1

Using two-dimensional electrophoresis and time-of-flight mass spectrometry, the proteomic profile of peritoneal fluid in external genital endometriosis was studied. Differential proteins that appear in external genital endometriosis have been identified: apolipoprotein A-IV, sex hormone-binding globulin, components of the complement system C3 and C4b. Proteins absent in external genital endometriosis include pigment epithelial differentiation factor, transthyretin, haptoglobin, α-1-antitrypsin, and apoptosis inhibitor 6. The possible role of the identified proteins in the development of major disorders in endometriosis is discussed. The detected difference proteins can be used as markers of this disease.

external genital endometriosis

peritoneal fluid

proteomic analysis

protein differences

1. Adamyan, L.V. Endometriosis: a guide for doctors / L.V. Adamyan, V.I. Kulakov, E.N. Andreeva // M.: Medicine, 2006. - 416 p.

2. Govorun, V.M. Proteomic technologies in modern biomedical science / V.M. Govorun, A.I. Archakov // Biochemistry. - 2002. - No. 10. - P. 1341-1359.

3. Grandfather, M.I. The system of proteolysis in blood serum and peritoneal fluid in the surgical treatment of patients with endometriosis / M.I. Grandfather, L.E. Radetskaya, L.N. Kirpichenok // News of Surgery. - 2006. - No. 3. - P.74-80.

4. Ishchenko, A.I. Endometriosis: diagnosis and treatment / A.I. Ishchenko, E.A. Kudrina // M.: GEOTAR-MED, 2002. - 104 p.

5. Linde, V.A. Proteomic technologies in the study of endometriosis / V.A. Linde, L.R. Tomai, V.O. Gunko and others // Med. vestn. South of Russia. - 2013. - No. 4. - P.12-16.

6. Minkevich, N.I. PEDF-noninhibitory serpin with neuroprotective and antiangiogenic activities / N.I. Minkevich, V.M. Lipkin, I.A. Kostanyan // ActaNaturae. - 2010. - No. 3. - P.74-84.

7. Bedaiwy, M.A. Peritoneal fluid environment in endometriosis. Clinicopathological implications / M.A. Bedaiwy, T. Falcone // Minerva Ginecol. -2003. - V.55, N 4. - P.333-345.

8. Bernard, K.R. Methods in functional proteomics: two-dimensionalpolyacrylamide gel electrophoresis with immobilized pH gradients, in-gel digestion and identification of proteins by mass spectrometry / K.R. Bernard, K.R. Jonscher, K.A. Resing, N.G.Ahn //Methods Mol. Biol. - 2004. -V. 250.– P. 263-282.

9. Hammond, G.L. Diverse roles for sex hormone-binding globulin in reproduction / G.L. Hammond // Biol. reproduction. - 2011. - V. 85, N 3. - P.431-441.

10. Kabut, J. Levels of complement components iC3b, C3c, C4, and SC5b-9 in peritoneal fluid and serum of infertile women with endometriosis / J. Kabut, Z. Kondera-Anasz, J. Sikora et al. // Fertile. Steril. -2007. - V.88, N 5. - P.1298-1303.

11. Richardson S.J. Cell and molecular biology of transthyretin and thyroid hormones / S.J. Richardson // Int. Rev. Cytol. - 2007. - V. 258. - P.137-193.

12 Sarrias M.R. A role for human Sp alpha as a pattern recognition receptor / M.R. Sarrias, S. Roselló, F. Sánchez-Barbero et al. // J. Biol. Chem. - 2005. - V. 280, N 42. - P. 35391-35398.

13. Spaulding H.L. Apo A-IV: an update on regulation and physiologic functions / H.L. Spaulding, E. Delvin, M. Lambert et al. // Biochim. Biophys. acta. - 2003. - V.1631, N 2. - P.177-187.

14. Wassell J. Haptoglobin: function and polymorphism / J. Wassell // Clin. Lab. –2000. - V. 46, N 11-12. – P.547-552.

The relevance of the study of external genital endometriosis (EGE) is associated with the high prevalence of this pathology among women of childbearing age and its significant impact on their reproductive health and standard of living. Currently, an important role of peritoneal fluid (PJ) in the pathogenesis of endometriosis has been shown, since it is in it that the development and growth of endometrioid foci occurs. The study of the protein composition of the pancreas using proteomic technologies aimed at studying the totality of proteins expressed by the genome creates qualitatively new opportunities for deepening the understanding of the molecular mechanisms of the development of endometriosis, its prediction and early diagnosis.

Objective. To study the proteomic spectrum of the pancreas of women with EGE and without EGE.

Material and research methods

The study included 20 patients of reproductive age (mean age 29.3 ± 0.3 years), including 10 patients with EGE with stages III-IV of the disease according to the r-AFS classification (main group) and 10 without endometriosis (control group). ). The study material was the pancreas obtained from the posterior uterine space during laparoscopy. Proteomic analysis of the pancreas was performed using two-dimensional polyacrylamide gel electrophoresis (Protein IEFCell and ProteanIIxiMulti-Cell, Bio-Rad, USA) followed by protein staining with silver ions. Proteins were identified after their trypsinolysis by time-of-flight MALDI mass spectrometry on an AutoflexII mass spectrometer (Bruker, Germany) using the MascotMSSearch program (MatrixScience, USA) and the NCBI and Swiss-Prot databases. The results of protein identification were taken as significant at the level of significance not less than 95% and sequence coverage not less than 60%.

The significance of differences in the proteomic spectrum of the pancreas of women in the control and main groups was determined using the c2-criterion (Statistica program version 6.0, StatSoft. Jnc.). The results were evaluated as statistically significant at p<0,05.

Research results and discussion

As a result of the proteomic analysis of the pancreas, a number of difference proteins were identified, the presence or absence of which occurs only in EGE (see Table, Fig.). So, in the pancreas of women of the main group, the appearance of the following proteins was established: apolipoprotein A-IV, sex hormone-binding globulin (SHBG), components of the complement system C3 and C4b, which were not found in patients of the control group.

Table 1

Identified proteins of the pancreas of women in the control and main groups

	protein name
	α-1-antitrypsin
	pigment epithelium differentiation factor
	Component of the C3 complement system
	Apolipoprotein A-IV
	Haptoglobin
	Globulin that binds sex hormones
	Apoptosis inhibitor 6
	Component of the C4-b complement system
	Transthyretin

Note: pI - isoelectric point, Mm-molecular weight, "+" - presence of protein, "-" - absence of protein, p - significance of differences between groups.

A B

Rice. 1. Proteomic maps of the peritoneal fluid of women in the control (A) and main (B) groups

Note. Protein numbering corresponds to that in the table

An increase in the production (and, as a result, the appearance in the pancreas) of apolipoprotein A-IV, which has antioxidant and anti-inflammatory properties, obviously has a compensatory value in conditions of oxidative stress and inflammation that accompany the development of this pathology.

An increased content of SHBG in endometriosis, which regulates the bioavailability of steroid hormones for endometrial cells, creates conditions for local hyperestrogenism. Under these conditions, it becomes possible to increase the proliferative potential of endometrioid heterotopia cells.

Increased secretion by peritoneal macrophages of the C3 and C4b complement system components involved in the inflammatory response, neutralization of apoptotic cells and immune complexes, makes a certain contribution to the mechanisms of development of endometriosis and endometriosis-associated infertility.

Along with these abnormalities in EGE, 5 proteins are absent in the pancreatic proteomic spectrum: pigment epithelium differentiation factor, transthyretin, apoptosis inhibitor 6, haptoglobin, and α-1-antitrypsin.

The pigment epithelium differentiation factor is one of the most powerful anti-angiogenic and anti-proliferative factors, therefore, inhibition of its expression may be one of the reasons leading to a decrease in endometrial apoptosis and an increase in angiogenesis, promoting implantation and growth of heterotopias.

The absence in the pancreas of women of the main group of transthyretin, which transports T3 and T4, apparently creates a local excess of thyroid hormones, the toxic effect of which leads to damage to the reproductive organs. Thyroid hormones, modulating the effects of estrogens at the cellular level, can contribute to the development of disorders in the histo- and organogenesis of hormone-sensitive structures and worsen the course of endometriosis.

Among the proteins not detected during EGE, an apoptosis inhibitor secreted by macrophages plays an important role in the regulation of the immune response 6 . It is possible that it is the violation of the expression of this protein that leads to the formation of an imbalance of immunocompetent cells in the pancreas (due to the suppression of apoptosis of T-lymphocytes and NK cells).

The absence of the non-enzymatic antioxidant haptoglobin in the pancreas may contribute to the increased oxidative stress that develops in endometriosis.

With this pathology, α-1-antitrypsin, an inhibitor of serine proteases that are directly involved in the processes of endometrial cell invasion, was also not found in the pancreas, which, apparently, causes an imbalance in the protease-inhibitor system, contributing to the implantation of endometrial cells.

The conducted studies indicate that the development of endometriosis occurs against the background of changes in the production of a number of important proteins involved in the regulation of hormone action, angiogenesis, apoptosis, redox processes, inflammation, and immune response.

conclusions

1. Modification of the proteomic spectrum of the pancreas is an important pathogenetic factor in the development of EGE.

2. Proteins that are absent or appear in the pancreas in endometriosis can serve as informative markers of this disease.

Reviewers:

Avrutskaya V.V., Doctor of Medical Sciences, Leading Researcher of the Obstetrics and Gynecology Department, Head of the Polyclinic Department of the Federal State Budgetary Institution "RNIIAP" of the Ministry of Health of Russia. Federal State Budgetary Institution "Rostov Research Institute of Obstetrics and Pediatrics" of the Ministry of Health of Russia, Rostov-on-Don;

Kaushanskaya L.V., Doctor of Medical Sciences, Chief Researcher of the Obstetrics and Gynecology Department of the Federal State Budgetary Institution "RNIIAP" of the Ministry of Health of Russia. Federal State Budgetary Institution "Rostov Research Institute of Obstetrics and Pediatrics" of the Ministry of Health of Russia, Rostov-on-Don.

Bibliographic link

Tomai L.R., Linde V.A., Ermolova N.V., Gunko V.O., Pogorelova T.N. THE ROLE OF PROTEMAL IMBALANCE OF PERITONEAL FLUID IN THE PATHOGENESIS OF EXTERNAL GENITAL ENDOMETRIOSIS // Modern problems of science and education. - 2014. - No. 6.;
URL: http://science-education.ru/ru/article/view?id=17171 (date of access: 01.02.2020). We bring to your attention the journals published by the publishing house "Academy of Natural History"

peritoneal fluid normally provides uniform lubrication of the surface of the abdominal organs and reduces friction between them. They are transparent and absolutely sterile - there are no bacteria in it. The amount is minimal - 5-20 ml. Consists of water, a minimum amount and trace elements.

Peritoneal fluid is formed during blood filtration and returns back to the same through the lymph. The balance of formation and reabsorption of peritoneal fluid keeps its constant amount in the abdominal cavity.

Various diseases lead to an increase in the volume of peritoneal fluid, in which case it is called ascitic.

Ascitic fluid - an indicator of pathology, the inability to maintain a balance between the formation and reabsorption of peritoneal fluid.

Peritoneal fluid is normal

• quantity - up to 50 ml
• color - from transparent to pale yellow
• and - normal values ​​depend on blood levels
• - the same level as in the blood
• cells - in a small amount

Analysis of ascitic fluid is

several types of studies aimed at diagnosing the causes of ascites - excessive accumulation of fluid in the abdominal cavity.

The procedure for taking ascitic fluid for analysis is called paracentesis.

Causes of ascites

• increased pressure in the vessels of the liver - cirrhosis of the liver, chronic heart failure
• inflammation of the peritoneum as a result of its damage - infection, tumors (stomach cancer, intestinal cancer, primary peritoneal cancer, lymphoma), pancreatitis, autoimmune diseases

The nature of the ascitic fluid will indicate the type of pathological process and help in choosing the best method of treatment.

Types of analyzes of peritoneal and ascitic fluid

• external assessment of ascitic fluid - quantity, color, smell
• biochemical analysis - LDH, total protein, albumin gradient calculation, glucose,
• sediment microscopy - to detect abnormal tumor cells
• sediment staining by the Gram method - allows you to see bacteria in a microscope
• bakposev on nutrient media with the determination of sensitivity to antibiotics

Types of ascitic fluid

• transudate
• exudate

transudate

transudate appears with increased pressure in the vessels of the liver and seepage of fluid into the space of the peritoneum, as well as a low level of albumin protein in the blood, which normally retains water inside the vessels.

Transudate occurs in chronic heart failure, nephrotic syndrome, cirrhosis of the liver.

Transudate properties

• ascitic fluid is clear or slightly straw-coloured
• total protein less than 3g/dl
• albumin - reduced, gradient over 1.1 g/dl
• LDH gradient blood/ascitic fluid - less than 0.6
• glucose - equal to the level in the blood
• sediment microscopy - a small number of lymphocytes
• specific gravity - less than 1.015

Exudate

Exudative ascitic fluid- the result of damage to the peritoneum by bacteria, malignant neoplasms, enzymes (for example, the pancreas), rupture of the abdominal organs (gall bladder with cholelithiasis or pancreatic cysts).

Properties

• color - from yellow to greenish
• total protein in ascitic fluid more than 3 g/dl
• albumin elevated, gradient less than 1.1 g/dl
• LDH gradient over 0.6
• glucose - below 3.3 mmol / l
• increased number of leukocytes, mainly in the sediment
• specific gravity - 1.015

Peritoneal fluid in normal and diseased conditions

External parameters

• normal peritoneal fluid is clear, slightly yellowish in color
• yellow color - with liver pathology with elevated levels of bilirubin
• milky color - blockage of the lymphatic vessels
• greenish - the presence of bile, which directly indicates a rupture of the bile ducts
• when blood enters, ascitic fluid becomes red, which happens with trauma and tumors
• turbidity - when bacteria multiply in the abdominal cavity

Biochemical analysis of ascitic fluid

• Normally, glucose in ascitic fluid is equal to the level in the blood
• amylase is examined only if inflammation of the pancreas (parcteratitis) is suspected
• tumor markers - to determine the type and possible primary localization of the tumor
• LDH - an indicator of cell decay

Sediment microscopy

Microscopy of the sediment of ascitic fluid is done if an infection or neoplasm is suspected. Under normal conditions, the sediment is extremely scarce, single leukocytes (mainly lymphocytes) are found in it, and there are no bacteria.

• neutrophils (a subspecies of leukocytes) are increased with bacterial lesions of the peritoneum, and lymphocytes - with peritoneal tuberculosis
• abnormal cells (irregular in shape, large size and atypical coloration) occur when the tumor spreads through the peritoneum

Tests for infections

• Gram stain - the precipitate is applied to a glass slide and stained according to the Gram, which allows the detection of bacteria and fungi
• bakposev on nutrient media with the cultivation of culture for several days and the determination of its sensitivity to antibiotics
• adenosine deaminase - protein, significantly increased in peritoneal tuberculosis

Analysis of peritoneal and ascitic fluid - the norm and changes in diseases was last modified: December 6th, 2017 by Maria Bodyan

The Douglas space, or retrouterine space, is an anatomical space located in the back of the woman's small pelvis. It is located between the posterior wall of the uterus, the cervix, the vaginal posterior fornix and the anterior wall of the rectum. In physiological terms, the space of Douglas is said to be free, that is, it does not contain fluid or tissue.

The presence of traces of fluid in the retrouterine space may indicate ovulation, in which case there is no cause for concern. A larger volume of fluid can be visualized during transvaginal ultrasound. It is always necessary to determine the nature of the detected secret - bloody fluid, peritoneal fluid (ascites), pus, etc. For this purpose, a diagnostic puncture of the retrouterine space is often performed to obtain material for research and determine the probable cause of fluid accumulation.

The causes of the presence of fluid in the Douglas space, as a rule, are diseases of the genital organs, but not always. If fluid in the retrouterine space appears on certain days of the menstrual cycle, there is no cause for concern.

Sexually mature women and girls regularly - especially immediately after ovulation (just after half the cycle) - have a small amount of free fluid. However, if the presence of fluid is detected in the first phase of the cycle or at the end of the second, and in large quantities, then pathology of the uterine appendages or abdominal cavity can be suspected.

Fluid in the retrouterine space causes

The most common causes of fluid behind the uterus are diseases:

• rupture of an ovarian cyst;
• dropsy of the ovary;
• endometriosis;
• rupture of an ectopic pregnancy;
• adnexitis;
• ovarian cancer;
• peritonitis;
• enteritis;
• cirrhosis of the liver;
• ovarian hyperstimulation (after hormonal stimulation).

Depending on the nature of the fluid behind the uterus:

The presence of bloody fluid behind the uterus can result from:

• bleeding into the abdominal cavity from the pelvic organs,
• rupture of an ectopic pregnancy,
• ruptured ovarian cysts
• foci of peritoneal endometriosis.

A large amount of ascitic (peritoneal) fluid may be due to:

• female genital cancer (cancer of the ovary, fallopian tube, cervix),
• cirrhosis of the liver,
• circulatory failure.

The presence of purulent fluid may indicate:

• inflammation of the small pelvis (for example, appendages);
• or abdominal cavity (eg, peritonitis, inflammatory bowel disease).

Diseases in which there is free fluid in the space of Douglas

Rupture of an ovarian cyst

An ovarian cyst is an abnormal space inside the ovary surrounded by a wall. There are several types of ovarian cysts: simple, serous fluid-filled, dermoid cysts, and endometrial cysts (chocolate cysts that form during endometriosis). Sometimes a cyst can form at the site of an unruptured follicle during ovulation - this type of cyst tends to spontaneously reabsorb. Unfortunately, it can also happen that a cyst in the ovary indicates the presence of cancer. Cysts may sometimes cause no symptoms and are discovered incidentally during a routine abdominal ultrasound. Sometimes, however, their presence can cause various ailments:

• menstrual irregularities,
• irregular bleeding not related to the menstrual cycle,
• abdominal pain,
• pain in the area of ​​the ovary where the cyst is located.

It happens that the cyst ruptures, then the woman feels severe pain, and during an abdominal ultrasound, fluid is found in the retrouterine space. Treatment of cysts, if they do not give any symptoms, can only consist in their systematic observation. However, if cysts cause problems or enlarge, they need to be removed (either laparoscopically or conventionally, depending on the type of cyst).

Rupture of an ectopic (ectopic) pregnancy

When does an ectopic pregnancy occur? An ectopic pregnancy occurs when fertilized eggs implant in a different location than the body of the uterus. The frequency of ectopic pregnancy is estimated at about 1% of all pregnancies. The most common site of an ectopic pregnancy is the fallopian tube. In fact, the embryo can implant almost anywhere: in the cervix, ovary or abdomen. The most dangerous for the health and life of a woman is an abdominal or cervical pregnancy, but, fortunately, they occur very rarely.

What are the symptoms of an ectopic pregnancy? During an ectopic pregnancy, abnormal discharge and bleeding may occur, in addition, there are abdominal pains, sometimes difficulty with defecation. In a situation where an ectopic pregnancy ruptures, there is sharp pain in the abdomen, while an ultrasound will reveal fluid in the pouch of Douglas. The treatment of an ectopic pregnancy is always surgical.

Inflammation of the appendages

For adnexitis, the so-called ascending path is characteristic - vaginal microbes enter the higher organs of the female reproductive system. Until recently, the most common pathogen causing inflammation of the appendages was gonococcus. Currently, due to a significant decrease in the incidence of gonorrhea, the bacterium is no longer the most common organism. The following pathogens are also included in the etiological factors of adnexitis:

• chlamydia;
• mycoplasma genitalis and other mycoplasmas;
• coli;
• group B streptococci and other streptococci;
• Gardnerella gardnerella vaginalis.

Chlamydia and gonococci have the largest share in the formation of infection leading to inflammation of the appendages.

What are the symptoms of adnexitis? First of all, there may be pain in the lower abdomen, usually the pain is bilateral. In addition, dyspareunia (pain during intercourse) may be present, as well as abnormal discharge from the genital tract associated with inflammation of the cervix or vagina. There is abnormal bleeding - intermenstrual bleeding or very heavy menstrual bleeding and fever above 38 C. Ultrasound examination may reveal the presence of fluid behind the uterus. Treatment of inflammation of the appendages is the use of antibiotics and symptomatic therapy.

ovarian cancer

This cancer does not cause any symptoms for a long time, the presence of symptoms such as pain in the lower abdomen, enlargement of the abdominal cavity or vaginal bleeding, unfortunately, indicates the severity of the cancer.

Peritonitis

The presence of purulent fluid in the retrouterine space may indicate the presence of peritonitis and requires clarification of the diagnosis and examination of the gastrointestinal tract and urinary tract.

Symptoms of Fluid in Douglas Space

Symptoms depend on the cause of fluid accumulation. For example, in the event of a rupture of an ovarian cyst, pain in the abdominal cavity may appear, which periodically become sharp and cutting, nausea and vomiting, diarrhea, and loss of appetite. With the rupture of an ectopic pregnancy - spotting and bleeding from the vagina, pain in the lower abdomen, pain in the ovaries, and sometimes a feeling of incomplete emptying of the intestine.

With inflammation of the appendages, there is a sudden cramping pain on both sides of the abdomen, aggravated during intercourse. Sometimes it radiates to the groin and thighs. Accompanied by weakness, fever or febrile state.

Diagnostic puncture through the posterior fornix of the vagina

Puncture of the retrouterine space is a simple invasive method, especially useful for diagnosing bleeding in the abdominal cavity of the pelvic organs and for detecting a disturbed ectopic pregnancy. The procedure is performed under general anesthesia in a hospital setting. The puncture of the Douglas pocket is performed through the vagina using a 20 ml syringe and a needle with a length of min. 20 cm and 1.5 mm in diameter. After inserting a speculum, the gynecologist inserts a needle through the posterior fornix of the vagina, and then aspirates its contents into a syringe.

Sometimes the puncture is performed under ultrasound guidance to avoid the risk of puncturing large pelvic vessels. After the needle is removed, the contents of the syringe are carefully examined. The obtained material can also be transferred for cytological or bacteriological examination. Detection of fragments of clots or bloody fluid may indicate bleeding into the abdominal cavity due to a disturbed ectopic pregnancy. This condition, with the presence of clinical, laboratory and ultrasound symptoms, is an indication for surgery to remove a disturbed ectopic pregnancy, most often using the laparoscopic method.

The lack of content obtained by puncture of the retrouterine cavity does not exclude bleeding into the peritoneal cavity or the existence of an ectopic pregnancy, especially when symptoms indicate peritoneal irritation. Bleeding may be minimal or there may be post-inflammatory adhesions that prevent collection of material for examination. The presence of bloody fluid can also indicate endometriosis. The bloody contents of the Douglas cavity can become infected (superinfection), worsening the condition of a patient with endometriosis. Treatment includes aspiration of hemolyzed blood from the pouch of Douglas and laparoscopic removal of the endometriosis.

Cytological examination of the fluid

The detection of an increased amount of peritoneal fluid may be sufficient reason for maintaining oncological activity. Ascitic fluid collected at the time of puncture of the retrouterine cavity should be sent for cytological examination to confirm or exclude a tumor. Detection of the presence of cancer cells in the fluid from the abdominal cavity provides valuable information for the doctor, as it may indicate the appearance of a primary malignant neoplasm of the female genital organs.

In women who have previously had cancer and have undergone surgery, this symptom may indicate a recurrence of the cancer. As a rule, the presence of tumor cells in the peritoneal fluid is associated with a high prevalence of female genital cancer, which is an unfavorable prognostic factor in these patients. It should be noted that cytological examination of fluid from the peritoneal cavity is only an auxiliary method in detecting malignant tumors of the ovary, fallopian tube, and cervix.

Cytological examination of the fluid sediment can also reveal an increased number of inflammatory cells that appear with various inflammations of the pelvic organs. Finally, an increased amount of peritoneal fluid is the result of other diseases, such as cirrhosis of the liver or circulatory failure.

When should you see a doctor?

Patients should seek immediate medical attention if, in addition to increased fluid in Douglas's cavity, the following symptoms are present:

• abdominal pain,
• painful intercourse,
• bleeding from the genital tract, not associated with menstruation, contact bleeding,
• nausea, vomiting,
• a rapid increase in the circumference of the abdominal cavity,
• fever, chills,
• weight loss.

Treatment

Treatment depends on the cause of fluid in the retrouterine space. For example, if an ovarian cyst ruptures, surgery is usually needed to remove the cyst. If an ectopic pregnancy ruptures, it must be removed laparoscopically.

The study of body fluids spans several analytical disciplines. It includes counting and differentiating cells and other particles. Cell counting and differentiation in various body fluids, such as cerebrospinal fluid, serous fluid, and synovial fluid, is possible with our XN-series analyzers and some X-class analyzers. Counting and differentiating cells in body fluids is an important aspect of the process of making a correct diagnosis. Such an analysis may be required for various reasons, which depend solely on the type of body fluid.

Automation of these manipulations with body fluids has several advantages over manual methods that involve the use of a traditional counting chamber. It's fast and convenient. At the same time, the quality of the analysis does not depend on the subjective level of training of the employee, in connection with which this method ensures the standardization of the differentiated calculation procedure. In addition, the number of time-consuming manual counts in the counting chamber is reduced.

Because cells in body fluids, especially neutrophils, degrade rapidly, the sample must be analyzed as quickly as possible.

Cerebrospinal fluid (CSF)

Cerebrospinal fluid is a clear body fluid in the form of saline with a low protein content due to ultrafiltration of the blood. It fills the space between the skull, brain, and ventricles and surrounds the spinal cord. CSF serves as a "shock absorber" for the brain and spinal cord, transports hormones and neurotransmitters, aids in the removal of toxic metabolites, and provides a constant environment for the brain. A healthy adult contains between 100 and 150 ml of this fluid, and daily production levels are typically around 500 ml.

Cellular analysis of cerebrospinal fluid is necessary to identify or exclude diseases that affect the central nervous system: infections caused by bacteria, viruses, fungi or protozoa; inflammation (eg, multiple sclerosis or acute idiopathic polyneuritis); meningitis (eg, cells from peripheral neoplasms).

Sampling is mainly done by lumbar puncture, less often by cisternal puncture. In patients with a ventricular shunt, such as after surgery or during treatment for hydrocephalus, samples may also be obtained from the shunt. CSF collection is a routine procedure, but still carries some risks, making CSF a valuable material.

Pleural fluid

Pleural fluid accumulates between the two pleural layers in the space around the lungs known as the pleural cavity. In the normal state of the body, the volume of pleural fluid does not exceed 10 ml. An excess of this fluid (pleural effusion) is considered a pathology. There are various causes for this condition, from acute heart failure (the most common cause) to pneumonia, pulmonary embolism, tuberculosis, etc.

Pleural fluid cells are counted and differentiated to determine the cause of the pleural effusion and to identify or rule out infection of the lungs or pleura by bacteria, viruses, or protozoa. A high concentration of neutrophils, for example, suggests an infection, but even a non-infectious pleural effusion may contain a significant number of leukocytes, although there may be more mononuclear cells in this case. In addition, mesothelial cells are constantly found, and cancer cells can be found in cancer. If the fluid is bloody, this is often the result of tumor invasion.

peritoneal fluid

Like pleural fluid, peritoneal fluid is considered abnormal when it exceeds a certain volume, usually 10 ml. Peritoneal fluid accumulates in the abdominal cavity. If this fluid accumulates in excess, the condition is known as ascites. In most cases, ascites is a consequence of cirrhosis of the liver, but also occurs with cancer, acute heart failure, and even with tuberculosis. Analysis of peritoneal fluid is performed to determine the cause of its presence and to detect or exclude peritonitis. A high concentration of neutrophils usually indicates the presence of infection, while bloody ascites is most often the result of tumor invasion.

pericardial fluid

Pericardial effusion is an abnormal accumulation of fluid in the pericardial cavity, the volume of which in the normal state does not exceed 20-50 ml. Pericardial effusion may be due to pericarditis, viral infections, inflammatory disorders, kidney failure, cardiac surgery, etc. As for other serous body fluids (pleural fluid, peritoneal fluid), their analysis mainly serves to determine their etiology or to detection or exclusion of infections.

synovial fluid

Synovial fluid is a clear body fluid found in the joint cavity that reduces friction between articular cartilage during movement. With arthritis and infection, the volume of this fluid increases. Cell count and differentiation can help identify the inflammatory or infectious nature of the joint effusion. A very high concentration of white blood cells (possibly more than 100,000/µl) with a predominance of neutrophils indicates the presence of an infection in the joint.

Fluid produced during continuous ambulatory peritoneal dialysis (CAPD)

The fluid produced by continuous ambulatory peritoneal dialysis (CAPD) is not a natural body fluid, because it is not produced physiologically or due to disease, but is solely a consequence of the treatment of the disease. The CAPD process is an alternative to hemodialysis for patients suffering from kidney disease. In this process, the patient's abdominal cavity is used as a membrane through which fluids and substances are removed from the blood by osmosis. This procedure is used for common infections of the abdominal cavity. Elevated white blood cells with a high neutrophil count may indicate peritonitis, while eosinophilia is usually considered a secondary effect of catheter use.

Peritoneal fluid for research is obtained by paracentesis. Paracentesis is performed using a trocar and cannula, which are inserted through the abdominal wall under local anesthesia. If the peritoneal fluid is evacuated for therapeutic purposes, the trocar cannula is attached to the drainage system. However, if only a small amount of peritoneal fluid is to be obtained for examination, both a trocar and an 18-gauge puncture needle can be used. When puncturing all four quadrants of the abdomen, peritoneal fluid is aspirated from each quadrant, which is important for diagnosing damage to the abdominal organs in trauma and timely surgical intervention.

• It should be explained to the patient that the study allows you to clarify the cause of ascites or diagnose damage to the abdominal organs during trauma.
• No restrictions on diet and diet are required.
• The patient should be informed that a sample of peritoneal fluid will be taken during the examination, an abdominal wall puncture will be performed under local anesthesia, which will reduce discomfort, and that the examination will usually take approximately 45 minutes.
• To relieve anxiety in the patient, he should be reassured, assuring that complications in this study are very rare.
• If the patient has ascites, he should be told that the evacuation of ascitic fluid will improve well-being and facilitate breathing.
• It is necessary to ensure that the patient or his relatives give written consent to the study.
• Before the study, the initial basic physiological parameters, body weight are determined and the abdominal circumference is measured.
• The patient should be warned that, if necessary, blood will be taken from him for research.
• Before the study, the patient must urinate, which is important to prevent accidental damage to the bladder by a puncture needle or trocar.
• Before the study, a survey x-ray of the abdomen is also performed.

• The patient is seated on a bed or in a chair so that his feet are comfortably placed on the floor, and his back is securely supported. If it is difficult for the patient to be out of bed, raise the head of the bed high (Fowler high position) and ask the patient to get comfortable.
• The patient is covered to prevent chills, leaving only the puncture site open.
• To prevent bedding from getting wet and the outflow of peritoneal fluid on the patient, a plastic oilcloth is used.
• The hair in the puncture area is shaved off, the skin is treated with a disinfectant solution and covered with sterile diapers.
• The puncture site is anesthetized with a local anesthetic solution.
• The doctor inserts a puncture needle or trocar with a cannula at a level of 2.5-5 cm below the navel, but the puncture of the abdominal wall can also be done in the iliac region, at the edge of the rectus abdominis, in the lateral region, or in each of the four quadrants of the abdomen.
• When using a trocar with a cannula, a small incision is made in the skin to facilitate their introduction through the abdominal wall. The penetration of the needle into the abdominal cavity is accompanied by a characteristic sound. After removing the trocar, the peritoneal fluid is aspirated using a 50 ml syringe. If more peritoneal fluid needs to be evacuated, the syringe is connected with a tubing from an IV line to a plastic bag. You can evacuate no more than 1500 ml of peritoneal fluid. If the fluid is difficult to drain, a trocar with a cannula or a puncture needle should be inserted elsewhere in the abdominal wall.
• After evacuation of the peritoneal fluid, the cannula or needle is removed and the puncture site is pressed down with a sterile napkin, sometimes one suture is applied to the skin wound.
• Peritoneal fluid samples are numbered in the order in which they were obtained. If the patient is receiving antibiotics, this is noted on the laboratory referral form.
• Carefully, in compliance with the existing instructions, the used tools are removed, the material for single use is packed in a special container for subsequent destruction.
• A sterile gauze bandage is applied to the puncture site. It must be multi-layered to absorb the outflowing peritoneal fluid. The dressing should be reviewed periodically (for example, at each vital signs test) and changed or secured as necessary.
• It is necessary to periodically determine the main physiological parameters, in the unstable condition of the patient, they are determined every 15 minutes. The patient is weighed and the circumference of the abdomen is measured, after which the results are compared with the original ones.
• The patient is provided with peace and, if possible, refrains from medical and other procedures that may cause him stress (for example, changing bed linen).
• Monitor diuresis for 24 hours, the presence of hematuria indicates damage to the bladder.
• With the evacuation of a significant amount of peritoneal fluid, the risk of collapse increases, so you should be especially careful about symptoms such as pallor of the skin, increased heart rate and respiration, lowering blood pressure and central venous pressure, impaired consciousness, complaints of dizziness. In such cases, if the patient is conscious, he is given fluids to drink.

In connection with the features noted above, by administering intravenous fluids and albumin to patients, the content of electrolytes (especially sodium) and protein in the serum is determined.

Features of the peritoneal fluid are normal.

General Features and Components	Meaning
General signs	Sterile, clear or pale yellow liquid, odorless, in an amount not exceeding 50 ml
red blood cells	Missing
Leukocytes	Less than 300 in 1 µl
Protein	0.3-4.1 g/dl (SI: 3-4.1 g/l)
Glucose	70-100 mg/dl (SI: 3.5-5 mmol/l)
Amylase	138-404 U/l
Ammonia	Less than 50 mg/dL (SI: less than 29 µmol/L)
Alkaline phosphatase	Men over 18: 90-239 U/l Women under 45: 76-196 U/l Women over 45: 87-250 IU/L
Tumor cells (cytological examination)	Missing
bacteria	Missing
Mushrooms	Missing

Turbid peritoneal fluid is observed with peritonitis caused by a primary bacterial infection, intestinal rupture as a result of trauma, pancreatitis, intestinal infarction, strangulation obstruction, appendicitis. Bloody peritoneal fluid is observed in benign and malignant tumors, hemorrhagic pancreatitis, or vessel damage when a trocar is inserted into the abdominal cavity Greenish peritoneal fluid, due to the presence of bile in it, indicates gallbladder rupture, acute pancreatitis, intestinal perforation or duodenal ulcer.

The presence in the peritoneal fluid of more than 100 erythrocytes per 1 μl (SI: 100-106 / l) indicates a tumor or tuberculosis in case of abdominal trauma with damage to internal organs, the number of erythrocytes exceeds 100,000 per 1 μl (SI: 100-109 / l) Increased number leukocytes in the peritoneal fluid, more than 25% of which are neutrophils, is observed in 90% of patients with spontaneous bacterial peritonitis and in 50% of patients with cirrhosis of the liver. A high content of lymphocytes is characteristic of patients with tuberculous peritonitis or chylous ascites. A large number of mesothelial cells in the peritoneal fluid is also characteristic of tuberculous peritonitis. A protein level of more than 3 g/dl (SI: 30 g/l) is observed in malignant tumors, more than 4 g/dl (SI: 40 g/l) in tuberculosis. Low glucose levels in the peritoneal fluid are observed in patients with tuberculous peritonitis and peritoneal carcinomatosis.

The activity of alkaline phosphatase in the peritoneal fluid increases by more than 2 times compared with the normal activity in the blood serum in patients with intestinal rupture and strangulation small bowel obstruction. A more than twofold increase in the level of ammonia compared to the normal level in the blood serum is observed with intestinal rupture and strangulation of the small intestine or large intestine, with perforated ulcer and perforated appendicitis The ratio of the protein content in the peritoneal fluid to its content in the blood serum, equal to or greater than 0.5 , characteristic of a malignant tumor, tuberculous or pancreatic ascites and indicates an extrahepatic cause of ascites. A ratio less than 0.5 indicates uncomplicated liver cirrhosis. A gradient between the level of albumin in ascitic fluid and serum more than 1 g/dl (SI: more than 10 g/l) indicates chronic hepatitis, a gradient of less than 10 g/l is characteristic of a malignant tumor. A cytological examination of the peritoneal fluid allows us to detect tumor cells, a microbiological examination - E. coli, anaerobes and enterococci that enter the abdominal cavity when a hollow organ ruptures, inflammatory processes of internal organs (appendicitis, pancreatitis), tuberculosis, ovarian diseases. Identification of gram-positive cocci usually indicates primary peritonitis. With histoplasmosis, candidiasis or coccidioidomycosis, fungi are found in the peritoneal fluid.