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Cryopreservation As a Way to Maintain Extracorporeal Photopheresis Regimen for Gvhd Treatment While Circumventing Patient Temporary Inability to Undergo Apheresis

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Cryopreservation As a Way to Maintain Extracorporeal Photopheresis Regimen for Gvhd Treatment While Circumventing Patient Temporary Inability to Undergo Apheresis Bone Marrow Transplantation (2017) 52, 167–170 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0268-3369/17 www.nature.com/bmt LETTER TO THE EDITOR Cryopreservation as a way to maintain extracorporeal photopheresis regimen for GvHD treatment while circumventing patient temporary inability to undergo apheresis Bone Marrow Transplantation (2017) 52, 167–170; doi:10.1038/ Nucleated cells were quantified using the pocH-100i device bmt.2016.240; published online 19 September 2016 (Sysmex, Villepinte, France). Cell viability was analyzed by flow cytometry using 7-aminoActinomycin D. CD3+ and CD14+ cells were quantified by FACS analysis, only in the Nancy center. Extracorporeal photopheresis (ECP) is a safe treatment in patients In both centers, the same treatment regimen was used with at with mild acute or chronic GvHD.1–3 However, its clinical use is least 3 ECP (fresh or ‘cryo-ECP’) per week for acute GvHD, and 1 sometimes limited by logistical problems that compromise or 2 ECP per week (depending on GvHD grade) for chronic or late repeated aphereses, especially venous access failure. acute GVHD, until clinical improvement. Merlin et al.4 previously concluded that cryopreservation The clinical severity of acute GvHD was determined as defined 5 before irradiation did not impair WBC functional capabilities. in the 1994 consensus conference criteria. The severity of chronic The hematopoietic stem cell transplantation (HSCT) centers of GvHD was determined as per the 2005 National Institutes of 6 Nancy and Clermont-Ferrand (CF) University Hospitals Health (NIH) consensus guidelines. Response was retrospectively investigated the use of frozen and thawed WBC (FTWBC, assessed at 1, 3 and 6 months in all patients. Clinical CR was ‘cryo-ECP’) in a series of GvHD patients. defined as GvHD disappearance, PR as a GvHD grade regression Patients were eligible for ECP as a second- to fourth-line and no remission (No R) as a GvHD progression on ECP treatment. treatment for acute or chronic GvHD. A portion of the WBC For each patient, 3 series corresponding to either ‘total fresh’, collected by apheresis was cryopreserved for patients presenting ‘divided fresh’ or ‘thawed’ treated WBC were distinguished. temporary inability to undergo apheresis, and used each time The mean of all series was analyzed using the non-parametric apheresis was not possible. Informed consent was obtained from Wilcoxon test. The correlation between variables was analyzed all patients and/or parents. The Ethics Committees of Nancy and using the non-parametric Spearman test. Statistical significance CF University approved the conduct of this retrospective study. was set for a P-value o0.05. ECP was performed using the off-line system as previously From 2009 to 2015, 231 ‘cryo-ECP’ (including 83 in Nancy described.3 Cells were collected on a commercially available Hospital and 148 in CF Hospital) were performed in 20 patients apheresis system (Spectra by Caridian BCT, Comtec by Fresenius including 10 males. Thirteen patients (8 children) were treated and Optia by Terumo BCT). The WBC-rich fraction collected was for acute GvHD (Patients 1–13). Seven patients (5 children) divided into two or three equal fractions, depending on the were treated for chronic GvHD (patients 14–20, Table 1). Skin was volume collected. involved in 15 patients. One fraction was treated, i.e., diluted with 0.9% NaCl to a Indications for FTWBC infusions were venous access failure hematocrit ⩽ 1%, and incubated for 3 min with 0.06 mg of (n=8), bacterial infections (n=6), very poor medical condition 8-methoxypsoralen (MOP) (Uvadex). Cells were transferred into (n=3), deep cytopenia (n=6), bad compliance (n=1) and patients an ultraviolet A (UVA)-permeable bag (Macopharma, Tourcoing, living too far away from the HSCT center (n=4) whose treated France) then exposed to UVA light (315-400 nm) at 2 J/cm2 for cells were transported to a hospital near their home (Table 1). The 15 min (Bio Genic Vilber Lourmat technology or Macogenic, median number of ‘classic fresh’ ECP before first cryopreservation Macopharma). The cellular product was immediately reinfused in was of 5 (0–85) sessions. The 20 patients received a median to the patient. number of 8.5 FTWBC infusions (1–36), corresponding to a median The remaining one or two fractions collected were frozen, rate of 30% (1–69%) of cryo-ECP (Table 2). The median time according the protocol used in each center for hematopoietic between cryopreservation and reinfusion was of 11.5 (1–116) days. stem cells (HSC) cryopreservation in clinical routine: in Nancy, cells ‘Split’ sessions (either thawed or fresh) represented a median were frozen in a cryoprotective solution at a final concentration of proportion of 68% of the total number of ECP sessions (2–100%). 10% of DMSO (WAK-chemie Medical GmbH, Steinbach/TS, No adverse event occurred after the 231 FTWBC infusions. Germany) and 40% of human serum albumin (4%, HSA, LFB, Les The mean number of FTWBC infused to each patient was of Ulis, France), exposed to a controlled cooling temperature 26.2 × 106 WBC/kg (±21.6), versus 33.6 × 106 WBC/kg (±24.7) program up to − 100 °C and then stored in nitrogen vapor at for fresh cells after cell product division (P=0.008), and versus − 150 °C. In CF, cells were frozen in a solution with final concen- 56.2 × 106 WBC/kg (±44.9) for total fresh cells (conventional trations of 3.5% DMSO, 1% HSA and 2.5% hydroxyethylamidon off-line ECP; P=0.003). (HES) (Voluven, Fresenius Kabi, Bad Homburg, Germany) for The mean WBC viability was of 98% ± 1.5% versus 84% ± 8.7% uncontrolled rate freezing and stored at − 80 °C in mechanical for fresh and thawed cells after UVA-irradiation, respectively freezers. (P=0.003). The mean number of CD3+ and CD14+ cells infused in On a subsequent day, cells were thawed in a 37 °C water bath, to each patient in Nancy center was similar before and after washed with a solution containing 50% of HSA (4%), 10% of A cryopreservation: 8.5 × 106 CD3+ cells/kg (±14.0) for FTWBC, versus formula acid citrate dextrose (ACD) solution and 40% of 0.9% NaCl, 10.4 × 106 CD3+ cells/kg (±14.1) for fresh cells after cell product with a cellular suspension rate of 33%, then centrifuged (400 g, for division (P=0.515); 7.1 × 106 CD14+ cells/kg (±8.6) 15 min at 10 °C). The cells were suspended in 250 ml of 0.9% NaCl for FTWBC versus 6.5 × 106 CD14+ cells/kg (±6.5) for fresh divided and UVA-irradiated in presence of 8-MOP, then immediately cells (P=0.260). The mean viability of treated CD3+ cells after reinfused in to the patient. cryopreservation was 92% ± 9.3%. Letter to the Editor 168 Table 1. ECP indications No Age GvHD Location Date of Pre-ECP gradea Date of ECP Line of Rationale for cryopreservation GvHD onset onset (days) treatment (days) 1 13.5 Late acute Gut 570 Grade II (0-1-0) 694 Third Poor peripheral venous access 2 53 Acute Skin 26 Grade II (3-0-0) 83 Third Recurrent septicemias, severe cytopenia 3 14 Recurrent Gut/skin 31 then 130 Grade II (2-1-0) 122 Second Severe cytopenia acute 4 49 Acute Skin/gut/liver 38 Grade IV (4-1-1) 70 Third Severe cytopenia Central line infection, poor medical condition (OMS score = 4) 5 63 Acute Skin/gut 76 Grade IV (4-1-0) 84 Second Severe cytopenia, poor medical condition (OMS score = 4) 6 41 Acute Skin/gut Skin 47 Grade IV (4-1-0) Grade II 65 Third Central line dysfunction and infection Recurrent 201 (2-0-0) 206 acute 7 12 Recurrent Gut 385 Grade II (0-1-0) 404 Second Central line dysfunction acute 8 5 Acute Skin/gut 10 Grade IV (4-4-0) 31 Third Central line infection 9 9 Recurrent Skin/gut 7 Grade II (3-2-0) 174 Third Central line dysfunction acute 10 9 Acute Skin/gut 41 Grade II (1-2-0) 44 Second Distant medical center Central line dysfunction 11 21 Acute Skin 14 Grade I (2-0-0) 83 Third Central line dysfunction 12 2 Acute Skin 12 Grade I (1-0-0) 46 Second Central line dysfunction 13 15 Acute Skin/liver/lung 28 Grade III (3-2-2) 28 Second Weak compliance to an intensive treatment 14 5 Chronic Skin/joints/liver 420 Severe chronic GvHD 724 Third Central line infection Distant medical (sclerodermic) center 15 58 Chronic Liver 350 Severe chronic GvHD 415 Second Pneumocystosis with Liver: grade 3 oxygenotherapy, deep fatigue 16 16 Chronic Liver 150 Severe chronic GvHD 182 Third Central line infection Liver: grade 3 17 4 Chronic Skin/joints MD Severe chronic GvHD (joints 1515 Third Central line dysfunction and fascia: 3) 18 12 Chronic Joints/muscles 150 Moderate chronic GvHD joints 370 Fourth Distant medical center and fascia: 2 19 11 Chronic Skin 47 Severe chronic GvHD 449 Third Distant medical center (fascia/joints: 3 and mouth: 1) 20 23 Chronic Skin 270 Severe chronic GvHD 479 Fourth Poor peripheral venous access (sclerodermic skin 3) Abbreviations: CyA = cyclosporin A; ECP = extracorporeal photopheresis; MD = missing data; MMF = mycophenolate mofetil. aAcute GvHD is graded according to the Glücksberg classification,5 and chronic GvHD is classified following NIH recommendations.6 We observed a significant correlation between the circulating (45% CR and 18% PR). Patients with lower grade acute GvHD monocyte and lymphocyte counts and the mean number of showed a better response to ECP than those with higher grade thawed CD14+ cells (P=0.019), and thawed CD3+ cells (P=0.002) acute GvHD.
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