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
Under physiological conditions, cerebrospinal fluid is a clear, colorless liquid. If there are colored substances in the cerebrospinal fluid, it 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 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
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:
 * Pathological changes in proteinorachia: :
 * When the blood-brain barrier is broken, a larger amount of protein penetrates pathologically into the cerebrospinal fluid. When the cerebrospinal fluid pathways 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 when the immune system is activated.
 * 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 determination of protein 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: :
 * Sp-Total protein (proteinorachia): 0,20–0,45 g/l
 * Pandy reaction: negative < 0,2 g/l of 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.

For a more accurate assessment of the barrier condition, the so-called albumin quotient Qalb is used, which takes into account the concentration of albumin in cerebrospinal fluid (AlbCSF) and serum (Albserum):


 * $$Q_{alb} = \frac{Alb_{CSF}}{Alb_{s\acute{e}rum}} $$


 * 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 albumin quotient: :
 * An increase in Qalb is found in a damaged blood-brain barrier, which we encounter in  inflammatory diseases of the CNS (meningitis of various origins), multiple sclerosis or obstruction in  the cerebrospinal fluid.


 * Methods for determination of albumin: :


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


 * Reference values: :
 * Sp-Albumin: 		120–300 mg/l
 * Albumin quotient – Qalb (depends on age):
 * 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
Immunoglobulins in cerebrospinal fluid can either come from the blood or arise intrathecally. Intrathecal synthesis of immunoglobulins 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:
 * Blood-brain barrier disorder;
 * Increased intrathecal synthesis;
 * Increased serum immunodlobulin 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
Determination of the concentration of immunoglobulins in cerebrospinal fluid alone is insufficient, as it is necessary to distinguish 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 immunoglobulinový index provides us with indicative information. It evaluates immunoglobulins and albumin in serum and in 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.


 * $$IgG_{index} = \frac{Q_{IgG}}{Q_{alb}} $$


 * $$IgG _{index} = \frac{IgG_{CSF}/IgG_{s\acute{e}rum}}{Alb_{CSF}/Alb_{s\acute{e}rum}} $$


 * Reference values: :


 * Immunoglobulin index IgG:
 * 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
250px|thumb|Obr. 3: Reiber diagram Reiber diagram allows rapid demonstration of intrathecal immunoglobulin synthesis. The calculated values ​​of QAlb and QIgG are output to 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: :

Reiber diagram (picture 3) is divided into 5 regions, that define the findings:
 * Region 1 – normal finding;
 * Region 2 – isolated disorder of the blood-brain barrier without local synthesis of Ig;
 * Region 3 – blood-brain disorder together with intrathecal Ig synthesis region 3;
 * Region 4 – isolated intrathecal Ig synthesis without blood-brain barrier disorder;
 * Region 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.

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.

It is believed that only a limited number of B-lymphocytes enters 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.

Evaluation:

Five different types  of isoelectrophoregrams are described (picture 4): náhled|260x260pixelů|Obr. 4: Základní typy izoelektroforegramů mozkomíšního moku
 * Type 1 – either in serum and in cerebrospinal fluid only polyclonal IgG are presented – normal finding;


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


 * Type 3 – oligoclonal bands in cerebrospinal fluid and  other oligoclonal bands in cerebrospinal fluid and serum - 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  serum 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 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. Glucose concentration in the cerebrospinal fluid – glycorachia monitors the concentration of glucose in the blood.

Thherefore, for the evaluationbof glycorachia, it is necessary to simultaneously determine the concentration in lthe cerebrospinal fluid and in the blood and calculate their Qglu quotient from the values (glycocharia / glycemia ratio) similarly to albumin or immunoglobulins:


 * $$Q_{glu} = \frac{Glu_{CSF}}{Glu_{s\acute{e}rum}} $$

Under physiological conditions, the ratio of glycorachia to glycemia is about 0,6;
 * A value below 0,45 is considered pathological.

Pathological changes of glycorachia and Qglu:


 * We encounter increased glycorachia – hyperglycorachia in diabetics.


 * Decreased values of glycorachia – hypoglycorachia (after excluding hypoglycaemia), which occur in the 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 (Qglu <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.
 * In subarachnoid hemorrhage, erythrocytes contribute to the increased glucose requirement.
 * 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
 * Qglu: 	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 infiltration of the meninges 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 indicatestuberculosis or fungi.

Related articles

 * The cerebrospinal fluid
 * Proteins in cerebrospinal fluid
 * Cerebrospinal fluid spectrophotometry
 * Cytological examination of cerebrospinal fluid
 * Cerebrospinal fluid syndromes