Biological examination of CSF

Biochemical examination of cerebrospinal fluid

Cerebrospinal fluid testing is one of the basic methods that contribute to the diagnosis of neurological diseases. The cerebrospinal fluid is most often removed by lumbar puncture (3 × 5 ml, between L4 – L5 or S1), the suboccipital approach is less common. The cerebrospinal fluid needs to be transported to the laboratory as quickly as possible, as the cells gradually break down, the glucose concentration decreases and the lactate increases.

Color

Physiological clear colorless cerebrospinal fluid Erythrochromic cloudy cerebrospinal fluid in purulent neuroinfection Xantochrome cerebrospinal fluid Under physiological conditions, cerebrospinal fluid is a clear, colorless liquid . If there are colored substances in the cerebrospinal fluid, this is a pathological condition. It is usually conditioned by the presence of hemoglobin , methaemoglobin and  bilirubin .

• The admixture of blood causes a pinkish to red coloration, referred to as erythrochromic (sanguinolent) . Blood can enter the cerebrospinal fluid artificially by injuring blood vessels during lumbar puncture. If the cerebrospinal fluid is collected in three test tubes during collection, then in this case the color of the liquid in the test tubes weakens and after centrifugation the cerebrospinal fluid is colorless. In intracranial hemorrhage, the staining is the same intensity in all tubes. During fresh bleeding, the supernatant is also colorless.

• The yellow, xanthochromic coloration of the cerebrospinal fluid is due to the presence of bilirubin caused by hemoglobin conversion . Xantochromy may persist for 3-4 weeks after the onset of bleeding (see cerebrospinal fluid spectrophotometry below ).

• The presence of methaemoglobin shows an ocher yellow to brown color .

Turbidity

Cerebrospinal fluid turbidity is usually caused by leukocytes , which are present in cerebrospinal fluid in purulent neuroinfections . The intensity of turbidity corresponds to the number of leukocytes. The admixture of erythrocytes can also manifest itself in turbidity.

Proteins in cerebrospinal fluid

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Pathological changes in proteinorachia

From a clinical point of view, the increased concentration of total protein in cerebrospinal fluid, so-called hyperproteinorachia , which can be caused by several mechanisms, is important:

• When the blood-brain barrier is broken, a larger amount of protein penetrates pathologically into the cerebrospinal fluid. When the cerebrospinal fluid blocks are blocked, a severe blood-brain barrier breakage occurs and plasma proteins  ( albumin and high-molecular-weight fibrinogen ) enter the fluid.
• Intrathecal synthesis of immunoglobulins in the activation of the immune system.
• The abnormal composition of plasma proteins is reflected in the composition of cerebrospinal fluid proteins, eg monoclonal gammopathy is manifested by the presence of the same immunoglobulins in cerebrospinal fluid.
• Increased structural proteins in CNS tissue damage .
• Tumor infiltration of the meninges .

The determination of total protein in cerebrospinal fluid is used primarily as a rapidly feasible examination, which provides basic information about the state of the blood- brain barrier .

Methods for protein determination in cerebrospinal fluid

• One of the recommended methods for the quantitative determination of total protein in cerebrospinal fluid is the reaction with pyrogallol red

• As a guide, a qualitatively increased amount of protein in the cerebrospinal fluid can be demonstrated by the Pandy reaction , in which globulins and partly also  albumin are denatured with an aqueous phenol solution.

Reference values

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  Sp-Total protein (proteinorachia): 0.20-0.45 g / l.
• Panda reaction: negative <0.2 g / l protein

Albumin in cerebrospinal fluid

Albumin in cerebrospinal fluid always comes from the blood, because it is not formed in the CNS. Its synthesis takes place in the liver and enters the cerebrospinal fluid by crossing the blood-brain barrier. Albumin accounts for about 57% of the total protein in CSF . Elevated cerebrospinal fluid albumin levels are always a sign of a blood-brain barrier disorder.

To more accurately assess her condition, the so-called albumin quotient of Q albums is used , which takes into account the concentration of albumin in cerebrospinal fluid (Alb CSF ) and serum (Alb serum )

Albumin quotient is used

• To assess the degree of involvement of the blood-brain barrier;
• For the calculation of intrathecal immunoglobulin synthesis.

Pathological values ​​of the albumin quotient

• An increase in Q albums is found in a damaged hematolevic barrier, which we encounter in inflammatory diseases of the CNS (meningitis of various origins), multiple sclerosis or obstruction in the cerebrospinal fluid.

Methods for albumin determination

• Albumin is determined in the CSF by sensitive immunochemical methods (immunoturbidimetry, immunonephelometry, ELISA ).

Reference values

• Sp-Albumin: 120-300 mg / l
• Albumin quotient - Q albums (age dependent):

up to 15 years: ≤  5 × 10 −3

up to 40 years: ≤  6.5 × 10 −3

up to 60 years: ≤  8 × 10 −3

Immunoglobulins in cerebrospinal fluid

The cerebrospinal fluid immunoglobulins can either come from the blood or are produced intrathecally . Intrathecal synthesis of antibodies takes place in perivascularly deposited B-lymphocytes , which differentiate into plasma cells.

Pathological changes in immunoglobulin concentration

The increase in the concentration of immunoglobulins in cerebrospinal fluid may be due to:

• Hematoliquor barrier barrier ;
• Increased intrathecal synthesis;
• Increased serum immunoglobulin levels  ;
• Cerebrospinal fluid circulation disorder .

Methods for determination of immunoglobulins

Individual classes of immunoglobulins are determined by more sensitive immunochemical methods such as immunoturbidimetry, immunonephelometry and ELISA.

Reference values:

• Immunoglobulin concentrations in cerebrospinal fluid:

Sp- IgG : 12.0–40.0 mg / l

Sp- IgM : 0.2–1.2 mg / l

Sp- IgA : 0.2–2.1 mg / l.

Demonstration of intrathecal immunoglobulin production

Mere determination of the concentration of immunoglobulins in  cerebrospinal fluid is insufficient, as it is necessary to differentiate the intrathecal or blood origin of immunoglobulins for differential diagnostic purposes. For this, the calculation of various indices, equations or evaluations using graphs is used.

Immunoglobulin index

• The immunoglobulin index will provide us with indicative information . Evaluates immunoglobulins and  albumin in serum and  cerebrospinal fluid . To calculate it, it is necessary to determine the concentrations of both analytes in the cerebrospinal fluid and at the same time in the serum. It is calculated on the basis of the quotient of the relevant immunoglobulin (IgG, IgA, IgM) and the albumin quotient

Reference value

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• IgG immunoglobulin index :

IgG index: <0.5 does not indicate intrathecal IgG synthesis

IgG index: 0.5-0.75 does not rule out intrathecal IgG synthesis

IgG index: > 0.75 indicates intrathecal IgG synthesis.

Reiber diagram

Giant. 3: Reiber diagram The Reiber diagram allows rapid demonstration of intrathecal immunoglobulin synthesis . The calculated Q Alb and Q IgG values ​​are plotted into it . According to the location of the plotted value in the graph, the origin of immunoglobulins and the disorder of the blood- brain barrier can be determined .

Evaluation

The Reiber diagram (Fig. 3) is divided into 5 areas that define the findings:

• Area 1 - normal finding;
• Area 2 - isolated disorder of the blood-brain barrier without local Ig region 2 synthesis;
• Region 3 - disorder of the blood-brain barrier together with intrathecal Ig synthesis region 3;
• Area 4 - isolated intrathecal Ig synthesis without blood-brain barrier disorder;
• Area 5 - area of ​​analytical errors.

The boundary between local immunoglobulin synthesis and their passive transfer is shown by the blue line. Values ​​above this line indicate intrathecal synthesis and the range is indicated by a dashed line and expressed as a percentage. A vertical dashed line separates the normal and disrupted hematoliquor barrier.

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See the Reiber Diagram page for more information .

Oligoclonal immunoglobulins in cerebrospinal fluid

The most sensitive method for detecting intrathecal antibody synthesis is the determination of oligoclonal immunoglobulins by isoelectric focusing followed by staining or immunofixation or immunoblotting. Physiologically, immunoglobulins in serum and cerebrospinal fluid are polyclonal in nature and express the heterogeneity of individual antibodies produced in response to a variety of antigens encountered by an individual.

Only a limited number of B-lymphocytes are thought to enter the CNS , which, upon activation by an antigen (eg, a particular microorganism or autoantigen), differentiate into antibody-secreting plasma cells. Intrathecally produced antibodies show only limited (oligoclonal) heterogeneity, which in isoelectric focusing appears as isolated bands that are not apparent in the serum analysis . This implies the need to perform cerebrospinal fluid and serum immunoglobulin analysis at the same time. The presence or absence of identical IgG bands in serum and fluid is compared; the number and location of the strips has no differential diagnostic significance.

Rating:

Five different types of isoelectrophoregrams are described (Fig. 4): Giant. 4: Basic types of cerebrospinal fluid isoelectrophoregrams

Type 1 - serum and fluid only polyclonal IgG - normal finding;

Type 2 - oligoclonal bands only in cerebrospinal fluid - local IgG synthesis (eg in  multiple sclerosis );

Type 3 - cerebrospinal fluid oligoclonal bands and other cerebrospinal fluid and serum oligoclonal bands - local IgG synthesis and antibody production in the body (eg chronic CNS infection, multiple sclerosis);

Type 4 - identical oligoclonal bands in serum and fluid (so-called "mirror" image of bands in era and cerebrospinal fluid - antibodies penetrate from blood into cerebrospinal fluid) - systemic immune activation without local IgG synthesis in the CNS;

Type 5 - identical monoclonal bands in serum and fluid in a short section of the pH gradient, it is the presence of a monoclonal paraprotein in cerebrospinal fluid of serum origin ( myeloma , monoclonal gammopathy ) - paraprotein image.

Glucose in the cerebrospinal fluid

Glucose, which is the main energy substrate for nerve tissue, is transported to the brain through the transport systems in the chorioid plexus. The concentration of glucose in the cerebrospinal fluid is determined by the capacity of the transporter systems, utilization in the nervous tissue and the rate of reabsorption in the cerebrospinal fluid. Concentration of glucose in cerebrospinal fluid - glycorachia monitors the concentration of glucose in the blood.

Therefore, for the evaluation of glycorachia, it is necessary to simultaneously determine the concentration in cerebrospinal fluid and blood and calculate their quotient Q glu (values ​​of glykorachie / glycaemia) from the values, similarly to albumin or immunoglobulins:

Under physiological circumstances, the ratio of glycorachia to glycemia is approximately 0.6 ;

A value below 0.45 is considered pathological .

Pathological changes of glycorachia and Q glu :

• We encounter increased glycocaemia - hyperglycocaemia in diabetics.

• Decreased values ​​of glycocaemia - hypoglycaemia (after excluding hypoglycaemia), which occur in cerebrospinal fluid with a higher number of cells, are important for diseases of the nervous system . Hypoglycorachia is demonstrated in bacterial meningitis, where it is explained by glucose consumption by bacteria and leukocytes (Q glu <0.4). It can drop to zero values. Successful treatment leads to a return of glycorachia to normal.
• Other causes of reduced cerebrospinal fluid glucose may be CNS cancer, where glucose is used by tumor cells, and cerebral ischemia, where oxygen deficiency limits aerobic glycolysis and requires more glucose to meet energy requirements with anaerobic glycolysis.
• In subarachnoid hemorrhage, erythrocytes contribute to the increased glucose requirement.

Method for determination of glycorachia:

• Methods for determining the concentration of glucose in cerebrospinal fluid are similar to those for determination in blood (plasma), ie. peroxidase or hexokinase reaction . If it is not possible to examine glucose within 30 minutes of collection, the cerebrospinal fluid should be stored on ice or sodium fluoride added to prevent undesired glucose loss due to glycolysis. It takes place in microorganisms, leukocytes or tumor cells, which may be part of the fluid under pathological conditions.

Reference values:

• Sp-Glucose: 2.2-4.2 mmol / l
• Q glu : 0.6

Lactate in cerebrospinal fluid

Lactate is formed by the breakdown of glucose by glycolysis under anaerobic conditions. A certain amount of lactate is physiologically produced in the brain tissue, which reflects the metabolic activity of the brain. It practically does not cross the blood-brain barrier and its concentration, unlike glucose, does not depend on the plasma concentration. CSF lactate levels have the opposite trend than glucose levels.

Determination of lactate in CSF is a more sensitive indicator than determination of glucose. Lactate is an important parameter that helps to distinguish between meningitis of bacterial and viral origin.

Pathological changes in lactate:

• Elevated lactate levels in bacterial meningitis are a manifestation of anaerobic glycolysis of bacteria and, to a lesser extent, leukocytes. With effective therapy, lactate decreases.
• All disorders of the brain's oxygen supply (eg anoxia, hypoxia, eg in stroke) due to increased anaerobic glucose metabolism are accompanied by increased lactate levels.
• In subarachnoid hemorrhage, the rise in lactate is caused by erythrocytes present in the cerebrospinal fluid, which also cover energy requirements by anaerobic glycolysis (they do not have mitochondria).
• It is also increased in malignant mening infiltration and in some metabolic diseases (mitochondrial encephalomyopathy).

Methods for determination of lactate:

Enzyme spectrophotometric methods based on the Warburg optical test principle are used .

Reference values:

• Sp-Lactate: 1.2–2.1 mmol / l

Chlorides in cerebrospinal fluid

The chloride concentration in the cerebrospinal fluid is normally 124 mmol / l .

A decrease below 100 mmol / l together with a decrease in glucose indicates tuberculosis or fungi.

Links

https://www.wikiskripta.eu/index.php?curid=13206

related articles

• Cerebrospinal fluid
• Proteins in cerebrospinal fluid
• Cerebrospinal fluid spectrophotometry
• Cytological examination of cerebrospinal fluid
• Cerebrospinal fluid syndromes

External links

• ADAM, P, C. ANDRÝS and B FRIEDECKÝ, et al. Recommendations of the Czech Society of Clinical Biochemistry and the Czech Society of Allergology and Clinical Immunology - Examination of cerebrospinal fluid  [online]. © 2005. Last revision 2005, [cited. September 8, 2009]. < http://www.cskb.cz/cskb.php?pg=doporuceni--vysetrovani-mozkomisniho-moku >.

• FIALOVÁ, L. and M VEJRAŽKA. Basic examination of cerebrospinal fluid  [online]. © 2005. Last revision 2008, [cited. September 8, 2009]. < https://el.lf1.cuni.cz >.

References

• ADAM, P, et al. Cerebrospinal fluid cytology (CD-ROM). 1st edition. Prague: SEKK, 2000. 

• AMBLER, Z, J BEDNAŘÍK and E RŮŽIČKA. Clinical neurology - general part. 1st edition. Prague: Triton, 2004.  ISBN ISBN 80-7254-556-6 .

• GLOSOVÁ, L.  Cytological atlas of cerebrospinal fluid. 1st edition. Prague: Galén, 1998.  ISBN 80-85824-70-1 .

• KALA, M. and J MAREŠ. Lumbar puncture and cerebrospinal fluid. 1st edition. Prague: Galén, 2008.  ISBN 978-80-7262-568-0 .

• MASOPUST, J.  Clinical Biochemistry. Requirements and evaluation of biochemical examinations I. and II. part. 1st edition. Prague: Karolinum, 1998.  ISBN 80-7184-650-3 .

• NEVŠÍMALOVÁ, S, E RŮŽIČKA and J TICHÝ, et al. Neurology. 1st edition. Prague: Galén, 2005.  ISBN 80-7262-160-2 .

• SCHNEIDERKA, Petr, et al. Chapters from clinical biochemistry. 2nd edition. Prague: Karolinum, 2004.  ISBN 80-246-0678-X .

• RACEK, J, et al. Clinical biochemistry. First edition. Prague: Galén - Karolinum, 1999.  ISBN 80-7262-023-1 .

• STERN, P, et al. General and clinical biochemistry for bachelor's fields of study. 1st edition. Prague: Karolinum, 2005.  ISBN 978-80-246-1025-2 .

• ZIMA, T, et al. Laboratory diagnostics. 1st edition. Prague: Galén - Karolinum, 2002.  ISBN 80-7262-201-3 .