Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical Diagnosis (990 page)

   The class I region contains the genes encoding the “classical” class I HLA antigens, HLA-A, HLA-B, and HLA-C. Class I antigens, made up of an alpha chain and a beta chain (beta-2 microglobulin encoded on chromosome 15), are expressed on almost all cells of the body at varying density except erythrocytes and trophoblasts. This region also contains other HLA class I genes such as HLA-E, HLA-F, and HLA-G; MHC class I chain–related (MIC-A and MIC-B) genes; and a variety of other genes, not all of which are immune related.

   The class II region contains the genes encoding the “classical” class II molecules, HLA-DR, HLA-DQ, and HLA-DP. Class II antigens are only expressed on B cells, dendritic cells, and monocytes and can be induced during inflammation on many other cell types that normally have little or no expression. The class II molecules also consist of an alpha and a beta chain both are encoded by genes within the MHC.

   Between class I and class II is the class III region. This region does not contain any of the HLA genes. It does, however, contain many genes of importance in the immune response, for example, complement, tumor necrosis factor, and heat shock protein.

   Clinical applications of HLA are associated with, but not limited to, the following: solid organ transplantation, stem cell transplantation, platelet transfusion, disease association, and drug sensitivity.

   Testing Methods

HLA Typing

   HLA typing was originally performed by serology. Due to the limited accuracy, the serologic HLA typing has largely been replaced by DNA PCR–based methods. Sequence-specific oligonucleotide probing (PCR-SSOP), sequence-specific primer amplification (PCR-SSP), and sequence-based typing (SBT) are commonly used in clinical HLA laboratories.

   
PCR-SSP:
Multiple pairs of PCR primers, designed to anneal within DNA regions present in certain alleles or groups of alleles, are used for PCR amplification. If the corresponding alleles are present, their specific amplicons can be detected by gel electrophoresis. The size of the PCR products needs to be known for interpretation, and the electrophoresis migration must be run longer to separate all PCR fragments.

   
PCR-SSOP:
Locus-specific PCR primers were selected to amplify the HLA locus of interest followed by the hybridization using sequence-specific oligonucleotide probes. The HLA genotype was assigned on oligonucleotide positive reactions. Currently, the Luminex platform is the most commonly used PCR-SSOP method. Oligonucleotide probes, covalently linked to a set of polystyrene carboxylated microbeads, are designed to specifically detect the nucleotide sequences at the polymorphic sites of the HLA specificities.

   
SBT:
This is the only technique that directly detects the nucleotide sequences of an allele, thus allowing exact assignment. Sequence analysis software generates final allele assignment by comparing the nucleotide sequence obtained with a database of all known alleles.

HLA Antibody Analysis

   The deleterious role of HLA antibodies has become more apparent with the development of sensitive assays identifying previously undetectable antibody specificities in the last few years, replacing the less sensitive lymphocyte target–based complement-dependent cytotoxicity (CDC) assays. Depending upon the clinical application needs, HLA antibody analysis by CDC, ELISA, flow cytometry, and Luminex platform can all be utilized, either individually or in combination, to characterize the HLA antibody.

T- and B-cell crossmatch

   The crossmatch is used to determine if the recipient has antibodies against the potential donor. The test is performed between the patient’s serum and the potential donor’s T and B cells. Complement-dependent cytotoxicity (CDC), anti–human globulin–enhanced CDC (AHG-CDC), and flow cytometric crossmatches are commonly used in the lab. The sensitivity of the crossmatch methods varies a lot; CDC as the least sensitive and flow as the most sensitive. A positive crossmatch is a strong indication of HLA incompatibility.