Estimation of serum Calcium

Table of Contents

Introduction

Calcium is one of the most important minerals in the human body.
About 99% of total body calcium is present in bones and teeth.
The remaining calcium is present in extracellular fluid and intracellular compartments.
Serum calcium plays an essential role in neuromuscular function, blood coagulation, enzyme activity, hormone secretion, and membrane stability.
Calcium exists in serum in three forms:
ionized calcium (physiologically active form)
protein-bound calcium
calcium complexed with anions
Serum calcium estimation is an important biochemical test used to assess bone metabolism, parathyroid function, renal disease, and metabolic disorders.

Principle

Serum calcium estimation is based on Arsenazo III method.
Arsenazo III dye combines with calcium ions in acidic medium to form a blue-purple colored complex.
The intensity of color formed is directly proportional to calcium concentration in the sample.
Absorbance is measured at 650 nm.

Reaction

Calcium + Arsenazo III → Calcium-Arsenazo III colored complex

Arsenazo III has high specificity for calcium and shows minimal interference from other serum ions.

Specimen

Sample Type

Serum is preferred specimen
Heparin plasma can also be used
Urine may also be analyzed

Precautions

Use non-hemolyzed sample
Avoid calcium contamination
Plastic tubes are preferred

Stability

Serum / Plasma

7 days at 20–25°C
3 weeks at 4–8°C
8 months at −20°C

Reagents

Reagent 1

Arsenazo III
Phosphate buffer (pH 7.8)

Standard

Calcium standard solution

Reagent Preparation

Reagents are liquid and ready to use

Materials Required

Test tubes
Micropipette
Pipette tips
Colorimeter / spectrophotometer
Cuvette
Timer
Calcium reagent kit

Procedure

Components	Blank	Standard	Test
Reagent 1	1000 µL	1000 µL	1000 µL
Distilled water	10 µL	—	—
Standard	—	10 µL	—
Sample	—	—	10 µL

Incubation

Mix properly
Incubate at 37°C for 1 minute

Reading

Measure absorbance of test and standard against blank
Read at 650 nm

Calculation

Formula

Calcium (mg/dL) = Absorbance of Test / Absorbance of Standard × Standard concentration

Unit Conversion

mg/dL × 0.25 = mmol/L

Normal Reference Values

Group	Normal Value
Adult	8.6 – 10.2 mg/dL
Child (2–12 years)	8.8 – 10.8 mg/dL

Clinical Significance

Increased Serum Calcium (Hypercalcemia)

Hyperparathyroidism
Paget disease of bone
Malignancy with bone metastasis
Vitamin D excess

Decreased Serum Calcium (Hypocalcemia)

Rickets
Osteomalacia
Malabsorption syndrome
Chronic renal disease
Hypoparathyroidism

Diagnostic Importance

Assesses calcium metabolism
Evaluates bone disorders
Helps diagnose parathyroid disease
Monitors renal and metabolic disorders

MCQs

1. Which method is commonly used for estimation of serum calcium?

A. GOD-POD method
B. Arsenazo III method
C. Jaffe method
D. Biuret method
Answer: B. Arsenazo III method

2. Serum calcium is measured mainly at which wavelength?

A. 340 nm
B. 405 nm
C. 650 nm
D. 540 nm
Answer: C. 650 nm

3. Arsenazo III reacts with calcium to form:

A. Red complex
B. Green complex
C. Blue-purple complex
D. Yellow complex
Answer: C. Blue-purple complex

4. The intensity of color formed is:

A. Inversely proportional to calcium concentration
B. Directly proportional to calcium concentration
C. Independent of calcium concentration
D. Related to protein concentration
Answer: B. Directly proportional to calcium concentration

5. Preferred sample for calcium estimation is:

A. Whole blood
B. Serum
C. CSF
D. Saliva
Answer: B. Serum

6. Anticoagulant suitable for calcium estimation:

A. EDTA
B. Oxalate
C. Heparin
D. Citrate
Answer: C. Heparin

7. EDTA is avoided because:

A. It increases calcium
B. It binds calcium
C. It changes color
D. It increases pH
Answer: B. It binds calcium

8. Main reagent used in calcium estimation:

A. Urease
B. Arsenazo III
C. Peroxidase
D. Lipase
Answer: B. Arsenazo III

9. Normal adult serum calcium level:

A. 4–6 mg/dL
B. 6–8 mg/dL
C. 8.6–10.2 mg/dL
D. 12–14 mg/dL
Answer: C. 8.6–10.2 mg/dL

10. Major body store of calcium:

A. Liver
B. Muscle
C. Bone
D. Blood
Answer: C. Bone

11. Physiologically active calcium form:

A. Protein bound
B. Ionized calcium
C. Complexed calcium
D. Stored calcium
Answer: B. Ionized calcium

12. Calcium is important for:

A. Blood coagulation
B. Muscle contraction
C. Nerve transmission
D. All of the above
Answer: D. All of the above

13. Hypercalcemia occurs in:

A. Hypoparathyroidism
B. Hyperparathyroidism
C. Rickets
D. Osteomalacia
Answer: B. Hyperparathyroidism

14. Hypocalcemia occurs in:

A. Vitamin D intoxication
B. Hyperparathyroidism
C. Rickets
D. Paget disease
Answer: C. Rickets

15. Incubation temperature for calcium estimation:

A. 25°C
B. 30°C
C. 37°C
D. 45°C
Answer: C. 37°C

16. Incubation time:

A. 10 min
B. 1 min
C. 20 min
D. 30 sec
Answer: B. 1 min

17. Blank contains:

A. Sample
B. Standard
C. Distilled water
D. Serum
Answer: C. Distilled water

18. Standard volume used:

A. 100 µL
B. 10 µL
C. 1 mL
D. 500 µL
Answer: B. 10 µL

19. Reagent volume used:

A. 500 µL
B. 1000 µL
C. 2000 µL
D. 100 µL
Answer: B. 1000 µL

20. Calculation is based on:

A. Test/Blank
B. Test/Standard
C. Standard/Blank
D. Blank/Test
Answer: B. Test/Standard

21. Calcium standard concentration is:

A. Kit dependent
B. Fixed always
C. 1 mg/dL
D. 100 mg/dL
Answer: A. Kit dependent

22. Unit conversion:

A. mg/dL × 2
B. mg/dL × 0.25
C. mg/dL × 10
D. mg/dL × 5
Answer: B. mg/dL × 0.25

23. Major interference avoided by:

A. Plastic tubes
B. Metal tubes
C. Colored tubes
D. EDTA tubes
Answer: A. Plastic tubes

24. Hemolysis affects:

A. Calcium estimation
B. Not significant if mild
C. Always absent
D. Standard only
Answer: B. Not significant if mild

25. Hypercalcemia may cause:

A. Tetany
B. Renal stones
C. Convulsions
D. Hypotension
Answer: B. Renal stones

26. Hypocalcemia causes:

A. Polyuria
B. Tetany
C. Hyperglycemia
D. Jaundice
Answer: B. Tetany

27. Calcium regulation hormone:

A. Insulin
B. Thyroxine
C. Parathyroid hormone
D. Cortisol
Answer: C. Parathyroid hormone

28. Vitamin affecting calcium absorption:

A. Vitamin C
B. Vitamin D
C. Vitamin K
D. Vitamin A
Answer: B. Vitamin D

29. Calcium estimation helps diagnose:

A. Bone disease
B. Parathyroid disease
C. Renal disease
D. All of the above
Answer: D. All of the above

30. Calcium in urine requires:

A. No dilution
B. Dilution
C. Heating
D. Freezing
Answer: B. Dilution