Christina Christersson

Evaluation of microparticles in whole blood by multicolour flow cytometry assay

Abstract Objective. To develop and evaluate a multicolour flow cytometry method for analysis of microparticles (MPs) in fresh whole blood without any centrifugation steps or freezing/thawing procedure. Materials and methods. Flow cytometry was performed using a FC500 MPL cytometer. The compensation in the protocol was performed based on the platelet population. Polystyrene microspheres 0.50–1.27 μm were used for size position, and the MP gate was set as particles 0.5–1.0 μm. Whole blood was incubated with annexin V and antibodies to tissue factor (TF), platelets (CD41 and CD62P), monocyte (CD14) and endothelial cells (CD144). For comparison, MPs from platelet free supernatant was used. The TF activity was evaluated by Calibrated Automated Thrombogram. Results. Annexin V was used to distinguish true events from background noise. For standardization, each analysis included 10,000 events in the gate of platelets. There were 622(462–1001) MPannV+/10,000 platelets and of these, 66 (49–82)/10,000 platelets expressed TF. After correction for the individual platelet counts, the amount of circulating MPannV+ was 17.1 (12.1–24.9) × 109/L in whole blood, and of these, 10% (6–12%) expressed TF. The majority of the MPs expressed CD41, and 5.6% (2.2–6.9%) of these co-expressed TF. The amount of CD41 + MPannV+ tended to correlate to the TF activity in whole blood. There was no correlation between the MPannV+ in whole blood and MPs derived from platelet free supernatant. Patients with pulmonary arterial hypertension and stable coronary artery disease had increased concentrations of CD41 + MPannV+ in whole blood. Conclusion. This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.

D-dimer and risk of thromboembolic and bleeding events in patients with atrial fibrillation – observations from the ARISTOTLE trial

D‐dimer is related to adverse outcomes in arterial and venous thromboembolic diseases.

Treatment with an oral direct thrombin inhibitor decreases platelet activity but increases markers of inflammation in patients with myocardial infarction

Abstract.  Christersson C, Oldgren J, Wallentin L, Siegbahn A (Uppsala University, Uppsala, Sweden). Treatment with an oral direct thrombin inhibitor decreases platelet activity but increases markers of inflammation in patients with myocardial infarction. J Intern Med 2011; 270: 215–223.

Height, weight and body mass index in adults with congenital heart disease

BACKGROUND High BMI is a risk factor for cardiovascular disease and, in contrast, low BMI is associated with worse prognosis in heart failure. The knowledge on BMI and the distribution in different BMI-classes in adults with congenital heart disease (CHD) are limited. METHODS AND RESULTS Data on 2424 adult patients was extracted from the Swedish Registry on Congenital Heart Disease and compared to a reference population (n=4605). The prevalence of overweight/obesity (BMI ≥ 25) was lower in men with variants of the Fontan procedure, pulmonary atresia (PA)/double outlet right ventricle (DORV) and aortic valve disease (AVD) (Fontan 22.0% and PA/DORV 15.1% vs. 43.0%, p=0.048 and p<0.001) (AVD 37.5% vs. 49.3%, p<0.001). Overt obesity (BMI ≥ 30) was only more common in women with AVD (12.8% vs. 9.0%, p=0.005). Underweight (BMI<18.5) was generally more common in men with CHD (complex lesions 4.9% vs. 0.9%, p<0.001 and simple lesions 3.2% vs. 0.6%, <0.001). Men with complex lesions were shorter than controls in contrast to females that in general did not differ from controls. CONCLUSION Higher prevalence of underweight in men with CHD combined with a lower prevalence of overweight/obesity in men with some complex lesions indicates that men with CHD in general has lower BMI compared to controls. In women, only limited differences between those with CHD and the controls were found. The complexity of the CHD had larger impact on height in men. The cause of these gender differences as well as possible significance for prognosis is unknown.

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression

INTRODUCTION High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs). MATERIAL AND METHODS DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI. RESULTS In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p=0.02) and to granulocytes (p=0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p=0.02-0.002). In the MI population, the DTI reduced the TF activity (p<0.001). CONCLUSION DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.

High incidence of infective endocarditis in adults with congenital ventricular septal defect

Objective Ventricular septal defects (VSDs), if haemodynamically important, are closed whereas small shunts are left without intervention. The long-term prognosis in congenital VSD is good but patients are still at risk for long-term complications. The aim of this study was to clarify the incidence of infective endocarditis (IE) in adults with VSD. Methods The Swedish registry for congenital heart disease (SWEDCON) was searched for adults with VSD. 779 patients were identified, 531 with small shunts and 248 who had the VSD previously closed. The National Patient Register was then searched for hospitalisations due to IE in adults during a 10-year period. Results Sixteen (2%) patients were treated for IE, 6 men and 10 women, with a mean age of 46.3±12.2 years. The incidence of IE was 1.7–2.7/1000 years in patients without previous intervention, 20–30 times the risk in the general population. Thirteen had small shunts without previous intervention. There was no mortality in these 13 cases. Two patients had undergone repair of their VSD and also aortic valve replacement before the episode of endocarditis and a third patient with repaired VSD had a bicuspid aortic valve, all of these three patients needed reoperation because of their IE and one patient died. No patient with isolated and operated VSD was diagnosed with IE. Conclusions A small unoperated VSD in adults carries a substantially increased risk of IE but is associated with a low risk of mortality.

Comparison of effects on coagulation and inflammatory markers using a duty-cycled bipolar and unipolar radiofrequency pulmonary vein ablation catheter vs. a cryoballoon catheter for pulmonary vein isolation

AIMS Thrombo-embolic events are one of the most feared complications related to atrial fibrillation (AF) ablation. Since radiofrequency (RF) energy is thought to be associated with a higher risk of thrombus formation than cryoenergy, the purpose of this study was to assess if the degree of activation of coagulation and inflammatory markers differed between ablation procedures performed with a cryoballoon catheter vs. a RF energy-based pulmonary vein ablation catheter (PVAC), respectively. METHODS AND RESULTS Thirty patients referred for AF ablation were randomized to pulmonary vein isolation with either the cryoballoon or the PVAC. Biomarkers were studied for endothelial damage (von Willebrand factor antigen), platelet activation (soluble P-selectin), and coagulation activity [prothrombin fragment 1 + 2 (F1 + 2) and D-dimer] at five different time points during the procedure. Troponin I (Trop I) and C-reactive protein were analysed to reflect myocardial destruction and inflammatory activity. Markers of endothelial damage and platelet activation increased after ablation in both the cryo and the RF group. Similarly, the D-dimer levels increased significantly (P = 0.001) in both groups, whereas the F1 + 2 levels increased after the transseptal puncture only (P = 0.001). The overall activation of the coagulation system was, however, comparable between the groups. The cryoballoon was associated with higher Trop I compared with the PVAC (P < 0.001), but the ratios between biomarkers and Trop I were higher with the PVAC than with the cryoballoon. CONCLUSION Even though the cryoballoon causes a higher degree of myocardial destruction than the PVAC, markers of coagulation, endothelial damage, and inflammation were comparable between the two techniques.

How to use D-dimer in acute cardiovascular care.

D-dimer testing is important to aid in the exclusion of venous thromboembolic events (VTEs), including deep venous thrombosis and pulmonary embolism, and it may be used to evaluate suspected aortic dissection. D-dimer is produced upon activation of the coagulation system with the generation and subsequent degradation of cross-linked fibrin by plasmin. Many different assays for D-dimer testing are currently used in routine care. However, these tests are neither standardized nor harmonized. Consequently, only clinically validated assays and assay specific decision limits should be used for routine testing. For the exclusion of pulmonary embolism/deep vein thrombosis, age-adjusted cut-offs are recommend. Clinicians must be aware of the validated use of their hospital’s D-dimer assay to avoid inappropriate use of this biomarker in routine care.

Left ventricular hypertrophy in adults with previous repair of coarctation of the aorta; Association with systolic blood pressure in the high normal range

BACKGROUND Arterial hypertension is common in adults with repaired coarctation of the aorta (CoA). The associations between the diagnosis of hypertension, actual blood pressure, other factors affecting left ventricular overload, and left ventricular hypertrophy (LVH) are not yet fully explored in this population. MATERIAL AND RESULTS From the national register for congenital heart disease, 506 adult patients (≥18years old) with previous repair of CoA were identified (37.0% female, mean age 35.7±13.8years, with an average of 26.8±12.4years post repair). Echocardiographic data were available for all patients, and showed LVH in 114 (22.5%) of these. Systolic blood pressure (SBP) (mmHg) (OR 1.02, CI 1.01-1.04), aortic valve disease, (OR 2.17, CI 1.33-3.53), age (years) (OR 1.03, CI 1.01-1.05), diagnosis of arterial hypertension (OR 3.02, CI 1.81-5.02), and sex (female) (OR 0.41, CI 0.24-0.72) were independently associated with LVH. There was an association with LVH at SBP within the upper reference limits [130, 140] mmHg (OR 2.23, CI 1.05-4.73) that further increased for SBP>140mmHg (OR 8.02, CI 3.76-17.12). CONCLUSIONS LVH is common post repair of CoA and is associated with SBP even below the currently recommended target level. Lower target levels may therefore become justified in this population.

Circulating cell-derived microparticles as biomarkers in cardiovascular disease

Cardiovascular diseases (CVDs) are a common cause of death, and a search for biomarkers for risk stratification is warranted. Elevated levels of cell-derived microparticles (MPs) are found in patients with CVD and in groups with risk factors for CVD. Subpopulations of MPs are promising biomarkers for improving risk prediction, as well as monitoring treatment. However, the field has been hampered by technical difficulties, and the ongoing development of sensitive standardized techniques is crucial for implementing MP analyses in the clinic. Large prospective studies are required to establish which MPs are of prognostic value in different patient groups. In this review, we discuss methodological challenges and progress in the field, as well as MP populations that are of interest for further clinical evaluation.