or
B(depending
on genotype),
H in serum
Weak A and/or B
depending on
genotype and
weak H
H, A
and/or
B
Weak IH,
anti-A and or
anti-B
Bombay blood group not available! Can I transfuse
O group cells in Parabombay phenotype?
•(In red cell H deficient secretors) ‘Anti-HI is unlikely to be active at
37oC. ABO-identical blood, compatible by ICT at 37oC, can be used for
transfusion.’
•(In H partially deficient non secretors) ‘little information exists on the
clinical significance of anti H. Ideally Oh (Bombay) phenotype should be
selected, but if not available red cells of the appropriate ABO group (A
for Ah, B for Bh) compatible by ICT at 37oC, may be used.’
Source: The clinical significance of blood group
alloantibodies and the supply of blood for transfusion. (NHSBT
specification SPN214/1.1)
Case 7- ABO HDN
ABO - HDN
 Most common cause of HDN
 Maternal ABO antibodies that are IgG can cross the placenta and
attach to the ABO incompatible antigens of the fetal RBCs.
 Occur more frequently as high-titered IgG antibodies in group O
individuals than in group A or B individuals.
 ABO HDN is nearly always limited to A or B infants of
group O mothers with potent anti-A,B.
 The mother’s history of prior transfusions or pregnancies -
unrelated to the occurrence and severity of the disease.
 ABO HDN may occur in the first pregnancy and in any,
subsequent pregnancies
•Disease is manifested by the onset of hyperbilirubinemia and
jaundice within 12 to 48 hours of birth.
• Microspherocytes and increased RBC fragility in the infant are
characteristic of ABO HDN
 The DAT result is positive in 90 percent of the cases complicated
by jaundice
 The bilirubin peak is at 1 to 3 days
 Phototherapy is usually sufficient for slowly rising bilirubin levels
 With rapidly increasing bilirubin levels - exchange transfusion
with group O RBCs may be required
Case 8 – Transfusion and transplantation





39 year old male with Chronic Myeloid Leukaemia
Underwent bone marrow transplant using stem cells from HLA
identical sibling
Blood group of patient – AB positive
Blood group of donor – O positive
Received 2 units of compatible O+ve red cells in CMC, 6 days and 3
days prior to transplant
 1 day after transplant blood request for 1 unit packed cells




received.
Forward typing of patient sample – AB+ve
Backward typing of patient sample –AB+ve
Antibody screen – Negative
Issued 1 unit of O positive blood crossmatched and
compatible till Coombs
 1 day after transfusion haemoglobinuria reported.
DD of haemolysis following haematopoietic
stem cell transplantation
 Major ABO incompatibility between donor and recipient
 Minor ABO incompatibility between donor and recipient
 Major incompatibility for other blood group antigens
 Minor incompatibility for other blood group antigens
 Transfusion of erythrocytes incompatible with donor or patient
 Other causes of immune haemolysis eg. auto immune
haemolytic anaemia, drug induced
 Non immune haemolysis - TTP
Major incompatibility between donor
and recipient
 Due to recipient-derived antierythrocyte antibodies
 Haemolysis immediately after transplant
 Should be suspected when DCT positive
 Eluate must be tested for anti A and anti B to identify
haemolysis due to ABO mismatch
Pure red cell aplasia
 Occurs in major mismatch
 Recipient lymphocytes may remain and later produce antibodies





to transplanted donor derived erythrocytes
Can occur early or late (>100 days)
Suppress erythropoiesis by destroying erythroid progenitors
Diagnosis – Based on reticulocytopenia persisting for more than
60 days with absence of erythroids in marrow
Delayed erythrocyte engraftment occurs in 20% of ABO
mismatched transplants
?related to haemagglutinins
Minor incompatibility
 Usually delayed haemolysis
 May be due to plasma or ‘passenger lymphocytes’
 Plasma in a bone marrow product to be depleted depending
on agglutinin tire.
 Diagnosis - DCT positive with eluate showing anti A or anti B
Passenger lymphocyte syndrome
 Donor derived lymphocytes in the HPC graft form blood group specific
antibodies to patient erythrocytes
 Usually ABO antibodies but can involve other blood group antibodies.
(Kell, Kidd, Duffy reported)
 Risk factors - Treatment with cyclosporine alone without an
antproliferative agent like methotrexate, low intensity conditioning and
?PBSCT (theoretically, since it contains more B lymphocytes)
 Usually begins 1-2 weeks after transplant, persists for 5-10 days, then
subsides
 Hemolysis can be severe
 Can involve transfused compatible erythrocytes (bystander
immune hemolysis)
 Management - Transfusion of compatible erythrocytes at a pace
that matches hemolysis
 In massive hemolysis - Exchange transfusion may be considered
Incompatibility due to other blood
group antibodies
 Supected when DCT is positive and antibody screen positive
for donor or recipient
 The eluate shows an irregular antibody
Investigations in our patient
 Grouping of the patient: AB
 Grouping of the donor: O
 DCT +ve (2+)
 Post transfusion sample – ICT +ve; antibody screen positive
(1+)
 Antibody ID – anti Jkb (3+)
 Transfused red cells – Jkb positive
• Phenotype of stem cell donor- D+; C4+; E-; c4+; e4+; K-;
Fya3+; Fyb -; Jka3+; Jkb3+
• Phenotype of patient – D+; C4+; E-; c4+; e4+; K-;
Fya3+mf; Fyb 3+mf; Jka3+mf; Jkb3+mf
 Inference: Delayed haemolytic transfusion
reaction due to anti-Jk(b) formed by patient
reacting with transfused cells.
 Subsequent transfusions with Jkb negative fully
compatible units.
 However, haemoglobinuria persisted until 54 days after
transplant, when antibody screen turned negative.
Transfusion support in mismatched
transplants
 Must be compatible with recipient and donor
 Such compatible blood must be selected prior to
transplantation (from the start of myeloablation) as red cells
may persist for weeks
3 phases of transplantation
RBC tx:DCT negative and recipient
isoagglutinins not detectable
Beginning of preparation
Phase 1
Phase 2
Transplant
Recipient group
Compatible with
both donor and recipient
Donor group
Phase 3
Plasma and platelets tx:
Recipient red cells not
detectable
Selection of blood products in phase 2
Recipient
RBC group
Donor's
RBC group
Category of ABO
mismatch†
Erythrocyte
transfusion
Platelet or plasma
transfusion
A
O
Minor
O
A, AB
B
O
Minor
O
B, AB
AB
O
Minor
O
AB
AB
A
Minor
A, O
AB
AB
B
Minor
B, O
AB
O
A
Major
O
A, AB
O
B
Major
O
B, AB
O
AB
Major
O
AB
A
AB
Major
A, O
AB
B
AB
Major
B, O
AB
A
B
Minor and major
O
AB
B
A
Minor and major
O
AB
A
A
None
A, O
A, AB
B
B
None
B, O
B, AB
AB
AB
None
AB, A, B, O
AB
O
O
None
O
O, A, B, AB
Blood group chimerism
 Forward or backward grouping discrepancies and mixed field
agglutination
 Intrinsic characteristic of mismatched transplantation eg. A group
recipient transplanted with O group donor
Anti Anti Anti A
-A
-B
-AB cell
s
MF
Neg MF
B
cell
s
Neg 4+
Neg Neg Neg 2+
4+
O
cell
s
Inference
Neg Mixed field on front type may be due to
transfused O cells/ conversion. Recipient
red cells still seen. Reverse type is
recipient group
Neg Front and reverse type donor group,
indicating full donor engraftment
Case 9 – ABO mismatch in kidney transplant
 A 21 year old gentleman of blood group O-positive, with end
stage kidney disease
 Only available related donor was mother whose blood group was
A-positive.
 HLA-crossmatch by Complement Dependent Cytotoxicity (CDC)
was negative
 Anti-A titre at presentation was 1:512.
 Desensitization protocol involving Rituximab, plasmapheresis
and Anti-thymocyte globulin initiated
 Column agglutination test used for monitoring
 Schedule of monitoring: Pre-pheresis, immediately post pheresis




and 24 hours later to detect rebound
A group FFP used for replacement in order to quench anti A in
recipient.
Rituximab interfered with HLA CDC crossmatch. HLA antibody
monitoring supplemented with solid phase assays
Anti A titre reduced to 1:2 against which transplant performed
successfully.
3 years post transplant - graft functional, patient well
Principles of ABOi KT
 Removal of existing antibody – plasma exchange, double
filtration plasmapheresis, immunoadsorption
 Preventing recurrence of antibody – IVIg,
immunosuppression, splenectomy/ Rituximab
 Waiting until titres achieve safe levels for transplantation
Range of Plasmapheresis Treatments required Before and After ABOiKT
based on initial antibody titre
Lipshutz GS et al. Arch Surg. 2011;146(4):453-458
Monitoring of antibody titre
 Baseline antibody levels (> 256) predict severity of
antibody mediated graft injury and graft survival.
Reports suggest this is no longer true in patients that
received tacrolimus or mycophenolate mofetil for
immunosuppression*
 Titre cutoff for transplant 16– empirically chosen
 Titre endpoint based on IgG or IgG and IgM.
*
H. Chung, J.Y. Lee, S. H. Kang et al., “Comparison of clinical outcome between high and low
baseline anti-ABO antibody titers in ABO-incompatible kidney transplantation,” Renal Failure, vol.
33, no. 2, pp. 150–158, 2011.
Summary
 ABO discrepancy recognition AND resolution is
imperative in the blood bank.
 ABO discrepancies can present themselves as a front type
problem, reverse type problem, or combination of both
 If ABO discrepancy resolution cannot be performed, and
blood is needed immediately, transfuse with O red cells
If we cannot end our differences, at least we can make
the world safe for diversity
John F. Kennedy
Acknowledgement : Dr. Mary Purna Chacko
Asst. Prof: Dept of Transfusion Medicine