Candida auris is a high-priority multidrug-resistant yeast. In New York, C. auris is reportable, and surveillance screening is recommended for high-risk patients. This study provides a retrospective review of the yield and characteristics of C. auris cases detected through active surveillance testing at a tertiary cancer care center. Testing for C. auris was performed using a laboratory-developed real-time PCR test on patients admitted to the intensive care unit. Sample sources included axilla, groin, and nares swabs. Candida auris-positive PCR samples were further tested by culture with recovered isolates identified and further characterized by whole-genome sequencing and antifungal susceptibility testing. From 2019 to 2023, 27,299 samples were tested, with 139 positive samples (0.5%) on 16 unique patients. Positive swabs included 40 of 139 (28.7%) axilla, 44 of 139 (31.7%) nares, and 55 of 139 (39.5%) groin. A total of 134 of 139 (96.4%) samples were cultured, and 76 of 134 (56.7%) were positive in culture. An increase in positive swabs was noted. Four patients developed disseminated infections following a positive surveillance swab. Whole-genome sequencing classified all isolates as clade I, except for one isolate identified as clade III. Resistance to fluconazole was detected in 80% of isolates. Although the positivity rate remained low in this patient population, the recent increase in cases of C. auris nationwide underlies the need for active surveillance to prevent spread of this multidrug-resistant organism.

Analyzing somatic mutations in liquid biopsies poses a real challenge in treating patients with breast cancer. Because of the high sensitivity required to detect circulating tumor DNA, which may be present at low levels, digital PCR analysis seems highly appropriate. However, new targeted next-generation sequencing solutions are now available, enabling highly sensitive multigene analysis that could benefit patients. This study compared the analytical performance of multiplex digital PCR and targeted next-generation sequencing in detecting somatic erb-b2 receptor tyrosine kinase 2 (ERBB2), estrogen receptor 1 (ESR1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) mutations in a series of 32 plasma samples from patients with metastatic breast cancer. Forty-four mutations were detected, with an overall concordance of 95% (90/95) and a high degree of correlation (R = 0.9786). Interestingly, two ESR1 mutations detected in multiplex drop-off digital PCR were also detected by targeted next-generation sequencing with comparable mutant allele frequencies, enabling the identification of these specific variants (p.D538N and p.536LYD>P). Moreover, an additional PIK3CA mutation (p.P539R) was first detected using targeted next-generation sequencing and later confirmed with a newly designed digital PCR assay. Although more expensive than multiplex digital PCR, these new types of small targeted next-generation sequencing gene panels could provide a rapid answer to a specific clinical question with a ready-to-use solution, which could benefit patients.

Conventional methods for spinal muscular atrophy (SMA) screening have been challenging in detecting SMN1/2 single-nucleotide variants (SNVs) and small insertions and deletions, SMN1 2 + 0 silent carrier, and the copy number (CN) of SMN2. To address these limitations, a long-read sequencing (LRS)-based approach termed comprehensive analysis of SMA 2 (CASMA2) was developed. CASMA2 was used to perform CN analysis by integrating Poisson distribution with an endogenous reference gene, the first such method developed for LRS platforms. The performance and clinical feasibility of CASMA2 were evaluated by using 414 retrospective peripheral blood samples and 303 prospective dried blood spot samples. CASMA2 displayed 100% accuracy in SMN1/2 CN analysis and identified the SNVs/insertions and deletions in SMN1/2. CASMA2 also showed the capability of screening for the SMN1 2 + 0 silent carrier with family-trio haplotype analysis. It achieved a 99.0% (410 of 414) first-attempt success rate for long-term peripheral blood samples and a 98.7% (299 of 303) rate for dried blood spot samples. CASMA2 offers a clinically feasible, precise, and efficient method for SMA carrier and newborn screening.

Cameroon is an epicenter of diverse HIV-1 strains, with challenges in diagnosis and disease management. The objective herein was to update the prevalence of HIV-1 non-M and compare diagnostic-performance of the two- versus three-test algorithms. A facility-based study was conducted in February 2024 on 2207 HIV-1 clinical-samples at the Chantal BIYA International Reference Centre (Yaoundé, Cameroon). Molecular phylogeny and rapid subtyping were performed for identifying HIV-1 non-M. Performances of rapid diagnostic tests (RDTs) used in the two-test (Determine and KHB) versus three-test (First Response, One Step, and KHB) algorithms were evaluated on non-M, with ACRO Rapid Test (HIV1/2 and p24) as independent RDT. No group N (0%) nor P (0%) was found, whereas nine group O strains were identified (0.4%; 95% CI, 0.2%-0.8%). For individuals harboring group O (mean age, 43 ± 12 years; 50% female), median [interquartile range (IQR)] duration since HIV diagnosis was 627 (423 to 775) weeks, median (IQR) viremia was 12,385 (5340 to 72,682) copies/mL, and median (IQR) CD4 count was 52 (39 to 228) cells/mm. One Step, KHB, and ACRO Rapid Test (HIV1/2 and p24) detected eight of eight group O (100% performance); First Response HIV1-2.0, seven of eight (87.5% performance); and Determine HIV1/2, six of eight (75% performance), P = 1.00. In this Cameroonian setting, HIV-1 group N and P are scarce, whereas group O remains low (<1%). Transitioning from the two-test (75% performance) to the three-test algorithm (87.5% performance) could lead to improved diagnostic performance on currently circulating HIV-1 group O, calling for updates in RDTs to adapt to viral dynamics.

Splice-site variants within the ABO gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized how four intron 6 splice-site variants-three in the A1 allele (c.374+4A>T, c.374+4A>G, and c.374+5G>A) and one in the ABO∗B.01 allele (c.374+2_374+3insT)-affect ABO pre-mRNA splicing. A combination of serologic, molecular genetic, bioinformatic, and minigene assays established a genotype-splicing-phenotype association model. Bioinformatics predictions showed that all variants impaired the recognition of the 5' splice site. Specifically, the A allele variant c.374+5G>A induced approximately 2.8% exon 6 retention, whereas the c.374+4A>G (A/A) and c.374+4A>T (A) variants preserved approximately 6.9% and 10.2% functional transcripts, respectively. The novel B allele insertion variant c.374+2_374+3insT (B subtype) retained approximately 4.7% exon 6-containing transcripts, sufficient for trace B glycosyltransferase expression. Quantitative real-time PCR analysis confirmed that the transcriptional levels of the ABO gene in the AB individual carrying the c.374+2_374+3insT variant were approximately 51.7% of those in the ABO∗A1.02/ABO∗B.01 control. This study demonstrated that splice-site variants in the ABO gene reduce the abundance of functional transcripts via altered transcriptional regulation, which, in turn, leads to decreased ABO glycosyltransferase expression, ultimately resulting in weak antigen phenotypes of varying intensity.

Breast cancer is the most common cancer among women, and metastasis to the lung is associated with poor prognosis. Reliable biomarkers for predicting lung metastasis are urgently needed to improve early detection and clinical decision-making. This study utilized microarray datasets comprising gene expression profiles and clinical data from primary breast cancer patients who were followed for lung metastasis outcomes. High-throughput screening, combined with Venn diagram analysis, was used to identify common candidate probes, and the least absolute shrinkage and selection operator (LASSO) method selected eleven genes for model development. Logistic regression was used to construct predictive models, and the final risk signature consisted of ten candidate genes (CDK19, GLUD1, GTPBP4, HLCS, HYI, KCND3, MAP2K1, NMUR1, PRKD3, and SLC16A3). The model achieved strong performance in training and validation cohorts (AUCs > 0.87) and generalized to the independent METABRIC dataset (AUC = 0.706). Subset analyses restricted to early-stage patients confirmed that the signature retained predictive value. Kaplan-Meier analyses demonstrated that patients with high-risk scores had shorter lung metastasis-free survival, recurrence-free survival, and overall survival. Multivariate Cox analysis confirmed that the risk signature provided independent predictive information from clinical variables. In conclusion, the risk signature accurately identifies breast cancer patients at risk of lung metastasis, enabling clinicians to better assess risk and tailor treatment strategies effectively.

Donor chimerism analysis is used for monitoring engraftment status and risk of disease relapse following allogeneic stem cell transplantation. Recently developed assays using next generation sequencing (NGS) have demonstrated enhanced sensitivity and accuracy compared to standard capillary electrophoresis methods. We report here our validation results using One Lambda Devyser Chimerism assay, an NGS-based test for monitoring donor chimerism. A total of 270 samples, including clinical and cell line DNA, were tested. There was a high correlation between chimerism results obtained with One Lambda Devyser and STR assays (Rˆ2 = 0.999). Determination of the limit of blank, limit of detection and limit of quantitation indicated that One Lambda Devyser Chimerism assay can reliably detect recipient DNA fractions as low as 0.1 %. Analytical specificity was > 99.9 %. Reproducibility, linearity, DNA library characteristics and sequencing metrics are presented. The suitability of DNA markers was verified in a population predominantly African American and Hispanic, comprising 30 recipient/donor pairs. The average number of informative markers per pair was 7, with a lower representation (5 markers) in related pairs. In conclusion, our results show that One Lambda Devyser Chimerism assay is a highly sensitive test for detecting donor chimerism in a diverse patient population. The assay performed remarkably well at low recipient concentrations, having the potential to detect early changes associated with disease relapse.

Epidermal Growth Factor Receptor variant III (EGFRvIII), a common oncogenic variant in glioblastoma (GBM) and other solid tumors, results from an in-frame deletion of exons 2-7 in the EGFR gene. Detection of EGFRvIII is crucial for understanding tumor biology, guiding targeted therapies, and development of personalized treatment strategies. In this study, two detection approaches based on DNA next-generation sequencing (NGS)-read depth (RD)-based and split read (SR)-based detection-were compared to evaluate their sensitivity and accuracy in identifying EGFRvIII. Thirty-one tumor samples, including glioblastoma (GBM) and pancreatic adenocarcinoma, were analyzed using both methods. The SR-based method detected EGFRvIII in 20 out of 31 samples, while the RD-based method identified it in only 12 samples, demonstrating that the SR-based method had significantly higher sensitivity (p < 0.001). RNA NGS confirmed EGFRvIII expression in most SR-positive cases. Additionally, the SR-based method identified multiple breakpoints in several tumors, revealing intratumor heterogeneity and the subclonal origins of EGFRvIII. The RD-based method was prone to false negatives, particularly in cases with high EGFR amplification or low tumor cell percentage, where copy number variations could be masked by background noise. The findings highlight the superior sensitivity and accuracy of SR-based detection in identifying EGFRvIII and capturing intratumor heterogeneity. SR-based analysis is recommended as the method for EGFRvIII detection in both clinical and research settings.

Optical genome mapping (OGM) enables high-resolution detection of structural variants (SVs) and copy number aberrations (CNAs) using ultra-long DNA molecules and minimal bioinformatics processing. Its diagnostic utility in solid tumors remains underexplored. Whole-genome sequencing (WGS) offers comprehensive variant detection but is resource-intensive. This study presents a technical benchmarking of OGM versus WGS for mesenchymal tumors of the gynecological tract. We prospectively analyzed 25 uterine mesenchymal tumors using matched WGS, transcriptome sequencing, and OGM. Detected SVs, CNAs, and fusion genes were compared across platforms. OGM identified structural driver events in 80% of cases and demonstrated high concordance with WGS for major CNAs and translocations. In select cases, OGM resolved complex rearrangements not clearly defined by WGS, including a PLAG1::RERE fusion and an embedded inversion in a RAD51B::HMGA2 event. Conversely, WGS uniquely detected a truncating NF1 translocation and a TSC2::SENP3 fusion, both clinically significant. OGM is a technically robust platform for SV and CNA detection in mesenchymal tumors, and may serve as an efficient alternative to sequencing-based cytogenomic approaches in selected clinical contexts, especially in tumors known to be driven by gross chromosomal rearrangements. WGS provides a comprehensive view of the cancer genome, suitable for tumors driven by both single nucleotide variants, SVs, and CNAs. The choice between platforms should be guided by clinical context, diagnostic needs, and available resources.

Liquid biopsy offers a promising noninvasive alternative for tissue sampling in solid cancers. Saliva, an easily accessible biofluid, can harbor tumor DNA, yet its clinical utility compared with plasma circulating tumor DNA (ctDNA) in head and neck squamous cell carcinoma (HNSCC) remains uncertain. Tumor samples from patients with HNSCC underwent next-generation sequencing to identify TP53 mutations. Matched plasma and saliva cell-free DNA (cfDNA) samples were analyzed for the presence of tumor-specific mutations. Pathologic features and survival data were evaluated in relation to mutation detectability in biofluids. Overall, TP53 mutations were detected in 64 of 85 tumors (75%). Liquid biopsy analysis included 36 plasma and 21 saliva samples from 40 patients. Plasma ctDNA was detected in 68% of oral cavity squamous cell carcinoma compared with 29% of laryngeal cancer cases, and it was associated with nodal metastases (P = 0.034). Detection of ctDNA showed a trend toward worse progression-free survival (37.4 versus 68.5 months; P = 0.134). Tumor mutation was identified in saliva cfDNA in 57% of cases, irrespective of disease stage or presence of regional metastases. Plasma ctDNA emerged as a potential prognostic marker in HNSCC, whereas mutated saliva cfDNA, although frequently detectable, lacked prognostic value or correlation with adverse pathologic features. Further research is warranted to elucidate the mechanisms governing tumor DNA shedding into saliva and to define its clinical applications.

RNA-based targeted sequencing aids the detection of several types of variants in hematologic and other malignancies, including splice-altering variants. However, accurately identifying clinically relevant mis-splicing events remains challenging because of the inherent complexity of the human transcriptome and the high prevalence of false-positive splice junctions in deep RNA-sequencing data. To address these challenges, SpliceChaser and BreakChaser were developed, which are bioinformatics tools designed to enhance the detection and characterization of relevant splice-altering events. SpliceChaser improves the identification of clinically relevant atypical splicing by analyzing read length diversity within flanking sequences of the mapped reads around the splice junctions. BreakChaser processes soft-clipped sequences and alignment anomalies to enhance the detection of targeted deletion breakpoints associated with atypical splice isoforms generated from intrachromosomal gene deletions. These tools were developed and validated using a cohort of >1400 RNA-sequencing samples from patients with chronic myeloid leukemia. Collectively, SpliceChaser and BreakChaser achieved a positive percentage agreement of 98% and a positive predictive value of 91% for the detection of clinically relevant atypical splice-altering variants or gene deletions in the targeted regions. By integrating splicing and breakpoint detection with robust filtering strategies, these tools facilitate precise identification of clinically relevant variants, paving the way for improved diagnostics and therapeutic strategies in chronic myeloid leukemia and other malignancies.

Gene expression signatures are important for classifying lymphoid malignancies, although routine diagnostic workflows predominantly use immunohistochemical staining and fluorescence in situ hybridization. These traditional methods are labor intensive and may misclassify the underlying oncogenic signatures, leading to inaccurate prognostication. To address this issue, an RNA expression panel was developed, the Lymphoma Expression Analysis (LExA120) 120 gene expression panel, using the NanoString platform for rapid, modular analysis of various lymphoma subtypes. The LExA120 panel targets 95 genes and 25 housekeeping genes to evaluate aggressive B-cell lymphomas, including: diffuse large B-cell lymphoma cell-of-origin, dark zone, and primary mediastinal large B-cell lymphoma signatures; Epstein-Barr virus (EBV) status; and a classical Hodgkin lymphoma posttransplant risk. Fifty-four formalin-fixed, paraffin-embedded tissue samples were tested with known diagnoses and 51 samples with known EBV status. The panel showed high concordance with previously validated methods according to Pearson correlation coefficients of the signature scores. The assay also displayed high reproducibility in repeated tests and across different clinical laboratories. This study confirmed the panel's ability to stratify EBV-positive and EBV-negative lymphomas with high diagnostic certainty. Although EBER in situ hybridization confirmation was needed in approximately 12% of cases, synergizing with traditional techniques may facilitate more rapid and cost-effective diagnoses. The LExA120 panel offers a multiplexed approach to lymphoma classification, enhancing the efficiency and accuracy for subtyping lymphomas.

In high-risk neuroblastoma, identification of ALK activating genetic alterations is considered for clinical decision-making in a relapse setting or more recently in frontline treatment. The accurate diagnosis of genetic alterations requires harmonization of molecular techniques and reporting, especially when these are considered as inclusion criteria for clinical trials. Analysis and validation were performed across the 21 SIOPEN (International Society of Paediatric Oncology Europe Neuroblastoma) molecular diagnostic laboratories, with 14 DNA samples harboring distinct ALK alterations. These included ALK mutations at or outside hotspots in the tyrosine kinase domain with variant allele frequencies (VAFs) ranging from 1% to 91% or ALK genomic amplification shared between the laboratories. Each laboratory used their own established techniques: ALK amplifications were detected by using either pan-genomic copy number techniques or fluorescence in situ hybridization, and ALK mutations were characterized by next-generation sequencing techniques. All laboratories correctly identified high-level ALK amplification and ALK mutations within the tyrosine kinase domain hotspots with VAF >5%, with the exception of two cases. Differences in interpretation and reporting were apparent for samples harboring mutations with a VAF <5% or outside known hotspots. These results highlight the importance of standard operating procedures, standardized reporting, and the robustness of ALK genetic testing in the SIOPEN laboratories, as well as the need for expert discussions regarding atypical ALK alterations, to validate eligibility for ALK targeted treatment in clinical trials.

Preanalytical tissue assessment is an important step in cancer molecular testing; however, its impact on molecular test results has not been systematically evaluated. This study describes a quality-improvement project in which routine histology review was implemented at a US molecular diagnostics laboratory. The effects of implementation on laboratory compliance and the analytical performance of a targeted POLE assay were measured as changes in tumor cellularity documentation, tumor sample enrichment (in samples with <40% tumor cellularity), POLE mutation rate, tumor signal intensity, and repeat-testing rate. Endometrial carcinoma samples (N = 1752) and tested for POLE mutations using a multiplex PCR assay. POLE mutation rates were 6.3% and 5.0% before and after intervention, respectively (P = 0.25), with the mutations most commonly detected being p.Pro286Arg (47%) and p.Val411Leu (21%). Documentation of tumor cellularity increased from 29% to 100%, and the rate of tumor enrichment increased from 1.4% to 31.5% (both, P < 0.0001). Mutation signal intensity increased from 0.32 to 0.58, and the repeat-testing rate decreased from 8.8% to 2.3% (P = 0.004 and <0.0001, respectively). Systematic preanalytical histology review was associated with improved analytical performance of a targeted POLE assay, accompanied by compliance in tumor cellularity documentation, increased tumor enrichment, and decreased repeated testing, supporting preanalytical assessment in improving somatic mutation detection in pathology specimens with low tumor content.

Liquid biopsies quantifying mutations in circulating tumor DNA by targeted next-generation sequencing have been gaining popularity. They are performed by various library preparation methods, each with distinct advantages and limitations. This work introduces Bridge Capture, a novel technology that goes beyond the advantages of market-leading liquid biopsy technologies, eliminating the need to compromise between scalability, cost-efficiency, sensitivity, or panel size. Twenty-four matched contrived colorectal biospecimens mimicking circulating tumor DNA were analyzed by Bridge Capture, Archer LIQUIDPlex, and AmpliSeq CHP version 2 for Illumina to compare variant allele frequency (VAF) detection. Bridge Capture was evaluated for sequencing depth requirement, interlaboratory reproducibility, automatization, and panel scalability. Of all methods, Bridge Capture detected the lowest VAF, and all VAFs strongly correlated with Archer LIQUIDPlex (R = 0.995) and AmpliSeq CHPv2 for Illumina (R = 0.988). Owing to its unique design, the Bridge Capture is compatible with the commonly used next-generation sequencing platforms and effectively uses sequencing capacity, permitting affordable and sensitive variant detection. The method demonstrated high reproducibility across independent laboratories and between automated and manual workflow. The panel size was increased by 300% and had negligible impact on performance and cross-reactivity of the probes, implying high multiplexing capabilities. Taken together, Bridge Capture is a cost-efficient, simple, rapid, and sensitive cancer diagnostics tool that has a potential to significantly improve the detection of mutations.

Despite the availability of various in silico prediction tools, accurately assessing the pathogenicity of splice-region variants remains limited. In this study, 18 splice-region variants of uncertain significance (VUSs) were functionally characterized using either in vitro (minigene) or in vivo (RT-PCR) experiments. The impacts of in-frame mutants on the indicated protein were further analyzed by modeling the three-dimensional protein structures. The accuracy of different in silico prediction tools (SpliceAI, varSEAK, and Splicing Prediction Pipeline) was also compared. The 18 VUSs included 3 canonical and 15 noncanonical splicing variants. Splicing abnormalities were observed in 16 variants (88.9%), with exon skipping (33.3%, 6/18) being the most prevalent event. Among them, 11 variants were predicted to generate premature termination codons, 3 caused in-frame alterations, and 2 resulted in both outcomes. Structural modeling revealed significant disruption of protein secondary structure in four of five in-frame alterations. Integrating functional and structural evidence, 15 VUSs (83.3%) were reclassified as pathogenic/likely pathogenic, enabling definitive diagnoses and informed reproductive decisions for affected families. Among the in silico tools, SpliceAI demonstrated the highest accuracy in predicting splicing anomalies; however, all tools showed decreased accuracy in analyzing specific splicing patterns. This study establishes an integrated workflow for assessing the pathogenicity of splicing VUSs in clinical diagnostics, offering a practical approach to overcome this critical diagnostic challenge.

This retrospective study was designed to demonstrate the clinical utility of two diagnostic tests, the FoundationOneCDx (F1CDx) and Exact Sciences Resolution homologous recombination deficiency (HRD; Resolution HRD assay) as clinical trial-enrollment assays to identify patients with metastatic castration-resistant prostate cancer harboring homologous recombination repair (HRR) gene alterations. Tumor tissue and plasma collected from patients with metastatic castration-resistant prostate cancer in the phase 3 MAGNITUDE study were tested using the F1CDx tissue assay and/or the Resolution HRD plasma assay. Patients with HRR alterations were randomized (1:1) to receive niraparib (NIRA) and abiraterone acetate + prednisone (NIRA + AAP) or placebo and AAP (NCT03748641). Efficacy was based on radiographic progression-free survival (primary end point), time to symptomatic progression, time to cytotoxic chemotherapy, and overall survival as secondary end points (interim analysis 1). Of 423 HRR patients, 291 (68.8%) were HRR positive (HRR) by F1CDx [BRCA: 162 (38.2%)], and 38 (8.9%) were HRR negative by F1CDx but HRR by Resolution HRD assay. Also, 277 of 423 (65.5%) were HRR by Resolution HRD assay [BRCA: 150 (35.5%)], and 124 (29.3%) were HRR negative by Resolution HRD assay but HRR by F1CDx assay. Clinically meaningful benefits for all end points were comparable for BRCA and HRR patients detected by either tissue or plasma assays. These results demonstrated the clinical utility of both tissue and plasma assays in identifying patients for NIRA + AAP treatment.

Next-generation sequencing (NGS) has become integral to the clinical workup of blood cancers. However, there remain barriers to implementing clinical NGS, including the separate workflows required for DNA and RNA sequencing, complex bioinformatics analyses, and long turnaround times. Duoseq has been developed as a comprehensive, kitted solution to these barriers and implemented as a genomic profiling tool for blood cancers to simultaneously detect single-nucleotide variants (SNVs), small insertions/deletions (indels), and structural variants (SVs; translocations and fusions). Additionally, Duoseq can evaluate numerous other blood cancer diagnostic markers, including gene expression, copy number alterations, cell of origin, oncogenic viral status (eg, Epstein-Barr virus), B-/T-cell receptor clonality, Ig heavy chain mutational status, and diffuse large B-cell lymphoma subtypes. Analytical and clinical validation of Duoseq was performed in parallel at two clinical institutions. Limit of detection was confirmed as 5% variant allele frequency for SNVs, 10% variant allele frequency for indels, and ≥20% tumor purity for SVs. Repeatability studies showed >99% intrarun and interrun positive predictive value and >96% percentage positive agreement. Duoseq was run on formalin-fixed, paraffin-embedded biopsy specimens (N = 197), using NGS and/or fluorescence in situ hybridization as orthogonal comparison. SNVs, indels, and SVs achieved accuracy of >95%. These results establish Duoseq as a genomic profiling tool for blood cancers that can enable laboratories to provide critical diagnostic information in a time- and cost-effective manner.

Fragile X syndrome (FXS) is the most common cause of intellectual disability. It is usually caused by the expansion of the CGG trinucleotide repeat (>200 repeats) in FMR1, resulting in DNA hypermethylation and gene silencing. Conventional FXS diagnosis is based on a combination of PCR and Southern blot (SB) analysis, which is technically challenging and labor-intensive. Methylation-specific triplet repeat-primed PCR (msTP-PCR) has been proposed to overcome these limitations in detecting FMR1 expansion and methylation status. However, its performance in FXS diagnosis in clinical laboratory settings has not been extensively studied. We validated and implemented an msTP-PCR assay and compared it versus the conventional diagnostic protocol in clinical samples. A total of 420 clinical samples (339 male subjects and 81 female subjects) previously genotyped for FMR1 CGG repeat expansion using reference methods (PCR-based methods and/or SB analysis) were investigated using msTP-PCR. The results showed high concordance with the reference results for allele categorization, repeat number correlation, and methylation status. However, discordant results were observed in rare cases of female patients with complex mosaic normal, premutation, and full mutation alleles, which need further confirmation via SB analysis. Nonetheless, these results confirm that the msTP-PCR method is a useful alternative technique for FXS diagnosis and prenatal testing, as it is rapid, reliable, cost-effective, and can potentially reduce the requirement for SB analysis.

Factor XIII (FXIII) is a heterotetramer that plays a crucial role in the coagulation cascade, facilitating fibrin crosslinking to stabilize clots. Congenital or acquired deficiency of the FXIII A or B subunits can lead to prolonged bleeding. Existing enzyme-linked immunosorbent assay-based and latex agglutination assays for measuring FXIII are limited to detecting only the FXIIIA subunit of the complex. In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to accurately quantify both FXIIIA and FXIIIB subunits in plasma. Peptides measured by this targeted assay were rigorously selected based on analytical robustness, with the method demonstrating excellent sensitivity (limit of detection, 1.56 to 3.67 mU/mL), linearity (r > 0.998), and precision (CV, <10%). Comparison with the conventional assay based on ammonia release demonstrated a strong correlation (r = 0.983) and agreement (Cohen κ = 0.846) for FXIIIA. On analysis of 98 clinical plasma samples, the assay accurately identified cases with FXIII deficiency based on significantly reduced FXIIIA with varying alterations in FXIIIB levels. These results establish the clinical utility of the LC-MS/MS assay, highlight its potential for improving diagnostic accuracy for FXIII deficiency, and lay the foundation for future research on FXIII dynamics in pathologic states.