CSF is a clear fluid found in the brain and spinal cord that serves several functions, including protection, nutrition, and waste removal. It is produced by the choroid plexus at a rate of 20 mL/hour in adults. Normal CSF is clear and colorless with a protein level of 15-45 mg/dL and glucose level of 50-80 mg/dL. Examination of CSF includes analysis of appearance, cell count, chemistry, and cytology to diagnose conditions like meningitis or hemorrhage. Increased cells typically indicate infection or inflammation while decreased glucose suggests bacterial or fungal meningitis.

2. CSF)(Cerebrospinal Fluid
DEFINITION OF CSF :
 CSF is a clear, colourless transparent fluid present in the
cerebral ventricles, spinal canal, and subarachnoid
spaces.
 CSF is the 3rd major fluid of the body
 Adult volume 90-150 mL
 Neonate volume 10-60 mL
FUNCTIONS OF CSF :
1. Protection (Buoyancy)
2. Nutrition
3. Removal of waste
4. Lubrication
DEFINITION OF CSF :
 CSF is a clear, colourless transparent fluid present in the
cerebral ventricles, spinal canal, and subarachnoid
spaces.
 CSF is the 3rd major fluid of the body
 Adult volume 90-150 mL
 Neonate volume 10-60 mL
FUNCTIONS OF CSF :
1. Protection (Buoyancy)
2. Nutrition
3. Removal of waste
4. Lubrication

3. FORMATION :
 CSF is largely formed by the choroid plexus of the
lateral ventricle and remainder in the third and
fourth ventricles.
 CSF is a selective ultrafiltrate of plasma.
 Small amount of the CSF is also formed from the
ependymal cells lining the ventricles and other brain
capillaries.
 CSF is largely formed by the choroid plexus of the
lateral ventricle and remainder in the third and
fourth ventricles.
 CSF is a selective ultrafiltrate of plasma.
 Small amount of the CSF is also formed from the
ependymal cells lining the ventricles and other brain
capillaries.

4. FORMATION. cont,
Rate of formation:
 About 20 ml/hour ( 0.3 - 0.4 ml/min)
 500 ml/day in adults. Turns over 3.7 times a day
 Total quantity: 90 - 150 ml in adults distributed as :


Rate of formation:
 About 20 ml/hour ( 0.3 - 0.4 ml/min)
 500 ml/day in adults. Turns over 3.7 times a day
 Total quantity: 90 - 150 ml in adults distributed as :



5. CHARACTERISTICS OF CSF:
Nature :
Color :- Clear, transparent fluid
Specific gravity : - 1.004 – 1.007
Reaction : Alkaline and does not coagulate
Pressure :
 60-180 mm of H2O (adult)
 10-100 mm of H2O (newborn)
Nature :
Color :- Clear, transparent fluid
Specific gravity : - 1.004 – 1.007
Reaction : Alkaline and does not coagulate
Pressure :
 60-180 mm of H2O (adult)
 10-100 mm of H2O (newborn)

6. CHARACTERISTICS OF CSF: cont,
Cells :
 Adults : 0 - 5 cells/cumm
 Infants : 0 - 30 cells/cumm
 1-4 years : 0 - 20 cells/cumm
 5-18 years : 0 -10 cells/cumm
Chemicals :
 Protein 15-45mg/dl
 Glucose 50-80mg/dl
 Chloride 113-130 mEq/L
Cells :
 Adults : 0 - 5 cells/cumm
 Infants : 0 - 30 cells/cumm
 1-4 years : 0 - 20 cells/cumm
 5-18 years : 0 -10 cells/cumm
Chemicals :
 Protein 15-45mg/dl
 Glucose 50-80mg/dl
 Chloride 113-130 mEq/L

7. INDICATIONS OF CSF EXAMINATION
1. Infections: meningitis, encephalitis.
2. Inflammatory conditions: Sarcoidosis,
Neurosyphilis, SLE.
3. Infiltrative conditions: Leukemia, lymphoma
4. Administration of drugs in CSF (Therapeutic aim):
 Antibiotics
 Anticancer drugs
 Anesthetic drugs
1. Infections: meningitis, encephalitis.
2. Inflammatory conditions: Sarcoidosis,
Neurosyphilis, SLE.
3. Infiltrative conditions: Leukemia, lymphoma
4. Administration of drugs in CSF (Therapeutic aim):
 Antibiotics
 Anticancer drugs
 Anesthetic drugs

8. CSF COLLECTION
LUMBAR PUNCTURE
 A lumbar puncture also called a spinal tap is a
procedure where a sample of cerebrospinal fluid is
taken for examination.
LUMBAR PUNCTURE
 A lumbar puncture also called a spinal tap is a
procedure where a sample of cerebrospinal fluid is
taken for examination.

9. ROUTINE & OPTIONAL CSF TESTS
Routine :
 Gross examination
 Cell Counts + Differential
 Glucose [60-70% plasma]
 Protein [15 – 40 mg/dL]
When Indicated :
 Cultures
 Stains [Gram, Acid Fast]
 Cytology
 Electrophoresis
 VDRL
 Fungal and bacterial antigen
 D –Dimers
 PCR
Routine :
 Gross examination
 Cell Counts + Differential
 Glucose [60-70% plasma]
 Protein [15 – 40 mg/dL]
When Indicated :
 Cultures
 Stains [Gram, Acid Fast]
 Cytology
 Electrophoresis
 VDRL
 Fungal and bacterial antigen
 D –Dimers
 PCR

10. Macroscopic Examination
 Normal CSF appearance is crystal clear and
colourless
 Pathological processes can cause fluid to appear
cloudy, turbid, bloody, viscous, or clotted.
 The clarity of the fluid is of little clinical use, except
to provide an immediate indication of abnormality of
the CSF.
 A very useful point to remember is that a large
number of cells can be present without affecting the
clarity.
 Normal CSF appearance is crystal clear and
colourless
 Pathological processes can cause fluid to appear
cloudy, turbid, bloody, viscous, or clotted.
 The clarity of the fluid is of little clinical use, except
to provide an immediate indication of abnormality of
the CSF.
 A very useful point to remember is that a large
number of cells can be present without affecting the
clarity.

11. APPEARANCE OF CSF
1. Traumatic Tap or CNS Hemorrhage
 ~20% of LPs result in bloody specimens.
 Pink-red CSF usually indicates the presence of
blood.
 It is extremely important to identify the source of the
blood
1. Traumatic Tap or CNS Hemorrhage
 ~20% of LPs result in bloody specimens.
 Pink-red CSF usually indicates the presence of
blood.
 It is extremely important to identify the source of the
blood

12. APPEARANCE cont,
2. Xanthochromia :
Causes :
 Subarachnoid and intracerebral hemorrhage.
 Traumatic tap.
 Jaundice.
 Elevated protein level (>150 mg/dl)
 Premature infants (with immature blood- CSF
barrier & elevated bilirubin ).
 Hypercarotenemia.
 Meningeal malignant melanoma.
2. Xanthochromia :
Causes :
 Subarachnoid and intracerebral hemorrhage.
 Traumatic tap.
 Jaundice.
 Elevated protein level (>150 mg/dl)
 Premature infants (with immature blood- CSF
barrier & elevated bilirubin ).
 Hypercarotenemia.
 Meningeal malignant melanoma.

13. Traumatic LP v.s Subarachnoid hemorrhage :
Subarachnoid
Hemorrhage
Traumatic LPCSF finding
Blood uniform in all
tubes, Blood does not
clot on standing
Blood more in initial
tubes, Blood clot on
standing
Gross
appearance
Pink or yellow
supernatent
Clear supernatantCentrifugation
RBC count uniform in all
tubes
Progrresive decrease
in RBC count in later
tubes
Microscopy
IncreasedNormalCSF Pressure
IncreasedNormalCSF Protein

14. APPEARANCE cont,
3. Thick viscous CSF.
 Severe meningitis.
 Cryptococcal meningitis.
 Metastatic mucinous adenocarcinoma.
4. Clot formation:(cob web)
 Increased proteins( >150 mg)
 Tuberculous meningitis.
 Spinal block
3. Thick viscous CSF.
 Severe meningitis.
 Cryptococcal meningitis.
 Metastatic mucinous adenocarcinoma.
4. Clot formation:(cob web)
 Increased proteins( >150 mg)
 Tuberculous meningitis.
 Spinal block

15. Differentiation on the basis of type of clot
 Delicate and fine clot – Tuberculous meningitis.
 Large clot - Purulent meningitis
 Complete and spontaneous clot – Spinal
constriction.
Differentiation on the basis of type of clot
 Delicate and fine clot – Tuberculous meningitis.
 Large clot - Purulent meningitis
 Complete and spontaneous clot – Spinal
constriction.

16. Microscopic Examinations
1. Cell counts
 Total Leukocyte count & Differential
 RBC
2. Cytology
1. Cell counts
 Total Leukocyte count & Differential
 RBC
2. Cytology

17. Total Leukocyte count
METHOD ( Total leukocyte count)
 Properly mix the CSF sample.
 Nine drops of CSF is diluted with one drop of CSF
diluting fluid (in the ratio 9: 1)
 The counting chamber is covered with a cover slip.
 Charge the counting chamber with fluid and allowed to
stand for 5 min for the cells to settle.
CSF Diluting Fluid: Add 10 ml of glacial acetic acid and
0.2 grams of crystal violet to a 100-ml volumetric flask.
Dilute to the mark with distilled water.
METHOD ( Total leukocyte count)
 Properly mix the CSF sample.
 Nine drops of CSF is diluted with one drop of CSF
diluting fluid (in the ratio 9: 1)
 The counting chamber is covered with a cover slip.
 Charge the counting chamber with fluid and allowed to
stand for 5 min for the cells to settle.
CSF Diluting Fluid: Add 10 ml of glacial acetic acid and
0.2 grams of crystal violet to a 100-ml volumetric flask.
Dilute to the mark with distilled water.

18. Total Leukocyte count cont,
Neubauer’s chamber : Cells are counted in all the
nine squares.
 Calculation: Number of cells counted x 10/9
(as neubauer’s chamber has a depth of 0.1 mm and
total counting area is 9 sq. mm).
Fuch’s rosenthal chamber: Cells are counted in
five large squares.
 Calculation = no. of cells counted x 10/5 x 2
(depth is 0.2 mm. and total counting area is 16
sq.mm).
Neubauer’s chamber : Cells are counted in all the
nine squares.
 Calculation: Number of cells counted x 10/9
(as neubauer’s chamber has a depth of 0.1 mm and
total counting area is 9 sq. mm).
Fuch’s rosenthal chamber: Cells are counted in
five large squares.
 Calculation = no. of cells counted x 10/5 x 2
(depth is 0.2 mm. and total counting area is 16
sq.mm).

19. Causes of increased cell count :
 Meningitis.
 Intracranial hemorrhage.
 Meningeal infiltration by malignancy.
 Multiple sclerosis.
Causes of increased cell count :
 Meningitis.
 Intracranial hemorrhage.
 Meningeal infiltration by malignancy.
 Multiple sclerosis.

20. Leukocyte count
Differential
 Performed on a stained smear made from CSF.
 It is recommended that stained smears be made even
when the total cell count is within normal limits.
 Count 100 cells in consecutive oil-power fields.
 Report percentage of each type of cell present.
Differential
 Performed on a stained smear made from CSF.
 It is recommended that stained smears be made even
when the total cell count is within normal limits.
 Count 100 cells in consecutive oil-power fields.
 Report percentage of each type of cell present.

21. Leukocyte count cont,
Predominant Neutrophils :-
A. Meningitis (bacterial, early viral ,early tubercular
and funga
B. Sub arachnoid hemorrhage.
C. Metastasis.
Predominant Lymphocytes :-
A. Meningitis (viral or tubercular)
B. Incompletely treated bacterial meningitis.
C. Toxoplasmosis and cysticercosis.
Predominant Neutrophils :-
A. Meningitis (bacterial, early viral ,early tubercular
and funga
B. Sub arachnoid hemorrhage.
C. Metastasis.
Predominant Lymphocytes :-
A. Meningitis (viral or tubercular)
B. Incompletely treated bacterial meningitis.
C. Toxoplasmosis and cysticercosis.

22. Leukocyte count cont,
Predominant Eosinophils :-
A. Parasitic and fungal infections.
B. Reaction to foreign material.
Mixed cell pattern :-
A. Tubercular meningitis.
B. Chronic bacterial meningitis.
Predominant Eosinophils :-
A. Parasitic and fungal infections.
B. Reaction to foreign material.
Mixed cell pattern :-
A. Tubercular meningitis.
B. Chronic bacterial meningitis.

23. Chemical Analysis of CSF
Glucose :
- Normal : (45-80 mg/dl)
 Need to know plasma value ( 2/3rd of plasma value )
Increased
 Hyperglycemia
 Traumatic tap
Decreased
 Hypoglycemia
 Meningitis (bacterial, tuberculous & fungal)
 Tumors (meningeal carcinomatous)
Note: CSF glucose normal in viral meningitis.
Glucose :
- Normal : (45-80 mg/dl)
 Need to know plasma value ( 2/3rd of plasma value )
Increased
 Hyperglycemia
 Traumatic tap
Decreased
 Hypoglycemia
 Meningitis (bacterial, tuberculous & fungal)
 Tumors (meningeal carcinomatous)
Note: CSF glucose normal in viral meningitis.

24. Chemical Analysis of CSF
Protein :
- Normal : (15-45 mg/dl)
 80% plasma derived
 20% intrathecal synthesis.
Qualitative Test :–
Pandy’s Test
Pandy’s Reagent : ( 30 gm phenol + 500 ml distilled
water ).
Procedure : CSF is added in concentrated solution of
phenol, appearance of cloudiness indicates increased
protein (globulins).
Protein :
- Normal : (15-45 mg/dl)
 80% plasma derived
 20% intrathecal synthesis.
Qualitative Test :–
Pandy’s Test
Pandy’s Reagent : ( 30 gm phenol + 500 ml distilled
water ).
Procedure : CSF is added in concentrated solution of
phenol, appearance of cloudiness indicates increased
protein (globulins).

25. Proteins cont,
Quantitative Test -
Turbidimetric method:
 Principle : In the presence of sulphosalicylic acid and
sodium sulphate, protein yields a uniform turbidity
which absorbs maximum at 520 nm or green filter and is
directly proportional to the concentration of proteins.
 Reagent :
 Sulphosalicylic acid-30 gms/lt.
 Sodium sulphate-70 gms/lt.
 Standard - Albumin fraction-100 mgs/ lt.
Quantitative Test -
Turbidimetric method:
 Principle : In the presence of sulphosalicylic acid and
sodium sulphate, protein yields a uniform turbidity
which absorbs maximum at 520 nm or green filter and is
directly proportional to the concentration of proteins.
 Reagent :
 Sulphosalicylic acid-30 gms/lt.
 Sodium sulphate-70 gms/lt.
 Standard - Albumin fraction-100 mgs/ lt.

26.  Note :- False elevation of protein occurs if CSF is
contaminated with blood, this can be corrected by
deducing 1 mg/ dl of protein for every 1000 RBC’s.

27. Causes of raised CSF protein
1. Lysis of contaminated blood from traumatic tap (each
1000 RBC/mm3 raise the CSF protein by 1mg/dl).
2. Increased Permeability of epithelial membrane(Blood
Brain Barrier)
 CNS Bacterial or fungal Infections(Meningitis)
 Cerebral Hemorrhages
3. Increased production by CNS tissue as in -
 Multiple Sclerosis, Neurosyphilis (Increased production
of local immunoglobulin)
 Subacute sclerosing panencephalitis (SSPE)
 Guillain Barre syndrome.
4. Obstruction as in cases of - Tumors or abscess.
1. Lysis of contaminated blood from traumatic tap (each
1000 RBC/mm3 raise the CSF protein by 1mg/dl).
2. Increased Permeability of epithelial membrane(Blood
Brain Barrier)
 CNS Bacterial or fungal Infections(Meningitis)
 Cerebral Hemorrhages
3. Increased production by CNS tissue as in -
 Multiple Sclerosis, Neurosyphilis (Increased production
of local immunoglobulin)
 Subacute sclerosing panencephalitis (SSPE)
 Guillain Barre syndrome.
4. Obstruction as in cases of - Tumors or abscess.

28. Microbiological examination:
 Direct wet mount :- Candida, Cryptococcus
infection, amoebic encephalitis.
 Indian ink preparation :- A drop of CSF and
Indian ink is placed on a slide and covered with
cover slip and observe it under 40x
- Cryptococcus appears as budding yeast surrounded
by unstained capsule.
 Direct wet mount :- Candida, Cryptococcus
infection, amoebic encephalitis.
 Indian ink preparation :- A drop of CSF and
Indian ink is placed on a slide and covered with
cover slip and observe it under 40x
- Cryptococcus appears as budding yeast surrounded
by unstained capsule.

29. Gram’s Stain :-
 Smear is made from the sediment and is air dried,
stain it with gram’s stain and observe it under oil
immersion.
Streptococcus pneumococci - Diplococci, gram
positive, lying end to end.
Neisseria meningitides - Diplococci gram negative,
lying side by side.
Haemophilus influenza - coccobacilli gram negative
Gram’s Stain :-
 Smear is made from the sediment and is air dried,
stain it with gram’s stain and observe it under oil
immersion.
Streptococcus pneumococci - Diplococci, gram
positive, lying end to end.
Neisseria meningitides - Diplococci gram negative,
lying side by side.
Haemophilus influenza - coccobacilli gram negative

30. Ziehl- Neelsen stain:
 AFB smears are negative in 70% of cases however
florescent auramine stain have better sensitivity.
Serologic test for Neurosyphilis :
 Combination of VDRL test in CSF and FTA-ABS test
in serum is diagnostic.
Ziehl- Neelsen stain:
 AFB smears are negative in 70% of cases however
florescent auramine stain have better sensitivity.
Serologic test for Neurosyphilis :
 Combination of VDRL test in CSF and FTA-ABS test
in serum is diagnostic.

31. CSF CULTURE- Gold standard
1. Appearance of bacteria on gram stained smears.
2. Increased proteins or cell count.
3. Inoculated on chocolate agar, Blood agar &
Mackonky agar.
4. Sensitivity -90%.
PCR :-
 Viral infection of CNS.
 Requires only a drop of CSF.
CSF CULTURE- Gold standard
1. Appearance of bacteria on gram stained smears.
2. Increased proteins or cell count.
3. Inoculated on chocolate agar, Blood agar &
Mackonky agar.
4. Sensitivity -90%.
PCR :-
 Viral infection of CNS.
 Requires only a drop of CSF.

32. Culture media, conditions of incubation
1- Sheep blood agar incubated at CO2 incubator (5-
10%) at 35-37 o C.
2- Chocolate agar incubated at CO2 incubator (5-10%)
at 35-37 o C.
3- MacConkey agar incubated in ordinary incubator at
35-37 o C.
4- for special diagnosis include special media for TB or
fungal infections.
1- Sheep blood agar incubated at CO2 incubator (5-
10%) at 35-37 o C.
2- Chocolate agar incubated at CO2 incubator (5-10%)
at 35-37 o C.
3- MacConkey agar incubated in ordinary incubator at
35-37 o C.
4- for special diagnosis include special media for TB or
fungal infections.

33. Interpretation of Cultures
 Examine the Blood Agar and Chocolate Agar plates
and the Fastidious Anaerobic Broth daily for 4 days
incubation.
 If no growth on the culture plates but evidence of
growth in Fastidious Anaerobic Broth, all isolates are
to be identified
Interpretation of Cultures
 Examine the Blood Agar and Chocolate Agar plates
and the Fastidious Anaerobic Broth daily for 4 days
incubation.
 If no growth on the culture plates but evidence of
growth in Fastidious Anaerobic Broth, all isolates are
to be identified

34. Meningitis & Subarchnoid hemorrhage (SAH)
SAHViral
meningitis
Bacterial
meningitis
CSF Parameter
Often elevatedNormal to mildly
elevated
Often elevatedOpening pressure
( NL : 60 – 200 mm)
Clear to bloodyOften clearClear to turbidAppearance
Often presentNegativeNegativeXanthochromia Often presentNegativeNegativeXanthochromia
> 50 per mm³< 5 per mm³< 5 per mm³RBCs ( NL : < 5 )
Slightly elevatedElevated ( many
lymp )
Elevated ( many
PMNs )
WBCs (0 – 5 )
Normal to lowNormallowGlucose ( 2/3 of
serum
ElevatedElevatedElevatedProtein ( 15 – 45 )
NegativeNegativeMay show positiveGram stain