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
