Transfusion and Apheresis Science 51 (2014) 188–192

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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

The loss of CD34+ cells in peripheral hematopoietic stem cell products cryopreserved by non-controlled rate freezing and stored at −80 °C after overnight storage Ayhan Donmez *, Fergun Yilmaz, Nur Soyer, Seckin Cagirgan, Bahar Arik, Murat Tombuloglu Department of Hematology, Ege University Medical School, Izmir, Turkey

A R T I C L E

I N F O

Article history: Received 12 May 2014 Accepted 19 August 2014 Keywords: Cryopreservation Peripheral blood stem cell Overnight storage CD34+ cells

A B S T R A C T

Although peripheral blood stem cell (PBSC) products cryopreserved by non-controlled rate freezing and stored at −80 °C after overnight storage are used frequently, data regarding the rate of loss of CD34+ cells in these products are limited. In this prospective study, CD34+ cells were counted at three (fresh, post-overnight and post-thaw) points in 83 PBSC products from 41 patients by flow cytometry. Compared to fresh products, the mean losses of post-overnight and post-thaw total CD34+ cells are 16.3% and 38.4% (p = 0.02), and the mean losses of post-overnight and post-thaw viable CD34+ cells are 16.5% and 48.5%, respectively (p < 0.001). The numbers of fresh viable, post-thaw total and post-thaw viable CD34+ cells were inversely correlated with the durations of neutrophil and platelet engraftment. Our results indicate that the mean loss of post-thaw total and viable CD34+ cells is approximately 20% higher than that observed in standard cryopreservation methods. In addition, fresh viable, post-thaw total and especially post-thaw viable CD34+ cell levels are valuable predictors of both neutrophil and platelet engraftments. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The standard cryopreservation method for peripheral blood stem cell (PBSC) products is immediate controlledrate freezing after collecting and storage in nitrogen. The range of loss of viable CD34+ cells is 13.6–38.9% [1–3], and the loss of total CD34+ cells is 22.6% [1] by the standard method, as counted by flow cytometry. The post-thaw viable CD34+ cell count is a valuable predictor of hematopoietic stem cell engraftment using this method [1–3]. Because of time, labor and cost effectiveness, PBSC products cryopreserved by no-controlled rate freezing and stored at −80 °C after overnight storage have been increasingly used in many transplantation centers, especially in Turkey. Although the clinical effectiveness of these products

* Corresponding author. Tel.: +902323903563; fax: +902323437876. E-mail address: [email protected] (A. Donmez). http://dx.doi.org/10.1016/j.transci.2014.08.013 1473-0502/© 2014 Elsevier Ltd. All rights reserved.

is documented by different studies [4–7], data regarding the rate of loss of CD34+ cells are limited. In this prospective study, the loss of CD34+ cells in PBSC products cryopreserved by non-controlled rate freezing and stored at −80 °C after overnight storage in the standard refrigerator was investigated. In addition, the relationships between the level of CD34+ cells and the durations of the neutrophil and platelet engraftments were analyzed. 2. Patients and methods 2.1. Subjects In this prospective study, 41 patients who underwent stem cell mobilization in the Hematology Department were enrolled. The subjects consisted of patients with multiple myeloma (n = 23), lymphoma (n = 15, [non-Hodgkin lymphoma = 10, Hodgkin lymphoma = 5]), acute leukemia (n = 2) and amyloidosis (n = 1). The characteristics of the

A. Donmez et al./Transfusion and Apheresis Science 51 (2014) 188–192

Table 1 Characteristics of the patients. Number of patients Sex (female/male) Age (years; median, range) Diagnosis Multiple myeloma Lymphoma (non-Hodgkin/Hodgkin) Acute leukemia Amyloidosis Mobilization method (GCF/GCF + chemotherapy) Disease status at mobilization Complete remission Partial remission Very good partial response Stable disease Conditioning regimen BEAM (carmustine, etoposide, ara-C, melphalan) High dose melphalan Total-body irradiation + cyclophosphamide Not transplanted Engraftment time (days, median; range) Neutrophil Thrombocyte

41 19/22 52 (19–64) 23 15 (10–5) 2 1 20–21 30 5 3 3 12 23 1 5 12 (9–19) 12 (0–35)

mobilized patients are presented in Table 1. The patients included 22 males and 19 females. The median age was 52 years (range, 19–64). The characteristics of the 36 patients who underwent stem cell transplantation are presented in Table 1. Informed consent was obtained from all patients. The study was approved by the local Ethics Committee (Ege University Research Ethics Committee Decision Number: 0.9– 2.1/1), and the procedures followed were in accordance with the Helsinki Declaration. 2.2. Previous therapies In patients with multiple myeloma and amyloidosis, VAD (vincristine, adriamycin, and dexamethasone) ± bortezomib were administered as the initial therapy. In patients with acute myeloid leukemia, cytarabine and idarubicin (7/3) were administered as the induction therapy. In patients with acute lymphoblastic leukemia, the Hoelzer protocol ± prophylactic cranial irradiation was performed as the induction therapy. In patients with non-Hodgkin lymphoma, CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) ± rituximab were administered as the initial therapy. In patients with Hodgkin lymphoma, ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) ± involved field irradiation was administered as the initial therapy. In all relapsed or refractory lymphoma patients, ESHAP (methylprednisolone, cisplatin and cytarabine) or ICE (ifosfamide, carboplatin and etoposide) were administered. Radiation therapies were performed in 12 (one acute lymphoblastic leukemia, two Hodgkin lymphoma, two nonHodgkin lymphoma, and seven multiple myeloma) patients. 2.3. PBSC mobilization Mobilization was performed using G-CSF (10 μg/kg/ day) with or without disease-specific chemotherapies

189

(cyclophosphamide [4 g/m2] in the six multiple myeloma patients; ESHAP [etoposide (40 mg/m2), methylprednisolone (500 mg/m2), cytosine arabinoside (2000 mg/m2), and platinum (25 mg/m 2 )] ± rituximab or ICE [ifosfamide (5000 mg/m2), carboplatin (500 mg/m2), and etoposide (100 mg/m 2 )] ± rituximab or 7704 [cyclophosphamide (1200 mg/m2), methotrexate (3000 mg/m2), vincristine (1.4 mg/m2), adriamycin (40 mg/m2), prednisone (40 mg/ m 2 )] in the 11 lymphoma patients; high dose ARA-C (6000 mg/m2) or hyperCVAD [(cyclophosphamide (600 mg/ m 2 ), vincristine (2 mg), doxorubicin (50 mg/m 2 ), and dexamethasone (40 mg)] in three acute leukemia patients). 2.4. Collection of PBSC In patients receiving only G-CSF, apheresis was initiated on the fifth day. In patients receiving chemotherapy plus G-CSF, apheresis was initiated when the circulating CD34+ cell count was higher than 10/μL. Leukapheresis was performed using a fully automated apheresis system (Comtec, Fresenius, Waltham, MA). 2.5. Overnight storage and PBSC cryopreservation Leukapheresis products were stored overnight in a commercial refrigerator without any added medium with gentle shaking every 2 h. The median overnight-storage time was 21 (range, 17–23.3) h (Table 2). After overnight storage, leukapheresis products were diluted with autologous plasma to obtain the target nucleated-cell concentration of 200 × 109/L. Cryopreservation media (15% dimethyl sulfoxide [WAK-Chemie Medical, Steinbach/Ts., Germany], 6% hydroxy ethyl starch [Expahes, Baxter], and 25% autologous plasma) were prepared separately. The diluted product and cryopreservation media were mixed at a ratio of 1:1 in the bags (Cryocyte Freezing Container, 500 mL, Nexcell; CryoMACS freezing bag 500, Miltenyl Biotech; Cryostore CS 500 ns, Origen Biomedical). The final products, including 7.5% dimethyl sulfoxide and 3% hydroxy ethyl starch in bags with nucleated-cell concentrations of 100 × 109/L, were placed in a −80 °C mechanical freezer (Sanyo, Japan) after the microbiological culture sample was obtained. The products were held (median 80 days, range 13−306) at −80 °C until reinfusion (Table 2). 2.6. High-dose chemotherapy BEAM (BCNU 300 mg/m2; etoposide 200 mg/m2/day, 4 days; ara-C 400 mg/m2/day, 4 days; melphalan 140 mg/ m2) was administered to 12 patients with Hodgkin and nonHodgkin lymphoma. High-dose melphalan (200 mg/m2) was administered to 22 multiple myeloma patients and one patient with amyloidosis. One patient with acute

Table 2 Features of peripheral blood stem cell products. Total number of products Duration of overnight storage (hour, median, range) Duration of storage at −80 °C (day; median, range)

83 21 (17–23.3) 80 (13–306)

A. Donmez et al./Transfusion and Apheresis Science 51 (2014) 188–192

leukemia was treated with cyclophosphamide (120 mg/ kg), etoposide (40 mg/kg) and total-body irradiation, with a total dose of 12 Gy. Five patients have not yet undergone transplant (Table 1). 2.7. Infusion of PBSCs and patient monitoring At 24–48 h after the end of conditioning, the patients were premedicated with antiemetic, corticosteroid and antihistaminic agents. The autologous cryopreserved bags were thawed in a 37 °C water bath at the bedside and the thawed products were immediately re-infused using standard transfusion filters. All of the patients received 5 μg/kg G-CSF per day, starting on the fifth day after transplantation. The irradiated blood components were infused to maintain hemoglobin and platelet levels above 8 g/dL and 20 × 109/L, respectively. Throughout the aplastic phase, all of the patients received antiviral and antifungal prophylaxis. Patients who developed neutropenic fever were treated according to the established guidelines. The day of neutrophil and platelet engraftment was defined as the first of three consecutive days on which the neutrophil and platelet counts exceeded 0.5 × 109/L and 20 × 109/L (without a need for platelet transfusion), respectively. 2.8. PBSC samples To obtain PBSC samples from the three different (fresh harvest, after overnight storage [before freezing] and postthaw) periods, 2 mL was drawn into tubes containing EDTA (Vacuette K2 EDTA). PBSC samples were analyzed without delay. 2.9. Viability measurement by trypan blue exclusion staining The cell suspension was diluted with normal saline, mixed with an equal volume of (0.4%) trypan blue (SigmaAldrich, St. Louis, MO, USA). At least 100 cells were microscopically analyzed for viability in a hemocytometer chamber. 2.10. CD34+ enumeration The single-platform flow cytometric absolute CD34+ cell counts were performed as previously described [8]. Briefly, 100 μL of the PBSC sample was mixed with 5 μL CD34/PE (clone 4H11), 5 μL CD45/FITC (clone 2D1) and 20 μL 7-amino-actinomycin D (7-AAD, BD Pharmingen) in TRUCOUNT tubes (BD Bioscience). Tubes were incubated for 20 min at room temperature in the dark. Then, 2 mL Pharm lyse (BD Biosciences) was added to lyse the red blood cells and were than incubated for an additional 10 min at room temperature in the dark. After incubation, the sample was analyzed by flow cytometry (FACSAria, BD Biosciences, 2007, USA), using BD FACS Diva Software 6.1.3. The results were reported as the absolute number of CD34+ cells. Total CD34+ cells were counted irrespective of 7-ADD, while viable CD34+ cells were counted as CD34-positive and 7-ADDnegative cells.

6000 Viable CD34+ cell count (/µL)

190

4000

2000 1056

882.8

544.1

0 Fresh

Post-overnight

Post-thaw

Fig. 1. The level of viable CD34+ cells (mean [—]), according to the cryopreservation period (p = 0.001).

2.11. Statistics Statistical analysis was conducted using Prism 4.03 (GraphPad Software, San Diego, CA, USA). The Kolmogorov– Smirnov and D’Agostino–Pearson tests showed that the results were not in a normal Gaussian distribution. Therefore, we used nonparametric tests. The two groups were compared by the Mann–Whitney test. Three or more groups were compared by Kruskal–Wallis test and Dunn’s posttest. Correlations were assessed by the Spearman test. P-values