ABO Discrepancies & other problems

cls.umc.edu/COURSES/CLS325/Week5/Discrepancies.ppt

Reneé Wilkins, PhD, MLS(ASCP)CM

CLS 325/435School of Health Related Professions

University of Mississippi Medical Center

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

PatientPatient Anti-AAnti-A Anti-BAnti-B AA11 Cells Cells B CellsB Cells

11 4+4+ 1+1+ 00 4+4+

22 00 4+4+ 1+1+ 00

33 4+4+ 4+4+ 1+1+ 00

44 00 3+3+ 00 00

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

• 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

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 contamination

Red 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

Rouleaux May 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-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

00 00 00 4+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 may type as group O

• 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-AAnti-A Anti-BAnti-B A1 A1 CellsCells

B B CellsCells

4+4+ 1+1+ 00 4+4+

• Acquired B • B(A) phenotype• Rouleaux• Polyagglutination• 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)

Problems with The Forward Grouping:

Extra ABO antigens Acquired ‘B’ Antigen

a) Microbial deacetylating enzymes such as E. coli cleave off the N-Acetyl of the Group A N-acetyl-D-galactosamine immunodominant sugar. The remaining D-galactosamine becomes similar enough to the Group B D-galactose immunodominant sugar that it DOES react with reagent anti-B.1) Secondary to bowel obstruction or carcinoma of

the bowel

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• TestTest patients serum with their own RBCs

– The patients own anti-B will not react with the acquired B antigen on their red cell (autologous (autologous testing)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-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

00 2+ mf2+ mf 4+4+ 00

• 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 NONO agglutination 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 (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 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)

*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

Anti-A1 from patient

+ A1 cells

+ A2 cells

AGGLUTINATION

NO AGGLUTINATION

Resolving anti-A1 discrepancy

Anti-AAnti-A Anti-BAnti-B A1 A1 CellsCells

B B CellsCells

4+4+ 00 2+2+ 4+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-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

3+3+ 00 00 1+1+

Problem:

Causes:

Resolution:

Example 2

Anti-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

3+3+ 1+1+ 00 4+4+

Problem:

Causes:

Resolution:

Example 3

Anti-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

2+2+ 0+0+ 1+1+ 4+4+

Problem:

Causes:

Resolution:

Example 4

Anti-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

00 00 00 3+3+

Problem:

Causes:

Resolution:

Example 4

Anti-A,BAnti-A,B

Patient RBCPatient RBC 1+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-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

00 2+mf2+mf 3+3+ 00

Problem:

Causes:

Resolution:

Example 6

Anti-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

4+4+ 4+4+ 00 1+1+

Problem:

Causes:

Resolution:

Example 7

Anti-AAnti-A Anti-BAnti-B A1 CellsA1 Cells B CellsB Cells

00 00 00 00

Problem:

Causes:

Resolution:

Example 6

Screening Screening Cells Cells (I and II)(I and II)

Autocontrol Autocontrol (AC)(AC)

ConclusionConclusion

Patient Patient Serum 1Serum 1

Pos Neg Cold alloantibody

Patient Patient Serum 2Serum 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.