1.0 INTENDED USE
This reagent is intended for the quantitative
determination of albumin in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Methodologies for the determination of serum albumin and
globulin include electrophoresis, salt fractionation, glyoxylic acid‑tryptophan
reaction, and dye binding techniques (10.1‑10.3). The electrophoresis and salt fractionation
methodologies are too involved for routine or stat work. The bromocresol green method for
determination of serum albumin is the most specific and sensitive of the dye
binding techniques (10.4). The glyoxylic
acid method measures tryptophan content which represents 8‑10% albumin
and 90‑91% globulin. Since the
bromocresol green method is specific and simple, it is the method of choice for
albumin determination.
2.2 TEST PRINCIPLE
Serum
albumin reacts with bromocresol green to form a colored complex. The
color is proportional to the amount of albumin present.
2.3 CLINICAL SIGNIFICANCE
Elevated levels of albumin are often indicative of
dehydration, rather than the presence of a pathological condition. Low albumin levels are often associated with
edema, and are also found in nephrotic conditions due to loss of albumin into
the urine.
Serum albumin is synthesized in the liver. Hepatic disorders, such as cirrhosis and
hepatitis, interfere with protein synthesis and will lower the level of albumin
in circulation.
3.0 SPECIMEN COLLECTION AND HANDLING
3.1 PATIENT PREPARATION
No special
patient preparation is required.
3.2 SPECIMEN COLLECTION.
Fresh, clear, unhemolyzed serum is the preferred specimen. Hemolyzed samples should not be used.
Use a
standard venipuncture tube to draw patient sample.
The amount of sample required will depend on the analyzer
used. The amount of serum required is in
the range of 5-25 µl. Call Biotron's
technical service department at 1-800-595-8766 for the recommended sample
volume for your analyzer.
Record the patient's name, date and time of sample
collection and preparation.
3.3 SPECIMEN STORAGE
In the absence of bacterial contamination, serum albumin
levels remain unchanged after 4 days at room temperature (18°-26° C,) 2 weeks
refrigerated (2°-8° C,) or for 6 months frozen (-20° C.) Frozen samples should be thawed at room
temperature and mixed thoroughly before analysis. Thawed samples should not be refrozen. (10.5)
It is recommended that testing be done as soon as
possible after sample collection and preparation. If testing cannot occur within 4 hours of
collection, store the sample properly using the guidelines above.
4.0 MATERIALS
(2 X 125 ml)
(1 X 500 ml)
Reagents necessary for the determination of albumin are included in the kit.
4.1 ALBUMIN REAGENT
Albumin
reagent contains:
bromocresol green sodium
salt 0.21
mM
sodium citric buffer 104
mM
preservatives and stabilizers
4.2 WARNINGS AND PRECAUTIONS
For In Vitro Diagnostic Use. Not for Internal use in Humans or
Animals. In Vitro Diagnostics reagents
may be hazardous. Avoid ingestion and
skin or eye contact.
4.3 REAGENT PREPARATION
The
reagent is ready to use as is.
4.4 REAGENT STORAGE AND STABILITY
When stored at room temperature (18‑26° C) the reagents are stable until the expiration
date stated on the labels.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance at 630 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 630 nm.
4.5.3 Test tubes
capable of holding 3 ml.
4.5.4 Pipettes
capable of delivering 2 ml and 10 µl.
4.5.5. Timer for a 1
minute incubation.
4.5.6 Calibrator
4.5.7 Normal and
abnormal control for quality control.
5.0 TEST PROCEDURE
The
following is a general procedure for use on a manual instrument.
5.1 PROCEDURE CONDITIONS
Wavelength 630
nm
Temperature 18‑26°
C or 37° C
Pathlength 1.0
cm
Mode endpoint
Reaction time 1
minute
Sample volume 10
µl
Reagent volume 2.0
ml
Total volume 2.01
ml
Sample to reagent
ratio 1/200
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 630 nm may be used. The band width should be 10 nm or less, stray
light 0.5% or less, and the wavelength accuracy within 2 nm.
5.3 CALIBRATION
5.4 The albumin assay is calibrated by referencing the
absorbance of the unknown sample to the absorbance of the calibrator.
5.4 PROCEDURE
5.4.1 Into separate
test tubes pipette 10 µl of distilled water, calibrator, or serum to be
assayed.
5.4.2 Add 2.0 ml of
albumin reagent and mix.
5.4.3 Incubate at
18‑26° C (room temperature) for 1 minute.
5.4.4 Determine the
absorbance of the calibrator (As) and of each unknown (A) at 630 nm using the
distilled water as the reagent blank.
5.5 PROCEDURE NOTE
The color
of the final reaction mixture is stable for 1 hour.
5.6 CALCULATION AND RESULTS
A
albumin (g/dl)
= ‑-‑‑‑ X
albumin value of calibrator
As
A =
absorbance of sample
As =
absorbance of calibrator
Example:
.650
albumin
concentration = -----‑‑‑‑ X 4.0 g/dl = 4.8 g/dl
.540
with A =
.650, As = .540, albumin value of calibrator = 4.0 g/dl
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.5)
The range
of expected values is: 3.2 ‑ 5.3 g/dl
These values are suggested guidelines. It is recommended that each laboratory
establish the normal range for the area in which it is located.
6.2 MEDICAL ALERT VALUES (10.7)
Each laboratory should establish low and high values
beyond which the patient would require immediate attention by a physician. If a "medical alert value" is
reached, always repeat the test to confirm the result and notify a physician if
the result is confirmed.
6.3 LIMITATIONS OF PROCEDURE
A comprehensive list of drugs and other substances which
can affect the albumin concentration in serum is given by Young. (10.6)
7.0 QUALITY CONTROL
Standard practice for quality control should be applied
to this system. Commercially available
lyophilized controls can be used to monitor the daily acceptable
variations. Normal and abnormal controls
should be assayed at the beginning of each run of patient samples, whenever a
new reagent or a different lot number is being used, and following any system
maintenance.
A satisfactory level of performance is achieved when the
analyte values obtained are within the "acceptable range" established
by the laboratory.
8.0 CALIBRATION PROCEDURES
The albumin assay is calibrated by referencing the
absorbance of the unknown sample to the absorbance of the calibrator. Refer to your instrument manual for more
details.
Calibration is required with the use of a new lot of
reagent, any system maintenance or whenever indicated by quality control data.
9.0 PERFORMANCE CHARACTERISTICS
9.1 PRECISION
The estimates of precision shown below were obtained from assays of human control serum.
Within-Run
In this
study, 15 replicates of 2 control sera were run.
Mean
(g/dl) SD (g/dl) CV
(%)
2.19 ± 0.02 1.14
3.73 ± 0.09 2.29
Between-Run
In this study, 5 runs were made, each run consisting of 5
replicates of 2 control sera.
Mean
(g/dl) SD (g/dl) CV (%)
2.96 ± 0.11 3.58
3.49 ± 0.16 2.96
9.2 CORRELATION
A correlation study was done on the Technicon RA-500
system at 37° C comparing this method and a similar albumin (BCG) method. The samples range between 0.3 g/dl and 5.8
g/dl.
Number Regression Equation Correlation
of
Samples y=Biotron, x=Comparative Coefficient
48 y = .905 x + .169 0.997
9.3 LINEARITY
This procedure is linear through 8 g/dl beyond which the
specimen should be diluted with an equal volume of deionized water. Reassay the
specimen and multiply the results by 2.
10.0 REFERENCES
10.1 Bigat, T.K.,
and Saifer, A., Clinical Chemistry 18:630,1972
10.2 Miyada, D.S.,
et.al., Clinical Chemistry, 18:52,1972
10.3 Reece, R.L.,
and Hobbie, R.K., American Journal of Clinical Pathology, 57:664
10.4 Doumas,
B.T.,and Biggs, H.G., Standard Methods of Clinical Chemistry, Vol. 7, Academic
Press, N.Y., 1972
10.5 Tietz, N.W.,
Fundamentals of Clinical Chemistry, W.B. Saunders
10.6 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed.,
10.7 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704