1. RESOLVING ABO DISCREPANCIES
by DR G P Saluja Alchemist Hospitals Ltd Panchkula

2. Case 1
Blood Sample of 20 years Male for Blood Grouping 20 years through OPD
He had donated blood earlier
He told that his blood group is “O Rh+ve”

3. Forward Grouping A Cells : Neg B Cells : Neg Rh : 4+
Blood Group : O Rh +ve

4. Forward & Reverse Grouping
A Cells : Neg ANTI A Neg
B Cells : Neg ANTI B
Rh :
Blood Group : O Rh +ve
There is Discrepancy between Forward & Reverse Grouping But as the reaction in Anti B is 4+, it shows presence of A antigen

5. Resolving Discrepancy
A1 Lectin : Neg Control : Neg
Anti H Lectin : ve O cells : Neg
Anti AB : Mf A2 Cells : Neg
Blood group is Neither A1 nor A2.

6. Absorption & Elution 5th wash gave negative reaction with A cells
Elute gave negative reaction with o cells
Elute gave Positive reaction with A cells
This confirms the presence of A Antigen & hence Weak A blood group Confirmed

7. CASE 2 Forward Grouping FORWARD GROUPING A CELLS +2 B CELLS +4 Rh NEG
BLOOD GROUP AB Rh-ve
Control NEG

8. Forward & Reverse Grouping
FORWARD GROUPING REVERSE GROUPING
A CELLS ANTI A
B CELLS ANTI B NEG
Rh NEG Du NEG
BLOOD GROUP AB Rh-ve BLOOD GROUP B Rh-ve
Control NEG

9. Antibody Screen
Three Cell Panel for Antibody screen is Negative

10. Resolving Discrepancy
Anti-A1 Lectin gave negative reaction.
Absorption and Elution technique confirmed the presence of A antigen.
The findings therefore are:
The blood group therefore is AB Rh-ve with Anti A1 Antibody present
Forward Grouping
Reverse Grouping
A antigen +ve
B antigen +ve
Anti A1 +ve
Anti B -ve

11. CASE 3
Patient Dharmender 33 yrs Male admitted with Dengue MR IP No 13/5627 Already operated at Fortis Hospital and blood group given was B Rh-ve
Forward Grouping
FORWARD GROUPING A CELLS
B CELLS
Rh NEG
BLOOD GROUP AB Rh-ve
Control NEG

12. Forward and Reverse Grouping
CASE 3
Forward and Reverse Grouping
FORWARD GROUPING REVERSE GROUPING
A CELLS ANTI A NEG
B CELLS ANTI B NEG
Rh NEG Du NEG
BLOOD GROUP AB Rh-ve BLOOD GROUP AB Rh-ve
Control NEG

13. Resolving Discrepancy
Anti-A1 Lectin gave negative reaction.
Absorption and Elution technique confirmed the presence of A antigen.
Blood group was confirmed to be A2B Rh-ve

14. CASE 4
Pre-operative (TKR) blood grouping was done for R M SINGLA 68 years Male
A Cells : ANTI A Neg
B Cells : Neg ANTI B Neg
Rh :
Blood Group : There is discrepancy between forward and
reverse grouping

15. Enhancement Method
With the Enhancement method the strength of reaction with “B” cells has increased to 2+. Hence it shows presence of Anti B antibodies and the blood group is confirmed as A RH+VE

16. RBC Precursor Structure
Glucose
Galactose
Precursor Substance (stays the same)
N-acetylglucosamine
Galactose

17. Formation of the H antigen
RBC
Glucose
H antigen
Galactose
H enzyme is fucosyltransferase
N-acetylglucosamine
Galactose
Fucose

18. H antigen The H antigen is the foundation upon
which A and B antigens are built
A and B genes code for enzymes that add
an immunodominant sugar to the H
antigen
Immunodominant sugars are present at the terminal ends of the chains and confer the ABO antigen specificity

19. A and B Antigen
The “A” gene codes for an enzyme (transferase) that adds N-acetylgalactosamine to the terminal sugar of the H antigen
N-acetylgalactosaminyltransferase
The “B” gene codes for an enzyme that adds
D-galactose to the terminal sugar of the H antigen
D-galactosyltransferase

20. Formation of the A antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
N-acetylgalactosamine
Fucose

21. Formation of the B antigen
RBC
Glucose
Galactose
N-acetylglucosamine
Galactose
Galactose
Fucose

22. Genetics
The H antigen is found on the RBC when you have the Hh or HH genotype, but NOT from the hh genotype
The A antigen is found on the RBC when you have the Hh, HH, and A/A, A/O, or A/B genotypes
The B antigen is found on the RBC when you have the Hh, HH, and B/B, B/O, or A/B genotypes

23. O>A2>B>A2B>A1>A1B
H antigen
Certain blood types possess more H antigen than others:
O>A2>B>A2B>A1>A1B
Greatest amount of H
Least amount of H

24. Why do Group O individuals have more H antigen than the other groups?
Group O individuals have no A or B genes to convert the H antigen to A or B antigens….that means more H antigen sites

25. ABO Subgroups
ABO subgroups differ in the amount of antigen present on the red blood cell membrane
Subgroups have less antigen
Subgroups are the result of less effective enzymes. They are not as efficient in converting H antigens to A or B antigens (fewer antigens are present on the RBC)
Subgroups of A are more common than subgroups of B

26. Subgroups of A The 2 principle subgroups of A are: A1 and A2
Both react strongly with reagent anti-A
To distinguish A1 from A2 red cells, the lectin Dolichos biflorus is used (anti-A1)
80% of group A or AB individuals are subgroup A1
20% are A2 and A2B

27. A2 Phenotype Why is the A2 phenotype important?
A2 and A2B individuals may produce an anti-A1
This may cause discrepancies when a crossmatch is done (incompatibility)
What’s the difference between the A1 and A2 antigen?
It’s quantitative
The A2 gene doesn’t convert the H to A very well
The result is fewer A2 antigen sites compared to the many A1 antigen sites

28. A1 and A2 Subgroups* A1 A2 4+ Anti-B 900 x103 3+ Anti-B & anti-A1
Anti-A antisera
Anti-A1 antisera
Anti-H lectin
ABO antibodies in serum
# of antigen sites per RBC A1 4+
Anti-B
900 x103 A2 3+
Anti-B & anti-A1
250 x103
*Adapted from Flynn, J. (1998). Essentials of Immunohematology

29. Other A subgroups There are other additional subgroups of A
Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu
A3 red cells cause mixed field agglutination when polyclonal anti-A or anti-A,B is used
Mixed field agglutination appears as small agglutinates with a background of unagglutinated RBCs
They may contain anti-A1

30. A 1 4+ - +,- A,H 810-1170 A 2 2+ + in 1%-8% 240-290 A3 2+ mf 3+
RBC Pheno-
types
Reaction of Red Blood Cells with known Antiserum
Reaction of Serum with Known Cells
Substances present in saliva of secretors
Presence of A Transferase in Serum
Number of antigen sites
RBC X 103
Anti A
Anti B
Anti A,B
Anti H
Anti A1
A1 Cells
B Cells
O Cells
A 1 4+ -
+,-
A,H
A 2 2+
+ in
1%-8% A3
2+ mf 3+
Sometimes 35 Ax
-/w
Almost always
A(trace),H
Rarely 5
Aend
W mf H No
3.5 Am
Yes 1 Ay
Trace
Ael +
0.7

31. CONCLUSION Always put up Reverse blood grouping.
Blood group should only be confirmed when the findings of the forward grouping match with those in reverse grouping.
There can be fatal reaction

32. Be a Life Guard