Search Results (115)

Type 1 diabetes (T1D) is driven by autoreactive CD4⁺ T-cell responses to pancreatic beta cell antigens presented by disease-associated human leucocyte antigen (HLA) class II molecules. However, the molecular mechanisms by which subtle antigenic modifications promote pathogenic immunity remain incompletely defined. Recent immunopeptidomic studies have identified a cysteine-to-serine substitution at position 19 of the insulin B chain, referred to as InsC19S, as a microenvironment-driven neoepitope that can be presented by HLA class II molecules, including HLA-DQ8, and is recognized by diabetogenic CD4⁺ T cells. In this study we explore potential structural and thermodynamic mechanisms that may contribute to the enhanced immunogenicity associated with this single-amino-acid modification. Using molecular dynamics simulations combined with coarse-grained free-energy-perturbation analyses, we compared HLA DQ8 complexes bound to wild-type (WT) insulin and InsC19S peptides. The InsC19S variant is predicted in simulations to exhibit enhanced binding stability, characterized by increased hydrogen bond occupancy, reduced peptide conformational mobility, and a more favorable binding free energy. In addition, the modified peptide is predicted to induce peptide-dependent conformational adjustments within the HLA-DQ8 peptide-binding groove, resulting in expansion of the conformational landscape and stabilization of distinct low-energy states that are not accessed by the WT complex. Principal component analysis and free-energy landscape mapping suggest that this mutation may promote altered collective motions within HLA DQ8 that are consistent with enhanced peptide major histocompatibility complex (MHC) persistence and optimized antigen presentation geometry. Together, these computational observations suggest a structural framework that may help explain the preferential presentation and pathogenic recognition of InsC19S reported in experimental studies. These findings provide a molecular-level framework that may help link microenvironment-driven insulin neoepitope formation to altered peptide–MHC stability and conformational dynamics in HLA-DQ8. Full article

Background: Only a subset of cannabis users develop persistent psychosis, implying that genetic vulnerability modulates risk. HLA-DR/DQ variation is a strong non-dopaminergic risk locus for schizophrenia, but its role in cannabis-related psychosis is unclear. Methods: We studied 296 cannabis users from Romanian psychiatric services, grouped as non-psychosis (0), non-schizophrenia psychosis (1) and schizophrenia (2). High-resolution HLA-DRB1, DRB3/4/5 and inferred DRB1-DQB1 haplotypes were tested using Fisher’s exact tests with FDR correction in a universal contrast (0 vs. 1+2) and 0-1-2 pairwise comparisons, with Firth logistic regression and resampling as supportive analyses. Results: In the universal analysis, DRB1*16, DRB5 and the DRB1*16-DQB1*05 haplotype were associated with roughly two- to threefold higher odds of psychosis, whereas DRB1*07 and DRB4/DRB3 paralogs showed protective effects or trends. In the 0-1-2 contrasts, DRB1*16 was enriched in non-schizophrenia psychosis. DRB4/DRB3 paralogs were under-represented in schizophrenia relative to both cannabis users without psychosis and those with non-schizophrenia psychosis, suggesting a schizophrenia-specific protective association. Firth models supported effect directions but were underpowered. Conclusions: HLA class II immunogenetic background may modify psychosis risk among cannabis users: DRB5/DRB1*16-containing backgrounds were associated with increased vulnerability, whereas DRB4/DRB3 paralogs were associated with reduced schizophrenia risk in this cohort. These findings are hypothesis-generating, do not establish causality, and warrant replication in larger cohorts. Full article

Celiac disease (CD) is an immune-mediated enteropathy triggered by dietary gluten in genetically predisposed individuals. Although HLA-DQ2 and HLA-DQ8 are the primary genetic determinants of susceptibility, they are not sufficient to explain disease onset and progression. A key molecular event in CD pathogenesis is the post-translational modification of gluten peptides by transglutaminase 2 (TG2), which enhances their binding to HLA-DQ molecules and promotes CD4⁺ T cell activation. TG2 also acts as the principal autoantigen, driving the production of anti-TG2 autoantibodies through linked recognition mechanisms. Beyond its enzymatic activity, TG2 is tightly regulated by gene regulatory mechanisms, including cytokine-driven transcription, epigenetic modulation, and stress-responsive signaling pathways. These processes determine TG2 expression and activity in the intestinal mucosa, thereby influencing the efficiency of gluten peptide modification and antigen presentation. Here, we propose that TG2 operates at the interface between gene regulation and antigen processing, linking transcriptional control of TGM2 to HLA-restricted immune activation. In this framework, disease susceptibility arises from the coordinated interaction between HLA-dependent peptide presentation, TG2-mediated modification of gluten epitopes, and regulation of TG2 expression within the intestinal mucosa. This integrated model provides a mechanistic basis for disease heterogeneity and identifies TG2 as a central regulatory node and potential therapeutic target in CD. Full article

Background/Objectives: Chronic lung allograft dysfunction (CLAD) remains the leading cause of late graft failure after lung transplantation (LuTX). De novo donor-specific anti-HLA antibodies (dnDSA), especially HLA-DQ, have been implicated; we assessed associations between dnDSA (class and specificity) and CLAD after LuTX. Methods: We retrospectively analyzed all LuTX recipients transplanted from 2005–2018 at a single center (n = 585). dnDSA were measured by Luminex single-antigen bead assays (MFI > 1000) at 1, 3, 6, and 12 months and at least annually thereafter. CLAD was defined by ISHLT criteria; time-to-event comparisons used log-rank testing. Results: dnDSA developed in 151/585 recipients (25.8%), predominantly class II (129/585; 22.1%); class I dnDSA occurred in 52/585 (8.9%). CLAD occurred more frequently in dnDSA-positive than dnDSA-negative recipients (64/151; 42.4% vs. 109/434; 25.1%; p < 0.0001). Rejection-attributed death was higher in dnDSA-positive recipients (19/151; 11.3% vs. 25/434; 5.3%; p = 0.01). Both class I and class II dnDSA were associated with higher CLAD rates (log-rank p < 0.001 each). Locus-specific analyses identified HLA-DQ dnDSA as strongly associated with CLAD (p < 0.0001); DQ7 was the most frequent specificity (n = 44) and showed the strongest association (p < 0.0001). Conclusions: dnDSA after LuTX were associated with increased CLAD incidence and rejection-attributed mortality, with a prominent association for HLA-DQ—particularly DQ7. Full article

Background: Celiac disease (CD) is a multifactorial autoimmune disorder strongly associated with specific HLA class II molecules, particularly HLA-DQ–encoding haplotypes. Although the genetic contribution of these loci is well established, the structural features accompanying allele-specific disease susceptibility remain incompletely explored. Methods: In this study, molecular HLA typing was integrated with in silico secondary structure analysis to examine the relationship between genetic predisposition and structural organization of HLA class II molecules in a Sardinian population. A total of 100 patients with CD and 100 healthy controls were genotyped for HLA-DR and HLA-DQ alleles, and allelic and haplotypic distributions were compared between groups. Secondary structure predictions were performed using PSIPRED on selected HLA class II alleles, focusing on groove-forming domains of HLA-DRB1 and HLA-DQA1. Results: CD patients showed a marked enrichment of the DR3–DQ2.5 haplotype, together with a population-specific predominance of DQ2.5 and a reduced contribution of DQ8. Secondary structure analysis of the HLA-DRB1 β1 domain revealed a largely conserved organization, with only modest allele-dependent variations. In contrast, comparative analysis of HLA-DQA1 identified localized differences within the α1 domain, with the DQ2.5 molecule displaying a more coherent secondary structure organization compared with the lower-risk DQ2.2 variant. Conclusions: By integrating genetic and in silico structural analyses, this study highlights that HLA-associated susceptibility to celiac disease reflects not only allele and haplotype distribution but also subtle, allele-specific features in the structural organization of peptide-binding regions. These findings provide a refined framework for interpreting HLA-DQ–mediated genetic risk and support the relevance of structural coherence as a complementary dimension in the assessment of disease susceptibility. Full article

Background and Objectives: Celiac disease (CD) is a major global public health problem that can occur at any age. Pediatric CD can be typical, atypical, or even asymptomatic. Early diagnosis and early initiation of treatment are essential for improving patients’ quality of life and preventing serious complications later in life. However, it is impossible to identify asymptomatic children and adolescents without screening. In this systematic review, we attempted to identify different mass screening programs that have been reported for CD in apparently healthy children and adolescents across the world, to highlight the advantages and disadvantages of such strategies, and to collect and synthesize data from these studies reporting the prevalence of CD. In addition, where data were available, we also attempted to evaluate the diagnostic accuracy of the tests used, their cost-effectiveness, the reported clinical benefits, and follow-up data from individuals identified through screening. Materials and Methods: Electronic databases, including PubMed and Scopus, were systematically searched. Initially, a total of 316 studies were retrieved. Finally, 55 studies met all inclusion criteria and were included in this review. The included studies were published between 1996 and 2023. Results: The reported age of participants ranged from 6 months to 23 years. Confirmation of CD by biopsy was reported in all but six studies. According to the studies that provided data, the (tTG IgA) seroprevalence of CD in apparently healthy children and adolescents, detected through different mass screening methods around the world, ranged from 0.20% (Turkey) to 3.11% (Italy). In addition, the prevalence of biopsy-confirmed CD ranged from 0.036% (Vietnam) to 3% (Sweden and Spain). Studies from 17 countries reported mass screening strategies based on finger-prick rapid tests. All rapid tests detected CD antibodies, except two, which detected HLA DQ2/DQ8 haplotypes. Rapid tests appeared to be no less sensitive and specific than other screening tests for CD and were probably less expensive, but further studies are needed for more reliable conclusions. Of the 55 studies in the review, only 10 reported follow-up data. After 3 months of a gluten-free diet, the general condition of the patients improved; after 6 months, tTG IgA and EMA IgA levels decreased and hemoglobin values increased; while after 1 year, tTG IgG levels also decreased, symptoms subsided, the children’s weight and height increased, school performance improved, episodes of upper respiratory tract infections decreased, and thyreoperoxidase antibodies that were positive at screening became negative. Conclusions: Mass screening for CD in asymptomatic children and adolescents is a challenge. Future research should provide more answers regarding the most appropriate target age, the frequency of screening, the optimal screening method, the cost-effectiveness, the clinical utility, and the long-term impact of mass screening on patients’ quality of life. Full article

Background/Objectives: While standard Luminex single antigen bead (SAB) detects total IgG antibodies, qualitative differences among IgG subclasses may influence their immunologic risk. In particular, complement fixing ability, assessed via C1q binding, is linked to poor transplant outcomes. This study aimed to evaluate the relationship between IgG subclasses and C1q-binding activity in HLA antibodies and to define clinically relevant subclass-specific mean fluorescence intensity (MFI) thresholds for predicting complement binding. Methods: We analyzed 4189 HLA IgG bead reactions from sera of 37 kidney transplant recipients using SAB assays for total IgG, IgG1-4 subclasses, and C1q-binding. IgG subclasses were assessed using a modified SAB assay with subclass-specific monoclonal secondary antibodies. Results: IgG reactivity (MFI ≥ 1000) was observed in 15.3% of beads (639/4189), with 31.0% (198/639) also positive for C1q binding. IgG⁺C1q⁺ beads exhibited significantly higher MFIs compared with IgG⁺C1q⁻ beads. IgG1 showed positive correlations with both total IgG (r_s = 0.5439, p < 0.0001) and C1q MFIs (r_s = 0.4042, p < 0.0001), with the strongest correlations at HLA-DQ. Among subclass-positive beads, IgG1 predominated and was strongly associated with C1q binding, whereas isolated IgG2 or IgG4 positivity was rarely C1q-binding. ROC analysis identified an IgG1 MFI threshold of >837 to predict C1q positivity with 73.2% sensitivity and 92.3% specificity, while the cutoff for total IgG MFI was >7881 with 85.4% sensitivity and 88.9% specificity. At the patient level, IgG1-positive immunodominant DSAs were more frequent in antibody-mediated rejection than in non-rejection biopsies Conclusions: IgG1 predominates among complement-fixing antibodies and correlates strongly with total IgG and C1q binding. Quantitative IgG subclass assessment, especially IgG1, may serve as a useful predictor of complement activation. Full article

Celiac disease is a chronic immune-mediated disorder triggered by the ingestion of gluten in genetically susceptible individuals, primarily those carrying HLA-DQ2 or HLA-DQ8 and, in rare cases, HLA DQ7 alleles. Although traditionally regarded as a gastrointestinal condition, celiac disease is now recognized as a multisystem disorder with a wide range of clinical presentations. It has been described as a “clinical chameleon” due to its variable manifestations, which may include non-specific symptoms, extraintestinal involvement, or even an asymptomatic course, often identified only through screening of high-risk groups. This narrative review provides a comprehensive overview of celiac disease, highlighting recent insights into its pathogenesis, including genetic predisposition, immune mechanisms, and the role of environmental and microbial factors. It emphasizes the importance of recognizing extraintestinal features, outlines current diagnostic approaches and their limitations, and discusses management strategies centered around the gluten-free diet. Furthermore, it explores emerging therapies aimed at improving patient outcomes and reducing dependence on dietary restriction. By synthesizing the latest developments, this review aims to present a fresh perspective on a condition with significant clinical relevance that is evolving. Full article

Celiac disease (CeD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals, with a heterogeneous clinical spectrum spanning classical gastrointestinal symptoms, extraintestinal manifestations, and subclinical forms. We synthesize contemporary epidemiology, immunopathogenesis, and the updated 2025 European Society for the Study of Coeliac Disease diagnostic framework. Adaptive responses to deamidated gliadin peptides presented by human leukocyte antigen (HLA)-DQ2/DQ8, together with interleukin-15-driven activation of intraepithelial lymphocytes (IELs), culminate in villous atrophy, crypt hyperplasia, and increased IELs. Serology centered on tissue transglutaminase immunoglobulin A (tTG-IgA) with total immunoglobulin A assessment remains first-line, complemented by standardized duodenal sampling (≥4 distal + 2 bulb biopsies) and selective HLA typing. The guidelines conditionally endorse a no-biopsy pathway for adults <45 years with tTG-IgA ≥10× upper limit of normal confirmed on a second sample, emphasizing shared decision-making and exclusion of red flags. We delineate differential diagnoses (tropical sprue, Crohn’s disease, common variable immunodeficiency, small intestinal bacterial overgrowth) and contrast CeD with non-celiac gluten sensitivity, which lacks villous atrophy, disease-specific serology, and HLA association. Emerging tools (immunohistochemistry, CD3/CD8/γδ IELs, video capsule endoscopy, confocal laser endomicroscopy) and the limitations of salivary/fecal assays are reviewed. Early detection improves quality of life and reduces healthcare utilization. Future directions include artificial intelligence-assisted imaging, molecular immunophenotyping, and non-dietary therapeutics. Full article

Objective: Ongoing debates focus on the role of human leukocyte antigen (HLA) class II expression in shaping clinical phenotypes of chronic inflammatory airway diseases. This study seeks to clarify the impact of class II HLA on chronic obstructive pulmonary disease (COPD) and asthma–COPD overlap (ACO). Method: The expression levels of HLA-DQ/DR in blood immune cells were analyzed in 116 participants: 41 with COPD, 37 with ACO, 20 with pure asthma, and 18 healthy subjects (HS). Results: In the COPD group, HLA-DR protein expression levels were significantly elevated on blood M2a monocytes (7695 ± 3743 vs. 5391 ± 3153 MFI, p = 0.026), helper T cells (2551 ± 956 vs. 1836 ± 531 MFI, adjusted p = 0.018), cytotoxic T cells (1591 ± 531 vs. 1360 ± 477 MFI, adjusted p = 0.036), and B cells (20,667 ± 7985 vs. 15,694 ± 2003 MFI, adjusted p = 0.031) compared to the HS group. Conversely, no significant changes were observed in the asthma group. In ACO patients, helper T cells showed increased HLA-DR protein expression (2416 ± 914 MFI; adjusted p = 0.016) compared with the HS group. Higher levels of HLA-DR expression correlated with reduced pulmonary function, frequent exacerbations, and more severe symptoms. Following one year of treatment in 14 COPD and 16 ACO patients, HLA-DR protein expression on blood helper T cells, cytotoxic T cells, M2a monocytes, and neutrophils significantly declined (all p < 0.05). In vitro experiments demonstrated that exposure of M2- or M1-polarized THP-1 cells to a stimulus mix containing cigarette smoke extract, house dust mite antigens, and lipopolysaccharide led to up-regulation of HLA-DR expression. This response was linked to increased apoptosis and reduced production of reactive oxygen species. Conclusions: Up-regulation of HLA-DR in COPD and ACO patients may represent a novel biomarker for assessing disease severity and treatment response. Additionally, it could serve as a useful tool to distinguish COPD and ACO from asthma. Full article

Coeliac disease (CeD) is a gastrointestinal enteropathy triggered by the consumption of gluten in predisposed individuals. A recent study showed that individuals were at more than 10% risk of having CeD if a first-degree relative also had the disease. However, only around 50% of CeD genetic heritability is attributable to specific loci, with the majority of this heritable risk attributed to the HLA loci, while the remaining 50% of disease risk is currently unidentified. We investigated the butyrophilin family of immunomodulators as novel CeD risk loci. We sequenced the butyrophilin loci of 48 CeD and 46 control patients and carried out gene-based burden testing on the captured single-nucleotide polymorphisms (SNPs). We found a significantly increased BTN2A1 gene burden in CeD patients. To validate these results, the SNP data of 3094 CeD patients and 29,762 control participants from the UK Biobank database were subjected to single-variant analyses. Fourteen BTN2A1, ten BTN3A1, and thirteen BTN3A2 SNPs were significantly associated with CeD status. These results are interesting, as BTN2A1 and BTN3A2 have not been associated with CeD risk previously but are known to modulate the activation of Vγ9+ γδ T cells and NK cells. Twenty of the 37 SNPs above were associated with CeD status independent of the risk-associated HLA genotypes. All twenty of these SNPs, alongside a novel SNP not included in the above SNPs, were associated with CeD in HLA-DQ2.5-matched case-control groups. We reaffirm the association of the BTN3A2 locus with CeD risk and identify BTN2A1 and BTN3A1 as putative novel CeD risk loci. Full article

Background: Celiac disease (CD) is a gluten-sensitive immune-related enteropathy of the small intestine characterized by villus atrophy, crypt hyperplasia, and increased intraepithelial lymphocytes (IELs). Objectives: To characterize the phenotype of IELs and immune cells of the lamina propria of small intestine control using immuno-oncology and immune-phenotype markers and test the most relevant marker, an immune checkpoint co-inhibitory receptor, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), in CD. Methods: Immunohistochemical analysis of CD3 (CD3E), CD4, CD8, CD103 (ITGAE), Granzyme B (GZMB), TCR beta (β), TCR delta (δ), CD56 (NCAM), CD16 (FCGR3A), LAIR1 (CD305), PD-L1 (CD274), PD1 (CD279), BTLA (CD272), TOX2, HVEM (TNFRSF14), CD163, HLA-DP-DQ-DR, IL4I1, and FOXP3 was performed using histological analysis. Gene expression analysis was performed using an independent dataset to expand and confirm the findings. Results: IELs exhibited a cytotoxic T-cell phenotype and were CD3+, CD8+, CD103+, TCR beta+, and LAIR1+. The lamina propria (LP) was abundant in CD163+, HLA-DP-DQ-DR+, BTLA+, PD-L1+, CD103+, CD56+, and LAIR1+ cells corresponding to macrophages and T- and B-lymphocytes. In CD, IELs and part of the inflammatory cells of the lamina propria cells were LAIR1+. CD was characterized by higher quantity of LAIR1+ IELs and LP immune cells than the small intestine control (p = 0.004). Higher intestinal lesions evaluated by Marsh scoring were correlated with higher LAIR1 (p < 0.001). Gene expression analysis confirmed the overexpression of the LAIR1 pathway in CD and highlighted BTLA. At the protein level, BTLA overexpression was confirmed in CD. Finally, as a proof-of-concept AI analysis, a convolutional neural network classified LAIR1-stained image patches between the three diagnoses of small intestine control, CD, and reactive tonsils with high accuracy (99.6%). Conclusions: IELs exhibit a cytotoxic T-cell phenotype and were found to be CD3+, CD8+, CD103+, TCR beta+, and LAIR1+ in the small intestine control. Increased numbers of LAIR1+ IELs and lamina propria immune cells characterize CD. Full article

Background/Objectives: Sera from patients before and after organ transplantation were tested at two different temperatures, 21 °C and 37 °C. Currently, organs are transported under normothermic conditions (37 °C). This observational pilot study was conducted to define the effect of the incubation at 37 °C, comparing the results to the usual temperature of 21 °C for serum–bead incubation. Methods: We used the Luminex-based assay for the identification and characterization of HLA-specific antibodies. The assays were performed using single antigen beads for HLA class I and HLA class II. A total of 42 sera were assessed and tested, and 38 were analyzed on the Luminex 200 platform at both temperatures. Results: We noted varying outcomes: both an increase and a decrease in mean fluorescence intensity values. A shift from negative to positive values (n = 6) and vice versa (n = 1) was observed. Several sera (n = 4 for HLA class I and n = 5 for HLA class II) exhibited no alterations. In general, we observed an increase in the mean fluorescence intensity values by incubation at 37 °C. The analysis at the bead level revealed a significant deviation (37 °C vs. 21 °C) for the bead carrying HLA-A80 (p = 0.0006) and two HLA-DQ beads, DQA1*05:01-DQB1*02:01 (p = 0.0438) and DQA1*01:03-DQB1*06:03 (p = 0.0438). Conclusions: Mimicking physiological temperature conditions for the testing of HLA-specific antibodies will lead to the better and more accurate interpretation of the results. This method shows potential for use in the delisting strategy for highly sensitized patients as well, thus allowing a better and more reliable option for the patient awaiting a suitable crossmatch-negative organ. Full article

Background/Objectives: Pharmacogenetics examines genome variability and its influence on drug efficacy and toxicity, forming the foundation for personalized medicine. Patients with inflammatory bowel disease (IBD) treated with azathioprine with thiopurine S-methyltransferase (TPMT) deficiency are at an increased risk of drug-related toxic effects. Variability in the HLA-DQA1 and DQB1 alleles may lead to an inadequate therapeutic response. This study aimed to determine the significance of TPMT and HLA-DQ Allelic Variants in therapy optimization planning. Methods: A retrospective study was conducted to determine TPMT gene polymorphism and the presence of HLA-DQA1 and HLA-DQB1 alleles in children diagnosed with IBD and treated at the Institute for Child and Youth Health Care of Vojvodina in May 2023. Results: The study included 104 children with a mean age of 13.71 ± 3.1 years, with a balanced gender distribution. A TPMT mutation was identified in only one child. The most common HLA-DQA1 alleles were *01 (49%) and *05 (28.8%), while the most frequent allele at the HLA-DQB1 locus was 03 (15.4%). The presence of the HLA-DQA105 allele was associated with the development of anti-drug antibodies against anti-TNF therapy (RR: 1.23; 95% CI: 1.03–1.50), while the presence of HLA-DQA101 was significantly more frequent in children on optimized therapeutic regimens (RR: 1.63; 95% CI: 1.13–2.10). Conclusions: Prior to the initiation of azathioprine therapy, TPMT genotyping should be performed to prevent adverse effects and ensure optimal drug dosing. Identification of the HLA-DQA105 and HLA-DQA101 alleles plays an important role in the planning of biological therapy regimens, including decisions on dose escalation or interval shortening. Full article

Coeliac disease (CD) is a chronic immune-mediated enteropathy triggered by gluten ingestion in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8 haplotypes, characterized by small intestinal mucosal damage and systemic manifestations. This systematic review and meta-analysis aimed to compare the effectiveness of urine versus stool GIPS testing for monitoring gluten intake in coeliac patients, providing evidence-based recommendations for clinical practice. A comprehensive literature search was conducted in databases like PubMed and Embase. Studies evaluating urine or stool GIPS testing in coeliac patients were included, focusing on sensitivity, specificity, and patient adherence. The meta-analysis included six studies with a total of 572 participants. The stool GIPS testing demonstrated a pooled sensitivity of 85.1% (95% CI: 79.0–89.9%) and a specificity of 92.5% (95% CI: 88.3–95.6%), making it highly reliable for detecting gluten exposure and ruling out false positives. It also achieved an AUC of 0.9853, indicating excellent diagnostic performance. In contrast, the urine GIPS testing showed a pooled sensitivity of 55.4% (95% CI: 49.6–61.2%) and a specificity of 73.0% (95% CI: 67.4–78.1%), with an AUC of 0.7898. The heterogeneity across the studies was significant (I² > 80%), driven by variations in the population characteristics, sample handling, and testing protocols. These findings emphasize the need for standardized methodologies to enhance the reliability and comparability of results. Full article