A Modified Cord Blood Collection Method Achieves Sufficient Cell Levels for Transplantation in Most Adult Patients
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《干细胞学杂志》
a Madrid Cord Blood Bank,
b Department of Hematology,
c Department of Immunology, Hospital Universitario 12 de Octubre, Madrid, Spain
Key Words. Antigens ? CD34 ? Cord blood banks ? Cord blood stem cell transplantation ? Hematopoietic stem cells ? Placental circulation
Correspondence: Rafael Bornstein M.D., Ph.D., Madrid Cord Blood Bank, Hospital 12 de Octubre, Avda. de Córdoba, s/n, Madrid 28041, Spain. Telephone: 34-91-390-8419; Fax: 34-91-390-8483; e-mail: rbornstein.hdoc@salud.madrid.org
ABSTRACT
In the last decade, hematopoietic cell transplantation (HCT) using umbilical cord blood (UCB) grafts has increasingly been used, particularly for pediatric but also for adult patients . As reported by NetCord, more than 2,500 unrelated umbilical cord blood transplants (UCBTs) have been performed, one-third in adult recipients . The data published so far indicate that UCB is a viable alternative source of hematopoietic stem cells (HSCs), and in certain situations may have advantages over unrelated donor marrow grafts .
Based on this extensive experience in UCBT, the total nucleated cell (NC) dose infused has emerged as the most critical factor in determining speed of engraftment and survival after UCBT . Among 0–2 antigen HLA-mismatched grafts, current data suggests for the same cell dose, survival is superior with better-matched grafts. Although, the negative effect of HLA-mismatch can be at least partially overcome by a higher cell dose . Therefore, the fixed cell content of a UCB unit represents the major limiting factor, particularly for adult recipients. However, the patient’s age does not appear to affect the UCBT outcomes, provided the cell dose is adequate ; a conception supported by the most recent UCBT series in adult patients in which engraftment and survival rates were comparable to those seen in child recipients .
Several reports have suggested that a threshold number of nucleated cells is needed for engraftment. Particularly poor results are seen after UCBT in both children and adults when the NC dose infused is less than 1.5 x 107/kg . Above this figure, there is a level—the "optimal" dose—which is associated with a distinct survival advantage. Gluckman et al. have shown that a graft NC dose > 3.7 x 107/kg was associated with shorter time to neutrophil recovery (25 versus 34 days) and higher engraftment rate (94% versus 76%). While both the minimum acceptable and the optimal UCB graft cell doses are yet to be unanimously agreed upon, most of the available data suggests there is a threshold effect somewhere within this range and that a target (advisable) cell dose must be between 1.5 and 2.5 x 107/kg. Many transplant centers would now recommend 2 x 107/kg as a reasonable target cell dose to obtain satisfactory UCBT outcomes .
As the finite number of HSCs in single UCB units may result in underutilization of this alternate stem cell source in larger pediatric and adult recipients, UCB banks should focus on the collection of larger units with greater numbers of cells . Using the standard collection technique, the mean number of NCs reported by the biggest UCB banks worldwide is about 10 x 108 per unit , and with this cell content, only 25% of UCB units contain enough cells to fulfill the target dose for transplantation in patients weighing 50–70 kg . Here we present data on the increase in UCB cell retrieval by using a modified placental/umbilical collection method. By means of these enriched UCB units, a cell dose of 2 x 107/kg would be achieved in most larger pediatric patients and in a significant proportion of heavier adult patients requiring HCT.
MATERIALS AND METHODS
Increase in UCB Cell Retrieval by Using a Modified Placental/Umbilical Collection Method
The technique for UCB collection used in this study comprises two separate blood harvestings. We hypothesized that a second blood fraction obtained after placental perfusion in addition to the standard umbilical venipuncture collection would result in higher blood volume and NC yield. Indeed, we observed a significant correlation between UCB volume and cell content (Fig. 2). By using this novel technique we obtained an average volume of 119.6 ml (range, 27–279 ml) and a NC content of 1.21 ± 0.52 x 109 (group I, Table 1). As compared to data reported by other banks , where the average volume of UCB units is 71–98 ml and the cell content ranges from 0.85 to 1.05 x 109, our results suggest that the additional blood collected after placental perfusion may increase the total number of NCs.
Figure 2. Correlation of umbilical cord blood volume and nucleated cells: Pearson’s correlation coefficient, r =.63 (n = 300).
Table 1. Volume, nucleated cell content, and mononucleated cell content of umbilical cord blood units
To determine more precisely the benefits of collecting a second fraction, we analyzed the contribution of both UCB fractions in 44 units from group I (Table 2). Although the umbilical venipuncture fraction provided most of the total volume and NC content, the second fraction contribution was 32% and 15%, respectively. However, the NC number provided by the second fraction was very variable between units (Fig. 3). In approximately one out of four (27%) units the second fraction had a contribution of more than 20% of the total cell content, whereas in one out of three (34%) units the cell profit was minimal (<10% of the total NCs). Of note, the second fraction represented over 30% to the total NC count in 9% of the UCB units collected.
Table 2. Volume and number of NCs in UCB collected by intrautero umbilical venipuncture (fraction 1) and placental perfusion after delivery (fraction 2)
Figure 3. Contribution of the second fraction to the NC content in the UCB units. Forty-four units were analyzed to determine the percentage of NCs contributed by the second fraction (in 5% intervals). This contribution was very variable. It represented more than 20% of the total number of NCs (columns 5 to 7) in 27% of the units, but less than 10% (columns 1 and 2) in 34% of the units. Abbreviations: NC, nucleated cell; UCB, umbilical cord blood.
Several reports have suggested a 2 x 107/kg NC as the target dose for transplantation. For adult patients weighing 50 and 65 kg it will be necessary to infuse 1 x 109 and 1.3 x 109 NC, respectively. With the standard venipuncture UCB collection method (first fraction), 50% and 30% of these units, respectively, would reach the target dose (Table 3). As a result of the second fraction collection, 70% and 41% of units exceeded 1 x 109 and 1.3 x 109 NC. Therefore, the two-fraction collection technique described here increases by 20% and 11% the number of UCB units clinically useful for adult patients.
Table 3. Frequency of UCB units with > 1 x 109 or > 1.3 x 109 NCs
HPC Analysis in First and Second UCB Fractions
To assess the hematopoietic potential of the first and second UCB fractions, we determined the number of HPC (CD34, CD133, CFU-GM, and BFU-E) present in each fraction. The analysis was performed on 10 units from group I. The number of CD34+ and CD133+ cells were analyzed by flow cytometry (Fig. 4). The proportion of CD34+ cells present in the first and second fraction was 0.36% ± 0.18% and 0.33% ± 0.2%, respectively. There were no significant differences in HPC subsets between both UCB fractions. Indeed, most of the CD34+ cells co-expressed the CD133 antigen, and there were nearly undetectable CD34–/CD133+ cells. Clonogenic assays were also performed in a similarly comparative way (Fig. 5). The CFU-GM and BFU-E absolute numbers in the first and second fractions were proportional to the total number of NCs. Thus, the second fraction represents 19% and 17% of the total CFU-GM and BFU-E, respectively, corresponding with the NC content (15%) of the second fraction (see Table 2). There were no differences in colony size between fractions (data not shown), suggesting that UCB clonogenic progenitors present in the first and second fractions have the same proliferative capacity. In summary, cells obtained by the standard umbilical venipuncture (first fraction) and cells collected after placental perfusion (second fraction) have similar HPC contents and in vitro hematopoietic potential.
Figure 4. Comparative analysis of CD34+/CD133+ cells content in first and second UCB fractions. The data show CD34+ or CD133+ (or both) cell counts (mean ± SD) in the first and second UCB fractions. The proportion of CD34+ cells in the fraction harvested by umbilical venipuncture (fraction 1) and in the fraction obtained after placental perfusion (fraction 2) was 0.36% ± 0.18% and 0.33% ± 0.2%, respectively. CD34+/CD133+ cells were 0.32% ± 0.17% and 0.33% ± 0.19%, whereas CD34+/CD133– cells were lower than 0.03% in both fractions. CD34–/CD133+ were barely detectable. Differences between both UCB fractions are not significant (paired-samples T test, n = 10). Abbreviation: UCB, umbilical cord blood.
Figure 5. Comparative analysis of CFU-GM and BFU-E (mean ± SD) in first and second umbilical cord blood (UCB) fractions. The number of CFU-GM in the fraction harvested by umbilical venipuncture (fraction 1) and in the fraction obtained after placental perfusion (fraction 2) was 39.56 ± 17.76 x 104 and 10.54 ± 8.28 x 104, respectively, whereas BFU-E numbers in each of these fractions were 83.77 ± 28.88 x 104 and 15.22 ± 9.98x 104 (n = 10). The CFU-GM and BFU-E absolute numbers in the first and the second fractions are proportional to the total number of nucleated cells (see Table 2). Abbreviations: CFU-GM, colony forming units–granulocyte macrophage.
Increase in Cell Content of the UCB Bank Inventory as a Result of a Processing Restricted to Units 0.8 x 109 NC
Since 1998, we have only processed UCB units with an NC count 0.8x109(group II). The restriction bar was established at this level after two considerations. The UCB units below these cell numbers would probably never be requested for grafting adult patients. Also, the processing restriction should not significantly increase the number of discarded UCB units because our two-fraction collection method provides more NCs per unit. Indeed, the units discarded by using the 0.8 x 109 NC restriction limit was just 16%. This rate would have been higher (27%) if we had not harvested the placental fraction. Group I NC content is 1.21 ± 0.52 x 109, whereas in group II it is 1.46 ± 0.52 x 109 (p < .001). As a result, the percentage of units with a cell count 1 x 109 and 1.3 x 109 in group II were 84% and 54% compared with 62% and 40% of group I, respectively (Table 4).
Table 4. NC content in UCB units stored prior to or since the use of the 0.8 x 109 NC processing restriction
Bacterial Contamination of UCB Collected by the Two-Fraction Method
The bacterial contamination detected in the stored UCB units from groups I and II (n = 1620) is 2.78% (aerobic, 1.97%; anaerobic, 0.81%). The rate is similar to the contamination obtained with the procedures used at other UCB banks , suggesting that the second fraction collection does not increase the risk of bacterial contamination.
No Increase in Risk of Maternal Cell Contamination with the Placental Perfusion Fraction
We used locus-specific amplification of the noninherited maternal DRB1 genes to determine the presence of maternal cells in UCB units collected by the two-fraction method. DNA samples from 20 units from group II were amplified with DRB1-specific and ?-globin (control) primers. The noninherited maternal genes analyzed were DRB1*07 (in five units), *04 (three units), *11 (three units), *14 (two units), *15 (two units), and *01, *08, *12, *13, and *17 (one unit each). The sensitivity of the technique was 1% (see Materials and Methods). Two representative cases are shown in Figure 6. Whereas cord blood DRB1–specific genes were amplified, we did not detect PCR products for the noninherited maternal DRB1 alleles. Therefore, no maternal DNA could be detected in any of the UCB units tested, indicating that the second fraction seems not to increase the level of cord blood contamination by maternal cells reported by other groups .
Figure 6. PCR amplification with sequence-specific primers for the noninherited maternal DRB1 allele. Two representative analyses are shown. (A): Cord blood and maternal DRB1 genes were *04, *07 and *07, *12, respectively. Lane 1: molecular size marker (123-bp ladder); lane 2: negative control (no DNA); lanes 3 and 4: amplifications using primers for DRB1*12 (noninherited maternal gene); lanes 5 and 6: amplifications using primers for DRB1*04 (UCB-specific gene). (B): Cord blood and maternal DRB1 genes were *10, *15 and *07, *10, respectively. Lane 1: molecular size marker; lane 2: amplification using primers for DRB1*10 (maternal and UCB genes); lanes 3 to 9: amplifications using primers for DRB1*07 (noninherited maternal gene); lane 10: negative control (no DNA). Cord blood DRB1*04 (A) and DRB1*10 (B) products were detected (arrows), whereas the noninherited maternal DRB1 alleles were not amplified. ?-globin amplification was used as a loading control (arrowheads). Abbreviations: PCR, polymerase chain reaction; UCB, umbilical cord blood.
Use of Specific UCB Collection Bags
To simplify the harvesting procedure, we recently introduced a specific bag for UCB collection (Stemflex, Maco Pharma) with two lines for the retrieval of each blood fraction into the same container (Fig. 1D). This new bag allows us to proceed with the protocol without substantial modifications. The volume of blood collected into these bags (group III, n = 319) was 118.9 ± 39 ml, and the NC content was 1.46±0.58 x109NC. These data are similar to the results obtained with the standard blood donor bags (Table 4). Bacterial contamination in the new bags was 2.19%, a rate slightly lower than the one observed with the standard bags.
DISCUSSION
The higher hematopoietic potential of UCB units harvested and processed according to the methodology proposed in this study leads to an increase in the number of grafts with a 2 x 107/kg NC dose. Thus, 84% and 54% of our UCB units would fulfill this target dose in recipients weighing 50 and 65 kg compared with less than 30% units from other UCB banks. This significant advance procured by our novel UCB collection technique gives larger pediatric and many adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.
ACKNOWLEDGMENTS
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b Department of Hematology,
c Department of Immunology, Hospital Universitario 12 de Octubre, Madrid, Spain
Key Words. Antigens ? CD34 ? Cord blood banks ? Cord blood stem cell transplantation ? Hematopoietic stem cells ? Placental circulation
Correspondence: Rafael Bornstein M.D., Ph.D., Madrid Cord Blood Bank, Hospital 12 de Octubre, Avda. de Córdoba, s/n, Madrid 28041, Spain. Telephone: 34-91-390-8419; Fax: 34-91-390-8483; e-mail: rbornstein.hdoc@salud.madrid.org
ABSTRACT
In the last decade, hematopoietic cell transplantation (HCT) using umbilical cord blood (UCB) grafts has increasingly been used, particularly for pediatric but also for adult patients . As reported by NetCord, more than 2,500 unrelated umbilical cord blood transplants (UCBTs) have been performed, one-third in adult recipients . The data published so far indicate that UCB is a viable alternative source of hematopoietic stem cells (HSCs), and in certain situations may have advantages over unrelated donor marrow grafts .
Based on this extensive experience in UCBT, the total nucleated cell (NC) dose infused has emerged as the most critical factor in determining speed of engraftment and survival after UCBT . Among 0–2 antigen HLA-mismatched grafts, current data suggests for the same cell dose, survival is superior with better-matched grafts. Although, the negative effect of HLA-mismatch can be at least partially overcome by a higher cell dose . Therefore, the fixed cell content of a UCB unit represents the major limiting factor, particularly for adult recipients. However, the patient’s age does not appear to affect the UCBT outcomes, provided the cell dose is adequate ; a conception supported by the most recent UCBT series in adult patients in which engraftment and survival rates were comparable to those seen in child recipients .
Several reports have suggested that a threshold number of nucleated cells is needed for engraftment. Particularly poor results are seen after UCBT in both children and adults when the NC dose infused is less than 1.5 x 107/kg . Above this figure, there is a level—the "optimal" dose—which is associated with a distinct survival advantage. Gluckman et al. have shown that a graft NC dose > 3.7 x 107/kg was associated with shorter time to neutrophil recovery (25 versus 34 days) and higher engraftment rate (94% versus 76%). While both the minimum acceptable and the optimal UCB graft cell doses are yet to be unanimously agreed upon, most of the available data suggests there is a threshold effect somewhere within this range and that a target (advisable) cell dose must be between 1.5 and 2.5 x 107/kg. Many transplant centers would now recommend 2 x 107/kg as a reasonable target cell dose to obtain satisfactory UCBT outcomes .
As the finite number of HSCs in single UCB units may result in underutilization of this alternate stem cell source in larger pediatric and adult recipients, UCB banks should focus on the collection of larger units with greater numbers of cells . Using the standard collection technique, the mean number of NCs reported by the biggest UCB banks worldwide is about 10 x 108 per unit , and with this cell content, only 25% of UCB units contain enough cells to fulfill the target dose for transplantation in patients weighing 50–70 kg . Here we present data on the increase in UCB cell retrieval by using a modified placental/umbilical collection method. By means of these enriched UCB units, a cell dose of 2 x 107/kg would be achieved in most larger pediatric patients and in a significant proportion of heavier adult patients requiring HCT.
MATERIALS AND METHODS
Increase in UCB Cell Retrieval by Using a Modified Placental/Umbilical Collection Method
The technique for UCB collection used in this study comprises two separate blood harvestings. We hypothesized that a second blood fraction obtained after placental perfusion in addition to the standard umbilical venipuncture collection would result in higher blood volume and NC yield. Indeed, we observed a significant correlation between UCB volume and cell content (Fig. 2). By using this novel technique we obtained an average volume of 119.6 ml (range, 27–279 ml) and a NC content of 1.21 ± 0.52 x 109 (group I, Table 1). As compared to data reported by other banks , where the average volume of UCB units is 71–98 ml and the cell content ranges from 0.85 to 1.05 x 109, our results suggest that the additional blood collected after placental perfusion may increase the total number of NCs.
Figure 2. Correlation of umbilical cord blood volume and nucleated cells: Pearson’s correlation coefficient, r =.63 (n = 300).
Table 1. Volume, nucleated cell content, and mononucleated cell content of umbilical cord blood units
To determine more precisely the benefits of collecting a second fraction, we analyzed the contribution of both UCB fractions in 44 units from group I (Table 2). Although the umbilical venipuncture fraction provided most of the total volume and NC content, the second fraction contribution was 32% and 15%, respectively. However, the NC number provided by the second fraction was very variable between units (Fig. 3). In approximately one out of four (27%) units the second fraction had a contribution of more than 20% of the total cell content, whereas in one out of three (34%) units the cell profit was minimal (<10% of the total NCs). Of note, the second fraction represented over 30% to the total NC count in 9% of the UCB units collected.
Table 2. Volume and number of NCs in UCB collected by intrautero umbilical venipuncture (fraction 1) and placental perfusion after delivery (fraction 2)
Figure 3. Contribution of the second fraction to the NC content in the UCB units. Forty-four units were analyzed to determine the percentage of NCs contributed by the second fraction (in 5% intervals). This contribution was very variable. It represented more than 20% of the total number of NCs (columns 5 to 7) in 27% of the units, but less than 10% (columns 1 and 2) in 34% of the units. Abbreviations: NC, nucleated cell; UCB, umbilical cord blood.
Several reports have suggested a 2 x 107/kg NC as the target dose for transplantation. For adult patients weighing 50 and 65 kg it will be necessary to infuse 1 x 109 and 1.3 x 109 NC, respectively. With the standard venipuncture UCB collection method (first fraction), 50% and 30% of these units, respectively, would reach the target dose (Table 3). As a result of the second fraction collection, 70% and 41% of units exceeded 1 x 109 and 1.3 x 109 NC. Therefore, the two-fraction collection technique described here increases by 20% and 11% the number of UCB units clinically useful for adult patients.
Table 3. Frequency of UCB units with > 1 x 109 or > 1.3 x 109 NCs
HPC Analysis in First and Second UCB Fractions
To assess the hematopoietic potential of the first and second UCB fractions, we determined the number of HPC (CD34, CD133, CFU-GM, and BFU-E) present in each fraction. The analysis was performed on 10 units from group I. The number of CD34+ and CD133+ cells were analyzed by flow cytometry (Fig. 4). The proportion of CD34+ cells present in the first and second fraction was 0.36% ± 0.18% and 0.33% ± 0.2%, respectively. There were no significant differences in HPC subsets between both UCB fractions. Indeed, most of the CD34+ cells co-expressed the CD133 antigen, and there were nearly undetectable CD34–/CD133+ cells. Clonogenic assays were also performed in a similarly comparative way (Fig. 5). The CFU-GM and BFU-E absolute numbers in the first and second fractions were proportional to the total number of NCs. Thus, the second fraction represents 19% and 17% of the total CFU-GM and BFU-E, respectively, corresponding with the NC content (15%) of the second fraction (see Table 2). There were no differences in colony size between fractions (data not shown), suggesting that UCB clonogenic progenitors present in the first and second fractions have the same proliferative capacity. In summary, cells obtained by the standard umbilical venipuncture (first fraction) and cells collected after placental perfusion (second fraction) have similar HPC contents and in vitro hematopoietic potential.
Figure 4. Comparative analysis of CD34+/CD133+ cells content in first and second UCB fractions. The data show CD34+ or CD133+ (or both) cell counts (mean ± SD) in the first and second UCB fractions. The proportion of CD34+ cells in the fraction harvested by umbilical venipuncture (fraction 1) and in the fraction obtained after placental perfusion (fraction 2) was 0.36% ± 0.18% and 0.33% ± 0.2%, respectively. CD34+/CD133+ cells were 0.32% ± 0.17% and 0.33% ± 0.19%, whereas CD34+/CD133– cells were lower than 0.03% in both fractions. CD34–/CD133+ were barely detectable. Differences between both UCB fractions are not significant (paired-samples T test, n = 10). Abbreviation: UCB, umbilical cord blood.
Figure 5. Comparative analysis of CFU-GM and BFU-E (mean ± SD) in first and second umbilical cord blood (UCB) fractions. The number of CFU-GM in the fraction harvested by umbilical venipuncture (fraction 1) and in the fraction obtained after placental perfusion (fraction 2) was 39.56 ± 17.76 x 104 and 10.54 ± 8.28 x 104, respectively, whereas BFU-E numbers in each of these fractions were 83.77 ± 28.88 x 104 and 15.22 ± 9.98x 104 (n = 10). The CFU-GM and BFU-E absolute numbers in the first and the second fractions are proportional to the total number of nucleated cells (see Table 2). Abbreviations: CFU-GM, colony forming units–granulocyte macrophage.
Increase in Cell Content of the UCB Bank Inventory as a Result of a Processing Restricted to Units 0.8 x 109 NC
Since 1998, we have only processed UCB units with an NC count 0.8x109(group II). The restriction bar was established at this level after two considerations. The UCB units below these cell numbers would probably never be requested for grafting adult patients. Also, the processing restriction should not significantly increase the number of discarded UCB units because our two-fraction collection method provides more NCs per unit. Indeed, the units discarded by using the 0.8 x 109 NC restriction limit was just 16%. This rate would have been higher (27%) if we had not harvested the placental fraction. Group I NC content is 1.21 ± 0.52 x 109, whereas in group II it is 1.46 ± 0.52 x 109 (p < .001). As a result, the percentage of units with a cell count 1 x 109 and 1.3 x 109 in group II were 84% and 54% compared with 62% and 40% of group I, respectively (Table 4).
Table 4. NC content in UCB units stored prior to or since the use of the 0.8 x 109 NC processing restriction
Bacterial Contamination of UCB Collected by the Two-Fraction Method
The bacterial contamination detected in the stored UCB units from groups I and II (n = 1620) is 2.78% (aerobic, 1.97%; anaerobic, 0.81%). The rate is similar to the contamination obtained with the procedures used at other UCB banks , suggesting that the second fraction collection does not increase the risk of bacterial contamination.
No Increase in Risk of Maternal Cell Contamination with the Placental Perfusion Fraction
We used locus-specific amplification of the noninherited maternal DRB1 genes to determine the presence of maternal cells in UCB units collected by the two-fraction method. DNA samples from 20 units from group II were amplified with DRB1-specific and ?-globin (control) primers. The noninherited maternal genes analyzed were DRB1*07 (in five units), *04 (three units), *11 (three units), *14 (two units), *15 (two units), and *01, *08, *12, *13, and *17 (one unit each). The sensitivity of the technique was 1% (see Materials and Methods). Two representative cases are shown in Figure 6. Whereas cord blood DRB1–specific genes were amplified, we did not detect PCR products for the noninherited maternal DRB1 alleles. Therefore, no maternal DNA could be detected in any of the UCB units tested, indicating that the second fraction seems not to increase the level of cord blood contamination by maternal cells reported by other groups .
Figure 6. PCR amplification with sequence-specific primers for the noninherited maternal DRB1 allele. Two representative analyses are shown. (A): Cord blood and maternal DRB1 genes were *04, *07 and *07, *12, respectively. Lane 1: molecular size marker (123-bp ladder); lane 2: negative control (no DNA); lanes 3 and 4: amplifications using primers for DRB1*12 (noninherited maternal gene); lanes 5 and 6: amplifications using primers for DRB1*04 (UCB-specific gene). (B): Cord blood and maternal DRB1 genes were *10, *15 and *07, *10, respectively. Lane 1: molecular size marker; lane 2: amplification using primers for DRB1*10 (maternal and UCB genes); lanes 3 to 9: amplifications using primers for DRB1*07 (noninherited maternal gene); lane 10: negative control (no DNA). Cord blood DRB1*04 (A) and DRB1*10 (B) products were detected (arrows), whereas the noninherited maternal DRB1 alleles were not amplified. ?-globin amplification was used as a loading control (arrowheads). Abbreviations: PCR, polymerase chain reaction; UCB, umbilical cord blood.
Use of Specific UCB Collection Bags
To simplify the harvesting procedure, we recently introduced a specific bag for UCB collection (Stemflex, Maco Pharma) with two lines for the retrieval of each blood fraction into the same container (Fig. 1D). This new bag allows us to proceed with the protocol without substantial modifications. The volume of blood collected into these bags (group III, n = 319) was 118.9 ± 39 ml, and the NC content was 1.46±0.58 x109NC. These data are similar to the results obtained with the standard blood donor bags (Table 4). Bacterial contamination in the new bags was 2.19%, a rate slightly lower than the one observed with the standard bags.
DISCUSSION
The higher hematopoietic potential of UCB units harvested and processed according to the methodology proposed in this study leads to an increase in the number of grafts with a 2 x 107/kg NC dose. Thus, 84% and 54% of our UCB units would fulfill this target dose in recipients weighing 50 and 65 kg compared with less than 30% units from other UCB banks. This significant advance procured by our novel UCB collection technique gives larger pediatric and many adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT.
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