1. Lok Samarpan Regional Blood Bank and Research Center,
A New Approach in Classification of ABO Blood Group Discrepancies. [with illustration on the cases investigated]
Sanmukh R. Joshi
Lok Samarpan Regional Blood Bank and Research Center,
Surat
November 24th, 2018, Session: Immunohematology , m

2. ABO blood grouping
ABO group is assigned as per Ag on RBC but verified by absence of corresponding Ab in serum.
Group
Forward Group: Cell
Reverse Group:Serum
Anti-A
Anti-B
A1-Cells
B-Cells O +
Although looks simple, it is not hassle-free!
Discrepancy does result due to several reasons.
Group
Forward Group: Cell
Reverse Group:Serum
Anti-A
Anti-B
A1-Cells
B-Cells
0? or B? O +

3. ABO Group Discrepancies
When ‘forward’ group does not tally with ‘reverse’
Group
Forward Group: Cell
Reverse Group:Serum
Anti-A
Anti-B
A1-Cells
B-Cells
0? or B? O +
Problem could be with RBCs or serum or both.
Must be investigated to assign proper group to prevent serious compatibility errors.

4. ABO Group Discrepancies
Rule out common source of errors by repeat test before embarking into investigation.
Clerical errors/ mixing up in the sample/
failed to add reagents or serum,
Out-dated/ contaminated reagents.
Fibrin clots formation,
background haemolysis, hinder viewing the result
Cell concentration, too heavy or too light
Uncalibrated centrifuge,
manufacturer-instruction not followed

5. Classification as to in 4 categories (after Harmengis)
Discrepancy
Observations
Condition
Group I
Unexpected Rxn in reverse group, weak, missing Ab,
Chimerism (BT, BMT feto maternal bleed), infants/ elderly, Hypogammaglobulinemia (leukemia, immunodeficiency diseases)
Group II
Unexpected Rxn in forward group, weak, missing Ag
Subgroups of A or B, Leukemia, Acq B (in gram neg septicemia, intestinal obstruction and cancer of colon or rectum).
Group III
Discrepancies in forward/ reverse group due to plasma protein abnormalities (rouleaux phenomena)
Elevated globulin level (MM, Waldenstrom’ macroglobulinemia, plasma cell dyscrasias, Hodgkin lymphoma), Plasma expanders like dextran, polyvinyl pyrrolidone, Wharton’s jelly (in cord blood)
Group IV
Discrepancies in forward/reverse group due to miscellaneous cause
Exposure crypt antigen (T-transformation, Polyagglutination), AIHA (Cold and warm type), unexpected alloantibody, (acriflavin antibody), cis-AB case.
A great deal of ambiguity persists with this classification

6. Classification, a Q-based approach
13-Qs based classification of discrepancies meant not only for blood group anomalies but also for problems in compatibility tests as associated with underlying cause for it. Jovana & Heddle (2014)
Diagnosis ?
Transfusion history?
Pregnancy history?,
Passive Anti-D,
the medication history ?
ABO group?
Rh type?
DAT positive? If yes, what were the results of eluate testing?
Ab reacting? temperature
React with enzyme-treated RBCs?
Ab reactive with mono-specific Anti-IgG DAT?
agglutination unusual appearance?
agglutination show ing variable reactivity?
Antibody react with umbilical cord cells?

7. A novel approach based on Etiology (proposed)
Intrinsic to RBCs
Extrinsic to RBCs
Weak ABH antigens:
Genetic variants
Acquired Loss leukemia
de novo transformation in
infections/ cancers/ pregnancy
Acquired B
Exposed cryptantigens, T
Acquired antigens in obstetric
patient (new observation)
Reagent Dependent Anomalies
Contaminant antibodies in anti-
sera (polyclonal)
Chemicals, promoting specific antibody to react
Chemicals, act as antigen to form complex attaching RBCs

8. Weak ABH antigens: Genetic Variants
Antigen showing weaker agglutination than normal A/ B with anti-A/ -B, are called "weaker variant”.
Weak H also gives weak A or B (para-Bombay)
Classified as per serological features
RBCs Tested with a battery of antisera, some of several may react
absorption-elution of antibody to detect weak antigen,
Secretor status to detect salivary antigen.
Missing iso-antibody in plasma
Family may show inheritance of the trait
have reduced survival of RBCs in gr O patient so
Recipient be consider as group O, so to transfuse group O

9. ABH weaker Variants….
Enormous heterogeneity, ambiguity in interpretation
were increased beyond one’s ability to grasp and keep track on.
People started naming as their own ‘will’ or ‘whim’, say ‘name of population, country’ etc. where was it first found!
….and then came the variants named like Afin and Aend
Peter Issitt wrote with a sigh of relief, stating “finally the time has come to finish them with variants like Afin and ended them with Aend)!’

10. Genetic variants of A: Serological features
Anti-A
Anti-A,B
Saliva Ag
Anti-A1 in serum A2 +4
A, H
Sometimes A3
+2-3(mf)
Abantu
+1-2 H
Yes Ax
+2-3
Often Am No Ay 0#
Ael / (Ao)
Aend +1
Afin
#anti-A can be absorbed to and eluted from cells in this subgroup,
Similarly, variants for B and H antigens were considered

11. Para-Bombay case VN, 60Y male; a case of renal calculus for surgery
Forward grouping
Reverse grouping
group -A -B
-A+B -H
-A1
A cell
B cell
O cell
Bombay? +m +4
+4-3
RBCs:
Test with panel of antisera:
Anti-A: all 12 did not react
Anti-B: 3 of 12 sera, 1-w+
Anti-A+B: 3 of 8 sera, w+
Anti-H (Oh): 2 of 6 +w
Anti-H (Ulex lectin): 0.
Serum: complement mediated haemolytic anti-H, Titer values
RBCs
37°C
4°C A 64 B 32 O
128

12. Absorption-Elution and Family study
Eluate: from anti-B reacted weak; stronger from anti-A+B
and the strongest from anti-A+B +H (Oh), that was
inhibited by B/H secretors; not by non-secretors.
Saliva secretor status: Non-secretor
Family:
No Bh in 25 members/ 3 generations
B in VN inhibited due to lack of H;
Children revealed B when got H
from the mother.

13. Acquired Loss of Antigens in Leukemia
Patient
Forward Gr
Reverse Gr
Group
Anti-A
Anti-B
A1-Cells
B-Cells
S.P.S.
0/+ (mf)* 4+
O/B? AK
O/A?
*78% RBCs unagglutinated, 22% RBCs agglutinated
Joshi et al, 1998;
Joshi et al, 2018.
A + H PS H
B + H
AB + H H
Mixed field agglutination reaction pattern

14. Acquired Gain of Antigens (in infections/ cancer)
Acq ‘B’ phenomenon:
Forward Grouping
Reverse Grouping
Group
Anti-A
Anti-B
A1-Cells
B-Cells
O-Cells 4+
W-1+
AB?
Mechanism Involved
B: D-galactose A: N-acetyl - D-galactosamine
Microbial enzymes
deacetylase
Mimics B

15. Exposed crypt-antigens: (T- transformation)
Acquired Gain of Antigens
(infections/ cancer)
Exposed crypt-antigens: (T- transformation)
Crypt-antigens are underneath structures in the cell membrane
Exposed through cleavage by bacterial enzymes
Antibodies to these antigens are naturally occurring and present in all human sera.
Grouping anti-sera of human origin with these antibodies may show an anomalous reaction.
With an advent of monoclonal antisera, such problems have historical significance.

16. Acquired Gain of Antigens
in obstetric patients
A new phenomenon yet to be recognized!
Please go through the poster: 123
My colleague will explain the concept behind the possible mechanisms involved.
Your feedback will be highly appreciated
The phenomenon features both intrinsic and extrinsic factors causing grouping anomaly.

17. Grouping Anomalies due to the Factors Extrinsic to RBCs
Associated with surrounding medium,
mainly reagent dependent anomalies
Antibody in serum interfering in reverse grouping
Cases
Forward Grouping
Reverse
Grouping
Antibody Specificity
Anti-A
Anti-B
A1 Cell [Le(a-b+)]
B Cells
[N+]
1. Bal. 4+
Allo-Anti-N
[1:32, 37C]
2. Azi.
Allo-Anti-Leb [1:256, HDN]
3. Sal.
[F/78yr] w+
Auto-anti-I
[>1:130,000]

18. Antiserum with contaminant antibodies interfere in cell grouping.
Anti-Mg present in anti-A reagent
Cases
Forward Grouping
Reverse Gr
Anti-A**
Anti-B
Anti-A,B
A1-Cell
B-Cell
NS/DM* 4+
Joshi et al,1972
* Reacted with 5 different anti-Mg reagents
** had contaminant anti-Mg
Other case: RBCs of an infant with HDN agglutinated by
a batch of anti-B, but not with 2 other anti-B/ anti-(A+B)
Primary anti-B had anti-Gm reacting with Gm on IgG
coated RBCs (DAT+).
Mother gr O, father gr A the baby gr O (later);
Simmons et al. (1971)

19. Blood grouping reagents
Reagent antiserum fortified with chemicals
Prelim group on slide as AB; in tube, forward/reverse as B
Anti-H in donor’s serum reacting in presence of citrate
present in grouping reagent
Test RBCs
Suspending
medium
Blood grouping reagents
Anti-A
Anti-B
Anti-AB
SRC. gr B
Autolog serum +
Homolog serum –
Normal saline
Group B
S.R.C.’s serum
Group O
Joshi (1997)

20. Antiserum had streptomycin, the patient had anti-streptomycin
Test RBCs
Suspending
medium
Monoclonal antisera
Anti-A
Anti-B
Anti-AB
SV, gr A
Autologous serum +
Homolog. serum –
Normal saline
Donor, gr A
Patient’s serum
Donor, gr O
Joshi SR. (2001)

21. Summary Blood group anomalies have variety of causes.
due to antibodies to RBC antigens requiring
certain chemicals to exhibit reaction
may or may not be of clinical significance, yet
incur nuisance in delayed blood supply.
A new way to classify the grouping anomalies is
proposed based on causative factors involved