Effect of Temperature and ph on Bloodstain Evidence

Effect of Temperature and pH on Bloodstain Evidence

Kannika Sutthapodjanarux

1,

Nathinee

Panvisavas'",

and Nopadol

Chaikum

1,3

1

For

e

n

sic

S

cie

n

ce

G

r

ad

u

at

e

Programm

e,

2

Departm

e

nt of Plant S

cie

n

ce,

3

D

e

partm

e

nt

of

C

h

e

mis

try

,

Fa

c

ult

y

of S

c

i

e

nce

,

Mahidol Universit

y,

Rama

V

I Road

,

Bangkok

,

Th

ai

la

nd.

Abstract

Bloodst

a

in

s

can b

e

successfully reco

v

ered through

s

earch

tec

hniques

,

s

o th

at

fo

rensic

examinat

ion

s

c

o

uld th

e

n b

e

pe

r

formed

to link the perpetrator

,

th

e v

i

c

tim

,

a

nd the

c

rim

e sce

n

e.

I

n t

h

is st

ud

y, t

h

e ef

f

ec

t

of

t

e

mp

e

rature and pH on alternat

e

light sour

ce sea

r

c

hin

g

(ALS

), pres

umpti

ve

chemi

ca

l t

e

st

s

,

a

nd DNA an

a

l

y

sis of bloodstain were investigated

.

Th

e

r

es

ult

s s

howed that ALS

-

se

ar

ch

for blood

s

tain on t

w

o different surfaces was not affected by tempe

ra

ture

and pH of th

e s

t

a

in

.

Pr

es

umpti

v

e

testing for blood

,

both Kastle Myer (KM) and leuco

-

malachite

green (LMG

)

test

s

,

ga

ve

n

eg

ati

ve

result

s

w

hen

the bloodstain

w

as treated

at temp

e

rature

higher

th

a

n 250

°

C

.

Con

ce

ntr

a

ted

a

ci

dic and alkali solutions did not affect the chemical presumpti

v

e

t

es

t

s

for blood

,

but

affe

cted DNA analysis

.

However

,

DNA analysis results could b

e

obtained

fr

om bloodstai

ns

treat

e

d

w

ith O

.

lM N

a

OH and temperature lower than 250

°

C

.

Key word: Blood

s

tain

,

For

e

n

s

i

c ex

amination

,

T

e

mperatur

e

,

pH

Introduction

Bloodstains found in the crime scenes often became crucial evidences to solve cases. Ifbloodstains are successfully detected and analyzed, then the link between the contributor of the bloodstain. Bloodstains in forensic science is used toidentify suspects, victims and persons who at the crime scene. And the crime scene

could then be established. The science of bloodstain identification is still in high ranking in forensic science

base 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, biological

evidence can also bedeteriorated by chemical insult. And bloodstains could still be recovered and identified

which had never been demonstrated before to show the effecting factor of pH for forensic examination. The result will beusefulness for forensic scientist when identify suspect stain incrime scene. Here, we reported

the effect of temperature and pH on bloodstain evidence using alternate light source (ALS), presumptive chemical tests, and DNA analysis.

Material and Methods

1

.

Preparation of stains

To 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 different

temperatures: 1) Low temperatures at O°C,25°C, 40°C and uncontrolled for 1day, 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 for 10 minutes.

Tostudy the effect ofpH, 0.5 ml. human blood was mixed with HCI or NaOH at 0.1,0.5, and 1 M. Then the blood ofdifferent pH were spotted on cotton fabric and tile, 6-9 replicates per surface per setwere

examined.

l

A

4

/'

21 CIFS

ACADEMIC DAY

2009

2

.

Search and E

x

amination 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 analysis

2.3.1 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 was quantified byspectrophotometry (NanoDropTM 1000 Software Version 3.6.0). The absorbance was measured

at thewavelengths of 260 and 280 nm.

2.3.2 DNAamplification

2.3.2.1 STR analysis, human genotype was generated by using the AmpliSTR@ Identifiler kit

(Applied Biosystems, USA). A number of 15STR 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<XlIdentifiler PCR Reaction Mix, 1.4 ~L of each primer set, 0.1 ~lL of AmpliTaq Gold, 1.1~L of H20,and I ~L DNA

Polymerase. The following thennocycle condition was applied; initial denaturation at incubation step at 95°C for 11 min; 28 cycles of denaturing at 94°C for I min, annealing at 59°C for 1 min and extension at 72°C for I min, final extension at60°C for 60 min.

2.3.2.2 Mitochondrial DNA analysis. Human cytochrome b, 160-bp, on the mitochondrial

DNA was PCR amplified. Primer sequence were Forwarded 5'-ATCTGAGGAGGCTACTCAGTAGACA-3',

Reverse 5'-ATCGGAATGGGAGGTGATTCCTAGG-3 PCR was carried out in a total volume of 25 ~L

containing 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 each

primer, 0.2 ~L of AmpliTaq DNA Polymerase, and 2 ng of DNA template. The following thennocycle condition was applied; initial denaturation at 95°C for3min; 30cycles of denaturing at 95°C for 30seconds, annealing at 60°C for 30seconds and extension at72°C for I min, finalextension at

noc

for 10 min.

2.3.3 Analysis of amplification products

2.3.3.1 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 ID version 3.1 software.

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

1/

4

CIFS 22

ACADEMIC DAY

:2009

Results and discussion

1

.

Th

e e

ffect of temperature

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

Treated bloodstains were then presumptively tested with KM and LMG reagents. All bloodstains left at low temperature showed positive results, giving a pink and blue green color change to the KM and LMG reagents, respectively, as the positive bloodstain control. However, no color change was observed

when the samples were exposed totemperatures higher than 250°C.

In the present of Fe2+ and phenolphthalein, LMG would be oxidized, resulting inthe color change of the chemical tests. High temperature caused oxidation of Fe2+ toFe3+. Therefore, no color change would

DNA was then extracted from the treated bloodstains for PCR-based DNA analysis. Full STR

profiles were obtained from allsamples treated in low temperature for up to 5weeks. PCR Amplification of

the human cytochrome b gene fragment was performed to check the bloodstain samples treated in high

temperature prior to STR typing. The results showed that the 160-bp DNA fragment of human cytochrome b

gene was amplified from samples treated in100, 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 the

group 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 with

different level of allele dropout, only one genetic locus was dropped out from the partial DNA profile

obtained from the bloodstain treated at 100°C, i.e. FGA. In contrast, only one allele was obtained from the

DNA profile generated from theblood treated at250°C.

The results suggested that the physical appearance of the bloodstains could be altered by high

temperatures (>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 blood

composition (heme) and destroy the genetic material, then affecting both chemical presumptive tests for

blood and DNA analysis.

SamD1e File Samole Name IPanel SQO ISQ

CS002BS-26-09-Q-02PM.fsa ICSOO2B lreennruer v1 10.87

I· 08S1179 B 021$11 11 07S820 11 CSF1PO 1 90 150 210 270 330 2:

t

, _I

I

Id

..

1000 I\{\

,

,

• 0

.

.

.

IT" 'i"' i! ,;;... r:;- r-; .4 _'0 .•. 11.2 ! [zs

J

[

m

J

w~

11

~

[204.60 227.63 263.50

~

1.3 [32.~ 1.1 11 . 144.79 218.32 275.50 .E§:.921

CS002B5-26-09-Q-02 PM.fsa CSOO2B Identifiler v1 0.87

1 03S1358 1 1 THOl ₁₁ 0135317 11 016S539 _I 1 0251338 I 90 150 210 270 330 2:

t

R

"

I , I , 1000 .1

I

.

0

•

.

</\

.,

J\. •I&' I r-: .•.

.

•

.

.

.

[i4 ' ·9 ~~5.321 _1;6~d~ IOL 1~q~611 183.74 '324.55 _J_ I --L _-=1 -.-18 9 OL 12 I OL 13,6-6:7 1~3.74 '227.63,. 280.6S: 324.55 CS002B5·26-09-Q-02 PM.fsa ICSOO2B IIdentifiler v1 I !O.87 1 019S433I ₁ VVVA ₁₁ TPOX ₁₁ 018S51 1 90 150 210 270 330 2~t

I

1000

1

\

_" I c-: 'I' " ",I' I

.,

, 0 _..--' --14

I

T

g

-

~

1

17 :1.2~.72 175.64 230~ ;30~.88 [;9.4;:]

-=

14 ~.181 17 L..!ll.72 303.88 C5002B5·26-09'{;-02 PM.fsa CSOO2B Identifiier v1 0.87 0 1 05S818 I

c=

FGA 1 90 150 210 270 330 2~t ;, il

I

1000

'

"

~

I

,

1\/\ .:11 f I 0

l

~

_

r~~6.151 122207'.63J

~

~

I

2755.6~

i

J li13.~ -~~-L -23' 1160.48. 239.38

Sample A:Reference DNA profile obtained for bloodstain

., ft,

In

¥

,

23 CIFS

ACADEMIC DAY

s.~ I.,..ne s.~ •Na.ne ,Panel ISQC) se> B"OOCf5-10-09--12_34 PM.'sa Bl00C IIdentifiler v., 10.SS I 0851'79 11 021511 I I 07S820 IC:==CSF1PO I '000 '00 ,SO 200 250 300 3SO 500~ I

11

III ~I A

j!

•

A

_•

_~

.

I

0

~~-qg

J

!

;~

.

4

~

!

1

1

~6

3.

,

J

I

~

i~

_

.~

[

~!4

.

s4

1

!

32

.

2

I

_I

_g

S

_.

₄₉

_]

_r

_~i~

_.

₇₄

₎

21B.1Sj el00C8-10-09_12_34 PM.'_ Bl00C tIdentifiler vt O.Sg IlS 03513$8 I I THO, 11 0135317 !I 0165539 I I

.

0251338 I '000 '00 -eeo 200 2SO 300 3SO

"",:1

0 A

~

~

A A AA

I

J ~6B.43J 1

14

9

10

OL 120.24 1~~.S2, 22_~_OB 32.9.~~

18

9

10

1

2

'·OL :1.36.34 183.52 225.08 280.45 324.4:1. e"100C6-10-09-12-34 PM.f_ IBl00C Identifiler vl 0.89 I 0195433 I I v'NA I1 TPOX "] I 018551 I

':

:

1

vco 'so 200 2SO 300 3SO

.

B

• •

4 ~

••

t

• 4

I

~. 9

r

'I!

I~,

.

I

14

_l

_l

_{_}

_~~.1

_~

_1~30.011

'

~~S

';

b

121.57

~

=

~

J

~:~~o1

1

14

17

_L

OL 121.57 179.23 303.64 -e100C6-10-09-12_34 PM.f_ Bl00C Identifiler vl 0.89

c:::

J

I 055818 I I FGA I

':

:

1

'00 '50 200 250 300 350

[

~'"

•

~~

.

.

'. I .": I I

III

•

I

~q~.1§J

1

~~~9~

y

13

112.73J ,160.24...J

Sample B:DNA profile obtained for bloodstain treated at ]00°C

Sam leFile Isam eName IPanel 500 ISQ

[ldentiliter v1 0.89 021S11

1

1

07S820

11

CSF1PO 250 35_,0 B250T6-1 0-09-11-59 AM.fsa B250T wo08S1179 100

Sample C:DNA profile obtainedfor bloodstain treated at 250°C

Sample A:Reference Amel 05 FGA 08 021 07 CSFI 03 THO) 013 016 02 019 Vwa TPOX 018 ONAprofile X 12 23 12 29 8 11 14 9 10 9 18 14 i6 8 17 Y 13 27 13 32 11 11 18 9 10 12 19 14 17 8 17 Sample B: DNA X 12 11 29 8 11 14 8.3 10 9 18 13 15.2 8 14.2 profile lOOQC Y 13 13 32 11 I1 17 8.3 10 12 19 13 17 8 17 Sample C: DNA X profile 250QC Y 13

Figure.I Partial or incomplete DNA profiles

~4

l'

CIFS

ACADEMIC DAY

-&009

2. The effect of pH

Blood 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 untreated bloodstain 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 when

the stains were chemically presumptively tested with KM and LMG reagents. The acidic and alkali pH

bloodstains were then subjected to PCR-based DNA analysis. The 160-bp human cytochrome b gene fragment was PCR amplified from only one, sample, i.e., bloodstain mixed with 0.1 M NaOH. Three samples were then selected for human STR analysis, one was the bloodstained mixed with 0.1 M HCI and

the other two was bloodstains mixed with 0.1 M, and 0.5 M NaOH. A partial DNA profiles were obtained

from the alkali pH bloodstain. Acidic conditions can cause hydrolysis of nucleic acids to their smaller

molecular units (bases, sugar, and phosphate) (6).Therefore the DNA strands would then be destroyed,

resulting in noDNA profile. Alkali pH denatures DNA by disrupting the hydrogen bonding. The effect of alkali in the pH range of 7-8, which is near the physiological range, would be more subtle on the DNA structure (6)resulting inthe presence of partial DNA profiles.

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

DNA analysis of the bloodstains.

Conclusion

Both temperature and pH didnot affect searching of bloodstains using alternate light source at 415 nm, but affected the downstream examinations, both presumptive testing and DNA analysis. Bloodstains

treated 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 weak alkali 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

t

i

4

l'

25 CIFS

ACADEMIC DAY

d

4

Y

CIFS 26

ACADEMIC DAY

2009

Reference

1. William D. Haglund, Marcella H. Sorg Forensic Taphonomy: The Postmortem Fate of Human Remains.

Danvers: CRC Press,cl996

2. Nicholas Vandenberg, B.Sc. (Hons) and Roland A. H. van Oorschot, Ph.D. The Use ofPolilights in the Detection of Seminal Fluid, Saliva, and Bloodstains and Comparison with Conventional Chemical-Based

. .

.

Screening Tests. 2006 March; 51(2):361-370

3. Shanan S.Tobe, M.Sc.; Nigel Watson, Ph.D.; and Niarnh Nic Dae'id, Ph.D.Evaluation of Six

Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA. 2007 Jan; 52(1):102 - 109

4. T. Spear and N. Khoshkebari, California Criminalistics Institute (CCI), Sacramento, CA. Analysis of Old Biological Samples: A Study on the Feasibility of Obtaining Body Fluid Identification and DNA Typing Results. 2001: 1-5

5. Richard Saferstein, Ph.D. Forensic Science Consultant, Mt. Layrel, New Jersey Lecturer, Widener University School of Law. Criminalistics: An Introduction to Forensic Science.8 th ed. Upper Saddle River; N.J: Prentice Hall, c2004

6. Stuart H. James, Jon J.Nordby. Forensic Science: An Introduction to Scientific and Investigative Techniques. Boca Raton, Fla: Taylor & Forensic/CRC Press, c2005

7. P.C. Turmer, A.G.Mclennan, A.D.Bates & M.R.H.White. Molecular Biology. Second edition: 38-44 8. Kenneth S. Saladin, Robin K. McFarland. Human Anatomy. Boston: McGraw-Hill Higher Education,