The Effect of Different Washing Treatment against Protein Quantity

and Agglutination Level in Bloodstain for Forensic Identification

Ardyan Pradana Putra

, Hartono Kahar

and Ahmad Yudianto

1,3

Master of Forensic Science School Postgraduate School, Universitas Airlangga, Surabaya, Indonesia

Department Clinical Pathology Faculty of Medicine Universitas Airlangga Surabaya

Department Forensic Medicine and Medicolegal, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia

Keywords: Agglutination level, Bloodstain, Forensic Identification, Protein Quantity, Washing

Abstract: Forensic examination of bloodstain provides important information to uncover inquired offense because

blood is easily scattered in almost all forms of violence, and its biological state has specific properties in

each person. In reality, perpetrators often attempt to conceal blood stained evidences to block revelation

during investigation by disposing, burying, burning & washing. A number of stained evidences account but

worn clothes during the incident are usually treated in either of the form, and when washed, chemicals such

as detergents and bleach solution (NaClO) are used. The purpose of this study was to investigate the effect

of washing on protein quantity and agglutination level on bloodstain in clothes for forensic identification.

This is a laboratory experimental study, in which 32 samples of bloodstained clothes were given different

treatments: tap water only, detergents and bleach solution with a control without washing. From the total of

32 samples, 16 samples were measured for protein quantity using UV spectrophotometer and 16 samples for

agglutination level. The results were then analyzed using statistical parametric One Way Anova with

significance level of 0.05. The result of statistical test obtained (p value <0.05) showing that there was a

difference between the mean of protein quantity and agglutination level on bloodstain sample in each

treatment. This research concluded that there is an effect on blood protein quantity and agglutination level

due to washing type used (tap water, detergent, and bleach solution) by its decreasing trend supported by

leaching and protein denaturation behavior.

1 INTRODUCTION

Along with the development of science and

technology in the field of molecular biology, in

efforts to solve case, especially the criminal, the law

enforcers i.e., the police, prosecutors, judges and

legal counsel would require assistance from the

experts. In accordance with their respective fields,

the experts will examine the evidence (corpus

delicti) scientifically so that the tragedy can illustrate

the case, and at the end, to determine a bright spot

about a crime act (Darmayani, 2011).

According to the data from Badan Pusat Statistik

(2016), crime rates in Indonesia fluctuate during the

period of 2013-2015. Of the many cases of crime,

murder case tops the list, followed by crime against

physical (violence). As per report of the Bureau of

Development and Operations of the National

‘MABES POLRI’, the total number of crime

incidents in 2013 was 342,084 cases which

increased to 352,936 cases in 2015. Meanwhile, the

risk of crime rate per 100,000 population is

estimated to be 140 people by 2015.

In cases of murder or violence, it is very

common to find evidence related to a crime both left

at a crime scene and attached to the body of the

perpetrator or victim. Physical evidence of body

material can be bloodstain, sperm, tissue, hair etc.

(Puspitaati et al, 2016). Forensic examination of

bloodstain provides important information to

uncover inquired offense because blood is easily

scattered in almost all forms of violence, and its

biological state has specific properties in each

person (Idries, 2013). Serological examination of

bloodstain can be done quickly and inexpensively

with a variety of methods using preference of

Absorption Elution Technique (Hoediyanto, 2012).

In reality, perpetrators often attempt to conceal

blood stained evidences to block revelation during

investigation by disposing, burying, burning &

446

Putra, A., Kahar, H. and Yudianto, A.

The Effect of Different Washing Treatment against Protein Quantity and Agglutination Level in Bloodstain for Forensic Identiﬁcation.

DOI: 10.5220/0007544704460452

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 446-452

ISBN: 978-989-758-348-3

washing. A number of stained evidences account but

worn clothes during the incident are usually treated

in either of the form, and when washed, chemicals

such as detergents and bleach solution (NaClO) are

used (Yudianto, 2013). Detergent & sodium

hypochlorite are some chemical examples and

household cleaning agents used as cleanser because

they contain surfactants, which is capable to clean

stains of dirt and blood spots, so they can dissolve in

water (Marcelisa, 2015). Based on the problem,

phenomena, and gap described above, it is important

to know the effect of different washing treatment

towards protein quantity and agglutination level on

bloodstained clothes for forensic identification.

2 METHODOLOGY

2.1 Research Design

This was a laboratory experimental study, posttest

only control group design. In which 32 samples of

bloodstain on cloth were given different treatments

which are without washing (control), washing using

tap water, detergents and bleach solution. From the

total of 32 samples, 16 samples were measured for

protein quantity using UV spectrophotometer and 16

samples for agglutination level. The result of protein

quantity measurement was then analyzed using

parametric One Way Anova with the requirement of

normal and homogeneous data, while the

agglutination level data was analyzed using

nonparametric Kruskal Wallis statistic test. This

research was conducted at Human Genetic

Laboratory, Institute of Tropical Disease,

Universitas Airlangga, Surabaya.

2.2 Materials and Method

2.2.1 Materials

Blood with blood group A (Rh+), cloth (cotton),

Detergent (active ingredient 19% Anionic

Surfactant), stain/bleach cleaner (5.25% NaCIO), tap

water & washing machine.

2.2.2 Reagent

Ether Alcohol, Anti A, tap water, 2% Red Blood

Cell Suspension, distilled water, Guanidine HCL,

Trizole reagent, Chloroform, Ethanol, Isopropanol,

1% SDS and 0.9% NaCl.

2.3 Research Procedures

2.3.1 Collection of Sample

Blood samples were obtained from a volunteer who

had blood type A (Rh+) and stored in an EDTA

vacutainer tube. Then, blood spots were made on

cotton clothes by way of blood drops as much as

300μ in each spotting. The number of blood spots on

each shirt was 2 spots of blood, 1 spot of blood for

the examination of protein level and the other one

for the measurement of level of agglutination. The

total number of blood spots formed was 32 spots.

2.3.2 Washing of Stains

The clothes were washed using a washing machine

(LG Inventer ®). The washing process was done

separately based on the shirt label. A stained labeled

shirt was not washed, for control. B labeled shirt that

has blood spots washed in a 10 liters of tap water

without detergent, C labeled clothes soaked in 10

liters washing machine that has been added 30

grams of powder detergent, while D labeled clothes

were soaked in a 10 liters of tap water which has

added 60 ml stain cleaning solution (5.25% NaCIO)

then washed with the washing machine with

automatic setting. Once the washing process is

completed, it was rinsed with tap water for 1 time.

Then all clothes were allowed to dry by hanging at

room temperature.

2.3.3 Sample Preparation

Each shirt labeled A, B, C & D that has dried after

washing process is cut into small pieces on the area

where the blood stains are marked. They were then

inserted into the test tube based on the label to read

protein content and the level of agglutination.

2.3.4 Examination of Protein Quality

The estimate of protein quantity in blood spots were

measured using a Spectrophotometer with a

wavelength of 280 nm, using an absorbance ratio of

280/260 to determine the correction factor present in

a table. The level of protein was determined by the

following formula: protein content (mg / ml) = A280

x correction factor x dilution. Finally, the protein

was isolated using Trizole reagent, Chloroform,

Ethanol, Isopropanol, SDS 1% & Guanidine HCL.

The Effect of Different Washing Treatment against Protein Quantity and Agglutination Level in Bloodstain for Forensic Identiﬁcation

447

2.3.5 Examination of Agglutination Level

Agglutination level examination was done by

soaking the pieces of cloth contained blood spots

into the reaction tube containing reagent anti A.

Then incubated at 4oC for at least 16 hours. After

the immersion, the anti-A reagent in the test tube

was removed and replaced with 0.9% NaCl.

Washing was performed with NaCl 0.9% 5 times

and centrifuged at 1000 rpm for 1 minute to remove

the rest of the reagent. After the last centrifuge,

0.9% NaCl was added and warmed for 15 minutes at

a temperature of 56oC. After the warming, 2% of

blood suspense was added and incubated at 4oC for

2 hours then shaken strongly. After the procedure,

the level of agglutination was recorded.

3 RESULT AND DISCUSSION

3.1 Bloodstain Protein Quantity Under

Varied Washing Treatment

32 formed bloodstains samples on 16 pieces of

cotton clothes spotted with blood drops of 300μ each

and washed under treatments without washing,

washing using only tap water, detergents, and bleach

solution gave results on protein quantity and

agglutination level as follows;

Protein content measurement using

spectrophotometer at UV 280 nm wavelength by

absorbance ratio of 280/260 determined the

correction factor that exist in a table. These levels of

protein were determined by the following formula:

protein content (mg/ml) = A280 x correction factor x

dilution.

Figure 1. Graph of protein content in blood spots with

different treatment

Blood protein levels shown in Figure 1 showed a

decrease in the mean difference of protein levels in

various groups, i.e. in the untreated group and the

tap water washed group. The decrease of protein

content was at 0.6 mg / ml. The group without

treatment and the detergent washed clothes was at

1.42 mg / ml; and the group without treatment with

bleach (NaClO) washed group was at 1.88 mg / ml.

Statistically; detergent leaching effect on protein

content, Anova parametric analysis responded

normally. According to the test, all data obtained

results at p value> 0.05, ie p value for protein

content based on the difference of treatment of

(0.150-0.95), this stated a normal distribution of

studied data. P value obtained, measured > 0.05.

This study variance assume homogeneous data. The

data in this study have qualified because of normal

distribution and homogeny, then statistical analysis

parametric Anova (One Way Anova) followed.

Table 1: One Way Anova parametric analysis of washing

effect on protein content

The result of statistical analysis of parametric

One Way Anova in Table 1 obtained the value of p

(sig.) = 0.001 because the p value (sig.) <0.05. Then

by this test, H0 is rejected and H1 is accepted which

means to effect of washing type on protein content at

spotting blood with UV spectrophotometer method.

Bloodstained samples under different treatments

as resulted in findings portray significant

information on the effect of washing as concealment

of evidence in forensic inquiries. Referring to blood

protein quantity shown in Figure 1, a decreased in

the mean of protein levels is observed in various

groups. The summary is given by: the group without

washing, the average protein content of 2.17 mg / ml

is presented, tap water at 1.57 mg / ml, washing

group detergent at 0.75 mg / ml, and washing group

using bleach (NaClO) at 0.29 mg / ml.

Interpretation of the findings of this study

showed that washing using bleach (NaClO) was

more likely to decrease protein levels compared to

detergents and tap water. The observed differences

ICPS 2018 - 2nd International Conference Postgraduate School

448

of protein group without treatment with washing

group using bleach (NaClO) were 1.88 mg / ml. The

group without treatment, washing group using

detergent of 1.42 mg / ml, the group without

treatment, and washing group using tap water of 0.6

mg / ml.

Decreased protein levels from the total blood

spots occurred due to washing and occurrence of

protein denaturation is due to exposure to detergent

chemicals and stain cleaning fluid. Denaturation of

proteins is the loss of higher structural properties by

disruption of hydrogen bonds and other secondary

forces required by the molecule. Factors that can

cause protein denaturation include temperature, pH,

physical factor (mechanical), and addition of

chemicals. Changes in temperature and pH can

disrupt the structure of the protein and cause loss of

function. The pH of the detergent alone > 10 also

certainly cause protein denaturation (Silverthorn,

2014).

Washing the sticking blood stains on clothes

requires three types of energy. The three energies

are: chemical energy supplied by detergent, thermal

energy provided by warm or hot water, and

mechanical energy derived from washing machine

or hand. The role of mechanical energy derived from

the movement of the washing machine causes

erasion of all blood spots sticking to the fabric. All

of these three energies must work in synergy so that

the washing process can be effective (Ophardt,

2013).

If leaching uses only water, the quantity of

protein content remaining is more than washing

using detergent because the detergent contains

chemical composition as one of its ingredients is

responsible for protein structural destruction. Similar

to Sopiah Nida (2015), the function of surfactant in

detergent is also to reduce surface tension so as to

increase the wetting power of the water, which in

return wet and erode the bloodstain, loosen and

remove the dirt and suspend the loose dirt (Sopida

Nida, 2015). Biological detergents contain enzymes

designed to hydrolyze protein molecules, and

therefore, are potential to further lower blood stains

and protein levels (Oldfield, 2017).

Tap water will not be able to remove blood spots

that have spread into the fabric fibers. In such cases,

detergent is used to remove stains spotted blood.

Structurally, protein remain active at 30oC-48oC,

above it to 50oC the biological structure will be

subjected to destruction hence denaturation process

occurs (Silverthorn, 2014). The water temperature

detergent added will usually increase. Therefore,

high temperature washing can remove traces of

blood to a greater extent than cold or lowered

temperatures (Oldfield, 2017).

Similar to previous experiments conducted by C.

Oldfield et. al. (2017), the interpretation of the

research findings showed that detergent and NaClO

are able to remove some blood traces. This finding

may be useful for investigations of offenses that

suspect the use of detergent and NaClO in

concealing the bloodstain evidence by washing

method, making it potentially useful to test

suspected items and/or perform additional analysis

on evident suspected material.

Research conducted by K.A. Harris in 2006

support this study by deriving that all of the cleaning

agents, bleach has the most damaging effect on the

quality of DNA profile obtained in blood spots.

Therefore, from the citation above; it is learnt that

the quantity measure of DNA in biological

substances treated with bleach decreases over time

due to degradation – congruent to the quality above.

This condition is not visible on the substrate being

cleaned with soap, detergent, and / or non-chlorine

disinfectant.

3.2 Bloodstain Agglutination at Varied

Washing Treatment

The result of measurement of blood group

agglutination level by using Absorption Elution

method of bloodstain on cloth in group without

treatment, washing group using tap water, washing

group using detergent and washing group using

bleach (NaClO) can be seen in table 2 below:

Table 2: Agglutination Rate Data on Blood Spot on Cloth

The Effect of Different Washing Treatment against Protein Quantity and Agglutination Level in Bloodstain for Forensic Identiﬁcation

449

Based on the result of blood group agglutination

level by using Absorption Elution method on the

blood spots in cloth, the change of agglutination

level (tend to decrease) in the group without

treatment, washing using tap water, washing with

detergent, and washing with bleach (NaClO). In the

washing with detergent and washing with bleach

(NaClO) group, there were 4 samples with negative

agglutination results.

However, to test whether there is a detergent

leaching effect on agglutination rates or not, a

nonparametric statistical analysis by Kruskal Wallis

was performed. The test resulted Kruskal Wallis’

nonparametric statistical analysis, which is portrayed

in Table 3.

Table 3. Kruskal Wallis Test for bloodstain agglutination

level

Agglutination Level

Chi-Square 13.551

Df 3

. Si

. .004

a. Kruskal Wallis Test

b. Grouping Variable: Treatments

In Table 3, p value (Asymp Sig.) = 0.004, since p

value <0.05, then H0 is rejected, and H1 is accepted.

It means that there is a detergent leaching effect on

the agglutination level in ABO blood group

determination on blood spots with absorption elution

method.

The data on blood group agglutination level in

the sample group without treatment were obtained

+4 by 100%. The group of water-washed samples

obtained +3 by 25% and +2 by 75%. The group

using detergent obtained +1 by 50% and negative by

50%. The group using bleach (NaClO) obtained +1

by 50% and negative by 50%.

Agglutination rate data in the Table 2 tended to

decrease in the trend of untreated group, wash using

tap water group, wash using detergent group, and

wash using bleach (NaClO) group. Thus, the protein

content of a bloodstain is directly proportional to the

level of agglutination. If the protein level decreases,

the agglutination rate also decreases.

As studded in the protein quantity above, the

tending effect was also contributed by the impacting

energy that directly or indirectly affects molecular

structure of protein. The force energy: chemical

energy, thermal energy, and mechanical energy are

counted several inclusive factors (Ophardt, 2013).

The other agglutinating factors (antibody-antigen

reaction), apart from those generated by friction due

to washing, are as discussed by Whitlock (2010).

they are antigen-antibody specificity complex

pairing, noncovalent bond, lock and key physical

mechanism, antigens and antibodies concentration

under prozone phenomenon, temperature, time, pH

(7.2-7.4) plus the surface net negative charge - zeta

potential surrounding red blood cells

3.3 Blood Protein Quantity and

Agglutination Level Effect

From the 16 bloodstain pairs (300μ each) made on

16 cotton pieces of cloth, one pair was assessed for

agglutination and the other pair for protein content.

The 32 blood drops (16 pairs) in total treated under

similar treatment gave corresponded reaction

according to the category of assessment. The

findings were obtained from the two factors

analyzed which was built on the potential impact.

Discussing the factors in aggregation, it conveys a

meaningful attribute of combined factors in

ascertaining the extent.

Referring to the 4 different types of treatment

used for both agglutination or protein content:

without washing (which referred as control), average

protein content was measured at 2.17 mg / ml with

+4 agglutination level at 100%. The control reaction

(without the use of treatment) portrays a set

benchmark of a 100% positive reaction to

agglutination with maximum protein content.

Washing using only tap water (treatment) present a

decreased amount of protein content to 1.57 mg / ml

with agglutination level of +3 by 25% and +2 by

75%. Detergent washing treatment resulted into a

continued lowering effect of protein content to 0.57

mg/ml when referred to control content. The

corresponding drops agglutinatinated a level of +1

and negative by 50%/50%. Washing by bleach

solution gave a protein content of 0.29 mg / ml at

agglutination level of +1 by 50% and negatively too.

In this study, the differences in washing blood

spots proved to affect protein content and

agglutination levels. There was a significant

decrease in the average level of protein in blood

spots washed using tap water, detergent and bleach.

Either washed using detergent and washed with

bleach, both give equal chance of either obtaining

agglutination at +1 or not obtaining at all (negative

agglutination reaction). The probability to

agglutination is counted and vice versa with the

content of protein (Refer Figure 1 and Table 2).

Likewise, various factors that affect the level of

protein in bloodstain will also affect the results of

agglutination examination level in bloodstain.

ICPS 2018 - 2nd International Conference Postgraduate School

450

Thereby, the aggregation of findings analyzed

from protein concentration and blood agglutination

showed a paralleling and complementing

dependency resulted by washing treatment.

This study is based in blood tissue cellular

content. Conceptually, protein content and

agglutination level determine the behavior and state

of the blood cellular composition. Focusing on red

blood cell (erythrocytes), the contained protein

integrates with the cell wall, which is antigenic. The

composition of the red blood cell membrane is 49%

protein, 43% lipids and 8% carbohydrates. This

variation in protein in erythrocytes, which results in

blood, is divided into several groups called blood

groups (Silverthorn, 2014).

Thus, each blood group has its own antigen on

the surface of the red blood cell membrane. Blood

type antigens consist of carbohydrates and proteins.

They together form glycoproteins and all are

attached to various components in the red blood cell

membrane. Protein is one of the macromolecules

that make up more than half of the cell (Silverthorn,

2014). Hence, the amount of protein contained

directly affect agglutinin reaction. Thus, the more

damaging and destruction to cellular protein, the

more it responds to weakened agglutination in order

to complicate in ascertaining and determination of

the forensic inquiry.

4 CONCLUSIONS

Forensic examination of bloodstain provides

important information to uncover inquired offenses

because blood is easily scattered in almost all forms

of violence. In this study, the difference of washing

treatment on bloodstain proved to affect protein

content and agglutination levels. There was a

gradual significant decrease in the average of protein

content and level of agglutination in blood spots

washed using tap water only, detergent, and bleach.

The decrease in the average of protein content and

the level of agglutination in this study was caused by

various factors, including mechanical factors derived

from washing machines, chemical factors provided

by detergents and bleach liquids, and thermal factors

provided by water with a detergent mixture. In

addition, the temperature factor, time, pH, and

antigens-antibodies concentration also affect the

decrease in the average protein content and the level

of agglutination in blood spots. In general, a

decreased protein content and agglutination level is

by protein denaturation and destruction due to

washing treatment and exposure to detergent

chemicals and stain cleaning fluids.

Hence, amount of protein contained is directly

affected by agglutinin reaction. Thus, the more

damaging and destruction to cellular protein, the

more it responds to weakened agglutination in order

to complicate in ascertaining and determination of

the forensic inquiry. The study opens up a new

approach to blood identification even after washed

with detergent, which could prove useful in solving

important forensic criminal cases

ACKNOWLEDGEMENTS

The authors would like to thank the technicians of

the Human Genetic of Tropical Diseases (ITD)

Laboratory of Airlangga University and all those

who have assisted in the completion of this research.

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