Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation.

BACKGROUND: Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient's response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. METHODS: A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. RESULTS: Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. CONCLUSIONS: Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

In this article, we evaluated the role of OCTN1, an organic cation transporter, in modifying gut microbiota and immune T cell populations, as well as its effects on experimental colitis and the response to infliximab treatment.

Genetic Characterization of a Cohort of Italian Patients with Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency.

INTRODUCTION: Defects in the steroid 21-hydroxylase gene (CYP21A2) cause 21-hydroxylase deficiency (21OHD), the main cause of congenital adrenal hyperplasia (CAH). The disease shows a broad spectrum of clinical forms, ranging from severe or classical (salt wasting, SW, and simple virilizing, SV), to mild late onset or nonclassical (NC). 21OHD affects 1 in 15,000 in its severe classic form and 1 in 200-1000 in its mild NC form. There are many studies reporting the frequency of CYP21A2 pathogenic variants in different populations; however, few of them provide comprehensive information about Italian patients. Here, we present genetic results from a cohort of 245 unrelated Italian individuals with clinical diagnosis of CAH due to 21OHD. METHODS: A specific polymerase chain reaction (PCR) protocol combined with Sanger sequencing was used for CYP21A2 analysis. The multiplex ligation-dependent probe amplification (MLPA) assay was employed for copy number variation (CNV) determination. RESULTS: One hundred fourteen (46.5%) of the index cases had the NC form, 57 (23.3%) had the SV form, and 74 (30.2%) presented the SW form of the disease. The most prevalent variant found in NC patients was the p.Val282Leu (51.3%), while the most frequent variants in the classical form were p.Ile173Asn (8.6%) and c.293-13C>G (26.0%). In our study, the frequency of large rearrangements was 15.3%, with CAH-X alleles representing 40% of all DEL/CONV. In addition, 12 alleles carried rare variants, and 1 had a novel variant p.(Arg342Gln). We observed phenotype-genotype correlation in 94.7% of cases. A complete concordance was observed in Groups 0 (enzyme activity completely impaired) where all patients had the SW form as expected. In Group A (0-1% residual enzyme activity), 78.4% of patients had the anticipated SW form while 21.6% were diagnosed with the SV form. Within Group B (~ 2% residual enzyme activity), 93.4% of patients exhibited SV form and 6.5% SW disease. Finally, 92.6% and 7.4% of patients belonging to Group C (enzyme partially impaired to ~ 20-60% residual activity) exhibited NC and SV phenotypes, respectively. CONCLUSION: This work, representing a comprehensive genetic study, expanded the CYP21A2 variants spectrum of Italian patients with 21OHD and could be helpful in prenatal diagnosis and genetic counseling.

Comparison of Fixed and Live Cell-Based Assay for the Detection of AChR and MuSK Antibodies in Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Live cell-based assay (CBA) can detect acetylcholine receptors (AChRs) or muscle-specific tyrosine kinase (MuSK) antibodies (Abs) in a proportion of patients with radioimmunoassay (RIA)-double seronegative myasthenia gravis (dSN-MG). A commercial fixed CBA for AChR and MuSK Abs has recently become available; however, comparative studies on fixed and live CBAs are lacking. In this study, we compared the performance of fixed and live CBAs in patients with RIA-dSN MG and assessed their sensitivity in RIA-positive MG samples and their specificity. METHODS: AChR and MuSK Abs were tested in 292 serum samples from 2 Italian MG referral centers by live and fixed CBAs: 192 from patients with MG and 100 from controls. All samples had been previously assessed by RIA: 66 were AChR positive, 40 MuSK positive, and 86 dSN. All controls were negative. Two independent raters assessed the CBA results. Fixed and live CBAs were compared with the McNemar test; interrater and interlaboratory agreement were assessed with Cohen's kappa or interclass correlation coefficient (ICC), as appropriate. RESULTS: In 86 RIA-dSN samples, fixed CBA detected Abs in 10 cases (11.6%, 95% CI 5.7-20.3), whereas live CBA detected Abs in 16 (18.6%, 95% CI 11.0-28.5) (p = 0.0143). Of these sera, those positive by fixed CBA were also positive by live CBA. In addition, live CBA could detect MuSK Abs in 4 and AChR Abs in 2 samples that were negative by fixed CBA, providing an 8% (95% CI 2.9-16.6) further increase in the Ab detection rate. These results were confirmed by flow cytometry. In the RIA-positive cohort, the sensitivity for AChR Abs was 98.5% (95% CI 91.9%-99.9%) for fixed CBA and 100% (95% CI 94.6-100) for live CBA (p = 0.1573). For both assays, the sensitivity for MuSK Abs was 100% (95% CI 91.2-100), and the specificity was 100% (95% CI 96.4-100). Interrater agreement was almost perfect for live and fixed CBAs (Cohen's kappa 0.972 and 0.978, respectively), alike interlaboratory agreement. Interrater agreement for the CBA score ranged from good to excellent (ICC: 0.832-0.973). DISCUSSION: Fixed CBA represents a valuable alternative to RIA for AChR and MuSK Ab detection in patients with MG and could be considered as a first-step diagnostic test. Live CBA can be useful in the serologic evaluation of RIA- and fixed CBA-negative samples.

A Novel GCK Large Genomic Rearrangement in a Patient with MODY-2 Detected by Clinical Exome Sequencing.

Maturity-onset diabetes of the young (MODY) is a rare form of non-autoimmune diabetes with an autosomal dominant inheritance. To date, 14 genes have been reported as genetic basis of MODY. GCK gene, encoding the glucokinase enzyme, was the first MODY gene to be identified. GCK heterozygous inactivating variants cause the GCK-MODY or MODY2 subtype. However, partial or whole gene deletions have been rarely identified, showing it to be a rare cause of GCK-MODY. We reported the molecular evaluation of a Ukrainian patient with clinical diagnosis of MODY2. We performed the Next generation sequencing of the clinical exome using the Clinical Exome Solution® kit (SOPHiA Genetics), followed by the design of a 14 genes virtual panel related to the suggestive diagnosis of MODY. Bioinformatics analysis was performed using the SOPHiA DDM platform (SOPHiA Genetics). The SALSA MLPA kit for MODY (MRC-Holland) was used for relative quantification of GCK exons. From the molecular evaluation, no pathogenic sequence variants were detected in the investigated genes. Copy Number Variation analysis was able to identify a large deletion involving the last three exons of the GCK gene. This result was confirmed by MLPA. To the best of our knowledge, the identified rearrangement has never been reported in the literature.

The biochemical diagnosis of acromegaly: revising the role of measurement of IGF-I and GH after glucose load in 5 questions.

INTRODUCTION: Acromegaly is a rare disorder characterized by the excessive secretion of growth hormone (GH), mostly caused by pituitary adenomas. While in full-blown cases the diagnosis is easy to establish, milder cases are more challenging. Additionally, establishing whether full cure after surgery is reached may be difficult. AREAS COVERED: In this article, we will review the challenges posed by the variability in measurements of GH and its main effector insulin-like growth factor I (IGF-I) due to both biological changes, co-morbidities, and assays variability. EXPERT OPINION: Interpretation of GH and IGF-I assays is important in establishing an early diagnosis of acromegaly, in avoiding misdiagnosis, and in establishing if cure is achieved by surgery. Physicians should be familiar with the variables that affect measurements of these 2 hormones, and with the performance of the assays available in their practice.

Does mild intrahepatic cholestasis of pregnancy require an aggressive management? Evidence from a prospective observational study focused on adverse perinatal outcomes and pathological placental findings.

OBJECTIVE: To ascertain the most effective approach in pregnancies complicated by mild intrahepatic cholestasis of pregnancy (mICP) by evaluating rates of adverse perinatal outcomes (APOs) and pathological placental findings. METHODS: A total of 89 pregnancies complicated by mICP (defined as total serum bile acids (TSBAs) levels <40 µmol/L) were included. One-drug (ursodeoxycholic acid [UDCA]) (n = 49, 55.1%) and combined (UDCA plus S-adenosyl methionine (SAMe)) (n = 40, 44.9%) therapies were compared. RESULTS: No differences were found in demographic, obstetric, and placental characteristics. In UDCA plus SAMe group, premature delivery was a common clinical decision (14.3 versus 25%, p-value = .201), with increased rates of instrumental vaginal delivery (VD; 28.6 versus 40%, p-value = .522), but similar cesarean section (CS) rates (26.5 versus 25%, p-value = .498). Mean placental weight was comparable (UDCA, mean 595.7 g, SD 213.1 g versus UDCA plus SAMe, mean 586.4 g, SD 102.9 g, p-value = .875). A total of 110 lesions were identified, 64 in 25 placentas of patients assigned to the UDCA and 46 in 15 placentas of patients managed by UDCA plus SAMe. Placental findings attributable to maternal malperfusion were found in 41/25 and 32/15 cases treated by UCDA and UDCA plus SAMe (165 versus 213%, p-value = .774), pathological fetal vascular supply in 17/25 and 8/15 placentas (68 versus 53%, p-value = .777), and inflammatory lesions in 6/25 and 6/15 cases (24 versus 40%, p-value = .757). CONCLUSIONS: Pregnancies complicated by mICP and managed by UDCA alone present similar APO rates and placental histopathology if compared with those treated by UDCA plus SAMe, failing to recognize advantages in the combined therapy. Further prospective studies and data sharing from ongoing RTCs could drive changes in therapeutic plan.

Genes and Pseudogenes: Complexity of the RCCX Locus and Disease.

Copy Number Variations (CNVs) account for a large proportion of human genome and are a primary contributor to human phenotypic variation, in addition to being the molecular basis of a wide spectrum of disease. Multiallelic CNVs represent a considerable fraction of large CNVs and are strictly related to segmental duplications according to their prevalent duplicate alleles. RCCX CNV is a complex, multiallelic and tandem CNV located in the major histocompatibility complex (MHC) class III region. RCCX structure is typically defined by the copy number of a DNA segment containing a series of genes - the serine/threonine kinase 19 (STK19), the complement 4 (C4), the steroid 21-hydroxylase (CYP21), and the tenascin-X (TNX) - lie close to each other. In the Caucasian population, the most common RCCX haplotype (69%) consists of two segments containing the genes STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB, with a telomere-to-centromere orientation. Nonallelic homologous recombination (NAHR) plays a key role into the RCCX genetic diversity: unequal crossover facilitates large structural rearrangements and copy number changes, whereas gene conversion mediates relatively short sequence transfers. The results of these events increased the RCCX genetic diversity and are responsible of specific human diseases. This review provides an overview on RCCX complexity pointing out the molecular bases of Congenital Adrenal Hyperplasia (CAH) due to CYP21A2 deficiency, CAH-X Syndrome and disorders related to CNV of complement component C4.

Iodine Absorption in Celiac Children: A Longitudinal Pilot Study.

Background: non-autoimmune thyroid disorder is a common finding in celiac patients, more frequent than in the general population. An impairment of iodine absorption has been hypothesized, but it has never been investigated so far. We aimed to evaluate the iodine absorption in children and adolescents with newly diagnosed celiac disease. Methods: 36 consecutive celiac patients (age 7.4 years, range 2.4-14.5 years) before starting a gluten-free diet (GFD) were enrolled. We assayed the urinary iodine concentration (UIC) in a 24-h urine sample, at baseline (T0) after 3 (T1) and 12 months (T2) of GFD. Results: UIC at T0 was 64 µg/L (IQR 45-93.25 µg/L) with an iodine deficiency rate of 77.8%. UIC was not different according to histological damage, clinical presentation (typical vs atypical); we found no correlation with the thyroid function tests and auxological parameters. UIC was not statistically different at T1 (76 µg/L) and T2 (89 µg/L) vs T0. UIC at T2 was similar between patients with positive and negative anti-transglutaminase antibodies at T2. No patients presented overt hypothyroidism during the study. Conclusions: We found that iodine absorption in celiac children is impaired compared to the general population; it increases slightly, but not significantly, during the GFD. We should regularly reinforce the need for a proper iodine intake in celiac disease patients to reduce iodine deficiency risk.

Cortisol circadian rhythm and jet-lag syndrome: evaluation of salivary cortisol rhythm in a group of eastward travelers.

PURPOSE: The activity of the hypothalamus-pituitary-adrenal axis plays a crucial role as an endogenous stress-reactive system. Lifestyle and work often interfere with the endogenous circadian rhythms and can modify the physiological patterns of stress-hormones secretion, including cortisol. We evaluated the cortisol circadian rhythm in the "jet-lag syndrome" that is the most known condition associated with the desynchronization of the circadian rhythm. METHODS: To assess the modifications of cortisol secretion after a long-haul flight, we compared baseline and post-travel salivary cortisol rhythm in a group of 28 healthy eastward travelers (from the U.S.A. or Canada to Italy). The salivary samples were collected about 1 week before the departure at 11 p.m. on day 0 and at 8 a.m., 12 a.m. (midday) and 11 p.m. on day 1 (R0). The same samples were obtained after the landing, the day they flew back home (R1). RESULTS: Statistical analysis showed a significant difference between R0 and R1 for each sample considered (p < 0.005). In particular, the post-travel salivary cortisol levels detected at 11 p.m. both on day 0 and on day 1, were significantly higher than at baseline. Post-travel morning salivary cortisol levels were lower compared with basal rhythm and increased during the morning, reaching the acrophase at 12 a.m. CONCLUSIONS: In eastward travelers, crossing more than five time zones, the cortisol circadian rhythm after the return to the East "remained behind," being synchronized with the West time. This impaired cortisol secretion can contribute to the pathogenesis of the jet-lag syndrome.

Evaluation and cost estimation of laboratory test overuse in 43 commonly ordered parameters through a Computerized Clinical Decision Support System (CCDSS) in a large university hospital.

BACKGROUND: Computerized Clinical Decision Support Systems (CCDSS) have become increasingly important in ensuring patient safety and supporting all phases of clinical decision making. The aim of this study is to evaluate, through a CCDSS, the rate of the laboratory tests overuse and to estimate the cost of the inappropriate requests in a large university hospital. METHOD: In this observational study, hospital physicians submitted the examination requests for the inpatients through a Computerized Physician Order Entry. Violations of the rules in tests requests were intercepted and counted by a CCDSS, over a period of 20 months. Descriptive and inferential statistics (Student's t-test and ANOVA) were made. Finally, the monthly comprehensive cost of the laboratory tests was calculated. RESULTS: During the observation period a total of 5,716,370 requests were analyzed and 809,245 violations were counted. The global rate of overuse was 14.2% ± 3.0%. The most inappropriate exams were Alpha Fetoprotein (85.8% ± 30.5%), Chlamydia trachomatis Nucleic Acid Amplification (48.7% ± 8.8%) and Alkaline Phosphatase (20.3% ± 6.5%). The monthly cost of over-utilization was 56,534€ for basic panel, 14,421€ for coagulation, 4,758€ for microbiology, 432€ for immunology exams. All the exams, generated an estimated avoidable cost of 1,719,337€ (85,967€ per month) for the hospital. CONCLUSIONS: The study confirms the wide variability in over-utilization rates of laboratory tests. For these reasons, the real impact of inappropriateness is difficult to assess, but the generated costs for patients, hospitals and health systems are certainly high and not negligible. It would be desirable for international medical communities to produce a complete panel of prescriptive rules for all the most common laboratory exams that is useful not only to reduce costs, but also to ensure standardization and high-quality care.

Immunoassay Interference on Thyroid Function Tests During Treatment with Nivolumab.

Background: Immune checkpoint inhibitors (ICIs) are associated with several endocrine side effects. In particular, the use of programmed cell death protein-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) inhibitors is related to a higher incidence of thyroid dysfunction. Patient Findings: An 85 years-old patient, diagnosed with a metastatic melanoma treated with nivolumab, presented to our hospital with severe ICI-related thyrotoxicosis. Diagnosis was complicated by a biochemical interference on thyroid hormones assay, probably induced by nivolumab. Summary: Baseline laboratory examination conducted before onset of anticancer therapy showed normal thyroid function test (TFTs). A few days after receiving the second nivolumab administration, the patient developed a severe thyrotoxicosis. According to destructive thyroiditis, in a short period thyrotropin (TSH) levels normalized and rapidly increased, but free thyroxine (fT4) levels were inappropriately elevated and did not decrease as expected. The sample was processed by using a Siemens Centaur® immunoassay. We reanalyzed the same sample at another laboratory and with a different immunoassay method (Roche Elecsys®). The results obtained from this assay confirmed severe hypothyroidism with appropriately low fT4 levels. We suspected a possible nivolumab-associated interference on the fT4 assay. Therefore, we subjected the same sample to a polyethylene glycol (PEG) 6000 precipitation, a simple method for the removal of macromolecules, before assaying for fT4 levels. Evaluation of the post-PEG-precipitation sample (Siemens Centaur immunoassay) revealed appropriately low fT4 levels. The patient was started on levothyroxine (LT4) therapy, with monthly TFT monitoring using the Roche immunoassay. Approximately 9 months after starting nivolumab therapy, the patient was advised treatment cessation. A month later, the TFTs were retested on a Siemens Centaur immunoassay, and appropriate fT4 levels were observed in accordance with normal TSH levels on adequate LT4 replacement therapy. Conclusions: We report a possible novel nivolumab-induced biochemical interference on assays of fT4 levels. The hypothesis of a biochemical drug-induced interference is further supported by the disappearance of the interference after the withdrawal of nivolumab. Further studies are needed to prove the biochemical mechanisms of this interference.

Head-to-head comparison of plasma cTnI concentration values measured with three high-sensitivity methods in a large Italian population of healthy volunteers and patients admitted to emergency department with acute coronary syndrome: A multi-center study.

BACKGROUND: The study aim is to compare cTnI values measured with three high-sensitivity (hs) methods in apparently healthy volunteers and patients admitted to emergency department (ED) with acute coronary syndrome enrolled in a large multicentre study. METHODS: Heparinized plasma samples were collected from 1511 apparently healthy subjects from 8 Italian clinical institutions (mean age: 51.5 years, SD: 14.1 years, range: 18-65 years, F/M ratio:0.95). All volunteers denied chronic or acute diseases and had normal values of routine laboratory tests. Moreover, 1322 heparinized plasma sample were also collected by 9 Italian clinical institutions from patients admitted to ED with clinical symptoms typical of acute coronary syndrome. The reference study laboratory assayed all plasma samples with three hs-methods: Architect hs-cTnI, Access hs-cTnI and ADVIA Centaur XPT methods. Principal Component Analysis (PCA) was also used to analyze the between-method differences among hs-cTnI assays. RESULTS: On average, a between-method difference of 31.2% CV was found among the results of hs-cTnI immunoassays. ADVIA Centaur XPT method measured higher cTnI values than Architect and Access methods. Moreover, 99th percentile URL values depended not only on age and sex of reference population, but also on the statistical approach used for calculation (robust non-parametric vs bootstrap). CONCLUSIONS: Due to differences in concentrations and reference values, clinicians should be advised that plasma samples of the same patient should be measured for cTnI assay in the same laboratory. Specific clinical studies are needed to establish the most appropriate statistical approach to calculate the 99th percentile URL values for hs-cTnI methods.

Evaluation of 99th percentile and reference change values of the hs-cTnI method using ADVIA Centaur XPT platform: A multicenter study.

BACKGROUND: According to quality specifications required by international guidelines, the evaluation of the 99th URL value is a very difficult task that is usually beyond the capacity of a single laboratory. The aims of this article are to report and discuss the results of a multicenter study concerning the evaluation of the 99th percentile URL and reference change (RCV) of the ADVIA Centaur High-Sensitivity Troponin I (TNIH), recently distributed to the Italian clinical laboratories. MATERIALS AND METHODS: The reference population evaluated with ADVIA XPT method for the calculation of cTnI reference distribution parameters consisted of 1325 healthy adults subjects (age range from 18 to 86 years), including 653 women (mean age 50.7 years, SD 14.5 years) and 672 men (mean age 50.9 years, SD 13.8 years), well matched for both age (P = .8112) and sex (F/M = 0.97). RESULTS: cTnI distribution values of reference population was highly skewed, while log-transformed cTnI values roughly approximated a log-normal distribution. Men have higher cTnI values than women throughout all the adult lifespan. Moreover, the subjects with age ≤ 55 years had significantly lower cTnI values than those with age > 55 years (p < .0001). Of note, 62% of women and 77% of men had equal or higher than cTnI values than the LoD value of the method (i.e., 2.2 ng/L). CONCLUSIONS: The results of the present study demonstrate that the ADVIA Centaur High-Sensitivity Troponin I using the XPT automated platform fits both the criteria and quality specifications required by the most recent international guidelines for high-sensitivity methods for cTnI assay.

Evaluation of 99th percentile and reference change values of a high-sensitivity cTnI method: A multicenter study.

BACKGROUND: The Italian Society of Clinical Biochemistry (SIBioC) and the Italian Section of the European Ligand Assay Society (ELAS) have recently promoted a multicenter study (Italian hs-cTnI Study) with the aim to accurately evaluate analytical performances and reference values of the most popular cTnI methods commercially available in Italy. The aim of this article is to report the results of the Italian hs-cTnI Study concerning the evaluation of the 99th percentile URL and reference change (RCV) values around the 99th URL of the Access cTnI method. MATERIALS AND METHODS: Heparinized plasma samples were collected from 1306 healthy adult volunteers by 8 Italian clinical centers. Every center collected from 50 to 150 plasma samples from healthy adult subjects. All volunteers denied the presence of chronic or acute diseases and had normal values of routine laboratory tests (including creatinine, electrolytes, glucose and blood counts). An older cohort of 457 adult subjects (mean age 63.0 years; SD 8.1 years, minimum 47 years, maximum 86 years) underwent also ECG and cardiac imaging analysis in order to exclude the presence of asymptomatic cardiac disease. RESULTS AND CONCLUSIONS: The results of the present study confirm that the Access hsTnI method using the DxI platform satisfies the two criteria required by international guidelines for high-sensitivity methods for cTn assay. Furthermore, the results of this study confirm that the calculation of the 99th percentile URL values are greatly affected not only by age and sex of the reference population, but also by the statistical approach used for calculation of cTnI distribution parameters.

A Black Swan in clinical laboratory practice: the analytical error due to interferences in immunoassay methods.

It is well known that the results of immunoassay methods can be affected by specific or non-specific interferences, ranging from 0.4% to 4.0%. The presence of interference may greatly compromise the accuracy of immunoassay analyses causing an error in the measurement, producing false-positive or false-negative results. From a clinical point of view, these analytical errors may have serious implications for patient care because they can cause misdiagnosis or inappropriate treatment. Unfortunately, it is a very difficult task to identify the irregular analytical errors related to immunoassay methods because they are not detectable by normal laboratory quality control procedures, are reproducible within the test system, may be clinically plausible and are relatively rare. The first line of defense against erroneous results is to use in laboratory practice only immunoassay systems with the highest level of robustness against interference. The second line of defense is always taking into account the possibility of interference in immunoassay results. A correct approach should be addressed on identification of samples at high risk of interference. The attainment of this goal requires a critical review of the test result in relation to patient's clinical conditions and literature data, taking into account the analytical characteristics of the immunoassay system. The experts in immunoassay systems should make every effort to find some specific and reliable quality indicators for irregular analytical errors in order to better detect and monitor erroneous immunoassay results due to specific or non-specific interferences.

CYP21A2 intronic variants causing 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase gene (CYP21A2). Most of CYP21A2 mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. Intronic variants represent only a little part of these but their effect on the protein is generally deleterious. The aim of this paper is to provide a comprehensive literary review regarding all intronic CYP21A2 pathological variants reported to date. In addition, we describe three novel causing disease variants in our patients affected by the classic form of CAH: IVS4-1G>A, IVS5-8T>A, IVS8-2A>G. In silico analysis revealed that all these substitutions affect the splicing process leading to a non-functional protein. Based on these results, we are able to classify them as pathological variants according to the patient's phenotype.

Harmonization protocols for TSH immunoassays: a multicenter study in Italy.

BACKGROUND: Systematic difference between thyroid-stimulating hormone (TSH) immunoassays may produce misleading interpretation when samples of the same patients are measured with different methods. The study aims were to evaluate whether systematic differences are present among TSH immunoassays, and whether it is possible to obtain a better harmonization among TSH methods using results obtained in external quality assessment (EQA) schemes. METHODS: Seven Italian clinical laboratories measured TSH in 745 serum samples of healthy subjects and patients with thyroid disorders. These samples were also re-measured by two reference laboratories of the study with the six TSH immunoassays most popular in Italy after 2 months of storage at -80 °C. Moreover, these data were compared to 53,823 TSH measurements, obtained by laboratories participant to 2012-2015 EQA annual cycles in 72 quality control samples (TSH concentrations from about 0.1 mIU/L to 18.0 mIU/L). TSH concentrations were recalibrated using a mathematical approach based on the principal component analysis (PCA). RESULTS: Systematic differences were found between the most popular commercially available TSH immunoassays. TSH concentrations measured by the clinical laboratories were very closely correlated to those measured with the same method by reference laboratories after 2 months of storage at -80 °C. After recalibration using the PCA approach the variation of TSH values significantly decreased from a median pre-calibration value of 13.53% (10.79%-16.53%) to 9.63% (6.90%-13.21%) after recalibration. CONCLUSIONS: Our data suggest that EQA schemes are useful to improve harmonization among TSH immunoassays and also to produce some mathematical formulas, which can be used by clinicians to better compare TSH values measured with different methods.

Multicenter evaluation of the new immunoassay method for TSH measurement using the automated DxI platform.

AIM OF THE STUDY: Recently, Beckman Coulter Diagnostics set up a new TSH immunoassay for the automated DxI platform. The aim of this study was to evaluate and compare the analytical performance and clinical results of this method with those of previous method. MATERIAL AND METHODS: A multicenter study (named TSH ELAS Study) was organized using 593 serum samples, collected from healthy subjects and patients with thyroid disorders, and 13 control samples, circulated in an External Quality Assessment (EQA) scheme. RESULTS: The values of LoB and LoD, and LoQ at 20% CV were 0.0004mIU/L, 0.001mIU/L and 0.0023mIU/L, respectively. Moreover, TSH concentrations >0.01mIU/L actually show imprecision values lower than 5% CV. This new TSH assay showed a systematic underestimation (on average of 6.25%) compared to old method, which is mainly due to larger differences between methods for samples with low TSH concentrations, related to the better analytical sensitivity of new compared to old method. In a reference population, including 279 apparently healthy adult subjects, Caucasian volunteers (mean age 43.6years, age 20-63years, 138 women and 141 males) the distribution of TSH concentrations was: mean (CI 95%) 1.694mIU/L (1.588-1.779), median 1.495mIU/L (1.412-1.588mIU/L), 97.5th percentile 3.707mIU/L. CONCLUSIONS: The new TSH immunoassay for DxI platform shows some relevant improvements compared to the previous one: use of the most recent WHO 3rd IRP 81/563 standard and monoclonal antibodies (instead of polyclonal antibodies of the old method), and better analytical sensitivities and reproducibility.