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Brazil, February 18, 2021.

Time since deposit. Fiction or reality?


      It can be said that blood is the most common biological trace in crime scenes having several forensic applications. Blood stains are also often associated with violent crimes. In addition to visual perception of a crime by its color, bloodstains are easy to obtain genetic profiles and to interpret criminal dynamics by analyzing bloodstains profiles. As if that weren't enough, there is a new potential for the use of this trace that stands out in terms of relevance, and that every day has become a reality for forensic sciences: the time since deposit (TDD).

     TDD refers to the time elapsed from the time it forms to the time it is collected for analysis. In most cases, such an estimate is indicative of the time of the crime itself. An estimate of this nature has applicability in the validation of evidence, in the confrontation of testimonies or even in the direction of investigative hypotheses. Obtaining a valid method for estimating the chronology of crime through the bloodstain is so important that it is considered the Holy Grail of forensic science by some researchers (ZADORA & MENZYK, 2018).

      For decades, in fact, methods of obtaining this chronology have been presented by the scientific community. Bergmann et al. (2017) report the existence of studies on this topic since 1901.  In practical terms, however, results obtained so far present high standard deviations for a reliable forensic application. In this issue, the need for further study of the variations of the environment in the face of denaturation of the blood stain, and the very influence of the surface on which this stain is deposited, stand out.

      However, in recent years research with the application of different weather conditions on bloodstains added to statistical tools and spectroscopic techniques refined products have shown promising results. There are also new proposals for studying the surfaces on which the blood stain is deposited. These approaches seem to bring to light a reality seen as unattainable in the past.


          _cc781905-5cde-3194 -bb3b-136bad5cf58d_    

Understanding the interaction of hemoglobin with oxygen

      Hemoglobin is a protein contained within the red blood cells, which transports oxygen and carbon dioxide within of the human body. It is also the chromophore responsible for the red color of blood. It consists of four polypeptide subunits: two α and two β where in each of these subunits there is an organic compound called heme, a protoporphyrin with an iron atom in the center. Hemoglobin is responsible for about 93% by mass of the constituents of a dried bloodstain and is perhaps for this reason the most studied blood component for the determination of TDD.

      Within a healthy human body, hemoglobin molecules are present in two forms: oxygen-free, called deshemoglobins. , and saturated with oxygen, called oxyhemoglobin (HbO2). In the blood circulation part of the blood (1%) undergoes autoxidation into methemoglobin (Met-Hb) when it is then reduced back to deoxyhemoglobin by an enzyme, cytrochrome b5. When outside the human body, however, HbO2 becomes saturated with the oxygen contained in the ambient air. Due to the absence of the cytrochrome b5 enzyme, methemoglobin cannot be reduced to deoxyhemoglobin again, causing its denaturation to Hemicrome (HC). This denaturation has a reaction kinetics dependent on temperature, relative humidity, thickness and incidence of sunlight to which the blood stain is being subjected, and which can be measured by spectroscopy.

spectroscopic techniques

       A técnica espectroscópica funciona por meio de ondas eletromagnéticas que incidem sobre a amostra submetendo their substances at different levels of vibration, reflection and/or absorption  allowing their identification and quantification. As it is a non-invasive, low-cost method capable of interacting with the kinetics of hemoglobin denaturation, it has been used in research to estimate TDD. 

     Doty_cc781905-5cde-3194-bad_bb3b-13 . (2017) and Takamura  et al. (2019) propose the use of Raman spectroscopy in addition to statistical methods in the determination of TDD, where the denaturation of hemicromes is identified against its kinetics. Takamura  et al. (2019) analyzed the Raman spectra against the variations of three temperatures, also determining the kinetics of hemoglobin denaturation and inferring that, if the temperature is not a constant, the chronology of the spots can be estimated based on the integration of the reference equation with the time.  Other spectroscopy methods were presented in the visible, ultraviolet and infrared spectrum. All are based on the absorption, transmittance or reflectance of the bloodstain in solid or even diluted form over a range of wavelengths.

      Spectroscopy is actually the oldest recorded method in history. It was Schwarzacher (1930), for example, who first reported the influence of sunlight on the aging of bloodstains. Kind  et al. (1972) presented work based on earlier studies by Patterson (1960) and by Sch warza cher (1930). The authors measured the influences of temperature, humidity, and incident light from the experimental environment on the bloodstains, in addition to the influence of their thickness. They created an α factor that relates α (576nm) and β (541nm) absorption peaks, a kind of dimensionless comparative of hemoglobin oxidation.  Hanson et al. (2010) also present a TDD estimation marker called blue band in UV-VIS (412nm), since this band undergoes chronological intensity changes. However, many of these methods are based on the absorbance of a blood sample diluted in a solvent, which makes the method destructive and susceptible to cross-contamination, in addition to the fact that when the interaction of the stain with the surface is not considered, it is difficult to predict which was the effective area of contact with air oxygen before collection.

     Zadora & Menzyk (2018) through a more recent review of the area recognized the estimation effort in the development of recognized estimation methods TDD and indicate that advances in methodology in spectroscopy have shown promise each year. They suggest studies with greater monitoring of the kinetics of hemoglobin denaturation, and of how these variations are influenced both by the support of the blood stain and by climatic conditions. Although there are many methodologies presented to determine TDD, the fact is that non-destructive, simple, inexpensive methods that can still be used at the crime scene stand out. Nowadays, this challenge proves to be feasible as the market offers  increasingly sophisticated portable spectrometers  (Canelas Neto; Souza, 2020).

    Works of this nature have also been carried out in the chemical engineering postgraduate department of the Federal University of Santa Catarina (UFSC) using diffuse reflectance spectroscopy (ERD). This method analyzes the trace without promoting physical contact with it. The support studied are simulations of the victim's clothing. Fabrics have the ability to maintain the thickness of the bloodstain and allow a mathematical modeling of its structure and porosity, which translates into a standardization for different garments.  A temperature control chamber , relative humidity and artificial sunlight was also designed at this university seeking to simulate climatic conditions of blood denaturation in tissues with the same parameters from different crime scenes. It seeks to establish prediction statistical equations for the different climatic conditions encountered.  Another important aspect is the reflectance data obtained by reading a blood stain deposited on the tissue. These can be mathematically treated by optical physics models that take into account the influence of this surface on the denaturation kinetics, resulting in a more reliable obtaining of HbO2, met-Hb and HC contents. This is not always possible on other surfaces. The equation below represents the relationship of the reflectance of the bloody surface (R) and the bloodless surface (R0). The remaining values of the equation are the absorption coefficient (µ) in l/ in the species i medium, the concentration of species i (Ci) in g/l and the effective photon penetration coefficient (τ) in mm.



          _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194- bb3b-136bad5cf58d_           _cc781905- 5cde-3194-bb3b-136bad5cf58d_     _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_                     _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_     _cc781905-5 cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b -136bad5cf58d_           _cc781905-5cde -3194-bb3b-136bad5cf58d_     (1)


      Due to the possibility of modeling any textile structure, the use of victim's or suspect's garments as support for the chromophores of hemoglobin would present a measurable value for Ro, including through the collection of tissue adjacent to the trace (white).   The values in parentheses (λ) indicate dependence on wavelength, and the value of τ indicates dependence on the effective scattering coefficient (µs´) and total blood absorption coefficient (µa ), and which are also dependent on wavelength (λ).  Studies on the denaturation of hemoglobin chromophores (BREMMER; DE BRUIN; et al., 2011) indicate that the hemoglobin contained in Blood spots are essentially made up of the chromophores HbO2, met-Hb and HCs. We then have that the global blood absorbance µa can be equated through the expression:

          _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194- bb3b-136bad5cf58d_           _cc781905- 5cde-3194-bb3b-136bad5cf58d_     _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_                     _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_     _cc781905-5 cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b -136bad5cf58d_           _cc781905-5cde -3194-bb3b-136bad5cf58d_      (2)


     Being x1, x2, x3 the respective fractions of HbO2, met-Hcde-5 and 8 of HC9-Hb-7 3194-bb3b-136bad5cf58d_ Substituting into equation (1) we then have:


          _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194- bb3b-136bad5cf58d_           _cc781905- 5cde-3194-bb3b-136bad5cf58d_     _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_                     _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_     _cc781905-5 cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b -136bad5cf58d_           _cc781905-5cde -3194-bb3b-136bad5cf58d_       (3)

      From the literature it is possible to extract the absorption coefficients of the forms HbO2, met-Hb and HC or even to determine them in the laboratory for different wavelengths. The values of x1, x2, x3 can be obtained through the squared error of the weighted sum between the measured reflectance and the model proposed in this equation. The effective penetration coefficient (τ) can be adjusted simultaneously according to the empirical model of penetration behavior of a photon proposed by Kanick  et al.   (2009).


          _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194- bb3b-136bad5cf58d_           _cc781905- 5cde-3194-bb3b-136bad5cf58d_     _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_                     _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_     _cc781905-5 cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b -136bad5cf58d_           _cc781905-5cde -3194-bb3b-136bad5cf58d_ (4)



   Where τ is the mod is the effective penetration coefficient of the model, d is an estimated length constant in millimeters, p1,p2,p3 and p4 fit parameters of this model in order to minimize the error of the effective penetration coefficients between the model and the measurement.  The estimation of these parameters can be performed by the Levenberg-Marquardt fit algorithm.



    The study of bloodstain deposit chronology is considered one of the most desired estimates by the forensic community. Such interest is justified because the mastery of a technique of this nature allows the examiner to better estimate the reality of a crime in terms of the elapsed time, helping the criminal investigation to obtain evidence and verify testimony. The estimation in methods of measuring the time since the deposit (TDD) of the blood is similar to what legal medicine proposes with the time of death, but much more comprehensive since a bloodstain does not need the presence of a body, it does not occur. only in homicide cases, and need not necessarily be found only at the immediate scene of the crime. More than that, it can be performed by a technology that lessens the influence of the examiner's perception and/or experience. 

      The techniques related to non-destructive spectroscopy are presented as the most promising in the validation of this estimate, since they are based on mostly in the denaturing behavior of hemoglobin the main compound  of a dried bloodstain. Hemoglobin has a reaction kinetics with oxygen in the air that generates a time-dependent structural rearrangement and can be quantified for chronological estimation. With current technology, this technique opens the possibility of estimating the chronology of blood com  portable spectroscopes at the crime scene and in an analysis of a few seconds duration. These measurements take into account different conditions of temperature, relative humidity and solar incidence, which would be predicted by mathematical models previously developed for these variations added to a statistical refinement. The study of the influence of the fabric surface on the scattering of electromagnetic waves makes it possible to standardize different surfaces contained in crime scenes since the fabrics have repeated structures in the manufacture of garments, are easy for mathematical modeling and even allow the removal of blank samples.

      _cc781905-5cde-3194-bb3b-136 view of various parts of the scientific world in summary on the subject certainly in a few years what was fiction can become an unbelievable reality.


          _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194- bb3b-136bad5cf58d_           _cc781905- 5cde-3194-bb3b-136bad5cf58d_     _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_                     _cc781905-5cde-3194 -bb3b-136bad5cf58d_           _cc781905 -5cde-3194-bb3b-136bad5cf58d_     _cc781905-5 cde-3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b -136bad5cf58d_           _cc781905-5cde -3194-bb3b-136bad5cf58d_         _cc781905-5cde-3194-bb3b- 136bad5cf58d_           _cc781905-5cde- 3194-bb3b-136bad5cf58d_ Antonio A. Canelas Neto


BERGMANN, T.; HEINKE, F.; LABUDDE, D. Towards substrate-independent age estimation of blood stains based on dimensionality reduction and k-nearest neighbor classification of absorbance spectroscopic data. Forensic Science International, v. 278, p. 1–8, Sept. 2017. Available at: <>.

BREMMER, RH; NADORT, A.; et al. Age estimation of blood stains by hemoglobin derivative determination using reflectance spectroscopy. Forensic Science International, v. 206, no. 1–3, p. 166–171, 2011. Available at: <>.

CANELAS NETO, AA; SOUZA, AAU DE. Chronological evaluation of blood stains on textile fabrics via color spectrophotometry and enzymatic washing. Brazilian Journal of Police Sciences, v. 11, p. 91–111, 2020.

DOTY, KC; MURO, CK; LEDNEV, IK Predicting the time of the crime: Bloodstain aging estimation for up to two years. Forensic Chemistry, v. 5, p. 1–7, Sept. 2017. Available at: <>.

HANSON, EK; BALLANTYNE, J. A blue spectral shift of the hemoglobin soret band correlates with the age (time since deposition) of dried bloodstains. PLoS ONE, v. 5, no. 9, p. 1–11, 2010.

KANICK, SC; STERENBORG, HJCM; AMELINK, A. Empirical model of the photon path length for a single fiber reflectance spectroscopy device. Optics Express, v. 17, no. 2, p. 860, 2009.

KIND, SS; PATTERSON, D.; OWEN, GW Estimation of the age of dried blood stains by a spectrophotometric method. Forensic Science, v. 1, no. 1, p. 27–54, 1972.

PATTERSON, D. Use of reflectance measurements in assessing the color changes of aging bloodstains. Nature, v. 187, no. 4738, p. 688–689, 1960.

SUN, H. et al. Accurate Age Estimation of Bloodstains Based on Visible Reflectance Spectroscopy and Chemometrics Methods. IEEE Photonics Journal, v. 9, no. 1, p. 1–14, Feb. 2017. Available at: <>.

SCHWARZACHER, PD Determination of the Age of Bloodstains. The American Journal of Police Science, vol. 1, no. 4, p. 374–380, 1930. Available at: <>.

TAKAMURA, A. et al. Comprehensive modeling of bloodstain aging by multivariate Raman spectral resolution with kinetics. Communications Chemistry, v. 2, no. 1, p. 1–10, 2019. Available at: <>.

TSURUGA, M. et al. The Molecular Mechanism of Autoxidation for Human Oxyhemoglobin. Journal of Biological Chemistry, v. 273, no. 15, p. 8607–8615, 1998.

ZADORA, G.; MENŻYK, A. In the pursuit of the holy grail of forensic science – Spectroscopic studies on the estimation of time since deposition of bloodstains. TrAC - Trends in Analytical Chemistry, v. 105, p. 137–165, Aug. 2018. Available at: <>.

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