16.6 Testing for HLA-specific antibodies - HLA typing and HLA serology

HLA-specific antibody screening and characterisation must comply with the relevant EFI Standards.

Sera containing HLA-specific antibodies may be interpreted in terms of specific antigens (i.e. whole gene products), cross-reactive groups, single epitopes, or any combination of these as long as standard and unequivocal nomenclature is used. Specificity characterisation may be helped by computer analysis but a final result must involve manual interpretation.

Solid phase techniques have now superseded cellular based methods for HLA antibody detection and identification. Commercial kits are available which consist of beads impregnated with differing ratios of 2 fluorochromes resulting in a unique signal for each bead and which have 1 or several types of HLA molecules attached.

The assay involves:

  • Incubation of a patient's serum with the beads.
  • If the patient has HLA antibodies, the serum will react with the bead expressing the appropriate HLA molecule.
  • After washing, the beads are incubated with a secondary antibody, usually with a phycoerythrin (PE)-labeled anti‑human IgG.

Three levels of testing are possible depending on requirements:

  1. The first level provides a positive/negative result with respect to a patient's antibody status. In this instance, the beads are bound with a large number of HLA class I or class II molecules derived from lymphoblastoid cell lines.
  2. Beads used in second level testing are bound with molecules derived from a single cell line and hence express 2 HLA molecules for each of the HLA loci (HLA-A, -B, -C for class I and HLA-DR, -DQ and -DP for class II).
  3. The third level of testing involves the use of beads bound with single HLA molecules produced by recombinant technology, so called single antigen beads (SAB). These beads provide a real advantage of this technology as complex mixtures of antibodies can be characterised and HLA specificities accurately determined. This technology is now considered essential for the pretransplant testing of sensitised patients.

The composition of the panel should be sufficient to discriminate the specificities (Class I, Class II, or both as appropriate) given in Tables 16.2.1 to 16.2.5. The full list of antigens comprising a panel should be supplied and typed to the higher level of resolution shown in the tables.

The detector reagent should be able to identify IgG and discriminate between IgG, IgA and IgM. Cut-off values for HLA antibody detection should be set in accordance with manufacturer's instructions and local clinical evaluation.

For DNA typed reagents the types should be supplied at the 4-digit (second field) level (e.g. HLA-A*02:01) and null alleles identified.

Table 16.2.1: Characterisation of HLA-specific antibodies – HLA-A
HLA-A broad specificities Splits
A1
Not applicable
A2
Not applicable
A3
Not applicable
A9
A23
A9
A24
A10
A25
A10
A26
A10
A34
A10
A66
A11
Not applicable
A19
A29
A19
A30
A19
A31
A19
A32
A19
A33
A19
A74
A28
A68
A28
A69
A36
Not applicable
A43
Not applicable
A80
Not applicable
Table 16.2.2: Characterisation of HLA-specific antibodies – HLA-B
HLA-B broad specificities Splits
B5
B51
B5
B52
B7
Not applicable
B8
Not applicable
B12
B44
B12
B45
B13
Not applicable
B14
B64
B14
B65
B15
B62
B15
B63
B15
B75
B15
B76
B15
B77
B16
B38
B16
B39
B17
B57
B17
B58
B18
n/a
B21
B49
B21
B50
B22
B54
B22
B55
B22
B56
B27
Not applicable
B35
Not applicable
B37
Not applicable
B40
B60
B40
B61
B41
Not applicable
B42
Not applicable
B46
Not applicable
B47
Not applicable
B48
Not applicable
B53
Not applicable
B59
Not applicable
B67
Not applicable
B70
B71
B70
B72
B73
Not applicable
B78
Not applicable
B81
Not applicable
Bw4
Not applicable
Bw6
Not applicable
Table 16.2.3: Characterisation of HLA-specific antibodies – HLA-C
HLC-C broad specificities Splits
Cw1
Not applicable
Cw2
Not applicable
Cw3
Cw9
Cw3
Cw10
Cw4
Not applicable
Cw5
Not applicable
Cw6
Not applicable
Cw7
Not applicable
Cw8
Not applicable
Cw12
Not applicable
Cw14
Not applicable
Cw15
Not applicable
Cw16
Not applicable
Cw17
Not applicable
Cw18
Not applicable
Table 16.2.4: Characterisation of HLA-specific antibodies – HLA-DR
HLC-DR broad specificities Splits
DR1
Not applicable
DR103
Not applicable
DR2
DR15
DR2
DR16
DR3
DR17
DR3
DR18
DR4
Not applicable
DR5
DR11
DR5
DR12
DR6
DR13
DR6
DR14
DR7
Not applicable
DR8
Not applicable
DR9
Not applicable
DR10
Not applicable
DR51
Not applicable
DR52
Not applicable
DR53
Not applicable
Table 16.2.5: Characterisation of HLA-specific antibodies – HLA-DQ
HLA-DQ broad specificities Splits
DQ1
DQ5
DQ1
DQ6
DQ2
Not applicable
DQ3
DQ7
DQ3
DQ8
DQ3
DQ9
DQ4
Not applicable

Last updated on 4 September 2023