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HLA Typing for Blood Bankers

HLA Typing for Blood Bankers. Kaaron Benson, M.D. Professor and Senior Member Departments of Oncologic Science, and Pathology and Cell Biology H. Lee Moffitt Cancer Center. Introduction to the HLA System. “HLA” – Human Leukocyte Antigens Key role in immunologic function

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HLA Typing for Blood Bankers

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  1. HLA Typing for Blood Bankers Kaaron Benson, M.D. Professor and Senior Member Departments of Oncologic Science, and Pathology and Cell Biology H. Lee Moffitt Cancer Center

  2. Introduction to the HLA System • “HLA” – Human Leukocyte Antigens • Key role in immunologic function • Part of the major histocompatibility complex (MHC) • HLA antigens encoded for by genes from 6 primary loci on chromosome 6

  3. Structure of HLA Class I and Class II Molecules HLA typing targets N Engl J Med 2000;343:702.

  4. HLA Class I Ribbon Structure

  5. Serology Ab reagents DR4 Molecular DNA amplification DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405… DRB1*0424 Nomenclature: Serology vs Molecular Typing

  6. Nomenclature HLA HLA complex HLA-A Locus HLA-A*02 Ag equivalent HLA-A*02:01 Allele specificity HLA-A*02:01N Null allele HLA-A*02:01:01 Silent mutation (coding region) HLA-A*02:01:01:01 Noncoding region mutation HLA-A*02:01:01L Low expression gene

  7. New Nomenclature: April 2010 • HLA-Cw becomes HLA-C • Cw*0202 now C*02:02, represents Cw2 Ag • Colons (:) to delimit separate fields • A*0301 becomes A*03:01 • A*2601 becomes A*26:01 • A*02 and A*92 to change • A*9201 becomes A*02:101 • A*9202 becomes A*02:102 • B*15 and B*95 to change • B*9501 becomes B*15:101 • B*9502 becomes B*15:102

  8. Nomenclature Confusion:One Example • HLA-DRB1*03:01 • HLA-DRB1*03 • HLA-DR17 • HLA-DR3 • This one allele could be written these four different ways. • All four do not refer to the same thing.

  9. GENES OF THE HUMAN MHC SYSTEM Chromosome 6 Class II Class III Class I DP DQ DR B C A B1 A1 B1 A1 B1 B3/4/5 A C4 TNF DRB1*0401 DRB1*0402 DRB1*0403 DRB1*0404 DRB1*0405 DRB1*0406 B*0702 B*0801 B*1301 B*1401 B*1501 A*0101 A*0201 A*0301

  10. http://www.ebi.ac.uk/imgt/hla/intro.html(1968: <200; 2007: >7000)

  11. Identification of New HLA Alleles

  12. Family Genotype Mother Father Possible combinations in children (4)

  13. Chance of Finding an HLA-Matched Sibling 1 – (0.75)n n = no. of siblings No. of sibsChance (%) 1 25 2 44 3 58 4 68 5 76 6 82 7 87 8 90 9 92 10 94

  14. Crossover Mother Father

  15. HLA Typing Methods • Serologic assays • Microlymphocytotoxicity test • Cellular assays • Mixed lymphocyte culture • Molecular assays • Sequence-specific primer (SSP) • Sequence-specific oligonucleotide probe (SSOP) • Sequence-based testing (SBT)

  16. All dead = Red Score =8 All alive = Green Score = 1

  17. HLA Typing by Molecular Methods Pros • More accurate and precise than serology • e.g. DRB1 >300 alleles but only 17 serotypes • Better matching between pt and donor • Less sample required, nonviable cells • Wider variety of samples can be used • Becoming easier + automated Cons • Does not account for genes that are present but not expressed: Genotype ≠ Phenotype • Rare alleles = growing list of ambiguities

  18. Sequence-Specific Primer PCR (SSP) Gel

  19. ML H G F E D C B A 1 2 3 4 * * * * * Class II DQB1 High Resolution Gel

  20. * *

  21. * DQB1*02:02

  22. Sequence-Specific Oligonucleotide Probe Hybridization (SSO, SSOPH) Multiple Microparticles (Luminex)

  23. SSO Pros and Cons • Pros • Fairly rapid • High volume • High(er) resolution • Reasonable cost/test • Cons • Single typing is more expensive • Requires more DNA • Expensive equipment

  24. Sequence-Based Typing (SBT) • Gold standard for HLA typing  • Detects novel alleles  • Expensive  • Sequencers are costly (but other uses) • Requires highly skilled technologists • Cis/trans polymorphisms - ambiguities result - require additional testing 

  25. DNA Typing Resolution • Low • serologic equivalent • DRB1*04, DRB1*13 • Intermediate • DRB1*04:01 or *04:03 or *04:04 • DRB1*04:01/03/04 • High • allele level • DRB1*04:01 • DRB1*04:01/36/45/52/56

  26. DNA Typing Resolution • Low • serologic equivalent • DRB1*04, DRB1*13 • Intermediate • DRB1*04:01 or *04:03 or *04:04 • DRB1*04:01/03/04 • High • allele level • DRB1*04:01 • DRB1*04:01/36/45/52/56 ambiguities

  27. High Resolution Typing • Type to a single “common” allele • Rare allele definition: • For Class I < 1:50,000 alleles • For DRB1 < 1:100,000 alleles • B*15:01, 35:01 • B*15:01/29/33/34, 35:01/40N/42

  28. Sequence-Based Typing (SBT) Sanger sequencing (chain-termination method)

  29. Sequence-Based Typing (SBT) Homozygous sequence Heterozygous sequence

  30. A*01:01, 02:01

  31. Confirmatory HLA Typing • New sample must be collected • Patient: prior to final donor selection • Donor: prior to stem cell collection • Both MRD and MUD transplants • NMDP donor typing counts as one typing • Only one typing must be at high resolution • Level of typing (LR, IR, HR) decided by program • MCC: IR/HR typing for original and repeat

  32. Clinical Applications of HLA Typing • Population studies • Disease associations • Pharmacogenomics • Platelet transfusion • TRALI risk reduction • Transplantation • hematopoietic stem cell • solid organ

  33. Population Studies:HLA Antigen Frequencies (%)

  34. HLA and Disease Associations Disease HLA RR Ankylosing spondolytis B*27 >100 Narcolepsy DRB1*15:01 94 Celiac disease DQB1*02:01 11 Rheumatoid arthritis DRB1*04 11 Multiple sclerosis DRB1*15:01 5 HIV slow progress DQB1*06:05 9 HIV fast progress B*07 3

  35. HLA-B27 and Ankylosing Spondylitis A.S. >90% B27+ HLA-B*27 ~8% of population ~2% of B*27+ dev AS

  36. Ingelman-Sundberg M. Pharmacogenomic Biomarkers for Prediction of Severe Adverse Drug Reactions NEJM 2008;358:637-639

  37. Platelet Transfusion • Platelet refractoriness: non-immune vs immune • Non-immune refractoriness • infection, splenomeg, BMT, DIC, bleed, meds • Immune platelet refractoriness • HLA Abs (other Abs: plt-specific, drug-induced, ABO) • HLA alloimmunization: due to allogeneic WBC exposure via prior transfusion or pregnancy (WBCs – cl. I and II, PLTs – cl. I only) • Management: more PLTs vs. histocompat. PLTs • Prevention: WBC-reduced blood

  38. Histocompatible Platelets • HLA-matched • Crossmatch-compatible • HLA antigen-negative • HSCT donor • Blood relative donor • Not for potential HSCT recipient

  39. “HLA-Matched” Platelets Patient: A1,2; B7,8 Matching grade A: perfect match; A1,2; B7,8 donor B: crossreactive (X) or unidentified (U) BIX, BIU; A1,3; B7,8 or A1,-; B7,8 donor B2X, B2U: A1,3; B7,27 or A1,-; B7,- donor C: one MM Ag; A1,2; B7,44 donor D: two MM Ag; A1,24; B7,44 donor

  40. ASCP, 2012

  41. Transfusion-Related Fatalities Reported to FDA FY2008-FY2012

  42. Transfusion-Related Fatalities Reported to FDA FY2002-FY2012 TRALI Cases

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