Cord Blood (CB) Hematopoietic Cord Blood (CB) Hematopoietic Progenitor Cell (HPC) Characterization Progenitor Cell (HPC) Characterization

and Correlation with Ethnicity: A Report and Correlation with Ethnicity: A Report From The COBLT STUDYFrom The COBLT STUDY

1M S Cairo, 2G Cohen, 2E.L. Wagner, 3J Fraser, 4L Jensen, 2S Carter, 5N.A. Kernan, 6J Kurtzberg

1Georgetown University, Washington DC, 2The Emmes Corporation, Rockville MD, 3University of California Los Angeles,

Los Angeles CA, 4National Heart, Lung and Blood Institute, Bethesda MD, 5Memorial Sloan Kettering Cancer Center, New

York NY, 6Duke University Medical Center, Durham NC

IntroductionIntroduction

• Advantages of CB and CBT

Ease and safe procurement Enriched HPC

Rapid availability Immaturity of T cell immunity

Decreased viral transmission Decreased severe AGVHD

Multi-ethnic representation Reduced chronic GVHD

• Disadvantages of CB and CBT

Decreased supply Delay in hematopoietic recovery

Expensive to develop Prolonged immune reconstitution

Limited cell dose Lack of available cells for ACI

Genetic/infectious transmission Increased infectious morbidity

IntroductionIntroduction

• CB CD34+/kg graft content has been significantly correlated with engraftment and survival following UCBT

Wagner et al, Blood, 2002Styczynski 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, 1993Hao 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

ObjectivesObjectives

• 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

Cord Blood Banks, Data Coordinating Cord Blood Banks, Data Coordinating Center and NHLBICenter 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

MethodsMethods

• 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

MethodsMethods

Phenotype Monoclonal 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/IgG1 FITC/CD45 PerCP

*ProCount/Isotype control Nucleic Acid Dye/IgG1 FITC/CD45 PerCP

* Duke & UCLA

MethodsMethods

• 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.

ResultsResultsMaternal Donor

Screened34,799(100%)

Maternal DonorConsented

20,710(60%)

Total Collections16,764(81%)

45% Pregnancy related29% Refused9% Medical History6% Risk Behavior11% Other

44% No Staff41% Exclusions15% Other

Long Term Storage8,731

53% of Collections

Low CountsPoor Viability

Positive CultureNo HPC

Positive HGB Screen

Cryopreserved11,077(66%)

64% CB Volume11% +ID7% History18% Other

Ethnic Distribution of Long Term Ethnic Distribution of Long Term Stored Cryopreserved UCBStored Cryopreserved UCB

White 3632 42%

Hispanic 1841 21%

African-American 1213 14%

Mixed 1018 12%

Asian 987 11%

Delivery, Gender, Gestational Age, Delivery, Gender, Gestational Age, Birth WeightBirth Weight

• Type of Delivery Gender

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%

Cord Blood Volume, TNC, TMNC, Cord Blood Volume, TNC, TMNC, CD34CD34++

Pre-Processing Post Processing

Volume 80 mL 25 mL

Total Nucleated cells 1.8 x 109 9.2 x 108 (-22%)

Total Mononuclear cells 4.7 x 108 3.8 x 108 (-19%)

Total CD34+ NA 3.4 x 106

Gate: CD34+

Gated Events: 908

Total Events: 6808

Gate Events % Gated

G1 908 100.00

G2 908 100.00

CD45+ 907 99.89

34+ Subset - 871 95.93

Dual + 1 0.11

CD34+ 908 100.00

R4

R5

Gate: CD34+

Gated Events: 905

Total Events: 7634

Gate Events % Gated

G1 905 100.00

G2 905 100.00

CD45+ 904 99.89

34+ Subset - 22 2.43

Dual + 72 7.96

CD34+ 905 100.00

R4

R5

Representative Flow Cytometry Dot Plots of Representative Flow Cytometry Dot Plots of CD34CD34++, CD34, CD34++/CD38/CD38--, CD34, CD34++/CD61/CD61++ and and

CD34CD34++/CD90/CD90++

CD34+ CD34+/CD38-

CD34+/CD61+ CD34+/CD90+

R4

R5

Gate: CD34+

Gated Events: 923

Total Events: 408361

Gate Events % Gated

G1 923 100.00

G2 923 100.00

CD45+ 922 99.89

34+ Subset - 772 83.64

Dual + 4 0.43

CD34+ 923 100.00

Gate: CD45+

Gated Events: 344734

Total Events: 408361

Gate Events % Gated

G1 1146 0.33

G2 1042 0.30

CD45+ 344734 100.00

34+ Subset - 976 0.28

Dual + 836 0.24

CD34+ 922 0.27

R2

R3

Cord Blood Hematopoietic Progenitor Cord Blood Hematopoietic Progenitor Cells (HPC)Cells (HPC)

CD34+ Subsets (x 105) In vitro HPC (x 105)

CD34+ 34 ± 42 Total CFU 23.2 ± 19.8

CD34+/CD38- 3.4 ± 4.8 BFU-E 13.3 ±13.1

CD34+/CD61+ 7.3 ± 9.5 CFU-GM 9.3 ± 8.6

CD34 +/CD90+ 0.3 ± 1.9 CFU-GEMM 0.5 ± 1.1

Significant Correlation of CB Log Significant Correlation of CB Log CD34CD34++ vs Log Total CFU vs Log Total CFU

Log CD34+ (x 104)

Lo

g A

ll C

FU

(x

104 )

Significant* Correlations of CB Log CD34Significant* Correlations of CB Log CD34++ vs CFU-GM, CFU-GEMM & BFU-Evs 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

Positive Correlation of Log Total Nucleated Positive Correlation of Log Total Nucleated Cell Count (TNCC) with Log CD34Cell Count (TNCC) with Log CD34++ Count in Count in

COBLT Processed Cord BloodCOBLT Processed Cord Blood

Significant* Correlations of CB Log Significant* Correlations of CB Log CD34CD34++ subsets with Log CFUs subsets with Log CFUs

Log CD34+/CD61+ Log CD34+/CD38- Log CD34+/CD90+

N R N R N R

Log Total CFU 5073 0.55 5048 0.33 3600 0.30

Log Total BFU-E 5069 0.49 5045 0.31 3597 0.27

Log Total

CFU-GM5075 0.51 5050 0.35 3600 0.27

Log Total

CFU-GEMM2689 0.41 2684 0.27 2281 0.26

* All p<0.001

Significant* Correlations of CB Log Significant* Correlations of CB Log CD34CD34++ Subsets with Each Other Subsets with Each Other

Log CD34+ Log CD34+/CD38-

Log CD34+/CD61+

Log CD34+/CD90+

Log CD34+ 1.00 0.69 0.64 0.47

Log CD34+/CD38- 0.69 1.00 0.35 0.40

Log CD34+/CD61+ 0.64 0.35 1.00 0.47

Log CD34+/CD90+ 0.47 0.40 0.47 1.00

* All p<0.0001

Significant* Reduction in CB CD34Significant* Reduction in CB CD34++/CD38/CD38-- and CD34and CD34++/CD61/CD61++ in African Americans & in African Americans &

Asians vs Caucasians & HispanicsAsians vs Caucasians & Hispanics

* All p<0.001

CD34+/CD38- CD34+/CD61+

White Black Asian Hispanic Other White Black Asian Hispanic Other

53

44

White African American Asian Hispanic Other White African American Asian Hispanic Other

63

44

21

14

Correlation of CB HPC with Birth Weight Correlation of CB HPC with Birth Weight and Gestational Ageand Gestational Age

Birth Weight Gestational Age

CD34+/CD61+

CD34+/CD38-

Adj

uste

d m

ean

CD

61+

(x

10e-

4)

Birth weight (kg)<=2.5 2.5-3.0 3.0-3.5 3.5-4.0 > 4.0

30

40

50

60

70

80

90

CD34+/CD61+

Adj

uste

d m

ean

CD

61+

(x

10e-

4)

Gestational age (weeks)<=37 38 39 40 > 40

30

40

50

60

70

80

90

CD34+/CD38-

Adj

uste

d m

ean

CD

38+

(x

10e-

4)

Birth weight (kg)

<=2.5 2.5-3.0 3.0-3.5 3.5-4.0 > 4.0

10

15

20

25

Adj

uste

d m

ean

CD

38+

(x

10e-

4)

Gestational age (weeks)

<=37 38 39 40 > 4010

15

20

25

Significant Correlation of CB HPC Significant Correlation of CB HPC with Delivery and Genderwith Delivery and Gender

Delivery Gender

C-Section vs Vaginal Male vs Female

Total CFU

+ 8.7% (5.8-11.7)

CD34+/CD61+

+ 6.5% (0.4-13.0)

p <0.001 p <0.035

SummarySummary

• Post processing CB log CD34+ was significantly correlated with log total CFU, BFU-E, CFU-GM and CFU-GEMM

• Post processing CB log CD34+ subsets (CD38-, CD61+, CD90+) were significantly correlated with log total CFU, BFU-E, CFU-GM and CFU-GEMM

• Post processing CB log CD34+ subsets were significantly correlated with each other (CD34+/CD38-, CD34+/CD61+, CD34+/CD90+)

SummarySummary

• African-American and Asian donors were associated with significantly less CD34+/CD38- and CD34+/CD61+ processed CB HPC subsets

• C-section delivery was significantly associated with increased total processed CB CFU

• Male gender was significantly associated with increased CB CD34+/CD61+ HPC subset

• Birth weight was significantly correlated with both CD34+/CD38- and CD34+/CD61+ and inversely correlated with gestational age.

SpeculationSpeculation

• 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

• The current COBLT UCBT study will determine the

significance of these observations.

AcknowledgmentsAcknowledgments

• 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