TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three pmtmls on CEM-C7 cells, a model of glucocorticoid-indud apoptosis. Samples were submitted to six modalities of fuation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter

SUMMARY TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, stepby-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

The study of the human virome is still in its infancy, especially with regard to the viral content of the blood of people who are apparently disease free. In this study, the genome of a new giant virus that is related to the amoeba-infecting pathogen Marseillevirus was recovered from donated blood, using high-throughput sequencing. Viral antigens were identified by an immunoconversion assay. The virus was visualized with transmission electron microscopy and fluorescence in situ hybridization and was grown in human T lymphocytes. Specific antibody reactions were used to identify viral proteins in blood specimens from polymerase chain reactive-positive donors. Finally, we tested 20 blood specimens from additional donors. Three had antibodies directed against this virus, and 2 had circulating viral DNA. This study shows that giant viruses, which are missed by the use of ultrafilters, are part of the human blood virome. The putative pathogenic role of giant viruses in humans remains undefined.

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