Chemical Examination of CSF

ROUTINE LABORATORY EXAMINATION OF CSF

-Estimation of total proteins in CSF. by Turbidimetric,

Chemical Examination of CSF

Trichloroacetic, Micro method,

-Estimation of Globulin in CSF by Pandy's test

Nonne-Apelt's- method, Radial immunodiffusion test,

-Clinical Significance CSF proteins,

-Desease condition that effect the CSF glucose levels,

-Bacteriological Examination of CSF

-Bacteriological examination of synovial fluid,

-Mucin Clot test evaluate Viscosity of Synovial Fluid

 

Chemical Examination of CSF

Chemical Examination of CSF, Estimation of total proteins in CSF. by Turbidimetric methods ,  Trichloroacetic Acid method ,Micromethod

Estimation of Globulin in CSF by Pandy's test, Nonne-Apelt's- method , Radial immunodiffusion test

Clinical Significance CSF proteins

Desease condition that effect the csf glucose levels

Bacteriological Examination of CSF

SYNOVIAL FLUID

Becterialogical examination of synovial fluid, 

Viscosity of Synovial Fluid

Mucin Clot test evaluate the Viscosity is in the laboratory  of Synovial Fluid

Proteins- Turbidimetric methods are commonly employed for the estimation of total proteins in CSF.

Turbidimetric methods

Principle- Proteins in CSF are precipitated by either dilute trichloroacetic acid or sulphosalicylic acid in sodium sulphate solution. The turbidity of the resultant uniform suspension is measured in a colorimeter against a known standard.

Trichloroacetic Acid method

Reagents

1. Trichloroacetic acid 3% aqueous solution.

2. Stock standard protein solution 5.0 g/L in 0.90% NaCI: Distribute in small volumes and store at -18°C. The standard can be prepared from diluted pooled serum, or making suitable dilutions of commercial control serum or from bovine albumin.

3. Working standard protein solution 0.5 g/L (50 mg %): Dilute the stock standard 1 in 10 with 0.9% NaCl. Fresh stock is to be prepared each week and stored in 1.5ml volumes at about -18°C. Thaw a sample for use each day.

Method

1. To 4.0 ml of trichloracetic acid, add 1.0 ml of CSF drop by drop with constant mixing.

2. In the same manner, add 1.0 ml of standard to 4.0 ml of trichloroacetic acid.

Preparation of calibration curve --Dilute the stock standard protein (5.0 g/L) 1 in 5 with 0.9% NaCl (saline) and prepare a series of tubes as follows:

3. Add 1.0 ml distilled water to 4.0ml trichloroacetic acid. This is the blank.

4. Stand at room temperature for 10 minutes and remix the turbid solutions.

5. Read the absorbances of the standard and test against the blank at 450 nm or by using a blue filter.

Calculations 

T= Absorbance of Test;

 S = Absorbance of standard

T/S x0.5 = g CSF protein per litre

To convert to mg/100 ml (mg %), multiply by 100.

Note

1. Use appropriate dilutions if the value of the test exceeds the upper limit of the method as established by a cali bration curve.

2. A calibration curve will show if a linear relationship holds for increasing protein concentration and should be prepared for each photometric in strument.

3. The method is temperature depend Ent

4. The size and shape of the particles affect the absorbance. The tubes must be shaken to mix constantly for up to 4 times.

Mix well; add 1.0 ml of each standard protein solution to 4.0 ml trichloroacetic acid. Read the absorbance at 450 nm (blue filter). A straight line calibration curve is obtained if the absorbance is linear.

Method using permanent standard This method is not very accurate but it is such a rapid and simple method that most bacteriology laboratories which are not usually equipped for chemical procedures, find it of adequate accuracy.

 Reagents 1. 3% Sulphosalicylic acid.

 2. Permanent protein standard marked in mg100 ml (10-100 mg/100 ml) supplied commercially.

Method

1. Add 1.0 ml of clear CSF to 3.0 ml of sulphosalicylic acid in a standard test tube.

2. Mix contents and leave for 5 minutes.

3. Compare the tube with the turbidity standards Results Normal CSF contains protein between 0.15-0.45 g/L ( 15 to 45 mg/100 ml). If the value of the CSF protein is higher than 100 mg/100 ml, dilute with saline and adjust the result accordingly.

Micromethod- This method is very useful when the volume of the CSF is small.

Reagent 3% aqueous trichloroacetic acid.

Method 1.

Set up 3 test tubes as follows:

Blank: 0.2 ml of saline Standard: 0.2 ml of standard protein

Test: 0.2 ml of CSF

2. Add to all tubes 0.8 ml of 3.0% trichloroacetic acid. Mix by inverting about 5 times. Allow to stand for 10 minutes. Read absorbance of the standard and test against the blank at 450 nm with glass microcuvettes of Icm light path.

Calculation

Globulin

Tests for globulin are performed only on blood-free CSF specimens.

1.     Pandy's test

Principle- In a saturated aqueous solution of phenol, globulin molecule absorbs water and the phenol is displaced from the solution, causing a fine persistent turbidity

Method:- Add 1 drop of CSF to 0.5 ml of saturated aqueous solution of phenol (must be very clear) in a small test tube. Cloudiness/turbidity against a dark background indicates increase in globulin content. Normal CSF remains clear.

Report as Nil, +, ++, +++ or ++++. 

2.     Nonne-Apelt's- method 

Principle- Globulin is precipitated by solution of saturated ammonium sulphate. Method Carefully layer 1.0 ml of clear CSF on to 1.0 ml of saturated ammonium sulphate. A thin white ring of precipitation appears at the interface of the two liquids. On mixing, the ring may disappear indicating a 1+ reaction. Heavy cloudiness indicates a 4+ reaction.

3. Radial immunodiffusion test -

--This is a useful test for the measurement of concentrations of albumin and globulin in CSF. This involves the diffusion of an antigen (IgG or albumin) in the CSF through a semi-solid medium containing an antibody (anti-IgG or anti albumin). thereby forming visible zones of precipitation. The measurement of the zones of precipitation corresponds to the concentration of the antigen. This method also determines IgG/albumin ratio which is of diagnostic importance in multiple sclerosis and other neurological disorders.

Note:Electrophoresis of CSF protein can also be carried out to evaluate albumin and globulin content in CSF. The Lange's colloidal gold test, though now obso lete, is a good substitute for electrophoresis.

Normal range: Normal CSF contains approximately 60% albumin, 8% immunoglobulins and 32% other proteins.

IgG/albumin ratio: 17.5

IgG index: <0.7

Clinical Significance

Increase in CSF proteins

(a) Mild increase can be seen in viral meningitis, neurosyphilis, subdural haematoma, cerebral thrombosis, brain tumour, multiple sclerosis.

(b) CSF globulins, especially the immunoglobulin IgG, are raised in cases of multiple sclerosis, neurosyphilis.

(c) Pronounced increase in CSF proteins is seen in acute bacterial meningitis, tuberculous meningitis, spinal cord tumour, cerebral haemorrhage and intracranial tumour.

Glucose CSF glucose is estimated in the same way as for blood glucose usallay csf glucose less then 40mg/100ml is considered as reduced level.

Desease condition that effect the csf glucose levels

Viral meningitis Neurosyphilis Brain or cord tumor Cerebral thrombosis Multiple sclerosis	no significance
Cns leukemia Subarachnoid Hemorrhage	Modernate reduction
Bacterial meningitis Tuberculosis meningitis Fungal meningitis	Marked reduction

It is essential that the glucose assay is carried out as soon as possible after collection in order to prevent the action of glycolytic enzymes in the CSF. This may result in the reduction of glucose level.

Bacteriological Examination of CSF

CSF for bacterial examination must reach the laboratory as soon as possible after collection in a sterile container. The examination involves mainly

(1) Grams stain and any other stain requested for,

 (2) Isolation of pathogens,

(3) Serology.

Bacteriological examination of CSF is discussed in the Microbiology Section of this book.

SYNOVIAL FLUID:-The synovial fluid (SF) is found in each large joint such as the knee, ankle, wrist, hip, elbow and shoulder. About Iml of the fluid is normally present.

To aspirate the fluid, aseptic precautions must be observed. The fluid is commonly sterile. It is collected into sterile. It is  collected in to sterle container and then sent to the laboratory without deley for the microbiological examnination. For serological tests a chemically clean tube should be used to prevent clotting.

Becterialogical examination of synovial fluid:- the procedure is similar to that used for csf.refer to the microbiology section of this book.

Viscosity of Synovial Fluid

Normal synovial fluid will form a tenacious strand about 4-6cm long if allowed to drop from the end of the needle. A strand of less than 3 cm indicates reduced viscosity. Viscosity is evaluated in the laboratory by the mucin clot test.

Mucin Clot Test

Principle This test is based on the polymerisation of hyaluronidase. Method Add synovial fluid drop by drop to about 20 ml of 5% acetic acid in a beaker.

A normal synovial fluid will form a firm clot with clear surrounding fluid.

A poor clot is surrounded by a cloudy fluid and may fragment easily. This indicates an inflammatory condition.

The clots can be graded as good, fair, poor or very poor depending on the firmness of the clot and the clarity of the surrounding fluid. If no clot forms, only flakes in a cloudy fluid are seen, it is regarded as a very poor clot.