Comparing modified Lee and White method against 20-minute whole blood clotting test as bedside coagulation screening test in snake envenomation victims

Abstract Background: Twenty-minute whole blood clotting test (20WBCT) and Modified Lee and White (MLW) method are the most routinely employed bedside tests for detecting coagulopathic snake envenomation. Our study compared the diagnostic utility of MLW and 20WBCT for snakebite victims at a tertiary care hospital in Central Kerala, South India. Methods: This single-center study recruited 267 patients admitted with snake bites. 20WBCT and MLW were performed simultaneously at admission along with the measurement of Prothrombin Time (PT). The diagnostic utility of 20WBCT and MLW was determined by comparing the sensitivity (Sn), specificity (Sp), positive and negative predictive values, likelihood ratios, and accuracy at admission with an INR value > 1.4. Results: Out of 267 patients, 20 (7.5%) patients had VICC. Amongst those who had venom-induced consumption coagulopathy (VICC), MLW was prolonged for 17 patients, (Sn 85% 95% confidence interval [CI]: 61.1-96.0) whereas 20WBCT was abnormal for 11 patients (Sn 55%, 95% CI: 32.04-76.17). MLW and 20WBCT were falsely positive for the same patient (Sp 99.6%, 95% CI: 97.4-99.9%). Conclusion: MLW is more sensitive than 20WBCT to detect coagulopathy at the bedside amongst snakebite victims. However, further studies are necessary for standardizing bedside coagulation tests in snakebite cases.

Prothrombin time and activated partial thromboplastin time reference intervals in dogs, by gender and age group using the Start®4 (Stago) / Intervalos de referência para o tempo de protrombina e tempo de tromboplastina parcial ativada no cão de acordo com a idade e o gênero utilizando o Start ® 4 (Stago)

Prothrombin time (PT) and the activated partial thromboplastin time (aPTT) are useful tools for the diagnosis and monitoring of coagulation disorders in Veterinary Medicine. Our objectives were: to establish reference intervals (RI) for PT and a PTT for the dog using the Start®4 (Stago), to compare the obtained RI with literature; to evaluate the effects of gender and age on the coagulation profile. Plasma samples of 122 healthy dogs (57 males; 65 females) aged between 4 months and 18 years, divided into three age groups (0-2 years old; 3-10 years old; > 10 years old) and grouped in to males and females were analysed. The RI were estimated following the ASVCP guidelines with the Reference Value Advisor software. The RI were: PT 6.7'' to 10.8''; aPTT 9.0'' to 14.8''. PT was significantly higher in females than in males. Dogs aged 10 years or older have significantly higher mean aPTT times than younger dogs. RI comparison showed a considerable percentage of cases outside the reference RI of the literature (PT - 79.3%; aPTT - 77.1%), demonstrating the need of each laboratory to calculate its own RI. The RI established in this study are applicable for the coagulation profile assessment in dogs.

O tempo de protrombina (TP) e o tempo de tromboplastina parcial ativada (TTPa) são ferramentas úteis para o diagnóstico e monitorização das alterações da coagulação em Medicina Veterinária. Os objetivos deste estudo foram: estabelecer intervalos de referência (IR) para TP e TTPa para o cão utilizando o Start®4 (Stago), de modo a comparar os IR obtidos com a literatura; avaliar os efeitos do sexo e da idade no perfil da coagulação. Foram usadas amostras de plasma de 122 cães saudáveis (57 machos; 65 fêmeas) com idades entre quatro meses e 18 anos, divididos em três grupos (0-2 anos; 3-10 anos; > 10 anos) e agrupados em machos e fêmeas. Os IR foram calculados seguindo as diretrizes da ASVCP com o software Reference Value Advisor. Os IR obtidos foram: PT 6,7 '' a 10,8 ''; TTPa 9,0 '' a 14,8 ''. O TP foi significativamente maior nas fêmeas do que nos machos. Os cães com 10 anos ou mais apresentaram tempos médios de TTPa significativamente maiores do que cães mais jovens. A comparação de IR mostrou uma percentagem considerável de casos fora do IR de referência da literatura (TP - 79,3%; TTPa - 77,1%), confirmando a necessidade de cada laboratório calcular seu próprio IR. Os IR estabelecidos neste estudo são aplicáveis na avaliação do perfil hemostático em cães.

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

Hemostatic evaluation of rabbits envenomed with Bothrops alternatus treated with anti-bothropic serum, desmopressin and tranexamin acid / Avaliação hemostática de coelhos envenenados com Bothrops alternatus e tratados com soro antibotrópico, desmopressina e ácido tranexâmico

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly composed of proteolytic enzymes. Although the antibothropic serum produced by the Brazilian Institutes is remarkably efficient, more studies are necessary, especially in veterinary medicine. The venom contain enzymes and non-enzymatic proteins that interfere with hemostasis leading to hemorrhage or even thrombosis. Possible treatment associations with known bothropic antivenom were the reason for the development of the present study. The aim of this study was to evaluate hemostasis alterations caused by Bothrops alternatus venom in rabbits followed by treatments with anti-bothropic serum, tranexamic acid and desmopressin. Twenty New Zealand rabbits were distributed into five groups (n=4) that were experimentally envenomed with 150mcg/kg of B. alternatus venom via intramuscular injection and treated as follow: Group 1 (G1) was the positive control and received venom and PBS/BSA; Group 2 (G2) was treated with tranexamic acid; Group 3 (G3) with desmopressin; Group 4 (G4) with tranexamic acid and anti-bothropic serum; and Group 5 (G5) with anti-bothropic serum and desmopressin. Blood samples were collected before venom administration, and one, four, eight and 12 hours after, for Partial activated partial thromboplastin time, Prothrombin Time, Thrombin Time and fibrinogen evaluation. Thrombin generation (TG) test was carried out with a pool of samples from final times (8 and 12h). At the end of 12h, all animals were euthanized and necropsy was conducted. Samples from muscle tissue, heart, lungs and kidney were analyzed. Classic coagulation tests showed no significant differences amongst groups and times. However, TG indicated that the venom causes a hypocoagulability state, which was not reversed by proposed treatments. Histology showed muscle inflammation, hemorrhage and necrosis, as well as hemorrhage in other tissues with no differences amongst groups. B. alternatus envenomation causes hypocoagulability detected by TG assay, but not through classical coagulation tests. The use of tranexamic acid and desmopressin for hemostasis stabilization after inoculation of the venom did not show advantage in coagulation restoration.(AU)

No Brasil, as serpentes do gênero Bothrops são responsáveis por milhares de acidentes, e seus venenos são compostos principalmente de enzimas proteolíticas. Embora o soro antiofídico produzido pelos institutos brasileiros seja notavelmente eficiente, mais estudos são necessários, especialmente na medicina veterinária. O veneno contem enzimas e proteínas não-enzimáticas que interferem com a hemostasia levando a hemorragias ou trombose. A associação de outros tratamentos ao soro antibotrópico foi a razão para o desenvolvimento do presente estudo. O objetivo deste estudo foi avaliar as alterações da hemostasia causadas pelo veneno de Bothrops alternatus em coelhos, após tratamento com soro antibotrópico, ácido tranexâmico e desmopressina. Vinte coelhos da Nova Zelândia foram distribuídos em cinco grupos (n = 4) que foram submetidos a experimentos com 150mcg/kg de veneno de B. alternatus por injeção intramuscular. O Grupo 1 (G1) foi o controle positivo e recebeu veneno e PBS / BSA, enquanto o Grupo 2 (G2) foi tratado com ácido tranexâmico, o Grupo 3 (G3) com desmopressina, o Grupo 4 (G4) com ácido tranexâmico e soro antibotrópico, e o Grupo 5 (G5) com soro antibotrópico e desmopressina. As amostras de sangue foram coletadas antes da administração do veneno, e uma, quatro, oito e 12 horas após os tratamentos para realização de tempo de tromboplastina parcial ativada parcial (TTPa), tempo de protrombina (TP), tempo de trombina (TT) e mensuração de fibrinogênio. Para o ensaio de geração de trombina (TG) foi realizado com um pool de amostras nos tempos finais (8 e 12h). Ao final das 12h, todos os animais foram sacrificados e a necropsia foi realizada. Amostras de tecido muscular, coração, pulmões e rins foram analisadas. Os testes TTPa, TP, TT e fibrinogênio não mostraram diferenças significativas entre os grupos e os tempos. No entanto, o TG indicou que o veneno causa um estado de hipocoagulabilidade, que não foi revertido pelos tratamentos propostos. Na histologia, foram observadas inflamação muscular, hemorragia e necrose, além de hemorragia em outros tecidos, sem diferenças entre os grupos. O envenenamento por B. alternatus causa hipocoagulabilidade detectada mais precocemente pelo teste de geração de trombina. O uso de ácido tranexâmico e desmopressina para estabilização da hemostasia após a inoculação do veneno não mostrou vantagem na restauração da coagulação.(AU)

The effect of activated protein C in the experimental disseminated intravascular coagulation model formed by lipopolysaccharide infusion

Purpose: In this experimental study, activated protein C (APC), which has anticoagulant, antithrombotic, profibrinolytic, anti-inflammatory and antiapoptotic properties, was used to prevent coagulopathy in a disseminated intravascular coagulation (DIC) model formatted with lipopolysaccharide (LPS) infusion. Methods: Twenty-five Wistar albino rats weighting 280 320 g each were used. They were randomly divided into three groups: sham, control and study groups. To sham group (n = 5), only normal saline was infused. To control (n = 10) and study groups (n = 10), 30 mg/kg LPS was infused for 4 h from femoral vein. After LPS infusion, 100 µg/kg recombinant APC was given during 4 h in study group. Eight hours later, blood samples were taken from abdominal aorta and the animals sacrificed. From these samples, platelet, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were studied. Results: Platelet counts and fibrinogen levels were significantly lower in control and study groups than sham group (p 0.05). The PT, aPTT and D-dimer levels were significantly higher in control and study groups than in sham group (p 0.05). When comparing control and study groups, platelet counts were not statistically different (p = 0.36). However, the difference of the fibrinogen levels was significant between these groups (p = 0.0001). While PT and aPTT were longer in the study group compared to the control group (p 0.05), D-dimer levels were lower in the study group than in control (p = 0.0001). Conclusion: Use of APC can prevent hypercoagulation and consumption coagulopathy in the DIC as a result of correcting hematological parameters other than prolongation of coagulation time.(AU)

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Sazonalidade e rotas de coleta influenciam a ocorrência de leite instável não ácido, a densidade e a crioscopia do leite fornecido a um laticínio no Norte de Minas Gerais / [Seasonality and collection routes influence the occurrence of non-acid unstable milk, the density and cryoscopy of milk supplied to a dairy industry in northern Minas Gerais]

Objetivou-se avaliar, nos meses, nas estações do ano e nas rotas de coleta, a ocorrência de leite instável não ácido (LINA) e de outras classes de leite, de acidez titulável, densidade e crioscopia em 10.654 resultados de laticínio localizado em Montes Claros, MG. Em rotas de coleta, agruparam-se:a)Bocaiúva; Brasília de Minas, Coração de Jesus e São João da Lagoa; b) Capitão Enéas e Janaúba; Francisco Sá; c) Icaraí de Minas e São Francisco; d)Juramento e Glaucilândia; e) Montes Claros; f) Pedras de Maria da Cruz, Japonvar, Lontra e São João da Ponte;e g) Ubaí. Foram avaliados frequências das classes de leite, teste do álcool, acidez titulável, densidade e crioscopiapor mês, estação do ano e rota. Com testes de qui-quadrado e análises de variância, verificou-se influência de mês, estação do ano e rota em acidez titulável, densidade e crioscopia. Análises de correspondência múltipla descreveram a associação de classes de leite e mês ou rotas e de teste do álcool, acidez titulável, densidade e crioscopia em relação às estações do ano.Por regressão logística, calculou-se probabilidade de ocorrência de LINA por grupos de meses e de rotas. Houve maior porcentagem de amostras dentro da normalidade. Resultados normais, LINA, amostras alcalinas e ácidas foram, respectivamente, 79,46%; 12,93%; 4,14% e 3,48% de 10.271 resultados. Positividade ao teste do álcool (n=10.561) foi mais frequente no final do inverno. LINA associou-se à transição da seca para chuva, com maior chance de ocorrência em setembro e janeiro. As rotas de Brasília de Minas, Icaraí de Minas e Ubaí apresentaram maiores desconformidades e probabilidades para ocorrência de LINA.(AU)

The purpose of this study was to evaluate in the months, seasons and collection routes, the occurrence of non-acidic unstable milk (UNAM) and other milk categories, titratable acidity, density and cryoscopy in 10,654 samples from a dairy industry located in Montes Claros, MG, Brazil. Municipalities were grouped in collection routes: a) Bocaiúva; Brasília de Minas, Coração de Jesus and São João da Lagoa; b) Capitão Enéas and Janaúba; c) Francisco Sá; Icaraí de Minas and São Francisco; d) Juramento and Glaucilândia; e) Montes Claros; f) Pedras de Maria da Cruz, Japonvar, Lontra and São João da Ponte and g) Ubaí. The milk category frequencies and alcohol test, titratable acidity, density and cryoscopy of milk by months, season and routes were evaluated. Chi-square tests and variance analysis showed the influence of month, season and route on titratable acidity, density and cryoscopy. Multiple correspondence analyzes described the association of milk and month or routes and alcohol test categories, titratable acidity, density and cryoscopy in relation to the seasons. The probability of occurrence of LINA by month and route groups was calculated by logistic regression. There were a higher percentage of samples within the normal range. Normal, UNAM, alkaline and acid milk results were respectively 79.46, 12.93, 4.14 and 3.48% of 10,271 results. Alcohol test positivity (n=10,561) was more frequent in late winter. UNAM was associated with the transition from drought to rainy season, most likely to occur in September and January. The Brasília de Minas, Icaraí de Minas and Ubaí routes presented higher nonconformities and probabilities for UNAM occurrence.(AU)

Sazonalidade e rotas de coleta influenciam a ocorrência de leite instável não ácido, a densidade e a crioscopia do leite fornecido a um laticínio no Norte de Minas Gerais / [Seasonality and collection routes influence the occurrence of non-acid unstable milk, the density and cryoscopy of milk supplied to a dairy industry in northern Minas Gerais]

Objetivou-se avaliar, nos meses, nas estações do ano e nas rotas de coleta, a ocorrência de leite instável não ácido (LINA) e de outras classes de leite, de acidez titulável, densidade e crioscopia em 10.654 resultados de laticínio localizado em Montes Claros, MG. Em rotas de coleta, agruparam-se:a)Bocaiúva; Brasília de Minas, Coração de Jesus e São João da Lagoa; b) Capitão Enéas e Janaúba; Francisco Sá; c) Icaraí de Minas e São Francisco; d)Juramento e Glaucilândia; e) Montes Claros; f) Pedras de Maria da Cruz, Japonvar, Lontra e São João da Ponte;e g) Ubaí. Foram avaliados frequências das classes de leite, teste do álcool, acidez titulável, densidade e crioscopiapor mês, estação do ano e rota. Com testes de qui-quadrado e análises de variância, verificou-se influência de mês, estação do ano e rota em acidez titulável, densidade e crioscopia. Análises de correspondência múltipla descreveram a associação de classes de leite e mês ou rotas e de teste do álcool, acidez titulável, densidade e crioscopia em relação às estações do ano.Por regressão logística, calculou-se probabilidade de ocorrência de LINA por grupos de meses e de rotas. Houve maior porcentagem de amostras dentro da normalidade. Resultados normais, LINA, amostras alcalinas e ácidas foram, respectivamente, 79,46%; 12,93%; 4,14% e 3,48% de 10.271 resultados. Positividade ao teste do álcool (n=10.561) foi mais frequente no final do inverno. LINA associou-se à transição da seca para chuva, com maior chance de ocorrência em setembro e janeiro. As rotas de Brasília de Minas, Icaraí de Minas e Ubaí apresentaram maiores desconformidades e probabilidades para ocorrência de LINA.(AU)

The purpose of this study was to evaluate in the months, seasons and collection routes, the occurrence of non-acidic unstable milk (UNAM) and other milk categories, titratable acidity, density and cryoscopy in 10,654 samples from a dairy industry located in Montes Claros, MG, Brazil. Municipalities were grouped in collection routes: a) Bocaiúva; Brasília de Minas, Coração de Jesus and São João da Lagoa; b) Capitão Enéas and Janaúba; c) Francisco Sá; Icaraí de Minas and São Francisco; d) Juramento and Glaucilândia; e) Montes Claros; f) Pedras de Maria da Cruz, Japonvar, Lontra and São João da Ponte and g) Ubaí. The milk category frequencies and alcohol test, titratable acidity, density and cryoscopy of milk by months, season and routes were evaluated. Chi-square tests and variance analysis showed the influence of month, season and route on titratable acidity, density and cryoscopy. Multiple correspondence analyzes described the association of milk and month or routes and alcohol test categories, titratable acidity, density and cryoscopy in relation to the seasons. The probability of occurrence of LINA by month and route groups was calculated by logistic regression. There were a higher percentage of samples within the normal range. Normal, UNAM, alkaline and acid milk results were respectively 79.46, 12.93, 4.14 and 3.48% of 10,271 results. Alcohol test positivity (n=10,561) was more frequent in late winter. UNAM was associated with the transition from drought to rainy season, most likely to occur in September and January. The Brasília de Minas, Icaraí de Minas and Ubaí routes presented higher nonconformities and probabilities for UNAM occurrence.(AU)

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1. Methods: Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins. Results: When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis Conclusion: These results suggest that the recombinant enzyme has no potential as a thrombolytic agent but can be applied in the prevention of thrombus formation in some pathological processes and as molecular tools in studies related to hemostasis.(AU)

Evaluation of coagulation parameters in dogs with overweight or obesity

Background: Hemostatic alterations have already been well characterized in humans with body fat excess, being includedin the list of obesity related comorbidities. Overweight and obesity are common in dogs; however, there is little informationabout the blood coagulation parameters in dogs with these conditions. The aim of this study was to compare hematologicaland coagulation parameters between lean dogs and overweight or obese dogs, including platelets count; prothrombin time(PT); activated partial thromboplastin time (aPTT), coagulation time (CT) and plasma fibrinogen concentration.Materials, Methods & Results: A total of 22 dogs (aged 1 to 10 years, neutered) were evaluated, 10 of them presentedideal body condition score (BCS 4-5) and formed the group 1 (control) and 12 were overweight or obese (BCS 7-9) andformed the group 2. The dogs were submitted to clinical evaluation and then to blood collection for the following laboratorytests: blood count (performed on automatic analyzer), quantification...

Functional and biological insights of rCollinein-1, a recombinant serine protease from Crotalus durissus collilineatus

Background:The prevalent class of snake venom serine proteases (SVSP) in Viperidae venoms is the thrombin-like enzymes, which, similarly to human thrombin, convert fibrinogen into insoluble fibrin monomers. However, thrombin-like serine proteases differ from thrombin by being unable to activate factor XIII, thus leading to the formation of loose clots and fibrinogen consumption. We report the functional and biological characterization of a recombinant thrombin-like serine protease from Crotalus durissus collilineatus, named rCollinein-1.Methods:Heterologous expression of rCollinein-1 was performed in Pichia pastoris system according to a previously standardized protocol, with some modifications. rCollinein-1 was purified from the culture medium by a combination of three chromatographic steps. The recombinant toxin was tested in vitro for its thrombolytic activity and in mice for its edematogenicity, blood incoagulability and effect on plasma proteins.Results:When tested for the ability to induce mouse paw edema, rCollinein-1 demonstrated low edematogenic effect, indicating little involvement of this enzyme in the inflammatory processes resulting from ophidian accidents. The rCollinein-1 did not degrade blood clots in vitro, which suggests that this toxin lacks fibrinolytic activity and is not able to directly or indirectly activate the fibrinolytic system. The minimal dose of rCollinein-1 that turns the blood incoagulable in experimental mice is 7.5 mg/kg. The toxin also led to a significant increase in activated partial thromboplastin time at the dose of 1 mg/kg in the animals. Other parameters such as plasma fibrinogen concentration and prothrombin time were not significantly affected by treatment with rCollinein-1 at this dose. The toxin was also able to alter plasma proteins in mouse after 3 h of injection at a dose of 1 mg/kg, leading to a decrease in the intensity of beta zone and an increase in gamma zone in agarose gel electrophoresis...(AU)

Evaluation of coagulation parameters in dogs with overweight or obesity

Background: Hemostatic alterations have already been well characterized in humans with body fat excess, being includedin the list of obesity related comorbidities. Overweight and obesity are common in dogs; however, there is little informationabout the blood coagulation parameters in dogs with these conditions. The aim of this study was to compare hematologicaland coagulation parameters between lean dogs and overweight or obese dogs, including platelets count; prothrombin time(PT); activated partial thromboplastin time (aPTT), coagulation time (CT) and plasma fibrinogen concentration.Materials, Methods & Results: A total of 22 dogs (aged 1 to 10 years, neutered) were evaluated, 10 of them presentedideal body condition score (BCS 4-5) and formed the group 1 (control) and 12 were overweight or obese (BCS 7-9) andformed the group 2. The dogs were submitted to clinical evaluation and then to blood collection for the following laboratorytests: blood count (performed on automatic analyzer), quantification...(AU)

Extraction, physical-chemical characterization and in vitro inhibitorypotential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds / Extração, caracterização físico-química e potencial inibitório in vitro da geração detrombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras

The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) wereanalyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p < 0.001) from 0.66±0.03% (C. racemosa) to 41.60±1.10% (Halymenia sp.) of crude SPs varying sulfate (8.41-42.60%) and total sugars (47.80-70.53%). Crude SPs showed difference in mobility and resolution pattern by agarose electrophoresis, while polyacrylamide analysis revealed SPs of > 100 kDa. These procedures,combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU(Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. Withrespect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TGby both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crudeSPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinctcrude SPs from Brazilian seaweeds.(AU)

O valor biotecnológico das macroalgas para ensaios de varredura de geração de trombina-GT pouco tem sido explorado usando polissacarídeos sulfatados-PSs como substitutos à heparina. Foramanalisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às característicasfísico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, aotempo de protrombina-TP e a GT. A extração com papaína rendeu (p < 0,001) de 0,66±0,03%(C. racemosa) a 41,60±1,10% (Halymenia sp.) de PSs brutos variando sulfato (8,41-42,60%) e açúcarestotais (47,80-70,53%). Os PSs brutos mostraram diferenças na mobilidade e resolução por eletroforese emagarose, enquanto pela análise em poliacrilamida revelou PSs brutos de >100 kDa. Esses procedimentos,combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. OsTTPAs foram dependentes da sulfatação dos PSs brutos e variaram de 2,81 (A. muscoides) a 21,30 UI (Halymenia sp.) vs. heparina (193 UI). O TP não foi alterado. Com respeito ao ensaio de GT, os PSs brutos modificaram, dependente de concentração e independentemente de massa molecular, GT pelas viasintrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca total usando PSs brutosde A. muscoides em paralelo à heparina (p < 0,05). A trombose in vitro é modulada diferencialmente porPSs brutos distintos de algas marinhas brasileiras.(AU)

Extraction, physical-chemical characterization and in vitro inhibitorypotential of thrombin generation of crude sulfated polysaccharides from Brazilian tropical seaweeds / Extração, caracterização físico-química e potencial inibitório in vitro da geração detrombina de polissacarídeos sulfatados brutos de algas marinhas tropicais brasileiras

The biotechnological value of macroalgae for screening assays of thrombin generation-TG using sulfated polysaccharides-SPs as substitutes to heparin has been poorly explored. Five Brazilian species of macroalgae (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides and C. racemosa) wereanalyzed and compared for their abundance, physical-chemical characteristics and in vitro anticoagulant assays of activated partial thromboplastin time-APTT, prothrombin time-PT and TG. Papain extraction yielded (p 100 kDa. These procedures,combined with the use of Stains-All, also indicated nonSPs. APTTs ranged from 2.81 (A. muscoides) to 21.30 IU(Halymenia sp.) vs. heparin (193 IU), and were dependent on sulfation of the crude SPs. PT was not altered. Withrespect to TG assay, crude SPs modified concentration-dependent and independently from molecular mass TGby both intrinsic/extrinsic pathways in 60-fold diluted human plasma, with total intrinsic inactivation using crudeSPs from A. muscoides in parallel to heparin (p < 0.05). Thrombosis in vitro is differentially modulated by distinctcrude SPs from Brazilian seaweeds.

O valor biotecnológico das macroalgas para ensaios de varredura de geração de trombina-GT pouco tem sido explorado usando polissacarídeos sulfatados-PSs como substitutos à heparina. Foramanalisadas e comparadas cinco espécies brasileiras de macroalgas (Gracilaria birdiae, Acanthophora muscoides, Halymenia sp., Caulerpa cupressoides e C. racemosa) quanto à abundância, às característicasfísico-químicas e os ensaios anticoagulantes in vitro de tempo de tromboplastina parcial ativada-TTPA, aotempo de protrombina-TP e a GT. A extração com papaína rendeu (p 100 kDa. Esses procedimentos,combinados ao uso de azul de toluidina/Stains-All, indicaram também polissacarídeos-não sulfatados. OsTTPAs foram dependentes da sulfatação dos PSs brutos e variaram de 2,81 (A. muscoides) a 21,30 UI (Halymenia sp.) vs. heparina (193 UI). O TP não foi alterado. Com respeito ao ensaio de GT, os PSs brutos modificaram, dependente de concentração e independentemente de massa molecular, GT pelas viasintrínseca/extrínseca no plasma humano diluído 60 vezes, com inativação intrínseca total usando PSs brutosde A. muscoides em paralelo à heparina (p < 0,05). A trombose in vitro é modulada diferencialmente porPSs brutos distintos de algas marinhas brasileiras.

Interpretation of coagulation tendency contributing to thrombosis in vector-borne diseases (ehrlichiosis, anaplasmosis, leishmaniosis, and dirofilariasis) among dogs

Background: Vector-borne infectious and zoonotic diseases are an important health problem that directly affects human and animal health negatively. Results through evaluation of coagulation disorders among vector-borne diseases should be of beneficial for both human and dogs studies. According to the present authors knowledge reports regarding changes in platelet (PLT) count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and D-dimer levels in dogs naturally infected with one or more vector-borne pathogens are lacking. Therefore, the present study was aimed to detecting those parameters for relation between diagnosis and prognosis of vector-borne diseases among dogs. Materials, Methods & Results: The material of this study was 46 dogs (36 were naturally infected with vector-borne diseases and 10 were healthy) from different breed, age and of both sexes. Venous blood samples were obtained to detect PLT counts, antibodies of ehrlichiosis, anaplasmosis, borreliosis, leishmaniosis and antigens of Dirofilaria immitis. The diagnosis of vector-borne diseases was performed by using a commercial ELISA assay kits. PLT count was performed with an automated blood cell counter analyser. In addition, PT, APTT and FIB concentrations were measured using a microcoagulometer. D-dimer concentrations were determined using fluorescence immunoassay rapid quantitave test analyser. Subgroups were formed according to the number of cases and the distribution of vector-borne agent. Statistically significant decreased PLT count was found in dogs mono infected with ehrlichiosis compared to healthy dogs (P < 0.001). Changes in mean PT value in the studied animals did not show statistically significant differences among the groups (P > 0.05). APTT values in the ehrlichiosis mono infection group were significantly higher than that of the healthy control (P < 0.01). A significant increase in FIB levels were detected […](AU)

Interpretation of coagulation tendency contributing to thrombosis in vector-borne diseases (ehrlichiosis, anaplasmosis, leishmaniosis, and dirofilariasis) among dogs

Background: Vector-borne infectious and zoonotic diseases are an important health problem that directly affects human and animal health negatively. Results through evaluation of coagulation disorders among vector-borne diseases should be of beneficial for both human and dogs studies. According to the present author’s knowledge reports regarding changes in platelet (PLT) count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and D-dimer levels in dogs naturally infected with one or more vector-borne pathogens are lacking. Therefore, the present study was aimed to detecting those parameters for relation between diagnosis and prognosis of vector-borne diseases among dogs. Materials, Methods & Results: The material of this study was 46 dogs (36 were naturally infected with vector-borne diseases and 10 were healthy) from different breed, age and of both sexes. Venous blood samples were obtained to detect PLT counts, antibodies of ehrlichiosis, anaplasmosis, borreliosis, leishmaniosis and antigens of Dirofilaria immitis. The diagnosis of vector-borne diseases was performed by using a commercial ELISA assay kits. PLT count was performed with an automated blood cell counter analyser. In addition, PT, APTT and FIB concentrations were measured using a microcoagulometer. D-dimer concentrations were determined using fluorescence immunoassay rapid quantitave test analyser. Subgroups were formed according to the number of cases and the distribution of vector-borne agent. Statistically significant decreased PLT count was found in dogs mono infected with ehrlichiosis compared to healthy dogs (P 0.05). APTT values in the ehrlichiosis mono infection group were significantly higher than that of the healthy control (P < 0.01). A significant increase in FIB levels were detected […]

Interpretation of Coagulation Tendency Contributing to Thrombosis in Vector-Borne Diseases (Ehrlichiosis, Anaplasmosis, Leishmaniosis, and Dirofilariasis) among Dogs

Background: Vector-borne infectious and zoonotic diseases are an important health problem that directly affects human and animal health negatively. Results through evaluation of coagulation disorders among vector-borne diseases should be of beneficial for both human and dogs studies. According to the present authors knowledge reports regarding changes in platelet (PLT) count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) and D-dimer levels in dogs naturally infected with one or more vector-borne pathogens are lacking. Therefore, the present study was aimed to detecting those parameters for relation between diagnosis and prognosis of vector-borne diseases among dogs.Materials, Methods & Results: The material of this study was 46 dogs (36 were naturally infected with vector-borne diseases and 10 were healthy) from different breed, age and of both sexes. Venous blood samples were obtained to detect PLT counts, antibodies of ehrlichiosis, anaplasmosis, borreliosis, leishmaniosis and antigens of Dirofilaria immitis. The diagnosis of vector-borne diseases was performed by using a commercial ELISA assay kits. PLT count was performed with an automated blood cell counter analyser. In addition, PT, APTT and FIB concentrations were measured using a microcoagulometer. D-dimer concentrations were determined using fluorescence immunoassay rapid qu

Argon plasma versus electrofulguration in the treatment of anal and perianal condylomata acuminata in patients with acquired immunodeficiency virus

Purpose: To compare the effectiveness of anal and perianal condylomata treatment using argon plasma and electrofulguration. Methods: From January 2013 to April 2014, 37 patients with anal and perianal condylomata, who had been diagnosed through proctological examination, oncotic cytology, polymerase chain reaction (PCR) and histology, underwent treatment with argon plasma and electrofulguration. The perianal and anal regions were divided into two semicircles. Each semicircle was treated using one of the methods by means of simple randomization. Therapeutic sessions were repeated until all clinical signs of infection by HPV were eliminated. The patients were evaluated according to several variables like the genotype of HPV, HIV infection, oncological potential per genotype, oncotic cytology and histology. Results: Among all the variables studied, only immunosuppression due to HIV influenced the results, specifically when the fulguration method was used. There was no significant difference in effectiveness between argon and fulguration based on lesion relapse (p > 0.05). However, among HIV-positive patients, fulguration presented worse results, with a significant difference (p = 0.01). Conclusion: Regarding treatment of anal and perianal condylomata acuminata, comparison between applying fulguration and argon demonstrated that these methods were equivalent, but use of fulguration presented more relapses among HIV-positive patients.(AU)

DETERMINAÇÃO DOS INTERVALOS DE REFERÊNCIA DAS VARIÁVEIS TROMBOELASTOGRÁFICAS SEM O USO DE ATIVADORES EM DIFERENTES AMOSTRAS SANGUÍNEAS DE CÃES HÍGIDOS

O sistema hemostático compreende um conjunto de eventos fisiológicos e complexos do organismo. Este previne o escape de sangue a partir dos vasos sanguíneos e a formação de trombos que afetarão o fluxo sanguíneo. Para manter a fluidez do sangue nos vasos sanguíneos, é necessária a interação em homeostase de vários componentes, como a parede vascular, plaquetas, fatores de coagulação, substâncias anticoagulantes naturais e substâncias fibrinolíticas, entre outros. A avaliação de desordens hemostáticas é realizada por meio de exames laboratoriais geralmente subdivididos em testes convencionais e em testes viscoelásticos, como a tromboelastografia (TEG). O presente estudo teve como objetivo evidenciar as variáveis tromboelastográficas de cães hígidos utilizando diferentes amostras sanguíneas, como o sangue total fresco (STF), sangue total com citrato de sódio a 3,2% (STC), plasma fresco (PF) e plasma fresco congelado (PFC). A finalidade foi estabelecer intervalo de referência (IR) para tais variáveis em cães hígidos e verificar se o tipo de amostra e o método de conservação da amostra alteram o traçado tromboelastográfico. Para este projeto, foram selecionados 22 cães sadios oriundos do serviço de atendimento clínico do Núcleo de Atendimento Veterinário do Centro Universitário Moura Lacerda, Ribeirão Preto/SP, que buscaram o atendimento para avaliações rotineiras anuais de sanidade. As diferentes amostras sanguíneas foram coletadas por meio de venopunção jugular e submetidas à análise tromboelastográfica em diferentes circunstâncias. Para determinação dos IR, foi realizado teste t Student com intervalo de confiança (IC) de 95%, valor-p 0,05 e teste de Fisher para verificar homogeneidade. Além disso, foi realizado teste de Shapiro Wilk para verificação de normalidade e com auxílio de gráficos box plot para detecção e exclusão de valores extremos (outliers). Os dados foram processados no software estatístico livre RGui versão 4.0.0 (R Core Team (2020). O conjunto de eventos do sistema hemostático, necessita de avaliação laboratorial robusta, como testes viscoelásticos, para melhor interpretação. Portanto, realizar tromboelastografia em diferentes amostras sanguíneas, bem como, determinar intervalo de referência para as variáveis TEG das diferentes amostras, mostra avanço para a realização do TEG em laboratórios clínicos veterinários. A obtenção de IR para diferentes tipos de amostras, possibilita o acesso ao exame tromboelastográfico em diferentes circunstâncias. O presente projeto mostrou ser possível e viável a realização da tromboelastografia em diferentes amostras sanguíneas e todas elas representam o paciente animal de maneira confiável.

The hemostatic system comprises a set of physiological and complex events in the body. This prevents the leakage of blood from the blood vessels and the formation of thrombi that will affect blood flow. To maintain the fluidity of blood in blood vessels, it is necessary to interact in homeostasis of various components, such as the vascular wall, platelets, coagulation factors, natural anticoagulant substances and fibrinolytic substances, among others. The evaluation of hemostatic disorders is performed through laboratory tests, usually subdivided into conventional tests and viscoelastic tests, such as thromboelastography (TEG). The present study aimed to evidence the thromboelastographic variables of healthy dogs using different blood samples, such as fresh whole blood (FTS), whole blood with 3.2% sodium citrate (STC), fresh plasma (FP) and fresh plasma frozen (PFC). The purpose was to establish a reference interval (RI) for such variables in healthy dogs and to verify whether the type of sample and the method of sample conservation alter the thromboelastographic tracing. For this project, 22 healthy dogs were selected from the clinical care service of the Center for Veterinary Care of the Moura Lacerda University Center, Ribeirão Preto/SP, which sought care for routine annual health assessments. Different blood samples were collected through jugular venipuncture and submitted to thromboelastographic analysis under different circumstances. To determine the RI, Student's t test was performed with a confidence interval (CI) of 95%, p-value 0.05 and Fisher's test to verify homogeneity. In addition, the Shapiro Wilk test was performed to verify normality and with the aid of box plot graphics for detection and exclusion of extreme values (outliers). Data were processed in the free statistical software RGui version 4.0.0 (R Core Team (2020). The set of events of the hemostatic system requires robust laboratory evaluation, such as viscoelastic tests, for better interpretation. Therefore, perform thromboelastography in different samples blood tests, as well as determining the reference interval for the TEG variables of different samples, shows advances in the performance of TEG in clinical veterinary laboratories. This project showed that it is possible and viable to perform thromboelastography in different blood samples and all of them represent the animal patient reliably.