Automatic Cell Analyzer Research Articles

Evaluation of a new automated cell analyzer (Sysmex UF-1000i) for bacteriological screening of urine

Introduction. Sysmex UF-1000 is a new flow cytometry for the analysis of urine based on a laser diode technology using specific compounds for the staining nucleic acids, characterized by a threshold of detection of bacteria equal to 1000 CFU/ml.The purpose of this study was to compare the positivity or negativity of the urine samples using standard procedures and with the response obtained from the instrument UF-1000i. Methods. During the period May and July 2011, 1024 urine samples obtained from the laboratory of the hospital in Genoa-Voltri were analyzed with the instrument Sysmex UF-1000i.The samples were stored at a temperature of 5°C during transport.The instrument after loading of the sample is able to assess the bacterial load in about 1 minute per sample. Results. 1024 samples were analyzed with Sysmex UF-1000 analyzed in parallel by the hospital laboratory in Genoa Voltri. 228 are positive results for Sysmex UF-1000 (bacterial loads in excess of 1000 CFU/ml).The data obtained were consistent with those recorded with traditional analysis. 18 samples were positive only for our instrument, but not with the traditional system used in the laboratory for comparison. Conclusion. The advantage of Sysmex UF-1000 is certainly due to obtain immediate results after the reading of each individual sample that is higher than any other instrument or method of analysis used. The slight discrepancy of the results obtained may be due to the extremely low calibration of UF-1000. This allows in a very short time to discard all negative samples with enormous saving of time and material. For positive samples and applies the criterion of the routine ie, the bacterial load has a meaning according to the type of patient considered, as in the case of pediatric or catheterized patients

Delta neutrophil index versus lactate clearance: Early markers for outcome prediction in septic shock patients

BackgroundThis study was carried out to evaluate, compare the clinical utility of delta neutrophil index (DNI) values and lactate-6h-clearance as early indicators for outcome prediction in septic shock patients and to find out and correlate the specificity and sensitivity of each to the patient's mortality. MethodsThe study was carried out in Alexandria Main University Hospital on 53 adult patients with severe sepsis/septic shock after taking an informed written consent and approval of the ethics committee. All patients received all standard therapy and resuscitation measures. Arterial blood samples were obtained for measurement of DNI using an ADVIA 2120 automated cell analyzer. Initial arterial serum lactate level and arterial lactate after 6h from admission were measured for lactate-6h-clearance calculation. Primary outcome measures were hospital mortality (non-survivors) and discharge or transfer to a general medical ward (survivors). Data were analyzed by SPSS® software using (“t” test and chi-square χ2) P<0.05 was considered significant. ResultsA total of 53 severe sepsis/septic shock patients were enrolled in the study, patients were (36) survivors and (17) non-survivors. This study demonstrated the reliability of DNI and lactate-6h-clearance as predictors of negative outcome. Variables identified by the backward logistic regression model as significantly correlated with negative outcome were lactate-6h-clearance <15%; (sensitivity, 92.5%; specificity, 85.0%; positive predictive value, 90.0%; and negative predictive value, 88.0%) and DNI value >5.2; (sensitivity, 95.0%; specificity, 90.0%; positive predictive value, 92.0%; and negative predictive value, 95.0%). ConclusionMonitoring of lactate-6h-clearance can be used to identify patients with severe sepsis/septic shock at high risk of mortality. DNI may serve as a more useful diagnostic and prognostic marker for early diagnosis of disease severity in patients with severe sepsis/septic shock, it is suggested that, increased DNI value should alert clinicians to apply more aggressive therapy.

Delta neutrophil index: Could it predict mortality in patients with bacteraemia?

Despite advances in therapy, sepsis still has a high mortality rate. To improve the treatment outcome, early diagnosis and treatment is essential, as reflected in many publications about biomarkers for sepsis. Recent models of automatic cell analyzers provide the delta neutrophil index (DN), which corresponds to the fraction of immature granulocytes in circulating blood. This study investigated DN as a prognostic marker in patients with bacteraemia. We retrospectively collected data on adult patients with bacteraemia admitted to an urban hospital between November 2009 and April 2010. The DN was measured at the onset of bacteraemia and at 24 and 72 h later. Factors associated with 28-day mortality were assessed using logistic regression. A total of 102 patients were included in the analysis, and 24 patients died within 28 days. In the univariate analysis, appropriate antibiotic treatment, multidrug-resistant bacterial infection, history of intensive care unit (ICU) stay before the onset of bacteraemia, healthcare-associated infection, DN at 72 h after the onset of bacteraemia (DN-72 h), and platelet count at the onset of bacteraemia were significantly associated with survival. In multivariate analysis, DN-72 h (odds ratio (OR) 1.246, 95% confidence interval (CI) 1.030-1.508; p = 0.023), platelet count at the onset of bacteraemia (OR 0.986, 95% CI 0.977-0.995; p = 0.003), and history of ICU stay before the onset of bacteraemia (OR 4.907, 95% CI 1.045-23.051; p = 0.044) were the independent factors associated with survival. DN at 72 h after bacteraemia may be valuable to assess the prognosis of patients with bacteraemia.

Gradient convergence filters and a phase congruency approach for in vivo cell nuclei detection

Computational methods used in microscopy cell image analysis have largely augmented the impact of imaging techniques, becoming fundamental for biological research. The understanding of cell regulation processes is very important in biology, and in particular confocal fluorescence imaging plays a relevant role for the in vivo observation of cells. However, most biology researchers still analyze cells by visual inspection alone, which is time consuming and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cells. While the classic approach for automatic cell analysis is to use image segmentation, for in vivo confocal fluorescence microscopy images of plants, such approach is neither trivial nor is it robust to image quality variations. To analyze plant cells in in vivo confocal fluorescence microscopy images with robustness and increased performance, we propose the use of local convergence filters (LCF). These filters are based in gradient convergence and as such can handle illumination variations, noise and low contrast. We apply a range of existing convergence filters for cell nuclei analysis of the Arabidopsis thaliana plant root tip. To further increase contrast invariance, we present an augmentation to local convergence approaches based on image phase information. Through the use of convergence index filters we improved the results for cell nuclei detection and shape estimation when compared with baseline approaches. Using phase congruency information we were able to further increase performance by 11% for nuclei detection accuracy and 4% for shape adaptation. Shape regularization was also applied, but with no significant gain, which indicates shape estimation was good for the applied filters.

MODULATING EFFECT OF THE −158 GΓ (C→T) XMN-1 POLYMORPHISM IN INDIAN SICKLE CELL PATIENTS

Xmn1 polymorphism is a known factor, which increases fetal haemoglobin production. Among the inherited disorders of blood, thalassaemia and Sickle Cell Diseases contributes to a major bulk of genetic diseases in India. Our aim was to verify the role of the Xmn1 polymorphism as a modulating factor in sickle cell patients and frequency of the polymorphism in Indian sickle cell patients. 60 sickle homozygous and 75 sickle beta thalassemia patients were included and 5 ml blood sample was collected from them. Screening of sickle patients was done by HPLC. An automated cell analyzer SYSMEX (K-4500 Model) was used to analyze the Complete Blood Count of patients. Xmn1 polymorphism analysis was done by PCR-RFLP and one-way ANOVA test was applied to analysis of variance between groups. Among the sickle patients 27 were heterozygous (+/−) and 19 were homozygous (+/+) while 30 were heterozygous (+/−) and 24 were homozygous (+/+) in sickle β-thalassemia patients. Extremely significant differences (p-value <0.001) of hematological parameters seen among patients with Xmn1 carrier and without the Xmn1 carrier. In our cases the clinical symptoms were barely visible and higher HbF level with Xmn1 carriers were found. Presence of Xmn1 polymorphism in sickle cell patients with higher HbF were phenotypically distinguished in the sickle cell patients. We conclude that the phenotypes of Indian sickle cell patients were greatly influenced by Xmn1polymorphism.

Prevalence of glutathione S-transferase gene deletions and their effect on sickle cell patients

BackgroundGlutathione S-transferase gene deletions are known detoxification agents and cause oxidative damage. Due to the different pathophysiology of anemia in thalassemia and sickle cell disease, there are significant differences in the pathophysiology of iron overload and iron-related complications in these disorders.ObjectiveThe aim of this study was to estimate the frequency of the GSTM1 and GSTT1 genotypes in sickle cell disease patients and their effect on iron status.MethodsForty sickle cell anemia and sixty sickle ß-thalassemia patients and 100 controls were evaluated to determine the frequency of GST gene deletions. Complete blood counts were performed by an automated cell analyzer. Hemoglobin F, hemoglobin A, hemoglobin A2 and hemoglobin S were measured and diagnosis of patients was achieved by high performance liquid chromatography with DNA extraction by the phenol-chloroform method. The GST null genotype was determined using multiplex polymerase chain reaction and serum ferritin was measured using an ELISA kit. Statistical analysis was by EpiInfo and GraphPad statistics software.ResultsAn increased frequency of the GSTT1 null genotype (p-value = 0.05) was seen in the patients. The mean serum ferritin level was higher in patients with the GST genotypes than in controls; this was statistically significant for all genotypes except GSTM1, however the higher levels of serum ferritin were due to blood transfusions in patients.ConclusionGST deletions do not play a direct role in iron overload of sickle cell patients.

Molecular characterization of hemoglobin D Punjab traits and clinical-hematological profile of the patients.

Hemoglobin (Hb) D hemoglobinopathies are widespread diseases in northwestern India and usually present with mild hemolytic anemia and mild to moderate splenomegaly. The heterozygous form of Hb D is clinically silent, but coinheritance of Hb D with Hb S or beta-thalassemia produces clinically significant conditions like thalassemia intermedia of moderate severity. Under heterozygous conditions with coinheritance of alpha and beta-thalassemia, patients show a degree of clinical variability. Thus, our aim was to molecularly characterize the Hb D trait among individuals who were clinically symptomatic because of co-inheritance of alpha deletions or any beta-globin gene mutations. This was a cross-sectional study conducted in an autonomous tertiary-care hospital. Complete blood count and red cell indices were measured using an automated cell analyzer. Quantitative assessment of hemoglobin Hb F, Hb A, Hb A2 and Hb D was performed by means of high performance liquid chromatography (HPLC). DNA extraction was done using the phenol-chloroform method. Molecular analyses on common alpha deletions and common beta mutations were done using the Gap polymerase chain reaction and Amplification Refractory Mutation System, respectively. We evaluated 30 patients and found clinical variation in the behavior of Hb D traits. In six patients, the Hb D traits were clinically symptomatic and behaved like those of thalassemia intermedia. Molecular characterization showed that three out of these six were IVS-1-5 positive. HPLC may not be the gold standard for diagnosing symptomatic Hb D Punjab traits. Hence, standard confirmation should include molecular studies.

Osteonecrosis and Leg Ulceration in Indian Sickle Cell Patients

Sir, Avascular necrosis of bone and cutaneous leg ulcers are the most common manifestation of patients with sickle cell disease (SCD) [1, 2]. The bone areas most frequently affected are cortical bone of the acetabulum, head of the femur, and head of the humerus. Osteonecrosis of the femoral head is common in patients with sickle cell disease. The hip and shoulder abnormalities are also seen in patients. Multifocal osteonecrosis is defined as disease of 3 or more anatomic sites. Patients diagnosed with osteonecrosis of the knee, shoulder and ankle should be evaluated for other joints for osteonecrosis. In patients with sickle cell disease, clinical complications including osteonecrosis can vary in frequency and severity, presumably due to the effects of genes that modify the pathophysiology initiated by the sickle mutation [3]. Baldwin et al. suggested genetic influence of BMP6 and ANXA2 SNPs with osteonecrosis in SCD [3]. The incidence of leg ulcers in patients with SCD ranges from 25.7% to 75% [4, 5]. Leg ulcers are a common sub-phenotype of sickle cell disease. Their cause is unknown, their prevention is impractical and their management, once present, is often difficult. Leg ulceration and avascular necrosis (AVN) in SCD has become serious problem in a few decades in India, however the incidence of leg ulcer and AVN in sickle cell patients has not been studied extensively in India till date. We had 150 sickle cell patients (60 sickle cell anemia, 75 sickle β-thalassemia and 15 sickle hemoglobin D) recruited from out patient department hematology AIIMS, New Delhi, India. Study period was 3 y. Signed consent form taken from all the patient and study was approved by institutional ethical committee. Diagnosis of patients was done by high performance liquid chromatography (Bio-Rad-VariantTMBio Rad, CA, USA). Complete blood count and red cell indices were measured by automated cell analyzer (SYSMEXK-4500, Kobe Japan). Osteonecrosis screening was done by X-ray and confirmation was done by magnetic resonance imaging (MRI) while leg ulceration was reported on physical examination. MRI is the most sensitive means of diagnosing AVN. MRI facilitates better response to treatment because with the use of MRI, avascular necrosis is diagnosed at an earlier stage and therapeutic measures are more successful the earlier they are begun. Out of 150 cases 14 (8 boys and 6 girls with mean age; 11.5±3.4 y) presented with osteonecrosis where seven were from HbSS, 6 from sickle beta thalassemia and one fromHbSD patients. Nine patients presented with femoral head ( 5 HbSS and 4 HbSβ-thal) while 3 hip (2 HbSS and 1HbSD) and 2 (HbSβ-thal)with shoulder osteonecrosis. Seven patients (4 boys and 3 girls with mean age; 12.3±2.6 y) presented with leg ulceration out of 150 SCD patients (5HbSS and 2 HbSβ-thal). None of the patients presented both complications i.e., leg ulceration and AVN together. Observation of the study concludes the increased risk of AVN (9.34%) and leg ulceration (4.67%) in Indian sickle cell patients where alpha thalassemia and high HbF is prevalent in patients.

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.

Sensitivity and specificity of the high fluorescent lymphocyte count-gate on the Sysmex XE-5000 hematology analyzer for detection of peripheral plasma cells

Plasma cells are one of the end products of the B-lymphocyte mediated immune response. These cells normally reside in the bone marrow or some peripheral lymphoid tissues. Increased numbers of plasma cells in the blood, usually indicates pathology such as infection, auto-immunity or haematological malignancy. Therefore, the ability to measure plasma cells (PCs) on an automated cell analyzer might be advantageous. The performance of the Sysmex XE-5000 leukocyte differential channel [high fluorescent lymphocyte count (HFLC) area] was evaluated for the ability to detect plasma cells in peripheral blood and compared to the detection of plasma cells by flow cytometric analyses. Our results show that the HFL count from the XE-5000 correlates (R(2)=0.8) with the number of PCs in peripheral blood, but detects PCs with moderate to good sensitivity (88.9%) and specificity (87.8%). The Sysmex XE-5000 is suitable for screening blood samples for the presence of elevated number of plasma cells in peripheral blood, but the actual quantification needs to be confirmed by flow cytometry.

Genotypic influence of α-deletions on the phenotype of Indian sickle cell anemia patients.

BackgroundSome reports have shown that co-inheritance of α-thalassemia and sickle cell disease improves hematological parameters and results in a relatively mild clinical picture for patients; however, the exact molecular basis and clinical significance of the interaction between α-thalassemia and sickle cell disease in India has not yet been described. There is little agreement on the clinical effects of α-thalassemia on the phenotype of sickle cell disease.MethodsComplete blood count and red cell indices were measured by an automated cell analyzer. Quantitative assessment of hemoglobin variants HbF, HbA, HbA2, and HbS was performed by high performance liquid chromatography (HPLC). DNA extraction was performed using the phenol-chloroform method, and molecular study for common α-deletions was done by gap-PCR.ResultsOut of 60 sickle cell anemia patients, the α-thalassemia genotype was found in 18 patients. Three patients had the triplicated α-genotype (Anti α-3.7 kb), and the remaining patients did not have α-deletions. This study indicates that patients with co-existing α-thalassemia and sickle cell disease had a mild phenotype, significantly improved hematological parameters, and fewer blood transfusions than the patients with sickle cell anemia without co-existing α-deletions.ConclusionCo-existence of α-thalassemia and sickle cell anemia has significant effects on the phenotype of Indian sickle cell patients.

Abstract P5-06-07: Artemisinin Disrupts the Barrier Function in Vascular Endothelial Cells, a Potential Implication in Improving Penetration of Therapeutic Drugs in the Central Nervous System

Abstract Introduction. The barrier functions of endothelial cells are primarily controlled by tight junctions in the vascular lining. Tight junctions are also the main cellular structure that makes up the Blood Brain Barrier (BBB) in the central nervous system (CNS). Treating brain tumours, primary and secondary, has been a difficult issue, one of the key reasons being drug permeability in the CNS by BBB. It has been shown recently that that in metastatic brain tumours, the Blood Brain Barriers remain intact. Thus, this presents a significant challenge for delivering sufficient anticancer agents in the CNS. Artemisinin is an anti-malarial drug that has been widely used. It is an effective modality in treating the cerebral type of malaria due to its ability to pass the BBB. The drug has been shown in recent years to be toxic to a variety of cancer cells and has been attempted in the treatment of certain solid tumours. Aims. To investigate if Artemisinin a direct effect on the tight junctions in vascular endothelial cells. Materials and methods. An established human vascular endothelial cell line was used. The effect of Artemisinin on the barrier function, migration and micromotion of the endothelial cells was investigated using an automatic barrier function assay, Electric cell-substrate impedance sensing (ECIS). Cells were treated with a broad range of concentrations over various periods. Barrier function, cell migration and cell micromotion functions were monitored and analysed using an automated cell analysis software. The effect of Artemisinin on the toxicity of the endothelial cells was also monitored. Results. Artemisinin had a marginal toxic effect on endothelial cells at concentrations over 500μM. Subsequent tests were conducted at concentrations below toxic level. Low concentrations of Artemisinin (0.5-10 μM) resulted in a significant reduction of the barrier function in the endothelial cells (for example, barrier function measured by electric resistance in non-treated cells being 75.3±3.4Ω compared with 48.9±2.2Ω when cell treated with Artemisinin at 4μM). Using an electric wounding assay, we failed to detect a significant change in cell motility when cells were treated with Artemisinin. However, it is interesting to note that Artemisinin consistently increased the cell membrane micromotion over a wide concentrations between 0.1-100 μM. For example, cell modelling analysis showed that micromotion Rb in non-treated cells was 5.8±1.2Ω compared with 9.7±4.3Ω of cells treated with Artemisinin. Our data further demonstrated that the effect of Artemisinin on the barrier function of the cells sustained over a period of 4 hours. Conclusion. Artemisinin is able to disrupt the barrier function of vascular endothelial cells without exerting significant cytotoxicity to the cells. The compound has a potential in being considered as a way to improve the permeability of endothelial cells during the delivery of anticancer drugs in the treatment of CNS malignancies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-06-07.

Cell Nuclei and Cytoplasm Joint Segmentation Using the Sliding Band Filter

Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and actin. Both image datasets present a difficult problem due to the high variability of cell shapes and frequent cluster overlap between cells. On the Drosophila dataset our approach achieved a precision/recall of 95%/69% and 82%/90% for nuclei and cytoplasm detection respectively and an overall accuracy of 76%.

Implementation of new software for fast screening of cell compatibility on surface modifications using low-contrast time-lapsed microscopy

Assessment of cell adhesion and cell size provides valuable information on surface biocompatibility. However, most investigations on cell morphology dynamics are time and resource consuming, of rather descriptive character and lack procedures for appropriate quantification. The aim of the study was to develop a software programme which allows automated cell segmentation and identification as well as calculation and further processing of cell size in low-contrast images. The software utilises modified edge detection and morphologic operations for automatic cell analysis in light microscopy images. In an application study, osteogenic cell-adhesion dynamics were quantified for the ECM proteins collagen type I (COL) and fibronectin (FIB) over a period of 12 hrs. Untreated tissue culture polystyrene (TCPS) served as control. The software programme proofed full function in automatic cell tracking and quantification of cell size. After 11 h, cell sizes were highest for COL (6391 ± 1167 µm(2)) and FIB (6036 ± 411 µm(2)) compared with TCPS (3261 ± 693 µm(2)). The developed software allows quantification of initial cell size changes on translucent surface modifications and is suitable as a reliable tool for fast biocompatibility screening. Osteogenic cell adhesion was significantly promoted by COL and FIB indicating the potential of respective functionalized biomaterial surfaces.

Automated cell analysis in 2D and 3D: A comparative study

Optical projection tomographic microscopy is a technique that allows 3D analysis of individual cells. Theoretically, 3D morphometry would more accurately capture cellular features than 2D morphometry. To evaluate this thesis, classifiers based on 3D reconstructions of cell nuclei were compared with 2D images from the same nuclei. Human adenocarcinoma and normal lung epithelium cells were used. Testing demonstrated a three-fold reduction in the false negative rate for adenocarcinoma detection in 3D versus 2D at the same high specificity. We conclude that 3D imaging will potentially expand the horizon for automated cell analysis with broad applications in the biological sciences.

Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein

Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.

Integrated microfluidic cell culture and lysis on a chip

We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated.

Correlation between erythrocytes deformability and size: A study using a microchannel based cell analyzer

The deformability of erythrocytes is of great importance for oxygen delivery in the microcirculation [ Lipowsky, H.H., 2005. Microvascular rheology and hemodynamics. Microcirculation 12, 5–15]. Aging of erythrocytes is associated with a reduction in deformability and also in size. The present work describes an automated cell analyzer which utilizes a glass microchannel and advanced image processing software. Erythrocytes suspended in a high viscosity medium are filmed flowing through the microchannel. Under these conditions, the cells assume different orientations and undergo varying deformations according to their location in the velocity profile. The cell analyzer enables the measurement of individual erythrocyte velocity, deformability and volume at varying depths within the microchannel. The volume of the cells is calculated based on the experimental data and a fluid mechanics model. The results obtained show that, on average, the deformability of the cells increases with increase in their size. Additionally, the behavior of RBCs in a microchannel is investigated, showing promising diagnostic possibilities.

The Association of Delta Neutrophil with Severity of Sepsis.

Background: The entrance of microbes into blood stream results in rapid spreading of infection through circulation system progressing to sepsis with accompanying fever, leukocytosis and circulatory collapse necessitating prompt recognition and treatment. We calculated the difference between white blood cell counts measured in myeloperoxidase and basophil channel, designated as “delta neutrophil”, and found that the value correlates the progression of sepsis.Methods: We categorized patients who were referred for automatic blood cell analysis according to the calculated value of delta neutrophil and analyzed positive rate of blood culture, mortality and laboratory data including hemoglobin, platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, fibrinogen degradation product, D-dimer, ESR, CRP. Delta neutrophil values were calculated as above mentioned from measured values from automatic blood cell analyzer, ADVIA® 120 automated Hematology System (Bayer Diagnostics, New York, USA). Among 1781 patients selected, those with delta neutrophil value less then 5 were categorized as group I, 5 to 10 as group II, 10 to 20 group III, 20 to 30 group IV, 30 to 40 group V, 40 to 50 group VI and greater than 50 as group VII, arbitrarily. Patients with hematologic malignancy or with Gynecologic malignancy were excluded in this study.Results: The mortality and blood culture rate in group VII were 75.0% and 57.1% and in group VI 76% and 46.7%, group V 46.2% and 37.9%, respectively. On the contrary for group I the mortality and isolation rate were 3.3% and 17.1%. (The means of hemoglobin concentration for group I and II were 13.2 g/dL and 12.1 g/dL compared with 10.5 g/dL and 10.2 g/dL in group VI and VII.) With regard to consumptive coagulopathy, the means of platelet count were 263,700/mL and 259,200/mL in group I and II while those for group VI and VII were 107,200/mL and 112,600/mL. Antithrombin activities were 5.20 % and 7.98% in group VII and VI in contrast to 66.6% and 75.8% for group I and II.Conclusion: The categorization by delta neutrophil value correlated with the isolation rate of bacteria from blood culture and mortality. The delta neutrophil value is expected to provide useful information for following up progression of sepsis.

Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96

Background: Differential counting of peripheral blood cells is an important diagnostic tool. Yet, this technique requires highly trained staff, is labour intensive and has limited statistical reliability. A recent development...