ABO Discrepancies

Betsy Cole
ABO Discrepancies
 Unexpected rxns in the forward and reverse grouping

Anti-A Anti-B Anti-D Rh Control A1 Cells B Cells

4+ 0 4+ 0 2+ 4+

 Front type: A
 Back type: O
 Most probable ABO/Rh for this patient?
› A pos
 Discrepancy??
› A1 cells in back type
Problem:
 Cannot report results until a reason for discrepancy
is uncovered.

 ABO type cannot be interpreted

 Group O, Rh compatible RBCs until resolved

 AB plasma
Technical Errors
 Cell suspensions  Forgot to drip
› Pro/post –zone
› Wash
 Contaminated/wrong
reagent added
 Clerical errors
› Check labeling
 Manufacturer’s
instructions
 Sample mix up
› Request new sample
 Contaminated reagents

 Missed hemolysis
Repeat ABO
• Most discrepancies can be resolved by identifying
technical errors, and repeating testing
Patient Information
• Patient history is important
• Age
• Diagnosis
• Txn/transplant history
• Multiple out of group products
• BMT
• Meds
• Pregnancy
• Genetics
Discrepancies
1) Additional reaction in forward grouping

2) Missing reaction in forward grouping

3) Additional reaction in reverse grouping

4) Missing reaction in reverse grouping

 Additional reaction in forward grouping

Anti-A Anti-B Anti-D Rh Cntrl A1 Cells B Cells

4+ 2+ 4+ 0 0 4+

Extra Weak Rxn

Additional Reactions in Forward Grouping
 Acquired B antigen on type A1 patient
› Diagnosis
 Tissue conditions allow colonic bacteria to enter circulation
 Colon cancer
› Acidified anti-B reagent
› Patient history confirmation
 Pt. RBCs/pt. plasma - AC
 If acquired B, pt. Anti-B and acquired B antigen should NOT agglutinate

 Rouleaux
› Washed cell suspension
› Microscopic examination

 Reagent antibodies
› Pt antibody to acriflavin in Anti-B
 Different reagents
 Washed cell suspension
Additional Reactions in Forward
Grouping
 Cold-reacting autoantibody
› Spontaneously agglutinate
› Wash red cell suspension
 Warm saline
 15 minutes in warm waterbath, or in incubator
› Repeat with warm washed red cells
› DAT positive

 MF agglutination
› Pt. received multible blood transfusions of different ABO type
› Pt. received BMT from different ABO type
› Patient is type A3
› Txn History/diagnosis
Missing reaction in forward grouping

Anti-A Anti-B Anti-D Rh Cntrl A1 Cells B Cells

0 0 4+ 0 0 4+

Missing Rxn
Missing Reactions in Forward Grouping
 A or B subgroups
› A: Anti-A1 lectin
› Dolichos biflorus
 Agglutinate A1 cells, but not A2
 BMTs
 ABO Blood group-specific soluble substances
(BGSS)
› Neutralize/inhibit desired rxn
› Stomach/Pancreatic cancers
› Washed RBC suspension
Rare Causes of Missing Front Type Rxns

• In plasma in disease states

• Carinomas of stomach and pancreas
• Neutralized anti-A or anti-B
• No antibody to bind with pt antigens
Missing Reactions in Forward Grouping
 Obtain pt. diagnosis
› Leukemia
 Weakened A/B antigens
› Hodgkin’s disease
 Weakened antigens
› Genetics
 Inherited variant alleles
 Wash pt. RBC suspension
› Anti-A and Anti-B to separate tubes
› Pt. RBC suspension
› Incubate 30” at RT
 If negative….
› Incubate 30” at 4°C
 AC – NO agglutination
Additional Reactions in Reverse Grouping

Anti-A Anti-B Anti-D Rh Cntrl A1 Cells B Cells

0 4+ 4+ 0 4+ 2+

Unexpected
Positive Rxn
Additional Reactions in Reverse
Grouping
 Rouleaux
› Excess protein
› Saline replacement
› Microscopic examination
 Rouleaux disappears
 True agglutination remains

 Cold Alloantibodies
› Test with different lot of A 1 and B cells
› Anti-A1
 A2 and A2B
 Serum tested with 3 lots of A 1, A2, O cells
 Anti-A1 will agglutinate A1 cells, but not A 2 or O cells
› Anti-H
 A1 and A1B
› Non-ABO
 IS, tube screen and panel
 M, N, Lewis, P1
 Find A1 and B cells antigen negative for antibody
Additional Reactions in Reverse Grouping
 Cold Autoantibodies
› Prewarm technique
 37°C 10” incubation of pt plasma, A1 reagent/ B reagent
› Wash with warm saline
› Adsorption

 Passively acquired ABO antibodies

› Massive transfusion
› Placental transfer
› Patient history
Missing Reactions in Reverse Grouping

Anti-A Anti-B Anti-D Rh Cntrl A1 Cells B Cells

4+ 0 3+ 0 0 0

Missing Rxn
Missing Reactions in Reverse Grouping
 Back type rxns are usually very strong (4+)

 Depressed antibody production

› No ABO antibodies
 Age
› Newborns
› Elderly
 Leukemias
› Hypogammaglobulinemia
 Antibody deficiency
› Lymphomas
 Stem cell transplant
› Immunosuppressive meds
 Immunodeficiency
 Diluted ABO abs due to plasma exchange
Missing Reactions in Reverse Grouping
• Chimerism: two cell populations in a single individual.
• MF
• True Chimerism:
• Twins
• Lifelong
• No anti-A or B due to in utero exchange of blood
• No back type abs
• Dispermy
• 2 sperms, 1 egg
• Mosaicism
• Resembling chimerism:
• Txn
• BMT or PBSC transplant
• Exchange txn
• Fetal-maternal bleed
Missing Reactions in Reverse Grouping -
Solutions
• Incubate pt serum with A1 and B cells at RT for 30”
• No rxn?

• Incubate at 4°C for 15-30”

• AC and O cell control
• No agglutination
 Review the following ABO incompatibility problems

Practice!
1
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
4+ 0 4+ 0 2+ 4+
ABO Testing Discrepancy Considerations

 Forward grouping?
 Back grouping?
 A1 Cells: Weak unexpected positive
 Possible Causes?
 Further testing:
› A1 Lectin
 Negative – Subgroup A
 Positive – A1 individual
2
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
0 0 4+ 0 0 2
ABO Typing Discrepancy Considerations

• Forward grouping?
• Back grouping?
• A1 Cells: Missing rxn
• Possible Causes?
• Further testing:
• Incubate @ RT 15-30 minutes
• Incubate at 4°C 15-30 minutes
3
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
0 4+ 4+ 0 3+ 1+
ABO Typing Discrepancy Considerations

AC: Negative

 Forward grouping?
 Back grouping?
 B cells: extra rxn in back type
 Possible Causes?
 Further testing:
› Ab screen: pos
› Ab panel: pos
› Find antigen negative B cells
4
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
4+ 1+ 4+ 0 0 3+
ABO Typing Discrepancy Considerations

• Forward grouping?
• Back grouping?
• Anti-B: Unexpected weak rxn
• Possible Causes?
• Further testing:
• Pt. history
• Acidified Anti-B
5
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
2+ 4+ 2+ 2+ 4+ 2+
ABO Typing Discrepancy Considerations
Autocontrol: 2+
Antibody Screen (AHG): negative

• Forward grouping?
• Back grouping?
• Anti-A, B cells: Extra weak rxns
• AC!!
• Possible Causes?
• Further testing:
• Saline replacement
6
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
0 0 0 0 0 0
ABO Typing Discrepancy Considerations

• Forward grouping?
• Back grouping?
• Possible Causes?
• Further testing:
• Repeat
7
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
0 1+/MF 3+ 0 3+ 1+
ABO Typing Discrepancy Considerations

Antibody Screen: negative

• Forward grouping?
• Back grouping?
• Possible Causes?
• Further testing:
• Txn history?
• Wash, repeat front type.
8
Anti-A Anti-B Anti-D Rh A1 Cells B Cells
Control
1+ 1+ 1+ 1+ 3+ 3+
ABO Typing Discrepancy Considerations
Autocontrol: 1+
Antibody Screen (AHG): negative

• Forward grouping?
• Back grouping?
• Possible Causes?
• Further testing:
• DAT
• Treat with EGA to remove antibodies