Analysis and cytologic characterization of hemocytes from freshwater mussels (Quadrula sp.)

Background: Freshwater mussels are among the most endangered taxa in North America and minimally invasive techniques to evaluate their health are needed. Objective: The objective of this study was to develop a standardized approach for identifying and enumerating the cellular components of freshwater mussel hemolymph. Methods: Hemocyte clumping, total hemocyte count, and hemocyte morphology were compared in untreated hemolymph or hemolymph treated with formalin, sodium citrate, sodium heparin, EDTA, water, or l ‐cysteine. Morphology was then used to categorize hemocytes and perform a 100‐cell differential. Results: Treatment with formalin or >25 mg/mL l ‐cysteine reduced hemocyte clumping, although only formalin significantly increased the total hemocyte count. However, formalin also induced crenation that impaired hemocyte identification. Both EDTA and sodium citrate‐induced hemocyte degranulation while sodium citrate and >40 mg/mL l ‐cysteine‐induced cell lysis. Hemocytes could be categorized into 2 groups of granulocytes (eosinophilic or basophilic) and 2 groups of agranulocytes (large or small) for performing a cytologic differential. The differential was not significantly altered by anticoagulant treatments providing cell morphology was adequate for obtaining a differential. Eosinophilic granulocytes predominated (59%) with fewer large agranulocytes (27%) and basophilic granulocytes (13%). Small agranulocytes comprised 2% of the total population. Conclusions: No single treatment provided an optimal method to evaluate freshwater mussel hemolymph. Maximal hemocyte counts were obtained following formalin treatment. l ‐cysteine reduced clumping and maintained hemocyte morphology for performing a cytologic differential. These techniques provide a standardized approach for the hematologic evaluation of freshwater mussels.