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

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

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

4. Questions for Discussion What is the frequency of culture positive products? What is the frequency of culture positive products? What are the sources for contamination in the products? What are the sources for contamination in the products? What is the clinical significance of infusion of culture positive products? What is the clinical significance of infusion of culture positive products? What can be done to decrease the occurrence of positive products? What can be done to decrease the occurrence of positive products?

5. Experience at Mayo 7233 HPC products collected from January 1998 through March 2006 7233 HPC products collected from January 1998 through March 2006 2118 transplants performed 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. 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.

6. Collection and Transplant Activity Total Collections7233 Auto BM131 Allo BM127 PBPC6838 DLI137 Transplants Auto1764 Transplants Allo354

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

8. Frequency of Positive Products Total ProductsApheresisBone Marrow All Collections72336975258 Culture positive (%)119 (1.6%)111 (1.6%)8 (3.1%) Excluding Culture Positive Donors 72016944257 Culture positive (%)87 (1.2%)80 (1.2%)7 (2.7%)

9. Questions for Discussion What is the frequency of culture positive products? What is the frequency of culture positive products? What are the sources for contamination in the products? What are the sources for contamination in the products? What is the clinical significance of infusion of culture positive products? What is the clinical significance of infusion of culture positive products? What can be done to decrease the frequency of positive culture products? What can be done to decrease the frequency of positive culture products?

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

12. Donor/Patient as the Source of contamination Preprocess sample=A Postprocess sample=B Positive Products Products from Culture Positive Patients Total119 32 (27%) Sample A 43 2 (5%) Sample A&B 29 21 (72%) Sample B 47 9 (19%)

13. 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. 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 The PATIENT is a major source of bacteria

14. Organisms from HPCs OrganismsIsolatesInfused Coagulase negative Staphylococci7357 Staphylococcus aureus85 Corynebacterium sp.75 Enterococcus faecalis66 Acinetobacter sp.31 Moraxella sp20 Micrococcus sp.32 Gram negative bacillus33 Stenotrophomonas maltophilia3

15. Organisms from HPCs OrganismsIsolatesInfused Pseudomonas aeruginosa22 Acid fast bacilli11 Escherechia coli11 Enterobacter cloacae11 Filamentus fungus11 Propionibacterium sp.11 Ralstonia pickettii11 Staphylococcus haemolyticus11 Staphylococcus lugdunensis11 Group A streptococcus11 Chaetomium sp.1 Chryseobacterium sp1 Clostridium perfringens 1 Methylobacterium sp.1 Pseudomonas fluorescens/putida1

16. Infusion of Positive Products Total Number of Patients with positive products collected 95 Total Positive Products collected 119 Total Patients Receiving Positive Products 69 Total Positive Products Infused 88

17. Acute Infusion Reactions

18. Figure 2

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

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

22. Overall Summary: 177 Reporting Institutions

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

24. Policies encompass these elements

25. Types of Products (92 Respondents)

26. Frequency of Positive Products 1.4%1.7%

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

28. Test Methods for Sterility

29. Distribution of Organisms Found

30. 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 32% of positive cultures reported to be associated with a positive culture in the patient/donor within 5 days before or after collection

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

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

33. Implemented new cleaning of BSCs, “robust” changeover procedures, increased BSC preventive maintenance Implemented new cleaning of BSCs, “robust” changeover procedures, increased BSC preventive maintenance Process in BSC, clean with 70% sterile alcohol between each product processed. Process in BSC, clean with 70% sterile alcohol between each product processed. More strict gowning procedures, including use of sterile gloves. More strict gowning procedures, including use of sterile gloves. Perform EM daily Perform EM daily Innoculate cultures for microbial testing in BSC in cell processing lab and then transport to microbiology. 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. 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. 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. Perform tracking and trending of all positive cultures. No effect on rates seen No effect on rates seen ISCT Survey Respondents Collection/processing changes introduced to reduce contamination

34. 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. (?) 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. (?) ISCT Survey Respondents Collection/processing changes introduced to reduce contamination

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

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

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

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

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

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

41. 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. 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 Closed remains a relative term

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

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

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

45. Events Requiring Hospitalization Within 7 days, or extension of hospitalization Risks of Collection

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

47. Summary Infusion of culture-positive HPC products appears to be associated with minimal toxicity when associated with usual clinical practice and precautions 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 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 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 Overall risk to the patient AND donor for recollection must be balanced against the assessed risk of infusion of culture-positive product