Int J Hematol

DOI 10.1007/s12185-014-1546-6

NOMBRES: Klver Bryan

APELLIDOS: Alejandro Prez
Grupo 1

ORIGINAL ARTICLE

Thromboelastometry profile in children with beta-thalassemia

zcan Bor O. Meltem Akay
Ays e B. Turhan O
Necat A. Akgun

Received: 22 October 2013 / Revised: 8 February 2014 / Accepted: 13 February 2014

The Japanese Society of Hematology 2014

Abstract Beta (b)-thalassemia is characterized by a

hypercoagulable state and an increased risk of thrombosis,
which can result in significant morbidity and mortality. The
coagulation pattern and determinants of thrombosis in
patients with b-thalassemia remain largely unknown. The
aim of this study was to evaluate the whole blood thromboelastometry (TEM) profile of b-thalassemic children by
ROTEM. ROTEM assays (INTEM, EXTEM) and traditional coagulation parameters (platelet count, prothrombin time, activated partial thromboplastin time, and
fibrinogen) were performed on blood samples from 17
subjects with b-thalassemia and 19 non-thalassemic controls. Maximum clot firmness (MCF) was significantly
higher in subjects with b-thalassemia than in controls on
EXTEM and INTEM analysis (p \ 0.001 and p \ 0.001,
respectively). Of the patients with b-thalassemia, MCF was
higher and clot formation time was shorter in splenectomized subjects than in non-splenectomized subjects on
EXTEM and INTEM (p = 0.026, p = 0.002, p \ 0.001,
p \ 0.001, respectively). TEM profiles in b-thalassemic
children were more hypercoagulable compared with controls. Larger prospective studies are needed to evaluate the
relevance of the association between ROTEM profile and
thromboembolic events in patients with b-thalassemia.

. Bor
N. A. Akgun
A. B. Turhan (&)
O
Division of Pediatric Hematology and Oncology, Department of
Pediatrics, Eskisehir Osmangazi University Faculty of Medicine,
Eskisehir 26480, Turkey
e-mail: aysebturhan@hotmail.com
O. M. Akay
Department of Hematology, Eskisehir Osmangazi University
Faculty of Medicine, Eskisehir, Turkey

Keywords Thromboelastometry
Thalassemia

Thrombosis
Children

Introduction
The b-thalassemia syndromes are a group of inherited
disorders of hemoglobin synthesis, characterized by various degrees of defective b-globin chain production, an
imbalance in a/b-globin chain synthesis, ineffective
erythropoiesis and anemia. Although the life expectancy of
thalassemia patients has improved markedly over the last
few decades, numerous severe complications involving
several organ systems have been described, among which
pulmonary hypertension and thromboembolic events
(TEEs) remain the most serious [1, 2]. The high prevalence
of TEEs, especially in thalassemia intermedia, has led to
the identification of a hypercoagulable state in these
patients.
Several factors that contribute to the hypercoagulable
state in patients with b-thalassemia have been identified.
The underlying mechanism of hypercoagulability primarily
involves chronic platelet activation and alteration of red
blood cell membranes [3, 4]. Other associated factors
involve endothelial activation and disturbances in the
coagulation system, collectively leading to a prothrombotic
state. In most cases, a combination of these abnormalities
leads to TEEs [5].
Unlike bleeding disorders, there are no laboratory assays
that can reliably screen for hypercoagulability or predict
the occurrence of de novo venous TEEs. Whole blood
rotation thromboelastometry (TEM) performed by the
ROTEM method (Tem International, Munich, Germany)
has been used to evaluate the hypercoagulable state in
individuals with acute or previous venous TEEs [6, 7]. This

123

A. B. Turhan et al.

method is based on a pin rotating within a heated cup. An

optical system detects the impedance of the rotation of the
pin and a plot system describes the trace produced by
viscoelastic changes associated with fibrin polymerization
[8]. The main advantages of TEM compared with traditional clotting assays are the simultaneous evaluation of
different components involved in clot formation (i.e.,
plasma factors, platelets, leukocytes and red blood cells)
and the continuous graphical representation of clot initiation, development and lysis [9]. As a first step toward
determining the utility of this assay in predicting TEEs, it
has been reported that TEM is highly sensitive to hypercoagulability [10].
Only limited data are available from previous research
regarding the use of TEM in detecting hypercoagulable
states in conditions, such as thalassemia, sickle cell anemia
and cancer [11, 12]. The aim of this casecontrol study was
to evaluate the sensitivity of TEM in detecting hypercoagulability in patients with thalassemia. We hypothesized
that thalassemia patients would exhibit abnormal TEM
profiles, consistent with a thrombogenic state compared
with healthy controls.

Materials and methods

Subjects
The study was conducted between November 2012 and
April 2013 at the Pediatric Hematology and Oncology
Department of Eskis ehir Osmangazi University Hospital in
Turkey. All of the patients with b-thalassemia who
received regular transfusion were enrolled into the study.
Patients with b-thalassemia, and their guardians were
interviewed to gather information on their clinical history
and their medical records were also reviewed. The patients
(n = 17) were classified as having b-thalassemia major
(n = 10) or b-thalassemia intermedia (n = 7). None of the
patients had used the hydroxycarbamide therapy. Nonthalassemic controls were recruited randomly from a group
of the patient population of the Pediatric Hematology
Clinic presenting for office appointments and visits due to
other illness. None of the control subjects had a personal or
first degree family history of bleeding or thrombosis, was
taking any medications or had any acute infection or
chronic illness.
This study was approved by the College of Medicine
Institutional Review Board. Informed consent was obtained
from the parents of each patient before his or her inclusion
in the study. The study was conducted according to the
Declaration of Helsinki.

123

Blood sampling
All blood samples were obtained during the same phlebotomy procedure required for routine testing at the time of
an office visit. The blood samples from thalassemia
patients were taken just before blood transfusions. In
splenectomized patients taking Aspirin, the tests were
performed 10 days after discontinuation of the drug.
Specimens for TEM and coagulation tests were drawn into
tubes containing 3.2 % buffered sodium citrate (0.129 mol/
L) as the anticoagulant (9:1). Specimens for complete
blood count were drawn into tubes containing ethylenediaminetetraacetic acid. Complete blood count was measured using a Beckman Coulter LH750 machine (Kraemer
Blud. Brea, CA, USA). Prothrombin time (PT, s), activated
partial thromboplastin time (aPTT, s), fibrinogen (mg/dL)
and dimerized plasmin fragment D (D-dimer) tests were
performed immediately on an Siemens BCS XP machine
(Tem International, Marburg, Germany).
TEM
Rotation TEM was performed according to the manufacturers guidelines using a Thromboelastometry Coagulation
Analyzer model Gamma 2500 (Tem International, Munich,
Germany). The two standard ROTEM assays, termed
INTEM and EXTEM, were performed; in these assays, the
intrinsic and the extrinsic coagulation pathways are triggered, respectively [6, 13]. In INTEM, coagulation is
activated with 20 lL of contact activator (partial thromboplastin-phospholipid from rabbit brain extract and ellagic acid, in-TEM; Tem International, Munich, Germany).
In EXTEM, coagulation is activated by 20 lL of tissue
factor (TF, tissue thromboplastin from rabbit brain extract,
ex-TEM; Tem International, Munich, Germany).
The mean parameters obtained were clotting time (CT,
s), clot formation time (CFT, s) and maximum clot firmness (MCF, mm). CT is the time from the beginning of the
coagulation analysis until an increase in amplitude of
2 mm, which reflects the initiation phase of the clotting
process. CFT is the time taken for the amplitude of the
thromboelastogram to increase from 2 to 20 mm and
reflects the propagation phase of whole blood clot formation. MCF is the maximal amplitude reached during
TEM [6] and correlates with platelet count and function as
well as with the concentration of fibrinogen [14]. A
hypercoagulable profile was defined as a shorter CT,
shorter CFT and/or higher MCF than the corresponding
values in healthy controls [6, 9]. In addition, laboratory
values for fibrinogen, platelets and hematocrit were
determined in the study group.

TEM profile of b-thalassemic children

Statistics

Table 2 Hematologic parameters in splenectomized and non-splenectomized patients with beta-thalassemia

Statistical analyses were performed using SPSS for Windows version 15.0 (SPSS, Inc., Chicago, IL, USA). The
baseline characteristics of the study population were
described by frequency and descriptive analysis. Whether
values were normally distributed was assessed using the
ShapiroWilk test. Unpaired t tests and analysis of variance
were used for statistical analysis. Multiple comparisons
were made using KruskalWallis analyses. When differences were detected, the MannWhitney U test with
Bonferroni correction (a = 0.0083) was used for analysis.
Two-tailed p values of \0.05 were regarded as significant.

Results

Splenectomized
patients (n = 5)

Non-splenectomized
patients (n = 12)

p value

Erythrocyte
(9106/lL)

3.13 0.3

3.32 0.8

0.627

Monocyte
(9103/lL)

1.14 0.4

0.63 0.4

0.025

Neutrophil
(9103/lL)

8.6 6.9

5.2 2.3

0.144

Platelet
(9103/lL)

795 670

295 67

<0.001

PT (s)

13.2 1.3

12.5 0.6

0.11

aPTT (s)

36.1 2.6

33.1 3.1

0.074

Fibrinogen
(mg/dL)

335 65

281 75

0.189

Bold values indicate statistical significance

A total of 36 patients were enrolled in the study; 17 patients

were in b-thalassemia group and 19 patients were in control group. While b-thalassemia group included 11 females
(64.7 %) with a mean age of 10.7 years, control group
included 8 females (42.1 %) with a mean age of 8.2 years.
There was no significant difference in age and sex between
the groups. b-Thalassemia group included 9 b-thalassemia
major and 8 b-thalassemia intermedia patients. None of the
patients had history of clinical thrombotic events. Five of
the patients (29.4 %) were splenectomized: two of b-thalassemia major patients, three of b-thalassemia intermedia
patients. All splenectomized patients were using Aspirin,
and, none of the patients had thrombocytopenia due to
hypersplenism.
Hematological parameters which affected the ROTEM
(erythrocytes, neutrophils and monocytes, platelet) were
compared in patients with b-thalassemia and controls
(Table 1). The mean erythrocyte count was significantly
lower in patient with b-thalassemia than controls
(p \ 0.001). Subgroup analysis of b-thalassemia patients
with splenectomized versus non-splenectomized was performed (Table 2). The mean monocytes and platelet counts
were higher in splenectomized patients than non-splenectomized patients (p = 0.025 and p \ 0.001, respectively).
There was no difference in erythrocyte counts, PT, aPTT
and fibrinogen levels between the groups.
Table 1 Hematologic characteristics of the study population
Beta-thalassemia
(n = 17)

Controls
(n = 19)

p value

Erythrocyte (9106/lL)

3.26 0.7

5.18 0.6

<0.001

Monocyte (9103/lL)

0.78 0.44

0.61 0.20

0.145

Neutrophil (910 /lL)

6.2 4.3

4.1 1.7

0.058

Platelet (9103/lL)

441 254

325 87

0.068

Bold value indicates statistical significance

PT prothrombin time, aPTT activated partial thromboplastin time

Table 3 Thromboelastometry parameters in the study population

Beta-thalassemia
(n = 17)

Controls
(n = 19)

p value

EXTEM
CT (s)

69.8 10.1

72.8 13.5

0.444

CFT (s)

91.2 27.6

88.4 19.5

0.729

MCF (mm)

69.4 4.3

64.0 3.6

<0.001

180.6 37.1

175.7 38.9

0.702

69.7 22.1
68.1 4.7

73.8 16.0
62.2 3.6

0.522
<0.001

INTEM
CT (s)
CFT (s)
MCF (mm)

Bold values indicate statistical significance

CT clotting time, CFT clot formation time, MCF maximum clot
firmness

When TEM parameters analyzed, it was shown that

MCF on both EXTEM and INTEM analysis was significantly higher in the subjects with b-thalassemia than in the
controls (p \ 0.001 and p \ 0.001, respectively; Table 3).
Furthermore, MCF was higher and CFT was shorter in
splenectomized patients than in non-splenectomized
patients on both EXTEM and INTEM (p = 0.026,
p = 0.002, p \ 0.001 and p \ 0.001, respectively;
Table 4). MCF on both EXTEM and INTEM analysis was
also significantly higher in non-splenectomized patients
than in the controls (p = 0.023, p = 0.02, respectively;
Table 4).
A significant correlation was found between platelet
count and CFT on EXTEM (Spearman coefficient = -0.9,
p \ 0.001) and INTEM (Spearman coefficient = -0.8,
p \ 0.001) and between platelet count and MCF on
EXTEM (Spearman coefficient = 0.6, p = 0.009) and

123

A. B. Turhan et al.
Table 4 Thromboelastometry
parameters in splenectomized,
non-splenectomized and control
patients with beta-thalassemia

Splenectomized
patients (A) (n = 5)

Non-splenectomized
patients (B) (n = 12)

Control patients
(C) (n = 19)

p value

73.4 13.1

68.3 8.9

72.8 13.5

AB: p = 0.708

EXTEM
CT (s)

AC: p = 0.560
BC: p = 0.999
CFT (s)

54.4 7.7

106.5 14.7

88.4 19.5

AB: p \ 0.001
AC: p = 0.001
BC: p = 0.018

MCF (mm)

73.2 3.5

67.0 4.7

64.0 3.6

AB: p = 0.026
AC: p \ 0.001
BC: p = 0.023

INTEM
CT (s)

173.4 31.4

183.7 40.1

175.7 38.9

AB: p = 0.871
AC: p = 0.843
BC: p = 0.992

CFT (s)

43.2 3.4

80.7 16.0

73.8 16.0

AB: p \ 0.001
AC: p = 0.001

Bold values indicate statistical

significance

BC: p = 0.437
MCF (mm)

73.2 2.8

CT clotting time, CFT clot

formation time, MCF maximum
clot firmness

INTEM (Spearman coefficient = 0.7, p = 0.002). No significant correlations were found between the TEM
parameters and PT/international normalized ratio, aPTT or
fibrinogen.

Discussion
Epidemiological data on TEE in thalassemia are limited.
An Italian study indicated TEE as the primary cause of
death in 2.5 % of 159 transfusion-dependent thalassemic
patients [15]. In nine pediatric thalassemia centers,
49.6 % of patients with b-thalassemia had experienced
TEE [16]. In another study, it was found that 24 thalassemic patients (29 %) developed deep vein thrombosis,
pulmonary embolism or portal vein thrombosis [17].
The hypercoagulable state in thalassemia is due to many
factors including chronic activation of platelets, enhanced
platelet aggregation, elevated levels of endothelial adhesion proteins (E-selectin, intercellular adhesion molecule-1
and von Willebrand factor) and various organ dysfunctions
(cardiac, hepatic and endocrine) [3, 1821]. In most cases,
a combination of these abnormalities leads to hypercoagulability [5, 22, 23]. However, traditional clotting parameters are unable to reflect these coagulation abnormalities.
In the case of PT and aPTT, for example, these assays were
designed to detect deficiencies of clotting factor concentrates and in general are sensitive to low concentrations of

123

66.0 3.6

62.2 3.6

AB: p = 0.002
AC: p = 0.020
BC: p = 0.020

such factors. They are not routinely utilized to screen for

thrombotic risk [10].
In recent years, new global coagulation tests, in particular thromboelastography/elastometry, have been applied
to the study of acquired coagulation equilibria [9, 24].
Whole blood rotation TEM, performed by the ROTEM
method, is a global test that measures clot formation and
stability as a consequence of the interaction between
plasma coagulation factors and blood cells. It provides
information on the kinetics of all stages of clot initiation,
formation and dissolution, and the stability and strength of
clots in whole blood or plasma [2527]. ROTEM has
several advantages compared with routine coagulation
tests. It is easy to use by non-laboratory personnel as a
point-of-care assay in the perioperative and emergency
setting, and provides rapid graphical and numerical information on hemostatic status [25].
Hypercoagulability, as detected on TEM assays/ROTEM, has been reported in a wide variety of medical and
surgical conditions [6, 9, 11, 12, 25]. To our knowledge,
this is the first report assessing the utility of ROTEM in
detecting hemostatic abnormalities in pediatric b-thalassemia patients. Our data show that MCF and CFT differed
significantly between b-thalassemia patients and controls.
These findings are consistent with the concept of a prothrombotic tendency in b-thalassemia patients and may
suggest that the differences we observed have clinical
significance in these patients. The differences did not seem

TEM profile of b-thalassemic children

to depend on fibrinogen levels. Although splenectomy is an

important risk factor for TEE, a combination of several of
the above-mentioned factors may account for our results.
Splenectomy is increasingly highlighted as an important
risk factor for arterial or venous TEEs [28, 29]. It has been
shown that the percentages of plateletneutrophil and
plateletmonocyte aggregates are elevated in thalassemia
genotypes, with marked increases in patients who have
undergone splenectomy [29]. These findings suggest that
platelets adhering to neutrophils and monocytes are activated, which correlate with our findings that splenectomized thalassemia patients had higher platelet and monocyte
count. Increased level of plateletmonocyte aggregates
may contribute to higher MCFs and shorter CFTs in splenectomized thalassemia patients.
Our results show that TEM detects significant differences in b-thalassemia patients versus controls and that
these differences are consistent with a hypercoagulable
state in the former group. These findings support the need
for continued follow-up of this patient cohort to determine
whether TEM abnormalities predict thrombotic complications. The main limitation of our study is the small size of
the study population. Secondly, because genetic analyses
of patients were not performed the association between
ROTEM data and gene mutations could not be studied.
Prospective and larger studies in subjects who are prone to
TEEs are warranted to determine whether ROTEM
parameters can predict the clinical outcome of these
patients.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.
Acknowledgments The authors have no conflicts of interest to
disclose and there are no sources of funding to declare. The authors
thank Fezan Mutlu, PhD for providing statistical analysis support.

References
1. Taher AT, Musallam KM, Cappellini MD, Weatherall DJ. Optimal management of b thalassaemia intermedia. Br J Haematol.
2011;152:51223.
2. Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood. 2002;99:3643.
3. Winichagoon P, Fucharoen S, Wasi P. Increased circulating
platelet aggregates in thalassaemia. Southeast Asian J Trop Med
Public Health. 1981;12:55660.
4. Shinar E, Rachmilewitz EA, Lux SE. Differing erythrocyte
membrane skeletal protein defects in alpha and beta thalassemia.
J Clin Invest. 1989;83:40410.
5. Musallam KM, Taher AT. Thrombosis in thalassemia: why are
we so concerned? Hemoglobin. 2011;35:50310.
6. Spiezia L, Marchioro P, Radu C, Rossetto V, Tognin G, Monica
C, Salmaso L, Simioni P. Whole blood coagulation assessment
using rotation thrombelastogram thromboelastometry in patients
with acute deep vein thrombosis. Blood Coagul Fibrinolysis.
2008;19:35560.
7. Hvitfeldt Poulsen L, Christiansen K, Sorensen B, Ingerslev J.
Whole blood thrombelastographic coagulation profiles using
minimal tissue factor activation can display hypercoagulation in

18.

19.

20.

21.
22.

23.

24.

25.

thrombosis-prone patients. Scand J Clin Lab Invest.

2006;66:32936.
Sorensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J.
Whole blood coagulation thrombelastographic profiles employing
minimal tissue factor activation. J Thromb Haemost.
2003;1:5518.
Rossetto V, Spiezia L, Senzolo M, Rodriguez-Castro KI, Maggiolo S, Simioni P. Whole blood rotation thromboelastometry
(ROTEM) profiles in subjects with non-neoplastic portal vein
thrombosis. Thromb Res. 2013;132:e1314.
Ko RH, Ji L, Young G. A novel approach for detecting hypercoagulability utilizing thromboelastography. Thromb Res.
2013;131:3526.
Yee DL, Edwards RM, Mueller BU, Teruya J. Thromboelastographic and hemostatic characteristics in pediatric patients with
sickle cell disease. Arch Pathol Lab Med. 2005;129:7605.
Papa ML, Capasso F, Pudore L, Torre S, Mango S, Russo V,
Delrio P, Palaia R, Ruffolo F, dEufemia MD, De Lucia D,
Napolitano M, Di Micco P, Parisi V. Thromboelastographic
profiles as a tool for thrombotic risk in digestive tract cancer. Exp
Oncol. 2007;29:1115.
Nielsen VG, Geary BT, Baird MS. Evaluation of the contribution
of platelets to clot strength by thromboelastography in rabbits: the
role of tissue factor and cytochalasin D. Anesth Analg.
2000;91:359.
Zuckerman L, Cohen E, Vagher JP, Woodward E, Caprini JA.
Comparison of thrombelastography with common coagulation
tests. Thromb Haemost. 1981;46:7526.
Zurlo MG, De Stefano P, Borgna-Pignatti C, Di Palma A, Piga A,
Melevendi C, Di Gregorio F, Burattini MG, Terzoli S. Survival
and causes of death in thalassaemia major. Lancet. 1989;2:2730.
Borgna Pignatti C, Carnelli V, Caruso V, Dore F, De Mattia D, Di
Palma A, Di Gregorio F, Romeo MA, Longhi R, Mangiagli A,
Melevendi C, Pizzarelli G, Musumeci S. Thromboembolic events
in beta thalassemia major: an Italian multicenter study. Acta
Haematol. 1998;99:769.
Cappellini MD, Robbiolo L, Bottasso BM, Coppola R, Fiorelli G,
Mannucci AP. Venous thromboembolism and hypercoagulability
in splenectomized patients with thalassaemia intermedia. Br J
Haematol. 2000;111:46773.
Cappellini MD, Poggiali E, Taher AT, Musallam KM. Hypercoagulability in b-thalassemia: a status quo. Expert Rev Hematol.
2012;5:50511.
Goldschmidt N, Spectre G, Brill A, Zelig O, Goldfarb A, Rachmilewitz E, Varon D. Increased platelet adhesion under flow
conditions is induced by both thalassemic platelets and red blood
cells. Thromb Haemost. 2008;100:86470.
Butthep P, Bunyaratvej A, Funahara Y, Kitaguchi H, Fucharoen S,
Sato S, Bhamarapravati N. Alterations in vascular endothelial cellrelated plasma proteins in thalassaemic patients and their correlation with clinical symptoms. Thromb Haemost. 1995;74:10459.
Taher AT, Otrock ZK, Uthman I, Cappellini MD. Thalassemia
and hypercoagulability. Blood Rev. 2008;22:28392.
Cappellini MD, Motta I, Musallam KM, Taher AT. Redefining
thalassemia as a hypercoagulable state. Ann N Y Acad Sci.
2010;1202:2316.
Ataga KI, Cappellini MD, Rachmilewitz EA. Beta-thalassaemia
and sickle cell anaemia as paradigms of hypercoagulability. Br J
Haematol. 2007;139:313.
Tripodi A, Cappellini MD, Chantarangkul V, Padovan L, Fasulo
MR, Marcon A, Mannucci PM. Hypercoagulability in splenectomized thalassemic patients detected by whole-blood thromboelastometry, but not by thrombin generation in platelet-poor
plasma. Haematologica. 2009;94:15207.
Afshari A, Wikkels A, Brok J, Mller AM, Wetterslev J.
Thrombelastography (TEG) or thromboelastometry (ROTEM) to

123

A. B. Turhan et al.
monitor haemotherapy versus usual care in patients with massive
transfusion. Cochrane Database Syst Rev. 2011;2011:007871.
26. Di Benedetto P, Baciarello M, Cabetti L, Martucci M, Chiaschi
A, Bertini L. Thrombelastography. Present and future perspectives in clinical practice. Minerva Anestesiol. 2003;69:50115.
27. Luddington RJ. Thrombelastography/thromboelastometry. Clin
Lab Haematol. 2005;27:8190.

123

28. Crary SE, Buchanan GR. Vascular complications after splenectomy for hematologic disorders. Blood. 2009;114:28618.
29. Keawvichit R, Khowawisetsut L, Chaichompoo P, Polsrila K,
Sukklad S, Sukapirom K, Khuhapinant A, Fucharoen S, Pattanapanyasat K. Platelet activation and plateletleukocyte interaction in b-thalassemia/hemoglobin E patients with marked
nucleated erythrocytosis. Ann Hematol. 2012;91:168594.