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Examination of cerebrospinal fluid
Examination of Cerebrospinal fluidDr.Pavulraj.S5246M.V.Sc., scholarDivision of pathologyIndian Veterinary Research InstituteIndia
Introduction Cerebrospinal Fluid - clear, colorless transparent, tissue fluid present in the cerebral ventricles, spinal canal and subarachnoid spaces.Almost no blood cells, little protein and more salt
Formation of cerebrospinal fluid CSF is largely formed by the choroid plexus of the lateral ventricle and remainder in the third and fourth ventricles.
30% of the CSF is also formed from the ependymal cells lining the ventricles and other brain capillaries.
The choroid plexus of the ventricles actively secrete cerebrospinal fluid.
The choroid plexuses are highly vascular tufts covered by ependyma.
Circulation of CSF
Function of CSFMechanical cushion to brain Source of nutrition to brain Excretion of metabolic waste products Intra-cerebral transport medium Control of chemical environment Auto-regulation of intracranial pressure
Indications Diagnostic purposeInfections:meningitis, encephalitisInflammatory conditionsInfiltrative conditions : Leukemia, lymphoma, carcinomatous - meningitisHistory of seizures or CNS diseases.Relief of abnormally high pressure and drainage of blood or exudate.As a prognostic tool for evaluation of CNS diseases.To assess the response to treatment.
Collection SiteCisterna magna or Atlanto-occipital puncture - Horse, cat & dog.Sub lumbar or Lumbosacral puncture - cow, sheep & goat. Only Lumbar puncture PigInstruments 12.5 cm long 14G needle with a stylet Large animal
3 inch long 16 G spinal needle Small animal
Site
Normal components of CSFNormal biochemical constituents of CSF: Lower in CSF than plasma Proteins Glucose Phosphorus Bicarbonate Potassium Sulfate Cholesterol Enzymes
Higher in CSF than plasma Sodium Chloride
CSF normally does not contain erythrocytes. Normal CSF consists of varying proportions of small lymphocytes and monocytes. Major protein in CSF is albumin. The major Ig in normal CSF is IgG, which normally originates from the serum. The normal CSF glucose level is about 60% to 80% of the blood glucose concentration.
Examination of CSFPhysical examinationChemical examinationCytological examinationBacteriological examination
Increased glucose level in the CSF- hyperglycorrhaciaDecreased glucose level in the CSF - hypoglycorrhaciaIncreased number of white blood cells in CSF - pleocytosis
Macroscopic examination Color Clear and colorless as distilled water NormalEncephalitis and meningitis associated with viral infectionsBright redPuncture of blood vesselsOld hemorrhage (yellow supernatant) Brown or dull redIntra cranial hemorrhageYellow Xanthochromic bilirubin from disintegration of RBC in subarachnoid space from old hemorrhageExcess bilirubin in plasma
Xanthochromia
Left to right, Normal CSF, mildly xanthochromicCSF, moderately xanthochromic CSF, redtingedturbid CSF caused by hemorrhage, and cloudyred-tinged fluid from a horse with bacterial meningitis.
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Turbidity Due to presence of cells - >500/lBacterial meningitis, hemorrhageCoagulation Normal CSF not coagulateIncreased protein- fibrinogenAcute suppurative meningitisSpecific gravity- 1.003-1.008Reaction Alkaline as like blood
Chemical examination Protein - Normal range 10-40mg/dlPresent in very small quantity albuminGlobulin pathological conditionsTotal proteinFoam test Sulfasalicylic acid testGlobulin Nonne-Apelt test 1ml ammonium sulfate +1ml CSF Gray ringPandys test 1ml phenol+1ml CSF TurbidityIncreased levelInflammation meningitis, encephalitisNeoplasiaHemorrhageUremiaTissue destruction
Glucose Decreased levelAcute pyogenic meningitisHypoglycemiaMetastatic meningeal carcinomaIncreased levelHyperglycemiaNormal levelViral encephalitisBrain tumor
SodiumIncreased salt poisoning swine
Chlorides Reduced pyogenic meningitis
Cytological ExaminationTotal cell count Collection of CSF in plastic or silicon coated glass tube is preferred.The total cell counts of the CSF must be estimated within 20 minutes of collection, since the cells degenerate rapidly. Storage can be done at 48C (short term) or at 20C (long term)
Cells counted with standard hemocytometer chamber with Neubauer ruling.The cells in 9 large squares counted & then multiplied by 0.6 to get number of cells per cu mm of CSF.
Normal counts Cattle, sheep and pig : 0 - 15 cells/ cu mm Dog : upto 25 cells/cu mm Horse : upto 23 cells/cu mmIncreased number of white blood cells (pleocytosis) occurs in inflammatory lesions or irritation of brain and spinal cord.WBC > 200 cells/mlRBC > 400 cells/ml
Marked increase 100-500/lAcute pyogenic meningitisBrain or spinal abscessModerate increase EncephalitisMild increaseNeoplasiaViral infections 10-100(rabies upto 500)
Hemacytometer grid. The large cells with slightly irregular cell margins are WBCs. RBCs are smaller, light tan in color and round
Differential count Indicated when total count is elevated Various methods : Centrifugation If the total cell count is less than 500 cells/l. Membrane filtration Even small Number of cells can be examined. Sedimentation technique.
For staining Romanowsky stains Wrights Wright-Geimsa Leishmans stain Also rapid staining methods : Diff-quik
Neutrophils- Not seen in CSFBacterial encephalitis/meningitisAbscessHemorrhageLymphocytesViral infectionsAbscessFungal infections Cryptococcus neoformansPost vaccinal inflammationChronic conditionsNeoplastic cells Large cells arranged in clusters
Wright-Giemsa. 100xPMNs, Lymphocytes LymphocytesMonocyte
Segmented neutrophil
Lymphocyte
100x Wright-Giemsa
Segmented neutrophilEosinophil
Neutrophilic pleocytosis
Mono, lympho, neutro27
Mononuclear pleocytosis
Granulomatous meningoencephalitis (Wright-Giemsa)
Macro. Lumpho, neuto28
Mononuclear (lymphocytic) pleocytosis
Necrotising meningoencephalitis Wright-Giemsa
Mixed cell pleocytosis
Granulomatous meningoencephalitis (Wright-Giemsa).
Eosinophilic pleocytosis in the CSF from a llama
Meningeal worm infection Parelaphostrongylus tenuis Wright-Giemsa
Macro, eosino31
Mixed inflammatory cell response in CSF horse with nonseptic meningoencephalitis. Small lymphocytes, neutrophils, eosinophil , and monocyte . (Wrights stain)
Subarachnoid hemorrhage
Subarachnoid hemorrhage - macrophageswith phagocytosed erythrocytes
Myelin fragment in CSF from a horse withnecrotizing encephalomyelitis. Large spherical structure near macrophage contains a long spiral fragment of myelin. (Wrights stain)
Lymphoma in the CSF
Medium to large lymphocytes with immature chromatin, prominent nucleoli and basophilic, vacuolated cytoplasm.(Wright- Giemsa)
Bacteriological examinationIt is carried out when the CSF cell count and protein contents are high.The organisms are isolated in CSF and identified by cultural methods.Organisms detected are toxoplasmas,trypanosomes, bacteria
Gram stained CSF showing gram positive Anthrax bacilli
Fungal infection - Cryptococcus neoformans
Many extracellular yeasts. Wright-Giemsa
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Foal with septic meningitis.Degenerate neutrophils withphagocytosed cocci.(Wrights stain)
Bacterial meningitis: granulocytes with phagocytosed diplococci - pneumococci
Thank you
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