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(Circulation. 2000;101:e239.)
© 2000 American Heart Association, Inc.

Circulation Electronic Pages

Significance of Myocytes With Positive DNA In Situ Nick End-Labeling (TUNEL) in Hearts With Dilated Cardiomyopathy

Antti Saraste Kari Pulkki; Liisa-Maria Voipio-Pulkki

Departments of Anatomy, Clinical Chemistry, and Medicine, University of Turku, Turku, Finland

	Introduction

 
To the Editor:

Recently, Kanoh et al¹ seriously questioned the reliability of the DNA in situ nick end-labeling (TUNEL) assay as a method of detecting cardiomyocyte apoptosis (CA) in dilated cardiomyopathy. Our experience with both failing² and infarcted³ human hearts is remarkably different. We agree that TUNEL positivity is not 100% specific to what is morphologically defined as apoptosis. However, if one accepts double-strand DNA breaks as one of the hallmarks of apoptotic cell death (which Kanoh et al must even question), our experience is that the detection of this preferred substrate by the TUNEL assay results in reasonable (0.1% to 1.0%) and reproducible (r=0.88) estimates of CA frequency in relevant samples. DNA ladders are demonstrable in the TUNEL-positive areas when the amount of positive cells exceeds 0.04%.

We think that the key reason for the findings by Kanoh et al is their lack of appropriate standardization of the TUNEL assay. As we have pointed out previously,^{2 3 4 5} one must go beyond the manufacturer’s instructions to avoid erroneously false-positive and false-negative results. This can be done by using adjacent tissue sections treated with DNase I as a positive control of apoptosis and by interrupting the staining reaction on the appearance of positive signal in these sections. This procedure confirms the optimal sensitivity of the assay and normalizes it for differences in tissue permeability.^{2 3 4} Using this approach, TUNEL positivity is never zero; rather, it is in the range of 0.003% to 0.01% in normal myocardium. Furthermore, the very high numbers . . . [Full Text of this Article]