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Increased content of CD34+CD38− hematopoietic stem cells in the last collected umbilical cord blood
Increased Content of CD34ⴙCD38ⴚ Hematopoietic Stem Cells in the Last Collected Umbilical Cord Blood W. Malangone, O. Belvedere, G. Astori, V. Adami, A. Donini, E. Falasca, P.G. Sala, G. Del Frate, M. Pittino, D. De Anna, and A. Degrassi
P
LACENTAL/umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem and progenitor cells for transplantation in hematologic malignant and nonmalignant disorders.1,2 Parameters commonly used to evaluate a UCB unit and predict the outcome of its clinical use are nucleated cell (NC) and CD34⫹ cell content.3 Furthermore, within the CD34⫹ cell population, the committed and uncommitted stem cell ratio is mostly important to predict short- and long-term engraftment, respectively, after transplantation. The expression of CD38 identifies already committed CD34⫹ cells,4 while the CD34⫹CD38⫺ phenotype identifies a UCB primitive subpopulation of hematopoietic stem cells.5,6 In this study, we evaluated these parameters within a single collection to address the question whether the last collected UCB is the same as the first collected one. We analyzed the kinetics of NC and CD34⫹ cell output and characterized different CD34⫹ cell subpopulations obtained in sequential UCB fractions during a single UCB collection. MATERIALS AND METHODS Umbilical Cord Blood Collections Within 3 minutes after placenta delivery, sequential 10-mL blood fractions were drained by gravity from the maternal end of the severed cord in tubes containing 3 mL citrate phosphate dextrose adenine (CPD-A) as anticoagulant. When UCB flow rate by gravity was ⱕ1 drop/30 seconds, and, following the traditional procedure, placenta is usually discarded, collection was continued using a recently developed device for the collection of UCB (patent pending).7 After collection, UCB samples were stored at room temperature and processed within 3 to 10 hours.
UCB Volume, Cell Number, and Flow Cytometric Analysis UCB volume from the sequential fractions was measured and NC number was evaluated using an automated cell counter. The percentage of hematopoietic stem cells in each fraction was evaluated by flow cytometric analysis following a direct immunofluorescence procedure using anti CD34-PE (Anti-HPCA-2, Becton Dickinson, Mountain View, Calif). To evaluate different CD34⫹ subpopulations in the first and last fraction, three-color analysis was performed by staining nucleated cells with anti-CD34 FITC, anti-CD38 PE (anti-Leu 17) and anti-HLA-DR PerCP (anti-HLA-DR), all purchased from Becton Dickinson. Analyses were performed using a FACScan flow cytometer (Becton Dickinson). 0041-1345/01/$–see front matter PII S0041-1345(00)02672-5
Normalization of Data and Statistical Analysis Given the variability of cell density and CD34⫹ cell percentage among different samples, data were normalized with respect to each sample’s first fraction and a value of 1 was given to results obtained from the first fraction. Significant differences between UCB sequential fractions were determined using a paired t test.
RESULTS Umbilical Cord Blood Collections
Forty-eight UCB samples were collected as described. The mean UCB volume collected by gravity was 46.7 ⫾ 25.6 mL (mean ⫾ SD), ranging from 8 to 134. After collection by gravity, the mean UCB volume further collected using the device was 14.3 ⫾ 7.8 mL, ranging from 2 to 31. The mean total collected volume was 60.9 ⫾ 26.2 mL, ranging from 17 to 141.5 mL. Volume calculations do not include the blood harvested for routine testing performed at birth (15 mL). NC and CD34⫹ Cell Number and Flow Cytometric Analysis
NC density evaluation in sequential blood fractions was performed in 38 UCB samples. No statistically significant difference was observed between the number of NC per milliliter in sequential collected fractions (Table 1). CD34⫹ cell percentage and absolute number in sequential blood fractions were also evaluated in 30 UCB samples. As shown in Table 1, there is a statistically significant increase in CD34⫹ percentage in successive collected fractions. Intrasample comparison of the first and last collected fraction CD34⫹ cell percentage shows a 2.07 ⫾ 1.18-fold increase From Consorzio Fenice (W.M., G.A., V.A., E.F., A.D.); Department of Experimental and Clinical Pathology and Medicine (A.D.), Medical Oncology (O.B.), and Department of Surgery (A.D., D.D.A.), University of Udine; Chem.-Clinical Lab, Udine General Hospital (P.G.S.), Udine, Italy; and Obstetric Division, S. Daniele General Hospital (G.D.F., M.P.), S. Daniele del Friuli, Italy. Supported in part by the Consiglio Nazionale delle Ricerche (CNR) Contributo di Ricerca n. 97.04090.CT04, from the Associazione Italiana per la ricerca sul Cancro (AIRC) and from Fondazione CA.RI.FE. Address reprint requests to Dr Alberto Degrassi, Consorzio FENICE, c/o Padiglione Petracco, P.le S.M della Misericordia, 33100 Udine, Italy. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1766
Transplantation Proceedings, 33, 1766–1768 (2001)
38 0.99 ⫾ 0.12 0.74 –1.19
30 1.13 ⫾ 0.46 0.63–2.9
38 1⫾0 1–1
30 1⫾0 1–1
2
92.04 ⫾ 2.85 86.34 –95.16
25 1.24 ⫾ 0.41* 0.76 –2.13
29 1.02 ⫾ 0.15 0.75–1.54
4
4.05 ⫾ 1.98 0.45–7.69
1.61 ⫾ 1.12 0.58 – 4.44
% CD38⫺DR⫹
First Fraction CD34⫹ Cells % CD38⫹DR⫺
22 1.52 ⫾ 1.06* 0.5–5.71
25 1 ⫾ 0.18 0.64 –1.34
5
19 1.54 ⫾ 0.71* 0.78 –3.25
19 0.96 ⫾ 0.33 0.66 –1.49
6
Fraction
9 1.58 ⫾ 0.8* 0.83–3.14
9 0.96 ⫾ 0.27 0.46 –1.39
7
1.63 ⫾ 1.05 0.52– 4.48
% CD38⫺DR⫺
59.83 ⫾ 21.88* 16.99 – 85.58
% CD38⫹DR⫹
8
3 1.8 ⫾ 1.15 1.02–3.13
3 1.13 ⫾ 0.05 1.09 –1.16
9
5.74 ⫾ 2.13** 1.58 –9.21
% CD38⫹DR⫺
1 1⫾0 1–1
10
% CD38⫺DR⫺
1 1.9 ⫾ 0 1.9 –1.9
1 1.15 ⫾ 0 1.15–1.15
11
15.45 ⫾ 11.89* 1.57–39.09
1 1.62 ⫾ 0 1.62–1.62
18.98 ⫾ 13.96* 5.22– 43.74
% CD38⫺DR⫹
Last Fraction CD34⫹ Cells
7 1.36 ⫾ 0.33* 1.05–1.96
7 1.03 ⫾ 0.25 0.6 –1.32
Table 2. CD34ⴙ Cell Subsets in the First and Last Collected UCB (n ⴝ 12)
30 1.35 ⫾ 0.91* 0.66 –5.8
35 1.06 ⫾ 0.19 0.83–1.7
3
*P ⬍ .001 for first vs last fraction; **P ⬍ .05 for first vs last fraction.
Mean ⫾ SD Range
% CD38⫹DR⫹
*P ⬍ .05 for comparison with the first fraction.
NC n Mean ⫾ SD Range CD34⫹ cells n Mean ⫾ SD Range
1
Table 1. Normalized Nucleated Cell Content and CD34ⴙ Cell Percentage in Subsequent UCB Fractions
CD34⫹CD38⫺ CELLS IN UCB 1767
1768
(mean ⫾ SD), ranging from 0.99 to 5.8 (P ⬍ .0001). To evaluate differences between CD34⫹ cells in sequential collected blood, the expression of CD38 was evaluated on CD34⫹ cells in the first and last collected fractions by flow cytometry. Based on the expression of these antigens, CD34⫹ cells can be divided into committed (CD34⫹CD38⫺) and pluripotent (CD34⫹CD38⫺) stem cells. The analysis of these cellular subpopulations shows that the composition of CD34⫹ cells in the first and last collected UCB is different both in terms of percentage and absolute number, the mean percentage of CD34⫹CD38⫹ cells ranging from 96.08 ⫾ 3.18 and 65.57 ⫾ 22.6 respectively, and the mean percentage of CD34⫹CD38⫺ cells ranging from 3.24 ⫾ 1.39 and 34.43 ⫾ 22.62, respectively. Furthermore, in the first and last fractions, the mean percentage of CD34⫹CD38⫺HLA-DR⫹ cells was 1.61 ⫾ 1.12 and 18.98 ⫾ 13.96, respectively (Table 1). DISCUSSION
In the present study we evaluated the kinetics of NC and CD34⫹ cells and characterized different CD34⫹ cell subpopulations obtained during a single UCB collection using a novel system developed for UCB collections from delivered placentas. Collection results show that when a traditional UCB collection is ended and the placenta is discarded, there is still residual placental blood that can be recovered using the device by the application of an appropriate pressure to the maternal and fetal surfaces of the placenta. The increased volume of UCB that can be harvested using the device is particularly relevant with respect to the observation that the content of CD34⫹ and
MALANGONE, BELVEDERE, ASTORI ET AL
CD34⫹CD38⫺ cells increase dramatically in the last collected UCB. Our results demonstrate that there is an increase in CD34⫹ cell content in the last collected UCB fractions without a significant variation in NC content. Moreover, in the last UCB collected fraction there is a mean 11.15 ⫾ 6.13-fold increase of the percentage of CD38⫺ cells within the CD34⫹ cells population compared to the first UCB collected fraction. Based on these results, the improvement of collection procedures might have important consequences in the clinical use of UCB, increasing significantly the availability of pluripotent stem cells capable of long-term engraftment. Moreover, the recovery of a higher number of CD34⫹CD38⫺ cells could provide a better starting population for ex vivo stem cell expansion. ACKNOWLEDGMENT The authors wish to thank Dr Valter Paderni for kindly providing the umbilical cord blood collection system used for this study.
REFERENCES 1. Cairo MS, Wagner JE: Blood 90:4665, 1997 2. Rubinstein P, Carrier C, Scaradavou A, et al: N Engl J Med 339:1565, 1998 3. Gratama JW, Orfao A, Barnett D, et al: Cytometry 34:128, 1998 4. Terstappen LWMM, Huang S, Safford M, et al: Blood 77:1218, 1991 5. Hao QL, Shah AJ, Thiemann FT, et al: Blood 86:3745, 1995 6. Huang S, Terstappen LWMM: Blood 83:1515, 1994 7. Belvedere O, Feruglio C, Malangone W, et al: Stem Cells 18:245, 2000
P
LACENTAL/umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem and progenitor cells for transplantation in hematologic malignant and nonmalignant disorders.1,2 Parameters commonly used to evaluate a UCB unit and predict the outcome of its clinical use are nucleated cell (NC) and CD34⫹ cell content.3 Furthermore, within the CD34⫹ cell population, the committed and uncommitted stem cell ratio is mostly important to predict short- and long-term engraftment, respectively, after transplantation. The expression of CD38 identifies already committed CD34⫹ cells,4 while the CD34⫹CD38⫺ phenotype identifies a UCB primitive subpopulation of hematopoietic stem cells.5,6 In this study, we evaluated these parameters within a single collection to address the question whether the last collected UCB is the same as the first collected one. We analyzed the kinetics of NC and CD34⫹ cell output and characterized different CD34⫹ cell subpopulations obtained in sequential UCB fractions during a single UCB collection. MATERIALS AND METHODS Umbilical Cord Blood Collections Within 3 minutes after placenta delivery, sequential 10-mL blood fractions were drained by gravity from the maternal end of the severed cord in tubes containing 3 mL citrate phosphate dextrose adenine (CPD-A) as anticoagulant. When UCB flow rate by gravity was ⱕ1 drop/30 seconds, and, following the traditional procedure, placenta is usually discarded, collection was continued using a recently developed device for the collection of UCB (patent pending).7 After collection, UCB samples were stored at room temperature and processed within 3 to 10 hours.
UCB Volume, Cell Number, and Flow Cytometric Analysis UCB volume from the sequential fractions was measured and NC number was evaluated using an automated cell counter. The percentage of hematopoietic stem cells in each fraction was evaluated by flow cytometric analysis following a direct immunofluorescence procedure using anti CD34-PE (Anti-HPCA-2, Becton Dickinson, Mountain View, Calif). To evaluate different CD34⫹ subpopulations in the first and last fraction, three-color analysis was performed by staining nucleated cells with anti-CD34 FITC, anti-CD38 PE (anti-Leu 17) and anti-HLA-DR PerCP (anti-HLA-DR), all purchased from Becton Dickinson. Analyses were performed using a FACScan flow cytometer (Becton Dickinson). 0041-1345/01/$–see front matter PII S0041-1345(00)02672-5
Normalization of Data and Statistical Analysis Given the variability of cell density and CD34⫹ cell percentage among different samples, data were normalized with respect to each sample’s first fraction and a value of 1 was given to results obtained from the first fraction. Significant differences between UCB sequential fractions were determined using a paired t test.
RESULTS Umbilical Cord Blood Collections
Forty-eight UCB samples were collected as described. The mean UCB volume collected by gravity was 46.7 ⫾ 25.6 mL (mean ⫾ SD), ranging from 8 to 134. After collection by gravity, the mean UCB volume further collected using the device was 14.3 ⫾ 7.8 mL, ranging from 2 to 31. The mean total collected volume was 60.9 ⫾ 26.2 mL, ranging from 17 to 141.5 mL. Volume calculations do not include the blood harvested for routine testing performed at birth (15 mL). NC and CD34⫹ Cell Number and Flow Cytometric Analysis
NC density evaluation in sequential blood fractions was performed in 38 UCB samples. No statistically significant difference was observed between the number of NC per milliliter in sequential collected fractions (Table 1). CD34⫹ cell percentage and absolute number in sequential blood fractions were also evaluated in 30 UCB samples. As shown in Table 1, there is a statistically significant increase in CD34⫹ percentage in successive collected fractions. Intrasample comparison of the first and last collected fraction CD34⫹ cell percentage shows a 2.07 ⫾ 1.18-fold increase From Consorzio Fenice (W.M., G.A., V.A., E.F., A.D.); Department of Experimental and Clinical Pathology and Medicine (A.D.), Medical Oncology (O.B.), and Department of Surgery (A.D., D.D.A.), University of Udine; Chem.-Clinical Lab, Udine General Hospital (P.G.S.), Udine, Italy; and Obstetric Division, S. Daniele General Hospital (G.D.F., M.P.), S. Daniele del Friuli, Italy. Supported in part by the Consiglio Nazionale delle Ricerche (CNR) Contributo di Ricerca n. 97.04090.CT04, from the Associazione Italiana per la ricerca sul Cancro (AIRC) and from Fondazione CA.RI.FE. Address reprint requests to Dr Alberto Degrassi, Consorzio FENICE, c/o Padiglione Petracco, P.le S.M della Misericordia, 33100 Udine, Italy. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1766
Transplantation Proceedings, 33, 1766–1768 (2001)
38 0.99 ⫾ 0.12 0.74 –1.19
30 1.13 ⫾ 0.46 0.63–2.9
38 1⫾0 1–1
30 1⫾0 1–1
2
92.04 ⫾ 2.85 86.34 –95.16
25 1.24 ⫾ 0.41* 0.76 –2.13
29 1.02 ⫾ 0.15 0.75–1.54
4
4.05 ⫾ 1.98 0.45–7.69
1.61 ⫾ 1.12 0.58 – 4.44
% CD38⫺DR⫹
First Fraction CD34⫹ Cells % CD38⫹DR⫺
22 1.52 ⫾ 1.06* 0.5–5.71
25 1 ⫾ 0.18 0.64 –1.34
5
19 1.54 ⫾ 0.71* 0.78 –3.25
19 0.96 ⫾ 0.33 0.66 –1.49
6
Fraction
9 1.58 ⫾ 0.8* 0.83–3.14
9 0.96 ⫾ 0.27 0.46 –1.39
7
1.63 ⫾ 1.05 0.52– 4.48
% CD38⫺DR⫺
59.83 ⫾ 21.88* 16.99 – 85.58
% CD38⫹DR⫹
8
3 1.8 ⫾ 1.15 1.02–3.13
3 1.13 ⫾ 0.05 1.09 –1.16
9
5.74 ⫾ 2.13** 1.58 –9.21
% CD38⫹DR⫺
1 1⫾0 1–1
10
% CD38⫺DR⫺
1 1.9 ⫾ 0 1.9 –1.9
1 1.15 ⫾ 0 1.15–1.15
11
15.45 ⫾ 11.89* 1.57–39.09
1 1.62 ⫾ 0 1.62–1.62
18.98 ⫾ 13.96* 5.22– 43.74
% CD38⫺DR⫹
Last Fraction CD34⫹ Cells
7 1.36 ⫾ 0.33* 1.05–1.96
7 1.03 ⫾ 0.25 0.6 –1.32
Table 2. CD34ⴙ Cell Subsets in the First and Last Collected UCB (n ⴝ 12)
30 1.35 ⫾ 0.91* 0.66 –5.8
35 1.06 ⫾ 0.19 0.83–1.7
3
*P ⬍ .001 for first vs last fraction; **P ⬍ .05 for first vs last fraction.
Mean ⫾ SD Range
% CD38⫹DR⫹
*P ⬍ .05 for comparison with the first fraction.
NC n Mean ⫾ SD Range CD34⫹ cells n Mean ⫾ SD Range
1
Table 1. Normalized Nucleated Cell Content and CD34ⴙ Cell Percentage in Subsequent UCB Fractions
CD34⫹CD38⫺ CELLS IN UCB 1767
1768
(mean ⫾ SD), ranging from 0.99 to 5.8 (P ⬍ .0001). To evaluate differences between CD34⫹ cells in sequential collected blood, the expression of CD38 was evaluated on CD34⫹ cells in the first and last collected fractions by flow cytometry. Based on the expression of these antigens, CD34⫹ cells can be divided into committed (CD34⫹CD38⫺) and pluripotent (CD34⫹CD38⫺) stem cells. The analysis of these cellular subpopulations shows that the composition of CD34⫹ cells in the first and last collected UCB is different both in terms of percentage and absolute number, the mean percentage of CD34⫹CD38⫹ cells ranging from 96.08 ⫾ 3.18 and 65.57 ⫾ 22.6 respectively, and the mean percentage of CD34⫹CD38⫺ cells ranging from 3.24 ⫾ 1.39 and 34.43 ⫾ 22.62, respectively. Furthermore, in the first and last fractions, the mean percentage of CD34⫹CD38⫺HLA-DR⫹ cells was 1.61 ⫾ 1.12 and 18.98 ⫾ 13.96, respectively (Table 1). DISCUSSION
In the present study we evaluated the kinetics of NC and CD34⫹ cells and characterized different CD34⫹ cell subpopulations obtained during a single UCB collection using a novel system developed for UCB collections from delivered placentas. Collection results show that when a traditional UCB collection is ended and the placenta is discarded, there is still residual placental blood that can be recovered using the device by the application of an appropriate pressure to the maternal and fetal surfaces of the placenta. The increased volume of UCB that can be harvested using the device is particularly relevant with respect to the observation that the content of CD34⫹ and
MALANGONE, BELVEDERE, ASTORI ET AL
CD34⫹CD38⫺ cells increase dramatically in the last collected UCB. Our results demonstrate that there is an increase in CD34⫹ cell content in the last collected UCB fractions without a significant variation in NC content. Moreover, in the last UCB collected fraction there is a mean 11.15 ⫾ 6.13-fold increase of the percentage of CD38⫺ cells within the CD34⫹ cells population compared to the first UCB collected fraction. Based on these results, the improvement of collection procedures might have important consequences in the clinical use of UCB, increasing significantly the availability of pluripotent stem cells capable of long-term engraftment. Moreover, the recovery of a higher number of CD34⫹CD38⫺ cells could provide a better starting population for ex vivo stem cell expansion. ACKNOWLEDGMENT The authors wish to thank Dr Valter Paderni for kindly providing the umbilical cord blood collection system used for this study.
REFERENCES 1. Cairo MS, Wagner JE: Blood 90:4665, 1997 2. Rubinstein P, Carrier C, Scaradavou A, et al: N Engl J Med 339:1565, 1998 3. Gratama JW, Orfao A, Barnett D, et al: Cytometry 34:128, 1998 4. Terstappen LWMM, Huang S, Safford M, et al: Blood 77:1218, 1991 5. Hao QL, Shah AJ, Thiemann FT, et al: Blood 86:3745, 1995 6. Huang S, Terstappen LWMM: Blood 83:1515, 1994 7. Belvedere O, Feruglio C, Malangone W, et al: Stem Cells 18:245, 2000