Flow cytometric validation of a commercial kit to assess the concentration and viability of bivalve hemocytes

[SHORT COMMUNICATION] Introduction: “Bivalve molluscs have an innate immune system which relies heavily on the defensive capabilities of hemocytes [2]. Accurate assessment of the quantity and condition of the hemocytes within the hemolymph provides important information related to the health of the individual [4]. The green-lipped mussel (Perna canaliculus, Gmelin 1791) is a marine bivalve species of commercial, cultural and ecological significance in New Zealand. Information relating to the concentration and viability of the hemocytes allows us to characterise how the immune system of this important aquaculture species responds to stressors such as ocean warming, exposure to pathogens, anthropogenic contaminants, and harmful algal blooms. Flow cytometry has been used for decades to assess the concentration and viability of bivalve hemolymph rapidly and accurately [1,5,9]. Recent developments in flow cytometers have led to the production of smaller machines, designed for ease of use and portability, with the potential to substantially increase the accessibility of flow cytometry technology. The versatility of these newer devices may be constrained by a need to utilise pre-assembled assay kits, raising concerns as flow cytometry assays have traditionally been developed for vertebrate immunological studies. The osmolality of the premixed buffers used in kits developed for vertebrate studies are likely unsuitable for marine invertebrate studies, which require an osmolality close to seawater (∼1000 mOsm). Exposing bivalve hemolymph to such hypo-osmotic conditions will likely impact the cellular morphology and possibly the viability of the hemocytes, causing problems for valid cell count and viability measurements. The hemocytes of P. canaliculus and many other marine bivalves also aggregate readily. As such, it is important to follow gentle sampling, handling and storage procedures when sampling the hemolymph [8], to obtain accurate measurements of cell concentration. It is therefore important to validate assay kits against established methods to ensure applicability to invertebrate hemolymph samples [3,6]. […]”

 

Keywords: Flow cytometry; Hemocytes; Bivalve; Perna canaliculus; Green-lipped mussel viability; Concentration