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Mitchell S. Cairo , Elizabeth Wagner, Geoff Cohen, John Fraser, LeeAnn Jensen, Shelly Carter, Carmella van de Ven, Nancy Kernan, Joanne Kurtzberg PowerPoint PPT Presentation


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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 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 Kernan, Joanne Kurtzberg

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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 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)


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Experimental Model of Human Hematopoiesis

CD34+

CD41+/61+


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Experimental Model of Early Hematopoiesis

Pluripotent Stem Cell

CD34+/CD90+

CD34+/CD38-

CD34+/CD38+

CFU-GEMM

CFU-L


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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


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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


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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


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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


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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


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Methods

* Duke & UCLA


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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.


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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


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Ethnic Distribution of Long Term Stored Cryopreserved UCB


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Delivery, Gender, Gestational Age, Birth Weight

  • Type of DeliveryGender

  • Vaginal74%Males52%

  • C-Section26%Females48%

  • Gestational Age (wks)Birth Weight (gms)

  • < 378%< 25001%

  • 3817%2501-300011%

  • 3930%3001-350037%

  • 4031%3501-400036%

  • 4114%>400015%


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Cord Blood Volume, TNC, TMNC, CD34+


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

CD34+

CD34+/CD38-

CD34+/CD61+

CD34+/CD90+


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Cord Blood

Hematopoietic Progenitor Cells (HPC)


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


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Log All CFU (x 104)

Log CD34+ (x 104)

Significant Correlation of CB Log CD34+

vs Log Total CFU


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


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


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


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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


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Significant* Correlations of CB Log CD34+ subsets with Log CFUs

* All p<0.001


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63

53

21

44

44

14

WhiteAfrican AmericanAsianHispanicOther

WhiteAfrican AmericanAsianHispanicOther

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


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CD34+/CD38-

CD34+/CD61+

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

Birth Weight


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Significant Correlation of CB HPC with Delivery and Gender


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


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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


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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


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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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