Identifying Antibodies

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Identifying Antibodies

• The Antibody Panel

CLS 422 Clinical Immunohematology I
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Objectives

• 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 technique
• b. Variation in strengths of reaction
• c. Phases of reaction
• d. Autocontrol
• e. Red blood cell antigen typing

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Objectives

• List testing that can be performed to confirm the
  identification of antibodies.
• Identify the antibodies present, when given panel
  results.

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When is an antibody identification panel
performed?

• When the antibody screen is positive.

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When to perform test

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

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Identification

• 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

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Confirmation

• Rule of 3 and 3
• Antigen type patients RBCs.
• Landsteiners Law!!!

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The 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

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Antigram
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Panel Antigram
Donor Cell number D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M 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
rr 4 0 0 0 0 0 0 0 0 0 0
rr 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
Patient Cells
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Auto Control

• Patients serum/plasma tested against a
  suspension of patients RBCs
• Optional
• Evaluate results in conjunction with patient
  history
• Autoantibody
• Newly forming alloantibody
• If patient has a positive DAT, auto control will
  be positive

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Test Method

• 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

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Interpreting Panel Results
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Cell D C c E e Cw K k Kpa Kpb J sa J sb F ya F yb J ka J kb L e a L eb P 1 M N S s L ua Lub X ga A H G 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
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Exclusion

• 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

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Exclusion
Cell D C c E e Cw K k Kpa Kpb J sa J sb F ya F yb J ka J kb L e a L eb P 1 M N S s L ua Lub X ga A H G 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
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Inclusion

• 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.

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Inclusion
Cell D C c E e Cw K k Kpa Kpb J sa J sb F ya F yb J ka J kb L e a L eb P 1 M N S s L ua Lub X ga A H G 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
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Other 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.

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Probability

• 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

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The Rule of 3 and 3
Cell D C c E e Cw K k Kpa Kpb J sa J sb F ya F yb J ka J kb L e a L eb P 1 M N S s L ua Lub X ga A H G 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
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Antigen Typing

• Confirms the antibody identification
• LANDSTEINERS LAW
• Test patients 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

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Selecting Controls for Antigen Typing
Cell D C c E e Cw K k Kpa Kpb J sa J sb F ya F yb J ka J kb L e a L eb P 1 M N S s L ua Lub X ga A H G 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
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Value 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

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Reporting

• 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

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The End