Here is the method for assessment of RBC osmotic fragility used in my lab and published in ASAIO Journal. 46(6):679-692, November-December 2000.

The washed RBCs were resuspended in PPP to provide a hematocrit of 40%. Our blood substitute, unmodified hemo- globin solution, and allogenic PPP (control) were added to the RBC suspensions in a concentration corresponding to 40% of the total blood volume without changing the osmolarity or pH. The RBC suspensions were then oxygenated and incubated in sterile Vacutainers (Becton Dickinson Vacutainer System) placed on an aliquot mixer (Model 4651; Ames Co., Div. Miles Labs., Inc., Elkhart, IN) at 37°C for 18 hours, after which the reaction was terminated. After centrifugation at 1,500g for 10 minutes, the supernates were collected and stored frozen at 􏰂90°C until use. The RBCs were washed four times with PBS, and their osmotic fragility was evaluated by the method of Parpart et al.44 In brief, 100 􏰊l of the washed packed RBCs were suspended in 2 ml of PBS, and 50 􏰊l of the RBC suspen- sion was incubated at 37°C for 30 minutes in 3 ml of a solution containing: 0.80, 0.70, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.10, or 0.00% NaCl-PO4, pH 7.4. After centrifugation at 1,500g for 5 minutes, the absorbance of the supernate vs. water was determined with a Coleman 575 spectrophotometer (Perkin-Elmer, Oak Brook, IL) at 540 nm, and the percentage of RBCs lysed in each solution was calcu- lated. The concentration at which 50% of the RBCs were lysed was considered the median osmotic fragility (MOF). The re- maining washed RBCs were subjected to assessment of the mechanical fragility or stored at 􏰂90°C until use.