|Year : 2018 | Volume
| Issue : 1 | Page : 10-18
Thrombin antithrombin complex assessment in patients with chronic hemolytic anemia as a marker for the activity of coagulation
Ibrahim Y Abdel-Messih1, Nevine G Andrawes2, Yasmin N El-Sakhawy1, Nesma A Safwat1, Yasmin H Ibrahim1
1 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||06-Sep-2017|
|Date of Acceptance||10-Sep-2017|
|Date of Web Publication||3-Aug-2018|
Yasmin N El-Sakhawy
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo
Source of Support: None, Conflict of Interest: None
Background Hypercoagulability in chronic hemolytic anemia, namely, β-thalassemia major (TM), β-thalassemia intermedia (TI), and sickle cell disease (SCD), is well recognized. Activation of coagulation results in thrombin formation which in turn is inactivated by complex formation with its major inhibitor thrombin antithrombin complex (TAT). As a result, TAT is considered a coagulation marker which confirms the hypercoagulability state.
Aim The aim of this study was to measure TAT as a coagulation activation marker in patients with β-TM, β-TI, and SCD and to correlate TAT levels with their clinicolaboratory parameters.
Patients and methods A total of 60 children and adolescents having β-thalassemia syndromes and SCD were recruited from pediatric hematology clinic, Ain Shams University. They underwent routine complete blood picture and hemolytic profile in addition to TAT.
Results The TAT was significantly higher in all patients (164.13±42.47 µg/l) compared with controls (106.75±21. 35 μg/l). In the thalasssemia group, the TAT level was 156.45±42.71 µg/l, whereas in patients with SCD, it was 179.50±38.52 µg/l. On comparing patients with thalassemia and those with SCD, there was a significant difference (P<0.05). However, measured TAT levels in patients with TM versus those with TI were 151.11±26.07l versus 167.54±65.25 µg/l, respectively. Comparison of TAT level between patients with TM and TI revealed no statistically significant difference. TAT was significantly correlated with mean corpuscular volume, mean corpuscular hemoglobin, and total and indirect bilirubin levels in all patients.
Conclusion β-Thalassemia syndromes and SCD are associated with increased coagulation activity, and this activity is correlated with the level of hemolysis.
Keywords: sickle cell disease, thrombin antithrombin complex, thalassemia
|How to cite this article:|
Abdel-Messih IY, Andrawes NG, El-Sakhawy YN, Safwat NA, Ibrahim YH. Thrombin antithrombin complex assessment in patients with chronic hemolytic anemia as a marker for the activity of coagulation. Egypt J Haematol 2018;43:10-8
|How to cite this URL:|
Abdel-Messih IY, Andrawes NG, El-Sakhawy YN, Safwat NA, Ibrahim YH. Thrombin antithrombin complex assessment in patients with chronic hemolytic anemia as a marker for the activity of coagulation. Egypt J Haematol [serial online] 2018 [cited 2022 May 19];43:10-8. Available from: http://www.ehj.eg.net/text.asp?2018/43/1/10/238542
| Introduction|| |
An increased incidence of thrombosis has been reported in different hemolytic anemias (HAs), particularly in sickle cell disease (SCD) and thalassemia. Although HAs have different pathophysiologies, hemolysis per se, whatever the cause seems to be a procoagulant condition .
Hemolysis contributes to coagulation abnormalities in HA in three ways: the first is red blood cell (RBC) membrane alterations and microparticles. An increased exposure of anionic phospholipids such as phosphatidylserine confers procoagulant properties to thalassemic RBC and sickle cells. In such condition, erythroid cells may act at activated platelets and enhance prothombin conversion to thrombin especially in splenectomized patients. Similarly, microparticles produced by RBC fragmentation during hemolysis have polynegative niches that activate thrombin generation . The second is erythrocyte/endothelium interaction. As a consequence of red cell destruction, plasma hemoglobin stimulates the expression of adhesion molecules on endothelial cells leading to vessel obstruction, in addition to abnormal expression of tissue factor on endothelial cells in sickle cell anemia. The third is nitric oxide (NO) deficiency. In severe HA, free plasma hemoglobin scavenges NO and depletes it. In addition, hemolysis releases erythrocyte arginase, which converts L arginine, the substrate for NO synthesis, to ornithine, leading to further NO reduction .
Thalassemia is an inherited hemolytic disorder caused by a partial or complete deficiency of α-globin or β-globin chain synthesis. Homozygous carriers of β-globin gene defects experience severe anemia and other serious complications from early childhood .
Among the clinical complications of this hematological disorder, thromboembolic episodes are particularly important and frequent, especially in splenectomized patients with the milder thalassemia intermedia (TI) than in those with the more severe thalassemia major (TM) .
Several factors that contribute to the hypercoagulable state in patients with β-thalassemia have been identified. In most cases, a combination of these abnormalities leads to thromboembolic events. Chronic activation of platelets and enhancement of platelet aggregation are observed in patients with thalassemia and confirmed by the increased expression of CD62P (P-selectin) and CD63. Metabolites of prostacyclin (PGI2) and thromboxane A2, which are markers of hemostatic activity, are increased four- to 10-fold in splenectomized patients with TM and nonsplenectomized patients with TI .
SCD is an inherited disorder characterized by the presence of sickle hemoglobin, which results from the substitution of glutamic acid by valine at the sixth position of the β-globin chain .
SCD is associated with a hypercoagulable state that may contribute to certain morbidities such as vaso-occlusion and cerebrovascular accidents. Decreased levels of natural anticoagulant proteins are observed in sickle cell anemia and even more so in vaso-occlusive crisis. Abnormal tissue factor expression on endothelial cells and inflammatory states in SCD may also contribute to hypercoagulability in SCD .
Thromboembolism is appreciated as the fifth most common adverse event ,,. This finding could have important therapeutic consequences and pave the way to the development of novel approaches to decrease the occurrence of thrombotic complications in HA .
| Aim|| |
The aim of this study was to measure TAT as a coagulation activation marker in the patients with chronic HA, namely, β-thalassemia major (β-TM), β-thalassemia intermedia (β-TI), and SCD, and to correlate TAT levels with the clinicolaboratory parameters among these patients.
| Patients and methods|| |
This study was conducted on 60 children and adolescents who were diagnosed as having β-thalassemia syndromes and SCD. They were recruited from Pediatric Hematology Clinic, Pediatric Hospital, Ain Shams University. A written consent was taken from the patients or their legal guardians.
Non-Egyptians, critically ill patients with concurrent infections, inflammations, or chronic illness, hemorrhagic fever, diabetes, malignancies, and renal failure were excluded from the study, in addition to any patient whose sample showed hemolysis after centrifugation.
The studied patients were divided into two groups: group I included patients with thalassemia and group II were patients with SCD. All were subjected to detailed history taking, clinical examination to evaluate the hemolytic manifestations and thrombotic events, and laboratory investigations. Overall, three blood samples were taken from each patient: one for hematological testing (complete blood count, stained blood film, and reticulocytic count), second sample for assessing hemolysis [lactate dehydrogenase (LDH) and total bilirubin level] and to measure serum level of TAT, and the third one was to assess the coagulation state (prothrombin time, international normalized ratio, and partial thromboplastin time).
Overall, 2 ml of peripheral venous blood was collected in tubes containing K2-EDTA for hematologic tests. Samples were analyzed using coulter LH 750 cell counter, USA. Moreover, 2 ml of peripheral venous blood was collected in sodium citrate vacutainer for performance of coagulation profile by Stago autoanalyzer using Neoplastin Cl plus supplied by Diagnostica Stago (France). Another 2 ml of peripheral venous blood was collected in serum-separating tubes for measuring serum level of LDH and total and indirect bilirubin levels using Synchron CX-5 autoanalyzer, USA. TAT was also measured using enzyme-linked immunosorbent assay kit supplied by Glory Science Co. (USA).
The SPSS 22.0 USA for Windows was used for data analysis. Quantitative parameter data were presented as mean±SD, and for comparison of the mean of two groups, independent t-test was used.
The association between two variables among normally distributed data was done by Pearson’s correlation (R).
The association between qualitative data was determined using the χ2-test. A P-value less than 0.05 was considered significant. The receiver operating characteristic curve was used to detect the best cutoff value.
| Results|| |
The studied patients were divided into two groups: group I (n=40) included patients with thalassemia (of which 27 patients had TM and 13 patients had TI), group II (n=20) comprised patients with SCD, and the control group (n=20) included healthy age-matched children.
Descriptive data of the studied groups are shown in [Table 1].
|Table 1 Descriptive data of the studied participants (control, thalassemia, and sickle cell disease groups)|
Click here to view
Laboratory data of the studied groups are shown in [Table 2].
|Table 2 Laboratory data of the studied participants (control, thalassemia, and sickle cell disease groups)|
Click here to view
Comparison of clinicolaboratory parameters among TM and TI is shown in [Table 3].
|Table 3 Comparison of clinicolaboratory parameters among patients with thalassemia major and those with thalassemia intermedia|
Click here to view
Comparison of thrombin antithrombin complex level among the different studied groups
The TAT level was significantly higher in all patients compared with control. Moreover, on comparing patients with thalassemia and those with SCD, there was a significant difference (P<0.05) ([Table 4] and [Table 5]).
|Table 5 Thrombin antithrombin complex level among the control, thalassemia, and sickle cell disease groups|
Click here to view
However, no statistically significant difference was found in TAT level between patients with TM and those with TI ([Table 6]).
|Table 6 Thrombin antithrombin complex among thalassemia major and intermediate groups|
Click here to view
Relation between thrombin antithrombin complex with quantitative and qualitative data
No significant relation was found regarding qualitative data studied, including age, sex, and splenectomy, except a borderline correlation with the history of thrombosis in SCD (P=0.053) ([Table 7],[Table 8],[Table 9]). However, a positive correlation was found between TAT and MCV, MCH, and total and indirect bilirubin levels in all patients ([Table 10]).
|Table 7 Relation of thrombin antithrombin complex with sex, splenectomy, and history of thrombosis in all patient groups|
Click here to view
|Table 8 Relation of thrombin antithrombin complex with sex, splenectomy, and history of thrombosis in thalassemia group|
Click here to view
|Table 9 Relation of thrombin antithrombin complex with sex, splenectomy, and history of thrombosis in sickle cell disease group|
Click here to view
|Table 10 Correlation analyses between thrombin antithrombin complex and different studied parameters in patients with thalassemia, patients with sickle cell disease groups, and all the patients|
Click here to view
Performance of thrombin antithrombin complex as a diagnostic marker
Receiver operator characteristic curves were prepared to establish cutoff levels of TAT for the diagnosis of thalassemia and SCD.
In thalassemia, the best cutoff value of TAT was 115 μg/l. This had a diagnostic sensitivity of 92% and specificity of 80% ([Table 11] and [Figure 1]). However, in SCD, the best cutoff value of TAT was 120 μg/l, with 95% sensitivity and 95% specificity ([Table 12] and [Figure 2]).
|Table 11 The diagnostic performance of thrombin antithrombin complex for discriminating thalassemic group from normal control group|
Click here to view
|Figure 1 Receiver operating characteristic curve for thrombin antithrombin complex (TAT) (µg/l) in the prediction of patients with thalassemia.|
Click here to view
|Table 12 The diagnostic performance of thrombin antithrombin complex for discriminating sickle cell disease group from normal control group|
Click here to view
|Figure 2 Receiver operating characteristic curve for thrombin antithrombin complex (TAT) (μg/l) in the prediction of patients with sickle cell disease.|
Click here to view
| Discussion|| |
Hypercoagulability in HAs is well recognized, resulting in an increased risk for thromboembolic events ,,, which is appreciated as the fifth most common adverse event, particularly in SCD and thalassemias. Despite prophylaxis, thrombotic events can continue and can result in severe physical or mental debilitation or death of the patients .
Activation of coagulation results in the formation of thrombin, which in turn is inactivated by complex formation with its major inhibitor AT. This process leads to the appearance and increase of TAT in peripheral blood . In this regard, this study aimed to assess serum TAT in a group of patients with hemoglobinopathies to evaluate its clinical utility in the assessment of hypercoagulability and the risk of thrombotic formation.
Regarding TAT, when considering all included patients with thalassemia in the study, it was found that TAT was significantly higher (156±42 µg/l) compared with the control group (106±21 µg/l).
This finding was in agreement with the evidence of a hypercoagulable state in β-thalassemia mentioned by many other studies ,. They demonstrated an increased level of TAT in a cohort of patients with β-TM and β-TI and thus increased coagulation activity. In addition, Gunes et al.  had documented that the chronic hypercoagulable state frequently observed in β-thalassemia exists since childhood.
Level of TAT in patients with SCD in our study was 179.5±38.52 µg/l compared with 106±21 µg/l in control group. This finding was in agreement with many other studies. By using TAT together with d-dimer, Chantrathammachart et al.  and Ataga et al.  evidenced the hypercoagulable state in SCD. The mean of TAT level in the study by Ataga et al.  was ∼5.63 µg/l for SS/S-β 0 thalassemia and 4.86 µg/l for SC/S-β+ thalassemia . There was no control group in that study. Moreover, in a study done by Hamid and colleagues, TAT level was significantly higher among SCD cases (2.55±2.04 µg/l) than controls (0.58±0.08 µg/l), with further elevation among patients in crisis than those in steady state (5.21±5.03 µg/l) . TAT level was much lower in their studies than the present one as they used plasma TAT level, whereas in this study, we used serum TAT level.
Furthermore, increased levels of TAT, F1.2, and d-dimers have also been reported by Solovey et al.  in patients with sickle cell trait, suggesting that a direct link may exist between carriage of the βs gene and activation of the coagulation system.
Looking into the effect of different laboratory variables (complete blood count, retics, ferritin, LDH, prothrombin time, partial thromboplastin time, and international normalized ratio) on TAT, no significant association was detected except for total and direct bilirubin levels as indicators of hemolysis. Similarly, Abdul-Fattah et al.  showed no significant relation between TAT and LDH. Setty et al.  positively correlate plasma level of TAT and d-dimer with measures of hemolysis and anemia (LDH, indirect bilirubin, and hemoglobin) and levels of soluble vascular cell adhesion molecule-1, as a marker of endothelial cell activation. Moreover, Ataga et al.  found a positive association between LDH and TAT level with borderline correlation between TAT and total and indirect bilirubin levels.
In the current study, 27.5% of thalassemia group patients and 15% of patients with SCD were splenectomized. However, no statistically significant difference was found in this study between TAT level and presence or absence of spleen. This was in agreement with the results of Abdul-Fattah et al.  where no statistically significant difference was found between patients with or without splenectomy as they reported 20% cases with splenectomy in all the patients. However, in the study by Taher et al. , 93% of the patients who experienced thromboembolic events were splenectomized
In our study, thromboembolic events constitute 2.5% among the 40 patients with β-thalassemia. However, one (3.7%) patient among the 27 patients with β-TM had evidence of thrombosis, and none of the 13 patients with β-TI had history of thrombosis. Taher et al.  demonstrated that thromboembolic events occurred in a clinically relevant proportion of 1.65% among 8860 patients with thalassemia from the Mediterranean and Iran. Thromboembolism occurred 4.38 times more frequently in TI than TM with more venous events occurring in TI and more arterial events occurring in TM. The most common thrombotic events in those patients were deep vein thrombosis (33.3%), stroke (17.4%), pulmonary vein thrombosis (16.0%), and pulmonary embolism (11.6%). Recurrent thrombosis was reported in 25% and 36% of patients with TM and those with TI, respectively.
In the study by Eldor and Rachmilewitz , the prevalence of thromboembolic events was 3.3% among 421 patients with β-TM and 16.2% among 74 patients with β-TI, although 15.3% of these patients had predisposing congenital or acquired factors contributing to the hypercoagulability state. Cappellini  observed a high incidence of venous thromboembolic events in a group of 83 patients with β-TI who were followed for 10 years.
The observation that thrombotic events are more frequent in β-TI or in patients with thalassemia who have undergone splenectomy was explained by Franchini and Mannucci  with two reasons: first, patients with TM are usually regularly transfused and thus the great majority of their circulating RBCs are donor-derived normal allogenic cells and second, the spleen, when present, helps to remove the structurally abnormal and rigid red cells that cause hypercoagulability, particularly in nontransfused or rarely transfused patients. These findings may have clinical implications mainly in splenectomized patients who should be considered at risk of thrombosis and should receive prophylaxis when they are exposed to transient thrombotic risk factors.
Risk factors for developing thrombosis in patients with β-TI are age more than 20 years, previous thromboembolic events, family history, and splenectomy . In this study, there was a nonstatistically significant difference between TM and TI related to the history of thrombosis as all of our patients were less than 20 years, no positive family history, and limited number of splenectomy in comparison with the excepted increase in the future with increasing in the risk in developing thromboembolic event.
The prevalence of thromboembolic events was 30% among 20 patients with SCD in this study: 10% experienced acute abdomen, 10% from sequestration crisis, 5% from avascular necrosis in hip bone, and 5% from stroke. In the study by Ataga et al. , which was done among 64 patients with SCD, there was a history of thrombosis in many sites, such as 7.8% stroke, 48% avascular necrosis, 18% leg ulcer, 85% acute chest syndrome, and 32% retinopathy. In that study, it was found that the level of TAT was significantly higher in patients with retinopathy compared with those without this complication.
| Conclusion|| |
It was found that the bulk of evidence demonstrates that there is increase in the coagulation activity in thalassemia and SCD measured by TAT level, and this coagulation activity is correlated with the level of hemolysis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Cappellini MD. Coagulation in the pathophysiology of hemolytic anemias. Hematology Am Soc Hematol Educ Program
Franchini M, Mannucci PM. Hypercoagulability in congenital hemolytic anemias. Blood Transfus
Ataga KI. Hypercoagulability and thrombotic complications in hemolytic anemias. Hematologica
Cappellini MD, Taher A, Mussalm K. Hypercoagulability in b thalassemia. Expert Rev Hematol
Ataga KI, Brittain JE, Desai P, May R, Jones S, Delaney J et al.
Association of coagulation activation with clinical complications in sickle cell disease. PLoS One
Hamid IM, Mahmoud RM, Merghani GM, Abdalla MH. Assessment of hypercoagulablity state among Sudanese sickle cell patients. J Biochem Pharmal Res
Succar J, Musallam KM, Taher AT. Thalassemia and venous thromboembolism. Mediterr J Hematol Infect Dis
Taher AT, Musallam KM, Karimi M, El-Beshlawy A, Belhoul K, Daar S et al.
Overview on practices in thalassemia intermedia management aiming for lowering complication rates across a region of endemicity: the OPTIMAL CARE study. Blood
Borgna-Pignatti C, Rugolotto S, De Stefano P, Zhao H, Cappellini MD, Del Vecchio GC et al.
Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine. Haematologica
Ataga KI, Cappellini MD, Rachmilewitz EA. Β-Thalassaemia and sickle cell anaemia as paradigms of hypercoagulability. Br J Haematol
Cappellini MD, Musallam KM, Marcon A, Taher AT. Coagulopathy in beta-thalassemia: current understanding and future perspectives. Mediterr J Hematol Infect Dis
Taher A, Ismaeel H, Cappellini M. Thalassemia intermedia; revisited. Blood Cells Mol Dis
Tatra G, Reinthaller A. Elevated thrombin-antithrombin III complex concentrations in patients with gynaecological malignancy. Klin Wochenschr
Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood
Del Vecchio GC, Nigro A, Giordano P, Schettini F, Altomare M, Pietrapertosa A, de Mattia D. Plasma protein Z and protein C inhibitors and their role in hypercoagulability of thalassemia. Acta Haematol
Gunes BT, Turker M, Gozmen S, Oymak Y, Ay Y, Ince D et al.
Procoagulant phospholipid activity, whole blood thromboelastography and thrombin generation assay to detect hypercoagulability in thalassemic children. Blood
Chantrathammachart P, Mackman N, Sparkenbaugh E, Wang JG, Parise LV, Kirchhofer D et al.
Tissue factor promotes activation of coagulation and inflammation in a mouse model of sickle cell disease. Blood J
Solovey A, Kollander R, Shet A, Milbauer LC, Panoskaltsis-Mortari A, Blazar BR et al.
Endothelial cell expression of tissue factor in sickle mice is augmented by hypoxia/reoxygenation and inhibited by lovastatin. Blood
Abdul-Fattah ME, Ghobrial AG, Soliman GT, Abdul-Aallah AR. Hypercoagulability and endothelial activation in B-thalassemia major: possible role of nitric oxide deficiency. El Minia Med Bull
Setty BN, Key NS, Rao AK. Tissue factor positive monocytes in children with sickle cell disease: correlation with biomarkers of haemolysis. Br J Haematol
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]