Cerebrospinal Fluid Analysis

Cerebrospinal fluid (CSF) is made by the choroid plexus at about 500 mL a day, so the 125 to 150 mL total volume turns over three to four times daily. It cushions the brain and spinal cord and reflects the state of the meninges and the central nervous system.

A lumbar puncture, usually at L3-L4 or L4-L5, gives a sample for pressure, appearance, cell count and differential, protein, glucose and microbiology. It also allows specialist tests: oligoclonal bands, cytology, flow cytometry, PCR and antibody assays.

The test is the key investigation for meningitis, encephalitis, subarachnoid haemorrhage not shown on CT, demyelination, Guillain-Barré syndrome, central nervous system involvement by lymphoma and leukaemia, and intracranial pressure disorders. Results direct urgent treatment, so the sample is handled as an emergency.

Bacterial meningitis affects about 2.5 million people a year worldwide and carries a mortality of 10 to 30% even with treatment, higher in low income settings and in pneumococcal disease. Viral meningitis is commoner and benign. Cryptococcal meningitis causes about 150 000 deaths a year, mostly in advanced HIV disease, and tuberculous meningitis kills a third or more of those affected.

Indications. Fever with headache, neck stiffness, photophobia or altered consciousness, unexplained encephalopathy or seizures, thunderclap headache with a negative CT, suspected demyelination or Guillain-Barré syndrome, neurosyphilis, cryptococcal antigen positivity, suspected central nervous system lymphoma or leukaemia, idiopathic intracranial hypertension, and normal pressure hydrocephalus.

Specimen. Collect four sterile tubes in order, with 1 to 2 mL each (more for mycobacterial culture and cytology).

• Tube 1: chemistry and serology.
• Tube 2: microbiology.
• Tube 3: cell count (the least affected by a traumatic tap).
• Tube 4: cytology or other tests.

Record the opening pressure with the patient lying on the side and legs relaxed.

Send a paired plasma glucose taken within 30 minutes, and plasma for oligoclonal bands when ordered. Process within 1 hour at room temperature (cells lyse and glucose is consumed), and never refrigerate samples for bacterial culture.

Before the puncture, check for raised intracranial pressure (focal signs, papilloedema, reduced consciousness, seizures) with imaging, for clotting abnormality (platelets under 50 x 10^9/L, INR over 1.5, anticoagulants) and for local infection.

Normal adult CSF.

• Opening pressure: 100 to 250 mm of water (7 to 18 cm) lying down. Over 250 mm is raised, and over 300 mm is markedly raised.
• Appearance: clear and colourless.
• White cells: 0 to 5 per mm3, all mononuclear. Neonates up to 20 to 30 per mm3 (mostly lymphocytes). Over 5 polymorphonuclear cells is abnormal at any age.
• Red cells: none. A traumatic tap is corrected with about 1 white cell per 700 red cells.
• Protein: 0.15 to 0.45 g/L in adults (up to 1.0 g/L in neonates, 0.6 g/L in people over 60 years). Lumbar protein is higher than ventricular.
• Glucose: 2.5 to 4.4 mmol/L, or 60% of the plasma value (CSF to plasma ratio 0.6, ratio under 0.4 abnormal, under 0.3 strongly abnormal).
• Lactate: under 2.1 mmol/L.
• IgG index: under 0.7. Oligoclonal bands: none, or the same bands in serum and CSF.

Patterns.

Key tests and their meaning.

• Gram stain: sensitivity 60 to 90% in untreated bacterial meningitis (lower after antibiotics), with specificity close to 100%.
• Culture: positive in 70 to 85% before antibiotics, falling to 40 to 60% after.
• Bacterial PCR or multiplex meningitis panel: useful after antibiotics, with high sensitivity and a risk of false positives.
• Lactate over 3.5 mmol/L favours bacterial over viral meningitis (sensitivity and specificity about 93%).
• Cryptococcal antigen (lateral flow or latex): sensitivity over 95%, and India ink about 70 to 85%. Opening pressure is measured and often raised.
• Tuberculous meningitis: acid fast stain sensitivity about 10 to 20%, rising with large volumes (10 mL) and repeat samples, Xpert MTB/RIF Ultra about 70%, culture 50 to 70%, adenosine deaminase over 10 U/L supports the diagnosis.
• Herpes simplex and varicella zoster PCR: sensitivity over 95% after day 3 of illness, and a negative PCR in the first 72 hours is repeated if suspicion remains.
• Venereal Disease Research Laboratory test (VDRL) in CSF: specific, but sensitivity only 30 to 70% for neurosyphilis.
• Oligoclonal bands: present in CSF and absent in matched serum in over 90% of multiple sclerosis, and also in neurosyphilis, infection and neuro inflammatory conditions.
• Cytology and flow cytometry: sensitivity for malignant cells 50 to 75% on first sample, rising to 85 to 90% with repeat samples.
• Xanthochromia: spectrophotometry for bilirubin and oxyhaemoglobin at 12 hours or more after onset, with high sensitivity for subarachnoid haemorrhage.
• Autoimmune encephalitis antibodies: NMDA receptor, LGI1, CASPR2 and others, tested in CSF and serum.
• 14-3-3 protein and real time quaking induced conversion for Creutzfeldt-Jakob disease.

Critical values and findings needing immediate action.

• Turbid CSF, a positive Gram stain, polymorph count over 1000 per mm3 or glucose ratio under 0.4: this is bacterial meningitis until proven otherwise, so treat without delay and notify.
• Opening pressure over 300 mm of water with headache, vision loss or cryptococcal infection: therapeutic drainage.
• Positive cryptococcal antigen: urgent antifungal treatment and pressure control.
• Xanthochromia or red cells that do not clear with CT negative: subarachnoid haemorrhage needs vascular imaging.
• Malignant cells, or CSF in leptomeningeal metastasis: oncology review.
• Positive herpes simplex PCR: start aciclovir at once and continue until the diagnosis is excluded.

Interferences and pitfalls.

• Antibiotics before lumbar puncture reduce culture yield but within hours the cell count, protein and glucose remain abnormal, so antibiotics are not withheld for the test.
• A traumatic tap raises red cells, white cells and protein (about 0.01 g/L for each 1000 red cells/mm3). Comparing red cells in the first and last tubes separates it from subarachnoid haemorrhage.
• Delay in processing destroys cells and consumes glucose, giving false results.
• Normal CSF in early bacterial meningitis and in immunocompromised or neonatal patients is possible, so clinical judgement is needed.
• Early viral meningitis can show a polymorph predominance, and repeat puncture after 24 to 48 hours shows the shift to lymphocytes.
• Hyperglycaemia or hypoglycaemia changes the CSF glucose with a lag of 2 to 4 hours, and the ratio is calculated with a paired plasma sample.
• High CSF protein (over 10 g/L) can cause xanthochromia and a Froin clotting syndrome in spinal block.
• Lumbar puncture in a patient with a mass lesion or raised pressure risks herniation.
• Antibody tests need paired serum to calculate the intrathecal index.

Special groups.

• Neonates: higher normal cell counts and protein, with Group B Streptococcus, Escherichia coli and Listeria the main bacterial causes, and enterovirus and parechovirus common viral causes.
• Children: a full septic workup is common, and clinical signs are less specific.
• Older adults: Listeria and pneumococcus, and atypical presentation.
• Immunocompromised and HIV patients: cryptococcus, tuberculosis, toxoplasmosis, cytomegalovirus, syphilis and lymphoma.
• Pregnancy: lumbar puncture is safe when indicated, and Listeria is considered.
• Regions: in the African meningitis belt, meningococcal epidemics occur in the dry season, and rapid tests and culture on site direct the response.

Follow up tests.

• Blood cultures (before antibiotics), blood glucose, full blood count, CRP, procalcitonin.
• HIV test, serum cryptococcal antigen, syphilis serology.
• CT or MRI of the brain, and EEG for encephalitis.
• Serum and CSF autoantibodies.
• Xpert MTB/RIF on CSF, and sputum or gastric aspirate for tuberculosis.
• Repeat lumbar puncture at 24 to 48 hours in doubtful cases, and CSF opening pressure monitoring.