Abstract:
A technique that uses hypotonic lysis of erythrocytes was optimized for the purification
of leukocytes from the peripheral blood and anterior kidney (pronephros) of rainbow trout
Oncorhynchus mykiss. Comparisons of initial blood dilution (1:2, 1:4, and 1:6) and the time of
exposure to hypotonic conditions (10, 20, and 40 s) revealed that a dilution of 1:2 provided the
most complete hemolysis after 20 or 40 s in a hypotonic solution. For pronephros, a 1:5 (w:v)
dilution and lysis in hypotonic solution for 10–40 s was effective in eliminating erythrocytes.
Total leukocyte yield from the blood and pronephros by use of the hypotonic lysis method was
comparable with that obtained by use of typical density gradient centrifugation, and cell viability
was 97% or greater. Differential cell counts showed that hypotonic lysis resulted in a distribution
of leukocyte cell types similar to that of density gradient separation. Hypotonic lysis of erythrocytes
is a simple, rapid, and inexpensive method of purifying leukocytes from salmonid fish blood and
pronephros.
