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Original Research

ABO Blood Group Detection in Extracted Teeth: A Forensic

Study
Mithilesh N. Mishra1, Vidyadevi Chandavarkar1, Deepak Bhargava1, Ritika Sharma1, Radhika Gupta2, Sahil Thakar3
1
Department of Oral Pathology, School of Dental Sciences, Sharda University, Greater Noida, 2Department of Periodontology, School of Dental Sciences, Sharda
University, Greater Noida, 3Department of Public Health Dentistry, School of Dental Sciences, Sharda University, Greater Noida, Uttar Pradesh, India

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

Received: 21-04-2020, Revised: 30-04-2020, Accepted: 20-08-2020, Published: 28-01-2021.

Introduction Blood group substances are secreted in numerous body

fluids including saliva, mucus, tears, seminal fluid, vaginal
Forensic odontology is a relevant and indispensable science
fluid, and gastric juice. In 1960, Kind came up with the
in medicolegal cases and identification of the deceased.
presence of ABO blood group substances in saliva by the
Dental tissues are well protected for a long time even after
absorption–elution (AE) method where he could detect
death; therefore, they remain as the most stable biological
the blood group form a dried stain. AE procedure was
evidence.[1,2] Teeth are invaluable sources of personal
foremostly concocted by Siracusa and is now used in
identification in catastrophe. Pulp within a tooth is the
all forensic laboratories because it is proven to be most
most protected oral tissue being surrounded by dental hard
sensitive, reliable, and consistent.[6,7] In the AE method,
tissues from all the sides. As pulp contains numerous blood
dried pulpal content is mixed with antisera A and B. Fresh
vessels; blood group agglutinogens, and agglutinins (A, B,
saline is added to elute the antibodies. This leads to
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 Address for correspondence: Dr. Mithilesh N. Mishra,
the blood group and Rhesus factor of an individual is Department of Oral Pathology, School of Dental Sciences,
established, it remains unchanged for the rest of life.[5] Sharda University, Knowledge Park III, Greater Noida,
Gautam Buddha Nagar 201308, Uttar Pradesh, India.
E-mail: mitsmishra1810@rediffmail.com

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DOI: How to cite this article: Mishra MN, Chandavarkar V, Bhargava

10.4103/jioh.jioh_149_20 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.

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Mishra, et al.: Blood grouping in teeth
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
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
Figure 1: Micromotor with carborundum disc to dissect tooth
94 94  Journal of International Oral Health ¦ Volume 13 ¦ Issue 1 ¦ January-February 2021
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
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Mishra, et al.: Blood grouping in teeth
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 [Figures 3 and 4].
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
Figure 2: Dental pulp in antisera A and B
Figure 3: Agglutination grossly
      Journal of International Oral Health ¦ Volume 13 ¦ Issue 1 ¦ January-February 2021
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
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.
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.
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).
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
Figure 4: Agglutination microscopically (x400)
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Mishra, et al.: Blood grouping in teeth

Table 1: Age-wise distribution of subjects in two groups

Age group (years) Group 1 (on the day of extraction) Group 2 (after 9 months of extraction) Total N = 60 (100%) χ2
N = 30 (50%) N = 30 (50%)
11–20 6 (20%) 1 (3.33%) 7 6.73
21–30 5 (16.66%) 6 (20%) 12 P = 0.15
31–40 7 (23.33%) 8 (26.66%) 15
41–50 6 (20%) 12 (40%) 18
51–60 6 (20%) 3 (10%) 9
Total 30 (100%) 30 (100%) 60
Mean 35.83 38.83
SD 14.26 10.30
Range 15–60 18-60
SD = standard deviation
* Not significant, P > 0.05

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

Gender Group 1 (on the day of extraction) Group 2 (after 9 months of extraction) N = 30 Total N = 60 (100%) χ2
N = 30 (50%) (50%)
Male 21 (70%) 18 (60%) 39 0.66
Female 09 (30%) 12 (40%) 21 P = 0.41
Total 30 (100%) 30 (100%) 60
* Not significant, P > 0.05

Table 3: Comparison of both groups with control groups

Blood Group 1 (at the time of extraction) with control Group 2 (after 9 months of extraction) with control
group Control Study group Control Study group
Positive Negative Positive Negative
A 12 (40%) 12 (40%) 0 (0.00%) χ2 = 0.00 07 (23.33%) 06 (20%) 1 (3.33%) χ2 = 2.16
B 08 (26.67%) 08 (26.67%) 0 (0.00%) P = 1.00 14 (46.67%) 10 (33.33%) 4 (13.33%) P = 0.0098
AB 04 (13.33%) 04 (13.33%) 0 (0.00%) 02 (6.67%) 02 (6.67%) 0 (0.00%)
O 06 (20%) 06 (20%) 0 (0.00%) 07 (23.33%) 06 (20%) 1 (3.33%)
Total 30 (100%) 30 (100%) 0 (0.00%) 30 (100%) 24 (80%) 6 (20%)
* Significant, P < 0.05

[Table 5]. A positive, linear, great strength of association

Table 4: Comparison of blood grouping between two study
(r = +0.71) and a significant relationship (P = 0.02) was
groups
found between both the groups using Pearson’s correlation
coefficient [Table 6]. Blood group Group 1 Group 2
A 12 (40%) 6 (20%)
B 08 (26.67%) 10 (33.33%)
Discussion AB 04 (13.33%) 02 (6.67%)
Forensic science has many maxims, perhaps the best known O 06 (20%) 06 (20%)
of which is every contact leaves its trace.[9] Forensic science Not found 0 (0.00%) 06 (20%)
is defined as any expert evidence given by special scientific, Total 30 (100%) 30 (100%)
or experts having particular knowledge and study in any *χ2 = 6.45, P = 0.17; not significant, P > 0.05
particular scientific subjects during the administration
sustain their characteristics even in the most untoward
of justice in the court of law. Pederson defined forensic
environmental conditions. Thence teeth are used for blood
odontology as the branch of dentistry that deals with
grouping and are a hallmark in identifying biological
proper handling and examination of dental evidence with
subjects in forensic investigation. Ramnarayan et  al.[10]
proper evaluation and presentation of dental findings in
reported that apart from ABO agglutinogens, adenylate
the interest of justice.[4,10,11]
kinase, adenosine deaminase, phosphoglucomutase-1, and
Teeth are the most stable biological clue materials G6PD (glucose-6-phosphate dehydrogenase) are naturally
encountered in forensic science, being made up of the found in teeth and could be used for various purposes.[12]
hardest and most stable chemical tissues in the body; they AE technique was originally devised by Siracusa. In 1960,

      
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Mishra, et al.: Blood grouping in teeth
Table 5: Association between Groups 1 (constant) and 2
using the multivariate linear regression analysis
Blood grouping Coefficient t P Value
predictor
A –3.09 –0.99
B 1.34 6.21
AB –0.22 2.47
O 2.33 –1.26
SD = standard deviation
* Significant, P < 0.05
Table 6: Correlation between knowledge, attitudes, and
practices using Pearson’s correlation test
Relationship Pearson’s coefficient CI
between of correlation (r)
Blood grouping 0.71 0–1
(Groups 1 and 2)
CI = confidence interval
*Significant, P < 0.05
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
      Journal of International Oral Health ¦ Volume 13 ¦ Issue 1 ¦ January-February 2021
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%
0.02* of A, 36.67% of B, 10% of AB, and 21.67% of O,
0.55 whereas 30% of A, 30% of B, 10% of AB, and 20% of O,
1.01 respectively, from the extracted teeth (dental pulp) [Table
0.32 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
P Value 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
0.02*
[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 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
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Mishra, et al.: Blood grouping in teeth
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
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
98 98  Journal of International Oral Health ¦ Volume 13 ¦ Issue 1 ¦ January-February 2021
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.
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