Molecular Biomarkers of Neurodegenerative Disorders: A Practical Guide to Their Appropriate Use and Interpretation in Clinical Practice.

Neurodegenerative disorders (NDs) represent a group of different diseases characterized by the progressive degeneration and death of the nervous system's cells. The diagnosis is challenging, especially in the early stages, due to no specific clinical signs and symptoms. In this context, laboratory medicine could support clinicians in detecting and differentiating NDs. Indeed, biomarkers could indicate the pathological mechanisms underpinning NDs. The ideal biofluid for detecting the biomarkers of NDs is cerebrospinal fluid (CSF), which has limitations, hampering its widespread use in clinical practice. However, intensive efforts are underway to introduce high-sensitivity analytical methods to detect ND biomarkers in alternative nonivasive biofluid, such as blood or saliva. This study presents an overview of the ND molecular biomarkers currently used in clinical practice. For some diseases, such as Alzheimer's disease or multiple sclerosis, biomarkers are well established and recommended by guidelines. However, for most NDs, intensive research is ongoing to identify reliable and specific biomarkers, and no consensus has yet been achieved.

Prostate Health Index (PHI) as a triage tool for reducing unnecessary magnetic resonance imaging (MRI) in patients at risk of prostate cancer.

INTRODUCTION: The aim of this study is to assess the usefulness of the Prostate Health Index (PHI) as a triage tool for selecting patients at risk of prostate cancer (PCa) who should undergo multiparametric Magnetic Resonance Imaging (mpMRI). MATERIAL AND METHODS: We enrolled 204 patients with suspected PCa. For each patient, a blood sample was collected before mpMRI to measure PHI. Findings on mpMRI were assessed according to the Prostate Imaging Reporting & Data System version 2.0 (PI-RADSv2) category scale. RESULTS: According to PI-RADSv2, patients were classified into two groups: PI-RADS < 3 (48 %) and ≥ 3 (52 %). PHI showed the best performance for predicting PI-RADS ≥ 3 [AUC: 0,747 (0,679-0,815), 0,680(0,607-0,754), and 0,613 (0,535-0,690) for PHI, PSA ratio, and total PSA, respectively]. The best PHI cut-off was 30, with a sensitivity of 90%. At the univariate logistic regression, total PSA (p = 0.007), PSA ratio (p = 0.001), [-2]proPSA (p = 0.019) and PHI (p < 0.001) were associated with PI-RADS ≥ 3; however, at the multivariate analysis, only PHI (p < 0.001) was found to be an independent predictor of PI-RADS ≥ 3. CONCLUSION: PHI could represent a reliable noninvasive tool for selecting patients to undergo mpMRI.

The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

OBJECTIVES: Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. METHODS: Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. RESULTS: We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. CONCLUSIONS: Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Monocyte distribution width (MDW) kinetic for monitoring sepsis in intensive care unit.

OBJECTIVES: Monocyte distribution width (MDW) is a measure of monocyte anisocytosis. In this study, we assessed the role of MDW, in comparison to C-reactive protein (CRP), procalcitonin (PCT), and lactate, as a screening and prognostic biomarker of sepsis in intensive care unit (ICU) by longitudinally measuring it in the first 5 days of hospital stay. METHODS: We considered all consecutive patients admitted to the ICU. At admission, patients were classified as septic or not according to Sepsis-3 criteria. MDW, CRP, PCT, and lactate were measured daily in the first 5 days of hospitalization. ICU mortality was also recorded. RESULTS: We included 193 patients, 62 with sepsis and 131 without sepsis (controls). 58% and 26 % of the patients, with and without sepsis respectively, died during ICU stay. MDW showed the highest accuracy for sepsis detection, superior to CRP, PCT, and lactate (AUC of 0.840, 0.755, 0.708, 0.622, respectively). At admission, no biomarker predicts ICU mortality in patients with sepsis. The kinetic of all biomarkers during the first 5 days of hospitalization was associated with ICU mortality. Noteworthy, above all, the kinetic of MDW showed the best accuracy. Specifically, an increase or decrease in MDW from day 1-4 and 5 was significantly associated with mortality or survival, respectively. CONCLUSIONS: MDW is a reliable diagnostic and prognostic sepsis biomarker, better than traditional biomarkers.

A machine learning strategy to mitigate the inappropriateness of procalcitonin request in clinical practice.

Aim: The aim of this study was to develop machine learning (ML) models to mitigate the inappropriate request of Procalcitonin (PCT) in clinical wards. Material and methods: We built six different ML models based on both demographical data, i.e., sex and age, and laboratory parameters, i.e., cell blood count (CBC) parameters, inclusive of monocyte distribution width (MDW), and C-reactive protein (CRP). The dataset included 1667 PCT measurements of different patients. Based on a PCT cut-off of 0.50 ng/mL, we found 1090 negative (65.4%) and 577 positive (34.6%) results. We performed a 70:15:15 train:validation:test splitting based on the outcome. Results: Random Forest, Support Vector Machine and eXtreme Gradient Boosting showed optimal performances for predicting PCT positivity, with an area under the curve ranging from 0.88 to 0.89. Conclusions: The ML models developed could represent a useful tool to predict PCT positivity, avoiding unusefulness PCT requests. ML models are based on laboratory tests commonly ordered together with PCT but have the great advantage to be easy to measure and low-cost.

Drainage fluid LDH and neutrophil to lymphocyte ratio as biomarkers for early detecting anastomotic leakage in patients undergoing colorectal surgery.

OBJECTIVES: In this study, we investigated the role of several circulating and drainage fluid biomarkers for detecting postoperative complications (PCs) and anastomotic leakage (AL) in patients undergoing colorectal surgery. METHODS: All consecutive patients undergoing colorectal surgery between June 2018 and April 2020 were prospectively considered. On postoperative days (POD) 1, 3, and 5, we measured lactate dehydrogenase (LDH) in drainage fluid, C-reactive protein (CRP) in serum and drainage fluid, and neutrophil to lymphocyte ratio (NLR). RESULTS: We enrolled 187 patients. POD1 patients with AL had higher serum CRP levels, while on POD3 and on POD5 higher NLR and serum CRP. LDH and CRP in drainage fluid were also significantly higher at both time points. The area under the curves (AUCs) of serum and drainage fluid CRP were 0.752 (0.629-0.875) and 0.752 (0.565-0.939), respectively. The best cut-off for serum and drainage fluid CRP was 185.23 and 76 mg/dL, respectively. The AUC of NLR on POD3 was 0.762 (0.662-0.882) with a sensitivity and specificity of 84 and 63 %, respectively, at a cut-off of 6,6. Finally, drainage fluid LDH showed the best diagnostic performance for AL, with an AUC, sensitivity, and specificity of 0.921 (0.849-0.993), 82 %, and 90 % at a cut-off of 2,186 U/L. Trends in serum parameters between patients with or without PCs or AL were also evaluated. Interestingly, we found that NLR decreased faster in patients without PCs than in patients with PCs and patients with AL. CONCLUSIONS: Drainage fluid LDH and NLR could be promising biomarkers of PCs and AL.

Analysis of annual distributions of hemoglobin A2 values as a method to test for HbA2 standardization.

BACKGROUND AND AIMS: The monitoring of yearly distributions of HbA2 measured has been indicated as a reliable indicator of worldwide standardization. MATERIALS AND METHODS: Measurements/year of HbA2 have been collected over three consecutive years in 15 Italian laboratories each using the same analytical method over three years period. HbA2 distributions, cleaned of replicated measurements, were compared by the overlapping area of the raw probability density functions expressed by coefficient eta (η), and by comparing the reference intervals for the central part of each distribution estimated by the indirect method refineR using the R package "refineR". RESULTS: According to the overlapping areas analysis the distributions/year of the data provided by 4 centers able to perform at least 1000 measurements/year were similar in 2 consecutive years. Moreover, the reference intervals provided by 2 centers using the same analytical methods in two separate locations over the three consecutive years, were very similar. The highest overlap (99.7 %) was observed in one center over two consecutive years. The overlapping areas were very high (93.6-95.7%) in 8 out of 9 inter-comparisons. CONCLUSION: Despite the limitations of this study the yearly distribution of the HbA2 measured in various centers appears a reliable tool to test HbA2 standardization over different centers using different analytical methods.

Monocyte distribution width (MDW) in sepsis.

Sepsis is a life-threatening syndrome due to a dysregulated host response to infection, which can be caused by bacterial, viral, or fungal infection. Thus, it is crucial to know how the different microorganisms influence the levels of a biomarker. In the last decade, monocyte distribution width (MDW) has emerged as a promising sepsis biomarker, especially in acute settings, such as the Emergency Department and Intensive Care Unit. In this article, we explore the relationship between MDW and the different pathogens causing infection. Noteworthy, MDW is not a biological molecule, but it is calculated by a mathematical formula based on monocyte characteristics. Monocytes represent the first line defence against microorganisms and undergo activation upon infection, independently from the invading pathogen. According to the knowledge on the biomarker biology and the few literatures evidence, MDW may be considered a biomarker of sepsis, independent of the causative pathogen. However, further investigations are warranted before drawing definite conclusion.

Detection of Antibodies against the Acetylcholine Receptor in Patients with Myasthenia Gravis: A Comparison of Two Enzyme Immunoassays and a Fixed Cell-Based Assay.

The detection of serum anti-acetylcholine receptor (AChR) antibodies is currently an important tool for diagnosing myasthenia gravis (MG) since they are present in about 85% of MG patients. Many serological tests are now available. Nevertheless, results from these tests can be different in some patients. The aim of this study is to compare the sensitivity of a commercially available fixed cell-based assay (F-CBA) to that of enzyme-linked immunosorbent assay (ELISA) kits for anti-AChR detection in patients with a diagnosis of MG. Overall, 143 patients with a confirmed MG diagnosis were included in the study. The detection and measurement of serum anti-AChR antibodies were performed by three analytical methods, namely, a competitive ELISA (cELISA), an indirect ELISA (iELISA), and an F-CBA, according to the manufacturers' instructions. Anti-AChR antibody titers were positive in 94/143 (66%) using the cELISA, in 75/143 (52%) using the iELISA and in 61/143 (43%) using the F-CBA (adult and/or fetal). Method agreement, evaluated by concordant pairs and Cohen's kappa, was as follows: cELISA-iELISA: 110/143 (77%), k = 0.53 (95%CI 0.40-0.66); cELISA-F-CBA: 108/143 (76%), k = 0.53 (95%CI 0.41-0.66); iELISA-F-CBA: 121/143 (85%), k = 0.70 (95%CI 0.57-0.80). Our findings show that the cELISA has better analytical performance than the iELISA and F-CBA. However, the iELISA and F-CBA show the highest concordance.

The effects of estradiol levels on crossmodal perception: a study on the sound induced flash illusion in healthy and menstrually related migraine individuals.

OBJECTIVE: The sound-induced flash illusion (SIFI) is a valid paradigm to study multisensorial perception. In the "fission" SIFI, multiple flashes are perceived when observing a single flash paired with two or more beeps. SIFI is largely dependent on visual and acoustic cortex excitability; in migraine, dysfunctional cortical excitability affects SIFI perception. Since estrogen peak occurring during ovulation can increase neuronal excitability, the present study aims to verify whether cortical excitability shifts linked to the menstrual cycle could influence SIFI. METHODS: In a comparative prospective study, we tested the effect of estrogens on crossmodal perception using the SIFI. We recruited 27 females in reproductive age, including 16 healthy and 11 menstrually related migraine females, testing their proneness to SIFI on day 14 (high estradiol) and day 27 (low estradiol) of menstrual cycle. RESULTS: Women on day 14 reported less flashes than on day 27 (p = 0.02) in the fission illusion, suggesting a pro-excitatory effect of estradiol on visual cortex excitability during ovulation. Moreover, we confirmed that migraine women perceived less flashes (p = 0.001) than controls, independently from cycle phase. Non-migraineurs women significantly reported more flashes on day 27 than on day 14 (p = 0.04). CONCLUSIONS: This study suggests that estradiol may influence the multisensory perception due to changes of visual cortex excitability, with high estradiol peak leading to increased visual cortical sensitivity during ovulation in non-migraineurs. Visual cortex hyperresponsiveness, here reflected by reduced SIFI, is not influenced by estradiol fluctuations in migraine women, as shown by reduced fission effects on day 14 and 27.

The Role of TAR DNA Binding Protein 43 (TDP-43) as a CandiDate Biomarker of Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis.

BACKGROUND: TAR DNA-binding protein 43 (TDP-43) aggregation in neuronal cells is recognized as a hallmark of amyotrophic lateral sclerosis (ALS). Although the literature strongly supports the pathogenetic role of TDP-43 in ALS pathogenesis, the role of TDP-43 as a biomarker of ALS is controversial. We performed a systematic review and meta-analysis to assess the diagnostic performance of TDP-43 for ALS. METHODS: Relevant publications were identified by a systematic literature search on PubMed and Web of Science from their inception to 8 April 2022. RESULTS: Seven studies, including 472 individuals, of whom 254 had ALS according to the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale, met the inclusion criteria for our meta-analysis. According to the random-effects model, CSF TDP-43 levels are higher in ALS patients compared with control groups. CONCLUSIONS: CSF TDP-43 could represent a biomarker of ALS, but further studies are mandatory before drawing conclusions.

Monocyte distribution width alterations and cytokine storm are modulated by circulating histones.

OBJECTIVES: Extracellular histone levels are associated with the severity of many human pathologies, including sepsis and COVID-19. This study aimed to investigate the role of extracellular histones on monocyte distribution width (MDW), and their effect on the release of cytokines by blood cells. METHODS: Peripheral venous blood was collected from healthy subjects and treated with different doses of a histone mixture (range 0-200 µg/mL) to analyze MDW modifications up-to 3 h and digital microscopy of blood smears. Plasma obtained after 3 h of histone treatment were assayed to evaluate a panel of 24 inflammatory cytokines. RESULTS: MDW values significantly increased in a time- and dose-dependent manner. These findings are associated with the histone-induced modifications of cell volume, cytoplasmic granularity, vacuolization, and nuclear structure of monocytes, promoting their heterogeneity without affecting their count. After 3 h of treatment almost all cytokines significantly increased in a dose-dependent manner. The most relevant response was shown by the significantly increased G-CSF levels, and by the increase of IL-1ß, IL-6, MIP-1ß, and IL-8 at the histone doses of 50, 100, and 200 µg/mL. VEGF, IP-10, GM-CSF, TNF-α, Eotaxin, and IL-2 were also up-regulated, and a lower but significant increase was observed for IL-15, IL-5, IL-17, bFGF, IL-10, IFN-γ, MCP-1, and IL-9. CONCLUSIONS: Circulating histones critically induce functional alterations of monocytes mirrored by MDW, monocyte anisocytosis, and hyperinflammation/cytokine storm in sepsis and COVID-19. MDW and circulating histones may be useful tools to predict higher risks of worst outcomes.

Microglial Activation and Priming in Alzheimer's Disease: State of the Art and Future Perspectives.

Alzheimer's Disease (AD) is the most common cause of dementia, having a remarkable social and healthcare burden worldwide. Amyloid ß (Aß) and protein Tau aggregates are disease hallmarks and key players in AD pathogenesis. However, it has been hypothesized that microglia can contribute to AD pathophysiology, as well. Microglia are CNS-resident immune cells belonging to the myeloid lineage of the innate arm of immunity. Under physiological conditions, microglia are in constant motion in order to carry on their housekeeping function, and they maintain an anti-inflammatory, quiescent state, with low expression of cytokines and no phagocytic activity. Upon various stimuli (debris, ATP, misfolded proteins, aggregates and pathogens), microglia acquire a phagocytic function and overexpress cytokine gene modules. This process is generally regarded as microglia activation and implies that the production of pro-inflammatory cytokines is counterbalanced by the synthesis and the release of anti-inflammatory molecules. This mechanism avoids excessive inflammatory response and inappropriate microglial activation, which causes tissue damage and brain homeostasis impairment. Once the pathogenic stimulus has been cleared, activated microglia return to the naïve, anti-inflammatory state. Upon repeated stimuli (as in the case of Aß deposition in the early stage of AD), activated microglia shift toward a less protective, neurotoxic phenotype, known as "primed" microglia. The main characteristic of primed microglia is their lower capability to turn back toward the naïve, anti-inflammatory state, which makes these cells prone to chronic activation and favours chronic inflammation in the brain. Primed microglia have impaired defence capacity against injury and detrimental effects on the brain microenvironment. Additionally, priming has been associated with AD onset and progression and can represent a promising target for AD treatment strategies. Many factors (genetics, environmental factors, baseline inflammatory status of microglia, ageing) generate an aberrantly activated phenotype that undergoes priming easier and earlier than normally activated microglia do. Novel, promising targets for therapeutic strategies for AD have been sought in the field of microglia activation and, importantly, among those factors influencing the baseline status of these cells. The CX3CL1 pathway could be a valuable target treatment approach in AD, although preliminary findings from the studies in this field are controversial. The current review aims to summarize state of the art on the role of microglia dysfunction in AD pathogenesis and proposes biochemical pathways with possible targets for AD treatment.

Deciphering the role of monocyte and monocyte distribution width (MDW) in COVID-19: an updated systematic review and meta-analysis.

The SARS-CoV-2 infection is characterized by both systemic and organ hyper-thromboinflammation, with a clinical course ranging from mild up-to critical systemic dysfunction and death. In patients with coronavirus disease 2019 (COVID-19) the monocyte/macrophage population is deeply involved as both trigger and target, assuming the value of useful diagnostic/prognostic marker of innate cellular immunity. Several studies correlated morphological and immunophenotypic alterations of circulating monocytes with clinical outcomes in COVID-19 patients, concluding that monocyte distribution width (MDW) may retain clinical value in stratifying the risk of disease worsening. Through an electronic search in Medline and Scopus we performed an updated literature review and meta-analysis aimed to explore the association between increased MDW levels and illness severity in COVID-19 patients, deciphering role(s) and function(s) of monocytes in the harmful network underlining SARS-CoV-2 infection. We found that significantly elevated MDW values were frequently present in COVID-19 patients who developed unfavorable clinical outcomes, compounded by a significant association between monocyte anisocytosis and SARS-CoV-2 outcomes. These findings suggest that blood MDW index and its scatter plot could represent useful routine laboratory tools for early identification of patients at higher risk of unfavorable COVID-19 and for monitoring the progression of viral infection, clinical outcomes, and therapeutic efficacy throughout hospitalization. According to this evidence, therapeutic decisions in patients with SARS-CoV-2 infection could benefit from monitoring MDW value, with administration of drugs limiting thrombo-inflammation due to monocyte hyper-activation in patients with severe/critical COVID-19 disease.

Evaluation of the analytical performance of three chemiluminescence serological assays for detecting anti-SARS-CoV-2 antibodies.

The serology surveillance of SARS-CoV-2 antibodies represents a useful tool for monitoring protective immunity in the population. We compared the performance of three SARS-CoV-2 antibody serological immunoassays in 600 vaccinated subjects after the BNT162b2 mRNA COVID-19 vaccine. All serum samples were evaluated by three different immunoassays for detecting anti-SARS-COV-2 antibodies. All SARS-CoV-2 antibody serological immunoassays could detect, when present, a post-vaccine humoral immune response. Median (interquartile range, IQR) anti-S-RBD IgG, Access SARS-CoV-2 IgG (1st IS) and Access SARS-CoV-2 IgG II levels of the subjects investigated were, respectively, 687 BAU/mL (131-2325), 419 IU/mL (58-1091) and 104 AU/mL (14-274). By studying a cohort of unvaccinated subjects, without previous COVID-19 infection, we found a high specificity for all methods. A high correlation was found between IgG titres. Considering the kinetics of subjects with multiple doses, we observed that percentage decreasing gradients were comparable across methods. Our results suggest that all the SARS-CoV-2 antibody serological immunoassays evaluated in this study are suitable for monitoring IgG titers over time. This study contributes to a better understanding of antibody response in vaccinated subjects using some currently available assays.

Distribution of KIR Genes and Their HLA Ligands in Different Viral Infectious Diseases: Frequency Study in Sicilian Population.

Natural killer (NK) cells play a role in defence against viral infections by killing infected cells or by producing cytokines and interacting with adaptive immune cells. Killer immunoglobulin-like receptors (KIRs) regulate the activation of NK cells through their interaction with human leucocyte antigens (HLA). Ninety-six Sicilian patients positive to Human Immunodeficiency Virus-1 (HIV) and ninety-two Sicilian patients positive to SARS-CoV-2 were genotyped for KIRs and their HLA ligands. We also included fifty-six Sicilian patients with chronic hepatitis B (CHB) already recruited in our previous study. The aim of this study was to compare the distribution of KIR-HLA genes/groups of these three different infected populations with healthy Sicilian donors from the literature. We showed that the inhibitory KIR3DL1 gene and the KIR3DL1/HLA-B Bw4 pairing were more prevalent in individual CHB. At the same time, the frequency of HLA-C2 was increased in CHB compared to other groups. In contrast, the HLA-C1 ligand seems to have no contribution to CHB progression whereas it was significantly higher in COVID-19 and HIV-positive than healthy controls. These results suggest that specific KIR-HLA combinations can predict the outcome/susceptibility of these viral infections and allows to plan successful customized therapeutic strategies.

High Cerebrospinal Fluid CX3CL1 Levels in Alzheimer's Disease Patients but Not in Non-Alzheimer's Disease Dementia.

Alzheimer's disease (AD) is the most common form of cognitive decline worldwide, occurring in about 10% of people older than 65 years. The well-known hallmarks of AD are extracellular aggregates of amyloid ß (Aß) and intracellular neurofibrillary tangles (NFTs) of tau protein. The evidence that Aß overproduction leads to AD has paved the way for the AD pathogenesis amyloid cascade hypothesis, which proposes that the neuronal damage is sustained by Aß overproduction. Consistently, AD cerebrospinal fluid (CSF) biomarkers used in clinical practice, including Aß 1-42, Aß 1-40, Aß 42/40 ratio, and pTau, are related to the amyloid hypothesis. Recently, it was suggested that the Aß deposition cascade cannot fully disclose AD pathogenesis, with other putative players being involved in the pathophysiology of the disease. Among all, one of the most studied factors is inflammation in the brain. Hence, biomarkers of inflammation and microglia activation have also been proposed to identify AD. Among them, CX3 chemokine ligand 1 (CX3CL1) has taken center stage. This transmembrane protein, also known as fractalkine (FKN), is normally expressed in neurons, featuring an N-terminal chemokine domain and an extended mucin-like stalk, following a short intra-cytoplasmatic domain. The molecule exists in both membrane-bound and soluble forms. It is accepted that the soluble and membrane-bound forms of FKN evoke differential signaling within the CNS. Given the link between CX3XL1 and microglial activation, it has been suggested that CX3CL1 signaling disruption could play a part in the pathogenesis of AD. Furthermore, a role for chemokine as a biomarker has been proposed. However, the findings collected are controversial. The current study aimed to describe the cerebrospinal fluid (CSF) levels of CX3XL1 and classical biomarkers in AD patients.