HLA typing and HLA serology
Preface
For the seventh edition we have made significant changes in both the structure and content to the previous version of the chapter concerning HLA typing and antibody testing. This obviously reflects the continuing scientific and technical development in the field. DNA-based testing for HLA alleles now predominates over serological phenotyping and antibody detection and characterization increasingly involve non-cell based methods. To reflect this, much of the guidance relating to HLA serology in the sixth edition has been removed. This HLA section is now constructed from three main parts, concerning reagents (Section 15.1), testing (Sections 15.2, 15.3 and 15.4), and application to donor and patient investigations (Section 15.5), respectively. For certain patient or donor investigations there is, of course, an overlap with the guidance given in this and the Granulocyte or Platelet immunology chapters (Chapters 16 and 17 respectively). This is particularly relevant to the laboratory investigations of platelet refractoriness and TRALI, so diagrams are included (Figures 15.1 and 15.2) to indicate how the different guidelines relate to each other.
Introduction
The transfusion or transplantation of blood products bearing allogeneic HLA (human leukocyte antigen) can stimulate clinically significant immunological responses. All cellular components except erythrocytes express HLA and any plasma-containing product may include HLA-specific antibodies which are potentially harmful to the recipient.
Prospective HLA typing of donors is undertaken for the platelet transfusion of immune refractory patients and those with disorders of platelet function and structure. Potential haemopoietic stem cell (HSC) donors are HLA typed to be placed on one of the national donor registries.
HLA typing or antibody investigations may be undertaken for diagnostic purposes or to investigate harmful consequences of transfusion. Thus the diagnosis of immune refractoriness requires the demonstration of HLA-specific antibodies (or other, platelet-specific antibodies) in the donor. As part of the investigation of transfusion-related acute lung injury (TRALI), implicated donors are screened for HLA (and HNA) -specific antibodies and the patient HLA typed if possible. The investigation of transfusion associated graft versus host disease (TAGvHD) requires HLA typing of patient and donors.
The European Federation for Immunogenetics (EFI) has established standards (currently version 5.3, www.efiweb.org)(1) for histocompatibility testing and where appropriate these
guidelines will defer to the relevant EFI standard and these will be stated in the text. In general guidance for practice is indicated by the term 'should'. The use of the term 'must' is mostly limited to circumstances where an EFI standard applies.
Terminology and nomenclature
All assignments, irrespective of the method, must comply with the current WHO Nomenclature Committee for Factors of the HLA System report,(2) Nomenclature for Factors of the HLA System, 2004. Marsh SGE et al(3) (and see EFI standards D1.000-D1.300, inclusive). Examples of acceptable HLA assignments are as follows:
HLA-B12, HLA-B44, HLA-B-44(12), HLA-B*44, HLA-B*4409
HLA may be serologically typed (to determine the phenotype) or by DNA analysis. The term genotype is properly used to describe the genetic (DNA) constitution determined by the pattern of inheritance (EFI standard D1.230).
Traditionally HLA typing has been performed using serological typing techniques using panels of alloantibodies derived primarily from multiparous women. This has allowed for the typing of HLA serological antigen specificities. Some HLA specificities, termed 'broad', have, since their first discovery, been further 'split' into two or more subtypes. For example, the 'broad' HLA-A9 specificity can be split into the A23 and A24 types, and similarly HLA-B15 can be split into B62, B63, B75, B76 and B77. In addition two HLA specificities, Bw4 and Bw6, have been defined which refer to public epitopes present as alternatives on all HLA-B antigens. Bw4 is also found on certain HLA-A antigens.
More recently HLA typing has been performed by DNA based molecular techniques, which employ either DNA based probes or primers, to type for the presence or absence of sequence motifs. Kits using this technology are able to define the HLA alleles present in an individual to a variable level of resolution dependent on a number of factors which may include; the number of probes or primers employed; the number of alleles defined for a given specificity; the HLA alleles present in the individual. Although it is possible to achieve a high resolution or allele level typing using these methods, it is common practice to type only to a low or medium level of resolution, and in so doing to present HLA typing results that include some ambiguity in the interpretation.
Each HLA antigenic specificity may be encoded by a number of different HLA alleles, and for many of the HLA alleles there is no determined serologically defined antigen. Thus it is not always possible to assign a serological equivalent to each HLA allele.(4) One consequence of this is that it is not practical to subject serological and DNA-based typing to the same standard as this would need to be unacceptably low (i.e. the lowest common denominator). Both techniques are in general use, each having specific advantages and disadvantages, and under these circumstances professional judgement together with the following guidelines should be used to delivery an appropriate standard of HLA typing.
Caution should be exercised therefore, if an HLA type assigned using DNA-based molecular techniques is converted into a serological equivalent and must always be avoided with alleles for which the phenotype has not been unequivocally defined.