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Forensic Science International 151 (2005) 139149

www.elsevier.com/locate/forsciint

Is there recent progress in the estimation of the postmortem


interval by means of thanatochemistry?
Burkhard Madea *
Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany
Received 25 August 2004; accepted 7 January 2005
Available online 21 April 2005

Abstract

Numerous methods have been proposed in the last 60 years for the determination of the time since death by chemical means.
Many of them were reviewed by Schleyer in his monograph on the determination of the time since death by means of
thanatochemistry about 40 years ago and none of these early methods has gained any practical value since they do not meet the
demands in practice (being precise, reliable, giving an immediate result).
While the earlier studies were mainly carried out on blood and cerebrospinal fluid (CSF) since the late 60s most
investigations have been performed on vitreous humor (VH). This is mainly due to the fact that vitreous humor is topographically
isolated and well protected, and thus, autolytic changes proceed slower compared to blood and CSF. The most studied parameter
in VH is potassium and even nowadays reports on the postmortem rise of vitreous potassium are published, proposing new
analytical methods or statistical evaluations.
Chemical parameters studied for the determination of the time since death have to be differentiated according to the
underlying process (catabolism, metabolic processes, pure autolysis and diffusion, putrefactive changes). In the present paper,
recent studies on thanatochemistry are discussed regarding the underlying process, the analytical methods (for instance H
magnetic resonance spectroscopy (1H MR spectroscopy), immunohistochemistry), the studied fluid compartment, the statistical
evaluation and the precision of death time estimation.
The value of chemical methods for the determination of the time since death is up to now very limited. This is supported by
the fact that field studies on the reliability and precision of death time estimation by chemical means are still scarce in the
literature.
# 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Estimation of the time since death; Postmortem chemistry; Potassium; Hypoxanthine; Vitreous humor; Immunohistochemistry;
DNA degradation; Magnetic resonance spectroscopy; Capillary electrophoresis

1. Introduction  purely physical processes (body cooling, hypostasis);


 metabolic processes (supravital reactions);
The postmortem changes used for estimating the time  autolysis (loss of selective membrane permeability, diffu-
since death are several and based on different processes sion);
[33,37]:  physicochemical processes (rigor mortis);
 bacterial processes (putrefaction).

* Tel.: +49 228 738330; fax: +49 228 738368. Scientific efforts should be made to replace the tradi-
E-mail address: B.madea@uni-bonn.de. tional methods of estimating the time since death by those

0379-0738/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2005.01.013
140 B. Madea / Forensic Science International 151 (2005) 139149

that calculate this parameter providing mean values and 3. Products of bacterial metabolism (increase of aminoa-
confidence limits, including the precision and accuracy of cids).
calculation. For some methods, these efforts were success- 4. Products of protein breakdown (decrease).
ful, for instance the nomogram method for cooling of the
body after death [16,17].
Methods of estimating the time since death can be divided These underlying principles cover, although overlapping,
in the following categories according to the type of measure- different postmortem intervals. Autolysis is seen in the early
ment, description of postmortem changes, influencing factors, postmortem interval while products of bacterial metabolism
precision of death time estimation and evidential value (rough increase in a comparably later postmortem interval.
estimation or sound scientific evidence) [33]. All the chemical methods on the determination of the time
since death have up to now in common that they are of very
1. Quantitative measurement, mathematical description, limited value in practice, since they are neither precise nor
taking into account influencing factors quantitatively, reliable nor giving an immediate result at the scene of crime.
declaration of precision, proof of precision on indepen- This is true for the recently published chemical methods
dent material. Examples: body cooling (nomogram of estimating the time since death as well. Nevertheless,
method) and potassium in vitreous humor (VH). some progress has been achieved, at least at the level of
2. Subjective description (grading), considering influencing methodology. In the present paper, recent studies on thana-
factors, declaration of precision, proof of precision on tochemistry will be discussed regarding the postmortem
independent material. Example: supravital reactions. processes, the analytical methods and the possible precision
3. Subjective description of postmortem changes, influen- of death time estimation.
cing factors known in principle, empiric estimations
instead of statistically evaluated reference values. Exam-
ples: rigor mortis, lividity. 2. Vitreous humor
4. Subjective description, analogous conclusions based on
empiricism and assumptions instead of statistically eval- 2.1. Vitreous potassium
uated reference values. Example: gastric content.
5. Subjective description, velocity of progression of post- Most chemical methods measure concentration changes
mortem changes entirely depending on ambient factors; (rise or fall) in different fluid compartments, for instance
due to the broad spectrum of ambient factors no sound autolysis with increase of potassium in the extracellular fluid
empirical estimation possible. Example: putrefaction. compartment or a decrease of sodium and chloride. Some of
these autolysis-induced concentration changes are even
Due to their nature, all analytes included in postmortem combined with changes resulting from metabolic processes
chemical changes of thanatochronological relevance should such as an increase of ammonia, lactic acid, hypoxanthine or
fall into category 1. There should be a quantitative measure- the decrease of glucose. Metabolic and autolytic processes
ment of the analyte over the postmortem interval, a mathema- are influenced by temperature, disease and cause of death,
tical description of the postmortem change should be possible length of terminal phase before death, site and method of
and influencing factors, such as temperature, should be taken sample acquisition and the resulting inter-individual varia-
into account quantitatively. The precision of the death time tion is so great that the concentration changes are of no value
estimation should be proved on an independent case material. in practice [32].
Numerous methods have been proposed in the last 60 Autolysis proceeds faster in blood than in cerebrospinal
years for the determination of the time since death by fluid (CSF) and slower in vitreous humor (due to its isolated
chemical means. Many of them were reviewed by Schleyer and confined topography) than in CSF. Thus, CSF and VH
[48,49] in his monograph on the determination of the time are the most studied fluid compartments as the speed and
since death by means of thanatochemistry about 40 years direction of change in blood was recognized as being largely
ago, further methods were reviewed later on by Coe [7]. unpredictable as long as 40 years ago.
Most chemical methods of estimating the time since Concentration gradients after loss of selective membrane
death are based on the following underlying principles: permeability disappear in blood within a few hours after
death, in CSF within 1520 h postmortem and in VH after
1. Autolysis with breakdown of cell membranes and diffu- 120 h postmortem. Therefore, the VH is the fluid compart-
sion due to the diffusion gradient according to Ficks law ment mainly studied for late postmortem chemical changes.
of diffusion. The higher the concentration gradient, the The postmortem increase of potassium in VH, first described
more suitable is the analyte for the estimation of the time by Sturner in 1963 [55], is the most extensively studied
since death, depending on the volume of distribution. parameter for estimating the postmortem interval [32]
2. Since metabolic processes do not cease immediately (Table 1). Factors governing the postmortem rise of vitreous
with death products of metabolism may be investigated potassium (temperature, chronic illness, urea retention) and
(lactate, hypoxanthine (Hx)). the range of scatter are known and can be taken partly into
B. Madea / Forensic Science International 151 (2005) 139149 141

Table 1 dl, and in a second step additionally cases with a terminal


Formulae for determining PMI (h) with [K+] (mmol/l) episode >6 h, the 95% limits of confidence could be reduced
Reference Equation obtained Formula proposed to 22 and 20 h [31,33].
+
y = [K ] Vitreous urea nitrogen is, therefore, a very suitable
Sturner y = 0.14x + 5.6 PMI = 7.14 [K+] 39.1 indicator of disturbed homeostasis of electrolyte metabolism
Adelson et al. y = 0.17x + 5.36 and depending on the urea values, the corresponding refer-
Hansson et al. y = 0.17x + 8 ence samples and formula can be chosen to extrapolate time
Coe y = 0.332x + 4.99 since death with different 95% limits of confidence. The
(x < 6 h) correlation of the precision of death time estimation with
Coe y = 0.1625x + 6.19 inner standards as critical levels of urea and/or creatinine
(x  6 h) was meanwhile confirmed by independent authors [40].
Adjutantis and y = 0.55x + 3.14
Coutselinis
Stephens and y = 0.238x + 6.342
2.1.2. Which is the dependent, which the independent
Richards variable
Madea et al. y = 0.19x + 5.88 PMI = 5.26 [K+] 30.9 Munoz et al. [40] further recommended not to use the
James et al. y = 0.23x + 4.2 PMI = 4.32 [K+] 18.35 PMI as the independent and potassium as the dependent
Munoz et al. y = 0.17x + 5.60 PMI = 3.92 [K+] 19.04 variable but potassium as the independent variable to esti-
Variable x is postmortem (h); from Munoz et al. [42]. mate the PMI. In their opinion, using PMI as the independent
variable the formula derived for calculating the time since
consideration; clear values on the precision of the method death lead to false estimations. By changing the variables,
are available. obtaining a new formula in which potassium is considered as
After Sturners original description subsequent authors the independent variable and the PMI as the dependent death
found a much wider range, especially when cases with time estimation becomes more precise.
abnormal electrolytes prior to death were included, and This statistical approach leads in fact to a more precise
different statistical parameters of the regression line were death time estimation as we could confirm on our own
described [32]. The slope of Sturners formula is one of the material. Using potassium as independent variable, the
flattest reported in the literature [34]. It should, therefore, accuracy of death time estimation with 95% limits of con-
only be used if the environmental temperature during the fidence was 23.27 h, i.e. more precise compared to that
postmortem interval has been near icebox levels. The slope with of 25.96 h using potassium as dependent variable
is increasing with increasing ambient temperature as has (time interval up to 130 h postmortem). However, this rise in
been shown experimentally by Rognum et al. [46]. precision is not statistically significant.

2.1.1. Urea as inner standard 2.1.3. Loess procedure


A second factor beside temperature determining rise and Lange et al. [28] reanalysed the data of six studies on the
scatter of vitreous potassium is the state of health or chronic rise of vitreous potassium comprising all together 790 cases.
illness preceding death. Potassium values from individuals This reanalysis revealed that:
dying after chronic illness are much more erratic than those
derived from individuals dying of acute trauma [32]. Addi-  the relationship between vitreous potassium and PMI is
tionally, the slopes of potassium increases are much steeper not completely linear;
in individuals having significant urea nitrogen retention  the residual variability of vitreous potassium as a function
(>100 mg/dl) (Table 2). of PMI is not constant.
In an entire sample including clinical and forensic
pathology cases, the 95% limits of confidence were Therefore, they developed a new approach for modelling
34 h. By eliminating cases with urea values >100 mg/ vitreous potassium and PMI that accommodates non-linear-

Table 2
Precision of time of death estimation using vitreous potassium in different random samples
Entire sample Urea < 100 mg/dl Change (%)
n 270 (170) 228 (138) 15.5
Intercept 6.10 (5.99) 6.02 (5.88) 1.3
Slope 0.20 (0.2033) 0.18 (0.1877) 10
Correlation coefficient 0.89 (0.86) 0.91 (0.89) +2.2
Variance (S2) 8.57 5.09 40.6
Standard deviation (Syx) 2.93 (3.42) 2.25 (2.62) 23.2
95% limits of confidence (h) 25.51 (34) 21.78 (22) 14.6
Urea was used as inner standard. Statistical parameters of the entire sample and subgroups; the data in brackets are from Madea et al. [31].
142 B. Madea / Forensic Science International 151 (2005) 139149

Fig. 1. The estimated relationship between vitreous potassium and PMI for six studies, with 95% lower and upper confidence bands from [28].

ities and changing residual variability. At first, a local regres- Table 3


sion model, specifically a Loess smooth curve, is fitted sepa- Estimated values of postmortem interval for various increasing
rately to the data from each of the six studies. The data of all values of potassium obtained by combining all 790 cases and using
six studies were then combined to yield a single Loess curve the loess procedure
with 95% confidence limits (Fig. 1). The estimated Loess Measured vitreous Estimated postmortem interval (h)
curve and confidence limits were then used in an inverse potassium concen-
Lower 95% Mean Upper 95%
prediction method to construct low, middle and high PMI tration (mml/l)
value value value
estimates at given values of vitreous potassium (Table 3).
5.9 2 3 4
The reliability of estimated PMI decreases with increas- 6.4 3 5 6
ing potassium concentration. However, according to the 7.0 6 7 8
authors, PMI estimates are more precise over the entire 7.5 8 10 12
range of vitreous potassium and PMI than those obtained 8.0 11 13 15
from any single study alone. For potassium values <7 mmol/ 8.5 14 16 19
l, the extent of the lower and upper 95% confidence limits is 9.1 18 21 22
1 h. For potassium >7 mmol/l but <12 mmol/l, the extent 10.1 23 25 27
of these confidence limits is 2 h. For a potassium value 11.1 29 30 32
>12 and <18 mmol/l, the extent is 3 h, while for a value 12.1 33 35 38
13.0 39 41 44
>18 mmol/l the extent is 5 h. For concentrations
13.9 44 47 50
>18 mmol/l, the extent is even greater. If these data were 14.9 51 54 57
reliable, nobody would further go to a scene of crime for 15.9 58 61 64
death time estimation but would wait for the autopsy and 17.0 66 69 72
rely on the vitreous values. 18.3 74 77 81
However, due to the much greater variability of the single 19.7 81 85 90
potassium concentrations of the included studies from the 21.1 89 94 100
single Loess curve with its 95% confidence limits, the 22.6 98 103 111
reliability of the statistical evaluation remains unclear. On 24.2 106 113 123
independent own cases, we could not confirm the precision From Lange et al. [28].
B. Madea / Forensic Science International 151 (2005) 139149 143

of death time estimation based on this statistical approach precise (3 h compared to 15 h). The time interval studied
but revealed very high false estimations (unpublished own was up to 200 h postmortem, however, the study group
data). Therefore, the use of the data in Table 3 cannot be consisted only of 61 cases.
recommended.
2.2.2. Methodology of vitreous humor analysisimproving
2.1.4. Multiple linear regression analysis precision of metabolite measurement
Multiple linear regression analysis uses, besides vitreous Coe and Apple [8] already published several years ago
potassium, further time dependent changing vitreous analytes. that vitreous chemical values differ according to the method
By using a multiple linear regression formula taking into applied. They compared flame photometry, colorimetry and
account beside potassium also sodium, urea and glucose, 95% ion-selective electrodes. With ion-selective electrodes they
confidence limits of 14 h could be achieved compared to revealed, e.g. higher values for chloride.
95% confidence limits on the same sample of 16.2 h using However, the variation of vitreous humor values is not
potassium alone. Therefore, it can be concluded that by using only due to instrumentation but the composition of vitreous
multiple linear regression analysis, a slight increase can be humor and the preanalytical handling.
achieved in the precision of death time estimation [32]. The main obstacle in vitreous humor analysis is up to now
Measuring vitreous humor by capillary zone electrophoresis that analytical methods are applied which are calibrated and
(CZE) (beside potassium also NH4, Na, Ba) and taking into validated for serum or urine but not for vitreous humor. Then
account all ions death time estimation becomes by the factor 5 differences in results obtained from left and right eyes and
more precise [56] (see also Section 2.2.1). different results when specimens have been analysed using
different instruments occur [8,31,45]. Even the reproduci-
2.2. Recent technology bility of vitreous humor electrolyte measurement with the
same analyzer is poor [38].
Also some new methodology has been proposed in the These problems seem to be due to the high viscosity of
last years, for instance capillary zone electrophoresis vitreous humor. Therefore, several attempts have been made
[2,13,56,57]. to reduce the viscosity by different sample pre-treatments as
heating or enzymatic digestion by hyaluronidase. For exam-
2.2.1. Capillary zone electrophoresis of potassium in ple, McNeil et al. [38] recommend heating of the vitreous
vitreous humor humor in capped glass tubes in a heating block, cooled at
Tagliaro et al. [56] described a reliable, simple and fast room temperature for 30 min and then centrifugation at
capillary electrophoresis method for potassium analysis in the 1800  g for 5 min. Most of the specimens were then
vitreous humor and presented data on the analytical reprodu- obviously less viscous on pipetting after the treatment and
cibility and accuracy. The results of potassium measurements some contained a visible precipitate. Table 4 is taken from
by CZE were strongly correlated to the results by flame photo- the original paper. The reproducibility of sodium and potas-
metry (r2 = 0.9333). They used no special pre-treatment of the sium measurements on untreated vitreous humor specimens
sample. Only very low amounts of vitreous humor are neces- was significantly worse on the Hitachi 911 (S.D. of dupli-
sary for measurement (50 ml). It seems that the separative cates, 20.8 and 1.97 mmol/l, respectively) compared with
mechanism CZE offers advantages to the analysis of complex the Hitachi 737 (S.D. of duplicates, 2.3 and 0.23 mmol/l,
matrices, such as postmortem samples, compared to conven- respectively). The heat treatment increased the mean results
tional methods as flame photometry or ion-selective electro- for sodium and potassium measured on the Hitachi 911 but
des. By CZE, not only potassium but NH4, Na and Ba are produced no change in the precision or mean values of urea
analysed. or creatinine measurements on either analyser. The authors
Prediction of the postmortem interval taking into account conclude that heating vitreous humor to 100 8C for 5 min is a
all ions compared to only potassium is by the factor 5 more simple and safe method for improving the precision of

Table 4
Precision of vitreous humor measurements
n Hitachi 737 Hitachi 911
Controla Treateda Controla Treateda
Sodium 40 150 (2.3) 148 (1.1) 132 (20.8) 147 (1.9)
Potassium 42 11.8 (0.23) 11.8 (0.44) 10.6 (1.97) 11.7 (0.22)
Chloride 39 125 (2.3) 122 (0.7)
Urea 35 10.55 (0.27) 10.21 (0.32) 9.94 (0.25) 9.79 (0.20)
Creatinine 35 0.11 (0.004) 0.11 (0.004) 0.10 (0.004) 0.10 (0.007)
From McNeil et al. [38].
a
Mean (S.D. of duplicates) (mmol/l).
144 B. Madea / Forensic Science International 151 (2005) 139149

electrolyte measurements on the Hitachi 911 and 737 ana- tide metabolism. Hx is formed by the action of several
lyzers without any adverse effects on the measurements of encymatic reactions and then diffuses along with the con-
urea, glucose or creatinine. Centrifuging vitreous humor centration gradient. In theory, it might be expected that a
specimens alone has not the same effect. Other authors parameter such as a postmortem increase which is solely due
recommended an enzymatic pre-treatment of vitreous humor to diffusion would correlate much more strongly with time
before analysis [14]. Enzymatic pre-treatment with hyalur- since death than would a parameter that increases due to
onidase revealed results differing much from the results in vital/postmortem degradation and diffusion. The higher
vitreous without pre-treatment. After pre-treatment the precision of death time estimation by vitreous potassium
results were reproducible. Obviously pre-treatment of compared to vitreous Hx becomes also apparent from data
vitreous humor specimens with hyaluronidase as a liquefy- published by Munoz et al. [41].
ing agent facilitates accurate pipetting by the instrument and
increases analytical precision in electrolyte testing, particu-
larly in the measurement of sodium and chloride. 3. Synovial fluid
Developing a calibrated and validated method for
vitreous humor analysis will be one of the tasks for the Synovial fluid is a well investigated fluid compartment in
future. rheumatology and handbooks of join fluid analysis are
available. However, only a few studies of medico-legal
2.3. Hypoxanthine in vitreous humor interest on synovial fluid have been published, dealing with
alcohol concentration, drug distribution into synovial fluid
Beside potassium hypoxanthine was proposed as a bio- and postmortem chemistry regarding the cause of death. An
chemical method for estimation of postmortem interval. A own study on the postmortem biochemical examination of
study by Rognum et al. [46] revealed: synovial fluid [36] revealed that synovial fluid can be used as
a postmortem examination tool compared to vitreous humor.
 a linear rise of Hx in vitreous humor in the postmortem The spread of values was comparable in all examined
interval up to 120 h postmortem; electrolytes. The evaluation of the data with regard to the
 a dependence of the slope of rise of vitreous Hx on time course over the postmortem period was not useful
temperature (the higher the ambient temperature, the except for glucose and potassium. Especially, the develop-
steeper the slope); ment of the potassium concentration over the time in the
 a strong correlation between vitreous Hx and vitreous synovial fluid is nearly the same as in vitreous humor. This
potassium values; may be of importance if vitreous humor is not available.
 a smaller range of scatter of the vitreous Hx than the
vitreous potassium values.
4. DecompositionH Magnetic resonance spectroscopy
However, in further studies (Table 5) the postmortem
increase of Hx could be confirmed, but the correlation of the When putrefactive changes are apparent only a rough
potassium values with the time since death was found to be estimation of the PMI is possible based on subjective experi-
much stronger than the Hx values. Therefore, the precision ence of the forensic pathologist, but not as a scientific sound
of death time estimation is more precise using vitreous method. About 30 years ago extensive experimental work was
potassium than with vitreous Hx [35]. The reason is that carried out on the protein degradation during putrefaction in
the postmortal rise of vitreous potassium is mainly due to various organs, e.g. the brain by Bonte [3] and Daldrup [9].
diffusion from the retina into the centre of the globe, while Daldrup [9] proposed a method of death time calculation on
Hx is a postmortem degradation product of adenine nucleo- the basis of amino acid concentrations in the brain. However,
all these methods never gained practical relevance.
Table 5 Recently there seems to be a change, since methods
Formulae for determining PMI (h) with [Hx] (mmol/l) developed in radiology like H magnetic resonance spectro-
Reference Equation obtaineda Formula proposed scopy have been applied for the identification of metabolites
emerging during decomposition of brain tissue as a step
y = [Hx]
towards quantitative determination of postmortem intervals
Rognum et al. y = 4.2x + 8.6 at 5 8C
y = 5.1x + 8.6 at 10 8C in putrefaction [4,1928,39,43,5054]. The worldwide lead-
y = 6.2x + 8.6 at 15 8C ing group is the collaborative research group of the Institute
y = 8.8x + 8.6 at 23 8C of Forensic Medicine and Department of Clinical Research
Madea et al. y = 1.29x + 3.69 (MR Spectroscopy and Methodology) at the University of
James et al. y = 3.2x 0.15 PMI = 0.31[Hx] + 0.05 Bern [1928,5054,58].
Munoz et al. y = 3.01x + 26.45 PMI = 0.17 [Hx] + 0.17 H MRS offers a non-invasive chemical analysis in situ
Variable x is postmortem (h). that can be performed on the same equipment as MR
From Munoz et al. [42]. imaging. Since brain tissue shows small interindividual
B. Madea / Forensic Science International 151 (2005) 139149 145

metabolic variations and is protected from environmental rate, proprionate), weighted according to their variances
influences by the skull, it is expected that decay and appear- with true times after death. The correlation coefficient of
ance of metabolites is following a reproducible time frame. the predicted time versus true time is r = 0.93 for the whole
The authors studied eight sheep heads as a model system time period up to 300 h postmortem. Values are shown with
supplemented by selected human cases. The sheep heads an error range of two standard deviations. As you can see
were kept in a closed plastic container after slaughtering and from the figure PMIs > 250 h are systematically underesti-
stored at a constant temperature (21  3 8C) for 18 days and mated in this model system.
were studied daily up to 18 days postmortem. In addition, As could be expected, comparison of the sheep brain
brain samples were taken at the end of the study and spectra with selected human postmortem cases shows the
investigated by high resolution NMR for identification of same metabolites and the same characteristics of appearance,
unknown metabolites. The method is able to quantify meta- thus validating the sheep model for postmortem studies of the
bolite concentrations as minimal as 1 mmol. human brain (Fig. 2A). Meanwhile by another group [1], pig
Brain decomposition resulted in reproducible concentra- brains have also been studied using the same technique and
tion changes of known metabolites and the appearance of similar time courses for some analytes were described.
decay products that had to be characterized first. Thirty The investigations on postmortem decompositions by H
metabolites could be identified and quantified in decompos- MRS may represent a real progress in research for the
ing brain tissue, 19 of which showed well defined time following reasons:
courses (Fig. 2A).
For instance, the neuronal marker N-acetylaspartate  A non-invasive chemical analysis in situ is possible with
decreases rapidly after death and may serve as indicator quantitation of analytes.
for a time period below 70 h (Fig. 2B). Butyrate or proprio-  Longitudinal studies of postmortem changes with repro-
nate, thought to arise from bacterial metabolism, start to ducible results are possible.
increase after 3050 h postmortem and reveal an unequi-  The sheep model seems to be valid for human brains as
vocal function up to 400 h (Fig. 2C). Fig. 2D compares for well.
every measurement of the series time predictions combined  With this model influencing factors like temperature can
from five metabolites (actetate, alanine, trimetylamine, buty- be easily studied.

Fig. 2. A Human and sheep spectra measured (a) 2 days and (b) 7 days postmortem. (B) Time course of (a) NA and (b) Ace concentration. (A)
and (B) reproduced by kind permission of the authors and ISMRM, original figures were published by [64]. (C) Concentration changes of
butyrate over time postmortem. (D) Correlation of predicted time vs. true time (2 S.D.). (C and D) reproduced by kind permission of authors
and ISMRM; original data published by [65].
146 B. Madea / Forensic Science International 151 (2005) 139149

 The definition of analytical functions for the time courses time limits may change in different environmental condi-
of 10 metabolites up to 400 h postmortem was successful. tions. Control studies on independent case material are still
 Prediction of PMI based on the combination of five missing.
metabolites correlate very well with true time postmortem
up to 250 hpm.
 These metabolic changes cover a PMI where no other 6. DNA degradation
method allows a quantitative calculation of the time since
death with any acceptable degree of certainty. DNA degradation as well as DNA incorporation as
biochemical or immunhistochemical methods of determin-
Last but not least this study shows that old questions in ing the time since death were published more than 30 years
forensic medicine like death time estimation can benefit ago [11,26]. By use of a cytophotometric scanning method, a
from recent developments in modern imaging systems [19 non-linear decrease of DNA in samples of human liver and
28,5054]. testicles was described. However, due to the great range of
scatter of values DNA degradation is of no practical value for
determining the time since death [29,30].
5. Immunohistochemical detection of insulin, DNA degradation as a predictor of the postmortem
thyroglobulin and calcitonin interval was studied again by flow cytometric evaluation
[5,11,12]. The preliminary data showed a correlation of
Although morphological methods of death time estima- DNA degradation and the time since death in ten cases with
tion are of no practical value in forensic medicine, at least decomposition of the tissue at room temperature. The author
some studies on immunohistochemistry should be men- indicated the possible influence of body and ambient tem-
tioned. perature, clothing, etc. [5]. Further reports with regard to
Wehner et al. [6062] studied if a positive immunoreaction these factors have not been published until now. In 1998, Di
to various antigens like insulin, thyroglobulin or calcitonin is Nunno et al. [11] published an additional study of another 35
correlated with the time since death (Table 6). The philosophy autopsy cases. They state in their introduction that they were
of these investigations is that with increasing postmortem looking for a method of estimating the time since death
interval the tertiary structure of the antigen undergoes post- which is not affected by external factors and conclude that
mortal changes and due to protein denaturation staining the DNA denaturation is such a process. They emphasise that
becomes negative. Their results (Table 5) for thyroglobulin the method shows significant different histograms only in the
are as follows: the colloid and the follicular cells of the thyroid first 72 h postmortem. Differences between the histograms
glands give a positive immunoreaction until 5 days postmor- of reference values (extirpated spleens from operation cases)
tem, whereas none of the corpses older than 13 days show such and autopsy cases are explained through biological differ-
a reaction. Negative reaction means, therefore, more than 6 ences which may be influenced by diseases and the putre-
days dead, positive reaction means less than 12 days dead. The faction process of the intestinal content in the autopsy cases.
pancreatic b-cells from up to 12-day-old corpses produce a Detailed data (body weight, storage time and temperature) of
positive immunoreaction towards insulin in all cases, whereas the autopsy cases are not given.
none of the corpses older than 30 days shows such a reaction. However, the authors assumption that degradation of
This means that in a case of a negative immunoreaction the DNA is independent of any external influence, especially of
time since death can be assumed to be more than 12 days the body and ambient temperature, is definitely wrong. Like
before the autopsy. When there is a positive stain the death any biological reaction the degradation of DNA is faster in
must lie a maximum of 29 days earlier. warmer surroundings than in colder conditions [29,30]. The
Calcitonin was always detectable in c-cells of the thyroid authors themselves state that autolysis and putrefaction of
up to 4 days, in bodies older than 13 days there was always a the spleen may be influenced by the intestinal content,
negative staining. conceivably bacteria-induced putrefaction. It is well known
Of course, the immunohistochemical detection of anti- to every forensic pathologists that this process (which can be
gens allows only a very rough estimation of the time since seen at the body surface by the greenish colour of the skin,
death, which may be helpful in single cases. However, these especially of the abdomen) is dependent on many conditions,
the most important being perhaps the ambient temperature
(faster greening of the abdominal skin in summer than in
Table 6
Immunhistochemical detection of insulin, thyroglobulin and calci-
winter and in smaller persons than in bigger ones). Experi-
tonin according to Wehner et al. [6062] ences with DNA degradation in virological samples show the
same result [27]. In addition, it should be mentioned that
Positive staining Negative staining
extensive older investigations on DNA degradation visua-
Insulin Up to 12 days in all cases >30 days in all cases lised by Feulgen staining revealed a temperature dependence
Thyroglobulin Up to 5 days in all cases >13 days in all cases of the process [29,30]. The dispersion of values in the time
Calcitonin Up to 4 days in all cases >13 days in all cases
interval from 0 to 100 h postmortem ranged so widely that
B. Madea / Forensic Science International 151 (2005) 139149 147

the 95% limits of confidence covered the whole investigated  further analytes in vitreous humor and CSF [10,24] or
postmortem period. The conclusion should be that DNA other body tissues [42];
degradation is of no value for the investigation of the time  protein degradation [47].
span since death. Especially because statements on the
precision of the estimation are missing in recent publica- They all have in common that they are of heuristic value
tions. understanding decomposition but are not of practical rele-
Nevertheless, Di Nunno et al. [12] present a case report vance.
demonstrating the method by examination of two corpses. A Therefore, for practical purposes there is no real break-
couple was found dead in one room, fully dressed. Accord- through in estimating the time since death by chemical
ing to the authors, it was assumed that the man first killed the methods. This is mainly due to the underlying processes
woman with two gunshots and then killed himself. The (metabolic processes, autolysis, putrefaction). However,
ambient temperature was 20 8C, the rectal temperatures with the novel methods as flow cytophotometry, capillary
26 8C (female) and 27.8 8C (male). Additionally, the corpse zone electrophoresis, H magnetic resonance spectroscopy,
of the woman was said to have shown chromatic putrefaction immunohistochemistry postmortem changes can be quanti-
at the trunk. The storage time prior to autopsy was 24 h at a fied and an extrapolation of the time since death is possible.
temperature of 1015 8C and 48 h at 4 8C. The splenic tissue But it should be kept in mind that the objective measurement
was taken at autopsy. According to the authors the histogram of postmortem changes contributes only to a small part to a
of the woman showed a similarity to the control peak for more precise death time estimation. Novel technology does
60 h, the histogram of the man for 72 h. Their resume of the not make death time estimation more precise on behalf of
results (. . . indicates a postmortem interval of 6072 h for itself. Only when in longlasting longitudinal studies influen-
both subjects.) is, therefore, misunderstandable. The results cing factors are taken into account as, e.g. ambient tem-
given in the figures indicate that the man who killed the perature or local temperature at the site of measurement,
woman died in fact earlier than she did. Following the case which affects all postmortem changes, death time estimation
description this is impossible. In consideration of the given may become more precise. Novel technology like H mag-
data (body height, body weight), this result is easy to netic resonance spectroscopy offers an excellent opportunity
explain. The body mass index of the woman was 22, of to study these influencing factors.
the man approximately 28. This indicates that the cooling of For vitreous humor, it has become apparent that we must
the mans body especially of the trunk was slower in the avoid analytical methods that have been validated for urine
same ambient temperature than that of the womans corpse. or serum but methods optimised just for this fluid must be
Regarding the temperature dependence of biological pro- developed for precise measurements.
cesses, the autolysis and putrefaction of the mans spleen Field studies are a good indicator for the practical value
proceeded faster compared to the womans tissue. of a method but to my knowledge field studies on chemical
Using the nomogram method by Henssge [16,17], which methods of estimating the time since death are nearly
is of predominant value for the early postmortem period, completely missing in the literature, compared for instance
with the data for ambient and rectal temperature given in the to body cooling or supravital reactions. Thus, chemical
paper, the time since death for the woman is 17.5  4.5 h, for methods at present seem to be still of academic interest.
the man 18  4.5 h. A differentiation of the assumed short Their importance may change in the future, when all criteria
time between the both times of death could not be achieved according to point one (page 2) will be fulfilled: quantitative
as might be expected. measurement, mathematical description, taking into account
In addition, it must be pointed out that the authors influencing factors quantitatively, declaration of precision,
describe a storage time for both corpses of 72 h which proof of precision on independent material.
means that the storage time is longer or as long as the
postmortem time resulting from the cytometric evaluation of
the DNA degradation. Acknowledgement
The necessary conclusion is that flow cytometric evalua-
tion of DNA degradation is not yet a reliable or precise I am indebted to Dr. Eva Scheurer from the Institute of
method to investigate the postmortem interval. Forensic Medicine University of Bern who provided me with
some of her original data and figures.

7. Conclusions
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