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The page below is a sample from the LabCE course Hematopoietic Stem Cell Transplantation. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Donor and Recipient Testing of Human Leukocyte Antigens (HLAs)

Prevention of graft rejection
There are two methods to prevent graft rejection from an allogeneic transplant. Immunosuppression of the recipient using drugs or antibodies and pretransplant testing of the donor to ensure HLA compatibility.
Serologic testing
Lymphocyte microcytotoxicity testing has been used by laboratories for detection of Class I and II molecules for many years. Lymphocytes are obtained from the peripheral blood and separated by centrifugation with a Ficoll-Hypaque gradient. The HLA phenotype is determined by incubating the lymphocytes in a microtiter plate with complement and antibodies to specific HLA antigens. HLA typing sera can be obtained from the sera of immunized individuals including women with multiple births, multi-transfused patients, and transplant recipients. Reagent manufacturers have developed monoclonal typing sera to improve specificity and reduce cross-reactivity, which may result in typing errors or difficulty in determining tissue type.
If the lymphocytes carry a molecule recognized by the antibody, the antibody will bind to the cell and the cell will be lysed. To detect lysis, a fluorescent compound such as ethidium bromide is added and fluorescence is measured. Reactions are graded based on the percentage of lysis. Although this seems fairly straightforward, the interpretation and assignment of the HLA type can be difficult and requires highly experienced laboratory personnel for accurate results.
Mixed lymphocyte culture
Mixed lymphocyte culture reactions can identify discrepancies in the HLA class II loci which traditional microcytotoxicity testing may not detect. The recipient’s lymphocytes are mixed with the potential donor’s lymphocytes and incubated. The donor’s lymphocytes have been treated so that they will not proliferate in the presence of the recipient’s lymphocytes. A radioactive DNA compound is added to the mixture. If the recipient’s lymphocytes react to the donor lymphocytes they will uptake the DNA and their radioactivity can be measured and is a measure of the responsiveness of the recipient’s lymphocytes to the donor’s cells. This technique has been used to identify the best donor among several potentially matched donors. Unfortunately this assay is very labor intensive and costly.
DNA-based HLA typing
HLA typing by PCR methods have become the standard testing methodology for genotyping hematopoietic transplant donors and recipients. The first methods utilized PCR-sequence specific oligonucleotide primers (SSOP) and sequence based typing. Newer methods using next generation sequencing (NGS) have increased the resolution and accuracy of HLA genotyping. They also enable detection of other classes of HLA antigens. Higher resolution is required for progenitor cell transplants because the entire immune system is being transferred from the donor to the recipient. As these methods have developed, more alleles of HLA antigens have also been discovered, increasing the ability to successfully match donors with recipients. Newer methods have also changed testing requirements for specimens. Early testing required blood samples from potential donors. This reduced the number of potential donors due to costs involved in collecting and storing specimens, and donor’s reluctance to have their blood drawn. Today, bone marrow registry drives use buccal swabs to collect prospective donor DNA.
Volunteers joining a bone marrow registry such as the National Marrow Registry are usually typed for HLA-A, B, C, and DRB1 loci using an intermediate resolution method. If they match a patient who is searching for a donor, the prospective donor is retested by a higher resolution method. This approach reduces the cost of initial genotyping of unrelated donors. However this also delays the transplantation process and may affect the success of a HSC transplant procedure. Newer methods under development may replace this two-step process and ensure that patients' receive their transplant as early as possible.