Please review the draft manuscript and send comments to Meera Chitlur (MCHITLUR@dmc.or!

with a
cop" to #u$committee Chairman on %actor &III and I'( %lora Pe"vandi (info@florape"vandi.eu!.
Recommendations for Throm$oelastoraph")Throm$oelastometr" in Patients with
*leedin disorders
Meera Chitlur
+
( *enn" #orensen
,
( -eores . Rivard
/
( 0avid Lillicrap
1
( 2en Mann
3
(
Midori #hima
4
( -u" 5oun
6
+ Children7s Hospital of Michian( 0etroit( MI( U#89 , -u":s ; #t Thomas: <H# %oundation Trust( London(
U29 / CHU #ainte=>ustine( Montr?al( @C( Canada( 1 @ueens Universit"( 2inston( A<( Canada9 3
Universit" of &ermont( Colchester( &T( U#89 4 <ara Medical Universit"( 2ashihara cit"( <ara( >apan9 6
Children7s Hospital Los 8neles( Los 8neles( C8( U#8.
IntroductionB
Hemostasis is maintained throuh compleC interactions $etween coaulant and anti=coaulant proteins(
cellular components and endothelium. Currentl" assessment of hemostasis involves plasma=$ased
assa"s of clottin proteins and stud"in platelets. There is now a rowin interest in the use of so=called
lo$al hemostasis assa"s liDe throm$elastoraph" and throm$oelastometr" which measure the
viscoelastic chanes occurrin durin clot formation( for the stud" of coaulation disorders. These assa"s
are performed usin whole $lood and provide the opportunit" to assess the Dinetics of clot formation in
the presence of the plasma and cellular $lood components.
The I#TH=##C EP on standardiFation of throm$oelastoraph" was esta$lished to determine a standard
methodolo" for performin throm$oelastoraph" in patients with hemophilia $oth at $aseline and
followin treatment with prohemostatic medications. In this manuscript the EP has put forth
recommendations for performin throm$oelastoraph") throm$oelastometr" in patients with hemophilia
and other coaulation factor deficiencies with the eCclusion of von Eille$rand factor deficienc"( as the test
ma" reGuire modification to increase its sensitivit" for von Eille$rand disease.
0ifferences $etween Throm$elastoraph")Throm$oelastometr"B
The two instruments currentl" availa$le that utiliFe the principles of throm$oelastoraph" are the
T.-H3III (Haemonetics Corp.( *raintree( M8( U#8! and the RAT.MH delta (Tem International -m$H(
Munich( -erman"!. The resultant anal"sis from the T.-H3III(T.-! is referred to as
Throm$elastoraph" while that from the RAT.MH delta (RAT.M! is called Throm$oelastometr". The
throm$oelastoraph consists of a heated c"lindrical sample cup into which is suspended a pin. In the
T.-( the cup oscillates at J1K137 ever" 3 seconds and the pin is suspended freel" into the cup $" a
torsion wire. In the RAT.M( the cup is stationar" while the pin transduction s"stem oscillates at J1K137
ever" 4 seconds. Eith the initiation of coaulation( the formin clot results in a ph"sical connection
(presuma$l" $" strands of fi$rin! $etween the cup and the pin( transferrin the torGue of the cup to the
pin. The rate of clot formation and its elastic strenth affect the manitude of motion of the pin its rane of
oscillation. In the T.-( a mechanical=electrical transducer is used to convert the rotation of the pin to an
electrical sinal which is then recorded $" a computer. In the RAT.M( an optical detection s"stem is
used to enerate the electrical sinal which is recorded $" a computer. Eith clot l"sis( the transfer of
motion from the cup to the pin is interrupted. %or $oth instruments( computer software produces $oth
Guantitative parameters (see $elow! and a raphical representation (fiure +! which allows one to
Please review the draft manuscript and send comments to Meera Chitlur (MCHITLUR@dmc.or! with a
cop" to #u$committee Chairman on %actor &III and I'( %lora Pe"vandi (info@florape"vandi.eu!.
evaluate the different phases of clottin and deduce the adeGuac" of the coaulation and fi$rinol"tic
pathwa"s. The sample volume in the T.- is /4ILL and /1I LL for the RAT.M. Coaulation ma" $e
initiated purel" $" contact activation with the cup (called MnativeN!( or with specific activators can either
taret the contact pathwa" or the eCtrinsic pathwa" to reduce the time to clot formation and add a
measure of relia$ilit" to the assa"s. . %or the T.-( the manufacturer provides a vial coated with Daolin for
activation of the intrinsic pathwa" while for the eCtrinsic pathwa"( the use of recom$inant human tissue
factor (T%( mostl" InnovinH( 0ade *ehrin( Miami( %L! has $een deplo"ed $" investiators in Mhome=
madeN reaents. %or the RAT.M( the manufacturer provides reaents for $oth intrinsic activation =I<T.M
(partial throm$oplastin phospholipid made from ra$$it $rain!( and .'T.M (throm$oplastin)recom$inant
tissue factor! for eCtrinsic factor activation. MiCin of the contents in the sample cup is done with the
oscillation of the cup over the pin in the T.- and $" re=aspiratin into the automated pipette in the
RAT.M.
The parameters measured in $oth s"stems are similar $ut have a slihtl" different nomenclature. The
measures commonl" used in the evaluation of patients with $leedin disorders areB
+. Coaulation timeB R on T.- and CT on RAT.M
,. Clot %ormation timeB 2 on T.- and C%T on RAT.M
/. MaC. Clot %irmnessB M8 on T.- and MC% on RAT.M
1. #hear .lastic Modulus #trenthB - on $oth instruments.
3. O)8nle=Rate of pol"meriFation of clotB O on $oth instruments.
Pre=anal"tical &aria$les affectin throm$oelastoraphic resultsB
8s with an" coaulation assa"( incorrect $lood collection techniGues and samplin result in activation of
the coaulation s"stem and therefore erroneous results on throm$oelastoraph". Pa"in attention to the
followin details will minimiFe these varia$lesB
1. *lood collection tu$esB %or 2aolin or I<T.M( standard ($uffered! sodium citrate (/.,P! $lood
collection tu$es should $e used. If .'T.M or T% is considered( it is important that the collection
tu$e contain corn tr"psin inhi$itor (CTI! prior to the $lood $ein placed in the tu$e as studies have
shown that clottin times are much shorter if there is an" contact activation. The addition of the
CTI in the collection tu$e( prior to collection of the $lood sample( to achieve a final concentration
of I.+m)ml decreases this activation (+!. The ideal method would involve testin a native
sample( however this is impractical. 8lthouh studies have shown that there is a tendenc"
towards h"percoaua$ilit" with citrated samples due to inadeGuate inhi$ition of throm$in
eneration (,!( most studies with throm$oelastoraph" have utiliFed citrated $lood. The addition
of CTI to the tu$e ma" therefore $e $eneficial to overcome this effect as well( there$" allowin for
the use of citrated samples with eCtrinsic pathwa" activation. Af note( the currentl" availa$le CTI
aent is not sterile and therefore collection tu$es should not $e directl" connected to the
individual underoin phle$otom".
+. 8pplication of a tourniGuet for venous samplinB &enous stasis durin $lood collection has $een
Dnown to result in increased varia$ilit" in coaulation test results(/!. In children it is sometimes
eCtremel" difficult to o$tain a peripheral venous $lood draw( and while the preferred method
would $e to o$tain the $lood without application of a tourniGuet( it ma" $ecome necessar" to
appl" a liht tourniGuet and release this once the vein is accessed.
,. <eedle siFeB 8 ,+- or larer needle is recommended to o$tain the specimen for
throm$oelastoraphic testin. #maller needles have $een demonstrated to result in platelet
activation (/!.
/. Multiple samplinB Repeated samplin from the same tu$e has $een Dnown to result in activation
of platelets as well as coaulation factors and therefore should $e avoided if possi$le(,!.
1. Restin timeB The sample ma" $e allowed to sit for /I minutes $ut not loner than , hours prior to
the runnin the test. If storae $e"ond /I minutes is reGuired( the tu$e should remain capped to
prevent the escape of CA, and chane in pH( and attempts should $e made to Deep the sample
at /6KC.
Eith the a$ove in mind( the EP here$" maDes the followin recommendations for the use of
throm$oelastoraph" in patients with coaulation factor deficienc"B
Recommendations for the 2aolin)I<T.M MethodB *lood should $e collected as descri$ed a$ove. %or the
T.-( + mL should $e transferred into the Daolin vial and /1IQL then transferred into the cup into which
,IQL of calcium chloride had $een placed. %or the RAT.M( the $lood is transferred directl" into the
device for automated pipettin. #ince $oth reaents are supplied $" the manufacturers( their potenc" is
standardiFed ensurin relia$le results.
Recommendations for the Tissue %actor methodB Most of the initial worD in patients with hemophilia was
performed usin T% as the activator since it was $elieved that this was a more ph"sioloic representation
of the in vivo coaulation process. These studies have used dilutions of the commerciall" availa$le T%(
InnovinH. 8 hih T% dilution of +B+6(III (approCimatel" I./3pM( final estimated concentration which ma"
var" slihtl" dependin the patient hematocrit! and low T% dilution (+B1,(III dilution( approCimatel"
I.+3pM! have $oth $een studied with some studies suestin the hiher concentration is effective while
others have shown the lower concentration to $e more effective(1( 3! This issue remains controversial.
Ee suest performin preliminar" studies with $oth dilutions to determine the most appropriate one for
the specific stud" one is performin. The +B+6(III dilution can $e prepared as followsB +I LL of InnovinH
is added to +4I LL of saline diluent (final concentration R +B+6!. +II LL of the +B+6 dilution of T% is then
added to SII LL of diluent (final concentration R +B+6I!. %inall" SSI LL of the +B+6I dilution of T% is added
to +III LL of diluent to o$tain the desired final concentration of a$out +B+6(III (assumin a final
hematocrit in the cup of a$out /IP!. 8 similar approach can $e followed for the +B1,(III dilution. Ance
prepared( ,ILL of the dilute T% is placed in the specimen cup( followed $" ,I LL of calcium chloride for
recalcification of the citrated $lood sample. #ince the maCimum volume in the specimen cup cannot
eCceed /4I LL( /,I LL of $lood is added instead of /1I LL as with 2aolin in the T.-. The same
procedure ma" $e applied to $oth the T.- and the RAT.M with manual pipettin. Atherwise( for the
RAT.M( the .'T.M reaent ma" $e used as the eCtrinsic pathwa" activator( $ut it is important to $e
aware that the results ma" differ from that o$tained with the T% dilution descri$ed a$ove as the
concentration of the activators are not identical.

Comparison of the T% and 2aolin methodsB This remains a much de$ated and controversial Guestion
since $oth activators have strenths and weaDnesses. The advantae of Daolin or I<T.M is that the
method is ver" simple and the reaents are standardiFed. The main disadvantae is the non=ph"sioloic
nature of contact s"stem activation. The advantae of T% is the ph"sioloic activation of clottin while the
disadvantaes include the lacD of standardiFation of the potenc" of the T% and the reGuirement with the
T.- of maDin a Mhome=madeN T% reaent usin commercial T% intended for other uses. 8lthouh with
the RAT.M( a T% reaent is provided (.'T.M! $" the manufacturer( there are concerns reardin the
potential varia$ilit" in different lots. 8ll of these issues with T% have led to sinificant varia$ilit" $etween
studies usin the same $rand of T%. In a stud" comparin Daolin to T%( Daolin performed as well as a low
concentration of T% and $etter than a hiher concentration(3!. In a recent stud"( it was shown that with a
well characteriFed T% reaent (*aCter Healthcare( H"land 0ivision! at a concentration of 3pM com$ined
with CTI and relipidated in 63PPC),3PP#( improved the reproduci$ilit" of throm$oelastoraphic
measurements sinificantl"(4!. %urther research will $e reGuired to determine which method will $e the
most relia$le in predictin clinical outcomes.
ConclusionB This EP has concluded that throm$oelastoraph" can $e performed with either of the
availa$le instruments and that $oth the contact activation and tissue factor activation methods continue to
$e used in clinical trials. %urther research to determine a correlation $etween the la$orator" results and
clinical outcomes are reGuired $efore these assa"s can $e recommended for clinical use.
*i$lioraph"B
1. Fenger-Eriksen C, Anker-Moller E, Heslop J, Ingerslev J, Sorensen B.
Thrombelasographi! "hole bloo# !lo $ormaion a$er e% vivo a##iion o$ plasma
s&bsi&es' improvemens o$ he in#&!e# !oag&lopah( "ih )brinogen
!on!enrae. Br J Anaesh*++, Mar-./012'1*/-..
*. 3ambr&ni A, Thalheimer 4, 5ean#ro 6, 7err( 8, B&rro&ghs A9.
Thromboelasograph( "ih !irae# bloo#' !omparabili( "ih naive bloo#, sabili(
o$ !irae sorage an# e:e! o$ repeae# sampling. Bloo# Coag&l Fibrinol(sis*++/
Jan-1,012'1+1-;.
1. 5ippi 6, Fran!hini M, Monagnana M, Salvagno 65, 7oli 6, 6&i#i 6C. <&ali(
an# reliabili( o$ ro&ine !oag&laion esing' !an "e r&s ha sample= Bloo#
Coag&l Fibrinol(sis*++> ?!-1;0;2',11-..
/. Sorensen B, Ingerslev J. Tailoring haemosai! reamen o paien
re@&iremens - an &p#ae on monioring haemosai! response &sing
hrombelasograph(. Haemophilia*++, Aov-11 S&ppl 1'1->.
,. Bo&ng 6, 3hang C, Miller C, Bassin 8, A&gen 8J. Comparison o$ kaolin an#
iss&e $a!or a!ivae# hromboelasograph( in haemophilia. Haemophilia
Ma(-1>012',1D-*/.
>. Fole( JH, B&enas S, Mann 96, Br&mmel-3ie#ins 9E. Meas&ring he
me!hani!al properies o$ bloo# !los $orme# via he iss&e $a!or pah"a( o$
!oag&laion. Anal Bio!hem Mar 1-/**012'/>-,1.
Fig&re 1' Thromboelasograph'