Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

CSF Protein - Pyrogallol Dye Binding Method

     Principle of the method

Protein molecules present in CSF bind quantitatively with pyrogallol red-molybdate complex at pH 2.0 to form a violet complex, the intensity of which is measured at 600nm and compared with the colour given by a set of human protein standards.

     Reagents

All chemicals must be Analar grade

     Pyragallol red dye

Dissolve 10 mg of disodium molybdate, 5.9g of succinic acid, 134mg of sodium oxalate and 430mg of sodium benzoate in about 800 ml of distilled water taken in a one-litre volumetric flask. To this add 25 mg of pyrogallol red dye and mix well till it is completely dissolved. Make up to the mark with distilled water. Store in an amber bottle. Stable at 2-80C for 3 months.

     Standards

Refer Total Protein(page 40 (c) Standard) for the preparation of protein standard from pooled serum. Note that there is always a risk of infection from pooled serum. After determining the concentration of the protein standard, dilute this to several levels as described below.

For example, if the total protein value of the pooled serum is6.7g/dl, then the volume of pooled serum to be diluted to 100ml to get a protein concentration of 20 mg/dl is found out using the dilution formula.

IV x IC = FV x FC

IV = Initial Volume of pooled serum.

IC = Initial Concentration of total protein (mg/dl).

FV = Final Volume.

FC = Final Concentration

IV x 6700 = 100 x 20

IV = 100 x 20 20
----------- ----- = 0.3 ml
6700 67

i.e. Dilute 0.3 ml of the pooled serum to 100ml with 0.9 g/dl sodium chloride containing 0.1 g/dl sodium azide. Similarly, 40, 80 and 120 mg/dl standards are prepared by diluting 0.6, 1.2 and 1.8 ml of the pooled serum each to 100 ml with the diluent. The standards are stable at 2-8° C for one month.

The following table summarizes the preparation of diluted standards.

Concentration of protein in pooled serum is taken as 6.7g/dl.

     Equipment, glassware and other accessories

Refer to Section A (2), Introduction to SOP.

     Procedure

The protocol of the procedure is described below.

Pipette the following into appropriately labelled 13 x 100 mm tubes

Mix all tubes well. Leave at 25-35⁰C for 15 minutes. Set the spectrophotometer /filter photometer to zero using blank at 600 nm/ red filter and measure the absorbance of standards, test and QC.

     Calculation and calibration graph

Since standards and test/ QC procedures are identical, the absorbance values of standards are plotted against their respective concentrations. The calibration curve should be linear up to 120 mg/dl, with a lower limit of 4-5mg/dl.

Plot the absorbance values of test on the calibration graph and read off protein concentrations in patients’ CSF. As 1: 100 diluted QC serum is analysed, read off the protein concentration in QC on the calibration graph and multiply the value by 100 to get the correct protein value in QC serum.

Once linearity is proved, it is not necessary to prepare the standard graph every time that patients' samples are analysed. It will be adequate if standard S4 is taken every time and patients' results are calculated using the formula.

Test absorbance
---------------------------- x 120 mg/dl.
Standard absorbance

     Analytical reliabilities

Refer to pages 7-9 of section 1 (General Introduction) on the use of internal QC and interpretation of daily QC data (for releasing patients’ results).

Include one internal QC every time a patient specimen is measured, irrespective of the number of samples in a batch. However, even if only a single QC sample is analysed in a day, this value can be pooled with the preceding 10 or 20 values obtained in the previous days and the between-day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e, 8%.

At least once a week analyse another QC serum from either a low QC or high QC pool.

"Assayed" QC sera with stated values (ranges) are available from several commercial sources, viz. Boehringer Mannheim, BioRad & Randox.

If a laboratory uses QC sera from a commercial source, it is important that the company certifies that their QC materials are traceable to international reference materials.

     Hazardous materials

This procedure uses sodium azide. Do not swallow, and avoid contact with skin and mucous membranes.

     Reference range and clinical interpretation

CSF protein - 15-45 mg/dl

Normally the protein present inCSF is entirely albumin, but in many disease states, CSF contains a mixture of albumin and globulins. Increase in proteins up to 400 mg/dl is observed in meningitis and up to several grams in spinal tumour. In inflammatory lesion, increase in protein is associated with increase in cells. A marked increase is also observed in paralysis and in disseminated sclerosis.

Abnormally increased total CSF protein may be found in conditions where there is an increased permeability of the capillary endothelial barrier through which ultrafiltration occurs. Examples of such conditions include; bacterial, viral and fungal meningitis, traumatic tap, multiple sclerosis, obstruction, neoplasm and cerebral infarction.

     Limitations

Any CSF protein value > 120 mg/dl should be diluted with 0.9% saline either 1:2 or 1:3 and reassayed, and the value obtained is multiplied by the appropriate dilution factor to get the correct value. The presence of blood and pus will increase CSF protein. The report to the requesting physician should include the appearance of CSF before and after centrifugation.

     Reference

1.      Watanabe et al (1986) Clin. Chem 32 : 1551-1554