Trilogy Biotechnology LLC

CALCIUM
225 Madinah St., 305 Commercial Complex, Amman, Jordan Quantitative Determination of Calcium
Post code 11954,
Tel: +962 79 8599872, Colorimetric OCC Method
Email: [email protected]
Website: www.trilogybiotech.com
For professional in vitro diagnostic use only.

Working Solution preparation: Mix 1 volume of R1 + 1 volume of R2.

INTENDED USE Stability: 5 days at 2-8ºC. Recap reagents immediately after use.
Quantitative determination of Calcium in Serum or Plasma urine. o-
Cresolphtalein Complexone (OCC) Colorimetric method. SAMPLE COLLECTION AND PREPARATION
GENERALITIES Serum, heparinized plasma, urine. Do not use EDTA plasma.
Calcium exists in the blood in three forms: ionized (13%), complexed The 24h Urine must be acidified with 10 mL of concentrated
(47%) and bound to protein, mainly albumin (40%). When calcium hydrochloric acid. The morning urine should be acidified with a few
determinations are performed, the total calcium concentration is drops of concentrated hydrochloric acid.
determined regardless of the amount of calcium present in each form. The urine sample should be diluted 1: 2 (1 + 1) with distilled water.
A depressed concentration of total calcium can be due to Stability in serum/plasma: 7 days at 15° - 25 °C, 3 weeks at 2°- 8 °C,
3 months at -20 °C.
hypoproteinemia, but the concentration of physiologically active
Stability in urine: 2 days at 15-25 °C, 4 days at 2-8°C, 3 weeks at -20 °C.
(ionized) calcium in such case may be normal. For this reason, protein
Discard contaminated samples.
determination should accompany each calcium analysis so that the
calcium value can be interpreted properly. Depressed serum calcium
TEST PROCEDURE
levels usually accompany hypoparathyroidism, some bone diseases,
Wavelength : 570 nm
certain kidney diseases, and low protein levels. Elevated serum
Light path : 1 cm
calcium levels occur in hyperparathyroidism, vitamin D poisoning,
Temperature : 37 °C
and sarcoidosis. The plasma level in calcium is greatly affected by the Measurement: : Against Reagent Blank
plasma level of inorganic phosphate. In most cases, there is an inverse Assay type : ENDPOINT
relationship between calcium and inorganic phosphate. Conditions Assay:
associated with hypercalcemia, such as primary Blank Assay
hyperparathyroidism, are usually associated with hypophosphatemia; Working Solution 1000 µL 1000 µL
the opposite is true as well. Urine calcium excretion parallels the
Sample/ Standard/ Cal / 10 µL
serum calcium level. Large amounts of calcium are excreted in the - Mix and incubate for 5 min at 37 °C.
urine in hyperparathyroidism, metabolic acidosis, renal tubular -Read the absorbance (A) of the samples and the standard
insufficiency, and multiple myeloma and bone malignancies. against the reagent blank.
TEST PRINCIPLE
CALCULATION
The method is based on the specific binding of Cresolphtalein
Serum/ Plasma:
complexone (OCC), a metallochromic indicator, and calcium at Abs Sample - Abs blank
Calcium Concentration= x Std/ Cal.
alkaline pH with the resulting shift in the absorption wavelength of the ∆ Abs Std/Cal - Abs blank
complex. The intensity of the chromophore formed is proportional to Urine:
the concentration of total calcium in the sample. Calculate as for the serum and multiply the result by 2 (initial sample
dilution).
REAGENT COMPOSITION Conversion Factor: [mg/dL] x 0.25 = [mmol/L]
Reagent 1 (OCC Buffer)
Ethanolamine 500 mmol/L
QUALITY CONTROL
Chloroform 15 mmol/L
Methanol 5.7 mmol/L Normal and abnormal control sera of known concentration should be
Reagent 2 (OCC Indicator) analyzed routinely with each group of unknown samples.
o-Cresolphtalein complexone 0.62 mmol/L
8-quinolinol 69 mmol/L EXPECTED VALUES
Standard Serum/plasma:
Calcium value on label
• Newborn: 8.0 - 13.0 mg/dL.
STORAGE, PREPARATION AND SHELFLIFE • Children: 8.5 - 12.0 mg/dL.
Liquid and ready to use reagents, stable until the expiry date shown, if • Adults: 8.5 - 10.5 mg/dL.
stored as indicated on the label and avoid contamination, evaporation
and prolonged exposure to direct light. Do not freeze the reagents. Urine :
Discard the reagent if signs of deterioration appear, such as the • Women: < 250 mg/24 h.
presence of particles and turbidity or failure to recover the values of • Men: < 300 mg/24 h
certified control sera. After opening the bottles, it is advisable to •
withdraw the necessary volume, to immediately close the bottles and Each laboratory should establish a range of expected values based on
store them in the fridge to avoid contamination, degradation from its patient population and, if necessary, determine its own reference
direct light and evaporation. The measurement is not influenced by interval. For diagnostic purposes, results should always be assessed
occasionally occurring color changes, as long as the absorbance of together with the patient's medical history, clinical examination and
the reagent is < 0.22 at 570 nm. other results.

CC-JO-COC-01/ REVISION A /DATE: 14/01/2025 Page | 1

PERFORMANCE USED SYMBOLS
PRECISION:
Low Level: Samples= 20; Average = 9.14; S.D. = 0.07; CV = 0.74%. In Vitro Diagnostic Medical Device
High Level: Samples = 20; Average = 16.02; S.D. = 0.11; CV = 0.68%. Manufacturer
ACCURACY: Date of Manufacture
A comparison between this method (x) and a certified method of trade
Catalogue Number
(y) gave the following correlation:
y=0.8234x + 1.55 r =0.990 Batch Code
Use by YYYY-MM (MM = end of month)
SENSITIVITY: 0.1 mg/dL.
LINEARITY: 35 mg/dL. Operator's Manual; Operating Instructions
Keep away from Sunlight
Keep away from Rain
PRECAUTIONS
Temperature Limit
R1: Harmful if swallowed, in contact with skin or inhaled. It causes
severe skin burns and eye damage. Causes damage to organs. Caution
R2: May be corrosive to metals. Causes severe skin burns and eye Do not use if Package is Damaged
damage. Do Not Re-Use
in case of contact of reagents with the operator, you must apply the
Contains Sufficient for <N> Tests
following first aid: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin, wash
immediately with plenty of water. Wear suitable protective clothing,
gloves and eye/face protection.
Note: Most of the detergents and water softening products used in the
labs contain chelating agents. A defective rinsing will invalidate the
procedure. Keep the glassware acid washed and thoroughly rinsed at
all times.

INTERFERENCES
No interference was observed by Hemoglobin up to 100 mg/dL,
Bilirubin up to 40 mg/dL, triglycerides up to 1.25 g/L.

LITERATURE
- Connerty, H.V. y Biggs, A.R. Am. J. Clin. Path. 45 : 290 (1966).
- Anderegg, G, Flashka, H., Sallmann, R. and Schwartzenbach, G.
Metallindikatoren VII. Fasciculus: 37: 113 (1954).
- Farell E C. Calcium. Kaplan A et al. Clin Chem The C.V. Mosby Co. St
Louis. Toronto. Princeton 1984; 1051-1255 and 418.
- Young DS. Effects of drugs on clinical laboratory tests, 5th ed. AACC
Press, 2000.
- Tietz N W et al. Clinical Guide to Laboratory Tests, 3rd ed. AACC
1995.

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