Laboratory diagnosis of neurological diseases
For the laboratory diagnosis of central nervous system diseases the assumption of four compartments suffices:
- Intravascular compartment, consisting of the lumen of the capillaries and venules of the parenchyma, the choroid plexus and the leptomeninges
- Intracellular space of nerve and glial cells
- Extracellular labyrinth of gaps between the interwoven processes of nerve and glial cells: the labyrinth is open to the CSF space
- CSF compartment, consisting of ventricles, basal cisterns, subarachnoid space and a narrow zone of adjacent extracellular space.
Between the four compartments there is an equilibrium based on exchange regulated by the following barriers:
- Blood-brain barrier: the main structures are the tight junctions (zonulae occludentes) of the brain capillaries. There is a carrier mediated trans endothelial exchange, preferentially of lipophilic substances
- Blood-CSF barrier: its functions are the filtration of the primary CSF in the plexus via the leaky epithelial junctions and ensuring constant CSF flow through the ventricles and subarachnoid space to the Pacchioni granulations
- Intra-/extracellular barrier of the nerve cells: there is a trans membranous exchange, preferentially of lipophilic substances, mediated by specific carriers.
Between the CSF space and the extracellular space there is a diffusion equilibrium for macromolecules as well. The intra-/extracellular barrier and the blood-brain barrier are essentially lipoprotein barriers which, for the most part, retain even small hydrophilic molecules, but allow the passage of lipophilic molecules up to a size of approximately 500 Da (e.g., phenytoin, ether, pentobarbital, caffeine, nicotine, alcohol, anesthetic and narcotic agents) .
The blood-brain barrier, also called CSF/serum barrier, is relatively permeable to hydrophilic macromolecules (e.g., α2-macroglobulin and IgM). The passage of smaller molecules, even those larger than 500 Da, is facilitated by their lipophilicity (e.g., antibiotics and cytostatic drugs) depending on the octanol/water partition coefficient . The composition of the extracellular fluid of the brain parenchyma is unknown. It resembles CSF only in a narrow margin of a few millimeters adjacent to the free CSF space, a zone into which limited diffusion of water soluble molecules is possible . The composition of CSF is well known since the subarachnoid space can be tapped at its lowest point. Despite its large distance from its site of production, the choroid plexus, CSF still shows all the characteristics of a filtrate, even in the lumbar sac ().
Thus, for hydrophilic molecules there is a clear correlation between the CSF/serum quotient and the hydrodynamic radius of the molecules (). This is only applicable in the presence of a steady state equilibrium (i.e., when serum concentrations are stable and exchange conditions at the blood-CSF barrier are undisturbed). The CSF/serum ratio for water, by definition, is 1.0. The concentration of chloride (smaller molecule than H2O) is higher in CSF than in serum; therefore, in blood-CSF barrier dysfunction, it decreases in comparison to other larger sized molecules. For the amino acids, active transport across the blood-CSF barrier is negligible. Changes of small, brain derived molecules in the CSF do not represent brain disorders because most of them are selectively removed from the CSF between the ventricles and the lumbar sac (e.g., glycine) or have been metabolized, (e.g., sorbitol). Molecules with an unexpectedly high CSF concentration (e.g., ascorbate, β2-microglobulin, neuron specific enolase), predominantly originate from the central nervous system or have reached the CSF space by carrier mediated transport.
Proteins synthesized in the leptomeningeal system are:
- Prostaglandin D synthetase (βtrace protein), MW 27 kDa, concentration approximately 1.0 mg/dL
- Transthyretin (prealbumin), MW 55 kDa, concentration approximately 1.7 mg/dL (plus 0.1 mg/dL from serum)
- τ-transferrin, MW 81 kDa, concentration approximately 0.6 mg/dL
- Cystatin C (γ-trace protein), MW 13 kDa, concentration approximately 0.6 mg/dL.
Prostaglandin D synthase: this potein is mainly synthesized in the choroid plexus epithelium and the leptomeninges. It is a reliable marker for the detection of CSF (e.g., for the diagnosis of CSF fluid leakage) .
τ-transferrin: this electrophoretically slow moving transferrin can also be used for the detection of CSF fluid leakage by means of immunoblotting . However, it is less sensitive than the detection of prostaglandin D synthetase.
Approximately 20% of the proteins in normal CSF are derived from brain parenchyma, such as neuron specific enolase (NSE), tau protein, β-amyloid 1-42 as well as protein 14-3-3 and S100 protein are of diagnostic significance. In the case of nerve tissue destruction, proteins of the brain parenchyma are liberated, but their CSF concentration remains below 1 mg/L. Proteins originating in brain tumors, such as carcinoembryonic antigen (CEA), rarely reach CSF concentrations above 100 μg/L. Brain derived proteins are not influenced by the CSF flow rate and are assessed by their absolute concentration. Examples of diseases with pure blood-brain barrier dysfunction are shown in .
The concentration of blood-derived proteins in CSF is the result of passive, molecular size dependent diffusion. With pathologically reduction of the CSF flow because of blood-CSF barrier dysfunction the level of plasma proteins increases in the CSF. Because of reduced CSF flow the concentration of plasma proteins increases in the CSF because of an enhanced net diffusion of proteins from the plasma into the CSF. The CSF/plasma quotient of a plasma protein follows a hyperbolic function. If the concentration of hydrophilic molecules in the CSF is higher than expected based on their molecular size, then intrathecal synthesis is assumed. Some CSF proteins originate predominantly in adjacent tissues (). During diagnostic evaluation, serum fractions < 1% can be ignored even in the setting of marked barrier dysfunction.
In healthy individuals, approximately 83% of CSF proteins originate from the serum, with medium sized proteins such as albumin, acidic α1-glycoprotein, α1-antitrypsin, hemopexin, α2-HS glycoprotein and transferrin dominating.
The blood-brain barrier is a physical barrier and determines the protein content of the CSF. Albumin is a good marker of the blood-brain barrier, because it originates exclusively from the serum. Albumin is produced only in the liver, has a radius of 35.8 Angstroms. The CSF/serum albumin quotient (QAlb) is the accepted reference value for the characterization of the blood-CSF barrier function and a reference for the evaluation of other blood-derived serum proteins in CSF . During mid-adult life the QAlb is less than 7 × 10–3. If the barrier permeability increases, for example as seen in purulent meningitis, the QAlb can elevate to values greater than 100 × 10–3 (barrier breakdown).
The QAlb is not influenced by intrathecal protein synthesis, is corrected for the plasma level of albumin, and is an integral part of intrathecal immunoglobulin synthesis formulae . However, the QAlb is not only dependent on the blood-brain barrier permeability but also on the fluid turnover, which normally is 14% per hour of the CSF space volume. If the turnover declines, as is the case of a spinal cord tumor, the QAlb can increase to values ≥ 100 × 10–3 (CSF block).
Between the blood-brain barrier permeability (P) and the fluid turnover (T) the relationship (R) exists for QAlb: R = P/(T + P). The implication of the equation is that both an increasing permeability as well as a decreasing turnover will result in the CSF concentration of albumin approaching its serum level because of the passive transfer of albumin through the blood-brain barrier . Identical concentrations (R = 1) are not quite reached, but the QAlb can increase to values ≥ 500 × 10–3.
In the case of space occupying processes (e.g., tumors, hemorrhage, prolapse of intervertebral discs) the extent of barrier dysfunction depends on the location and extent of the underlying process (). The more the CSF flow is impaired between the ventricles and the lumbar sac, the higher the increase in the albumin concentration in the CSF.
The blood-brain barrier of newborns is significantly more permeable than that of adults . The QAlb continuously decreases during the course of the first months of life, reaches the lowest values between 1 and 3 years of age, and then slowly rise again. It is therefore advisable to consider age when assessing the blood-brain barrier, especially during infancy and old age.
Immunoglobulines of the isotypes IgA, IgG and IgM are produced from B cells locally in central nervous system and can be measured in the CSF. The upper limit of the reference interval (Qlim) refers to the upper reference interval value which involves 99% of all patients without an intrathecal immunoglobulin (Ig) synthesis. This is shown as the bold line in the quotient diagram according to Reiber (Reibergram for IgG).
- IgLoc: the locally synthesized Ig concentration in CSF; e.g., IgGLoc = [QIgG – QLim(IgG)] × IgG (mg/L) in serum
- IgIF: the intrathecal fraction (%) of total CSF Ig concentration e.g., IgGLoc/total CSF IgG × 100 (%) or IgIF = (1 – QLim/QIgG) × 100 (%)
The intrathecal fractions of IgG, IgA or IgM with reference to QLim can be directly read from the diagram (). These calculated values represent the minimal amount of intrathecal synthesis of Ig. The following relations exist :
- Intrathecal IgG synthesis: QIgG > QAlb(in spite IgGIF > 0%)
- Intrathecal IgA synthesis: QIgA > QIgG(in spite IgAIF > 0%)
- Intrathecal IgM synthesis: QIgM > QIgA(in spite IgMIF > 0%).
The relative values for the intrathecal fraction takes into account that IgG, IgA and IgM are synthesized in different amounts and always more IgG is synthesized than IgA and IgM. With a pattern IgMIF > IgGIF >IgAIF (three class reaction), IgM is the dominant intrathecal fraction. The graphical evaluation in quotient diagrams (Reibergrams) present both informations about immune response pattern and barrier function recognizable at one glance. Refer to :
- AI = Qspec/QIgG for (QIgG < Qlim)
- AI = Qspec/QIgG for (QIgG > Qlim).
Assessment of the antibody index (AI):
- Reference interval: 0.6–1.3
- Intrathecal synthesis of specific antibodies: ≥ 1.5
- Values below 0.5 are an indication of non-matched CSF/serum sample or of analytical faults.
- Using conventional assays (complement binding assay, hemagglutination test), an AI ≥ 4 is considered pathologic. Measurement of pathogen specific antibodies using ELISA improves the accuracy of antibody determination and the calculation of the AI is as follows:
The dynamic pattern of intrathecal antibody synthesis during the course of an infectious disease is shown in . Intrathecal antibody synthesis can be mimicked following plasmapheresis or extensive hemorrhage. It takes a few days before a new steady state equilibrium between the compartments is reached.
Blood contamination, be it either artificially induced or disease related, renders the assessment of the ratio diagram impossible.
In some infectious diseases, the humoral immune response within the CNS tends to resolve very slowly even if the patient was treated successfully and has fully recovered from the illness. However, in some cases, still marked intrathecal antibody synthesis with pathologic AI values even several years after herpes encephalitis and years after successfully treated progressive paralysis are found. Repeat treatment is not required if cell counts are normal.
In some diseases caused by pathogens, AI values > 20 are found (e.g., in subacute sclerosing pan encephalitis or neurosyphilis).
A pathologic AI in diseases caused by pathogens is approximately twice as sensitive as the detection of an oligoclonal pattern of the CSF.
In infections caused by viruses of the herpes group, the intrathecally synthesized immunoglobulins mainly consist of specific antibodies against the pathogen, for instance in Herpes simplex virus encephalitis, Herpes zoster virus ganglionitis, Varicella virus cerebellitis, and Cytomegalovirus encephalitis. The AI calculation almost always reveals intrathecal antibody synthesis.
However, there are also poly specific immune responses in which the specific antibody to be determined is only one of several. In multiple sclerosis for example, 80–90% of the CSF IgG may consist of such non definable antibodies, and AI values for individual antibody specificities of falsely < 1.0 may be found.
Autoantibodies have led to the identification of new neurological syndromes, especially
- Onconeural antibodies are a diagnostic approach to autoimmune encephalitis /, /
- Paraneoplastic autoantibodies are a diagnostic approach to paraneoplastic neurological syndromes /, /.
For autoantibodies refer to:
In some conditions where the IgG antibody index is determined, it can be particularly useful to determine intrathecally IgG after isoelectric focussing because this method is 50-fold more sensitive than the quantitative IgG determination. Oligoclonal bands allow the detection of intrathecal IgG with as little as 0.5% of total CSF IgG. Intrathecally synthesized IgG represents qualitatively characteristic patterns on electrophoresis, also called oligoclonal bands .
The detection of intrathecal IgG in the CSF is based on a comparison with parallel focussing of serum IgG. Serum IgG molecules are polyclonal and reflect the virtually infinite heterogeneity of the individual antibodies (i.e., the end product of a patient’s numerous immune responses). In contrast, intrathecal antibodies reflect the limited immune response to one or few pathogen(s) or an auto antigen. In these cases the immune response is mediated only by a limited spectrum of plasma cells of the leptomeninges. This immune response is not polyclonal, but oligoclonal in nature. Therefore, the IgG produced intrathecally differs from the serum IgG by the kappa/lambda ratio, the pattern of electrophoretic charge, the IgG subclass composition, and the antigen specificity. The CSF IgG predominate significantly over those originating in serum and, due to limited heterogeneity, produce a limited antibody response and limited oligoclonal banding .
Because of their singularity compared to the antibodies originating from the serum, oligoclonal antibodies in the CSF can be recognized as additional bands not present in the serum, especially in the highly alkaline range, on isoelectric focusing (IFE). The number and location of these additional bands are, however, of no differential diagnostic significance.
Causes of oligoclonal banding
Oligoclonal IgG can be found in:
- Acute central nervous system infections with a very specific immune response to a single pathogen (virus, bacterium, parasite)
- Infections in the distant past with a persisting “anamnestic” immune response to the pathogen in the CSF (e.g., TPHA antibodies in neurosyphilis)
- Chronic inflammatory diseases or autoimmune diseases of the CNS. There is a poly specific immune response and production of IgG with specificities e.g. against Measle virus, Rubella virus, and Herpes zoster virus (MRZ response) in multiple sclerosis and in cerebral lupus erythematosus without the presence of the corresponding antigens.
A monoclonal immunoglobulin band in the CSF immunofixation electrophoresis in the absence of blood-brain barrier disruption is rare and generally non diagnostic.However in the case of leptomeningeal myelomatosis due to relapsed multiple myeloma was this the case .
The results of the emergency tests must be available within 2 h.
In patients with acute disease of the CNS primary care physicians frequently perform lumbar puncture, because CSF is a diagnostic window to the central nervous system. Commonly performed tests are: CSF color, glucose and protein levels, cell count and differential, microscopic examination, and culture.
Diagnostic puncture refers to the timing of puncture as performed by the experienced clinician with certain diagnostic findings in mind. Based on experience, punctures are performed on the following days of the disease:
- Purulent meningitis: days 1 and 2
- Viral meningitis: days 3–5
- Tuberculous meningitis: weeks 1–3
- Herpes encephalitis: days 5–7 after the start of the flu-like prodromal stage
- Neuroborreliosis: weeks 2–4 after the start of the myalgic stage.
Acute and subacute inflammation within the central nervous system is usually associated with an impairment of the blood-CSF barrier and an increase in cell numbers ≥ 5/μL (pleocytosis). Neither alteration is proof of the presence of inflammation because pleocytosis and an increase in QAlb are also present in numerous non inflammatory disorders.
Besides being associated with inflammation, pleocytosis can also occur with tumors, trauma, parenchymal hemorrhages, or after lumbar puncture (irritant pleocytosis) (). Therefore, a cell differential is required to distinguish between inflammatory and non inflammatory pathology .
The following cell responses are differentiated:
- Granulocytic (> 50% of WBCs are polymorphonuclear granulocytes); present in bacterial and the early phase of acute viral meningitis. In the post acute phase a mononuclear transformation occurs.
- Lymphocytic (> 85% of WBCs are lymphocytes); associated with viral meningitis (meningoencephalitis) with pleocytosis of below 100 to higher than 1,000 WBCs/μL. Lymphocytosis also occurs in fungal and tuberculous infections of the CNS.
- Mixed-cell type (granulocytes, lymphocytes, monocytes in approximately equal proportions); associated e.g., with tuberculous or syphilitic meningitis and concurrent pleocytosis of below 100 to a few hundred WBCs/μL.
- Mononuclear cells (monocytes and lymphocytes); associated with meningeal irritation secondary to various causes such as hemorrhage. The majority of patients with Guillain-Barré syndrome will have fewer than 10 monocytes per μL.
- Eosinophilic meningitis; defined as more than 10 eosinophils per μL or a total CSF cell count of more than 10% eosinophils.
- Further diagnostically relevant cell types are listed in .
The National Comprehensive Cancer Network of the USA recommends the use of flow cytometric analysis (FCA) in evaluating for possible CNS lymphoma. The data of a study support a policy in which CSF FCA is only performed when atypical lymphocytes or blasts are seen on Wright-stained slides or a history of a prior hematologic malignancy is present.
- Neutrophil cell response
- Lymphocytic cell response
- Humoral tertiary phase.
A cellular response with proliferation of polymorphonuclear granulocytes develops rapidly in bacterial meningitis. Within a few hours, 10,000 to 20,000 leukocytes/μL can be measured in the CSF. The blood-CSF barrier breaks down and serum proteins pass into the CSF. If antibiotic treatment is administered early, the leukocytosis usually resolves rapidly. The cell count declines to one half within 24 h. Following the exudative phase of bacterial meningitis, a lympho-monocytic cell profile prevails in the proliferation and regeneration phase.
In viral meningitis, granulocytes also prevail during the first 3 days, although there are rarely more than 700 cells/μL. After 10 days of disease, there are no more granulocytes in viral infections.
Lymphocytic responses are typical of viral meningeal infections. They are associated with a significantly less extensive blood-CSF barrier dysfunction than that seen in bacterial meningitis.
Among the lymphocytes, lympho-monocytic types and plasma cells are frequently found.
In bacterial meningitis intrathecal immunoglobulin synthesis takes place if treatment is delayed or complications arise. A humoral immune response may indicate underlying cortical or fissural abscess formation.
In viral meningitis, local antibody synthesis in the CNS starts during the second week of disease. It can persist for months to years and usually occurs in response to the following microorganisms and diseases:
- Paramyxovirus infection (mumps and measles meningoencephalitis)
- Herpes virus infection (Herpes simplex and Herpes zoster encephalitis)
- Coxsackievirus infection (early summer meningoencephalitis viruses and the meningoencephalitides and central European encephalitis).
In some cases of viral meningitis, especially during childhood, intrathecal Ig synthesis may begin as early as during the first week of disease. Encephalitic courses, as seen in mumps encephalitis or Herpes zoster virus encephalitis, can result in intense humoral immune responses, which are markedly more pronounced than those associated with herpes zoster ganglionitis.
The detection of pathogen specific nucleic acid sequences by polymerase chain reaction (PCR) is of importance in the CSF diagnosis of infectious diseases of the CNS. The finding of nucleic acid sequences during the early phase of disease, before antibodies are produced, are indicative of florid infection ().
The qualitative demonstration of viral DNA or RNA in the CSF is almost always indicative of florid infection. Patients with positive viral genome are 88 times as likely to have an infection of the CNS as those with a negative PCR result .
PCR can be an important adjunct test, but is less reliable compared to viral tests. If bacterial meningitis is suspected and microbiological results were negative, PCR can detect microbial DNA of meningitis pathogens in the CSF.
PCR gains value in the diagnosis of:
- Neuroborreliosis ().
- Neurosyphilis ().
- Central nervous system tuberculosis (). Although the diagnostic sensitivity of PCR assay (M. tuberculosis DNA in CSF) has significant discrepancies (65–83%) between each type of measuring methods and different laboratories
Locally synthesized CEA can be a marker of malignant CNS tumors. CEA and IgA are of similar molecular size, allowing the IgA quotient diagram to be used for the detection of intrathecal CEA synthesis. An intrathecal fraction occurs in :
- 90% of all carcinomatous meningeal involvements
- 45% of all intraparenchymal metastases.
The chance of identifying an intraparenchymal metastasis by means of CSF CEA declines with increasing distance from the ventricles. Although intracortical metastases communicate with the subarachnoidal space of the pallium, the latter is connected with the lumbar sac only in the basal (temporal) sections. The largest portion of the supra cortical CSF space (fronto-parietal) directly drains into the blood via the Pacchioni granulations. The intact dura is impermeable to proteins.
Markers of neurodegeneration and dementia are brain specific proteins which are released in response to acute brain damage, such as neuron specific enolase or S100 protein.
In response to chronic damage to the CNS the following biomarkers are of diagnostic and prognostic value:
- Hydrophobic β-amyloid 1-42 peptide as a component of senile plaques in the brain
- Tau protein as an indicator of damage to neuronal axons
- Neurofilament light chains as an indicator of inflammatory demyelinating disease.
Enolase (EC 22.214.171.124) is one of eleven cytosolic glycolytic enzymes and catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate . The enzyme is a dimer composed of two out of three possible non species specific subunits (α, β, γ subunits of 39 kDa, total MW of 100 kDa). The subunits have different immunological, biochemical and organ specific properties.
There are five isoforms (αα, ββ, γγ, αγ, αβ), of which the subunits are present in the following tissues:
- The α-subunit in cells ubiquitous in the body; non neuronal enolase
- The β-subunit in muscle cells (cardiac muscle αβ, striated muscle ββ)
- The γ-subunit in nerve cells and neuroendocrine cells (APUD cells), e.g. in the intestine, lungs and endocrine organs such as the thyroid, pancreas and pituitary gland.
γγ-Enolase, also known as neuron specific enolase (NSE), is mainly found in neuronal tissues, glial tissue and neuroendocrine tissue and accounts for 1.5% of soluble proteins in the CSF. Elevated CSF levels of NSE can be found in 18% of a nonselective patient population consisting, in particular, of patients with polyneuropathy, metabolic myopathy, hepatic encephalopathy, multiple sclerosis, and convulsive diseases . The upper reference interval value for NSE in serum and CSF is 15 μg/L.
S100 is an acidic, thermolabile 21-kDa protein (see ). Its name derives from its biochemical property of being soluble in 100% saturated ammonium sulfate solution at neutral pH. S100 belongs to the multi gene family of calcium binding proteins and exists as a dimer composed of two isomeric subunits (α, 10.4 kDa; β, 10.5 kDa). It occurs in three isoforms: S100B (ββ), S100A (αβ), and S100A1 (αα).
S100A is expressed especially by malignant melanoma cells. It is also present in the CNS, but accounts for only 5% of total S100.
S100A1 is localized in keratinocytes, melanocytes, smooth muscle, cardiomyocytes, and the kidneys.
The upper reference interval value for S100 in serum and CSF is 0.1 μg/L. Most immunoassays measure all forms of S100, but are calibrated against S100B (ββ).
S100 in neurodestruction and neurodegeneration
Neurodestruction and neurodegeneration leads to the release of S100 from astrocytic glial cells and initially to an increase in CSF S100 levels. If the blood-CSF barrier is compromised, S100 may spread into the systemic circulation and consequently lead to elevated serum S100 levels. High serum S100 levels are measured in particular after traumatic brain injury, cerebral ischemia or infection, hypoxic brain damage following cardiac arrest, or after cardiopulmonary bypass surgery .
In the case of acute brain injury, levels begin to rise after a few hours to reach a peak after 1–3 days, and subsequently return to healthy control levels within approximately 1 week with a half life of 2–3 h, provided there are no further complications. The interval between the acute episode and peak levels after ischemic infarctions is longer than that following traumatic or hypoxic lesions; hemorrhages cause earlier and steeper increases in S100 than ischemic infarctions.
Both trauma patients and patients with ischemias and hemorrhages show a correlation between S100 levels and the severity of organic brain damage as objectified on a CT scan, the clinical status, and the prognosis for short- and medium-term rehabilitation .
Elevated S100 levels in CSF are seen in Creutzfeldt-Jakob disease (CJD) and variant CJD (vJCD). Serum levels of S100 are also markedly higher in CJD than in other dementia illnesses or in controls without dementia. No elevated S100 levels are found in the serum of patients with various neurodegenerative, autoimmune and psychiatric diseases. However, some studies report mildly elevated S100 levels in CSF in Alzheimer’s disease, in exacerbated multiple sclerosis, Guillain-Barré syndrome, bacterial inflammatory cerebral diseases, and various psychiatric diseases.
In conclusion, early measurement of S100 after acute cerebral injury is recommended for determining the prognosis and for assessing cerebral involvement if CT findings are negative. Frequent serial measurements may be useful as an additional criterion for monitoring.
In major depression S100 plasma levels (0.095 ± 0.065 μg/L) were significantly higher than in healthy controls (0.048 ± 0.024 μg/L) and positively correlated with treatment response after 4 weeks of treatment .
Micro tubules are hollow cylinders of up to 25 nm in diameter. They are part of the structural network of the cytoplasm (cytoskeleton) of cells. Dimers of tubulin polymerize end to end to form the basic structure of a microtubule. Tau proteins are microtubule associated proteins that bind with high affinity to tubulin, the main protein of micro tubules, to promote tubulin assembly into micro tubules and to stabilize the micro tubules. The binding of tau to the micro tubules occurs through phosphorylation of the isoforms at specific binding sites. The phosphorylation of tau protein leads to the dissociation of the tubules. There are six isoforms of tau protein in the adult human brain, which derive from alternatively spliced products of a gene located on chromosome 17q21-22. Tau is not an essential protein, but is required for the polymerization and stabilization of the micro tubules. Tau is a characteristic component of the neuronal axons in the CNS.
It is thought that in Alzheimer’s disease sulfated glycosaminoglycans induce the assembly of tau into filaments. Tau is highly hyper phosphorylated and assembled into paired helical or linear filaments, thus losing its ability to stabilize the micro tubules. Elevated levels of tau protein or phosphorylated tau are associated with Alzheimer’s disease, but are also found in other neurodegenerative diseases such as frontotemporal dementia, progressive supranuclear paralysis, corticobasal degeneration and dementia, and prion disease .
In Alzheimer’s disease, there is hyper phosphorylation of tau protein. Of particular diagnostic importance is the phosphorylated threonine 181.
The 40 and 42 amino acid isoforms of Aβ result from intracellular proteolytic cleavage of the β-amyloid precursor protein (AβPP). Aβ play an important morphological and biochemical role in Alzheimer’s disease. Aβ1-42 is insoluble and is bound in plasma to lipoproteins, especially those containing apo E. Aβ1-40 is more readily soluble, but is also bound to triglyceride rich lipoproteins. The hydrophobic Aβ1-42 is the main component of senile plaques. Mutations in the gene AβPP and the Presenilin 1 and Presenilin 2 genes, which are present in 7% of patients with Alzheimer’s disease, influence the metabolism of the protein AβPP by increased synthesis of the amyloid forming Aβ1-42 .
14-3-3 proteins are a family of at least seven proteins, each with a MW of 30 kDa, which are thought to play a role in cellular signal transduction, in particular of the kinases.
The 14-3-3 proteins are detected by sodium dodecyl sulfate polyacryamid gel electrophoresis and immunoblotting using an antibody which recognizes all seven human isoforms, since these have a common N-terminal amino acid sequence.
Neurofilaments are neuron-specific heteropolymers considered to consist of a triplet of light chain (68 kDa), medium (145 kDa) and heavy chain (about 200 kDa) according to their molecular weight. Peripherin in the peripheral nervous system and α-internexin in the central nervous system can also be included in the neurofilament polymers. These five proteins co-assemble into the 10 nanometer intermediate filament in different combinations and concentations depending on the type of neuron, location in the axon and stage of development /, /.
Each of the neurofilament proteins consists of an amino-terminal domain, a variable carboxy-terminal domain, and a central helical domain. Following synthesis and assembly in the neuron, tetramers of neurofilament proteins are transported bidirectionally along axons by the microtubular apparatus prior to forming continuously overlapping array that runs parallel to axons. Once formed, in the healthy state, they are remarkably stable for months to years. In mature myelinated axons, neurofilaments are the single most abundant protein.
Neurofilament light chain (NF-L) is the most promising biomarker in inflammatory demyelinating disease of the central nervous system (CNS). NF-L reflects ongoing neuronal damage and gradual progressive decline and neurological dysfunction. NF-L is detected in cerebrospinal fluid (CSF). CSF sampling provides a direct approach to the CNS and requires a lumbar puncture. NF-L in serum reflects leakage or diffusion through the blood-brain barrier into venous blood. Concentrations in serum were 42- fold lower than in CSF . However, NF-L concentration is not reliably measured with conventional immunoassays. The development of Single Molecule Assay (Simoa) however, is sufficiently sensitive to measure NF-L (refer to .
A variety of neurological disease states which can be followed longitudinally and conditions that require monitoring e.g.,
- Multiple sclerosis
- Amyotrophic lateral sclerosis
- Creutzfeldt-Jacobs disease
- Acquired peripheral neuropathies
- In healthy individuals the serum median NF-L value was 6.3 pg/ml (range 2.1–19.1 pg/mL
- In serum samples from246 healthy controls (median age 44.3 years) the median serum NF-L was 22.9 pg/mL .
Quantitative measurement of NF-L provides an indication of axonal damage in the CNS. Increased NF-L level reflects ongoing neuronal damage, irrespective on the underlying pathology. Overall NF-L appears useful as a biomarker and as a predictor of outcome in the early clinical stages prior to definite multiple sclerosis. Strengths and weaknesses of NF-L as a biomarker in the clinical management CNS diseases are shown in . Serum NF-L is called jokingly the neurologist’s troponin.
Comments and problems
For the determination of NF-L Simoa is more sensitive than regular ELISA (126-fold in CSF and 25-fold in serum).
Serum NF-L levels correlate with CSF levels, however the levels in serum have been evaluated to be a 42-fold lower than in CSF.
NF-L concentrations depend on age, irrespective of the underlying pathology.
GAD-Ab are found in serum in up to 90% of patients with type 1 diabetes mostly at low concentration.The autoantibodies are found in the CSF in severe neurological conditions associated to altered synthesis of gamma-aminobutyric acid (GABA) from glutamate, where GAD is the rate-limiting enzyme. GAD is an intracellular enzyme found in presynaptic neurons of central nervous system.
Neuronal disorders, now encompassed in the phenotype GAD antibody-spectrum disorders comprehend the diseases, Stiff person syndrome and variants e.g., cerebellar ataxia (CA), progressive encephalomyelitis with rigidity and myoclonus (PERM), and limbic encephalitis (LE) .
- Stiff person syndrom
- Variants of Stiff person syndrom, e.g., cerebellar ataxia (CA), progressive encephalomyelitis with rigidity and myoclonus (PERM), and limbic encephalitis (LE).
Method of determination
Cerebrospinal fluid: 0.5 mL
Stiff person syndrome (SPS)
SPS has an onset of axial muscle stiffness progressively involving proximal and distal limbs. The prevalence of SPS is 1 to 2 cases per million population and mostly develops in persons of 30–50 years of age. Up to 80% of patients with SPS have GAD65 antibodies.
Cerebellar ataxia (CA)
The disease is characterized by gait and limb ataxia, dysarthria, and nystagmus. In about 80% of cases women are affected. CA is the second most frequent GAD65 antibody-associated neurological disease.
Limbic encephalitis (LE)
LE is an encephalitis and inflammatory disorder of the brain. LE is caused by autoimmunity. Some cases are associated with cancer. Although the disease is known as LE, it is seldom limited to the limbic system and usually involved are other parts of the brain.
The exclusion of organic disease as the cause of a neurologic or neuropsychiatric syndrome can be difficult. In this context, among serum biological tests, biological tests in CSF are indicated, because many diseases of the brain are associated with a blood-CSF barrier (blood-brain barrier) disorder or an increased cell count. Both findings, sensitive yet nonspecific, are suggestive of a CNS disease. Apart from these two nonspecific alterations, targeted CSF analysis is an important additional tool, depending on the clinical setting.
Every patient with an acute meningitic syndrome (headache, vomiting, fever, neck stiffness) must undergo lumbar puncture unless there are signs of possible cerebral herniation. Although the course of purulent meningitis is usually more dramatic and the cortex becomes involved in the inflammatory process more rapidly than in viral meningitis (seizures, decreased level of consciousness), the differential diagnosis between bacterial and viral etiology in the early stages cannot be made without CSF examination.
According to a systematic review and meta-analysis serum PCT is a highly accurate diagnostic test that can be used for rapid differentiation between bacterial and viral causes of meningitis in adults. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio (DOR) for PCT were 0.90 (95% CI 0.84–0.94), 0.98 ((95% CI 0.97–0.99), 27.3 (95% CI 8.2–91.1), 0.13 (95% CI 0.07–0.26), and 287 (95% CI 58.5–149). The cutoff range of PCT was 0.25–2.13 ng/mL, the most used cutoff was 0.50 ng/mL.
- Cloudy or purulent CSF
- Neutrophil pleocytosis up to ≥ 1,000 cells/μL and > 60% polymorphonuclear granulocytes
- Severe blood-CSF barrier dysfunction (Qalb > 25 × 10–3)
- Total protein in CSF > 2 g/L
- Detection of bacteria or bacterial antigens in CSF
- CSF/serum glucose ratio < 0.40
- Lactate in CSF > 32 mg/dL (3.5 mmol/L).
Microscopic detection of bacteria following methylene blue staining and differentiation according to the gram stain should be attempted. Latex agglutination tests for detecting bacterial antigens are available for the following pathogens: Haemophilus influenzae, Streptococcus pneumoniae, E. coli, Neisseria meningitides, Streptococcus group B. Latex agglutination tests can also detect antigens shortly after starting antibiotic treatment, when pathogen cultivation is no longer possible. However, latex agglutination tests are less sensitive than the detection by culture.
The most common etiologic pathogens of bacterial meningitis are listed in . Neonatal meningitis and neonatal sepsis are associated with long term neurological and cognitive impairment; primarily impairment of hearing, vision or motor function, cerebral palsy, and epilepsy .
Erroneous diagnoses with disastrous consequences are possible in non purulent bacterial meningitis . Although the CSF has a cloudy appearance and the blood-CSF barrier is completely broken down, there is only a slight increase in cells. The stained microscopic preparation shows a pure culture of bacteria, most commonly S. pneumoniae.
The cause of this lack of leukocytosis is probably a consumption leukopenia within the CSF space, possibly presenting on the 3rd or 4th day of undiagnosed bacterial meningitis.
The very common occurrence of intrathecal IgA synthesis does not always result in the upper cutoff limit being surpassed of the CSF/serum quotient diagram for IgA (). The relative intrathecal IgA synthesis, however, often becomes greater than the IgG formation.
Even immediately following bacterial invasion of the subarachnoidal space during N. meningitidis sepsis, the protein and cell count may still be normal. However, very soon thereafter the typical picture of a rampant purulent meningitis develops.
When performing a lumbar puncture because of suspected meningitis, a blood sample should be obtained and swabs from throat, ears and wounds should be taken for immediate culture. If the microscopic and culture based detection of bacteria in CSF is unsuccessful (e.g., in patients with preceding antibiotic treatment) the detection of bacterial antigens or detection of pathogens with molecular biological methods is often still possible.
The following findings are suggestive of this type of meningitis, which usually has a benign course:
- Transparent CSF
- Mononuclear pleocytosis with up to several hundred cells per μL
- At the most a moderate blood-CSF barrier disturbance
- Normal lactate levels.
Occasionally, during the first lumbar puncture, a polymorphonuclear pleocytosis is encountered, which may precede the lymphocytic phase. Culture based detection is most commonly accomplished for enteroviruses (Coxsackievirus, Echovirus) and Mumpsvirus, however, this has no therapeutic relevance. Herpes simplex virus and Varicella zoster virus can rarely be cultured from CSF. The detection of viral DNA is the method of choice during the acute phase of disease.
During the second week the antibody index rises above 1.5, even if the humoral response is weak, as is the case in Herpes zoster virus meningitis in adults or in post infectious encephalomeningitis in children following infection with Varicella zoster virus, Measle virus, and Rubella virus. A surprisingly frequent finding is the intrathecal synthesis of antibodies directed against the Herpes simplex virus.
Worldwide, rabies and Japanese encephalitis virus are responsible for an estimated annual mortality of 60,000 and 17,000 people, respectively. Rabies and Herpes simplex virus encephalitis are present worldwide .
In the United states 7 of 100,000 population are hospitalized for encephalitis each year. Encephalitis is characterized by altered mental status and various combinations (e.g., acute fever, seizures, neurologic deficits), pathologic CSF results (e.g., pleocytosis), and abnormalities in neuroimaging and in electroencephalography. About 20–50% of cases are attributed to viruses: Herpes simplex virus accounts for 50–75% of cases. Initial testing in immunocompetent hosts includes Polymerase chain reaction (PCR) as reverse-transcriptase (PCR/RT-PCR) assays of a CFS specimen for HSV-1, HSV-2, VZV, enteroviruses, and in children younger than 3 years of age human parechoviruses. If these initial tests (tear 1 tests) fail to establish a diagnosis, additional testing (tier 2 and tier 3 tests) can be undertaken. Tier 2 tests often include PCR for cytomegalovirus, human herpes virus 6 and 7, Epstein-Barr virus and HIV. Serologic tests, including tests of serum specimens, are also essential parts of the diagnostic evaluation of arboviruses. Serologic testing of CSF-IgM may help diagnose encephalitis due to arboviruses, VZV, EBV, measles virus, mumps virus, rubella virus, rabies virus or other viruses .
Depending on the disease, HSV-1 and HSV-2 are involved with different prevalences:
- Encephalitis: with an incidence of 2–4 per million population, HSV encephalitis is the most common form of viral encephalitis. Over 90% of herpes encephalitis cases are due to HSV-1, only 5–10% of cases are due to HSV-2.
- Meningitis: approximately 5–10% of meningitis cases are caused by HSV-2.
- Myelitis and radiculitis are rarely due to HSV. If they are, then HSV-2 is the cause.
The onset of herpes encephalitis is clinically characterized by a flu-like, meningitic prodromal stage lasting a few days. Subsequently, temporal lobe symptoms such as Wernicke’s aphasia, confusion, and complex focal seizures occur which are combined with the CSF picture of viral meningitis. Magnetic resonance imaging reveals abnormalities as early as during the first week of illness, while early CT scans may still be normal.
The following rule applies: if a temporal lobe syndrome with lymphocytic pleocytosis occurs after a flu like prodromal stage, the presence of HSV encephalitis must be presumed until proven otherwise.
Using PCR, HSV DNA detection in the CSF is almost always successful during the first week of illness . Initially there is pleocytosis < 300/μL and a moderate increase of Qalb, usually < 20 × 10–3. During the second week, an increase of IgG, IgA, IgM in the CSF is evident, with a marked predominance of IgG. There is intrathecal HSV antibody synthesis (AI > 1.5), and oligoclonal IgG is detectable by isoelectric focusing. IgG, IgA and IgM decrease gradually over the course of months and years.
In the early stage of disease, the differential diagnosis must include:
- Vascular neurosyphilis; specific syphilis reactions are positive
- Glioblastoma of the temporal lobe; CT finding
- Other types of viral encephalitis, e.g. Coxsackievirus, Varicella zoster virus, Mumpsvirus; specific antibodies are positive
- Tuberculous meningitis; mycobacteria DNA detection
- Temporal lobe phlegmonous abscess; history, ear-nose-throat findings, CT.
VZV infection is harmless in childhood, and most individuals will have had an infection or, in 30–60% of cases, even manifest infection, by adulthood. Later in life, VZV infection can lead to meningitis, meningoencephalitis, myelitis, cranial neuritis, ganglionitis, and radiculitis.
During the acute phase of disease, the detection of VZV DNA in CSF is the method of choice for detecting an infection.
The following findings are suggestive of this diagnosis:
- Transparent CSF
- Lymphocytic pleocytosis in the range of 30–300 cells/μL
- Blood-CSF barrier dysfunction in meningitis; common in meningoencephalitis, not in ganglionitis
- Total protein > 0.5–1 g/L in ganglionitis.
An immune response, although weak, is always present. Only about 15% of patients with acute VZV infection show intrathecal IgG synthesis. Due to cross-reactivity between HSV and VZV in chronic inflammation of the CNS, a measles-rubella-zoster (MRZ) response may occur. An isolated elevated VZV antibody index requires careful clinical interpretation.
VZV establishes a latent infection. Reaction usually results from suppression of cell mediated immunity, most age related immunosenescence. CNS reactivation is relatively uncommon, but reactivation in a dorsal root ganglion can lead to herpes zoster .
The ESME virus is predominantly transmitted by ticks, but the clinical presentation can hardly be mistaken for a Borrelia burgdorferi infection of the CNS. If encephalitis develops after a tick bite, the diagnosis can be confirmed by the increase in specific serum and CSF antibodies. The rate of manifestation following an ESME infection is 50%, the remainder of cases are clinically asymptomatic. Of the symptomatic cases, 50% are meningitis, 40% encephalitis, and 10% myelitis.
The following findings are indicative of acute ESME infection:
- Transparent CSF
- Lymphocytic pleocytosis in the range of 30–1500 cells/μL, granulocytic cell profile during the initial phase
- Total protein 0.25–2.2 g/L
- Blood-CSF barrier dysfunction in 70% of cases.
Demonstration of specific IgM in serum 7–10 days after the infection; increase of specific IgG antibodies during the 2nd week. A pathologic antibody index for IgG can be measured 2 weeks after the start of the infection. A specific immune response in the CSF to ESME is unlikely after vaccination.
The infection can occur at any age, even during the embryonic period. Congenital CMV infection is the most common acquired cause of hearing loss in children in the United states. In adults, the antibody prevalence is over 70%. The pathogen persists in endothelial cells and reactivation can occur at any time. In immunocompromised patients CMV infection can lead to encephalitis, retinitis or colitis.
The following findings are suggestive of acute CMV infection:
- In immunocompetent patients, the presence of specific IgM antibodies or an increase in the serum IgG titer are indicative of viral replication
- Transparent CSF
- Lymphocytic pleocytosis (median of 150 cells/μL)
- Total protein variably elevated
- Blood-CSF barrier dysfunction.
To differentiate latent from active, florid CMV, the viral load is determined using PCR on serum (see also ). The detection of CMV genome in CSF is also indicative when encephalitis is suspected. In immunosuppressed individuals, the determination of the antibody index is rarely useful.
- Primary HIV infection: causes acute aseptic (viral) meningitis or meningoencephalitis
- Secondary opportunistic infection causing neurodegenerative conditions characterized by HIV associated neurocognitive disorders (HAND), motor and behavioral abnormalities
- Treatment related neurologic disease.
Primarily neurological manifestations of HIV infection include encephalopathy, myelopathy, peripheral neuropathy, and myopathy. Focal lesions of the CNS in AIDS include primary lymphoma and cerebrovascular diseases. Approximately 2 weeks following infection, not only the CD4+T cells but also the cells of the CNS are affected. This occurs via infected monocytes/macrophages, which infect the neuronal structures, in particular the microglia, with neurotoxic substances. HIV infection can affect the entire CNS and cause meningitis, myopathy, polyneuropathy, encephalopathy, and myelopathy . Classifications of HIV according to the CDC refer to:
This aseptic meningitis occurs in 5–10% of HIV patients. Of particular diagnostic importance is the manifestation of seroconversion which, in 30–40% of cases, occurs concomitantly with a clinical picture comparable to infectious mononucleosis. CSF analysis shows mild pleocytosis in the range of 20–80 cells/μL and mildly elevated total protein.
HIV myopathy includes HIV induced polymyositis, azidothymidine (AZT) induced toxic myopathy, polymyositis, and wasting syndrome in advanced stages of AIDS. Polymyositis is immune mediated and occurs during the asymptomatic phase in combination with hypergammaglobulinemia on serum protein electrophoresis. Patients may have acute paresis with an up to 10-fold increase in serum creatine kinase (CK) activity. Up to 30% of patients on long term treatment with AZT develop painful myopathies with elevated serum CK and lactate.
Various types of neuropathies occur, depending on the stage of disease. During the seroconversion phase, predominantly inflammatory, demyelinating neuropathies are observed, e.g. Guillain-Barré syndrome, chronic demyelinating polyneuropathy or sensory ataxic neuropathy. Once AIDS has developed, the classic distal sensorimotor neuropathy, mononeuritis multiplex, autonomous neuropathy, lumbosacral Cytomegalovirus polyradiculopathy or lymphomatous neuropathy become manifest.
Primary HIV encephalopathy
This disease, also known as subacute AIDS encephalitis or AIDS dementia complex, is the most common primary HIV associated neurological disease in adults and children. It occurs late in the stages of HIV infection and is generally accompanied by symptoms of immune deficiency. HIV encephalopathy follows a variable course, with some patients showing continuous progression while others show periods of stability or even improvement. Some children have static encephalopathy in the form of learning and language difficulties as well as pyramidal symptoms.
HIV associated myelopathy
Myelopathies can occur as a direct consequence of:
- Primary HIV infection in the form of vacuolar myelopathy
- Myelitis due to an opportunistic infection
- Neurosyphilis or a lymphoma.
Vacuolar myelopathy occurs in approximately 4% of AIDS patients, usually in the late stages of the disease. Patients typically present with progressive gait impairment, pareses of the lower limbs, imbalance, and sphincter dysfunction.
Staging of HIV infection
HIV infection is staged according to the CDC classification and laboratory categories relating to the CD4+T cell count.
Approximately 40–80% of patients with asymptotic HIV infection are found to have CSF abnormalities in the form of mild lymphocytic pleocytosis. Blood-CSF barrier function is normal in most cases, oligoclonal banding is found in up to 70% of cases. About 80% of both asymptomatic patients and patients with neurologic symptoms have an intrathecal synthesis of IgG, IgA and IgM. In late stage AIDS, the humoral and cellular immune responses and thus the pathologic findings in CSF decrease. The occurrence of intrathecal antibody synthesis during the late stage is often indicative of an opportunistic infection with viruses (CMV, HSV, VZV), mycobacteria, fungi, or toxoplasma.
Tuberculous meningitis occurs in approximately 1% of all tuberculosis cases and in less than 3% of the estimated cases of bacterial meningitis in the United States . About 50% of tuberculous meningitis result in severe disability or death. Individuals with increased risk for tuberculous meningitis include young children with primary tuberculosis and patients with immunodeficiency caused by aging, malnutrition, or disorders such as HIV and cancer . Tuberculous meningitis is associated with a high frequency of neurologic sequelae and mortality if not treated promptly.
- Lymphocytic pleocytosis. Total leukocyte count 100–500 cells/μL. Occasionally different lymphocyte populations. Very early in the disease, lower counts and neutrophil predominance may be present.
- Elevated protein concentration, typically 1–5 g/L
- CSF/serum glucose ratio usually < 0.5 or below 45 mg/dL (2.5 mmol/L)
- Lactate is significantly higher in comparison with the serum value
- QAlb higher than 25 × 10–3
- In approximately 85% of cases intrathecal IgA synthesis (IgA index > IgG index) at the time of the initial diagnosis
- Intrathecal IgG synthesis can be detected in approximately 50% of patients during the course of the disease
- Acid-fast smear; important caveat is that a single sample has only a diagnostic sensitivity in the order of 20–40%. After starting treatment, the sensitivity of CSF smear decreases rapidly.
- M. tuberculosis culture, diagnostic sensitivity 40–80%. Refer to . After starting treatment, the sensitivity of CSF culture decreases rapidly.
- Nucleic acid amplification . PCR tests which amplify several target genes simultaneously have sensitivities in the range of 85–95% . Currently, most experts conclude that commercial PCR tests can confirm tuberculous meningitis but cannot rule it out. Thus, a negative CSF examination neither excludes the diagnosis of tuberculous meningitis nor obviates the need for empirical therapy if the clinical suspicion is high /, /. After starting treatment of a positive result mycobacterial DNA may be detectable for up to a month.
- Adenosin deaminase (EC 126.96.36.199). According to a study CSF adenosin deaminase greater than 6 U/L attains a diagnostic specificity of 95% but a sensitivity of only 55%. With a positive likelihood ratio of 10.7 and a pretest probability of 38%, the post-test probability of having tuberculous meningitis was 87%, and thus treatment was offered.
Opportunistic pathogens such as Toxoplasma sp., Cryptococcus sp., Candida sp. and Aspergillus sp. cause severe infections of the CNS almost exclusively in immunosuppressed patients. Cryptococcal meningitis is a leading cause of mortality in low- and middle-income countries where access to antiretroviral therapy is limited. Diagnostically relevant investigations are listed in . Patients having chronic fungal meningitis typically present with some combination of mental confusion, fever, headache, nausea, stiff neck and vomiting. These fungi include Acremonium sp., Aspergillus amstelodami, A. flavus, A. fumigatus, A. oryzae, A. terreus, Blastomyces dermatitides, Candida albicans, C. tropicalis, C. viswanathii, Coccidioides immits, Cryptococcus albidus, C. neoformans, Histoplasma capsilatum, Paecilomyces variotii, Paracoccidiodis brasiliensis, Pseudoallescheria boydii, Schizophyllum spp., and Sporothrix schenckii .
Cerebral toxoplasmosis is a reactivated latent infection. During the initial infection, which may be contracted (e.g., by consuming raw meat) the pathogen penetrates through the intestinal wall and, via hematogenous dissemination, invades the skeletal muscle and the CNS where it forms cysts. Infected individuals usually have no symptoms (see also ).
Due to a compromised immune response, in non immunocompetent individuals, tachyzoites are released and neurons are infected, leading to the development of centrally necrotizing granulomatous lesions in the CNS.Seroprevalence for Toxoplasma gondii in individuals with HIV ranges from 10 to 80% with the highest proportion in African countries .
Laboratory findings: PCR on serum or CSF for the detection of Toxoplasma gondii genome. Serology on serum is of little value due to the high level of infection in the population. Approximately 50% of patients have a pathologic antibody index due to intrathecal antibody synthesis. Some patients have blood-CSF dysfunction, a smaller number have pleocytosis.
This is the most common type of mycosis with selective involvement of the CNS. The infection is acquired by inhaling Cryptococcus sp. (e.g., from bird droppings). The disease affects predominantly patients with AIDS or those on long-term corticosteroid therapy and chemotherapy. Cryptococcosis can lead to acute, subacute and chronic meningitis or, less commonly, meningoencephalitis.
Neurocandidiasis is usually caused by Candida albicans. The fungal pathogen is present on the skin and in mucous membranes. CNS infection occurs via hematogenous dissemination. Candida sp. often produces small subcortical micro abscesses; meningitis or vasculitis is rare. Refer to
Fungi of the Aspergillus sp. are often present in composted soil. In hospitals, the fungi are present in the air mainly during construction work. Aspergillus sp. is inhaled and manifests primarily in the lungs, from where it spreads to the CNS via hematogenous dissemination, often resulting in solitary or multiple brain abscesses, less frequently in granulomas and rarely in meningitis and vasculitis. Aspergillosis runs a subacute to chronic course. Refer to .
Laboratory findings in fungal meningitis: pleocytosis that is often lymphocytic, elevation of total protein up to 250 mg/dL, glucose concentration either normal or increased, lactate concentration can be increased. Because there are typically a small number of fungal cells, if any, fungi are rarely detected using microscopy (india ink 26%), latex agglutination assay (60%) and culture (first lumbar culture 63%, second lumbar culture 89%) for detection of Cryptococcus sp. . Nucleic acid amplification is recommended.
Definition of chronic meningitis is inflammation of meninges, with signs and symptoms persisting for at least 4 weeks without alleviation. Symptoms are headache, lethargy, mental status changes, and fever. The headache is typically constant but nonspecific in location, quality, and temporal pattern. Infectious causes are bacterial, fungal, parasitic, viral neoplastic, chemically, autoimmune and parameningeal infectious causes .
Laboratory findings: CSF cell count is elevated with predominant lymphocytosis. Eosinophils may indicate parasitic or coccidiodal meningitis. The CSF protein level is elevated. Commercially available multi-organism PCR test of CSF for the diagnosis of acute meningitis and encephalitis is less useful.
Chronic infectious diseases of the CNS are frequently not recognized until a CSF investigation is performed; both humoral and cellular responses are observed. Pleocytosis may be absent, as is the case in subacute sclerosing pan encephalitis, for example.
The humoral immune response is frequently predominant in chronic inflammatory processes. The specificity of the diagnostically relevant intrathecal antibodies depends on the underlying cause of the disorder. In chronic infective processes, the antibodies are exclusively directed against the pathogens, e.g. in HIV encephalitis, chronic neuroborreliosis, and neurosyphilis .
In chronic autoimmune diseases of the central nervous system, the immune response is poly specific. For example, antibodies are produced simultaneously against antigens of Measle virus, Rubella virus and Herpes zoster virus (MRZ response) as, for example, in multiple sclerosis and cerebral lupus erythematosus. The antibody synthesis against pathogens can lose its specificity during the late stages of the disease, as is seen frequently in AIDS encephalopathy (e.g., HSV type I), rarely in HTLV-1 myelitis (tropical spastic paraparesis), or in chronic neuroborreliosis.
The clinical presentation of chronic inflammatory disorders of the CNS depends on the location of the lesions and can therefore be quite variable. Occasionally there is a predominance of psychopathological symptoms, which may occur at any age. In general, a wide range of diagnostic tests are necessary to identify the cause of the inflammatory process:
- Is the process limited to the nervous system or is it part of a systemic disorder?
- Is it caused by a pathogen or is there an autoimmune disease?
- Does it primarily affect the cerebral white matter, the gray matter, the meninges, or the peripheral components of the nervous system?
Infection of the CNS with Treponema pallidum occurs via hematogenous spread during the second stage or latency period of the disease. The clinical presentation of neurosyphilis depends on the stage of the disease :
- During stage II (secondary stage), approximately 5% of infected individuals experience a mostly clinically silent isolated meningitis with occasional cranial nerve paresis, vascular syndrome and polyradiculitis.
- During the latent stage, chronic treponemal related encephalomyelitis (asymptomatic neurosyphilis) may develop, leading to inflammatory abnormalities in the CSF with a mixed cellular and humoral response. The first, rather variable symptoms of neurosyphilis usually do not appear until years later, in stage III of the disease.
- In stage III, the meningo vascular form of neurosyphilis is distinguished from the parenchymal form (tabes dorsalis and progressive paralysis). The meningo vascular form occurs after a latency period of 4–6 years and causes meningeal irritation, circulatory disturbances or infarctions. The tabes dorsalis of the parenchymatous form is a chronically progressive radiculoganglionitis with typical clinical symptoms (ataxic gait, areflexia of the lower limbs, lancinating pain). The progressive paralysis corresponds to a cortical encephalitis with the typical symptoms of personality changes, dementia, ataxia, and epileptic seizures.
Since the pathogen reaches all organs via the bloodstream, neurosyphilis is not an isolated infection of the CNS. Therefore, if there is clinical suspicion of neurosyphilis, a negative serum screening test is sufficient for excluding this condition. To confirm CNS involvement in the infection, however, concurrent examination of serum and CSF samples obtained on the same day is mandatory.
Isolated examination of the CSF does not provide any diagnostic information, since the immunoglobulin content and thus the CSF concentration of T. pallidum specific antibodies is influenced by three factors:
- The functional condition of the blood-CSF barrier. Increased barrier permeability leads to increased passage of serum proteins into the CSF and thus a relative increase in these CSF proteins
- The intrathecal synthesis of immunoglobulins can cause a relative increase in the concentration of these proteins in the CSF regardless of the function of the blood-CSF barrier
- The serum immunoglobulin concentration. Any increase in the immunoglobulin level or increase in the titers of specific antibodies leads to an elevation in the concentration of these proteins in the CSF.
Intrathecal T. pallidum specific antibodies
To determine whether there is intrathecal synthesis of T. pallidum specific antibodies, the CSF/serum index is calculated (ITpa, intrathecal antibody index) (). In the samples (serum and CSF), the IgG concentration (mg/L) and the T. pallidum specific IgG antibodies (titer or ELISA absorption) are determined based on the assumption that the ratio of T. pallidum specific IgG to total IgG is identical in serum and CSF if the antibodies originate exclusively from the serum.
A ratio of 1.0 with a variation of 0.5–2.0 results in the normal case. If there is intrathecal synthesis of pathogen specific IgG in the CNS, this value increases to ≥ 3.0.
The formulas can also be used to determine whether there is intrathecal IgM antibody synthesis provided that sufficiently sensitive tests are used for determining total IgM and pathogen specific IgM in the CSF.
The detection of a specific intrathecal IgG antibody synthesis does not imply the diagnosis of active neurosyphilis, since this phenomenon remains detectable for years, and in many patients for life, even after adequate therapy. An overview of common constellations and their interpretation is shown in .
For assessment of disease activity, non-immunologic CSF parameters must also be considered.
The CSF findings depend on the stage of disease:
- Stage II: lymphocytic pleocytosis up to approximately 300 cells/μL, normal blood- CSF barrier function or mild barrier dysfunction, low rate of intrathecal immunoglobulin synthesis
- Stage III: cell count may be normal, total protein may be markedly elevated due to blood-CSF barrier dysfunction and a high rate of intrathecal IgG synthesis, sometimes with a three class response (IgG, IgA, IgM).
The diagnosis of neurosyphilis is confirmed by the detection of intrathecal treponemal antibody synthesis.
Differences between the two forms of neurosyphilis are found in the extent of the intrathecal Ig synthesis:
- Meningo vascular form; intrathecal IgG synthesis, frequently with normal blood-CSF brain barrier function
- Parenchymal form (progressive paralysis); intrathecal IgG synthesis is more intense than that associated with the meningo vascular form and usually there is also marked intrathecal IgM synthesis.
Lyme disease is the most common tick borne zoonotic disease in Europe and North America (see also ). While in North America only Borrelia sensu stricto is found, in Europe there are three human pathogenic genospecies: B. burgdorferi sensu strictu, B. garinii and B. afzelii. In 75% of cases, an infected tick bite will lead to infection. In 95% of infected individuals the infection is clinically inapparent and resolves spontaneously, although some patients develop specific antibodies. Approximately 5% of those infected become ill, the first clinical symptom usually being erythema migrans. Lyme disease is a multi systemic disease that runs in stages. It may either go through all stages (which is rare), skip stages, or show initial symptoms in any stage .
- Stage I: early localized infection, with organ manifestations of erythema migrans in up to 75% of cases or borrelial lymphocytoma in up to 3% of cases. Some infected individuals have unspecific symptoms such as fever, myalgias and arthralgias due to hematogenous spread of the pathogen, while others are symptom free. Stage I resolves completely without antibiotic treatment in 90% of cases.
- Stage II: this stage involves acute organ manifestations and commences 2–10 weeks after initial infection. After the erythema migrans rash has spontaneously resolved, the infection disseminates to the CNS (meningitis, meningoradiculitis, meningoradiculo-myeloencephalitis, cerebrovascular forms), skeletal muscle (myositis), eyes (ocular borreliosis) and internal organs (arthritis, carditis, hepatitis). Stage II usually resolves spontaneously within six months.
- Stage III: if there was no spontaneous recovery in stage II and the course of the disease was chronically progressive, then stage III is present. This stage is characterized by chronically destructive disease processes without a tendency to spontaneously resolve.
The following nerves and organs are affected:
- Joints (Lyme arthritis) with a frequency of organ manifestation of 30% in the USA and 8% in Europe
- Skin (acrodermatitis chronica atrophicans) with a frequency of organ manifestation of 1–2%.
- Meningoradiculitis is a prominent feature of Lyme neuroborreliosis and the most extracutaneous manifestation. Meningoradiculitis includes meningitis, radicular pain, and palsies of cranial nerves, in particular the peripheral facial nerve.
The criteria include
- neurological symptoms
- cerebrospinal pleocytosis
- presence of intrathecally produced antibodies
- if two criteria are fulfilled the Lyme neuroborreliosis is considered positive.
Many neurological clinics order serum test and CSF analysis of Borrelia infection in addition to the recommended criteria. In a prospective follow-up study of patients investigated for Lime neuroborreliosis showed elevated CSF levels of the inflammation markers sTrem2 (soluble triggering receptor expressed on myeloid cells) and the CXCL13 which were elevated in Lyme neuroborreliosis and declined in at follow-up after antibiotic treatment.
Borrelia infection can be demonstrated indirectly by determining specific antibodies in the serum using an ELISA test 4–6 weeks following initial infection. A positive ELISA screen is followed by an immunoblot test. This procedure is necessary, because the commercial ELISAs from different manufacturers have different analytical sensitivities and specificities. The criteria for a positive interpretation of immunoblots using full antigen of B. burgdorferi are shown in .
Antibodies can persist for decades and are therefore not an indicator of acute infection. Approximately 5% of the population have specific antibodies. Serologic tests cannot differentiate between recent infection and a residual titer, because neither the presence of IgM antibodies nor the antibody titer level are critical. In approximately 50% of cases the antibody test may be negative during stages I and II due to a delayed immune response.
Cell count of 30–1200/μL, lymphocytic, partly with plasma cells. Blood-CSF barrier dysfunction and IgM dominant immune response. An intrathecal Ig response must always be evaluated together with other protein analytical and CSF serologic results (presence of blood-CSF barrier dysfunction, lymphocytic pleocytosis). A specific intrathecal antibody synthesis can persist for months or even years (CSF residual titer), even after the neuroborreliosis has been sufficiently treated and resolved. By the same token, patients with a short disease duration (< 4–6 weeks) may still be antibody negative in the CSF despite having blood-CSF barrier dysfunction and inflammatory pleocytosis. It must also be taken into account that in particular the B. garinii as the main causative agent of neuroborreliosis may not be detected equally well by all serologic assays, especially not by those that only use B. afzelii or B. burgdorferi as an antigen source.
During antibiotic therapy a significant decrease in pleocytosis is a sign of recovery. An intact blood-CSF barrier, in particular, is an important indication that no disease is present which requires therapy. Intrathecal Borrelia antibody synthesis and the humoral three class response with IgM predominance can still be detected years after the neuroborreliosis has completely resolved.
The essential question arising during the investigation of a round lesion is whether inflammation or a tumor is present. Frequently the CSF examination can provide the answer. In acute inflammatory processes the findings depend on the stage of the disease.
A brain abscess is among the differential diagnosis of any round lesion. During the early stage, however, only a limited inflammatory infiltrate (phlegmon) is visible. A ring structure with a dense margin is not visible until tissue destruction with liquefaction has occurred. The bacteria may be included in infected emboli or migrate in from surrounding tissue. Both the mode of transmission of the infection as well as the stage of development have an impact on the CSF findings.
The clinical suspicion of brain inflammation is strengthened by the finding of an increased serum C-reactive protein (CRP) level which makes necessary the immediate start of antibiotic treatment.
A forming abscess initially causes a purely cellular response in the CSF which can be granulocytic but also lymphocytic or monocytic in nature. Pleocytosis may be mild or initially even be absent, especially if the infiltrate is remotely located i.e., far from the CSF space accessible by (lumbar) puncture, for instance within the frontal cerebral lobe. Only after break through into the subarachnoidal space will purulent meningitis be present. At that point it will already be too late for optimal antibiotic treatment. The local production of immunoglobulin cannot be detected until the second week.
If evidence of intrathecal antibody synthesis is already present despite the acute onset of symptoms, it is presumed that bacteria have slowly invaded the parenchyma and caused a plasma cellular response. IgA in particular is occasionally synthesized at a remarkably high rate, particularly in association with tuberculomas.
These abscesses can result in a lymphocytic response in the CSF space, occasionally however only a bood-CSF barrier disturbance can be detected, especially if the abscess is in the spinal cord.
Primary brain tumors
Primary brain tumors rarely cause leptomeningeal involvement and thus barrier dysfunction. Most commonly, this is the case with medulloblastomas, ependymomas, pineal or suprasellar germinomas, and pinealoblastomas.
Secondary brain tumors or CNS metastases are brain tumors that have metastasized from another primary tumor. Carcinomatous meningitis is present when the tumor has metastasized to the leptomeninges. The incidence is 40–50% in breast cancer, 25% in lung cancer, and 10% in melanoma. Clinical symptoms are headache, focal or generalized seizures, and impaired vigilance.
Laboratory findings in primary brain tumors
CSF cell count is variable; tumor cytology is the most important investigation. Total protein is elevated, especially in intracranial and spinal neurinomas. Lactate is also frequently elevated; intrathecal immunoglobulin synthesis, especially of IgG and IgA, is seen in 20% of patients. Selective IgM synthesis raises the suspicion of non Hodgkin lymphoma.
Tumor marker levels are usually higher in CSF than in serum. Other relevant tumor marker findings are as follows:
- LD in germinoma
- β-hCG and α1-fetoprotein in embryonic cancer and yolk sac tumors
- Serum tests of importance for the differential diagnosis of brain tumors include prolactin, TSH, hGH, FSH and LH.
Laboratory findings in brain metastases
Most patients have a blood-CSF barrier dysfunction and markedly elevated total protein. Often there is an accompanying inflammatory response with an elevated CSF/serum IgG ratio and oligoclonal banding. Of importance is the detection of tumor cells in the CSF. The detection of intrathecal synthesis of CEA is highly specific for a carcinoma, but not for the organ localization of the primary tumor.
Intrathecal production may also be detected for:
- CEA in breast cancer and colorectal cancer
- CA 15-3 in breast cancer
- NSE in small cell lung cancer
- CYFRA 21-1 in non small cell lung cancer.
Immediately following a brain infarction the CSF is normal. Only on the 2nd–4th day are signs indicative of mild to moderate blood-CSF barrier disturbance found. If inflammatory CSF changes are present, a purely cellular response suggests a septic embolism in which the first embolus led to the occlusion of a large vessel. However, if a humoral response is already present during the acute stage, it is likely that a clinically silent inflammatory process preceded the infarction. In this context it is important to consider:
- Multiple micro emboli in clinically silent regions of the brain as sequelae of bacterial endocarditis (embolic focal encephalitis)
- Arteritis with secondary thrombosis, such as is seen in vascular syphilis or autoimmune vasculitis.
- Refer to ).
Polyneuropathies are due to acquired or hereditary disorders. Acquired disorders can be metabolic (diabetes mellitus), immune mediated (Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy), infectious (Herpes zoster virus), toxic (alcohol), or paraneoplastic (sensory neuropathy).
GBS predominantly is a motor polyneuritis that evolves over 10–14 days, and progressive flaccid tetra paresis with areflexia . GBS is likely triggered by an infection, since most patients have a history of respiratory or gastrointestinal infection several weeks prior to onset. There are associations with infectious pathogens such as Campylobacter jejuni, Cytomegalovirus, Epstein Barr virus, Mycoplasma pneumoniae and HI virus.
- Acute inflammatory demyelinating polyneuropathy (AIDP), the classic sensorimotor GBS, which is the most common form of the disease in Europe and North America
- Acute motoric axonal neuropathy (AMAN), a primary axonal GBS which is frequently seen in China and Japan
- Acute motoric axonal and sensoric neuropathy (AMSAN), a primary axonal GBS with sensory involvement, which is frequently seen in China and Japan
- Miller-Fisher syndrome (MFS), which is characterized by ophthalmoplegia, ataxia and areflexia.
Blood-CSF barrier dysfunction with marked to extreme elevation of total protein with normal or initially only mildly elevated cell count up to 50 leukocytes/μL, predominantly lymphocytes and monocytes. Qalb > 50 × 10–3, can be normal during the first week of disease, often reaching its peak only during the third week. CSF glucose and lactate levels are normal. Serum often shows anti-ganglioside antibodies in GBS .
CIDP is characterized by an ascending, progressive, symmetric sensorimotor weakness of the distal muscles . It evolves over 8 weeks. There is decreased sensitivity and hypo- or areflexia. Nerve conduction studies provide the main criteria for diagnosing demyelination.
Total protein in the CSF > 45 mg/dL with a leukocyte count < 10/μL.
Parkinson's disease is a neurodegenerative disorder characterized by the inoperability of dopamine producing neurons of the substantia nigra. Clinical trials for neurodegenerative diseases are increasingly utilizing measurements of posttranslational modifications and pathological isoforms as surrogate markers of target engagement. Using a panel of high-sensitivity single molecule array (Simoa), refer to and comparing Parkinson's disease to healthy controls alpha synuclein, pSer129 alpha synuclein, DJ-1 and C-reactive protein were reduced in patients with Parkinson’s disease in CSF .
- Multiple sclerosis (MS)
- Aquaporin 4 IgG seropositive neuromyelitis optica spectrum disorder
- Myelin oligodentrocyte glycoprotein antibody-associated disease (MOGAD)
MS is the most common chronic inflammatory and demyelinating disease of the CNS and has an onset between the age of 15 to 50 years. 2.8 million people live with MS diagnosis around the world. Suggestive features of the disease include periods of attacks and periods of clinical remission or a progressive course from onset with a gradual decline in walking distance over time.
- radiologically isolated syndrome (MRI findings without clinical symptoms)
- clinically isolated syndrome (representing the first attack of MS)
- relapsing remitting MS (in 85% of patients MS presents in the form of an attack with acute or subacute symptoms that later improve)
- primary progressive MS
- secondary progressive MS (in a time frame between 10 to 30 years from MS onset, most patients will develop a slow decline in the ability to walk usually from myelopathy).
- myelitis (numbness with or without mild weakness, imbalance or bowel/bladder dysfunction)
- optical neuritis (ON) (monocuclar vision loss with pain with eye movements)
- brainstem or cerebellar syndrome (diplopia that is painless and resolves with unilateral eye closure or imbalance from ataxia).
An important pathogenic principle of MS is the migration of autoreactive leukocytes into the perivascular cerebrospinal fluid spaces, where the disease is most active. The inflammation and the presence of autoantibodies against myelin and other antigens of the nerve cells cause damage to the myelin sheaths, resulting in reduced conduction of electrical impulses along the nerves .
Convergent epidemiological and laboratory results are consistent with a polygenic model of inheritance, while the data also support the view that MS susceptibility rests on the action of polymorphisms common in the population. The strong HLA association with MS supports the idea that MS is, at its core, an antigen-specific autoimmune disease. HLA-DRB1*15:01 has the strongest effect with an average odds ratio of 3.08.
CSF cell count
Half the patients do not have an elevated cell count, others have mild pleocytosis in the range of 5–30 cells/μL, predominantly lymphocytes and monocytes.
Glucose, lactate: normal.
Total protein is normal or elevated up to approximately 0.8 g/L, a Qalb up to 10 × 10–3 indicates mild blood-CSF barrier dysfunction in some cases.
Intrathecal IgG synthesis
The hallmark for the early diagnosis of MS is the finding of a constant intrathecal synthesis of IgG . The latter is the sign of chronic inflammation of the CNS and is analyzed quantitatively in the CSF/serum quotient diagram ().
Oligoclonal bands are, however, also detected in infectious and autoimmune diseases of the CNS () and therefore have low specificity. In MS, the presence of oligoclonal bands indicates a non specific intrathecal immune response.
The intrathecal IgG fraction contains IgG antibodies directed against pathogen associated antigens of Measle virus, Rubella virus and Varicella virus without a specific stimulus. The rate of intrathecal synthesis of these antibodies is determined by calculating the pathogen specific antibody index (AI). In 10–30% of cases, a pathologic antibody index against Chlamydia sp., Borrelia sp., Herpes simplex virus and Toxoplasma gondii can be detected. The diagnostic sensitivity of the MRZ response and other tests for MS is shown in .
The CSF changes are very constant and are present even in remissions. As is the case in all chronic inflammatory processes of the CNS, including MS, there is no relationship between the extent of the changes in the CSF and the severity or the progression of the disorder. Thus, marked intrathecal IgG synthesis may be related with only mild symptoms of the disease, while normal CSF findings may occur despite severe, progressive, disease.
In rare cases the inflammatory demyelination process is limited to the hemispheres, and psychiatric symptoms predominate, such as endogenous or organic psychoses, changes in personality and dementia. Epileptic seizures occur more frequently. In the encephalitic form of MS, more cases with pleocytosis of up to 200 leukocytes/μL and blood-CSF barrier dysfunction with a Qalb of up to 20 × 10–3 are common.
If, in the case of monophasic disseminated encephalomyelitis, doubts remain about whether it represents a viral infection or the first flare-up of MS, the CSF should be re-examined a year later. If the intrathecal IgG synthesis is quantitatively unchanged, MS can be reliably confirmed.
In cases of CNS involvement in systemic autoimmune disorders, such as SLE or Sjögren’s syndrome, inflammatory changes in the CSF are usually present. In individual cases, however, MS cannot be differentiated from other conditions by CSF analysis. Intrathecal DNA antibodies are found in MS while the MRZ response is found in systemic autoimmune disorders, although more rarely.
The diagnosis of MS is generally doubtful in the face of the following CSF findings:
- A cell count > 40/μL
- A pure blood-CSF barrier dysfunction without oligoclonal banding
- A higher rate of IgA or IgM relative to IgG synthesis.
NMOSD is a condition that falls under inflammatory CNS demyelating diseases, defined by the aquaporin 4-antibody positive neuromyelitis optica spectrum disorders. CSF and/or serum samples are tested for AQP4 antibodies if the MS presentation is atypical. In NMOSD clinical attack types are most commonly tranverse myelitis, optical neuritis, and area postrema syndrome. Attacks seem to be more severe than MS with less recovery, and thus a single attack can lead to permanent blindness or paraplegia. Disability (intractable nausea, vomiting) accumulates with each attack, but it differs from MS in that it lacks a progressive course .
MOGAD is a condition that falls under inflammatory CNS demyelating diseases, defined by the myelin oligodentrocyte glycoprotein (MOG) antibodies. Manifestations include optical neuritis, acute disseminated encephalomyelitis, myelitis or cerebral encephalitis or cortical encephalitis.The attacks are more severe than MS but recover better. About half of the cases do not develop a recurrence. MOGAD lacks the progressive course of MS .
Worldwide AD is the most common of the neurodegenerative diseases. Typically the symptoms of disease begin with mild memory difficulties and evolve towards cognitive impairment, dysfunctions in daily activities and several aspects of cognition. By the time that AD is clinically diagnosed, neuronal loss and neuropathologic lesions occur in many brain regions.Neuropathological hallmarks include amyloid β (Aβ)-containing plaques and tau-containing neurofibrillary tangles throughout the brain .
A distinction is made between:
- The familial form, which accounts for 5–10% of AD cases. It usually develops between the ages of 30–50 years and is due to autosomal dominant mutations in the gene APP, which encodes the amyloid precursor protein APP, and mutations in the genes PSEN1 and PSEN2, which encode the protein presenilin.
- The sporadic form, which accounts for 90% of AD cases. The genetic variant encoding apolipoprotein e4 (APOE) is a mutation associated with the late onset form of AD .
AD is a progressive disease, affecting about 14 million people in Europe and the United States of America, including 43% of the population aged > 85 years . From age 65, the incidence and prevalence of AD are clearly associated with age. AD has a 10–20 year preclinical phase, during which the neurodegenerative processes progress.
- Pre-symptomatic: an insidious and silent preclinical phase. AD pathology may be present for up to 20 years before clinical disease onset. Therefore, healthy individuals and non-AD dementia patients may have AD pathologic features.
- Prodromal: clinically characterized by mild cognitive impairment (MCI). Approximately 10–20% of cases with MCI progress to AD per year. The MCI can be seen as transitional zone between the cognitive decline in normal aging and cognitive dysfunctions of AD dementia. However, other causes of MCI must be differentiated e.g., cerebrovascular disease, polypharmacy, depression, excessive alcohol/drug use and neurodegeneration .
- Symptomatic with dementia: implies the existence of multiple cognitive symptoms severe enough to interfere with daily functioning e.g., memory, language, spatial orientation, behavior, and personality. Atypical AD presents with logopenic aphasia, posterior cortical atrophy. About 6–14% of AD cases are the proportion of the frontal variant.
The core CSF biomarkers of neurodegeneration (T-tau, P-tau, and Aβ42) are strognly associated with Alzheimer’s disease.
A key neuropathologic feature of AD is extracellular amyloid plaques comprising β-amyloid (Aβ) peptides including lengths of 42 and 40 amino acids (Aβ42 and Aβ40, respectively). The biomarkers determine whether or not an individual is in the Alzheimer’s continuum. Low CSF Aβ42 is the best considered marker of a pathologic state that is associated with amyloid plaque formation and not a marker of amyloid plaque load. The Aβ42 concentration or the Aβ42/Aβ40 ratio is a valid indicator of the abnormal pathologic state associated with cerebral Aβ.
Exploratory analyses of cognitively normal cohort followed up for a median of 3.1 years suggest that elevation in baseline brain amyloid level compared to with normal brain amyloid level, is associated with higher likelihood of cognitive decline .
Total tau protein
Total tau (T-tau) is not specific for AD and a nonspecific indicator of damage that may derive from a variety of etiologies, for example cerebrovascular injury (e.g., in stroke, brain trauma, non-AD dementia, and Creutzfeldt-Jacob disease). T-tau reflects the intensity of neuronal damage at a specific point, whereas elevated P-tau reflects an abnormal pathologic state associated with hyper phosphorylated tau formation.
P-tau (hyper phosphorylated tau): is best considered a biomarker of a pathologic state that is associated with hyper phosphorylated tau formation and not a pathologic measure of pathologic tau deposits. The only disorder that consistently shows an increase in CSF P-tau is AD. The combination of a decrease in CSF concentration of Aβ42 and an increase in P-tau can discriminate AD from controls with a sensitivity and specificity of over 85%.
- The lumbar puncture should be performed in the morning after an overnight fast
- The CSF is collected in polypropylene (instead of glass or polystyrene) tubes to minimize protein adsorption to the walls of the collection vessels
- The CSF sample should be stored at room temperature for a short period only. If the analysis is not performed on the same day, the sample should be deep frozen at –70 °C or –80 °C.
Cerebrospinal fluid (CSF) samples measured with immunoassays, e.g., enzyme linked immunosorbent assay (ELISA) are used . The variability of CSF biomarker concentrations between the laboratories is 20–35%. Large interassay and interlaboratory variations have serious consequences, because results from different laboratories cannot be directly compared. An automated system is the second-generation Elecsys CSF immunoassay system that measures β-amyloid (1–42), Phospho-Tau (181P) and Total Tau. All three immunoassays have potential utility in clinical routine to aid diagnosis of Alzheimer disease .
- Aβ42: 675 (182–1879) ng/L
- T-tau: 280 (42–915) ng/L
- P-tau: 51 (16–156) ng/L
There is a large variation how different laboratories establish cutoffs for biomarker concentrations to differentiate patients with Alzheimer’s disease from controls. In a systematic review from 4,521 records from PubMed and 624 from Web of Science and 231 articles comprising 15,699 patients with AD and 13,018 controls the accuracy of biomarkers for the diagnosis of AD was investigated. The generation of change (i.e., the ratio of the mean biomarker concentration between cohorts) was used. The study showed that the biomarkers of neurodegeneration (T-tau, P-tau, and Aβ42) were strongly associated with AD. Refer to .
According to the National Institute on Aging and Alzheimer’s Association work group (NIA-AA) a scheme which is labeled AT(N) recognizes three general groups of biomarkers based on the nature of the pathologic process that each measures. Only biomarkers that are specific for hallmark Alzheimer’s disease proteinopathies (i.e., Aβ and pathologic tau) are considered.
- Biomarkers of Aβ plaques (labeled A) are cortical amyloid PET ligand binding or low CSF Aβ42.
- Biomarkers of fibrillar tau (labeled T) are elevated CSF phosphorylated tau (P-tau) and cortical tau PET ligand binding.
- Biomarkers of neurodegeneration or neuronal injury (labeled N) are CSF T-tau or FDG PET hypometabolism and atrophy on magnetic resonance imaging (FDG = 18F-Fluorodesoxyglucose, Tracer in Positron-Emission-Tomography).
The AT(N) system is designed with both a CSF and an imaging biomarker in each of the three biomarker groups. Refer to:
The proportion of amyloid PET-positive clinically normal individuals by age nearly perfectly paralleles the increasing age specific prevalence of individuals clinically diagnosed as AD dementia 15–20 years later. The first biomarkers to become abnormal in carriers of deterministic AD mutations are those of Aβ. A causal upstream role for Aβ in the pathogenesis of AD is suggested .
- Alzheimer’s pathologic change: abnormal amyloid PET scan or low CSF Aβ42 or Aβ42/Aβ40 ratio
- Alzheimer’s disease: Aβ and pathologic tau is present.
Alzheimer’s pathologic change and AD are not regarded as separate entities but earlier and later phases of Alzheimer’s continuum.
Aβ biomarkers determine whether or not a person is in Alzheimer’s continuum. Pathologic tau biomarkers determine if someone who is in Alzheimer’s continuum has Alzheimer’s disease because both Aβ and tau are required for a neuropathologic diagnosis of disease .
Reports have demonstrated that high precision assays for plasma Aβ42/Aβ40 are strongly predictive of brain amyloidosis . According to a study plasma Aβ42/Aβ40, especially when combined with age and APOE e4 status, accurately diagnoses brain amyloidosis and can be used to screen cognitively normal individuals for brain amyloidosis. Individuals with a negative PET scan and positive plasma Aβ42/Aβ40 are at increased risk for converting to amyloid PET imaging positive. Plasma Aβ42/Aβ40 could be used in prevention trials to screen for individuals likely to be amyloid PET positive and at risk for Alzheimer disease dementia.
Dementia has a complex pathologic and clinical heterogeneity. Neurodegeneration associated with Alzheimer’s disease accounts for 50–60% of cases of dementia in elderly patients, neuropathologic autopsy studies reported dementia with Lewy bodies in 15–25% of cases and vascular dementia is considered responsible for most remaining cases .
Lewy bodies are intracytoplasmic spherical, eosinophile neuronal inclusion bodies. The areas of predilection for Lewy bodies are brainstem, subcortical nuclei, and limbic cortex. Some Alzheimer pathology, predominantly β-amyloid deposition and diffuse plaque formation, is a common feature of most demented cases with cortical Lewy bodies. The central features required for dementia with Lewy bodies are described in Ref. . shows the concentrations of AD biomarkers in selected forms of dementia.
Creutzfeldt-Jacob disease (CJD) is a representative of a group of rare transmissible and genetic neurologic disorders caused by prions (proteinaceous infectious only), small misfolded proteins that alter the physical conformation of adjacent proteins and lead to neurological dysfunction. The major process underlying the pathogenesis of prion diseases is conformational change of the normal neuronal host protein to its pathologic form .
- aggregates of abnormal host-derived prion protein
- histopathological triad of spongiform changes (small, round or oval, vacuolated empty spaces in the neuropil surrounding neurons)
- neuronal loss
- gliosis without inflammation.
Prion diseases arise sporadically, genetically, or by infectious transmission. Sporadic CJD accounts for 80–90% of annual human prion disease mortality (about 1–2 per million individuals) . The average age of patients is 65 years.
The main features of CJD is rapidly progressive cognitive decline and myoclonus. During the early phase of the disease, individuals experience personality changes as well as visual disturbances and cerebellar symptoms. In advanced stages patients are akinetic with severe spasticity and rigidity.
Variant CJD (vCJD)
The prototype of all prion diseases, scrapie in sheep causes bovine spongiform encephalopathy (BSE). The emergence of a new variant form of von Creutzfeldt-Jacob disease (vCJD) in young people indicates that BSE has spread to humans by dietary exposure .
The variant type differs from vCJD in its clinical course and pattern of changes in the brain. The variant type typically affects younger patients (average age of 29 years) and has a duration of illness that is almost twice as long (14 months) as that of classic vCJD. The early stage is characterized by psychiatric symptoms such as behavioral disturbances, depression and anxiety. All patients show ataxia and develop progressive dementia.
- 14-3-3 proteins, a family of regulatory proteins (diagnostic sensitivity 85%, specificity 84%)
- Tau protein, associated with microtubules in neuronal and glial cells (diagnostic sensitivity 86%, specificity 88%)
- Neurofilament light chains.
The diagnostic accuracy of CSF protein markers for sporadic CJD are shown in:
Encephalitis an often serious neurological disease that is characterized by the rapid development of cognitive decline, focal neurological deficits, psychiatric symptoms and/or seizures. Although classically attributed to infection, epidemiologic data reveal that an autoimmune basis for encephalitis occurs with similar frequency. Neural antibody testing for suspected autoimmune encephalitis is most often considered for patients with subacute-onset central nervous system dysfunction of unclear etiology
- Antibodies targeting intracellular neuronal proteins. They are classically found in patients with immune mediated neurological dysfunction that is triggered by underlying malignancy. This type of neurological disease is referred to as a paraneoplastic neurological syndrome. A typical intracellular neuronal antibody is the Anti-Neuronal Nuclear Antibody (ANNA).
- Antibodies that bind to extracellular cell-surface or synaptic neural antigens. The antibodies are pathogenic and can directly cause neurological dysfunction . As example N-methyl-D-aspartate receptor (NMDAR), glial fibrillary acidic protein (GFAP), LGI 1, and Contactin associated protein-like 2 (CASPR2) antibodies are all associated with autoimmune encephalitis.
In general, antibody detection in CSF has higher clinical specificity than serum. With few exceptions submision of both serum and CSF for neural antibody testing in cases of suspected autoimmune encephalitis is recommended. Ordering neural antibodies as a panel rather than individually is usually preferable, because phenotypic overlap among these antibodies can be substantial .
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% of CSF
AChE, acetylcholinesterase; NSE, neuron specific esterase
* Model diseases for the construction of empirical immunoglobulin differentiation curves.
CNS, central nervous system; ND, not determined; 1) Usually associated with oligoclonal banding; 2) Neurosarcoidosis; 3) Prominent IgA synthesis in adrenoleukodystrophy
Group of diseases
* The values are recommendations only. Reference intervals in the true sense only exist for the CSF/serum ratios.
Typical constellation of CSF parameters
Clinical and laboratory findings
Diagnostic specificity (%)
Diagnostic sensitivity (%)
* Immunocompromised patients (AIDS, organ transplantation)
The disease is classified into the clinical categories A, B,C and the clinical stages 1, 2, 3. A, asymptomatic; B, symptomatic; C, AIDS. Stage 1, asymptomatic or generalized lymphadenopathy; stage 2, recurrent infections (herpes zoster), recurrent ulcerations; stage 3, weight loss > 10%, acute necrotizing ulcerations. Patients of categories A3, B3 and C1–C3 are considered to have AIDS. The above are only examples of the categories and stages.
Clinical and laboratory findings
Explanations: E, elevated; N, negative; P, positive; AI, antibody index; * Or comparable screening test; ** fractionated IgM FTA-ABS test or other IgM antibody assay with comparable sensitivity and specificity; TPHA, Treponema pallidum hemagglutination test
IEF, isoelectric focusing
Ratio of AD/C
The ratio of the mean biomarker concentration between the two cohorts is formed
Only A: pathological Alzheimer changes and non-specific neurodegeneration, but no Alzheimer’s disease (AD);
A+ T+ (N)+ or –: all criteria of AD
A+ T– (N)+: Alzheimer changes, non-specific neurodegeneration, no AD
A– T+ (N)– or A– T– (N)+ or A– T+ (N)>: No Alzheimer changes, no AD
Data expressed as median and 95% CV; LR, likely hood ratio
Clinical and laboratory findings
Figure 46-1 Concept of a functionally independent blood-CSF barrier which is basically a filtration barrier for hydrophilic molecules. The blood-CSF barrier is practically impermeable to hydrophilic molecules but can very easily be crossed by lipophilic molecules of less than 500 Da. The CSF composition does not allow a statement to be made on the permeability of the blood-brain barrier; CSF and extracellular fluid are entirely different in their composition. The parenchymal area from which proteins diffuse into the free CSF space and are detectable in the lumbar region by sensitive techniques, is at the most 30 mm wide (shaded area). Each barrier has specific carriers for transfer (e.g., amino acids, metabolites, glucose).
Figure 46-2 Plasma filtration as the basis for CSF production. The steady state CSF/serum concentration ratios of lumbar CSF are depicted. The curve shows the correlation of the concentration decrease of hydrophilic components between circulating blood and CSF as a function of the hydrodynamic molecular radius. Abbreviations: Asp, asparagine; Glu, glutamine; Lys, lysine; Meth, methionine; Ser, serine; α1Ach, α1-antichymotrypsin; α1At, α1-antitrypsin; Alb, albumin; Cp, ceruloplasmin; Hpx, hemopexin; α2HSGp, α2Hs-glycoprotein; R, ratio
Figure 46-4 CSF/serum quotient diagram for IgG, IgA, IgM according to Reiber . The upper cutoff limit between intrathecal Ig synthesis and Ig passing from the serum is depicted as a continuous line. Values above this line are indicative of intrathecal Ig synthesis. The extent of intrathecal synthesis in % is represented by the broken lines. Because of the age dependence of the blood-CSF barrier, the thick vertical lines show the upper reference limit of Qalb × 10–3 in relationship to age; Qalb = 5 (up to 15 years), Qalb = 6.5 (up to 40 years), Qalb = 8.0 (up to 60 years). Determination of the cutoff limit lines see .
The following ranges are represented in the upper diagram:
1 = reference range
2 = blood-CSF barrier dysfunction
3 = local Ig synthesis with blood-CSF barrier dysfunction
4 = local Ig synthesis without blood-brain barrier dysfunction
5 = analytical error
Figure 46-5 Determination of the antibody index (AI) in relationship to total IgG by means of titration. Serum and CSF are diluted to a level of 1 mg/dL and the equivalent IgG concentrations are read out at an ELISA absorption (A) of 0.6 corresponding to an AI of 4.5.
Figure 46-6 CSF/serum quotient diagram for IgG, IgA, IgM over a course of time in a patient with neuroborreliosis . The patient underwent (lumbar) puncture six times during the course of 83 weeks. The dynamics of decrease of the Borrelia specific IgM antibodies, which originally accounted for 80%, is pronounced.
■ = primary diagnostic puncture, ● = subsequent punctures.
Figure 46-7 Immunoblots of CSF and serum after isoelectric focusing (IEF) for local IgG synthesis. Typical pattern in multiple sclerosis: there are IgG bands in both CSF and serum. Several additional bands are present in the CSF (oligoclonal band pattern).
Figure 46-8 Prototypical course of acute inflammatory diseases of the central nervous system. PN, polynuclear leukocytes per μL; MN, mononuclear leukocytes per μL; Qalb, CSF to serum albumin quotient; IgG (mg/dL)