The document discusses examination of cerebrospinal fluid (CSF). It describes how CSF is formed by the choroid plexus and circulates through the ventricles and subarachnoid spaces. Examination of CSF includes physical, chemical, cytological and bacteriological analysis. The normal components and indications for examination are provided. Collection methods and sites are outlined. Macroscopic, chemical and cell analysis are described to identify abnormalities that may indicate infections, inflammation or other conditions.

1. Examination of Cerebrospinal
fluid
Dr.Pavulraj.S
5246
M.V.Sc., scholar
Division of pathology
Indian Veterinary Research Institute
India

2. 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

4. 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.

6. Circulation of CSF
Subarachnoid space of Brain and Spinal cord
Foramen of Megendie and foramen of Luschka
Fourth ventricle
Cerebral aqueduct of Sylvius
Third ventricle
Foramen of Monro [Interventricular foramen]
Lateral ventricle

7. Function of CSF
• Mechanical 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

8. Indications
• Diagnostic purpose
– Infections: meningitis, encephalitis
– Inflammatory conditions
– Infiltrative conditions : Leukemia, lymphoma,
carcinomatous - meningitis
 History 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.

9. Collection
Site
• Cisterna magna or Atlanto-
occipital puncture - Horse,
cat & dog.
• Sub lumbar or Lumbosacral
puncture - cow, sheep &
goat.
• Only Lumbar puncture – Pig
Instruments
• 12.5 cm long 14G needle
with a stylet – Large animal
• 3 inch long 16 G spinal
needle – Small animal

10. Site

11. Normal components of CSF
Normal 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.

12. Examination of CSF
Physical examination
Chemical examination
Cytological examination
Bacteriological examination
• Increased glucose level in the CSF-
hyperglycorrhacia
• Decreased glucose level in the CSF -
hypoglycorrhacia
• Increased number of white blood cells in CSF -
pleocytosis

13. Macroscopic examination
• Color
– Clear and colorless as distilled water
• Normal
• Encephalitis and meningitis associated with viral
infections
– Bright red
• Puncture of blood vessels
• Old hemorrhage (yellow supernatant)
– Brown or dull red
• Intra cranial hemorrhage
– Yellow
• Xanthochromic – bilirubin from disintegration of
RBC in subarachnoid space from old hemorrhage
• Excess bilirubin in plasma
Xanthochromia

16. • Turbidity
– Due to presence of cells - >500/μl
– Bacterial meningitis, hemorrhage
• Coagulation
– Normal CSF – not coagulate
– Increased protein- fibrinogen
– Acute suppurative meningitis
• Specific gravity- 1.003-1.008
• Reaction – Alkaline as like blood

17. Chemical examination
• Protein - Normal range – 10-40mg/dl
– Present in very small quantity – albumin
– Globulin – pathological conditions
• Total protein
• Foam test
• Sulfasalicylic acid test
• Globulin
• Nonne-Apelt test – 1ml ammonium sulfate +1ml CSF – Gray ring
• Pandy’s test – 1ml phenol+1ml CSF – Turbidity
• Increased level
– Inflammation – meningitis, encephalitis
– Neoplasia
– Hemorrhage
– Uremia
– Tissue destruction

18. Glucose
• Decreased level
• Acute pyogenic meningitis
• Hypoglycemia
• Metastatic meningeal carcinoma
• Increased level
• Hyperglycemia
• Normal level
• Viral encephalitis
• Brain tumor
• Sodium
• Increased – salt poisoning – swine
• Chlorides
• Reduced – pyogenic meningitis

19. Cytological Examination
Total 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 4–8 C (short term) or
at −20 C (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.

20.  Normal counts
 Cattle, sheep and pig : 0 - 15 cells/ cu mm
 Dog : upto 25 cells/cu mm
 Horse : upto 23 cells/cu mm
 Increased number of white blood cells (pleocytosis) occurs in
inflammatory lesions or irritation of brain and spinal cord.
WBC > 200 cells/ml
RBC > 400 cells/ml

21. • Marked increase – 100-
500/μl
– Acute pyogenic meningitis
– Brain or spinal abscess
• Moderate increase
– Encephalitis
• Mild increase
– Neoplasia
– Viral 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

22. Differential count
 Indicated when total count is elevated
 Various methods :
Centrifugation – If the total cell count is less than 500 cells/μl.
 Membrane filtration – Even small Number of cells can be
examined.
 Sedimentation technique.
 For staining – Romanowsky stains
 Wrights
 Wright-Geimsa
 Leishman’s stain
 Also rapid staining methods : Diff-quik

23. • Neutrophils- Not seen in CSF
oBacterial encephalitis/meningitis
oAbscess
oHemorrhage
• Lymphocytes
oViral infections
oAbscess
oFungal infections – Cryptococcus neoformans
oPost vaccinal inflammation
oChronic conditions
• Neoplastic cells
oLarge cells arranged in clusters

24. Wright-Giemsa. 100x
PMNs, Lymphocytes Lymphocytes
Monocyte

25. Segmented neutrophil
Lymphocyte
100x Wright-Giemsa

26. Segmented neutrophil
Eosinophil

27. Neutrophilic pleocytosis

28. Mononuclear pleocytosis
Granulomatous meningoencephalitis
(Wright-Giemsa)

29. Mononuclear (lymphocytic)
pleocytosis
Necrotising meningoencephalitis
Wright-Giemsa

30. Mixed cell pleocytosis
Granulomatous meningoencephalitis
(Wright-Giemsa).

31. Eosinophilic pleocytosis in the CSF
from a llama
Meningeal worm infection
Parelaphostrongylus tenuis
Wright-Giemsa

32. Mixed inflammatory cell response in CSF
horse with nonseptic meningoencephalitis.
Small lymphocytes, neutrophils, eosinophil ,
and monocyte . (Wright’s stain)

33. Subarachnoid hemorrhage
Subarachnoid hemorrhage - macrophages
with phagocytosed erythrocytes

34. Myelin fragment in CSF from a horse with
necrotizing encephalomyelitis. Large spherical
structure near macrophage contains a long
spiral fragment of myelin. (Wright’s stain)

35. Lymphoma in the CSF
Medium to large lymphocytes with immature
chromatin, prominent nucleoli and basophilic,
vacuolated cytoplasm.(Wright- Giemsa)

36. Bacteriological examination
It 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

37. Fungal infection - Cryptococcus
neoformans
Many extracellular yeasts. Wright-Giemsa

38. Foal with septic meningitis.
Degenerate neutrophils with
phagocytosed cocci.
(Wright’s stain)

39. Bacterial meningitis: granulocytes with
phagocytosed diplococci - pneumococci