Effect of Temperature and pH on Bloodstain Evidence

Kannika Sutthapodjanarux1, Nathinee Panvisavas'", and Nopadol Chaikum1,3

1 Forensic Science Graduate Programme, 2 Department of Plant Science, 3 Department of

Chemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, Thailand.

AbstractBloodstains can be successfully recovered through search techniques, so that forensic

examinations could then be performed to link the perpetrator, the victim, and the crime scene.In this study, the effect of temperature and pH on alternate light source searching (ALS), presumptivechemical tests, and DNA analysis of bloodstain were investigated. The results showed that ALS-searchfor bloodstain on two different surfaces was not affected by temperature and pH of the stain.Presumptive testing for blood, both Kastle Myer (KM) and leuco-malachite green (LMG) tests,gave negative results when the bloodstain was treated at temperature higher than 250°C.Concentrated acidic and alkali solutions did not affect the chemical presumptive tests for blood,but affected DNA analysis. However, DNA analysis results could be obtained from bloodstainstreated with O.lM NaOH and temperature lower than 250°C.

Key word: Bloodstain, Forensic examination, Temperature, pH

Introduction

Bloodstains found in the crime scenes often became crucial evidences to solve cases. Ifbloodstainsare successfully detected and analyzed, then the link between the contributor of the bloodstain. Bloodstainsin forensic science is used to identify suspects, victims and persons who at the crime scene. And the crime scenecould then be established. The science of bloodstain identification is still in high ranking in forensic sciencebase on the fact.

Uncontrolled conditions of crime scenes affect the quality of biological evidence, including bloodstains.Degradation of biological trace can be caused by heat, moisture, microorganisms, etc. Moreover, biologicalevidence can also be deteriorated by chemical insult. And bloodstains could still be recovered and identifiedwhich had never been demonstrated before to show the effecting factor of pH for forensic examination.The result will be usefulness for forensic scientist when identify suspect stain in crime scene. Here, we reportedthe effect of temperature and pH on bloodstain evidence using alternate light source (ALS), presumptivechemical tests, and DNA analysis.

Material and Methods

1. Preparation of stainsTo study the effect of temperature, 6-9 replicates of 20 ul, of human blood were spotted on two

different surfaces, i.e., cotton fabric, and tile. The blood was left overnight to dry, and treated in differenttemperatures: 1) Low temperatures at O°C, 25°C, 40°C and uncontrolled for 1 day, 1, 2, 3, and 5 weeks.2) High temperatures at 100, 150, 200, 250, 300, and 350°C by leaving the bloodstains in the oven for10 minutes.

To study the effect of pH, 0.5 ml. human blood was mixed with HCI or NaOH at 0.1,0.5, and 1 M.Then the blood of different pH were spotted on cotton fabric and tile, 6-9 replicates per surface per set wereexamined.

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2. Search and Examination of bloodstains

2.1 Bloodstain was investigated using the alternate light source (mini crime scope MC-400)at 450 nm.

2.2 Chemical presumptive tests were performed using the Kastler-Myer (KM) and Leucomalachite

Green (LMG) reactions. Untreated bloodstain and negative control were also included.

2.3 DNA analysis2.3.1 DNA extraction & quantification. DNA was extracted from the treated bloodstains using

QIAamp® DNA Mini Kit (QIAGEN, CA, USA), following the manufacturer protocol. The DNA extract wasquantified by spectrophotometry (NanoDropTM 1000 Software Version 3.6.0). The absorbance was measured

at the wavelengths of 260 and 280 nm.

2.3.2 DNAamplification2.3.2.1 STR analysis, human genotype was generated by using the AmpliSTR@ Identifiler kit

(Applied Biosystems, USA). A number of 15 STR loci and the anelogenin sex- determination marker were

analysed. PCR was carried out in a total volume of 6.25 ~L containing 2.6 ~L of AmpliSTR<XlIdentifilerPCR Reaction Mix, 1.4 ~L of each primer set, 0.1 ~lL of AmpliTaq Gold, 1.1 ~L of H20, and I ~L DNAPolymerase. The following thennocycle condition was applied; initial denaturation at incubation step at95°C for 11 min; 28 cycles of denaturing at 94°C for I min, annealing at 59°C for 1 min and extension at72°C for I min, final extension at 60°C for 60 min.

2.3.2.2 Mitochondrial DNA analysis. Human cytochrome b, 160-bp, on the mitochondrialDNA was PCR amplified. Primer sequence were Forwarded 5'-ATCTGAGGAGGCTACTCAGTAGACA-3',Reverse 5'-ATCGGAATGGGAGGTGATTCCTAGG-3 PCR was carried out in a total volume of 25 ~Lcontaining 2.5 ~L of 10X PCR buffer, 1.5 ~L of 1.5 mM MgCh, 0.5 ~L of 10 mM dNTPs, 1 ~L of eachprimer, 0.2 ~L of AmpliTaq DNA Polymerase, and 2 ng of DNA template. The following thennocyclecondition was applied; initial denaturation at 95°C for 3 min; 30 cycles of denaturing at 95°C for 30 seconds,annealing at 60°C for 30 seconds and extension at 72°C for I min, final extension at noc for 10 min.

2.3.3 Analysis of amplification products2.3.3.1 Amplification products of nuclear STRs were analysed using the AB! 310 Genetic

analyzer. PCR fragment sizes and genotype (allele types) were determined using the Gene mapper IDversion 3.1 software.

2.3.3.2 Amplification products of the human cytochrome b gene fragment were analysed byethidium bromide stained-Agarose gel electrophoresis.

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Results and discussion

1. The effect of temperatureIn this study, 2 ranges of temperatures were investigated: low (0, 25, and 40°C, and uncontrolled),

and high temperatures. Bloodstains left at low temperatures could be easily distinguished from the surfacedeposited by the naked eyes. In contrast, when bloodstains on cotton fabric were treated at hightemperatures, both bloodstains and surface became charred, thus it was difficult to distinguish the stain fromthe surface by the naked eyes. Then alternate light source at 415 nm was used to expose the treatedbloodstains. The results showed that the position of the stain could be located in all treatments, includingthe samples treated at high temperature.

Treated bloodstains were then presumptively tested with KM and LMG reagents. All bloodstainsleft at low temperature showed positive results, giving a pink and blue green color change to the KM andLMG reagents, respectively, as the positive bloodstain control. However, no color change was observedwhen the samples were exposed to temperatures higher than 250°C.

In the present of Fe2+ and phenolphthalein, LMG would be oxidized, resulting in the color change

of the chemical tests. High temperature caused oxidation of Fe2+ to Fe3

+. Therefore, no color change wouldbe observed in the absence ofFe2

+.

DNA was then extracted from the treated bloodstains for PCR-based DNA analysis. Full STRprofiles were obtained from all samples treated in low temperature for up to 5 weeks. PCR Amplification ofthe human cytochrome b gene fragment was performed to check the bloodstain samples treated in hightemperature prior to STR typing. The results showed that the 160-bp DNA fragment of human cytochrome bgene was amplified from samples treated in 100, 150, and 200°C; but not 250,300, and 350°C. Then 2 samples,one from the sample group which PCR-amplified the 160-bp cytochrome b fragment and the other from thegroup which gave no PCR- product, were selected for STR typing.

As depicted in Figure 1, partial or incomplete DNA profiles were obtained from both samples withdifferent level of allele dropout, only one genetic locus was dropped out from the partial DNA profileobtained from the bloodstain treated at 100°C, i.e. FGA. In contrast, only one allele was obtained from theDNA profile generated from the blood treated at 250°C.

The results suggested that the physical appearance of the bloodstains could be altered by hightemperatures (> 100°0, and made the observation by naked eyes difficult. Alternate light source could aid

the detection of stain in this case. Moreover, the temperature higher than 250°C could alter the bloodcomposition (heme) and destroy the genetic material, then affecting both chemical presumptive tests for

blood and DNA analysis.

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Sample A: Reference DNA profile obtained for bloodstain

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Figure.I Partial or incomplete DNA profiles

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2. The effect of pHBlood was clotted and separated from the liquid serum when mixed with HCl and NaOH

solutions. Different concentration of acidic and alkali pH bloodstains reacted similar to the untreatedbloodstain when exposed to the alternate light source at 415 nrn. (Fig. 2)

Blood mix HCI Blood mix HCI

Figure 2. The result of bloodstain samples treated with HCl (upper) or NaOH (lower)when exposed to the alternate light source at 415 nm.

However, the intensity of the stains was slightly in the control. Also positive color change whenthe stains were chemically presumptively tested with KM and LMG reagents. The acidic and alkali pHbloodstains were then subjected to PCR-based DNA analysis. The 160-bp human cytochrome b genefragment was PCR amplified from only one, sample, i.e., bloodstain mixed with 0.1 M NaOH. Threesamples were then selected for human STR analysis, one was the bloodstained mixed with 0.1 M HCI andthe other two was bloodstains mixed with 0.1 M, and 0.5 M NaOH. A partial DNA profiles were obtainedfrom the alkali pH bloodstain. Acidic conditions can cause hydrolysis of nucleic acids to their smallermolecular units (bases, sugar, and phosphate) (6).Therefore the DNA strands would then be destroyed,

resulting in no DNA profile. Alkali pH denatures DNA by disrupting the hydrogen bonding. The effect ofalkali in the pH range of 7-8, which is near the physiological range, would be more subtle on the DNAstructure (6) resulting in the presence of partial DNA profiles.

The results suggested that elevated pH of the bloodstains did not affect the search and chemicalpresumptive testing. However, because acidic and alkali pH destroyed the DNA structure, thus affectingDNA analysis of the bloodstains.

ConclusionBoth temperature and pH did not affect searching of bloodstains using alternate light source at 415

nm, but affected the downstream examinations, both presumptive testing and DNA analysis. Bloodstainstreated in temperature higher than 250°C could not be presumptively tested with KM and LMG reagents.Partial STR profiles were obtained from bloodstains treated in temperature higher than 100°C and weakalkali pH (0.) M NaOH). In conditions which temperature is over 250°C, acidic and alkali pH, no DNA

profile would be generated from the bloodstain sample.

Acknowledgments

Ms. Kannika Suthapodjanarux was supported by the Australian Federal Police (AFP) Scholarship2008

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