Despite the fact that only the Ca++ fraction is physiologically active, the Ca status of animals has typically been based on evaluation of serum total Ca concentration, as total serum Ca concentration has been assumed to be directly proportional to Ca++. In dogs, total serum Ca concentrations are also routinely “corrected” or “adjusted” relative to the serum total albumin, whenever the albumin concentration is below 3.5 mg/dL. However, a question has remained about whether total serum Ca can be used to accurately predict Ca++. A recent retrospective study of over 1500 cases found diagnostic discordance between total Ca and Ca++ of 27-37% and between 36-54% of the subpopulation of dogs with chronic renal failure. Thus, hypercalcemia is overestimated, and hypocalcemia is underestimated. In a parallel retrospective review of > 400 feline cases, discordancy between total Ca and Ca++ was ~25%. Thus, serum Ca++ must be measured to accurately assess Ca status.

Accurate determination of Ca++ requires that samples be collected and processed correctly. Acidic pH favors dissociation of Ca from protein and increases the amount of Ca++ in the sample, whereas alkaline pH decreases the amount of Ca++. Mixing of serum with air results in decreased Ca++ and increased pH.

While Ca++ can be measured in heparinized whole blood, this is not recommended as heparin may interfere with measurement of Ca++. Silicone-separator tubes should not be used because ionized Ca concentration is increased due to release of Ca from the silicone gel.

Serum should be collected via vacutainer directly into a serum vacutainer tube. Blood should be allowed to clot, and then separated by centrifugation. To remove serum from the tube, the tube must not be opened. A needle attached to an empty syringe should be used to withdraw serum from the vacutainer tube, through the red top. This serum is then transferred to a second serum vacutainer tube through the needle to avoid introducing any air into the sample. Serum collected this way (anaerobically) and stored at 4C is stable for up to 7 days, which permits time for shipment to a reference laboratory for measurement of Ca++.