Sunday, December 20, 2015

Cerebrospinal fluid (CSF) : Formation, composition, function, pathological state and biochemical analysis

A. Formation

Cerebrospinal fluid (CSF) is a clear, colourless fluid filling the ventricles and subarachnoid space. CSF production is a result of the combined processes of diffusion, pinocytosis and active transfer. The majority is produced by selective dialysis of blood plasma by a specialized sponge-like structure called the "choroid plexus" of third, lateral and fourth ventricles.

Fig. CSF collection
The anatomy of the ventricular system allows for movement of CSF in and around all the major structures of the brain. From the lateral ventricles located within the cerebral hemisphere, it circulates through the foramina of Monro into the third ventricle. At its caudal end, the third ventricle is connected by aqueduct of Sylvius to the fourth ventricle. CSF then flows into the basal cisterns and subarachnoid space by two lateral foramina of Lusckha and median foramina of Magendie. From the cisterns the CSF flows / throughout the subarachnoid space and over the hemispheric convexities and around the spinal cord.

The total volume of CSF is about 150 ml and the rate of CSF production is about 550 ml per day thus, turnover rate is about 3.7 times a day. CSF is reabsorbed into the venous system by numerous microscopic arachnoid villi and larger but less common arachnoid granulations (pacchinian bodies). Villi and granulations act as valves, which permit single directional flow of CSF into the venous blood. The reabsorption of CSF occurs along the entire neuraxis. In the SA space CSF comes in contact with perivascular spaces around the blood vessels entering and leaving the brain where cells and protein leak during inflammation. It must be remembered that there is no lymphatic drainage system in the central nervous system CNS), hence only 2 pathways are available for the elimination of wastes - capillary drainage and excretion via CSF. CSF secretion is an active process overall but production is independent of intraventricular pressure and resorption is proportional to it. A blood CSF barrier exists for many substances like bilirubin and certain drugs, so that their concentration in CSF is lower than in plasma.

B. Composition 

The composition of CSF is essentially same as that of brain ECF and is largely determined at the cell surfaces on which it is produced (choroid plexus), where it is absorbed (arachnoid villi). Its ionic composition is the same as that of plasma for some components and different for others. In general CSF glucose concentration is 60% of serum, sodium chloride and magnesium are same or greater than serum but potassium, calcium and glucose are lower than serum. Active transport in and out of the CSF space is probably responsible for maintaining this difference.

Total volume    =             150 ml
Specific gravity =            1.006-1.008
pH                     =             7.31 -7.40 (7.33)
Normal pressure   =         110-130 mm Ringer's solution, or 7-10mm Hg
Color                  =            colorless
Transparency     =            clear,  free of clots ;  Osmolarity = 292-297mOsm/l
Cellularity         =             nil or less than 5 lymphocyte or monocyte / mm3
Glucose             =             40-70 mg/dl
Protein               =             15-50 mg/dl
Bilirubin            =             nil
Na+                    =            138-150 mEq/L
CI -                    =             116-122 mEq/L ;     HCO3 = 20-24 mmol/L

The normal A:G ratio in CSF proteins is 3: 1.
Ratio of serum: CSF protein is 200: 1.

C. Functions

1.       mechanical support (cushion effect)
2.       removal of metabolic waste products
3.       transport of biologically active compounds
4.       maintenance of the chemical environment of the brain

D. Pathological states in which examination of CSF may be required:

A wide range of disorders can produce change in CSF composition and the commonest indication for a lumbar puncture to remove CSF is meningitis or infection of the coverings of the brain.

(1)    Infections:
(a)   Meningitis (Bacterial commonest)
(b)   Encephalitis
(c)    Neurosyphylis (acute)
(d)   TB meningitis
(2)    Cerebrovascular
(a)   Sub-arachnoid haemorrhage
(3)    Dementia and degenerative
(a)   Alzheimer’s disease
(4)    Neoplastic
(a)   Meningial carcinomatosis
(b)   Secondary deposits
(5)    Demyelinating 
(a)   Multiple sclerosis
(6)    Autoimmune    
(a)   Sarcoidosis

E. Acquisition

Quinke first developed the technique of LP or spinal tap in 1891. CSF is collected by lumbar puncture in which a fine bore needle (22 or 24 L.P needle) is passed between the 3rd and 4th lumbar vertebrae into the subarachnoid space with the patient lying in the lateral position and the fluid allowed to flow automatically. The bevel of the needle should be parallel to the long axis of the spine. The whole procedure is done under strict asepsis. The first few drops of the fluid are discarded and the rest of the fluid is collected in sterile containers. There are specific indications and contraindications for lumbar puncture.

The specimen is divided into 3 aliquots for:
a)      Chemistry and Serology
b)      Bacteriology
c)       Microscopy
Protocol for investigation:
(1)    Pressure (Opening and closing pressure)
(2)    Appearance
(a)   color
(b)   turbidity
(c)    coagulum
(3)    Cytological examination: :
(a)   direct examination
(b)   staining of the centrifuged deposit (e.g.Leishman's stain). .
(4)    Microbiological investigations:
(a)   staining of centrifuged deposit (gram stain, AFB)
(b)   culture and sensitivity.
(5)    Biochemical investigations.-
(a)   Total proteins (Lowry method or turbidimetry)
(b)   Qualitative test for gamma globulin
(c)    Quantitation of glucose
(d)   Quantitation of chloride
(e)    Enzymes like LDH
(f)     Bicarbonate, folate levels
It must be borne in mind that CSF samples must always be centrifuged prior to analysis in order to precipitate any cells otherwise falsely high values for CSF protein will be obtained.

F. Changes in CSF in diseased states

Physical Analysis

Pressure: Normally 60-150 mm of water in recumbent position.
Low opening pressure: 10- 20 cm H2O normal- CSF leak or spinal SA obstruction. Elevated opening pressure: More than 20 cm H2O - space occupying lesion, diffuse cerebral inflammation.

Appearance: Normal CSF is clear and colorless and gives no coagulum or sediment on standing.

Color: Changes only in pathological conditions, whereas the term xanthochromia means yellow colour. It has been used for the presence of other colours as well
Yellowish tinge --markedly increased protein >200%.
Yellowish --bilirubin
Blood may be present due to bleeding from L.P. site, pathological subarachnoid hemorrhage, ventricular hemorrhage, or neurosurgical operations. When hemolysis occurs in CSF the hemoglobin liberated is converted to bilirubin and that gives a yellow coloration to the CSF (more visible after centrifugation) called xanthochromia. Bilirubin is detected after 10 hours of subarachnoid bleeding.

Turbidity: CSF may occasionally clot if the ratio of blood to CSF is high. Usually due to fibrin clot (e.g., tubercular meningitis a cobweb coagulum appears by keeping CSF for 12-15 hours). Turbidity can also be due to microscopic fat globules (fat embolism).

Cell count : Normal CSF should contain no more than 5 lymphocytes or monocytes / mm3. Nature and number of cells are noted. Presence of RBCs indicate hemorrhage. Presence of WBCs predominantly polymorphs indicate bacterial meningitis. In viral infection and chronic infections a lymphocytic response is obtained.

Biochemical analysis


CSF proteins ordinarily originate from serum and reach the cerebral space by endocytosis across the capillary endothelium. An increase in total proteins is the commonest chemical abnormality in CSF and results from a breakdown of the blood CSF and brain-CSF barriers usually as a consequences of an inflammatory reaction but on occasion, if the flow of CSF is obstructed. Albumin is the predominant protein to be increased, globulins appear in varying amount. If the permeability of the barriers is markedly increased, fibrinogen is present which in the test tube forms a clot or coagulum.

Examination of CSF protein is done mainly to detect :
  • Increased blood-brain barrier permeability to plasma protein (80%)
  • Increased intrathecal IgG secretion (20% ).

Increase in CSF protein occurs in

Hemorrhage (trauma, neoplasm, ruptured aneurysm). Note: A false result may occur from a "bloody tap" -rupture of a blood vessel during LP (presence of 1000RBCs -increase protein I mg / ml).

Inflammation, meningitis especially bacterial (meningococcal), may be as high as 2000mg/dl.

Other causes: encephalitis, polio.


CSF glucose concentration is ~ 60% of the normal plasma glucose. Blood and CSF glucose equilibrate only after a lag period of 4hours so that CSF glucose at a given time reflects blood glucose level during the past 5 hrs. When glucose determination is critical, CSF and blood glucose should be obtained only after the patient has been fasted for the last 4 hrs. Equilibrated CSF glucose is definitely abnormal when it is less than 40% of simultaneously determined blood glucose-values of 40 mg/dl are almost always abnormal.

Decreased CSF glucose (Hypoglycorrachia):

Markedly decreased in pyogenic meningitis ( e.g., 10-20 mg/dl); in tuberculous meningitis it is 30-50mg/dl; in viral meningitis it is normal.
Specialised Tests:
Increased lactate              Bacterial meningitis
Increased glutamine         Hepatic encephalopathy
Increased LDH                  Bacterial meningitis, metastatic carcinoma                                 Increased CK-BB               Parenchymal damage
Increased adenosine         Tuberculous meningitis deaminase    


Protocol for Protein Estimation in CSF

Add I ml each of test CSF, standard and distilled water in respective tubes. Then add 4ml of 3% TCA in all tubes. Mix. keep for 10 mins. Read at 450nm Standard protein concentration - 50mg/dl

Protocol for estimation of CSF glucose

Fig. Normal CSF interperetation and pathological states
Place 0.1ml of CSF into 7.8ml of isotonic solution in a centrifuge tube. Mix well. Add 0.1ml of sodium tungstate solution. Mix and centrifuge at 2000 rpm for 10 min. Take 2ml each of the supernatant, standard and isotonic solution in the respective tubes. Then add 2ml of alkaline reagent in all tubes. Mix well and heat in boiling water for 10 min, cool and add 2ml of color reagent in all tubes. Mix and wait for 5 min. Read at 540 nm.Concentration of standard: 1.25 mg/dl.

(cobweb coagulum)
< 5/ mm3
< 5/mm3
40-70 mg/dl
30-60 mg/dl
20-30 mg/dl
20-40 mg/dl
15-50 mg/dl
20-80 mg/dl
50-200 mg/dl or  more
20-100 mg/dl

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