Andrea Siegel IDEXX Laboratories, Inc, New York, NY, USA Cerebrospinal fluid (CSF) analysis combined with the history, physical and neurological exam, as well as other diagnostic tests, is an important component of the diagnostic evaluation of patients with central and peripheral neurological disease. CSF should be collected whenever an inflammatory, infectious, traumatic, neoplastic, or degenerative disorder of the brain and spinal cord is suspected. However, analysis of CSF often only supports the diagnosis of a central nervous system (CNS) disorder and is rarely definitively diagnostic. Similar to a complete blood count, CSF analysis has good sensitivity for disease detection but low specificity. However, CSF analysis is not always abnormal in a horse with central nervous disease if the lesion is extradural, collection occurred early or late in the course of disease, or the CSF collection site is far away from the lesion. The CSF is a clear, colorless fluid that surrounds and permeates the entire CNS and therefore protects, supports, and nourishes it. The CSF is produced by ultrafiltration and active transport mechanisms in the choroid plexuses. Fluid formed in the lateral ventricles flows into the third ventricle and then into the fourth ventricle, where the majority exits into the subarachnoid space. The CSF also moves caudally, surrounding the spinal cord and leaving the CNS via the dural reflections of the spinal nerve roots. The CSF is absorbed by the arachnoid villi and ultimately directed into the venous system. The CSF functions include physical support of the neural structures, excretion of potentially toxic by‐products of cerebral metabolism, and intracerebral transport of biologically active substances [1]. The diagnostic quality of CSF depends on collection site and handling. CSF can be collected in horses from the atlantooccipital (AO) or lumbosacral sites depending on the neurological exam and location of the lesion. An intracranial or cervical lesion is more likely to yield a diagnostic sample if the CSF is obtained from an AO tap. In horses with clinical signs of brain herniation, the AO space should be avoided. Lumbosacral aspirates are indicated for lower spinal cord lesions. AO collection requires general anesthesia, whereas lumbosacral collection usually can be performed in standing horses. Ultrasound‐guided thecal puncture techniques to collect CSF on the standing horse using the AO and C1–C2 locations have been described [2, 3]. In foals, CSF can be collected from either site with the animal in lateral recumbency [4]. Techniques of cerebromedullary cisternal and lumbosacral CSF collection are aptly covered in neuroanatomy and clinical neurology textbooks. Specific risks of CSF collection include iatrogenic brainstem trauma or spinal cord trauma due to needle puncture, extradural hemorrhage, and herniation of the cerebrum or cerebellum through the foramen magnum in patients with increased intracranial pressure [5]. An aseptic technique is necessary due to the potential risk for introducing infectious agents into the CNS. A study that compared sequential samples of CSF in horses suggested that in order to minimize blood contamination, a minimum of three 2 mL samples should be collected before submitting the final sample for analysis, even if there is no gross evidence of blood contamination [6]. Artefacts like deteriorated cells and contaminants (including iatrogenic hemodilution) may affect the cytological interpretation. The routine laboratory analysis of CSF includes gross inspection, cell counts, protein concentration, chemistry assays, and cytological examination (Table 19.1). Other tests, such as bacterial and fungal culture, serology, and polymerase chain reaction (PCR), are added depending on the results of the cytological examination, clinical presentation, and neurological exam. Due to the low CSF protein and lipid concentrations, cells degenerate and lyse rapidly, causing distortion of cellular morphology and reduction of the total nucleated cell count. For this reason, cell counts and cytological preparations should be performed within 30 minutes of collection [7]. Cells can be preserved by mixing autologous serum with the sample, which will conserve cell morphology for 24 hours when stored at 4 °C [8]. The addition of hetastarch at a ratio of 1:1 (vol:vol) or fetal calf serum at a concentration of 20% volume can help to stabilize cells in CSF [7]. Samples containing additives to preserve cellular morphology should not be used to measure protein or for antibody titers. Generally, 1 mL of fluid is sufficient for protein and cellular examination [9]. CSF samples should be collected in sterile plastic or silicon‐coated tubes, especially when not processed immediately, because monocytes will adhere to glass and activate [1]. EDTA tubes are not recommended because the additive can falsely elevate the total protein concentration, although submission in an EDTA tube may be required if the sample is for PCR analysis [10, 11]. The normal CSF is clear and colorless, has a viscosity similar to water, and does not clot due to the absence of fibrinogen. Turbidity is measured subjectively from 0 or clear to +4, which is cloudy enough to preclude reading through the sample. Causes of CSF turbidity include elevated cell count (WBC or RBC or both), presence of microorganisms, markedly increased protein concentration, or aspiration of epidural fat [12]. Nucleated cell counts greater than 400–500 cells/μL are necessary to produce a slightly turbid CSF. Table 19.1 Reference values for CSF in adult horses. Source: Modified from [10]. Figure 19.1 Macroscopic examination of CSF. Tubes are viewed against a lined white index card to assess colour and turbidity. (Left to right) Normal CSF, mildly xanthochromic CSF, moderately xanthochromic CSF, red‐tinged turbid CSF caused by hemorrhage, and cloudy red‐tinged fluid from a horse with bacterial meningitis. Abnormal CSF colors on visual inspection include red‐tinged, xanthochromic, and cloudy white (Figure 19.1). Pink or red discoloration suggests the presence of blood, which could result from iatrogenic blood contamination, intrathecal hemorrhage, or diapedesis secondary to blood–brain barrier (BBB) disruption. A clear supernatant after centrifugation indicates iatrogenic hemorrhage or peracute hemorrhage in the subarachnoid space. On the other hand, a red‐tinged CSF with a xanthochromic supernatant (yellow‐orange coloration) supports prior hemorrhage, likely associated with trauma, neoplasia, vascular disorders, or infectious disease [13]. Xanthochromia is caused by the erythrocyte degradation products oxyhemoglobin and bilirubin. Markedly increased CSF protein (>400 mg/dL) and severe icterus are other causes of xanthochromia. Normal neonates may have slightly xanthochromic CSF [5]. Cytological evidence of pathological hemorrhage includes the presence of hemosiderin, hematoidin, and/or erythrophagocytosis. Increased fluid viscosity usually results from a very high protein concentration, especially fibrinogen. Cryptococcosis may increase the CSF viscosity due to the polysaccharide capsule of the yeast [1]. Reference intervals for CSF total protein concentration can vary with the laboratory and testing method used. Therefore, laboratory‐established reference ranges should be used. Normal CSF has very low protein concentration compared to plasma and other body cavity fluids. Most of the protein in normal CSF is albumin. Refractometric methods are not accurate for the measurement of CSF protein content. Refractive index of CSF can be artefactually increased by suspended particles (cells, bacteria), hemoglobin, or radiographic contrast media [13, 14]. Urine protein dipsticks have been used to estimate CSF protein concentration; however, they are highly specific for albumin detection and less specific for globulins [15]. The Pandy and Nonne‐Apelt tests are qualitative methods that screen for the presence of globulins. These tests have been replaced by quantitative methods that measure immunoglobulins. Quantitative methods that measure both albumin and globulin in CSF include turbidimetric procedures (trichloroacetic acid) and dye‐binding assays (Coomassie brilliant blue, Ponceau S acid red, and pyrogallol red). Dye‐binding protein methods are simple, rapid, and more accurate for the measurement of CSF protein concentration [1, 13]. The pyrogallol red assay gives a more uniform response to albumin and globulin and therefore is the preferred method [16]. Reported equine CSF protein values range from 10 to 120 mg/dL (generally 20–80 mg/dL), which are much higher than those reported in other domestic animals. CSF protein concentration in foals less than one week old ranges from 90 to 180 mg/dL, decreasing gradually to adult horse levels by two weeks of age [4]. Enhanced transport of albumin into CSF from blood or increased permeability of the BBB may be the reason for the difference between adult and neonatal CSF concentration [4, 17]. Nonpathological elevation of protein in CSF occurs with blood contamination during collection. Pathological causes of elevated CSF protein include damage to the BBB, intrathecal hemorrhage, increased local synthesis of immunoglobulins, degeneration of neural tissue, or obstruction of the CSF circulation (e.g., cervical vertebral malformations, abscess, or tumor). Because albumin is not synthesized intrathecally, increased CSF albumin indicates damage to the BBB, iatrogenic blood contamination, or intrathecal hemorrhage. If hemorrhage or blood contamination is not present, the ratio between CSF albumin and serum albumin (albumin quotient) can be used to assess the functionality of the BBB: BBB damage in horses is suggested by CSF albumin concentration greater than 2.45 with an albumin quotient greater than 2.35. However, due to the normal variability of CSF albumin in horses, the use of this index is limited [17]. Electrophoretic techniques define the gamma‐globulins as a heterogeneous group of proteins with similar migration rates; this fraction contains the immunoglobulins. High‐resolution agarose gel electrophoresis (HRE) produces sharper bands and definition of CSF protein fractions [18]. Electrophoresis should be performed on CSF and serum simultaneously to compare the distribution of proteins. Three major immunoglobulins are found in CSF: IgG, IgM, and IgA. IgG is the major CSF immunoglobulin present in normal CSF. Since CSF IgG originates from plasma, elevated concentrations of IgG in CSF may indicate an abnormal BBB or increased intrathecal synthesis by inflammatory cells. An IgG index is used to determine whether the IgG present is due to local synthesis or originates from the plasma (alteration in the blood–brain CSF barrier, intrathecal hemorrhage, or iatrogenic blood contamination): An IgG index greater than 0.3 with a normal albumin quotient suggests intrathecal IgG production, which is generally caused by infectious inflammatory diseases [19]. The IgG index may be useful in differentiating inflammatory lesions from noninflammatory diseases. Other techniques such as the antibody index and C‐value, that use antigen‐specific antibody titers rather than total IgG, are believed to be more accurate than the IgG index [20]. The antibody index relates a specific IgG titer to the total IgG concentration, allowing the detection of a fraction of specific antibody made in the CNS. Movement of a specific antibody into the CSF due to leakage across a porous barrier or blood contamination during sample collection would not be expected to affect the antibody index [21, 22]. The C‐value theory is that the ratio of antigen‐specific antibody to total IgG in CSF is equal to the ratio in the serum. Values >1.7 are suggestive of intrathecal antibody production [19]. The presence of IgM is an abnormal finding in the CSF and is considered more specific than IgG or total immunoglobulin levels for detection of active or recent infectious disease [14]. Serum IgM capture ELISA (MAC‐ELISA) is the preferred method for the diagnosis of West Nile virus infection in vaccinated and unvaccinated horses due to high sensitivity and specificity. Because there is 100% agreement between serum and CSF results, there is no clear advantage in testing CSF rather than serum. However, future introduction of live attenuated vaccines that may elicit a serological IgM response suggests that CSF testing may become the gold standard for diagnosing West Nile disease in vaccinated horses [23]. Elevated CSF protein concentration may be found in cases of encephalitis, neoplasms, meningitis, and trauma. Typically, neoplasms or trauma produce an increase of albumin, because of disruption of the BBB. In contrast, in inflammatory diseases such as encephalitis and meningitis, globulins are the primary protein increased, due to intrathecal production [23]. The CSF nucleated cell count and CSF protein concentration tend to increase proportionately. However, in some disorders the cell count remains normal, whereas the total protein concentration may be increased. This condition is called albuminocytological dissociation, which has been described in viral nonsuppurative encephalomyelitis such as equine herpes virus meningoencephalitis (EHV‐1), neoplastic disease, and with traumatic, vascular, degenerative, and compressive spinal cord lesions [14]. A variety of antibody and antigen tests are available for viruses, fungi, rickettsia, protozoa, and parasites [24]. In order to evaluate antibody titers, two samples taken two weeks apart should be analyzed. Antibodies detected in CSF may be derived from vaccination, antigen exposure, or actual disease. Interpretation of CSF antibody titers should be done in light of the vaccination history, serum titers, BBB integrity, intrathecal immunoglobulin production (IgG index), and CSF IgM levels. Antibody titers for EHV‐1, togavirus, West Nile virus, and equine protozoal myelitis (EPM) (Sarcocystis neurona) can be measured in CSF [5, 25, 26]. Titers can be measured using Western blot, a semiquantitative antibody‐based test, or indirect immunofluorescence indirect fluorescent antibody test (IFAT
19
Cerebrospinal Fluid
19.1 Formation, Circulation, Absorption, and Function
19.2 Collection
19.3 Laboratory Analysis
19.3.1 Gross Appearance
Macroscopic evaluation
Reference values
Color
Colorless
Turbidity
Clear
Erythrocytes
0 to small number
Total nucleated cell count
0–8 cells/L
Total protein
30–80 mg/dL
Microscopic evaluation
Differential cell count
Small lymphocytes (70%)
Monocytoid cells (30%)
Rare erythrocytes
19.3.2 Protein Concentration
19.3.3 Antibody Titers
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