Cerebrospinal Fluid - Analysis

Introduction

Typical tests carried out on cerebrospinal fluid involve examining its overall physical characteristics such as color and cloudiness, performing a quantitative analysis to determine the number of red blood cells, total nucleated cell count (TNCC), and protein concentration, as well as a cytologic analysis to study the distribution and characterization of leukocytes, and to detect the presence of other cells or infectious agents.

Ideally, it is recommended to send cerebrospinal fluid (CSF) to a commercial laboratory that has the necessary equipment to perform the required tests, established reference intervals, and experienced clinical pathologists who can interpret cytologic preparations. However, if sending it to a commercial laboratory is not feasible, it is possible to approximate many, if not all, of these tests using standard equipment available in most clinics. A recent study has validated the use of a manual cytocentrifuge developed to obtain in-clinic cytospins of a variety of fluid samples.

It is important to note that cell counts and cytologic analysis of CSF require practice and expertise, so caution must be exercised while performing these tests.

Macroscopic Evaluation

During the macroscopic examination of cerebrospinal fluid (CSF), it is important to check for clots and assess its color against a white background. Good clarity is indicated by being able to read black print through the tube. Turbidity can occur when the total nucleated cell count (TNCC) exceeds 500/µL.

Protein Concentration

Evaluation of protein in cerebrospinal fluid, also known as microprotein concentration, is typically very low and consists mostly of albumin. The concentration of protein is typically higher in the lumbar sample than the CMC one. Unlike cytologic analysis, the protein analysis is not time-dependent. To accurately measure the protein concentration, specialized techniques and reagents such as Coomassie blue or pyrogallol red are required. The protein concentration in CSF is too low to be quantified by refractometry, but an in-house semi-quantitative approximation of the protein content of CSF can be performed using a standard urine dipstick protein pad. It's essential to keep in mind that dipsticks are more efficient at detecting albumin and may give a false negative result in cases of increased globulins. Protein can reliably be considered elevated based on a dipstick reading of 2+ or greater.

Table 1 - Using a urine dipstick for CSF protein analysis

Cell Counts

Total nucleated cell counts (TNCC) are most commonly performed using the standard hemocytometer technique. Coating the cells with new methylene blue using steps 1 through 5 below does not affect the cell counts, and it makes it easier to distinguish red blood cells from white blood cells. Although the data has not been published, this approach is very practical and feasible for a rapid approximation of red and white blood cell counts in emergencies.

Creation of a sedimentation technique for cytological analysis of CSF

Materials Required:

• Standard hole puncher

• Two binder-type clips

• Filter paper

• Glass microscope slide

• 1ml syringe, plunger removed, cut in half

Step-by-Step Technique:

1. Punch a hole in the middle of the filter paper and place it on the glass slide.

2. Center the flanged end of the syringe over the hole in the filter paper.

3. Clamp the flanges of the syringe barrel onto the microscope slide using the clips.

4. Load 0.25 to 0.5ml of CSF into the open end of the barrel.  

5. Allow the fluid to diffuse for 30 minutes.

6. Air dry the slide (do not heat fix).

7. Send the slide to a commercial laboratory or stain with a Romanowsky-type stain (such as Wright-Giemsa) for in-house examination.

Normal Values

Normal cerebrospinal fluid may contain various types of cells including those that line the CNS such as ependymal cells, choroid plexus cells, meningeal cells, and occasionally neurons. Rarely, hematopoietic cells may also be present, but these are likely to be collected from the bone marrow during the lumbar puncture and are not considered significant. Squamous epithelial cells may also be present as contaminants from the skin.

Effects of Blood Contamination

Cerebrospinal fluid normally does not contain red blood cells. However, when collecting CSF, especially from the lumbar cistern, it is common to get peripheral blood contamination. This can result in fluid that looks red but clears after centrifugation. There are other indicators of pathologic hemorrhage that will be discussed later.

The presence of peripheral blood in the sample affects the quantitative CSF analysis by increasing the erythrocyte count, total nucleated cell count, and total protein concentration. Although various ratios have been proposed to account for the extra leukocytes and protein in the CSF due to iatrogenic hemorrhage, one study has shown these to be unreliable. The red blood cells from hemorrhage are usually accompanied by little to no protein and no leukocytes. However, when the hemorrhage contributes leukocytes in excess of 10,000 per µl, these values may be altered, and it is better to repeat the procedure. The tap can be repeated in 24 hours to obtain a new sample.

Blood contamination (>500 RBC/µl) significantly increases the percentage of neutrophils, the total protein concentration, and the presence of eosinophils. However, it does not affect the presence of activated macrophages or reactive lymphocytes. This suggests that the presence of these cells may be a more specific indicator of neurologic disease in patients with normal TNCC and blood contamination.

Interpretation of Abnormal Cerebrospinal Fluid Results

Table 2 - Gross Appearance of CSF

Increased Total Protein

Elevation in cerebrospinal fluid protein concentration is a sensitive indicator of CNS disease, but it is the least specific change observed on CSF analysis and accompanies increased leukocyte counts virtually every time that abnormality is seen.  The concentration of protein in the CSF can be increased due to abnormalities in the blood-brain barrier or to intrathecal globulin production. Albuminocytologic dissociation is the term used to describe an elevation in the microprotein concentration with a normal leukocyte count.  This occurs with diseases that cause intrathecal production of protein, obstruct the flow of CSF and allow protein accumulation, or disturb the blood-brain barrier enough to allow protein from peripheral blood to enter the CNS. These conditions tend to be non-inflammatory in aetiology, though there are exceptions to this generalization.

Increased Total Nucleated Cell Count

Mixed Cell pleocytosis - no single cell type predominates; it contains a mixture of mostly lymphocytes and large mononuclear cells with smaller numbers of macrophages, neutrophils, and occasionally plasma cells and eosinophils.  Numerous conditions can be associated with a mixed pleocytosis, the most notable being granulomatous meningoencephalomyelitis (GME), in which the cell counts, are often markedly elevated.

Lymphocytic pleocytosis - characterized by an elevated TNCC with >50% lymphocytes.  It is the most common form of mononuclear pleocytosis and usually consists of a majority of small, mature lymphocytes with smaller numbers of reactive lymphocytes and larger monocytoid cells.  It occurs commonly in cases of viral diseases, notably canine distemper virus, in which the cell count is typically less than 50 cells/µl, but can also be associated with other chronic CNS infections.  Necrotizing meningoencephalitis and necrotizing leukoencephalitis cause a strongly lymphocytic (>80%) pleocytosis with a moderately to markedly elevated cell count. Rather than being mixed, the pleocytosis in GME frequently has a predominance of lymphoplasmacytic cells.

Neutrophilic pleocytosis - defined as an elevated TNCC with a predominance of neutrophils.  That being said, diseases known to cause neutrophilic pleocytosis should be considered when neutrophils constitute greater than two percent of the nucleated cells whether or not neutrophils are the predominant cell type.  In general, when there is a very high nucleated cell count, and neutrophils are the predominant cell type present, suppurative inflammatory processes (steroid responsive meningitis-arteritis, bacterial or fungal meningoencephalitis/meningoencephalomyelitis) must be heavily considered.  A mild to moderate neutrophilic pleocytosis can be associated with acute inflammatory disorders, including trauma, post-myelographic aseptic meningitis, encephalitis of unknown aetiology, haemorrhage, necrosis, infarction, and neoplasia. Historically, meningiomas have been associated with a neutrophilic pleocytosis, but various other tumor types can cause this change. Degenerate neutrophils may or may not be observed in cases of bacterial infection.

Eosinophilic pleocytosis - While the presence of eosinophils in the cerebrospinal fluid in the absence of significant peripheral blood contamination is not normal, it is often a nonspecific change.  A differential cell count with >10-20% eosinophils is considered an eosinophilic pleocytosis.  This is a rare finding and has been associated with an eosinophilic meningoencephalomyelitis of unknown etiology (EME) in both dogs and cats, aberrant parasitic migration, various infectious agents particularly protozoa and fungi, and, rarely, intervertebral disc disease.  The total nucleated cell count can be markedly elevated (>1000 cells/µl) in cases of EME, cryptococcosis, and Baylisascaris migration.