Int J Legal Med (2016) 130:1557–1566
DOI 10.1007/s00414-016-1395-3
ORIGINAL ARTICLE
Estimating the postmortem interval of human skeletal remains
by analyzing their optical behavior
V. Sterzik 1,2 & T. Jung 2 & K. Jellinghaus 2 & M. Bohnert 2
Received: 17 March 2016 / Accepted: 27 May 2016 / Published online: 4 June 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract The aim of this study was to figure out a new prac-
tically applicable method to distinguish between historical and
recent human skeletal remains. Therefore, the optical behavior
of bone cross sections was investigated using the combination
of two methods: a modification of an already established test
(UV-induced fluorescence) and a new method (490 nm-in-
duced fluorescence). We evaluated the areal extent of fluo-
rescence of 30 bone cross sections with known postmortem
interval (PMI) using ultraviolet light and 490 nm light. For
analysis, the areal extend of fluorescent surface was deter-
mined using photos of the samples and an image editing
software. The results prove that there is a correlation be-
tween PMI and the areal extent of fluorescent surface in
both tests. Furthermore, the combination of both methods
is a good indicator to distinguish within the forensic rele-
vant post mortem interval between PMI < 30 years and
PMI > 30 years.
Keywords Lumatec Superlite 410 . Forensic light source .
Alternative light source (ALS) . Human skeletal remains .
Death time estimation . Postmortem interval (PMI) . Luminol .
Forensic photography . Forensic osteology . Fluorescence
* V. Sterzik
vera.sterzik@kssg.ch
1
Institute of Forensic Medicine, Kantonsspital St. Gallen, Rorschacher
Strasse 95, 9007 St. Gallen, Switzerland
2
Institute of Forensic Medicine, Julius-Maximilians-University,
Versbacher Str. 3, 97078 Würzburg, Germany
Introduction and literature review
Determining the postmortem interval of human skeletal
remains is a challenging part in daily practice of forensic
osteology. In order to clarify if further criminal investigations
by law enforcement agencies are necessary, the postmortem
interval (PMI) plays an important role. Skeletal remains can
either be historical or recent. In a historical case, there will
most likely be no interest in criminal investigations. On the
contrary, further criminal investigations might be needed if
human skeletal remains turn out to be recent. Therefore, it is
of criminal interest to distinguish between historical and re-
cent human skeletal remains by estimating their postmortem
interval [1]. To do so, different methods have been used and
described so far.
Radionuclide tests
Furthermore, methods analyzing radionuclides exist like
the determination of radiocarbon 1 4 C [2–10], 9 0 Sr
[11–13], 210Pb [13, 14], 210Po [14], plutonium [15] as well
as 228Th, and 228Ra [16]. Initially, analysis of radiocarbon
seemed to be inaccurate because of its long half-life period
(5730 years) [5]. Subsequently, analysis of radiocarbon
content and thoughtful comparison with bomb-curve
values could distinguish human remains from individuals
who died before 1950 from those who were alive after that
date [2, 5–7, 10]. The relationship of values of the cortical bone
and the trabecular bone assists in the determination of whether
the values fall before 1963 or after [7]. Furthermore, radiocar-
bon analysis of dental enamel and different bone tissues can
provide valuable information relating to both the date of birth
and the date of death [8]. The advantage of these methods is
that they mostly seem to be independent of external influencing
1558
factors like e.g., the climate [4, 9]. Clear disadvantages are high
costs and an elaborate handling [9].
Spectroscopy methods
Using Fournier transform infrared spectroscopy to determine
the crystallinity index and carbonate-phosphate index ap-
peared extremely useful to distinguish ancient and recent bone
fragments [17, 18]. Once you have access to Fournier trans-
formation infrared spectroscopy, it is a cheap and fast tech-
nique [18]. Raman spectroscopy analysis of buried turkey
bone showed time-dependent results within an interval of
12–62 days [19].
Luminol tests
Inside the bone, hemoglobin can remain protected and under
relatively stable conditions as long as the bone’s structure
integrity is given during the postmortem period [1]. This is
why analyzing the hemoglobin content within human skeletal
remains can be a viable method in determining the postmor-
tem interval [1]. First used to detect bloodstains [20], luminol
has later on been used to estimate the PMI of human skeletal
remains [21, 22]. Craemer and Buck showed that it is possible
to distinguish between historical and recent skeletal remains
by spraying luminol on powdered bone, defining Bhistorical^
with PMI > 100 years [1]. Analysis with a photomultiplier
tube revealed a significant difference between recent
(<100 years PMI) and historical bone samples (>100 years)
with a cutoff at 11 % chemiluminescence intensity [1].
Ramsthaler et al. [22] showed that a negative luminol test
result (providing a PMI without forensic relevance) is
superior to a positive luminol test result indicating a short
PMI of <100 years. Thirty percent of the samples >100 years
gave a false-positive reaction [22]. Other authors claim that
human skeletal remains have a historical status already up
from a postmortem interval of 50 years, though [9, 22–25].
A different luminol test described by Ramsthaler et al. was
considered not to be valid as an only method [26]. Cappella et
al. combined luminol tests with microscopically examinations
and had 41.1 % of ancient bones reacting false positive to
luminol [27]. Nevertheless, the combination with macroscop-
ic analysis decreased the false positives remarkably and there-
fore may represent a promising solution to cases where many
fragments need to be tested quickly [27].
UV-induced fluorescence test
The blue fluorescence has in former years been described as
an indicator for PMI < 100 years [28] and PMI < 150 years
[29]. Later on, Ramsthaler et al. showed that UV-induced
fluorescence of a cross cut bone sample can be an indicator
for a forensic relevant PMI (<50 years) [22]. Following Hoke
Int J Legal Med (2016) 130:1557–1566
et al., there is a correlation between UV-induced fluorescence
color and PMI [30]. However, they could not find a correlation
between PMI and fluorescence intensity [30]. Summarizing,
the UV-induced fluorescence test is considered to be more
suitable to exclude historical material than to accurately deter-
mine the PMI [22, 30, 31].
Besides, other procedures have been investigated to ad-
vance the PMI estimation of human skeletal remains. Those
ended without satisfying results or brought along other disad-
vantages. Macroscopic and microscopic examinations e.g.,
can reveal important information in histomorphological
changes [25, 31]. However, analyzing the microstructure of
human skeletal remains could not satisfy as an only method to
estimate the PMI [27, 32–34]. Analyzing the citrate content of
bone seems to be a promising tool as long as the skeletal
remains were never exposed to temperatures below 0 °C
[35]. Besides the temperature limitation, the method is expen-
sive and time-consuming, though [35]. The Combur test, the
Hexagon OBTI test, and the RSID test have proven not to be
of any help in estimating the postmortem interval [22, 36, 37].
Analyzing the adipocere formation seems not to be suitable
either [38, 39] as well as analyzing the length of DNA frag-
ments [40].
To be usable in daily practice, methods to estimate the PMI
of human skeletal remains have to be cheap and easily viable.
Both the luminol test and UV-induced fluorescence test fulfill
these criteria. Results could be more precise though, especial-
ly to determine a PMI of less than 50 years, which was the aim
of our study. Therefore, we used a new method: The analysis
of bone cross sections with a forensic light source of 490 nm
and a dark red filter. To improve the validity in PMI estima-
tion, the new method was combined with a modified analysis
of UV-induced fluorescence.
Material and methods
Material
Bones
The femoral bones of 30 individuals with known PMI were
investigated. Two samples were taken in the context of post-
mortem examinations at the Institute of Forensic Medicine
Würzburg. Twenty-eight bones were collected from aban-
doned graves at six different cemeteries. The postmortem in-
terval for each grave was well documented and ranged from 1
to 49 years. There were different burial environments and four
different types of soil (clay, silt, sand, and keuper). Most of the
samples were affected by adipocere formation. Residues of
soil and soft tissue were removed, and the bones were cleaned
with a brush and warm water. Cross sections from each bone
were prepared using an oscillating autopsy saw (Buehler).
Int J Legal Med (2016) 130:1557–1566
One side of the cross section was then ground with fine sand-
paper (1200 grit) to avoid reflection artifacts during the inves-
tigation. Each sample was given a case number and was stored
in a dark, dry place with constant temperature (18–20 °C) in
special acid-free cardboard boxes. The latency time until
investigation was added to the known PMI. A forensic
light source and an UV lamp were used to analyze the cross
section’s optical behavior. The results were photographically
documented.
Forensic light source
The Lumatec Superlite 410 is a portable forensic light source
with gas discharge lamp. The following wavelengths have
been used to evaluate the cross section’s optical behavior in
a pre-study: 415, 440, 460, 490, 550, and 570 nm. Each wave-
length was combined with additional colored filters: yellow,
orange, and dark red. The most impressive fluorescence reac-
tion was caused by 490 nm in combination with a dark red
colored filter.
UV light source
For the UV-induced fluorescence test, a UV analysis lamp
(Herolab) with two 8-W tubes emitting long-wave (365 nm)
or short-wave (254 nm) UV light has been used. Just the long-
wave UV light has been applied the study presented.
Photography
The 490 nm pictures have been taken with a Nikon D7000 and
a 50 mm Nikkor lens. For settings were chosen ISO-100, lens
opening F-9, and length of exposure 5 s. To document the UV-
induced fluorescence, we used a Canon EOS 600D with the
standard lens (EF-S 18–55 mm 1:3, 5-5,6 IS II). The settings
were ISO-100, lens opening F-36, and length of exposure
25 s. In order to take vibration-free pictures, the cameras have
been mounted on a tripod and focused manual.
Methods
Three bone samples were placed in the center of a box lined
with black fabric (a Bphoto box^). In the middle, the 30 cross
sections (the ones to be investigated) were placed one by one.
Fig. 1 Arrangement of the
samples inside the Bphoto box^.
Left to right: negative sample
(PMI > 150 years), sample to be
examined, and positive sample
(PMI < 14 days)
1559
Additionally, a negative sample (known PMI > 150 years) was
placed left, and a positive sample (PMI < 14 days) was placed
right. The light sources were arranged in an angle of approx.
45° to the cross sections surface and caused fluorescence (a
spontaneous emission of light). After focusing, the room light
has been switched off and a photograph was taken, showing
all three samples in one picture (Fig. 1).
First, the photos of UV-induced fluorescence were ana-
lyzed as to their areal extend of blue fluorescence surface.
Therefore, the image editing software Adobe Photoshop CS
5.1 was used. The following steps have been performed
(Fig. 2):
& After loading the image by the software, it was adjusted
with the tool Bauto levels^ for visual assessment. In a first
step, the size of the image was determined. Therefore, a
square with fixed size (1130 pixels * 1130 pixels = 1,276,
900 pixels) was used (tool Brectangular marquee^). The
result was copied in a new tab for further studies.
& The tool Bmagic wand^ (tolerance 50) enabled to identify
areas, which do not belong to the sample by simply
clicking on a black area. The size could be read in the
Bhistogram^. Subtracting this value from the overall size
of the square gave the size of the sample in pixels. The
results were documented in a table.
& In a second step, the proportion of blue fluorescent surface
has been determined. Using the tool Beyedropper,^ a color
selection in the positive sample was made (red 0, green
186, blue 203). To figure out how many pixels in the
investigated sample showed the same blue fluorescent col-
or, we used the tool Bcolor range^ (all > > color range…).
The program calculated the number of pixels with a toler-
ance of 75. The result was shown in the histogram.
& Dividing the number of fluorescent pixels by the total
sample size calculated in the first step, you get the percent-
age of blue fluorescent surface.
By visual inspection, the use of 490 nm light in combina-
tion with a dark red filter seemed to show the most interesting
optical changes within the use of Lumatec Superlite. For that
reason, this particular combination was chosen and analyzed
using a similar method as described above. The color selection
was made using the tool Beyedropper^ (red 201, green 152,
blue 159).
1560
Fig. 2 Step by step analysis (schematic) of the sample using Adobe Photoshop CS 5.1
The overall results were documented with Excel 2013 and
analyzed with SPSS (statistical package of social sciences,
version 22, IBM).
Results
A total of 30 cases were analyzed. The PMI ranged between 1
and 49 years, and therefore, all cases had a forensic relevant
PMI (<50 years). Every sample showed a certain proportion of
blue fluorescent (UV light) and/or red fluorescent surface
(490 nm). The measured data are shown in Fig. 3.
Int J Legal Med (2016) 130:1557–1566
UV-induced fluorescence
There was a significant correlation (p = 0.009) between PMI
and the areal extend of blue fluorescent surface (Fig. 4). The
linear regression line shows the blue fluorescent surface
decreases over time (Fig. 4). The correlation coefficient
was weak (Pearson correlation r = 0.47, coefficient of de-
termination r2 = 0.221).
In the following, a formula was created to calculate the
postmortem interval of each cross section. Therefore, X- and
Y-axis were changed, and a linear regression analysis was
used to determine the confidence interval [95 %] improving
Int J Legal Med (2016) 130:1557–1566 1561

Fig. 3 Measured percentage of

UV-induced fluorescence and
490 nm-induced fluorescence in
relation to the total surface of each
sample

the formulas accuracy: samples correctly (Fig. 5). Because we only had samples
available with a known PMI below 50 years, we used a PMI
y ¼ −1:046x½0:76 þ 32:651½5:33 of 30 years as the forensic relevant cutoff value. Among the
samples with PMI < 30 years, 78.9 % showed more than 1 %
areal extend of blue fluorescent surface (Fig. 6). By contrast,
y PMI interval in years samples with PMI > 30 years showed more than 1 % areal
x measured fluorescent surface of the bone cross extend of blue fluorescent surface in 54.5 %.
section
brackets confidence interval 490 nm-induced fluorescence

To test the equation, we calculated the upper and lower There was a statistical correlation (p = 0.002) between PMI
bound of each sample and compared the results with the and the areal extend of red fluorescent surface as well
known PMI. With the formula, we estimated 70 % of the (Fig. 7). Again, the linear regression line shows a decreasing
1562 Int J Legal Med (2016) 130:1557–1566

Fig. 4 Results of the UV-induced

fluorescence test. X-axis: PMI in
years; Y-axis: UV-induced
fluorescent surface in percent.
Regression line with
determination formula and
coefficient (R2)

red fluorescent surface with increasing PMI, Pearson coeffi-
cient r = 0.55 and coefficient of determination r2 = 0.3038.
Like described above, a formula was created to calculate
the postmortem interval of each cross section:
y ¼ −0:632½0:368x þ 33:14½4:92
y PMI interval in years
x measured red surface of the bone cross section
brackets confidence interval
We used the same method outlined above to test the
equation. The PMI of 18 samples (60 %) was estimated
correctly (Fig. 5). In the group PMI < 30 years, 16 out of
19 samples (84.2 %) showed an areal extend of red fluorescent
surface >1 % (Fig. 8). On the contrary, samples with
PMI > 30 years showed red fluorescent surface of >1 % in
only 3 out of 11 samples (27.3 %).
Finally, both methods were combined. Thereby, in the
group PMI < 30 years, 14 out of 19 samples (73.7 %) showed
an areal extend of blue and red fluorescent surface above 1 %
(Fig. 9). By contrast, in the group PMI > 30 years only 2 out of

Fig. 5 Results of PMI

determination without cutoff. The
results calculated with formula for
both tests; each were compared to
the known PMI. The figure shows
how many samples were assigned
correctly (blue) and incorrectly
(orange)
Int J Legal Med (2016) 130:1557–1566 1563

Fig. 6 Number of samples with

UV-induced fluorescence above
or below 1 % in the categories
PMI < 30 years and
PMI > 30 years

11 samples (18.2 %) showed an areal extend of blue and red
fluorescent surface above 1 % (Fig. 9). Furthermore, in the
group PMI > 30 years, samples showed blue and red fluores-
cent surface or at least one value (blue or red) below 1 % in
81.8 % of the cases.
to former works, we examined the proportion of fluorescent
surface (areal extent, percent of the surface). The second test
was performed at 490 nm: a completely new method not
published before.
& Evaluation of the UV-induced fluorescence:

Discussion
The aim of our study was to determine the forensic relevant
PMI (<50 years) in human skeletal remains more precisely.
Therefore, we investigated a series of samples with known
PMI ranging from 1 to 49 years using two different methods.
In previous studies, the UV-induced fluorescence has been an-
alyzed as to its intensity and color [22, 30]. In contradistinction
Each sample had a forensic relevant PMI (<50 years) and
showed a certain areal extent of blue fluorescent surface.
Therefore, we can confirm that the existence of blue fluo-
rescence is a sign for forensic relevant PMI (<50 years)
[22, 25, 28]. We were able to show a significant but weak
correlation between areal extend of blue fluorescent sur-
face on the one hand and PMI on the other. The fluorescent
surface decreased linearly with increasing PMI. The two

Fig. 7 Results of the 490 nm-

induced fluorescence test. X-axis:
PMI in years; Y-axis: 490 nm-
induced fluorescent surface in
percent. Regression line with
determination formula and
coefficient (R2)
1564 Int J Legal Med (2016) 130:1557–1566

Fig. 8 Number of samples with

490 nm-induced fluorescence
above or below 1 % in the
categories PMI < 30 years and
PMI > 30 years

samples with the lowest PMI showed the largest fluorescent
surface, while the older samples displayed a wide variation.
The developed formula assigned 70 % of the samples correct-
ly (n = 21) and therefore proved to be a first approach to dis-
tinguish between recent and historical samples. In addition to
analyzing the linearity, we considered the blue fluorescent
surfaces above and below a cutoff value of 30 years. We were
able to show that a blue fluorescent surface above 1 % could
be an indicator for samples with PMI < 30 years (78.9 % of the
samples in group PMI < 30 years). However, the majority of
samples in the class with PMI > 30 years also showed more
than 1 % blue fluorescent surface (54.5 %). So, taken by itself,
the UV-induced fluorescence test was only of little help deter-
mining the PMI.
& Evaluation of the 490 nm-induced fluorescence:
There was a significant correlation between the areal extent
of red fluorescent surface and the PMI. The areal extend of red
fluorescent surface decreased linearly with increasing PMI.
The calculated formula assigned 60 % of the samples correctly
(n = 18). Using the cutoff value of 30 years PMI, the PMI
correlation was much stronger than in the UV-induced
fluorescence test. So, we were able to show that an areal
extend of red fluorescent surface above 1 % is a good
indicator for PMI < 30 years (84.2 % of the samples in group
PMI < 30 years). By contrast, in the group of PMI > 30 years
only 27.3 % of the samples showed an areal extend of
red fluorescent surface above 1 %. So, taken by itself, the

Fig. 9 Number of samples with

UV/490 nm-induced fluorescence
surface above and/or below 1 %
in the categories PMI < 30 years
and PMI > 30 years
Int J Legal Med (2016) 130:1557–1566
fluorescent red surface turned out to be a much more precise
method than the UV-induced fluorescence test.
In both groups there are some outliers, which have a short
PMI and a small proportion of fluorescent surface. These sam-
ples are from different cemeteries with various types of soil. A
reason could be the so-called sandwich effect. This is known
as reduction or inhibition of the UV-reflection in bones with a
short PMI due to remaining saturation with body fat [22]. On
the other hand, one sample (PMI 28 years) was very much
affected by adipocere and showed very high fluorescence.
Consequently, there have to be other factors that influence
the fluorescence in addition to the burial environment. Due
to the fact that we only had 30 samples available, the calcu-
lated formulas have a high confidence interval. To improve
the formulas, a higher number of bones have to be
photographed and analyzed.
& Combination of both methods:
Both methods combined provided the best results. Among
the samples with PMI < 30 years the majority (14 out of 19
samples) showed the combination of blue and red surface
fluorescence above 1 % (73.7 %). On the contrary, in the class
PMI > 30 years, samples showed the combination of blue and
red surface fluorescence above 1 % in just 18.2 %. Here, the
large majority of samples showed blue and red surface fluo-
rescence or at least one value below 1 % (81.8 %).
Our results are in accordance with Hoke et al. who showed
that there is a correlation between UV-induced fluorescence
color and PMI [30]. On the contrary, Hoke et al. could not find
a correlation between fluorescence intensity and PMI [30].
However, our results prove the fluorescence intensity to be
an important factor (if the fluorescence is first linked to a
special color and then analyzed by its areal extent).
It is currently uncertain by which component the fluores-
cence is caused. Most likely, the reason is collagen fibers as
recently described by Swaraldahab and Christensen [41]. In a
different context, Bohnert et al. showed that collagenous scar
tissue of the myocardium can be detected under UV light
because of its fluorescence. So, the fluorescence of collage-
nous tissue can be used to diagnose a myocardial fibrosis [42].
If collagen fibers are the reason for the measured fluorescence,
in our study as well is currently investigated.
The results are promising considering the comparatively
small sample size in our study. For more significant results,
larger test series need to be performed. In future works, human
skeletal remains with a PMI > 50 years should be included,
and 50 years should be used as the cutoff value. In our
study, the cutoff value of PMI 30 years was investigated
since we only had access to human skeletal remains with a
PMI < 50 years. Nevertheless, the surrogating cutoff value
of 30 years indicates a detectable optical change over time,
which is worth further investigations.
1565
Acknowledgments This study is supported by a grant of the
Interdisciplinary Centre for Clinical Research IZKF of Julius-
Maximilians-University of Würzburg. Thanks to Katharina Jellinghaus
for providing the bone collection. Thanks to Viktoria Rücker, Institute for
Clinical Epidemiology and Biometry, Julius-Maximilians-University of
Würzburg, for statistic advice.
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