Cerebrospinal fluid (CSF) is formed within the ventricles and circulates in the subarachnoid space and ventricles. CSF examination is important for diagnosing conditions like meningitis, tumors, and multiple sclerosis. A lumbar puncture is performed to collect CSF specimens, which are then examined for properties like color, clarity, cell count, glucose and protein levels, and presence of microorganisms. Abnormal CSF findings can indicate conditions such as bacterial meningitis (elevated white blood cells and proteins, low glucose) or viral meningitis (elevated lymphocytes and proteins, normal or slightly low glucose).

1. INTRODUCTION
Cerebrospinal fluid (CSF) is formed
within the ventricles and circulates in
the subarachnoid space and ventricles
Total volume of CSF
Adults - 90 to 150 ml
Neonates – 10-60 ml
 Functions of CSF
Protection of brain and spinal cord
from injury (Acts a cushion, lubricant
and shock absorber)
Supply nutrients and removal of
waste products from brain
10/29/2021 Examination of CSF 1
CEREBROSPINAL FLUID

2. CLINICAL SIGNIFICANCE /Importance of CSF
examination
Diagnostic importance in
conditions
 Meningitis
Subarachnoid haemorrhage
Spinal cord tumour
Multiple sclerosis
Benign intracranial
hypertension
Metastatic tumor of CNS with
CSF involvement
10/29/2021 Examination of CSF 2

3. 10/29/2021 Examination of CSF 3

4. SPECIMEN COLLECTION
 Specimen should be collected by a physician, a specially trained technician or nurse
 Sterile lumbar puncture needle is inserted between the 4 and 5 lumbar vertebra to a
depth of 4 to 5 cm
 Sites
 Lumbar puncture
 Cisternal puncture
 Ventricular puncture
 CSF Pressure - Normal Range
Adults – 90-100mm of water in lateral positions
Infants & children – 10-100mm of water reaching in adults level by 6-8 Years
 Increased – Meningitis, cerebral edema
Decreased – Dehydration
10/29/2021 Examination of CSF 4

5. Method of collection
Tube 1: Estimation of protein, glucose and serology
 Tube 2: Gram stain or other stains ,culture and sensitivity
Tube 3: Cell count & Differential counts
Tube 4:Special tests – Cryptococcus antigen, serologic test
for syphilis, molecular test , serological studies & Cytology
INDICATIONS FOR LUMBAR PUNCTURE
Diagnostic and therapeutic indications are
Bacterial, viral & fungal infection
Primary or metastatic malignancy
Spinal anesthesia
• Subarachnoid hemorrhage
10/29/2021 Examination of CSF 5

6. NORMAL COMPOSITION OF CSF
COLOR – Colorless
pH 7.3-7.4
Appearance – clear
Clot formation – no clot formation on standing
Specific Gravity – 1.003-1.008
Total solids – 0.85 – 1.70g/dl
Protein - 15 to 45mg/dl(albumin = 50 -70%,globulins = 30 -50%)
Glucose = 40 -80mg/dl
 Chlorides = 700 – 750mg/dl
10/29/2021 Examination of CSF 6

7. NORMAL COMPOSITION OF CSF
 Sodium = 144 - 154mEq/1
 Potassium = 2.0 – 3.5mEq/1
 Creatinine = 0.5 – 1.2 mg/dl
 Cholesterol = 0.2 -0.6mg/dl
 Urea = 6-16g/dl
 Uric acid = 0.5 – 4.5mg/dl
 Cells – 0-8 lymphocytes/per cumm(µl)
 Neutrophils :Absent
10/29/2021 Examination of CSF 7

8. IMPORTANT PRECAUTIONS
Collected CSF specimen must be examined immediately
Specimen collected for bacterial culture should not be stored in the
refrigerator
Specimen meant for biochemical tests only, may be stored at 2- 8 c for
2 to 3 hrs
Cells and trypanosomes are rapidly lysed after the collection of CSF
Urgent analysis of CSF is necessary
Specimen is difficult to collect
Once it is collected it is necessary to analyze the specimen carefully and
economically
Specimen may contain virulent organisms & necessary to handle it
carefully
10/29/2021 Examination of CSF 8

9. ROUTINE EXAMINATION OF CSF
PHYSICAL EXAMINATION
 Observe the specimen and note down for the following aspects
color
Appearance
Presence of blood
Presence of clot or fibrin web - Fibrin clot formation takes place due to the
presence of fibrinogen
Use pH paper to determine Ph
Specific gravity by the weight method by using a hand refractometer
CSF pressure
10/29/2021 Examination of CSF 9

10. MICROSCOPIC EXAMINATION
Requirements
1.Glass slides
2.Improved Neubauer counting chamber with coverslip
3.Pasteur Pipettes
4.Leishman stain and Buffer solution ph 7.0
5.CSF diluting fluid
6.Gram’s staining reagent & Acid fast staining reagent
7.centrifuge
8.Microscope
10/29/2021 Examination of CSF 10

11. TOTAL LEUKOCYTE COUNT
Mix the CSF sample carefully
Fill the neubauer chamber with the CSF sample
If CSF appears clear ,use it undiluted
If CSF appears cloudy ,make 1:20 dilution by using a WBC pipette or
pipette 9.95 ml of CSF into it
Mix well
Place the chamber on the microscope stage & count the cells in 5
squares = cells counted x 10/9
If specimen is diluted 1:20 then leukocytes in CSF/per cumm = cells
counted x 10 x 20/9
 CLINICAL SIGNIFICANCE
Increased Neutrophil - Acute pyogenic meningitis
Increased Lymphocytes – Viral,tuberculous and fungal menigitis
10/29/2021 Examination of CSF 11

12. Determination of differential leukocyte count
PROCEDURE
 If the cells count is low centrifuge CSF specimen and prepare a thin smear
of the sediment
 If the cell count is more than 500WBC/cumm
 Prepare a thin smear, of uncenrtifuged CSF
 Add 10 -15 drops of leishman’s stain on the smear
 Wait for 1 minute
 Add equal number of drops of buffer solution pH7.0
 Mix well and keep for 10 minutes
 Wash under running tap water,air dry and observe under oil immersion
CLINICAL CONDITIONS
 Increased lymphocyte count - Viral infection
 Increased Neutrophil count - Bacterial infection
10/29/2021 Examination of CSF 12

13. CHEMICAL COMPOSITION OF CSF
 QUALITATIVE TEST - GLUCOSE TEST
 O.5ml of benedicts reagent in a test tube and dilute to 5 ml by adding 4.5 ml of distilled water
 Add0.5 ml of csf
 Boil for 2 mintues on a flame
 Allow to cool
 Turbid greenish yellow – normal
 No color change – very low/absent color
 Elevated csf protein –colour changes from blue to purple due to the reaction with the alkaline
copper reagent
 QUANTITATIVE TEST - Folinwu test
10/29/2021 Examination of CSF 13

14. PROTEIN TEST
• Pandy’s test
• Prepared by dissolving 10 g of phenol in 150 ml of distiiled water
• Should be clean and colourless
PROCEDURE
• Pipette 2 ml of pandy’s reagent in a small test tube
• Add 2 r 3 drops of clean CSF specimen , do not mix
• Observe for the formation of turbidity
OBSERVATION
• No formation of precipitate –globulin :normal
• Formation of precipitate ring – globulin :increased ( protein elevated in meningitis)
• Grade positive result as trace,+,++,+++ and ++++
• Based on the degree of formation of the precipitate
10/29/2021 Examination of CSF 14

15. CHLORIDES TEST
1ml of CSF + 1 or 2 drops of 5%potassium chromate being used as
indicators
Add into a small conical flask
Titer with silver nitrate reagent from 25ml biuret
Gently swirl and mix during titration after each drop
When the end point reached a sudden change from yellow to
orange colour
Note the volume of silver nitrate solution added
OTHER TEST -Mercuric nitrate solution
Chloride reduction in tuberculosis meningitis
CLINICAL SIGNIFICANCE
Increase in globulin – bacterial infection
10/29/2021 Examination of CSF 15

16. LABORATORY OBSERVATIONS OF ROUTINE CSF EXAMINATION IN VARIOUS CLINICAL CONDITIONS
CLINICAL
CONDITION
APPEARANCE CELLS/cumm(µl) GLUCOSE CHLORIDES PROTEINS
Bacterial infection Cloudy >500 neutrophils Low values (0-40
mg/dl)
Marked decrease
600 -700mg/dl
High values(45-
500mg/dl),increas
e in globulins
Viral infection Clear Mostly lymphocytes (10-
200)
Slightly low or
normal
Moderate decrease High values (45-
300mg/dl)
Fungal infection
(very rare
)Cryptococcus
neoformans
Clear (0-5)Lymphocytes Low values (0-
40mg/dl)
Normal or slight
decrease
Normal
Acute purulent
meningitis
Cloudy to purulent
clot
Very high count(500-
20,000)per
cumm,mostly
lymphocytes
Very low values
(0-40 mg/dl)
Low values (600-
700mg/dl)
Very high(45-1000
mg/dl)increase in
globulins
10/29/2021 Examination of CSF 16

17. LABORATORY OBSERVATIONS OF ROUTINE CSF EXAMINATION IN VARIOUS CLINICAL
CONDITIONS
CLINICAL
CONDITIONS
APPEARANCE CELLS/CUMM(µl) GLUCOSE CHLORIDES PROTEINS
Tuberculous
meningitis
Cloudy to purulent
clot
High count(10 -500)
mostly lymphocytes
Very low
values(0-
40mg/dl)
Very low
values(500-
600mg/dl)
High values (45-500
mg/dl),increase in
globulins
Acute syphilitic
mengitis
Clear or turbid High count (20-
2000)mostly
lymphocytes
Very low
values(0-
40mg/dl)
Normal or slightly
decreased
Normal
globulin:normal
Brain tumour clear 0-5 increased normal Increasedglobulin:I
ncreased
Cerebral
hemorrhage
Xanthochromic 0-5 variable normal Increased,globulin:
Normal
Encephalitis
lethargica
clear 10-100 all lymphocytes Slightly
increased 80-
120 mg/dl
Normal Normal or
increased
10/29/2021 Examination of CSF 17

18. QUESTIONS
1.CSF findings in tuberculosis meningitis( 5 & 10 Marks )
2.CSF findings in pyogenic meningitis ( 5 & 10 Marks )
3.CSF findings in Viral meningitis ( 5 & 10 Marks )
4.Analysis of CSF ( 5 & 10 Marks )
5.Examination of CSF ( 5 & 10 Marks )
6.Indications of Lumbar puncture ( 3 Marks )
10/29/2021 Examination of CSF 18