Calcium is found in three major forms in circulation: protein-bound, anion-bound, and ionized or free. To begin on a side note, the term “ionized” calcium is somewhat misleading, as even calcium bound to protein or anions is in fact ionized; from here on I’ll refer to unbound calcium as free. Semantics aside, the best indicator of a patient’s overall circulating calcium status is free calcium. Free calcium is the form of the mineral that binds to G-protein coupled calcium-sensing receptors and is tightly regulated by PTH and 1,25OH vitamin D. If free calcium is so great, why are there so many orders for total calcium? The majority of the time, a total calcium measurement is consistent with free calcium levels providing sufficient information for patient management. The advantage of total calcium is that it has less stringent sample collection requirements than free calcium and can be added on to most routine blood draws.
When is free calcium testing useful? Total calcium can be misleading in critically ill patients, patients receiving transfusions (with citrate blood), those with late stage CKD, and neonates among a number of other clinical situations (references to these and other issues with calcium testing can be found in a Sept. 2011 CLN article by Dr. John Toffaletti). The challenge of free calcium measurement is preanalytical variation. Accurate free calcium measurement requires anaerobic sample collection and handling, and rapid analysis or inhibition of cellular metabolism. These requirements are necessary because the amount of free calcium is directly affected by pH. As pH decreases, proteins (particularly albumin) bind additional hydrogen ions. Hydrogen ions effectively compete with free calcium for available negative charges on proteins. Thus, an increase in circulating hydrogen ions leads to a decrease in protein-bound calcium and increased circulating free calcium.
This relationship between pH and calcium has led to the practice of correcting free calcium levels for pH. If a patient has a pH of 7.4, but over the course of collection and transport the pH of the sample has drifted down to 7.2, it makes sense that the result should be corrected to 7.4. Free calcium measurement should be reported at the pH of the patient. A problem occurs when the patient’s pH is not 7.4. The free calcium concentration cannot be accurately corrected when the patient’s true pH is unknown. Thus the question of whether or not to correct free calcium for pH demands knowledge of the patient’s pH. Another problem with pH correction is that free calcium is buffered by lactate (the anionic pool). pH correction in a sample with high lactate can result in overcorrection of free calcium. Did I mention that there are more than 5 different formulas to correct free calcium for pH? Each of these formulae is subject to variation in the concentration of protein and anion pools and each was derived within a limited pH range of pH.
Despite these issues, some have advocated for pH-adjusted free calcium measurements in specific clinical scenarios. In community outpatient settings, where most patients should not have an acid-base disorder, one could assume that patients will have a “normal” pH; but that word assume is there. A more practical approach is to order total calcium in patients who do not need a free calcium measurement (see above), and to follow the stringent sample collection requirements for free calcium in patients who do need free calcium measurement. Rather than make a correction using a potentially flawed equation based on an assumption, it is safer to avoid the problem with total calcium and good laboratory practice.