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Subject: HLA and Stem Cell Transplant
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Allogeneic hematopoietic stem cell transplantation (HSCT) has been established as the top treatment of choice for hematologic malignancies and other hematologic or immune disorders. HSCT is also emerging as the most common cell-based immunotherapy to treat solid tumors. Since human leukocyte antigen (HLA), the major histocompatibility complex (MHC) in humans, can elicit an immune response either by presentation of variable peptides or by recognition of polymorphic fragments of foreign HLA molecules, selection of an HLA identical or near-identical donor is preferred. HLA disparity has been associated with graft failure, delayed immune reconstitution, graft versus host disease (GVHD), and mortality.
HLA is one of the most polymorphic gene systems in the human genome. Consequently, many patients lack HLA-matched donors. In recent years, advances in HLA testing and matching, extensive research on the role of each HLA locus mismatch on clinical outcome, and further knowledge of donor selection factors have made it easier to search for and select a partially matched donor. While the role of donor-specific HLA antibodies (DSA) in solid organ transplantation is well established, their importance in HSCT is only recently emerging. HLA antibody assessment should be incorporated into the HSCT donor selection process.
The requirements for HLA testing can be very different based on various transplant protocols and conditioning regimens. A joint agreement, between the transplant program and the HLA lab, detailing the testing algorithm, which is customized to meet the needs of different programs and patient cohorts, is very important. Based on the requirements of ASHI, AABB, CAP, and FACT, a recommended testing algorithm is listed below:
New transplant candidates:
High-resolution typing of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci and intermediate-resolution typing of HLA-DQA1 and -DRB345 loci
HLA antibody screen and specificity identification using a highly sensitive method, for example, Luminex single antigen–bead (SAB) assay.
If a patient is identified as having strong DPB1 antibodies at the time of evaluation by Luminex SAB assay, the patient and potential donors should be typed for DPB1 to confirm DPB1 antibody specificity and avoid selecting donors with corresponding DPB1 antigens. Matching DPB1 can also improve the transplant outcome.
Recent transfusions should be documented. At least 2–3 weeks should pass prior to collecting a new sample for HLA antibody analysis after sensitizing events.
Related donor typing
Intermediate-resolution typing of HLA-A, HLA-B, and HLA-DRB1 loci
If matched with the recipient, high-resolution typing of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci and intermediate-resolution typing of HLA-DQA1 and HLA-DRB345 loci.
Identity confirmation by intermediate-resolution typing of HLA-A, HLA-B, and HLA-DRB1 loci with a new sample
Unrelated donor search and typing
If a suitable matched related donor cannot be found, an unrelated donor search will be initiated. Transplant physicians, coordinators, and HLA lab staff will be involved in the donor search and selection. NMDP, BMDW, and individual donor registries will be searched using the patient's high-resolution typing data.
High-resolution typing of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci and intermediate-resolution typing of HLA-DQA1 and HLA-DRB345 loci are performed for selected unrelated donors. Additional DPB1 matching can improve transplant outcome.
A 10/10 allele-matched unrelated donor is preferred. Mismatched donors will be evaluated on a case-by-case basis. If the patient has HLA antibodies, especially the class II antibodies, a final T- and B-cell crossmatch with the unrelated donor may be necessary. The most current patient serum on file is preferred for the final crossmatch.
Cord blood search and typing
If a suitable matched related donor cannot be found, a cord blood unit search will be initiated. NMDP, BMDW, and individual cord blood registries will be searched using the patient's high-resolution typing data.
High-resolution typing of HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 loci and intermediate-resolution typing of HLA-DQA1 and -DRB345 loci on the selected cord(s), attached segments preferred if available, cord bag can be used as well for retrospect typing. If resources are limited, whether financial or sample material, only high-resolution typing of HLA-A, B, and DRB1 loci will be performed.
When a suitable family donor is not available, the choice of an unrelated donor or cord blood will depend on the degree of HLA match, cell dose of the cord blood unit, the urgency of the transplant, and other variables (donor age, sex, ABO incompatibility, HLA antibody, etc.) that may affect the transplant outcome.
Patient and donor identity confirmation
Repeat HLA typing of recipient using a new sample such that the individual's HLA typing is confirmed prior to final donor selection for related or unrelated transplantation.
Repeat HLA typing of a related or unrelated stem cell donor using a new sample such that the donor's HLA typing is confirmed prior to stem cell collection. For unrelated donors, registry data are acceptable as the first of these two samples.
Identity typing can be performed by intermediate-resolution HLA-A, HLA-B and HLA-DRB1 typing by PCR-SSOP.
Transplant program coordinators are responsible for requesting the second sample for identity typing before final donor selection is made. For donors, the registry typing will be considered as the first typing and the extended high-resolution typing will be considered as the second typing.
To ensure maximum accuracy:
Two sample collections on different dates, the second date should be prior to the final donor selection for related or unrelated donors or cords.
Two sample types (blood and buccal swab) if the first sample is blood. Buccal swabs are acceptable as the first or second or both collections. Buccal swab is recommended for patients with any acute diagnosis, for example, AML and ALL with blasts.
Two testing methods, PCR-SSOP/SSP or SBT
HSCT for leukemia can play a major role in reducing the risk of relapse by inducing a graft versus leukemia (GVL) effect. The effectiveness of mismatching inhibitory killer cell immunoglobulin–like receptors (KIR) on donor natural killer (NK) cells as a mechanism for GVL is being studied extensively. Together, the gene content of KIR, the T-cell and NK cell components of the graft, the graft source, the conditioning regimen, and mechanisms to reduce graft versus host disease (GVHD), will improve the overall benefit of HSCT.
Generic KIR typing, to detect the presence/absence of the inhibitory KIR genes, can be performed on selected patients and donors. The ligand matching information and the B-haplotype content can provide assistance in selecting the best possible donors/cords. The matching and mismatching of inhibitory KIR in the graft verse host direction can be determined based on the patient HLA ligand and donor inhibitory KIR.
Null allele typing
Alleles that have been shown not to be expressed, “null” alleles, have been given the suffix “N.” Based upon regulations required by the American Society of Histocompatibility and Immunogenetics (ASHI) and the National Marrow Donor Program (NMDP), guidelines for null allele typing have been established. The HLA lab is required to test for the following null alleles when alleles and/or haplotypes that have been associated with the specific null alleles exist. Additional CWD null alleles are constantly being updated and incorporated into the required typing list.
Engraftment monitoring (EM) testing
Following HSCT, patients are monitored closely for early engraftment, evidence of graft rejection, or recurrence of the original disease. HSCT creates a donor/recipient chimerism in the patient, which can be quantitatively measured through short tandem repeat (STR) analysis of peripheral whole blood, lineage-specific subsets, whole marrow, or CD34 progenitor cells in the marrow to determine the percent chimerism.
The pretransplant samples used in the EM assay can be the samples used for HLA typing. No additional pretransplant samples from the patient or the donor are necessary. The most common postsamples are, whole blood, T cells (CD3), B cells (CD19/20), myeloid cells (CD15/CD33/CD66b), NK cells (CD56), whole marrow, and CD34 marrow.
Results are commonly reported as the percentage of donor chimerism within the post-HSCT samples. The assay sensitivity is a key element and has to be considered in results interpretation and reporting, for example, for a postsample with no patient DNA detected and assay sensitivity is 3%, the result should be reported as more than 97% donor DNA, no patient DNA detected, full engraftment.