Calatzis, A.

Schiffl, Helmut

Schramm, W.

Spannagl, M.

Toepfer, Marcel

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Letter to the EditorNephron 2001;89:233–236Citrate Anticoagulation for Extracorporeal Circuits:Effects on Whole Blood Coagulation Activation andClot FormationA. Calatzisa M. Toepferb W. Schramma M. Spannagla H. SchifflbDivisions of aHemostaseology and bNephrology, Medizinische Klinik Innenstadt, Ludwig Maximillians University,Munich, GermanyM. Toepfer, MDMedizinische Klinik, Ludwig Maximilians UniversityZiemssenstrasse 1, D–80336 Munich (Germany)Tel. +49 89 5160 2111, Fax +49 89 5160 4406E-Mail toepfer@fbs.med.uni-muenchen.deABCFax + 41 61 306 12 34E-Mail karger@karger.chwww.karger.com© 2001 S. Karger AG, Basel0028–2766/01/0892–0233$17.50/0Accessible online at:www.karger.com/journals/nefDear Sir,Adequate anticoagulation is a precondi-tion for any effective hemodialysis therapyto prevent clotting in the extracorporeal cir-cuit. While systemic anticoagulation withunfractionated heparin has been widelyused, it is associated with several disadvan-tages, such as an increase in bleeding risk.Hemodialysis of patients who are at high riskfor hemorrhages is complicated by a 10–30%incidence of bleeding [1, 2]. Alternatives toconventional heparin anticoagulation thatare designed to reduce bleeding complica-tions include limited-dose heparinization,low-molecular-weight heparins, neutraliza-tion of heparin with protamine, prostacyclininfusion or saline-flush dialysis. Althoughthe incidence of bleeding complications canbe reduced, each of these methods has itsown limitations [3, 4].The use of regional citrate anticoagula-tion for hemodialysis and continuous renalreplacement therapies in patients at high riskfor bleeding offers unique advantages overother anticoagulation alternatives [3–6]. Byvirtue of chelating ionized calcium, citrate isa powerful anticoagulant. Free calcium ionsare required for activation of coagulation(fig. 1) but are also reported to have concen-tration-dependent effects on clot polymeri-zation [7–9].Citrate anticoagulation is usually moni-tored by the determination of activated clot-ting time (ACT). However, this method isaffected by factor deficiencies and other an-ticoagulants. Measuring ionized calcium byion-selective calcium electrodes may be aninexpensive alternative way for monitoringcitrate effects in regional citrate anticoagula-tion.The aim of this study was to assess thedose-response relationship of citrate anticoa-gulation on coagulation activation and clotformation in whole blood. In addition, thevalue of direct determination of ionized cal-cium by ion-selective calcium electrodes formonitoring of citrate anticoagulation wasevaluated.Venous blood was taken from 6 healthyvolunteers after informed consent and bloodsamples we immediately analyzed. Coagula-tion studies were performed with tubes con-taining anticoagulant citrate dextrose-A(ACD-A) in different amounts (0–5.65mmol citrate/l of whole blood). ACD-A is anisotonic mixture of dextrose, citrate acid,and trisodium citrate (38 mmol/l anhydrouscitric acid, 75 mmol/l trisodium citrate) ascommonly used by blood banks for bloodsampling and cell storage.Whole blood ACT was analyzed with aKC4-coagulometer (Sigma-Amelung, Lem-go, Germany) using kaolin as surface activa-tor (100 ng of kaolin added to 0.3 ml ofwhole blood). This test serves as a model forcontact activation (intrinsic pathway) anddetermines the time until an initial forma-tion of fibrin fibers is detected. Further, weperformed thromboelastography (TEG) witha RoTEG analyser (Nobis, Endingen, Ger-many) [10]. With this test, fibrin formationkinetics and clot firmness are continuouslyassessed (fig. 2). Results are given as clot for-mation time (CFT; time from onset of clotformation until a clot firmness of 20 mm isattained) and maximum clot firmness(MCF). To reveal the effects of citrate onfibrin formation and clot polymerization, weperformed clotting times and thrombelasto-grams using either kaolin as contact activa-tor or the snake venom ecarin, which directlyactivates prothrombin independently fromionized calcium and earlier stages of thecoagulation cascade (0.2 U of ecarin addedto 0.3 ml of whole blood) (fig. 3). [11]. Ion-ized calcium and blood pH were determinedby ion-selective electrodes on a Synthesis1720 analyzer (Instrumentation Laboratory,Kirchheim, Germany).Studies using contact activation withkaolin revealed a dose-dependent inhibitionof coagulation with increasing citrate concen-trations (table 1). The correlation betweenconcentrations of ionized calcium and clot-ting times revealed almost no anticoagulanteffect when ionized calcium levels were 10.50mmol/l, while clotting times showed a steepincrease when calcium levels were furtherdecreased (fig. 4). 5.65 mmol/l citrate in-duced clotting times of infinity in all samples.In contrast to these findings, in coagulationstudies activated by ecarin, hemostasis pa-rameters were not influenced at all (table 1).Activation of clotting with kaolin234 Nephron 2001;89:233–236 Calatzis/Toepfer/Schramm/Spannagl/SchifflFig. 1. The importance of ionized calcium in the activation of coagu-lation: ionized calcium is required for the formation of both tenaseand prothrombinase complex, which lead to thrombin activation andfinally to clot formation. Citrate acts by complexing ionized calciumand thus it inhibits the activation of coagulation.Fig. 2. Assessment of whole blood coagulation. a Coagulometrictechnique: The blood sample is placed into a rotating cup. The move-ment of a steel ball is detected magnetically. As soon as fibrin fibersare formed, the viscosity of the sample rises and the movement of thesteel ball is stopped. By this technique the time interval from start ofthe test to the initial formation of the fibrin fibers is detected.b Thrombelastography: the blood sample is placed into a stationarycup. A cylindrical pin oscillates. The blood clot reduces the move-ment dependent on its firmness. With this technique, blood dynam-ics of clot formation and clot firmness are assessed.Table 1. Clotting time, clot formation time and clot firmness of blood samples anticoagulated with increasingamounts of citrateCitratemmol/lIonized Ca2+mmol/lBloodpHKC4: clottingtime, sTEG: clotformation time, sTEG: clotfirmness, mm0.00 1.18B0.05 7.39B0.02 101B9 82B15 65B41.13 0.67B0.07 7.38B0.03 108B13 81B17 66B52.26 0.40B0.04 7.35B0.04 157B51 102B28 63B43.39 0.28B0.03 7.32B0.04 327B175 167B64 56B64.52 !0.25 7.29B0.04 681B267 423B245 42B135.65 !0.25 7.25B0.04 no clotting no clotting no clottingActivation of clotting with ecarin0.00 1.20B0.08 7.40B0.03 101B9 74B16 68B35.65 !0.25 7.24B0.05 104B8 79B12 67B6Citrate Anticoagulation for ExtracorporealCircuitsNephron 2001;89:233–236 235Fig. 3. To evaluate the effect of citrate anti-coagulation on clot formation and polymeri-zation, coagulation studies were performedusing either kaolin (contact activation) or thesnake venom ecarin (direct prothrombin ac-tivation). Effects of citrate anticoagulationwere assessed by clot formation time (coagu-lometric detection) or clot firmness (throm-belastographic analysis).Fig. 4. Correlation of clotting time (a) and clot firmness (b) with ionized calcium. Coagulation triggered by contactactivation (addition of kaolin to non-anticoagulated whole blood).236 Nephron 2001;89:233–236 Calatzis/Toepfer/Schramm/Spannagl/SchifflIn summary, citrate anticoagulation in-hibits coagulation, in a dose-dependentmanner. In concentrations used for regionalcitrate anticoagulation, citrate inhibits acti-vation of coagulation but does not directlyinfluence fibrin formation and clot polymer-ization. The clinically relevant effects of ci-trate anticoagulation rely alone on the dis-turbed formation of the calcium-dependentcoagulation factor complexes. The anticoag-ulant effects of citrate can be monitored eith-er by methods that quantify clot formation(i.e. ACT) or by direct assessment of ionizedcalcium levels. Measuring concentrations ofionized calcium by ion-selective electrodescan be used as an inexpensive and precisemethod of monitoring regional citrate anti-coagulation.References1 Schwarz RD: Hemorrhage during high-risk he-modialysis using controlled heparinization.Nephron 1981;28:65–69.2 Remuzzi G: Bleeding in renal failure. Lancet1988;105:1205–1208.3 Ward DM, Mehta RL: Extracorporeal manage-ment of acute renal failure patients at high riskof bleeding. Kidney Int 1993;43:237–244.4 Pinnick E, Wiegmanson TE, Diederich DA:Regional citrate anticoagulation for hemodial-ysis in a patient at high risk for bleeding. NEngl J Med 1983;308:258–261.5 Flanigan MJ, Pillsbury L, Sadewasser G, LimVS: Regional hemodialysis anticoagulation:Hypertonic tri-sodium citrate or anticoagulantcitrate dextrose-A. Am J Kidney Dis 1996;27:519–524.6 Janssen MJ, Deegens JK, Kapinga TH, Beuk-hof JR, Huijgens PC, van Loenen AC, van derMeulen J: Citrate compared to low-molecular-weight heparin anticoagulation in chronic he-modialysis patients. Kidney Int 1996;49:806–813.7 Francis JL, Watson NJ, Simmonds VJ: Therole of sodium citrate in the dysfibrinogenaem-ia of liver disease. Thromb Res 1984;34:187–197.8 Carr ME, Powers PL: Differential effects ofdivalent cations on fibrin structure. Blood Coa-gul Fibrinolysis 1991;2:741–747.9 Sinauridze EI, Volkova RI, Krasotkina YV,Sarbash VI, Ataullakhanov FI: Dynamics ofclot growth induced by thrombin diffusing intononstirred citrate human plasma. Biochim Bio-phys Acta 1998;25:607–616.10 Mallett SV, Cox DJ: Thrombelastography. Br JAnaesth 1992;60:307–313.11 Kornalik F, Blomback B: Prothrombin activa-tion induced by Ecarin – a prothrombin con-verting enzyme from Echis carinatus venom.Thromb Res 1975;6:57–63.

Ataullakhanov FI: Dynamics of clot growth induced by thrombin diffusing into nonstirred citrate human plasma. Biochim Biophys Acta

Bleeding in renal failure.

Blomback B: Prothrombin activation induced by Ecarin – a prothrombin converting enzyme from Echis carinatus venom. Thromb Res 1975;6:57–63.

Citrate compared to low-molecularweight heparin anticoagulation in chronic hemodialysis patients. Kidney Int

Hemorrhage during high-risk hemodialysis using controlled heparinization.

Mehta RL: Extracorporeal management of acute renal failure patients at high risk of bleeding.

Powers PL: Differential effects of divalent cations on fibrin structure. Blood Coagul Fibrinolysis

Regional citrate anticoagulation for hemodialysis in a patient at high risk for bleeding.

VJ: The role of sodium citrate in the dysfibrinogenaemia of liver disease. Thromb Res

VS: Regional hemodialysis anticoagulation: Hypertonic tri-sodium citrate or anticoagulant citrate dextrose-A.

Citrate anticoagulation for extracorporeal circuits: Effects on whole blood coagulation activation and clot formation

A. Calatzis

M. Toepfer

W. Schramm

M. Spannagl

H. Schiffl

Crossref

Citrate Anticoagulation for Extracorporeal Circuits: Effects on Whole Blood Coagulation Activation and Clot Formation

Universität München: Elektronischen Publikationen

Letter to the Editor
Nephron 2001;89:233–236
Citrate Anticoagulation for Extracorporeal Circuits:
Effects on Whole Blood Coagulation Activation and
Clot Formation
A. Calatzisa M. Toepferb W. Schramma M. Spannagla H. Schifflb
Divisions of aHemostaseology and bNephrology, Medizinische Klinik Innenstadt, Ludwig Maximillians University,
Munich, Germany
M. Toepfer, MD
Medizinische Klinik, Ludwig Maximilians University
Ziemssenstrasse 1, D–80336 Munich (Germany)
Tel. +49 89 5160 2111, Fax +49 89 5160 4406
E-Mail toepfer@fbs.med.uni-muenchen.de
ABC
Fax + 41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
© 2001 S. Karger AG, Basel
0028–2766/01/0892–0233$17.50/0
Accessible online at:
www.karger.com/journals/nef
Dear Sir,
Adequate anticoagulation is a precondi-
tion for any effective hemodialysis therapy
to prevent clotting in the extracorporeal cir-
cuit. While systemic anticoagulation with
unfractionated heparin has been widely
used, it is associated with several disadvan-
tages, such as an increase in bleeding risk.
Hemodialysis of patients who are at high risk
for hemorrhages is complicated by a 10–30%
incidence of bleeding [1, 2]. Alternatives to
conventional heparin anticoagulation that
are designed to reduce bleeding complica-
tions include limited-dose heparinization,
low-molecular-weight heparins, neutraliza-
tion of heparin with protamine, prostacyclin
infusion or saline-flush dialysis. Although
the incidence of bleeding complications can
be reduced, each of these methods has its
own limitations [3, 4].
The use of regional citrate anticoagula-
tion for hemodialysis and continuous renal
replacement therapies in patients at high risk
for bleeding offers unique advantages over
other anticoagulation alternatives [3–6]. By
virtue of chelating ionized calcium, citrate is
a powerful anticoagulant. Free calcium ions
are required for activation of coagulation
(fig. 1) but are also reported to have concen-
tration-dependent effects on clot polymeri-
zation [7–9].
Citrate anticoagulation is usually moni-
tored by the determination of activated clot-
ting time (ACT). However, this method is
affected by factor deficiencies and other an-
ticoagulants. Measuring ionized calcium by
ion-selective calcium electrodes may be an
inexpensive alternative way for monitoring
citrate effects in regional citrate anticoagula-
tion.
The aim of this study was to assess the
dose-response relationship of citrate anticoa-
gulation on coagulation activation and clot
formation in whole blood. In addition, the
value of direct determination of ionized cal-
cium by ion-selective calcium electrodes for
monitoring of citrate anticoagulation was
evaluated.
Venous blood was taken from 6 healthy
volunteers after informed consent and blood
samples we immediately analyzed. Coagula-
tion studies were performed with tubes con-
taining anticoagulant citrate dextrose-A
(ACD-A) in different amounts (0–5.65
mmol citrate/l of whole blood). ACD-A is an
isotonic mixture of dextrose, citrate acid,
and trisodium citrate (38 mmol/l anhydrous
citric acid, 75 mmol/l trisodium citrate) as
commonly used by blood banks for blood
sampling and cell storage.
Whole blood ACT was analyzed with a
KC4-coagulometer (Sigma-Amelung, Lem-
go, Germany) using kaolin as surface activa-
tor (100 ng of kaolin added to 0.3 ml of
whole blood). This test serves as a model for
contact activation (intrinsic pathway) and
determines the time until an initial forma-
tion of fibrin fibers is detected. Further, we
performed thromboelastography (TEG) with
a RoTEG analyser (Nobis, Endingen, Ger-
many) [10]. With this test, fibrin formation
kinetics and clot firmness are continuously
assessed (fig. 2). Results are given as clot for-
mation time (CFT; time from onset of clot
formation until a clot firmness of 20 mm is
attained) and maximum clot firmness
(MCF). To reveal the effects of citrate on
fibrin formation and clot polymerization, we
performed clotting times and thrombelasto-
grams using either kaolin as contact activa-
tor or the snake venom ecarin, which directly
activates prothrombin independently from
ionized calcium and earlier stages of the
coagulation cascade (0.2 U of ecarin added
to 0.3 ml of whole blood) (fig. 3). [11]. Ion-
ized calcium and blood pH were determined
by ion-selective electrodes on a Synthesis
1720 analyzer (Instrumentation Laboratory,
Kirchheim, Germany).
Studies using contact activation with
kaolin revealed a dose-dependent inhibition
of coagulation with increasing citrate concen-
trations (table 1). The correlation between
concentrations of ionized calcium and clot-
ting times revealed almost no anticoagulant
effect when ionized calcium levels were 10.50
mmol/l, while clotting times showed a steep
increase when calcium levels were further
decreased (fig. 4). 5.65 mmol/l citrate in-
duced clotting times of infinity in all samples.
In contrast to these findings, in coagulation
studies activated by ecarin, hemostasis pa-
rameters were not influenced at all (table 1).
Activation of clotting with kaolin
234 Nephron 2001;89:233–236 Calatzis/Toepfer/Schramm/Spannagl/
Schiffl
Fig. 1. The importance of ionized calcium in the activation of coagu-
lation: ionized calcium is required for the formation of both tenase
and prothrombinase complex, which lead to thrombin activation and
finally to clot formation. Citrate acts by complexing ionized calcium
and thus it inhibits the activation of coagulation.
Fig. 2. Assessment of whole blood coagulation. a Coagulometric
technique: The blood sample is placed into a rotating cup. The move-
ment of a steel ball is detected magnetically. As soon as fibrin fibers
are formed, the viscosity of the sample rises and the movement of the
steel ball is stopped. By this technique the time interval from start of
the test to the initial formation of the fibrin fibers is detected.
b Thrombelastography: the blood sample is placed into a stationary
cup. A cylindrical pin oscillates. The blood clot reduces the move-
ment dependent on its firmness. With this technique, blood dynam-
ics of clot formation and clot firmness are assessed.
Table 1. Clotting time, clot formation time and clot firmness of blood samples anticoagulated with increasing
amounts of citrate
Citrate
mmol/l
Ionized Ca2+
mmol/l
Blood
pH
KC4: clotting
time, s
TEG: clot
formation time, s
TEG: clot
firmness, mm
0.00 1.18B0.05 7.39B0.02 101B9 82B15 65B4
1.13 0.67B0.07 7.38B0.03 108B13 81B17 66B5
2.26 0.40B0.04 7.35B0.04 157B51 102B28 63B4
3.39 0.28B0.03 7.32B0.04 327B175 167B64 56B6
4.52 !0.25 7.29B0.04 681B267 423B245 42B13
5.65 !0.25 7.25B0.04 no clotting no clotting no clotting
Activation of clotting with ecarin
0.00 1.20B0.08 7.40B0.03 101B9 74B16 68B3
5.65 !0.25 7.24B0.05 104B8 79B12 67B6
Citrate Anticoagulation for Extracorporeal
Circuits
Nephron 2001;89:233–236 235
Fig. 3. To evaluate the effect of citrate anti-
coagulation on clot formation and polymeri-
zation, coagulation studies were performed
using either kaolin (contact activation) or the
snake venom ecarin (direct prothrombin ac-
tivation). Effects of citrate anticoagulation
were assessed by clot formation time (coagu-
lometric detection) or clot firmness (throm-
belastographic analysis).
Fig. 4. Correlation of clotting time (a) and clot firmness (b) with ionized calcium. Coagulation triggered by contact
activation (addition of kaolin to non-anticoagulated whole blood).
236 Nephron 2001;89:233–236 Calatzis/Toepfer/Schramm/Spannagl/
Schiffl
In summary, citrate anticoagulation in-
hibits coagulation, in a dose-dependent
manner. In concentrations used for regional
citrate anticoagulation, citrate inhibits acti-
vation of coagulation but does not directly
influence fibrin formation and clot polymer-
ization. The clinically relevant effects of ci-
trate anticoagulation rely alone on the dis-
turbed formation of the calcium-dependent
coagulation factor complexes. The anticoag-
ulant effects of citrate can be monitored eith-
er by methods that quantify clot formation
(i.e. ACT) or by direct assessment of ionized
calcium levels. Measuring concentrations of
ionized calcium by ion-selective electrodes
can be used as an inexpensive and precise
method of monitoring regional citrate anti-
coagulation.
References
1 Schwarz RD: Hemorrhage during high-risk he-
modialysis using controlled heparinization.
Nephron 1981;28:65–69.
2 Remuzzi G: Bleeding in renal failure. Lancet
1988;105:1205–1208.
3 Ward DM, Mehta RL: Extracorporeal manage-
ment of acute renal failure patients at high risk
of bleeding. Kidney Int 1993;43:237–244.
4 Pinnick E, Wiegmanson TE, Diederich DA:
Regional citrate anticoagulation for hemodial-
ysis in a patient at high risk for bleeding. N
Engl J Med 1983;308:258–261.
5 Flanigan MJ, Pillsbury L, Sadewasser G, Lim
VS: Regional hemodialysis anticoagulation:
Hypertonic tri-sodium citrate or anticoagulant
citrate dextrose-A. Am J Kidney Dis 1996;27:
519–524.
6 Janssen MJ, Deegens JK, Kapinga TH, Beuk-
hof JR, Huijgens PC, van Loenen AC, van der
Meulen J: Citrate compared to low-molecular-
weight heparin anticoagulation in chronic he-
modialysis patients. Kidney Int 1996;49:806–
813.
7 Francis JL, Watson NJ, Simmonds VJ: The
role of sodium citrate in the dysfibrinogenaem-
ia of liver disease. Thromb Res 1984;34:187–
197.
8 Carr ME, Powers PL: Differential effects of
divalent cations on fibrin structure. Blood Coa-
gul Fibrinolysis 1991;2:741–747.
9 Sinauridze EI, Volkova RI, Krasotkina YV,
Sarbash VI, Ataullakhanov FI: Dynamics of
clot growth induced by thrombin diffusing into
nonstirred citrate human plasma. Biochim Bio-
phys Acta 1998;25:607–616.
10 Mallett SV, Cox DJ: Thrombelastography. Br J
Anaesth 1992;60:307–313.
11 Kornalik F, Blomback B: Prothrombin activa-
tion induced by Ecarin – a prothrombin con-
verting enzyme from Echis carinatus venom.
Thromb Res 1975;6:57–63.


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Citrate anticoagulation for extracorporeal circuits: Effects on whole blood coagulation activation and clot formation

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