Analysis of CSF, Gucose, Protien, Albumin, Globulin, Function of CSF, Secretion of CSF, Absorption of CSF, Bacterial Infection, Clinical Significance, Normal Composition of CSF, Microscopic examination of CSF, COOMASSIE BRILLIANT BLUE(CBB), REVERSE BIURET METHOD
Cerebrospinal fluid (CSF) is a clear, colourless fluid formed in the ventricles of
Mainly by choroid plexus (meshwork of tiny small blood vessels in lateral third
and fourth ventricles).
An ultrafiltrate of plasma.
CSF is contained within the cerebral ventricles, the spinal canal and the
subarachnoid space (space between arachnoid externally and pia mater internally)
surrounding the brain and spinal cord.
CSF is reabsorbed into the blood through the arachnoid villi of dural venous
Cerebrospinal fluid (CSF) is present
within the subarachnoid space
surrounding the brain in the skull and
the spinal cord in the spinal column.
Secreted by choroid plexuses into
Choroid plexus are areas where the
lining wall of the ventricle is very
thin and has a profusion of
Selective ultrafiltration of plasma
Active secretion by epithelial
some originates from the
ependymal cells lining the
ventricles and from the brain
Rate 0.35-0.40 ml/min OR
Total Volume:100-150ml & 10-60ml in New Born
0.25% of total vol replace each minute
Turn over time for total CSF vol 5-7 hours
= 4 times / day
40%-70% enters macroscopic spaces via CP
30%-60% enters across ependyma and pia
CIRCULATION OF CSF
Circulation: CSF is mainly formed in choroid plexus of the lateral ventricle.
CSF passes from the lateral ventricle to the third ventricle through the interventricular
foramen (foramen of Monro).
From third ventricle it passes to the fourth ventricle through the cerebral aqueduct. The
circulation is aided by the arterial pulsations of the choroid plexuses.
From the fourth ventricle (CSF) passes to the subarachnoid space around the brain and
spinal cord through the foramen of magendie and foramina of luschka.
CIRCULATION OF CSF
Foramen of Monro [Interventricular foramen]
Subarachnoid space of Brain and Spinal cord
Foramen of megendie and formen of luschka
FUNCTIONS OF CSF
A shock absorber
A mechanical buffer
Act as cushion between the brain and cranium
Act as a reservoir and regulates the contents of the cranium
Serves as a medium for nutritional exchange in CNS
Transport hormones and hormone releasing factors
Removes the metabolic waste products through absorption
CLINICALAPPLICATION OF CSF
In the diagnosis of
a. Bacterial, viral or fungal meningitis.
c. Malignant infiltrates like in acute leukemia, lymphoma.
d. Subarachnoid hemorrhage.
e. Spinal canal blockage leading to elevated intracranial tension.
f. Sub acute sclerosing pan encephalitis (SSPE)
Routine Laboratory CSF Analysis
At interspace of vertebrate Lumber 3rd-4th or
With complete aseptic techniques
3-5 ml of CSF is collected
It requires certain precautions and careful
technique to prevent the introduction of
infection or the damaging of neural tissue.
Collected CSF sample is immediately divided into three
Tube 1: Chemical Investigation (kept in freezer till
Tube 2: Microbiology Investigation (kept in room
Tube 3: Microscopic Investigation (Cellular Counting –
Tube 4: Cytology and Special Studies
Normal Physical appearance
Appearance & Color Clear ,Colorless
Daily Secretion 450-500 ml
Specific Gravity 1.006-1.007
Normal Microscopic cells
Lymphocytes 1-5 /H.P.F
Normal Chemical appearance
Protein 15-45 mg/dl
Glucose 50-80 mg /dl
Chloride 115-130 mmol /L
Calcium 1.0-1.40 mmol/L
Phosphorus 0.4-0.7 mmol/L
Magnesium 1.2-1.5 mmol/L
Potassium 2.6-3.0 mmol/L
Normal Microbiological appearance
No pathogenic microorganisms
Normal CSF Composition
PHYSICAL EXAMINATION OF CSF
Normal CSF: Clear & colorless
Viscosity: equal to water (increased with increased proteins)
Color and/or turbidity of CSF: observed only in pathological circumstances.
WBCs cells or pus cells: suggestive of a CNS infection (menigitis or encephalitis)
Blood : suggestive of hemorrhage: subarachnoid or artifactual traumatic tap.
Red & brown color :
1- Jaundice (bilirubin in CSF)
2- Xanthochromia (hemoglobin breakdown pigments in CSF)
Xanthochromic CSF suggests that a subarachnoid hemorrhage has recently occurred
(at least within two hours prior to tapping).
The yellow color is due to bilirubin generated in the CNS by the breakdown of hemoglobin
released from RBC's. (so jaundice should be excluded).
MICROSCOPIC EXAMINATION OF CSF
Normal Total WBCs count: 1-5 lymphocytes /HPF
Normal differential WBCs Count: (in centrifuged cells)
- 62% lymphocytes
- 36% monocytes
- 2% neutrophils
Increased neutrophils: bacterial meningitis
Increased lymphocytes: aseptic and viral meningitis
Normally CSF is blood free
RBCs in CSF: subarachnoid hemorrhage & malignancy
Artifact: traumatic tap (should be excluded)
(Traumatic tap bright red color RBCS in decreasing number as the
fluid is sampled) 31-05-2017 23
CHEMICAL EXAMINATION OF CSF
In addition to the major ions, CSF contains oxygen, sugars (e.g.
glucose, fructose), lactate, proteins (e.g. albumin, globulins) and
ANALYSIS OF PROTEIN IN CSF
Coomassie Brilliant Blue Method
Pyrogallol Red Method
Reverse Biuret method
Protein molecules which are normally soluble in solution can be made to precipitate by the
addition of 3% Trichloro acetic Acid(TCA).
Protein precipitation causes the solution to become turbid.
Thus the concentration of protein can be determined by measuring the degree of turbidity.
The absorbance of turbidity is measured at 490nm.
Precipitation can be achieved by sulfosalicylic acid and with sulfosalicylic acid in
combination with sodium sulphate/TCA.
0.5% w/v stock protein standard
S.No. Reagent Test Std Blank
1 CSF Sample 0.5ml - -
2 Std - 0.5ml -
3 D.W. - - 0.5ml
4 3% TCA 1.5ml 1.5ml 1.5ml
Working protein standard
Normal saline 10m
Mix and keep at RT for 10-15 min.
Read at 490nm
𝑇 − 𝐵
𝑆 − 𝐵
𝑥 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑠𝑡𝑑
COOMASSIE BRILLIANT BLUE(CBB)
PRINCIPLE: Dye-binding methods are based on the ability of proteins to
bind dyes such as Amido black 10B and Coomassie brilliant blue (CBB). The
dye-binding method is particularly used for assay of Total protein in CSF,
This is simple and fast method but linearity is less. Leaves stain on glassware.
Ability of proteins to bind with CBB G-250 & read at 700nm.
Particularly for total protein in CSF and Urine.
It leaves stain on the glassware. Imparts color to the cuvette also.
This dye combined with the molybdate, when they combined with albumin
of CSF, they form a complex with albumin & give red color. Read at
This dye also react with basic amino acid & gives red purple color
REVERSE BIURET METHOD
A method of protein determination has been developed which combines the biuret
reaction and the copper(I)-bathocuproine chelate reaction.
Protein in the specimen forms a C𝑢2+-protein chelate complex (biuret reaction)
during the first step.
is reduced to C𝑢+ by ascrobic acid, allowing the C𝑢+ to form a C𝑢+-
bathocuproine chelate complex during the second step.
The amount of C𝑢+-bathocuproine chelate complex formed is inversely proportional
to the protein concentration.
The sensitivity of this method was higher than that of the original Lowry, pyrogallol
red and commercially available Coomassie Brilliant Blue G.250 methods.
INCREASED PROTEIN: CSF protein may rise to 500 mg/dl in
A more moderate increase (150-200 mg/dl) occurs in inflammatory
diseases of meninges (meningitis, encephalitis), intracranial tumors,
subarachnoid hemorrhage, and cerebral infarction.
A more severe increase occurs in the Guillain-Barre syndrome and acoustic
and spinal schwannoma.
CSF protein is normal or mildly increased.
Increased IgG in CSF, but not in serum [IgG/albumin index normally
90% of MS patients have oligoclonal IgG bands in the CSF.
Oligoclonal bands occur in the CSF only not in the serum.
The CSF in MS often contains myelin fragments and myelin basic
MBP can be detected by radioimmunoassay. MBP is not specific for
MS. It can appear in any condition causing brain necrosis, including
Proteins (globulin and albumin) are precipitated by a saturated solution of phenol(7%) in
The reagent used is phenol (carbolic acid crystals dissolved in water) or, pyrogallic
acid or, cresol, usually termed as Pandy's reagent or Pandy's solution.
One drop of CSF sample (collected from the patient by lumbar puncture technique), is
added to about 1ml of Pandy's solution.
The turbid appearance signifies the presence of elevated levels of globulin protein in the
CSF and is regarded as positive Pandy's reaction.
The CSF from a normal adult shows no turbidity or precipitates and this is a negative
Half saturated ammonium sulphate are common use which
demonstrate the presence of an increase of globulin in CSF.
Carefully layer 1ml clear CSF over 1ml of saturated solution of
A thin white ring appearing at the junction of the liquids which
disappears on mixing indicates a 1+ reaction.
Heavy cloudiness persisting after mixing is 4+ reaction.
Analysis Of Protein Fractions: (Albumin & IgG)
Albumin of CSF is obtained from blood by means of blood-
brain barrier (as it is produced solely by the liver).In cases of
increased permeability of BBB, albumin is increased in CSF.
IgG of CSF can be obtained: from blood (By BBB) : increase in
cases of increase permeability of BBB & by local synthesis from plasma
cells within CSF (increased in cases of MS)
So, it is essential to determine the source of IgG
FIRST: CHECK INTEGRITY OF BLOOD BRAIN BARRIER (BBB)
BY CSF / serum albumin index calculation
CSF serum albumin index = CSF albumin (mg/dl) / serum albumin (g/dl)
Index less than 9 indicates intact BBB (no increased permeability of BBB)
SECOND: CSF IgG INDEX IS CALCULATED
𝐼𝑔𝐺 𝐼𝑛𝑑𝑒𝑥 =
𝐶𝑆𝐹 𝐼𝑔𝐺 × 𝑆𝑒𝑟𝑢𝑚 𝐴𝑙𝑏𝑢𝑚𝑖𝑛
𝑆𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 × 𝐶𝑆𝐹 𝐴𝑙𝑏𝑢𝑚𝑖𝑛
Normal : less than 0.7
Increased in cases of demyelinating diseases of CNS as : Multiple sclerosis (MS)
The enzyme glucose oxidase catalyses the oxidation of glucose to gluconic
acid and hydrogen peroxide.
The enzyme peroxidase and a chromogenic oxygen acceptor, such as o-
dianisidine, results in the formation of a colored compound that is
Glucose oxidase is highly specific for β-d-glucose.
36% and 64% of glucose in solution are in α- and β- forms, respectively.
The second step, involving peroxidase, is much less specific than the
glucose oxidase reaction.
SCHALES AND SCHALES TITRATION
PRINCIPLE: PFF of CSF is titrated with mercuric nitrate solution. Diphenyl
Carbazone is used as indicator. Chloride ions in CSF react with mercuric ions. At
the end point, mercuric ions in excess react with Diphenyl carbazone indicator to
blue-violet colored complex.
Specimen with low proteins content can be titrated directly without
𝐻𝑔2+ ions have more affinity for chloride ions as compared to DPC .
INCREASED CSF GLUCOSE IS OF NO CLINICAL
Causes of decreased CSF glucose
• Meningitis-Bacterial, fungal tubercular and syphilitic
• Tumors involving the meninges.
• Subarachnoid hemorrhage.
• Cerebral ameobiasis.
Test Appearance Pressure WBC/μL Protein mg/dL Glucose mg/dL Chloride
Normal CSF Clear 90 – 180 mm 0-8 lymph. 15-45 50-80 115-130 mEq/L
Turbid Increased 1000 -10000 100 – 500 < 40 Decreased
Viral meningitis Clear Normal to
Normal to mild
50-300 due to
Clear Increased 40-400 mixed 50-300 Decreased Decreased
Acute syphilitic Clear Increased About 500