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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.