SARS-CoV-2 Infection in Health Workers: Analysis from Verona SIEROEPID Study during the Pre-Vaccination Era.

BACKGROUND: To report the baseline phase of the SIEROEPID study on SARS-CoV-2 infection seroprevalence among health workers at the University Hospital of Verona, Italy, between spring and fall 2020; to compare performances of several laboratory tests for SARS-CoV-2 antibody detection. METHODS: 5299 voluntary health workers were enrolled from 28 April 2020 to 28 July 2020 to assess immunological response to SARS-CoV-2 infection throughout IgM, IgG and IgA serum levels titration by four laboratory tests. Association of antibody titre with several demographic variables, swab tests and performance tests (sensitivity, specificity, and agreement) were statistically analyzed. RESULTS: The overall seroprevalence was 6%, considering either IgG and IgM, and 4.8% considering IgG. Working in COVID-19 Units was not associated with a statistically significant increase in the number of infected workers. Cohen's kappa of agreement between MaglumiTM and VivaDiagTM was quite good when considering IgG only (Cohen's kappa = 78.1%, 95% CI 74.0-82.0%), but was lower considering IgM (Cohen's kappa = 13.3%, 95% CI 7.8-18.7%). CONCLUSION: The large sample size with high participation (84.7%), the biobank and the longitudinal design were significant achievements, offering a baseline dataset as the benchmark for risk assessment, health surveillance and management of SARS-CoV-2 infection for the hospital workforce, especially considering the ongoing vaccination campaign. Study results support the national regulator guidelines on using swabs for SARS-CoV-2 screening with health workers and using the serological tests to contribute to the epidemiological assessment of the spread of the virus.

Evaluation of three immunochromatographic tests in COVID-19 serologic diagnosis and their clinical usefulness.

Results of three rapid immunochromatographic tests (ICTs) were compared with those obtained with two automated immunoassays for evaluation of their usefulness. One hundred fifty-nine patients and 67 healthy volunteers were included. Different assays demonstrate 41-45% of diagnostic sensitivities and 91-98% of specificities, with substantial agreement (89.3-91.2%), but a high percentage of weak positive results (13-22%) was observed with ICTs. ICTs performances were comparable to those of automated immunoassays. ICTs could have a role as screening approach due to their easy usability. Subjective interpretation, significant rate of uncertain results, uncertainty on viral antigens source are undoubtedly drawbacks.

Venous stasis and whole blood platelet aggregometry: a question of data reliability and patient safety.

The assessment of platelet function by multiple electrode aggregometry (MEA) (Multiplate, Roche Diagnostics GmbH) is common in laboratory hematology. As regards the ISO 15189:2012 international standard, appropriate use of laboratory equipment requires appropriate pre-examination activities (e.g., blood collection). Venous stasis can influence several blood analytes, but the tourniquet time is rarely regarded as a source of variability. Aim of the present study was to evaluate the impact of venous stasis on platelet function by MEA. A total of 6 ml of blood was collected from 20 volunteers into two 3.0 ml Hirudin vacuum tube (Roche Diagnostics GmbH), and subjected to two procedures: procedure 1 (no stasis) - collection after localization of forearm vein by subcutaneous tissue transilluminator device without tourniquet; procedure 2 (stasis) - collection after localization of vein by prior 60 s tourniquet application. Samples were processed on Multiplate, for: ADP-test (without prostaglandin E1), ADP HS-test (with prostaglandin E1), ASPI-test, COL-test, RISTO H-test (high concentration, 0.77 mg/ml), RISTO L-test (low concentration, 0.20 mg/ml), and TRAP-test. The significance of the differences between samples was assessed by Wilcoxon ranked-pairs test. Surprisingly, the results of ADP HS-test, ASPI-test, COL-test, and RISTO H-test appeared unbiased by venous stasis. RISTO L-test, ADP-test, and TRAP-test were significantly biased; the mean percent difference between stasis and no stasis were -7.2% (P = 0.040), -28.4% (P = 0.015), and 1.1% (P = 0.031), respectively. In conclusion, the tourniquet should be avoided when assessing platelet function by MEA.

Processing of diagnostic blood specimens: is it really necessary to mix primary blood tubes after collection with evacuated tube system?

BACKGROUND: The preanalytical phase is considered the most vulnerable phase in biopreservation, biobanking, and laboratory diagnostics. Accurate mixing after blood collection is claimed to be important and recommended by the manufacturers. OBJECTIVE: To evaluate whether it is really necessary to mix the primary blood tubes immediately after blood collection by means of evacuated tube systems. MATERIAL AND METHODS: Blood from 300 outpatients was equally and randomly divided into three groups: G1, sodium citrate vacuum tubes; G2, lithium heparin vacuum tubes; and G3, K2EDTA vacuum tubes. All vacuum tubes were processed using three different procedures. Procedure 1: Gold Standard (P1): All specimens mixed gently and carefully by inverting five times as recommended; Procedure 2: Rest time (P2): All specimens remained 5 min in the upright position, followed by gentle careful mixing by inverting five times; Procedure 3: No mix (P3): All specimens were left in upright position without mixing afterwards. The influence of the primary mixing tube procedure was evaluated for clinical chemistry, hematology, and coagulation parameters by paired t-test. The bias from the mixing procedure was also compared with quality specifications derived from biological variation. RESULTS: Significant differences (p<0.017) were found for: i) red blood cell count and hematocrit when P1 was compared with P2; ii) alanine aminotransferase and erythrocyte sedimentation rate when P1 was compared with P3; iii) red blood cell count, hematocrit, and hemolysis index when P2 was compared with P3. Surprisingly, clinically significant differences were found only for sodium when P1 was compared with P2, and P1 was compared with P3. No fibrin filaments or microclots were observed in any samples. CONCLUSION: Primary blood tubes mixing after collection with evacuated tube system appears to be unnecessary.

Rapid stimulation of tyrosine phosphorylation signals downstream of G-protein-coupled receptors for thromboxane A2 in human platelets.

Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-27632. Although low doses of U46619 or adrenaline alone were unable to trigger platelet aggregation and integrin alphaIIbbeta3 activation, the combination of the two stimuli effectively induced these responses. PP2, a tyrosine kinase inhibitor, and Y-27632 inhibited platelet activation induced by low doses of U46619 plus adrenaline and, when used in combination, totally suppressed this platelet response. In addition, the two inhibitors selectively blocked tyrosine kinases and the Rho/Rho-kinase pathway respectively. These findings suggest that both tyrosine phosphorylation and the Rho/Rho-kinase pathway are required to activate platelet aggregation via G(12/13) plus G(z) signalling.

Acute hyperhomocysteinemia induces a reduction in arterial distensibility and compliance.

OBJECTIVE: The aim of the study was to evaluate the effects of acute hyperhomocysteinemia on distensibility and compliance of large peripheral arteries. Isoprostanes generation and antioxidant vitamins were used to assess the role of oxidative stress. DESIGN: A cross-over, double-blind study on distensibility (DC: distensibility coefficient) and compliance (CC: cross-sectional compliance) of common femoral and brachial arteries was performed in 12 healthy young male volunteers by means of a wall track system before and 4 h after a single oral methionine (100 mg/kg) or placebo administration. The effects of methionine load were investigated also after oral administration of vitamin C (1g/day) and vitamin E (800 mg/day) for 8 consecutive days. RESULTS: Oral methionine induced a significant increase in plasmatic levels of homocysteine. Distensibility and compliance of brachial and femoral arteries were significantly reduced after methionine load in comparison to placebo. This acute impairment of arterial wall mechanical properties was associated to endothelial dysfunction, since altered flow-dependent vasodilatation (P < 0.05 versus placebo) was observed in the same arterial districts. A significant increase in urinary 8-iso-prostaglandin F2alpha was observed after methionine. Pretreatment with vitamins C and E prevented the effects of methionine on femoral and brachial arteries as well as on urinary 8-iso-prostaglandin F2alpha excretion. CONCLUSIONS: Hyperhomocysteinemia seems responsible for altered arterial wall elasticity and for endothelial dysfunction. A pivotal role can be attributed to oxidative stress.

Determinants of platelet activation in human essential hypertension.

Experimental data suggest that oxidative stress might be enhanced in hypertension and contribute to platelet activation. We hypothesized that both oxidative stress and platelet activation could be related to the clinical characteristics of hypertensive patients. The urinary excretion of 11-dehydrothromboxane (TX) B2, reflecting in vivo platelet activation, was measured in 75 patients with mild to severe essential hypertension and 75 pair-matched, healthy controls. The urinary excretion of 8-iso-prostaglandin (PG) F2alpha was determined as an index of in vivo lipid peroxidation. Urinary 11-dehydro-TXB2 was significantly higher in essential hypertensives compared with controls. Although no statistically significant difference in urinary 8-iso-PGF2alpha was observed between patients and controls, plasma vitamin C was lower and plasma homocysteine higher in hypertensive patients than in controls. Both urinary 11-dehydro-TXB2 and 8-iso-PGF2alpha were higher in patients with advanced hypertensive retinopathy compared with patients without retinopathy. Multivariate linear regression analysis identified urinary 8-iso-PGF2alpha, plasma fibrinogen, homocysteine, and vitamin E as the only variables independently correlated with urinary 11-dehydro-TXB2. Logistic regression analysis showed that high urinary 8-iso-PGF2alpha, plasma fibrinogen, and homocysteine, as well as low plasma vitamin E, advanced retinopathy, elevated diastolic blood pressure, and the absence of antihypertensive treatment, were predictors of high urinary 11-dehydro-TXB2. We demonstrated increased oxidative stress and persistent platelet activation in essential hypertensives with advanced vascular lesions. These findings might help identify hypertensive patients who are at increased risk of cardiovascular events and who might benefit from long-term antiplatelet therapy.

Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease.

BACKGROUND: Hypertensive patients with renovascular disease (RVD) may be exposed to increased oxidative stress, possibly related to activation of the renin-angiotensin system. METHODS AND RESULTS: We measured the urinary excretion of 8-iso-prostaglandin (PG) F2alpha and 11-dehydro-thromboxane (TX) B2 as indexes of in vivo lipid peroxidation and platelet activation, respectively, in 25 patients with RVD, 25 patients with essential hypertension, and 25 healthy subjects. Plasma renin activity in peripheral and renal veins, angiotensin II in renal veins, cholesterol, glucose, triglycerides, homocysteine, and antioxidant vitamins A, C, and E were also determined. Patients were also studied 6 months after a technically successful angioplasty of the stenotic renal arteries. Urinary 8-iso-PGF2alpha was significantly higher in patients with RVD (median, 305 pg/mg creatinine; range, 124 to 1224 pg/mg creatinine) than in patients with essential hypertension (median, 176 pg/mg creatinine; range, 48 to 384 pg/mg creatinine) or in healthy subjects (median, 123 pg/mg creatinine; range, 58 to 385 pg/mg creatinine). Urinary 11-dehydro-TXB2 was also significantly higher in RVD patients compared with healthy subjects. In RVD patients, urinary 8-iso-PGF2alpha correlated with 11-dehydro-TXB2 (r(s)=0.48; P<0.05) and renal vein renin (r(s)=0.67; P<0.005) and angiotensin II (r(s)=0.65; P=0.005) ratios. A reduction in 8-iso-PGF2alpha after angioplasty was observed in RVD patients with high baseline levels of lipid peroxidation. Changes in 8-iso-PGF2alpha were related to baseline lipid peroxidation (r(s)=-0.73; P<0.001), renal vein angiotensin II (r(s)=-0.70; P<0.01) and renin (r(s)=-0.63; P<0.05) ratios. CONCLUSIONS: Lipid peroxidation is markedly enhanced in hypertensive patients with RVD and is related to activation of the renin-angiotensin system. Moreover, persistent platelet activation triggered or amplified by bioactive isoprostanes may contribute to the progression of cardiovascular and renal damage in this setting.

Functional role of p38 mitogen activated protein kinase in platelet activation induced by a thromboxane A2 analogue and by 8-iso-prostaglandin F2alpha.

We investigated similarities in the signaling pathways elicited by the F2 isoprostane 8-iso-PGF2alpha and by low doses of U46619 to induce platelet activation. Both 0.01-0.1 micromol/L U46619 and 0.01-1 micromol/L 8-iso-PGF2alpha triggered shape change and filopodia extension, as well as adhesion to immobilized fibrinogen of washed platelets. At these doses the two platelet agonists failed to trigger secretion and aggregation, which were however induced by higher doses of U46619 (0.1-1 micromol/L). SB203580 (1-10 micromol/L), a specific inhibitor of the p38 mitogen activated protein (MAP) kinase blunted platelet shape change and adhesion induced by 0.05-1 micromol/L 8-iso-PGF2alpha and by 0.01 micromol/L U46619. These platelet responses were also inhibited by 20 micromol/L cytochalasin D, an inhibitor of actin polymerization, and 50 micromol/L piceatannol, an inhibitor of the Syk tyrosine kinases. Both 8-iso-PGF2alpha and U46619-induced p38 MAP kinase phosphorylation in suspended platelets and this was inhibited by piceatannol, indicating that Syk activation occurs upstream of p38 MAP kinase phosphorylation. These findings suggest that the signaling pathway triggered by both 8-iso-PGFalpha and low concentrations of U46619 to induce platelet adhesion and shape change implicates Syk, the p38 MAP kinase, and actin polymerization.