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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.

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Presentation on theme: "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."— Presentation transcript:

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

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

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 ( events) –ProCount ® /FACSort™ — Duke and UCLA IgG 1 FITC isotype control –ISHAGE/Epics XL-MCL —Georgetown University IgG 1 PE isotype control

9 Methods PhenotypeMonoclonal Antibodies CD34 + /CD61 + CD34 PE/CD61 FITC/CD45 PerCP CD34 + /CD90 + CD34 PE/CD90 FITC/CD45 PerCP CD34 + /CD38 - CD34 PE/CD38 FITC/CD45 PerCP CD34 + /Isotype control CD34 PE/IgG 1 FITC/CD45 PerCP *ProCount/Isotype control Nucleic Acid Dye/IgG 1 FITC/CD45 PerCP * Duke & UCLA

10 Methods In vitro HPC Colony Assays –Processed CB (1 x 10 5 nucleated cells /mL) in IMDM, 2% FBS in Methocult ® media –Incubated at 37°C, 5% for 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%) Maternal Donor Consented 20,710 (60%) Total Collections 16,764 (81%) 45% Pregnancy related 29% Refused 9% Medical History 6% Risk Behavior 11% Other 44% No Staff 41% Exclusions 15% Other Long Term Storage 8,731 53% of Collections Low Counts Poor Viability Positive Culture No HPC Positive HGB Screen Cryopreserved 11,077 (66%) 64% CB Volume 11% +ID 7% History 18% Other

12 Ethnic Distribution of Long Term Stored Cryopreserved UCB White363242% Hispanic184121% African- American % Mixed101812% Asian98711%

13 Delivery, Gender, Gestational Age, Birth Weight Type of DeliveryGender Vaginal74%Males52% C-Section26%Females48% Gestational Age (wks)Birth Weight (gms) < 378%< 25001% 3817% % 3930% % 4031% % 4114%>400015%

14 Cord Blood Volume, TNC, TMNC, CD34 + Pre-ProcessingPost Processing Volume80 mL25 mL Total Nucleated cells1.8 x x 10 8 (-22%) Total Mononuclear cells4.7 x x 10 8 (-19%) Total CD34 + NA3.4 x 10 6

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) CD34 + Subsets (x 10 5 )In vitro HPC (x 10 5 ) CD ± 42Total CFU23.2 ± 19.8 CD34 + /CD ± 4.8BFU-E13.3 ±13.1 CD34 + /CD ± 9.5CFU-GM9.3 ± 8.6 CD34 + /CD ± 1.9CFU-GEMM0.5 ± 1.1

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

18 Significant Correlation of CB Log CD34 + vs Log Total CFU Log CD34 + (x 10 4 ) Log All CFU (x 10 4 )

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-GMCD34 + vs CFU-GEMMCD34 + vs BFU-E r = 0.68 P = n = 8696 r = 0.52 p = n = 4189 r = 0.61 p = n = 8689 * All p<0.001

23 Significant* Correlations of CB Log CD34 + subsets with Log CFUs Log CD34 + /CD61 + Log CD34 + /CD38 - Log CD34 + /CD90 + NRNRNR Log Total CFU Log Total BFU-E Log Total CFU-GM Log Total CFU-GEMM * All p<0.001

24 Significant* Reduction in CB CD34 + /CD38 - and CD34 + /CD61 + in African Americans & Asians vs Caucasians & Hispanics * All p<0.001 CD34 + /CD38 - CD34 + /CD61 + White BlackAsianHispanic Other White BlackAsianHispanic Other WhiteAfrican AmericanAsianHispanicOther

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

26 Significant Correlation of CB HPC with Delivery and Gender DeliveryGender C-Section vs VaginalMale vs Female Total CFU + 8.7% ( ) CD34+/CD % ( ) p <0.001p <0.035

27 Recipient weight (kg)PercentNumber 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 and N01-HB 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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