![Schematic depiction of the workflow used generate the results presented in this work. (a) Blood samples were taken from humans and pigs. Subsequently, human blood was treated either with or without EDTA. After the blood has been left to age for a defined amount of time, UV/VIS spectra were taken. (b) Reference spectra of oxyhemoglobin (HbO), methemoglobin (MetHb) and hemichrome (HC) were retrieved from literature ([30, 31, 32]). The reference spectra were used to separate the signals from the measured spectra into calculated pseudo concentrations (CPC).](https://www.researchgate.net/profile/Tommy-Bergmann/publication/352511961/figure/fig2/AS:1094578773991425@1637979324300/Schematic-depiction-of-the-workflow-used-generate-the-results-presented-in-this-work-a_Q320.jpg)
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Analysis of the influence of EDTA-treated reference
samples on forensic bloodstain age estimation
Tommy Bergmann∗, Christoph Leberecht, Dirk Labudde
Forensic Science Investigation Lab (FoSIL)
University of Applied Sciences Mittweida, Technikumplatz 17, 09648 Mittweida
Abstract
The age estimation of blood traces provides important leads for the chronological
assessment of criminal events and their reconstruction. To determine bloodstain
age, experimental comparative data from a laboratory environment are used.
Under these conditions the utilization of anticoagulants such as EDTA helps
to suppress the blood clotting mechanism to allow the examination over a
longer time period. This unnatural prevention of blood coagulation is highly
questionable when estimating bloodstain age, since the blood’s pysical and
chemical properties are altered. For this reason, the authors determined actual
influence of EDTA on blood spectra over time in order to formulate a statement
as to whether this effect can be measured. Human and porcine blood samples
were aged under controlled conditions. The resulting UV/VIS spectra were
separated into their individual components using signal separation techniques,
allowing the changes in the ratios of the individual hemoglobin derivatives to
be observed over time. The results show a significant influence of EDTA on the
conversion of oxyhemoglobin to methemoglobin and a minor influence on the
conversion of methemoglobin to hemichrome within the relevant time range of
5 hours to 100 hours. The use of EDTA thus slows down the aging process of
blood spots. To illustrate the great influence of EDTA, spectra of untreated
pig blood samples were included as comparison data. These show that the
difference between EDTA-treated and untreated blood samples is as great as
the difference between human blood and pig blood. As a consequence of our
findings experimental comparative data for the age estimation of bloodstains
should never result from EDTA-treated blood.
Keywords: Blood, Age estimation, Hemoglobin, UV/VIS absorbance
spectroscopy, Anticoagulants, EDTA, Criminal event, Crime scene
reconstruction
∗Corresponding author. E-Mail.: bergmann@hs-mittweida.de
Preprint submitted to Elsevier June 15, 2021
1. Introduction
The forensic analysis of the morphology (shape and arrangement) of blood
drops is called blood pattern analysis and can provide useful information about
the crime scene. This includes the type, number and direction of the violent acts
committed as well as the positions and direction of movement of objects used
and persons involved. Testimonies of accused persons and witnesses can thus
be evaluated and, if necessary, be falsified. A comprehensive and educational
review of this topic was given by Peschel et al. [1].
1.1. Forensic bloodstain age estimation
A subfield of forensic blood pattern analysis is the age estimation of blood
stains. This involves the estimation of the time that has elapsed from the creation
of a blood stain until analysis. This allows not only the evaluation of witness
statements, but also the determination of the sequence of events at the scene.
Irrelevant evidence can be excluded from the investigation process. For 120 years,
the research community has been discussing which methods are most suitable
for bloodstain age examination. Bremmer et al. provide a overview of the most
important publications in this field up to 2012 [
2
] and Sharma and Kumar up to
2018 [3].
Probably the most widely used method for forensic bloodstain age estimation
is the spectroscopic analysis of blood stain color. Blood stains found at the
crime scene are either dissolved and examined by UV/VIS spectroscopy [
4
,
5
,
6
]
or examined untreated by reflection spectroscopy [
7
,
8
]. For age estimation
characteristic peaks in the blood spectra within the visible range between 400
nm and 700 nm are used. In particular, the presence and ratio of the derivatives
of hemoglobin can be detected in the wavelength range between about 400 nm
and 700 nm. Once blood exits the human body (ex vivo) deoxyhemoglobin is
oxidized to methemoglobin. Additionally, methemoglobin is transformed into
hemichrome (see Fig. 1).
The proportions of these three hemoglobin derivatives in the ex vivo blood
thus change over time in a fixed and well-studied pattern. Under laboratory
conditions, more than half of this transformation occurs within the first 5 days
(120h) [
9
].Spectroscopy allows for the determination of these proportions and,
subsequently an estimation of the blood stain age with high precision up to about
3 weeks after its formation. This period of time is of high forensic relevance.
Other methods are precise in other time ranges [2].
The spectroscopic method is fast and inexpensive in comparison to other
methods such as the determination of the mRNA/tRNA ratio of white blood cells
[
10
], aspartic acid racemisation [
11
], and the analysis of the activity of various
enzymes such as lactate dehydrogenase and glutamate-oxaloacetate transaminase
[
12
]. Recently, we could show that the spectroscopy is independent of the surface,
if the blood samples used are measured in the solution [4].
2
Figure 1: Schematic depiction of the transformation of the hemoglobin derivatives in vivo
and ex vivo. The color of fresh blood stains change over time as a result of changes in the
concentration ratio of hemoglobin and its intermediates HbO2, metHb, and HC. This is
caused by the autoxidation of Hb into metHb which is irreversible ex vivo and the partially
denaturation of metHb into HC.
1.2. Factors influencing bloodstain aging
In addition to the surface, there are other environmental factors that can influ-
ence the aging of bloodstains ex vivo. These include, among others, temperature,
humidity and direct solar radiation. The influence of these factors is complex
and has been analyzed in several publications [
13
,
14
,
15
,
6
,
16
,
17
], but to date
there is no method that takes all factors into account. This is an important
challenge for future generations of researchers [
3
]. The use of anticoagulants also
influences bloodstain aging, but mostly intentionally. Since fresh blood begins
to clot within a minute [
18
], anticoagulants such as ethylenediaminetetraacetic
acid (EDTA), heparin or citrate are often used in medical examinations. Also
in forensic blood tests, such as DNA extraction, EDTA is usually applied to
preserve the sample as long as possible. However, this unnatural prevention of
blood coagulation is highly questionable when estimating bloodstain age, since
the natural behaviour of native human blood is distorted. To date there is
no clear evidence that anticoagulants have a significant influence on the aging
process or on the hemoglobin derivatives spectra [
2
,
19
]. Of course, forensically
relevant blood samples from crime scenes should not be treated with EDTA.
3
Nevertheless, the spectroscopic bloodstain age estimation is based on reference
samples previously prepared in the laboratory. These comparative samples could
distord the age estimation results if they were prepared with EDTA-treated
blood.
For this reason, the authors attempted to determine the influence of EDTA
on blood spectra over time.
1.3. Hemostasis and Work Mechanism of anticoagulants
Anticoagulants are used to inhibit the clotting of the blood. This transformation
of the fluid into a gel-like state is a vital body function that helps to seal injured
vessels and reduce blood loss. When treating thrombosis, it is useful to suppress
this mechanism in order to prevent the formation of dangerous blood clots.
The physiological processes that cause a bleeding to stop is called hemostasis
and is triggered by vascular injury or tissue trauma. In the presence of calcium
ions, a tissue factor forms a complex together with factor VIIa and breaks down
factors X and IX into their active forms Xa and IXa. With their help, the
prothrombinase complex can be built up, which cuts the prothrombin (factor
II) into thrombin (so called factor IIa). With the help of this highly active
enzyme, fibrinogen is broken down into fibrin monomers, which connect the
blood platelets to each other (thrombogenesis) and, thus, ensure clotting (see
Fig. 2). [20]
This thrombogenesis can be prevented by anticoagulants altering certain
pathways within the clotting cascade, inhibiting cofactors or targeting the
thrombin directly. Heparin inactivates the factors IXa, Xa and two others which
prevents the formation of thrombin. Hirudin and argatroban are direct thrombin
inhibitors, which bind either univalent (hirudin) to the active or univalent
(argatroban) to the active and passive side of thrombin. In turn, citrate binds
existing calcium reversibly which means that complexes with tissue factors can
no longer be built up. By adding calcium, this effect can be dissolved again.
EDTA works in the same way, but has an irreversible bond to calcium. Heparin,
citrat and EDTA are often used for the preparation of blood samples in the
laboratory, where rapid clotting is undesirable. [21]
1.4. Porcine blood as a reliable substitute for human blood
According to Number 6.1.2 Paragraph 1 of TRBA 100 "Schutzmaßnahmen
für gezielte und nicht gezielte Tätigkeiten mit biologischen Arbeitsstoffen in
Laboratorien", human sample materials (body fluids, tissues, cell cultures, etc.)
whose infection status has not been further characterized are to be regarded as
potentially infectious [
22
]. For this reason, work with human blood that has not
been tested for contamination is only permitted in laboratories of protection
level 2 or higher.
In many research areas, an adequate substitute for human blood had to be
found in order to be able to perform experiments with blood in environments that
do not belong to protection level 2. Similarly, when developing drugs animals
4
Figure 2: A overview of the coagulation cascade including possible points of attack for
anticoagulants. In the interaction of an intrinsic and an extrinsic pathway, coagulation factors
are activated cascade-like so that ultimately thrombogenesis can be initiated by separation by
fibrin into firbrin monomers. The different representations of the elements of the figure represent
inactive factors (thin), activated factors (bold), complexes (framed) and anticoagulants (white
writing on grey frames). EDTA inhibits the building of tissue-factor-complex which activates
Factor X. Because of that the Prothrombinase complex cant be build up which in turn cant
activate Prothrombin into Thrombin. Without this important coagulation factor the formation
of a thrombus and thus wound closure is prevented. (modified from [20]).
are use, which have to be close to humans. The substitute should be very close
to humans and at the same time easier to obtain.
In forensic experiments, pigs are often used as an effective substitute for
humans because they share many similarities in size, weight, and anatomy
[
23
,
24
,
25
]. In the case of blood, the choice is more difficult because there are
already enormous differences in composition among mammals. However, for
economic and ethical reasons, only slaughter animals, such as cattle, horses, and
pigs, can be considered. At the end of the 19th century, Abderhalden examined
blood of several mammals for their constituents (including water, hemoglobin,
protein, iron oxide, magnesium, chlorine, and inorganic phosphoric acid) and
recognized a high degree of agreement between the human blood composition and
that of omnivorous mammals, such as pigs, horses, and rabbits [
26
]. Since a great
deal of attention is paid to red blood cells and the blood pigment hemoglobin in
spectroscopic blood analysis, a commonality in the red blood cells is particularly
important. The mean absolute hemoglobin content of humans (Hb content/1
mm
3
blood) is about 32.5
·
10
−12
g. Besides the dog (24
·
10
−12
g), the pig is
the closest here with 22
·
10
−12
g (rabbit: 20
·
10
−12
g, cattle: 19
·
10
−12
g,
5
horse: 18
·
10
−12
g, goat: 7
·
10
−12
g) [
27
]. Thus, the relatively easy availability,
low cost, and clear similarities to humans suggest the use of porcine blood for
forensic experiments and, therefore, it is often used.
The three bloods used in this work are untreated human blood, EDTA-treated
human blood, and untreated porcine blood. While untreated human blood is
anatomically different from untreated pig blood, there are no external differences
between untreated human blood and EDTA-treated human blood. However,
since EDTA delays the process of coagulation, there are rheological differences
between these two bloods during the first hours of storage. The influence of this
on the spectroscopic age determination is to be investigated.
2. Methods
2.1. Blood acquisition, sample pretreatment and spectroscopic analysis
Both human and porcine blood was used for the present results. Human
blood was collected during several blood donation campaigns of the „Deutsches
Rotes Kreuz“ (DRK) from volunteers by expert personnel. This resulted in a
total of nine venous blood samples that were immediately transferred to airtight
tubes. Six of these blood samples (three female, three male) were not treated and
three of these blood samples (one female, two male) were treated with EDTA. 50
µl
of each sample where applied to strips of cotton (2
cm
x 2
cm
). This volume
was chosen because it is considered the mean drop size [
28
]. No more than two
minutes passed between blood collection and application.
The controlled aging of the bloodstains was carried out in a forensic labora-
tory. The temperature was between 21
◦
C and 25
◦
C and the relative humidity
was between 35% and 50% during the whole period. These values were con-
trolled because it is known that they have an enormous influence on the rate
of blood ageing [
29
]. Triplicates of the samples were taken at pre-defined times
and analysed by UV/VIS spectroscopy. The measuring points of the different
experiment series could not be synchronized completely due to public holidays
and weekends. On average, there were 12 measurements per experimental series
with most measuring points within the first two weeks (168
hours
). To prepare
the samples for spectroscopy, the blood-soaked cotton strips were dissolved in 100
ml
distilled water for one hour. During that time the samples were shaken twice.
The distilled water lysed the blood cells, which allowed better spectroscopic
detection of the hemoglobin derivatives [4].
Afterwards, 3
ml
of each dissolved blood sample were transferred into a macro-
UV cuvette with a diameter of 1
cm
from Rotilabo and analysed with the UV-VIS
spectrometer UV-1800 from Shimadzu. The wavelength range from 450
nm
to
650
nm
was examined with one measuring point per wavelength. This range
includes the soret band and all bands for the hemoglobin derivatives relevant for
spectroscopic analysis of blood aging: oxyhemoglobin (HbO), methemoglobin
(MetHb) and hemichrome (HC).
For the porcine blood samples blood from different individuals from a nearby
butcher shop was obtained. It was mixed and stirred but otherwise unadulterated.
6
Sample preparation for spectroscopic examination was performed as described
previously. Fig. 3 give a schematic depictionof the workflow.
Figure 3: Schematic depiction of the workflow used generate the results presented in this work.
(a) Blood samples were taken from humans and pigs. Subsequently, human blood was treated
either with or without EDTA. After the blood has been left to age for a defined amount of time,
UV/VIS spectra were taken. (b) Reference spectra of oxyhemoglobin (HbO), methemoglobin
(MetHb) and hemichrome (HC) were retrieved from literature ([
30
,
31
,
32
]). The reference
spectra were used to separate the signals from the measured spectra into calculated pseudo
concentrations (CPC).
2.2. Data Analysis
The recorded spectra were analysed in R [
33
]. Reference spectra of isolates of
oxyhemoglobin, methemoglobin, and hemichrome were used to reduce the number
of significant wavelengths for further analysis (spectral data from [
30
,
31
,
32
]).
The reduction resulted in spectra consisting of 71 wavelengths from 450
nm
to
630
nm
. To fit the data series, we also reduced the wavelengths of our recorded
blood spectra to the same 71 wavelengths. Reference spectra were grouped by
chemical compound and min-max normalized to fit the range [0,1] in order to
reduce concentration-related fluctuations of the peaks.
We assume that the majority of the absorption in the UV-VIS range of
blood results from hemoglobin and its derivatives [
9
]. Therefore, it is possible
7
to decompose the spectrum of the mixture to its individual components using
signal separation techniques. We consider the signal separated, if the residual
sum of squares:
RSS =X
λ∈Λ
(Iλ−αAλ+βBλ+γCλ)2(1)
is minimal. Where
λ
is the wavelength and Λis the set of reference wavelengths.
Iλ
is the intensity of the target spectrum at wavelength
λ
.
α
,
β
,
γ
are parameters
for optimization.
Aλ
,
Bλ
,
Cλ
are the reference spectra at wavelength
λ
for oxyhe-
moglobin, methemoglobin, and hemichrome. Broyden-Fletcher-Goldfarb-Shanno
algorithm as implemented in optimx [
34
] was used to solve the optimization
problem and predict values for
α
,
β
, and
γ
constrained to a range of [0,1]. The
predicted parameters can be thought of as pseudo concentrations where 0 and 1
are the minimal and maximal concentration of the respective compound.
In order to determine the inaccuracy of the determined values, the standard
deviation of the measured values was calculated for every measure point and the
confidence interval was derived using the formula
CI =−z∗σ
√N,+z∗σ
√N(2)
where
σ
is the standard deviation and
N
the number of samples. We used
z∗= 1.96 for a confidence level of 95%.
3. Results
A sample of the spectra recorded during this analysis is shown in Fig. 4.
The calculated pseudo concentrations (CPC ) of the haemoglobin derivatives
over time is shown in Fig. 5a.
Here a clear increase in HbO and a clear decrease in MetHb is visible as time
increases. The HC CPC increases only slightly. It can also be seen that EDTA
has an influence on the derivative ratios over time. In EDTA-treated blood
samples the HbO CPC decreases more slowly and the MetHb CPC increases
faster (Fig5a). For HC no significant influence can be identified. This EDTA-
induced difference in the CPC seems to be most pronounced between 1
hour
and 500 hours. Before and after that time the CPC -values are comparable.
The confidence intervals of the CPC per time stamp show a clear trend for
HbO and MetHb (Fig5b). The CPC of EDTA-treated blood are initially more
stable (lower) than the CPC of untreated blood, then become more unstable
(higher). At about 10h they overtake the untreated blood CPC. This trend can
no longer be observed after 500
hours
as the CPC of both blood types are again
approximately the same. The graph of the confidence intervals of the CPC of
HC is similar, however, it is not as distinct as for HbO and MetHb.
Fig. 6 illustrates the influence of EDTA on blood aging compared to porcine
blood. In addition to untreated human blood and EDTA-treated human blood,
the data of the porcine blood spectra are integrated here. While in EDTA-treated
8
Figure 4: Overview of the measured blood spectra. The age of the underlying blood is identified
through colors. Blue spectra are younger and red ones are older.
Figure 5: Results of the spectral separation analysis. The first row of plots (a) shows the
calculated pseudo concentrations (CPC) of the hemoglobin derivatives HbO, MetHb and HC
over time. The second row (b) represents the confidence intervals of the regression and the
third row (c) indicates whether the difference of the CPC within a time range of 5 to 100 hours
is significant or not. Red data points represent EDTA-treated blood and blue data points
represent untreated blood.
human blood samples the conversion of HbO to MetHb proceeds more slowly
than in untreated human blood, it proceeds more rapidly in porcine blood than
in untreated human blood. The extent of the deviations of these derivative
9
conversions is about the same for the first 100 hours.
Figure 6: Comparison of the influence of EDTA-treated human and unadulterated porcine blood.
HbO and MetHb show the biggest differences in aging. Calculated pseudo concentrations
(CPC) are shown over time. Interestingly, in porcine blood (blue, dashed line) MetHb is
converted faster than in untreated blood (blue line). In treated blood (red line) the conversion
slowed even further. The difference is most distinct in the first 100 hours of ageing.
4. Discussion
The results show that the hemoglobin derivatives of blood samples treated
with EDTA age more slowly. In the range between 5
hour
and 100
hours
blood
from the crime scene would have been predicted one hundred hours older on
average. This would be a major source of error if experimental data from
EDTA-blood was used to estimate the age of blood samples from the crime scene.
The confidence intervals show that the CPC of EDTA-blood do not scatter
strongly in the first 10 h. This could mean that the inhibition of hemostasis
also slows down the conversion of hemoglobin derivatives. Therefore, the tight
distribution of the CPC could be explained by the small real change in the
derivative components. However, in a period of time that is highly relevant for
forensic blood age estimation (about 10
hours
to 500
hours
), the CPC of EDTA
blood scatter significantly more than those of untreated blood (p < 0.001 for
Hbo and MetHb and p < 0.01 for HC). This can be explained by a temporal shift
in hemostasis and conversion of hemoglobin derivatives. Where the conversion is
fastest, the CPC also scatter the most. Even under this assumption, it is not
advisable to use EDTA-blood in the preparation of experimental comparative
data for blood ageing. EDTA-treated blood tends to be more spread from about
10 hours onwards and the aging process is delayed.
A delay in the conversion of hemoglobin derivatives of porcine blood compared
to human blood can also be deduced from the available data. The differences
in composition responsible for this are well documented [
26
]. The difference
between porcine blood and native human blood may also be due to the fact that
porcine blood is usually stirred to stop the clotting process. This means that a
large part of the fibrin network is skimmed off, which can no longer cross-link.
10
Similar to the use of EDTA, hemostasis is inhibited. Nevertheless, porcine blood
is still used as a substitute for human blood in research, but researchers are
usually aware of the dangers. Interestingly, the CPC of untreated human blood
differ about as much from porcine blood as from EDTA-blood.
The authors recommend that in future studies the influence of other coagula-
tion factors or inhibition points (see Fig. 2) should also be considered in order
to determine whether only EDTA or EDTA-like substances cause such changes
or whether the use of anticoagulants in this area is generally not advisable. A
conceivable extension of the present approach is to increase the sample size.
5. Conclusions
Researchers should be aware of the influence of EDTA addition to blood within
the first 100 hours. As mentioned above, more than half of the hemoglobin
derivative conversion processes responsible for spectroscopic bloodstain age
estimation occur within the first 5 days. As a consequence, the increased
deviation of the values should be calculated, similar to the use of porcine blood.
Although most forensic scientists are probably aware of the effects of EDTA
on blood spot aging, the danger is elsewhere. Not only the blood traces found
at the crime scene should be examined untreated, but also the preparation of
necessary comparison samples in advance must be untreated, otherwise there
can be large deviations in the blood spot age estimate. Even if EDTA is very
helpful in the examination of blood samples in many other areas, it is advisable
to do without this anticoagulant in forensic blood pattern analysis, even if this
makes the generation of data more difficult.
6. Acknowledgement
This projekt was founded by the „Sächsische Aufbaubank“ (SAB) within the
framework of the „Europäischer Sozialfonds“ (ESF).
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