1. 1 Summary September 25-26, 2006 FDA Workshop Molecular Methods in Immunohematology 89 th Meeting of BPAC April 27, 2007 Sheryl A. Kochman Chief, Devices Review Branch, DBA/OBRR/CBER/FDA

2. 2 Molecular Methods in Immunohematology Workshop September 25 & 26, 2006 Lister Hill Auditorium Building 38A National Institutes of Health Bethesda, MD 20894 Sponsored by FDA/CBER/OBRR DHHS OS/Office of Public Health and Science NIH/National Heart, Lung, and Blood Institute

3. 3 Why Did We Need a Workshop? Growing body of knowledge on the basis of blood group genotypes Growing use of various molecular methods in transfusion medicine –Tests for IVD use available in Europe –Home brew and RUO testing in US Showing promise of addressing current problems in transfusion medicine

4. 4 Current Problems in Immunohematologic Testing Lack of reagent grade antibodies, polyclonal and monoclonal –This leads to difficulties in Patient and donor phenotyping Characterization of Reagent Red Blood Cells for antibody detection and identification Variability of reactivity of monoclonal antibodies as compared to each other and to polyclonal antibodies Weak reactivity of clinically significant antibodies –E, K, Jk a, Jk b –Leads to failure to detect and identify May erroneously allow electronic crossmatch –Serologic crossmatch may not detect incompatibilities CBER is aware of fatalities

5. 5 Current Problems in Immunohematologic Testing Weak expression of antigens Lack of a single “universal test method” for antibody detection and identification –Different methods optimum for different antibodies –No single method detects all antibodies optimally Inherent limitations in the hemagglutination test –Limit of detection –Subjective nature of the test performance, reading, and interpretation –Usually single “analyte” per test –Not always able to automate

6. 6 Goals of workshop Provide FDA with sufficient info to: –Frame dialogue with manufacturers wishing to proceed to market –Identify potential issues of importance for manufacturers –Identify potential issues of importance for users of molecular methods Blood establishment Transfusion service Reference laboratory

7. 7 Agenda The International Experience ISBT/ICSH International Workshops & Proficiency Test On Molecular Blood Group Genotyping The BloodGen Project Blood Group Genotyping in Germany Molecular Genetic Blood Group Typing By The Use Of PCR SSP Technique

8. 8 Agenda (cont) The Americas Experience Overview of Molecular Methods in Immunohematology Fetal Blood Group Genotyping Rh Complexities: Serology and DNA Genotyping The Kidd Blood Group System The Duffy System The Kell and Kx Blood Group Systems Consortium for Blood Group Genes – CBBG

9. 9 Agenda (cont) Human Erythrocyte Antigen (HEA) determinations by DNA Analysis Application of Genotype Analysis to the Quality Assurance of Reagent RBCs Applications of Blood Group Antigen Expression Systems for Antibody Detection and Identification Donor Genotyping Proficiency Testing for Molecular Assays Overcoming Limitations In Current Pre- Transfusion Compatibility Testing Methods Using Phage Display

10. 10 Agenda (cont) Current FDA Processes For Bringing Products To Market Review of Current FDA Guidance

11. 11 Key Points Using molecular methods in donor screening will –Allow testing more donors for more antigens because of automation and multiplex testing –Assist in the management of rare donor units Using molecular methods in patient testing will allow –Testing when cells are sensitized with antibodies –Testing when there are multiple cell populations –Resolution of unusual serologic findings –Determination of a more rational approach to transfusion practices involving multi-transfused and transfusion dependent patients:

12. 12 Key Points (cont) Using molecular methods for fetal genotyping aids in predication and management of HDN Using molecular methods in the manufacture of Reagent Red Blood Cells could –Provide probable genotype when antisera are not available to determine phenotype –Assist in selection of homozygous cells to increase the chance of detecting weak but clinically significant antibodies, e.g. Jk a and Jk b Molecular Methods cannot completely replace serological methods –Antibody detection and identification cannot be done –May need to confirm serologic phenotype, at least for a while –Need to keep the crossmatch (but how long?)

13. 13 Concerns 1 st ISBT/ICSH workshop in 2004 –40 participants –6 DNA samples 34 errors (5%) –Most in Rh system – due to its complexity –Some clerical errors –Failure to detect silencing SNPs, esp. D and DY –7 recommendations for controls and testing schemes

14. 14 Concerns (cont) 2005 ISBT/ICSH Quality Assurance Exercise –Two DNA samples distributed to 29 labs –ABO, Rh D, C, c, E, e ( including RHD zygosity ), Kk, Fy a /Fy b /Fy/Fy x, Jk a /Jk b, Do a /Do b –496 tests only 3 clearly incorrect results (<1%)

15. 15 Concerns (cont) 2nd Workshop 2006 41 laboratories participated. Six samples were distributed –# of tests unknown –~52 errors (% unknown)

16. 16 Concerns (cont) BloodGen Project - GeneChip –3 year project ending Sept 2006 –Funded by Framework Five of EU - 2.5 million euros Progenika – 1 million euros –Major goal to look for mechanism for high- throughput molecular testing –Studies in 5 sites 685 samples analyzed 531 samples “OK” (ave = 77.5%, range = 60% - 89.1%) 154 samples show discrepancy between genotype and phenotype (ave.= 22.4%, range = 10.9% - 40%) –DNA quality issue? –Software issue?

17. 17 Remaining Questions How much pre-market testing is needed to evaluate these methods? –Number cited for BloodGen project are consistent with FDA’s Draft Guidance for Field Trials 3,000-5,000 randoms (plus known selected variants, conditions, etc.) for ABO/D 1,000 randoms (plus known selected variants, conditions, etc.) for all others Do we know enough about the limitations to adequately inform the user? Can the unexpected problems seen in the early development and use of monoclonal antibodies be anticipated and avoided?

18. 18 Remaining Questions (cont) How should the technology be used? –Mandatory vs. voluntary? –Blood establishment vs. transfusion service vs. reference laboratory? –In every transfusion service? –In every blood center? –Mandatory for manufacturers of Reagent Red Blood Cells? Education of users –How to perform and interpret these tests –How to recognize when something needs further investigation by a reference laboratory.

19. 19 Did We Meet Ours Goals? Partially –We have had discussions with one kit manufacturer –It is clear that proficiency testing will be needed –Tests for some RBC antigens are closer to marketing than others ABO/Rh far too complex at this point MN, Ss, Kk, Fy a /Fy b /Fy/Fy x, Jk a /Jk b, Do a /Do b appear well-defined and closer to market

20. 20 For More Info Presenters’ Slides http://www.fda.gov/cber/summaries.htm Transcripts (one for each day) http://www.fda.gov/cber/minutes/workshop-min.htm