The impact of citrate concentration on adhesion of platelets and leukocytes to adsorbents in whole blood lipoprotein apheresis

Abstract

Lipoprotein apheresis is applied to deplete low density lipoprotein and other apolipoprotein B containing lipoproteins in patients with severe familial hypercholesterolemia, hypertriglyceridemia associated pancreatitis, or lipoprotein (a)-hyperlipoproteinemia. Anticoagulation of the extracorporeal circuit may influence cellular activation, as evidenced by a reduction of inflammatory parameters during regional citrate anticoagulation with acid citrate dextrose A (ACD-A) commonly used in whole blood lipid apheresis. While the citrate concentration in the extracorporeal circuit has to ensure efficient anticoagulation, citrate infusion into the patient should be limited to avoid citrate overload. We assessed the influence of citrate concentration on cellular activation during in vitro circulation of whole blood containing 2.8 mM citrate (ACD-A 1:40), 5.6 mM citrate (ACD-A 1:20), or 13 mM citrate over polyacrylate-based adsorbents for lipoprotein apheresis. We found increased platelet adhesion for anticoagulation with 2.8 mM citrate as compared to 5.6 or 13 mM citrate, as shown by cell counting and confirmed by scanning electron microscopy of adsorbent beads as well as by elevated levels of platelet activation markers and of platelet-derived microvesicles. Leukocytes showed an equivalent adhesion pattern, while red blood cells remained unaffected at all citrate concentrations. Passage of blood over two consecutive columns resulted in enhanced platelet adhesion to the second column, presumably due to upstream preactivation. In conclusion, citrate influences activation and adhesion of platelets and leukocytes in a concentration-dependent manner, and ACD-A 1:20, equivalent to a citrate concentration of 5.6 mM in whole blood, ensures minimal cellular activation during passage of whole blood over polyacrylate-based adsorbents.

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

“…While platelet-derived EVs increased significantly during in vitro dialysis for both citrate groups, their levels remained significantly lower in the 4 mM vs . the 3 mM citrate group, providing further evidence for reduced cellular activation at higher citrate concentrations and confirming previous findings on reduced release of platelet-derived EVs at higher citrate concentrations in an in vitro set-up of lipoprotein apheresis[ 9 ].…”

“…The reduction in platelet counts observed in our study was likely caused by formation of platelet aggregates rather than by platelet deposition on the dialyzer, as evidenced by the absence of macroscopic clotting and by the fact that platelets were only scarcely detected on dialyzer fibers using scanning electron microscopy. As an additional marker of platelet activation, we quantified the release of platelet-derived extracellular vesicles, which have been associated with cellular activation in extracorporeal circuits [ 9 , 21 ]. In addition to their pro-coagulant potential, which is mediated by their exposure of phosphatidylserine and, depending on the physiological setting, by the expression of tissue factor, EVs may exhibit pro-inflammatory effects, and their elevation has been confirmed in a number of pathological conditions [ 27 – 29 ].…”

“…By chelating calcium and magnesium, RCA minimizes the risk of clotting in the extracorporeal circuit inherent to heparin-free dialysis and enables safe maintenance hemodialysis. Citrate is thought to have benefits beyond anticoagulation [ 4 ] and has been reported to reduce complement activation [ 5 ] as well as platelet and leukocyte activation [ 6 – 9 ], thereby dampening inflammatory reactions in the extracorporeal circuit and ensuring an extended filter lifetime during continuous treatment [ 10 , 11 ]. There is evidence that many of the inhibitory effects of citrate in the extracorporeal circuit are dose-dependent [ 12 ], and clinical data suggest that higher doses of citrate are required to improve blood compatibility [ 6 , 7 , 13 ].…”

Differential Interaction of Platelet-Derived Extracellular Vesicles with Leukocyte Subsets in Human Whole Blood

“…Our data firstly demonstrate the critical impact of anticoagulation, showing that chelation of Ca ++ by citrate limits the release of EVs 26 , 37 , 57 , 58 . Secondly, they indicate that platelets constitute the main source of EVs in stored whole blood, as evidenced by the exposure of CD41 on the large majority of released EVs 46 , 59 , 60 . Based on these findings, we chose heparinized whole blood as source for all further experiments to follow the association of released EVs with immune cells over time.…”

“…Beyond the quantification of EVs in a size range of 200 nm to 1,000 nm 7 , flow cytometry yields information on the cellular origin of EVs based on the expression of markers derived from their parent cells. While positive staining of immune cells for lactadherin or annexin V 46 , 60 during flow cytometry can provide indirect evidence for their association with phosphatidylserine exposing EVs 26 , imaging flow cytometry allows for the direct visualization of labeled EVs associated with immune cells 39 . Our imaging flow cytometry data suggest that the large majority of EVs released in whole blood were platelet-derived and associated with granulocytes and monocytes.…”