Internucleosomal cleavage of DNA is insufficient evidence to conclude that cell death is apoptotic.

In a recent volume of Blood, Yuan et a l l demonstrated internucleosomal cleavage of DNA in erythroid precursors obtained from the bone marrow (BM) of a patient with @-thalassemia major, “lesser” (but unquantitated) amounts of intemucleosomal cleavage in those from a @-thalassemia trait donor, and no evidence of such cleavage in erythroid precursors separated from normal BM. Although no other evidence was presented, the investigators concluded that 8thalassemic progenitors exhibit apoptosis, or “programmed cell death.” Apoptosis is a form of cell death distinct from necrosis. Morphologic criteria for the defbition of cell death as apoptotic include rapid chromatin condensation with aggregation of chromatin along the nuclear envelope and plasma membrane blebbing followed by nuclear condensation and separation into small, apoptotic bodies. When it accompanies these features, intemucleosomal fragmentation of chromatin DNA is useful as a criterion to help define cell death as apopto t i~ .~~ We recently identified artifactual internucleosomal cleavage of DNA after its extraction from normal rat kidneys. Extrcction of DNA from tissue processed in buffer containing low concentrations of EDTA resulted in extensive internucleosomal fragmentation of DNA (Fig 1) despite the fact that these tissues showed no evidence of apoptosis morphologically. This artifact was not prevented simply by the addition of a protease inhibitor (phenylmethyl-sulfonylfluoride [PMSFI), of 25 mmolR. EGTA to inhibit endogenous calciumdependent nuclease activity, or of spermidine to stabilize chromatin structure. However, the artifact was prevented by an increased NaCl concentration combined with 25 mmoVL EDTA. It is of note that the buffer used by Yuan et al for isolation of fragmented DNA (10