Rene Wilkins, PhD, MLS(ASCP)CM CLS 325/435 School of Health Related Professions University of Mississippi Medical Center

ABO DISCREPANCIES & OTHER PROBLEMS

IMPORTANCE
It is important for students 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 1 2 3 4

Anti-A Anti4+ 0 4+ 0

Anti-B A1 Cells B Cells Anti1+ 4+ 4+ 3+ 0 1+ 1+ 0 4+ 0 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 patients 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 Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Reagents not added Manufacturers directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination

Reagent or equipment problems

Procedural errors

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 OR, tubes containing EDTA can be used

CONTAMINATED SAMPLES OR REAGENTS

Sample contamination
Microbial

growth in tube

Reagent contamination
Bacterial

growth causes cloudy or discolored appearancedo not use if you see this! Reagents contaminated with other reagents (dont 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
Anti-A,

binding

anti-B, anti-H, and anti-Lea

Bacterial

contamination
Red supernatant

ABO DISCREPANCIES

ABO DISCREPANCIES

Problems with RBCs

Weak-reacting/Missing Extra

antigens

antigens Mixed field reactions

Problems with SERUM

Weak-reacting/Missing Extra

antibodies

antibodies

Grouping Forward
Missing/Weak Extra Mixed Field

Reverse
Missing/Weak
Young Elderly
Immunocompromised

Extra

A/B Subgroup

Acquired B

O Transfusion

Cold Autoantibody

Disease (cancer)

B(A) Phenotype

Bone Marrow Transplant

Cold Alloantibody

Rouleaux May cause all + reactions

Rouleaux

Anti-A1

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 ABO Subgroups Disease (leukemia, Hodgkins disease)

AntiAnti-A 0
Group O

Anti-B Anti0

A1 Cells 0

B Cells 4+

Group A

Since the forward and reverse dont match, there must be a discrepancy (in this case, a missing antigen in the forward grouping)

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 antiA,B for A subgroups

FORWARD GROUPING:
EXTRA ANTIGENS

Acquired B B(A) phenotype Rouleaux Polyagglutination Whartons Jelly Anti- AntiAnti-A Anti-B 4+ 1+ A1 Cells 0 B Cells 4+

EXAMPLE

ACQUI

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.
Acquired B Phenotype

Group A individual

N-acetyl galactosamine

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 Whartons 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 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)
AntiAnti-A 0 Anti-B Anti2+ mf A1 Cells B Cells 4+ 0

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 patients 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 antibody activity

Elderly
Weakened

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 4C for 5 minutes with autologous control (a.k.a. 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 37C and no agglutination at AHG phase Associated with:

Multiple meloma Waldenstroms 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)

*some procedures suggest only 2 drops of saline (UMMC)

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 Cells

+ A1 cells Anti-A1 from patient + A2 cells

AGGLUTINATION NO AGGLUTINATION

RESOLVING ANTI-A1 DISCREPANCY

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 Anti- AntiAnti-A Anti-B 4+ 0 A1 Cells 2+ B Cells 4+

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 (Bombays dont have H and will not react)

FI

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 patients history:

age Recent

transplant Recent transfusion Patient medications The list goes on.

LETS PRACTICE !

EXAMPLE
AntiAnti-A 3+ Anti-B Anti0 A1 Cells 0 B Cells 1+

Problem: Causes: Resolution:

EXAMPLE
AntiAnti-A 3+ Anti-B Anti1+ A1 Cells 0 B Cells 4+

Problem: Causes: Resolution:

EXAMPLE
AntiAnti-A 2+ Anti-B Anti0+ A1 Cells 1+ B Cells 4+

Problem: Causes: Resolution:

EXAMPLE
AntiAnti-A 0 Anti-B Anti0 A1 Cells 0 B Cells 3+

Problem: Causes: Resolution:

EXAMPLE
AntiAnti-A,B Patient RBC
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)

1+

EXAMPLE 5
AntiAnti-A 0 Anti-B Anti2+mf A1 Cells 3+ B Cells 0

Problem: Causes: Resolution:

EXAMPLE 6
AntiAnti-A 4+ Anti-B Anti4+ A1 Cells 0 B Cells 1+

Problem: Causes: Resolution:

EXAMPLE 7
AntiAnti-A 0 Anti-B Anti0 A1 Cells 0 B Cells 0

Problem: Causes: Resolution:

EXAMPLE 6
Screening Autocontrol Cells (AC) (I and II) Pos Neg Pos Pos Conclusion

Patient Serum 1 Patient Serum 2

Cold alloantibody 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.