Bone Marrow Transplantation (2014) 49, 1127–1128 © 2014 Macmillan Publishers Limited All rights reserved 0268-3369/14 www.nature.com/bmt

LETTER TO THE EDITOR

Automated washing of autologous hematopoietic stem cell grafts after thawing does not impair engraftment Bone Marrow Transplantation (2014) 49, 1127–1128; doi:10.1038/ bmt.2014.111; published online 2 June 2014

Autologous hematopoietic SCT (AHSCT) remains widely used to support the administration of high-dose chemotherapy to a subset of patients affected by malignant blood diseases, mostly lymphoid malignancies. Nowadays, patients are almost exclusively transplanted with peripheral blood grafts, collected by apheresis, after mobilization of hematopoietic progenitor with various regimens. Cryopreservation and storage of collected cell products in liquid- or gas-phase nitrogen are mandatory before reinfusion. The historical but still topical technique of cryopreservation uses DMSO as cryoprotectant in order to prevent intracellular ice crystal formation, thus preserving cell viability and function. Reinfusion of the graft thawed at the bedside might be associated with various side effects,1 ranging from mild to severe. The etiology of these adverse effects is generally attributed to DMSO, damaged cells or cell lysis products. In order to remove such byproducts, biomedical devices like Cytomate (Baxter, Deerfield, IL, USA) or Sepax (Biosafe, Eysins, Switzerland) have been developed for washing thawed grafts before reinfusion. Additionally, cell therapy facilities are under increasing regulatory pressure to better characterize and control the cell product to be reinfused, which can only be done to a limited extent when thawing at the bedside. However, washing thawed grafts before infusion remains controversial2 since CD34+ progenitor cell loss is unavoidable and might induce delayed engraftment.3 This is in opposition to the process of bedside thawing followed by direct infusion, where cell loss is supposed to be lower but remains unknown due to the absence of quality control on the thawed graft. This controversy therefore remains hypothetical, as there is, to our knowledge, no matched analysis comparing the end point of both procedures, that is, hematopoietic reconstitution. In order to determine if cell loss associated with the washing step has a detrimental effect on engraftment, we retrospectively compare two cohorts of carefully matched patients, who received either washed or unwashed autologous grafts. Between January 2000 and December 2004, 1360 AHSCTs were performed at our institution; we selected 364 AHSCTs for which (a) grafts were thawed at the bedside in a 37 °C water bath and directly infused and (b) hematopoietic reconstitution raw data were available in the electronic data management system. We then picked 218 AHSCTs that fall within four diagnosis groups for which intensive chemotherapy regimens were homogeneous (see Table 1); among this group we retained the 65 unwashed AHSCTs for which cryopreserved CD34+ cell doses were within the therapeutic range of 2.0–5.0 × 106/kg. Then, among the 1570 AHSCTs performed between January 2005 and December 2010, we selected 580 AHSCTs for which (a) hematopoietic reconstitution raw data was available, (b) patients received the same four intensive regimens as the unwashed group and (c) grafts were homogeneously processed (dry thawing at 37 °C followed by washing on the CytoMate device); among these, we retained the 260 washed AHSCTs with cryopreserved CD34+ cell doses between 2.0–5.0 × 106 CD34+/kg. Engraftment data were controlled twice for each group.

For both groups, hematopoietic progenitors were collected by apheresis after mobilization by either chemotherapy and rhG-CSF (Neupogen, Amgen, Thousand Oaks, CA, USA or Granocyte, Chugai Pharmaceutical, Tokyo, Japan) or rhG-CSF alone. Cryopreservation was performed following the addition of an equal volume of 20% DMSO in either 4% human albumin or hydroxyethyl starch (Voluven, Fresenius Kabi AG, Bad Homburg, Germany). After controlled-rate freezing, grafts were stored in gasphase nitrogen. Following AHSCT, only those patients who had o2 × 106 viable CD34+/kg received rhG-CSF. In order to compare these two groups of AHSCT, we used a case-match design with one-to-two matching between the 65 unwashed AHSCTs and the 260 matches from the washed AHSCT cohort using the macro ‘%match’ developed by the Mayo Clinic.4 Briefly, for each unwashed case (i) two unwashed control cases (j) closest to the case in terms of Dij were selected. Dij is the weighted sum of the absolute differences between the case and control matching factors (that is, Dij = SUM{W.k*ABS(X.ik − X.jk)}, where the sum is over the number of matching factors X (with index k), W.k is the weight assigned to matching factor k and X.ik the value of variable X(k) for subject i). The unwashed controls (j) selected for a washed case (i) are the two with the smallest Dij. The higher the weight of a matching factor, the more likely it is that the case and control will be matched on the factor. We used four matching factors: diagnosis, cryopreserved CD34+ cell dose, age at treatment and sex, with relative weights of 200, 10, 2 and 1, respectively. Descriptive statistics are based on the frequencies and percentages for categorical variables and on the mean (s.d.), median and extreme for continuous variables. Categorical data were compared using a χ2 test and continuous data through a Wilcoxon test. The case-match analysis exhibits, as expected, no significant difference in terms of age, sex ratio, diagnosis and CD34+ cryopreserved cell dose (Table 1). Median time to reach Table 1.

Patients, autografts and clinical characteristics

Sex (M/F) Age (median (range))

Unwashed (n = 65)

Washed (n = 130)

P-value

30/35 55.3 (17–71)

52/78 56.7 (23–71)

0.41 0.74

97 (74.6)

0.15

23 (17.7) 2 (1.5) 8 (6.2) 3.8

0.29

Diagnosis/high-dose chemotherapy (n (%)) Plasma cell disorders/ 48 (73.8) melphalan Lymphoma/BEAM 8 (12.3) Acute leukemia/BU-MEL 5 (7.7) Solid tumors/CY-MEL 4 (6.2) + 3.7 Number of CD34 cells cryopreserved (106/kg) Patients according to CD34+ cell dose (n) (2; 3.5) 27 (3.5; 5) 38 Days to neutrophils >0.5 g/L 12.4 ± 1.4 (median ± s.d. (range)) (10–15)

44 86 12.5 ± 1.6 (8–17)

Abbreviations: F = female; M = male; MEL = melphalan.

0.29 0.67

Letter to the Editor

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0.5 × 109/L circulating neutrophils between the two groups is comparable, with 12.4 (10–15) and 12.5 (8–17) days in the unwashed and washed groups, respectively (P = 0.67). Our study highlights that washing peripheral blood cell products after thawing does not impair hematopoietic engraftment as compared to bedside thawing. This observation is not surprising if we consider that progenitor cell loss occurs even without washing, while the thawed cell products are being infused in to the patient, owing to the detrimental effect of the toxicity of DMSO at room temperature.2 Therefore, the latter is probably comparable to cell loss associated with the washing step. Although the size of our groups is rather small, the approach we developed, based on a rigorous one-to-two matching analysis, allowed us to compare two matched cohorts issued from 2930 AHSCTs performed over 10 years. This accurate matching leads us to conclude that processing hematopoietic stem cell grafts in the laboratory does not compromise hematopoietic engraftment as previously reported in unmatched smaller series of AHSCT.3,5,6 Moreover, using automated devices such as Cytomate or Sepax allows for better standardization and improved stability of thawed cell products,7 as well as precise determination of infused CD34+ cell dose, and might therefore be preferred over bedside thawing. CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Frederick Christy at the Département de la Recherche Clinique et de l'Innovation, Institut Paoli-Calmettes, for data management, and the staff of the Centre de Thérapie Cellulaire, Institut Paoli-Calmettes, for careful management of cell products.

AUTHOR CONTRIBUTIONS BC and CC conceived and designed the study; AA, BC and AD analyzed and interpreted the data; BC, AD, CH, CM, CL, A-MS, RB and DC provided study materials or patients; BC, AD and CC wrote the manuscript; all authors reviewed and approved the manuscript.

Bone Marrow Transplantation (2014) 1127 – 1128

B Calmels1,2,3,7, A Drezet3,7, C Huynh1, A Autret4, A-M Stoppa5, R Bouabdallah5, D Coso5, C Malenfant1, C Lemarié1,2 and C Chabannon1,2,3,6 1 Centre de Thérapie Cellulaire, Département de Biologie du Cancer, Institut Paoli-Calmettes, Marseille, France; 2 Inserm CBT-510, Centre d’Investigations Cliniques en Biothérapie, Marseille, France; 3 Inserm UMR 1068, CNRS UMR 7258, AMU 105, Centre de Recherche en Cancérologie de Marseille, Marseille, France; 4 Unité de Biostatistique et de Méthodologie, Département de la Recherche Clinique et de l’Innovation (DRCI), Institut Paoli-Calmettes, Marseille, France; 5 Département d’Onco-Hématologie, Institut Paoli-Calmettes, Marseille, France and 6 Aix-Marseille Université (AMU), Marseille, France E-mail: [email protected] 7 These authors contributed equally to this work. REFERENCES 1 Cox MA, Kastrup J, Hrubisko M. Historical perspectives and the future of adverse reactions associated with haemopoietic stem cells cryopreserved with dimethyl sulfoxide. Cell Tissue Bank 2012; 13: 203–215. 2 Shu Z, Heimfeld S, Gao D. Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant 2013; 49: 469–476. 3 Akkok CA, Holte MR, Tangen JM, Ostenstad B, Bruserud O. Hematopoietic engraftment of dimethyl sulfoxide-depleted autologous peripheral blood progenitor cells. Transfusion 2009; 49: 354–361. 4 Bergstralh EJ, Kosanke JL, Jacobsen SJ. Software for optimal matching in observational studies. Epidemiology 1996; 7: 331–332. 5 Sanchez-Salinas A, Cabanas-Perianes V, Blanquer M, Majado MJ, Insausti CL, Monserrat J et al. An automatic wash method for dimethyl sulfoxide removal in autologous hematopoietic stem cell transplantation decreases the adverse effects related to infusion. Transfusion 2012; 52: 2382–2386. 6 Syme R, Bewick M, Stewart D, Porter K, Chadderton T, Gluck S. The role of depletion of dimethyl sulfoxide before autografting: on hematologic recovery, side effects, and toxicity. Biol Blood Marrow Transplant 2004; 10: 135–141. 7 Lemarie C, Calmels B, Malenfant C, Arneodo V, Blaise D, Viret F et al. Clinical experience with the delivery of thawed and washed autologous blood cells, with an automated closed fluid management device: CytoMate. Transfusion 2005; 45: 737–742.

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Automated washing of autologous hematopoietic stem cell grafts after thawing does not impair engraftment.

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