Abstract: Background: Identification of a deceased subject can be established not only from the skeletal remains (including the congenital peculiarities in setting of teeth) but it can also be precisely established by blood grouping from the teeth with the help of the absorption-elution technique. Objectives: Our study was conducted to evaluate if dental pulp can be used as a reliable source for determination of ABO blood group and rhesus factor in an individual. Materials & Method: This double-blinded randomized controlled trial was conducted on a sample of 250 extracted teeth. An attempt to establish the blood group from pulp was made by absorption-elution method. The collected data were coded, and statistical analysis for comparison of ABO blood groups was done using Cronbach's alpha to check the reliability of the absorption-elusion method in the detection of blood group from dental pulp. Results: On comparison of capillary blood group (slide agglutination method) with pulp blood group (absorption elution technique) of the subjects, we found that positive results were obtained in 235 cases while 15 cases showed negative results. Hence, the sensitivity (ability to measure) of pulp in establishment of blood group was found to be 94%. Conclusion: On the basis of the results obtained from the present study, it could be concluded that pulp is a reliable source of blood group determination for ABO blood grouping where teeth happen to be the only remnants available for personal identification.
Key Words: Pulp, ABO blood group, forensic odontology

Introduction:

Forensic is a Latin word derived from "forensis" meaning public, and forensic science refers to areas of endeavour that can be used in judicial setting and accepted by the court and general scientific community to separate truth from untruth.[1]

Keiser-Neilson defined forensic dentistry as "that branch of forensic dentistry that in the interest of justice deals with the proper handling and examination of dental evidence and the proper evaluation and presentation of dental findings".[2] It is an investigative aspect of dentistry that analyses dental evidence for human identification.[3]

The use of teeth as evidence is not recent. There are historical reports of identification by recognizing specific dental features as early as 49 A.C. However, Forensic Odontology, as a science, did not appear before 1897 when Dr. Oscar Amoedo wrote his doctoral thesis entitled 'L' Art Dentaire en Medecine Legale' describing the utility of dentistry in forensic medicine with particular emphasis on identification.[4]

Identification of a deceased subject can be established not only from the skeletal remains (including the congenital peculiarities in setting of teeth) but it can also be precisely established by blood grouping from the teeth with the help of the absorption-elution technique.[5] Blood grouping has been one of the cornerstones of identification of biological material. The term blood group is applied to inherited antigens detected on the red blood cell surface by specific antibodies. The ABO blood group system, first described by Karl Landsteiner in the year 1900, remains the bulwark of forensic blood group investigation since it is the primary, most common, conspicuous and easily detectable system.[3]

Lattes has aptly said "the fact that belonging to a definite blood group is a fixed character of every human being and can be altered neither by the lapse of time nor by intercurrent disease". Blood group like fingerprint is an unalterable primary character.[6,7]

In the dental pulp, vascular endothelium and red blood cells are regarded as a source of ABH antigenicity.[3] Pulpal tissue is one of the most protected oral tissues being surrounded from all sides by the dental hard tissues.[8] Hence, our study was conducted to evaluate if dental pulp can be used as a reliable source for determination of ABO blood group and rhesus factor in an individual.

Material and Methods:

The study was a double-blinded randomized control trial conducted at Department of Oral Pathology & Microbiology, School of Dental Sciences, Sharda University. Ethical clearance was obtained from the Institutional Ethical Committee of the University. Study sample comprised of 250 extracted teeth, indications for extraction being periodontal disease or orthodontic or prosthetic reasons. The exclusion criteria used was carious teeth, root canal treated teeth and restored teeth. A brief case history was recorded from all the patients selected for the study after obtaining due consent. The blood groups were determined for the study participants using capillary blood by slide agglutination method, and duly entered in the records. The investigator and the statistician were blinded regarding the blood group of the individual.

For sample collection, the extracted teeth were washed under running water to remove debris and kept in numbered bottles. The teeth were sectioned into two halves using a micromotor with a carborundum disc and pulp was scooped with a spoon excavator. Absorption-elution test was used to identify blood groups from the extirpated pulp, which was later compared with the recorded blood group of the patients. When blood grouping from the pulp correlated with the blood group of the person, the result was recorded as positive and if they did not match, the result was recorded as negative. The obtained results were statistically analyzed. Cronbach's alpha was applied to check the reliability of the absorption-elusion method in the detection of blood group from dental pulp.

Results

The age for our study samples ranged from 20 years to 70 years. Among them, the maximum numbers of samples were in the age group of 41-60 years. Blood Group B was found to be the most common amongst all blood groups. On comparison of capillary blood group (slide agglutination method) with pulp blood group (absorption elution technique) of the subjects, we found that positive results were obtained in 235 cases while 15 cases showed negative results (Table 1). Hence, the sensitivity (ability to measure) of pulp in establishment of blood group was found to be 94%.

Discussion

Human identification is the mainstay of civilization and the identification of unknown individuals has always been of paramount importance to the society. The use of blood group substances in medicolegal examination is based on the fact that once a blood group is established in an individual, it remains unchanged throughout life.[3] The proofs of individual identification by use of blood group testing gained legal value in the German courts in 1920, being legally accepted in the United States only in 1935. In Brazil, these exams were given legal value with the first paternity investigation in 1948.[9]

Although advances have been made in Deoxyribonucleic acid (DNA) analysis, fingerprinting, etc., blood grouping still has a major role in the forensic practice in the field of personal identification, paternity disputes, and other scenarios. ABO blood grouping is examined in suspects of criminal cases and paternity disputes before DNA profiling. This is attributed to the fact that genetic and antigenic constituents of an individual remain not affected by environmental conditions.[10]

Teeth being the hardest of all tissues can be preserved intact for long periods of time after death. They are stable chemically and they retain their characteristic even in the most adverse condition. The presence of blood group substances and other genetic markers such as enzymes in soft and hard dental tissues makes it possible to assist in the identification of highly decomposed bodies.[6] The possible distribution of ABO substances from the pulpal cavity wall to the dentin edge and to the enamel gradually decreases because of fewer possibilities of diffusion of antigens from both blood and saliva.[2]

The basic technique in identification of the antigens and antibodies of blood groups is the agglutination test using glass slide or white porcelain tile. Other manual techniques are glass test tube test and microplate test. Newer techniques are column technique and solid phase tests. Blood grouping from dried stain by elution procedure was described more than 50 years ago but not employed widely in forensic serology, until 1960, when Kind refined this technique. Absorption Elution procedure devised by Vittorio Siracusa in 1923 is now employed in all forensic laboratories because it is proven to be most sensitive, reliable and reproducible in identifying blood group of deceased individuals.[11]

In our study, sample comprised of extirpated pulp from extracted teeth of subjects, from which patient's blood group was determined and compared with the blood group obtained using capillary blood. The subjects taken for our study ranged from 18 years to 70 years of age. Among them, the maximum numbers of samples were in the age group of 41-60 years. We found the sensitivity of pulp in establishment of blood group to be 94%. This was in accordance with the study conducted by Shetty and Premalatha who found that the sensitivity of pulp in determining blood grouping is significantly high (96.7%), even after storing dry for 6 months.[12] Similar results were also obtained by Saxena V, et. al. who performed a study on 30 extracted teeth to check the credibility of dental pulp in human blood group identification. They observed that the sensitivity of pulp in determining blood groups was 80%.[13] Prasath & Athanari found 100% accurate results in all 40 samples and concluded that blood grouping, by using a tooth pulp, might be a great aid in individual identification even after different time intervals of extraction or in dead individuals.[14]

Nayar AK, et. al., also found a 100% accuracy of blood group determination from pulp after 2 days of extraction, in normal environmental condition and under the saline water.[15] Motawei SM, et al. investigated the possibility of detecting the blood groups antigens in the hard dental material and the pulp obtained by splitting the teeth without heat using a new Hand-Held Pulp Isolator instrument and using a modified absorption elution technique. They found that overall sensitivity of the pulp for ABO blood groups detection was 92.31% for fresh teeth and 80.77% for aged teeth.[10]

Sidhu S, et al., concluded that although age, sex, and blood group are more reliably determined in freshly extracted teeth, these variables may be of significant help in identification even after a period of 6 weeks post-extraction.[16] Ramnarayan B conducted a study on 60 teeth to detect ABO blood grouping from hard and soft tissues of teeth by modified absorption-elution technique and obtained significant positive results of ABO blood groups from dental pulp in their study.[6]

Aswath N, et al., found that out of 60 samples, 85% showed accuracy to determine the correct blood group, from normal pulp which was processed immediately whereas, pulp placed under hydrostatic pressure for 48 hours showed marginally 79.1% accuracy.[17] Smeets B, et al., determined ABO blood group from pulp, dentin and enamel of 35 teeth using the adsorption-elution technique. Twenty teeth were examined within 6 weeks after extraction and fifteen teeth were examined 6-10 months after extraction. It was found, that blood grouping on pulp gives fairly good results, whereas the possibilities for correct blood grouping seem to be limited for dentin and debatable for enamel. Similar results were found in both groups of teeth.[18]

We found that most of teeth that showed negative results in pulp were teeth in the age group of 60 years and above. This is similar to a study done by Motawei SM, et al., who found that most of teeth that showed negative results in pulp were in the age group of 40 years and above. Aswath N, et al., found negative results in 3 out of 60 samples. The negative results of the pulp for blood grouping can be attributed to insufficient quantity of pulp, loss of the pulp tissue with increasing age, or due to increased calcification of the pulp canal.[8,10]

Conclusion

The remarkable character of teeth as one of the most enduring integral remnants of the human body empowers its effective use in forensic sciences. Pulp tissue is surrounded by dental hard tissues from all sides, making it one of the most shielded tissues of the human body, and easily available for examination. Blood grouping from pulp could assist in identification of individuals by narrowing the search via exclusion of people with different blood groups. Limitations of this study can be ascribed to the fact that tooth with obliterated canals and regressive alterations do not provide sufficient pulpal tissue. Also, the process is time-consuming and technique sensitive. Hence utmost care is needed while doing the procedure.

Financial support: We would like to thank Sharda University for funding this research study.

Conflicts of interest: There are no conflicts of interest.

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