RU2265223C1 - Method for predicting external genital endometriosis - Google Patents

Abstract

FIELD: medicine, clinical laboratory diagnostics.

SUBSTANCE: in the sample of peripheral venous blood one should determine relative content of CD45RO+ lymphocytes and at its value being equal to 31% or lower it is possible to diagnose external genital endometriosis. The method is atraumatic and enables to diagnose external genital endometriosis at high accuracy.

EFFECT: higher efficiency of diagnostics.

3 ex, 1 tbl

Description

The invention relates to medicine, namely to gynecology, and can be used to diagnose external genital endometriosis.

The relevance of the invention is determined by the high frequency of genital endometriosis. According to various authors, about 15% of all women of childbearing age and up to 50% of women with infertility suffer from endometriosis [1, 2]. The diagnosis of external genital endometriosis is made on the basis of the results of an invasive traumatic instrumental examination using laparoscopy or laparotomy followed by histological examination of the removed tissue, which excludes a complex of pathogenetic therapeutic measures without surgical intervention. In this regard, the development of non-traumatic reliable and affordable laboratory diagnostic criteria for external genital endometriosis is an urgent task for practical public health.

There is a method of non-invasive diagnosis of external genital endometriosis, based on the analysis of patient questionnaire data on the severity of pain, menstrual irregularities, as well as a gynecologist and ultrasound examination using ATL Color Doppler HDI 3000, using a combination of positive ultrasound data with a high degree of symptomatology diagnosed with endometriosis of the ovaries with an accuracy of 100% [3]. However, this method has several disadvantages:

1) the proposed method was low informative for other forms of external genital endometriosis, so its accuracy in the diagnosis of endometriosis of the peritoneum of the pelvis was only 12%;

2) the method is based on the use of patient survey data, which implies the subjectivity of assessing the severity of symptoms and reduces the accuracy of the method;

3) there is evidence that endometrioid ovarian cysts of even large sizes may not give any symptoms [1], therefore, the asymptomatic course of endometriosis precludes the possibility of its diagnosis by the proposed method.

Closest to the technical solution to the claimed method is a method for the diagnosis of external genital endometriosis, based on an immunological study of blood serum, characterized in that the concentration of interleukin-6 (IL-6) in the blood serum and at a serum IL-6 content of more than 2 pg / ml it is proposed to diagnose external endometriosis in women. The sensitivity of the proposed method is 90%, specificity is 67% [4].

This method was chosen by us as the closest analogue (prototype). However, it has several disadvantages:

1) the specified threshold value at which the authors propose to diagnose endometriosis is only 2 pg / ml. Commercial test systems proposed by various companies for determining IL-6 by enzyme-linked immunosorbent assay have a different sensitivity threshold, and in some cases, diagnostically significant values of the indicator may be outside the sensitivity zone of the test system;

2) the time of the enzyme immunoassay (ELISA) is long and ranges from 4 to 6 hours;

3) the ELISA method has low reproducibility and requires the formulation of the studied samples in duplicates or triplicates (depending on the recommendations of the manufacturer), which significantly increases the cost of the method;

4) the specificity of this method is low, since a high content of IL-6 in the blood serum is also observed in other pathological conditions, for example, with liver cirrhosis of viral etiology [5] chronic viral hepatitis [6]. These disadvantages are proposed to eliminate in the present method.

The claimed technical result is achieved by the fact that the relative content of CD45RO + lymphocytes is determined in the peripheral blood of women and, with values of this indicator equal to or less than 31% or less, external genital endometriosis is diagnosed with an accuracy of 79%, sensitivity 81%, specificity 77%.

The novelty of the proposed method lies in the fact that for the first time it is proposed to diagnose external endometriosis in women by definition in the peripheral blood of the content of CD45RO + lymphocytes.

We did not find information on the use of indicators of the content of CD45RO + lymphocytes for the diagnosis of pathological conditions.

It is known that after antigenic stimulation of lymphocytes on their surface during differentiation, the isotypes of the CD45 molecule are switched and its isoform CD45RO is expressed only on mature effector cells, as well as on memory T cells, being a marker of the final stage of cell differentiation [7]. Apparently, the low content of CD45RO + lymphocytes is associated with a violation of the response of peripheral lymphocytes to antigenic stimulation in women with external endometriosis [8].

The method is as follows:

1) Isolation of lymphocytes from peripheral venous blood is carried out according to standard methods [9].

2) 100 μl of lymphocytes at a concentration of 1 × 10 ⁶ cells / ml are incubated with 20 μl of monoclonal anti-CD45CO labeled with phycoerythrin (PE) (CALTAG LABORATORIES, USA, registration certificate of the Ministry of Health of the Russian Federation No. 2003/584 dated April 17, 2003) in for 20 minutes at room temperature in the dark, after which the cells are washed 1 time with 1 ml of physiological solution by centrifugation at 1500 rpm for 2 minutes. Cells are resuspended in 500 μl of saline. The reaction is taken into account on a flow cytometer, determining the content of CD45PO-positive cells in the lymphocyte gate.

Using the proposed method allows with high accuracy 79%, sensitivity 81%, specificity 77% to diagnose external endometriosis.

Distinctive features of the method are:

- a diagnostic parameter was established for the relative content of CD45RO + lymphocytes in the peripheral blood of women, with quantitative values of which 31% or less, external endometriosis is diagnosed.

The essence of the proposed method is illustrated by the following examples:

Example 1. Patient P. 28 years. Appeared about secondary infertility for 3 years. A history of 2 medical abortions. Repeatedly carried out anti-inflammatory therapy for exacerbation of chronic 2-sided adnexitis without effect. With vaginal examination: the uterus is of normal size and position. Uterus appendages without features. Palpation of the sacro-uterine ligaments is painful. Examined: cycle - two-phase (tests of functional diagnostics and zug-endometrium); according to hysterosalpingography - the uterus is not changed, the fallopian tubes are freely passable. Husband examined - healthy. The post-coital test is positive.

Clinical diagnosis: infertility II, external genital endometriosis.

According to the immunological examination according to the claimed method, the relative content of CD45RO + lymphocytes in the peripheral venous blood is 22.3%, which is lower than the claimed parameter. Conclusion: external endometriosis is diagnosed.

On the 9th day of the menstrual cycle, laparoscopy was performed, during which it was found: the uterus and fallopian tubes are not changed, freely passable during chromohydrotubation. On the surface of the right ovary, the peritoneum of the posterior leaves of the wide uterine and sacro-uterine ligaments, multiple superficial and deep foci of endometriosis with an area of more than 3 cm ^{2 were found} . Coagulation and excision of foci of endometriosis by a monopolar coagulator were performed. Histological examination of the removed tissue - peritoneal endometriosis, ovarian endometriosis.

Endoscopic and histological studies indicate the correctness of the diagnosis made by the present method.

Example 2. Patient A., 33 years old, with primary infertility for 8 years, was admitted to the endoscopic department of the gynecological clinic to clarify the causes of infertility. Married, denies gynecological diseases. During the previous examination, the presence of a two-phase menstrual cycle was revealed (according to tests of functional diagnostics and zug-endometrium), according to hysterosalpingography, the uterus was not changed, the fallopian tubes are moderately tortuous, freely passable, the post-coital test is positive. With a vaginal examination, the body of the uterus is slightly larger than normal, with painful heaviness in the lateral arches.

Clinical diagnosis: infertility I. Chronic salpingitis.

According to the immunological examination according to the claimed method, the relative content of CD45RO + lymphocytes in the peripheral venous blood is 43%, which is higher than the claimed parameter for external endometriosis.

Conclusion: the diagnosis of endometriosis is not diagnosed.

On laparoscopy, the uterus and fallopian tubes are not changed, freely passable with chromohydrotubation, the pelvic peritoneum without pathological inclusions. The ovaries are normal in size. The performed intraoperative postcoital test revealed the cause of infertility: in the aspirated peritoneal fluid and the posterior third of the cervical canal, no movable spermatozoa were found.

An endoscopic examination indicates the correctness of the diagnosis made by the claimed method.

Example 3. Patient N., 25 years old. Appeared about secondary infertility. A history of one medical abortion without apparent complications. He is in his first marriage, there was no history of gynecological diseases, he does not present specific complaints.

During the examination (tests of functional diagnostics and zug-endometrium), a two-phase menstrual cycle was established. According to hysterosalpingography, the uterine cavity has the correct shape, the fallopian tubes are not filled with contrast from the corners. The post-coital test is positive. With an objective examination, the uterus is of normal size and position. Appendages are not determined, arches are free, mucous discharge.

Conclusion: infertility II, chronic salpingitis, proximal tubal occlusion.

According to an immunological examination according to the present method, the relative content of CD45RO + lymphocytes in the peripheral venous blood is 31%.

Conclusion: external endometriosis is diagnosed.

When laparoscopy revealed that the uterus and fallopian tubes are not changed, freely passable during chromohydrotubation. Multiple deep foci of endometriosis with a total area of more than 3 cm ^{2 were} found on the peritoneum of the wide uterine ligaments. The result of a histological examination of a peritoneal biopsy sample is peritoneal endometriosis, which confirms the correctness of the diagnosis established by the claimed method.

In this way, 29 women were examined, the research data are shown in table 1.

Table 1 Indicator The number of examined Total women 29th true positive result 10 False positive 3 true negative result thirteen False negative result 3

Total:

the accuracy of the proposed method is 79%

the sensitivity of the proposed method is 81%

the specificity of the proposed method is 77%.

The advantages of the proposed method:

1. Can be used as an independent or screening diagnostic method.

2. Not traumatic.

3. High accuracy - 79%, sensitivity - 81%, specificity - 77%.

4. The determination of the CD45RO + lymphocyte index by flow cytometry has good reproducibility and high accuracy, since the results are calculated automatically on a large number of cells (5-10 thousand) with an accuracy of 0.01%.

5. The duration of the methodology for determining CD45RO + lymphocytes does not exceed 1.5 hours.

Sources of information

1. Adamyan L.V., Kulakov V.I. Endometriosis: A Guide for Physicians. - M.: Medicine. - 1998 .-- 320 s.

2. Vinatier D., Orazi G., Cosson M., Dufour P. Theories of endometriosis // European Journal of Obstetrics & Gynecology and Reproductive Biology. - 2001.- V.96. - P.21-34.

3. Eskenazi B., Warner M., Bonsignore L., Olive D., Samuels S., Vercellini P. Validation study of nonsurgical dianosis of endometriosis // Fertility and Sterility. - 2001. - V.76. - N5. - P.929-935.

4. Bediway M.A., Falcone T., Sharma R.K., Goldberg J.M., Attaran M., Nelson D.R., Agarwal A. Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial // Hum Reprod. - 2002. - V.17. - N2. - P.426-431.

5. Shapiro V.Ya., Sec. Ok Sleep, Korning B.E. Clinical and prognostic significance of indicators of the immune and cytokine status in cirrhosis of the liver of viral etiology // Medical Immunology. - 2002. - T. 4, No. 2, P.169.

6. Lugovskaya S.A., Lukina E.A., Sysoeva E.L., Levina A.A., Mayevskaya M.V. The study of the content of cytokines in the blood serum of patients with chronic viral hepatitis // Medical immunology. - 2000. - T. 2, No. 2. - S.175.

7. Yarilin A.A. Fundamentals of Immunology: A Textbook. M .: Medicine, 1999 .-- 608 p.

8. Dmowski W.P., Steele R.N., Baker G.F. Deficient cellular immunity in endometriosis // Am. J. Reprod. Immunol. - 1984. - V.6. - P.33

9. Bourn A. Isolation of mononuclear cells and granulocytes from human blood and bone marrow // Scand.J. Clin. Invest. - 1968. - V.21. - N97. - P.77.

Claims (1)

A method for diagnosing external endometriosis in women by examining peripheral venous blood, characterized in that the relative content of CD45RO + lymphocytes is determined in a blood sample and, when its value is 31% or less, external genital endometriosis is diagnosed.