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A huge amount of experimental and clinical evidences clearly demonstrate that the measurement of cardio-specific biomarkers [cardiac natriuretic peptides (CNP), and cardiac troponins (cTns)] can significantly and independently improve the diagnostic accuracy and risk stratification in cardiovascular diseases. Furthermore, several recent studies report that the measurement of cardio-specific biomarkers has a beneficial impact also on management and outcome of patients with cardiovascular diseases. Considering the significant and independent information associated to cardio-specific biomarkers, several studies have recently reported that the combined assay of CNP and cTns may be cost effective not only for diagnosis, prognosis and treatment of cardiac disease, but also for screening in general population of individuals at high cardiovascular risk. Due to the higher cost of their measurement in comparison to other laboratory tests, the clinical appropriateness of the combined measurement of CNP and cTns should be accurately evaluated. Accordingly, an increase in clinical utilization of a laboratory test should be based not only on the peculiar pathophysiological characteristics of a biomarker, but also on the high performance of immunoassay methods used for the biomarker measurement. From a pathophysiological point of view, elevated CNP values indicate that some stressor substances or situations are having an adverse impact on cardiac function; while an increase in cTn levels above the cut-off value indicates that a sustained or powerful stress has actually produced a damage on cardiomyocytes (i.e. a myocardial injury). Consequently , the combined measurement of CNP and cTns gives complementary and distinct rather than redundant clinical information. These two distinct pathophysiological mechanisms also explain why cardiovascular risk is strongly increased in patients with both CNP and cTns elevated. In conclusions, the combined measurement of CNP and cTns is a useful tool for diagnosis, follow-up, and stratification of risk in all patients with suspected cardiac disease, especially those admitted to the emergence department.

The most recent international guidelines recommend that high-sensitivity (hs) methods should be preferred for the measurement of cardiac troponin I (cTnI) and T (cTnT) for the detection of myocardial injury and the differential diagnosis of acute coronary syndromes. Furthermore, these guidelines state that there is no significant difference in accuracy among hs cTnI and cTnT methods for diagnosis of acute myocardial infarction. Myocardial injury is a prerequisite for the diagnosis of acute myocardial infarction, but it is also a distinct entity. The 2018 Fourth Universal Definition of Myocardial Infarction states that myocardial injury is detected when at least one value above the 99th percentile upper reference limit is measured in a patient by high-sensitivity methods for cTnI or cTnT. Occasionally, discrepancies between hs-cTnI and hs-cTnT test results can be found, when tested in the same blood sample. Several studies have reported discrepancies between hs-cTnI and hs-cTnT test results in some clinical conditions (especially chronic neuromuscular diseases) or even in apparently healthy subjects. This review, summarizes and discusses the biochemical, pathophysiological and analytical possible mechanisms causing discrepancies between hs-cTnI and hs-cTnT test results.

Background: the development of improved methods for cardiac troponins measurement, characterized by higher analytical sensitivity, allows to adopt rapid rule-in and rule-out strategies by increasing diagnostic accuracy and optimizing the clinical and organizational pathways.
Methods: the efficiency achieved with the adoption of a high sensitive troponin I method, defined by its analytical characteristics a “new generation” method (STAT high sensitive troponin I, hs-TnI, Abbott Diagnostics), in place of a previous assay (LOCI CTNI, Siemens Health Care Diagnostics), has been evaluated. The results obtained in 1000 consecutive patients admitted to the Emergency Department (ED) with acute chest pain, in 2015 (before the new method) have been compared with those obtained in 1004 consecutive patients admitted in 2017 (after theintroduction of the hs-TnI method).
Results: in 2017 an increased number of troponin I baseline values higher than 99th percentile (10.7 versus8.3% in 2015, p = 0.089) has been observed. Furthermore, an improvement on the following clinical and organizational aspects has been detected in 2017: the adoption of an accelerated algorithm, being the median interval between two consecutive measurements 3 h and 18 min in 2017 and 4 h and 30 min in 2015 (p <0.001); in the 75% of patients showing either positive or negative value at admission, the second measurement has been carried out within 180 min and 225 min (p = 0.024), respectively (in 2015 the timings were 239 e 337 min, p <0.001, respectively). Finally, in 2017 a third troponin I measurement has been carried out in 0.4% of patients only (it was 3.5% in 2015, p = 0.044).
Conclusions: These data demonstrate a significant improvement in the patient management as well as an optimization of human and organizational resources with the adoption of a method with improved analytical performances to measure troponin I in ED.

Background: the effects of caloric restriction (CR) on oxidative stress in obesity has been previously studied using markers that not always were able to describe all the components of the oxidative-inflammatory picture.
Methods: in this study, the redox state of 20 obese was evaluated at baseline and after 30 and 60 days of CR using a complete panel of markers: the majority of them were determined using HPLC methods.
Results: before CR (V0), serum peroxides (dROMs) were very high, total antioxidant barrier (BAP) was at the lower limit of the reference interval and C-reactive protein (hsCRP) was increased; on the opposite, glutathione was well within the reference intervals in both total and reduced form. Despite the imbalance of the dROMs/BAP equilibrium, the markers of oxidative damage, such 3-nitrotyrosine (3NT) and 8-hydroxy-deoxyguanosine (8OHdG), index of a mild oxidative-inflammatory imbalance, were not particularly relevant.
After 30 days of CR (V30), in addition to the slight improvements of glucose, fructosamine and HOMA-IR, hsCRP was decreased, while BAP and total glutathione were increased, with consequent improvement of the oxidative stress-inflammatory balance (oxidative-inflammation). After 60 days of CR (V60) the improvements observed at V30 appeared to be slowing down for glucose and fructosamine, in slight inverse tendency for HOMA-IR and hsCRP, and decreasing for BAP and glutathione. The slight increase of inter-quartile range (IQR) of 3NT showed a lower counter-regulatory antioxidant response capacity, as if the ameliorative effects of CR in the first period had turned off.
Conclusion: the improvements of the oxidative-inflammatory equilibrium appear to be transient in the course of CR. The rearrangements of the gut microbiota during CR and the consequent epigenetic modulations could be responsible for this peculiar trend.

Background: monoclonal gammopathies include a broad spectrum of pathologies, and free light chains (FLC) measurement is recommended by guidelines for the diagnosis, follow up and prognosis of plasma cell dyscrasia. The aim of the study is to compare the measurement of FLC performed with two different analytical platforms, a nephelometer and a turbidimeter, in order to assess the analytical and diagnostic agreement.
Methods: 87 consecutive samples received by the laboratory with a request of FLC measurement were analyzed on nephelometer Immage 800 (Beckman Coulter, USA) and turbidimeter Optilite (The Binding Site, UK), using the same antibodies (Freelite, The Binding Site, UK).
Results: by applying the Passing-Bablok regression and the Bland-Altman analysis, a proportional and constant systematic error but non-significant bias for FLC ratio (rFLC) has been observed. By applying the Weighted Cohen’s Kappa (WK) test to the rFLC values, an excellent diagnostic agreement between the two instruments has been shown, considering both the normal range (0.26 – 1.65) (WK=0.87) and the diagnostic range for multiple myeloma (<0.01 or >100) (WK=0.84).
Conclusions: Although there are statistical differences between the measurements performed by the two instruments, these do not affect the diagnostic agreement, that is excellent. Nevertheless, the turbidimeter is provided with a software that can automatically detect the antigen excess; by diluting further the samples automatically, it performs fewer dilutions than the nephelometer and provides a wider range of measurement, especially for low concentrations. These characteristics assist the operator both during the analysis and the validation phases of the results, saving time and resources. On the basis of the results of the study, it can be concluded that the turbidimeter shows better performances compared to the nephelometer.

Background: Gestational Diabetes Mellitus (GDM) is diagnosed by the oral glucose tolerance test (OGTT) using Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) study-derived definite cut-off values, where reference glucose is determined in sodium fluoride (NaF) containing tubes immediately centrifuged. The aim of the present study is to evaluate the effect of the use of the recommended citrate containing tubes, as immediate glycolisis inhibitor, for OGTT as screening for GDM, comparing them with NaF containing tubes, used in routine laboratory conditions.
Methods: a total of 83 pregnant women were enrolled in the study; OGTT (75 g) was carried out in the Clinical Laboratories of Brescia and Vicenza in all the subjects for screening of GDM. All subjects signed written informed consent to participate in the study. Glucose determination was performed using three different types of glycolysis inhibitor: [sodium fluoride (NaF, in both laboratories), a lyophilized acidified mixture (FC-MIX, in Vicenza), and a liquid acidified mixture (GlucoEXACT, in Brescia)]. The glucose concentration was measured by an hexochinase method on Dimension Vista systems from Siemens Healtheers. The International Association of Diabetes and Pregnancy Study Groups (IAPDGS) criteria, derived from the HAPO study, were used for the diagnosis of GDM.
Results: Using NaF and FC-mix tubes, 6/51 (11.8%) and 12/51 (23.5%) women respectively, were diagnosed having GDM in Vicenza. Using NaF and GlucoEXACT tubes 2/31 (6.5%) and 6/31 (18.8%) women respectively, were diagnosed having GDM in Brescia. There was a statistically significant difference (p<0.05) between NaF and citrate acidified mixture in the glucose measurements at all time points.
Conclusions: The use of the tubes containing a mixture of NaF, EDTA and citrate is a useful and necessary pre-analytical tool for an accurate OGTT, when utilized for the screening of GDM. Tubes containing NaF alone should no longer be used for screening of GDM because their use results in underdiagnosis of GDM.

Background: this article provides an updated overview on the current epidemiologic burden of ischemic heart disease (IHD) in Italy.
Methods: an electronic search was carried out in the Global Health Data Exchange (GHDx) registry, using the keyword “ischemic heart disease”, for retrieving recent information (year 2017) on incidence, prevalence, health loss (expressed as disability-adjusted life year, DALYs) and mortality of IHD in Italy.
Results: the cumulative incidence, prevalence and mortality of IHD in Italy are 0.38%, 3.47% and 0.16% respectively, whilst the impact on health loss is 1.11 million DALYs (6.73% of total DALYs). Incidence, prevalence and DALYs are higher in men than in women, whilst mortality is only marginally higher in males. Overall, IHD is the first cause of death in Italy (15.5% of all deaths). During the last three decades the incidence has constantly increased (+30%), whilst DALYs have declined in both sexes by ~30%. Mortality for IHD steadily decreased in men (-13%), whilst it remained almost unchanged in women. The curve of incidence of IHD in Italy peaks between 60-84 years in men and between 75-94 years in women. The mortality curve of IHD in Italy peaks in both sexes between 85-94 years. Hypertension, hypercholesterolemia and diabetes are the leading risk factors for IHD in Italy.
Conclusions: IHD still poses a substantial clinical, social and economic burden in Italy.

Introduction: this study compares the cardiac troponin I (cTnI) values measured with three high-sensitivity (hs) different methods in apparently healthy volunteers enrolled in a multicenter study.
Methods: heparinized plasma samples were collected from 1511 volunteers in 8 Italian clinical institutions (mean age 51.5 years, SD 14.2, range 18-86, female to male ratio 0.94). All volunteers denied chronic or acute diseases and had normal values of routine laboratory tests and ECG. The reference laboratory of the study (Laboratorio Fondazione CNR Regione Toscana G. Monasterio, Pisa, Italy) assayed all plasma samples with three hs-methods: Architect hs-cTnI, Access hs-cTnIand ADVIA Centaur XPT hs-cTnI. After the exclusion of outlier values, calculation of 99th percentile (Upper Reference Limit, URL) values was performed using both robust nonparametric and bias corrected and accelerated bootstrap methods.
Results: large between-method differences were found. ADVIA Centaur measured higher cTnI values (up to 2-fold) than the two other methods. cTnI values were significantly higher in men than in women, and progressively increased with age over 55 years. Moreover, 99th percentile URL values also depended on the statistical approach used for calculation (robust non-parametric versusbootstrap). All 99th percentile URL values calculated with non-parametric robust method were on average slightly lower than those suggested by manufacturers (mean difference 4.2 ng/L, standard error 1.7, p=0.0273).
Conclusion: clinicians should be advised that plasma samples from the same patient should be measured for hs-cTnI in the same laboratory. Specific clinical studies are needed to establish the most appropriate statistical approach to calculate 99th percentile URL values for hs-cTnI methods.

Introduction: the Working Groups “Hematology” and “Extra-Analytical Variability” of the Italian Society of Clinical Biochemistry (SIBioC) promoted a survey investigating the quality of the complete blood count reporting among Italian Laboratories.
Methods: the survey included 36 questions and was sent to all the SIBioC members. 251 laboratories participated in the survey.
Results: there is a full concordance in reporting the traditional parameters (leukocytes, erythrocytes and platelet count, hemoglobin, hematocrit and the calculated indices, plus the leukocytes differential count), while other relatively new parameters, like the mean platelet volume (MPV) and the platelet distribution width (PDW) are reported by 70% of the laboratories. A low percentage of laboratories (20-30%) do not report the presence of abnormal cell populations, if detected (blasts, immature granulocytes, plasma cells, prolymphocytes and erythroblasts). 70% of laboratories do not report the erythrocyte and leukocyte related parameters available on the new analyzers. Specific reference intervals for gender and age are adopted by 68% of the laboratories, but only 50% have instrument-specific intervals. 83% of the laboratories include interpretative comments in the report, but only in less than half of them these are harmonized according to the recent available recommendations. 83% of the laboratories have a shared document to manage critical values, that are communicated to the requesting physician by 90% of the laboratories.
Discussion: activities promoted by the SIBioC Hematology working group to harmonize the hematological report have been effective on traditional parameters reporting with a substantial improvement compared to the 2014 survey. Two issues remain however unresolved: the inclusion of interpretative comments and of the recent available parameters in the report.

Systemic amyloidosis is an increasingly recognized cause of heart failure. The two most common forms of amyloidosis involving the heart are AL (caused by monoclonal immunoglobulin light chains) and ATTR (caused by wild-type or variant transthyretin) amyloidosis. Involvement of the heart is by far the most important prognostic determinant, and its severity is easily and accurately stratified by cardiac biomarkers (natriuretic peptides and troponins). In AL amyloidosis, cardiac troponins are also useful in the design of the therapeutic strategy (excluding fragile patients from autologous stem cell transplantation) and in identification of cardiac progression. Response and progression criteria are still lacking in ATTR amyloidosis, and studies addressing this topic are warranted.

The evolution of the biochemical diagnosis of cardiac diseases, represents a paradigm of the laboratory medicine evolution in the recent years.
Starting from the use of poor specific and sensitive biomarkers, the “so-called” cardiac enzymes (aspartate aminotransferase; lactate dehydrogenase; creatine kinase) recommended by World Health Organization for the acute myocardial infarction (AMI) diagnosis, a fundamental development in biochemical knowledge has been obtained, providing new biomarkers (CK-MB, myoglobin) for a more specific and early diagnosis according to the clinical and therapeutic needs. However, the revolutionary biochemical issue has been represented by the discovery of cardiac troponins and by the implementation of methods allowing their measurement in emergency setting in patients with acute chest pain. Cardiac troponins, are characterized by an absolute cardiac specificity and by a high sensitivity that allow to carry out a timely and safe diagnosis of AMI, being recognized as “gold standard” in all clinical and biochemical guidelines. In patients with acute chest pain and in ischemic clinical setting, a typical kinetic release of biomarker concentration may be suggestive of AMI even if ECG typical patterns are lacking. The actual improvement in analytical performance of troponins methods, particularly in the analytical sensitivity, allows to extend the measurement also in diagnosis of minor myocardial damage in patients suffering from different cardiac disease, to monitor the efficacy of therapy, the progression of the disease and to provide prognostic information and risk-stratification in addition to the clinical pathway.

A hemostatic assessment is usually included in the evaluation of patients undergoing surgical procedures. The aim of this work is to present a protocol shared with the Intensive Care, Hematology and Clinical Laboratory Units of the San Giacomo Hospital (Castelfranco Veneto, Treviso, Italy) to investigate the causes of the prolonged coagulation tests in these patients. In case of altered coagulation screening tests, a form is filled in with the pertinent anamnestic information and sent to the laboratory together with an adequate amount of blood. Following a pre-defined algorithm, the clinical laboratory performs the second level tests. The most relevant one for the purpose is the mixing test that permits an easy identification of the appropriate subsequent tests. The results of these tests allow clinicians to take the appropriate decisions. In case of a negative bleeding anamnesis, and/or in presence of a factor XII deficiency or a lupus anticoagulant positive test, the patient undergoes the surgical intervention; in other cases, the hematologist carefully evaluates the hemorrhagic risk of the specific patient.
In 2017, the protocol was applied to 37 cases of prolonged PT/APTT. In 8% of the patients the alteration was not confirmed, in 11% of cases the investigations could not identify the cause of the alterations. In the remaining 81% ofcases, the identification of the specific coagulative defect was possible.
The application of this protocol can achieve a number of benefits: it reduces the number of accesses to the hospital for the patients, only the appropriate tests are performed on the basis of reflex testing procedures, the clinician is provided with all the relevant tests at one time with no need to request the tests one after another. The value of the mixing test in this clinical scenario is confirmed.

A large body of evidence supports that quality improvement efforts tailored to the analytical phase only, are less likely to generate further clinical and economical progresses. Actually, most diagnostic errors made within the laboratory diagnostics emerge in the extra-analytical domains of testing, especially within the preanalytical phase. It is now clear that the underlying causes are most frequently due to system errors or to the implementation of poorly standardized procedures for collection, handling, transportation, preparation and storage of biospecimens. Some of these problems could generate a number of issues related to the quality of clinical samples, ending up with the reception by the laboratory of unsuitable samples. The identification and the management of unsuitable samples represent thus unavoidable practices in clinical laboratories to guarantee the quality of test results throughout the total testing process. Due to the ongoing evolution of the in vitro diagnostic market and the availability of new evidence, this paper provides a revision of the national recommendations issued by the Italian Society of Clinical Biochemistry and Clinical Molecular Biology in 2007 for detection and practical management of unsuitable specimens in clinical laboratories.

Only very recently the set-up of some immunoassay methods with high-sensitivity analytical performance allowed the accurate detection of cardiac troponin I (cTnI) and T (cTnT) levels in healthy adult subjects. Several studies have demonstrated the association between the risk of major cardiovascular events and cardiac troponin concentrations even for biomarker values within the reference intervals. High-sensitivity cTnI and cTnT methods enable to monitor myocardial renewal and remodelling, and to promptly identify patients at highest risk to heart failure development. An early and effective treatment of individuals at higher cardiovascular risk may revert the initial myocardial remodelling and slow down heart failure progression. Dedicated trials are needed also in Italian population to demonstrate the efficiency of general population screening by means of cost benefit analysis for individuals at higher risk for heart failure progression.

We describe the case of a 47 years old male patient who underwent cardiac surgery for valve sparing root replacement (known as David procedure), in September 2018. In the post-operative period and during the rehabilitation stage, the patient was constantly monitored with the measurement of cardiac troponin I (cTnI), performed in different hospital facilities using different cTnI analytical methods. In the rehabilitation clinic, cTnI [measured with Access AccuTnI+3 method (Beckman Coulter)] was found steadily raising, so the patient was readmitted to the hospital to undergo a coronary angiography. The coronary arteries were found free from critical lesions and the cTnI [measured with Advia Centaur cTnI Ultra method, (Siemens Healthcare)] was repeatedly negative. This finding rose the suspect of an analytical interference in the Access AccuTnI+3 method. This was actually the case: heterophile and anti-alkaline phosphatase antibodies were demonstrated in the patient sample; they both were responsible for the elevated values of cTnI.

Serum monoclonal components, Bence-Jones proteinuria (PBJ) and free light chains (FLC) are clonal biomarkers for diagnosis and response assessment in light chain (AL) amyloidosis. Two clinical reports are presented here toi llustrate the utility of these biomarkers. The first case is a patient with AL κappa renal amyloidosis. Serum and urine immunofixation were negative and the FLC ratio was abnormal. Immunoelectron microscopy on tissue biopsy was negative. Amyloid typing was achieved by mass spectrometry on fat pad aspirate. The second case is a patient with AL cardiac amyloidosis with PBJ lambdaand low concentration of amyloidogenic FLC (32 mg/L). Urine capillary electrophoresis was used to assess response to treatment. The progressive reduction of PBJ after treatment was accompanied by reduction of NT-proBNP and improvement of clinical conditions. Clonal biomarkers are irreplaceable tools in management of AL amyloidosis. There is a need for more sensitive techniques for identification of monoclonal FLC on serum and urine.

Monoclonal gammopathy of renal significance (MGRS) is a condition defined by the presence of a small-B cell clone causing a renal disease trough deposition in renal tissues of the monoclonal component (MC) secreted by the B cells. Since MGRS is associated with several types of renal diseases, characterization of renal damage caused by protein deposition is important to define the correct diagnosis as well as the identification of the MC. Adult Fanconi Syndrome (FS) is characterized by the presence of a MC and damage in the proximal tubule with impaired small molecules transport. We report the case of a 32 years old man with moderate kidney failure, normoglycemic glycosuria and hypouricemia. Further investigations revealed hypophosphoremia and phosphaturia; an IgGκ MC was detected by immunofixation. The kidney biopsy confirmed FS suspect. This case underlines that the results of the biochemical analysis carried on for the diagnosis of FS, need to be confirmed by histopathologic analysis.