Analysis of annual distributions of hemoglobin A2 values as a method to test for HbA2 standardization.

BACKGROUND AND AIMS: The monitoring of yearly distributions of HbA2 measured has been indicated as a reliable indicator of worldwide standardization. MATERIALS AND METHODS: Measurements/year of HbA2 have been collected over three consecutive years in 15 Italian laboratories each using the same analytical method over three years period. HbA2 distributions, cleaned of replicated measurements, were compared by the overlapping area of the raw probability density functions expressed by coefficient eta (η), and by comparing the reference intervals for the central part of each distribution estimated by the indirect method refineR using the R package "refineR". RESULTS: According to the overlapping areas analysis the distributions/year of the data provided by 4 centers able to perform at least 1000 measurements/year were similar in 2 consecutive years. Moreover, the reference intervals provided by 2 centers using the same analytical methods in two separate locations over the three consecutive years, were very similar. The highest overlap (99.7 %) was observed in one center over two consecutive years. The overlapping areas were very high (93.6-95.7%) in 8 out of 9 inter-comparisons. CONCLUSION: Despite the limitations of this study the yearly distribution of the HbA2 measured in various centers appears a reliable tool to test HbA2 standardization over different centers using different analytical methods.

Weak correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects.

Plenty of serologic tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas® by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we firstly compared two serologic tests on serum samples collected at two different time points from 46 laboratory-confirmed coronavirus disease-2019 (COVID-19) subjects. Secondly, 85 negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8% and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity evaluated for the two tests ranges from 96.5% to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study. These data further shed light on both potentials and possible limitations related to SARS-CoV-2 serology. In this context, great efforts are still necessary for investigating antibody kinetics to develop novel diagnostic algorithms. Moreover, further investigations on the role of neutralizing antibodies and their correlate of protection will be of paramount importance for the development of effective vaccines.

Routine blood analysis greatly reduces the false-negative rate of RT-PCR testing for Covid-19.

BACKGROUND: The COVID-19 outbreak is now a pandemic disease reaching as much as 210 countries worldwide with more than 2.5 million infected people and nearly 200.000 deaths. Amplification of viral RNA by RT-PCR represents the gold standard for confirmation of infection, yet it showed false-negative rates as large as 15-20% which may jeopardize the effect of the restrictive measures taken by governments. We previously showed that several hematological parameters were significantly different between COVID-19 positive and negative patients. Among them aspartate aminotransferase and lactate dehydrogenase had predictive values as large as 90%. Thus a combination of RT-PCR and blood tests could reduce the false-negative rate of the genetic test. METHODS: We retrospectively analyzed 24 patients showing multiple and inconsistent RT-PCR, test during their first hospitalization period, and compared the genetic tests results with their AST and LDH levels. RESULTS: We showed that when considering the hematological parameters, the RT-PCR false-negative rates were reduced by almost 4-fold. CONCLUSIONS: The study represents a preliminary work aiming at the development of strategies that, by combining RT-PCR tests with routine blood tests, will lower or even abolish the rate of RT-PCR false-negative results and thus will identify, with high accuracy, patients infected by COVID-19.

Retrospective analysis of the cholesterol levels in a European mid-latitude population between 2007 and 2018: controversies and therapeutic implications.

BACKGROUND AND AIM OF THE WORK: Coronary heart disease is the leading cause of morbidity and mortality in developed countries and its association with LDL-cholesterol has been well established. During the last decades statin therapy, and an healthy lifestyle, has proven highly effective in reducing cardiovascular event rates. Yet, there is still controversy among national guidelines and clinical practice with regard to LDL-cholesterol measurements. The aim of this study was to verify whether significant changes in cholesterol levels were observed over a relatively long (12 years) period. We also compared the Friedewald-calculated with directly-measured LDL cholesterol in order to verify whether the two methods were equivalent. METHODS: We retrospectively analyzed 288000 data (total cholesterol, HDL, LDL both directly-measured and Friedewald-calculated, and triglycerides), from 57600 individuals, collected at the San Raffaele Hospital in Milan between 2007 and 2018. Data were statistically analyzed using a linear regression over the 12 years long period of time. RESULTS: We observed a general decreasing trend in total cholesterol (and an increasing trend in HDL) and a decrease in triglycerides only in the male group. Directly-measured  LDL did not correlate well with calculated LDL. CONCLUSION: The total cholesterol decrease and the corresponding increment in HDL was attributed to either an increased use of statin and/or an improvement of the general population dietary habits. The latter was further confirmed by the decrease in triglycerides observed in the male group. Based on our data, an alternative method for assessing atherogenics lipoproteins level has been proposed.

A Possible Antioxidant Role for Vitamin D in Soccer Players: A Retrospective Analysis of Psychophysical Stress Markers in a Professional Team.

The health benefits of physical activity are recognized, however, high levels of exercise may lead to metabolic pathway imbalances that could evolve into pathological conditions like the increased risk of neurological disease observed in professional athletes. We analyzed the plasma/serum levels of 29 athletes from a professional soccer team playing in the Italian first league and tested the levels of psychophysical stress markers (vitamin D, creatine kinase, reactive oxygen species (ROS) and testosterone/cortisol ratio) during a period of 13 months. The testosterone/cortisol ratio was consistent with an appropriate training program. However, most of the athletes showed high levels of creatine kinase and ROS. Despite the large outdoor activity, vitamin D values were often below the sufficiency level and, during the "vitamin D winter", comparable with those of the general population. Interestingly, high vitamin D values seemed to be associated to low levels of ROS. Based on the results of our study we proposed a vitamin D supplementation as a general practice for people who perform high levels of physical exercise. Beside the known effect on calcium and phosphate homeostasis, vitamin D supplementation should mitigate the high reactivity of ROS which might be correlated to higher risk of neurodegenerative diseases observed in professional athletes.

Routine blood tests as a potential diagnostic tool for COVID-19.

Objectives The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to date, the epidemic has gradually spread to 209 countries worldwide with more than 1.5 million infected people and 100,000 deaths. Amplification of viral RNA by rRT-PCR serves as the gold standard for confirmation of infection, yet it needs a long turnaround time (3-4 h to generate results) and shows false-negative rates as large as 15%-20%. In addition, the need of certified laboratories, expensive equipment and trained personnel led many countries to limit the rRT-PCR tests only to individuals with pronounced respiratory syndrome symptoms. Thus, there is a need for alternative, less expensive and more accessible tests. Methods We analyzed the plasma levels of white blood cells (WBCs), platelets, C-reactive protein (CRP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), alkaline phosphatase and lactate dehydrogenase (LDH) of 207 patients who, after being admitted to the emergency room of the San Raffaele Hospital (Milan, Italy) with COVID-19 symptoms, were rRT-PCR tested. Of them, 105 tested positive, whereas 102 tested negative. Results Statistically significant differences were observed for WBC, CRP, AST, ALT and LDH. Empirical thresholds for AST and LDH allowed the identification of 70% of either COVID-19-positive or -negative patients on the basis of routine blood test results. Conclusions Combining appropriate cutoffs for certain hematological parameters could help in identifying false-positive/negative rRT-PCR tests. Blood test analysis might be used as an alternative to rRT-PCR for identifying COVID-19-positive patients in those countries which suffer from a large shortage of rRT-PCR reagents and/or specialized laboratory.

Biochemical, immunochemical and serology analytes validation of the lithium heparin BD Barricor blood collection tube on a highly automated Roche COBAS8000 instrument.

BACKGROUND: Recently developed blood tubes with a barrier to provide plasma are becoming widespread. We compared 43 biochemical, 35 immunochemical and 7 serology analytes in a BD-Vacutainer® Barricor tube for local clinical validation of this lithium-heparin tube with a barrier. METHODS: Samples from 70 volunteers were collected in different BD-tubes: a clot-activator tube with gel (SST), a lithium-heparin tube with gel (PST), and a lithium-heparin tube with barrier (BAR). Biases from Bland-Altman plots and 95% confidence intervals were compared with the desirable specification from the Ricos database in order to verify whether measurements from different tubes were significantly different. RESULTS: For most of the analytes tested, the measurements using SST, PST or BAR tubes were equivalent. Only BIC, GLU, K, LAD, LPA, P, TP, CTX, Ferritin, HGH, vitD3 and ANTIS showed statistically significant, between-tubes, differences which might have clinical implication. CONCLUSIONS: The study demonstrates that SST, PST and BAR can be used interchangeably for most of the analytes tested, including serology analytes. This allows the use of the same tube for assaying multiple analytes, increasing the laboratory efficiency while decreasing patients discomfort by minimizing blood withdrawal.

Association between solar ultraviolet doses and vitamin D clinical routine data in European mid-latitude population between 2006 and 2018.

The major source of vitamin D in humans is the ultraviolet radiation-dependent cutaneous synthesis of cholecalciferol; however, low vitamin D status is common in Europe even at mid-latitudes. The UV-radiation that reached the Earth's surface near Milan between May 2006 and December 2018 was retrieved from the TEMIS database and matched with the serum vitamin D levels measured in 30 400 people living in the same area. The results showed a high percentage of insufficient vitamin D levels (measured as 25-hydroxy-vitamin D) throughout the years. During the "vitamin D winter" (November-March) up to 60-90% of the population shows deficient/insufficient (<20-30 ng mL-1) levels of vitamin D and it is explained by the difficulty in obtaining the recommended UV vitamin D doses. In contrast, the warm season provides plenty of UV-radiation, but still 30-50% of the population shows deficient/insufficient vitamin D levels. The circannual vitamin D variations were less evident in the female groups which, in the cold season, show values higher than the corresponding male groups. An age group analysis explained this difference by the strongly recommended vitamin D intake for post-menopausal women. In conclusion, increasing the medical advice for vitamin D intake is strongly recommended to improve the vitamin D status at European mid-latitudes. Our findings suggest that UV availability alone cannot explain the vitamin D status of the population which instead is likely to be influenced by several other factors related to both the people's lifestyle and their personal characteristics. A desirable vitamin D range considering the time of the year and sun exposure, but also including factors not related to UV-radiation, would probably result in a more accurate diagnosis of the patients' vitamin D status. Despite the relatively large time interval, no evident effects due to climate changes were observed in the vitamin D levels during the almost 13 years of analysis.

Calibration by commutable control materials is able to reduce inter-method differences of current high-performance methods for HbA2.

BACKGROUND: Most of the current methods used for the determination of HbA2 seem not well aligned. A comparison among the best performing techniques and the commutability of some control materials currently available and under development has been evaluated. METHODS: Forty blood samples were analyzed in duplicate over two separate days by different HPLC and capillary electrophoresis systems. The commutabilities of different control materials (NIBSC WHO reagent, Bio-Rad Lyphochek, and home prepared lyophilized controls RP1-3) have been assessed by analyzing the controls in quadruplicate over two consecutive days together with the blood samples. RESULTS: The mean within-run imprecision of HbA2 measurement on blood samples (CV, %) was between 0.6% and 10.1% for HbA2 values <3.5%, and between 1.1 and 3.1 for HbA2≥3.5%. The different methods were highly correlated (r between 0.9941 and 0.9995) although biased each other. The NIBSC WHO reagent was found not commutable in 15 over 28 comparisons, the Lyphochek 2 in 18/28, and RP3 in 4/28. Recalibration of all methods by RP1 and RP2 materials was able to reduce the overall variability from 6.8% to 3.4% at HbA2≤3.0% and from 6.7% to 3.0% at HbA2≥4.6%. CONCLUSION: The use of adequate commutable control materials as calibrators may reduce the inter-method variability of routine methods to an extent closer to the current analytical goals of bias based on biological variability.

Glycation gap: An additional tool for glycometabolic monitoring.

BACKGROUND: The determination of glycated albumin (GA) has been suggested as an additional parameter having an independent added value respect to that of HbA1c. The determination of glycation gap (gg) has also been proposed, but few studies have addressed its potential impact in the routine evaluation of glycometabolic control. METHODS: A total of 157 subjects presenting normal whole blood cell count, no hemoglobin variants, normal creatinine levels and serum protein electrophoresis patterns were studied. In a second phase, a total of 205 subjects with no restrictions as those of the first phase study, were analyzed. HbA1cwas measured by capillary electrophoresis, glycated albumin by an enzymatic method and their gg were then calculated. RESULTS: The correlation between HbA1c and GA for the subjects of phase 1 was strong (r=0.8927) and significant correlation between gg and age was remarked (r=0.4486). We found 17.1% of phase 2 subjects with gg falling outside the 95% prediction intervals. Various clinical conditions seemed to affect these subjects, in our experience mostly often related to impaired renal function. CONCLUSION: The glycation gap may be useful to alert clinicians about patients under unstable glycemic control or when various pre-analytical conditions my affect the reliability of the measurement of GA or HbA1c.

Sample collections from healthy volunteers for biological variation estimates' update: a new project undertaken by the Working Group on Biological Variation established by the European Federation of Clinical Chemistry and Laboratory Medicine.

BACKGROUND: Biological variation (BV) data have many fundamental applications in laboratory medicine. At the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) the reliability and limitations of current BV data were discussed. The EFLM Working Group on Biological Variation is working to increase the quality of BV data by developing a European project to establish a biobank of samples from healthy subjects to be used to produce high quality BV data. METHODS: The project involved six European laboratories (Milan, Italy; Bergen, Norway; Madrid, Spain; Padua, Italy; Istanbul, Turkey; Assen, The Netherlands). Blood samples were collected from 97 volunteers (44 men, aged 20-60 years; 43 women, aged 20-50 years; 10 women, aged 55-69 years). Initial subject inclusion required that participants completed an enrolment questionnaire to verify their health status. The volunteers provided blood specimens once per week for 10 weeks. A short questionnaire was completed and some laboratory tests were performed at each sampling consisting of blood collected under controlled conditions to provide serum, K2EDTA-plasma and citrated-plasma samples. RESULTS: Samples from six out of the 97 enroled subjects were discarded as a consequence of abnormal laboratory measurements. A biobank of 18,000 aliquots was established consisting of 120 aliquots of serum, 40 of EDTA-plasma, and 40 of citrated-plasma from each subject. The samples were stored at -80 °C. CONCLUSIONS: A biobank of well-characterised samples collected under controlled conditions has been established delivering a European resource to enable production of contemporary BV data.

Methyl-CpG binding protein 2 (MeCP2) localizes at the centrosome and is required for proper mitotic spindle organization.

Mutations in MECP2 cause a broad spectrum of neuropsychiatric disorders of which Rett syndrome represents the best defined condition. Both neuronal and non-neuronal functions of the methyl-binding protein underlie the related pathologies. Nowadays MeCP2 is recognized as a multifunctional protein that modulates its activity depending on its protein partners and posttranslational modifications. However, we are still missing a comprehensive understanding of all MeCP2 functions and their involvement in the related pathologies. The study of human mutations often offers the possibility of clarifying the functions of a protein. Therefore, we decided to characterize a novel MeCP2 phospho-isoform (Tyr-120) whose relevance was suggested by a Rett syndrome patient carrying a Y120D substitution possibly mimicking a constitutively phosphorylated state. Unexpectedly, we found MeCP2 and its Tyr-120 phospho-isoform enriched at the centrosome both in dividing and postmitotic cells. The molecular and functional connection of MeCP2 to the centrosome was further reinforced through cellular and biochemical approaches. We show that, similar to many centrosomal proteins, MeCP2 deficiency causes aberrant spindle geometry, prolonged mitosis, and defects in microtubule nucleation. Collectively, our data indicate a novel function of MeCP2 that might reconcile previous data regarding the role of MeCP2 in cell growth and cytoskeleton stability and that might be relevant to understand some aspects of MeCP2-related conditions. Furthermore, they link the Tyr-120 residue and its phosphorylation to cell division, prompting future studies on the relevance of Tyr-120 for cortical development.

MeCP2 post-translational modifications: a mechanism to control its involvement in synaptic plasticity and homeostasis?

Although Rett syndrome (RTT) represents one of the most frequent forms of severe intellectual disability in females worldwide, we still have an inadequate knowledge of the many roles played by MeCP2 (whose mutations are responsible for most cases of RTT) and their relevance for RTT pathobiology. Several studies support a role of MeCP2 in the regulation of synaptic plasticity and homeostasis. At the molecular level, MeCP2 is described as a repressor capable of inhibiting gene transcription through chromatin compaction. Indeed, it interacts with several chromatin remodeling factors, such as HDAC-containing complexes and ATRX. Other studies have inferred that MeCP2 functions also as an activator; a role in regulating mRNA splicing and in modulating protein synthesis has also been proposed. Further, MeCP2 avidly binds both 5-methyl- and 5-hydroxymethyl-cytosine. Recent evidence suggests that it is the highly disorganized structure of MeCP2, together with its post-translational modifications (PTMs) that generate and regulate this functional versatility. Indeed, several reports have demonstrated that differential phosphorylation of MeCP2 is a key mechanism by which the methyl binding protein modulates its affinity for its partners, gene expression and cellular adaptations to stimuli and neuronal plasticity. As logic consequence, generation of phospho-defective Mecp2 knock-in mice has permitted associating alterations in neuronal morphology, circuit formation, and mouse behavioral phenotypes with specific phosphorylation events. MeCP2 undergoes various other PTMs, including acetylation, ubiquitination and sumoylation, whose functional roles remain largely unexplored. These results, together with the genome-wide distribution of MeCP2 and its capability to substitute histone H1, recall the complex regulation of histones and suggest the relevance of quickly gaining a deeper comprehension of MeCP2 PTMs, the respective writers and readers and the consequent functional outcomes.