A hampered chemoattractant-induced cytoskeletal rearrangement in granulocytes of patients with unexplained severe chronic and relapsing infections of the upper and lower airways. In vitro restoration by G-CSF exposure.

Granulocytes play a major role in host defense against bacterial infections. Severe inborn defects in granulocyte function are associated with fulminant bacterial infections in early childhood. Subtle disturbances in granulocyte function might contribute to an enhanced susceptibility to bacterial infections in adulthood. We investigated chemoattractant (N-formyl-methionyl-leucyl-phenylalanine, fMLP and casein) induced cytoskeletal rearrangements (polarization) of blood granulocytes in 77 adults with chronic and recurrent therapy-resistant infections of the upper and lower airways. These infections could not be explained by B- and/or T-cell defects or local anatomic abnormalities. Besides polarization, chemotaxis of blood granulocytes was measured in 33 patients, as well as granulocyte superoxide production in eight patients. The chemoattractant-induced cytoskeletal rearrangement in patient blood granulocytes was significantly lower as compared to healthy control values with both fMLP and casein as stimuli. About two-thirds of the patients showed a defective polarization response to fMLP. Granulocyte colony-stimulating factor (G-CSF) when added in vitro corrected the defective polarization responses; responses in the normal range were not enhanced. The chemotactic motility of patient blood granulocytes was also slightly, but significantly lowered. However, it did not correlate to the lowered polarization. Granulocyte superoxide production was comparable in patients and in healthy controls. Our data thus show that subtle abnormalities in chemoattractant-induced cytoskeletal and motile function of blood granulocytes are frequent in patients with severe therapy-refractory bacterial infections of the upper and lower airways.

An abnormal adherence of monocytes to fibronectin in thyroid autoimmunity has consequences for cell polarization and the development of veiled cells.

Blood monocytes of patients with thyroid autoimmune disease (TAID) display defects in rearranging their cortical actomyosin cytoskeleton ('polarize') in response to chemoattractants. Such rearrangements also take place after the adherence of monocytes to the extracellular matrix (ECM). It is therefore not surprising that monocytes are primed after fibronectin (FN) adherence, displaying an enhanced polarization toward chemoattractants. We investigated the integrin expression and chemoattractant-induced polarization of monocytes of TAID patients before and after FN adherence. Since cytoskeletal rearrangements are also required during the transition of monocytes into veiled antigen-presenting cells (VCs), we investigated such transition of FN-adherent monocytes of TAID patients. Adherent and nonadherent monocyte populations from TAID patients and healthy controls were subjected to a polarization test with the chemoattractant fMLP (or MCP-1), FACS analyses (FITC-labelled FN, CD29, CD49e, d, b and a) and tested for their capability to develop into veiled APC. Monocytes of healthy individuals showed an improved chemoattractant-induced cell polarization after FN adherence, not reflected by TAID monocytes, in which chemoattractant-induced polarization worsened. Monocytes of healthy individuals up-regulated CD49e and d integrins and their capability to bind FITC-labelled FN after adherence to a FN-coated plate, as well as enhancing their capability to generate T cell-stimulatory VCs. Monocytes of TAID patients did not. These data indicate that integrin- (and chemokine-) mediated functions are hampered in monocytes of TAID patients. Because integrin action is pivotal to processes such as monocyte adherence to endothelial cells, uropod formation, migration into tissues and differentiation into APC and macrophages, these defects might underly immune dysbalances important in thyroid autoimmune development.

Glucocorticoids hamper the ex vivo maturation of lung dendritic cells from their low autofluorescent precursors in the human bronchoalveolar lavage: decreases in allostimulatory capacity and expression of CD80 and CD86.

Dendritic cells (DCs) were prepared from human bronchoalveolar lavage (BAL) cells. We previously reported that, in particular, the CD1a fraction of the low autofluorescent (LAF) cells contains the precursors for DCs: after overnight culture, 40% of the LAF cells change into functionally and phenotypically prototypic dendritic/veiled cells. There are, as yet, no data on the modulatory effects of glucocorticoids (GC) on the maturation and function of such DCs isolated from the human lung. Functional tests (allogeneic mixed lymphocyte reaction: allo-MLR) were therefore performed with CD1a+ LAF cells at different stimulator-to-T-cell ratios and after preincubation with different dexamethasone (DEX) concentrations. DEX caused suppression of the T-cell stimulatory capacity of CD1a+ LAF cells, which was dose-dependent, and more evident at the higher stimulator-to-T-cell ratios. Here, we also show that CD80 and CD86 are normally expressed at low levels on CD1a+ LAF cell-derived DCs compared to other DC populations. This low-level expression of costimulatory molecules is discussed here in relation to the previously reported low-level expression of CD80 (and CD86) on lung DCs in experimental animals. This appears to play a role in a predominant Th2 cell stimulating potential of DC from the lung environment. DEX exposure of CD1a+ LAF cells prevented the upregulation of even this low-level expression of CD80 and CD86. The veiled/dendritic morphology and the expression of other relevant cell surface markers and adhesion molecules was not affected by DEX exposure. It is concluded that DEX hampers the maturation of CD1a+ LAF cells into active lung DCs.

Opposing effects of dehydroepiandrosterone and dexamethasone on the generation of monocyte-derived dendritic cells.

BACKGROUND: Dehydroepiandrosterone (DHEA) has been suggested as an immunostimulating steroid hormone, of which the effects on the development of dendritic cells (DC) are unknown. The effects of DHEA often oppose those of the other adrenal glucocorticoid, cortisol. Glucocorticoids (GC) are known to suppress the immune response at different levels and have recently been shown to modulate the development of DC, thereby influencing the initiation of the immune response. Variations in the duration of exposure to, and doses of, GC (particularly dexamethasone (DEX)) however, have resulted in conflicting effects on DC development. AIM: In this study, we describe the effects of a continuous high level of exposure to the adrenal steroid DHEA (10 M) on the generation of immature DC from monocytes, as well as the effects of the opposing steroid DEX on this development. RESULTS: The continuous presence of DHEA (10 M) in GM-CSF/IL-4-induced monocyte-derived DC cultures resulted in immature DC with a morphology and functional capabilities similar to those of typical immature DC (T cell stimulation, IL-12/IL-10 production), but with a slightly altered phenotype of increased CD80 and decreased CD43 expression (markers of maturity). The continuous presence of DEX at a concentration of 10 M in the monocyte/DC cultures resulted in the generation of plastic-adherent macrophage-like cells in place of typical immature DC, with increased CD14 expression, but decreased expression of the typical DC markers CD1a, CD40 and CD80. These cells were strongly reactive to acid phosphatase, but equally capable of stimulating T cell proliferation as immature DC. The production of IL-12 by these macrophage-like cells was virtually shut down, whereas the production of IL-10 was significantly higher than that of control immature DC. CONCLUSION: The continuous presence of a high level of GC during the generation of immature DC from monocytes can modulate this development away from DC towards a macrophage-like cell. The combination of a low CD80 expression and a shutdown of IL-12 production suggests the possibility of DEX-generated cells initiating a Th2-biased response. These effects by DEX on DC development contrast with those by DHEA, which resulted in a more typical DC although possessing a phenotype possibly indicating a more mature state of the cell.

Preparation of leukocyte-poor platelet concentrates via a short, hard spin of a pool of buffy coats.

BACKGROUND AND OBJECTIVES: A new method for the preparation of leukocyte-poor platelet concentrates was developed, based on a short, hard spin of a pool of 5 buffy coats (BCs) combined with automated collection of the platelets. MATERIALS AND METHODS: The characteristics of platelet concentrates (PCs) were studied as a function of the total g force applied to a pool of 5 BCs. Pools of BCs were centrifuged for 1 min with a total g force ranging from about 3,300 to 5,000 gmin (n = 7-9 per applied g force). Deceleration took place without the means of a brake. The total centrifugation time was about 11 min. The platelet-rich plasma (PRP) fraction above the cell layer was separated by an automated component preparation device. RESULTS: A short, hard spin with a total g force of between 3,400 and 4,600 gmin resulted in PCs that contained on average more than 290x10(9) platelets and less than 5x10(6) leukocytes without the use of a leukocyte filter, provided that the transfer of PRP was electronically checked and terminated. The cell concentrations in the PCs are a function of the total g force. Both the platelet and leukocyte levels in the concentrate decreased with an increase in the total g force applied to the pool. CONCLUSION: The preparation of PCs via a short hard, spin of BCs, combined with automated collection of the PRP, may be an alternative method for the preparation of leukocyte-poor PCs.

CD1a+ and CD1a- accessory cells from human bronchoalveolar lavage differ in allostimulatory potential and cytokine production.

Recently, we described the isolation through fluorescent-activated cell sorting (FACS) of low autofluorescent (LAF) cells from human bronchoalveolar lavage (BAL). These LAF cells displayed an immunophenotype comparable with that of dendritic cells (DC), and showed a high potency to stimulate naive T cells. In the study reported here we investigated the capability of LAF cells to produce interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-alpha), and the role of these cytokines in allogeneic T-cell stimulation by LAF cells. Lipopolysaccharide (LPS)-stimulated LAF cells released biologically active IL-1, IL-6, and TNF, and also showed intracellular immunoreactivity for IL-1, IL-6, and TNF-alpha. A neutralizing antibody against IL-1 slightly but statistically significantly (P < 0.05, Wilcoxon's test) inhibited the ability of the LAF cells to stimulate allogeneic T-cell proliferation (89% of stimulation in the absence of the antibody). Neutralizing antibodies against IL-6 and TNF-alpha had no effect. An antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF) also interfered with the accessory function of the LAF cells (79% of stimulation in the absence of the antibody, P < 0.05). We also investigated whether subsets of LAF cells (i.e., positive or negative for CD1a and purified by FACS sorting) differed in T-cell stimulatory capacity and in the ability to produce IL-1, IL-6, TNF-alpha, and S100. CD1a+ LAF cells were positive for and produced S100, CD1a- LAF cells were negative in this respect. The CD1a+ subset exhibited a clearly higher and very strong accessory capability as compared with the CD1a- subset. Despite this, CD1a+ LAF cells were poor producers of IL-1, IL-6, and TNF-alpha. The neutralizing antibody to IL-1, however, inhibited the ability of CD1a+ cells to stimulate allogeneic T-cell proliferation (43% of stimulation in the absence of the antibody, P < 0.01). Anti-IL-6 and alpha-GM-CSF had no effects. CD1a- LAF cells were potent producers of IL-1, IL-6, and TNF-alpha, and antibodies to IL-1, IL-6, and GM-CSF strongly interfered with their weaker accessory capability. In conclusion, two different subsets of LAF cells could be identified on the basis of accessory capability and cytokine profile. CD1a+ LAF cells (S100+; very potent T-cell stimulators, poor cytokine producers) are the "Langerhans cells" of the lung. CD1a- LAF cells (S100-; lower T-cell stimulatory capability, potent producers of IL-1, IL-6, and TNF-alpha) displayed a marker pattern intermediate between that of monocytes and monocyte-derived DC.

Dendritic cells and their precursors isolated from human bronchoalveolar lavage: immunocytologic and functional properties.

Human bronchoalveolar lavage (BAL) has been described to contain, besides a large number of alveolar macrophages (AM) (approximately 95%), small numbers of monocyte-like cells (approximately 2%) and dendritic cells (DC) (approximately 0.4%). To separate AM (high autofluorescence) from DC, we used a fluorescence activated cell sorter (FACS) to separate BAL cells into a low autofluorescent (LAF) fraction and a high autofluorescent (HAF) fraction. Immunocytologic and functional properties of these fractions were investigated. The LAF fraction was composed of acid phosphatase (APh)- and RFD9-negative cells, which were strongly positive for HLA-DR, L25, RFD1, and CD68. A portion of these cells expressed CD1a (22%) and My4 (60%). The marker pattern of these cells is reminiscent to that of intraepithelial bronchial DC and to that of blood DC. The majority of the LAF cells had a monocyte-like morphology, but after overnight culture the percentage of LAF cells with long cytoplasmic extensions (DC morphology) was strongly augmented (from 18 to 51%). The HAF fraction contained 100% AM, strongly positive for APh, HLA-DR, CD68, RFD7, and RFD9. In culture, the LAF cells formed clusters with T cells and vigorously stimulated the proliferation of allogeneic T cells and naive (CD45RO-negative) T cells. BAL and LAF cells produced higher responses in nonsmokers than in smokers. In contrast, HAF cells did not form clusters with T cells and did not stimulate allogeneic T cell proliferation. HAF cells even suppressed mitogen-driven T cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution and immunophenotype of mononuclear phagocytes and dendritic cells in the human lung.

Mononuclear phagocytes and dendritic cells (DC) play an important role in the immune response in the lung. DC act in the afferent phase of the immune response by presenting antigen to T cells, while macrophages play a role in the efferent phase by exerting phagocytic/cytotoxic functions. We investigated the localization and the marker pattern of these cells in the human lung. Macrophages, identified as large, rounded, acid phosphatase-positive cells, were mainly detected in the alveolar spaces, in the lumen of the bronch(iol)us, and in the bronchoalveolar lavage (BAL). They were positive for major histocompatibility complex (MHC) class II antigens (DR, DQ), CD68, RFD7, RFD9, and partly positive for RFD1. Irregularly shaped cells with a marker pattern comparable to that of blood-derived DC (positive for DR, DQ, L25, RFD1, and CD68) were predominantly observed in the epithelium and subepithelial tissue of the bronch(iol)us and in the bronchus-associated lymphoid tissue. In the epithelium, approximately 30% of these cells were positive for CD1a (OKT6). In the subepithelial tissue, these DC formed characteristic small clusters with T cells. The BAL, the alveolar spaces, and the alveolar walls contained only a small number of DC. These immunohistologic data suggest that the bronch(iol)us is well equipped to initiate immune responses. The high number of macrophages in the alveolar compartment, which have been described to suppress T cell proliferation, together with low numbers of DC, makes the alveolar compartment less suited for mounting an immune response.

Effect of thyroid hormones and other iodinated compounds on the transition of monocytes into veiled/dendritic cells: role of granulocyte-macrophage colony-stimulating factor, tumour-necrosis factor-alpha and interleukin-6.

Stimulation of human peripheral blood monocytes with the thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) enhanced their ability to mature into cytologically and functionally characteristic veiled/dendritic cells. Veiled/dendritic cell transition induced by T3 and T4 was dependent on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6) in the culture, since the addition of antibodies specific for GM-CSF, TNF alpha and IL-6 to the culture system had blocking effects. The addition of antibodies to macrophage colony-stimulating factor and IL-1 had no effects. Contaminating T cells and B cells did not contribute to the transition of monocytes to veiled/dendritic cells, and it is therefore likely that the GM-CSF, TNF alpha and IL-6 produced in the culture system were derived from the monocytes themselves. Stimulation of the blood monocytes with an optimal concentration of metrizamide (14.5%), reverse T3 (rT3; 2 x 10(-10) M) or highly iodinated thyroglobulin (Tg; 2 x 10(-11) M) also resulted in an increased transition of monocytes to veiled/dendritic cells, but to a lesser extent in comparison with the thyroid hormones (T3, 31 +/- 6% and T4, 25 +/- 5% vs rT3, 22 +/- 8% and Tg with an iodination grade of 0.37%: 20 +/- 4% veiled/dendritic cells). Administration of anti-GM-CSF, anti-TNF alpha and anti-IL-6 to the culture system also had blocking effects on the transition from monocytes to veiled/dendritic cells induced by the iodinated compounds. The mechanisms by which such iodinated compounds act on the monocyte to veiled/dendritic cell transition can only be speculated on (interference H2O2-generating system?).

A high iodine intake in Wistar rats results in the development of a thyroid-associated ectopic thymic tissue and is accompanied by a low thyroid autoimmune reactivity.

Evidence is accumulating that dietary iodine intake is an important modulator of autoimmune thyroid reactions. To study this role of iodine intake further, female Wistar rats were kept on an enriched iodine diet (EID, iodine intake 100 micrograms iodine/day) for a period of up to 18 weeks. Control rats were either on a normal iodine diet (NID, iodine intake 7 micrograms iodine/day) or a low iodine diet (LID, 2 days of 1% KClO4 followed by iodine-deficient drinking water/pellets). During the first 6 weeks of the EID rats developed a thyroid-associated ectopic thymic tissue (50-57% of the animals on EID versus 7-14% of NID rats and 0% of LID rats). This thyroid-associated ectopic thymic tissue showed a similar histology (cortex and medulla) and a similar marker pattern as normal rat thymus concerning TdT expression (positive cells in the cortex) and CD4/CD8 positivity (double-positive cells in the cortex, single-positive cells in the medulla). The excessive iodine diet also resulted in a lowered thyroid autoimmune reactivity as compared to the NID and LID, viz. (1) in a lower incidence of anti-colloid antibodies in serum (12.5% positivity in EID rats versus 36% in NID and 60% in LID rats at 18 weeks) and (2) lower numbers of intrathyroidal lymphoid cells, viz. lower numbers of dendritic cells and lower numbers of CD4 and CD8 positive lymphocytes. It is hypothesized that the development of the thyroid-associated ectopic thymic tissue in the EID rats is related to their low thyroid autoimmune responsiveness; the tissue might play a role in tolerance induction to thyroidal autoantigens.

Development and small-scale production of a severely heated factor VIII concentrate.

The small-scale production of a severely heated factor VIII concentrate is described. As starting material for the production 48 units of fresh frozen plasma from whole-blood donations or 24 units of plasma from apheresis are used. During a glycine and sodium chloride fractionation, contaminating proteins are removed from a pooled extract of single-donor cryoprecipitates. The resulting factor VIII-rich precipitate is redissolved in freeze-drying buffer and this solution is desalted by diafiltration. After filtration, this solution is dispensed into 10 vials and frozen. The product is freeze dried, and heat treated at 80 degrees C for 72 h. The mean yield of the heat-treated product improved from 160 IU factor VIII per liter plasma at the start of the production to 215 IU per liter plasma after a modification of the purification process. The validation of the virus inactivation during freeze-drying and 80 degrees C heat treatment for 72 h showed a reduction of > or = 7.6 log PFU/ml for Sindbis and a reduction of > or = 6.4 log TCID50/ml for HIV-1. The factor VIII concentrate was clinically tested in 6 patients. It possessed a normal half-life, showed a high recovery and no side effects. A Dutch license was granted in June 1991 and since then this product has been routinely manufactured in three Dutch blood banks.

Effect of monosaccharides during severe dry heat treatment of coagulation factor VIII concentrates.

During product development of a factor VIII concentrate (Dutch blood banks) the conversion from unsterilized to autoclaved freeze-drying buffer caused impaired product characteristics after severe dry heat treatment (80 degrees C for 72 h). Analysis of the freeze-drying buffers showed the presence of fructose and glucose in heated buffers, resulting from hydrolysis of sucrose. The detrimental effect of glucose and fructose on solubility, yield of factor VIII and color of the heat-treated product was confirmed by freeze-drying and heating products spiked with increasing levels of these monosaccharides. The effect of the use of freeze-drying buffers autoclaved with and without sucrose was examined in two other factors VIII concentrates, S(8) and Z8 (Protein Fractionation Centre, Edinburgh, UK). If sucrose was present during autoclaving of the buffer, a slightly lower yield over freeze-drying and 80 degrees C heat treatment was observed. Since glucose is present as a substrate in the medium for the host cells during cultivation of viruses, its potential effect on the 80 degrees C heated product (Dutch blood banks) was examined during the validation study of the inactivation of HIV-1 and Sindbis. The cultivation cycles for the virus inocula were simulated and residual glucose levels measured. In the supernatant medium of the host cell culture used for the propagation of Sindbis no residual glucose was found. Glucose however was found in the supernatant medium of the host cell culture for propagation of HIV-1, and therefore small molecular weight substances were removed from the actual HIV-1 inoculum by ultrafiltration.(ABSTRACT TRUNCATED AT 250 WORDS)

An excess of dietary iodine accelerates the development of a thyroid-associated lymphoid tissue in autoimmune prone BB rats.

Previous studies have shown that dietary iodine enhances the severity and incidence of focal thyroiditis in autoimmune BB rats and OS chickens. However, which lymphoid cells are involved in the development of the iodine-induced focal thyroiditis and what the consequences are for the anticolloid antibody production have not been studied in detail. We therefore performed a study in which 3-week-old female BB rats were kept on either an enriched iodine diet (EID; iodine intake, 100 micrograms iodine/day) or a normal iodine diet (NID; iodine intake, 7 micrograms iodine/day) for a period of 18 weeks. The development of the focal thyroiditis was immunohistologically studied. Immunohistological data were compared to the thyroid hormone status and anti-colloid antibody production. Our data confirm that a high dietary iodine intake results in an accelerated development of the focal lymphoid cell infiltrates in the thyroid of the BB rat. After 12-18 weeks of an EID 50% of the BB rats developed these infiltrates. Our data additionally show that: (a) the process starts with increases in the number of infiltrating MHC class II-positive dendritic cells and a clustering of these cells with T cells, B cells, and some macrophages and (b) the focal infiltrates are highly organized and consist of central B cell follicle-like structures surrounded by rims and areas of T cells. The architecture of the focal thyroiditis is hence very similar to mucosa-associated lymphoid tissue and secondary lymphoid organs (spleen and lymph node). Only minor signs of thyrocyte destruction were observed. We therefore consider the term "thyroiditis" as inappropriate and prefer the term "thyroid-associated lymphoid tissue." Since the thyroiditis component was small, it is also not surprising that the BB rats on the EID remained euthyroid. The presence of the thyroid-associated lymphoid tissue in the BB rats was positively correlated to the presence of anti-colloid antibody in the serum of the BB rats. We speculate that the dietary iodine might have direct effects on cells of the immune system or on cells forming the microenvironment of lymphoid tissue (reticulum cells). A role for highly iodinated thyroglobulin in the accelerated development of thyroid-associated lymphoid tissue is also possible.

Iodine deficiency induces thyroid autoimmune reactivity in Wistar rats.

The last 2 decades it has become clear that iodine deficiency has a modulating effect on the thyroid autoimmune response in humans. Also, in animals that spontaneously develop autoimmune thyroid disease, evidence is accumulating that a low iodine intake can modulate thyroid autoimmune reactivity. However, it is still not clear what the effect of a low iodine intake on thyroid autoimmune reactivity is in normal nonautoimmune animals. To study the relationship of a dietary low iodine intake on the thyroid autoimmune reactivity in nonautoimmune animals, normal Wistar rats (female) were kept on an enriched iodine diet (daily iodine intake of 100 micrograms iodine), a "for our area normal" (conventional) diet (COD; daily iodine intake of 7 micrograms iodine), a low iodine diet (LID; 2 days of 1% KCLO4, followed by iodine-deficient drinking water/pellets), or an extremely low iodine diet (LID+; 1% KCLO4 continuously in the drinking water and iodine-deficient pellets). The enriched iodine diet rats were euthyroid (T3, approximately 8 nM/liter: T4, approximately 50 nM/liter; TSH, approximately 2 ng/ml), had a normal thyroid weight (approximately 12.5 mg), and showed only minimal signs of local thyroid immune reactivity; low numbers of intrathyroidal dendritic cells (DC; approximately 35 DC/mm2), CD4+ cells (approximately 2 cells/mm2), and CD8+ cells (approximately 2.5 cells/mm2) were found in combination with low anticolloid antibody production (incidence of positive animals, 12.5%). The COD resulted in a normal thyroid function. The rats were euthyroid (range of T3, 1.6-1.2 nM/liter; T4, approximately 50 nM/liter; TSH, approximately 2 ng/ml) and had a normal thyroid weight (approximately 12.5 mg). However, some signs of thyroid autoimmune reactivity were found [number of intrathyroidal DC, approximately 40/mm2; approximately 3 CD4-positive (CD+) cells/mm2; approximately 3 CD8+ cells/mm2; together with a 30% incidence of anticolloid antibodies]. The LID and LID+ not only induced goiter formation [thyroid weight, 27.3 +/- 4.2 mg (mean +/- SD) after 12 weeks of LID and 38.4 +/- 5.3 mg after 4 weeks of LID+] and low production of T4 by the thyroid [28 +/- 3 nM/liter (mean +/- SD)] after 12 weeks of LID and 14 +/- 3 nM/liter after 2 weeks of LID+], but also induced various signs of thyroid autoimmune reactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of the Organon Teknika Plasmapur system with new software and two types of filter.

A convenient plasmapheresis apparatus is the Plasmapur system (Organon Teknika). Recently the software of the Plasmapur monitor has been changed. We evaluated the modified Plasmapur monitor and two types of Plasmapur separators containing polypropylene membranes with a mean maximum pore size of 0.5 mum and 0.6 mum respectively. 50 plasmaphereses with each separator were performed; during 10 procedures donor blood samples and samples from the plasma obtained were drawn. No hypersensitivity reactions were observed, the operator "hands on" time was less than 5 min, the mean procedure time was 45 min to collect 650 mL of plasma with both types of filters. Biochemical analysis of the samples indicated that with both separators the plasma obtained was of good quality with respect to Factor VIII and other proteins and that no significant activation of the complement or clotting cascades occurred.