Role of plasma phosphorylated neurofilament heavy chain (pNfH) in amyotrophic lateral sclerosis.

The phosphorylated neurofilament heavy chain (pNfH) is a promising biomarker in amyotrophic lateral sclerosis (ALS). We examined plasma pNfH concentrations in order to corroborate its role as a diagnostic and prognostic biomarker in ALS. Incident ALS cases enrolled in a population-based registry were retrospectively selected and matched by sex and age with a cohort of healthy volunteers. Plasma pNfH levels were measured by an ELISA kit and correlated with clinical parameters. Discrimination ability of pNfH was tested using receiving operating characteristic (ROC) curves. Kaplan-Meier (KM) analysis and Cox proportional hazard models were used for survival analysis. Plasma pNfH was significantly higher in patients compared to controls. An optimal cut-off of 39.74 pg/ml discriminated cases from controls with an elevated sensitivity and specificity. Bulbar-onset cases had higher plasma pNfH compared to spinal onset (p = 0.0033). Furthermore, plasma pNfH positively correlated with disease progression rate (r = 0.19, p = 0.031). Baseline plasma pNfH did not influence survival in our cohort. Our findings confirmed the potential utility of plasma pNfH as a diagnostic biomarker in ALS. However, further studies with longitudinal data are needed to corroborate its prognostic value.

Low-Dose of Rocuronium During Thyroid Surgery: Effects on Intraoperative Nerve-Monitoring and Intubation.

BACKGROUND: Intraoperative Neurophysiological Monitoring (IONM) reduces the incidence of Recurrent Laryngeal Nerve (RLN) injuries during thyroid surgery. To preserve nerve function, long acting neuromuscular blocking agents (NMBA) should be avoided. However, NMBA are necessary for laryngoscopy and endotracheal intubation. We designed this double-blinded, randomized, placebo-controlled trial to assess if a low-dose of rocuronium given at intubation would affect the IONM data recorded before the thyroid dissection. METHODS: Hundred patients undergoing elective thyroid surgery were randomized to receive either 0.3 mg kg-1 of low dose rocuronium (intervention) or no-NMBA (control). Intubation was performed with video-laryngoscopy. IONM was placed on RLN and nerve stimulation was performed before and after thyroid dissection. The presence of a valid amplitude prior to dissection was defined when the IONM signal was >100 µV. Occurrence of difficult laryngoscopy was reported together with intubation details including time, difficulty and failure. The lowest peripheral saturation (SpO2) and the number of desaturation episodes during the intubation were also registered. RESULTS: No patients showed impaired IONM signal before dissection in both groups. Cormack-Lehane grade was higher in the intervention group (11;2) compared to control one (11;1; P = 0.046). No-NMBA patients had increased number of difficult laryngoscopies (21% versus 6%, P = 0.041) and intubations (34% versus 8%; P = 0.003) as well as a longer time to intubation (78 [55; 175] versus 55 [31; 110] sec; P = 0.006). Lower values of peripheral SpO2 during intubation attempt were registered in the no NMBA group (99 [97; 100] versus 99 [99; 100] %; P = 0.020). However, the number of intubation failure was similar between groups (p=0.495). CONCLUSIONS: Low-dose of rocuronium does not compromise pre-dissection IONM signal and improves intubation condition when compared to a relaxant free strategy.

Evaluation of Biomarkers of Bone Metabolism on Salivary Matrix in the Remodeling of Periodontal Tissue during Orthodontic Treatment.

The aim of this study was to evaluate changes in the concentration of N-terminal type I collagen extension pro-peptide (PINP), tartrate-resistant acid phosphatase (TRAcP), and parathyroid hormone-related protein (PTHrP) in saliva during orthodontic treatment in order to evaluate whether changes in bone turnover marker (BTM) concentration can help highlight the effects of orthodontic mechanical loading in the absence of clinical evidence of tooth movement in terms of tooth movement. Saliva samples from 25 apparently healthy young subjects (10 females and 15 males) were collected using Salivette® (Sarstedt) with cotton swabs and the concentrations of PTHrP, TRAcP 5b, and PINP were analyzed at time 0 (T1), 25 days (T2), and at 45 days (T3). Differences in the median value of biomarker levels between baseline T1 and follow-up of the different groups (T2 and T3) were assessed using the non-parametric Mann-Whitney U test. Trough concentrations of P1NP, PTHrP, and TRAcP were 0.80 µg/L, 0.21 ng/mL, and 0.90 U/L above the method LOD. The non-parametric Mann-Whitney U test confirmed a statistically significant difference in T1 versus concentrations of T2 and T3. All subjects evaluated had a statistically significant difference between T1 vs. T3. when compared with the specific critical difference (RCV) for the analyte The results obtained demonstrate that the evaluation of BTM changes in saliva can help the evaluation of orthodontic procedures and the monitoring of biomechanical therapy.

Biomarkers and Biochemical Indicators to Evaluate Bone Metabolism in Preterm Neonates.

The purpose of the present study was to evaluate the concentrations of some bone turnover markers in preterm neonates with uncomplicated clinical course in the first month of life. Samples from 13 preterm neonates were collected at three different times: at birth (T0) from umbilical cord blood (UCB); and at 15 (T1) and 30 (T2) days of life from peripheral blood (PB). The concentrations of calcium (Ca), phosphate (P), total alkaline phosphatase (ALP), Collagen Type 1 Amino-terminal Propeptide (PINP), osteocalcin (OC), Collagen Type 1 Carboxyl-Terminal Telopeptide (CTX) and Leptin were assessed. A statistically significant difference for ALP concentration at birth versus T1 and T2 was found. An evident increase in the median concentrations of CTX, OC and PINP from T0 to T2 were observed. A significant difference was also found for Leptin concentration at T0 compared to T1. In preterm infants, in the absence of acute or chronic medical conditions and without risk factors for metabolic bone disease (MBD) of prematurity, there is a significant increase in bone turnover markers during the first month of life. The knowledge of the variations in these markers in the first weeks of life, integrated by the variations in the biochemical indicators of bone metabolism, could help in recognizing any conditions at risk of developing bone diseases.

Analysis of Quality Indicators of the Pre-Analytical Phase on Blood Gas Analyzers, Point-Of-Care Analyzer in the Period of the COVID-19 Pandemic.

AIM OF THE STUDY: We evaluated and compared blood gas analysis (EGA) non-conformities (NC) considered operator-dependent performed in Point-Of-Care (POC) analyzer as quality indicators (IQ) of the pre-analytical phase. To this end, four different NC registered in the resuscitation departments of the Hospital Polyclinic Bari from the beginning of the pandemic (March 2020) until February 2022 were evaluated. The results obtained were compared with those recorded in the pre-COVID period (March 2018-February 2020) to check if there were differences in number and type. MATERIAL AND METHODS: GEM 4000 series blood gas analyzers (Instrumentation Laboratory, Bedford, MA, United States) are installed with integrated Intelligent Quality Management (iQM®), which automatically identify and log pre-analytical errors. All blood gas analyzers are connected to the company intranet and interfaced with the GEM Web Plus (Werfen Instrumentation Laboratory, Bedford, MA, United States) data management information system, which allows the core laboratory to remotely supervise all decentralized POC stations. The operator-dependent process NC were expressed in terms of absolute and relative proportions (percentiles and percentage changes). For performance evaluation, the Mann-Whitney U test, Chi-squared test and Six-Sigma Metric calculation for performance classification were performed. RESULTS: In the COVID period, 31,364 blood gas tests were performed vs. 16,632 tests in the pre-COVID period. The NC related to the suitability of the EGA sample and manageable by the operators were totals of 652 (3.9%) and 749 (2.4%), respectively, in the pre-COVID and COVID periods. The pre-analytical phase IQs used did not show statistically significant differences in the two periods evaluated. The Sigma evaluation did not show an increase in error rates. CONCLUSIONS: Considering the increase in the number of EGAs performed in the two periods, the training procedures performed by the core laboratory staff were effective; the clinical users of the POC complied with the indications and procedures shared with the core laboratory without increasing the operator-dependent NCs. Furthermore, the core laboratory developed monitoring activities capable of guaranteeing the maintenance of the pre-analytical quality.

Neurosensory Alterations and Interleukins Cascade in SARS-CoV-2 Infection - Results from a Retrospective Cohort of COVID-19 Inpatients.

OBJECTIVES: The aim of this study was to relate IL-6 and IL-1ß serum levels with the severity of olfactory disorders and with the type of unperceived odors. METHODS: 82 inpatients (45 men aged 62.3 ± 14.2 and 37 women aged 57.1 ± 12.8) with only smell dysfunctions were divided into two groups. The evaluation of the smell disorder was carried out with a questionnaire to define which sensitivity is most compromised in COVID-19 patients. Cytokine levels were measured with chemiluminescence and ELISA assay. Statistical analyses were performed with the Wilcoxon Rank test, Welch's T-test, and Mann-Whitney test (p < 0.05). RESULTS: Statistically significant differences in IL-6 and IL-1 ß levels were found in moderate disease patients when there was an impairment of trigeminal sensitivity (p <0.05) and trigeminal and olfactory sensitivity. CONCLUSIONS: The results obtained showed that in COVID-19 patients the impairment of trigeminal sensitivity in association with olfactory sensitivity was more prevalent in moderate than in mild forms.

Bone Remodeling Markers in Children with Acute Lymphoblastic Leukemia after Intensive Chemotherapy: The Screenshot of a Biochemical Signature.

PURPOSE: to investigate the effects of intensive chemotherapy and glucocorticoid (GC) treatment on bone remodeling markers in children with acute lymphoblastic leukemia (ALL). METHODS: A cross-sectional study was carried out in 39 ALL children (aged 7.64 ± 4.47) and 49 controls (aged 8.7 ± 4.7 years). Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), osteocalcin (OC), C-terminal telopeptide of type I collagen (CTX), bone alkaline phosphatase (bALP), tartrate-resistant acid phosphatase 5b (TRACP5b), procollagen type I N-terminal propeptide (P1NP), Dickkopf-1 (DKK-1), and sclerostin were assessed. Statistical analysis was conducted using the principal component analysis (PCA) to study patterns of associations in bone markers. RESULTS: ALL patients showed significantly higher OPG, RANKL, OC, CTX, and TRACP5b than the controls (p ≤ 0.02). Considering ALL group, we found a strong positive correlation among OC, TRACP5b, P1NP, CTX, and PTH (r = 0.43-0.69; p < 0.001); between CTX and P1NP (r = 0.5; p = 0.001); and between P1NP and TRAcP (r = 0.63; p < 0.001). The PCA revealed OC, CTX, and P1NP as the main markers explaining the variability of the ALL cohort. CONCLUSIONS: Children with ALL showed a signature of bone resorption. The assessment of bone biomarkers could help identify ALL individuals who are most at risk of developing bone damage and who need preventive interventions.

Determination of iFGF23 Upper Reference Limits (URL) in healthy pediatric population, for its better correct use.

Background: The measurement of Fibroblast growth factor 23 (FGF23) may be useful in the diagnosis and management of abnormal phosphate metabolism in both patients with preserved renal function or with chronic kidney disease (CKD). FGF-23 tests differ considerably by molecule assayed (iFGF23 or cFGF23), analytical performance and reference ranges. We establish iFGF23 Upper Reference Limits (URL) in apparently healthy pediatric individuals using automated immunochemiluminescent assay. Methods: We measured the levels of plasma iFGF23 from 115 samples from apparently healthy pediatric subjects [59 (51.3%) individuals were male; median age 10 years (range 1-18)] included in an observational study conducted at Policlinico University Hospital of Bari. The method used for the iFGF23 assay was immunochemiluminescent sandwich assay developed by DiaSorin on the Liaison XL platform. Statistical calculation of 95% reference interval, right-sided (CLSI C28-A3) and verification of age and sex covariables was performed for the calculation of the URL. Results: The URL concentration of iFGF23 was 61.21 pg/mL (58.63 to 63.71, 90% CI). No significant differences were found between the median concentrations of iFGF23 differentiated by sex and age. Conclusions: The dosage of iFGF23 is important both for the differential diagnosis of the various forms of rickets, and for the subsequent monitoring of the effectiveness of drug treatment. We have established the URL for the iFGF23 Liaison test in apparently healthy pediatric subjects. The availability of iFGF23 pediatric reference values will allow a better clinical use of the test.

Parathyroid Hormone-Related Peptide (PTHrP): Evaluation of Pediatric, Covariate-Stratified Reference Intervals.

Parathyroid hormone-related peptide (PTHrP) is expressed at a wide range of sites in the body and performs different functions including vasodilation, relaxation of smooth muscle cells, and regulation of bone development. PTHrP also mediates hypercalcemia related to neoplastic diseases. However, reference ranges specific method and age were not evaluated. We establish PTHrP reference ranges in apparently healthy, normocalcemic, normophosphatemic pediatric individuals. In this observational prospective, study we measured PTHrP in serum from 178 samples (55.06% male 44.94% female) from apparently healthy pediatric subjects [median age 10 years (range 1-18)] subunit ELISA method The statistical analysis performed provided for the calculation of the 95% reference interval, right-sided, with a non-parametric percentile method (CLSI C28-A3). Upper reference limits (URL) for PTHrP was 2.89 ng/mL (2.60 to 3.18; 90% CI). No significant differences were found between the median PTHrP concentrations in males vs females and in the age range categories selected. Comprehensive normal values for PTHrP are indispensable to the assessment of calcium phosphorus dysfunction in children. Severe hypercalcemia is a rare, but clinically significant condition, in infancy and childhood. PTHrP values higher than the reference value may help to distinguish the hypercalcemic product of a malignancy, paraneoplastic syndromes mediated by PTHrP, from other causes.

The Role of Age on Beta-Amyloid1-42 Plasma Levels in Healthy Subjects.

Beta-amyloid (Aß) plaques have been observed in the brain of healthy elderlies with frequencies strongly influenced by age. The aim of the study is to evaluate the role of age and other biochemical and hematological parameters on Aß1-42 plasma levels in cognitively and neurologically normal individuals. Two-hundred and seventy-five normal subjects stratified by age groups (<35 years, 35-65 years, and >65 years) were included in the study. Aß1-42 plasma levels significantly correlated with age (rs = 0.27; p < 0.0001) in the whole sample, inversely correlated with age in the first age group (rs = -0.25, p = 0.01), positively correlated in the second group (rs = 0.22, p = 0.03), while there was no significant correlation in the older group (rs = 0.02, p = 0.86). Both age (ß-estimate = 0.08; p < 0.001) and cholesterol (ß-estimate = 0.03; p = 0.009) were significantly associated with Aß1-42 plasma level in multivariable analysis. However, only the association with age survived post hoc adjustment for multiple comparisons. The different effects of age on the Aß level across age groups should be explored in further studies to better understand the age-dependent variability. This could better define the value of plasma Aß as a biomarker of the Alzheimer neuropathology.

Comparative evaluation of two immunoassays for cerebrospinal fluid ß-Amyloid1-42 measurement.

AIM OF THE STUDY: Beta-Amyloid 1-42 peptide (ßA42) is a cerebro-spinal fluid (CSF) biomarker, key element of the NIA Alzheimer's disease diagnostic criteria. The enzyme-linked immunosorbent assay (ELISA) has been the mainstay method for ßA42 measurement on cerebrospinal fluid (CSF). Recently, a new ßA42 measurement method in chemiluminescence enzyme immunoassay (CLEIA) is available on Lumipulse G 600 II automatic platform. The aim of the work was to evaluate the concordance of the ELISA and the new method (CLEIA) in the CSF ßA42 levels measurement. MATERIALS AND METHODS: CSF ßA42 levels were assayed in 49 samples using the ELISA method (Innotest ß- amyloid 1-42, Fujirebio Europe N.V., Gent, Belgium) and CLEIA method on Lumipulse G600II fully automatic platform (Lumipulse G ß- amyloid 1-42, Fujirebio Europe N.V., Gent, Belgium). We compared values of the two methods using acceptability interval based on Inherent Combined Imprecision (ICI), the Passing-Bablok regression analysis, the Pearson correlation coefficient (r) and the Bland-Altman plot. RESULTS: The analysis of the ICI showed that the two methods differ substantially. The regression equation (y = -103.04 + 1.52×) highlighted the presence of proportional systematic difference, without significant deviation from linearity (p = .42). The Pearson correlation coefficient was 0.826. The Bland-Altman plot analysis showed a significant systematic difference in the two methods: ELISA measurements were in average -27.06% (95% CI -31.89 to -22.23%) lower compared to CLEIA ones. CONCLUSIONS: Our study highlighted a difference between the two methods. Therefore, the cut-off for the normal levels of ßA42 should be reviewed in the laboratory report.

Plasma ß-amyloid1-42 reference values in cognitively normal subjects.

BACKGROUND: In clinical practice, the use of plasma ß-Amyloid1-42 (Aß1-42) as biomarker for Alzheimer's disease is largely limited by the absence of reference values. The aim of this study was to evaluate Aß1-42 plasma concentrations in cognitively normal subjects and to propose reference values. METHODS: Plasma samples were obtained from 245 subjects, with a wide age-range (19-89 years), enrolled at the Unit of Laboratory Medicine of the "Azienda Ospedaliera Cardinale G. Panico" (younger subjects) and from a population-based study on aging (GreatAGE study) (older subjects). Three different age-groups were established: young (≤ 34), adult (35 ≤ age ≤ 64) and old (>64). The Innogenetics Elisa kit for plasma Aß1-42 was used for the analysis. RESULTS: The mean (SD) concentration of plasma Aß1-42 was 17.65 (5.71) pg/mL. A positive trend was found in Aß1-42 levels across the three age groups (p < .0001): young subjects showed values of Aß1-42 significantly lower than the adult group (p < .0001) and than the old one (p < .0001 overall); no significant differences were found between the adult and the old groups (p = 1.0000). For the entire cohort the lower limit of 90% Reference Interval, double-sided, was 8.12 pg/mL (95% CI 6.77-9.45) and the upper limit was 29.00 pg/mL (95% CI 27.01-31.00). CONCLUSION: The present study proposes reference values for plasma Aß1-42. Nevertheless, further studies are needed to confirm these results and corroborate the use of these reference values in clinical practice.

A Novel Splice-Acceptor Site Mutation in GRN (c.709-2 A>T) Causes Frontotemporal Dementia Spectrum in a Large Family from Southern Italy.

Heterozygous loss of function mutations in granulin represent a significant cause of frontotemporal lobar degeneration with ubiquitin and TDP-43 inclusions (FTLD-TDP). We report a novel GRN splice site mutation (c.709-2 A>T), segregating with frontotemporal dementia spectrum in a large family from southern Italy. The GRN c.709-2 A>T is predicted to result in the skipping of exon 8, leading to non-sense mediated mRNA decay. Moreover, the PGRN plasma levels in the GRN c.709-2 A>T carriers were significantly lower (24 ng/ml) compared to controls (142.7 ng/ml) or family members non-carriers (82.0 ng/ml) (p-value = 0.005, Kruskal Wallis), suggesting progranulin haploinsufficiency. We do not report any potential pathogenic GRN mutation in a follow-up cohort composed of 6 FTD families and 43 sporadic FTD cases, from the same geographic area. Our study suggests that GRN (c.709-2 A>T) is a novel and likely very rare cause of FTD in this Italian cohort. Finally, in line with previous studies, we show that GRN haploinsufficiency leads to a heterogeneous clinical picture, and plasma progranulin levels may be a reliable tool to identify GRN loss of function mutations. However, given that a) genetic and environmental factors, gender, and age may regulate PGRN plasma levels and b) plasma progranulin levels may not reflect PGRN levels in the central nervous system, we suggest that the measurement of progranulin in the plasma should always be coupled with genetic screening of GRN for mutations.

Anti-cyclic citrullinated peptide autoantibodies measured by an automated enzyme immunoassay: analytical performance and clinical correlations.

BACKGROUND: Autoantibodies against cyclic citrullinated peptide (anti-CCP) are considered to be a sensitive and specific marker for rheumatoid arthritis (RA). This study evaluated the analytical performance and clinical correlation of an automated enzyme immunoassay (DSX, DINEX Technologies), for the detection of anti-CCP autoantibodies (DIASTAT anti-CCP, Axis-Shield, DUNDEE UK). METHODS: Commercial controls and serum pools were used to determine its precision, analytical sensitivity, functional sensitivity and linearity. Sera from 83 patients with established RA and from 140 controls, including patients with various autoimmune diseases, viral infections and cancer, as well as sex- and age-matched healthy subjects, were studied. The rheumatoid factor (RF) was also assayed in each sample, and the results were compared to the anti-CCP findings. RESULTS: The total imprecision (CV%) was 4.7-7.2% for concentrations ranging between 1.98 and 71.81 U/mL. The lower detection limit was 0.038 U/mL. At a cut-off of 5 U/mL, the sensitivity and specificity for RA were 67.5% and 99.3%, respectively. The RF had a sensitivity of 66.3% and a lower specificity 82.1% than anti-CCP. When the two antibodies were used together, the specificity was 99.1%. CONCLUSIONS: The anti-CCP assay we examined on a fully automated system showed a good analytical performance (analytical and functional sensitivity, linearity) and good clinical correlation. We conclude that this system can provide rapid, useful data.

Application of a combined protocol for rational request and utilization of antibody assays improves clinical diagnostic efficacy in autoimmune rheumatic disease.

CONTEXT: Because of a marked increase in the number of requests for antinuclear antibodies, anti-extractable nuclear antigen antibodies, and anti-double-stranded DNA antibodies for the diagnosis of autoimmune rheumatic disease, guidelines have been proposed for their appropriate use. OBJECTIVE: To evaluate in terms of clinical efficacy and cost-benefit ratio the outcome of applying a protocol for the diagnosis of autoimmune rheumatic disease. DESIGN: A diagnostic protocol for the rational utilization of second-level tests (anti-extractable nuclear antigen antibodies and anti-double-stranded DNA antibodies) was applied at Hospital Polyclinic beginning January 2004. The appropriateness of 685 consecutive requests received at the clinical pathology laboratory from January to June 2004 was assessed. Patients who underwent these laboratory tests were followed up for 12 months after blood sample drawing. RESULTS: Introduction of the protocol led to a significant reduction in the number of second-level tests prescribed (27.9% vs 49.5% for anti-extractable nuclear antigen antibodies; 27.5% vs 56.6% for anti-double-stranded DNA antibodies). After the period of observation, none of the 163 patients who had negative results on the first-level test and were asymptomatic, for whom second-level tests had not therefore been performed, were found to have autoimmune rheumatic disease. In 90.5% (77/85) of patients positive for the second-level tests, clinical confirmation of autoimmune rheumatic disease was obtained. CONCLUSIONS: Not only did application of the diagnostic protocol reduce the number of second-level tests performed but it also increased their specificity. Our data thus indicate that the use of shared guidelines by clinical and laboratory specialists yields satisfactory results.