Cerebrospinal Fluid Analysis

Characteristics of normal spinal fluid are below:[1, 2, 3, 4]

• Total volume: 150 mL                                     
• Color: Colorless, clear, like water
• Pressure: < 20 cm H ₂O                 
• Osmolarity at 37°C: 281 mOsm/L
• Specific gravity: 1.006 to 1.008
• Acid-base balance:                                                            

  • pH: 7.28-7.32                                                
  • Pco₂: 47.9 mm Hg                                          
  • HCO₃-: 22.9 mEq/L                                         
• Sodium: 135-150 mmol/L                                                
• Potassium: 2.7-3.9 mmol/L                                                       
• Chloride: 700-750 mg/dL                                                      
• Calcium: 2.0-2.5 mEq/L (4.0 to 5.0 mg/dL)                                  
• Magnesium: 2.0-2.5 mEq/L (2.4 to 3.1 mg/dL)                              
• Lactic acid: 10-25 mg/dL                                                    
• Lactate dehydrogenase: 40 U/L or less (adults); 70 U/L or less (neonates)                                      
• Glucose: 50-75 mg/dL in cerebrospinal fluid (CSF) or 60-70% of blood glucose concentration                                                  
• Glutamine: 6-15 mg/dL
• Proteins: 20-40 mg/dL

  • At different levels of spinal tap:     
    • Lumbar: 20-40 mg/dL                
    • Cisternal: 15-25 mg/dL
    • Ventricular: 15-45 mg/dL  
  • Normal CSF proteins concentration in children:
    • Up to 6 days of age: 70 mg/dL
    • Up to 4 years of age: 24 mg/dL
• Electrophoretic separation of spinal fluid proteins (% of total protein concentrations)

  • Prealbumin: 2-7%
  • Albumin: 56-76%
  • Alpha1-globulin: 2-7%
  • Alpha2-globulin: 4-12%
  • Beta-globulin: 8-18%
  • Gamma-globulin: 3-12%
• Oligoclonal bands - absent
• Immunoglobulins

  • IgG: 10-40 mg/L
  • IgA: 0-0.2 mg/L
  • IgM: 0-0.6 mg/L
  • k/l ratio: 1
• Erythrocyte count:

  • Newborn: 0-675/mm³
  • Adult: 0-10/mm³
• Leukocyte count:
• Children: 

  • Younger than 1 year: 0-30/mm³
  • Age 1-4 years: 0-20/mm³
  • Age 5 years to puberty: 0-10/mm³
• Adult: 0-5/mm ³
• Antibodies, viral DNA: None
• Bacteria (Gram stain, culture, VDRL): Negative
• Cancerous cells: None
• Cryptococcal antigen: None

Conditions associated with an abnormal CSF analysis include (but not limited) the following:

• Infectious diseases (eg,  encephalitis,  meningitis)
• Intracranial hemorrhage
• Inflammatory or autoimmune diseases (eg,  multiple sclerosis,  Guillain-Barre syndrome,  meningeal sarcoidosis)
• Primary or metastatic CNS malignancies

Conditions associated with changes in the appearance of CSF

Conditions associated with changes in the appearance of CSF include the following:

• Infectious meningitis – Turbid, milky, cloudy CSF samples
• Hemorrhage or traumatic tap – Xanthochromic CSF samples with increased hemoglobin
• Kernicterus - Xanthochromic CSF samples with increased bilirubin
• Meningeal melanosarcoma – Xanthochromic CSF samples with increased melanin
• Disorders affecting blood-brain barrier - Cloudy CSF samples with increased proteins (above 150 mg/dL) and albumin and IgG

Xanthochromia is the term used for any kind of discoloration of CSF (pink, yellow, orange). Multiple conditions are associated with xanthochromia: traumatic tap, presence of carotene, melanoma and increased bilirubin concentration (bilirubin concentration will also be elevated in serum and patients are often jaundiced) due to liver diseases, hemolytic diseases (also increased free hemoglobin concentration) and inborn errors of metabolism.[2, 3, 5]

Note that normal CSF samples should be colorless, clear, like water. If CSF samples are centrifuged immediately, xanthochromia due to traumatic tap should not occur. However, if CSF samples are carefully centrifuged immediately and the supernatant is still xanthochromic, this indicates that bleeding may have occurred 2-4 hours before sample collection. Furthermore, in about 10% of patients with subarachnoid hemorrhage, the CSF samples might be clear if the samples are collected 12 hours after the hemorrhage occurred.

Typically, high levels of oxyhemoglobin occur in CSF fluid obtained through a traumatic lumbar puncture, in which red blood cells enter the subarachnoid space via direct needle puncture. This frustrates the ability to determine whether xanthochromia, and thus subarachnoid hemorrhage, is present. However, a retrospective study by China et al found that when a repeat lumbar puncture was performed on patients after the initial, traumatic one, it was possible to determine that xanthochromia was absent, thereby ruling out the possibility of subarachnoid hemorrhage. According to the study, the timing of the second puncture must be determined on a case-by-case basis, with repeat punctures in the report being performed an average of 2.4 days after the traumatic puncture. The investigators stated that performing the repeat lumbar puncture too soon (eg, less than 12 hours) after the first could still produce equivocal results, while performing the second puncture too long after the initial one could put the patient at greater risk for morbidity and mortality, due to a missed diagnosis.[6]

Conditions associated with changes in the biochemical composition of CSF

Conditions associated with changes in glucose concentration of CSF include the following:

• Bacterial, fungal, and tuberculous meningitis (but not viral)
• Primary or metastatic meningeal malignancy
• Subarachnoid hemorrhage
• Hypoglycemia

Note that normal concentration of glucose in CSF samples is 45-80 mg/dL or 60-80% of that in the plasma (for glucose plasma concentrations less than 400 mg/dL). Absolute decreased CSF glucose level and especially decreased CSF glucose level in relation with serum are usually associated with bacterial or fungal meningitis. However, in patient with a normal CSF glucose concentration but with increased number of WBC, viral meningitis should be suspected. For accurate interpretation of CSF glucose concentration, serum glucose should be evaluated in serum samples collected about 2 hours prior to spinal tapping (allow time for equilibrium) and all specimens (CSF and serum) should be tested immediately to avoid glycolysis.[2, 3]

Conditions associated with elevated CSF lactate include any condition associated with decreased blood flow or hypoxia (eg, head trauma), such as the following:

• Intracranial hemorrhage
• Cerebral arteriosclerosis
• Hypotension
• Metastatic cancer
• Trauma
• Seizures
• Bacterial meningitis
• Mycoplasma meningitis

Evaluation of lactic acid concentration in CSF is useful for the diagnosis and management of different types of meningitis. Generally, the following guidelines can be applied:

• CSF lactate >35 mg/dL is seen in patients with bacterial meningitis
• CSF lactate 25-35 mg/dL is seen in patients with tubercular and fungal meningitis
• CSF lactate < 25 mg/dL is seen in patients with viral meningitis

RBCs contain high concentration of lactate and LDH. Therefore, xanthochromic CSF samples with elevated hemoglobin and/or RBC can lead to falsely elevated lactate and LDH results.[3]

Conditions associated with elevated CSF LDH include the following:

• Intracranial hemorrhage
• Bacterial meningitis

Conditions associated with elevated CSF glutamine include the following:

Glutamine results from the amination of a-ketoglutarate with ammonia and it represents the main way in which the toxic metabolite ammonia is removed from the CNS. In the conditions in which ammonia accumulates, such as in liver disease, inherited urea cycle disorders, or Reye syndrome, glutamine concentration will raise as well. The normal glutamine concentration in CSF is 8-18 mg/dL. Increased concentration of glutamine in CSF is followed rapidly by signed and symptoms, while at concentration of 35 mg/dL or above, strong seizures and coma can occur. Evaluation of glutamine in CSF is a common practice in the case of patients, especially children, with coma of unknown origin.[2]

Conditions associated with cerebrospinal protein variation

The protein concentration in CSF varies with age and level of tapping (eg, lumbar, ventricular, etc). It correlates well with the concentration of total proteins and different fractions in serum, but they are significantly lower. The predominant fraction in CSF is albumin, similarly as in serum. Decreased total protein concentration in CSF is generally associated with CSF leakage, while elevation of proteins in CSF can be seen in a multitude of conditions.

In addition to medical conditions, protein concentration can be falsely elevated in CSF due to traumatic tap and increased RBC and hemoglobin. Therefore, corrections are commonly applied: if CSF sample is xanthochromic, for every 103 RBC counted, 1.1 mg/dL should be subtracted from the measured total protein concentration of CSF.[2, 3] Online calculators, which calculate corrected protein concentration in CSF taking into account the counted RBC, hematocrit and serum protein concentration, are also available: http://reference.medscape.com/calculator/csf-protein-concentration-correction.

CSF protein fractions and CSF IgG

In certain conditions, such as in multiple sclerosis, evaluation of total proteins in CSF is not sufficient. Evaluation of different protein fractions and various immunoglobulins is necessary.

IgG immunoglobulins can be produced by the plasma cells on both sides of the blood-brain barrier: within CNS and in serum. When IgG fraction of CSF is elevated, the immediate question targets the integrity of the blood-brain barrier. Therefore, evaluations of serum albumin and serum IgG and normalization of IgG concentration of CSF, taking into account these serum concentrations, are necessary.[2, 3, 7]

Quotient of albumin

Albumin is synthetized in the liver and can reach CSF via diffusion. Normal albumin concentration in CSF is about 500 times lower than that of serum. Abnormal concentration of albumin in CSF is most often associated with disruption in the blood-brain barrier (eg, trauma, inflammation). Quotient of albumin (Qalb) is a calculated parameter that normalizes the CSF concentration of albumin to the concentration of albumin in serum:

          Q-Alb=(AlbCSF/AlbSerum) X 1000

Normally, the Quotient of albumin is less than 9 and reflects intact blood-brain barrier. However, the higher the quotient of albumin, the higher the blood-brain barrier damage and vice-versa.[2, 3, 7]

IgG Index

IgG index is a calculated parameter that normalizes the IgG concentration in CSF, taking into account the concentration of albumin (Qalb) and IgG in serum:

          IgG index = (IgGCSF/IgGSerum) / Q-Alb

IgG index provides a better idea regarding IgG molecules entering CSF via damaged blood-brain barrier. Variations exist between laboratories regarding the normal value of IgG index (generally 0.25-0.7). However, generally if IgG index is greater than 0.7, the patient is actively producing IgG within CSF, while the blood-brain barrier is intact. A decreased IgG index reflects damaged blood-brain barrier, which allows IgG crossing (eg, stroke, tumors, some meningitis).[2, 3, 7]

CSF isoelectric focusing electrophoresis (IEF) and testing for multiple sclerosis: oligoclonal banding

The CSF oligoclonal bands represent a population of gamma-migrating globulins with similar electrophoretic mobility. IEF is significantly more sensitive for optimal separation of CSF oligoclonal bands than the regular CSF protein electrophoresis.

Detection of oligoclonal bands is associated with multiple neurological conditions. Up to 90% of patients with multiple sclerosis present oligoclonal band upon CSF IEF evaluation, while the blood-brain barrier is intact (normal Qalb) and IgG index can be within normal range.[2, 3, 7]

For correct interpretation of CSF IEF results, serum IEF should be run in parallel. Few patterns/clinical situations can be encountered:

Although detection of oligoclonal bands is most often associated with multiple sclerosis, other causes of oligoclonal banding should be excluded. Multiple myeloma and other monoclonal gammopathies, as well as some viral infections, are characterized by the presence of immunoglobulin banding in serum. Upon disruption of the blood-brain barrier or after introduction of blood into the CSF samples during a traumatic tapping, banding can be detected in a matching pattern in both CSF and serum. Integrity of the blood-brain barrier should be always evaluated (eg, Qalb). Therefore, for correct interpretation of CSF IEF results, serum immunofixation should be considered for all positive cases with matching pattern. In addition, some neurological disorders, such as encephalitis, neurosyphilis, some forms of meningitis and Guillain-Barre syndrome can also produce CSF-specific banding. Clinical correlations should always be considered. Oligoclonal banding will remain positive during multiple sclerosis remission, but it will disappear in other disorders.[2, 3, 7]

CSF-specific transferrin and evaluation of CSF leakage

Transferrin is present in serum, in normal circumstances, only as a sialated isoform. However, CSF contains the specific, desialated isoform, also known as tau protein or tau transferrin. Normal serum transferrin (sialated isoform) migrates upon electrophoresis anodically and make up most of the beta-1 electrophoretic band. However, the CSF-specific desialated isoform (tau transferrin) is more positive and, therefore, it migrates more cathodically, as a distinct band, designated as “beta-2 transferrin.” Note that this is a CSF-specific band/isoform and it is not detected in serum in normal circumstances. These electrophoretic properties of transferrin isoforms have diagnostic application in rhinorrhea or otorrhea (leakage of CSF into the nose or ear canal, usually as a result of head trauma, tumor, congenital malformation, or surgery). Beta-2 transferrin is used as an endogenous marker of CSF leakage.

Upon electrophoresis of ear or nose fluid samples, common transferrin migrates in the beta-1 electrophoretic fraction (“beta-1 transferrin”), while beta-2 transferrin, which is the CSF-specific variant of transferrin, if present in the ear or nose fluid samples, will migrate as an additional distinctive band. Detection of beta-2 transferrin in ear or nose fluid samples is an indication of CSF leakage.[7]

Summary of chemistry evaluation of CSF in different clinical conditions

Chemistry evaluation of CSF in different clinical conditions is summarized below.[2]

Table. Changes in Analytes With Various CNS Disease (Open Table in a new window)

Conditions associated with changes in microscopic/cellular findings of CSF

Conditions associated with a reactive CSF lymphocytosis include the following:

• Meningitis
• Syphilitic meningoencephalitis
• Parasitic CNS infection
• Multiple sclerosis
• Guillain-Barré syndrome
• Meningeal sarcoidosis
• Polyneuritis
• Subacute sclerosing panencephalopathy (SSPE)

Conditions associated with CSF monocytosis include the following:

• Chronic or treated bacterial meningitis
• Syphilitic, viral, fungal, amebic meningitis
• Intracranial hemorrhage
• Cerebral infarct
• CNS malignancy
• Foreign body reaction

Conditions associated with increased CSF polymorphonuclear neutrophils include the following:

• Bacterial meningitis
• Acute viral meningitis
• Tuberculous and fungal meningitis
• Amebic encephalomyelitis
• Brain abscess
• Subdural empyema
• CNS hemorrhage
• Cerebral infarct
• Malignancies
• Previous lumbar puncture
• Intrathecal chemotherapy
• Seizure

Specimen collection

CSF samples are usually collected through lumbar puncture between the 3rd, 4th or 5th lumbar vertebrae. Other rarely used methods include collection from a ventricular shunt or drain, cisternal puncture, and ventricular puncture.

CSF samples are collected in four sterile tubes (no preservatives) designated as “tube 1,” “tube 2,” “tube 3,” and “tube 4” in order in which they are withdrawn.

 

Tube 1 is generally used for Chemistry (including electrophoresis and testing for oligoclonal banding) and some hematology (eg, cell count) testing.

In the case of a traumatic tap for CSF collection, the first collection tube will contain CSF with a high number of RBC and high concentration of hemoglobin. Presence of these compounds can lead to inaccurate measurements for some chemistry analytes. Therefore specific corrections (eg, for total proteins concentration) must be considered. (Alternatively, CSF collection from additional tubes (3-4) can be used for chemistry testing.

Similarly, if cell count is evaluated using first tube CSF collection from a traumatic tap, the RBC count will be significantly elevated resulting in misleading results. Therefore, in these circumstances, tube 4 should be used instead.

Tube 2 is generally used for microbiology testing. It can also be used for molecular testing, and viral or serological testing.

Tube 3 is generally used for cytology.

Tube 4 is generally used for hematology (cell count and differentiation) and flow cytometry.

Additional tubes can be collected and used for more esoteric testing (eg, immunologic tests).

Lumbar puncture is contraindicated in patients with neurological signs indicating increased intracranial pressure (eg, papilledema).[3]

Specimen volume

Depending on the test and number of tests required, different CSF volumes are necessary for each of these tubes (ie, tubes 1-4) (eg, 0.5 mL for most of the chemistry test, 3 mL for cytology).[3]

Specimen stability

Most of the CSF tests are performed on a STAT basis. If this is not possible, the CSF sample for hematology testing (tube 4) needs to be refrigerated, CSF sample for microbiology testing (tube 2) can remain at room temperature, and CSF sample for chemistry testing (tube 1 or 3-4) needs to be frozen. Additional/specific requirements can be set by laboratories for particular tests to ensure the stability of the analytes of interest within the specimen.

CSF testing

Few general steps are usually followed when routine CSF analysis is requested in clinical chemistry labs. Samples are usually examined for their appearance (eg, color), chemical analysis (eg, glucose, total proteins) and microscopic evaluation (cell count and differential count).

Description

Cerebrospinal fluid (CSF) represents the fluid that is produced in the choroid plexuses of the ventricles of the brain. The CSF is contained within the ventricular system and the subarachnoid space of the whole CNS (brain and spinal cord). The total CSF volume is about 150 mL (this volume is maintained constant), but it is continuously resorbed and produces at a rate of about 500 mL/day or approximately 0.35 mL/min, being replaced at every 6-8 hours. Blockages or pressure (eg, meningeal inflammation, bacterial meningitis, subarachnoid hemorrhage) within any of the brain ventricles or within the foramina between these ventricles leads to accumulation of CSF and hydrocephalus.

The main functions of CSF are mechanical support for brain, transporter of different biochemical compounds, especially neuromodulators and removal of various metabolites, and maintenance of biochemical homeostasis at the CNS level.

CSF is a clear colorless fluid, free of any pigmentation. The biochemical composition of CSF is slightly different from that of plasma and tightly regulated. However, changes in the plasma can trigger changes in the CSF within a short interval of time (eg, electrolytes, glucose).

The biochemical composition of CSF changes with different diseases or medication. Also, various laboratory findings after CSF testing can provide valuable information for diagnosis and association with different diseases. Analysis of cerebrospinal fluid is used in the diagnosis of a wide variety of diseases and conditions affecting the central nervous system. CSF analysis includes measurement of normal CSF compounds (eg, proteins, glucose), as well as examining for abnormal elements (eg, cells, pathogens, abnormal proteins, serology).[1, 8, 9, 2]

CSF glucose is in equilibrium, and, in normal circumstances, the glucose concentration in CSF is 60-80% of that in the plasma.

Most of the proteins present in CSF originate from serum and cross the capillary endothelium of the blood-brain barrier via pinocytosis. The normal ratio of proteins in serum versus CSF is 200:1.[2, 3]

Indications/Applications

CSF analysis may be indicated in patients whose history or examination suggests a CNS process. Such symptoms and signs may include the following:

• Changes in mental status and consciousness

• Sudden, severe, or persistent headache or a stiff neck

• Confusion, hallucinations, or seizures

• Muscle weakness or lethargy, fatigue

• Nausea (severe or prolonged)

• Flu-like symptoms that intensify over a few hours to a few days

• Fever or rash

• Sensitivity to light

• Numbness or tremor

• Dizziness

• Difficulties with speech

• Difficulty walking, lack of coordination

• Mood swings, depression

• Infants: Persistent irritability, body stiffness, poor feeding, or bulging fontanel

Considerations

The aspect, biochemical composition, and microscopic content of CSF change with different diseases, conditions, or medication. Also, various laboratory findings after CSF testing can provide valuable information for diagnosis and association with different diseases. Analysis of cerebrospinal fluid is used in the diagnosis of a wide variety of diseases and conditions affecting the central nervous system.

Related Tests

Related tests include the following:[1, 8, 9]

• Serum glucose
• Serum albumin
• Serum total proteins
• Serum IgG
• Serum protein electrophoresis
• Complete blood count
• Serum lactate
• Acid-fast bacillus (AFB) smear and culture
• Blood culture
• Herpes
• Lyme disease
• Rubella
• Syphilis
• West Nile virus
• Toxoplasmosis
• Epstein-Barr virus (EBV) antibodies
• Fungal tests
• CSF elements (eg, cells, pathogens, abnormal proteins, serology)