Identifying AntibodiesIdentifying Antibodies

The Antibody Panel

CLS 422

Clinical Immunohematology I

ObjectivesObjectives Discuss clinical situations when it is appropriate to

perform antibody identification. Define a panel of cells. Explain how the following factors aid in the

interpretation of antibody panels:a. Cross-out techniqueb. Variation in strengths of reactionc. Phases of reactiond. Autocontrole. Red blood cell antigen typing

ObjectivesObjectives

List testing that can be performed to confirm the identification of antibodies.

Identify the antibodies present, when given panel results.

When is an When is an antibody antibody

identification panel identification panel performed?performed?

When the antibody screen is positive.

When to perform testWhen to perform test

The panel red blood cells (RBCs) are tested against the patient’s serum or plasma in order to identify the unexpected antibody or antibodies present.

IdentificationIdentification

Antibody screen – positive Run antibody panel to identify antibody (-ies). If original panel does not provide a clear-cut

ID, test additional RBCs. Selected cells Alternate methods

ConfirmationConfirmation

Rule of 3 and 3 Antigen type patient’s RBCs.

Landsteiner’s Law!!!

The PanelThe Panel

Series of 8 to 20 Group O RBCs Various distribution of the most common RBC

antigens Suspended in a preservative to protect antigen

integrity for 2 -4 weeks Packaged with a lot-specific antigram

AntigramAntigram

Donor

Cell number

D C c E eCw K k

Kpa

Kpb

Jsa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua

Lub

Xga

RZR1 1 + + 0 + + 0 0 + 0 + 0 + 0 0 0 + 0 + 0 0 + 0 + 0 + +

R1wR1 2 + + 0 0 + + + + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + +

R2R2 3 + 0 + + 0 0 0 + 0 + 0 + + + 0 + + 0 + + + 0 + 0 + 0

r’r 4 0 + + 0 + 0 0 + 0 + 0 + 0 + 0 + + 0 + + + + + 0 + +

r’’r 5 0 0 + + + 0 0 + 0 + 0 + + 0 + 0 0 + 0 + + 0 + 0 + +

rrK 6 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + +

rrFya 7 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + 0 0 + 0 + + + 0 + +

Ror 8 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + +

rr 9 0 0 + 0 + 0 + + 0 + 0 + + + + 0 0 + + + 0 + 0 + + +

R2r 10 + 0 + + + 0 + 0 0 + 0 + + + + 0 0 + + + + + + + + +

R1R1 11 + + 0 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + + 0 + 0 + +

PatientCells

Panel AntigramPanel Antigram

Auto ControlAuto Control

Patient’s serum/plasma tested against a suspension of patient’s RBCs Optional Evaluate results in conjunction with patient history

Autoantibody Newly forming alloantibody

If patient has a positive DAT, auto control will be positive

Usually the same as was used for the antibody screen

Must include incubation at 37oC Must include an AHG phase with reagent

containing anti-IgG

Test MethodTest Method

Interpreting Interpreting Panel ResultsPanel Results

Cell D C c E e Cw K k Kpa Kpb J

sa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua Lub X

ga

AHG

CC

1 + + 0 0 + 0 + + 0 + 0 + + + + 0 0 + + + + 0 + 0 + + 3+

2 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 0 + + 0 2+

3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 2+

4 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + + 0 2+

5 0 + + 0 + 0 0 + 0 + 0 + 0 + + + 0 + + + + 0 + 0 + + 0 2+

6 0 0 + + + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + 0 + + 0 2+

7 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + 0 3+

8 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 0 + + + 0 0 2+

9 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + 0 2+

10 + + + 0 + 0 + + 0 + 0 + + + 0 + 0 0 + + 0 + + 0 + + 3+

11 + w + + w 0 0 + 0 + 0 + 0 0 + 0 0 + + 0 + + 0 0 + + 0 2+

Auto 0 2+

ExclusionExclusion

Begin with the RBCs that failed to react The antibody in the serum is not directed

against the antigens on these RBCs, so we can eliminated these antibody specificities

Look at alleles to avoid problems with dosage! Exclusion should be done using RBCs having

homozygous antigen expression. Exceptions are low prevalence antigens

Cell D C c E e Cw K k Kpa Kpb J

sa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua Lub X

ga

AHG

CC

1 + + 0 0 + 0 + + 0 + 0 + + + + 0 0 + + + + 0 + 0 + + 3+

2 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 0 + + 0 2+

3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 2+

4 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + + 0 2+

5 0 + + 0 + 0 0 + 0 + 0 + 0 + + + 0 + + + + 0 + 0 + + 0 2+

6 0 0 + + + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + 0 + + 0 2+

7 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + 0 3+

8 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 0 + + + 0 0 2+

9 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + 0 2+

10 + + + 0 + 0 + + 0 + 0 + + + 0 + 0 0 + + 0 + + 0 + + 3+

11 + w + + w 0 0 + 0 + 0 + 0 0 + 0 0 + + 0 + + 0 0 + + 0 2+

Auto 0 2+

ExclusionExclusion

InclusionInclusion

Of the antibody specificities that have not been excluded, match the pattern of positive and negative reactions with the pattern of antigen positive and antigen negative cells.

There must be an explanation for each positive reaction seen.

Cell D C c E e Cw K k Kpa Kpb J

sa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua Lub X

ga

AHG

CC

1 + + 0 0 + 0 + + 0 + 0 + + + + 0 0 + + + + 0 + 0 + + 3+

2 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 0 + + 0 2+

3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 2+

4 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + + 0 2+

5 0 + + 0 + 0 0 + 0 + 0 + 0 + + + 0 + + + + 0 + 0 + + 0 2+

6 0 0 + + + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + 0 + + 0 2+

7 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + 0 3+

8 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 0 + + + 0 0 2+

9 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + 0 2+

10 + + + 0 + 0 + + 0 + 0 + + + 0 + 0 0 + + 0 + + 0 + + 3+

11 + w + + w 0 0 + 0 + 0 + 0 0 + 0 0 + + 0 + + 0 0 + + 0 2+

Auto 0 2+

InclusionInclusion

Other Points to ConsiderOther Points to Consider

In what phase(s) of testing is the antibody reactive? May give clue as to antibody identity and clinical

significance. Is the strength of reaction the same for each cell that

reacts, or is there variation in strength? Dosage, antigen variability, or multiple antibodies.

Is the antibody reacting only with “homozygous” cells of a certain specificity? Weak antibody showing dosage.

Did the autologous control react? Autoantibody or newly forming alloantibody.

ProbabilityProbability

Rule of 3 and 3 For each antibody specificity, are there 3

antigen positive cells that reacted and 3 antigen negative cells that did not react? May use screen cells in addition to panel cells to

fill this rule Cells do not need to have homozygous antigen

expression to fill this rule

Cell D C c E e Cw K k Kpa Kpb J

sa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua Lub X

ga

AHG

CC

1 + + 0 0 + 0 + + 0 + 0 + + + + 0 0 + + + + 0 + 0 + + 3+

2 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 0 + + 0 2+

3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 2+

4 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + + 0 2+

5 0 + + 0 + 0 0 + 0 + 0 + 0 + + + 0 + + + + 0 + 0 + + 0 2+

6 0 0 + + + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + 0 + + 0 2+

7 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + 0 3+

8 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 0 + + + 0 0 2+

9 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + 0 2+

10 + + + 0 + 0 + + 0 + 0 + + + 0 + 0 0 + + 0 + + 0 + + 3+

11 + w + + w 0 0 + 0 + 0 + 0 0 + 0 0 + + 0 + + 0 0 + + 0 2+

Auto 0 2+

The Rule of 3 and 3The Rule of 3 and 3

Antigen TypingAntigen Typing

Confirms the antibody identification LANDSTEINER’S LAW

Test patient’s RBCs (unknown antigen) against appropriate anti-sera (known antibody)

Results should be negative

Run positive and negative controls for anti-sera A positive DAT or recent transfusion may invalidate

the typing results

Cell D C c E e Cw K k Kpa Kpb J

sa

Jsb

Fya

Fyb

Jka

Jkb

Lea

Leb

P

1M N S s

Lua Lub X

ga

AHG

CC

1 + + 0 0 + 0 + + 0 + 0 + + + + 0 0 + + + + 0 + 0 + + 3+

2 + + 0 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + + + 0 + 0 + + 0 2+

3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 2+

4 + 0 + 0 + 0 0 + 0 + + 0 0 0 + 0 0 + + 0 + 0 0 0 + + 0 2+

5 0 + + 0 + 0 0 + 0 + 0 + 0 + + + 0 + + + + 0 + 0 + + 0 2+

6 0 0 + + + 0 0 + 0 + 0 + + + + + + 0 + 0 + 0 + 0 + + 0 2+

7 0 0 + 0 + 0 + + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + 0 3+

8 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 0 + + + 0 0 2+

9 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 + 0 0 + + 0 2+

10 + + + 0 + 0 + + 0 + 0 + + + 0 + 0 0 + + 0 + + 0 + + 3+

11 + w + + w 0 0 + 0 + 0 + 0 0 + 0 0 + + 0 + + 0 0 + + 0 2+

Auto 0 2+

Selecting Controls for Antigen TypingSelecting Controls for Antigen Typing

Value of Patient HistoryValue of Patient History

The following additional information may assist in determining the identity of the antibody: History of antibodies Transfusion, transplant, pregnancy (how many and

how long ago) Medications Diagnosis Ethnicity

ReportingReporting

Panel results are reported as “anti-” and then the specificity Anti-K

If specificity cannot be determined at this point, additional testing must be performed

The End