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                      Calcium Examination Procedure
**1.Purpose of examination:** 
Calcium estimation from serum or plasma by Arsenazo III Method.

**2.Responsibility and Authority:** 
  * Calibration: Technician 
  * Quality Control: Technician 
  * Routine operation: Technician 
  * Overall Monitoring: Quality Manager

**3.Sample Details:** 
  * Type of Sample: Serum,  Plasma
  * Type of container and additives: Plain without any additives 
  * Patient Preparation: As per Primary Sample Collection Manual [[sample_collection_manual|]]
  * Stability: At Room temperature 18°–28°C (64°–82°F) stability ≤ 24 hours
  * Handling and transport: As per Primary Sample collection manual 
  * Storage: 24 hours at 2-8° C

**4.Required Equipment:** 
Centrifuge, Auto-Pipette, Disposable Tips, Disposable sample cups, FullyAuto Chemistry Analyzer

**5.Required reagents:**
R1 Arsenazo-III dye 0.94 mmol/L
Sodium acetate 271 mmol/L

**6.Reagent Handling**
Remove any air bubbles present in the reagents with a new applicator stick, or allow the reagents to settle at the appropriate storage temperature to allow the bubbles to dissipate. To minimize volume depletion, do not use a transfer pipette to remove bubbles

**7.Reagent Storage and stability**
  * Unopened reagent stable at 2-8°C until expiration date.
  * On board System temperature reagent is  stable for 30 days
  * Instability or deterioration should be suspected if there are precipitates, visible signs of leakage or
  * contamination, turbidity, or if calibration or controls do not meet the appropriate criteria.

**8.Calibration Procedure:**
  * Multiconstituent Consolidated Calibrator(MCC)
  * Frequency:
  - Reagent lot change
  - QC out of range
  - After service or maintenance
  - Replacement in any parts of Instrument

**Procedure:**
  - Start the equipment.WDI abbotte fully.docx
  - Calibrators are ready to use.
  - Put calibrator 15-20 minutes at room temperature  
  - Prior to use, mix the contents by inverting the vial. Do not shake the vial to prevent foam formation. 
  - Take a 150 µl both level of calibrator solution in to separate aliquot.
  - Go to the calibration and give the calibration order.
  - Verify calibration with at least two levels of controls
  - If control results fall outside acceptable ranges,root cause analysis or recalibration may be necessary.

**9.Quality control Procedure:**
**Name**: Biorad Level 1 & 2
**Frequency:** As per Quality Control Procedure

**Procedure for Reconstitution of IQC** 
  - Reconstitution of QC material with 5 ml Distilled water by using calibrated fixed volume pipette.
  - Leave to stand for 30 min in the dark place.
  - Swirl gently several times during the reconstitution period to ensure that the contents are completely dissolved.
  - Prior to use, mix the contents by inverting the vial. Do not shake the vial as the information of foam should be avoided. Ensure that no lyophilized material remains un-reconstituted.
  - Prepare aliquots of 150 µl from the reconstituted QC material.
  - Store these aliquots at -15° C to -20° C.
  - Prior to use, make sure that aliquots should be at room temperature for at least 15 min.

**Procedure to run IQC**
  * Press Control order 
  * Select Assay /Panel, to be run.
  * Select the control/s and its level/s
  * Give Carrier Number and Position number
  * Press F3 / Add order
  * Put respected carrier in RSH rack
  * Check IQC results, in case outliers call residents.

**10.Principle of the procedure used for examinations:**
Arsenazo-III dye reacts with calcium in an acid solution to form a blue-purple complex. The color developed is measured at 660 nm and is proportional to the calcium concentration in the sample.

**11.Sample Preparation:**
  - Required SampleVolume: 150 µl of the sample 
  - Temperature: 37° 
Take 150-200µl of the sample from Primary tube to the secondary aliquot. Write the sample ID on the aliquot. 

**Procedure to run Patient sample**
  * Press Patient order 
  * Select Assay /Panel, to be run.
  * Give Carrier Number and Position number
  * Press F3 / Add order
  * Put respected carrier in RSH rack

**12.Performance Characteristics:** 
  * Linearity: 2.0 to 24.0 mg/dL 
  * Limit of Detection (LOD): 0.5 mg/dL
  * Limit of Quantitation (LOQ):1.0 mg/dL
  * Unit: mg/dl

**Automated Dilution Protocol:**
When using the Automated Dilution Protocol, the system performs a dilution of the specimen and automatically corrects the concentration by multiplying the result by the appropriate dilution factor.

**Manual Dilution Procedure:**
Dilute the specimen with saline (0.85% to 0.90% NaCl). Enter the dilution factor in the Patient or Control order screen.

**Normal and critical ranges**:

**Calcium, total**
^Child and adult^8.6-10.2^mg/dL^


**13.Laboratory Clinical interpretation:**
  * Hypercalcemia can result from hyperparathyroidism, hypervitaminosis D, multiple myeloma, and some neoplastic diseases of bone.1 Long- term lithium therapy has been reported to cause hyperparathyroidism in some individuals, with resulting hypercalcemia.
  * Hypocalcemia can result from hypoparathyroidism, hypoalbuminemia, renal insufficiency, and pancreatitis.1

**14.Interference and cross reaction:**
  * Bilirubin 30 mg/dL (513 μmol/L) 
  * Hemoglobin 1,000 mg/dL
  * Intralipid 500 mg/dL

**15.Potential source of variation:** 
**Turn around time (TAT):**
  * Routine: 6.0 hours
  * Urgent: 2.0 hours

**16.Recording of observation:**
  * Software backup 
  * Machine raw data


**17.Storage & Disposal of waste**: Follow storage & discard procedure

**18.Environmental & Safety control:**
**Reagent R1** contains sufamic acid that Causes severe skin burns and eye Damage

**19.Precautions:**
  * Avoid breathing mist / vapors / spray.
  * Wash hands thoroughly after handling.
  * Contaminated work clothing should not be allowed out of the workplace.
  * Wear protective gloves / protective clothing / eye protection.

**20.Response**
  - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
  - If eye irritation persists: Get medical advice / attention.
  - IF ON SKIN: Wash with plenty of water.If skin irritation or rash occurs: Get medical advice / attention. Take off contaminated clothing and wash it before reuse.

**21.References:**
  - Bendz H, Sjödin I, Toss G, et al. Hyperparathyroidism and long-term lithium therapy—a cross-sectional study and the effect of lithium withdrawal. J Intern Med 1996;240:357–365.
  - US Department of Labor, Occupational Safety and Health Administration, 29 CFR Part 1910.1030, Bloodborne pathogens.
  - US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, DC: US Government Printing Office; December 2009.
  - World Health Organization. Laboratory Biosafety Manual. 3rd ed. Geneva: World Health Organization; 2004.

|**Printed copy of this document is considered uncontrolled.** It should be compared with controlled electronic copy before use|

^ Name of Laboratory : Laboratory Services Sir T. Hospital (LSSTH),Bhavnagar ^^^^
^Document No.1^**Document Name**: Calcium Examination Procedure^**Unique ID**:LSSTH /BIOCHEM/ SOP-5^^
^Issue No. : 01^Issue Date :30/04/2024^Page No.^^
^Amend No.^ Amend Date ^Prepared by: Section Incharge^Approved & Issued by: HOD,Biochemistry^