Analysis of cerebrospinal fluid( Cerebrospinal fluid) - Causes, symptoms and treatment. MF.

Liquor( cerebrospinal or cerebrospinal fluid, CSF) is a biological fluid necessary for the functioning of the central nervous system. His research is one of the most important types of laboratory research. It consists of a pre-analytic stage( preparation of the subject, collection of material and its delivery to the laboratory), analytical( actual execution of the research) and post-analysis( decoding of the result).Only the correct execution of all manipulations at each of these stages determines the quality of the analysis.

Cerebrospinal fluid( cerebrospinal fluid) is formed in the vascular plexuses of the ventricles of the brain. In an adult human, 110-160 ml of CSF circulates simultaneously in the subarchnoid spaces and in the ventricles of the brain, 50-70 ml in the spinal canal. Liquor is formed continuously at a rate of 0.2-0.8 ml / min, which depends on the intracranial pressure. A day in a healthy person, 350-1150 ml of cerebrospinal fluid is formed.

Liquor is obtained by puncture of the spinal canal, more often - lumbar - in accordance with the procedure, well-known neuropathologists and neurosurgeons. Its first drops are removed( "pathway" blood).Then the cerebrospinal fluid is collected in at least 2 tubes: in a conventional test tube( chemical, centrifugal) for general clinical and chemical analysis, and sterile for bacteriological testing. On the form of referral for the study of CSF, the doctor should indicate not only the name of the patient, but also the clinical diagnosis and the purpose of the study.

It should be remembered that samples of liquor delivered to the laboratory should be protected from overheating or cooling, and samples designed for detection of bacterial polysaccharides in serological tests should be heated in a water bath for 3 minutes.

The laboratory laboratory examination of the cerebrospinal fluid( analytical stage) is carried out according to all the rules adopted in clinical laboratory diagnostics in the analysis of any biological fluids and includes the following steps:

- macroscopic analysis - assessment of physical and chemical properties( volume, color, nature),
- counting the number of cells,
- microscopy of the native preparation and cytological study of the colored preparation;
- biochemical study,
- microbiological examination( according to indications).

We find it expedient and informative in some cases to supplement the study of CSF with immunological and, possibly, other tests, the significance of which is discussed in the specialized literature.

Decoding of CSF indicators

Normal CSF is colorless and porous( like distilled water, in comparison with which the physical properties of liquor are usually described).

Grayish or gray-green color of the liquor is usually due to an admixture of microbes and leukocytes. The red color of CSF of different intensity( erythrochromia) is due to the admixture of erythrocytes, which occur with fresh hemorrhages or brain trauma. Visually the presence of red blood cells is detected when their content is more than 500-600 in μl.

In pathological processes, the liquid can be xanthochromic - colored in yellow or yellow-brown color by the products of the decomposition of hemoglobin. It is necessary to remember and false xanthrochromy - the color of the liquor caused by medicinal preparations. Less often we meet the greenish color of CSF( purulent meningitis, brain abscess).In the literature, the crust color of the cerebrospinal fluid is also described - with the breakthrough of the craniopharyngioma cyst in the cerebrospinal fluid.

Turbidity of the cerebrospinal fluid can be due to impurity of blood cells or microorganisms. In the latter case, the turbidity can be removed by centrifugation. When the CSF contains an increased amount of coarsely dispersed proteins, it becomes opalescent.

Relative density of cerebrospinal fluid obtained with lumbar puncture, 1.006-1.007.With inflammation of the meninges, brain traumas, the relative density of the cerebrospinal fluid increases to 1.015.It decreases with hyperproduction of cerebrospinal fluid( hydrocephalus).

With a high content of fibrinogen in the CSF, a fibrinous film or clot develops, which is more common in tuberculous meningitis. Sometimes a test tube with a liquid is left at room temperature for a day( if it is necessary to determine exactly - did the film form?).In the presence of a fibrinous film, it is transferred by a dissecting needle onto a slide and stained by Tsil-Nielsen or by another method to detect mycobacteria. A normal CSF is 98-99% water.

Nevertheless, the study of its chemical composition is an important task. It includes the determination of protein, glucose and chloride levels, and in some cases is supplemented by other indicators.

Protein in liquor

More than 80% of CSF protein comes from the plasma by ultrafiltration. The protein content is normal in different portions: in ventricular - 0.05-0.15 g / l, cistern 0.15-0.25 g / l, luminal 0.15-0.35 g / l. Any of the standardized methods( with sulfosalicylic acid and ammonium sulfate, etc.) can be used to determine the protein concentration in the liquor. Increased protein content in cerebrospinal fluid( hyperproteinarchy) may be due to various pathogenetic factors( Table 1).

The study of CSF proteins allows us not only to clarify the nature of the pathological process, but also to assess the state of the hemato-encephalic barrier. The indicator for these purposes can serve as albumin provided that its level in the CSF is determined by immunochemical methods. The determination of albumin is carried out in connection with the fact that it, being a protein of blood, is not synthesized locally and therefore can be a "marker" of immunoglobulins that have penetrated from the bloodstream due to impaired barrier permeability. Simultaneous determination of albumin in serum( plasma) of blood and CSF makes it possible to calculate the albumin index:

With an intact hemato-encephalic barrier, this index is less than 9, with its moderate damage is 9-14, with a noticeable 14-30, in case of severe damage, 30-100, and an increase of more than 100 indicates a complete damage to the barrier.

In recent years, interest in CNS-specific CSF proteins - neuron-specific enolase, S-100 protein, myelin basic protein( MBM) and some others has been increasing. One of the promising among them for clinical purposes is OBM.In normal liquor it is practically absent( its concentration does not exceed 4 mg / l) and appears only in conditions of pathology. This laboratory attribute is not specific for certain nosological forms, but reflects the extent of the lesion( associated primarily with the destruction of white matter).Some authors consider promising the definition of MBP in cerebrospinal fluid for the monitoring of neurospida. Unfortunately, today there are still problems related to the direct determination of the concentration of this protein.

Glucose in liquor

Glucose is contained in normal liquor in a concentration of 2.00-4.18 mmol / l. This value is subject to significant fluctuations, even in a healthy person, depending on the diet, exercise, other factors. For a correct assessment of the glucose level in the cerebrospinal fluid, it is recommended to simultaneously determine its level in the blood, where it is normally 2 times higher. Elevated levels of blood glucose( hyperglycosurgy) occurs in diabetes mellitus, acute encephalitis, ischemic circulatory disorders and other diseases. Hypoglycopic archiocytosis is noted with meningitis of different etiology or aseptic inflammation, tumor damage to the brain and membranes, less often - with herpetic infection, subarachnoid hemorrhage.

Some advantage over glucose as a diagnostic marker is lactate( lactic acid), since its concentration in the CSF( 1.2-2.1 mmol / l) does not depend on that in the blood. Its level is significantly increased at various conditions associated with a violation of energy metabolism - meningitis, especially - caused by gram-positive flora, brain hypoxia and some others.

Chlorides in liquor

Chlorides - content in normal liquor - 118-132 mmol / l. Increased concentration in CSF is observed when there is a violation of their excretion from the body( kidney disease, heart disease), with degenerative diseases and tumors of the central nervous system. Reduction of the chloride content is observed with encephalitis and meningitis.

Enzymes in liquor

Liquor is characterized by low activity of the enzymes contained in it. The change in enzyme activity in CSF for various diseases is mostly non-specific and parallel to the described shifts in blood in these diseases( Table 2).Another interpretation deserves an interpretation of changes in the activity of creatine phosphokinase( CKF).This enzyme is represented in tissues by three fractions, characterized not only by molecular differences, but also by the nature of the distribution in tissues: CKF-MB( myocardium), CK-MM( muscle), CK-BB( brain).If the total activity of CKK in CSF is not of fundamental diagnostic value( it can be increased in case of tumor, cerebral infarction, epilepsy and other diseases), the fraction of CK-BB is a rather specific marker of damage to the brain tissue and its activity in CSF correlates with the Glasgow scale.

Number of cells and cytogram of CSF

In the study of biological fluids, including CSF, the number of cells and the cytogram are usually counted in smears stained with azureosin( Romanovsky-Giemsa, Nohtu, Pappenheim).Counting of cellular elements in the cerebrospinal fluid( determination of cytosis) is performed with the help of a Fuchs-Rosenthal camera, previously diluted with a Samson reagent 10 times. Use of this particular dye, not any other.allows the cells to stain for 15 minutes and keep the cells unchanged up to 2 hours.

The number of cells in the whole chamber is divided by 3, so the cytosis is obtained in 1 μl. For greater accuracy, cytosis is considered in three chambers. In the absence of a Fuchs-Rosenthal camera, you can use the camera Goryaeva, counting the cells on the entire grid also in three chambers, multiplying the result by 0.4.Until now, there are differences in the units of measurement of cytosis - the number of cells in the chamber, in 1 μl or 1 liter. It is probably advisable to nevertheless express the cytosis by the number of cells in μL.To calculate the number of leukocytes and erythrocytes in CSF, automated systems can also be used.

An increase in the content of cells in the CSF( pleocytosis) appears more often in inflammatory diseases, to a lesser extent - in the stimulation of the meninges. The most pronounced pleocytosis is noted with bacterial infection, fungal brain damage and tuberculous meningitis. With epilepsy, arachnoiditis, hydrocephalus, dystrophic processes and some other diseases, the CNS cytosis remains normal.

Cell staining of the native preparation with Samson's reagent allows to sufficiently differentiate the cells. But their morphological characteristics are more accurate after fixation and coloration of the prepared cytological preparations. A modern approach to the preparation of such drugs involves the use of a cytocentrifuge. However, even in the US they are equipped with only 55% of laboratories. Therefore, in practice, a simpler method is used - the deposition of cells on a slide. Drugs should be well dried in the air, and then painted.

In the colored preparation, cell counts are counted. They are mainly represented by blood cells( more often - lymphocytes and neutrophils, rarely monocytes, eosinophils, basophils), plasma and mast cells, macrophages, granular spheres( degenerative forms of a special kind of macrophages - lipofagas in the state of fatty degeneration), arachnoendotelia cells, epindyma. The morphology of all these cellular elements is usually well known to laboratory diagnostics physicians and is described in detail in many manuals. The level of pleocytosis and the character of the cytogram of CSF make it possible to clarify the nature of the pathological process( Table 3).

Neutrophilic leukocytosis often accompanies an acute infection( local and diffuse meningitis).Eosinophilia CSF is observed quite rarely - with echinococcosis of the brain, eosinophilic meningitis. Eosinophilia of CSF does not correlate, as a rule, with the number of eosinophils in the blood. Lymphocytic pleocytosis cerebrospinal fluid occurs in viral meningitis, multiple sclerosis, in the chronic phase of tuberculous meningitis, after operations on the membranes of the brain. With pathological processes from the side of the central nervous system, there is a polymorphism of lymphocytes, among which there are activated ones. They are characterized by the presence of abundant pale cytoplasm with individual azurophilic granules, some cells have a ligature or fragmentation of the cytoplasm( clasmatosis).Plasma cells appear in the cytogram for viral or bacterial meningitis, slow inflammatory processes, during recovery with neurosyphilis. Monocytes exposed to CSF ​​in the CSF are faster than lymphocytes, are observed with multiple sclerosis, progressive panencephalitis, chronic low-intensity inflammatory processes. Macrophages - "sanitari" liquor, appear with hemorrhages, infections, traumatic and ischemic necrosis.

Sometimes in the CSF there are atypical cells - elements, which in their morphological features can not be attributed to certain cellular forms. Atypical cells are found in chronic inflammatory processes( tuberculous meningitis, multiple sclerosis, etc.), and often they are tumor cells. The probability of finding tumor cells in the cerebrospinal fluid is small( no more than 1.5%).The detection of blast cells in CSF in hemoblastosis allows us to talk about neuroleukemia.

When analyzing the composition of the CSF, it is important to evaluate the ratio of protein and cell elements( dissociation).In cell-protein dissociation, pronounced pleocytosis is observed with a normal or slightly increased protein content. This is characteristic of meningitis. Belkovokletochnaya dissociation is characterized by hyperproteinarchy in normal cytosis. This condition is typical for stagnant processes in the cerebrospinal fluid( tumor, arachnoiditis, etc.).

Clinical situations sometimes require counting the number of erythrocytes in the bloody cerebrospinal fluid( for objectivizing the volume of hemorrhage).Counting red blood cells is the same as in blood. As indicated above, the color of the CSF varies, if 1 μl contains more than 500-600 red blood cells, a noticeable coloration occurs when there are about 2000, and it becomes hemorrhagic when the erythrocyte level is more than 4000 / μl.

Microbiological investigation of CSF

One of the frequent CNS diseases is purulent meningitis. In such cases, mycorobiological research becomes particularly relevant. It includes an orientation test - bacterioscopy of drugs and classical culture techniques. Bacterioscopy of cerebrospinal fluid has a limited diagnostic value, especially when obtaining a clear CSF.A smear prepared from the precipitate of liquor obtained by centrifugation is powdered with methylene blue or Gram, although some authors believe that the latter variant of coloring "traumatizes" the uniform elements and creates artifacts. With meningitis and abscesses, a diverse flora is found that corresponds to the nature of the disease. Regardless of the results of microscopy, the diagnosis of bacterial meningitis must necessarily be confirmed by a culture test, which becomes determinant in the diagnosis of this group of diseases and the choice of adequate therapy. It is conducted in accordance with Order No. 375 of the Ministry of Health of the Russian Federation of 23.12.98 "On measures to strengthen epidemiological surveillance and prevention of meningococcal infection and purulent bacterial meningitis."The most common cause of bacterial meningitis is Gram-negative diplococcus Neisseria meningitidis, which in 80% of cases can be detected already with bacterioscopy.

CSF liquor

Normally, only lymphocytes and monocytes are present in the CSF. With different diseases and pathological conditions in the cerebrospinal fluid, other types of cells may appear.

Lymphocytes in size are similar to red blood cells. Lymphocytes have a large nucleus and a narrow, unpainted cytoplasm rim. Normally, the liquor contains 8-10 cells of lymphocytes. The number of them increases with tumors of the central nervous system. Lymphocytes are found in chronic inflammatory processes in the membranes( tuberculous meningitis, cysticercosis arachnoiditis).

Plasmatic cells in liquor. Cells are larger than lymphocytes, the nucleus is large, eccentrically located, a large amount of cytoplasm with a relatively small size of the nucleus( cell size - 6-12 microns).Plasmatic cells in CSF are found only in pathological cases with long-term inflammatory processes in the brain and membranes, with encephalitis, tuberculous meningitis, cysticercosis arachnoiditis and other diseases, in the postoperative period, with slow healing of the wound.

Tissue monocytes in liquor. The size of the cells is from 7 to 10 μm. In a normal fluid, sometimes they can occur in the form of single specimens. Monocytes are detected in cerebrospinal fluid after surgical intervention on the central nervous system, with long-term inflammatory processes in the membranes. The presence of tissue monocytes suggests an active tissue reaction and normal healing of the wound.

Macrophages in liquor. Can have nuclei of various forms, more often the nucleus is located on the periphery of the cell, the cytoplasm contains inclusions and vacuoles. In normal liquor, macrophages are not found. The presence of macrophages with a normal number of cells in the cerebrospinal fluid is observed after bleeding or in the inflammatory process. As a rule, they occur in the postoperative period, which has prognostic value and speaks about active purification of the CSF.

Grainy balls in liquor. Cells with fatty infiltration - macrophages with the presence of droplets of fat in the cytoplasm. In colored liquor preparations, the cells have a small peripherally located nucleus and a large cell cytoplasm. The size of the cells is different and depends on the included droplets of fat. Granular balls are found in a pathological fluid obtained from brain cysts in the foci of decay of brain tissue, with tumors.

Neutrophils in liquor. In the chamber, neutrophils of peripheral blood are identical in appearance. The presence of neutrophils in the cerebrospinal fluid, even in minimal amounts, indicates either a former or an existing inflammatory reaction. The presence of altered neutrophils indicates an attenuation of the inflammatory process.

Eosinophils in liquor. Determine in the cerebrospinal fluid by the uniform, shiny granularity. Eosinophils are found in subarachnoid hemorrhages, meningitis, tuberculosis and syphilitic brain tumors.

Epithelial cells in CSF.Epithelial cells, which limit the subarachnoid space, are found in the cerebrospinal fluid quite rarely. These are large round cells with small round or oval cores. Discovered by neoplasms, sometimes with inflammatory processes.

Tumor cells and complexes in the cerebrospinal fluid. They are found in a cell and a colored preparation of liquor. Malignant cells can refer to the following types of tumors:

• medulloblastoma;
• spongioblastoma;
• astrocytoma;
• cancer.

Crystals in liquor. Occur in the cerebrospinal fluid rarely, in the case of tumor decay.

Echinococcus elements in cerebrospinal fluid, hooks, scolexes, scraps of chitinous membrane - are rarely found in cerebrospinal fluid.

PCR diagnostics of liquor

In recent years, certain prospects in the etiologic diagnosis of neuroinfections have been associated with the development of molecular genetic mechanisms for the detection of infectious disease causative agents in cerebrospinal fluid( PCR diagnostics).

Thus, the cerebrospinal fluid is the environment that reacts clearly to the pathological processes in the central nervous system. The depth and nature of its changes are due to the depth of pathophysiological disturbances. A correct evaluation of laboratory liquorological symptoms makes it possible to clarify the diagnosis and evaluate the effectiveness of treatment.

V.V.Bazarian professor of UGMA, deputy chief doctor OKB number 1