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);

The postmortem changes used for estimating the time
since death are several and based on different processes
[33,37]:
 metabolic processes (supravital reactions);
 autolysis (loss of selective membrane permeability, diffu-
sion);
 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
confidence limits, including the precision and accuracy of
calculation. For some methods, these efforts were success-
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
in the following categories according to the type of measure-
ment, description of postmortem changes, influencing factors,
precision of death time estimation and evidential value (rough
estimation or sound scientific evidence) [33].
1. Quantitative measurement, mathematical description,
taking into account influencing factors quantitatively,
declaration of precision, proof of precision on indepen-
dent material. Examples: body cooling (nomogram
method) and potassium in vitreous humor (VH).
2. Subjective description (grading), considering influencing
factors, declaration of precision, proof of precision on
independent material. Example: supravital reactions.
3. Subjective description of postmortem changes, influen-
cing factors known in principle, empiric estimations
instead of statistically evaluated reference values. Exam-
ples: rigor mortis, lividity.
4. Subjective description, analogous conclusions based on
empiricism and assumptions instead of statistically eval-
uated reference values. Example: gastric content.
5. Subjective description, velocity of progression of post-
mortem changes entirely depending on ambient factors;
due to the broad spectrum of ambient factors no sound
empirical estimation possible. Example: putrefaction.
Due to their nature, all analytes included in postmortem
chemical changes of thanatochronological relevance should
fall into category 1. There should be a quantitative measure-
ment of the analyte over the postmortem interval, a mathema-
tical description of the postmortem change should be possible
and influencing factors, such as temperature, should be taken
into account quantitatively. The precision of the death time
estimation should be proved on an independent case material.
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
[48,49] in his monograph on the determination of the time
since death by means of thanatochemistry about 40 years
ago, further methods were reviewed later on by Coe [7].
Most chemical methods of estimating the time since
death are based on the following underlying principles:
1. Autolysis with breakdown of cell membranes and diffu-
sion due to the diffusion gradient according to Ficks law
of diffusion. The higher the concentration gradient, the
more suitable is the analyte for the estimation of the time
since death, depending on the volume of distribution.
2. Since metabolic processes do not cease immediately
with death products of metabolism may be investigated
(lactate, hypoxanthine (Hx)).
3. Products of bacterial metabolism (increase of aminoa-
cids).
4. Products of protein breakdown (decrease).
These underlying principles cover, although overlapping,
different postmortem intervals. Autolysis is seen in the early
postmortem interval while products of bacterial metabolism
increase in a comparably later postmortem interval.
All the chemical methods on the determination of the time
since death have up to now in common that they are of very
limited value in practice, since they are neither precise nor
reliable nor giving an immediate result at the scene of crime.
This is true for the recently published chemical methods
of estimating the time since death as well. Nevertheless,
some progress has been achieved, at least at the level of
methodology. In the present paper, recent studies on thana-
tochemistry will be discussed regarding the postmortem
processes, the analytical methods and the possible precision
of death time estimation.
2. Vitreous humor
2.1. Vitreous potassium
Most chemical methods measure concentration changes
(rise or fall) in different fluid compartments, for instance
autolysis with increase of potassium in the extracellular fluid
compartment or a decrease of sodium and chloride. Some of
these autolysis-induced concentration changes are even
combined with changes resulting from metabolic processes
such as an increase of ammonia, lactic acid, hypoxanthine or
the decrease of glucose. Metabolic and autolytic processes
are influenced by temperature, disease and cause of death,
length of terminal phase before death, site and method of
sample acquisition and the resulting inter-individual varia-
tion is so great that the concentration changes are of no value
in practice [32].
Autolysis proceeds faster in blood than in cerebrospinal
fluid (CSF) and slower in vitreous humor (due to its isolated
and confined topography) than in CSF. Thus, CSF and VH
are the most studied fluid compartments as the speed and
direction of change in blood was recognized as being largely
unpredictable as long as 40 years ago.
Concentration gradients after loss of selective membrane
permeability disappear in blood within a few hours after
death, in CSF within 1520 h postmortem and in VH after
120 h postmortem. Therefore, the VH is the fluid compart-
ment mainly studied for late postmortem chemical changes.
The postmortem increase of potassium in VH, first described
by Sturner in 1963 [55], is the most extensively studied
parameter for estimating the postmortem interval [32]
(Table 1). Factors governing the postmortem rise of vitreous
potassium (temperature, chronic illness, urea retention) and
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
scatter of vitreous potassium is the state of health or chronic
illness preceding death. Potassium values from individuals
dying after chronic illness are much more erratic than those
derived from individuals dying of acute trauma [32]. Addi-
tionally, the slopes of potassium increases are much steeper
in individuals having significant urea nitrogen retention
(>100 mg/dl) (Table 2).
In an entire sample including clinical and forensic
pathology cases, the 95% limits of confidence were
34 h. By eliminating cases with urea values >100 mg/
Lange et al. [28] reanalysed the data of six studies on the
rise of vitreous potassium comprising all together 790 cases.
This reanalysis revealed that:
 the relationship between vitreous potassium and PMI is
not completely linear;
 the residual variability of vitreous potassium as a function
of PMI is not constant.
Therefore, they developed a new approach for modelling
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
of death time estimation based on this statistical approach
but revealed very high false estimations (unpublished own
data). Therefore, the use of the data in Table 3 cannot be
recommended.
2.1.4. Multiple linear regression analysis
Multiple linear regression analysis uses, besides vitreous
potassium, further time dependent changing vitreous analytes.
By using a multiple linear regression formula taking into
account beside potassium also sodium, urea and glucose, 95%
confidence limits of 14 h could be achieved compared to
95% confidence limits on the same sample of 16.2 h using
potassium alone. Therefore, it can be concluded that by using
multiple linear regression analysis, a slight increase can be
achieved in the precision of death time estimation [32].
Measuring vitreous humor by capillary zone electrophoresis
(CZE) (beside potassium also NH4, Na, Ba) and taking into
account all ions death time estimation becomes by the factor 5
more precise [56] (see also Section 2.2.1).
2.2. Recent technology
Also some new methodology has been proposed in the
last years, for instance capillary zone electrophoresis
[2,13,56,57].
2.2.1. Capillary zone electrophoresis of potassium in
vitreous humor
Tagliaro et al. [56] described a reliable, simple and fast
capillary electrophoresis method for potassium analysis in the
vitreous humor and presented data on the analytical reprodu-
cibility and accuracy. The results of potassium measurements
by CZE were strongly correlated to the results by flame photo-
metry (r2 = 0.9333). They used no special pre-treatment of the
sample. Only very low amounts of vitreous humor are neces-
sary for measurement (50 ml). It seems that the separative
mechanism CZE offers advantages to the analysis of complex
matrices, such as postmortem samples, compared to conven-
tional methods as flame photometry or ion-selective electro-
des. By CZE, not only potassium but NH4, Na and Ba are
analysed.
Prediction of the postmortem interval taking into account
all ions compared to only potassium is by the factor 5 more
Table 4
Precision of vitreous humor measurements
n Hitachi 737
Controla
Sodium 40 150 (2.3)
Potassium 42 11.8 (0.23)
Chloride 39 125 (2.3)
Urea 35 10.55 (0.27)
Creatinine 35 0.11 (0.004)
From McNeil et al. [38].
a
Mean (S.D. of duplicates) (mmol/l).
143
precise (3 h compared to 15 h). The time interval studied
was up to 200 h postmortem, however, the study group
consisted only of 61 cases.
2.2.2. Methodology of vitreous humor analysisimproving
precision of metabolite measurement
Coe and Apple [8] already published several years ago
that vitreous chemical values differ according to the method
applied. They compared flame photometry, colorimetry and
ion-selective electrodes. With ion-selective electrodes they
revealed, e.g. higher values for chloride.
However, the variation of vitreous humor values is not
only due to instrumentation but the composition of vitreous
humor and the preanalytical handling.
The main obstacle in vitreous humor analysis is up to now
that analytical methods are applied which are calibrated and
validated for serum or urine but not for vitreous humor. Then
differences in results obtained from left and right eyes and
different results when specimens have been analysed using
different instruments occur [8,31,45]. Even the reproduci-
bility of vitreous humor electrolyte measurement with the
same analyzer is poor [38].
These problems seem to be due to the high viscosity of
vitreous humor. Therefore, several attempts have been made
to reduce the viscosity by different sample pre-treatments as
heating or enzymatic digestion by hyaluronidase. For exam-
ple, McNeil et al. [38] recommend heating of the vitreous
humor in capped glass tubes in a heating block, cooled at
room temperature for 30 min and then centrifugation at
1800  g for 5 min. Most of the specimens were then
obviously less viscous on pipetting after the treatment and
some contained a visible precipitate. Table 4 is taken from
the original paper. The reproducibility of sodium and potas-
sium measurements on untreated vitreous humor specimens
was significantly worse on the Hitachi 911 (S.D. of dupli-
cates, 20.8 and 1.97 mmol/l, respectively) compared with
the Hitachi 737 (S.D. of duplicates, 2.3 and 0.23 mmol/l,
respectively). The heat treatment increased the mean results
for sodium and potassium measured on the Hitachi 911 but
produced no change in the precision or mean values of urea
or creatinine measurements on either analyser. The authors
conclude that heating vitreous humor to 100 8C for 5 min is a
simple and safe method for improving the precision of
Hitachi 911
Treateda Controla Treateda
148 (1.1) 132 (20.8) 147 (1.9)
11.8 (0.44) 10.6 (1.97) 11.7 (0.22)
122 (0.7)
10.21 (0.32) 9.94 (0.25) 9.79 (0.20)
0.11 (0.004) 0.10 (0.004) 0.10 (0.007)
144 B. Madea / Forensic Science International 151 (2005) 139149
electrolyte measurements on the Hitachi 911 and 737 ana-
lyzers without any adverse effects on the measurements of
urea, glucose or creatinine. Centrifuging vitreous humor
specimens alone has not the same effect. Other authors
recommended an enzymatic pre-treatment of vitreous humor
before analysis [14]. Enzymatic pre-treatment with hyalur-
onidase revealed results differing much from the results in
vitreous without pre-treatment. After pre-treatment the
results were reproducible. Obviously pre-treatment of
vitreous humor specimens with hyaluronidase as a liquefy-
ing agent facilitates accurate pipetting by the instrument and
increases analytical precision in electrolyte testing, particu-
larly in the measurement of sodium and chloride.
Developing a calibrated and validated method for
vitreous humor analysis will be one of the tasks for the
future.
2.3. Hypoxanthine in vitreous humor
Beside potassium hypoxanthine was proposed as a bio-
chemical method for estimation of postmortem interval. A
study by Rognum et al. [46] revealed:
 a linear rise of Hx in vitreous humor in the postmortem
interval up to 120 h postmortem;
 a dependence of the slope of rise of vitreous Hx on
temperature (the higher the ambient temperature, the
steeper the slope);
 a strong correlation between vitreous Hx and vitreous
potassium values;
 a smaller range of scatter of the vitreous Hx than the
vitreous potassium values.
However, in further studies (Table 5) the postmortem
increase of Hx could be confirmed, but the correlation of the
potassium values with the time since death was found to be
much stronger than the Hx values. Therefore, the precision
of death time estimation is more precise using vitreous
potassium than with vitreous Hx [35]. The reason is that
the postmortal rise of vitreous potassium is mainly due to
diffusion from the retina into the centre of the globe, while
Hx is a postmortem degradation product of adenine nucleo-
Table 5
Formulae for determining PMI (h) with [Hx] (mmol/l)
Reference Equation obtaineda Formula proposed
y = [Hx]
Rognum et al. y = 4.2x + 8.6 at 5 8C
y = 5.1x + 8.6 at 10 8C
y = 6.2x + 8.6 at 15 8C
y = 8.8x + 8.6 at 23 8C
Madea et al. y = 1.29x + 3.69
James et al. y = 3.2x 0.15 PMI = 0.31[Hx] + 0.05
Munoz et al. y = 3.01x + 26.45 PMI = 0.17 [Hx] + 0.17
Variable x is postmortem (h).
From Munoz et al. [42].
tide metabolism. Hx is formed by the action of several
encymatic reactions and then diffuses along with the con-
centration gradient. In theory, it might be expected that a
parameter such as a postmortem increase which is solely due
to diffusion would correlate much more strongly with time
since death than would a parameter that increases due to
vital/postmortem degradation and diffusion. The higher
precision of death time estimation by vitreous potassium
compared to vitreous Hx becomes also apparent from data
published by Munoz et al. [41].
3. Synovial fluid
Synovial fluid is a well investigated fluid compartment in
rheumatology and handbooks of join fluid analysis are
available. However, only a few studies of medico-legal
interest on synovial fluid have been published, dealing with
alcohol concentration, drug distribution into synovial fluid
and postmortem chemistry regarding the cause of death. An
own study on the postmortem biochemical examination of
synovial fluid [36] revealed that synovial fluid can be used as
a postmortem examination tool compared to vitreous humor.
The spread of values was comparable in all examined
electrolytes. The evaluation of the data with regard to the
time course over the postmortem period was not useful
except for glucose and potassium. Especially, the develop-
ment of the potassium concentration over the time in the
synovial fluid is nearly the same as in vitreous humor. This
may be of importance if vitreous humor is not available.
4. DecompositionH Magnetic resonance spectroscopy
When putrefactive changes are apparent only a rough
estimation of the PMI is possible based on subjective experi-
ence of the forensic pathologist, but not as a scientific sound
method. About 30 years ago extensive experimental work was
carried out on the protein degradation during putrefaction in
various organs, e.g. the brain by Bonte [3] and Daldrup [9].
Daldrup [9] proposed a method of death time calculation on
the basis of amino acid concentrations in the brain. However,
all these methods never gained practical relevance.
Recently there seems to be a change, since methods
developed in radiology like H magnetic resonance spectro-
scopy have been applied for the identification of metabolites
emerging during decomposition of brain tissue as a step
towards quantitative determination of postmortem intervals
in putrefaction [4,1928,39,43,5054]. The worldwide lead-
ing group is the collaborative research group of the Institute
of Forensic Medicine and Department of Clinical Research
(MR Spectroscopy and Methodology) at the University of
Bern [1928,5054,58].
H MRS offers a non-invasive chemical analysis in situ
that can be performed on the same equipment as MR
imaging. Since brain tissue shows small interindividual
 B. Madea / Forensic Science International 151 (2005) 139149 145

metabolic variations and is protected from environmental
influences by the skull, it is expected that decay and appear-
ance of metabolites is following a reproducible time frame.
The authors studied eight sheep heads as a model system
supplemented by selected human cases. The sheep heads
were kept in a closed plastic container after slaughtering and
stored at a constant temperature (21  3 8C) for 18 days and
were studied daily up to 18 days postmortem. In addition,
brain samples were taken at the end of the study and
investigated by high resolution NMR for identification of
unknown metabolites. The method is able to quantify meta-
bolite concentrations as minimal as 1 mmol.
Brain decomposition resulted in reproducible concentra-
tion changes of known metabolites and the appearance of
decay products that had to be characterized first. Thirty
metabolites could be identified and quantified in decompos-
ing brain tissue, 19 of which showed well defined time
courses (Fig. 2A).
For instance, the neuronal marker N-acetylaspartate
decreases rapidly after death and may serve as indicator
for a time period below 70 h (Fig. 2B). Butyrate or proprio-
nate, thought to arise from bacterial metabolism, start to
increase after 3050 h postmortem and reveal an unequi-
vocal function up to 400 h (Fig. 2C). Fig. 2D compares for
every measurement of the series time predictions combined
from five metabolites (actetate, alanine, trimetylamine, buty-
rate, proprionate), weighted according to their variances
with true times after death. The correlation coefficient of
the predicted time versus true time is r = 0.93 for the whole
time period up to 300 h postmortem. Values are shown with
an error range of two standard deviations. As you can see
from the figure PMIs > 250 h are systematically underesti-
mated in this model system.
As could be expected, comparison of the sheep brain
spectra with selected human postmortem cases shows the
same metabolites and the same characteristics of appearance,
thus validating the sheep model for postmortem studies of the
human brain (Fig. 2A). Meanwhile by another group [1], pig
brains have also been studied using the same technique and
similar time courses for some analytes were described.
The investigations on postmortem decompositions by H
MRS may represent a real progress in research for the
following reasons:
 A non-invasive chemical analysis in situ is possible with
quantitation of analytes.
 Longitudinal studies of postmortem changes with repro-
ducible results are possible.
 The sheep model seems to be valid for human brains as
well.
 With this model influencing factors like temperature can
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
of 10 metabolites up to 400 h postmortem was successful.
 Prediction of PMI based on the combination of five
metabolites correlate very well with true time postmortem
up to 250 hpm.
 These metabolic changes cover a PMI where no other
method allows a quantitative calculation of the time since
death with any acceptable degree of certainty.
Last but not least this study shows that old questions in
forensic medicine like death time estimation can benefit
from recent developments in modern imaging systems [19
28,5054].
5. Immunohistochemical detection of insulin,
thyroglobulin and calcitonin
Although morphological methods of death time estima-
tion are of no practical value in forensic medicine, at least
some studies on immunohistochemistry should be men-
tioned.
Wehner et al. [6062] studied if a positive immunoreaction
to various antigens like insulin, thyroglobulin or calcitonin is
correlated with the time since death (Table 6). The philosophy
of these investigations is that with increasing postmortem
interval the tertiary structure of the antigen undergoes post-
mortal changes and due to protein denaturation staining
becomes negative. Their results (Table 5) for thyroglobulin
are as follows: the colloid and the follicular cells of the thyroid
glands give a positive immunoreaction until 5 days postmor-
tem, whereas none of the corpses older than 13 days show such
a reaction. Negative reaction means, therefore, more than 6
days dead, positive reaction means less than 12 days dead. The
pancreatic b-cells from up to 12-day-old corpses produce a
positive immunoreaction towards insulin in all cases, whereas
none of the corpses older than 30 days shows such a reaction.
This means that in a case of a negative immunoreaction the
time since death can be assumed to be more than 12 days
before the autopsy. When there is a positive stain the death
must lie a maximum of 29 days earlier.
Calcitonin was always detectable in c-cells of the thyroid
up to 4 days, in bodies older than 13 days there was always a
negative staining.
Of course, the immunohistochemical detection of anti-
gens allows only a very rough estimation of the time since
death, which may be helpful in single cases. However, these
Table 6
Immunhistochemical detection of insulin, thyroglobulin and calci-
tonin according to Wehner et al. [6062]
Positive staining Negative staining
Insulin Up to 12 days in all cases >30 days in all cases
Thyroglobulin Up to 5 days in all cases >13 days in all cases
Calcitonin Up to 4 days in all cases >13 days in all cases
time limits may change in different environmental condi-
tions. Control studies on independent case material are still
missing.
6. DNA degradation
DNA degradation as well as DNA incorporation as
biochemical or immunhistochemical methods of determin-
ing the time since death were published more than 30 years
ago [11,26]. By use of a cytophotometric scanning method, a
non-linear decrease of DNA in samples of human liver and
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].
DNA degradation as a predictor of the postmortem
interval was studied again by flow cytometric evaluation
[5,11,12]. The preliminary data showed a correlation of
DNA degradation and the time since death in ten cases with
decomposition of the tissue at room temperature. The author
indicated the possible influence of body and ambient tem-
perature, clothing, etc. [5]. Further reports with regard to
these factors have not been published until now. In 1998, Di
Nunno et al. [11] published an additional study of another 35
autopsy cases. They state in their introduction that they were
looking for a method of estimating the time since death
which is not affected by external factors and conclude that
the DNA denaturation is such a process. They emphasise that
the method shows significant different histograms only in the
first 72 h postmortem. Differences between the histograms
of reference values (extirpated spleens from operation cases)
and autopsy cases are explained through biological differ-
ences which may be influenced by diseases and the putre-
faction process of the intestinal content in the autopsy cases.
Detailed data (body weight, storage time and temperature) of
the autopsy cases are not given.
However, the authors assumption that degradation of
DNA is independent of any external influence, especially of
the body and ambient temperature, is definitely wrong. Like
any biological reaction the degradation of DNA is faster in
warmer surroundings than in colder conditions [29,30]. The
authors themselves state that autolysis and putrefaction of
the spleen may be influenced by the intestinal content,
conceivably bacteria-induced putrefaction. It is well known
to every forensic pathologists that this process (which can be
seen at the body surface by the greenish colour of the skin,
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
winter and in smaller persons than in bigger ones). Experi-
ences with DNA degradation in virological samples show the
same result [27]. In addition, it should be mentioned that
extensive older investigations on DNA degradation visua-
lised by Feulgen staining revealed a temperature dependence
of the process [29,30]. The dispersion of values in the time
interval from 0 to 100 h postmortem ranged so widely that
 B. Madea / Forensic Science International 151 (2005) 139149
the 95% limits of confidence covered the whole investigated
postmortem period. The conclusion should be that DNA
degradation is of no value for the investigation of the time
span since death. Especially because statements on the
precision of the estimation are missing in recent publica-
tions.
Nevertheless, Di Nunno et al. [12] present a case report
demonstrating the method by examination of two corpses. A
couple was found dead in one room, fully dressed. Accord-
ing to the authors, it was assumed that the man first killed the
woman with two gunshots and then killed himself. The
ambient temperature was 20 8C, the rectal temperatures
26 8C (female) and 27.8 8C (male). Additionally, the corpse
of the woman was said to have shown chromatic putrefaction
at the trunk. The storage time prior to autopsy was 24 h at a
temperature of 1015 8C and 48 h at 4 8C. The splenic tissue
was taken at autopsy. According to the authors the histogram
of the woman showed a similarity to the control peak for
60 h, the histogram of the man for 72 h. Their resume of the
results (. . . indicates a postmortem interval of 6072 h for
both subjects.) is, therefore, misunderstandable. The results
given in the figures indicate that the man who killed the
woman died in fact earlier than she did. Following the case
description this is impossible. In consideration of the given
data (body height, body weight), this result is easy to
explain. The body mass index of the woman was 22, of
the man approximately 28. This indicates that the cooling of
the mans body especially of the trunk was slower in the
same ambient temperature than that of the womans corpse.
Regarding the temperature dependence of biological pro-
cesses, the autolysis and putrefaction of the mans spleen
proceeded faster compared to the womans tissue.
Using the nomogram method by Henssge [16,17], which
is of predominant value for the early postmortem period,
with the data for ambient and rectal temperature given in the
paper, the time since death for the woman is 17.5  4.5 h, for
the man 18  4.5 h. A differentiation of the assumed short
time between the both times of death could not be achieved
as might be expected.
In addition, it must be pointed out that the authors
describe a storage time for both corpses of 72 h which
means that the storage time is longer or as long as the
postmortem time resulting from the cytometric evaluation of
the DNA degradation.
The necessary conclusion is that flow cytometric evalua-
tion of DNA degradation is not yet a reliable or precise
method to investigate the postmortem interval.
7. Conclusions
Several other methods for death time estimation using
chemical methods could be addressed:
 enzyme activity in various tissues and organs [15,18];
 morphological changes [6,44,59,63];
147
 further analytes in vitreous humor and CSF [10,24] or
other body tissues [42];
 protein degradation [47].
They all have in common that they are of heuristic value
understanding decomposition but are not of practical rele-
vance.
Therefore, for practical purposes there is no real break-
through in estimating the time since death by chemical
methods. This is mainly due to the underlying processes
(metabolic processes, autolysis, putrefaction). However,
with the novel methods as flow cytophotometry, capillary
zone electrophoresis, H magnetic resonance spectroscopy,
immunohistochemistry postmortem changes can be quanti-
fied and an extrapolation of the time since death is possible.
But it should be kept in mind that the objective measurement
of postmortem changes contributes only to a small part to a
more precise death time estimation. Novel technology does
not make death time estimation more precise on behalf of
itself. Only when in longlasting longitudinal studies influen-
cing factors are taken into account as, e.g. ambient tem-
perature or local temperature at the site of measurement,
which affects all postmortem changes, death time estimation
may become more precise. Novel technology like H mag-
netic resonance spectroscopy offers an excellent opportunity
to study these influencing factors.
For vitreous humor, it has become apparent that we must
avoid analytical methods that have been validated for urine
or serum but methods optimised just for this fluid must be
developed for precise measurements.
Field studies are a good indicator for the practical value
of a method but to my knowledge field studies on chemical
methods of estimating the time since death are nearly
completely missing in the literature, compared for instance
to body cooling or supravital reactions. Thus, chemical
methods at present seem to be still of academic interest.
Their importance may change in the future, when all criteria
according to point one (page 2) will be fulfilled: quantitative
measurement, mathematical description, taking into account
influencing factors quantitatively, declaration of precision,
proof of precision on independent material.
Acknowledgement
I am indebted to Dr. Eva Scheurer from the Institute of
Forensic Medicine University of Bern who provided me with
some of her original data and figures.
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