Comparison of performance for leukocyte differential counting of the Technicon H6000 system with a manual reference method using the NCCLS standard

The National Committee for Clinical Laboratory Standards (NCCLS) has published a tentative standard for leukocyte differential counting, by means of which a manual or automated method for leukocyte differential counting can be compared with a manual reference method. The performance of the Technicon H6000 system was evaluated using the standard at Stamford and Overlook Hospitals. A total of 502 patient samples were analyzed: 315 from Overlook and 187 from Stamford. The H6000 system was found to be approximately four times more precise than the 200-cell manual reference method for each cell type. Correlation of the H6000 system with the manual method was good, with correlation coefficients of 0.98 for neutrophils and lymphocytes, 0.96 for eosinophils, 0.72 for monocytes, and 0.5 for basophils. The clinical sensitivity of the H6000 system, measured in terms of false normals and false abnormals, was similar to that of the manual reference method when measured against itself. There were no clinically significant discrepancies in results from the H6000 system, except for possibly one case where a patient was already on antibiotic therapy. The NCCLS standard was found to be a useful but rather complex and involved method for evaluating the performance of the H6000 system, the major problem being the amount of work needed to count manually the number of cells required for the manual reference method.     

The Coulter Counter leukocyte differential

The leukocyte differential counting capability of the Coulter Counter model S-PLUS IV was evaluated for precision, accuracy, and clinical sensitivity. The evaluation used procedures described in the NCCLS tentative standard (H20-T) when these were appropriate for this technology. Within its inherent constraints, the system was found to be more reproducible than the stained-film reference method and at least as accurate. Clinical sensitivity was equivalent to that of a four-slide, microscope differential count. Reference ranges for the cell classes identified by the instrument were similar to those given by the stained-film method.     

Comparison of Venous and Capillary Differential Leukocyte Counts Using a Standard Hematology Analyzer and a Novel Microfluidic Impedance Cytometer

Capillary blood sampling has been identified as a potentially suitable technique for use in diagnostic testing of the full blood count (FBC) at the point-of-care (POC), for which a recent need has been highlighted. In this study we assess the accuracy of capillary blood counts and evaluate the potential of a miniaturized cytometer developed for POC testing. Differential leukocyte counts in the normal clinical range from fingerprick (capillary) and venous blood samples were measured and compared using a standard hematology analyzer. The accuracy of our novel microfluidic impedance cytometer (MIC) was then tested by comparing same-site measurements to those obtained with the standard analyzer. The concordance between measurements of fingerprick and venous blood samples using the standard hematology analyzer was high, with no clinically relevant differences observed between the mean differential leukocyte counts. Concordance data between the MIC and the standard analyzer on same-site measurements presented significantly lower leukocyte counts determined by the MIC. This systematic undercount was consistent across the measured (normal) concentration range, suggesting that an internal correction factor could be applied. Differential leukocyte counts obtained from fingerprick samples accurately reflect those from venous blood, which confirms the potential of capillary blood sampling for POC testing of the FBC. Furthermore, the MIC device demonstrated here presents a realistic technology for the future development of FBC and related tests for use at the site of patient care.     

The routine differential leukocyte count vs automated differential counts

The clinical use of the proposed performance standards for differential leukocyte counts is determined by multiple factors. Their use must be considered according to the specific use of the differential count, the sources of variability in differential counting, the relation between specific use and sources of variability, the role of analytic errors in the detection of nonspecific changes, the use of qualitative vs quantitative data, the sensitivity and specificity of the routine eye count, the role of disease or specific cell prevalence in determination of predictive value, the effect on use of automated instruments for screening, and whether abnormal specimen flagging can be done. The routine eye-count differential method, as performed by a well-trained technologist or technician, seems to lack both sensitivity and specificity. Because of the magnitude of technique- and method-related and biologic sources of variability, the 100- or 200-cell eye-count differential method is not a good screening method for detection of hematologic illnesses, particularly those that are uncommon. The automated differential leukocyte instruments address many of the technique- and method-related errors and are thus able to equal or exceed the performance of the routine eye-count differential method.     

Soy isoflavones prevent the ovarian hormone deficiency-associated rise in leukocytes in rats.

Recent reports indicate that ovariectomy (ovx) increases lymphopoiesis. Ipriflavone, a synthetic isoflavone, has been reported to reduce lymphocytes in postmenopausal women. The aim of this study was to investigate whether naturally occurring isoflavones also affect lymphopoiesis in ovarian hormone deficiency. The present study was carried out using an ovariectomized (ovx) rat model. To mimic early menopause, forty-eight 12-month-old Sprague-Dawley rats were either sham-operated (sham; 1 group) or ovx (3 groups) and were fed a standard semi-purified diet for 120 days. Thereafter, the ovx groups received one of the three doses of isoflavones: 0 (ovx), 500 (ISO500), or 1000 (ISO1000) mg/kg diet for 100 days. Ovariectomy increased total leukocyte counts significantly (p < 0.05) as a result of increased (p < 0.05) lymphocyte, monocyte, eosinophil, and basophil differential counts. Isoflavones at 500 and 1000 mg/kg diet returned the total leukocyte counts, as well as leukocyte subpopulations, to levels comparable to that of sham-operated rats. No other hematological parameters, e.g., red blood cell counts or red cell indices, were affected by ovariectomy or isoflavones. We conclude that soy isoflavones restore normal leukocyte counts elevated in ovarian hormone deficiency.     

Leukocyte isolation by sedimentation: the effect of rouleau-promoting agents on leukocyte differential count.

The rouleau-promoting agents dextran and polyvinylpyrrolidone (PVP) were used to accelerate erythrocyte sedimentation in order to harvest the leukocyte rich plasma (LRP). The objective of the work was to determine if agent concentration or blood: agent ratio had any effect on the leukocyte differential count and if so at what agent concentration and agent:blood ratio did the LRP leukocyte differential count most closely match the whole blood leukocyte differential count. With both sedimentation agents the data clearly indicate that both parameters effect LRP differential counts and that low concentrations of sedimentation agents are most important in obtaining LRP differential counts which most closely match the whole blood differential counts.     

Use of a fluid dispensing apparatus for bronchoalveolar lavage in mice

The volume of the mouse lung is small, so bronchoalveolar lavage (BAL) in mice is generally performed with 1 ml syringes to infuse smaller volumes of fluid. Multiple infusions are required to obtain enough recovered fluid for multiple analyses. This paper introduces the use of one type of a simple fluid dispensing apparatus as an infusion device. It proved to be a faster and a less tedious method than the syringe infusion method. The results of studies in normal mice using both infusion techniques showed no differences between the two with respect to the recovery of cells and protein and to differential leukocyte counts. Thus, the results obtained with this device can be compared with those previously obtained with syringes.     

Performance of the LARC classifier in clinical laboratories.

As part of the installation procedure of the LARC leukocyte differential classifier in a clinical laboratory, a 100-slide protocol is carried out to establish the performance of the classifier in the laboratory. The detailed make-up of this protocol and its relationship to key performance parameters for the leukocyte differential are described in detail. Data from the first ten of these protocols are presented which establish the (a) normal ranges, (b) reproducibility, (c) accuracy, (d) false-positive/false-negative rates for the detection of left shifts and (e) false-positive/false-negative rates for the detection of bloods with abnormal cells.     

Determination of bronchoalveolar lavage leukocyte populations by flow cytometry in patients investigated for respiratory disease

BACKGROUND: Characteristic changes in the proportions of leukocyte populations in bronchoalveolar lavage (BAL) reflect different disease states in the lung. The standard method for examination of BAL leukocytes is by microscopy of cytospin preparations. This method may not be the optimum technique due to difficulties in distinguishing cell types morphologically and due to the low number of cells routinely counted. We hypothesized that flow cytometry (FCM) may be a more precise tool for investigating BAL. METHODS: 100 BALs were performed on 92 patients. All samples were stained using the pan-leukocyte marker (CD45) in combination with a granulocyte marker (CD15) and a cell viability marker (7-aminoactinomycin D). Selected samples were also stained with an eosinophil marker (CD23). These samples were run on an FCM and the results compared with leukocyte differentials obtained by light microscopy of parallel cytospin preparations. RESULTS: Close correlations between the two methods were demonstrated for the enumeration of all leukocyte subsets, but the coefficient of variation was considerably lower by FCM than by cytospin. CONCLUSIONS: These findings, combined with the speed of FCM and the ability to perform simple lymphocyte phenotyping, argue in favor of this becoming the method of choice for investigating BAL.     

Observations on the Technique for the Study of Human Chromosomes-by the Culture of Leukocytes from Peripheral Blood

A critical analysis of the various features of blood leukocyte culture for the study of human chromosomes was carried out. The following observations were made: (1) Fasting blood was essential for effective separation of leukocytes. (2) These cells are easily obtained by differential centrifugation and RBC sedimentation. (3) Non-specific agglutination of leukocytes was prevented by the use of Eagle''s medium for suspension cultures. (4) No contamination occurred in a series of 50 leukocyte cultures to which antibiotics were not added. (5) Addition of an experimental Phaseolus vulgaris extract at concentration of 1 × 10^?4 to cultures resulted in a 12 to 15% mitotic index. (6) Desacetyl-methyl-colchicine (Colcemid) had optimal effect at concentration of 0.1 μg./ml. (7) Distilled water added to cell suspension in culture medium (5: 1) was an effective hypotonic agent.A simplified technique of leukocyte culture for chromosome preparations is proposed.     

A critical evaluation of the manual/visual differential leukocyte counting method

The performance of differential leukocyte counting by 73 technologists and technicians working in 5 different laboratories in a large medical center was evaluated. Good correlation with the reference method was found for neutrophils, normal lymphocytes, and eosinophils. More variability was noted in the estimation of stab neutrophils and variant (atypical) lymphocytes and monocytes. The sensitivity of this method for clinically important conditions ranged from 100% to 34%, depending upon the abnormality.     

Hematologic and serum biochemical values for Yucatan miniature swine

Hematologic and serum biochemical values were determined for healthy, mature Yucatan miniature swine, Sus scrofa. These values were similar to those reported for other breeds of swine. There was no effect on erythrocyte count, hematocrit, MCV, MCH, MCHC, RDW, platelet count, or leukocyte count attributable to sex (p greater than 0.05). Differential leukocyte counts generated on an automated multichannel blood cell counter, having a three part leukocyte differential capability, were compared to 100-cell manual leukocyte differentials. Determination of lymphocyte and non-lymphocyte fractions on this system were not significantly different from microscopic differentials (p less than 0.001). However, the mononuclear cell count did not correlate well with the percentage of monocytes determined manually (r = 0.084, p greater than 0.5). Leukocytes, erythrocytes, and platelets behaved properly with respect to counting thresholds as modified for counting cells of other common domestic species on this automated cell counter.     

Leukocyte depletion for safe blood transfusion

Leukocytes have ability to distinguish between self cells (body own cells) and foreign (allogenic) cells on the basis of human leukocyte antigen (HLA) proteins that are present on the cell membrane and are effectively unique to a person. During allogenic blood transfusion a person receives large number of allogenic donor leukocytes and these are recognized as foreign cells by the recipient immune system which leads to several adverse reactions. To avoid such leukocyte-mediated adverse reactions leukodepleted blood transfusion is required. Leukocytes can be separated on the basis of size, dielectric properties, by affinity separation, freeze-thawing and centrifugation but all these methods are time consuming and costly. Filtration is another method for leukocyte depletion that is comparatively less expensive and more efficient as it gives more than 90% leukodepletion of blood along with minimal cell loss. However, present filtration procedures also have some limitations as they work efficiently with blood components but not with whole blood and show non-specific adhesion of large number of platelets and red blood cells along with leukocytes. All the currently available filters are costly, which has been a major reason for their limited application. Therefore, demand for a more efficient and cost-effective filter is high in medical community and scientists are attenpting to improve the efficiency of currently available filters. The present review gives an overview of the significance of leukodepleted blood transfusion and focuses on different methods for leukocyte depletion and challenges involved in all these technologies.     

LeukocyteMask: An automated localization and segmentation method for leukocyte in blood smear images using deep neural networks

Digital pathology and microscope image analysis is widely used in comprehensive studies of cell morphology. Identification and analysis of leukocytes in blood smear images, acquired from bright field microscope, are vital for diagnosing many diseases such as hepatitis, leukaemia and acquired immune deficiency syndrome (AIDS). The major challenge for robust and accurate identification and segmentation of leukocyte in blood smear images lays in the large variations of cell appearance such as size, colour and shape of cells, the adhesion between leukocytes (white blood cells, WBCs) and erythrocytes (red blood cells, RBCs), and the emergence of substantial dyeing impurities in blood smear images. In this paper, an end‐to‐end leukocyte localization and segmentation method is proposed, named LeukocyteMask, in which pixel‐level prior information is utilized for supervisor training of a deep convolutional neural network, which is then employed to locate the region of interests (ROI) of leukocyte, and finally segmentation mask of leukocyte is obtained based on the extracted ROI by forward propagation of the network. Experimental results validate the effectiveness of the propose method and both the quantitative and qualitative comparisons with existing methods indicate that LeukocyteMask achieves a state‐of‐the‐art performance for the segmentation of leukocyte in terms of robustness and accuracy .     

A mathematical model for the leukocyte filtration process

Leukocyte filters are applied clinically to remove leukocytes from blood. In order to optimize leukocyte filters, a mathematical model to describe the leukocyte filtration process was developed by modification of a general theoretical model for depth filtration. The model presented here can be used to predict the time-dependent leukocyte filtration as a function of cell-cell interaction in the filter, filter efficiency, filter capacity, filter dimensions, and leukocyte concentration in the suspension applied to the filter. The results of different leukocyte filtration experiments previously reported in the literature could be well described by the present model. (c) 1995 John Wiley & Sons, Inc.     

A Single 9-Colour Flow Cytometric Method to Characterise Major Leukocyte Populations in the Rat: Validation in a Model of LPS-Induced Pulmonary Inflammation

The rat is a commonly used model for immunological investigation. Yet basic research and characterisation of leukocyte populations and sub-sets lags far behind murine research, with inconsistency on reported leukocyte markers and their overlap. These shortcomings limit the opportunity for more complex and advanced rat immunology research. In this study, we developed a robust 9-colour flow-cytometric protocol to elucidate the major blood and tissue rat leukocyte populations, and validated it in a model of LPS-induced pulmonary inflammation. Blood and tissues (lung, BALF, spleen, liver, bone marrow) from naïve Sprague-Dawley rats were collected and analysed by flow cytometry (FCM). Rats were exposed to aerosolised saline or LPS (1mg/mL), at 3 and 24hrs thereafter blood, lung and BALF were collected and analysed using FCM and ELISA. Neutrophils, two monocyte subsets, NK Cells, B Cells, CD4+, CD8+ T Cells and alveolar macrophages can be identified simultaneously across different tissues using a 9-colour panel. Neutrophils and monocytes can be distinguished based upon differential expression of CD43 and His48. Neutrophils and CD43Lo/His48Hi monocyte-macrophages are elevated in the lung at 3 and 24hrs during LPS-induced pulmonary inflammation. This validated method for leukocyte enumeration will offer a platform for greater consistency in future rat immunology and inflammation research.     

Testing the Hardy-Weinberg equilibrium in the HLA system

A new method of testing the Hardy-Weinberg equilibrium in the human leukocyte antigen (HLA) system is proposed and applied to real data. The derivation is based on the maximum likelihood method and closely related to standard regression theory. The test statistic has a closed representation of residual sum of squares by a projection mapping of data onto the estimated regression plane. Under the Hardy-Weinberg law the noniterative estimates for the gene frequencies are suggested by the use of the projection mapping. The test statistic and gene frequency estimates are shown to be asymptotically equivalent to the maximum likelihood method and to be more efficient than the other suggested test statistic when there are more than two identified alleles.     

Leukocyte Populations in Human Preterm and Term Breast Milk Identified by Multicolour Flow Cytometry

Background

Extremely preterm infants are highly susceptible to bacterial infections but breast milk provides some protection. It is unknown if leukocyte numbers and subsets in milk differ between term and preterm breast milk. This study serially characterised leukocyte populations in breast milk of mothers of preterm and term infants using multicolour flow cytometry methods for extended differential leukocyte counts in blood.

Methods

Sixty mothers of extremely preterm (<28 weeks gestational age), very preterm (28–31 wk), and moderately preterm (32–36 wk), as well as term (37–41 wk) infants were recruited. Colostrum (d2–5), transitional (d8–12) and mature milk (d26–30) samples were collected, cells isolated, and leukocyte subsets analysed using flow cytometry.

Results

The major CD45+ leukocyte populations circulating in blood were also detectable in breast milk but at different frequencies. Progression of lactation was associated with decreasing CD45+ leukocyte concentration, as well as increases in the relative frequencies of neutrophils and immature granulocytes, and decreases in the relative frequencies of eosinophils, myeloid and B cell precursors, and CD16- monocytes. No differences were observed between preterm and term breast milk in leukocyte concentration, though minor differences between preterm groups in some leukocyte frequencies were observed.

Conclusions

Flow cytometry is a useful tool to identify and quantify leukocyte subsets in breast milk. The stage of lactation is associated with major changes in milk leukocyte composition in this population. Fresh preterm breast milk is not deficient in leukocytes, but shorter gestation may be associated with minor differences in leukocyte subset frequencies in preterm compared to term breast milk.     

The reaction of leukocytes of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> to hormone-induced stress

The influence of cortisone on the part of leukocytes in the differential leukocyte count of peripheral blood of sterlet is described. The changes in particular groups of leukocytes are characterized quantitatively. To the hormone, the fish reacted by a decrease in leukocyte count, blastic (juvenile) cells, and eosinophils and an increase in neutrophils and monocytes. It is concluded that the stress hormone suppresses lymphopoiesis by the hormone-induced apoptosis of lymphocytes while, on the contrary, myelopoiesis activates it.