Measurement of red cell sickling: a method for studying the efficacy of antisickling drugs under physiological conditions.

A method was developed to study sickling in vitro under physiological conditions using a small amount of blood (0.1 mL). The diluted blood suspension (2.1 mL) was placed in a flask and flushed with a gas mixture containing 5% CO2. In deoxygenation experiments, samples were withdrawn anaerobically into a microslide (optical path 0.1 mm) and red cell morphology was studied directly under a light microscope after both ends of the microslide were sealed. The blood suspension with a hematocrit value of 1% can be deoxygenated in less than 10 min, but it takes 30 min for the sickling of cells to reach a plateau. The degree of sickling increases with increasing osmolality of the medium, or with a decrease of the pH. With a citrate-phosphate-dextrose-adenine solution, sickle blood may be stored for this type of study for about 10 days at 4 degrees C. The blood may be stored for about 5 days with a noncitrate preservative. This method was found useful in examining the antisickling activity of various drugs.