HLA typing

Serologic and cellular methods

Historically, HLA typing has been performed by using a combination of the serologic microcytotoxic-ity and, in special cases, mixed lymphocyte reaction (MLR) assays. In the microcytotoxicity assay, which can be used to type both class I and II antigens, an antiserum (or monoclonal antibody) is mixed with live lymphocytes and allowed to bind to the cell surface molecules. Specific binding is then detected with the addition of complement which lyses the cells and allows the uptake of a dye. The microcytotoxicity assay requires viable cells and uses antisera obtained primarily from individuals who have been sensitized to HLA differences such as multiparous women (women who have had multiple pregnancies) or individuals who have received multiple transfusions. Consequently, these reagents are limiting in quantity and difficult to standardize. Although, as noted above, >150 DRB1 alleles (different amino acid sequences) have been identified, serologic reagents can distinguish only 15 different groups of DR molecules encoded by these alleles.

DNA-based techniques

Over the last decade, molecular genetic techniques have been used to isolate the genes encoding the HLA class I and class II molecules and to characterize their genomic organization. The initial approach to HLA typing at the DNA level involved the restriction fragment length polymorphism (RFLP) method; provided that several restriction enzymes were used, this technique could often subdivide HLA class II serotypes. However, RFLP typing failed to distinguish much of the HLA class II sequence polymorphism.

The development of the PCR in the mid-1980s greatly facilitated the analysis of sequence polymorphism at both the class I and II loci. By generating billions of copies of the target sequence, this technique enabled the use of simple, nonradioactive methods to analyze sequence information. Based on the available database of class II allelic sequence diversity, a variety of relatively simple and rapid PCR-based methods has been developed to carry out HLA class II typing at the DNA level. The first approaches utilized labeled sequence-specific oligonucleotide (SSO) probes to hybridize to PCR products amplified from the sample and immobilized on a nylon or nitrocellulose filter, the 'dot blot' method. Under appropriate hybridization and wash conditions, these SSO probes would bind only to the complementary sequence in the amplified DNA and were able to distinguish single nucleotide differences. Given enough primers and probes, the SSO method is, in principle, capable of distinguishing all of the alleles at a given HLA locus. It has now been applied to typing all of the class II loci and, more recently, to the class I loci.

Another PCR-based approach, based on the specificity of primer extension rather than probe hybridization, has also been applied to HLA typing. This method is known variously as allele-specific amplification (ASA), sequence-specific priming (SSP), and the amplification refractory mutation system (ARMS). Here, a specific primer pair is designed for each polymorphic sequence motif or pair of motifs and the presence of the targeted polymorphic sequence in a sample is detected as a positive PCR, typically identified as a band on a gel. If the PCR is negative, the sample is assumed to lack one or both of the specific motifs. Allele-specific amplification can be used in conjunction with SSO probe typing for high-resolution typing. Other PCR-based methods such as PCR-RFLP, PCR-SSCP (single-

strand conformation polymorphism), and PCR-DHA (directed heteroduplex analysis), have also been developed but are not widely used.

Recently, a new approach to SSO probe analysis of HLA polymorphism has been developed that facilitates DNA-based HLA typing. The conventional dot bot involved an immobilized PCR product that is hybridized to each of many labeled SSO probes. The 'reverse blot' (or immobilized probe) method is based on the hybridization of PCR product, labeled with biotinylated primers during the amplification, to an array of immobilized probes on a membrane. This procedure requires only a single PCR and a single hybridization reaction to obtain information from the entire SSO probe panel; all of the probe reactivity information is contained on a single membrane, making it amenable to automated data interpretation.

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