ABO discrepancy in transfusion medicine

1. ABO DISCREPANCY
Presented by: Dr. Bharat Ninggo
Moderator: Prof. A. Barindra Sharma

2. Introduction:
• ABO Discrepancy means when the Red cell grouping does not match the
serum grouping results
• In other words the forward does not match the reverse
• It may be due to followings:-
• Technical errors
• Clinical discrepancy:-
Problems with the patient’s serum(Reverse Grouping)
Problems with the patient’s RBC(Forward Grouping)
Problems with both the serum & cells
An extra positive reaction or a weak or missing reaction in the forward and
reverse grouping
• In general, mostly RBC & serum grouping reactions are very strong (3+ to 4+)
and the weaker reactions typically represent the discrepancy.

3. Road map for ABO discrepancy workout

5. Categories of ABO Discrepancies:
1. Technical Discrepancy
2. Clinical Discrepancy
i) Group I Discrepancy
ii) Group II Discrepancy
iii) Group III Discrepancy
iv) Group IV Discrepancy

6. Technical Discrepancy:
Common sources are
• Incorrect or inadequate identification of blood specimens, test tubes, or
slides
• Inadequate washing of cells
• Pro-zone & Post-zone effect. Cell suspension either too heavy or too
light
• Clerical errors or incorrect recording of results
• A mix up in samples
• Missed observation of hemolysis
• Failure to add reagents
• Failure to add samples
• Uncalibrated centrifuge
• Over-centrifugation or under-centrifugation
• Contaminated reagents, dirty glassware

7. Resolution of Technical Error:
• All technical factors should be reviewed carefully and corrected.
• A new sample must be taken for incorrect sampling & repeat the
testing.
• Repeat the testing using a saline suspension of RBCs after adequate
washing of cells, if the initial testing was performed using suspended
RBCs and serum.
• Antigen-Antibody ratio:
Pro-zone effect: Excess of antibody lead to unbound immunoglobulin.
Post-zone effect: Excess of antigen causes surplus of antigen binding
sites.
To avoid this usually 2 volumes of serum and 1 volume of 2-5% red
cell suspension in NS are used for optimum antigen-antibody reaction.

8. Clinical Discrepancy:
• Here the problem lies with the patients
• Clinical history is critical in solving these types
• Age , diagnosis, transfusion history, h/o pregnancy and medication

9. Group I Discrepancy:
 Most common
 Unexpected reactions in reverse grouping
 Due to weakly reacting or missing antibodies
 The patients has depressed antibody production or can’t produce the
ABO antibodies

10. Common Causes of Group-I Discrepancy:
 Newborns (ABO antibody production is not detectable until 3 to 6
months of age)
 Elderly patients (Production of ABO antibodies is depressed)
 leukemia(eg.CLL)or malignant lymphoma or hypogammaglobunemia
 Patients using immunosuppressive drugs
 Patients with congenital or acquired agammaglobulinemia
 Immunodeficiency diseases
 Bone marrow or hematopoietic progenitor stem cell transplants
 Plasma or exchange transfusion
 ABO subgroups

11. Resolution of common Group-I Discrepancies:
 For newborns only forward grouping is done till 4months of age
 Resolves by enhancing the serum grouping reactions
 By incubating the reagent cell-serum mixture at room temperature for
15-30 mins
 If still no reaction occurs after centrifugation then the cell-serum
mixtures can be incubated at 4℃ for 15-30 mins
 An auto control and O cell control must always be tested concurrently

12. Rare Group-I Discrepancies:
CHIMERISM
 The presence of two cell populations in a single individual
 It was first discovered in twins born to a Gr-O mother and Gr-B
father with a mixture of both B & O cells instead of expected group of
either B or O
 Detecting a separate cell population may be easy or difficult,
depending on what percentage of cells of the minor population are
present
 Reaction from Chimerism are typically mixed-field

13. True Chimerism:
 Occurs only in twins and is rarely found
 Exist throughout the life
 Due to in utero exchange of blood because of vascular anastomosis
 As a result two cell populations emerge, both of which are recognized
as self
 Hence expected iso-agglutinins are not present in the reverse
grouping
 If the patient or donor has no h/o a twin, then the Chimera may be
due to Dispermy (Two sperm fertilizing one egg) and indicates
mosaicism

14. Artificial Chimerism:
 More common then True Chimerism
 This yield mixed cell populations as a result of the following:
• Different ABO type blood transfusions; eg: Group O cells given to an A
or B patient
• Transplanted bone marrow or hematopoietic progenitor stem cells of a
different ABO type
• Exchange transfusions
• Feto-maternal bleeding

15. Group II Discrepancies:
 Unexpected reactions in the forward grouping
 Due to weakly reacting or missing antigens
 This group is least frequently encountered
Common causes are:-
Subgroup of A & B
 Leukemia and lymphoma which yield weakened A or B antigens.
 Excess antigen of blood group soluble substances
 ‘Acquired B’ phenomenon most often associated with cancer of colon
or gram negative septicemia

16. Resolution of Gr-II Discrepancies:
Repeat testing of same sample using a saline suspension of RBCs after
adequate washing should be done
This will also wash out blood group-specific soluble substances
present in the plasma
The agglutination reaction can be enhanced by incubating the test
mixture at room temperature for up to 30mins
 If reaction is still negative, incubate the test mixture at 4℃ for 15-
30mins. Include group-O & autologous cells as control
 RBCs can also be pretreated with enzymes (eg. papain) and retested
with reagent antisera
 To solve discrepancy caused by anti A1 in a group A individual, red
cells should be tested with Dolichos biflorus lectin, which agglutinates
group-A1 only but not A2 and weaker A subgroups.

17. Acquired B Phenomenon:
 The acquired B antigen arises when bacterial enzymes modify the
immunodominant group A sugar N-acetyl-D-galactosamine into D-
galactosamine, which is similar to the group B sugar D-galactose to
cross react with anti-B antisera
 Most often associated with disease of digestive tract (eg: Ca Colon)
& gram negative septicemia
 This pseudo-B antigen is formed at the expense of A1 antigen and
disappears following the patient’s recovery
 The reaction of the appropriate antisera with these acquired antigens
demonstrates a weak reaction, often yielding a mixed-field
appearance.

18. Resolution of Acquired B Phenomenon:
 Testing the patient’s serum against autologous RBCs gives a negative
reaction, because the anti-B in serum does not agglutinates the
patient’s RBC with the acquired B antigen
 The acquired B antigen is also not agglutinated when reacted with
anti-B that has a pH greater than 8.5 or less than 6
 Secretor studies can also be performed, if the patient is a secretor
only A substance is secreted in the acquired B phenomenon
 Treating RBCs with acetic anhydride re-acetylates the surface
molecule, that markedly decreases the reactivity of acquired B cells
with anti-B. The reactivity of normal B cells is not effected

19. Group-III Discrepancies:
 These discrepancies are caused by protein or plasma abnormalities
and result in rouleaux formation or pseudoagglutination
 Causes are :-
i) Elevated levels of globulin from certain disease states, such as
Multiple myeloma, Waldenstrom’s macroglobulinemia, other plasma
cell dyscrasias, and certain moderately advanced cases of Hodgkin’s
lymphomas
ii) Elevated level of fibrinogen
iii) Plasma expanders, such as dextran and polyvinylpyrrolidone
iv) Wharton’s jelly in cord blood samples

20. Resolution of common Group-III Discrepancies:
 Rouleaux formation can be solved by washing the cells with normal
saline 6-8 times
If reverse grouping is affected, perform saline replacement technique
in which serum is removed and replaced by an equal volume of saline
(saline disperses cell)
 Wharton’s jelly is a viscous mucopolysaccharide material present on
cord blood cells that may cause red cell aggregation
Thoroughly washing cord cells 6-8 times with saline should alleviate
rouleaux formation due to Wharton’s jelly

21. Group IV Discrepancy:
 These discrepancies are due to miscellaneous problems that have the
following causes-
i) Poly-agglutination resulting from inherited or acquired
abnormalities of the red cell membrane, with exposure of ‘cryptic auto
antigens’. All human sera contains naturally occurring antibodies to
such cryptic antigens. Those abnormal red cells are agglutinated by sera
from group A, B, & AB individuals
ii) RBCs which are so heavily coated with Cold Autoantibodies can
spontaneously agglutinate, independent of the specificity of reagent
antibody
iii) Unexpected ABO iso-agglutinins
iv) Unexpected non-ABO alloantibodies
v) Cis AB

22. Resolution of common group IV Discrepancies:
Cold Autoantibodies:
• The cells with cold auto-antibodies often yield a positive DAT
• If the antibody in serum reacts with all adult cells (eg. anti-I), the
reagent A1 & B cells used in reverse grouping also agglutinates
• To solve this, the patient’s RBCs could be incubated at 37℃ for a
short period, then washed with warm saline(37℃) 3 times & retyped
• If this is not successful then patient’s RBC can be treated with 0.01M
dithiothreitol (DTT) to disperse IgM related agglutination
• As for the serum, the reagent RBCs and serum can be warmed to
37℃ for 10-15mins, mixed and tested
• Cold auto-adsorption technique could be performed to remove the
cold autoantibodies from the patient’s serum

23. Poly-agglutination:
• RBCs are agglutinated by almost all samples of human sera from
adults but not by autologous serum
• Poly-agglutination state may be transient or persistent
• Transient state results from the exposure of cryptic antigens by
bacterial enzymatic activity during the course of an infection
• Persistent state may be a consequence of somatic mutation leading to
an enzyme deficiency that results in exposure of a normally cryptic
antigen, Tn. Most human sera contain anti-Tn. Tn poly-agglutination
is usually associated with leukemia
• In poly-agglutination state auto control and DAT will be negative

24. Pan-agglutination:
• Occurs when the patient’s serum react with all the reagent red cells
sample in both the screening and identification panel
• It can musk clinically significant alloantibodies
• Evaluations involve the assessment of the intensity of reactivity with
the reagent red cells used.
• And whether the auto control is positive or not.

26. Unexpected ABO iso-agglutinins:
• This type of discrepancy include A2 & A2B individuals who can
produce naturally occurring anti-A1.
• In these cases serum grouping can be done using A1, A2, B, O and
autologous control cells. If the antibody agglutinates only A1 cells, it
is most likely anti-A1.
• The patient’s RBCs can be tested with Dolichos biflorus to confirm
the presence of ABO subgroup.
• Dolichos biflorus will only agglutinate A1 cells but not A2 and weaker
A subgroups.

27. Unexpected non-ABO alloantibodies:
• Unexpected alloantibodies in patient’s serum (eg. anti-M) may cause
discrepancy.
• Reverse grouping cells possess other antigens in addition to A1 & B,
and it is possible that unexpected antibodies present in the patient’s
serum will react with these cells.
• In this situation an antibody identification panel should be performed
with the patient’s serum.
• Once unexpected alloantibodies are identified, reagent A1 & B cells
negative for the corresponding antigen should be used in reverse
grouping.

28. Cis-AB:
• Cis-AB refers to the inheritance of both AB genes from one parent
carried on one chromosome and an O gene inherited from the other
parent
• This results in the offspring inheriting three ABO genes instead of two
• RBCs with the Cis-AB phenotype express a weakly reactive A & B
antigen
• Most Cis-AB individuals having weak anti B in the serum leads to
ABO discrepancy in reverse grouping. This weak anti-B reacts with all
ordinary B RBCs but not with cis-AB RBCs

29. Other Rare Trouble Shooting Situations in Red Cell Serology:
• Small fibrin clots in plasma or incompletely clotted serum can be
mistaken for red cell agglutinates
• Unexpected absence of anti-A in patients receiving equine-derived
immunoglobulins
• Unexpected absence of anti-B in children who are sterile, free of
bacteria and result from long term parenteral and enteral nutrition.
• Recent infusion of IV immunoglobulin, which may contain ABO iso-
agglutinins
• Individuals having unexpected antibody against acriflavine, the yellow
dye used in some commercial anti-B reagents can cause false
agglutination in forward grouping.

30. To summarise causes of ABO discrepancy

31. THANK YOU