The function, composition and analysis of cerebrospinal fluid in ...

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The Veterinary Journal 180 (2009) 15–32

TheVeterinary Journal

Review

The function, composition and analysis of cerebrospinal fluidin companion animals: Part II – Analysis

Roberta Di Terlizzi a,*, Simon R. Platt b

a Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USAb Department of Small Animal Medicine, College of Veterinary Medicine, University of Georgia, GA 30602-7371, USA

Accepted 25 November 2007

Abstract

Accurate analysis of cerebrospinal fluid (CSF) provides a wide range of information about the neurological health of the patient. CSFcan be withdrawn from either of two cisterns in dogs and cats using relatively safe techniques. Once CSF has been collected it must beanalysed immediately and methodically. Evaluation should consist of macroscopic, quantitative and microscopic analyses. As part of aquantitative analysis, cell counts and infectious disease testing are the most important and potentially sensitive indicators of disease.Although certain pathologies can be described, microscopic analysis will rarely be specific for any disease, emphasising the adjunctivenature of this diagnostic modality.Published by Elsevier Ltd.

Keywords: Cerebrospinal fluid; Analysis; Dog; Cat; Neurological disease

Introduction

Accurate analysis of cerebrospinal fluid (CSF) providesa wide range of information about the neurological healthof a patient. Similar to a complete blood count, CSF hashigh sensitivity but low specificity for the detection of dis-ease. The possible abnormalities of CSF are relatively lim-ited given the varieties of neurological disease that exist.CSF analysis is not always abnormal with neurological dis-eases (Braund, 2003) but occasionally it will help to providea specific diagnosis. For these reasons, accurate anamnesis,physical and neurological examinations, imaging studiesand other diagnostic tests are essential for an accurateand correct interpretation of CSF changes in an individualcase (Chrisman, 1992).

The first part of this paper discussed the function andcomposition of CSF (Di Terlizzi et al., 2006). This review

1090-0233/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.tvjl.2007.11.024

* Corresponding author. Tel.: +1 515 450 6385; fax: +1 515 294 6906.E-mail address: [email protected] (R. Di Terlizzi).

will address the collection, sample processing and completeanalysis of CSF in companion animals.

CSF collection

CSF analysis may be useful as an important componentof the diagnostic evaluation of patients with central andperipheral neurological disease. CSF should be collectedwherever an inflammatory, infectious, traumatic, neoplas-tic or degenerative disorder of the brain and the spinal cordis suspected. However, the analysis of CSF may only sup-port the diagnosis of a central nervous system (CNS) disor-der and is rarely definitively diagnostic.

CSF can be collected from the cerebellomedullary cis-tern (CMC) or the caudal lumbar subarachnoid space.Because the fluid flows predominantly in a rostro-caudaldirection, it is more diagnostic and therefore preferableto collect it from a site caudal to the suspected lesion(Thomson et al., 1990). Collection of CSF requires thepatient to be under general anaesthesia with the site ofcollection clipped and aseptically prepared. No more than

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16 R. Di Terlizzi, S.R. Platt / The Veterinary Journal 180 (2009) 15–32

1 mL of CSF per 5 kg bodyweight (in dogs, cats andhorses) should be collected (Carmichael, 1998). The equip-ment required for sampling includes sterile plain collectiontubes (polymerase chain reaction [PCR], will requireEDTA tubes), 20–22 G 40–90 mm (1.5–3.5 in.) spinal nee-dles, and sterile surgical gloves. Small hypodermic needlesfor CSF collection (22 or 25 G) may be useful in small dogsand cats and are safer if the operator is inexperienced inassessing the depth of the relative cisterns from the skinsurface. In such patients there may not be an obvious ‘sen-sation’ that accompanies penetration of the dura; a needlewithout a stylette, such as a hypodermic needle, will helpthe user identify the time of puncture of the dura with a‘flash’ of CSF that simultaneously appears in the hub. Aspinal needle may be used, removing the stylette after pierc-ing the skin, but the larger size needle makes the procedurecumbersome in a small dog.

Risks and contraindications

General anaesthesia is required for CSF collection, andanaesthesia needs to be considered as a risk for everypatient with intracranial disease. A specific risk of CSF col-lection is iatrogenic brainstem trauma or spinal cordtrauma due to needle puncture (Platt et al., 2005; LujanFeliu-Pascual et al., 2006). An aseptic technique is requiredbecause of the potential for introducing infectious agents tothe CNS (Cook and DeNicola, 1988).

A frequently documented specific contraindication toCSF collection is that of increased intracranial pressure(ICP). Increased ICP is generally a non-specific findingand may be associated with space occupying lesions, cere-bral trauma, hydrocephalus, and inflammatory CNS dis-eases (Braund, 2003). Normal values of pressure are<170–180 mm H2O for dogs and <100 mm H2O in cats(De Lahunta, 1983; Braund, 1986; Oliver and Lorenz,2005). However, practically, ICP and associated cerebralperfusion pressures are rarely measured and so reliance isplaced upon clinical signs suggestive of increased ICP.

The neurological signs associated with increased ICPare unfortunately non-specific and include altered menta-tion (depression progressing to stupor and coma), pare-sis, poor pupillary response to light, abnormal and/orunequal pupil size, decerebrate and decerebellate rigidityand vertical nystagmus (Smith and Madsen, 2004). Someof these signs may only appear terminally as a conse-quence of brain herniation. Caudal transtentorial hernia-tion results in pressure exerted downward through themidbrain with subsequent compression of the oculomo-tor nerve nucleus resulting in pupil dilation and poorresponse to light (Kornegay et al., 1983). With unilateralherniation, an ipsilateral dilated pupil, unresponsive tolight stimulation, may be seen. These signs may be toounreliable to assist with the detection of increased ICPand subsequent herniation as oculomotor palsy specifi-cally occurred in only 14% of 54 dogs with brain herni-ation (Walmsley et al., 2006).

Increased ICP may sometimes be associated with sys-temic manifestations of increased blood pressure anddecreased heart rate as part of the so-called Cushing’sresponse (Jones, 1989). In dogs and cats, heart rate andblood pressure may be more accurate indices of a suddenchange in ICP. Suggested heart rates <60 and blood pres-sures of >180 mm Hg may indicate elevated ICP in people,but there is currently no evidence for this in veterinarymedicine. In patients with elevated ICP, a needle insertedin the subarachnoid space in either the cisterna magna orthe lumbosacral space may produce a pressure gradientsufficient to cause a caudal shift of intracranial structures(herniation) (Evans, 1988; Rand et al., 1994b). If thereare clinical signs suggestive of elevated ICP, and advancedimaging is not available prior to CSF collection, consider-ation should be given to pre-administration of an osmoticdiuretic such as intravenous mannitol. Further aggressivetreatment may include hyperventilation but it is essentialto keep the patient’s pCO2 level above 35 mm Hg to avoidvasoconstriction induced ischaemia. The overall risk of cis-ternal puncture in patients with underlying intracranial dis-ease has not been documented but is low in the authors’experience.

Other contraindications to CSF collection include thesuspicion of an underlying coagulopathy, atlanto-axialsub-luxation, Chiari-like malformation or cervical trauma.As with any procedure the risk and benefit of CSF collec-tion should be considered in each case.

Technique

CMC collection

Patients are positioned in lateral recumbency, with theskull and cervical vertebrae at the edge of the table, andthe skull fully flexed to create a 90� angle with the cervicalspine. A reinforced endotracheal tube may be used toreduce the chance of ‘kinking’, and the cuff may be deflatedat the point of maximum neck flexion to reduce the chanceof tracheal trauma. The nose is slightly elevated in order toposition the long axis of the muzzle parallel to the table.The entry site for the spinal needle is at the intersectionof imaginary lines drawn from the occipital protuberanceto the dorsal arch of C2 horizontally and along the rostralaspect of the wings of the atlas vertically. The needleshould be kept perpendicular to the dorsal laminae of thevertebral column, at the level of the atlanto-occipital space,and advanced very slowly through the skin (Fig. 1).

Other methods of determining the site of puncture havebeen described, but the aforementioned method is pre-ferred by the authors. As it is advanced, resistance maybe felt just before the needle pierces through the atlanto-occipital ligament, the meninges (dura mater and arach-noid) and into the cisterna magna. The stylette of the spinalneedle is removed at this point, and the fluid is allowed toflow into plain sterile collection tubes. Many times thisresistance is not felt, particularly in small dogs and cats.

Fig. 1. CSF collection from the cerebro-medullary cistern (CMC). Thedog is in right lateral recumbency with the head to the right of the picture(arrow). Gloves are worn by the operator when handling the needle. Theoperator’s left hand then palpates the wings of the first cervical vertebra,whilst the right hand simultaneously palpates the occipital protuberanceand carefully inserts the needle. The insertion point is at the intersection ofa vertical line delineating the cranial aspect of the wings of C1 (arrowhead)and a horizontal line joining the occipital protuberance and the dorsalarch of C2.

Fig. 2. CSF collection from the lumbar cistern. The dog is in right lateralrecumbency with the caudal end indicated by the arrow. A sterile area isprepared over the L5 to S2 vertebrae as shown. Sterile gloves are worn tohandle the needle but drapes are not required to ensure sterility. Theinsertion point is at the cranial aspect of the dorsal spinous process of L6(arrowhead) and the needle is inserted perpendicular to the spinal column.

R. Di Terlizzi, S.R. Platt / The Veterinary Journal 180 (2009) 15–32 17

To prevent cord damage, reliance should not be placed onthe presence of this sensation and, instead, either the sty-lette can be withdrawn once the needle is in the muscleor a hypodermic needle can be used. In each case, CSF will‘flash’ back into the hub as soon as the cisterna is entered.If the needle hits bone during its passage, the needle may beslightly withdrawn and redirected more caudally. If bloodappears in the hub of the needle, the needle should be with-drawn at once and the procedure repeated following re-assessment of the anatomical landmarks.

Generally, 0.75–2 mL fluid is sufficient for protein andcellular examinations (Cook and DeNicola, 1988). In theauthors’ experience, most laboratories can comfortablyassess protein levels, cytology and cell counts when suppliedwith 0.5 mL of CSF. A few drops should be saved in separateplain tubes for microbial culture and sensitivity if infection issuspected, and for virological and immunological studies, ifneeded. An EDTA tube is necessary if the sample is for PCRanalysis. Once collection is completed, the needle is gentlyremoved from the site of collection, and if further fluid isrequired, the needle should be placed over the collection tubeas it empties out its contents.

Lumbar collection

Technically, lumbar collection is more difficult to per-form than the CMC collection and more likely to resultin iatrogenic blood contamination. The patient is posi-tioned in lateral recumbency, with the pelvic limbs fullyflexed (Fig. 2). The appropriate intervertebral space isL5–L6 in dogs and L6–L7 in cats (Oliver and Lorenz,

1997). At these spaces, the spinal cord has tapered intothe conus medullaris and is surrounded by nerve roots orthe cauda equina, which are much less likely to be damagedby needle insertion than the cord itself. The subarachnoidspace rarely extends to the lumbosacral junction in dogs(Oliver et al., 1987), whereas collection may sometimes bemade from the lumbosacral space in cats.

The needle is inserted just caudal to the space of interest,perpendicular to the dorsal laminae of the vertebrae, alongthe cranial border of the caudal dorsal spinous process. Ifthe needle hits bone, it should be moved a few millimetrescranially or caudally. In most medium to large sized dogs,it is necessary to use 20 G needles to reduce needle bendingfollowing the necessary manipulation. A slight twitch ofthe tail or leg may occur upon insertion of the needle insidethe canal due to stimulation of the nerve roots or cauda equ-ina following irritation or penetration by the needle. The nee-dle is often inserted until it contacts the bone of the ventralaspect of the spinal canal; the stylette is then removed andthe fluid collected as above. If no fluid appears, very slightrotation and/or withdrawal of the needle should encourageCSF flow. The rate of fluid flow is usually slower than fromthe CMC, and the fluid quantity retrieved less.

Sample processing

CSF samples should be collected in sterile plain tubes.EDTA tubes are not recommended for routine sampling,as the additive can falsely elevate the total protein concen-tration (Parent and Rand, 1994).

A significant distortion of cell structure and reduction oftotal nucleated cell count (TNCC) from cell lysis may occurif processing is delayed (Fry et al., 2006). This fragility is


partly related to the typically low CSF protein levels, lipidconcentration and tonicity, factors which in other fluidscontribute to stabilisation of cell membranes (Steeleet al., 1986). If processing is delayed for longer than 1 h,cellular changes occur, which include nuclear pyknosis,lysis and disintegration of the cytoplasmic and nuclearmembranes. At room temperature, neutrophils degeneratemost rapidly (within 1 h) and lymphocytes and monocytesstart to degenerate after 3 h (Kjeldsberg and Knight, 1986).However, a recent study on CSF stored at 4 �C withoutadditives and analysed at 2, 4, 8, 12, 24, and 48 h demon-strated that small mononuclear cells deteriorate most rap-idly, followed by large mononuclear cells, neutrophilsand then eosinophils (Fry et al., 2006). Eosinophil structurewas frequently unchanged after 48 h of storage. Cells of thesame type started to degenerate with considerable terminalvariation, and after 48 h cells tended to clump or adhere toeach other (Fry et al., 2006).

Delaying analysis of canine CSF by 4–8 h is unlikely toalter diagnostic interpretation if the protein concentrationis P50 mg/dL (Fry et al., 2006). Therefore, addition offetal calf serum (FCS) with a protein concentrationof 3.7 g/dL (Biuret method) to CSF at a concentration of20% vol, or the addition of hetastarch to CSF at a ratioof 1:1 (vol:vol) can help to stabilise cells in CSF (Fryet al., 2006). FCS appears to stabilise mononuclear cellsmore effectively than hetastarch 48 h after collection (Fryet al., 2006). The addition of 1 drop of 10% formalin to1–2 mL of CSF may be used to preserve cell concentrationand structure for up to 8 h after collection (Evans, 1988;Carmichael, 1998). Alternatively, cellular stability can beincreased for up to 24 h from the time of collection by addi-tion of fresh or frozen autologous clear plasma or serum(11% by volume) (Bienzle et al., 2000).

If the analysis requires a delay, two aliquots of CSF canbe collected and placed in sterile plain tubes. In one of thetubes, a preservative should be used and the sample can besubmitted for cell counts and cytological evaluations. Thesample without preservative can be used for protein quan-tification and antibody titre analysis.

Macroscopic evaluation

Colour

Normal CSF is colourless. Any change in colour gener-ally represents an abnormality. Pink or red colourationsuggests the presence of blood. If, after centrifugation, ared cellular pellet is present at the bottom of the tubeand the supernatant is colourless, the colouration wasdue to the presence of intact erythrocytes from iatrogenicperipheral blood contamination or recent (few hours)haemorrhage in the subarachnoid space (Cook and DeNi-cola, 1988). If the supernatant is xanthochromic (yellow toyellow-orange discolouration), previous haemorrhage andaccumulation of oxyhaemoglobin or methaemoglobinderived from erythrocyte degradation is likely (Kjeldsberg

and Knight, 1986; Cook and DeNicola, 1988). The inten-sity of the colour peaks 24 h after haemorrhage and disap-pears by 4–8 days (Jamison and Lumsden, 1988).Xanthochromia has also been reported when there is anincrease in total protein concentration, hyperbilirubina-emia, and also with CNS inflammation and neoplasia(Krieg, 1979; Cook and DeNicola, 1988) (Table 1).

Turbidity

Normal CSF is translucent. An increased turbidity isattributed to particles in the fluid and typically is due toincreased cellularity of the sample. Mild to moderate eleva-tions in TNCC rarely alter CSF clarity (Chrisman, 1992),whereas a nucleated cell count of >500 cells/lL is associ-ated with an increase of turbidity (Coles, 1986).

Quantitative analysis

Protein concentration and total nucleated cell count

CSF has an extremely low protein concentration relativeto serum (Fishman, 1992). In dogs and cats, as well as inpeople, the total protein concentration increases alongthe neuraxis rostro-caudally, and for this reason lumbarfluid typically has a higher protein and lower nucleated cellconcentration when compared with the CMC fluid (Baileyand Higgins, 1985). The reasons for the difference in cellconcentration are not well understood: the lower cell con-centration may result from increased cell lysis as the CSFflows in a caudal direction (Fishman and Chan, 1980).Higher protein concentration is also thought to arise fromthe slower circulation of CSF in the lumbar region withsubsequent local protein accumulation (Thomson et al.,1990). Other studies suggest that the increasing concentra-tion of protein from the ventricles to the CMC, and fromthe CMC to the lumbar region is due to the higher perme-ability of the blood–CSF barrier to proteins in the lumbarregion (Fishman et al., 1958) (Table 2).

Total protein concentration

Almost all of the proteins normally present in CSF arederived from plasma (Reiber, 1998; Reiber, 2003). In nor-mal CSF, protein levels consist almost entirely of albumin.There are minor concentrations of transthyretin (TTR orprealbumin), retinol-binding protein (RBP) and transfer-rin, which are synthesised by the choroid plexi (Aldredet al., 1995); in addition, there are traces of beta andgamma globulins, tau protein (a fraction of modified trans-ferrin), glial fibrillary acidic protein, and myelin basic pro-tein, which appear to be synthesised intrathecally(Thompson, 1988). There are quantitative and qualitativedifferences between plasma and CSF TTR and RBP indogs, suggestive of a selective and controlled transfer ofretinol into CSF and a local synthesis of TTR and RBPin the choroid plexus (Forterre et al., 2006).

Table 1Component of CSF evaluation necessary in all routine analyses andcommonly cited reference intervals (see text)

Macroscopicevaluation

CSF reference intervals

Colour ColourlessTurbidity ClearErythrocytes 0 to small numberTotal nucleated

cell count0–5 cells/lL (dog) 0–8 cells/lL (cat)

Total protein <30 mg/dL (cerebellomedullary) <45 mg/dL(lumbar cistern)

Microscopicevaluation

Differential cellcount

Lymphocytes and monocytes predominate, fewsegmented non-degenerate neutrophils, and rareerythrocytes

Table 2Other CSF tests that might be performed in particular conditions (see text)

Test CSF referenceintervals

Condition

Albumin quotient (AQ) <2.35 " BBB permeabilityImmunoglobulin G index

(IgG)<0.272 with normalAQ

Intrathecal IgG

Antibody index Negative Specific antibodiesIgA Negative SRMAProtein electrophoresis N/A Intrathecal IgGFlow cytometry N/A ImmunophenotypingMyelin basic protein Negative Demyelinating

diseaseCSF culture Negative Infectious diseasesPCR organisms Negative Infectious diseasesMatrix metalloproteinase Negative Research analyte

SRMA, steroid-responsive meningitis–arteritis; BBB, blood-brain barrier;CSF, cerebrospinal fluid; PCR, polymerase chain reaction.


Reference intervals for CSF total protein concentrationcan vary with the laboratory and testing method used.Refractometer evaluation is not accurate for the measure-ment of CSF total protein concentration (Chrisman, 1992).Urine protein reagent strips may be useful for an initial pre-liminary screening test to estimate CSF protein concentra-tion, however it is highly specific for albumin detection andless specific for globulin detection, only providing a crudequantification (Jacobs et al., 1990). False-positive andfalse-negative test results may occur at dipstick readings oftrace or 1+. However, dipstick readings of 2+ or above reli-ably represent a true increase of total protein concentration(Jacobs et al., 1990). The Pandy test is a screening test for thepresence of globulins. It is performed by adding a few dropsof CSF to 1 mL of Pandy reagent (10% carboxylic acid). Tur-bidity indicates the presence of globulin and has a sensitivityof approximately 50 mg/dL or 0.5 g/L.

For an accurate evaluation of CSF protein concentra-tion, special analytical techniques are necessary (Chrisman,1992; Parent and Rand, 1994). The most common proteinassays use the specific dyes, Coomassie blue and pyrogallolred (Marshall and Williams, 2000). Pyrogallol red is con-sidered the most specific technique for CSF total proteindetermination (Marshall and Williams, 2000), but it under-estimates CSF total protein in dogs due to 20% lower affin-ity for globulins than for albumins (Behr et al., 2003). Ahuman immunoturbidimetric assay (microalbumin) hasrecently been validated to measure canine albumin concen-tration in urine and CSF (Gentilini et al., 2005).

Albumin and albumin quotient

The major protein in the CSF is albumin (80–95%), which isonly synthesised in the liver (Evans, 1988). The concentrationof CSF albumin is much lower than the concentration ofserum albumin, but the ratio is kept constant in healthy states;for this reason the calculation of the ratio between CSF andserum albumin, called albumin quotient (AQ), may be usefulto evaluate a disruption of the blood-brain barrier (BBB). Athorough discussion of AQ and its calculations and interpreta-tions has been given elsewhere (Sorjonen, 1987).

Gamma-globulins and IgG index

Electrophoretic techniques define the gamma-globulinsas a heterogeneous group of proteins with similar migrationrates. The gamma-globulin fraction contains the immuno-globulins (Ig). Three major immunoglobulins are found inCSF: IgG, IgM, and IgA (Bailey and Vernau, 1997). Themajor immunoglobulin in normal CSF is IgG, which nor-mally originates from the plasma. Small amounts of IgGare usually found in the CSF of normal dogs and cats (Baileyand Vernau, 1997). Increased IgG levels may occur in a num-ber of inflammatory CNS disorders. In these conditions,gamma globulin may enter the CSF through dysfunctionalblood–brain/CSF barriers, or be synthesised intrathecallydue to the local disease process (Tipold et al., 1994).

Serum IgG concentrations are at least 1000� higherthan those in the CSF, but there is a strong associationbetween the two values. A ratio of the values can be moreuseful than the absolute CSF IgG concentration alone(Tipold et al., 1993). IgG ratio and IgG index equationsand their interpretation have been already described in pre-vious reviews (Bichsel et al., 1984; Vandevelde et al., 1986;Tipold et al., 1993; Tipold, 1995b). Based on the authors’experience, these calculations are usually reserved for spe-cific cases where an infectious disease is suspected.

In addition to the IgG index, recent studies havedescribed other techniques to measure intrathecal antibodyproduction: the Antibody index (Knopf et al., 1998) andanother similar technique called the Goldman–Witmer coef-ficient (C-value) (Potasman et al., 1988). Both techniquesare believed to be more accurate than the IgG index, asthey use antigen-specific antibody titres rather than totalIgG (Furr, 2002).

The presence of IgM is not a normal finding in the CSFand its presence can have diagnostic significance (Krak-owka et al., 1981). IgM is detected in the earliest stage ofthe general humoral immune response of the body andit is the first immunoglobulin that returns to normal


concentration when the antigen disappears. The presenceof IgM in serum and/or CSF is considered more specificthan IgG or total immunoglobulin levels for detection ofactive infectious diseases (Chrisman, 1992).

IgA is normally present in low concentrations in serum,but is involved as a major first-line defensive role in infec-tions that enter via mucosal surfaces. The highest concentra-tions of intrathecal as well as systemic IgA have been foundin dogs with steroid responsive meningitis–arteritis (SRMA)(Tipold, 1995a) and so are used as an adjunctive diagnosticprocedure for this condition. In this instance, IgA appearsto play a central role in the CNS humoral response (Tipoldet al., 1994). Unfortunately, increased IgA levels in CSFcan also be found in other inflammatory diseases such asgranulomatous meningoencephalomyelitis and canine dis-temper encephalitis (Tipold, 1995a). Based on our experi-ence, IgA levels in the CSF should be routinely measuredin suspected cases of SRMA and is clinically useful.

Other proteins which may be measured in the CSFinclude myelin basic protein (Summers et al., 1987; Ojiet al., 2007), S-100 and C-reactive protein, the latter ofwhich may help to differentiate bacterial from viral menin-goencephalitis (Fishman, 1992; Stearman and Southgate,1994). Myelin basic protein (MBP) was studied by someauthors (Oji et al., 2007) who found an increase in MBPconcentration in CSF collected from the lumbar cisternin dogs with degenerative myelopathy, but not in theCSF collected from the cisterna magna or from either sitein control dogs. These findings suggested that an activedemyelinating lesion in the spinal cord was present. Also,the same study confirmed the correlation between MBPand the severity of the demyelination (Oji et al., 2007).These tests currently have limited availability, and remainof academic interest in veterinary medicine.

Cell count

The cellular concentration of normal and sometimesabnormal CSF is too low to be detected by standard haema-tological analysis. For this reason, cell counts are performedusing a haemocytometer (Neubauer chamber or Fuchs-Rosenthal chamber). The chamber is placed in a humidifiedenvironment (e.g., Petri dish with damp tissue) for up to 10–15 min to allow cells to settle to the surface of the glass. Theerythrocytes and the nucleated cells are counted separately.In the Neubauer chamber, the total cells present in all ninelarge squares of the chamber are counted. The calculationsare performed using the average of the two sets of ninesquares:

Number of cells� 10=9

¼ cells=lL; cells=mm3 or cells� 106=L:

In the Fuchs-Rosenthal chamber the cell the total cellspresent in 16 � 1 mm2 areas are counted and the followingformula is applied:

Number of cells� 10=9

¼ cells=lL; cells=mm3 or cells� 106=L:

In this case, the cells are counted and divided by 3instead of 3.2 and the 10/9 dilution is disregarded. Thetwo fractions will compensate for each other so that theremaining error will be negligible.

Normal CSF should not contain erythrocytes, but theymay be seen in low numbers due to iatrogenic blood con-tamination. Erythrocytes can also be seen associated withpathological acute haemorrhage. Practice is needed to dif-ferentiate RBCs from WBCs in the haemocytometer cham-ber using phase microscopy, especially small maturelymphocytes because of their similar size. In general, RBCslack a nucleus and internal structure, while WBCs are lar-ger and have a granular appearance. Sometimes crenationof RBCs can be helpful in their identification (Meinkothand Crystal, 1999). When an unstained specimen is exam-ined, it is necessary to lower the microscope condenser toreduce the light intensity. Alternatively, the cells may bestained with a small amount of new methylene blue stainwhilst in a microhaematocrit tube, and the stained CSFcan be drawn into the haemocytometer chamber (Mein-koth and Crystal, 1999).

Abnormalities in cell type or morphology may be pres-ent even when CSF nucleated cell count are within normallimits (Christopher et al., 1988). In many disease processes,the CSF cell count and CSF total protein concentrationtend to increase in parallel (Carmichael, 1998). In some dis-orders the cell count remains normal, whereas the totalprotein concentration may be markedly increased; this con-dition is called albuminocytological dissociation or pro-tein-cytological dissociation (Evans, 1988; Carmichael,1998).

Elevated CSF protein concentrations without increasesin CSF nucleated cell count have been described in viralnon-suppurative encephalomyelitis (Bichsel et al., 1984;Sorjonen, 1987; Sorjonen, 1990), neoplastic disease, trau-matic, vascular, degenerative, and compressive spinal cordlesions (Evans, 1988; Chrisman, 1992).

Other tests

Antibody titres

CSF infectious disease titres may be more reliable thanserum titres (Matsuki et al., 2004). A variety of antibodyand antigen tests are available for detecting viral, fungal,protozoal, and rickettsial agents (Matsuki et al., 2004).Interpretation of CSF antibody titres, when performed inisolation, may not be accurate, because intrathecal anti-body production can be difficult to differentiate from a dis-ruption of the blood brain barrier or false elevation ofserum antibodies due to blood contamination (Bailey andVernau, 1997).

Intrathecal production of antigen-specific antibody canbe determined with an antibody index in the same way as


intrathecal IgG production is detected with the IgG index(Reiber and Lange, 1991). Antibody indices have been cal-culated in human patients with a variety of diseases (Reiberand Lange, 1991), but they need further study for applica-tion in veterinary medicine.

The prevalence of autoantibodies in the CSF from dogswith various CNS diseases has recently been documented(Matsuki et al., 2004). Anti-astrocytic autoantibodies incanine CSF were detected and considered highly specificfor necrotising meningoencephalitis (NME) and granulom-atous meningoencephalomyelitis (GME); however, anti-astrocytic autoantibodies have also been detected in casesof brain tumours (Matsuki et al., 2004).

Recent studies (Boettcher et al., 2007) demonstratedthat diseases associated with severe vasculitis like FIPmay disrupt and impair the blood–brain barrier and CSFflow rate. In those cases, the measurement of the CSF anti-bodies may be increased due to increased vascular perme-ability, therefore reflecting the concentration ofantibodies in the plasma rather than estimating the intra-thecal production of antibodies. The calculation of AQand specific antibodies index may be useful in these casesas an indicator of intrathecal antibody production (Boett-cher et al., 2007).

CSF culture

When an infectious organism is suspected to be thecause of a CNS disease, both aerobic and anaerobic bacte-rial cultures of CSF may be performed. However, positivebacterial culture results in confirmed cases of bacterialmeningitis are extremely uncommon (Tipold, 1995a; Fen-ner, 1998; Radaelli and Platt, 2002). A negative culturemay be the result of inappropriate sample handling or cul-ture media, in addition to a low number of organisms in theCSF (Fenner, 1998; Garges et al., 2006). Additionally,some bacteria are prone to rapid autolysis in collectiontubes (Peters et al., 1995). PCR techniques have been usedto detect the presence of bacterial DNA in people (Peterset al., 1995), but are not routinely used in veterinary med-icine. Fungal culture of CSF can be used to isolate Crypto-

coccus spp. and virus isolation has also been successful indiagnosing cases of distemper meningoencephalitis (Kaiet al., 1993; Fenner, 1998).

Polymerase chain reaction (PCR)

PCR can identify an infectious agent’s DNA or RNA,and is extremely useful when the organisms cannot be cul-tured (Sharp, 1998). Single-round PCR amplification isused for the majority of infectious agent’s such as caninedistemper virus (CDV), Toxoplasma gondii, Neospora cani-

num, Ehrlichia canis, Rickettsia rickettsii, Bartonella spp.,Borrelia burgdorferi, feline leukaemia virus (FeLV), andfeline immunodeficiency virus (Stiles et al., 1996).

Some studies have shown that a multiplex PCR assaywith built-in control reactions may be useful as a comple-

mentary clinical tool for the ante-mortem diagnosis oftoxoplasmosis or neosporosis associated with the CNS(Schatzberg et al., 2003). Reverse transcription-PCR (RT-PCR) is used to amplify the RNA template nucleoproteinof canine distemper virus (CDV) in the CSF (Frisk et al.,1999). Reverse transcriptase-PCR represents a sensitiveand specific method for an ante-mortem diagnosis ofCDV if serum or whole blood are examined at the sametime (Frisk et al., 1999). In our experience, however, a neg-ative PCR assay does not rule out the presence of the infec-tious agent.

Research analyses

The following analyses are currently not clinically eval-uated and as such are research tools but their potential util-ity is discussed.

Matrix metalloproteinases (MMPs)

MMPs form a large group of zinc-dependent endopro-teinases (endopeptidases) that are able to degrade compo-nents of the extracellular matrix (ECM) (Birkedal-Hansen et al., 1993). Matrix MPs can be divided into fourgroups based on substrate specificity: collagenases, strom-elysin, gelatinases, and membrane-type MMPs. The ECMis important in maintaining the structure of the CNS inaddition to its role in transport of ions, cell migrationand delivery of growth factors (Perides et al., 1998). MMPshave several roles in wound healing, angiogenesis, andembryologic development. They are also involved in neuro-pathological processes such as tumour migration, axonaldegeneration, lumbar disk herniation, multiple sclerosisand acute spinal cord injury (Matsui et al., 1998).

MMPs have been characterised in dogs. Specifically, theenzyme activities of MMP2 and MMP9 have been found ina variety of tissues (Lana et al., 2000) including synovialfluid, brain, myocardium as well as osteosarcomas andmast cell tumours in dogs (Gilbert et al., 1997; Coughlanet al., 1998; Leibman et al., 2000). In people, the pro-enzyme form of MMP (proMMP)-2 has been found in nor-mal brain and CSF, whereas MMP-9 has been found onlyduring pathological processes (Mun-Bryce and Rosenberg,1998). Many cells in the CNS produce MMPs (neurons,microglia, oligodendrocytes, and astrocytes) (Yong et al.,1998). Leukocytes from the peripheral blood may also con-tribute to the concentration of MMP-9 during CNS dis-eases (Bergman et al., 2002).

CNS expression of MMP-2 has been documented inhumans without CNS inflammation or BBB disturbancesas well as in healthy experimental animals (rats and rab-bits); concentrations do not increase dramatically duringpathological conditions (Paul et al., 1998; Azeh et al.,1998; Yushchenko et al., 2000). MMP-9 has not beenfound in the CSF of healthy animals or controls in otherstudies (Gijbels et al., 1992; Perides et al., 1998; Kolbet al., 1998), but a high correlation exists with the nucleated


cell count (Yushchenko et al., 2000). Granulocytes andmacrophages are strong producers of MMP-9, whereaslymphocytes are weak producers, thus CSF microscopicanalysis maybe predictive of the MMP-9 levels. Bacterialmeningitis in people results in high levels of MMP-9 whichmay be due to the invasion of large numbers of granulo-cytes (Gijbels et al., 1992; Paul et al., 1998; Kieseieret al., 1999). Levels of MMP-9 are low to moderatelyincreased in viral meningoencephalitis in the presence ofhigh concentrations of lymphocytes (Yushchenko et al.,2000). If CSF is dominated by lymphocytes, the presenceof detectable MMP-9 may be due to the presence of mac-rophages (Yushchenko et al., 2000).

The increase of MMP-9 activity in CSF of dogs may nothelp to make a diagnosis of a specific neurological disease,but it may be useful as a marker of the various neuroin-flammatory diseases of dogs (Bergman et al., 2002). Forinstance, elevated MMP-9 activity in the CSF and serumhas been reported in dogs with acute spinal cord traumafrom intervertebral disc disease (IVDD) (Levine et al.,2006). This study showed that dogs with IVDD oftenexpressed MMP-9 in the CSF when neurological dysfunc-tion had been present for <24 h. Dogs with paraplegiadue to IVDD, more frequently expressed MMP-9 thanthose with paraparesis and ataxia or no neurological defi-cits (Levine et al., 2006). Further investigations arerequired of CSF and serum MMP expression in dogs withIVDD as well as other neurological diseases to better assessthe prognostic value of MMP-9 in the CSF and to evaluatethe potential benefit of MMP-9 inhibitors (Levine et al.,2006).

Protein electrophoresis

Qualitative CSF protein analysis is performed to charac-terise the distribution of protein in the CSF, and often isvery useful to compare the distribution with the ones foundin serum for a better understanding of the pathological sta-tus of the patient. Several techniques are available, espe-

Fig. 3a. Flow cytometry plots of CSF from a 7-year-old, female, spayed, GoldeUniversity with hind limb ataxia and weakness. CSF collected from CMC wTNCC 3175 cell/lL. A lymphocytic pleocytosis was present on cytologicalconjugated antibodies to specific cell markers (CD3 for T cells and CD21 for B clabelled with the negative control antibodies and is used to identify populationsintensity (log scale). In this case, 86% of the cell population expressed CD3, anDr. Melinda Wilkerson, Manhattan, KS).

cially in human laboratories (agarose gel electrophoresis,immunoelectrophoresis, and isoelectric focusing) (Sorjonenet al., 1991; Reiber et al., 2003); however, high resolutionagarose gel electrophoresis (HRE) produces sharper bandsand definition of CSF protein fractions in people (Fish-man, 1992), horses (Furr et al., 1997), and dogs (Sorjonen,1987; Behr et al., 2006).

Abnormalities in CSF protein electrophoretic patternshave been reported to be useful in the identification ofinflammatory, neoplastic, and degenerative disease (Sorjo-nen, 1987; Sorjonen et al., 1989). Dogs with canine distem-per viral disease often have absolute elevation in CSFgamma globulins (probably due to intrathecal production)(Sorjonen et al., 1989), and dogs with granulomatousmeningoencephalomyelitis may have an increase in betaand gamma globulins (Chrisman, 1992). CSF protein elec-trophoresis may be helpful to differentiate dogs withinflammatory disease that produce mild BBB impairmentfrom those that have intrathecal production of gammaglobulins (Sorjonen et al., 1989) However, in general,CSF electrophoresis cannot be confirmed as a valuableancillary diagnostic tool for neurological disease (Behret al., 2006).

Flow cytometry

Flow cytometric analysis of CSF is largely performedin human medicine to demonstrate the presence of lym-phocyte clones in the diagnosis of lymphomas and lym-phoproliferative disorders (Babusikova and Zeleznikova,2004) (Subira et al., 2002). In veterinary medicine, fewstudies have reported the use of flow cytometry andimmunophenotyping to identify mononuclear cells inthe CSF of inflammatory conditions (Duque et al.,2002). Flow cytometric methods can provide informa-tion on the cellular phenotypes present in the CSF.Recently, two cases of CNS lymphoma were immuno-phenotyped as B- or T-cell lymphomas using CSF sam-ples and flow cytometric techniques in place at one of

n Retriever presented to the College of Veterinary Medicine, Kansas Stateas colourless, hazy with a total protein concentration of 145 mg/dL andexamination. Flow cytometry of CSF cells labelled with fluorochrome-ells) are listed on the X-axis. A vertical line is placed to the right of the cellsof cells that expressed CD molecules and shifted to the right in fluorescentd only 2% expressed CD21, consistent with a T-cell lymphoma (Courtesy


the author’s institutes (Fig. 3). This technique allowsone to identify the lineage of the neoplastic cells evenwhen the distinction between monocytes and lympho-cytes is difficult via microscopy. The use of this diagnos-tic modality is hindered by the practical issue that largervolumes (about 4–5 mL) of CSF than are usually avail-able from dogs and cats are necessary unless the cellcount is very high.

Microscopic evaluation

Cytological microscopic slide preparation

Because of the low numbers and extreme fragility ofCSF cells, concentration of the cells is required for cyto-logical evaluation. Several procedures are available forthe morphological study of CSF cells. Most laboratoriesuse a cytocentrifuge to prepare cells for evaluation, (Gar-ma-Avina, 2004). A cytocentrifuge uses a slow speed cen-trifugation and acceleration (around 100–150 g based onthe one we use, a Cytospin 2 [Shandon], for 4–6 min) toconcentrate the cells from 200–400 lL (depending on thenumbers of cells in the fluid) into a small circular areaon a glass slide (Christopher et al., 1988). The cellulardetail is excellent because the elements are gently spreadout on the slide; however, there are some artifactualchanges in the cells such as increased vacuolation andalteration in structure, particularly of monocytoid cellsand macrophages.

Other techniques available including sedimentation andmembrane filtration have already been described (Garma-Avina, 2004). An inexpensive in-house sedimentationchamber has been described to obtain cytological speci-mens of CSF. This device may be very useful when accessto a laboratory is difficult, or when a cytocentrifuge isnot available. The results obtained using this techniqueare consistent, and it also permits retrieval of the cell-freefluid for its use in chemical or immunological procedures(Garma-Avina, 2004).

Fig. 3b. Flow cytometry plots of CSF from a 10-year-old, female, spayed, BeaVMTH with cervical pain. CSF collected from CMC was opaque and cloudy, wlymphocytic pleocytosis was present (>95% lymphoblasts) on cytological examfluorochrome-conjugated antibodies to specific cell markers (CD3 for T cells aright of the cells labelled with the negative control antibodies and is used to idright in fluorescent intensity (log scale). In this case, 90% of the cell populalymphoma (Courtesy of Dr. Melinda Wilkerson and Dr. Mehrdad Ameri, Ma

Cytocentrifuge and sedimentation preparations are mostcommonly air-dried and stained with Romanowsky stains(May-Grunwald-Giemsa Wright-Giemsa or Diff-Quick).Special stains are indicated in some cases: Gram stainingmay be useful for confirmation and identification of bacte-ria (Cook and DeNicola, 1988; Evans, 1988). India ink ornew methylene blue could be helpful in identification offungal infections, especially cryptococcosis (Cook andDeNicola, 1988). Periodic acid-Schiff stain may be usefulto demonstrate positive intracellular material in animalswith storage diseases such as globoid cell leukodystrophyor mucopolysaccharidosis (Roszel et al., 1972). Luxol fastblue can be helpful to confirm the presence of myelin inCSF samples (Mesher et al., 1996).

Cytological microscopic examination

Cytological CSF examination is essential even if theTNCC is within normal limits because it is still possibleto have abnormalities in cell type or structure (Evans,1988). The absence of any abnormality in CSF does notrule out the possibility of neurological disease. In all cases,cytocentrifugation is recommended to concentrate cellswith minimal cell loss.

Blood contamination

CSF does not normally contain erythrocytes (Cook andDeNicola, 1988; Chrisman, 1992). The presence of erythro-cytes in a sample is most commonly iatrogenic in origin(contamination during collection), but otherwise suggestsa pathological subarachnoid haemorrhage. Numerous fac-tors and formulas have been used to correct protein con-centration and TNCC for the effect of bloodcontamination in CSF, but most are considered inaccurate(Sweeney and Russell, 2000). In feline CSF, a red blood cellcount >30 cells/lL can have effect on the total and differen-tial cell count (Rand et al., 1990). However, in a recentcanine study, the RBC count was not significantly

gle presented at Kansas State University, College of Veterinary Medicineith a total protein concentration of 117 mg/dL and TNCC 9387 cell/lL. Aination. Flow cytometry of cells obtained from CSF were labelled with

nd CD21 for B cells) as listed on the X-axis. A vertical line is placed to theentify populations of cells that expressed CD molecules and shifted to thetion expressed CD21, and 27% expressed CD3, consistent with a B-cellnhattan, KS).


associated with TNCC or protein concentrations in CSFfrom clinically normal dogs or those with evidence of neu-rological disease, even if the sample contained up to13,200 RBC/lL (Hurtt and Smith, 1997).

Normal nucleated cells in the CSF

Mononuclear cells predominate in the CSF of healthydogs and cats and consist of normal looking small lympho-cytes, which are very similar to the same cells seen in theblood, and larger cells classified as monocytoid cells (Cookand DeNicola, 1988; Rand et al., 1990; Chrisman, 1992;Parent and Rand, 1994). Low numbers (up to 10% ofTNCC) (Chrisman, 1992) of mature, non-degenerate neu-trophils are occasionally seen in normal CSF and probablyresult from systemic blood contamination at the time ofsampling. Some authors, however, have suggested thatthe presence of neutrophils or eosinophils in the CSF isindicative of an abnormality (Christopher et al., 1988). Incats, on the other hand, up to 8% of nucleated cells canbe neutrophils in non-blood contaminated CSF (Randet al., 1990). Occasionally, ependymal cells, choroid plexuscells, meningeal lining cells, or mitotic figures may be seenin samples of normal individuals (Cook and DeNicola,1988; Rand et al., 1990; Chrisman, 1992; Parent and Rand,1994). Published reference intervals for canine CSF TNCCare reported to be <5 cells/lL (Cook and DeNicola, 1988;Oliver and Lorenz, 1997).

Lymphocytes

Small lymphocytes (Fig. 6) are the most common celltype in normal CSF of dogs; they may also be called smallmononuclear cells. They are 9–15 lm in diameter, withdark, compact, round to slightly oval or indented nucleiand usually have a narrow border of pale-blue cytoplasm.A nucleolus is occasionally visible. Medium and large lym-phocytes (measured based on nuclear size) are not usually aconstituent of normal CSF (Cook and DeNicola, 1988;Chrisman, 1992). Their nuclei are often larger with a lessdense and more scattered chromatin pattern than the smalllymphocytes. The cytoplasm is much more abundant andmay contain a few dense azurophilic granules.

The presence of medium and large lymphocytes sug-gest the possibility of a pathological process, even ifthe TNCC is not increased, especially if there is a localantigenic stimulation within the subarachnoid space(Grevel and Machus, 1992). Occasionally plasma cellsmay be observed with active or resolving infectious dis-eases, neoplastic processes, and potential immune-medi-ated diseases (Cook and DeNicola, 1988). The origin oflymphocytic cells in the CSF is not well known; themajority may be derived from leptomeningeal stem cellsand migrate from the blood to the CSF (Sayk, 1962).It is also possible that the lymphocytes return to theblood, accounting for a regular circulation and exchange(Herndon and Brumback, 1989).

Monocytes

Monocytes (Fig. 7a) are less common than lymphocytesin the normal CSF of dogs, whereas in cats these cells seemto be the predominant components of normal CSF (Cookand DeNicola, 1988; Rand et al., 1990; Parent and Rand,1994). Monocytoid cells compose 69–100% of the TNCC,lymphocytes 0–27%, neutrophils 0–9%, macrophages 0–3%, and eosinophils 0 to <1% of the CSF in healthy cats.Those cells may also be called large mononuclear or mon-ocytoid cells.

The cells are large (12–15 lm) in diameter and haveoval, sometimes kidney-shaped, or slightly lobulated nucleithat stain less intensely than the nuclei of small lympho-cytes (Cook and DeNicola, 1988; Chrisman, 1992). Theyhave moderate to abundant light-blue cytoplasm, whichis foamy or finely vacuolated. In pathological conditions,monocytes are transformed into macrophages that containvarying amount of phagocytised material, such as lipiddroplets, erythrocytes, microorganisms, and cell debris invarious stages of digestion. Activated monocytoid cellsand macrophages may appear in clusters mimicking epithe-lial cells (Chrisman, 1992).

Uncommon, incidental components and other findings

Leptomeningeal lining cells, choroid plexus cells, andependymal cells can occasionally be found in normalCSF. They are more commonly observed if aspirationhas been used to collect the fluid (Cook and DeNicola,1988). The leptomeningeal lining cells often appear in smallclusters and consist of mononuclear cells with moderate toabundant amounts of pale basophilic cytoplasm, round tooval eccentric nuclei and indistinct cytoplasm margins.

Choroid plexus cells and ependymal cells are difficult todistinguish; both elements are very fragile, small, columnarto cuboidal and of uniform size and morphology (DeMay,1996) with round, often pyknotic-appearing nuclei and awide border of pink or blue-grey cytoplasm. These cells,which usually appear in groups, derive from the epitheliumof the cerebral ventricles or from the choroid plexus andcan appear frequently in the CSF of small children associ-ated with hydrocephalus (DeMay, 1996). These cells arerarely seen in veterinary medicine, but it is important notto interpret them as neoplastic cells when identified.

It is possible to find elements that originate from thepath of the spinal needle. These often include cartilage cellsand epithelial cells. Bone marrow elements may be foundas contaminants in canine CSF associated with bone mar-row aspiration during lumbar cistern collections of CSF(Christopher, 1992).

Intracellular myelin (within macrophage cytoplasm) hasbeen found in the CSF from a dog with myelomalacia(Mesher et al., 1996). Macrophages on the cytocentrifugepreparation stained positively with Luxol fast blue(LFB), which specifically stains myelin. The myelin-likestructures described in other cases reported may be referred

Fig. 4. Myelin figures in CSF from a dog. Extracellular myelin fragmentsare the grey-blue ribbon structures which are phospholipids from damagedaxons (Wright-Giemsa; �100) (Courtesy of Dr. Steve Stockham,Manhattan, KS).


to as either myelin ‘figures’ or myelin ‘fragments’, whichappear to be very similar on light microscopy (Baueret al., 2006). A consistent difference between these twostructures is that myelin ‘figures’ originate from a non-spe-cific lipid source; they are laminated lipids arranged in coilsor stacks derived from cell membrane phospholipids andcellular organelles as a result of cell death or injury. Myelinfigures may be found extracellularly (Fig. 4) or as engulfedmaterial within macrophages (Ghadially, 1988).

Myelin ‘fragments’ specifically denote a collection ofmyelin found only extracellularly, and are thus are moreindicative of demyelination (Ghadially, 1988). However,it is important to remember that during demyelination,the lipid originated from myelin undergoes a denaturationchange including the formation of free neutral fats whichwill not stain with LFB (Jones and Hunt, 1983). The onlyreliable way to differentiate between myelin figures andmyelin fragments is to examine the laminated structureon electronic microscopy specifically for the appearance,periodicity and location of the myelin bands relative tothe cells (Fallin et al., 1996).

Other rare or occasional findings in CSF samplesinclude neural elements such as neuroglial cells, ventricleassociated cells (ependymal and choroidal plexus cells),and meningeal lining cells (Chrisman, 1992). Nervous tis-sue has also been rarely identified in a few cases after rou-tine CSF cytological analysis, and reported ascontamination due to accidental puncture of the spinalcord during cerebello-medullary cisternal collection (Fallinet al., 1996).

Interpretation of cytological abnormalities

TNCCs may be within normal reference intervals butthe cytocentrifuged CSF may demonstrate increased per-

centages of cells such as neutrophils and eosinophils in avariety of neurological disorders (Freeman and Raskin,2001). If blood contamination is not the cause, increasedneutrophil percentages of >10–20% (Chrisman, 1992;Meinkoth and Crystal, 1999) and eosinophil percentagesof >1% should be considered abnormal (Meinkoth andCrystal, 1999). An increased percentage of neutrophilswithout increases in TNCC may indicate a mild or earlyCNS inflammation, a lesion that does not contact themeninges or ependymal cells or previous use of drugs suchas glucocorticoids and antibiotics, which may reduce thepresence of an inflammatory response (Meinkoth andCrystal, 1999). An increase in the neutrophil percentagewith normal TNCC has been reported in acute IVDD, ver-tebral fractures (Thomson et al., 1989), cerebrovascularaccidents, (infarct/haemorrhage) and in fibrocartilagenousthromboembolism in dogs (Bailey and Vernau, 1997).

Eosinophils are considered an abnormal finding incanine and feline CSF. An increased eosinophil percentagewithout increased TNCC has been described concurrentlywith either parasite migration (e.g., Parelaphostrongylus

tenuis in llamas) or protozoal disease (Neospora spp. indogs) (Chrisman, 1992).

An increase in TNCC in the CSF is referred to as pleo-cytosis, which may be considered mild (<25 cells/lL), mod-

erate (26–100 cells/lL), or marked (>100 cells/lL) and isfurther defined by the predominant cell type in the sampleas neutrophilic, eosinophilic, mononuclear, or mixed (Free-man and Raskin, 2001). The degree of pleocytosis is due toseveral factors, including the cause, the severity and thelocation of the lesion with respect to the subarachnoidspace or ventricular system (Cook and DeNicola, 1988).As mentioned above, it is important to remember that nor-mal CSF results do not exclude the presence of disease(Kjeldsberg and Knight, 1986; Fishman, 1992), especiallywith deep parenchymal lesions which do not communicatewith the leptomeninges, the subarachnoid space or theependymal surfaces (Cook and DeNicola, 1988). Cytologi-cal changes must always be interpreted in light of thepatient’s history and neurological findings. However,abnormal CSF findings always indicate the presence of apathological abnormality if blood contamination has beenruled out.

Neutrophilic pleocytosis

Neutrophilic pleocytosis (Fig. 5) has been described in awide range of acute inflammatory diseases that includetrauma, haemorrhagic diseases, post myelographic reac-tions as well as bacterial and fungal infections, immune-mediated disease, and neoplasia (Coles, 1986; Cook andDeNicola, 1988; Chrisman, 1992; Meinkoth and Crystal,1999). In dogs, steroid-responsive meningitis–arteritis(SRMA) and necrotising vasculitis is usually associatedwith a neutrophilic response (Meric, 1988). The neutrophilsare well-preserved, generally non-degenerate or hyperseg-mented, and CSF cultures are negative for bacteria (Mein-

Fig. 5. Neutrophilic pleocytosis in the CSF of a 1-year-old mixed breeddog with steroid responsive meningo-arteritis (SRMA). Many non-degenerate neutrophils (long arrow), a few monocytes (short arrow) andscattered small lymphocytes (arrowhead) are present (Wright-Giemsa).

Fig. 6. Mononuclear pleocytosis (lymphocytic) in the CSF of an 8-month-old Pug with necrotising meningoencephalitis. Many small lymphocytes(long arrow), few monocytes (short arrow) and rare neutrophils (arrow-head) are present (Wright-Giemsa).


koth and Crystal, 1999). SRMA may be associated withneutrophilic pleocytosis >500 cells/lL, with a neutrophilicpercentage of between 75% and 100% (Chrisman, 1992).

Recent retrospectives of dogs with bacterial meningitisshowed that 98% had neutrophilic pleocytosis (Tunkeland Scheld, 1993). However, when a bacterial or fungalmeningitis has been treated with antibiotics for a week ormore, mononuclear cells may be found in elevated percent-ages replacing neutrophils as the predominant cell (Korne-gay et al., 1978; Bullmore and Sevedge, 1978; Dow et al.,1988). Cytological evaluation of the CSF for the presenceof intracellular rods or cocci is essential in cases of suspectbacterial CNS disease.

Feline infectious peritonitis (FIP) is another commoncause of marked neutrophilic pleocytosis (>50% neutro-phils) with elevated TNCC (>100 lL�1) in cats and is oftenassociated with increased CSF total protein (usually>200 mg/dL) (Foley et al., 1998). Necrosis associated withinflammation induced by neoplasia within the brain andsevere seizure activity may also cause a mild neutrophilicpleocytosis or an increased neutrophil percentage with nor-mal TNCC (Bailey and Higgins, 1986).

CNS neoplasia may induce tumour necrosis, thus a reac-tive neutrophilic inflammation may be found in the CSF(Vandevelde and Spano, 1977). Several studies (Baileyand Higgins, 1986; Grevel et al., 1992) have describedabnormal CSF in cases of intracranial meningiomas indogs, and that the majority of the samples had a neutro-philic pleocytosis (neutrophils >25%); however, a recentstudy showed that 16% (12/77) of dogs with intracranialmeningiomas had normal CSF. Pleocytosis was detectedin only 27% (20/77) of the dogs, and pleocytosis with a pre-dominance of neutrophils was found in only 19% (15/77) ofthe dogs (Dickinson et al., 2006). The location of thetumour plays a significant role in the CSF changes and

neutrophilic pleocytosis may not be detected in CSF sam-ples from dogs with meningiomas located within the middleor rostral portion of the cranial fossae (Dickinson et al.,2006). Practically, most intracranial neoplasia is detectedwith advanced imaging and so CSF analysis may not beperformed due to the concerns of subsequent parenchymalshift or due to its inability to add further information.

Mononuclear pleocytosis

Mononuclear pleocytosis occurs with an increase in thepercentage or concentration of small and mature lympho-cytes (>70%) (Fig. 6) and/or an increase in the number ofmonocytoid/macrophage cells (Figs. 7a and 7b). The pres-ence of reactive lymphocytes with normal TNCC may be asign of CNS disease (Coles, 1986; Christopher et al., 1988).

An alteration in both the number and structure of lym-phocytes occurs in a variety of diseases such as CNS viralinfection in dogs (Vandevelde and Spano, 1977) and cats(Rand et al., 1994a). Canine distemper virus (CDV) infec-tion is typically associated with a mild to moderate lym-phocytic pleocytosis. The CSF in CDV cases may showan increase in macrophages, and occasionally intracyto-plasmic CDV inclusion bodies are recognised (Abateet al., 1998). Granulomatous meningo-encephalomyelitis(GME) is another frequent cause of mononuclear pleocyto-sis, even though it may have an extremely variable spec-trum of cytological findings. The CSF in GME may havea mild to moderate lymphocytic, neutrophilic or mixedcells pleocytosis (Chrisman, 1992; Munana and Luttgen,1998).

In Pugs, Yorkshire terriers and Maltese terriers, a mod-erate to marked lymphocytic pleocytosis with >80% lym-phocytes is consistent with a necrotising meningo- orleuko-encephalitis, although a mixed cell pleocytosis may

Fig. 7a. Mononuclear pleocytosis (monocytic/macrophagic) in the CSF ofa 4-year-old Weimaraner with granulomatous meningoencephalitis(GME). Many monocytoid cells (long arrow), small lymphocytes (shortarrow), and rare neutrophils are present (Wright-Giemsa).

Fig. 7b. Mononuclear pleocytosis (monocyto/macrophagic) in the CSF ofa 4-year-old Weimaraner with GME. Many monocytoid cells (longarrow), few small lymphocytes (short arrow), and occasional mitoticfigures (arrowhead) are present. (Wright-Giemsa).

Fig. 8. Mixed cell pleocytosis in the CSF of a 6-year-old English Springerspaniel with GME. A mixture of non-degenerate neutrophils (long arrow),monocytes/macrophages (short arrow) and small lymphocytes (shortarrow) are present in roughly equal numbers (Wright-Giemsa).


be seen (Freeman and Raskin, 2001). Large numbers ofgranular lymphocytes have also been described in a dogwith necrotising meningo- or leuko-encephalitis (Garma-Avina and Tyler, 1999).

Marked lymphoid pleocytosis has been seen in some ani-mals with a CNS lymphoma (Long et al., 2001). Whenthese cells represent immature lymphocytes (lymphoblasts),lymphoma is easily differentiated cytologically (Freemanand Raskin, 2001; Long et al., 2001). However, well-differ-entiated lymphoid malignancies may not be easily differen-tiated from a lymphocytic pleocytosis (Freeman andRaskin, 2001). Other diseases in which lymphocytic pleocy-tosis is seen include ehrlichiosis, toxoplasmosis, neosporo-

sis, and in some cases of bacterial meningitis followingantibiotic treatment (Fishman, 1992; Rand et al., 1994b;Thomas, 1998; Radaelli and Platt, 2002).

Mixed cell pleocytosis

Mixed cell pleocytosis (Fig. 8) describes a mixed cellpopulation of lymphocytes, monocytoid/macrophages,neutrophils and a few to rare eosinophils and plasmacells. The most common disease demonstrating CSFmixed cell pleocytosis is canine GME, but all the otherpossible differentials cannot be ruled out. The chronicphase of SRMA should also be considered as well asfungal infections such as cryptococcosis (Fig. 9), histo-plasmosis, blastomycosis and aspergillosis, ehrlichiosis,toxoplasmosis, neosporosis, and protothecosis (Chrisman,1992; Meadows et al., 1992).

Mild to moderate mixed cell pleocytosis may result fromnecrosis or inflammation secondary to a variety of diseasessuch as disc disease, haemorrhagic myelomalacia, ischae-mia, or infarction (Chrisman, 1992; Freeman and Raskin,2001). A mixed cell pleocytosis with mononuclear atypiahas been reported in a dog with primary CNS malignanthistiocytosis (Zimmerman et al., 2006).

Eosinophilic pleocytosis

Eosinophils are not present in normal CSF unlessblood contamination has occurred. Peripheral bloodeosinophilia is not always associated with CSF eosino-philic pleocytosis, and when present there is no associa-tion between the magnitude of the peripheraleosinophilia and the severity of the CSF eosinophilia(Smith-Maxie et al., 1989). Eosinophilic pleocytosis con-

Fig. 9. Cryptococcosis in the CSF of a 1-year-old Miniature Schnauzer.Many extracellular yeasts consistent with Cryptococcus neoformans arepresent (Wright-Giemsa).

Fig. 11. Meningioma in the CSF of a 13-year-old mixed breed dog. Nestsof large cohesive cells of a meningoepitheliomatous appearance arepresent. Cells contain eosinophilic secretory material (Wright-Giemsa;�100) (Courtesy of Dr. Steve Stockham, Manhattan, KS).


sisting predominantly of eosinophils is infrequent (Chris-man, 1992). Non-specific acute inflammatory responsesmay increase the number of the eosinophils in theCSF. This condition has been described as steroidresponsive eosinophilic meningoencephalitis, and hasbeen reported in dogs (Smith-Maxie et al., 1989) and cats(Schultze et al., 1986). Some breeds of dogs, such asGolden retrievers and Rottweilers may be predisposed(Smith-Maxie et al., 1989; Bennett et al., 1997).

Eosinophilic pleocytosis has also be seen in protozoalinfections such as toxoplasmosis, neosporosis, and fungaldiseases such as cryptococcosis (Cook and DeNicola,1988; Chrisman, 1992). Eosinophilic pleocytosis has alsobeen reported in animals with aberrant parasitic migrations

Fig. 10. Eosinophilic pleocytosis in the CSF from a llama. Manyeosinophils (long arrow) and occasional macrophages (short arrow) arepresent. The llama was infected by the meningeal worm Parelaphostrongy-

lus tenuis (Wright-Giemsa; �100) (Courtesy of Dr. Karen Young,Madison, WI).

as is found, for example, with Parelaphostrongylus tenuis inllamas (Fig. 10), protothecosis, and very rarely in CDVinfection, and rabies (Chrisman, 1992).

Conclusions

CSF analysis forms a vital part of the panel of diag-nostic tests required for the assessment of neurologicalcases. It infrequently provides a definitive diagnosis,but if the analysis is performed thoroughly it may assistin the diagnosis of underlying diseases in conjunctionwith advanced imaging and systemic health assessmentsto better define a more specific differential diagnosis(see Figs. 11 and 12).

Fig. 12. Lymphoma in the CSF of a 9-year-old Labrador Retriever.Medium to large lymphocytes with immature chromatin, prominentnucleoli and basophilic, often vacuolated, cytoplasm are present (Wright-Giemsa; �100) (Courtesy of Dr. Karen Young, Madison, WI).


Acknowledgements

The authors are grateful to Dr. Steve Stockham and Dr.Melinda Wilkerson, veterinary clinical pathology profes-sors at Kansas State University College of Veterinary Med-icine, for their advice, guidance, suggestions, and criticalreview of the manuscript.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.tvjl.2007.11.024.

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