Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.     

Modulation of granulocyte responses in three-spined sticklebacks Gasterosteus aculeatus infected with the tapeworm Schistocephalus solidus

Leukocytes isolated from the head kidney and peripheral blood of 3-spined sticklebacks Gasterosteus aculeatus L. were analysed by means of flow cytometry during infection with the tapeworm Schistocephalus solidus (Müller, 1776). Although parasites increased their body weight continuously throughout the observation period (98 d), proportions of granulocytes increased in blood and head kidney only up to Day 63 post-infection (p.i.). Thereafter, declining proportions of granulocytes were observed in both organs. Thus the relative decrease in granulocyte number was not correlated to a decline in the parasitic load of the fish. To investigate a possible modulatory impact of S. solidus on granulocyte function, head kidney leukocytes were isolated at times before Day 63 p.i. and tested in vitro for their capacity to produce reactive oxygen species (ROS). Head kidney leukocytes from S. solidus-infected fish, analysed immediately after isolation (ex vivo, Day 40 p.i.), exhibited a higher ROS production when stimulated with phorbol myristate acetate (PMA), than leukocytes from naive, sham-treated control fish and fish that had resisted or cleared the infection (exposed but not infected). The latter showed an increased spontaneous ROS production that was not correlated to the numbers of granulocytes present in the head kidney isolates. In infected sticklebacks, spontaneous and PMA-induced ROS production was significantly correlated with numbers of granulocytes present in the head kidney isolates, suggesting that elevated ROS production was due to higher numbers of responding cells rather than an increased capacity of single cells. In vitro, after cultivation for 4 d in the presence of pokeweed mitogen (PWM) or extracts from S. solidus, head kidney leukocytes from control fish showed an increased ROS production and phagocytic activity compared with non-stimulated control cultures. In contrast, head kidney leukocytes from infected fish isolated on Days 48 and 44 p.i., failed to respond to S. solidus antigens in vitro. During S. solidus infection, granulocyte mobilisation resulted in elevated numbers of these cells in head kidneys, but the lack of an in vitro response to S. solidus antigens indicates an in vivo priming of granulocytes by the parasite. These observations may reflect the ability of S. solidus to impair the host's immune response once the parasite is developing in the body cavity of G. aculeatus.     

Leukocyte kinetics in the microcirculation

The transport of leukocytes in the microcirculation is specific for the type, size, and the rheological and adhesive properties, the microanatomy of the host organ, and the hemodynamics. The adhesion to the endothelium is determined largely by the degree of activation via chemotactic factors. Leukocyte motion differs from that of red cells or platelets in several respects. When granulocytes enter into capillaries, they are deformed just like red cells. Under normal flow conditions, the time to deform at the entry to capillaries is typically 1,000 times larger than for the red cell, leading to temporary obstruction of the capillaries. After entry, granulocytes move with lower velocity than red cells which causes a cell train formation inside the capillary. At the venular side, the granulocyte is displaced from the center stream toward the endothelium by faster moving red cells. This process leads to systematic attachment of the granulocytes to the endothelium. At a reduced perfusion pressure or in the presence of locally elevated levels of chemotactic factors, the granulocytes may not be able to pass through the capillary network, which leads to microvascular obstruction. Organs with a narrow capillary network may thereby become filters for circulating granulocytes. This event is accompanied in many situations with damage to the host organ.     

Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice]

We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG-CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms.     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Alterations by leukocytes of erythrocyte flow in microchannels

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.     

Cellular Interrelationships during in vitro Granulopoiesis

Long-term production of fully differentiated granulocytes can be maintained in vitro in a liquid system of cultured bone marrow. Marrow is cultured in medical flasks and allowed to form an adherent layer over a three-week period, and then recharged with fresh marrow resulting in continued mature granulocyte production for several months.
During the initial establishment of the adherent layer, three attached populations become apparent: phagocytic monocytes, an attached epithelial cell type, and aggregations of epithelial cells swollen to enormous proportions by the presence of numerous lipid-containing vacuoles. Without the formation of these aggregations, granulocyte production is not maintained beyond an initial period and the culture converts to phagocytic mononuclear cell production alone. Thus not only is the presence of the fat-containing aggregations necessary for continued granulopoiesis, but cultures in full granulocyte production show a characteristic clumping of granulocytes around these aggregates. Electron microscopy has shown that the epithelial cells from the adherent layer form a layer covering some of the attached cells in these areas and thus may provide the necessary in vitro microenvironment for granulopoiesis to occur. Pinocytotic vesicles and gap junctions have been observed between the adjacent membranes of the undifferentiated granulocytes (possibly stem cells) and the epithelial cells themselves.     

Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.     

Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF)

A standardized four-step assay for the flow cytometric determination of the oxidative activity of human polymorphonuclear leukocytes (PMNL) from normal human individuals and from septic patients was developed, using 2,7-dichlorofluorescin-diacetate (DCFH-DA) as indicator for the intracellular formation of H2O2 and free radicals. Spontaneous H2O2 and free radical formation was measured by preincubation of buffy coat PMNLs from fresh peripheral venous blood at 37 degrees C and pH 7.4 with 10 microM DCFH-DA. Intracellular peroxidase activity was determined by addition of 1 mM external H2O2 to this assay. A maximum of granulocyte oxidative burst activity was elicited by the addition of 150 nM phorbol-myristate-acetate (PMA). A physiological burst was generated by incubating buffy coat PMNLs together with E. coli bacteria. The DNA of dead cells was in all instances simultaneously counterstained with propidium iodide (PI). Quiescent or H2O2 or bacteria treated granulocytes moved as a single cell cluster to higher fluorescences. Stimulation with PMA, in contrast, generated always a bimodal distribution of granulocyte fluorescence with the high activity cell cluster being approximately sevenfold more active than the low activity cell cluster. Roughly half of the granulocytes in normal individuals had high fluorescence. An increase of the high activity granulocytes was observed in septic patients. Model experiments with the nonfluorescent DCFH-DA cleavage product DCFH (2,7-dichlorofluorescin) showed that DCFH was quickly photo-oxidized to fluorescent DCF (2,7-dichlorofluorescein) by UV-light and to a lower degree by daylight. DCFH even slowly autooxidized in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

S100A12 is expressed exclusively by granulocytes and acts independently from MRP8 and MRP14.

Changes in cytosolic calcium concentrations regulate a wide variety of cellular processes, and calcium-binding proteins are the key molecules in signal transduction, differentiation, and cell cycle control. S100A12, a recently described member of the S100 protein family, has been shown to be coexpressed in granulocytes and monocytes together with two other S100 proteins, MRP8 (S100A8) and MRP14 (S100A9), and a functional relationship between these three S100 proteins has been suggested. Using Western blotting, calcium overlays, intracellular flow cytometry, and cytospin preparations, we demonstrate that S100A12 expression in leukocytes is specifically restricted to granulocytes and that S100A12 represents one of the major calcium-binding proteins in these cells. S100A12, MRP8, and MRP14 translocate simultaneously from the cytosol to cytoskeletal and membrane structures in a calcium-dependent manner. However, no evidence for direct protein-protein interactions of S100A12 with either MRP8 or MRP14 or the heterodimer was found by chemical cross-linking, density gradient centrifugation, mass spectrometric measurements, or yeast two hybrid detection. Thus, S100A12 acts individually during calcium-dependent signaling, independent of MRP8, MRP14, and the heterodimer MRP8/MRP14. This granulocyte-specific signal transduction pathway may offer attractive targets for therapeutic intervention with exaggerated granulocyte activity in pathological states.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

Human neutrophil lipocalin (HNL) is a specific granule constituent of the neutrophil granulocyte. Studies in bronchial and lung parenchymal tissue and peripheral blood cells

 The neutrophilic granulocyte is a cytotoxic and potentially tissue-injuring cell participating in the destructive processes and symptoms seen in a variety of inflammatory diseases. Sensitive immunoassays have been introduced to measure the levels of specific secretory proteins of various inflammatory cells in blood and other body fluids. The aim has been to develop highly specific markers for each cell type. The results obtained by immunoassay have indicated that human neutrophil lipocalin (HNL) is a protein unique to the neutrophil. The present study investigated the specificity of HNL as a neutrophil marker in peripheral blood and lung tissue by using flow cytometry and immunocytochemistry. Flow cytometry and immunocytochemistry on peripheral blood showed that monoclonal antibodies to HNL only react with neutrophils and not with other types of leukocytes. Immunocytochemistry on plastic-embedded sections and on frozen sections of lung tissue showed that a cocktail of six monoclonal antibodies to HNL specifically reacts with neutrophils and not with epithelial cells or macrophages. By immunoelectron microscopical studies performed on healthy human neutrophils after low temperature embedding in Lowicryl K4M following aldehyde fixation and partial dehydration, it could be shown that HNL colocalized with lactoferrin (a known marker for secondary or specific granules) and that myeloperoxidase was localized in the primary or azurophil granules. The results confirm that HNL is a unique component of the secondary granules of the neutrophil granulocyte. Accepted: 8 January 1997     

Dynamic characteristics of the fluorescence of human peripheral blood neutrophilic polymorphonuclear leukocytes and lymphocytes intravitally fluorochrome-stained with acridine orange]

Neutrophil segmentonuclear leukocytes and lymphocytes of the human peripheral blood vitally stained with Acridine Orange (AO) in concentrations of 250 and 330 mcg/ml show different fluorescence dynamics. The number of neutrophil segmentonuclear granulocytes with green fluorescence of nuclei decreases, whereas the number of cells with red fluorescence of nuclei increases. As a criterion of this process, time T1/2 is taken during which the number of green-fluorescent cells decreases twofold. With AO concentrations of 250 and 330 mcg/ml, T1/2 is equal to 40 or 5 minutes, resp. The nuclei of lymphocytes within a 60 minutes observation show green fluorescence. This effect is likely to be due to structural-functional peculiarities of neutrophil segmentonuclear granulocytes and lymphocytes.     

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.     

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.     

Bronchiolar pathology. II. Light and electron microscopical changes of bronchiolar epithelial cells in rabbits repeatedly injected intratracheally with a detergent solution

Intratracheal administration in rabbits of a detergent solution (Blue Perlan) determined the progressive swelling of bronchiolar epithelial cells, mainly of non-ciliated secretory ones, with hypertrophy of cytoplasms, frequent bleb ruptures and partial cell necroses. Mucoprotein synthesis was not enhanced. Ultrastructurally, the non-ciliated Clara cells were predominating; their cytoplasms were hypertrophied, prominent in bronchiolar lumina, and contained a few mitochondria and numerous dark-stained secretory granules with a thin membrane; glycogen was present in cytosol, and the apical zones of cytoplasms were locally balloonized; nuclei were chromatin-monomorphous and had an evident membrane. Disrupted blebs presented the same granules and glycogenrich structure as the cytoplasms. Intermingled ciliated cells presented small mitochondria, sometimes modified, and some secretory granules; cilia and basal corpuscles were rarely damaged. Some microvilli intermingled among cilia, but they were extremely rare in non-ciliated secretory hypertrophied cells. Some light junctions were observed between bronchiolar cell cytoplasms. The evolution to partial necrotizing bronchiolitis was obvious mainly after the third intratracheal injection of the detergent solution.     

Cytological response of hemocytes in the European flat oyster, Ostrea edulis, experimentally exposed to mercury

Molluscs bivalves have been widely used as bioindicators to monitor contamination levels in coastal waters. In addition, many studies have attempted to analyze bivalve organs, considered pollutant-targets, to understand the bio-accumulation process and to characterize the effects of pollutants on the organisms. Here we analyzed the effects of mercury exposure on flat oyster hemocytes. Optical and electronic microscope procedures were used to characterize hemocyte morphology. In addition, cell solutions treated with acridine orange were analyzed by flow cytometry and laser scanning cytometry in order to evaluate the variations of cytoplasmic granules (red fluorescence, ARF) and cell size (green fluorescence, AGF) of hemocyte populations over time. Light and electron microscopical studies enabled us to differentiate four hemocyte subpopulations, agranulocytes (Types I and II) and granulocytes (Types I and II). Slight morphological differences were observed between control and Hg-exposed cells only in granulocytes exposed to Hg for 30 days, where condensed chromatin and partially lysed cytoplasmic regions were detected. Flow and laser scanning cytometry studies allowed us to differentiate three hemocyte populations, agranulocytes (R1) and granulocytes (R2 and R3). The exposure time to Hg increased the average red fluorescence (ARF) of agranulocytes and small granulocytes, while there was no change in large granulocytes, which showed a loss of membrane integrity. In control oysters, the three hemocyte populations showed an increase of ARF after 19 days of exposure although initial values were restored after 30 days. The average green fluorescence (AGF) was more stable than the ARF throughout the experiment. In Hg-exposed oysters, the values of AGF of agranulocytes showed an increase at half Hg-exposure period while the AGF values of large granulocytes decreased throughout the experiment, confirming the instability of these types of cells. The relative percentage of small granulocytes and granulocytes showed time variations in both control and exposed oysters. However, the values of small granulocytes remained constant during the whole experiment. The fact that there were only changes in agranulocytes and large granulocytes suggested a possible relationship between these two types of cells. In a quantitative study, we found a significant linear relationship between the agranulocytes and large granulocytes.     

Cell analysis in cerebrospinal fluid (CSF) using Sysmex? hematology analyzers XT-4000i and XE-5000: evaluation with CSF controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine (DGKL)

In cerebrospinal fluid (CSF) analysis, hematology analyzers (HAs) Sysmex? XT-4000i and XE-5000, equipped with flow cytometry (FCM), were used to count cells and differentiate leukocytes into mononuclear and polymorphonuclear cells (MNCs, PMCs) applying body fluid mode. FCM was evaluated with 20 DGKL CSF controls containing viable human leukocytes and erythrocytes. HA values were compared with reference values by Passing/Bablok regression analysis to reveal conformity. Conformity of white blood cells (WBCs) was obtained with native leukocytes, counted in calibrated Fuchs-Rosenthal chamber as reference; red blood cell counts proved inaccurate. CV <40% with WBC counts <20 per μL impairs accuracy. Reference WBC differentiation was assayed using FACS Canto II? and FC-500 SN with anti-CD45, anti-CD14, anti-CD16, anti-CD16/56 [Becton Dickinson (BD); Beckman Coulter (BC)]. BD FACS lysing solution?-no-wash-procedure was applied. BC pretreatment with Versalyse lysing solution was not recommended. MNCs (lymphocytes + monocytes) were significantly lower (～14%) on both HAs; PMCs (granulocytes or sum of neutrophils + eosinophils + basophils: range 1-86 M/L) were significantly higher (～2.2-fold). WBC HA differentiation is not reliable because MNC/PMC differentiation yielded lower and higher values than FACS-FCM references, respectively. This is attributed to incorrect discrimination of leukocytes with rounded/nonrounded nuclei; adding leukocytes with nonrounded nuclei to too low HA MNCs (about 40% not-activated) yielded P/B conformity; subtraction of leukocytes with nonrounded nuclei from elevated HA PMCs showed conformity (about 85% activated). Nucleus/activation state of leukocytes was assessed using microhistology. Sysmex XT-4000i and XE-5000 HAs systems are inappropriate for complete CSF cell analysis.