[Evaluation of morphologically determined apoptotic index]

Stimulated by the huge scientific interest in programmed cell death, the sophisticated but expensive methods of apoptosis detection, occasionally even in various stages of the apoptotic process, have been developed in the last two decades. These methods are quite demanding and time consuming, e.g., TUNEL, flow cytometry and its modifications, immunohistochemistry, hybridization in situ, etc. Irrespective of considerable technical improvements, these methods are associated with a relatively high rate of false-positive and false-negative results. Reading off the results obtained by these methods is mostly left to investigators inexperienced in morphological analysis. It is well known that the basic morphological characteristics of apoptotic cells and apoptotic bodies have been described on the basis of classic light microscopy of histologic hemalaun-eosin (HE) stained slides. As a rule, apoptotic cell has a shrunken, condensed, dark red cytoplasm and dark purple picnotic nucleus, and is surrounded by light, 'hollow' space (halo). Apoptotic bodies are fragments of the disintegrated apoptotic cell and each fragment of dense cytoplasm usually contains a fragment of disintegrated nucleus. It is known that, unlike necrosis, there is no inflammatory reaction in the vicinity of apoptotic cells and apoptotic bodies. During more than 25 years of research into apoptosis, the Zagreb Group for the Study of Apoptosis (Apoptosis Section, Department of Basic Medical Sciences, Academy of Medical Sciences of Croatia) have found it possible to determine the number of apoptotic cells and apoptotic bodies (apoptotic index) in daily routine on classic HE stained histologic slides by counting in 10 large fields under light microscope, following the methodology of mitotic index determination. Of course, the method is not absolutely accurate (the sophisticated and expensive methods mentioned above are not so either), but it is rapid and inexpensive. Thus, we believe that each pathologist should be professionally obliged to determine apoptotic index when assessing mitotic index. Expression of these indices as a mitotic-apoptotic ratio, e.g., 1:1, 1:2, 2:1, etc., may better reflect the growth potential of the examined, usually tumor tissue than determination of the mitotic index alone.