Assessing post-analytical phase harmonization in European laboratories: a survey promoted by the EFLM Working Group on Harmonization.

OBJECTIVES: Harmonization of the laboratory total testing process (TTP) is critical to improving patient outcome. In 2016, an EFLM survey on the harmonization of TTP underlined the serious shortcomings pertaining to the post-analytical phase. In 2023, the WG-H conducted a new survey aiming to update information in the 2016 harmonization report in order to ascertain whether countries that had declared they were keen to adopt SI units had continued with this program, the aim being to verify the state-of art in harmonization units in areas of laboratory medicine not included in the previous survey. METHODS: Questionnaires were distributed to the Presidents and National Representatives of EFLM Full Member Societies and EFLM affiliate Members. The survey questions were grouped into three categories: measurement units, reference intervals, and nomenclature/terminology, and results were evaluated using Survey Monkey software and Excel. RESULTS: A total of 123 questionnaires from 31 countries were analyzed. A trend (+19.3 %) was observed toward a wider use of SI units for general clinical biochemistry parameters. The results for tests not included in the 2016 survey (i.e., endocrinology diagnostics and coagulation panels), demonstrated that for reports on hormones, responses were satisfactory, 70-90 % of the responders adopting the recommended units, whereas for coagulation test panels, a serious lack of harmonization was found, "seconds", which are inaccurate and not recommended, being widely used units (91 %). CONCLUSIONS: The findings made in the 2023 survey demonstrated a progressive, albeit slow, improvement in harmonization reports. However, further efforts at improvement are mandatory.

Real-world usage of Chronic Kidney Disease - Mineral Bone Disorder (CKD-MBD) biomarkers in nephrology practices.

Background: Chronic kidney disease mineral bone disorder (CKD-MBD) is a condition characterized by alterations of calcium, phosphate, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF-23) metabolism that in turn promote bone disorders, vascular calcifications, and increase cardiovascular (CV) risk. Nephrologists' awareness of diagnostic, prognostic, and therapeutic tools to manage CKD-MBD plays a primary role in adequately preventing and managing this condition in clinical practice. Methods: A national survey (composed of 15 closed questions) was launched to inquire about the use of bone biomarkers in the management of CKD-MBD patients by nephrologists and to gain knowledge about the implementation of guideline recommendations in clinical practice. Results: One hundred and six Italian nephrologists participated in the survey for an overall response rate of about 10%. Nephrologists indicated that the laboratories of their hospitals were able to satisfy request of ionized calcium levels, 105 (99.1%) of both PTH and alkaline phosphatase (ALP), 100 (94.3%) of 25(OH)D, and 61 (57.5%) of 1.25(OH)2D; while most laboratories did not support the requests of biomarkers such as FGF-23 (intact: 88.7% and c-terminal: 93.4%), Klotho (95.3%; soluble form: 97.2%), tartrate-resistant acid phosphatase 5b (TRAP-5b) (92.5%), C-terminal telopeptide (CTX) (71.7%), and pro-collagen type 1 N-terminal pro-peptide (P1NP) (88.7%). As interesting data regarding Italian nephrologists' behavior to start treatment of secondary hyperparathyroidism (sHPT), the majority of clinicians used KDOQI guidelines (n = 55, 51.9%). In contrast, only 40 nephrologists (37.7%) relied on KDIGO guidelines, which recommended referring to values of PTH between two and nine times the upper limit of the normal range. Conclusion: Results point out a marked heterogeneity in the management of CKD-MBD by clinicians as well as a suboptimal implementation of guidelines in Italian clinical practice.

Therapeutic drug monitoring of monoclonal antibodies in chronic inflammatory diseases: A snapshot of laboratories and applications across Europe.

The European Cooperation in Science and Technology (COST) action ENOTTA (The European Network on Optimising Treatment with Therapeutic Antibodies in chronic inflammatory diseases) was launched in 2022. To pave the way for harmonization of analytical methods for quantitation of serum levels of therapeutic antibodies in research and clinical settings, ENOTTA recently performed an online survey mapping laboratories in the field. The survey, which contained 30 questions surrounding therapeutic drug monitoring of relevant drugs and anti-drug antibodies, was distributed via the ENOTTA and European Federation of Clinical Chemistry and Laboratory networks. Among 63 respondents across Europe, 45 reported analytical activity, with a range of utilized methods. Future engagement of as many sites as possible will enable comparison of methodologies and facilitate progress in the field.

Tubular phosphate transport: a comparison between different methods of urine sample collection in FGF23-dependent hypophosphatemic syndromes.

OBJECTIVES: Tubular maximum phosphate reabsorption per glomerular filtration rate (TmP/GFR) is used to evaluate renal phosphate reabsorption and it is a useful tool for the differential diagnosis of hypophosphatemic syndromes. TmP/GFR is typically calculated from fasting plasma and second morning void urine samples, obtained 2 h after the first void (TmP/GFR 2 h). The purpose of this study was to evaluate if TmP/GFR calculated from 24 h urine collection (TmP/GFR 24 h) can be used as an alternative for TmP/GFR 2 h in patients with urine phosphate wasting. METHODS: We enrolled adult patients with X-linked hypophosphatemia (XLH) or tumor-induced osteomalacia (TIO). All patients underwent blood and urine sample collections, to calculate TmP/GFR 24 h and TmP/GFR 2 h. RESULTS: Twenty patients (17 XLH and 3 TIO), aged 24-78 years, were included. All patients had low TmP/GFR 2 h (0.35 mmol/L, IQR 0.24-0.47 mmol/L) and TmP/GFR 24 h (0.31 mmol/L, IQR 0.22-0.43 mmol/L). The concordance correlation coefficient between TmP/GFR 2 h and TmP/GFR 24 h was 0.86 (95 % CI: 0.69-0.93), with a systematic bias of 0.05 mmol/L (95 % limits of agreement: -0.10 to 0.20). Furthermore, in 70 % (i.e., 14 patients out of 20) and 80 % (i.e., 16 patients out of 20) of cases the difference between TmP/GFR 2 h and TmP/GFR 24 h was within ±30 % and ±35 %, respectively. CONCLUSIONS: Despite TmP/GFR 2 and 24 h show a relatively suboptimal agreement, the difference between the two parameters appears to be small and not clinically significant in the setting of adult patients with FGF23-dependent urine phosphate wasting and secondary hypophosphatemia.

Analytical and clinical evaluations of SNIBE Maglumi chemiluminescent immunoassay for the detection of SARS-CoV-2 antigen in salivary samples.

OBJECTIVES: In this study, we describe the analytical and clinical performances of the SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay (MAG-CLIA) on salivary samples. METHODS: Limit of detection (LOD), linearity and precision were tested for values close to or below the declared LOD. Clinical performance of MAG-CLIA was evaluated on leftover salivary samples from the healthcare workers (HCW) surveillance program, at the University-Hospital of Padova. Salivary samples were analyzed by Lumipulse G SARS-CoV-2 Ag, and in case where the values exceeded 0.41 ng/L, further testing was conducted using TaqPathTM COVID-19 RT-PCR (Applied Biosystems, Thermo Fisher Scientific). RESULTS: The estimated MAG-CLIA LOD was 3 ng/L, with repeatability of 7.5 %. Good linearity was demonstrated by diluting two samples at 52.7 ng/L and 211.4 ng/L. Of the 228 HCW samples, 59/228 (25.9 %) were positive, 169/228 (74.1 %) were negative. MAG-CLIA SARS-CoV-2 sAg median level (and interquartile range [IQR]) was 5.03 ng/L (<0.001-35.8 ng/L) for positive and <0.001 ng/L (<0.001 ng/L) for negative samples. MAG-CLIA AUC was 0.795 (95 % CI: 0.720-0.871). Using the best cut-off, 3.5 ng/L, sensitivity and specificity were 57.1 % (95 % CI: 42.2-71.2 %) and 97.0 % (95 % CI: 93.2-99.0 %), respectively. The agreement with the molecular assay was 88.1 % (Cohen's kappa 0.606 [SE=0.066, p<0.001]). CONCLUSIONS: The analytical performances of MAG-CLIA are satisfactory, also when values below LOD were tested. In saliva samples, although specificity was elevated, clinical performance was not comparable with that on nasopharyngeal swabs (NPS).

Troponin T in spinal and bulbar muscular atrophy (SBMA).

Serum biomarkers that might detect clinical progression are currently lacking for Spinal and bulbar muscular atrophy (SBMA), thus limiting the effectiveness of possible future pharmacological trials. Elevation of cardiac troponin T (cTnT) unrelated to myocardial damage in a motor neuron (MN) disease as amyotrophic lateral sclerosis (ALS) was associated to disease severity. We enrolled 47 SBMA patients and 5 Spinal muscular atrophy (SMA) type 3 adult patients as control group; each SBMA patient was evaluated at baseline and at one-year follow-up visit. Demographic and clinical data including functional scores (SBMAFRS) were collected; serum was collected as standard of care and tested for cardiac troponins. Levels of cTnT but not cTnI were increased in SBMA with respect to reference values; unlike other neuromuscular diseases, SMA patients had overall normal cTnT values. Median cTnT concentrations did not change after one year and values were correlated to motor function, particularly with lower limb subdomain, at baseline only. Variations of cTnT and of SBMAFRS were unrelated. The cautiously promising results of cTnT as potential biomarker should undergo a more extensive clinical validation, including studies with longer follow-up period. When evaluating SBMA patients for a potential cardiac damage cTnI testing should be coupled or preferred to cTnT.

Measurement of Cardiac-Specific Biomarkers in the Emergency Department: New Insight in Risk Evaluation.

The aim of this article review is to analyze some models and clinical issues related to the implementation of accelerated diagnostic protocols based on specific cardiac biomarkers in patients admitted to the emergency department (ED) with symptoms compatible with acute cardiac disorders. Four specific clinical issues will be discussed in detail: (a) pathophysiological and clinical interpretations of circulating hs-cTnI and hs-cTnT levels; (b) the clinical relevance and estimation of the biological variation of biomarkers in patients admitted to the ED with acute and severe diseases; (c) the role and advantages of the point-of-care testing (POCT) methods for cardiac-specific biomarkers in pre-hospital and hospital clinical practice; and (d) the clinical role of specific cardiac biomarkers in patients with acute heart failure (AHF). In order to balance the risk between a hasty discharge versus the potential harms caused by a cardiac assessment in patients admitted to the ED with suspected acute cardiovascular disease, the measurement of specific cardiac biomarkers is essential for the early identification of the presence of myocardial dysfunction and/or injury and to significantly reduce the length and costs of hospitalization. Moreover, specific cardiac biomarkers (especially hs-cTnI and hs-cTnT) are useful predictors of mortality and major adverse cardiovascular events (MACE) in patients admitted to the ED with suspected acute cardiovascular disease. To guide the implementation of the most rapid algorithms for the diagnosis of Non-ST-Elevation Myocardial Infarction (NSTEMI) into routine clinical practice, clinical scientific societies and laboratory medicine societies should promote collaborative studies specifically designed for the evaluation of the analytical performance and, especially, the cost/benefit ratio resulting from the use of these clinical protocols and POCT methods in the ED clinical practice.

Serum biomarkers of remodeling in severe asthma with fixed airway obstruction and the potential role of KL-6.

Over 3% of asthmatic patients are affected by a particularly severe form of the disease ("severe asthma", SA) which is often refractory to standard treatment. Airway remodeling (AR), which can be considered a critical characteristic of approximately half of all patients with SA and currently thought to be the main mechanism triggering fixed airway obstruction (FAO), seems to be a key factor affecting a patient's outcome. Despite the collective efforts of internationally renowned experts, to date only a few biomarkers indicative of AR and no recognizable biomarkers of lung parenchymal remodeling have been identified. This work examines the pathogenesis of airway and lung parenchymal remodeling and the serum biomarkers that may be able to identify the severe asthmatic patients who may develop FAO. The study also aims to examine if Krebs von den Lungen-6 (KL-6) could be considered a diagnostic biomarker of lung structural damage in SA.

SARS-CoV-2 specific T-cell humoral response assessment after COVID-19 vaccination using a rapid direct real-time PCR amplification.

OBJECTIVES: The SARS-CoV-2 immune response is mediated by both humoral and cellular immunity. In this study, SARS-CoV-2 specific cellular immunity was tested by a novel direct real-time PCR (dRT-PCR) assay, targeting mRNA of CXCL10, and compared with respect to an ELISA measuring interferon gamma (IFN-γ) release. METHODS: Whole blood (Li-He) and serum samples were collected from 92 healthcare workers (HCW), with three doses of homologous (Pfizer/BioNTech, n=74) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n=18) vaccinations. Li-He samples were incubated with SCV2 PANEL-1-T-ACTIVATION (Hyris srl, Lodi, Italy), or CoV-2 IGRA TUBE ELISA (Euroimmune, Lubeck, Germany). CXCL10 mRNA expression was analyzed by bCube/bApp (Hyris), while IFN-γ was evaluated by quant-T-Cell SARS-CoV-2 ELISA (Euroimmune). Anti-SARS-CoV-2 S-RBD IgG levels were measured in sera using a CLIA assay (Snibe, Shenzen, China). RESULTS: Imprecision of dRT-PCR assay was found to be satisfactory, and the two methods for measuring T cell immunity to SARS-CoV-2 peptides agreed in 82/87 (94.2%) of results. At qualitative dRT-PCR analyses, 81 subjects (93.2%) resulted as reactive to SARS-CoV-2 peptides, 3 (3.4%) were borderline and 3 were negative (3.4%). At univariate and multivariate analyses of quantitative dRT-PCR mRNA of CXCL10 and IFN-γ release results showed no difference between HCW with previous infection, homologous/heterologous vaccination, or demographical features. Anti-SARS-CoV-2 S-RBD IgG was associated with the previous infection and the time between the last vaccination or positivity. CONCLUSIONS: Direct RT-PCR appeared accurate for determining the presence or absence of immunoreactivity of SARS-CoV-2 specific T cells, especially when rapid analyses are required, such as for organ transplantation.

Variability of cardiac troponin levels in normal subjects and in patients with cardiovascular diseases: analytical considerations and clinical relevance.

In accordance with all the most recent international guidelines, the variation of circulating levels of cardiac troponins I and T, measured with high-sensitivity methods (hs-cTnI and hs-cTnT), should be used for the detection of acute myocardial injury. Recent experimental and clinical evidences have demonstrated that the evaluation of hs-cTnI and hs-cTnT variations is particularly relevant: a) for the differential diagnosis of Acute Coronary Syndromes (ACS) in patients admitted to the Emergency Department (ED); b) for the evaluation of cardiovascular risk in patients undergoing major cardiac or non-cardiac surgery, and in asymptomatic subjects of the general population aged >55 years and with co-morbidities; c) for the evaluation of cardiotoxicity caused by administration of some chemotherapy drugs in patients with malignant tumors. The aim of this document is to discuss the fundamental statistical and biological considerations on the intraindividual variability of hs-cTnI and hs-cTnT over time in the same individual. Firstly, it will be discussed in detail as the variations of circulating levels strictly depend not only on the analytical error of the method used but also on the intra-individual variability of the biomarker. Afterwards, the pathophysiological interpretation and the clinical relevance of the determination of the variability of the hs-cTnI and hs-cTnT values ​​ in patients with specific clinical conditions are discussed. Finally, the evaluation over time of the variation in circulating levels of hs-cTnI and hs-cTnT is proposed for a more accurate estimation of cardiovascular risk in asymptomatic subjects from the general population.

Diagnostic and prognostic performance of urinary neutrophil gelatinase-associated lipocalin in patients with cirrhosis and acute kidney injury.

BACKGROUND AND AIMS: Acute kidney injury (AKI) commonly occurs in patients with decompensated cirrhosis. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) could help discriminate between different etiologies of AKI. The aim of this study was to investigate the use of uNGAL in (1) the differential diagnosis of AKI, (2) predicting the response to terlipressin and albumin in patients with hepatorenal syndrome-AKI (HRS-AKI), and (3) predicting in-hospital mortality in patients with AKI. APPROACH AND RESULTS: One hundred sixty-two consecutive patients with cirrhosis and AKI were included from 2015 to 2020 and followed until transplant, death, or 90 days. Standard urinary markers and uNGAL were measured. Data on treatment, type, and resolution of AKI were collected. Thirty-five patients (21.6%) had prerenal AKI, 64 (39.5%) HRS-AKI, 27 (16.7%) acute tubular necrosis-AKI (ATN-AKI), and 36 (22.2%) a mixed form of AKI. Mean values of uNGAL were significantly higher in ATN-AKI than in other types of AKI (1162 ng/ml [95% CI 423-2105 ng/ml] vs. 109 ng/ml [95% CI 52-192 ng/ml]; p  < 0.001). uNGAL showed a high discrimination ability in predicting ATN-AKI (area under the receiver operating characteristic curve, 0.854; 95% CI 0.767-0.941; p  < 0.001). The best-performing threshold was found to be 220 ng/ml (sensitivity, 89%; specificity, 78%). The same threshold was independently associated with a higher risk of nonresponse (adjusted OR [aOR], 6.17; 95% CI 1.41-27.03; p  = 0.016). In multivariable analysis (adjusted for age, Model for End-Stage Liver Disease, acute-on-chronic liver failure, leukocytes, and type of AKI), uNGAL was an independent predictor of in-hospital mortality (aOR, 1.74; 95% CI 1.26-2.38; p  = 0.001). CONCLUSIONS: uNGAL is an adequate biomarker for making a differential diagnosis of AKI in cirrhosis and predicting the response to terlipressin and albumin in patients with HRS-AKI. In addition, it is an independent predictor of in-hospital mortality.

Seroprevalence of SARS-CoV-2 antibodies in Italy in newborn dried blood spots.

OBEJCTIVES: Serosurveys can be used to monitor COVID-19 seroprevalence and conduct surveillance. Dried blood spot (DBS), used increasingly as a valuable sample to assay severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies (Ab), has several advantages, particularly in infants, due to the limited amount of blood required and its utility in testing a large number of samples in a limited time-frame. We evaluated SARS-CoV-2 IgG Ab prevalence in newborn DBS in the Trentino region of Italy, during the time period January 2020 - December 2021. METHODS: Anti-SARS-CoV-2 IgG levels were determined in DBS by means of Anti-SARS-CoV-2 QuantiVac IgG ELISA assay (Euroimmun, Lubeck, Germany). RESULTS: Analyses included 2,400 DBS from newborns (54% M, 46% F), samples being collected 2-3 days after birth. The first DBS that tested positive for anti-SARS-CoV-2 IgG antibodies was found in March 2020 and, up to May 2020, only 4 positive results were detected overall. Starting from June 2020, the positivity thresholds increased according to the epidemiological waves of the COVID-19 pandemic in Italy, with a robust increment in the winters of 2020 and 2021. The percentage of positive DBS rose from 0 to 6% to 10-47%, in 2020 and 2021, respectively. CONCLUSIONS: This study demonstrates DBS is a suitable tool for both epidemiological purposes and surveillance in the SARS-CoV-2 pandemic, particularly in newborns and pregnant women, saving blood waste and sparing patients any discomfort.

The harmonization issue in laboratory medicine: the commitment of CCLM.

The analytical quality of the clinical laboratory results has shown a significant improvement over the past decades, thanks to the joint efforts of different stakeholders, while the comparability among the results produced by different laboratories and methods still presents some critical issues. During these years, Clinical Chemistry and Laboratory Medicine (CCLM) published several papers on the harmonization issue over all steps in the Total Testing Process, training an important number of laboratory professionals in evaluating and monitoring all the criticisms inherent to the pre-analytical, as well as analytical and post analytical phases: from the consensus statement on the most informative testing in emergency setting, to the prevention and detection of hemolysis or to patients identification and tube labeling procedures, as far as to different approaches to harmonize hormones measurements or to describe new reference methods or to harmonize the laboratory report. During these years the commitment of the journal, devoted to the harmonization processes has allowed to improve the awareness on the topic and to provide specific instruments to monitor the rate of errors and to improve patients safety.

Evaluation of the cardiovascular risk in patients undergoing major non-cardiac surgery: role of cardiac-specific biomarkers.

Major adverse cardiovascular events are frequently observed in patients undergoing major non-cardiac surgery during the peri-operative period. At this time, the possibility to predict cardiovascular events remains limited, despite the introduction of several algorithms to calculate the risk of adverse events, mainly death and major adverse cardiovascular events (MACE) based on the clinical history, risk factors (sex, age, lipid profile, serum creatinine) and non-invasive cardiac exams (electrocardiogram, echocardiogram, stress tests). The cardiac-specific biomarkers natriuretic peptides (NPs) and cardiac troponins (cTn) have been proposed as additional tools for risk prediction in the peri-operative period, particularly for the identification of myocardial injury in patients undergoing major non-cardiac surgery. The prognostic information from the measurement of BNP/NT-proBNP and hs-cTn is independent and complementary to other important indicators of risk, also including ECG and imaging techniques. Elevated levels of cardiac-specific biomarkers before surgery are associated with a markedly higher risk of MACE during the peri-operative period. BNP/NT-proBNP and hs-cTn should be measured in all patients during the clinical evaluation before surgery, particularly during intermediate- or high-risk surgery, in patients aged >65 years and/or with comorbidities. Several questions remain to be assessed in dedicated clinical studies, such as how to optimize the management of patients with raised cardiac specific biomarkers before surgery, and whether a strategy based on biomarker measurement improves patient outcomes and is cost-effective.

Time for Revival of Bone Biopsy with Histomorphometric Analysis in Chronic Kidney Disease (CKD): Moving from Skepticism to Pragmatism.

Bone Biopsy (BB) with histomorphometric analysis still represents the gold standard for the diagnosis and classification of different forms of renal osteodystrophy. Bone biopsy is the only technique able to provide comprehensive information on all bone parameters, measuring static and dynamic parameters of turnover, cortical and trabecular microarchitecture, and mineralization defects. In nephrological practice, bone biopsy yields relevant indications to support therapeutic choices in CKD, heavily impacting the management and prognosis of uremic patients. Unfortunately, the use of bone biopsy has decreased; a lack of expertise in performing and interpreting, perceived procedure invasiveness and pain, and reimbursement issues have all contributed to this decline. Nevertheless, both bone biomarkers and instrumental images cannot be considered reliable surrogates for histological findings, being insufficiently accurate to properly evaluate underlying mineral and bone disorders. This is a multidisciplinary position paper from the Nephrology and Osteoporosis Italian Scientific Societies with the purpose of restating the role of bone biopsy in CKD patient management and of providing strong solutions to allow diffusion of this technique in Italy, but potentially also in other countries. The Italian approach through the optimization and standardization of bone biopsy procedure, the construction of the Italian Hub and Spoke network, and a request for adjustment and national homogenization of reimbursement to the Italian Health Ministry has led the way to implement bone biopsy and to improve CKD patient management and prognosis.

The Role of Vitamin K in CKD-MBD.

PURPOSE OF REVIEW: We describe the mechanism of action of vitamin K, and its implication in cardiovascular disease, bone fractures, and inflammation to underline its protective role, especially in chronic kidney disease (CKD). RECENT FINDINGS: Vitamin K acts as a coenzyme of y-glutamyl carboxylase, transforming undercarboxylated in carboxylated vitamin K-dependent proteins. Furthermore, through the binding of the nuclear steroid and xenobiotic receptor, it activates the expression of genes that encode proteins involved in the maintenance of bone quality and bone remodeling. There are three main types of K vitamers: phylloquinone, menaquinones, and menadione. CKD patients, for several conditions typical of the disease, are characterized by lower levels of vitamin K than the general populations, with a resulting higher prevalence of bone fractures, vascular calcifications, and mortality. Therefore, the definition of vitamin K dosage is an important issue, potentially leading to reduced bone fractures and improved vascular calcifications in the general population and CKD patients.