CEREBROSPINAL FLUID CHAPTER 9

Learning Objectives Upon completing this chapter, the reader will be able to State the three major functions of cerebrospinal fluid (CSF). Distribute CSF specimen tubes numbered 1, 2, 3, and possibly 4 to their appropriate laboratory sections and correctly preserve them.   Describe the appearance of normal CSF and the causes of abnormally appearing CSF. Define xanthochromia and state its significance. Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap.

Learning Objectives (cont’d) Calculate CSF total, white blood cell (WBC), and red blood cell (RBC) counts when given the number of cells seen, amount of specimen dilution, and the squares counted in the Neubauer chamber. Describe the leukocyte content of the CSF in bacterial, viral, tubercular, and fungal meningitis. Describe and state the significance of macrophages in the CSF. Differentiate between the appearance of normal choroidal cells and malignant cells.

Learning Objectives (cont’d) State the reference values for CSF total protein and name three pathologic conditions that produce an elevated CSF protein. Determine whether increased CSF albumin or immunoglobulin is the result of damage to the blood-brain barrier or central nervous system production. Discuss the significance of CSF electrophoresis, immunophoresis, and isoelectric focusing findings in multiple sclerosis and the identification of CSF. State the reference values for CSF glucose and name the possible pathologic significance of a decreased CSF glucose.

Learning Objectives (cont’d) Discuss the diagnostic value of CSF lactate and glutamine determinations. Name the microorganism associated with a positive India ink preparation. Discuss the diagnostic value of the bacterial and cryptococcal antigen tests. Determine whether a suspected case of meningitis is of bacterial, viral, fungal, or tubercular origin, when presented with pertinent laboratory data. Describe the role of the Venereal Disease Research Laboratories test and fluorescent treponemal antibody-absorption test for syphilis in CSF testing.

Formation and Physiology Brain and spinal cord lined by meninges Three layers of meninges Dura mater: outer Arachnoid: middle Pia mater: surfaces of brain and spinal cord Cerebrospinal fluid (CSF) produced in choroid plexuses of the four ventricles 20 mL/min produced in adults Volume adults 90 to 150 mL, neonates 10 to 60 mL

The Meninges

Formation and Physiology CSF flows through subarachnoid space between arachnoid and pia mater Reabsorbed into blood in arachnoid granulations/villae (one-way valves) Formation by selective filtration Hydrostatic pressure and active transport Not an ultrafiltrate Very tight-fitting endothelial cells, prevent filtration of large molecules—called the blood-brain barrier

Flow of Spinal Fluid Through the Brain

Formation and Physiology Blood-brain barrier Essential to protect brain Chemicals and harmful substances do not pass Antibodies and medications are excluded CSF composition differs from plasma Meningitis, multiple sclerosis disrupt membrane Test for substances that pass through: cells, protein, bacteria, immunoglobulins

Specimen Collection & Handling CSF collected between third to fifth lumbar vertebrae Three sterile tubes in this order Chemistry/serology Microbiology (avoid skin contamination) Hematology (avoid cells from tap) Save leftover fluid/fourth tube for additional tests Volume removed based on patient volume and opening pressure

CSF Specimen Collection Tubes

Specimen Collection & Handling Usually STAT requests Handle carefully to avoid repeat taps Preservation Hematology Refrigerate Microbiology Room temperature Chemistry/serology Frozen

Appearance Crystal clear, cloudy/turbid, milky, xanthochromic, hemolyzed/bloody Cloudy = infection; milky = lipid or protein Xanthochromic Pink, orange, yellow RBC degradation products Also jaundice, ↑ ↑ protein, carotene Pathologic = cerebral hemorrhage

Tubes of CSF

Traumatic Tap Blood vessel punctured during tap Differentiate from cerebral hemorrhage Uneven blood distribution in tubes with traumatic tap Erythrophagocytosis, hemosiderin granules Hemorrhage = even distribution in all tubes Traumatic tap = decreasing tubes 1 through 3

Traumatic Tap (cont’d) Clot formation Clots present = traumatic tap (plasma) Hemorrhage does not have enough fibrinogen Other causes of clot formation Nonbloody CSF = damage to blood-brain barrier TB meningitis: web-like pellicle after refrigeration Xanthochromia Not present in a recent traumatic tap Indicates older hemorrhage D-dimer test for hemorrhage

Cell Count White blood cell (WBC) and total cell count Red blood cell (RBC) count seldom done Granulocytes lyse within 1 hour; STAT Normal adult 0 to 5 WBCs/µL Neonates up to 30 mononuclear cells/µL Neubauer counting chamber Automated cell counters can be used Body fluid specific automation is available

Neubauer Counting Chamber

Calculating CSF Cell Count Standard Neubauer calculation formula (cells/µL) Number of cells counted × dilution Number of cells counted × volume of 1 square = cells/µL Can be used for diluted and undiluted samples

Calculating CSF Cell Count (cont’d) Example # cells counted × dilution × 1 μL = cells/µL 1 μL (0.1 × 10) (volume counted) </exeq)

Calculating CSF Cell Count (cont’d) Total cell count Clear specimens count undiluted unless overlapping cells are seen Load with transfer pipette Dilute with normal saline if necessary WBC count Dilute with 3% acetic acid; methylene blue helps to see cells; undiluted rinse transfer pipette with acetic acid, gently rotate pipette

Quality Control Commercial cell controls are available Check diluents for contamination biweekly Monthly check on cytocentrifuge speed and timing Soak nondisposable chambers in bactericidal solution for 15 minutes; rinse; clean with isopropyl alcohol

Differential Count Valuable diagnostic aid Stained smear only Must concentrate specimen Sedimentation, filtration, centrifugation, and cytocentrifugation 100 cells should be counted, classified, and reported in terms of percentage

Cytocentrifugation Cytocentrifuge Forces cells onto a slide in a monolayer Filter paper absorbs moisture 0.1 mL CSF to 1 drop 30% albumin Albumin increases the cell yield and decreases the cellular distortion Positively charged slides to attract cells Daily control of 0.2 mL saline and two drops of albumin stained for bacterial contamination

Cellular Constituents Normal lymphocytes and monocytes Adults: normal lymphocytes:monocytes = 70:30 Children’s ratio is reversed Occasional neutrophils are normal Pleocytosis: increased amounts of normal cells Pleocytosis of normal cells is valuable in determining the cause of meningitis Neutrophils = bacterial Lymphocytes = viral, tubercular, fungal, parasitic

Prominent Cells Seen in CSF Type of Cell Major Clinical Significance Microscopic Findings Lymphocytes Normal All stages of development may be found   Viral, tubercular, and fungal meningitis Multiple sclerosis Neutrophils Bacterial meningitis Granules may be less prominent than in blood Early cases of viral, tubercular, and fungal meningitis Cells disintegrate rapidly Cerebral hemorrhage Monocytes Found mixed with lymphocytes Table 9-3

Prominent Cells Seen in CSF (cont’d) Type of Cell Major Clinical Significance Microscopic Findings Macrophages RBCs in spinal fluid from hemorrhage Contrast media May contain phagocytized RBCs appearing as empty vacuoles or ghost cells, hemosiderin granules, and hematoidin crystals Blast forms Acute leukemia Lymphoblasts, myeloblasts, or monoblasts Lymphoma cells Disseminated lymphomas Resemble lymphocytes with cleft nuclei Plasma cells Multiple sclerosis Traditional and classic forms seen   Lymphocyte reactions Reactive lymphs Ependymal, choroidal, and spindle-shaped cells Diagnostic procedures Seen in clusters with distinct nuclei and distinct cell walls Malignant cells Metastatic carcinomas Primary central nervous system carcinoma Seen in clusters with fusing of cell borders and nuclei Table 9-3

Neutrophils Primarily in bacterial meningitis Often contain phagocytized bacteria Increased early viral, fungal, tubercular, parasitic Vacuoles may be present

Cellular Constituents

Nucleated RBCs (NRBC) Seen with bone marrow contamination from tap in 1% of specimens Neutrophils with pyknotic nucleii may resemble NRBCs Capillary structures and epithelial cells from traumatic taps

Cellular Constituents

Lymphocytes and Monocytes Lymphs and monos in viral, fungal, tubercular Reactive lymphocytes with viral Multiple sclerosis has 50 or fewer lymphocytes/μL, both normal and reactive Seen in HIV and AIDS

Eosinophils Parasitic and fungal infections (primarily Coccidioides immitis) Medications and shunts into the central nervous system

Macrophages Purpose is to remove cellular and other debris May be seen after repeated taps Hemorrhage: enter CSF within 2 hours to phagocytize RBCs RBCs degraded to hematoidin crystals representing unconjugated bilirubin

Macrophages (cont’d)

Nonclinically Significant Cells Seen after diagnostic procedures Choroidal cells Epithelial lining of choroid plexus, singular and in clumps, uniform cells Ependymal cells lining ventricles and neural canal; less defined cell membranes in clumps Spindle cells lining arachnoid seen in clumps

Malignant Cells of Hematologic Origin Leukemias Lymphoblasts, monoblasts, and myeloblasts Nucleoli may be more prominent than in blood Lymphomas Dissemination from lymph organs Cleaved nucleii and prominent nucleoli

Malignant Cells of Hematologic Origin (cont'd)

Malignant Cells of Hematologic Origin (cont'd)

Malignant Cells of Nonhematologic Origin Metastatic carcinoma cells Lung, breast, renal, gastrointestinal, and melanoma Fused cell walls, nuclear irregularities, and hyperchromatic nucleoli Primary tumors Astrocytomas, retinoblastomas, medulloblastomas

Chemistry Tests CSF formed by plasma filtration Normal values differ from plasma because of selectivity of blood-brain barrier Abnormal values result Alterations in the permeability of the blood-brain barrier Increased production or metabolism by the neural cells in response to a pathologic condition

Cerebrospinal Protein Total protein is the most common test Normal 15 to 45 mg/dL (mg, not grams) Method dependent Increased in infants and persons >40 Albumin is predominant, prealbumin is second Alpha globulins-haptoglobin and ceruloplasmin Transferrin is major beta globulin TAU, carbohydrate-deficient transferrin seen in CSF, not in blood; used to identify CSF IgG major gamma globulin

Clinical Significance Decreased protein levels = fluid leakage Elevated levels = damage to blood-brain barrier, IG production within CNS, decreased clearance, degeneration of neural tissue Meningitis/hemorrhage most common causes of increased damage to blood-brain barrier Find abnormal results on clear fluid with low cell counts from neurologic disorders

Clinical Significance of Elevated Protein Values Elevated Results • Meningitis • Hemorrhage • Primary CNS tumors • Multiple sclerosis • Guillain-Barré syndrome • Neurosyphilis • Polyneuritis • Myxedema • Cushing disease • Connective tissue disease • Diabetes • Uremia Decreased results • CSF leakage/trauma • Recent puncture • Rapid CSF production • Water intoxication * Reference values for protein are usually 15 to 45 mg/dL, but are method dependent, and higher values are found in infants and people older than 40 years. Table 9-4

Methodology Turbidity Automated instrumentation available Nephelometry

Protein Fractions Comparisons between serum and CSF levels of albumin and IgG CSF/serum albumin index Blood-brain barrier integrity CSF IgG index Comparison of the CSF/serum albumin index with the CSF/serum IgG index Values for CSF albumin and globulin adapted for automated instruments

Protein Fractions (cont’d) IgG index = CSF IgG (mg/dL)/serum IgG (g/dL) CSF albumin (mg/dL)/serum albumin (g/dL) Values >0.70 indicate IgG production within the CNS

Electrophoresis and Immunophoretic Techniques Detection of oligoclonal bands Represent inflammation within the CNS Located in the gamma region of the protein electrophoresis Simultaneous serum electrophoresis must be performed

Electrophoresis and Immunophoretic Techniques (cont'd) Multiple sclerosis (MS) = no bands in serum, bands in CSF Leukemia, lymphoma, viral, HIV: bands in both Primary purpose for MS, compare also to IgG index Encephalitis, neurosyphilis, Guillain-Barré, and neoplasms may give same pattern Consider symptoms

Myelin Basic Protein Presence in CSF indicates demyelination of myelin sheath around axons of neurons Monitors the course of multiple sclerosis Effectiveness of treatment Immunoassay procedures available

CSF Glucose Selective transport across blood-brain barrier Approximately 60% to 70% plasma glucose Plasma = 100 mg/dL; CSF = 65 mg/dL Draw blood 2 hours before spinal tap Significance Values that are decreased relative to plasma values Elevated CSF glucose values are always a result of plasma elevations

CSF Glucose Clinical Significance Markedly decreased with increased neutrophils in bacterial meningitis Tubercular meningitis decreased with increased lymphocytes Viral/fungal meningitis, normal glucose and increased lymphocytes

CSF Lactate Diagnosis and management of meningitis Bacterial, TB and fungal levels >25 mg/dL Viral <25 mg/dL More reliable than CSF glucose Levels remain elevated until treatment becomes effective, then fall rapidly Can result from any condition that decreases oxygen flow to the tissues Monitor severe head injuries

CSF Glutamine Produced by brain cells from ammonia and α-ketoglutarate to remove toxic ammonia Indirect test for the presence of excess ammonia in the CSF Normal: 8 to 18 mg/dL Elevated in liver disease Elevated in children with Reye syndrome Disturbance of consciousness when glutamine levels are more than 35 mg/dL More reliable than direct CSF ammonia

Microbiology Tests Gram stain and cultures must be performed on sediment from centrifuged CSF; cytocentrifuge helps Gram stains Blood cultures also must be drawn Difficult to interpret Gram stains, few organisms and often debris Organisms: S. pneumocystis<<AU: Should this be Streptococcus pneumocystis or pneumoniae?>>, Haemophilus influenza, Escherichia coli, Neisseria meningitidis, Listeria monocytogenes, S. agalactiae

Microbiology Tests (cont’d) Cultures also plated on chocolate agar TB smears very important because of growth time delay Latex agglutination tests are available for Group B streptococcus, Haemophilus influenzae, S. pneumocystis<<AU: Should this be Streptococcus pneumocystis or pneumoniae?>>, Neisseria meningitidis, and Escherichia coli Gram stain is the best for detection Compare with hematology and chemistry results

Parasites Naegleria fowleri Found in ponds, lakes, and some pools Enters nasal passages and migrates to the brain Motile amoeba seen in wet preps, nonmotile in cytospin preps Elongated with tapered posterior

Serologic Testing Primary test is for neurosyphilis, third stage Performed less now that people have been treated early with penicillin Detect active cases within the CNS The Venereal Disease Research Laboratories (VDRL) produces the recommended test for specificity Should be accompanied by a positive serum Fluorescent Treponemal Antibody Absorption (FTA-ABS) <<AU: defined FTA-ABS: ok?>>