Albumin Assay – Bromocresol Green method

Practical Documentation:

Aims

To perform serum albumin determinations on samples, explain the principle behind the Bromocresol Green method for albumin measurement and to list the factors that will cause interference with this method.

Principle

Albumin is known for its ability to bind many types of organic compounds, including organic dyes. When albumin selectively binds with Bromcresol Green (BCG) it causes a change in the absorbance maximum of BCG. The intense blue-green complex that is formed has an absorbance max of 670nm. Bromocresol reagent at pH 4.3 is negatively charged. The pI of albumin is 4.7.

For all spectrometric assays, always use a Reagent
blank. It usually contains all diluents and reagent in the reaction
solution, but no sample. Some reagent blanks do contain the sample as well, but
they lack one crucial reagent component needed to produce a colour-yielding
reaction. This is different from the water used to zero a spectrophotometer
(set 100% T).

Materials:

  1. Albumin Stock Solution (100g/L)
    or Cobas calibrator
  2. Serum patient samples (record
    the albumin results off track)
  3. Cobas albumin controls
  4. Bromocresol green reagent (BCG)
    – obtain Cobas reagent or make up as follows:
  5. 7.5mg of
    BCG
  6. 250mg
    EDTA
  7. 250µL
    Tween 20
  8. Bring up
    to 25mL with water

Methods:

  1. Generate a calibration curve of
    at least 8 standards (0- 80 g/L) by diluting the Albumin Stock (100 g/L).
  2. In labelled tubes, set up a
    calibration, controls and test samples as follows:
Sample Water (µL) Std (µL) Control (µL) Sample (µL) BCG Reagent (µL)
Blank 310 – – – 300
Calibrators 300 10 – – 300
Control 300 – 10 – 300
Serum Sample 300 – – 10 300
Figure 1. Setting up the standards for the calibration curve.

To set up a standard curve with more points along the usable range, I chose more points in the concentration range 20 – 40 g/L, which in my experience constitutes the bulk of albumin measurements.

3. Mix well read immediately at 630nm and record absorbances

Layout of
wells
Dilution as
per manual		   Dilution of
1:2 with H2O		  
  1 2 3 4
A Standard 0 Unknown 1 Standard 0 Unknown 1
B Standard 1 Unknown 2 Standard 1 Unknown 2
C Standard 2 Unknown 3 Standard 2 Unknown 3
D Standard 3 Unknown 4 Standard 3 Unknown 4
E Standard 4 Unknown 5 Standard 4 Unknown 5
F Standard 5 QC Low Standard 5 QC Low
G Standard 6 QC High Standard 6 QC High
H Standard 7   Standard 7  
Layout of wells
Layout of wells
Absorbance Absorbance
Standard concentration (g/L) No dil Dil 1:2 (150uL reagent mix + 150uL H2O
0 0.095 0.065
20 0.506 0.245
25 0.719 0.345
30 0.779 0.395
35 0.784 0.411
40 0.999 0.504
50 1.242 0.593
80 1.477 0.68

4. Plot a standard curve and determine experimental concentrations of controls and serum samples

Figure 2 – Deriving the formula was done with a statistical Calculator by entering the respective X- and Y-values into a table and obtaining a slope and y-intercept by linear regression. Only the neat reagent mix’s formula was done manually with a calculator. Slope (b) was calculated to be 0.01769 and y-intercept (a) was 0.206.
These values corresponded to the values when plotting an X-Y plot on Microsoft Excel (see 2 standard curve data plots above).

The figure below illustrates the formula used to determine the unknown concentrations.

Unknown concentrations were calculated as follows:

Unknowns
Sample no.		 Absorbance Absorbance
(Diluted 1:2)		   Calculated concentration Calculated concentration
(Diluted 1:2)		 Reference albumin values (from Roche Cobas 6000)
1 0.893 0.48 Hemolysed 38.8 44.2 29.1
2 1.057 0.55   48.1 53.0 44.2
3 0.614 0.334   23.1 26.0 22
4 0.997 0.5 Hemolysed 44.7 46.7 38.6
5 0.894 0.479   38.9 44.1 33.2
Lo 0.843 0.453   36.0 40.9 31.9
Hi 1.161 0.593   54.0 58.4 49.4

Compare results to expected results and comment on any differences between the manual BCG vs automated BCP assays.

Figure 3 – Manual BCG method vs. Automated BCP method (Roche). It can be seen that the manual BCG method overestimated the albumin concentration in all serum samples.
A possible explanation for this difference is:
1. Pipetting error
2. Interferring substances in the serum which absorb light at 670nm.
3. It may be due to the fact that albumin stock solution was made up with water as opposed to physiological albumin-free serum matrix.
4. Hemolysis – Evidenced by one of the hemolysed samples which clearly measured falsely high.

A haemolysed sample is brought to the laboratory for albumin analysis. Can the sample be used? Discuss.

Yes. Hemoglobin does not absorb light at 670 nm, therefor will not interfere significantly with the analysis. See figure below:

It does however interfere in the following manner:

Hemoglobin decreases the apparent albumin concentration by 1 g/L for each 100 g/L added. Blanking does not correct this interference, and the negative bias is therefore caused by interference with the dye binding rather than hemoglobin color. For the BCP method, a blank correction is required on icteric sera and on grossly hemolyzed and grossly lipemic sera to correct for an underestimation of albumin caused by these agents. Heparin causes a positive interference with BCP and BCG methods. This interference can be eliminated by the addition of hexadimethrine bromide to a concentration of 50 mg/L in the BCP reagent

Kaplan’s Methods

Why is it not desirable to incubate the reaction before measuring the absorbance?

Incubating the sample can give rise to other non-specific binding of analytes in the sample to the chromophore dye and a falsely elevated reading can be obtained.

Some analyzers can measure the absorbance of the BCG reaction within 30 seconds after adding sample. Does this tend to increase the specificity of the reaction?

Yes. The less time there is for other interfering substances to bind to BCG, potentially the more specific it will be to albumin, as albumin is the more specific binding to BCG.

Probably the most promising adaptation of the BCG reaction for albumin analysis utilizes fast reaction readings. Gustafsson reported that measuring the absorbance of the BCG-protein complex at 629 nm at a time shortly after mixing improves the specificity of the assay. Interference by other proteins such as ceruloplasmin and orosomucoid becomes significant at times greater than 5 minutes.

Kaplan’s Methods