ABO DISCREPANCIES & OTHER PROBLEMS

Reneé Wilkins, PhD, MLS(ASCP)CM

CLS 325/435School of Health Related ProfessionsUniversity of Mississippi Medical Center

IMPORTANCE It is important to recognize discrepant

results and how to (basically) resolve them

Remember, the ABO system is the most important blood group system in relation to transfusions

Misinterpreting ABO discrepancies could be life threatening to patients

DISCREPANCIES A discrepancy occurs when the red

cell testing does NOT match the serum testing results

In other words, the forward does NOT match the reverse

WHY? Reaction strengths could be weaker

than expected Some reactions may be missing in the

reverse or forward typings Extra reactions may occur

Patient Anti-A Anti-B A1 Cells B Cells

1 4+ 1+ 0 4+

2 0 4+ 1+ 0

3 4+ 4+ 1+ 0

4 0 3+ 0 0

WHAT DO YOU DO? Identify the problem Most of the time, the problem is

technical Mislabeled tube Failure to add reagent Either repeat test on same sample, request

a new sample, or wash cells Other times, there is a real discrepancy

due to problems with the patient’s red cells or serum

DISCREPANCY ? If a real discrepancy is encountered,

the results must be recorded

However, the interpretation is delayed until the discrepancy is RESOLVED

ERRORS

TECHNICAL ERRORS Clerical errors

Mislabeled tubes Patient misidentification Inaccurate interpretations recorded Transcription error Computer entry error

Reagent or equipment problems Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures

Procedural errors Reagents not added Manufacturer’s directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination

CLOTTING DEFICIENCIES Serum that does not clot may be due to:

Low platelet counts Anticoagulant therapy (Heparin, Aspirin, etc) Factor deficiencies

Serum that does not clot completely before testing is prone to developing fibrin clots that may mimic agglutination

Thrombin can be added to serum to activate clot formation

CONTAMINATED SAMPLES OR REAGENTS

Sample contamination Microbial growth in tube

Reagent contamination Bacterial growth causes cloudy or

discolored appearance…do not use if you see this!

Reagents contaminated with other reagents (don’t touch side of tube when dispensing)

Saline should be changed regularly

EQUIPMENT PROBLEMS Routine maintenance should be

performed on a regular basis (daily, weekly, etc)

Keep instruments like centrifuges, thermometers, and timers calibrated Uncalibrated serofuges can cause false

results

HEMOLYSIS Detected in serum after

centrifugation (red) Important if not documented Can result from:

Complement binding Anti-A, anti-B, anti-H, and anti-Lea

Bacterial contaminationRed

supernatant

ABO DISCREPANCIES

ABO DISCREPANCIES Problems with RBCs

Weak-reacting/Missing antigens Extra antigens Mixed field reactions

Problems with SERUM Weak-reacting/Missing antibodies Extra antibodies

Grouping

Forward Reverse

Missing/Weak Extra Mixed Field Missing/Weak Extra

A/B Subgroup

Disease (cancer)

Acquired B

B(A) Phenotype

O Transfusion

Bone Marrow Transplant

YoungElderly

Immunocompromised

Cold Autoantibody

Anti-A1

Rouleaux

Cold Alloantibody

RouleauxMay cause all + reactions

FORWARD GROUPING PROBLEMS

RED CELL PROBLEMS Affect the forward grouping results

Missing or weak antigens Extra antigens Mixed field reactions

FORWARD GROUPING:MISSING OR WEAK ANTIGENS

Anti-A Anti-B A1 Cells B Cells

0 0 0 4+

ABO Subgroups Disease (leukemia, Hodgkin’s

disease)

• Since the forward and reverse don’t match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

Group O Group A

SUBGROUPS OF A (OR B) Subgroups of A account for a small

portion of the A population (B subgroups rarer)

These subgroups have less antigen sites on the surface of the red blood cell

As a result, they show weakened (or missing) reactions when tested with commercial antisera

Resolution: test with Anti-A1, Anti-H, and anti-A,B for A subgroups

FORWARD GROUPING:EXTRA ANTIGENS

Anti-A Anti-B A1 Cells

B Cells

4+ 1+ 0 4+

Acquired B B(A) phenotype Rouleaux Wharton’s Jelly

EXAMPLE

ACQUIRED B PHENOTYPE Limited mainly to

Group A1 individuals with: Lower GI tract

disease Cancer of

colon/rectum Intestinal

obstruction Gram negative

septicemia (i.e. E. coli)

ACQUIRED B Bacteria (E. coli) have a deacetylating

enzyme that effects the A sugar….

Group A individual

N-acetyl galactosamine

Acquired B

Phenotype

Bacterial enzyme removes acetyl group

Galactosamine now resembles

D-galactose (found in Group B)

RESOLVING ACQUIRED B Check patient diagnosis: Infection? Some manufacturers produce anti-B

reagent that does not react with acquired B

Test patients serum with their own RBCs The patients own anti-B will not react

with the acquired B antigen on their red cell (autologous testing)

B(A) PHENOTYPE Similar to acquired B Patient is Group B with an apparent

extra A antigen The B gene transfers small amounts of

the A sugar to the H antigen Sometimes certain anti-A reagents will

detect these trace amount of A antigen Resolution: test with another anti-A

reagent from another manufacturer

OTHER REASONS FOR “EXTRA” ANTIGENS

Polyagglutination – agglutination of RBCs with human antisera no matter what blood type Due to bacterial infections Expression of hidden T antigens react with

antisera Rouleaux – extra serum proteins Wharton’s Jelly – gelatinous substance

derived from connective tissue that is found in cord blood and may cause false agglutination (Remember: only forward typing is performed on cord blood) Wash red cells or request new sample from heel,

etc

FORWARD GROUPING: MIXED FIELD AGGLUTINATION

Anti-A Anti-B A1 Cells B Cells

0 2+ mf 4+ 0

Results from two different cell populations

Agglutinates are seen with a background of unagglutinated cells All groups transfused with Group O cells Bone marrow/stem cell recipients A3 phenotype (sometimes B3)

MIXED FIELD AGGLUTINATION (POST TRANSFUSION)~ (ABO Testing) Can be seen in A, B and AB individuals who have received O units. The antisera reacts with the patient’s RBCs, but not with the transfused O cells.~ (Antibody screen) Can also be seen post transfusion if a person makes an antibody to antigen on donor cells; antibody agglutinates with donor cell, but not their on cells.

REVERSE GROUPING PROBLEMS

REVERSE GROUPING Affect the reverse grouping results

Missing or weak antibodies Extra antibodies

REVERSE GROUPING:MISSING OR WEAK ANTIBODIES

Newborns Do not form antibodies until later

Elderly Weakened antibody activity

Hypogammaglobulinemia Little or no antibody production (i.e.

immunocompromised) Often shows NO agglutination on

reverse groupings

RESOLVING WEAK OR MISSING ANTIBODIES

Determine patients age, diagnosis Incubate serum testing for 15 minutes

(RT) to enhance antibody reactions If negative, place serum testing at 4°C

for 5 minutes with autologous control (Autocontrol, AC)

This is called a “mini-cold” panel and should enhance the reactivity of the antibodies

REVERSE GROUPING:EXTRA ANTIBODIES

Cold antibodies (allo- or auto-) Cold antibodies may include anti-I, H, M,

N, P, Lewis Rouleaux Anti-A1 in an A2 or A2B individual

COLD ANTIBODIES Sometimes a patient will develop cold-

reacting allo- or auto-antibodies that appear as “extra” antibodies on reverse typing

Alloantibodies are made against foreign red cells

Autoantibodies are made against ones own red cells. Cold reacting antibodies cause agglutination with red cells at room temperature and below. The autocontrol will be positive. Resolution: warming tube to 37° and washing

red cells can disperse agglutination; breaking the IgM bonds with 2-ME will also disperse cells

ROULEAUX Can cause both extra antigens and extra

antibodies “stack of coins” appearance May falsely appear as agglutination due to

the increase of serum proteins (globulins) Stronger at IS and weak reaction at 37°C and

no agglutination at AHG phase Associated with:

Multiple meloma Waldenstrom’s macroglobulinemia (WM) Hydroxyethyl starch (HES), dextran, etc

RESOLVING ROULEAUX Remove proteins! If the forward grouping is affected, wash

cells to remove protein and repeat test If the reverse grouping is affected,

perform saline replacement technique (more common) Cells (reagent) and serum (patient) centrifuged

to allow antigen and antibody to react (if present)

Serum is removed and replaced by an equal volume of saline (saline disperses cells)*

Tube is mixed, centrifuged, and reexamined for agglutination (macro and micro)

ANTI-A1

Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody

A2 (or A2B) individuals have less antigen sites than A1 individuals

The antibody is a naturally occurring IgM

Reacts with A1 Cells, but not A2 CellsAnti-A1 from

patient

+ A1 cells

+ A2 cells

AGGLUTINATION

NO AGGLUTINATION

RESOLVING ANTI-A1 DISCREPANCY

Anti-A Anti-B A1 Cells

B Cells

4+ 0 2+ 4+

2 steps: Typing patient RBCs with Anti-A1 lectin Repeat reverse grouping with A2 Cells

instead of A1 Cells Both results should yield NO

agglutination

OTHERS… The Bombay phenotype (extremely

RARE) results when hh is inherited These individuals do not have any

antigens and naturally produce, anti-A, anti-B, anti-A,B, and anti-H

Basically, NO forward reaction and POSITIVE reverse

Resolution: test with anti-H lectin (Bombay’s don’t have H and will not react)

FINDING THE PROBLEM… Forward type tests for

the antigen (red cell) Reverse type tests for

the antibody (serum) Identify what the

patient types as in both Forward & Reverse Groupings

Is there a weaker than usual reaction?

Is it a missing, weak, or extra reaction??

RESOLVING ABO DISCREPANCIES Get the patient’s history:

age Recent transplant Recent transfusion Patient medications The list goes on….

LET’S PRACTICE !

EXAMPLE 1

Anti-A Anti-B A1 Cells B Cells

3+ 0 0 1+

Problem:Causes:Resolution:

EXAMPLE 2

Anti-A Anti-B A1 Cells B Cells

3+ 1+ 0 4+

Problem:Causes:Resolution:

EXAMPLE 3

Anti-A Anti-B A1 Cells B Cells

2+ 0+ 1+ 4+

Problem:Causes:Resolution:

EXAMPLE 4

Anti-A Anti-B A1 Cells B Cells

0 0 0 3+

Problem:Causes:Resolution:

EXAMPLE 4Anti-A,B

Patient RBC 1+

• Probably a subgroup of A (Ax)• if the result was negative (0), adsorption or

elution studies with anti-A could be performed (these will help determine what A antigens)

EXAMPLE 5

Anti-A Anti-B A1 Cells B Cells

0 2+mf 3+ 0

Problem:Causes:Resolution:

EXAMPLE 6

Anti-A Anti-B A1 Cells B Cells

4+ 4+ 0 1+

Problem:Causes:Resolution:

EXAMPLE 7

Anti-A Anti-B A1 Cells B Cells

0 0 0 0

Problem:Causes:Resolution:

EXAMPLE 6

Screening Cells (I and II)

Autocontrol (AC)

Conclusion

Patient Serum 1

Pos Neg Cold alloantibody

Patient Serum 2

Pos Pos Cold autoantibody

• if alloantibody – antibody ID techniques• if autoantibody – special procedures (minicold panel, prewarming

techniques); no prior transfusions. If they have had a recent transfusion, then it could be an alloantibody.

REFERENCES Rudmann, S. V. (2005). Textbook of Blood Banking and

Transfusion Medicine (2nd Ed.). Philadelphia, PA: Elsevier Saunders.

Blaney, K. D. and Howard, P. R. (2009). Basic & Applied Concepts of Immunohematology. St. Louis, MO: Mosby, Inc.