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Expanding Role Of HLA Labs

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Vol. 19 • Issue 5 • Page 16
The Molecular Edge

Histocompatibility laboratories (also known as HLA laboratories) have increasingly incorporated molecular methods into their workflow. This rather recent transformation of the HLA laboratory has perhaps blurred the distinction between HLA and other laboratories utilizing molecular methods.

For example, some tests such as engraftment monitoring after stem cell transplantation or parentage studies are often performed in both HLA and non-HLA molecular laboratories. Also, non-HLA molecular laboratories have begun to test HLA loci for drug hypersensitivity or disease association (e.g., HLA-B*5701 testing for abacavir hypersensitivity or HLA-DQB1 for celiac disease genetic susceptibility). Nevertheless, HLA laboratories provide services that clearly distinguish them from all other laboratories.

Evolution of the Field

The field of human histocompatibility perhaps began in 1952 with Jean Dausset, who discovered an antibody defining a leukocyte group. Over the following 12 years, interest in histocompatibility gained international attention and serological typing techniques improved.1In 1964, the first international histocompatibility workshop was held to compare tissue typing methods. In 1967, the first WHO nomenclature committee was convened and named the leukocyte antigen system Human Leukocyte Antigen, or HLA. In 1969, two landmark papers demonstrated the clinical utility of histocompatibility testing: Singal et al.2showed a dramatic improvement in kidney graft survival in matched donor-recipient pairs as compared to mismatched pairs. Patel and Terasaki3verified a dramatic improvement in kidney graft survival in crossmatch negative donor recipient pairs as compared to crossmatch positive pairs. With these two studies, the importance of HLA typing and antibody testing to kidney graft survival was realized, and the clinical HLA lab became indispensable to solid-organ transplantation programs.

Addressing Challenges

The first hematopoietic stem cell transplant (HSCT) was performed in 1968 in an immunocompromised patient; the first unrelated HSCT was performed in 1973. However, finding suitable unrelated donors for HSCT was difficult until the establishment of registries such as the Anthony Nolan Trust in 1978 (www.anthonynolan.org.uk) and the National Marrow Donor Program in 1987 (www.marrow.org).

Because HSCT requires allele-level typing for the best possible outcomes, the establishment of these unrelated donor registries created a need for higher resolution typing methods. Advances in molecular methods such as PCR and sequencing coincided with the establishment of these registries and filled the void. As a result, since the late 1980s, virtually all the newly identified HLA alleles have been characterized using molecular methods. The convenience and improved accuracy of molecular genotyping methods has largely replaced serological typing.

Emergence of Accreditation

In 1974, the American Association of Clinical Histocompatibility Testing was established to organize laboratories involved in HLA testing. From 1973 to 1985, over 5,000 papers were published that studied the association of HLA with non-transplant related diseases.4Given the interest in HLA gene function and its role in histocompatibility, the name of the organization was changed to the American Society for Histocompatibility and Immunogenetics (ASHI).

ASHI began accrediting laboratories on a voluntary basis in 1974 and presently accredits 221 laboratories in one or more of seven areas-hematopoietic stem cell transplantation (related donors and unrelated donors), solid organ transplantation (related donors and unrelated donors), parentage testing, histocompatibility testing for non-transplant clinical purposes and transfusion support. In 1992 and 1995, National Marrow Donor Program (NMDP) and the Centers for Medicare and Medicaid Services (CMS) granted deemed status to ASHI accredited labs. This is important to note since the NMDP has not granted deemed status to any other organization (including CAP with 148 accredited HLA laboratories), which effectively restricts unrelated HSCT to ASHI accredited laboratories.

Tools, Technologies

HLA laboratories combine techniques and technologies of immunology and genetics. These generally include:

• serology for HLA typing, antibody detection, antibody characterization and crossmatching;

• flow cytometry for crossmatching, antibody detection, antibody characterization, immunophenotyping and functional assays;

• cellular assays to test immunological function; and

• molecular methods for HLA typing, genotyping of other immunologically important genes, engraftment monitoring and parentage studies.

The molecular methods most often used for genotyping include direct sequencing, sequence-specific priming, sequence-specific oligonucleotide probes by microarray or bead microarray. For engraftment monitoring and parentage studies, the methods are analysis of short tandem repeats, variable nucleotide tandem repeats or quantitative PCR for single nucleotide polymorphisms.

These methods are not unique to HLA laboratories. Indeed, many non-HLA molecular laboratories use the same methods for engraftment monitoring and parentage testing as well as many other applications. However, for laboratories genotyping the HLA genes, an understanding of the complexity of this region is essential. There are 21 HLA class I and class II genes with as many as 1,543 alleles per gene (4,447 alleles total) and 14 pseudogenes. Moreover, the number of newly identified alleles continues to grow so a primer set that may appear specific for a targeted allele or group of alleles (e.g., HLA-B*5701 or HLA-B27) may no longer be specific after a given time. Given the rate of change, HLA databases need to be updated at least every three months. This complexity makes HLA genotyping particularly challenging.

An Important Role

HLA laboratories are also unique in the support role they serve to transplant services. The unique application of immunologic and genetic methods allows HLA laboratory directors to be well equipped to advise clinicians on their transplant patients. Evaluation of a crossmatch result, for example, requires careful consideration of the patient's sensitization history, HLA antibody history, antibody specificity and HLA genotypes, as well as the potential donor's HLA genotype. Thus, transplant support virtually defined the HLA laboratory throughout its history and even today.

HLA laboratories utilize a combination of immunologic and genetic methods primarily to support transplant services. While other aspects of HLA testing often spill over into other laboratories using similar methods such as drug hypersensitivity or disease susceptibility, testing of the HLA loci can prove quite challenging due to their inherent complexity.

Dr. Jennings is the director of Molecular Diagnostic and HLA Laboratories at Children's Memorial Hospital, Chicago.

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