Citrate Blood a Century of Artifact.

Sodium citrate has been used as an anticoagulant to prepare blood and fractions for over 100 years. Its use is sufficiently common that many investigators do not differentiate anticoagulated blood from blood per se. To evaluate the influence of citrate anticoagulation on the dynamics of the blood coagulation process, we compared the properties of blood, platelet rich plasma (PRP) and platelet poor plasma (PPP) prepared and stimulated using standard citrate/Ca++ reconstitution methodologies with matching samples in which transient anticoagulation without disruption of Ca++ homeostasis was achieved by blockade of the contact pathway (using corn trypsin inhibitor (CTI) or inhibitory antibodies to factor XIa). Phlebotomy blood was collected into citrate, CTI or CTI supplemented with citrate, the latter combination establishing conditions in which the effects of citrate anticoagulation in the absence of contact pathway contributions could be studied. Coagulation reactions in citrated milieus were initiated in typical fashion by Ca++ addition or following preincubation with Ca++ prior to tissue factor (Tf) addition in CTI citrate plasma; blood/products stabilized by CTI alone were initiated via Tf addition. Reaction dynamics were analysed using either calibrated automated thrombography (CAT) or thromboelastography (TEG). Platelet aggregation studies compared the reactivity of a panel of agonists. Significant differences were seen in the response of samples exposed to citrate. These results included: CTI plasma and blood displayed a more potent response to Tf when compared to their respective CTI citrate products. Specifically shorter lag phases (~120 s) and higher thrombin levels (1.5–2 fold higher maximum thrombin levels) are observed in CTI plasma with CAT and similarly shorter r (lag) times [(mean ± SD)CTI blood, r = 7.1 ± 0.9 min (n=21, 9 individuals) vs CTI citrate blood, r = 9.1 ± 1.5 min (n=10, 5 individuals)] with TEG. Reconstitution of prechelation Ca++ in CTI citrate plasma or blood prior to Tf addition only partially restored thrombin generation to that of its prechelation product. For example preincubation with Ca++ resulted in altered TEG profiles with prolonged “K” (rate) values (mean ± SD) for CTI citrate blood (7.5 ± 0.6 min (n=5)) compared to CTI blood (3.3 ± 0.5 min (n=5)) from the same individuals. Vitamin K dependent protein conformational transitions in response to chelation/Ca++ reconstitution were important contributors to the differences observed between CTI and citrated blood/plasmas, including the process of protein S polymerization and depolymerization Platelet function studies in citrate containing PRP showed significant enhancement in reactivity toward epinephrine and ristocetin with lesser effects with collagen; in 3 of 4 donors CTI PRP showed no sensitivity to epinephrine. The magnitude of alterations induced by citrate chelation were in the order epinephrine> ristocetin> ADP > collagen. Historically, the regimen of citrate chelation followed by Ca++ addition has proven useful in the interpretation of the integrity of the coagulation system and thereby in clinical diagnosis. However, our results suggest that the procedure of Ca++ chelation/recalcification results in artifactual representations of the dynamics and biology of the blood coagulation process.