Abstract
Objective—To describe ultrasonographic landmarks for use in collection of CSF from the lumbosacral region in equids.
Design—Prospective study.
Animals—37 equids (27 with neurologic disease and 10 with nonneurologic disease).
Procedures—Standing equids (n = 17) were sedated with detomidine hydrochloride (0.006 to 0.01 mg/kg [0.003 to 0.005 mg/lb], IV) followed by butorphanol tartrate (0.01 mg/kg, IV) and restrained with a nose twitch for collection of CSF. The CSF was collected from 20 laterally recumbent equids (10 sedated and 10 immediately after euthanasia). Anatomic landmarks were identified ultrasonographically. Height at the dorsal point of the shoulders, body weight, depth of the spinal needle, number of attempts to collect CSF, and cytologic evaluation of CSF were recorded.
Results—Lumbosacral puncture cranial to the cranial border of the most superficial location of both tuber sacrale along the midline was consistently successful for CSF collection (35/37 equids). Two horses had anatomic abnormalities that precluded CSF collection. Mean number of attempts to collect CSF per animal was 1.1. Height and body weight were strongly correlated with needle depth for CSF collection. Pelvic and sacral displacement was observed in several laterally recumbent animals, which resulted in discrepancies of the midline between the cranial and caudal aspects of the vertebral column. In most equids, the spinal needle was aligned on the midline of the caudal aspect of the vertebral column.
Conclusions and Clinical Relevance—Ultrasonography was a useful aid for collection of CSF from the lumbosacral space and decreased the risk of repeated trauma and contamination in equids.
Collection and examination of CSF is a commonly used diagnostic procedure in the evaluation of neurologic disorders in horses. However, contamination of CSF with tissue or blood is a major limiting factor in the interpretation of cytologic examination and biochemical analyses, as well as other tests such as western blotting, for the diagnosis of equine protozoal myeloencephalitis.1–4 Blood contamination of CSF was not evaluated in newer tests for equine protozoal myeloencephalitis, such as the indirect fluorescent antibody test.5 The albumin quotient and IgG index have been used as indicators of increased permeability of the blood-brain barrier and intrathecal IgG production, respectively.3 An increase in the albumin quotient above the reference range suggests blood contamination of CSF during collection but could also indicate compromise of the blood-brain barrier.6 Equations to correct nucleated cell count and total protein concentration during traumatic CSF collection have been used but are inaccurate.7 In 1 study,7 iatrogenic blood contamination decreased in sequential CSF samples with a minimum of 3 samples (2 mL/sample) collected before submission of the final sample. However, sequential aspirations may also induce trauma.
The risk of trauma and contamination with tissue or blood may increase as the result of multiple attempts to obtain CSF from the LS space. Horses with abnormal osseous conformation, heavy musculature, or fat deposition in the LS area may represent anatomic challenges. Furthermore, anatomic positional-dependent changes in the alignment of the vertebral column and pelvis in recumbent horses may increase the number of attempts needed to collect CSF from the LS space.
Collection of CSF from the atlanto-occipital space may yield less contamination of samples, compared with results for LS puncture.8 Ultrasonographic studies9,10 of the atlanto-occipital area in horses have been reported. Ultrasound-guided myelography of the atlanto-occipital area allows reduction of potential complications associated with blind percutaneous puncture.10 However, when there are limiting factors (such as financial constraints, client compliance, risk of anesthesia, and anatomic location of the lesion in a horse), collection from the LS space may be preferred. Although there have been ultrasonographic evaluations11,12 of the pelvis and sacral regions, use of ultrasonography for collection of CSF from the subarachnoid LS space has not been evaluated. Thus, the objective of the study reported here was to describe ultrasonographic anatomic landmarks and assess the value of ultrasonography for use in the successful collection of CSF from the LS space in standing and laterally recumbent horses.
Materials and Methods
Animals—Thirty-seven equine patients (36 horses and 1 mule) from the Veterinary Medical Teaching Hospital at the University of California–Davis were included in the study. Breeds represented included Quarter Horse and associated breeds (n = 15), Thoroughbred (5), Arabian (5), Clydesdale (4), Warmblood (2), mixed breed (2), Standardbred (1), Morgan (1), and American Miniature Horse (1). There were 24 geldings, 2 stallions, and 11 mares. Age ranged from 1 to 30 years (median, 11 years). Twenty-six horses and the mule were examined because of neurologic disease; the remaining 10 horses were euthanized because of advanced nonneurologic disease. Height at the dorsal point of the shoulders and body weight were recorded for all animals.
Ultrasonographic examination—The intersection of lines joining the caudal borders of each tuber coxae on the dorsal midline was clipped (approx 16 cm [6.3 inches] wide and 20 cm [8.0 inches] long). The intersection of the lines was in the center of the clipped area. The skin was cleaned and prepared with alcohol, and ultrasonographic coupling gel was applied.8 Ultrasonographic examinations were performed by 2 investigators (MA and AB). An ultrasound machinea with 7.5MHz microconvex and 3.5-MHz convex linear probes was used. To ensure adequate quality of images for publication, all ultrasonographic images used in this report were obtained from live horses by use of another ultrasound machineb with a 2- to 5-MHz blended curvilinear probe.
Because collection of CSF was assisted by, but not guided with, ultrasonography, the identified site for puncture was marked on each animal with adhesive tape. The clipped area was aseptically prepared prior to LS puncture. Structures located at the LS area were identified and described in the transverse and longitudinal planes. Ultrasonographic examination and LS puncture were not performed simultaneously for safety reasons (to protect the collector, ultrasonographic operator, and ultrasound equipment from unpredictable reactions of an equid when the dura mater was penetrated). For horses that were euthanized, ultrasonographic examination and collection of CSF were performed within 5 minutes after each animal was euthanized. Position of the needle in relation to the tuber sacrale, tuber coxae, and midline was investigated.
A line connecting the palpable most caudal border of both tuber coxae was drawn with adhesive tape on each animal to indicate this landmark. The perpendicular distance between this line and the site of needle placement was recorded. Because the caudal border of the tuber coxae has been used as one of the landmarks for collection of CSF,8 cranial or caudal deviation from the tuber coxae was identified when the perpendicular distance was ≥ 0.5 cm (≥ 0.2 inches).
Midline was defined as the sagittal axis of the spinous processes of the vertebral column. Cranial and caudal midline were defined as axes on the midline cranial and caudal to the LS puncture site, respectively. In animals that stand with a perfectly square stance, the cranial and caudal midline of the vertebral column should be in a straight line. However, in animals that shift their weight or that are in lateral recumbency, discrepancies between the cranial and caudal midline may be observed. Thus, we assessed whether needle placement coincided with the cranial or caudal midline in equids in which discrepancies were detected. For statistical purposes, needle placement on the cranial and caudal midline was assigned a score of 0 (no discrepancy), whereas any discrepancy between the aforementioned features was assigned as score of 1.
Collection of CSF from the LS space—Collection of CSF from the LS space was performed in 17 standing and 10 laterally recumbent equine patients with neurologic disease, whereas CSF was collected immediately after 10 equids with nonneurologic disease were euthanized. Animals were placed in stocks and sedated by administration of detomidine hydrochloride (0.006 to 0.01 mg/kg [0.003 to 0.005 mg/lb], IV) followed by butorphanol tartrate (0.01 mg/kg, IV). In addition, a nose twitch was applied to all standing horses immediately before collection of CSF. In live laterally recumbent horses, collection of CSF was performed with the animal in its stall. Animals were sedated by use of the same agents as for the standing position. The puncture site was locally anesthetized by injection of 1 mL of lidocaine hydrochloride. The primary clinician for the animal and 2 of the investigators (MA and AB) collected CSF. An 18-gauge, 15.2-cm (6-inch) spinal needlec was used for all equids, except for Warmblood and draft horses, for which an 18-gauge, 20-cm (8-inch) spinal needled was used. The number of attempts to collect CSF and needle depth were recorded.
CSF analysis—After collection, CSF samples were immediately submitted for cytologic assessment. Variables such as total number of RBCs and WBCs, total protein concentration, and cytologic interpretation were recorded. A grading system was used for cytologic examination of CSF (grade 1, normal; grade 2, abnormal). Because of the objectives of the study, other diagnostic tests performed on the CSF samples were not included.
Statistical analysis—Number of attempts to collect CSF, needle position, needle depth, and results of cytologic evaluation of CSF were compared among the 10 euthanized animals with no obvious neurologic disorders and 25 animals examined because of neurologic disease. These same variables were compared among 17 equids in the standing position and 18 laterally recumbent equids, regardless of neurologic status. Results were reported as mean, SD, and range values. Bivariate relationships between categoric variables were assessed by use of the Fisher exact test. Variables with continuous outcomes were compared by use of an exact MannWhitney test or Student 2-group t test. The number of animals in which needle placement deviated from the midline was evaluated by use of a binomial test with the null hypothesis that the distribution would be equal. Linear relationships among continuous variables were evaluated by use of least squares regression and the Pearson correlation coefficient. Values were considered to differ significantly at P ≤ 0.05.
Results
Animals—We were unable to collect CSF from 2 live equids. Thus, results reported represent values for only 35 animals. One of these animals had a complex displaced pelvic fracture, and the other had severe general abnormal conformation that precluded clear observation of the LS area (eg, several bone interfaces).
Ultrasonographic examination—The ultrasonographic examination began in a transverse plane on the midline, and the most superficial (dorsal) location of both tuber sacrale was identified (Figure 1). Ultrasonographic images of the reference point in the transverse and longitudinal views were identified (Figure 2). The examination included areas along the midline cranial and caudal to the most superficial location of the tuber sacrale reference point in the transverse and longitudinal planes. The ultrasound probe was advanced approximately 0.5 cm (0.2 inches) cranially (range, < 0.5 to 1.2 cm [< 0.2 to 0.5 inches], depending on conformation and size of each animal) to the reference point, and the LS area (puncture site) was examined in the transverse and longitudinal planes (Figures 3 and 4). At this location in a transverse plane, the tuber sacrale were still visible in addition to the intertransverse facets of S1. In a longitudinal plane, the LS area was easily identified as a U-shaped structure formed by the caudal border of L6, the LS space, and the cranial border of S1. When the ultrasound probe was advanced cranially to the LS area, the spinous process of L6 was the only bony structure visible (Figure 5). The distance between the LS space and spinous process of L6 was highly variable depending on the animal and was not useful for LS puncture; therefore, that value was not included. When an image includes the spinous process of L6, it should alert a clinician that the LS space is farther caudal. Conversely, when a clinician scans in a transverse plane caudally from the reference point (most superficial [dorsal] location of the tuber sacrale) and can identify both tuber sacrale and the spinous process of S1, it should alert that clinician to the fact that the LS space is farther cranial (Figure 6).
Longitudinal photographic view of a dissected specimen of the LS area of a horse. Notice the reference point (asterisk) for LS puncture to collect CSF. The reference point is the most superficial (dorsal) location of the tuber sacrale. The cranial (CR) and caudal (CD) directions of the vertebral column are indicated. VC = Vertebral canal (in this specimen, the spinal cord was removed).
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Transverse (A) and longitudinal (B) ultrasonographic images of the LS area of a horse. Notice that in the transverse view, the tuber sacrale (TS) are evident as convex, thin, hyperechoic lines on the left (L) and right (R) sides of the midline. Marks on the right side of each image are at intervals of 1 cm (0.39 inches). DSIL = Dorsal sacroiliac ligament. TLF = Thoracolumbar fascia. See Figure 1 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Caudal transverse photographic view of an anatomic dissection of the LS area of a horse with the skin, subcutaneous muscle, and fat removed. An 18-gauge, 15.2-cm (6-inch) spinal needle was placed as a reference. 1 =TLF. 2 = Supraspinosus ligament.3=Longissimus lumborum muscle. 4 = Multifidus lumborum muscle. 5 = Interspinosus ligament. 6 = Interarcuate ligament. 7 = Sacral articular process. 8 = LS intertransverse joint. 9 = VC. Bar = 1 cm. See Figures 1 and 2 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Transverse (A) and longitudinal (B) ultrasonographic images of the LS area (puncture site) approximately 0.5 cm (0.2 inches) cranial to the reference point. See Figures 1 and 2 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Transverse ultrasongraphic image obtained 3 cm (1.18 inches) cranial to the reference point in the LS area. SP6 = Spinous process of L6. LL = Longissimus lumborum muscle. See Figure 2 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Transverse ultrasonographic image obtained 0.5 cm caudal to the reference point in the LS area. Double arrows = Spinous process of S1. See Figures 1 and 2 for remainder of key.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Deviation from the tuber coxae was recorded in 19 of 35 equids from which CSF was collected (caudal deviation in 18 animals and cranial deviation in 1 animal). Deviation ranged from 0.5 to 2.1 cm (0.2 to 0.8 inches), with a mean ± SD of 0.83 ± 0.45 cm (0.33 ± 0.18 inches). In the other 16 horses, the puncture site was at the connecting line of the caudal border of the tuber coxae. There was no significant difference between equids in the standing and laterally recumbent positions.
Cranial and caudal aspects of the vertebral column were assessed (Figure 7). Eleven of 17 animals stood with a perfectly square stance. In those 11 animals, there were no midline discrepancies between the cranial and caudal midline of the vertebral column. Midline discrepancies were recorded in 23 of 35 animals (6 in a standing position and 17 in a laterally recumbent position). Animals in lateral recumbency had significantly (P < 0.001) more midline discrepancies between the cranial and caudal midline of the vertebral column, compared with results for the animals in the standing position. Needle placement was on the caudal midline of the vertebral column in 20 of 23 equids (5/6 in the standing position and 15/17 in the laterally recumbent position). In 2 horses, the needle was on the cranial midline (1 each for the standing and laterally recumbent positions), and in 1 horse, the needle was slightly off midline (cranial and caudal). The number of animals with needle placement on the caudal midline was significantly (P < 0.001) higher than those with needle placement on the cranial midline. The discrepancy distance between cranial and caudal midlines ranged from 0.4 to 3 cm (0.16 to 1.2 inches; mean ± SD, 1.61 ± 0.8 cm [0.64 ± 0.3 inches]).
Diagram of a standing (top) and laterally recumbent (bottom) horse (A) and photograph of a horse on which tape was applied (B) to mark the site for LS puncture (asterisk) to collect CSF. In most standing horses, the LS space is located cranial to the tuber sacrale at the point where both solid lines cross. In panel A, the cranial border of the most superficial (dorsal) aspect of each tuber sacrale is indicated (black circles), whereas in a laterally recumbent horse, the midline is shifted (dashed line), as are the tuber sacrale (dashed circles). In panel B, notice the discrepancy of the midline between the cranial and caudal aspects of the vertebral column. The perpendicular piece of tape depicts the caudal border of the tuber coxae (TC). CrM = Cranial midline. CdM = Caudal midline.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
Collection of CSF from the LS space—Cerebrospinal fluid was collected in 35 horses at a midline location cranial (mean distance, 0.5 cm) to the most superficial (dorsal) point of the tuber sacrale. The collection was achieved on the first attempt in 33 horses. In the other 2 horses, adjustments of needle placement were necessary to obtain CSF. The mean number of attempts to collect CSF was 1.1/animal. Height (r = 0.81) and logarithm of the body weight (r = 0.91) were significantly (P < 0.001) correlated with needle depth (Figure 8).
Regression analysis between needle depth for collection of CSF and height at the dorsal point of the shoulders (A) and between needle depth for collection of CSF and body weight (B). Each symbol represents results for 1 animal. Needle depth was significantly (P < 0.001) correlated with height (r = 0.81) and logarithm of the body weight (r = 0.91). To convert centimeters to inches, divide value by 2.54. To convert meters to inches, multiply value by 39.37. To convert kilograms to pounds, multiply value by 2.2.
Citation: Journal of the American Veterinary Medical Association 230, 3; 10.2460/javma.230.3.378
CSF analysis—The CSF was grossly normal for 33 animals, xanthochromic for 1 animal, and hemorrhagic for 1 animal. Investigators in another study3 reported that as few as 8 RBCs/μL were sufficient to contaminate CSF and interfere with the interpretation of results of western blot analysis for the detection of antibodies against Sarcocystis neurona. In the study reported here, 10 of 25 equine patients with neurologic disease had > 8 RBCs/μL (range, 17 to 1,000 RBCs/μL), whereas all equine patients in the nonneurologic group had 0 to 2 RBCs/μL. The RBC counts were significantly (P = 0.018) higher for the neurologic group, compared with counts for the nonneurologic group. Other variables such as number of WBCs, total protein concentration, and cytologic interpretation (normal vs abnormal) were not significantly different between standing and recumbent and between neurologic and nonneurologic animals.
Discussion
The study reported here provided ultrasonographic landmarks to assist with collection of CSF from the LS space by use of a simple and nontraumatic approach in standing and recumbent equine patients. It was our intention to provide clinicians with an alternative method for collection of CSF. The study revealed that locating the most superficial (dorsal) point of both tuber sacrale (reference point) in the transverse plane on the midline was easily achieved. However, the cranial border of the spinous process of S1 prevented advancement of the needle for collection of CSF at this reference point. The LS area for collection of CSF was < 0.5 to 1.2 cm (mean, 0.5 cm) cranial to the reference point on the midline. At this location, both tuber sacrale were still visible in a transverse plane in addition to the intertransverse facets of S1. In a longitudinal plane, the LS space was easily identified. Identifying exclusively the spinous process of L6 in either plane indicated that the scan plane was too far cranial for puncture of the LS space.
Position of an equine patient is an important factor to consider when defining midline as an anatomic landmark for collection of CSF. Midline should not be determined by visual assessment of the skin surface; instead, it should be determined by assessment of the vertebral column. Discrepancy on the midline between the cranial and caudal aspects of the vertebral column may be observed, especially in recumbent animals. In the equids reported here, pelvic and sacral displacement as the result of gravitational forces and body weight may have caused a difference in midline discrepancy of as much as 3 cm. Midline of the caudal aspect of the vertebral column may be closer to or farther ventral than midline of the skin; in most horses in this study, midline of the vertebral column was more ventral. In general, when displacements were observed in standing or laterally recumbent animals, placement of the spinal needle typically coincided with the caudal midline of the vertebral column. Displacement of palpable bony structures in laterally recumbent animals was also reported in another study,8 but specific details are lacking. To our knowledge, discrepancies between the cranial and caudal midline of the vertebral column have not been reported.
The anatomic approach for collection of CSF from the LS space described by other investigators is commonly used in clinical settings and is dependent on identification of external landmarks.8 However, in horses for which the landmarks are not easily identified, the use of ultrasonography would be beneficial, independent of the breed, height, body weight, and body position of the horse, as was reported here. Height and weight must be considered when choosing the length of the spinal needle to be used because an inappropriate length will contribute to collection failure. Draft horses and certain Warmblood breeds will require a longer needle than standard-sized breeds. It is estimated that traumatic lumbar punctures occur in approximately 20% of human patients.13 Local hemorrhage resulting from injury of venous plexuses and spinal and perivertebral veins, pain in the LS region, disk herniation, trauma to ligaments and periostium, damage to the spinal cord, or entrapment of nerve roots in the spinal needle are some of the complications that result from traumatic puncture in humans.14 The prevalence of traumatic LS punctures in horses is unknown. The number of attempts required to collect CSF from each of the animals of our report was 1.1. Requiring fewer attempts may result in a lower possibility of traumatic puncture.
In addition, requiring fewer LS punctures will also decrease the likelihood of contamination with tissue or blood. In our nonneurologic group, the number of RBCs in CSF ranged from 0 to 2 RBCs/μL. In the neurologic group, the number of RBCs was significantly higher than for the nonneurologic group. This could have resulted from the neurologic condition rather than traumatic puncture because only 1 clean attempt was made to obtain CSF. In addition, 3 of the neurologic patients had previous CSF collections within a few days preceding the study. On the other hand, CSF was collected within 5 minutes after nonneurologic patients were euthanized. In our experience, horses may continue to hemorrhage when puncture occurs shortly after euthanasia. However, this confounding feature was considered. Analyzing the number of RBCs in live, healthy, standing and laterally recumbent animals would have been ideal.
Collection of CSF is routinely performed without the use of ultrasonography. The study reported here does not refute what has been done routinely. Rather, it simply provides an alternative method for obtaining CSF with fewer attempts, which minimizes the potential for contamination that may result in invalidation of results or make it difficult to interpret diagnostic tests. Sophisticated ultrasound equipment is not required to view the anatomic landmarks mentioned in our study. Ultrasound-assisted collection of CSF can be especially useful in horses with excessive fat deposition and heavy musculature on which anatomic landmarks are not readily palpable or in horses with anatomic abnormalities (conformation or trauma). In addition, our study revealed that body position affects definition of the midline. More importantly, we provided additional information to enable better success for collection of CSF, even without the use of ultrasonography.
The information reported here should prove useful for successfully obtaining CSF from the LS space during the first attempt in standing and laterally recumbent equine patients with less risk of trauma and contamination. It is important to emphasize that in most equids, the defined anatomic landmarks for collection of CSF were appropriate, but differences among animals may exist. Length of the spinal needle must be selected on the basis of the height and body weight of the patient. Pelvic and sacral displacement must be considered when defining midline, especially in laterally recumbent animals. When discrepancies of the midline are apparent and ultrasound equipment is not available, puncture to obtain CSF should be performed at a site approximately 0.5 cm cranial to the palpable most dorsal area of both tuber sacrale on the midline of the caudal aspect of the vertebral column.
ABBREVIATIONS
| LS | Lumbosacral |
MegaES FD570A, Biosound ESAOTE Inc, Indianapolis, Ind.
ATL5000, Philips, Andover, Mass.
Spinal needle, Monoject Tyco Healthcare Group, Mansfield, Mass.
Spinal needle, Mila International Inc, Florence, Ky.
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