Automated separation of cord blood units in top and bottom bags using the Compomat G4

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1 Clin. Lab. Haem. 2006, 28, P. SOLVES, V. MIRABET, F. CARBONELL- UBEROS, M. A. SOLER, R. ROIG Summary Keywords Automated separation of cord blood units in top and bottom bags using the Compomat G4 Umbilical Cord Blood Bank, Valencia Transfusion Centre, Valencia, Spain Cord blood (CB) has become a real alternative source of haematopoietic stem cells for bone marrow reconstitution in a variety of malignant disorders. As a response to this increasing activity, CB banks have been developed to guarantee the quality of processed CB units. Volume reduction of CB units maximizes storage space and also has other advantages. The aim of this study was to develop a program for the volume reduction of CB in the Compomat G4 device. We also compared two different top and bottom systems for CB fractionation (Compomat G4 and Optipress II). We empirically designed three different programs for volume reduction of CB with Compomat G4: two for final BC volume of 41 ml (CB1 and CB2) and the other one for buffy coat (BC) volume of 25 ml (CB3). Significantly worse recoveries were achieved for CB processed with program CB3. A RBC depletion of 50%, 60% and 70% were achieved for 67%, 39% and 9% of all units respectively. When comparing Compomat G4 and Optipress II, total nucleated cell recovery was similar for both methods, while lymphocytes recovery was significantly better for Optipress II. Cord blood, volume reduction, top and bottom doi: /j x 202 Introduction Since 1988 when human umbilical cord blood (CB)- derived stem cells were transplanted for the first time as an alternative to marrow or circulating blood-derived progenitor cells (Gluckman et al., 1989), umbilical CB transplantation has been performed in more than 4000 patients worldwide (Ballen, 2005). In fact, CB has become a real alternative source of haematopoietic stem cells for bone marrow reconstitution in a variety of genetic, haematological and oncological disorders. As a response to this increasing activity, CB banks have been developed to guarantee the quality of processed CB units (Stanworth et al., 2001). One of the major problems with long-term CB banking is the required storage space. Red blood cell (RBC) depletion of CB not only maximizes storage space, but also has other advantages like the reduction of the potential side-effects of dimethylsulfoxide (DMSO) and the reduction Received 14 November 2005; accepted for publication 6 March 2006 Correspondence: Pilar Solves, Valencia Transfusion Centre, Avda del Cid 65-A, Valencia, Spain. Tel.: ; Fax: ; of side-effects of ABO-mismatched or haemolysed red blood cells in the unfractionated infusates (Oliveira et al., 2004). Various methods have been employed for the CB volume reduction purpose. To date, most CB banks have used the method developed by the New York Blood Centre consisting of hydroxyethylstarch (HES) sedimentation of RBC (Rubinstein et al., 1994; Adorno et al., 1998). To standardize the process and improve cell recovery, other approaches with automated or semi-automated systems have also been developed (Armitage et al., 1999; Zingsem et al., 2003). Our CB bank performs the volume reduction of CB by top and bottom methodology with the Optipress II machine. It is a semi-automated system that allows good cell recoveries. However, the transfusion centre with which our bank is integrated decided to change to Compomat G4 fractionation machines, so we had to do the same. The Compomat G4 is a fully automated machine for the separation of centrifuged whole blood into components in transfusion centres. The Compomat G4 has been developed to process double, triple and quadruple blood bags systems (Loos, 1986). The principle is that two electronically directed presses can variably express blood Journal compilation Ó 2006 Blackwell Publishing Ltd

2 P. Solves et al. 203 components in the upper and lower part of the blood bag. The aim of this study was to develop a program for the separation of centrifuged CB into components, reducing the final volume of CB for cryopreservation and getting good cell recoveries. We also compared two different top and bottom techniques (Compomat G4 and Optipress II). Material and methods Cord blood collection The obstetrical team evaluated maternal and neonatal pairs during the prepartum period in the maternity wards collaborating with the CB program. Donors signed informed consent before delivery, during the last months of pregnancy. For collection, the umbilical cord was clamped immediately after baby delivery and cleaned with 70% alcohol and an iodine swab. CB was collected from the umbilical vein by gravity in a triple bag system (R MQT 2205PU; Maco Pharma, Tourcoing Cedex, France) containing 21 ml of ACD. CB collections were stored at 4 C until processing, always performed before 48 hours from collection. Only CB units containing total nucleated cell (TNC) count were volume reduced and finally banked. Volume reduction of CB with the Compomat G4 using various programs The CB units collected in triple bag system were centrifuged in oval buckets at 3000 g for 12 min at 22 C, ensuring that the bags were well supported to prevent disruption of the buffy coat (BC) layer. CB collections were separated into plasma, red blood cells concentrate (RBCC) and BC containing haematopoietic progenitors. On the basis of the parameters for blood fractionation, program CB1 was empirically developed to reach a BC volume of 41 ml. Taking into account the preliminary results, program CB2 was developed for separation of CB collections with higher volume ( 100 ml) to reach a BC volume of 41 ml. Program CB3 was developed to standardize the BC volume to 25 ml. In this program, the upper press is extended to 58 mm, creating a very small upper compartment. The plasma/cell interface is pushed upwards until detector A6 is reached, resulting in a small BC volume. All steps of these programs are detailed in Table 1. Volume reduction of CB with Optipress II The CB units were centrifuged in the same conditions as those described before for Compomat G4. A standard Table 1. Summary of the steps used in various programs for separation of cord blood units, using the Compomat G4 protocol programmed into the Optipress II (Baxter Healthcare, Deerfield, IL, USA), together with the standard backplate for BC preparation was used to process the CB units. The program was set with the following parameters: BC volume of 40 ml, a BC level of 5.5 and a force of 25. Cell counts Nucleated cell counts were performed with an haematology analyser micros 60 (ABX diagnostics, Montpellier Cedex, France) and TNCs were calculated. The counts were not corrected for nucleated cells. CD34 assay CD34 + cells were quantified by flow cytometry. CB ( cells) was incubated using monoclonal antibodies-conjugated CD45 fluorescein and CD34 phycoerythrin (Becton Dickinson, San Jose, CA, USA) and 7-amino actomicin D as the marker of DNA staining. Flow cytometric analysis was performed using Cell Quest software (Becton Dickinson) ProCount progenitor cell enumeration kit was used in comparison with our standard protocol, giving similar results (Sutherland et al., 1996). Clonogenic assays Program CB1 Program CB2 Program CB3 Check head 1,2,3,6 1,2,3,6 1,2,3,6 Open head 1,2,3,6 1,2,3,6 1,2,3,6 Both presses out DET A1 + B A1 + B A1 + B Upper press to mm Close head 1,6 1,6 1,6 Speed % Open head Speed % Lower press out DET A3 + B A3 + B A6 + B Close head 1,2,3 1,2,3 1,2,3 Slide out Open head Lower press to mm Seal head Wait for start Balance B B B End of program Clonogenic assays were performed using a commercially prepared complete methylcellulose medium Methocult GF H4434 (Stem Cell Technologies Inc, Vancouver, BC,

3 204 Automated CB units separation using the Compomat G4 Canada), containing recombinant cytokines and supporting growth of burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte, macrophage (CFU-GM) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). For development study, cultures were plated at nucleated cells/ml. Samples for routine clonogenic assays were drawn from a thawed cryovial, taking a volume previously calculated according to the total nucleated and CD34 cells content of CB units before cryopreservation. Cultures were plated in duplicate 35-mm diameter Petri dishes and incubated for 14 days at 37 C with 5% CO 2 in a humidified atmosphere. Colonies defined as aggregates of more than 40 cells were counted under an inverted microscope. Colony forming units (CFU) were calculated by the sum of BFU-E, CFU-GM and CFU-GEMM. Viability Ethidium bromide and acridine orange were used to assess cell viability. The nonviable cells stain deep orange. Viability was performed after volume reduction and before cryopreservation. Statistical analysis Data were processed using the Statistical Package for Social Sciences (SPSS 10.0; SPSS Inc, Chicago, IL, USA). Descriptive statistics are presented for CB variables. The Kolmogorov Smirnov test was employed to investigate the normality distribution of the variables. The correlation between the cell counts of CB units before and after volume reduction was analysed by means of Spearman correlation coefficient (rho). The Mann Whitney U-test and Kruskal Wallis test for continuous variables were used to compare the groups when applicable. Multivariate analysis was performed by backwards stepwise regression. A P < 0.05 (two sided) was considered to be significant. Results Cells recovery and RBC depletion Monitoring the TNC, RBC, CD34 + cells and CFU content in both preprocess and postprocess CB units assessed the volume reduction process during the development phase of the study, using the program CB1. These results are shown in Table 2. When program CB1 was introduced into routine banking, we observed some differences in cell recoveries according to the volume of CB units (Table 3). CB collections with volume 100 ml had worse cell Table 2. Results of volume reduction processing for initial development group using program CB1 Pre-process Postprocess Recovery (%) Volume (ml) 89.5 ± ± 6.0 NA TNC ( 10 8 ) 9.9 ± ± ± 9.4 RBC ( ) 3.1 ± ± ± 12.5 CD34 + ( 10 6 ) 3.2 ± ± ± 13.6 CFU ( 10 4 ) ± ± ± 41.2 Results are shown as mean ± SD. NA, not applicable. Table 3. Cells recovery after separation of cord blood units with Compomat G4 according to the initial volume of the units was <100 ml (n ¼ 71) or 100 ml (n ¼ 59) Mean ± SD Final volume <100 ml 39.5 ± 11.2 < ml 48.1 ± 6.2 TNC recovery (%) <100 ml 79.7 ± 8.4 < ml 76.2 ± 8.7 RBC recovery (%) <100 ml 57.1 ± 13.5 < ml 41.2 ± 12.1 Lymphocyte recovery (%) <100 ml 80.1 ± 6.6 < ml 76.5 ± 8.6 TNC, total nucleated cell. recoveries than those with volume <100 ml. These results led us to design a program CB2 for CB units with volume 100 ml. These two programs were designed to achieve a BC volume of 41 ml with the aim of cryopreserving each CB collection in two bags containing 60% and 40% of TNC respectively. We also designed a program CB3 for reducing the BC volume to 25 ml with the aim of cryopreserving CB collections in only one bag. This program achieved worse cell recoveries than the other ones. Table 4 shows the volumes and cell counts of whole CB and components prepared on the three different programs. For all units, total CD34 + cells content, viability and CFU after volume reduction were 46 ± , 93 ± 5% and 80 ± respectively. RBC removal was higher for greater units (>100 ml) as shown in the Table 3. We reached a RBC depletion of 50%, 60% and 70% for 67%, 39% and 9% of all units respectively. A RBC depletion of 80% was not reached for any case. Multivariate analysis confirmed that RBC depletion was significantly influenced by initial volume and RBC counts. Lymphocytes recovery was negatively influenced by initial volume and TNC counts. P

4 P. Solves et al. 205 Table 4. Volumes and cell counts of cord blood units and their components prepared with Compomat G4 using the different programs in routine cord blood banking. Cord blood component Program CB1 (n ¼ 150) Program CB2 (n ¼ 50) Program CB3 (n ¼ 35) P Cord blood Volume (ml) (%) ± ± ± 19.9 CB1 vs. CB2 P < 0.001; CB2 vs. CB3 P < TNC ( 10 7 ) ± ± ± 44.9 CB1 vs. CB2 P < 0.001; CB2 vs. CB3 P < RBC ( ) 4.0 ± ± ± 0.9 Lymphocytes (%) 36.7 ± ± ± 8.0 NS BC for cryopreservation Volume (ml) 49.4 ± ± ± 1.4 CB1 vs. CB3 and CB2 vs. CB3 P < TNC ( 10 7 ) ± ± ± 31.0 CB1 vs. CB2 P < 0.005; CB1 vs. CB3 P < 0.05; CB2 vs. CB3 P < TNC recovery (%) 78.2 ± ± ± 6.7 CB1 vs. CB3 P < 0.001; CB2 vs. CB3 P < RBC ( ) 1.8 ± ± ± 0.2 CB1 vs. CB3 P < 0.001; CB2 vs. CB3 P < RBC depletion (%) 51.0 ± ± ± 10.4 CB1 vs. CB2 P < 0.05; CB1 vs. CB3 P < Lymphocytes (%) 37.0 ± ± ± 8.2 NS Lymphocytes recovery (%) 77.3 ± ± ± 8.2 CB1 vs. CB3 P < 0.001; CB2 vs. CB3 P < TNC, Total nucleated cells; BC, buffy coat; CB, cord blood; NS, not significant. Table 5. Cells recoveries of cord blood units processed with Compomat G4 (n ¼ 150 for CB1 and n ¼ 50 for CB2) and Optipress II (n ¼ 190) Comparison between Compomat G4 and Optipress II We compared 190 CB units processed with Optipress II to 200 CB units processed with program CB1 (n ¼ 150) and CB2 (n ¼ 50) of Compomat G4. Initial values of volume, TNC, Htc and lymphocytes percentages were ± 26.5 ml, ± , 37.4 ± 4.7% and 36.5 ± 8.9% respectively for CB processed with Optipress II and ± 23 ml, ± , 39 ± 4.4% and 36.5 ± 8.3% respectively for units processed with Compomat G4 (P ¼ NS for all variables). Cells recoveries are shown in Table 5. CD34 + cells content after volume reduction was similar for both groups (44.8 ± for Optipress II and 44.0 ± for Compomat G4, P ¼ NS). Discussion Optipress II (n ¼ 190) Compomat G4 (n ¼ 200) P TNC recovery (%) 78.5 ± ± 7.2 NS RBC depletion (%) 55.6 ± ± 14.9 <0.005 Lymphocyte recovery (%) 79.6 ± ± 7.5 <0.001 Results are expressed as mean ± standard deviation. TNC, total nucleated cell; NS, not significant. The main goal of CB banks is to provide quality-processed units that can be transplanted into the largest possible number of patients. CB stored as unmanipulated whole blood requires vast amount of storage space in liquid nitrogen tanks. To reduce the storage requirements in large-scale CB banks and to decrease the infusion-related haemolysis and DMSO toxicity, reduction of the volume and RBC content of CB collections is widely used in CB banking (Stanworth et al., 2001). Many techniques have been tested for the volume reduction purpose but to date, most CB banks use the HES methodology developed by the New York Cord Blood Center (Kogler, Sarnowski & Wernet, 1998; Stanworth et al., 2001). The use of automated blood processors, such as Optipress or Compomat, in combination with top and bottom containers has been found to improve the quality of blood components in transfusion centres (Van Rhenen et al., 1998; Van der Meer et al., 1999; Hurtado et al., 2000). This methodology has also been used for CB fractionation (Armitage et al., 1999). To our knowledge, there are no previous studies analysing the volume reduction process of CB units with the Compomat G4 device. There are only two studies processing CB units with Optipress II (Armitage et al., 1999; Godinho et al., 2000). In these studies, BC volume was standardized to ml, achieving TNC recoveries of more than 80% and CD34 + cell recoveries of more than 90%. We also used Optipress II with good cell recoveries but adjusting BC volume to 41 ml (Solves et al., 2005). Our CB bank is integrated with a transfusion centre and we recently changed the top and bottom technique to Compomat G4, so we had to design a program for CB processing with this top and bottom device. We initially designed a program to standardize the BC volume to 41 ml (CB1). Our first objective was to get a

5 206 Automated CB units separation using the Compomat G4 high cells yield. With this program we found similar TNC, CD34 + cells and CFU recoveries after volume reduction to those reported previously with sedimentation methods (Regidor et al., 1999; Dal Cortivo et al., 2000; Tsang et al., 2001; Aroviita et al., 2003) and with automatic systems (Zingsem et al., 2003). However, when we introduced this program into routine CB banking, we observed worse cell recoveries for those units with volumes equal or higher than 100 ml. These results led us to design the program CB2 for these higher units, achieving TNC recoveries similar to that of program CB1. The only two differences between these two programs are that for CB2 we increased upper press to 54.8 mm and decreased the lower press to 58.6 mm. To cryopreserve the CB units in a single bag, we also designed a program for a BC volume of 25 ml. We observed worse TNC and lymphocyte recoveries with program CB3 than with the other two programs. The loss of cells was due to the transference of BC cells to the RBCC. In fact, the problem with top and bottom techniques for reducing volume of CB is the difficulty to achieve a low final BC volume maintaining good cell yields (more than 75%). Another problem of top and bottom fractionation of CB collection is the RBC depletion that usually is about 50% (Armitage et al., 1999), significantly less than for sedimentation methods using HES or similar methods that achieve a RBC depletion of 80% or more (Tsang et al., 2001). Compomat failed to achieve this RBC depletion level as reported by some authors using automatic devices (Zingsem et al., 2003). In addition to this, CB processed with the program CB3 contained higher RBC contents than those processed with the other two programs. Residual RBC can make the washing of CB units difficult after thawing and before transplantation and can influence the results of clonogenic assays (De Kreuk et al., 2001). This is a reason why top and bottom systems are not suitable for CB volume reduction purpose. We finally compared two different top and bottom devices for volume reduction of CB (Optipress II and Compomat G4), finding that Optipress II allows similar TNC recoveries but significantly better lymphocytes recovery. However, CD34 + cells content after volume reduction was not different, i.e. mean haematopoietic content of CB units is similar for both cases. RBC depletion was also higher for CB processed with Optipress II. Taking into account that conditions of centrifugation are the same for both groups, we think these differences are due to the different top and bottom methodologies of these devices. In conclusion, the volume reduction method with Compomat G4 is a semi-automated system that allows acceptable cell recoveries, although insufficient RBC depletion and requires adaptation of programs for CB with higher volumes. Reducing the BC final volume implies a significant diminution of cell recoveries. Compared to Optipress II, this system allows worse lymphocyte recovery and RBC depletion. References Adorno G., Bruno A., Caravita T., Venditti A., Ballatore G., Santinelli S., Postorino M., Monaco I., Piazza A., Calugi A., Araco P., Tribalto M. & Amadori S. (1998) Red blood cell depletion of cord blood using hydroxyethylstarch double sedimentation: analysis of 40 cases. Clinical and Laboratory Haematology 20, Armitage S., Fehily D., Dickinson A., Chapman C., Navarrete C. & Contraras M. (1999) Cord blood banking: volume reduction of cord blood units using a semi-automated closed system. Bone Marrow Transplantation 23, Aroviita P., Teramo K., Westman P., Hiilesmaa V. & Kekomaki R. (2003) Associations among nucleated cell, CD34+ cell and colony-forming cell contents in cord blood units obtained through a standardized banking process. Vox Sanguinis 84, Ballen K.K. (2005) New trends in umbilical cord blood transplantation. Blood 105, Dal Cortivo L., Robert I., Mangin C., Sameshima T., Kora S., Gluckman E., Benbunan M. & Marolleau J.P. (2000) Cord blood banking: volume reduction using ÔProcordÕ Terumo filter. Journal of Hematotherapy and Stem Cell Research 9, De Kreuk A.M., Zevenbergen A., van Oostveen J.W., Schuurhuis G.J., Huijgens P.C. & Jonkhoff A.R. (2001) A single-step colony-forming unit assay for unseparated mobilized peripheral blood, cord blood, and bone marrow. Journal of Hematotherapy and Stem Cell Research 10, Gluckman E., Broxmeyer H.E., Auerbach A.D., Friedman H.S., Douglas G.W., Devergie A., Esperou H., Thierry D., Socie G. & Lehn P. (1989) Hematopoietic reconstitution in a patient with Fanconi s anemia by means of umbilical-cord blood from an HLA-identical sibling. New England Journal of Medicine 321, Godinho M.I., de Sousa M.E., Carvalhais A. & Barbosa I.L. (2000) Umbilical cord blood processing with the Optipress II blood extractor. Cytotherapy 2, Hurtado C., Bonanad S., Soler M.A., Mirabet V., Blasco I., Planelles M.D. & de Miguel A. (2000) Quality analysis of blood components obtained by automated buffy-coat layer removal with Top & Bottom system (Optipress II). Haematologica 85, Kogler G., Sarnowski A. & Wernet P. (1998) Volume reduction of cord blood by hetastarch for long-term stem cell banking. Bone Marrow Transplantation 22, S14 S15. Loos J.A. (1986) Automation in blood component preparation. In: Transfusion Medicine: Recent Technological Advances (eds K. Murawski, F. Peetoom), pp Karger Publishers, New York. Oliveira O.M.W., Vieira M.J., Bastos E.M.S.C., Delbuono E., Ginani V.C., Gordan L., Gouveia R.V., Cecyn K.Z., Carvalho M.L. & Lee M.L.M. (2004) DMSO removal reduces stem-cell infusion-related toxicity and allows excellent engraftment of

6 P. Solves et al. 207 cryopreserved unrelated cord blood and autologous stem cells. Biology of Blood and Marrow Transplantation 10(S1), 66. Regidor C., Posada M., Monteagudo D., Garaulet C., Somolinos N., Forés R., Briz M. & Fernández M.N. (1999) Umbilical cord blood banking for unrelated transplantation: Evaluation of cell separation and storage methods. Experimental Hematology 27, Rubinstein P., Taylor P.E., Scaradavou A., Adamson J.W., Migliaccio G., Emanuel D., Berkowitz R.L., Alvarez E. & Stevens C.E. (1994) Unrelated placental blood for bone marrow reconstitution: organization of the placental blood program. Blood Cells 20, Solves P., Mirabet V., Planelles D., Blasco I., Perales A., Carbonell-Uberos F., Soler M.A. & Roig R. (2005) Red blood cell depletion with a semi-automated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study. Transfusion 45, Stanworth S., Warwick R., Fehily D., Persaud C., Armitage S., Navarrete C. & Contreras M. (2001) An international survey of unrelated umbilical cord blood banking. Vox Sanguinis 80, Sutherland D.R., Anderson L., Keeney M., Nayar R. & Chin-Yee I. (1996) The ISHAGE guidelines for CD34 + cell determination by flow cytometry. International Society of hematotherapy and graft engineering. Journal of Hematotherapy 5, Tsang K.S., Li K., Huang D.P., Wong A.P., Leung Y., Lau T.T., Chang A.M.Z., Li Chi K., Fok T.F. & Yuen P.M.P. (2001) Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood. Transfusion 41, Van der Meer P.F., Pietersz R.N.I., Hinloopen B., Dekker W.J.A. & Reesink H.W. (1999) Automated separation of whole blood in top and bottom bags into components using the Compomat G4. Vox Sanguinis 76, Van Rhenen D.J., Vermeig J., de Voogt J., Bernes J.C. & Payrat J.M. (1998) Quality and standardization in blood component preparation with an automated blood processing technique. Transfusion Medicine 8, Zingsem J., Strasser E., Weisbach V., Zimmermann R., Ringwald J., Goecke T., Beckmann M.W. & Eckstein R. (2003) Cord blood processing with an automated and functionally closed system. Transfusion 43,

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