Influence of the concentration of EDTA, time and temperature of storage on hematological parameters of dogs in count blood cells automated and manual

O estudo teve como objetivo identificar os efeitos do tempo, da temperatura de armazenamento e do excesso de anticoagulante sobre parâmetros hematológicos de cães. Foram utilizadas amostras do sangue de dez cães de raças variadas, clinicamente hígidos. As alíquotas foram colhidas com 1,8 mg, 3,6 mg, 7,2 mg e 14,4 mg de ácido etilenodiaminotetraacético (EDTA) por mL de sangue, distribuídas em dois grupos: de 2ºC a 8ºC e temperatura ambiente.Após a coleta, foram avaliadas em quatro tempos: 0, 12, 24 e 48 h. Usando um contador automático de células, foram avaliados leucócitos, hemácias, hemoglobina, hematócrito, volume corpuscular médio (VCM), hemoglobina corpuscular média (HCM), concentração de hemoglobina corpuscular média (CHCM), índice de anisocitose eritrocitária (RDW), plaquetas, plaquetócrito (PCT), volume plaquetário médio (VPM) e índice de anisocitose plaquetária (PDW). Simultaneamente à análise realizada no contador automático, foi determinado o hematócrito, por meio da técnica do microhematócrito. Também se procedeu à dosagem da hemoglobina com espectrofotômetro, contagem global de leucócitos, em câmara de Neubauer, e contagem diferencial de leucócitos por microscopia de esfregaços corados, além da dosagem da proteína plasmática, por refratometria. Na análise com contador automático, o VCM diminuiu nas maiores concentrações de EDTA (7,2 mg/ml e 14,4 mg/ml) atingindo o máximo de 2,36%, na maior concentração. A temperatura e o tempo também influenciaram este parâmetro, que apresentou decréscimo no tempo de 12 horas, temperatura de 2ºC a 8ºC e aumento nos tempos 24 horas e 48 horas, na temperatura ambiente (p<0,05). HCM sofreu alterações com relação ao tempo e à temperatura e a CHCM, com relação ao tempo, à temperatura e a concentração de EDTA (p<0,05). O VPM e o PDW apresentaram discreto aumento, ao longo do tempo. A temperatura de conservação influenciou discretamente a contagem de leucócitos e eritrócitos, que apresentaram valores menores na temperatura ambiente. Hemoglobina, hematócrito, plaquetas e PCT não apresentaram alteração significativa. As alterações observadas, não comprometeram os resultados obtidos no contador automático, mostrando que as amostras sanguíneas, conservadas por dois dias, se mantiveram em boas condições para processamento, principalmente quando armazenadas sob refrigeração. Nos dados obtidos sem automação, o hematócrito diminuiu de forma significativa com o aumento da concentração de EDTA atingindo na maior concentração um decréscimo médio de 25,35%. O tempo também influenciou o hematócrito, que apresentou aumento (p<0,05) no tempo 48 horas. O fator tempo influenciou positivamente a proteína, que teve um aumento médio de 11%, no tempo 48 horas. A proteína também foi influenciada pela concentração de EDTA, apresentando aumento considerável em seu valor médio, na concentração 14,4 mg/mL de sangue. Na contagem diferencial à microscopia óptica foram detectadas alterações no número de neutrófilos e monócitos, com relação ao tempo e à concentração de EDTA (p<0,05). A contagem de bastonetes sofreu alterações mais significativas, relacionadas com o tempo e a temperatura (p<0,05). Leucócitos, hemoglobina, linfócitos, eosinófilos e basófilos não apresentaram alteração relevante (p<0,05). O decréscimo do hematócrito nas amostras com alta concentração de EDTA demonstrou a importância da relação sangue/EDTA para a qualidade do hemograma, com a utilização da técnica do micro-hematócrito.The study aimed to identify the effects of time, temperature of storage and excess of anticoagulant on hematological parameters of dogs. Blood samples of ten, clinically healthy dogs, of different breeds were utilized. Aliquots were taken with 1.8 mg, 3.6 mg, 7.2 mg and 14.4 mg of ethylenediaminetetraacetic acid (EDTA) per mL of blood, divided into two groups: 2°C to 8ºC and room temperature. Right after collection, they were evaluated in four times: 0h, 12h, 24h and 48 h. White blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) platelet, thrombocrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDW) were evaluated in the automatic cell counter. Hematocrit was determined using the microhematocrit technique, simultaneously with the analysis carried out in the automatic blood cell counter. The hemoglobin determination in the spectrophotometer, the total leukocyte count in the Neubauer chamber, the leukocyte count by microscopy of stained smears and the plasma protein dosage by refractometry were also done. In automatic cell counter analysis, the MCV increased significantly in higher concentrations of EDTA (7.2 mg / ml and 14.4 mg / mL) peaking at2.36%, in the highest concentration. Temperature and time also influenced the MCV, which showed a decrease in time 12 hours, in the temperature of 2 º C to 8 º C and an increase in time 24 hours and 48 hours at room temperature. MCH showed minor changes related to time and temperature and MCHC, related to time, temperature and EDTA. MPV and PDW showed a slight increase over time. Storage temperature influenced slightly in the leukocytes anderythrocytes counts, which showed lower values at room temperature. Hemoglobin, hematocrit, platelet and PCT did not change significantly. The changes observed did not compromise the results obtained automatic cell counter, showing that the blood samples stored for two days remained in good condition for processing, especially when stored under refrigeration. In the data obtained without automation, the hematocrit decreased significantly with the increasing concentration of EDTA: in the highest concentration (14.4 mg of EDTA per mL of blood), it showed an average decrease of 25.35%. The time also affected the hematocrit, which increased (p<0.05) in 48 hours time. The time factor has positively influenced the protein, which had an average increase of 11% in 48 hours time. The protein was also influenced by the concentration of EDTA, with considerable increase in the average value, in the concentration of 14.4 mg/mL of blood. In the differential count, made by microscopy, small changes in the number of neutrophils and monocytes were detected, related to time and the concentration of EDTA. Rods counting suffered the most significant changes related to time and temperature. WBC, hemoglobin, lymphocytes, eosinophils and basophils did not present relevant changes. The hematocrit decrease in the samples with high concentration of EDTA demonstrated the importance of the blood / EDTA proportion to the quality of the blood count, using the microhematocrit technique

Density gradient centrifugation, molecular biology and hematological changes in the diagnosis of canine hemoparasitoses

Neste estudo foram analisadas 100 amostras de sangue de cães com trombocitopenia importante (plaquetas < 100.000/μL), oriundas da rotina laboratorial de atendimento do Hospital Veterinário da Universidade Federal de Viçosa (UFV), Viçosa, Minas Gerais. Todas as amostras de sangue foram submetidas ao analisador hematológico automático, à confecção de esfregaços sanguíneos de sangue total, à capa de leucócitos e à técnica de centrifugação por gradiente de densidade. Além disto, as amostras foram submetidas à extração de DNA e posteriormente à reação em cadeia da polimerase (PCR), para o diagnóstico molecular de Ehrlichia spp., Hepatozoon sp., Babesia spp., Anaplasma spp. e Leishmania spp. Os resultados da PCR confirmaram amostras positivas para Ehrlichia spp. (69%), Hepatozoon sp. (25%), Babesia spp. (16%) e Anaplasma platys (4%), com alguns animais apresentando até três patógenos em coinfecção. Verificou-se ainda estreita relação entre a ocorrência da trombocitopenia e erliquiose e a alta frequência de coinfecções. A centrifugação por gradiente de densidade mostrou-se bastante sensível na identificação do Hepatozoon canis, com resultados superiores aos das técnicas tradicionais (esfregaço sanguíneo e capa de leucócitos) e concordância substancial com a PCR. Foram observadas diferenças significativas nos parâmetros sanguíneos entre os animais com e sem hematozoários, como a presença de desvio nuclear de neutrófilos à esquerda maior nos animais positivos para Babesia spp. e alterações no volume plaquetário médio e PDWc nos animais com erliquiose. No sequenciamento realizado, identificou-se a presença de várias espécies, incluindo Ehrlichia canis, Ehrlichia chaffeensis, Hepatozoon canis, Babesia canis vogeli, Anaplasma platys, Anaplasma phagocytophilum e Rangelia vitalii, sendo os patógenos Ehrlichia chaffeensis e Anaplasma phagocytophilum relacionados a infecções em humanos.In this study we analyzed blood samples from 100 dogs with important thrombocytopenia (Platelets < 100.000/μL), of the routine care of the Veterinary Hospital from Universidade Federal de Viçosa (UFV). All Samples were processed on automated hematology analyzer and subjected to analysis of their blood smears, buffy coat, and the density gradient centrifugation technique. In addition, samples were subjected to procedure for DNA extraction and subsequent polymerase chain reaction (PCR) to Ehrlichia spp., Hepatozoon sp., Babesia spp., Anaplasma spp. and Leishmania spp. The PCR results confirmed positive samples to Ehrlichia spp. (69%), Hepatozoon sp. (25%), Babesia spp. (16%) and Anaplasma platys (4%), with some animals having up to three concomitant pathogens. We also found a close relationship between the occurrence of thrombocytopenia and ehrlichiosis and the high frequency of coinfection. The density gradient centrifugation showed to be more sensitive at detecting Hepatozoon canis than traditional tecniques (blood smears, buffy coat) and the results are in agreement to those by PCR. Significant differences were observed in blood parameters between animals with and without hemoparasite, such as presence of a nuclear deviation of neutrophils on the left bigger in positive animals for Babesia spp. and changes in mean platelet volume and PDWc in animals with ehrlichiosis. In the sequencing performed we identified the presence of several species, including Ehrlichia canis, Ehrlichia chaffeensis, Hepatozoon canis, Babesia canis vogeli, Anaplasma platys, Anaplasma phagocytophilum and Rangelia vitalii, being the Anaplasma phagocytophilum and Ehrlichia chaffeensis related pathogens infections in humans.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Plasma rico em plaquetas para reparação de falhas ósseas em cães

As plaquetas chegam rapidamente ao local da ferida e liberam múltiplos fatores de crescimento (FC) e citocinas que contribuem para a reparação óssea e aumentam a vascularização local. O Plasma Rico em Plaquetas (PRP) concentra as plaquetas e os FC liberados por elas, aceleram a formação óssea e melhora a qualidade do trabeculado. Este trabalho apresenta um protocolo para confecção de PRP e demonstra alguns aspectos da sua utilização na reparação óssea de cães. O protocolo foi desenvolvido a partir de sangue coletado por punção jugular em três cães adultos, pesando em média 20kg. Para avaliação da aplicação clínica e dos aspectos da reparação óssea, foram criadas duas falhas mediais no terço proximal de cada tíbia. Assim, a falha 1 não foi preenchida, constituindo o controle, a falha 2 foi preenchida com 3mg de enxerto ósseo autógeno da crista da tíbia, a falha 3 com gel de plaquetas (PRP) e falha a número 4 com a associação PRP e 3mg de enxerto ósseo autógeno. O protocolo laboratorial proposto mostrou-se de fácil execução e de baixo custo e possibilitou a concentração adequada de plaquetas no PRP final, cujo número foi dependente da contagem inicial no sangue total de cada animal. A comparação da radiopacidade na região da falha, em todos os tratamentos, e ao longo do tempo demonstrou que o PRP associado ao enxerto determinou maior precocidade e uniformidade de radiopacidade, quando comparado à falha preenchida pelo PRP e ao enxerto usados isoladamente, e sendo que ambos determinam melhores resultados de preenchimento que a falha mantida sem tratamento.The platelets arrive quickly at the injury site and release several growth factors (GF) and citokines that contribute to bone repair and increasing local vascularization. The Platelet-rich Plasma (PRP) concentrates the platelets and their growth factors, increasing the rate of bone formation and better quality of trabecular bone. This research presents a protocol to PRP formulation and demonstrates some aspects about the use in canine bone repair. In this protocol blood was obtained from the jugular ven of tree adult dogs with medium weight of 20kg to produce PRP. Two defects in the medial aspect of proximal third of the tibia were surgically created to evaluate the clinical and radiographic aspect of PRP. The control defect wasn’t treated.The defect 2 was filled with 3 mg of autogenous bone graft from the tibia crest. The defect 3 was filled with PRP alone and the number 4 with PRP in the combination with 3mg autogenous bone graft. The proposed laboratory protocol demonstrated to be easy to execute at low cost. Further, it was adequate to concentrate platelets in final PRP, whose number was dependent on the blood from each dog. Comparing the defect regions, was concluded that the association of PRP and bone graft showed greater precocity and uniform radiopacity than the PRP or bone graft isolated, although both determine better results than the defect without treatment

Platelet-rich plasma in the treatment of induced tendinopathy in horses: histologic evaluation

This study was carried out to evaluate the effect of platelet-rich plasma (PRP) on the treatment of tendinopathy induced in the superficial digital flexor tendon (TFDS) of horses, by using histologic evaluation. Six healthy crossbred geldings aged 8 to 15 years (12 ± 3) were used. The TFDS tendinopathy was provoked in both forelimbs, by intratendinous administration of 2.5 mg collagenase (2.5 mg/mL), and this procedure was considered as the beginning of the experimental phase. At 12 days after induction of the tendinopathy, the animals were subjected to the following treatments: (1) in the lesion caused in the right superficial digital flexor tendon (PRP-treated group), 2.5 mL PRP activated with calcium chloride at 0.0125 mol/L at concentrations from 320,000 to 500,000 platelets/μL, were injected; (2) in the tendinopathy of the left SDFT (control group), 2.5 mL 0.9% saline solution was administrated. Thirty-six days after the treatments, a biopsy of the injured area was performed for histologic evaluation. In both groups, the histologic analysis showed an increase in the fibroblastic density, as well as the presence of neovascularization, lymphocytes, and plasmocytes infiltrate and tissue organization at variable intensity. In the PRP-treated group, the SDFT was more organized, with the collagen fibers and fibroblasts being better arranged on the tendon matrix. The numbers of the fibroblasts and blood vessels did not differ between the groups. Histologic evaluation 36 days after tendinopathy showed that injuries under a single PRP treatment present a more uniform and organized tissue repair when compared with the control group

Concentração de anticoagulante, tempo e temperatura de armazenagem sobre os parâmetros hematológicos no hemograma automatizado Anticoagulant concentration, time and storage temperature on hematological parameters in automated blood count

O estudo teve como objetivo identificar os efeitos do tempo de estocagem, da temperatura de armazenamento e da quantidade de anticoagulante sobre parâmetros hematológicos de cães. Foram utilizadas amostras do sangue de dez cães de raças variadas, clinicamente hígidos. As alíquotas foram colhidas com 1,8mg; 3,6mg; 7,2mg e 14,4mg de ácido etilenodiaminotetracético (EDTA) por mL de sangue, distribuídas em dois grupos: de 2&deg;C a 8&deg;C e temperatura ambiente. Após a coleta, foram avaliadas em quatro tempos: 0, 12, 24 e 48 horas. Usando um contador automático de células, foram avaliados leucócitos, eritrócitos, hemoglobina, hematócrito, volume corpuscular médio (VCM), índice de anisocitose eritrocitária (RDW), plaquetas e plaquetócrito (PCT). O valor do VCM diminuiu nas maiores concentrações de EDTA (7,2mg mL-1 e 14,4mg mL-1), com decréscimo de 2,36% na maior concentração. A temperatura e o tempo de armazenagem também ocasionaram alteração nesse parâmetro, ou seja, houve decréscimo no tempo 12 horas à temperatura de 2 a 8&deg;C e aumento nos tempos 24 e 48 horas à temperatura ambiente (P<0,05). A temperatura de conservação influenciou discretamente a contagem de leucócitos e eritrócitos, que apresentaram valores menores na temperatura ambiente. Hemoglobina, hematócrito, plaquetas e PCT não apresentaram alteração significativa. As alterações observadas não comprometeram os resultados obtidos no contador automático, mostrando que as amostras sanguíneas, conservadas por 48 horas, mantiveram-se em boas condições para análise, principalmente quando armazenadas à temperatura de 2 a 8&deg;C.<br>The study aimed to identify the effects of time, temperature of storage and excess of anticoagulant on hematological parameters of dogs. Blood samples of ten, clinically healthy dogs, of different breeds were utilized. Aliquots were stored with 1.8mg, 3.6mg, 7.2mg and 14.4mg of ethylenediaminetetraacetic acid (EDTA) per mL of blood, divided into two groups: 2&deg;C to 8&deg;C and room temperature. Right after collection, they were evaluated in four times: 0h, 12h, 24h and 48h. White blood cells, red blood cells, hemoglobin, packed cell volume (PCV), mean corpuscular volume (MCV), red cell distribution width (RDW), platelet and thrombocrit (PCT) were evaluated in the automatic cell counter. In the automatic cell counter analysis, the MCV increased significantly with higher concentrations of EDTA (7.2mg mL-1 and 14.4mg mL-1) peaking at 2.36%, in the highest concentration. Temperature and time also influenced the MCV, which showed a decrease in time 12 under temperature of 2&deg;C to 8&deg;C and an increase in time 24 and 48 at room temperature. Storage temperature influenced leukocytes and erythrocytes counts, which showed lower values at room temperature. Hemoglobin, PCV, platelet and PCT did not change significantly. The changes observed did not compromise the results obtained by automatic cell counter, showing that blood samples stored for two days remained in good condition for processing, especially when stored under refrigeration

Direct antiviral therapy for treatment of hepatitis C: A real-world study from Brazil

Introduction and objectives: Direct antiviral agents (DAAs) including sofosbuvir (SOF), daclatasvir (DCV), simeprevir (SIM) and ombitasvir, paritaprevir and dasabuvir were introduced 2015 in Brazil for treatment of hepatitis C virus (HCV) infection. The aims of this study were to assess effectiveness and safety of HCV treatment with DAA in real-life world in a highly admixed population from Brazil. Materials and methods: All Brazilian reference centers for HCV treatment were invited to take part in a web-based registry, prospectively conducted by the Brazilian Society of Hepatology, to assess outcomes of HCV treatment in Brazil with DAAs. Data to be collected included demographics, disease severity and comorbidities, genotype (GT), viral load, DAA regimens, treatment side effects and sustained virological response (SVR). Results: 3939 patients (60% males, mean age 58 ± 10 years) throughout the country were evaluated. Most had advanced fibrosis or cirrhosis, GT1 and were treated with SOF/DCV or SOF/SIM. Overall SVR rates were higher than 95%. Subjects with decompensated cirrhosis, GT2 and GT3 have lower SVR rates of 85%, 90% and 91%, respectively. Cirrhosis and decompensated cirrhosis in GT1 and male sex and decompensated cirrhosis in GT3 were significantly associated with no SVR. Adverse events (AD) and serious AD occurred in 18% and 5% of those subjects, respectively, but less than 1% of patients required treatment discontinuation. Conclusion: SOF-based DAA regimens are effective and safe in the heterogeneous highly admixed Brazilian population and could remain an option for HCV treatment at least in low-income countries