SU1654748A1 - Method for determination of immuno-defficiency states - Google Patents

Abstract

The invention relates to medicine and can be used in clinical immunology to assess the immune status of patients and during clinical examination of the population. The aim of the invention is to simplify and improve the accuracy of the analysis. The method of determining immunodeficiency states is to determine the level of expression of receptors for growth factors (interleukins) on the surface of immunocompetent cells using the chemiluminescent method. Mononuclear cells from human peripheral blood are cultured for 16-18 h in the presence of a mitogen, then the level of activation of these cells by exogenous interleukins is determined compared to the level of spontaneous chemiluminescence in a cell suspension in the presence of pyuminol. In immunodeficient states, a sharp decrease in the activation of chemiluminescence by interleukins 1 and 2 is noted (for interleukin-1, 18.5% and below, for interleukin-2, 17% and below). W fe

Description

The invention relates to medicine and can be applied as a laboratory method for determining the immunological status of a patient.

The purpose of the invention is to simplify and improve the accuracy of the method by determining the immunodeficiency states, based on an assessment of the expression of growth factor receptors (interleukins) on the cell membrane.

The method is carried out as follows.

Mononuclear cells from peripheral venous blood collected sterilely from the cubital vein and heparinized (10-25 U / ml of blood) were isolated by centrifugation on a density gradient of ficollarografin using the Wi-Fi method. Mononuclear cells are cultured in N: 199 medium (Institute of Polio and Viral Encephalitis) at a concentration of 1-2 x 106 ml with PHA or KOH A (Serva, HGF) at a dose of 5-10 μg / ml at 37 ° C. for 14-16 h in centrifuge tubes. After cultivation, the cells were centrifuged at 500 ° C. The supernatant was removed and the cells were resuspended in 0.1 ml of medium No. 199. The cell suspension was transferred to a cuvette containing 0.8 ml of the incubation medium of the following composition: 110 mM NaCI, 5 mM glucose, 2.5 mM MgCl2. 0.65 mM luminal, 10 mM tris HCI. pH 7.4. Determine the level of spontaneous chemiluminescence using a luminometer (LKB, Sweden). Do not disturb the light insulation of the system, in a cuvette

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0.1 ml of medium containing interleukin-1 or interleukin-2 was added, and the level of activated chemiluminescence was determined. Does the ratio calculate

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values of activated and spontaneous chemiluminescence (activation index). Activation indices characteristic of healthy donors and for patients with immunopathological conditions confirm the invention. Immunodeficiency states were diagnosed in a known manner.

. Interleukin-1 and interleukin-2 were obtained from the culture medium of mononuclear cells of the peripheral blood of healthy donors. Mononuclear cells were divided into adherent and non-adherent glass by cultivation in penicillin vials at a concentration of 2-4x10 / ml with 20% fetal calf serum at 37 ° C for 45-60 minutes. Non-adherent cells were transferred to new vials. Adherent cells were removed with a cold Versen solution (4-6 ° C), centrifuged and resuspended in medium No. 109. To obtain a medium containing interleukin-1, adherent cells were cultured at a concentration of 0.2-0.4x10 / ml in medium No. 199 s 10% fetal bovine serum. After 72 h, the supernatant was collected and stored at -20 ° C. To obtain a supernatant containing interleukin-2, non-adherent cells at a concentration of 1x10 / ml were cultured in medium No. 199 with 10% fetal calf serum and 10 µg / ml PHA. After 72 h, the supernatant was collected and stored at -20 ° C. When performing the method, it is possible to use commercial preparations of interleukin-1 or interleukin-2.

PRI me R 1. Patient B., 36 years old, was admitted to the Department of Reconstructive Eye Surgery of the All-Union Scientific Research Institute of the USSR Ministry of Public Health with complaints of burning pains in the right eye, lightening and deterioration of vision. On examination, it was found that the cornea was infiltrated, the transparency was disturbed, and there were many eruption bubbles. Herpetic keratouveitis has been diagnosed in the exacerbation stage. On the basis of a long relapsing course of the disease, it has been suggested that the patient has an immunodeficiency condition aggravating the course of the disease. For parallel evaluation of the expression of receptors for interleukins, the patient was bled. When using the proposed method to reduce the chemiluminescence activation index by interleukin-1 to 10.2%, interleukin-2 to 9.10% was diagnosed with an immunodeficiency state 18 hours after blood collection. After 80 h, these data were confirmed using a known method for assessing the expression of interleukin receptors.

Example 2: Patient I., 26 years old, was admitted to the radiological department of the Moscow Scientific Research Institute of RRI with complaints of tumor-like formations.

0 in the neck, fever, feeling unwell. On a case-by-case examination of the neck, the armpits, palpable lymph node packages are palpated. A biopsy revealed Berezovsky5 Sternberg cells, on the basis of which a diagnosis of lymphogranulomatosis was made. Blood was collected from the patient to evaluate the expression of interleukin receptors. When using this method in a patient

0 was diagnosed with an immunodeficiency state by reducing the chemiluminescence activation index to 15% for interleukin-1 and to 17% for interleukin-2. Results were obtained 18 h after

5 blood sampling. The obtained results were confirmed using a known method 80 hours after blood collection. In this way; diagnosis of immunodeficiency was reduced by 62 hours.

0P e rm 3. Patient G., 6 years old, was

on treatment at the Children's Clinical Hospital No. 10 of Moscow. There were no complaints. Objectively - signs of Down syndrome. Entered about the lengthy in the lottery

5 diseases of the upper respiratory tract. When assessing the expression of receptors for interleukins, the patient was diagnosed with an immunodeficiency state. With concurrent assessment of receptor expression

By the proposed method, an immunodeficiency state was also diagnosed with a decrease in the chemiluminescence activation index by interleukin-1 to 18.5%, interleukin-2 to 14.2%. Diagnostic time

5 was also reduced by 62 h.

Thus, the proposed method has several advantages compared with the previously known method for diagnosing immunodeficiency states: less

0 laboriousness and duration (shortening the diagnosis by 62 hours), high accuracy and reproducibility (the value of the activation index calculated from the chemiluminescent method,

5 ranges from 2–5% in five parallel samples), no need for expensive labeled reagents, progressiveness (using the modern highly sensitive method of biophysical analysis).

Claims (1)

Invention Formula A method of determining immunodeficiency states by activating mononuclear blood cells using interleukl-1 or interleukin-2, followed by an assessment of the mitogenic effect on cells, characterized in that, in order to simplify and improve the accuracy of the method. additionally, before activation by interleukins, mononuclear cells are cultivated for 16–18 h in the presence of mitogen, then the level of activation by mitogens and interleukins is compared with chemiluminescence and with decreased chemiluminescence with interleukin-1 to 18.5% and below and interleukin-2 up to 17% and below, the immunodeficiency state is determined.