1. CITRATE ANTICOAGULATION IMPROVES BLOOD COMPATIBILITY IN DIALYSIS AND APERESIS – DATA FROM IN VITRO EXPERIMENTS
S. Harm, J. Hartmann
Center for Biomedical Technology, Department for Health Sciences and Biomedicine Danube University Krems, Austria

2. Overview Citrate vs Heparin: mechanism of action
Citrate/Heparin: advantages & disadvantages
In vitro experiments dialysis setup
In vitro experiments apheresis setup
Citrate and Endotoxins
Conclusion
citrate

3. Heparin as an anticoagulant-
Heparin
STOP

4. Heparin as an anticoagulant
Most frequently used anticoagulant  dosage is well known
Low cost
Short half-life
Reversible with protamine
Systemic anticoagulation
Higher risk of bleeding
Can induce heparin-induced thrombocytopenia (HIT)
Reduced effect in patients with lower antithrombin (AT)
Inhibits the anti-inflammatory effect of AT

5. Citrate as an anticoagulant
STOP
iCa ≤ 0.35 mM
Ca++

6. Citrate as an anticoagulant
Biocompatible (lower cell attachment)
Regional anticoagulation
HIT
Inhibit extracorporeal complement system activation and inflammation
Longer filter survival time compared to heparin
No systemic anticoagulation  lower risk of internal bleeding
Citrate accumulation (low citrate clearance)  metabolic Alkalosis
Higher treatment costs
Intensive Care Med. 2004;30(2):260-5.
Monchi M. et al
96 patient were treated with CVVH with heparin & citrate, dialyzer live time was determined.

7. In vitro Dialysis setup Citrate vs Heparin
Run time
120 min
Blood volume
210 ml
Blood flow rate
30 ml/min
Dialysate flow rate
80 ml/min
Sample time
0/15/30/60/120/180/240 min

8. In vitro Dialysis setup Citrate vs Heparin
Platelets activation: Thromboxan B2 (TXB2)
β-Thromboglobuline (β-TG)
Platelet factor 4 (PF4)
Leukocytes activation: TNF-α
IL-6
IL-8
IL-1β
IL-17
Cell count: blood count (Sysmex)
Microvesicles: Gallios flow cytometer
Cell adhesion: SEM images

9. Results of in vitro Dialysis Citrate vs Heparin
Platelet activation:

10. Results of in vitro Dialysis Citrate vs Heparin
Leukocyte activation:

11. Results of in vitro Dialysis Citrate vs Heparin
Blood cell count:

12. Results of in vitro Dialysis Citrate vs Heparin
Detection of Microvesicles
Gallios flow cytometer (Beckman Coulter)
Lactadherin: MV marker (Ca++-independent detection of phosphatidylserine)
Antibodies: Lactadherin FITC (2.5 µl); CD41 PC7 (5 µl)
R. Weiss
 Most MVs are Platelets derived!

13. Results of in vitro Dialysis Citrate vs Heparin
After the experiment the filters were washed with 0.9 % NaCl solution and cut with a saw.
6 mM Citrate
2 IU/ml Heparin

14. Results of in vitro Dialysis Citrate vs Heparin
SEM-Images
Heparin
Citrate

15. In vitro biocompatibility test Citrate vs Heparin
Tested adsorbents:
Alteco® LPS adsorber
Toraymyxin™
CytoSorb®

16. Results of in vitro apheresis Citrate vs Heparin
Platelet activation:

17. Results of in vitro apheresis Citrate vs Heparin
 Higher cytokine levels in blood anticoagulated with heparin!

18. Results of in vitro apheresis Citrate vs Heparin

19. Results of in vitro Apheresis Citrate vs Heparin
Alteco®
Heparin
Citrate

20. Results of in vitro apheresis Citrate vs Heparin
Toraymyxin™
Heparin
Citrate

21. Results of in vitro Dialyse Citrate vs Heparin
Cytosorb™
Heparin
Citrate

22. Endotoxins and Citrate
Endotoxines – Lipopolysacharides (LPS), responsible for inflammation and gramnegative sepsis
Outer membrane of Gram negative bacteria
Release of LPS:
Cell division
Cell growth
Cell death (some antibiotics!)

23. Endotoxins and Citrate
Endotoxin activity in citrated plasma:
+ Ca++/Mg++
- Ca++/Mg++
(Citrate, EDTA) Ca Mg
LPS-Aggregates
< 100 kDa
LPS-Micelles
> 100 kDa Y
… Plasma proteins
… Immunoglobulins
… Endotoxin
Water
Plasma

24. Endotoxins and Citrate
Endotoxin activity in blood which was citrate anticoagulated and “recalcificated”:
blood
+ LPS
blood
+ LPS
+ citrate (6 mM)
blood
+ LPS
+ citrate (6 mM)
+ CaCl2/MgCl2 to reach physiological level of iCa and iMg
 Citrate anticoagulation reduces endotoxin activity!

25. Conclusion
Citrate anticoagulation, compared to heparin anticoagulation leads to lower blood cell activation! (leucocytes, platelets, microvesicles)
Citrate anticoagulation reduces the cell adhesion on material surfaces and makes materials, used in dialysis and apheresis setups, more biocompatible!
Citrate anticoagulation may reduces endotoxin activity in blood!

26. Acknowledgements Jens Hartmann Jasmine Sadler René Weiss
Ute Fichtinger
Claudia Schildböck