The Effects of Environmental Temperature Exposure of Blood

Spatter Towards Protein and Agglutinations Level in ABO Blood

Typing

Amanovitasari, D.

, Kahar, H.

, Prasetya, R.A.

, Lestari, T.P.

and Yudianto, A.

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

Clinical Pathology Department, Faculty of Medicine, Airlangga University, Indonesia

Surabaya Pharmacy Academy

Forensic Medicine and Medicolegal Department, Faculty of Medicine, Airlangga University, Indonesia

Keywords: Blood spatter, Environmental temperature, Protein level, Agglutination level, ABO Blood Typing

Abstract: The time needed for gathering evidence of a crime is an obstacle in the process of identifying the blood type

in the blood spots on the cloth. The blood will dry out after contact with the outside air within 3-5 minutes

and change the color from red to dark brown. Hence, the aim of this study was to investigate the effect of

exposure to environmental temperature on the levels of protein and agglutination in ABO blood typing of

blood spots after 0 (1 hour after blood drops on cloth), 5, 10, 15, and 20 days. Time series design was used

in this study using 30 blood spots on cotton from 1 individual subject (blood type A). Blood samples were

incubated at room temperature (23-24

C) (15 samples) and other ones were incubated at ambient

temperature (exposed to sunlight and rain). Determination of protein level was performed with a UV

spectrophotometer using trizole reagent. The agglutination level was examined by elution absorption

method using antisera A and read macroscopically. One Way ANOVA and Kruskal Wallis test were

performed in this trial and conclude that there was no significant effect to protein level (p>0.01). Based on

the trial ABO blood typing can still be performed for all blood ages (0, 5, 10, 15, 20 days).

1 INTRODUCTION

Murder cases in Indonesia tended to increase from

1,277 cases in 2014 to 1,491 cases in the next year

(BPS, 2016). Examining the crime scene, the

evidence commonly found in cases of murder is

blood spots. Blood spots on the objects around the

victim are often disguised or even removed by the

perpetrator by throwing the victim’s clothes away.

Hence, the blood spots could become vague or

unseen (Yudianto, 2013). The blood will dry out

within 3-5 minutes after contact with the outside air.

Once blood dries up, the color changes from red to

dark brown (Princess and Ketut, 2015). In the

forensic investigation, one of the most common

blood spot examinations is determining the blood

type (Knight, 2001). Blood typing can be done

quickly and cheaply; however, it can provide

accurate data to assist an investigation process

(Yudianto, 2013).

The human blood type is grouped according to

several blood typing systems. In 1900, the man who

first discovered the ABO blood typing was

Landsteiner. With the development of medical

science, many blood typing systems are found,

namely Rhesus (Rh), M and N, Kell, Duffy, and

Lewis (Knight, 2001). ABO blood type examination

of a sample of blood spots is still an important

technique for the identification of corpses in

criminal cases. Blood type examination in blood

spots can be done by direct agglutination or elution

absorption method (Nishi et al., 2005). The former

method will become more difficult when the red

blood cells lysis because of the sunlight exposure.

However, as long as the antigen in the blood spots is

still attached to the clothes, the antigen can still be

detected.

Different levels of protein in dried blood over

periods of time can be affected by several factors

such as the occurrence of microbes that lead to cell

degradation, UV exposure of the sun, and ambient

temperature. Putri and Ketut (2015) stated that the

D., A., K., K., R.A., P., T.P., L. and A., Y.

The Effects of Environmental Temperature Exposure of Blood Spatter Towards Protein and Agglutinations Level in ABO Blood Typing.

DOI: 10.5220/0007542303290332

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 329-332

ISBN: 978-989-758-348-3

329

decreasing protein level in dried blood can occur due

to the activity of microorganisms as time goes by.

Protein in the dried blood can stand up to four

months, but there is no further data regarding the

effect of environmental temperature exposure to the

dried blood. Hence, the aim of this study was to

determine the effect of the exposure on the level of

protein and agglutination in ABO blood typing of

blood spots after 0 (2 hours after blood drops on a

cloth and exposed to the room temperature), 5, 10,

15, and 20 days. The maximum exposure time of 20

days was chosen based on Article 24 paragraph (1)

of Indonesian Criminal Procedural Code, which

regulates the detention duration of a suspect in the

interest of the examination shall only be valid for a

maximum period of twenty days.

2 MATERIAL AND METHOD

This study was laboratory experiments using control

time series design. In this study, the independent

variable is defined as the exposure time of 0 days (1

hour after blood drops on the cloth), 5 days (5x24

hours), 10 days (10x24 hours), 15 days (15x24

hours) and 20 days (20x24 hours), while the

dependent variable is the protein and agglutination

level in ABO blood typing. The confounding

variable is room temperature (23–24

C) and the

ambient temperature (exposed to sunlight and rain).

Samples of 30 blood spots on cotton were obtained

from one individual subject (blood type A). Blood

samples were incubated at room temperature (15

samples) and the others were incubated at ambient

temperature. This study was conducted at Human

Genetic Laboratory Institute of Tropical Disease

Center Airlangga University Surabaya. Samples

which had been exposed to room or ambient

temperature at a given period of time were then

tested for the protein level with a UV

spectrophotometer using trizole reagent. While the

agglutination level was examined by elution

absorption method using antisera A and read

macroscopically. The data from samples incubated

at room temperature was analyzed using One Way

ANOVA, while the others were analyzed using

Kruskall Wallis.

3 RESULT AND DISCUSSION

Measurement of protein concentration using UV

spectrophotometer from sample of blood spatter of

both room (23-24

C) and climate exposure (exposed

to sun and rain) for 0 days (1 hour after blood drip

on cloth), 5th day, 10th day, 15th day and 20th day.

Shapiro Wilk normality test is performed to

determine whether the data is normally distributed or

not.

Table 1: Means of protein concentration at room

temperature. (23 – 24

Figure 1: Protein concentration graphic at room

temperature. (23 – 24

Based on Shapiro Wilk normality test, results

obtained all times of exposure significance (0 =

0.484, 5 = 0.888, 10 = 0.618, 15 = 0.502, 20 =

0.619) variables show the significance value (Sig)>

0.01, all the is normally distributed

Based on the homogeneity test, the value of

significance (sig) was 0.109> 0.01, it can be

concluded that the protein content from the sample

at room temperature (23-240C) from all times of

exposure (0, 5, 10, 15, 20 days) fulfills the

assumption of homogenity in One Way Anova test.

The results of One Way Anova test, prove that

the significance value (sig) is 0.119> 0.01.

Alternative hypothesis is rejected, it can be

concluded that there is no effect of long exposure at

room temperature (23-24

C) significantly to protein

concentration for all time variables (0, 5, 10, 15, 20

days).

ICPS 2018 - 2nd International Conference Postgraduate School

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Table 2: Means of Agglutination level at room

temperature (23-24

Shapiro Wilk normality test is performed to

determine whether there is correlation of exposure

time’s effect on the sample of blood spatter on the

cotton fabric left at ambient temperature (exposed

to sun and rain) to the protein content.

Table 3: Means of protein concentration at ambient

temperature (exposed to sun and rain)

Figure 2: concentration graphic at ambient temperature

(exposed to sun and rain)

Shapiro Wilk normality test shows all the

variable exposure times value significance (sig)>

0.01, (0 = 0.054, 5, 10 = 0.172, 15 = 0.605, 20 =

0.301). The data distribution of this research is

normally distributed.

Based on homogeneity test, the value of

significance (sig) is 0.003. Protein level from the

sample of blood spatter left at ambient temperature

(exposed to sunlight and rain) at 0, 5, 10, 15, 20 days

is heterogeneous. The assumption of homogenicity

in One Way Anova test is not fulfilled. The results

of protein concentration were tested by non-

parametric test using Kruskall Wallis test.

Kruskall Wallis test obtained significance value

(sig) of 0.833> 0.01, then null hypothesis is accepted

and alternative hypothesis is rejected, it can be

concluded that there is no effect of exposure time at

ambient temperature (exposed to sunlight and rain)

protein levels in the blood spots on the cloth left

within 0, 5, 10, 15, 20 days.

Table 4: Means of agglutination level at ambient

temperature (exposed to sun and rain)

The result show that the protein level of blood

spots exposed to the room temperature slightly

decreased over the given period of time. However, a

statistical test of One Way Anova did not show

significant difference (p > 0,01). It means that

protein denaturation process can occur at room

temperature very slowly; therefore, the decrease will

not be significant indeed.

On the other hand, the other samples, which are

exposed to the environmental temperature depicted a

fluctuation level of protein. Thus, Kruskall Wallis

statistical test yielded no significant difference

(p > 0,01), which means there is no correlation

between exposure time and the protein level of

blood spots. The uncertain environmental

temperature in the presence of exposure to sunlight

and rain can accelerate the process of decay. The

most optimal degradation process occurs at a

temperature of 70-100

F or equivalent to 21-38

At those temperatures, the chemical breakdown

process of tissue and the development of

microorganisms will help the occurrence of decay.

At temperatures below 50°F (0°C) or above 100°F

(45°C) the decomposition process becomes slower

due to inhibition of microorganism growth (Aziz,

2014).

The Effects of Environmental Temperature Exposure of Blood Spatter Towards Protein and Agglutinations Level in ABO Blood Typing

331

4 CONCLUSIONS

From the discussion above, we can conclude that the

room temperature exposure did not affect the protein

level of the blood spots significantly. Furthermore,

there is no correlation between ambient temperature

exposures to the blood spots. The blood type of the

samples can still be detected after 20 days of

exposure both at room and ambient temperature.

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