T R A N S P L A N TAT I O N A N D C E L L U L A R E N G I N E E R I N G Cryopreserved stem cell products containing dimethyl sulfoxide lead to activation of the coagulation system without any impact on engraftment Andreas Holbro,1,2 Lukas Graf,1 Maria Topalidou,1 Christoph Bucher,1 Jakob R. Passweg,1 and Dimitrios A. Tsakiris1
BACKGROUND: Dimethyl sulfoxide (DMSO) is extensively used as a cryoprotectant in stem cell preservation. Little is known on direct hemostatic changes in recipients of hematopoietic stem cell transplantation (HSCT), immediately after DMSO administration. The objectives of the current study were to measure hemostatic changes during HSCT. STUDY DESIGN AND METHODS: In this prospective analysis, changes in plasma biomarkers, platelets (PLTs), or endothelial cells (D-dimers, thrombin– antithrombin complex [TAT], microparticle activity as thrombin-generation potential [MPA], whole blood aggregation, von Willebrand factor) were measured before and immediately after HSCT. Furthermore, associations with clinical complications were recorded. RESULTS: A total of 54 patients were included in the study. Mean MPA and TAT increased significantly immediately after HSCT, returning to baseline the day after the procedure (p < 0.01). No significant differences in engraftment for neutrophils and PLTs were found in patients presenting a high increase of TAT or MPA compared with those presenting with a smaller increase. Patients with a high increase in TAT and MPA had received a greater number of total mononucleated cells (p < 0.001) and higher transplant volumes (p = 0.002). CONCLUSIONS: Infusion of stem cells containing DMSO reversibly activated coagulation, measured as thrombin generation. This finding was not associated with acute adverse events and did not influence engraftment. Further studies are needed to compare variable DMSO concentrations as well as DMSO-free products, to better address the influence of DMSO on hemostasis.
1508
TRANSFUSION Volume 54, June 2014
H
ematopoietic stem cell transplantation (HSCT), both autologous and allogeneic, remains the only curative approach for many malignant and nonmalignant diseases.1 Although hematopoietic stem cells (HSCs) can be stored at room temperature or cooled at 2 to 8°C, a progressive loss of viable HSCs occurs. Cryopreservation avoids this progressive loss and allows the elective storage of HSCs for subsequent transplantation. As the formation of ice crystals during cooling is the primary cause of mechanical cell damage, inclusion of cryoprotectants is essential.2 However, cryopreservation has its own toxic effects, such as dehydration during cooling and induction of apoptosis.3 Other factors such as prefreezing processing, plasma protein and solvent content, cooling process, and storage conditions must be considered. An important breakthrough was achieved with the discovery that HSCs could be frozen and thawed using dimethyl sulfoxide (DMSO).4-6 DMSO is extensively used as a cryoprotectant in HSC preservation.7 The optimal concentration of DMSO for this purpose is 5% to 10%.8-10 ABBREVIATIONS: AA = arachidonic acid; DD = D-dimers; FBG = fibrinogen; HSCT = hematopoietic stem cell transplantation; MPA = microparticle activity; TAT(s) = thrombin–antithrombin complex(-es); TRAP = thrombin receptor–activating peptide; VWF:Ag = von Willebrand factor antigen. From the 1Department of Hematology and Diagnostic Hematology, University Hospital Basel; and the 2Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland. Address reprint requests to: Andreas Holbro, MD, Department of Hematology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland; e-mail: [email protected]. Received for publication July 17, 2013; revision received October 14, 2013, and accepted October 22, 2013. doi: 10.1111/trf.12511 © 2013 AABB TRANSFUSION 2014;54:1508-1514.
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
The in vitro properties and toxic effects of DMSO include rapid penetration across biologic membranes, free radical scavenging, and histamine release by mast cells.11,12 Toxicity of DMSO on stem cells, even at concentrations as high as 10%, has been found to be low.13 However, adverse reactions in patients after transfusion have been described, including nausea, vomiting, chills, and fever.14,15 A high incidence of transient and mild pulmonary and cardiovascular events was described by one study.16 A few small-scale studies have also described isolated serious adverse events, such as multiorgan failure, neurologic toxicity, transient ischemic attacks, cardiac arrest, and arrhythmia.17-22 The effects on cell membranes and free radical scavenging implicate an influence of hemostatic mechanisms because circulating cells as well as endothelial cells can be damaged. An in-vitro study showed that DMSO inhibits tissue factor expression and vascular smooth muscle cell proliferation and migration, in a concentration-dependent manner. The same study reported that DMSO prevented thrombus formation in a mouse model.23 Platelet (PLT) recovery and PLT function after cryopreservation in DMSO have been studied, but not in the setting of HSCT.24-27 Little is known about the direct changes in hemostatic and coagulation mechanisms in recipients, immediately after DMSO administration. With the increasing use of cryopreserved stem cell transplants—including single or multiple cord blood stem cell transplants—we aimed to study the direct effects of cryopreserved transplants on hemostatic mechanisms. Thus, the objectives of this study were: 1) to evaluate changes in markers of activation of coagulation, PLTs, and/or endothelial cells (D-dimers [DD], thrombin– antithrombin complex [TAT], microparticle activity [MPA] as thrombin generation potential, whole blood aggregation, von Willebrand factor [VWF]), before and immediately after HSCT, and 2) to investigate any association of these changes with complications of HSCT, including delayed engraftment, as a possible indicator of stem cell damage.
MATERIALS AND METHODS Patients This report is a prospective single-center study of 54 consecutive adult patients who received an autologous or allogeneic HSCT from a DMSO-containing graft at our institution. Patients were transplanted according to the current guidelines or based on a protocol.1 The institutional ethics review board approved the study and all patients signed an informed consent form allowing data collection and analysis. Patients were separately informed and asked about any additional testing that was planned, as described in the informed consent form for the
transplantation. No additional costs were generated for the patients.
Stem cell mobilization and collection Stem cells for patients undergoing autologous HSCT were collected directly after chemotherapy or after vinorelbine and granulocyte–colony-stimulating factor (G-CSF; 5 μg/kg every 12 hr subcutaneously) treatment.28,29 For the four patients who underwent allogeneic HSCT, stem cells from the donors were mobilized using G-CSF (5 μg/kg every 12 hr subcutaneously). The graft was collected using an apheresis machine (Cobe Spectra, TerumoBCT, Lakewood, CO), by large-volume apheresis. In the four patients with allogeneic HSCT, the graft (related familial donor, n = 3; matched unrelated donor, n = 1) was cryopreserved and not directly infused because of patient or donor factors. For the allogeneic unrelated graft, cryopreservation was done after consulting the donor registry (Swiss Blood Stem Cells, Bern, Switzerland).
Graft processing Cell counts and stem cell counts were performed by fluorescence-activated cell sorting using a flow cytometer (FACSCanto, BD Biosciences, Franklin Lakes, NJ) as described elsewhere.30 Peripheral blood stem cells, collected by apheresis and processed within a closed system, were washed with phosphate-buffered saline. The grafts were initially stored for a maximum of 30 minutes at 4°C. Subsequently, DMSO was added to achieve a final concentration of 7.5%. Freezing was achieved with an automated rate controller (1°C/min), and grafts were stored deepfrozen at −194°C in the vapor phase of liquid nitrogen. All procedures were performed according to the standard operating procedure of our accredited laboratory (ISO/IEC 17025 and ISO 15189).
Stem cell transplantation For all patients, transplantation was conducted in an inpatient setting. The conditioning regimen was chosen based on the underlying disease. Grafts were thawed at 37°C at the bedside in the transplant ward and infused within a maximum of 15 minutes without additional processing steps, other than particle filtration. The graft was administered over a central venous catheter (jugular or subclavian vein). No specific pretransplantation medication was applied. Toxicity was monitored continuously until 6 hours after graft infusion and then daily.
Laboratory testing Blood samples were collected in 0.12 mol/L sodium citrate immediately before, 15 minutes after, and 16 to 18 Volume 54, June 2014
TRANSFUSION
1509
HOLBRO ET AL.
hours after HSCT. Plasma aliquots were prepared after centrifugation at 4500 × g for 5 minutes and kept frozen at −70°C for further analysis. Testing included fibrinogen (FBG; Clauss functional method), DD (Liatest, Roche Diagnostics, Rotkreuz, Switzerland), TAT (ELISA Enzygnost TAT micro, Siemens AG, Switzerland), VWF antigen (VWF:Ag; Liatest, Roche Diagnostics), and cell membrane MPA (Zymutest Hyphen, Endotell AG, Basel, Switzerland) expressed as thrombin generation. The latter is a functional assay for the measurement of microparticles’ procoagulant activity in human plasma. Microparticles are immobilized on annexin V and are used as phospholipids for thrombin generation in a purified system. There is a direct relationship between the phospholipid concentration and the amount of thrombin generation in the system. PLT activity was analyzed immediately using whole blood impedance aggregometry (multiplate), in blood samples collected in hirudin as the anticoagulant. We tested PLT aggregation before and immediately after HSCT, with the following agonists (end concentration): adenosine diphosphate (ADP; 6.4 μmol/L), collagen (3.2 μg/mL), thrombin receptor–activating peptide (TRAP; 32 μmol/L), and arachidonic acid (AA; 0.5 mmol/ L), as described elsewhere.31 Results were recorded as area under the curve for the different agonists used.
age at transplantation was 58 years (range, 20-72 years). Multiple myeloma was the primary indication for HSCT (n = 29). Other underlying diseases included lymphoma (n = 14), acute leukemia (n = 5), and others (n = 6). Patient characteristics are summarized in Table 1.
Other supportive measures
Stem cell transplantation
Red blood cells were used to maintain a hemoglobin level of more than 80 g/L, and PLTs were transfused for a count of fewer than 10 × 109/L or fewer than 20 × 109/L in case of fever or infection or bleeding manifestations. All blood products were leukoreduced and irradiated.
Fifty patients (93%) underwent autologous HSCT, while four patients (7%) underwent allogeneic HSCT. Acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin’s lymphoma (n = 1, each) were the underlying diseases among patients receiving allogeneic HSCT. Median volume of the graft was 108 mL (range, 20-280 mL), median total cell count was 35.2 × 109/L (range, 0.44 × 109-99.3 × 109/L), and median CD34 positive cell count was 4.52 × 109/L (range, 1.69 × 109-23.22 × 109/L). Ten patients (19%) experienced immediate infusionrelated adverse events, which included fever and/or urticaria. None of the patients experienced serious adverse events related to the transplant procedure.
Engraftment Neutrophil engraftment after HSCT was defined as the first of 3 consecutive days of an absolute neutrophil count exceeding 0.5 × 109/L. PLT recovery was defined as the first of 3 days with a blood PLT count exceeding 100 × 109/L.
Statistical analysis Nonparametric testing was used for paired (Wilcoxon test) and unpaired (Mann-Whitney test) comparisons or correlations (Pearson). Categorical variables were compared by a chi-square test. Analyses were performed with computer software (IBM SPSS Statistics, Version 19, IBM copyright 1989, 2010, SPSS, Inc., IBM Corp., Armonk, NY).
TABLE 1. Patient characteristics* Number of patients Male Female Disease Multiple myeloma Non-Hodgkin’s lymphoma Acute myelogenous leukemia Systemic sclerosis Hodgkin’s lymphoma Acute lymphoblastic leukemia Amyloidosis CIDP Germ cell tumor Age at transplantation (median, range) Type of HSCT Autologous Allogeneic†
54 33 (61) 21 (39) 29 (54) 14 (26) 4 (7) 2 (4) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 58 (20-72) 50 (93) 4 (7)
* Data are reported as number (%). † Diseases in patients with allogeneic HSCT were as follows: acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin’s lymphoma, n = 1 each. CIDP = chronic inflammatory demyelinating polyneuropathy.
Cell membrane MPA Mean MPA increased significantly immediately after HSCT, returning to baseline the day after (mean ± SD, 16 ± 9 nmol/L at baseline before HSCT, 27 ± 18 nmol/L 15 min after HSCT, and 14 ± 8 nmol/L 16 to 18 hr after HSCT, respectively, p < 0.01; Fig. 1A).
RESULTS Patient characteristics
TATs
Over a time period of 3 years, 54 patients were included in the study, of whom 33 (61%) patients were male. Median
Similar to MPA, the mean TAT significantly increased immediately after HSCT (mean ± SD, 4 ± 2 μg/L at
1510
TRANSFUSION Volume 54, June 2014
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
panied by higher VWF (p = 0.008) and higher DD (p = 0.01) but not MPA, FBG, or TAT changes (calculated as the relative difference from the baseline value). Similarly, clinical events did not correlate with graft volume and hence absolute amount of DMSO.
Engraftment Neutrophil engraftment was seen after a mean ± SD of 15 ± 7 days. PLT recovery Fig. 1. Cell membrane MPA expressed as thrombin generation in nmol/L thrombin was recorded in 34 patients and (A) and TAT in μg/L (B) at baseline (I), 15 minutes after HSCT (II), and 16 to 18 hours occurred after a mean ± SD time of after HSCT (III). Box plots represent median, 50th, and 25th percentiles, and whis37 ± 34 days. No significant differences kers, 5th and 95th percentiles of the distribution, respectively; (○) outliers. Numbers in engraftment for both cell lines were are outliers and refer to patient serial numbers. found in patients presenting a high increase of TAT or MPA (values above the median), meabaseline, 17 ± 13 μg/L immediately after HSCT, p < 0.001) sured as the relative change of the baseline value comand returned to baseline values the day after (mean ± SD, pared with patients with a lower increase (values below 4 ± 4 μg/L, Fig. 1B). median) of these two variables. Patients with a high relative increase in TAT had received significantly greater FBG, DD, and VWF:Ag number of total nucleated cells (29 × 109 ± 17 × 109/L vs. FBG was found slightly lower immediately after transplan53 × 109 ± 21 × 109/L, p < 0.001) and higher transplant tation (mean ± SD, 3.35 ± 0.58 g/L at baseline, 3.22 ± volumes (91 ± 43 mL vs. 137 ± 61 mL, p = 0.002). The same 0.56 g/L immediately after HSCT). DD and VWF:Ag were was seen in patients with high MPA changes (34 × 109 ± slightly higher immediately after transplantation (mean 23 × 109/L vs. 44 × 109 ± 18 × 109/L, p < 0.001; and 101 ± ± SD, 1.02 ± 0.9 μg/mL at baseline, 1.21 ± 1.09 μg/mL 61 mL vs. 123 ± 47 mL, p = 0.32). immediately after HSCT for DD; and 177.34 ± 58.64% at In line with these findings, the relative changes in TAT baseline, 185.66 ± 59.58% immediately after HSCT for and DD correlated weakly but significantly with the total VWF:Ag; Fig. 2). number of infused mononucleated cells (r = 0.5, p < 0.001; and r = 0.5, p < 0.001, respectively).
Whole blood aggregation
Whole blood PLT aggregation was performed in 34 patients. Only a trend for reduction of PLT aggregation with ADP was found immediately after HSCT (mean area under the curve ± SD, 479 ± 258 before HSCT and 425 ± 282 after HSCT). No changes were observed for the other three agonists (collagen, TRAP, and AA; Fig. 3). Table 2 summarizes all our findings. To account for the absolute amount of DMSO, we investigated the above-described variables according to the graft volume. There was no significant difference in MPA and VWF between patients receiving a low volume graft (i.e., lower than the mean volume) and patients receiving a high volume graft (i.e., higher than the mean volume). In contrast, DD and TAT were significantly higher in patients receiving higher graft volume and thus higher absolute DMSO amounts. However, no clear correlation could be ascertained (Pearson [r] = 0.246 and 0.276, respectively).
Adverse events Ten of 54 (19%) patients presented fever and/or urticaria immediately after HSCT. These side effects were accom-
DISCUSSION The discovery of DMSO as a cryoprotectant allowed longterm storage of autologous and allogeneic stem cells.6-8 This widened the opportunities and options for patients needing HSCT and for cellular therapies, but concerns regarding toxicity and side effects were raised because DMSO is volatile, can penetrate cells rapidly, and can cause direct toxic effects on cell membranes. Side effects have been described, including nausea, vomiting, fever and chills, and cardiac events, ranging from mild to severe and life-threatening.14,16 Thrombotic events are well-known complications associated with the acute or late phase of HSCT.32,33 Because DMSO can cause a direct damage to cell membranes, including endothelial cells, it is possible that DMSO activates the coagulation processes after infusion. Large-scale studies on hemostasis, investigating the direct effects of DMSO-containing stem cells, have not been performed in recipients of stem cells. In this single-center study, we observed an activation of hemostasis, measured as increase in MPA (expressed Volume 54, June 2014
TRANSFUSION
1511
HOLBRO ET AL.
Fig. 2. FBG (g/L; A), DD (μg/mL; B), and VWF:Ag (%; C) at baseline (I), 15 minutes after HSCT (II), and 16 to 18 hours after HSCT (III). Box plots represent median, 50th, and 25th percentiles, and whiskers, 5th and 95th percentiles of the distribution, respectively; (○) outliers. Numbers are outliers and refer to patient serial numbers.
the measured MPA. However, having observed that this effect was short-lived, there may not be any permanent damage to the recipient’s endothelium or to other cellular structures. On the other hand, the positive association between the increase in MPA and the number of total cells infused and the volume of the transplant indicates that the latter may be the trigger, because after the expected rapid elimination of the cell particles, thrombin generation ceased. This effect on coagulation did not end in massive or relevant fibrin formation because DD were only marginally increased and FBG was only slightly decreased, excluding a relevant consumption coagulopathy. Unfortunately, we cannot attribute thrombin generation to an in vivo effect of DMSO after infusion because MPA Fig. 3. Whole blood PLT aggregation before (I) and immediately after (II) HSCT using and TAT lack specificity for this phethe following agonists (end concentration): ADP (6.4 μmol/L), AA (0.5 mmol/L), colnomenon. However, DMSO may have lagen (COL; 3.2 μg/mL), and TRAP (32 μM). AUC = area under the curve in arbitrary contributed to the damage of cell strucunits over time. Box plots represent median, 50th, and 25th percentiles, and whistures in the stem cell unit during freezkers, 5th and 95th percentiles of the distribution, respectively. ing and thawing, thus predisposing to thrombin generation. Further, it is not known whether thrombin generation as functional thrombin generation induced by cell could cause damage to the transfused stem cells or to the microparticles) and TAT (expressed as TATs), immediately stem cell niches, thus impairing the homing of stem cells. (i.e., 15 min) after HSCT. This effect was short-lived and We did not observe any indication for such an effect. Both disappeared within 16 hours. Changes in MPA and TAT neutrophil and PLT engraftment were not affected by could mean activation of coagulation and thrombin thrombin generation and remained in the expected range. generation, which in turn can induce cytokine release Adverse reactions to transfusion of cryopreserved with unknown effects on stem cell homing. Increase in HSCs, such as fever and/or urticaria, have been occasioncellular microparticles containing phospholipids, after ally described in the past with DMSO-containing the transfusion of stem cells, indicates cell damage. This products.14-16 An observational multicenter study on this damage can be induced by a soluble component of the transfused product or by the transfused cells themselves. subject, performed by the European Group for Blood and Unfortunately, we cannot assign the clear origin of Marrow Transplantation, has recently concluded and the 1512
TRANSFUSION Volume 54, June 2014
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
TABLE 2. Descriptive statistics of all variables measured (mean ± SD) Variable Before (I) Plasma assays (n = 54) MPA (nmol/L) 16 ± 9 TAT (μg/L) 3.8 ± 2.1 VWF (%) 177 ± 59 DD (μg/mL) 1.0 ± 0.9 APTT (s) 31 ± 9 FBG (g/L) 3.4 ± 0.6 Whole blood aggregation (n = 34) ADP (AUC) 479 ± 258 AA (AUC) 649 ± 262 COL (AUC) 528 ± 267 TRAP (AUC) 750 ± 305
After 15 min (II)
p value* (I-II)
After 16 hr (III)
p value* (I-III)
Normal range
27 ± 18 16.8 ± 13.2 186 ± 60 1.2 ± 1.1 27 ± 3 3.2 ± 0.6
0.002 0.001 0.463 0.224 0.635 0.234
14 ± 8 4.4 ± 4.2 196 ± 62 1.2 ± 1.0 29 ± 3 3.2 ± 0.5
0.209 0.628 0.239 0.363 0.033 0.105
BACKGROUND: Dimethyl sulfoxide (DMSO) is extensively used as a cryoprotectant in stem cell preservation. Little is known on direct hemostatic changes in recipients of hematopoietic stem cell transplantation (HSCT), immediately after DMSO administration. The objectives of the current study were to measure hemostatic changes during HSCT. STUDY DESIGN AND METHODS: In this prospective analysis, changes in plasma biomarkers, platelets (PLTs), or endothelial cells (D-dimers, thrombin– antithrombin complex [TAT], microparticle activity as thrombin-generation potential [MPA], whole blood aggregation, von Willebrand factor) were measured before and immediately after HSCT. Furthermore, associations with clinical complications were recorded. RESULTS: A total of 54 patients were included in the study. Mean MPA and TAT increased significantly immediately after HSCT, returning to baseline the day after the procedure (p < 0.01). No significant differences in engraftment for neutrophils and PLTs were found in patients presenting a high increase of TAT or MPA compared with those presenting with a smaller increase. Patients with a high increase in TAT and MPA had received a greater number of total mononucleated cells (p < 0.001) and higher transplant volumes (p = 0.002). CONCLUSIONS: Infusion of stem cells containing DMSO reversibly activated coagulation, measured as thrombin generation. This finding was not associated with acute adverse events and did not influence engraftment. Further studies are needed to compare variable DMSO concentrations as well as DMSO-free products, to better address the influence of DMSO on hemostasis.
1508
TRANSFUSION Volume 54, June 2014
H
ematopoietic stem cell transplantation (HSCT), both autologous and allogeneic, remains the only curative approach for many malignant and nonmalignant diseases.1 Although hematopoietic stem cells (HSCs) can be stored at room temperature or cooled at 2 to 8°C, a progressive loss of viable HSCs occurs. Cryopreservation avoids this progressive loss and allows the elective storage of HSCs for subsequent transplantation. As the formation of ice crystals during cooling is the primary cause of mechanical cell damage, inclusion of cryoprotectants is essential.2 However, cryopreservation has its own toxic effects, such as dehydration during cooling and induction of apoptosis.3 Other factors such as prefreezing processing, plasma protein and solvent content, cooling process, and storage conditions must be considered. An important breakthrough was achieved with the discovery that HSCs could be frozen and thawed using dimethyl sulfoxide (DMSO).4-6 DMSO is extensively used as a cryoprotectant in HSC preservation.7 The optimal concentration of DMSO for this purpose is 5% to 10%.8-10 ABBREVIATIONS: AA = arachidonic acid; DD = D-dimers; FBG = fibrinogen; HSCT = hematopoietic stem cell transplantation; MPA = microparticle activity; TAT(s) = thrombin–antithrombin complex(-es); TRAP = thrombin receptor–activating peptide; VWF:Ag = von Willebrand factor antigen. From the 1Department of Hematology and Diagnostic Hematology, University Hospital Basel; and the 2Blood Transfusion Centre, Swiss Red Cross, Basel, Switzerland. Address reprint requests to: Andreas Holbro, MD, Department of Hematology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland; e-mail: [email protected]. Received for publication July 17, 2013; revision received October 14, 2013, and accepted October 22, 2013. doi: 10.1111/trf.12511 © 2013 AABB TRANSFUSION 2014;54:1508-1514.
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
The in vitro properties and toxic effects of DMSO include rapid penetration across biologic membranes, free radical scavenging, and histamine release by mast cells.11,12 Toxicity of DMSO on stem cells, even at concentrations as high as 10%, has been found to be low.13 However, adverse reactions in patients after transfusion have been described, including nausea, vomiting, chills, and fever.14,15 A high incidence of transient and mild pulmonary and cardiovascular events was described by one study.16 A few small-scale studies have also described isolated serious adverse events, such as multiorgan failure, neurologic toxicity, transient ischemic attacks, cardiac arrest, and arrhythmia.17-22 The effects on cell membranes and free radical scavenging implicate an influence of hemostatic mechanisms because circulating cells as well as endothelial cells can be damaged. An in-vitro study showed that DMSO inhibits tissue factor expression and vascular smooth muscle cell proliferation and migration, in a concentration-dependent manner. The same study reported that DMSO prevented thrombus formation in a mouse model.23 Platelet (PLT) recovery and PLT function after cryopreservation in DMSO have been studied, but not in the setting of HSCT.24-27 Little is known about the direct changes in hemostatic and coagulation mechanisms in recipients, immediately after DMSO administration. With the increasing use of cryopreserved stem cell transplants—including single or multiple cord blood stem cell transplants—we aimed to study the direct effects of cryopreserved transplants on hemostatic mechanisms. Thus, the objectives of this study were: 1) to evaluate changes in markers of activation of coagulation, PLTs, and/or endothelial cells (D-dimers [DD], thrombin– antithrombin complex [TAT], microparticle activity [MPA] as thrombin generation potential, whole blood aggregation, von Willebrand factor [VWF]), before and immediately after HSCT, and 2) to investigate any association of these changes with complications of HSCT, including delayed engraftment, as a possible indicator of stem cell damage.
MATERIALS AND METHODS Patients This report is a prospective single-center study of 54 consecutive adult patients who received an autologous or allogeneic HSCT from a DMSO-containing graft at our institution. Patients were transplanted according to the current guidelines or based on a protocol.1 The institutional ethics review board approved the study and all patients signed an informed consent form allowing data collection and analysis. Patients were separately informed and asked about any additional testing that was planned, as described in the informed consent form for the
transplantation. No additional costs were generated for the patients.
Stem cell mobilization and collection Stem cells for patients undergoing autologous HSCT were collected directly after chemotherapy or after vinorelbine and granulocyte–colony-stimulating factor (G-CSF; 5 μg/kg every 12 hr subcutaneously) treatment.28,29 For the four patients who underwent allogeneic HSCT, stem cells from the donors were mobilized using G-CSF (5 μg/kg every 12 hr subcutaneously). The graft was collected using an apheresis machine (Cobe Spectra, TerumoBCT, Lakewood, CO), by large-volume apheresis. In the four patients with allogeneic HSCT, the graft (related familial donor, n = 3; matched unrelated donor, n = 1) was cryopreserved and not directly infused because of patient or donor factors. For the allogeneic unrelated graft, cryopreservation was done after consulting the donor registry (Swiss Blood Stem Cells, Bern, Switzerland).
Graft processing Cell counts and stem cell counts were performed by fluorescence-activated cell sorting using a flow cytometer (FACSCanto, BD Biosciences, Franklin Lakes, NJ) as described elsewhere.30 Peripheral blood stem cells, collected by apheresis and processed within a closed system, were washed with phosphate-buffered saline. The grafts were initially stored for a maximum of 30 minutes at 4°C. Subsequently, DMSO was added to achieve a final concentration of 7.5%. Freezing was achieved with an automated rate controller (1°C/min), and grafts were stored deepfrozen at −194°C in the vapor phase of liquid nitrogen. All procedures were performed according to the standard operating procedure of our accredited laboratory (ISO/IEC 17025 and ISO 15189).
Stem cell transplantation For all patients, transplantation was conducted in an inpatient setting. The conditioning regimen was chosen based on the underlying disease. Grafts were thawed at 37°C at the bedside in the transplant ward and infused within a maximum of 15 minutes without additional processing steps, other than particle filtration. The graft was administered over a central venous catheter (jugular or subclavian vein). No specific pretransplantation medication was applied. Toxicity was monitored continuously until 6 hours after graft infusion and then daily.
Laboratory testing Blood samples were collected in 0.12 mol/L sodium citrate immediately before, 15 minutes after, and 16 to 18 Volume 54, June 2014
TRANSFUSION
1509
HOLBRO ET AL.
hours after HSCT. Plasma aliquots were prepared after centrifugation at 4500 × g for 5 minutes and kept frozen at −70°C for further analysis. Testing included fibrinogen (FBG; Clauss functional method), DD (Liatest, Roche Diagnostics, Rotkreuz, Switzerland), TAT (ELISA Enzygnost TAT micro, Siemens AG, Switzerland), VWF antigen (VWF:Ag; Liatest, Roche Diagnostics), and cell membrane MPA (Zymutest Hyphen, Endotell AG, Basel, Switzerland) expressed as thrombin generation. The latter is a functional assay for the measurement of microparticles’ procoagulant activity in human plasma. Microparticles are immobilized on annexin V and are used as phospholipids for thrombin generation in a purified system. There is a direct relationship between the phospholipid concentration and the amount of thrombin generation in the system. PLT activity was analyzed immediately using whole blood impedance aggregometry (multiplate), in blood samples collected in hirudin as the anticoagulant. We tested PLT aggregation before and immediately after HSCT, with the following agonists (end concentration): adenosine diphosphate (ADP; 6.4 μmol/L), collagen (3.2 μg/mL), thrombin receptor–activating peptide (TRAP; 32 μmol/L), and arachidonic acid (AA; 0.5 mmol/ L), as described elsewhere.31 Results were recorded as area under the curve for the different agonists used.
age at transplantation was 58 years (range, 20-72 years). Multiple myeloma was the primary indication for HSCT (n = 29). Other underlying diseases included lymphoma (n = 14), acute leukemia (n = 5), and others (n = 6). Patient characteristics are summarized in Table 1.
Other supportive measures
Stem cell transplantation
Red blood cells were used to maintain a hemoglobin level of more than 80 g/L, and PLTs were transfused for a count of fewer than 10 × 109/L or fewer than 20 × 109/L in case of fever or infection or bleeding manifestations. All blood products were leukoreduced and irradiated.
Fifty patients (93%) underwent autologous HSCT, while four patients (7%) underwent allogeneic HSCT. Acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin’s lymphoma (n = 1, each) were the underlying diseases among patients receiving allogeneic HSCT. Median volume of the graft was 108 mL (range, 20-280 mL), median total cell count was 35.2 × 109/L (range, 0.44 × 109-99.3 × 109/L), and median CD34 positive cell count was 4.52 × 109/L (range, 1.69 × 109-23.22 × 109/L). Ten patients (19%) experienced immediate infusionrelated adverse events, which included fever and/or urticaria. None of the patients experienced serious adverse events related to the transplant procedure.
Engraftment Neutrophil engraftment after HSCT was defined as the first of 3 consecutive days of an absolute neutrophil count exceeding 0.5 × 109/L. PLT recovery was defined as the first of 3 days with a blood PLT count exceeding 100 × 109/L.
Statistical analysis Nonparametric testing was used for paired (Wilcoxon test) and unpaired (Mann-Whitney test) comparisons or correlations (Pearson). Categorical variables were compared by a chi-square test. Analyses were performed with computer software (IBM SPSS Statistics, Version 19, IBM copyright 1989, 2010, SPSS, Inc., IBM Corp., Armonk, NY).
TABLE 1. Patient characteristics* Number of patients Male Female Disease Multiple myeloma Non-Hodgkin’s lymphoma Acute myelogenous leukemia Systemic sclerosis Hodgkin’s lymphoma Acute lymphoblastic leukemia Amyloidosis CIDP Germ cell tumor Age at transplantation (median, range) Type of HSCT Autologous Allogeneic†
54 33 (61) 21 (39) 29 (54) 14 (26) 4 (7) 2 (4) 1 (2) 1 (2) 1 (2) 1 (2) 1 (2) 58 (20-72) 50 (93) 4 (7)
* Data are reported as number (%). † Diseases in patients with allogeneic HSCT were as follows: acute myelogenous leukemia, acute lymphoblastic leukemia, multiple myeloma, and non-Hodgkin’s lymphoma, n = 1 each. CIDP = chronic inflammatory demyelinating polyneuropathy.
Cell membrane MPA Mean MPA increased significantly immediately after HSCT, returning to baseline the day after (mean ± SD, 16 ± 9 nmol/L at baseline before HSCT, 27 ± 18 nmol/L 15 min after HSCT, and 14 ± 8 nmol/L 16 to 18 hr after HSCT, respectively, p < 0.01; Fig. 1A).
RESULTS Patient characteristics
TATs
Over a time period of 3 years, 54 patients were included in the study, of whom 33 (61%) patients were male. Median
Similar to MPA, the mean TAT significantly increased immediately after HSCT (mean ± SD, 4 ± 2 μg/L at
1510
TRANSFUSION Volume 54, June 2014
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
panied by higher VWF (p = 0.008) and higher DD (p = 0.01) but not MPA, FBG, or TAT changes (calculated as the relative difference from the baseline value). Similarly, clinical events did not correlate with graft volume and hence absolute amount of DMSO.
Engraftment Neutrophil engraftment was seen after a mean ± SD of 15 ± 7 days. PLT recovery Fig. 1. Cell membrane MPA expressed as thrombin generation in nmol/L thrombin was recorded in 34 patients and (A) and TAT in μg/L (B) at baseline (I), 15 minutes after HSCT (II), and 16 to 18 hours occurred after a mean ± SD time of after HSCT (III). Box plots represent median, 50th, and 25th percentiles, and whis37 ± 34 days. No significant differences kers, 5th and 95th percentiles of the distribution, respectively; (○) outliers. Numbers in engraftment for both cell lines were are outliers and refer to patient serial numbers. found in patients presenting a high increase of TAT or MPA (values above the median), meabaseline, 17 ± 13 μg/L immediately after HSCT, p < 0.001) sured as the relative change of the baseline value comand returned to baseline values the day after (mean ± SD, pared with patients with a lower increase (values below 4 ± 4 μg/L, Fig. 1B). median) of these two variables. Patients with a high relative increase in TAT had received significantly greater FBG, DD, and VWF:Ag number of total nucleated cells (29 × 109 ± 17 × 109/L vs. FBG was found slightly lower immediately after transplan53 × 109 ± 21 × 109/L, p < 0.001) and higher transplant tation (mean ± SD, 3.35 ± 0.58 g/L at baseline, 3.22 ± volumes (91 ± 43 mL vs. 137 ± 61 mL, p = 0.002). The same 0.56 g/L immediately after HSCT). DD and VWF:Ag were was seen in patients with high MPA changes (34 × 109 ± slightly higher immediately after transplantation (mean 23 × 109/L vs. 44 × 109 ± 18 × 109/L, p < 0.001; and 101 ± ± SD, 1.02 ± 0.9 μg/mL at baseline, 1.21 ± 1.09 μg/mL 61 mL vs. 123 ± 47 mL, p = 0.32). immediately after HSCT for DD; and 177.34 ± 58.64% at In line with these findings, the relative changes in TAT baseline, 185.66 ± 59.58% immediately after HSCT for and DD correlated weakly but significantly with the total VWF:Ag; Fig. 2). number of infused mononucleated cells (r = 0.5, p < 0.001; and r = 0.5, p < 0.001, respectively).
Whole blood aggregation
Whole blood PLT aggregation was performed in 34 patients. Only a trend for reduction of PLT aggregation with ADP was found immediately after HSCT (mean area under the curve ± SD, 479 ± 258 before HSCT and 425 ± 282 after HSCT). No changes were observed for the other three agonists (collagen, TRAP, and AA; Fig. 3). Table 2 summarizes all our findings. To account for the absolute amount of DMSO, we investigated the above-described variables according to the graft volume. There was no significant difference in MPA and VWF between patients receiving a low volume graft (i.e., lower than the mean volume) and patients receiving a high volume graft (i.e., higher than the mean volume). In contrast, DD and TAT were significantly higher in patients receiving higher graft volume and thus higher absolute DMSO amounts. However, no clear correlation could be ascertained (Pearson [r] = 0.246 and 0.276, respectively).
Adverse events Ten of 54 (19%) patients presented fever and/or urticaria immediately after HSCT. These side effects were accom-
DISCUSSION The discovery of DMSO as a cryoprotectant allowed longterm storage of autologous and allogeneic stem cells.6-8 This widened the opportunities and options for patients needing HSCT and for cellular therapies, but concerns regarding toxicity and side effects were raised because DMSO is volatile, can penetrate cells rapidly, and can cause direct toxic effects on cell membranes. Side effects have been described, including nausea, vomiting, fever and chills, and cardiac events, ranging from mild to severe and life-threatening.14,16 Thrombotic events are well-known complications associated with the acute or late phase of HSCT.32,33 Because DMSO can cause a direct damage to cell membranes, including endothelial cells, it is possible that DMSO activates the coagulation processes after infusion. Large-scale studies on hemostasis, investigating the direct effects of DMSO-containing stem cells, have not been performed in recipients of stem cells. In this single-center study, we observed an activation of hemostasis, measured as increase in MPA (expressed Volume 54, June 2014
TRANSFUSION
1511
HOLBRO ET AL.
Fig. 2. FBG (g/L; A), DD (μg/mL; B), and VWF:Ag (%; C) at baseline (I), 15 minutes after HSCT (II), and 16 to 18 hours after HSCT (III). Box plots represent median, 50th, and 25th percentiles, and whiskers, 5th and 95th percentiles of the distribution, respectively; (○) outliers. Numbers are outliers and refer to patient serial numbers.
the measured MPA. However, having observed that this effect was short-lived, there may not be any permanent damage to the recipient’s endothelium or to other cellular structures. On the other hand, the positive association between the increase in MPA and the number of total cells infused and the volume of the transplant indicates that the latter may be the trigger, because after the expected rapid elimination of the cell particles, thrombin generation ceased. This effect on coagulation did not end in massive or relevant fibrin formation because DD were only marginally increased and FBG was only slightly decreased, excluding a relevant consumption coagulopathy. Unfortunately, we cannot attribute thrombin generation to an in vivo effect of DMSO after infusion because MPA Fig. 3. Whole blood PLT aggregation before (I) and immediately after (II) HSCT using and TAT lack specificity for this phethe following agonists (end concentration): ADP (6.4 μmol/L), AA (0.5 mmol/L), colnomenon. However, DMSO may have lagen (COL; 3.2 μg/mL), and TRAP (32 μM). AUC = area under the curve in arbitrary contributed to the damage of cell strucunits over time. Box plots represent median, 50th, and 25th percentiles, and whistures in the stem cell unit during freezkers, 5th and 95th percentiles of the distribution, respectively. ing and thawing, thus predisposing to thrombin generation. Further, it is not known whether thrombin generation as functional thrombin generation induced by cell could cause damage to the transfused stem cells or to the microparticles) and TAT (expressed as TATs), immediately stem cell niches, thus impairing the homing of stem cells. (i.e., 15 min) after HSCT. This effect was short-lived and We did not observe any indication for such an effect. Both disappeared within 16 hours. Changes in MPA and TAT neutrophil and PLT engraftment were not affected by could mean activation of coagulation and thrombin thrombin generation and remained in the expected range. generation, which in turn can induce cytokine release Adverse reactions to transfusion of cryopreserved with unknown effects on stem cell homing. Increase in HSCs, such as fever and/or urticaria, have been occasioncellular microparticles containing phospholipids, after ally described in the past with DMSO-containing the transfusion of stem cells, indicates cell damage. This products.14-16 An observational multicenter study on this damage can be induced by a soluble component of the transfused product or by the transfused cells themselves. subject, performed by the European Group for Blood and Unfortunately, we cannot assign the clear origin of Marrow Transplantation, has recently concluded and the 1512
TRANSFUSION Volume 54, June 2014
CRYOPRESERVED STEM CELLS AND HEMOSTASIS
TABLE 2. Descriptive statistics of all variables measured (mean ± SD) Variable Before (I) Plasma assays (n = 54) MPA (nmol/L) 16 ± 9 TAT (μg/L) 3.8 ± 2.1 VWF (%) 177 ± 59 DD (μg/mL) 1.0 ± 0.9 APTT (s) 31 ± 9 FBG (g/L) 3.4 ± 0.6 Whole blood aggregation (n = 34) ADP (AUC) 479 ± 258 AA (AUC) 649 ± 262 COL (AUC) 528 ± 267 TRAP (AUC) 750 ± 305
After 15 min (II)
p value* (I-II)
After 16 hr (III)
p value* (I-III)
Normal range
27 ± 18 16.8 ± 13.2 186 ± 60 1.2 ± 1.1 27 ± 3 3.2 ± 0.6
0.002 0.001 0.463 0.224 0.635 0.234
14 ± 8 4.4 ± 4.2 196 ± 62 1.2 ± 1.0 29 ± 3 3.2 ± 0.5
0.209 0.628 0.239 0.363 0.033 0.105
