Laten Blood Detection Test Sensitifity of Lizard (Varanus salvator)

Using Leuco Malachite Green (LMG) with Different Dilution

Farah Aidah Nurreza

, Bilqisthi Ari Putra

, Nadia Yohana

and Djoko Legowo

Forensic Science, Postgraduate School Airlangga University, Surabaya, Indonesia 60115

Veterinary Pathology Department, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia 60115

Keywords: Forensic, Forensic veterinary, Lizard, Blood Pattern staining, Leuco Malachite Green, LMG

Abstract: This study was undertaken to investigate reaction sensitivity blood detection Leuco Malachite Green (LMG)

of Varanus salvator’s blood. Forensic veterinary may be requested in both criminal and civil cases. Blood

tests at crime scene can provide useful information for the investigation process. Indonesia is home to several

varanid species. Varanus salvator known as water lizard. This animal is an endemic lizard species of

Indonesia. Leuco Malachite Green is one of latent blood staining method that commonly used to detect blood

pattern on the crime scene or tools. The aim of research is to compare the sensitivity of LMG reaction to each

different blood varanus salvator dilution (10

, 10

). 35 total samples were used in this study.

The staining results show different sensitivity to different dilutions. The higher the dilution rate becomes less

visible.

1 INTRODUCTION

Blood tests at crime scene can provide useful

information for the investigation process (Idries et al,

2011). Blood is the most common and perhaps the

most important form of evidence in today's criminal

justice world (Tobe et al, 2007). An investigator can

interpret certain basic patterns at the scene, such as

drag marks, smears, or blood trails. It is important to

keep in mind that an injured animal may be mobile

and may shake his head or body, causing spatter

(Merck, 2007). Mature reptile erythrocytes are

generally larger than bird erythrocytes or mammals.

The reptile erythrocytes are ellipsoid cells with an

oval or rounded middle core. The color of the

cytoplasm is more pink uniform with the staining of

Wright's Stain (Bijanti et al, 2010).

Presumptive tests for blood utilize a variety of

chemicals to identify the presence of blood through a

reaction with the haemoglobin molecule (Spalding,

2006). They are described as presumptive because

there are substances other than haemoglobin which

may cause a false positive reaction and in forensic

settings further testing is required to confirm the

result. These are rapid tests that are used to identify

whether an unknown substance is likely blood and to

identify areas of a crime scene that should be

investigated in more detail. The benefit of utilizing

these tests is the rapidity of results and the ease of

interpretation (Colotelo, 2009). Leuco Malachite

Green (LMG) is widely used for presumptive testing

in casework at crime scene investigations and in the

laboratories. A presumptive test will indicate if a

biological substance such as blood is present in a stain

found (Andersson, 2017). Hemoglobin makes up the

greater share of the solid content of the red blood cell

(Dessauer, 1970). Heme-reacting chemicals react

with the heme group in haemoglobin present in blood.

These chemicals, also known as peroxidase reagents,

are colourless dyes that are oxidised to form a

coloured product. The reaction between LMG and

blood results in a green colour (Farrugia et al, 2010).

Research on blood detection in animals that

have similarities with reptiles has already been done

on fish blood using fluorescein, Bluestar©,

phenolphthalein, Hemastix®. The result conclude

that fluorescein was found to be the best (i.e., low rate

of false positives, detected highest proportion of true

positives). Based on this information, fluorescein was

investigated further to refine its application in

fisheries research. Using fluorescein, injuries could

be detected up to 5 hours after the injury occurs and

once fluorescein is applied, there is significantly less

detectable fluorescein after one hour (Colotelo,

2009). The research on reptil’s blood using LMG to

Nureza, F., Putra, B., Yohana, N. and Legowo, D.

Laten Blood Detection Test Sensitiﬁty of Lizard (Varanus salvator) Using Leuco Malachite Green (LMG) with Diffrent Dilution.

DOI: 10.5220/0007542903610364

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 361-364

ISBN: 978-989-758-348-3

361

see the quality and reaction results has never been

done before.

2 MATERIALS AND METHODS

Materials:

LMG, H

, Filter paper, Pipette, Reptile’s blood,

container, Aquades, EDTA.

Forensic Photography:

Photographs are taken in close-up

(photomacrography) of the labeled bloodstain to give

an interpretation of the using a Digital Single-lens

Reflex (DSLR) Camera and 50mm fixed lens

Sample:

Lizard’s blood (Varanus salvator) was taken and lied

in EDTA tube. Lizard’s blood is placed on the ice box

and brought to pathology laboratory, Faculty of

Veterinary Medicine, Airlangga University.

Blood Dilution:

The blood was diluted with aquadest with dilution 10

, 10

-2

, 10

-3

, 10

-4

and 10

-5

Figure 1: blood dilution scheme.

Reagen Test and Interpretation

Adapted from

Andersson (2017), The LMG solution

was prepared by adding 150 ml of aquadest and 100

ml of concentrated acetic acid to 1 g of Leuco

Malachite Green into a brown chemical bottle. The

solution was stirred until all the Leuco Malachite

Green was dissolved and the solution was filtrated.

The prepared LMG solution was afterwards

transferred from the brown chemical bottle with LMG

solution into a smaller separate tube. A 30 % H

(MERCK®) was diluted with deionised water to a 10

% H

solution and the H

was then transferred to

a smaller separate tube. A volume of 25 μl of LMG

solution and 25 μl of H

solution was used

throughout this study. When not in use, all solutions

where kept in a refrigerator.

Methods:

Leuco Malachite Green (LMG) and H

were

prepared according to Manufacturer guideline and

Forensic Laboratory of Indonesian’s Police

Department as well. All reagents were used according

to the manufacturer’s guidelines. Positive controls

were taken by applying the reagent to a bloodstained

piece of ﬁlter paper. Negative controls were

performed by applying the reagents to a fresh piece of

ﬁlter paper with no trace of blood.

Sensitivity Testing Autoclaved bottles and

distilled H

O were used. Water was measured using a

graduated cylinder and blood was added using a

Gilson pipette. Based on Webb et al (2006), differing

low concentrations of blood were achieved by making

a stock solution of blood and distilled water.

Solutions of 10

-1

, 10

-2

, 10

-3

, 10

-4

and 10

-5

were

prepared. A set of 35 3cmx3cm pieces of ﬁlter paper

were placed in each of the diluted blood solutions for

each of the presumptive reagents tested. The pieces of

ﬁlter paper were then removed and allowed to dry for

10 minutes. Each of the pieces of ﬁlter paper was then

tested with its corresponding reagent to see whether

the blood present was detectable. The time taken for

the reagent to register a positive result was

determined and recorded. Tests were considered

negative if reagents failed to react within 4min of

exposure to the blood-stained ﬁlter paper.

ICPS 2018 - 2nd International Conference Postgraduate School

362

3 RESULT AND DISCUSSION

Figure 2: LMG reaction toward lizard’s blood dilution.

Sensitivity

Blood

Dilution

-1

-2

-3

-4

-5

Lizar

+ + + + -

The table above shows that LMG in lizard’s blood can

still react to dilution 10

-4

Descriptive

Mean SD Min. Max.

Lizar

35 1.743 1.6688 .0 5.0

Quality

Kruskal-Wallis Test

Dilution N Mean Rank

Lizard 10

-1

7 32.00

-2

7 25.00

-3

7 17.71

-4

7 8.29

-5

7 7.00

Total 35

All the data was analyzed by Kruskal-Wallis test with

significancy p<0.05 for all groups.

The result of this study shows that different

dilution resulted on different interpretation of lizard’s

blood. Dilution of 10

-1

, 10

-2

,10

-3

and 10

-4

obtained

positif (+) result and 10

-5

obtained negative (-) result.

Hemoglobin concentration can affect the quality

of the LMG test reaction. At the higher dilutions it

causes a decrease in hemoglobin concentration.

Based on Gul and Tosunoglu (2011), the amount of

hemoglobin lizard ranges from 8-13 g/dL (Mean:

10.54 g/dL). It is estimated that hemoglobin can no

longer be detected by LMG at dilutions of more than

-4

, but can be re-examined in the narrower range

between 10

-4

to 10

-5

4 CONCLUSIONS

The conclusions of this study show that the level of

dilution affects the level of sensitivity of LMG test to

blood Varanus salvator. Sensitivity LMG in lizard’s

blood can still react to dilution 10

-4

ACKNOWLEDGEMENTS

The author would like to thank Bilqisthi Ari Putra,

DVM, Djoko Legowo, DVM and Nadia Yohana,

DVM as my partner team for this research.

REFERENCES

Andersson, A., 2017. An Evaluation of Two Presumptive

Blood Tests and Three Methods to Visualise Blood.

Linköping University.

Bijanti, R., Yuliani, M.G.A., Wahjuni, R.S and Utomo,

R.D., 2010. Patologi Klinik Veteriner. Airlangga

University Press. Surabaya.

Colotelo, A.H., 2009. Evaluation and Application of

Presumptive Tests for Blood for Fish Epithelial Injury

Detection. Carleton University Ottawa, Ontario.

Dessauer H. C., 1970. Blood chemistry of reptiles:

physiological and evolutionary aspects. In Biology

ofthe Reptilia (Edited by Gans C. and Parsons T. S.),

Vol. C, pp. l-72. Academic Press, London.

Farrugia, K.J., NicDaéid, N., Savage, K.A., Bandey, H and

Ciuksza, T., 2010. Chemical enhancement of footwear

impressions in blood on fabric — part 2: peroxidase

reagents. Elsevier.

Gul, H. And Tosunoglu, M., 2011. Hematological

Reference Intervals of Four Agamid Lizard Species

from Turkey. Herpetozoa Journal.

Idries, A.M and Tjiptomartono, A.L., 2011. Penerapan

Ilmu Kedokteran Forensik Dalam Proses Penyidikan.

Jakarta: CV Sagung Seto.

Marck, M.D., 2007. Veterinary Forensics Animal Cruelty

Investigations, Blackwell Publishing. USA, 1

edition.

Spalding, R.P., 2006. The identification and

characterization of blood and bloodstains. In: James

SH, Nordby JJ (eds) Forensic science: an introduction

to scientific and investigative techniques, 2nd edn. CRC

Press, Boca Raton, FL, p 237–260

Tobe, S.S., Watson, N., and Dae’id, N.N., 2007. Evaluation

of Six Presumptive Tests for Blood, Their Specificity,

Sensitivity, and Effect on High Molecular-Weight DNA.

Journal Forensic Science. Vol. 52; p: 102-109.

Laten Blood Detection Test Sensitiﬁty of Lizard (Varanus salvator) Using Leuco Malachite Green (LMG) with Diffrent Dilution

363

Webb, J.L., Creamer, J.I. and Quickenden, T.I., 2006. A

comparison of the presumptive luminol test for blood

with four non-chemiluminescent forensic techniques.

University of Western Australia

ICPS 2018 - 2nd International Conference Postgraduate School

364