Izumi Tsuda

Effects of heparin on polymerase chain reaction for blood white cells

Polymerase chain reaction (PCR) has been used with increasing frequency to diagnose infectious and genetic diseases. In this study, the effects of heparin on PCR were investigated, because heparinized blood may sometimes be used in PCR studies. HLA‐DQA1 gene amplification was used as a model. PCR was clearly interfered with when heparinized blood was used as a source of template DNA, and the degree of interference was affected by the following three factors; (1) type of Taq DNA polymerase; (2) leukocyte count in blood; and (3) concentration of heparin contained. When additional tests were conducted with additions of definite heparin concentrations to a PCR reaction mixture, specimens with large amounts of DNA tended to exhibit less interference by heparin. The addition of ≥ 0.1 to 0.0016 U of heparin per reaction mixture (50 μl) suppressed DNA amplification in a dose‐dependent fashion. We therefore concluded that much care should be taken when heparinized blood is used as a PCR material. J. Clin. Lab. Anal. 13:133–140, 1999.

DNA extraction from human urinary sediment

DNA was extracted from urinary sediments and was sufficient for polymerase chain reaction (PCR) and enzymatic analysis, even if DNA from microorganisms coexisted. From urine samples, the yield of DNA ranged from trace levels to 20 μg per 10 mL urine. When urinary sediment was stored in ethanol, DNA remained stable for 2 weeks or more. Individual identification and sex determination could easily be performed using either fresh or ethanol‐fixed urine. In conclusion, urine can be used as a source for PCR‐based investigations and genetic studies. J. Clin. Lab. Anal. 12:88–91, 1998.

A new direction in automated laboratory testing in Japan: five years of experience with total laboratory automation system management.

The introduction of integrated laboratory systems has proceeded rapidly in Japan in these 15 years, but they require large initial investment for installation and do not always succeed in reducing laboratory cost. We also experienced three major events that taught us that total laboratory systems are not always effective: these were an earthquake, a nerve gas attack, and an outbreak of food poisoning. Political changes in the national health care system in Japan have forced the cutting of expenses for laboratory testing. In this context, cost-effective laboratory testing has been considered, and many hospitals have replaced total laboratory systems with small laboratory systems. Our University Hospital introduced a mini-lab system consisting of compact instruments to increase laboratory efficiency, and we have begun point-of-care testing education for medical students. This combination enables rapid and convenient testing, and is responsive to the political changes in the Japanese health care system.

Maturity of reticulocytes in various hematological disorders.

To the Editor: The reticulocyte count is clinically important in detecting erythropoietic activity in patients. However, the manual method of obtaining the reticulocyte count is neither accurate nor precise (1, 2). Recently, an automated reticulocyte counter was developed that provides rapid, precise and accurate counting of reticulocytes (3). This instrument also gives parameters for the degree of cell maturity by measurement of the fluorescence intensity of an RNA-binding dye. We measured these parameters for healthy subjects and patients with hematological disorders. Venous blood was collected in a bottle containing EDTA-2K. For the establishment of normal reference values, samples were obtained (n

Usefulness of determining reticulated and large platelets in idiopathic thrombocytopenic purpura

This article is also accessible online at: http://BioMedNet.com/karger In 1969, Ingram and Coopersmith [1] identified reticulated platelets (RP) as those displaying coarse, punctate condensations (reticulum) when stained supravitally with the new methylene blue dye. They suggested that RP may provide valuable clinical information regarding thrombocytopoiesis, as reticulocyte counts do for erythropoiesis. However, this method had not been applied routinely in the clinical laboratory. Watanabe et al. [2] have recently counted RP using an automated reticulocyte counter (R-3000, Toa Medical Electronics Co., Kobe, Japan) equipped with special software. This instrument performs flow cytometry of reticulocytes after rapid staining of RNA by a fluorescent dye, auramine O [3]. The software used by Watanabe et al. contains a computer algorithm to discriminate the platelet population by the intensity of forward light scatter (cell size) and fluorescence (RNA content). A typical fluorescence scattergram of platelets from a normal control obtained with this system is shown in figure 1. Line C is predefined in the R-3000. Cells in the region between lines A and D were determined using

Automated Enumeration of Cellular Composition in Bone Marrow Aspirate with the CELL-DYN 4000TM Automated Hematology Analyzer

The present study was designed to evaluate the automated analysis of bone marrow aspirates with the CELL-DYN 4000 (CD4000) hematology analyzer. Bone marrow aspirates were diluted twice with phosphate-buffered saline and assayed with the CD4000. The percentages of subpopulations including lymphocytes, neutrophils, and erythroblasts were obtained with the CD4000, and as a reference, differential counts by microscopic observation of May-Grünwald-Giemsa-stained films of bone marrow aspirate were performed (n = 48). Significant correlations (p < 0.0001) between the results with the two methods were obtained for total nucleated cell count, lymphocytes, neutrophils, erythroid cells, and the myeloid/erythroid ratio. The present method can provide quantitative data of bone marrow aspirate and will be useful in bone marrow screening.

Measurement of the zeta potential of human platelets by the use of laser-light scattering

An instrument was developed to detect the shift in scattering of laser light that occurs when particles in suspension move in a chamber with an electrical load. The instrument measures the zeta potential of particles. We applied the instrument to study human blood cells. Platelet-rich plasma was used because of the stability of the suspension, without the sedimentation or autoaggregation that is often seen with red or white blood cells. The reproducibility of the measurements was satisfactory when there were enough platelets in the suspension. Platelets from healthy controls (n = 136) had a potential of -14.20 +/- 1.64 mV at the detection angle of 17.1 degrees. Platelets from patients with essential thrombocytosis (n = 16) or polycythemia vera (n = 8) had higher potentials than the healthy controls.

CTAD as a universal anticoagulant

The feasibility of CTAD (a mixture of citrate, theophylline, adenosine and dipyridamole) as a new anticoagulant for medical laboratory use was studied prospectively. Whole blood anticoagulated with CTAD exhibited results very similar to those of blood anticoagulated with EDTA on complete blood count and automated white cell differential except for a slight decrease in platelet count and mean platelet volume. Chemistry test data for plasma obtained from CTAD whole blood were close to those obtained for matched sera. Among coagulation tests, prothrombin time, activated partial thromboplastin time and fibrinogen concentrations were close to those obtained with citrate plasma. Based on the results, CTAD was judged to be a good candidate as a new anticoagulant.

First basic performance evaluation of the XE-2100 haematology analyser

The newly developed XE-2100 haematology analyser can provide complete blood counts, leukocyte differentials, perform reticulocyte analysis, and obtain quantitative data on nucleated red blood cells (NRBCs). In this study, we evaluated the basic performance of this instrument using routinely obtained blood specimens treated with ethylenediaminetetraacetic acid-2k. Reproducibility, carryover, stability during storage at 4°C and room temperature, and accuracy were evaluated. In this evaluation, reproducibility was good and little carryover was found. Accurate measurements were possible for up to 48h of storage. A good correlation between findings with the XE-2100 and SE-9000 haematology analysers was found for complete blood count on 210 samples tested. The leukocyte differential obtained with the XE-2100 correlated well with eye counts and with the results obtained with the SE-9000 automated haematology analyser, with r values over 0.9 for the percentages of neutrophils, lymphocytes and eosinophils. The precision and accuracy of VRBC and reticulocyte counts by the XE-2100 were satisfactory. We used the XE-2100 to obtain differential counts for bone marrow aspirates, and good correlations with manual differentials were obtained for total nucleated cell count, percentage of myeloid cells and percentage of erythroid cells. The performance of the XE-2100 was excellent, and this instrument should be able to provide reliable data to clinical laboratories.

Automated bone marrow analysis using the CD4000 automated haematology analyser

At present, bone marrow analysis is performed microscopically, but is time consuming and labour intensive. No automated methods have been successfully applied to classification of bone marrows cells because automated blood cell analysers have been incapable of identifying erythroblasts. The present study was designed to evaluate automated analysis of bone marrow aspirates with the CELL-DYN 4000 (CD4000) haematology analyser, which enables automated determination of erythroblast counts in both the normal mode (haemolytic time; 11.5s) and the resistant RBC mode (34.0s). The percentages of subpopulations including lymphocytes, neutrophils and erythroblasts were obtained with the CD4000, and as a reference, differential counts by microscopic observation of May–Grünwald–Giesa-stained films of bone marrow aspirates were performed (n=98). Significant correlations (P < 0.01) between the results obtained with the two methods were observed for total nucleated cell count and lymphocytes, neutrophils, erythroblasts and myeloid/erythroid (M/E) ratio. However, there were biases in the average percentages of erythroblasts, lymphocytes and M/E ratio obtained using the normal mode with the CD4000 toward values lower than those obtained with the microscopic method. Using the RBC resistant mode with the CD4000, the average percentages of erythroblasts, lymphocytes and M/E ratio approximated those obtained with the microscopic method. In conclusion, the CD4000 in resistant RBC mode is more useful for analysis of bone marrow aspirates than is the normal mode, because the former better approximates the M/E ratio than the latter.