witt 2012 isovolaemic hemodilu

ORIGINAL PAPER

Isovolaemic hemodilution with gelatin andhydroxyethylstarch 130/0.42: effects on hemostasisin pigletsLars Witt1, Wilhelm Alexander Osthaus1, Wiebke Jahn1, Niels Rahe-Meyer2, Alexander Hanke1,Florian Schmidt3, Martin Boehne3 & Robert Sumpelmann1

1 Clinic of Anaesthesiology, Hannover Medical School, Hannover, Germany

2 Department of Anaesthesiology, St. Franziskus Hospital Bielefeld, Bielefeld, Germany

3 Department for Paediatric Cardiology and Intensive Care, Hannover Medical School, Hannover, Germany

Introduction

Prevention of acute or delayed hemorrhagic shock with

artificial colloids to maintain blood volume and tissue

perfusion is a standard procedure during major pediat-

ric surgery. However, all artificial colloids provoke

dilutional coagulopathy and interact specifically with

the coagulation system (1,2). Hydroxyethylstarch

(HES) solutions and gelatin preparations, frequently

used in pediatric anesthesia (3), have negative impact

on blood coagulation in vivo and in vitro, although

third-generation 6% HES 130/0.4 with a lower molec-

ular weight and a lower molar substitution seems to

have a significantly reduced effect on hemostasis (4–8).

Until now, only a few studies have investigated

the effect of moderate doses (10–20 mlÆkg)1) of

Keywords

gelatin; hydroxyethyl starch; i.v. infusions;

multiple electrode impedance

aggregometry; thrombelastometry

Correspondence

Lars Witt, Clinic of Anaesthesiology,

Hannover Medical School,

Carl-Neuberg-Str.1, 30625 Hanover,

Germany

Email: [email protected]

Section Editor: Andrew Davidson

L. W. and W. A. O. contributed equally to

the study.

Accepted 20 December 2011

doi:10.1111/j.1460-9592.2012.03798.x

Summary

Objectives: Artificial colloids, frequently used to prevent hemorrhagic shock

in children, impair blood coagulation. To determine the impact of acute

isovolaemic hemodilution with artificial colloids on clot formation, we con-

ducted an experimental study in a pediatric animal model.

Methods: Fifteen piglets underwent hemorrhage by withdrawing 40 mlÆkg)1

of blood volume in steps of 10 mlÆkg)1 each within 1 hour. After each

withdrawal, the blood loss was randomly compensated by administering

4% gelatin (GEL) or hydroxylethyl starch 130/0.42 (HES) in a ratio of

1 : 1, or isotonic crystalloid solution (ICS) in a ratio of 1 : 4 for isovolae-

mic hemodilution. Quality of clot formation and platelet function was mea-

sured using Thrombelastometry (ROTEM�) and Multiple electrode

impedance aggregometry (Multiplate�) after 10, 20, and 40 mlÆkg)1 blood

replacement.

Results: Moderate hemodilution (10–20 mlÆkg)1 blood replacement) caused

no significant differences among groups (e.g. INTEM�-MCF after

20 mlÆkg)1 blood replacement (ICS vs GEL vs HES, P > 0.05). Profound

hemodilution with 40 mlÆkg)1 blood replacement showed a significant dif-

ference between ICS and both colloids (P < 0.05), but no significant dif-

ferences between GEL and HES.

Conclusions: Impairment of clot formation by moderate isovolaemic he-

modilution did not significantly differ between ICS, GEL, and HES. Pro-

found hemodilution of more than 50% of the estimated blood volume with

GEL and HES caused significant impairment of clot formation in compari-

son to ICS and has to be considered when using high amounts of these

synthetic colloids.

Pediatric Anesthesia ISSN 1155-5645

ª 2012 Blackwell Publishing Ltd 379Pediatric Anesthesia 22 (2012) 379–385

third-generation 6% HES 130/0.4 and gelatin on he-

mostasis in children (9,10). The effect of high doses

(e.g. 40 mlÆkg)1) of artificial colloids on hemostasis in

children (including platelet aggregation) have never

been studied. Therefore we conducted a prospective,

randomized experimental animal study with a focus on

thrombelastometry (ROTEM�) and multiple electrode

impedance aggregometry (Multiplate�) in piglets to

determine the influence of acute isovolaemic hemodilu-

tion with moderate and high doses (40 mlÆkg)1) of arti-

ficial colloids on hemostasis in an established pediatric

animal-model (11). We hypothesized that both, 6%

HES 130/0.42 and 4% gelatin influence ROTEM� and

Multiplate� parameters in the same manner and to the

same extent.

Methods

After approval of the study by the local animal experi-

mentation committee (Protocol No. 42502-04-09/1794),

fifteen 4-week-old female German landrace piglets were

included in the study. The used pediatric animal model

is well established in our study group and the anesthetic

and perioperative management has been described pre-

viously (12). Heart rate, body temperature, and endtid-

al carbon dioxide were continuously measured using a

patient monitoring system (Cardiocap 5; Datex-Ohme-

da, Freiburg, Germany). Using standard cut down

techniques, 5 F percutaneous sheath introducer sets

(Arrow, Reading, PA, USA) were inserted in the right

jugular vein and the right common carotid artery.

Through the venous introducer set, a 4 F two-lumen

central venous catheter (Arrow) was placed in the supe-

rior vena cava for central venous pressure (CVP)

recording. Through the arterial introducer set a 4F

thermodilution catheter (Pulsiocath, 4 F PV 2024L;

Pulsion, Munich, Germany) was inserted to determine

continuously mean arterial pressure (MAP) and cardiac

output (CO) with a standard hemodynamic monitor

system (PiCCO plus; Pulsion). Cardiac index (CI)

adapted to the piglets body surface was calculated by

using the formula of Mack (K equals the piglets’ body

surface area constant):

CI ¼ CO

K �ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffibodyweight23

p ½l �min�1m�2�

(13). After insertion of the cervical catheters and sutur-

ing the neck the first blood samples for blood gas anal-

ysis using a standard blood gas oximetry system (ABL

735; Radiometer, Copenhagen, Denmark) were col-

lected into heparinised syringes. In each sample,

pH, paO2, paCO2, base excess, bicarbonate, sodium,

chloride, potassium, lactate, hemoglobin, and hemato-

crit were measured. A further blood sample was drawn

in a citrated tube (S-Monovette; Sarstedt, Numbrecht,

Germany) for thrombelastometry coagulation analysis

(ROTEM�; Pentapharm, Munich, Germany). In addi-

tion, blood was drawn into tubes containing recombi-

nant hirudin for multiple electrode impedance

aggregometry analysis (Multiplate�; Dynabyte Medi-

cal, Munich, Germany). All blood samples were drawn

from the right carotid artery without stasis, whereby

the first 5 ml of blood were discarded. The animals

subsequently underwent hemorrhage by withdrawing

40 mlÆkg)1 of the estimated total blood volume

(70 mlÆkg)1) within 1 hour (in steps of 10 mlÆkg)1

each). After each blood withdrawal, the blood loss was

compensated by administering either 4% gelatin (GEL,

Gelafundin 4%�; Braun, Melsungen, Germany) in a

ratio of 1 : 1 or hydroxylethyl starch 130/0.42 (HES,

Tetraspan 6%�; Braun) in a ratio of 1 : 1 or as a con-

trol group isotonic crystalloid solution (ICS, Sterofun-

din ISO�; Braun) in a ratio of 1 : 4. A computer-

generated randomization list was used for the alloca-

tion to the treatment groups. Further blood samples

for blood gas-, ROTEM�- and Multiplate� -analysis

were collected after 10 mlÆkg)1, 20 mlÆkg)1, 40 mlÆkg)1

blood replacement, respectively. When the infusion

was stopped, the last blood sample was collected and

the piglets were euthanized by intravenous injection of

pentobarbital.

Measurements

Thrombelastometry

ROTEM� was used for assessing changes in coagula-

tion. It was carried out according to the manu-

facturer’s guidelines in samples of citrated blood at

37�C. The following tests were performed: INTEM�

(Pentapharm) to measure clot formation triggered by

phospholipids and EXTEM� (Pentapharm) to measure

clot formation triggered by the activation of the extrin-

sic, tissue factor dependent pathway, and FIBTEM�

(Pentapharm), which is based on EXTEM�, but

contains cytochalasin D to inhibit the contribution

of platelets to measure the contribution of fibrin/

fibrinogen to the clot firmness.

By digital data processing, the following typical vari-

ables are obtained: clotting time (CT), which is the

time from start of measurement until the onset of clot-

ting; clot formation time (CFT), which is the time

between onset of clotting and the moment when clot

firmness reaches an amplitude of 20 mm; and maxi-

mum clot firmness (MCF), which corresponds to the

maximum amplitude of the curve.

Hemodilution effects of colloids on hemostasis L. Witt et al.

380 ª 2012 Blackwell Publishing Ltd

Pediatric Anesthesia 22 (2012) 379–385

Multiple electrode impedance aggregometry

Multiplate technology is based on impedance aggre-

gometry, assessing platelet aggregation in whole blood

after activation with adenosine diphosphate (ADP),

collagen (COL) or thrombin receptor-activating pro-

tein (TRAP) in the presence of the direct thrombin

inhibitor hirudin (14,15). Three hundred micolitres of

saline and 300 ll of blood were pipetted into single use

test cells. After incubation the impedance change

caused by the adhesion of the platelets onto the sensor

surfaces was plotted against time. The area under the

aggregation curve was used to measure the aggregation

response, quantified in arbitrary aggregation units (U).

Statistical analyses

The power calculation was carried out using the nQue-

ry Advisor software 6.0 (Statistical solutions, Cork, Ire-

land) with a power of 90% and a significance level (a)of 0.05. This showed that a sample size of five piglets

per group would allow detection of a difference of 10%

in the ROTEM�- and Multiplate� values. For demo-

graphic data and measured values before, during and

after hemodilution, nonparametric statistical tests were

performed. The Kruskal–Wallis test (H test) was used

to determine differences between the groups at baseline

and 10 mlÆkg)1, 20 mlÆkg)1 respectively 40 mlÆkg)1

blood replacement. Wilcoxon’s test and the Mann–

Whitney U test were used as appropriate to compare

within-group and between-group differences. The level

of statistical significance was set at P < 0.05. Values

are expressed as medians and ranges. Recorded data

were analysed using the SPSS 16.0 software for Windows

(SPSS Software, Munich, Germany).

Results

All fifteen piglets used in the study received their

intended treatment and were analysed as reported. The

three groups were comparable for weight [GEL- group

12.8 (range 5.4) kg, HES-group 13.2 (5.4) kg, ICS-

group 12.8 (5.6) kg] and length [GEL-group 74 (range

12) cm, HES-group 74 (9] cm, ICS-group 77 (11) cm.

At baseline, no significant differences with regard to

hemodynamics, acid-base-balance and hemostasis were

observed among groups (Tables 1 and 2, Figures 1–3).

The intrinsic thrombelastometry (INTEM�; Penta-

pharm) showed a significant increase in CFT in the

HES-group, whereas gelatin caused a significant exten-

sion of CFT not until 40 mlÆkg)1 blood replacement

(Figure 1). The ICS-group revealed a small, but signifi-

cant increase of CFT after 20 and 40 mlÆkg)1 blood

replacement (Figure 1). The INTEM� clotting time Tab

le1

Com

parison

of

hem

oglo

bin

,hem

ato

crit

and

hem

odynam

icpara

mete

rsat

baselin

eand

diffe

rent

pro

port

ions

of

blo

od

repla

cem

ent,

gela

tin

(GE

L)

ratio

of

1:

1,

hydro

xyle

thyl

sta

rch

6%

(HE

S)

ratio

of

1:

1,

isoto

nic

ele

ctr

oly

tesolu

tion

(IC

S)

ratio

of

1:

4

Blo

od

repla

cem

ent

Baselin

e10

mlÆk

g)

120

mlÆk

g)

140

mlÆk

g)

1

Variable

GE

LH

ES

ICS

P1

GE

LH

ES

ICS

P2

GE

LH

ES

ICS

P3

GE

LH

ES

ICS

P4

Hem

oglo

bin

(gÆd

l)1)

8.9

(2.1

)7.8

(2.3

)7.8

(1.3

)ns

6.7

(1.2

)6.2

(1.6

)6.4

(1.5

)ns

5.3

(1.4

)5.0

(1.0

)5.4

(1.4

)ns

3.8

(0.8

)*3.5

(0.6

)*4.1

(2.2

)*ns

Hem

ato

crit

(%)

27.7

(6.4

)24.4

(6.9

)24.3

(4.1

)ns

20.9

(3.6

)19.3

(4.9

)20.2

(4.4

)ns

16.8

(4.2

)16.0

(2.8

)17.0

(4.3

)ns

12.2

(2.3

)*11.5

(1.9

)*13.2

(6.6

)*ns

Pla

tele

ts(1

09

lÆ)1)

498

(610)

298

(216)

377

(92)

ns

385

(536)

225

(241)

302

(62)

ns

305

(337)

225

(169)

270

(85)

ns

304

(214)

106

(135)

278

(166)

ns

Fib

rinogen

(mgÆd

l)1)

101

(10)

130

(90)

120

(20)

ns

90

(10)

95

(60)

90

(20)

ns

70

(10)

80

(50)

80

(40)

ns

60

(10)*

60

(40)*

70

(80)*

ns

Mean

art

erialpre

ssure

(mm

Hg)

69

(21)

75

(20)

75

(14)

ns

67

(20)

81.0

(16)

67

(18)

ns

70

(14)

67

(36)

63

(13)

ns

66

(27)

68

(14)

58

(13)*

0.0

3

Card

iac

index

(mlÆm

in)

1m

)2)

3.0

(1.7

)3.8

(1.6

)3.9

(1.2

)ns

3.4

(1.7

)3.9

(1.5

)4.2

(0.9

)ns

3.9

(1.2

)3.9

(2.2

)4.2

(1.1

)ns

4.4

(0.8

)*4.9

(1.5

)*4.4

(1.1

)*ns

Glo

balend-d

iasto

licvolu

me

(ml)

144

(66)

198

(77)

175

(87)

ns

181

(84)

185

(118)

170

(100)

ns

208

(94)

194

(95)

175

(120)

ns

214

(84)

196

(79)

162

(86)

ns

Centr

alvenous

pre

ssure

(mm

Hg)

8(5

)6

(3)

6(2

)ns

9(5

)9

(6)

7(4

)ns

8(2

)8

(6)

7(3

)ns

9(2

)9

(6)

8(2

)*ns

Data

isexpre

ssed

as

media

ns

(range),

P1

valu

eat

baselin

e,

GE

Lvs

HE

Svs

ICS

,P

2valu

eat

10

mlÆk

g)

1blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

,P

3valu

eat

20

mlÆk

g-1

blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

,P

4valu

eat

40

mlÆk

g)

1blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

.*P

<0.0

5,

baselin

evs

40

mlÆk

g)

1blo

od

repla

cem

ent.

L. Witt et al. Hemodilution effects of colloids on hemostasis


showed no significant alterations, whereas MCF

decreased significantly in all groups (Figure 1).

EXTEM� revealed a prolonged clotting time in the

HES-group at study end and as well a continuous

decrease of MCF in all groups (Figure 2). The HES

application caused additionally a prolonged CFT from

10 mlÆkg)1 blood replacement upward, whereas ICS

showed a significant increase at 40 mlÆkg)1 blood

replacement (Figure 2). The effect of isovolaemic he-

modilution on FIBTEM was a significant prolonged

clotting time in the GEL- and the HES-group at study

end, respectively, after 20 and 40 mlÆkg)1 blood

replacement (HES-group), whereas CFT showed no

major alterations. The MCF decreased again both,

after HES and GEL application and at 40 mlÆkg)1

blood replacement even in the control group. Induc-

tion of platelet aggregation by TRAP and ADP

showed no significant changes during blood replace-

ment, whereas the COL-induced aggregometry revealed

a significant reduction by all solutions at 40 mlÆkg)1

blood replacement (Figure 3).

At the end of the study hemoglobin, hematocrit,

and fibrinogen decreased significantly in all groups,

whereas platelets and acid-base parameters did not

change significantly. Even hemodynamics remained

stable or even increased (cardiac index) during the

course of the study, with the exception of a small, but

significant decrease of the MAP in the ICS-group after

40 mlÆkg)1 blood replacement.

Discussion

Appropriate fluid replacement is essential for patient’s

safety during pediatric anesthesia. Although synthetic

colloids are commonly used during major pediatric

surgery, the effects of high doses of these solutions on

blood coagulation and platelet aggregation in infants

and children have never been studied.

The objective of this experimental animal study was

therefore to determine the impact of isovolaemic

hemodilution on hemostasis by artificial colloids, com-

monly used in pediatric anesthesia. In accordance to

our hypothesis, key findings were a comparable, dose–

dependent impairment of clot formation with 4% gela-

tin and 6% HES 130/0.42 in comparison to the control

group. The experimental study design was chosen to

illustrate profound blood replacement under standard-

ized conditions in an approved pediatric animal model

(11), which is also applicable for hemostatic analyses

(5,11) and provides comparable blood volumes with

infants (16). Furthermore, similarities in the human

and porcine coagulation system favor the use of

porcine models when researching blood coagulation.Tab

le2

Com

parison

of

ele

ctr

oly

te,

acid

-base

para

mete

rsand

glu

cose

at

baselin

eand

diffe

rent

pro

port

ions

of

blo

od

repla

cem

ent,

gela

tin

(GE

L)

ratio

of

1:

1,

hydro

xyle

thyl

sta

rch

6%

(HE

S)

ratio

of

1:

1,

isoto

nic

ele

ctr

oly

tesolu

tion

(IC

S)

ratio

of

1:

4

Blo

od

repla

cem

ent

Baselin

e10

mlÆk

g)

120

mlÆk

g)

140

mlÆk

g)

1

Variable

GE

LH

ES

ICS

P1

GE

LH

ES

ICS

P2

GE

LH

ES

ICS

P3

GE

LH

ES

ICS

P4

Sodiu

m(m

M)

137

(4)

136

(2)

138

(3)

0.0

3137

(2)

137

(4)

137

(2)

ns

137

(3)

136

(4)

138

(2)

0.0

4138

(1)

135

(3)

138

(2)

0.0

4

Pota

ssiu

m(m

M)

3.5

(0.8

)3.6

(0.6

)3.5

(1.0

)ns

3.7

(0.5

)4.1

(0.7

)3.5

(0.4

)ns

3.7

(0.5

)4.2

(0.9

)3.6

(0.7

)ns

3.8

(0.5

)3.9

(1.0

)3.7

(0.6

)ns

Calc

ium

(mM

)1.4

(0.2

)1.5

(0.2

)1.3

(0.2

)ns

1.4

(0.3

)1.4

(0.3

)1.3

(0.2

)ns

1.4

(0.2

)1.4

(0.2

)1.4

(0.3

)ns

1.4

(0.2

)1.4

(0.2

)1.4

(0.2

)ns

Chlo

ride

(mM

)101

(8)

98

(4)

99

(3)

ns

101

(3)

98

(4)

102

(3)

ns

100

(3)

98

(6)

103

(2)

0.0

3101

(3)

99

(5)

105

(3)*

0.0

1

Glu

cose

(mM

)4.7

(2.3

)3.7

(1.5

)5.0

(4.1

)ns

3.8

(2.9

)3.9

(1.4

)4.4

(2.9

)ns

4.4

(3.3

)5.0

(1.9

)4.1

(3.4

)ns

5.0

(3.4

)5.5

(1.4

)*4.7

(3.5

)ns

pH

7.5

(0.2

)7.5

(0.2

)7.4

(0.1

)ns

7.5

(0.1

)7.5

(0.1

)7.5

(0.1

)ns

7.5

(0.1

)7.5

(0.0

)7.5

(0.1

)ns

7.5

(0.1

)7.5

(0.1

)7.5

(0.1

)ns

Base

excess

(mM

)6.3

(6.9

)5.9

(4.3

)4.9

(7.0

)ns

6.2

(4.7

)7.2

(4.7

)5.0

(3.8

)ns

5.8

(4.1

)6.9

(5.6

)5.3

(3.9

)ns

5.0

(4.3

)6.1

(5.4

)3.9

(4.6

)ns

Bic

arb

onate

(mM

)29.4

(6.0

)30.2

(4.0

)29.0

(7.6

)ns

30.0

(4.2

)30.9

(5.9

)28.4

(3.8

)ns

29.7

(4.2

)30.7

(5.6

)28.7

(3.5

)ns

28.9

(4.3

)29.8

(4.7

)27.9

(3.5

)0.0

4

Lacta

te(m

M)

1.4

(1.8

)1.3

(1.5

)3.0

(1.9

)ns

1.1

(1.6

)1.3

(1.4

)2.5

(1.5

)ns

1.3

(1.5

)1.6

(1.4

)1.6

(1.5

)ns

1.7

(1.6

)2.0

(1.0

)*2.6

(1.4

)ns

Data

isexpre

ssed

as

media

ns

(range),

P1

valu

eat

baselin

e,

GE

Lvs

HE

Svs

ICS

,P

2valu

eat

10

mlÆk

g)

1blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

,P

3valu

eat

20

mlÆk

g)

1blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

,P

4valu

eat

40

mlÆk

g)

1blo

od

repla

cem

ent

GE

Lvs

HE

Svs

ICS

.*P

<0.0

5,

baselin

evs

40

mlÆk

g-1

blo

od

repla

cem

ent.




Velik-Salchner et al. (17) demonstrated the applicabil-

ity of ROTEM� analysis for porcine blood, even

though they found a potentially methodical bias for

the FIBTEM� MCF value, which should therefore be

interpreted with caution (17). Despite the fact that

Multiplate� has not been validated for porcine blood,

the suitability of the domestic swine as a model for

impedance aggregometry was demonstrated previously

(18).

Although circulating blood volume was not directly

measured, changes in preload parameters like global

end-diastolic volume and CVP as well as hemodynamic

changes were comparable between groups (with the

exception of MAP, Table 1) and made a comparable

hemodilution in this study setting likely. The signifi-

cant lower MAP in the ICS-group after 40 mlÆkg)1

blood replacement may point to an insufficient hemo-

dynamic effect of isovolaemic ICS infusion in compari-

son to colloids.

Hypovolaemia is the most common cause of circula-

tory failure in children and can lead to critical tissue

perfusion. Unlike crystalloids, colloids may be used to

rapidly treat or prevent hypovolaemia with the advan-

tage of markedly reducing the total volume of the

administered infusion and simultaneously the need of

blood transfusion (2). For several years, the impact of

Figure 1 Intrinsic thrombelastometry (INTEM�) analysis: coagula-

tion time (CT); clot formation time (CFT); maximal clot firmness

(MCF) at baseline and different proportions of blood replacement,

gelatin (GEL) ratio of 1 : 1, hydroxylethyl starch 6% (HES) ratio of

1:1, isotonic electrolyte solution (ICS) ratio of 1 : 4. Data are

expressed as medians, 25% and 75% percentile, whiskers are high-

est and lowest values that are not outliers, circles are extreme val-

ues (more than three times the interquartile range), *P < 0.05,

baseline vs 10 mlÆkg)1, 20 mlÆkg)1, 40 mlÆkg)1 blood replacement,

respectively, #P < 0.05 GEL vs HES vs ICS.

Figure 2 Extrinsic thrombelastometry (EXTEM�) analysis: CT, CFT

maximal clot firmness (MCF) at baseline and different proportions

of blood replacement, gelatin (GEL) ratio of 1 : 1, hydroxylethyl

starch 6% (HES) ratio of 1 : 1, ICS ratio of 1 : 4. Data are expressed

as medians, 25% and 75% percentile, whiskers are highest and

lowest values that are not outliers, circles are extreme values (more

than three times the interquartile range), *P < 0.05, baseline vs

10 mlÆkg)1, 20 mlÆkg)1, 40 mlÆkg)1 blood replacement, respectively,

#P < 0.05 GEL vs HES vs ICS.



gelatin infusion on coagulation system appeared lim-

ited due to dilution of coagulation factors and platelets

until the reduction in clot quality with gelatin-based

colloids was demonstrated in vitro (19). Despite this,

the clinical relevance of the impairment of hemostasis

after gelatin infusion remains uncertain. Perioperative

infusion of moderate doses of gelatin in children does

not alter coagulation to an extent above the effect of

hemodilution (10) and thrombelastographic parameters

remain within the reference range (9). In our study,

isovolaemic hemodilution with gelatin did not signifi-

cantly differ from the control group at 10 and

20 mlÆkg)1 blood replacement. It was only the high

doses of 40 mlÆkg)1 blood replacement seems to impair

clot formation including platelet function beyond the

effect of hemodilution.

First generation hydroxyethyl starch (HES) prepara-

tions were manufactured with high average molecular

weight (MW 450–650 kDa, hetastarch), but were soon

found to have significant adverse effects regarding

coagulation. This was due not only to hemodilution

but also to inhibition of blood platelet function,

decrease of coagulation factors, such as von Wille-

brand factor, factor VIII, and fibrinogen (8). The sec-

ond generation of HES with a molar substitution of

around 0.5 (MW 200 kDa, pentastarch) led to fewer

adverse effects. Finally the third generation HES prep-

arations with molar substitution of around 0.4 (MW

130 kDa, tetrastarch) present even more favorable

physicochemical properties and are approved for use

in children with a maximal daily dose of 50 mlÆkg)1

(2). Pooled analysis of prospective and randomized

studies comparing second generation HES 200/0.5 with

the new third generation HES 130/0.4 reporting

decreased effects on coagulation by the latest HES

generation (20). A recent in vitro study on coagulation

effects of balanced, nonbalanced HES 130/0.42, and

gelatin revealed pronounced inhibitory effects of these

colloids compared with crystalloids on blood coagula-

tion following 33% and 66% hemodilution, but no

significant differences between the colloids (4). A study

of infants and toddlers compared the impact of

15 mlÆkg)1 HES 130/0.4, albumin or gelatin on hemo-

stasis (9). For all tested colloids, thrombelastographic

parameters and routine coagulation tests were signifi-

cantly altered from baseline values. These changes

were very similar for albumin and gelatin, but signifi-

cantly more pronounced following HES (9). In con-

trast, a more recent clinical trial investigating

alterations in thrombelastographic parameters in

children receiving moderate doses of HES 130/0.42 or

gelatin, reported thrombelastographic parameters

within the reference range (10). In our study, moderate

isovolaemic hemodilution with the third generation

HES 130/0.42 impaired thrombelastographic parame-

ters and platelet function, but only the blood replace-

ment of 40 mlÆkg)1 caused significant differences to the

control group. However, a recent clinical study in chil-

dren undergoing cardiac surgery revealed a comparable

blood loss following 50 mlÆkg)1 of HES 130/0.4 or

albumin, which has minimal effects on hemostasis (21).

The study even reported a reduced requirement for

allogenic blood transfusion after HES 130/0.4 infusion

indicating, that marked changes in clot formation

Figure 3 Multiple electrode impedance aggregometry (Multiplate�)

analysis: Platelet aggregation after activation with adenosine diphos-

phate (ADP), collagen (COL) or thrombin receptor-activating protein

(TRAP) coagulation at baseline and different proportions of blood

replacement, gelatin (GEL) ratio of 1 : 1, hydroxylethyl starch 6%

(HES) ratio of 1 : 1, ICS ratio of 1 : 4. Data are expressed as medi-

ans, 25% and 75% percentile, whiskers are highest and lowest val-

ues that are not outliers, circles are extreme values (more than

three times the interquartile range), *P < 0.05, baseline vs

10 mlÆkg)1, 20 mlÆkg)1, 40 mlÆkg)1 blood replacement, respectively,

#P < 0.05 GEL vs HES vs ICS.




because of high amounts of HES 130/0.4 do not auto-

matically imply a clinical impact on blood coagulation

and blood loss. In addition, a recent multicenter safety

study revealed no clinical serious adverse effects (e.g.

clotting disorders or anaphylactoid reactions) after

moderate doses of HES 130/0.42 in 316 children

during pediatric anesthesia (22).

In conclusion, the impairment of clot formation by

isovolaemic hemodilution up to 20 mlÆkg)1 blood

replacement did not significantly differ between iso-

tonic crystalloid solution and 4% gelatin, respectively,

HES 130/0.42 in this pediatric animal model. There-

fore, a severe impact on clot formation with this infu-

sion volume in a clinical setting seems to be negligible.

In addition, profound hemodilution of more than 50%

of the estimated blood volume with 4% gelatin and

HES 130/0.42 caused significant impairment of clot

formation in comparison to isotonic electrolyte solu-

tion (ICS). This has to be considered when using high

amounts of these synthetic colloids, even though an

increased blood loss or transfusion need is not neces-

sarily implied.

Acknowledgments

The authors thank B. Buchalik for providing excellent

laboratory assistance. This research was carried out

without funding.

Conflict of interest

No conflicts of interest declared.

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