Tissue-specific cell-free DNA (cfDNA) offers promise as a minimally invasive biomarker of organ injury. However, current methods for cfDNA tissue-of-origin analysis often depend on genome-wide sequencing approaches, limiting their clinical scalability. We developed a streamlined workflow for translating publicly available DNA methylation datasets into PCR-compatible biomarker assays, using kidney-derived cfDNA as a model. DNA methylation microarray data were mined to identify kidney-specific hypermethylated regions, and candidate biomarkers were screened in silico and experimentally. A digital PCR assay targeting a PAX2-associated DMR was evaluated for analytical performance and assessed in a small exploratory clinical cohort (healthy controls n = 9; transplant recipients n = 5 pre-transplant, n = 7 at 24 h, n = 2 at day 7). The selected PAX2 methylation marker demonstrated high analytical specificity for kidney tissue and was not detected in healthy donor plasma or pre-transplant samples. In contrast, the marker was robustly detected in post-transplant plasma samples, consistent with acute kidney injury due to ischemia-reperfusion. This proof-of-concept study outlines a scalable method for developing PCR-based tissue-specific cfDNA biomarkers. Our kidney-specific assay demonstrates the potential for rapid, cost-effective organ injury monitoring and could be adapted for other tissue types. This strategy may support future diagnostic applications in nephrology, oncology, and transplant medicine.

Currently, C5 complement inhibitors serve as the first-line therapy for classic paroxysmal nocturnal hemoglobinuria (PNH), effectively controlling hemolysis and enhancing patients' quality of life. However, the impact of this treatment on patients' immune status remains unclear. A retrospective analysis was conducted on 27 PNH patients treated with C5 complement inhibitors, focusing on the dynamic changes and recovery of key immune cell subsets, including T lymphocytes, B lymphocytes, natural killer (NK) cells, and dendritic cells (DCs). Among the 27 patients, 70.3% (19/27) achieved transfusion independence. Treatment responses were categorized as follows: complete response (7.4%, 2/27), major response (3.7%, 1/27), good response (25.9%, 7/27), partial response (22.2%, 6/27), minor response (29.6%, 8/27), and non-response (11.1%, 3/27). Before treatment, the proportion of CD4+ T lymphocytes was significantly lower in PNH patients than in normal controls [(38.96 ± 8.270)% vs. (48.57 ± 5.760)%, p < 0.001], and it gradually recovered toward normal levels at 12 months [(42.48 ± 5.623)%] and 24 months [(43.71 ± 6.204)%] post-treatment. In contrast, the percentage of CD8+ T lymphocytes was markedly elevated in pre-treatment patients compared with controls [(49.38 ± 7.259)% vs. (41.75 ± 4.744)%, p < 0.01], and it returned to near-normal levels at 12 months [(42.97 ± 7.074)%]. The proportion of B lymphocytes was significantly reduced in pre-treatment PNH patients [(6.535 ± 1.854)% vs. (12.70 ± 2.256)% in controls, p < 0.001] and partially recovered at 6, 12, and 24 months post-treatment [(8.266 ± 1.756)%, (8.626 ± 1.649)%, and (8.559 ± 1.697)% respectively], though it did not reach normal levels. No significant changes were observed in Treg cells, NK cells, and DCs after treatment. This is the first study to systematically analyze immune status alterations following C5 complement inhibitor treatment in PNH patients, suggesting that the complement system may be involved in the development of immune escape mechanisms in PNH.

Y. Liu, J. Tian, D. Qin, J. Liu, Y. Xie, "AKIP1 Expression in Tumor Tissue as a New Biomarker for Disease Monitoring and Prognosis in Non-Small Cell Lung Cancer: Results of a Retrospective Study," Journal of Clinical and Laboratory Analysis 34, no. 4 (2019): e23128, https://doi.org/10.1002/jcla.23128. The above article, published online on 29 November 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Rong Fu; and Wiley Periodicals, LLC. The retraction has been agreed due to concerns raised by a third party, which identified compromised data and systematic issues in the published article, specifically, inappropriate image duplications in Figure2 (panel (A)) that had been previously published by a different author group. In addition, Figures3 and 4 were found to show unattributed similarities to figures in another article published around the same time by a different author group. The authors and their institutes were unresponsive when these concerns were brought to their attention. Accordingly, the article is retracted as the editors have lost trust in the reliability of the data and the results presented.

UGT1A1 is involved in glucuronidation of drugs and plays a large role in metabolism. Genotyping variants of differing dinucleotide TA repeat length within the TATA box of the UGT1A1 region (dbSNP ID rs3064744) are utilized in the dosing of irinotecan and atazanavir for pharmacogenomics purposes. Here we describe a CLIA-validated PCR and fragment analysis assay that distinguishes between UGT1A1*36 (TA)5, UGT1A1*1 (TA)6, UGT1A1*28 (TA)7, and UGT1A1*37 (TA)8, as well as potential heterozygous combinations. This development offers a more determinant genotype than assays like PharmacoScan can accurately establish alone. PCR was performed using 2X Platinum SuperFi PCR Master Mix, 5X SuperFi GC Enhancer, a FAM-labeled forward primer, reverse primer, and 0.5 ng/μL-20 ng/μL DNA. MiniGene plasmids for each variant served as controls. QC was performed using the Agilent Bioanalyzer 2100 or TapeStation 4200; fragment analysis was performed with HiDi Formamide and LIZ 500 size standard and processed via capillary electrophoresis. Data were analyzed using the GeneMapper version 5 software and peaks with the largest area under the curve (AUC) were identified and compared with known sizes of controls. Per CLIA regulations, the assay passed validation with 100% concordance for all parameters: accuracy, precision, reportable range, reference range, analytical specificity, and analytical sensitivity. Following validation, clinical assay implementation for 940 samples run on PharmacoScan showed disagreement for 74 samples at UGT1A1 rs3064744. Clinical implementation as a supplemental assay to other pharmacogenomics offerings resulted in a correction rate of the loci within patient samples of 7.8%, with 2.77% caused by miscalling UGT1A1*37 variant.

Turnaround time refers to the time it takes from receiving a sample in the lab to delivering test results to clinicians or stakeholders. This study aimed to determine the magnitude of prolonged turnaround time in the hematology laboratory of three selected hospitals in the South Gondar zone, North West Ethiopia. An institutional-based cross-sectional study was conducted from September to December 2023 in the hematology laboratory of selected South Gondar zone hospitals. Debre Tabor's Comprehensive Specialized Hospital was selected purposefully as the sole referral hospital, while Addis Zemen and Nefas Mewcha primary hospitals were randomly selected. The final sample size was 2401. Missing values (n = 70) were managed using the deletion method, assuming data were Missing completely at Random (MCAR). The final dataset included 2331 dataset. Of the 2331 samples, 807 (34.6%: 95% CI 32.8%-36.6%) had prolonged turnaround time. Poor sample condition caused delays in 382 (16.4%) samples. Workday of the weeks, clotted blood samples, and blood fill levels were significantly associated with prolonged TAT. The magnitude of prolonged TAT in hematology laboratories is high. The delays are primarily driven by preanalytical factors, specifically poor sample condition (clotting and improper fill levels) and timing of collection (Fridays). Interventions should focus on enhancing preanalytical sample quality management and optimizing laboratory workflows to reduce prolonged TAT.

Diabetic Foot Ulcers (DFUs) represent a prevalent and serious complication of diabetes, frequently resulting in persistent wounds and delayed healing. This research explores the healing effects of mesenchymal stem cells (MSCs) on DFUs and examines the involvement of Notch signaling in this therapeutic process. BALB/c mice were induced with diabetes using streptozotocin (STZ) and divided into five groups: Control, Model, MSCs Treatment, MSCs + Notch Inhibition, and MSCs + Notch Activation. Wound healing was monitored, and tissue samples were analyzed using histological and molecular techniques. Our results demonstrated that MSCs treatment significantly accelerated wound healing, as evidenced by improved histopathology and enhanced expression of CD31, VEGF, FGF, and PECAM-1 in MSCs-treated groups. Notch activation further enhanced MSCs-mediated healing, as shown by increased Notch1 and Jagged-1 protein levels. Conversely, the MSCs + Notch Inhibition Group showed reduced healing and similar results to the Model Group, with lower expression of Notch1 and Jagged-1. These findings suggest that MSCs promote DFU healing, and Notch signaling plays a crucial role in enhancing MSC-mediated repair. Our results highlight the potential of MSC-based therapies combined with Notch pathway modulation as a promising strategy for improving DFU treatment and wound healing in diabetic patients.

Immune-mediated bone marrow failure (AA) is characterized by T cell-mediated destruction of hematopoietic stem and progenitor cells (HSPCs). The JAK-STAT signaling pathway plays a crucial role in regulating immune responses, and JAK inhibitors have shown potential in treating immune-mediated disorders. This study investigates the effects of MMB, a JAK inhibitor, in a mouse model of immune-mediated bone marrow failure to evaluate its impact on hematopoiesis and immune cell function. In this study, immune-mediated AA was induced in B6D2F1 female mice through irradiation and lymphocyte infusion. The mice were treated with MMB monotherapy at doses of 6.25 and 12.5 mg/kg for 14 days. Peripheral blood cell counts, bone marrow cell counts, plasma cytokine levels, T lymphocyte subsets, Fas/FasL expression, and hematopoietic stem and progenitor cell (HSPC) levels were analyzed to assess the effects of MMB treatment on immune function and hematopoiesis. MMB treatment exacerbated the reduction in peripheral blood cell counts in marrow failure mice and decreased bone marrow hematopoietic cell numbers. Concurrently, MMB also reduced cytokine levels. The treatment also led to a significant reduction in CD4+ T cells and Tregs in both the spleen and bone marrow, while the low-dose MMB group exhibited a decrease in CD8+ T cell count. Additionally, MMB did not significantly improve HSPC levels, and a decrease in the more primitive LSK and LT-HSC populations was observed. However, later-stage stem and progenitor cells, such as ST-HSCs, MPPs, and CMPs, showed an increase in numbers. The treatment also resulted in downregulation of Fas expression on non-T cells, while FasL expression on lymphocytes increased. MMB treatment exacerbated peripheral blood cell reduction and impaired bone marrow hematopoiesis in the immune-mediated AA mouse model. Although MMB modulated T lymphocyte subsets, it did not significantly improve hematopoietic stem and progenitor cell function. These findings highlight the need for further research to explore the potential and limitations of JAK inhibitors like MMB in the treatment of immune-mediated bone marrow failure.

Flow cytometry (FC) enables rapid identification of cell lineages based on their fluorescent and light-scattering properties, playing a critical role in diagnosing and monitoring oncohematological diseases. Standardization of procedures in spectral FC is essential for method validation studies. We designed a comparative study using 21 blood samples from healthy donors. Three commercial lysing solutions used in spectral FC immunophenotyping were evaluated. Samples were stained with a panel of eight antibody-conjugated fluorochromes and aliquoted for assessment with each lysing solution (Excellyse I, Excellyse Easy, and Tombo RBC Lysis Buffer), following the manufacturers' protocols. Data acquisition was performed using a full-spectrum flow cytometer. An average cell loss of 31.4% was observed, with no statistically significant differences between lysing solutions, although Excellyse I showed lower cell loss under the no-wash protocol. This reagent also achieved superior resolution in distinguishing lymphocytes from debris. It demonstrated the lowest coefficients of variation in both scatter and fluorescence parameters of leukocyte populations, highlighting its ability to preserve the optical characteristics of stained cells. RBC-T performed well in specific channels such as APC and PE-Cy7, whereas Excellyse Easy produced a resolution comparable to Excellyse I, albeit with greater signal dispersion. Excellyse I demonstrated the best overall performance, with greater cell preservation, clearer population resolution, and lower variability in scatter and fluorescence. Nonetheless, Excellyse Easy may still be considered a viable alternative.

ABSTRACTBackgroundBronchiectasis, a leading chronic airway disease, often worsens due to infections, making rapid pathogen detection crucial. This study aims to evaluate the diagnostic value of bronchoalveolar lavage fluid (BALF) metagenomic next‐generation sequencing (mNGS) in identifying pathogens in moderate‐to‐severe bronchiectasis and compare its advantages to conventional methods.MethodsFifty‐two hospitalized patients initially diagnosed with moderate‐to‐severe bronchiectasis at Foshan Hospital of Traditional Chinese Medicine from May 2022 to March 2024 were enrolled. Clinical data and BALF samples were collected and subjected to both mNGS and conventional pathogen detection methods. The differences and concordance in pathogen distribution between mNGS and conventional methods, as well as their diagnostic performance, were compared.ResultsThe positive detection rate of pathogens by mNGS was significantly higher than that by conventional methods (p < 0.01). Both methods predominantly identified bacterial pathogens, with Pseudomonas aeruginosa being the most common bacterium and Aspergillus fumigatus the most frequent fungus. However, mNGS detected a broader range of pathogens and demonstrated superior sensitivity in identifying mixed infections (p < 0.01). The sensitivity of mNGS was 66% higher than that of conventional methods (p < 0.01), and the complete concordance rate between the two methods in double‐positive cases was 41.18%. Additionally, mNGS‐guided anti‐infection treatment significantly improves patient symptoms, reduces hospital stays, and lowers costs (p < 0.05).ConclusionsCompared with conventional methods, BALF mNGS demonstrates higher sensitivity, a greater positive detection rate, superior capability in identifying mixed infections, improved diagnostic performance, and a better guiding effect on anti‐infection treatment in moderate‐to‐severe bronchiectasis.