Prozone Research Articles

Fuzzy-Based Anti-Swing Control for Variable-Length Cable-Suspended Aerial Transportation Systems Considering the Hook Effect

Fuzzy-Based Anti-Swing Control for Variable-Length Cable-Suspended Aerial Transportation Systems Considering the Hook Effect

Performance Evaluation of Newly-Developed Tumor Markers Assay Kit Based on Flow Fluorescence Assay.

The purpose of this study is to evaluate the performance of recently developed tumor marker clinical kits in China, with the aim of encouraging local medical technology innovation and thus narrowing the research and development gap with foreign kits. The newly established reagent kits were analyzed on the TESMI F3999-Luminex200 flow lattice instrument to verify precision, sensitivity (blank limit), linearity, anti-interference ability, carry-over contamination rate, hook effect, and reference interval verification. Additionally, the newly established reagent kits were compared to other commercially available detection kits (reference reagent kits) to analyze the correlation between the two types of kits. The intra-assay and inter-assay precision had coefficients of variations (CVs) less than 3.50% and 6.91%, respectively. The tumor marker blank limits were lower than the manufacturer's statement. The newly established reagent kits demonstrated excellent linearity (r > 0.99). Rheumatoid factor, triglycerides, bilirubin, and hemoglobin did not have significant interference with the determination of tumor markers. The carry-over contamination rates were all much lower than 3%. At extremely high concentrations of AFP (277,335 ng/mL and 1,031,424 ng/mL), the measured tumor marker values were higher than the upper limit of the linear range and no hook effect occurred. The reference interval was suitable for use in clinical laboratory settings. Correlation analysis indicated a satisfactory relevance and consistency between the newly developed reagent kits and reference reagent kits, with correlation coefficients of r > 0.967 among 654 patients and healthy individuals. The newly developed reagent kits for tumor markers performed well in all evaluated parameters, having the potential for clinical promotion and application.

Abstract B144: Discovery and preclinical evaluation of novel oral WEE1 degraders

Abstract Background: WEE1 is a key component of cell cycle checkpoints as it inhibits cell cycle progression through phosphorylation of CDK1. Inhibition of WEE1 could induce premature mitotic entry of cells with damaged DNA, leading to mitotic catastrophe and cell death. WEE1 small molecule inhibitors (SMIs) have demonstrated clinical benefits particularly in gynecological malignancies. However, limited antitumor activity and multifaceted toxicity profile from SMIs warranted the discovery of novel WEE1-targeting modalities to afford better therapeutic index towards a greater patient population. Here, we report the discovery of a novel oral, heterobifunctional WEE1 degrader NXD01 and its preclinical evaluation. Methods: We discovered NXD01 through our AI-empowered, prediction-guided compound optimization platform neoDegrader. We quantified NXD01-mediated WEE1 protein degradation by western blot and evaluated the anti-proliferative activity of NXD01 by CellTiter-Glo assay in multiple human cancer cell lines. We further assessed downstream effects of WEE1 degradation on phosphorylation of its target CDK1, and markers of cell cycle and apoptosis by western blot and flow cytometry. We studied the in vivo antitumor activity of NXD01 by oral gavage in the SK-UT-1 (human uterine leiomyosarcoma cell) xenograft model, followed by PK/PD analysis of the tumor tissues. Results: In SK-UT-1 cells, NXD01 potently degraded WEE1 protein at a DC50 of 1.4 nM with no obvious hook effect and achieved complete degradation within 4 h of treatment (DC90 = 4.5 nM). Marked reduction in CDK1-Y15 phosphorylation and increased γ-H2AX levels (a marker for DNA damage) and PARP cleavage (apoptosis) were all detected for NXD01 at 100 nM, one-tenth the effective concentration for the WEE1 SMI azenosertib (ZN-c3). Treatment of NXD01 (but not ZN-c3) at 100 nM greatly increased the proportion of cells in the G2/M phase indicating mitotic arrest. These results translated into a more potent anti-proliferative effect by NXD01 compared to ZN-c3 in vitro: the IC50 was 1.3 nM vs 37 nM for MOLT4, a T cell leukemia line, as well as across 10 gynecological cancer cell lines (NXD01 IC50: 10-210 nM; ZN-c3 IC50: 100-975 nM). NXD01 exhibited good exposure after oral administration in mice (dose normalized AUC0-t: 765 mg∙h/mL, t1/2: 3 h, F%: 15.7%). NXD01 showed strong in vivo antitumor activity in the SK-UT-1 xenograft model, achieving 96% tumor growth inhibition (TGI) at 30 mg/kg BID, as compared to 81% TGI for ZN-c3 at 60 mg/kg QD. Analysis of tumor samples suggested that 85% of in-tumor WEE1 protein had been degraded by NXD01 at 12 h post dose. Conclusion: We have discovered a structurally-novel, orally bioavailable, cereblon-mediated, heterobifunctional degrader of WEE1 protein NXD01. NXD01 displayed stronger in vitro and in vivo antitumor activity than currently the most advanced WEE1 small molecule inhibitor azenosertib, making NXD01 a differentiated candidate with strong potential for clinical development. Citation Format: Weiqiang Xing, Meixin Tao, Fan Huang, Liuge Gu, Min Cao, Chunhao Wang, Jianxiong Diao, Chunming Du, Xinyu Bai, Mingchen Chen, Xin Zheng, Ying Lei, Yang Xie, Taylor B. Guo. Discovery and preclinical evaluation of novel oral WEE1 degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B144.

The Ameliorative Effects of Phyllanthus gomphocarpus Hook. F. on Sperm Parameters of Rats Induced with Bisphenol A

Infertility due to poor sperm quality is a common problem nowadays, particularly in developed countries. Herbs are one of the natural treatment options that have been proposed. Phyllanthus gomphocarpus Hook. F is a tropical plant that has traditionally been used in Malaysia to treat male infertility. Antioxidant characterization of its root aqueous extraction was performed using ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), total phenolic content (TPC), and total flavonoid content (TFC). Male rats were administered two different concentrations of the extract orally along with BPA for 21 days, and their sperm was evaluated using Computer-Assisted-Sperm-Analysis (CASA). The results showed that this plant has a high antioxidant capacity, with 78.6 ± 0.65 %, 0.14 ± 0.01 mg/ml, 28.52 ± 0.13 mg GAE/g and 164.73 ± 23.13 mg QE/g, for DPPH, FRAP, TPC and TFC respectively. The ability of this plant to enhance sperm concentration, motility, velocity, progression ratio, elongation, and percentages of normal sperm morphology significantly with p<0.05, appears to be due to its high antioxidant capacity. Furthermore, this plant was found to have sperm-protective effects against the endocrine disruptor BPA. A thorough investigation is required to determine the full mechanism of this plant's beneficial effects on male fertility and human health.

False Negative Results of First-Tier Treponemal Antibodies in an HIV-Infected Patient

Abstract Introduction/Objective Syphilis diagnosis relies on detecting Treponema pallidum antibodies in serum or CSF. In the reverse syphilis screening algorithm, an automated enzyme immunoassay first detects serum antibodies against treponemal antigens. A negative result halts further testing; however, a positive result prompts manual testing for antibodies against non-treponemal antigens such as cardiolipin. The sensitivity of first-tier automated assays is typically high. We present a case of an HIV patient who initially tested negative for syphilis in the first-tier assay but was subsequently diagnosed with the infection. Methods/Case Report A 30-year-old HIV-positive female (CD4+ count 24/mm3), previously diagnosed with chlamydia and gonorrhea, experienced blurred vision, eye pain, fever, and a palmoplantar rash. Despite clinical suspicion of syphilis, the initial first-tier chemiluminescence immunoassay (CIA) for treponemal antibodies (LIAISON Treponema, DiaSorin, Stillwater, MN) returned a negative result, precluding any further syphilis testing at that time. However, a skin biopsy revealed interface dermatitis and spirochetes. CSF analysis showed pleocytosis, increased proteins, and borderline low glucose, with both VDRL and IFA assays negative. Based on these findings, we retested for serum antibodies by repeating the CIA test and performing RPR and TP-PA assays outside the algorithm sequence. Despite reconfirmation of the CIA negativity, both RPR (titer 1:64) and TP-PA were positive, leading to a secondary syphilis diagnosis and penicillin G treatment. Subsequent investigations aimed to explain the initial false negative CIA result. Serial dilution of the patient's serum up to 1:1000 did not alter the result, excluding the 'hook effect' caused by an excessive concentration of antibodies. Further serum testing at a different laboratory using another type of first-tier immunoassay yielded a positive result, pointing toward differences in the composition of treponemal antigens utilized across various manufacturer assays as the likely cause of the discrepancy. Results (if a Case Study enter NA) NA Conclusion Given their high sensitivity, automated first-tier treponemal assays rarely yield false negative results, whereas false positives are the most common ‘lab error’ and are typically resolved with second-tier assays. Nevertheless, this case highlights that false negatives can occur and, thus, stresses the need to re-evaluate the approach in diagnosing syphilis, especially in patients with antibody production abnormalities, such as those with HIV.

Discovery of BCL-XL Heterobifunctional Degrader with Potentially Improved Therapeutic Window and Minimal Platelet Toxicity for Hematological Malignancies

Background In hematologic malignancies, apoptosis evasion promotes tumor formation/progression and is driven by the overexpression of anti-apoptotic proteins, including BCL-2 and BCL-XL. BCL-2-targeting small molecule inhibitor venetoclax won FDA approval for chronic lymphocytic leukemia and acute myeloid leukemia. Interests in developing BCL-XL-selective inhibitors arise as BCL-XL overexpression is implicated in venetoclax resistance. However, these efforts failed in clinical development due to dose-limiting thrombocytopenia, as platelets (PLT) also depend on BCL-XL for survival. To mitigate this on-target toxicity, a degrader strategy has been proposed, whereby BCL-XL protein can be selectively degraded by the von Hippel-Lindau protein (VHL) E3 ligase in tumor cells, but not in PLT which minimally express VHL. The first such BCL-XL degrader (DT2216) was developed and is now in early clinical trials. Despite negligible PLT toxicity in vitro, DT2216 was still toxic to PLT in vivo. We reasoned that since earlier BCL-XL degraders used strong BCL-XL inhibitor as warhead, e.g. the warhead for DT2216 is ABT263, they could readily bind to and inhibit BCL-XL in PLT causing thrombocytopenia. Warhead-containing metabolites could also form and behave as an inhibitor, exacerbating PLT toxicity in vivo. Utilizing our artificial intelligence-empowered neoDegrader platform, which accurately characterizes the landscape of the degrader-mediated, metastable interactions between E3 and its target protein, we were able to computationally design molecules that were relatively weak BCL-XL binders but could maintain the ternary complex at metastable states. Herein, we report the preclinical profile of the lead candidate NXD02. Methods We assessed the binding affinity of NXD02 to BCL-XL by surface plasmon resonance. We conducted flow cytometry to quantify NXD02-mediated BCL-XL protein degradation and apoptotic effects in MOLT4 cells. We also quantified the anti-proliferative activity of NXD02 by CellTiter-Glo assay. We measured protein level changes of apoptosis markers by western blot. We simultaneously determined plasma concentration of NXD02 and PLT at various time points following a single injection of NXD02 in rodents and monkeys. Finally, we evaluated the anti-tumor activity of NXD02 in an orthotopicMOLT4-luc mouse xenograft (CDX) model by bioluminescence imaging. Results The binding affinity of NXD02 to BCL-XL was > 40-fold weaker than that to DT2216 (K D 28.7 vs 0.6 nM). Despite this, NXD02 strongly degraded BCL-XL protein (DC 50 6.6 nM) with no obvious hook effect, which was more potent than DT2216 (DC 50 17.4 nM) in MOLT4 cells. At 100 nM, NXD02 markedly increased levels of active caspase 3 and cleaved PARP, which was consistent with evidence of massive apoptosis. This was translated to a potent anti-proliferative effect comparable to DT2216 (IC 50 27.8 vs 42.7 nM). NXD02 did not affect PLT viability up to a concentration of 10 μM in vitro. Notably, a single dose of NXD02 at 10 mg/kg i.v. achieved an exposure >4-fold than that of DT2216 (dose-normalized AUC 68.5 vs 15.3 μg*h/mL/(mg/kg)) in CD1 mice, while producing similar levels of peak PLT reduction (58% vs 65%) at ~24 h, which recovered by day 3. Comparable or less PLT reduction was also observed in rats (64% vs 66%) at 15 mg/kg and in cynomolgus monkeys (63% for NXD02 vs 87% for DT2216) at 3 mg/kg, all of which fully recovered by days 5-6, supporting the possibility for weekly dosing. Furthermore, NXD02 demonstrated stronger in vivo anti-tumor activity than DT2216 in a CDX model. Weekly i.v. dosing of NXD02 at 3 and 10 mg/kg achieved 80% and 100% tumor growth inhibition (TGI) by day 28 respectively, as compared to 36% TGI for DT2216 at 10 mg/kg. There was no significant body weight loss for all groups. Conclusions We have discovered a structurally-novel, VHL-mediated, heterobifunctional BCL-XL degrader NXD02 with “Weak Inhibitor, Strong Degrader” properties. NXD02 displayed, at the same dose, a much higher drug exposure, better anti-tumor activity and comparable platelet toxicity profile in vivo than the most advanced BCL-XL degrader DT2216. The results suggest that NXD02 may have a more favorable safety margin. In vitro toxicity profiling of NXD02 including Ames test and safety panel revealed no concerns so far. In vivo safety assessment is ongoing for NXD02 as a promising candidate for clinical development in liquid and potentially select solid tumors.

Ambulatory human chorionic gonadotrophin (hCG) testing: a verification of two hCG point of care devices.

Quantitative human chorionic gonadotropin (hCG) measurements are used to manage women classified with a pregnancy of unknown location (PUL). Two point of care testing (POCT) devices that quantify hCG are commercially available. We verified the i-STAT 1 (Abbott) and the AQT 90 FLEX (Radiometer) prior to use in PUL triage. Tests for precision, external quality assurance (EQA), correlation, hook effect and recovery were undertaken alongside a POCT usability assessment during this prospective multi-center verification. Coefficients of variation ranged between 4.0 and 5.1 % for the three i-STAT 1 internal quality control (IQC) solutions and between 6.8 and 7.3 % for the two AQT IQC solutions. Symmetric differences in POCT EQA results when compared with laboratory and EQA stock values ranged between 3.2 and 24.5 % for the i-STAT 1 and between 3.3 and 36.9 % for the AQT. Correlation coefficients (i-STAT 1: 0.96, AQT: 0.99) and goodness of fit curves (i-STAT 1: 0.92, AQT: 0.99) were excellent when using suitable whole blood samples. An hCG hook effect was noted with the i-STAT 1 between 572,194 and 799,089 IU/L, lower than the hook effect noted with the AQT, which was between 799,089 and 1,619,309 IU/L. When hematocrit concentration was considered in sample types validated for use with each device, hCG recovery was 108 % with the i-STAT 1 and 98 % with the AQT. The i-STAT 1 scored lower on usability overall (90/130) than the AQT (121/130, p<0.001, Mann-Whitney). Both hCG POCT devices were verified for use in clinical practice. Practical factors must also be considered when choosing which device to use in each unit.

NY-ESO-1 antigen-antibody interaction process based on an TFBG plasmonic sensor.

Autoantibodies against New York esophageal squamous cell cancer 1 (NY-ESO-1) play a crucial role in the diagnosis of esophageal cancer. In this work, a surface plasmonic tilted fiber Bragg grating (TFBG) biosensor is proposed for the detection of NY-ESO-1 antibody, as well as the investigation of the hook effect (which refers to the false negative result in some immunoassays when the concentration of antibodies in the sample is very high) during biomolecular binding between NY-ESO-1 antigen and antibody. The biosensor is made by an 18° TFBG coated with a 50-nm-thick gold film over the fiber surface together with NY-ESO-1 antigens attached to the metallic surface serving as bio-receptors. This biosensor can provide a limit of detection at a concentration of 2 × 10-7 µg/ml with a good linearity in the range from 2 × 10-7 to 2 × 10-5 µg/ml. For a concentration higher than 2 × 10-3 µg/ml, the performance of the sensor probe is reduced owing to the hook effect. Furthermore, experimental results have also demonstrated the repeatability of the proposed biosensor. This proposed biosensor features label-free, compactness, and fast response, which could be potentially applied in the diagnosis of esophageal cancer.

FRI351 Pituitary Stalk Epidermoid Cyst: A Rare Cause Of Diabetes Insipidus

Abstract Disclosure: J.T. Batch: None. F. Warda: None. P. Natter: None. R. Makary: None. G.Y. Gandhi: None. Introduction: Epidermoid cysts are benign squamous epithelial cysts filled with keratin. They are rarely found in the central nervous system, constituting 0.2-1.8% of intracranial masses, with the majority located in the cerebellopontine angle. Case Report: A 34-year-old female presented with blurred vision, debilitating headaches, milky breast discharge, and no menstrual periods for three years. Prolactin was mildly high on two occasions at 40.3 and 52.8 ng/mL (3.0-30.0 ng/mL). Estradiol was low at 15 pg/ml in the setting of inappropriately normal FSH (10.1 mIU/mL) and LH (5.8 mIU/mL). ACTH, cortisol, TSH, free thyroxine, and IGF-1 were in the normal range. A pregnancy test was negative. A 1:100 diluted prolactin did not detect the hook effect. MRI revealed a 1.8 x 1.4 x 1.4 cm suprasellar mass centered along the pituitary stalk with peripheral enhancement and central cystic vs. necrotic components. It caused a mass effect on the optic chiasm. Formal visual field testing revealed a left inferior temporal defect. Subsequently, the patient reported increased thirst (consuming 4-5 L of water daily) and urination (confirmed with a 24-hour urine output of 4 L). Following 12 hours of overnight water deprivation, morning labs confirmed diabetes insipidus with high serum sodium of 146 mmol/L, high serum osmolality of 306 mOsm/kg, and low urine osmolality of 204 mOsm/Kg. Initiation of oral DDAVP 0.1 mg nightly resulted in a resolution of nocturia and dramatic improvement in thirst. The patient underwent transsphenoidal resection, during which the neurosurgeon identified a suprasellar cystic lesion with classic epidermoid pearl white material. The lesion and capsule were resected, and patient started on dexamethasone 6 mg IV every 6 hours to prevent chemical meningitis. Pathology was consistent with an epidermoid cyst. Prolactin stained several cells, possibly from compression of the pituitary stalk by the epidermoid cyst. Postoperatively, the patient had severe headaches, and photophobia thought to be associated with chemical meningitis. Lumbar puncture revealed elevated CSF neutrophils and aseptic culture. The patient was continued on dexamethasone with slow taper upon discharge. DDAVP was increased to 0.1 mg twice daily as polyuria and polydipsia worsened. Discussion: There have been six reported cases of epidermoid cysts involving the pituitary stalk. Visual disturbance and headaches are common, while galactorrhea and amenorrhea are uncommon. Preoperative polyuria can be a frequent presenting symptom that can evolve into permanent postoperative diabetes insipidus. Surgical resection is the treatment of choice. It may be difficult to achieve complete excision due to the adherence of the cyst wall to the adjacent structures. The most common complications after resection include chemical meningitis (noted in our patient), hydrocephalus, infectious meningitis, and cranial nerve palsies. Presentation: Friday, June 16, 2023

FRI319 Diagnostic And Management Challenges In A Patient With A Giant Pituitary Lactosomatotroph Adenoma

Abstract Disclosure: C.R. Peixe: None. D. López-Presa: None. M. Bugalho: None. P. Marques: None. Introduction: Giant prolactinomas are rare, accounting for only 2–3% of all prolactinomas. Although benign, several challenges may emerge at diagnosis and during their management. Case: A 38-yr man was incidentally diagnosed with a large skull base tumor on CT-scan undertaken due to head trauma. MRI showed a 55x33mm skull base mass, involving the clivus and sellar/parasellar regions, with bilateral cavernous sinus invasion. A chordoma was suspected leading to a transnasal biopsy, which revealed a PIT1+-plurihormonal pituitary adenoma. His serum prolactin levels were then assessed, which were markedly elevated at 50,414ng/mL (NR:4-15), with concomitant raised IGF-1 at 323ng/mL (NR:101-221). He was then referred to our Endocrine department. Retrospectively, there was a 3-yr history of erectile dysfunction, decreased libido, and infertility, although his testosterone levels were within reference range (307ng/dL; NR:240-830), and sperm analysis parameters were normal. He commenced on low dose cabergoline (0.5mg/week) and two months later clinical improvement was noted, accompanied by a marked reduction of prolactin (830ng/mL) and tumor shrinkage. He remains on cabergoline for ten months, and his latest prolactin and IGF-1 levels were, respectively, 496 and 224ng/mL. In the light of patient’s normal spermogram and restored sexual function, the couple has been referred to the Fertility clinic. Conclusions: The differential diagnosis of large skull base tumors, particularly those affecting sellar/clival regions, must always include prolactinoma, hence serum prolactin should be measured keeping in mind the possible occurrence of hook effect. Most giant prolactinomas respond to dopamine agonists, which should be considered as a first-line therapy, with marked reductions in both prolactin and tumor size often seen within the first weeks of treatment. GH-cosecretion may also ameliorate/normalize with dopamine agonists. In prolactinoma patients, infertility may or may not be directly related with hyperprolactinemia, hence a careful assessment is required, and its management may imply assisted reproductive techniques. Presentation: Friday, June 16, 2023

SAT604 An Extensively Invasive Giant Prolactinoma Associated With CDH23 Mutation

Abstract Disclosure: B. Alghamdi: None. L.A. Alobaid: None. M. Alswailem: None. E.O. Othman: None. M. Dababo: None. O. Alsagheir: None. A.S. Alzahrani: None. Pituitary adenomas (PA), renamed recently pituitary neuroendocrine neoplasms, are common and prolactinomas are the most common subtype of these tumors. The vast majority of PA are sporadic but they can also be due to germline (∼ 5%), mosaic (&amp;lt;1%) or somatic mutations (∼40%). PA with germline mutations might be part of a syndrome such as MEN1 or apparently sporadic, either due to de novo mutations or because of low penetrance of the underlying gene (e.g. AIP). Among the several genes that have been identified, CDH23 was reported to be associated with familial PA in a single study that included 12 families with PA (33% of them had CDH23 variants) and 125 sporadic PA (15 (12%) of them harbored CDH23 variants) compared to only 0.8% of 260 healthy controls. In this report, we describe a middle-aged woman who was incidentally found to have a huge macroprolactinoma with extensive invasion. Genetic testing revealed a novel germline CDH23 variant. Case report: A 53-year-old woman who with hypertension, diabetes and chronic renal insufficiency diagnosed 4 years before. She accidentally fell down on the back of her head. A CT and subsequent MRI scans of the head showed a huge mass extensively involving the Sella, suprasellar space, the cavernous sinuses and middle fossae. This mass extends inferiorly to the sphenoid and parasellar sinuses, nasopharynx, and appears at the nostrils. The patient reported chronic headaches, snoring and congested nose, and worsening hearing and vision over the last 4 years. She denied history of galactorrhea and her periods ceased 8 years before. She never got married and has no children. Her family history is negative for any pituitary or other tumors. The mass was initially thought to be a nasopharyngeal cancer. However, a bedside biopsy from the protruding mass in the right nostril revealed a pituitary tissue strongly positive for prolactin and weakly positive for growth hormone (GH). An undiluted serum prolactin level was 47 ug/l (3.4-24). After several dilutions for the hook effect, the level was 578,000 ug/l (3.4-24). IGF-1 253 ng/ml (93-245), random GH 4.7 ng/ml, TSH 1.9 mU/l (0.4-4.2), FT4 11.4 pmol/l (12-22), AM cortisol 235 nmol/l (159-620), ACTH 27.6 (5-60). Whole exome sequencing followed by Sanger sequencing revealed a novel variant in CDH23 (c.2621C&amp;gt;A, p.Ala874Asp). Several in silico analyses suggest that this variant is pathogenic and it is assigned variant of unknown significance (VUS) according to American College of Medical Genetics (ACMG) classification. It is not reported in EXAC and 1000G databases. All other known PA-associated genes and cancer-associated genes revealed no pathogenic variants. Conclusion: We report a massive prolactinoma, likely due to a novel CDH23 variant. This is the second report on the likely pathogenic role of this gene in PA. In the case reported here, it is associated with a huge and highly invasive pituitary macroadenoma. Presentation: Saturday, June 17, 2023

B-067 Performance Assessment of a Newly Developed Lateral Flow Assay for the Quantification of Faecal Pancreatic Elastase

Abstract Background Pancreatic exocrine insufficiency (PEI) can be defined as an insufficient secretion of pancreatic enzymes and bicarbonate to maintain a normal digestion (1). PEI is associated to fat malabsorption, weight loss and malnutrition-related complications. Due to its important role in the digestive process, as a proteolytic enzyme and the high stability of the enzyme during intestinal transit, fecal pancreatic elastase (fPELA) is an ideal biomarker for the assessment of pancreatic function in patients suffering from PEI. The aim of this work was to develop and optimize a new rapid test for the quantification of fPELA. Methods A quantitative lateral flow assay was designed for the selective measurement of elastase antigen concentrations from stool extracts. The BÜHLMANN Quantum Blue® fPELA is a sandwich immunoassay. A monoclonal detection antibody is conjugated to gold nanoparticles. Elastase present in the sample binds to this gold conjugate. A polyclonal capture antibody, highly specific for elastase, is immobilized on the analytical membrane and captures the complex of elastase bound to the anti-elastase gold conjugate, resulting in a coloring of the test line. The remaining free anti-elastase gold conjugate binds to the control line. The signal intensities of the test line and the control line are measured quantitatively by the Quantum Blue® Reader. Pancreatic elastase is quantified in extracts of human stool samples collected with the BÜHLMANN CALEX® Cap device. Several analytical studies, such as sensitivity, linearity, High Dose Hook Effect (HDHE), were performed during optimization phase to assess the performance of the assay. Moreover, a method comparison with the commercially available high throughput turbidimetric BÜHLMANN fPELA® turbo assay was also performed. Results Both analytical and clinical performance were evaluated. Preliminary results of the assay performance were obtained during the optimization phase. Analytical sensitivity was determined as 12.8 µg/g LoB, 16.6 µg/g LoD and 17.2 µg/g LoQ. The linearity evaluation revealed a linear range of 10 to 472 µg/g. No HDHE was observed in native samples up to 4000 µg/g. Method comparison of the new lateral flow assay with BÜHLMANN fPELA® turbo assay, with 80 clinical samples, revealed good analytical agreement with a slope of 1.04 (Passing-Bablok) and a bias at cutoff (200 µg/g) of 5.6%. Relative bias (Bland-Altman) of the two assays was 6.9%. Considering the turbidimetric assay as the reference method, the sensitivity and specificity were 92% and 83%, respectively. Conclusion The newly developed quantitative lateral flow assay, BÜHLMANN Quantum Blue® fPELA, is an accurate, non-invasive and rapid test to determine fPELA levels in fecal extracts. The lateral flow assay is an attractive alternative to ELISA with a turn-around time of only 15 minutes enabling patients’ management in a more patient near clinical environment.

A-023 Performance Evaluation of the Atellica IM High-Sensitivity Troponin I Assay on the Atellica CI 1900 Analyzer

Abstract Background The Atellica® CI 1900 Analyzer is an automated, high-throughput, integrated chemistry and immunoassay analyzer employing both Atellica CH and Atellica IM assays. This study was designed to evaluate the analytical performance of the Atellica IM High Sensitivity Troponin I (TnIH) assay* on the Atellica CI 1900 Analyzer*. Methods Precision and method comparison were used as performance indicators for the Atellica CI 1900 Analyzer. Precision studies were performed according to CLSI EP05-A3 and method comparison according to CLSI EP09-A3. Precision studies used a panel of four native and one contrived serum/lithium-heparin plasma samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 h apart on each analyzer for &amp;gt;20 days with two reagent lots on two systems. For the method comparison, individual native human lithium-heparin plasma samples were analyzed using the Atellica IM TnIH assay on both the Atellica IM and Atellica CI 1900 Analyzers. Detection capability studies including limit of blank (LoB), limit of detection (LoD), and 20-day functional sensitivity (LoQ) were performed according to CLSI EP17-A2 guidelines. Expected values of normal healthy individuals and hook effect were also verified on the Atellica CI 1900 Analyzer. Results Repeatability and within-lab %CVs ranged from 0.7% to 4.4% and 1.3% to 5.1%, respectively, at TnIH concentrations ranging from 13.73 to 23 197.12 ng/L. Method comparison between the Atellica IM TnIH assay on the Atellica CI 1900 Analyzer vs the Atellica IM Analyzer showed a regression slope of 1.00 and an intercept of 0.04 ng/L (n = 170). In detection capability studies, four lots of the Atellica IM TnIH assay on two Atellica CI 1900 analyzers showed a LoB of 0.35 ng/L and an LoD of 0.76 ng/L. LoQ, defined as the lowest amount of analyte in a sample at which within-lab %CV = 20%, was 1.10 ng/L; %CV was approximately 10% at a TNIH concentration of 2.57 ng/L. In addition, &amp;gt;50% of healthy individuals tested had TnIH values &amp;gt;LoD. The 99th percentile for the Atellica IM TnIH assay (45.20 ng/L) was verified on Atellica CI 1900 Analyzer and was consistent with the Atellica IM Analyzer. No interference from biotin was observed up to a concentration of 3500 ng/mL, and no hook effect was observed up to a concentration of 500 000 ng/L. Conclusion Evaluation of the Atellica IM TnIH assay using the Atellica CI 1900 Analyzer demonstrated reproducible assay precision, sensitive detection capabilities, and equivalent performance compared to the same assay on the Atellica IM Analyzer. This evaluation shows that the Atellica IM TnIH assay on Atellica CI 1900 Analyzer meets the definition of a true high-sensitivity troponin I assay as defined by the International Federation of Clinical Chemistry (IFCC) Task Force on Clinical Applications of Cardiac Biomarkers. *The products/features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed.

A-167 Repeatedly Positive Serum and Urine Beta-HCG Results in Non-Pregnant Patients—Application of Clinical Decision Support

Abstract Background In January of 2020, a 48-year-old woman, status post bilateral tubal ligation was postponed colonoscopy due to a positive urine and serum pregnancy point of care (POC) test. The quantitative serum HCG levels were normal at 4.9 mIU/mL (reference range 0–8 mIU/mL). Follow-up showed that over the prior year there were other 6 similar incidents when nonpregnant patients showed positive qualitative pregnancy results with quantitative HCG levels within “nonpregnancy” normal range being brought to the attention of the laboratory. Methods We assessed the possibility of a detection/measurement error including possible interfering antibodies. The likelihood of interfering substances/antibodies that would interact with 2 different types of POC detection systems (urine qualitative—Clinitest HCG Pregnancy test from Siemens Medical Solutions USA, serum qualitative—Pregnancy HCG Rapid Test from Cardinal Health) and serum quantitative—is very low, especially given that both serum and urine specimens were affected, and interfering antibodies are not normally excreted into urine. Testing of the diluted sample showed results like the neat specimen which further excludes interferences in the quantitative reaction as well as a possible hook effect. Based on this we concluded that the results were accurate.Chart review showed that 6 months prior, she had another quantitative HCG test performed using the same instrumentation obtaining a value of 6.6 mIU/mL. At this time a transvaginal ultrasound excluded the presence of any masses or abnormal findings. FSH (47.8 mIU/ml) and LH (49.8 mIU/ml) were found to be within the perimenopausal range. Qualitative serum and urine test were found to be repeatedly positive, while the quantitative tests were within nonpregnancy range, over several months. Retrospective chart review of the other 6 cases revealed similar pattern. Pregnancy, certain tumors (gestational trophoblastic neoplasia, ovarian germ cell tumor), kidney failure and menopause will manifest with increased HCG production. In (peri)menopausal women the pituitary produces HCG up to 14 mIU/mL in association with increased levels of FSH. The qualitative tests were repeatedly positive over several months while the quantitative results remained unchanged, excluding the diagnosis of pregnancy or tumors, and supporting the conclusion that these results are menopause related. Results To avoid similar future incidents we added the following comment to the POC test results: “If the result does not fit the clinical picture, please repeat testing on a new sample collected 48–72 h later or order serum HCG quantitative” We also added for the quantitative HCG result, the following comment: “Peri- and postmenopausal patients may have increased HCG of pituitary origin up to 14 mIU/mL. If the result does not fit the clinical picture, FSH measurement can be used for additional information.” During the 12 months preceding the implementation of the comments we had 6 incidents of positive qualitative HCG results in non-pregnant (peri)menopausal patients brought for investigation, and none during the 24 months after that. Conclusion The addition of the interpretative comment as clinical decision support increased the efficiency of the medical service, staff engagement and most importantly, patient satisfaction and safety.

B-367 Analytical Validation of New CLIA Based TPS Assay as a Biomarker in Cancer Management

Abstract Background Tissue polypeptide-specific antigen (TPS) has been described as a potentially useful serum marker of tumor activity in adult epithelial tumors such as breast, ovarian, prostate, and other malignancies. Here we developed and analytically validated Chemiluminescence immunoassay (CLIA) for TPS on the CIA series analyzer and FOSUN series analyzer in collaboration with Jiangsu ZECEN Biotech Co., Ltd (Jiangsu and Beijing, China). Methods The TPS-CLIA relies on two monoclonal antibodies generated against peptides that bind to cytokeratin 18 (CK18). The TPS-CLIA assay has been analytically validated and a reference range was established by using sera from blood donors (n = 101), and potential applicability was demonstrated in sera from patients with different malignant diseases (n = 54). The same serum samples were also analyzed with TPS-ELISA for comparison. Results The TPS-CLIA had a limit of the blank of 6 U/L with a limit of detection of 10 U/L and a dynamic range of 10–1200 U/L. The total assay precision was below 20% and no hook effect was observed up to 3265 U/L. Linear dilution of high endogenous TPS sera at 1:4 to 1:256 had shown a recovery in the range of 85%–115%. No significant interference was observed with bilirubin, hemoglobin, and triglycerides (&amp;lt;10%, Table 1). The assay binds to CK18 and no cross-reactivity was observed with CK8 or CK19. The cut-off value (95% CI) based on the blood donor sera was 80 U/L (range = 10–116). There was a significant correlation between TPS-CLIA and TPS-ELISA assay (r = 0.93; P &amp;lt; 0.0001) in different malignancies. More analytical parameters of TPS-CLIA are summarized as shown in Table 1. Conclusion The new TPS-CLIA provides a unique, robust, and convenient method for measuring cytokeratin 18 levels in serum. This can improve the clinical applications of TPS as a biomarker in cancer management.

Effect and safety of Lasso suture hook in transvaginal sacrospinous ligament fixation

To investigate the feasibility and safety of the Lasso suture hook in transvaginal sacrospinous ligament fixation, a total of 38 patients with vaginal vault prolapse at or above stage Ⅱ, and aged 46-75(62.7±12.5)years, who underwent transvaginal sacrospinous ligament fixation at the Second Affiliated Hospital of Soochow University from January 2018 to January 2021 were retrospectively analyzed. After complete exposure of the right sacrospinous ligament, the cervical/uterosacral ligament was sutured to the sacrospinous ligament using Lasso hook and polypropylene sutures. The completion rate of the operation, intraoperative complications, operation time, blood loss, and postoperative situations were observed, and the objective cure rate and subjective satisfaction were followed up. Transvaginal sacrospinous ligament fixation was successfully performed in all 38 patients using Lasso suture hooks. There were no bladder or rectum injuries during the operations, and no pelvic hematoma occurred. The operation time was 15-40 (24±9.5) min; the intraoperative bleeding was 20-60 (40±12.5) ml; the visual analogue scale(VAS)score was 3-5 (3.2±1.4) points on the first day of postoperative, and 2-4 (2.2±1.8) points on the third day of postoperative. No numbness or pain in buttocks and lower limbs after the operation. The 3-month follow-up results showed that the objective surgical success rate of the postoperative pelvic organ prolapse quantitation (POP-Q) score was 100% (38/38). The 1-year follow-up results showed that the objective surgical cure rate of postoperative POP-Q score was 92.1% (35/38). The score of Pelvic Floor Distress Inventory-Short Form 20 (PFDI-20)was 42-180(120.4±44.9)before operation and 8-75 (28.0±14.3) after operation(t=15.90, P<0.001); The score of Pelvic Floor Function Impact Questionnaire-Short Form7 (PFIQ-7) was 52-214(112.8±44.5)before operation and 5-29 (14.3±6.0) after operation (t=14.40, P<0.001), and the subjective satisfaction rate is 89.5% (34/38) conducted by Patient Global Impression of Improvement (PGI-I). Transvaginal sacrospinous ligament fixation with Lasso suture hook is simple, safe, and feasible.

The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device

BackgroundRapid and accurate diagnosis of individuals with SARS-CoV-2 infection is an effective way to prevent and control the spread of COVID-19. Although the detection of SARS‐CoV‐2 viral RNA by RT‐qPCR is the gold standard for COVID-19 testing, the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) is emerging as a complementary surveillance tool as Omicron case numbers skyrocket worldwide. However, the results from Ag-RDTs are less accurate in individuals with low viral loads.ResultsTo develop a highly sensitive and accurate Ag-RDT, 90 monoclonal antibodies were raised from guinea pigs immunized with SARS CoV-2 nucleocapsid protein (CoV-2-NP). By applying a capture antibody recognizing the structural epitope of the N-terminal domain of CoV-2-NP and a detection antibody recognizing the C-terminal tail of CoV-2-NP to an automated chemiluminescence flow-through membrane immunoassay device, we developed a novel Ag-RDT, CoV-2-POCube. The CoV-2-POCube exclusively recognizes CoV-2-NP variants but not the nucleocapsid proteins of other human coronaviruses. The CoV-2-POCube achieved a limit of detection sensitivity of 0.20 ~ 0.66 pg/mL of CoV-2-NPs, demonstrating more than 100 times greater sensitivity than commercially available SARS-CoV-2 Ag-RDTs.ConclusionsCoV-2-POCube has high analytical sensitivity and can detect SARS-CoV-2 variants in 15 min without observing the high-dose hook effect, thus meeting the need for early SARS-CoV-2 diagnosis with lower viral load. CoV-2-POCube is a promising alternative to currently available diagnostic devices for faster clinical decision making in individuals with suspected COVID-19 in resource-limited settings.

BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4+, CD8+, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

Advances in the preclinical and clinical research of proteolysis targeting chimera

Proteolysis targeting chimera (PROTAC) refers to heterobifunctional small molecules that can simultaneously bind an E3 ubiquitin ligase and a target protein, enabling specific degradation of the target protein with the aid of the ubiquitin proteasome system. At present, most PROTAC drugs are in the clinical trial stage, and the ligands are mainly non-covalent compounds. PROTAC drugs have the advantage of overcoming drug resistance and degrading "undruggable" target proteins, but non-covalent ligands could lead to the hook effect that undermines drug efficacy. With its own advantages, covalent ligands can avoid the occurrence of this phenomenon, which is of great help to the development of PROTAC. This review summarizes the progress in preclinical and clinical research and application of PROTAC molecules targeting three different classes of protein targets, including intranuclear, transmembrane, and cytosolic proteins. We also offer perspective discussions to provide research ideas and references for the future development of PROTAC.

Vertical Flow Immunoassay Based on Carbon Black Nanoparticles for the Detection of IgG against SARS-CoV-2 Spike Protein in Human Serum: Proof-of-Concept.

Point-of-care tests play an important role in serological diagnostics of infectious diseases and post-vaccination immunity monitoring, including in COVID-19. Currently, lateral flow tests dominate in this area and show good analytical performance. However, studies to improve the effectiveness of such tests remain important. In comparison with lateral flow tests, vertical flow immunoassays allow for a reduction in assay duration and the influence of the hook effect. Additionally, the use of carbon black nanoparticles (CNPs) as a color label can provide a lower detection limit (LOD) compared to conventional colloidal gold. Therefore, we have developed a vertical flow immunoassay for the detection of IgG against SARS-CoV-2 spike protein in human serum samples by applying a conjugate of CNPs with anti-human IgG mouse monoclonal antibodies (CNP@MAb). The vertical flow assay device consists of a plastic cassette with a hole on its top containing a nitrocellulose membrane coated with spike protein and an absorbent pad. The serum sample, washing buffer, and CNP@MAb flow vertically through the nitrocellulose membrane and absorbent pads, reducing assay time and simplifying the procedure. In positive samples, the interaction of CNP@MAb with anti-spike antibodies leads to the appearance of black spots, which can be visually detected. The developed method allows for rapid visual detection (5-7 min) of IgG vs. spike protein, with a LOD of 7.81 BAU/mL. It has been shown that an untrained operator can perform the assay and visually evaluate its results. Thus, the presented assay can be used in the further development of test systems for the serological diagnostics of COVID-19 or post-vaccination immunity monitoring.