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The extraordinary advances in clinical hematology, biology, and oncology in the last decades would not have been possible without discovering how to identify and count the cells circulating in the blood. For centuries, scientists have used slides, counting chambers (hemocytometers), and diluting and staining solutions for this task. Then, automated hemocytometry began. This science, now linked to the daily routine of laboratory hematology, has completed an overwhelming path over a few decades. Our laboratories today operate with versatile multiparameter systems, ranging from complex single-channel instruments to bulky continuous flow machines. In terms of clinical information obtained from a simple routine blood test, the full exploitation of their potential depends on the operators' imagination and courage. A comprehensive review of the scientific publications that have accompanied the development of hemocytometry from the 1950s to today would require entire volumes. More than seven hundred contributions that authors worldwide have published in Clinical and Laboratory Haematology until 2007 and then the International Journal of Laboratory Hematology are summarized. Such journals have represented and hopefully will continue to represent the privileged place of welcome for future scientific research in hemocytometry. Improved technologies, attention to quality, new reagents and electronics, information technology, and scientist talent ensure a more profound and deeper knowledge of cell properties: current laboratory devices measure and count even minor immature or pathological cell subpopulations. Full-field hemocytometry includes the analysis of nonhematic fluids, digital adds to the microscope, and the development of effective point-of-care devices.

Objective: Total white blood cell (WBC) count in body fluids (BF) is important in diagnosis and treatment of inflammatory and infectious diseases. We aimed to compare hemogram and BF modes of Mindray BC-6800 hematology analyzer with the manual hemocytometry method for determination of total WBC count in peritoneal and pleural fluids. Method: Our study consisted of a total of 143 specimens, 109 peritoneal fluid and 34 pleural effusion fluid. Each sample was analyzed consecutively twice, first with the manual method and, later with both hemogram mode and the BF mode of Mindray BC-6800 automated hematology analyzer. Results: There was a statistically significant difference between manual count and hemogram mode (for peritoneal fluids p< 0.001, for pleural fluids p< 0.001). There was a statistically significant difference between the hemogram mode and BF mode too. (for peritoneal fluids p< 0.001, for pleural fluids p< 0.001). However, there was no statistically significant difference between the manual count and BF mode (for peritoneal fluids p=0.236, for pleural fluids p=0.627). Less than100 cells/mL, there was a statistically significant difference between each of the counting methods (Manual count vs hemogram mode p< 0.001, manual count vs BF mode p= 0.012, hemogram mode vs BF mode p< 0.001).More than 100 cells/mL, there was no statistically significant difference between manual count and the BF mode (p= 0.332). However, there were statistically significant differences between manual count and hemogram mode, and hemogram mode and BF mode (p= 0.003 and p< 0.001 respectively). Conclusion: The use of manual method instead of hematology analyzer is more convenient in samples 100 cells/mL. Amaç: Vücut sıvılarında (BF) toplam beyaz kan hücre (WBC) sayısı, enflamatuar ve bulaşıcı hastalıkların tanı ve tedavisinde önemlidir. Peritoneal ve plevral sıvılarda toplam WBC sayısının belirlenmesi için Mindray BC-6800 hematoloji analizörünün hemogram ve BF modlarını manuel hemositometri yöntemiyle karşılaştırmayı amaçladık. Yöntemler: Çalışmamız 109 periton sıvısı ve 34 plevral efüzyon sıvısı olmak üzere toplam 143 örnekten oluşuyordu. Herbir örnek, önce manuel yöntemle ve daha sonra Mindray BC-6800 otomatik hematoloji analizörünün hemogram ve BF modları ile iki kez art arda analiz edildi. Bulgular: Manuel sayım ve hemogram modu arasında istatistiksel olarak anlamlı farklılık vardı (periton sıvıları için p< 0.001, plevral sıvılar için p< 0.001). Hemogram modu ile BF modu arasında da istatistiksel olarak anlamlı bir fark bulundu (periton sıvıları için p< 0.001, plevral sıvılar için p< 0.001). Ancak manuel sayım ve BF modu arasında istatistiksel olarak anlamlı bir sonuç bulunamadı (Periton sıvıları için p= 0.236, plevral sıvılar için p= 0.627). 100 hücre/mL’den daha az olduğunda, her bir sayma yöntemi arasında istatistiksel olarak anlamlı farklılık mevcuttu (Manuel sayım & hemogram modu p< 0.001, manuel sayım & BF modu p= 0,012, hemogram modu & BF modu p< 0.001). 100 hücre/mL’den fazla olduğunda, manuel sayım ve BF modu arasında istatistiksel olarak anlamlı bir farklılık yoktu (p= 0,332). Ancak manuel sayım ve hemogram modu arasında ve hemogram modu ve BF modu arasında arasında istatistiksel olarak anlamlı bir farklılık vardı (sırasıyla p= 0,003 ve p< 0.001). Sonuç: Hematoloji analizörü yerine manuel metodun kullanılması, 100 hücre / mL'lik numunelerde bir tarama aracı olarak kullanılabilir.

BACKGROUND: Dried blood spots (DBSs) have gained recent popularity as a sampling method for therapeutic drug monitoring. For patients, DBS sampling has several advantages over venous blood sampling. However, technical issues primarily influenced by hematocrit levels, interfere with the implementation of this method in daily clinical practice. The results of concentration measurements of drugs that are influenced by hematocrit should be corrected for hematocrit levels. In this article, we developed a fast, nondestructive, near-infrared (NIR)-based method for measuring the hematocrit in DBSs. METHOD: Using a partial least squares algorithm, an NIR-based quantification method was developed for measuring hematocrit levels of 0.19-0.49 L/L. Residual venous blood of 522 patients was used to build this partial least squares model. The validity of the method was evaluated using 40 patient samples. DBSs were created by adding a small amount (50 µL) of blood on a Whatman filter paper and drying for 24 hours in a desiccator cabinet. The robustness was evaluated by measuring 24 additional samples with a high hemolysis, icterus, and lipemia (HIL) index. The hematocrit values obtained using a Sysmex XN hemocytometry analyzer were used as reference. RESULTS: The difference between hematocrit measurements obtained with NIR spectroscopy and a hemocytometry analyzer was

INTRODUCTION: While single-cell analysis technology has flourished, obtaining single cells from complex tissues continues to be a challenge. Current methods require multiple steps and several hours of processing. This study investigates chemical and mechanical methods for clinically relevant preparation of single-cell suspension from frozen biopsy cores of complex tissues. The developed protocol can be completed in 15 min. METHODS: Frozen bovine liver biopsy cores were normalized by weight, dimension, and calculated cellular composition. Various chemical reagents were tested for their capability to dissociate the tissue via confocal microscopy, hemocytometry and quantitative flow cytometry. Images were processed using ImageJ. Quantitative flow cytometry with gating analysis was also used for the analysis of dissociation. Physical modeling simulations were conducted in COMSOL Multiphysics. RESULTS: A rapid method for tissue dissociation was developed for single-cell analysis techniques. The results of this study show that a combination of 1% type-1 collagenase and pronase or hyaluronidase in 100 U/µL HBSS solution is the most effective at dissociating 2.5 mm thawed bovine liver biopsy cores in 15 min, with dissociation efficiency of 37-42% and viability >90% as verified using live MDA-MB-231 cancer cells. Cellular dissociation is significantly improved by adding a controlled mechanical force during the chemical process, to dissociate 93 ± 8% of the entire tissue into single cells. CONCLUSIONS: Understanding cellular dissociation in ex vivo tissues is essential to the development of clinically relevant dissociation workflows. Controlled mechanical force in combination with chemical treatment produces high quality tissue dissociation. This research is relevant to the understanding and assessment of tissue dissociation and the establishment of an automated preparatory workflow for single cell diagnostics. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s12195-021-00667-y) contains supplementary material, which is available to authorized users.

Objectives COVID-19 is an ongoing global pandemic. There is an urgent need for identification and understanding of clinical and laboratory parameters related to progression towards a severe and fatal form of this illness, often preceded by a so-called cytokine-storm syndrome (CSS). Therefore, we explored the hemocytometric characteristics of COVID-19 patients in relation to the deteriorating clinical condition CSS, using the Sysmex XN-10 hematology analyzer. Methods From March 1st till May 16th, 2020, all patients admitted to our hospital with respiratory complaints and suspected for COVID-19 were included (n=1,140 of whom n=533 COVID-19 positive). The hemocytometric parameters of immunocompetent cells in peripheral blood (neutrophils [NE], lymphocytes [LY] and monocytes [MO]) obtained upon admission to the emergency department (ED) of COVID-19 positive patients were compared with those of the COVID-19 negative ones. Moreover, patients with CSS (n=169) were compared with COVID-19 positive patients without CSS, as well as with COVID-19 negative ones. Results In addition to a significant reduction in leukocytes, thrombocytes and absolute neutrophils, it appeared that lymphocytes-forward scatter (LY-FSC), and reactive lymphocytes (RE-LYMPHO)/leukocytes were higher in COVID-19-positive than negative patients. At the moment of presentation, COVID-19 positive patients with CSS had different neutrophils-side fluorescence (NE-SFL), neutrophils-forward scatter (NE-FSC), LY-FSC, RE-LYMPHO/lymphocytes, antibody-synthesizing (AS)-LYMPHOs, high fluorescence lymphocytes (HFLC), MO-SSC, MO-SFL, and Reactive (RE)-MONOs. Finally, absolute eosinophils, basophils, lymphocytes, monocytes and MO-FSC were lower in patients with CSS. Conclusions Hemocytometric parameters indicative of changes in immunocompetent peripheral blood cells and measured at admission to the ED were associated with COVID-19 with and without CSS.

Sorting and measuring blood by cell type is extremely valuable clinically and provides physicians with key information for diagnosing many different disease states including: leukemia, autoimmune disorders, bacterial infections, etc. Despite the value, the present methods are unnecessarily costly and inhibitive particularly in resource poor settings, as they require multiple steps of reagent and/or dye additions and subsequent rinsing followed by manual counting using a hemocytometer, or they require a bulky, expensive equipment such as a flow cytometer. While direct on-paper imaging has been considered challenging, paper substrate offers a strong potential to simplify such reagent/dye addition and rinsing. In this work, three-layer paper-based device is developed to automate such reagent/dye addition and rinsing via capillary action, as well as separating white blood cells (WBCs) from whole blood samples. Direct onpaper imaging is demonstrated using a commercial microscope attachment to a smartphone coupled with a blue LED and 500 nm long pass optical filter. Image analysis is accomplished using an original MATLAB code, to evaluate the total WBC count, as well as differential WBC count, i.e., granulocytes (primarily neutrophils) vs. agranulocytes (primarily lymphocytes). Only a finger-prick of whole blood is required for this assay. The total assay time from finger-prick to data collection is under five minutes. Comparison with a hemocytometry-based manual counting corroborates the accuracy and effectiveness of the proposed method. This approach could be potentially used to help make blood cell counting technologies more readily available, especially in resource poor, point-of-care settings.

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.

Cytometry is a basic method to determine cell populations and morphology. Flow cytometry and hemocytometry are the two most common methods among cytometric technologies. However, flow cytometry needs bulky and expensive equipment as well as professional operations, while hemocytometry is limited by its simple function. Both of them are not suitable for real-time monitoring of the morphological changes of single cells. Here, we developed a fixed cytometer chip with two functional modes for both identification of cell populations (I-mode) and real-time monitoring of single-cell morphological changes (M-mode). In I-mode, the fixed cytometer chip was employed to evaluate the cell populations, the results were in accordance with those from the hemocytometer counting and flow cytometry. Besides that, the cell populations were further precisely identified by measuring two-color fluorescence intensities of single cells, which were consistent with the dual parameter analysis in flow cytometry. In M-mode, the chip was applied to real-time monitoring of the single-cell apoptosis under gradient UV radiation, generated by a novel stair-like UV shield. The dynamic apoptotic morphologies of a large number of single cells were monitored in real-time by time-lapse imaging. In addition, we integrated eight parallel channels on a 60 mm × 30 mm chip, and the chip could achieve scalable single-cell capture and analysis capability. This fixed cytometer chip is bifunctional, easy-to-handle, universal, and scalable.

Purpose To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. Methods B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. Results All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). Conclusion FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells. Electronic supplementary material The online version of this article (10.1007/s11095-018-2422-5) contains supplementary material, which is available to authorized users.

Microalgae have attracted attention for several industrial applications, but all such applications demand culture quality because of their sensitivity to environmental changes. Although simplicity, speed, and accuracy are important to assess algal cultures, researchers have expended vast amounts of labor to monitor algal health using hemocytometry. Along with its user bias, quantifying the cell status aside from the cell density is not easy. This paper describes the easy and rapid evaluation of algal number and status using an image-based cell counter (Countess II FL; Thermo Fisher Scientific Inc.) with a fluorescent filter for chlorophyll. Unlike mammalian cultured cells larger than microalgae, it is not easy for a low-resolution camera alone to distinguish microalgae from grimy spots and microbubbles on counting plates. To assess this method’s performance, freshwater/marine microalgae and environmental samples were evaluated using the instrument. Results reveal that an instrument with a fluorescence filter can distinguish microalgae from other particles more precisely than a device with no filter. Values obtained using the instrument were not significantly different from those obtained using hemocytometry. Moreover, the cell counter, but not hemocytometry, can qualify the algal status. Results demonstrate that this system, which has no user bias, can contribute to algal assessment.

OBJECTIVES: Intestinal ischemia/reperfusion often leads to acute lung injury and multiple organ failure. Ischemic preconditioning is protective in nature and reduces tissue injuries in animal and human models. Although hematimetric parameters are widely used as diagnostic tools, there is no report of the influence of intestinal ischemia/reperfusion and ischemic preconditioning on such parameters. We evaluated the hematological changes during ischemia/reperfusion and preconditioning in rats. METHODS: Forty healthy rats were divided into four groups: control, laparotomy, intestinal ischemia/reperfusion and ischemic preconditioning. The intestinal ischemia/reperfusion group received 45 min of superior mesenteric artery occlusion, while the ischemic preconditioning group received 10 min of short ischemia and reperfusion before 45 min of prolonged occlusion. A cell counter was used to analyze blood obtained from rats before and after the surgical procedures and the hematological results were compared among the groups. RESULTS: The results showed significant differences in hematimetric parameters among the groups. The parameters that showed significant differences included lymphocyte, white blood cells and granulocyte counts; hematocrit; mean corpuscular hemoglobin concentration; red cell deviation width; platelet count; mean platelet volume; plateletcrit and platelet distribution width. CONCLUSION: The most remarkable parameters were those related to leukocytes and platelets. Some of the data, including the lymphocyte and granulocytes counts, suggest that ischemic preconditioning attenuates the effect of intestinal ischemia/reperfusion on circulating blood cells. Our work contributes to a better understanding of the hematological responses after intestinal ischemia/reperfusion and IPC, and the present findings may also be used as predictive values.

Complete blood count (CBC) is a basic and important diagnostic procedure in pathological laboratories. However, current technologies such as hemocytometry, impedancemetry, or flow-cytometry, are either time-consuming or requiring bulky and high-cost of instrumentation. To address this issue we demonstrate a compact, cost-effective, and automated blood cell counting method based on the lens-free shadow imaging technique. For that a lens-free shadow imaging technique based blood cell counting device was fabricated using a blue light emitting diode (LED) and a complementary metal oxide semiconductor (CMOS) image sensor. The shadow images of the blood cells, i.e., diffraction patterns, were captured by the device and processed automatically using a custom developed algorithm to count and differentiate the cells. The comparative study from 21 outpatient whole blood samples was carried out with the fully automated hematology analyzer (LH 750, Beckman Coulter). The comparison between the lens-free shadow imaging device and the standard reference system showed correlation indices of 0.878 for red blood cell (RBC) and 0.927 for white blood cell (WBC). The linearity comparison gave a statistical trend of y = 0.9432x with R2 = 0.99 for RBC and y = 0.7395x with R2 = 0.92 for WBC. Also the correlation coefficient for WBC subpopulations, i.e., neutrophil, lymphocyte, and monocyte, for each individual sample was >0.90. This cost-effective and compact blood cell counting system can be utilized as a powerful point of care (POC) tool, which could play an important role in primary healthcare, especially in areas with limited resources. Since the system is capable to send the images wirelessly and process them on a moderate smart phone, therefore, the proposed system possesses great potential in telemedicine applications.

In order to develop effective cell based therapies for treating several human diseases, it is necessary to increase the limited number of stem cells harvested from a single patient to be used in autologous transplantation. Along these lines, mathematical modeling may represent a useful tool for interpreting and rationalizing the in vitro expansion techniques, thus helping to find the optimal operative conditions. Specifically, this work addresses the mathematical simulation of the proliferation kinetics of sheep bone marrow mesenchymal stem cells (phenotypic characterized by flow-cytometric analysis), as model similar to the human one, seeded at different initial concentrations in petri dishes and expanded up to confluence. The sigmoidal temporal profiles of total counts obtained through classic hemocytometry are quantitatively interpreted by a novel model based on population balance approach capable to take into account the contact inhibition at confluence. First, the adjustable parameters of the proposed model are fitted against experimental data on population expansion starting from one single seeding concentration. Then, the reliability of the mathematical model is successfully tested through the prediction of cell proliferation kinetics carried out starting from different seeding concentrations.

Aims: To test Fountain FlowTM Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. Methods and Results: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R-phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light-emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid-phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0·06 amoebae ml−1, using a flow rate of 15 ml min−1. Conclusions: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. Significance and Impact of the Study: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real-time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.

The development of new technologies and software that are routinely used in laboratories has now allowed for a more diverse novel range of methods to determine sperm concentrations more rapidly. The aim of this study was to compare 3 such novel methods developed in our laboratory, including a new flow cytometry approach, image analysis, and a fluorescent plate reader, with more conventional methods (hemocytometry, spectrophotometry, and Microcell analysis). Fifteen ejaculates were collected from 13 bulls at an artificial insemination center. The semen samples were analyzed for sperm concentration using a spectrophotometer, hemocytometry, and a novel flow cytometry technique based on counting a fixed volume of fluid. The raw ejaculate was also diluted fivefold in a long-term diluent and sent overnight to another laboratory, where sperm numbers were assessed using Microcells, an image analysis system, and a fluorescent plate reader. Each ejaculate was assessed 5 times using each of the methods described in order to determine the coefficient of variation for each method. Comparisons between methods were determined using correlation and limits of agreement. The flow cytometry results showed the lowest coefficient of variation (2.3%), with the plate reader showing the highest coefficient of variation (20.0%). There was no significant difference between any of the methods used, and none of them consistently over- or underestimated numbers when compared against each other. It is concluded that flow cytometry showed the highest repeatability of results. However, the method employed by each laboratory should be determined based on a range of factors, including cost, convenience, sample size, and number of ejaculates to be assessed.

Image cytometry centrifugation (ICC) method has been developed to enumerate residual leukocytes in transfusion blood products. Leukocyte counts by the ICC method were found to be consistent with those by the conventional Nageotte hemocytometry.

Counting cells is often a necessary but tedious step for in vitro cell culture. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, a survey (1) of 400 researchers who count cells revealed that 71% use a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse that results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of error in hemocytometry include uneven cell distribution in the sample; too many or too few cells in the sample; subjective decisions as to whether a given cell falls within the defined counting area; contamination of the hemocytometer; user-to-user variation; and variation of hemocytometer filling rate (2). To alleviate the tedium associated with manual counting, 29% of the researchers in the survey count cells using automated cell counting devices; these include vision-based counters—systems that detect cells using the Coulter principle—and flow cytometers (1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments (1). The ScepterTM cell counter (Millipore) combines the ease of automated instrumentation and the accuracy of Coulter impedance-based particle detection (3) in an affordable, handheld format (Figure 1). The instrument, which is the size of a pipet, uses a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip (Figure 1, inset) has a microfabricated, cell-sensing zone that can discriminate cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter graphically displays cell population statistics as histograms. Counting is typically completed in less than 20 seconds.

BACKGROUND: A multicenter study was conducted to evaluate the performance characteristics of flow cytometry and microfluorimetry for counting low concentrations of WBCs and to compare the results with Nageotte hemocytometry. STUDY DESIGN AND METHODS: A two-phase study involving 10 centers located in the United States and in Europe was performed. Coded samples of RBCs and platelets were distributed by 24-hour (Phase 1) or 2-day (Phase 2) courier service to each test site for analysis. Samples were prepared to include concentrations of WBCs slightly above and below the concentration corresponding to the threshold standards for WBC-reduced RBCs and platelets. All centers tested samples by Nageotte hemocytometry plus one or both of two automated methods. RESULTS: Both flow cytometry and microfluorometry gave better results than Nageotte hemocytometry in testing freshly prepared samples. At WBC concentrations >5 per μL (RBCs) or >3 per μL (platelets), the intersite CV was 30 percent for the Nageotte hemocytometer method (p

Background Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest. Study Design and Methods Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2−ΔΔCt of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples. Results Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 106 WBCs/L. Conclusion The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses. Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed(1) who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate(2). To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry(1). For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments(1). The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection(3) in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

Background : To enumerate leukocyte count in cerebrospinal fluid (CSF) is important for diagnosing bacterial meningitis. Using automated hematology analyzer for enumeration of leukocyte in CSF is below the sensitivity, so microscopic hemocytometric method is standard method. But this requires sufficient practical experience and has limitation of accuracy and stability. So we developed new manual method and evaluated it. Methods : We designed new method using transparent ruler tape. We performed correlation, accuracy and precision test by counting leukocyte in diluted EDTA blood with three methods: new method, Neubauer and Nageotte hemocytometry. Twenty two CSF were used for stability test, which determines leukocyte count according to time (within one hour and after 2, 4 and 12 hr), by new method and Neubauer hemocytometry at room temperature. Results : There was no clinical significant difference between three methods in correlation test, whereas Neubauer and Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with new method in case with low leukocyte count. The new method showed the lowest CV and most accurate result. In stability test, leukocyte counts decreased being 44.4%, 72.1% of initial values after 2 hr, 14.8%, 31.1%, after 4 hr and 4.2%, 8.7%, after 12 hr, by Nageotte hemocytometry and new method, respectively. Conclusions : The new method we devised is simple, easy and applicable to use in a laboratory and offers advantages of improved precision and stability. It may be sufficient for replacing standard methods for leukocyte counting in CSF.

The increasing importance of nanomaterial-related applications has given rise to concerns pertaining to their impact on human health. In vitro mammalian cell-based assays can provide a quick and simple estimate of the possible adverse effects of the nanomaterials. However, recent studies have questioned the efficacy of traditional assays such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, in evaluating cell-nanomaterial interactions, implying the need for alternate methods. We applied image analysis to enumerate the DAPI (2-[4-(Aminomethyl) phenyl]-1H-indole-6-carboximidamide, dihydrochloride) – stained cellular nuclei. Image analysis, being non-destructive, capable of automation, and applicable over a wide range of cell seeding densities, offers several advantages compared to older methods like the MTT assay and hemocytometry. Using image analysis, the impact of singlewalled carbon nanotubes (SWNT) on rat aortic smooth muscle cell (SMC) growth kinetics, were examined. Despite the carbon nanomaterial presence, the fluorescent signal from the nuclei was not noticeably impacted over the SWNT range examined (0.00-0.10 mg/ml). We anticipate that this method can also be applied to evaluate the biological impact of other nanomaterials.

importance, for example in the diagnosis and treatment of anemia and in transfusion protocols [1-3]. Despite the fact that it would be preferable to determine hemoglobin in a hospital using only one method, frequently different methods are used. Necessarily, hemoglobin results are reported into a hospital information system regardless of the sample type and method used. Therefore a good correlation between the methods used is mandatory. Deviations between different methods for the measurement of hemoglobin are described in the literature [4-6]. However, to our knowledge, this is the first published comparison of hemoglobin measured on a blood gas analyzer and a hemocytometry analyzer. For this study we used the Rapidlab 865 blood gas analyzer (Bayer Health Care, Tarrytown, NY, USA) and the Cell-Dyn 4000 hemocytometry analyzer (Abbott Laboratories, Abbott Park, IL, USA). The Cell-Dyn 4000 measures hemoglobin using absorption spectrophotometry. A cyanide-free hemoglobin reagent is used to lyse all cells and convert hemoglobin to a single chromagen with an absorption peak at 544 nm. A 540 nm LED illuminates the sample, and a photodetector measures the amount of light that passes through the sample. The Rapidlab 865 also measures hemoglobin using absorption spectrophotometry. Cells are lysed using ultrasound. Total hemoglobin and several clinically significant derivatives of hemoglobin are determined using an algorithm based on absorption spectrophotometry data using 40 different wavelengths. During 1 year, from April 2002 until March 2003, we tested samples from 1864 patients from our emergency department. Two samples were drawn at the same time from each patient, an arterial blood gas sample (3-mL heparin blood gas syringes [Rapidlyte; Bayer, Mijdrecht, the Netherlands]) and a venous blood sample (3-mL K3EDTA vacuum tubes [BD vacutainer, Plymouth, UK]). The results are shown in Figure 1. There is a linear relation with good correlation between the 2 methods using different sample types (y = 1.014x + 0.164; r = 0.926). In Figure 2 a bias analysis is shown. The Rapidlab 865 has a mean bias of 0.354 g/dL compared with the Cell-Dyn 4000. Despite the fact that the actual difference between the 2 methods is very small, clinical decisions are often based on a LETTER TO THE EDITOR

Microfluidic image cytometry is developed and validated both theoretically and experimentally, which is a method for the simultaneous measurement of the number and sizes of particles flowing through a microchannel using image sequence analysis and micro particle image velocimetry technique. Theoretical considerations on image formation in this method predict the image profile of a known particle and suggest that corrections are required in particle size measurement in order to cancel the effects of both diffraction and out-of-focus location. A dilution series of 2 µm polystyrene particle suspensions were measured and compared with the results obtained by conventional Burker–Turk hemocytometry for validation of the particle counting. For the particle diameter measurements, the diameters of 2, 5, 10 and 20 µm particles were measured and compared with the official values of the manufacturer. The results of the number and sizes of the particles measured by the proposed method agreed well with the reference values. We hope that the proposed method will be applicable to the quantitative study of platelet aggregation in blood flow and become a powerful diagnostic tool in the future.

The number of mammalian cells in a defined volume of medium can be measured using a hemocytometer. Automated methods using cell-counting devices such as those produced by Coulter are desirable when large numbers of individual samples are to be counted. A hemocytometer contains two chambers, each of which when filled and coverslipped contains a total volume of 9 µL. Each chamber is ruled into nine major squares, and each square is 1 × 1 mm with a depth of 0.1 mm. Thus, when coverslipped, the volume of each square is 0.1 mm3 or 0.1 µL. Additional subdivisions of the major nine squares are not necessary for counting and can be ignored.

A protocol for the laboratory preparation of a multiparameter hemocytometry control is given. Human platelets, stabilized by a basically simplified and inexpensive fixation procedure, are added to our previously described white and red blood cell control. Evaluation of this multiparameter control shows good precision characteristics and acceptable mechanical stability for at least 7 weeks, as measured in the Coulter counter Model S Plus-II. The control can basically contribute to the realization of the essence of internal quality control: continuous self-auditing and continuous attempts at improvement of performance.

Quantitation of red cell agglutination can be made by counting the residual free cells remaining after agglutination and comparing the count with the pre-agglutination red cell count. Most previous studies have used visual hemocytometry for this purpose. The use of the Coulter Model B electronic particle counter has been investigated and is shown to be a practical alternative. The problem of counting a free cell population overlapped in size by small aggregates is approached by two methods. In the first, a correction is made for aggregates present in the size range of single red cells based on the change in mean particle volume which results from the presence of cell clumps. In the second, red cells are counted in a size range in which aggregates are unlikely to occur. The methods are evaluated and their extension to provide further data on the pattern of red cell aggregation in agglutination is indicated.

Outdated, buffy-coat depleted, CPDA-1 blood and redundant buffy-coats were used as sources for the laboratory preparation of controls for Coulter-type (resistive-particle) hemocytometry. Deteriorated white blood cells and platelets and potentially interfering microaggregates with volumes not exceeding 400 fl are shown to be virtually completely removed by centrifugation and filtration. Addition of fixed red blood cells as white blood cell substitutes and of isolated, fixed platelets enable the preparation of multiparameter controls of short-to-medium-term stability. The availability of these simple, inexpensive controls can contribute significantly to optimal internal quality control in hemocytometry.

Publisher Summary This chapter examines the electronic enumeration and sizing of primary tissue cells. A simple, quick, and reproducible method for determining cell yield from primary tissue has become a necessity for large-scale tissue culture production. Primary cell suspensions are prepared from the freshly harvested chicken embryos, and rhesus and cercopithecus monkey kidneys. Following trypsinization, the cells are resuspended in appropriate growth media, thoroughly mixed, and samples are removed for enumeration. The remaining cell suspension is diluted, inoculated into culture bottles, and incubated at 36°C. Confluent monolayers developed in 16 to 18 hours for chicken embryo cultures and in 7 days for monkey kidney cultures. The results of a study discussed in the chapter showed that the electronic counter is suitable for routine tissue culture practices, because it is more consistent than the hemocytometer and possesses excellent reproducibility. It is evident that the degree of variation existing in hemocytometry could eventually be responsible for the establishment of erroneous planting rates causing either a decrease or increase in the number of total cells available, which would directly affect the number of cultures obtained from a primary suspension.

To the Editor.— Shaw et al1advocate using leukocyte esterase-measuring dipsticks for "routine urinalyses" and reading the results at five minutes to increase the sensitivity of detecting abnormal urine sediment. They define abnormal sediment as any red blood cells, casts, yeast, or more than three white blood cells (WBCs) per high-power field. These recommendations are based upon several assumptions regarding the use of clinical tests that need to be examined more closely. The authors have chosen spun-urine microscopy as the "gold standard" for measuring formed elements in the urine. However, for pyuria, spun-urine microscopy is unreliable and correlates poorly with leukocyte excretion rates (number of WBCs per hour) and hemocytometer chamber techniques (number of WBCs per cubic millimeter).2Leukocyte esterase dipsticks correlate better with pyuria by hemocytometry than by spun-urine microscopy3and may be more reliable than spun-urine microscopy. The author's definition of abnormal urine sediment appears