Study On The Mobilization And Collection Of Peripheral Hematopoietic Stem Cells

Background and ObjectiveIn malignant blood disease, chemotherapy and radiotherapy is the most effective methods to kill tumor cells, with the discovery of the new chemotherapeutics, radiotherapy technology improvement and support therapy,which increased their effectiveness.Theoretically,there are positive corelation between chemotherapy, radiation dose and killed tumor cells, the treatment effect,but the side effects of radiotherapy and chemotherapy limit the dose increase, which affect curative effect and prognosis of patients.Hematopoietic stem cell transplantation can solve this problem to some extent, and improve the therapeutic effect of blood and some malignant tumors,which significantly improve the prognosis of patients. Hematopoietic stem cell transplantation refers to provide the normal donor or autologous hematopoietic stem cells with the patients who undertake the process of chemotherapy, radiation therapy and immune inhibitor via vascular infusion,which help the patients reconstruct their normal hematopoiesis and immune function to treat primary disease (blood disease, malignant tumor, etc).According to the different hematopoietic stem cell collection way, the hematopoietic stem cell transplantation can be composed of bone marrow transplantation (BMT), peripheral blood hematopoietic stem cell transplantation (PBSCT) and umbilical cord blood stem cell transplantation (UCBT), Combined with the bone marrow transplantation,the peripheral blood hematopoietic stem cell transplantation has the following advantages:First of all,collection hematopoietic stem cells is relatively simple, donors do not need hospitalization, anesthesia, easy to accept, and less complications;Second,with bone marrow metastasis or bone marrow in patients undergoing radiotherapy damage can also be collected hematopoietic stem cells;Third,hematopoietic system reconstruction is faster than bone marrow transplantation, implantation rate is high, early immune function reconstruction;At last,in the short term,the donors can be separated in the second time.As the early1980s,with cell separator used in collection of peripheral blood hematopoietic stem cells and the improvement of technology of peripheral blood hematopoietic stem cell mobilization, separation and acquisition,the peripheral blood hematopoietic stem cell transplantation which has gradually taken the placed of BMT become the main method of treatment of hematologic malignancies.The basic principle of blood cell separator apheresis are that there are different proportion of all kinds of blood cells.We can set the centrifugal separation condition, under the effect of a certain centrifugal force,all kinds of cells in a certain distribution in centrifugal container after the centrifugal balance, such as the plasma in the top,red blood cells in the lower level,distribution among the various component blood cells. Without destroying the balance conditions,by adjusting settings of the collection interface,we can collect cells needed relatively.The density,biophysics characteristics and size of peripheral blood stem cells (PBSC) are similar to the peripheral blood Mononuclear cells (PBMNCs). Therefore, with the cell separator collecting the PBSC, the PBSC mainly exists in PBMNCs group.There were highly positive correlation between the number of the CD34+cells and the PBMNCs, so the PBMNCs can indirectly reflect the PBSC.Currently in clinically the separators for collecting the hematopoietic stem cells separator are as follows:Fresenius COM.TEC, Fenwal COBE Spectra and Fenwal CS3000plus. To collect stem cells in our hospital are Fresenius COM.TEC and Fenwal CS3000plus stem cell separators.The quality and quantity of the PBSC is one of the most critical factors to make peripheral blood hematopoietic stem cell transplantation successful, how to increase the collection efficiency of PBSC and to avoid adverse reactions in the collection process,which become the key problems. We retrospectively analyzed the influence factors for the collection of PBSC, and compare different characteristics of the blood cell separator or the differences in healthy donors and autogenous donors, and study the measures of preventing adverse reactions. We do that to optimize the mobilization and acquisition conditions, improve the efficiency of collection, ensure the quality, so as to prevent and reduce adverse reactions.Part I Study on the factors influencing the mobilization and collection of peripheral hematopoietic stem cellsObjectiveWe made a retrospective analysis on the collection factors such as different age, sex, body mass index (BMI), white blood cell counts,mobilization plans on the healthy donors before the separation.Method1.In this part we retrospectively analyzed the peripheral blood stem cells yield in40healthy donors in three years from January2009to June2012in our department (Zhujiang hospital, the Southern Medical University).The median age was33years(range:14-52years),the median height was166cm (range:137-180cm), the median body weight(BW) was65kg (range:43-100kg).2.30donors received lenograstim,6donors received filgrastim,and4donors received lenograstim and filgrastim.2donors received treatment for3days,11donors received treatment for4days,23donors received treatment for5days,and4donors received treatment for6days.Apheresis was performed using the Fresenius COM.TEC cell separator’s (Fresenius HemoCare GmbH,BadHomburg, Germany) with Auto-mononuclear cell(MNC) collection program,and the blood flow rate for apheresis was30-60ml/min,and the acid citrated dextrose-A(ACD-A) was used as the anticoagulant.The cycle media volume was2-4times the body volume of donors (range8880-12600ml),During apheresis10%grape sour calcium was intermittent used via intravenous injection or oral potassium chloride in order to prevent sodium citrate toxicity and hypokalemia, and the adverse events were observed closely.The MNC and CD34+cells were numbered after apheresis by picking up an aliquot of the apheresis products.3.Data were analyzed with an SPSS program (SPSS13.0.Inc.Chicago,IL).The non-normal distribution data were expressed as the median±quartile range,and the normal distribution data were expressed as the mean±standard deviation(SD).Two pairs were compared by using the Mann-Whitney U-test,the Bivariate Correlations.Statistical significance was set at p<0.05.ResultWe obtained an adequate number of peripheral blood mononuclear cells and CD34+from40donors,meeting the requirement of the peripheral blood stem cell transplantation The median number of PBMNCs and CD34+cells per kilogram of receiptor body weight were (9.77±2.74)×108/kg、(5.07±6.5)×106/kg respectively. There was positive correlation between pre-WBC and PBMN、CD34+cells.There was no correlation between age,sex,BMI,different methods of mobilization and PBMNCs, CD34+cells.Conclusion It can successfully collect PBSC with there kinds of mobilization plans, pre-WBC is the main factors influencing the collection of PBMNC and CD34+cells,when to collection PBMNC and CD34+cells is up to the time that the pre-WBC were mobilized to3×109/L.Part II Comparison of the peripheral blood stem cell collection on the Fenwal CS-3000plus and Fresenius COM.TECObjectiveWe researched retrospectivly the collection effect of PBMNC on Fresneius COM. TEC and Fenwal CS3-000plus to compare their collection efficiency.Method1.In this part we retrospectively analyzed the peripheral blood stem cells yield on77healthy donors from2004to2012in our department.There were41donors in the Fresenius COM.TEC(Fresenius HemoCare GmbH,BadHomburg, Germany) group,72consecutive leukapheresis procedures were performed.There were total36healthy donors in the Fenwal CS-3000plus (Baxter,Deerfleld, IL, USA) group, G-CSF(10μg·kg-1-·d-1)were administered to stimulate PBSC of the two groups,they received treatment4to6days according to the preWBC counts.2.Auto-MNC program were used for the collection of the Fresenius COM.TEC cell separator, the cycle volume was8595-12402ml,and the blood flow rate was30to60ml/min;l-special program were used for the collections of Fenwal CS-3000plus cell separator,the cycle volume was7614-11711ml,and the blood flow rate was30to60ml/min used;acid citrated dextrose-A(ACD-A) used as the anticoagulant of the both groups.During apheresis10%grape sour calcium was intermittent used via intravenous injection or oral potassium chloride in order to prevent sodium citrate toxicity and hypokalemia, and the adverse event was observed closely.The white blood cell counts,the mononuclear cell counts,the percentages and collection efficiency of the mononuclear cells in the apheresis products were numbered after apheresis by picking up an aliquot of the apheresis products.3.Data were analyzed with an SPSS program (SPSS13.0.Inc.Chicago,IL).The normal distribution data were expressed as the mean±standard deviation(SD).Two pairs were compared by using the Mann-Whitney U-test,the Bivariate CorrelationsStatistical significance was set at p<0.05.ResultThe PBMNC of Fresenius COM.TEC were (51.10±12.56)×109个,Fenwal CS-3000plus (25.57±10.38)×109个, the former was significantly higher than the latter, the differences between two groups was statistically significant (P=0.000). The percentages of the PBMNC are as follows, Fresenius COM.TEC (61.99±23.22)%, Fenwal CS-3000plus (73.71±19.84)%, and the former is lower than the latter, there were significant difference between them (P=0.000). The collection efficiency of two groups are respectively (0.52±0.21)%,(0.53±0.31)%,there were no significant difference between them (P=0.91).There were no correlation between pre-MNC and the collection efficiency of PBMNC (r=0.174, P=0.302>0.05, r=0.093, P=0.093>0.05).ConclusionFor the healthy donors, two kinds of blood cell separators can collect enough numbers of PBSC, which were needed for PBSCT.There were no significant difference between them,there was no correlation between pre-WBC and CE of PBMNC of the two groups. The PBMNC of the Fresenius COM. TEC is higher than the Fenwal CS-3000pllus, but the percentage of the PBMNC of former is lower than the latter,and we can see the product of former in the apheresis process,the volume of the product become bigger with the increase of the cycle volume. We can not see the product in the apheresis progress of the Fenwal CS-3000plus,and the volume is50ml,whether the activity of PBSC will be effected remains to be further researched.PartⅢ Comparison of the Fenwal CS-3000plus and Fresenius COM.TEC cell separators for autologous peripheral blood stem cell collectionObjectiveWe analyzed retrospectively the collection effect of PBMNC on Fresneius COM. TEC and Fenwal CS3-000plus of patients to compare their collection efficiency. Method1. In this part we retrospectively analyzed the peripheral blood stem cells yield on40patients from2004to2012in our department.In Fresenius COM.TEC group,there were twenty-two patients (7males,22females) underwent autologous PBPC leukapheresis.Of these,11had a diagnosis of AML,6NHL and5MM.The median age was41years;eighteen patients (11males,7females) underwent autologous PBPC leukapheresis.Of these,12had a diagnosis of AML,3NHL and3MM.The median age was38years old.2.47apheresis procedures were performed on the Fresenius COM.TEC with Auto-MNC program,the cycle volume was (7099-14100) ml,and the blood flow rate was30to60ml/min,38apheresis procedures were performed on the Fenwal CS-3000with1-special program,the cycle volume was7614-11711ml,and the blood flow rate was30to60ml/min used.acid citrated dextrose-A(ACD-A) used as the anticoagulant of the both groups.During apheresis10%grape sour calcium was intermittent used via intravenous injection or oral potassium chloride in order to prevent sodium citrate toxicity and hypokalemia, and the adverse event was observed closely.Harvest product was analyzed for volume,the numbers of collected total MNCs,PLT contamination and the percentage of PBPLT decrease were also evaluated.In addition,the CE of PBMNCs were calculated based on patient’s blood volume:CE%=total cells obtained from the apheresis×100/(peripheral cells/mL×apheresis volume processd mL)3.Data were analyzed with an SPSS program.The non-normal distribution data were expressed as the median±uartile range,and the normal distribution data were expressed as the mean±standard deviation(SD).Two pairs were compared by using the Mann-Whitney U-test or Independent-Samples T test,the Bivariate Correlations.Statistical significance was set at p<0.05.ResultThe product volume of Fenwal CS-3000plus was ixed50ml,and the product volume of Fresenius COM. TEC (129±25)ml, the former was significantly less than the latter, the difference had statistical significance (P=0.0000). The other results of two groups are as follows:PBMNC(4.31±6.4,3.87±3.29)×109个platelet numbers (138.85±120,227.97±274.04)×109个the collection efficiency of PBMNC (0.59±0.59,0.68±0.22)%, the percentage of PBPLT decrease (0.22±0.16,0.34±0.29)%,there were no differences between them.ConclusionFor patients with autologous hematopoietic stem cell transplantation, the product volume of the Fenwal CS-3000was only50ml, so the effect of the cycle volume were much smaller,and it is more convenient to keep cryopreserved cells and revive cells than Fresenius COM.TEC cell separator.Part IV The analysis and countermeasures of the adverse events on the peripheral blood hematopoietic stem cell collection ObjectiveThe adverse events and the reasons on the collection of the PBSC were researched to evaluate the safety, prevention and effect of the treatment measures,so we can improve measures of the prevention and treatment and security.MethodThe kinds and numbers of the adverse effects of the collection of the PBSC were researched on donors in our department from2004to2012,the reasons of the side effects,measures of prevention and treatment,the effect on the donors were also studied.117consecutive leukapheresis procedures were performed. There were58males and59females,the median age was41years (14-67years).APBSCT patients were40,Allo-PBSC donors were77,40males and37females.1-special program were used on54donors in Fenwal CS-3000plus group,Auto-MNC program on63donors in Fresenius COM.TEC.The blood flow rate was30to60ml/min,acid citrated dextrose-A(ACD-A) were used as the anticoagulant of the both groups.The ratio of the blood volume and ACD-A was (9-10) to1.During apheresis10%grape sour calcium was intermittent used via intravenous injection or oral potassium chloride.Result1case appeared body chills;1case appeared chills, no fever;1case of a double lower limbs numbness, cramps;2cases with chest tightness, heart palpitations, nausea, vomiting;1case appeared venipuncture arm pins and needles.For the donors who appeared the side effects.For the adverse reaction,we give additional intravenous push10%grape sour calcium injection,oral potassium chloride solution and symptomatic treatment, adverse symptoms and signs disappeared soon, the apheresis were successfully completed,there were no follow-up adverse consequences. ConclusionThe occurrance rate of the collection of the PBSC is very low,and adverse reactions were mild,the side effects can be avoided through routine preventive measures.We can also reduce the incidence rate of the side effects by carefully observed,early detection, early treatment.So the PBSC collection was a relatively safe procedure.