Resolving ABO Discrepancy

Mina Hur
Department of Laboratory Medicine Hallym University College of Medicine

ABO discrepancy
Cell typing Front typing Forward typing Serum typing Back typing Reverse typing

Possible causes
Red cell
ABO subgroup, cis-AB, acquired B, recent ABO-incompatible transfusion, chimerism, DAT-positive red cells, weakened antigen, cells antigen polyagglutination, non-specific agglutination, etc.

Serum

Alloantibody or autoantibody, age-related, ABO subgroup, hypogammaglobulinemia, g p, yp g g , Paraproteinemia, etc.

Technical

Clerical errors Reagent or equipment problems Procedural errors

Possible test results

Red cell weak/missing reactivity Extra red cell reactivity Mixed-field red cell reactivity Serum weak/missing reactivity / g y Serum extra reactivity

Weak

or

Missing

or

Extra

Resolving ABO discrepancy

Identify the problem M l the problem is technical Mostly, h bl i h i l
Mislabeled tube Failure to add reagent Either 1) repeat test on same sample, 2) request a new sample, or 3) wash cells

Persistent discrepancy
Diagnosis, clinical/transfusion/drug histories Real discrepancy d/t problems with the patients red cells or serum

Errors

Technical errors
Mislabeled tubes Patient misidentification Inaccurate interpretation recorded Transcription or computer entry error

Clerical errors

Using expired reagents Using an uncalibrated centrifuge Incorrect storage temperatures

Reagent/equipment problems

Reagents not added Manufacturers directions not followed Incorrect concentration in RBC suspension Cell buttons not resuspended before grading agglutination

Procedural errors P d l

Clotting deficiencies
Serum that does not clot maybe d/t:
L platelet counts Low l t l t t Anticoagulant therapy (heparin, aspirin, etc.) Factor deficiencies

Serum that does not clot completely before testing:

Prone to developing fibrin clots that may mimic agglutination g y gg

Thrombin can be added to serum to activate clot formation Tubes containing EDTA can be used

Contaminated samples/reagents
Sample contamination
Mi bi l growth in tube Microbial g th i t b

Reagent contamination
Bacterial growth causes cloudy or discolored appearance Reagents contaminated with other reagent g g Saline should be changed regularly

Equipment problems
Routine maintenance should be performed on a regular basis (daily, weekly, etc) (daily weekly Keep instruments calibrated
Centrifuges, thermometers and timers Uncalibrated serofuges can cause false results g

Hemolysis
Detected in serum after centrifugation (red) C result from: Can l f
Complement binding Anti A anti B anti H and anti-Lea Anti-A, anti-B, anti-H, anti Le Bacterial contamination

Red supernatant

ABO discrepancy
Grouping Forward Reverse

Missing/Weak

Extra

Mixed Field

Missing/Weak

Extra

A/B Subgroup

Acquired B

O Transfusion

Young Elderly
Immunocompromised

Cold Autoantibody

Disease (cancer)

B(A) Phenotype

Bone Marrow Transplant

Cold Alloantibody

Rouleaux

Rouleaux

Anti-A1

Forward grouping p problems

Missing or weak antigens Missing Extra antigens Mixed field agglutination

Forward grouping problems F d i bl

Missing or weak antigens
ABO subgroup Disease (leukemia, Hodgkins disease)

Anti-A 0

Anti-B 0

A1 cells 0

B cells 4+

Group O

Group A

A missing antigen in the forward grouping

Subgroups of A (or B)
Subgroups of A account for a small portion of the A p p population (B subgroups rarer) ( g p ) Less antigen sites on the surface of the red blood cell Weakened (or missing) reactions when tested with commercial antisera

Resolution:
test with anti-A1, anti-H and anti-A B for A subgroups anti A anti H, anti A,B

Detection of weakly expressed Ag

Incubate washed red cells with anti-A, anti-B, anti-A,B
15 min at RT 15-30 min at 4 Autocontrol in parallel

Proteolytic enzyme treatment (ficin, papain, or bromelin)

Wi hi 30 min at RT Within i Enzyme-treated group O cells and autocontrol in parallel

Adsorption and elution

Aliquot of red cells with anti-A or anti-B at RT or 4 Group A or B and O red cells in parallel (PC and NC) p p ( ) Test the eluate against group A1, B, and O cells

Saliva test for the presence of H and A or B substances

Forward grouping problems F d i bl

Extra antigens
Acquired B B(A) phenotype Rouleaux Polyagglutination Whartons jelly

Anti-A 4+

Anti-B 1+

A1 cells 0

B cells 4+

Group AB?

Group A

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)

Acquired B phenotype
Bacteria (E. coli) have deacetylating enzyme that affects the A sugar
Acquired B Phenotype

Group A individual

N-acetyl galactosamine

Galactosamine now resembles D-galactose (found in Group B)

Bacterial enzyme removes acetyl group

Resolving Acquired B
Check patients diagnosis: infection? Some anti B reagents do not react with acq ired B anti-B ith acquired 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) Test the red cells with anti-B reagent acidified to pH 6.0

B(A) phenotype
Similar to acquired B g p pp g Patient is group B with an apparent extra A antigen Varying reactivity with anti-A reagent Excessively high levels of B allele-specified allele specified galatosyltransferase Anti-A reagents containing p g g particular murine monoclonal antibody, MHO4 clone Resolution: Testing with an anti-A reagent without MHO4 clone

B(A) phenotype
ABO genotyping 526 A1 B B(A) C (Arg) G (Gly) 657 C T C 703 G (Gly) A (Ser) G 796 C (Leu) A (Met) 803 G (Gly) C (Ala)

Forward grouping problems F d i bl

Mixed field agglutination
Results from two different cell population Agglutinates with a background of unagglutinated cells
All groups transfused with group O cells t f d ith ll Bone marrow/stem cell recipients A3 phenotype

Anti-A 0

Anti-B 2+mf

A1 cells 4+

B cells 0

Group B and ?

Group B

Reverse grouping p problems

Missing or weak antibodies Mi i g k tib di Extra antibodies

Reverse grouping problems R i bl

Missing or weak antibodies
Newborns Elderly Hypogammaglobulinemia: immunocompromised Often shows NO agglutination on reverse groupings

Anti-A 4+

Anti-B 0

A1 cells 0

B cells 0

Group A

Group AB

Resolving weak or missing Ab

Determine patients age & diagnosis Incubate serum with A1 and B red cells
At RT for 15-30 min at 4 for 15-30 min Autocontrol & group O red cells in parallel to control for reactivity of common cold autoagglutinins

Proteolytic enzyme treatment of the A1 and B reagent cells

Ficin, papain, or bromelin Enzyme-treated autocontrol and group O red cells in parallel

Reverse grouping problems R i bl

Extra antibodies
Cold antibodies (allo- or auto-) Rouleaux Anti-A1 in an A2 or A2B individual

Anti-A Anti A 4+

Anti-B Anti B A1 cells B cells O cells 4+ 2+

?

Auto 2+

2+

2+

Group AB

Cold autoantibody i g C ld t tib d in group AB individual i di id l Rouleaux

Cold antibodies
Cold antibodies (allo- or auto-)
Anti-I, IH, IA, IB, M, N, P, Lewis

Resolution:
Warm the serum and reagent red cells to 37 before mixing and testing One-hour incubation & settled reading (without centrifugation) Breaking the IgM bonds with 2-ME will also disperse cells g g p

Panagglutination P l i i
autoantibody

Polyagglutination P l l i i
Polyagglutinable RBC

Rouleaux
C cause b th extra antigens and extra antibodies Can both t ti d t tib di Stack of coins appearance Falsely appear as agglutination d/t the increase of serum f proteins (globulins) St g at IS and weak reaction at 37 and no Stronger t d k ti t d agglutination at AHG phase Associated with:
Multiple myeloma Waldenstroms macroglobulinemia g Hydroxyethyl starch (HES), dextran, etc.
Agglutination Rouleaux

Resolving Rouleaux
Remove proteins If the forward grouping is affected wash cells to remove affected, protein and repeat test If the reverse grouping is affected perform saline affected, replacement technique

Anti A Anti-A1
Sometimes A2 (or A2B) individuals will develop an anti-A1 antibody A2 (or A2B) individuals have less antigen sites than A1 individuals d dua s The antibody is naturally-occurring IgM React with A1 cells but not with A2 cells
+ A1 cells Anti-A1 from patient + A2 cells

AGGLUTINATION NO AGGLUTINATION

Resolving Anti A1 discrepancy Anti-A

2 steps: Typing patients RBCs with anti A1 lectin patient s anti-A Repeat reverse grouping with A2 cells instead of A1 cells Both results should yield NO agglutination

Anti-A Anti-B A1 cells B cells 4+

Group A

Anti-A1 A2 cells 0 0

2+
Group O?

4+

Cis-AB AB
Comparison of ABO transferase gene
Base (Amino a.) A1 B Cis-AB Cis AB 261 G G G 297 A G A 467 (156) C/T (Pro/Leu) C T (Leu) 526 (176) C (Arg) G (Gly) C 657 C T C 703 (235) G (Gly) A (Ser) G 796 (266) C (Leu) A (Met) C 803 (268) G (Gly) C (Ala) C (Ala) 930 G A G

Cis-AB AB
Weakened antigen and antibody to the weakened antigen Variable phenotypes according to its counterpart gene

Phenotype A1B3 A2B3 A2B

Genotype Cis-AB/A1 Cis-AB/O Cis-AB/B

ABO discrepancy
Grouping Forward Reverse

Missing/Weak

Extra

Mixed Field

Missing/Weak

Extra

A/B Subgroup

Acquired B

O Transfusion

Young Elderly
Immunocompromised

Cold Autoantibody

Disease (cancer)

B(A) Phenotype

Bone Marrow Transplant

Cold Alloantibody

Rouleaux

Rouleaux

Anti-A1

Thank you for your attention