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ASHI Governance
Standards for Histocompatibility Testing Section H - Antibody Screening H1.000
Techniques H1.100
A complement-dependent cytotoxic technique
must be used for the detection of antibody to HLA antigens. Other
techniques may be used as an adjunct to the lymphocyte-based technique
if they have been demonstrated by the laboratory, or established in
publications, to identify HLA-specific antibody with a specificity
equivalent or superior to that of the lymphocyte-based technique or at
a level appropriate for the clinical indication. H1.120
To detect antibodies to HLA Class II antigens, a method must be used
that distinguishes them from antibodies to Class I antigens. H1.130
Techniques that detect lymphocyte-dependent antibody or test for
cellular sensitization may be used to supplement the laboratory’s
technique that meets the requirements of H1.100. H1.140
Techniques to identify non-HLA alloantibodies such as those using
monocytes or cells from specific tissues may be used to supplement the
laboratory’s that meets the requirements of H1.100. H1.150
Reports of results of antibody screening must include identification
of the technique. H1.200
Sera H1.210
Sera must be tested at a concentration determined to be optimal for
detection of antibody to HLA antigens. The dilution(s) must be
documented. H1.220
Negative control sera must include a serum from non-alloimmunized
human donor(s). Each assay must include negative control(s). H1.230
Positive control sera should be from highly alloimmunized individuals
and documented to react with HLA antigens. The antibodies must be of
the appropriate isotype for each assay. Each assay must include
positive control(s). H1.300
Panel H1.310
The panel of HLA antigens must include sufficient panel cell donors to
ensure that they are appropriate for the population served and for the
use of the data. H1.320
For assays intended to provide information on antibody specificity,
documentation of the Class I and Class II phenotypes of the donors of
the panel cells must be provided. H1.330
To identify the specificity of an antibody with certainty, the
laboratory should test the serum with additional cells expressing and
lacking the candidate antigen and cross-reactive antigens. H2.000
Antibody screening by complement-dependent cytotoxicity H2.100
Positive and negative controls for the activity of complement and the
viability of the test cells must be included on each tray. H2.200
Sera must be tested undiluted. H.2.300
Target Cells H2.310
Target cells may be mononuclear cells from peripheral blood, lymph
nodes, spleen or cell lines or CLL. H2.320
To detect antibodies to HLA Class II antigens, B lymphocytes, B
lymphoblastoid cell lines or CLL may be used. H2.400
The specificity of serum to be used as a reagent must be validated in
other laboratories. Specificity determinations should include
supporting statistical analysis. H3.000
Antibody screening by Flow Cytometry H3.100
Laboratories performing assays using flow cytometry must conform to
the Standards in Section Q1.000 Instrument Standardization/Calibration
and in Section Q2.000 Flow Cytometric Crossmatch Technique. H3.200
If cells pooled from multiple individuals are used for a present/not
present detection of antibody, the cells used must cover the major
antigen specificities or CREG. The laboratory must cite the
publication used to define major antigen specificities or CREG. H3.300
For assignment of antibody specificity, cells from a sufficient number
of individuals must be used to cover appropriate specificities. To
assign specificity for major antigen specificities or CREG, sufficient
pools of individual cells must be used. The laboratory must cite the
publication used to define major antigen specificities or CREG. H4.000
Antibody screening by ELISA H4.100
Laboratories using ELISA techniques for antibody screening must
conform to Standards in Sections R. Enzyme Linked Immunosorbent Assay
(ELISA). H4.200
To control for non-specific binding of antibody, each serum must be
assayed in a test system which lacks HLA antigen. H4.300
Antigens obtained from pooled cells may be used for a present/not
present detection of antibody. Cells from a sufficient number of
individuals must be used to cover major antigen specificities. The
number of individuals must be documented. H4.400
Sera must be tested at a concentration determined to be optimal for
detection of antibody to HLA antigens. The dilution must be
documented. H4.500
The panel for HLA antigens must include sufficient panel cell donors
to ensure that they are appropriate for the population served and for
the use of the data. H4.510
Antigens obtained from pooled cells may be used for a present/not
present detection of antibody. H4.520 For assays intended to provide information on antibody specificity, the manufacturer must provide documentation of the Class I and Class II phenotypes of the donors of the panel cells. |
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