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Mitchell S. Cairo , Elizabeth Wagner, Geoff Cohen, John Fraser, LeeAnn Jensen, Shelly Carter, Carmella van de Ven, Nancy

Cord Blood Log CD34+ and CD34+ Subsets (CD38-, CD61+, CD90+) are Significantly Correlated to Log Total CFU, CFU-GEMM, CFU-GM and BFU-E: A Report From The COBLT/NHLBI Program.

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Mitchell S. Cairo , Elizabeth Wagner, Geoff Cohen, John Fraser, LeeAnn Jensen, Shelly Carter, Carmella van de Ven, Nancy

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  1. Cord Blood Log CD34+ and CD34+ Subsets (CD38-, CD61+, CD90+) are Significantly Correlated to Log Total CFU, CFU-GEMM, CFU-GM and BFU-E: A Report From The COBLT/NHLBI Program Mitchell S. Cairo, Elizabeth Wagner, Geoff Cohen, John Fraser, LeeAnn Jensen, Shelly Carter, Carmella van de Ven, Nancy Kernan, Joanne Kurtzberg Cord Blood Transplant (COBLT) Program National Heart Lung and Blood Institution (NHLBI)

  2. Experimental Model of Human Hematopoiesis CD34+ CD41+/61+

  3. Experimental Model of Early Hematopoiesis Pluripotent Stem Cell CD34+/CD90+ CD34+/CD38- CD34+/CD38+ CFU-GEMM CFU-L

  4. Introduction • We and others have demonstrated that unrelated cord blood (UCB) is an effective alternative source of allogeneic stem cells in pediatric and selected adult recipients • Kurtzberg et al, NEJM, 1996 • Cairo et al, Blood, 1997 • Rubinstein et al, NEJM, 1998 • Laughlin et al, NEJM, 2001 • Rubinstein et al at the NYBC and the COBLT/NHLBI working group have established procedures for maternal donor screening and consent and UCB collection, processing, cryopreservation, thawing and shipping • Rubinstein et al, PNAS, 1995 • Fraser et al, J Hematotherapy, 1998

  5. Introduction • CB CD34+/kg graft content has been significantly correlated with engraftment and survival following UCBT • Wagner et al, Blood, 2002 • Styczynski et al, BMT, 2004 • CB vs BM CD34+/38- has a higher cloning efficiency, proliferate more rapidly and generate more HPC • Cardoso et al, PNAS, 1993 • Hao et al, Blood, 1995 • CB CD34+/CD90+(Thy-1+) possess a high clonogenic cell expansion capacity and increased HPP-CFC • Mayani et al, Blood, 1994 • PB CD34+/CD41+ (CD61+) subsets are significantly correlated with platelet recovery following Auto PBSCT • Derecksen et al, JCO, 1998

  6. Objectives • To characterize processed CB HPC subsets including CD34+, CD34+/CD38-, CD34+/CD90+ and CD34+/CD61+ • To correlate processed CB HPC subsets with in vitro CB HPC colony formation including total CFU, CFU-GEMM, CFU-GM and BFU-E • To determine the relationship of birth weight, gestational age, ethnicity, gender and type of delivery with processed CB HPC

  7. Cord Blood Banks, Data Coordinating Center and NHLBI Cord Blood Bank Duke University J. Kurtzberg PI COBLT Study Committee N.A.Kernan, Chair/L Jensen, NHLBI Data Coordinating Center (Emmes) S. Carter & E.L. Wagner Cord Blood Bank Georgetown University M.Cairo, PI Cord Blood Bank University of California, LA J Fraser, PI

  8. Methods • Cord blood collection and processing were performed as described by COBLT • Fraser,Cairo, Kurtzberg J. Hematotherapy, 1998 • Cord Blood CD34+ and CD34+ subset characterization (300-1200 events) • ProCount®/FACSort™ — Duke and UCLA • IgG1 FITC isotype control • ISHAGE/Epics XL-MCL —Georgetown University • IgG1 PE isotype control

  9. Methods * Duke & UCLA

  10. Methods • In vitro HPC Colony Assays • Processed CB (1 x 105 nucleated cells /mL) in IMDM, 2% FBS in Methocult® media • Incubated at 37°C, 5% for 11-14 days in 24 well tissue culture plates. • BFU-E • Bright red ≥ 50 cells/burst, tightly packed, multicentered • CFU-GM • Colorless, granular, ≥ 30 cells/colony, dispersed formation • CFU-GEMM • “Fried egg” appearance, ≥ 40 cells/colony, compact, spherical hemoglobinized area at center or one side.

  11. Results Maternal Donor Screened 34,799 (100%) Long Term Storage 8,731 53% of Collections 45% Pregnancy related 29% Refused 9% Medical History 6% Risk Behavior 11% Other Maternal Donor Consented 20,710 (60%) Low Counts Poor Viability Positive Culture No HPC Positive HGB Screen 44% No Staff 41% Exclusions 15% Other Cryopreserved 11,077 (66%) Total Collections 16,764 (81%) 64% CB Volume 11% +ID 7% History 18% Other

  12. Ethnic Distribution of Long Term Stored Cryopreserved UCB

  13. Delivery, Gender, Gestational Age, Birth Weight • Type of DeliveryGender • Vaginal 74% Males 52% • C-Section 26% Females 48% • Gestational Age (wks) Birth Weight (gms) • < 37 8% < 2500 1% • 38 17% 2501-3000 11% • 39 30% 3001-3500 37% • 40 31% 3501-4000 36% • 41 14% >4000 15%

  14. Cord Blood Volume, TNC, TMNC, CD34+

  15. Representative Flow Cytometry Dot Plots of CD34+, CD34+/CD38-, CD34+/CD61+ and CD34+/CD90+ CD34+ CD34+/CD38- CD34+/CD61+ CD34+/CD90+

  16. Cord Blood Hematopoietic Progenitor Cells (HPC)

  17. Positive Correlation of Log Total Nucleated Cell Count (TNCC) with Log CD34+ Count in COBLT Processed Cord Blood

  18. Log All CFU (x 104) Log CD34+ (x 104) Significant Correlation of CB Log CD34+ vs Log Total CFU

  19. Positive Correlation of Log Total Nucleated Cell Count (TNCC) with Log Nucleated RBC Count in NHLBI/COBLT Processed Cord Blood

  20. Significant* Correlation of Post Processing CB Log Nucleated RBC vs Total CFU

  21. Significant* Correlation of Post Processing CB Log Nucleated RBC vs Log BFU-E

  22. Significant* Correlations of CB Log CD34+ vs CFU-GM, CFU-GEMM & BFU-E CD34+ vs CFU-GM CD34+ vs CFU-GEMM CD34+ vs BFU-E r = 0.68 P = 0.000 n = 8696 r = 0.52 p = 0.000 n = 4189 r = 0.61 p = 0.000 n = 8689 * All p<0.001

  23. Significant* Correlations of CB Log CD34+ subsets with Log CFUs * All p<0.001

  24. 63 53 21 44 44 14 White African American Asian Hispanic Other White African American Asian Hispanic Other Other Other White White Black Black Asian Asian Hispanic Hispanic Significant* Reduction in CB CD34+/CD38- and CD34+/CD61+ in African Americans & Asians vs Caucasians & Hispanics CD34+/CD38- CD34+/CD61+ * All p<0.001

  25. CD34+/CD38- CD34+/CD61+ Correlation of CB CD34+/61+ and CD34+/38- with Birth Weight Birth Weight

  26. Significant Correlation of CB HPC with Delivery and Gender

  27. Proportions of Stored CBUs Suitable for Transplant to Recipients of Given Weights

  28. Summary • Post processing CB log nucleated RBC was significantly correlated with log CD34+, TNCC, total CFU and BFU-E • Post processing CB log CD34+ was significantly correlated with log TNCC, CD34+/CD38-, CD34+/CD61+ and CD34+/CD90+ HPC subsets • Post processing CB log CD34+ and CD34+ subsets were significantly correlated with log total CFU, CFU-GEMM, CFU-GM and BFU-E HPC subsets

  29. Speculation • Post processing CB HPC CD34+ subsets and/or in vitro CB HPC CFUs may potentially correlate with the kinetics of lineage specific CB engraftment post CBT • Ethnicity, gender, delivery, birth weight and /or gestational age may have a significant effect on CB HPC and therefore, may be a factor in the future selection of a processed CB unit for CBT

  30. Acknowledgments • NHLBI Support • N01-HB-67136, N01-HB-67142, N01-HB-67132 and N01-HB-67141 • Cord Blood collection, processing and flow cytometry personnel at Carolina, UCLA and Georgetown University Cord Blood Banks • Maternal donors and newborns for their public generosity

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