MICROPHOTOHEMOLYSIS TO MEASURE ERYTHROCYTE FRAGILITY

ERYTHROCYTE MEMBRANE FRAGILITY MAY BE ALTERED BY DISEASE, AGE, AND DRUGS AND PLAYS AN IMPORTANT ROLE IN THE SURVIVAL AND FUNCTION OF THE ERYTHROCYTE. THE ONLY COMMONLY USED TECHNIQUE FOR ASSESSING THIS PARAMETER, THE OSMOTIC FRAGILITY TEST, IS VERY TIME CONSUMING, GIVES AN END-POINT ANALYSIS AND REQUIRES CONSIDERABLE EXPERTISE TO AVOID MANY SOURCES OF ERROR (PH, DILUTIONS, OSMOLALITY, CONTROL CELLS). USING ANIMAL ERYTHROCYTES WE HAVE DEVELOPED A TECHNIQUE, MICROPHOTOHEMOLYSIS, WHICH IS RAPID AND EASY TO USE AND GIVES BOTH THE RATE AND MAGNITUDE OF HEMOLYSIS WITH VERY SMALL VARIANCES. LIGHT THROUGH A MICROSCOPE IS USED FOR ACTIVATION OF A FLUOROCHROME-ERYTHROCYTE MIXTURE AND THE RESULTING HEMOLYSIS IN THE FIELD OF VIEW IS QUANTITATED BY A DETERMINATION OF THE INTENSITY OF TRANSMITTED LIGHT PASSING THROUGH THE SAMPLE. IN PHASE I, OUR AIMS ARE TO DETERMINE IF THIS TECHNIQUE CAN BE USED TO DISTINGUISH 1) NORMAL HUMAN ERYTHROCYTES FROM THOSE OF PATIENTS WITH SICKLE CELL ANEMIA OR HEREDITARY SPHEROCYTOSIS, 2) NORMAL CELLS AND FROM THOSE THAT HAVE BEEN TREATED WITH DRUGS WHICH ALTER MEMBRANE FRAGILITY AND, 3) SUBPOPULATIONS OF SICKLE CELLS OR OF DRUG TREATED NORMAL CELLS WITHIN THE SAME SAMPLE. OUR LONG TERM GOAL IS THE DEVELOPMENT OF AUTOMATED EQUIPMENT WHICH CAN BE USED TO SCREEN FOR DISEASE OR DRUG TREATMENTS AFFECTING ERYTHROCYTES.