Pesq. Vet. Bras. 36(5):431-435, maio 2016 DOI: 10.1590/S0100-736X2016000500013 731 RESUMO.- [Anticoagulante Na2EDTA danifica amostras de sangue do teleósteo Piaractus mesopotamicus.] O presente estudo teve por objetivo avaliar os efeitos da he- parina sódica e Na2EDTA no sangue de Piaractus mesopota- micus (360,7±42,4g, 26,4±1,0cm). Foram amostrados vinte peixes em dois ensaios experimentais, sendo dez peixes utilizados para análise da fragilidade dos eritrócitos e dez peixes para análise dos parâmetros hematológicos e estu- do bioquímico do plasma. O sangue coletado por punção veno-caudal foi aliquotado e armazenado em diferentes so- luções anticoagulantes: Na2EDTA 10%, Na2EDTA 3%, hepa- rina sódica 5.000 UI e heparina sódica 100 UI. Níveis plas- máticos de cálcio apresentados nas amostras armazenados em Na2EDTA diminuíram cerca de 80% em relação aos dois grupos armazenados com heparina. Amostras de sangue de pacus armazenados com Na2EDTA demonstraram aumen- to do hematócrito e VCM, e diminuição na CHCM. O efeito dose-resposta foi observado neste estudo. Estes resulta- dos são reforçados pelos níveis mais elevados de proteína plasmática e hemólise apresentado no sangue armazenado com Na2EDTA 10%, o que confirma o efeito deletério des- se tratamento anticoagulante na qualidade de amostras de sangue. Na2EDTA não é indicada para armazenar amostras de sangue de P. mesopotamicus, e heparina sódica a 100 UI é o anticoagulante mais recomendado, uma vez que este tratamento apresentou a menor taxa de alterações no san- gue armazenado. Na2EDTA anticoagulant impaired blood samples from the teleost Piaractus mesopotamicus1 Thaís Heloisa Vaz Farias2, Nycolas Levy Pereira2, Santiago Benites de Pádua3, Lindomar de Oliveira Alves2, Róberson Sakabe4, Marco Antonio de Andrade Belo5 and Fabiana Pilarski2* ABSTRACT.- Farias T.H.V., Pereira N.L., Pádua S.B., Alves L.O., Sakabe R., Belo M.A.A. & Pilar- ski F. 2016. Na2EDTA anticoagulant impaired blood samples from the teleost Piarac- tus mesopotamicus. Pesquisa Veterinária Brasileira 36(5):431-435. Centro de Aquicultura da Universidade Estadual Paulista, Rodovia Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil. E-mail: lapoa_caunesp@hotmail.com The present study aimed to evaluate the effects of Na heparin and Na2EDTA on blood of Piaractus mesopotamicus (360.7±42.4g, 26.4±1.0cm). Twenty fishes were sampled in two experiment trials, ten for erythrocyte fragility analysis and ten for hematologic and plas- ma biochemical study. The blood collected by venous-caudal puncture was fractioned and stored in anticoagulants solution: Na2EDTA 10%, Na2EDTA 3%, Na heparin 5000 IU and Na heparin 100 IU. Plasmatic levels of calcium presented in the Na2EDTA stored samples were about 80% lower than both heparin groups. Blood samples of P. mesopotamicus stored with Na2EDTA demonstrated increase in the hematocrit and MCV, and decrease in MCHC. The dose-response effect was observed in this study. The results are reinforced by the higher levels of plasmatic protein and hemolysis presented in the Na2EDTA 10% stored blood, confirming the deleterious effect of this anticoagulant treatment on the quality of blood samples. Na2EDTA is not indicated to store P. mesopotamicus blood samples, but sodium heparin at 100 IU is the most recommended anticoagulant, since this treatment presented the lower rate of alterations in the stored blood. INDEX TERMS: Fish hematology, blood clotting, EDTA, Heparin, Piaractus mesopotamicus. 1 Received on October 3, 2015. Accepted for publication on January 15, 2016. 2 Laboratório de Patologia de Organismos Aquáticos (Lapoa), Centro de Aquicultura da Unesp (CAUnesp), Campus de Jaboticabal, Rodovia de Acesso Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil. *Coresponding author: lapoa_caunesp@hotmail.com 3 AquiVet Saúde Aquática, Rua Emília Joaquina de Jesus Castro 525, Complemento 33, Cidade Nova, São José do Rio Preto, SP 15085-310, Brazil. 4 Departamento de Zootecnia e Desenvolvimento Sustentável, Faculdade de Veterinária, Universidade Federal Fluminense (UFF), Rua Vital Brasil Filho 64, Niterói, RJ 24230-340, Brazil. 5 Departamento de Medicina Veterinária Preventiva, Universidade Es- tadual Paulista (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, Zona Rural, Jaboticabal, SP 14884-012, Brazil. Pesq. Vet. Bras. 36(5):431-435, maio 2016 732 Thaís Heloisa Vaz Farias et al. TERMOS DE INDEXAÇÃO: Hematologia de peixes, coagulação san- guínea, EDTA, Heparina, pacus, Piaractus mesopotamicus. INTRODUCTION Although blood parameters are routinely used to determi- ne the health status of fish and to monitor their physiolo- gical conditions, almost the whole methodology is adapted from other animals such as mammals, resulting in some certain difficulties since the blood sampling until the analy- sis processes. Also, it is know that fish blood parameters can be influenced by several factors, such as seasonal varia- tions, genetics, age, gender, stock density, stress and water quality (Ekanem et al. 2012, Belo et al. 2013, Castro et al. 2014a), what could explain the lack of information on the reference values of health and ill fish. Among the alterations, several can be due to post- -sampling proceedings, in especial the misuse of anticoa- gulants. The blood coagulation is one of the main causes of the quality deterioration of a hematological analysis, also being responsible for alterations in the immunological and biochemical parameters (Pádua et al. 2012). The most used anticoagulants for fish are the sodium heparin and the so- dium ethylendiamintetracetic acid (NA2EDTA). The heparin acts accelerating the action of antithrombin III, inhibiting the action of thrombin and, consequently, the continuity of the coagulation cascade. On the other hand, the EDTA is able to chelate ions, including calcium (Ca2+), important in several steps of the blood clotting (Harr et al. 2005). Ho- wever, according to Ishikawa et al. (2010), both anticoa- gulants can promote changes on the blood parameters in teleost fish. The pacu (Piaractus mesopotamicus), an emergent species in the world aquaculture and considered a good experimental model, is a native teleost fish of the Parana- -Paraguay Basin, and is of importance in the South America for human consumption, angling and aquaculture (Belo et al. 2005, 2012). The pacu has proven to be a good bioindi- cator of water quality, and in accordance with Castro et al. (2014b) this species has been used in ecotoxicity studies for registration of chemicals in Brazil, and no information about the effects caused by the use of anticoagulants on the hematological and biochemical parameters has been rela- ted in the literature. Hence, the present study aimed to eva- luate the effects of different concentrations of Na2EDTA and sodium heparin on the osmotic fragility of erythrocytes and some routinely blood analysis in order to determine which anticoagulant is more indicated to this species. MATERIALS AND METHODS Fish and experimental conditions. In the present study were used 20 specimens of Piaractus mesopotamicus juveniles (360.7g±42.0 g) obtained from a commercial fish farm (Sertão- zinho, São Paulo, Brazil). The animals were maintained in 500 L tanks for one month in order to acclimate to the experimental conditions, with continuous aeration and water change, under constant water quality parameters (temperature 26.0±2.0°C, pH 7.9±0.1, oxygen 6.7±0.69mg L-1, ammonium 0.1±0.1mg L-1). The animals were fed a commercial feed (32% of crude protein), twice a day, until the apparent satiation. Compliance with Ethical Standards. The experimental pro- tocol was approved by Ethics Committee on the Use of Animals from São Paulo State University, process nº22.518/10. Blood sampling and anticoagulant test assay. Ten fish were captured using a net and mechanically immobilized in a wet pie- ce of fabric. The sampling of the blood of each fish was realized through veno-caudal puncture, using sterile syringes (3mL) and hypodermic needles (25x7mm), containing no anticoagulants, as recommended by Ishikawa et al. (2010). Trying to not promote any hemolitic effects besides the possibly caused by the anticoa- gulants, we decided to use no anesthetic chemicals (Korcock et al. 1988). The blood collected, circa of 0.5mL, was quickly placed in four polyethylene tubes (1.5mL): The first and second contained 15μL of NA2EDTA, at a concentration of 3% (0.3mg mL-1 of blood) and 10% (1mg mL-1 of blood), respectively. At last, the third and fourth tubes contained 15 μL of sodium heparin at concentration of 5.000 UI (75 UI mL-1 of blood) and 100 UI (1.5 UI mL-1 of blood), obtained from the dilution of the heparin 5.000 UI in physiological solution (0.65% of NaCl, 1:50) (Ishikawa et al. 2010). Total blood NBT assay. The reactive oxygen species (ROS) production of the blood leukocytes was evaluated using nitro- blue tetrazolium (NBT) reduction following the method of Biller- -Takahashi et al. (2013). Briefly, 100 µL of blood mixed with the anticoagulant was added to 100 µL of phosphate-buffered-saline (pH 8.4) containing NBT (Sigma, USA). After 30 minutes of incu- bation in the dark, 50 µL of this solution was added to 1mL of N,N-dimethylformamide (Sigma, USA), shaken, and centrifuged at 700 g for 10 minutes. The optical density of the supernatant was measured in a conventional spectrophotometer, at a wave length of 540nm. Plasmatic protein and ionic analysis. The remaining blood from the hematological analysis was centrifuged at 300g during 10 minutes and the plasma obtained was used to the determina- tion of the plasmatic protein and plasmatic chloride (Labtest®, Lagoa Santa, MG, Brazil). In order to determine the plasmatic concentration of calcium, sodium and potassium was used a selective ion analyzer (Iselab® Drake, São José do Rio Preto/SP, Brazil). Osmotic fragility of erythrocytes and coagulation effi- cacy assay. To the determination of the osmotic fragility of the erythrocytes were used the ten remaining animals in the tank, and blood sampling procedure followed the same as described above. Dilutions were made in serial from the stock solution (PBS, pH 7.4, 10.5%), in the following concentrations: 0.65, 0.54, 0.43, 0.32, 0.21 e 0.10% of NaCl-PO4, as described by Parpart et al. (1947). The blood was diluted in 1:100 concentration in each one of the NaCl-PO4 solutions, stored at room temperature for 30 min and homogenized each 10 min. After this, the solution was cen- trifuged at 2000 rpm for 5 min and the supernatant hemoglobin was determined according to Collier (1944). The rest of the blood was maintained at 6°C for 10h and, after this, visually evaluated in order to classify the occurrence of coagulation and/or hemolysis (Hattingh & Smith 1976). Hematology. After maintained at room temperature (25°C) during 15 minutes, the blood was used to determination of the he- matocrit following the technic of the microhematocrit, hemoglo- bin by the method of the cyanomethahemoglobin, and red blood cell count (RBC) in hemocytometer after dilution of the blood in citrate formaldehyde (1:200) solution. The hematimetric indexes were calculated according to the equations of Wintrobe. Diffe- rential leukocyte count was performed on blood smears stained with May-Grünwald-Giensa-Wright counting 200 cells. For the quantification of total leukocytes and thrombocytes were coun- ted the number of erythrocytes, leukocytes and thrombocytes in 10 fields for each blood smear. The values for total leukocytes and Pesq. Vet. Bras. 36(5):431-435, maio 2016 733Na2EDTA anticoagulant impaired blood samples from the teleost Piaractus mesopotamicus thrombocytes were determined by the equation: Total number of leukocytes or thrombocytes (mL) = [(number of leukocytes or thrombocytes counted in the smear) X (erythrocyte global count per mL)]/ Number of erythrocytes counted in the blood smear (Belo et al. 2014). Statistical analysis. To the statistical analysis of the data was performed a variance analysis (ANOVA) and, when significant (p<0.05), the means were compared using the Tukey’s test. The data was transformed when necessary and all the statistical pro- cedure was realized in R Software. RESULTS In the osmotic fragility of erythrocytes assay, the cells of the heparinized blood and containing NA2EDTA 3% presented a higher resistance to the hypotonic solution when compa- red to the NA2EDTA 10% group, which showed significant increase (p<0.05) in the cell lysis between 0.35 to 0.65% NaCl-PO4 solution. These analyze showed dose-response effect (Figure 1). Blood containing NA2EDTA 10% showed higher scores (p<0.0001) of hemolysis after 10 hours at 6° C (Table 1). Blood biochemical parameters presented significant differences according to the anticoagulant used (Table 1). The values of plasmatic protein were higher in the samples containing NA2EDTA 10% (p=0.,0088) when compared to NA2EDTA 3% and Heparin 100 IU, but showed no differen- ces when compared to the Heparin 5000 IU group. The samples containing Heparin 100 IU showed signi- ficant increase (p<0.0001) in the plasmatic chloride values when compared to NA2EDTA 3 and 10%, and blood contai- ning heparin 5000 IU presented no differences from any of the groups (Table 1). On the other hand, blood containing NA2EDTA 10% presented the highest (p<0.0001) plasmatic sodium concentration, followed by the NA2EDTA 3% and Heparin 5000 IU, while the lower values of plasma sodium were observed in blood containing Heparin 100 IU. Samples with Heparin 100 IU showed low levels of plas- ma potassium when compared blood containing NA2EDTA 3% (p=0.0198). The NA2EDTA groups of samples (3 and 10%) presented significant decrease (p<0.0001) in the plasmatic calcium when compared to the Heparin groups. No differences were found among the groups of samples containing different anticoagulants in the total blood NBT assay (p=0.6640). All the anticoagulants investigated in the present stu- dy were efficient in the prevention of the blood coagula- tion of Piaractus mesopotamicus for longer than 10 hours Table 1. Mean values (± SEM)a and ANOVAb observed in the plasmatic protein, ions, NBT and hemolysis study of Piaractus mesopotamicus blood stored with different anticoagulants Parameters Na2EDTA Heparin p 10% 3% 5000 UI 100 UI Plasmatic protein (g/dL) 3.31±0.54A 2.8±1.0B 3. 09±0.24AB 2.92±0.5B 0.0088 Chlorede (meq L-1) 99.43±4.8C 103.1±8.3BC 106.4±3.4AB 109.2±4.4A <0.0001 Na (meq L-1) 150.2 a±5.96A 139±5.6B 137.5±5.42B 129.2±3.31C <0.0001 K (meq L-1) 3.44±0.33AB 3.59±0.35A 3.42±0.55AB 2.99±0.45B 0.0198 Ca (meq L-1) 0.200±0.08C 0.226±0.09C 1.032±0.11B 1.216±0.05A <0.0001 NBT assay (OD) 0. 3402±0.06 0.3615±0.14 0.3347±0.02 0.346±0.04 0.6640 Hemolysis Score (1-5) 2.70±0.25 A 0.42±0.15 B 0.50±0.17 B 0.50±0.17 B <0.0001 a Mean values of 10 samples; SEM (standard error of the mean¬), b Means compared in the lines, with at least one letter in common do not differ from each other through Tukey’s test (p>0.05). Table 2. Mean values (± SEM)a and ANOVAb observed in the hematological study of Piaractus mesopotamicus blood stored with different anticoagulants Parameters Na2EDTA Heparin p value 10% 3% 5000UI 100UI Ht (%) 36.9±3.1A 35.9±2.6A 31.9±1.7B 31.5±2.4B < 0.0001* Hemoglobin (g.dL-1) 10.2±1.0 10.3±0.8 10.5±0.8 10.2±0.8 0.8430 MCV (fL) 173.5±28.8A 147.5±20.5B 134.1±12.0B 124.2±18.0B < 0.0001* MCHC (g dL-1) 27.8±1.0B 28.6±1.3B 33.0±1.3A 32.5 ±2.0A < 0.0001* RBC (x 106 µL-1) 2.2±0.4 2.5±0.4 2.4±0.2 2.6±0.4 0.0993 Throm (x 103 µL-1) 71.7±15.5 73.3±11.2 63.6±11.6 68.4±7.3 0,1840 Leuk (x 103 µL-1) 28.9±5.9 36.3±9.0 37.4±5.4 33.7±2.2 0,3060 a Mean values of 10 samples; SEM (standard error of the mean¬), b Means compared in the lines, with at least one letter in common do not differ from each other through Tukey’s test (p>0.05). Fig.1. Osmotic fragility of erythrocytes of Piaractus mesopotamicus blood stored in different anticoagulants (p<0.05). Pesq. Vet. Bras. 36(5):431-435, maio 2016 734 Thaís Heloisa Vaz Farias et al. (Data not show). At the same time, thrombocyte, leukocyte and erythrocyte counts did not present difference (p>0,05) among treatments (Table 2). Blood samples containing NA- 2EDTA (3% and 10%) presented increase in the hematocrit values (p < 0.0001) and decrease in the MCHC (p<0.0001) when compared to fish blood containing heparin (100 and 5000 IU). The same response profile was observed for MCV analyzes, but only the samples stored with NA2EDTA 10% presented a significant increase on MCV (p<0.0001). DISCUSSION Despite the effectiveness of anticoagulants in the control of Piaractus mesopotamicus blood clotting process for longer than 10 hours, the results obtained in the present study shows that the hematological and biochemical parameters of P. mesopotamicus suffered considerable changes accor- ding to the anticoagulant used to the samples preservation. Plasmatic levels of calcium presented in the Na2EDTA stored samples of pacus were about 80 % lower than both heparin groups. The Na2EDTA acts as a chelating to the an- ticoagulant factor VI (Ca2+), responsible by the stability and permeability of the cell membrane (Harr et al. 2005), cau- sing an increase of erythrocytes volume. Kaestner (2011) and Bogdanova et al. (2013) described the importance of Ca2+ as universal signaling molecule involved in regulating cell cycle, metabolism, structural integrity and volume, and changes in the extracellular levels of Ca2+ result in serious dysregulation of hydromineral balance. Since mechanisms of transport are calcium dependent such as Gardos chan- nel recognized in erythrocytes. In this study, increase in sodium levels and decrease in choride was observed in samples stored with Na2EDTA 10%, confirming this hypo- thesis. Hemolysis were observed in birds, reptiles (Hattin- gh & Smith 1976) and carps (Orlov et al. 2005) with poor extracellular levels of calcium, emphasizing the importance of this ion to the preservation of the membrane integrity of nucleated erythrocytes. Blood samples of pacus stored with Na2EDTA demons- trated increase in the hematocrit and MCV, and decrease in MCHC. The dose-response effect was observed and sam- ples stored with Na2EDTA 10% showed more significant re- sults. In samples stored with Na2EDTA 10% were observed decrease in RBC counts, although statistical difference did not occur among treatments. These results are reinforced by the higher levels of plasmatic protein and hemolysis presented in the Na2EDTA 10 % stored blood, confirming the deleterious effect of this anticoagulant treatment on the quality of blood samples. In addition, the acidic nature of the EDTA salts can change the pCO2 and pH of the blood (Witeska & Wargoka 2009) altering the Na+/K+ pump func- tion, an important protein that regulates the osmotic gra- dient in cell (Högman et al. 1997, Högman 1998). Also, in heparin 5000 IU samples, the increase of plasmatic protein probably comes from the fact that heparin is a protein, and this group received 50 times more of this anticoagulant then the heparin 100 IU samples. The evaluation of the osmotic fragility of erythrocytes shows the resistance of these cells in front of osmotic stress simulated by different NaCl concentration solutions, and the degree of hemolysis is measured by the amount hemo- globin liberated by the rupture of the cells (Sarkar et al. 1999). Blood samples of pacus stored with Na2EDTA 10 % presented increase in the cell lysis between 0.35 to 0.65% NaCl-PO4 solution. Walencik & Witeska (2007) observed higher fragility in common carp erythrocytes to hipotonic solution since the lesser concentration of Na2EDTA tests (0.01mg ml-1) and a positive relation of the osmotic fragility and the increase of the anticoagulant concentrations. Simi- lar results were described in Blennius pholis (Mainwaring & Rowley 1985), Oncorhynchus mykiss (Korcock et al. 1988), Oreochromis niloticus (Ekanem et al. 2012), Pseudoplatys- toma reticulatum x P. corruscans (Ishikawa et al. 2010) and Colossoma macropomum (Pádua et al. 2012). Reactive Oxygen Species (ROS) are substances with the ability to degrade membranes and are extremely aggressi- ve to pathogens during the process of phagocytosis (John et al. 2002), and their production was not influenced by the anticoagulant levels in P. mesopotamicus blood, in the same way as observed by Nielsen (1985) in human blood sam- ples stored in heparin, EDTA and oxalate. However, some studies have showed an increase in the ROS production due to the phagocytosis of the cellular debris resulted from the erythrocytes lysis when the blood was added to NA2EDTA or heparin (Walencik & Witeska 2007, Wan et al. 1992). Al- though other studies have reported changes in leukocyte and thrombocyte counts in blood samples of Cyprinus car- pio (Walencik & Witeska 2007) and Blenius pholis (Mainwa- ring & Rowley 1985) stored with EDTA and heparin, blood samples of pacus did not present difference in thrombocyte and leukocyte counts. CONCLUSIONS Na2EDTA is not indicated to store Piaractus mesopota- micus blood samples, causing hematological and serum biochemistry alterations and hemolysis, principally when in a high concentration (1mg mL-1). On the other hand, sodium heparin at 100 IU is the most recommended anticoagulant, since this treatment presen- ted the lower rate of alterations in the stored blood. Both anticoagulants can be used in total blood NBT assay. Acknowledgements.- Thanks to CAUnesp (Aquaculture Center of São Paulo State University) for the facilities. This study was financed by CAPES Foundation, Ministry of Education from Brazil. REFERENCES Belo M.A.A., Schalch S.H.C., Moraes F.R., Soares V.E., Otoboni A.M.M.B. & Moraes J.R.E. 2005. Effect of Dietary Supplementation with Vitamin E and Stocking Density on Macrophage Recruitment and Giant Cell For- mation in the Teleost Fish, Piaractus mesopotamicus. J. Comp. Pathol. 133:146-154. Belo M.A.A., Moraes J.R.E., Soares V.E., Martins M.L., Brum C.D. & Moraes F.R. 2012. Vitamin C and endogenous cortisol in foreign-body inflamma- tory response in pacus. Pesq. Agropec. Bras. 47:1015-1021. Belo M.A.A., Souza D.G.F., Faria V.P., Prado E.J.P., Moraes F.R. & Onaka E.M. 2013. Haematological response of curimbas Prochilodus lineatus, na- turally infected with Neoechinorynchus curemai. J. Fish Biol. 82:1403- 1410. Belo M.A.A., Moraes F.R., Yoshida L., Prado E.J.R., Moraes J.R.E., Soares V.E. & Silva M.G. 2014. Deleterious effects of low level of vitamin E and high Pesq. Vet. Bras. 36(5):431-435, maio 2016 735Na2EDTA anticoagulant impaired blood samples from the teleost Piaractus mesopotamicus stocking density on the hematology response of pacus, during chronic inflammatory reaction. Aquaculture 422:124-128. Biller-Takahashi J.D., Takahashi L.S., Saita M.V., Gimbo R.Y. & Urbinati E.C. 2013. Leukocytes respiratory burst activity as indicator of innate immu- nity of pacu Piaractus mesopotamicus. Braz. J. Biol. 73:425-429. Bogdanova A., Makhro A., Wang J., Lipp P. & Kaestner L. 2013. Calcium in Red Blood Cells: a perilous balance. Int. J. Mol. Sci. 14:9848-9872. Castro M.P., Claudiano G.S., Bortoluzzi N.L., Garrido E., Fujimoto R.Y., Belo M.A.A., Shimada M.T., Moraes J.R.E. & Moraes F.R. 2014a. Chromium car- bochelate dietary supplementation favored the glucocorticoid response during acute inflammation of Piaractus mesopotamicus. Aquaculture 432:114-118. Castro M.P., Moraes F.R., Fujimoto R.Y., Cruz C., Belo M.A.A. & Moraes J.R.E. 2014b. Acute toxicity by water containing hexavalent or trivalent chro- mium in native Brazilian fish, Piaractus mesopotamicus: Anatomopatho- logical alterations and mortality. Bull. Environ. Cont. Toxicol. 92:213- 219. Collier H.B.1944. The standardizations of blood haemoglobin determina- tions. Can. Med. Assoc. J. 50:550-552. Ekanem A.P., Udoh A.J. & Inyang-Etoh A.P. 2012. Effect of different anticoa- gulants on hematological parameters of Oreochromis niloticus. Int. J. Sci. Adv. Technol. 2:17-20. Harr K.E., Raskin R.E. & Heard D.J. 2005. Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons. Vet. Clin. Pathol. 34:383- 388. Hattingh J. & Smith E.M. 1976. Anticoagulants for avian and reptilian blood: heparin and EDTA. Pflug. Arch. Eur. J. Physiol. 363:267-269. Högman C.F., Erickson L., Wallvik J. & Payrat J.M. 1997. Clinical and labora- tory experience with erythrocyte and platelet preparations for 0.5 CPD Erythro - Sol opti systems. Vox Sang. 73:212-219. Högman C.F. 1998. Preparation and preservation of red cells. Vox Sang. 74:177-187. Ishikawa M.M., Padua S.B., Satake F., Hisano H., Jeronimo G.T. & Martins M.L. 2010. Heparin and Na2EDTA as anticoagulants for hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans): efficacy and he- matological. Ciência Rural 40:1557-1561. John M.B., Chandran M.R., Aruna B.V. & Anbarasu K. 2002. Production of superoxide anion by head-kidney leucocytes of Indian major carps im- munized with bacterins of Aeromonas hydrophila. Fish Shellfish Immun. 12:201-207. Kaestner L. 2013. Cation channels in erythrocytes-historical and future perspective: calcium signalling. Springer, Berlin, p.223-233. Korcock D.E., Houston A.H. & Gray J.D. 1988. Effects of sampling conditions on selected blood variables of rainbow trout, Salmo gairdneri Richard- son. J. Fish Biol. 33:319-330. Mainwaring G. & Rowle A.F. 1985. The effect of anticoagulants on Blennius pholis L. leucocytes. Comp. Biochem. Physiol. 80A:85-91. Nielsen H. 1985. Influence of five different anticoagulants on human blood monocyte isolation and functional activities. Acta Pathol. Microbiol. Im- munol. Scand. 93:49-52. Orlov S.N., Aksentse V.S.L. & Kotelevtsev S.V. 2005. Extracellular calcium is required for the maintenance of plasma membrane integrity in nuclea- ted cells. Cell Calcium 38:53-57. Pádua S.B., Pilarski F., Sakabe R., Dias-Neto J., Chagas E.C. & Ishikawa M.M. 2012. Heparin and K3EDTA as anticoagulants for tambaqui (Colossoma macropomum Cuvier, 1816). Acta Amazonica 42:293-298. Parpart A.K., Lorenz P.B., Parpart E.R., Gregg J.R. & Chase A.M. 1947. The os- motic resistance (fragility) of human red cells. J. Clin. Invest. 26:636-640. Sarkar M., Barari S.K., Mandal D.B., Nandankar U.A., Basu A., Mohanty T.K. & Ray S. 1999. The effects of anti-coagulants on the osmotic fragility of erythrocytes in the Yak (Poephagus grunniens). Vet. J. 157:91-93. Walencik J. & Witeska M. 2007. The effects of anticoagulants on hemato- logical indices and blood cell morphology of common carp (Cyprinus carpio L.). Comp. Biochem. Physiol. 146C:331-335. Wan T.S., Tam A.Y. & Yeung C.Y. 1992. Effects of anticoagulants and incuba- tion time on neutrophil nitroblue tetrazolium score. Biol. Signals 1:167- 172. Witeska M. & Wargocka W. 2011. Disodium EDTA used as anticoagulant cau- ses hemolysis in commom carp blood. Turk. J. Vet. Anim. Sci. 35:99-104.