# Presentation: 914144

## 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

## 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**

## 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**

## 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**

## 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**

## 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 **

## 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**

## 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**

## 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**

## Agenda (cont)

**Current FDA Processes For Bringing Products To Market**
**Review of Current FDA Guidance**

## 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:**

## 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?)**

## 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**

## 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%)**

## Concerns (cont)

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

## 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?**

## 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?**

## 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.**** **

## 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**

## 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