Free ionized calcium

Physiology

Total calcium as measured on the chemistry panel includes a large component that is protein-bound and does not necessarily reflect the status of the biologically active ionized form. Free ionized calcium (iCa2+) is the form of calcium that is readily available to cells, and measurement of iCa2+ is a more accurate reflection of the physiological calcium state than total calcium. This is also the form of calcium that stimulates or suppresses hormones involved in calcium homeostasis, such as PTH, vitamin D and calcitonin. Approximately 50% (dogs 56%, horses 49%) of total calcium is free ionized, with the remainder (40-45%) being bound to proteins (dogs 34%, horses 47%), principally albumin, and 5-10% (dogs 10%, horses 4%) to anions such as citrate, lactate, bicarbonate, phosphate or lactate. Protein-or anion-bound bound calcium is not involved in calcium homeostasis; this is mediated by the hormones that control free ionized calcium. Proteins do not serve as a reservoir for calcium that the body can readily access, i.e. if free ionized calcium is low, calcium is not released from albumin or anions to compensate for the decrease in free ionized calcium, rather there is stimulus of parathyroid hormone (PTH) synthesis and release (PTH acts to increase free ionized calcium by stimulating osteolysis and calcium resorption in the renal tubules). Even if free ionized calcium is transiently affected (which is unlikely) by changes in anions or albumin, free ionized calcium concentrations are quickly normalized by hormones involved in calcium homeostasis. Also, changes in albumin that lead to changes in total calcium usually occur slowly, giving time for the body to adjust.

Methods

Free ionized calcium is measured directly using ion-selective electrodes (direct potentiometry). These electrodes are not provided with a routine chemistry analyzer but are available with blood gas analyzers or point of care analyzers (e.g. iStat).

Procedure

With this technique, an electrode containing an internal electrolyte solution is immersed in the patient sample, which is separated from the internal solution by a membrane that can detect the electromotive force (EMF) generated by the ions in both solutions. This EMF is determined by the difference in concentration of the test ion in the test solution and internal filling solution (test ion at fixed concentration). The EMF is predicted by the Nernst equation (see Techniques for more details on the method).

Units of measurement

The concentration of free ionized calcium is measured in mg/dL (conventional units), mEq/L (conventional units), and mmol/L (SI units). The conversion formulas are identical to total calcium:

mg/dL x 0.2495 = mmol/L 

mEq/L x 0.5 = mmol/L

Sample considerations

Sample type

Serum (preferred), heparinized plasma (sodium heparin, lithium heparin)

Anticoagulant

EDTA or citrate cannot be used for free ionized calcium measurement (these anticoagulants chelate calcium). Heparin can be used, but it is a polyanion (negatively charged) and will bind free ionized calcium (falsely decreasing concentrations). This will occur even when liquid heparin is drawn into a syringe, which is then expelled to eliminate most of the heparin. Studies have shown that this method leaves variable amounts of heparin, which can falsely lower free ionized calcium concentrations. Ideally, heparin should not exceed 4% of total blood volume in the tube. However, this is hard to control, thus specific electrolyte-balanced heparin syringes are the best-choice to use for free ionized calcium measurement in whole blood (they prevent calcium chelation), but are expensive. Hence, serum is  preferred over regular lithium heparin tubes (green top tubes) and the expulsion method with liquid heparin. Serum-separator tubes or gel tubes contain calcium in the silicon gel, spuriously increasing values from the release of free ionized calcium from the gel. Zinc heparin interferes with the assay.

Stability

The stability of free ionized calcium with storage differs depending on the species. Samples should not be frozen. Freezing falsely decreases free ionized calcium values (related to an increase in pH or alkalinity that develops during frozen storage). When stored at refrigerated or room temperature, free ionized calcium values increase due to the drop in pH that comes with cell metabolism.

  • Canine:
    • Whole blood: Stable for 3 hours at 4°C. After this time, free ionized calcium values increase (due to decreased pH as stated above). Free ionized calcium is not stable in whole blood kept at room temperature.
    • Serum/heparin plasma: Stable for 240 hours at 4°C, serum also is stable for 72 hours at 20°C. Samples should not be frozen (not stable at 240 hours when stored at -20°C).
  • Bovine: In blood, serum or heparinized plasma, it is stable for 48 hours at 4°C, but unstable frozen at 240 hours.
  • Equine: In whole blood, serum or heparinized plasma, it is stable for 48 hours at 4°C, but unstable frozen at 240 hours.

Interferences

Free ionized calcium is affected by anticoagulant (as stated above), pH, storage of the sample and exposure to air.

  • Anticoagulant: see above.
  • pH: Acidosis can increase the proportion of free ionized calcium (by causing release from protein), whereas alkalosis can decrease it (increases protein binding). This seems to have more implications for sample storage than in vivo changes secondary to acidosis or alkalosis in the animal, although brief changes in free ionized calcium have been reported in experimentally induced acidosis and alkalosis. The pH is altered by sample storage (usually decreases at refrigerated temperatures and increases at frozen temperatures), which will affect the free ionized calcium result (see above).
  • Storage conditions: This varies with species (see above). If free ionized calcium is not measured immediately, serum or plasma should be separated from cells (cells will be undergoing anaerobic metabolism in the tube, leading to lactic acid accumulation, which decreases the pH and falsely increases free ionized calcium values).
  • Air exposure: Will decrease free ionized calcium values. Exposure of the surface of serum briefly to air does not alter values, but mixing serum with air decreases free ionized calcium and increases pH in dogs (due to loss of pCO2, which is an acid – the subsequent increase in pH will decrease free ionized calcium). Another study indicated that free ionized calcium in dogs can be measured 10 minutes after exposure to air, after which time values decreased (both aerobically and anaerobically), although medians were lower in aerobic tubes.

Recommendations for sample collection for measurement of free ionized calcium

  • Collect blood into red top tubes (preferred to lithium heparin or green top tubes). Note, that some laboratories recommend collection of blood into green top tubes for free ionized calcium measurements, but associated reference intervals are presumably generated from similar samples (and may have lower reference interval limits than intervals based on serum samples). In the past our reference intervals were based on evacuated heparin-coated syringes, however our current intervals are based on serum samples handled as anaerobically as possible.
  • Allow samples to clot, then spin down.
  • Maintaining anaerobic conditions, collect the serum (can uncap tube briefly to remove serum or using a syringe and needle, withdraw serum through the cap, making sure all air is expelled from the syringe and needle). Do not mix serum with air.
  • Place serum in another red top tube, maintaining anaerobic conditions as much as possible (go through the cap with needle and syringe, if using).
  • Keep cool and submit to laboratory ASAP.

Test interpretation

Aberrations in free ionized calcium indicate a disruption in calcium homeostasis. Free ionized calcium cannot be predicted reliably from total calcium (this is particularly true for animals with renal disease) and correction formula (or adjusting calcium values for albumin concentration) are not recommended for use. Thus, if a disorder of calcium homeostasis is suspected, free ionized calcium should be directly measured (with no attempts made to predict values from total calcium or adjusted calcium). 

Increased concentrations (free ionized hypercalcemia)

Deleterious effects of increased free ionized calcium include defective organ function from mineralization of tissues (metastatic calcification), decreased contractility of muscles (gastrointestinal tract, heart) and nerves and kidney damage. To see more on these effects, see the total calcium page. The most common causes of increased free ionized calcium in small animals are cancer (particularly lymphoma and anal sac apocrine gland carcinoma, but also sarcomas, such as osteosarcoma in dogs, and hematopoietic tumors, like multiple myelomas), hyperparathyroidism and hypoadrenocorticism in dogs (n=1641) and cancer, renal disease (acute and chronic) and idiopathic hypercalcemia in cats (n=119) (Coady et al 2019). In another study of 238 cats, acute or chronic kidney disease (including obstructive urolithiasis), cancer (T cell lymphoma, leukemia not specified, epithelial tumors, such as pulmonary carcinoma), idiopathic hypercalcemia  were the most common causes of high free ionized calcium > 1.41 mmol/L. Infrequent causes included hyperparathyroidism, iatrogenic from excess calcium or vitamin D supplementation or calcium gluconate infusion to treat hyperkalemia), vitamin D toxicity, granulomatous inflammation (feline infectious peritonitis, mycobacterial infection), and transient hypercalcemia (Broughton et al 2023). Cancer, hyperparathyroidism and vitamin D toxicity and cancer and idiopathic hypercalcemia being associated with moderate to severe increases in calcium in dogs (>1.4 mmol/L) and cats (>1.5 mmol/L), respectively (Coady et al 2019).

  • Artifact: With storage of samples, particularly whole blood not separated from cells, pH will decrease, which may falsely increase free free ionized calcium (see above).
  • Iatrogenic: Administration of calcium-rich solutions, particularly intravenously, can cause a high free ionized calcium. This is the most common cause of hypercalcemia in dairy cattle.
  • Physiologic: Cats under 1 year of age can have free ionized calcium >1.41 mmol/L and < 1.54 mmol/L (n=5) (Broughton et al 2023).
  • Pathophysiologic: The mechanisms that could lead to increased free ionized calcium are increased osteolysis (PTH, PTHrP, localized tumors), increased gastrointestinal absorption, and decreased renal excretion. Anion- or protein-binding causes of high total calcium are generally associated with normal free ionized calcium.
    • Primary and secondary hyperparathyroidism:Increased PTH due to parathyroid disease or secondary to nutritional imbalance of calcium phosphate (nutritional secondary hyperparathyroidism) or renal disease (renal secondary hyperparathyroidism) may increase free ionized calcium.
    • Neoplasia: Mechanisms include tumor production of parathyroid hormone-related peptide (PTHrP) (e.g. lymphoma and apocrine anal sac adenocarcinoma in dogs, squamous cell carcinoma in horses), tumor production of vitamin D (e.g. histiocytic sarcoma, lymphoma in dogs) and localized osteolysis (e.g. multiple myeloma). Note that although osteosarcoma is an osteolytic and osteoproductive neoplasm, high total or free ionized calcium is not typically seen in animals with this bone tumor.
    • Renal disease: In dogs with chronic renal disease, a high total calcium is usually attributed to increases in anion-complexed calcium. Free ionized calcium is rarely increased, i.e. only 9% of dogs with renal disease with high total calcium had high free ionized calcium – most had normal or low free ionized calcium (cannot assume free ionized calcium will be high, just because total calcium is high; this is true even if albumin is normal). Horses with chronic renal disease frequently have high total calcium with a normal albumin, so presumably free ionized calcium is also high (free ionized calcium is not frequently measured in horses). Cats with acute kidney injury and chronic renal disease can have high free ionized calcium (Broughton et al 2023).
    • Hypoadrenocorticism: Some dogs with Addison’s disease can have high total and free ionized calcium (up to 1.98 mmol/L). Variable increases in parathyroid hormone and calcidiol and calcitriol are seen in affected dogs (Gow et al 2009).

Decreased concentrations (free ionized hypocalcemia)

Clinical signs of hypocalcemia in many species include muscle tremors, convulsions, ataxia, and weakness. In horses, hypocalcemia is associated with synchronous diaphragmatic flutter and signs of tetany including stilted gait, muscle tremors, flared nostrils, inability to chew, recumbency, convulsions, and cardiac arrhythmias. In cows, hypocalcemia is usually manifested as weakness and recumbency. Signs of hypocalcemia develop when free ionized calcium is too low for normal muscle and nerve function. The most common causes of hypocalcemia in one study were critical illness, kidney disease, and toxicity (chelation by citrate from transfusions, ethylene glycol toxicity, furosemide administration) in dogs (n=1467) and acute and chronic kidney disorders (including urethral obstruction) and critical illness in cats (n=450). Moderate to severe hypocalcemia (<1.00 mmol/L) was seen mostly in hypoparathyroidism, kidney disease, eclampsia and critical illness in dogs and with renal disorders, soft tissue damage and critical illness in cats. The cause of severe hypocalcemia was not determined in 9 and 4% of dogs and cats, respectively. Hypocalcemia was more common than hypercalcemia in cats than the latter study, whereas the reverse was true for dogs (although the difference in numbers was small) (Coady et al 2019).

  • Artifact: Excessive heparin in blood sample (chelates calcium), EDTA or citrate anticoagulant (chelates calcium), frozen storage (increased pH), aerobic sample handling (mixing with air increases pH).
  • Iatrogenic: Administration of large amounts of citrate-containing fluids (massive blood or plasma transfusions) can result in free ionized hypocalcemia and associated clinical signs of weakness in dogs. Administration of furosemide is also associated with low free ionized calcium, which can be severe (Coady et al 2019).
  • Pathophysiologic: The following mechanisms could lead to decreased free ionized calcium: Decreased gastrointestinal absorption, increased renal excretion, movement into muscle, and increased loss in milk.
    • Chronic renal disease: In dogs and cats with chronic renal disease, free ionized calcium can be low (total calcium can be normal, increased or high, i.e. cannot predict free ionized calcium from total calcium). This will stimulate a secondary renal hyperparathyroidism (low ionized calcium is a stimulus for PTH secretion, which will result in osteolysis in an attempt to correct the low free ionized calcium). This is why animals with chronic renal disease can develop “rubber jaw” (from prolonged osteolysis in an attempt to maintain free ionized calcium values) but happens in a very low percentage of dogs with this disease. Low free ionized calcium in dogs with renal disease has been attributed more to lack of vitamin D production (this is produced in the kidney) or unresponsiveness of the renal tubules to PTH than increased anion-complexing, although low free ionized calcium is seen in patients with high phosphate (e.g. > 15 mg/dL). A mild increase in phosphate is unlikely to affect free ionized calcium levels. Urinary tract obstruction in cats can also result in low free ionized but normal total calcium (mechanism unknown).
    • Hypoparathyroidism: This has been rarely reported in dogs and cats.
    • Hyperthyroidism: Cats with overactive thyroids can have low free ionized but normal total calcium. This seems to be pathologic (although the mechanism is unknown), because the cats have  hyperparathyroidism (likely as a consequence of low free ionized calcium).
    • Lactation-associated hypocalcemia: Milk fever in cattle and eclampsia in other species (for more, see total calcium page).
    • Other causes: Idiopathic hypocalcemia in foals, excess sweating in horses (e.g. endurance rides – total calcium may be normal), dietary deficiency (most species), cantharidin toxicosis in horses, oxalate toxicity in grazing animals, hypercalcitonism in dogs, horses and cattle, gastrointestinal disorders in horses (loss), transport tetany, sepsis in all animals, and hyperthermia (movement into muscle).
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