Introduction
Haematological parameters are valuable indicators of fish health status. Blood reflects the overall physiological status of the animal and has been widely used as a diagnostic tool to assess health status in fish in response to changes related to nutrition, water quality and pathological conditions. Haematology combined with other routine diagnostic methods can be used to identify and assess conditions that cause stress and/or diseases that affect fish production and performance. Moreover, blood samples can be collected without euthanizing the animals, providing an additional advantage for both ethical and economic reasons. Fish blood haematology analysis is currently performed mainly by manual methods as automated analysers have been scarcely validated and used in fish. Automated methods present significant advantages compared to manual techniques, offering time- and cost-effective options, eliminating operator-tied errors and, consequently, increasing repeatability and reliability of the obtained results. The present study has two main objectives: to validate the automated blood cell analyser (Sysmex XN-1000V) for its potential use with blood samples of the teleost fish Oncorhynchus mykiss (rainbow trout) and to establish reference values for the species since those currently available are scarce.
Material and methods
Fish used in the study were netted and anesthetized with tricaine methanesulfonate (MS-222). Blood was sampled from the caudal vein using heparinized needles (25 G) and 2 mL syringes and immediately transferred to Eppendorf tubes with heparin. Samples were analysed within 12 h after the extraction using the Sysmex XN-1000V, Ver. 3 (3.05-00), employing the settings for bird blood samples. Precision, linearity and carry-over were calculated for the results obtained from the analyser in red blood cell count (RBC), haemoglobin (HGB), haematocrit value (HCT), mean corpuscular volume (MCV), white blood cell count (WBC), and platelet count (PLT). Precision was calculated for RBC and WBC counts performed by manual method. A comparison was made between the results obtained by the analyser and the manual method for erythrocytes, leukocytes, haematocrit value and percentages of heterophils and mononuclear cells. Blood from 71 healthy fish was analysed using the Sysmex XN-1000V for the determination of the haematological reference values for rainbow trout.
Results
Precision obtained with the blood cell analyser for low-normal and high cell counts were 2.71% and 0.83% for RBC, 1.43% and 3.94% for HGB, 1.07% and 1.78% for HCT, 4.82% and 4.66% for WBC, and 0.35 and 0.12 for PLT, respectively. For MCV, heterophils (%) and mononuclear (%) the precision was only calculated with blood with normal cell counts and it was 1.39 %, 15.08 % and 2.24 % respectively. Pearson’s correlation coefficient (r) for the linearity of RBC, HGB, HCT, WBC, heterophils cells (%), mononuclear cells (%) and PLTs were over 0.99 for all parameters. Carry-over for RBC and PLT were 0, but WBC was 0.57 %. Precision of the manual method was 21.7% for RBC and 17.57 % for WBC. Comparison between Sysmex XN1000V and manual method was good for most parameters with a Pearson’s correlation coefficient of 0.78 for RBC, 0.87 for HCT, 0.51 for WBC, 0.68 for mononuclear cells (%) and 0.76 for heterophils (%). Reference interval range for the haematological parameters obtained from healthy rainbow trout was established (Table 1).
Discussion
Present results show that the Sysmex XN-1000V produce more accurate and reproducible haematological data than the manual methods. Moreover, the use of the automated analyser overcome one of the main limitations of blood haematology in fish that it is the time consuming and great variability of the manual method. Reference values interval obtained using the Sysmex XN-1000V are similar to those described by the manual methods by other authors with trout blood (Zinkl et al., 1991, Jan et al., 2021) allowing its use in future haematological studies. In conclusion, with this study we validate a new tool that will contribute to the non-lethal and fast evaluation of the health status of wild and farmed fish.
References
Zinkl JG, Cox WT and Kono CS (1991). Morphology and cytochemistry of leukocytes and thrombocytes of six species of fish. Comp Hematol Intl 1:87-195.
Jan K, Ahmed I, Dar NA (2021). Haematological and serum biochemical reference values of snow trout, Schizothorax labiatus habiting in river Sindh of Indian Himalayan region. J Fish Biol. 98(5):1289-1302.