Cytological examination of synovial fluid. The study of synovial fluid: how the analysis is performed and what it shows

The procedure, which is called "the study of synovial fluid", is necessary for the diagnosis of various degenerative and inflammatory diseases of the joints.

Synovial fluid is an exudate that produces articular sheath, consisting of connective tissue and lining the bone and cartilage surfaces. It performs the following functions in the joint:

• locomotor;
• metabolic;
• barrier;
• trophic.

Joint fluid quickly responds to all inflammatory processes that occur in the joint, synovial membrane and cartilage tissue. This substance is one of the most important articular components, which determines the morphofunctional state of the articulation.

In a normal, healthy joint, fluid volume is moderate. But with the development of some articular ailments, a so-called articular effusion is formed, which is subject to investigation. More often than others, an analysis of a sample of synovial fluid of large joints (elbow, knee) is done.

Synovial fluid can be obtained by puncture. The most important condition for taking a puncture is the sterility of the joint.

The standard analysis of a synovial fluid sample includes:

1. Macroscopic analysis of punctured fluid (color, volume, turbidity, viscosity, mucin clot).
2. Counting the number of cells.
3. Microscopy of the native preparation.
4. Cytological analysis of the stained preparation.

At healthy person synovial fluid is light yellow (straw) in color. However, in both arthritis and ankylosing spondylitis (), the color of the test fluid remains yellow. At inflammatory processes the color of the joint fluid may become different, depending on the characteristic changes in the synovial membrane.

If you have psoriatic or rheumatoid arthritis the color of the examined exudate can vary from yellow to green. In traumatic or bacterial diseases, the color of the synovial fluid ranges from burgundy to brown.

synovial fluid healthy joint transparent, but in the presence of psoriatic, rheumatoid or septic arthritis, its turbidity is observed.

The nature of the viscosity depends on:

1. pH level;
2. salt concentration;
3. the presence of previously administered drugs;
4. degree of polymerization of hyaluronic acid.

An increased level of viscosity is noted when:

• systemic lupus erythematosus;
• various traumatic changes.

A decrease in viscosity is observed when:

1. rheumatism;
2. arthrosis;
3. ankylosing spondylitis;
4. various arthritis (psoriatic, gouty, rheumatoid).

One of key features synovial fluid is the ability to produce a mucin clot as a result of mixing with acetic acid.

In this case, the presence of a loose clot indicates inflammatory processes occurring in the joints.

The main analysis that determines the pathology of the articulation

The main study that diagnoses a particular pathology is a microscopic analysis of a sample of synovial fluid.

First of all, doctors pay attention to counting the number of cells in the preparation. The norm is up to 200 cells/µl. A significant increase in the number of cells is called cytosis. Cytosis allows diagnosing dystrophic and inflammatory diseases, clearly assessing the development of inflammatory processes.

During the acute stage of the course of any type of arthritis, the patient has a pronounced cytosis (the number of cells ranges from 30,000 to 50,000).

1. With microcrystalline arthritis, the patient has a slight cytosis.
2. In Reiter's syndrome, pseudogout, or psoriatic arthritis, cytosis is moderate (20,000 to 30,000 cells).
3. If the cell count exceeds 50,000, the patient is diagnosed with bacterial arthritis.

Careful analysis can reveal the presence of a large number of various crystals in a patient, but only two of their types are important for diagnosis. In pseudogout, the patient has crystals of calcium dihydropyrophosphate, and the presence of sodium urate crystals indicates gout. These deposits can be detected using polarizing microscopy.

A healthy synovial fluid contains blood elements (lymphocytes, monocytes, neutrophils) and a variety of tissue cells (histiocytes, synoviocytes).

In inflammatory processes in the articular exudate, it is possible to detect special form neutrophils - rhagocytes. Such cells have a cellular structure formed due to the incorporation of immune complexes into the cytoplasm. The presence of ragocytes is mainly indicative of rheumatoid arthritis.

The detection of mononuclear cells in the synovial fluid is characteristic of tuberculous processes, allergic synovitis and arthritis that developed against the background of neoplasms.

It should be noted that inflammatory joint diseases are characterized by an increase in acute phase parameters and the level of lactate dehydrogenase.

Microscopic examination of the smear can detect gram-positive cocci, chlamydia or gonococci. Often, fungal bacteria are detected in patients. To accurately determine the nature of the infectious process and establish sensitivity to antibiotics, doctors inoculate the synovial fluid for pathogenic microflora.

It is possible to puncture the articular exudate only as prescribed by a rheumatologist. In conclusion, the video in this article will raise the very interesting question of synovial fluid prosthetics.

Cytological examination of synovial fluid (SF) is a simple analysis that allows for any joint disease with effusion to learn about the inflammatory or non-inflammatory nature of the pathology. In the case of inflammation, the analysis allows diagnosing a group of metabolic arthropathy, represented mainly by gout and chondrocalcinosis, when intra-articular microcrystals (MK) cause inflammation. With these ailments, SF testing makes it possible to carry out a quick diagnosis, accurate and reliable.

Cytological studies of synovial fluid are not expensive, minimally invasive, fast, and technically available. In this case, two diagnostic features are of particular interest:

• The presence of a large number of cells is a true reflection of inflammatory joint disease;
• The presence or absence of MC allows you to confirm or exclude microcrystalline arthropathy within a few minutes and determine its nature. Synovial fluid is usually present in very small amounts in each joint cavity. Its role is twofold: it acts as a lubricant, reducing friction between articular surfaces during movement, and provides nourishment to cartilage tissue that has no blood supply.

Cytological examination of synovial fluid provides useful information about the pathology responsible for joint effusion.

• Features of microcrystals in SF - valuable information in metabolic arthropathy
• 500 but<1500, то считают процентное содержание отдельных типов лейкоцитов, обращая особое внимание на их необычные формы.">The cellularity and formula of the cell population make it possible to distinguish five types of SF (Table 1). This feature is only of relative importance, but is valuable to the clinician.

Table 1 - different types SF according to cytology

• Normal fluid is clear, colorless or yellowish. It becomes more opaque with increasing cellularity.
• The viscosity of the fluid is related to the content of hyaluronic acid. It is high in normal or non-inflammatory SF. Viscosity is easily assessed by stretching a drop of SF between two fingers protected by a glove or fingertips and measuring the length of the filament thus obtained. With all inflammations, the viscosity of the fluid decreases.
• Clot formation is characteristic of abnormal synovial fluid. Indeed, normal SF is free of fibrinogen and does not coagulate. However, any pathological SF, especially if it is inflammatory, contains fibrin and forms a loose thrombus that surrounds the cellular elements in a mesh. Therefore, it is very important to prevent SF coagulation by using anticoagulants.

Synovial Fluid Cytology Technique

1. Taking SF

Any volume is sufficient for analysis, as the test requires only a negligible amount of sample. In the fresh state, a drop is sufficient for analysis. SF is collected in a sterile plastic tube. Cytological examination of the synovial fluid requires the use of an anticoagulant. The best choice- sodium heparinate; calcium oxalate and lithium heparinate should be excluded as they contain UAs that can be phagocytosed by polymorphonuclear cells. For the same reason, particles such as talc or starch from gloves should be avoided.

2. Cytological examination of fresh synovial fluid

This step of cytological examination of the SF should be performed as soon as possible after collection, without delay a drop (0.05 ml) of the sample is viewed under a coverslip. Centrifugation is not required because low cell SF is rarely pathological. Identification of MCs is difficult and requires a polarizing microscope, possibly with a compensator and a turntable, which makes it easier to see MCs, especially if there are few of them. In the absence of a polarizing microscope, the analysis can be carried out with a very closed aperture of the microscope, when elements are detected that have a refractive index different from that of the cellular components. This study can be supplemented by determining the number of cells in the Goryaev chamber, which is important for determining the nature of joint damage (Table 2).

Main pathological conditions and types of fluid

If an immediate cytological examination of the synovial fluid sample is not possible, then it can be delayed up to 24 hours, provided that the SF is kept at 4 °C or, even better, in the freezer at -20 °C. It is also possible to prepare a swab and air dry. K can be viewed either directly, in polarized light, or after staining according to May-Grunwald-Jiams (MGJ).

3. Coloring according to MGZH

The results of counting the number of cells in fresh SF are supplemented by the count of individual cell types after cytocentrifugation, smear preparation and staining for MGF, which makes it possible to clarify the nature of the cells present in the SF.

results

Cytological examination of synovial fluid is a useful test for clinicians, as it allows the examination to be focused on a specific pathology from the many that a patient may suffer from. For metabolic or microcrystalline lesions of the joints, such an analysis is the key to the examination (Table N 3).

Table 3 - Cytological parameters of SF in microcrystalline arthritis

Infectious arthritis

Cytological examination of the synovial fluid reveals very a large number of cells with a predominance of polynuclear cells, often mutated. If the infection is suppressed by antibiotics, then the number of PMNs is less numerous and evidence of infection should be based on bacteriological analysis of the SF or on histological examination synovial biopsy. If SJ is rich eosinophils, must be sought microfilariae.

Inflammatory rheumatic diseases

Here again cellularity has importance, and polymorphonuclear leukocytes (PMNs) are the most numerous. This group of joint pathologies does not have a specific cytological profile that reflects a specific rheumatism. But SF is statistically more inflammatory in rheumatoid and reactive arthritis (Fissinger-Leroy-Reiter spondyloarthritis) than in SLE polyarthritis, rheumatic pelviospondylitis, or psoriatic arthritis.

Metabolic or microcrystalline arthritis

It is for this group of diseases that the cytological examination of the synovial fluid is reasonable and interesting. It should be noted that the detection of MK does not eliminate infection, and that bacteriological examination remains desirable in all cases.

Gout

Gout is always associated with the presence of countless UA in the joint cavity. monosodium urate, very characteristic form and physical characteristics. These are elongated MK, needle-shaped, 5-20 microns in length, with a conical end that pierces cell membranes. The fluid is typically highly inflammatory and rich in neutrophils (Figure 1).

Very small crystals (1-2 µm) can be seen in asymptomatic effusions between gout flares. Now they are often extracellular. Sodium urate MCs are soluble in water, they are not preserved and are not detected in smears stained for BMF.

Chondrocalcinosis of the joints

This pathology is due to the presence in the cartilage and / or synovial membrane of deposits of calcium pyrophosphate dihydrate MK, which in the acute period causes symptoms of either pseudogout or inflammatory rheumatoid or osteoarthritis. Key to diagnostics – identification of characteristic MC in the SF. MK is 5-10 microns long, inside leukocytes, in the form of a straight or oblique parallelepiped. Their birefringence is less than the K of uric acid. These FAs are slightly soluble in water and persist after staining for MGJ. Sometimes analysis of SF in a fresh state or after staining of MGJ shows both urate and pyrophosphate UA. These mixed joint lesions are not exclusive.

Joint Diseases and Calcium Phosphates

In some patients, often suffering from calcification of the rotator cuff shoulder joint, destructive arthropathy and signs of SF inflammation may develop. Symptoms of SF inflammation are associated with the presence of UA, owned various forms calcium phosphate. The most common is hydroxyapatite, but octacalcium phosphate and tricalcium phosphate can also be found. The length of these crystals is 0, l - 0.2 µm, they are invisible in an optical microscope.

Other microcrystals

When examining fresh SF, other types of MK are sometimes detected, which include:

• Calcium oxalate, which can be observed in patients on hemodialysis. They form

MK sometimes pyramidal, sometimes irregular shape or sticks that can be mistaken for calcium pyrophosphate;

• MK cholesterol - large, rectangular, flat, often with a beveled corner. They are observed in rheumatoid arthritis or in age-related bursitis;
• Cortisone derivatives that are injected into the joint for therapeutic purposes crystallize there and UA can persist for several weeks or months. They can call real

microcrystalline arthritis. Their shape is variable and they are all birefringent;

• Charcot-Leiden MC are rare and can be seen in eosinophil-rich fluid. Their shape is similar to a compass needle, weakly birefringent.

Conclusion

Cytological examination of the joint fluid is a valuable diagnostic test, little invasive, fast, affordable, in which:

The abundance of cells is a true reflection of the inflammatory nature of the disease of the joints,

The presence or absence of MC allows confirmation or exclusion, within minutes, of microcrystalline arthropathy, and often determines the nature of metabolic arthropathy.

Standardized analytical technology for clinical laboratory analysis of synovial fluid.

1. Purpose of the study

Technology "Clinical laboratory analysis synovial fluid” is performed to diagnose diseases of the joints, as well as to monitor the course of the disease and the effectiveness of treatment.

The study of synovial fluid has great importance at:

All employees must comply with the instructions and safety regulations set out in the technical data sheets for electrical devices used in the technology (photometers, microscopes, centrifuges); personnel working with reagents must be trained in handling them, use the means personal protection observe the rules of personal hygiene.

To prevent fires, it is necessary to comply with fire safety rules in accordance with current regulations.

Thus, it is necessary to strictly adhere to all points of the instructions for safety, fire safety and biological safety.

2.3 Conditions for the implementation of the technology of clinical laboratory analysis of synovial fluid and functional purpose

Clinical laboratory analysis of synovial fluid is performed in clinical diagnostic laboratories of specialized outpatient and inpatient healthcare facilities (rheumatology and arthrology centers).

Functional purpose of the service: carried out for the purpose of diagnosing diseases of the joints, monitoring the course and progression of the disease and the effectiveness of treatment.

2.4. Material resources necessary for the implementation of technology: instruments, measuring instruments, laboratory equipment

2.4.1. Binocular microscope with immersion and built-in illuminator.

2.4.2 Polarizing microscope.

2.4.3. Laboratory centrifuge (with cooling: 5-8 °C).

Synovial fluid pellets should be prepared with centrifuges running at 1000 rpm. When using a centrifuge, the manufacturer's instructions must be strictly followed.

2.4.4. Counter-calculator for counting leukocyte formula blood (for counting synoviocytes).

2.4.5. Stand for test tubes.

2.4.6. Containers and cuvettes for staining and fixing smears.

2.4.7. Device for drying smears.

2.4.8. Glass (plastic) products.

2.4.8.1. Centrifuge tubes (10 ml).

For macroscopic study of SF, it is better to use transparent glass tubes. For centrifugation of SF, plastic centrifuge tubes are used, which should have a conical shape to concentrate the sediment, a graduation to determine the amount of synovial fluid obtained during puncture of the joint, and be closed with lids to reduce the risk of splashing. Tubes must be chemically clean and labeled for proper patient identification. Application possible vacuum tubes.

2.4.8.2. Goryaev's camera.

2.4.8.3. Slides and coverslips for microscopy of the native preparation.

Object glass (preferably with a frosted field for marking, size 26 x 76 x 1.1 mm.) for microscopy of the stained preparation.

Glass slide with a ground edge (size 26 x 76 x1.1 mm.) or a plastic spatula for preparing a smear.

2.4.8.4. Pipettes for transferring synovial fluid. Currently, plastic Pasteur pipettes with a finely drawn end and a spray can are used, designed for standardization sediment droplet volume and reduce the risk of biohazards associated with resuspension or transfer of synovial fluid. They must be dry and chemically clean.

2.4.8.5 Glass rods.

2.5 Reagents

2.5.1 Solutions of dye fixatives and other necessary reagents for the preparation of stained smears (see GOST R Cytological examination of bone marrow punctate);

2.5.2 solution acetic acid 5%;

2.5.3 EDTA (dicalcium or disodium salt).

2.5.4. alizarin red solution 2%.

2.6 Other Consumables

2.6.1. Rubber gloves.

2.6.2. Disinfectants.

3. Characteristics of the technique for performing the technology for the study of synovial fluid

3.1 Obtaining samples of synovial fluid

For the correct conduct of the preanalytical stage, it is necessary to comply with the requirements of the GOST R 53079.4-2008 standard. .

The puncture of the joint is performed by a clinician.

The rules for storage and transportation of synovial fluid samples are set out in

appendix A.

When puncturing the joint, SF is collected in sterile centrifuge tubes (2-3 or more, depending on the amount of SF obtained) and immediately transferred to the clinical diagnostic laboratory. One of the tubes (or more, depending on the number of tubes received) is sent to the microbiological laboratory (department) for microbiological studies, and the rest are used to conduct a clinical laboratory study of SF (determination of physicochemical properties and microscopic examination of native and stained with azure-eosin preparations with counting synoviocytogram, counting cellular elements in 1 μl (cytosis), as well as performing biochemical and immunological studies.Biochemical and immunological studies are carried out in the supernatant after centrifugation of the SF, and the precipitate is used to search for crystals in the native preparation using a polarizing microscope, as well as for counting synoviocytogram in a stained smear.To count cells, you can collect SF in a tube containing an anticoagulant (disodium or dipotassium EDTA), special vacuum tubes with K2EDTA are available, which can be used to take SF.

If there are appropriate indications (suspicion of the presence of neoplasm cells), the stained smear is sent to the cytology laboratory.

3.2 Sample identification

The following information should be included in the referral for research: the patient's surname and initials, age or date of birth, sex, department medical institution and ward (in a hospital), room medical card(identification number), diagnosis, date and time of collection of the synovial fluid sample, time of delivery of the sample to the laboratory. All indicators that need to be defined should be listed. If necessary, indicate the drugs injected into the punctured joint.

Unlabeled or mislabeled specimens are not suitable for testing and should be notified to the clinician ordering the test.

3.3 Sample acceptance

Since the accuracy of the synovial fluid test results largely depends on the quality of the delivered sample, it is necessary to strictly follow the rules for storing and transporting synovial fluid (Appendix A).

After the synovial fluid sample is delivered to the laboratory, the laboratory employee receiving the material must check the correctness of the referral for analysis, the labeling of the dishes (the patient's code or surname and other data must be identical to the data indicated in the referral form) and register the received material.

The synovial fluid collected in a tube with K2 EDTA should also be examined within 30 minutes, and when stored in a refrigerator (temperature 3-50C) - no later than 24 hours (only for examining stained smears).

N o t e ─ Long-term storage of the SF supernatant is allowed at -70°C for biochemical and immunological studies.

Postponement of analysis and use of sample cooling are noted on the answer sheet.

Samples should be brought to room temperature before testing.

3.4 Macroscopic evaluation and examination of the physicochemical properties of the synovial fluid

3.4.1. The amount of synovial fluid normally varies from 0.2 to 2.0 ml (depending on the size of the joint). In various diseases of the joints, the amount of SF can reach 100 ml or more.

3.4.2. Synovial fluid color.

The color of synovial fluid is normal - light yellow

NOTE Light yellow or yellow synovial fluid is seen in degenerative joint disease; bloody - with traumatic arthritis; for inflammatory joint diseases (rheumatoid arthritis (RA), reactive arthritis (ReA), ankylosing spondylitis, psoriatic arthritis) characterized by different shades of yellow and brown (light yellow, yellow, lemon, light brown, brown, amber or orange); with gout, a light yellow, greenish-yellow, milky-white, milky-yellow, pinkish-white color of the SF is observed; with pyrophosphate arthritis and chondrocalcinosis - yellow or milky yellow, with septic arthritis - grayish-yellow, greenish-yellow or bloody.

3.4.3. transparency of the synovial fluid.

Normal synovial fluid is completely transparent. Turbidity is usually due to an increase in the number of cellular elements, the presence of crystals or microorganisms.

Transparency score.

There are 4 degrees of transparency: transparent, translucent, moderately cloudy and intensely cloudy.

Note - in degenerative joint diseases (osteoarthrosis), SF is transparent and translucent; in inflammatory diseases (RA, seronegative arthritis, gout, pyrophosphate arthritis) - translucent, moderately cloudy or intensely cloudy; with septic arthritis - intensely cloudy, thick.

3.4.4. Presence of sediment.

Normally, there is no sediment in the SF. It appears only in pathology and, as a rule, is fragments of cell membranes, fibrin filaments, tissue fragments resulting from destruction cartilage and synovium, as well as crystals.

N o t e ─ In degenerative diseases of the joints, an amorphous precipitate is found in the SF in amyloidosis. In inflammatory diseases of the joints, sediment is almost always found. In the SF of patients with RA, especially often in children with juvenile RA, one can observe a granular sediment resembling rice grains or "rice bodies" formed from microscopic fragments of fibrin-saturated necrotic synovial membrane. Such a precipitate may be an indicator of the high inflammatory activity of the process.

3.4.5. Viscosity

The most important feature of SF, which distinguishes it from other biological fluids, is the presence of hyaluronic acid, a high molecular weight polymer. It is hyaluronic acid, having a high viscosity, that mainly ensures the fulfillment of the main functions of the SF. There is a direct relationship between the content, molecular weight of hyaluronic acid and the viscosity of the fluid.

Methods for determining viscosity.

Quantitative characteristics of the viscosity of the fluid is determined using a viscometer.

In routine studies, the glass rod method is usually used:

the glass rod is lowered into the SF and then removed. Viscosity is estimated by the length of the mucin threads, there are three degrees of viscosity:

with a thread length above 5 cm - high viscosity, up to 5 cm - medium, less than 1 cm - low.

It is possible to express viscosity in point units: 1 - high, 2 - medium, 3 - low. Normally, the viscosity of the fluid is high.

The intensity of viscosity depends on the concentration of crystals, the degree of polymerization of hyaluronic acid and temperature.

N o t e ─ Use instrumental methods with the use of various viscometers requires (in addition to the availability of the device) a number of additional operations and, therefore, a significant investment of time, while not providing any fundamentally new information compared to the available laboratory test.

4.4.6. Determination of the density of the mucin clot in the synovial fluid.

Hyaluronic acid in SF exists in a complex with proteins known as mucin. The definition of a mucin clot is of great diagnostic value in inflammatory diseases. The test for mucin in synovial fluid correlates well with viscosity.

Methods for studying the density of a mucin clot.

The principle of the method: when acetic acid is exposed to the SF, a mucin clot is formed.

Definition progress:

A drop of SF is added to a test tube containing 3 ml of a 5% solution of acetic acid (CH3COOH). Shake the contents of the tube vigorously for 1 minute, a precipitate forms. There are 4 degrees of sediment density: dense (the sediment looks like a dense lump), moderately dense (a kind of branched, but not broken up structure), moderately loose and loose - more or less breaks down into tiny particles. The formation of a dense mucin clot indicates a significant content of mucin.

Normally, the sediment is dense.

Note 1 ─ In non-inflammatory arthropathy, the mucin clot is usually dense or moderately dense. In inflammatory diseases of the joints, moderately friable and friable.

NOTE 2 Determination of the viscosity and density of the mucin clot is essential for differentiating between "non-inflammatory" and inflammatory processes in the joint. These methods can be mutually controlling: the indicators of one method strictly correspond to those of the other. High viscosity corresponds to a dense, medium - to a moderately dense, low - to a moderately loose and loose mucin clot.

3.5 Microscopic examination of synovial fluid

3.5.1. Requirements for a sample of synovial fluid for microscopic examination.

Before conducting a microscopic examination, the physician should have information about the time of obtaining the synovial fluid and the results of the assessment of the physicochemical properties.

Currently, vacuum tubes containing an anticoagulant (K2EDTA) are produced for taking biological fluids, which is also a preservative for cellular elements and does not affect them. morphology.

Note 1─ Synovial fluid stabilized with K2EDTA cannot be used to detect rhagocytes.

There are three types of microscopic examination:

counting cells in the native synovial fluid in the Goryaev chamber (cytosis), examination of the native preparation and the preparation stained with azure-eosin with the calculation of the synoviocytogram.

3.5.2 Counting the number of cellular elements in 1 µl of synovial fluid in the Goryaev chamber (determination of cytosis).

Research progress.:

The study is carried out in native or stabilized K2EDTA synovial fluid.

Pour 0.4 ml of isotonic or hypotonic NaCl solution into a test tube.

Filter the suspension and store in a refrigerator in a dark glass bottle. Immediately before the study, filter the required amount of dye through a millipore filter.

Mix 20 µl of dye with an equal volume of SF or sediment obtained after centrifugation. It is better to prepare a native preparation and microscopically in a polarizing microscope: ovoid-shaped crystals, 2-3 microns in diameter, saturated red color with a pink halo.

NOTE 4 These crystals are found in hydroxyapatite arthropathy.

Crystals of calcium oxalate, cholesterol, lipids, Charcot-Leiden, etc. can also be found in the synovial fluid.

NOTE 5 Calcium oxalate (C2CaO4 · H2O) crystals are usually cubic in shape but can form colorless, lustrous, highly refractive crystals of various sizes in the form of octahedrons or rectangles resembling postal envelopes. Sometimes there are crystals of calcium oxalate rounded and intercepted, resembling hourglass, gymnastic weights or bows (C2CaO4 2H2O). These crystals can be phagocytosed by polymorphonuclear leukocytes (neutrophils).

NOTE 6 Liquid crystals of lipids are shown in dark field as black Maltese crosses dividing each lipid drop into four white lustrous segments. Drops neutral fat do not have the effect of two-beam refraction of light.

Cholesterol, sodium oxalate, and liquid lipid crystals are not specific to any particular joint disease and may occur in a variety of arthropathies, reflecting a metabolic disorder.

NOTE 7 Amyloid clumps can be found in the SF. These are colorless formations of a rounded shape, a layered structure, resembling a saw cut of a tree, with a characteristic sheen. They are identified in native preparations at x400 magnification, as well as with immersion at x1000 magnification. Amyloid can be detected in native SF stained with Congo red. The resulting preparation can be viewed both in a light and in a polarizing microscope.

Amyloid lumps are found in diseases associated with amyloid arthropathy.

Hematoidin crystals.

Hematoidin crystals are formed by the breakdown of hemoglobin in hematomas without oxygen. These are slightly elongated diamonds and / or golden yellow needles. Hematoidin crystals are well distinguishable both in native and in azure-eosin-stained preparations. Since these crystals are usually quite small in SF, it is recommended to microscopically examine native preparations by immersion. In the focus of inflammation, these crystals can be phagocytosed by macrophages or located on the surface of cellular elements.

NOTE 8 In case of trauma and intra-articular bleeding, conditions are created in the joint cavity under which hematoidin crystals can form.

Charcot-Leiden crystals.

Charcot-Leyden crystals are shaped like a compass needle or a diamond sharply elongated in length. Usually Charcot-Leiden crystals are located on the background of detritus or in combination with a large number of eosinophils and are formed during the breakdown of eosinophils from eosinophilic granularity, these crystals can be found in the SF of patients suffering from allergic synovitis.

Medicinal Crystals

Steroids. Intra-articular injections steroid drugs lead to their crystallization inside the joints, where they can persist for up to 10 weeks. Detection of these crystals during microscopic examination of native preparations and subsequent incorrect differentiation can lead to erroneous conclusions.

Non-cellular and non-crystalline elements in SF.

Fragments of cartilage and damaged ligaments may be found in the SF. Fragments of cartilage in the native preparation can be recognized by their characteristic silky sheen. Fragments of cartilage are also found containing clusters of chondrocytes and fragments of the meniscus, which are represented by wavy collagen fibers and also chondrocytes; fragments of ligaments are represented by long thin fibrils and parallel strands of collagen

NOTE 9 Occurs most frequently in the SF following knee injury.

NOTE 10 Despite the high sensitivity of the polarization microscopy method, serious errors are possible when using it, which usually arise due to the insufficient resolution of a particular microscope, the presence of foreign crystal-like impurities and damage to the slide or coverslip. glass. The microscopist must be aware of the possibility of interference and be familiar with the principles of crystal recognition.

3.5.5. Microscopic examination of preparations of synovial fluid stained with azure-eosin (with counting synoviocytogram).

Preparation of SF smears and methods for their staining (section 5.5.2).

Cellular composition of synovial fluid (synoviocytogram).

Determination of the cellular composition of the SF is the most important stage in its study, which allows clarifying the diagnosis, determining the degree of inflammatory activity of the process and prognosis. Determination of the quantitative distribution of cells (synoviocytogram) is the most important indicator for differential diagnosis joint diseases. The calculation of the percentage of cells is carried out in the same way as the calculation of the leukocyte blood formula. (100 cells in a smear are counted and the percentage of each cell type is calculated).

Normally, cells of tissue origin (synoviocytes and histiocytes) predominate in the SF - up to 65%. Lymphocytes make up about 30%, and monocytes and neutrophils - 1-2%.

Blood cells in the SF.

Neutrophils (polymorphonuclear leukocytes).

Neutrophils are 1.5-2 times larger than an erythrocyte, in diameter (14-16 microns). The ratio of the nucleus and cytoplasm is shifted towards the nucleus. The cytoplasm is lilac in color, filled with fine, dusty granularity, which has the color of the cell nucleus. The nuclei consist of 3-4 segments, with a clear division into oxychromatin and basichromatin. With dystrophy, the number of segments in neutrophils increases sharply to 5-7 (hypersegmentation). During apoptosis in the neutrophil, fragments of the nucleus merge into one or two hyperchromic homogeneous, structureless masses of the correct round shape.

In normal SF, the number of neutrophils does not exceed 1-2% in the formula.

NOTE 1 In rheumatoid arthritis, the neutrophil count reaches 90% and the lymphocyte count drops to 10%. A similar picture is observed in ankylosing spondylitis. In inflammatory diseases and intra-articular bleeding, neutrophils make up 60-80% in the SF formula, and more than 95% in septic arthropathy.

Lymphocytes.

These cells are up to 12 microns in diameter. The ratio of cytoplasm and nucleus is shifted towards the nucleus (9:1). The nucleus has a roughly clumpy structure, the basophilic cytoplasm surrounds the nucleus with a narrow rim, sometimes an area of ​​enlightenment around the nucleus is visible.

In normal SF, the number of lymphocytes ranges from 8 to 30%.

NOTE 2 In inflammatory diseases, neutrophils predominate, while in degenerative diseases, lymphocytes predominate. With degenerative diseases of the joints and traumatic arthritis in the SF, the content of lymphocytes reaches 85%. Lymphocytes predominate in the formula also in toxic-allergic synovitis and synovial form of tuberculosis. For arthritis viral etiology, for example, caused by the HTLV-1 virus - atypical lymphocytes appear, the number of which reaches 20%.

Monocytes.

NOTE 3 Monocytes are found in various articular arthropathies, including viral arthritis and monocytic arthritis, as well as damage to implant prostheses.

In addition to these cells in the SF (in pathology), other blood cells can be detected in a small amount: eosinophils, basophils, plasma cells.

Note 4 - Eosinophils are extremely rare in the SF, identical to peripheral blood eosinophils.

Note 5 - Basophils are found in small numbers in inflammatory arthritis, seronegative arthropathy, non-inflammatory arthropathies associated with trauma.

NOTE 6 Plasma cells are found in the SF in inflammatory arthropathies. The detection of plasma cells is typical, in particular, for rheumatoid arthritis, i.e., for a long, sluggish inflammatory process.

Tissue cells in SF.

synoviocytes.

These cells belong to the single-layer flattened epithelium covering the synovial membranes of the joints. In their morphology, they are identical to mesothelial cells. Synoviacytes - epithelial cells with a diameter of 18-25 microns, with a different nuclear/cytoplasmic ratio. They contain centrally or eccentrically located nuclei of a round or oval shape, small-lumpy or looped structure, surrounded by a wide rim of basophilic cytoplasm, sometimes with a "frill" along the periphery. The cytoplasm in the perinuclear zone of some synoviocytes contains fine grains. Synoviocytes are torn away from the surface of the synovial membrane of the joint and are found in the SF in arthropathies. Synovial cells may contain 2 or more nuclei (multinuclear).

There are three types of synoviocytes:

type A - macrophage synoviocytes capable of phagocytosis;

type B - synovial fibroblasts capable of synthesizing and secreting hyaluronic acid;

type AB - transitional forms of cells that combine these two properties.

Histiocytes.

Tissue macrophages are micron-sized cells with a rounded or monocytoid compact nucleus surrounded by fine-grained or non-granular cytoplasm.

NOTE 7 Histiocytes are always present in the SF during inflammatory processes.

NOTE 8 Multinucleated cells can be found in SF, which are synoviocytes or plasma cells and have the same significance as the mononuclear variants of these cells.

NOTE 9 Detection of LE-cells containing inclusions of homogenized nuclear material in the cytoplasm in SF, unlike in peripheral blood, is not a direct indication of SLE. However, the combination of LE cells with a large number of lymphocytes in the SF makes it possible to suspect the presence of SLE in a patient.

NOTE 10 - Cells in mitosis.

Mitotic figures have no diagnostic value. Synoviocytes in a state of division confirm the process of proliferation of cells lining the articular bag.

undifferentiated cells.

Undifferentiated cells are observed in almost all synoviograms.

In thin, well-made smears of SF, fixed with fixatives or dye fixatives and stained with azure-eosin, all cellular elements are amenable to differentiation. Only in thick smears prepared by an inexperienced hand of a laboratory assistant from viscous, hypercellular and previously undiluted SF, cells are encountered that cannot be differentiated. It can be any cellular elements - both tissue and blood. It is almost impossible to detect crystals and microorganisms in such preparations.

4. Registration of synovial fluid analysis results

Each laboratory employee should use the same forms (forms of test results) to report the results. The form must contain the name of the laboratory and medical organization; information about the patient sufficient to identify him; the name of the biological material and all the studied indicators; date of receipt of the sample and, if applicable, time of receipt; research results; reference intervals; the name and signature of the employee who performed the study. The procedure for issuing results should be determined by the instruction approved by the head of the medical organization

5. Quality assurance of synovial fluid analysis technology performance

5.1. Quality assurance programs

Quality Assurance Programs include consistent monitoring of every aspect of the procedure to ensure sufficient diagnostic and patient follow-up capabilities. Quality assurance programs should include all stages of work and establish links between all components of the process (patient, laboratory, clinician). Control is also necessary at the stages of sampling, storage, delivery, manual processing, registration, and issuance of documents. The technical competence of the staff, continuous continuation of education also needs to be controlled. For the successful implementation of all control activities, it is necessary to follow the rules set out in the GOST R ISO 15189 -2006 standard. .

5.2. Record keeping of control measures

Registration of the control should be carried out at all levels: pre-analytical, analytical and post-analytical, for each stage, rules for conducting all procedures should be developed and documented.

A test request form should be developed for clinicians, including the date of appointment and sample collection, patient identification information, diagnosis, medication information, or diagnostic procedures if they can influence the results of the study.

The technique for taking SF samples should be standardized and described in detail in the relevant instructions for doctors and nurses. surgical departments producing punctures of the joints.

The instructions for sample delivery should include the conditions and terms of storage of samples and rules for safe transportation.

Criteria for accepting and refusing to accept samples, requirements for sample registration, handling, labeling and sample storage prior to analysis should be defined for laboratory personnel. The analytical stage is carried out in accordance with research methods. At the post-analytical stage, it is necessary to develop rules for assessing the acceptability of the results of the analysis, which should include an assessment of drug interference, comparison of results with a reference interval, and verification of registration accuracy. The form for issuing results must be approved by the institution and agreed with the treatment departments.

5.3. Instructions for the methods used laboratory research

The methodology for the process of performing laboratory research should be documented and be available at the workplace. The methodology should be based on guidelines or other documents approved in the prescribed manner. It should include criteria for accepting or rejecting SF samples (taking into account the duration of the sample storage period after sampling, a sufficient amount of SF for research); reference intervals; method of registration of results; precautions related to the biological hazard of the test material; reasons for false positive and false negative results.

5.4. Quality control of microscopic examinations.

When developing requirements for the analytical reliability of a visual method, the results of a study of biomaterial samples produced by a researcher with extensive experience in visual examination of images, correct detection and classification of the studied components of biomaterials should be used as a guide.

5.5. Continuing education specialists

To ensure the quality of the analysis, the qualifications of the staff should be appropriate to the complexity of the study being performed. All laboratory personnel must periodically (once every five years) undergo training in refresher cycles, which are conducted by medical educational institutions that have the appropriate license. Each specialist must engage in self-education. The laboratory must have accessible contemporary literature, including periodicals on laboratory diagnostics and atlases. Laboratory specialists need to participate in conferences and seminars.

6. Requirements for the regime of work and rest, diet and restrictions in the preparation of the patient

For the personnel carrying out the sampling, an instruction should be developed containing, in addition to the sampling procedure, the conditions for preparing the patient. The influence of drugs is especially important, for example, the introduction of steroid hormones into the joint, which can crystallize (Appendix A.2).

7. Labor costs for the implementation of the technology of clinical laboratory analysis of synovial fluid

Table 1 - Labor costs in UET for the implementation of the technology "Clinical laboratory analysis of synovial fluid"

APPENDIX A

(reference)

Collection of SF samples, storage and delivery conditions (preanalytical stage)

A.1 Introduction

Joint puncture is performed by clinicians.

The pre-analytical stage is carried out in medical department and after delivery of the biomaterial to the laboratory - in the laboratory itself. Clinicians prepare applications for research. The application must indicate the patient's full name, gender, age or year of birth, the method of obtaining the biomaterial, the joint to be punctured, the time of puncture, the number of tubes filled with SF, native and with K2 EDTA. The clinical diagnosis and influencing analysis must be indicated. medications. The absence in the order of the diagnosis or medications taken by the patient that affect the results may lead to incorrect interpretation of the results and an error in the diagnosis. Nursing staff of the department is responsible for the preparation of the patient, the urgent delivery of test tubes with SF to the clinical diagnostic laboratory.

The continuation of the preanalytical stage in the laboratory consists in receiving and registering the incoming biomaterial, storing it, if necessary, until the study, processing and preparing for the study.

The preparation of requests for tests by clinicians is very important point, since the correctness of the diagnosis largely depends on the correct application.

A.2 Patient preparation

Preparation of the patient for joint puncture should be standardized.

Steroids injected into the joint capsule can crystallize and interfere with the diagnosis of the pathological process or lead to misdiagnosis, so intra-articular steroid injection should be canceled at least 5-7 days before the joint puncture. If the introduction of steroids into the joint bag cannot be canceled in advance, the clinician should note the introduction of these drugs in the application for the study. In the application, in addition to the patient's passport data, it should be noted which joint was punctured, the number of tubes filled with SF, the time of puncture, and the clinical diagnosis must be indicated, at least at the level of a diagnostic assumption.

A.3 Storage and delivery.

For general analysis SF is usually delivered to the laboratory immediately after the puncture. A study of a native preparation prepared from unstabilized SF is being carried out to detect ragocytes and crystals, as well as to determine cytosis. The study of the stained smear can be carried out by storing a test tube with SF stabilized by K2 EDTA in a refrigerator at a temperature of +3-+50C for 24 hours.

Long-term storage of SF is allowed at a temperature of -70 ° C; these samples are used for biochemical and immunological studies.

N o t e ─ Currently, special vacuum tubes and disposable containers for collecting biological fluids of 100 ml are produced from unbreakable material without reagents, with K2EDTA or other directives.

Bibliography

1.Zakharova synovial fluid. In: Rheumatology. National guides. Ed. Academician of the Russian Academy of Medical Sciences and Academician of the Russian Academy of Medical Sciences. M., "GEOTAR-Media", 2008, pp. 62-66.

2.Instruction on measures to prevent the spread infectious diseases when working in clinical diagnostic laboratories of medical institutions, Moscow, 1991.

3. Rules for the collection, storage and disposal of waste from medical institutions. SanPiN 2.1.1.728-99., Moscow, 1999

4. GOST R ISO 52095 -2:2003) Safety requirements.

5. GOST R 53079.4-2008 Medical laboratory technologies. Ensuring the quality of clinical laboratory research. Part 4 Rules for conducting the preanalytical stage of clinical laboratory studies.

6. GOST R ISO 15189 -2006 Medical laboratories. Particular requirements for quality and competence.

The draft standardized technology was prepared by:

, (MMA named after I.M. Sechenov), (Research Institute of Rheumatology of the Russian Academy of Medical Sciences), (RMAPE), (RCSC named after the Russian Academy of Medical Sciences), (polyclinic No. 000 of Moscow).

Why do synovial fluid analysis?

In primary care settings, synovial fluid (SF) data can help determine the specialist to whom the patient should be referred.

• If non-inflammatory SF - to the orthopedist.
• If inflammatory - to the rheumatologist.

Diagnostic value of synovial fluid analysis

• Inflammatory or non-inflammatory pathology
• Crystalline inflammation or sepsis or aggravation
• Assistance in identifying groups of diseases based on the number of cells and their type
• Determination of the type of prosthetic insufficiency
• predictive value
• Orthopedic intervention
• Stage of a specific disease
• Therapy monitoring. In particular, the rejection of monoclonal antibody therapy.

On fig. Figures 1 and 2 reflect the algorithm for diagnosing joint diseases based on synovial fluid analysis data.

Pathological changes in the tissues surrounding the diseased joint are reflected in the volume, cellular composition and the presence of solid particles in the fluid. Inflammatory diseases joints that differ in etiology have characteristic cell models that can be recognized and used in the diagnosis of a particular disease or group of diseases (Fig. 1, 2). In order to identify these differences, it is necessary to correctly select and properly store SF in order to minimize autolytic changes and degradation of characteristic CL. EDTA is used as an anticoagulant. Storage at 4°C is well tolerated by SF and gives excellent diagnostic results. Practically adequate results can be obtained up to 48 hours from aspiration, but longer storage, even at 4°C, usually allows only crystals and particles to be identified. The majority of Cl undergoes lysis.

Cytological analysis of synovial fluid

Obesity Cl can be found in the analysis of SF in most patients with joint disease, but they are most often observed in inflammatory arthritis in patients with seronegative spondyloarthropathies and in non-inflammatory joint lesions associated with trauma.

This type of Cl is often found in the analysis of SF of patients with intra-articular hemorrhage or arthrography, as well as in allergic reaction to injectables such as artificial SF.