11 - ANTIBODY SCREENING AND IDENTIFICATION

Immune alloantibodies

Naturally occurring alloantibodies

Passively acquired antibodies

Autoantibodies

Unexpected/Irregular Antibodies (4)

Clinically significant alloantibodies

cause decreased survival of RBCs

IgG

37

Clinically significant alloantibodies

typically _ antibodies that react at _°C

Antibody Detection

Detect any potentially clinically significant antibodies in a donor’s or recipient’s sample

patient serum

Antibody Detection

Involves the reaction between _ _ with 2 or 3 reagents phenotyped for multiple antigens

R1R1, R2R2 or R1R1, R2R2, rr

Reagent RBC (2)

Reagent RBC

O cell with: C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, Lea, Leb, P1, M, N, S, and s antigens

Homozygous or Heterozygous

Reagent RBC expressions

M, N

M+N+

Antigen present?

Heterozygous

M+N+

Homozygous or heterozygous?

Fya

Fy(a+b-)

Antigen present?

Homozygous

Fy(a+b-)

Homozygous or heterozygous?

Immediate Spin

37C Phase

AHG Phase

3 Phases in Antibody Screening

Negative for unexpected antibodies

Positive for unexpected antibodies

Cold reacting antibody

Positive for unexpected antibodies

Warm reacting antibody

Autologous Control

The patient’s RBCs tested against the patient’s serum in the same manner as the antibody screen

Single antibody

Interpretations

_ _ specificity should be suspected when all screen cells yielding a positive reaction react at the same phase(s) and strength

Multiple antibodies

Interpretations

_ _ are most likely present when screen cells react at different phases or strengths

Autoantibodies

Interpretations

_ should be suspected when the autologous control or DAT is positive and all screen cells tested yield a positive reaction

anti-Lea

anti-Leb

anti-PP1Pk

anti-Vel

Antibodies associated with Hemolysis (in vitro) (4)

anti-Sda

anti-Lu

Antibodies associated with Mixed-Field (Mf) (2)

Rouleaux

Cells have a “stacked coin” appearance when viewed microscopically

Rouleaux

Observed in all tests containing the patient’s serum, including the autologous control and the reverse ABO grouping

washed away

Rouleaux

Does not interfere with the AHG phase of testing in tube method or solid-phase technology because the patient’s serum is _ _ prior to the addition of the AHG reagent

1-3

saline

Rouleaux

Disperses by adding _-_ drops of _ to the test tube when performing tube testing

Single Alloantibody

Probably IgG

Multiple Alloantibodies

Probably IgG

Single or multiple Alloantibody

Probably IgM

Multiple alloantibodies

Probably IgG or IgM

Warm Autoantibody

Probably IgG

Antibody Identification

Identification of an antibody to red cell antigen(s) requires testing the serum against a panel of selected red cell samples with known antigen composition

Antibody Identification Panel

collection of 11 to 20 group O reagent RBCs

antigen expression should be diverse

Exclusion or “Rule-Outs”

First Step

RBCs that gave a negative reaction in all phases of testing are examined

The antigens on these negatively reacting cells are probably not the antibody’s target

Rule of three

must be met to confirm the presence of the antibody

Positive

Negative

Rule of three

Patient serum MUST be:

_ with 3 cells with the antigen

_ with 3 cells without the antigen

Selected Panel

Enzymes

Neutralization

Adsorption

Additional Techniques for Resolving Antibody Identification (4)

Selected Panel

Additional Techniques for Resolving Antibody Identification

Simplest step

The cells selected for testing should have minimal overlap in the antigens they possess

Enzymes

Additional Techniques for Resolving Antibody Identification

Modify the RBC surface by removing sialic acid residues and by denaturing or removing glycoproteins

Results in the destruction of certain antigens and the enhanced expression of others

Rh

Kidd

Lewis

P1

I

ABO

Additional Techniques for Resolving Antibody Identification

Enzymes

Enhanced (6)

Duffy

MNS

Xga

Additional Techniques for Resolving Antibody Identification

Enzymes

Inactivated (3)

Neutralization

Additional Techniques for Resolving Antibody Identification

Used to neutralize the corresponding RBC antibodies in serum

Anti-P1

Anti-Lewis

Anti-Chido, anti-Rodgers

Anti-Sda

Anti-I

Adsorption

Additional Techniques for Resolving Antibody Identification

Process of removing antibody from the serum

RBC or another antigen-bearing substance

Additional Techniques for Resolving Antibody Identification

Adsorption

Adsorbent

Human platelet concentrate

Additional Techniques for Resolving Antibody Identification

Adsorption

adsorb Bg-like antibodies

Rabbit erythrocyte stroma (RESt)

Additional Techniques for Resolving Antibody Identification

Adsorption

Cold-reacting autoantibodies

two

one

one

20, 3400

Immediate Spin Saline

Procedure

1. Label four tubes as SC I, SC II, SCIII, and AC.

2. Add _ drops of serum into each of the tubes.

3. Add _ drop of the corresponding screening cell to each of the tubes.

4. Add _ drop of patient’s 5% red cell suspension to the autocontrol tube (AC).

5. Gently mix then centrifuge for _ seconds at _ rpm.

6. After centrifugation, gently dislodge the red cells and observe for agglutination or hemolysis.

7. Grade and record the results as “_ _ _”.

two

37, 15-30

20

37C albumin

Procedure

1. Add _ drops of bovine serum albumin to each of the four tubes. Mix gently.

2. Incubate the tubes at _°C for _-_ minutes.

3. After incubation, centrifuge the tubes for _ seconds.

4. Repeat step 6.

5. Grade and record the results as “_ _”.

isotonic saline, 3

two

20

AHG

2

20

CC

Procedure

1. Wash the serum-cell mixtures with _ _ for _ times.

2. Add _ drops of antihuman globulin reagent to each of the four tubes.

3. Gently mix and centrifuge for _ seconds.

4. Repeat step 6.

5. Grade and record results as “_”.

6. Add _ drops of check cells to all negative tubes.

7. Gently mix then centrifuge for _ seconds.

8. Repeat step 6.

9. Grade and record results as “_”.