Collection & Handling

Before we talk about a single test, we must discuss the sample itself. Of all the specimens that arrive in the clinical laboratory, cerebrospinal fluid is perhaps the most precious and the most unforgiving

Why?

1. It is difficult and invasive to obtain.: A lumbar puncture is not a simple blood draw. It is a sterile, medical procedure with inherent risks to the patient, including headache, bleeding, and nerve damage. Repeat collections are highly undesirable
2. The volume is extremely limited.: We may only receive a few milliliters of fluid to answer life-or-death questions
3. The results are often critical and time-sensitive.: The difference between a diagnosis of viral and bacterial meningitis can be the difference between a patient going home in a few days and suffering permanent neurological damage or death

As laboratory scientists, we are the guardians of this specimen from the moment it leaves the patient until the final result is reported. Our actions in the first 30 minutes after collection can dictate the entire clinical course for the patient. There is no room for error

Procedure: Lumbar Puncture (LP)

While we do not perform the LP, understanding the procedure is essential for interpreting potential pre-analytical issues

• Patient Positioning: The patient is typically in a lateral decubitus (fetal) position to open the vertebral spaces
• Site: The needle is inserted under sterile conditions into the subarachnoid space, usually between the L3-L4 or L4-L5 vertebrae. This is below the termination of the spinal cord to prevent injury
• “Opening Pressure”: A manometer is often attached to the needle to measure the intracranial pressure before any fluid is removed. This is a vital diagnostic sign
• Collection: CSF is allowed to drip passively into sterile, numbered collection tubes. It should never be aspirated with a syringe, as this can cause a pressure shift and damage nerve roots

The Sacred Order: CSF Tube Collection

This is the single most important concept in CSF collection. The tubes are collected sequentially, and each tube has a designated purpose based on the potential for contamination introduced during the puncture itself

Standard Collection: 3 to 4 Tubes

• Tube #1
  • Primary Department(s): Chemistry / Serology
  • Rationale: This tube is the most likely to be contaminated with blood from the puncture (a “traumatic tap”), tissue fluid, or skin cells. These contaminants have the least impact on protein and glucose measurements
  • Potential Consequences of Error: Using this tube for cell counts would yield a falsely elevated RBC and WBC count. Using it for microbiology could result in a false-positive culture from skin flora
• Tube #2
  • Primary Department(s): Microbiology
  • Rationale: By the second tube, any initial skin contaminants from the needle have likely been flushed out. This provides a cleaner sample for Gram stain and culture, reducing the risk of a false-positive result
  • Potential Consequences of Error A bloody Tube #2 can inhibit bacterial growth. Using Tube #1 could lead to an incorrect diagnosis of skin-contaminant meningitis
• Tube #3
  • Primary Department(s): Hematology (Cell Count)
  • Rationale: This tube is the “cleanest” of the first three. It is least likely to have peripheral blood contamination from the tap itself, providing the most accurate reflection of the cells actually present in the patient’s CNS
  • Potential Consequences of Error Using a bloody Tube #1 would make it impossible to determine if the RBCs are from a hemorrhage or the tap, leading to a major diagnostic error
• Tube #4
  • Primary Department(s): Special Studies / Hold
  • Rationale: This tube can be used for additional, specialized tests like cytology (cancer cells), oligoclonal banding (MS), VDRL (neurosyphilis), or simply held in case add-on tests are ordered
  • Potential Consequences of Error: N/A

Mnemonic: Think of it like a faucet. The first splash of water (Tube 1) might have some rust from the pipes, the next (Tube 2) is cleaner, and the next (Tube 3) is the purest

Specimen Handling: The STAT Imperative

Once collected, the clock starts ticking. All CSF specimens must be hand-delivered to the laboratory immediately and treated as STAT. The word STAT is not a suggestion

Why the Urgency?

1. Cell Lysis: Both RBCs and WBCs begin to disintegrate rapidly after collection, especially at room temperature
   • WBCs: Neutrophils are particularly fragile. Up to 40% of WBCs can lyse within 2 hours. A delay can falsely lower the cell count, potentially masking a bacterial meningitis diagnosis
   • RBCs: Lysis of RBCs leads to the formation of xanthochromia in vitro, which can be confused with a true subarachnoid hemorrhage
2. Microbiology Integrity
   • Fastidious organisms like Neisseria meningitidis or Haemophilus influenzae may die if left at room temperature or refrigerated
   • Contaminating bacteria, if present, can multiply, leading to a falsely high colony count

Laboratory Processing & Storage Protocol

Upon arrival in the lab, a CSF specimen triggers a specific workflow:

1. Accessioning: The specimen is logged in, and labels are meticulously checked against the requisition. Time of collection and receipt are documented
2. Immediate Distribution: The tubes are immediately delivered to their respective departments
   • Tube #1 (Chemistry) -> Chemistry Dept
   • Tube #2 (Microbiology) -> Microbiology Dept
   • Tube #3 (Hematology) -> Hematology Dept
3. Storage Guidelines (If immediate testing is impossible)
   • Hematology: Must be analyzed within 1 hour. If a delay is unavoidable, refrigerate at 4°C to preserve cell morphology
   • Microbiology: Keep at room temperature. Refrigeration can kill sensitive organisms
   • Chemistry/Serology: Can be stored frozen or refrigerated. Centrifuge and separate supernatant to prevent cellular contamination of results

Safety: Universal Precautions on High Alert

CSF is potentially one of the most infectious specimens you will handle. Assume every CSF is infectious until proven otherwise

• Personal Protective Equipment (PPE): Gloves, lab coat, and a face shield/goggles are mandatory
• Biological Safety Cabinet (BSC): All processing that can create aerosols (e.g., vortexing, opening tubes, making smears) must be performed in a Class II BSC
• High-Risk Pathogens: Be mindful of Neisseria meningitidis, Cryptococcus neoformans, and the prions that cause Creutzfeldt-Jakob Disease (CJD)
  • CJD: If CJD is suspected, the specimen requires special handling protocols, and all equipment must be treated with specific prion-inactivating disinfectants (e.g., sodium hydroxide, concentrated bleach) or incinerated. Standard autoclaving is not sufficient

Pre-Analytical Complications: The Art of Troubleshooting

Traumatic Tap vs. Subarachnoid Hemorrhage (SAH)

This is the most common and most critical pre-analytical challenge. The lab plays a pivotal role in differentiation

Traumatic Tap

• RBC Count
  • Uneven distribution of blood across the collection tubes
  • The RBC count is highest in Tube #1 and progressively decreases in Tubes #2 and #3
• Clot Formation
  • The sample may form clots.
  • This occurs because the puncture introduced plasma fibrinogen from the peripheral blood into the CSF
• Supernatant (after centrifugation)
  • Clear and colorless.
  • The red blood cells are from a fresh bleed and have not had time to lyse
• Microscopic Examination
  • Shows fresh, intact RBCs
  • Erythrophages and hemosiderin-laden macrophages are not present

Subarachnoid Hemorrhage (SAH)

• RBC Count
  • Even distribution of blood across the collection tubes
  • The RBC count is consistent in Tubes #1, #2, and #3
• Clot Formation
  • The sample will not clot.
  • The blood has been in the subarachnoid space long enough for in-vivo defibrination to occur
• Supernatant (after centrifugation)
  • Xanthochromic: (pink, orange, or yellow)
  • This is the definitive finding, caused by the breakdown of RBCs that occurred inside the body before the tap. Xanthochromia typically appears 2-4 hours after the initial bleed
• Microscopic Examination
  • May show erythrophages: (macrophages that have engulfed RBCs)
  • After 12-24 hours, hemosiderin-laden macrophages: may be present, which is conclusive evidence of a previous bleed

Insufficient Volume (Quantity Not Sufficient - QNS)

• When a “short draw” occurs, the lab must prioritize testing. This requires communication with the clinical team
• Standard Prioritization
  1. Microbiology: An undiagnosed infection is the most immediate threat to the patient’s life
  2. Hematology (Cell Count/Differential): Essential for differentiating the type of infection
  3. Chemistry: While important, results are often less acute than the first two

Conclusion

Mastering CSF handling is a hallmark of a competent laboratory scientist. It is a discipline of precision, urgency, and critical thinking. By respecting the specimen, adhering strictly to procedure, and understanding the “why” behind every step, you ensure that the laboratory provides not just a result, but a clear, accurate, and potentially life-saving diagnosis. Never forget: for this specimen, every drop counts and every minute matters