Pathology

Genotyping

IC uses state-of-the art, robust, accurate and reliable molecular methods for genotyping HLA Class I and Class II and MICA antigens, and NK cell receptors (KIR).
Individual methods for HLA typing result in varying levels of resolution- low, intermediate or high (Table 1).

Table 1. HLA Typing Methods, Resolution, Results and Potential Applications

Method

Resolution

Result

Potential Applications

SSO

Intermediate

A*02:AGFGE1
  • Screening potential donors for Bone Marrow Drives
  • Typing for solid organ transplant
  • Confirmatory Typing for BMT/stem cell transplant

NGS

High:

Class I- Full Gene sequence

Class II- Near Full Gene sequence

A*02:02:01:01
  • Typing of stem cell transplant patients and donors
  • Typing for experimental allogeneic cellular therapies
  • Assessment of HLA molecular eplet mismatch
  • When full gene sequencing is indicated

1. Intermediate resolution HLA typing results by SSO include a NMDP string that defines the list
of alleles that cannot be excluded AGFGE:A*02:01/A*02:01L/A*02:01Q/A*02:07/A*02:09/A*02:15N/A*02:18/A*02:20
/A*02:24/A*02:29/A*02:30/A*02:31/A*02:33/A*02:42/A*02:43N/A*02:45/A*02:49/A*02:53N
/A*02:59/A*02:60/A*02:64/A*02:66/A*02:67/A*02:68/A*02:74/A*02:75/A*02:76/A*02:83N
/A*02:88N/A*02:89/A*02:93/A*02:94N/A*02:95

PCR-SSO

Reverse sequence specific oligonucleotide (SSO) hybridization is used to determine HLA-A, -B, -C, -DRB, -DQA, -DQB, -DPA and -DPB locus types at intermediate and higher than intermediate levels of resolution. Our laboratory utilizes an SSO method that is able to resolve Common Well Documented (CWD) HLA alleles. This technology is used for high volume testing allowing for relatively low-cost typing for bone marrow donor drives or other applications involving large sample numbers. Special volume pricing and terms may apply.

NGS

High Resolution HLA Typing by Next Generation Sequencing (NGS) provides unambiguous, phase-defined, first pass HLA typing results. Our strategy for HLA typing with NGS allows for full gene sequencing for HLA-A, B, C, DQA1 and DPA1 loci, and long-range sequencing from exon 2 through partial-exon 4 (including introns) for HLA-DR, DQ and DP. In addition, full gene sequencing for HLA-DR, DQ and DP antigens is currently under validation. This method of typing enables reporting of allele level results to a minimum of three nomenclature fields for HLA Class I, DQA1 and DPA1 genes and two nomenclature fields for Class II genes, or minimum G group allele level typing. The long range NGS typing provides the highest HLA typing resolution that is important for donor selection for stem cell transplants. In addition, it can be used to define HLA alleles of solid organ transplant donors to aid in the understanding of allele specific antibodies displayed by the recipient.

Disease Association Testing

HLA typing for disease associations and drug hypersensitivity can be performed at intermediate or high-resolution typing.

  • Ankylosing Spondylitis, rheumatoid arthritis, uveitis: HLA-B27
  • Behcet’s: HLA-B51
  • HIV controller/progressor: HLA-B57, B35
  • Drug Hypersensitivity: (abacavir, allopurinol): HLA-B*57:01, B*58:01, B*15:02
  • Celiac Disease: HLA-DQA1*05/DQB1*02, HLA-DQA1*03/DQB1*03:02 (DQ8), homozygous HLA-DQA1*02/DQB1*02
  • Narcolepsy: HLA-DRB1*15, DQB1*06:02

MICA Gene Typing

MICA genotyping targets MICA sequence polymorphisms by SSO. MICA typing can be helpful in determining whether patients are matched or mismatched for MICA with their donors, and to facilitate identification of donor specific MICA antibodies.

KIR Gene Typing

Killer Cell Ig-Like Receptors (KIRs) are used by NK cells to distinguish abnormal cells from healthy cells. KIR genotyping performed by SSO identifies the presence and absence of 15 distinct KIR genes. Based on the result, the KIR A or B haplotypes can be assigned. Donor KIR B content is associated with positive outcomes after stem cell transplant. Presence of maternal KIR AA haplotypes are also associated with preeclampsia, fetal growth restriction and recurrent miscarriage in pregnant women.

Antigen-based and Allogeneic Cellular Therapies

HLA genotyping is informative in many allogeneic cellular therapy settings.

  • CAR-T therapies using genetically modified T cells may be specific for neoantigens in an HLA restricted manner.
  • Allogeneic mesenchymal or other stem cell-based therapies may carry a risk of alloimmunization of the patient.
  • Tolerogenic cellular therapies or tolerizing epitope-specific vaccines in autoimmune disease rely on presentation by appropriate HLA alleles.
  • Our laboratory has extensive experience in CLIA-accredited HLA typing of patients, clinical trial subjects, and allogeneic cell products, along with monitoring of alloantibody quantity and breadth over the course therapy, for assessment of inclusion criteria (such as HLA matching) and secondary safety endpoints (expansion of alloimmunity) in these settings.
  • For research and clinical trials, HLA genotyping results are available in a batched electronic file output, that can be tailored to the investigators’ requirements.