Stages of Decomposition: Effect of Time and Temperature

Stages of Decomposition: Effect of Time and Temperature Chapter I INTRODUCTION Watson, can you determine cause and time of death? I knelt over the woman and began a cursory examination â€Å"Rigor mortis has set in, so Id estimate shes been dead about 10 to 12 hours. Holmes stood up and brushed himself off with his hands. So, that puts her death between midnight and 2 am†(Anonymous 2007). After the question of cause of death; the question of time of death is the most sought after piece of information associated with a medical death investigation. As a consequence, death investigators find themselves in need of a means of ascertaining the period of time between when an individuals body is found and when they died, sometimes referred to as the post mortem interval. Establishing the time of death through the determination of post mortem interval may have a direct bearing on the legal questions of guilt or innocence by confirming that a suspects alibi covers the period when the victim died, or demonstrating that it does not. If the time of death can be established to within hours, days, months or even years, an individual may be able to prove that they were at some other place at that time. On the other hand, if the suspect is known to have been in the vicinity of the victim during the appropriate time period, then they can be shown to have had an opportunity to commit th e crime. Currently, there are multiple techniques for determining post mortem interval that incorporate methods in almost every discipline of forensic science. Depending on the circumstances, these techniques can yield results that vary from a narrow accurate estimate (video of the victim, the victims stopped watch etc.) to a wide range estimate (counting tree rings on trees growing over or through the remains). Regardless of the of the method used, the calculation of post mortem interval is at best an estimate and should not be accepted as accurate without considering all of the factors that can potentially impact the result. Post Mortem Interval Estimation â€Å"For everything there is a season, And a time for every matter under heaven: A time to be born, and a time to die†¦Ã¢â‚¬  Ecclesiastes 3:1-2 The techniques currently utilized for estimating post mortem interval can be broken down into two broad categories based upon the methodology used. The first of these categories are the concurrence-based methodologies. Concurrence based methods relate or compare the occurrence of a known event, which took place at a known time, with the occurrence of death, which took place at an unknown time. Examples of concurrence-based methods include the determining the years of manufacture of clothing found on a body, tree ring development, dates on personal effects, etc. Concurrence based methods rely on both evidence associated with the body, and anamnestic evidence such as the deceaseds normal pattern of movements. The second grouping of techniques include rate of change methodologies. Rate of change-based methodologies measure some aspect of a evidence, directly associated with the body, that changes at a known or predictable rate and is started or stopped at the time of death. Examples of the rate of change based methods include body temperature, tissue decomposition, insect succession and bone weathering. Some of these methodologies can be considered to fall into both categories. Examples of these would be tree ring development (Coyle, Lee et al. 2005) and insect succession. Previous post mortem interval Estimation Methods The variety of approaches for estimating post mortem interval spring from the varied expertise and experiences of their proponents as such the different methods tend to be focused on the immediate needs of the investigator, and limited to a particular stage of the post mortem interval or type of observation. As a consequence, the period of time for which a procedure is effective will overlap others. Algor, Rigor and Liver Mortis â€Å"Tis after death that we measure men.† James Barron Hope The earliest recorded methods for estimating early post mortem interval were a rate of change methodology based on the most easily observed changes. The cooling of the body after death (algor mortis), the gradual stiffening of the body (rigor mortis) and the fixed pooling of the blood resulting in discoloration of the lower portions of the body (livor mortis) can be easily assessed with minimal or in some instances no instrumentation. Since the time of the ancient Greeks when the following rule of thumb was developed: Warm and not stiff: Not dead more than three hours; Warm and stiff: Dead between 3 and 8 hours; Cold and stiff: Dead between 8 and 36 hours; Cold and not stiff: Dead more than 36 hours; (Starkeby 2004) until modern times, the basis of most temperature based post mortem interval analyses is the assumption that the human body, which averages 98.2 oF +/- 1.3 oF (Mall and Eisenmenger 2005), was at 98.6 oF (Mackowiak, Wasserman et al. 1992) at death and that after death the body looses heat in a predictable manner. There have been many temperature based methods for estimating post mortem interval. As early as the 1800s, Dr. John Davy had developed a method using the fall in body temperature (algor mortis), measured rectally, to determine the post mortem interval (Henssge and Knight 2002). This method was refined by De Saram by recording detailed temperature measurements collected from executed prisoners (De Saram G. 1955). More recent approaches to this technique have included measuring rectal temperature, body surface temperature, ear canal temperature, eye socket temperature and liver temperature (Simonsen, Voigt et al. 1977; Henssge and Knight 1995; Baccino, De Saint Martin et al. 1996; Kanetake, Kanawaku et al. 2006). Improvements to these techniques have included multiple progressive sampling, and the introduction of concepts such as the initial temperature plateau, core temperature, heat gradients, the effects of insulation, the ratio of surface area to volume, the effects of humidity and the effect of conductive surfaces, Microclimates and postmortem skin cooling (Green and Wright 1985; Nokes, Flint et al. 1992; Nelson 2000). However, most methods that attempt to use body temperature changes to determine the post mortem interval are hampered, as most methods are, by individual variability. Even when complex calculations and algorithms have been designed to model for tissue density, initial temperature distribution, post mortem exothermic reactions and heat loss, these refinements have not appreciably narrowed the estimate window for post mortem interval. Multiple studies outlining instances of initial temperature increase of a body soon after death (Hutchins 1985) associated with post mortem chemical changes such as rigor mortis, cell lysis and the conversion of cellular energy production to anaerobic respiration (Nelson 2000); variations in the core body temperature ranging from 0.5 1.2  °C during a 24 hour period (Chisholm 1911; Mackowiak, Wasserman et al. 1992); the effect of variable environmental temperatures (Green and Wright 1985; Green and Wright 1985); and the effect of environmental temperatu re on overall body surface temperatures (Mall, Hubig et al. 2002) have all contributed to limit the usefulness temperature as a consistent indicator of post mortem interval. Additionally, once the body has reached ambient temperature temperature ceases to be a factor. Marshall said it best when he said ‘‘It would seem that the timing of death by means of temperature can never be more than an approximation(Henssge and Knight 1995). Soft and Hard Tissue Decomposition â€Å"Now, a corpse, poor thing, is an untouchable and the process of decay is, of all pieces of bad manners, the vulgarest imaginable†¦Ã¢â‚¬  Aldous Huxley Cadaveric decomposition is a complex process that begins immediately following death and proceeds beyond the time when recognizable human remains have ceased to exist. Decomposition can be broken down into two major stages. The first stage, soft-tissue decomposition, is caused by autolysis and putrefaction. Autolysis is the digestion of tissue by cellular enzymes and digestive processes normally present in the organism. Putrefaction is the digestion of whole tissues systems caused by the enzymatic activity of fungi and bacteria that are either present in the organism or the environment that opportunistically invade the tissue. Both autolysis and the microorganisms responsible for putrefaction are normally held in check in living organisms. However, when an organism dies the cellular and systemic mechanisms responsible for regulating autolysis and inhibiting putrefying microorganisms stop. â€Å"Without these controlling processes the body becomes fancy (bacterial) culture media† (Carayannopoulos 1992). These early postmortem changes in soft tissues can be used to provide an estimate of the post mortem interval from death until skeletonization. However, the rate of soft tissue decomposition can be dramatically affected by both internal and external factors that affect the body (i.e. ambient temperature, cause of death, scavenging, trauma, environmental conditions, clothing, body size, mummification and adipocere formation) (Rodriguez and Bass 1985; Micozzi 1986; Mant 1987; Vass, Bass et al. 1992; Komar 1998; Campobasso, Di Vella et al. 2001). There are reported instances of rapid decomposition associated with acute illness (Frisch 2001) and the author is personally aware of an instance of a post mortem interval of less than eleven days resulting in complete skeletalization of an individual that died of complications related to Acquired Immunodeficiency Syndrome (Watson 1994). Additionally, there are a number of examples of bodies remaining intact for year s after death (Bass and Jefferson 2003). Beyond gross observation for assessing decomposition, researchers have developed multiple morphometric and chemical methods for assessing soft tissue decomposition. These have ranged from early (ca.1800s) methods such as the Brouardel method which examined the shift in flammability of putrefaction gases in the early post-mortem interval, and the Westernhoffer-Rocha-Valverde method examining the formation of crystals in the blood formed after the third day of putrefaction (Cengage 2006); to more modern methods such as ultrasound assessments of organ condition (Uchigasaki, Oesterhelweg et al. 2004) and the use of electron microscopy to examine measurable physical changes in mitochondria (Munoz, de Almeida et al. 1999) and platelet count (Thomsen, Kaatsch et al. 1999). Chemical methods used to assess time since death include the assessment of volatile organic compound formation (Vass, Bass et al. 1992; Statheropoulos, Spiliopoulou et al. 2005; Statheropoulos, Agapiou et al. 2007; Dekeir sschieter, Verheggen et al. 2009); the concentrations of non-protein nitrogen (Sasaki, Tsunenari et al. 1983; Gallois-Montbrun, Barres et al. 1988) and creatinine (Gallois-Montbrun, Barres et al. 1988; Brion, Marc et al. 1991). Bony tissue decomposition, the second major stage of decomposition, consists of a combination of surface weathering due to environmental conditions (temperature, humidity, sunlight) and erosion from soil conditions (pH, mineral content, etc.) (Behrensmeyer 1978; Janjua and Rogers 2008). While not much detailed study has been done on the environmental factors that affect bony tissue breakdown, it has been established that environmental factors such as pH, oxygenation, hydrology and soil flora and fauna can affect the long term stability of collagen (Garlick 1969; Henderson 1987; Bell, Skinner et al. 1996). Collagen, the primary protenatious component of bone, slowly hydrolyzes to peptides and then to amino acids leading to the breakdown of the collagen-mineral bonds which weakens the overall bone structure leaving it more susceptible to environmental weathering (Henderson 1987). By examining the effects of related changes (cracking, flaking, vacuole formation, UV-fluorescence of compa ct bone) the investigator can estimate the period of time a bone sample has been exposed to weathering (Yoshino, Kimijima et al. 1991; Bell, Skinner et al. 1996; Janjua and Rogers 2008; Wieberg and Wescott 2008). Current methods of assessing time since death using bone weathering rely heavily upon the experience of the investigator (Knight and Lauder 1969) and are limited to immediately post skeletalization to 10 to 100 years based on environmental conditions (Haglund and Sorg 1997). As with the assessment of soft tissue decomposition for time since death, investigators examining bone decomposition have supplemented observational methods with quantifiable testing techniques that analyze changes that are not directly affected by the physical environment (Lundquist 1963). Radiocarbon dating of carbon-14 and strontium-90 have been used to group remains pre and post 1950 (Taylor, Suchey et al. 1989; Maclaughlin-Black, Herd et al. 1992). Neis suggested that, with further study of strontium-90 distributions, determination of times since death should be possible (Neis, Hille et al. 1999). Bradley suggested that measuring the distribution of 210Pb and 210Po in marrow and calcified bone could prove forensically significant (Bradley 1993). This work was built upon by Swift who evaluated using 210Pb and 210Po distribution in conjunction with trace element analysis to provide a meaningful estimate of the post-mortem interval (Swift 1998; Swift, Lauder et al. 2001). Maclaughl in demonstrated that chemical changes due to environment could measurably affect isotope levels (Maclaughlin-Black, Herd et al. 1992). In addition to radionucleotide studies, investigators have also measured the changes in both organic (amino acids, urea, proteins, DNA) and inorganic compounds (nitrogen, potassium, sulphur, phosphorous) in bone. (Jarvis 1997; Prieto-Castello, Hernandez del Rincon et al. 2007). Stomach Contents/Rate of Digestion â€Å"Govern well thy appetite, lest sin surprise thee, and her black attendant Death.† John Milton The presence or absence of food in the stomach is often used as an indicator of post mortem interval. Its use as an indicator of post mortem interval is predicated on the assumption that under normal circumstances, the stomach digests and empties at a predictable rate taking from two to six hours to eliminate a full meal (Jaffe 1989). If a person had eaten a light meal the stomach would empty in about 1.5-2 hours. For a medium-sized meal the stomach would be expected to take about three to four hours to empty. Finally, a large meal would take about four to six hours to exit the stomach. Regardless, it would take from six to eight hours for the initial portion of the meal to reach the large intestine (Hallcox 2007). This information, coupled with reliable ante-mortem information relating to when an individual last ate is used by some pathologists when providing an estimate of the times since death. It is for this reason, among others, that comprehensive autopsies usually include an ex amination of the stomach contents (Batten 1995; Siegel 2006). Although it provides another useful indicator of time since death, there are serious limitations to the assessment of the stomach contents as an accurate indicator of time since death. Its reliance on reliable anamnestic evidence such as eating habits, the extent to which the victim chews their food (Pera, Bucca et al. 2002), the physiological state of the victim (Troncon, Bennett et al. 1994; Jayaram, Bowen et al. 1997; Lipp, Schnedl et al. 1997; Phillips, Salman et al. 1997) and the state of mind of the victim (Jaffe 1989); as well as verifiable antemortem evidence such as what the last meal consisted of (protein vs. fiber vs. fat)(Dubois 1985; Tomlin, Brown et al. 1993), the amount of liquid consumed with the meal, alcohol consumption and the time when it was consumed limits its usefulness to a small number of cases (Jaffe 1989). These factors combined with evidence that digestion can continue after death (Koersve 1951) makes the estimation of post mortem interval using stomach co ntents difficult at best. Insect Succession â€Å"Buzzards gotta eat, same as worms.† Clint Eastwood from the Outlaw Josey Wales Insect colonization of a body begins within hours of death and proceeds until remains cease to be a viable insect food source. Throughout this period, multiple waves of colonization by different insect species, as well as multiple generations of previously established species can exist. Forensic entomologists can use the waves of succession and generation time to estimate the postmortem interval based on the variety and stage of development of the insects, or insect remnants, present on the body (Archer and Elgar 2003). In addition to information regarding time since death, forensic entomology can provide useful information about the conditions to which the body was exposed. Most insects have a preference for specific conditions and habitats when colonizing a body and laying their eggs. Modifications to that optimal habitat can interrupt the expected insect colonization and succession. The presence of insects or insect larva that would typically be found on bodies colonized indoors o r in shade on a body discovered outside in direct sunlight may indicate that the body was moved after death (Sharanowski, Walker et al. 2008). Aquatic insects found on bodies discovered on land could indicate the body was originally in water (Wallace, Merritt et al. 2008; Proctor 2009). Although insect succession varies by season, geographical location and local environmental conditions, it is commonly assumed to follow a predictable sequence within a defined habitat. While there are a multitude of studies that have examined regional succession patterns (Archer and Elgar 2003; Tabor, Brewster et al. 2004; Tabor, Fell et al. 2005; Martinez, Duque et al. 2007; Eberhardt and Elliot 2008; Sharanowski, Walker et al. 2008) these studies use different approaches towards defining habitat and assessing insect succession making cross-comparisons of their data difficult. Also, the majority of these studies do not rigorously address the statistical predictability of a species occurrence making their results of limited use as post mortem interval indicators (Michaud and Moreau 2009). Additionally, beyond the presence or absence of clothing, the majority of the post mortem entomological studies conducted do not examine non-habitat external factors that may affect succession. For example, only a few studies have been conducted that assess the affect of drug ingestion (George, Archer et al. 2009) or the presence of chemicals (bleach, lye, acid etc.) used to cover-up evidence (Charabidze, Bourel et al. 2009) on the insect life cycle. As with other means of assessing time since death, more extensive studies with different insect species and drugs in a wider variety of habitats is necessary. Electrolyte Concentration â€Å"Death is a low chemical trick played on everybody†¦Ã¢â‚¬  J.J. Furnas Cellular activity does not immediately cease when an organism dies. Rather, individual cells will continue to function at varying metabolic rates until the loss of oxygen and metabolic substrates caused by the cessation of blood flow results in hypoxia (low oxygen). As cell metabolism shifts from aerobic to anaerobic, oxidative phosphorylation and ATP generation, the cellular processes keeping autolysis in check, begin to decrease and eventually cease all together. Without energy to maintain osmotic gradients membranes begin to fail. As lysosomal membranes begin to fail the enzymes within are released and begin consuming the cell from the inside out. With autolysis comes a cascade of metabolic chemicals, released ions, originally bound up in various cellular processes begin to diffuse due to the diffusion gradient according to Ficks law into the intracellular spaces (Madea 2005). Forensic researchers have used the presence, absence or effects of inorganic ions such as potassium, phos phorous, calcium, sodium and chloride as a means of estimating time since death (Schleyer and Sellier 1958). In most instances the higher the concentration gradient, the more suitable is the analyte for the estimation of the time since death. When analyzing body fluids for the purposes estimating post mortem interval, early researchers tended to focus their studies on body fluids such as, cerebrospinal fluid, blood and pericardial fluid (Schleyer and Brehmer 1958; Coe 1972; Henssge and Knight 1995; Yadav, Deshpande et al. 2007) with a few others examining other compartmentalized bodily fluids (Madea, Kreuser et al. 2001) and the largest numbers focusing on vitrious humor (Madea, Henssge et al. 1989; Ferslew, Hagardorn et al. 1998; Madea and Rodig 2006; Kumagai, Nakayashiki et al. 2007; Thierauf, Musshoff et al. 2009). Chemical methods used to assess these analytes in blood and spinal fluid as an indicator of post mortem interval have failed to gain general acceptance because, for th e most part, they failed to produce precise, reliable, and rapid results as required by the forensic community (Lundquist 1963). Current chemical methods which have primarily focused on vitreous fluid tend to suffer from the same limitations demonstrated by the fact that with notable exceptions (Pounder 1995) very few statistically rigorous field studies on the reliability and precision of estimating post mortem interval are available in the literature (Coe 1993; Madea 2005). Enzyme Activity As previously discussed, cellular activity does not cease when clinical death occurs. In any circumstances where the cellular metabolism shifts from a homeostatic balanced state to an imbalanced state biochemical changes occur. Changes in the levels and/or activity of enzymes (i.e. cardiac troponin, c-reactive proteins, and G proteins) have long been used as indicators of cellular stress (Li, Greenwood et al. 1996; Katrukha, Bereznikova et al. 1998; Tsokos, Reichelt et al. 2001; Uhlin-Hansen 2001). Assessing similar changes in cellular biochemistry as a function of time since death provides investigators with a wide variety of tissues, testing methods and analytes for consideration. As a consequence, forensic investigators have assessed and suggested enzymes from heart, pancreas, muscle, blood and brain as potentially suitable markers for time since death (Wehner, Wehner et al. 1999; Wehner, Wehner et al. 2001; Kang, Kassam et al. 2003; Jia, Ekman et al. 2007; Poloz and ODay 2009). C omparisons of total proteins analyzed ante and post mortem analyzed using two dimensional gel electrophoresis and Matrix Assisted Laser Desorption/Ionization Time-of-Flight have demonstrated changes in metabolic enzymes, (Jia, Ekman et al. 2007; Hunsucker, Solomon et al. 2008). Assessing the changes in enzyme activity provides examiners a means to assess time since death, in many instances long before visible cellular changes. However, in at least a few of these studies results indicate that enzyme degradation during extraction and partial enzyme activity observed with degradation products these markers better suited to qualitative analysis rather than quantitative analysis (Sabucedo and Furton 2003). Muscle/Nerve Excitation Both neurons and myocytes retain the ability to respond to electrical stimulation for at least a short period of time after organism death. (Sugioka, Sawai et al. 1995; Briskey, Kastenchmidt et al. 2002; Sams 2002). The response of nervous and muscle tissue to external electric stimulation has also been investigated and proposed as means to estimate time since death (Kline and Bechtel 1990; Straton, Busuttil et al. 1992). Methods developed to investigate myocyte excitability assess the relative magnitude and duration of the muscle contraction during the application of external stimulation. To assess the contractile response, a combination of observational based assessments (Madea 1990; Jones, James et al. 1995) and measurement based assessments (Henssge, Lunkenheimer et al. 1984; Madea 1992) have been suggested and reported. Similar investigations have examined post mortem excitation of nervous tissue by measuring a variety of neurological reactions to stimuli. These include the alteration of Compound Muscle Action Potential (Nokes, Daniel et al. 1991; Elmas, Baslo et al. 2001; Elmas, Baslo et al. 2002), lengthen of the refractory or non-propagating period immediately following the CMAP (McDowall, Lenihan et al. 1998), the extracellular impedance/resistance (Querido 2000), the chronaxie measurement or the time over which a current double that necessary to produce a contraction is applied before the contraction occurs (Straton, Busuttil et al. 1992) and the changes in the amplitude of the F-wave (the secondary CMAP observed after the initial CMAP) have all been examined, and been suggested as potential indicators of time since death. The results of studies examining the response of excitable tissue to electric stimulation have been consistent in that the stimulation response varies predictably over time. However, suitability for absolute indicators of time since death remains in questions as investigators have reported contradictory results related to the effect of the manner of death on the stimulation response (Madea and Henssge 1990; Elmas, Baslo et al. 2002). RNA Degradation RNA degradation, both antemortem and postmortem, is a complex process that is not well understood. Unlike with DNA degradation, continuous degradation of inducible mRNAs by native ribonucleases is used as a means of translational control. After cell death these ribonucleases, no longer kept in check by the mechanisms of cellular homeostasis, combine with exogenous ribonucleases from bacteria and fungi to begin un-inhibited digestion of all cellular RNA. Investigators have noted extensive variability in RNA degradation rates in different tissues (Bauer 2007). Not surprisingly such variability appears to be related to the antemortem ribonuclease activity of the tissue; with relatively ribonuclease poor tissues such as brain and retina exhibiting greater RNA stability (Johnson, Morgan et al. 1986; Malik, Chen et al. 2003) when compared to ribonucleases rich tissues such as liver, stomach and pancreas (Humphreys-Beher, King et al. 1986; Finger, Mercer et al. 1987; Bauer, Gramlich et al. 2003). Additionally, but also not surprisingly, some constitutively expressed mRNAs have been shown to be more stable, or perhaps simply more prevalent, than inducible mRNAs (Inoue, Kimura et al. 2002). Additionally, while intrabrain mRNA levels are fairly constant, interbrain levels vary considerably (Preece, Virley et al. 2003). As a consequence of these observations, the degradation of RNA (total and/or mRNA) have been suggested as a potential analyte to assess time since death. Researchers examining the effect of post mortem interval on RNA stability have examined a variety of targets (mRNA, both tissue specific and constitutively expressed, and total RNA) with an assortment of methods including Reverse Transcriptase (RT) PCR(Ohshima and Sato 1998; Fleige, Walf et al. 2006; Haller, Kanakapalli et al. 2006; Zhao, Zhu et al. 2006), RNA (cDNA) microarrays (Bahn, Augood et al. 2001; Catts, Catts et al. 2005; Son, Bilke et al. 2005; Popova, Mennerich et al. 2008) and quantitative RT-qPCR (VanGuilder, Vrana et al. 2008). Based on these studies, there are indications that beyond time and temperature, factors such as hypoxia, tissue pH, antemortem physiological conditions (coma, seizure activity and injury) postmortem transcriptional activity and RNA sequence can dramatically affect the stability and measurable levels of RNA (Burke, OMalley et al. 1991; Harrison, Heath et al. 1995; Ohshima and Sato 1998; Catts, Catts et al. 2005; Bauer 2007). When examining the sem inal question regarding time since death and temperature some researchers have reported temperature and time as significant factors affecting mRNA levels (Burke, OMalley et al. 1991), while others have reported the reverse (Harrison, Heath et al. 1995; Preece and Cairns 2003). These contradictory data are not surprising given the changes in the specificity, sensitivity and application of the assays used; however, the ultimate question has not been resolved. What is clear from the research is that RNA degradation (mRNA or total) is a complex process (Preece and Cairns 2003; Preece, Virley et al. 2003; Heinrich, Lutz-Bonengel et al. 2007) effected by multiple factors indicating more study will be required before RNA degradation can be considered a reliable indicator of time since death. DNA Degradation and its Effect on DNA Typing Since the initial application of molecular biology techniques to samples of forensic significance in the latter half of the 1980s, forensic scientists have noted that increased exposure to environmental insults can negatively impact DNA quality. Developmental validation studies performed to evaluate the efficacy of new typing techniques (SWGDAM 2008) have found that environmental variables such as heat, high humidity, direct moisture, fungal/bacterial contamination and ultraviolet light can impact the quantity or quality of the DNA sample making them unsuitable for DNA analysis (McNally, Shaler et al. 1989; Graw, Weisser et al. 2000; Takayama, Nakamura et al. 2003; Bender, Farfan et al. 2004; Schneider, Bender et al. 2004; Niemcunowicz-Janica, Pepinski et al. 2007). During transitions in technology from Restriction Fragment Length Polymorphism (RFLP) analysis to Polymerase Chain Reaction (PCR) based testing, researchers noted that samples too degraded to produce an RFLP pattern could still produce profiles using a variety of PCR based markers that evaluated loci shorter in length (Hochmeister, Budowle et al. 1991). This finding supports the hypothesis that degradation in the forensic setting is (not surprisingly) processive. Additional research found that while the DNA in some samples like cadaveric blood and kidney tissue could degrade to the point where it was no longer suitable for DNA fingerprinting after as little as a week (Ludes, Pfitzinger et al. 1993); other samples such as bone (Hochmeister, Budowle et al. 1991; Frank and Llewellyn 1999) and teeth (Schwartz, Schwartz et al. 1991; Pfeiffer, Huhne et al. 1999) could, under most conditions, provide typeable DNA for months. The fact that DNA degradation has a detrimental effect on larger genetic loci, and affects different tissues at different rates is considered to be of extraordinary forensic significance is evidenced by the numbers of studies that seek to examine, and overcome this effect (42 validation studies specifically mentioning DNA degradation from 1995-2009 in PubMed). This makes perfect sense when the observer considers the impact that degradation can have on selecting suitable samples and evaluating the resultant DNA profiles. However, a number of researchers have looked beyond the simple question of how degradation affects the typing of samples to broader questions such as the mechanisms of postmortem degradation (De Marà ­a and Arruti 2004; Foran 2006) and synthesis (Oehmichen, Frasunek et al. 1988) and how that knowledge can be used to assist in the assessment of time since death. DNA degradation by RFLP: Since Sir Alec Jeffreys first applied Southern blotting (Southern 1975) techniques to the testing of forensically significant samples in 1985 (Jeffreys, Brookfield et al. 1985) DNA analysis has revolutionized forensic science. Restriction Fragment Length Polymorphism DNA analysis relies on variations in the lengths of DNA fragments generated by enzyme restriction. With restriction fragments ranging from approximately from 2 33 kilobases (Baird, Balazs et al. 1986) successful typing and analysis requires high quality (un-fragmented) DNA. Researchers noted from the outset that in some cases involving older and/or postmortem samples that DNA degradation, tied to the exposures of higher temperatures, resulted in the gradual disappearance of the longer fragments reducing the evidentiary value of older samples (Bar, Kratz