Reliability of white blood cell counting

The Laboratory Proficiency Testing Program has successfully operated mandatory proficiency testing for 13 years in Ontario, Canada, using committees of volunteer peers. This is an alternative to gubernatorial proficiency testing. Using white blood cell (WBC) count determinations as a focus, we present our results. Ninety-six percent of participants attained WBC count results within +/- 10% of an all-methods mean and 77% attained results within +/- 5%. Ninety-six percent displayed satisfactory precision. Details of the program, ie, testing material, computer analysis, reference results, and scope of committee action, are discussed. In 1975, clinical expectations for WBC count determinations greatly exceeded most laboratories' capabilities. By 1987, participants were able to produce reliable, clinically useful WBC count results. The paramount reason for this improvement is the transition from manual to automated counting methods resulting from the stimulus of proficiency testing and the concomitant education.     

Coincidence correction in red blood cell counting

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.     

A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.     

A simple method for electronic counting of rosette forming human blood lymphocytes.

We investigated the possibility of using a standard electronic laboratory cell counter, of a type which permits manual settings of discriminator and sensitivity levels, for the quantitation of E rosette forming human blood lymphocytes. The electronic counting was performed on rosette specimens prepared from 10 healthy donors, nine lymphoma patients and eight patients with chronic lymphocytic leukaemia (CLL) of B cell type. The simple procedure which was earlier developed for the electronic counting of thymocyte rosettes in guinea-pigs was used and now supplemented with a method for rapid, graphical determination of rosette frequency. The low level of E rosettes in CLL patients was easily distinguished from the higher level seen in the other groups and there was a good agreement between electronic and visual determinations. In samples with a high rosette frequency, the cell counter initially gave 10-30% lower values than when recorded visually. This discrepancy was corrected, however, by a minor adjustment of discriminator level and the introduction of a correction procedure for the presence of double rosettes. We conclude that the electronic counter can well be used for the counting of human rosette forming blood lymphocytes and, due to a high precision and large capacity, this approach would prove to be useful when screening of a large number of samples is required.     

Identification and red blood cell automated counting from blood smear images using computer-aided system

Red blood cell count plays a vital role in identifying the overall health of the patient. Hospitals use the hemocytometer to count the blood cells. Conventional method of placing the smear under microscope and counting the cells manually lead to erroneous results, and medical laboratory technicians are put under stress. A computer-aided system will help to attain precise results in less amount of time. This research work proposes an image-processing technique for counting the number of red blood cells. It aims to examine and process the blood smear image, in order to support the counting of red blood cells and identify the number of normal and abnormal cells in the image automatically. K-medoids algorithm which is robust to external noise is used to extract the WBCs from the image. Granulometric analysis is used to separate the red blood cells from the white blood cells. The red blood cells obtained are counted using the labeling algorithm and circular Hough transform. The radius range for the circle-drawing algorithm is estimated by computing the distance of the pixels from the boundary which automates the entire algorithm. A comparison is done between the counts obtained using the labeling algorithm and circular Hough transform. Results of the work showed that circular Hough transform was more accurate in counting the red blood cells than the labeling algorithm as it was successful in identifying even the overlapping cells. The work also intends to compare the results of cell count done using the proposed methodology and manual approach. The work is designed to address all the drawbacks of the previous research work. The research work can be extended to extract various texture and shape features of abnormal cells identified so that diseases like anemia of inflammation and chronic disease can be detected at the earliest.     

Platelet counting using the Coulter electronic counter

A method for counting platelets in dilutions of platelet-rich plasm using the Coulter electronic counter is described.(1) The results obtained show that such platelet counts are at least as accurate as the best methods of visual counting. The various technical difficulties encountered are discussed.     

An evaluation of the Celloscope 401 electronic blood cell counter

For counting erythrocytes the instrument was precise, with a mean coefficient of variation of 1.21%.Erythrocyte counts showed close agreement with results obtained on a Coulter A electronic counter of proven accuracy.When the Celloscope 401 was modified by the manufacturers to eliminate electrical interference from other laboratory equipment, satisfactory precision and accuracy for white cell counting was obtained. Using cetrimide diluent the coefficient of variation was 1.6% but when using saponin/saline diluent the coefficient of variation was 3.5%. For leucocyte counting there was close agreement between duplicate tests performed on the Celloscope 401 and the Coulter S.The instrument was capable of satisfactory precision and accuracy in platelet counting, provided that the sedimentation method was used to obtain a platelet-rich plasma. The best results were obtained if a two-step dilution was carried out with a first dilution in 10% EDTA and a second in 2.5 mM cocaine in water. Using this method the precision study indicated a coefficient of variation of 3.11%. Close agreement was obtained between platelet counts on the Celloscope 401 when compared with the results obtained either by phase-contrast microscopy or using another electronic counter.Allowing for predilution and duplicate counts on each sample, the rate of throughput was approximately 32 samples per hour.Throughout the test period, the instrument remained electronically and mechanically stable.     

Further methodological problems in cell counting

Although Coleman and Flood discuss a number of important methodological issues in the “Interpretive Cautions” section, several additional points may also deserve consideration.     

A parallel comparison of three automated multichannel blood cell counting systems for analysis of blood of laboratory animals

A parallel evaluation was performed on three automated haematology analysers, the Sysmex K-4500 and Coulter Counter S-PLUS STKR impedance analysers, and the Techinicon H*1E flow cytometry analyser. The same blood samples from animals from three different species were analysed on the same day. An analytical -comparison of haemograms from healthy monkeys, dogs, rats and phlebotomised rats was made from paired blood samples anticoagulated with dipotassium ethylenediaminetetra-acetate. Each instrument was calibrated with commercially available material based on human blood products. The precision of each system was good, but whereas the Coulter Counter S-PLUS STKR had better precision for white blood cell counts, platelet counts showed greater variability. When compared to spun packed cell volume (PCV), or to each of the other instruments, the Coulter system consistently gave lower haematocrit values. The magnitude of relative bias of spun PCV values was about −7% in monkey, −5% in dogs and −10% in rats. It was deemed necessary with this instrument to make adjustments to the calibration, especially for rats. The Coulter also gave consistently higher white blood cells (WBC) counts in monkeys, and lower platelet counts in rats compared to the other two instruments. The biases may be due to inherent physical differences between the analytical methods and/or the calibration techniques. With few exceptions, each instrument provides reliable results for all major animal species encountered in routine experimental and toxicological haematology.     

A new assay for leukocyte chemotaxis using cell retrieval,electronic particle counting and flow cytometry

A new micropore membrane assay for leukocyte migration has been devised. It permits the complete retrieval in monodisperse suspension of functionally intact cells that have traversed the membrane, thus allowing the application of precise, automated techniques, including flow cytometry and electronic particle counting. Hemocytometers may also be used. Direct comparison with 2 different conventional membrane methods showed that the new method performed superiorly. It was also much more economical with regard to time and labor. This technique permitted detection of functional differences between leukocytes isolated from blood in different ways. Data on the duration of concentration gradients in chemotaxis chambers are also presented.     

The impact of a closed-loop electronic blood transfusion system on transfusion errors and staff time in a children's hospital

ObjectivesTo assess the impact of a closed-loop electronic blood transfusion system on transfusion errors and staff time.Materials and methodsBefore and after study in all wards of a children's hospital, involving patients and staff of all the wards. The changes were closed-loop electronic blood transfusion, barcode patient identification, electronic blood transfusion administration records and error pop-up warning. The main outcome measures were percentage of blood transfusion errors, time spent on transfusion tasks.ResultsTransfusion errors were identified in 3.87% of 2556 blood transfusion orders pre-intervention and 0.78% of 2577 orders afterwards (P < 0.01). Phlebotomists, nurses, and physicians may make mistakes, including wrong blood type when apply for blood, wrong patient when blood draw or transfusion, wrong dose when apply for blood and the wrong tube label when blood draw or cross-matching, which are significantly reduced after change (1.09% vs 0.31%, 1.13% vs 0%, 0.31% vs 0%, 1.33% vs.0.78%, P < 0.01). Time spent on blood apply was 5.3 ± 1.2 min, hand over blood bag at the transfusion department was 14.9 ± 1.4 min and blood transfusion was 15.8 ± 2.4 min. Time per transfusion round decreased to 2.6 ± 1.0 min, 6.3 ± 1.6 min and 9.3 ± 2.2 min respectively (P < 0.01).ConclusionsA closed-loop electronic blood transfusion, barcode patient identification and error pop-up warning reduced transfusion errors, and increased confirmation of patient and blood types identity before transfusion. Time spent on blood transfusion tasks reduced.     

Bias in counting procedures for white blood cells

Summary To increase the precision of low white blood counts with traditional counting procedures often an initial count is performed in 0.1 cu.mm. Only if this pilot count is low, additional zones of 0.1 cu.mm. are counted and the results of the pilot count and the additional count then averaged. This counting procedure should be avoided as it gives biased estimates of the white blood count. The bias is avoided if the result of the initial count is discarded and an independent count is performed in a larger volume.In analogous counting situations — such as radioactivity counts — bias can arise and be prevented in analogous ways.     

Electrical sizing and counting of platelets in whole blood

Hydrodynamic focusing with a jet close to the orifice of a Coulter counter is used as a detector for particle sizing, with a substantial increase in resolution and size range. To maintain these improvements during analogue-digital conversion, zero crossover triggering for sample and hold and a pulse-shape dependent inhibition before address transfer to the memory of a multichannel pulse-height analyser have been performed. Results are given showing size distributions of polystyrene latex particles, fixed red cells and living red cells with platelets from whole blood.