1.0 INTENDED USE
This
reagent is intended for the quantitative determination of
total
calcium concentration in serum.
2.0 BACKGROUND
2.1 METHOD AND HISTORY
Historically, serum calcium has been determined by a
variety of procedures including flame photometry, atomic absorption
spectrophotometry and recently specific dye binding such as o-cresolphthalein
complexone. Accurate and precise measurement
of calcium in biological fluids has traditionally been difficult (10.1.) The introduction of Arsenazo III by
Michaylova and Illkova (10.3) provided a highly sensitive and very specific
reagent for calcium determination. The
King method is based on this dye-binding method.
2.2 TEST PRINCIPLE
This method for the determination of calcium presented
here uses Arsenazo III (3,6-bis ((2-Arsonophenyl) Azo)-4, 5-dihydroxy-2,
7-naphthalenedisulfonic acid) (10.2); CAS registry number: 1668-00-4.
Arsenazo III is chemically stable and has a very high affinity
for calcium in a neutral pH range. In
this assay system, the Arsenazo III forms a 1:1 violet Arsenazo III: calcium
complex with an absorbance maximum at 650nm (10.3.) The concentration of calcium is proportional
to the absorbance of the violet colored Arsenazo III: calcium complex. The color produced by this complex is stable
for at least 8 hours at room temperature (18-26°C).
2.3 CLINICAL SIGNIFICANCE (10.8)
The normal calcium concentration of serum is maintained
by hormones in the parathyroid gland.
Decreased levels occur in hypoparathyroidism, vitamin D deficiency,
rickets, osteomalacia and renal tubular acidosis. Increased levels are found in
hyperparathyroidism, vitamin D intoxication, and are associated with neoplasms,
especially those of bone.
A significant fraction of serum calcium is bound to
protein. Hyperproteinemia is associated
with an increased level of serum calcium and hypoproteinemia is associated with
an decreased level of serum calcium.
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. Heparinized plasma may also be
used. Plasma prepared using EDTA,
oxalate, citrate, which function by removal of calcium, obviously must 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
It is recommended that testing be done as soon as
possible after sample collection and preparation. If testing cannot occur immediately, the
serum sample can be stored refrigerated (2-8°C) for up to 7 days.
4.1 MATERIALS (1 X 500 ml)
Reagents necessary for the determination of calcium are
included in the kit.
4.1 REAGENT
Calcium reagent contains:
Arsenazo III 290
mmol/L
buffer solution 75
mmol/L
preservative and a stabilizer
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.
4.2.1 DO NOT pipet
by mouth. DO NOT ingest.
4.2.2 Organic
arsenic compounds have been classified as potentially carcinogenic; therefore,
safe laboratory practices should be carefully observed.
4.2.3 Components of
the reagent may be irritating to the skin and mucous membranes; avoid
contact. If contact occurs, wash with
copious amounts of water.
4.3 REAGENT PREPARATION
The
reagent is ready to use as is.
If the absorbance of the reagent alone (without sample
added) in a 1 cm cuvette is greater than 0.500 when measured against distilled
water at 650 nm, do not use the reagent.
4.4 REAGENT STORAGE AND STABILITY
The reagent is stable at room temperature (18-26°C) until
the expiration date on the label.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance at 650 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 650 nm.
4.5.3 Test tubes
capable of holding 2 ml.
4.5.4 Pipettes
capable of delivering 1 ml and 25 µl.
4.5.5 Timer for 1
minute incubation.
4.5.6 Distilled or
deionized water.
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 650
nm
Temperature 18‑26°
C or 37° C
Pathlength 1.0
cm
Mode endpoint
Reaction time 1
minute
Sample volume 25
µl
Reagent volume 1
ml
Total volume 1.025
ml
Sample to reagent
ratio 1/40
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 650 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
The calcium assay is calibrated by referencing the absorbance
of the unknown sample to the absorbance of the calibrator.
5.4 PROCEDURE
5.4.1 Prepare the
required volume of calcium reagent.
5.4.2 Into separate
calcium free test tubes pipette 25 µl of distilled water, calibrator or serum
to be assayed.
5.4.3 Add 1 ml of
calcium reagent and mix.
5.4.4 Incubate for
1 minute at room temperature and determine the absorbance of the calibrator
(As) and of each serum (A) at 650 nm using the distilled water sample as the
reagent blank.
5.5 PROCEDURE NOTE
The color
is stable for 8 hours.
A major source of difficulty with the assay of calcium is
contaminated glassware employed in the performance of the test. Many detergents and water supplies contain
calcium and incompletely rinsed containers used in this test will lead to
inaccurate results.
5.6 CALCULATION AND RESULTS
A
Calcium =
-------- x concentration of calibrator
As
where A =
absorbance of sample, As = absorbance of calibrator.
0.581
Example: Calcium = --------- x 10 mg/dl = 9.0 mg/dl
0.645
where A =
0.581, As = 0.645, concentration of calibrator = 10 mg/dl.
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES (10.5,10.6)
The range
of expected values is: 8.5 to 10.5 mg/dl (2.1 to 2.6 mmol/L)
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 (10.9)
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
Any substance which either chelates calcium or contains
calcium will interfere with the assay.
Magnesium does not interfere in this assay system. The affinity of Arsenazo III for magnesium is
essentially zero at the pH at which the assay is performed (10.2.)
When magnesium concentrations in incremental amounts of
1,2,4 and 6 mg/dL (magnesium expected range value: 1.8 to 2.9 mg/dL) were added
to 12 serum samples with calcium values ranging from 7.2 to 9.8mg/dL, there was
no increase in the calcium values. There
was also no increase due to magnesium in the calcium value (9.8 ± 0.9 mg/dl) in
12 assays of a control serum to which magnesium was added in concentrations
ranging from 0.5 to 15mg/dL (10.4.)
Young et al. (10.4) have published a comprehensive list
of drugs and substances which may interfere with in vitro diagnostic assays,
including that for serum calcium.
Interference from lipemia is minimized because of the
small amount of sample used.
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 calcium 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, 10 replicates of 3 control sera were run.
Mean
(mg/dl) SD
(mg/dl) CV (%)
Serum 1 6.2 ±
0.09 1.5
Serum 2 15.2 ±
0.15 .98
Serum 3 21.0 ±
0.34 1.6
Between-Run
In this study, 5 runs were made, each run consisting of 5
replicates of 3 control sera.
Mean
(mg/dl) SD
(mg/dl) CV (%)
Serum 1 6.1 ±
.12 1.9
Serum 2 15.2 ±
.22 1.5
Serum 3 20.6 ±
.29 1.4
9.2 CORRELATION
A correlation study was done comparing this method (y)
with a calcium o-cresolphthtalein method as the reference method (x1). 76 samples were run with a range between 6.2
mg/dl to 18.6 mg/dl. A second
correlation study was done comparing this method (y) with an atomic absorption
method as the reference method (x2).
20 samples were run with a range between 6.2 mg/dl to 19 mg/dl. The results are summarized below.
Number Regression Equation Correlation
of
Samples y=Biotron,
x=Comparative Coefficient
76 y = .993 x1 +
.060 0.994
20 y = .960 x2 +
.364 0.997
9.3 LINEARITY
The assay is linear to 15 mg/dl calcium. Samples with calcium concentrations exceeding
15 mg/dl should be diluted with an equal volume of distilled or deionized water
and the assay repeated. Multiply results
by 2.
9.4 SENSITIVITY
Using a 1:40 sample to reagent ratio and reading at
650nm, a 1 mg/dl calcium sample will produce a net absorbance of approximately
0.051.
10.0 REFERENCES
10.1 Textbook of
Clinical Chemistry, Edited by N.W. Tietz, p. 1342. W.B. Saunders Company,
Philadelphia, 1986.
10.2 Bauer, P.J.
Anal. Biochem. 110, 61-72, 1981.
10.3 Michaylova,
V., and Illkova, P., Anal Chim Acta 53, 194-198, 1971.
10.4 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC
Press (1990).
10.5 Todd, Sanford
and Davidsohn, Clinical Diagnosis and Management by Laboratory Methods, Edited
by Henry, J.B., W.B. Saunders Company, Philadelphia, 1979.
10.6 Tietz, N.W.,
Clinical Guide to Laboratory Tests, p. 92, W.B. Saudners Company, Philadelphia,
1983.
10.7 Hoffman,
W.S., The Biochemistry of Clinical Medicine, 4th Ed., 548-613, Year Book
Medical Publishers, Inc., Chicago, 1970.
10.8 Todd, Sanford
and Davidsohn, Clinical Diagnosis and Management by Laboratory Methods, Edited
by Henry, J.B., W.B. Saunders Company, Philadelphia, 1969, p.575.
10.9 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704