Infusion of Culture Positive Stem Cell Products FDA Liaison Meeting June 16, 2006

1. Infusion of Culture Positive Stem Cell ProductsFDA Liaison Meeting June 16, 2006 D. A. Gastineau Mayo College of Medicine

2. Overview • Background-historical perspective • Questions to be addressed • Specific Experience at Mayo • ISCT Survey • Changes to practice of respondents • influence on sterility results • Closed Systems • Conclusions

3. Background • Human bone marrow and PBPC transplant products have traditionally been sampled for bacterial contamination • A small percentage of these products have positive cultures • Known culture positive products have commonly been infused • However, strict interpretation of the new GTP regulations prohibit this practice

4. Experience at Mayo • 7233 HPC products collected from January 1998 through March 2006 • 2118 transplants performed • Review of medical records for evidence of adverse reactions, toxicity and post-transplant blood cultures for the 69 patients (3% of total) who received positive products.

5. Collection and Transplant Activity

6. Before 3/04 1 ml was injected in an isolator tube and incubated for 28 days After 3/04 1 ml was injected in peds Bactec bottle and incubated for 5 days. Samples were taken from the product upon arrival in the laboratory and after all processing before freezing Sterility Testing at Mayo

7. Frequency of Positive Products

8. Sources of Bacteria in Product Cultures • Donor (patient) • Introduced during collection • Introduced during processing • Introduced during sampling • Introduction during thawing process • Did not examine

10. Donor/Patient as the Source of contamination

11. Culture Positive Products • When two cultures are positive (the “A” and “B” cultures), 21 of 29 or 72% of patients had concomitant bacteremias documented by blood culture. • The PATIENT is a major source of bacteria

12. Organisms from HPCs

13. Organisms from HPCs

14. Infusion of Positive Products

15. Figure 2

16. Conclusion • Clinical Significance of infusion of positive products • Minimal acute toxicity or adverse reactions • Equivalent short-term and long-term survival

17. Survey Sponsored by ISCT • Goals • Obtain general information about the current practice of hematopoietic stem cell therapy • Obtain preliminary information concerning experience with safety of culture-positive products

18. Overall Summary: 177 Reporting Institutions

19. Survey Question: Does your institution have a policy for handling culture-positive products?

20. Policies encompass these elements

21. Types of Products (92 Respondents)

22. Frequency of Positive Products 1.7% 1.4%

23. Infusion of positive products: Institutional Responses (67%)

24. Test Methods for Sterility

25. Distribution of Organisms Found

26. Association With Donor Bacteremia • 32% of positive cultures reported to be associated with a positive culture in the patient/donor within 5 days before or after collection

27. Deaths Associated with Infusions • Of 91 respondents • 4 reported deaths likely related to infusion • 0 were felt to be due to microbial contamination • 1 response was excluded as it reported 6 deaths but fewer than 100 infusions and was otherwise very incomplete

28. ISCT Survey Respondents Collection/processing changes introduced to reduce contamination • Clean room facilities introduced • No effect • Second cultures sent from frozen samples • If negative, presumed to be false positive • All products since April 2003 have been in cGMP facility, Grade A critical area, Grade B background • No change in rates • Validated blood culture process, discontinued use of multi-use heparin vials • Reduction from 4% to 1.9% between 2003 and 2005

29. ISCT Survey Respondents Collection/processing changes introduced to reduce contamination • Implemented new cleaning of BSCs, “robust” changeover procedures, increased BSC preventive maintenance • Process in BSC, clean with 70% sterile alcohol between each product processed. • More strict gowning procedures, including use of sterile gloves. • Perform EM daily • Innoculate cultures for microbial testing in BSC in cell processing lab and then transport to microbiology. • GMP/GTP training performed for all staff in Cell Processing and Microbiology departments including using sterile technique. • Rearrangement of microbiology laboratory to accommodate microbial cultures for cellular products and to incubate all of these cultures in an isolated incubator. • Perform tracking and trending of all positive cultures. • No effect on rates seen

30. ISCT Survey Respondents Collection/processing changes introduced to reduce contamination • We used to culture every product immediately after collection and after processing. We reduced cost of culture and removed an unnecessary step. We have implemented additional training regarding the line connections to reduce possible contamination. We had our infection control department observe all steps of the process to make suggestions to improve our handling and reduce possible contamination in the laboratory. Overall our number of positive cultures has dropped from approximately 5.5% to less than 4%. In 2005 we did not have any contaminated products. (?)

31. Changes Introduced to Reduce Infection Risk and Effects • Introduction of sterile sleeves • Sterile gloves • No longer perform separate fungus culture. Reduced the number of positive cultures related to contamination by reference lab. (no numbers given)

32. What is a Closed System? • Closed • “ . . . an isolated system having no interaction with an environment.” Dictionary of Cybernetics and Systems • To be completely closed a product should not be exposed to the external environment from beginning to end of the processing. • Is this feasible? • Collection: Venipuncture • Sterile docking—adding reagents • Sampling • Infusion

33. What is a Closed System? • What would be a closed system? • A container with all necessary additives and vessels for processing attached to an original collection container • Final vessel disconnected from original set of containers • BUT: How to sample in process (also separate sample bags?)

34. What is a Semi-closed System? • Definition varies widely • Generally refers to processes which have been adapted to bags from “open” processes such as ficoll-hypaque • Tsang, Transfusion, 2001

35. What is a Functionally-closed System? • Functionally-closed systems • Varies as well • Processing cord blood with bags attached to central processing kit, valves to open/close for each process. • Sepax-S100, Biosafe S.A. • Zingsem, Transfusion, 2003 • Product positive rate—7.5%, similar to that of literature • Unable to demonstrate superiority of “functionally-closed system”

36. Functionally Closed System • Fluids intrinsic to sterilized kit • No flexibility of anticoagulation • Sterile filters used for added reagents

37. How Closed is Closed? A matter of degree, with nothing completely closed as some air and starting material (blood) enter the system and may not be sterile. Closed remains a relative term

38. Clinical Balance • Risk of Infusion of Positive Products • Very low with the current clinical practice of often giving antibiotics, but even without antibiotics, symptoms are few • Vast majority of programs have SOPs addressing culture-positive products • Alternatives to use of positive products • No treatment • Recollection

39. Risk of Recollection • Healthy donor may need catheter re-inserted • Autologous donor may have underlying disease increasing donation risk

40. Risks of Collection • During period of report • Two patients with amyloid died during mobilization/collection (not while on machine) • One central catheter migrated to pericardial space • One central catheter created AV fistula in thorax

41. Strategies for Reducing Positive Culture Rates • Improved screening of donors • Drawing blood cultures 48 hours prior to collection • Cost: 100-200 blood cultures to detect a bacteremia, and difficult to measure benefit • Some bacteremias are transient (clostridium) and won’t be detected • Clean room environment does not have a clear benefit • “Closed” systems-some additional protection for post-donor sources

42. Summary • Infusion of culture-positive HPC products appears to be associated with minimal toxicity when associated with usual clinical practice and precautions • The type of bacterium detected may affect the clinical decision whether or not to use the product • HPCs are not analogous to blood products in ease or risk of replacement • Overall risk to the patient AND donor for recollection must be balanced against the assessed risk of infusion of culture-positive product