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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