Establishment the time of death by determining the changes in neutrophil elastase activity blood plasma in persons who died from ischemic heart disease and mechanical injuries



In order to establish the time of death, a method has been developed to determine the changes in elastase activity of blood plasma in people who died as a result of ischemic heart disease and mechanical injuries. Asignificant decrease has been established in elastase activity in persons who died due to ischemic heart disease and mechanical injuries within one day, from the moment of occurrence of death. The data obtained to determine the elastase activity of neutrophils in the blood plasma can be used as a diagnostic criterion for determining the time of death within one day of death.

Keywords: elastase activity, time of death (prescription of death), ischemic heart disease, mechanical injuries.

Introduction . When investigating crimes against human life and health, it is often necessary to establish the time of death. This information is important for law enforcement agencies and is often crucial for identifying the individual perpetrator.

Therefore, one of the main directions in the development of this problem is the research of postmortem phenomena that occur in organs, tissues and biological fluids.

New research methods are being developed in the area of forensic medicine, including for diagnosing the time of death- (TOD)/(prescription of death), these factors contribute and improve the quality of the examinations conducted. So thus, the problem of establishing the time of death — (TOD)/(prescription of death) has not lost it’s relevance in the present time.

Оne of the most important factors of immunity is neutrophil elastase (NE), the main human serine protease. This enzyme is able to degrade a wide range of substrates extracellular matrix, including elastin, collagen, fibronexin and proteoglycans. The action of NE is controlled by inhibitors serine proteinase, including SKALP/elafin and SLPI, which are present in extracellular matrix fluids. High levels of NE have been detected in various forms of pathology such as bronchiectasis, chronic obstructive pulmonary disease, type 2 diabetes mellitus, acute respiratory distress syndrome, atherosclerosis, arterial hypertension.

The method we have chosen for this study to define NE is new and is, insufficiently studied in forensic science, including for establishment for diagnosing the time of death.

Purpose of the study. Study changes in the elastase activity of blood serum during the postmortem period in people who died from ischemic heart disease (IHD) and mechanical injuries.

Material and methods. The material for the study was whole blood that was collected from 50 cadavers belonging to both genders (32 Male and 18 Female), the specimen was collected from right half of the heart and large vessels of the lower extremity, in a volume of 10ml. The blood was taken with syringes at intervals, starting from the time of death, then 4; 12; 18; 24 hours. In order for the data to be statistically processed, groups were formed at intervals of 2–6; 7–11; 12–16 and 17–21 hours.

The serum was selected, frozen, and stored at — 25 ⁰ С.

From the time of death to the collection of material, the bodies were in the morgue at ambient temperatures of 15 to 20°C (autumn-winter) and 20 to 25°C (spring-summer) and relative humidity of 40–60 %.

The time period during which death may have occurred is procured from sources of the preliminary investigation or inquiry, as well as tentatively determined by the degree of manifestation of the cadaver, by methods generally accepted in expert practice (state of cadaver stains, severity of rigor mortis, rectal thermometry). In some cases, the death was confirmed by an emergency medical team.

The cause of death was determined on the basis of a section study and additional results (forensic, histological and forensic-chemical examinations) as well as, in some cases, based on the medical records of the inpatient. In all deceased persons, the associated pathology was analyzed for elimination: autoimmune, infectious and oncological diseases. According to the literature, the conditions listed may be accompanied by a marked change in immunological parameters.

To determine the elastase activity of the cadaveric blood, the blood was centrifuged before use for 7 minutes (10 thousand rpm/min; MICRO 120 centrifuge). To set up the method, we used elastin-Congo red (particle diameter 37–75 micron, Sigma production) at a concentration of 0.8 mg per 1 ml of buffer, as a substrate for serum enzyme and buffer solution (0.2 M saline tris buffer) with pH 7.4. Elastase would split the elastin, and the Congo would turn red into a solution, changing its color from colorless to red with a maximum absorption spectrum of 495 nm. For convenience of setting instead of test tubes, eppendorf were used.

The eppendorf were sequentially injected with 400 μs of a solution of elastin-Congo red on tris-HCL buffer pH 7.4 and 100 μl of serum. The control was performed by samples containing a buffer solution in the amount of 400 μl and 100 μl of blood serum. Then the Incubation of samples in the thermostat at t=37°C within 20 hours. The samples were then extracted from the thermostat and centrifuged for 7 minutes (10 thousand rpm/min; MICRO 120 centrifuge) to precipitate the remaining elastin-Congo red in the form of undamaged particles. From the backpack was taken in doubles of 150 μl of solution and transferred to the wells of 96 wells polystyrene tablet. The tablet was placed in a multichannel spectrophotometer F 300, where at a wavelength of 492 nm (maximum close to 495) the optical density in the holes was determined. The result was calculated as the difference between the optical density of the test samples and the corresponding control samples.

To recalculate the final result into picocatals used the formula:

Where X is the desired result;

Еоп — (optical density unit) — the difference between the optical density of the sample and the optical control density.

The formula derived after constructing a calibration graph for diluted Congo red, which reflected the dependence of the enzyme activity on the optical density of the solution, assuming that when one substrate molecule breaks down, 1 Congo red molecule goes into the solution.

The comparison group included relevant indicators of elastase blood plasma activity in 10 practically healthy persons (donors) of the Vitebsk regional blood transfusion station.

The analysis of the obtained data was carried out with the help of non-parametric methods of statistical research using application packages of statistical programs: «Statgraphics» (Statistical graphics Corp.) and «Pimer of Biostatistics». Methods of descriptive statistics and correlation analysis were used. Pre-determined the distribution of each topic. Since the distribution of the values studied differed from the normal one, for descriptive statistical analysis the feature was characterized through its median, interquartile distance, and magnitude. In this case, the validity of the differences between the features was assessed according to the Mann-Whitney criterion [6]. The differences were statistically valid at p<0.05.

Research results : when assessing elastase activity blood serum of donors, it was found that its average level is 0.03 (0.23; 0.037) pcat.

The results of the stud of elastase activity of blood serum in post-moretm period in people died as a result of coronary artery disease and mechanical injuries are presented in Table 1.

Table 1

Dependence of elastic activity in the plasma of the blood on at the time of death, of the deceased from IDH and mechanical injuries

Cause of death	Time elapsed since the onset death (hours)
	2–6	7–11	12–16	17–21
IDH (n=26)	0,035(0,029–0,042)	0,02(0,016–0,025) ***	0,013(0,010–0,017) **	-
Mechanical injury (n=24)	0,03(0,033–0,027)	0,019(0,016–0,022) ***	0,011 (0,008–0,014) **	-
Control group (n=10)	0,03(0,023–0,037) pcat

Note: * — р<0,05, ** — р<0,01, *** — р<0,001.

When estimating elastic activity, it was found that after 2–6 hours in the first group of deaths from IDH (n=26) elastic activity amounted to 0.035 (0.029;0.042) pcat, which turned out to be somewhat higher than in the control group of donors — 0.03 (0.023;0.037) pcat. Further, the figures declined reliably after 7–11 hours and amounted to 0.02 (0.016;0.025) pcat (p<0.001), and after 12–16 hours there was a sharp and reliable decline to 0.013 (0.010;0.017) pcat (p<0.01).

Analyzing the obtained data, it can be noted that in plasma blood of those who died from coronary artery disease in the period from 2–6 hours after death there was slight increase in elastase activity in plasma of the deceased people. Then starting from 7 hours after onset of death, this indicator significantly decreased and by 7–21 hours hours reached almost zero.

When estimating elastic activity, it was found that after 2–6 hours in the second group of deceased from mechanical injuries (n=24) elastic activity amounted to 0.03 (0.033;0.027) pcat, which is almost the same as the control group of donors — 0.03 (0.023;0.037) pcat. Further, the figures decreased reliably after 7–11 hours to 0.019 (0.016;0.022) pcat (p<0.001), and after 12–16 hours there was a reliable decline to 0.011 (0.008;0.014) pcat (p<0.01). After 17–21 hours, elastic activity reached almost zero.

Regression analysis of the data obtained using the Statgraphics program.

Based on elastase activity, it is possible to determine the time of death using the Statgraphics equation: t = 18.7476–391.36*a, where t is the time of death; a — elastase activity level in the blood plasma.

Construction was carried out in linear regression dependence. Correlation coefficient = -0.82901; p<0.001.

Here is an example. A 62-year-old man died unexpectedly from the IDH. Examined the blood about 12 hours after death. Elastic activity was detected, which amounted to 0,014 pcat. Using the equation dependence of elastase activity in the plasma, we establish that about 13.27 hours have passed since death.

For ease of use, it is possible to use a graph of the dependence of elastase activity in the blood serum from the time of death in the deceased as a result of IDH and mechanical injuries.

Conclusion: The data obtained for the determination of elastase activity in blood plasma of corpses can be used as diagnostic criteria to establish the time of death within one day from the time of onset.

For convenience of using the method you can use graph dependence of elastase activity in blood plasma in those whose cause of death was due to IDH and mechanical injury.

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