Preliminary Study On Safety And Efficiency Of Peripheral Blood Stem Cells Collected By Different Blood Cell Separators

Background:Granulocyte-colony-stimulating factor(G-CSF)-mobilized peripheral blood stem cell apheresis products have become the preferred stem cell source for both allogeneic and autologous transplantation, so it has become more frequent than BMT for reconstituting hematopoiesis in many patients with malignant diseases undergoing myeloablative regimens of chemotherapies throughout the world. For example.in Germany, peripheral blood stem cells (PBSCs) mobilized by G-CSF were used for1739(86.7%of all) allogeneic hematopoietic stem cell transplantations in2006.The imperative of autologous patient and allogeneic donor safety requires optimization of apheresis procedures, to maximize stem cell yield and to minimize donor exposure and depletion of nontarget blood components. More efficient collection of PBPCs with fewer risks of apheresis are the most important focus for autologous patients and allogeneic donors. These goals may require opposing modifications of the collection process and will need to be balanced against each other.Donor safety and graft procurement cost mandate that as often as possible a single apheresis should suffice to generate a graft of adequate cellularity; that is, collection efficiency should be maximized. Apheresis procedures should be terminated when achievement of the target dose can be safely assumed. Platelet (PLT) and Hb loss should be restricted, both by providing PLT-sparing technologies and by limiting apheresis duration. A variety of apheresis devices are now available on the market to collect PBPCs. The Fenwal CS-3000Plus cell separator (Baxter Healthcare.Deerfield. IL. USA)、the COBE Spectra(CaridianBCT) and the COM.TEC cell separator’s (Fresenius HemoCare GmbH. Bad Homburg. Germany) mononuclear cell (MNC) collection program are currently available for clinical use. Occasional serious adverse events during apheresis collection have been reported. A more e□cient collection of PBPCs with fewer apheresis related risks is important for autologous patients. However, there are scant data regarding separation statistics and content of the harvest with three machines. Additionally, there is no published study comparing the Amicus and the Fresenius COM.TEC cell separators used for autologous and allogeneic PBPCs.Objective:The primary aim was to test whether the Bater CS-3000Plus and the COM.TEC were at least equivalent to what is considered by many the"gold standard" stem cell apheresis device, that is. the COBE Spectra MNC. with respect to collection efficiency. Secondary aims included comparative assessment of red blood cell (RBC) and PLT loss. Moreover, the study was to test prospectively whether the novel,electronically managed device might address some shortcomings of apheresis procedures performed with the established manual apheresis system. Predictability of apheresis outcomes would enable more accurate timing of apheresis duration and volume needing to be processed to achieve the desired stem cell dose. As we are showing, a greater stem cell yield and significantly lower PLT reinfusion rate were achieved. Finally, we retrospectively compared the efficiency and/or quality of collected components and collection variables between three apheresis machines to evaluate which machine is suitable for collecting PBPCs.Materials and methods:Patient and donor:129consenting autologous patients and allogeneic donors determined eligible for G-CSF-mobilized stem cell apheresis in the blood center of Zhejiang province were randomly assigned to apheresis collection with Fenwal CS-3000Plus (47)、the COM.TEC (22) or the COBE Spectra MNC (60) between October2005and January2012. All patients and donors received high-dose G-CSF to mobilize PBPCs (Amgen-Roche)(10ug/kg/day. beginning on day5until completion of the PBPCs collection). PBPCs were harvested when the peripheral blood(PB) leukocyte count was5×109/L.Apheresis collections:Patients underwent leukapheresis using a16G backeye access needle in an antecubital fossa vein; a20G venous cannula was placed in the opposite arm for the return line.Central venous catheters (12F, Arrow) were used in patients with an inadequate peripheral venous access. The target dose of CD34+/kg BW was3×106. In order to avoid severe hypocalcaemia during the procedure, all patients and donors received an oral10%calcium gluconate (20-40mL). Senior apheresis technicians performed all procedures. Vital signs were monitored at the beginning and end of each procedure, and patients were monitored for adverse events during the apheresis procedures. Written informed consent was obtained from all patients and donors after procedural risks were explained in detail before each procedure.MNC collections with CS-3000Plus、the COM.TEC and the COBE Spectra were controlled with computer software according to the manufacturer’s instructions. CS-3000Plus was set for a flow rate of40to55mL per minute,and a9:1-12:1ratio of whole blood to ACD-A (ACD-A solution, Terumo,Tokyo).The Fresenius COM.TEC instrument parameters were set as follows:whole blood flow rate set at40-60mL/min. cycle volume at300-500mL, the number of cycles varied from25to40, anticoagulant/whole blood ratio=1:10-14. COBE spectra (software version6.1for MNC) was set for a flow rate of40to55mL per minute, a9:1-12:1ratio of whole blood to ACD-A, and a collecting volume rate of1.5mL per minute (range,1.0-2.0). The three instruments processed equivalent volumes of whole blood per apheresis and per minute (4000-12000mL). Run time and volumes of ACD used were equivalent (260-300min,500-700mL,respectively). Laboratory tests:Pre-and/or postapheresis WBC,MNC,Hb and platelet counts were performed with a hemocytometer (Model XT-1800i,Sysmex. Norderstedt. Germany).CD34+cells were enumerated in peripheral blood before and after apheresis and on an apheresis product sample (SCE kit. BectonDickinson [BD]. Heidelberg. Germany).Statistical analysisData were expressed as the means±standard deviation (SD). Data were analyzed with an SPSS program (SPSS13.0,Inc.,Chicago, IL).We compared the CS-3000Plus> COM.TEC and the COBE Spectra machines by using an unpaired t-test in terms of percent decrease of Hb level、%decrease of PLT count, and leukapheresis product variables (harvest content).Linear association between two variables was ascertained with the Pearson’s r test. Statistical significance was set at p<0.05.Results:One hundred and twenty-nine PBPCs collections with the CS-3000Plus (patients and donors=47. procedure=70).the COM.TEC (patients and donors=22. procedure=33) and the COBE Spectra(patients and donors=60. procedure=89) were performed within study period.Pre-and post-apheresis PB data are shown inTable1.There was a higher mean%decrease in PB platelet count in collections on the COM.TEC compared to the CS-3000Plus (28.7±4.88%vs.11.59±4.09%;p<0.001) and the COBE Spectra (28.7±4.88%vs.17.09±3.36%;p<0.001). Further, the mean%decrease in PB Hb level was statistically lower with the COM.TEC compared to the CS-3000Plus instrument (7.33±1.37%vs.11.21±3.03%;p<0.001) and the COBE Spectra (7.33±1.37%%vs.12.27±3.02%;p <0.001). Spectra; p=0.4940, CS-3000Plus versus Spectra+P<0.001, COM.TEC versus CS-3000Plus;P<0.001, COM.TEC versus COBESpectra;0.0773, CS-3000Plus versus COBE SpectraThere was a statistically significant difference between the mean volume of products for the CS-3000Plus、the COM.TEC and the COBE Spectra device (54.43mL vs.218.55mL vs.49.75mL; p<0.001), however, there was no statistical difference in terms of total CD34+cell count per component(p=0.8890,0.9442,0.7323). Leukapheresis collection data are shown Table2. *Data are reported as mean±SD+P<0.001, COM.TEC versus CS-3000Plus and COBE Spectra++p=0.8890, CS-3000Plus versus COM.TEC; p=0.9442, COM.TEC versus COBESpectra; p=0.7323, CS-3000Plus versus COBE SpectraThere was a positive correlation between total CD34+cell count per component MNCs before apheresis and processed volume (r=0.7714,0.3971;separately). Data are shown Fig.1and Fig.2We encountered no severe adverse eE□ects necessitating interruption or cessation of the PBPC collection process. Discussion:Clinical outcome of patients with malignancy has shown significant improvement with hematopoietic stem cell transplantation. PBPC has essentially replaced BM in restoring marrow function after myeloablative chemotherapy. PBPCs can be harvested using an automated blood cell separator. Further, PBPCs can be collected safely and e□ciently by new cell separators, which increase the number of CD34+cells in the product, decrease product volume, reduce the number of procedures,and improve the purity of the product. To date there is only one published study concerning PBPC harvesting with the COM. TEC machine. Additionally, there is no published data comparing the COM.TEC and the Amicus devices in terms of PBPC collection. One of the problems which arise during the collection of PBPCs is the large volume of leukapheresis product that often requires volume reduction prior to cryopreservation. A study comparing the Amicus and the AS.TEC instruments showed that blood volume processed was higher in PBPC collections with the Amicus than with the AS.TEC device (10,091mL vs.9895mL), and separation time was longer in the Amicus than in the AS.TEC machine (251min vs.236min). However, the product volumes were not statistically di□erent between machines (129mL vs.111mL). Ikeda et al., compared the Amicus and the Spectra machine in a prospective crossover trial and reported that the Amicus took longer to process the same volume than did the Spectra (p<0.05)[25]. Schwella et al., reported that the median blood volume processed was12L(10.7-13.3L) with the COM.TEC device equipped with theMNC program. In this study, the mean blood volume processed was significantly higher with the Amicus compared to the COM.TEC cell separator (13,422±2156mL vs.10,092±1688mL)(p<0.001). Therefore, the mean separation time was significantly longer in the Amicus than in the COM.TEC instrument (290±30min vs.226±41min)(p<0.001). On the other hand, the median product volume was significantly lower with the Amicus compared to the COM.TEC instrument (125mL vs.300mL; p<0.001). This di□erence in product volumesmay be associated with the development of the toxicity of dimethyl sulfoxide (DMSO),which is a cryoprotective agent, after infusion of PBPCs product in patients in an autologous setting.Thus, one might expect that a decreased product volume obtained by the Amicus would result in low frequency of DMSO-related toxicity because less cryopreserved product with DMSO is to be infused. Therefore, the Amicus device may be selected to avoid the risk of DMSO-related toxicity in autologous patients.Schwella et al., reported that the median CD34+cells/kg BW was5.5-106with COM.TEC device. Moog reported that the number of CD34+cells in products was higher in the Amicus compared to the AS.TEC device (p=0.056). Snyder et al., reported that the number of CD34+cells/kg BW was higher with the Amicus compared to the CS3000machine (10.2±106vs.8.4·106), when the same blood volume was processed in patients who did not have significantly di□erent pre-apheresis CD34+cell counts [21].Ikeda et al., reported a similar result for nucleated cell counts, CD34+cells and colony forming units with the Amicus and the Spectra machine.In this study, although product volume was significantly higher with the COM.TEC compared to the Amicus device, there was no statistically significant di□erence between the CD34+cell count/kg BW for the Amicus and the COM.TEC (3.0·106/kg vs.4.1·106/kg)(p=0.129); this may be related to a more concentrated yield obtained with the Amicus device. Briefly, this study showed that PBPCs could be e□ciently collected with the COM.TEC and the Amicus machines in patients in an autologous setting. The other important challenge of PBPC collection is PLT-related product contamination.The literature suggests that high PLT contamination in progenitor cell concentrates results in significantly lower CD34+yield after immunoselection。 However, an increase of CD34+cells in the product can be achieved by reducing PLTs in the product.Additionally, the PLT contamination of the products results in the PLT loss in PB; this is important because these patients are often thrombocytopenic due to their underlying disease and the chemotherapy used for PBPCs mobilization. Kazuhiko Ikeda et al.,reported there was significantly more efficiency and/or numbers of platelet contamination in all patients/donors with the Spectra than with the Amicus suggests that the Spectra may cause more apheresisrelated thrombocytopenia. Thrombocytopenia, one of the most common complications of apheresis, sometimes requires a transfusion, so we should be aware of the possible profound thrombocytopenia caused by using Spectra. The Amicus is recommended for patients with thrombocytopenia or with less than-normal platelet counts. On the other hand, Green et al. compared software versions4.7and6.0of the Spectra. They reported that although samples collected using versions4.7and6.0had mean numbers of1.28and0.68of contaminating platelets,respectively, this difference was not significant. However, this can be significant if compared with a cross-over study design. We need further evaluation to compare the contaminating platelets using the Amicus and the Spectra software version6.0with a cross-over study design to see whether the version6.0can decrease platelet contamination significantly. The level of Hb contamination in collected components was more with the Amicus than with the Spectra. However, the decrease of Hb concentration in patients after the procedures was not different with the Amicus and the Spectra(<10%with both instruments), suggesting that RBC transfusion would not be required after apheresis. In addition, although transplantation-related hemolytic complications may occur more frequently when PBPCs collected by the Amicus are used, the level of RBC contamination is much less than with marrow, and RBCs in the collected samples can be removed by sedimentation or centrifugation. Schwella et al., reported that PLT loss in patients with PBPC collected with the COM.TEC device was35.9%(19.2-66.1%). Ikeda et al., reported that products collected by the Amicus had fewer PLTs than those collected by the Spectra device (p <0.001); and, the decrease of patient PB PLT counts was more prominent using the Spectra than the Amicus instrument (p<0.0001). They concluded that the Amicus was superior to the Spectra in terms of apheresis-induced thrombocytopenia caused by PLTs contaminating the PBPC products.Snyder et al., reported that the PLT content of the Amicus collections was significantly lower than that of the CS3000+collections (4.35·1010vs.6.61-1010)(p<0.05)[21]. Additionally, Jeanne et al.,reported that PLT contamination in Amicus components was twice as low compared to the CS3000+[26]. In another study, the PLT content of the products was lower in the Amicus than in products from the AS.TEC machine (0.17·1011vs.0.65·1011)(p<0.001).In addition, the Amicus machine resulted in lower PB PLT loss compared to the AS.TEC device (12.5%vs.34.4%). In this study, There was a higher mean%decrease in PB platelet count in collections on the COM.TEC compared to the CS-3000Plus (28.7±4.88%vs.11.59±4.09%;p<0.001) and the COBE Spectra (28.7±4.88%vs.17.09±3.36%;p<0.001). Further, the mean%decrease in PB Hb level was statistically lower with the COM.TEC compared to the CS-3000Plus instrument (7.33±1.37%vs.11.21±3.03%; p<0.001) and the COBE Spectra (7.33±1.37%%vs.12.27±3.02%;p<0.001). There was a statistically significant di□erence between the mean volume of products for the CS-3000Plus、the COM.TEC and the COBE Spectra device (54.43mL vs.218.55mL vs.49.75mL; p<0.001), however, there was no statistical di□erence in terms of total CD34+cell count per component(p=0.8890,0.9442,0.7323). There was a positive correlation between total CD34+cell count per component、MNCs before apheresis and processed volume (r=0.7714,0.3971;separately).In conclusion, According to situation of different patient/donor establishing each kind of gathering parameter in reason,chosing suitable puncture spot and gathering once or twice,can collect high quality PBSC by using three instruments and use in transplanting safely to meet clinical requirement. However, COM.TEC has the shortage of more decrease in PB platelet count with higher product volume.