A Multidisciplinary Approach to CSF Analysis
By Marian J. Cavagnaro, MS, MT(ASCP)DLM
A 54-year-old female was admitted with a suspected diagnosis of central nervous system (CNS) hemorrhage. A Wright's stained cytocentrifuge smear on cerebrospinal fluid (CSF) demonstrated the presence of siderophages (hemosiderin-laden macro-phages) and hematin crystals.
A newborn female presented to the emergency room (ER) with a suspected diagnosis of meningitis. Again, a spinal tap was done and a Wright's stained cytocentrifuge smear demonstrated the presence of choroid plexus cells. These cells are commonly seen in newborns and should not be mistaken for malignant cells. They may occur singularly but more often in clusters or sheets.
A 29-year-old male arrived at the ER with a cough and headache. He informed the clinician that he was infected with the human immunodeficiency virus (HIV). A Wright's stained cytocentrifuge smear on CSF demonstrated the appearance of Cryptococcus neoformans, a frequent cause of fungal meningitis. The organism resembles mononuclear cells.
A 12-year-old male presented to the ER with a two-week history of headaches, weakness and dizziness. The child had been previously treated for lymphoblastic leukemia and suffered from a CNS relapse. A Wright's stained cytocentrifuge smear demonstrated the presence of blasts with characteristic immature chromatin and small nucleoli.
A clinical history and physical examination was obtained on the four ER patients. The CSF specimens were collected by lumbar puncture between the third and fourth or fourth and fifth lumbar vertebrae. The specimens were analyzed using a multidisciplinary approach that cuts across both laboratory medicine and pathology.
Importance of CSF Analysis
Tests performed on CSF are critical to patient care, yet many technologists involved in body fluid analysis are not comfortable with the procedures and techniques used. This article confirms the importance of correlating the results of hematologic and cytologic findings with chemical, microbiologic and serologic/immunologic findings that assist clinicians with their diagnosis.
CSF diagnoses encompass a wide array of conditions. Examination contributes to diagnosis and sometimes management of various disease entities of the CNS, including meningitis, encephalitis, neurosyphilis, brain abscess, subarachnoid and intracerebral hemorrhages, leukemias and lymphomas, primary or metastatic brain or spinal cord neoplasms, degenerative cord or brain diseases, autoimmune diseases involving CNS and demyelinating diseases (e.g., multiple sclerosis, acute demyelinating polyneuropathy).1
Anatomy and Pathophysiology
CSF is normally a clear, colorless fluid. It provides a protective cushion that protects the brain and CNS from sudden changes in pressure and pH, maintains stable chemical environment, supplies nutrients and removes metabolic waste products. CSF is formed by ultrafiltration of plasma through the choroid plexus. Endothelium of the choroid plexus vessels and ependymal cells lining the ventricles act as a barrier to the passage of metabolites, protein and drugs. CSF circulates in the subarachnoid space and ventricles of the brain.2 The total volume of CSF present is 90 mL-150 mL in adults and 60 mL in neonates.3
Laboratory Test Findings
Specimen Collection--The protocol for proper sample collection requires that an aliquot of CSF be placed in, sequentially numbered and properly labeled, sterile tubes, as the fluid is collected. In a three-tube collection, the first aliquot is sent to the chemistry department, since it may be contaminated with blood or skin flora; the second is sent to microbiology and immunology and the third aliquot is sent to hematology and cytology. Without immediate processing, degeneration of cells within the spinal fluid begins occuring within 20-30 minutes at room temperature.
Gross Appearance--The gross appearance of CSF may indicate the difference between pathology and traumatic tap. Turbidity is first assessed on an uncentrifuged tube and is graded clear to opaque. Causes of a cloudy CSF are increased protein, microorganisms, more than 200/mL white blood cells (WBCs), more than 400/mL red blood cells (RBCs), contrast media and contamination with epidural fat.3 Blood specimens may be indicative of a traumatic tap or malignant condition.
Color is assessed after the tube is centrifuged--Colorless is normal and any amount of discoloration, pathologic. Xan-thochromia is any pink, yellow or orange color. It may be caused by a subarachnoid hemorrhage, free hemoglobin, carotene, melanin, increased CSF protein (greater than 150 mg/dL) or bilirubin.3 The bilirubin may be caused by a CNS hemorrhage, a prior traumatic tap or hepatic disease.
In a bloody tap, lysis of red cells begins within four hours, so it is important to process the specimen quickly to prevent a false positive detection of xanthochromia. Bilirubin formed after a CNS bleed produces a yellow color that disappears in two and four weeks. Methemoglobin gives a brown color and is formed in subdural or intracerebral hematomas.4
A combination of methods and observations usually differentiate a traumatic tap from a subarachnoid hemorrhage. Indica-tions of a traumatic tap include clearing of the fluid as it is aspirated, a colorless supernatant after centrifugation and a presence of a clot in the sample. Xanthochromia, erythrophagocytosis and a positive cross-linked fibrin-derivative D-dimer, measured by a latex agglutination immunoassay, are clear evidence of a subarachnoid bleed if the fluid is tested in a timely manner.3 Within eight hours after hemorrhage, macrophages enter the CSF and remove RBCs by phagocytosis (erythro-phagocytes). After several days these cells are digested, forming hemosiderin (siderophages), which appear as black granules, and hematin, which appears as flat yellow rhomboid crystals.
Hematologic and Cytologic Analysis
Hematologic examination includes the cell count and differential. The cell count routinely performed on CSF is the WBC count. The presence of RBCs can usually be ascertained from the appearance of the specimen. RBC counts have limited diagnostic value, they are usually performed when a traumatic tap is expected. The normal WBC count for adults is 0-5/mL, children 5 years to puberty, 0-10/mL; children 1-4 years, 0-20/mL and children less than 1 year, 0-30/mL.3
A hemacytometer is used for cell counting. Electronic cell counters are not recommended because they lack precision and validity and have variation in background counts resulting in falsely elevated counts.5 A cell count should not be performed on a clotted specimen.
Microscopic examination of the CSF involves hematology with air-dried Wright-Giemsa-stained (or any other Romanowsky type stain) slides and cytology with alcohol-fixed PAP-stained slides.4 A differential cell count (cytocentrifuge method) should be performed on every specimen, regardless of the cellularity, since "acellular" and "hypocellular" specimens contain cells that may be malignant in some cases.
CSF taken from an adult normally contain only lymphocytes and monocytes in a ratio of 70:30. In neonates, monocytes predominate.3 Macrophages may be seen in normal CSF with large numbers often visible after a subarachnoid hemorrhage.
Cells lining the ventricles, choroid plexus or ependymal cells may occasionally be seen in both normal and abnormal CSF. They are not considered clinically significant, but should be differentiated from malignant cells. In the CSF of adults, the polymorphonuclear neutrophils (PMNs) should be less than 10 percent and less than 5/mL.
A high WBC count in the CSF is pleocytosis. Neutrophils predominate in bacterial meningitis causing a neutrophile pleocytosis, while lymphocytes predominate in viral meningitis causing a lymphocytic pleocytosis. Fungal meningitis produces a pleocytosis of mixed cellularity (neutrophils and lymphocytes). Hemorrhage, traumatic tap and malignancy may cause an increase in neutrophils. An eosinophilic pleocytosis often occurs in parasitic and fungal infections. Malignant cells--from tumors, leukemias and lymphomas--may also be found in the CSF. Contaminants may originate from the path of the needle used to aspirate the CSF or ventricular fluid, including bone marrow cells, cartilage cells, capillaries and fibrose or adipose tissue.
CSF glucose is derived solely from the serum. Normal adult values range from 50-75 mg/dl (60 percent to 70 percent of blood glucose level).6 Newborns have slightly higher levels, due to the immaturity of glucose exchange mechanisms. CSF protein is a non-specific indicator of pathology. Normal adult values range from 15-45 mg/dL--up to 70 mg/dL in children and elderly adults.6 Newborns have increased protein up to two-fold due to immaturity of blood brain barrier.
Subarachnoid hemorrhage, malignancy and multiple sclerosis are conditions associated with low CSF glucose and high CSF protein-bacterial meningitis. Increased CSF glucose is of no diagnostic importance--it simply indicates previous hyperglycemia. Low CSF protein can occur in four situations: with the removal of large amounts of CSF, in some leukemic patients, when there is a CSF leak and in some patients with benign intracranial hypertension.
Additional tests may be ordered to assist in the differential diagnosis, including creatinine kinase (CK), elevated in cortical strokes; C-reactive protein (CRP), elevated in inflammation and early bacterial meningitis; lactate dehydrogenase (LD), significantly elevated in bacterial meningitis; lactate, increased in septic meningitis; and glutamine levels, elevated in hepatic encephalopathy and coma.3
Microbiologic, Serologic and Immunologic Analysis
The gram stain is the primary diagnostic tool and may give the clinician preliminary information about the causative infectious agent.6 The major test to be performed on the CSF for meningitis is the bacteriologic culture. Blood cultures are often positive with meningitis.5 Acid fast stains are indicated if monocytosis is present. The gram stain and culture would be performed on the sediment of the second or third aliquot of CSF after it is centrifuged. Fungal cultures require a minimum of 5 mL of fluid. Blood and chocolate agar and an anaerobic should be used and, if sterile, held for a minimum of three days.
The most common pathogens of bacterial meningitis isolated include: Escherichia coli and group B Streptococcus (Streptococcus agalactiae) in neonates, Hemophilus influenzae in children between the ages of 2 months and 6 years, Neisseria meningitides in school children and Streptococcus pnuemoniae in the elderly.3 Trauma, ICU and neurosurgery patients may be infected with Staphylococcus aureus and Staphylococcus epidermidis. Cryptococcus neoformans, the most common fungus isolated from the CSF, may also produce meningitis and is found in patients in which multiple opportunistic infections occur or who have AIDS.
Latex particle agglutination tests are available for detecting bacterial antigens; however, these should not be used as a substitute for culture and Gram stain. The latex particle test is more useful as an adjunct in selected cases of partially treated meningitis and differentiating from suspected cases of early viral meningitis when antibiotic therapy is not going to be initiated. Cryptococcal antigen titers of serum and CSF provide rapid diagnosis and have a greater sensitivity than an India Ink preparation.7
Additional tests that may be ordered to assist in the differential diagnosis include CSF VDRL (positive for neurosyphilis) and a combination of the CSF gamma globulin (IgG) index and oligoclonal bands for confirming the presence of multiple sclerosis. Tumor markers may be of value in detecting disease, suggesting a primary source for metastatic disease, monitoring response to therapy or in predicting relapse.3 *
Marian J. Cavagnaro is director, Laboratory Services, Memorial Hospital West, Pembroke Pines, FL.
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2. Walters J. Laboratory technology: Examining three common body fluids. ADVANCE for Medical Laboratory Professionals. Sept., 1996:73.
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7. Werner V and Kruger RL, MD. Value of the bacterial antigen test in the absence of CSF fluid leukocytosis. Laboratory Medicine, 22 (11): 787-789.