Di Nunno, Nunzio R. M.D.; Costantinides, Fulvio M.D.; Bernasconi, Paola M.D.; Bottin, Cristina; Melato, Mauro M.D.
From the Institute of Forensic Medicine, (N.R.D., F.C., P.B.) and the Department of Biomedical Science (C.B., M.M.), University of Trieste, Trieste, Italy.
Received October 17, 1996; accepted January 31, 1997.
Address correspondence and reprint requests to Dr. Nunzio R. Di Nunno, Istituto di Medicina Legale dell'Univerzsità di Trieste, Via del Molino a Vento no. 123, 34137 Trieste, Italy.
Determination of the time of death has always been one of the primary goals of forensic medicine. Various methods have been used to define the length of the postmortem interval with reasonable approximation. Corpse temperature is probably the first method ever used (1); this method is based on the assumption that body temperature after death will tend to be equal to the temperature of the environment in which it is found (2); however, many factors may influence this process (3). For example, the temperature of the body at the moment of death could be higher or lower than the normal level of 37°C, or the temperature of the environment could be higher than 37°C. Thermic conductivity of the material on which the corpse is lying, the presence of garments on the body, and cause of death may also affect this process.
Observation of cadaveric phenomenology can be helpful in the attempt to determine the time of death, but hypostasis, rigor mortis, and the state of decomposition are all easily influenced by external factors.
Gauging of the K+ content of vitreous humor (4,5) is strongly dependent on the conditions of the eyeball, which can be seriously damaged by external factors such as the mechanism of death, environmental temperature, length of agony, or the presence of ethanol at the time of death (6). Vitreous fluid K+ content changes rapidly in children after death but more slowly in adults (7); thus, results using this method are reliable only in the first 12 hours after death (8).
It is evident that definition of the length of the postmortem interval requires a parameter that evolves constantly from the time of death with a linear process that is not affected by external factors.
The DNA molecule seems to be a valuable parameter to study to determine the time of death, because DNA denaturation by autolysis begins immediately postmortem and continues for 3 days at a constant rate that seems to be unaffected by environmental temperature and the mechanism of death. A new method based on the study of spleen DNA denaturation has been proposed by Cina (9). Our study is an attempt to confirm the reliability of this method in autopsy practice.
MATERIALS AND METHODS
Portions of human spleens collected during autopsy were preserved in nonsterile phosphate-buffered saline (PBS) at −20°C until cytofluorimetric reading was performed. The time of death was known for all corpses. We chose human spleen according to the method of Cina (9) and in consideration of the fact that the spleen is rich in nucleated cells and easily homogenized.
Our reference sample was made up of serial samples of two human spleens removed during organ exploration at time 0 and stored in PBS at room temperature.
Italian legislation requires the corpse be observed for 24 hours after death to confirm biologic death. During this period, corpses are placed in special storage at a temperature of 15°C. After 24 hours, corpses are moved to cold storage at 2°C, and after postmortem examination the corpses are available to relatives for burial.
During our experimental study we had some difficulty in taking spleen tissue using biopsy needles because of the organ's small size, so we were forced to collect the organs during autopsy.
Because the autopsies quite frequently were performed a few days after death, we were forced to observe the process of decomposition of the spleen inside cold storage, taking samples every 12 hours until 144 hours after death. These conditions allow partial putrefaction.
We compared the data reported by Cina (9) with those obtained from 35 autopsies of 16 women (mean age, 72.7 years; range, 34-91 years) and 19 men (mean age, 57.7 years; range, 20-93 years). Causes of death included neoplasia (7 women, 4 men), cardiovascular (6 women, 5 males), pneumonia related to lung disorders (6 women, 5 men), and liver failure and cirrhosis (2 men); 9 of the cases were gathered by the Institute of Forensic Medicine and 19 by the Institute of Pathology of the University of Trieste. Distribution of the specimens according to time of collection ranged from 24 to 126 hours postmortem.
Flow cytometry was performed on 0.5-cm3 spleen specimens maintained in PBS and stored at −20°C. The tissue was placed in a Petri dish, minced with a syringe, diluted in PBS, and filtered through 50-μm mesh nylon cloth. Cell suspension was collected in a test tube, centrifuged for 5 minutes at 400 × G at 4°C, and the cell pellet was resuspended in 500 μL of PBS. The cells were stained with propidium iodide (20 μL in 1 mL of staining solution) and analyzed after 2 hours. The suspension was run on the Coulter Epics Elite ESP Flow Cytometer using Elite version 4.1 software. A fresh peripheral blood sample was used as a control. Cells were gated on peak red fluorescence versus integrated fluorescence to exclude cellular aggregates. A histogram was generated, plotting the number of cells on the Y axis versus the red fluorescence intensity on the X axis.
Histograms obtained from the first part of our study showed a progressive denaturation of the DNA molecule in the range of 24 to 60 hours after death. From this time, denaturation was accelerated and, after 60 hours, histograms tended to overlap showing massive denaturation of DNA from 72 hours to 144 hours. However, comparing the two histograms from 72 and 144 hours, there is a considerable difference.
In regard to autopsy samples, we observed a general agreement with the previously reported histograms (Figs. 1 through 3), although not all histograms overlapped. The method was particularly reliable in the first range, 24 to 36 hours, in which results from autoptic samples were comparable to reference results; in following time periods, variability among histograms increased with time. Our study did not detect any relations among weight of the spleen, size of the subject, and DNA denaturation.
The method proposed by Cina (9) is interesting partly because it allows the possibility of repeating the observation. After conducting the initial assay, the cytofluorimetric reading samples can be kept in cold storage at −80°C and later retested by other pathologists to ensure quality and accuracy of the result. This is the only method reported thus far that makes it possible to reuse the same samples for many determinations.
Results showed a slight variability in the range of the constant relation between the time of death and the process of denaturation of DNA molecules. This method is useful to determine a postmortem period not exceeding 72 hours. This appears to be the limit of reliability, because beyond 72 hours, DNA denaturation is massive and cytofluorimetric readings are not significant.
The small differences among peaks obtained from the spleens taken during autopsy and those obtained from the two explanted spleens making up the reference samples probably result from the fact that all samples studied at autopsy came from different subjects and were therefore biologically different. Other differences in some samples are probably the result of residual viability of the organ or infective pathologic conditions. Other differences can be assigned to variations in the organ and in the agonic period. Variability is also linked to the variance of the putrefactive process related to intestinal content. The large bowel is situated near the spleen, and the fullness of the bowel may influence the rapidity of corruption of the spleen.
It should be noted that reference samples were obtained from the same subject, whereas experimental samples came from different subjects; therefore, some differences were expected because the various organs had begun the process of putrefaction at different times. Moreover, spleen tends to putrefy quickly because of its structure.
Use of this method, which was shown to be a reliable means to determine time of death, could be justified for on-the-spot investigation provided that a more suitable organ could be found that does not tend to corrupt as quickly as the spleen.
Flow cytometry findings in autopsy subjects correlate well with in vitro studies, suggesting that this method may be practically applied to autopsy cases; furthermore, our data point out a constant relation between the time of death and DNA denaturation, particularly within the first 72 hours after death. The choice of the spleen for tissue sampling led to some practical problems, presumably because it tends to colliquate easily and rapidly. For this reason, we are testing other organs which may prove more suitable for research on DNA degradation and determination of the time of death.
Acknowledgments: We thank Dr. Ilaria Capozzi for her technical support and Dr. Mario Linassi for his support in translation of this paper. The research was performed at the Centro Grandi Strumenti, polo di Citofluorimetria (proff. Melato & Sava)" of the University of Trieste, Trieste, Italy.
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