Method for predicting external genital endometriosis

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

 

The invention relates to medicine, namely to gynecology, and can be used for the diagnosis of external genital endometriosis.

The urgency of the invention is determined by a high frequency of genital endometriosis. According to different authors, about 15% of all women of reproductive age and up to 50% of women with infertility suffer from endometriosis [1, 2]. Diagnosis of external genital endometriosis is based on the results of invasive traumatic instrumental studies using laparoscopy or laparotomy with subsequent histological examination of tissue removed, which eliminates the complex pathogenetic treatment measures without surgical intervention. In this regard, the development of non-traumatic reliable and available laboratory diagnostic criteria of external genital endometriosis is an actual task for healthcare practice.

The known method of non-invasive diagnosis of endometriosis based on the data analysis of the survey of patients about the severity of pain, menstrual disorders, as well as gynecologist examination and ultrasound examination using ATL Color Doppler HDI 3000, the combination of a positive ultrasound data with a high degree of viragen the STI symptoms diagnosed endometriosis of the ovaries with 100% accuracy [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 pelvic peritoneum was only 12%;

2) method based on the use of survey data of patients that involves the subjective evaluation of the severity of symptoms and reduces the accuracy of the method;

3) there is evidence that endometrioid ovarian cysts even larger sizes may not produce any symptoms [1], therefore, asymptomatic endometriosis eliminates the possibility of diagnostics on the proposed method.

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

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

1) the specified threshold value at which the authors suggest to diagnose endometriosis is l is nil 2 PG/ml Offered by different companies commercial test systems for determination of IL-6 enzyme-linked immunosorbent assay have different threshold, while in some cases diagnostically significant indicator values can be outside the zone of sensitivity of the test system;

2) the timing of enzyme-linked immunosorbent assay (ELISA) long and ranges from 4 to 6 hours;

3) ELISA method has a low reproducibility and requires setting of the investigated samples in duplicate or triplicate (depending on the recommendations of the manufacturer), which significantly increases the cost method;

4) low specificity of this method, because the high content of IL-6 in serum is observed also in other pathological conditions, such as cirrhosis of viral etiology [5] chronic viral hepatitis [6]. These drawbacks are proposed to be eliminated in the present method.

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

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

We have not found information about the use of indicators content CD45RO+ lymphocytes for the diagnosis of pathological conditions.

It is known that after antigenic stimulation of lymphocytes on their surface during differentiation switch isotypes molecule CD45 and its isoform CD45RO is expressed only on Mature effector cells, and T-memory cells, as a marker of end-stage differentiation of cells [7]. Apparently, low content of CD45RO+ lymphocytes is associated with impaired 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 in a concentration of 1×106cells/ml are incubated with 20 μl of monoclonal anti-D45 labeled with phycoerythrin (PE) (CALTAG LABORATORIES, USA, registration certificate of Ministry of health of the Russian Federation No. 2003/584 of 17 April 2003) for 20 minutes at room temperature in the dark, after which the cells washed 1 time with 1 ml of saline by centrifugation at 1500 rpm for 2 minutes. Cells resuspended in 500 μl of saline. The reaction accounting is performed on a flow cytometer, specifying content D45-positive cells in the lymphocyte gate.

COI is whether the proposed method with high accuracy 79%, sensitivity 81%, specificity 77% to diagnose external endometriosis.

Distinctive features of the method are:

- set diagnostic parameter of the relative content of CD45RO+ lymphocytes in the peripheral blood of women in the quantitative values of which 31% and less external endometriosis is diagnosed.

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

Example 1. Patient P. 28 years. Asked about secondary infertility for 3 years. History 2 medical abortion. Repeatedly held anti-inflammatory therapy regarding exacerbation of chronic 2-sided adnexitis without effect. At vaginal examination: the uterus is normal in size and position. The appendages of the uterus without features. Palpation Sacro-uterine ligaments painful. Surveyed: cycle two - phase (tests of functional diagnostics and Zug-endometrium); according hysterosalpingography - the uterus is not changed, the fallopian tube is freely passable. Husband examined healthy. Postcoital test - positive.

Clinical diagnosis: infertility II, external genital endometriosis.

According to immunological examination by the present method the indicator of the relative content of CD45RO+ lymphocytes in the peripheral venous blood of 22.3%, which is lower than the proposed option. Bookmark the increase: external endometriosis is diagnosed.

On day 9 of the menstrual cycle produced laparoscopy, during which discovered: the uterus and the fallopian tubes are not changed freely passable by chromopertubation. On the surface of the right ovary, peritoneum posterior leaves of the broad uterine and Sacro-uterine ligaments found multiple superficial and deep foci of endometriosis, an area of more than 3 cm2. Produced by coagulation and excision of endometriosis monopolar coagulator. Histological examination of remote tissue - endometriosis peritoneal endometriosis of the ovary.

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

Example 2. A patient, 33 years of age, with primary infertility for 8 years, he entered the Department of gynecologic endoscopic clinic to clarify the causes of infertility. Married, gynecological diseases denies. During previous surveys revealed the presence of two-phase of the menstrual cycle (on tests of functional diagnostics and Zug-endometrium), according to hysterosalpingography uterus is not changed, the fallopian tubes moderately convoluted, freely passable, postcoital test is positive. At vaginal examination the body of the uterus a little more normal size in the lateral arches painful tictacti.

Clinical diagnosis: infertility I. Chronic salpingitis.

According to immunological examination by the present method the indicator of the relative content of CD45RO+ lymphocytes in peripheral venous blood of 43%, which is higher than the proposed option for external endometriosis.

Conclusion: the diagnosis of endometriosis cannot be diagnosed.

At laparoscopy the uterus and the fallopian tubes are not changed freely passable by chromopertubation, pelvic peritoneum without pathological inclusions. The ovaries are normal in size. Conducted intraoperative postcoital test revealed a cause of infertility: aspirated peritoneal fluid and the rear third of the cervical canal of motile sperm was not found.

Endoscopic examination proves the correctness of the diagnosis made by the present method.

Example 3. Patient N., 25. Asked about secondary infertility. History of one abortion without apparent complications. Is the first marriage, gynecological diseases in anamnesis no specific no complaints.

During examination (tests of functional diagnostics and Zug-endometrium) is a two-phase menstrual cycle. According to hysterosalpingography the uterus has a normal form, the fallopian tube is not filled with contrast edges. On totally test positive. At objective examination, the uterus is normal in size and position. The appendages are not defined, the arches of free allocation mucous.

Conclusion: infertility II, chronic salpingitis, proximal occlusion of the fallopian tubes.

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

Conclusion: external endometriosis is diagnosed.

When laparoscopy revealed that the uterus and the fallopian tubes are not changed freely passable by chromopertubation. On the peritoneum of the broad uterine ligament has multiple deep foci of endometriosis with a total area of more than 3 cm2. The result of histological examination of a biopsy of the peritoneum - peritoneal endometriosis, which confirms the correctness of the diagnoses made by the claimed method.

In this way were examined 29 women, these studies are shown in table 1.

Table 1
IndexThe number of the examined
Total female29
true-positive result10
False-positive result3
true-negative results is 13
A false-negative result3

Total:

the accuracy of the proposed method - 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 a separate or diagnostic screening method.

2. Retraumatized.

3. High accuracy of 79%, a sensitivity of 81%, specificity of 77%.

4. Indicator definition CD45RO+ lymphocytes by flow cytometry has good reproducibility and high accuracy, because the calculation of results is performed automatically on a large number of cells (5-10 thousand) with a precision of 0.01%.

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

Sources of information

1. L.V. Adamyan, Kulakov V.I. Endometriosis: a Guide for physicians. - M.: Medicine. - 1998. - 320 S.

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

3. Eskenazi Century, Warner M., L. Bonsignore, 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., J.M. Goldberg, 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 VA, S OK to Sleep, Knowing BE Clinical Prognos the practical significance of immune and cytokine status in liver cirrhosis of viral etiology// Medical immunology. - 2002. - So 4, No. 2, S.

6. Lugovskaya S.A., Lucy E., Sysoeva AL, Levin, A.A., Mayevskaya M.V. investigation of the content of cytokines in the serum of patients with chronic viral hepatitis// Medical immunology. - 2000. - So 2, no 2. - S.

7. The Yarylo A.A. fundamentals of immunology: a Textbook. M.: Medicine, 1999. - 608 S.

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.

Method for the diagnosis of external endometriosis in women through the study of peripheral venous blood, wherein the blood sample to determine the proportion of CD45RO+ lymphocytes and when it is equal to 31% or less, diagnose external genital endometriosis.



 

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