The primary purpose of HLA testing is to identify suitable donors for patients being considered for hematopoietic cell transplantation (HCT). Potential donors can be categorized as follows:
- HLA Identical Sibling (“Genotypic Match”). The ideal donor is a healthy sibling who has inherited the same HLA determinants as the patient. The HLA genes reside on chromosome 6 within the major histocompatibility complex (MHC). Five HLA genes are defined for matching purposes (A, B, C, DRB, and DQB) and are usually inherited as a block or “haplotype.” HLA-A, B, and C are referred to as Class I genes and HLA-DRB and DQB are referred to as Class II genes. Any two full siblings have a 25% chance of inheriting the same HLA genes (genotypic match) because they have inherited the same two haplotypes from their parents.
- HLA Phenotypic Match. Because some HLA alleles are common in certain populations, there may be a family member who has the same HLA alleles as the patient, despite having inherited one different haplotype. In this case, patient and the phenotypically matched donor would have the same HLA type, but may differ for other so called minor histocompatibility antigens.
- HLA Haplotype Match. A patient’s parent or child will be a haplotype match with the patient because they will share one haplotype due to inheritance. The other unshared haplotype may, due to chance, carry one or more HLA alleles in common with the patient, but usually the unshared haplotype will carry different HLA alleles.
- HLA Matched Unrelated. An unrelated donor (URD) who has the same HLA alleles as the patient at HLA-A, B, C, DRB, and DQB. If a patient cannot find a suitable family donor, an unrelated search will attempt to identify a fully matched URD for 10 out of 10 HLA alleles.
- HLA Mismatched Unrelated. An unrelated donor who is mismatched for one, two, or even three HLA alleles.
- Unrelated Umbilical Cord Blood. Frozen umblicial cord blood (UCB) units are an alternative source of hematopoietic cells. Cord blood units are typed to the allele level at HLA-DRB and to an intermediate (antigen) level at HLA-A and B but are not typed at HLA-C and DQB.
- High Resolution/Allele Level Typing/Sequencing. The gold standard for HLA typing is high resolution molecular typing at the allele level. This test determines the exact DNA sequences at the HLA-A, B, C, DRB1, and DQB1 loci. DNA is isolated from nucleated cells (peripheral blood lymphocytes or, in some cases, buccal swabs) and the DNA within the HLA region is amplified using PCR (polymerase chain reaction) methods. This amplified DNA is then sequenced and analyzed using sophisticated software to identify the HLA alleles encoded. Allele typing is reported using four or more digits preceded by an asterisk (HLA-B*4501, for example). Depending on the HLA alleles involved, there may be ambiguous combinations of alleles. If this is the case, a second amplification and sequencing assay is required to remove the ambiguity.
- Medium Resolution Level Typing. Medium resolution molecular typing also uses DNA and PCR methods, but does not determine an exact DNA sequence at HLA-A, B, C, DRB1, and DQB1. Instead it takes a more general approach using a panel of DNA probes to identify groups of HLA alleles. Such probes are called “sequence specific oligonucleotide probes” or SSOPs. A medium resolution result is usually expressed as a number followed by a letter code (B*45CKB, for example). The letter code is translated in the body of the report (e.g. B*45CKB = B*4501/*03/*07 means that the sample could be either a B*4501, a B*4503, or a B*4507). Medium resolution typing is faster and cheaper than sequencing, and thus is used to screen potential donors. Donors who are matched with the patient by medium resolution typing can then be tested by sequencing.
- Serological Typing/Antigen Level Typing. Serology (or microcytotoxicity) is the original HLA typing method. This method requires intact viable cells (usually lymphocytes) that express HLA antigens on their cell surface. These cells are incubated with known HLA antisera and then treated with complement to create a pattern of cytotoxicity. By interpreting this pattern, the HLA antigens present on the cells can be deduced. Serological results are not preceded by an asterisk (“B45” in our example).
- Antibody Screening (“PRA”). Another traditional test, PRA (for “Panel Reactive Antibody”) is a general measure of a patient’s antibody status regarding anti-HLA antibodies (or alloantibodies). Patient sera is tested against a panel of cells expressing a diverse set of HLA antigens. A patient who has never been pregnant and has never had a transfusion or transplant should not have any circulating alloantibodies; this patient’s serum would not react against any of the cells in the panel and would have a PRA of “0%.” Patients who have undergone multiple transfusions may have many anti-HLA antibodies in their circulation –- their sera will react with many of the cells on the standard panel and so could have a high PRA (70 – 100%).
- Crossmatch Studies. Crossmatch tests are designed to identify specific alloantibodies directed against a particular donor. These donor specific antibodies may be directed against Class I molecules (detected in T or B cell crossmatches) or against Class II molecules (detected in B cell crossmatches). In addition, the alloantibodies may be further characterized as IgG or IgM in flow cytometry crossmatches.
Samples received in CIL must be accompanied with an SCCA requisition, available here. (PDF)
A. New Patient Initial Typing (No Prior Typing):
1) Patients without prior typing, their full siblings, or other potential family donors are typed in a two step process:
a) HLA-A, B, and DRB by medium resolution. If a family member is matched, then HLA-C and DQB by medium resolution and HLA-DRB1 by high resolution.
b) If no family match is found and the clinician plans to proceed with an alternate donor search, then full typing on the patient (HLA-A, B, C, and DQB by medium resolution and HLA-DRB1 by high resolution) may be requested.
B. Recommended Testing Following Review of Previous Typing (Prior Typing done by an outside laboratory)
If HLA testing if requested for a patient with prior HLA typing, test plans are determined on a case-by-case basis following a review of the existing HLA typing results and acceptability of the existing results for the treatment plan being considered at SCCA (allo transplant, URD search, etc). Usual reasons for requesting HLA typing to be done by CIL:
- To confirm reported HLA match between patient and reportedly matched family member(s)
- To complete incomplete typing of patient (for example, only HLA-A and B typing results available) to be able to predict availability of potential unrelated donors
- To confirm patient’s reported type and HLA type family members that have not yet had HLA typing completed
- To update reported HLA typing results with molecular-based typing methods
- To resolve reported discrepancies and inconsistencies with external HLA typing results.
C. Modified Typing of Patients or Family Members
For some patients, circumstances may require modification of either the Standard Typing Plan (No Prior Typing) or the Recommended Typing Plan (Prior Typing).
Examples of circumstances include (1) expedited typing needed in cases of clinical urgency, (2) unusually large immediate family available, or (3) exceptional financial considerations present. For each patient, following the collection of family information and determination of HLA typing benefits, the CCO/Transplant Coordinator will determine if modified typing is needed. If modified typing is needed, details of typing to be completed are provided to CIL.