The diagnosis of hypercalcaemia is primarily made on the basis of serum calcium and preferably serum Alb-Ca or, whenever possible, Ca2+ levels (Figure 1). When serum calcium is above the normal range of the laboratory, the first step should be to confirm hypercalcaemia by a second measurement.
Fig1. Differential diagnosis of hypercalcaemia. PTH, parathormone; FHH, familial hypocalciuric hypercalcaemia; LH, Hodgkin’s lymphoma; NHL, non- Hodgkin’s lymphoma.
It is important to know that serum calcium results are affected by important preanalytical, analytical, and postanalytical factors. Fasting blood samples should be collected, avoiding venous stasis. Haemoconcentration and haemodilution are the most common preanalytical causes of misleading results. Haemoconcentration is usually due to dehydration and venous stasis and may increase serum calcium concentration. Other factors can be posture, abnormal proteins levels, temperature, anions, and pH. Moreover, some analytical variables should be considered: the most common method for total serum calcium measurement is the spectrometry analysis, using metallochromic indicators or an ion- specific electrode (ISE). The within- and between subjects’ variation of total serum calcium ranges between 1.9 and 2.8%, and the desirable value for inaccuracy, imprecision, and total assay error are 0.8%, 1%, and 2.4%, respectively. It is very important to validate the assay using appropriate quality control (QC) methods and have adequate laboratory coefficients of variation. Finally, post- analytical interpretation is also very important. The large majority of laboratories that use high accuracy methods for total calcium measurement have a reference interval of approximately 8.8– 10.2 mg/ dl (Use the formula serum calcium mg/ dl: 2.5 = serum calcium mmol/ L.). The reference interval is generally established at the central 95% of the Gaussian distribution, so at the extreme 2.5% two sites of the curve values obtained in normal individuals will be interpreted as hyper- or hypocalcemia. In bordeline cases we recommend using the serum Alb-Ca obtained by the previously reported formula. However, values derived from this formula does not take into account the binding of calcium to proteins other than albumin and therefore do not accurately reflect the free serum calcium concentration.
Wherever available, serum Ca2+ should be measured, because it provides a more accurate assessment of the calcium status. It has been demonstrated that serum Ca2+ has a higher sensitivity for malignant hypercalcaemia and for the diagnosis of PHPT.
Moreover, in particular conditions, the measurement of serum Ca2+ is critical. In patients with kidney failure, comparison of results of total and Alb- Ca with Ca2+ demonstrated that the former measurements improperly classified as hypercalcaemic 20% of patients. The correct interpretation of calcium levels in those patients is important for the appropriate treatment [13]. In patients with critical diseases there is a redistribution of free, protein- bound, and complexed calcium and, therefore, the measurement of serum Ca2+ should be preferred. The measurement of serum Ca2+ is also preferable in neonates and infants. Exercise and hyperventilation may influence the concentration of serum Ca2+: exercise may increase serum Ca2+ largely because exercise- induced acidosis decreases the binding of calcium to serum proteins, but reduction of plasma volume and in flux form extracellular sources may also contribute; conversely hyper ventilation may decrease Ca2+ because of hyperventilation- induced alkalosis increases the binding of calcium to serum proteins.
Once the diagnosis of hypercalcaemia is confirmed plasma PTH levels should be measured to distinguish PTH- dependent and PTH- independent hypercalcaemia. Second and third generation immunoradiometric PTH assays demonstrated a higher sensitivity (88– 97%) in the diagnosis of PHPT compared to first generation assays and are the most commonly used. When PTH measurement is not easily available serum phosphate may be used to guide the differential diagnosis, being usually normal in PTH- independent hypercalcaemia and low or in the lower normal range in PTH- dependent hypercalcaemia. Serum creatinine and kidney function should also be evaluated.
A 24- hour urine collection for calcium and creatinine measurements should be obtained to calculate the calcium to creatinine clearance ratio and rule out familial hypocalciuric hypercalcaemia (FHH) (see next). Indeed, a calcium to creatinine ratio of less than 0.01 is suggestive of FHH. The measurement of serum 25(OH) vitamin D (25(OH)D) and, especially, 1,25(OH)2D may help in the diagnosis of vitamin D- dependent forms of hypercalcaemia.
