ABO BLOOD GROUP

ABO DISCREPANCIES
- When the forward and reverse typing does not match
- Usually resolved by repeating the test using RBCs suspended in saline
- Make sure that the discrepancy is not due to technical errors (Box 6 – 5)

Technical Errors in ABO Grouping (GOAL: REDUCE ERRORS)

A. Clerical Errors
1. Sample or patient misidentification
2. Mislabeling of test tubes
3. Error in recording results
4. Transcription error
5. Computer entry error
6. Sample mix – up

- A technologist should accept only accurately and legibly labeled samples

- If Manufacturer’s directions are not followed, then results are invalid
Critical elements in the test system:

Serum/cell ratios

Media

Temperature

Incubation time

Sample type

Centrifuge rpm
- Should develop careful, routine, standard method of performing a test

B. Reagent/ Equipment Variable

1. Use of outdated reagents
2. Inappropriate storage temperatures
3. Contaminated reagents
4. Use of uncalibrated centrifuge
5. Warming of sample during centrifugation
Equipments that should be monitored for performance
o Thermometer, timer, centrifuge
C. Procedural Errors
1. Improper cell suspensions
2. Reagents not added
3. Failure to recognize or record hemolysis
4. Misinterpretation of results
5. Failure to follow manufacturer’s directions

o Patients with clotting deficiency

Serum should be allowed to clot completely or add protamine sulfate to neutralize heparin
therapy or thrombin to induce coagulation. Otherwise, a clear, gel – like clot may form and mimic
agglutination
o Contaminated samples
o Hemolysis is also a positive reaction but should eliminate the possibility of bacterial contamination when
doing so
 Acquire patient information
o Age
o Diagnosis
o Transfusion history
o Medications
o History of pregnancy
- Results may be recorded but reporting should be delayed until the blood type is resolved
- If the discrepancy is from the donor, do not use the blood

Group I Discrepancy
- Most common discrepancy
- Unexpected reactions in reverse grouping due to missing or weakly reacting or missing antibodies

Population with decreased Antibody Production

1. Newborn
2. Elderly patients
3. Patients with leukemia (CLL/ Lymphoma)
4. Patients using immunosuppressive drugs
5. Patients with immunodeficiency diseases
6. Patients with BM transplants
7. Recently transfused (diluted antibodies)
8. ABO subgroups

Resolution
o Enhance weak antibodies
o Incubate patient serum with reagent A1, & B cells at RT for 15 – 30 minutes
o If no reaction, incubate at 4°C for 15 – 30 minutes with autocontrol (patient’s serum with patient’s RBC) and
O control (patient’s serum with O cells) to eliminate false positive reactions due to cold agglutinins (anti – I)

Other possible causes:

• Chimerism – the presence of 2 cell populations in a single individual (usually mixed field reaction)

a. True chimerism
o Twins, vascular anastomosis in utero; both blood groups recognized as self

b. Mosaicm
o Dispermy (2 sperms + 1 egg)

c. Blood transfusion ( example: O to A)

d. Transplanted BM or peripheral blood stem cells
e. Exchange transfusion
f. Fetal – maternal bleeding

Group II Discrepancy
- Unexpected reactions in forward grouping due to weakly reacting or missing antigens
- Least frequently encountered

Possible Causes:
1. A or B subgroups
2. Weakened A or B due to leukemia or Hodgkin’s disease
3. Acquired B phenomenon associated with diseases of digestive tract
Resolution of Group 2
o Enhance the antigen
o Incubate patient antigen + reagent antisera at RT for 15 – 30 minutes
o If negative, incubate at 4°C for 15 – 30 minutes with auto and O control cells
o Acquired B

When bacterial enzymes modify the immunodominant blood group A sugar (N-
acetylgalactosamine) into D – galactosamine which is almost similar to B sugar D – galactose thus
causing a cross – reaction

Formed from A1

Disappears after recovery

Reaction with anti – B gives mixed field appearance

Check:

Reaction with monoclonal anti – B clone (ES4) = strong agglutination (lowered pH)

Patient’s plasma (anti – B) + patient’s RBC (A with acquired B) = no agglutination

Anti – B (>8.5 or <6.0 pH) + patient’s RBC = no agglutination

Secretor studies

Reactivity of D – galactosamine is decreased by treatment with acetic anhydride but has no effect
on B sugar (normal)

Rare Group II
a. Excess blood group – specific soluble substances (BGSS) present in association with diseases like
carcinoma or the stomach and pancreas (increased secretions)

BGSS neutralizes anti – A or anti – B (false negative or weak reaction in forward)

Check: wash patient RBC with saline
b. Presence of antibodies to low – incidence antigens in reagent anti – A or anti – B
Check: use reagent with different lot number
c. Chimerism = 2 cell population

Group III Discrepancy

- Discrepancies between forward and reverse groupings caused by protein or plasma abnormalities that result in
rouleaux or pseudoagglutination

Possible Causes:
1. Elevated globulins – MM, Waldenstrom’s, plasma cell dyscrasias, HD
2. Elevated fibrinogen
3. Plasma expanders – dextran and polyvinylpyrrolidone
4. Wharton’s jelly in cord blood samples

Resolutions:
o Wash patient RBC with NSS
o Add NSS to reverse typing: true agglutination will remain
o Wash cord cells 6 – 8 times with NSS - reverse may not correlate due to antibodies from mother (baby’s abs
detectable at 3 – 6 months)

Group IV Discrepancy
- Discrepancies between forward and reverse due to miscellaneous problems
Possible Causes
1. Cold reactive autoantibodies coating RBCs causing spontaneous agglutination
2. Patient has 2 groups of RBCs due to transfusion / transplantation
3. Unexpected isoagglutinins
4. Unexpected non – ABO alloantibodies
Resolution:

For Cold – reactive antibodies:

Forward: Patient RBC coated with antibodies causing agglutination
Reverse: Patient serum contains anti – I present in all adults cells which is also present in reagent A1 and B cells

To Correct:
Forward:
a. Incubate patient’s RBC at 37°C, wash with saline at 37°C 3 times and retype OR/AND
b. Add 0.01 M dithiothreitol (DTT) to disperse IgM – related agglutination

Reverse:
a. Warm reagent cells and serum at 37°C, mix, test and read at 37°C
b. Convert to AHG (add AHG) – weakly reactive anti – A / anti – B may not react at 37°C (IgM)
c. Autoadsorption may be performed: Px RBC (antigen) + patient serum (auto-antibodies) ----
spin and separate. Test using absorbed serum at RT

For Unexpected Isoagglutinins

o Isoagglutinin in patient serum reacts with antigen on Reagent RBC

Example: anti – A1 in the serum of A2 or A2B individuals or anti – H in A1B individuals
To Correct:

Reverse type using at least 3 examples of A1, A2, B, and O and autologous (control) cells and
observe the pattern of reactivity

Test patient’s RBC with Dolichos biflorus to confirm A1

For Unexpected antibodies in patient’s serum against ags in reagent cells

o Reverse grouping
o Antigens in reagent cells

To Correct:

Test patient serum with a panel of reagent cells and identify the antibody present

Repeat using reagent cells negative for the antigen

Rare Group IV
o Antigen – antibody complex adsorbed onto patient RBC
o Example: antibodies against acriflavine (yellow dye of anti – B) ab + acriflavine + RBC = agglutination

To correct: Wash RBC 3x with saline and retype