CALCIUM (CPC) PROCEDURE

Intended for the Quantitative Determination of Calcium in Serum and Urine

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).

While several methodologies have been introduced for calcium assay, the one which offers maximum sensitivity and specificity with ease of handling involves the use of o-cresolphthalein complexone to bind calcium (2). The EAGLE CALCIUM PROCEDURE is based on a modification of this method. A reaction accelerator has been included to assure reaction completion in less than one minute.

PRINCIPLE

O-Cresolphthalein complexone binds calcium in an alkaline medium to form a purple colored complex which absorbs maximally at 570 nm. Magnesium interference is minimized by the addition of 8-hydroxyquinoline. Stabilizers are included to produce a low blank absorbance and increased Working Reagent stability. A reaction accelerator assures reaction completion in less than one minute.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 2400 provides:

CALCIUM COLOR REAGENT - (Cat. No. 2401)

REACTIVE INGREDIENTS:

0.082 mM o-cresolphthalein complexone and 17.2 mM 8-hydroxyquinoline. Stabilizer and reaction accelerator 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. PROTECT FROM LIGHT.

DETERIORATION:

The reagent should be a clear, yellow solution. Turbidity or a dark orange color would indicate deterioration.

CALCIUM BASE REAGENT - (Cat. No. 2402)

REACTIVE INGREDIENTS:

0.51 M diethylamine and 7.7 mM potassium cyanide.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water. Contains cyanide. Do not mix with acid.

STORAGE AND STABILITY:

Store at 15 - 30° C. Stable until expiration date if sealed tightly.

DETERIORATION:

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

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 570 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

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

2. Prolonged contact between the serum and blood clot will result in falsely low calcium values.

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

4. Grossly hemolyzed or lipemic samples should not be used.

5. Urine may be analyzed by this method.

SAMPLE STORAGE:

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

ADDITIVES:

No special additives or preservatives are needed for serum. An aliquot of the urine collection should be acidified to pH = 4 with concentrated hydrochloric acid before testing. Mix the urine well before sampling to assure an even suspension of any precipitated calcium.

INTERFERING SUBSTANCES:

Specimens from patients receiving EDTA or bromsulfophtalein (BSP) should not used. Young et al (3) have reviewed drug effects on serum and urine calcium.

PROCEDURE

MATERIALS PROVIDED:

CALCIUM COLOR REAGENT (Cat. No. 2401), CALCIUM BASE REAGENT (Cat. No. 2402) and CALCIUM CALIBRATOR (Cat. No. 2403).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.01 mL micropipettor

2. 0.5 and 1.0 mL pipettor

3. Test tubes and rack

4. Timer

REACTION CONDITIONS:

Wavelength 340 nm

Incubation Temperature 30° C or 37° C

Prewarm Substrate 5 minutes

Preincubation Time 1 minute

Incubation Time 1 minute

Reagent Volume 1.0 mL

Total Volume 1.1 mL

Low Normal 8.5 mg/dL

High Normal 11.0 mg/dL

Calibrator Value 10.0 nm

PREPARATION OF WORKING REAGENT:

Use a plastic container or acid-washed glass container. Add one part CALCIUM COLOR REAGENT to one part CALCIUM BASE REAGENT. Swirl to mix. Use within one hour of preparation. The Working Reagent contains a small amount of cyanide and should not be mixed with acid. Discard by flushing with water. The Working Reagent should have an absorbance of .250 when measured against D-H2O at 570 nm. Absorbance greater than 0.250 indicates contamination or deterioration and the reagent cannot be used.

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume of >>1.0mL, increase CALCIUM WORKING REGENT volume to 2.0 mL and Sample volume to 0.02mL. Proceed as outlined.

1. Dispense 1.0 mL of Working 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.

NOTE: Dilute urine samples X 2 with deionized water before testing.

3. Permit the test samples to stand at room temperature for one minute.

4. Adjust instrument to zero absorbance at 570 nm using the Reagent Blank.

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

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

PROCEDURAL NOTE:

Linearity extends to 15 mg/dL. Specimens exceeding this value should be diluted with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain the final answer.

STABILITY OF FINAL REACTION PRODUCT:

The test samples should be read within 15 minutes after color development.

CALIBRATION:

It is not necessary to determine a calibration curve for this purpose 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 which is provided in the reagent set for this purpose or any other suitable calibration material.

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 and quantities:

SERUM

Unknown (mg/dL) =

Unk. Abs.

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

Cal. Abs.

EXAMPLE:

A 10.0 mg/dL Calibrator had Abs. = 0.223, while the Unknown Abs. = 0.204. The calcium concentration of the Unknown is:

0.204

-------- X 10.0 mg/dL = 9.1 mg/dL

0.223

URINE

Unknown (mg/24 hr.) =  

Unk. Abs.

-------------  X Cal. Conc. X 2 X 10 mg/L

Cal. Abs.

X Urine Volume (L/24 hr.)

The factor X 2 corrects for sample dilution during testing and the factor X 10 converts mg/dL to mg/L.

EXAMPLE:

A 24 hour urine collection had Abs. = 0.166 and volume = 1.4000 L. The quantity of calcium excreted is:

0.166  

-------- X 10.0 mg/dL X 2 X 10 dL/L X 1.4000 L/24 hr. = 208 mg/24 hr

0.223

LIMITATIONS

Plastic or acid-washed containers should be used for preparation of the Working Reagent and in performance of test.

EXPECTED VALUES

Serum: 8.5 - 11.0 mg/dL

Urine: 50 - 150 mg/24 hr

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 10.7 / 0.12 / 1.1

Abnormal 8.1 / 0.08 / 1.0

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

Normal 10.5 / 0.16 / 1.5

Abnormal 8.1 / 0.11 / 1.4

SPECIFICITY:

A comparison of this CALCIUM PROCEDURE with another widely used commercial method showed a 99% correlation with samples in the normal and abnormal range.

SENSITIVITY:

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

REFERENCES

1. W.B. Saunders Co., Philadelphia, 1976, p. 901.

2. B.,Clin.Chem.Acta, 46, 49 (1973).

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