Abo discrepancies

1. ABO Discrepancies

2. Blood Grouping
• There are 2 components to blood typing:
o Test unknown cells with known antibodies
:forward grouping
o Test unknown serum/plasma with known red
cells: reverse grouping
• The patterns are compared and the blood group
is determined.

3. Cell grouping (Forward grouping)
– Tests the patients red cells with known Anti-A
& Anti-B to determine the antigen expressed
Serum grouping (Reverse grouping)
– Test the patients serum with known A & B cells
to determine the presence of antibody

9. Red Cell Suspensions for Blood Grouping
• 2-5%: Test Tube Method
• 0.8-1%: Gel technology
• 1%: Microplate

11. 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:-
1.Technical errors
2.Clinical discrepancy:-
• Problems with the patient's serum(Reverse
Grouping)

13. • 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.

15. Common sources of error
• 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

16. • Failure to add reagents
• Failure to add samples
• Uncalibrated centrifuge
• Over-centrifugation or under-centrifugation
• Contaminated reagents, dirty glassware

17. 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.

18. 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

19. 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

20. O group.
B group

21. 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

22. Resolution of group
1discrepancy
➤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°C for 15-30

23. Rare Group-I Discrepancies
CHIMERISM
➤ The presence of two cell populations in a
single individual
➤ Reaction from Chimerism are typically mixed-
field

24. Mixed field reaction

25. 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 antibodies are not present in the
reverse
grouping

26. 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 B patient
• Transplanted bone marrow or hematopoietic
progenitor stem cell different ABO type
• Exchange transfusions
• Feto-maternal bleeding.

27. 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.
➤'Acquired B' phenomenon most often associated
with cancer of
colon or gram negative septicemia

28. Group AB. Group A

29. Resolution of Gr-II
Discrepancies:
• Repeat testing of same sample using a saline
suspension of RBCs after adequate washing
should be done
• 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°C for 15- 30mins.
• Include group-O & autologous cells as control

30. • 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-Al only but not A2 and
weaker A subgroups.

31. 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

35. 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

36. ➤ 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

37. 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

38. ii) Elevated level of fibrinogen
iii) Plasma expanders, such as dextran and
polyvinylpyrrolidone
iv) Wharton's jelly in cord blood samples

39. 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)

40. 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

41. • In poly-agglutination state auto control and
DAT will be negative

42. • 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

43. 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 Al & B cells
used in reverse grouping also agglutinates
• To solve this, the patient's RBCs could be
incubated at 37°C for a short period, then
washed with warm saline(37°C) 3 times & retyped

44. • 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°C for 10-15mins, mixed and
tested
• Cold auto-adsorption technique could be
performed to remove the cold autoantibodies
from the patient's serum

48. Ans: C

49. A.A sub group
B.Excess antigen
c.rouleaux formation
d.Acquired B antigen
ans. A

50. Which typing results are most likely to occur
when a patient has an acquired B antigen?
A. Anti-A 4+, anti-B-3+, A1 cells neg, B cells
neg
B. Anti-A 3+, anti-B neg, A1 cells neg, B cells
neg
C. Anti-A 4+, anti-B 1+, A1 cells neg, B cells
4+
D. Anti-A 4+, anti-B 4+, A1 cells 2+, B cells
neg

51. Thank you