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

16.6: Testing for HLA-specific antibodies

16.6.1: General guidance

HLA-specific antibody screening and characterisation must comply with EFI Standards Section F (Antibody Screening and Crossmatching) and Section M (Flow Cytometry).

All commercial HLA antibody test kits should be CE and in vitro device (IVD) marked and validated for use. Each batch of commercial test kit or in-house panel should be evaluated against a minimum of three sera of known HLA specificity from different cross-reacting groups.

HLA-specific antibodies may be detected using reagent lymphocytes (or cell lines), solid-phase bound, purified HLA molecules, or particle bound, purified HLA molecules. If such techniques are used for screening (i.e. not characterisation of specificity) the following apply:

  • There should be discrimination between HLA Class I and Class II-specific antibodies.
     
  • Overall the target cells or molecules should cover either all the known HLA immunogenic epitopes or all HLA specificities (Class I, Class II, or both as appropriate) found in the population at over 0.5%.

16.6.2: Characterisation of antibody specificity

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.

Panels of HLA typed cells or purified HLA molecules are used for identification. 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.

There are many techniques available for the detection of HLA antibodies such as those developed for the detection and identification of HLA antibodies utilising Luminex microspheres. These assays are highly sensitive, leading to the detection of very low levels of HLA antibodies. 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

 

 

 

 

 

 

 

16.6.2.1: HLA antibody characterisation by complement dependent cytotoxicity

Rabbit complement

Rabbit complement used for detection of HLA antibodies by complement dependent cytotoxicity should comply with guidelines in section 16.4.4.

Instructions for use

In addition to the information required in section 16.6.2, the instructions for use should include the following information on each individual preparation of HLA reagent lymphocytes or set of HLA reagent lymphocytes:

  • the HLA phenotype of the reagent lymphocytes
     
  • the nature of the HLA reagent lymphocytes (e.g. normal peripheral lymphocytes, separated peripheral B lymphocytes, separated peripheral T lymphocytes, chronic lymphocytic leukemia (CLL) cells, splenic lymphocytes, lymph node lymphocytes, lymphoblastoid cell line)
     
  • the concentration of the lymphocyte suspension should be stated in the instructions for use for HLA reagent lymphocytes issued in individual immediate containers, or on the phenotype listing of batches issued as multi-immediate container products
     
  • HLA reagent lymphocyte sets issued in multi-well trays should include a representation of the tray or reservoir layout indicating the location of the various HLA reagent lymphocytes in the wells of the tray
     
  • for HLA reagent lymphocyte sets issued in multi-well trays or reservoirs the phenotype information may take the form of a listing of the phenotypes of each of the individual donations comprising the set
     
  • the shelf life of the HLA reagent lymphocytes following recovery from long-term storage and subsequent storage in conditions recommended by the manufacturer should be stated in the instructions for use
     
  • when HLA reagent lymphocytes are provided suspended in preservative or medium, the components of the preservative or the name of the medium should be stated in the instructions for use.

Reagent lymphocytes

Freshly isolated or previously frozen lymphocytes should have a viability of at least 80% and should contain less than 1% platelets or granulocytes.

Reagent B lymphocytes isolated for the identification of Class II antibodies should contain less than 10% of non-B cells.

The background incidence of spontaneous cell death, as assessed by a negative control serum, should be less than 30%.

Reagent lymphocytes supplied as previously frozen in test trays should contain 1000 to 2000 lymphocytes per well, after recovery following manufacturer’s instructions.

The manufacturer should specify in the instructions for use those antigens known to be present or absent, and those for which no testing has been performed. HLA-A, HLA-B, HLA-C, HLA-DR and HLA-DQ serologically defined specificities should be included in this statement.

16.6.2.2: HLA antibody characterisation by solid-phase and particle bound methods

Purified HLA captured onto a microtitre well, nylon membrane or microparticles can be used as sensor molecules for characterising sera containing HLA-specific antibodies.

Antibody binding can be detected by ELISA or fluorescence. The detector reagent should be able to identify IgG and discriminate between IgG, IgA and IgM.

Human material

If a product is prepared from human source material then the guidance in section 11.1.4.10 must be followed.

Instructions for use

The instructions for use must comply with the requirements of EN ISO 18113:2009 and the information required in section 16.6.1. In addition, the instructions for use should include the following information on each individual preparation or component of a set of HLA screening product:

  • the HLA antigens represented in each container
     
  • the concentration of any cells or particles in suspension should be stated in the instructions for use of HLA screening product issued in individual immediate containers or on the antigen information table of batches issued as multi-immediate container products or multi-well trays or reservoirs
     
  • HLA screening products issued in multi-well trays should include a representation of the tray or reservoir layout indicating the location of the HLA antigens in the wells of the tray
     
  • the expiry life of the HLA screening product following reconstitution or preparation and subsequent storage in conditions recommended by the manufacturer should be stated
     
  • when components of an HLA screening product contains preservatives the name of the chemical preservatives and the components which contain them should be stated.

Validation

Kits for the detection of HLA antibodies should be validated for sensitivity and specificity on a batch basis using a panel of clinically representative HLA antisera. A panel of sera shown to be inert for HNA and HLA antibodies should also be used.