CALCIUM (ARSENAZO) PROCEDURE

Intended for the Quantitative Determination of Calcium in Serum

SUMMARY AND EXPLANATION

Calcium in serum is distributed about equally as the ionized form and the protein-bound non-ionized form. The ionized form plays a physiologically active role in blood coagulation and enzyme activation. Increased calcium levels result from hyperparathyroidism, multiple myeloma and bone neoplasms. Hypoparathyroidism, nephroses, steatorrhea and Rickets decrease serum calcium. A reciprocal relationship appears to exist between calcium and phosphorus in serum. As one goes up, the other usually decreases (1).

Several methodologies have been introduced for calcium assay, including flame photometry, flourescent and titrimetric procedures, ion selective electrodes and atomic absorption. Specific dye binding methods such as methylthymol blue and o-Cresolphthalein Complexone have also been widely used. The EAGLE CALCIUM (ARSENAZO) PROCEDURE uses arsenazo III as the chromagen in a stable, liquid form (2).

PRINCIPLE

Arsenazo III binds with calcium in an alkaline medium to form a purple colored complex which absorbs maximally at 650 nm. Magnesium interference is minimized by the addition of 8-hydroxyquinoline. The intensity of the color is proportional to the calcium concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2410 provides:

CALCIUM (ARSENAZO) REAGENT - (Cat. No. 2411)

REACTIVE INGREDIENTS:

Arsenazo III 0.15 mM and 5.0 mM 8-Hydroxyquinoline Sulfonate. Buffer and stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes 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 sealed tightly.

DETERIORATION:

The reagent should be a clear and dark purple in color. Turbidity would indicate deterioration and the reagent should not be used.

CALCIUM CALIBRATOR - (Cat. No. 2403)

REACTIVE INGREDIENTS:

10.0 mg/dL calcium as calcium carbonate in dilute hydrochloric acid.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes 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 sealed tightly.

DETERIORATION:

The calibrator should be a clear, colorless solution. Turbidity would indicate deterioration.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 650 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Use fresh, unhemolyzed serum separated from the blood clot as soon as possible. Heparinized plasma may also be used.

2. Prolonged contact between the serum and the blood clot will result in falsely low calcium values as red cells can absorb calcium.

3. EDTA, citrate and oxolate bind calcium and should not be used as anticoagulants.

SAMPLE STORAGE:

Calcium appears stable in serum for one week at 2 - 8° C and up to one year frozen.

ADDITIVES:

No special additives or preservatives are needed.

NTERFERING SUBSTANCES:

Specimens from patients receiving EDTA or bromsulfophtalein (BSP) should not used. Bilirubin up to 20 mg/dL and hemoglobin to 500 mg/dL do not interfere. Severe lipemia may cause elevated results and a serum blank should be run for greatest accuracy (see Serum Blank). Young et al (3) have reviewed drug effects on serum calcium.

PROCEDURE

MATERIALS PROVIDED:

CALCIUM (ARSENAZO) REAGENT (Cat. No. 2411), CALCIUM CALIBRATOR (Cat. No. 2403).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.01 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. Test tubes and rack

4. Timer

REACTION CONDITIONS:

Wavelength 650 nm

Filter Selection 600 - 670 nm

Reaction Type Endpoint

Incubation Temperature 15 - 30° C

Incubation Time 1 minute

Sample Volume 0.01 mL

Reagent Volume 1.00 mL

Total Volume 1.01 mL

Low Normal 8.5 mg/dL

High Normal 11.0 mg/dL

Calibrator Value 10.0 mg/dL

PREPARATION OF WORKING REAGENT:

Use CALCIUM (ARSENAZO) REAGENT as supplied.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume of 1.0 mL, increase CALCIUM (ARSENAZO) REAGENT volume to 2.0 mL and sample volume to 0.02 mL. Proceed as outlined.

1. Dispense 1.0 mL of Calcium (Arsenazo) 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. Let stand at room temperature for one minute.

4. Adjust instrument to zero absorbance at 650 nm using the Reagent Blank.

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

NOTE: For a direct read-out instrument, set read-out concentration of Calibrator and read the Unknown concentrations directly.

PREPARATION OF A SERUM BLANK:

Add 0.01 mL of lipemic sample to 1.0 mL D-H20. Adjust instrument to zero with D-H20 at wavelength used for reading of assays. Read and record absorbance of sample. Subtract this reading from the test absorbance reading. Use this corrected reading in calculation of results.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 60 minutes after color development.

CALIBRATION:

It is not necessary to determine a calibration curve with this procedure since the reaction is linear in the range of 0.5 - 15.0 mg/dL serum calcium. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use the CALCIUM CALIBRATOR provided with the test set or other suitable calibration material for this purpose. Specimens exceeding 15.0 mg/dL should be diluted with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain the final answer. Severe lipemic samples require a serum blank.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same mannner used 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 equations are used to determine Unknown concentrations:

Uknown (mg/dL) =  

Unk. Abs.

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

Cal. Abs.

EXAMPLE:

A 10.0 mg/dL Calibrator had Abs. = 0.447, while the Unknown Abs. = 0.394. The calcium concentration of the Unknown is:

0.394

---------  X 10.0 mg/dL = 8.8 mg/dL

0.447

LIMITATIONS

Plastic or acid-washed containers and test tubes should be used as contaminated glassware will adversely affect the test results.

EXPECTED VALUES (1)

Serum 8.5 - 11.0 mg/dL

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

PERFORMANCE CHARACTERISTICS

LINEARITY:

This method is linear in the range of 0.5 - 15.0 mg/dL.

PRECISION:

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

MEAN / ST. DEV. / %CV

Normal 10.0 / 0.14 / 1.3

Abnormal 14.3 / 0.13 / 0.9

Run to Run - Normal and abnormal control sera were assayed for 10 working days to establish run to run precision.

Normal 10.0 / 0.09 / 1.1

Abnormal 14.3 / 0.18 / 1.3

SPECIFICITY:

A comparison of this CALCIUM (ARSENAZO) PROCEDURE with a commercial method using o-Cresolphthalein Complexone showed a 98.9% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This CALCIUM (ARSENAZO) PROCEDURE has a sensitivity of 0.02 mg/dL per 0.001 absorbance unit.

REFERENCES

1 Tiez, N.W., Fundamentals of Clinical Chemistry, 2nd ed.,W.B. Saunders Co., Philadelphia, 1976, p.901

2 Thomas, M.V., Biophysics Journal 25, 1979, p. 541

3. Young, D.S. , Pestaner, L.C. and Gibberman, V., Clin Chem.,Vol. 21, p.272D (1975)