Resolving ABO Discrepancy

8/11/2019 Resolving ABO Discrepancy

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Resolving ABO Discrepancy

Dr. Sheikh Ahmed .W.

Section In charge Blood bankIMC

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Recognition and resolution are two very

important skills that blood bank

technologists must possess.

Of all the blood group systems, ABO is

THE MOST IMPORTANT.

ABO misinterpretation can lead to serious

transfusion complications in

patients.

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ABO Discrepancies MUST be Resolved

In PATIENTS, an ABO discrepancy must be

resolved before any blood component is

transfused.AABB policy 5.12.1 & 5.12.2 says blood shall not be released for a patient until

any discrepancy in patient ABO grouping is resolved.

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In DONORS, the discrepancy must be resolved

before any blood is labeled .


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Landsteiners Rule

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What IS an ABO Discrepancy?

Definition:When the results of the forward grouping

(patients cells) does not correspond to the

reverse grouping (patients plasma/serum

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What CAUSES an ABO

Discrepancy?

Weak or Missing antigens in theFRONT type

Weak or Missing antibodies in the

REVERSE type

Additional antigens in the

FRONT type

Additional antibodies in the

REVERSE type

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Clerical Errors

Mislabeled tubesPatient misidentification errors

Inaccurate interpretations recorded

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Reagent or equipment

problems

Using expired reagents

Using an un-calibrated

centrifugeContaminated or hemolyzed

reagents

Incorrect storage temperatures

Types of Errors

Procedural errors

Reagents not added

Manufacturers

directions not followed

RBC suspensions

incorrect concentration

Cell buttons not re-suspended before

grading agglutination

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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

YoungElderly

Immunocompromised

Cold

Autoantibody

Anti-A1

Rouleaux

Cold

Alloantibody

Rouleaux

May cause all + reactions

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Mixed Field (mf) ReactionsSmall agglutinates with many un-agglutinated cells

Result of:

Mixed cell population from a massive transfusion of another blood

group. non-O individual receiving O red blood cells)

Bone marrow transplants having both the original type and donor

marrow cells.

The inheritance of weak ABO subgroups such as A3, Ax and B3 and

B can traditionally present a mixed field reaction.

Chimerism

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Resolving Mixed Field (mf)ReactionsDetermine the CAUSE of the mixed field

reaction

Checking the patients transfusion history andclinical history

e.g. HPC transplant

If it is determined that it is a weak subgroup,

perform

specialized testse.g. adsorption and elution.

Molecular testing

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Weak or Missing Antigen

Result of:Inheritance of a weak ABO subgroup

Malignancies may result in the loss of ABH

transferases

H d ki di

Forward Grouping Problems

Hodgkins diseaseLymphomas

Leukemias

Massive transfusion with group O blood to a

non-group O

person

e.g. a group A person receiving lots of group O

blood.

Bone marrow transplant and chemotherapy

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Resolving Weak or Missing Antigens

Check the patients transfusion and clinical history

Read the forward group microscopically

Use anti-A,B and monoclonal antisera that is known to react with Ax and

A3 weak subgroups.

Perform adsorption and elution studies 17


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Additional Antigens

Result of:Bacterial enzymes deacetylate the A antigen to a B

antigen and the patient front types as an AB and reverses as an A.Acquired B antigens are observed in patients with

recurring GI or colon infections with GI bacteria

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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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This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but

still technically it is extra). Patients should receive Group A units. Acquired

B usually goes away when the condition resolves


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Resolving Additional Antigens

Check clinical history for evidence of colon

infections with

Gram negative sepsis

Auto control is negative- that proves the blood

group as A.

As the patients own anti-B will not react with

their own AB cells

Acidify the anti-B to a p.H. of 6 and retest

Acquired B antigens will not react in acidified

antiserum, whereas as normal B antigens will

react.

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Spontaneous Agglutination

Result of:

Strong potent cold auto antibodies

Forward Grouping ProblemsWould appear as AB in the front type and O in

the reverse type

Strong positive DAT with IgG, C3d and saline

control

Whartons jelly in cord blood

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Resolving Spontaneous

AgglutinationIncubate plasma and cells (separately) at 37C

for 5 to15 minutes

Wash cells 5 to 6 times with warm saline


Retest warm washed cells with warm plasma

Retest the DAT and saline control

Treat cells with 0.01M DTTWash cord blood a minimum of 6 times with

saline

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PolyagglutinationResult of:

Inheriting acquired abnormalities of the red

cell membranes with

exposure to crypt antigens

e g T activationBacterially contaminated sample

Resolve by:

Avoid testing with human antisera; use

monoclonal antisera.

Collection of a new sample

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patientserum-plasma

(with anti-B Ab)

AHGreagent

red cells (type

B)

Reverse Grouping

Problems

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Plasma or serum ABO discrepancies are

more common than red cell discrepancies.

Weak/Missing

Additional Antibodies

Rouleaux


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Weak/Missing Antibodies

Newborns

Antibodies are not present at birth and only develop after 3

to 6 months of age.

Elderly

Weakened antibody activity

Hypogammaglobulinemia

Little or no antibody production

(immune-compromised patient)

NO agglutination on reverse grouping

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Reverse Grouping Problems

Resolving Weak/Missing

AntibodiesDetermine patients age and diagnosisIncubate serum testing for 15 minutes at room

temperature or 18 C to enhance antibody

reactions

If negative, incubate serum testing at 4C for

15 minute

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Extra AntibodiesResult of:

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

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Extra Antibodies: Rouleaux

Result of:

Abnormal concentrations of serum proteins

Altered serum/protein ratios

High-molecular-weight volume expanders

Associated with:

Multiple myeloma

Waldenstromsmacroglobulinemia (WM)

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Extra Antibodies: Anti-A1Result of:

A2 (or A2B) individuals development of anti-A1

antibody

A2 (or A2B) individuals have less antigen sites

than A1 individuals.

anti-A1 is a naturally occurring IgM antibody

Antibody reacts with A1 cells, but not A2 cells

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A sub-groups

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Reverse Grouping Problems

Resolution Extra Antibodies: Anti-A1

Type patient red blood cells with Anti-A1 lectin

Test patient serum with A1 , A2 and O cells

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CASE 1

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Case Study #1: Patient History

88 year old male

Diagnosis: ImmunocompromisedMedications: Corticosteroids

Transfusion History: Massive plasma infusion

Lab: Hemoglobin: 6.5 g/dL


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What is the problem?

Both a front AND reverse typing issue

Investigation

Patient age: 88 years oldDiagnosis: Hypogammaglobulinemia

Medications: Immunosuppressive drugs

Resolution:

weak front type, weak reverse

type

A B AB AntiD D

CONTROL

A1cells A2cells Bcells

Patient 0 0 0 3+ 0 W+ W+ 0

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A B AB AntiD D

C

A1cells A2 cells Bcells

patient 0 0 0 3+ 0 W+ W+ 030 RT 0 1+ 2+ 1+ 1+ 0

Weak front type and weak reverse type resolution:

Enhance forward typeIncubate patient cells with antisera (per manufacturers directions)

Enhance reverse type

Incubate reverse type cells with patient serum for 15 to 30 minutes

Room temperature or 18C (per manufacturers directions)

4C for 15 minutes

test concurrently with autologous cells and group O screening cells

Enzyme treat reverse type cells with FICIN.

Final diagnosis---B +ve38


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Case Study #2: Patient History

33 year old female

Diagnosis: Anemia

Medications: NoneTransfusion History: 2 units of packed red

blood

cells 5 years ago

Lab: Hemoglobin: 9.1 g/dL

Case 2

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Reverse typing issue

Additional antibodies: cold

Investigation

Patient age: 33 years oldDiagnosis: Anemia

Medications: None

Resolution:

Additional antibody in reverse

Anti-A Anti-B AntiD D C A1

Cells

A2

cells

B

cells

patient 0 4+ 4+ 0 3+ 3+ 1+

Screening cells IS IAT

1 1+ 0

2 1+ 0

3 1+ 0

DAT Anit-

IgG

Anti C3d

c3b

cont

Patient 0 2+ 0

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Additional antibody in reverse type resolution:

Prewarm Technique

Incubate the serum and red cells separately at 37C before testing

Cold Adsorption

Incubate equal amounts of red cells (adsorbing cells) and patient

serum at 4C for 30 to 60 minutes

Test adsorbed serum against reverse typing cells.

FINAL DIAGNOSISB+ve

Screening cells IS 4c adsorbed

serum

1 0

2 0

3 0

A1

Cells

A2

cells

B cells

Patient 3+ 3+ 1+

4C adsorbed serum 3+ 3+ 0

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Anti-A Anti-

B

Anti-

AB

Anti-

D

D

contro

l

A1

cells

A2

cells

B

cells

Patient 4+ 0 4+ 4+ 0 2+ 0 3+

Screening

cells

IS IAT

1 0 0

2 0 0

3 0 0

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Reverse typing

issue

DAT -NEGATIVE

Patient

Investigation

Patient age: 36 years old

Diagnosis: PREGNANTMedications: VITAMINS

Resolution:

Additional antibody in reverse type


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Patientserum

A1 cells 3+

AI cells 3+

A2 cells 0

Anti

A1Lecitin(Dolic

hous biflorus

Patient 0

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Resolution: Patient is (probable)type A2 with anti-A1 in her serum


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73 YEAR OLD MALE

DIAGNOSIS: LYMPHOMA AND SEVERE ANEMIA

MEDICATIONS: ASPIRIN

TRANSFUSION HISTORY: 3 UNITS 6 MONTHS AGO

LABORATORY: HEMOGLOBIN: 4.5 G/DL

Case Study #6

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Forward type:

Weak Reactivity

Weak ABO subgroup? Malignancy?

Spontaneous agglutination?.

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Spontaneous agglutination was not resolved

with warm washed cells.

DTT (0.01M) treatment of the red blood cells

should be performed to

resolve the ABO/Rh discrepancy and disperse

the spontaneous

agglutination.

:

Patient is B PositivePositive DAT and autocontrol was further

investigated by a reference

laboratory, and it was discovered the patient

had a warm autoantibody

in his plasma


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Example 7

Anti-A Anti-B A1 Cells B Cells

3+ 1+ 0 4+

Problem:

Causes:

Resolution:

T k H M


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Take Home Message

ABO discrepancy recognition & resolution is imperative in the blood bank

ABO discrepancies can present themselves as a

front type problem, reverse type problem, or

combination of both.

If ABO discrepancy resolution cannot be performed, and blood is

needed immediately, transfusion of type O blood may be necessary

.

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Relation of H substance (antigen)


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Relation of H-substance (antigen)

and ABO groups

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OhPhenotype (Bombay)

Occurs when two hh genes are inherited atthe Hh locus.

Possess normal A or B genes (if they were

inherited) but unable to express. Must have H on red cell membrane

Can transmit A or B gene to offspring

Term Bombay used since first discovered in

Bombay, India

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Symbol Oh denotes this phenotype

RBCs not agglutinated by anti-A, -B orA,B

Serum/plasma agglutinates A and B cells

Not recognized until serum tested against group Ocells and causes strong agglutination.

Have anti-A, -B, -A,B andH

Can only be transfused with Bombay blood


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Blood group

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Confirmatory testing

Anti-H lectin (Ulex europaeus)negative

Agglutination of A, B, AB and O cells

Serum/plasma will not agglutinate Oh cells.

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Antigen / Antibody

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Reading in tube method

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Principle of Gel Technology

J The sephadex gel matrix acts as asieve.

J Large agglutinates remain on or near

the top of the gel interface.

J Smaller agglutinates pass partway

through the gel, depending on size.

J Unagglutinated cells pass to the base of

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Result of:


Altered serum/protein ratios

Reverse Grouping ProblemsHigh-molecular-weight volume expanders

Associated with:

Multiple myeloma

Waldenstromsmacroglobulinemia (WM)

Hydroxyethyl starch (HES), dextran, etc


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Result of:


Altered serum/protein ratiosReverse Grouping Problems


Associated with:

Multiple myeloma


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Result of:


Altered serum/protein ratiosReverse Grouping Problems


Associated with:

Multiple myeloma


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Waldenstromsmacroglobulinemia (WM)Hydroxyethyl starch (HES), dextran, etc

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Resolving ABO Discrepancy

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