Q&A column

Editor: Frederick L. Kiechle, MD, PhD

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Q. The classic literature on collection tube order for cerebrospinal fluid testing usually says tube one is for chemistry, tube two is for microbiology, tube three is for hematology, and tube four, if collected, is extra. But I have seen some sources that say cell count testing should be done from tube four. In this scenario, tube one is extra, tube two is chemistry, tube three is microbiology, and tube four is hematology. What is the current standard practice?

A. July 2019—Normal CSF is a clear, colorless fluid, produced by the choroid plexus of the ventricles, that fills the ventricles and surrounds the brain and spinal cord. It delivers nutrients, removes waste, and cushions the brain and spinal cord from acute pressure changes. CSF is a valuable fluid for diagnosing infections, malignancy, and a variety of neurological and other conditions. Collection of CSF most commonly involves inserting a needle into the intervertebral space between the L3 and L4 lumbar vertebrae and removing five to 15 milliliters of fluid. The fluid is collected into sequentially labeled sterile tubes.

Procedures vary slightly between laboratories, but there are three areas of consensus. The first point is that a minimum of three tubes should be collected. The second point is that cell counts are most accurate when performed on the last tube collected. Thus, cytopathology and flow cytometry are typically also performed on the last tube collected so these results can be correlated with the cell count data. The reasoning behind this is that the last tube collected would be the least likely to be contaminated by blood or debris from the collection process. The third point of consensus is that testing should be performed as quickly as possible to ensure that the most accurate data are collected.

Review of multiple large reference laboratories’ procedures posted online and the literature showed the most common testing protocols for three collected tubes involved performing chemistry and immunological testing on tube one, cultures and PCR testing on tube two, and cell counts on tube three. If four adequate samples were collected, tube one was held in reserve (as it is the most likely to have blood or debris contamination), tube two was used for chemistry and immunological testing, tube three was used for microbiologic testing, and tube four was used for cell counts and other analyses. This implies that the samples should be evaluated for adequacy if four tubes are used to ensure that ample sample is present for the needed analysis. If the fourth tube is scanty, the laboratory may want to revert to using the first tube for chemistry and immunological testing rather than possibly compromising the data collected from the CSF collection procedure.

1. Morgenlander JC. Lumbar puncture and CSF examination. Postgrad Med. 1994;95(8):125–131.
2. Karcher DS, McPherson RA. Cerebrospinal, synovial, serous body fluids, and alternative specimens. In: McPherson RA, Pincus MR, eds. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 23rd ed. St. Louis: Elsevier; 2017:481.
3. Perkins SL, Couturier MR, Grenache DG, Kjeldsberg CR. Cerebrospinal fluid. In: Hussong JW, Kjeldsberg CR, eds. Kjeldsberg’s Body Fluid Analysis. Chicago: ASCP Press; 2015:45.

Roberta L. Zimmerman, MD
Clinical Laboratory Director
Northern Pathology Services
Grand Rapids, Minn.
Member, CAP Hematology/Clinical
Microscopy Committee