The aim of this study was to develop flow cytometric (FC) methods to enumerate rainbow trout (Oncorhynchus mykiss) whole blood cells and New Zealand freshwater crayfish (Paranephrops planifrons) haemocytes as non-lethal endpoints in the evaluation of physiological status.
In the FC method development for rainbow trout, heparin was found to be superior to neutralised EDTA as a blood anticoagulant, because the use of EDTA resulted in significant lysis and shrinkage of erythrocytes. Leishman's-Giemsa and May Grunwald-Giemsa yielded comparable differential staining of leukocytes, and were superior to Wright-Giemsa staining. Morphological ambiguity between thrombocytes and lymphocytes in smears could not be resolved using Romanowsky or cytochemical staining. Use of FC was demonstrated to be a rapid, more accurate alternative to manual total cell counting procedures. Phosphate-buffered saline (PBS) was found to be superior to IsoTon II as a FC sheath fluid; IsoTon II induced lysis of erythrocytes. Characterisation of fish blood cell types and differentiation of leukocytes using FC could be achieved using 50 nM concentrations of the fluorescent lipophilic dye dihexyloxacarbocyanine iodide (DiOC6(3)), but inconsistent fluorescent behaviour exhibited by thrombocytes between specimens prevented clear resolution of these cells from erythrocytes and lymphocytes. Higher concentrations of DiOC6(3) did not enhance resolution and became cytotoxic, particularly to leukocytes. Resolution between thrombocytes and lymphocytes could only be achieved with a fluorescent-labelled thrombocyte monoclonal antibody (mAb). The results suggest that the application of FC and mAb to fish blood cells is the most accurate approach to differential counting of leukocytes.
The second FC method objectively characterised and enumerated New Zealand freshwater crayfish haemocytes. Haemocyte populations were isolated by FC sorting based on differential light scatter properties, followed by morphological characterisation by light microscopy and software image analysis. Cells were identified as hyaline, semi-granular and granular haemocytes based on established invertebrate haemocyte classification. A characteristic decrease in nuclear size and increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential haemocyte counts from three lake crayfish populations and between wild and captive specimens. Differences in total and differential haemocyte counts were not observed between wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total haemocyte count, while the relative haemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of sub-acute stressor effects in selected decapod crustacea
