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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 93-99

ABO blood group detection in extracted teeth: A forensic study


1 Department of Oral Pathology, School of Dental Sciences, Sharda University, Greater Noida, India
2 Department of Periodontology, School of Dental Sciences, Sharda University, Greater Noida, India
3 Department of Public Health Dentistry, School of Dental Sciences, Sharda University, Greater Noida, Uttar Pradesh, India

Date of Submission21-Apr-2020
Date of Decision30-Apr-2020
Date of Acceptance20-Aug-2020
Date of Web Publication28-Jan-2021

Correspondence Address:
Dr. Mithilesh N Mishra
Department of Oral Pathology, School of Dental Sciences, Sharda University, Knowledge Park III, Greater Noida, Gautam Buddha Nagar 201308, Uttar Pradesh.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_149_20

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  Abstract 

Aim: The aim of this study was to ascertain ABO blood group from extracted teeth using pulp and dentin tissues with the help of the absorption–elution (AE) technique. Materials and Methods: The study was conducted using an experimental study design and included 60 patients who underwent extraction due to periodontal and therapeutic purposes. Blood group antigens were ascertained for all the study participants using capillary blood by slide agglutination method (Controls). AE technique was used to check blood grouping using powdered dentine and dental pulp immediately after extraction and after 9 months. The study group was compared with the control group for blood group determination at different time intervals to find the sensitivity of dental pulp and a significant difference between those values at different time intervals. The statistical tests used were the Shapiro–Wilk test, chi-squared test, multivariate linear regression, and the Pearson correlation coefficient. Results: A total of 60 study subjects, 39 males and 21 females, were taken. In the estimation of blood group, 54 teeth, that is, 90% of total sample, were positive. We found an inverse relationship between the result for the blood grouping and time intervals, that is, 100% and 80% test results, done on the day of extraction and after 9 months. Conclusion: It could be inferred that the antigens from pulp are biologically stable for long time. This study brings a spotlight on the time duration for which teeth can remain as the prominent source for the detection of blood group.

Keywords: ABO Blood Group, Dentin, Pulp


How to cite this article:
Mishra MN, Chandavarkar V, Bhargava D, Sharma R, Gupta R, Thakar S. ABO blood group detection in extracted teeth: A forensic study. J Int Oral Health 2021;13:93-9

How to cite this URL:
Mishra MN, Chandavarkar V, Bhargava D, Sharma R, Gupta R, Thakar S. ABO blood group detection in extracted teeth: A forensic study. J Int Oral Health [serial online] 2021 [cited 2021 Feb 27];13:93-9. Available from: https://www.jioh.org/text.asp?2021/13/1/93/308347


  Introduction Top


Forensic odontology is a relevant and indispensable science in medicolegal cases and identification of the deceased. Dental tissues are well protected for a long time even after death; therefore, they remain as the most stable biological evidence.[1],[2] Teeth are invaluable sources of personal identification in catastrophe. Pulp within a tooth is the most protected oral tissue being surrounded by dental hard tissues from all the sides. As pulp contains numerous blood vessels; blood group agglutinogens, and agglutinins (A, B, and O) are most likely to be present in it.[3],[4] Blood grouping has been one of the major factors for identification of biological materials in forensic investigations. Once the blood group and Rhesus factor of an individual is established, it remains unchanged for the rest of life.[5]

Blood group substances are secreted in numerous body fluids including saliva, mucus, tears, seminal fluid, vaginal fluid, and gastric juice. In 1960, Kind came up with the presence of ABO blood group substances in saliva by the absorption–elution (AE) method where he could detect the blood group form a dried stain. AE procedure was foremostly concocted by Siracusa and is now used in all forensic laboratories because it is proven to be most sensitive, reliable, and consistent.[6],[7] In the AE method, dried pulpal content is mixed with antisera A and B. Fresh saline is added to elute the antibodies. This leads to agglutination of respective agglutinins to the blood group agglutinogens present on the cell surface of red blood cells (RBCs).

It is speculated that blood group substances in dentin are located in dentinal tubules, which are filled with dentinal fluid. Load of ABO substances from the pulp cavity wall to the dentin edge and then to the enamel gradually diminishes because of minimal chances of diffusion of antigens from blood and saliva. Presence of blood group antigens in tooth dentin and enamel has been authenticated by infusion–sedimentation phenomena along with the naturally present antigens. This hypothesis elucidates the infusion of water-soluble antigens from saliva into the tooth tissues.[8] Usually, teeth and bones are the only relevant tissues that might remain in mass disasters and they contain ABO blood group and Rhesus factor antigens. So, the aim of this study was to ascertain ABO blood group from dentin and pulp of extracted teeth with the help of “AE technique” in freshly extracted teeth, and after a storage period of 9 months.


  Materials and Methods Top


Setting and design

This study adopted an experimental study design and recruited patients aged 12–60 years reporting to Department of Oral Medicine and Radiology over a span of 2 years (convenience sampling). The consent form was also available in local language (Hindi) so as to avoid any communication bias between the researchers and the patients.

Based on the results of the pilot study to determine the feasibility of the study (done on 10 patients (5 in each group), a certified statistician calculated a minimum sample of 52 subjects (26 in each group) keeping the confidence interval (CI) as 95% and power of the study(1 – β) as 80%. Therefore, 60 extracted teeth (30 in each group) were chosen.

Patients were randomly allocated into two groups (based on flip of a coin). Patients allocated in Group 1 were taken for ABO blood grouping immediately after extraction, whereas the extracted teeth of patients belonging to Group 2 were stored for 9 months before performing their blood grouping. Tooth/teeth were indicated for extraction due to poor periodontium and/or for therapeutic causes were included in the study. Patients having carious and grossly decayed teeth were excluded from the study.

There was no bias in the selection samples observed. At the time of extraction of teeth, blood was obtained by prick on patient’s finger under aseptic condition and blood group was examined using the slide agglutination method. The extracted teeth were water-washed to clear out blood, saliva, or any debris attached to it. After that, they were air-dried and arranged in tagged plastic cassettes. As a part of the study protocol, the patients were provided refreshments postextraction and were asked to avoid any strenuous activity for the day. No drop of the study was noted.

Laboratory procedure

The teeth were split at its constricted margin with the help of a carborundum disc attached to the micromotor [Figure 1]. To moisten the pulp, saline was added to pulp chamber and root canals. Afterwards, pulp was extirpated with the help of a spoon excavator. Extirpated pulp was stored in sterile, tagged test tubes. After that pulp was fragmented into two parts and placed in the test tubes. Then anti-A or anti-B serum was added to each of the test tubes containing pulp tissues, respectively, and was blended thoroughly. Test tubes were plugged by cotton and were well-kept at 4°C for overnight to allow the absorption to take place [Figure 2]. Then test-tube samples were mixed with saline and subjected to centrifugation. Centrifugation was done for 10 min at 3000 rpm. Clear fluid above precipitate was offloaded with the help of a Pasteur pipette. Then two drops of 10% BSA (Sigma) in saline were stirred inside the test tubes and test tubes were settled in a water bath at around 50°C for approximately 10 min to break loose the agglutinins. Subsequently a drop of 0.5% A and B RBC suspensions were descended into the test tubes. Later on samples in test tubes were agitated and incubated at 37°C for half an hour to intensify antigen-antibody reaction. Evaluation of extent of agglutination was done grossly and microscopically [Figure 3] and [Figure 4].
Figure 1: Micromotor with carborundum disc to dissect tooth

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Figure 2: Dental pulp in antisera A and B

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Figure 3: Agglutination grossly

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Figure 4: Agglutination microscopically (x400)

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Statistical analysis

The analysis was done using IBM Statistical Package for the Social Sciences (SPSS) for Windows, version 21.0 (IBM, Armonk, New York) and the significance level (P value) was kept at P ≤ 0.05. The data analysis included the Shapiro–Wilk test (to check the data for normality), chi-squared test, multivariate linear regression, and the Pearson’s correlation coefficient. The data were first coded and then sent to the statistician to ensure the confidentially of the data.


  Results Top


In this study, the mean age of Group 1 was 35.83 ± 14.26 (15–60) and Group 2 was 38. 83 ± 10.30 (18–60). The two study groups indicated statistically not significant with χ2 = 6.73, P = 0.15 (P > 0.05), as shown in [Table 1]. Distribution of study population according to Gender was statistically calculated in two groups. Group 1 comprised 21 (70%) male and 09 (30%) female, whereas Group 2 included 18 (60%) male and 12 (40%) female. This was found to be statistically not significant with χ2 = 0.66, P = 0.41 (P > 0.05), as shown in [Table 2].
Table 1: Age-wise distribution of subjects in two groups

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Table 2: Gender-wise distribution of patients in two groups

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When comparing Group 1 with control, all the teeth showed 100% positive result, which indicated that it is statistically not significant with χ2 = 0.00, P = 1.00 (P > 0.05). After comparing Group 2 with control, except for six teeth, the rest showed positive result which comprised 80%; this indicated that it is statistically significant with χ2 = 2.16, P = 0.0098 (P < 0.05), as shown in [Table 3].
Table 3: Comparison of both groups with control groups

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In [Table 4], comparison between the two study groups showed a change in the pattern of result with the increase in time duration, that is, percentage of negative result is 20% at 9 months of interval. It was zero when teeth were subjected to ABO blood grouping immediately after extraction. So, it was statistically not significant with χ2 = 6.45, P = 0.17 (P > 0.05).
Table 4: Comparison of blood grouping between two study groups

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The multiple linear regression model to analyze blood group antigens revealed a statistical difference with respect to the A blood group (P = 0.02) between both the groups [Table 5]. A positive, linear, great strength of association (r = +0.71) and a significant relationship (P = 0.02) was found between both the groups using Pearson’s correlation coefficient [Table 6].
Table 5: Association between Groups 1 (constant) and 2 using the multivariate linear regression analysis

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Table 6: Correlation between knowledge, attitudes, and practices using Pearson’s correlation test

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  Discussion Top


Forensic science has many maxims, perhaps the best known of which is every contact leaves its trace.[9] Forensic science is defined as any expert evidence given by special scientific, or experts having particular knowledge and study in any particular scientific subjects during the administration of justice in the court of law. Pederson defined forensic odontology as the branch of dentistry that deals with proper handling and examination of dental evidence with proper evaluation and presentation of dental findings in the interest of justice.[4],[10],[11]

Teeth are the most stable biological clue materials encountered in forensic science, being made up of the hardest and most stable chemical tissues in the body; they sustain their characteristics even in the most untoward environmental conditions. Thence teeth are used for blood grouping and are a hallmark in identifying biological subjects in forensic investigation. Ramnarayan et al.[10] reported that apart from ABO agglutinogens, adenylate kinase, adenosine deaminase, phosphoglucomutase-1, and G6PD (glucose-6-phosphate dehydrogenase) are naturally found in teeth and could be used for various purposes.[12] AE technique was originally devised by Siracusa. In 1960, Kind refined this technique and is now used in all forensic laboratories.[1] Further, Sunitha and Vidya,[13] Ramnarayan et al.,[10] Haertig et al.,[14] Ballal and David,[15] Motawei et al.,[l6] and Vala et al.[17] obtained positive results on tooth by using AE technique. In the last 60 years with various modifications it is proven to be most sensitive, reliable and reproducible.

In this study, a total number of 60 permanent teeth were collected from the Department of Oral and Maxillofacial Surgery. The blood group estimation was done at different time intervals to examine if blood grouping on teeth remains possible after relatively long storage periods. Blood group was determined from pulp and dentin; and was compared with blood grouping from controls to verify the study result. We found that a total of 54 teeth of 60 showed a positive result for ABO blood grouping from the dental pulp after storing them for 9 months. This showed a 90% sensitivity [Table 4]. Our study was in accordance with the studies done by Dil and Ahmed,[7] Metgud et al.,[8] and Ramnarayan et al.[10] showed that agglutinates for dentine and pulp weakened as the time period increased.[10] It was asserted that existence of blood group agglutinogens in dental hard tissue is based on the infusion sedimentation phenomenon sorbed with intrinsic antigens. In our study, we found that antigenicity of pulp and dentin deteriorated over the period of time. This was in congruence with the study conducted by Vala et al.[17]

The age of the study subjects was recorded. They ranged from 15 to 60 years with the mean age of 37.33 years [Table 1]. In this study, we found that the negative results recognized in six subjects were of 40,43, 45,45, 48, and 60 years [Table 3]. Thus, we can say that age had a slight effect on blood group assessment. As age increased, the pulp tissue became more fibrosed or reduced in bulk due to calcified canals, which led to more negative results. This is in accordance with study done by Vala et al.[17]

The blood group of 60 study subjects which was determined from the patient’s finger belonged to 31.67% of A, 36.67% of B, 10% of AB, and 21.67% of O, whereas 30% of A, 30% of B, 10% of AB, and 20% of O, respectively, from the extracted teeth (dental pulp) [Table 3]. In a study carried out by Shetty and Premlatha,[5] blood grouping belonging to O were highest. In contrast, a study conducted by Ballal and David[15] showed that blood group B was of highest percentage. In this study, blood group B comprised the highest percentage which was 36.67% of total study subjects, whereas blood group AB was present in only 10% subjects. Of six negative results blood group A was present in one tooth, blood group B was present in four teeth and blood group O was present in one tooth [Table 3].

In studies conducted by Ballal et al.,[15] Motawei et al.,[16] and Ramnarayan et al.[10] on ABO blood group, identification from dental pulp on the same day of extraction using AE technique was positive for 90%, 80%, and 100%, respectively. A study carried out by Shetty and Premlata[5] reported that sensitivity to pulp in relation to control was 96.7%. In this study, we correlated blood group determination from the dental pulp of permanent teeth at different time intervals that is on same day and 9 months after extraction, which were 100% and 80%, respectively [Table 4]. To accomplish the elution of antibodies and agglutination process, 10% BSA was used. BSA has specific property of overriding ionic repulsive forces among erythrocytes and thus it potentiates precise bonding with their related agglutinins.[10] In this study, Group 1 showed higher percentage of positive results as compared to Group 2. This finding is consistent with the studies done by Shetty and Premlata[5] and Motawei et al.[16] In the existing study, there were negative reports in four cases and mistyping in two. This was probably due to lysis of cells, contamination of the tooth, insufficient pulp tissue, increased fibrosis of the pulp with higher age and calcification of the canals.[2]

Gram-negative aerobes are dwelling in the oral cavity in good numbers. They are existing in saliva and on teeth surfaces.[1],[3] It has been inferred from various studies that contamination of teeth by gram-negative aerobes (mainly  Escherichia More Details coli and Serratia maracescens) constitutes blood group like agglutinogens, simulating a B type blood group. It suggests that only one subspecies of E. coli and S. maracescens are able to form blood group simulating antigens. Gram-negative aerobes along with yeast grow rapidly in nonsterile containers with tooth specimens. This process further leads to the overgrowth of other species. The massive growth of such bacteria tends to obscure the pulpal blood group antigens and it can produce a negative result.[10],[17] In addition, negative results can also occur from specimens of post-mortem dental pulp because of hemolysis of RBCs, putridness and embalmment of the body during post-mortem time-period. All these procedures are contributary to mistyping of blood groups. In this study, we deduced that blood group typing from dental pulp of stored permanent teeth after 9 months showed success rate of 80%, whereas in 20% it showed negative results. The 9 months’ duration was chosen as the previous studies had 6 months’ duration interval between two groups and this study tried to evaluate the changes if any after 9 months. Based on the results of this study, the AE technique was found to give dependable results after 9 months and thus, leading to the acceptance of the null hypothesis.

Essentiality of blood group typing in medicolegal or forensic studies is braced by the concept that every individual has a predetermined blood group which remains unchangeable throughout life. After death too, teeth are the last to show any post-mortem changes because pulp is well protected within the calcified walls of the teeth. So they can volunteer for postmortem identification of the individual at the crime scene or disaster sites.[18] Studies have been able to show positive findings using pulp for ABO blood grouping up to 12 months. It is further needed to study the possibility of ABO blood group antigen positivity from the dental pulp at an extended time interval and with other novel techniques.[1],[3],[17]


  Conclusion Top


Teeth are said to be one of the clues in forensic examinations. In this study, the AE technique was used for blood grouping from the dental pulp and slide agglutination technique was used for blood grouping by finger prick method. It was inferred from this study that gender has no significance on blood group determination. Our study detected blood group from tooth material after 9 months of storage period with minimal cost. In view of the results obtained from this study, it can be gathered that teeth are the corroborative tool to authenticate personal identification in cases where it is the only remnant available. Further endeavors are required to conduct similar research with larger sample size, longer storage period with sophisticated techniques.

Future scope

It is recommended to conduct experimental studies on teeth samples under different environmental conditions with longer storage periods for a large sample size. Different laboratory materials should be tried to elute the antibodies. Limitation in the study is contamination by plants, animals, bacteria, and fungi during the storage period which can bring antigens similar to blood group-like agglutinogens leading to blood groups mistyping. This study will benefit researchers to endeavor newer techniques with different laboratory materials in detecting blood group from teeth. We hope that such experimental trials conducted in future will be worthy to society in cataclysmic situations.

Acknowledgement

We are thankful to Dr. Prerit Verma who accomplished the work of data collection from the Department of Oral and Maxillofacial Surgery and Department of Oral Medicine and Radiology.

Financial support and sponsorship

The study was self-funded.

Conflicts of interest

There are no conflicts of interest.

Author contributions

Mithilesh Mishra and Vidyadevi Chandavarkar: Study conception, data collection, data acquisition and analysis, data interpretation, and manuscript writing; Ritika Sharma: Data collection and analysis, data interpretation, manuscript writing, and manuscript reviewing; Dr. Deepak Bhargava: Data interpretation and manuscript reviewing; Dr. Radhika Gupta: Data interpretation and manuscript reviewing; Dr. Sahil Thakar: Data interpretation and statistical analysis.

Ethical policy and institutional review board statement

This experimental study was approved by the Institutional Ethics Committee of Sharda University, Greater Noida in January 2015. All the procedures have been performed as per the ethical guidelines laid down by Declaration of Helsinki (1964).

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Data availability statement

Data set used in this study is available in the Central Library of School of Dental Sciences, Sharda University.

 
  References Top

1.
Kumar PV, Vanishree M, Anila K, Hunasgi S, Suryadevra SS, Kardalkar S Determination of ABO blood grouping and Rhesus factor from tooth material. J Oral Maxillofac Pathol 2016;20:540-4.  Back to cited text no. 1
    
2.
Saxena V, Jain M, Tiwari V, Santha B, Khare A, Shah R The credibility of dental pulp in human blood group identification. J Forensic Dent Sci 2017;9:6-9.  Back to cited text no. 2
    
3.
Aswath N, Selvamuthukumar SC, Karthika B Role of dental pulp in identification of the deceased individual by establishing ABO blood grouping and rhesus factor. Indian J Dent Res 2012;23:811-3.  Back to cited text no. 3
    
4.
Inamdar P, Inamdar P, Rai K, Mirajkar AM, Venkatesh S Teeth-hidden treasure of blood group. Indian J Forensic Med Pathol 2011;4:113-18.  Back to cited text no. 4
    
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Shetty M, Premlata K ABO blood grouping from tooth material. J Indian Acad Forensic Med 2010;32:336‑8.  Back to cited text no. 5
    
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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. 6
    
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Dil F, Ahmed A The reliability of dental pulp in human blood group identification. Pakistan Oral Dent J 2018;38: 42-4.  Back to cited text no. 7
    
8.
Metgud R, Khajuria N, Mamta , Ramesh G Evaluation of the secretor status of ABO blood group antigens in saliva among southern rajasthan population using absorption inhibition method. J Clin Diagn Res 2016;10:ZC01-3.  Back to cited text no. 8
    
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Queiroz CL, Silva RF, Silva RH Computed tomography use on age estimation in forensic dentistry: A review. J Forensic Sci Criminol 2016;4:105.  Back to cited text no. 9
    
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Ramnarayan B, Manjunath M, Joshi AA ABO blood grouping from hard and soft tissues of teeth by modified absorption-elution technique. J Forensic Dent Sci 2013;5:28-34.  Back to cited text no. 10
    
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Narang D, Nayyar AS, Gandhi P Assessment of correlation of abo blood grouping and impacted third molars: A blind trial. Int J Res Health Allied Sci 2016;2:28-30.  Back to cited text no. 12
    
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Sunitha S, Vidya GD Determination of blood group from the tooth material - an medico legal investigative procedure. RRJDS 2017;5:45-8.  Back to cited text no. 13
    
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Haertig A, Krainic K, Vaillant JM, Derobert L [Medicolegal identification: Teeth and blood groups (author’s transl)]. Rev Stomatol Chir Maxillofac 1980;81:361-3.  Back to cited text no. 14
    
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Ballal S, David MB Determination of ABO blood grouping from dentin and pulp. Pak Oral Dent J 2011;31:3-6.  Back to cited text no. 15
    
16.
Motawei SM, El-Zehary RR, Shteiwi A ABO blood grouping from dentin and pulp of fresh and aged teeth by modified absorption elution technique. Egypt Dent J 2018;64:2251-61.  Back to cited text no. 16
    
17.
Vala D, Nayyar AS, Pooja VK, Kartheeki B, Patel N, Vala D, Tanmay P Determination of ABO blood grouping from dentine and pulp by absorption-elution technique. Int J Orofac Biol 2017;1:70-80.  Back to cited text no. 17
    
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Nayar AK, Parhar S, Thind G, Sharma A, Sharma D Determination of age, sex, and blood group from a single tooth. J Forensic Dent Sci 2017;9:10-4.  Back to cited text no. 18
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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