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PubMed1979

Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes.

The role of serum factors in the intracellular killing of bacteria by monocytes was studied on the basis of an assay independent of phagocytosis. After 3 min of phagocytosis of preopsonized bacteria and removal of noningested bacteria, the monocytes containing bacteria are reincubated for various periods and the number of unkilled bacteria is determined by a microbiological method after lysis of the cells. Evidence that this assay measures the killing of ingested bacteria was provided by scanning electron microscopy, lysostaphin treatment, and the effect on the rate of intracellular killing of inactivated serum lacking specific opsonic activity. Intracellular killing of Staphylococcus aureaus, S. epidermidis, and Escherichia coli by human monocytes does not occur or is low in the absence of serum, and maximal killing is only reached when fresh serum is present; intermediate values are obtained in the presence of heat-inactivated serum. These findings indicate that complement stimulates intracellular killing. Isolated heterogeneous immunoglobulin (Ig)G, pFc fragments of heterogeneous IgG, and both IgG1 and IgG3 stimulate intracellular killing of S. aureaus by monocytes to the same degree as heat-inactivated serum. Sphingomyelinase, which decreases the number of Fc receptors, and neuraminidase, which increases these receptors, respectively, decreased and increased the intracellular killing, whereas anti-monocyte serum completely abolished the stimulation of intracellular killing by inactivated serum. These results prove that interaction of the Fc receptor with the Fc part of IgG is required for the intracellular killing. Inhibition of the activation of complement components via the alternative pathway gave a considerable reduction in the intracellular killing of S. aureaus; impairment of the activation via the classical pathway had no effect. The addition of complement components to heat-inactivated serum showed that intracellular killing is maximal only when C3b is generated. Reduction of the number of C3b receptors in the membrane by trypsin or pronase decreased intracellular killing in the presence of fresh serum; anti-monocyte serum completely abolished the stimulation of intracellular killing by fresh serum. These results lead to the conclusion that intracellular killing is also dependent on the interaction between C3b and its receptor in the membrane.

Blood Bactericidal Activity↗

PubMed1988

Evidence that the phagocytosis mediated by the peanut agglutinin-like activity of IgG(Fc) receptors of human monocytes is selectively modulated by estradiol and natural estrogens.

The percentage of human monocytes (MCs) that are able to form rosettes with, and to phagocytose, IgG-coated sheep red blood cells (IgG-SRBCs) has been first determined in vitro by a classical rosette assay in 12 postmenopausal (PM) women. Half of them never received any suppletive estrogen (E) therapy at the time of testing, whereas the other six were chronically treated with E. Three different preparations of the same anti-SRBC IgG antibody batch were coated to SRBCs: the first one was the starting antibody preparation [IgG(total] and the other two were purified by affinity chromatography either on Sepharose-concanavalin A (Con A) or on agarose-peanut agglutinin (PNA) columns specifically recognizing terminal, and/or accessible, alpha-mannosyl [IgG(Con A)] or beta-galactosyl [IgG(PNA)] residues of the Fc domain, respectively. The three IgG preparations exhibited similar hemagglutinating antibody titers (1/100). All experiments were conducted using a coating range of 5000 to 6000 IgG antibody molecules per SRBC. In PM women with E, the rosetting capacity of autologous MCs (percentage of MCs rosetting at least three IgG-SRBCs), their phagocytosing capacity (percentage of MCs ingesting at least three IgG-SRBCs), and the phagocytosis index (number of SRBCs ingested/100 MCs) were similar for each IgG-SRBC preparation considered. In contrast, in PM women without E, the capacity of MCs to phagocytose IgG(PNA)-SRBCs, as well as the phagocytosis index measured with those SRBCs, was strongly reduced (P less than 0.01 at least), when compared to the same parameters determined using IgG(total)-SRBCs and IgG(Con A)-SRBCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

Cytofluorometric and cytomorphologic analysis of human bone marrow cells derived from stromal cultures stimulated by granulocyte-macrophage colony-stimulating factor, interferon-gamma and splenopentin pentapeptide.

We studied the influence of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF), human recombinant interferon-gamma (hrIFN-gamma) and splenopentin pentapeptide (Sp-5), either alone or in combination, on the proliferation and differentiation of human bone marrow cells in modified Dexter's cultures. After 10, 14 and 21 days cells were analyzed by classical staining according to Pappenheim and by cytofluorometry with a set of different monoclonal antibodies. IFN-gamma inhibited the proliferation of progenitor cells and provided signals promoting monocytic differentiation, whereas GM-CSF induced the proliferation of blastoid elements which expressed HLA-DR and M2 (VIM-2 monoclonal antibody), but progressively lost surface CD34. Furthermore, an increase of CD15+ cells was also observed. When GM-CSF was tested in combination with IFN-gamma, it abolished the inhibitory effect of IFN-gamma and both cytokines synergized to promote the expression of CD11c, CD14 and M2 surface antigens. Sp-5 alone had only a marginal activity, but it potentiated the effects of GM-CSF. These findings suggest that GM-CSF may induce the transition from stem cells to committed myeloid progenitors. In contrast to IFN-gamma, Sp-5 can serve as an additional proliferative signal with negligible effects on cell maturation.

Amino Acid Sequence↗

PubMed1979

Deficiency of the fifth component of complement in human subjects. Clinical, genetic and immunologic studies in a large kindred.

The discovery of a large kindred with a heritable deficiency of the fifth component of complement (C5) has permitted the accumulation of new clinical, genetic and immunologic data concerning the role of C5 in human subjects. The proband, who has had nine episodes of disseminated gonococcal infection, has a hemolytic C5 level of approximately 0.5 per cent of normal. No C5 protein was detectable, but low levels of functional C5 activity could be found using a sensitive bactericidal assay. The proband's twin as well as another sister also had extremely low levels of hemolytic C5(approximately 0.5 per cent normal), but both these subjects have been healthy. Hemolytic complement and bacteriolytic activity could be restored by the addition of purified C5. No chemotactic activity for polymorphonuclear leukocytes could be generated in the C5-deficient serums upon activation of either the classic or alternative pathways, again demonstrating the importance of C5 in human subjects for the production of chemotactic factors. The chemotactic responsiveness of the patients' polymorphonuclear leukocytes and monocytes to preformed chemotactic factors was not depressed. Twenty-two of 32 other family members from three generations had depressed whole hemolytic complement levels. In 19 of 30 family members, levels of hemolytic C5 ranged from 13 to 64 per cent of normal. No linkage for C5 deficiency and the A or B loci of the major histocompatibility complex could be found. These data suggest an autosomal codominant mode of inheritance of C5 deficiency. Deficiency of C5 is compatible with good health, but it can be associated with repeated disseminated gonococcal infection.

Adult↗

PubMed1986

Sympathetic ophthalmia. Immunopathological findings.

Ocular tissue from six patients with a clinical diagnosis of sympathetic ophthalmia (SO) was examined using immunohistochemical techniques. All patients presented with a history of bilateral panuveitis after penetrating ocular injury or multiple intraocular surgeries and clinical features of SO. In four cases, classic histopathological features of SO were observed, including granulomatous uveal tract infiltration and subretinal pigment epithelium (RPE) collections of inflammatory cells (Dalen-Fuchs nodules). Bone marrow derived monocytes were the major cellular components in these granulomas. In two cases, histopathology failed to demonstrate typical Dalen-Fuchs nodules or granulomas in the choroid. However, the choroidal infiltrates were composed primarily of T-helper and B lymphocytes, without macrophages or epithelioid cells. The eyes examined in this report indicate that a varied spectrum of immunopathological and histopathological findings may occur in clinically diagnosed SO.

Adult↗

PubMed2000

A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo.

The strongest susceptibility genes for the development of systemic lupus erythematosus (SLE) in humans are null mutants of classical pathway complement proteins. There is a hierarchy of disease susceptibility and severity according to the position of the missing protein in the activation pathway, with the severest disease associated with C1q deficiency. Here we demonstrate, using novel in vivo models of apoptotic cell clearance during sterile peritonitis, a similar hierarchical role for classical pathway complement proteins in vivo in the clearance of apoptotic cells by macrophages. Our results constitute the first demonstration of an impairment in the phagocytosis of apoptotic cells by macrophages in vivo in a mammalian system. Apoptotic cells are thought to be a major source of the autoantigens of SLE, and impairment of their removal by complement may explain the link between hereditary complement deficiency and the development of SLE.

Animals↗

PubMed2003

Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide.

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.

Animals↗

PubMed1989

Relation between immunocytological features of bronchoalveolar lavage fluid and clinical indices in sarcoidosis.

This study was designed to determine whether cell populations in bronchoalveolar lavage fluid represent a reflection of disease activity in sarcoidosis. Bronchoalveolar lavage fluid cells were obtained from 22 patients with sarcoidosis and from 10 normal control subjects and investigated by immunocytological methods. A panel of monoclonal antibodies was used to determine the relative proportions of phenotypically distinct subsets of macrophages and lymphocytes in the patients with sarcoidosis and to correlate them with clinical indices, such as disease duration, serum angiotensin converting enzyme, the chest radiograph, and results of pulmonary function tests. Patients with sarcoidosis had a higher percentage than the normal subjects of macrophage like cells expressing RFD1 (a class II associated antigen preferentially expressed by dendritic cells), an epithelioid cell antigen (RFD9), and a circulating monocyte antigen (UCHMI). The increase in RFD1+ cells appeared to be due to detection of antigen by this antibody on cells that were also expressing phenotypic markers of classical tissue macrophages (RFD7). The lymphocytes in lavage fluid from patients with sarcoidosis were characterised by increased expression of activation markers, such as interleukin-2 receptors (anti-Tac+), HLA-DR (RFDR+), and "blast" forms (expressing above normal concentrations of CD7 antigen). This was associated with increased proportions of the CD4+ (helper-inducer) T cell subset. Patients with sarcoidosis whose clinical indices suggested activity showed an increased number of macrophages coexpressing RFD1 and RFD7 antigens, of macrophages expressing UCHM1 and lymphocytes expressing activation markers. The expression of these markers was also increased on lavage cells from patients with radiographic evidence of widespread disease (chest radiographic stage II and III), but there was no relation with disease duration, pulmonary function, or serum angiotensin converting enzyme activity. Immunocytological analysis of lavage cells offers a probe for studying the pathogenesis of sarcoidosis and may be of value in monitoring disease activity.

Adult↗

PubMed1990

Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways.

Endotoxemia was evoked by bolus injection of Escherichia coli endotoxin (2 ng/kg body weight) in six healthy subjects to investigate the early kinetics of cytokine release in relation to the development of clinical and hematologic abnormalities frequently seen in gram-negative septicemia. The plasma concentration of tumor necrosis factor (TNF) increased markedly after 30 to 45 minutes, and reached a maximal level after 60 to 90 minutes. In each volunteer, the initial increase of plasma interleukin 6 (IL-6) concentrations occurred 15 minutes after the initial TNF increase, and maximal IL-6 concentrations were reached at 120 to 150 minutes. A transient increase in body temperature and pulse rate occurred simultaneously with the initial TNF and IL-6 increases, whereas a significant decrease in blood pressure occurred after 120 minutes. These changes were proportional to the changes in TNF and IL-6 concentrations. Coagulation activation, as assessed by a rise of prothrombin fragments and thrombin-antithrombin III complexes, was noted after 120 minutes, in the absence of activation of the contact system. A two- to sixfold increase in the concentrations of tissue plasminogen activator (t-PA) and von Willebrand factor antigen indicated endothelial cell activation. This increase started at 120 and 90 minutes, respectively. The release of t-PA coincided with activation of the fibrinolytic pathway, as measured by plasmin-alpha 2-antiplasmin complexes. The fibrinolytic activity of t-PA was subsequently offset by release of plasminogen activator inhibitor, observed 150 minutes after the endotoxin injection, and reaching a peak at 240 minutes. No complement activation was detected. These results show that in humans endotoxin induces an early, rapidly counteracted fibrinolytic response, and a more long-lasting activation of thrombin by a mechanism other than contact system activation. In addition, our data suggest that endotoxin-induced leukopenia and endothelial cell activation are mediated by TNF.

Adult↗

PubMed1979

Neonatal cellular and humoral immunity to group B streptococci.

The mechanisms of host resistance to group B streptococci have not been defined precisely. In the studies reported here we have assessed the contributions of both humoral and cellular factors in protection against strains of this group. With assays of specific opsonic activity based upon the production of polymorphonuclear leukocyte chemiluminescence and radiolabeled bacterial uptake, we have demonstrated that specific heat-stable antibody and the classic complement pathway are major factors in opsonization of these organisms. In the absence of specific antibody, fresh serum resulted in markedly reduced bacterial uptake indicating, at best, a minor role for the alternative complement pathway. Additional studies have indicated that strain-specific antiphagocytic factors as well as type-specific ones may play a role in the virulence of these organisms. Neonates who developed group B streptococcal sepsis usually lacked opsonic activity in their infecting strain. In addition, polymorphonuclear leukocytes from normal term and stressed neonates showed impaired metabolic activation as measured in the chemiluminescence assay following exposure to opsonized group B streptococci. These results suggest that neonates who develop group B streptococcal disease may have defects in both the humoral and cellular aspects of their acute inflammatory response which may contribute to the high mortality observed in this most fulminant of bacterial infections.

Antibody Formation↗

PubMed2000

Induction of tissue factor procoagulant activity in myelomonocytic cells inoculated by the agent of human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy. The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors. We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA). Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages. Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h. HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h. By comparison, E. Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course. Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells. Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism. Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants. We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition. An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

Blood Coagulation↗

PubMed1987

Immuno-electron microscopic characterization of large granular lymphocytes (natural killer cells) from rat liver.

The immunocytochemical characteristics of large granular lymphocytes (LGL), isolated from the liver sinusoids of euthymic and athymic (nude) rats, were investigated in electron microscopy by the immunoperoxidase technique. The LGL were found positive for MRC OX-8 (natural killer cells and cytotoxic/suppressor T cells) and negative for MRC OX-19 (pan-T marker) in both rat strains. The LGL were heterogeneous in the expression of the natural killer cell marker asialo-GM1 which was found on 56% of the LGL from euthymic and on 71% of the LGL from athymic rats. LGL were easily distinguished from the other cells in the preparations, "conventional" lymphocytes and monocytes, thanks to their highly characteristic ultrastructural features, in particular by the presence of specific electron-dense cytoplasmic granules and rod-cored vesicles. These features have been described formerly for the so-called "pit cells" and are more reliable than the classical LGL characteristics at the light microscopic level, i.e. the presence of azurophilic granules. Our results give further support for the existence of an important population of natural killer cells in the liver sinusoids.

Animals↗

PubMedDate not supplied

Regulation of beta-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor alpha.

Amyloid deposition within the brains of Alzheimer's Disease patients results in the activation of microglial cells and the induction of a local inflammatory response. The interaction of microglia or monocytes with beta-amyloid (A beta) fibrils elicits the activation a complex tyrosine kinase-based signal transduction cascade leading to stimulation of multiple independent signaling pathways and ultimately to changes in proinflammatory gene expression. The A beta-stimulated expression of proinflammatory genes in myeloid lineage cells is antagonized by the action of a family of ligand-activated nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs). We report that THP-1 monocytes express predominantly PPAR gamma isoform and lower levels of PPAR alpha and PPAR delta isoforms. PPAR mRNA levels are not affected by differentiation of the cells into a macrophage phenotype, nor are they altered following exposure to the classical immune stimulus, lipopolysaccharide. Previous studies have found that PPAR gamma agonists act broadly to inhibit inflammatory responses. The present study explored the action of the PPAR alpha isoform and found that PPAR alpha agonists inhibited the A beta-stimulated expression of TNFalpha and IL-6 reporter genes in a dose-dependent manner. Moreover, the PPAR alpha agonist WY14643 inhibited macrophage differentiation and COX-2 gene expression. However, the PPAR alpha agonists failed to inhibit A beta-stimulated elaboration of neurotoxic factors by THP-1 cells. These findings demonstrate that PPAR alpha acts to suppress a diverse array of inflammatory responses in monocytes.

Amyloid↗

PubMed2003

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes.

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor Gö6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.

Adjuvants, Immunologic↗

PubMed1981

Uptake of chylomicron remnants by the native LDL receptor in human monocyte-derived macrophages.

Human chylomicron remnants were taken up by cultured human monocyte-derived macrophages. Competition studies using 125I-labeled and unlabeled lipoproteins demonstrated that the remnant particles were not taken up by the modified LDL (acetyl LDL) receptor in these cells, which also contain a receptor for native LDL. The data thus suggest that the apolipoprotein E- and B-containing remnant particles are mainly taken up through an extra-hepatic E and B receptor (the classical LDL receptor pathway) in macrophages as is the case in cultured human skin fibroblasts.

Biological Transport↗

PubMed2003

Identification of a new SERCA2 splice variant regulated during monocytic differentiation.

Sarco/endoplasmic reticulum-type calcium transport ATPases (SERCA enzymes) pump calcium ions from the cytosol into the endoplasmic reticulum. We report that in addition to the ubiquitously expressed SERCA2b isoform, a new splice variant of SERCA2 can be detected (SERCA2c) that arises from the inclusion of a short intronic sequence located between exons 20 and 21 of the SERCA2a isoform. Sequence analysis revealed classical splice donor and acceptor sites, as well as a branch-point site. Due to the presence in the new exon of an in-frame stop codon that is preceded by a 17 bp coding sequence, this mRNA potentially codes for a protein with a truncated C-terminus containing a short unique C-terminal peptide stretch. SERCA2c message was detected in epithelial, mesenchymal, and hematopoietic cell lines, as well as in primary human monocytes. Moreover, we found that during monocytic differentiation total SERCA2 ATPase expression is induced on the protein and mRNA level and that the novel SERCA2c messenger is also up-regulated during this process. These data indicate that the alternative splicing pattern of the 3(') region of the SERCA2 primary transcript is more complex than that previously thought and that this enzyme may be involved in the process of monocyte differentiation.

Alternative Splicing↗

PubMed2005

Discoidin domain receptor 1: a new class of receptor regulating leukocyte-collagen interaction.

Previous studies demonstrated that type I collagen, a major component of the extracellular matrix, could influence the differentiation and function of leukocytes; however, it is not clear whether those effects of collagen were based on its interaction with the classic collagen receptors, alpha1beta1 and alpha2beta1 integrins. We recently detected significant upregulation of discoidin domain receptor 1 (DDR1), a new class of collagen receptor, in human leukocytes, including neutrophils, monocytes, and lymphocytes, in vitro, leading to the hypothesis that the leukocyte-activating effects of collagen might be owing to its interaction with DDR1. In this review, we summarize our recent findings demonstrating that DDR1-collagen interaction facilitates the adhesion, migration, differentiation/maturation, and cytokine/chemokine production of leukocytes. We also describe the intracellular signaling pathways activated by DDR1 interaction with collagen.

Cell Adhesion↗

PubMed1990

Quantitative buffy coat analysis in haematological patients compared to standard laboratory methods.

We have tested a Quantitative Buffy Coat (QBC) instrument (Becton Dickinson, USA), and compared results obtained with this instrument to results obtained with standard methods in a haematology clinic. The basic principle of the method is a classical haematocrit centrifuge. The analysis provides a haematocrit value, platelet count, a total white blood count, and separates the white blood cells in granulocytes and mononuclear cells (lymphocytes plus monocytes). The instrument is easy to use but requires a trained observer. All results are available in 15 min. We have found the accuracy of the method good for all parameters. The precision of the instrument is good but for estimation of granulocytes and lymphocytes plus monocytes we did not find the high sensitivity claimed by the manufacturer (manufacturer's lower limit 0.02 x 10(9)/l; standard deviation for low levels of granulocytes: 0.300 x 10(9)/l and lymphocytes plus monocytes: 0.235 x 10(9)/l). A large fraction of samples (leukocytes 27%, platelets 40%) from a haematological clinic falls beyond the limits for reliable results set by the manufacturer, which reduces the utility of the instrument in such patients. Furthermore, in 21% and 10% of samples within the recommended range for leukocytes and platelets, respectively, QBC results could not be read owing to ill-defined boundaries. For granulocytes and lymphocytes plus monocytes 25% and 34% of the samples, respectively, could not be read owing to ill-defined boundaries. The instrument is not constructed to protect against blood contamination of the centrifuge and, therefore, the safety of the instrument is not satisfactory.

Blood Cell Count↗