methods of immunologic research

2.5.1. Indicators of the general immunity

The analysis of indicators of the general immunity spent at the primary reference of patients, the analysis of indicators of local immunity carried out prior to the beginning of treatment and in three months of therapy at patients of 1 and 2 groups.

For an estimation of a condition of system (general) immunity at patients defined following indicators of blood:

• Total T-limfotsitov (CD3 +), %, a g/l

• "active" lymphocytes (CD4 +/25 +), %

• T-helpery (CD4 +), %

• TSTL ^D3 +/CD8 +), %

• Balance immunoreguljatornyh cells (CD8 +/CD4 +), %

• Number V-limfotsitov (CD22 +), %

• blood Immunoglobulins, a g/l:

o IgA

o IgM

o IgG

• Phagocytic activity of neutrophils of peripheric blood:

About the Phagocytic index (FI)

About Phagocytic number (FCH)

About KZF

Definition of total T-limfotsitov, their subpopulation structure, and also number V-limfotsitov was spent with the help
Monoclonal antibodies (MKAT) a method of flowing laser cytometry. It is one of modern methods of research of immunity at which carrying out MKAT react it is strictly specific with one type of receptors on a cell surface that allows to receive exact results. For definition of quantity mature T-limfotsitov were used MKAT specificity CD3 +. The subpopulation analysis immunoreguljatornyh T-limfotsitov defined by means of MKAT specificity CD4 + (helperno-induktornye) and CD8 + (supressorno-cytotoxic). The estimation of quantity V-limfotsitov was spent by means of MKAT specificity of CD 19 + and CD22 +.

Functional activity V-limfotsitov was defined on level of immunoglobulins cosecreted by them by an immunodiffusion method in an agar with use of monospecific antiserums to serumal IgA, IgM, IgG on Mancini G. (1965), in Chernohvostovoj E. V's updating (1987) [71, 139].

The essence of a method consists that in an agar where preliminary dispergirovana a monospecific antiserum, bring reference Serum of the person with the known maintenance of immunoglobulins and examinees of blood serum of the person. The precipitation rings which diameters are directly proportional to quantity of the brought antigen are thus formed. Measure diameters of the formed rings of a precipitation and build schedules for each immunoglobulin separately.

Phagocytic function of neutrophils was studied with bacteriemic the test - culture. The percent of englobing neutrophils - a phagocytic index (FI) and quantity fagotsitirovannyh microbes in one neutrophil from 100 counted cells - phagocytic number (FCH) was counted up.

2.5.2 Indicators of local immunity

The immune system of an oral cavity includes two subsystems, one of which is presented by cellular components of various departments of an oral cavity and immunoglobulins synthesised by them, another - the cellular and humoral factors of the general immunity arriving in an oral cavity through desnevuju a sulcus from the general blood flow.

For an estimation of a condition of local immunity in an oral cavity carried out the analysis of a saliva of patients (secretory factors) and the analysis of washouts from an oral cavity (cellular factors). For research used nestimulirovannuju the admixed saliva in number of 3-5 ml which collected in a plastic test tube in 1 hour after meal. For reception of washout from an oral cavity to the patient suggested to rinse an oral cavity of 0,9 % of a solution of Sodium chloridum of 10 ml within 2-3 minutes, washout collected in a plastic test tube.

A test tube with washout cells tsentrifugirovali, a deposit washed solution Henksa, then solution Henksa added to concentration of cells 2х106 kl/ml. Viability of cells defined on a staining of 0,3 % a solution tripanovogo dark blue (dead cells are painted only). In reaction it was supposed having washed off with the maintenance not less than 60 % of teleorgánic neutrophils.

Defined following indicators:

• the General number of neutrophils in washout from an oral cavity, %

For definition of quantity of neutrophils in washouts spent cytology of washouts with calculation of cells.

• Subpopulation structure of neutrophils:

About Late neutrophils (EN-PO), %. These are the mature neutrophils possessing the highest phagocytic activity and a set
Bactericidal fibers and the enzymes participating in destruction of cells damaged at an inflammation and tissues of an organism.

o Double neutrophils (DRON), %. They are precursors of the late neutrophils which quantity should be enlarged at reduction of the maintenance of late neutrophils and, accordingly, depression of cellular mechanisms of protection.

The estimation of subpopulation structure of neutrophils was spent by means of erythrocyte markers in reactions rozetkoobrazovanija (I.V.Petrova and co-workers., 1984) with revealing of number EN-PO in reaction spontaneous rozetkoobrazovanija and numbers DRON in reaction komplementarnogo rozetkoobrazovanija.

Directly ahead of reaction statement prepared suspension of erythrocyte markers. For this purpose spent washing up by a normal saline solution of erythrocytes of a ram (EB) a centrifugation until nadosadochnaja the liquid did not become colourless. From the washed deposit prepared 0,5 % suspension of erythrocytes on the medium 199.

Definition of number of late neutrophils (EN-PO) by means of reaction spontaneous rozetkoobrazovanija. Neutrophils have on the surface receptors to erythrocytes a ram (Eb-receptors), at bracing of erythrocytes of a ram "rosella" formation occurs by them. To 0,1 ml of a deposit of cells added 0,1 ml of 0,5 % of a solution of erythrocytes of a ram, inkubirovali an admixture at t - 37 C 5 minutes, further in a refrigeration cabinet 2 hours for formation of rosellas. Then added for bracing of rosellas of 0,6 % of a solution of 0,05 ml gljutaraldegida and left for 20 minutes at a room temperature. Washed from the further influence gljutaraldegida a normal saline solution 2 times by a centrifugation, then did smears, dried, fixed in alcohol, painted on Romanovsky - Gimza and counted up number rozetkoobrazujushchih neutrophils (E - an erythrocyte, N - a neutrophil, RO - rozetkoobrazovanie). The rosella the cell which has attached not less 3х of erythrocytes is considered.

Definition of quantity of double neutrophils (DRON) spent to reactions komplementarnogo rozetkoobrazovanija. Early neutrophils, besides Eb -
Receptors on the surface have receptors to a complement () and, therefore, are designated as double neutrophils (DRON). In reaction komplementarnogo rozetkoobrazovanija besides erythrocytes of a ram standard hemolitic Serum with antibodies () to erythrocytes of a ram in delution 1:200 is used. For carrying out of reaction of hemolitic Serum of 2,0 ml bridged about 2,0 ml of erythrocytes of a ram (), inkubirovali at t - 37 C 30 minutes for complex formation (EA). A standard dry complement of the mouse planted 1:10 and to 2,0 ml EA added 2,0 ml of a complement (), inkubirovali at t - 37 C for formation of complex EAS. An admixture washed in medium twice from excess of a complement by a centrifugation. Further 0,1 ml of complex EAS bridged to a deposit of neutrophils, inkubirovali at t - 37 C 30 minutes and tsentrifugirovali 5 minutes for planting EAS to neutrophils. Following stages did not differ from reaction spontaneous rozetkoobrazovanija.

• Phagocytic function of neutrophils:

About the Phagocytic index (FI) is the percent of englobing neutrophils which are an indicator of activity of a phagocytosis.

About Phagocytic number (FCH) is a number of microbes, fagotsitirovannyh on the average one neutrophil. The given indicator reflects intensity of a phagocytosis.

For definition of phagocytic function of neutrophils the test with bacteriemic culture on I.JA.Serebrijsky's method and co-workers was carried out. (1950): to 0,1 ml of a deposit of cells of washout added 0,1 ml of a suspension bacteriemic the test - the culture containing 2 billion of microbic bodies in 1 ml of a normal saline solution. The admixture inkubirovali at t - 37 C within 30 minutes, was done by smears, dried up, fixed in alcohol and painted on Romanovsky - Gimza in delutions 1:20. In smears counted up percent of englobing neutrophils - FI, and also counted up number of microbes, fagotsitirovannyh on the average one neutrophil - FCH.

• Parities for an estimation of secretory mechanisms of protection:

About S-IgA/IgG - a parity of level secretory S-IgA, formed in an oral cavity as a result of interaction In - lymphocytes (synthesising subedinitsy IgA) and the epithelial cells producing a secretory component (S), to IgG. The given indicator specifies in prevalence protective secretory S-IgA, or prevalence of an inflammatory component (IgG).

About S-IgA/IgA - the parity which is an indicator

Abilities of epithelial cells of a mucosa of an oral cavity to synthesise a secretory S-component,

Necessary for synthesis S-IgA.

• Parities for an interrelation estimation between secretory, humoral and cellular components of protection in an oral cavity:

About IgG/ФИ

About IgG/ФЧ

Concentration of immunoglobulins in a saliva was defined a method radial by immunodiffusions (READ) in gel on Manchini (Mancini G. et al., 1965) with use of monospecific antiserums to S-IgA, IgA, IgM, IgG. As concentration of immunoglobulins in cosecreted liquids much more low, than in blood serum, at their definition in an agar dispergirovali antiserums in concentration in 3-4 times smaller, than at definition of serumal immunoglobulins.

Also carried out calculation of indexes of an allergization and an intoxication. Normal value of a leukocytic index of an intoxication (LII) is in a limit from 0,5 to 1,0. The formula for calculation LII:

In a basis of an index allergizations (news agency) the sum parity was necessary
Lymphocytes and eosinocytes to other cells of white blood. In norm the allergization index makes 0,7-1,0.


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Scientific source ZASLAVSKAYA NATALIA ALEKSANDROVNA. OPTIMIZATION of PREVENTIVE MAINTENANCE And TREATMENT BISFOSFONATNYH of OSTEONECROSES of JAWS At PATIENTS WITH MALIGNANT NEOPLASMS. The dissertation on competition of a scientific degree of the candidate of medical sciences. Moscow - 2014. 2014

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