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 Table of Contents  
Year : 2020  |  Volume : 12  |  Issue : 3  |  Page : 275-279

Blood group analysis from cigarette butts by absorption inhibition method: An experimental study

1 Department of Forensic Odontology, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia
2 Diploma 3 Technology Laboratory Medic, Institut Ilmu Kesehatan Bahakti Wiyata, Kediri, Indonesia

Date of Submission29-Aug-2019
Date of Decision13-Dec-2020
Date of Acceptance19-Dec-2019
Date of Web Publication02-Jun-2020

Correspondence Address:
Prof. Mieke Sylvia Margaretha Amiatun Ruth
Department of Forensic Odontology, Faculty of Dental Medicine, Universitas Airlangga, Jalan, Moestopo 47, Surabaya 60132.
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JIOH.JIOH_219_19

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Aim: In red blood cells (RBCs), antigens such as A, B, D, and H, are found to be present. The antigens are secreted in various body secretions such as semen, sweat, amniotic fluid, saliva and also blood. At the crime scene, saliva samples are very valuable, which can be collected from dry or wet cigarette butts. Blood group antigens in saliva can be found by absorption inhibition method. This study aimed to investigate blood group antigens on the cigarette butts based on time. Materials and Methods: Eighteen cigarette butt samples, used in accordance with the inclusion criteria, were placed at room temperature for 1, 3, and 6h. The estimation of blood group antigens on the cigarette butts was carried out by absorption inhibition method. Results: All samples indicated the presence of antigens in the ABO blood group. Blood group antigens in saliva on cigarette butts, after incubation from 1h until 6h, could be examined by absorption inhibition method (100%). Conclusion: Cigarette butt samples that contain saliva can be used for forensic investigations in narrowing down the suspect pool by blood group determination.

Keywords: Absorption Inhibition Method, Blood Group, Cigarette Butts, Saliva

How to cite this article:
Ruth MS, Purnadianti M, Marini MI. Blood group analysis from cigarette butts by absorption inhibition method: An experimental study. J Int Oral Health 2020;12:275-9

How to cite this URL:
Ruth MS, Purnadianti M, Marini MI. Blood group analysis from cigarette butts by absorption inhibition method: An experimental study. J Int Oral Health [serial online] 2020 [cited 2021 Jan 16];12:275-9. Available from:

  Introduction Top

Forensic identification is an investigator’s attempt to reveal the victim’s identity or the perpetrators of a crime. In the evidence of cigarette butts found in the murder case that occurred on May 14, 2013, in Bangkingan Madura, East Java, Indonesia, the habit of smoking patterns, what brands are consumed, lip prints, fingerprints, and even saliva leftovers could be observed.[1] The remaining saliva left on the cigarette butts can be used as a blood type examination specimen, which is a secondary identification component.[2] Saliva will dry out at room temperature within 1h and 40 min, and it will dry out in less than 3h with the help of various other factors.[3] To assist in the process of identifying a criminal case with the evidence in the form of cigarette butts, it is necessary to determine the blood type using saliva examination.

Blood group has an important place in genetics, immunology, anthropology, clinical medicine, and forensic medicine, as a major part of forensic investigations, and can be used in narrowing down the suspect pool.[4],[5] The term “blood group” is used for inherited antigens that are identified by unique antibodies on the red blood cell (RBC) surface.[6] At the scene of medicolegal cases such as child abuse, rape, robbery, and murders, blood may be missing; however, other evidence may be present and available. These evidences, such as fingerprints, hair, semen, and saliva, play an important role in criminal investigations as they may help to link the suspect, victim, and crime scene, or act in accordance with the Locard’s Principal of Exchange.[7],[8] Blood group antigens can be found not only on the RBCs, but also on lymphocytes, platelets, tissue cells, body fluids (except cerebrospinal fluid), and secretions. The existence of blood group substances in different body secretions depends on whether the individual is a secretor or a nonsecretor.[9],[10] Yamakani discovered water-soluble substances in human saliva that are present in approximately 80% of people, which are called secretory, and the remaining 20% are called nonsecretory.[11],[12],[13]

Saliva samples at the crime scene can aid in the typing of deoxyribonucleic acid (DNA), sex identification, bite mark analysis, and blood grouping. Human bite marks, cigarette butts, envelope flaps, dental devices, toothpicks, and plastic casings can be used to acquire a dry or wet source of saliva.[9],[12] In forensic practice, cigarette butts are one of the common carriers of saliva traces, which the people smoke when they are nervous, for example, when they involved in a crime.[14] However, owing to the unpredictable time lapse between the occurrence of the crime and the start of the investigation, dry form of saliva is often collected more than the wet form.[7]

This study aimed to investigate blood group antigens on the cigarette butts based on time.

  Materials and Methods Top

This was an experimental analytical study, conducted for 8 months in the Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia, following the review and approval of the study protocol by the institutional ethics committee of the Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia (253/HRECC.FODM/X/201). The participants were explained the essence of the research, and subsequently their written consent was obtained.

An experimental laboratory study was carried out with time series design (1, 3, and 6h) at a room temperature of 20°C. With a time series design,[15] eighteen cigarette butt samples were used by following the inclusion criteria. Blood specimens were obtained from each subject, and screening was carried out to obtain blood type by slide method before checking secretor and nonsecretor blood groups. The total subjects were six, consisting of two subjects in each blood group (A, B, and AB).

Cigarette butts, collected from each subject, were placed at room temperature for 1, 3, and 6h. The standard absorption inhibition method was used to estimate antigens in the blood group.

Procedure for absorption inhibition method

Cigarette butts, collected from each subject, were placed at room temperature for 1, 3, and 6h [Figure 1]. Test tubes with cigarette butts were added aq. dest. ± 5 mL, and then were centrifuged at 3000 rpm for 5 minutes. After 24h, the samples were centrifuged; the supernatant was diluted to 1:4 until 1:64 and was placed in the microplate. ABO antigen was added to each microplate. The microplate was incubated at 4°C for 2 days. Then one drop of 2% RBC suspension was added to the microplate, mixed, and incubated for ±3 min.
Figure 1: Procedure for absorption inhibition. (A) Cigarette butts collected from the subjects. (B) Test tubes. (C) ABO antigen added to each microplate

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The sample either macroscopically or microscopically [Figure 2] was considered as positive if agglutination was not seen, suggesting that the reaction between antigen and antibody had taken place between saliva and antiserum, and RBCs did not have antibody to respond, suggesting the existence of blood group. The result of the negative reaction was reassessed using the same technique, and if the second result was negative, it was considered negative. Examination of protein levels in cigarette butts was carried out using spectrophotometry at a wavelength of 280 nm.
Figure 2: Results of blood group based on photomicrograph. (A) Photomicrograph showing large masses of agglutinates (3+, visual). (B) Photomicrograph showing small masses of agglutinates (2+, visual). (C) Photomicrograph showing clumps of 8–12 cells (good weak)

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Statistical analysis

To find out the relationship between the effect of exposure time and the amount of protein in the saliva of cigarette butts, the research data were analyzed using IBM® SPSS® Statistics version 23.0 (IBM, Armonk, New York, USA), by descriptive analysis with nonparametric statistical test (paired sample t test).

  Results Top

[Table 1] shows that ABO blood group antigens were present in all samples. This means that until 6h, ABO blood type antigen carrier in saliva was still present and had not been totally denatured. The aforementioned means that blood group in saliva from 1h until 6h after incubation in cigarette butts could be examined (100%) by absorption inhibition method. In this study, samples indicated that 100% of the subjects were secretors.
Table 1: Blood group agglutination results by absorption inhibition method

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[Table 2] shows 100% positive 3 agglutination after 1h of incubation in 1:4 titrations. After 3 and 6h incubation, 50% of positive 3 agglutination and 50% of positive 2 agglutination were observed in 1:8 titration.
Table 2: Microscopic result of blood group agglutination

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[Table 3] shows a significant difference in protein level between the samples, which were exposed to the room temperature in 3–6, 1–6, and 1–3h. The statistical analysis with paired sample t test showed significant P value at ≤0.05.
Table 3: Statistical analysis of time exposure and protein level with paired sample t test

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  Discussion Top

Blood group determination has a major role in forensic science in the field of personal identification, paternity dispute, and other scenarios besides DNA analysis, fingerprinting, and so on.[16] This study showed that blood group can be detected from all of the samples. Blood group can be determined from the cigarette butts of those subjects who have antigen in the saliva (secretors) with 100% accuracy. This means that up to 6h of room temperature exposure, the ABO blood group antigen-carrying protein in saliva is still present and has not been fully denatured. Protein denaturation can occur by drying or reducing the water content in the carrier material. In saliva, denaturation of ABO blood group antigen-carrying proteins can occur if the saliva is drained. Study in Germany found that saliva would dry up on the stone at room temperature, within 1h 40 min.[3] However, it could not be determined from nonsecretor subjects.[9],[17]

This research used the method of absorption inhibition to reduce the concentration of antigens in the stains.[18] A previous study showed that absorption inhibition method is better than other methods for blood group determination from saliva.[9] Thrumiaya et al.[19] found that the process of absorption inhibition is a more effective technique for recognizable proof and secretor determination.

There are many issues in forensic cases, such as insufficient content, low antigen concentration, and degradation of substances in the blood group by temperature, humidity, and aging.[20] This study proved that blood group can be detected from all of the samples until 6h. However in this study, there were some limitations because of the lack of research samples, the absence of climate variations, and because the blood type identification can only be performed on individuals who have secretor genes, whereas individuals who do not have secretor genes cannot be used as samples.

At the crime scene, saliva can be found on various objects, care must be taken not to ignore or mismanage this evidence. The findings can prove an accused person’s blood stain or secretion in a crime scene.[6]

The samples revealed that 100% of the subjects were secretors. ABO blood group antigen carrier was still present in saliva and had not been totally denatured until 6h by absorption inhibition method. Cigarette butt samples that contain saliva can be used for forensic investigations in narrowing down the suspect pool by blood group determination.

Data availability statement

The data set used in the current study is available on request from Mieke Sylvia Margaretha Amiatun Ruth/ [email protected].

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Sen MP, Vanishree M, Hunasgi S, Surekha R, Koneru A, Manvikar V. A comparison of absorption inhibition and absorption elution methods for estimation of ABO blood groups in saliva. J Med Radiol Pathol Surg 2015;1:1-4.  Back to cited text no. 13
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[PUBMED]  [Full text]  
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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3]


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