Egyptian Journal of Forensic Sciences (2014) xxx, xxx–xxx

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Egyptian Journal of Forensic Sciences

journal homepage: www.ejfs.org

A study on salivary hemagglutinins in a Central Indian

population

Ashish Badiye *, Neeti Kapoor, Vaishali Badiye

Govt. Institute of Forensic Science, Nagpur, Maharashtra 440001, India

Received 20 August 2013; revised 2 November 2013; accepted 11 December 2013

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KEYWORDS

Salivary grouping;

Hemagglutinins in saliva;

Forensic serology;

Saliva;

Forensic biology

Corresponding author. A

ience, Institute of Science Ca

aharashtra 44001, India. Tel

-mail address: Badiye.Ashis

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Abstract Purpose: To study the presence of salivary hemagglutinins in a Central Indian Popula-

tion.

Method: A drop of supernatant of centrifuged saliva samples was placed in cavity tiles. A drop of

0.2% cell suspensions of twice washed cells of known A and B blood groups was added into the

respectively marked cavities. After mixing, the tiles were kept in a moist chamber for 30 min and

examined microscopically. Clumps formed of at least 3–4 cells were taken as positive.

Result: A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has

been developed. Presence of salivary hemagglutinins in Maharashtrians was quite high i.e. 69.41%

as compared to Gonds 17%1 and the Punjabis 35.98%2.

Conclusion: Saliva in the form of stains is encountered as physical evidence in many cases such as

anonymous letters, secret writing, sexual assault, rape, murder, disputed paternity, cigarette butt

ends, etc. If analysed properly saliva can not only help in the elimination of the innocents but also

in the actual identification of a specific individual. Like blood grouping, this can also be used for

forensic purposes.ª 2014 Production and hosting by Elsevier B.V. on behalf of Forensic Medicine Authority.

1. Introduction

Salivary fluid is an exocrine secretion consisting of approxi-mately 99% water and the other 1% is a complex of organicand inorganic molecules, containing a variety of electrolytes

Govt. Institute of Forensic

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87490889.

.com (A. Badiye).

nsic Medicine Authority.

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02

adiye A et al. A study on salirg/10.1016/j.ejfs.2013.12.002

(sodium, potassium, calcium, chloride, magnesium, bicarbon-ate, phosphate) and proteins, represented by enzymes, immu-

noglobulins and other antimicrobial factors, mucosalglycoproteins, traces of albumin and some polypeptides andoligopeptides of importance to oral health. The carbohydrates

of salivary glycoprotein carry the ABH blood group antigens,expressing in the salivary glands and secreting in saliva.

Iso-agglutinins to the ABH antigens are naturally occurring

allo-antibodies regularly found in plasma or serum of anindividual lacking the corresponding antigen on red cells.The reciprocal relationship of these antibodies in serum to

corresponding antigen on red cells helps in confirming theABO blood group of a person and warrants the use ofhomologous blood in transfusion. Besides plasma, the ABOiso-antibodies have also been detected in other body fluids

orensic Medicine Authority.

vary hemagglutinins in a Central Indian population, Egypt J

Figure 1 No hemagglutination (250·).

2 A. Badiye et al.

and secretions.3–8 ABO iso-antibodies are not only confined toserum but are also present in various secretions includingsaliva,1,2,4 cervical secretion,5 tears,6 and milk.9 Badakere

and Bhatia10 found the low titre ABO agglutinins in salivaof Indians from Bombay.

Saliva is an accessible fluid that can easily be collected using

non invasive collection procedure(s) that is neither painful nortraumatic.

Saliva was first analysed for the presence of anti-A and

anti-B hemagglutinins by Yosida,11 when 58 different salivasamples were examined to determine the ABO blood groupof each donor. Since then many investigators have confirmedthe presence of hemagglutinins in saliva that have specificity

for the A and B red blood cells. Although these hemagglutininsprovide information concerning the ABO blood group of theindividual from whom the saliva has been obtained, salivary

hemagglutinins have not been used for forensic science pur-poses primarily as a result of their low concentration and thelack of a sufficiently sensitive procedure to detect and study

them.Kobilinsky and Harrington12 studied that the ability to

blood type an individual by analysis of his or her saliva is

important, especially when cigarette butts, envelope flaps, orother articles with saliva stains are available. Harringtonet al.13 developed a new assay method that includes five com-plementary strategies to increase assay sensitivity for detection

of hemagglutinins in dried saliva stains and their potential usein blood typing.

Similar work on the presence and absence of salivary agglu-

tinins has been done by Sharma and Chattopadhyay2 on Pun-jabis and on Gond Population by Chatterjee and Badiye1 Theyobserved that the frequency of persons having salivary agglu-

tinins varies from one population to another. In the presentstudy 340 Saliva samples have been analysed for the presenceof salivary hemagglutinins with a view to study the percentage

of salivary agglutinins in the Maharashtrian population.

2. Materials and methods

2.1. Ethical clearance

All of the experiments and techniques were conducted in

accordance with principles and procedures approved by theR.T.M. Nagpur University Departmental ResearchCommittee.

2.2. Collection of samples

Subjects were asked to rinse their mouth with water, to remove

any traces of any eatables that they may have had earlier. Ster-ile cotton swabs were placed below their tongue for around3 min. The swabs were then taken out and squeezed into ster-

ilized glass bottles. The vials were closed, sealed kept in thedeep freezer (�4 �C) till their analysis.1 The samples were ana-lysed within 15 days of collection.

2.3. Analysis

The samples were removed from refrigeration and kept forsome time at room temperature (28–32 �C) for thawing. The

saliva samples were then centrifuged at 5000 rpm for 15 min.

Please cite this article in press as: Badiye A et al. A study on salForensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.002

The supernatant was used for testing the presence of aggluti-nins. In a cavity tile, a drop of saliva sample was placed withthe help of a dropper in each of the cavities. A 0.2% cell sus-

pension of twice washed cells of known ‘A’ and ‘B’ bloodgroups was made with normal saline. Neat and clean cavityslides were taken and each of cavities was marked as A and

B along with the serial number of the sample marked on theside. A drop of ‘A’ blood group cell suspension was addedto the cavities marked A, while a drop of ‘B’ blood group cell

suspension was added to the cavities marked B. The contentsof the cavity tile were mixed thoroughly by gentle shaking.The tiles were then kept in a moist chamber (in a Petri dishwith wet cotton around and placed in a refrigerator at 4 �C)for about half an hour.1 The tiles were then taken out, shakengently and examined, first visually followed by microscopicexamination for any possible agglutination reaction. Clumps

formed of at least 3–4 cells were taken as positive reaction.Photomicrographs of the same were taken using a digital cam-era (Kodak M 531 Easyshare) and analysed digitally on a

computer.

3. Observations and results

Saliva samples of 340 Maharashtrian adults have been ana-lysed for the presence of salivary hemagglutinins. On micro-scopic examination absence of hemagglutinins was marked

by the presence of free cells in the cavity tile (Fig. 1). Presenceof hemagglutinins was marked by agglutination (black arrows)(Fig. 2).

Out of all the samples analysed (n = 340), hemagglutina-

tion with A cells was found in 52 samples, hemagglutinationwith B cells was present in 112 samples, hemagglutination withboth A and B cells was present in 72 samples, no hemaggluti-

nation was found in 104 samples either with A or Bcells(Table 1; Fig. 3).

Percentage wise, ‘A’ hemagglutinins were found in 15.29%,

‘B’ hemagglutinins in 32.94%, both ‘A’ and ‘B’ hemagglutininsin 21.18% of the samples. 30.59% of the samples showed noagglutination hence no hemagglutinins (Table 2; Fig. 4).

4. Discussion

In the current work a sensitive method for the detection of

anti-A and anti-B hemagglutinins in fresh saliva has been

ivary hemagglutinins in a Central Indian population, Egypt J

Figure 2 Hemagglutination (250·).

A study on salivary hemagglutinins in a Central Indian population 3

developed. Salivary anti-A and anti-B hemagglutinins are

potentially important since it is possible to determine an indi-vidual’s ABO blood group by analysis of these antibodies.Forensic serologists have thus far been unable to exploit these

52

112

7

0

20

40

60

80

100

120

Agglu�na�on withA Cells

Agglu�na�on withB Cells

Agglu�naA and

Saliva Sa

Figure 3 No. of samples wit

Table 2 Percentage distribution of salivary hemagglutinins among

Hemagglutinins present

Population with

A salivary hemagglutinins

Population with

B salivary hemagglutinins

15.29% 32.94%

69.41%

The bold value denotes the sum total of the percentage of population.

Table 1 Total number of samples with and without hemagglutinin

Presence of hemagglutinins

A B AB

Number of Samples 52 112 72

239

The bold values represent the total number of samples in which agglutin

Please cite this article in press as: Badiye A et al. A study on saliForensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.002

molecules for the purpose of blood grouping for several rea-sons: low concentration of IgA molecules in saliva; lack of asufficiently sensitive detection method; lack of knowledge con-

cerning hemagglutinin stability in wet and dried states; andlack of a database with which to interpret observations.

Harrington et al.12 developed a new assay method that in-

cludes five complementary strategies to increase assay sensitiv-ity for the detection of hemagglutinins in dried saliva stainsand their potential use in blood typing. Badakere and Bhatia6

found the low titre ABO agglutinins in saliva of Indians fromBombay. By contrast, for our studies we modified the tech-nique used by Sharma and Chattopadhyay,7 for fresh salivasamples.

The percentage of population with the presence and/orabsence of salivary hemagglutination in the Maharashtrianpopulation was calculated. Population with A salivary hemag-

glutinins were 15.2 percent, population with B salivary hemag-glutinins were 32.9 percent, population with A and B salivaryhemagglutinins were 21.1 percent, population with no salivary

hemagglutinins were 30.5 percent. Similar studies were done byChatterjee and Badiye1 on Gonds of Betul District of Madhya

2

104

�on withB Cells

No Agglu�na�onwith Either Aand/or B Cells

mples

Saliva Samples

h/without hemagglutinins.

the Maharashtrian population.

Hemagglutinins Absent

Population with A and

B salivary hemagglutinins

Population with no

salivary hemagglutinins

21.18% 30.59%

s.

Absence of hemagglutinins Total sample size

104 340

ation is present.

vary hemagglutinins in a Central Indian population, Egypt J

30.59%

21.18%

32.94%

15.29%

0.00% 5.00% 10.00% 15.00% 20.00% 25.00% 30.00% 35.00%

Absent

A & B both Hemagglu�nins

B Hemagglu�nins

A Hemagglu�nins

Present

Figure 4 Percentage distribution of salivary hemagglutinins among the Maharashtrian population.

Table 3 Percentage of Maharashtrian population with the

presence and absence of salivary hemagglutinins.

Samples with

hemagglutinins

Samples without

hemagglutinins

Total sample

69.41% 30.59% 100%

30.59%

69.41%

Hemagglu�nins in Maharashtrian Popula�on

Absent

Present

Figure 5 Percentage of Maharashtrian population with the

presence and absence of salivary hemagglutinins.

4 A. Badiye et al.

Pradesh, for the presence of salivary hemagglutinins. They

found that the percentage of individuals with salivary aggluti-nins in Gonds is quite low as compared to the Punjabis with35.98 percent (Sharma and Chattopadhyay).2 Our observa-

tions suggest that the percentage of individuals with salivaryhemagglutinins in Maharashtrians was quite high (69.41 per-cent) as compared to Gonds (17 percent)1 and the Punjabis

(35.98 percent).2

5. Conclusion

The percentage of population with the presence and/orabsence of salivary hemagglutination in the Maharashtrian(Central Indian) population was calculated. Salivary hemag-glutinins were present in 69.41% and absent in 30.59% of

Please cite this article in press as: Badiye A et al. A study on salForensic Sci (2014), http://dx.doi.org/10.1016/j.ejfs.2013.12.002

the population (Table 3; Fig. 5). Population with A salivaryhemagglutinins were 15.2 percent, population with B salivary

hemagglutinins were 32.9 percent, population with A and Bsalivary hemagglutinins were 21.1 percent, population withno salivary hemagglutinins were 30.59 percent.

A sensitive method for the detection of anti-A and anti-Bhemagglutinins in fresh saliva has been developed. Salivaryanti-A and anti-B hemagglutinins are potentially importantsince it is possible to determine an individual’s ABO blood

group by analysis of these antibodies. Forensic serologists havethus far been unable to exploit these molecules for the purposeof blood grouping for several reasons: low concentration of

IgA molecules in saliva; lack of a sufficiently sensitive detec-tion method; lack of knowledge concerning hemagglutinin sta-bility in wet and dried states; and lack of a database with

which to interpret observations.12

The results of the hemagglutinin assay are not affected bythe age or sex of the sample donor.12 Since salivary hemagglu-

tinins can be used to determine ABO blood type, analyses ofthis kind can serve as an important confirmatory test whichthe forensic serologist can use in conjunction with salivaryagglutinogen determinations. The implication of the current

work is almost similar to that of blood grouping, as it maybe used in cases of genetic inheritance,14 individualization, sex-ual abuse, disputed parentage,15 etc. Even with the latest

advancements in DNA analysis and DNA fingerprinting,blood grouping still continues to be a basis for the forensiccaseworks. Likewise, saliva or saliva stains when recovered

from scene of crime may be tested preliminarily for the pres-ence or absence of salivary hemagglutinins for elimination pur-poses (e.g. narrowing down of suspect pool like in case of

blood grouping). Further work in this topic considering otherfactor(s) in a larger set of population is expected so as to in-crease the database and usage of salivary hemagglutinins inroutine forensic case work and examination.

Funding

None.

ivary hemagglutinins in a Central Indian population, Egypt J

A study on salivary hemagglutinins in a Central Indian population 5

Conflict of interest

None declared.

Ethical approval

Necessary ethical approval was obtained from the institute

ethics committee

Informed consent

Informed consent were enrolled for the study

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