Review of Postmortem Interval Estimation Using Vitreous Humor: Past,Present,and Future

Introduction

In cases of unwitnessed death, forensic investigators and family members often want to know the time between death and discovery of a body. Family members may ask whether some intervening action could have prevented death, and if so, whether something or someone is to blame for this death. Forensic investigators could use an accurate postmortem interval to corroborate a witness's statements, prove or disprove alibis, ease tension between family members, and ideally as evidence in a court of law (1-10). Despite decades of research, no forensic scientist has found a metric to determine the postmortem interval with sufficient accuracy to be adopted universally. This article reviews past and present attempts to estimate the postmortem interval by analyzing samples collected during postmortem examination.

Background

In the immediate postmortem period, determination of the postmortem interval by examination of the body relies on subjective methods such as assessment of livor and rigor mortis and objective measurements of body cooling, rectal temperature, and changes in the vitreous chemistries (1, 7, 8, 11-15). Also described, mostly in European literature, analysis of the supravitality of tissues with skeletal muscle stimulation or pupillary response to chemical stimuli has been applied in the immediate postmortem period (usually less than 20 hours postmortem) (1, 16, 17). Most published work has focused on postmortem analysis of vitreous humor. Vitreous humor is a unique source of physical evidence in forensic pathology owing to its location, relative protection from bacterial contamination, ease of sampling, and general availability during postmortem examination (5, 6, 9, 15, 18-26). The vitreous sample is typically collected via a scleral puncture and aspiration of gelatinous contents of the posterior chamber of the eye, with care to avoid blood or retinal cell contamination, which can yield a variety of false results (3, 12, 23, 27-31). Common practice during postmortem examination is to collect vitreous humor samples at one time point from both eyes and combine them into one sample (3, 11, 32-36). Few authors have demonstrated variability between the eyes, assuming that the eyes appear to be in a similar state of preservation; however, recent work has shown that the variability is minimal during repetitive sampling from a single corpse over the course of many hours (5, 15, 30, 37, 38).

Discussion

The loss of active ATP-mediated ion transport, loss of barrier integrity between cells, membranes, and fluids, as well as general cellular degradation and autolysis during decomposition have allowed researchers to examine a plethora of ion kinetics in the postmortem period using vitreous humor with varying success (1, 6, 7, 9, 15, 36, 39-46). With the knowledge that the baseline potassium concentration in the vitreous humor is within a physiological range of 3.5 to 5.0 mmol/L and that potassium leaks into the vitreous humor from neighboring cells via diffusion, many researchers proposed that the concentration of potassium in the vitreous humor changed at a predictable rate following death (32, 33, 35, 47-56). These various researchers plotted data and obtained a linear relationship between the postmortem interval and the rise in vitreous potassium concentration in the postmortem period spanning a few hours to a few days. They then used linear regression to determine a formula that they proposed would allow an individual to determine the postmortem interval in a new case by measuring the concentration of potassium in the vitreous humor and entering this number as the independent variable into their linear regression formula. A more recent publication by Zilg et al. demonstrated a nonlinear correlation that approached asymptotic after five days (1). Few studies have evaluated the use of vitreous potassium in the late postmortem period, as correlation weakens beyond a few days postmortem, likely due to decomposition and volume changes (1,48). Some researchers are evaluating inorganic content of bone in their attempt to determine the postmortem interval when a body has entered the stage of putrefactive decay (57,58).

Estimating the postmortem interval from the concentration of potassium in vitreous humor sounds like an elegantly simple method of estimating the time of death, so why does it not work? Analysis of a sample of vitreous humor obtained at postmortem examination is subject to all the sources of error that affect clinical laboratory analyses – pre-analytical error, analytical error, and post-analytical error. Tight though the control of the potassium concentration in the body is, it still varies from 3.5-5.0 mmol/L, meaning that a normal concentration of 5.0 mmol/L exceeds a normal concentration of 3.5 mmol/L by 40%. On that basis alone, one could argue that an estimated postmortem interval of three hours could vary by up to roughly 75 minutes in either direction, providing an estimated time of death ranging between 1.75-4.25 hours prior to collection of the vitreous humor. This range may be longer than family members and investigators would find useful and is little better than making an educated guess based on reliable evidence that shows the last time that the decedent was known to be alive.

Another source of variability are factors that affect the degree and speed at which potassium leaks from the inner cell mass of the eye into the vitreous humor. Health conditions such as renal failure and diabetes mellitus can affect the overall homeostasis of electrolytes throughout the body, and thereby alter baseline values, leading to erroneous estimations of time of death (1,59). More recent publications have looked at the effect on potassium diffusion of ambient temperature and time within that environment, including the controlled temperature environments of the morgue, room temperature, and elevated environmental temperatures (1, 34, 46, 60-64). It appears that the relative increase in potassium is affected by higher temperatures and the duration that a body is at those temperatures. Obviously, a corpse left at room temperature or higher for a considerable amount of time will also progress through the stages of decomposition quickly and further complicate the ability to utilize vitreous humor for testing. Interestingly, some investigators report that a decedent's age affects the concentration of vitreous potassium, although this is not universally agreed upon (1, 3, 7, 65, 66). The effect of age is hypothesized to be due to globe size, vitreous composition, and cell mass or volume (1). It may be assumed that the agonal period and numerous neurotransmitters released during this period may cause shifts in cell ion concentrations; however, few have demonstrated that this affects concentration of potassium seen in the vitreous including samples collected from victims of hanging, burns, trauma, or poisoning (1, 49, 59).

Additional errors in accuracy and precision have been shown due to sources of pre-analytic variability including sample preparation and pretreatment as well as analytic variation regarding instrumentation available and technique utilized (15, 17-19, 29, 34, 36, 37, 67-73). It is currently recommended to centrifuge the sample in order to assist with liquefying the vitreous sample for testing and for each institution to develop standardized values since there are no normalized ranges for postmortem vitreous chemistries established at this time (29, 74, 75). More important still is that different researchers used different techniques to analyze the potassium concentration in vitreous humor. Because the concentration of potassium depends in part upon the technique used for analysis, a given regression formula determined by a given researcher is only truly valid if one uses the same analytical process as was used in the original research. Thus, entering a potassium concentration into a regression formula determined by a different technique creates post-analytical error. Moreover, any regression formula should be published with a confidence interval, typically a 95% confidence interval, and this confidence interval must also be allowed for when using a formula to estimate the postmortem interval, further decreasing the accuracy of the estimate.

Determining the postmortem interval from the concentration of potassium in vitreous humor was very attractive when it was proposed because the determination is easily made and the formula easily calculated, but the formula proved too simple to accommodate variability from decedent to decedent and from scene to scene, rendering controversial conclusions (6, 17, 33, 59, 75, 76). That does not mean that the work already done is not useful. Science advances in this sort of way – simple theories are proposed, rejected because their simplicity is inadequate to accurately model circumstances, and eventually replaced by more complicated models that do accurately model the behavior of the system being studied. A simple test that would accurately estimate the postmortem interval would be useful, but it is simplistic to think that one test will answer a medical question. A diagnosis of myocardial infarction based solely on an elevated troponin concentration is less convincing than a diagnosis based on a history of chest pain with tingling in the left arm, ST segment elevation on an electrocardiogram, and the appropriate elevation of troponin. In the same way, vitreous humor analysis alone cannot be used for estimating the postmortem interval with accuracy. Forensic pathologists can expect that the most accurate estimation of the postmortem interval will incorporate information from the combination of data retrieved during investigation, postmortem examination, and ancillary testing (77).

Conclusion

In order for postmortem laboratory analysis to provide a useful estimation of the postmortem interval, the analysis must be valid, reproducible, accurate, accessible, and quick. To achieve this, researchers must continue to provide relevant data details, utilize varied populations with adequate power in each group, utilize validated testing parameters, provide confidence intervals, and rigorously test the mathematical model in a blinded and controlled fashion (1, 15, 29, 75). Researchers can begin this testing themselves by using whatever formula they develop for estimating the postmortem interval in a blinded fashion on a new group of cases for which one individual knows the postmortem interval by means of alternate information (e.g., a witnessed death). If a person blinded to the correct answer can use the formula or algorithm to provide an accurate estimate of the time between examination and death, then the researchers can be confident that they are reporting a useful model. If the proposed method for estimating the postmortem interval does not predict the time of death accurately for a control group where the time of death is known, then the researchers will know that they must continue their work and temper their enthusiasm for the usefulness of their current model when they describe it in presentations and publications.