ABO Discrepancies

Practical Blood Bank

2

ABO Discrepancy When the results of the forward grouping (patient

cells) do not match to the results of the reverse grouping (patient serum) or abnormal reactivity is present (i.e. Mixed Field) then we called this ABO discrepancy.

The Discrepancy will be noticed by: Strength of reaction

Weak or missing.

Additional reactions Abnormal reactions

HINT ABO forward and reverse reactions are typically

very strong: 3+ to 4+. Weaker reactions should immediately send up red flags indicating that something is wrong.

Since production of ABO antigens is genetically controlled they are less vulnerable to problems than does the production of ABO antibodies.

Therefore we see more problems in which grouping: Forward or Reverse?

Patient Anti-A Anti-B A1-Cells B-Cells

A 4+ 1+ 0 4+

B 0 4+ 1+ 0

C 4+ 4+ 1+ 0

D 0 3+ 0 0

Patient A: Additional reaction with anti-B and patients cells.

Patient B: Weak reaction with patients serum and A1-cells.

Patient C: Additional reaction with patients serum and A1-cells.

Patient D: Missing reactions with patients serum A1-cells

Forward Grouping

Problems

Missing or Weak antigens

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

Subgroups of A and B. Solution: test with Anti-A1, Anti-H, and anti-A,B for A

subgroups

Extra Antigens

Anti-A Anti-B A1 Cells

B Cells

4+ 1+ 0 4+

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

EXAMPLE

Solutions: 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 Test with another anti-A reagent from another manufacturer

Polyagglutination, Rouleaux, Wharton’s Jelly

Wash red cells or request new sample from heel, etc

Can be seen in A, B and AB individuals who have received O units. Can also be seen post transfusion if a person makes an antibody to

antigen on donor cells.

Mixed Field agglutination

Mixed Field Agglutination (Post transfusion)

Reverse Grouping

Problems

Unexpectedly Weakened Antibodies

Immunodeficient due to therapy or disease Immunosuppressive drugs Certain leukemia’s (CLL) or lymphoma’s (malignant

lymphomas) have hypogammaglobulinemia (Little or no antibody production)

Age related Very young: <6 months of age (Newborns) Very old: >65 years of age (Weakened Abs Activity)

Dilutional Effect Plasma Exchange, Transfusion, etc. dilutes out

patient antibodies

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

Extra Antibodies Cold antibodies (allo- or auto-)

Cold antibodies may include anti-I, H, M, N, P, Lewis

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 Stronger at IS and weak reaction at 37° C and no

agglutination at AHG phase Solutions

Anti-A1 in an A2 or A2B individual

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

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)

Popular LAB CAUSES Of ABO Discrepancies

1. Poorly labeled specimen OR test tubes

2. Patient RBC suspension too heavy or light

3. Wrong specimen put in Patient’s labeled test tubes

4. Oh? Is hemolysis really a Pos. Rx’n?

5. Wrong results recorded on Pt. Form

6. Didn’t follow manufacturer’s instructions

7. Poor centrifugation: over or under!

Popular LAB CAUSES Of ABO DiscrepanciesDidn’t add:

1. Patient Serum

2. Reagents

3. Correct Reagent

Reaction Reading:1. Shaking tubes while looking elsewhere

2. Shaking tubes too hard

3. Shaking tubes too gently or not completely re- suspending cell button

ABO DiscrepancyWhen an ABO Discrepancy is encountered:

1. Results must be recorded, but interpretation of the ABO group must be delayed until the discrepancy is resolved…by you!

2. Begin follow up by getting an accurate patient history – age, medications, diagnosis, etc.

3. Repeat testing to rule out tech errors such as mislabeling, adding reagents, wrong patient sample, etc.

Resolving ABO Discrepancies1. Repeat testing on the

same sample…

2. Repeat testing using saline suspended and/or washed patient red blood cell’s. Saline Replacement.

1. From the beginning: re-label tubes, re-drop patient and reagent drops, etc.

1. Many labs make the patients red blood cell suspension with the patient’s serum/plasma. If the patient has increased plasma proteins it can cause non-specific red cell aggregation.

3. Weak or missing reactions?

4. Mislabeled or contaminated specimen:

3. Incubate test system at room temperature for 15-30 minutes! Get patient history.

4. Redraw Patient!!

a) ALL of the above: any labeling error may account for the problem and needs to be redrawn.

b) Drawn above an IV?

Resolving ABO Discrepancies

Call the floor!!!

1. Get patient history.a) Recent transplant: two cell populations

b) Recent transfusion: two cell populations and/or dilutional effect

c) Patient medication

d) etc., etc., etc.

5. Test patient cells with anti-A1 (Dolichos biflorus), anti-A,B or anti-H (Ulex europaeus)

6. Test patient serum with A1 or A2 cells

5. For suspected subgroups of A

6. Ditto!

7. Review Antibody Screening tests

1. Allo antibody or cold reactive allo or auto Ab

8. Incubate tests and controls for 10-30 minutes room temperature

7. Can react with reagent A1 and B cells

8. Should strengthen weakened ABO antibody reactivity! WHY?

Anti-A Anti-B A1-Cells B-Cells

3+ 0 0 1+

Resolution: Incubate Room Temperature 15-30 minutes and respin. Check Patient history.

Problem: Reverse grouping - weakened patient antibody

Causes: Age related (>65, infant), immunosuppressed or immunocompromised,

Anti-A Anti-B A1-Cells B-Cells

3+ 1+ 0 4+

Problem: 1+ Reaction with Anti-B. Appears to have additional antigens.

Causes: Acquired ‘B’ antigen.

Resolution: Patient history – bowel obstruction, carcinoma of the bowel. (E. coli deacetylation of the Group A antigen.)

Anti-A Anti-B A1-Cells B-Cells

2+ 0 1+ 4+

Problem: Weak forward anti-A and 1+ reaction with A1 Cells.

Causes:

1. Subgroup of A – A2 with anti-A1.

2. Unexpected cold reacting antibody to antigen on reagent A1 cells.

Resolution:

1. Test patient cells with anti-A1 lectin and with patient serum test A2 cells

2. Antibody screen should demonstrate unexpected cold reacting antibody.

Let’s

practice!

EXAMPLES of ABO Discrepancies and Possible Resolution

Forward: Reverse:Screening

Anti-A Anti-B A1 Cells B Cells Cells Autocontrol: Possible Causes Possible Resolutions

1 0 0 0 0 0 0

Group O newborn; elderly patient; low

immunoglobulin levels

Incubate tests at 4°C, check age of patient

2 4+ 4+ 2+ 2+ 2+ 2+

Rouleaux; cold autoantibody

Wash RBCs and repeat testing; test for cold

antibodies

3 4+ 0 1+ 4+ 0 0

Probable A2 subgroup with anti-A1

Test with anti-A1 and anti-H lectins and A2 cells

4 3+ 4+ 1+ 0 0 0

Probable A2B subgroup with anti-A1

Test with anti-A1 and anti-H lectins and A2 cells

5 0 0 4+ 4+ 4+ 0

Probable Oh (Bombay) Test with anti-H lectin; may sent to reference lab

for confirmation

6 4+ 2+ 0 4+ 0 0

Probable acquired B phenotype

Investigate patient history; test with anti-B lectin if

available

7 4+ 4+ 2+ 0 2+ 0

Probable alloantibody Perform antibody identification (antibody

panel)

8 0 4+ 4+ 1+ 1+ 1+

Probable group B with cold autoantibody

Test for cold antibodies and identify if appropriate

Adapted from Table 3-11: Flynn, J. C. (1998). Essentials of Immunohematology. Philadelphia: W.B. Saunders Company.

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 4

Anti-A,B

Patient RBC 1+

Problem:

Causes:

Resolution:

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: