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ASHI Governance
Standards for Histocompatibility Testing Section F - Serologic Typing - HLA Class II F1.000
HLA-DR region antigens. F1.100
Typing for DR locus antigens is highly recommended. F1.200
If DR locus typing is done, the laboratory must be able to type for
all HLA-DR specificities for which sera are readily available, and
should make continuing efforts to type for all recognized HLA-DR
antigens. F2.000
HLA-DQ region antigens. F2.100
Typing for DQ locus antigens is not mandatory. F2.200
If DQ locus typing is done, the laboratory must be able to type for
all HLA-DQ specificities for which sera are readily available and
should make continuing efforts to type for all recognized HLA-DQ
antigens. F3.000
HLA-DP region antigens. F3.100
Typing for DP locus antigens is not mandatory. F3.200
If DP locus typing is done, the laboratory must be able to type for
those HLA-DP specificities which do not have a “w” prefix, and
should make continuing efforts to type for all recognized HLA-DP
antigens. F4.000
Serologic typing techniques – HLA Class II F4.100
Techniques used must be those which have been established to define
HLA Class II specificities optimally. F4.200
Techniques used should employ minimal amounts of rare reagents. In
general, only 1 microliter of each typing serum should be used in each
serological test. When monoclonal antibodies are used, the amount
should be adequate to ensure accuracy of the assay employed. F4.300
Control sera. F4.310
Each typing must include at least one positive control serum,
previously shown to react with all cells expressing Class II antigens. F4.311
Typing results may be invalid if the positive control fails to react
as expected. F4.320
Each typing must include at least one negative control serum. The
negative control should either be one previously shown to lack
antibody or should be from a healthy male with no history of blood
transfusion. F4.321
Cell viability in the negative control well at the end of incubation
must be sufficient to permit accurate interpretation of results. For
most techniques, viability should exceed 80%. F4.322
In assays in which cell viability is not required, results on positive
and negative controls must be sufficiently discriminatory to permit
accurate interpretation of results. F4.400
Target cells. F4.410
Cells may be obtained from peripheral blood, bone marrow, lymph nodes
or spleen, or cultured cells. F4.411
If the cell donor has been transfused within the previous seven days,
results are acceptable only if antigens are unequivocally defined,
with no more than two antigens per locus. F4.420
Typing for Class II antigens usually requires B lymphocyte-enriched
preparations. The proportion of B lymphocytes in each preparation must
be confirmed and should usually be at least 80%. F4.421
Separation of B lymphocytes is not required if a technique is used
which distinguishes between T and B lymphocytes or in assays in which
antibodies with well-defined specificity are used which only define
HLA class II molecules. F4.500
Each HLA-Class II antigen should be defined by at least three sera, if
all are operationally monospecific. If multispecific sera must be
used, at least five partially non-overlapping sera should be used to
define each HLA-Class II antigen. F4.510
If monoclonal antibodies are used, each DR, DQ, DP antigen should be
defined by at least two antibodies with private epitope specificity or
one antibody with private epitope specificity and two with public
epitope specificity or at least three partially non-overlapping
antibodies with public epitope specificities. F4.600
Each monoclonal antibody used for alloantigen assignment must be used
at a dilution and with a technique in which it demonstrates: 1)
specificity comparable to antigen assignment by alloantisera on a
well-defined cell panel or 2) specificity officially recognized by the
W.H.O. F5.000
Internal quality control. F5.100
Cell panels of known HLA Class II type must be available to prove the
specificity of new antibodies. The panel cells should include at least
one example of each HLA antigen the laboratory should be able to
define, and be from a variety of ethnic groups. Storage of at least
some panel cells at -80°C or in liquid nitrogen may be necessary to
insure availability of required antigens. F5.200
Typing sera. F5.210
It is recommended that the specificity of typing sera obtained locally
be confirmed in at least one other HLA laboratory. F5.220
Specificity of individual sera received from other laboratories or
commercial sources must be confirmed to ensure that they reveal the
same specificities in the receiving laboratory. F5.230
Each lot of new commercial typing trays must be evaluated by testing
either with at least five different cells of known phenotype
representing major specificities or in parallel with previously
evaluated trays. F5.300
Complement. F5.310
Each batch of complement must be tested to determine that it mediates
cytotoxicity in the presence of specific antibody, but is not
cytotoxic in the absence of specific antibody. F5.311
The test should employ multiple dilutions of complement to ensure that
it is maximally active at least one dilution beyond that intended for
use. F5.312
The test should be carried out with at least two antibodies which
should react with at least two different test cells and at least one
cell which should not react. A strong and a weak antibody should be
selected for the test, or serial dilutions of a single serum may be
used. F5.313
Complement should be tested separately for use with each type of
target cell, since a different dilution or preparation may be required
for optimal performance. F6.000
External quality control. F6.100
At least one form of external quality control must be used to ensure
that local definition of HLA antigens agrees with that in other
laboratories. F6.200
The external quality control may consist of comparison of results
using typing sera tested by others or typing of cells typed by others.
Preferably, both approaches should be used. F6.300 External quality controls may be carried out through local or regional arrangements and by participation in the ASHI/CAP or another equally acceptable proficiency test. |
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