Cerebro Spinal Fluid Analysis

Indications of Cerebro Spinal Fluid Analysis

CSF analysis can help to diagnose the following diseases of the brain and the spinal cord:

Infectious diseases

• Encephalitis
• Meningitis

CSF analysis determines the number and types of white blood cells, presence or absence of bacteria, etc.

Intracranial hemorrhage (bleeding in the brain)

• Subarachnoid hemorrhage

CSF analysis examines the physical appearance of CSF along with the presence of red blood cells.

Autoimmune disorders

• Guillain-Barré Syndrome
• Multiple sclerosis

CSF tests for these diseases look for high levels of certain proteins in the CSF. These include albumin and immunoglobulins.

CNS malignancies (brain tumors)

• Primary brain tumor
• Metastatic brain tumor

The presence of malignant cells in CSF is confirmed by microscopic examination of the fluid.

Read more about Metastasis or Metastatic Disease

Sample Collection and Testing

 

CSF samples are usually collected through lumbar puncture between the 3^rd, 4^th or 5^th lumbar vertebrae.

Read more about Lumbar Puncture or Spinal Tap-Indications, Procedure and Complications

The sample is collected in four sterile tubes in order in which they are withdrawn.

• Tube 1 – For biochemical tests such as glucose and protein

• Tube 2 – For microbiology and immunology testing

• Tube 3 – For total and differential cell counts

• Tube 4 – Hold for a repeat cell count with differential, if needed (or for other tests not initially ordered)

Under certain conditions, this order may be altered. For example, if tube 1 is hemorrhagic because of a traumatic tap, it should not be used for protein studies ( especially when they are the most important aspect of the analysis as in suspected multiple sclerosis). In such cases, tube 3 or 4 may be used for this purpose.

Depending on the indication of the procedure and the number of tests required, different CSF volumes are collected in each of these tubes.

The tests on CSF sample should be performed as soon as possible, preferably within one hour of collection.

If this is not possible, CSF sample for biochemistry testing (tube 1 or 3-4) should be frozen, CSF sample for microbiology testing (tube 2) should remain at room temperature, and the CSF sample for cell count testing (tube 3) needs to be refrigerated.

Physical Appearance

Normal CSF is crystal clear, colorless, and water-like.

Xanthochromia is a yellow, orange, or pink discoloration of the CSF. It occurs due to the lysis of RBCs that causes the hemoglobin to breakdown into oxyhemoglobin, methemoglobin, and bilirubin. To detect xanthochromia, the CSF should be centrifuged and the supernatant fluid compared with a tube of distilled water. Discoloration of CSF starts after RBCs have been present in the spinal fluid for about two hours, and remains for about 15 to 30 days.

• Turbid, milky, cloudy
  • Infections (meningitis and encephalitis)
  • Increased proteins (above 150 mg/dL) and albumin and IgG
• Pink or red
  • Subarachnoid or intracerebral hemorrhage
  • Traumatic tap
• Yellow
  • Blood breakdown products
  • Hyperbilirubinemia/ Kernicterus
• Orange
  • Blood breakdown products
  • High carotenoid ingestion
• Green
  • Hyperbilirubinemia
  • Purulent CSF
• Black or brown
  • Melanoma

Causes of blood within CSF

A traumatic tap may occur in about 20% of lumbar punctures. It is important to differentiate whether blood present in CSF is due to trauma during the spinal puncture or due to actual subarachnoid hemorrhage.

• In a traumatic tap, the amount of blood within the CSF keeps on decreasing so that the CSF collected in the third tube is almost clear of blood. However, the blood within CSF remains almost constant in case of subarachnoid hemorrhage.
• Xanthochromia, microscopic evidence of erythrophagocytosis, or presence of hemosiderin­laden macrophages indicate a subarachnoid bleed.  All these features of RBC lysis, however, may also be seen in case of a traumatic tap, if the sample is examined 1–2 hours after the procedure. Hence it is important to evaluate the sample as soon as possible.
• High levels of the enzyme lactate dehydrogenase are seen in subarachnoid or intracerebral hemorrhage. In the case of traumatic tap with intact RBCs, the levels will not be significantly increased.

Biochemical Examination

Glucose

Derived from blood glucose, fasting CSF glucose levels are normally 50–80 mg/dL or about 60-80 % of plasma values.

Blood glucose level must be determined along with CSF glucose level for proper interpretation. The blood sample should be collected about 2 hours prior to lumbar puncture. Also both the blood and CSF sample must be tested immediately to avoid glycolysis and hence prevent erroneous readings.

Causes of decreased CSF glucose level (<40 mg/dl) include:

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

Decreased CSF glucose level (and especially decreased CSF glucose level in relation to blood glucose level) is usually associated with bacterial or fungal meningitis. However, 50 percent of patients that have bacterial meningitis may have normal CSF glucose levels.

A normal CSF glucose level along with an increased number of WBCs may indicate viral meningitis.

The only cause of increased glucose level in CSF is an elevated level of glucose in the blood. It carries no other clinical significance.

Protein

The CSF protein concentration varies with age and level of the spinal tap (eg, lumbar, ventricular, etc). Similar to blood, the predominant fraction in CSF is albumin.

Causes of increased CSF protein

• Infections: Highest elevation is seen in tubercular infections followed by bacterial and viral infections
• Subarachnoid and intracerebral hemorrhage
• Multiple sclerosis
• Guillain Barré syndrome
• Malignancies
• Endocrine and metabolic disorders
  • Milk–alkali syndrome with hypercalcemia
  • Diabetic neuropathy
  • Hereditary neuropathies and myelopathies
  • Decreased endocrine function (thyroid, parathyroid)
  • Other disorders (uremia, dehydration)
• Overdose of certain medicines
  • Ethanol, phenothiazines, phenytoin

The presence of RBCs in a traumatic tap causes false elevation of protein levels. This can be corrected by subtracting 1.1 mg/dL of protein for every 1,000 RBCs per mm³. This correction should however be applied only if the same tube is used for both protein and cell count estimation.

Causes of low CSF protein levels

• Chronic CSF leak
• Repeated lumbar puncture
• Acute water intoxication
• Some children between 6 months to 2 years
• Few patients with idiopathic intracranial hypertension (due to an increased rate of protein resorption by the arachnoid villi)

Low levels of protein in the blood (hypoproteinemia) do not cause reduced CSF protein levels.

The permeability of the blood ­brain barrier can be assessed by CSF/ Serum albumin index which is calculated as follows:

CSF/ Serum albumin index= CSF albumin (mg dL)  ⁄  Serum albumin (g dL)

An index value <9 is consistent with an intact barrier. An index value of 9–14 indicates a slight impairment, 14–30 indicates a moderate impairment while values greater than 30 are indicative of severe impairment. The index is slightly elevated in infants up to 6 months of age, reflecting the immaturity of the blood­brain barrier. The index also gradually increases after 40 years of age.

Increased intrathecal IgG synthesis is reflected by an increase in the CSF/serum IgG ratio:

CSF/Serum IgG index: CSF IgG (mg/dl) / Serum IgG (g/dl)

The CSF/serum IgG index can be increased by intrathecal IgG syn­thesis or increased plasma IgG crossover from the breakdown of the blood­ brain barrier. Its normal range is 3.0–8.7.

Certain conditions, such as in multiple sclerosis, require the evaluation of different protein fractions and various immunoglobulins in addition to total protein estimation.

Patients of multiple sclerosis show CSF oligoclonal bands (that represent a population of gamma-migrating globulins with similar electrophoretic mobility). Isoelectric focusing electrophoresis (IEF) is superior to regular CSF protein electrophoresis for oligoclonal banding.

Lactate

Lactate levels can be used to differentiate viral meningitis from bacterial, mycoplasma, fungal, and tuberculous meningitis (in which routine parameters yield equivocal results).

In patients with viral meningitis, lactate levels are usually below 25 mg/dL and are almost always less than 35 mg/dL, whereas bacterial meningitis usually has levels above 35 mg/dL.
Persistently increased CSF lactate levels indicate a poor prognosis in patients with a severe head injury.

Enzymes

Although CSF enzyme levels are not routinely used in the diagnosis of brain diseases, there are certain diseases in which they may have a role.

• Adenosine deaminase: increased in tubercular meningitis
• Creatine kinase: increased in hydrocephalus, cerebral infarction, primary brain tumors, and subarach­noid hemorrhage
• Lactate dehydrogenase: increased in CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid hemorrhage. It is also useful in differentiating a traumatic tap from intracranial hemorrhage because a traumatic tap with intact RBCs will not have significantly high levels of lactate dehydrogenase.
• Lysozyme: increased in tubercular and bacterial meningitis

Cell Counts

The normal leucocyte count of CSF is 0-5 cells//µL. It is higher in neonates, ranging from 0–30 cells/µL. No RBCs are present in normal CSF.

The leucocytes in normal adult CSF consist of about 70% lymphocytes and 30% monocytes. Small numbers of neutrophils may be seen in “normal” CSF samples, most likely due to minor hemorrhage that occurs during the procedure.

Causes of increased CSF neutrophils

• Meningitis
  • Bacterial meningitis
  • Early viral, tubercular, fungal meningitis
  • Amebic encephalomyelitis
• Other infections
  • Brain abscess
  • Subdural empyema
• Following seizures
• Following brain hemorrhage
  • Subarachnoid
  • Intracerebral
• Following cerebral infarct
• Reaction to repeated lumbar punctures
• Injection of foreign material in subarachnoid space (like methotrexate, contrast media, etc)
• Malignancies

Causes of increased CSF lymphocytes

• Meningitis
  • Viral meningitis
  • Tubercular meningitis
  • Fungal meningitis
  • Syphilitic meningoencephalitis
  • Leptospiral meningitis
  • Early bacterial meningitis
  • Parasitic infections (like cysticercosis, trichinosis, toxoplasmosis)
• Degenerative disorders
  • Subacute sclerosing panencephalitis
  • Multiple sclerosis
  • Drug abuse encephalopathy
  • Guillain-Barré syndrome
• Other inflammatory disorders
  • Sarcoidosis
  • Polyneuritis

Causes of increased CSF eosinophils

• Acute polyneuritis
• Reaction to foreign material
• Fungal infections
• Idiopathic eosinophilic meningitis
• Parasitic infections
• Malfunctioning ventriculoperitoneal shunts
• Infrequently associated with
  • Bacterial, tubercular, viral meningitis
  • Neurosarcoidosis
  • Leukemia/lymphoma
  • Primary brain tumors

Eosinophilic meningitis is defined as >10 eosinophils/µl or a total CSF cell count comprising of >10% eosinophils.

Causes of increased CSF monocytes

• Chronic or treated bacterial meningitis
• Syphilitic, viral, fungal, amebic meningitis
• Brain hemorrhage
• Brain infarct
• Brain tumors
• Foreign body reaction

Microscopic Examination

It helps to detect the

• Presence of RBCs
• The number and types of WBCs
• Infection by specific organisms using special stains
  • Gram stain for bacterial meningitis
  • Acid-fast staining for suspected tubercular meningitis
  • Fungal stains for detecting hyphae in case of fungal meningitis.
  • India ink preparation for Cryptococcus neoformans.
  • Wright or Giemsa stain for Toxoplasma infection.
  • A simple wet preparation of CSF under a coverslip for a variety of protozoan and helminthic infections.
• Presence of malignant cells in case of brain involvement by leukemia or lymphoma.

Read more about Light Microscope-Parts, Usage, Handling, and Care

Read more about Complete Blood Count or CBC

Microbiological Examination

• In the case of meningitis, specific organisms can be assessed using special stains.
  • Gram stain for bacterial meningitis
  • Acid-fast staining for suspected tubercular meningitis
  • Fungal stains for detecting hyphae in case of fungal meningitis.
  • India ink preparation for Cryptococcus neoformans.
  • Wright or Giemsa stain for Toxoplasma infection.
  • A simple wet preparation of CSF under a coverslip for a variety of protozoan and helminthic infections.
• Culture of CSF specimen to detect the type of organism responsible for causing the infection. It is the gold standard for determining the cause of bacterial meningitis.
• Polymerase Chain Reaction (PCR): PCR has high sensitivity and specificity in cases of meningitis. It is fast and can be done with small volumes of CSF. It is especially useful in the diagnosis of viral meningitis.

References

• McPherson RA, Matthew R. Pincus MR. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders; 2011. 254-5.
• Kaplan, Pesce. Clinical Chemistry: Theory, Analysis, Correlation. 5th ed. St. Louis, MI: Elsevier Inc; 2010. 904-928.
• Burris CA, Ashwood ER, Burns DE. 4th ed. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. St. Louis: Elsevier Saunders; 2006. 1633:962-967
• Wallach J. Interpretation of Diagnostic Tests. 6th ed. New York: Little, Brown; 1996. 717.
• Clarke W. Contemporary Practice in Clinical Chemistry. 2nd ed. AACC; 233-246.