Routine Specimens Research Articles

The Clinical Impact of Urinalysis Screened by Automated Microscopy Compared to Reference Manual Analysis.

Clinical laboratories have replaced conventional manual urine microscopy with automated urinalysis; however, concerns persist regarding its validity in detecting specific elements of urinary sediment crucial for evaluating kidney diseases. This study aimed to assess the accuracy of urinary sediment analysis performed by a large hospital laboratory compared to a standardized microscopic review, focusing on patients both with and without kidney disease. Urine samples were randomly selected from routine laboratory specimens at a university hospital. Laboratory analysis was performed using LabUmat 2 and Urised 3 PRO equipment (Abbott Diagnostics). In the automated analysis, technicians had the option to reclassify urinary sediment elements and, if needed, conduct manual microscopic evaluations to validate findings. The laboratory's analysis was compared with a "reference" analysis, which was double-blinded and conducted by two experienced technicians using bright-field and phase-contrast microscopy. 503 samples were selected, with 52.3% originating from nephrology outpatient clinic patients. Overall agreement between the laboratory results and the reference analysis was 42.1%. The sensitivity (SN) of the laboratory examination for detecting pathological casts, lipiduria, and renal tubular cells was low (<50%), while specificity (SP) was high (>98%). However, for hyaline casts (SN: 50.4%; SP: 80.9%) and dysmorphic red blood cells (SN: 62.3%; SP: 96.2%), accuracy was intermediate. Performance was better for hematuria (SN: 86.1%; SP: 82.3%; ICC: 0.703; R: 0.828) and leukocyturia (SN: 84.9%; SP: 95.1%; ICC: 0.807; R: 0.861). In patients with kidney disease (N=248) and in samples manually reviewed by the laboratory (N=115), accuracy for each urinary element was comparable to the overall sample findings. However, when assessing the ability to identify elements suggestive of nephropathy, only samples manually reviewed by the laboratory showed statistically similar results to those obtained by the reference analysis (p= 0.503, McNemar's test). Employing automated urinalysis seems to be accurate for detecting hematuria and leukocyturia, as well as for screening patients without kidney diseases. However, clinical laboratories attending complex patients should employ personalized strategies to help decide when to perform manual review, thus avoiding misleading urinalysis results.

The utility of disc space and vertebral body specimens cell count differential for the diagnosis of native vertebral osteomyelitis: a prospective cohort study.

Diagnostic methods for native vertebral osteomyelitis (NVO) often yield inconclusive results. Image-guided spine biopsies for culture are specific but diagnose NVO in only 50% of cases. Pre-exposure to antimicrobials further reduces diagnostic yield. Our study assesses the value of neutrophil percentage in disc space fluid and vertebral body (DS/VB) samples for diagnosing NVO. Adults referred for spine biopsy at Mayo Clinic from August 2022 to September 2023 were consented and enrolled at the time of biopsy. Following routine specimen collection, the biopsy needle was rinsed in saline into an EDTA tube for cell analysis. NVO diagnosis required organism identification in spine tissue or blood and/or positive histopathology, and consistent symptoms and imaging. Sixty-eight patients were prospectively enrolled, comprising 14 with NVO and 54 with alternative diagnoses. The median biopsy sample polymorphonuclear (PMN) percentage for NVO patients was 80.5% (IQR 72.5-85.2), compared to 64.5% (IQR 54.0-69.0) for those without NVO (p < 0.001). Nine (64.3%) NVO patients received antibiotics within 10 days prior to spine biopsy. As a continuous measure, PMN differential showed a moderately strong ability in classifying NVO status with an area under ROC curve of 0.795; an optimal point on the curve of 71.5% corresponded to a sensitivity of 78.6%, specificity of 79.6%, negative predictive value of 93.5% and positive predictive value of 50.0%. PMN differential in DS/VB biopsies may serve as an effective diagnostic tool in the evaluation of patients with NVO particularly in ambiguous cases with an initially negative spine biopsy. Future efforts will aim to implement these findings within routine clinical practice.

Tonsillectomy Under the Microscope: Is Routine Histopathological Analysis Necessary?

Introduction Tonsillectomy is the most common surgery in otorhinolaryngology worldwide, with many indications in adults and children. Traditionally, all tonsillectomy specimens are routinely submitted for histopathological examination, even in the absence of clinical suspicion of malignancy. This practice has been debated due to its resource implications and the low yield of malignancy in routine cases. In this study, we aim to determine the prevalence of occult malignancy in our population and assess the necessity of histopathology examination after routine tonsillectomies. Methodology This retrospective chart review was conducted on patients who underwent tonsillectomy between January 2018 and December 2019 at a single tertiary care center. The patients were divided into two groups: those undergoing routine tonsillectomy without clinical suspicion of malignancy and those with suspected malignancy based on preoperative evaluation. Results A total of 1857 tonsillectomy cases were analyzed, comprising 1839 routine cases with no suspicion of malignancy and 18 cases where malignancy was suspected. Among the routine cases, 1609 (87.5%) were pediatric patients, and 230 (12.5%) were adult patients. All cases in the suspected malignancy group were adults. In the routine tonsillectomy group, recurrent tonsillitis was the most common indication for surgery, observed in 821 (51.1%) pediatric patients and 160 (69.6%) adult patients. Reactive lymphoid hyperplasia was the most common histopathological finding, present in 1589 (98.8%) pediatric cases and 187 (81.3%) adult cases. No unexpected malignancies were identified in the routine tonsillectomy group (0%). In the suspected malignancy group, reactive lymphoid hyperplasia was identified in 13 cases (72.2%), lymphoma in four cases (22.2%), and squamous cell carcinoma (SCC) in one case (5.6%). All patients diagnosed with lymphoma presented with visible tonsillar lesions and palpable neck lymph nodes, three (75%) exhibited tonsillar asymmetry, and two (50%) reported weight loss. The single patient with SCC had asymmetrical tonsils, a visible tonsillar lesion, and palpable neck lymph nodesand experienced weight loss. Conclusion The study findings demonstrated no cases of unexpected malignancy in routine tonsillectomy specimens, reinforcing the argument that routine histopathological examination may not be necessaryin the absence ofclinical suspicion of malignancy. These results, along with considerations of cost-effectiveness and resource optimization, suggest that histopathological analysis maybe reserved only for cases with clinical indicators of malignancy.

Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis

The use of cold formalin fixation (CFF; i.e., fixating tissue samples with 4 °C precooled formalin) recently attracted further attention owing to its putative improved ability to preserve nucleic acid compared with standard room temperature formalin (SFF). In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2/DV1, DNA λ ratio 260/230, and ABL gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2/DV1, RNA λ ratio 260/230, RNA QuBit concentration, DV100/200, RNA electrophoresis concentration and absolute quantity, and ABL copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.

Cost Analysis of Routine Histopathologic Evaluation of Specimens Following Total Ankle Arthroplasty.

Routine histopathologic examination of orthopaedic surgical specimens is a standard practice at many institutions. Previous studies have demonstrated that this practice seldom altered patient management for several orthopaedic procedures. As a result, the value of such practices has come into question. The purpose of this study is to determine the cost-effectiveness of routine histopathologic analysis of specimens obtained during total ankle arthroplasty (TAA). A retrospective analysis was performed of patients who underwent uncomplicated primary TAA at a large, academic, health system between January 2015 and December 2021. The postoperative histopathologic diagnoses were compared with the respective patient's preoperative clinical and intraoperative diagnoses. The prevalence of concordant, discrepant, and discordant diagnoses was determined. Cost-effectiveness analysis was conducted to assess the financial implications of obtaining routine specimens for histopathologic examination for TAA. A total of 85 TAAs were identified in 85 individual patients and were included in the present study. A total of 172 specimens were sent for routine histopathologic review. On histopathologic analysis, a final diagnosis was confirmed in 82 (96.5%) of the total specimens reviewed. A discrepant diagnosis was discovered in 3 (3.5%; 2 cases of gout/pseudogout and 1 case of osteonecrosis) cases and 0 (0%) discordant diagnoses were discovered, corresponding to positive and negative predictive values of 97% and 100%, respectively The total estimate of costs incurred for the routine analysis of all specimens included in the study was between $12 299.20 and 17 846.00. The estimated cost to establish each discrepant diagnosis ranged between $4099.73 and $5948.67, and the cost for a discordant diagnosis was unable to be established. Routine histopathologic analysis of specimens obtained during TAA rarely revealed a discordant diagnosis and resulted in no alterations to patients' plan of care. Furthermore, the additional costs of routine histopathologic examination are significant. As such, it is recommended that such interventions in TAA should be performed on a per-case basis at the operating surgeon's discretion.

Reflex Xpert MTB/XDR Testing of Residual Rifampicin-Resistant Specimens: A Clinical Laboratory-Based Diagnostic Accuracy and Feasibility Study in South Africa.

The World Health Organization-approved Xpert MTB/XDR test detects Mycobacterium tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and injectable drugs directly in specimens. This pragmatic, laboratory-based study assessed the diagnostic accuracy and feasibility of a reflex testing approach, where Xpert MTB/XDR was performed on residual specimens previously processed for Xpert MTB/RIF Ultra. Routine respiratory specimens, processed for Xpert MTB/RIF Ultra, were stored in sample reagent buffer at 2°C-8°C. If rifampicin resistant, the residual specimen was assessed for adequate volume (≥2 mL) and tested with Xpert MTB/XDR, with storage time recorded. A second specimen was used for routine and reference standard testing (culture and sequencing). Specimens (99% sputum) from 763 participants submitted to 2 large routine laboratories were included. Xpert MTB/XDR yielded valid resistance detection results in 639 (84%), compared with 507 (66%) for routine testing (difference [95% CI], 18% [13%-22%]). The median turnaround time for results was 23 hours for Xpert MTB/XDR and 15 days for routine testing. While 748 specimens (98%) were ≥2 mL, only 102 (13%) were stored for ≤4 hours. By the reference standard, 284 of 394 (72%) were isoniazid resistant, and 57 of 380 (15%) were fluroquinolone resistant. The sensitivities of Xpert MTB/XDR were 94% (95% CI, 91%-97%) for isoniazid and 91% (81%-97%) for fluoroquinolone resistance detection. The specificities were 98% (94%-100%) and 100% (98%-100%), respectively. Xpert MTB/XDR performed favorably compared with the reference, and the reflex testing approach increased results availability over routine testing, while dramatically decreasing turnaround time from weeks to hours. Laboratory workflow precluded testing within the manufacturer-recommended 4-hour storage time, but longer storage did not appear detrimental.

The EFLM European Urinalysis Guideline 2023.

The EFLM Task and Finish Group Urinalysis has updated the ECLM European Urinalysis Guidelines (2000) on urinalysis and urine bacterial culture, to improve accuracy of these examinations in European clinical laboratories, and to support diagnostic industry to develop new technologies. Graded recommendations were built in the following areas. Strategies of urine testing are described to patients with complicated or uncomplicated urinary tract infection (UTI), and high or low-risk to kidney disease. Patient preparation, and urine collection are supported with two quality indicators: contamination rate (cultures), and density of urine (chemistry, particles). Measurements of both urine albumin and α1-microglobulin are recommended for sensitive detection of kidney disease in high-risk patients. Performance specifications are given for urine protein measurements and quality control of multiproperty strip tests. Procedures for microscopy are reviewed for diagnostic urine particles, including urine bacteria. Technologies in automated particle counting and visual microscopy are updated with advice how to verify new instruments with the reference microscopy. Chromogenic agar is recommended as primary medium in urine cultures. Limits of significant growth are reviewed, with an optimised workflow for routine specimens, using leukocyturia to reduce less important antimicrobial susceptibility testing. Automation in bacteriology is encouraged to shorten turn-around times. Matrix assisted laser desorption ionization time-of-flight mass spectrometry is applicable for rapid identification of uropathogens. Aerococcus urinae, A. sanguinicola and Actinotignum schaalii are taken into the list of uropathogens. A reference examination procedure was developed for urine bacterial cultures.

Scanning Electron Microscopy.

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM Alternate Protocol 1: Practical considerations for the preparation of soft tissues Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates Alternate Protocol 3: Fixation of colonies grown on agar plates Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence Support Protocol 3: Microwave processing of biological specimens for examination by SEM Basic Protocol 2: Critical point drying of specimens Alternate Protocol 6: Chemical alternative to critical point drying Basic Protocol 3: Sputter coating Alternate Protocol 7: Improved bulk conductivity through "OTOTO" Basic Protocol 4: Immune-labeling strategies Alternate Protocol 8: Immune-labeling internal antigens with small gold probes Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques Basic Protocol 5: Exposure of internal structures by mechanical fracturing Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images.

Accurate and reproducible enumeration of CD4 T cell counts and Hemoglobin levels using a point of care system: Comparison with conventional laboratory based testing systems in a clinical reference laboratory in Cameroon.

Measurements of CD4 T cells and hemoglobin (Hb) are conventionally used to determine the immunological state and disease progression for HIV-infected patients. We obtained a small lightweight point-of-care device, the BD FACSPrestoTM in order to demonstrate its ability to deliver CD4 and Hb analysis in comparison with two larger clinical machines the BDFACSCantoTM analyzer and Sysmex XN 1000 haematology analyzer. The advantages of using the POC device include access to HIV patient data in remote and in resource limited settings. The analytical performance of the BD FACSPrestoTM, compared with the FACSCantoTM II flow cytometer and the Sysmex XN 1000 haematology analyzer was evaluated by testing 241 routine clinical specimens collected in EDTA tubes from patients attending the Immunology and Microbiology laboratory of Chantal BIYA International Reference Centre (Yaounde, Cameroon) between January and May 2016. The mean in absolute counts and percentage of CD4 T cells was 606 cells/mL and 25% respectively via the FACSPrestoTM, and 574 cells/mL and 24% respectively via the BD FACSCantoTM II. The mean concentration of Hb levels was 11.90 on the Sysmex XN 1000 and 11.45 via the BD FACSPrestoTM, A high correlation (R2 = 0.95, P < 0.001) of Hb level measurements was noted between the BD FACSPrestoTM and Sysmex XN 1000 hematology analyzer. Overall, a Bland-Altman plot of the differences between the two methods showed an excellent agreement for absolute and percentage CD4 counts and hemoglobin measurements between POC and conventional methods evaluated here. Furthermore, the study demonstrated the ease of use of the BD FACSPrestoTM POC technology in remote areas. The BD FACPrestoTM is a suitable tool for CD4 enumeration in resource-limited settings, specifically providing a deployable, reliable POC testing option. The BD FACSPrestoTM performed appropriately in comparison to the conventional reference standard technologies. The BD FACSPrestoTM, system provides accurate, reliable, precise CD4/%CD4/Hb results on venous blood sampling. The data showed good agreement between the BD FACSPrestoTM, BD FACSCantoTM II and Sysmex XN 1000 XN 1000 systems.

Underdetected dispersal and extensive local transmission drove the 2022 mpox epidemic

The World Health Organization declared mpox a public health emergency of international concern in July 2022. To investigate global mpox transmission and population-level changes associated with controlling spread, we built phylogeographic and phylodynamic models to analyze MPXV genomes from five global regions together with air traffic and epidemiological data. Our models reveal community transmission prior to detection, changes in case reporting throughout the epidemic, and a large degree of transmission heterogeneity. We find that viral introductions played a limited role in prolonging spread after initial dissemination, suggesting that travel bans would have had only a minor impact. We find that mpox transmission in North America began declining before more than 10% of high-risk individuals in the USA had vaccine-induced immunity. Our findings highlight the importance of broader routine specimen screening surveillance for emerging infectious diseases and of joint integration of genomic and epidemiological information for early outbreak control.

Bioinformatic analysis reveals prognostic value and immunotherapy potential of Siglec-15 in laryngeal squamous cell carcinoma

BackgroundLaryngeal squamous cell carcinoma (LSCC) is the ultimate common malignant head and neck cancer with dismal prognosis. The expression pattern and clinical significance of Siglec-15 (Sialic acid-binding immunoglobulin-like lectin 15) in LSCC are poorly understood. In order to lay the groundwork for future immune-related research on Siglec-15 in LSCC, we set out to study its expression and prognostic importance in the disease, as well as to use bioinformatics to investigate the immune features modulated by Siglec-15 in LSCC. Methods① In order to get the gene expression profile and clinical data for TCGA head and neck cancer (TCGA-HNSC), you may access the relevant data from UCSC xena and use 110 cases of laryngeal cancer as a training set. Two datasets, GSE27020 and GSE25727, were obtained from the GEO databank and utilized as validation sets. These datasets include expression profiles and clinical information. The Siglec-15 gene and immune characteristics were analyzed by bioinformatics methods. ② Retrospectively collected routine paraffin specimens from patients with pathological diagnosis of squamous cell carcinoma from December 2012 to November 2015 in Sun Yat-sen Memorial Hospital and fresh frozen tissue of patients from June 2021 to March 2022. Immunohistochemistry method, immunofluorescence technique and real-time quantitative PCR was used to examine the difference of Siglec-15 appearance in LSCC tissue and adjacent tissue, and its correlation of prognosis, clinic pathological characteristics and CD8+T lymphocyte infiltration. Using human laryngeal cancer cell line (LCC), we studied the influence of Siglec-15 in cell proliferation and invasion. ResultsWe identified Siglec-15 was upregulated in LSCC. The patients in Siglec-15 high expression group had a poor overall survival (OS) based on the clinical information from TGCA and 111 LSCC patients that hospitalized in Sun Yat-sen Memorial Hospital. The COX regression analysis indicated Siglec-15 as an independent predictor for poor prognosis of LSCC. Bioinformatic analysis suggested that the high expression of Siglec-15 shape an immune suppressive tumor microenvironment (TEM), leading to poor response to immunotherapy in LSCC. Siglec-15 enhanced cell invasion and proliferation, as we showed in vitro. ConclusionOur study support Siglec-15 as a potential predictor for LSCC prognosis and an attractive target for LSCC immunotherapy.

Technical evaluation of a novel digital PCR platform for detecting EGFR/KRAS mutations in NSCLC archived plasma specimens

IntroductionKRAS p.G12C hot spot mutations has rapidly modified diagnostic algorithm for lung cancer patients electing Non-Small Cell Lung Cancer (NSCLC) patients to target treatment. As regards, patients that harbor this hallmark showed a clinical benefit in terms of progression-free survival (PFS) and overall survival (OS) in comparison with control group under target treatment. In this scenario, KRAS p.G12C mutation requires optimized testing strategy in diagnostic routine practice. Although the widespread diffusion of NGS platforms, a not negligible percentage of Italian diagnostic institutions adopt singleplex technology (RT-PCR, dPCR) for molecular testing. Here, we aim to technically validate a novel dPCR system (QIAcuity™ Digital PCR System, Qiagen; Hilden, Germany) on a retrospective series of cfDNA samples from previously tested with a custom NGS system. (1,2)Methods: n = 50 liquid biopsy specimens (n = 25 KRAS/EGFR mutated and n = 25 wild type for actionable KRAS/EGFR mutations) from diagnostic routine NSCLC patients previously tested with a custom NGS panel were retrieved from our internal archival. Each sample was tested by adopting n = 5 KRAS and n = 3 EGFR commercially available dPCR assays on QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany); an ultra-deep dPCR walk-away platform that automatizes molecular analysis. Technical sensitivity, technical specificity, and concordance rate between “gold standard” NGS system and QIAcuity™ Digital PCR System (Qiagen; Hilden, Germany) were assessed. ResultsOverall, all specimens were successfully analyzed with dPCR system. In details, 24 out of 25 mutated and 21 out of 24 wild type cases were detected. A technical sensitivity, specificity, and a concordance rate of 96.0 % (24/25), 88.0 % (22/25) and 92.0 % (46/50) were evaluated taking into account a MAF cut-off ≥ 0.2 % and a partition number of 100 positive partitions in wild-type channel. ConclusionQiacuity (Qiagen; Hilden, Germany) platform enables accurate molecular analysis of diagnostic routine specimens. Optimized technical workflow is required to technically implement this platform in diagnostic routine setting.

Incidental precancerous and cancerous neoplasms in routine cholecystectomy specimens – A fascinating series of cases in a tertiary care center

Laparoscopic cholecystectomy is one of the most commonly performed surgeries for benign gall bladder pathologies. Pre-cancerous and malignant gall bladder neoplasms often have overlapping non-specific clinical manifestation in their early stages. Radiological findings are also non-specific in the early stages with the presence of cholelithiasis in a significant proportion of cases. Cholelithiasis is said to be one of the important comorbid risk factors for gall bladder carcinoma (GBC) as a source of constant irritation to biliary mucosa. In such cases, cholecystectomy done for benign indications may lead to a histopathological diagnosis of incidental gall bladder carcinomas. Pancreaticobiliary type of adenocarcinoma is the most common subtype of GBC with two described cases in this series. Invasive foci need to be ruled out in. All cases of high-grade biliary intraepithelial neoplasia as were seen in the third case. Intracholecystic papillary neoplasm is a recently described entity with one reported case in this series that was associated with invasive adenocarcinoma. Signet ring adenocarcinoma is a rarely encountered adenocarcinoma in gall bladder with worse outcome. One such case has been described here where the patient has succumbed to death within 3 months of surgery and during the course of adjuvant therapy.

Abstract B010: Spatially-resolved prediction of gene expression signatures in H&amp;E whole slide images using additive multiple instance learning models

Abstract Background: Newly developed molecular technologies, such as spatial multiplexed assays and single-cell sequencing, have provided increased resolution and output for tumor analysis. However, these assays are often cost-prohibitive, making them inadequate ways to detect clinical biomarkers. In contrast, hematoxylin and eosin (H&amp;E) staining is routine for cancer diagnostics but does not provide molecular information, potentially limiting its utility in the targeted therapy era. Machine learning models could augment the information revealed by H&amp;E, potentially allowing molecular information to be inferred. Here, we describe a novel approach to predict gene expression signatures (GES) in H&amp;E-stained whole slide images (WSI) using an additive multiple instance learning (aMIL) end-to-end model (1). We present results in breast cancer predicting spatially resolved levels of a TGFb GES, a proposed biomarker for TGFb antagonists and immunotherapy. Methods: H&amp;E-stained WSI from the TCGA BRCA cohort (N=1090) were split into training (60%), validation (20%), and test (20%) sets. TGFb-CAF GES (2) were computed, and median expression cut-off on training data was used to define “high” and “low” TGFb-CAF levels. aMIL models were optimized in training data for the binary classification of TGFb-CAF levels. Top-performing model iterations were compared on the validation set, and the optimal model was deployed on the held-out test set. aMIL heatmaps were merged with PathExplore tumor microenvironment (TME) model heatmaps to characterize cell, tissue, and nuclear spatial distributions and morphology in terms of human interpretable features (HIFs). HIFs were extracted from high-importance patches (top 25% of aMIL scores) for both TGFb-CAF-high and -low. Results: Our model accurately predicted TGFb-CAF-high vs. -low BRCA samples (test AUROC=0.80). Also, model deployment on WSI provided interpretable heatmaps depicting TGFb-CAF predictions in tissue, providing spatial resolution to TGFb-CAF expression. Patches contributing most to TGFb-CAF-high prediction were enriched for cancer stroma, as well as cancer-infiltrating and stromal fibroblasts. Furthermore, significant differences in HIFs relating to fibroblast nucleus size and lymphocyte nucleus shape were observed between patches contributing most to TGFb-CAF-high and -low predictions. Conclusions: We have developed a method to predict GES with spatial resolution in H&amp;E-stained WSI. aMIL models provide exact marginal contributions of each patch towards every class prediction, allowing downstream analysis of tissue, cell, and nuclear features and providing biological interpretability not found in typical black-box models. The ability of our method to detect GES in H&amp;E-stained WSI allows complex molecular information to be detected in routine clinical specimens with spatial specificity, providing a means for GES to potentially be realized as clinical biomarkers.

Presence of tissue schistosomiasis in KwaZulu-Natal, South Africa: a retrospective histopathologic review

BackgroundSchistosomiasis affects many parts of the human body including those not usually accessible during routine clinical follow-up. We investigated the presence of schistosomiasis in routine tissue specimens sent to the only public histopathology laboratory in KwaZulu-Natal, South Africa.MethodsThe catchment area for the Department of Anatomical Pathology constitutes 11 million people in 10 districts. We retrospectively reviewed all the histopathology reports for occurrence of schistosomiasis between 1 January 2015 and 30 June 2020.ResultsSchistosomiasis was identified in the appendix, uterine cervix, urinary bladder, lung, liver, fallopian tubes and prostate. During the study period, 725 cases had a diagnosis of schistosomiasis confirmed on histopathology, which equals 0.3% of the total number of specimens sent to the laboratory. Female genital schistosomiasis represented 49.1% (356/725) of the schistosomiasis cases of which 25.1% (182) were from the uterine cervix and 24% (174) from the fallopian tubes. The appendix had 39.7% (289) of all the cases of schistosomiasis. Other organs were urinary bladder (4.4%, 32), lung (3.2%, 23) and liver (2.6%, 19). There were two cases of schistosomiasis in the prostate and four cases in the anorectal region. The main three indications for taking biopsies were acute appendicitis, cervical intraepithelial neoplasia, and sterilization. Majority of the schistosomiasis cases (312) were from eThekwini/Durban metropolitan district, however this represented only 1.2% (312/25 111) of the specimens received from eThekwini/Durban. The districts with the highest percentage positive cases were uMkhanyakude (43/965, 4.5%), followed by Ugu (129/5 251, 2.6%), and King Cetshwayo districts (132/5 360, 2.5%).ConclusionClinicians in the KwaZulu-Natal public health sector hospitals did not suspect schistosomiasis when they submitted patient samples for histopathological investigations. The study indicates the prevalence and the diversity of the body organs affected by schistosomiasis.

1287. Assessing the Utility of Vertebral Body / Disc Space Fluid Cell Differential in the Diagnosis of Native Vertebral Osteomyelitis

Abstract Background Current approaches to diagnosing suspected native vertebral osteomyelitis (NVO) rely on imaging and microbiological workup, including blood and CT-guided biopsy cultures. These existing diagnostic methods often result in ambiguity, necessitating additional tests to distinguish NVO from its mimics. Our study aims to validate pilot findings demonstrating that an elevated neutrophil differential (PMN) in vertebral bone biopsy/ disc space fluid aspirate is associated with NVO. This may allow stratification of patients with suspected NVO into those who can be monitored closely versus those who may benefit from further invasive testing or empiric antibiotic therapy (Fig 1). Figure 1: Proposed workup of patients with suspected native vertebral osteomyelitis guided by disc space/ vertebral body neutrophil (PMN) differential. Methods This analysis examines a subset of patients from a prospective cohort study, enrolling adults referred to Mayo Clinic's neuroradiology department for spine biopsy. After collecting routine clinical specimens, the needle is rinsed with saline into an EDTA tube for automated cell count and differential analysis. To evaluate the predictive capacity of neutrophil (PMN) differential for NVO, logistic regression models were employed, and receiver operating characteristic (ROC) curve analyses were conducted for various PMN percentage cutoff points. Results In this preliminary analysis, 39 patients were included, comprising 7 patients with NVO and 32 patients with alternative diagnoses. Baseline characteristics are presented in Table 1. All NVO patients received antibiotics within two weeks of the spine biopsy. The median biopsy sample PMN percentage for NVO patients was 83.0 (77.5-88.0), compared to 65.0 (56.5-69.5) for those without NVO (p=0.005) (Fig 2A). The estimated relationship between the probability of NVO and each measure across the full range of percentages is illustrated in Fig 2B. The optimal ROC curve point corresponds to a PMN differential of 72.5% (sensitivity=0.86, specificity=0.81) (Fig 3).Table 1:Baseline Characteristics of Patients Included in the Preliminary AnalysisFigure 2:A) Distribution of spine biopsy neutrophil percent by native vertebral osteomyelitis (NVO) status, B) Loess nonparametric smoother showing the relationship between the probability of NVO and biopsy sample neutrophil percentage.Figure 3:ROC curve analysis of neutrophil percent and NVO Conclusion This preliminary analysis indicates that an elevated PMN differential in spine biopsy samples may serve as a valuable diagnostic tool for NVO, with results seemingly unaffected by recent antibiotic intake. The PMN differential cutoff of 72.5% exhibited notable sensitivity and specificity. Investigations are ongoing to further validate these findings Disclosures Brett Freedman, MD, Clear Choice Therapeutics: Stocks/Bonds|Medtronic: Grant/Research Support|Neuroinnovations: Stocks/Bonds Aaron J. Tande, M.D., Musculoskeletal Infection Society: Board Member|Wolters Kluwer Health - Lippincott Williams &amp; Wilkins: Publishing royalties, financial or material support

Prediction of Adjuvant Gemcitabine Sensitivity in Resectable Pancreatic Adenocarcinoma Using the GemPred RNA Signature: An Ancillary Study of the PRODIGE-24/CCTG PA6 Clinical Trial.

GemPred, a transcriptomic signature predictive of the efficacy of adjuvant gemcitabine (GEM), was developed from cell lines and organoids and validated retrospectively. The phase III PRODIGE-24/CCTG PA6 trial has demonstrated the superiority of modified folinic acid, fluorouracil, irinotecan, and oxaliplatin (mFOLFIRINOX) over GEM as adjuvant therapy in patients with resected pancreatic ductal adenocarcinoma at the expense of higher toxicity. We evaluated the potential predictive value of GemPred in this population. Routine formalin-fixed paraffin-embedded surgical specimens of 350 patients were retrieved for RNA sequencing and GemPred prediction (167 in the GEM arm and 183 in the mFOLFIRINOX [mFFX] arm). Survival analyses were stratified by resection margins, lymph node status, and cancer antigen 19-9 level. Eighty-nine patients' tumors (25.5%) were GemPred+ and were thus predicted to be gemcitabine-sensitive. In the GEM arm, GemPred+ patients (n = 50, 30%) had a significantly longer disease-free survival (DFS) than GemPred- patients (n = 117, 70%; median 27.3 v 10.2 months, hazard ratio [HR], 0.43 [95% CI, 0.29 to 0.65]; P < .001) and cancer-specific survival (CSS; median 68.4 v 28.6 months, HR, 0.42 [95% CI, 0.27 to 0.66]; P < .001). GemPred had no prognostic value in the mFFX arm. DFS and CSS were similar in GemPred+ patients who received adjuvant GEM and mFFX (median 27.3 v 24.0 months, and 68.4 v 51.4 months, respectively). The statistical interaction between GEM and GemPred+ status was significant for DFS (P = .008) and CSS (P = .004). GemPred+ patients had significantly more adverse events of grade ≥3 in the mFFX arm (76%) compared with those in the GEM arm (40%; P = .001). This ancillary study of a phase III randomized trial demonstrates that among the quarter of patients with a GemPred-positive transcriptomic signature, survival was comparable with that of mFOLFIRINOX, whereas those receiving adjuvant gemcitabine had fewer adverse events.

Incidental gallbladder carcinoma in routine cholecystectomy specimens at tertiary care hospital

Background: Incidental gallbladder carcinoma (IGBC) refers to gallbladder cancer which is not suspected clinically or radiologically and is detected for the first time on histopathological examination (HPE). Aims and Objectives: To find the incidence of IGBC and determine the pathological staging of IGBC accurately as it decides the mode of surgical intervention and thereby increases the quality of life. Materials and Methods: Three years of retrospective study were done from January 2020 to December 2022 at Sheth L. G. General Hospital. Routine histopathological evaluation of 857 such gallbladder specimens was carried out. Fifteen IGBC cases were reported whose pathological staging was done according to American Joint Committee on Cancer and were further analyzed in terms of demographic status, grading, lymph node status, margins, perineural, and lymphovascular invasion. Results: IGBC incidence was 1.75%. Out of 15 IGBC cases, 73% were females. Cholelithiasis was present in 86% of such cases. Gross inspection of all the IGBC-reported specimens revealed thickened gallbladder wall. All cases were adenocarcinoma – pancreaticobiliary type and the most common histologic grade reported was G2. Tumor cells invading the muscularis propria were observed in 7 cases (pT1b), and peri-muscular connective tissue in 7 cases (pT2). Tumor cells invading the serosa were seen in 1 case. (pT3). Conclusion: It is crucial to correctly report the pathologic T stage which determines patient’s survival. The differentiation between pathologic stage pT1a and pT1b should be made cautiously, as this decides the surgical mode of intervention required for increasing the quality of life. In our study, we found the incidence of IGBC to be 1.75%. HPE of all routine cholecystectomy specimens is the gold standard for IGBC detection and is highly recommended irrespective of the radiological diagnosis or macroscopic findings.

Tackling a global epidemic threat: Nipah surveillance in Bangladesh, 2006-2021.

Human Nipah virus (NiV) infection is an epidemic-prone disease and since the first recognized outbreak in Bangladesh in 2001, human infections have been detected almost every year. Due to its high case fatality rate and public health importance, a hospital-based Nipah sentinel surveillance was established in Bangladesh to promptly detect Nipah cases and respond to outbreaks at the earliest. The surveillance has been ongoing till present. The hospital-based sentinel surveillance was conducted at ten strategically chosen tertiary care hospitals distributed throughout Bangladesh. The surveillance staff ensured that routine screening, enrollment, data, and specimen collection from suspected Nipah cases were conducted daily. The specimens were then processed and transported to the reference laboratory of Institute of Epidemiology, Disease Control and Research (IEDCR) and icddr,b for confirmation of diagnosis through serology and molecular detection. From 2006 to 2021, through this hospital-based surveillance platform, 7,150 individuals were enrolled and tested for Nipah virus. Since 2001, 322 Nipah infections were identified in Bangladesh, 75% of whom were laboratory confirmed cases. Half of the reported cases were primary cases (162/322) having an established history of consuming raw date palm sap (DPS) or tari (fermented date palm sap) and 29% were infected through person-to-person transmission. Since the initiation of surveillance, 68% (218/322) of Nipah cases from Bangladesh have been identified from various parts of the country. Fever, vomiting, headache, fatigue, and increased salivation were the most common symptoms among enrolled Nipah patients. Till 2021, the overall case fatality rate of NiV infection in Bangladesh was 71%. This article emphasizes that the overall epidemiology of Nipah virus infection in Bangladesh has remained consistent throughout the years. This is the only systematic surveillance to detect human NiV infection globally. The findings from this surveillance have contributed to early detection of NiV cases in hospital settings, understanding of Nipah disease epidemiology, and have enabled timely public health interventions for prevention and containment of NiV infection. Although we still have much to learn regarding the transmission dynamics and risk factors of human NiV infection, surveillance has played a significant role in advancing our knowledge in this regard.

B-109 Outcome of a Large Multicenter Prospective Clinical Study Aimed at Validating a Newly Developed VlsE1/pepC10 IgG/IgM Chemiluminescent Immunoassay (CLIA) in Conjunction with a New Automated Analyzer

Abstract Background Lyme borreliosis is the fastest growing vector-borne illness in North America, with recent estimates as high as 476 000 new cases annually in the U.S. alone. Measurement of Borrelia-specific antibodies via serologic testing remains an important diagnostic aid. Since 1994, serologic testing for Lyme disease in the United States has consisted of a two-stage algorithm, termed standard two-tiered testing (STTT). In the STTT algorithm, specimens equivocal/positive by first tier screening methodologies are subsequently tested via western blots. In recent years, the FDA has cleared several alternatives for replacing western blots with more sensitive second-tier tests, termed modified two-tiered testing (MTTT). We recently developed a VlsE1/pepC10 IgG/IgM chemiluminescent immunoassay (CLIA), to be used in conjunction with a new automated analyzer. The goal of these studies was to conduct a multi-site validation for this new assay and analyzer as a first-tier screening test within an STTT algorithm. Methods Retrospective cohort: 280 clinically characterized samples were provided from the U.S. Centers for Disease Control and Prevention (90 cases, 190 controls). The CDC provided VlsE1/pepC10 IgG/IgM ELISA data derived from their own testing. VlsE1/pepC10 IgG/IgM CLIA and western blotting data were also generated for this panel at ZEUS Scientific. Sensitivity and specificity values were calculated for the retrospective cohort as follows:VlsE1/pepC10-CLIA vs existing VlsE1/pepC10 IgG/IgM-ELISA, and VlsE1/pepC10-CLIA + Western blots, vs VlsE1/pepC10 IgG/IgM-ELISA + Western blots, interpreted as STTT algorithms. All testing of the retrospective cohort was performed at ZEUS Scientific. Prospective Cohort: 600 routine Lyme serology specimens were prospectively collected at three locations (1800 total): Mayo Clinic (MOC), Rochester MN.; Marshfield Clinic (MC), Stevens Point, WI; Health Network Laboratories (HNL), Allentown PA. The samples were assayed and analyzed in the same combinations as described above for the retrospective cohort; however, instead of sensitivity and specificity, the results were reported as positive percent agreement (PPA) and negative percent agreement (NPA) since the prospective samples were not clinically characterized. Testing of the prospective cohort was evenly split across three different laboratories, one of which was ZEUS Scientific. Results Retrospective cohort: [VlsE1/pepC10 CLIA, Sensitivity = (77/90) = 85.56%, Specificity = (177/190) = 93.16%; VlsE1/pepC10 ELISA, Sensitivity = (80/90) = 88.89%, Specificity = (179/190) = 94.21%], [STTT-VlsE1/pepC10-CLIA + Western blots, Sensitivity = (72/90) = 80.00%, Specificity = (190/190) = 100.00%; STTT-VlsE1/pepC10-ELISA + Western blots, Sensitivity = (70/90) = 77.78%, Specificity = (190/190) = 100.00%]. Prospective Cohort (1 sample out of the 1800 collected was omitted due to an invalid western blot result):[VlsE1/pepC10-CLIA vs VlsE1/pepC10-ELISA, PPA = (196/235) = 86.51%, NPA = (1452/1564) = 92.84%; STTT-VlsE1/pepC10-ELISA vs STTT-VlsE1/pepC10-ELISA, PPA = (160/167) = 95.81%, NPA = (1607/11 632) = 98.47%]. Conclusion These studies demonstrate that the newly developed VlsE1/pepC10 IgG/IgM CLIA, in conjunction with a new automated analyzer, yielded accurate results as a first-tier screening assay for both the retrospective and prospective multi-site validation cohorts. Future studies, utilizing the same sample cohorts, will be aimed at validating additional second tier CLIA tests as part of an MTTT algorithm.