自体纯化CD34~+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床研究

目的分析自体纯化CD34+细胞移植治疗中/高危淋巴细胞来源恶性肿瘤的临床疗效。方法 10名中/高危组淋巴细胞来源恶性肿瘤患者行自体纯化CD34+细胞移植治疗,细胞分选采用cliniMACS系统。计算并统计分选纯度和CD34+细胞回收率,观察移植相关并发症及患者生存情况。结果纯化后CD34+细胞纯度为(87.79±3.73)%,回收率达到(65.74±10.37)%。10名患者全部顺利造血重建,感染发生率50%(5/10例),复发率为20%(2/10例)。结论利用CliniMACS系统进行外周血CD34+细胞分选,CD34+细胞纯度、回收率均满意,自体移植后造血功能重建顺利,近期疗效满意。     

CD34+ cell enrichment for autologous peripheral blood stem cell transplantation by use of the CliniMACs device

Several devices for selection of CD34+ peripheral blood stem cells (PBSC) have been used during the last years for reducing tumor cell contamination of the graft. The new CliniMACS system (magnetic-activated cell separation system by Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) was recently approved for clinical use in Europe. To evaluate its purging efficiency and engraftment data in the autologous transplant, PBSC from 28 adult patients with various malignant diseases (non-Hodgkin's lymphoma, n = 17; chronic lymphocytic leukemia, n = 5; multiple myeloma, n = 4; acute lymphocytic leukemia, n = 1; medulloblastoma, n = 1) were mobilized by chemotherapy and granulocyte colony-stimulating factor (G-CSF) (10 microg/kg per day). Thirty leukapheresis products from 28 patients with a median of 4.4 x 10(8) nucleated cells/kg body weight (bw)(range 0.6-10.8 x 10(8)/kg bw) and a median of 7.1 x 10(6) CD34+ cells/kg bw (range 2.8 to 18.8 x 10(6)/kg bw) were selected using the Cobe spectra cell separator (Cobe BCT Inc., Lakewood, CO). After the CliniMACS procedure, the median yield of CD34+ selected cells was 4.5 x 10(6)/kg (range 2.2-11.1 X 10(6)/kg bw) with a median recovery of 69.5% (range 46.9-87.3%) and a median purity of 97.7% (range 89.4-99.8%). The procedure did not alter viability of selected cells, which was tested by propidium iodide staining. So far, purified PBSC were used for autologous transplantation in 15 out of 28 patients after total body irradiation and/or high-dose chemotherapy. Median time to reach an absolute neutrophil count > 500/microl was 12 days (range 10-18 days), platelet recovery >50,000/microl occurred at day + 16 (range 11-22). With a median follow-up time of 12 months (range 3-19), 5 patients died of relapse. We confirmed the feasibility and safety of the CliniMACS CD34+ cell enrichment procedure in adult patients with autologous PBSC transplantation.     

A controlled comparison of two different clinical grade devices for CD34+ cell selection of autologous blood stem cell grafts

Six patients who were to undergo autologous PBSC transplantation with positively selected CD34+ cells were included in this study to compare the efficiency of two devices for clinical grade stem cell selection, the Isolex 300i (Baxter, Munich, Germany) and CEPRATE SC (CellPro, Bothell, WA). PBSC were mobilized by chemotherapy and G-CSF and were collected by leukapheresis on a CS3000 cell separator on 2 consecutive days. The two apheresis products were pooled for CD34 selection. The pooled apheresis products from each patient were divided into two equal portions to be separated on each of the two devices. Cell selection was performed according to the manufacturers' instructions. Enumeration of CD34+ cells was performed by flow cytometry using the HPCA-2 MAb. Purity and yield were significantly better with Isolex than with CEPRATE. Median purity was 93.0% (range 80%-98%) for Isolex and 61.5% (range 27%-72%) for CEPRATE (p = 0.03); median yields for Isolex and for CEPRATE were 48.0% (range 18%-73%) and 23.0% (range 17%-29%), respectively (p = 0.03). The number of CD34+ cells/kg body weight was also significantly higher with Isolex (median 3.8x10(6), range 1.7-5.2) compared with CEPRATE (median 2.35x10(6), range 0.7-4.3) (p = 0.03). Thus, the Isolex 300i device gave products of higher purity and recovered a higher proportion of the CD34+ cells in the harvest before separation. The yield was still poor with both devices, however, and further optimization of the technique for clinical grade stem cell selection is warranted.     

Isolation of CD34+ progenitor cells from peripheral blood by use of an automated immunomagnetic selection system: factors affecting the results

BACKGROUND: The isolation of CD34+ cells from mobilized peripheral blood is being increasingly used in the setting of allogeneic or autologous hematopoietic cell transplantation. Investigation of variables that may influence the effectiveness of CD34+ cell selection is of interest. STUDY DESIGN AND METHODS: Fifty-one CD34+ cell selections from peripheral blood progenitor cells (PBPCs) (39 allogeneic and 12 autologous) were performed using a magnetic cell separator (Isolex 300i, Baxter), including version 2.0 software. The results obtained were analyzed for different processing variables. The feasibility of transplanting these isolated CD34+ cells was also analyzed. RESULTS: The isolated CD34+ cell fraction had a median purity of 88.9 percent (range, 47.8-98.3). The median recovery of CD34+ cells was 45.1 percent (13.8-76.2), and the median colony-forming unit- granulocyte-macrophage (CFU-GM) content was 17. 2 percent (0.8-58.6). Logarithms of T- and B-cell depletion had median values of 3.7 and 2.8, respectively. The version 2.0 software of the Isolex 300i gave a higher CD34+ cell recovery in the enriched cell fraction (median 57.8%) than did version 1.11 (39.4%) or 1.12 (44.4%) (p = 0.01). The use of recombinant human deoxyribonuclease I during cell processing yielded more CD34+ cells (53% vs. 41%, p = 0. 01) and higher purity (92.8% vs. 87%, p = 0.03). There was a correlation between the percentage of CD34+ cells labeled with the monoclonal antibody 8G12 clone and the percentage of CD34+ cells labeled with the monoclonal antibody used during the processing technique (9C5 clone) in the initial, enriched, and depleted CD34+ cell fractions (R(2) = 0.95; 0.92; 0.78, p< 0.005, respectively). Median times for recovering >0.5 x 10(9) per L of granulocytes and >20 x 10(9) per L of platelets were 13 and 16 days in the allograft patients and 13 and 14 days in the autograft patients. CONCLUSION: CD34+ cells can be highly and effectively isolated from allogeneic and autologous grafts by use of this automated technique, with a high grade of T- and B-cell depletion. These purified CD34+ cell components can engraft normally.     

Immune reconstitution after autologous selected peripheral blood progenitor cell transplantation: comparison of two CD34+ cell-selection systems

BACKGROUND: Selection of CD34+ PBPCs has been applied as a method of reducing graft contamination from neoplastic cells. This procedure seems to delay lymphocyte recovery, while myeloid engraftment is no different from that with unselected PBPC transplants. STUDY DESIGN AND METHODS: Lymphocyte recovery was studied in two groups of patients who underwent autologous CD34+ PBPC transplant with two different technologies (Ceprate SC, Cellpro [n = 17]; CliniMACS, Miltenyi Biotech [n = 13]). The median number of CD34+ cells transfused was 3.88 x 10(6) per kg and 3.32 x 10(6) per kg, respectively. Residual CD3 cells x 10(6) per kg were 4.97 and 0.58, respectively (p = 0.041). Residual CD19 cells x 10(6) per kg were 1.33 and 0.73, respectively (NS). RESULTS: No differences were found between the two groups in total lymphocyte recovery to >0.5 x 10(9) per L, which achieved a stable count by Day 30. During the study period, the CD4+ cell count remained below 0.2 x 10(9) per L, and the B-cell subset showed a trend toward normalization. CD3/HLA-DR+ and CD16/56 increased markedly in both groups by Day 30. An increase in CMV (13%) and adenovirus (17.4%) infection was found in both groups. CONCLUSION: Both CD34+ cell selection technologies used here determined an excellent CD34+ cell purity and an optimal depletion of T cells. The high rate of viral complications is probably due to the inability of residual T cells left from the CD34+ cell selection to generate, immediately after transplant, an adequate number of virus-specific lymphocytes.     

自体纯化CD34+细胞移植治疗严重自身免疫性疾病的初步研究

目的探讨自体外周血CD34+细胞移植治疗严重自身免疫性疾病的干细胞动员、细胞采集和分选、预处理和并发症处理等问题.方法 10例重度自身免疫性疾病患者接受自体外周血CD34+细胞移植治疗.采用环磷酰胺(CTX)+rhG-CSF方案动员外周血干细胞,并以CliniMACS细胞分选仪分选CD34+细胞,适时用CTX+抗胸腺细胞球蛋白(7例)或CTX+全身照射(3例)两种预处理方案后,进行CD34+细胞回输的方法治疗.结果经CTX+rhG-CSF方案动员并以CliniMACS细胞分选仪分选后,可获得(1.98±0.95)×108的CD34+细胞,其纯度为(91.4±10.6)%,回收率为(60.5±19.8)%.在回输(2.14±1.05)×106/kg的CD34+细胞后,ANC ≥0.5×109/L的时间为(8.6±2.5)d,血小板升至20×109/L的时间为(9.0±5.2)d.在造血恢复后,所有CD3+细胞、CD19+细胞和CD16+CD56+细胞均未恢复至移植前状态.在造血和免疫抑制时,巨细胞病毒感染的发生率较高.2例患者死于移植相关并发症.所有患者近期疗效满意,6例系统性红斑狼疮患者DAI评分由移植前的平均17分降为移植后的4分;类风湿关节炎患者DAS28评分由6.4分降至1.8分;干燥综合征患者的症状和体征均明显缓解.结论对常规治疗无效的严重自身免疫性疾病,自体外周血CD34+细胞移植是可选择的治疗方法之一.     

自体纯化CD34+细胞移植治疗自身免疫性疾病14例的临床观察

目的 探讨自体纯化CD34+细胞移植治疗自身免疫性疾病(AID)的疗效.方法 对14例自身免疫性疾病患者进行自体纯化CD34+细胞移植.采用环磷酰胺(CTX)+G-CSF动员外周血干细胞,通过CliniMACS细胞分选仪分选CD34+细胞并冻存.预处理方案:8例采用氟达拉滨(FDB)+抗胸腺细胞球蛋白(ATG)+CT...     

动员外周血造血干细胞移植物中同时高效去除T／B细胞的体外研究

单倍体相合造血干细胞移植时，去除移植物中的T细胞和B细胞可减少移植物抗宿主病和EBV相关的淋巴细胞增殖性疾病的发生。本研究在国内首次应用CliniMACS系统，通过CD3／CD19磁珠抗体同时有效地去除动员外周血造血干细胞中的T细胞和B细胞。用流式细胞术检测移植物中T细胞和B细胞的去除效率，以集落形成实验评价去除后的干细胞功能。结果表明：T细胞和B细胞去除之前单个核细胞总数为4．88×10^10，去除之后移植物中T细胞比例为0．02％，去除4．4Log；B细胞比例不到0．01％，至少去除3．3Log。移植物中除CD34^＋细胞外。还包括NK细胞，单核细胞和粒细胞。CD34^＋细胞纯度为0．98％，计数1．84×10^8，回收率为69．7％；NK细胞计数2．54×10^9，回收率为71．7％。集落形成实验显示CD3／CD19去除对造血干细胞功能无影响。结论：应用CliniMACS系统可有效地同时去除动员外周血干细胞中的T细胞和B细胞，且不影响造血干细胞的生物学功能，此项研究为去除T／B细胞的单倍体相合造血干细胞移植提供了技术平台。     

How do I perform hematopoietic progenitor cell selection?

Graft‐versus‐host disease remains the most important source of morbidity and mortality associated with allogeneic stem cell transplantation. The implementation of hematopoietic progenitor cell (HPC) selection is employed by some stem cell processing facilities to mitigate this complication. Current cell selection methods include reducing the number of unwanted T cells (negative selection) and/or enriching CD34+ hematopoietic stem/progenitors (positive selection) using immunomagnetic beads subjected to magnetic fields within columns to separate out targeted cells. Unwanted side effects of cell selection as a result of T‐cell reduction are primary graft failure, increased infection rates, delayed immune reconstitution, possible disease relapse, and posttransplant lymphoproliferative disease. The Miltenyi CliniMACS cell isolation system is the only device currently approved for clinical use by the Food and Drug Administration. It uses magnetic microbeads conjugated with a high‐affinity anti‐CD34 monoclonal antibody capable of binding to HPCs in marrow, peripheral blood, or umbilical cord blood products. The system results in significantly improved CD34+ cell recoveries (50%‐100%) and consistent 3‐log CD3+ T‐cell reductions compared to previous generations of CD34+ cell selection procedures. In this article, the CliniMACS procedure is described in greater detail and the authors provide useful insight into modifications of the system. Successful implementation of cell selection procedures can have a significant positive clinical effect by greatly increasing the pool of donors for recipients requiring transplants. However, before a program implements cell selection techniques, it is important to consider the time and financial resources required to properly and safely perform these procedures.     

Rapid and effective CD3 T-cell depletion with a magnetic cell sorting program to produce peripheral blood progenitor cell products for haploidentical transplantation in children and adults

BACKGROUND: Effective T-cell depletion is a prerequisite for haploidentical peripheral blood progenitor cell (PBPC) transplantation. This study was performed to investigate the performance of magnetic cell sorting-based direct large-scale T-cell depletion, which is an attractive alternative to standard PBPC enrichment procedures. STUDY DESIGN AND METHODS: PBPCs were harvested from 11 human leukocyte antigen (HLA)-haploidentical donors. T cells labeled with anti-CD3-coated beads were depleted with a commercially available magnetic separation unit (CliniMACS, Miltenyi Biotec) with either the Depletion 2.1 (D2.1, n=11) or the novel Depletion 3.1 (D3.1, n=12) program. If indicated, additional CD34+ selections were performed (n=6). Eleven patients received T-cell-depleted grafts after reduced-intensity conditioning. RESULTS: The median log T-cell depletion was better with the D2.1 compared to the D3.1 (log 3.6 vs. log 2.3, p<0.05) and was further improved by introducing an immunoglobulin G (IgG)-blocking step (log 4.5 and log 3.4, respectively). The D3.1 was superior to the D2.1 (p<0.05) in median recovery of CD34+ cells (90% vs. 78%) and in median recovery of CD3- cells (87% vs. 76%). The median processing times per 10(10) total cells were 0.90 hours (D2.1) and 0.35 hours (D3.1). The transplanted grafts (directly T-cell-depleted products with or without positively selected CD34+ cells) contained a median of 10.5 x 10(6) per kg CD34+, 0.93x10(5) per kg CD3+, and 11.6x10(6) per kg CD56+. Rapid engraftment was achieved in 10 patients. The incidences of acute graft-versus-host disease were less than 10 percent (Grade I/II) and 0 percent (Grade III/IV). CONCLUSION: The novel D3.1 program with IgG blocking enables highly effective, time-saving large-scale T-cell depletion. Combining direct depletion techniques with standard CD34+ selection enables the composition of grafts optimized to the specific requirements of the patients.     

P-Selectin coated microtube for enrichment of CD34+ hematopoietic stem and progenitor cells from human bone marrow

BACKGROUND: Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematologic disorders and in basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion on selectins than mature CD34- mononuclear cells (MNCs). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow by perfusing MNCs through selectin-coated microtubes. METHODS: Bone marrow MNCs were perfused through the cell-capture microtubes coated with adhesion molecules. We washed the device lumen and visualized and estimated captured cells by video microscopy. Adherent cells were eluted by high shear, calcium-free buffer, and air embolism. We used immunofluorescence staining followed by flow cytometry to analyze CD34+ HSPCs. RESULTS: CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow MNCs [mean (SE) 2.5% (0.8%)]. P-selectin-coated surfaces yielded 16% to 20% CD34+ cell purity, whereas antibody-coated surfaces yielded 12% to 18%. Although CD34+ cell purity was comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (approximately 5.7 x 10(4) to 7.1 x 10(4)) than antibody devices (approximately 1.74 x 10(4) to 2.61 x 10(4)). CONCLUSIONS: P-selectin can be used in a compact flow device to capture HSPCs. Selectin-mediated capture of CD34+ HSPCs resulted in enrichment approximately 8-fold higher than the CD34+ cell population from bone marrow MNCs. This study supports the hypothesis that flow-based, adhesion molecule-mediated capture may be a viable alternative approach to the capture and purification of HSPCs.     

The excessive numbers of total nucleated cells does not affect the performance of the CliniMACS

This prospective study was carried out in healthy donors and patients. The performance of the CliniMACS was evaluated with the comparison of the numbers of total nucleated cell (TNC) within and over the capacity of the normal scale column. In addition, large vs. normal scale column and manual vs. automated washing systems were also compared. A total of 44 selections were done. Eighteen normal scale selections were done with initial TNC numbers over 6 x 10(10) and 14 selections were performed below this number. None of the cases had CD34+ cell numbers over the capacity. Flow cytometry was used to check each separation performance for purity and recovery of CD34+ cells along with T- and B-cell depletion level parameters. All healthy donors had significantly better mean purity and recovery of CD34+ cells, and T- and B-cell depletion status than that of patients with values 95 vs. 85%, P: 0.006; 77 vs. 58%, P: 0.004; 4.55 log vs. 4.06 log, P: 0.004; 3.19 log vs. 2.63 log, P: 0.01, respectively. However, the performance of the system was not dependent on using the normal or large-scale column; automated or manual washing systems; and initial TNC numbers above (>6 x 10(10), range: 7.05-21.84 x 10(10), mean: 12.32 x 10(10)) or within (<6 x 10(10), range: 0.86-5.89 x 10(10), mean: 4.15 x 10(10)) the column capacity. In conclusion, the performance of the CliniMACS is more efficient in healthy donors than in patients. However, the performance of the system did not change as long as the numbers of CD34+ cells (range: 0.34-5.87 x 10(8)) were not exceeding the column capacity despite that more than 6 x 10(10) TNCs were used.     

Large-volume leukapheresis using femoral venous access for harvesting peripheral blood stem cells with the Fenwal CS 3000 Plus from normal healthy donors: predictors of CD34+ cell yield and collection efficiency

The current paper reports on the predicting factors associated with satisfactory peripheral blood stem cell collection and the efficacy of large-volume leukapheresis (LVL) using femoral vein catheterization to harvest PBSCs with Fenwal CS 3000 Plus from normal healthy donors for allogeneic transplantation. A total of 113 apheresis procedures in 57 patients were performed. The median number of MNCs, CD3+ cells, and CD34+ cells harvested per apheresis was 5.3 x 10(8)/kg (range, 0.3-11.0 x 10(8)/kg), 3.0 x 10(8)/kg (range, 0.2-6.6 x 10(8)/kg), and 7.9 x 10(6)/kg (range, 0.1-188.9 x 10(6)/kg), respectively. The median collection efficiency of MNCs and CD34+ cells was 49.8% and 49.7%, respectively. A highly significant correlation was found between the collected CD34+ cell counts and the pre-apheresis WBC counts in the donors (P = 0.013), and between the collected CD34+ cell counts and the pre-apheresis peripheral blood (PB) CD34+ cell counts (P<0.001). Harvesting at least >4 x 10(6)/kg CD34+ cells from the 1st LVL was achieved in 44 (77.2%) out of 57 donors and in 19 (90.5%) out of 21 donors with a PB-CD34+ cell count of >40/microl. There was no significant difference in the harvested MNC and CD34+ cell counts between the 1st and 2nd apheresis. The catheter-related complications included catheter obstruction (n = 2) and hematoma at the insertion site (n = 3). Accordingly, LVL using femoral venous access for allogeneic PBSC collection from normal healthy donors would appear to be safe and effective.     

Combined isolation of CD34+ progenitor cells and reduction of B cells from peripheral blood by use of immunomagnetic methods

BACKGROUND: Malignant cells may contribute to relapse after autologous hematopoietic cell transplantation The effectiveness of a double immunomagnetic purging strategy combining CD34-positive with B-negative cell selection to purge peripheral blood progenitor cells (PBPCs) from patients with chronic lymphoproliferative disorders has been analyzed. STUDY DESIGN AND METHODS: Twenty-two CD34+ cell selections from patients with follicular lymphoma (n = 14), chronic lymphocytic leukemia (n = 6), mantle cell lymphoma (n = 1), and splenic marginal zone lymphoma (n = 1) were performed by use of a magnetic cell selector followed by a negative cell selection step with anti-CD19 monoclonal antibody bound to immunomagnetic beads. RESULTS: The PBPC components contained median CD34+ cells of 1.24 percent (range, 0.38-3.92%) and CD19+ cells of 1.83 percent (range, 0.06-69.7%). After positive selection (n = 22), 49 percent (range, 16-72%) of CD34+ cells were recovered with a purity of 93 percent (range, 24-99%). The double-positive and -negative selections (n = 20) yielded 57.5 percent of CD34+ cells (range, 33.4-79.4%) with a purity of 95 percent (range, 63-99%). Logarithms of B-cell reduction in the CD34+-cell-enriched B-cell-depleted component had a median value of 3.63 (range, 2.74-4.84 log) and CD19+ and CD5+ cells for chronic lymphocytic leukemia patients with more than 4.56 log (>3.6-5.6 log). Of 13 PBPC components that had a tumor-specific clonal signal, 10 became PCR negative after the double-selection procedure. CONCLUSION: Combined positive and negative magnetic cell selection achieves a high grade of tumor cell reduction with up to 77 percent of the grafts being negative for tumor-specific clonal signal by PCR analysis. This technique preserves an adequate recovery of progenitor cells able to engraft.     

CD34+ cell mobilization for allogeneic progenitor cell transplantation: efficacy of a short course of G-CSF

BACKGROUND: G-CSF-mobilized PBPCs are considered the richest source of HPCs for both autologous and allogeneic transplantation, but, despite their wide use, the best dose and schedule for G-CSF administration have not been definitively established. STUDY DESIGN AND METHODS: With a target of collecting from the peripheral blood > or = 4 x 10(6) CD34+ cells per kg of body weight of the recipient, the short-course administration of glycosylated G-CSF (gly-G-CSF) in 30 healthy donors for an allogeneic transplantation was investigated. Gly-G-CSF was given subcutaneously at a dose of 10 microg per kg per day in two divided doses over 3 days and was followed by a leukapheresis (on the 4th day) 12 hours after the last dose. RESULTS: A median of 53.5 circulating CD34+ cells per microL (range, 19-190) was found in the 30 donors on the day of first leukapheresis, which allowed a median CD34+ cell collection of 6.0 x 10(6) per kg of body weight of the donor and 6.5 x 10(6) per kg of body weight of the recipient. In 25 (83%) of 30 donors, a single procedure was sufficient to collect the target CD34+ cells, while in the other 5, two leukapheresis procedures were required. Hematologic reconstitution was observed in all patients at a median of 14 days (range, 10-23) for neutrophils and 14.5 days (range, 11-46) for platelets. With a median infusion of 3.9 x 10(8) CD3+ T-lymphocytes per kg of body weight of the recipient (range, 1.3-7.8), acute and chronic GVHD occurred in 13 (43%) of 30 and 15 (60%) of 25 evaluable patients, respectively. After a median follow-up of 337 days from transplant, 22 (73%) of 30 patients are alive in complete remission. CONCLUSION: A schedule consisting of 3-day administration of gly-G-CSF followed by a single leukapheresis can be proposed and widely accepted by healthy donors, as 84 percent of them reach the target in the estimated time with a reduced drug exposure. The cost of the procedure is reduced, in terms of both the growth factor administration and the number of leukapheresis procedures. The search for the optimum methods of donor management may improve the acceptability of this procedure and increase the number of allogeneic transplantations from PBPCs.     

Leukapheresis after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation: a novel approach to harvest a second autograft

BACKGROUND: Autologous peripheral blood progenitor cells (PBPCs) are usually collected after the administration of conventional-dose chemotherapy (CDCT) and growth factors. However, there are no data available concerning the collection of PBPCs after high-dose chemotherapy (HDCT) and autologous hematopoietic transplantation in a larger series. STUDY DESIGN AND METHODS: Patients (n = 30) underwent leukapheresis for PBPC harvest after CDCT. After HDCT and autografting, the collection of a second PBPC autograft was attempted. RESULTS: Leukapheresis was performed after CDCT in all cases at a median of 118 CD34+ cells per microL (range, 18-589) and resulted in 6.4 x 10(6) CD34+ cells per kg (range, 1.7-29.0). After HDCT and autografting, 24 patients (80%) underwent secondary leukapheresis, although they had a significantly lower median of peripheral blood (PB) CD34+ cells (30/microL; range, 10-171; p < 0.001). In these patients a median of 3.6 x 10(6) CD34+ cells per kg (range, 1.6-10.1) was collected in the post-transplantation course. In the remaining six patients (20%) with PB CD34+ cells < 10 per microL, no PBPC harvesting was performed. These so-called poor mobilizers had received significantly less CD34+ cells for autologous transplantation than patients with successful post-HDCT mobilization (median, 2.5 x 10(6)/kg [range, 1.7-3.0] vs. 6.5 x 10(6)/kg [range, 3.2-19.6]; p < 0.001). CONCLUSION: Collection of PBPCs is possible in most patients during the recovery phase of hematopoiesis after HDCT plus autografting, and the number of circulating PBPCs may be related to the CD34+ cell dose transfused by the preceding autograft.     

Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known. METHODS: Forty-three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component. RESULTS: The mean purity of the final components was 84.3 percent (range, 27-99%), and the mean yield was 51.4 percent (range, 9.4-80. 4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, -0.704; p = 0. 001). CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.     

Effective collection of peripheral blood stem cells in children weighing 20 kilogram or less in a single large‐volume apheresis procedure

Introduction : Peripheral blood stem cell (PBSC) transplantation has become a routine procedure in pediatric oncology. A special group of PBSC donors are children weighing 20 kg or less. Limited vascular access and low blood volume puts them at a higher risk. Central line placement and a priming apheresis machine are recommended to avoid these complications. Patients and Methods : PBSC collections performed from July 2006 to May 2013 in children weighing less than 20 kg were included. All donors had a central venous catheter (CVC). An apheresis machine was primed with packet red blood cells. Results : Twenty‐seven PBSC collections were performed in 22 children weighing 20 kg or less, 14 for allogeneic and 8 for autologous transplantation, in order to collect at least 2 × 10⁶ CD34+ cells/kg. In the allogeneic group, median age and weight were 3 years (0.8–7) and 15.5 kg (8–20). In the autologous group, median age and weight were 3 years (2–7) and 15.35 kg (12.5–19.5). A single large‐volume apheresis was sufficient to obtain the CD34+ cells needed in 78.5% and 75% of the allogeneic and autologous groups, respectively, with a median 11.84 × 10⁶ and 5.79 × 10⁶ CD34+ cells collected per kilogram of weight of the recipient. No serious complications related to the apheresis procedure or CVC placement occurred. Conclusion : PBSC collection in a single large‐volume apheresis for allogeneic and autologous transplants in children weighing 20 kg or less is a safe and effective procedure when based on standardized protocols. J. Clin. Apheresis 30:281–287, 2015. © 2014 Wiley Periodicals, Inc.     

Positive selection of CD34+ cells by immunoadsorption: factors affecting the final yield and hematopoietic recovery in patients with hematological malignancies and solid tumors.

There is a progressive increase in the use of selected hematopoietic progenitor cells after myeloablative therapy in patients affected by malignancies. Our goal was to determine which blood parameters, in the starting cell population, influence the concentration of CD34+ progenitors and the removal of unwanted cells in the final product. Also, we evaluated the hematopoietic recovery and toxicity associated with peripheral blood stem cell infusion. We retrospectively reviewed 53 procedures of positive selection of CD34+ cells, performed with the Ceprate SC immunoadsorption system, in 47 paticnts affected by various hematologic malignancies and solid tumors. An increased percentage of CD34+ cells in the starting fraction was associated both with the final purity and enrichment of CD34+ cells and with a decreased percentage of CD3+ and CD19+ cells in the final product. A low platelet count before selection had a borderlinc influence on the recovery of CD34+ cells. Forty patients received a median of 5 x 10(6) CD34+ cells per kg; the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l in a median of 10 days whereas a PLT count above 20 x 10(9)/l was observed in 14 days. The reinfusion of selected CD34+ cells, containing a very low amount of dymethylsulfoxide. was well tolerated and no adverse reactions were observed. Autologous transplantation with selected CD34+ cells is a safe and well-tolerated procedure in patients affected by hematologic malignancies and solid tumors. Positive selection of CD34+ cells seems to be related to the quality of the apheresis products, particularly to the initial CD34+ cell and PLT content.     

Prediction of engraftment after autologous peripheral blood progenitor cell transplantation: CD34, colony‐forming unit–granulocyte‐macrophage,or both?

BACKGROUND: The rate of hematologic recovery after peripheral blood progenitor cell (PBPC) transplantation is influenced by the dose of progenitor cells. Enumeration of cells that express CD34+ on their surface is the most frequently used method to determine progenitor cell dose. In vitro growth of myeloid progenitor cells (colony-forming unit-granulocyte-macrophage [CFU-GM]) requires more time and resources, but may add predictive information. STUDY DESIGN AND METHODS: A series of 323 patients, who underwent autologous PBPC transplantation for multiple myeloma, malignant lymphoma, or locally advanced breast cancer, were studied for the effect of CD34+ dose and CFU-GM dose on hematologic recovery. Measures for engraftment were days to absolute granulocyte and platelet (PLT) counts to greater than 500 per muL and than 20 x 10(9) per L, respectively, and number of PLT transfusions and red cell units required. RESULTS: The CD34+ dose had a median of 8.4 x 10(6) per kg, and the CFU-GM dose a median of 84.9 x 10(4) per kg. The CD34+ and CFU-GM doses showed significant correlation (R = 0.63; p < 0.0001) but a wide variation in the ratio of CD34+ and CFU-GM. Both CD34+ and CFU-GM doses had significant correlation with the measures of engraftment, but for all measures the relationship of CD34+ was stronger. Multivariate analysis and subgroup analysis of patients receiving CD34+ doses of less than 5 x 10(6) per kg also did not reveal an independent predictive value for CFU-GM. CONCLUSION: For prediction of hematologic recovery after autologous PBPC transplantation, determination of CFU-GM dose does not add to the predictive value of the CD34+ dose.