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FORMATION AND PHYSIOLOGY CSF Supply nutrients to N.

N.S Remove metabolic waste Cushion the brain and spiral cord against trauma

*excess fluid should not be discarded but must be frozen until there is no further use *tests are performed in a STAT basis, if this is not possible, specimens are maintained by: HEMATOLOGY tubes refrigerated MICRO. tubes remain @ roomtemp CHEMISTRY & SEROLOGY frozen APPEARANCE normally crystal clear CSF examination occurs first at the BEDSIDE. TERMINOLOGIES to describe CSF - crystal clear - cloudy - turbid - milky - xanthochromic - hemolyzed/bloody cloudy ,turbid or milky specimen = caused by inc. protein or lipid concentration, may be indicative of infection (cloudiness caused by WBCs) use gloves,face shields, and fluid for centrifugation must be in capped tubes. TRAUMATIC COLLECTION (Tap) 3 VISUAL EXAMINATIONS: - use to determine whether the blood is the result of hemorrhage or a traumatic tap. 1. UNEVEN DISTRIBUTION OF BLOOD Cerebral Hemorrhage - blood will be evenly distributed throughout the 3 CSF specimen tubes. Traumatic Tap - have the heaviest concentration of blood in tube 1 with gradually diminishing amounts in tubes 2 and 3 - Streaks of blood are may be seen

MENINGES lining of brain and spinal cord 3 LAYERS: a. DURA MATER - outer layer, lines the skull and vertebral canal b. ARACHNOID - spider like, filamentous inner membrane c. PIA MATER - is a thin membrane lining the surface of the brain and spinal cord. CHOROID PLEXUSES OF THE 2 LUMBAR VENTRICLES & THE 3rd and 4th VENTRICLES - where CSF produced 20mL- is produce every hour SUBARACHNOID SPACE - where the csf flow s through & located between Arachnoid & Pia Mater 90-150mL adult 10-60mL neonate ARACHNOID GRANULATIONS VILLAE - reabsorption of the circulating fluid.

HYDROSTATIC PRESSURE & ACTIVER TRANSPORT - mechanisms in formation of CSF BLOOD BRAIN BARRIER - tight-fitting structure of the endothetical cell in the choroid plexus SPECIMEN COLLECTION & HANDLING Lumbar puncture - way of collection , in between the 3rd, 4th or 5th lumbar vertebrae elevated pressure withdraw fluid slowly COLLECTED IN THREE TUBES: Tube 1 chemical and serologic test, least affected by blood/bacteria Tube 2 microlab Tube 3 used for cell count Tube 4 (optional) for microbiology laboratory to provide better exclusion of skin contamination or for additional serologic tests. Supernatant Fluid that is left over may be used for additional chemical or serologic tests

2. CLOT FORMATION Traumatic tap - may form clots owing to the introduction of plasma fibrinogen into the specimen Intracranial hemorrhage - bloody CSF but does not contain enough fibrinogen to clot. Diseases in which damage in blood- brain barrier allows increased filtration of protein and coagulation factors also cause clot but doesnt produce a bloody fluid. Meningitis

Froin Syndrome Blockage of CSF circulation through the subarachnoid space 3. XANTHOCHROMIC SUPERNATANT RBCs must remain in the CSF for approximately 2hrs. before noticeable hemolysis begins. Result of blood that has been present longer than that introduced by traumatic tap Recent hemorrhage = clear supernatant Introduction of serum protein to traumatic tap could appear xanthochromic TEST FOR THE PRESENCE OF XANTHOCHROMA: Fluid is centrifuged in a microhematocrit tube & supernatant examined against white background Microscopic examination presence of ingested RBCs by macrophages (erythrophagocytosis) or hemosiderin granules (indicates intracranial hemorrhage) D-dimer test by latex agglutination immunoassay, presence of D-dimer (a fibrin degradation product) indicates formation of fibrin at a hemorrhage site

*This formula can be used for both diluted and undiluted specimens *Eliminates the need to correct for the volume counted by counting the 4 large corner squares (0.4uL) & the large center (0.1uL) Ex. No. of cells counted x dilution x .1uL = cells/ul 1mL (0.1 x 10) volume counted TOTAL CELL COUNTS Normal Saline mixed by inversion and loaded into hemocytometer w/ a Pasteur pipette Cells are counted: 4 corner squares and the center square on both sides of the hemocytometer WBC COUNT Lysis of RBCs must be obtained 3% of glacial acetic acid to lyse the RBC, used in diluting Add methylene blue stains WBCs provides diff. bet. Neutrophils & mononuclear cells. Preparing a clear specimen w/o dilution: 1. Place 4 drops of mixed specimen in a clean tube 2. Rinse a Pasteur pipette w/ 3% glacial acetic acid

CELL COUNT WBC Count routinely performed on CSF specimen RBC count - are determined only when a traumatic tap has occurred - Correction for leukocytes - Or protein is desired - CALCULATED BY: Total cell count RBC = TC WBC count Cell count should be performed immediately because WBCs & RBCs begin to lyse w/in 1 hour, w/ 40% of the leukocytes disintegrating after 2 hrs. METHODOLOGY Normal Reference range: - ADULT 0-5 wbcs/ul - CHILDREN or NEONATES 30 mononuclear cells/ul CALCULATION OF CELL COUNTS: # of cells counted x dilution # of squares counted x vol.of 1 square

3. Draw the 4 drops of CSF 4. Allow the pipette to sit 1 minute 5. Mix the solution 6. Discard the 1st drop & load the hemocytometer Counted: 4 corner squares & the center on both sides of hemocytometer Cells counted x dilution factor = WBC / uL If different squares is counted, use neubauer formula.

CORRECTIONS FOR CONTMINATION: Determination of CSF RBC count and the blood RBC & WBC counts is used to perform correction Read the book to fully understand this topic and calculations.

= cells/ul

QUALITY CONTROL OF CSF & OTHER BODY FLUID CELL COUNTS : Commercial Liquid control for CSF RBC & WBC are available Can be purchased in two levels of connection Biweekly basis = all diluents are checked by examination in a counting chamber under 4x magnification Monthly basis = speed of cytocentrifuge w/ tachometer Nondisposable counting chambers must be soaked w/ bactericidal solution for 15mins. then rinsed w/ water & cleaned w/ isopropyl alcohol

Addition of Albumin inc. the cell yield and decreases the cellular distortion + charged coated slide is available to attract cells. daily control slide for bacteria - prepared using 0.2 mL. saline & 2 drops of 30% albumin. The slide is stained and examined. The chamber count should be repeated if too many cells are seen on the slide, and a new slide should be prepared if not enough cells are seen on the slide. CYTOCENTRIFUGE RECOVERY CHART No. of WBCs counted # of cells counted on in chamber cytocentrifuge slide 0 0-40 1-5 20-100 6-10 60-150 11-20 150-250 20 250

DIFFERENTIAL COUNT ON A CSF SPECIMEN: Performed on a stained smear not in a counting chamber In the counting chamber = only the percentage of mononuclear & polynuclear cells present. Specimen should be concentrated Methods for concentrating specimen : Sedimentation Filtration Centrifugation Cytocentrifugation Sedimentation & Filtration - Not routinely used - Do produce less cellular distortion Lab. w/o cytocentrifuge concentrates specimen w/ routine centrifugation - Centrifuged (5-10mins.) - Supernatant fluid is saved. Slides made from suspended sediment are stained w/ wrights stain 100 cells should be counted reported in percentage If below 100, report only the numbers of the cell types seen.

CSF CELLULAR CONSTITUENTS Normal CSF: primarily lymphocytes and monocytes are found ADULTS - 70:30 ratio, lymphocytes to monocytes. In children the ratio is reversed. occasionally neutrophils is present. PLEOCYTOSIS presence of increased numbers of these normal cells. This is considered abnormal, as is the finding of immature leukocytes,eosinophils, plasma cells, macrophages, increase tissue cells & malignant cells. CSF Diff. Count provides diagnostic information about the type of microorganism that is causing an infection of the meninges (meningitis) Bacterial Meningitis elevated neutrophils and phagocytized bacteria Viral,tubercular, fungal or parasitic Meningitis - moderately elevated CSF wbc count w/ high % of lymphocytes and monocytes.

CYTOCENTRIFUGATION Fluid is added to the conical chamber Cells present in the fluid are forced into a monolayer w/in 6-mm diameter circle on the slide. Fluid is absorbed by filter paper blotter, producing a more conc. area of cells. 0.1ml of CSF combined w/ 1 drop of 30% albumin (produces an adequate cell yield when processed w/ the cytocentrifuge.

NEUTROPHILS INCREASED are seen in early stages (1-2days) of viral, fungal,tubercular and parasitic meningitis. CNS hemorrhage repated lumbar punctures injection of medications or radiographic dye may contain cytoplasmic vacuoles following cytocentrifugation. Granules are lost rapidly in CSF Neutrophils w/ pyknotic nuclei - indicate degenerating cells - resembles NRBCs if single nucleus is present but usually have multiple nuclei. NRBCs -seen as a result of bone marrow contamination during the spinal tap. - found in approx. 1% of specimens Capillary structures & endothelial cells - may be seen in a traumatic tap

Hemosiderin granules - from the degradation od phagocytized RBCs - dark blue or black, contains iron Yellow hematoidii crystals - represent further degeneration - they are iron-free, consisting of Hb and inconjugated bilirubin

LYMPHOCYTES & MONOCYTES common in cases of viral, tubercular, and fungal meningitis. Reactive lymphocytes w/ increased dark blue cytoplasm and clumped chromatin - frequently seen in viral infections INCREASED LYMPHOCYTES - asymptomatic HIV infection - AIDS Multiple sclerosis or other neurologic disorders - <50 wbcs/ul with increased normal and reactive lymphocytes and plasma cells EOSINOPHILS INCREASED - parasitic infections - fungal infections (Coccidioides immitis) - intro. of foreign mtrl. - medications - shunts into the CNS MACROPHAGES its purpose in CSF is to remove cellular debris & foreign objects such as RBCs. appear w/n 2-4hrs after RBCs enter the CSF. frequently seen following repeated taps - indicative of previous hemorrhage

NONPATHOLOGICALLY SIGNIFICANT CELLS seen in: - pneumoencephalography diagnostic procedures - fluid obtained from ventricular taps - during neurosurgery cell appear in clusters & can be distinguished from malignant cells by their uniform appaearance Choroidal cells - are from the epithelial lining of the choroid plexus - singularly and in clumps - nucleoli are absent & nuclei have a uniform appearance Ependymal cells - from the lining of ventricles and neral canal - have less defined cell membranes - in clusters, nucleoli are often present Spindle-shaped cells - lining cells from the arachnoid - seen in clusters - may be seen w/ systemic malignancies MALIGNANT CELLS OF HEMATOLOGIC ORIGIN Lymphoblasts, myeloblasts, monoblasts in CSF - seen in acute leukemias - nucleoli are prominent than in blood smears LYMPHOMA CELLS - indicates dessimination from the lymphoid tissue - resemble large and small lymphocytes - appear in clusters of large, small or mixed cells. - nuclei appear cleaved and prominent nucleoli. MALIGNANT CELLS OF NONHEMATOLOGIC ORIGIN Metastatic carcinoma cells - from lung, breast, renal & gastrointestinal malignancies.

CNS tumors - astrocytomas, retinoblastomas and medulloblastomas Fusing of cell walls and nuclear irregularities and hyperchromatic nucleoli are seen in clusters of malignant cells. Slides w/ abnormal cells PATHOLOGY

METHODOLOGY PRIINCIPLES USE: - turbidimetry (nephelometry) - dye binding PROTEIN FRACTIONS Routine CSF protein measure total protein conc. For neurologic disorder CSF protein fractions measurement is used. Damage in BBB - contains proportional fractions to those in plasma - albumin highest in conc. Multiple sclerosis - stimulat immunocompetent cells in the CNS - High IgG To accurately determine whether IgG is : - comparisons bet, serum& CSF levels of albumin and IgG must be made. CSF / serum albumin index -to evaluate the integrity of the BBB & the CSF IgG index to measure IgG synthesis w/in CNS FORMULA: CSF/serum albumin index = CSF albumin (mg/dl) Serum albumin (g/dl)

CHEMISTRY TESTS CEREBROSPINAL PROTEIN most performed chemical test on CSF protein determination. norm. protein = 15-45mg/Dl - higher in infants and older persons - reported in mg/Dl alpha-globulins haptoglobin, ceruloplasmin Transferrin major beta globulin tau- a separate carbohydrate deficient transferring fraction. Seen in CSF not in serum. Gamma globulin IgG, IgA IgM, fibrinogen and beta lipoprotein are not found in normal CSF CLINICAL SIGNIFICANCE OF ELEVATED PROTEIN VALUES Pathologic conditions damage to the blood brain barrier production of Igs w/in CNS decreased clearance of normal protein from the fluid degeneration of neural tissue meningitis & hemorrhage most common neurologic disorders LOW VALUES present when fluid is leaking from the CNS. ARTIFICIALLY INDUCED PLASMA PROTEINS p.190 Formula used to correct:

CSF / serum IgG index -compensates for any IgG entering the CSF via the BBB. FORMULA: IgG index = CSF IgG (mg/dl) / serum IgG (g/dl) CSF albumin (mg/dl) / serum albumin (g/dl)

*>0.70 indicative of IgG production w/n CNS ELECTROPHORESIS for the detection of oligoclonal bands representing inflammation w/n CNS. method of choice when determining if a fluid is CSF Gamma region (oligoclonal bands) SERUM BANDING - Leukemia - lymphoma - viral infections w/c can appear in CSF as: - BBB leakage - traumatic tap

Serum & CSF w/ HIV -banding representing both systemic & neurologic involvement. Multiple sclerosis - 2 or more oligoclonal bands in the CSF not in serum. Accompanied by increased IgG index. w/ oligoclonal banding that may not be present in serum - neurologic disorders (encephalitis,neurosyphilis) - Guillain-Barre syndrome - Neoplastic disorders Agarose gel electrophoresis followed by Coomassie brilliant blue staining -most frequentlt used BETTER RESOLUTION:s the axons of the neuron (demyelination) - Immunofixation electrophoresis (IFE) - Isoelectric focusing (IEF) followed by silverstaining

Myelin Basic Protein indicative of recent destruction of the myelin sheath that protect used to monitor the course of multiple sclerosis Immunoassay used for measurement

CEREBROSPINAL FLUID GLUCOSE