Influence of citrate concentration on the activation of blood cells in an in vitro dialysis setup

Abstract

BackgroundRegional citrate anticoagulation has been associated with enhanced biocompatibility in hemodialysis, but the optimal dose of citrate remains to be established. Here, we compared parameters related to cellular activation during in vitro dialysis, using two doses of citrate.MethodsHuman whole blood, anticoagulated with either 3 mM or 4 mM of citrate, was recirculated in an in vitro miniaturized dialysis setup. Complement (C3a-desArg), soluble platelet factor 4 (PF4), thromboxane B2 (TXB2), myeloperoxidase (MPO), as well as platelet- and red blood cell-derived extracellular vesicles (EV) were quantified during recirculation. Dialyzer fibers were examined by scanning electron microscopy after recirculation to assess the activation of clotting and the deposition of blood cells.ResultsIncreases in markers of platelet and leukocyte activation, PF4, TXB2, and MPO were comparable between both citrate groups. Complement activation tended to be lower at higher citrate concentration, but the difference between the two citrate groups did not reach significance. A strong increase in EVs, particularly platelet-derived EVs, was observed during in vitro dialysis for both citrate groups, which was significantly less pronounced in the high citrate group at the end of the experiment. Assessment of dialyzer clotting scores after analysis of individual fibers by scanning electron microscopy revealed significantly lower scores in the high citrate group.ConclusionsOur data indicate that an increase in the citrate concentration from 3 mM to 4 mM further dampens cellular activation, thereby improving biocompatibility. A concentration of 4 mM citrate might therefore be optimal for use in clinical practice.

Biocompatibility Parameters with Standard and Increased Dose of Citrate in Hemodialysis—A Randomized Trial

“…Reduced complement activation with citrate anticoagulation was shown in apheresis [12] and during dialysis with cellulose membranes [21], but not with modern synthetic membranes [11]. Although our previous in-vitro data showed a trend towards lower complement activation at higher doses of citrate [17], the results of the present clinical study confirm that no significant activation of complement occurs during RCA even at the standard dose.…”

“…A significant reduction in clotting was reported with citrate compared to low molecular weight heparins and moderate doses of unfractionated heparin [20]. Our previous in vitro results also showed a significantly better score with an increased (4 mmol/L) dose of citrate, although the results were already very good at the lower dose [17]. The results of the present randomized study could not confirm the in vitro findings and showed no further improvement of the SEM clotting score with the increased dose of citrate.…”

“…To comprehensively and visually evaluate platelet adhesion as well as activation of coagulation and deposition of fibrin, electron microscopy of the inner surface of dialyzer fibers has been used effectively [17,20]. A significant reduction in clotting was reported with citrate compared to low molecular weight heparins and moderate doses of unfractionated heparin [20].…”

“…For evaluation of SEM images, we used a slightly modified, previously used semi quantitative scoring system [17,20], where we evaluated: (a) fiber area covered by adherent material/cells, (b) fibrin network deposition, (c) erythrocyte aggregates, (d) platelet adhesion, and (e) obstruction of fiber lumen. All five parameters were quantified on a semiquantitative scale of 0-4 corresponding to the estimated covered surface area: 0-no signs of adhesion, 1-<25% of visible surface, 2-25%-50%, 3-50%-75% and 4->75% of visible surface.…”

“…In vitro evidence shows that many inhibitory effects of citrate in the extracorporeal circuit are dose-dependent [17] and are a result of the level of ionized hypocalcemia achieved in the circuit [18], i.e., more severe ionized hypocalcemia more intensely inhibits the blood cells' interactions with the dialysis membrane. Clinical data also suggest that increased doses of citrate are required to abolish complement and blood cells activation [9,11], but there are no studies directly comparing different doses of citrate in a clinical setting.…”