Enhanced harvest performance predictability through advanced multivariate data analysis of mammalian cell culture particle size distribution.

The industry's pursuit for higher antibody production has led to increased cell density cultures that impact the performance of subsequent product recovery steps. This increase in cell concentration has highlighted the critical role of solids concentration in centrifugation yield, while recent product degradation cases have shed light on the impact of cell lysis on product quality. Current methods for measuring solids concentration and cell lysis are not suited for early-stage high-throughput experimentation, which means that these cell culture outputs are not well characterized in early process development. This article describes a novel approach that leveraged the data from a widely-used automated cell counter (Vi-CELL™ XR) to accurately predict solids concentration and a common cell lysis indicator represented as lactate dehydrogenase (LDH) release. For this purpose, partial least squares (PLS) models were derived with k-fold cross-validation from the particle size distribution data generated by the cell counter. The PLS models showed good predictive potential for both LDH release and solids concentration. This novel approach reduced the time required for evaluating the solids concentration and LDH for a typical high-throughput cell culture system (with 48 bioreactors in parallel) from around 7 h down to a few minutes.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

Red Cell Distribution Width to Platelet Count Ratio Reference Intervals in Premature Infants Beyond the First Week of Life.

Objective: Red cell distribution width (RDW) is a parameter of complete blood count (CBC). The RDW to platelet count ratio (RPR) is a new index that has been shown to reflect the severity of inflammation. We aim to determine the reference interval (RI) of RPR for premature newborns. Study design: The medical records of preterm infants who were followed up between January 2016 and December 2018 were reviewed. CBC levels were measured in 144 infants at <72 hours of age. Results: CBCs of infants (gestational age from 28 to 35weeks) had a RI of 0.038-0.126 for the RPR. The RI for RPR in infants with a gestational age of 32-35weeks was 0.042-0.129; and the RI for infants at 28-31weeks was 0.022-0.121. Conclusion: Establishment of RI for RPR in premature infants will allow clinical correlation of RPR alterations in this population.

Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma.

The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4 to 15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei-regardless of genotype-became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.

The relationship of bovine milk somatic cell count to neutrophil level in samples of cow's milk assessed by an automatic cell counter.

This research communication describes the application of a fluorescent automatic cell counter Lactoscan SCC for simultaneous determination of somatic cell count and neutrophils in bovine milk. The obtained results were compared with results obtained by a flow cytometer and a light microscope. The Pearson correlations between the methods were calculated. A comparison between the main characteristics of the three kinds of analysis was made - the assay duration and the intra-assay precision. A relation between the SCC and neutrophil cells was observed in 55 milk samples. The obtained results confirm that the simultaneous determination of SCC and neutrophil analysis are necessary and support the early diagnosis of mastitis, the timely treatment of the animal and the avoidance of major economic losses.

Evaluation of 3 esterase tests for the diagnosis of subclinical mastitis at dry-off and freshening in dairy cattle.

Subclinical mastitis (SCM) and intramammary infection (IMI) increase esterase activity in the glandular secretions of dairy cattle. Our objective was to evaluate the clinical performance of 3 commercially available esterase tests for diagnosing SCM and IMI. Foremilk samples were collected from 380 quarters (96 cows) at dry-off and from 329 quarters (83 cows) within 4 to 7 d after calving. Quarter somatic cell count (SCC) was measured using the reference method (DeLaval cell counter; De Laval International AB, Tumba, Sweden) with SCM defined as SCC >200,000 cells/mL. Bacterial culture of foremilk samples was used to diagnose IMI based on the growth of ≥100 cfu/mL. The SCC was estimated using 3 PortaSCC tests (PortaCheck, Moorestown, NJ) from the measured esterase activity and the California Mastitis Test (CMT). Clinical performance was evaluated using logistic regression to determine the area under the receiver operating characteristic curve (AUC) and identify test sensitivity (Se) and specificity (Sp) at the optimal cut-point for diagnosing SCM and IMI. Test agreement was also evaluated using the kappa coefficient (κ) and weighted κ. The PortaSCC color test was the best-performing PortaSCC test for diagnosing SCM at dry-off (AUC = 0.90, Se = 0.91, Sp = 0.81, κ = 0.71) and at freshening (AUC = 0.86, Se = 0.74, Sp = 0.95, κ = 0.72), at an optimal cut-point of ≥250,000 cells/mL but required 45 min to produce a result. For comparison, the CMT required 2 min to produce a result and a CMT score of trace or higher was superior to the PortaSCC color test for diagnosing SCM at dry-off (AUC = 0.95, Se = 0.95, Sp = 0.86, κ = 0.81) and freshening (AUC = 0.88, Se = 0.79, Sp = 0.95, κ = 0.76). The PortaSCC quick test was the best-performing PortaSCC test for diagnosing IMI at dry-off (AUC = 0.81, Se = 0.81, Sp = 0.78 κ = 0.40) and required 5 min to produce a result, whereas the PortaSCC color test was the best performing PortaSCC test for diagnosing IMI at freshening (AUC = 0.80, Se = 0.75, Sp = 0.79 κ = 0.38). For comparison, the CMT was inferior to the PortaSCC quick test for diagnosing IMI at dry-off (AUC = 0.73, Se = 0.76, Sp = 0.60, κ = 0.20) but was equivalent to the PortaSCC color test at freshening (AUC = 0.79, Se = 0.58, Sp = 0.93, κ = 0.50). The PortaSCC color and quick tests and CMT were considered good tests for diagnosing SCM and IMI because clinically useful tests typically have an AUC >0.80 and κ >0.6. Based on the test sensitivity, cost, and analysis time, there does not appear to be a persuasive reason to select the PortaSCC tests over the traditional CMT for diagnosing SCM and IMI.

Routine Management of Microalgae Using Autofluorescence from Chlorophyll.

From a high-potential biomass perspective, microalgae have recently attracted considerable attention due to their extensive application in many areas. Although studies searching for algal species with extensive application potential are ongoing, technical development for their assessment and maintenance of quality in culture are also critical and inescapable challenges. Considering the sensitivity of microalgae to environmental changes, management of algal quality is one of the top priorities for industrial applications. Helping substitute for conventional methods such as manual hemocytometry, turbidity, and spectrophotometry, this review presents an image-based, automated cell counter with a fluorescence filter to measure chlorophyll autofluorescence emitted by algae. Capturing chlorophyll-bearing cells selectively, the device accomplished precise qualification of algal numbers. The results for cell density using the device with fluorescence detection were almost identical to those obtained using hemocytometry. The automated functions of the device allow operators to reduce working hours, for not only cell density analysis but simultaneous multiparametric analysis such as cell size and algal status based on chlorophyll integrity. The automated device boldly supports further development of algal application and might contribute to opening up more avenues in the microalgal industry.

Evaluation of a method to measure HHV-6B infection in vitro based on cell size.

BACKGROUND: Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. Another method used in the field relies on the identification of enlarged cells in the culture; this method requires little sample processing and is relatively fast. However, visual inspection of cell cultures can be subjective and it can be difficult to establish clear criteria to decide if a cell is enlarged. To overcome these issues, we explored a method to monitor HHV-6B infections based on the systematic and objective measurement of the size of cells using an imaging-based automated cell counter. RESULTS: The size of cells in non-infected and HHV-6B-infected cultures was measured at different times post-infection. The relatively narrow size distribution observed for non-infected cultures contrasted with the broader distributions observed in infected cultures. The average size of cultures shifted towards higher values after infection, and the differences were significant for cultures infected with relatively high doses of virus and/or screened at longer times post-infection. Correlation analysis showed that the trend observed for average size was similar to the trend observed for two other methods to measure infection: amount of virus DNA in supernatant and the percentage of cells expressing a viral antigen. In order to determine the performance of the size-based method in differentiating non-infected and infected cells, receiver operating characteristic (ROC) curves were used to analyze the data. Analysis using size of individual cells showed a moderate performance in detecting infected cells (area under the curve (AUC) ~ 0.80-0.87), while analysis using the average size of cells showed a very good performance in detecting infected cultures (AUC ~ 0.99). CONCLUSIONS: The size-based method proved to be useful in monitoring HHV-6B infections for cultures where a substantial fraction of cells were infected and when monitored at longer times post-infection, with the advantage of being relatively fast and easy. It is a convenient method for monitoring virus production in-vitro and bulk infection of cells.

The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells.

The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.

Useful information provided by graphic displays of automated cell counter in hematological malignancies.

BACKGROUND: Automated cell counters have become more and more sophisticated with passing years. The numerical and graphic data both provide useful clues for suspecting a diagnosis especially when the workload is very high. AIM: We present our experience of useful information provided by graphic displays of an automated cell counter in hematological malignancies in a cancer hospital where a large number of complete blood count (CBC) requests are received either before or during chemotherapy. This study was conducted to assess the usefulness of hematology cell counter, viz. WBC-Diff (WBC differential), WBC/BASO (WBC basophil) and IMI (immature myeloid information) channel scatter plots, and the flaggings generated in various hematological malignancies. MATERIAL & METHODS: The graphic displays have been compiled over a period of 1 year (October 2015-September 2016) from blood samples of various solid and hematological malignancies (approximately 400 per day) received for routine CBC in the laboratory. Approximately 50 000 scattergrams have been analyzed during the study period. The findings were confirmed by peripheral blood smear examination. RESULTS: The scattergram analysis on XE-2100 is very sensitive as well as specific for diagnosing acute leukemia, viz. acute myeloid leukemia, acute lymphoblastic leukemia; chronic myeloproliferative disorders, viz. chronic myeloid leukemia; and chronic lymphoproliferative disorder especially chronic lymphocytic leukemia. CONCLUSION: It is suggested that the laboratories using the hematology analyzers be aware of graphic display patterns in addition to flaggings generated which provide additional information and give clue toward the diagnosis even before peripheral smear examination.

On-Chip Cell Staining and Counting Platform for the Rapid Detection of Blood Cells in Cerebrospinal Fluid.

Counting blood cells in cerebrospinal fluid (CSF) is indispensable for diagnosing several pathological conditions in the central nervous system, such as meningitis, even though collecting CSF samples is invasive. Cell counting methods, such as hemocytometer chambers and flow cytometers, have been used for CSF cell counting, but they often lack the sensitivity to detect low blood cell numbers. They also depend on off-chip, manual sample preparation or require bulky, costly equipment, thereby limiting their clinical utility. Here, we present a portable cell counting platform for simple, rapid CSF cell counting that integrates a microfluidic cell counting chamber with a miniaturized microscope. The microfluidic chamber is designed not only to be a reagent container for on-chip cell staining but also to have a large control volume for accurate cell counting. The proposed microscope miniaturizes both bright-field and fluorescence microscopy with a simple optical setup and a custom cell-counting program, thereby allowing rapid and automated cell counting of nucleated white blood cells and non-nucleated red blood cells in fluorescence and bright-field images. Using these unique features, we successfully demonstrate the ability of our counting platform to measure low CSF cell counts without sample preparation.

Application of a portable microscopic cell counter for the counting of residual leukocytes in leukoreduced apheresis platelet concentrates in a hospital blood bank.

While a portable microscopic cell counter has been evaluated to enumerate residual white blood cells (WBCs) in red blood cells and platelet concentrates at blood centers, it has not yet been assessed in a hospital blood bank. We investigated the performance of this device and evaluated its accuracy, along with its benefits in time management. Residual WBCs from each of 100 apheresis platelet specimens were measured manually using a Nageotte chamber, along with flow cytometry methods and an ADAM-rWBC automated instrument (NanoEnTek, Seoul, South Korea). The efficiency was calculated by measuring the time required for the analysis of one specimen ten times consecutively. Flow cytometry and the ADAM-rWBC were able to detect four sporadic cases that had residual WBCs exceeding 1/µL that were not detected by the manual method. Analysis time was the shortest with the ADAM-rWBC, followed by flow cytometry and the manual method. Our data suggest that hospital blood banks require quality control of residual WBCs; among the methods evaluated in this study, the portable microscopic cell counter offers the best time efficiency.

Application of image cytometry to characterize heterologous lipid flippases in yeast.

Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number of lipid flippases has been identified but key features of their activity remain to be elucidated. A well-established method to characterize ATP-driven flippases is based on their heterologous expression in yeast, followed by incubation of the cells with fluorescent lipids. Internalization of these probes is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical flow cytometric evaluation of lipid-labeled cells. In addition, we demonstrated that expression of fluorescently tagged flippase complexes can be directly co-related with fluorescent lipid uptake using the image-based cell counter system. The method extends the number of techniques available for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry.

RetFM-J, an ImageJ-based module for automated counting and quantifying features of nuclei in retinal whole-mounts.

The present article introduces RetFM-J, a semi-automated ImageJ-based module that detects, counts, and collects quantitative data on nuclei of the inner retina from H&E-stained whole-mounted retinas. To illustrate performance, computer-derived outputs were analyzed in inbred C57BL/6J mice. Automated characterization yielded computer-derived outputs that closely matched manual counts. As a method using open-source software that is freely available, inexpensive staining reagents that are robust, and imaging equipment that is routine to most laboratories, RetFM-J could be utilized in a wide variety of experiments benefiting from high-throughput, quantitative, uniform analyses of total cellularity in the inner retina.

A Cross-Sectional Study of Hematological Parameters in the Geriatric Population Using Peripheral Smear Examination and a Five-Part Cell Counter.

BACKGROUND: "Healthy aging" is a major public health challenge, as the prevalence and incidence of diseases are much higher in older people. Among them, diabetes mellitus, hypertension (HTN), and cardiovascular illnesses are the most prevalent chronic ailments. A complete blood count test can give an overall picture of a patient's health status because abnormal counts might indicate the presence of many different types of disease. Using advanced hematology analyzers, a typical microscopic examination of a peripheral blood smear can yield vital information about the clinical state of the patient. The objective of the study was to investigate the hematological parameters in the elderly population in a tertiary care hospital, utilizing a five-part cell counter and a peripheral smear test. METHOD: A cross-sectional study was conducted at a tertiary care institute on 188 patients aged 60 years and above attending the outpatient department for two years. The routine complete blood count and differential count were determined on the Siemens Advia 2120 analyzer. All the data were collected and entered into the data sheets. Appropriate statistical analysis was carried out to interpret the results. RESULTS: HTN, diabetes mellitus, cardiovascular disease (CVD), and generalized weakness were the most common conditions affecting our senior group. Decreased Hb was positively correlated with widespread weakness. Total leukocyte count (TLC) was found to be more prevalent in people with CVD and HTN. A sizable share of the elderly population who had diabetes mellitus and CVD showed an elevated red cell distribution width (RDW) percentage. CONCLUSION: The results indicated a significant deviation from normal hematological parameters, which were associated with various health issues. These parametric associations can be used as risk indicators, which can help healthcare professionals ensure the good health of the geriatric population.

Graphic type differentiation of cell count data for diagnosis of early and late periprosthetic joint infection: A new method.

BACKGROUND: Graphic type differentiation of cell count data of synovial aspirates is a new method for the diagnosis of early and late periprosthetic joint infection. OBJECTIVE: The aim of the study was to analyse if the same 6 LMNE-types can be differentiated in the new Yumizen H500 cell counter as it was the case for the old cell counter ABX Pentra XL 80 of previous publications, to verify if the erythrocyte and thrombocyte curves of the new device give additional information and to calculate the difference of cell count in LMNE-type I and III (with abrasion) in the cell counter and in the manual counting chamber (Neubauer improved). METHODS: 450 aspirates of 152 total hip arthroplasties and 298 knee arthroplasties obtained for the diagnosis of periprosthetic joint infection were analysed with the Yumizen H500. RESULTS: All LMNE-matrices of the 450 aspirates could assigned to one of the six LMNE-types. There were 76 LMNE-type I, 72 LMNE-type II, 14 LMNE-type III, 241 LMNE-type IV, 36 LMNE-type V and 12 LMNE-type VI. The erythrocyte and thrombocyte distribution curves were very helpful for differentiation of hematoma and infection. The cell count in the manual counting procedure was lower than in the cell counter: for the LMNE-type I (abrasion type) the median of the difference was 925/µL (median) and for the LMNE-type III (combined type of infection and abrasion) 3570/µL (median). CONCLUSION: The described graphic type differentiation is a new and helpful method for differentiation of hematoma and early PJI as well as abrasion and late PJI.

Factors Affecting the Number of Pollen Grains per Male Strobilus in Japanese Cedar (Cryptomeria japonica).

Japanese cedar (Cryptomeria japonica) is the most important timber species in Japan; however, its pollen is the primary cause of pollinosis in Japan. The total number of pollen grains produced by a single tree is determined by the number of male strobili (male flowers) and the number of pollen grains per male strobilus. While the number of male strobili is a visible and well-investigated trait, little is known about the number of pollen grains per male strobilus. We hypothesized that genetic and environmental factors affect the pollen number per male strobilus and explored the factors that affect pollen production and genetic variation among clones. We counted pollen numbers of 523 male strobili from 26 clones using a cell counter method that we recently developed. Piecewise Structural Equation Modeling (pSEM) revealed that the pollen number is mostly affected by genetic variation, male strobilus weight, and pollen size. Although we collected samples from locations with different environmental conditions, statistical modeling succeeded in predicting pollen numbers for different clones sampled from branches facing different directions. Comparison of predicted pollen numbers revealed that they varied >3-fold among the 26 clones. The determination of the factors affecting pollen number and a precise evaluation of genetic variation will contribute to breeding strategies to counter pollinosis. Furthermore, the combination of our efficient counting method and statistical modeling will provide a powerful tool not only for Japanese cedar but also for other plant species.

[A comparative analysis of two methods for assessing the proinflammatory activity of monocytes in patients with juvenile depression]. / Sravnitel'nyi analiz dvukh sposobov otsenki provospalitel'noi aktivnosti monotsitov pri depressii u bol'nykh yunosheskogo vozrasta.

OBJECTIVE: To show that the results of evaluation of monocyte pro-inflammatory activity (PA) in patients with juvenile depression and healthy donors, obtained using a new method developed by us for counting the relative number of large monocytes on a multifunctional counter and cell analyzer, are similar to the results obtained using a standard assessment of the level of proinflammatory CD14+/CD16+ - monocytes on a flow cytofluorimeter. MATERIAL AND METHODS: The PA of monocytes, isolated from the peripheral venous blood of 18 patients with juvenile depression and 12 mentally and somatically healthy age and gender-matched persons was evaluated in two ways: using the generally accepted method of determining the relative number of monocytes with the proinflammatory phenotype CD14+/CD16+ on a flow cytofluorometer FC-500 and by counting the relative number of large monocytes on a multifunctional counter and cell analyzer Multisizer MS-4. PA of monocytes in patients was studied by using both methods in different variants: in the general group and in the subgroups of patients with low and high levels of active monocytes. RESULTS: The levels of monocyte PA determined in patients using the two methods did not statistically differ from each other in all variants of the analysis (p=0.6). The equivalence of the obtained results was confirmed by the Chi-square test (r=0.77, p=0.05), as well as by the detection of a statistically significant positive correlation between the number of monocytes with the pro-inflammatory CD14+/CD16+ phenotype, on the one hand, and the relative number of large monocytes, on the other hand (Spearman r=0.75; p<0.05). At the same time, a comparative analysis of the level of monocyte PA in the general groups of patients and healthy controls revealed significantly higher values of this indicator in patients compared with healthy persons when evaluated by both methods (p<0.05). Definition of monocytes PA using the new method developed by us for counting the relative number of large monocytes on the analyzer and cell counter is more economical and easier to perform, since it does not require the use of expensive devices and reagents, as well as complex device settings and a high level of operator qualification, as in the common method, and is carried out only by two parameters: by counting the number of large monocytes with a diameter of 12.5 to 15 microns and the total number of monocytes with a diameter of 9 to 15 microns. CONCLUSION: The proposed method for assessing monocyte PA by counting the relative number of large monocytes on the cell counter and analyzer can be used to analyze the activity of monocytes for research purposes.

Quantitative Detection of Plasmodium falciparum Using, LUNA-FL, A Fluorescent Cell Counter.

The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, in this study, we investigated if a commercially available fluorescent cell counter, LUNA-FL, was useful for the detection of Plasmodium parasite and the estimation of parasitemia. Whole blood samples from uninfected persons, spiked with P. falciparum-infected erythrocytes, were analysed. Most of the leucocytes and platelets were removed from whole blood samples with SiO2-nanofiber filters set on spin columns. The filtered samples were stained with acridine orange, and automatic detection, as well as counting of erythrocytes and parasites, were performed using LUNA-FL. Whole blood, with various levels of parasites, was analysed by Giemsa microscopy or with LUNA-FL to estimate parasitemia, and a comparative analysis was performed. The coefficient determination value of the regression line was high (R2 = 0.98), indicating that accurate quantitative parasite detection could be performed using LUNA-FL. LUNA-FL has a low running cost; it is compact, fast, and easy to operate, and may therefore be useful for point-of-care testing in the endemic areas.

Abnormal dot plots on current automated blood cell analyzer helped to yeast detection.

Compromised data are usually flagged by instruments. This is the first report of yeast detection using the new launched Sysmex XN analyzer.