Cerebrospinal Fluid

Common Neurologic Problems

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Cerebrospinal Fluid

James R. Cook, Jr., DVM,* and Dennis B. DeNicola, DVM, PhDt

Because the central nervous system (CNS) is almost entirely encased in bone, direct diagnostic evaluations of the CNS, such as biopsies and radiographic examination, take on a degree of complexity that is much greater than for most other organ systems. Currently, the diagnostic test for CNS disease that provides the most information with least invasiveness is the collection and analysis of cerebrospinal fluid (CSF) . With some practice, the procedures are safe, quick, and cost-effective in terms of both time and supplies. The time-dependent portions ofCSF analysis (cell counts and cytology preparation) are within the capabilities of most veterinary practices. More complex analyses (protein, electrolyte, and chemical determinations) are, with few exceptions, not particularly time-dependent and can be delivered to the appropriate laboratory faCility when convenient. In this article, we will describe principles of CSF collection and analysis. The physiology of CSF, indications and contraindications for collection, and correlation of abnormalities of CSF to clinical disease states will be briefly discussed as well. Although specific diagnosis of CNS disease should not be based entirely on results of CSF analysis, it is still invaluable in detection and management of most CNS diseases.

PHYSIOLOGY AND FUNCTION Cerebrospinal fluid is produced primarily by the choroid plexus in the ventricular system of the brain; smaller amounts are contributed by the ependymal lining of the ventricular system, the external pial-glial membrane of the brain surface, and blood vessels in the pia-arachp.oid. The blood*Diplomate, American College of Veterinary Internal Medicine (Neurology); Assistant Professor, Department of Veterinary Clinical Sciences, and Staff Neurologist, Veterinary Teaching Hospital. Purdue University School of Veterinary Medicine, West Lafayette, Indiana tDiplomate, American College of Veterinary Pathologists (Clinical Pathology); Associate Professor, Department of Veterinary Pathobiology, and Director, Veterinary Clinical Pathology Laboratory, Purdue University School ofVeterina.ry Medicine, West Lafayette, Indiana Veterinary Clinics of North America: Small Animal Practice-Vol. 18, No.3, May 1988

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CSF barrier is a semipermeable membrane formed by the vascular endothelium and tall columnar ependymal cells of the choroid plexus. The CSF is a result of both ultrafiltration of plasma and active transport mechanisms across this membrane. Compared with plasma, CSF contains slightly more chloride, sodium, and magnesium; slightly less potassium, calcium, and glucose; and significantly less protein. 4 The CSF is relatively acellular, normally containing only a few small lymphocytes (see the section "Analysis"). The rates of CSF formation are approximately 0.05 cc per minute, 0.02 cc per minute, and 0.5 cc per minute in dogs, cats, and man, respectively. This rate is independent of CSF pressure or blood hydrostatic pressure, but is reduced by increased blood osmotic pressure. 4 The CSF circulates through the ventricular system and enters the subarachnoid space through the lateral apertures at the fourth ventricle of the brain. Once in the subarachnoid space, it percolates between the arachnoid and pial membranes of the spinal cord and brain, thus bathing all surfaces of the CNS. The CSF is believed to flow primarily in a caudal direction, although there is some evidence in man of craniad movement as well. 4 It is absorbed mostly through arachnoid villi in the venous sinuses and cerebral veins . These structures act as a one-way (CSF to blood) flap or ball-valve mechanism that is open when CSF pressure exceeds venous pressure (the normal situation) and collapses closed if venous pressure increases, thus preventing retrograde flow of venous blood into the subarachnoid space. IO Small amounts of CSF are absorbed by veins and lymphatics found around spinal nerve roots and the first and second cranial nerves, where they exit the skull. If the intraventricular CSF pressure is increased, some CSF may enter the ependymal cells and be absorbed by blood vessels in the brain parenchyma. 4 Functionally, the CSF suspends the brain to physically protect it from injury. It helps modulate normal variations in intracranial pressure (ICP) and together with cerebral blood flow helps to regulate ICP. By its proximity to brain parenchyma and extracellular fluid, it provides a more stable, closely controlled, and chemically responsive ionic environment than blood plasma. This is important because the pH of CSF has a direct effect on brain function. The CSF has some antibacterial properties and contains some antibodies. It may also provide a medium for transport of metabolites, nutrients, neurohormones, and neurotransmitters. 4 • 10 PathologiC changes in CNS tissue that happen at or near the tissuefluid interface frequently cause changes in CSF composition. Although there is no functional brain-CSF barrier, processes deep in the CNS parenchyma may not cause CSF abnormalities.

RISKS AND CONTRAINDICATIONS FOR CSF COLLECTION It is safe to say that virtually any diagnostic plan for CNS disease, including pre myelographic evaluation, should include CSF analysis, so particular indications will not be individually discussed. There are, however, some attendant risks and specific contraindications that must be taken into account. The major risk is anesthesia, which is an absolute necessity for

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safe CSF collection in veterinary patients. Even a routine anesthetic procedure is something of a calculated risk; patients undergoing CSF collection obviously have overt or suspected CNS disease, adding a further risk factor and necessitating careful monitoring during all phases of the anesthetic episode. A somewhat smaller but nonetheless important consideration is iatrogenic injury to the CNS by an overeager or inexperienced clinician. Accidents can and do happen to anyone regardless of experience, but this hazard can be minimized by expert supervision and repeated practice episodes on fresh cadavers until the clinician is proficient and confident of his or her ability. There is also a slight chance of introduction of infectious agents to the CNS, but if proper technique is observed, this risk should be virtually eliminated (see the section "Technique"). Specific contraindications include any unstable CNS or systemic conditions that will not permit the patient to be anesthetized;20 as previously mentioned, the procedure should never be attempted without general anesthesia. In addition, CSF collection should be avoided in any situation in which ICP is significantly elevated. These situations include but are not limited to acute head trauma, active or decompensated hydrocephalus, cerebral edema, and expanding large mass lesions such as subdural hematomas, aneurysms, neoplasms, or abcesses. Clinicians should be alert for a history of head trauma or intracranial signs that have progressed relatively rapidly over hours to a few days . If the signs, history, and signalment suggest any of the aforementioned abnormalities, CSF collection should be bypassed in favor of less invasive techniques such as computerized tomography or cerebral contrast angiography. In most instances, this will necessitate referral to a university or private specialty practice. If there is access to an electroencephalograph, the presence of diffuse high-voltage slowfrequency activity may be detected with diseases that can cause elevated ICP. 9 If ICP is elevated, removal of CSF from either a cisterna magna or lumbar puncture will create a lower pressure area in the spinal CSF compartment relative to the intracranial compartment, which may then trigger tentorial or foramen magnum herniation. 2o It has been suggested that a transcranial tap of the lateral ventricle may be beneficial in relieving increased CSF pressure from outflow obstruction. The difficulty of this procedure is such that it should only be attempted by experienced individuals; in addition, if the outflow obstruction is from a mass lesion in the posterior fossa, the decrease in anterior fossa pressure may result in forward herniation of the cerebellum under the tentorium. 17 TECHNIQUE This section will cover all aspects of CSF collection, concentrating on cisterna magna collection. Lumbar collection will be discussed separately at the end of the section. Noncritical specific details will usually reflect the personal preference of the author and will be noted as such; these may be changed at the discretion of the operator. However, the basic principles identified that govern aseptic technique and patient safety should be constant every time this procedure is performed.

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Equipment The following items are required: sterile surgical gloves, a sterile disposable or resterilizable spinal needle with a stylet, and equipment for sterile preparation of the collection site. A 11/2-inch, 22-gauge needle is sufficient for cisternal puncture in most patients; large dogs may require' a 21/z-inch or longer needle. Under no circumstances should a needle without a stylet be used, Disposable needles may be cleaned and resterilized but will quickly become very dull and should not be used more than twice. Use of a dull needle will result in excessive tissue drag and dangerous loss of operator controL Optional items include a sterile drape for isolating the collection area and a pressure measurement device, The author does not normally employ a drape, because if it is used to truly isolate the collection site, important landmarks will be covered and head position cannot be easily observed. If care is taken and an adequate area prepared, contamination should not be a problem even without a drape. Pressure measurements are usually made using a three-way stopcock and a glass or disposable spinal manometer column graduated in millimeters of H 20. Patient Preparation and Positioning General anesthesia is required for CSF collection; the type of induction and maintenance is at the discretion of the clinician or as dictated by the patient's condition, Tracheal intubation is always performed to ensure a patent airway during the procedure. For a cisterna magna collection, the hair is clipped from the dorsal midline of the head and neck in a rectangle, starting from the anterior margin of the pinna and extending caudally to the level of the third cervical vertebra (C3); the lateral margins are the pinnae on either side (Fig. 1), The author prefers to clip a large area to prevent contamination and ensure that the prepared area will be over the tap site without having to resort to excessive pulling and stretching of the skin during positioning and collection. The clipped area is surgically scrubbed with tamed iodine and alcohol or similar compounds, The patient is placed in lateral recumbency with the head flexed at approximately 90° to the vertebral column. More extreme flexion is unnecessary and may cause occlusion of the airway.19 Righthanded operators will probably find right lateral recumbency to be most comfortable, but the procedure can be performed easily from either direction. The assistant holds the head firmly, with the long axis of the nose parallel to the table (Fig, 2). The nose should not be allowed to rotate in either direction; a line drawn between the base of each ear should be perpendicular to the long axis of the vertebral column (;ee Fig. 1), The assistant should also retract the pinnae and keep the prepared area centered for the operator. Cisternal Collection Using the passive hand (the hand not actively advancing the needle), the operator should palpate the external occipital.protuberance and the craniodorsal tip of the dorsal spine of C2, This will delineate the midline, which can be palpated as a shallow horizontal groove formed by the

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Figure 1. Patient prepared for cisterna magna CSF collection. The short axis of the head (vertical line drawn between the ears) is perpendicular to the long axis of the vertebral column. o is the external occipital protuberance, C is the craniodorsal tip of the dorsal spine of C2, and the arrow points to the cranial margin of the wing of C1. The dot represents the midline pOint for insertion of the spinal neede. (Photograph courtesy of David Pekarek, Pur4ue University. )

Figure 2. The hand is positioned with the long axis of the nose parallel to the table. The pinnae are retracted and held by the assistant. (Photograph courtesy of David Pekarek, Purdue University. )

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insertion of the dorsal cervical muscles. The author normally uses just these two bony landmarks and visually selects a point on the midline halfWay between them for insertion of the needle. If the operator desires, the anterior margins of the wings of Cl can be palpated to help locate the insertion point (see Fig. 1). Only the ring and little fingers of the passive hand should be used for this because they will probably be contaminated in the process. Once the insertion point is selected, the needle is inserted through the skin, with the bevel directed cranially. The needle is grasped firmly where it enters the skin by the thumb and forefinger of the passive hand to stabilize it and control its advance (Fig. 3). Once inserted, the needle is never released by the passive hand until it is withdrawn at the end of the procedure. The needle is held perpendicular to the skin surface and advanced gradually by the active hand, taking care that the stylet remains in place. The needle should never be advanced without the stylet completely inserted. The depth of the subar;lchnoid space will vary depending on the position, size, and body type of the patient, so periodically the stylet should be withdrawn by the active hand. If the subarachnoid space has been entered, fluid should appear in the hub almost immediately. If not, the stylet is replaced and the needle is advanced further. "Popping" or "snapping" sensations represent penetration of fascial planes and are not a reliable indicator of meningeal penetration. Occasionally, a sudden loss of resistance is felt when the subarachnoid space is entered; this feeling is not consistent, but with practice can be detected in most instances. The only truly reliable indicator of subarachnoid penetration is appearance of CSF in the needle when the stylet is withdrawn. 19 If bone is encountered, the needle is withdrawn and redirected slightly either cranially or caudally.' If bone is encountered again at approximately the same depth, the needle is probably against the dorsal arch ofCI and should be directed more cranially. If bone is encountered at a different depth, the needle is being moved along the posterior aspect of the occipital bone and should be redirected caudally (Fig. 4). If the operator believes that the needle has been inserted too deeply, the stylet should be removed and the needle carefully withdrawn a few millimeters at a time, watching for the appearance of fluid in the hub. It is acceptable to withdraw the needle without the stylet in place, but the stylet must be replaced if the needle is to be advanced again. Once the subarachnoid space is entered, fluid samples are collected. If pressure is to be measured, the three-way stopcock and manometer are carefully attached to the needle hub and the stopcock manipulated so that . fluid flows into the manometer. When the fluiq level is stable, the opening pressure is noted and the stopcock is then adjusted to allow external flow of fluid for laboratory samples. Once these are obtained, fluid flow is again directed into the manometer to record closing pressure. The needle and stopcock are then gently disengaged before the needle is withdrawn. Another assistant may be required for safe handling of the stopcock and manometer. If pressure is not measured, samples can be collected directly from the spinal needle hub with a syringe or test tube. If a syringe is used, drops are aspirated as they collect at the hub. Collection should not be

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Figure 3. The operator uses the thumb and forefinger of the passive hand to stabilize the needle during the procedure. (Photograph courtesy of David Pekarek, Purdue University.)

Figure 4. Proper positioning of the spinal needle in relationship to the skull and vertebrae. 1 is Cl, and 2 is C2. (Photograph courtesy of David Pekarek, Purdue University.) <

o is the external OCcipital protuberance,

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attempted by attaching the syringe to the spinal needle and applying suction, The arachnoid and pial membranes are connected by numerous fine trabeculi separated by meningeal lining cells. 6, 10 Suction may draw these tissues into the lumen of the needle, causing obstruction to CSF flow or contamination with blood and/or meningeal cells. If CSF flow is slow, the needle can be gently rotated to clear the lumen at the tip, If this is not effective, compression of the jugular veins may improve flow by expanding the venous sinuses and increasing CSF pressure. Some blood may be mixed initially with the fluid from damaged meningeal vessels or contamination of the needle during advancement through the dorsal cervical tissues , In these cases, the first few drops should be allowed to fall , after which the fluid will usually clear. If blood contamination persists, it may be due to the pathologic process and the desired samples should be collected. Pure blood indicates that the point of the needle is off the midline and has entered a venous sinus. The needle should immediately be withdrawn and discarded. Because the venous sinuses are extradural, more careful placement of a clean needle should produce a clean CSF sample. Usually 0.75 to 2.0 cc of fluid is sufficient for cell and protein examinations. A few drops should be saved in a separate container for microbial culture and sensitivity if infection is suspected. Other samples for virology, immunology, and so forth are collected as needed. Once collection is completed, the needle is withdrawn in one smooth motion. The stylet need not be replaced prior to removal of the needle. All containers should be appropriately sealed to prevent contamination and delivered immediately to the laboratory. Lumbar Collection Lumbar subarachnoid puncture in small animals is usually used for thoracolumbar myelography. For a purely diagnostic CSF sample, lumbar collection is technically more difficult and more likely to result in iatrogenic blood contamination. Frequently, no fluid can be obtained at all at a lumbar site owing to the small size of the subarachnoid space. 1O For this reason, the author frequently performs a cisterna magna collection before thoracolumbar myelography to ensure that a diagnostic CSF sample will be obtained. If fluid is then obtained from the lumbar puncture, both should be analyzed. OccaSionally, however, circumstances require lumbar collection for diagnostic samples. Proliferative or infiltrative diseases may obliterate the subarachnoid space at the cisterna magna. This situation is suspected when fluid cannot be obtained from cisternal puncture despite careful attention to needle placement and advancement. Also, diseases centered in the thoracolumbar spinal cord may not produce changes in the cisternal fluid due to the primarily caudal flow of CSF. 20 . For lumbar puncture, the dorsal midline is prepared between the midsacrum and L3, extending laterally to the wing of the ilium on either side. With the patient in left lateral recumbency (for a right-handed operator), the assistant flexes the back slightly to open the space between the dorsal laminae of the vertebrae (Fig. 5). In dogs, the L5-L6 or L6-L7 interspace is normally used; the subarachnoid space" rarely extends to the lumbosacral junction in dogs, although in cats collection can frequently be

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Figure 5. Patient prepared and positioned for lumbar CSF collection. The location of the dorsal spinous processes of L6 and L7 are shown relative to the wings of the iliums (dots). (Photograph courtesy of David Pekarek, Purdue University.)

made from the lumbosacral space. A 1V2-inch needle is usually sufficient for cats, but a 2V2-inch or longer needle will be needed for all but the smallest dogs. 19 The most reliable landmark is the dorsal spinous process of L7, which lies between the wings of the iliums and is usually noticeably smaller than that of L6 just cranial to it (Fig. 5). In order to collect from the L5-L6 interspace, the author prefers to place the point of the needle just off the midline at the caudal aspect of the L6 dorsal spinous process. The needle is advanced at an angle cranioventrally and slightly medially to enter the spinal canal between the dorsal laminae of L5 and L6 (Fig. 6A and B). There is rarely a true opening between the dorsal laminae, especially in older dogs with bony proliferative changes of the vertebrae. The operator should identifY the desired space by its slightly "woody" feel as compared with the surrounding bone. Once this space is encountered, the needle is wedged through into the spinal canal. If the needle is advanced to the hub without ever encountering bone, either the tip has been misdirected laterally into the paralumbar muscles or else the operator has sadly underestimated the length of needle required. The operator may attempt to locate and collect from the dorsal subarachnoid space, but it is usually easier and more productive to find the ventral space instead. This is accomplished by advancing the needle through the spinal cord until it strikes the floor of the spinal canal (Fig. 7). A slight twitch of the tail may be seen when the cord is penetrated. Once the needle contacts the floor of the canal, the stylet is removed . It may be necessary to carefully withdraw the needle a few millimeters to obtain fluid flow. The rate of fluid flow is usually slower than from the cisterna magna and may be enhanced by jugular compression: Penetration of the spinal cord during collection from the ventral subarachnoid space rarely causes postprocedural neurologic deficits unless the operator allows excessive needle motion to Occur within the spinal canal. 2

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Figure 6. A. Lateral view demonstrating the position of the spinal needle for CSF collection at L6-L7. B, Dorsolateral view showing the spinal needle entering the spinal canal between the dorsal laminae of L6 and L7. (Photographs courtesy of David Pekarek. Purdue University. )

ANALYSIS

Pressure

~easurernent

Pressure recording, if desired, is measured during the collection procedure (see the section "Technique"). Normal values are less than 170 to 180 mm H 20 for dogs and less than 100 mm H 20 in cats. 2. 4. 17,20 Elevated pressure is a nonspecific finding and may be encountered with spaceoccupying lesions, cerebral edema, hydrocephalus, inflammatory CNS diseases, and vitamin A deficiency, 4 Significant fluctuation in pressure may occur in a normal individual over a 24-hour period,17 and larger dogs tend to have a higher normal pressure,4 Positioning and anesthesia can have a variable effect on CSF pressure, so the procedure should be standardized

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Figure 7. Anterior view of L6 showing the position of the spinal needle for lumbar CSF collection from the ventral subarachnoid space. (Photograph courtesy of David Pekarek, Purdue University.)

for each clinician. 4. 17 An abnormally high opening pressure with a much lower closing pressure suggests the presence of a space-occupying mass that is causing reduced volume of eSF.9. 17 If opening pressure is greater than 200 mm H 20, samples should be collected and the procedure terminated as quickly as possible. 17 The measurement of eSF pressure adds technical difficulty to the collection procedure and increases the risk of both iatrogenic hemorrhage in the sample and trauma to the eNS parenchyma. As noted above, elevation of pressure is a nonspecific abnormality found in many pathologic conditions. For this reason, many veterinary neurologists (the author included) do not routinely measure eSF pressure, believing that the information gained is not worth the risk. 19 In addition, as previously mentioned, eSF collection should not be performed if significantly elevated pressure is likely. The increases in pressure seen in inflammatory disease are mild, so suspected inflammation of the eNS is not a contraindication for eSF collection. 20

LABORATORY ANALYSIS OF CEREBROSPINAL FLUID Relatively complete eSF analysis can be accomplished in the average veterinary practice. The overall analysis should include physical, cytologic (quantitative and qualitative), and chemical evaluation. Once the fluid is collected, it should be delivered to the laboratory (either in-house or reference) as soon as possible. As mentioned previously, protein and other analyte analyses have no special time-dependent requirements. In fact,

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CSF samples can be handled just like any other fluid , including serum, for transport to a reference laboratory for evaluation. Cytologic analysis, however, is very time-dependent. Cell counts and slide preparation for qualitative analysis must be completed within 30 minutes of fluid collection. If not performed immediately, the fluid should be refrigerated or placed on ice until processing. Physical Examination Characterization of the physical properties of CSF is extremely simple, yet it can often provide valuable insight into the etiopathogenesis of a particular neurologic disorder. This evaluation generally consists of gross inspection of the collected fluid; therefore, fluid collection should be made into a transparent, noncolored glass tube. Normal CSF from any of the domestic animals with which we deal is colorless and clear, resembling water. In fact, direct visual comparison with a similar tube containing water will optimize the evaluators ability to detect even subtle changes from normal. Any increase in turbidity is attributed to particles in the fluid . In general, this directly correlates with increased cellularity of the sample. Fluids containing greater than 200 nucleated cells or 400 erythrocytes per cubic millimeter can be detected as a slight turbidity by the average person.7, 11 Identification of turbidity may be facilitated by inspecting a printed page through the tube of fluid. Because this physical change could be due to accumulations of different cell types, including erythrocytes, inflammatory cells, microorganisms, and neoplastic cells, microscopic evaluation of the specimen is required for diagnostic specificity. Because normal CSF is colorless, any detectable color is generally easy to recognize and represents an ·abnormality. As mentioned before, comparing the fluid with water is helpful in identifying subtle coloration. As with other fluids, a pink or red tint to a CSF sample suggests blood. If, after centrifugation, there is a red cellular pellet at the bottom of the tube and the supernatant is colorless, this coloration is due to the presence of intact erythrocytes. This represents either peripheral blood contamination or recent (within a few hours) hemorrhage into the subarachnoid space. If the supernatant is xanthochromic (yellow to yellow-orange discoloration), this generally is an indication of previous hemorrhage and an accumulation of oxyhemoglobin or methemoglobin derived from erythrocyte degradation . 11 However, xanthochromia may also occur with situations in which there is an increase in total protein, hyperbilirubinemia, and in certain cases of hydrocephalus and CNS neoplasia. 14 , An additional component of CSF physical analysis is the determination of the specific gravity of the specimen. This is a simple procedure requiring only a few drops of CSF and a good refractometer, which is commonly available in most practices. However, the value of determining specific gravities on CSF samples has been questioned. The majority of "abnormal" samples fall within the reference range of 1. 004 to 1. 006. 23 Only when there are relatively marked increases in total protein 'ire changes in specific gravity detectable.

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Cytologic Examination Cytologic analysis of all CSF samples is strongly recommended; currently, this component of the laboratory analysis is among the most helpful in the routine workup of an animal with neurologic disease. Oftentimes, the original differential diagnosis list established for a patient can be limited following evaluation of the cellular composition of CSF. In certain circumstances, specific diagnoses are possible. Total erythrocyte (RBC) and nucleated cell (WBC) counts are quickly made with the use of a standard hemacytometer. With a little practice, one can accurately determine total RBC and WBC counts following direct loading of the hemacytometer with well-mixed CSF without the use of any particular stain . Nuclei give cells a granular character and, with experience, one can recognize nuclear morphology. Erythrocytes are often crenated (multiple short, sharp projections on their surface) or refractile, making them easily distinguishable from the nucleated cells . Both RBCs and WBCs are enumerated following inspection of the large nine squares of the hemacytometer. Because this area represents only 0.9 mm 3 , the number of cells in the nine large squares multiplied by 1.1 gives the total number of cells per cubic millimeter. Until the individual doing the cell counts is comfortable with the preceding technique, cell counts should be made following standard procedures using a stain to help identify nucleated cells. With these procedures, a white cell diluting pipette is filled to the" 1" mark with a special diluting solution (0.2 gm crystal violet dissolved in 100 ml of a 10 per cent glacial acetic acid solution) and then to the "u" mark with the CSF sample. This solution will lyse the erythrocytes and stain the nuclei of all nucleated cells, making enumeration of these cells very simple . Again , cells in all large nine squares of the hemacytometer are counted. Because the sample is diluted by a factor of 1.1, the number of cells per cubic millimeter is calculated by multiplying the total count by 1.2 (1.1 XLI) . Normal RBC and WBC counts for canine and feline CSF are reported to be 0 and less than 5 to 8 cells per mm 3 , respectively.3-5 An increase in CSF cellularity is termed "pleocytosis." The degree of pleocytosis in patients with various neurologic disorders is directly related to several factors, including the severity of the disease, the inciting etiology, and the degree of communication of the CNS lesion with the subarachnoid space. Central space-occupying lesions may result in severe neurologic disorders yet present with minimal to no cellular changes in the CSF. It is important to follow up the quantitative analysis with a detailed microscopic evaluation to determine the types of cells p.resent. In our experience, abnormal cells or abnormalities in morphology of cells normally found in the CSF occur frequently, even when pleocytosis is not detected. If qualitative analysis is only performed on those fluids with obvious increased cellularity, potential valuable diagnostic information is lost. Various methods have been proposed for the preparation of cytologic specimens for this qualitative analysis. However, the majority of these result in poor-quality cytologic preparations or are tao expensive or cumbersome for application in the average veterinary practice. Because of the

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low total cellularity and the low protein content (no support for suspended cells) of CSF, centrifugation methods that are used for cellular fluids such as exudates collected from other body cavities are not helpful in preparing specimens from CSF. A membrane filter technique which can be performed in the practice without the need for special expensive equipment has been proposed for cytologic evaluation of CSF samples. 21 However, routine Romanovski-type stains cannot be used, and cytomorphology is reported to be inferior to alternative methods available to the average veterinarian. 5 At most reference laboratories, cytocentrifuges are used for preparing CSF cytologic specimens. These specialized centrifuges allow the rapid preparation of air-dried or alcohol-fixed specimens with good cytomorphologic preservation. They use centrifugal force to concentrate cells onto glass slides. In the majority of situations, they are cost-prohibitive to the veterinarian. Similar good-quality preparations are possible for the practitioner through the use of various sedimentation systems. 17 Several of these procedures are awkward or complicated, making their practical application difficult. We have developed a simple method that provides cytomorphologic detail comparable to the original sedimentation procedure designed by Sayk and later modified by Kolar. 13 Equipment required includes a tuberculin syringe, a V4-inch paper punch, filter paper, a few scraps of wood, several bolts and wing nuts, and clean glass slides (Fig. 8). The end of the barrel of the syringe where the needle is normally attached is cut off and discarded. The scraps of wood are assembled as demonstrated in Figure 8, such that two narrow strips of wood can be fastened to the larger platform piece. These strips should attach parallel to one another, with the space between them being slightly larger than the outside diameter of the barrel of the syringe. Strips of filter paper are

Figure 8. Partially assembled sedimentation apparatus. A strip of filter paper with an appropriately punched hole is sandwiched between the plastic column (syringe barrel) and a clean glass slide. Note the positioning of the strip of wood resting on the lip of the end of the syringe.

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prepared in advance by punching holes using the paper punch. These holes are slightly greater than the inside diameter but less than the outside diameter of the barrel of syringe. The unit is assembled by sandwiching a piece of filter paper (punched hole lined up with the barrel of the syringe) between a glass slide and the syringe. The strips of wood when positioned over the lips of the end of the syringe (end where the plunger would normally enter the barrel) will hold the syringe in place and allow the application of pressure onto the paper. The apparatus should be tested and "primed" before introducing a CSF sample. This is accomplished by the addition of a few milliliters of physiologic saline to the syringe and watching the flow of fluid onto the filter paper. If flow is not even or if the fluid rapidly is absorbed onto the paper, repositioning of the wood strips or adjustment of the pressures can be done before loading the CSF sample. Because of the low protein content of CSF, it is recommended that the cells be supported in a protein solution for added cellular stability. This can be most simply accomplished by using either fresh or frozen cell-free serum or plasma. Immediately prior to loading the sedimentation system, 200 ·to 300 mm 3 of serum or plasma (5 to 8 gm per dl) are mixed with 200 to 300 mm 3 of fresh, well-mixed CSF. Once loaded, the sedimentation process should optimally take 45 minutes for completion. This allows slow settling of the cells onto the glass slide as the fluid phase of the CSF is absorbed onto the filter paper. If the process is not complete within 45 minutes, excess fluid is aspirated out of the syringe barrel and the slide is allowed to air dry. After drying, any routine Romanovsky-type stain may be used to prepare the slide for microscopic evaluation. In our hands, this type of apparatus produces preparations similar in quality to those prepared by commercially available cytocentrifuges that cost more than $5000 . . The normal distribution of cell types in the CSF is similar for the dog and cat. Mononuclear cells predominate and consist of a mixture of normalappearing lymphocytes and larger cells classified as "monocytoid" cells. The percentage of lymphocytes and monocytoid cells varies with the type of method used for preparing cytologic specirriens; however, most references state that lymphocytes are the predominating nucleated cell in normal dog and cat CSF. Lymphocytes are morphologically similar to those present in the peripheral blood. They range from 9 to 15 I..l. in diameter and have scant to only moderate amounts of pale basophilic cytoplasm and a round to ovoid, slightly indented nucleus (Fig. 9). When there is local antigenic stimulation within the subarachnoid space, reactive lymphocytes may be seen. Reactivity can be demonstrated by finding lymphocytes with greater amounts of cytoplasm, more deeply basophilic cytoplasm, potentially prominent perinuclear clear zones, and possibly nuclei with coarse chromatin patterns . On occasion, plasma cells may be observed. Examples of various stages of reactivity are shown in Figure lOA, B, and C. No particular significance can be attached to the finding of reactive lymphocytes. They may be seen with active or resolving infectious diseases, neoplastic processes, and potential immune-mediated diseases. The normal "monocytoid" cell is a relatively large cell ranging from 12

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Figure 9. Lymphocyte in CSF from a clinically normal dog (sedimentation preparation). Normal lymphocytes are morphologically similar to typical small lymphocytes observed in peripheral blood films (x lOoo, Wright's stain).

B

A

c Figure 10. A, Lymphocyte in CSF from a dog. The centrally located reactive lymphocyte is surrounded by two erythrocytes and a neutrophil. Reactivity of the lymphocyte is indicated by the increased cell size, cytoplasmic basophilia, and presence of a perinuclear clear zone (x 1000, Wright's stain). B, Lymphocyte in CSF from a dog. This lymphocyte is slightly more reactive than the cell described in part A. Note the coarse chromatin pattern in the eccentric oval nucleus (x 1000, Wright's stain). C, Lymphocyte in CSF from a dog. The markedly reactive lymphocyte is surrounded by erythrocytes and a much less reactive lymphocyte. The deeply basophilic cytoplasm gives this cell a plasmacytoid appearance (x 1000, Wright's stain).

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to 15 f.I. in diameter. Cytoplasm is pale basophilic, moderate in amount, and often finely foamy in nature. Nuclei are quite variable in shape and often ameboid. Chromatin patterns are open and lacey (Fig. llA and B). These cells have many features of the normal peripheral blood monocyte and in fact are derived from blood monocytes. During periods ofleptomeningeal irritation or in situations in which there is need for local cleanup of an inflammatory or degenerative process, these cells undergo activation and a series of morphologic changes toward macrophage development. Activated monocytoid cells are larger than normal and have greater amounts of cytoplasm that is often paler than normal and possibly vacuolated. Nuclei become round to oval and eccentric in location. Chromatin patterns are slightly more coarse than in the typical monocytoid cell (Fig. 12). Once phagocytosis of material is evident, the cells are identified as macrophages (Fig. 13A and B). In human medicine, these cells are classified according to the material being phagocytized (erythrophage, hemosiderophage, lipophage). The significance of their presence is often directly related to the material being phagocytized. For example, the finding of erythrophagocytosis indicates recent hemorrhage. Beyond the lymphocytes and monocytoid cells, low numbers of mature nondegenerate neutrophils may be present in normal CSF.5 In one of our recent investigations (unpublished data), evaluation of CSF from 16 clinically normal dogs revealed a total nucleated cell count of less than 3 cells per mm 3 , and although the majority of these samples had no neutrophils, certain samples had up to 25 per cent neutrophils. In general, when there is a very high nucleated cell count (to the point of finding a turbid CSF sample) and neutrophils are the predominant cell type present, bacterial infectious processes must be very strongly considered; however, a neutrophilic pleocytosis is not diagnostic for bacterial meningitis. Mild to moderate

A

B

Figure 11. A, A single normal-appearing monocytoid cell in CSF from a dog with an eccentric, indented nucleus , open chromatin pattern, and pale basophilic cytoplasm (x 1000, Wright's stain). B, Two normal monocytoid cells in CSF from a dog with ameboid nuclei and irregular cytoplasmic margins (x 1000, Wright's stain).

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Figure 12, A cluster of eight activated monocytoid cells with increased amounts of cytoplasm, round to ovoid eccentric nuclei, and pale vacuolated cytoplasm (X 1000, Wright's stain).

A

B

Figure 13. A, An erythrophage with phagocytized erythrocytes clearly identifiable within cytoplasmic vacuoles (X 1000, Wright's stain). B, A macrophage surrounded by two neutrophils, two lymphocytes, and a single monocytoid cell (x 1000, Wright's stain).

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neutrophilic pleocytosis can be associated with a wide variety of acute inflammatory disorders, including trauma, postmyelographic aseptic meningitis, hemorrhage, and neoplasia (Fig. 14).18 Other cells that may be found in a normal CSF sample include cells lining the leptomeninges, choroid plexus cells, and ependymal lining cells. The incidence of finding these cells is increased when CSF is collected during aspiration with a syringe. The leptomeningeal lining cells often appear in small clusters and consist of mononuclear cells with moderate to abundant amounts of pale basophilic cytoplasm, rounded to oval eccentric nuclei, uniform and often delicate nuclear chromatin patterns, and indistinct cytoplasmic margins. Choroid plexus and ependymal lining cells are indistinguishable and present as uniform round to cuboidal mononuclear cells found individually or in cohesive clusters of varying sizes. These cells have eccentric rounded nuclei, uniformly granular to coarse chromatin patterns, and moderate amounts of finely granular cytoplasm (Fig. 15A and B). If one finds almost any other cell type, there is usually an abnormality. Eosinophils may be a component of a nonspecific acute inflammatory response; however, one must consider that their presence may be indicative of parasitic, hypersensitivity, or neoplastic processes involVing the CNS. Neoplastic processes (either primary or metastatic) involving the CNS have the potential of presenting with neoplastic cells in the CSF if the process is communicating with the subarachnoid space (Fig. 16). Beyond host cells, occasional microscopic evaluation of CSF samples allows the identification of specific etiologic agents such as bacteria and fungi . Special staining procedures may be indicated for certain infectious processes. For example, Gram staining may provide added information about bacterial processes,

Figure 14. Neutrophils and normal-appearing monocytoid cells in CSF from a dog with aseptic meningitis 24 hours after a myelogram (x 1000, Wright's stain).

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Figure 15. A, A cluster of choroid plexus epithelial cells surrounded by erythrocytes ( x 1000, Wright's stain). B, Pleomorphic, elongate mononuclear cells in a cohesive cluster in

CSF from a dog with choroid plexus carcinoma. Note the anisocytosis, anisokaryosis, prominent nucleoli, coarse chromatin patterns, basophilic cytoplasm, and variable nuclear/cytoplasmic ratios (x 1000, Wright's stain).

Figure 16. Malignant lymphoblasts in CSF from a dog with multicentric lymphosarcoma. Note the single prominent nucleolus, irregular chromatin patterns, anisocytosis, variable nuclear/cytoplasmic ratios, deeply basophilic cytoplasm, and occasionally vacuolated cytoplasm (x 1000, Wright's stain).

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A Figure 17. A. A multinucleated giant cell surrounded by six eosinophils and a single monocytoid cell in CSF from a dog with cryptococcosis. A phagocytized fungal form with a prominent unstained area around the organism is seen (x 1000. Wright's stain). B. A cluster of budding yeast forms are seen in the CSF from a dog with cryptococcosis (x 100. India ink preparation).

and india ink preparations may be helpful in identifying fungal infections such as cryptococcosis (Fig. 17A and B) . Chemical Examination Probably the most helpful component of a CSF chemical profile is determination of total protein content. Increased total protein is among the most frequent alterations observed with human CSF samples. II Increases can be associated with a wide variety of conditions, including peripheral blood contamination during collection, alterations in the blood-brain barrier (inflammation), obstruction of CSF flow, increased synthesis of immunoglobulins within the CNS, and accumulation of proteins associated with a local destructive lesion. Although many situations present with both an increased CSF total protein and pleocytosis, it should be pointed out that protein changes may be seen in the absence of cytologic abnormalities and vice versa. Because of the low protein concentrations of CSF, even under most abnormal conditions, special sensitive methods unavailable to most practitioners are required. However, CSF samples can be delivered to both veterinary and human reference laboratories for these sensitive determinations. Many laboratories use either one of several turbidometric or certain dye-binding methods for microprotein quantitation. A common problem with all of the available methods is the lack of 100 per cent sensitivity to all types of proteins. Some of the reported variability in reference ranges from different laboratories is directly related to this variable sensitivity and the use of different types of standards (100 per cent albumin versus an albumin/globulin mixture). Consistency in the method and the type of protein standard utilized is potentially more important to the clinician than

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the accuracy of the method employed. 8 Therefore, it is important to utilize one particular laboratory for CSF protein determination and to maintain good communications with the individuals responsible for these tests to assure notification if there is a change in methodology. As with any of the components of the CSF analysis, knowing the results of the total protein content determination as soon as possible is important for the early initiation of the appropriate therapy. Something that can be done in-house while waiting for the more accurate protein estimate from the reference laboratory is the use of a common urine reagent test strip. These test strips provide relatively accurate protein estimates of fluid samples, and although they are more sensitive to albumin than other proteins, they can give an immediate indication of normal or increased protein content. Use of the Ames Multistix* allows the detection bf various ranges of protein concentrations that are within the normal and abnormal working ranges for most canine and feline CSF specimens (Table 1). Normal canine and feline CSF has a reported total protein of less than 25 to 40 mg per dl. 4. 5, 17 In our laboratory, both species have values less than 20 to 25 mg per dl. Therefore, a normal CSF yields a trace protein result using the urine reagent test strip. Mild increases in protein give a 1 + test result, and moderate to marked increases in protein give a 2 + to 3 + result. Table 2 demonstrates the relatively good correlation between the test strip and a standard dye-binding microprotein determination (data not previously reported). The mere identification of increased protein content alone is not helpful in differentiating the various pathogenic mechanisms of its increase. For this, measurement of specific protein fractions, namely albumin and immunoglobulin G (IgG), and comparison with serum levels are necessary. Four general categories of CSF protein patterns can be characterized if these measurements are made. They include normal, alteration in the blood-brain barrier, local IgG synthesis, and a combination of local IgG synthesis and alteration in the blood-brain barrier. 1, 22 Currently, reference laboratories do not routinely perform these testing procedures, which require modified immunologic and electrophoretic methodologies. However, the use of these methods on CSF samples from dogs with space-occupying, vascular, traumatic, inflammatory, and degenerative lesions of the CNS has allowed the documentation of protein patterns like those observed in human patients with similar conditions. 1, 15.22 In the near *Ames Division, Miles Laboratories Inc., Elkhart, Indiana

Table 1. Ames Multistix Results and Correlation to Actual Protein Concentrations AMES MULTlSTlX

PROTEIN

RESULT

CONCENTRATlON

trace 1+ 2+

3+ 4+

<30 30 .100 300 >2000

mg/dl mg/dl mg/dl mg/dl mg/dl

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Table 2. Comparison of Quantitative Dye-Binding (Coomassie Brilliant Blue G-2S0) and Qualitative (Ames Multistix) Microprotein Determination Methods for CSF Protein Determination in the Dog CSF

COOMASSIE BLUE

AMES MULTISTIX

SAMPLE*

(mg/dl)

RESULTS

1 2 3 4 5 6

7 8 9 10

11 12 13 14 15

24

TRACE TRACE TRACE TRACE TRACE

28 35 48 75 115 190 240 470 520 590

1+ 1+ 1+ 1+ - 2+ 2+ 2+ 2+ 3+ 3+ 3+

10

12 18 20

'Canine CSF samples presented to the clinical pathology laboratory of the Purdue University School of Veterinary Medicine for routine analysis.

future, these techniques may be as commonplace in veterinary medicine as they presently are in human medicine. Beyond total and specific protein quantitation, qualitative protein analysis for the presence of globulins is often included in a complete CSF analysis. The Pandy test, which merely consists of mixing a few drops of fresh CSF with 1.0 ml of Pandy reagent (10 gin carbolic acid crystals in 100 ml distilled water) , can be performed in the average practice situation. Globulins are selectively precipitated, giving the test solution a slight to marked cloudy appearance dependent upon the amount of globulins present. 3 Because normal CSF protein primarily consists of albumin, the Pandy test in the dog and cat is normally negative (no turbidity development). Although not specific for any particular type of globulin, the Pandy test is relatively sensitive and shows good correlation with more sophisticated globulin and IgG quantitation methods in the dog. I Another relatively common component to the chemical analysis of CSF is glucose quantitation. In general, these determinations are made at a reference laboratory; however, a practice equiped with even limited serum chemistry capabilities can process their own CSF sample, because methodologies for both serum and CSF are the same. Cerebrospinal fluid glucose concentration is dependent upon the serum concentration, because CSF levels are attained through a facilitated transport mechanism across the blood-brain barrier. Therefore, determination of the serum levels at the time of CSF analysis is imperative. Values of 60 to 80 per cent of the serum glucose concentration are generally reported for most domestic animals. 17 Sudden changes in serum glucose concentrations are not immediately reflected" in the CSF concentrations, because there is an approximately 30- to gO-minute lag time for

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equilibration. The most common alteration observed with CSF glucose concentration is a decrease that can be seen in cases in which there is hypoglycemia, impaired transport across the blood-brain barrier, increased metabolism by the brain parenchyma, or increased metabolism by inflammatory cells and microorganisms within the CNS. In human medicine, 60 to 80 per cent of children with bacterial meningitis have decreased CSF glucose concentrations. 12 Other, less commonly measured analytes in CSF include various enzyme activities and electrolyte levels. The reference laboratory should be contacted before sending samples for analysis to ensure proper handling of specimens. Increases in creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotrasnferase (ALT) , and aspartate aminotransferase (AST) activities have been documented with various CNS disorders in veterinary medicine; however, no particular specificity has been attributed to these changes. 5 , 24 In man, quantitation of the specific CK 1 (BB) isozyme of CK shows relatively good correlation with the degree of damage to the CNS and appears to be of prognostic value. 16 More thorough investigation of its potential value in veterinary medicine is needed. SUMMARY It is hoped that we have demonstrated that collection, handling, and limited analysis of CSF samples from the dog and cat are relatively simple. No special equipment or handling is required, and the procedures are within the capabilities of any veterinarian interested in performing them. In addition, although this article was not intended to present a detailed discussion on the interpretation of the analysis of CSF, we have demonstrated some very practical interpretations to the various aspects of a routine CSF analysis . When combined with a signalment, complete history, and thorough general physical and neurologic examination, CSF analysis can prove invaluable in the workup of an animal with a neurologic disorder. Relatively simple laboratory procedures can be helpful in differentiating peripheral blood contamination from true intrathecal hemorrhage, in identifying an active inflammatory process, in potentially characterizing an etiologic agent, and, on rare occasions, in identifying primary or metastatic neoplastic disease involving the CNS. In many cases, the above is not directly possible, because the changes observed in our routine analysis are nonspecific. Yet, documenting and following these "nonspecific" alterations are helpful in determining if there is progression or regression of the 8isease process. In turn, these changes or lack of changes are helpful in identifying if the proper therapy has been instituted and if additional or different therapy is required. REFERENCES 1. Bischel P, Vandevelde M, Vandevelde E, et al: Immunoelectrophoretic determination of albumin and IgG in serum and cerebrospinal fluid in dogs with neurological diseases. Res Vet Sci 37:101-107, 1984

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2. Braund KG: Clinical Syndromes in Veterinary Neurology. Baltimore, Williams & Wilkins Co, 1986, p 207 3. Coles EH: Cerebrospinal fluid. In Veterinary Clinical Pathology. Edition 3. Philadelphia, WB Saunders Co, 1980 4. de Lahunta A: Veterinary Neuroanatomy and Clinical Neurology. Edition 2. Philadelphia, WB Saunders Co, 1983, p 30 5. Duncan JR, Oliver JE, Mayhew IG: Laboratory examination. In Oliver JE, Hoerlein BF, Mayhew IG (eds): Veterinary Neurology. Philadelphia, WB Saunders Co, 1987, pp 5764 6. Evans HE, Christensen GC: Miller's Anatomy of the Dog. Edition 2. Philadelphia, WB Saunders Co, 1979, p 935 7. Fishman RA: Cerebrospinal fluid . In Clinical Neurology. New York, Harper & Row, 1971 8. Gerbaut L, Macart M: Is standardization more important than methodology for assay of total protein in cerebrospinal fluid? Clin Chern 32:353-355, 1986 9. Hoerlein BF: Canine Neurology. Edition 3. Philadelphia, WB Saunders Co, 1978, pp 136, 150 10. Jenkins TW: Functional Mammalian Neuroanatomy. Edition 2. Philadelphia, Lea & Febiger, 1978, p 85 11. Kjeldsberg CR, Knight JA: Cerebrospinal fluids . In Kjeldsberg CR (ed): Body Fluids. Edition 2. American Society of Clinical Pathology Press, 1986 12. Knight JA, Dudek SM, Haymond RE: Early (chemical) diagnosis of bacterial meningitis: Cerebrospinal fluid glucose, lactate, and lactate dehydrogenase compared. Clin Chern 27:1431-1434, 1981 13. Kolar 0, Zeman V: Spinal fluid cytomorphology. Arch Neurol 18:44-51, 1968 14. Krieg AF: Cerebrospinal fluid and other body fluids. In Henry JB (ed): Toss-Sanfor Davidson Clinical Diagnosis and Management by Laboratory Methods. Volume 1. Philadelphia, WB Saunders Co, 1979, pp 653-679 15. Long JF , Jacoby RO, Olson M, et al: Beta-glucuronidase activity and levels of protein and protein fractions in serum and cerebrospinal fluid of dogs with distemper associated demyelinating encephalopathy. Acta Neuropathol 25:179-187, 1973 16. Matias-Guiu J, Martinez-Vasquez J, Ruibal A, et al: Myelin basic protein and creatine kinase BB isozyme as CSF markers of intracranial tumors and stroke. Acta Neurol Scand 73:461-465, 1986 17. Mayhew IG, Beal CR: Techniques of analysis of cerebrospinal fluid . Vet Clin North Am 10:155, 1980 18. Oehmichen M: Cerebrospinal Fluid Cytology: An Introduction and Atlas. Philadelphia, WB Saunders Co, 1976 19. Oliver JE , Hoerlein BF, Mayhew IG : Veterinary Neurology. Philadelphia, WB Saunders Co, 1987, p 57 20. Oliver JE , Lorenz MD: Handbook of Veterinary Neurologic Diagnosis. Philadelphia, WB Saunders Co, 1983, p 106 21. Roszel JF: Membrane filtration of canine and feline cerebrospinal fluid for cytologic evaluation. J Am Vet Med Assoc 165:165, 1974 22. Sorjonen DC, Warren IN, Schultz RD: Qualitative and quantitative determination of albumin , IgG, IgM and IgA in normal cerebrospinal fluid of dogs . J Am Anim Hosp Assoc 17:833, 1981 23. Wilson JW, Stevens JB: Analysis of cerebrospinal fluid specific gravity. J Am Vet Med Assoc 172:911-913, 1978 24. Wright JA: Evaluation of cerebrospinal fluid in the dog. Vet Rec 103;48-51, 1978 Department of Veterinary Clinical Sciences School of Veterinary Medicine Purdue University West Lafayette, Indiana 47907