Studies on Spontaneous In Vitro Autohemolysis in Hemolytic Disorders

Abstract

Abstract Measurements of spontaneous lysis (autohemolysis) of red cells in sterile defibrinated blood after 48 or more hours of incubation at body temperature were found useful in the investigation of certain hemolytic states. Abnormally rapid autohemolysis was demonstrated most consistently in hereditary spherocytosis, but was also found in other types of spherocytosis and in several examples of non-spherocytic hemolytic anemia. Autohemolysis above the normal range can be regarded as strong presumptive evidence of a hemolytic disorder, but a normal rate of autohemolysis by no means excludes the possibility of a hemolytic process in any given case. Abnormalities of hereditary spherocytes causing increased autohemolysis were shown to be correlated with those responsible for spherocytosis as reflected in osmotic fragility tests. There appeared to be closer correlation with abnormalities causing increased osmotic fragility of the cells after 24 hours incubation. The autohemolysis test and the osmotic fragility test on incubated red cells were found to be equally sensitive in their capacity to detect spherocytosis of the hereditary type. Addition of adenosine, guanosine or inosine caused moderate to marked reduction in autohemolysis of nearly all types of red cells tested. Lysis of incubated spherocytes from active cases of autoimmune hemolytic disease was more markedly inhibited by a small amount of adenosine than was the lysis of hereditary spherocytes. Glucose regularly caused marked inhibition of autohemolysis of hereditary spherocytes and of red cells from patients with acute leukemia. The effect of glucose on HS red cells might have been due in part to a lowering of pH by formation of lactic acid since acidification of the blood with lactic, citric or hydrochloric acid also caused substantial reduction in autohemolysis. Glucose caused slight to marked increase in lysis of red cells in certain other cases of spherocytosis, notably in autoimmune hemolytic disease and in myeloid metaplasia. Addition of lactic acid to the blood from several of these patients had similar effect. In 2 atypical cases of chronic spherocytosis, with hematologically normal relatives, autohemolysis was accelerated by addition of glucose to the blood samples. Adenosine had little effect until after splenectomy when both glucose and adenosine inhibited hemolysis, much the same as in blood from typical cases of hereditary spherocytosis. It seems likely that when the abnormalities of carbohydrate metabolism of the red cells in certain hemolytic disorders are better understood, measurements of autohemolysis may be modified so as to enhance their usefulness in detecting and differentiating the various hemolytic states. It also seems likely that further studies on in vitro autohemolysis will help to elucidate some hemolytic mechanisms operating in vivo.