Reagent Preparation

Reagents are ready to use. Protect from bright light.

Sample Material

Serum/heparinized plasma. Calcium is reported to be

stable in serum for 7 days at 2–8°C.

Procedure

Wavelength/filter : 650 nm (Hg 623 nm)/red

Temperature : RT

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Calcium reagent (L1 ) 1.0 1.0 1.0

Distilled water 0.01

Calcium standard(s) - 0.01

Sample - - 0.01

Mix well and incubate at RT (25°C) for 5 minutes.

Measure the absorbance of the standard (Abs S), and test

sample (Abs T) against the blank, within 60 minutes.

Calculations

 Abs T

Calcium in mg/dL = _______ × 10 Abs S

Linearity

This procedure is linear upto 15 mg/dL. If values exceed

this limit, dilute the sample with distilled water and repeat

the assay. Calculate the value using the proper dilution

factor.

Note

As calcium is a very widely distributed ion, care should

be taken to avoid any contamination. All glassware being

used for the test should first be rinsed with 1% or 0.1 N HCI

and then with good quality deionized water before use.

It is suggested that after the rinsing of the tubes with HCI

the reagent be pipetted in their respective tubes and the

tubes be rinsed with the reagent. The reagent then should

be pooled together in the ‘blank’ tube and repipetted out

into the ‘standard’ and ‘test’ test tubes. This will ensure that

any remaining contamination will be carried over equally

in all the tubes. For flow cell cuvettes it is suggested that

some reagent be aspirated before the blank to take away any

contamination in the flow through tubing or cuvette which

may cause a higer than the actual blank of the reagent.

Chelating agents such as EDTA, present even in

traces, prevent the formation of the color complex, hence

necessary care should be taken during the assay.

System Parameters

Reaction : End point Interval :

Wavelength : 650 nm Sample volume : 0.01 mL

Zero setting :  Reagent

blank

Reagent volume : 1.00 mL

Incubation

temperature

: RT Standard : 10 mg/dL

Incubated time : 5 min Factor :

Delay time : — React slope : Increasing

Read time : — Linearity : 15 mg/dL

No. of read : — Units : mg/dL

Normal Values

Adults

18–60 years 8.6–10.5 mg/dL 2.15–2.62 mmol/L

60–90 years 8.8–10.7 mg/dL 2.20–2.67 mmol/L

> 90 years 8.2–9.6 mg/dL 2.05–2.40 mmol/L

Children

Cord blood 8.2–11.2 mg/dL 2.05–2.80 mmol/L

Premature infant 6.2–11.0 mg/dL 1.55–2.75 mmol/L

< 10 days 7.6–10.4 mg/dL 1.90–2.60 mmol/L

10 days–2 years 9.0–11.0 mg/dL 2.25–2.75 mmol/L

2–12 years 8.8–10.8 mg/dL 2.20–2.70 mmol/L

12–18 years 8.4–10.5 mg/dL 2.10–2.62 mmol/L

Panic levels

Tetany < 7 mg/dL < 1.75 mmol/L

Coma > 12 mg/dL > 2.99 mmol/L

Possible death < 6 mg/dL < 1.50 mmol/L

Specimen Collection and Storage

1. Fresh, unhemolyzed serum is the preferred specimen.

2. Heparinized plasma may also be used.

3. Anticoagulants other than heparin should not be used.

4. Remove serum from clot as soon as possible since red

cells can absorb calcium.

5. Older serum specimens containing visible precipitate

should not be used.

6. Serum calcium is stable for 24 hours at room temperature, one week at 2–8°C, and up to 5 months frozen

(–15 to –25°C.) and protected from evaporation.

Specimens should not be thawed and refrozen.

496 Concise Book of Medical Laboratory Technology: Methods and Interpretations Clinical Relevance

Normal levels of total calcium combined with other

findings

1. Normal calcium levels with overall normal findings

in other tests indicate that there are no problems with

calcium metabolism.

2. Normal calcium and abnormal phosphorus indicate

impaired calcium absorption due to alteration of

parathyroid hormone activity or secretion. In rickets,

the calcium level may be normal or slightly lowered

and the phosphorus level is depressed.

3. Normal calcium and elevated BUN indicates

 a. Possible secondary hyperparathyroidism. Initially

lowered serum calcium results from uremia and

acidosis. The lower calcium level stimulates the

parathyroid to release parathyroid hormone, which

acts on bone to release more calcium.

 b. Possible primary hyperparathyroidism. Excessive

amounts of parathormone cause elevation in

calcium levels, but secondary kidney disease would

cause retention of phosphate and concomitant

lower calcium.

4. Normal calcium and decreased serum albumin. This

is indicative of hypercalcemia, since, there should

be a decrease in calcium when there is a decrease in

albumin because of the 50% of serum calcium that is

protein-bound.

Hypercalcemia (Increased Total Calcium)

Hypercalcemia is associated with many disorders, but its

greatest clinical importance rests in its association with

cancer, including multiple myeloma, parathyroid tumors,

nonendocrine tumors producing a parathormone-like substance, and cancers metastasizing to the bone. Increased

calcium levels are caused by or associated with.

1. Hyperparathyroidism due to

 a. Parathyroid adenoma associated with hypophosphatemia

 b. Hyperplasia of parathyroid glands associated with

hypophosphatemia.

2. Cancer

 a. Metastatic cancers involving bone cancers of lung,

breast, thyroid, kidney, and testes may metastasize

to bone

 b. Hodgkin’s disease other lymphomas

 c. Multiple myeloma in which there is extensive bone

destruction

 d. Lung and renal cancers may produce parathormone

resulting in symptoms of hypercalcemia

 e. Sarcoidosis due to increased IgG or IgA

 f. Leukemia.

3. Addison’s disease

4. Hyperthyroidism

5. Paget’s disease of bone (also accompanied by high

levels of alkaline phosphatase)

6. Prolonged immobilization

7. Bone fractures combined with bed rest

8. Excessive intake of vitamin D

9. Prolonged use of diuretics, thiazides

10. Respiratory alkalosis

11. Milk alkali syndrome (history of peptic ulcer could

indicate excessive intake of milk and antacids).

Hypocalcemia (Decreased Total Calcium Levels)

Commonly caused by/associated with

1. Pseudohypocalcemia (hyperproteinemia). Actually,

what looks like hypocalcemia is really a reflection of

diminished albumin (as revealed by a serum protein

electrophoresis). It is the reduced protein that is

responsible for the low calcium, since 50% of the

calcium total is protein-bound. (Excessive use of IV

fluids will decrease albumin levels and thus, decrease

the amount of calcium).

2. Hypoparathyroidism (primary is very rare) may be due

to accidental removal of parathyroid glands during

a thyroidectomy, irradiation, hypomagnesemia, GI

disorders, renal wasting.

3. Hyperphosphatemia

 Due to renal failure, laxatives, cytotoxic drugs

4. Malabsorption

 Due to sprue,