Which CSF tube should be sent first and what is the proper order of investigations for a CSF sample?

CSF Sample Collection Order and Tube Allocation

Send the first tube collected for bacterial culture and Gram stain, as it has the highest contamination risk and should NOT be used for microbiology studies; the second and third tubes should be sent for microbiology.

Standard Collection Order and Tube Allocation

The optimal collection sequence allocates CSF tubes as follows:

• Tube 1 (2-2.5 mL): Cell count with differential and red blood cell count 1, 2

  • This tube has the highest risk of blood contamination from the procedure itself and should be used for tests where minor contamination is least problematic 2
  • If traumatic tap occurs, white cell count and protein can be corrected by subtracting 1 white cell per 7,000 red blood cells and 0.1 g/dL protein per 100 red blood cells 1

• Tube 2 (2-2.5 mL): Gram stain, bacterial culture, and antimicrobial susceptibility testing 1, 2

  • This tube should be prioritized for microbiology studies as it has lower contamination risk than tube 1 2, 3
  • Gram stain has 92% sensitivity when patients have not received prior antibiotics and 0.1% false-positive rate 3

• Tube 3 (1-2 mL): Protein and glucose 1, 2

  • Must be paired with plasma glucose drawn just before lumbar puncture for accurate interpretation 1
  • Normal CSF glucose is >35 mg/dL with CSF-to-blood ratio >0.23 2

• Tube 4 (2 mL minimum): Virology PCR and storage for future testing 1, 2

  • Should include HSV-1, HSV-2, VZV, and enterovirus PCR, which identifies 90% of viral encephalitis cases 1, 2
  • Store frozen for additional testing as clinical picture evolves 2

Critical Volume and Processing Requirements

Collect adequate volumes to prevent false-negative results:

• Minimum 5 mL total for standard testing, with 8-10 mL required for comprehensive panels 2
• Larger volumes (5-10 mL) significantly increase sensitivity for mycobacterial and fungal cultures 2
• Process within 30 minutes to prevent cellular degradation; if immediate processing is impossible, fix with ethanol/Carbowax (1:1 ratio) 2
• Insufficient volume is a leading cause of false-negative results 2

Additional Tubes for Specific Clinical Scenarios

When tuberculosis or fungal infection is suspected:

• Tube 5 (6 mL): Mycobacterium tuberculosis culture and acid-fast bacillus smears 1, 2
  • Indicated in immunocompromised patients or when CSF protein >100 mg/dL with subacute onset 2

When immunocompromised:

• Additional testing should include cryptococcal antigen, fungal cultures, CMV PCR, JC virus PCR, and Toxoplasma gondii testing 2

Essential Opening Pressure Measurement

Record opening pressure before collecting any CSF:

• Opening pressure is critical for detecting elevated intracranial pressure and guiding management 2
• This measurement must be obtained before fluid removal begins 1, 2

Common Pitfalls to Avoid

Never delay antibiotics for CSF collection in suspected bacterial meningitis:

• If lumbar puncture is delayed for any reason (including CT scan), obtain blood cultures and start empirical antibiotics immediately 1, 2
• Delay in therapy increases morbidity and mortality from rapidly fatal pathogens like S. pneumoniae 1

Do not send tube 1 for bacterial culture:

• The first tube has the highest procedural contamination risk and will increase false-positive rates 2
• This practice reduces unnecessary workup of contaminants 3

Ensure paired serum glucose is drawn:

• CSF glucose interpretation is very difficult without simultaneous plasma glucose 1
• Draw blood glucose just before lumbar puncture 1, 2

Repeat lumbar puncture if initial results are non-diagnostic:

• Perform second LP at 24-48 hours, particularly important for HSV encephalitis where initial CSF may be normal in 5-10% of cases 1, 2