ABO Discrepancies & other problems - PowerPoint Presentation

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ABO Discrepancies & other problems

Reneé Wilkins, PhD, MLS(ASCP)CMCLS 325/435School of Health Related ProfessionsUniversity of Mississippi Medical Center

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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

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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

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Why?

Reaction strengths could be

weaker

than expected

Some reactions may be

missing

in the reverse or forward typings

Extra

reactions may occur

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Patient

Anti-A

Anti-B

A

1

Cells

B Cells

1

4+

1+

0

4+

2

0

4+

1+

0

3

4+

4+

1+

0

4

0

3+

0

0

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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

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Discrepancy ?

If a real discrepancy is encountered, the results must be recorded

However, the interpretation is delayed until the discrepancy is RESOLVED

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Errors

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Technical Errors

Clerical errorsMislabeled tubesPatient misidentificationInaccurate interpretations recordedTranscription errorComputer entry errorReagent or equipment problemsUsing expired reagentsUsing an uncalibrated centrifugeContaminated or hemolyzed reagentsIncorrect storage temperaturesProcedural errorsReagents not addedManufacturer’s directions not followedRBC suspensions incorrect concentrationCell buttons not resuspended before grading agglutination

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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

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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

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Equipment problems

Routine maintenance should be performed on a regular basis (daily, weekly, etc)Keep instruments like centrifuges, thermometers, and timers calibratedUncalibrated serofuges can cause false results

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Hemolysis

Detected in serum after centrifugation (red)Important if not documentedCan result from:Complement bindingAnti-A, anti-B, anti-H, and anti-LeaBacterial contamination

Red supernatant

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ABO discrepancies

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ABO Discrepancies

Problems with

RBCs

Weak-reacting/Missing antigens

Extra antigens

Mixed field reactions

Problems with

SERUM

Weak-reacting/Missing antibodies

Extra antibodies

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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

Young

Elderly

Immunocompromised

Cold

Autoantibody

Anti-A

1

Rouleaux

Cold

Alloantibody

Rouleaux

May cause

all

+ reactions

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Forward Grouping Problems

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Red Cell Problems

Affect the

forward grouping

results

Missing or weak antigens

Extra antigens

Mixed field reactions

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Forward Grouping:Missing or Weak antigens

Anti-AAnti-BA1 CellsB Cells0004+

ABO SubgroupsDisease (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

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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-A

1

, Anti-H, and anti-A,B for A subgroups

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Forward Grouping:Extra Antigens

Anti-AAnti-BA1 CellsB Cells4+1+04+

Acquired B B(A) phenotypeRouleauxWharton’s Jelly

EXAMPLE

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Acquired B Phenotype

Limited mainly to Group A1 individuals with:Lower GI tract diseaseCancer of colon/rectumIntestinal obstructionGram negative septicemia (i.e. E. coli)

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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)

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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)

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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

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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

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Forward Grouping: Mixed Field Agglutination

Anti-AAnti-BA1 CellsB Cells02+ mf4+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

A

3

phenotype (sometimes B

3

)

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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.

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Reverse Grouping Problems

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Reverse Grouping

Affect the reverse grouping results

Missing or weak antibodies

Extra antibodies

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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

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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

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Reverse Grouping:Extra Antibodies

Cold antibodies (allo- or auto-)

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

Rouleaux

Anti-A

1

in an A

2

or A

2

B individual

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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

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Rouleaux

Can cause both extra antigens and extra antibodies“stack of coins” appearanceMay 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 phaseAssociated with:Multiple melomaWaldenstrom’s macroglobulinemia (WM)Hydroxyethyl starch (HES), dextran, etc

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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)

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Anti-A1

Sometimes A2 (or A2B) individuals will develop an anti-A1 antibodyA2 (or A2B) individuals have less antigen sites than A1 individualsThe antibody is a naturally occurring IgMReacts with A1 Cells, but not A2 Cells

Anti-A

1 from patient

+ A

1

cells

+ A2 cells

AGGLUTINATION

NO AGGLUTINATION

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Resolving anti-A1 discrepancy

Anti-AAnti-BA1 CellsB Cells4+02+4+

2 steps:

Typing patient RBCs with Anti-A

1

lectin

Repeat reverse grouping with A

2

Cells instead of A

1

Cells

Both results should yield NO agglutination

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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)

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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 GroupingsIs there a weaker than usual reaction?Is it a missing, weak, or extra reaction??

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Resolving ABO Discrepancies

Get the patient’s history:

age

Recent transplant

Recent transfusion

Patient medications

The list goes on….

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Let’s practice !

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Example 1

Anti-AAnti-BA1 CellsB Cells3+001+

Problem:

Causes:

Resolution:

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Example 2

Anti-AAnti-BA1 CellsB Cells3+1+04+

Problem:

Causes:

Resolution:

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Example 3

Anti-AAnti-BA1 CellsB Cells2+0+1+4+

Problem:

Causes:

Resolution:

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Example 4

Anti-AAnti-BA1 CellsB Cells0003+

Problem:

Causes:

Resolution:

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Example 4

Anti-A,BPatient RBC1+

Probably a subgroup of A (A

x

)

if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)

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Example 5

Anti-AAnti-BA1 CellsB Cells02+mf3+0

Problem:

Causes:

Resolution:

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Example 6

Anti-AAnti-BA1 CellsB Cells4+4+01+

Problem:

Causes:

Resolution:

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Example 7

Anti-AAnti-BA1 CellsB Cells0000

Problem:

Causes:

Resolution:

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Example 6

Screening Cells (I and II)Autocontrol (AC)ConclusionPatient Serum 1PosNegCold alloantibodyPatient Serum 2PosPosCold 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.

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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.

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