Automatic interface-controlled apheresis collection of stem/progenitor cells: results from an autologous donor validation trial of a novel stem cell apheresis device

BACKGROUND: Cryopreserved hematopoietic progenitor cells collected by apheresis from granulocyte–colony‐stimulating factor with or without chemotherapy–mobilized patients have become the preferred type of autograft to support treatment of diseases amenable to high‐dose chemotherapy. A novel apheresis system, the Spectra Optia v.5.0 (CaridianBCT), was constructed to meet certain shortcomings of manual apheresis systems such as the COBE Spectra MNC (CaridianBCT), including the need for continuous optical or manual monitoring and readjustment of buffy coat position and sensitivity to inconsistent blood flow. By use of optical sensors, which provide real‐time automatic interface (buffy coat) and collection line control, the Spectra Optia promises to automatically guide apheresis procedures, potentially freeing up operator time and reducing variability in collection efficiency (CE2). STUDY DESIGN AND METHODS: In a two‐center clinical trial, 35 autologous stem cell donors were subjected to apheresis with the Spectra Optia to validate feasibility and effectiveness of apheresis procedures. Results were compared to data from 80 autologous apheresis procedures with the COBE Spectra MNC. RESULTS: Usability and function of the automatic interface management were excellent. CD34+ cell quality, assessed by viability staining, colony‐forming unit–culture frequency, and engraftment kinetics, was equally good with both systems. CE2 of the Spectra Optia, calculated as CD34+ contents in the product divided by the number of CD34+ cells presented to the collection port, exceeded that of the COBE Spectra MNC. Spectra Optia product volumes were significantly smaller. Very high white blood cell and platelet counts modestly reduced CE2 with the Spectra Optia. CONCLUSION: The Spectra Optia is a novel automatic apheresis system supporting autologous stem cell collection with at least equal efficiency and superior user‐friendliness compared to the COBE Spectra MNC.     

Comparison of two mononuclear cell program settings on two apheresis devices intended to collect high yields of CD14+ and CD3+ cells

BACKGROUND: In cancer and transplantation therapy apheresis devices and software of optimum standards are required for the collection of high cell yields with high purity of the desired cell fraction. STUDY DESIGN AND METHODS: In a paired study, 15 healthy blood donors underwent four 10-L leukapheresis procedures (197 +/- 33 min) with an inlet blood flow rate of 60 mL per minute by use of two different MNC program settings of the COBE Spectra (Gambro BCT) and the AS.TEC 204 (Fresenius Hemocare) cell separators. RESULTS: Use of the standard MNC program of both apheresis devices resulted in significantly higher (p < 0.01) collection efficiencies of CD14+ monocytes, CD3+ cells, CD4+ cells, CD8+ T cells, CD16+ CD56+ natural killer (NK) cells, and residual PLTs (p < 0.001), owing to higher centrifuge speed. The mean MNC purity of all components was more than 90 percent. By use of standard programs of either device, significant correlations (p < 0.01) between donor monocytes and preleukapheresis NK cell counts and the corresponding component cell yields were found. CONCLUSION: Compared to the program modifications with lower centrifuge velocities the standard MNC programs were significantly more efficient regarding CD14+, CD3+, and CD16+ CD56+ cells. Enhanced centrifuge speed and inlet blood flow rate in MNC programs resulted in higher, similar composed MNC concentrations of the products.     

Evaluation of the spectra optia apheresis system for mononuclear cell (MNC) collection in G‐CSF mobilized and nonmobilized healthy donors: Results of a multicenter study

The Spectra Optia apheresis system is a newer centrifugation‐based device that in comparison with the COBE Spectra includes features that enhance procedure automation and usability. In this FDA‐approved three‐center two‐arm observational study we characterized the performance of the Spectra Optia for collection of MNCs and CD34+ cells from nonmobilized and granulocyte‐colony stimulating factor (G‐CSF) mobilized healthy donors, respectively. There were a total of 15 evaluable subjects in each arm. Key performance indicators included collection efficiency of MNCs/CD34+ cells, product purity and cellular viability. For nonmobilized donors, median MNC collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 57%, 12%, 4%, and 1.7%, respectively. For mobilized donors, median MNC collection efficiency, CD34+ cell collection efficiency, platelet collection efficiency, product hematocrit and granulocyte contamination were 61%, 77%, 19%, 4%, and 15%, respectively. Average WBC viability in the mobilized products was 99%. There was one severe (grade 3) adverse event related to citrate toxicity. This study demonstrates that the Spectra Optia can be used for safe and efficacious collection of MNCs, and results obtained are in line with expectations on collection efficiency and product characteristics. Adverse events were limited to those that are well documented in the stem‐cell mobilization and leukapheresis process. As of the time of this writing, FDA 510(k) approval for use of the Spectra Optia device for MNC collection was achieved in the US based partly on the results of this study. J. Clin. Apheresis 29:273–280, 2014. © 2014 Wiley Periodicals, Inc.     

Comparison of two blood cell separators in collecting peripheral blood stem cell components

To ensure that a sufficient number of CD34+ cells are collected for an allogeneic blood progenitor cell transplant, the most effective blood cell separator should be used to collect peripheral blood stem cell (PBSC) components. We compared the effectiveness of two blood cell separators. We gave 29 healthy people 7.5 or 10 μg kg^?1 of granulocyte colony stimulating factor (G-CSF) daily for 5 days and collected one PBSC component with either a Fenwal CS3000 (n = 15) or a Cobe Spectra (n = 14) blood cell separator. The volume of blood processed was the same for each machine (8.4 ± 1.0 L; range = 4.9–9.4 L for the CS3000 and 8.9 ± 1.0 L; range 6.7–10.9 L; P = 0.71). The components collected with the CS3000 contained more mononuclear cells (39.6 ± 21.9 × 10⁹ compared with 26.9 ± 5.6 × 10⁹, P = 0.02) and fewer neutrophils (1.38 ± 1.88 × 10⁹ compared with 5.53 ± 8.71 × 10⁹, = 0.001). The total number of CD34+ cells collected with the two instruments was the same (470 ± 353 × 10⁶ for the CS3000 and 419 ± 351 × 10⁶ for the Spectra; P = 0.64) as was the number of CD34+ cells collected per litre of whole blood processed (55.9 ± 42.0 × 10⁶ L^?1 compared with 45.9 ± 37.9 × 10⁶ L^?1; P = 0.59). The mononuclear cell collection efficiency was greater for the CS3000 (82.4 ± 54.9% compared with 53.3 ± 14.1; P = 0.04) but the CD34+ cell collection efficiencies were the same (87.4 ± 61.1% for the CS3000 compared with 56.3 ± 23.5% for the Spectra; P = 0.07). In conclusion, both blood cell separators collected components which contained large numbers of CD34+ cells, but those collected with the CS3000 contained fewer neutrophils and the CS3000 was more efficient at collecting mononuclear cells.     

Comparison of hematopoietic progenitor cell collections using the COBE Spectra version 7 and Amicus version 3.1 for patients with AL amyloidosis

To address concerns about infused fluid volume during HPC collections in patients with AL amyloidosis, our institution has used a 26:1 anticoagulant (AC) ratio on the COBE Spectra and on the Fenwal Amicus. In this study, in a cohort of AL amyloid patients, we compared the Amicus version 3.1 to the Spectra version 7 MNC collections with regard to infused fluid volume, CD34+ cell yield, lymphocyte yield, cross‐cellular content, and adverse reactions. Both instruments used a 26:1 AC ratio but the Amicus delivered significantly less AC per procedure (Amicus 678 mL vs. Spectra 753 mL). With comparable baseline CD34+ cell counts (Amicus 33 cells/μL vs. Spectra 27 cells/μL); Amicus collected significantly more CD34+ cells (3.1 vs. 1.5 × 10⁶/kg) and equivalent lymphocytes (18.7 vs. 14.5 × 10⁹). Amicus collected significantly fewer WBC (51.8 vs. 72.7 × 10⁹), granulocytes (15.1 vs. 27.5 × 10⁹), and PLT (2.3 vs. 3.9 × 10¹¹) per procedure with equivalent RBC content (26 vs. 30 mL). CD34+ cell (5.0 vs. 4.4 × 10⁶/kg) and lymphocyte doses (32.7 vs. 33.9 × 10⁹) were equivalent in infused products collected on the Amicus and Spectra but the frequency of high volume products was lower for Amicus. Frequency and severity of adverse reactions during collection and infusion were similar for both. In this group of AL amyloid patients, Amicus was superior to Spectra with regard to fluid infused, CD34+ cell yield, and cross‐cellular contamination with equivalent lymphocyte yield and reaction incidence. © 2011 Wiley‐Liss, Inc.     

Terumo spectra optia leukapheresis of cynomolgus macaques for hematopoietic stem cell and T cell collection

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen‐specific lymphocytes for in‐depth immunological analysis, and latently‐infected CD4+ T‐cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T‐cells from cynomolgus macaques as small as 3 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 10⁹ total nucleated cells from mobilized animals and 1.2 × 10⁹ total nucleated cells from nonmobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.     

Donor safety in triple plateletpheresis: results from the German and Austrian Plateletpheresis Study Group multicenter trial

BACKGROUND: The objective was to investigate potential risks for apheresis donors associated with a triple‐plateletpheresis (TP) program. STUDY DESIGN AND METHODS: Eleven hemapheresis centers randomly assigned 411 repeat donors (ratio, 1:1.2) to either double plateletpheresis (DP; 185 donors) or TP (226 donors) with a platelet (PLT) target content of at least 5.0 × 10¹¹ PLTs/DP and at least 7.5 × 10¹¹ PLTs/TP. The primary endpoint was procedure‐related postapheresis PLT count of at least 150 × 10⁹/L (probability, ≥98%). Secondary endpoints were apheresis characteristics and donor adverse reactions. RESULTS: In 6 of 1133 DPs (0.5%) in 4 of 185 donors (2.2%) and in 20 of 1020 TPs (2.0%) in 14 of 226 donors (6.2%), postapheresis PLT counts were below 150 × 10⁹/L. There were marginal but significant differences in collection efficiency (DP, 69.2 ± 9.1%; TP, 70.9 ± 9.0%; p ≤ 0.0001) and collection rate (DP, 10.4 × 10⁹ ± 2.3 × 10⁹ PLTs/min; TP, 10.8 × 10⁹ ± 2.3 × 10⁹ PLTs/min; p ≤ 0.005). The PLT yields were 5.9 × 10¹¹ ± 0.8 × 10¹¹ PLTs for DP and 8.3 × 10¹¹ ± 0.9 × 10¹¹ PLT for TP (p ≤ 0.0001) at processing times of 59 ± 13 minutes (DP) versus 80 ± 16 minutes (TP; p ≤ 0.0001). Significant PLT recruitment (1.10 ± 0.14 vs. 1.20 ± 0.23; p < 0.0001) was seen for both DP and TP. DP and TP did not differ with regard to venous access problems (VAPs) without discontinuation (3.8% for both), but DP induced fewer VAPs with discontinuation (1.1% vs. 3.0%; p < 0.01). Mild citrate toxicity (1.7% vs. 3.9%; p < 0.01) and circulatory reactions (0.4% vs. 2.2%; p < 0.01) were more often noticed in TP, but caused no increase in discontinuations. CONCLUSIONS: TP results in an increase in mild donor reactions but does not significantly impair donor safety or product quality.     

Evaluation of a new protocol for peripheral blood stem cell collection with the Fresenius AS 104 cell separator

In this report we analyzed sixty leukapheresis procedures on 35 patients with a new protocol for the Fresenius AS 104. Yields and efficiencies for MNC, CD 34+ cells, and CFU-GM indicate that the new protocol is able to collect large quantities of hemopoietic progenitors. Procedures were performed processing 8.69 ± 2.8 liters of whole blood per apheresis and modifying 3 parameters: spillover-volume 7 ml, buffy-coat volume 11.5 ml, centrifuge speed 1,500 rpm; blood flow rate was 50 ml/min and the anticoagulant ratio was 1:12. No side effects were observed during apheresis procedures except for transient paresthesia episodes promptly resolved with the administration of calcium gluconate. Yields show a high capacity of the new program to collect on average MNC 17.28 ± 10.85 × 10⁹, CD 34+ 471 ± 553.5 × 10⁶ and CFU-GM 1278.7 ± 1346.3 × 10⁴ per procedure. Separator collection efficiency on average was 49.91 ± 23.28% for MNC, 55.1 ± 35.66% for CFU-GM, and 62.97 ± 23.09% for CD 34+ cells. Particularly interesting are results for MNC yields and CD 34+ efficiency; these results make the new program advantageous or similar to the most progressive blood cell separators and capable to collect a sufficient number of progenitor cells for a graft with a mean of 1.80 ± 0.98 procedures per patient. J. Clin. Apheresis 12:82–86, 1997. © 1997 Wiley-Liss, Inc.     

Allogeneic donor peripheral blood "stem cell" apheresis: prospective comparison of two apheresis systems

BACKGROUND: Granulocyte–colony‐stimulating factor–mobilized peripheral blood stem cells, collected by white blood cell apheresis, are used for more than 80% of allogeneic and most autologous hematopoietic stem cell transplantations. Optimal donor and recipient outcomes require maximized stem cell collection efficiency and minimized non–target cell contamination. Therefore, improved apheresis technology is desirable. The safety and feasibility of apheresis collections with the novel, electronics‐assisted apheresis system Spectra Optia v.5.0 (CaridianBCT) were recently demonstrated. An unpublished optimization trial had furthermore determined that different settings than manufacturer‐installed default might result in improved apheresis yields. STUDY DESIGN AND METHODS: The first prospective comparison of allogeneic peripheral blood stem cell apheresis with the Spectra Optia versus the COBE Spectra (CaridianBCT) mononuclear cell (MNC) in a routine clinical setting is reported here; “optimized” machine settings were used. Assessed variables included collection efficiency, product characteristics, donor outcomes, and frequency of operator interventions. Outcomes were additionally compared with historical data from the Spectra Optia in default mode. RESULTS: The mean CD34+ cell collection efficiency CE1 was 7.9% greater with the Spectra Optia than with the COBE Spectra MNC. Variability of outcomes was equally great. Reduced platelet (PLT) attrition necessitated 90% fewer autologous PLT reinfusions. Spectra Optia products contained 50% fewer red blood cells, but 50% more granulocytic lineage cells. Less operator input was required, although 26% of Spectra Optia apheresis procedures required triggering of the first chamber flush. Apheresis yield and collection efficiency were also markedly greater than in default‐mode Spectra Optia collections. CONCLUSION: Using optimized machine settings, peripheral blood stem cell apheresis outcomes with the automated apheresis system Spectra Optia exceed results with the COBE Spectra MNC or the Spectra Optia in the default mode.     

The use of hematocrit level for predicting the efficiency of peripheral blood CD34+ cell collection after G‐CSF Mobilization in Healthy Donors

Recently, peripheral blood stem cell (PBSC) has been widely used and replaced bone marrow (BM) as the stem cell source in allogeneic hematopoietic stem cell transplantation (HSCT) because of a more rapid engraftment, easier accessibility, and lower risk of donor complications. We, therefore, report the predicting factors for the high PBSC harvest yields in 50 healthy donors. Among the 50 donors, median collected CD34⁺ cell number was 4.6 × 10^6/kg (1.5–16.3 × 10⁶/kg). Number of circulating CD34+ cells and hematocrit (HCT) level increased parallelly whereas peripheral CD34+ cell numbers were decreased with increasing donor age. In univariate analysis, HCT level≥ 35.5% at the time of PBSC collection was significantly associated with high PBSC number (≥ 5.0 × 10⁶ cells/kg) and donor aged <30 years was significantly associated with collected CD34+ cells ≥ 6.0 × 10⁶/kg, P = 0.03. HCT level ≥35.5% was an independent parameter for high WBC count (≥50 × 10⁹/L), P < 0.05. None of donor who had both HCT < 35.5% and WBC < 50 × 10⁹/L had circulating CD34+ cells ≥ 5.0 × 10⁶/kg. Platelet count ≥ 200 × 10⁹/L was found significantly in donors with WBC ≥ 40 × 10⁹/L (P = 0.03) and HCT ≥ 35.5%, P < 0.05. Collected PBSC number tended to be higher in our donors with high levels of HCT, WBC, and platelet. We also found that HCT and platelet levels in our donors decreased after receiving G‐CSF administration compared with the initial complete blood counts (CBC) results. We, therefore, concluded that HCT level at the time of initiation leukapheresis was an important predictor for PBSC collection yields. J. Clin. Apheresis 30:329–334, 2015. © 2015 Wiley Periodicals, Inc.     

Evaluation of a new continuous mononuclear cell collection procedure in a single transplant center cohort enriched for AL amyloidosis patients

Background

The Spectra Optia continuous mononuclear cell (CMNC) program is newly available, and herein validated in a single-center cohort enriched with AL amyloidosis patients to collect a target CD34+ yield of 2.5?×?10⁶ cells/kg within 2 days.

Comparison of COBE® Spectra™ software version 4.7 PBSC and version 6.0 auto PBSC™ program

Until recently, the collection of peripheral blood progenitor cells (PBPC) has been semi-automated by using the COBE® Spectra™, with the operator manually maintaining the position of the white cells being collected. The COBE® Spectra™ Version 6.0 apheresis device offers the user an automated program for the collection of PBPC. In this study, we compared the new software Version 6.0 to that of Version 4.7. Patients (n=46) undergoing PBPC collection were allocated to cell processing with either Version 4.7 (n=24) or Version 6.0 (n=22). The CD34+ cell count, mononuclear cell (MNC) count, white cell count (WCC), hemoglobin (Hb), and platelet content in the autograft product by using the two versions were compared. We divided the analysis into three subsets according to peripheral blood (PB) CD34 content: <10×10⁶/L, 10–50×10⁶/L and >50×10⁶/L. Analysis of the three subsets showed no statistical difference between results obtained when the starting PB CD34+ cell count was 10–50×10⁶/L (P=0.08) or >50 ×10⁶/L (P=0.4065). At lower starting PB CD34+ cell counts of <10×10⁶/L, Version 4.7 was superior (P=0.0167). However, autograft platelet contamination of the autograft was significantly higher using Version 4.7 (P=<0.0001). J. Clin. Apheresis 14:26–30, 1999. © 1999 Wiley-Liss, Inc.     

CD 14+ cell collection in non-cytokine-stimulated donors with the COM.TEC cell separator

BACKGROUND: The COM.TEC cell separator (Fresenius Hemocare), equipped with the MNC program (single-stage chamber) and the PBSC-LYM program (dual-stage chamber), was evaluated for CD 14+ cell collection regarding cell yields, collection efficiencies (CEs), and the content of residual cells in harvests. STUDY DESIGN AND METHODS: Twenty-four non-cytokine-stimulated donors underwent 5-L mononuclear cell (MNC) collections on the COM.TEC device to compare both programs. Two software versions (v02.03.05 vs. v 03.00.04) were investigated for optimization of the CD 14+ cell collection process. Blood counts of donors and products were analyzed for CD 14+ cells by flow cytometry and for platelets (PLTs), white blood cells, and red blood cells (RBCs) by a blood cell counter. RESULTS: In 5-L collections, the MNC program resulted in high CEs (83+/- 23%) and yields (1.2 x 10(9)+/- 0.6 x 10(9) per unit) of CD 14+ cells, but the products showed high residual PLTs. The use of a dual-stage chamber in the PBSC-LYM program produced a low content of residual PLTs (0.7 x 10(11) +/- 0.3 x 10(11) per unit) and RBCs but failed to reach a target of 1 x 10(9) CD 14+ cells. Modulated light to stabilize the buffy-coat detection by the interface monitor significantly improved CD 14+ cell enrichment. By use of the PBSC-LYM program, higher centrifuge velocity (1700 rpm [382 x g] vs. 1500 rpm [297 x g]) improved significantly CD 14+ cell yields (0.7 x 10(9) vs. 0.5 x 10(9) cells). CONCLUSION: A pure CD 14+ cell product with low numbers of residual cells was obtained by the PBSC-LYM program, which could be useful for good manufacturing practice-conformed production within closed systems. The MNC program offers the collection of high CD 14+ cell yields with excellent CEs but also high residual PLT counts.     

Comparison of the in vitro storage properties of Amicus apheresis platelets collected using single- and double-needle procedures from the same donors

BACKGROUND: Amicus apheresis platelets (PLTs) can be collected using either a single‐ (SN) or a double‐needle (DN) procedure. To investigate whether the method of PLT collection using the same instrument influences PLT quality, the in vitro storage properties of Amicus PLTs were evaluated in the same donors collected by SN and DN procedures. STUDY DESIGN AND METHODS: Single apheresis PLT collections with concurrent plasma were performed on donors using the Amicus with a target yield of 4 × 10¹¹. A PLT unit was collected from a donor assigned to either a SN or a DN procedure; a subsequent donation from the same individual was collected by the other procedure (n = 10). Units were stored at 20 to 24°C with continuous agitation, assayed for 19 PLT storage variables, and analyzed by paired t test, with differences between values obtained with SN and DN collections considered significant with p values of less than 0.001. RESULTS: PLT units collected by SN procedure had contents and concentrations similar to those collected by DN procedures (4.1 × 10¹¹ ± 0.3 × 10¹¹ vs. 4.0 × 10¹¹ ± 0.3 × 10¹¹ and 1396 × 10⁹ ± 131 × 10⁹ vs. 1367 × 10⁹ ± 110 × 10⁹ PLTs/L). On Day 7, SN and DN PLTs had comparable pH values (7.07 ± 0.09 vs. 6.99 ± 0.17), morphology (52.4 ± 18.7% vs. 56.0 ± 13.3% discoid), aggregation (87.1 ± 11.5% vs. 91.3 ± 5.4%), and activation (45.8 ± 11.9% vs. 48.2 ± 8.7% CD62P), as well as all other variables (p > 0.05; Day 7 CO₂, p = 0.0304). CONCLUSION: The in vitro storage properties of apheresis PLTs collected from the same donors using a SN and DN procedure with the Amicus instrument were maintained through 7 days of storage and yielded comparable results.     

Unstimulated apheresis for chimeric antigen receptor manufacturing in pediatric/adolescent acute lymphoblastic leukemia patients

Autologous unstimulated leukapheresis product serves as starting material for a variety of innovative cell therapy products, including chimeric antigen receptor (CAR)-modified T-cells. Although it may be reasonable to assume feasibility and efficiency of apheresis for CAR-T cell manufacture, several idiosyncrasies of these patients warrant their separate analysis: target cells (mononuclear cells [MNC] and T-cells) are relatively few which may instruct the selection of apheresis technology, low body weight, and, hence, low total blood volume (TBV) can restrict process and product volume, and patients may be in compromised health. We here report outcome data from 46 consecutive leukaphereses in 33 unique pediatric patients performed for the purpose of CD19-CAR-T-cell manufacturing. Apheresis targets of 2×10⁹ MNC/1×10⁹ T-cells were defined by marketing authorization holder specification. Patient weight was 8 to 84 kg; TBV was 0.6 to 5.1 L. Spectra Optia apheresis technology was used. For 23 patients, a single apheresis sufficed to generate enough cells and manufacture CAR-T-cells, the remainder required two aphereses to meet target dose and/or two apheresis series because of production failure. Aphereses were technically feasible and clinically tolerable without serious adverse effects. The median collection efficiencies for MNC and T-cells were 53% and 56%, respectively. In summary, CAR apheresis in pediatric patients, including the very young, is feasible, safe and efficient, but the specified cell dose targets can be challenging in smaller children. Continuous monitoring of apheresis outcomes is advocated in order to maintain quality.     

Collection of peripheral blood mononuclear cells as a byproduct of plateletpheresis with two different blood cell separators

Peripheral blood mononuclear cells (PBMC) were collected as a byproduct of plateletpheresis of normal blood cell donors using modifications to standard automated protocols on either the CS-3000 or Spectra blood cell separator machine. Comparison of the PBMC products obtained showed X ± SD WBC yields of 5.3 ± 3.4 vs. 3.8 ± 2.0 × 10⁹ with the CS-3000 and Spectra, respectively (P < .0001). The majority of the cells were lymphocytes, with 13–15% monocytes with both machines. Sixteen percent of the WBC collected with the Spectra, but only 1% of those collected with the CS-3000, were granulocytes. The CS-3000 PBMC product contained fewer RBC (0.2 ± 0.1 × 10¹¹ vs. 2.4 ± 0.6 × 10¹¹) and more platelets (1.6 ± 0.6 × 10¹¹ vs. 0.35 ± 0.39 × 10¹¹) in a smaller volume (40 ± 14 ml vs. 229 ± 37 ml) than the Spectra products. Comparison of the platelet collections harvested when PBMC were also collected to platelets harvested using standard procedures on the same machine showed no change in platelet. WBC, or RBC yields for the Spectra. A significant increase in mean WBC contamination from 40 ± 56 × 10⁷ to 112 ± 205 × 10⁷ and a small, but statistically insignificant, decrease in platelet yield from 4.1 ± 1.2 × 10¹¹ to 3.9 ± 1.8 × 10¹¹ was observed in the CS-3000 platelet collections when PBMC were harvested. There was no sustained change in donor lymphocyte counts and no change in acute donor side effects or time requirements when PBMC were collected. The procedural modifications were easily learned by multiple operators, and required about 15 and 5 min additional operator time on the CS-3000 and Spectra, respectively. Thus, harvest of large numbers of PBMC as a byproduct of plateletpheresis can be readily accomplished with either of 2 different blood cell separator machines. The cells obtained have been useful for a variety of purposes in both clinical hospital and basic research laboratories. © 1992 Wiley-Liss, Inc.     

Automated CD14+ monocyte collection with the autoMNC program of the COM.TEC cell separator

BACKGROUND: The standard mononuclear cell (MNC) program of the COM.TEC device (Fresenius HemoCare GmbH) showed excellent collection efficiency of CD14+ monocytes. A major disadvantage was high content of residual cells in MNC harvests, which could influence dendritic cell (DC) culture. STUDY DESIGN AND METHODS: The autoMNC program (COM.TEC) was compared with the standard MNC program (n = 12). Additionally, two cycle volumes (300 mL vs. 450 mL, n = 19) were compared (standard MNC program). Samples were assayed for white blood cells (WBCs), red blood cells (RBCs), granulocytes (PMNs), hematocrit, and platelets (PLTs) on an automated blood cell counter (Sysmex K 4500, TAO Medical). CD14+ cells were analyzed by flow cytometry (FACSCalibur, BD). RESULTS: The autoMNC program produced 1.33 x 10(9) +/- 0.36 x 10(9) CD14+ cells, 5.60 x 10(11) +/- 0.97 x 10(11) PLTs, and 1.43 x 10(11) +/- 0.37 x 10(11) RBCs. Compared to the standard MNC program, significantly higher PLT yields but lower RBC yields and product volume were harvested. Increasing the CV from 300 to 450 mL dropped the product volume, residual PLTs, and RBCs significantly, whereas WBC and monocyte yields did not change. The WBC predonation counts of donors correlated significantly with monocyte yields. CONCLUSIONS: The autoMNC program reduced the buffy coat (BC) volume and RBC yields in products compared to the standard MNC program. Increasing the CV (standard MNC program) reduced residual PLTs, RBCs, and the BC volume of MNC harvests. The donor WBC predonation count was a good predictor for the monocyte yield of products.     

Mononuclear cell variability and recruitment in non-cytokine-stimulated donors after serial 10-liter leukapheresis procedures

BACKGROUND: We introduced monitoring of mononuclear cell (MNC) counts to obtain enhanced donor control and a stable quality of MNC products, because there are limited data available about blood donors after serial leukapheresis (LP) procedures. STUDY DESIGN AND METHODS: In a prospective paired study, 13 male healthy blood donors underwent 10-L LP procedures performed on two apheresis devices by use of two MNC program settings (COBE Spectra, Gambro BCT, SF 250 vs. SF 500; and AS.TEC 204, Fresenius Hemocare, CP 129 vs. CP 194). Donors' pre- and postdonation MNC counts were analyzed by fluorescence-activated cell sorting. RESULTS: After each 10-L LP procedure, a transient decline (p < 0.05) of CD14+ monocyte and platelet counts appeared in donors. Loss of donors' CD3+ T cells, CD19+ B cells, and CD16+56+ natural killer (NK) cells during MNC collection was partly compensated by cell recruitment. The MNC recruitment factor (RF) seems to be higher with high-yield MNC program settings. Negative correlations (p < 0.01) were noticed between predonation counts and RFs of CD3+ T cells and CD16+56+ NK cells. Four serial 10-L LP procedures did not result in long lasting MNC depletion for donors. CONCLUSION: MNC recruitment seems to depend on MNC program settings and collected cell yields. Low MNC counts could result in high cell recruitment that may contribute to stable collection results to some degree. Nevertheless, there seems to be a considerable individual variation of MNC recruitment in donors that should be investigated in more detail.     

Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters

BACKGROUND: Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1 × 10³‐4 × 10³/mL), but their number trapped in one LRF after filtration of 400 to 450 mL of blood is high (0.4 × 10⁶‐1.6 × 10⁶). STUDY DESIGN AND METHODS: To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500‐mL continuous or after sequential elution (50‐ or 20‐mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. RESULTS: The 20‐mL back‐flushing of LRFs allowed the most efficient WBC recovery. The next steps (110 × g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76 ± 10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4 × 10⁵ to 6 × 10⁵ cells containing approximately 85% of functional CD34+ cells were obtained. CONCLUSION: This procedure allows the easy, rapid (<5 hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.     

Blood counts in healthy donors 1 year after the collection of granulocyte-colony-stimulating factor-mobilized progenitor cells and the results of a second mobilization and collection

BACKGROUND : Granulocyte–colony-stimulating factor (G–CSF)-mobilized blood cells are being used for allogeneic transplants, but the long-term effects of G–CSF on healthy individuals are not known. Furthermore, it is not certain how many CD34+ cells can be collected in a second mobilization and collection procedure. STUDY DESIGN AND METHODS : Nineteen people were given 2, 5, 7.5, or 10 μg of G–CSF per kg per day for 5 days, and blood progenitor cells were collected by apheresis on the sixth day; this was done on two occasions separated by at least 12 months. Blood counts obtained before and after each course of G–CSF and the quantity of cells collected were compared. RESULTS : There were no differences in white cell (WBC), platelet, red cell, and WBC differential counts measured before each course of G–CSF, and all the values were in the normal range. In a subset of 12 people who received 7.5 or 10 μg of G–CSF per kg per day for both courses, the numbers of neutrophils, mononuclear cells, and CD34+ cells in the blood after each course were similar (34.1 ± 7.31 × 10⁹/L vs. 36.4 ± 12.3 × 10⁹/L, p = 0.24; 6.59 ± 2.28 × 10⁹/L vs. 5.63 ± 2.11 × 10⁹/L, p = 0.24; and 92.0 ± 55.6 × 10⁶/L vs. 119.2 ± 104.6 × 10⁸/L; p = 0.48, respectively), as were the quantities of mononuclear cells (31.0 ± 8.4 × 10⁹ vs. 31.0 ± 6.1 × 10⁹; p = 0.64) and CD34+ cells (417 ± 353 × 10⁶ vs. 449 ± 286 × 10⁶; p = 0.53) collected in the two apheresis procedures. Furthermore, there was a positive correlation between the quantity of CD34+ cells collected from each of the 12 people per liter of whole blood processed in the two procedures (r² = 0.86, p<0.001). CONCLUSION : One year after the administration of G–CSF to healthy people, their blood counts were normal and unchanged from pretreatment counts. If healthy people donate blood progenitor cells after a second G–CSF course, the quantity of CD34+ cells collected will be similar to that obtained in the first collection.