RU2211452C2 - Method for in vitro detecting allergization phenomena on administering captopryl - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves determining increase in amoeboid activity of neutrophils when incubating blood taken from an organism sensitized with Captopryl. The reaction was set as follows. 0.1 ml of allergen is added to case test tube in required concentration, 0.1 ml of 5% sodium nitrate is added to control test tube. Then, blood under test is added into both test tubes. The case and control tubes are incubated for 2 h at 37 C. Blood smears are sequentially stained with Schiff reactant and Ehrlich hematoxylin. Neutrophil injury index is determined from formula (H₁-H₂):100, where H₁ is the injured neutrophil number in case, and H₂ is the injured neutrophil number in control tube, 100 is examined in smear. Diagnostic value is assumed to be equal to 0.1 and higher. EFFECT: high accuracy and sensitivity of the method. 2 dwg, 4 tbl

Description

 The invention relates to medicine, for the use of an in vitro diagnostic method for detecting allergization with captopril, which can be used as a laboratory test for the timely assessment of body sensitization and the development of allergic complications in the treatment of this drug.

 It is known that in vitro methods have been developed to assess the status of delayed-type hypersensitivity in patients who are not recommended to have direct skin reactions. In addition, it was found that there is a direct correlation between skin tests and similar tests both for chronic infections and for allergic reactions to medications (Fradkin V.A. Allergodiagnosis in vitro - M. - 1975. - 143 p.).

 It has been proven that captopril containing a free sulfohydryl group is able to easily form covalent disulfide bonds with serum albumin. Drug-specific antibodies are synthesized on the conjugate in the body (Coleman JW, Jeung J.HK., Tingli HD, Park BK Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to protein-reactive drugs and metabolites: criteria for indentification of antibody activity // J. Imrnunol. Methods. - 1986. - V.88. - N. 1. - P.37-44.).

 It is known that captopril therapy can cause certain changes in the blood picture in the form of neutropenia, leukopenia, agranulocytosis. In addition, a number of adverse reactions of an immunological nature are known in the treatment of patients with this drug (Vilchinskaya M.Yu., Nasonov E.L., Zharova EA.The immunological effect of captopril and ramipril in patients with hypertension // Clin. Honey. - 1990 . - .8. - S. 56-57.).

 In the studied literature, we did not come across data on the developed tests for detecting allergization in the treatment of captopril patients. This issue becomes relevant primarily because antihypertensive therapy with captopril is carried out for a long time, and often for life.

 The aim of the present invention is to develop a method for detecting in vitro the effect of allergization on the administration of captopril.

 To achieve this goal, an indicator of neutrophil damage was used (PPN Test). The principle of the method is based on enhancing the amoeboid activity of neutrophils during incubation of the blood of a sensitized organism, which manifests itself in the appearance of several protrusions in the leukocyte - pseudopodia or lobodopia (Fig. 1). An allergenic dose was selected in preliminary experiments with the blood of intact animals. The optimal concentration of the drug was adopted, which ensured the absence of a pronounced amoeboid reaction from neutrophils or intracellular degenerative changes. Intact animals were also used for control. The specificity of the reaction was determined on guinea pigs immunized with other drugs - dinacil, penicillin, bovine serum albumin (BSA).

The reaction was carried out by a known method: 0.1 ml of allergen was added to the test tube in the required concentrations, and 0.1 ml of 5% sodium citrate was added to the control tube. Then, 0.4 ml of test blood was added to both tubes. The experimental and control tubes were incubated for 2 hours at 37 ^o C. Blood smears were stained sequentially with Schiff's reagent and Erlich hematoxylin (V. Fradkin Allergodiagnostika in vitro - M. - 1975. - 143 S.). The neutrophil damage index was determined by the formula - (H ₁ -H ₂ ): 100, where H ₁ is the number of damaged neutrophils in the experiment, N ₂ in the control, 100 - viewed in the smear. For the diagnostic value, indicators from 0.1 and higher were taken.

 Guinea pigs were used in the experiment, the concentration of captopril 250 μg / ml of the studied blood was chosen as the optimal allergenic dose for the PPN test. An increase in the amount of the drug had a toxic effect, since the PPN in the blood of control animals exceeded 0.1 (Table 1). When selecting an allergizing dose in people, it turned out to be even less - 125 μg / ml.

 As a result of the study, it was found that the values of the PPN test with blood neutrophils of the sensitized organism significantly exceeded those in the control samples, and this method can be used as an in vitro laboratory test to assess the body allergization with captopril.

 Examples of specific applications.

 Example 1. The study of the effect of allergization in guinea pigs after parenteral administration of captopril.

 In the experiment, guinea pigs (n = 25) were used, who were injected subcutaneously with captopril at a dose of 5 mg / kg body weight on Freund's adjuvant, two injections with an interval of 6 days, blood was taken sequentially on days 2, 7, 14, 21, and 45 from re-immunization.

 The neutrophil damage index was positive on the 2nd day after re-immunization, that is, on the 9th after the first injection of the drug. The PPN test (> 0.3) reached its maximum value on the 7-14th day (Fig. 2 and Table 2), subsequently gradually decreased, but allergization, quite moderate, continued to persist for up to 45 days (maximum observation period). Heterologous preparations (benzylpenicillin, dinacil and BSA) were administered to the control groups of animals according to the same schedule and in the same doses as captopril. Blood sampling and formulation of the PPN test was performed on the 2nd and 7th day (Table 3). The results in all three groups of animals with captopril were negative, that is, the test values did not exceed 0.08, which indicates a rather high specificity of the chosen method.

 To characterize the variational series of indicators in table. 2 and 3 did not use the arithmetic mean, but the median (Me), a variant less than half of the frequencies. The main advantage of Me compared to the arithmetic mean is that, with a small sample, its size is not affected by the magnitude of the extreme values of the variant in the variational series.

 From the above tables it is seen that captopril is capable of causing sensitization of the body, and its level with a sufficient degree of clarity can be estimated by the proposed PPN test.

 Example 2. Identification of the allergic effect in patients with arterial hypertension who took captopril.

 In patients taking captopril (16 patients with hypertension), the PPN values were 0.14-0.32, while in the blood of healthy people who had never taken ACE inhibitors (10 people), they did not exceed 0.08 (significance of differences in indicators in these groups> 95%). An attempt to establish the role of the duration of drug treatment on the level of allergization (Table 4) did not reveal statistically significant differences between the indicators in the groups (p> 0.05). Although there is a tendency to increase the index in the case of a longer intake of captopril.

 Thus, we can say that captopril has an allergenic ability in patients who are treated with captopril, which is easily detected by an in vitro allergy diagnostic test.

Claims (1)

A method for detecting the in vitro effect of allergization on the administration of captopril, based on the determination of neutrophil damage during incubation of the blood of a sensitized organism with captopril, while 0.1 ml of captopril in a pre-selected allergizing dose is added to the test tube, 0.1 ml of 5% in the control sodium citrate, and then both tubes making study a 0.4 ml blood was incubated for 2 hours at 37 ^o C and smears were stained sequentially with hematoxylin and reagent Schiff Ehrlich and neutrophils damage index is determined by formula rmule (H ₁ -H _2): 100, wherein N ₁ - number of damaged neutrophils in experiment 100 viewed in a smear, H ₂ - the same in the control, and a value of 0.1 or above is determined sensitization effect.

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