The cerebrospinal fluid (CSF), part of the central nervous system (CNS), is clear and colorless (“rock water”); it contains numerous solutes, a very low concentration of proteins, and is isosmotic with plasma, due to a higher concentration of chlorides. The concentration of proteins, under physiological conditions, is 20–45 mg/dL. Its total volume is 140 mL, although about 500 mL is produced in the course of a day.
CSF is produced in the ventricles by the choroidal plex uses, vascular formations of pial origin, projecting into the ventricles. The inner part of the ventricles, as well as the central canal of the spinal cord, is lined by a thin epithelial mem brane called the ependyma. The choroid plexuses are equipped with unidirectional transport systems of ions from the periphery to the CSF (this involves by osmosis a movement of water) and, by dedicated mechanisms, glucose, and amino acids. Finally, the choroid plexuses synthesize the proteins of the CSF, such as transthyretin and asialotransferrin. The CSF passes from the ventricles to the subarachnoid spaces, where Pacchioni’s granulations reabsorb it and ends up in the venous system and, according to recent studies, also in the lymphatic system.
The blood-liquor barrier (BLB) allows the passage of plasmatic molecules, especially proteins, into the CSF and should not be confused with the blood-brain barrier (BBB), which is, instead, impermeable, like a cell membrane, due to the presence of tight junctions that “weld” endothelial cells together.
CSF is enriched with solutes. Indeed, albumin, a protein used as a model for the passage from plasma to CSF because it is exclusively synthesized by the liver, has a concentration 2.5 times higher in the lumbar CSF than in the ventricular one.
CSF also has a cellular component (lymphocytes and monocytes) due to previously unknown connections between the meninges and the lymphatic vessels.
Circulating in a rigid bone structure, in case of increased production or reduced reabsorption, the amount of CSF cannot increase much, but its pressure increases, creating an endocranial hypertension that pathologically compresses the CNS. The CNS is reduced in volume and the CSF volume increases, a phenomenon called hypertensive hydrocephalus. Normotensive hydrocephalus occurs when CSF volume increases due to a primitive reduction of the cerebral mass, generally due to atrophic phenomena.
CSF is collected by lumbar puncture, usually performed by the neurologist through the intervertebral space between L4 and L5 or L5 and S1, where there is no spinal cord. This maneuver frequently results in blood contamination due to accidental rupture of capillaries in the needle path. For every 600 red blood cells present in the sample due to collection, there is one additional leukocyte in the CSF, and for every 1000 red blood cells, there is a 1 mg/dL increase in total proteins.
To discriminate between the presence of blood in the sample due to traumatic lumbar puncture and blood from sub arachnoid hemorrhage, the CSF should be collected, whatever the quantity taken, in three tubes after having numbered them: in case of blood appearance from puncture, this will tend to disappear passing from the first to the third tube.
CSF is currently studied in the suspicion of the following diseases:
• Inflammatory meningitis on an infectious basis, or due to carcinomatosis by metastatic localization of tumors outside or within CNS (not to be confused with meningiomas, which are primary tumors of the meninges).
• Encephalitis, which can be infectious, inflammatory, auto immune, often as a result of previous infection of the CNS or degenerative type, either on a genetic basis or acquired sporadic.
• Polyradiculoneuritis, inflammation of spinal and cranial nerve roots, acute or chronic.
• Myelitis, diseases confined to the spinal cord.
• Acute hemorrhagic phenomena, such as subarachnoid hemorrhages.
The molecules reach the CSF by simple passage through the blood-liquor barrier, or by secretion from the choroid plexuses, or by drainage from the central nervous tissue. Exit, not at all selective, occurs from the arachnoid granulations and most likely from the lymphatic structures of the meninges, as recently proposed.
CSF proteins fall into three categories according to their origin:
1. Plasma, by simple passage from the plasma to the CSF through the BLB, in an amount dependent on the state of the barrier.
2. Brain, only from the CNS; they increase in case of tissue damage (e.g., tau proteins and phosphorylated tau in Alzheimer’s disease).
3. Mixed (plasma-cerebral).
In CSF diagnostics, the use of proteins has a three- pronged approach: assaying proteins as markers of barrier status, assaying proteins of brain origin, and assaying proteins of intrathecal synthesis.
The total protein assay is the most widely used test to diagnose barrier damage. As damage increases, its selectivity decreases. However, the test cannot detect simultaneous or exclusive increases in brain-derived proteins. The recommended method for assessing barrier damage is to assay albumin, either in CSF or serum, calculate the ratio, and multiply the result by 1000. This gives the albumin quotient (QAlb). The reference values of total protein and QAlb change with age: they are higher in infants, lower in children, and rise with age. It was determined that the reference value (RV) of QAlb, for subjects aged 11–80 years, should be calculated using the formula: QAlb = age/25 + 8.
The QAlb is a dimensionless figure, which does not require standardization of the albumin assay. The dosage of proteins of brain origin is expressed with absolute values, generally in mass concentration, with thresholds discriminating sick from healthy.
The dosage of intrathecal synthesis proteins is fundamental, because they are pathognomonic of CNS pathology. Electrophoretic fractionation is very useful, especially with iso electrophoresis (IEF), as in the case of IgG of intrathecal synthesis, for which the patterns have long been encoded. The formulas quantifying the intrathecal fraction, the more complex ones, such as the Reiber equation, take into account the greater pas sage of specific proteins as the barrier damage increases and are to be preferred because they are more specific.
Analyses can be of the following types:
• Macroscopic analysis, usually performed by the neurologist at the time of the lumbar puncture
• Cytometric and cytological examination to count and identify the CSF cells
• Biochemical examination (metabolites, proteins, etc.)
Macroscopic Evaluation and Cellular Analysis
Under pathological conditions, the CSF loses its transparent and clear appearance, becoming increasingly turbid. The cornerstone of cellular analysis is not only cytometric analysis, by cell counting and differential counting of lymphocytes, monocytes, and granulocytes, but also qualitative evaluation by cytological examination for the search and reporting of particularly voluminous cells, probably of neoplastic origin, especially if in clusters. The cytometric examination is fundamental for diagnosing meningitis and allowing to define the type. Bacterial meningitis are characterized by a high number of neutrophil granulocytes (typically greater than 1000 × 106/L), while viral meningitis are characterized by a lower number of mononuclear, typically lymphocytes.
In the resolving stages of subarachnoid hemorrhages, siderophages may also be present: macrophages with iron inclusions.
Biochemistry
Glucose is used to estimate the extent of the presence of bacteria and cells; it should be reported as CSF to serum glucose ratio; reference value >0.4–0.5.
Lactates, a product of anaerobic glucose metabolism, increase under all conditions in which glucose is metabolized in the CSF. Since they are not affected by plasma concentration, they are reported in mmol/L; reference value <2.8.
Total protein is a crude indicator of blood-liquor barrier damage.
Immunoglobulin G, when intrathecally synthesized, has long been used, and still is, to make the diagnosis of multiple sclerosis (MS).
M-immunoglobulins have been defined as indicators of the type of CNS disease and not markers of recent infections: However, epidemiological studies and case reports have shown that they appear first and alone in acute infections.
Immunoglobulin A is a marker of purulent meningitis and neurotuberculosis.
Immunoglobulin free light chains are candidates to become the best indicator of immune activation in the CNS.
Specific antibodies should be investigated using criteria that allow their intrathecal synthesis fraction to be identified exclusively by calculating the Antibody Index (AI): the numerator is the quotient of the specific antibody and the denominator is the quotient of the total immunoglobulins of the same class. The search for specific liquid antibodies to infectious agents, especially viral, is also extremely useful in the era of molecular diagnostics. A particular application of the search for specific IgG antibody response is the so-called MRZ (measles, rubella, zoster) reaction search to discriminate MS patients from patients with viral infection, or affected by other CNS inflammatory diseases.
The search for tumor markers in the CSF is very useful for diagnosing meningeal carcinomatosis together with the search for tumor cells. CEA and mucin markers such as CA15.3, CA125, and CA19.9 are used. β-hCG is a specific marker of cerebral germinoma, but it may be present in highly undifferentiated tumors.
Autoantibodies against the CNS are responsible for well- defined neurological diseases. They are mainly to be sought in serum. Intrathecal synthesis can be demonstrated in the CSF, but the synthesis is not of particular clinical significance. Only anti-MNADr autoantibodies are to be sought in the CSF, because they are often higher in the CSF than in the serum, where sometimes they may be absent.
Biomarkers of Alzheimer’s disease (AD), a neurodegenerative disease accounting for 50–60% of clinically diagnosed forms of dementia, are exclusively in the CSF. They include β-amyloid protein, resulting from the proteolysis of an amyloid precursor and constituting amyloid plaques; total tau protein (T-tau), associated with microtubules and mainly located in the axon, which has higher values in AD patients; phosphorylated tau protein (p-tau), a hyperphosphorylated form of tau protein and expression of neurofibrillary degeneration, which has higher values in AD patients.
Creutzfeldt-Jakob disease, the best known prion disease, does not have a specific biomarker. For its diagnosis, the protein 14-3-3, not related to the prion, but typical of extensive neuronal necrosis, is used.
Markers of subarachnoid hemorrhage are indicative of a rupture of a vessel in the subarachnoid space, representing an acute and dramatic event with low probability of survival for the patient. The reference examination is computed tomography of the brain, which demonstrates the presence of a hemorrhagic infarction. The CSF examination is performed only in doubtful cases, 2% of the total, especially in the post-acute phase, in search of hemoglobin and bilirubin, the main product of hemoglobin metabolism.
The markers of CSF, presence of CSF outside its physio logical sites, indicate an abnormal communication of the cerebral spaces with the external environment, with a strong risk of infection. The most frequent form is rhinoliquorrhoea, a non-acute episode, often post-traumatic, of otorhinolaryngological scope. Post-neurosurgical CSF may represent an urgent complication of a recent neurosurgical procedure. The search for asialotransferrin, isoform of transferrin produced in the CNS, is the reference test, but the β-trace protein (β-TP), produced in the CNS, present in the CSF in concentrations much higher than in plasma, and rap idly measured on automatic analyzers is now the test of choice for these urgent requests.
CSF chlorides, which are higher in CSF than in plasma, decrease in chronic meningitis, such as tuberculosis.
CSF LDH is the only enzyme currently assayed, because it is produced by CSF cells. It is an absolutely non-specific marker of inflammation, if its isoenzymes are not assayed. Thus, it has a high negative predictive value. Physiological values are <21 U/L.
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