Cord blood stem cells Comparison between two strategies for umbilical cord blood collection

Transcription

1 (2003) 31, & 2003 Nature Publishing Group All rights reserved /03 $ Cord blood stem cells Comparison between two strategies for umbilical cord blood collection P Solves 1, R Moraga 2, E Saucedo 2, A Perales 3, MA Soler 1, L Larrea 1, V Mirabet 1, D Planelles 1, F Carbonell-Uberos 1, J Monleo n 2, T Planells 1, M Guille n 1, A Andrés 1 and E Franco 1 1 Valencia Cord Blood Bank, Processing and Cryopreservation Service, Valencia Transfusion Centre, Valencia, Spain; 2 La Fe University Hospital, Valencia, Spain; and 3 Dr Peset Hospital, Valencia, Spain Summary: The use of cord blood (CB) for transplantation has increased greatly in recent years. The collection strategy is the first step in collecting good-quality CB units. There are two main techniques for collecting CB from the umbilical vein: in the delivery room while the placenta is still in the uterus by midwives and obstetricians or in an adjacent room after placental delivery by CB bank trained personnel. In this study, the benefits and disadvantages between the two different CB collection strategies were evaluated, in order to improve CB bank methodology. Valencia CB bank maintains the two different collection strategies. CB was obtained from 569 vaginal and 70 caesarean deliveries and obstetrical and clinical charts were reviewed. Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation samples were drawn for cell counts, CD34+cell analysis, viability, clonogenic assays and microbiology were drawn directly from the bags. We compared the efficiency of the two collection techniques. Obstetric data and umbilical CB were obtained from 569 vaginal (264 collected in utero and 305 collected ex utero) and 70 caesarean deliveries. The proportion of excluded CB units before processing was 33% for vaginal ex utero, 25% for vaginal in utero and 46% for caesarean deliveries. Differences were statistically significant. For vaginal deliveries a larger volume and a higher number of nucleated cells, percentage of CD34+ cells and colony-forming units (CFUs) were harvested in the in utero collection group. There was no statistical difference between CB collected after placental expulsion from vaginal and caesarean deliveries. Comparison between all vaginal and caesarean deliveries did not show any difference. We conclude that the mode of collection influences the haematopoietic content of CB donations. Collection before placental delivery is the best approach to CB collection and allows optimisation of CB bank methodology. Caesarean deliveries seem to contain similar progenitor content to vaginal deliveries. Correspondence: Dr P Solves, Valencia Transfusion Centre, Avda del Cid 65-A, Valencia, Spain Received 23 May 2002; accepted 02 October 2002 (2003) 31, doi: /sj.bmt Keywords: cord blood; obstetric factors; cord blood collection Cord blood (CB) is recognised as a rich source of haematopoietic stem and progenitor cells and its use has increased greatly in recent years. Since the first CB transplantation was performed in Paris in 1988, knowledge of the biologic characteristics of umbilical CB has improved, and the benefits of using CB stem cells have become apparent. 1 In fact, CB has been shown to have several advantages as compared to bone marrow: easy availability, lower risk of infectious disease transmission such as CMV and EBV and lower risk of graft-versus-host disease. However, the progenitor content of CB units is usually only sufficient for small recipients, usually children weighing less than 40 kg. Gluckman et al 2 showed grafting occurring in 85% of patients receiving 37 million or more nucleated cells per kilogram of body weight 2. In the last few years, use of CB in larger recipients is increasing and an effort to improve CB quality is mandatory. The collection strategy is the first step for collecting good-quality CB units and varies among banks and among collection sites for the same CB bank. 3 In this sense, different collection methods have been proposed to optimise volume and total nucleated cells content of CB units. There are two main techniques for collecting CB from the umbilical vein: in the delivery room while the placenta is still in the uterus by midwives and obstetricians or in an adjacent room after placental delivery by CB bank trained personnel. Both methods have some advantages and some disadvantages. The advantage of the first method is that the volume of cells collected is usually higher if the cord is clamped early and collection is started immediately. However, this can disrupt the normal process of delivery. After delivery, CB collection is easier and it can be performed by trained personnel. However, fewer cells may be collected and there may be an increase in the risk of bacterial contamination. Feasibility of obstetricianbased collection has been shown by many CB banks. 4 On the other hand, open systems, which were initially used for CB collection, have been replaced by closed systems with blood collection bags to minimise the risk of microbiological contamination. 5

2 270 In this study, the benefits and disadvantages between two different CB collection strategies were evaluated, in order to improve CB bank methodology. Material and methods Collection strategies Valencia CB bank maintains two different collection strategies: before placental delivery by Maternal Hospital staff and after placental expulsion by CB bank trained personnel. Before placental delivery (in utero) collection Donors must sign informed consent before delivery, during the last months of pregnancy. After delivery, the umbilical cord is clamped immediately and cleaned with 70% alcohol and an iodine swab. Umbilical CB is collected from the umbilical vein by gravity in a closed sterile collection 350 ml double bag (R 1315, Baxter) containing 23 ml CPDA anticoagulant. Previously, 26 ml of CPDA has been removed from the collection bag. This process is performed by trained midwives or the obstetric staffs. CB collection is stored at 41C until processing. After placental delivery (ex utero) collection After informed consent is obtained from the mother, CB collection is performed by trained umbilical CB bank personnel. Once the placenta is delivered, it is transported quickly to the collection area, which contains necessary supplies and equipment. There is no time limit set between placental delivery and the CB collection. The placenta is placed on a specially designed tray with a central hole that allows the umbilical cord to hang down. After stringently cleaning the umbilical cord with 70% alcohol and an iodine swab, CB is collected from the umbilical vein by gravity in a sterile collection 350 ml double bag (R 1315, Baxter) containing 23 ml CPDA anticoagulant. CB collection is stored at 41C until processing. All caesareans were processed in this way. Obstetric data For both methods, obstetric and clinical maternal chart was reviewed by CB bank medical staff. Delivery information (route of delivery, gestation length, gestation number, placental weight, amniotic fluid characteristics and newborn characteristics), sex and weight were reviewed. In all cases, placental weight was calculated by adding the CB collection weight to the after collection placental weight. Red cell depletion and cryopreservation Within 48 h after collection, CB is processed. More than total nucleated cells (TNC) were required to accept CB units for the bank. Volume reduction is performed by the method developed by Rubinstein et al. 6 Briefly, a 6% hydroxy-ethyl-starch (HES, Grifols) solution is mixed with the umbilical CB in the ratio of one volume of HES to five volumes of blood in the collection bag (1.2% HES final concentration). Then, the bag is centrifugated at 45 g for 7 min at 101C (Varifuge 3.ORS, Heraeus Instruments). The removed supernatant leucocyte-rich plasma and 12 g of red blood cells are centrifuged again at 600 g for 15 min at 101C. After discarding the supernatant, CB is separated into two 50 ml cryopreservation bags (Cryocyte R4R9951, Nexwell) containing 20 ml each. Cryopreservation of CB is performed by adding 50% dimethylsulphoxide (DMSO) solution with dextran (Rheomacrodex, Ibyss) for 15 min to reach a 10% DMSO final concentration. The cryocyte bags are placed in aluminium cassettes for immediate freezing. Biological studies Before processing CB units, volume was calculated and samples were drawn for cell counts. After processing and before cryopreservation, samples were drawn for cell counts, CD34 analysis, viability, HLA typing, clonogenic assays and microbiology directly from the bags. Cells counts were made with an autoanalyser Sysmex K800 (Toa Medical Electronics, Kobe, Japan). TNC was calculated. CD34+ cells were quantified by flow cytometry. FACScalibur cytometer was used with Procount software (Becton Dickinson). Cell viability was assessed by ethidium bromide and acridine orange. Clonogenic assays were performed using a commercially prepared complete methylcellulose medium (Methocult GF H4434), supporting growth of CFU-GM, BFU-E and CFU-GEMM. Cultures were plated at viable cells in duplicate 35 mm diameter Petri dishes and incubated for 14 days at 371C with 5% CO 2 in a humidified atmosphere. Colonies defined as aggregates of more than 40 cells were counted under an inverted microscope. Sterility controls were performed using an automated blood culture system (BacT/Alert: Organon Teknica) at 351C for 14 days. Statistical analysis Groups were compared regarding maternal, neonatal and CB units parameters. Statistical Package for Social Sciences (SPSS) v. 10 was used to perform the statistical analysis. The groups were compared by the w 2 test or the Fisher test for categorical variables, the Kruskal Wallis and Mann Whitney U-test for continuous variables. A Po0.05 was considered to be significant. The correlations between the TNC, CD34+ cells and CFUs of CB collected were analysed by means of Pearson s correlation coefficient. Results Obstetric data and umbilical CB were obtained from 569 vaginal and 70 caesarean deliveries. In Table 1, we summarise the obstetric data according to collection by vaginal and caesarean deliveries. There was no statistical difference between the groups for obstetric variables except for the infant weight that was greater for group 3 (Po0.05). The proportion of CB units excluded before processing was 33% for vaginal ex utero, 25% for vaginal in utero and 46% for caesarean deliveries. Differences were statistically

3 Table 1 Obstetric characteristics of deliveries according to the umbilical CB harvested before or after placental delivery and route of delivery 271 Route of delivery Vaginal deliveries Caesarean Mode of collection Group 1 In utero (n=264) Group 2 Ex utero (n=305) Group 3 Ex utero (n=70) Maternal age Gestational age (weeks) Gestation number Infant weight (g) Placental weight (g) Newborn sex Male Female Amniotic fluid Normal Meconium stained Continuous variables are expressed as mean and s.d. There were no statistical differences among groups except for the variable infant weight that was greater for group 3 (Po0.05). Table 2 Umbilical CB units data of vaginal and caesarean deliveries Route of delivery Vaginal deliveries Caesarean Mode of collection Group 1 In utero (n=264) Group 2 Ex utero (n=309) Group 3 Ex utero (n=70) Volume (ml) WBC 10 6 /ml TNC a p TNC TNC recovery (%) CD34+cells% CD34+cells CFUs b Viability (%) a Total nucleated cells. b Colony-forming units. Volume and ptnc were calculated from CB units before processing. The other variables were calculated after volume reduction of CB units. There was a statistical difference for all variables except for total CD34+ cells (P: 0.057) and viability between groups 1 and 2. There was no statistical difference between groups 2 and 3. significant. The main reasons for discarding these units were low volume and low total cell count. Microbiological studies were positive for three cases in before placental delivery collections and for 19 cases in after placental delivery collection (Po0.05). In ex utero collections, clots were present in five cases that were not discarded (Po0.05). CB units data are shown in Table 2. In utero vs ex utero CB collections from vaginal deliveries. A larger volume and a higher number of nucleated cells (before and after volume reduction), CD34+ cells, percentages and CFUs were harvested in the in utero collection group. There was also a trend for a higher number of absolute CD34+ cells, although the difference did not reach statistical significance (P ¼ 0.06). The WBC concentration was similar for both groups. The difference also was significant for TNC recovery after volume reduction (Po0.05). Ex utero CB collection from vaginal deliveries vs caesareans. There was no statistical difference between CB collected after placental expulsion from vaginal and caesarean deliveries. In utero CB collections from vaginal deliveries vs caesareans. A higher number of nucleated cells (before and after volume reduction) and percentage of CD34+ cells percentages were harvested in the in utero collection group (Po0.05). A better TNC recovery after volume reduction was also observed (Po0.05). There was also a trend for a larger volume (P ¼ 0.074). All vaginal deliveries versus caesareans. Comparison between all vaginal deliveries and caesarean did not show any differences. Volume and TNC correlate with haematopoietic content. For vaginal deliveries, a significant correlation was found between TNC and CD34 + total cells (r: 0.693; Po0.01), between TNC and total CFUs (r: 0.537; Po0.01), and between CD34+cells and CFUs (r: 0.641; Po0.01). These correlations were maintained for caesarean deliveries. Discussion Since umbilical CB banking requires important financial investment and organisational effort, banking efficiency

4 272 should be optimised. Many CB banks have a minimum volume limit as the first selection criterion for CB donations in order to store clinically useful CB units. Some banks have reported more than one-half of CB collections are discarded for banking, mainly because of their low volume and/or cell counts. 7 CB banks must develop strategies to increase the TNC content of stored units. CB collection is the first step in this. In this sense, it is of major concern to know which collection strategy allows the bestquality CB units. The impact of obstetric factors on quality of CB units has been investigated by some authors. Donaldson et al 8 concluded that significant factors affecting volume and total nucleated cells count were length of gestation, time from delivery of the infant to cord clamping and total length of labour. Although these obstetric variables have not been analysed, in our maternity ward, cord clamping is always performed immediately after delivery. Other authors have shown that babies of longer gestational age had higher cell counts, but lower CD34+ cell counts and CFU-GM. Bigger babies had higher cell counts, more CD34+ cells and more CFU-GM. 9 Longer duration stress during delivery increases the number of nucleated cells, granulocytes, CD34+ cells and haematopoietic progenitors. 10 In our study, no differences in maternal or neonatal characteristics were found between groups. Obstetric factors are beyond the control of the CB collector, but collection mode can be controlled. At present, there is no international consensus on the procedure of UCB collection in the maternity ward. 11 Surbek et al 12 compared both strategies in vaginal deliveries and concluded that in utero collection yielded a significant higher volume and total number of mononuclear cells. 12 Results from a recent study demonstrated that the median concentrations of nucleated cells and total CFUs were significantly lower in CB collected after placental delivery when compared to their counterparts collected before placental delivery. The incidence of macroscopic clots was also higher in ex utero collections. The reduction of stem and progenitor cells is possibly because of clotting activities developed with time. 13 Our results are in agreement with those presented by Surbeck, and show also higher numbers of total nucleated cells and CFUs for in utero CB collections. The presence of haemorrhage in the maternal and fetal areas of delivered placenta and the clots formed in fetal placental vessels could explain the loss of haematopoietic progenitor cells in ex utero collections. In our cases, total nucleated cells correlate with both CFUs and the number of CD34+ cells, indicating the TNC reflects haematopoietic content of CB units as shown by Lim et al. 14 Besides, a smaller number of CB units was discarded for banking compared to ex utero vaginal and caesarean deliveries, and the only five cases with macroscopic clots belonged to the ex utero collection group. Time from cord clamping to collection also could contribute to differences between in utero and ex utero methods; while in the first case collection is performed immediately after CB clamping, in the second case it is necessary to wait until placental expulsion. Other authors have confirmed a larger CB volume and more total nucleated cells were obtained in the samples collected before placental expulsion for caesarean deliveries. 15 However, our conclusions differ from those recently reported by Sparrow et al who did not find differences between the two ways of collection for vaginal deliveries, or between vaginal and caesarean deliveries, although there was a trend towards a higher cell concentration and collected volume for in utero collections 16. The limited number of cases (99 ex utero vs 58 in utero) could account for a lack of statistical power. Yamada et al 17 compared 29 caesarean deliveries with 126 vaginal deliveries and found that umbilical CB volume and CD34+ cells following caesarean sections and collected after placenta expulsion were greater, although the gestational age and the weight of the newborn were lower. 17 The difference was thought to be because of the position of the umbilical cord and the infant prior to clamping. Placing the newborn on the maternal abdomen after delivery increases the volume and CD34+ cell content in the umbilical CB. 18 In our study, obstetric practice was not modified in any case of CB collection. We performed all CB collections with a closed bag system. Open systems have mostly been discarded because they result in excessive microbiological contamination, as shown by some authors. 5 Our results show microbiological contaminations were higher for ex utero collections. This could be explained by manipulation of placenta after delivery as compared to in utero collections. For both groups, neither a second puncture nor other additional collection procedure was used. However, some authors have tried different systems in order to maximise the yield of haematopoietic cells in UCB units. The syringeassisted sodium chloride solution flush collection method with a blood bag has been found to be most effective, although it resulted in high bacterial contamination rate and a lower CD34+ cell percentage. Also, the potential risk of maternal cell contamination could be increased. 5 Based on our findings, we conclude that the mode of collection influences the haematopoietic content of CB donations. Collection before placental delivery is the best approach for CB collection to allow optimisations of CB bank methodology. These results show the feasibility of an obstetrician-based collection network that has other advantages. The close relation that the delivering physician has with the prospective donors facilitates the process of obtaining quality consent for the banking. Obstetricians can easily decide not to collect the CB if there are some maternal/infant problems. This strategy avoids the financial investment that generates the presence of CB banking personnel in the maternity ward. Taking into account our results, caesarean deliveries seem to contain similar progenitor content to vaginal deliveries. References 1 Gluckman E, Broxmeyer HE, Auerbach AD et al. Haematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989; 321:

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