Intercomparison of pitch measurements for one-dimensional-grating standards (240 nm pitch), one of the widely used reference standards for nanometric lateral scales, was performed by three different methods, optical diffraction, critical dimension scanning electron microscopy and nanometrological atomic force microscopy. Average pitch values obtained by the three methods deviated by a maximum of only 0.67 nm with expanded uncertainties (k = 2) of less than 1.2 nm. The calculated E n number, the index of measurement quality, of less than 1 indicates consistency of the measured pitch values and subsequent uncertainty analyses performed by three methods.
We described a fully automated measurement of reticulated platelets using a fluorescent dye, auramine O, and a reticulocyte counter, the R‐3000, equipped with special software. Reproducibility and linearity were shown to be good. In the normal subjects studied (n = 60), the mean value for reticulated platelets was 0.98% ± 0.41% and the mean absolute count was 2.12 ± 0.69 × 109/l. The absolute count for reticulated platelets was significantly lower (p < 0.05) in patients with reduced thrombopoiesis as seen in acute myeloblastic leukemia, aplastic anemia or chemotherapy‐induced thrombocytopenia and it was elevated (p < 0.05) in essential thrombocythemia and in chronic myelocytic leukemia with thrombocytosis. All 20 patients with chronic idiopathic thrombocytopenic purpura had a high percentage of reticulated platelets. The percentage of reticulated platelets was significantly increased (p < 0.05) in patients with impaired thrombopoiesis despite the reduction in the absolute count. In 2 leukemic patients, an apparent rise was noticed in the percentage of reticulated platelets which preceded by several days a progressive increase in the platelet count at the recovery phase of thrombocytopenia. The results suggest that an automated measurement of reticulated platelets can be applied to routine laboratories for clinical use.
This report presents a flow cytometer devised by the authors and equipped with a CCD camera to obtain images of selected particles. Each image is linked to a point in the scattergram from which it can be retrieved from memory. The system uses an argon ion laser for flow-cytometric analysis and a diode-pumped pulse laser (532nm) to generate CCD images. Aggregated platelets in a blood sample and casts in a urine sample can be detected by this system. A fast flow rate is suitable for efficient flowcytometric detection, and image resolution is sufficiently adequate for recognizing aggregated platelets and casts. Flow-cytometric and morphological data are used together to distinguish different types of cells in the same region on the scattergram and identify cells present in extremely small numbers among the majority. o 1995 Wiley-Liss, Inc. Key terms: Flow cytometry, image cytometry, cell imaging, aggregated platelet, platelet clumps, castNumerous attempts have been made to detect abnormal cells in blood or urine using flow cytometry with cytochemical and/or electrical methods (2,3,13,15,18). However, it is quite difficult to distinguish cells similar in chemical and physical properties by such flow-cytometric methods without morphological data. This situation is encountered when trying to distinguish aggregated platelets from large platelets in the blood and to identify casts in small numbers in the urine.Aggregated platelets detection with suspected flagging of "platelet clumps" has so far been conducted by flow cytometry-based hematology analyzers ( 2,18). EDTA-induced platelet clumping that causes pseudothrombocytopenia is a clinical problem in hematology, and its reliable detection is difficult with hematology analyzers (3). Aggregated and large platelets are similar in size and possess the same physical properties, so that distinction by flow-cytometric parameters alone is difficult. There is a similar difficulty in identification of casts (e.g., hyaline casts) whose refractive indices are very low and approximately the same as in mucus (4). Difficulty in the detection of casts exists not only due to such similarity but also due to their scarcity. At most clinical laboratories, a microscope alone is used to detect casts present in very small numbers, but this method is laborious and the results may not be reliable.A flow cytometer used in conjunction with an imaging device to obtain morphological data in addition to flow-10 c d s . This resolution does not permit observation of small cells (e.g., platelets) and the flow rate is too slow to detect cells present in small numbers (e.g., casts). The authors have developed a flow cytometer that obtains images with sufficiently high resolution and flow rate for detecting aggregated platelets in blood samples and casts in urine samples. Flow-cytometric data on the scattergram are used to select images and retrieve images from memory for display. This system makes it possible to distinguish aggregated platelets in blood samples and casts in urine samples. cytometric d...
A method has been developed for accurate correction of red blood cell count for coincidence in aperture-impedance electronic blood cell counters. It is based on extrapolation of the slope of regression of the counts which are obtained with sequential dilutions of the samples.
Laser-based flow cytometry and aperture-impedance methods are still the dominant technologies used for cell analysis in haematology, but both are limited to areas such as morphological analysis of red cell shape and high-sensitivity detection of platelet agglutinates and aggregates. Flow cytometry alone does not provide precise measurement of red cell volume without chemical pretreatment before detection and aperture-impedance is still considered the gold standard in the field of particle volume analysis.In the present study, an experimental prototype instrument called the imaging-combined flow cytometer (IFC) was evaluated. The IFC is equipped with an imaging device consisting of a pulse laser, lens units and a charge-coupled device (CCD) camera in addition to the flow-cytometric optical set-up. A personal computer was attached to the instrument to handle images derived from the imaging device. Laser illumination was triggered so that the image of an object was captured for each exposure of the CCD camera. Objects in the images were used to calculate size and shape information and to compute fractal texture features by image processing after each measurement.The advantage of the IFC is that it can capture images of selected cells of interest at the same time as flow-cytometric detection. Estimation of red cell volume, discrimination of red cells and platelets, and detection of platelet agglutinates and aggregates were attempted using the IFC in combination with image processing, It was found that image analysis on the IFC could provide a substitutional function for mean corpuscular volume (MCV) estimation and detection of platelet agglutinates and aggregates. The additional information generated by the IFC may be useful in diagnostic haematology.
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