WBC Counting Research Articles

Accuracy of Haematology Analyzer in the Diagnosis of Malaria in Comparison with Gold Standard Microscopy

Objective: To assess the accuracy of Haematology analyzer in the diagnosis of malaria in comparison with gold standard microscopy.
 Methodology: A cross-sectional study was conducted at the Pakistan Institute of Medical Sciences and the Pakistan Atomic Energy Commission in Islamabad, Pakistan. The study was conducted for around six months, from May 2023 to October 2023. The Mindray BC-6200 haematology analyzer was utilised to evaluate a total of 191 samples, comprising 127 samples from that were infected with malaria and 64 samples from healthy controls. When the presence of malaria parasites, identified as Plasmodium vivax and Plasmodium falciparum, was detected in dyed thick blood film, a microscopy examination was carried out as a reference. Analyse-it v4.92.3 was used to create the receiver operating characteristic (ROC) curve analysis. The agreement between BC-6200 and light microscopy was assessed using the Kappa value.
 Results: The InR by BC-6200's sensitivity and specificity for P. falciparum and P. vivax infections. The sensitivity of the InR by BC-600 for P. falciparum and P. vivax was 27.9% and 85.5%, respectively. The specificity of the InR by BC-6200 for P. vivax and P. falciparum was 82.7% and 86.5%, respectively. The infection densities in microscopy varied statistically significantly between the various InR groups (χ2 = 14.50, P < 0.005).An analysis was conducted on the correlation between the cell blood count and the count of InR in both the P. vivax and P. falciparum-infected patient groups. The results showed a clear correlation between InR (P. vivax group R2 = 0.87) and ΔWBC (WBCDIFF–WBCBASO). In the Mindray BC-6200 haematology analyzer, WBCBASO represents the number of WBC counting in the BASO channel with severe membrane degradation, while WBCDIFF represents the number of WBC counting in the DIFF channel with mild lyse. The volume distribution widths of RBC, HGB, and red blood cells did not differ substantially. The reticulocyte characteristics of the P. vivax/P. falciparum patient group and control group differed significantly (P<0.02), although RBC, HGB, and red blood cell volume distribution width (RDW) did not differ significantly (P > 0.05). There was a significant difference (P<0.02) in the PLT count between the P. vivax/P. falciparum patient groups and the control group.
 Conclusion: The results imply that malaria might be screened for in a clinical setting using the BC-6200 haematology analyzer's "InR" flag and "InR#/InR‰" parameters.

ACCURACY OF AUTOMATED CELL ENUMERATION METHOD FOR VARYING CONCENTRATION OF WBCS FOR VARIOUS BODY FLUID SAMPLES

Background: Body fluids (BF) including peritoneal, pericardial, pleural, synovial and cerebrospinal fluids are now being analyzed by fully automated methods that are replacing the manual methods. The aim of this study is to assess the accuracy of automated instrument at varying concentrations of WBCs of various body fluid samples. Material and Methods: This cross-sectional study was conducted at the section of Hematology, Department of Pathology and Laboratory Medicine, the Aga Khan University, Karachi, Pakistan from November 2020 to April 2021. Forty body fluid samples with suspicion of infection including peritoneal, pericardial, pleural, synovial and cerebrospinal fluids (CSF) were analyzed to verify accuracy of white blood cells counts on fully automated XN-1000TM hematology analyzer (Sysmex Corporation, Kobe, Japan) against Neubauer chamber method (a manual method of differentiating WBCs via microscopy). The culture results of the body fluid samples were also analyzed. EP Evaluator version 10.3.0.556 (Data Innovations, LLC, VT, US) was used for statistical analysis with other supporting statistics such as Correlation Coefficient (R), Bias, Mean and Standard Deviation were included. Results: Eleven (n=11) of forty (n=40) body fluid samples showed high WBC count above the normal range. Samples with normal WBC (n=29) were also included in accuracy study to assess instrument performance at varying concentration of analyte. An average Error Index of -0.27 (-0.99 to 0.74) for WBC was obtained. Microbiological cultures grew Escherichia. Coli in 1 and Acinetobacter species in 1 CSF samples and Staphylococcus Aureus in 1 pleural sample. Conclusion: The study verified accuracy of varying concentration of WBC counting by fully automated Sysmex XN-1000 analyzer for various body fluid samples. Keywords: Body fluids, Validation, Automated analyzer, Performance specifications, Accuracy verifications, High white blood count, Infectious.

Оценка эффективности и безопасности препарата Аденопросин® у пациентов с хроническим простатитом IIIA категории.

Introduction. Chronic prostatitis (СР) is an actual problem of modern urology as it leads to significant deterioration of life quality of able-bodied male population. Frequent relapses of the disease in spite of timely prescribed therapy leads to the necessity to search for alternative drugs. Objective of the study. To evaluate efficacy and safety of Adenoprosin® in patients with CP/chronic pelvic pain syndrome (CРРS) of IIIA category according to USA National Health Institute classification. Materials and methods. The prospective observational study of efficacy and safety of Adenoprosin® was carried out from 10.2021 till 10.2022 on the basis of the Center of Urology and Andrology of the State Research Center named after Burnazyan, Federal Medical and Biological Center of the Russian Federation. Men aged 35 to 60 with diagnosed CKD (IIIA according to the US National Institutes of Health classification) took part in the study. Patients were divided into 2 comparable groups. The control group was treated with an alfa1-adrenoblocker (Tamsulosin 0.4 mg 1 once daily), antibacterial agent (Levofloxacin 500 mg 1 once daily) for 30 days. In the main group, in addition to the standard therapy, Adenoprosin® 150 mg, one suppository a day was administered rectally for 30 days. The study requirements were included 4 Visits: Visit 0-1 – the baseline visit; Visit 2 on Day 14 from the therapy start; Visit 3 on Day 30 from the therapy start; Visit 4 on Day 90 from the therapy start. Clinical monitoring was performed with standardized questionnaires «International system of summarizing prostate disease symptoms» (IPSS), «Chronic prostatitis symptoms index (NIH-CPSI)», WBC counting in prostate secretion. Results. Statistical analysis of NIH-CPSI, IPSS questionnaires, as well as WBC count in the prostate secretion before and after the beginning of treatment, showed that the therapy was significantly effective in both groups (p <0,05). In the main group (Adenoprosin® group) the results were statistically better (p<0,05) if all questionnaires and WBC count in the prostate secretion were evaluated after 90 days from the therapy start as compared to the control group. Conclusions. The results of investigation showed that Adenoprosin® preparation is effective and safe as a part of combination therapy of CP/CPTB and leads to significant improvement of urination and pain syndrome relief according to NIH-CPSI, IPSS questionnaires and decrease of WBC count in prostate secretion.

Geometric Feature Extraction for Identification and Classification of Overlapping Cells for Leukaemia

This paper describes the study of overlapping leukaemia cells based on geometric features for identification and classification. Geometric features of blood cells are proposed to identify and classify overlapping cells into groups based on different overlapping degrees and the number of overlapped cells. In the proposed method, the percentage of average accuracy for identifying overlapping cells reached 98 percent. The proposed method can segment white blood cells from the overlapping cells with an overlapping degree of 70 percent. Improved Watershed Algorithm successfully increased 36.89 percent of accuracy in WBC segmentation. It reduced 46.12 percent of the over-segmentation problem. Tests of cell counting are conducted in the two stages, which are before and after the process of identification and classification of overlapping cells. The average percentage of total cell count is 83.31 percent, the average percentage of WBC counting is 84.8 percent, and the average percentage of RBC counting is 60.55 percent. The proposed method is efficient in identifying and classifying overlapping cells for increasing the accuracy of cell counting.

WBC-Net: A white blood cell segmentation network based on UNet++ and ResNet

The counting and identification of white blood cells (WBCs, i.e., leukocytes) in blood smear images play a crucial role in the diagnosis of certain diseases, including leukemia, infections, and COVID-19 (corona virus disease 2019). WBC image segmentation lays a firm foundation for automatic WBC counting and identification. However, automated WBC image segmentation is challenging due to factors such as background complexity and variations in appearance caused by histological staining conditions. To improve WBC image segmentation accuracy, we propose a deep learning network called WBC-Net, which is based on UNet++ and ResNet. Specifically, WBC-Net designs a context-aware feature encoder with residual blocks to extract multi-scale features, and introduces mixed skip pathways on dense convolutional blocks to obtain and fuse image features at different scales. Moreover, WBC-Net uses a decoder incorporating convolution and deconvolution to refine the WBC segmentation mask. Furthermore, WBC-Net defines a loss function based on cross-entropy and the Tversky index to train the network. Experiments on four image datasets show that the proposed WBC-Net achieves better WBC segmentation performance than several state-of-the-art methods.

The results of external quality assessment programme on urine leukocyte and erythrocyte counting in Poland.

IntroductionUrine particle analysis is an important diagnostic tool. The aim of this study was to evaluate the quality of urine leukocyte (WBC) and erythrocyte (RBC) counting results obtained with manual and automated methods in Polish laboratories participating in the international external quality assessment (EQA) programme.Materials and methods1400 WBC and RBC counting results were obtained from 183 laboratories in EQA surveys organised by Labquality (Helsinki, Finland) from 2017 to 2019. The between-laboratory coefficient of variation (CV), the percentage difference between the laboratories' results and target values (Q-score (%)), as well as modified Youden plots were analysed.ResultsFor automated method groups, the medians of inter-laboratory CVs varied from 14% to 33% for WBC counting and from 10% to 39% for RBC counting. For manual method groups, the medians of CV varied from 53% to 71% (WBC) and from 55% to 70% (RBC), and they were significantly higher, in comparison to CVs for most automated method groups (P < 0.001). The highest percentage of results outside the target limits (36%) and the highest range of Q-score (%) (from - 93% to 706%) were observed for laboratories which participated in the surveys for the first or second time. The percentage of deviating results and the ranges of Q-score decreased with an increased frequency of laboratories’ participation in the surveys.ConclusionsThe quality of manual methods of urine WBC and RBC counting is unsatisfactory. There is an urgent need to take actions to improve laboratories’ performance and to increase harmonisation of the results.

White blood cell counting at point-of-care testing: A review.

White blood cells, which are also called leukocytes, are found in the immune system that are involved in protecting the body against infections and foreign invaders. Conventional methods of leukocyte analysis provide valuable and accurate information to medical specialists. Analyzing and diagnosing of a disease requires a combination of multiple biomarkers, in some cases, however, such as personal health care, this will occupy some medical resources and causes unnecessary consumption. Traditional method (such as flow cytometer) for WBC counting is time and labor consuming. Compared to gold standard (flow-based fraction/micropore filtration) or improved filtration methods for WBC counting, this is still a lengthy and time consuming process and can lead to membrane fouling due to the rapid accumulation of biological materials. Therefore, the analysis of WBC counts requires more compact and efficient equipment. The microfluidic technologies, powered by different field (force, thermal, acoustic, optical, magnetic) and other methods for leukocyte counting and analysis, are much cost-efficient and can be used in in-home or in resource-limited areas to achieve Point-of-Care (POC). In this review, we highlight the mainstream devices that have been commercialized and extensively employed for patients for WBC counting, Next, we present some recent development with regards to leucocyte counting (mainly microfluidic technologies) and comment on their relative merits. We aim to focus and discuss the possibility of achieving POC and help researchers to tackle individual challenges accordingly. Finally, we offer some technologies in addition to previous detection devices, such as image recognition technology and cloud computing, which we believe have great potential to further promote real-time detection and improve medical diagnosis.

Development of Blood-Cell-Selective Fluorescent Biodots for Lysis-Free Leukocyte Imaging and Differential Counting in Whole Blood.

Complete blood count with leukocyte (white blood cell, WBC) differential is one of the most frequently ordered clinical test for disease diagnosis. Herein, multifunctional fluorescent carbon dots derived from biomolecules (biodots) for rapid lysis-free whole blood analysis are developed. Specifically, two types of biodots are molecularly engineered through hydrothermal synthesis for differential blood cells labeling. Type I biodots synthesized from amino acid (serine/threonine) precursors and passivated with polyethylenimine can label both red blood cells (RBCs) and WBCs with excellent contrast in fluorescence intensity, enabling direct counting of leukocytes in whole blood samples without a tedious RBC lysis step. It also allows three-part leukocyte differential counting by flow cytometry without using expensive fluorophore-conjugated antibodies. On the other hand, Type II biodots synthesized from the same amino acid precursors but passivated with a biopolymer (chitosan) are able to selectively lyse RBCs with greater than 98% efficiency to allow simultaneous fluorescent labeling of leukocytes for WBC counting in whole blood. It is envisioned that these novel nanoreagents, which eliminate the cumbersome lysis and antibody conjugation steps for selective labeling of different blood cells, would revolutionize disease diagnostics toward achieving faster, cheaper, and more accurate whole blood analyses in one test.

Monitoring internal contamination from OEW of an 18F-FDG production plant: WBC or head counting setup?

The production 18F-FDG for positron emission tomography (PET) has consistently increased over the past two decades. The risk of internal contamination at 18F-FDG production facilities exists. A setup for evaluation of the 18F-FDG activity incorporated into the OEW brain, called Head Counting System (HCS), was presented in previous works. In this study, the whole body counter setup (WBC) was evaluated for monitoring 18F incorporations. The Monte Carlo Virtual Software (VMC in-vivo) and the MCNPx code were used to assess the system calibration coefficient (CC). Three 18F distributions were simulated: i) uniformly distributed in soft tissue (UDST); ii) Na18F biodistribution (NAFB); and iii) 18F-FDG biodistribution (FDGB). The calibration coefficient of WBC was compared to the current head counting system CC under the same biodistribution conditions. The ICRP male reference voxelized phantoms (RCP_AM) was used in the simulations. The results showed that the WBC setup was more efficient than the head counter for all the studied 18F distributions: UDST = 1060 %, FDGB = 488 % and NAFB = 340 %. Despite this, especially for 18F-FDG, the possibility of bladder voiding before measurement can lead to considerable uncertainties when the WBC setup is used. On the other hand, bladder activity does not show great influence the calibration coefficient of the head counting system. Future work will evaluate the WBC sources of uncertainties in the measurement of 18F incorporated activity.

Detection of BCR-ABL gene in Chronic Myeloid Leukemia patients treated with Tyrosine Kinase Inhibitors Drugs by Gene Expert System

Chronic myeloid leukemia (CML) is one of human malignancies caused by genetic mutation and chromosomal translocation, a BCR-ABL fusion gene and as result Philadelphia chromosome is formed. The irregular tyrosine kinase activity of encoded protein by this gene causes the establishment of the disease. Nilotinib are potent and well inhibitor for BCR-ABL tyrosine kinase. This study was conducted at the period from September 2016 to February 2017,100 Iraqi CML patients were divided into two groups, first group of 50 patients were received Imatinib 400-800 mg/day, second group of another 50 patients were received 800 mg/day Nilotinib, WBC were microscopically counted using improved Neubauer ruled hemocytometer counting chamber.BCR-ABL gene RNA transcript and endogenous control (house keeping gene ) RNA transcript were extracted and purified and then reverse transcripted to cDNA after that the product was amplified and quantified by q RT-PCR.&#x0D; The results first group patients distribution according to the gender were 56% and 44% for males and females respectively while the mean age of the patients was 45.82 ± 16.17,the result of WBC counting of this group in regard to disease duration showed that the highest value was observed in newly diagnosed and advanced stage 98.28 ± 89.28,77.11± 2.98 respectively.The WBC count return to normal level after the period of treatment with Imatinib with significant reduction after one year at p≤ 0.0001.The results of molecular technique and BCR-ABL analysis in newly diagnosed, advanced stage and cytogenetic failure patients were 10.05 ± 4.7,3.03± 0.94 and 28.4±0.09 respectively with significant decrease after one year of treatment at p ≤ 0.002.On the other hand the results of the second group of CML patients in relation to the gender were 45% and 55% of males and femalesrespectively,while the mean age group was 36.68 ±13.51. The results of WBC count according to disease distribution in newly diagnosed and advanced stage were 87.5 ± 8.71 and43±21.72 respectively. WBC count was return to normal level after one year of treatment with Nilotinib with significant decrease at p≤0.0001. While the molecular technique and BCR-ABL analysis in newly diagnosed, advanced stage and cytogenetic failure group were 7.77±4, 1, 16.17 ± 3.78 and 2.02±0.53 after one year of treatment with Nilotinib with significant decrease at p≤ 0.0001. We conclude that treatment with Imatinib was found tough in a high extent of patients, Nilotinib is extending specific tyrosine kinaseinhibitor possess greater and selectively activity for BCR-ABL.

Clinical value of high expression level of CD71 in acute myeloid leukemia.

CD71 (transferrin receptor 1, TfR-1) is a type II membrane glycoprotein and associated closely with tumors. It was recognized as an indication for diagnosing acute erythroid leukemia (AEL). High expression level of CD71 has been identified as a negative prognostic marker for many solid tumors. However, whether CD71 should be identified as an adverse marker in acute myeloid leukemia (AML) remained conflicting. We studied 214 AML patients for analysis of clinical and laboratory data. Taking the CD71 expression level of 60% as a standard, we divided our patients into two groups. We discovered that AML with high expression level of CD71 was prone to linked with severe anemia (P=0.004), thrombocytopenia (P<0.001) and complex karyotype (P=0.024) and had increasing expression level of CD117 (P=0.001). No statistically significant correlations in age, gender, WBC counting, molecular markers between the two groups. And moreover, high expression level of CD71 did not alter the pattern of survival time.

Comparison of Some Hematological Parameters Between Horses in an Endurance Competition

This study conducted to comparison of some blood constituents between horses participated an endurance racing. Jugular vein puncture was done to collect blood from15 horses (1 gelding, 2 mares, and 12 male). These horses had been classified to three breeds include 12Kurd, 1 pure Arab, 2cross-breed and participated in the endurance racing of 0 (before competition), 15 (After first loop) and 30 km (final loop). Horses were classified in three age-groups include below 5(n=1), 5-10 (n=10) and above 10years of age (n=4). Hematocrit and Hemoglobin percentage, Total protein concentration, Red blood cell and White blood cell counting, glucose and cortisol level were measured at before starting competition (0), 15 and 30 km. Data was analyzed by SPSS (version 22). Results showed significant difference in glucose level after 30 km (P 0.05). No significant difference was observed between cortisol concentration and age groups (P>0.05) but, there was significant difference between cortisol concentration and gender (P 0.05). HT and WBC counting were higher in Arab, cross breed and Kurd respectively. Hemoglobin percentage was found higher in Arab, Kurd and Cross breed respectively. Total protein concentration in Arab was higher than Cross breed and Kurd. In conclusion, in short distance endurance competition of horses, stress is not effect on metabolism of animal, furthermore, gender and breed could important in blood parameters alterations.

The study of clinical applying continuous hemofiltration in children severe hand-foot-and-mouth disease with cardiopulmonary failure

Objective To analyze the clinical value of continuous veno-venous hemofiltration (CVVH) treatment in children with severe hand-foot-and-mouth disease (HFMD) complicated with cardio-pulmonary failure, via the prognostic comparison of the general comprehensive treatment and CVVH add-on treatment. Methods Fifty-one cases of severe HFMD with cardiopulmonary failure were divided into a CVVH group (n=19) and a control group (n=32) based on whether CVVH add-on or not. Their physiological and biochemical indicators were recorded and pediatric critical illness score (PCIS) and pediatric risk of mortality score (PRISM III) were calculated within 24 hours, when they were diagnosed with neurogenic pulmonary edema (NPE) /pulmonary hemorrhage. Both groups were treated with endotracheal intubation, mechanical ventilation with high PEEP, corticosteroids, ulinastatin, actively lowering the intracranial pressure, fluid resuscitation, milrinone, dopamine and other vasoactive drugs, high-dose intravenous gamma globulin, the CVVH group were added with CVVH treatment (duration>12 h). Prognosis difference of CVVH add-on treatment after diagnosed with NPE/pulmonary hemorrhage by tracking indicators of the third day. Survival analysis between two groups were compared by 3-day survival rates, 7-day survival rates, 28-day survival rates and the finally survival rates. Results (1) The overall conditions of two groups were comparable when diagnosed with NPE/pulmonary hemorrhage. PCIS, PRISM III, WBC counting, lactic acid, micro-blood sugar, myocardial enzymes and liver enzymes showed no significant difference between two groups. Three days after treatment, WBC and lactic acid decreased, but there was no significant difference (P>0.05), the remaining indicators had significantly improved in the CVVH group than those in the control group (P<0.05). (2) The 3-day survival rate, 7-day survival rate, 28-day survival rate and the finally survival rates in control group and CVVH group were 40.63 % vs.84.21%, 37.50% vs.73.68%, 25.00% vs.63.16%, 18.75% vs.52.63%, the survival rate in CVVH group were significantly higher (P<0.05). (3) The survival curve indicated that the survival time of CVVH group was significantly longer than that of the control group, the median survival time were 17 d and 2 d, respectively, and the difference was statistically significant (P<0.05). (4) In the CVVH group, 15 cases received CVVH after diagnosed with NPE/pulmonary hemorrhage within 12 hours, of which 10 cases (66.67%) ultimately survived, while the other 4 cases received CVVH after 12 h were all end to death, the difference was statistically significant (P<0.05). Further analysis of the impact of the timing of blood purification on the prognosis of children showed that the mortality rates of children received CVVH within 6 hours, 6 to 12 hours, after 12 hours of diagnosis of NPE/pulmonary hemorrhage, were 2/8, 3/7, 4/4, respectively. Conclusion Continuous hemofiltration can significantly improve the prognosis of children with severe HFMD, and may be preferable to perform in early stage. Key words: Continuous hemofiltration; Severe hand-foot-mouth disease; Cardiopulmonary failure; Children

Automated Evaluation of Synovial and Ascitic Fluids With the Advia 2120 Hematology Analyzer

The purpose of this study was to determine if the Advia 2120 hematology analyzer (Siemens Healthcare Diagnostics, Deerfield, IL) can accurately quantify both WBCs and the proportion of neutrophils from synovial and ascitic body fluids. We analyzed 60 samples on the Advia and compared the results with manual counts and smear reviews. We also assessed the effect of adding hyaluronidase to the samples. WBC counts and the proportion of neutrophils reported by the hematology analyzer were harmonized and highly correlated with manual counts and fluid smear reviews. The addition of hyaluronidase to the synovial fluid consistently increased the WBC counts on both manual and automated analysis (P < .001). We conclude that the Advia hematology analyzer can be used for WBC and neutrophil counting of cells in synovial and ascitic fluids. Hyaluronidase should be added before manual or automated counting of cells in synovial fluids.

A prospective randomized study on digestive tract cleaning before ERCP for the prevention of post- procedure cholangitis

Objective To evaluate the effectiveness of mouth care combined with intestinal and en- doscopic working channel washing for ERCP related cholangitis. Methods A total of 573 patients diagnosed as having obstructive jaundice were randomized into three groups, 190 cases in the control group, 192 in the saline group and 191 in the amikacin group. Clinical and laboratory data were collected before ERCP and ERCP related cholangitis were recorded. Results There were no significant differences among the three groups in sex, age, the level of obstruction, the category of obstruction, total bilirubin or WBC counting. The incidences of ERCP related cholangitis were 21.1% （40/190） , 13.5% （26/192） and 4. 7% （9/191） in control group, saline group and amikacin group respectively, which was significantly different （X2 = 22. 409, P = 0. 000 ）. The incidences of ERCP related cholangitis were 19.5% （65/333） and 4.2% （10/240） in patients diagnosed as having hilar duct obstruction and low positioned biliary obstruction re- spectively （X2 =27. 175, P =0. 000）. There was no significant difference in ERCP related cholangitis be- tween benign and malignant biliary obstruction. Subgroup of hilar duct obstruction showed the incidences of ERCP related cholangitis were 29. 7% （33/111 ）, 20. 5% （24/117） and 7.6% （8/105） in the control group, the saline group and the amikacin group, respectively （X2 = 16. 905, P = 0. 000）. Conclusion The inci- dence of ERCP related cholangitis is relatively higher in patients with hilar duct obstruction. Mouth carecombined with intestinal and endoscopic working channel washing can effectively reduce the incidence of ER- CP related cholangitis, especially in the amikacin group. Key words: Infection control; Cholangiapancreatography, endoscopic retrograde ; Biliary tract

Un nouvel automate de cytologie hématologique : l’Excell 2280

Excell 2280 analyser is a new automated haematology analyser manufactured by Drew Scientific Inc, Texas, USA, and distributed in France by MAXMAT S.A., Montpellier. It can achieve 80 complete blood cell counts per hour, with leukocyte differential counts. Three sampling possibilities are included: a direct one (open tubes, 180 microL), a blood saver one (80 microL) and an automatic, through-the-cap one (180 microL). The analytic principles are: electrical impedance for cell counting (WBC, RBC, platelets, MCV) and RBC/platelet sizing; and a new multidimensional optical system using a laser light scattering flow cytometer for WBC counting and classification. We evaluated the Excell 2280 in our laboratory: we quantified intra-run and within-run variations, correlations between the automatic and the direct sampling method, stability of the results over time, linearity of the detections and finally correlation between results obtained with this analyzer and the Gen'S one from Beckman-Coulter Inc. The obtained results were within the theoretical ranges given by the manufacturer. The presence of any abnormal result, or of any flag, must systematically lead to check the blood smear. This new automated haematology analyser appears to be convenient for emergency room-related laboratories, and for routine small-to-medium laboratories.

Performance evaluation of the Z2 coulter counter for WBC and RBC counting

The Z2 Coulter Counter (Z2) is a new, single-channel semi-automated electronic counter using the Coulter principle, providing both count and concentration results of cells, as well as providing size distribution of the cell population. The Z2 was evaluated according to guidelines published by the International Committee for Standardisation in Haematology. The results demonstrated excellent linearity for WBC and RBC (r > 0.999). The Z2 showed excellent precision, with a within-run, between-run and overall coefficient of variance less than 2%. Carryover was generally acceptable (<1%). WBC and RBC were stable after blood storage for 48 h at 4 degrees C and room temperature. There was almost no interference for WBC, RBC at high concentrations of bilirubin (178.5 mumol/l) or triglyceride (14.8 mmol/l) (P > 0.05). The bias of coincidence correction in WBC and RBC counting with the Z2 was less than 1%. Correlation between the Z2 and Sysmex F-820 or SE-9500 was excellent (r > 0.996) for WBC and RBC counting. Our results indicate that the Z2 has good features of precision, accuracy, linearity and good comparison with other haematology analysers, and could be used as a calibrating instrument for WBC and RBC counting.

The effect of whole‐blood storage time on the number of white cells and platelets in whole blood and in white cell–reduced red cells

Whole blood (WB) can be stored for some time before it is processed into components. After introduction of universal white cell (WBC) reduction, it was observed that longer WB storage was associated with more residual WBCs in the WBC-reduced red cells (RBCs). Also, weak propidium iodide (PI)-positive events were observed in the flow cytometric WBC counting method, presumably WBC fragments. The effect of storage time on the composition of WB and subsequently prepared WBC-reduced RBCs was studied. WB was collected in bottom-and-top collection systems with inline filters, obtained from Baxter, Fresenius, or MacoPharma. Units were stored at room temperature and separated into components in 4-hour intervals between 4 and 24 hours after collection. RBCs were WBC-reduced by inline filtration (approx. 50/group). Platelet (PLT) counts were lower in WB stored for 4 to 8 hours compared to 20 to 24 hours (mean +/- SD): 79 +/- 31 versus 102 +/- 30 for Baxter (p < 0.01); 91 +/- 31 versus 101 +/- 35 for Fresenius (not significant); and 73 +/- 47 versus 97 +/- 31 (all x 10(9) per unit) for MacoPharma (p < 0.01), respectively. The median residual WBC counts in WBC-reduced RBCs for WB stored for 4 to 8 and 20 to 24 hours were 0.03 versus 0.17 for Baxter (p < 0.001), 0.00 versus 0.06 for Fresenius (p < 0.001), and 0.13 versus 0.26 (all x 10(6) per unit) for MacoPharma (not significant), respectively. All WBC-reduced RBCs contained fewer than 5 x 10(6) WBCs per unit. A longer storage time of WB was associated with more weak PI-positive events, irrespective of the filter. Longer storage of WB before processing results in counting higher numbers of PLTs in WB, higher numbers of WBCs in WBC-reduced RBCs, and more weak PI-positive events.

Automated analysis of pleural fluid total and differential leukocyte counts with the Sysmex XE-2100

Determination of leukocyte (WBC) counts in pleural fluid is routinely performed by microscopic examination. In this study, we evaluated the performance of automated (differential) WBC counting in comparison with manual counting. Pleural fluid samples (n=45) were obtained from patients undergoing diagnostic thoracocentesis. The manual total WBC count was determined after Samson staining in a Fuchs-Rosenthal hemocytometer; microscopic differential counts were performed on May-Grünwald Giemsa-stained cytospin slides. The Sysmex XE-2100 hematology analyzer was used for automated (differential) WBC counting. The functional detection limit was determined by serial dilution of continuous ambulatory peritoneal dialysis (CAPD) fluid and replicate measurements of each dilution. The automated WBC count (x10(6)/L) was highly correlated with that of the microscopic reference method (r(2)=0.95; WBC-analyzer=0.97 x WBC-reference method+16; n=45). Good agreement was also observed for the absolute lymphocyte count (r(2)=0.92; WBC-analyzer=0.99 x WBC-reference method+32; n=36), neutrophil count (r(2)=0.94; WBC-analyzer=0.91 x WBC-reference method+6; n=35), and monocyte count (r(2)=0.73; WBC-analyzer=0.83 x WBC-reference method+6; n=38). The functional detection limit for WBCs was calculated at 50 x 10(6)/L (coefficient of variation 20%). With some limitations, total and differential WBC counts in pleural fluid can be reliably determined using the Sysmex XE-2100 instrument.

The origin and identification of unknown events associated with low-level leucocyte counting by flow cytometry.

Flow cytometric enumeration of residual leucocytes (WBC) in leucocyte-depleted components occasionally reveals a population of events that stain with propidium iodide, but which are outside the main counting region. These extraregional events may result in discrepancies in residual WBC counts. To identify the origin of these unknown events, separate populations of mononuclear cells and polymorphonuclear neutrophils (PMNs) (prepared by density-gradient centrifugation) were spiked into leucocyte-depleted red cell concentrates (RCC) to a concentration of 150 cells/ microl and assessed, using LeucoCount and DNA Prep reagents, by flow cytometry. In addition, isolated WBC nuclei, DNA in saline, or enzymatically digested free DNA, was spiked into diluted whole blood (final concentration 5 microg/ml). Isolated WBC nuclei fell in the main counting region of the dot plot. Extraregional events were generated in leucocyte-depleted RCC spiked with PMN (17/ microl by day 4), but not with mononuclear cells, and increased with sample storage (4 days). A significantly (P < 0.01) greater number of extraregional events was observed using the LeucoCount reagent, compared with the DNA Prep reagent. Enzymatically digested WBC nuclei or free DNA, added to diluted whole blood, generated extraregional events using LeucoCount, but not the DNA Prep reagent. When free DNA was enzymatically digested, no extraregional events were observed. These previously unidentified events are probably fragmented nuclei or free DNA originating from PMN owing to a combination of ageing and reagent addition. Currently, in our protocols, the region used for WBC enumeration counts intact WBC nuclei. To achieve WBC counting consistency, flow cytometric gating protocols must be standardized, and a decision taken as to whether to include extraregional events in the count.