Testing for HLA-specific antibodies - HLA typing and HLA serology - Chapter 16

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Solid phase techniques have now superseded cellular based methods for HLA antibody detection and identification.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 two fluorochromes resulting in a unique signal for each bead and which have one 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 two 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 characterized and HLA specificities accurately determined. This technology is now considered essential for the pretransplant testing of sensitized 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 four-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 n/a
A2 n/a
A3 n/a
A9 A23
A9 A24
A10 A25
A10 A26
A10 A34
A10 A66
A11 n/a
A19 A29
A19 A30
A19 A31
A19 A32
A19 A33
A19 A74
A28 A68
A28 A69
A36 n/a
A43 n/a
A80 n/a
Table 16.2.2: Characterisation of HLA-specific antibodies – HLA-B
HLA-B broad specificities Splits
B5 B51
B5 B52
B7 n/a
B8 n/a
B12 B44
B12 B45
B13 n/a
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 n/a
B35 n/a
B37 n/a
B40 B60
B40 B61
B41 n/a
B42 n/a
B46 n/a
B47 n/a
B48 n/a
B53 n/a
B59 n/a
B67 n/a
B70 B71
B70 B72
B73 n/a
B78 n/a
B81 n/a
Bw4 n/a
Bw6 n/a
Table 16.2.3: Characterisation of HLA-specific antibodies – HLA-C
HLC-C broad specificities Splits
Cw1 n/a
Cw2 n/a
Cw3 Cw9
Cw3 Cw10
Cw4 n/a
Cw5 n/a
Cw6 n/a
Cw7 n/a
Cw8 n/a
Cw12 n/a
Cw14 n/a
Cw15 n/a
Cw16 n/a
Cw17 n/a
Cw18 n/a
Table 16.2.4: Characterisation of HLA-specific antibodies – HLA-DR
HLC-DR broad specificities Splits
DR1 n/a
DR103 n/a
DR2 DR15
DR2 DR16
DR3 DR17
DR3 DR18
DR4 n/a
DR5 DR11
DR5 DR12
DR6 DR13
DR6 DR14
DR7 n/a
DR8 n/a
DR9 n/a
DR10 n/a
DR51 n/a
DR52 n/a
DR53 n/a
Table 16.2.5: Characterisation of HLA-specific antibodies – HLA-DQ
HLA-DQ broad specificities Splits
DQ1 DQ5
DQ1 DQ6
DQ2 n/a
DQ3 DQ7
DQ3 DQ8
DQ3 DQ9
DQ4 n/a