Identifying Antibodies The Antibody Panel CLS 422 Clinical Immunohematology I

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

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

When is an antibody identification panel performed? When the antibody screen is positive.

When 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. May also test the panel cells against an eluate made from the patient’s RBCs when the patient has a positive DAT with IgG, in order to determine the identity of the antibody coating the RBCs.

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

Confirmation Rule of 3 and 3 Antigen type patient’s RBCs. Landsteiner’s Law!!! Rule of 3 and 3 - The probability that the reactions observed are due to a given antibody specificity and are not due to random chance. 3 antigen positive cells react with the serum; 3 antigen negative cells fail to react with the serum. This gives 95% confidence that the antibody ID is correct. In order to form an antibody, the patient’s RBCs should lack the antigen (type as antigen negative).

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 Designed for each specificity to have a unique pattern of positive and negative cells.

Antigram The panel is accompanied by an antigen profile sheet, which lists the antigens present on each cell, and provides a place to record results. The profile sheet is lot specific.

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 + R1wR1 2 R2R2 3 r’r 4 r’’r 5 rrK 6 rrFya 7 Ror 8 rr 9 R2r 10 R1R1 11 Patient Cells Which cell is homozygous for K antigen? 10 (It is somewhat unusual to have a K+k- cell on a panel since k is a high prevalence antigen. Which cell is heterozygous for C antigen? 4 Which cell will react best with anti-Jka? 5, 6, 8, 9, 10 because they have homozygous antigen expression Which cell is probably from a Black donor? 1 or 8 because they are Fy (a-b-) Which donor is a secretor? 1, 2, 5, 6, 8, 9, 10 (positive for Leb)

Auto 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 The auto control is not required by regulation. Some institutions will test the auto control in conjunction with the antibody screen or the panel. Some institutions opt not to test an auto control. A DAT may be tested in place of the auto control.

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 Application of the indirect antiglobulin test.

Interpreting Panel Results

Cell D C c E e Cw K k Kpa Kpb J sa sb F ya yb ka kb L a eb P 1 M N S s ua Lub X ga A H G CC + 3+ 2 2+ 3 4 5 6 7 8 9 10 11 w Auto If the antibody in the serum is directed against one of the antigens present on a panel cell, that cell should test positive. If the antibody is not directed against an antigen on the panel cell, the cell should give a negative reaction.

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 Dosage – An antibody may not react with a cell that has both alleles present, if the antibody is showing dosage. By using RBCs with homozygous antigen expression, you avoid eliminating an antibody showing dosage. Low prevalence antigens are K, Kpa, Jsa, and Lua. Since antibodies of the Kell system do not show dosage, it is not necessary to use homozygous cells in order to exclude antibodies to Kell antigens. A second exception to the “homozygous” rule is when anti-D is present is a patient’s serum. It may be difficult to find a cell that is D negative and has homozygous expression of C or E. In this situation ONLY, C and E may be excluded using a cell with heterozygous antigen expression (i.e. r’r for C and r”r for E) When needed, screen cells can be used for exclusions too!

Exclusion Cell D C c E e Cw K k Kpa Kpb J sa sb F ya yb ka kb L a eb P 1 M N S s ua Lub X ga A H G CC + 3+ 2 2+ 3 4 5 6 7 8 9 10 11 w Auto Cell 1 cannot be used for exclusion, as it reacted with the patient’s serum. The specificity of the antibody should match one of the antigens present on Cell 1. Cell 2 can be used to exclude: D, C, e, Cw, k, Kpb, Jsb, Fyb, Jkb, Leb, P1, s, Lub, and Xga. Cell 3 excludes: c, E, Fya, and M. Cell 4 excludes: Jsa, Jka, and N. Cell 5 could be used for exclusion, but in this case does not exclude any new specificities. Cell 6 excludes Lea. Cell 7 cannot be used for exclusion because it yielded a positive result. Cell 8 can be used to exclude Lua. Cell 9 excludes S. Cell 10 cannot be used for exclusion because it reacted with the serum. Cell 11 could be used for exclusion, but in this case, does not exclude any additional specificities.

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.

Inclusion Cell D C c E e Cw K k Kpa Kpb J sa sb F ya yb ka kb L a eb P 1 M N S s ua Lub X ga A H G CC + 3+ 2 2+ 3 4 5 6 7 8 9 10 11 w Auto Only K and Kpa remain. The pattern of reactivity (inclusion) matches anti-K. Kpa has NOT been eliminated, i.e. we cannot say that anti- Kpa is not present because none of these panel cells were Kpa positive.

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.

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

The Rule of 3 and 3 Cell D C c E e Cw K k Kpa Kpb J sa sb F ya yb ka kb L a eb P 1 M N S s ua Lub X ga A H G CC + 3+ 2 2+ 3 4 5 6 7 8 9 10 11 w Auto Cells 1, 7 and 10 are K positive and gave a positive reaction. Cells 2-6, 8, 9, and 11 are K negative and they did not react. We can be 95% confident that anti-K is present in this specimen.

Antigen 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 Landsteiner’s Law states that a person should not make an antibody against an antigen that their cell possesses. The positive control should be a cell with a single dose of the antigen (heterozygous) in order to prove sensitivity (the reagent is potent enough to detect even small amounts of the antigen). Many of the typing anti-sera rely on an IAT procedure.

Selecting Controls for Antigen Typing Cell D C c E e Cw K k Kpa Kpb J sa sb F ya yb ka kb L a eb P 1 M N S s ua Lub X ga A H G CC + 3+ 2 2+ 3 4 5 6 7 8 9 10 11 w Auto For anti-K typing serum, cells 1, 7 or 10 could serve as a positive control. Cells 2 -6 , 8, 9, or 11 could serve as a negative control.

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

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

The End