Electronic Cell Counter Research Articles

Correlations between Transcranial Doppler Velocities and Laboratory Markers of SCA Severity

Introduction. Stroke is one of the main complications of Sickle Cell Anemia (SCA), occurring in about ten percent of the affected individuals before the age of twenty. Hydroxyurea (HU) currently used to treat SCA, although its exact mechanism of action remains not fully understood. The transcranial Doppler (TCD) is used to detect the risk of stroke in children with SCA, which measures the time-averaged mean of the maximum velocity (TAMMV) in the internal carotid artery and the middle cerebral arteries. TAMVV above 200 centimeters per second (cm/s) is associated with a high risk of stroke. In the present work, we evaluated the correlations between laboratory markers and TCD velocities and we also studied the influence of HU in different TCD groups. Material and methods. A cross-sectional study including 164 HbSS patients with average age 7.51±4.14 was conducted at the Pediatric Professor Hosannah de Oliveira Hospital (HUPES/UFBA) and in the Sickle Cell Disease Reference Center, hospitals located in Salvador and in Itabuna, both cities in northeastern Brazil. The risk of stroke was assessed by TCD, for three outcomes were considered: TCD1 for the normal group (TAMMV <170 cm/s), TCD2 for the conditional group (TAMMV 170-199 cm/s) and TCD3 for the high risk (TAMMV> 200 cm/s). The legal representatives signed the informed consent terms. The study was approved by the Research Ethics Committee and was conducted in accordance with the guidelines of the Declaration of Helsinki and its revisions. Hemoglobin profiles were confirmed by high-performance liquid chromatography (HPLC). Hematological analyses were carried out using an electronic cell counter. Biochemical and inflammatory analyses were performed by immunochemistry assays and immunoassays. Statistical analyses were performed on Graphpad prism 6.0. The distribution of quantitative variables was analyzed using the Shapiro-Wilk test. The median of quantitative variables between three or more groups was compared using One-way Anova/Kruskall-Wallis for nonparametric data. When comparing two groups, Mann-Whitney t-tests were performed. The correlation analyses were performed between variables using Spearman coefficient (r). Results. We observed negative correlations (with Spearman r< -0.2 and p<0.05) between left and right TAMMV and red blood cell (RBC) count, hemoglobin levels and hematocrit. There were positive correlations (Spearman r> 0.2 and p<0.05) with mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Red Cell Distribution Width (RDW), white blood cell (WBC), lymphocytes, reticulocytes percentage, aspartate aminotransferase (AST) and total bilirubin. By comparing the three TCD groups, excluding the stroke group, nitric oxide (NO) levels (p=0.157) and mean platelet volumes (MPV) (p=0.0134) varied between groups, with the lower numbers observed for the TCD2 and TCD3 groups. WBC count is progressively increased in DTC groups (DTC3>2>1), but not significantly. The lymphocyte count (p=0.006) was increased in the TCD3 group, as compared to the other two groups. Ferritin median values were increased (p=0.005) in TCD2 and TCD3 groups. The MPV values were lower in TCD2 and TCD3 groups both for HU treated and untreated patients (p=0.032), with the lowest MPV values observed for TCD3. WBC count was significantly different among TCD groups of untreated patients (p=0.009) but not among TCD groups of patients treated with HU. Patients in use of HU showed decreased WBC counts in all TCD groups, but only in a significant way for TCD3 (p=0.002). In the same line, the lymphocyte counts were significantly decreased by HU in TCD 1 and TCD3, (p=0.04 and p=0.006) but not for TCD2.Conclusions. The current results show significant correlations between TCD-TAMMV and laboratory markers commonly used to monitor SCA pediatric patients. These laboratory parameters included reticulocyte percentage and white blood cell counts, which are known markers of SCA severity, but their correlations with TAMMV has never been shown before. We also show that hydroxyurea treatment had different impacts on laboratory parameters such as MPV, lymphocyte and leukocyte numbers in the different TCD groups evaluated here. We speculate that the association of these markers with TCD should be further studied and carefully analyzed along with TCD velocities. DisclosuresNo relevant conflicts of interest to declare.

Quantification of persister formation of Escherichia coli leveraging electronic cell counting and semi-mechanistic pharmacokinetic/pharmacodynamic modelling.

Persister formation of Escherichia coli under fluoroquinolone exposure causes treatment failure and promotes emergence of resistant strains. Semi-mechanistic pharmacokinetic/pharmacodynamic modelling of data obtained from in vitro infection model experiments comprehensively characterizes exposure-effect relationships, providing mechanistic insights. To quantify persister formation of E. coli under levofloxacin exposure and to explain the observed growth-kill behaviour, leveraging electronic cell counting and pharmacokinetic/pharmacodynamic modelling. Three fluoroquinolone-resistant clinical E. coli isolates were exposed to levofloxacin in static and dynamic in vitro infection model experiments. Complementary to plate counting, bacterial concentrations over time were quantified by electronic cell counting and amalgamated in a semi-mechanistic pharmacokinetic/pharmacodynamic model (1281 bacterial and 394 levofloxacin observations). Bacterial regrowth was observed under exposure to clinically relevant dosing regimens in the dynamic in vitro infection model. Electronic cell counting facilitated identification of three bacterial subpopulations: persisters, viable cells and dead cells. Two strain-specific manifestations of the levofloxacin effect were identified: a killing effect, characterized as a sigmoidal Emax model, and an additive increase in persister formation under levofloxacin exposure. Significantly different EC50 values quantitatively discerned levofloxacin potency for two isolates displaying the same MIC value: 8 mg/L [EC50 = 17.2 (95% CI = 12.6-23.8) mg/L and 8.46 (95% CI = 6.86-10.3) mg/L, respectively]. Persister formation was most pronounced for the isolate with the lowest MIC value (2 mg/L). The developed pharmacokinetic/pharmacodynamic model adequately characterized growth-kill behaviour of three E. coli isolates and unveiled strain-specific levofloxacin potencies and persister formation. The mimicked dosing regimens did not eradicate the resistant isolates and enhanced persister formation to a strain-specific extent.

Rapid Quantification of Microalgae Growth with Hyperspectral Camera and Vegetation Indices.

Spectral cameras are traditionally used in remote sensing of microalgae, but increasingly also in laboratory-scale applications, to study and monitor algae biomass in cultures. Practical and cost-efficient protocols for collecting and analyzing hyperspectral data are currently needed. The purpose of this study was to test a commercial, easy-to-use hyperspectral camera to monitor the growth of different algae strains in liquid samples. Indices calculated from wavebands from transmission imaging were compared against algae abundance and wet biomass obtained from an electronic cell counter, chlorophyll a concentration, and chlorophyll fluorescence. A ratio of selected wavebands containing near-infrared and red turned out to be a powerful index because it was simple to calculate and interpret, yet it yielded strong correlations to abundances strain-specifically (0.85 < r < 0.96, p < 0.001). When all the indices formulated as A/B, A/(A + B) or (A − B)/(A + B), where A and B were wavebands of the spectral camera, were scrutinized, good correlations were found amongst them for biomass of each strain (0.66 < r < 0.98, p < 0.001). Comparison of near-infrared/red index to chlorophyll a concentration demonstrated that small-celled strains had higher chlorophyll absorbance compared to strains with larger cells. The comparison of spectral imaging to chlorophyll fluorescence was done for one strain of green algae and yielded strong correlations (near-infrared/red, r = 0.97, p < 0.001). Consequently, we described a simple imaging setup and information extraction based on vegetation indices that could be used to monitor algae cultures.

Evidence for biological effects in the radiosensitization of leukemia cell lines by PEGylated gold nanoparticles

Gold nanoparticles (Au NPs) are promising radiosensitizers for cancer therapy. While the radiosensitizing effects of Au NPs have been shown in many epithelial cancers, there are no documented cases of their use in cells of hematopoietic origin, which constitute ~ 10% of all cancer cases and are frequently treated using radiation therapy. In this work, we measure the uptake of polyethylene glycol-coated (PEGylated) Au NPs (5 nm core diameter) in HL-60 II and Jurkat D1.1 cells using flow cytometry and ICP-AES. Electronic cell counting, metabolic activity assays, and DNA synthesis assays reveal cell line–specific radiosensitization that is independent of the number of internalized nanoparticles. The high SER value for the HL-60 II cell line (1.33 at 5 Gy) points to a dominant biological mechanism.

Vergelijking van twee California Mastitis Testen met de elektronische celgetalbepaling voor de detectie van intramammaire infecties in mengmelkstalen van melkvee

Het celgetal van de melk is een indicator voor de aanwezigheid van een intramammaire infectie bij melkvee. Het doel van deze studie was om van 179 mengmelkstalen de resultaten van de elektronische celgetalbepaling via het Fossomatic™ toestel te vergelijken met deze van twee commerciële California Mastitis Testen (CMT) op basis van verslijming, al dan niet in combinatie met kleurverandering. De meest voorkomende major pathogenen bij bacteriologisch onderzoek waren Streptococcus uberis en Staphylococcus aureus en de vaakst geïsoleerde minor pathogenen waren de niet-aureus stafylokokken. Het celgetal geschat aan de hand van de CMT-testen kwam goed overeen met het elektronisch bepaalde celgetal bekomen met de Fossomatic™. De testkarakteristieken van beide CMT-testen waren vergelijkbaar, zowel onderling als met deze bekomen voor de elektronische celgetalbepaling. Op basis van de bevindingen kan geconcludeerd worden dat beide CMT-testen in de praktijk kunnen gebruikt worden om koeien met een intramammaire infectie veroorzaakt door major pathogenen te helpen detecteren.

Flow cytometric analysis of lymphocyte subsets in alcoholic liver disease

Background: The World Health Organization defines alcohol-related diseases as the third major cause of death and disability worldwide accounting for approximately 2.5 million deaths per year. Immune dysregulation has possible role for pathogenesis of alcohol liver injury. We aimed to study lymphocyte subsets in alcoholic liver disease (ALD). Materials and Methods: A prospective cross-sectional study was done over 2 years in a tertiary care hospital in Western Maharashtra. The cases were classified into four groups: nonalcoholics (n = 18), fatty liver (n = 13), alcoholic hepatitis (n = 11), and alcoholic cirrhosis (n = 51). ALD was confirmed by abdominal ultrasonography and liver function tests. Blood sample collected in ethylenediaminetetraacetic acid was run on electronic cell counter for total leukocyte count and on flow cytometer for CD45, CD3+, CD4+, and CD8+ counts. Results: Hemoglobin and platelet count showed a statistically significant reduction (P Conclusion: In this study, we found a decrease in the concentration of lymphocytes and its subpopulation (CD3+, CD4+, and CD8+) while as CD4+/CD8+ ratio was comparatively increased in ALD patients in comparison to nonalcoholics. These findings signify an immune dysregulation in ALD.

Pattern of Red Cell Count and Red Cell Distribution Width (RDW %) in Beta Thalassaemia Trait in Adults

Microcytic hypochromic anemia is one of the commonest hematological abnormalities in Bangladeshi population. Iron deficiency anemia and beta thalassemia traits are the most frequent causes of microcytic hypochromic anemia which are sometimes difficult to differentiate clinically and by routine laboratory examinations due to similar blood picture. To differentiate between patients of beta thalassemia trait and iron deficiency anemia, physicians need a group of investigations including peripheral blood film, estimation of HbA2, serum ferritin, serum iron, total iron binding capacity and transferrin saturation. But these tests are relatively expensive, time consuming and need sophisticated techniques. The aim of this study was to evaluate the pattern of red cell count and RDW-CV (%) in beta thalassemia trait in adults and thereby determine the role of these parameters in differentiation between patients of beta thalassemia trait and iron deficiency anaemia. In this study 50 confirmed cases of beta thalassemia trait aged 18 to 60 years, both male and female were included as cases and 50 age- and sex-matched iron deficiency subjects were included as control. RBC count and RDW-CV (%) were measured by an electronic cell counter device. The present study revealed that RBC count was higher in patients with beta thalassemia trait than that in iron deficiency anemia and RDW-CV(%) was significantly higher in patients with iron deficiency anemia than that in beta thalassemia trait.Bangladesh J Med Biochem 2016; 9(1): 31-35

Long-term changes in the properties of skin-derived fibroblasts following irradiation of the head and neck.

The tumor stroma performs an important role in carcinogenesis. It predominantly consists of fibroblasts and the connective tissue produced by them, and undergoes a multitude of interactions with the surrounding cancer cells. Since irradiation is part of the majority of therapeutic strategies for head and neck squamous cell carcinoma, more information regarding the effects of a previous irradiation on the tumor stroma is desirable. In the present study, fibroblasts were cultivated from human non-irradiated and pre-irradiated skin of the neck for 48 h. Subsequently, analyses of cell viability, apoptosis, necrosis and motility were conducted via MTT assay, Annexin V/propidium iodide staining, electronic cell counting for 4 consecutive days, and scratch assay. Pre-irradiated fibroblasts exhibited a significantly slower growth rate as well as increased rates of apoptosis and necrosis. They also exhibited significantly decreased motility compared with non-irradiated fibroblasts. These results indicated the long-term effects of irradiation on fibroblasts, which may affect cancer recurrence in the irradiated region via the tumor stroma. More information, such as that regarding the secretory capacities of pre-irradiated fibroblasts, is required to evaluate the possible therapeutic implications of these findings.

Lamellar Body Count With Cell Dyn Emerald And Cell Dyn Ruby Methods On Preterm Birth

Background: Lamellar bodies are produced by pneumocyte type II cells in the lung alveoli. Lamellar bodies are present in amniotic fluid in increasing quantities as gestation advances, 1 – 5 µm in size, similar in size to small platelets and can be counted on most electronic cell counters in hematology analyzer. Lamellar body count is useful for prediction of fetal lung maturity and neonatal respiratory distress syndrome. The current gold standard for determination of fetal lung maturity is the evaluation of phospholipids in amniotic fluid samples by thin-layer chromatography, but it is time-consuming and not continuously available at most institutions. In this study we compare Cell Dyn Emerald and Cell Dyn Ruby method, which is expected to be a review for lamellar body count method. Purpose: The aim of this study was to analyze lamellar body count with Cell Dyn Emerald and Cell Dyn Ruby method on preterm birth. Methods : This was a cross sectional study. Thirty three samples study were inpatient’s amniotic fluid with premature rupture of membranes in Obstetry and Gynecology ward emergency room Dr. Soetomo Hospital Surabaya. Lamellar body count was counted with Cell Dyn emerald and Cell Dyn Ruby method. The statistical differences were assessed using the ANOVA test . Results : The results showed significant differences (t=49,04), lamellar body count with Cell Dyn Ruby method was much lower than Cell Dyn Emerald method. The lowest result with Cell Dyn Ruby method was 3.38 x 103/µL and 17 x 103/ µL with Cell Dyn Emerald method. The highest results with Cell Dyn Ruby method was 98,2 x 103/ µL and 221 x 103/ µL with Cell Dyn Emerald method. Conclusion : Lamellar body count with impedance method (Cell Dyn Emerald) is significantly higher than optic method (Cell Dyn Ruby).

Serum Haptoglobin and Hemopexin Levels in Pediatric SS and SC Disease Patients: Biomarker of Hemolysis and Inflammation

▪INTRODUCTION: Hemolysis is a hallmark of sickle cell disease (SCD) pathogenesis, with the release of hemoglobin (Hb) and heme into the plasma that can promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin (Hp) and hemopexin (Hpx) tightly bind free Hb and heme, respectively, thwarting these clinical sequelae. Hp-Hb and Hpx-heme complexes bind to CD163 and CD91 receptors found primarily on macrophages and hepatocytes, respectively, and are taken up by receptor-mediated endocytosis. In SCD patients it is generally believed that chronic hemolysis depletes plasma haptoglobin and hemopexin levels, however we found only one paper in the literature that has reported plasma Hp and Hpx levels in SCD patients, albeit in a limited number of SS (n=5) and SC (n=3) patients (Muller-Eberhard U et al. Blood 1968). Here we report serum Hp and Hpx levels in 272 Brazilian SS, SC, and AA children along with systemic biomarkers of hemolysis, iron metabolism, inflammation leukocyte and platelet counts, and lipid metabolism. We hypothesize that serum Hp and Hpx levels will be significantly lower and biomarkers of hemolysis, iron metabolism, and inflammation will be significantly higher in SS and SC children compared to AA children. METHODS: The present study used blood samples collected prospectively from 272 unrelated steady-state SCD children with SS (n=179) and SC (n=93) disease. Patients were followed at Bahia Pediatrics Centers, during January 2010 to November 2015. The study included 28 healthy AA volunteers randomly selected from the Clinical Laboratory of the Faculdade de Farmácia da Universidade Federal da Bahia. The study was approved by the Fundação de Pesquisa Oswaldo Cruz research board, and all procedures were in accordance with the Declaration of Helsinki of 1975, as revised in 2000. Biochemical and inflammatory analyses were performed by immunochemistry assay and immunoassay. Hematological analyses were carried out using an electronic cell counter and the Hb profile was investigated by high performance liquid chromatography. Serum Hp and Hpx levels were measured by enzyme-linked immunoassay following the manufacturer’s instructions. RESULTS: Evaluating the Hp and Hpx levels, we found that SS and SC patients have lower Hp and Hpx levels (SS<SC<AA) than AA controls (p<.001, Table 1). Similarly, SS and SC patients have lower hemoglobin and hematocrits (SS<SC<AA) and increased reticulocyte counts and serum LDH (SS>SC>AA) than AA controls (p<.001, Table 1). Total, direct, and indirect bilirubin, markers of heme degradation, followed a similar pattern (SS>SC>AA, p<.001, Table 1). These data may reflect more intravascular hemolysis in the following pattern: SS>SC>AA. Likewise, serum ferritin levels, a marker of body iron stores, were higher in SS and SC patients than AA controls (SS>SC>AA, p<.001, Table 1). CRP levels and white blood cells count, markers of inflammation, also reflected the same pattern (SS>SC>AA, p<.001, Table 1). In a different pattern, platelet counts were highest in SS patients and lowest in SC patients (SS>AA>SC). Finally, Total, HDL, and LDL cholesterol levels were lowest in patients with more hemolysis (SS<SC<AA, p<.001, Table 1). CONCLUSIONS: We report lower levels of Hp and Hpx in children with SCD (SS<SC<AA). These data support Muller-Eberhard’s 1968 brief report on Hp and Hpx levels in hemolytic conditions and demonstrate significant correlations of serum Hp and Hpx with biomarkers of hemolysis, inflammation, and cholesterol. We speculate that serum Hp and Hpx levels in SCD may serve as clinical indicators of hemolysis, inflammation, and disease severity. [Display omitted] DisclosuresVercellotti:Imara: Research Funding. Belcher:Cydan/Imara: Research Funding; CSL-Behring: Research Funding.

Preparation and stability of milk somatic cell reference materials

Our objectives were to develop a method to produce milk somatic cell count (SCC) reference materials for calibration of electronic somatic cell count (ESCC) using gravity separation and to determine the effect of refrigerated storage (4°C) and freeze-thaw stability of the skim and whole milk SCC reference materials. Whole raw milk was high-temperature short-time pasteurized and split into 2 portions. One portion was gravity separated at 4°C for 22 h and the second portion was centrifugally separated to produce skim milk that was also gravity separated with somatic cells rising to the surface. After 22 h, stock solutions (low SCC skim milk, high SCC skim milk, high SCC whole milk) were prepared and preserved (bronopol). Two experiments were conducted, one to compare the shelf-life of skim and whole milk SCC standards at 4°C and one to determine the effect of freezing and thawing on SCC standards. Both experiments were replicated 3 times. Gravity separation was an effective approach to isolate and concentrate somatic cells from bovine milk and redistribute them in a skim or whole milk matrix to create a set of reference materials with a wider and more uniformly distributed range of SCC than current calibration sets. The liquid SCC reference materials stored using the common industry practice at 4°C were stable (i.e., fit for purpose, no large decrease in SCC) for a 2-wk period, whereas frozen and thawed reference materials may have a much longer useful life. A gradual decrease occurred in residual difference in ESCC (SCC × 1,000/mL) versus original assigned reference SCC over duration of refrigerated storage for both skim and whole milk SCC samples, indicating that milk ESCC of the preserved milks was gradually decreasing during 28 d of storage at 4°C by about 15,000 SCC/mL. No difference in the ESCC for skim milk was detected between refrigerated and frozen storage, whereas for whole milk the ESCC for frozen was lower than refrigerated samples. Future work is needed to determine the time and temperature of longer term frozen storage over which the SCC results are stable.

Evaluation of the Diagnostic Accuracy of Twelve Discrimination Indices for Differentiating β-thalassemia Trait from Iron Deficiency Anemia

The two most frequently encountered microcytic and hypochromic anemias are iron deficiency anemia (IDA) and β-thalassemia trait (βTT) need expensive laboratory tests to differentiate. Several mathematical formulae have been proposed for differentiation of these two entities, based on red blood cell indices generated by electronic cell counters. The diagnostic reliability of the twelve discrimination indices (RBC, MCV, RDW, RDW index, Mentter index, England & Fraser index, Srivastava & Bevington index, Shine & Lal index, Green & King index, Ricerca index, Ehsani index and Sirdah index) was assessed. One hundred twenty four (124) children aged between 4.0 and 17.3 years were enrolled in this study, out of which, 53 cases were β-thalassemia trait and 71 were iron deficient. The receiver operative characteristic (ROC) curve was constructed for each index to calculate the area under the curve (AUC), in addition, sensitivity, specificity, and likelihood ratios were calculated. None of the above mentioned discrimination indices found to be 100% sensitive and specific, though some of these indices (RDW, MCV, S&LI) showed more than 80% sensitivity, but their specificity for the detection of IDA was not satisfactory. EI and G&KI showed largest AUC (0.773 and 0.771 respectively) with both sensitivity and specificity above 70%. It was concluded that EI and G&KI could be used as a screening tool for differentiation of these two conditions.

Evaluation of Platelet and Red Blood Cell Parameters with Proposal of Modified Score as Discriminating Guide for Iron Deficiency Anemia and β-Thalassemia Minor.

Iron Deficiency Anaemia (IDA) and β-Thalassaemia Minor (BTM) are considered to be important cause of microcytic hypochromic anaemia. Studies have evaluated various red cell parameters which are easily available on electronic cell counters for discrimination of IDA and BTM in different ethnic populations. The analysis of previously established red cell discriminative indices with new cut-off have also been done by studies which may be relevant in their set of population for differentiation. The study was conducted to propose a modified score considering the established red blood cell indices with a new cut off and to formulate index taking into consideration Red Blood Cell (RBC) and platelet parameters for early differentiation of IDA and BTM. The prospective study included cases with MCV< 80 fl and new modified score of 11 was proposed by statistically analysing the previous discriminative indices with new cut-off by giving score 0 for IDA and score 1 for BTM. The summation of all scores gave modified 11 T score. A new cut off for differentiation of IDA and BTM was proposed in the study by using ROC curve and analysing AUC which statistically corresponded to highest accuracy. An attempt to formulate a new index using the RBC and platelet parameters was also made for initial discrimination. The study included 153 cases and in addition to red blood cell parameters, mean platelet volume and platelet distribution width also showed statistical significant difference between IDA and BTM (p<0.05). Modified new 11 T score was 87.6% specific for BTM while proposed index showed 80.4% negative predictive value for BTM and correctly identified 75% of cases. The proposed new index and modified 11T score may be used for initial discrimination of BTM and IDA especially in resource limited regions. Apart from RBC parameters, mean platelet volume and platelet distribution width may also be useful in early differentiation. It is essential to determine cut off of every index in given population for differentiation of these two conditions.

Effectiveness of naked eye single tube osmotic fragility test for screening of beta-thalassemia trait from north Maharashtra region, India

Background: Thalassemia’s are serious public health problem in all over the world. Different techniques are used for the diagnosis of different types of thalassemia’s which are costly and time consuming. This study was aimed to determine the efficacy of naked eye single tube osmotic fragility test as a screening test for β-thalassemia trait and to standardize a saline concentration which could give best results with maximum sensitivity and specificity. Methods: Three different buffered saline concentrations (0.32%, 0.34% and 0.36%) were used. The test was applied to blood samples of 1702 family members from 130 β-thalassemia major patients. Results: The test results was compared and on the basis of our findings, recommend the use of 0.36% buffered saline. The test was positive in 589/629 subjects with β-thalassemia traits. The sensitivity of the test was 93.6% and specificity was 89.1%. There were 10.9% false positive results in hematologically normal subjects. The test also gave positive results with 3/13 α-thalassemia and 20/24 double heterozygous sickle/β-thalassemia. Conclusion: The test was proved to be useful in areas with high frequency of hemoglobinopathies and found simple, cheap and adaptable in under-resourced laboratories. However, it cannot replace red cell indices generated by automatic electronic cell counters.

Effect of Iron Deficiency Anemia on Intellectual Performance of Primary School Children in Islamabad, Pakistan

Purpose: To assess the differences, if any, in the intellectual performance scores of children who have iron deficiency anemia and those who are neither anemic nor iron-deficient in New Kashmir Model School, Islamabad, Pakistan.Methods: A total of 108 primary New Kashmir Model School children of Islamabad, Pakistan between the ages of 7 to 9 years were included in the study. They were divided into two groups based on Iron deficiency anemia and Non-anemia children’s. A brief clinical history and physical examination was performed. All the 5 mandatory subjects of WISC-R were administered to the children of both groups. Electronic cell counter was used for the evaluation of hemoglobin, packed cell volume (PVC), mean cell volume (MCV), Mean cell hemoglobin (MCH) while serum parameters were determined after first separating serum from blood. Giemsa stain was used to evaluate red cell morphology.Results: Seventy six of these children had iron deficiency anemia based on their hemoglobin, packed cell volume, serum iron, total iron binding capacity and serum ferritin levels. The remaining 32 children were neither anemic nor iron-deficient based on their profiles. Intellectual performance scores which comprised of central nervous system (CNS) impact, as well as hemoglobin, packed cell volume, serum iron, total iron binding capacity and serum ferritin levels and also clinical parameters for the iron deficiency anemia group, were significantly lower than those of the non-anemia group.Conclusion: Iron deficiency anemia appears to be associated with lower intellectual performance scores in school children. These results support previous findings that iron depletion leads to psychological and developmental effects in children.Keywords: Intellectual performance, Anemia, Children, Psychological and developmental effects

Mutagenic potential of the isoflavone irilone in cultured V79 cells

After consumption of red clover-based dietary supplements, plasma concentrations of the isoflavone irilone (IRI) equal that of the well-investigated daidzein. Since some isoflavones are genotoxic, the potential of IRI to induce mutations was investigated.Gene mutations were determined by hypoxanthine-guanine phosphoribosyltransferase (HPRT) assay and sequencing of mutant cDNA, chromosome and genome mutations by micronucleus assay complemented by immunochemical staining of centromere proteins and microtubules in cultured V79 cells. Cell proliferation was monitored by electronic cell counting, flow cytometry and fluorescence microscopy.IRI did not affect the mutant frequency in the Hprt locus but altered the mutation spectrum by increasing the proportion of deletions and decreasing that of base pair substitutions. Induction of chromosome mutations was supported by a slight but significant increase in the number of micronucleated cells containing chromosomal fragments despite activation of three cell cycle checkpoints possibly interfering with micronuclei formation. Moreover, IRI exhibited a strong aneugenic potential characterized by disrupted mitotic spindles, mitotic arrest, and asymmetrical cell divisions leading to chromosome loss, nuclear fragmentation as well as mitotic catastrophe. Thus, IRI might be another isoflavone to be taken into account in safety assessment of dietary supplements.

Paraoxonase 1 Activity and Pon 1 Gene Polymorphisms As a Potential Prognostic Biomarker in Sickle Cell Disease

Background: Sickle cell disease (SCD) has a clinical heterogeneity and vasoocclusive events are an important clinical characteristic among patients. The paraoxonase 1 (PON 1) is a serum glycoprotein associated with the high-density lipoproteins cholesterol (HDL-C) and the variability of the PON1 activity has been associated with the PON1 genotypes. We tested a possible relevance of the PON1 in SCD patients in steady and crisis state, studying its activity and genotype, investigating M55L (rs854560) and R192Q (rs662) polymorphisms, and their association with markers of hemolysis, inflammation and organ dysfunction.Methods: The casuistic of the study was compound by 373 individuals, 154 SCD patients in steady state, 23 SCD patients in crisis, and 196 healthy controls. Chemistry, inflammatory and hematological biomarkers were investigated by chemiluminescence, immunoassay and electronic cell counter respectively. Molecular biology analyses were performed by PCR and PCR-RFLP techniques.Results: The allelic frequency of PON1192R among SCD patients group was 0.38 and of PON1192Q was 0.62. The allelic frequency of the control group individuals was 0.45 to the PON1192R and 0.55 to PON1192Q. The allelic frequency of PON1M55L gene polymorphism was 0.91 to the PON155L and 0.09 to the PON155M among the SCD patients group. The allelic frequency of the PON155L was 0.85 and for the PON155M was 0.15 among individuals of the control group. PON1 gene polymorphisms allelic frequencies were in Hardy-Weinberg equilibrium. Patients with genotype RR of PON1192 (p=0.0015) and MM of PON155 (p=0.0013) showed less PON1 activity than others genotypes. PON1 activity was higher among SCD patients in crisis and steady states than in control group individuals (p<0.0001). The PON1 activity was higher among SCD patients with higher concentrations of high density lipoprotein cholesterol (HDL-C) (p-0.03), higher iron serum levels (p=0.007), and lower ferritin levels (p<0.0001). SCD patients with the PON1c.55M allele had lower platelet count (p=0.011), creatinine (p=0.033), and C Reactive Protein (p=0.014), and patients with the PON1192R alleles had higher triglycerides (p=0.013) and very low lipoprotein cholesterol (VLDL-C). Related to clinical characteristics SCD patients that experienced stroke (p=0.045) had lower PON1 activity and patients that made splenectomy (0.011) had highest PON1 activity.Conclusion: The PON1 activity and genetic alteration were not explored among SCD patients and may represent a new insight to explain endothelial dysfunction and oxidative stress reaction among these patients. Also, PON1 may represent an important molecule in SCD pathogenesis and the associated mechanisms need to be evaluated once it seems to be delivery from the HDL-C structure under a very intense hemolytic and inflammatory environment as happened in SCD, and may be explore as a further therapeutic strategy in disease. DisclosuresNo relevant conflicts of interest to declare.

Phytoplankton Communities in Green Bay, Lake Michigan after Invasion by Dreissenid Mussels: Increased Dominance by Cyanobacteria

Biological invasions of aquatic systems disrupt ecological communities, and cause major changes in diversity and ecosystem function. The Laurentian Great Lakes of North America have been dramatically altered by such invasions, especially zebra (Dreissena polymorpha) and quagga (D. rostriformis bugensis) mussels. Responses to mussel invasions have included increased water clarity, and decreased chlorophyll and phytoplankton abundance. Although not all systems have responded similarly, in general, mussels have changed nutrient dynamics and physical habitat conditions. Therefore examination of different impacts can help us further understand mechanisms that underlie ecosystem responses to biological invasions. To aid our understanding of ecosystem impacts, we sampled established locations along a well-studied trophic gradient in Green Bay, Lake Michigan, after the 1993 zebra mussel invasion. A strong trophic gradient remained during the period sampled after the mussel invasion (2000–2012). However, mean summer chlorophyll increased and other measures of phytoplankton biomass (microscope and electronic cell counting) did not change significantly. Multivariate analyses of phytoplankton community structure demonstrate a significant community shift after the invasion. Cyanobacteria increased in dominance, with Microcystis becoming the major summer taxon in lower Green Bay. Diatom diversity and abundance also increased and Chlorophyta became rare. Phytoplankton responses along the trophic gradient of Green Bay to zebra mussel invasion highlight the importance of mussel effects on nutrient dynamics and phytoplankton diversity and function.

Etiologia e fatores de risco para a infecção mamária de cabras leiteiras em seis propriedades do Estado de São Paulo

The aim of this study was to identify etiologic agents and risk factors for mastitis in dairy goats. For this, two aliquots were collected from 257 milk samples, between April and September, 2012, from 136 Saanen goats from six intensive management farms, with manual and mechanical milking system located in the state of São Paulo. The first aliquot of milk samples was obtained in vials containing preservative bronopol for electronic somatic cell count (SCC), and the second in sterile bottles for bacteriological examination. For risk factors evaluation, epidemiologic questionnaires were performed by the research team by visual observation and survey with handler and owners. Analyzes univariate and multivariate were performed using multiple logistic regression. Was observed prevalence of 28.79% of mammary infection, and predominance of coagulase-negative Staphylococcus (CNS). The SCC median was 4,7x105 cells per mL (cells mL-1) of milk. In univariate analysis, was observed risk for mammary infection in places where, the goat was not the main activity of the property (p=0.03); when the diagnosis of clinical mastitis by proof of dark background was not performed (p=0.07); when chlorine was not used in the cleaning of the teats (p=0.29); improper cleaning of the milking parlor (p= 0.06); inadequate hygiene in the milking process (p=0.03) and inadequate hygiene of the milkers (p=03). The main group of pathogen isolated in this study was CNS. The biggest risk associated with mammary infection was the hygiene of ceilings, room, milker and also the process of milking resulting from these microorganisms. Thus, despite indications of hygienic measures in the programs of milk quality and mastitis control, the applicability and awareness of employees involved in the production chain of milk goats are not sensitized. Therefore, the personnel training is the principal measure recommended for the control of mastitis in dairy goats.

PS-090 Hereditary Spherocytosis And Red Cell Indices Mchc, Mcv, Rdw

Background Hereditary spherocytosis (HS) is a common inherited disorder that is characterised by anaemia, jaundice, and splenomegaly. Clinical severity is variable with most patients having a well-compensated hemolytic anaemia. The primary lesion in HS is loss of membrane surface area, leading to reduced deformability due to defects in the membrane proteins ankyrin, band 3, beta spectrin, alpha spectrin, or protein 4.2. Many isolated mutations have been identified in the genes encoding these membrane proteins; common hereditary spherocytosis-associated mutations have not been identified. The classic laboratory features of HS include minimal or no anaemia, reticulocytosis, an increased mean corpuscular haemoglobin concentration (MCHC), spherocytes on the peripheral blood smear, hyperbilirubinemia, and abnormal results on the osmotic fragility test. Aim Of the study is to evaluate the role of MCV, MCHC as a screen test to diagnose spherocytosis Methods In our study are included 60 subjects, 30 children with HS and 30 children-control groups. Our patients with anaemia, jaundice, and splenomegaly are diagnose with HS by incubated osmotic fragility test, performed after incubating RBCs for 18–24 h under sterile conditions at 37°C. Results We found that 25% of pts. have mild HS, 20% moderate HS, 30% moderate to severe HS and 25% severe HS. In peripheral blood smear 7% of pts. had 0–5 spherocites for field, 30% had 5–10 spherocites for field and 63% had 10–15 spherocites for field. 70% of pts. With HS have MCHC > 38%. There are a positive correlation between MCHC and spherocites in peripheral blood smear (r = 0,898, p Conclusion The dedication of hiperdense erythrociyte today is used as a new tool in diagnosing HS. The determination of MCHC constantly growing with other red cell index, MCV 15 obtained from an electronic cell counter usually is enough to suggest for HS. Key Words Spherocytosis, MCHC, anaemia, children.