Journal of Clinical Pathology

BackgroundUnited Kingdom Standards for Microbiology Investigations limits the pre-analytical delay of blood cultures to a maximum of four-hours between collection and incubation. Compliance with this delay standard is a measure of the ability of a microbiology service to support the management of sepsis which is a life-threatening complication of infection. A positive blood culture confirms the infection and an early result is critical to the effective management of the condition. Delayed results lead to the prolongation of empiric broad spectrum antimicrobial therapy which is considered a causal factor in the emergence of antimicrobial resistance. This retrospective observational study documents compliance with the standard by microbiology services in England in 2022/23. The impact of laboratory centralisation on the ability of microbiology services to comply with this standard is examined. MethodsFreedom of Information requests were submitted to 116 National Health Service Trusts/administrative units in England requesting retrospective audit data showing compliance with the recommended pre-analytical delay standard. Data relating to service configuration and cost were also requested. ResultsResponses were received from 89 Trusts (76.7%) managing 146 hospitals. Overall, the rate of compliance was low, with only four hospitals (2.7%) showing full compliance and 31.5% showing >80% compliance. ConclusionsPoor rates of compliance with the PAD standard are a concern as prompt attention to blood cultures improves patient outcomes from sepsis and supports antimicrobial stewardship. Laboratory centralisation has resulted in withdrawal of staff and facilities from some hospitals with insufficient investment in others, leading to a demonstrable inability of many hospitals to comply with this standard. Compliance will require investment in microbiology services. The financial implications of the improvements proposed should be evaluated in the context of overall health care and community benefits.

Background: External Quality Assurance (EQA) is an essential component of modern laboratory medicine. Current scientific evidence on EQA focuses primarily on the analyses carried out by EQA providers while relatively little research has been conducted in individual clinical laboratories. Methods: In this retrospective single-center observational study in a clinical laboratory, EQA results were analyzed over a period of four years (2021-2024). The evaluation was based on EQA action reports documented in the institutes internal quality management system. Deviations were classified according to department, type of discrepancy, root cause category (analytical, preanalytical, systemic, unidentifiable), and measures taken. Results: A total of 7226 EQA participations were evaluated during the observation period. The overall error rate remained consistently low, ranging between 0.8% and 1.6%, with no significant change over time (p = 0.87). Most deviations occurred in the departments of clinical chemistry and immuno/autoimmune diagnostics (p < 0.001). These were predominantly quantitative discrepancies (false low/false negative or false high/false positive). Root cause analysis showed a clear dominance of analytical causes (p < 0.001), while preanalytical and systemic causes were identified less frequently. In most cases, corrective measures, such as re-analyses, recalibrations, process adjustments, or staff training, were implemented promptly. Hard structural measures, such as changing methods or discontinuing tests, were rarely necessary. Conclusion: In a clinical laboratory, EQA is an important tool for structured error analysis and continuous quality improvement. Consistent processing of deviating EQA results goes hand in hand with stable analytical performance and a low error rate.

BackgroundOptimal pre-analytical management of breast tissue specimens, particularly formalin fixation, is essential for accurate immunohistochemical (IHC) biomarker assessment in invasive breast cancer. Although international guidelines suggest using 4% neutral buffered formalin with controlled fixation time, many laboratories in low-resource settings deviate from these standards. This study aimed to determine whether fixative preparation (4% neutral buffered formaldehyde vs. 4% non-buffered formaldehyde) and cold ischemia time impact the preservation and evaluation of tissue biomarkers in invasive breast cancer. MethodsWe conducted an experimental study using fresh mastectomy tissue from a 34-year-old patient with invasive ductal carcinoma (pT4, hormone receptor-positive, HER2-negative, Ki67=40%) who had not received neoadjuvant chemotherapy. Fifty microsamples (5-15 mm in length, 1 mm in width) were obtained and divided into four cohorts: (1) 19 samples fixed in 4% neutral buffered formaldehyde for 0.5 to 144 hours; (2) 19 samples fixed in 4% non-buffered formaldehyde for 0.5 to 144 hours; (3) 6 samples with delayed fixation (0.5 to 8 hours) then fixed in neutral buffered formaldehyde for 10 hours; (4) 6 samples with delayed fixation (0.5 to 8 hours) then fixed in non-buffered formaldehyde for 10 hours. Hormone receptors (estrogen receptor-ER, progesterone receptor-PR) and Ki67 expression were evaluated by IHC using the Allred scoring system and current international recommendations. ResultsFixative preparation had a statistically significant, yet minimal, biological impact on biomarker evaluation. The mean percentage of ER-positive cells was 96.89{+/-}0.74% with neutral buffered formaldehyde compared to 94.32{+/-}1.51% with non-buffered formaldehyde (p=0.011). Similar trends were seen for PR (94.89{+/-}0.95% vs. 92.63{+/-}1.67%, p=0.027) and staining intensity. However, Allred scores remained constant. Cold ischemia time was strongly correlated with decreased biomarker expression regardless of fixative preparation. Hormone receptor expression and Ki67 remained stable with minimal Allred score changes for up to 2 hours of cold ischemia, but significantly decreased after 2 hours, with scores decreasing in proportion to the duration of ischemia (p<0.05). ConclusionsNon-buffered formaldehyde preserves tissue biomarkers almost as effectively as neutral buffered formaldehyde for IHC analysis. Following guidelines, a cold ischemia time of up to 1 hour is still a wise standard to guarantee accurate biomarker assessment. These results are significant for pathology laboratories in resource-limited settings where neutral buffered formalin may not be easily accessible.

BackgroundBehavioral telemetry--the analysis of clinical actions NOT taken--may identify care process failures associated with adverse outcomes. While missed nursing care predicts outcomes in survey-based studies, objective EHR-derived measures are lacking. We hypothesized that missing routine cognitive assessment in ICU patients with low acute physiologic derangement would predict mortality independent of illness severity. MethodsRetrospective cohort study using MIMIC-IV (2008-2022, Beth Israel Deaconess Medical Center) with external assessment of documentation practices in eICU (208 US hospitals). We identified ICU admissions with SOFA 0-2 (low acute physiologic derangement), excluding neurological ICUs. Orientation documentation was classified within 24 hours. Primary outcome was in-hospital mortality. Multivariable logistic regression adjusted for age, sex, SOFA, and Charlson Index. ResultsAmong 46,004 ICU patients with SOFA 0-2, 4,737 (10.3%) had no orientation documentation within 24 hours. These patients had 24.68% mortality versus 7.57% early-assessed and 4.56% late-assessed. After adjustment, missing orientation was associated with 4.29-fold higher odds of death (95% CI 3.95-4.65; E-value 8.0). In SOFA=0 patients (N=23,670), the signal strengthened (OR 5.65, 95% CI 5.03-6.35; E-value 10.8). Late-assessed patients had the LOWEST mortality (OR 0.65), arguing against reverse causation. Patients without orientation had 22% MORE chart events (1,600 vs 1,309), arguing against neglect. External assessment revealed that among 166 eICU hospitals with [&ge;]100 eligible patients, only 5% documented orientation routinely--92% lack the infrastructure to detect this signal. ConclusionsIn ICU patients with low acute physiologic derangement, absence of orientation assessment is associated with 4-6 fold increased mortality. This association may identify care process failures not captured by severity scores, though prospective studies are needed to establish causality. What is Already Known on This TopicMissed nursing care--care omissions--predicts patient mortality in survey-based studies. Nurse staffing ratios are associated with mortality, but the mechanism is poorly understood. No objective, EHR-derived measure exists to detect care process omissions in real time. What This Study AddsFirst EHR-based operationalization of the missed nursing care construct, enabling objective, real-time detection. Missing orientation assessment associated with 4-6 fold increased mortality (OR 4.29 in SOFA 0-2; OR 5.65 in SOFA=0). Signal strengthens in SOFA=0 patients (E-value 10.8), suggesting finding is not driven by acute illness severity. Argues against reverse causation: late assessment has BETTER outcomes than early or no assessment. Argues against neglect: patients without assessment had MORE documentation, not less. Argues against immortal time bias: Never Documented patients had LONGER ICU stays (7.58 vs 3.09 days). Quantifies association: 10.3% of patients account for 27.2% of deaths. Reveals systemic gap: 92% of US ICUs lack the documentation infrastructure to detect this signal. Key PointsO_ST_ABSQuestionC_ST_ABSDoes absence of routine orientation assessment predict mortality in ICU patients with low acute physiologic derangement (SOFA 0-2), independent of illness severity? FindingsIn this cohort study of 46,004 ICU patients with SOFA 0-2, those without orientation documentation within 24 hours had 4.29-fold higher adjusted odds of death (95% CI 3.95-4.65). In SOFA=0 patients, the signal strengthened to OR 5.65 (E-value 10.8). Patients assessed late (6-24h) had the LOWEST mortality (OR 0.65), arguing against reverse causation. Among 166 eICU hospitals, only 5% document orientation routinely-- 92% lack the infrastructure to detect this signal. MeaningMissing routine cognitive assessment may identify care process failures associated with increased mortality. The finding that 92% of US ICUs lack the documentation infrastructure to detect this signal reveals a systemic gap in care process monitoring.

Background: Classification of central nervous system (CNS) tumors has become increasingly complex, raising concerns about the sustainability of comprehensive molecular diagnostics. We have evaluated nanopore whole genome sequencing (nWGS) as a single workflow to replace multiple diagnostic assays. Methods: We performed nWGS on DNA extracted from 90 adult CNS tumor samples (58 retrospective, 32 prospective) and compared the results to findings from standard of care (SoC) diagnostic work-up. Analysis was done through an automated workflow that consolidated diagnostically and therapeutically relevant genomic alterations, including copy-number variation, structural, and single-nucleotide variants, chromosomal aberrations, gene fusions, and methylation-based classification. Results: nWGS supported final diagnostic classification in all samples with >15% tumor cell content, requiring ~3 hours of hands-on library preparation, parallel sample processing, and sequencing times within 72 hours. Methylation-based classification was available within 1 hour and was concordant with the integrated final diagnosis in 89% of cases (80/90). All diagnostically relevant copy-number variations, single-nucleotide variants, and gene fusions were concordant with SoC testing. MGMT promoter methylation status matched in 94% of cases. In addition, nWGS identified prognostic and potentially actionable variants that were not reported or covered by SoC. Conclusions: nWGS delivers comprehensive genetic and epigenetic results with a fast turn-around compared to standard methods. This enables efficient, accurate, and scalable molecular diagnostics of CNS tumors using a single platform. This data supports its implementation in routine clinical practice and may be extended to other cancer types requiring complex genomic profiling.

BackgroundPlacental growth and function are imperative for healthy fetal growth; data on placentas can inform research and clinical care. Measuring placental size after delivery should be easy, but current methods are hard to standardize and error prone. We developed PlacentaVision using artificial intelligence (AI)-based models, to automatically, accurately, and precisely measure placentas from digital photographs. ObjectiveWe aimed to compare placental disc morphology between gross pathology examination (human measurements) and our automated PlacentaVision model (AI measurements). MethodsPlacentaVision is a multi-site study to assess placental morphology, features, and pathologies from digital photographs. We built a large dataset of digital placenta photographs and clinical data from singleton births at three large hospitals: Northwestern Memorial (Chicago; n=24,933), UPMC Magee-Womens (Pittsburgh; n=1198) and Mbarara Regional Referral (Uganda, n=1715). Data and images were from the medical record for Northwestern, part of a biobank study for Magee, and from our prospective studies for Mbarara. We compared long and short disc axis length (defined by Amsterdam criteria) between human and AI-based PlacentaVision measurements by calculating the difference and using Bland-Altman; we stratified by site, disc shape, infant sex, and term/preterm birth. ResultsMean (SD) disc length was 19.2 (3.1) and 18.6 (3.1) cm from PlacentaVision and human measurement, respectively, with a difference of 0.57 (2.19) cm. Disc width was 16.3 (2.3) cm and 16.1 (2.4) cm from PlacentaVision and human measurement, respectively, with a difference of 0.25 (1.85) cm. Bland-Altman limits of agreement were -3.7 to 4.9 cm for length and -3.4 to 3.9 cm for width. Irregularly-shaped placentas had a greater difference between PlacentaVision and human measurements compared to those with round/oval shapes (length differences of 1.53 and 0.45 cm respectively). Further, there were length differences by site (Northwestern 0.6, Magee 0.0, and Mbarara 0.4) and gestational age at birth (preterm 0.71, term 0.53 cm), but similar results for male and female placentas. Results for width were similar to length. ConclusionsAI-based measurements were less than a cm from human measurements overall. Our findings of larger differences for irregular shapes and preterm may indicate it is difficult for humans to measure irregular or small placentas according to protocol. PlacentaVision can automate and standardize the process.

Digital pathology, coupled with advanced image recognition algorithms, represents a transformative frontier in histopathological diagnosis. This sub-Saharan African laboratorys exploratory study investigates the application of a Convolutional Neural Network (CNN) model, specifically leveraging the VGG16 architecture with transfer learning, for automated analysis and classification of selected gastrointestinal (GIT) and liver tissue samples, incorporating both routine and specialized staining protocols. The study utilized a dataset comprising 114 samples (18 liver, 96 GIT images) derived from archival formalin-fixed paraffin-embedded tissue blocks at University College Hospital, Ibadan, Nigeria. Specialized staining techniques included Alcian Yellow for GIT mucin visualization and Massons Trichrome for liver fibrosis assessment, alongside conventional H&E staining. Model performance was evaluated using statistical methodologies including Wilson Score confidence intervals (CI), Bayesian probability assessment, and effect size analysis. Results reveal a striking dichotomy in model performance. The GIT tissue model achieved perfect classification accuracy (100% test accuracy) with exceptional statistical significance (Z=10.0, p<0.0001), Wilson CI [96.29%, 99.99%], Cohens h=1.571, and Bayesian probability >99.99%. Conversely, the liver tissue model demonstrated diagnostic failure (42.86% test accuracy), with Z=-1.428, p=0.9236, Wilson CI [33.59%, 52.65%], Cohens h=-0.144, and Bayesian probability of 7.64%. This performance divergence correlates with training data availability, as the liver dataset fell far below empirically established thresholds (>100-200 samples) for reliable classification. The liver models failure reveals limitations in transfer learning with insufficient data. These findings underscore critical implications for AI-enhanced digital pathology, demonstrating potential deployment of the GIT model as a promising one that supports tissue-specific model development.

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-related death in sub-Saharan Africa (SSA). Differentiating primary HCC from metastatic liver tumors remains a significant diagnostic challenge. Understanding the prevalence and clinical predictors of HCC is crucial for improving diagnosis and patient care. This study examined the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), and HCC, and clinical predictors of HCC. MethodsWe used immunohistochemical markers on archived liver tumor biopsies and analyzed the data using descriptive and logistic regression analysis. ResultsAmong 58 liver carcinoma cases, 37.9% had HCC, and 62% had metastatic liver carcinoma (MLC). HCC was most common (61.5%) among middle-aged adults (50-59 years). HCC was more frequent in males (47.2%) than in females (22.7%). Over half of the patients (51.7%) tested positive for HBV. HCC was more prevalent in HBV-positive patients than HBV-negative ones (43.3% vs 32.1%). Hepatic fibrosis was identified in 27.6% of cases. HCC was more common in patients with fibrosis (56.2%) than in those without (31%). HCV infection was rare (6.9%) in this study. In multivariable logistic regression analysis, none of the examined predictors reached statistical significance (P>0.05). Patients aged 50-59 years, males, those with HBV infection, and hepatic fibrosis showed higher odds of HCC. Hepatocyte Paraffin-1 (Hep Par-1) demonstrated 97% specificity and a 95% positive predictive value (PPV) for differentiating HCC from MLC. The combined marker pattern of Hep Par-1 positive and AE1/AE3 negative was highly predictive of HCC (100% specificity, 100% PPV, and 93.2% diagnostic accuracy). ConclusionsOur findings indicate that while the assessed risk factors tend to show directional association with HCC, as expected, larger studies are needed to determine their independent effects. The combined Hep Par-1 AE1/AE3 immunophenotype is more accurate than either marker alone. Therefore, this combined test is a valuable diagnostic tool for confirming HCC in resource-limited settings.

BackgroundWilms tumour (WT) relapse occurs more frequently in patients with blastemal-type WTs. The presence of cancer stem cells (CSCs) is linked to tumour survival and relapse, and CSCs may be found in greater numbers in blastemal cell foci. CSC-associated phenotypes have been described in untreated WT, but their persistence, organisation and relevance after neoadjuvant chemotherapy is unknown. MethodsWe analysed 23 formalin-fixed paraffin-embedded blocks from 18 chemotherapy-treated patients where WTs were enriched for viable blastema, using human fetal kidney as developmental control. Immunohistochemistry and -fluorescence analysis determined progenitor (PAX2, SIX2, CITED1) and CSC-associated (NCAM, ALDH1, CD133) marker expression. We qualitatively and semi-quantitatively evaluated spatial expression patterns and co-localisation across tumour compartments. ResultsPAX2 and SIX2 were co-expressed in blastema in most cases (15/18), with PAX2 expression higher at the periphery of blastemal foci and SIX2 expression found uniformly in central aspects. CITED1 expression was also associated with SIX2 in blastema tissues (14/18). NCAM was blastema-enriched (15/18) with higher central intensity, frequently adjacent to PAX2-expressing peripheral zones. ALDH1 expression was present across blastema and epithelium while NCAM-, ALDH1-double-positive cells were rarely observed (4/18). CD133 expression was less commonly seen (2/18), localising near epithelial/nephrogenic structures. ConclusionsAfter neoadjuvant chemotherapy, WT blastema retained overlapping but non-identical progenitor/CSC-associated marker landscapes with reproducible peripheral-centre gradients. These spatial arrangements suggest a blastemal niche for CSCs that may sustain a therapy-resistant state. Our analysis provides the foundation for future functional validation and molecular profiling to define key lineage relationships and therapeutic vulnerabilities in post-chemotherapy WT. [250/250 words]

BackgroundIn 2004, South Africas public health system faced the dual challenge of rapidly scaling up antiretroviral therapy (ART) while reducing the cost of laboratory monitoring. At the time, conventional CD4 testing methods were expensive, labour-intensive, and impractical for sustaining a national testing network. This study aimed to assess the financial impact and cost savings associated with the implementation of the PanLeucogated CD4 (PLG/CD4) enumeration method between 2004 and 2024 in the public-sector in South Africa. MethodsA longitudinal cost analysis was conducted using annual test volumes and state tariffs for PLG/CD4 testing and the 4-colour CD3/CD4/CD8/CD45 T-cell enumeration reference method. Annual cost savings were calculated in United States Dollars (USD) by applying historical South African Rands (ZAR) to United States Dollars (USD) exchange rates. The state prices for tariff codes PLG/CD4 and the reference method were provided by calendar year in ZAR and converted to USD based on the prevailing exchange rate. The USD test prices were multiplied by annual test volumes. Cost savings were calculated by multiplying annual test volumes and the difference in test prices in USD (difference between PLG/CD4 and the reference method). ResultsThere were 50,745,848 PLG/CD4 tests performed over 20-years. The cost-per-test of PLG/CD4 was consistently lower than the reference method, ranging from $4,06 to $9,40, compared to $13,06 to $28,21. Cumulative national savings amounted to USD 626 million. The peak annual savings of $64,6 million occurred in 2011, coinciding with the height of ART enrolment. Cost savings persisted despite a doubling in the exchange rate over the study period. ConclusionThe PLG/CD4 implementation enabled cost-efficient, scalable, quality-assured CD4 testing as part of the national HIV response, reducing reliance on complex/costly technologies while improving coverage. These findings support the critical role of context-specific diagnostic innovation to strengthen health system resilience.

T-cell lymphomas are often histologically indistinguishable from benign T-cell infiltrates. Clonality testing is frequently required for diagnosis. It lacks the spatial context and is slow and expensive, relying on complex, multiplexed PCR reactions, interpreted by experienced scientists or pathologists. We previously published details of a pair of highly specific monoclonal antibodies against the two alternatively used, but very similar, T-cell receptor {beta} constant regions, TCR{beta}1 and TCR{beta}2. We demonstrated the feasibility of immunohistochemical detection of TCR{beta}1 and TCR{beta}2 in formalin-fixed, paraffin-embedded (FFPE) tissue as a novel diagnostic strategy for T-cell lymphomas. Here we validate an improved pairing of TCR{beta}1/2 rabbit monoclonal antibodies, and demonstrate their utility for single and double immunostaining, including with a chimeric mouse anti-TCR{beta}2 antibody. Finally, we show that this staining is amenable to automated cell counting, permitting accurate calculation of the TCR{beta}2:TCR{beta}1 ratio.

Sepsis is defined as a dysregulated response to infection, leading to life-threatening organ dysfunction that particularly affects parenchymal organs. Clinical studies remain inconclusive regarding the impact of biological sex on sepsis, and preclinical studies are predominantly performed in male animals. We examined early (8 h) septic responses in male and female mice using a fecal-induced peritonitis (FIP) model. Blood biochemical parameters, body temperature, and murine sepsis scores provided evidence of a septic response in animals randomized to FIP compared to controls, but showed no physiological differences between male and female mice. Transcriptomic analysis of the liver, kidney, and lung showed consistent inflammatory activation in response to sepsis as compared to controls. Notably, in the kidney and lung, female mice exhibited stronger immune activation and a heightened inflammatory response compared to males. Thus, biological sex differences in the septic response can be detected in early acute sepsis without apparent physiological differences.

Myeloproliferative neoplasms (MPNs) are characterized by progressive myelofibrosis that drives morbidity and mortality. Liquid biopsy approaches to noninvasively monitor fibrotic progression remain limited. We performed comparative transcriptomic profiling of CD45-depleted platelet-enriched and CD45+ leukocyte-enriched fractions from matched peripheral blood samples of 76 individuals (27 primary myelofibrosis, 17 polycythemia vera, 14 essential thrombocythemia, 18 healthy controls). Platelet RNA sequencing was performed in 2018-2020 on Illumina HiSeq 4000, while WBC RNA sequencing was conducted in 2023 on Illumina NovaSeq 6000 from cryopreserved CD45+ enriched fractions of specimens obtained at the identical time and from the same blood sample as the platelet RNA. Despite comparable library preparation protocols and higher sequencing depth in WBC samples, platelet transcriptomes exhibited 5.1-fold more differential expression in myelofibrosis (3,453 versus 681 genes, adjusted p<0.05, |log2FC|>1). Platelet signatures were enriched for proteostasis pathways including endoplasmic reticulum stress and unfolded protein response, reflecting megakaryocyte dysfunction in the fibrotic bone marrow niche. WBC signatures predominantly featured immune activation and proliferative pathways, indicating systemic inflammatory responses. Multinomial LASSO classification demonstrated superior performance of platelet-based models for myelofibrosis diagnosis (AUROC 0.85) compared to WBC-based (AUROC 0.77) or clinical models (AUROC 0.59). Combined platelet+WBC models did not improve performance (AUROC 0.80), indicating complementary but non-additive information. These findings establish platelet transcriptomic profiling as a superior noninvasive biomarker platform for monitoring myelofibrosis in MPNs, capturing megakaryocyte-driven fibrogenesis with greater sensitivity than peripheral leukocyte-based approaches. HighlightsUsing matched WBC and platelet RNA-seq from MPN patients, we identify myelofibrosis-associated transcriptomic signatures specifically enriched in platelets. Multinomial LASSO modeling highlights platelet-derived gene expression as a dominant and predictive biomarker of myelofibrosis, outperforming clinical parameters and WBC signatures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/714941v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d695aborg.highwire.dtl.DTLVardef@fc250forg.highwire.dtl.DTLVardef@1e52e8eorg.highwire.dtl.DTLVardef@15378e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundHistologic descriptors such as lymphovascular invasion (LVI), visceral pleural invasion (VPI), spread through air spaces (STAS), and grading system have each been associated with adverse outcomes in lung adenocarcinoma (LUAD). However, with the exception of VPI, these features are not formally incorporated into the TNM staging system. We evaluated the prognostic value and incremental contribution of these histologic descriptors within the framework of the 9th edition TNM staging system. MethodsIn total, 1,745 individuals diagnosed with stage I-III invasive non-mucinous lung adenocarcinoma (NM-LUAD) were included in this study, comprising 1139 French-Canadian patients who underwent surgical resection at IUCPQ-Universite Laval (discovery cohort) and 606 patients from the National Cancer Center Hospital in Tokyo, Japan (validation cohort). The objective of this study was to assess the prognostic contribution of histologic descriptors, including STAS, and LVI, as complements to conventional 9th edition TNM staging. ResultsGrade 3 tumors, LVI, and STAS were identified in 880 (50.4%), 809 (46.4%), and 775 (44.4%) of 1745 cases, respectively. Histologic grade and LVI demonstrated the strongest associations, particularly in early-stage disease, while STAS exhibited a stage-dependent effect, being more impactful in stages II-III. VPI showed less consistent prognostic value. Incorporating these histologic descriptors into TNM staging improved prognostic model performance, with the largest gains driven by histologic grade and LVI, while STAS provided additional, complementary prognostic refinement. ConclusionThese findings demonstrate that key histologic descriptors--including grading system, LVI, and STAS--represent robust and reproducible prognostic parameters. Importantly, these descriptors provide complementary, stage-dependent information that may enhance risk stratification and inform refinement of future TNM staging frameworks, including the forthcoming 10th edition.

Background: Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome) is a fatal neurodegenerative lysosomal storage disorder caused by impaired degradation of heparan sulfate (HS). Despite rapid advances in gene and enzyme therapies, there remains a critical need for an analytically validated, quantitative biomarker that accurately reflects central nervous system (CNS) substrate burden. Such biomarker would be a valuable tool in assessing disease progression and monitoring therapeutic efficacy. Objective: This study describes the method development, fit for purpose validation, and preliminary clinical application of a quantitative liquid chromatography-mass spectrometry (LC-MS/MS) assay for the HS-derived disaccharide N-sulfoglucosamine-glucuronic acid (GlcNS-GlcUA) in human cerebrospinal fluid (CSF), a critical biomarker for diagnosis, disease monitoring, and regulatory evaluation of emerging MPS IIIA therapies. Methods: A structurally defined GlcNS-GlcUA reference standard and its [13C6]-labeled internal standard were used in a derivatization and detection workflow employing 1-phenyl-3-methyl-5-pyrazolone labeling, and LC-MS/MS. Results: The method exhibited acceptable linearity across 0.005-0.500 nmol/mL (r[&ge;]0.9976), with intra- and inter-assay imprecision [&le;]3.5%CV and accuracy within 95%-110% of nominal concentrations. No matrix or hemolysis interference or carryover was observed, and the analyte remained stable during freeze-thaw storage conditions. Application of the method to 12 CSF samples from patients with MPS IIIA demonstrated quantifiable GlcNS-GlcUA levels ranging from 0.0054 to 0.106 nmol/mL, confirming suitability for clinical and regulatory use. Comparison of the MPS IIIA sample results between the development laboratory and the contract research organization laboratory support robust inter-lab assay transfer. Conclusions: This validated LC-MS/MS method establishes a regulatory-grade quantitative assay for measurement of CSF HS in MPS IIIA. Its high analytical sensitivity and reproducibility enable reliable assessment of CNS substrate reduction and pharmacodynamic response, supporting biomarker-driven therapeutic development and accelerated approval pathways for neuronopathic mucopolysaccharidoses.

BackgroundUterine atony ([~]70%), lacerations ([~]20%) and placenta-related problems ([~]10%) are assumed main reasons for postpartum hemorrhage genesis. Coagulation components predictive for postpartum blood loss can be identified prepartum and before traditionally assumed main reasons are observed. ObjectivesTo better understand postpartum hemorrhage genesis, we prospectively researched prepartum clinical information, presence of assumed main reasons and peripartum coagulation changes in parturient women. Study designIn 676 women with vaginal deliveries, age, BMI, parity, gestation age, duration of second stage of labor and presence and type of assumed main reasons (uterine atony, lacerations and placenta-related problems) were recorded. Measured blood loss within 24h postpartum defined no, non-severe or severe PPH (<500ml, [&ge;]500ml to <1000ml, [&ge;]1000ml). Hemoglobin, platelet count, fibrinogen, factor II and factor XIII activity were measured at admission and 24-48h postpartum. ResultsOf 191 women developing postpartum hemorrhage, 53.9% did not show assumed main reasons (expected <5%, p<.001). Of 45 women with severe postpartum hemorrhage, 15.5% were without assumed main reasons (<5%, p<.001). Sole atony occurred less frequently than expected (8.2% in non-severe and 35.5% in severe PPH, p<.001). FXIII showed the largest decrease of coagulation factors by far, from no (-12%) to non-severe (-20%) and severe postpartum hemorrhage (-32%, p<.001). Duration of the second stage of labor was longer in women developing postpartum hemorrhage later on (71 vs. 46 minutes, p=.004), but was not different between women with or without assumed main reasons. ConclusionUterine atony frequency is low in non-severe postpartum hemorrhage, but progresses from non-severe to severe postpartum hemorrhage. It can thus not be the frequent reason for postpartum hemorrhage it is assumed to be, as all postpartum hemorrhages start as non-severe. A prolonged second stage of labor together with an ongoing (likely self-reinforcing) consumptive coagulopathy helps to explain postpartum hemorrhage genesis. FXIII is a prepartum predictor of postpartum blood loss and shows the most pronounced peripartum coagulation factor loss in any setting. This might allow to identify new treatment pathways.

Objective The decline of the perinatal demise rate is slowing and demises are often unexplained. Significant research has been done regarding diagnostic yield and genetic causes of demise, but little is known about how Geneticist involvement impacts outcomes. The goal of the study was to evaluate post-mortem genetic testing practices and effects of the geneticists involvement. Methods Retrospective data from 111 perinatal demise cases was examined, including rates of prenatal genetic counseling, post-delivery genetics consult, genetic testing, and autopsy investigation. Results In this cohort 54% received genetic testing and 25% received a genetics consult. When compared to those without, cases with genetic specialist involvement were associated with significant increases in testing uptake (p=0.007), diagnostic yield (p<0.001), and patient education (p<0.001). Second trimester stillbirths and those with fewer ultrasound (US) abnormalities were less likely to receive genetic testing (both p values <0.001) and consults (p<0.001, p=0.020). Conclusion Though it was not possible to avoid ascertainment bias, this data demonstrates that geneticist involvement correlates with a higher rate of testing, greater diagnostic yield, and more thorough counseling. These findings underscore the importance of integrating genetics providers into perinatal postmortem healthcare teams.

BackgroundPreeclampsia, which is a leading cause of maternal and perinatal mortality and morbidity, represents a biologically heterogeneous syndrome. First-trimester screening with the Fetal Medicine Foundation competing-risks model enables prevention of preterm preeclampsia through aspirin prophylaxis but depends on Doppler velocimetry and biochemical measurements that limit scalability and offer limited discrimination for term disease. A unified, molecular first trimester test capable of stratifying risk across the full clinical spectrum of preeclampsia has not been established. ObjectiveTo determine whether multi-modal, tissue-resolved analysis of first trimester circulating cell-free DNA (cfDNA), obtained during routine non-invasive prenatal testing (NIPT), enables early prediction of both preterm and term preeclampsia. Study DesignThis nested case-control study included 125 singleton pregnancies sampled at 11-14 weeks gestation after quality control (48 controls, 30 preterm preeclampsia, 47 term preeclampsia). For 80 pregnancies, matched placental villi and maternal buffy coat samples were available to derive tissue reference profiles. Plasma cfDNA underwent multi-modal sequencing using Oxford Nanopore Technologies, enabling tissue-resolved analysis of fragmentomic and epigenetic signatures. Separate ensemble machine-learning classifiers were developed for preterm (<37 weeks) and term ([&ge;]37 weeks) preeclampsia using stratified 10-fold cross-validation. Model discrimination was evaluated using area under the receiver operating characteristic curve (AUROC), sensitivity at predefined specificity thresholds, and comparison with the FMF first-trimester risk score. A population-level simulation of 100,000 pregnancies, applying incidence point estimates of 2.5% for preterm and 7.5% for term PE, was used to derive predictive values and likelihood ratios. ResultsThe multi-modal cfDNA classifier achieved an AUROC (95% CI) of 0.85 (0.77-0.91) for preterm preeclampsia and 0.84 (0.76-0.91) for term preeclampsia. The FMF score yielded an AUROC of 0.80 (0.70-0.89) for preterm and 0.53 (0.43-0.63) for term PE. At 80% specificity, cfDNA sensitivity was 70.5% for preterm and 72.1% for term preeclampsia, demonstrating improved discrimination for term disease compared with FMF screening. In simulated population-level analysis, positive likelihood ratios were 4.25 (preterm) and 3.83 (term), with negative likelihood ratios of 0.21 and 0.34, respectively, supporting meaningful post-test risk stratification and strong rule-out performance. ConclusionFirst-trimester multi-modal, tissue-resolved cfDNA analysis enables early risk stratification across the full clinical spectrum of preeclampsia from a single routine blood sample. Compared with FMF screening, this approach can potentially improve discrimination for term preeclampsia while providing incremental improvement for preterm disease. The potential for integration into existing NIPT workflows offers a scalable pathway toward unified precision prevention, supporting timely aspirin prophylaxis for preterm preeclampsia and risk-adapted surveillance strategies for term disease.

BackgroundElectronic prescribing registries are widely used for antimicrobial stewardship surveillance. Existing indicators predominantly measure structure or process, while validated outcome indicators remain rare. The present study evaluates how well rule-based measures capture clinically meaningful postdiagnostic antibiotic decision making in pediatric febrile urinary tract infection. MethodsWe conducted a retrospective, multicenter validation study including all empirically treated febrile UTI episodes across three Swedish pediatric emergency departments. Prescribing outcomes were classified using registry rules and compared with outcomes determined by clinician review and laboratory findings. Guidance Ratio (GR) and Discontinuation Ratio (DR) were calculated monthly and in aggregate for both clinically validated- and registry rule classifications. ResultsIn total, 909 febrile UTI episodes were included across all sites. The rule-based GR was 49%. GR increased consistently with stronger diagnostic evidence. Among the 431 episodes with clinician-adjudicated follow-up, 63% resulted in guided treatment; 28% discontinued treatment, and 9% lacked follow-up documentation. The rule-based algorithm showed a sensitivity of 0.78 and a specificity of 1.00 for identifying guided outcomes. Monthly rule-based GR tracked validated temporal patterns but underestimated absolute values. A calibration function substantially improved agreement. ConclusionsRule-based indicators captured overall prescribing patterns but underestimated the level of prescribing concordant with guidelines. Validation against clinician reviewed reference data enabled calibration and improved the interpretability of indicators based on registry data for antimicrobial stewardship.

Equine endometrosis is a major cause of subfertility in mares characterized by fibrotic remodeling of the endometrium. Although transforming growth factor beta 1 (TGF-{beta}1) is implicated in fibrogenesis, the relationship between endometrosis severity and transcripts associated with tissue maintenance and proliferation remains incompletely defined. Present study evaluated endometrial mRNA expression of IGF1, MKI67, TGFB1, and ACTA2 in relation to endometrosis severity and defined histopathological features. Forty-seven endometrial samples were graded according to the modified Kenney and Doig (KD) categories. Relative mRNA expression was quantified by RT-qPCR and histopathology was extended using a standardized feature-based assessment. TGFB1 mRNA expression was higher in category I+ than in categories I and III (p = 0.041) and in samples with glandular basal lamina disruption (p = 0.020). MKI67 mRNA expression was lower in samples with luminal epithelial erosion (p = 0.049). IGF1 mRNA expression correlated negatively with KD category ({rho} = -0.401, p = 0.015), glandular degeneration ({rho} = -0.340, p = 0.043), overall inflammatory infiltration ({rho} = -0.387, p = 0.020), lymphocytic infiltration ({rho} = -0.426, p = 0.010), and neutrophilic infiltration ({rho} = -0.448, p = 0.006). MKI67 correlated positively with ESR1 ({rho} = 0.887, p < 0.001). These findings indicate that early endometrosis-compatible lesions are associated with increased TGFB1 transcription and that epithelial damage is accompanied by reduced MKI67 expression. The inverse associations between IGF1 expression and both lesion severity and inflammatory infiltration support a link between progressive histopathological changes and reduced expression of a growth factor involved in tissue maintenance.