JPAC Joint United Kingdom (UK) Blood Transfusion and Tissue Transplantation Services Professional Advisory Committee

16.6: Testing for HLA-specific antibodies

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Solid phase techniques have now superseded cellular based methods for HLA antibody detection and identification.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 Table 16.2. The full list of antigens comprising a panel should be supplied and typed to the higher level of resolution shown in Table 16.1.

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 Characterisation of HLA-specific antibodies

HLA-A broad specificities Splits HLA-B broad specificities Splits HLA-C broad specificities Splits HLA-DR broad specificities Splits HLA-DQ broad specificities Splits

A1

 

B5

B51

Cw1

 

DR1

 

DQ1

DQ5

A2

 

B5

B52

Cw2

 

DR103

 

DQ1

DQ6

A3

 

B7

 

Cw3

Cw9

DR2

DR15

DQ2

 

A9

A23

B8

 

Cw3

Cw10

DR2

DR16

DQ3

DQ7

A9

A24

B12

B44

Cw4

 

DR3

DR17

DQ3

DQ8

A10

A25

B12

B45

Cw5

 

DR3

DR18

DQ3

DQ9

A10

A26

B13

 

Cw6

 

DR4

 

DQ4

 

A10

A34

B14

B64

Cw7

 

DR5

DR11

 

 

A10

A66

B14

B65

Cw8

 

DR5

DR12

 

 

A11

 

B15

B62

Cw12

 

DR6

DR13

 

 

A19

A29

B15

B63

Cw14

 

DR6

DR14

 

 

A19

A30

B15

B75

Cw15

 

DR7

 

 

 

A19

A31

B15

B76

Cw16

 

DR8

 

 

 

A19

A32

B15

B77

Cw17

 

DR9

 

 

 

A19

A33

B16

B38

Cw18

 

DR10

 

 

 

A19

A74

B16

B39

 

 

 

 

 

 

A28

A68

B17

B57

 

 

 

 

 

 

A28

A69

B17

B58

 

 

DR51

 

 

 

A36

 

B18

 

 

 

DR52

 

 

 

A43

 

B21

B49

 

 

DR53

 

 

 

A80

 

B21

B50

 

 

 

 

 

 

 

 

B22

B54

 

 

 

 

 

 

 

 

B22

B55

 

 

 

 

 

 

 

 

B22

B56

 

 

 

 

 

 

 

 

B27

 

 

 

 

 

 

 

 

 

B35

 

 

 

 

 

 

 

 

 

B37

 

 

 

 

 

 

 

 

 

B40

B60

 

 

 

 

 

 

 

 

B40

B61

 

 

 

 

 

 

 

 

B41

 

 

 

 

 

 

 

 

 

B42

 

 

 

 

 

 

 

 

 

B46

 

 

 

 

 

 

 

 

 

B47

 

 

 

 

 

 

 

 

 

B48

 

 

 

 

 

 

 

 

 

B53

 

 

 

 

 

 

 

 

 

B59

 

 

 

 

 

 

 

 

 

B67

 

 

 

 

 

 

 

 

 

B70

B71

 

 

 

 

 

 

 

 

B70

B72

 

 

 

 

 

 

 

 

B73

 

 

 

 

 

 

 

 

 

B78

 

 

 

 

 

 

 

 

 

B81

 

 

 

 

 

 

 

 

 

Bw4

 

 

 

 

 

 

 

 

 

Bw6

 

 

 

 

 

 

 

 

Last updated 04/09/2023