Centrifugal Cell Separator Research Articles

Clinical implications of leukocytapheresis using a centrifugal cell separator for steroid‐resistant pyoderma gangrenosum associated with inflammatory bowel disease

Clinical implications of leukocytapheresis using a centrifugal cell separator for steroid‐resistant pyoderma gangrenosum associated with inflammatory bowel disease

Stool decay‐accelerating factor as a marker for monitoring the disease activity during leukocyte apheresis therapy in patients with refractory ulcerative colitis

We have shown previously that concentrations of stool decay-accelerating factor (DAF; CD55), a complement regulatory protein, in patients with ulcerative colitis (UC) are increased in relation to the severity of the colonic mucosal inflammation. In the present study, we evaluated the usefulness of stool DAF as a marker for monitoring disease activity in patients with steroid-resistant active UC being treated with leukocyte apheresis performed with a centrifugal cell separator. Twenty-one patients with active and steroid-resistant UC were treated with leukocyte apheresis once a week for 4 weeks, and stool DAF concentrations were determined weekly by immunoassay. After treatment, 11 (52%) of the 21 UC patients went into remission. Stool DAF concentrations decreased promptly and steadily in the responsive group. The difference reached statistical significance as soon as after the second apheresis session (P < 0.003), compared with values before the therapy and corresponding values in the non-responsive group (P = 0.024). The reduction in stool DAF concentrations after the second apheresis session was significantly greater in the responsive group (median 90%, range 22-90%) than in the non-responsive group (median -13%, range -307-94%) (P = 0.008). Hematological tests, that is, white blood cell (WBC) count and C-reactive protein, declined significantly during the apheresis therapy, but not in relation to therapeutic response. Stool DAF concentration is a useful marker in the clinical response of UC patients to treatment with leukocyte apheresis.

Leukocyte apheresis using a centrifugal cell separator in refractory ulcerative colitis: a multicenter open label trial.

Recently, successful results of ulcerative colitis (UC) treatments with leukocyte apheresis have been reported by several institutes. To certify the efficacy of leukocyte apheresis in refractory UC patients, a multicenter open label trial was conducted, and results were analyzed. Fifty patients diagnosed with active steroid-resistant UC were enrolled in this study from 14 medical centers. Using a centrifugal cell separator (Component Collection System, Haemonetics), leukocyte apheresis was performed once a week for 5 weeks. General conditions and abdominal symptoms were recorded daily, and laboratory tests were followed weekly. Changes of colonoscopic and histological manifestations of luminal activity through the study period were evaluated. At the end of the study period, stool frequency was decreased to less than 4 times a day in 68.4% (26 of 38) and serum C-reactive protein (CRP) concentration was normalized in 56.7% (17 of 30) of the patients. Colonoscopic remission was achieved in 57.7% (26 of 45), and histological improvement was noted in 54.1% (20 of 37) of the patients tested. Improved disease activity was demonstrated in 74% (37 of 50) of the patients by general assessment criteria. Analysis of the trial data confirmed the valid clinical efficacy of leukocyte apheresis by centrifugal cell separator in refractory UC patients.

Design optimization and characterization of a small-scale centrifugal cell separator

A cell separator design is presented for rapid, efficient separation of analytical quality plasma samples from whole blood for use in a continuous segmented flow point-of-care testing system. Sedimentation theory for multiple species suspensions, modified to account for continuity and rouleau formation, is used to model the separation process and optimize the separation chamber geometry. Model predictions, relating the sample separation time to a chamber taper angle, indicate respective separation times of 37–28 s for a chamber with a taper angle ranging from 16 to 20°, while maintaining sample volumes in the 1–1.5 ml range. Experimental observations of separation time agree to within 25% of theoretical predictions resulting in complete separation in 40 s, showing the ability to separate small 1–1.5 ml samples in less than 1 min. Results of characterization experiments conducted using the separator indicate indiscernible hemolysis when compared with hemoglobin levels measured from samples prepared in a bench-top centrifuge. Results show that cellular carryover is reduced in seven clean-in-place wash steps to 0.00063% of the original cellular content, and theory based on these results predicts no carryover in 11 steps.

Future directions in blood management and apheresis: the Dideco perspective

Dideco has been present in the blood management field since 1979 with a centrifugal cell separator and filters for plasma production and treatments. The first apheresis system, Progress BT 790, was based on Latham bowl technology and dedicated to the production of hemocomponents and to therapeutic procedures (plasma exchange). The first European intraoperative blood salvage system for autologous washed blood reinfusion was directly derived from Progress and commercialized as Dideco Autotrans 795-A. This was a good example of the utilization of apheresis technology in a different application field. From the beginning of the 80s up to now the development of Dideco apheresis and auto-transfusion systems was oriented to improve the quality of the collected hemocomponents and to increase the global easiness of the systems for a wide and cost-effective usage. In the apheresis field this strategy brought to change the technology from the classical bowl system to a double stage soft belt that has been able to provide better results in terms of purity and efficiency. This new technology, together with the umbilical cord for closed system processing of blood, allows the continuous processing of blood with a small extracorporeal volume. The Dideco Excel Pro version 4.1 is the present expression of Dideco apheresis: a multipurpose closed system, for production of hemocomponents with high efficiency and purity, with an extremely low extracorporeal volume and the choice of a double or single needle operation. At the same time the system has been improved in order to perform therapeutic procedures such as plasma exchange and in the last years, stem cell collection, in a faster and more efficient way. The new clinical and economic concerns bring us to re-evaluate the apheresis systems in a different way: the system must be clinically efficient and at the same time the procedure must be cost-effective. A good example of the development in this direction is the dry platelet Excel procedure. In the latest version of the system, the user can collect platelets in a small volume of plasma, maintaining the good efficiency and purity of the system. This procedure brings two advantages: from a clinical standpoint the recipient is less subject to immunological reactions due to the reinfusion of plasma, and from an economic standpoint the simultaneous collection of apheresis platelets, real units of plasma and/or red cells is possible, going towards a total multicomponent procedure. In the therapeutic field, the development is even faster due to the not yet completely exploited potential of the systems in the stem cell field. Today the stem cell collection, the purification and the expansion are performed in three different steps and are considered as three different activities. More and more the integration of these phases will be important to increase the number of procedures and to reduce the handling required today for this processing. In particular the apheresis technology can help other fields, for instance, contributing to solve the problem of cord blood banking by reducing the volume of the product while maintaining a high stem cells recovery efficiency. The presence of several techniques for plasma treatments like plasma exchange, cascade filtration and immunoadsorption can be developed in order to find, once again, a good compromise between efficiency and costs. One direction Dideco is following is to guarantee the highest flexibility of the apheresis cell separator, in order to provide a performing system for all the different plasma treatment procedures allowing the blood center to choose patient by patient the best clinical strategy. Many other application fields can be evaluated, but only a direct and constant discussion between the industry and the scientific world can bring a fast development of these possibilities.

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones.

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.

Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma.

Ethanol-fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80-90%) and macrophages (65-75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (rho = 1.121 g/ml) gradient fractions and results in a 2.4-fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (rho = 1.172 g/ml) gradient fractions and results in a 1.2-fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaou-stained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma.

The insulin receptor is an age-dependent integral component of the human erythrocyte membrane.

Human erythrocytes were separated according to age to determine whether there is heterogeneity in the specific binding of 125I-insulin to red cells. The mean cell age of erythrocyte isolates was determined from the cumulative distribution frequency of the cells in an isotonic (290 milliosmolar) density gradient and confirmed by assay for pyruvate kinase, an age-dependent red cell enzyme. An IBM 2997 centrifugal cell separator was used to obtain larger quantities of younger erythrocytes from normal subjects. 125I-insulin binding to red cells including reticulocytes was found to decrease exponentially as a function of their mean cell age in 8 normal subjects. A change in receptor number rather than affinity appeared to account for the observed change in 125I-insulin binding. An exponential, age-dependent change in binding of a hormone to its cell membrane bound receptor has not previously been observed. Consistent with these results is the possibility that regulation of the red cell insulin receptor concentration takes place only in the younger red cells.

Centrifugal separation of cells in sputum specimens from patients with adenocarcinoma.

A Ficoll density gradient centrifugation technique was used to separate fixed cells from the sputa of patients with bronchogenic adenocarcinoma. The selective enrichment of leukocytes, squamous or cancerous cells was achieved while retaining the diagnostically significant cell morphologic features. This technique is shown to typically eliminate most of the leukocytes (96.9%) macrophages (86.7%), squamous cells (70.0%) and necrotic debris from specimen fractions containing the majority of cancerous cells. The maximum purity of cancer cells (14.6%) occurs in high density (p = 1.35 g/ml) gradient fractions and results in an average 12.2-fold enrichment for cancer cells in contrast to their relative frequency of occurrence in unprocessed specimens. Differential cell count analyses of serial density centrifugation fractions show that this technique produces comparable enrichment rates for material from bronchogenic adenocarcinoma and squamous cell carcinoma.

Characterization of β-hydroxybutyrate transport in rat erythrocytes and thymocytes

A method was developed for study of β-hydroxybutyrate transport in erythrocytes and thymocytes. Critical to the method was a centrifugal separation of cells from medium which took advantage of β-hydroxybutyrate transport's temperature dependence and inhibition by phloretin and methylisobutylxanthine, all of which are demonstrated in this work. These properties suggested mediated transport, as did saturation kinetics and inhibition by several agents including pyruvate and α-cyanocinnamate. Most conclusive in this regard was a 2-fold preference for d- over l-β-hydroxybutyrate. Entry was not Na + dependent. It was stimulated by substitution of SO 4 2− for most of the Cl −. The equilibrium β-hydroxybutyrate space was much higher than the Cl − space of thymocytes, suggesting that β-hydroxybutyrate entry is not associated with net inward negative current and is not coupled to outward Cl − or inward K + movement (assuming that K + is at electrochemical equilibrium). Coupling to H + entry or OH − exit is compatible with the result. These findings are consistent with β-hydroxybutyrate entry by the carboxylate transport site which has been studied extensively with pyruvate and lactate as permeants. The Cl −/HCO 3 − exchange carrier did not appear to contribute significantly to β-hydroxybutyrate transport.

Design principles for a counterflow centrifugation cell separation chamber

Design principles for a counterflow centrifugation cell separation chamber

X-ray study of competence development in Bacillus subtilis.

Pre-competent and competent cultures of Bacillus subtilis were x-irradiated before and after centrifugal separation of cells in a Renografin density gradient. Pre-competent cultures have no cells at the radiosensitivity of the cells in the bulk competent culture, but there is a substantial fraction of cells in a multi-target state, with heterogeneous target numbers. On reaching maximal competence, the survival becomes entirely linear in radiosensitivity. Irradiation of the separated competent cells shows that competence development correlates with disappearance of multi-target cells from the non-competent band of cells and the appearance of single-target cells in the competent band at a radiosensitivity equal to that of the bulk competent culture. Thus the multi-target state may be a required stage in the development of competence in this system.