ANALYSIS OF URINE AND OTHER BODY FLUIDS CEREBROSPINAL FLUID ANALYSIS OUTLINE • Introduction • Cerebrospinal Fluid o Appearance o Intracranial Hemorrhage vs. Traumatic Tap Collection o Cell Count o Differential Count o Chemistry Tests o Laboratory Results for Differential Diagnosis of Meningitis o Microbiology Tests o Serologic Tests INTRODUCTION • The cerebrospinal fluid (CSF) provides protection and support for the brain and tissues of the central nervous system (CNS). It also provides nutrients to the tissues. Analysis of the CSF for physical, chemical, and microscopic properties provides useful information regarding the status of the CNS. CEREBROSPINAL FLUID FUNCTION • CSF is the third major fluid of the body. • It provides a physiologic system to supply nutrients to the nervous tissue. • It removes metabolic wastes. • It produces a mechanical barrier to cushion the brain and spinal cord against trauma. FORMATION AND PHYSIOLOGY • The brain and the spinal cord are lined by the meninges consisting of three layers: the dura mater, arachnoid mater and pia mater. • CSF flows through the subarachnoid space • Approximately 20 mL of fluid is produced every hour in the choroid plexuses. • The fluid is then reabsorbed in the arachnoid villi to maintain a total volume of 140 – 170 mL in adults and 10 to 60 mL in nonates. • Production of CSF is by filtration under hyrostatic pressure across the choroidal epithelial cells. • Blood-Brain Barrier is use to represent the control and filtration of blood components to the CSF and then to the brain. SPECIMEN HANDLING AND COLLECTION • CSF is routinely collected by lumbar puncture between the 3rd, 4th or 5th lumbar vertebra. • Specimens are usually collected in three sterile tubes, labeled 1, 2, and 3. o Tube 1 is used for chemical and serological tests, (glucose, LDH, antibodies, etc.) o Tube 2 is for microbiology section, o Tube 3 is for cell count because it is the least likely to contain cells introduced by the spinal tap collection. • Excess fluid should not be discarded until there is no further use for test. • Ideally, tests are performed on a STAT basis. • Mechanical method of specimen preservation are as follows: o Tube 1 : Chemistry and Serology tubes are frozen. o Tube 2 : Microbiology tubes remain at room temperature. o Tube 3 : Hematology tubes are refrigerated. APPEARANCE • Normally a crystal clear liquid. • Major terminology used to describe CSF appearance are crystal clear, cloudy or turbid, milky, xanthochromic and hemolyzed or bloody. • A cloudy, turbid, or milky specimen may be due to increased protein or lipid concentration or due to infection with the cloudiness caused by the presence of WBC. • Xanthochromia is used to describe CSF supernatant that is pink, orange or yellow. o Most common cause of xanthochromia is the presence of RBC degradation products. o The color vary depending on the amount of time it has been present. ▪ pink – very slight amount of oxyhemoglobin ▪ orange – heavy hemolysis ▪ yellow – conversion of oxyhemoglobin to unconjugated bilirubin o Other causes of xanthochromia include elevated serum bilirubin, presence of the pigment carotene, markedly increased protein concentrations and melanoma pigment. o Xanthochromia due to immature liver function is commonly seen in infants, particularly in those who are premature. CLINICAL SIGNIFICANCE OF CSF APPEARANCE Appearance Cause Major Significance Crystal clear Normal Hazy, turbid, milky, cloudy WBCs Meningitis Microorganisms Meningitis Protein Disorders affecting blood – brain barrier Production of IgG within the CNS Oily Radiographic contrast media
Appearance Cause Major Significance Bloody RBCs Hemorrhage Traumatic tap Xanthochromic Hemoglobin Old hemorrhage Lysed cells from traumatic tap Bilirubin RBC degradation Elevated serum bilirubin level Carotene Increased serum levels Protein Disorders affecting blood – brain barrier Melanin Meningeal melanosarcoma Clotted Protein Disorders affecting blood – brain barrier Clotting factors Introduced by traumatic tap Pellicle Protein Disorders affecting blood – brain barrier Clotting factors Tubercular meningitis INTRACRANIAL HEMORRHAGE VS TRAUMATIC TAP COLLECTION Intracranial Hemorrhage Traumatic Tap Collection Distribution of Blood Even Uneven Color of Supernatant Xanthochromic Clear Clot Formation (-) negative (+) positive D-dimer test (+) positive (-) negative CELL COUNT METHODOLOGY • Normal adult CSF contains 0 to 5 WBCs/L. • WBCs are higher in children, and as many as 30 mononuclear cells/μL can be considered normal in newborns. • Improved Neubauer counting chamber is routinely used for CSF cell counts. • Standard Neubauer calculation formula Number of cells counted X dilution = cells/μL Number of squares counted X volume of 1 square o This formula can be used for both diluted and undiluted specimens. • Formula to correct for the volume counted: Number of cells counted X dilution X 1 μL = cells/μL 1 μL (0.1 x 10) (volume counted) TOTAL CELL COUNT • Sample Dilution Method Clarity Dilution Amount of Sample Amount of Diluent Slightly hazy 1:10 30 μL 270 μL Hazy 1:20 30 μL 570 μL Slightly cloudy 1:100 30 μL 2970 μL Slightly bloody 1:200 30 μL 5970 μL Cloudy Bloody 1:10 000 0.1 mL of a 1:100 dilution 9.9 mL • Dilutions for total cell counts are made with Normal Saline, mixed by inversion, and loaded into the hemocytometer w/ a Pasteur pipette. • Cells are counted in the four corner squares and the center square on both sides of the hemocytometer. WHITE BLOOD CELL COUNT • Lysis of RBCs must be obtained prior to performing the WBC count on either diluted or undiluted specimens. • Dilution is done by substituting 3% acetic acid to lyse RBCs. Addition of Methylene Blue will stain the WBCs for better identification. • Methodology 1. Place four drops of mixed specimen in a clean tube. 2. Rinse a Pasteur pipette with glacial acetic acid, and draw the four drops of CSF into the rinsed pipette. 3. Allow the pipette to sit for 1 minute. 4. Mix the solution in the pipette. 5. Discard the first drop and load the hemocytometer • Corrections for Contamination o Calculations are possible to correct for WBCs and protein artificially introduced as a result of a traumatic tap. o WBC (added) = WBC (blood) X RBC (CSF) RBC (blood) DIFFERENTIAL COUNT • Differential count are performed on stained smears. • Specimen should be concentrated prior to preparation of the smear. • Methods available for specimen concentration include sedimentation, filtration, centrifugation, and cytocentrifugaton. • Automated body fluid microscopy also is available on the IRIS Model 500.
CHEMISTRY TESTS CEREBROSPINAL FLUID PROTEIN • The CSF protein fractions similar to those found in serum. • Transferrin is the major beta globulin present. • A separate carbohydrate-deficient transferrin fraction, called “tau”, is seen in CSF and not in serum. • Normal concentration is 15 – 45 mg/dL. • Elevated values are frequently seen in patients with meningitis, hemorrhage and multiple sclerosis. • CSF gamma globulin is primarily IgG, with only a small amount of IgA. IgM, fibrinogen and beta lipoprotein are not found in normal CSF. • Methodology o Turbidimetric methods rely on the precipitation of protein by either sulfosalicylic acid (SSA) or trichloroacetic acid. o Trichloroacetic acid precipitates both albumin and globulin equally. Unless SSA is combined with sodium sulfate , albumin will contribute more to the turbidity than globulin. o Dye-binding techniques offer the advantages of smaller sample size and less interference from external sources. o This method uses the dye Coomassie brilliant blue because it will bind to a variety of proteins rather than just albumin. • Protein Fractions o Comparisons between serum and CSF levels of albumin and IgG must be made to accurately determine whether IgG is increased because it is being produced in the CNS or is elevated due to a defect in the blood-brain barrier. o Methods include the CSF/serum albumin index to evaluate the integrity of the blood-brain barrier and the CSF IgG index to measure IgG synthesis within the CNS. • Formula o CSF/serum albumin index = CSF albumin (mg/dL) Serum albumin (g/dL) ▪ An index value less than 9 represents an intact blood-brain barrier. The index increases relative to the degree of damage to the barrier. o IgG index = CSF IgG (mg/dL) / serum IgG (g/dL) CSF albumin (mg/dL) / serum albumin (g/dL) ▪ Greater than 0.77 are indicative of IgG production within the CNS. IgG index greater than 0.77 suggests multiple sclerosis. • Electrophoresis o The primary purpose for performing CSF protein electrophoresis is for detection of oligoclonal bonds representing inflammation within the CNS. o Disorders, including leukemia, lymphoma, and infections, may produce serum banding, which can appear in the CSF as a result of blood-brain barrier leakage or traumatic introduction of blood into the CSF specimen. o Other neurologic disorders including encephalitis, neurosyphilis, Guillain-Barré syndrome, and neoplastic disorders also produce oligoclonal banding that may not be present in the serum. CEREBROSPINAL FLUID GLUCOSE • Normal value is 60 – 70% of the plasma concentration (50 - 80 mg/dL). • Decreased Levels are seen in patients with bacterial, tubercular and fungal meningitis. • CSF glucose is analyzed using the same procedures employed for blood glucose. • Markedly decrease CSF glucose with an increase in WBC count and a large percentage of neutrophils is indicative of bacterial meningitis. • If WBCs are lymphocytes, tubercular meningitis is suspected
• If a normal CSF glucose value is found with an increased number of lymphocytes, diagnosis would favor viral meningitis. CEREBROSPINAL FLUID LACTATE • The determination of CSF lactate levels can be a valuable aid in the diagnosis and management of meningitis cases. • Levels >35mg/dL are seen in patients with bacterial meningitis. • Levels > 25 mg/dL are found in patients with tubercular and fungal meningitis. • Lower levels are seen in patients with viral meningitis. • Destruction of tissue within the CNS owing to oxygen deprivation (hypoxia) causes the production of increased CSF lactic acid levels. CEREBROSPINAL FLUID GLUTAMINE • Glutamine is produced in the CNS by the brain cells from ammonia and α-ketoglutarate. • Normal concentration is 8 – 18 mg/dL. • Levels >35 mg/dL are associated with some disturbance of consciousness. • Elevated levels are found in association with liver disorders that result in increased blood and CSF ammonia. • Approximately 75 % of children with Reye’s syndrome have elevated CSF glutamine levels. CEREBROSPINAL FLUID ENZYMES • Creatinine Kinase CK-BB Isoenzyme o Elevated levels in patients post cardiac arrest indicate a poor prognosis. • Measurement of the creatinine kinase isoenzyme CK-BB in CSF after resuscitation from cardiac arrest has been shown to reliably predict recovery when levels are less than 17 mg/mL. LABORATORY RESULTS FOR DIFFERENTIAL DIAGNOSIS OF MENINGITIS Bacterial Viral Tubercular Fungal Elevated WBC count Neutrophils present Elevated WBC count Lymphocytes present Elevated WBC count Lymphocytes & Monocytes present Elevated WBC count Lymphocytes & Monocytes present Marked protein elevation Marked protein elevation Moderate to Marked protein elevation Moderate to Marked protein elevation Markedly decreased Glucose level Normal Glucose level Decreased Glucose Level Normal to Decreased Glucose Level Lactate level >35 mg/dL Normal Lactate Level Lactate level >25 mg/dL Lactate level >25mg/dL Positive Limulus Lysate test result with gram-negative organisms Pellicle formation Positive India Ink with Cryptococcus neoformans Positive Gram stain and bacterial antigen tests Positive immunologic test for C. neoformans MICROBIOLOGY TESTS GRAM STAIN • The gram stain is routinely performed on CSF from all suspected cases of meningitis. • All smears and cultures should be performed on concentrated specimens because often only a few organisms are present at the onset of disease. • Organisms most frequently encountered: o Neisseria meninditidis - causative agent of meningococcemia o Neisseria gonorrheae - causative agent of gonorrhea • Acid Fast o Acid-fast of fluorescent antibody stains are not routinely performed on specimens, unless tubercular meningitis is suspected. • The India Ink preparation is performed to detect the presence of thickly encapsulated Cryptococcus neoformans. • Latex agglutination and enzyme-linked immunosorbent assay (ELISA) methods provide a rapid means for detecting and identifying microorganisms in CSF. • Latex agglutination tests to detect the presence of Cryptococcus neoformans antigen in serum and CSF provide a more sensitive method than India Ink preparation. SEROLOGIC TESTS • Serologic testing of the CSF is performed to detect the presence of neurosyphilis. • The Venereal Disease Research Laboratories (VDRL) is the recommended procedure to diagnose neurosyphilis even though it is not as sensitive as Fluorescent Treponemal Antibody-Absorption (FTAABS) test for syphilis. • If the FTA-ABS is used, care must be taken to prevent contamination with blood, because the FTAABS remains positive in the serum of treated cases of syphilis.
ADDITIONAL MATRIX FROM BOOK 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 Normal Found mixed with lymphocytes Viral, tubercular, and fungal meningitis Multiple sclerosis Macrophages RBCs in spinal fluid 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, & spindle-shaped cells Diagnostic procedures Seen in clusters with distinct nuclei and distinct cell walls Malignant cells Metastatic carcinomas Seen in clusters with fusing of cell borders and nuclei Primary central nervous system carcinoma •