Single Blood Sample Research Articles

Morphometric and Myelin Basic Protein Expression Changes in Arcuate Nucleus Kisspeptin Neurons Underlie Activation of Hypothalamic Pituitary Gonadal-axis in Monkeys (Macaca Mulatta) during the Breeding Season

ABSTRACT Introduction Kisspeptin is involved in the hypothalamic pituitary gonadal-axis’ seasonal regulation in rodents and sheep. Studies of kisspeptin signaling in regulating the transition between breeding and nonbreeding seasons have focused on kisspeptin expression, myelin basic protein (MBP) expression around kisspeptin-ir cells, and quantifying the synaptic connections between kisspeptin and gonadotropin-releasing hormone (GnRH) neurons in various animal models; however, the role of kisspeptin in regulating the seasonal breeding of primates has not been explored yet. Objective This study investigated changes in kisspeptin signaling during breeding and a non-breeding season in a non-human primate model, the rhesus monkey. Methods Three adult male monkeys (n = 3) from the breeding season and two monkeys (n = 2) from the non-breeding season were used in this study. After measuring the testicular volume and collecting a single blood sample, all animals were humanely euthanized under controlled conditions, and their hypothalami were collected and processed. Two 20 µm thick hypothalamic sections (mediobasal hypothalamus) from each animal were processed for kisspeptin-MBP and kisspeptin-GnRH immunohistochemistry (IHC). One section from each animal was used as a primary antibody omitted control to check the nonspecific binding in each IHC. Results Compared to the non-breeding season, plasma testosterone levels and testicular volumes were significantly higher in monkeys during the breeding season. Furthermore, compared to the non-breeding season, increased kisspeptin expression and a higher number of synaptic contacts between kisspeptin fibers and GnRH cell bodies were observed in the arcuate nucleus of the breeding season monkeys. In contrast, enlarged kisspeptin soma and higher MBP expression were observed in non-breeding monkeys. Conclusion Our results indicated enhanced kisspeptin signaling in primate hypothalamus during the breeding season. These findings support the idea that kisspeptin acts as a mediator for the seasonal regulation of the reproductive axis in higher primates.

Plasma Copy Number Alteration-Based Prognostic and Predictive Multi-Gene Risk Score in Metastatic Castration-Resistant Prostate Cancer.

Simple SummaryAt a genomic level metastatic castrate resistant prostate cancer state is highly heterogeneous and no clear genome-based prognostic or predictive biomarkers exist in practice. We evaluated multiple copy number somatic alterations in two castrate resistant patient cohorts to determine if a genome-based risk score at the copy number level can predict clinical outcomes. The first cohort included patients in a prospective clinical-trial in which abiraterone acetate was given and the other comprised of a real-world hospital-registry. We extracted plasma cell free DNA in both cohorts and performed low pass whole genome sequencing. Copy number alterations were identified for 24 candidate genes and a final composite score developed from 11 genes. This risk score was able to predict survival in castrate resistant patients after adjusting for known clinical biomarkers. Additionally, the multi-gene copy number alteration based risk score algorithm also predicted if abiraterone acetate would be effective in castrate resistant patients.: BackgroundA plasma cell-free DNA (cfDNA) multi-gene copy number alteration (CNA)-based risk score was evaluated to predict clinical outcomes in metastatic castrate resistant prostate cancer (mCRPC) patients. Methods: Plasma specimens from two independent mCRPC patient cohorts (N = 88 and N = 92 patients) were used. A treatment-naïve mCRPC cohort (prospective clinical-trial cohort) included plasma samples before treatment with abiraterone acetate/prednisone and serially at 3-months. A separate real-world hospital-registry (RWHR) mCRPC cohort included a single blood sample collected prior to mCRPC treatments in 92 mCRPC patients following ADT failure. Low pass whole genome sequencing was performed on plasma cell-free DNA (cfDNA) and copy number alterations (CNAs) were identified for 24 candidate genes of interest. Associations of individual gene CNAs with 3 month primary resistance to therapy, progression-free survival (PFS) in the prospective trial cohort and overall survival (OS) in both cohorts was evaluated by Cox regression. A multi-gene risk score was determined for significantly associated candidate CNAs for predicting clinical outcomes. Clinical factors were included in the risk model for survival. Statistical significance for all tests was set at 0.05. Results: In the prospective trial cohort, patients responding to treatment were observed to have a significant copy number decrease in AR (p = 0.001) and COL22A1 (p = 0.037) at 3 months, while the non-responder group showed a significant CNA decrease in NKX3.1 (p = 0.027), ZBTB16 (p = 0.025) and CNA increases in PIK3CB (p = 0.006). Based on the significance level of each gene, CNAs in 11 of the 24 genes (AR, COL22A1, MYC, NCOR1, NKX3.1, NOTCH1, PIK3CA, PIK3CB, TMPRSS2, TP53, ZBTB16) were selected to develop a Cox-regression coefficient-based weighted multi-gene risk score for predicting mCRPC outcomes in both cohorts. A higher multi-gene risk score was observed to have poor OS in mCRPC patients in the prospective trial cohort (p = 0.00019) and for the RWHR cohort, (p < 0.0001). A higher risk score was also associated with poor PFS in the prospective cohort (p = 0.0043). Conclusions: A multi-gene CNAs-based risk score derived from plasma cfDNA may predict treatment response and prognosticate survival in mCRPC and warrants prospective validation of risk-based algorithms.

Testosterone status following short-term, severe energy deficit is associated with fat-free mass loss in U.S. Marines.

The objective of this study was to determine metabolic and physiological differences between males with low testosterone (LT) versus those with normal testosterone (NT) following a period of severe energy deficit. In this secondary analysis, 68 male US Marines (mean ± SD, 24.6 ± 2.4 y) were dichotomized by testosterone concentration (< or ≥ 10.5 nmol/L as determined from a single blood sample collected between 0600–0630 after an 8–10 h overnight fast by automated immunoassay) following 7 days of near complete starvation (~300 kcal consumed/d, ~85% energy deficit) during Survival, Evasion, Resistance, and Escape (SERE) training. Dietary intake was assessed before (PRE) SERE. Body composition (dual‐energy x‐ray absorptiometry and peripheral quantitative computed tomography) and whole‐body protein turnover (15N alanine) were assessed before (PRE) and after (POST) SERE. Mean testosterone concentrations decreased PRE (17.5 ± 4.7 nmol/L) to POST (9.8 ± 4.0 nmol/L, p < 0.0001). When volunteers were dichotomized by POST testosterone concentrations [NT (n = 24) 14.1 ± 3.4 vs. LT (n = 44): 7.5 ± 1.8 nmol/L, p < 0.0001], PRE BMI, total fat mass, trunk fat mass, and testosterone were greater and the diet quality score and total carbohydrate intake were lower in NT compared to LT (p ≤ 0.05). LT lost more fat‐free mass and less fat mass, particularly in the trunk region, compared to NT following SERE (p‐interaction≤0.044). Whole‐body protein synthesis, net balance, and flux decreased and whole‐body protein breakdown increased from PRE to POST in both groups (p‐time ≤0.025). Following short‐term, severe energy deficit, Marines who exhibited low testosterone had greater fat‐free mass loss than those who maintained normal testosterone concentrations. Altering body composition and dietary strategies prior to physical training that elicits severe energy deficit may provide an opportunity to attenuate post‐training decrements in testosterone and its associated effects (e.g., loss of lean mass, performance declines, fatigue).

NEW EXPERIMENTAL APPROACH IN BIODOSIMETRY: EX VIVO APOPTOSIS DETECTION.

This study establishes a new experimental approach for retrospective biodosimetric assessment by apoptosis detection ex vivo. For this purpose, we used mononuclear blood leukocytes isolated from the peripheral blood of irradiated Wistar rats and cultured them ex vivo for posterior analysis. Using flow cytometry, we distinguished apoptotic lymphocyte subsets individual biodosimetric potential at different time periods after exposure: B-lymphocytes 6-8h (0-7Gy), natural killer cells 24h (0-7Gy) and T-lymphocytes 24h (0-1Gy). This novel experimental design innovates through the need of a single blood sample from irradiated individuals for a complete biodosimetric assessment.

Plasma metabolomic profiling reflects the malnourished and chronic inflammatory state in recessive dystrophic epidermolysis bullosa

BackgroundRecessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder characterized by skin fragility, chronic inflammation, malnutrition, and fibrosis. Metabolomics is an emerging investigative field that helps elucidate disease pathophysiology and identify biomarkers. However, previous metabolomic studies in RDEB are limited. ObjectiveTo investigate the plasma metabolomic profiles in RDEB patients. MethodsWe recruited 10 RDEB patients and 10 age-/gender-matched healthy controls. Peripheral blood samples were collected and plasma metabolomic profiling was performed by LC-MS/MS analysis. MS data processing and compound identification were executed by MS-DIAL. Enrichment analysis was performed by MetaboAnalyst 5.0. ResultsMetabolomic analyses demonstrated that most amino acid levels were downregulated in RDEB patients, and the extent of insufficiency correlated with clinical severity. Several metabolites were dysregulated in RDEB, including glutamine and glutamate metabolism, tryptophan-to-kynurenine ratio, phenylalanine-to-tyrosine ratio, and succinate accumulation. LimitationsThe study was limited by small case numbers and the unrepresentativeness of a single time-point blood sample. ConclusionOur study demonstrated the altered metabolomic profiles in RDEB, reflecting the disease severity, the chronic inflammatory and malnourished status, while the fibrotic signatures were not evident.

The role of LCN2 and LCN2-MMP9 in spondylitis radiographic development: gender and HLA-B27 status differences

BackgroundMale HLA-B27-positive radiographic-axial spondyloarthritis (r-axSpA) patients are prone to have severe spinal radiographic progression, but the underlying mechanisms are unclear. We recently showed that persistently elevated Lipocalin 2 (LCN2; L) reflects sacroiliac joint (SIJ) inflammation. LCN2 binds to MMP9. Concomitant elevation of L and LCN2-MMP9 (LM) was detected in many inflammatory diseases. We asked whether L and LM play similar roles in r-axSpA pathogenesis.MethodsWe analyzed 190 axSpA patients (123 radiographic and 67 non-radiographic axSpA) who had no detectable circulating Oncostatin M, to avoid complications due to cross-talk between pathways. L and LM levels from a single blood sample of each patient were measured and were correlated with MRI and modified stoke AS (mSASS) scoring. Association of elevated L (L+) or concurrent L+ and elevated LM (LM+) patterns with B27 status and gender were assessed.ResultsIn L+LM+ axSpA patients, both L and LM levels correlated with MRI SPARCC SIJ scores, but only LM levels correlated with MRI Berlin Spine Scores, suggesting LM is a biomarker for both SIJ and spinal inflammation. Among patients with minimal spinal ankylosis (mSASSS < 10), 65% of male r-axSpA patients are L+LM+, while 30% and 64% of female patients are L+LM+ and L+, respectively, supporting the role of LM with disease progression. In B27+ L+LM+ male patients, both L and LM (but not CRP) levels correlate with mSASSS. B27 positivity and maleness have additive effects on spondylitis progression, suggesting concurrent high L and LM elevations are associated with B27+ male patients having more significant radiographic damage. L+ B27-negative male patients or L+ female patients are more likely to have milder disease.ConclusionL and LM are informative biomarkers for SIJ and spinal inflammation, as well as for ankylosing development in r-axSpA patients. Distinctive L+LM+ or L+ patterns not only could distinguish clinically aggressive vs milder course of disease, respectively, but also provide an explanation for B27-positive male patients being the most susceptible to severe ankylosis.

Estimation of Early Graft Function Using the BETA-2 Score Following Clinical Islet Transplantation.

Little is known about how early islet graft function evolves in the clinical setting. The BETA-2 score is a validated index of islet function that can be calculated from a single blood sample and lends itself to frequent monitoring of graft function. In this study, we characterized early graft function by calculating weekly BETA-2 score in recipients who achieved insulin independence after single transplant (group 1, n = 8) compared to recipients who required a second transplant before achieving insulin independence (group 2, n = 7). We also determined whether graft function 1-week post-transplant was associated with insulin independence in individuals who received initial transplant between 2000-2017 (n = 125). Our results show that graft function increased rapidly reaching a plateau 4-6weeks post-transplant. The BETA-2 score was higher in group 1 compared to group 2 as early as 1-week post-transplant (15 + 3 vs. 9 + 2, p = 0.001). In an unselected cohort, BETA-2 at 1-week post-transplant was associated with graft survival as defined by insulin independence during median follow up of 12months (range 2-119months) with greater survival among those with BETA-2 score >10 (p < 0.001, log-rank test). These findings suggest that primary graft function is established within 4-6weeks post-transplant and graft function at 1-week post-transplant predicts long-term transplant outcomes.

Absence of NOTCH1 mutation and presence of CDKN2A deletion predict progression of esophageal lesions.

Currently, surveillance for esophageal squamous cell carcinoma (ESCC) runs a risk of underestimation of early lesions which show absence of iodine staining, with no or only mild histologic changes. The development of molecular markers that indicate risk of progression is thus warranted. We performed whole-exome sequencing on biopsies from two sequential endoscopies of a single esophageal lesion and matching blood samples. There were 27 pairs of age-, gender-, pathologic stage-, and sampling interval-matched progressors and non-progressors identified in a prospective community-based ESCC screening trial. Putative molecular progression markers for ESCC were first evaluated by comparing somatic mutation, copy number alteration (CNA), and mutational signature information among progressors and non-progressors. These markers were then validated with another 24 pairs of matched progressors and non-progressors from the same population using gene alteration status identified by target sequencing and quantitative PCR. Progressors had more somatic mutation and CNA burden, as well as apolipoprotein B mRNA editing catalytic polypeptide-like and age-related signature weights compared with non-progressors. A gene score consisting of somatic NOTCH1 mutation and CDKN2A deletion is predictive of risk of progression in lesions which show absence of iodine staining under endoscopy but have no or only mild dysplasia. This gene score was also validated in an external cohort of matched progressors and non-progressors. Absence of NOTCH1 mutation and presence of CDKN2A deletion are markers of progression in squamous lesions of the esophagus. This gene score would be an ideal indicator for assisting the pathologist in the identification of high-risk individuals who could be potentially 'missed' or subject to a risk underestimation by histologic analysis, and might improve the performance of ESCC surveillance. © 2022 The Pathological Society of Great Britain and Ireland.

The Shape of Human Red Blood Cells Suspended in Autologous Plasma and Serum.

(1) Background: In almost all studies of the shape of the human red blood cell (RBC), the suspending medium was a salt solution supplemented with albumin. However, the ratio of thickness across the dimple region to the thickness of the rim (THR) depends on the albumin concentration. Values of the THR in the literature range from 0.27 to 0.627 whereas in the present work it was 0.550 or 0.601 whether measured in plasma or serum. (2) Methods: 9911 RBCs of eight donors were suspended in autologous plasma or serum. Sedimented RBCs were observed under bright field illumination at 416 nm. From the profiles of gray value, the THR was determined. (3) Results: The THR displays a wide distribution within a single blood sample. A direct correlation of THR and spontaneous curvature of the membrane is likely. The variation of the mean THR between different donors is large. The aspect ratio of RBCs viewed face-on ranged on average from 1 to 1.48. In oval RBCs, the rim is thicker along the major axis than along the minor axis, an effect increasing with increasing aspect ratio. Remodeling of the membrane skeleton occurs in vivo with a characteristic time () on the order of 1 h. (4) Conclusions: Consideration of these data in models of RBC behavior might improve the agreement with observations. h suggests a more general type of reference configuration of the membrane skeleton than a stress free shape.

Abstract 1976: Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA®)

Abstract Bicycles are fully synthetic constrained peptides with antibody-like affinities that target selectively, readily penetrate tumor tissue, have relatively short half-lives, and can be chemically linked together to generate multifunctional molecules. BT7480 is a Bicycle TICA® being developed as a first-in-class CD137 therapeutic for the treatment of human cancers associated with Nectin-4 expression which is currently being investigated in an ongoing phase I/II clinical trial. Monitoring target engagement for a given therapeutic can be a key factor in recommending the phase II dose. While flow cytometry-based receptor occupancy (RO) assays are commonly used to monitor target engagement in the clinic, a CD137-specific RO assay presents several important challenges that have historically hampered monitoring RO in the clinic including the dynamic expression of CD137 on unstimulated and stimulated T cells, the low frequency of CD137+ cells in human blood and limited reagents to confidently detect CD137+ cells in the presence of CD137-targeting drugs. To address these challenges, a fit-for-purpose 14-plex flow cytometry panel was developed that incorporates a fluorescently labelled CD137-specific binding Bicycle®. This CD137 Bicycle® was shown to directly compete with BT7480 for binding to CD137, but not with fluorescently labelled anti-CD137 antibody, thereby enabling simultaneous detection of various CD137+ immune cell types as well as receptor occupancy by BT7480 in a single blood sample. Panel performance was tested across blood-based sample matrices routinely used in the clinic including EDTA and Cyto-Chex® blood collection tubes and Cell Preparation Tubes (CPT) (n=3 each). CPT were selected as the optimal sample matrix based on sample viability and highest detection of CD137 antibody+ and CD137 Bicycle® + cells. Ex vivo RO assessments in anti-CD3 stimulated and unstimulated healthy human blood, demonstrated dose-dependent detection of CD137 RO by BT7480 and the detection of &amp;gt;1000 CD137+ cells with sample viability &amp;gt;70% (n=5 each). The optimized method and dose-dependent detection of CD137+ cells and RO by BT7480 was further verified in unstimulated lung cancer patient whole blood samples (n=5). Results from this study represent the first report of a clinic-ready CD137 RO assay and the first flow cytometry assay using fluorescently labelled Bicycle® reagents and demonstrate the utility of the Bicycle® CD137 RO assay to monitor target engagement in the BT7480 first-in-human clinical trial. Citation Format: Heather Cohen, Drasti Kanakia, Johanna Lahdenranta, Punit Upadhyaya, Kristen Hurov, Julia Kristensson, Chintan Jobaliya, Greg Bannish, Adam Cotty, Kevin McDonnell, Sandra Hirschberg, Phil Brandish, Sebastien Hazard, Dominic Smethurst, Nicholas Keen, Stephen J. Blakemore. Development of a CD137 receptor occupancy assay to support the phase I/II study of BT7480, a Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA®) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1976.

Optimizing rat and human blood cells sampling for in silico morphometric analysis

Measurements of Morphometric Parameters of the Blood Cells (MPBC) are key for the diagnosis of both mental and metabolic diseases. Several manual approaches or computational methodologies are useful to provide reliable clinical diagnosis. The sample processing and data analysis is relevant, however the sample handling on the pre-analytical phase remains scarcely evaluated. The main goal of this study was to favor the preservation of blood smear using a histological resin. This strategy lead us two practical approaches, give a detailed morphometric description of white blood cells and establish reference intervals in male Wistar rats, which are scarcely reported. Blood smears from male Wistar rats (n = 120) and adult men were collected at room temperature. The integrity of Wright-stained cells was evaluated by an in silico image analysis from rat and human blood smear preserved with a toluene-based synthetic resin mounting medium. A single sample of human blood was used as a control of procedure. The reference intervals was established by cell counting. Based on the results of segmentation algorithm followed by an automatic thresholding analysis, the incorporation of resin favor the conservation of cell blood populations, and lead to identify morphologic features such as nucleus/cytoplasmic shape, granules presence and DNA appearance in nucleus of white blood cells. The use of a histological resin could favor a fast and efficient sample handling in silico MPBC measurements both in the species studied as in wild animals.

Tracking changes in circulating stromal cells and circulating tumor cells predicts responsiveness of new line induction in metastatic breast cancer after 1 cycle of therapy.

3056 Background: In metastatic Breast Cancer (mBC), Circulating Tumor Cells (CTCs) are established prognostic indicators of patients (pts) not responding to new lines of therapy and who have poor clinical outcomes. However, CTCs are typically found in &lt; 20% of mBC pts and many pts without CTCs will also progress. Recently an inflammatory pro-tumorigenic macrophage emanating from tumor stroma (i.e. Cancer associated macrophage-like cell [CAML]) was found in &gt; 90% of mBC pts and were also indicative of poor clinical outcomes. As CTCs &amp; CAMLs are isolated in conjunction from a single blood sample, and both are prognostic for therapy response, we evaluated CTCs &amp; CAMLs before and after initiation of new therapies in mBC to determine their combined prognostic/predictive values. Methods: An observational prospective 2-year multi-institutional study was undertaken to evaluate CTCs &amp; CAMLs before, and after, induction of any new line of therapy in pts with diagnosed mBC (n = 101). Anonymized and blinded blood samples were taken at baseline (BL), prior to starting a new systemic therapy, and a 2nd sample (T1) taken after therapy initiation (̃30 days). Blood was filtered by CellSieve filtration. The quantities and subtypes of CTCs &amp; CAMLs were analyzed based on RECIST v1.1 for progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) by censored univariate &amp; multivariate analysis at 2 years. Results: CTCs were identified in 35% (n = 35/101) of pts at BL &amp; 24% (n = 24/101) at T1, with a single CTC at T1 being highly prognostic for worse PFS HR = 6.2 95%CI 3.0-13.2, p &lt; 0.001 &amp; OS (HR = 5.1 95%CI 2.0-13.4, p = 0.002. In parallel, CAMLs were found in 93% of BL and 86% of T1 samples, and whose decreases were significantly prognostic for improved PFS (HR = 2.7, 95%CI 1.4-5.1, p = 0.006) and OS (HR = 4.4 95%CI 1.5-13.2, p = 0.018) when CTCs were absent. Overall ≥1 CTC at T1 (n = 24) had median PFS = 2.4 &amp; mOS = 4.8 months (mos), however, in pts without CTCs plus an increase in CAMLs (n = 36) had mPFS = 5.9 &amp; mOS = 14.1 mos, while in pts without CTCs plus a decrease in CAMLs (n = 41) had mPFS = 14.8 &amp; mOS = 18.8 mos. Conclusions: Our data confirms that pts with persistent CTCs have the worst clinical outcomes. Further, simultaneous CAML quantification provided a new dynamic predictive blood based biomarker in pts without detectable CTCs which may be useful to better individualize therapy and improve outcomes, though future studies are need to validate these findings.

Circulating tumor DNA profile in pancreatic ductal adenocarcinoma (PDAC) and potential targeted therapy.

4152 Background: Despite huge efforts patients (pts) with advanced PDAC, still have a dismal long-term prognosis. The lack of available good-quality tissue samples for next generation sequencing (NGS) prevents from finding actionable alterations that could guide personalized treatments. Circulating tumor DNA (ctDNA) provides a non-invasive method for obtaining molecular information with therapeutic potential. Here, we present prospective data of ctDNA sequencing in pts with PDAC. Methods: We collected a single blood sample from advanced PDAC pts at any line of treatment (Tx) and at least 10 days apart from their last therapy. The samples were analyzed by Guardant360 plasma-based NGS, a standardized assay which covers microsatellite instability [MSI] evaluation and analysis of all four types of somatic alterations in 74 genes. Alterations were reported either as pathogenic or non-pathogenic. We classified each gene alteration according to the different OncoKB therapeutic levels of evidence [LE]. Additionally, we gathered the dates of both blood collection and molecular report. In case the molecular report showed no tumor-related alterations, it was interpreted as either absence of detectable mutations or low ctDNA levels, which would translate low disease burden or pts responding to therapy. Demographic and clinical data have been accessed from the medical records. Results: From 08/2019 to 09/2021 we collected blood samples from 94 PDAC pts. 56% of them were male and 53% were &gt;65 years old. At the time of sample collection, nearly all pts were metastatic (98%). Regarding previous lines of Tx, 57% pts were Tx naïve; 10% were receiving active systemic Tx and 33% had experienced disease progression. Molecular reports were available in an average of 9.1 days. A total of 243 gene alterations were detected, having 94% of pts at least 1 genomic alteration, which was pathogenic in 69% of the cases (see Table). There were no pathogenic alterations ranked as OncoKB LE 1-2 ( MSI or NTRK). Seventy alterations (30%) were ranked as OncoKB LE 3A and 4 ( ARID1A, BRAC2, CDKN2A, KRAS). Three of these pts were treated with PARP inhibitors due to the presence of BRCA2 mutations. No ctDNA was detected in 15 pts (16%), despite being the sample collected &gt;10 days from receiving their last Tx. Of these, 11 had low tumor burden (only peritoneal or lung metastases) and 4 had documented response to the Tx by the time the samples were collected. Conclusions: Real-time and prospective genomic profiling of pts with advanced PDAC using ctDNA is feasible and conveniently fast, which would allow its role in identifying and developing therapeutic targets for approved Tx or clinical trial treatments. [Table: see text]

"Knowing" Can Be the Medicine for Expecting Mothers.

RNA signatures from a single blood sampling during pregnancy can detect a pathologic condition before its onset.<br><br>Sun-Kyung Lee*. Mol. Cells 2022;45:291-3. https://doi.org/10.14348/molcells.2022.0030

Circulating neutrophil activation in dogs with naturally occurring spinal cord injury secondary to intervertebral disk herniation.

To investigate the time course of circulating neutrophil priming and activity in dogs with spinal cord injury secondary to intervertebral disk herniation that undergo decompressive surgery. 9 dogs with spinal cord injury and 9 healthy dogs (controls). For dogs with spinal cord injury, blood samples were collected on the day of hospital admission and 3, 7, 30, and 90 days after injury and decompressive surgery. A single blood sample was collected from the control dogs. Flow cytometry analysis was performed on isolated neutrophils incubated with antibody against CD11b and nonfluorescent dihydrorhodamine 123, which was converted to fluorescent rhodamine 123 to measure oxidative burst activity. Expression of CD11b was increased in dogs with spinal cord injury 3 days after injury and decompressive surgery, relative to day 7 expression. Neutrophils expressed high oxidative burst activity both 3 and 7 days after injury and decompressive surgery, compared with activity in healthy dogs. For dogs with spinal cord injury, high CD11b expression 3 days after injury and decompressive surgery was consistent with findings for rodents with experimentally induced spinal cord injury. However, the high oxidative burst activity 3 and 7 days after injury and decompressive surgery was not consistent with data from other species, and additional studies on inflammatory events in dogs with naturally occurring spinal cord injury are needed.

Predictors of laminitis development in a cohort of nonlaminitic ponies.

Quantifying risk factors for laminitis development requires improvement. To identify the most useful physical examination, metabolic and management factors to predict laminitis development in client-owned, nonlaminitic ponies. Prospective cohort study. Physical examination, metabolic and management data were collected from a pony cohort 6 monthly for up to 4years. Ponies were monitored for the development of laminitis. Metabolic data included basal plasma concentrations of ACTH ([ACTH]), adiponectin ([adiponectin]), triglycerides and glucose. Serum insulin concentrations ([insulin]) were measured in the unfasted basal state ([insulin]T0) and 60minutes ([insulin]T60) after administration of corn syrup (0.3ml/kg). Separate multivariable Cox proportional-hazards models were developed for physical, management/signalment and metabolic data and later combined into two final models. Low-, medium- and high-laminitis risk categories were defined based on basal or T60 [insulin]. Overall, 374 ponies (age 5-32years) and 891 pony-years were included in the main analysis. Laminitis incidence (95% confidence interval (CI)) was 4.8 (3.5-6.5) cases/100 pony-years. Laminitis development was associated with numerous univariable factors. Significant (P<.05) factors retained in the final multivariable models included [insulin]T0, [insulin]T60, [adiponectin] and divergent hoof growth. [ACTH] was not independently associated with laminitis. Based on [Insulin]T0, low- (<21.6 µIU/ml), medium- (21.6-45.2 µIU/ml) and high-risk (>45.2µIU/ml) categories encompassed 70, 20 and 10% of the population and had estimated 4-year laminitis incidences (95%CI) of 6 (2-9)%, 22 (10-33)% and 69 (48-82)% respectively. Based on [Insulin]T60 the low- (<53.4µIU/ml), medium- (53.4-153µIU/ml) and high-risk (≥153µIU/ml) categories comprised 60, 30 and 10% of the population and had estimated 4-year laminitis incidences (95%CI) of 3 (0-6)%, 20 (10-29)% and 73 (52-84)% respectively. Results may not apply to different insulin assays, geographical regions, breeds or management types. [Insulin]T0 or [insulin]T60 best quantify the risk of future laminitis development in nonlaminitic ponies.

A proof of concept of a machine learning algorithm to predict late-onset 21-hydroxylase deficiency in children with premature pubic hair

In children with premature pubarche (PP), late onset 21-hydroxylase deficiency (21-OHD), also known as non-classical congenital adrenal hyperplasia (NCCAH), can be routinely ruled out by an adrenocorticotropic hormone (ACTH) test. Using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a quantitative assay of the circulating steroidome can be obtained from a single blood sample. We hypothesized that, by applying multivariate machine learning (ML) models to basal steroid profiles and clinical parameters of 97 patients, we could distinguish children with PP from those with NCCAH, without the need for ACTH testing. Every child presenting with PP at the Trousseau Pediatric Endocrinology Unit between 2016 and 2018 had a basal and stimulated steroidome. Patients with central precocious puberty were excluded. The first set of patients (year 1, training set, n = 58), including 8 children with NCCAH verified by ACTH test and genetic analysis, was used to train the model. Subsequently, a validation set of an additional set of patients (year 2, n = 39 with 5 NCCAH) was obtained to validate our model. We designed a score based on an ML approach (orthogonal partial least squares discriminant analysis). A metabolic footprint was assigned for each patient using clinical data, bone age, and adrenal steroid levels recorded by LC-MS/MS. Supervised multivariate analysis of the training set (year 1) and validation set (year 2) was used to validate our score. Based on selected variables, the prediction score was accurate (100%) at differentiating premature pubarche from late onset 21-OHD patients. The most significant variables were 21-deoxycorticosterone, 17-hydroxyprogesterone, and 21-deoxycortisol steroids. We proposed a new test that has excellent sensitivity and specificity for the diagnosis of NCCAH, due to an ML approach.

PLASMA SEPARATOR TUBES DO NOT HAVE ANY OVERT EFFECTS ON ROUTINE PLASMA CHEMISTRY DATA OF GREEN SEA TURTLES (CHELONIA MYDAS).

Plasma separator tubes (PSTs) are a variant of lithium heparin blood tube containing a polymer gel, which, when centrifuged, creates a physical barrier between plasma and blood cells. Their use is common in laboratory procedures of reptilian species. This study aimed to determine whether the use of plasma separator tubes impacts plasma biochemistry data in green sea turtles (Chelonia mydas) at time of collection and after 24 hr of contact time with the separator gel after centrifugation at refrigerator temperature. A single blood sample was collected from 42 rehabilitating green sea turtles at the Sea Turtle Healing Center, Brevard Zoo, Melbourne, Florida, USA and divided into one lithium heparin tube [LHT (0 hr)] and two PSTs. After immediate centrifugation of all three tubes, plasma was transferred from the LHT (0 hr) and one PST (0 hr) into tubes without additive. The plasma was left in contact with the separator gel in the second PST (24 hr). After 24 hr of refrigeration, all three plasma aliquots were analyzed for the following 23 analytes: sodium, potassium, chloride, carbon dioxide, calcium, phosphorus, magnesium, iron, total protein, albumin, globulin (calculated), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, creatine kinase, glucose, urea nitrogen, creatinine, uric acid, triglyceride, and cholesterol. No statistically significant differences were found for any biochemical analytes between LHT (0 hr), PST (0 hr), and PST (24 hr). The use of PST does not appear to impact routine plasma biochemical analytes in green sea turtles and analytes appear stable in refrigerated plasma for up to 24 hr after centrifugation when using PSTs.

Hemagglutination Detection with Paper-Plastic Hybrid Passive Microfluidic Chip.

Hemagglutination is a critical reaction that occurs when antigens expressed on red blood cells (RBCs) react with the antibodies used for blood typing. Even though blood typing devices have been introduced to the market, they continue to face several limitations in terms of observation by the eye alone, blood manipulation difficulties, and the need for large-scale equipment, particularly process automated machines. Thus, this study aimed to design, fabricate, and test a novel hybrid passive microfluidic chip made of filter paper and polymer using a cost-effective xurography manufacturing technique. This chip is referred to as the microfluidic paper–plastic hybrid passive device (PPHD). A passive PPHD does not require external sources, such as a syringe pump. It is composed of a paper-based component that contains dried antibodies within its porous paper and a polymer component that serves as the detection zone. A single blood sample was injected into the chip’s inlet, and classification was determined using the mean intensity image. The results indicated that embedded antibodies were capable of causing RBC agglutination without a saline washing step and that the results could be classified as obviously agglutination or nonagglutination for blood typing using both the naked eye and a mean intensity image. As a proof-of-concept, this study demonstrated efficiency in quantitative hemagglutination measurement within a passive PPHD for blood typing, which could be used to simplify blood biomarker analysis.

Circulating Tumor DNA in Oncology

When somatic cells in the human body undergo apoptosis or necrosis, the released DNA enters the bloodstream. This type of DNA is called cell-free DNA (cfDNA). In patients with cancer, DNA released from tumor cells is called circulating tumor DNA (ctDNA), which carries genetic alterations specific to tumor cells. In recent years, ctDNA has attracted particular attention in terms of the concept of liquid biopsy in cancer care. Conventionally, tissue biopsy is required for the definitive diagnosis of cancer, and imaging examinations, such as CT, are performed for evaluating recurrence and residual lesions. Although the treatment burden on cancer patients is being slightly reduced due to advances in medicine, invasive examinations and medical exposure are still unavoidable. In addition, the prognosis of cancer varies considerably depending on the degree of progression at the time of detection. Therefore, the early detection of cancer is of utmost importance. With the increase in health consciousness, more people undergo regular health checkups, and it becomes necessary to diagnose cancer in a larger number of patients at an earlier stage. Although the accuracy of early detection has been improved by new imaging tests and examination techniques, each organ must be examined separately, and some organs are more difficult to examine than others in a regular health checkup. The process of cancer screening, diagnosis, and detection of recurrence after treatment is extensive. It can also be expensive, and some of the examinations may be invasive. If all of these processes can be replaced by the analysis of ctDNA in liquid biopsy, only a single blood sample is required. Under these circumstances, various studies are currently in progress on the use of ctDNA in clinical practice as an approach that may greatly reduce such burden. We present an overview of the current situation of ctDNA, as well as its future issues and prospects.