To Evaluate the Sensitivity of ABO Antigen Determination from Dental Pulp at Various Temperatures over a Duration of 3 Months - A Diagnostic Study

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Introduction
Identification of forensic evidence includes multidisciplinary team which relies on methodologies which are either presumptive or exclusionary in nature 1 . Dr. Oscar Amoedo was considered as the father of forensic odontology 2 . According to Federation Dentaire Internationale in the year 1940, forensic dentistry was defined as "that branch of forensics which is in interest of justice deals with proper handling and examination of dental evidence with proper evaluation and presentation of dental findings" 3 . Human identification is significant not only to identify the deceased individual but also to play a major role in certain issues like, criminal investigations, insurance settlements etc. 4 . Rh blood factor antigen was discovered by Landsteiner and Alexander S. Wiener in the year 1940 5,6 . Blood grouping among the individual once established remains unchanged throughout the life. Blood group of an individual can be used as a biological material in the field of forensics 7 .
Teeth are a major source of evidence in the field of forensics for identification purpose. Teeth are considered to be the hardest substance in the human body and can bear a temperature up to1600°C without significant loss of microstructure 7 . In conditions like mass disaster, adverse environmental grounds, teeth form a vital source of personal identification, while other means of identification like facial and dermatoglyphic features, tattoos, marks etc. fail due to mutilation, decomposition and charring 8 .
Pulp is soft connective tissue present along the central hollow part of tooth. Pulp surrounded by dental hard tissues is considered to be the most protected oral tissue 9 . Thus in blood grouping, dental pulp tissues could be considered as a hall mark for identifying biological materials in forensic studies.
India is a country of paradoxes. On one hand, we have sophisticated laboratories in the big metropolitan cities and on the other hand in rural areas there is absence of even basic laboratory facilities to handle the routine procedures. In such case scenarios, DNA analysis for forensic purposes may not be possible or it may take a long time 8,9 . Hence ABO blood group analysis for preliminary investigations may be essential in forensic odontology. Adsorption-Elution method is one of the commonly used techniques to determine ABO blood group from dental pulp tissues 10,13,14 . This technique was devised by Siracusa in 1923 and has been refined by Kind 11,12 . In highly decomposed bodies where teeth and the bones are the only source remaining, the presence of ABO blood group antigen in the dental soft tissue helps in the process of identification 10,12 . ABO blood grouping has been done by many investigators at room temperatures but in spite of extensive literature there were no studies on ABO blood group analysis where the teeth are exposed to high temperatures like fire victims, electrical burns, aircraft fire accidents, victims of volcanic eruptions etc. or low temperature like in snow slide accidents.
The present study was done to evaluate the efficacy of ABO blood group antigens retrieval from pulp tissue at various temperatures like freezing temperature, extreme temperature 100°C and 200°C with room temperature being the baseline of the study and at various time intervals (same day, 14 th , 30 th and 90 th day) post extraction.

Materials and Methods
A diagnostic study of 80 cases were included of age group 13 years and above with both male and female. The cases were obtained after surgical extraction of the teeth from the concerned department along with permission and written consent of the patient and bystander. The blood soaked cotton/gauze was used to determine the ABO blood group to avoid bias and was used as control. The extracted teeth were exposed to various temperatures, i.e. room temperature, freezing temperature (Remi Deep Freezer), 100°C and 200°C (Innovative incubator) and was stored for ABO antigen determination on same day, 14 th , 30 th and 90 th day post extraction. The Inclusion criteria were male and female patients of age 13 years and above, permanent teeth, teeth extracted for orthodontic purpose and periodontal problems. The exclusion criteria were carious teeth, endodontically treated teeth, grossly decayed teeth, grossly destructed with exposed pulp cavities and calcified root canals and deciduous teeth.
Dental extraction procedures were performed under local anesthesia following aseptic measures. After extraction, the sockets were compressed with sterile cotton/gauze and blood was collected. Normal saline and anticoagulant were added, later diluted to this blood in a sterile test tube. Three glass slides were arranged and to each slide single drop of diluted blood was placed, followed by a drop of anti-serum A, B and D. Presence of agglutination after addition of antiserum indicated that the particular blood group was present and if agglutination occurred in a slide with antiserum D, it indicated its positivity. This was considered control for the study.
Running water was used to wash the extracted teeth followed by removal of debris with the help of the probe. Later the teeth were wiped with gauze and were stored in bottles, labelled with numbers. The tooth was subjected to different degree temperatures i.e. at room temperature, freezing temperature ( Figure 1) 100°C and 200°C for 60 minutes respectively and stored for different time intervals. The extracted teeth were assessed for ABO blood group by the method of Adsorption-Elution test using dental pulp, which was later compared to the control blood group.
The tooth was embedded on a block shaped modelling wax. Vertical split was placed parallel to the long axis of the tooth (Figure 2) with the help of carborundum disc. Later the dental pulp was scooped out with a sterile spoon excavator and was placed in a test tube containing a drop of saline. To each test tube 3 drops of anti A and anti B serum was added and left at room temperature for three hours. The test tube was centrifuged and the supernatant (antiserum) was pipetted out. Later each sample was washed 3 times with cold saline. Two drops of fresh saline was added to the sample, later the test tube was heated in a water bath at a temperature of 50-55°C for ten minutes to elute the antibodies. A drop of 0.5% A or B group red cell suspension was added and incubated for 30 min, later it was centrifuged at 1500-2000 RPM for 1 min. (flow chart Figure 3). Gentle shaking of the test tube was done, indicating the presence ( Figure 4) or absence ( Figure 5) of red blood cell agglutination, which was seen under the bright field light microscope at a magnification of 100x. (OLYMPUS BX40).

Results
The ABO blood group distribution of total 80 cases was tabulated according to temperature and time interval   were made for same day, 14th day, 30 th day and 90 th day of post extraction and the results were tabulated and subjected to Goodman Krusal Gamma test. Goodman Kruskal Gamma test was done for analysis to obtain the correlation between blood groups at different temperatures. The following formula was used: Goodman Kruskal Gamma = Nc-Nd/Nc+Nd Nc -stands for number of concordant pairs and Ndstands for number of discordant pairs.
The correlation between the study and control groups of Room and Freezing Temperatures at various storage intervals were in good correlation (statistically significant) (Tables no 1 to 8). However the correlation between the study and control groups of high temperatures i.e. 100°C and 200°C at various storage intervals had negative correlation (statistically insignificant) (Tables no 9 to 16).
The sensitivity of pulp of various blood groups (ABO antigen) revealed that at room and freezing temperatures at various time storage intervals (the sensitivity was almost 80 to 100%) i.e. antigens can be calculated, while at higher temperatures (sensitivity was hardly 20%) it was difficult to detect the ABO antigens. (Figure 6/Graph)

Discussion
Forensic odontology is that speciality of dentistry which deals with application of dental evidence to the field of law 35,36 . Blood groups have been a foundation for identification of biological materials in forensic medicine 10,13 . In most of the adverse environmental conditions, the teeth serve as a vital biological trace material in the process of identification 7,8,10,28,29 . ABO blood group antigens are present in dental tissues and thus makes its possible in identification of putrefied bodies in cases where only teeth and bone are the vital remains 10,14,15,20,32 , According to Rothwell B.R, blood group substance used in forensic procedures are based on the point that once the blood group of an individual is established, it remains unaltered through the life. The dental pulp tissues used for blood grouping are considered authentic for identifying biological material in medicolegal procedures 4,6,15,16,17,33 .
Forensic experts for several years have been exploring a definitive method for blood typing of teeth. Shimura et al employed conventional adsorption inhibition technique for hard tissue but the problem was complicated pretreatments and the results were inadequate. Later after few years adsorption inhibition technique was replaced by adsorption elution method which was solely used in forensic laboratories 10,18,19,26 . Although the procedure was described 50 years ago, it was not widely used for forensic serology until 1960. Later clarified the technique. Vittorio Siracusa developed the adsorption elution procedure and is now employed in all forensic laboratories due to its proven sensitivity, reliability and reproducibility in identifying blood group of deceased individuals 6,35 .
Mahabalesh Shetty conducted a study on ABO blood grouping from tooth material where pulp and dentine were used for ABO blood grouping using adsorption Overall 5(100%) 0(0%) 1(100%) 0(0%) >0.5 -1 OVERALL 5(100%) 0(0%) 0(0%) 0(0%) --1 elution method 8 . Sushmini Ballal found that the correlation of blood grouping from dentine and dental pulp with the blood collected from the extraction socket of respective teeth can be determined 29 . Success rate was better when ABO blood group was determined at room temperatures 7,12,22 . Researchers have suggested that dental pulp tissue can be used for ABO blood typing of victims 7 . However in spite of extensive search of literature, there were no studies on ABO blood group analysis exposing teeth to high temperatures like fire victims, electrical burns, aircraft fire accidents, victims of volcanic eruptions etc. or low temperature like in snow slide accidents. Therefore in present study, we determined ABO blood group from individuals to examine that if the blood group antigens on the pulp remains possible even after subjecting the teeth to various temperatures and storing for up to 3 months.
In the present study, ABO blood group antigens at room temperature at different time intervals i.e. 18 out of 20 teeth showed 100% sensitivity rates, while two cases showed false positive results.
On the same day and 30 th day post extraction, the sensitivity rate was 100%, whereas on 14 th and 90 th day post extraction it was found as 80%. The results correlated with the study done by Inamdar et al 7 . Blood grouping of dental pulp carried out on 30 th , 90 th and 180 th day of extraction showed only 80% success rates 7 . Similar findings have been reported by Takata and Smeets showed 86% success rate up to 21 months post extraction 22,24 .
ABO blood grouping in dentine and pulp showed 97% sensitivity in pulp 8 whereas in the study conducted by Sushmini 30 i.e. 27 out of 30 teeth showed positive results in pulp and revealed 90% sensitivity. Negative results for ABO blood group antigen on 14 th day post extraction could be due to cross contamination with aerobic gram negative bacteria present in saliva/tooth or due to increased number of foreign antigen borne by bacteria in teeth 8,27,28,29,30 . The negative results on 90 th day of extraction for ABO blood group antigen could be attributed to autolysis, dehydration, loss of pulp antigens or even invasion of certain bacteria in damaged teeth which pass on their random antigenic activities to them 8,29 . It was observed by many authors (Inambdar and Ramanayaka) that storing pulpal tissue more than 90 th day of extraction can result in decrease in sensitivity rates 7,23,34 . According to Heartig et al. the decrease in the success rate of their study at room temperature could be due to cell lysis, tooth contamination or time lapse for the procedure 7, 16, 17 . Thus we can hypothesize that the false positive results in the samples could be due to the massive growth of aerobic gram negative bacteria that tends to obscure the pulpal blood group antigen and it can produce cross reaction.
Negative results could also be due to inadequate and fibrosed tissues in the pulp with increase in age and calcification 26,28 . Negative results can also occur from the specimen of post mortem dental pulp due to haemolytic erythrocytes, putrefaction and mummification or skeletonised of the body during post mortem interval according to Nalini 21,32 . This finding was also consistent with the study done by Yumi Nakayama and Yasuhiro Aoki 23 .
In present study when considering the higher temperatures (100°C and 200°C) at 10 °C on the day of extraction, 30 th day and 90 th day of extraction the sensitivity was found to be 0%, whereas on 14 th day of extraction it showed 20% sensitivity which means 1 out of 5 cases were true positive, whereas on 14 th day of extraction it also showed 2 false positive results. Hence caution should be exercised while considering the ABO blood group result in teeth exposed to such high temperatures. While at 200°C on the day of extraction, 14 th day, 30 th day and 90 th day of extraction, ABO blood grouping did not show any results and the sensitivity was found to be 0%. This is in accordance with Mondal et al. who mentions that heating at 200°C over destroys blood group substances 25,31 . Therefore we hypothesize that this finding may be due to effect of high temperatures that cause heat denaturation of antigens resulting in lack of reaction. In denaturation of antigens proteins lose the quaternary, tertiary and secondary structure. The characteristic variability of the denatured proteins shows loss of solubility to communal aggregation of the hydrophobic proteins to come closer and form bonding between them, so as to reduce the total area exposed to water [36][37][38] .
When the ABO blood grouping was analyzed at freezing temperature, it was found that on the day of extraction and 14 th day of extraction sensitivity was found 100% .While as time intervals progressed from 30 th day to 90 th day of extraction, the sensitivity was reduced from 80% to 60% respectively. Several investigators have found contradicting results to our findings, showing that at lower temperature there will be denaturation of antigens 36 .
Cold denaturation can spontaneously unwind proteins. Therefore we hypothesize that at lower temperatures the structure and antigenicity may be preserved for limited period i.e. possibly till a fortnight after which there could be loss or denaturation of antigens. The denaturation of proteins is temperature dependent as described by the Molar Gibbs free-energy change. The mechanism behind the temperature induced denaturation is not clear and it could be due to changes in the structure of water 39 .
The equilibrium between enthalpic (amount of internal energy in a compound) and entropic (amount of intrinsic disorder in a compound) effects are responsible for the stability of the proteins. Heat and cold denaturation are mainly due to increased entropic effects and hydration of internal non polar groups. So in either temperature level the proteins get denatured 38 . The heat and cold induced denaturation temperatures define the temperature at which Gibbs free energy of unfolding equals zero. This free energy contributed by both enthalpic and entropic, change with temperature and gives rise to heat denaturation and to some, cold denaturation. Therefore before forming any tight bonding, antigens and antibodies in a solution have to overcome this large entropic barriers [39][40][41] .
From our results we infer that there is not much sensitivity deviation observed between freezing and room temperature cases on the same day of extraction and 14 th day of extraction. But on 30 th and 90 th day of extraction sensitivity was found decreasing which could be due to cold denaturation or cross contamination. And at higher temperature, the lower sensitivity rate which could be due to heat denaturation. Therefore, we can say that, it might be tricky for us to identify ABO blood group of the deceased exposed to high temperatures that is 100 and 200 degrees. Hence temperature and time duration, to which the deceased individual is exposed to, has to be considered during ABO blood grouping. In present study the positive results of ABO blood group obtained from dental pulp can conclude that pulps possess high potential value for application at high and freezing temperatures in forensic odontology.
Hence these results accentuates that the identification of individuals even after the span of 3 months of death by the virtue of Adsorption-Elution method can be relatively determined by blood typing of tooth pulp.

Conclusion
The present study depicts the ABO blood group at different temperatures and different time intervals reflect antigen sensitivity. The results of the study show significant reduction in sensitivity at higher temperatures and with increasing the time of storage of pulp antigen. This indicates that ABO blood grouping can contribute in identification of deceased individual at room temperature, in lower temperatures for limited time, but it has restricted role at higher temperatures. However further diagnostic studies on larger series of cases are required to determine the practical use of ABO blood grouping in forensic odontology.