The alternative Thomas-plot: A new tool for effective anemia diagnostics.

INTRODUCTION: The Thomas-plot has proven to be a helpful tool to discriminate between different types of anemia. This plot combines the reticulocyte hemoglobin content (Ret-He) with the soluble transferrin receptor (sTfR)/log ferritin (fer) ratio. In this study, we designed an alternative Thomas-plot in which Ret-He is combined with the transferrin (Tf)/log ferritin ratio. We validated both Thomas-plots in a population of anemic patients and compared the performance to the current laboratory diagnostics of anemia. METHODS: A total of 536 anemic patients were included. The first 188 patients were used to generate ROC curves to define the optimal cut-off values for both Thomas-plots. With the following 348 patients included we studied the performance of the alternative and classical Thomas-plots compared to current anemia diagnostics. RESULTS: Cut-off values were defined (Ret-He: 31.2 pg, sTfR/log(fer): 0.91, and Tf/log(fer): 1.71). With both Thomas-plots the amount of e causa ignota (ECI) cases dropped from 39% to 27%. A more in depth analysis on the iron status of anemia of chronic disease (ACD) patients and a subdivision between latent and classical iron deficiencies could be made with the help of both plots. A shift from classical iron deficiency anemia (IDA) cases according to the classical Thomas-plot toward functional IDA according to the alternative Thomas-plot was observed. CONCLUSION: The alternative Thomas-plot is an effective tool that gives a more in depth view on the iron status of anemic patients. In addition, it is easier to implement due to the use of transferrin rather than the soluble transferrin receptor.

Chest CT for triage during COVID-19 on the emergency department: myth or truth?

PURPOSE: We aimed to investigate the diagnostic performance of chest CT compared with first RT-PCR results in adult patients suspected of COVID-19 infection in an ED setting. We also constructed a predictive machine learning model based on chest CT and additional data to improve the diagnostic accuracy of chest CT. METHODS: This study's cohort consisted of 319 patients who underwent chest CT and RT-PCR testing at the ED. Patient characteristics, demographics, symptoms, vital signs, laboratory tests, and chest CT results (CO-RADS) were collected. With first RT-PCR as reference standard, the diagnostic performance of chest CT using the CO-RADS score was assessed. Additionally, a predictive machine learning model was constructed using logistic regression. RESULTS: Chest CT, with first RT-PCR as a reference, had a sensitivity, specificity, PPV, and NPV of 90.2%, 88.2%, 84.5%, and 92.7%, respectively. The prediction model with CO-RADS, ferritin, leucocyte count, CK, days of complaints, and diarrhea as predictors had a sensitivity, specificity, PPV, and NPV of 89.3%, 93.4%, 90.8%, and 92.3%, respectively. CONCLUSION: Chest CT, using the CO-RADS scoring system, is a sensitive and specific method that can aid in the diagnosis of COVID-19, especially if RT-PCR tests are scarce during an outbreak. Combining a predictive machine learning model could further improve the accuracy of diagnostic chest CT for COVID-19. Further candidate predictors should be analyzed to improve our model. However, RT-PCR should remain the primary standard of testing as up to 9% of RT-PCR positive patients are not diagnosed by chest CT or our machine learning model.

Progression of subclinical atherosclerosis in subjects with rheumatoid arthritis and the metabolic syndrome.

BACKGROUND AND AIMS: Rheumatoid arthritis (RA) has been associated with an increased risk of atherosclerosis. We aimed to evaluate the progression of carotid intima media thickness (cIMT) in RA patients subject to a cardiovascular treat-to-target intervention. In addition, the presence of the metabolic syndrome (MetS) on cIMT outcomes was evaluated. METHODS: We performed a cohort analysis of FRANCIS, in which RA patients ≤70 years without CVD or diabetes mellitus were randomized for either a treat-to-target intervention or usual care concerning CVD risk factors. MetS was scored at baseline. RESULTS: Three-year data was available in 212 well-controlled RA patients. The treat-to-target intervention resulted in a lower cIMT progression over three years compared to the usual care. However, there was no difference in cIMT at three years between groups. MetS was present in 40.1% of RA patients. Baseline cIMT was significantly higher in RA patients with MetS compared to those without (0.619 (0.112) versus 0.557 (0.104) mm; p < 0.001). After three years, cIMT progression was comparable (0.043 (0.071) versus 0.043 (0.072) mm; p = 0.96). In RA patients with MetS, the presence of plaques increased over three years from 12.9% to 23.5% (p = 0.01). The type of intervention had no effect on cIMT progression in RA patients with MetS. However, in subjects without MetS, treat-to-target resulted in a lower progression. CONCLUSIONS: RA patients with MetS showed an increased CVD risk profile based on both a higher prevalence of CVD risk factors and structural vascular changes. A treat-to-target approach of CVD risk factors reduced cIMT progression only in RA patients without MetS.

Effect of a Single Dose of Vitamin D3 on Postprandial Arterial Stiffness and Inflammation in Vitamin D-Deficient Women.

CONTEXT: Cholecalciferol (vitamin D3) improves vascular function and inflammation, potentially providing an explanation for the proposed cardiovascular protection of vitamin D. OBJECTIVE: We investigated whether cholecalciferol supplementation reduces postprandial arterial dysfunction and inflammation. DESIGN: Randomized, 1:1, double-blind trial. SETTING: Diabetes and Vascular Center, Franciscus Gasthuis, Rotterdam, The Netherlands. PATIENTS: Twenty-four healthy, premenopausal, overweight or obese, vitamin D-deficient women. INTERVENTIONS: A single high (300,000 IU) or low dose (75,000 IU) of cholecalciferol. MAIN OUTCOME MEASURES: The effect of low- and high-dose cholecalciferol on postprandial leukocyte activation markers, pulse wave velocity (PWV), and augmentation index (AIx) during an oral fat loading test, expressed as area under the curve (AUC). RESULTS: High- and low-dose supplementation increased vitamin D by 163% ± 134% (P < 0.001) and 66% ± 59% (P < 0.001), respectively. Monocyte CD11b-AUC slightly increased after low but not high dose (6% ± 2%, P = 0.012, and 4% ± 1%, P = 0.339, respectively). There were no significant effects on postprandial PWV or AIx by high- or low-dose vitamin D. Fasting complement component 3 (C3) levels decreased by 5.9% (P = 0.004) in the high-dose group and by 4.0% (P = 0.018) in the low-dose group. CONCLUSION: A single dose of vitamin D does not seem to reduce arterial stiffness and leukocyte activation in overweight, vitamin D-deficient women. Vitamin D may decrease fasting C3. Possibly, higher vitamin D concentrations may be needed to decrease inflammation and improve vascular function in overweight or obese vitamin D-deficient women.

Leucocyte-bound apolipoprotein B in the circulation is inversely associated with the presence of clinical and subclinical atherosclerosis.

BACKGROUND: Atherosclerosis is a pro-inflammatory condition, in which leucocyte activation plays an important role. The interaction between circulating leucocytes and apolipoprotein (apo) B-containing lipoproteins results in pro-inflammatory changes of these cells. We aimed to evaluate the relationship between apo B bound to circulating leucocytes and atherosclerosis. METHODS: Apo B on circulating leucocytes was measured by flow cytometry in subjects with and without cardiovascular disease (CVD), expressed as mean fluorescent intensity in arbitrary units (au). Carotid intima-media thickness (cIMT) was measured using B-mode ultrasound. Data are given as median (interquartile range). RESULTS: A total of 396 subjects were included, of whom 183 had a history of CVD. Compared to subjects without CVD, patients with CVD had lower apo B bound to neutrophils (12·7 au (9·8-16·2) and 14·2 au (10·1-17·5), respectively, P = 0·038) and to monocytes (2·5 au (1·7-3·1) and 2·7 (1·9-3·6) au, respectively, P = 0·025). No differences were found for lymphocyte-bound apo B. Neutrophil- and monocyte-bound apo B were inversely correlated with cIMT (Spearman's rho: -0·123, P = 0·017 and -0·108, P = 0·035, respectively). Both monocyte- and neutrophil-bound apo B were inversely associated with different factors related to the metabolic syndrome, such as body mass index, triglycerides and complement C3. There was a positive association between erythrocyte-bound apo B and apo B bound to each of the leucocyte classes, possibly reflecting a similar mechanism. Discontinuation of statins in 54 subjects did not influence leucocyte-bound apo B. CONCLUSION: Unexpectedly, the presence of noninternalized apo B-containing lipoproteins on circulating neutrophil and monocyte membranes may represent a protective mechanism against atherosclerosis.

In vivo evidence for chylomicrons as mediators of postprandial inflammation.

BACKGROUND AND AIMS: The postprandial situation is a pro-inflammatory condition most likely linked to the development of atherosclerosis. We evaluated the relationship between apolipoprotein (apo) B48 and fasting and postprandial leukocyte activation markers. METHODS: Leukocyte activation markers and apo B48 were determined in 80 subjects with and without coronary artery disease (CAD). Twelve healthy subjects underwent an oral fat loading test (up to 8 h). RESULTS: Fasting apo B48 was significantly higher in patients with CAD (n = 47, 8.1 ± 5.2 mg/L) than in subjects without CAD (n = 33, 5.9 ± 3.9 mg/L, p = 0.022). Fasting apo B48 and triglycerides correlated positively with fasting monocyte CD11b and neutrophil CD66b expression. Plasma apo B48 and leukocyte activation markers increased after an oral fat load. No correlations were found between fasting or postprandial triglycerides and postprandial leukocyte activation markers. We observed no correlations between postprandial apo B48 and postprandial neutrophil CD11b or CD66b expression. CONCLUSION: This study suggests that chylomicron remnants may be responsible for postprandial leukocyte activation in the circulation. The postprandial chylomicron response may be a stronger mediator of postprandial inflammation than postprandial triglyceridemia.

Immature platelet fraction measured on the Sysmex XN hemocytometer predicts thrombopoietic recovery after autologous stem cell transplantation.

OBJECTIVES: A period of thrombocytopenia is common after stem cell transplantation (SCT). To prevent serious bleeding complications, prophylactic platelet transfusions are administered. Previous studies have shown that a rise in immature platelets precedes recovery of platelet count. Our aim was to define a cutoff value for immature platelets predicting thrombopoietic recovery within 2 d. METHODS: Hematological parameters were measured on the Sysmex XN hemocytometer. We calculated reference change values (RCV) for platelets in eight healthy individuals as marker for platelet recovery. To define a cutoff value, we performed ROC analysis using data from 16 autologous SCT patients. RESULTS: RCV for platelet concentration was 14.1%. Platelet recovery was observed 13 (median; range 9-31) days after SCT. Increase in immature platelet fraction (IPF) before platelet recovery was seen in all autologous SCT patients. Optimal cutoff IPF was found to be 5.3% for platelet recovery within 2 d (specificity 0.98, sensitivity 0.47, positive predictive value 0.93). CONCLUSIONS: We identified an optimal cutoff value for IPF 5.3% to predict platelet recovery after autologous SCT within 2 d. Implementing this cutoff value in transfusion strategy may reduce the number of prophylactic platelet transfusions.

[The diagnostic benefits of lipase values in acute pancreatitis]. / Diagnostische waarde lipase bij acute pancreatitis.

Serum lipase is a biochemical marker used for diagnosing acute pancreatitis. Lipase has largely replaced amylase in terms of diagnostic value, although both markers are still commonly used. In this publication, we discuss the diagnostic superiority of lipase compared to amylase in acute pancreatitis. Two cases are discussed; both patients presented with elevated lipase values but due to different causes. The first patient, who had recently experienced cholecystitis, presented to the emergency room with abdominal pain and high lipase levels. The second patient, who had received a renal transplant a few years earlier, visited the gastroenterological outpatient clinic with abdominal pain and only a slightly elevated lipase level.

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

In vivo high-resolution structural imaging of large arteries in small rodents using two-photon laser scanning microscopy.

In vivo (molecular) imaging of the vessel wall of large arteries at subcellular resolution is crucial for unraveling vascular pathophysiology. We previously showed the applicability of two-photon laser scanning microscopy (TPLSM) in mounted arteries ex vivo. However, in vivo TPLSM has thus far suffered from in-frame and between-frame motion artifacts due to arterial movement with cardiac and respiratory activity. Now, motion artifacts are suppressed by accelerated image acquisition triggered on cardiac and respiratory activity. In vivo TPLSM is performed on rat renal and mouse carotid arteries, both surgically exposed and labeled fluorescently (cell nuclei, elastin, and collagen). The use of short acquisition times consistently limit in-frame motion artifacts. Additionally, triggered imaging reduces between-frame artifacts. Indeed, structures in the vessel wall (cell nuclei, elastic laminae) can be imaged at subcellular resolution. In mechanically damaged carotid arteries, even the subendothelial collagen sheet (approximately 1 microm) is visualized using collagen-targeted quantum dots. We demonstrate stable in vivo imaging of large arteries at subcellular resolution using TPLSM triggered on cardiac and respiratory cycles. This creates great opportunities for studying (diseased) arteries in vivo or immediate validation of in vivo molecular imaging techniques such as magnetic resonance imaging (MRI), ultrasound, and positron emission tomography (PET).

Molecular MRI of early thrombus formation using a bimodal alpha2-antiplasmin-based contrast agent.

OBJECTIVES: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal alpha(2)-antiplasmin-based contrast agent (CA). BACKGROUND: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links alpha(2)-antiplasmin to fibrin, indicating the potential of alpha(2)-antiplasmin-based CAs in the detection of early thrombus formation. METHODS: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an alpha(2)-antiplasmin-based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of alpha(2)-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. RESULTS: In vitro-generated thrombi exposed to the alpha(2)-antiplasmin-based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the alpha(2)-antiplasmin-based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the alpha(2)-antiplasmin-based CA bound to fibrin. Immunohistochemistry demonstrated substantial alpha(2)-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 +/- 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs -0.14 +/- 0.55, p = 0.003, n = 6) and alpha(2)-antiplasmin-based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 +/- 0.23, p = 0.006, n = 6). CONCLUSIONS: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.

Nanoparticles for optical molecular imaging of atherosclerosis.

Molecular imaging contributes to future personalized medicine dedicated to the treatment of cardiovascular disease, the leading cause of mortality in industrialized countries. Endoscope-compatible optical imaging techniques would offer a stand-alone alternative and high spatial resolution validation technique to clinically accepted imaging techniques in the (intravascular) assessment of vulnerable atherosclerotic lesions, which are predisposed to initiate acute clinical events. Efficient optical visualization of molecular epitopes specific for vulnerable atherosclerotic lesions requires targeting of high-quality optical-contrast-enhancing particles. In this review, we provide an overview of both current optical nanoparticles and targeting ligands for optical molecular imaging of atherosclerotic lesions and speculate on their applicability in the clinical setting.

Two-photon lifetime imaging of fluorescent probes in intact blood vessels: a window to sub-cellular structural information and binding status.

Fluorescence lifetime imaging (FLIM) provides a complementary contrast mechanism to fluorescence intensity and ratio imaging in intact tissue. With FLIM the time-resolved decay in fluorescence intensity of (interacting) fluorophores can be quantified by means of time correlated single photon counting (TCSPC). Here we focus on fluorescence lifetime imaging in intact blood vessels. Requisites for imaging in intact tissue are good penetration depth and limited tissue damage. Therefore, in this pilot-study, we performed TCSPC-FLIM using two-photon laser scanning microscopy to determine, with sub-cellular resolution, the fluorescence lifetime of two fluorescent probes. First, we focused on the nucleic acid dye SYTO41 in the various compartments of cells in vitro and in situ in the wall of intact mouse carotid arteries. Second, it was assessed whether the interaction of the lectin WGA-FITC with the endothelial glycocalyx affects its fluorescence lifetime. Results showed comparable mono-exponential fluorescence lifetimes of SYTO41 in the nuclei of cells in vitro and in situ. The slightly shorter fluorescence lifetime observed in the cytoplasm allowed discrimination of the nuclei. SYTO41 displayed strong mitochondrial staining, as was verified by the mitochondrion-specific probe CMXRos. In addition, mitochondrial staining by SYTO41 was accompanied by a green shift in emission. In the mitochondrial region, SYTO41 showed a highly bi-exponential and relatively fast decay, with two distinct lifetime components. It is hypothesized that the fitted bi-exponential decay can either be contributed to (1) the mathematical approximation of the fluorescence intensity decay or (2) the presence of free and DNA-bound SYTO41 in the mitochondrial compartment, leading to two lifetime components. The fluorescence lifetime of WGA-FITC decreased by approximately 25% upon binding to the endothelial glycocalyx. From this study, we conclude that FLIM offers an additional contrast mechanism in imaging intact tissue and provides information on binding status between a probe and its ligand.

Optical and magnetic resonance imaging of cell death and platelet activation using annexin a5-functionalized quantum dots.

A quantum-dot-based nanoparticle is presented, allowing visualization of cell death and activated platelets with fluorescence imaging and MRI. The particle exhibits intense fluorescence and a large MR relaxivity (r1) of 3000-4500 mM-1 s-1 per nanoparticle due to a newly designed construct increasing the gadolinium-DTPA load. The nanoparticle is suitable for both anatomic and subcellular imaging of structures in the vessel wall and is a promising bimodal contrast agent for future in vivo imaging studies.