Isocitrate as calcium ion activity buffer in coagulation assays

Ca(2+) activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca(2+) activity at approximately 1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca(2+) buffering improves diagnostic efficacy in global diagnostic coagulation tests.

Buffering was investigated by mixing CaCl(2) and 11 candidate compounds and determining Ca(2+) activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca(2+) activity to approximately 1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved.

Two suitable Ca(2+) buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca(2+) buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca(2+) activity of 1.60 +/- 0.07 mmol/L, compared with 2.73 +/- 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 +/- 5.7, compared with 3.6 +/- 5.0 (P <0.005) for the original APTT.

Isocitrate can be used to buffer Ca(2+) activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca(2+) activity shows signs of improved diagnostic efficacy.