- Email: [email protected]
Umbilical cord blood processing with the Optipress II blood extractor
Cytotherapy (2000) Vol. 2, No. 6, 439–443
Umbilical cord blood processing with the Optipress II blood extractor MI Godinho, ME de Sousa, A Carvalhais and IL Barbosa Serviço de Imuno-Hemoterapia, Instituto Português de Oncologia de Francisco Gentil, Porto Portugal
Background
We have previously shown that UC blood (UCB) units can be volumereduced manually, in a closed system, without major losses of nucleated and CD34 cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the ‘buffy-coat’ collection in a standardized volume.
Group I < 80 mL and Group II ≥ 80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34 cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%. Discussion
Methods
After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34 cells, was set at 30 mL. The nucleated and CD34 cell content of the UCB collections and the resulting BC were evaluated.
This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor-cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.
Results
Keywords
The UCB units were grouped according to the volume collected:
cord blood, processing, volume reduction.
Introduction
Different methods of volume reduction have been used, such as red cell lysis [5] density gradient separation [6], gelatine, dextran or starch sedimentation [2,5–9] and fractionation by centrifugation [10–13]. Following our work on manual volume reduction [10] our aim was to standardize the process using the Optipress II blood extractor, which allows the automatic separation of the centrifuged unit into the plasma, red cell and nucleated fractions [12,13]. We were particularly interested in evaluating volume reduction of units with < 80 mL, as almost half of our family-related UCB units were within this volume (12/26 units, unpublished data). In the present study, we report on the efficiency of the Optipress II, fully automated blood extractor, in UCB volume reduction, by grouping the units as volumes of 40–80 mL (Group I) and ≥ 80 mL (Group II). Thus, we
The clinical success of UC blood (UCB) transplantation, associated with reduced incidence of GvHD, has led to cord-blood banking worldwide [1–3]. There are several UCB banks, in the USA and in Europe [2–4], that have established guidelines concerning volume, NC content and processing of the UCB units. The majority of the UCB banks first froze UCB units without manipulation. However, with the increase in the number of cryopreserved samples, it became necessary to develop methods for volume reduction of UCB units, aiming to minimize storage space. This UCB processing must be achieved without significant losses of the nucleated and hematopoietic progenitor cells, to ensure engraftment with full hematopoietic reconstitution of the recipient.
Correspondence to: IL Barbosa, Serviço de Imuno-Hemoterapia, IPO – Porto 4200 Porto – Portugal © 2000 ISHAGE
439
440
MI Godinho et al.
compare the efficiency of volume reduction in low (Group I) and high volume (Group II) UCB units.
Methods UCB collections Upon informed consent from the mother and prior to the delivery of the placenta, 51 UCB units were collected into a collection bag containing 25 mL of anticoagulant (CPD). The units were stored at 4ºC and processed within 24 h of collection.
Volume reduction The minimum volume for processing was established at 40 mL. These units were well-mixed and transferred under sterile conditions to the top and bottom Optipac triple bag system (Baxter-Portugal), which had previously been emptied of anticoagulant. The differential sedimentation of the unit was done by centrifugation at 3300 g for 10 min at 20ºC, in oval buckets with the bag well-supported to avoid folding. The unit was then carefully placed onto the Optipress II extractor (Baxter-Portugal) for the automatically-controlled transference of the plasma and erythrocyte fractions to the transfer packs, with the nucleated fraction ‘buffy coat’ (BC) kept in a pre-set volume of 30 mL in the original bag. The Optipac bag was weighed before and after the transference of UCB unit and the correct volume collected was determined by: weight (g) 0.95 volume (mL).
Hematological evaluation All UCB units and corresponding BC fractions were sampled for hematological counts in a Sysmex 900.
Progenitor cell determination by flow cytometry Enumeration of CD34 cells was by flow cytometric analysis of UCB and BC samples labelled with FITCconjugated CD45 (Becton-Dickinson, Enzifarma Portugal) and PE-conjugated CD34 (Becton-Dickinson,
Enzifarma Portugal) MAbs, lysed with 2 mL FACS lysing solution, washed and fixed with 300 µL Cell Fix. Analysis of the percentage of CD34 cells was on a BD FACSort cytometer with Lysis II software. The final results were expressed as absolute numbers.
Statistical analysis The results are expressed as median values. The correlation of UCB and BC for cell content was analysed by the Spearman Rank Order Correlation test. For the intragroup analysis the Wilcoxon matched pairs test was used and the Mann–Whitney U test for the analysis between groups. Tests with p < 0.05 were considered statistically significant. Values > 100% are presented as 100%.
Results The characteristics for the UCB units processed in each group are presented in Table 1. In both groups the pre- and post-processing nucleated cell content and CD34 cell values significantly correlate with the volume collected (p < 0.005). The cellular content of all these units is similar to others previously reported by different groups [11,13]. After volume reduction, the cellular content of the BC fractions was evaluated and the results are summarized in Table 2. The volume of the UCB units was significantly reduced, by 51% in Group I and 70% in Group II, with an equally significant reduction in red cell content of 34% and 56% respectively (p < 0.0001). These parameters were found to be significantly higher in Group II compared with Group I (p < 0.0001). Recovery values for the NC and the CD34 cell populations in Group I were 88% and 99%, and in Group II were 80% and 97% respectively. Evaluation of the RBC fraction containing the lost NC showed the predominance of granulocytes (data not shown). With the exception of CD34 cell recovery, the wider ranges of variation for volume reduction, RBC depletion and NC recovery observed in Group I, compared with Group II, may be due to the low volume collections (Table 2).
Table 1. UCB characteristics as median values for each UCB Group
Group I (n = 31) Group II (n = 20)
Volume (mL) (min–max)
RBC (108) (min–max)
61 (40–79) 102 (84–141)
1.64 (1.0–2.7) 3.30 (2.1–5.9)
NC (108) (min–max)
CD34 (106) (min–max)
5.0 (1.9–9.8) 10.4 (7.7–21.5)
0.7 (0.04–3.9) 2.4 (0.9–15.4)
UCB volume reduction
441
Table 2. Post-processing results expressed as percentage of the initial values
Group I (n = 31) Group II (n = 20)
Volume reduction (min–max)
RBC depletion (min–max)
NC recovery (min–max)
CD34 recovery (min-max)
51 (25–62) 70 (64–79)
34 (5–77) 56 (32–75)
88 (32–100) 80 (64–100)
99 (49–100) 97 (47–100)
The NC and CD34 cell content of each individual collection and its recovery of BC is shown in Figure 1. We have previously reported [14] some difficulties in CD34 cell quantification of unmanipulated UCB units, which could be better evaluated in the BC fraction. Thus, post-processing CD34 yields > 100% are a reflection of the difficulty in evaluating their initial content. A good linear correlation was found for each group between the initial content of NC (a), or CD34 cells (b), and the final values obtained in the BC (p < 0.001). The analysis of the data per group showed significant losses of nucleated cell values (p < 0.001) but no significant difference in CD34 cell content of the BC (p > 0.2). Several centers established a threshold of 2 107 NC/kg body weight required for transplantation [15], thus a unit with 4 108 NC would be suitable for a patient with 20 kg body weight. If we analyse our UCB collections according to this criteria, 20 of the 31 units in
Group I had the minimum NC number (4 108) required for transplantation, whereas in Group II all units had higher NC content. After processing, 15/20 units from Group I and all units from Group II had values ≥ 4 108 NC.
Discussion Efficient UCB banking means long-term storage of as many units as possible, making UCB volume reduction a mandatory procedure. We have previously reported on a significant volume reduction (56%), by manual separation of the different blood components after sedimentation by centrifugation, with good CD34 and nucleated cell recovery (87% and 72% respectively) [10]. The relevance of this study is the selective analysis of cell recovery in low (Group I) and high (Group II) UCB volumes, following standardized volume reduction using the Optipress II automated system.
2,4e9
6e6 1,5e7 Group I Group II
1,6e9
1,2e9
8e8
4e8
0 a
7,9e6 7,1e6
Group II
5e6 CD34cells in BC fraction
2e9
NC in BC fraction
Group I
4e6
3e6
2e6
1e6
0
4e8
8e8
1,2e9
1,6e9
NC in UCB unit
2e9
0
2,4e9 b
0
1e6
2e6
3e6
4e6
5e6
CD34 cells in UCB unit
Figure 1. (a) NC and (b) CD34+ cell content of the UCB units and corresponding BC fractions, for Group I and Group II.
6e6
442
MI Godinho et al.
Our results on RBC depletion in Group II (56%) were similar to those described by other groups [12], whereas in Group I the reduction was only 34%. However, part of the RBC content will lyse when the units are thawed and free hemoglobin removed by washing the cells prior to transplantation, decreasing possible transfusion reactions. We obtained a good recovery of CD34 cell numbers for both UCB groups (99% and 97% for Group I and Group II respectively). Although we observed a decrease in the NC content of the BC fractions, 75% of the units in Group I and all units in Group II, maintained NC values above the threshold of 4 108 NC. These results, obtained with the Optipress II, showed a marked improvement of post-processing yields in comparison with our manual method [10]. With respect to the CD34 cell recoveries, our results are similar to those reported by other groups [11–13], particularly Armitage et al. [12]. The results for NC recovery are contradictory, some groups reported good recoveries [11,13] whereas Carlton et al. [13], like us, found a decrease in the NC content. In summary, in our experience, the Optipress II is a fast and efficient method for processing both low and high volume UCB units to be cryopreserved without significant loss of hematopoietic progenitor cells. The cryoprotectant will be added to these volume-reduced UCB units, followed by immediate freezing in a controlled-rate freezer and storage in liquid nitrogen tanks. The use of the newly-developed BioArchive System (Thermogenesis), a fully automatic controlled rate freezing and storage system, curently uses freezing bags that hold a final volume of 25 mL (BC fraction plus the cryoprotectant solution) [16]. Thus, further optimization of the Optipress II system is required to achieve smaller BC fractions. This aim has to be achieved while preserving cell content at a level associated with successful engraftment [1], making cell-yield a determining parameter. However, this limitation may be overcome by the ex vivo expansion of CB cells [17–19], thus enhancing the importance of CD34 cell recovery.
Acknowledgements We are grateful to the medical doctors and midwives of the Obstetrics Department at the Hospital de S. JoãoPorto, for the collection of the UCB units. This work was supported by grant SAU 260/97 from the Portuguese Health Ministry.
References 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–81. Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995;92:10119–22. Querol S, Gabarró M, Amat L et al. The placental blood program of the Barcelona Cord Blood Bank. Bone Marrow Transplant 1998;22(Suppl 1):3–5. Fraser JK, Cairo MS, Wagner E et al. Cord blood transplantation study (COBLT): cord blood bank standard operating procedures. J Hematother 1998;7:521–61. Falkenburg JHF, Van Luxemberg-Heijs SAP, Zijlmans JM et al. Separation enrichment and characterization of human hematopoietic progenitor cells from umbilical cord blood. Ann Haematol 1993;67:231–6. Perutelli P, Catellani S, Scarso L et al. Processing of human cord blood by three different procedures for red blood cell depletion and mononuclear cell recovery. Vox Sang 1999;76:237–40. Tanavde VM, Desai SS, Rao SG. Erythrocyte depletion of human umbilical cord blood using dextran sedimentation. Indian J Med Res 1997;106:16–19. Bertolini F, Lazzari L, Lauri E et al. Comparative study of different procedures for the collection and banking of umbilical cord blood. J Hematother 1995;4:29–36. Regidor C, Posada M, Monteagudo D et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hematol 1999;27:380–5. Sousa T, Sousa ME, Godinho MI et al. Umbilical cord blood processing: volume reduction and recovery of CD34 cells. Bone Marrow Transplant 1997;19:311–3. Ademokun JÁ, Chapman C, Dunn J et al. Umbilical cord blood collection and separation for haematopoietic progenitor cell banking. Bone Marrow Transplant 1997;19:1023–8. Armitage S, Fehily D, Dickinson A et al. Cord blood banking: volume reduction of cord blood units using a semi-automated closed system. Bone Marrow Transplant 1999;23:505–9. Carlton KE, Dobell K, Piras L et al. Evaluation of Baxter Optipress® II to reduce the volume of cord blood donations. Cytotherapy 1999;1:230a. Barbosa IL, Sousa ME, Godinho MI et al. Single-versus dual platform assays for human CD34 cell enumeration. Cytometry 1999;38:1–6. Kogle G., Sarnowski A, Wernet P. Volume reduction of cord blood by Hetastarch for long term stem cell banking. Bone Marrow Transplant 1998; 22(Suppl 1):14–15. Armitage S. Collection and processing of cord blood units. In: Cohen SBA, Gluckman E, Rubinstein P, Madrigal JA, editors. Cord blood characteristics: role in stem cell transplantation. London: Martin Dunitz Ltd; 2000;129–48.
UCB volume reduction
17 Denning-Kendall PA, Horsley H, Nicol A et al. Clinical application of in vitro expansion of cord blood. Bone Marrow Transplant 1998;22(Suppl, 1):63–5. 18 Kögler G, Numberger W, Fischer J et al. First application of ex-vivo expanded CB cells at the time point of
443
transplantation. Blood 1998:92(Suppl. 1):312b. 19 Shpall EJ, Quinones R, Hami L et al. Transplantation of cancer patients receiving high dose chemotherapy with ex vivo expanded cord blood cells. Blood 1998;92(Suppl 1):646a.
Umbilical cord blood processing with the Optipress II blood extractor MI Godinho, ME de Sousa, A Carvalhais and IL Barbosa Serviço de Imuno-Hemoterapia, Instituto Português de Oncologia de Francisco Gentil, Porto Portugal
Background
We have previously shown that UC blood (UCB) units can be volumereduced manually, in a closed system, without major losses of nucleated and CD34 cells and without the addition of exogenous material. Our aim was to use an automated method for the separation of the UCB components using the Optipress II, extractor, with the ‘buffy-coat’ collection in a standardized volume.
Group I < 80 mL and Group II ≥ 80 mL. Standardization of the BC at 30 mL resulted in significant volume reduction for both groups, with median values of 51% in Group I and 70% in Group II. The nucleated and CD34 cell recoveries in the BC from Group I were 88% and 99% respectively; for Group II they were 80% and 97%. Discussion
Methods
After centrifugation, the 51 UCB units were separated into the three blood components, plasma, buffy coat (BC) and red cells, using the Optipress II. The final volume of the BC fraction, rich in nucleated and progenitor CD34 cells, was set at 30 mL. The nucleated and CD34 cell content of the UCB collections and the resulting BC were evaluated.
This semi-automated method of volume reduction efficiently reduces low, as well as high volume UCB units, with good nucleated- and progenitor-cell yields. Being a closed system and free of external material, the risk of contamination is minimized. The resulting fractions are then available for validation studies of the unit, effectively fulfilling the main requisites for UCB banking.
Results
Keywords
The UCB units were grouped according to the volume collected:
cord blood, processing, volume reduction.
Introduction
Different methods of volume reduction have been used, such as red cell lysis [5] density gradient separation [6], gelatine, dextran or starch sedimentation [2,5–9] and fractionation by centrifugation [10–13]. Following our work on manual volume reduction [10] our aim was to standardize the process using the Optipress II blood extractor, which allows the automatic separation of the centrifuged unit into the plasma, red cell and nucleated fractions [12,13]. We were particularly interested in evaluating volume reduction of units with < 80 mL, as almost half of our family-related UCB units were within this volume (12/26 units, unpublished data). In the present study, we report on the efficiency of the Optipress II, fully automated blood extractor, in UCB volume reduction, by grouping the units as volumes of 40–80 mL (Group I) and ≥ 80 mL (Group II). Thus, we
The clinical success of UC blood (UCB) transplantation, associated with reduced incidence of GvHD, has led to cord-blood banking worldwide [1–3]. There are several UCB banks, in the USA and in Europe [2–4], that have established guidelines concerning volume, NC content and processing of the UCB units. The majority of the UCB banks first froze UCB units without manipulation. However, with the increase in the number of cryopreserved samples, it became necessary to develop methods for volume reduction of UCB units, aiming to minimize storage space. This UCB processing must be achieved without significant losses of the nucleated and hematopoietic progenitor cells, to ensure engraftment with full hematopoietic reconstitution of the recipient.
Correspondence to: IL Barbosa, Serviço de Imuno-Hemoterapia, IPO – Porto 4200 Porto – Portugal © 2000 ISHAGE
439
440
MI Godinho et al.
compare the efficiency of volume reduction in low (Group I) and high volume (Group II) UCB units.
Methods UCB collections Upon informed consent from the mother and prior to the delivery of the placenta, 51 UCB units were collected into a collection bag containing 25 mL of anticoagulant (CPD). The units were stored at 4ºC and processed within 24 h of collection.
Volume reduction The minimum volume for processing was established at 40 mL. These units were well-mixed and transferred under sterile conditions to the top and bottom Optipac triple bag system (Baxter-Portugal), which had previously been emptied of anticoagulant. The differential sedimentation of the unit was done by centrifugation at 3300 g for 10 min at 20ºC, in oval buckets with the bag well-supported to avoid folding. The unit was then carefully placed onto the Optipress II extractor (Baxter-Portugal) for the automatically-controlled transference of the plasma and erythrocyte fractions to the transfer packs, with the nucleated fraction ‘buffy coat’ (BC) kept in a pre-set volume of 30 mL in the original bag. The Optipac bag was weighed before and after the transference of UCB unit and the correct volume collected was determined by: weight (g) 0.95 volume (mL).
Hematological evaluation All UCB units and corresponding BC fractions were sampled for hematological counts in a Sysmex 900.
Progenitor cell determination by flow cytometry Enumeration of CD34 cells was by flow cytometric analysis of UCB and BC samples labelled with FITCconjugated CD45 (Becton-Dickinson, Enzifarma Portugal) and PE-conjugated CD34 (Becton-Dickinson,
Enzifarma Portugal) MAbs, lysed with 2 mL FACS lysing solution, washed and fixed with 300 µL Cell Fix. Analysis of the percentage of CD34 cells was on a BD FACSort cytometer with Lysis II software. The final results were expressed as absolute numbers.
Statistical analysis The results are expressed as median values. The correlation of UCB and BC for cell content was analysed by the Spearman Rank Order Correlation test. For the intragroup analysis the Wilcoxon matched pairs test was used and the Mann–Whitney U test for the analysis between groups. Tests with p < 0.05 were considered statistically significant. Values > 100% are presented as 100%.
Results The characteristics for the UCB units processed in each group are presented in Table 1. In both groups the pre- and post-processing nucleated cell content and CD34 cell values significantly correlate with the volume collected (p < 0.005). The cellular content of all these units is similar to others previously reported by different groups [11,13]. After volume reduction, the cellular content of the BC fractions was evaluated and the results are summarized in Table 2. The volume of the UCB units was significantly reduced, by 51% in Group I and 70% in Group II, with an equally significant reduction in red cell content of 34% and 56% respectively (p < 0.0001). These parameters were found to be significantly higher in Group II compared with Group I (p < 0.0001). Recovery values for the NC and the CD34 cell populations in Group I were 88% and 99%, and in Group II were 80% and 97% respectively. Evaluation of the RBC fraction containing the lost NC showed the predominance of granulocytes (data not shown). With the exception of CD34 cell recovery, the wider ranges of variation for volume reduction, RBC depletion and NC recovery observed in Group I, compared with Group II, may be due to the low volume collections (Table 2).
Table 1. UCB characteristics as median values for each UCB Group
Group I (n = 31) Group II (n = 20)
Volume (mL) (min–max)
RBC (108) (min–max)
61 (40–79) 102 (84–141)
1.64 (1.0–2.7) 3.30 (2.1–5.9)
NC (108) (min–max)
CD34 (106) (min–max)
5.0 (1.9–9.8) 10.4 (7.7–21.5)
0.7 (0.04–3.9) 2.4 (0.9–15.4)
UCB volume reduction
441
Table 2. Post-processing results expressed as percentage of the initial values
Group I (n = 31) Group II (n = 20)
Volume reduction (min–max)
RBC depletion (min–max)
NC recovery (min–max)
CD34 recovery (min-max)
51 (25–62) 70 (64–79)
34 (5–77) 56 (32–75)
88 (32–100) 80 (64–100)
99 (49–100) 97 (47–100)
The NC and CD34 cell content of each individual collection and its recovery of BC is shown in Figure 1. We have previously reported [14] some difficulties in CD34 cell quantification of unmanipulated UCB units, which could be better evaluated in the BC fraction. Thus, post-processing CD34 yields > 100% are a reflection of the difficulty in evaluating their initial content. A good linear correlation was found for each group between the initial content of NC (a), or CD34 cells (b), and the final values obtained in the BC (p < 0.001). The analysis of the data per group showed significant losses of nucleated cell values (p < 0.001) but no significant difference in CD34 cell content of the BC (p > 0.2). Several centers established a threshold of 2 107 NC/kg body weight required for transplantation [15], thus a unit with 4 108 NC would be suitable for a patient with 20 kg body weight. If we analyse our UCB collections according to this criteria, 20 of the 31 units in
Group I had the minimum NC number (4 108) required for transplantation, whereas in Group II all units had higher NC content. After processing, 15/20 units from Group I and all units from Group II had values ≥ 4 108 NC.
Discussion Efficient UCB banking means long-term storage of as many units as possible, making UCB volume reduction a mandatory procedure. We have previously reported on a significant volume reduction (56%), by manual separation of the different blood components after sedimentation by centrifugation, with good CD34 and nucleated cell recovery (87% and 72% respectively) [10]. The relevance of this study is the selective analysis of cell recovery in low (Group I) and high (Group II) UCB volumes, following standardized volume reduction using the Optipress II automated system.
2,4e9
6e6 1,5e7 Group I Group II
1,6e9
1,2e9
8e8
4e8
0 a
7,9e6 7,1e6
Group II
5e6 CD34cells in BC fraction
2e9
NC in BC fraction
Group I
4e6
3e6
2e6
1e6
0
4e8
8e8
1,2e9
1,6e9
NC in UCB unit
2e9
0
2,4e9 b
0
1e6
2e6
3e6
4e6
5e6
CD34 cells in UCB unit
Figure 1. (a) NC and (b) CD34+ cell content of the UCB units and corresponding BC fractions, for Group I and Group II.
6e6
442
MI Godinho et al.
Our results on RBC depletion in Group II (56%) were similar to those described by other groups [12], whereas in Group I the reduction was only 34%. However, part of the RBC content will lyse when the units are thawed and free hemoglobin removed by washing the cells prior to transplantation, decreasing possible transfusion reactions. We obtained a good recovery of CD34 cell numbers for both UCB groups (99% and 97% for Group I and Group II respectively). Although we observed a decrease in the NC content of the BC fractions, 75% of the units in Group I and all units in Group II, maintained NC values above the threshold of 4 108 NC. These results, obtained with the Optipress II, showed a marked improvement of post-processing yields in comparison with our manual method [10]. With respect to the CD34 cell recoveries, our results are similar to those reported by other groups [11–13], particularly Armitage et al. [12]. The results for NC recovery are contradictory, some groups reported good recoveries [11,13] whereas Carlton et al. [13], like us, found a decrease in the NC content. In summary, in our experience, the Optipress II is a fast and efficient method for processing both low and high volume UCB units to be cryopreserved without significant loss of hematopoietic progenitor cells. The cryoprotectant will be added to these volume-reduced UCB units, followed by immediate freezing in a controlled-rate freezer and storage in liquid nitrogen tanks. The use of the newly-developed BioArchive System (Thermogenesis), a fully automatic controlled rate freezing and storage system, curently uses freezing bags that hold a final volume of 25 mL (BC fraction plus the cryoprotectant solution) [16]. Thus, further optimization of the Optipress II system is required to achieve smaller BC fractions. This aim has to be achieved while preserving cell content at a level associated with successful engraftment [1], making cell-yield a determining parameter. However, this limitation may be overcome by the ex vivo expansion of CB cells [17–19], thus enhancing the importance of CD34 cell recovery.
Acknowledgements We are grateful to the medical doctors and midwives of the Obstetrics Department at the Hospital de S. JoãoPorto, for the collection of the UCB units. This work was supported by grant SAU 260/97 from the Portuguese Health Ministry.
References 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord blood transplantation from related and unrelated donors. N Engl J Med 1997;337:373–81. Rubinstein P, Dobrila L, Rosenfield RE et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci USA 1995;92:10119–22. Querol S, Gabarró M, Amat L et al. The placental blood program of the Barcelona Cord Blood Bank. Bone Marrow Transplant 1998;22(Suppl 1):3–5. Fraser JK, Cairo MS, Wagner E et al. Cord blood transplantation study (COBLT): cord blood bank standard operating procedures. J Hematother 1998;7:521–61. Falkenburg JHF, Van Luxemberg-Heijs SAP, Zijlmans JM et al. Separation enrichment and characterization of human hematopoietic progenitor cells from umbilical cord blood. Ann Haematol 1993;67:231–6. Perutelli P, Catellani S, Scarso L et al. Processing of human cord blood by three different procedures for red blood cell depletion and mononuclear cell recovery. Vox Sang 1999;76:237–40. Tanavde VM, Desai SS, Rao SG. Erythrocyte depletion of human umbilical cord blood using dextran sedimentation. Indian J Med Res 1997;106:16–19. Bertolini F, Lazzari L, Lauri E et al. Comparative study of different procedures for the collection and banking of umbilical cord blood. J Hematother 1995;4:29–36. Regidor C, Posada M, Monteagudo D et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hematol 1999;27:380–5. Sousa T, Sousa ME, Godinho MI et al. Umbilical cord blood processing: volume reduction and recovery of CD34 cells. Bone Marrow Transplant 1997;19:311–3. Ademokun JÁ, Chapman C, Dunn J et al. Umbilical cord blood collection and separation for haematopoietic progenitor cell banking. Bone Marrow Transplant 1997;19:1023–8. Armitage S, Fehily D, Dickinson A et al. Cord blood banking: volume reduction of cord blood units using a semi-automated closed system. Bone Marrow Transplant 1999;23:505–9. Carlton KE, Dobell K, Piras L et al. Evaluation of Baxter Optipress® II to reduce the volume of cord blood donations. Cytotherapy 1999;1:230a. Barbosa IL, Sousa ME, Godinho MI et al. Single-versus dual platform assays for human CD34 cell enumeration. Cytometry 1999;38:1–6. Kogle G., Sarnowski A, Wernet P. Volume reduction of cord blood by Hetastarch for long term stem cell banking. Bone Marrow Transplant 1998; 22(Suppl 1):14–15. Armitage S. Collection and processing of cord blood units. In: Cohen SBA, Gluckman E, Rubinstein P, Madrigal JA, editors. Cord blood characteristics: role in stem cell transplantation. London: Martin Dunitz Ltd; 2000;129–48.
UCB volume reduction
17 Denning-Kendall PA, Horsley H, Nicol A et al. Clinical application of in vitro expansion of cord blood. Bone Marrow Transplant 1998;22(Suppl, 1):63–5. 18 Kögler G, Numberger W, Fischer J et al. First application of ex-vivo expanded CB cells at the time point of
443
transplantation. Blood 1998:92(Suppl. 1):312b. 19 Shpall EJ, Quinones R, Hami L et al. Transplantation of cancer patients receiving high dose chemotherapy with ex vivo expanded cord blood cells. Blood 1998;92(Suppl 1):646a.