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Is hydroxyethyl starch necessary for sedimentation of bone marrow?
ARTICLE IN PRESS Transfusion and Apheresis Science ■■ (2014) ■■–■■
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Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Is hydroxyethyl starch necessary for sedimentation of bone marrow? Margriet J. Dijkstra-Tiekstra *, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, Janny de Wildt-Eggen Department Transfusion Monitoring, Sanquin Blood Supply, P.O. Box 1191, 9701 BD Groningen, The Netherlands
A R T I C L E
I N F O
Article history: Received 13 June 2014 Received in revised form 3 October 2014 Accepted 8 December 2014 Keywords: Hydroxyethyl starch Bone marrow HPC WBC-enriched product RBC depletion MNC recovery Volume Gravity Centrifugation
A B S T R A C T
Hydroxyethyl starch (HES) is used to separate hematopoietic progenitor cells after bone marrow (BM) collection from red blood cells. The aims were to study alternatives for HAESsteril (200 kDa; not available anymore) and to optimize the sedimentation process. Using WBC-enriched product (10 × 109 WBC/L), instead of BM, sedimentation at 10% hematocrit using final 0.6 or 0.39% Voluven (130 kDa) or without HES appeared to be good alternatives for 0.6% HAES-steril. MNC recovery >80% and RBC depletion >90% was reached. Optimal sedimentation was reached using 110–140 mL volume. Centrifugation appeared not suitable for sedimentation. Additional testing with BM might be necessary to confirm these results. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hematopoietic progenitor cells (HPC) from patients or donors can be collected either after stimulation using growth factors (e.g. G-CSF) by apheresis or by bone marrow (BM) collection. Although apheresis is the most common method for adult patients [1,2], for some indications the transplant centers prefer BM [3]. Hydroxyethyl starch (HES) can be used to separate the HPC after BM collection from RBC. Sedimentation can be done by gravity or automated machine processing [4–9]. HES with molecular weights ≥200 kDa will become less available on the market because of its adverse effects when used as plasma volume expander. Adverse effects of HES when used as plasma expander are impairment of
* Corresponding author. Sanquin Blood Supply, Division Research, Department Transfusion Monitoring, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Tel.: +31 50 3610817; fax: +31 50 3610630. E-mail address: [email protected] (M.J. Dijkstra-Tiekstra).
coagulation, renal function impairment, inhibitory effect on platelet function, deterioration of rheological parameters, and accumulation in tissues [8–10]. Although the volume effect is prolonged for higher molecular weight HES compared to lower molecular weight HES, adverse effects of HES are more pronounced for higher molecular weight HES [8–10]. However, no adverse effects are described for HES when used for sedimentation of BM, probably because of the low volumes of HES that are transfused. For HPC from BM, RBC depletion is needed especially in case of AB0 incompatibility, to avoid AB0 induced transfusion reactions. Another reason for RBC depletion is volume reduction to reduce cryogenic storage space or in the case of allogeneic transplantation in the pediatric setting, irrespective of the blood group [11–13]. The standard procedure in our processing facility is as follows: BM is filtrated and diluted to a concentration of 10 × 109 WBC/L and divided over 400-mL bags with 140– 200 mL per bag, after which 9:1 (v/v) HAES-steril 6% (final 0.6%) is added. After sedimentation for 60–90 min the plasma and buffy coat are pressed into a satellite bag (Table 1).
http://dx.doi.org/10.1016/j.transci.2014.12.003 1473-0502/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Margriet J. Dijkstra-Tiekstra, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, Janny de Wildt-Eggen, Is hydroxyethyl starch necessary for sedimentation of bone marrow?, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.12.003
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Table 1 Values for BM processing (n = 11).
Volume, mL WBC, ×109/L Hematocrit, % Dilution factor Volume after dilution, mL WBC after dilution, ×109/La Hematocrit after dilution, %a Number of bags processed Volume per bag, mLb MNC recovery, % RBC depletion, % a b
Mean ± SD
Range
1386 ± 373 17.6 ± 7.9 31.4 ± 6.7 0.6 ± 0.2 2432 ± 1087 9.8 ± 0.9 19.9 ± 7.9 14 ± 6 168 ± 13 88 ± 15 95 ± 2
458–1891 9.7–36.7 21.5–41.7 0.3–1.0 948–4509 7.3–11.1 7.7–34.3 6–24 146–188 67–119 93–99
Calculated from values before dilution and dilution factor. Calculated from total volume and number of bags processed.
The objective of this study was to investigate whether sedimentation of BM using 0.6% HAES-steril (200 kDa/0.5; standard) can be substituted by sedimentation using 0.6% or 0.39% Voluven (130 kDa/0.4) or by sedimentation without HES. The second objective was to optimize the sedimentation process of BM in relation to the volume of the product before sedimentation and sedimentation by centrifugation instead of by gravity.
HES or without HES. For the 140 mL WBC-enriched product 15.6 mL or 9.7 mL HES was added with continuous gentle mixing, to reach a final 0.6% or 0.39% HES respectively. This resulted in four bags, containing (1) WBC-enriched product and 0.6% HAES-steril (standard), (2) WBC-enriched product and 0.6% Voluven, (3) WBC-enriched product and 0.39% Voluven, and (4) WBC-enriched product without HES. The products with 0.6% HAES-steril and 0.39% Voluven have the same molarity for the HES (3 μM). All bags were allowed to sediment by gravity for 90–105 minutes and were placed into a manual press. The supernatant including the buffy coat was pressed into an empty 400 mL transfer bag. This bag was disconnected from the bag containing the sediment that was discarded. After weighing, the bag containing the supernatant was sampled (1 mL sample).
2.3. The influence of the volume of the product before sedimentation Four WBC-enriched products were pooled, mixed and split into 15 portions, three of 110 mL, three of 140 mL, three of 170 mL, three of 200 mL and three of 230 mL. To each bag HES or no HES was added resulting in the variations 0.6% HAES-steril, 0.39% Voluven or without HES for each volume. These bags were allowed to sediment by gravity and the supernatant was collected as described above.
2. Materials and methods 2.1. WBC-enriched product
2.4. Sedimentation by centrifugation or gravity
Because BM derived HPC products are not readily available for research purposes, a WBC-enriched product from buffy coats and plasma, with WBC, RBC and platelet counts in the same range as real BM, but absence of CD34+ cells or immature cells, was used in this study. The preparation of a WBC-enriched product was as follows: Five buffy coats and one plasma unit, derived from overnight stored whole blood, were pooled and centrifuged using a soft spin (930 g for 4.5 min, brake 3). The platelet rich plasma was used to form a therapeutic platelet concentrate. The sediment was stored overnight at RT on a flat bed shaker. Next day, a 2 mL sample was taken and WBC count was determined. The sediment was diluted using plasma until a WBC concentration of about 10 × 109/L and a hematocrit of 7–11% in a total volume of about 600 mL were obtained resulting in the WBC-enriched product. If necessary the plasma was pressed in opposite direction over a WBC reduction filter (Compostop, Fresenius Hemocare; Emmer Compascuum, The Netherlands) that was used for the preparation of the platelet concentrate, to increase the number of WBC.
Two WBC-enriched products were pooled, mixed and split into three portions of 350 mL. To each bag HES or no HES was added resulting in the variations 0.6% HAESsteril, 0.39% Voluven or without HES. Subsequently, each bag was split in a bag containing 185 mL WBC-enriched product with additional HES or without HES and a bag containing 140 mL WBC-enriched product with additional HES or without HES. The bags with 185 mL WBC-enriched product and HES or without HES were centrifuged at 250 g for 10 min at RT and brake at 2. Volume and forces were optimized in pilot experiments (not shown). The bags containing 140 mL WBC-enriched product and HES or without HES were allowed to sediment by gravity and the supernatant of all bags was collected as described above.
2.2. The influence of using HAES-steril, Voluven or no HES The WBC-enriched product was equally divided over four 400-mL transfer bags (about 140 mL per bag; R4R2074; Fenwal, Inc. Lake Zurich, USA). After sampling and weighing the product its volume was calculated and from this the amount of HES necessary to result in either 0.6% or 0.39%
2.5. Tests Samples were taken of the WBC-enriched product before adding HES and after sedimentation from the supernatant. WBC counts (including differentiation), RBC counts and hematocrit were determined using the Sysmex XT 1800i (Sysmex, TOA, Japan). If the cell count was out of the linear range, samples were diluted using Cell Pack (Sysmex). The percentages of MNC recovery and RBC depletion were calculated. The MNC consists of monocytes and lymphocytes. A visual inspection of the distinction between sediment and supernatant after sedimentation for 90–105 minutes was performed.
Please cite this article in press as: Margriet J. Dijkstra-Tiekstra, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, Janny de Wildt-Eggen, Is hydroxyethyl starch necessary for sedimentation of bone marrow?, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.12.003
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2.6. Statistics
3
†
100
The comparison of four HES/without HES variations was performed 15 times. All other experiments were performed six times. Results are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA for repeated measurements with Dunnett’s post-test for comparison of the HES variations, with 0.6% HAES-steril as reference. Since there is no standard volume for the product, for comparison of volumes a Tukey posttest was used. For comparison of gravity and centrifugation a paired t-test was performed. A p-value <0.05 was used to indicate statistical significance.
MNC recovery, %
* 90
80
70
110
RBC depletion, %
140
170
200
230
Volume WBC-enriched product before sedimentation, mL
a
100
The WBC-enriched product for the experiments to compare four HES/without HES variations had a WBC count of 10.1 ± 0.5 × 109/L, a hematocrit of 8.6 ± 0.9% and a volume of 137 ± 3 mL per bag before addition of HES. Sedimentation of the WBC-enriched product with using 0.6% HAESsteril, 0.6% or 0.39% Voluven or without HES resulted in MNC recoveries varying between 88 ± 6% (0.39% Voluven) and 90 ± 3% (0.6% HAES-steril; p > 0.05; Fig. 1). The RBC depletion varied between 94 ± 2% (0.6% Voluven) and 97 ± 1% (without HES; p < 0.05 for 0.6% HAES-steril compared to 0.6% and 0.39% Voluven; Fig. 1). In one of these experiments the used plasma was fatty. In this experiment MNC recovery and
*
60
3. Results 3.1. The influence of using HAES-steril, Voluven or no HES
*
*
*
90
80
70
60 110 b
140
170
200
230
Volume WBC-enriched product before sedimentation, mL
Fig. 2. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.39% Voluven (striped), and without HES (gray) for various WBC-enriched product volumes. Results are shown as mean ± SD. *p < 0.05.
100
RBC depletion were decreased for the WBC-enriched product in 0.6% and 0.39% Voluven (77 and 73% MNC recovery, and 90 and 92% RBC depletion, respectively), but less for the WBC-enriched product in 0.6% HAES-steril or without HES (89 and 83% MNC recovery, and 95 and 94% RBC depletion, respectively). From these experiments it was decided to continue in the following experiments with 0.6% HAES-steril (reference), 0.39% Voluven and without HES.
MNC recovery, %
90
80
70
60
a
100
*
RBC depletion, %
90
80
70
b
60
Fig. 1. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.6% Voluven (white), 0.39% Voluven (striped), and without HES (gray). Results are shown as mean ± SD. *p < 0.05.
3.2. The influence of the volume of the product before sedimentation The WBC-enriched product used for the experiments to study the influence of volume of the product had a WBC count of 9.0 ± 1.1 × 109/L and a hematocrit of 10.1 ± 0.6% before addition of HES. The results are summarized in Fig. 2. For MNC a trend for lower recovery with increasing volume is shown, but there was only for WBC-enriched product without HES between 110 mL and 200 mL a significant difference. Further 0.6% HAES-steril showed a significantly better recovery compared to 0.39% Voluven at the 200 and 230 mL variations and compared to that without HES at the 140 mL variation. For RBC depletion the volume had no effect. But when the variations in HES were compared it was found that 0.6% HAES-steril showed a significantly higher RBC
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depletion compared to 0.39% Voluven at the 110 and 170 mL variations. In one of the six experiments there was no clear distinction between sediment and supernatant after sedimentation for all HES and volume variations. Since this may occur also in routine circumstances these results were included in the analysis. For this experiment MNC recovery was decreased to 61 ± 10% versus 79 ± 13% for total (all results), whereas RBC depletion was normal (93 ± 4% versus 92 ± 4% for total). There was no clear distinction between sediment and supernatant after sedimentation for the 200 and 230 mL variations independent of the (no) HES variation. 3.3. Sedimentation by centrifugation or gravity Sedimentations by centrifugation and gravity were compared for WBC-enriched product (8.4 ± 1.5 × 109 WBC/L, 9.9 ± 0.1% hematocrit) with 0.6% HAES-steril, 0.39% Voluven and without HES. As shown in Fig. 3, MNC recovery was significantly lower after sedimentation by centrifugation, with a mean of 33–37% versus 83–95% (p < 0.05). For RBC depletion no significant differences were found between both sedimentation methods, with a mean of 89–93% for centrifugation and 79–93% for gravity (p > 0.05). No significant differences were observed between the HES variations. No clear distinction between sediment and supernatant was found for the WBC-enriched product with 0.39% Voluven after sedimentation using centrifugation and in two out of
* 120
MNC recovery, %
100
* *
80 60 40 20 0
a
Centrifugation
Gravity
Centrifugation
Gravity
120
RBC depletion, %
100 80 60 40 20 0 b
Fig. 3. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.39% Voluven (striped), and without HES (gray) for sedimentation using centrifugation or gravity. Results are shown as mean ± SD. *p < 0.05.
six times for the WBC-enriched product with 0.39% Voluven and without HES after sedimentation using gravity. 4. Discussion In this study an alternative was sought for HAES-steril that is used for RBC depletion of BM. As alternatives for HAES-steril Voluven was studied and also sedimentation without HES. The second objective was to optimize the sedimentation process. We used a WBC-enriched product, instead of real BM, which allowed us to perform sedimentations under circumstances which usually would not be tested. Nevertheless, it has to be kept in mind that although the WBC-enriched product has similar cell values it remains different from real BM. However, three times we had the opportunity to perform some experiments with the sediment of real BM, of which two contained a lot of the fatty substance yellow marrow. WBC from whole blood derived buffy coats was added to the sediment. The results of these experiments (not shown) seem to confirm the results with the WBC-enriched product: (1) 0.6% HAES-steril and without HES gave better WBC recovery than 0.39% or 0.6% Voluven. (2). Yellow marrow decreased the WBC recovery. From these results we think the WBC-enriched product is a good and easier available product than BM for use in the optimization of the sedimentation procedure. Additionally, although for this study two-day old material was used, results for MNC recovery and RBC depletion fall in the same range of that for BM products processed in our own processing facility (Table 1) or as is described in literature. For sedimentation using HES, recovery of MNC was between 67% and 90% and RBC depletion between 81% and 98% [6,7,12–15]. All tested HES variations resulted in good MNC recovery and RBC depletion, only small (statistical) differences were shown between sedimentation using 0.6% HAESsteril, 0.6% Voluven, 0.39% Voluven and without HES. However, results of the volume experiments for 140 mL showed lower MNC recovery. As described in the results, in one of six experiments no clear distinction was seen between sediment and supernatant. When excluding the results of this experiment MNC recovery was 89%, 85% and 83% respectively for using HAES-steril, Voluven and without HES respectively, which is close to the comparison experiments. Further, incidentally a low recovery or depletion is found without explanation, which also might explain differences between the experiments. From this it can be concluded that 0.6% HAES-steril can be easy substituted by Voluven, a HES with a lower molecular weight, and even one may ask whether HES is needed for sedimentation. Although adverse effects of HES are not expected for HPC products, because of the low amount that is administered [9], no adverse effects will be found when no HES is used. MNC recovery seemed to be best for sedimentation with 110–140 mL, which is a quite low volume. Probably the smaller surface in the bag is important for a better separation. A disadvantage for sedimentation in small volumes is that more bags are needed for separation, thus a procedure will become more time consumable, and work load will become higher. On the other hand, while for our
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circumstances with a hematocrit of 10% a low volume appeared to be best, in literature sedimentation using larger amounts of BM with a higher hematocrit has been described [12,15]. From this it can be suggested that for sedimentation a combination of volume, hematocrit, used bags and HES might influence the results. In this study, sedimentation by centrifugation did not seem to be an alternative for sedimentation by gravity. Although sedimentation by centrifugation will go about three times faster than sedimentation by gravity, this advantage will have less value when more bags are processed for one patient. In literature in at least two papers centrifugation as a method for sedimentation is described, excluding the cell separators. Linch et al. found with the same centrifugation program (250 g, 10 min) a total nucleated cell (TNC) recovery of about 66–94% (mean 80%) and a RBC depletion of 56–85% (mean 73%). They explained the broad range by a bad distinction between plasma and red cells in half of the cases [16]. Although they found a higher cell recovery, RBC depletion is less compared to our study. This might be due to the method of collecting the HPC. Linch et al. removed the RBC, leaving the HPC in the original bag by centrifigation with the bag turned up-side down, and we collected plasma and buffy coat and kept the RBC in the original bag. Mayer et al. processed BM at 50% hematocrit with two centrifugation steps (g forces and time unknown). They found 84% TNC recovery and about 99% RBC depletion (mean 7.5 mL RBC). In 5 of 42 preparations this had be repeated because of low depletion or recovery [17]. Furthermore, these methods are very subjective, which also explains differences between the studies. In this study we tried to optimize the sedimentation procedure for BM derived HPC. However, further optimization might still be possible. The amount of WBC or TNC might be of influence for the recovery. Also the hematocrit in combination with used volume and form and/or material of the transfer bag might influence this. By using a higher volume and/or hematocrit the number of bags to process will be reduced which will also reduce the workload. Further, for fatty products optimization might be necessary, as we have seen in some experiments. Maybe a method has to be found to get rid of this yellow marrow before starting the sedimentation. Also it might be possible to optimize automatic processing of HPC using the conditions found in this study. In conclusion, sedimentation of BM using HAES-steril (200 kDa), which is no longer available, can be substituted by sedimentation using Voluven (130 kDa) or even by sedimentation without HES. Using a WBC-enriched product MNC recovery seemed to be best for small volumes (110–140 mL), whereas the RBC depletion was not dependent on the volume. Finally, it was found that sedimentation by gravity resulted in a far better MNC recovery and similar RBC depletion compared to sedimentation by centrifugation. However, additional testing using real BM derived HPC might still be necessary to confirm these results.
5
Acknowledgements The authors are grateful to Paul Thijssen, head of the stem cell laboratory, for critical review of the manuscript. The specific contributions of all authors are as follows: AS, MK and EG designed the study under supervision of MD and JW; AS, MK and EG performed the research and analysed the data; MD wrote the paper; AS, MK, EG and JW critically reviewed the paper.
References [1] Korbling M, Champlin R. Peripheral blood progenitor cell transplantation: a replacement for marrow auto- or allografts. Stem Cells 1996;14:185–95. [2] Korbling M, Freireich EJ. Twenty-five years of peripheral blood stem cell transplantation. Blood 2011;117:6411–6. [3] Aurran-Schleinitz T, Chabannon C, Faucher C, Viens P, Cournier S, Maraninchi D, et al. Bone marrow and blood cells as alternative sources of hematopoietic progenitors after failure of a first collection. A single institution retrospective study. Transplantation 1996;61:518–22. [4] Baker PK, Rhodes EG, Duguid JK. Continuous flow cell separator use for bone marrow processing. Transfus Sci 1991;12:183–7. [5] Braine HG, Sensenbrenner LL, Wright SK, Tutschka PJ, Saral R, Santos GW. Bone marrow transplantation with major ABO blood group incompatibility using erythrocyte depletion of marrow prior to infusion. Blood 1982;60:420–5. [6] Ho WG, Champlin RE, Feig SA, Gale RP. Transplantation of ABH incompatible bone marrow: gravity sedimentation of donor marrow. Br J Haematol 1984;57:155–62. [7] Letheby B, Jackson J, Greally J, Warkentin P, Smith D, Vose J, et al. Comparison of two methods of bone marrow processing for autologous bone marrow transplantation. Prog Clin Biol Res 1992;377:345–51. [8] Liu FC, Liao CH, Chang YW, Liou JT, Day YJ. Hydroxyethyl starch interferes with human blood ex vivo coagulation, platelet function and sedimentation. Acta Anaesthesiol Taiwan 2009;47:71–8. [9] Treib J, Baron JF, Grauer MT, Strauss RG. An international view of hydroxyethyl starches. Intensive Care Med 1999;25:258–68. [10] Langeron O, Doelberg M, Ang ET, Bonnet F, Capdevila X, Coriat P. Voluven, a lower substituted novel hydroxyethyl starch (HES 130/0.4), causes fewer effects on coagulation in major orthopedic surgery than HES 200/0.5. Anesth Analg 2001;92:855–62. [11] Booth GS, Gehrie EA, Bolan CD, Savani BN. Clinical guide to ABO incompatible allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2013;19(8):1152–8. [12] Dragani A, Angelini A, Iacone A, D’Antonio D, Torlontano G. Comparison of five methods for concentrating progenitor cells in human marrow transplantation. Blut 1990;60:278–81. [13] Truly M, Williams J, Stearnes L, Wood L, Bowman WP, Friedman DJ. Volume depletion of bone marrow using gravity sedimentation in hydroxyethyl starch for the pediatric allogeneic transplant patient. Prog Clin Biol Res 1992;377:327–32. [14] Smith RJ, Roath S. Bone marrow processing for transplantation using a Fenwal CS3000 Plus cell separator and a revised version of the Georgetown University procedure: comparison with a starch sedimentation method. Prog Clin Biol Res 1994;389:705–10. [15] Warkentin PI, Hilden JM, Kersey JH, Ramsay NK, McCullough J. Transplantation of major ABO-incompatible bone marrow depleted of red cells by hydroxyethyl starch. Vox Sang 1985;48:89–104. [16] Linch DC, Knott LJ, Patterson KG, Cowan DA, Harper PG. Bone marrow processing and cryopreservation. J Clin Pathol 1982;35:186–90. [17] Mayer G, Wernet D, Northoff H, Schneider W. A simple technique for red blood cell removal in major ABO-incompatible bone marrow transplantation. Vox Sang 1994;66:112–6.
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Contents lists available at ScienceDirect
Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Is hydroxyethyl starch necessary for sedimentation of bone marrow? Margriet J. Dijkstra-Tiekstra *, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, Janny de Wildt-Eggen Department Transfusion Monitoring, Sanquin Blood Supply, P.O. Box 1191, 9701 BD Groningen, The Netherlands
A R T I C L E
I N F O
Article history: Received 13 June 2014 Received in revised form 3 October 2014 Accepted 8 December 2014 Keywords: Hydroxyethyl starch Bone marrow HPC WBC-enriched product RBC depletion MNC recovery Volume Gravity Centrifugation
A B S T R A C T
Hydroxyethyl starch (HES) is used to separate hematopoietic progenitor cells after bone marrow (BM) collection from red blood cells. The aims were to study alternatives for HAESsteril (200 kDa; not available anymore) and to optimize the sedimentation process. Using WBC-enriched product (10 × 109 WBC/L), instead of BM, sedimentation at 10% hematocrit using final 0.6 or 0.39% Voluven (130 kDa) or without HES appeared to be good alternatives for 0.6% HAES-steril. MNC recovery >80% and RBC depletion >90% was reached. Optimal sedimentation was reached using 110–140 mL volume. Centrifugation appeared not suitable for sedimentation. Additional testing with BM might be necessary to confirm these results. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hematopoietic progenitor cells (HPC) from patients or donors can be collected either after stimulation using growth factors (e.g. G-CSF) by apheresis or by bone marrow (BM) collection. Although apheresis is the most common method for adult patients [1,2], for some indications the transplant centers prefer BM [3]. Hydroxyethyl starch (HES) can be used to separate the HPC after BM collection from RBC. Sedimentation can be done by gravity or automated machine processing [4–9]. HES with molecular weights ≥200 kDa will become less available on the market because of its adverse effects when used as plasma volume expander. Adverse effects of HES when used as plasma expander are impairment of
* Corresponding author. Sanquin Blood Supply, Division Research, Department Transfusion Monitoring, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. Tel.: +31 50 3610817; fax: +31 50 3610630. E-mail address: [email protected] (M.J. Dijkstra-Tiekstra).
coagulation, renal function impairment, inhibitory effect on platelet function, deterioration of rheological parameters, and accumulation in tissues [8–10]. Although the volume effect is prolonged for higher molecular weight HES compared to lower molecular weight HES, adverse effects of HES are more pronounced for higher molecular weight HES [8–10]. However, no adverse effects are described for HES when used for sedimentation of BM, probably because of the low volumes of HES that are transfused. For HPC from BM, RBC depletion is needed especially in case of AB0 incompatibility, to avoid AB0 induced transfusion reactions. Another reason for RBC depletion is volume reduction to reduce cryogenic storage space or in the case of allogeneic transplantation in the pediatric setting, irrespective of the blood group [11–13]. The standard procedure in our processing facility is as follows: BM is filtrated and diluted to a concentration of 10 × 109 WBC/L and divided over 400-mL bags with 140– 200 mL per bag, after which 9:1 (v/v) HAES-steril 6% (final 0.6%) is added. After sedimentation for 60–90 min the plasma and buffy coat are pressed into a satellite bag (Table 1).
http://dx.doi.org/10.1016/j.transci.2014.12.003 1473-0502/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Margriet J. Dijkstra-Tiekstra, Airies C. Setroikromo, Marcha Kraan, Effimia Gkoumassi, Janny de Wildt-Eggen, Is hydroxyethyl starch necessary for sedimentation of bone marrow?, Transfusion and Apheresis Science (2014), doi: 10.1016/j.transci.2014.12.003
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Table 1 Values for BM processing (n = 11).
Volume, mL WBC, ×109/L Hematocrit, % Dilution factor Volume after dilution, mL WBC after dilution, ×109/La Hematocrit after dilution, %a Number of bags processed Volume per bag, mLb MNC recovery, % RBC depletion, % a b
Mean ± SD
Range
1386 ± 373 17.6 ± 7.9 31.4 ± 6.7 0.6 ± 0.2 2432 ± 1087 9.8 ± 0.9 19.9 ± 7.9 14 ± 6 168 ± 13 88 ± 15 95 ± 2
458–1891 9.7–36.7 21.5–41.7 0.3–1.0 948–4509 7.3–11.1 7.7–34.3 6–24 146–188 67–119 93–99
Calculated from values before dilution and dilution factor. Calculated from total volume and number of bags processed.
The objective of this study was to investigate whether sedimentation of BM using 0.6% HAES-steril (200 kDa/0.5; standard) can be substituted by sedimentation using 0.6% or 0.39% Voluven (130 kDa/0.4) or by sedimentation without HES. The second objective was to optimize the sedimentation process of BM in relation to the volume of the product before sedimentation and sedimentation by centrifugation instead of by gravity.
HES or without HES. For the 140 mL WBC-enriched product 15.6 mL or 9.7 mL HES was added with continuous gentle mixing, to reach a final 0.6% or 0.39% HES respectively. This resulted in four bags, containing (1) WBC-enriched product and 0.6% HAES-steril (standard), (2) WBC-enriched product and 0.6% Voluven, (3) WBC-enriched product and 0.39% Voluven, and (4) WBC-enriched product without HES. The products with 0.6% HAES-steril and 0.39% Voluven have the same molarity for the HES (3 μM). All bags were allowed to sediment by gravity for 90–105 minutes and were placed into a manual press. The supernatant including the buffy coat was pressed into an empty 400 mL transfer bag. This bag was disconnected from the bag containing the sediment that was discarded. After weighing, the bag containing the supernatant was sampled (1 mL sample).
2.3. The influence of the volume of the product before sedimentation Four WBC-enriched products were pooled, mixed and split into 15 portions, three of 110 mL, three of 140 mL, three of 170 mL, three of 200 mL and three of 230 mL. To each bag HES or no HES was added resulting in the variations 0.6% HAES-steril, 0.39% Voluven or without HES for each volume. These bags were allowed to sediment by gravity and the supernatant was collected as described above.
2. Materials and methods 2.1. WBC-enriched product
2.4. Sedimentation by centrifugation or gravity
Because BM derived HPC products are not readily available for research purposes, a WBC-enriched product from buffy coats and plasma, with WBC, RBC and platelet counts in the same range as real BM, but absence of CD34+ cells or immature cells, was used in this study. The preparation of a WBC-enriched product was as follows: Five buffy coats and one plasma unit, derived from overnight stored whole blood, were pooled and centrifuged using a soft spin (930 g for 4.5 min, brake 3). The platelet rich plasma was used to form a therapeutic platelet concentrate. The sediment was stored overnight at RT on a flat bed shaker. Next day, a 2 mL sample was taken and WBC count was determined. The sediment was diluted using plasma until a WBC concentration of about 10 × 109/L and a hematocrit of 7–11% in a total volume of about 600 mL were obtained resulting in the WBC-enriched product. If necessary the plasma was pressed in opposite direction over a WBC reduction filter (Compostop, Fresenius Hemocare; Emmer Compascuum, The Netherlands) that was used for the preparation of the platelet concentrate, to increase the number of WBC.
Two WBC-enriched products were pooled, mixed and split into three portions of 350 mL. To each bag HES or no HES was added resulting in the variations 0.6% HAESsteril, 0.39% Voluven or without HES. Subsequently, each bag was split in a bag containing 185 mL WBC-enriched product with additional HES or without HES and a bag containing 140 mL WBC-enriched product with additional HES or without HES. The bags with 185 mL WBC-enriched product and HES or without HES were centrifuged at 250 g for 10 min at RT and brake at 2. Volume and forces were optimized in pilot experiments (not shown). The bags containing 140 mL WBC-enriched product and HES or without HES were allowed to sediment by gravity and the supernatant of all bags was collected as described above.
2.2. The influence of using HAES-steril, Voluven or no HES The WBC-enriched product was equally divided over four 400-mL transfer bags (about 140 mL per bag; R4R2074; Fenwal, Inc. Lake Zurich, USA). After sampling and weighing the product its volume was calculated and from this the amount of HES necessary to result in either 0.6% or 0.39%
2.5. Tests Samples were taken of the WBC-enriched product before adding HES and after sedimentation from the supernatant. WBC counts (including differentiation), RBC counts and hematocrit were determined using the Sysmex XT 1800i (Sysmex, TOA, Japan). If the cell count was out of the linear range, samples were diluted using Cell Pack (Sysmex). The percentages of MNC recovery and RBC depletion were calculated. The MNC consists of monocytes and lymphocytes. A visual inspection of the distinction between sediment and supernatant after sedimentation for 90–105 minutes was performed.
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2.6. Statistics
3
†
100
The comparison of four HES/without HES variations was performed 15 times. All other experiments were performed six times. Results are presented as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA for repeated measurements with Dunnett’s post-test for comparison of the HES variations, with 0.6% HAES-steril as reference. Since there is no standard volume for the product, for comparison of volumes a Tukey posttest was used. For comparison of gravity and centrifugation a paired t-test was performed. A p-value <0.05 was used to indicate statistical significance.
MNC recovery, %
* 90
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RBC depletion, %
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Volume WBC-enriched product before sedimentation, mL
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The WBC-enriched product for the experiments to compare four HES/without HES variations had a WBC count of 10.1 ± 0.5 × 109/L, a hematocrit of 8.6 ± 0.9% and a volume of 137 ± 3 mL per bag before addition of HES. Sedimentation of the WBC-enriched product with using 0.6% HAESsteril, 0.6% or 0.39% Voluven or without HES resulted in MNC recoveries varying between 88 ± 6% (0.39% Voluven) and 90 ± 3% (0.6% HAES-steril; p > 0.05; Fig. 1). The RBC depletion varied between 94 ± 2% (0.6% Voluven) and 97 ± 1% (without HES; p < 0.05 for 0.6% HAES-steril compared to 0.6% and 0.39% Voluven; Fig. 1). In one of these experiments the used plasma was fatty. In this experiment MNC recovery and
*
60
3. Results 3.1. The influence of using HAES-steril, Voluven or no HES
*
*
*
90
80
70
60 110 b
140
170
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230
Volume WBC-enriched product before sedimentation, mL
Fig. 2. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.39% Voluven (striped), and without HES (gray) for various WBC-enriched product volumes. Results are shown as mean ± SD. *p < 0.05.
100
RBC depletion were decreased for the WBC-enriched product in 0.6% and 0.39% Voluven (77 and 73% MNC recovery, and 90 and 92% RBC depletion, respectively), but less for the WBC-enriched product in 0.6% HAES-steril or without HES (89 and 83% MNC recovery, and 95 and 94% RBC depletion, respectively). From these experiments it was decided to continue in the following experiments with 0.6% HAES-steril (reference), 0.39% Voluven and without HES.
MNC recovery, %
90
80
70
60
a
100
*
RBC depletion, %
90
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Fig. 1. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.6% Voluven (white), 0.39% Voluven (striped), and without HES (gray). Results are shown as mean ± SD. *p < 0.05.
3.2. The influence of the volume of the product before sedimentation The WBC-enriched product used for the experiments to study the influence of volume of the product had a WBC count of 9.0 ± 1.1 × 109/L and a hematocrit of 10.1 ± 0.6% before addition of HES. The results are summarized in Fig. 2. For MNC a trend for lower recovery with increasing volume is shown, but there was only for WBC-enriched product without HES between 110 mL and 200 mL a significant difference. Further 0.6% HAES-steril showed a significantly better recovery compared to 0.39% Voluven at the 200 and 230 mL variations and compared to that without HES at the 140 mL variation. For RBC depletion the volume had no effect. But when the variations in HES were compared it was found that 0.6% HAES-steril showed a significantly higher RBC
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4
depletion compared to 0.39% Voluven at the 110 and 170 mL variations. In one of the six experiments there was no clear distinction between sediment and supernatant after sedimentation for all HES and volume variations. Since this may occur also in routine circumstances these results were included in the analysis. For this experiment MNC recovery was decreased to 61 ± 10% versus 79 ± 13% for total (all results), whereas RBC depletion was normal (93 ± 4% versus 92 ± 4% for total). There was no clear distinction between sediment and supernatant after sedimentation for the 200 and 230 mL variations independent of the (no) HES variation. 3.3. Sedimentation by centrifugation or gravity Sedimentations by centrifugation and gravity were compared for WBC-enriched product (8.4 ± 1.5 × 109 WBC/L, 9.9 ± 0.1% hematocrit) with 0.6% HAES-steril, 0.39% Voluven and without HES. As shown in Fig. 3, MNC recovery was significantly lower after sedimentation by centrifugation, with a mean of 33–37% versus 83–95% (p < 0.05). For RBC depletion no significant differences were found between both sedimentation methods, with a mean of 89–93% for centrifugation and 79–93% for gravity (p > 0.05). No significant differences were observed between the HES variations. No clear distinction between sediment and supernatant was found for the WBC-enriched product with 0.39% Voluven after sedimentation using centrifugation and in two out of
* 120
MNC recovery, %
100
* *
80 60 40 20 0
a
Centrifugation
Gravity
Centrifugation
Gravity
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RBC depletion, %
100 80 60 40 20 0 b
Fig. 3. MNC recovery (a) and RBC depletion (b) for sedimentation using HAES-steril (black), 0.39% Voluven (striped), and without HES (gray) for sedimentation using centrifugation or gravity. Results are shown as mean ± SD. *p < 0.05.
six times for the WBC-enriched product with 0.39% Voluven and without HES after sedimentation using gravity. 4. Discussion In this study an alternative was sought for HAES-steril that is used for RBC depletion of BM. As alternatives for HAES-steril Voluven was studied and also sedimentation without HES. The second objective was to optimize the sedimentation process. We used a WBC-enriched product, instead of real BM, which allowed us to perform sedimentations under circumstances which usually would not be tested. Nevertheless, it has to be kept in mind that although the WBC-enriched product has similar cell values it remains different from real BM. However, three times we had the opportunity to perform some experiments with the sediment of real BM, of which two contained a lot of the fatty substance yellow marrow. WBC from whole blood derived buffy coats was added to the sediment. The results of these experiments (not shown) seem to confirm the results with the WBC-enriched product: (1) 0.6% HAES-steril and without HES gave better WBC recovery than 0.39% or 0.6% Voluven. (2). Yellow marrow decreased the WBC recovery. From these results we think the WBC-enriched product is a good and easier available product than BM for use in the optimization of the sedimentation procedure. Additionally, although for this study two-day old material was used, results for MNC recovery and RBC depletion fall in the same range of that for BM products processed in our own processing facility (Table 1) or as is described in literature. For sedimentation using HES, recovery of MNC was between 67% and 90% and RBC depletion between 81% and 98% [6,7,12–15]. All tested HES variations resulted in good MNC recovery and RBC depletion, only small (statistical) differences were shown between sedimentation using 0.6% HAESsteril, 0.6% Voluven, 0.39% Voluven and without HES. However, results of the volume experiments for 140 mL showed lower MNC recovery. As described in the results, in one of six experiments no clear distinction was seen between sediment and supernatant. When excluding the results of this experiment MNC recovery was 89%, 85% and 83% respectively for using HAES-steril, Voluven and without HES respectively, which is close to the comparison experiments. Further, incidentally a low recovery or depletion is found without explanation, which also might explain differences between the experiments. From this it can be concluded that 0.6% HAES-steril can be easy substituted by Voluven, a HES with a lower molecular weight, and even one may ask whether HES is needed for sedimentation. Although adverse effects of HES are not expected for HPC products, because of the low amount that is administered [9], no adverse effects will be found when no HES is used. MNC recovery seemed to be best for sedimentation with 110–140 mL, which is a quite low volume. Probably the smaller surface in the bag is important for a better separation. A disadvantage for sedimentation in small volumes is that more bags are needed for separation, thus a procedure will become more time consumable, and work load will become higher. On the other hand, while for our
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circumstances with a hematocrit of 10% a low volume appeared to be best, in literature sedimentation using larger amounts of BM with a higher hematocrit has been described [12,15]. From this it can be suggested that for sedimentation a combination of volume, hematocrit, used bags and HES might influence the results. In this study, sedimentation by centrifugation did not seem to be an alternative for sedimentation by gravity. Although sedimentation by centrifugation will go about three times faster than sedimentation by gravity, this advantage will have less value when more bags are processed for one patient. In literature in at least two papers centrifugation as a method for sedimentation is described, excluding the cell separators. Linch et al. found with the same centrifugation program (250 g, 10 min) a total nucleated cell (TNC) recovery of about 66–94% (mean 80%) and a RBC depletion of 56–85% (mean 73%). They explained the broad range by a bad distinction between plasma and red cells in half of the cases [16]. Although they found a higher cell recovery, RBC depletion is less compared to our study. This might be due to the method of collecting the HPC. Linch et al. removed the RBC, leaving the HPC in the original bag by centrifigation with the bag turned up-side down, and we collected plasma and buffy coat and kept the RBC in the original bag. Mayer et al. processed BM at 50% hematocrit with two centrifugation steps (g forces and time unknown). They found 84% TNC recovery and about 99% RBC depletion (mean 7.5 mL RBC). In 5 of 42 preparations this had be repeated because of low depletion or recovery [17]. Furthermore, these methods are very subjective, which also explains differences between the studies. In this study we tried to optimize the sedimentation procedure for BM derived HPC. However, further optimization might still be possible. The amount of WBC or TNC might be of influence for the recovery. Also the hematocrit in combination with used volume and form and/or material of the transfer bag might influence this. By using a higher volume and/or hematocrit the number of bags to process will be reduced which will also reduce the workload. Further, for fatty products optimization might be necessary, as we have seen in some experiments. Maybe a method has to be found to get rid of this yellow marrow before starting the sedimentation. Also it might be possible to optimize automatic processing of HPC using the conditions found in this study. In conclusion, sedimentation of BM using HAES-steril (200 kDa), which is no longer available, can be substituted by sedimentation using Voluven (130 kDa) or even by sedimentation without HES. Using a WBC-enriched product MNC recovery seemed to be best for small volumes (110–140 mL), whereas the RBC depletion was not dependent on the volume. Finally, it was found that sedimentation by gravity resulted in a far better MNC recovery and similar RBC depletion compared to sedimentation by centrifugation. However, additional testing using real BM derived HPC might still be necessary to confirm these results.
5
Acknowledgements The authors are grateful to Paul Thijssen, head of the stem cell laboratory, for critical review of the manuscript. The specific contributions of all authors are as follows: AS, MK and EG designed the study under supervision of MD and JW; AS, MK and EG performed the research and analysed the data; MD wrote the paper; AS, MK, EG and JW critically reviewed the paper.
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