Digital droplet PCR versus quantitative PCR for lipoprotein (a) kringle IV type 2 repeat polymorphism genetic characterization.

BACKGROUND: Lipoprotein(a) [Lp(a)] level variability, related to atherothrombotic risk increase, is mainly attributed to LPA gene, encoding apolipoprotein(a), with kringle IV type 2 (KIV2) copy number variation (CNV) acting as the primary genetic determinant. Genetic characterization of Lp(a) is in continuous growth; nevertheless, the peculiar structural characteristics of this variant constitute a significant challenge to the development of effective detection methods. The aim of the study was to compare quantitative real-time PCR (qPCR) and digital droplet PCR (ddPCR) in the evaluation of KIV2 repeat polymorphism. METHODS: We analysed 100 subjects tested for cardiovascular risk in which Lp(a) plasma levels were assessed. RESULTS: Correlation analysis between CNV values obtained with the two methods was slightly significant (R = 0.413, p = 0.00002), because of the wider data dispersion in qPCR compared with ddPCR. Internal controls C1, C2 and C3 measurements throughout different experimental sessions revealed the superior stability of ddPCR, which was supported by a reduced intra/inter-assay coefficient of variation determined in this method compared to qPCR. A significant inverse correlation between Lp(a) levels and CNV values was confirmed for both techniques, but it was higher when evaluated by ddPCR than qPCR (R = -0.393, p = 0.000053 vs R = -0.220, p = 0.028, respectively). When dividing subjects into two groups according to 500 mg/L Lp(a) cut-off value, a significantly lower number of KIV2 repeats emerged among subjects with greater Lp(a) levels, with stronger evidence in ddPCR than in qPCR (p = 0.000013 and p = 0.001, respectively). CONCLUSIONS: Data obtained support a better performance of ddPCR in the evaluation of KIV2 repeat polymorphism.

Anti-SARS-CoV-2 vaccination does not influence anticoagulation levels in stable long-term warfarin treatment.

BACKGROUND: Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, administration of the currently available vaccines has mostly been recommended for subjects at high risk, including elderly populations on long-term oral anticoagulation therapy (OAT) with warfarin. However, there is no clear evidence of the stability of the International Normalised Ratio (INR) after vaccine administration in those subjects on long-term OAT. The present study aimed to investigate the effects of COVID-19 vaccination on anticoagulation levels in patients on long-term OAT. MATERIALS AND METHODS: INR values of patients on long-term OAT who had undergone anti-SARS-CoV-2 vaccination from January to June 2021 were monitored for a total of 90 days follow-up after the first vaccination dose. These were then compared with INR values before vaccination. The second dose, when required, was administered during follow-up. Inclusion criterion was stable long-term INR for at least 6 months before vaccination. Exclusion criteria were recent surgery, intercurrent diseases, or treatment with medication that could compromise findings in the 3 months before vaccination and during follow-up. RESULTS: No differences were observed in the anticoagulation levels before and after COVID-19 vaccination in any of the patients studied: mean INR values were 2.39 (range 2.20-2.63) before vaccination and 2.40 (range 2.16-2.76) after vaccination (p=0.5). There was no difference in anticoagulation levels in relation to age, sex, indication for OAT, or type of vaccine (p>0.5). No bleeding or thrombotic complications were documented during follow-up. DISCUSSION: These are the first data to be reported on anticoagulation levels in patients on stable OAT after COVID-19 vaccination. No influence on the quality of OAT was detected after the vaccination; no bleeding or thrombotic complications were recorded in the follow-up. No difference between the four available COVID vaccines was found. Dose adjustment was only required in a few cases, thus confirming the stability of anticoagulation levels.

Trandolapril, but not verapamil nor their association, restores the physiological renal hemodynamic response to adrenergic activation in essential hypertension.

The purpose of this study was to evaluate the effects of antihypertensive drugs on renal hemodynamics in hypertensive patients during an adrenergic activation by mental stress (MS), which induces renal vasoconstriction in healthy subjects. Renal hemodynamics was assessed twice in 30 middle-aged essential hypertensive patients (57±6 years)-after 15 days of pharmacological wash-out and after 15 days of treatment with Trandolapril (T, 4 mg, n=10), Verapamil (V, 240 mg, n=10), or both (T 2 mg+V 180 mg, n=10). Each experiment consisted of 4 30-min periods (baseline, MS, recovery I and II). Renal hemodynamics was evaluated with effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) from plasminogen activator inhibitor and inulin clearance, respectively. MS increased blood pressure (BP) to a similar extent before and after each treatment. Before treatment, the increasing BP was not associated with any modification of ERPF in the 3 groups. Renal vascular resistances (RVR) markedly increased during MS (+23% in the T group, +21.6% in the V group, and +32.9% in the T+V group); GFR remained constant during the whole experiment. After treatment, ERPF decreased significantly during MS in the T group (-15%, P<0.05) and in the V group (-11.7%, p<0.01); in the T+V group, ERPF modifications were not statistically significant (P=0.07). In the T group, ERPF reverted to baseline values at the end of the stimulus, whereas in the V group, renal vasoconstriction was more prolonged. Only in hypertensive patients treated with 4 mg of T, RVR reverted to baseline during the recovery I, whereas in the V group, RVR remained elevated for the whole experiment. No modifications of GFR were observed in all groups. The kidney of hypertensive patients cannot react to a sympathetic stimulus with the physiological vasoconstriction. A short-term antihypertensive treatment with 4 mg of T restores the physiological renal response to adrenergic activation.

Prognostic value of serum liver enzymes levels in type 2 diabetic patients.

BACKGROUND: Elevated liver enzymes are associated with cardiovascular disease, while their relationship with cancer-related mortality has not been assessed so far in diabetic patients. METHODS: An observational cohort study was performed on a consecutive series of 1952 type 2 diabetic patients. The association of liver enzymes with all-cause and cause-specific mortality was assessed. Information on all-cause mortality was obtained by the City of Florence Registry Office. RESULTS: The average duration of follow-up was 6.4 +/- 2.7 years. Over that period, 362 deaths were recorded (26.9%), with a yearly mortality rate of 4.2%. Age- and sex-adjusted HR of all-cause mortality for gamma glutamyl-transpeptidase (gamma-GT) gamma-GT > 40 U/l was 1.610 [1.245-2.082]. An increased cardiovascular mortality rate was observed in patients with elevation of gamma-GT, and gamma-GT and/or alanine aminotransferase (ALT), when compared with the rest of the sample (15.3 vs 10.8%, p < 0.05; and 15.2 vs 10.7%, p < 0.05, respectively). Similar results were observed when considering cancer-related mortality. The association of higher gamma-GT levels with all-cause, cardiovascular, and cancer-related mortality was confirmed at a multivariate analysis after adjustment for potential confounders. CONCLUSIONS: The present study shows that, in type 2 diabetic patients, higher gamma-GT, but not ALT, is associated with increased mortality for cardiovascular disease and malignancies.

Are comorbidity indices useful in predicting all-cause mortality in Type 2 diabetic patients? Comparison between Charlson index and disease count.

BACKGROUND AND AIMS: Several studies have shown that comorbidity is important in predicting morbidity and mortality in the general population. However, few studies have assessed the validity of comorbidity indices in diabetic patients. The aim of the present study was to compare the predictive value of disease count and Charlson's Comorbidity Index (CCI) for 3-year mortality in type 2 diabetic (T2D) patients. METHODS: The study was performed on a consecutive series of 1667 T2D outpatients. Comorbidity was assessed using Charlson's index, whereas the diseases used to calculate Charlson's score were taken into account for disease count. Information on all-cause mortality over the 3-year follow-up period was obtained from the City of Florence Registry Office. RESULTS: Mean duration of follow-up (+/-SD) was 31.4+/-10.6 months. One hundred and ninety-nine (11.9%) patients died during follow-up, with a yearly mortality rate of 4.7%. At multivariate analysis, after adjustment for sex and age, each additional disease was associated with a 54 [37-77]% increase in all-cause mortality. Mortality increased by 31 [21-41]% for each incremental point of Charlson's comorbidity score. CONCLUSIONS: A simple disease count is as predictive of mortality in T2D patients as the more complex Charlson's index. The possible usefulness of specific comorbidity indices in predicting incident disability in diabetic subjects needs to be further investigated.