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2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 480 29 CEREBROSPINAL, SYNOVIAL, SEROUS BODY FLUIDS, AND ALTERNATIVE SPECIMENS Donald S. Karcher, Richard A. McPherson CHAPTER CEREBROSPINAL FLUID, 480 Specimen Collection and Opening Pressure, 481 Indications and Recommended Tests, 481 Gross Examination, 481 Xanthochromia, 482 Differential Diagnosis of Bloody CSF, 482 Microscopic Examination, 482 Total Cell Count, 482 Differential Cell Count, 483 Chemical Analysis, 485 Proteins, 485 Glucose, 489 Lactate, 489 F2-Isoprostanes, 489 Enzymes, 489 Ammonia, Amines, and Amino Acids, 490 Electrolytes and Acid-Base Balance, 490 Tumor Markers, 490 Microbiological Examination, 490 Bacterial Meningitis, 490 Spirochetal Meningitis, 491 Viral Meningitis, 491 Human Immunodeficiency Virus, 491 Fungal Meningitis, 491 Tuberculous Meningitis, 491 Primary Amebic Meningoencephalitis, 492 SYNOVIAL FLUID, 492 Specimen Collection, 492 Recommended Tests, 492 Gross Examination, 493 Microscopic Examination, 493 Total Cell Count, 493 Differential Leukocyte Count, 493 Crystal Examination, 494 Chemical Analysis, 495 Mucin Clot Test, 495 Glucose, 495 Protein, 495 Enzymes, 495 Organic Acids, 495 Uric Acid, 496 Lipids, 496 Immunologic Studies, 496 Microbiological Examination, 496 PLEURAL FLUID, 496 Specimen Collection, 496 Transudates and Exudates, 496 Recommended Tests, 496 Gross Examination, 497 Microscopic Examination, 498 Cell Counts, 498 Differential Leukocyte Count and Cytology, 498 Chemical Analysis, 499 Protein, 499 Glucose, 499 Lactate, 499 Enzymes, 499 Interferon-γ, 499 pH, 499 Lipids, 499 C-Reactive Protein, 500 Tuberculostearic Acid (10-Methyloctadecanoic Acid), 500 Tumor Markers, 500 Immunologic Studies, 500 Microbiological Examination, 500 PERICARDIAL FLUID, 500 Specimen Collection, 501 Gross Examination, 501 Exudates and Transudates, 501 Microscopic Examination, 501 Chemical Analysis, 501 Protein, 501 Glucose, 501 pH, 501 Lipids, 501 Enzymes, 501 Interferon-γ, 501 Polymerase Chain Reaction, 501 Immunologic Studies, 501 Microbiological Examination, 502 PERITONEAL FLUID, 502 Transudates and Exudates, 502 Specimen Collection, 502 Paracentesis, 502 Diagnostic Peritoneal Lavage, 502 Peritoneal Dialysis, 502 Peritoneal Washings, 503 Recommended Tests, 503 Gross Examination, 503 Microscopic Examination, 503 Chemical Analysis, 503 Protein, 503 Glucose, 504 Enzymes, 504 Fibronectin, 504 Lactate, 504 Creatinine and Urea, 504 Bilirubin, 504 pH, 504 Cholesterol, 504 Interleukin-8, 504 Tuberculostearic Acid (10-Methyloctadecanoic Acid), 504 Tumor Markers, 505 Microbiological Examination, 505 ALTERNATIVE SPECIMENS, 505 Saliva, 505 Hair and Nails, 505 Tissue Aspirates, 505 Billing for Tests in Nonstandard Specimens, 505 CHEMICAL MEASUREMENTS IN BODY FLUIDS, 506 SELECTED REFERENCES, 506 KEY POINTS • Determining the etiologic cause of fluid accumulation in various body cavities (i.e., joints, chest, abdomen) is critical for proper treatment of these disorders. • Appropriate laboratory examination of these fluids is therefore critical for the diagnosis of numerous diseases (i.e., bacterial, viral and fungal infections; distinction between various arthritides; primary [i.e., mesothelioma] and metastatic malignancies; among others). • Accurate test interpretation depends on appropriate specimen collection, turnaround time, physician/laboratory communication, and reliable reference values. Cerebrospinal Fluid In adults, approximately 500 mL of cerebrospinal fluid (CSF) is produced each day (0.3–0.4 mL/min). The total adult volume varies from PA R T 3 481 parameters vary at different sites. The necessity for a simultaneous serum glucose should also be considered. This is best obtained 2–4 hours before lumbar puncture because of the delay in serum–CSF equilibrium. The CSF specimen is usually divided into three serially collected sterile tubes: Tube 1 for chemistry and immunology studies; tube 2 for micro biological examination; and tube 3 for cell count and differential. An additional tube may be inserted in the No. 3 position for cytology if a malignancy is suspected. However, under certain conditions, some variations are critical. For example, if tube 1 is hemorrhagic because of a traumatic puncture, it should not be used when protein studies are the most important aspect of the analysis (i.e., suspected multiple sclerosis). Indeed, tube 3 should be examined for the major purpose of CSF collec tion. Perhaps the only definite statement one can make is that tube 1 should never be used for microbiology because it may be contaminated with skin bacteria. If questions arise, communication between the labora tory and the clinician before CSF analysis is critical. Glass tubes should be avoided because cell adhesion to glass affects the cell count and differential. Specimens should be delivered to the laboratory and processed quickly to minimize cellular degradation, which begins within 1 hour of collection. Refrigeration is contraindicated for culture specimens because fastidious organisms (e.g., Haemophilus influenza, Neis- seria meningitidis) will not survive. INDICATIONS AND RECOMMENDED TESTS Indications for lumbar puncture can be divided into four major disease categories: meningeal infection, subarachnoid hemorrhage, primary or metastatic malignancy, and demyelinating diseases (American College of Physicians, 1986). Identification of infectious meningitis, particularly bac terial, is the most important indication for CSF examination (Table 291). Recommended laboratory tests are directed toward identification of these disorders (Table 292). CSF examination for other diseases is generally less helpful but often provides supportive evidence of a clinical diagnosis or helps to rule out other diseases (Irani, 2009). Limited routine studies fol lowed by reflexive ordering of more focused tests (as needed) on the stored specimen have been advocated as a way of improving test efficiency (Albright, 1988). GROSS EXAMINATION Normal CSF is crystal clear and colorless and has a viscosity similar to that of water. Abnormal CSF may appear cloudy, frankly purulent, or pigment tinged. Turbidity or cloudiness begins to appear with leukocyte (white blood cell [WBC]) counts over 200 cells/µL or red blood cell (RBC) counts of 400/µL. However, grossly bloody fluids have RBC counts greater than 6000/µL. Microorganisms (bacteria, fungi, amebas), radiographic contrast material, aspirated epidural fat, and a protein level greater than 150 mg/ dL (1.5 g/L) may also produce varying degrees of cloudiness. Experienced observers may be able to detect cell counts of less than 50 cells/µL with the unaided eye by observing for Tyndall’s effect (Simon, 1978). Here, 90–150 mL, about 25 mL of which is in the ventricles and the remainder in the subarachnoid space. In neonates, the volume varies from 10–60 mL. Thus, the total CSF volume is replaced every 5–7 hours (Wood, 1980). An estimated 70% of CSF is derived by ultrafiltration and secretion through the choroid plexuses. The ventricular ependymal lining and the cerebral subarachnoid space account for the remainder. CSF leaves the ventricular system through the medial and lateral foramina, flowing over the brain and spinal cord surfaces within the subarachnoid space. CSF resorption occurs at the arachnoid villi, predominantly along the superior sagittal sinus. The CSF has several major functions: (1) It provides physical support because the 1500g brain weighs about 50 g when suspended in CSF; (2)it confers a protective effect against sudden changes in acute venous (respi ratory and postural) and arterial blood pressure or impact pressure; (3) it provides an excretory waste function because the brain has no lymphatic system; (4) it is the pathway whereby hypothalamus releasing factors are transported to the cells of the median eminence; and (5) it maintains central nervous system ionic homeostasis. The concept of the bloodbrain barrier (BBB) is derived from dye exclusion (tryphan blue) studies. It consists of two morphologically distinct components: A unique capillary endothelium held together by intercellular tight junctions; and the choroid plexus, where a single layer of specialized choroidal ependymal cells connected by tight junctions overlies fenestrated capillaries. The CSF ionic components (e.g., H+, K+, Ca++, Mg++, bicarbon ate) are tightly regulated by specific transport systems, whereas glucose, urea, and creatinine diffuse freely but require 2 or more hours to equili brate. Proteins cross by passive diffusion at a rate dependent on the plasmatoCSF concentration gradient and inversely proportional to their molecular weight and hydrodynamic volume (Fishman, 1992). Thus, the BBB maintains the relative homeostasis of the central nervous system environment during acute perturbations of plasma components. SPECIMEN COLLECTION AND OPENING PRESSURE Cerebrospinal fluid may be obtained by lumbar, cisternal, or lateral cervical puncture or through ventricular cannulas or shunts. Details of the perfor mance of lumbar puncture are described elsewhere (Herndon, 1989; Ward, 1992). Respiratory compromise may occur in infants if the head is flexed (Ward, 1992). A manometer should be attached before fluid removal to record the opening pressure. CSF pressure varies with postural changes, blood pres sure, venous return, Valsalva maneuvers, and factors that alter cerebral blood flow. The normal opening adult pressure is 90–180 mm of water in the lateral decubitus position with the legs and neck in a neutral position. It may be slightly higher if the patient is sitting up and varies up to 10 mm with respiration. However, the pressure may be as high as 250 mm of water in obese patients. In infants and young children, the normal range is 10– 100 mm of water, with the adult range attained by age 6–8 years (Fishman, 1992). Opening pressures above 250 mm H2O are diagnostic of intracra nial hypertension, which may be due to meningitis, intracranial hemor rhage, and tumors (Seehusen, 2003). If the opening pressure is greater than 200 mm H2O in a relaxed patient, no more than 2.0 mL should be withdrawn. Idiopathic intracranial hypertension is most commonly seen in obese women during their childbearing years. When an elevated opening pres sure is noted, CSF must be removed slowly and the pressure carefully monitored. Additional CSF should not be removed if the pressure reaches 50% of the opening pressure (Conly, 1983). Elevated pressures may be present in patients who are tense or straining and in those with congestive heart failure, meningitis, superior vena cava syndrome, thrombosis of the venous sinuses, cerebral edema, mass lesions, hypoosmolality, or conditions inhibiting CSF absorption. Opening pres sure elevation may be the only abnormality in cryptococcal meningitis and pseudotumor cerebri (Hayward, 1987). Decreased CSF pressure may be present in spinalsubarachnoid block, dehydration, circulatory collapse, and CSF leakage. A significant pressure drop after removal of 1–2 mL suggests herniation or spinal block above the puncture site, and no further fluid should be withdrawn. Up to 20 mL of CSF may normally be removed. However, the clinician not only should be aware of the quantity of CSF required for the requested tests to ensure that a sufficient sample is submitted, but should also provide an appropriate clinical history to the laboratory. The sample site (i.e., lumbar, cisternal, etc.) should be noted because cytologic and chemical From American College of Physicians, Health and Public Policy Committee: The diagnostic spinal tap. Ann Intern Med 1986;104:880, with permission. TABLE 29-1 Diseases Detected by Laboratory Examination of CSF High sensitivity, high specificity* Bacterial, tuberculous, and fungal meningitis High sensitivity, moderate specificity Viral meningitis Subarachnoid hemorrhage Multiple sclerosis Central nervous system syphilis Infectious polyneuritis Paraspinal abscess Moderate sensitivity, high specificity Meningeal malignancy Moderate sensitivity, moderate specificity Intracranial hemorrhage Viral encephalitis Subdural hematoma *Sensitivity is the ability of a test to detect disease when it is present; specificity is the ability of a test to exclude disease when it is not present. CSF, Cerebrospinal fluid. 2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 482 direct sunlight directed on the tube at a 90degree angle from the observer will impart a “sparkling” or “snowy” appearance as suspended particles scatter the light. Clot formation may be present in patients with traumatic taps, com plete spinal block (Froin’s syndrome), or suppurative or tuberculous men ingitis. It is not seen in patients with subarachnoid hemorrhage. Fine surface pellicles may be observed after refrigeration for 12–24 hours. Clots may interfere with cell count accuracy by entrapping inflammatory cells. Viscous CSF may be encountered in patients with metastatic mucin producing adenocarcinomas, cryptococcal meningitis due to capsular poly saccharide, or liquid nucleus pulposus resulting from needle injury to the annulus fibrosus. Pink-red CSF usually indicates the presence of blood and is grossly bloody when the RBC count exceeds 6000/µL. It may originate from a subarachnoid hemorrhage, intracerebral hemorrhage, or cerebral infarct, or from a traumatic spinal tap. Xanthochromia Xanthochromia commonly refers to a pale pink to yellow color in the supernatant of centrifuged CSF, although other colors may be present (Table 293). To detect xanthochromia, the CSF should be centrifuged and the supernatant fluid compared with a tube of distilled water. Xanthochro mic CSF is pink, orange, or yellow owing to RBC lysis and hemoglobin breakdown. Pale pink to orange xanthochromia from released oxyhemo globin is usually detected by lumbar puncture performed 2–4 hours after the onset of subarachnoid hemorrhage, although it may take as long as 12 hours. Peak intensity occurs in about 24–36 hours and then gradually disappears over the next 4–8 days. Yellow xanthochromia is derived from bilirubin. It develops about 12 hours after a subarachnoid bleed and peaks at 2–4 days, but may persist for 2–4 weeks. Visible CSF xanthochromia may also be due to the following: (1) oxy hemoglobin resulting from artifactual red cell lysis caused by detergent contamination of the needle or collecting tube, or a delay of longer than 1 hour without refrigeration before examination; (2) bilirubin (bilirhachia) in jaundiced patients; (3) CSF protein levels over 150 mg/dL, which are also present in bloody traumatic taps (>100,000 RBCs/µL) or in pathologic states such as complete spinal block, polyneuritis, and meningitis; (4) merthiolate disinfectant contamination; (5) carotenoids (orange) in people with dietary hypercarotenemia (i.e., hypervitaminosis A); (6) melanin (brownish) from meningeal metastatic melanoma; and (7) rifampin therapy (redorange). Although spectral absorbance scans provide an objective record of xanthochromia, careful gross CSF inspection has comparable sensitivity (Britton, 1983). Spectrophotometrycan also help to differentiate hemoglobinderived substances from other xanthochromic pigments with different maximal absorption peaks. Modified from Kjeldsberg CR, Knight JA. Body fluids: laboratory examination of amniotic, cerebrospinal, seminal, serous, and synovial fluids. 3rd ed. Chicago: © American Society for Clinical Pathology; 1993, with permission. TABLE 29-2 Recommended CSF Laboratory Tests Routine Opening CSF pressure Total cell count (WBC and RBC) Differential cell count (stained smear) Glucose (CSF/plasma ratio) Total protein Useful under certain conditions Cultures (bacteria, fungi, viruses, Mycobacterium tuberculosis) Gram stain, acid-fast stain Fungal and bacterial antigens Enzymes (LD, ADA, CK-BB) Lactate Polymerase chain reaction (TB, viruses) Cytology Electrophoresis (protein, immunofixation) Proteins (C-reactive, 14-3-3, τ, β-amyloid, transferrin) VDRL test for syphilis Fibrin-derivative D-dimer Tuberculostearic acid ADA, Adenosine deaminase; CK-BB, creatine kinase-BB; CSF, cerebrospinal fluid; LD, lactate dehydrogenase; RBC, red blood cell; TB, tuberculosis; VDRL, Venereal Disease Research Laboratories; WBC, white blood cell. TABLE 29-3 Xanthochromia and Associated Diseases/Disorders CSF supernatant color Associated diseases/disorders Pink RBC lysis/hemoglobin breakdown products Yellow RBC lysis/hemoglobin breakdown products Hyperbilirubinemia CSF protein >150 mg/dL (1.5 g/L) Orange RBC lysis/hemoglobin breakdown products Hypervitaminosis A (carotenoids) Yellow-green Hyperbilirubinemia (biliverdin) Brown Meningeal metastatic melanoma CSF, Cerebrospinal fluid; RBC, red blood cell. Differential Diagnosis of Bloody CSF A traumatic tap occurs in about 20% of lumbar punctures. Distinction of a traumatic puncture from a pathologic hemorrhage is, therefore, of vital importance. Although the presence of crenated RBCs is not useful, the following observations may be helpful in distinguishing the two forms of bleeding. 1. In a traumatic tap, the hemorrhagic fluid usually clears between the first and third collected tubes but remains relatively uniform in subarachnoid hemorrhage. 2. Xanthochromia, microscopic evidence of erythrophagocytosis, or hemosiderinladen macrophages indicate a subarachnoid bleed in the absence of a prior traumatic tap. RBC lysis begins as early as 1–2 hours after a traumatic tap. Thus, rapid evaluation is necessary to avoid falsepositive results. 3. A commercially available latex agglutination immunoassay test for crosslinked fibrin derivative ddimer is specific for fibrin deg radation and is negative in traumatic taps (Lang, 1990). However, falsepositive results might be expected in disseminated intra vascular coagulation, fibrinolysis, or trauma from repeated lumbar punctures. MICROSCOPIC EXAMINATION Total Cell Count Although the traditional manual method for cell counting in CSF samples, using undiluted CSF in a manual counting chamber, continues to be a useful approach, because of the low cell counts frequently encountered in CSF, the precision of manual counting in these samples is inherently limited (Barnes, 2004). For example, using 18 large squares (1 mm2 each) in a FuchsRosenthal–type chamber with a depth of 0.2 mm, a total volume of 3.6 µL (18 × 0.2 µL/square) is examined. With 5 cells/µL, a total of 18 cells is counted. The coefficient of variation (CV), defined as 100 divided by the square root of the number of cells counted, is 24%; ±2 CV is about 48%. A Neubauer hemocytometer with nine 1mm2 squares with a depth of 0.1 mm has a CV of 45% (+90% for 2 CV) with the same cell concentration. Improvements in the hardware and software in flow cytometers now allow reliable use of these instruments in performing automated total WBC counts and WBC differential counts (Hoffman, 2002; Aune, 2004), and even in detecting bacteria (Nanos, 2008) in CSF samples; however, the low clinical decision levels for total WBC count and enumeration of some WBC types in CSF and limitations in detecting small numbers of RBCs continue to represent challenges in the use of these instruments with CSF (Hoffman, 2002; Andrews, 2005; Glasser, 2009; Kleine, 2009). Because of these limitations, laboratories should exercise caution in utilizing automated cell counters with CSF samples and should carefully follow manufacturer and Clinical and Laboratory Standards Insti tute guidelines (CLSI Approved Guideline H56A, 2006) when imple menting these automated methods. The normal leukocyte cell count in adults is 0–5 cells/µL. It is higher in neonates, ranging from 0–30 cells/µL, with the upper limit of normal decreasing to adult values by adolescence. No RBCs should be present in normal CSF. If numerous (except a traumatic tap), a pathologic process is probable (e.g., trauma, malignancy, infarct, hemorrhage). Although red cell counts have limited diagnostic value, they may give a useful approxi mation of the true CSF WBC or total protein in the presence of a trau matic puncture by correcting for leukocytes or protein introduced by the traumatic puncture. To be valid, all measurements (WBC, RBC, protein) PA R T 3 483 must be performed on the same tube. This procedure also assumes that the blood is derived exclusively from the traumatic tap. The corrected WBC count is as follows: WBC WBC WBCcorr obs added= − where WBC WBC RBC RBCadded BLD CSF BLD= × and WBCobs = CSF leukocyte count WBCadded = leukocytes added to CSF by traumatic tap WBCBLD = peripheral blood leukocyte count RBCCSF = CSF erythrocyte count RBCBLD = peripheral blood erythrocyte count. An analogous formula may be used to correct for added total protein (TP): TP TP HCT RBC RBCadded serum CSF BLD= × −( )[ ] ×1 In the presence of a normal peripheral blood RBC count and serum protein, these corrections amount to about 1 WBC for every 700 RBCs and 8 mg/dL protein for every 10,000 RBCs/µL. This latter RBC correc tion factor is reasonably accurate as long as the peripheral WBC count is not extremely high or low. Moreover, the accuracy of these corrections is limited by the precision of the CSF RBC count, which can significantly limit its value. An observed/expected (added) WBC count ratio greater than 10 has a sensitivity of 88% and a specificity of 90% for bacterial meningitis. When the predicted WBC is below the observed count, the probability of bacte rial meningitis appears to be low (Mayefsky, 1987; Bonadio, 1990). Differential Cell Count Suggested differential count reference ranges are presented in Table 294. A differential performed in a counting chamber is unsatisfactory because the low cell numbers give rise to poor precision, and identifying the cell type beyond granulocytes and “mononuclears” is difficult in a wet prepara tion. Direct smears of the centrifuged CSF sediment are also subject to significant error from cellular distortion and fragmentation. The cytocentrifuge method is rapid, requires minimal training, and allows Wright’s staining of airdried cytospins. Indeed, it is the recom mended method for differential cell counts in all body fluids (Rabinovitch, 1994). Cell yield and preservation are better than with simple centrifuga tion. From 30–50 cells can be concentrated from 0.5 mL of “normal” CSF. Variable artifactual distortions may be seen, but they are minimized when the specimen is fresh, albumin is added to the specimen (2 drops of 22% bovine serum albumin), and the cell concentration is adjusted to about 300 WBCs/L prior to centrifugation (Kjeldsberg, 1993). Manual differen tial cell counting on a cytocentrifuge preparation of CSF continues to be the most reliable method, even with low cell numbers. Although auto mated differential counts may be safely performed on CSF samples using flowcytometers (Hoffman, 2002; Aulesa, 2003), confirmation of the auto mated differential count by manual examination of a cytocentrifuged smear is recommended with some instruments (Aune, 2004) and is a requirement with specimens at risk of containing neoplastic cells. Filtration and sedimentation methods are too cumbersome for routine use. Filtration does, however, allow concentration of large volumes of CSF for cytologic examination or culture, while retaining the fluid filtrate for additional studies. In adults, normal CSF contains small numbers of lymphocytes and monocytes in an approximate 70 : 30 ratio (Fig. 291). A higher proportion of monocytes is present in young children, in whom up to 80% may be Figure 29-1 Cerebrospinal fluid cytology (lymphocyte to monocyte distribution ratio 70:30). Figure 29-2 Choroid plexus cells in cerebrospinal fluid. Figure 29-3 Cluster of blast-like cells in cerebrospinal fluid from premature newborn. (From Kjeldsberg CR, Knight JA. Body fluids: laboratory examination of amni- otic, cerebrospinal, seminal, serous and synovial fluids. 3rd ed. Chicago: © American Society for Clinical Pathology; 1993, with permission.) TABLE 29-4 CSF Reference Values for Differential Cytocentrifuge Counts Cell type Adults, % Neonates, % Lymphocytes 62 ± 34 20 ± 18 Monocytes 36 ± 20 72 ± 22 Neutrophils 2 ± 5 3 ± 5 Histiocytes Rare 5 ± 4 Ependymal cells Rare Rare Eosinophils Rare Rare CSF, Cerebrospinal fluid. normal (Pappu, 1982). Erythrocytes due to minor traumatic bleeding are commonly seen, especially in infants. Small numbers of neutrophils (PMNs) may also be seen in “normal” CSF specimens, most likely as a result of minor hemorrhage (Hayward, 1988) and improved cell concentra tion methods. No general consensus regarding an upper limit of normal for PMNs has been established. We accept up to 7% neutrophils with a normal WBC count. Over 60% neutrophils has been reported in highrisk neonates without meningitis (Rodriguez, 1990). The number of PMNs may be decreased by as much as 68% within the first 2 hours after lumbar puncture owing to cell lysis (Steele, 1986). Traumatic puncture may result in the presence of bone marrow cells, cartilage cells, squamous cells, ganglion cells, and soft tissue elements. In addition, ependymal and choroid plexus cells may rarely be seen (Fig. 292). Moreover, blastlike primitive cell clusters, most likely of germinal matrix origin, are sometimes found in premature infants with intraven tricular hemorrhage (Fig. 293). Increased CSF neutrophils occur in numerous conditions (Table 295). In early bacterial meningitis, the proportion of PMNs usually exceeds 60%. However, in about one quarter of cases of early viral men ingitis, the proportion of PMNs also exceeds 60%. Viralinduced neutro philia usually changes to a lymphocytic pleocytosis within 2–3 days. A total PMN count of over 1180 cells/µL (or more than 2000 WBCs/µL) has a 99% predictive value for bacterial meningitis (Spanos, 1989). Persistent neutrophilic meningitis (over 1 week) may be noninfectious or due to less common pathogens such as Nocardia, Actinomyces, Aspergillus, and the zygo mycetes (Peacock, 1984). 2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 484 Increased CSF lymphocytes have been reported in various diseases/ disorders (Table 296). Lymphocytosis (>50%) may occur in early acute bacterial meningitis when the CSF leukocyte count is under 1000/µL (Powers, 1985). Reactive lymphoplasmacytoid and immunoblastic variants may be present, particularly with viral meningoencephalitis. Blastlike lymphocytes may be seen admixed with small and large lymphocytes in the CSF of neonates. Plasma cells, not normally present in CSF, may appear in a variety of inflammatory and infectious conditions (Table 297), along with large and small lymphocytes, and in association with malignant brain tumors TABLE 29-5 Causes of Increased CSF Neutrophils Meningitis Bacterial meningitis Early viral meningoencephalitis Early tuberculous meningitis Early mycotic meningitis Amebic encephalomyelitis Other infections Cerebral abscess Subdural empyema AIDS-related CMV radiculopathy Following seizures Following CNS hemorrhage Subarachnoid Intracerebral Following CNS infarct Reaction to repeated lumbar punctures Injection of foreign material in subarachnoid space (e.g., methotrexate, contrast media) Metastatic tumor in contact with CSF AIDS, Acquired immunodeficiency syndrome; CMV, cytomegalovirus; CNS, central nervous system; CSF, cerebrospinal fluid. TABLE 29-6 Causes of CSF Lymphocytosis Meningitis Viral meningitis Tuberculous meningitis Fungal meningitis Syphilitic meningoencephalitis Leptospiral meningitis Bacterial due to uncommon organisms Early bacterial meningitis where leukocyte counts are relatively low Parasitic infestations (e.g., cysticercosis, trichinosis, toxoplasmosis) Aseptic meningitis due to septic focus adjacent to meninges Degenerative disorders Subacute sclerosing panencephalitis Multiple sclerosis Drug abuse encephalopathy Guillain-Barré syndrome Acute disseminated encephalomyelitis Other inflammatory disorders Handl syndrome (headache with neurologic deficits and CSF lymphocytosis) Sarcoidosis Polyneuritis CNS periarteritis CNS, Central nervous system; CSF, cerebrospinal fluid. Modified with permission from Kjeldsberg CR, Knight JA. Body fluids: laboratory examination of amniotic, cerebrospinal, seminal, serous and synovial fluids. 3rd ed. Chicago: © American Society for Clinical Pathology; 1993. TABLE 29-8 Causes of CSF Eosinophilic Pleocytosis Commonly associated with Acute polyneuritis CNS reaction to foreign material (drugs, shunts) Fungal infections Idiopathic eosinophilic meningitis Idiopathic hypereosinophilic syndrome Parasitic infections Infrequently associated with Bacterial meningitis Leukemia/lymphoma Myeloproliferative disorders Neurosarcoidosis Primary brain tumors Tuberculous meningoencephalitis Viral meningitis CNS, Central nervous system; CSF, cerebrospinal fluid. TABLE 29-7 Inflammatory and Infectious Causes of CSF Plasmacytosis • Acute viral infections • Guillain-Barré syndrome • Multiple sclerosis • Parasitic CNS infestations • Sarcoidosis • Subacute sclerosing panencephalitis • Syphilitic meningoencephalitis • Tuberculous meningitis CNS, Central nervous system; CSF, cerebrospinal fluid. Figure 29-4 Eosinophils in cerebrospinal fluid from a child with malfunctioning ventricular shunt. (Fishman, 1992). Multiple myeloma may also rarely involve the meninges (Oda, 1991). Although eosinophils are rarely present in normal CSF, they may be increased in a variety of central nervous system (CNS) conditions (Table 298). For example, eosinophilia is frequently mild (1%–4%) in a general inflammatory response, but in children with malfunctioning ventricular shunts, it may be marked (Fig. 294). A suggested criterion for eosinophilic meningitis is 10% eosinophils (Kuberski, 1981); parasitic invasion of the CNS is the most common cause worldwide. Coccidioides immitis is a signifi cant cause of CSF eosinophilia in endemic regions of the United States (Ragland, 1993). Increased CSF monocytes lack diagnostic specificity and are usually part of a “mixed cell reaction” that includes neutrophils, lymphocytes, and plasma cells. This pattern is seen in tuberculous and fungal meningitis, chronic bacterial meningitis (i.e., Listeria monocytogenes and others), lepto spiral meningitis, ruptured brain abscess, Toxoplasma meningitis, and amebic encephalomeningitis. A mixed cell pattern withoutneutrophils is characteristic of viral and syphilitic meningoencephalitis. Macrophages with phagocytosed erythrocytes (erythrophages) appear from 12–48 hours following a subarachnoid hemorrhage or traumatic tap. Hemosiderin laden macrophages (siderophages) appear after about 48 hours and may persist for weeks (Fig. 295). Brownish yellow or red hematoidin crystals may form after a few days. Figure 29-5 Hemosiderin-laden macrophages (siderophages) from the cerebro- spinal fluid of a patient with subarachnoid hemorrhage. Hemosiderin crystals (golden-yellow) are also present. (From Kjeldsberg CR, Knight JA. Body fluids: labora- tory examination of amniotic, cerebrospinal, seminal, serous and synovial fluids. 3rd ed. Chicago: © American Society for Clinical Pathology; 1993, with permission.) PA R T 3 485 are significantly less common (Bigner, 1992; Walts, 1992). T cells predomi nate in normal and inflammatory conditions, whereas most lymphomas, especially those occurring in immunocompromised hosts, are of B cell lineage. Lymphoblastic lymphoma, the most common T cell lymphoma, can be detected by terminal deoxynucleotidyl transferase stain. Multiparameter flow cytometric immunophenotypic studies and/or molecular DNA analysis may significantly improve diagnostic sensitivity and specificity in CSF samples involved by leukemic or lymphoma cells (Rhodes, 1996; Finn, 1998; Scrideli, 2004; Bromberg, 2007; Quijano, 2009), and these are used increasingly as part of a complete evaluation in patients being worked up for these conditions. Amebae, fungi (especially Cryptococcus neoformans), and Toxoplasma gondii organisms may be present on cytocentrifuge specimens but may be difficult to recognize without confirmatory stains. CHEMICAL ANALYSIS Reference values for lumbar cerebrospinal fluid in adults are listed in Table 299. Proteins Total Protein More than 80% of the CSF protein content is derived from blood plasma, in concentrations of less than 1% of the plasma level (Table 2910). Morphologic cerebrospinal fluid examination for tumor cells has mod erate sensitivity and high specificity (97%–98%) (Marton, 1986). Sensitiv ity depends on the type of neoplasm. CSF examination of leukemic patients has the highest sensitivity (about 70%), followed by metastatic carcinoma (20%–60%) and primary CNS malignancies (30%). Sensitivity may be optimized by using filtration methods with larger fluid volumes or by performing serial punctures in patients in whom a neoplasm is strongly suspected. Processing of CSF samples using liquidbased thinlayer methods also increases sensitivity in the detection of neoplastic cells and enhances preservation of these cells for potential immunocytochemical analysis (Sioutopoulou, 2008). These liquidbased methods are now com monly used for cytopathologic examination of CSF and other body cavity fluid specimens. Leukemic involvement of the meninges is more frequent in patients with acute lymphoblastic leukemia (Fig. 296) than in those with acute myeloid leukemia (Fig. 297); both are significantly more common than CNS involvement in the chronic leukemias. A leukocyte count over 5 cells/µL with unequivocal lymphoblasts in cytocentrifuged preparations is commonly accepted as evidence of CSF involvement. The incidence of CNS relapse in children with lymphoblasts but cell counts lower than 6 cells/µL appears to be low and is not significantly different from cases in which no blasts are identified (Odom, 1990; Gilchrist, 1994; Tubergen, 1994). NonHodgkin lymphomas involving the leptomeninges are usually highgrade tumors (lymphoblastic, large cell immunoblastic, and Burkitt’s lymphomas) (Fig. 298); lowgrade lymphomas and Hodgkin lymphoma Figure 29-6 Acute lymphoblastic leukemia in cerebrospinal fluid. Note uniformity of the blast cells. Figure 29-7 Acute myeloid leukemia in cerebrospinal fluid. Figure 29-8 Burkitt’s lymphoma in cerebrospinal fluid. The cells are characterized by blue cytoplasm with vacuoles and slightly clumped chromatin pattern. (From Kjeldsberg CR, Knight JA. Body fluids: laboratory examination of amniotic, cerebrospinal, seminal, serous and synovial fluids. 3rd ed. Chicago: © American Society for Clinical Pathology; 1993, with permission.) TABLE 29-9 Adult Lumbar CSF Reference Values Analyte Conventional units SI units Protein 15–45 mg/dL 0.15–0.45 g/L Prealbumin 2%–7% Albumin 56%–76% α1-Globulin 2%–7% α2-Globulin 4%–12% β-Globulin 8%–18% γ-Globulin 3%–12% Electrolytes Osmolality 280–300 mOsm/L 280–300 mmol/L Sodium 135–150 mEq/L 135–150 mmol/L Potassium 2.6–3.0 mEq/L 2.6–3.0 mmol/L Chloride 115–130 mEq/L 115–130 mmol/L Carbon dioxide 20–25 mEq/L 20–25 mmol/L Calcium 2.0–2.8 mEq/L 1.0–1.4 mmol/L Magnesium 2.4–3.0 mEq/L 1.2–1.5 mmol/L Lactate 10–22 mg/dL 1.1–2.4 mmol/L pH Lumbar fluid 7.28–7.32 Cisternal fluid 7.32–7.34 pCO2 Lumbar fluid 44–50 mm Hg Cisternal fluid 40–46 mm Hg pO2 40–44 mm Hg Other Constituents Ammonia 10–35 µg/dL 6–20 µmol/L Glutamine 5–20 mg/dL 0.3–1.4 mmol/L Creatinine 0.6–1.2 mg/dL 45–92 µmol/L Glucose 50–80 mg/dL 2.8–4.4 mmol/L Iron 1–2 µg/dL 0.2–0.4 µmol/L Phosphorus 1.2–2.0 mg/dL 0.4–0.7 mmol/L Total lipid 1–2 mg/dL 0.01–0.02 g/L Urea 6–16 mg/dL 2.0–5.7 mmol/L Urate 0.5–3.0 mg/dL 30–180 µmol/L Zinc 2–6 µg/dL 0.3–0.9 µmol/L CSF, cerebrospinal fluid; pCO2, partial pressure of carbon dioxide; pO2, partial pres- sure of oxygen. 2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 486 Prealbumin (transthyretin), transferrin, and small quantities of nerve tissue–specific proteins are the major qualitative differences that normally exist between CSF and plasma proteins. Although some authors have argued against routine measurement of total protein (American College of Physicians, 1986), it is the most common abnormality found in CSF. Thus, an increased CSF protein serves as a useful, albeit nonspecific, indicator of meningeal or CNS disease. Reference Values. CSF total protein reference values vary considerably between laboratories owing to differences in methods, instrumentation, and type of reference standard used (College of American Pathologists CSF Chemistry Survey, Set MB, 1991*; Gerbaut, 1986). CSF protein levels of 15–45 mg/dL have long been accepted as the “normal” reference range (Silverman, 1994). Using the classic Lowry method, the reported adult range was 24.1–48.5 mg/dL (Tibbling, 1977). Others reported a reference range of 14–49 mg/dL using a trichloroacetic acid–Ponceau S method (Breebaart, 1978), and of 22.3–50.3 mg/dL with a biuret method (Ahonen, 1978). Reference levels were also compared using three different methods: (1) A modified biuret technique; (2) a Dupont aca method in which protein was precipitated and then reacted with trichloroacetic acid; and (3) a Kodak Ektachem colorimetric slide technique (Lott, 1989). All three methods gave similar, although significantly higher, levels than those previously reported (i.e., 14–62 mg/dL, 16–61 mg/dL, and 12–60 mg/dL, respectively). Although discrepancies in gender and in those over the age of 60 years have been reported, the differences are probably not significant. However, infants have significantly higher CSF protein levels than older children and adults. Thus, mean levels of 90 mg/dL for term infants and 115 mg/ dL for preterm infants were reported; the upper levels were 150 mg/dL and 170 mg/dL, respectively (Sarff, 1976). Similarly, others recently noted that the CSF protein concentration fell rapidly from birth to 6 months of age (mean levels, 108 mg/dL–40 mg/dL), plateaued between 3 and 10 years (mean, 32 mg/dL), and thenrose slightly from 10–16 years (mean, 41 mg/dL) (Biou, 2000). Elevated CSF protein levels may be caused by increased permeability of the bloodbrain barrier, decreased resorption at the arachnoid villi, mechanical obstruction of CSF flow due to spinal block above the puncture site, or an increase in intrathecal immunoglobulin (Ig) synthesis. Common conditions associated with elevated lumbar CSF protein values (>65 mg/ dL) are summarized in Table 2911. Low lumbar CSF total protein levels (<20 mg/dL) normally occur in some young children between 6 months and 2 years of age and in patients with conditions associated with increased CSF turnover. These include the following: (1) Removal of large CSF volumes; (2) CSF leaks induced by trauma or lumbar puncture; (3) increased intracranial pressure, probably due to an increased rate of protein resorption by the arachnoid villi; and (4) hyperthyroidism (Fishman, 1992). Protein electrophoresis of concentrated normal CSF reveals two dis tinct differences from serum: A prominent transthyretin (prealbumin) band and two transferrin bands. Transthyretin is relatively high because of its dual synthesis by the liver and the choroid plexus. The second trans ferrin band, referred to as β2transferrin, migrates more slowly than its Adapted from Felgenhauer K. Klin Wochenschr 1974;52:1158, with permission. TABLE 29-10 Mean Concentrations of Plasma and CSF Proteins Protein CSF, mg/L Plasma/CSF ratio Prealbumin 17.3 14 Albumin 155.0 236 Transferrin 14.4 142 Ceruloplasmin 1.0 366 Immunoglobulin (Ig)G 12.3 802 IgA 1.3 1346 α2-Microglobulin 2.0 1111 Fibrinogen 0.6 4940 IgM 0.6 1167 β-Lipoprotein 0.6 6213 CSF, Cerebrospinal fluid. *College of American Pathologists, 325 Waukegan Road, Northfield, Ill. TABLE 29-11 Conditions Associated With Increased CSF Total Protein Traumatic spinal puncture Increased blood–CSF permeability Arachnoiditis (e.g., following methotrexate therapy) Meningitis (bacterial, viral, fungal, tuberculous) Hemorrhage (subarachnoid, intracerebral) Endocrine/metabolic disorders Milk–alkali syndrome with hypercalcemia Diabetic neuropathy Hereditary neuropathies and myelopathies Decreased endocrine function (thyroid, parathyroid) Other disorders (uremia, dehydration) Drug toxicity Ethanol, phenothiazines, phenytoin CSF circulation defects Mechanical obstruction (tumor, abscess, herniated disk) Loculated CSF effusion Increased immunoglobulin (Ig)G synthesis Multiple sclerosis Neurosyphilis Subacute sclerosing panencephalitis Increased IgG synthesis and blood–CSF permeability Guillain-Barré syndrome Collagen vascular diseases (e.g., lupus, periarteritis) Chronic inflammatory demyelinating polyradiculopathy CSF, Cerebrospinal fluid. serum equivalent owing to cerebral neuraminidase digestion of sialic acid residues. Methods. Turbidimetric methods, commonly based on trichloroacetic acid (TCA) or sulfosalicylic acid and sodium sulfate for protein precipita tion, are popular because they are simple and rapid, and require no special instrumentation. However, they are temperature sensitive and require much larger specimen volumes (about 0.5 mL). Moreover, some methods are prone to significant variation from changes in the albumin/globulin ratio (Schriever, 1965). A false protein elevation may be observed using TCA methods in the presence of methotrexate (Kasper, 1988). Benzethonium chloride and benzalkonium chloride have been used as precipitating agents in automated methods and micromethods (Luxton, 1989; Shephard, 1992). Colorimetric methods include the Lowry method, dyebinding, methods using Coomassie brilliant blue (CBB) or Ponceau S, and the modified Biuret method. The CBB method is rapid and highly sensitive, and can be used with small sample sizes. Immunologic methods measure specific proteins, require only 25–50 µL of CSF, and are relatively simple to perform once conditions and reagents have been standardized. Auto mated methods are also commonly used and usually show good correlation with standard methods (Lott, 1989). Albumin and IgG Measurements The permeability of the bloodbrain barrier may be assessed by immuno chemical quantification of the CSF albumin/serum albumin ratio in grams per deciliter (g/dL). The normal ratio of 1 : 230 (Tourtellotte, 1985) yields an unwieldy decimal of 0.004, which prompted the use of the CSF/serum albumin index, which is arbitrarily calculated as follows: CSF Serum albumin index CSF albumin mg dL Serum albumin g dL = ( ) ( ) (29-1) An index value less than 9 is consistent with an intact barrier. Slight impairment is considered with index values of 9–14, moderate impairment with values of 14–30, and severe impairment with values greater than 30 (Silverman, 1994). The index is slightly elevated in infants up to 6 months of age, reflecting the immaturity of the bloodbrain barrier, and increases gradually after age 40 years. A traumatic tap invalidates the index calculation. Increased intrathecal IgG synthesis is reflected by an increase in the CSF/serum IgG ratio: CSF Serum IgG ratio CSF IgG mg dL Serum IgG g dL = ( ) ( ) (29-2) PA R T 3 487 High-resolution agarose gel electrophoresis of concentrated CSF from patients with MS often shows discrete populations of IgG, the oligoclonal bands. Although these discrete IgG populations are normally absent, two or more bands are necessary to support the diagnosis of MS; a single band is not considered a positive result. Using this technique, oligoclonal bands have been reported in 83%–94% of patients with defi nite MS, 40%–60% of those with probable MS, and 20%–30% of possible MS cases. However, they are also frequently present in patients with subacute sclerosing panencephalitis, various viral CNS infections, neuro syphilis, neuroborreliosis, cryptococcal meningitis, GuillainBarré syn drome, transverse myelitis, meningeal carcinomatosis, glioblastoma multiforme, Burkitt’s lymphoma, chronic relapsing polyneuropathy, Behçet’s disease, cysticercosis, and trypanosomiasis, among others (Trotter, 1989; Chalmers, 1990; Fishman 1992; Hall, 1992). Subsequent studies indicate that agarose gel electrophoresis sensitivity for MS is less than previously reported (see later). Oligoclonal light chains (both κ and λ) are present in about 90% of MS patients (Gallo, 1989; Sindie, 1991). They have also occasionally been identified in the CSF of those who are negative for IgG oligoclonal bands. However, because of their uncommon occurrence in the absence of IgG and costineffectiveness, as well as the ready availability of magnetic resonance imaging, it is unlikely that this technique will become common. Coomassie brilliant blue or paragon violet stains can resolve oligoclonal bands in only 5 µg of IgG (Silverman, 1994). However, silver staining is 20–50 times more sensitive than CBB and can be used on unconcentrated CSF. It is important to note that these electrophoretic techniques must be simultaneously carried out on the patient’s serum to be certain that a polyclonal gammopathy is not present (e.g., liver disease, systemic lupus, rheumatoid arthritis, chronic granulomatous disease) because these disorders may be accompanied by Ig diffusion into the CSF, yielding falsepositive results. Immunofixation electrophoresis (IFE) is more sensitive than agarose gel electrophoresis and does not require CSF concentration (Cawley, 1976). A subsequent study reported a sensitivity of 74% using this tech nique compared with 57% for agarose gel electrophoresis (Cavuoti, 1998). More recently, using a semiautomated immunofixationperoxidase tech nique, the sensitivity was 83% and the specificity was 79% in patients with clinically definite MS (Richard, 2002). However, IFE provides fewer bandsthan isoelectric focusing and IgG immunoblotting (IgGIEF). More over, the bands obtained by IFE tend to be more diffuse. IgGIEF performed on paired CSF and serum samples is the most sensitive method for the detection of oligoclonal bands (Andersson, 1994). One study showed that IgGIEF detected 100% of definite MS, but only about 50% were positive by agarose electrophoresis (Lunding, 2000). Others detected 91% of MS cases but only 68% with agarose (Seres, 1998). Similarly, a semiautomated IgGIEF technique identified 90% of MS cases compared with 60% for agarose electrophoresis (Fortini, 2003). In 2005, an international consensus standard for the diagnosis of MS established IgGIEF as the method of choice for qualitative detection of oligoclonal IgG bands as evidence of intrathecal synthesis of IgG (Freedman, 2005). This method is more sensitive and specific than quantitative methods. In summary, the diagnosis of MS, as with many other neurologic dis orders, is ultimately a clinical one based on neurologic history and physical examination. Nevertheless, advanced laboratory results in CSF, such as elevated IgG indices and particularly the detection of oligoclonal IgG bands, as well as neuroimaging techniques, have proved invaluable in the diagnosis of MS. Other CSF Proteins Approximately 300 different proteins have been identified in CSF using twodimensional electrophoresis, the first dimension being isoelectric focusing and the second polyacrylamide gel in the presence of sodium dodecyl sulfate (Harrington, 1986). Using this technique, four abnormal proteins were identified in patients with CreutzfeldtJakob disease (CJD). Two of these proteins (molecular mass about 40 kilodaltons [kDa] each) were present in some, but not all, patients with herpes simplex encephalitis, Parkinson’s disease, GuillainBarré syndrome, and schizophrenia. They were not present in various other neurologic disorders, nor in 100 normal CSF control specimens. However, these and two other proteins (molecular masses about 26 and 29 kDa) were present in all cases of CJD and in 5 of 10 cases of herpes simplex encephalitis. Neither of these latter proteins was present in any other neurologic disease or in controls. Increased concentrations of various specific CSF proteins have been associated with several CNS diseases (Table 2912). The normal ratio is 1/390 or 0.003 (Tourtellotte, 1985). Similar to the albumin index, the CSF/serum IgG index may be obtained by using mil ligrams per deciliter for the CSF IgG value. The CSF/serum IgG index normal range is 3.0–8.7. The CSF/serum IgG index can be elevated by intrathecal IgG syn thesis or increased plasma IgG crossover from breakdown of the blood brain barrier. Ig derived from plasma crossover may be corrected by dividing the CSF/serum IgG index by the CSF/albumin index to yield the CSF IgG index. CSF IgG index CSF IgG mg dL Serum IgG g dL CSF albumin mg dL Serum = ( ) ( ) ( ) aalbumin g dL( ) (29-3) or CSF IgG index CSF IgG mg dL Serum albumin g dL Serum IgG g dL CS = ( ) × ( ) ( ) × FF albumin mg dL( ) (29-4) The normal reference range for the IgG index varies, reflecting varia tions in determination of the four index components. A reasonable normal upper limit is 0.8 (Souverijn, 1989). However, each laboratory should determine its own critical ratio. The IgG synthesis rate is calculated by an empirical formula (Tourtellotte, 1985): IgG synthesis rate mg day CSF IgG Serum IgG CSF albumin S ( ) = −( ) −[ − 369 eerum albumin Serum IgG Serum albumin dL day 230 0 43 5 ( ) × ( ) × × ]. (29-5) All protein concentrations are expressed in milligrams per deciliter. The first bracketed term represents the difference between measured CSF IgG and the IgG expected from diffusion across a normal bloodbrain barrier; 369 is the normal serum/CSF ratio. The second bracketed term represents the difference between measured CSF albumin and expected albumin if the bloodbrain barrier is intact; 230 is the normal serum/CSF albumin ratio. The CSF albumin excess is multiplied by the IgG/albumin ratio and the molecular weight ratio of IgG to albumin (0.43) to correct for changes in CSF IgG due to increased barrier permeability. The number 5 converts the result from a concentration to a daily amount, assuming an average daily CSF production of 500 mL (i.e., 5 dL). The formula does not consider variations in CSF production or Ig consumption. It assumes that the IgG/albumin ratio remains constant over various degrees of blood brain barrier impairment—a concept that may lead to variable error (Lefvert, 1985). The normal reference interval for the synthesis rate is −9.9 to +3.3 mg/day. Values greater than 8.0 mg/day indicate an increased rate (Silverman, 1994). CSF IgG is normally 3%–5% of total CSF protein, but in multiple sclerosis (MS), the concentration approaches that of plasma (15%–18%) (Hersey, 1980). The CSF IgG index and the IgG synthesis rate have a sensitivity of 90% in patients with definite MS, but the sensitivity is lower in patients with possible MS, in whom accuracy is most needed (Marton, 1986). In addition, the specificity for MS is only moderate because increased intrathecal IgG synthesis occurs in many other inflammatory neurologic diseases. The Ig index and synthesis rate calculations may also be applied to IgM, IgA, Ig light chains, and specific antibodies to infectious microorganisms. For example, increased synthesis of IgM and free κ light chains have been suggested as markers for MS (Rudick, 1989; Lolli, 1991). Electrophoretic Techniques. Although the diagnosis of MS is ulti mately a clinical one, significant advances have been made in laboratory testing for this disorder. CSF total protein is increased in less than 50% of patients with MS. Indeed, if the CSF protein exceeds 100 mg/dL, the patient probably does not have MS. However, the γglobulin fraction, as determined by CSF electrophoresis, is often increased in MS. Thus, the CSF total protein/γglobulin ratio exceeds 0.12 in about 65% of cases (Johnson, 1977). Using electroimmunodiffusion, a CSF IgG/albumin ratio greater than 0.25 is present in about 75% of cases (Tourtellotte, 1971). Furthermore, levels greater than the mean CSF IgG index + 3SD are present in 80%–85% of MS cases. However, this upper reference level varies significantly between laboratories, and 0.58, 0.66, and 0.77 have been reported as cutoff values (Olsson, 1976; Tibbling, 1977; Markowitz, 1983, respectively). Therefore, laboratories should establish their own reference values. 2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 488 In children with acute lymphoblastic leukemia, elevated CSF fibronec tin levels are associated with a poor prognosis, presumably due to leukemic involvement of the CNS (Rautonen, 1989). Significant CSF elevations have also been reported in Burkitt’s lymphoma (Rajantie, 1989), some metastatic solid tumors, astrocytomas, and bacterial meningitis (Weller, 1990; Torre, 1991). Decreased levels have been reported in viral meningitis and in the acquired immunodeficiency syndrome (AIDS)–dementia complex (Torre, 1991, 1993). β-Amyloid Protein 42 and τ Protein. The diagnosis of Alzheimer’s disease (AD) is based on the presence of dementia and a specific clinical profile (i.e., from medical history, clinical examination) suggestive of AD, together with the exclusion of other causes of dementia. Pathologically, the disease is characterized by the presence of neurofibrillary tangles and amyloid plaques. Recent studies indicate that measurement of biochemical markers increases diagnosticaccuracy, especially early in the course of the disease, when clinical symptoms are mild and vague and overlap with cognitive changes that accompany aging and ischemic dementia. Thus, increased CSF levels of microtubuleassociated τ protein and decreased levels of βamyloid protein ending at amino acid 42 have been shown to significantly increase the accuracy of AD diagnosis (Andreasen, 2001; Riemenschneider, 2002; Sunderland, 2003). Indeed, the predictive value for early AD is greater than 90% (Andreasen, 2001). Others have found that the calculated ratio of phosphorylated τ protein to βamyloid peptide is superior to either measure alone (Maddalena, 2003). The results were as follows: Distinguishing patients with AD from healthy controls—sensitivity 96%, specificity 97%; patients with AD from those with nonAD dementia—sensitivity 80%, specificity 73%; and patients with AD from those with other neurologic disorders—sensitivity 80%, specificity 89%. Protein 14-3-3. The transmissible spongiform encephalopathies consti tute a group of uniformly fatal neurodegenerative diseases. Of these, CJD is the major spongiform disease in humans. Two proteins, designated 130 and 131, have been detected in low concentrations in CSF from CJD patients. These proteins have the same amino acid sequence as protein 1433 (Hsich, 1996). Moreover, in patients with dementia, a positive immunoassay for the 1433 protein in CSF strongly supports a diagnosis of CJD. In a subsequent study of patients with suspected CJD, the sensitiv ity of the 1433 protein determined by immunoassay was 97%, and the specificity was 87% (Lemstra, 2000). Falsepositive results were seen pri marily in patients with stroke and meningoencephalitis. Others, using a modified Western blot technique, reported a 94.7% positive predictive value and a 92.4% negative predictive value for CJD (Zerr, 1998). Falsepositive results from a single CSF analysis were seen in patients with herpes simplex encephalitis, atypical encephalitis, meta static lung cancer, and hypoxic brain damage. Transferrin and CSF Leakage. Cerebrospinal fluid leakage usually presents as otorrhea or rhinorrhea following head trauma, in some cases beginning months to years after the injury. Recurrent meningitis is a serious complication, making accurate identification of the leaking fluid very important. In this regard, protein and glucose measurements are too nonspecific to be of value. Transferrin, an ironbinding glycoprotein with a molecular mass of about 77 kDa, is synthesized primarily in the liver. However, two transferrin isoforms are present in the CSF; the major isoform (β1transferrin) is present in all body fluids. The second isoform (β2transferrin), present only in the central nervous system, is produced in the central nervous system by the catalytic conversion of β1transferrin by neuraminidase. Immunofixation electrophoresis readily identifies both isoforms. Protein electrophoresis with transferrin immunofixation is a noninva sive, rapid, and inexpensive test of high sensitivity and specificity that requires as little as 0.1 mL of fluid (Ryall, 1992; Normansell, 1994). Several reports have demonstrated the value of this technique in the diagnosis of CSF otorrhea and rhinorrhea—conditions in which both isoforms are readily identified (Irjala, 1979; Rouah, 1987; Zaret, 1992). Others have stressed the importance of β2transferrin identification in both CSF and inner ear perilymphatic leakage, as well as possible sources of error due to the presence of a transferrin allelic variant (Skedros, 1993a,b; Sloman, 1993). Methemoglobin and Bilirubin. Although most cases of subarachnoid and intracerebral hemorrhage are readily identified by computed Myelin Basic Protein. Myelin basic protein (MBP), a component of the myelin nerve sheath, is released during demyelination as a result of various neurologic disorders, especially MS. Thus, MBP has been shown to posi tively correlate with CSF leukocyte count, intrathecal IgG synthesis, and the CSF/serum albumin concentration quotient (Sellebjerg, 1998). These results support the use of MBP in CSF as a surrogate disease marker during acute MS exacerbations. Others have found that analysis of antibody against MBP in patients with a clinically isolated syndrome is a rapid and precise method for predicting early conversion to clinically definite MS (Berger, 2003). However, increased CSF levels have also been reported in GuillainBarré syndrome, lupus erythematosus, subacute sclerosing panencephalitis, and various brain tumors, and following CNS irradiation and chemotherapy (Brooks, 1989; Mahoney, 1984). Measurement of CSF levels has also been proposed as a prognostic marker in patients with serious head injury (Noseworthy, 1985). α2-Macroglobulin. Except for a small amount transported across the BBB in pinocytic vesicles, α2macroglobulin (A2M) is normally excluded from the CSF because of its large size. The number of these vesicles is increased in certain polyneuropathies, resulting in an increased CSF A2M level. Significant elevation reflects subdural hemorrhage or breakdown of the BBB, as occurs in bacterial meningitis. A2M measurement alone, or in relationship to albumin and IgG, may assist in the evaluation of neurologic disorders and increased CSF protein, and in the rapid differentiation between bacterial and aseptic meningitis (Meucci, 1993; Kanoh, 1997). β2-Microglobulin. This protein is part of the human leukocyte antigen class I molecule on the surfaces of all nucleated cells. CSF levels above 1.8 mg/L are associated with leptomeningeal leukemia and lymphoma but are not highly specific (Weller, 1992), in that they have a maximal positive predictive value of 78% in cases with a positive cytology (Jeffrey, 1990). β2microglobulin (B2M) was also recently shown to be a marker for neuroBehçet’s syndrome (Kawai, 2000). Moreover, viral infections, including human immunodeficiency virus (HIV)1, other inflammatory conditions, and various malignancies have also been associated with ele vated levels. However, the measurement of B2M remains primarily investigational. C-Reactive Protein. Early studies indicated that CSF Creactive protein (CRP) is useful in differentiating viral (aseptic) meningitis from bacterial meningitis (Corral, 1981; Abramson, 1985; Stearman, 1994). Others have reported that CSF CRP is a more useful screening test for viral versus bacterial meningitis, especially in children (Sormunen, 1999). A meta analysis of CRP studies since 1980 suggested that a normal CSF or serum CRP has a high probability of ruling out bacterial meningitis (i.e., negative predictive value about 97%) (Gerdes, 1998). Moreover, a recent study found not only that CSF CRP levels were increased in bacterial meningitis, but that these levels were significantly higher in patients with gram negative bacterial meningitis than in those with grampositive bacterial meningitis (Rajs, 2002). Fibronectin. This large glycoprotein (molecular mass about 420 kDa) is normally present in essentially all tissues and body fluids. Its primary func tion is its role in cell adhesion and phagocytosis (Ruoslahti, 1981). Thus, cell adhesion allows leukocytes to adhere to and pass through the vascular endothelia and migrate to the inflammatory site. TABLE 29-12 CSF Proteins and Central Nervous System Diseases Protein Major diseases/disorders α2-Macroglobulin Subdural hemorrhage, bacterial meningitis β-Amyloid and τ proteins Alzheimer’s disease β2-Microglobulin Leukemia/lymphoma, Behçet’s syndrome C-reactive protein Bacterial and viral meningitis Fibronectin Lymphoblastic leukemia, AIDS, meningitis Methemoglobin Mild subarachnoid/subdural hemorrhage Myelin basic protein Multiple sclerosis, tumors, others Protein 14-3-3Creutzfeldt-Jakob disease Transferrin CSF leakage (otorrhea, rhinorrhea) AIDS, Acquired immunodeficiency syndrome; CSF, cerebrospinal fluid. PA R T 3 489 Adenosine Deaminase Adenosine deaminase (ADA) catalyzes the irreversible hydrolytic deamina tion of adenosine to produce inosine. Because ADA is particularly abun dant in T lymphocytes, which are increased in tuberculosis, its measurement has been recommended in the diagnosis of pleural, peritoneal, and men ingeal tuberculosis. Higher ADA levels are present in tuberculous infec tions than in viral, bacterial, and malignant diseases (Blake, 1982; Mann, 1982, Choi, 2002), although the degree of increase indicative of tubercu lous infection varies depending on the specific assay used. ADA appears to have limited utility in HIVassociated neurologic disorders (Corral, 2004). Creatine Kinase Brain tissue is rich in creatine kinase (CK) because it participates in main taining an adequate supply of adenosine triphosphate. Increased CSF CK activity has been reported in numerous CNS disorders, including hydro cephalus, cerebral infarction, various primary brain tumors, and subarach noid hemorrhage, among others (Savory, 1979). In patients with head trauma, CSF CK levels correlate directly with the severity of the concus sion (Florez, 1976). CSF CKMM and CKMB are not normally present; when identified, they are due to blood contamination (CKMM) and an equilibrium between CKBB and CKMM to produce CKMB. Because the CKBB isoenzyme accounts for about 90% of brain CK activity and mitochondrial CK (CKmt) the other 10%, CK isoenzyme measurements are more spe cific than total CK for CNS disorders (Chandler, 1984). CSF CKBB is increased about 6 hours following an ischemic or anoxic insult. Global brain ischemia following respiratory or cardiac arrest results in diffuse cerebral injury with peak CKBB levels in about 48 hours (Chandler, 1986). Here, CSF CKBB activity less than 5 U/L (upper normal level) indicates minimal neurologic damage; 5–20 U/L indicates mild to moderate CNS injury; and levels between 21 and 50 U/L are commonly correlated with death. Death occurs in essentially all patients with levels above 50 U/L. Increased CSF CKBB levels are also associated with the outcome fol lowing a subarachnoid hemorrhage (Coplin, 1999). Here, a CKBB level greater than 40 U/L increased the chance of an unfavorable early or late outcome to 100%. The death rate was 13% when the CSF CKBB level was less than 40 U/L. Lactate Dehydrogenase Lactate dehydrogenase (LD) activity is high in brain tissue, with a pre dominance of the electrophoretically fastmoving isoenzyme fractions LD1 and LD2. Total LD activity of 40 U/L is a reasonable upper limit of normal for adults and 70 U/L for neonates (Donald, 1986; Engelke, 1986). LD is useful in differentiating a traumatic tap from intracranial hemorrhage because a current traumatic tap with intact RBCs does not significantly elevate the LD level (Engelke, 1986). Sensitivity and specificity are about 70%–85% depending on the cutoff value. As with lactate, LD activity is significantly higher in bacterial meningitis than in aseptic meningitis (Donald, 1986; Engelke, 1986). Using a cutoff of 40 U/L, the sensitivity is about 86% and the specificity about 93%. Total CSF LD levels are also increased in patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid hemorrhage (Kjeldsberg, 1993). CSF LD isoenzymes have been shown to add considerable specificity in the evaluation of various metastatic brain tumors (Fleisher, 1981). Thus, the LD5/total LD ratio is increased (i.e., above 10%–15%) in patients with leptomeningeal metastases from carci noma of the breast and lung and malignant melanoma. Isoenzyme analysis also shows a distinct pattern in young children with infantile spasms (Nussinovitch, 2003a) and febrile convulsions (Nussinovitch, 2003b). Compared with controls, both disorders are characterized by decreased LD1, increased LD2 and LD3, and no changes in LD4 and LD5. CT is of limited value in estimating recovery potential and neurologic outcome during the early stages of ischemic brain injury. However, com pared with controls (mean LD, 11.2 U/L), patients with an early stroke had a mean level of 40.9 U/L, and those with a transient ischemic attack (TIA) had a mean value of 11.8 U/L (Lampl, 1990). Moreover, in patients with hypoxic brain injury, an increased LD level 72 hours following resus citation indicates a poor prognosis (Karkela, 1992). Lysozyme Lysozyme (muramidase) catalyzes the depolymerization of mucopolysac charides. Because the enzyme is particularly rich in neutrophil and mac rophage lysosomes, its activity is very low in normal CSF. However, CSF lysozyme activity is significantly increased in patients with both bacterial tomography (CT), patients with mild subarachnoid hemorrhage, small subdural or cerebral hematomas, and blood seepage from an aneurysm or neoplasm and from small cerebral infarcts often are not identified by this technique. In these cases, CSF spectrophotometric analysis has been shown to detect methemoglobin in colorless CSF (<0.3 µmol/L) (TrbojevicCepe, 1992). However, an increase in CSF bilirubin is now recognized as the key finding supporting the diagnosis of subarachnoid hemorrhage (UK National External Quality Assessment Scheme for Immunochemistry Working Group, 2003). Thus, a single net bilirubin absorbance cutoff point of >0.007 absorbance units is recommended in the decision tree for interpretation and reporting of results. Glucose Derived from blood glucose, fasting CSF glucose levels are normally 50–80 mg/dL (2.8–4.4 mmol/L)—about 60% of plasma values. Results should be compared with plasma levels, ideally following a 4hour fast, for adequate clinical interpretation. The normal CSF/plasma glucose ratio varies from 0.3–0.9, with fluctuations in blood levels caused by the lag in CSF glucose equilibration time. CSF values below 40 mg/dL (2.2 mmol/L) or ratios below 0.3 are considered to be abnormal. Hypoglycorrhachia is a characteristic finding of bacterial, tuberculous, and fungal meningitis. However, sensitivity can be as low as 55% for bacterial meningitis (Hayward, 1987), so a normal level does not exclude these conditions. Some cases of viral meningoen cephalitis also have low glucose levels, but generally not to the degree seen in bacterial meningitis. Meningeal involvement by a malignant tumor, sarcoidosis, cysticercosis, trichinosis, ameba (Naegleria), acute syphilitic meningitis, intrathecal administration of radioiodinated serum albumin, subarachnoid hemorrhage, symptomatic hypoglycemia, and rheumatoid meningitis may also produce low CSF glucose levels (Fishman, 1992). Decreased CSF glucose results from increased anaerobic glycolysis in brain tissue and leukocytes and from impaired transport into the CSF. Bacteria are usually present in insufficient quantities to be a major con tributor. CSF glucose levels normalize before protein levels and cell counts during recovery from meningitis, making it a useful parameter in assessing response to treatment. Increased CSF glucose is of no clinical significance, reflecting increased blood glucose levels within 2 hours of lumbar puncture. A trau matic tap may also cause a spurious increase in CSF glucose. Lactate CSF and blood lactate levels are largely independent of each other. The reference interval for older children and adults is 9.0–26 mg/dL (1.0–2.9 mmol/L) (Knight, 1981). Newborns have higher levels, ranging from about 10–60 mg/dL (1.1–6.7 mmol/L) for the first 2 days, and from 10–40 mg/dL (1.1–4.4 mmol/L) for days 3 to 10 (McGuinness, 1983). Elevated CSF lactate levels reflect CNS anaerobic metabolism due to tissue hypoxia.Lactate measurement has been used as an adjunctive test in differentiat ing viral meningitis from bacterial, mycoplasma, fungal, and tuberculous meningitis, in which routine parameters yield equivocal results. In patients with viral meningitis, lactate levels are usually below 25 mg/dL (2.8 mmol/L) and are almost always less than 35 mg/dL (3.9 mmol/L), whereas bacterial meningitis typically has levels above 35 mg/dL (Bailey, 1990; Cameron, 1993). Using 30–36 mg/dL as the cutoff value for bacterial meningitis, the sensitivity and specificity are about 80% and 90%, respectively. Viral men ingitis, partially treated bacterial meningitis, and tuberculous meningitis often have intermediate lactate levels that overlap each other, limiting the use of lactate measurements in this differential diagnosis. Persistently elevated ventricular CSF lactate levels are associated with a poor prognosis in patients with severe head injury (DeSalles, 1986). F2-Isoprostanes F2isoprostanes are increased in diseased regions of the brain in patients with AD (Pratico, 1998). Compared with agematched controls, CSF F2isoprostanes are also elevated in patients with probable AD (Montine, 1999). Therefore, in conjunction with CSF τ and βamyloid protein, the measurement of CSF F2isoprostanes appears to enhance the accuracy of the laboratory diagnosis of AD (Montine, 2001). Enzymes A wide variety of enzymes derived from brain tissue, blood, or cellular elements have been described in the CSF. Although CSF enzyme assays are not commonly used in the diagnosis of CNS diseases, there are diseases/disorders in which they may prove useful. 2 9 C ER EB RO SP IN A L, S YN O VI A L, S ER O U S BO D Y FL U ID S, A N D A LT ER N AT IV E SP EC IM EN S 490 the value of most of these tests in routine clinical practice has not been established. Carcinoembryonic Antigen Carcinoembryonic antigen (CEA) is an oncofetal protein produced by a variety of carcinomas. An early study found increased CEA levels in 44% of patients with metastatic brain tumors (Suzuki, 1980). Others reported that CSF levels of CEA have a sensitivity of only about 31%, although the specificity is about 90% for detecting metastatic carcinoma of the leptomeninges (Klee, 1986; Twijnstra, 1986). Recently, CSF levels of CEA in patients with benign, primary malignant, and metastatic brain tumors were 0.31 ng/mL, 0.92 ng/mL, and 6.3 ng/mL, respectively (Batabyal, 2003). Other oncofetal proteins include human chorionic gonadotropin (hCG), produced by choriocarcinoma and malignant germ cell tumors with a trophoblastic component, and α-fetoprotein, a glycoprotein pro duced by yolk sac elements of germ cell tumors. Results of a recent study revealed that both βhCG and αfetoprotein may be useful in the diagnosis and monitoring of the response to therapy in patients with CNS germ cell tumors (Seregni, 2002). Elevation of CSF ferritin is a sensitive indicator of CNS malignancy but has very low specificity because it is also increased in patients with inflammatory neurologic diseases (ZandmanGoddard, 1986). MICROBIOLOGICAL EXAMINATION A thorough and prompt examination of cerebrospinal fluid is essential for the diagnosis of CNS infection because an inaccurate or delayed report may result in significant mortality or morbidity. Although changes in opening pressure, total cell and differential counts, total protein, and glucose suggest an infectious origin (Table 2913), Gram stain and culture are critical for a definitive diagnosis. Bacterial Meningitis The most common agents of bacterial meningitis are group B streptococ cus (neonates), Neisseria meningitidis (3 months and older) (Fig. 299), Streptococcus pneumoniae (3 months and older), Escherichia coli and other gramnegative bacilli (newborn–1 month), Haemophilus influenzae (3 months–18 years), and Listeria monocytogenes (neonates, elderly, alcoholics, and immunosuppressed) (Graves, 1989; Wenger, 1990). H. influenzae, once and tuberculous meningitis. Thus, discriminant analysis demonstrated that 97% of patients with bacterial meningitis had increased lysozyme levels (Ribeiro, 1992). Others found that patients with tuberculous meningitis had significantly higher CSF lysozyme levels than those with bacterial meningitis, partially treated bacterial meningitis, and controls (Mishra, 2003). The diagnostic sensitivity and specificity for tuberculous meningitis were 93.7% and 84.1%, respectively. Increased levels are also present in cerebral atrophy, various CNS tumors, multiple sclerosis, intracranial hemorrhage, and epilepsy (Kjeldsberg, 1993). Ammonia, Amines, and Amino Acids CSF ammonia levels vary from 30%–50% of blood values. Elevated levels are generally proportional to the degree of existing hepatic encephalopathy but are difficult to quantify. Moreover, because hepatic encephalopathy generally correlates with blood ammonia levels, the measurement of CSF ammonia has little, if any, clinical value. However, cerebral glutamine, synthesized from ammonia and glutamic acid, serves as the means for CNS ammonia removal. Thus, CSF glutamine levels reflect the concentration of brain ammonia. Glutamine reference intervals are method dependent; the upper reference level is about 20 mg/dL. Values over 35 mg/dL are usually associated with hepatic encephalopathy (Fishman, 1992). Elevated CSF glutamine levels have also been reported in patients with encepha lopathy secondary to hypercapnia and sepsis (Mizock, 1989). A major etiologic theory of schizophrenia involves dopamine. The cornerstone for this theory is the fact that neuroleptic drugs that block dopamine receptors are effective in the treatment of this disorder. Thus, it has been reported that CSF levels of homovanillic acid (HVA), a metabo lite of the biogenic amines, are related to the severity of schizophrenic psychosis (Maas, 1997). However, HVA concentration varied as a function of psychosis rather than being related to the diagnosis of schizophrenia per se. Others reported decreased CSF levels of 5hydroxyindoleacetic acid, a metabolite of serotonin, in schizophrenic patients with suicidal behavior (Cooper, 1992). This report adds support for a possible relation ship between suicide and CNS serotonin metabolism. Although free CSF amino acids are relatively high in infants younger than 30 days of age, their concentration is further increased in those with febrile convulsions and bacterial meningitis. γAminobutyric acid (GABA), a major inhibitory brain transmitter, is significantly decreased in basal ganglia neurons, and is very low or undetectable in the CSF of patients with Alzheimer’s disease and Huntington’s disease (Achar, 1976; Dubowitz, 1992). In addition, CSF GABA was detected in all patients with migraine attacks, but not in those with tension headaches or in the control group without headaches (Welch, 1975). Conversely, infants with startle disease, a rare inherited autosomal dominant disorder characterized by seizures or the socalled stiff baby syndrome, have significantly decreased CSF GABA levels (Dubowitz, 1992; Berthier, 1994). Electrolytes and Acid-Base Balance There are no clinically useful indications for the measurement of CSF sodium, potassium, chloride, calcium, or magnesium. Measurements of CSF pH, partial pressure of carbon dioxide (pCO2), and bicarbonate are also not practical for patient care (Fishman, 1992). Tumor Markers Numerous studies have shown that various tumor markers are increased in the CSF of patients with both primary and metastatic tumors. However, TABLE 29-13 Typical Lumbar CSF Findings in Meningitis Test Bacterial Viral Fungal Tuberculous Opening pressure Elevated Usually normal Variable Variable
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