Sample Collection

CSF can be collected from either the atlanto-occipital or the lumbosacral region of the spine.^1,⁴ The neurologic examination and clinical condition of the horse determine the collection site.⁵ Lesions localized to the brain and cervical spine are most likely to produce abnormalities in CSF collected from the atlanto-occipital site. Lumbosacral collection is most appropriate for lesions in the caudal segments of the spinal cord. For some horses, comparison of the results for CSF collected from both sites can be helpful in lesion localization. Collection of CSF from either site requires correct positioning, adequate restraint, appropriate needles, surgical preparation of the skin, and availability of specimen tubes. Atlanto-occipital collection is limited to comatose horses or those under general anesthesia because immobilization and lateral recumbency are necessary for collection. Collection of CSF from the lumbosacral site can be achieved in the standing adult horse. In foals, CSF can be collected from either site with the animal in lateral recumbency.⁶ People involved in the collection and laboratory analysis should exercise extreme caution if rabies is being considered in the differential diagnosis.

CSF should be collected into sterile, clear-glass or plastic-capped vials. An aliquot can be removed aseptically from the tube and used for cell counts, total protein concentration, and cell identification. The remainder of the specimen can be used for microbial culture or chemistry assays if indicated. Although most CSF specimens do not clot, a tube containing EDTA should be readily available in case the fluid is bloody or purulent. If the fluid is bloody on initial penetration, several tubes should be available so that multiple aliquots can be collected to see if the fluid clears. A recent study in horses revealed that blood contamination diminished significantly as three or four 2-ml aliquots of CSF were removed sequentially from the lumbosacral site.⁷

Atlanto-Occipital Technique

For atlanto-occipital collection the horse is placed in lateral recumbency with the neck flexed such that the longitudinal axis of the head and cervical spine are at a right angle.^4,⁸ The nose is elevated to a horizontal plane. The subarachnoid space is located at a depth of 5 to 8 cm in adults and 2 to 4 cm in foals. A 9-cm, 18- to 20-gauge styletted spinal needle is used for adult horses. A 4-cm, 20-gauge disposable needle is appropriate for foals. The needle is inserted through the skin at the intersection of a line drawn between the cranial border of the wings of the atlas and the dorsal midline, which is identified by palpating the external occipital protuberance (Fig. 11-1). The needle is directed perpendicular to the cervical spine toward the rostral end of the mandible. Some resistance is encountered as the needle passes through the funicular part of the ligamentum nuchae. The needle is advanced until the atlanto-occipital membrane and dura mater are penetrated, which is perceived as a “popping” sensation or as a sudden absence of resistance. When either sensation is detected, the stylet is removed to allow CSF to flow. Position of the needle should not be adjusted without the stylet in place. The neck or head should not be moved while the needle is close to the subarachnoid space.

Fig. 11-1 Anatomic landmarks for collection of CSF from atlanto-occipital site. Needle is placed at the intersection of an imaginary line between the cranial border of the wings of the atlas (arrows) and the midline, which is designated by the external occipital protuberance (O).

Lumbosacral Technique

The lumbosacral site can be accessed in most standing adult horses with a 15- to 20-cm, 18-gauge styletted spinal needle after infiltration of the skin and subcutis with local anesthetic. For large horses (12 to 17 hands tall) a 20-cm styletted spinal needle is suggested. A 20- to 23-cm needle may be required for draft horses over 17 hands tall.^4,⁸ Anatomic landmarks are the tuber sacrale of the ilium and the dorsal midline. The needle is inserted through a stab wound located at the intersection of a line drawn between the cranial edges of each tuber sacrale and the midline, which is localized by palpating the spinous processes of the sixth lumbar vertebra and the second sacral vertebra (Fig. 11-2). A depression in the skin is often located in this area. The needle is directed perpendicular to the long axis of the vertebral column and advanced with stylet in place until the interarcuate ligament, dura mater, and arachnoid membrane are penetrated, as identified by a popping sensation or decreased resistance. Average depth of the subarachnoid space is 13 cm in adults. Entry of the needle into the subarachnoid space may be accompanied by excitement, tail movement, flexion of the pelvic limbs, or contraction of the axial muscles.¹

Fig. 11-2 Anatomic landmarks for collection of CSF from lumbosacral site. Needle is inserted perpendicular to the long axis of the spine, at the intersection of the midline and the cranial edges of the tuber sacrale (T) of each ilium. The midline is identified by the spinous processes of the sixth lumbar (L) and second sacral (S) vertebrae.

Laboratory Assessment

As with other body fluids, the laboratory analysis of CSF consists of inspection of gross appearance, cell counts, measurement of protein content, cell identification, and special chemistry assays.⁹ Cells, especially neutrophils, degenerate and lyse rapidly in CSF because of its low protein content and its electrolyte concentrations, which are different from corresponding plasma values. Therefore, procedures for cell counts and preparation of smears on glass slides for cell identification should be done within 30 minutes of collection. Cells can be preserved for preparation of glass slides by mixing 8 to 10 drops (0.25 ml) of fresh CSF with 1 ml of 40% ethanol or by adding autologous serum to the sample. The addition of 1 drop of autologous serum to 0.25 ml of CSF will preserve cell morphology and differential counts for 48 hours when stored at 4° C.¹⁰ Measurement of protein content and special assays can be done on CSF that is refrigerated or frozen without additives.

Macroscopic appearance

Visual inspection of the color and turbidity of CSF is best done by viewing the specimen through natural sunlight or against a white background.⁸ A white index card with lines provides a background for accurate color assessment and fine lines to detect turbidity.

Normal CSF is clear, colorless, and watery and does not form a clot.^3,⁸ Any deviation in color or turbidity is considered abnormal, and the cause should be determined. Causes of CSF turbidity include elevated cell count, presence of microorganisms, or a marked increase in protein content.^1,⁸ Cell counts greater than 400 to 500 μl are necessary before CSF becomes turbid. The presence of myelographic contrast media or fat aspirated during collection may cause turbidity.⁵ Abnormal colors encountered in the macroscopic assessment of CSF are red tinged, bright red, xanthochromic, and opaque white (Fig. 11-3). Cloudy CSF with a white tinge indicates a marked increase in nucleated cells. Red-tinged or bright-red fluid indicates pathologic or iatrogenic hemorrhage.^1,^4,⁸