Guidelines on Standard Operating Procedures for CLINICAL CHEMISTRY

Albumin - BCG Dye Binding Method

     Introduction

Serum albumin consists of a single species of proteins representing approximately 60% of the total protein. It is synthesized exclusively in the liver and functions as a regulator of blood oncotic pressure, as a carrier for many cations and water insoluble substances, and as a pool of aminoacids for caloric or synthetic purposes.

     Principle of the method

Albumin binds quantitatively with bromocresol green at pH 4.15 resulting in the formation of a green colour which can be measured at 630nm/red filter.

     Specimen type, collection and storage

Either serum or plasma may be used, but serum is preferred. A fasting specimen is not required but may be desired to decrease lipaemia. Avoid haemolysis. Tightly stoppered samples are stable for 24 hours at room temperature (25-35⁰C), one week at 2-8⁰C and for 3 months at -20⁰C.

     Reagents

All chemicals must be Analar grade

     Sodium hydroxide 1 M :

Weigh out 4.0 g of sodium hydroxide (NaOH), dissolve and make up to 100ml with distilled water. This solution is stable for several months at room temperature (25-35⁰C) in a polypropylene container.

     Brij - 35........... 30g/dl

Readily available at the above concentration from S.D Fine chemicals or Loba Chemical Company, in India.

Solid Brij can also be obtained from Sigma Co. In this case, warm 30g solid Brij in a beaker in a small volume of distilled water to dissolve and make up to 100ml with distilled water.

     Bromo Cresol Green (BCG) dye solution

Transfer 25ml of I M NaOH into a one-litre volumetric flask containing 600ml distilled water. Add 5.6g succinic acid and then add 56 mg of BCG powder. Mix and then make up to 1 litre with distilled water. Check the pH. If it is less than 4.15, adjust to 4.15 + 0.05 by the dropwise addition of 1 M NaOH.

Add 100 mg sodium azide and 3.5ml 30 g/dl Brij-35 to the reagent. Check the absorbance of the reagent at 630 nm/ red filter against distilled water. It should be less than 0.2. If it is greater than 0.2, add some more Brij to bring down the absorbance. Store ina polyethlyene container. Stable for 6 months at room temperature (25-35⁰C).

     Standard

In many biochemical estimations direct standardization is employed; for example, glucose urea and creatinine are available in pure forms, and can be directly weighed and used, whereas, in the case of human albumin, this is not readily available. The laboratory can obtain albumin standard from commercial firms or prepare the standard in-house.

The method of in-house preparation of the standard is described below:

Prepare pooled serum as described in Section 1 General Introduction under ‘Preparation of QC Pool’. The laboratory should pool daily leftover patients' sera. For albumin standard, only the use of human serum is recommended, since it has been documented that the affinity of BCG to bovine albumin is different from its affinity to human albumin beyond 3g/dI. After aliquoting and freezing the pooled serum, analyse albumin in the aliquots daily for a period of 20 days using a reliable albumin standard (commercial source or non-commercial source such as WHO). Calculate the mean value and assign this as the standard value.

Note: There is always a risk of infection from this material.

     Equipment, glassware and other accessories

Refer Section A (2), Introduction to SOP.

     Procedure

The protocol of the procedure is described below.

     Dilution of Standards (S1 -S4), Test & QC

Pipette the following into appropriately labelled 13 x 100mm tubes

Mix well

     Colour Development

Pipette the following into another set of appropriately labelled 18 x 50mm tubes

Mix all tubes well. Incubate at room temperature (25-35⁰C) for 10 minutes. Set the spectrophotometer /filter photometer to zero using blank at 630 nm/ red filter and measure the absorbance of standards, test & QC.

     Calculation and calibration graph

Since the protocol for standard tube S2 and the test is identical, standard S2 will represent the actual concentration of the pooled serum used as standard. Standard S1 contains half the volume of S2 , S3 has 1½times and S4 has twice the volume of S2. Therefore albumin concentrations represented by S1, S3 and S4 will be ½, 1 ½ and 2 times of S2 concentration respectively. For example, if S2 concentration is 3g/dl, SI will be 1.5g/dl, S3 will be 4.5g/dl and S4 will be 6g/dI.

Plot the absorbance values of standards against their respective concentrations. The measurable range with this graph is from 0.5 to 6.0 g/dl. Plot the absorbance values of test/QC on the calibration graph and read off the concentrations.

Once linearity is proved, it isnot necessary to prepare the standard graph every time when patients' samples are analysed. It will be adequate if standard S2 is taken every time and patients' results are calculated using the formula :

Test absorbance
-------------------- x Concentration of S2 g/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 in every batch of samples analysed every day irrespective of the number of samples in a batch. Since albumin is analysed in a single batch once a day in an intermediate laboratory, it will not be possible to analyse several QC samples and calculate within-day precision. 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 between-day precision can be calculated and expressed as %CV. Ensure that this is well within the acceptable limit, i.e, 6%.

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

Sodium hydroxide used in this procedure is a strong alkali and is caustic. Do not swallow, and avoid contact with skin and mucous membranes.

     Reference range and clinical interpretation

Serum albumin - 3.5-5.0 g/dl

Serum levels of albumin are used to assess nutritional status and have important influences on the metabolism of endogenous substances such as calcium, bilirubin and fatty acids and on the effect of drugs and hormones. Hyperalbuminemia has little diagnostic significance except in dehydration. Hypoalbuminemia is very common in many illnesses like impaired synthesis (liver disease), increased catabolism, reduced aminoacid absorption, protein loss in urine, malnutrition and protein losing enteropathy, and in hospital patients with acute illness.

     Limitations

Haemolysed and lipaemic sera interfere strongly with the measurement of albumin. Set up appropriate blank for such samples by adding 0.1 ml of serum to 4.5 ml of sodium chloride diluent (Refer Total Protein).The absorbance obtained is then subtracted from its test absorbance before calculating the test result.

     Reference

1.      Spencer K and Price C.P. (1977). Ann. Clin. Biochem 14, 105-115.