Evaluation of zebu nellore cattle blood samples using the Cell-Dyn 3500 hematology analyzer

The Cell-dyn 3500 is a multiparameter flow cytometer, which may analyze samples from several species performing several simultaneous analyses. It is able to perform white blood cells, red blood cells and platelet counts, besides differential leukocyte counts, packed cell volume and hemoglobin determination. Cell-Dyn 3500 performs total leukocyte count both optically and by impedance. The equipment may choose one or other method, based on the reliability of the results. Erythrocyte and platelet counts are determined by impedance. Leukocyte differentiation is based on an optical principle, using separation in multiangular polarized light. The objective of this study was to compare the results of complete blood count of Zebu Nellore heifers from Cell-dyn 3500, with those obtained from a semi-automated cell counter (Celm CC 510) and the manual technique. Blood samples were collected from the jugular vein in 5 mL EDTA vacuum tubes from 58 Nellore heifers, at 24 months of age. Samples were processed in parallel in the three different techniques. Results were analyzed using paired t test, Pearson's correlation and the Bland-Altmann method. There was a strong correlation for all parameters analyzed by Cell-Dyn 3500, manual method and semi-automated cell counter, except for basophils and monocytes counts. These results confirm that this analyzer is reliable for blood samples analysis of zebu cattle.

Valores da Amplitude de Distribuição do Tamanho dos Eritrócitos (RDW) em eqüinos Puro Sangue Inglês (PSI) submetidos a exercícios de diferentes intensidades

In order to evaluate the influence of exercise of different intensities on Red Blood Cell Distribution width (RDW) and Packed Cell Volume (VG) in Thoroughbred horses, blood was collected from 60 animals, 30 males and 30 females, subdivided in groups of horses with 24 to 36 months of age and not in training, and after 12 months of training, and horses with 36 to 48 months of age in training. Blood samples where collected before and after trot and gallop. Samples where analyzed with a automatic cell counter (Cell-Dyn 3500R, Abbott Diagnostic). Red Blood Cell Distribution width (RDW) values increased significantly after trot and gallop demonstrating a variation in the size of red blood cells, while Packed Medium Cell Volume (VGM) values did not show variations before or after exercise.

Standardization of platelet parameters determination in blood samples collected with EDTA

The Abbott Cell-Dyn 3000 automated hematological analyzer prepares a histogram of platelet volume, which is measured by a technique using electrical impedance, inferring from its platelet count (PLT), mean platelet volume (MPV) and platelet distribution width (PDW). This equipment also calculates the plateletcrit (PCT). These platelet parameters may be important to evaluate platelet function, but they require standardization, because platelets swell when in contact with ethylenediaminetetra-acetic acid salts, hence an increase in blood sample storage time produces artificially increased results. To assess the effect of storage time on MPV, PLT, PDW and PCT, blood samples from 23 sickle cell anemia patients during steady state (Group I) and 50 from healthy controls (Group II) were placed in Vacutainer® tubes with dipotassium ethylenediaminetetra-acetic acid and measured over a period of 1440 minutes (24 h) at the following times: immediately after the venipuncture (time 0), 15, 30, 60, 120, 240, 360, 480 and 1440 minutes. The mean values of MPV and PCT were significantly increased (p<0.00001) along the storage time in both groups. The mean values of PLT and PDW were practically stable (p>0.05) throughout the storage time in both groups.

Contagem automática de plaquetas : ação de um aminoglicosídeo na pseudotrombocitopenia induzida pelo ácido etilenodiaminotetracético tripotássico

Introdução: A pseudotrombocitopenia dependente de EDTA (EDTA-PTCP) consiste numa falsa diminuição na contagem de plaquetas (PLT), em amostras de sangue colhidas em EDTA, devido a aglutinação plaquetar. Estudos publicados referem que a aglutinação plaquetar na presença de EDTA se pode evitar e dissociar com a adição de um aminoglicosídeo. Objectivos: 1) avaliar a eficácia do aminoglicosídeo gentamicina (Gent) na dissociação de aglutinados plaquetares em amostras de sangue total colhidas em EDTA; 2) verificar se existe alterações nos outros parâmetros hematológicos e na morfologia do sangue periférico, após adição de Gent; 3) detetar o envolvimento das frações GPllb ou GPllIa do complexo GPllb/llIa no mecanismo de formação dos aglutinados plaquetares induzidos pelo EDTA. Material e Métodos: 145 amostras de sangue periférico em EDTA-K3 e com trombocitopenia, processadas no CELL-DYN Sapphire TM. De acordo com padrão gráfico característico em Mono-Poly I, amostras reprocessadas após adição de 20 μL de Gent, com a mesma metodologia. Esfregaços de sangue periférico (ESP) corados por May-Grünwald-Giemsa. 13 amostras processadas com contagem imunológica de PLT (CD61), antes e após Gent. Comparação dos valores dos diferentes parâmetros antes e após adição de Gent: Teste T- student emparelhado, significado estatístico: p<0,05. Resultados: 138 amostras com diferença estatisticamente significativa na contagem de PLT antes e após Gent, com desaparecimento do padrão inicial em Mono-Poly I: 97 EDTA-PTCP (p<0,0001) e 41 trombocitopenias reais (p<0,0001). Sete amostras sem aumento na contagem de PLT (P=0,15) com Gent, nem alteração em Mono-Poly I, sugerindo presença de microcoágulos, confirmados em ESP. Principais parâmetros hematológicos sem diferenças significativas (p>0,05), após adição de Gent, com exceção do VPM (P<0,0001) e Ptc (p<0,0001). Morfologia do sangue periférico sem alterações. As treze amostras processadas com CD61 antes de Gent tinham um número de PLT superior ao obtido na contagem ótica antes de Gent. Conclusões: A adição de Gent às amostras em EDTA induz uma rápida dissociação dos aglutinados, sem causar alterações morfológicas nem dos principais parâmetros hematológicos. Apenas a GPllb parece estar envolvida no mecanismo de formação dos aglutinados plaquetares. O uso de Gent revelou-se muito útil, rápido e com baixo custo nas EDTA-PTCP, permitindo rentabilizar a tecnologia existente, com grande benefício para o doente.

Deteção/quantificação de eritrócitos com hemoglobina fetal na gravidez e pós parto utilizando um analisador hematológico

Introdução: As células F(CF) são eritrócitos contendo hemoglobina (Hb) F e outros tipos de hemoglobina. São encontradas em indivíduos de todas as idades, ao contrário dos eritrócitos fetais, só encontrados em fetos e recém-nascidos. Nestes eritrócitos fetais, a Hb F é o tipo dominante de Hb. Estudos publicados indicam que a gravidez pode levar a um aumento progressivo de células com Hb F no sangue materno, devido à presença de CF e/ou de células fetais, estas últimas frequentemente associadas a hemorragia feto–materna (HFM). Objetivo: (1) Determinação da percentagem (%) de células com Hb F em sangue materno, usando um anticorpo Anti-Hb F num analisador hematológico. (2) Quantificação de CF e/ou células fetais na gravidez e pós o parto. (3) Elaboração de algoritmo para a triagem de HFM. Material e Métodos: Estudadas 168 amostras de sangue materno: 29 no 1º trimestre da gravidez (1ºT); 43 no segundo (2º T); 82 no terceiro (3ºT), 14 pós-parto (PP) (amostras entre dia 0 e dia 7, após parto); 32 controlos negativos (Ctl N) com homens adultos saudáveis e 30 controlos positivos (Ctl P), obtidos por mistura de sangue do cordão com sangue do adulto, AB0 compatíveis. Amostras processadas no analisador hematológico Cell-Dyn Sapphire tm, em modo RBC Flow, após ajuste de parâmetros IAS, FL1 e FL3, utilizando um reagente com iodeto de propídio e 2,5 uL de anticorpo monoclonal anti-Hb F FITC. Imagens analisadas pelo software FCS Express V3. Análise estatística com Kruskal-Wallis e teste t-Student (significância estatística p <0,05). Cut-off para HFM obtido pelo valor mínimo, em %, em que se detetam células fetais na amostra Ctl P. Resultados: foram encontradas diferenças estatisticamente significativas na % células com Hb F (P<0,0001) nos grupos estudados. % CF aumenta com a gravidez:1ºT vs Ctl N - p <0,0217 e também durante a gravidez 1ºT Vs 3ºT – P=0,0007. Mesmo depois do PP a % CF está aumentada Ctl N vs PP: - p<0,0001. Valores médios de % CF residuais em adultos saudáveis: 0,53. Maioria das amostras nos diferentes grupos estudados apresenta % CF acima do valor residual (>0,53%): 66% no grupo 1ºT, 83 % no 2º T e 91% no 3º T. Valor cut-off para suspeita de HFM de 1,70% de células com Hb F. Teste preciso (CV+- 4%) para baixas % de células com Hb F. Discussão/Conclusão: Há um aumento células com Hb F durante a gravidez e esse aumento permanece no período PP. Em duas amostras do 3ºT obteve-se % células com Hb F elevada, superior ao cut-off (≥1,70%), sendo detetada uma população de prováveis células fetais. A presença células fetais nestas amostras foi confirmada por citometria de fluxo com Anti-Hb F/ Anti-CA, com subsequente diagnóstico de HFM. Esta metodologia é simples, rápida e não dispendiosa, quando aplicada a um analisador hematológico, representa uma mais-valia no rastreio da HFM. No futuro, pode integrar o protocolo de análises de rotina das grávidas, permitindo detetar as HFM silenciosas, que são a origem de muitas anemias de causa desconhecida em recém-nascidos.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Solid Lipid Nanoparticles For Hydrophilic Biotech Drugs: Optimization And Cell Viability Studies (caco-2 & Hepg-2 Cell Lines).

Insulin was used as model protein to developed innovative Solid Lipid Nanoparticles (SLNs) for the delivery of hydrophilic biotech drugs, with potential use in medicinal chemistry. SLNs were prepared by double emulsion with the purpose of promoting stability and enhancing the protein bioavailability. Softisan(®)100 was selected as solid lipid matrix. The surfactants (Tween(®)80, Span(®)80 and Lipoid(®)S75) and insulin were chosen applying a 2(2) factorial design with triplicate of central point, evaluating the influence of dependents variables as polydispersity index (PI), mean particle size (z-AVE), zeta potential (ZP) and encapsulation efficiency (EE) by factorial design using the ANOVA test. Therefore, thermodynamic stability, polymorphism and matrix crystallinity were checked by Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD), whereas the effect of toxicity of SLNs was check in HepG2 and Caco-2 cells. Results showed a mean particle size (z-AVE) width between 294.6 nm and 627.0 nm, a PI in the range of 0.425-0.750, ZP about -3 mV, and the EE between 38.39% and 81.20%. After tempering the bulk lipid (mimicking the end process of production), the lipid showed amorphous characteristics, with a melting point of ca. 30 °C. The toxicity of SLNs was evaluated in two distinct cell lines (HEPG-2 and Caco-2), showing to be dependent on the concentration of particles in HEPG-2 cells, while no toxicity in was reported in Caco-2 cells. SLNs were stable for 24 h in in vitro human serum albumin (HSA) solution. The resulting SLNs fabricated by double emulsion may provide a promising approach for administration of protein therapeutics and antigens.

Autologous Platelet Gel: Five Cases Illustrating Use On Sickle Cell Disease Ulcers.

Leg ulcers represent a particularly disabling complication in patients with sickle cell disease (SCD). Platelet gel (PG) is a novel therapeutic strategy used for accelerating wound healing of a wide range of tissues through the continuous release of platelet growth factors. Here, we describe the use of PG preparation according to Anitua's PRGF (preparations rich in growth factors) protocol for treating chronic nonhealing ulcers in patients with SCD. A positive response occurred in 3 patients with an area reduction of 85.7% to 100%, which occurred within 7 to 10 weeks, and a 35.2% and 20.5% of area reduction in 2 other patients, who however, had large ulcers. After calcium chloride addition, the platelet-rich plasmas demonstrated enhanced platelet-derived growth factors-BB (P < .001), transforming growth factor-β1 (P = .015), vascular endothelial growth factors (P = .03), and hepatocyte growth factors (nonsignificant) secretion. Furthermore, calcium chloride addition induced a significant decrease in platelet number (P = .0134) and there was no leukocyte detection in the PG product. These results demonstrate that PG treatment might impact the healing of leg ulcers in sickle cell disease, especially in patients with small ulcers.

Impaired Compensatory Beta-cell Function And Growth In Response To High-fat Diet In Ldl Receptor Knockout Mice.

In this study, we investigated the effect of low density lipoprotein receptor (LDLr) deficiency on gap junctional connexin 36 (Cx36) islet content and on the functional and growth response of pancreatic beta-cells in C57BL/6 mice fed a high-fat (HF) diet. After 60 days on regular or HF diet, the metabolic state and morphometric islet parameters of wild-type (WT) and LDLr-/- mice were assessed. HF diet-fed WT animals became obese and hypercholesterolaemic as well as hyperglycaemic, hyperinsulinaemic, glucose intolerant and insulin resistant, characterizing them as prediabetic. Also they showed a significant decrease in beta-cell secretory response to glucose. Overall, LDLr-/- mice displayed greater susceptibility to HF diet as judged by their marked cholesterolaemia, intolerance to glucose and pronounced decrease in glucose-stimulated insulin secretion. HF diet induced similarly in WT and LDLr-/- mice, a significant decrease in Cx36 beta-cell content as revealed by immunoblotting. Prediabetic WT mice displayed marked increase in beta-cell mass mainly due to beta-cell hypertrophy/replication. Nevertheless, HF diet-fed LDLr-/- mice showed no significant changes in beta-cell mass, but lower islet-duct association (neogenesis) and higher beta-cell apoptosis index were seen as compared to controls. The higher metabolic susceptibility to HF diet of LDLr-/- mice may be explained by a deficiency in insulin secretory response to glucose associated with lack of compensatory beta-cell expansion.

Integrated Proteomics Identified Up-regulated Focal Adhesion-mediated Proteins In Human Squamous Cell Carcinoma In An Orthotopic Murine Model.

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Interplay Between Cell Growth And Cell Cycle In Plants.

The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.