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.

Comparison of hemostatic efficacy of topical Ankaferd Blood Stopper on heparinized and nonheparinized rats in bleeding related to liver injury

Purpose: In this study, hemostatic efficacy of Ankaferd Blood Stopper (ABS), a new generation hemostatic agent, was compared in the presence of heparin effect. Methods: Forty-eight Wistar albino rats were divided into two main groups as heparinized and nonheparinized, and these two main groupswere divided into six subgroups as control, Surgicel and ABS (n = 8). Grade 2 liver injury was performed on rats as standard. All groups were compared in terms of weight, laceration surface area, prothrombin time (PT), activated partial thromboplastin time (aPTT), international normalized ratio (INR), bleeding time, bleeding amount, hemoglobin (Hb) levels, macroscopic and microscopic reactions to the agent used. Results: Whereas there was no statistically significant difference between weight, laceration surface area, PT, INR and preoperative Hb values in the heparinized and nonheparinized groups, postoperative Hb, bleeding time, bleeding amount and aPTT values were statistically different (p 0.05). In the heparin-hemostat interaction, the ABS group had the lowest bleeding in the heparinized group in terms of the amount of bleeding compared to the control and Surgicel groups (F = 0.764; p = 0.047). In macroscopic and microscopic comparison, there was no difference between the groups in terms of cell necrosis andfresh bleeding (p > 0.05), it was found that the Surgicel group had statistical significantly higher reaction scores (p < 0.05) than the other groups in terms of other parameters. Conclusions: Ankaferd Blood Stopper can be safely and effectively used in surgical practice and in patients with additional diseases requiring heparinization, since it causes minimal reaction in the liver and decreases the amount of bleeding especially in the heparinized group.(AU)

Comparison of experimental wound healing in quail using two types of commercial hemostatic dressings / Comparação da cicatrização experimental de feridas em codornas usando dois tipos de curativos hemostáticos comerciais

The use of powdered hemostatic products and propolis-based preparations has become increasingly common to control bleeding and aid healing. This study evaluated two hemostatic dressings, one based on propolis (Alveolex®) and the other on ferric subsulfate powder (Quick Blood Stopper®), on experimental wound in quails. The experiment was conducted with 36 quails assigned to three groups: control, propolis-based hemostatic dressing and ferric subsulfate-based hemostatic dressing, on two types of wounds, superficial (skin and subcutaneous) and deep (skin, subcutaneous and muscle). Pre- (day-7) and post-procedure (days 2 and 10) examinations showed a tendency to reduce hematocrit values on day 2 and a tendency to increase fibrinogen levels and total leukocyte count on the same day. Visual assessment of wound healing indicated that birds using Quick Blood Stopper® showed signs of poor healing, while the group treated with Alveolex® had uncomplicated healing within the expected time. The groups treated with hemostatic based on ferric subsulfate showed a tendency to increase the values of fecal corticosterone metabolites on day 10, suggesting an increased level of stress in these animals. During the surgical procedure, both hemostatic products controlled bleeding, but Alveolex®was more efficient and easy to handle.

A adoção de hemostáticos em pó e preparações à base de própolis tem se tornado cada vez mais comum para realizar o controle de sangramentos e auxiliar na cicatrização. Neste trabalho foram avaliados dois curativos hemostáticos, um à base de própolis (Alveolex®) e outro em pó à base de subsulfato férrico (Quick Blood Stopper®), após lesão experimental em codornas. O experimento foi realizado com 36 codornas, separadas em três grupos: con-trole, hemostático à base de própolis e hemostático à base de subsulfato férrico, submetidos a dois tipos de feridas, superficiais (pele e subcutâneo) e profundas (pele, subcutâneo e musculatura). Os exames pré (dia -7) e pós proce-dimento (dias 2 e 10) demonstraram uma tendência à redução dos valores de hematócrito no dia 2 e tendência ao aumento dos níveis de fibrinogênio e contagem de leucócitos totais neste mesmo dia. A avaliação visual da cicatriza-ção da ferida demonstrou que as aves submetidas ao Quick Blood Stopper® exibiram sinais de dificuldade na cicatri-zação, enquanto o grupo tratado com Alveolex® apresentou cicatrização sem complicações e dentro do tempo espe-rado. Os grupos tratados com hemostáticos baseados em subsulfato férrico mostraram uma tendência a aumentar os valores dos metabólitos de corticosterona fecal no 10º dia, sugerindo um aumento do nível de estresse nestes animais. Durante o procedimento cirúrgico, ambos os produtos hemostáticos controlaram o sangramento, porém o Alveolex®foi mais eficiente e de fácil manipulação

Comparison of experimental wound healing in quail using two types of commercial hemostatic dressings / Comparação da cicatrização experimental de feridas em codornas usando dois tipos de curativos hemostáticos comerciais

The use of powdered hemostatic products and propolis-based preparations has become increasingly common to control bleeding and aid healing. This study evaluated two hemostatic dressings, one based on propolis (Alveolex®) and the other on ferric subsulfate powder (Quick Blood Stopper®), on experimental wound in quails. The experiment was conducted with 36 quails assigned to three groups: control, propolis-based hemostatic dressing and ferric subsulfate-based hemostatic dressing, on two types of wounds, superficial (skin and subcutaneous) and deep (skin, subcutaneous and muscle). Pre- (day-7) and post-procedure (days 2 and 10) examinations showed a tendency to reduce hematocrit values on day 2 and a tendency to increase fibrinogen levels and total leukocyte count on the same day. Visual assessment of wound healing indicated that birds using Quick Blood Stopper® showed signs of poor healing, while the group treated with Alveolex® had uncomplicated healing within the expected time. The groups treated with hemostatic based on ferric subsulfate showed a tendency to increase the values of fecal corticosterone metabolites on day 10, suggesting an increased level of stress in these animals. During the surgical procedure, both hemostatic products controlled bleeding, but Alveolex®was more efficient and easy to handle.(AU)

A adoção de hemostáticos em pó e preparações à base de própolis tem se tornado cada vez mais comum para realizar o controle de sangramentos e auxiliar na cicatrização. Neste trabalho foram avaliados dois curativos hemostáticos, um à base de própolis (Alveolex®) e outro em pó à base de subsulfato férrico (Quick Blood Stopper®), após lesão experimental em codornas. O experimento foi realizado com 36 codornas, separadas em três grupos: con-trole, hemostático à base de própolis e hemostático à base de subsulfato férrico, submetidos a dois tipos de feridas, superficiais (pele e subcutâneo) e profundas (pele, subcutâneo e musculatura). Os exames pré (dia -7) e pós proce-dimento (dias 2 e 10) demonstraram uma tendência à redução dos valores de hematócrito no dia 2 e tendência ao aumento dos níveis de fibrinogênio e contagem de leucócitos totais neste mesmo dia. A avaliação visual da cicatriza-ção da ferida demonstrou que as aves submetidas ao Quick Blood Stopper® exibiram sinais de dificuldade na cicatri-zação, enquanto o grupo tratado com Alveolex® apresentou cicatrização sem complicações e dentro do tempo espe-rado. Os grupos tratados com hemostáticos baseados em subsulfato férrico mostraram uma tendência a aumentar os valores dos metabólitos de corticosterona fecal no 10º dia, sugerindo um aumento do nível de estresse nestes animais. Durante o procedimento cirúrgico, ambos os produtos hemostáticos controlaram o sangramento, porém o Alveolex®foi mais eficiente e de fácil manipulação(AU)

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as "fibrin glue" or "fibrin sealants", are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was "heterologous fibrin sealant". The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Fibrin biopolymers, previously referred as “fibrin glue” or “fibrin sealants”, are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was “heterologous fibrin sealant”. The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.(AU)

Evaluation of hemostatic parameters in tumor-bearing dogs

Background: Hemostatic alterations are commonly detected in canine cancer patients. However, few studies have described hemostatic dysfunction in dogs with different tumor subtypes. In Veterinary Medicine, the state of hypercoagulability is hardly diagnosed alive, since laboratory exams for evaluate hemostatic function are not always requested. Due to importance of homeostatic disorders in cancer patients, this study aimed to evaluate hemostatic alterations such as platelet count, activated partial thromboplastin time (aPTT), prothrombin time (PT) and fibrinogen in tumor-bearing dogs.Materials, Methods & Results: From the 55 dogs evaluated, 30 had mammary carcinoma, 6 visceral hemangiosarcoma, 9 high-grade cutaneous mast cell tumor and 10 multicentric lymphoma. The results were compared to a control group composed by 10 Beagle dogs. Thrombocytosis was observed in 26.6% (8/30) of mammary carcinoma group and thrombocytopenia in 10% (3/30). The patients with hemangiosarcoma and mast cell tumor did not reveal thrombocytosis, however, thrombocytopenia was present in 16.6% (1/6) and 33% (3/9), respectively. Three dogs with multicentric lymphoma showed thrombocytopenia and other three showed thrombocytosis. From patients with thrombocytosis, one was classified as severe thrombocytosis (1077 x 10³/µL). Therefore, there were no statistically significant associations between neoplasia group with control group (P > 0.05). Regarding the aPTT and PT evaluation, mammary carcinoma (P = 0.0005), hemangiosarcoma (P = 0.033) and mast cell tumor (P = 0.012) patients showed statistical difference for aPTT, while the evaluation for PT was not significant (P > 0.05). We grouped all patients as a “tumor group” and compared to the control group. It was possible to observe increased aPTT and PT in 89% (49/55) and 50.90% (28/55) respectively, in tumor group compared to normal.[...]

Evaluation of hemostatic parameters in tumor-bearing dogs

Background: Hemostatic alterations are commonly detected in canine cancer patients. However, few studies have described hemostatic dysfunction in dogs with different tumor subtypes. In Veterinary Medicine, the state of hypercoagulability is hardly diagnosed alive, since laboratory exams for evaluate hemostatic function are not always requested. Due to importance of homeostatic disorders in cancer patients, this study aimed to evaluate hemostatic alterations such as platelet count, activated partial thromboplastin time (aPTT), prothrombin time (PT) and fibrinogen in tumor-bearing dogs.Materials, Methods & Results: From the 55 dogs evaluated, 30 had mammary carcinoma, 6 visceral hemangiosarcoma, 9 high-grade cutaneous mast cell tumor and 10 multicentric lymphoma. The results were compared to a control group composed by 10 Beagle dogs. Thrombocytosis was observed in 26.6% (8/30) of mammary carcinoma group and thrombocytopenia in 10% (3/30). The patients with hemangiosarcoma and mast cell tumor did not reveal thrombocytosis, however, thrombocytopenia was present in 16.6% (1/6) and 33% (3/9), respectively. Three dogs with multicentric lymphoma showed thrombocytopenia and other three showed thrombocytosis. From patients with thrombocytosis, one was classified as severe thrombocytosis (1077 x 10³/µL). Therefore, there were no statistically significant associations between neoplasia group with control group (P > 0.05). Regarding the aPTT and PT evaluation, mammary carcinoma (P = 0.0005), hemangiosarcoma (P = 0.033) and mast cell tumor (P = 0.012) patients showed statistical difference for aPTT, while the evaluation for PT was not significant (P > 0.05). We grouped all patients as a “tumor group” and compared to the control group. It was possible to observe increased aPTT and PT in 89% (49/55) and 50.90% (28/55) respectively, in tumor group compared to normal.[...](AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.(AU)

Envenoming by Viridovipera stejnegeri snake: a patient with liver cirrhosis presenting disruption of hemostatic balance

Background In most cases of envenoming by the green habu Viridovipera stejnegeri in Taiwan coagulopathy is not observed. Case presentation Herein, we describe the case of a patient with liver cirrhosis who developed venom-induced consumptive coagulopathy after V. stejnegeri bite. Laboratory investigation revealed the following: prothrombin time > 100 s (international normalized ratio > 10), activated partial thromboplastin time > 100 s, fibrinogen < 50 mg/dL, and fibrin degradation product > 80 μg/mL. The patient recovered after administration of bivalent hemorrhagic antivenom, vitamin K, fresh frozen plasma and cryoprecipitate. Conclusion The liver, directly involved in the acute phase reaction, is the main responsible for neutralization of animal toxins. Any patient with history of liver cirrhosis bitten by a venomous snake, even those whose venoms present low risk of coagulopathy, should be very carefully monitored for venom-induced consumptive coagulopathy (VICC), since the hemostatic balance may be disrupted.(AU)

Envenoming by Viridovipera stejnegeri snake: a patient with liver cirrhosis presenting disruption of hemostatic balance

Background In most cases of envenoming by the green habu Viridovipera stejnegeri in Taiwan coagulopathy is not observed. Case presentation Herein, we describe the case of a patient with liver cirrhosis who developed venom-induced consumptive coagulopathy after V. stejnegeri bite. Laboratory investigation revealed the following: prothrombin time > 100 s (international normalized ratio > 10), activated partial thromboplastin time > 100 s, fibrinogen < 50 mg/dL, and fibrin degradation product > 80 μg/mL. The patient recovered after administration of bivalent hemorrhagic antivenom, vitamin K, fresh frozen plasma and cryoprecipitate. Conclusion The liver, directly involved in the acute phase reaction, is the main responsible for neutralization of animal toxins. Any patient with history of liver cirrhosis bitten by a venomous snake, even those whose venoms present low risk of coagulopathy, should be very carefully monitored for venom-induced consumptive coagulopathy (VICC), since the hemostatic balance may be disrupted.(AU)

Paraneoplastic hematological, biochemical, and hemostatic abnormalities in female dogs with mammary neoplasms / Anormalidades hematológicas, bioquímicas e hemostáticas de origem paraneoplásica em fêmeas caninas com neoplasia mamária

Paraneoplastic laboratory abnormalities are identified in several types of cancers in dogs and cats. In veterinary medicine, particularly in mammary cancer, there are few studies that correlate abnormal laboratory findings with tumor type and staging. The aim of this study was to evaluate hematological, biochemical, and hemostatic abnormalities and correlate them with mammary tumor staging in female dogs with mammary cancer. Blood samples from 24 female dogs were evaluated, and the hematological, biochemical, and hemostatic parameters were correlated with tumor staging obtained by physical examination, imaging exams, and histopathological surgical biopsies. The groups were organized according to tumor staging: group 1 (stages I and II), group 2 (stage III), and group 3 (stages IV and V). Anemia, neutrophilic leukocytosis, monocytosis, eosinophilia, thrombocytosis, hypoalbuminemia, hypocalcemia, hypoglycemia, and low blood urea were observed. The variables MCHC, TPP, and RDW were correlated with tumor staging with no clinical relevance. Thrombin time and fibrinogen were significant between the groups in the coagulation test, being associated with tumor staging. The findings suggest influence of the proinflammatory cytokines released during tumor growth.(AU)

Alterações laboratoriais de origem paraneoplásica são identificadas em diversos tipos de câncer de cães e gatos. Na medicina veterinária, existem poucos estudos que correlacionam os achados laboratoriais anormais com o tipo e estadiamento tumorais, principalmente em cadelas com neoplasia mamária. O objetivo deste estudo foi avaliar as alterações hematológicas, bioquímicas e hemostáticas em cadelas com neoplasia mamária e relacioná-las com o estadiamento tumoral. Foram coletadas amostras de sangue de 24 fêmeas caninas, e os parâmetros hematológicos, bioquímicos e hemostáticos obtidos foram relacionados com o estadiamento tumoral, realizado através do exame físico, exames de imagem e avaliação histopatológica após remoção cirúrgica. Os grupos foram organizados de acordo com o estadiamento tumoral em: Grupo 1 (estádios I e II), grupo 2 (estádio III) e grupo 3 (estádios IV e V). Observou-se anemia, leucocitose neutrofílica, monocitose, eosinofilia, trombocitose, hipoalbuminemia, hipocalcemia, hipoglicemia e diminuição de ureia sanguínea. As variáveis CHCM, PPT e RDW foram relacionadas com o estadiamento tumoral, porém sem relevância clínica. Nos testes de coagulação, o TT e o fibrinogênio apresentaram diferença significativa entre os grupos, sendo associado com estadiamento tumoral. Os resultados sugerem influência das citocinas pró-inflamatórias liberadas durante o crescimento do tumor.(AU)

Paraneoplastic hematological, biochemical, and hemostatic abnormalities in female dogs with mammary neoplasms / Anormalidades hematológicas, bioquímicas e hemostáticas de origem paraneoplásica em fêmeas caninas com neoplasia mamária

Paraneoplastic laboratory abnormalities are identified in several types of cancers in dogs and cats. In veterinary medicine, particularly in mammary cancer, there are few studies that correlate abnormal laboratory findings with tumor type and staging. The aim of this study was to evaluate hematological, biochemical, and hemostatic abnormalities and correlate them with mammary tumor staging in female dogs with mammary cancer. Blood samples from 24 female dogs were evaluated, and the hematological, biochemical, and hemostatic parameters were correlated with tumor staging obtained by physical examination, imaging exams, and histopathological surgical biopsies. The groups were organized according to tumor staging: group 1 (stages I and II), group 2 (stage III), and group 3 (stages IV and V). Anemia, neutrophilic leukocytosis, monocytosis, eosinophilia, thrombocytosis, hypoalbuminemia, hypocalcemia, hypoglycemia, and low blood urea were observed. The variables MCHC, TPP, and RDW were correlated with tumor staging with no clinical relevance. Thrombin time and fibrinogen were significant between the groups in the coagulation test, being associated with tumor staging. The findings suggest influence of the proinflammatory cytokines released during tumor growth.(AU)

Alterações laboratoriais de origem paraneoplásica são identificadas em diversos tipos de câncer de cães e gatos. Na medicina veterinária, existem poucos estudos que correlacionam os achados laboratoriais anormais com o tipo e estadiamento tumorais, principalmente em cadelas com neoplasia mamária. O objetivo deste estudo foi avaliar as alterações hematológicas, bioquímicas e hemostáticas em cadelas com neoplasia mamária e relacioná-las com o estadiamento tumoral. Foram coletadas amostras de sangue de 24 fêmeas caninas, e os parâmetros hematológicos, bioquímicos e hemostáticos obtidos foram relacionados com o estadiamento tumoral, realizado através do exame físico, exames de imagem e avaliação histopatológica após remoção cirúrgica. Os grupos foram organizados de acordo com o estadiamento tumoral em: Grupo 1 (estádios I e II), grupo 2 (estádio III) e grupo 3 (estádios IV e V). Observou-se anemia, leucocitose neutrofílica, monocitose, eosinofilia, trombocitose, hipoalbuminemia, hipocalcemia, hipoglicemia e diminuição de ureia sanguínea. As variáveis CHCM, PPT e RDW foram relacionadas com o estadiamento tumoral, porém sem relevância clínica. Nos testes de coagulação, o TT e o fibrinogênio apresentaram diferença significativa entre os grupos, sendo associado com estadiamento tumoral. Os resultados sugerem influência das citocinas pró-inflamatórias liberadas durante o crescimento do tumor.(AU)

AVALIAÇÃO DE PERFIL HEMOSTÁTICO EM CÃES EM QUADRO DE INJÚRIA RENAL AGUDA NÃO SUBMETIDOS À TERAPIA DIALÍTICA

A injúria renal aguda (IRA) representa uma grande casuística na medicina veterinária e está associada à elevada mortalidade. Em humanos, sabe-se que os quadros de IRA podem levar a alterações hemostáticas, as quais podem aumentar o tempo de permanência do paciente em hospitalização e elevar a mortalidade. Entretanto, a ocorrência de distúrbios coagulatórios em cães com IRA ainda não é bem compreendida. O objetivo deste estudo foi avaliar o perfil hemostático de cães em IRA, devidamente estadiados e se esses pacientes apresentam distúrbios de hemostasia. Além disso, correlacionar as variáveis testadas com a intensidade da azotemia e com o risco de óbito. O estudo contou com 42 pacientes, sendo 14 no grupo controle, 14 no grupo IRIS III e 14 no grupo IRIS IV, de diferentes, sexos, raças e faixas etárias. Os marcadores hemostáticos testados foram Dímero-D, TP, TTPa, TT e fibrinogênio, sendo que foram comparados um kit para Dímero-D em humanos com um kit para cães. O Dímero-D e os demais marcadores hemostáticos apresentaram-se aumentados nos pacientes em IRA de estágios III e IV, porém não houve correlação entre esses marcadores e a intensidade da azotemia. Entretanto, o Dímero-D apresentou forte correlação positiva com as enzimas hepáticas e com a RPCU. Todos os marcadores hemostáticos testados apresentaram correlação positiva com o risco de óbito do paciente. Além disso, os dois testes para Dímero-D avaliados, foram eficazes e apresentaram correlação positiva. O aumento dos marcadores hemostáticos e Dímero-D e a correlação entre RPCU e Dímero-D, sugerem uma tendência ao estado de hipercoagulabilidade nos cães avaliados, com IRA em estágio III e IV. O Dímero-D apresentou forte correlação positiva com o risco de morte dos pacientes, evidenciando seu potencial como marcador de prognóstico.

Acute kidney injury (AKI) represents a large sample in veterinary medicine and is associated with a high mortality. In humans it is known that AKI can lead to hemostatic changes which can increase the length of stay of the patient in hospital and increase mortality. However, the occurrence of coagulation disorders in dogs with AKI is still not well understood. The aim of this study was to evaluate the hemostatic profile of dogs in AKI duly staged and whether these patients present hemostasis disorders. In addition, correlate the variables tested with the intensity of azotemia and the risk of death. The study included 42 patients, 14 in the control group, 14 in the IRIS group III and 14 in the IRIS group IV with different genders, races and age groups. The tested hemostatic markers were D-Dimer, TP, TTPa, TT and fibrinogen and a kit for D-Dimer in humans was compared with a kit for dogs. D-Dimer and other hemostatic markers were increased in patients with stage III and IV but there was no correlation between these markers and azotemia intensity. However, D-Dimer showed a strong positive correlation with liver enzymes and with UPCR. All tested hemostatic markers were positively correlated with the patient's risk of death. In addition, the two tests for D-Dimer evaluated were effective and showed a positive correlation. The increase in hemostatic and D-Dimer markers and the correlation between UPCR and D-Dimer suggest a tendency to a hypercoagulable state in the evaluated dogs, with IRIS stage III and IV. The D-Dimer showed a strong positive correlation with the risk of death of patients, showing its potential as a prognostic marker

Thromboelastometric evaluation of horses submitted to experimental thrombosis and jugular thrombectomy / Avaliação tromboelastométrica de equinos submetidos a trombose e trombectomia jugular experimental

Jugular thrombosis in horses occurs commonly in iatrogenic situations, secondary to endotoxemic clinical condition and disseminated vascular coagulation, potentially leading to death. Thus, hemostatic evaluation becomes necessary and extremely important for monitoring the risks of systemic hypercoagulability and for the efficiency of allopathic and surgical treatment. This paper describes the hemostatic behavior in experimental jugular thrombosis of ten healthy equines, subsequently submitted to two thrombectomy techniques and receiving heparin sodium as anti-rethrombosis therapy. These animals were evaluated for 20 days by thromboelastometry (TEM), platelet count, hematocrit and fibrinogen, at four moments: pre-induction to phlebitis (D0-MPF); three days after thrombophlebitis induction (D3-MFM); 6 days after, - moment of thrombophlebitis - (D9-MT); and 54 (D16) and 126 (D19) hours after thrombectomies (PTM). Thrombectomy was performed via a Vollmar Ring (group 1, n=5) and Fogarty catheter (group 2, n=5). All the animals received heparin (150 UI/kg, SC) every 12 hours, for ten days after the respective thrombectomies. Through the blood samples were evaluated TEM, activated partial thromboplastin time (aPTT) and prothrombin time (PT), dosing of fibrinogen, hematocrit and platelet count at the abovementioned moments. For comparison between groups and moments the t test was applied at 5% significance level. No significant difference was verified between treatment groups at any of the moments. There were reductions in clotting time (CT) and clot formation time (CFT), with increase in maximum lysis (ML) until the moment D9-MT. Evaluation through INTEM® reagent presented prolongations of CT and CFT with reduction of α angle and ML starting from D16 and D19. Similarly, aPTT presented significant differences between moments pre- (D0, 3 and 9) and post- (D16 and 19) anticoagulant and surgical treatment. The platelet numbers were diminished at moments D16 and D19. In evaluation with EXTEM® reagent, prolongation of CT and CFT occurred only between the moments D0 vs. D3 and vs. D9. O PT did not present significant differences. The results obtained demonstrate that experimental jugular thrombophlebitis leads to local clinical alterations, with impairment of tissue and of the extrinsic coagulation pathway (EXTEM® ), but without evidence of systemic hypercoagulability status, since there was no increase of the alpha angle or maximum clot firmness (MCF). Furthermore, TEM was shown useful and more sensitive than conventional coagulation tests (PT, aPTT and fibrinogen) for the monitoring of anticoagulant therapy, as demonstrated in other works.(AU)

A trombose jugular nos equinos ocorre comumente em situações iatrogênicas, secundárias a quadros endotoxêmicos e a coagulação vascular disseminada, podendo levar ao óbito. Por isso, avaliação hemostática se faz necessária e de extrema importância para monitorar os riscos de hipercoagulabilidade sistêmica e também a eficiência do tratamento alopático e cirúrgico. Este trabalho descreve o comportamento hemostático na trombose jugular experimental de dez equinos hígidos, submetidos posteriormente a duas técnicas de trombectomia e recebendo heparina sódica como terapia anti retrombosante. Estes animais foram avaliados durante 20 dias por tromboelastometria (TEM), contagem de plaquetas, hematócrito e fibrinogênio, em quatro momentos: pré-indução à flebite (D0-MPF); três dias após a indução da tromboflebite (D3-MFM); 6 dias após, - momento de tromboflebite - (D9-MT); e 54 (D16) e 126 (D19) horas após as trombectomias (MPT). A trombectomia foi realizada com Anel de Vollmar (grupo 1, n=5) e cateter de Fogarty (grupo 2, n=5). Todos os animais receberam heparina (150 UI/Kg, SC) a cada 12 horas, durante dez dias após as respectivas trombectomias. Através de amostras de sangue, foram avaliadas a TEM, o tempo de tromboplastia parcial ativada (TTPa) e tempo de protrombina (TP), a dosagem de fibrinogênio, hematócrito e contagem de plaquetas nos momentos descritos acima. Para a comparação entre os grupos e momentos foi aplicado teste t, com nível de significância de 5%. Não foi verificada diferença significativa entre os grupos de tratamento em nenhum dos momentos. Houve redução do tempo de coagulação (CT) e do tempo de formação do coágulo (CFT), com aumento da lise máxima (LM) até o momento D9-MT. A avaliação com o reagente intem apresentou prolongamento do CT e do CFT e redução do ângulo α e da LM a partir do D16 e D19. Da mesma forma, o TTPa apresentou diferenças significativas entre os momentos pré (D0, 3 e 9) e pós (D16 e 19) tratamento cirúrgico e anticoagulante. Houve diminuição do número de plaquetas nos momentos D16 e D19. Na avaliação com reagente extem ocorreu apenas o prolongamento do CT e CFT entre os momentos D0 e o D3 e D9. O TP não apresentou diferenças significativas. Os resultados obtidos demonstram que a tromboflebite jugular experimental leva a alterações clínicas locais, com comprometimento tecidual e da via extrínseca da coagulação (extem), porém sem evidências de um estado sistêmico de hipercoagulabilidade, pois não houve aumento do ângulo alfa e da firmeza máxima do coágulo (MCF). Além disso, a TEM se mostrou útil e mais sensível que os testes convencionais de coagulação (TP, TTPa e fibrinogênio) para o acompanhamento da terapia anticoagulante, conforme demonstrado em outros trabalhos.(AU)

Thromboelastometric evaluation of horses submitted to experimental thrombosis and jugular thrombectomy / Avaliação tromboelastométrica de equinos submetidos a trombose e trombectomia jugular experimental

Jugular thrombosis in horses occurs commonly in iatrogenic situations, secondary to endotoxemic clinical condition and disseminated vascular coagulation, potentially leading to death. Thus, hemostatic evaluation becomes necessary and extremely important for monitoring the risks of systemic hypercoagulability and for the efficiency of allopathic and surgical treatment. This paper describes the hemostatic behavior in experimental jugular thrombosis of ten healthy equines, subsequently submitted to two thrombectomy techniques and receiving heparin sodium as anti-rethrombosis therapy. These animals were evaluated for 20 days by thromboelastometry (TEM), platelet count, hematocrit and fibrinogen, at four moments: pre-induction to phlebitis (D0-MPF); three days after thrombophlebitis induction (D3-MFM); 6 days after, - moment of thrombophlebitis - (D9-MT); and 54 (D16) and 126 (D19) hours after thrombectomies (PTM). Thrombectomy was performed via a Vollmar Ring (group 1, n=5) and Fogarty catheter (group 2, n=5). All the animals received heparin (150 UI/kg, SC) every 12 hours, for ten days after the respective thrombectomies. Through the blood samples were evaluated TEM, activated partial thromboplastin time (aPTT) and prothrombin time (PT), dosing of fibrinogen, hematocrit and platelet count at the abovementioned moments. For comparison between groups and moments the t test was applied at 5% significance level. No significant difference was verified between treatment groups at any of the moments. There were reductions in clotting time (CT) and clot formation time (CFT), with increase in maximum lysis (ML) until the moment D9-MT. [...] (AU)

A trombose jugular nos equinos ocorre comumente em situações iatrogênicas, secundárias a quadros endotoxêmicos e a coagulação vascular disseminada, podendo levar ao óbito. Por isso, avaliação hemostática se faz necessária e de extrema importância para monitorar os riscos de hipercoagulabilidade sistêmica e também a eficiência do tratamento alopático e cirúrgico. Este trabalho descreve o comportamento hemostático na trombose jugular experimental de dez equinos hígidos, submetidos posteriormente a duas técnicas de trombectomia e recebendo heparina sódica como terapia anti retrombosante. Estes animais foram avaliados durante 20 dias por tromboelastometria (TEM), contagem de plaquetas, hematócrito e fibrinogênio, em quatro momentos: pré-indução à flebite (D0-MPF); três dias após a indução da tromboflebite (D3-MFM); 6 dias após, - momento de tromboflebite - (D9-MT); e 54 (D16) e 126 (D19) horas após as trombectomias (MPT). A trombectomia foi realizada com Anel de Vollmar (grupo 1, n=5) e cateter de Fogarty (grupo 2, n=5). Todos os animais receberam heparina (150 UI/Kg, SC) a cada 12 horas, durante dez dias após as respectivas trombectomias. Através de amostras de sangue, foram avaliadas a TEM, o tempo de tromboplastia parcial ativada (TTPa) e tempo de protrombina (TP), a dosagem de fibrinogênio, hematócrito e contagem de plaquetas nos momentos descritos acima. Para a comparação entre os grupos e momentos foi aplicado teste t, com nível de significância de 5%. Não foi verificada diferença significativa entre os grupos de tratamento em nenhum dos momentos. Houve redução do tempo de coagulação (CT) e do tempo de formação do coágulo (CFT), com aumento da lise máxima (LM) até o momento D9-MT. A avaliação com o reagente intem apresentou prolongamento do CT e do CFT e redução do ângulo α e da LM a partir do D16 e D19. Da mesma forma, o TTPa apresentou diferenças significativas entre os momentos pré (D0, 3 e 9) e pós (D16 e 19) tratamento cirúrgico e anticoagulante.[...] (AU)

The effects of ankaferd blood stopper and microporous polysaccharide hemospheres on epidural fibrosis in rat laminectomy model

PURPOSE: To investigate whether topically administered hemostatic agents ankaferd blood stopper and microporous polysaccharide hemospheres can decrease epidural fibrosis after laminectomy in rats. METHODS: Eighteen adult male Sprague-Dawley rats were equally and randomly divided into three groups. In the treatment groups, ankaferd blood stopper and microporous polysaccharide hemospheres topically administrated upon duramater surface after laminectomy. Fibroblast count, epidural fibrosis and arachnoidal involvement were evaluated and graded histopathologically. RESULTS: Our data revealed that the count of fibroblasts, the grading of epidural fibrosis and arachnoideal involvement in the rats treated with microporous polysaccharide hemospheres were significantly less than the control group. Although the arachnoideal involvement in ankaferd blood stopper group were significantly less than the control group, there were no statistical differences when comparing the grading of epidural fibrosis and the fibroblasts count between the treatment groups and the control group. CONCLUSION: The ankaferd blood stopper and microporous polysaccharide hemospheres reduced epidural fibrosis and arachnoideal involvement after laminectomy in rats.(AU)

DESCRIÇÃO DO STATUS HEMOSTÁTICO DE EQUINOS COM ABDOME AGUDO POR TÉCNICAS CONVENCIONAIS E TROMBOELASTOGRÁFICAS

Alterações hemostáticas são anormalidades suspeitadas em equinos com obstruções intestinais, estando associadas a elevadas taxas de morbidade e mortalidade. Contudo, as técnicas e/ou testes usados rotineiramente para avaliar a hemostasia apresentam grandes limitações, pois avaliam fases ou componentes isolados do processo hemostático. Em contraste, a tromboelastografia é uma técnica atual que avalia a dinâmica do processo de coagulação sanguínea em conjunto, tornando possível a obtenção de informações acuradas. Como estudos recentes relataram que o uso de plasma fresco ou congelado estaria apresentado resultados satisfatórios tanto em seres humanos como em animais, o presente estudo teve como objetivo inicial realizar testes hemostáticos convencionais (TP, TTPA, Fibrinogênio) e viscoelásticas (tromboelastografia) do plasma de 20 equinos hígidos, 20 equinos com obstrução simples (compactação de colo) e de 20 com obstrução estrangulativa (vólvulo) de alças intestinais de ocorrência natural. Na interpretação dos resultados, verificou-se que o uso da Análise por Componentes Principais foi efetiva na caracterização dos indíviduos a partir dos resultados dos testes realizados. Ademais, ao transformar a interação entre os componentes do processo hemostático em uma visualização gráfica, a tromboelastografia possibilitou uma interpretação e entendimento efetivos do que está acontecendo na coagulação do indivíduo. Assim sendo, pode-se concluir que os testes por métodos convencionais e a avaliação viscoelástica do plasma citratado foram eficazes na caracterização individual do status hemostático de equinos com obstrução intestinal de ocorrência natural, revelando que as obstruções simples também podem causar distúrbios hemostáticos relevantes.

Haemostatic changes are suspected abnormalities in horses with intestinal obstructions, being associated with high rates of morbidity and mortality. However, the techniques and / or tests routinely used to evaluate hemostasis have major limitations, since they evaluate isolated phases or components of the hemostatic process. In contrast, thromboelastography is a current technique that evaluates the dynamics of the blood coagulation process together, making it possible to obtain accurate information. As recent studies have reported that the use of fresh or frozen plasma would present satisfactory results in both humans and animals, the present study aimed to perform conventional hemostatic (PT, APTT, Fibrinogen) and viscoelastic (thromboelastography) plasma analyzes of 20 healthy equines, 20 equines with simple obstruction (colon impactation), and 20 with strangulative obstruction (volvulus) of naturally occurring intestinal loops. In the interpretation of the results, it was verified that the use of Principal Component Analysis was effective in the characterization of the individuals from the results of the tests performed. In addition, by transforming the interaction between the components of the hemostatic process in a graphical view, thromboelastography enabled an effective interpretation and understanding of what is happening in the coagulation of the individual. Thus, it can be concluded that tests by conventional methods and the viscoelastic evaluation of citrated plasma were effective in the individual characterization of hemostatic status of horses with naturally occurring intestinal obstruction, revealing that simple obstructions can also cause relevant haemostatic disorders.

PESQUISA DE Bartonella spp. E Mycoplasma spp. E AVALIAÇÃO HEMOSTÁTICA, HEMATOLÓGICA E BIOQUÍMICA SANGUÍNEA DE PRIMATAS DO GÊNERO Alouatta

Os parâmetros sanguíneos de animais domésticos e selvagens diferem em relação às localidades no mundo. Este trabalho teve como objetivo pesquisar a presença de hematozoários e determinar os parâmetros hemostáticos, hematológicos e bioquímicos sanguíneos de macacos bugios de cativeiro no Estado de São Paulo, sudeste do Brasil. Para realização do hemograma e das análises moleculares dos macacos bugios foi colhido sangue em tubos contendo K2EDTA de 68primatas Alouatta, sendo (Alouatta guariba clamitans, Alouattta caraya e Alouatta spp. de cativeiro). Para as análises bioquímicas e eletroforese de proteínas, foi colhido sangue em tubos contendo ativador de coágulo de 40 macacos bugios para o proteinograma e 29 para as demais análises bioquímicas. Por fim, para avaliação dos parâmetros hemostáticos foi colhido sangue de 30 macacos Alouatta em tubos contendo citrato de sódio a 3,2%. Os resultados obtidos no hemograma de 27 Alouatta caraya e 15 Aloautta guariba clamitans saudáveis foram estatisticamente diferentes na contagem global de hemácias. Nas análises bioquímico-séricas dos bugios hígidos das espécies estudadas, os resultados referentes às dosagens de creatinina, ureia e fosfatase alcalina não apresentaram diferenças estatísticas, porém em relação às dosagens de ALT, os valores foram significativamente diferentes. Quanto ao eletroforetograma das espécies de bugios hígidos, ocorreram diferenças estatísticas apenas na dosagem de proteína total. Nos parâmetros hemostáticos entre espécies de bugios saudáveis, os resultados referentes a contagem de plaquetas, concentração de fibrinogênio plasmático, tempo de tromboplastina parcial ativada e tempo de protrombina não foram significativamente diferentes. Nesta pesquisa, dos 68 macacos bugios avaliados através da PCR convencional, 18 (26,47%) foram positivos para Mycoplasma spp. para o gene 16S rRNA e 1 (5,55%) para o gene RNAseP. Mas, na cPCR para Bartonella spp. para o gene glta a população amostrada de bugios foi negativa. O estudo mostrou que a infecção dos macacos bugios aproximou-se de um genótipo filogeneticamente associado a Candidatus Mycoplasma kahanei. Nas condições do presente estudo, os resultados obtidos dos primatas Alouatta caraya e Alouatta guariba clamitans hígidos podem ser úteis como fonte de consulta para outros pesquisadores, quanto aos parâmetros hematológicos, bioquímicos sanguíneos e hemostáticos. Os resultados obtidos pela PCR convencional mostraram que espécies de hemoplasmascirculam entre primatas Alouatta de cativeiro no Estado de São Paulo, Brasil.

The blood parameters of domestic and wild animals differ in relation to the localities of the world. The objective of this study was to investigate the presence of hematozoars and to determine the hemostatic parameters, hematological parameters and blood biochemical of howler monkeys of captive in the State of São Paulo, Southeastern Brazil. For realizationhematological and molecular analyzis of howler monkeys, blood was collected in K2EDTA from 68 captive primatesAlouatta (Alouatta guariba clamitans, Alouattta caraya and Alouatta spp.). Blood was collected in tubes containing clot activator, of 40 healthy howler monkeys for the proteinogram and 29 for the blood biochemical analyzis. Finally, to evaluate the hemostatic parameters, blood was collected from 30 healthy howler monkeys in tubes containing 3.2% sodium citrate. In the biochemical-serum analyzes of the healthy howler monkeys of the species studied, the results regarding the creatinine, urea and alkaline phosphatase dosages showed no differences statistics, but in relation to the dosages of ALT the values were significantly different. As for the electrophoretogram of the healthy monkeys, the results were statistical differences only in the total protein. In the hemostatic parameters between species of healthy howler monkeys, the results about platelet count, plasma fibrinogen concentration, activated partial thromboplastin time and prothrombin time were not significantly different. In this study, of the 68 howler monkeys evaluated through conventional PCR, 18 (26.47%) were positive for Mycoplasma spp. for the 16S rRNA gene and 1 (5.55%) for the RNAse P gene. But in cPCR for Bartonella spp. for the glta gene the sampled population of howler monkeys was negative. The study showed that the howler monkeys infection approached a genotype phylogenetically associated with "Candidatus Mycoplasma kahanei". Under the conditions of the present study, the results obtained from the Alouatta caraya and Alouatta guariba clamitans healthy can be useful as a source of consultation for other researchers about parameters hematological, blood biochemical and haemostatic. The results obtained by conventional PCR showed that hemoplasm species circulate among Alouatta captive in the State of São Paulo, Brazil.