BCP ALBUMIN PROCEDURE

Intended for the Quantitative Determination of Albumin in Human Serum by the Bromcresol Purple Method

SUMMARY AND EXPLANATION

Albumin is the most abundant of the serum proteins. Its importance in body function is its ability to act as a carrier of substances such as drugs, antibiotics, bilirubin and fatty acids (1). Albumin also helps maintain the bodys osmotic pressure and serves as a storehouse of structural proteins that are used for tissue growth. Decreases in serum albumin levels occur commonly in a variety of diseases such as nephrotic syndrome, liver diseases, acute infections and malnutrition, making accurate albumin determinations of prime significance.

Many methods have been described for determining serum albumin. In 1965, Rodkey (2) introduced a simple dye-binding method based on albumins reaction with bromcresol green (BCG). The BCG method is used widely and many modifications have been described. Recently, after more intensive studies, deficiencies in the BCG methodology have become apparent. The BCG method overestimates serum albumin concentration because of its non-specific reaction with other serum proteins, reacting rapidly with albumin and then more slowly with ceruloplasmin, C3 component of complement, haptoglobin and transferrin (3). This non-specific reaction results in poor agreement of the BCG methods with the reference procedure of immunoelectrophoresis (4). Consequently, the IFCC Expert Panel on Proteins has recommended that BCG methods should be used only for screening purposes (5).

In 1968, Louderback (6) introduced a method for albumin based on reaction with bromcresol purple (BCP). BCP is quite specific for human albumin and binds instantly with no increase in color upon standing. The BCP method also shows excellent agreement with electroimmunoassay {Y(BCP) = 0.95 X(EIA) + 1.72} (6). In a study conducted by Walsh (7), it was noted that the BCP method correlated well with immunochemical procedures while no BCG method compared favorably with this reference technique. When considering cost and handling, the BCP method is recommended as the one of choice (7).

PRINCIPLE

The EAGLE BCP ALBUMIN PROCEDURE is based on the dye-binding capabilities of serum albumin with bromcresol purple. The absorbance at 600 nm of bromcresol purple increases with binding to albumin and is proportional to the concentration of albumin present.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2070 provides:

BCP ALBUMIN REAGENT - (Cat. No. 2071)

REACTIVE INGREDIENTS:

0.08 mM bromcresol purple buffered to pH = 4.9. Surfactant and antimicrobial agents added.

PRECAUTIONS:

Causes irritation. Avoid contact with eyes, skin and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 15-30° C. Stable until expiration date if kept tightly sealed.

DETERIORATION:

The reagent should be a clear yellow-green solution. Turbidity or presence of a precipitate would indicate deterioration.

ALBUMIN CALIBRATOR - (Cat. No. 2072)

REACTIVE INGREDIENTS:

Human albumin in saline. Preservative added. Consult vial label for assigned value.

PRECAUTIONS:

Do not pipet by mouth.

STORAGE AND STABILITY:

Store at 15-30° C. After opening vial, store at 2-8° C. Stable until expiration date if sealed tightly.

DETERIORATION:

Turbidity would indicate deterioration.

INSTRUMENTS

Use a suitable spectrophotometer or colorimeter calibrated at 600 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Serum is the specimen of choice.

2. Avoid gross hemolysis.

3. Highly icteric, grossly hemolyzed and lipemic samples require Serum Blank.

4. Avoid venostasis in specimen collection because hemo-concentrations will increase apparent concentrations of albumin.

SAMPLE STORAGE:

Albumin is stable for one week at 25° C or one month at 4° C (8). Avoid microbial contamination.

ADDITIVES:

No special additives or preservatives are required.

INTERFERING SUBSTANCES:

Young et al (9) have reviewed drug effects on serum albumin levels.

PROCEDURE

MATERIALS PROVIDED:

BCP ALBUMIN REAGENT (Cat. No. 2071) and ALBUMIN CALIBRATOR (Cat. No. 2072).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.02 mL micropipettor

2. 3.0 mL pipettor or dispenser

3. Test tubes / rack

4. Timer

REACTION CONDITIONS:

Wavelength 600 nm

Filter Selection 580 - 620

Reaction Type Endpoint

Incubation Temperature 15-30° C

Incubation Time 5 minutes

Sample Volume 0.01 mL

Reagent Volume 1.0 mL

Total Volume 1.01mL

Low Normal 3.5 g/d

High Normal 5.0 g/dL

Calibrator Value See Vial Label

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE:

For instruments requiring a total volume of >>1.0 mL, increase BCP ALBUMIN REAGENT volume to 2.0 mL and sample volume to 0.02 mL. Proceed as outlined.

1. Dispense 1.0 mL BCP ALBUMIN REAGENT into tubes labeled: Blank, Calibrator, Control, Sample 1, etc.

2. Place 0.01 mL Specimen into appropriately labeled tube. Mix well. Use deionized water as specimen for Reagent Blank.

3. Permit test samples to stand at room temperature (15-30° C) for 1 minute.

4. Adjust instrument to zero absorbance at 600 nm using Reagent Blank.

5. Read and record absorbance values for Calibrator, Controls and Unknowns.

NOTE: For a direct read-out instrument, zero with Regent Blank and set read-out to concentration of Calibrator. Read the Unknown concentration directly.

STABILITY OF FINAL REACTION PRODUCT:

The final color is stable for at least 60 minutes.

SERUM BLANK:

High icteric, grossly hemolyzed and lipemic samples require blank correction.

1. Dispense 1.0 mL of 0.9% Sodium Chloride solution into a tube.

2. Add 0.01 mL Specimen. Mix well.

3. Zero instrument at 600 nm with 0.9% Sodium Chloride solution.

4. Read and record Serum Blank absorbances.

5. Subtract the Serum Blank absorbance from the Unknown absorbance obtained in Step 5 of Performance of Test. Use this value in calculating the Unknown concentration.

CALIBRATION:

It is not necessary to determine a calibration curve for this procedure since the reaction is linear in the range of 0.5 - 8.0 g/dL serum albumin. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use the ALBUMIN CALIBRATOR which is provided in the reagent set for this purpose.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner employed for the unknowns. EAGLE DIAGNOSTICS offers CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera should be thoroughly investigated before patient values are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentration:

Unknown (g/dL) =

Abs. Unk

--------------- .X Cal. Conc. (g/dL)

Abs. Cal.

EXAMPLE:

A 4.0 g/dL Calibrator had an Abs. = 0.471 while the Unknown Abs. = 0.508. The albumin concentration of the Unknown is:

0.508  

----------- X 4.0 = 2.1 g/dL

0.471

LIMITATIONS

Calibrators and controls containing human albumin should be used with this method since dye-binding properties of albumin differ among species.

EXPECTED VALUES                 3.5 - 5.0 g/dL

This range represents the 95% confidence interval obtained from a clinically normal population. Each laboratory should establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

The reaction is linear in the range of 0.5 - 8.0 g/dL.

PRECISION:

Within Run - Normal and Abnormal sera were assayed 20 times each for within run precision.

MEAN / ST. DEV. / %CV

Normal 4.24 / 0.076 / 1.80

Abnormal 3.02 / 0.056 / .86

Run to Run - Normal and Abnormal sera were assayed for 10 working days to establish run to run precision:

MEAN / ST. DEV. / %CV

Normal 4.31 / 0.104 / 2.41

Abnormal 3.09 / 0.061 / 1.97

SPECIFICITY:

It has been well established that BCP methods are more specific for human albumin than BCG methods (3, 5, 6). To establish what a laboratory should expect when comparing the EAGLE method to their BCG method, a comparison was made to a widely used BCG commercial method. Results showed a 98.6% correlation for 20 samples in the normal and abnormal ranges, where y (BCP) = 1.061 x (BCG) - 0.448

SENSITIVITY:

This BCP ALBUMIN PROCEDURE has a sensitivity of 0.01 g/dL per 0.001 absorbance unit.

REFERENCES

1. Lynch, M.J., Medical Laboratory Technology and Clinical Pathology,2nd ed., W.B. Saunders Co., Philadelphia, 1969, p. 219.

2. Rodkey, F.L., Clin, Chem. 11, 478 (1965).

3. Duggan, J., and Duggan, P.F., Clin. Chem.28, 1407 (1982).

4. Webster, D., Bignell, A.H.C., and Attwood, E.C., Clin. Chem. Acta 55, 101 (1974).

5. Pinnell, A.E., and Northam, B.E., Clin. Chem. 24, 80 (1978).

6. Louderback, A., Mealy, E.H., and Taylor, N.A., Clin. Chem. 14, 793 (1968).

7. Walsh, R.L., Clin. Biochem. 16, 178 (1983).

8. Henry, R.J., Clinical Chemistry Principles and Techniques, 2nd ed., Harper & Row, Hagerstown, 1974, p. 413.

9. Young, D.S., Pestaner, L.C., and Gibberman, V., Clin. Chem. 21, 244D (1975).