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Objective: Salivary alpha-amylase (sAA) has been recently proposed as biomarker of stress responsiveness within the sympathetic nervous system, preferable to cortisol, since limitations related to cortisol measurement (e.g. diurnal and seasonal rhythms, drugs interferences). Several factors, as age, collection device and analytical methods, also influence sAA levels and interpretation. This study aims to assess whether sAA could be useful to evaluate the stress response, also verifying some sources of variability. Methods: To identify any sources of sAA variability, saliva samples were collected from eight healthy subjects at five different times (8:00, 10:00, 12:00, 14:00 and 16:00) over five consecutive days using two different collection devices. Saliva was then collected from 35 obstetric residents before and after a simulated shoulder dystocia scenario, one of the most traumatic birth events in the professional life of gynecologists, to assess the stress levels. Samples were analysed throughout two different assays. Heart rate (HR) of residents was also measured before and after simulation scenario. Residents self-collected two saliva samples 10 min apart on a typical day. Results: Overall, sAA levels increased significantly from morning to afternoon. Levels varied between subjects, but no differences were found between days or sampling devices. sAA activity levels in samples from 35 residents were significantly higher than those obtained before the scenario took place (about ten minutes later). These results were confirmed by two different assays. Moreover, a similar trend was observed when HR was considered. No differences were observed between samples collected 10 minutes apart on a typical day. Conclusions: Salivary alpha-amylase activity is a reliable, quick, and efficient marker for stress response, then its measurement will be of potential interest in the field of stress-related disorders. However, it is important to consider the timing of sample collection before introducing sAA in a clinical setting. [ABSTRACT FROM AUTHOR]

Cystinuria is one of the unrecognized causes of nephrolithiasis. As other stones, cystine stones can present with flank pain, haematuria, dysuria, as well as systemic symptoms such as nausea and vomiting. This report describes the case of a 36-years-old pregnant woman, admitted at Emergency Department for severe vomiting and weight loss (6 kilograms in one month), soon after being treated for hyperemesis gravidarum. Failure of response to conventional management prompted further clinical and laboratory investigations. Urinary sediment analysis by automated analyser and intelligent verification rules, allowed to highlight cysteine casts, recognized by well trained staff. Cooperation between laboratory staff and clinicians allowed diagnosing cystinuria and starting therapy with vomiting remission. [ABSTRACT FROM AUTHOR]

In the last few decades, quality in laboratory medicine has evolved in concert with the transformation and the changes (technological, scientific and organizational) in this sector. Laboratory professionals have faced great challenges, at times being overwhelmed, yet also involved in this progress. Worldwide, laboratory professionals and scientific societies involved in laboratory medicine have raised awareness concerning the need to identify new quality assurance tools that are effective in reducing the error rate and enhancing patient safety, in addition to Internal Quality Control (IQC) procedures and the participation in the External Quality Assessment Schemes (EQAS). The use of Quality Indicators (QIs), specifically designed for laboratory medicine are effective in assessing and monitoring all critical events occurring in the different phases of Total Testing Process (TTP), in particular, in the extra-analytical phases. The Model of Quality Indicators (MQI), proposed by the Working Group "Laboratory Errors and Patient Safety" (WG-LEPS) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and validated by experts in consensus conferences, is an important window of opportunity for the medical laboratory to demonstrate the use of an effective quality assurance tool fit for this purpose. Aim of this paper is to provide an update of the state-of-the-art concerning the most used QIs data collected in 2021 and the Quality Specifications (QSs) proposed for their evaluation. Moreover, a strategy for the future is proposed in order to improve the MQI and encourage its use in medical laboratories throughout the world. [ABSTRACT FROM AUTHOR]

The rapid, accurate and safe detection of SARS-CoV-2 is the key to improving surveillance and infection containment. The aim of the present study was to ascertain whether, after heat/chemical inactivation, SARS-CoV-2 N antigen chemiluminescence (CLEIA) assay in saliva remains a valid alternative to molecular testing. In 2022, 139 COVID-19 inpatients and 467 healthcare workers were enrolled. In 606 self-collected saliva samples (Salivette), SARS-CoV-2 was detected by molecular (TaqPath rRT-PCR) and chemiluminescent Ag assays (Lumipulse G). The effect of sample pre-treatment (extraction solution-ES or heating) on antigen recovery was verified. Salivary SARS-CoV-2 antigen assay was highly accurate (AUC=0.959, 95% CI: 0.943–0.974), with 90% sensitivity and 92% specificity. Of the 254 antigen positive samples, 29 were false positives. We demonstrated that heterophilic antibodies could be a cause of false positive results. A significant antigen concentration decrease was observed after ES treatment (p=0.0026), with misclassification of 43 samples. Heat had a minimal impact, after treatment the correct classification of cases was maintained. CLEIA SARS-CoV-2 salivary antigen provides accurate, timely and high-throughput results that remain accurate also after heat inactivation, thus ensuring a safer work environment. This supports the use of salivary antigen detection by CLEIA in surveillance programs. [ABSTRACT FROM AUTHOR]

The reliable identification of individuals with SARS-CoV-2 infection is the cornerstone for containing viral spread. Rapid molecular point-of-care testing (POCT) of saliva might reduce analysis time, thus increasing the efficacy of contact tracing. In this study, a new POCT RT-PCR assay for the detection of SARS-CoV-2 RNA in saliva was evaluated and compared with an already validated CE-IVD method. An evaluation was made of 160 left-over salivary samples (27 frozen, kept at −80 °C and 133 fresh), collected using Salivette (Sarstedt, Germany). Samples were analyzed by TaqPath COVID-19 CE-IVD RT-PCR kit, QuantStudio5 Real-Time (Applied Biosystems, USA) (TaqPath) and bKIT Virus Finder COVID-19 Saliva (Hyris Global Diagnostics, Italy). Performances of three- and fivefold pooling strategies were also evaluated. Blood assay interference in saliva was also tested with Hyris. On using TaqPath, SARS-CoV-2 positivity was detected in 35 samples. Another 10 positive samples were artificially-generated by blind mixing of positive with negative samples. Hyris positive and negative percentages of agreement were 97.6 (95% CI: 87.2–99.9%) and 100 (95% CI: 97.0–100%), respectively. Seventeen positive pools, evaluated for threefold strategy, were all correctly determined by both systems. For the 5-pool strategy, 94.7% (18/19) of samples resulted positive with the Hyris system, and 100% with TaqPath. The presence of 1% of blood (v/v) in saliva did not interfere with the accuracy of Hyris assay. The sensitivity and specificity of the bKIT Virus Finder COVID-19 Saliva were optimal with respect to TaqPath. In view of the safe and straightforward pre-analytical procedure involved, and the small size of the Hyris bCube, the Hyris system can be used for POCT. [ABSTRACT FROM AUTHOR]

Objectives: The direct identification of SARS-CoV-2 RNA in nasopharyngeal swabs is recommended for diagnosing the novel COVID-19 disease. Pre-analytical determinants, such as sampling procedures, time and temperature storage conditions, might impact on the end result. Our aim was to evaluate the effects of sampling procedures, time and temperature of the primary nasopharyngeal swabs storage on real-time reverse-transcription polymerase chain reaction (rRT-PCR) results. Methods: Each nasopharyngeal swab obtained from 10 hospitalized patients for COVID-19 was subdivided in 15 aliquots: five were kept at room temperature; five were refrigerated (+4 °C); five were immediately mixed with the extraction buffer and refrigerated at +4 °C. Every day and for 5 days, one aliquot per condition was analyzed (rRT-PCR) for SARS-CoV-2 gene E and RNaseP and threshold cycles (Ct) compared. To evaluate manual sampling, 70 nasopharyngeal swabs were sampled twice by two different operators and analyzed separately one from the other. Results: A total of 6/10 swabs were SARS-CoV-2 positive. No significant time or storage-dependent variations were observed in SARS-CoV-2 Ct. Re-sampling of swabs with SARS-CoV-2 Ct lower than 33 resulted in highly reproducible results (CV=2.9%), while a high variability was observed when Ct values were higher than 33 (CV=10.3%). Conclusions: This study demonstrates that time and temperature of nasopharyngeal swabs storage do not significantly impact on results reproducibility. However, swabs sampling is a critical step, and especially in case of low viral load, might be a potential source of diagnostic errors. [ABSTRACT FROM AUTHOR]

Background: In a medical laboratory, changes may be made to the analytical phase of diagnostic testing whenever a new test or the issue of a 'new generation' kit or new diagnostic system is required. In such cases, ISO 15189:2012 accreditation can assist laboratory professionals. The aim of the present study was to propose a working pathway for introducing new examination procedures into clinical practice in accordance with the ISO 15189:2012 standard, through the exemplars of 17-hydroxy progesterone, dehydroepiandrosterone sulphate and vitamin D. Methods: The working pathway includes the following steps: (i) analysing examination procedures under evaluation, (ii) analysing examination procedures currently in use, (iii) verifying metrological traceability, (iv) verifying examination procedures and (v) evaluating comparability of results. Results: The analysis of instructions for use issued by manufacturers revealed that metrological traceability was reported only for vitamin D. The imprecision verification was satisfactory, the imprecision obtained by the laboratory in terms of total imprecision always being less than the specified total imprecision. In only one case (IQC level 1, 17-hydroxy progesterone), the total upper verification limit was calculated. The trueness verification was satisfactory for all examination procedures, except for 17-hydroxy progesterone (second material). Passing–Bablok regression analyses in the comparability study demonstrated significant differences for all the examination procedures. Conclusions: The working pathway described for examination procedures in routine practice is in accordance with the requirements of ISO 15189:2012 accreditation and takes feasibility into account (as its main goal), based on the cost/patient benefit ratio. [ABSTRACT FROM AUTHOR]

A large portion of the human genome transcribes RNA sequences that do not code for any proteins. The first of these sequences was identified in 1993, and the best known noncoding RNAs are microRNA (miRNAs). It is now fully established that miRNAs regulate approximately 30% of the known genes that codify proteins. miRNAs are involved in several biological processes, like cell proliferation, differentiation, apoptosis and metastatization. These RNA products regulate gene expression at the post-transcriptional level, modulating or inhibiting protein expression by interacting with specific sequences of mRNAs. Mature miRNAs can be detected in blood plasma, serum and also in a wide variety of biological fluids. They can be found associated with proteins, lipids as well as enclosed in exosome vesicles. We know that circulating miRNAs (C-miRNAs) can regulate several key cellular processes in tissues different from the production site. C-miRNAs behave as endogenous mediators of RNA translation, and an extraordinary knowledge on their function has been obtained in the last years. They can be secreted in different tissue cells and associated with specific pathological conditions. Significant evidence indicates that the initiation and progression of several pathologies are "highlighted" by the presence of specific C-miRNAs, underlining their potential diagnostic relevance as clinical biomarkers. Here we review the current literature on the possible use of this new class of molecules as clinical biomarkers of diseases. [ABSTRACT FROM AUTHOR]

A large body of evidence collected in recent years demonstrates the vulnerability of the extra-analytical phases of the total testing process (TTP) and the need to promote quality and harmonization in each and every step of the testing cycle. Quality indicators (QIs), which play a key role in documenting and improving quality in TTP, are essential requirements for clinical laboratory accreditation. In the last few years, wide consensus has been achieved on the need to adopt universal QIs and common terminology and to harmonize the management procedure concerning their use by adopting a common metric and reporting system. This, in turn, has led to the definition of performance specifications for extra-analytical phases based on the state of the art as indicated by data collected on QIs, particularly by clinical laboratories attending the Model of Quality Indicators program launched by the Working Group "Laboratory Errors and Patient Safety" of the International Federation of Clinical Chemistry and Laboratory Medicine. Harmonization plays a fundamental role defining not only the list of QIs to use but also performance specifications based on the state of the art, thus providing a valuable interlaboratory benchmark and tools for continuous improvement programs. [ABSTRACT FROM AUTHOR]

Objectives: We investigated whether polymorphisms (SNPs) in the promoter region of TNFA, or in the autoinflammatory TNFRSF1A and MEFV genes, concur with HLA-B27 in enhancing the risk of Spondyloarthritis (SpA) and/or in predicting the response to anti-TNFα treatment. Methods: 373 controls and 137 SpA (82 with Psoriatic Arthritis-PsA and 55 with Ankylosing Spondylitis- AS; 98/137 under TNFα inhibitor therapy) from the Veneto Region (Italy) were studied. TNFA polymorphisms (-1031T>C;-857C>T;-376G>A;-308G>A;-238G>A) and HLA-B27 were assayed by RT-PCR. Direct sequencing of MEFV (exons 2,3,5 and 10) and TNFRSF1A (exons 2,3,4 and 6) genes were performed. Results: HLA-B27 was associated with AS (χ2 = 120.1; p = 0.000). Only the TNFA -1031T>C was singly associated with SpA, and the haplotype C/G, resulting from -1031T>C/-308G>A combination, was significantly associated with a reduced risk of SpA (OR: 0.67, CI: 0.46–0.97; p = 0.035). Two SNPs were identified in TNFRSF1A, the R92Q (Minor allele frequency-MAF = 0.034) and c.625+10A>G (MAF = 0.479). None of them was associated with SpA (p>0.05). The TNFRSF1A c.625+10 G allele was associated with late response to anti-TNFα therapy (p = 0.031). Twenty-one SNPs were identified in MEFV gene, 10 with a known potential functional significance. Variant alleles were extremely rare in our population (MAF<0.025) except for R202Q (MAF = 0.27). None was associated with SpA diagnosis (p>0.05). Conclusion: TNFRSF1A and MEFV gene SNPs are not associated with SpA in the North-East of Italy. AS risk appears to depend not only on HLA-B27, but also on the protective TNFA haplotype -1031C/-308G. The TNFRSF1A c.625+10A>G impacts on the response to anti-TNFα therapy. [ABSTRACT FROM AUTHOR]

The term spondyloarthritis (SpA) is used to describe a group of multifactorial chronic inflammatory diseases characterized by a predisposing genetic background and clinical manifestations typically involving the sacroiliac joint. The absence of pathognomonic clinical and/or laboratory findings generally results in a delay in diagnosis and, consequently, in treatment. In addition, 20–40% of SpA patients are non-responders to tumor necrosis factor (TNF) inhibitor therapies. Given these considerations, it is important to identify biomarkers that can facilitate the diagnosis and assessment of disease activity. As inflammation plays a key role in the pathogenesis of SpA, inflammatory mediators have been investigated as potential biomarkers for diagnosing the disease and predicting response to therapy. Some investigators have focused their attention on the role of matrix metalloproteinases (MMPs), which are known to be markers of synovial inflammation that is generated in the joint in reaction to inflammatory stimuli. Several studies have been carried out to verify if serum MMPs levels could be useful to diagnose SpA, to assess disease severity, and to predict response to TNF inhibitor therapy. The current review focuses on MMPs’ role in SpA pathogenesis, diagnosis and therapeutic implications. [ABSTRACT FROM AUTHOR]

Background: TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner. Methods: 511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence). Results: Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations. Conclusion: TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk. [ABSTRACT FROM AUTHOR]

Background: Tumor stage predicts pancreatic cancer (PDAC) prognosis, but prolonged and short survivals have been described in patients with early-stage tumors. Circulating microRNA (miRNA) are an emerging class of suitable biomarkers for PDAC prognosis. Our aim was to identify whether serum miRNA signatures predict survival of early-stage PDAC. Methods: Serum RNA from archival 15 stage I-III PDAC patients and 4 controls was used for miRNAs expression profile (Agilent microarrays). PDAC patients with comparable age, gender, diabetes, jaundice and surgery were classified according to survival: less than 14 months (7/15 pts, group A) and more than 22 months (8/15 pts, group B). Bioinformatic data analysis was performed by two-class Significance Analysis of Microarray (SAM) algorithm. Binary logistic regression analyses considering PDAC diagnosis and outcome as dependent variables, and ROC analyses were also performed. Results: 2549 human miRNAs were screened out. At SAM, 76 differentially expressed miRNAs were found among controls and PDAC (FDR = 0.4%), the large majority (50/76, 66%) of them being downregulated in PDAC with respect to controls. Six miRNAs were independently correlated with early PDAC, and among these, hsa-miR-6821-5p was associated with the best ROC curve area in distinguishing controls from early PDAC. Among the 71 miRNAs differentially expressed between groups A and B, the most significant were hsa-miR-3135b expressed in group A only, hsa-miR-6126 and hsa-miR-486-5p expressed in group B only. Eight miRNAs were correlated with the presence of lymph-node metastases; among these, hsa-miR-4669 is of potential interest. hsa-miR-4516, increased in PDAC and found as an independent predictor of survival, has among its putative targets a series of gens involved in key pathways of cancer progression and dissemination, such as Wnt and p53 signalling pathways. Conclusions: A series of serum miRNAs was identified as potentially useful for the early diagnosis of PDAC, and for establishing a prognosis. [ABSTRACT FROM AUTHOR]

Quality indicators (Qls) measure the extent to which set targets are attained and provide a guantitative basis for achieving improvement in care and, in particular, laboratory services. A body of evidence collected in recent years has demonstrated that most errors fall outside the analytical phase, while the pre- and post-analytical steps have been found to be more vulnerable to the risk of error. However, the current lack of attention to extra-laboratory factors and related Qls prevent clinical laboratories from effectively improving total quality and reducing errors. Errors in the pre-analytical phase, which account for 50% to 75% of all laboratory errors, have long been included in the 'identification and sample problems' category. However, according to the International Standard for medical laboratory accreditation and a patient-centered view, some additional Qls are needed. In particular, there is a need to measure the appropriateness of all test request and reguest forms, as well as the quality of sample transportation. The Qls model developed by a working group of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) is a valuable starting point for promoting the harmonization of available Qls, but further efforts should be made to achieve a consensus on the road map for harmonization. [ABSTRACT FROM AUTHOR]

The estimation of measurement uncertainty (MU) in clinical laboratories is of crucial importance in improving laboratory testing quality and correctly interpreting results. However, it is difficult for clinical laboratories to reliably estimate MU since current guidelines and standards fail to clearly define and harmonize methods to be used for this purpose. To propose a model for MU estimation in relation to test interpretation on the basis of three different scenarios. MU estimation was evaluated regard to the inclusion of imprecision and bias components for different test purposes. Three scenarios were identified. The expanded uncertainty values were: 0.56 ng/L and 1.86 ng/L for troponin I at levels 4.95 ng/L and 20.60 ng/L, respectively; 9.4 μmol/L and 27.2 μmol/L for creatinine at levels 121.0 μmol/L and 390.0 μmol/L, respectively. 0.2 mmol/L and 0.3 mmol/L for potassium at levels 4.03 mmol/L and 6.35 mmol/L, respectively; 0.36 mmol/L for glucose at level of around 6 mmol/L. These values represent MU results estimated for the three scenarios, which contemplate test results used for 1a) short term patient monitoring, 1b) longer term patient monitoring, 2) comparison with reference intervals and 3) comparison with a clinical decision point. Goal-based measurement uncertainty estimation in clinical laboratories provides an opportunity to improve laboratory testing quality and reduce risk in the interpretation of clinical results. [ABSTRACT FROM AUTHOR]

Quality indicators (QIs) are key tools for improving the quality of laboratory services, by reducing error rates and safeguarding patient safety. A body of accumulated evidence confirms the relevance of QIs and their impact on the overall quality of laboratory information. The consensus achieved on a list of "harmonized" QIs, along with the system used for data collection and reporting throughout an international benchmarking programme, has enabled achieving realistic performance targets, based on knowledge of the state-of-the-art. Data collected in 2017 and 2018 have been analyzed and performance measures obtained by laboratories participating in the project are summarized in the present article. The laboratory performance measures have been classified into three levels (optimum, desirable or minimum) in agreement with the widely accepted model of analytical quality specifications. [ABSTRACT FROM AUTHOR]