Does plateletpheresis induce a hypercoagulable state? A global assessment of donor's hemostatic system by ROTEM.

Since there is still debate on the effects of plateletpheresis on coagulation system, we aimed to perform a global assessment of donor's hemostatic function undergoing plateletpheresis by rotation thromboelastometry (ROTEM) analysis and to clarify if plateletpheresis procedure induces a hypercoagulable state. Thirty male plateletpheresis donors were included in the study. Four blood samples were drawn at different time intervals: before the beginning of the apheresis procedure; immediately after the completion of the apheresis procedure; 24 h and 7 days after the apheresis procedure. "Hypercoagulability" was diagnosed readily by having an accelerated clot formation, as evidenced by shortening of CFT and an increase of the clot strength, as evidenced by increasing of MCF. In INTEM assay, CFT value after apheresis was significantly prolonged compared with baseline value while CFT value 7 days after apheresis was significantly shortened compared with values immediately and 24 h after apheresis (p < 0.001). However, CFT-INTEM still did not show any shortening in any of the measurements when compared to pre-apheresis value. MCF value after apheresis was significantly shortened compared with baseline value while MCF value 7 days after apheresis was significantly prolonged compared with values immediately and 24 h after apheresis (p < 0.001). However, MCF-INTEM still did not show any increase in any of the measurements when compared to pre-apheresis value. There was no significant difference in CT value between four measurements (p = 0.064). In EXTEM assay, CFT value after apheresis was significantly prolonged compared with baseline value while CFT value 7 days after apheresis was significantly shortened compared with values immediately and 24 h after apheresis (p < 0.001). However, CFT-EXTEM still did not show any shortening in any of the measurements when compared to pre-apheresis value. MCF values immediately and 24 h after apheresis were significantly shortened compared with baseline value while MCF value 7 days after apheresis was significantly prolonged compared with values immediately and 24 h after apheresis (p < 0.001). However, MCF-EXTEM still did not show any increase in any of the measurements when compared to pre-apheresis value. We found no differences in CT value between four measurements (p = 0.208). Since ROTEM tracings on both INTEM and EXTEM assays did not reveal any significant shortening of CFT and increasing of MCF in any of the measurements after apheresis procedure, we concluded that plateletpheresis does not induce a hypercoagulable state in healthy donors.

Can we predict thrombotic tendency in rheumatoid arthritis? A thromboelastographic analysis (with ROTEM).

The higher incidence of arterial and venous events is well established in patients with rheumatoid arthritis (RA). Our aim here was to investigate whether there is a prothrombotic state in RA patients by using rotational thromboelastometry (ROTEM) method and to demonstrate whether the disease variables play a role in this process. A total of 85 patients who met the 2010 RA classification criteria were consecutively included in the study. The patients with RA who have been using antiaggregant, anticoagulant, or nonsteroidal anti-inflammatory drugs (NSAIDs) and had a history of arterial or venous thromboembolism were excluded from the study. Their complete blood count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fibrinogen, D-dimer, and lipid profiles were measured, DAS-28 disease activation scores were calculated, and simultaneous ROTEM analysis was performed to determine the predisposition to thrombosis. Of the ROTEM parameters, clotting time (CT, seconds (s)), clot formation time (CFT, s), and maximum clot firmness (MCF) were evaluated. Having a shorter CT and/or CFT in intrinsic (I) or extrinsic (E) pathway and/or a longer MCF compared to the healthy controls was considered as "predisposition to hypercoagulability". The mean age of the 85 RA patients were 54.12 ± 13 years, and 77.6% of the patients were female (n = 66). Of the patients, 52.9% (n = 45) were using methotrexate (MTX) ± hydroxychloroquine (HCQ) ± corticosteroid (CS), while 43.5% (n = 37) were using anti-tumor necrosis factor (TNF) ± MTX. Active steroid usage was ongoing in 64.7% of the patients (n = 55). When evaluated according to DAS-28, in those with higher disease activity, a shorter I-CFT and greater I-MCF were determined (p = 0.020 and p = 0.033, respectively). In those with higher disease activity based on the correlation analysis, I-CFT and E-CFT were shorter and I-MCF and E-MCF were longer, indicating a higher predisposition to thrombosis. Using linear regression, variables with a major effect on ROTEM parameters were identified as DAS-28, CRP, and platelet count. As the first study in the literature, we identified that disease activation is the most important risk factor for prothrombotic state in RA patients irrespective of the drugs used. ROTEM can be used in clinical practice to predict thrombotic events in RA patients.

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression.

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival.

Thromboelastometry profile in children with beta-thalassemia.

Beta (ß)-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 ß-thalassemia remain largely unknown. The aim of this study was to evaluate the whole blood thromboelastometry (TEM) profile of ß-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 ß-thalassemia and 19 non-thalassemic controls. Maximum clot firmness (MCF) was significantly higher in subjects with ß-thalassemia than in controls on EXTEM and INTEM analysis (p < 0.001 and p < 0.001, respectively). Of the patients with ß-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 ß-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 ß-thalassemia.

The role of flow cytometry in the diagnosis of non- Hodgkin's lymphoma, Hodgkin's lymphoma, granulomatous inflammation and reactive lymph node specimens.

PURPOSE: In this study we aimed to compare the flow cytometry (FC) results of patients with B cell lymphoma, T cell lymphoma, Hodgkin's lymphoma, granulomatous inflammation and reactive lymph node and investigate the role of FC in malignant or non malignant conditions. METHODS: Ninety patients were divided into 5 groups according to histopathology results. Patients were compared according to cytokeratin and positivity percentage of the following surface markers: CD45, CD19, CD5, CD19-CD5, CD4, CD8, CD3,CD16-CD56, CD10, CD10-CD19, CD23, CD20, CD4-CD8, CD3-CD16-56, CD30, CD38, kappa and lambda light chains, CD20-CD23. Patients were also compared according to the intensity of the expression (exp) of same markers. ROC curve analysis was performed for CD19+ cell percentage, CD38 exp, kappa/lambda and lambda/kappa ratios. RESULTS: 1) Kappa/lambda and lambda/kappa ratios can distinguish B cell lymphoma from T cell lymphoma, Hodgkin's lymphoma, granulomatous inflammation and reactive lymph node; 2) CD19+ cell percentage can distinguish T cell lymphoma from Hodgkin's lymphoma, granulomatous inflammation and reactive lymph node; 3) CD38 exp can partly distinguish B cell lymphoma from T cell lymphoma, Hodgkin's lymphoma, granulomatous inflammation and reactive lymph node and T cell lymphoma from granulomatous inflammation, T cell lymphoma from reactive lymph node, Hodgkin's lymphoma from reactive lymph node. CONCLUSION: Flow cytometry has a role in distinguishing lymphomas from non malignant lesions.

The role of hemostatic mechanisms in the development of thrombosis in Behcet's disease: an analysis by modified rotation thromboelastogram (ROTEM).

Behcet's disease (BD) is a multisystemic disorder characterised by recurrent oral and genital ulcers. Vasculitis and thrombotic events are the most important causes of mortality. Thrombosis is the major clinical finding in patients with BD, but the cause of the thrombosis is still unclear. Thromboelastography is an alternative method to evaluate almost all steps of the hemostatic system. Today, the modified rotation thromboelastogram (ROTEM) is a newer coagulation test and a more powerful technique. Our aim in this study was to investigate whether hemostatic mechanisms play a role in the development of thrombosis in BD patients by using ROTEM. Thirty BD patients, 20 ankylosing spondylitis patients, and 14 healthy controls who are not taking anti-aggregant or anti-coagulant therapy were included in the study. Whole blood count, protrombin time, activated protrombin time, fibrinogen, D-dimer levels, and ROTEM parameters (clotting time, clot formation time (CFT), and maximum clot formation(MCF)) were determined by INTEM and EXTEM analysis. Of the 30 patients with BD, 19 were women and 11 were men, and mean age was 40.6 ± 11.2. Two of the BD patients had vascular involvement, but none of them were in active phase of the disease during the study. In INTEM assay, MCF (p < 0,001) value was significantly increased, and CFT (p>0.05) value was decreased in BD patients compared with the control group. In the EXTEM assay, there was a similar significant increase in MCF (p=0.002) value and a decrease in CFT (p=0.002) value in BD patients compared with the control group. The results of our study indicated that primary hemostatic mechanisms which can be detected by ROTEM may play a role in the development of thrombosis in patients with BD.

Evaluating coagulation disorders in the use of bevacizumab for metastatic colorectal cancer by thrombelastography.

The aim of this study was to evaluate coagulation disorders in patients with metastatic colorectal cancer treated with bevacizumab by using rotation thrombelastogram (ROTEM(®)) and correlate ROTEM(®) parameters with routine coagulation tests. A total of 18 colorectal cancer patients who received bevacizumab combined with chemotherapy were included. There was no statistically significant difference between results of platelet count, prothrombin time (PT) and activated partial thromboplastin time (APTT), fibrinogen, and D-Dimer obtained at baseline and on day 1 of chemotherapy cycles 4, 8, and 12. CFT value was significantly increased on day 1 of cycle 12 compared with baseline value by both INTEM and EXTEM assays while CT and MCF showed no significant difference. Correlation analysis revealed significant correlation between laboratory parameters and ROTEM(®) parameters. Platelet count showed a positive correlation with MCF in INTEM (r = 0.627) and EXTEM (r = 0.699) assays while showed a negative correlation with CFT in EXTEM (r = -603). There was a significant negative correlation between fibrinogen levels and CFT in INTEM (r = -0.617) and EXTEM (r = -0.512). Our data demonstrated the value of TEG over conventional coagulation tests in evaluating antiangiogenesis agents-induced coagulation disorders.

The effect of calcineurin inhibitors on endothelial and platelet function in renal transplant patients.

BACKGROUND/AIM: Posttransplant cardiovascular mortality is still an important problem in renal transplant patients. In addition to conventional coronary risk factors, coagulation abnormalities play a key role in the hypercoagulable state observed in transplanted patients. Though renal transplantation eliminates cardiovascular disease risk factors by restoring renal function, it introduces new cardiovascular risks derived, in part from immunosupressive medications. We aimed to assess the effect of calcineurin inhibitors on endothelial function, platelet activation and aggregation in renal transplant patients. METHODS: 62 renal transplant were studied. Staging was performed according to immunosuppression regimen. Group 1 (n = 37) were treated with cyclosporine/mycophenolate mofetil/methylprednisolone and Group 2 (n = 25) were treated with tacrolimus/mycophenolate mofetil/methylprednisolone. The control group consisted of 16 healthy subjects (Group 3). Hematological and biochemical tests, asymmetric dimethyl arginine (ADMA), sP-selectin levels and platelet aggregation tests were studied. RESULTS: ADMA levels were higher in Group1 and statistically significant differences were observed compared with those of Group 2 and Group 3 (p < 0.05). Platelet aggregation values induced by all agonists (Adenosine diphosphate (ADP), epinephrine, ristocetin, collagen) were lower in Group 1 than Group 2 and Group 3, but the difference did not reach statistical significance (p > 0.05). There was a negative correlation between cyclosporine level and platelet aggregation values induced by ADP (r = -0.43, p < 0.01), ristocetin (r = -0.40, p < 0.05), epinephrine (r = -0.41, p < 0.05), and collagen (r = -0.43, p < 0.01). sP-selectin levels were appreciably higher in Group 1 and statistically significant differences were observed compared with those of Group 2 (p < 0.05) and Group 3 (p < 0.01). CONCLUSION: The results of our study suggest that CsA induces platelet activation without inducing platelet aggregation. Endothelial dysfunction due to vascular endothelial damage reflected by increases in ADMA values may increase the tendency for thrombotic events in patients who had undergone renal transplantation.

Aspirin-resistance frequency: a prospective study in 280 healthy Turkish volunteers.

This study reports the frequency of aspirin resistance and its correlation with clinical and biochemical parameters among 280 healthy Turkish volunteers (179 men, 101 women) who were taking 100 mg of aspirin 7 days or more. Aspirin resistance was detected by optical platelet aggregometry, using adenosine diphosphate and arachidonic acid, and defined as a mean aggregation of 64% or more with 5AmicroM adenosine diphosphate and a mean aggregation of 20% or more with 0.5-mg/mL arachidonic acid. Of the study population, 27.5% (26 women [25.5 %] and 51 men [28.5 %]) were aspirin resistant. The current findings indicate that aspirin resistance is an important and real laboratory diagnosis given its frequency of 27.5% in the study population. These results of this large trial evaluating the frequency of aspirin resistance in healthy subjects indicate that aspirin resistance should be diagnosed so that individuals with no response can receive alternative or additional antiplatelet therapy.

Laboratory investigation of hypercoagulability in cancer patients using rotation thrombelastography.

The goal of this study was laboratory testing for hypercoagulability in patients with solid tumors using rotation thrombelastogram (ROTEM) and correlate ROTEM parameters with routine coagulation tests. A total of 78 untreated patients with cancer were included: 28 gastrointestinal system tumors (group 1), 27 respiratory system tumors (group 2), and 23 miscellaneus group of ovarian, renal, nasopharyngeal, mesothelioma, and unknown origin (group 3). Platelet count was significantly increased in group 2 in respect to group 3 (P < 0.05) and fibrinogen level was significantly increased in group 2 in respect to group 1 (P < 0.05). There was no statistically significant difference between subgroups in respect to TEG parameters. Tumor-node-metastasis (TNM) stages of patients was not also associated with either of TEG parameters. Correlation analysis revealed significant correlation between laboratory parameters and ROTEM parameters. Fibrinogen showed the strongest correlation with MCF (r > 0.7) and CFT in all assays (INTEM, EXTEM, FIBTEM, APTEM). There were also statistically significant correlations between platelet number and other ROTEM parameters (INTEM-CFT, -MCF, EXTEM-CFT, -MCF, FIBTEM-MCF, APTEM-CFT, -MCF). In conclusions, our data demonstrates thromboelastographic signs of hypercoagulability in patients with solid tumors. ROTEM is able to identify the contribution of fibrinogen and platelets to clot strength in this patient population.

Prognostic impact of chromosome alterations detected by FISH in Turkish patients with B-cell chronic lymphocytic leukemia.

The goal of this study was to evaluate the relation of chromosomal abnormalities detected by fluorescence in situ hybridization (FISH) in the prognosis of B-cell chronic lymphocytic leukemia (B-CLL) patients. We evaluated the common recurrent chromosomal aberrations in 79 B-CLL patients (51 men, 28 women; mean age 64.3+/-1.2) by FISH analysis using 11q22.3 (ATM), 13q14.3 (13S319 and 13S25), CEP12, and 17p13.1 (TP53) specific probes. Of the 79 patients analyzed by FISH, 40 or 50.6% had at least one aberration. In particular, 34 (43%) patients had a single abnormality and 6 (7.6%) patients had 2 abnormalities. The most frequent abnormality was 13q14.3 deletion, which was detected in 26 (32.9%) patients. Trisomy 12 was seen in 12 (15.2%) cases, and was followed by 17p13.1 (TP53) deletions and 11q22.3 (ATM) deletions in 6 (7.6%) and 4 (5.1%) patients, respectively. When the overall frequencies of these chromosomal aberrations were distributed according to RAI stages, the majority of patients with 13q14.3 deletion (55%), trisomy 12 (70%), and ATM or TP53 deletions (66.7 %) were in advanced stages of disease (RAI II-IV). The overall survival durations in good, intermediate, and poor prognostic groups were 51+/-1.3, 50.9+/-8.6, and 12+/-3.3 months, respectively. Our data suggests that FISH analysis of B-CLL patients provides important diagnostic, clinical, and prognostic information which may help clinicians assess the prognosis and make appropriate treatment decisions.

Successful treatment of nephrotic syndrome due to systemic AL amyloidosis after autologous stem cell transplantation: renal response is an important therapeutic end point.

Primary systemic (AL) amyloidosis involves vital organs from the early phase of illness, resulting in poor prognosis. Today, high-dose melphalan followed by autologous peripheral blood stem cell transplantation is an effective treatment for systemic AL amyloidosis. We report a patient with nephrotic syndrome due to systemic AL amyloidosis, who was successfully treated with autologous peripheral blood stem cell transplantation. At follow-up 36 months from ASCT, the patient showed a significant improvement in the signs of peripheral neuropathy and reduction in proteinuria without further organ involvement. Due to poor prognosis with conventional therapy, autologous stem cell transplantation should be considered for treatment in patients with systemic AL amyloidosis, and favorable outcome is ensured with achievement of renal response after ASCT.

Platelet function testing during 5-day storage of single and random donor plateletpheresis.

Platelet concentrates are routinely manufactured from whole blood by differential centrifugation (random donor platelets-RDP) or by plateletpheresis (single donor platelets-SDP). These platelet concentrates have a storage period of 5 days and many different approaches exist to measure the condition of platelets during their storage. In this study, platelet aggregation testing using adenosine diphosphate (ADP) and collagen and flow cytometric platelet activation analysis using CD41 FITC and CD62 PE before and after ADP was performed on days 1, 3 and 5 of storage of platelet preparations. Thirty three RDPs, stored in Baxter and Kansuk blood bags and 18 SDPs stored in Fresenius blood bags were evaluated. In RDPs and in SDPs; ADP and collagen induced PA responses were decreased significantly on the 3rd and 5th days compared to 1st day. CD62 positive platelet percentage after ADP were decreased significantly on the 3rd and 5th days compared to the 1st day in Kansuk bags. Flow cytometric analysis revealed minor changes in CD41 expression after ADP on the 3rd day compared to 1st day and on the 5th day compared to 3rd day. Differences in CD62 positive platelet percentage were not significant between the RDPs and SDPs. Our results suggest that: (1) ADP and collagen induced PA responses decrease both in RDPs and SDPs during storage. (2) Flow cytometric analysis does not show major significant changes in platelet activation after ADP during storage. (3) Continous shaking on the agitator does not cause a significant change in CD62 positive platelet percentage during storage. (4) Platelet aggregation responses in RDPs stored in Baxter and Kansuk blood bags do not differ during storage.

Evaluation of inherited and acquired platelet function disorders in iron deficient women with menorrhagia by whole blood lumi-aggregometer.

The commonest cause of iron deficiency anemia (IDA) in premenopausal women is often menstrual blood loss. However, no organic pathology is identified in more than 50% of menorrhagic women. We therefore investigated inherited and acquired bleeding disorders among women with unexplained menorrhagia who developed IDA. In vitro whole blood platelet aggregation (PA) with ADP, arachidonic acid (AA), ristocetin and collagen was studied in addition to full blood count, serum iron levels, serum iron binding capacity, transferrin saturation, ferritin, prothrombin and activated partial thromboplastin time, fibrinogen, D-Dimer, Factor VIII, Factor IX, Factor XI, ristocetin cofactor activity, blood type and bleeding time in 67 women before and after therapy. Before therapy; decreased agonist induced PA was observed in 20% of women by ADP, in 12% by AA, in 2% by ristocetin and in 6% by collagen. After oral iron therapy, decreased platelet aggregation was shown in 8% of women with ADP and 2% of women with AA while initial abnormal ristocetin and collagen induced platelet aggregation responses became normal. Also there was a statistically significant increase of ristocetin cofactor activities and FXI levels after iron repletion. We conclude that; rather than von Willebrand disease, platelet function abnormalities and FXI deficiency are the most common hemostatic disorders in women with unexplained menorrhagia and significant portion of these disorders can be reversed by iron therapy.