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
The source of color reagents presently used for the
colorimetric determination of calcium came from the early titrimetric
determinations of Pollard (10.1) and Schwarzenbach (10.2). The primary source of interference with
calcium color reactions has been magnesium which also reacts to some extent
with the same complexones. In order to
obviate this problem Connerty and Briggs (10.3) described a procedure in which
cresolphthalein complexone (CPC) was used to determine calcium while 8‑hydroxy‑quinoline
was employed to complex with magnesium effectively removing it from the
system. Zak et. al.(10.4) have recently
reviewed calcium methodologies and have recommended the direct cresolphthalein
complexone procedure for serum calcium.
This procedure for serum calcium is a modification of the Connerty and
Briggs procedure employing CPC as a color developer and 8‑hydroxy‑quinoline
to mask the presence of magnesium.
2.2 TEST PRINCIPLE
Cresolphthalein complexone (CPC) reacts with calcium to
form a purple colored complex.
Cresolphthalein
Complexone + 2Ca++ ‑‑‑> CPC (Ca++)2
The formation of the purple colored causes an increase in
absorbance at 570 nm which is directly proportional to the concentration of
calcium in the sample.
2.3 CLINICAL SIGNIFICANCE (10.6)
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.
4.0 MATERIALS (2 X 125 ml)
(4 X 125 ml)
Reagents necessary for the determination of calcium are
included in the kit.
4.1 REAGENT
4.1.1 Calcium Color
Reagent contains 0.10 mM cresolphthalein complexone and 17.2 mM 8‑hydroxyquinoline.
4.1.2 Calcium Color Developer contains 0.6 M AMP buffer and a
surfactant.
4.1.3 Standard/Control/Calibrator
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 working reagent is prepared by mixing equal volumes
of the color reagent and color developer.
4.4 REAGENT STORAGE AND STABILITY
The reagent is stable at room temperature (18-26°C) until
the expiration date on the label. The
working reagent is stable for 7 days at room temperature.
4.5 ADDITIONAL MATERIALS REQUIRED
4.5.1 Spectrophotometer
or colorimeter capable of reading absorbance at 570 nm.
4.5.2 1 cm cuvettes
or a flow cell capable of transmitting light at 570 nm.
4.5.3 Test tubes
capable of holding 3 ml.
4.5.4 Pipettes
capable of delivering 2.5 ml and 25 µl.
4.5.5 Timer for 5
minute incubation.
4.5.6 Distilled or
deionized water.
4.5.7 Calibrator
4.5.8 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 570
nm
Temperature 18‑26°
C or 37° C
Pathlength 1.0
cm
Mode endpoint
Reaction time 5
minutes
Sample volume 25
µl
Reagent volume 2.5
ml
Total volume 2.525
ml
Sample to reagent
ratio 1/100
5.2 INSTRUMENT
Any instrument capable of reading absorbance accurately
with a sensitivity of 0.001 absorbance at 570 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 working reagent. (See
4.3 Reagent Preparation section.)
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 2.5 ml of
working reagent and mix.
5.4.4 Incubation
for 5 minutes at the desired temperature and determine the absorbance of the
calibrator (As) and of each serum (A) at 570 nm using the distilled water
sample as the reagent blank.
5.5 PROCEDURE NOTE
The color
is stable for 1 hour.
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
(mg/dl) = ‑‑‑-‑ X concentration of calibrator
As
A =
absorbance of sample, As = absorbance of calibrator
Example:
.359
Calcium
concentration = --‑‑‑‑-
X 10 mg/dl = 8.9 mg/dl
.405
with A =
.359 and As = .405, concentration of calibrator = 10 mg/dl
6.0 INTERPRETATION OF RESULTS
6.1 EXPECTED VALUES
The range
of expected values is: 8.7 ‑ 10.7 mg/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
Any substance which either chelates calcium or contains
calcium will interfere with the assay.
Young et al. (10.5) have published a comprehensive list
of drugs and substances which may interfere with in vitro diagnostic assays,
including that for serum calcium.
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 2 control sera were run.
Mean
(mg/dl) SD (mg/dl) CV
(%)
9.6 ±
0.06 0.6
13.8 ± 0.11 0.8
Between-Run
In this
study, 10 runs were made on a pooled serum sample.
Mean
(mg/dl) SD (mg/dl) CV
(%)
9.5 ±
0.10 1.1
9.2 CORRELATION
A correlation study was done comparing this method (y)
with a similar calcium o-cresolphthalein method (x). The study yielded a regression curve of y =
1.045 x ‑ 0.0812.
9.3 LINEARITY
This procedure is linear through 20 mg/dl beyond which
the specimen should be diluted 1 to 1 with 0.9% saline. Reassay the specimen and multiply the results
by 2.
10.0 REFERENCES
10.1 Pollard,
F.H., J.V., Analyst 81, 348‑353 (1956).
10.2 Schwarzenbach,
G., Analyst 80, 713‑729 (1955).
10.3 Connerty,
H.V., Briggs, A.R., Am. J. Clin. Path. 45, 290‑296 (1966).
10.4 Zak, B.,
Epstein, E., Babinski, E.S., Annals of Clinical and Laboratory Science 5, 195‑215
(1975).
10.5 Young, D.S.,
Effects of Drugs on Clinical Laboratory Tests, 3rd ed., Washington DC, AACC
Press (1990).
10.6 Todd, Sanford
and Davidsohn, Clinical Diagnosis and Management by Laboratory Methods, Edited
by Henry, J.B., W.B. Saunders Company, Philadelphia, 1969, p.575.
10.7 G.J. Kost,
"Critical Limits for Urgent Clinician Notification at U.S. Medical
Centers"; JAMA, Feb. 2, 1990; Vol 263, No.5, p.704
Rev
10//99