Pesq. Vet. Bras. 36(5):431-435, maio 2016 DOI: 10.1590/S0100-736X2016000500013

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: [email protected]

The present study aimed to evaluate the effects of Na heparin and Na2EDTA on blood of Piaractus mesopotamicus experiment trials, ten for analysis and ten for hematologic and plas- ma biochemical study. The (360.7±42.4g, blood collected 26.4±1.0cm). by venous-caudal Twenty puncture fishes were was sampled fractioned in twoand

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 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 presented in the Na2EDTA 10% stored blood,

samples. Na2EDTA is not indicated to store P. mesopotamicus blood samples, but sodium heparinconfirming at 100 the IUdeleterious is the most effect recommended of this anticoagulant anticoagulant, treatment since this on treatmentthe quality presented of blood the lower rate of alterations in the stored blood. INDEX TERMS: Fish , blood clotting, EDTA, Heparin, Piaractus mesopotamicus.

RESUMO.- [Anticoagulante Na2EDTA danifica amostras peixes para análise dos parâmetros hematológicos e estu- de sangue do teleósteo Piaractus mesopotamicus.] O do bioquímico do plasma. O sangue coletado por punção presente estudo teve por objetivo avaliar os efeitos da he- veno-caudal foi aliquotado e armazenado em diferentes so- parina sódica e Na2EDTA no sangue de Piaractus mesopota- luções anticoagulantes: Na2EDTA 10%, Na2EDTA 3%, hepa- micus (360,7±42,4g, 26,4±1,0cm). Foram amostrados vinte rina sódica 5.000 UI e heparina sódica 100 UI. Níveis plas- peixes em dois ensaios experimentais, sendo dez peixes máticos de cálcio apresentados nas amostras armazenados utilizados para análise da fragilidade dos eritrócitos e dez em Na2EDTA diminuíram cerca de 80% em relação aos dois grupos armazenados com heparina. Amostras de sangue de 1 Received on October 3, 2015. pacus armazenados com Na2EDTA demonstraram aumen- Accepted for publication on January 15, 2016. to do hematócrito e VCM, e diminuição na CHCM. O efeito 2 Laboratório de Patologia de Organismos Aquáticos (Lapoa), Centro dose-resposta foi observado neste estudo. Estes resulta- de Aquicultura da Unesp (CAUnesp), Campus de Jaboticabal, Rodovia de dos são reforçados pelos níveis mais elevados de proteína Acesso Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil. *Coresponding author: [email protected] plasmática e hemólise apresentado no sangue armazenado 3 AquiVet Saúde Aquática, Rua Emília Joaquina de Jesus Castro 525, com Na2 - se tratamento anticoagulante na qualidade de amostras de 4 Departamento de Zootecnia e Desenvolvimento Sustentável, Faculdade sangue. NaEDTA 10%, o que confirma o efeito deletério des Complemento 33, Cidade Nova, São José do Rio Preto, SP 15085-310, Brazil. 2 de Veterinária, Universidade Federal Fluminense (UFF), Rua Vital Brasil de sangue de P. mesopotamicus, e heparina sódica a 100 UI Filho 64, Niterói, RJ 24230-340, Brazil. EDTA não é indicada para armazenar amostras 5 Departamento de Medicina Veterinária Preventiva, Universidade Es- tadual Paulista (Unesp), Rodovia de Acesso Paulo Donato Castellane s/n, tratamento apresentou a menor taxa de alterações no san- Zona Rural, Jaboticabal, SP 14884-012, Brazil. gueé o anticoagulantearmazenado. mais recomendado, uma vez que este

731 732 Thaís Heloisa Vaz Farias et al.

TERMOS DE INDEXAÇÃO: Hematologia de peixes, coagulação san- Compliance with Ethical Standards. The experimental pro- guínea, EDTA, Heparina, pacus, Piaractus mesopotamicus. tocol was approved by Ethics Committee on the Use of Animals from São Paulo State University, process nº22.518/10. INTRODUCTION Blood sampling and anticoagulant test assay. captured using a net and mechanically immobilized in a wet pie- Although blood parameters are routinely used to determi- Ten fish were - through veno-caudal puncture, using sterile syringes (3mL) and gical conditions, almost the whole methodology is adapted hypodermicce of fabric. Theneedles sampling (25x7mm), of the containing blood of each no anticoagulants, fish was realized as fromne the other health animals status ofsuch fish as and mammals, to monitor resulting their physiolo in some 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. certain difficulties since the blood sampling until the analy- 1988). The blood collected, circa of 0.5mL, was quickly placed in tions,sis processes. genetics, Also, age, gender,it is know stock that density, fish blood stress parameters and water EDTA, at a concentration of 3% (0.3mg mL-1 of blood) can be influenced by several factors, such as seasonal varia 2 quality (Ekanem et al. 2012, Belo et al. 2013, Castro et al. andfour 10%polyethylene (1mg mL tubes-1 of blood), (1.5mL): respectively. The first and At last,second the containedthird and 2014a), what could explain the lack of information on the 15μL of NA of 5.000 UI (75 UI mL-1 of blood) and 100 UI (1.5 UI mL-1 of blood), Among the alterations, several can be due to post- obtainedfourth tubes from contained the dilution 15 ofμL the of sodiumheparin heparin 5.000 UI at in concentration physiological -samplingreference valuesproceedings, of health in and especial ill fish. the misuse of anticoa- solution (0.65% of NaCl, 1:50) (Ishikawa et al. 2010). gulants. The blood is one of the main causes of Total blood NBT assay. The reactive oxygen species (ROS) the quality deterioration of a hematological analysis, also production of the blood leukocytes was evaluated using nitro- being responsible for alterations in the immunological and blue tetrazolium (NBT) reduction following the method of Biller- biochemical parameters (Pádua et al. 2012). The most used - (pH-Takahashi 8.4) containing et al. (2013). NBT Briefly,(Sigma, 100 USA). µL After of blood 30 minutes mixed with of incu the- dium ethylendiamintetracetic acid (NA2EDTA). The heparin anticoagulant was added to 100 µL of phosphate-buffered-saline actsanticoagulants accelerating for the fish action are the of sodiumantithrombin heparin III, and inhibiting the so N,N-dimethylformamide (Sigma, USA), shaken, and centrifuged at the action of thrombin and, consequently, the continuity 700bation g for in 10the minutes. dark, 50 The µL opticalof this densitysolution of was the addedsupernatant to 1mL was of of the coagulation cascade. On the other hand, the EDTA is measured in a conventional spectrophotometer, at a wave length able to chelate ions, including calcium (Ca2+), important in of 540nm. several steps of the blood clotting (Harr et al. 2005). Ho- Plasmatic protein and ionic analysis. The remaining blood wever, according to Ishikawa et al. (2010), both anticoa- from the hematological analysis was centrifuged at 300g during 10 minutes and the plasma obtained was used to the determina- gulants can promote changes on the blood parameters in tion of the plasmatic protein and plasmatic chloride (Labtest®, Lagoa Santa, MG, Brazil). In order to determine the plasmatic The pacu (Piaractus mesopotamicus), an emergent concentration of calcium, sodium and potassium was used a speciesteleost fish. in the world aquaculture and considered a good selective ion analyzer (Iselab® - Brazil). -Paraguay Basin, and is of importance in the South America Osmotic fragility of erythrocytes Drake, São and José coagulation do Rio Preto/SP, effi- forexperimental human consumption, model, is a anglingnative teleost and aquaculture fish of the (BeloParana et cacy assay. To the determination of the osmotic fragility of the al. 2005, 2012). The pacu has proven to be a good bioindi- erythrocytes were used the ten remaining animals in the tank, cator of water quality, and in accordance with Castro et al. and blood sampling procedure followed the same as described above. Dilutions were made in serial from the stock solution (PBS, (2014b) this species has been used in ecotoxicity studies pH 7.4, 10.5%), in the following concentrations: 0.65, 0.54, 0.43, for registration of chemicals in Brazil, and no information 0.32, 0.21 e 0.10% of NaCl-PO4, as described by Parpart et al. about the effects caused by the use of anticoagulants on the (1947). The blood was diluted in 1:100 concentration in each one hematological and biochemical parameters has been rela- of the NaCl-PO4 solutions, stored at room temperature for 30 min ted in the literature. Hence, the present study aimed to eva- and homogenized each 10 min. After this, the solution was cen- trifuged at 2000 rpm for 5 min and the supernatant luate the effects of different concentrations of Na2EDTA and sodium heparin on the osmotic fragility of erythrocytes and was determined according to Collier (1944). The rest of the blood some routinely blood analysis in order to determine which was maintained at 6°C for 10h and, after this, visually evaluated in anticoagulant is more indicated to this species. order to classify the occurrence of coagulation and/or hemolysis (Hattingh & Smith 1976). Hematology. After maintained at room temperature (25°C) MATERIALS AND METHODS during 15 minutes, the blood was used to determination of the he- Fish and experimental conditions. In the present study matocrit following the technic of the microhematocrit, hemoglo- were used 20 specimens of Piaractus mesopotamicus juveniles bin by the method of the cyanomethahemoglobin, and red blood - cell count (RBC) in hemocytometer after dilution of the blood in zinho, São Paulo, Brazil). The animals were maintained in 500 L citrate formaldehyde (1:200) solution. The hematimetric indexes tanks(360.7g±42.0 for one g)month obtained in order from to a acclimatecommercial to fishthe farmexperimental (Sertão were calculated according to the equations of Wintrobe. Diffe- conditions, with continuous aeration and water change, under rential leukocyte count was performed on blood smears stained constant water quality parameters (temperature 26.0±2.0°C, pH with May-Grünwald-Giensa-Wright counting 200 cells. For the 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 ted the number of erythrocytes, leukocytes and thrombocytes in a day, until the apparent satiation. quantification of total leukocytes and thrombocytes were coun

10 fields for each blood smear. The values ​​for total leukocytes and Pesq. Vet. Bras. 36(5):431-435, maio 2016 Na2EDTA anticoagulant impaired blood samples from the teleost Piaractus mesopotamicus 733 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

(p<0.05), the means were compared using the Tukey’s test. The dataperformed was transformed a variance when analysis necessary (ANOVA) and and, all the when statistical significant 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- Fig.1. Osmotic fragility of erythrocytes of Piaractus mesopotamicus red to the NA2 blood stored in different anticoagulants (p<0.05). increase (p<0.05) in the cell lysis between 0.35 to 0.65%

NaCl-PO4 solution.EDTA These 10% group,analyze which showed showed dose-response significant NA2EDTA 10% presented the highest (p<0.0001) plasmatic effect (Figure 1). Blood containing NA2EDTA 10% showed sodium concentration, followed by the NA2EDTA 3% and higher scores (p<0.0001) of hemolysis after 10 hours at 6° Heparin 5000 IU, while the lower values of plasma sodium C (Table 1). were observed in blood containing Heparin 100 IU. Samples with Heparin 100 IU showed low levels of plas- differences according to the anticoagulant used (Table 1). ma potassium when compared blood containing NA2EDTA TheBlood values biochemicalof plasmatic protein parameters were presentedhigher in the significant samples 3% (p=0.0198). The NA2EDTA groups of samples (3 and containing NA2EDTA 10% (p=0.,0088) when compared to NA2EDTA 3% and Heparin 100 IU, but showed no differen- plasmatic calcium when compared to the Heparin groups. ces when compared to the Heparin 5000 IU group. No10%) differences presented were significant found among decrease the (p<0.0001)groups of samples in the The samples containing Heparin 100 IU showed signi- containing different anticoagulants in the total blood NBT assay (p=0.6640). when compared to NA2EDTA 3 and 10%, and blood contai- All the anticoagulants investigated in the present stu- ningficant heparin increase 5000 (p<0.0001) IU presented in the no plasmatic differences chloride from valuesany of - the groups (Table 1). On the other hand, blood containing tion of Piaractus mesopotamicus for longer than 10 hours dy were efficient in the prevention of the blood coagula 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 a b Leuk Mean (x values 103 µL-1) of 10 samples;28.9±5.9 SEM (standard 36.3±9.0 error of the mean¬),37.4±5.4 Means compared33.7±2.2 in the lines,0,3060 with at least one letter in common do not differ from each other through Tukey’s test (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 the degree of hemolysis is measured by the amount hemo- and erythrocyte counts did not present difference (p>0,05) globin liberated by the rupture of the cells (Sarkar et al. among treatments (Table 2). Blood samples containing NA- 1999). Blood samples of pacus stored with Na2EDTA 10 % 2EDTA (3% and 10%) presented increase in the hematocrit presented increase in the cell lysis between 0.35 to 0.65% values (p < 0.0001) and decrease in the MCHC (p<0.0001) NaCl-PO4 solution. Walencik & Witeska (2007) observed higher fragility in common carp erythrocytes to hipotonic

solution since the lesser concentration of Na2EDTA tests when compared to fish blood containing heparin (100 and -1 analyzes, but only the samples stored with NA2EDTA 10% (0.01mg ml ) and a positive relation of the osmotic fragility 5000 IU). The same response profile was observed for MCV and the increase of the anticoagulant concentrations. Simi- lar results were described in Blennius pholis (Mainwaring & presented a significantDISCUSSION increase on MCV (p<0.0001). Rowley 1985), Oncorhynchus mykiss (Korcock et al. 1988), Despite the effectiveness of anticoagulants in the control of Oreochromis niloticus (Ekanem et al. 2012), Pseudoplatys- Piaractus mesopotamicus blood clotting process for longer toma reticulatum x P. corruscans (Ishikawa et al. 2010) and than 10 hours, the results obtained in the present study Colossoma macropomum (Pádua et al. 2012). shows that the hematological and biochemical parameters Reactive Oxygen Species (ROS) are substances with the of P. mesopotamicus suffered considerable changes accor- ability to degrade membranes and are extremely aggressi- ding to the anticoagulant used to the samples preservation. ve to pathogens during the process of phagocytosis (John

Plasmatic levels of calcium presented in the Na2EDTA stored samples of pacus were about 80 % lower than both anticoagulant levels in P. mesopotamicus blood, in the same wayet al. as2002), observed and their by Nielsen production (1985) was in not human influenced blood by sam the- heparin groups. The Na2EDTA acts as a chelating to the an- ticoagulant factor VI (Ca2+), responsible by the stability and ples stored in heparin, EDTA and oxalate. However, some permeability of the cell membrane (Harr et al. 2005), cau- studies have showed an increase in the ROS production due sing an increase of erythrocytes volume. Kaestner (2011) to the phagocytosis of the cellular debris resulted from the and Bogdanova et al. (2013) described the importance of erythrocytes lysis when the blood was added to NA2EDTA Ca2+ as universal signaling molecule involved in regulating or heparin (Walencik & Witeska 2007, Wan et al. 1992). Al- cell cycle, metabolism, structural integrity and volume, and though other studies have reported changes in leukocyte changes in the extracellular levels of Ca2+ result in serious and thrombocyte counts in blood samples of Cyprinus car- dysregulation of hydromineral balance. Since mechanisms pio (Walencik & Witeska 2007) and Blenius pholis (Mainwa- of transport are calcium dependent such as Gardos chan- ring & Rowley 1985) stored with EDTA and heparin, blood nel recognized in erythrocytes. In this study, increase in samples of pacus did not present difference in thrombocyte sodium levels and decrease in choride was observed in and leukocyte counts. samples stored with Na2 - thesis. Hemolysis were observed in birds, reptiles (Hattin- CONCLUSIONS gh & Smith 1976) and carpsEDTA (Orlov 10%, etconfirming al. 2005) thiswith hypo poor Na2EDTA is not indicated to store Piaractus mesopota- extracellular levels of calcium, emphasizing the importance micus blood samples, causing hematological and serum of this ion to the preservation of the membrane integrity of biochemistry alterations and hemolysis, principally when nucleated erythrocytes. in a high concentration (1mg mL-1).

Blood samples of pacus stored with Na2EDTA demons- On the other hand, sodium heparin at 100 IU is the most trated increase in the hematocrit and MCV, and decrease in recommended anticoagulant, since this treatment presen- MCHC. The dose-response effect was observed and sam- ted the lower rate of alterations in the stored blood. Both ples stored with Na2 - anticoagulants can be used in total blood NBT assay. sults. In samples stored with Na2EDTA 10% were observed decrease in RBC counts,EDTA although 10% showed statistical more differencesignificant didre Acknowledgements.- Thanks to CAUnesp (Aquaculture Center of São not occur among treatments. These results are reinforced Foundation, Ministry of Education from Brazil. by the higher levels of plasmatic protein and hemolysis Paulo State University) for the facilities. This study was financed by CAPES presented in the Na 2 REFERENCES the deleterious effect of this anticoagulant treatment on Belo M.A.A., Schalch S.H.C., Moraes F.R., Soares V.E., Otoboni A.M.M.B. & the quality of blood samples.EDTA 10 In % addition, stored blood, the acidic confirming nature Moraes J.R.E. 2005. Effect of Dietary Supplementation with Vitamin E of the EDTA salts can change the pCO2 and pH of the blood and Stocking Density on Macrophage Recruitment and Giant Cell For- (Witeska & Wargoka 2009) altering the Na+/K+ pump func- mation in the Teleost Fish, Piaractus mesopotamicus. J. Comp. Pathol. tion, an important protein that regulates the osmotic gra- 133:146-154. dient in cell (Högman et al. 1997, Högman 1998). Also, in Belo M.A.A., Moraes J.R.E., Soares V.E., Martins M.L., Brum C.D. & Moraes heparin 5000 IU samples, the increase of plasmatic protein - tory response in pacus. Pesq. Agropec. Bras. 47:1015-1021. probably comes from the fact that heparin is a protein, and F.R. 2012. Vitamin C and endogenous cortisol in foreign-body inflamma this group received 50 times more of this anticoagulant 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- then the heparin 100 IU samples. turally infected with Neoechinorynchus curemai. J. Fish Biol. 82:1403- The evaluation of the osmotic fragility of erythrocytes 1410. shows the resistance of these cells in front of osmotic stress Belo M.A.A., Moraes F.R., Yoshida L., Prado E.J.R., Moraes J.R.E., Soares V.E. simulated by different NaCl concentration solutions, and & Silva M.G. 2014. Deleterious effects of low level of vitamin E and high

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