1096 Introduction: ITP is an autoimmune disorder characterized by increased platelet destruction and/or decreased platelet production. In addition to risks of bleeding, an increased risk of thrombosis has been identified, despite low platelet counts. Reasons behind this remain unclear. Elevated levels of VWF and decreased levels of ADAMTS13 have been observed in patients with myocardial infarction and ischaemic stroke. Given the interaction of these plasma proteins with platelets, we hypothesized that changes in VWF/ADAMTS13 levels may contribute to the thrombotic risk in patients with ITP. Methods: Adult patients (>18 years) with a diagnosis of ITP were included. ELISAs were used to measure the concentrations of VWF, ADAMTS13 and IL-6. Platelet count and C-Reactive protein (CRP) levels were recorded. Control samples for VWF and ADAMTS13 were taken from a previous study (n=626) (Andersson et al. Blood 2012; 119(6):1555–60). Published data from a population-based study (Marques-Vidal et al. PLoS One 2011; 6(6): e21002) enabled comparison of IL-6 levels to normal controls. Results: 93 samples were taken from 48 patients with ITP between March-June 2012. All samples were analyzed for VWF:Ag levels. Plasma concentrations of ADAMTS13 and IL-6 were analyzed in 58 and 34 plasma samples respectively. Plasma VWF antigen (VWF:Ag) levels were elevated in patients with ITP compared to control subjects (p=0.0369) (Figure 1). Levels of ADAMTS13 in patients with ITP were lower than those for control subjects (p=0.0050) (Figure 2). Plasma VWF levels did not correlate with platelet counts. Rather high VWF levels were specific to certain patients. Two patients with VWF:Ag levels >40 μg/mL had a past history of thrombosis; both had additional co-morbidities. Two further patients with acute disease (one recent acute intracranial bleed and one new relapse of ITP) also had high VWF levels. In these two patients, VWF, ADAMTS13 and platelets change over time (example shown in Figure 3). CRP levels did not correlate with platelet count (p=0.6091) or with VWF:Ag levels (p=0.5170). Median IL-6 levels were raised in patients with ITP (3.785 pg/mL) compared to healthy controls (1.47 pg/ml) (p=<0.0001). IL-6 correlated positively with CRP levels (p=0.0183, r=0.4211) however no association between IL-6 and VWF, ADAMTS13 or platelet count was established. Conclusion: The increased ratio of VWF: ADAMTS13 may be contributory in the pathogenesis of thrombosis, in particular in patients with acute and unstable disease. Increased VWF:ADAMTS13 ratio may also result in an increased VWF activity, even without a rise in total VWF. Future studies comparing VWF:Ag to collagen binding are planned. The mechanism behind fluctuating levels of VWF and ADAMTS13 remains unclear. We investigated whether inflammatory mediators associated with acute disease could play a role. CRP levels were elevated in many patients with ITP, but this did not correlate with VWF levels. Levels of IL-6 were also significantly raised in patients compared to healthy controls and correlated with CRP but did not correlate with VWF or with platelet count. In summary, ITP appears to be a heterogeneous disease. In some patients, autoimmunity may be associated with an inflammatory process, and in other patients, low platelets may interfere with other aspects of the coagulation system. Either may predispose to thrombosis or bleeding. Further investigation of the interactions of platelets, with inflammatory cytokines and the coagulation system may help us to better understand the disease, and to recognize those patients at risk of bleeding, or conversely thrombosis. Disclosures: Cooper: Amgen: Consultancy; GSK: Consultancy; Easai: Consultancy.
SYNOPSIS Carcinoembryonic antigen (CEA) has been measured in parallel with seven serum proteins and seromucoids in the sera of patients with malignant neoplasia and non-neoplastic disease. In the total group significant correlations were found between CEA and seromucoids and between CEA and several serum proteins. However, with two exceptions, when the individual disease groups were examined no correlation was seen. It is concluded that abnormal concentrations of the specific proteins measured do not consistently interfere in the CEA radioimmunoassay and do not explain the high CEA levels in patients with non-neoplastic diseases.A major defect in the application of carcinoembryonic antigen (CEA) assay to diagnostic clinical practice lies in the lack of specificity for cancer as against inflammatory disease. This finding has been widely
The pathology of immune thrombocytopenia (ITP) is not fully understood. Although antiplatelet antibodies (APAs) are thought to be causative, these are not detected in up to 30% of patients. In addition, T cell directed therapy is frequently successful. Patients with ITP display cytotoxic activity towards platelets, and abnormalities in T cell repertoire. However, there is incomplete understanding of the interactions between T cells and megakaryocytes within the bone marrow. We hypothesized that if T cell or NK cell cytotoxicity against megakaryocytes contributes to thrombocytopenia, then T and/or NK cells should be in direct contact with the megakaryocyte in the bone marrow. We therefore further explored the interactions between T cells and megakaryocytes in bone marrow trephine biopsies - taken at or near to diagnosis - in patients with ITP. To further explore apoptotic pathways, serum TRAIL levels were also assessed at different time points in disease. Methods Bone marrow trephine samples were taken as clinically indicated at or around the time of diagnosis and processed as per department protocol. Samples from patients with known ITP (n=9) were further processed and stained with CD3, CD8, CD4 and CD56. Immunohistochemistry was performed using Leica Bond-max automated immunohistochemistry system. The density of MK cells, CD3, CD8 and CD4 was recorded and the numbers of MK in contact with T or NK cells were recorded on multiple sites on the trephine. Control bone marrows from staging marrows for lymphoma were used for comparison (n=6). For further analysis of the non-antibody component of ITP, serum TRAIL levels were measured in 63 samples from 43 patients at different stages of disease. Patients had all consented for research. Results Results of the bone marrow examination are shown in the table. Bone marrow samples from patients with ITP had higher density of megakaryocytes than control bone marrows (median 41/mm3 compared to 20/mm3 - p=0.03). Megakaryocytes appeared normal in morphology. CD3 density was also higher in patients with ITP, although this was not significant (p=0.17). There was however increased interaction between CD3 and MK cells in patients with ITP compared to controls (median 50% of MK cells in contact with CD3 cells in ITP compared to 32% in controls - p=0.02) – figure 1a. There was no correlation between MK cell density, T cell density and MK-T cell interaction, making this finding unlikely to have occurred by chance. In ITP patient samples further analysis of T cell type was undertaken and shown in the table showing interactions occur between both CD4s and CD8s and the MK cells (figure 1b). Four patients had reverse CD4:8 ratios in the bone marrow. Interaction between MK and CD56+ cells were less frequent (0 to 8%). There was no association with response to treatment and MK or T cell findings, although patients who responded to rituximab appeared to have lower MK density. Patients who responded to thrombopoietic (TPO) agonists - and by virtue of their use, had not responded to other treatments – had a high density of MK cells at diagnosis (bone marrows were taken at diagnosis, and before TPO agonists were started). Serum TRAIL levels were higher in patients with chronic ITP and with platelets <50 when compared to patients in remission (platelets > 150) (p<0.1). In patients with serial samples, there was an inverse relationship between TRAIL and platelet count. Conclusion Preliminary evidence suggests increased interactions between T cells and megakaryocytes in the bone marrow of patients with ITP. The demonstration of interactions between CD3 cells and MK cells in both patients and controls shows the importance of the MK cell in the immune system. Further investigation is under way to explore whether CD8 or NK cell interaction is only found in patients with ITP, and whether these interactions result in MK cell death, or reduction in platelet production. High TRAIL levels in patients with chronic disease, refractory to first and second line therapy further suggests that increased apoptosis plays an important role in the ongoing thrombocytopenia in chronic disease. Disclosures: No relevant conflicts of interest to declare.
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