BCG ALBUMIN PROCEDURE

Intended for the Quantitative Determination of Albuminin

SUMMARY AND EXPLANATION

Albumin is the most abundant of the serum proteins. Its importance in body function is its ability to transport substances such as drugs, antibiotics, bilirubin and fatty acids (1). Albumin also helps maintain body osmotic pressure and serves as a storehouse of structural proteins that are used for tissue growth. Hyperalbuminemia is of little diagnostic significance except in cases involving dehydration. Lowered 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 albumin reaction with bromcresol green (BCG). The BCG method is widely used and several modifications have been described (3).

PRINCIPLE

The EAGLE BCG ALBUMIN PROCEDURE is based on the dye-binding properties of serum albumin with bromcresol green. The absorbance of BCG at 630 nm increases with binding to albumin and is proportional to the albumin concentration present.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2050 provides:

BCG ALBUMIN REAGENT - (Cat. No. 2051)

REACTIVE INGREDIENTS:

0.066 mM bromcresol green buffered to pH = 4.2. Surfactant and antimicrobial agent 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 15-30ºC. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear yellow-green solution. Turbidity would indicate deterioration.

ALBUMIN CALIBRATOR - (Cat. No. 2072)

REACTIVE INGREDIENTS:

4.0 g/dL human albumin in saline. Preservative added.

PRECAUTIONS:

Do not ingest.

STORAGE AND STABILITY:

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

DETERIORATION:

Turbidity would indicate deterioration.

INSTRUMENTS

Use a suitable spectrophotometer or colorimeter calibrated at 630 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 25ºC or one month at 4ºC (4). Avoid microbial contamination.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Young et al (5) have reviewed drug effects on serum albumin.

PROCEDURE

MATERIALS PROVIDED:

BCG ALBUMIN REAGENT (Cat. No. 2051) and ALBUMIN CALIBRATOR (Cat. No. 2072).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.01 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. Test tubes / rack

4. Timer

REACTION CONDITIONS:

Wavelength 630 nm

Filter Selection 610 - 650 nm

Reaction Type Endpoint

Incubation Temperature 15-30ºC

Incubation Time 5 minutes

Sample Volume 0.01 mL

Reagent Volume 1.0 mL

Total Volume 1.01 mL

Low Normal 3.5 g/d

High Normal 6.0 g/dL

Calibrator Value 4.0 g/dL

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume of 1.0 mL, increase BCG ALBUMIN REAGENT volume to 2.0 mL and samle volume to 0.02

mL. Proceed as outlined.

1. Dispense 1.0 mL BCG ALBUMIN REAGENT into tubes labeled: Reagent 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 (15-30ºC) for 5 minutes.

4. Adjust instrument to zero absorbance at 630 nm using Reagent Blank.

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

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

STABILITY OF FINAL REACTION PRODUCT:

The final color is stable for at least 60 minutes.

SERUM BLANK:

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

2. Add 0.01 mL Specimen and mix well.

3. Zero the instrument at 630 nm with 0.9% Sodium Chloride solution.

4. Read and record Serum Blank absorbance.

5. Subtract the Serum Blank absorbance from the Unknown absorbance obtained in Step 5 of MANUAL PROCEDURE. 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 - 6.0 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 has Abs. = 0.230 while the Unknown Abs. = 0.195. The albumin concentration of the Unknown is:

0.295  

----------- X 4.0 g/dL = 3.4 g/dL

0.330

LIMITATIONS

1. Calibrators and controls containing human albumin should be used with this  method since dye-binding properties of albumin differ among species (6).

2. The binding of BCG to albumin is not wholly specific, some dye binds to a2 and b2 - globulins, although more slowly than albumin (8).

EXPECTED VALUES 3.5 - 5.5 g/dL

This range represents the 95% confidence interval obtained from a clinically normal population (7). It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear in the range of 0.5-6.0 g/dL.

PRECISION:

Normal and Abnormal sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

WITHIN RUN:

MEAN / STD. DEV. / %CV

Normal 4.05 / 0.065 / 1.60

Abnormal 2.91 / 0.054 / 1.86

RUN TO RUN:

Normal 4.11 / 0.104 / 2.53

SPECIFICITY:

A comparison of this BCG ALBUMIN PROCEDURE with another widely accepted BCG albumin commercial method showed a 98% correlation for 28 samples in the normal and abnormal range.

SENSITIVITY:

This BCG ALBUMIN PROCEDURE has a sensitivity of 0.017 g/dL per 0.001 absorbance units.

REFERENCES

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

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

3. Doumas, B.T., Watson, W.A., and Biggs, H.G., Clin. Chem. Acta 31, 87 (1971).

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

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

6. Spencer, K., and Price, C.P., Anals Clin. Biochem. 14, 105 (1977).

7. Tietz, N.W., Textbook of Clinical Chemistry, W.B. Saunders Co., Philadelphia, p. 26, 1986.

8. Tietz, N.W., Fundamentals of Clinical Chemistry, 2nd ed, W.B. Saunders Co., Philadelphia, 1986, p. 589.