Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Bilirubin – Jendrassik & Grof method

     Introduction

Bilirubin is formed from the haem fragment of haemoglobin released by aged or damaged red blood cells. Liver, spleen and bone marrow are the sites of bilirubin production. Bilirubin formed in spleen and bone marrow is transported to the liver. In the liver it is converted into bilirubin conjugates – bilirubin mono and diglucuronides. Any liver disease affects the above systems, and hence bilirubin accumulates in serum leading to jaundice.

     Principle of the method

Conjugated (direct) bilirubin in serum is coupled with diazotised sulphanilic acid to form a red coloured compound. Ascorbic acid is used to stop the coupling reaction, and to eliminate interference by haemoglobin. Caffeine benzoate solution is used to split the unconjugated bilirubin protein complex releasing the bilirubin so that it can react with diazotised sulphanilic acid.

The tartrate buffer makes the mixture alkaline and converts the red acid bilirubin to a green coloured compound which shows peak absorbance at 607 nm. At this wavelength the absorbance due to haemoglobin or carotene is minimal.

     Specimen type, collection and storage

Use only clear, non-haemolysed samples of serum. Bilirubin is unstable and light sensitive and therefore the assay should be carried out within 2 hours of sample collection. If a longer delay is unavoidable, refrigerate the sample. Samples can be frozen at –20⁰C, to keep bilirubin stable for 2 months.

     Reagents

All chemicals must be Analar grade

     Caffeine-benzoate

Dissolve 100g caffeine sodium benzoate and 25 g sodium benzoate together in 800 ml of distilled water. Heat the solution to 60⁰C and then add 125g of hydrated sodium acetate and 1g of EDTA. Mix to dissolve and then make up to 1 litre with distilled water. Filter the solution. Store at room temperature (25-30⁰C). Stable for 6 months.

     Sulphanilic acid

Dissolve 5 g sulphanilic acid in 500ml distilled water with heating. Add 15ml of conc. HCl and when cool make up to 1 litre. Store at room temperature (25-30⁰C). Stable for 6 months.

     Sodium nitrite

Dissolve 500 mg sodium nitrite in about 80ml distilled water and then make up to 100 ml. Store at 2-8⁰C. Prepare fresh once a month.

     Diazo reagent

Mix 10ml sulphanilic acid with 0.25ml sodium nitrite. The solution is stable for approximately 3 hours at room temperature (25-30⁰C) and 24 hours at 2-8⁰C.

     Alkaline tartrate

Dissolve 100g NaOH and 350 g sodium potassium tartrate in distilled water and make up to 1 litre. Store at room temperature (25-30⁰C). Stable for 6 months.

     Ascorbic acid (4 g/dl)

Dissolve 200 mg of ascorbic acid in 5ml of distilled water. This solution must be freshly prepared each day.

     Equipment, glassware and other accessories

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

     Procedure

The protocol of the procedure is described below.

Add reagents, standards and test samples/QC in the order indicated into appropriately labelled tubes (18 x 150mm)

Mix all tubes. Set the spectrophotometer/ filter photometer to zero with distilled water at 607nm/ orange filter and read the absorbance in the order of assay tubes mentioned in the Table.

     Preparation of bilirubin standard

Commercially available bilirubin is water insoluble but the addition of a small amount of dimethyl sulphoxide (DMSO) and NaOH will dissolve it. As diluent, non-icteric and non-lipaemic human pooled serum tested negative for HIV antibodies and Hbs antigen could be used. However, there is always a risk of infection from this material.

Pool daily leftover normal human sera until about 120 ml are collected. Take an aliquot for screening for the presence of HIV antibodies and Hbs antigen and ensure that both are negative. Centrifuge the pooled serum twice at 3500 rpm for 10 minutes and collect the serum in a clean container. Measure the total bilirubin of the pooled serum to ensure that it is < 0.5 mg/dl.

Weigh accurately 10mg bilirubin in a small stoppered glass tube. Add 2.0 ml DMSO and 0.5 ml of 0.4M NaOH and shake the tube in dim light until the bilirubin has dissolved (It may be necessary to warm the tube in a 37⁰C water-bath to speed up this step).

Transfer about 75ml of the pooled serum into a 100 ml volumetric flask. Add the bilirubin solution slowly with continuous mixing to the serum. Rinse the tube with a few drops of DMSO and then with pooled serum and add to the flask. Make up to the mark with the pooled serum. Froth can be dispersed by touching with a glass rod minimally smeared with silicone or by adding a trace of capryl alcohol (octan –2-ol). Wrap the flask with carbon paper and store it at 2-8⁰C until the standarization procedure is complete. This bilirubin standard will have a concentration of about 10-11 mg/dl. The exact value will be determined after carrying out the procedure outlined in 3.5.7

     Calculation and calibration graph

     Extrapolation method

Prepare standards (S₁-S₅) as shown below

Proceed with bilirubin estimation in the usual way as for total bilirubin. Measure the absorbance of all standards against distilled water in a spectrophotometer at 607 nm/ orange filter in a filter photometer. Construct a calibration graph by plotting the theoretical bilirubin concentrations against the corresponding absorbance values. This graph will not pass through the origin, instead it will intercept the ‘y’ axis at a specific point, indicating that the blank tube contains a certain amount of bilirubin corresponding to the ‘y’ intercept absorbance value. Upon extrapolation of the calibration graph, this will read a specific value of bilirubin on the negative side of the x axis (concentration axis). In the sample graph given below (graph I), the bilirubin content of the blank is shown as 0.5 mg/dl. This value must be added to the amount of bilirubin dissolved in 100 ml of pooled serum. Therefore, in this example the actual bilirubin content in the standard S5 will be 10+0.5 = 10.5 mg/dl. Hence the levels of bilirubin in the diluted standards from S4 down to S1 will vary accordingly. This is explained in the Table given below.

The calibration graph with corrected bilirubin values should then be plotted against absorbance values as shown in graph II. The test/ QC absorbance values should be plotted on this graph and concentrations read off. The measurable range with this graph is from 0.2 to 20.0 mg/dl.

Once linearity is proved, it will be enough if a single standard is set up every time that patients’ samples are analysed and the results are calculated using the formula:

Test absorbance - Test blank absorbance (TBK Concentration
---------------------------------------------------------- x of Bilirubin
Std. Absorbance – Std blank absorbance (SBK) standard

Test (total) absorbance-TBK Concentration
1. Total Bilirubin mg/dl = ----------------------------------- x of Bilirubin
Standard absorbance –SBK standard

Test (direct) absorbance -TBK Concentration
2. Direct Bilirubin mg/dl = ---------------------------------- x of Bilirubin
Standard absorbance –SBK standard

     Storage of standard

Aliquot small volumes of standard into screw-capped vials and store in the freezer on the same day it is prepared. Stable for 2 months at -20⁰C. Do not refreeze leftover standard after use.

     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 in every batch of samples analysed every day irrespective of the number of samples in a batch. Since bilirubin is analysed in a single batch in a day in an intermediate laboratory, it will not be possible to analyse several QC samples and calculate within-day precision. However, even if 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 between-day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e, 10%.

Once a week it is good to 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 method uses sulphanilic acid and sodium hydroxide. Avoid contact with eyes, skin and mucous membranes.

     Reference range and clinical interpretation

Serum Direct Bilirubin - up to 0.5 mg/dl

Serum Total Bilirubin - 0.2 – 1.0 mg/dl

Hyperbilirubinaemia is characteristic of jaundice. Increase in unconjugated bilirubin is observed in haemolytic and neonatal jaundice.

In viral and toxic hepatitis there is impaired hepatocellular conjugation and excretion of bilirubin with a major rise in conjugated and a lesser rise in unconjugated bilirubin in serum. In cirrhosis there is overall damage to liver cells and hence the ability of the liver to form conjugated bilirubin, resulting in an increase in unconjugated bilirubin in serum. In obstructive jaundice there is an increase in predominantly conjugated bilirubin in serum.

     Limitations

Samples with bilirubin concentrations higher than 20mg/dl should be diluted with an equal volume of distilled water and the result obtained should be multiplied by 2. There is no interference in the assay by haemoglobin up to a concentration of 1.0g/dl; however, strong haemolysis will interfere negatively with measurement. Do not report results for specimens with suspected interference. Inform the requesting physician of the problem.

     Reference

1.      Doumas BT, Kwok-Cheung PP, Perry BW, et al. Clin. Chem.

2.      (1985) 31 : 1779-89.

3.      Tietz. NW. Fundamentals of Clinical Chemistry.

4.      Published by WB Saunders Company. 1986. page 1388-1390.