Madrid Cord Blood Bank, b Department of Hematology, c Department of Immunology, Hospital Universitario 12 de Octubre, Madrid, Spain

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1 Original Article A Modified Cord Blood Collection Method Achieves Sufficient Cell Levels for Transplantation in Most Adult Patients Rafael Bornstein, a,b Ana I. Flores, a M. Angeles Montalbán, b Manuel J. del Rey, c Javier de la Serna, b Florinda Gilsanz a,b 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 Abstract Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about , and only 25% of these units reach the target cell dose of /kg in UCBT patients weighing kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 ± 50.1 ml and 1.26 ± NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2% 100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over NCs, resulting in a 36% cell increment (1.46 ± NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of /kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT. Stem Cells 2005;23: Introduction 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 [1 11]. As reported by NetCord, more than 2,500 unrelated umbilical cord blood transplants (UCBTs) have been performed, one-third in adult recipients [ de.netcord.org/index.html]. 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 [1 5, 8, 10]. 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 Correspondence: Rafael Bornstein M.D., Ph.D., Madrid Cord Blood Bank, Hospital 12 de Octubre, Avda. de Córdoba, s/n, Madrid 28041, Spain. Telephone: ; Fax: ; rbornstein.hdoc@salud.madrid.org Received March 2, 2004; accepted for publication November 9, AlphaMed Press /2005/$12.00/0 doi: /stemcells Stem Cells 2005;23:

2 Bornstein, Flores, Montalbán et al. 325 survival after UCBT [3, 4, 6, 7, 10, 11]. 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 [10, 12]. 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 [13]; 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 [14 16]. 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 /kg [17 19]. Above this figure, there is a level the optimal dose which is associated with a distinct survival advantage. Gluckman et al. [3] have shown that a graft NC dose > /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 /kg. Many transplant centers would now recommend /kg as a reasonable target cell dose to obtain satisfactory UCBT outcomes [8, 14, 17, 20 22]. 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 [23]. Using the standard collection technique, the mean number of NCs reported by the biggest UCB banks worldwide is about per unit [24 26], and with this cell content, only 25% of UCB units contain enough cells to fulfill the target dose for transplantation in patients weighing kg [27]. 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 /kg would be achieved in most larger pediatric patients and in a significant proportion of heavier adult patients requiring HCT. Materials and Methods Two-Fraction UCB Collection Umbilical cord blood was obtained from healthy term newborns after vaginal delivery at Hospital 12 de Octubre. A consent form approved by the Institutional Review Board was signed by all mothers whose medical and family history obtained prior to collection did not reveal any exclusion criteria for UCB donation. The blood was obtained using a two-fraction collection protocol. The first blood fraction was obtained with the placenta in utero, according to the procedure used in most of the UCB banks. Briefly, the cord was double clamped, transected, and cleaned with iodine and alcohol. A 16-gauge needle from the collection bag was inserted into the umbilical vein; blood was allowed to flow by gravity until the blood flow ceased (Fig. 1A). The second blood fraction was collected from the delivered placenta by flushing the placental vessels with 50 ml of 0.9% saline (Baxter Healthcare, Round Lake, IL; plus 5,000 units of preservative-free heparin (Rovi, Madrid, Spain; rovi.es). The placenta was placed over a sterile cloth, and the vessels around the cord insertion were cleaned as above. Two large-size vessels were cannulated, trying to select an artery and a vein whenever they could be distinguished, for the saline infusion and the blood collection, respectively (Fig. 1B). The saline bag was hung on a dripstand to allow the saline solution to enter the placental circulation, and the blood was recovered into the collection bag. The umbilical venipuncture and placental perfusion fractions are named the first and second UCB fractions, respectively. Figure 1. Umbilical cord blood collection by a two-fraction harvest procedure. First and second blood fractions were obtained by (A) umbilical venipuncture and (B) placental perfusion. Both fractions were collected (C) into separate standard blood donor bags and then pooled prior to processing or (D) directly into a single Stemflex bag. Units from groups I and II were collected using the standard blood donor bags (C), and units from group III were collected into Stemflex bags (D).

3 326 Increased UCB Retrieval by Placental Perfusion The Madrid Cord Blood Bank came into operation in From 1996 to 2003, standard blood donor bags (Grifols, Barcelona, Spain; containing 63 ml citrate phosphate dextrose (CPD) anticoagulant with adenine were used for the collection of UCB units (groups I and II, see below). The original volume of CPD-adenine was reduced to 23 ml. Two separate bags were used for the collection of the first (Fig. 1A) and second blood fractions (Fig. 1B), and the two fractions were pooled in a transfer bag (Fig. 1C). After July 2003 the first and second UCB fractions were collected using a single bag (Stemflex, Maco Pharma, Tourcoing, France; com) with two collection lines for the retrieval of each blood fraction into the same container (group III, Fig. 1D). These Sternflex bags contain 21 ml CPD. To recover all the blood, the collection lines were washed with 8 ml CPD from a satellite bag, making the total amount of anticoagulant in the collection bag 29 ml. From 1996 to 1998, units containing or more NCs were processed within 48 hours of collection (group I: 351 units). Since 1998 UCB processing was restricted to units over NCs (group II: 1,269 units; group III: 319 units). UCB Processing and Sampling The UCB volume was estimated by subtracting the tare weight of the bag and the volume of anticoagulant from the total weight of the blood-containing bag, assuming that 1 g of blood is equivalent to 1 ml. To count the total number of nucleated and progenitor cells, 2 ml of sample were removed from the blood-containing bags. Nucleated cell count was assessed with an automated hematology analyzer (A C T diff; Coulter, Miami, Florida, and the total number of cells was calculated by multiplication with the blood volume contained in the bag. The UCB was processed with hydroxyethyl starch, as described [28]. Bacterial contamination in the entire unit was determined on the sedimented red blood cells. Samples were removed for aerobic and anaerobic bacterial cultures in BacT/ Alert media (Biomérieux, Durham, NC; biomerieux.com). To determine the contribution of the second UCB fraction to the total volume and number of NCs, the first and second fractions of 44 units from group I were analyzed separately. Also, hematopoietic progenitor cells (HPCs) were assessed on each blood fraction from an additional subset of 10 units from group I (see below). Hematopoietic Progenitor Cell (HPC) Assays CD34 + cell enumeration was performed by flow cytometry using the ISHAGE gating strategy [29]. Cells were incubated with CD34 phycoerythrin (PE) and CD45 fluorescein isothiocyanate (FITC) antibodies (Becton, Dickinson, Erembodegem, Belgium; for 20 minutes. After washing, stained cells were analyzed on a FACSort flow cytometer using the CellQuest version 3.3 software (both from Becton, Dickinson). The expression of CD133 was assessed by a three-color assay with CD45 peridinin-chlorophyll-protein complex (PerCP), CD34 FITC, and CD133 PE (Myltenyi Biotec, Bergisch Gladbach, Germany; biotec.com). Prior to data acquisition, a CD34 + /CD45 dim gate was established for the analysis of CD133 + cells. A minimum of 500 events was acquired in list mode. Colony-forming cells [colony-forming units granulocytemacrophage (CFU-GM), blast-forming units erythrocyte (BFU-E)] were determined in 24-well culture dishes (Nunc, Roskilde, Denmark; Cells at a density of 50,000/ml were cultured in a medium containing 30% fetal bovine serum, 1% bovine serum albumin, 10 4 M 2-mercaptoethanol, 2 mm L-glutamine, 10% agar leukocyte conditioned medium, 3 U/ml erythropoietin, and 0.9% methyl cellulose in Iscove s modified Dulbecco s medium (IMDM; Methocult H4431, StemCell Technologies, Vancouver, BC, Canada; Triplicate 0.3-ml cultures were incubated at 37 C in 100% humidified 5% CO 2 in air for 14 days prior to scoring as CFU-GM and BFU-E colonies. Detection of Maternal DNA in UCB Samples Collected by the Two-Fraction Method The level of cord blood contamination by maternal cells was determined by locus-specific amplification of noninherited maternal HLA-DRB1 genes using the HLA Micro SSP kit (One Lambda, Canoga Park, CA; com). Twenty UCB units from group II were selected for the analysis. Briefly, genomic DNA from cord and maternal blood samples was isolated by a DNA purification system (Gentra, Minneapolis, MN; and the DRB1 alleles were determined by Reverse Dot Blot (INNO-LiPA; Innogenetics, Gent, Belgium; Sequence-specific primer-polymerase chain reaction (PCR) amplification of the noninherited maternal DRB1 allele was performed on cord blood samples as recommended by the manufacturer. A pair of β-globin primers was used as loading control. The PCR products were run on 2% agarose gels and stained with ethidium bromide (0.5 μg/ml). The sensitivity of the Micro SSP technique was performed by diluting DRB1*04 + and DRB1*07 + blood samples into a negative blood sample at different proportions. Mononucleated cells from positive samples were diluted at 10%, 5%, 2%, 1%, and 0.5%; genomic DNA was then extracted, and DRB1*04 or DRB1*07 genes were amplified. Specific bands were clearly visible in all samples except for the 0.5% dilution. Thus, the limit of sensitivity of the DRB1 detection using SSP- PCR and electrophoresis gels was 1% (data not shown).

4 Bornstein, Flores, Montalbán et al. 327 Statistical Analysis For the comparative analysis of volume, NC and HPC content in first and second UCB fractions, the paired-samples T test was used. Similar comparisons between different units were done with the nonparametric Mann-Whitney U test. The Pearson s correlation coefficient was used to estimate a correlation between volume and NCs and between NCs and CD34 + cell content. A two-sided p <.05 was considered to be significant. Calculations were performed with SPSS 7.5 for Windows (SPSS Inc, Chicago, IL) statistical software package. Results 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 ml (range, ml) and a NC content of 1.21 ± (group I, Table 1). As Figure 2. Correlation of umbilical cord blood volume and nucleated cells: Pearson s correlation coefficient, r =.63 (n = 300). compared to data reported by other banks [24 26, 30 35], where the average volume of UCB units is ml and the cell content ranges from 0.85 to , our results suggest that the additional blood collected after placental perfusion may increase the total number of NCs. 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. Several reports have suggested a /kg NC as the target dose for transplantation. For adult patients weighing 50 and 65 kg it will be necessary to infuse and 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 and NC. Therefore, the two-fraction collection technique described here increases by 20% and 11% the number of UCB units clinically useful for adult patients. 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 Table 1. Volume, nucleated cell content, and mononucleated cell content of umbilical cord blood units Nucleated cell counts Mononucleated cell counts Volume a Relative Absolute Percentage Relative Absolute (ml) ( 10 9 /L) ( 10 9 ) ( 10 9 /L) ( 10 9 ) Mean SD b Range Data obtained from the analysis of the first 351 UCB units (group I) harvested at Madrid UCB Bank. a Umbilical cord blood net volume (total volume anticoagulant). b Standard deviation.

5 328 Increased UCB Retrieval by Placental Perfusion Table 2. Volume and number of NCs in UCB collected by intrautero umbilical venipuncture (fraction 1) and placental perfusion after delivery (fraction 2) Fraction 1 Fraction 2 Whole UCB units Fraction 2/UCB units (%) Volume (ml) 81.7 ± 35.2* 40.9 ± ± 50.1* 32.2 ± 17.3 NC ( 10 9 ) 1.07 ± 0.45* 0.19 ± ± 0.52* 15.5 ± 9.8 Results (mean ± SD) show volume and NCs of first and second fractions analyzed separately and the same parameters in the whole UCB unit after combining both fractions (n = 44). The last column shows the proportional volume and NC content that the second fraction provided for the whole unit. *Significantly different (p <.001, paired-samples T test). Abbreviation: UCB, umbilical cord blood. 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 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. after placental perfusion (second fraction) have similar HPC contents and in vitro hematopoietic potential. Increase in Cell Content of the UCB Bank Inventory as a Result of a Processing Restricted to Units NC Since 1998, we have only processed UCB units with an NC count (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 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 ± , whereas in group II it is 1.46 ± (p <.001). As a result, the percentage of units with a cell count and in group II were 84% and 54% compared with 62% and 40% of group I, respectively (Table 4). 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 [30, 31, 36, 37], suggesting that the second fraction collection does not increase the risk of bacterial contamination. Table 3. Frequency of UCB units with > or > NCs UCB collection fraction NCs NCs Venipuncture Venipuncture + placental perfusion The data show the percentage of UCB units (n = 44) in which the NCs in the first fraction (venipuncture) and in the whole UCB unit once the first and second fractions were pooled (venipuncture + placental perfusion) are equal to or higher than and , respectively. Abbreviations: NC, nucleated cell; UCB, umbilical cord blood.

6 Bornstein, Flores, Montalbán et al. 329 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 β-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 [38, 39]. 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 ± 39 ml, 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 ± and ± , respectively, whereas BFU-E numbers in each of these fractions were ± and ± (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. Table 4. NC content in UCB units stored prior to or since the use of the NC processing restriction No. of units (%) NC ( 10 9 ) NCs NCs Units pnr (group I) 1.21 ± 0.51* 219 (62) 140 (40) Units pr (group II) 1.46 ± 0.52* 1066 (84) 692 (54) Results (mean ± SD) show the NCs of the first 351 UCB units (group I) processed before setting the cell size restriction at NCs for UCB unit acceptance into the bank (pnr = processing not restricted), and the same parameters in the following 1,269 UCB units (group II) processed when the NC threshold was already operative (pr = processing restricted). Columns two and three show the number (percentage in parentheses) of UCB units with an NC content equal to or higher than the indicated values. *Significantly different (p <.001, Mann-Whitney U test). Abbreviations: NC, nucleated cell; UCB, umbilical cord blood.

7 330 Increased UCB Retrieval by Placental Perfusion and the NC content was 1.46 ± NC. 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 Many reports have documented the feasibility and efficacy of mismatched unrelated UCBT in pediatric patients [3, 6, 7, 11, 36, 40]. Since unrelated donor bone marrow transplantation in adults is associated with a high risk of graftversus-host disease (GVHD) and treatment failure [41 44], UCBT may also have an advantage in adult recipients due to potentially decreased risk of GVHD. There have been more than 1,000 transplants done in adults, with long-term disease-free survival particularly seen in younger patients with earlier stages of disease [8, 14 16, 20, 22]. However, as cell dose is associated with survival, this considerably limits the pool of eligible UCB grafts for adult patients. Several strategies to increase the nucleated/cd34 + cell dose are being investigated, including ex vivo expansion of UCB hematopoietic cells [45 47] and multiunit UCB transplant [48]. There are other potential approaches, such as UCBT combined with infusion of mesenchymal stem cells or haploidentical HSCs [49] and UCBT after a nonmyeloablative preparative regimen [50, 51]. Although initial results are encouraging, further studies are required to show a clinical benefit associated with any of these approaches. In the meantime, the importance of cell dose for transplantation outcomes provides the most compelling argument for focusing on the collection of UCB grafts with greater number of cells than units obtained by current protocols. Based on the harvest technique proposed by Turner et al. [52], we designed a two-phase collection method in which a blood fraction obtained by umbilical venipuncture, similar to the procedure used in most banks, is followed immediately upon placental delivery by a 0.9% saline perfusion. This method allows retrieving additional blood from placental vessels, which results in an increase in the total number of NCs with no extra risk of bacterial contamination. Because the two-fraction collection method provides more NCs per unit, we also introduced a processing restriction that should 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. β-globin amplification was used as a loading control (arrowheads). Abbreviations: PCR, polymerase chain reaction; UCB, umbilical cord blood. Figure 7. Increase in the nucleated cell content in the umbilical cord blood (UCB) units stored at the Madrid UCB Bank due to the two-fraction collection method and the processing restriction to units with a nucleated cell (NC) count For this comparison the cell content of the first fraction was considered as 100% (V [pnr]; n = 44). The second fraction collection produces a 13% increase in the total number of NCs (V + PP [pnr]; n = 351). Besides the second fraction, the restriction in the processing of units with an NC count produces an additional 20% increase in the total number of NCs per unit (V + PP [pr]; n = 1,269). The global increase achieved by the sum of the twofraction collection method and the processing restriction is 36%. Abbreviations: pnr, processing not restricted by cell content; PP, placental perfusion (fraction 2); pr, processing restricted to units NCs; V, venipuncture (fraction 1).

8 Bornstein, Flores, Montalbán et al. 331 enrich the cell content of the banked UCB units. In fact, the optimized collection method described here, together with the NC processing limit, have resulted in a 36% increase in the NC content compared with the standard collection method (Fig. 7). The improvement in the NC content would result in an increment in the number of units clinically useful for adult patients. Indeed, 84% and 54% of our UCB units (compared with less than 30% units from other UCB banks) would fulfill the target dose of NC for adult patients weighing 50 and 65 kg. Beside the improvement in the total cell content, this method provides around 15% increase in the number of committed HPC (CD34 + cells, CFU-GM and BFU-E) per unit. It is critical for the transplant centers to know if there is a correlation between CD34+ cells (or clonogenic progenitor cells) and NCs because the number of infused HPCs is more predictive of the time to neutrophil recovery and survival after UCBT than the total NCs [10, 53]. In the units stored in our bank we have found a linear correlation between CD34 + cells and NCs (r =.67, p =.01; data not shown). In addition, there is an increase in the number of CD133 + cells, a progenitor cell population that seems to be more enriched in pluripotent quiescent HSCs than CD34 + cells [54]. As a more primitive cell subset, functionally closer to the human long-term repopulating stem cell in vivo [55, 56], the higher CD133 + cell number may increase the hematopoietic reconstituting potential of our UCB units. Indeed, the preliminary results on eight patients transplanted with UCB units from our bank suggest that the two-fraction collection method may improve the clinical outcomes after UCBT. Four children and four adults were transplanted between September 2001 and February The children were 1 10 years of age (mean body weight, 19 kg), and the adults were years of age (mean body weight, 61 kg). Total NCs infused were /kg (range ) for children and /kg (range ) for adult patients. The number of CD34 + cells infused was 2.15 ( ) 10 5 /kg in children and 1.14 ( ) 10 5 /kg in adult patients. One adult patient died at day +14 from multiple organ failure. Neutrophil engraftment of /1 was observed in the other seven patients with an average time of 23 days (range, 10 47). Five patients (three adults and two children) are alive with a median clinical follow-up of 15 months (range, 3 24 months). The overall survival at 2 years is 56% ± 20%. These preliminary results compare favorably to the overall survival rate (~ 45%) described with UCB units collected by the standard method [3, 4, 6 10]. The second fraction collected by placental perfusion may give rise to higher maternal cell contamination of UCB units that could cause life-threatening GVHD. In our study, three out of seven patients developed grades II IV acute GVHD (43%). One of them had grade IV acute GVHD (14%). These incidences are similar to those previously reported in patients transplanted with UCB collected by standard methods [3, 4, 6, 10], suggesting that the two-fraction collection method does not increase the risk of GVHD. We also determined the level of cord blood contamination by maternal cells in 20 UCB units stored at our bank. Locus-specific amplification of noninherited maternal HLA-DR genes was performed using polymerase chain reaction amplification followed by gel electrophoresis [57]. With a sensitivity limit of 1%, we did not detect maternal DNA in any of the 20 samples analyzed. Other studies using different techniques with a similar sensitivity have detected maternal cells in 0% 2% UCB units [38, 39]. Therefore, although we cannot rule out maternal cell contamination below 1%, it seems that the two-fraction collection method does not increase the frequency of cord blood contamination by maternal cells. The majority of UCB units stored in our bank were collected using a standard blood donor bag for each fraction, but the recent introduction of specific bags for UCB collection has greatly simplified the procedure. First, the volume of anticoagulant does not have to be reduced in advance. Second, both fractions are combined in the same container, thereby preventing possible errors in pooling fractions from different donors during subsequent processing. These advantages do not compromise the efficacy and higher UCB yields procured by the collection method described here. The two-fraction collection method and the processing restriction led to a NC content of 1.46 ± per unit. This result is 39% 75% higher than UCB units collected by standard methods at several operative banks [24 26, 30 35, 58]. Other studies have tried to identify the optimal UCB collection techniques assessing the influence of mode of delivery (vaginal vs. cesarean) and harvest timing (before vs. after placental delivery) [31, 59 63]. Despite improved cell recovery claimed for certain procedures, the NC yields did not exceed the numbers reported by UCB banks [24 26, 30 35, 58]. One study successfully attempted to harvest larger units using a syringe-assisted flush and drain technique followed by umbilical arterial cathetherization and placental perfusion [58]. However, the associated bacterial contamination rate (19%) would prevent the use of this technique as a UCB harvest protocol for banking purposes. Of note, the volume and number of NCs in this study (174 ml and ) were higher than ours, probably due to the increased amount of saline infused into the placenta (100 ml). Thus, it would be possible to retrieve even more blood just by flushing the placenta with larger saline perfusions. We will address this issue in a further cohort of UCB collections.

9 332 Increased UCB Retrieval by Placental Perfusion Conclusion 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 /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 This work was supported by grant nos. FIS 02/1877 from the Ministry of Health in Spain and 08.3/0037/2001 from the Education Department of the Autonomic Government of Madrid. We are grateful for the collaboration of the Department of Obstetrics of the Hospital 12 de Octubre, where all umbilical blood collections were performed. 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