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Solid phase techniques have now superseded cellular based methods for HLA antibody detection and identification.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