Analysis of C-reactive protein from finger stick dried blood spot to predict high risk of cardiovascular disease

C-reactive protein (CRP) is an acute-phase protein involved in inflammation. Furthermore, CRP is an important biomarker used in diagnostics to predict risk of cardiovascular disease (CVD) in addition to monitoring bacterial and viral infections. To measure plasma CRP, venipuncture is still necessitated and has to be performed by trained phlebotomists. As a solution, dried blood spots (DBS) are used for minimally invasive at-home sampling of blood and can be send to diagnostic laboratories by regular mail. In this study, we included 53 patients that presented to the outpatient clinic of the University Medical Center Utrecht. Capillary finger stick was used to spot blood on a filter paper card and allowed to dry. After extraction of DBS, CRP was analyzed on an automated high-throughput chemistry analyzer. Additional validation steps regarding stability, effect of hematocrit, precision, and limits of blank and quantitation were conducted according to corresponding Clinical and Laboratory Standards Institute standards. An excellent regression analysis of R2 (95% confidence interval) = 0.986 (0.982–0.989) was found. This enabled correct classification for high CVD risk of all 25 cases with sensitivity (95% CI) of 1.00 (1.00–1.00) and specificity (95% CI) of 0.96 (0.89–1.03) and correct diagnosis of inflammation of 12/13 cases with sensitivity (95% CI) of 0.92 (0.77–1.07) and specificity (95% CI) of 1.00 (1.00–1.00). Furthermore, CRP was found to be stable for 31 days and observed hematocrit variation amongst patients was clinically acceptable. CRP from DBS can be accurately measured on an automated high-throughput chemistry analyzer and used to diagnose inflammation and classify high CVD risk. This method enables individuals to engage in at-home sampling of blood on DBS for (tele)diagnostics, screening programs, patient follow-up, and medication management

Relation of arterial geometry to luminal narrowing and histologic markers for plaque vulnerability: the remodeling paradox

AbstractObjective. To relate local arterial geometry with markers that are thought to be related to plaque rupture.Background. Plaque rupture often occurs at sites with minor luminal stenosis and has retrospectively been characterized by colocalization of inflammatory cells. Recent studies have demonstrated that luminal narrowing is related with the mode of atherosclerotic arterial remodeling.Methods. We obtained 1,521 cross section slices at regular intervals from 50 atherosclerotic femoral arteries. Per artery, the slices with the largest and smallest lumen area, vessel area and plaque area were selected for staining on the presence of macrophages (CD68), T-lymphocytes (CD45RO), smooth muscle cells (alpha-actin) and collagen.Results. Inflammation of the cap or shoulder of the plaque was observed in 33% of all cross sections. Significantly more CD68 and CD45RO positive cells, more atheroma, less collagen and less alpha-actin positive staining was observed in cross sections with the largest plaque area and largest vessel area vs. cross sections with the smallest plaque area and smallest vessel area, respectively. No difference in the number of inflammatory cells was observed between cross sections with the largest and smallest lumen area.Conclusion. Intraindividually, pathohistologic markers previously reported to be related to plaque vulnerability were associated with a larger plaque area and vessel area. In addition, inflammation of the cap and shoulder of the plaque was a common finding in the atherosclerotic femoral artery

Potential of mesenchymal- and cardiac progenitor cells for therapeutic targeting of B-cells and antibody responses in end-stage heart failure

Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF

Lowering Low-Density Lipoprotein Particles in Plasma Using Dextran Sulphate Co-Precipitates Procoagulant Extracellular Vesicles

Plasma extracellular vesicles (EVs) are lipid membrane vesicles involved in several biological processes including coagulation. Both coagulation and lipid metabolism are strongly associated with cardiovascular events. Lowering very-low- and low-density lipoprotein ((V)LDL) particles via dextran sulphate LDL apheresis also removes coagulation proteins. It remains unknown, however, how coagulation proteins are removed in apheresis. We hypothesize that plasma EVs that contain high levels of coagulation proteins are concomitantly removed with (V)LDL particles by dextran sulphate apheresis. For this, we precipitated (V)LDL particles from human plasma with dextran sulphate and analyzed the abundance of coagulation proteins and EVs in the precipitate. Coagulation pathway proteins, as demonstrated by proteomics and a bead-based immunoassay, were over-represented in the (V)LDL precipitate. In this precipitate, both bilayer EVs and monolayer (V)LDL particles were observed by electron microscopy. Separation of EVs from (V)LDL particles using density gradient centrifugation revealed that almost all coagulation proteins were present in the EVs and not in the (V)LDL particles. These EVs also showed a strong procoagulant activity. Our study suggests that dextran sulphate used in LDL apheresis may remove procoagulant EVs concomitantly with (V)LDL particles, leading to a loss of coagulation proteins from the blood

Increased circulating IgG levels, myocardial immune cells and IgG deposits support a role for an immune response in pre- and end-stage heart failure

The chronic inflammatory response plays an important role in adverse cardiac remodelling and the development of heart failure (HF). There is also evidence that in the pathogenesis of several cardiovascular diseases, chronic inflammation is accompanied by antibody and complement deposits in the heart, suggestive of a true autoimmune response. However, the role of antibody-mediated immune responses in HF progression is less clear. We assessed whether immune cell infiltration and immunoglobulin levels are associated with HF type and disease stage, taking sex differences into account. We found IgG deposits and increased infiltration of immune cells in the affected myocardium of patients with end-stage HF with reduced ejection fraction (HFrEF, n = 20). Circulating levels of IgG1 and IgG3 were elevated in these patients. Furthermore, the percentage of transitional/regulatory B cells was decreased (from 6.9% to 2.4%) compared with healthy controls (n = 5). Similarly, increased levels of circulating IgG1 and IgG3 were observed in men with left ventricular diastolic dysfunction (LVDD, n = 5), possibly an early stage of HF with preserved EF (HFpEF). In conclusion, IgG deposits and infiltrates of immune cells are present in end-stage HFrEF. In addition, both LVDD patients and end-stage HFrEF patients show elevated levels of circulating IgG1 and IgG3, suggesting an antibody-mediated immune response upon cardiac remodelling, which in the early phase of remodelling appear to differ between men and women. These immunoglobulin subclasses might be used as marker for pre-stage HF and its progression. Future identification of auto-antigens might open possibilities for new therapeutic interventions

Dysregulated innate and adaptive immune responses discriminate disease severity in COVID-19

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive pro-inflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis reveals no specific inflammatory endotypes in COVID-19 patients. Functional assays reveal abrogated adaptive cytokine production (interferon-gamma, interleukin-17 and interleukin-22) and prominent T cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlight potential biomarkers of disease severity

The contribution of plaque and arterial remodeling to de novo atherosclerotic luminal narrowing in the femoral artery

AbstractBackground: Atherosclerotic luminal narrowing is caused by plaque growth and arterial remodeling. In peripheral arteries, a role for constrictive remodeling in luminal narrowing has been recognized, but the impact on lumen decrease has not yet been assessed. We studied to what extent arterial remodeling and plaque formation contribute to luminal narrowing in the superficial femoral artery. Methods: Elderly subjects (n = 79) were studied. Post mortem, pressure-fixed femoral arteries (n = 125) were dissected and divided into 0.5-cm segments (n = 3266). In each cross section, we measured lumen area, plaque area, and the area encompassed by the internal elastic lamina (IEL area). For each artery, the cross section with the least amount of plaque was considered the reference segment. In cross sections with a decrease in lumen area compared with the reference, we determined the contributions of both plaque increase and IEL area change. Results: A decrease in lumen area was found in 2193 cross sections. In cross sections with >50% lumen stenosis, plaque increase (accompanied by IEL area increase) fully explained lumen decrease in 80 of 280 cross sections (29%). In the remaining 200 of 280 cross sections (71%), both plaque increase and IEL area decrease contributed to lumen stenosis. In 57 of 280 cross sections (20%), IEL area decrease was the major determinant of lumen decrease, dominating over plaque increase. In 143 of 280 cross sections (51%), plaque increase was the major determinant, dominating over IEL area decrease. Conclusion: The results of this post mortem study suggest that in a substantial part (20%) of severely stenotic lesions in the femoral artery, constrictive remodeling, not plaque size, is the major determinant of lumen decrease. Further serial studies are needed to confirm these results. (J Vasc Surg 2002;36:1194-7.

Morphometric and immunohistochemical characterization of the intimal layer throughout the arterial system of elderly humans

The purpose of the present study was to obtain insight into the natural development of adaptive intimal thickening and atherosclerosis in the arterial tree of human species. The morphometry and composition of the intimal layer were studied in the arterial system of elderly individuals. Post mortem, a total of 703 arterial segments were dissected from 24 subjects (age 81.9 ± 9.9 years). From each subject, segments were dissected from 31 different arteries. Area stenosis [(plaque area/vessel area) × 100%] was determined in each segment. By (immuno)histochemistry, lipid content and the presence of inflammatory cells (macrophages) were assessed in the coronary, common carotid, brachial, radial and internal iliac arteries. Adaptive intimal thickening or advanced atherosclerosis was observed in all studied artery types. Area stenosis was highest in the coronary arteries (median, 30%) and lowest in the arteries supplying the brain (median, ≤ 7%). Plaques hiding a lipid-rich core and plaques with macrophage infiltration were observed in all five selected artery types. In summary, the present observation demonstrates that intimal thickening is a systemic process involving most artery types. Within elderly humans, features of advanced atherosclerotic disease, a lipid-rich core and macrophages, can be observed in the intimal layer of artery types that are recognised for their relation with clinical syndroms as well as artery types that remain clinically silent

The influence of apical periodontitis on circulatory inflammatory mediators in peripheral blood: A prospective case–control study

Aim: To explore the influence of apical periodontitis (AP) on inflammatory markers in blood of otherwise healthy individuals and to depict the inflammatory profile of the healing after dental extraction. Methodology: This is a prospective case–control intervention study, during which, individuals with a diagnosis of AP of one affected tooth were included, along with a control group matched for age and gender. A broad panel of blood inflammatory mediators was examined longitudinally in all subjects during six visits. In the case of the AP subjects, the tooth with AP was extracted at the third visit. Results were analysed by linear regression analyses and linear mixed-model analyses. Results: A total of 53 subjects were included in the study, 27 with AP and 26 without. Fifteen females and 12 males were included in the AP group, and 14 females and 12 males in the control group. At baseline, granulocyte colony-stimulating factor (p <.001), interleukin (IL)-1β (p =.03) and IL-4 (p =.01) were significantly lower in AP subjects than in controls. Comparison of the differences between baseline and the last visit, i.e. 3 months after the tooth extraction, showed a significant reduction in IL-10 (p =.03) and IL-12p70 (p =.01). Conclusions: The immunologic profile of chronic AP in one tooth and its healing profile reveals a systemic low-grade inflammation through compensatory immunosuppression. A larger lesion or multiple lesions could disrupt the balance that the system is trying to maintain, resulting in loss of homeostasis

A Preliminary Analysis of Thyrotropin Measurement from Finger Stick Dried Blood Spot with an Automated High-Throughput Immunoassay Analyzer

Background: Hyper- and hypothyroidism are prevalent in Western countries and often go unnoticed for long periods. Thyrotropin (TSH) as a biomarker of thyroid dysfunction is regularly measured in venous plasma/serum. In newborn screening for congenital hypothyroidism, TSH is measured from dried blood spots (DBSs). DBS enables minimally invasive (at-home) sampling of a small blood volume that can be sent to diagnostic laboratories by regular mail. Methods: In this study, we included 109 patients who presented to the outpatient clinic of the University Medical Center Utrecht. Capillary finger stick was used to spot blood on a filter paper card and was dried. After extraction of TSH from DBS, method comparison with venous TSH was performed on an automated high-throughput immunoassay analyzer. Additional validation steps regarding stability, effect of hematocrit (Hct), precision, and limits of blank and quantitation were conducted according to corresponding Clinical and Laboratory Standards Institute evaluation protocol. Results: Method comparison of TSH from venous plasma versus finger stick DBSs showed an R2 [95% confidence interval] = 0.988 [0.986-0.990]. This enabled correct diagnosis of hypothyrotropinemia and hypothyroidism in 12 of 14 and 6 of 7 cases, respectively, with no false positives. Furthermore, TSH from DBS was stable for at least 4 days at temperatures between -20°C and +30°C, and the maximum decrease of eluate TSH was 1.13% for 1% increase in Hct. Conclusions: TSH from DBS may be accurately measured on an automated high-throughput immunoassay analyzer and could be used to diagnose hypothyroidism and, for the first time, hypothyrotropinemia. This method, when confirmed in larger field studies, may enable individuals to engage in (at-home) sampling of blood on DBSs for telediagnostics, screening programs, patient follow-up, and medication management