Edmund S. Cibas

Anatomy and Physiology

The brain contains four ventricles, fluid-filled cavities that communicate with each other and the subarachnoid space surrounding the brain and spinal cord. The ventricles are lined by a cuboidal ciliated cell layer called the ependyma. In some areas the ependyma differentiates into a complex villous structure called the choroid plexus, composed of a single layer of cuboidal nonciliated cells overlying a vascular core. The choroid plexus produces most of the CSF by filtering plasma across capillary walls and actively secreting fluid. After leaving the ventricles via the midline foramen of Magendie and the two lateral foramina of Luschka, CSF circulates in the subarachnoid space, formed by the leptomeninges (composed of the pia mater and arachnoid mater), over cerebral and spinal surfaces. Fluid is then reabsorbed by the arachnoid granulations into the venous system, and the cycle begins anew.8,9

The total volume of CSF in the adult is about 150 mL. Because 500 mL/day is produced (0.35 mL/min), CSF is renewed three or four times daily.

Obtaining and Preparing the Specimen

Most CSF specimens are obtained by LP, in which a needle is passed through the intervertebral space at L3 to L4 or L4 to L5. Rarely, because of inflammation at these sites or a bony abnormality, the specimen must be obtained from the cisterna magna at the base of the brain. Cisternal CSF sometimes yields positive results when LP is negative.¹⁰ CSF is sometimes aspirated directly from a lateral ventricle during a neurosurgical procedure; such specimens often contain microscopic fragments of normal brain. In patients undergoing chemotherapy for leptomeningeal metastasis, a silicone pouch (Ommaya reservoir) is implanted in subcutaneous tissue. A cannula leads from the pouch into a lateral ventricle through a 3-mm burr hole (Fig. 6.1). This is an efficient way to introduce chemotherapeutic drugs and withdraw CSF periodically for examination.

A minimum of 1 mL should be collected for cytologic evaluation, but 3 mL or more is preferable,¹¹ and at least 10 mL is considered ideal.¹² Surprisingly, the amount of CSF removed does not affect the likelihood that a patient will develop a headache.^13,14

Fluid should be collected fresh and delivered to the laboratory as quickly as possible to prevent cellular deterioration. If the specimen cannot be prepared immediately, it should be refrigerated at 4° C. If a delay of more than 48 hours is anticipated, cytomorphology can be preserved by adding an equal volume of 50% ethanol or RPMI.¹⁵ A CSF sample for cytologic examination should never be frozen.

The most common ways to prepare CSF specimens are by cytocentrifugation (Cytospin) and thinlayer preparation. Cytocentrifugation has greater flexibility because both alcohol-fixed and air-dried slides can be prepared using this method. Lymphoid cells are best evaluated using air-dried preparations, thus it is advisable to prepare an air-dried Romanowsky-stained slide in addition to the traditional alcohol-fixed Papanicolaou-stained slide. Depending on the volume and cellularity of the specimen, additional unstained slides can be prepared and set aside for immunocytochemical studies if needed. Unstained air-dried slides can be kept at room temperature for 1 to 2 weeks without compromising cytomorphology or antigenicity.¹⁶

Blood is a common contaminant. The needle lacerates the veins that course along the crowded nerve roots of the cauda equina in 75% of LPs,¹⁷ and this can create diagnostic problems. Neutrophils and eosinophils, if accompanied by red blood cells, should not necessarily be interpreted as evidence of meningitis. Similarly, leukemic blasts from peripheral blood can contaminate the fluid, leading to an erroneous impression of meningeal involvement by leukemia. It takes only a minute amount of blood to alter the cellular composition of CSF significantly, an amount that can be invisible to the naked eye.¹⁸ Because alcohol fixation lyses red blood cells, contamination is best detected on air-dried preparations. If red blood cells are present, the possibility of contamination by peripheral blood blasts should be raised in a patient with leukemia.

Reporting Terminology

As with most cytologic specimens, CSF cytology results are commonly reported as either “negative for malignancy,” “atypical” (connoting a low degree of suspicion for malignancy), “suspicious” (connoting a high degree of suspicion of malignancy), or “positive.” Over 90% of CSF specimens are assigned a cytologic diagnosis of “negative for malignant cells.”¹⁹ Many show only a small number of lymphocytes and monocytes, essentially a normal CSF.

The primary role of CSF cytology is to exclude circulating malignant cells in CSF pathways. Although a specific diagnosis of some benign diseases (e.g., cryptococcosis) can be made cytologically, in most nonmalignant diseases of the central nervous system (CNS), CSF cytology is frustratingly unrevealing. The myriad diseases that cause aseptic meningitis, for example, have as their final common denominator nothing more than a lymphocytic and/or monocytic pleocytosis. The cause is identified by other clinical and laboratory methods.

Accuracy

The sensitivity of CSF cytology for detecting malignant cells is about 60%,20–22 but sensitivity depends on several factors. First and foremost, a positive CSF sample will occur only if a malignancy actually invades into a ventricle or the subarachnoid space (Fig. 6.2).

The sensitivity of a single cytologic examination is 54% but increases to 84% with a second sample.²² Smaller incremental increases in sensitivity are observed with more than two specimens.²³ Sensitivity is dependent of sample volume; sensitivity is greater if 10 mL are submitted rather than 3 mL.¹² Sensitivity also depends on the extent of leptomeningeal disease: 38% for focal and 66% for disseminated leptomeningeal tumor.²⁰ Similarly, only 50% of patients with early meningeal involvement by acute lymphoblastic leukemia (ALL) have a suspicious or positive CSF.²⁴ The site from which the sample is obtained can also affect the sensitivity; if LP specimens are negative, a tap from the cisterna magna sometimes yields malignant cells.¹⁰

The specificity of CSF cytology is high. False-positive diagnoses are estimated at 2% to 3%.²⁵ The most common cause is the overdiagnosis of lymphoma or leukemia, particularly in patients with herpes zoster meningitis,²⁰ cryptococcal meningitis,^26,27 Lyme disease,²⁸ viral meningitis,²⁶ and in LP specimens contaminated with blood.²⁶

In light of these data, the American College of Physicians determined that cytologic examination of CSF for meningeal malignancy has moderate sensitivity and high specificity.²⁹

Normal Elements

Normal CSF is sparsely cellular and, in adults, contains less than five cells/mm³ (equivalent to 5000 cells/mL). In newborns the fluid is more cellular.³⁰

Normal CSF is composed of lymphocytes and monocytes (Fig. 6.3). Only small numbers of mature lymphocytes are present in normal CSF, but their number can increase markedly, particularly in viral meningitis and other inflammatory or infectious conditions. Such specimens can be highly cellular, with a minor population of so-called atypical lymphoid cells that are large and may show irregular nuclear contours, with clefting, blebs, and nucleoli.

Monocytes are also present in normal CSF. Larger than lymphocytes, they have folded, kidney bean–shaped nuclei and a moderate amount of cytoplasm.

Choroid plexus and ependymal cells are seen in less than 0.5% of LP specimens.³¹ These cells have a round to oval nucleus and a moderate amount of cytoplasm; they are isolated or in small clusters (Fig. 6.4). Microscopic brain fragments have a fibrillary texture and contain glial cells, neurons, and capillaries (Fig. 6.5). They are seen in samples taken directly from the ventricles, because the needle traverses brain parenchyma; they are not seen in LP samples. Rarely, isolated neurons are present (Fig. 6.6A and B).

In CSF from neonates, most commonly those born prematurely, one occasionally sees small, immature cells of germinal matrix origin.32,33 Germinal matrix cells lie beneath the ependyma in the wall of the lateral ventricles and exfoliate when there is subependymal and intraventricular hemorrhage (Fig. 6.7). They are often clustered and molded to one another, thus mimicking a small cell malignancy like medulloblastoma. Because of their frequent association with hemorrhage, these cells are often accompanied by hemosiderin-laden macrophages.

If the LP needle is inserted too far anteriorly, CSF can be contaminated by chondrocytes (Fig. 6.8) or bone marrow cells (Fig. 6.9) from the intervertebral disc or vertebral body, respectively. These cells, seen in less than 1% of CSF specimens,^34,35 should not be mistaken for malignant cells.³⁶ Although mitoses are more common in malignant specimens, they are occasionally seen in benign conditions such as bacterial and viral meningitis.³⁷

Abnormal Inflammatory Cells

Macrophages, plasma cells, and eosinophils are abnormal findings in CSF. They may accompany malignancy, but are also seen in a variety of non-neoplastic conditions.

Macrophages have abundant, vacuolated cytoplasm that sometimes contains ingested cells, organisms, or pigment (Fig. 6.10A and B).

Plasma cells are also an abnormal but nonspecific finding in CSF (Fig. 6.11).

Polymorphonuclear leukocytes are a normal finding if there is contamination by peripheral blood, but numerous neutrophils unaccompanied by a proportionate increase in red blood cells raise the possibility of acute meningitis (Fig. 6.12). In a patient with acquired immunodeficiency syndrome (AIDS), numerous neutrophils are highly suggestive of cytomegalovirus (CMV) radiculopathy. Viral cytopathic inclusions, however, are not seen.³⁹

Like polymorphonuclear leukocytes, eosinophils are a normal finding if there is contamination by peripheral blood. If peripheral blood contamination is not apparent, eosinophils, especially in large numbers (Fig. 6.13), suggest a parasitic infection, particularly Taenia solium and Angiostrongylus cantonensis.^40,41

Eosinophilic meningitis is defined as 10 or more eosinophils/μL or10% or more of the total CSF leukocyte count.⁴¹ The most common cause is CNS invasion by helminthic parasites. Worldwide, the most common cause of eosinophilic meningitis is Angiostrongylus cantonensis. Other parasites that cause eosinophilic meningitis include Gnasthostoma spinigerum, Baylisascaris procyonis, and Taenia solium. Meningitis caused by the fungus Coccidioides immitis occurs in up to 50% of patients with disseminated disease, and the meninges can be the only site of infection. Malignancies, especially Hodgkin and non-Hodgkin lymphoma and leukemia, have been associated with eosinophilic meningitis. Medications, either systemic or intrathecal, and ventriculoperitoneal shunts have been implicated in some cases of eosinophilic meningitis.⁴¹

Non-Neoplastic Disorders

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