BASIC ANTIBODY IDENTIFICATION. Jean Purcelli, MT (ASCP)SBB Blood Centers of the Pacific May 2010 Version 2 May 2012. There are two types of unexpected red blood cell antibodies. Alloantibodies which may occur as a result from: Transfusion Pregnancy Transplantation
Jean Purcelli, MT (ASCP)SBB
Blood Centers of the Pacific
Version 2 May 2012
Alloantibodies which may occur as a result from:
Injections of immunogenic material
I.V. or I.M. material such as IVIg or RhIg (passively acquired)
Autoantibodies which occur with certain diseases or medical treatments.
Alloantibodies can be detected in about 0.3%-5% of the general population.
The incidence increases if the transfused or previous pregnancy populations are studied.
The incidence is affected by:
Prevalence of the antigen in the population
Status of the immune system
Anti-A and Anti-B are examples of naturally occurring antibodies and they are expected.
Lewis (Anti-Lea, Leb), -P1, -M, -N are unexpected antibodies, but can occur naturally.
Naturally occurring antibodies have resulted from environmental, bacterial, or viral antigens that are similar to blood group antigens.
We concentrate on identifying 13 clinically significant alloantibodies that are commonly found:
Rh system antibodies: D C E c e f
Kell system antibodies: K
Duffy system antibodies: Fya Fyb
Kidd system antibodies: Jka Jkb
MNSs system antibodies: S s sometimes M
(-M may be significant if it reacts at 37 C or by IAT)
Several clinically insignificant alloantibodies and autoantibodies are commonly found.
Alloanti-M (reacting only below 37ºC), -N, -P1, -Lea, -Leb, generally react below 37ºC and usually are not clinically significant.
It is usual to transfuse with pre-warmed IAT, crossmatch compatible red cells. The IS crossmatch is dropped.
Autoanti-I and –IH are commonly found when testing is conducted at room temperature or colder.
A red cell panel is merely an expanded antibody detection set with 8-10 cells designed to identify and rule out the commonly found antibodies.
Make copies of both sides of the Master Lists when they arrive, a new set will arrive every 2-4 weeks.
The panel sheets are used as work sheets.
The lot number of the panel should match the master sheet.
Use in-dated cells whenever possible.
The panel is designed to have some homozygous expressions of antigens, except P1. In addition K, Kpa, Jsa, and Lua antigens rarely exist as homozygous expressions on a panel.
Next to the Vial Number is a column for Special Types.
There are columns for four High Frequency antigens: k, Kpb, Jsb, Lub. Cells negative for these antigens rarely exist in panels.
There is a note in the lower left listing several more high frequency antigens for which each cell was typed: I, Ge, Yta, Tja, Vel, Coa, Dib.
There are columns for five Low Frequency antigens: V, Cw, Kpa, Jsa, Lua.
In addition, there is a note in the lower left corner listing several more antigensof low frequency that each cell has been typed and found negative for Mg, Vw, Dia, Wra.
On the reverse side is another list of Extended Typings and notations.
Obtain Medical and Transfusion History
Each institution should develop a plan for antibody identification.
A plan establishes consistency and a general guide to antibody identification.
An example plan:
Steps 4-7 may be performed simultaneously.
Most test systems include enhancement media and antiglobulin reagent. Testing with panel cells is conducted at 37ºC, examined for agglutination or hemolysis and converted to the antiglobulin test.
A separate cold screen with Screening Cells and an auto control (in saline) offers valuable information.
Exclude specificities with negative reactions using a cross-out method.
A single antibody usually produces a clear pattern.
If the patient’s plasma is reactive with 2 cells positive for the antigen andnon-reactive with 2 cells negative for the antigen the minimum requirement for identification has been met.
Additional reactive antigen +cells and non-reactive antigen =cells increase the probability of correct identification of the antibody.
To exclude other possible antibodies, at least two cells with a homozgous expression of the antigen should be non-reactive, whenever possible.
The exclusion method by crossing out is a tentative and imperfect method to use until criteria have been met with a selected cell panel using the most sensitive enhancement media.
If a person’s cells type positive for the antigen, that person will not make the corresponding antibody.
Exceptions: There are many instances of D+ people producing anti-D and many auto-antibodies show specificities in the Rh system, particularly -e.
Monoclonal anti-Rh antisera show discrepant results when the antibody is made from different clones or sources.
In general a person who types negative for an antigen is capable of producing the antibody.
Lewis system antibodies are an exception: Only Le(a-b-) persons can make Lewis antibodies of any specificity.
Multiple antibodies: D+C, or D+E, or E+K
An antibody that demonstrates dosage:
anti-M,-N,-S,-s,-Jka,-Jkb, -C, -E,-c,-e
Some antigens have variable expression in different individuals: P1, I, “HTLA”
Unwanted reactions from cold or warm autoantibodies. Refer to the DAT or autocontrol and the cold antibody screen for clues.
There are several different approaches to resolving multiple antibodies :
We will concentrate on the most efficient way: a combination of phenotyping and using selected cell panels.
Two stage techniques using either ficin or papain to pre-treat red cells are the most useful.
Enzymes are used to enhance or destroy certain blood group antigens.
Enzyme technique is useful in sorting out multiple antibodies, enhancing weak antibodies, providing clues to the identity of certain antibodies to high prevalence antigens, and can be used in certain adsorption procedures.
Pre-treating panel cells takes about 10 minutes, then the serum is added and placed at 37º for 30 minutes.
With papain, “pan-agglutination” is often seen at the 37º stage and is disregarded if it is seen in all cells, including the auto control.
Unwanted reactions due to cold allo or autoantibodies and warm autoantibdies may be enhanced.
Antigens destroyed: M, N, S, Fya, Fyb. Small s is variableand should be QC’d with anti-s.
Antigens enhanced: Rh: D, E, c, e, f, and all Rh antigens. Jka, Jkb. Lewis, I.
Antigens unchanged: K, k and all Kell antigens.
IgG coated cells (positive DAT) cannot be typed with antisera requiring the antiglobulin test.
An elution is performed and eluate tested if the patient was recently transfused.
If the patient was not recently transfused, an elution is not performed, unless requested by a physician.
Cold autoantibodies are usually non-pathological and considered “normal” if they react only at 4ºC.
Normal cold autoantibodies may show anti-I or anti-IH specificity, but identity is not important.
Pathological cold autoantibodies have a wider thermal range, reacting at RT, 37º, and at AHG.
Patients with Cold Agglutinin Disease have red cells coated with complement components (C3).
Pathological cold agglutinins may present difficulty with proper cell typing. Warm a bottle of saline to 37º in a water bath. Pre-wash the red cells several times with warmed normal saline before typing.
Cold agglutinins with a wide thermal range may require Cold Autoabsorption and use of Pre-warmed saline techniques.
Patients become severely anemic and may require transfusions.
The DAT is positive for IgG or both IgG and C3.
The plasma reacts with all panel cells.
Warm autoantibodies may “mask” or obscure underlying alloantibodies.
DAT positive red cells can only be typed with monoclonal reagents. To type antiserum requiring the antiglobulin test, the red cells first have to be altered with Glycine-HCI-EDTA.
Warm auto adsorption is used to remove autoantibody.
There are several methods available.
PEG Autoadsorption is the most efficient, most effective, and least expensive.
Autoadsorption can be used only if the patient has not been recently transfused.
Results of the antibody identification, difficulty in typing, transfusion reactions, and special needs should be kept on file in the hospital Transfusion Service and the Blood Center
Antibodies may become undetectable over time, but antigen negative units should continue to be provided.