RU2330286C1 - Method of viral herpes gestationis diagnostics by herpes bodies estimation in peripheral blood - Google Patents

Abstract

FIELD: medicine; diagnostic methods.

SUBSTANCE: method of herpes gestationis diagnostics implies that leukocytic suspension in amount 1-2 drops resulted of whole blood centrifugation in test tube is added with 0.5 ml of blood serum. It is kept within 15 minutes in thermostat at t=37°C and is added with 0.5 ml of 0.5% neutral red made on physiologic saline at pH=7.4. Produced mix is kept within 5 min in thermostat at t=37°C thereafter mixture drop is applied on slide followed with smear production by centrifugation within 4 seconds. After being dried smear is analysed for herpes bodies number per 100 leukocytes, thus herpes bodies are leukocytes closed owing to increased ganglioside charge. If herpes bodies content is within 3-5±0.3% severe viral herpes infection is diagnosed while content value 1-2.0±0.2% indicates mediate severity, and healthy pregnant women practically do not have any bodies found.

EFFECT: invention application provides considerable reduction of analysis time.

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Description

The invention relates to medicine and can be used to diagnose herpes virus infection by counting the percentage of herpes bodies in the peripheral blood of pregnant women.

The disadvantages of the known methods:

Herpes bodies have been identified to date by the following cytological and ELISA methods:

1. Taking prints, scrapings from the selected organs: the urethral mucosa, the walls of the vagina, the cervical canal. The material taken by biopsy was poured into paraffin and studied on histological sections (Karpova, O.B., Avrorova, N.F., 1988; Tsinserling A.V., 1993; Tsinserling A.V. et al., 1993).

2. By ELISA (immunobiochemical reaction - Isakov V.A., 2004).

Using the above cytological and ELISA methods, it is not possible to identify the mechanism of formation of bodies formed by white blood cells that are brought together due to increased charge of gangliosides in peripheral blood.

The applied method drastically reduces the time for detection in an easily accessible medium - peripheral blood - of the formation of bodies formed by white blood cells that are brought together due to increased charge of gangliosides when exposed to an infection pathogen on their cell membranes.

The proposed method is as follows:

1. In a sterile tube moistened with heparin, 2 ml of blood is drawn from a pregnant woman’s vein. After the deposition of the shaped elements by centrifugation at 1,500 rpm, the leukocyte film is carefully aspirated by a pipette and placed in a volume of 2 drops in a test tube containing 0.5 ml of the blood serum of the same patient (t = 37 ° C), obtained by centrifugation whole blood. The suspension is shaken and kept for 15 minutes in a thermostat at t = 37 ° C. After that, 0.5 ml of a 0.5% solution of neutral red prepared in physiological saline at pH 7.4 is added to 0.5 l of this suspension. The mixture should stand for 5 minutes in a thermostat at t = 37 ° C, after which it is shaken and a drop is carefully applied to a glass slide and a smear is prepared instantly on a Diff Spin. Slide Sprinter centrifuge (4 sec). On this smear, after it dries up, herpes bodies are counted per 100 leukocytes.

2. After the preparation of the smear, herpes bodies formed by white blood cells at various stages of formation (see drawing) are detected from the proximity of the white blood cells to each other (A, B) to their tight assembly (C) and the formation of a common membrane around them (D).

The number of herpes cells increases with an increase in the titer of antibodies to the herpes virus: with a titer of 1: 3200 - up to 1.0 ± 0.06%; with an antibody titer of 1: 6400 - up to 2.0 ± 0.03%, and with an antibody titer of 1: 12800 - up to 5.0 ± 0.30% and higher. The antibody titer was determined by enzyme-linked immunosorbent assay on a Stax-Fax-2100 spectrophotometer (USA).

3. The proposed method has been clinically tested on the basis of the obstetric and gynecological department of the Far Eastern Scientific Center of Physiology and Respiratory Pathology SB RAMS. Using the proposed method, the detection of herpes bodies in the peripheral blood of 60 pregnant women with an outbreak of herpes virus infection from 75 examined. Thus, the proposed method can be effectively used for the diagnosis of herpes virus infection in pregnant women in 80% of cases.

Example 1: E., 23 years. Upon initial admission to the ward with a gestational age of 19 weeks, no signs of herpes infection were detected by ELISA. In the peripheral blood of herpes cells was not found. Repeated admission to the department. Pregnancy is 24 weeks. An ELISA detected an outbreak of herpes infection. The antibody titer is 1: 12800. In the peripheral blood per 100 leukocytes, 4% of herpes cells are observed, formed by the convergence of leukocytes to each other, in the membranes of which an intense reaction to gangliosides is detected. The diagnosis is a severe stage of herpes virus infection.

Example 2: 20 years. Upon admission to the department in peripheral blood by ELISA, a titer of antibodies to the herpes virus 1: 3200 is noted. In peripheral blood, there are 1.5% of the bodies formed by close white blood cells, in the membranes of which there is an increased reaction to gangliosides. The diagnosis is a moderate severity of herpes infection.

Literature

1. Karpova O. B., Avrorova N. F. Herpes simplex etiology, diagnosis, clinical and anatomical manifestations. - L., 1988, pp. 86-91.

2. Zinserling A.V. Modern infections. Pathological anatomy and questions of pathogenesis - Sob .; Sotis, 1993, 287 pp.

3. Tsinserling A.V., Popova E.D., Baykov V.V., Kuzmin V.V., Karpova O. B., Masing Yu.A., Kozhuhar V.G., Sorokina L.V. An experimental model of generalized herpetic infection of the newborn. Archive of Pathology, Moscow, Medicine, V. 55, No. 5, 1993, p. 28-32.

4. Isakov V.A. et al. Modern therapy of herpes virus infections. A guide for doctors - St. Petersburg., M., 2004.

Claims (1)

A method for the diagnosis of herpes virus infection in pregnant women, including a study of venous peripheral blood, characterized in that to 1-2 drops of leukocyte suspension taken after centrifugation of the patient’s whole blood in a test tube, add 0.5 ml of patient’s blood serum, incubated for 15 minutes in an thermostat at t = 37 ° C and add 0.5 ml of 0.5% neutral red, prepared in physiological saline at pH 7.4; the resulting mixture is kept for 5 min in a thermostat at t = 37 ° C, after which a drop of the mixture is applied to a glass slide and a smear is made by centrifugation for 4 s, after drying of the smear, herpes bodies are counted per 100 leukocytes, while the herpes bodies are close in the power of increasing the charge of gangliosides leukocytes, with a content of herpes bodies of 3-5 ± 0.3% - they judge the severe stage of herpes virus infection, with 1-2.0 ± 0.2% - they judge the middle stage of the disease, in healthy pregnant herpes almost no bodies tsya.

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