Research Article: Impact of experimental hypercalcemia on routine haemostasis testing

Date Published: March 31, 2017

Publisher: Public Library of Science

Author(s): Giuseppe Lippi, Gian Luca Salvagno, Giorgio Brocco, Matteo Gelati, Elisa Danese, Emmanuel J. Favaloro, Pablo Garcia de Frutos.


The blood to anticoagulant ratio is standardized according to the physiological calcium concentration in blood samples conventionally used for hemostasis testing. Specifically, one fixed volume of 0.109 mmol/L sodium citrate is added to 9 volumes of blood. Since little is known about the impact of hypercalcemia on the calcium-binding capacity of citrate, this study was planned to investigate the effect of experimental hypercalcemia on routine hemostasis testing.

Fifteen pooled citrated plasmas with matching lithium-heparin pooled plasma from patients with different values of prothrombin time (PT) were divided in three aliquots of 0.6mL each. The first paired aliquots of both citrate and lithium-heparin plasma were supplemented with 60μL of saline, the second paired aliquots with 30μL of saline and 30μL of calcium chloride and the third paired aliquots with 60μL of calcium chloride. Total and ionized calcium was measured in all aliquots of citrate and lithium-heparin plasma, whereas PT, activated partial thromboplastin time (APTT) and fibrinogen were measured in citrate plasma aliquots.

Total calcium concentration gradually increased in both lithium-heparin and citrate plasma aliquots 2 and 3 compared to baseline aliquot 1. The concentration of ionized calcium also gradually increased in lithium-heparin plasma aliquots 2 and 3, whereas it remained immeasurable (i.e., <0.10 mmol/L) in all citrate plasma aliquots. No significant differences were observed for values of PT, APTT and fibrinogen in citrate plasma aliquots 2 and 3 compared to the baseline aliquot 1, with a mean bias was always comprised within the desirable quality specifications derived from biological variability data. Hypercalcemia, up to severe hypercalcemia does not generate significant bias in results of first-line coagulations tests, so that hypothetical consideration of adjusting citrate-blood ratio is unjustified in hypercalcemic patients.

Partial Text

Laboratory testing is vital for screening, diagnosing and monitoring treatment related to hemostasis disturbances, as associated with both thrombotic and bleeding risk [1,2]. Coagulation tests are usually classified according a conventional hierarchy of complexity, entailing first-line (i.e., screening), second-line (i.e., diagnostic) and third-line tests, the last of which are usually performed to help defining the specific nature and severity of an underlying hemostatic disorder [3]. Due to the crucial role of haemostasis testing in diagnosing and managing hemostasis disturbances, a high degree of accuracy must be assured throughout the total testing process, i.e., from sample collection to testing and clinical interpretation of data [4–7].

A total number of 30 routine coagulation samples were identified according to their PT value, and concomitant availability of a paired lithium-heparin specimen referred for routine calcium measurement. The samples were selected as follows: 10 routine samples with values of PT comprised between 0.97–1.09 (set A; routine ‘normal haemostasis’ samples), 10 samples from patients on oral anticoagulant therapy (OAT) with values of PT comprised between 1.50–2.50 (set B; modestly ‘abnormal’ or ‘anticoagulated’ samples) and 10 samples of patients on OAT with values of PT comprised between 2.5–3.7 (set C; highly abnormal or anticoagulated samples). These were selected in order to assess any potential effect of hypercalcemia on both normal and abnormal samples, including patients being monitored for therapy, and where an effect might affect their clinical management. All routine samples were collected in evacuated blood tubes containing either 0.109 mmol/L buffered sodium citrate (citrate plasma; Vacutest Kima, Padova, Italy) or lithium-heparin (lithium-heparin plasma; Vacutest Kima) and were then separated by standard centrifugation at 1500 g per 10 min at room temperature.

The main results of this study are shown in Table 1.

The first evidence that a concentration of 0.01 mmol/L of sodium citrate may be sufficient to inhibit the coagulation process by neutralizing a physiological concentration of ionized calcium was provided by Quick and Stefanini nearly 70 years ago [15]. Since then, only few studies have been published regarding the potential impact of hypercalcemia on citrate-binding capacity and hemostasis testing. More than 40 years ago, Hilgard evaluated the effect of experimental hypercalcemia on blood coagulation in mice [16]. Hypercalcemia was induced by transplanting solid Walker 256 cancer and intraperitoneally injecting calcium gluconate. The effect on blood coagulation was then assessed by measuring whole blood clotting times in polystyrene and glass test tubes. A significant shortening of clotting times was observed at serum calcium concentrations between 5.1–5.7 mmol/L, thus being virtually incompatible with life, which was also found to be more pronounced in polystyrene (-44%) than in glass (-25%) tubes. Additional data suggesting that calcium may actually influence blood coagulation was conveyed by Bristow et al, who evaluated the impact of calcium supplements on thromboelastography (TEG) in post-menopausal women [17]. Interestingly, an increase of coagulation index was only noticed 4 h after ingestion of 1 g of calcium citrate, yielding a significant shortening of the time of clot initiation, thus reflecting a greater tendency toward hypercoagulability. No effects were instead observed at other different time points (i.e., 2, 6 and 8 hours).