Cerebrospinal Fluid

Cerebrospinal fluid is an ultrafiltrate of plasma which circulates in the subarachnoid space, located between the pia and arachnoid membranes. It is largely produced by the choroid plexuses, while lesser amounts are produced by the ependymal cells lining the ventricles. The choroid plexus epithelium and associated capillary endothelium make up the blood-brain barrier and regulate the passage of substances into the CSF from blood. The arachnoid villi reabsorb the fluid.

The function of the CSF includes cushioning and lubrication of the central nervous system, circulation of nutrients, and waste collection.

Total CSF volume is 90-150 mL in adults and 10-60 ml in neonates. There is a constant turnover of about 14% of the volume per hour. About 300-500 mL of CSF are formed per 24 hours (0.35 mL per minute).

CSF is obtained by lumbar puncture, with removal of fluid from the lumbar sac, located at the L3-4 or L4-5 interspace. At this level, the needle cannot injure the spinal cord, which in adults ends at L1. Between 5 and 20 mL are typically collected.

The indications for lumbar puncture include:

Suspected conditions

• Meningitis, encephalitis, syphilis, absces
• Subarachnoid hemorrhage, intracerebral hemorrhage, subdural hematoma
• Multiple sclerosis
• Acute leukemia or lymphoma with CNS involvement
• Spinal cord tumor

Therapy

• Chemotherapy for leukemia or lymphoma
• Introduction of anesthetics, radiographic contrast media
• Amphotericin therapy of fungal meningitis

The opening pressure is 90-180 mm water with the patient lying in the lateral position. It is slightly higher in the sitting position. Coughing, straining, and breathing can cause fluctuations in the pressure. Abnormally high pressures (>180 mm) indicate impaired absorption of fluid or a mass. Up to 20 mL of fluid can be withdrawn if CSF pressure is with normal limits. The causes of CSF pressure changes include:

 

Contraindications to lumbar puncture include neurological signs indicating increased intracranial pressure, including papilledema. Withdrawal of fluid in this case can promote downward herniation of the cerebellar vermis through the foramen magnum with brainstem compression. Patients with increased intracranial pressure should be tapped by a neurologist or neurosurgeon.

Three to four tubes of CSF should be collected aseptically. Each tube should contain 2 to 4 mL of fluid. The tubes should be labeled with a number indicating the order in which they were collected. Each tube goes to a different section of the laboratory for testing:

• Tube #1 Hold in reserve
• Tube #2 Chemistry and Immunology
• Tube #3 Microbiology & molecular testing
• Tube #4 Cell count, flow cytometry and cytopathology

Tube #1 is most likely to be contaminated with blood and debris from the collection process. Cell counts are most accurate when performed on the last tube collected. If the fourth tube contains insufficient volume, Tube #1 may be used for Chemistry and Immunology; Tube 2 for Microbiology; and Tube #3 for Cell count, flow cytometry and cytopathology. 

Specimens need to be received in the laboratory and processed quickly, preferably within one hour after collection. Refrigeration is contraindicated for culture specimens because some organisms like Haemophilus influenzae and Neisseria meningitidis will not survive.

Routine Tests include:

• Color and appearance
• Protein and glucose
• Gram stain and bacterial culture
• Total leukocyte count and differential

Gross Appearance

CSF is normally clear and colorless and has a viscosity similar to water. Turbidity (cloudiness) is graded 1+ through 4+. It can be due to:

• Increased leukocytes (meningitis)
• Increased RBC
• Microorganisms
• Radiographic contrast media
• Increased protein

Brown color indicates methemoglobin from a hematoma. Pink-red CSF indicates the presence of blood. Blood may originate from a traumatic tap, subarachnoid hemorrhage, intracerebral hemorrhage, or cerebral infarct.

It is important to distinguish a traumatic tap from pathologic bleeding.

 

The changes which occur in CSF following hemorrhage include:

Gross exam:

• 2 to12 hours; pink to orange xanthochromia
• 12 to 24 hours; yellow xanthochromia (lasts 2-4 weeks)

Microscopic exam:

• 2 to 24 hours; RBC’s, neutrophils, monocytes, lymphocytes
• 12 to 48 hours; monocytes, lymphocytes, erythrophagocytosis
• Greater than 48 hours; monocytes, erythrophagocytosis, siderophages

Traumatic taps are fairly common, occurring with an estimated frequency of 10 to 20%. Unfortunately, there is no specific threshold for the number of RBCs in CSF to differentiate subarachnoid hemorrhage from traumatic tap. The most common practice is to perform a RBC count on the first and last CSF tubes collected. CSF samples from a traumatic tap generally show clearing of RBCS with successive tubes, whereas those from a true hemorrhage show a more stable RBC count.

Other CSF findings are also helpful in distinguishing traumatic tap from subarachnoid hemorrhage. Opening pressure is often elevated in subarachnoid hemorrhage, but not traumatic tap. The presence of xanthochromia, erythrophagocytosis and hemosiderin-laden macrophages is indicative of subrarachnoid hemorrhage as long as there has not been a prior traumatic tap.  Subarachnoid hemorrhage can usually be rule out if tube 4 has an RBC count of less than 2000/uL and lacks xanthochromia. 

Xanthochromia

Xanthochromia is a yellow, orange, or pink color of the CSF. Two to four hours after hemorrhage, RBC’s lyse and release oxyhemoglobin giving a pink to orange color. By 24 hours hemoglobin is metabolized to bilirubin, resulting in a yellow color. Bilirubin concentration peaks at 36 hours, but persists for several weeks. The causes of xanthochromia include:

• Subarachnoid hemorrhage
• Jaundice
• Increased protein (>150 mg/dL)
• Meningeal melanoma
• Hypercarotenemia
• Rifampin therapy
•  Previous traumatic tap

Cell Counts

Normal CSF is essentially acellular, although a few cells may be seen due to leakage from injured blood vessels. WBC’s are counted in a manual counting chamber, using undiluted CSF. The WBC reference range varies with age:

CSF Differential Counts

A stained smear is made on all CSF specimens, even when the total leukocyte count is within normal limits. Smears are made using a cytocentrifuge to concentrate the cells. Using 0.5 ml of CSF, between 30 and 50 cells will be seen in normal specimens. The differential reference ranges are as follows:

 

In adults the ratio of lymphocytes to monocytes is about 70 to 30. Young children have more monocytes.

In early bacterial meningitis the proportion of neutrophils usually exceeds 60%. Lymphocytosis is not uncommon in bacterial meningitis when the CSF WBC count is under 1000/mL.

Ependymal and choroid plexus cells may be seen in both normal and abnormal CSF’s. They are present in increased numbers after trauma, neurosurgery, and brain infarcts. Cartilage cells may be seen if a vertebral body is punctured. Squamous epithelial cells from the skin may be seen.

Correction For Bloody Fluids

If a specimen is bloody due to a traumatic tap, the WBC count must be corrected. If the peripheral blood WBC count is normal, subtract 1-2 leukocytes per 1000 RBC. Protein may be corrected by subtracting 8 mg/dl for every 10,000 RBC/mL. .

Abnormal Cells

Reactive lymphocytes occur commonly in viral meningitis. They are also increased in:

• Treated bacterial meningitis
• Tuberculous meningitis
• Fungal meningitis
• Syphilitic meningoencephalitis
• Parasitic infestation of the CNS
• Multiple sclerosis
• Guillain-Barre syndrome
• Meningeal sarcoidosis
• Polyneuritis
• SSPE

Lymphoblasts can be seen in viral meningitis and in leukemias or lymphomas. Up to 25% of lymphoma cases involve the meninges. They are present in normal neonatal CSF.

Plasma cells are not present in normal fluids and suggest acute or chronic inflammation such as viral or mycobacterial meningitis, syphilis, sarcoid, SSPE, or MS.

Monocytes are increased in:

• Chronic bacterial meningitis
• Treated bacterial meningitis
• Syphilitic, viral, fungal, amebic meningitis
• CNS hemorrhage
• Cerebral infarct
• Multiple sclerosis
• Foreign body reaction
• CNS malignancies

Siderophages are monocytes with hemosiderin inclusions and indicate previous hemorrhage. Hematoidin inclusions may also be present at later stages.

Lipophages are lipid-laden monocytes. They occur after traumatic liquefaction necrosis, myelogram, intrathecal chemotherapy, and radiation therapy.

Neutrophils indicate inflammation. In bacterial meningitis the leukocyte count may reach 50,000/ml. Neutrophils may also predominate in the first few hours to days of viral meningitis. They will then subside and be replaced by reactive lymphocytes, monocytes and macrophages.

Other causes of increased neutrophils include:

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

Eosinophils occur rarely in normal CSF. Increased numbers are seen with malfunctions of intracranial shunts, parasitic infections, Coccidiodes immitis and other fungal infections, drug reactions, rarely in lymphomas and bacterial meningitis.

Basophils are seen only in abnormal CSF’s secondary to inflammation, foreign bodies, parasites, and chronic myelogenous leukemia.

The CSF of patients with drug induced meningitis typically shows a leukocytosis, with a median cell count of 200 cells per uL. Neutrophils predominate in three fourths of the cases and eosinophils are occasionally noted.  The degree of leukocytosis correlates with the severity of fever and inversely with the time interval from drug exposure. Laboratory values usually return to normal within 5 days after drug withdrawal. 

The term “Blasts” indicates immature cells that cannot be differentiated. Eighty percent of ALL cases and 60% of AML cases involve the CSF. A diagnosis of leukemia requires:

1. An increase in total WBC count
2. Blasts comprising >40% of total cells

Cytochemistry and flow cytometry can be used to determine the blasts lineage.

Malignant cells can arise from brain tumors or metastases. Medulloblastoma cells are the most commonly seen brain tumor cells; glioblastoma and meningioma cells are less common. The most common metastatic tumors seen in the CSF are melanoma, lung and breast.

Effects of Invasive Procedures

Eight to 12 hours after an LP there may be increased numbers of neutrophils, monocytes, macrophages, and erythrophagocytes. After myelography, increased monocytes and macrophages may persist up to 2-3 weeks. Intracranial shunts result in persistently increased numbers of eosinophils, monocytes, and macrophages

CSF Chemistry

Protein

Spinal fluid is an ultrafiltrate of plasma that lacks high molecular weight proteins such as beta lipoprotein, alpha-2 macroglobulin, IgM, and polymeric haptoglobins. Unlike plasma, CSF has a prominent prealbumin peak and a split beta peak, which contains transferrin and b2-transferrin, which is also called tau-protein. The protein concentration of spinal fluid is less than 1% of plasma proteins.The normal range for lumbar punctures varies with age:

 

 

Infants have higher protein levels (60–150 mg/dL) due to increased blood brain barrier permeability.Cisternal and ventricular fluids have lower total protein concentration than fluid obtained by lumbar puncture.

Elevation of spinal fluid protein is the most frequently encountered abnormality. The most common causes of altered protein concentration are listed below.

 

CSF protein may be normal or mildly increased in viral meningitis.In most cases of viral meningitis the protein concentration is <100 mg/dL.In contrast, acute bacterial meningitis is usually associated with a CSF protein concentration between 100 and 500 mg/dL. CSF protein is almost always high in tuberculous meningitis. Approximately 20% of patients with primary syphilis and 30-70% with secondary syphilis have elevated CSF protein. Approximately 50% of asymptomatic HIV patients exhibit mildly increased CSF protein levels.

Lumbar disc herniation with sciatica may be associated with increased CSF total protein. CSF protein concentration is slightly higher in males with depressive disorders than in women with similar diagnoses. CSF protein is within normal limits in 66% of patients with multiple sclerosis. Protein levels >100 mg/dL are usually not associated with primary neurologic disease.

Four groups of drugs have been associated with drug induced aseptic meningitis: nonsteroidal anti-inflammatory drugs (NSAID), antibiotics, intravenous immunoglobulins (IVIG), and OKT3 monoclonal antibody.  Most patients present with an abrupt onset of headache, fever, meningismus and altered mental status. This clinical presentation is not very helpful in differentiating drug induced from infectious meningitis. The interval between drug intake and onset of meningitis varies between several minutes and 4 months.  Approximately, one third of patients report prior use of the medication in question. The single most frequent underlying disease associated with drug induced meningitis is systemic lupus erythematosis (SLE).

IgG Index

Since CSF is an ultrafiltrate of plasma, it has much lower concentrations of the highest molecular weight proteins such as IgG, IgA and IgM. Elevated CSF IgG levels can either be the result of diffusion of plasma IgG across an altered blood brain barrier or intrathecal synthesis. Patients with multiple sclerosis and other demyelinating disorders often have elevated CSF IgG concentrations due to intrathecal synthesis.

Several tests have been devised to determine if IgG synthesis is increased. The percent of IgG present is more useful than the absolute concentration, because it compensates for increased blood brain barrier permeability. The IgG index is the most sensitive indicator of increased CNS IgG synthesis. The reference range is 0-0.85.

CSF IgG Index = CSF IgG X serum albumin/CSF albumin X serum IgG

Reference range is 0-8.1 mg/dL for CSF IgG and 0–27.0 mg/dL for CSF albumin

Oligoclonal Bands

One of the best methods to detect intrathecal IgG synthesis has been to examine CSF for the presence of oligoclonal bands after separation of proteins by electrophoresis. IgG in normal CSF migrates as a faint diffuse zone, but in demyelinating diseases, IgG migrates as discrete oligoclonal bands.

During the summer of 2003, the FDA approved a new method for the detection of oligoclonal bands that uses isoelectric focusing plus immunofixation (IEF) instead of electrophoresis. The Consortium of Multiple Sclerosis Centers has endorsed IEF because of its increased sensitivity (>95%). With IEF, oligoclonal bands may be detected while the total CSF IgG concentration is still within the normal range.

Oligoclonal bands are reported as present when there are 4 or more bands in the CSF than there are in serum. A red top tube of blood must accompany all CSF specimens submitted for a multiple sclerosis profile in order for this study to be interpreted. In addition to demyelinating diseases, oligoclonal bands may be seen in the CSF of patients with lymphoma, CLL, malignancies, chronic active hepatitis, rheumatoid factor, and viral illnesses.

Myelin Basic Protein

Demyelinating disorders release myelin basic protein into the CSF. Effective June 19, 2009, Mayo Medical Laboratories (MML) discontinued testing for myelin basic protein on spinal fluid.  From a quality perspective, MML determined that it was no longer possible to obtain satisfactory reagents on a regular basis to meet their standards. Also, recent revisions to the diagnostic criteria for multiple sclerosis include CSF oligoclonal IgG bands and/or increased IgG index, but not myelin basic protein.

Glucose

Glucose enters the CSF from the plasma by 2 mechanisms, diffusion and active transport. The level of CSF glucose is influenced by the concentration and duration of the plasma glucose level. CSF glucose levels are 60-70% of plasma glucose levels. The normal fasting CSF glucose is 40-70 mg/dl. In children the nonfasting reference range is 45-100 mg/dl. The CSF glucose level lags behind the plasma level by 30-90 minutes. An increase in CSF glucose means the patient was hyperglycemic 30 to 90 minutes before and has no special clinical significance. A decreased CSF glucose is seen in:

• Bacterial (60-80% of cases), fungal, and tuberculous meningitis
• Primary or metastatic meningeal malignancies
• Subarachnoid hemorrhage
• Hypoglycemia

A normal glucose level is seen in:

• Viral meningitis
• Brain abscess
• Neurosyphilis

Lactate

The normal CSF lactate range varies slightly with age:

 

Lactate increases in any condition associated with decreased blood flow or hypoxia such as:

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

Lactate levels are used mostly to distinguish early bacterial meningitis from viral meningitis. Lactate levels are usually >35 mg/dl in bacterial meningitis and <25 mg/dl in viral meningitis. Levels between 26-30 mg/dl are equivocal. Mycoplasma meningitis will usually give elevated lactate levels, even if cultures are negative. All xanthochromic fluids will have elevated lactate levels, so it is best not to measure lactate in these fluids.

Lactate Dehydrogenase

LD is normally present in CSF with 40 U/L a reasonable upper limit of normal for adults and 70 U/L for neonates. LD values may help in differentiating traumatic tap from intracranial hemorrhage because fresh traumatic taps with intact RBC’s do not significantly elevate the LD level. LD activity is significantly higher in bacterial meningitis than in aseptic meningitis. Elevated LD levels 76 hours following resuscitation predict a poor outcome in patients with hypoxic brain injury.

Ammonia

Normal CSF values of ammonia are about one half of serum values. Ammonia values are increased in Reyes syndrome, hypercapnea, and liver disease with hepatic encephalopathy.

Electrolytes And Acid-Base Balance

There are no clinically useful indications for the measurement of sodium, potassium, chloride, calcium, or magnesium in CSF. Measurements of CSF, pH, pCO2, and bicarbonate are not practical for patient care.

CSF Ventriculoperitoneal Shunt Fluid

CSF in the ventricles is different from CSF in the lumbar region. Protein levels in the ventricles are 66% lower (mean 15 mg/dL in ventricles versus 45 mg/dL in lumbar area) and glucose levels are 10% higher in ventricles. Clumps of choroidal cells are observed more commonly in VP fluid.  Normal CSF from both regions should contain less than 5 WBCs/uL.

The placement of a ventriculoperitoneal (VP) shunt adds another complication because a shunt is a foreign body, which can induce reactive changes, such as increased cell counts.  Leukocyte counts average 43 cells/uL in patients without shunt dysfunction or infection. Patients with shunt malfunction, without infection, have an average of 49 leukocytes/uL and >5% eosinophils. Patients with infection often have an average of 2200 leukocytes/uL, with >10% neutrophils.

Neurosyphilis Serology

The CSF VDRL test has a sensitivity of only 50-60% but is highly specific. A positive test rules in neurosyphilis. CSF VDRL is inappropriate as a screening test for neurosyphilis; it should be performed only if serum FTA-ABS tests are positive.

The RPR test is unsuitable for CSF because it has more false positives than the VDRL. CSF FTA-ABS is essentially 100% sensitive, but false positives may be caused by increased blood-brain barrier permeability due to inflammation, or as little as 0.8 mL of blood per mL of CSF (4000 CSF RBC’s). A negative serum FTA-ABS test rules out any syphilitic infection, whereas a positive serum FTA-ABS with a nonreactive CSF FTA-ABS excludes the diagnosis of active neurosyphilis.

CSF in HIV Infection

A wide variety of CSF abnormalities may be found in HIV-positive patients with or without neurologic symptoms. These include increased lymphocytes, elevated IgG indexes, and oligoclonal bands. Identification of opportunistic infections is the most important reason for examining the CSF. Serious fungal infections may exist in the presence of little or no CSF abnormalities.

Virology

Most community-acquired aseptic meningitis is caused by viruses. PCR is more sensitive than virus culture for enterovirus group, and is also the gold standard for detection of herpesviruses, including herpes simplex (HSV) and varicella (VZV). The only commonly encountered meningitis/encephalitis virus for which PCR is not the test of choice is West Nile virus, which is more reliably diagnosed by antibody (IgG & IgM) testing of CSF.

Lab test utilization for acute aseptic meningitis can be optimized by requesting tests for the most common viruses initially, while considering patient history and which seasonal viruses are being reported locally. Adenovirus, CMV, EBV & HHV-6 are usually only encountered in immuno-compromised patients; routine testing for these viruses is unnecessary in suspected community-acquired seasonal aseptic meningitis. The minimum CSF volume required for viral testing is 0.5 mL per test.  

Likelihood Ratios for Bacterial Meningitis

Based on a review of large studies of laboratory findings in patients with bacterial meningitis, the following likelihood ratios have been established for the most common tests.

 

The higher the positive LR, the more likely a patient has meningitis and the lower the negative LR the less likely it becomes. If the results of multiple tests are obtained, the appropriate likelihood ratios can be multiplied together to obtain the overall likelihood ratio.

For example, if a CSF had a WBC count of 700/uL, CSF/blood glucose ratio of 0.2, CSF Lactate of 32 mg/dL and a CSF protein of 42 mg/dL, then the overall likelihood ratio is 729, which is calculated by multiplying 15 x 18 x 3.0 x 0.9.