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The Past, Present and Future of HLA Typing

The Human Leukocyte Antigen (HLA) region, located on the short arm of chromosome 6 (6p21.3), is a highly polymorphic region containing about 200 genes. The HLA region is the human equivalent of the Major Histocompatibility Complex (MHC), and as such contains a set of genes that serve as the backbone of antigen presentation. The Class I proteins, classically involved in presenting endogenous antigens to CD8+ T-cells, are expressed by genes located in the HLA-A, -B and C loci. In contrast, the Class II proteins, which associate with and present exogenous antigens to CD4+ T-cells, are expressed by the HLA DR, -DQ and DP loci. Each locus is highly polymorphic; for example, there appear to be 300+ alleles in the HLA-B or DRB1 loci. A clear understanding of the differences between HLA polymorphisms has provided ample insight into why and how foreign tissue is rejected by the host, and as such been a critical enabler of the field of transplantation. Today, a variety of techniques are applied for HLA tissue typing, providing an important tool to increase the success rate of human transplants.

For many years, HLA polymorphisms were typed by serological responses HLA antigens; sera containing antibodies to Class I and II proteins were collected from multiparous women, or individuals who had received multiple blood transfusions. In addition, polymorphisms in Class II proteins were analyzed by T-cell responses in the Mixed Lymphocyte Reaction (MLR). However, the advent of recombinant DNA technology, which paved the way to identifying genetic differences among the HLA loci directly, has led many laboratories to abandon classic serological typing methods. Today, several different types of HLA DNA typing methods exist and are commonly used by clinical laboratories. In general, HLA DNA can be typed either by hybridizing labeled, sequence specific oligonucleotide probes to HLA loci amplified by the polymerase chain reaction (PCR), or by using PCR to amplify th e HLA DNA directly through differential primer extension.

One of the most commonly employed strategies in HLA DNA typing is to first amplify a given HLA locus, then hybridize it with a labeled, sequence specific oligonucleotide probe. The original methods, described in the 1980s, first blotted PCR amplified DNA onto nitrocellulose or nylon filters. These dot blots could then be incubated with radioactively labeled, sequence specific oligonucleotide probes. These probes would bind only to complementary HLA DNA amplified by PCR. Under appropriate conditions, sensitivity and specificity could be high enough to detect single nucleotide differences consistently. Several probes could be used to identify an array of HLA alleles. Today, radioactive probes are seldom used, and sequence specific oligonucleotides are now labeled with fluorescent dyes, biotin, digoxigenin, or directly with enzymes such as horseradish peroxidase. The appropriate substrate can be added to produce a fluorometric (or colorimetric) readout without the problems associated with radioactivity. Variations of the technique have also risen; for example, it is possible to construct PCR primers pre labeled with biotin, and then amplify biotin labeled HLA loci. These PCR products are then incubated with unlabeled, sequence specific oligonucleotide probes immobilized on the membrane. After the incubation and wash steps, the hybridization products can be detected by streptavidin horseradish peroxidase and chromogenic substrate. Modifications such as this allow the HLA typing to be completed in two steps with high accuracy.

The second HLA DNA typing technique is to use the PCR amplification reaction directly to detect HLA polymorphisms. In this technique, primers can be constructed specifically to complement HLA polymorphisms; if the primers bind the complementary polymorphism and amplify the gene segment, then the PCR product can be detected by standard techniques. If the p rimer is not complementary to the polymorphism, then it cannot bind and facilitate amplification: no PCR product is present. By constructing an array of PCR primers complementary to the range of HLA polymorphisms, it is possible to detect the HLA alleles directly by PCR. In fact, this technique was used in the first commercially available PCR test (AmpliType HLA-DQ-alpha Forensic assay system).

Of course, each HLA DNA typing technique has its advantages and disadvantages, and clinical laboratory personnel must choose the most appropriate test for their needs based on cost, average turnaround time, required resolution, and number of average samples per day. For those laboratories with access to Sequencers, it may be the most sensitive, specific and cost effective option to sequence certain HLA loci directly. And as more and more diseases are linked to specific HLA polymorphisms, emerging technologies in molecular diagnostics will be applied to HLA typing with increasing frequency.

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