Saliva as a Diagnostic Tool for AB0 Blood Group Determination

The simplicity and convenience of collecting saliva, as well as its component
composition, make this biological fluid a common object of laboratory research, in
particular for detecting the nucleic acids of viruses and bacteria, monitoring certain
clinical conditions. The range of clinical situations where to use saliva as a diagnostic
tool is becoming increasingly widespread and also includes determination of secretory
status. The aim of this paper is to characterize the secretory status of the oral fluid,
depending on the presence of group-specific antigens of the AB0 system.


Introduction
Constancy of internal environment of an organism is a necessary condition for normal functioning and vital activity of all its systems. Homeostasis maintenance is based on numerous physiological mechanisms, one of which is the presence of histohematic barriers. For the first time the term "histo-hematic barrier" was used in 1929 by the Soviet scientist L.S. Stern who defined it as a plastic, mobile mechanism taking part in maintaining the constancy of the internal environment and function, which can be regulated by exogenous and endogenous biologically active substances [1].
By means of histo-hematic barriers the passage of electrolytes, proteins with small molecular weight, products of metabolism, factors of specific and non-specific protection from blood to tissue fluid is carried out, which promotes breathing, trophism, cells proliferation and differentiation, elimination of unnecessary components formed during metabolism from organs and tissues [2]. Histo-hematic barriers maintain their functions due to their selective permeability, which is determined by the peculiarities of their structure. Hemato-encephalic (blood-brain barrier), hematoplacental, hemato-ophthalmic (blood-ocular barrier), and hematosalivary barriers are distinguished by their localization [3].
We are interested in studying the structure and properties of the hemato-salivary barrier. It represents a mechanism regulating selective intake of substances from blood into oral liquid [4].
The hemato-salivary barrier is formed by endothelium lining the capillary wall, myoepithelial, secretory cells, cells of salivary gland outlet ducts [5]. In recent years, interest in rapid and less invasive diagnostic tests has increased in progression, resulting in extensive studies of oral fluid as an object of clinical diagnosis. Oral fluid has some advantages over blood and urine, the two most commonly used biological fluids in the laboratory. Oral fluids can be obtained easily and noninvasively, with lower protein content and a less complex component composition compared to blood serum [6]. Oral fluid is successfully used in kidney disease monitoring, cardiometabolic risk prevention, forensic medicine, and drug abuse monitoring, as well as in the diagnosis of Human Immunodeficiency Virus (HIV) and other nucleic acids of bacteria and viruses, even including COVID-19. According to the latest data, there is a significant correlation between AB0 blood group affiliation and predisposition to COVID-19. It was shown in the study including more than 2000 patients that A(II) blood group carriers are more likely to have the disease [7]. The objective of this study is to characterize the secretory status of oral fluid depending on the presence of groupspecific antigens according to the AB0 system.

Materials and Methods
The study was conducted in the clinical diagnostic laboratory The average age of the study group was 27.5±5.1 years. Voluntary informed consent to participate in the study of blood and oral fluid was obtained. The study material was venous blood obtained using vacuum blood collection systems and oral fluid collected in a sterile plastic disposable container by spitting. Before oral fluids were taken, all participants were instructed with the rules of preparation and the procedure for collecting oral fluids. Oral fluid was collected in the morning hours, on an empty stomach, not earlier than 15 minutes after teeth cleaning. One hour before collecting oral fluid, the mouth was rinsed with boiled water. Before collecting oral fluid, physical and emotional stress and smoking were excluded. Samples with blood were not accepted for examination [8].

Results
In our study, we found that out of 89 subjects, 42.5% had blood group 0(I), 31.5% had blood group A(II), 16.5% had blood group B(III), and 9.5% had blood group AB(IV), with Rh-positive 82.7% and Rh-negative 17.3% (Figure 1). Since the group agglutinogens A and B are mainly localized on the erythrocyte membrane, we have studied the dependence of erythrocyte parameters on the group belonging by the system AB0. The obtained results are presented in Table 1 The presence of antigens A or B in the oral fluid was determined in 44.1 % of cases, while the number of non-secreting antigens is 55.9 %. It should be noted that 42.5% of the examined individuals have 0(I) blood group, so they cannot be antigen A and B secretors due to their absence on the membrane surface of red blood cells.
Thus, the absence of antigens A and B in individuals with "nonzero" blood group is observed in 13.4% of subjects, 9% of whom are blood group B(III) carriers. The presence of antigen A in the oral fluid was found in 27% and antigen B in 7.6%. In 9.5% of the individuals examined, both antigens are secreted (Figure 2). Table 1: Variability of erythrocytic parameters depending on group affiliation according to AB0 system (n=89).

Parameter Statistical indicator 0 (I) A(II) B(III) AB (IV)
RBC, x10 12    the oral fluid has been described by Yamakami [11], noting also that the antigens in the oral fluid is fully consistent with the antigenic composition of the blood in the case if a person is the secretor. In a recent study of oral fluid proteome [12] it was noted that the composition of glycans differed significantly when comparing secretors and non-secretors. This is explained by the fact that non-secretors have inactive forms of fucosyl transferase, which provides the construction of a glycanic framework for further transformation of epitopes of molecules. Therefore, non-secretors have less fucosylated derivatives, however, they have a higher content of sialated molecules [13]. In addition, the study of glycome -a set of glycoproteins -of oral fluid in people with different blood groups according to AB0 system revealed different glycan composition depending on time of day and physical activity [14]. c) The number of "true" non-secretors -individuals with "non-zero" blood group, who do not have either A or B antigens in oral fluid was 13.4%, and 9% of them clinically determined blood type B(III).
Antigens of the AB0 system are traditionally considered in the context of immunohematology and transfusiology. Recently, however, more and more data on the significance of study in oral fluid have been appearing. These results are of interest not only for practical medicine as a convenient and non-invasive method of diagnosis, but also for solving fundamental problems. Determining the patient's oral fluid glycome as an individual parameter of a particular individual will help to create a qualitatively new personalized approach in preclinical diagnosis and to develop preventive measures for many known diseases.