Transfusion medicine

• Each case of ABO discrepancy must be evaluated on an individual basis
• First step is to thoroughly check for technical errors and repeat testing with the same or fresh sample if required; further workup should be done if the discrepancy persists
• Reaching out to previous institutions for any records of past ABO typing or blood transfusions can provide additional insights
• Molecular testing can be used if available when serologic testing does not fully resolve the discrepancy (Transfusion 2018;58:2232)
• Patient's blood group should only be reported once the discrepancy has been fully resolved and documented
• Miscellaneous causes
  • Incidence of ABO discrepancies has reduced considerably with the automation and standardization of testing procedures; these can be avoided by regular training of lab staff and quality checks of testing
• Forward typing
  • To address weak forward typing reactions, prolonging the incubation time is often effective, as it strengthens the binding of antibodies to weakly expressed antigens on red cells
  • Specific reagents (lectins, monoclonal agents, etc.) can be used to distinguish ABO subgroups
    • Dolichos biflorus lectin binds to A1 cells but not A2
    • Ulex europaeus lectin can be used to assess the degree of H antigen expression
    • Monoclonal anti-A,B reagent can be used to detect weak A antigens (see Enzyme treatment)
  • In cases where a B(A), A(B) phenotype or acquired B phenotype is suspected, using different reagents may confirm the patient's blood type
  • Increase reactions workup involves washing the red cells to remove any residual plasma that could be causing pseudoagglutination
  • Observing the sample under the microscope allows for the identification of mixed field agglutination patterns, which might indicate a recent transfusion or the presence of 2 cell populations (fetomaternal hemorrhage, intrauterine fetal transfusion, chimerism and mosaicism)
• Reverse typing
  • To address weak reserve typing reactions, prolonging incubation time can enhance the reaction, as it allows weak antibodies to bind more effectively
  • In certain situations, incubation at a lower temperature, such as 4 °C, may further enhance weak reactions of antibodies that are reactive at cooler temperatures
  • For increased reactions, the saline replacement technique can be used to eliminate excess protein interference, providing a clearer result by removing excess protein
  • Performing a direct Coombs test may help detect the presence of autoantibodies
    • For cases involving cold autoantibodies or cold reactive alloantibodies, incubating the red cells at 37 °C can help prevent cold agglutination
    • Additional steps, such as saline washing of RBCs at 37 °C and autoabsorption techniques, can further reduce the impact of interfering antibodies
• References: Cohn: Technical Manual, 21st Edition, 2023, Immunohematology 2017;33:76, Immunohematology 2017;33:76

	Forward type			Reverse type
	Anti-A	Dolichos biflorus lectin (anti-A1)	Anti-B	A1 cells	B cells
Initial testing	3+	-	0	1+	4+
Further workup	3+	0	4+	1+	4+

• Reverse typing issue: the forward testing indicates blood group A but there is an unexpected reaction in reverse typing with A1 cells
  • This weak, unexpected reaction usually indicates ABO subgroup and can be resolved using appropriate lectins
  • In this case, dolichos biflorus lectin is used and no reaction is seen with RBCs, suggesting A2 subgroup

	Forward type		Reverse type
	Anti-A	Anti-B	A1 cells	B cells
Initial testing	1+	4+	4+	0
Further workup	0+	4+	4+	0

• Forward typing issue: an unexpected reaction in forward typing with the anti-A1 reagent
  • This type of discrepancy may be caused by autoagglutinins / excess proteins coating RBCs or plasma proteins reacting with the reagent
  • The discrepancy is resolved by washing the RBCs

	Forward type			Reverse type
	Anti-A	Anti-B	A1 cells	B cells
Previous testing	4+	0	0	4+
Current testing	0	0	4+	4+

• Discrepancy between previous and current ABO typing: in this kind of distinct blood group changes between samples or patient’s previously documented blood type, the first step should be to check for technical errors and review the patient’s chart
  • This case is resolved after reviewing the patient's chart, which revealed that the blood group A patient had previously undergone a bone marrow transplant (fully engrafted) from a blood group O donor

	Forward type		Reverse type
	Anti-A	Anti-B	A1 cells	B cells
Previous testing	0	4+	4+	0
Current testing	0	4+	1+	0
Further workup	0	4+	2+	0

• Reverse typing issue: there is weaker than expected reactivity in reverse typing with A1 cells
  • In elderly patients, reverse typing strength may decrease over time
  • Incubating for a longer period can enhance the reaction and resolve the issue, provided there is no other explanation for the discrepancy

	Forward type		Reverse type
	Anti-A	Anti-B	A1 cells	B cells
Initial testing	w+	4+	4+	4+
Further workup
Immediate spin	4+	4+	4+	4+
4 °C	4+	4+	4+	4+
Room temperature	4+	4+	4+	4+

In which clinical setting could you see the following ABO discrepancy along with a positive direct antibody test (DAT)?

1. 5 year old child with diabetes mellitus type 1
2. 20 year old woman with common variable immunodeficiency
3. 50 year old woman with out of group heart transplantation
4. 72 year old man with coronary heart disease
5. 80 year old man with IgM multiple myeloma and cold agglutinins

E. 80 year old man with IgM multiple myeloma and cold agglutinins. In this case, a reaction appears in every tube, making interpretation difficult. Additional testing at different temperatures did not resolve the issue. Given the positive DAT, the most likely explanation is the presence of autoantibodies, with reactions at lower temperatures suggesting a cold autoantibody. The result should be reported as undetermined and universal donor blood products should be provided. Answer B is incorrect because, in cases of common variable immunodeficiency, ABO typing typically shows a lack of expected antibodies in the serum. Answer D is incorrect because immunoglobulin titers tend to decrease with age and elderly patient results can show weak or missing reverse typing. Answer C is incorrect because solid organ transplantation can cause passenger lymphocyte syndrome in severe cases; however, only weak discrepant activity on the reserve typing would be expected. Answer A is incorrect because ABO discrepancy is not expected in diabetes mellitus patients.

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Reference: ABO discrepancies

	Forward type		Reverse type
	Anti-A	Anti-B	A1 cells	B cells
Patient result	3+	0	w	4+

Which of the following transfusion scenarios could account for the forward and reverse typing results shown above?

1. Transfusion of a group A recipient with group O red blood cells
2. Transfusion of a group A recipient with group O platelet
3. Transfusion of a group B recipient with group A platelet
4. Transfusion of a group B recipient with group O red blood cells
5. Transfusion of a group O recipient with group AB fresh frozen plasma

B. Transfusion of a group A recipient with group O platelet. In the present case, there is a strong (> 2+) reaction in forward typing, indicating the presence of A antigens; however, in reverse typing, both anti-A and anti-B antibodies are present, creating an ABO discrepancy. The most likely explanation is that a group A recipient was transfused with a large volume of group O platelets, which resulted in a weak reaction with A1 red cells due to O type plasma presence in the platelet product. Answer A is incorrect because the discrepancy observed in the reverse typing and transfusing O group red blood cells will not cause reactions in reverse typing. Answers C and D are incorrect because forward type shows strong reactivity with anti-A, making the recipient most likely group A. Answer E is incorrect because transfusing AB fresh frozen plasma will not cause a reaction to anti-A in forward typing.

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Reference: ABO discrepancies