Comparação de métodos de determinação da concentração de hemoglobina e sua estabilidade analítica em três tempos em Bothrops atrox (Linnaeus, 1758) (Ophidia, Viperidae) mantidas em cativeiro

Abstract

This study aimed to compare alternative methods for measuring plasma hemoglobin (Hematoclin 2.8 VET, Hb=Hc/3 and calculated by linear regression) with the cyanomethemoglobin methodology, considered the gold standard for reptiles; verify the analytical stability of this measurement in samples stored at 4ºC; infer whether the anticoagulant has an influence on the findings and propose a mathematical expression capable of estimating the plasma hemoglobin concentration in adult individuals of the Bothrops atrox species. For this purpose, 16 healthy adult northern jararaca (Bothrops atrox) were analyzed. For the measurement of hemoglobin by the cyanomethemoglobin method, 10 µl of whole blood with EDTA or sodium heparin was added to 2.5 ml of Drabkin's solution, the dilution was centrifuged and 300 µl of the supernatant was transferred to microplates, read at 540 nanometers. This process was repeated at 24h, 48h and 72h. In addition, the Hematoclin 2.8 VET hematology analyzer was used to read samples packed with EDTA K2. Cyanomethemoglobin and linear regression methods were statistically different from calculated hemoglobin Hb=Hc/3 and Hematoclin 2.8 VET (p < 0.0001). Despite that, they showed a strong positive correlation verified by the correlation coefficients 0.9564, 0.9371 and 0.9371, with p < 0.0001. Linear regression to correct the values obtained by the hematological analyzer was represented by the formula HbC (g/dL) = 0.8370 X HbH + 0.5708 (r2 = 0.8781). The hematocrit versus plasma hemoglobin concentration ratio was significant (r2 = 0.8781), so the present study proposed the expression HbC = 0.2459 X Hc + 1.266 as a technique to estimate hemoglobin (g/dL) in this species. The means for each anticoagulant did not differ (p = 0.0831) and those resulting from the sampling times were statistically equal (EDTA K2, p = 0.6598; heparin sodium, p = 0.0589). The data suggest that the conventional formula does not present scientific validity for the species and to avoid the use of toxic and time-consuming methodologies, the proposed formulas can be used to estimate the plasma hemoglobin concentration in Bothrops atrox.