Clinical Assays Research Articles

Assessment of Optical Genome Mapping for Front-Line Diagnostic Evaluation of Acute Leukemia: A Canadian Single-Center Evaluation of Added Yield in 69 Informative Cases

Optical genome mapping (OGM) is a novel method currently being evaluated internationally as a diagnostic assay for genomic profiling of malignancies. OGM uses imaging and reference alignment of fluorescently labeled ultra-long DNA molecules to detect structural and copy number variants genome-wide at significantly higher resolution than is attainable with current standard-of-care (SOC) cytogenetic analysis. OGM is nearing deployment as a first-line diagnostic test for patients with acute leukemia at Vancouver General Hospital, the largest tertiary care hospital in British Columbia. Here, we describe our experience in the evaluation of OGM as a diagnostic assay relative to SOC karyotyping and fluorescence in situ hybridization (FISH) in newly diagnosed and relapsed acute leukemia. A selected cohort of 69 bone marrow specimens produced adequate quality OGM data for analysis: 52 acute myeloid leukemia (AML), 12 B-lymphoblastic leukemia (B-ALL), 4 mixed phenotype acute leukemia (MPAL; 2 B/myeloid, 1 B/T, and 1 T/myeloid), and 1 blast phase (T) myeloid/lymphoid neoplasm with tyrosine kinase gene fusion (M/L-N-Eo). Variants were filtered using population thresholds and with pan-cancer (669 genes) and disease targeted (myeloid: 185; B-lymphoid: 130) gene lists to facilitate detection of variants of interest. OGM results were compared to SOC to identify additional diagnostic yield and changes to risk stratification or diagnosis resulting from OGM findings. In these 69 acute leukemias, filtering to remove common polymorphisms using a manufacturer-provided control dataset yielded a median of 53 variants (interquartile range (IQR) 39-86); using a pan-cancer list yielded a median of 6 (IQR 2-16); and disease-specific lists a median of 4 (IQR 1-10.5). In 11% of cases, diagnosis or risk stratification was altered by OGM (13% of AML; 8% of B-ALL), including a complex karyotype being resolved as normal, downgrading cytogenetic risk; identification of several cryptic KMT2A rearrangements, changing diagnostic classification; and identification of cryptic MECOM::RUNX1 (class-modifying), NUP98::NSD1 (risk-upgrading), and NUP214::ABL1 (potentially targetable and class- and risk-modifying) fusions. In 26% of cases, driver rearrangements were either newly identified by OGM or novel partner genes were resolved (23% of AML; 33% of B-ALL; 40% of others), including recurrent rearrangements of the IGH locus with MIR125B1/ BLID in B-ALL; KMT2A partial tandem duplication; novel MECOM::PAN3, JAKMIP2::PDGFRB, and NUP214::FRMD4B fusions; and a cryptic deletion juxtaposing FLT3 and PAN3 known to result in FLT3 overexpression. Additional findings, including copy number changes and clarifications of complex events, were seen in 54% of cases (44% of AML; 83% of B-ALL; 80% of others), mostly accounted for by small deletions or disruptive rearrangements in key disease genes,likely resulting in loss of function, including but not limited to RUNX1, GATA2 and ETV6 in AML; CDKN2A, PAX5, and RB1 in B-ALL; and TP53 in MPAL. Several cases harboured small MLLT4 copy number abnormalities that have been reported to be linked to cancer predisposition, and one potentially germline FANCA deletion was observed. Details of comparison between SOC methods (karyotyping and pertinent FISH where applicable) are detailed in the Figure and Table (bold text: newly resolved or clarified potential driver or resolution of normal karyotype; underline: change in diagnosis or risk stratification resulting from OGM findings). Notably, OGM failed to identify one hypodiploid B-ALL clone masked by endoreduplication, and further evaluation of this technology's performance in this context and in prospective practice is required. This study demonstrates the significant potential added diagnostic yield of OGM relative to SOC as a clinical diagnostic and research assay. High-resolution genome-wide evaluation not reliant on cell culture substantially expands the scope of detectable variants, for many of which determining the biologic and clinical implications will require larger scale prospective study. Importantly, application of OGM in the clinical setting is likely to result in clinically meaningful disease reclassification and restratification of risk, and in some cases may permit patients access to previously inaccessible targeted therapies (as in the instance of cytogenetically cryptic tyrosine kinase gene fusions).

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Genetic Basis of Relapse after GPRC5D-Targeted CAR T Cells

GPRC5D-targeted CAR T cell therapy was recently shown to be safe and active in patients with relapsed or refractory multiple myeloma with limited or no other therapeutic options, such as those with triple-refractory disease (to a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody) and disease progression on a BCMA antibody-drug conjugate, bi-specific T-cell engager, and/or CAR T cell therapy (Mailankody S. et al. NEJM 2022). Among the 17 patients treated in our phase 1 study of the GPRC5D-targeted CAR T cell therapy MCARH109, 12 patients had an objective response and 6 patients relapsed after an initial response of 3-9 months. While there are currently no validated clinical assays to assess GPRC5D expression, we reported decreased or loss of protein expression using immunohistochemistry in all 6 patients who relapsed. This finding suggests that genetic alterations may be an important mechanism of antigen escape after GPRC5D-directed CAR T cell therapy. With BCMA-directed CAR T cell therapies recently approved for multiple myeloma, antigen loss due to genetic alterations is a rare mechanism of resistance. We first investigated the genetic status of GPRC5D in a patient (Patient 15) who underwent biopsies of a left posterior chest wall plasmacytoma at baseline and a recurrent plasmacytoma at the same site at relapse with nearly 100% tumor. This patient received the maximum tolerated dose of 150×10 6 GPRC5D-directed CAR T cells and achieved a best response of stringent complete response without minimal residual disease in the bone marrow. Unfortunately, 6 months after infusion, he relapsed with recurrent FDG-avid lesions in similar locations on PET, extensive bone marrow involvement (90-95%), rapidly rising lambda free light chain in the serum, and significantly decreased but still detectable circulating CAR T cells (CAR vector copies per mL: 666) ( Figure A). We performed droplet digital PCR (ddPCR) on extracted DNA from the plasmacytoma biopsies and estimated GPRC5D copy number to be two at baseline and 0.1 at relapse, consistent with bi-allelic loss in a significant majority of plasma cells. We then performed whole exome sequencing which revealed two balanced copies of GPRC5D at baseline and bi-allelic loss at relapse consisting of a broader deletion (~8756 kb) in chromosome 12p in one allele and a more focal deletion (~145 kb) encompassing the GPRC5D gene locus in the second allele ( Figure B). Beyond this patient, we identified that relapses after GPRC5D-directed CAR T cell therapy are associated with decreased or loss of GPRC5D mRNA expression. We measured GPRC5D mRNA expression by ddPCR on patient bone marrow or plasmacytoma samples at baseline prior to CAR T cell infusion and at disease progression. The results corresponded with those from immunohistochemistry, and all patients who relapsed showed reduced or absent GPRC5D mRNA and protein expression at disease progression. Given that two patients who responded and relapsed had low or no GPRC5D expression at baseline (Patients 10 and 13), further studies are warranted to characterize the heterogeneity of GPRC5D expression and how well immunohistochemistry and ddPCR capture its expression. Here we report that antigen escape after GPRC5D-directed CAR T cell therapy can be mediated by structural chromosome alterations resulting in bi-allelic loss of the GPRC5D gene locus in a patient with initial excellent response. We also note transcriptional downregulation of GPRC5D in other patients at relapse, but it remains to be determined whether this is transient or durable and whether it can be explained by similar on-target genetic alterations or non-genetic mechanisms. Our patients with heavily pretreated multiple myeloma may harbor greater tumor heterogeneity and genomic instability than previously described, which can facilitate clonal outgrowth of antigen-negative tumor cells under continuous selective pressure of GPRCRD-targeted CAR T cells. Potential strategies to mitigate antigen escape-mediated relapse in myeloma patients receiving T-cell engaging therapies including earlier use of these therapies, multi-antigen targeting, or combination approaches are being evaluated in ongoing trials.

1742. Predictors of Antiviral Receipt for Influenza in Hospitalized U.S. Children, New Vaccine Surveillance Network (NVSN), 2016–2020

Abstract Background Children hospitalized with suspected or confirmed influenza should promptly receive influenza antivirals, as recommended by the Infectious Diseases Society of America, the American Academy of Pediatrics, and the Centers for Disease Control and Prevention (CDC). However, despite these recommendations, antiviral receipt remains suboptimal. We assessed predictors of antiviral receipt for influenza in hospitalized children. Methods We conducted active, population-based surveillance of children presenting with fever or respiratory symptoms from December 1, 2016, to March 31, 2020, at seven children’s hospitals in the CDC NVSN. The cohort consisted of children hospitalized with influenza confirmed by research molecular testing. We assessed predictors of antiviral receipt, selected a priori, using logistic regression, and included all in the final model. Results We enrolled and tested 16,853 hospitalized children for influenza, identifying 1,120 laboratory-confirmed cases (6.6%). Of these laboratory-confirmed cases, clinicians ordered influenza testing for 693/1,120 (61.9%), and among those tested, 599/693 (86.4%) tested positive by clinical assays. Overall, 545/1,120 children (48.7%) received influenza antivirals. Of those with a positive clinical test, 405/599 (67.6%) received antivirals. Factors associated with higher odds of antiviral receipt included an underlying oncologic or immunocompromising disorder, symptom onset ≤ 2 days, intensive care unit (ICU) admission at presentation, use of influenza antivirals for this illness prior to hospitalization, and a positive clinical test (Table). Additionally, we found that a negative clinical test was associated with lower odds of antiviral receipt, and receipt varied significantly by study site. Table Logistic regression model of influenza-specific antiviral receipt among hospitalized children testing positive for influenza by research testing, December 1, 2016, and March 31, 2020 (N=1,120). Conclusion More than half of children hospitalized with influenza in our surveillance population did not receive antivirals, despite treatment recommendations. Efforts to improve antiviral receipt are important for optimizing care in all children hospitalized with influenza, and clinician education should continue to highlight the use of antivirals across the full spectrum of children hospitalized with influenza, including children not admitted to the ICU and those without underlying conditions. Disclosures Marian G. Michaels, MD, MPH, Merck: Grant/Research Support|Viracor: Grant/Research Support John V. Williams, MD, Merck: Grant/Research Support|Quidel: Board Member Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Geoffrey A. Weinberg, MD, Merck & Co: Honoraria Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support Flor M. Munoz, MD, MSc, CDC respiratory virus surveillance: Grant/Research Support|Gilead: Grant/Research Support|Moderna, sanofi, aztra zeneca, Merck, GSK: Advisor/Consultant|NIH: DSMB|NIH COVID-19 vaccines in pregnancy: Grant/Research Support|Pfizer Pediatric COVID-19 vaccines: Grant/Research Support|Pfizer, Dynavax, Monderna, Meissa, NIH: DSMB Mary A. Staat, MD, MPH, CDC: Grant/Research Support|Cepheid: Grant/Research Support|Merck: Grant/Research Support|NIH: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria Elizabeth P. Schlaudecker, MD, MPH, Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support

Burden, determinants, consequences and care of multimorbidity in rural and urbanising Telangana, India: protocol for a mixed-methods study within the APCAPS cohort

IntroductionThe epidemiological and demographic transitions are leading to a rising burden of multimorbidity (co-occurrence of two or more chronic conditions) worldwide. Evidence on the burden, determinants, consequences and care of...

Transcription factor LHX9 (LIM Homeobox 9) enhances pyruvate kinase PKM2 activity to induce glycolytic metabolic reprogramming in cancer stem cells, promoting gastric cancer progression

BackgroundGlycolytic metabolic reprogramming is a phenomenon in which cells undergo altered metabolic patterns during malignant transformation, mainly involving various aspects of glycolysis, electron transport chain, oxidative phosphorylation, and pentose phosphate pathway. This reprogramming phenomenon can be used as one of the markers of tumorigenesis and development. Pyruvate kinase is the third rate-limiting enzyme in the sugar metabolism process by specifically catalyzing the irreversible conversion of PEP to pyruvate.PurposeThis study aimed to reveal the critical mediator(s) that regulate glycolytic metabolism reprogramming in gastric cancer and their underlying molecular mechanism and then explore the molecular mechanisms by which LHX9 may be involved in regulating gastric cancer (GC) progression.MethodsFirstly, we downloaded the GC and glycolysis-related microarray datasets from TCGA and MSigDB databases and took the intersection to screen out the transcription factor LHX9 that regulates GC glycolytic metabolic reprogramming. Software packages were used for differential analysis, single gene predictive analysis, and Venn diagram. In addition, an enrichment analysis of the glycolytic pathway was performed. Immunohistochemical staining was performed for LHX9 and PKM2 protein expression in 90 GC patients, and the association between their expressions was evaluated by Spearman's correlation coefficient method. Three human GC cell lines (AGS, NCI-N87, HGC-27) were selected for in vitro experimental validation. Flow cytometry was utilized to determine the stem cell marker CD44 expression status in GCSCs. A sphere formation assay was performed to evaluate the sphere-forming capabilities of GCSCs. In addition, RT-qPCR and Western blot experiments were employed to investigate the tumor stem cell markers OCT4 and SOX2 expression levels in GCSCs. Furthermore, a lentiviral expression vector was constructed to assess the impact of downregulating LHX9 or PKM2 on the glycolytic metabolic reprogramming of GCSCs. The proliferation, migration, and invasion of GCSCs were then detected by CCK-8, EdU, and Transwell assays. Subsequently, the mutual binding of LHX9 and PKM2 was verified using chromatin immunoprecipitation and dual luciferase reporter genes. In vivo experiments were verified by establishing a subcutaneous transplantation tumor model in nude mice, observing the size and volume of tumors in vivo in nude mice, and obtaining fresh tissues for subsequent experiments.ResultsBioinformatics analysis revealed that LHX9 might be involved in the occurrence and development of GC through regulating glycolytic metabolism. High LHX9 expression could be used as a reference marker for prognosis prediction of GC patients. Clinical tissue assays revealed that LHX9 and PKM2 were highly expressed in GC tissues. Meanwhile, GC tissues also highly expressed glycolysis-associated protein GLUT1 and tumor cell stemness marker CD44. In vitro cellular assays showed that LHX9 could enhance its activity and induce glycolytic metabolic reprogramming in GCSCs through direct binding to PKM2. In addition, the knockdown of LHX9 inhibited PKM2 activity and glycolytic metabolic reprogramming and suppressed the proliferation, migration, and invasive ability of GCSCs. In vivo animal experiments further confirmed that the knockdown of LHX9 could reduce the tumorigenic ability of GCSCs in nude mice by inhibiting PKM2 activity and glycolytic metabolic reprogramming.ConclusionThe findings suggest that both LHX9 and PKM2 are highly expressed in GCs, and LHX9 may induce the reprogramming of glycolytic metabolism through transcriptional activation of PKM2, enhancing the malignant biological properties of GCSCs and ultimately promoting GC progression.

Clinical Chemistry and Haematology Values of a Captive Population of Humboldt Penguins (Spheniscus humboldti).

Reference intervals for clinical chemistry and haematology analytes are essential for the interpretation of blood results, but limited data are available for Humboldt penguins (Spheniscus humboldti) in zoological collections as well as in the wild. The aim of the present study was therefore to establish reference intervals for a series of clinical chemistry and haematology analytes in a single zoological population of Humboldt penguins. Furthermore, possible variations of the analytes influenced by the age and sex of the individuals were investigated. Lithium heparinized plasma and whole blood samples from 39 animals were analysed and statistically evaluated. The following clinical chemistry analytes were significantly higher in females: glutamate dehydrogenase (p = 0.0003), alanine transaminase (p = 0.0005), alkaline phosphatase (p = 0.0245), aspartate aminotransferase (p = 0.0043), creatine kinase (p = 0.0016), lactate dehydrogenase (p < 0.0001), inorganic phosphorus (p = 0.0223), and sodium (p = 0.0415). No significant differences between males and females were found for any haematology analyte. Cholesterol (p = 0.0233; -0.39), white blood cell count (p = 0.0208; -0.40), and absolute heterophiles (p = 0.0148; -0.42) had significant negative correlations with the age of the penguins. The results indicate that the factors of sex and age should be considered when interpreting the results of haematological and clinical biochemistry assays, and further studies are needed to determine whether there are more differences in other age groups or seasons.

CTNI-51. A RANDOMIZED DOUBLE-BLIND PHASE 3 STUDY OF VORASIDENIB VS PLACEBO IN PATIENTS WITH MUTANT IDH1/2 DIFFUSE GLIOMA (INDIGO): EXPLORATORY ANALYSIS OF VARIANT ALLELE FREQUENCY AND PROGRESSION-FREE SURVIVAL

Abstract INTRODUCTION Vorasidenib is an oral, brain-penetrant, inhibitor of mutant isocitrate dehydrogenase (mIDH) 1/2 enzymes. The INDIGO study (NCT04164901) showed significantly improved radiographic progression-free survival (PFS) by blinded independent review committee (BIRC) with vorasidenib, compared with placebo, in patients with mIDH1/2 adulttype diffuse glioma (hazard ratio [HR] 0.39, 95% CI 0.27–0.56; one-sided P = 0.000000067 [Mellinghoff N Engl J Med 2023]). The key secondary endpoint of time-to-next-intervention was also met. METHODS In this double-blind Phase 3 study, patients aged ≥ 12 years with residual/recurrent grade 2 mIDH1/2 oligodendroglioma or astrocytoma, measurable non-enhancing disease, and no prior treatment for glioma were randomized 1:1 to receive vorasidenib 40 mg or placebo daily in 28-day cycles. An investigational clinical trial assay centrally confirmed IDH1 R132H/C/G/S/L or IDH2 R172K/M/W/S/G mutation variants. Pretreatment (archival) tumor tissue was analyzed by next-generation sequencing for CDKN2A/B homozygous deletion and other co-mutations (ACE Extended Cancer Panel). RESULTS As of Sep 6, 2022 (preplanned second interim analysis), 168 patients were randomized to vorasidenib and 163 to placebo (median age, 40.0 years; Karnofsky performance scale = 100, 53.5%; oligodendroglioma, 172; astrocytoma, 159; mIDH1, 315; mIDH2, 16). Two patient subgroups were defined by baseline IDH1/2 variant allele frequency (VAF): lower/higher than the median value (0.377). The median PFS results favored vorasidenib over placebo in both subgroups (low VAF, one-sided P = 0.0116; high VAF, one-sided P &amp;lt; 0.0001). Vorasidenib and placebo groups had similar and a low number of co-mutations in known/likely oncogenic genes at baseline (median number of co-mutations across both groups was 4). CDKN2A homozygous deletion was detected in only two participants (both in the placebo group). Additional data will be presented. CONCLUSION In the first randomized Phase 3 study of a targeted therapy in grade 2 mIDH1/2 glioma, vorasidenib prolonged median PFS by BIRC, relative to placebo, irrespective of IDH1/2 VAF.

Impact of early and empirical cryoprecipitate administration on the transfusion requirements in trauma patients with hemorrhagic shock

Abstract BACKGROUND: Fibrinogen is the earliest coagulation factor falling to the critical levels in severely bleeding trauma patients. Early fibrinogen supplementation in these patients has been recommended to improve the patient outcomes. However, its role in reducing the overall transfusion requirement remains equivocal. AIMS: This study aimed to evaluate the effect of early cryoprecipitate (CRYO) administration on the transfusion requirement and the coagulation assays in trauma patients with hemorrhagic shock. SETTINGS AND DESIGN: An interventional study was conducted in two phases at level I trauma center. During the 1st phase (Group I), patients received blood components in accordance with current practices, whereas in the 2nd phase (Group II), patients received an additional CRYO (pool of 6 units) empirically within 90 min of admission. METHODS: Demographics, clinical, injury characteristics, transfusion requirements, blood counts, and coagulation assays were assessed for both the study groups. STATISTICAL ANALYSIS: The comparative analysis was done for blood component utilization and coagulations assays (prothrombin time, activated partial thromboplastin time, and fibrinogen levels) in both study groups. RESULTS AND CONCLUSIONS: A total of 50 patients in Group I and 51 patients in Group II were recruited. Demographics, clinical, injury characteristics, and laboratory investigations were comparable, except for heart rate, blood pressure, and injury scores. Significant reductions in red blood cell and platelet transfusions were observed, whereas the fresh-frozen plasma transfusions remained unaffected. The results of coagulation assays at 18–24 h were similar for both groups. Although no significant differences in fibrinogen levels were observed despite CRYO administration, decreased requirement for transfusions indicated an improved hemostasis in CRYO group.

Increased Risk of Thrombosis in Patients with Polycystic Ovarian Syndrome

Polycystic Ovarian Syndrome (PCOS) is the most common endocrine-metabolic disorder in reproductive age women, with an estimated prevalence of 6-12% of assigned female at birth women in the US. We identified a pattern in our coagulation diagnostic management team, wherein women with PCOS had increased rates of and repeated histories of thrombotic events, such as strokes, heart attacks, pulmonary embolisms, and deep vein thromboses. However, clinical coagulation assays on these PCOS patients with repeated thrombotic events were all within the normal reference range. Our goal was to investigate the validity of this pattern and its occurrence in both local and national populations. This was accomplished by 1) Determining the incidence rate of thrombosis in PCOS and 2) Analyzing clinical presentations of thrombosis in PCOS. Methods/Study populations : Analyses on the rate of thrombotic events in PCOS patients locally at UTMB and in the US were performed on the TriNetX health research network. Using the incidence and prevalence and compared outcomes analytic functions on TriNetX, we determined the rate of thrombosis from 2013-2018 and explored patient characteristics. We further analyzed thrombosis in PCOS with the Symptom-Disease Pair Analysis of Diagnostic Error (SPADE) method. Through the compare outcomes analysis on the TriNetX platform, we looked at PCOS and control patients who experienced thrombotic events between 2013-2018 and analyzed clinical presentations that occurred in the 90 days prior to the index event. Patient queries included the diagnostic code for PCOS, ICD10 E28.2, women age 15-75, who had a clinical encounter since 2013. Exclusion criteria included inherited thrombotic disorders, smoking history, and human immunodeficiency virus. Events of interest included myocardial infarctions, cerebral infarctions, pulmonary embolisms, arterial embolisms and thrombosis, and portal vein thromboses. Statistical analysis: For evaluating statistical significance of the rate of thrombosis, we performed a compare outcome analysis on the UTMB network, with propensity score matching for current age. A Kaplan-Meier Analysis and a student's t-test were performed on the number of instances/events, and statistical significance was defined as a p-value &amp;lt;0.0001. Results:Between 2013-2018, the incidence rate of thrombosis in PCOS patients was 4.35% with 5.36% prevalence, this was statistically significantly higher than the control cohort at 2.66% incidence and 3.5% prevalence. The incidence rate of thrombosis in PCOS increased dramatically in post-menopausal women. Of the 60 outcomes investigated, 24 were determined to be statistically significant between control and PCOS, 22 symptoms and 2 procedures. Discussion: This is the first study to analyze the rate of thrombotic events in PCOS. We identified significant presentations and gaps that could assist in the early detection of thrombotic events in PCOS. However, the mechanisms of thrombosis in PCOS are unknown and are poorly detected with current clinical assays. Future studies will examine clinical assays in the detection of thrombosis in PCOS and investigate thrombotic mechanisms in PCOS.

Resistance to CD38 Antigen Targeted Therapy in Multiple Myeloma May Result from Underrecognized Chromosome 4 Gene Deletions

Anti-CD38 monoclonal antibodies (mAbs), daratumumab (dara) and isatuximab (isa), are approved to treat multiple myeloma (MM) in the first line and relapsed/refractory settings. Resistance portends a poor prognosis, yet underlying mechanisms have not been well characterized. Despite generally high CD38 expression on most malignant plasma cells (PCs), antigen density may impact the clinical efficacy of targeted therapy. Unfortunately, therapeutic anti-CD38 mAbs interfere with detection and quantification of CD38 surface level by most conventional clinical flow cytometry assays, limiting characterization of CD38 antigen escape. Tumor cell evasion of antigen specific therapeutics can occur with target loss or downmodulation, a phenomenon conventionally referred to as antigen escape. To elucidate mechanisms of CD38 antigen escape, we evaluated MM patients treated with anti-CD38 mAbs who underwent a marrow evaluation between 1/2017 and 10/2022 at a single institution. Eighty-one of 161 (50%) patients had become clinically refractory over their course. Among 82 (51%) patients with ≥1 chromosomal genomic array test (CGAT) performed, 10 (12%) were found to harbor a CD38 deletion ( Table). With a CGAT coverage of 31%, the estimated prevalence of CD38 deletions was 39%. The median interval from diagnosis to CD38 deletion was calculated as 6.7 years (IQR 2.8 to 11.6) for CD38-deleted patients and estimated as 12.9 years (95% CI 12 to NA; Figure) for all patients. The presence of a CD38 deletion was associated with an increasing number of chromosomal abnormalities on concurrent FISH (OR 2.00, 95% CI 1.33 to 3.29, p=0.002). Specific FISH and flow cytometry findings occurred in conjunction with a CD38 deletion. In 50% of patients, flow cytometry detected a bimodal distribution of CD38 fluorescence intensity, reflecting two populations of malignant plasma cells with distinct CD38 surface levels. No patient showed a uniform malignant plasma cell population with a normal surface CD38 level. When the CD38 (4p15.32) deletion was identified in the context of a large 4p deletion spanning the FGFR3 locus, the FGFR3 (4p16) probe could detect the relevant loss of 4p. This was seen in 6 of 8 patients with a concurrent FGFR3 deletion by CGAT. The proportion of clones harboring a CD38 deletion could be quantified by FISH over time when testing was performed on a CD138-enriched sample. Low-level CD38 deletions (≤25%) did not confer resistance to dara-containing multi-drug regimens. In two cases where such deletions were present at diagnosis, disease control is ongoing in response to dara-Pd and dara-Rd. We suggest that acquired CD38 gene deletions appear to be common, the proportion of patients harboring them increases over time irrespective of prior exposure to CD38 mAbs, and their presence facilitates tumor antigen escape. We demonstrate that CGAT can identify CD38 deletions. Conventional clinical flow cytometry assays are unable to quantify the CD38 surface level due to interference from anti-CD38 mAbs. However, we propose that the finding of a bimodal distribution of CD38 fluorescence by flow cytometry could serve as an initial screen to identify at-risk patients warranting confirmatory CD38 mutational analysis. Following characterization of a deletion, FISH could be used to monitor the clonal level longitudinally. Our suggestion that the presence of a CD38 deletion in &amp;gt;25% of malignant PC clones predicts for reduced CD38 mAb efficacy requires prospective validation.

WU-NK-101 (W-NK), a Memory-like (ML) NK Cell, Naturally Overcomes Tumor Microenvironment (TME) Metabolic Challenges, Retaining Anti-Tumor Potency

Cellular engineering revolutionized adoptive cell therapy (ACT) through improved antigen recognition and cellular activation. Cancer cells are metabolically active and extensively consume nutrients from the TME, rendering it nutrient-deprived, acidic, and hypoxic, and resulting in intratumoral metabolic demands which negatively impact ACT antitumor activity. This is central to T cells, however, some of these challenges can be overcome through engineering, an ideal solution would be to have a cellular product that is inherently adept at overcoming these challenges. W-NK is a cytokine-reprogrammed, expanded, cryopreserved, ML-NK cell product derived from healthy human peripheral blood mononuclear cells; it comes with naturally effective potency towards tumor cell killing, and importantly can overcome metabolic challenges present in the TME. We aim at deciphering what metabolic features make W-NK particularly potent in adverse TME. W-NK characterization of metabolic fitness/adaptability was with transcriptomics (sc-RNA-seq; 10X Genomics) flow cytometry, bioenergetics (Seahorse Real-Time Cell Metabolic Analysis; Agilent), and proteomic (Sciex Zeno TOF 7600 tandem-mass spectrometry). The aforementioned assays evaluated the effect of differing media/TME on W-NK: 1) conventional (N) media (pH 7, glucose 11 mM); 2) TME-aligned media (pH 5.9, glucose 6 mM, immune suppressive agents, e.g., PGE2, TGFb1); and 3) ascites derived from patients with malignancy. Media composition was analyzed biochemically using clinical assays (Core Lab Clinical Studies at Washington University, St. Louis, USA), and by immune secretome profiling (Nomic Bio, Quebec, CA). At baseline, W-NK cells have a unique metabolic phenotype, with higher expression of cell surface nutrient transporters, e.g., GLUT1, CD98, ASCT2, MCT1 and PiT1, compared to conventional NK cells (cNK); additionally, W-NK metabolism was consistent with aerobic (“Warburg”) glycolysis, with ~80% of ATP coming from glycolysis vs. ~30% for cNK. In TME/ascites media, W-NK surface nutrient receptor expression adapted to meet metabolic demand. For example, transporters for amino acids, lactate and pyruvate are upregulated in these hypoglycemic medias. This coincided with a 50% shift in ATP manufacturing from glycolysis to mitochondria, suggesting that nutrients bypassed glycolysis to enter tricarboxylic acid cycle and oxidative phosphorylation. Proteomic analyses of W-NK cells cultured with either TME-aligned media or ascites supported this hypothesis, with up-regulation of metabolic pathways (ShinyGO) capturing amino-acid and lipid metabolism, and mitochondrial function. Overall, W-NK retained cytotoxic function in TME and ascites, as well as in native TME-aligned 3D assays from primary surgical tumor samples. Conversely, cNKs did not adapt to TME media, whether in nutrient transporter expression or ATP manufacturing, and demonstrated reduced survival and function, including cytotoxicity. W-NK has enhanced metabolic fitness, flexibility, and plasticity. This enables W-NK to utilize diverse macronutrients, engage different metabolic pathways, and maintain optimal ATP manufacturing. Overall, W-NK inherently survives and maintains function in the TME, a limiting factor for immune cell-based ACT. These data herald the promise of NK cell therapy; a Phase 1 clinical study of W-NK in acute myeloid leukemia is currently open and enrolling patients (NCT# 05470140).

Optical Genome Mapping Allows Detection and Characterization of Cytogenetically Cryptic Oncogenic Fusions in Pediatric Acute Myeloid Leukemia

Background: Abnormal gene fusions are predominant oncogenic drivers in pediatric acute myeloid leukemia (AML) and are important diagnostic and prognostic markers. However, several oncogenic fusions specific for or enriched in pediatric AML (like CBFA2T3::GLIS2, NUP98::NSD1, ETV6::MNX1 and others) are not detectable by conventional cytogenetic analysis (cytogenetically cryptic) and may not be included in fluorescence in situ hybridization (FISH) or molecular fusion panels designed for adult patients. Optical genome mapping (OGM) is a powerful new method for genome-wide, high-resolution detection of copy number abnormalities and balanced chromosome rearrangements. This study determined the frequency of cryptic oncogenic fusions in an institutional cohort of pediatric AML cases and evaluated OGM as a method for their detection and characterization. Methods: Retrospective review of genetic testing results was performed for 93 consecutive de novo or relapsed pediatric AML cases tested at the Children's Hospital Los Angeles (CHLA) Center for Personalized Medicine between March 2016 and July 2023. Genetic testing was performed as part of clinical care following standard procedures and consisted of karyotype analysis, FISH testing, chromosomal microarray (CMA) analysis, CHLA custom sequencing panel (OncoKids®) (June 2017 onwards) and RNA-Seq fusion assay (March 2022 onwards). Cases with normal or inconclusive karyotype in which an oncogenic fusion was detected by another clinical assay were selected for OGM testing. High molecular weight DNA from peripheral blood or bone marrow samples was labeled, processed and imaged in a Saphyr System and analyzed in the Bionano Access software following the manufacturer's recommendations (BionanoGenomics, San Diego CA). Results: Out of 93 pediatric AML cases included in the study, a gene fusion was identified as the primary oncogenic driver in 54 (58%) and a DNA sequence variant in 11 (12%). In 28 cases (30%), the primary genetic abnormality remained undetermined after comprehensive genetic testing. In 17/54 (31%) fusion-driven cases [17/93 (18%) overall cases], the oncogenic fusion remained undetected by karyotype analysis (cryptic). OGM was completed in 13/17 fusion positive cases with non-informative karyotype; it could not be performed in 4 cases due to lack of samples (n=2) or poor DNA quality (n=2), and is still in progress for a subset of 28 unknown cases with available material. OGM analysis successfully revealed all cryptic fusions previously detected by FISH, CMA, OncoKids® and/or RNA-Seq, including NUP98::NSD1 (n=3), KMT2A::MLLT10 (n=3), KMT2A::MLLT3 (n=2)and one case each of CBFA2T3::GLIS2, NUP98::KDM5A, DEK::NUP214, MYB::GATA1 and ETV6::MNX1. Chromosomal mechanism and structure of cryptic fusions were resolved by OGM and included: intrinsically cryptic fusions due to chromosome location of partner genes ( NUP98::NSD1, n=4; CBFA2T3::GLIS2, NUP98::KDM5A and ETV6::MNX1, n=1 each); complex rearrangements due to incompatible gene orientation ( KMT2A::MLLT10 n=2; MYB::GATA1, n=1); three-way rearrangements involving an additional chromosome ( KMT2A::MLLT3 n=2) and small insertions ( KMT2A::MLLT10 and DEK::NUP214, n=1 each). OGM also matched the performance of chromosomal microarray (CMA) analysis in detection of copy number variants. Conclusions: We confirm the significant contribution of cryptic chromosome rearrangements to pathogenesis of pediatric AML, and highlight the importance of tailored testing approaches that effectively detect highly heterogeneous fusion drivers. Our preliminary results show potential of OGM to identify clinically significant cryptic fusions in pediatric AML, and to thus markedly increase diagnostic yield compared to karyotype analysis. As a single assay that detects abnormalities currently interrogated by conventional cytogenetics, FISH and CMA, OGM may allow streamlined and more cost-effective genetic evaluation for pediatric AML.

Distribution of Chromosome-Arm Specific Telomere Length in Patients with Telomere Biology Disorders

Introduction: Telomere biology disorders (TBD) are multisystem diseases often associated with bone marrow failure (BMF) and pulmonary fibrosis. TBD are clinically diagnosed when mean telomere lengths (TL) in blood cells measured by clinical flowFISH assays fall &amp;lt;1 st percentile, in comparison to age-matched reference controls. This information has limitations, e.g. TL in blood cells may not represent TL in other organs (e.g. lung, liver) and moreover, individuals with high clinical suspicion of TBD can show borderline average TL which complicates their diagnosis. The likely reason for this is that cell senescence can be triggered by shortening of a subset, or even a single chromosome arm telomere, a situation that may be missed when measuring average TL. Previous approaches to measure individual chromosome-arm telomere length (CA-TL) are only available for a small number of chromosome arms or can only estimate the number of shortened telomeres without matching them to specific chromosome arms. Taking advantage of recent developments in long-read next generation sequencing (NGS) and the availability of the recently published telomere-to-telomere (T2T) reference human genome, our group developed Telogator, (Stephens Z. et al, 2022) a bioinformatic tool that estimates CA-TL for most chromosome arms using long-read NGS data. Here we use Telogator to describe for the first time the CA-TL pattern in TBD patients in comparison with a control non-TBD cohort. Methods: TBD patients were identified through the Pre-Myeloid and Bone Marrow Failure Specialty Clinic at Mayo Clinic. All patients underwent flowFISH and genetic testing. Age-matched non-TBD patients identified at the Center for Individualized Medicine (Mayo Clinic) were used as reference controls. Informed consent was obtained from all individuals with approval from the Mayo Clinic Institutional Review Board. DNA from peripheral blood mononuclear cells was sequenced using PacBio Sequel II following the manufacturer indications for HiFi sequencing. BAM files were aligned to the T2T reference and CA-TL was calculated using Telogator as described in Stephens Z. et al. 2022. Average TL, CA-TL, and percent of short telomeres (both defined as &amp;lt;5 kb and &amp;lt;3.2 kb) were calculated. Comparisons of average TL and percent of short telomeres were performed using t-test, while Wilcoxon rank-sum test was used to compare CA-TL. Results: Four TBD patients [median age = 58.5 (52-60), 50% female] and 6 non-TBD controls [Median age = 56 (50-63), 33% female] were included in the analysis. All TBD patients presented average TL below the 1 st centile in both lymphocytes and granulocytes, and TBD-related phenotype (100% with pulmonary fibrosis, 50% with BMF, 25% with liver fibrosis). Three had a pathogenic variant in a TBD related genes [2 in TERT (NM_198253.3: c.2030G&amp;gt;A, p.(Gly677Asp) and c.2515_2533del, p.(Thr839Alafs*29)) and 1 in NHP2 (NM_017838.3: c.459_460delAT; p.(X154Argext29))]. The average TL calculated by Telogator showed high correlation with flowFISH values (r 2 = 0.9883, p = 0.0059) and was lower in TBD patients than controls (p = 0.0012). Next, when comparing individual CA-TL between groups, only half of CA-TL was statistically different in the TBD group compared to controls (1p, 3q, 4p, 5p, 5q, 6q, 7p, 7q, 8q, 9p, 9q, 10q, 11p, 12q, 12q, 16p, 16q, 18q, 19p, 19q, 20p, 20q, 21q - Figure 1). The percentage of telomeres &amp;lt;5kb and &amp;lt;3 kb was significantly increased in TBD patients compared to controls (p= 0.014 and 0.0333, respectively). Discussion: Using a novel informatic tool, we replicate previous results obtained using classic approaches and were able to expand on these studies by measuring specific CA-TL in TBD patients. We identified statistically significant differences at the CA-TL level in these patients when compared to age-matched controls, while other CA-TL were not statistically different. Future studies and the inclusion of additional TBD patients will help define the clinical relevance of the differential CA-TL shortening and its prevalence among TBD patients. While limited by the small number of samples in the study, we show the feasibility of using this approach to study TL with unprecedented resolution and show that Telogator offers important advantages compared to classic methods to study telomere biology that can be used in other relevant fields like aging and/or cancer. Our results pave the way to advance the mechanistic understanding of TBD.

Identification and validation of novel prognostic signatures based on m5C methylation patterns and tumor EMT profiles in head and neck squamous cell carcinoma

The role of 5-methylcytosine (m5C) in tumor initiation and progression has been increasingly recognized. However, the precise association between the regulation of m5C and the progression, metastasis, and prognosis of head and neck squamous cell carcinoma (HNSCC) has not yet been fully explored. Data from 545 HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database were analyzed. Unsupervised cluster analysis was conducted using the expression levels of m5C regulatory genes. Additionally, gene set variation analysis (GSVA), single-sample gene set enrichment analysis (ssGSEA), and Cox regression analysis were utilized. Quantitative reverse transcription polymerase chain reaction (RT-qPCR), colony formation assay, transwell experiments and western blots were performed in the HNSCC cell line UM-SCC-17B to assess the expression and functional role of one of the novel signatures, CNFN. Significant expression differences were found in m5C regulatory genes between tumor and normal tissues in HNSCC. Two distinct m5C modification patterns, characterized by substantial prognostic differences, were identified. Cluster-2, which exhibited a strong association with epithelial-mesenchymal transition (EMT), was found to be associated with a poorer prognosis. Based on the m5C clusters and EMT status, differentially expressed genes (DEGs) were identified. Using DEGs, an 8-gene signature (CAMK2N1, WNT7A, F2RL1, AREG, DEFB1, CNFN, TGFBI, and CAV1) was established to develop a prognostic model. The performance of this signature was validated in both the training and external validation datasets, demonstrating its promising efficacy. Furthermore, additional investigations using RT-qPCR on clinical specimens and experimental assays in cell lines provided compelling evidence suggesting that CNFN, one of the genes in the signature, could play a role in HNSCC progression and metastasis through the EMT pathway. This study highlighted the role of m5C in HNSCC progression and metastasis. The relationship between m5C and EMT has been elucidated for the first time. A robust prognostic model was developed for accurately predicting HNSCC patients’ survival outcomes. Potential molecular mechanisms underlying these associations have been illuminated through this research.

Hepatitis B virus (HBV) serum biomarkers for screening and monitoring infections at Rio de Janeiro, Brazil

HBV is a short-term disease and is one of the major causative agents of chronic liver illness. For others, it can become a long-term, chronic infection like liver disease or liver cancer. This paper was aimed to investigate the blood-based serum biomarkers for screening and monitoring of Brazilian patients collected in the All Lab at Rio de Janeiro city. This research were carried out using the chemiluminescence method. The presence (reactive) or absence of anti-HBV antibodies (non-reactive) was also investigated by the electrochemiluminescence. The serological diagnosis of hepatitis B involves the evaluation of antigens and antibodies. Our findings had been showed which it is possible to determine the different phases of the infection (reactive – positive or non-reactive – negative) and some samples can be resulted indeterminates or inconclusive by the choice method and the immune system or susceptible of the patients. The sum of numbers, average, standard deviation and coefficient of variation were calculated among the 2222 samples collected from 1660 patients and 477 samples from 411 patients from the year 2022 and 2023, respectively. So, irrespective of HBV genotype or infection phase, this paper provides a classic way fast-track to advance the HBV serum biomarkers detected in blood samples. Novel challenges of serum HBV biomarkers should be more discussed to mitigated weaknesses points and eliminate cross-reaction in clinical assays.

Invertebrate C1q Domain-Containing Proteins: Molecular Structure, Functional Properties and Biomedical Potential.

C1q domain-containing proteins (C1qDC proteins) unexpectedly turned out to be widespread molecules among a variety of invertebrates, despite their lack of an integral complement system. Despite the wide distribution in the genomes of various invertebrates, data on the structure and properties of the isolated and characterized C1qDC proteins, which belong to the C1q/TNF superfamily, are sporadic, although they hold great practical potential for the creation of new biotechnologies. This review not only summarizes the current data on the properties of already-isolated or bioengineered C1qDC proteins but also projects further strategies for their study and biomedical application. It has been shown that further broad study of the carbohydrate specificity of the proteins can provide great opportunities, since for many of them only interactions with pathogen-associated molecular patterns (PAMPs) was evaluated and their antimicrobial, antiviral, and fungicidal activities were studied. However, data on the properties of C1qDC proteins, which researchers originally discovered as lectins and therefore studied their fine carbohydrate specificity and antitumor activity, intriguingly show the great potential of this family of proteins for the creation of targeted drug delivery systems, vaccines, and clinical assays for the differential diagnosis of cancer. The ability of invertebrate C1qDC proteins to recognize patterns of aberrant glycosylation of human cell surfaces and interact with mammalian immunoglobulins indicates the great biomedical potential of these molecules.

Biological age estimation using circulating blood biomarkers

Biological age captures physiological deterioration better than chronological age and is amenable to interventions. Blood-based biomarkers have been identified as suitable candidates for biological age estimation. This study aims to improve biological age estimation using machine learning models and a feature-set of 60 circulating biomarkers available from the UK Biobank (n = 306,116). We implement an Elastic-Net derived Cox model with 25 selected biomarkers to predict mortality risk (C-Index = 0.778; 95% CI [0.767–0.788]), which outperforms the well-known blood-biomarker based PhenoAge model (C-Index = 0.750; 95% CI [0.739–0.761]), providing a C-Index lift of 0.028 representing an 11% relative increase in predictive value. Importantly, we then show that using common clinical assay panels, with few biomarkers, alongside imputation and the model derived on the full set of biomarkers, does not substantially degrade predictive accuracy from the theoretical maximum achievable for the available biomarkers. Biological age is estimated as the equivalent age within the same-sex population which corresponds to an individual’s mortality risk. Values ranged between 20-years younger and 20-years older than individuals’ chronological age, exposing the magnitude of ageing signals contained in blood markers. Thus, we demonstrate a practical and cost-efficient method of estimating an improved measure of Biological Age, available to the general population.

A microfluidic hemostatic diagnostics platform: Harnessing coagulation-induced adaptive-bubble behavioral perception

Clinical viscoelastic hemostatic assays, which have been used for decades, rely on measuring biomechanical responses to physical stimuli but face challenges related to high device and test cost, limited portability, and limited scalability.. Here, we report a differential pattern using self-induced adaptive-bubble behavioral perception to refresh it. The adaptive behaviors of bubble deformation during coagulation precisely describe the transformation of viscoelastic hemostatic properties, being free of the precise and complex physical devices. And the integrated bubble array chip allows microassays and enables multi-bubble tests with good reproducibility. Recognition of the developed bubble behaviors empowers automated and user-friendly diagnosis. In a prospective clinical study (clinical model development [n= 273]; clinical assay [n= 44]), we show that the diagnostic accuracies were 99.1% for key viscoelastic hemostatic assay indicators (reaction time [R], kinetics time [K], alpha angle [Angle], maximum amplitude [MA], lysis at 30min [LY30]; n= 220) and 100% (n= 44) for hypercoagulation, healthy, and hypocoagulation diagnoses. This should provide fresh insight into existing paradigms and help more clinical needs.

Illumination of Hydroxyl Radical in Kidney Injury and High-Throughput Screening of Natural Protectants Using a Fluorescent/Photoacoustic Probe.

The hydroxyl radical (•OH) is shown to play a crucial role in the occurrence and progression of acute kidney injury (AKI). Therefore, the development of a robust •OH probe holds great promise for the early diagnosis of AKI, high-throughput screening (HTS) of natural protectants, and elucidating the molecular mechanism of intervention in AKI. Herein, the design and synthesis of an activatable fluorescent/photoacoustic (PA) probe (CDIA) for sensitive and selective imaging of •OH in AKI is reported. CDIA has near-infrared fluorescence/PA channels and fast activation kinetics, enabling the detection of the onset of •OH in an AKI model. The positive detection time of 12 h using this probe is superior to the 48-hour detection time for typical clinical assays, such as blood urea nitrogen and serum creatinine detection. Furthermore, a method is established using CDIA for HTS of natural •OH inhibitors from herbal medicines. Puerarin is screened out by activating the Sirt1/Nrf2/Keap1 signaling pathway to protect renal cells in AKI. Overall, this work provides a versatile and dual-mode tool for illuminating the •OH-related pathological process in AKI and screening additional compounds to prevent and treat AKI.