Abstract
It is well recognized and accepted that the free or ionized component of calcium in the blood is the biologically important fraction. Consequently, the measurement of ionized calcium should play a critical role when investigating changes in calcium homeostasis, particularly in the diagnosis of both parathyroid-dependent and, less frequently, parathyroid-independent hypercalcaemia.1,2 For many previously cited reasons, including method availability, analytical performance, standardization, practicality of sample handling and lack of automated analysis, ionized calcium is not as frequently measured as total calcium and most consensus statements and guidelines fail to emphasize the utility of this measurement. 3 Are these valid reasons in 2013 for laboratories not to measure ionized calcium more frequently?
A number of correction factors can be used when measuring total calcium in an attempt to estimate the ionized calcium fraction. Approximately 50% of calcium circulates as a free fraction; described as the diffusible fraction by early investigators using dialysis experiments. The non-diffusible fraction represents 35–40% of total calcium, mainly bound to albumin and a smaller fraction to globulin, with the remaining 10–15% being complexed to anions including bicarbonate, phosphate, lactate and citrate. Consequently, nomograms have been developed which correct for changes in protein and utilize in the simplest form, an adjustment for serum albumin concentration. However, interindividual variation of calcium-albumin binding, changes in the binding affinity constant especially when the albumin concentration is less than 30 g/L and the importance of anion complexed calcium limits the utility of albumin-adjusted total calcium measurement. More complex formulae utilize albumin, globulin, anion gap or pH. Some laboratories are even able to estimate ionized calcium-based on the use of multiple laboratory analytes rather than direct measurement of ionized calcium by ion selective electrode methods. Others have been unable to reproduce those results and do not recommend estimated calculations for ionized calcium. 4 It is worth revisiting some of the preanalytical limitations as well as postanalytical benefits of the direct measurement of ionized calcium in this context. Lam et al. 5 in this issue of the journal, provide a welcome update on one of those preanalytical limitations, the use of pH adjustment for ex vivo sample changes, when measuring ionized calcium.
Preanalytically, three important issues regarding the direct measurement of ionized calcium are worth considering: sample type, expediency of measurement and sample pH. 6 Placing the relevance of whole blood, serum or plasma sample measurement aside, the importance of sample storage and sample pH are worth addressing. Ideally, ionized calcium should be measured in an anaerobically collected and stored sample with care being required to ensure the sample remains sealed until just before analysis. Ex vivo changes in sample pH, which occur when the sample has not been kept under anaerobic conditions, can be corrected by the use of a pH correction of the sample and adjusting the ionized calcium to the expected value when the ambient pH is 7.4. Such pH-adjusted ionized calcium measurements are possible as most early studies reported a linear association between ionized calcium and pH. In this issue, Lam et al. 5 investigate the use of pH adjustment to ionized calcium measurement utilized to correct for ex vivo pH changes. Their data question the validity of currently published algorithms that correct for pH, provide a welcome addition to the literature in this area and offer a reason to re-open discussion. Whether these data are reproducible since only one sample was analysed per person, relevant to other populations and applicable to other ion selective electrode methods awaits further studies. The solution to this problem is either the provision of an improved correction nomogram, which may or may not be method-specific, or to change sample handling in the laboratory to expedite analysis. The latter is preferable, especially when directly measuring ionized calcium in patients with a pre-existing acid-base disorder.
The diagnostic discordance between total and ionized calcium is estimated to be around 20–30% according to several earlier studies comparing both measurements. Two specific factors were identified in one study that explained this discordance: the concentrations of albumin and parathyroid hormone (PTH). The former may help explain why albumin-adjusted calcium estimation can be discordant with direct ionized calcium measurement. The latter alludes to data that have been verified in subsequent studies of subpopulations with parathyroid-dependent hypercalcaemia which confirm that the direct measurement of ionized calcium is more sensitive than either total calcium or albumin-adjusted total calcium; 20–25% of cases of primary hyperparathyroidism could be missed using either of the latter measurements.7,8 Recent data in surgically confirmed and histologically proven cases of primary hyperparathyroidism have substantiated that ionized calcium continues to offer improved diagnostic sensitivity.9,10 One study confirmed postoperative normocalcaemia according to repeat assessment of ionized calcium after surgical neck exploration and successful parathyroidectomy. 9 The latter finding is important as postoperative normocalcaemia excludes the possibility of familial hypocalciuric hypercalcemia (FHH), an important disorder in the differential diagnosis of parathyroid-dependent hypercalcaemia. Surgical cure is not possible or desirable in FHH which is due to a mutation in the calcium sensing receptor gene and not due directly to parathyroid disease. These findings could also explain a significant proportion of cases that have been characterized as normocalcaemic primary hyperparathyroidism according to total calcium results which are consistently within reference limits but PTH concentrations that are increased and where secondary causes of hyperparathyroidism have been excluded. 11
The direct measurement of ionized calcium also plays an important role in the diagnosis of pseudohypercalcaemia, an infrequent but important condition in myeloma where monoclonal immunoglobulins can bind calcium so total but not ionized calcium concentration is increased. Calcium bound to citrate may also occasionally cause significant discordance between total and ionized calcium especially when rapid and extensive blood transfusion is required or where clearance is delayed by hypothermia or liver transplantation. 12 Other potential benefits of measuring ionized calcium directly include among critically ill patients where discordance between total adjusted and ionized calcium being can be partially explained by hypoalbuminaemia and/or metabolic acidosis. 13 In chronic kidney disease (CKD), ionized calcium measurement has been demonstrated to be superior to total calcium measurement in the diagnosis of hypercalcaemia in renal transplant recipients 14 and CKD stages 3–5. 15 The inverse sigmoidal association between PTH and calcium is more evident for ionized calcium compared with total-adjusted calcium and haemodialysis patients show a discordance of 40% between ionized calcium and albumin-adjusted total calcium. 16
The maintenance of extracellular calcium homeostasis is crucial to many cellular processes within the body and depends on the integrated transport of calcium across the intestinal wall, renal tubule and into and out of skeletal mineral stores. The calcium sensing receptor orchestrates this regulatory role and utilizes several important hormones in this process including PTH and vitamin D. Changes in extracellular calcium concentration therefore only occur when there are genetic or pathological changes which perturb this regulatory system. The ability to directly measure ionized calcium offers several diagnostic advantages, particularly among patients with primary hyperparathyroidism, CKD and acute critical illness. Many laboratories already offer the facility to measure ionized calcium. One recent study which verified routine use of ionized calcium as a superior diagnostic test for primary hyperparathyroidism involved testing that was performed across several sites by several different pathology providers with samples being collected under routine laboratory conditions. 10 Consequently, improved diagnostic sensitivity is achievable in routine practice and is not restricted to single laboratory sites or research centres of excellence. Measurement of ionized calcium by direct electrode potentiometry has improved considerably with advancing technological developments and a reference method is established: there are no compelling analytical arguments against the use of this methodology. 17 Manual handling of samples and lack of full automation of analysis seems a small price to pay for superior analytical performance. With appropriate sample handling, including a change in laboratory processing, ionized calcium can be measured first and in a timely fashion in most laboratory settings. The time has finally come to question why we are not advising our clinical colleagues to make the transition to measuring ionized calcium more frequently.