1. Cord Blood Collection and Banking: Overview and Efforts to Minimize Microbial Contamination John P. Miller, M.D., Ph.D. VP and Senior Medical Director, NMDP May, 2009

2. Background: Cord Blood Banking and Transplantation Recruitment: prenatal or at L&D suite Collection: in utero or ex utero Processing: manual or automated plasma depletion +/- RBC depletion Cryopreservation: 6%HES/10% DMSO Storage: liquid or vapor phase of LN 2 Transport: Dry Shipper -196 to -150 C Thawing: with or without washing Infusion: ASAP

3. Cord Blood Recruitment and Collection

4. Collection Set-up and Preparation Ex Utero

5. Venipuncture and Completing the Collection

6. Final Product

7. Steps in cord blood processing Receipt, inspection Initial sampling: TNC, ABO/Rh Centrifuge soft spin: sedimentation of RBCs with HES Hard spin to concentrate HPCs Product analysis: TNC, viability, culture, CD34, CFU Cryopreservation Distribution/shipping Thawing and washing Infusing

8. Cord blood receipt and inspection Rinse into transfer bag with HES Inspection: Product intact, with no leaks clots or discoloration

9. Soft spin to pellet RBCs Express “buffy coat” from RBC pellet

10. RBCs separated from Buffy Coat and Plasma After Soft Spin

11. Hard spin to pellet RBCs and WBCs Express cell poor plasma from RBC pellet

12. Transfer to Freezing Bag and Place in CBU Freezing Cassette

13. Controlled Rate Freezing and Storage in LN2

14. Potential Sources of Microbial Contamination of Cord Blood Cross-placental transmission Contamination of cord or placenta during delivery Contamination during the collection procedure

15. Procedures to Minimize the Risk of Microbial Contamination Organizational level NMDP Standards NMDP Participation Criteria NMDP membership process AABB and FACT Standards and accreditation

16. Procedures to Minimize the Risk of Microbial Contamination At the CBU level: Maternal health history screening for RCD Maternal IDM testing for RCD Review maternal prenatal and delivery history/exam for risk of transmissible disease, e.g. chorioamnionitis, sepsis Review of infant history and exam Examination of cord and placenta, e.g. tears, infection Preparation of cord for venipuncture Aseptic Processing- one unit at a time Time from delivery to cryopreservation Bacterial and fungal cultures of final product prior to cryopreservation Post-discharge infant follow-up (bact and genetic) Adverse event reporting and investigation

17. Procedures to Minimize the Risk of Microbial Contamination Specific to Neisseria and Chlamydia: HHQ Donor is eligible with history of treatment for chlamydia for either type of delivery

18. Microbial contamination: Literature Review Contamination of CBU occurs at 2-5% 1-3 Culture Method: BacT-Alert or Bactec, Culture NOS organisms include: –Staphylococcus sp. –Streptococcus sp., including enterococcus –Corynebacterium sp. –E. coli –Bacteroides fragilis No Neisseria gonorrhea or Chlamydia trachomatis 1 Bertolini 1995, 2 M-Reboredo 2000, 3 Donaldson 2000

19. Microbial contamination: NMDP banks through March 2004 356 CBUs had positive cultures (0.7%, n=51,842) Culture Method: BacT-Alert or Bactec Common organisms include: –Staphylococcus sp. –Streptococcus sp., including enterococcus –Corynebacterium sp. –Bacillus sp. –E. coli –Bacteroides fragilis –No ID Aerobes and Anaerobes –Mixed flora No Neisseria gonorrhea or Chlamydia trachomatis

20. Microbial contamination: NMDP banks through March 2004 33.0 11.3 11.5 17.2 8.5 6.2 4.8 2.8

21. Summary Steps to minimize bacterial contamination of CBUs include maternal and infant screening, IDM testing, examination of the cord and placenta, preparation for venipuncture, aseptic processing and culture Rates of cord blood bacterial contamination are lower than reported in the literature (about 0.7%) No cases of contamination with Neisseria or Chlamydia have been reported

22. Questions? ?????