Serum stratifin measurement is useful for evaluating disease severity and outcomes in patients with acute exacerbation of interstitial lung disease: a retrospective study.

BACKGROUND: Serum levels of stratifin (SFN), a member of the 14-3-3 protein family, increase in patients with drug-induced lung injury associated with diffuse alveolar damage. Therefore, we hypothesised that SFN levels would be higher in those experiencing acute exacerbation of interstitial lung disease (AE-ILD). A secondary analysis was also planned to determine whether SFN levels could discriminate survival in those with AE. METHODS: Thirty-two patients with clinically stable ILD (CS-ILD) and 22 patients with AE-ILD were examined to assess whether high serum SFN levels were associated with AE-ILD and whether SFN levels reflected disease severity or prognosis in patients with AE-ILD. RESULTS: Serum SFN levels were higher in the AE-ILD group than in the CS-ILD group (8.4 ± 7.6 vs. 1.3 ± 1.2 ng/mL, p < 0.001). The cut-off value of the serum SFN concentration for predicting 90-day and 1-year survival was 6.6 ng/mL. SFN levels were higher in patients who died within 90 days and 1 year than in patients who survived beyond these time points (13.5 ± 8.7 vs. 5.6 ± 5.3 ng/mL; p = 0.011 and 13.1 ± 7.5 vs. 3.1 ± 1.9 ng/mL; p < 0.001, respectively) in the AE-ILD group. When this cut-off value was used, the 90-day and 1-year survival rates were significantly better in the population below the cut-off value than in those above the cut-off value (p = 0.0017 vs. p < 0.0001). CONCLUSIONS: High serum SFN levels are associated with AE-ILD and can discriminate survival in patients with AE-ILD.

Differential associations between selenoprotein P and distal sensorimotor polyneuropathy in people with and without diabetes: KORA F4/FF4 study.

BACKGROUND: Oxidative stress is a risk factor for distal sensorimotor polyneuropathy (DSPN). Selenoprotein P is a protein with antioxidant properties but has not been investigated in the context of DSPN. This study aimed to assess the associations between selenoprotein P and DSPN in people without and with type 2 diabetes (T2D). METHODS: Cross-sectional and prospective analyses were based on 1053 (including 217 with T2D) and 513 participants (including 79 with T2D), respectively, aged 61-82 years from the population-based KORA F4 survey. DSPN at baseline (KORA F4) and in the follow-up survey KORA FF4 was defined based on the Michigan Neuropathy Screening Instrument. Serum levels of full-length selenoprotein P were quantified by ELISA. Associations between selenoprotein P and prevalent or incident DSPN were estimated using logistic regression analysis adjusting for multiple confounders. RESULTS: Selenoprotein P levels were not associated with prevalent DSPN in the total sample. However, there was a significant interaction by diabetes status. Higher levels of selenoprotein P were associated with lower odds of prevalent DSPN in individuals without T2D (fully adjusted model: OR 0.825 [95 % CI 0.682, 0.998], p = 0.0476), but not in those with T2D (OR [95 % CI] 1.098 [0.829, 1.454], p = 0.5132; pinteraction = 0.0488). Selenoprotein P levels were not associated with incident DSPN over a follow-up of 6.5 years. CONCLUSION: In individuals without T2D from the older general population, lower selenoprotein P levels were associated with a higher prevalence of DSPN. Whether the antioxidant properties of selenoprotein P are responsible for the observed associations remains to be elucidated in future research.

Modeled Hepatic/Plasma Exposures of Omeprazole Prescribed Alone in Cytochrome P450 2C19 Poor Metabolizers Are Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database.

Omeprazole, a gastric acid pump inhibitor, is repeatedly administered and is oxidatively metabolized mainly by polymorphic cytochrome P450 2C19. The prescribed dosage of omeprazole was discontinued or reduced in 47 of the 135 patients who received omeprazole alone in this survey, as recorded in the Japanese Adverse Drug Event Report database. The days to onset of omeprazole-related disorders were 3-4 d (median) and 16 d for intravenous 20-40 mg and oral 20 mg daily doses, respectively, in 34 patients for whom relevant data were available. The maximum plasma concentration of omeprazole was pharmacokinetically modeled after a single oral 40-mg dose in P450 2C19-defective poor metabolizers and was 2.4-fold higher than that in extensive metabolizers. The modeled area under the hepatic concentration curves of omeprazole in P450 2C19 poor metabolizers after virtual daily 40-mg doses for 7 d was 5.2-fold higher than that in the extensive metabolizers. Omeprazole-induced P450 2C19 (approx. 2-fold), resulting in increased hepatic intrinsic clearance in repeated doses, was considered after the second day. Virtual plasma/hepatic exposure estimated using pharmacokinetic modeling in subjects with P450 2C19 poor metabolizers indicated that these exposure levels virtually estimated could be one of causal factors for unexpected hepatic disorders induced by prescribed omeprazole, such as those resulting from drug interactions with repeatedly co-administered medicines.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation.

Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics.

Modeled Hepatic/Plasma Exposures of Fluvastatin Prescribed Alone in Subjects with Impaired Cytochrome P450 2C9*3 as One of Possible Determinant Factors Likely Associated with Hepatic Toxicity Reported in a Japanese Adverse Event Database.

Fluvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor that competitively inhibits human cytochrome P450 (P450) 2C9 in vitro. Drug interactions between a variety of P450 2C9 substrates/inhibitors and fluvastatin can increase the incidence of fluvastatin-related hepatic or skeletal muscle toxicity in vivo. In this survey, the prescribed dosage of fluvastatin was reduced or discontinued in 133 of 164 patients receiving fluvastatin alone, as recorded in the Japanese Adverse Drug Event Report database of spontaneously reported events. The median days to onset of fluvastatin-related disorders were in the range 30-35 d in the 87 patients. Therefore, we aimed to focus on fluvastatin and, using the pharmacokinetic modeling technique, estimated the virtual plasma and hepatic exposures in subjects harboring the impaired CYP2C9*3 allele. The plasma concentrations of fluvastatin modeled after a virtual oral 20-mg dose increased in homozygotes with CYP2C9*3; the area under the plasma concentration curve was 4.9-fold higher than that in Japanese homozygotes for wild-type CYP2C9*1. The modeled hepatic concentrations of fluvastatin in patients with CYP2C9*3/*3 after virtual daily 20-mg doses for 7 d were 31-fold higher than those in subjects with CYP2C9*1/*1. However, heterozygous Chinese patients with CYP2C9*1/*3 reportedly have a limited elevation (1.2-fold) in plasma maximum concentrations. Virtual hepatic/plasma exposures in subjects harboring the impaired CYP2C9*3 allele estimated using pharmacokinetic modeling indicate that such exposure could be a causal factor for hepatic disorders induced by fluvastatin prescribed alone in a manner similar to that for interactions with a variety of co-administered drugs.

Relationship Between the Lipidome Profile and Disease Activity in Patients with Rheumatoid Arthritis.

Lipid mediators have been suggested to play important roles in the pathogenesis of rheumatoid arthritis (RA). Lipidomics has recently allowed for the comprehensive analysis of lipids and has revealed the potential of lipids as biomarkers for the early diagnosis of RA and prediction of therapeutic responses. However, the relationship between disease activity and the lipid profile in RA remains unclear. In the present study, we performed a plasma lipidomic analysis of 278 patients with RA during treatment and examined relationships with disease activity using the Disease Activity Score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR). In all patients, five lipids positively correlated and seven lipids negatively correlated with DAS28-ESR. Stearic acid [FA(18:0)] (r = -0.45) and palmitic acid [FA(16:0)] (r = -0.38) showed strong negative correlations. After adjustments for age, body mass index (BMI), and medications, stearic acid, palmitic acid, bilirubin, and lysophosphatidylcholines negatively correlated with disease activity. Stearic acid inhibited osteoclast differentiation from peripheral blood monocytes in in vitro experiments, suggesting its contribution to RA disease activity by affecting bone metabolism. These results indicate that the lipid profile correlates with the disease activity of RA and also that some lipids may be involved in the pathogenesis of RA.

Interlaboratory evaluation of LC-MS-based biomarker assays.

Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.

Clinical Profiles of Japanese Patients with Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis Collected by a Nationwide System from 2006 to 2023.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are potentially life-threatening severe cutaneous adverse drug reactions. These diseases are rare, and their onset is difficult to predict because of their idiosyncratic reactivity. The Japan Severe Adverse Reactions Research Group, led by the National Institute of Health Sciences, has operated a nationwide to collect clinical information and genomic samples from patients with SJS/TEN since 2006. This study evaluated the associations of clinical symptoms with sequelae and specific causative drugs/drug groups in Japanese patients with SJS/TEN to identify clinical clues for SJS/TEN treatment and prognosis. Acetaminophen, antibiotics, and carbocisteine were linked to high frequencies of severe ocular symptoms and ocular sequelae (p < 0.05). For erythema and erosion areas, antipyretic analgesics had higher rates of skin symptom affecting <10% of the skin than the other drugs, suggesting narrower lesions (p < 0.004). Hepatic dysfunction, was common in both SJS and TEN, and antiepileptic drugs carried higher risks of hepatic dysfunction than the other drug groups (p = 0.0032). This study revealed that the clinical manifestations of SJS/TEN vary according to the causative drugs.

[The Role of Supersulfide in Methylmercury Detoxification].

Methylmercury is a ubiquitous neurotoxic substance present in the environment, and health concerns, especially through the consumption of seafood, remain. Glutathione (GSH)-mediated detoxification and the excretion of methylmercury are known metabolic detoxification pathways. We have also discovered a mechanism by which endogenous super-sulfides convert methylmercury to nontoxic metabolites such as bis-methylmercury sulfide. However, these metabolites are present in very small quantities, and the significance of the detoxification of methylmercury by super-sulfides is not well understood. Methylmercury binds to thiol groups in vivo but can also react with highly reactive selenols (selenocysteine residues). Such covalent bonds (S-mercuration and Se-mercuration) are broken by nucleophilic substitution reactions with other thiol and selenols, however, the contribution of super-sulfides to this substitution reaction is not well understood. Interestingly, a recent study suggested that selenoprotein P, the major selenium transport protein in plasma, binds to methylmercury, however, Se-mercuration was not determined. In this review, we introduce these series of reactions and discuss their involvement with super-sulfides in methylmercury toxicity.

Selenoprotein P expression in glioblastoma as a regulator of ferroptosis sensitivity: preservation of GPX4 via the cycling-selenium storage.

Glioblastoma (GBM) is one of the most aggressive and deadly brain tumors; however, its current therapeutic strategies are limited. Selenoprotein P (SeP; SELENOP, encoded by the SELENOP gene) is a unique selenium-containing protein that exhibits high expression levels in astroglia. SeP is thought to be associated with ferroptosis sensitivity through the induction of glutathione peroxidase 4 (GPX4) via selenium supplementation. In this study, to elucidate the role of SeP in GBM, we analyzed its expression in GBM patients and found that SeP expression levels were significantly higher when compared to healthy subjects. Knock down of SeP in cultured GBM cells resulted in a decrease in GPX1 and GPX4 protein levels. Under the same conditions, cell death caused by RSL3, a ferroptosis inducer, was enhanced, however this enhancement was canceled by supplementation of selenite. These results indicate that SeP expression contributes to preserving GPX and selenium levels in an autocrine/paracrine manner, i.e., SeP regulates a dynamic cycling-selenium storage system in GBM. We also confirmed the role of SeP expression in ferroptosis sensitivity using patient-derived primary GBM cells. These findings indicate that expression of SeP in GBM can be a significant therapeutic target to overcome anticancer drug resistance.

Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. METHODS: Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. RESULTS: Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. CONCLUSIONS: The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Cholesterol is more readily oxidized than phospholipid linoleates in cell membranes to produce cholesterol hydroperoxides.

Cholesterol is an essential component of cell membranes and serves as an important precursor of steroidal hormones and bile acids, but elevated levels of cholesterol and its oxidation products have been accepted as a risk factor for maintenance of health. The free and ester forms of cholesterol and fatty acids are the two major biological lipids. The aim of this hypothesis paper is to address the long-standing dogma that cholesterol is less susceptible to free radical peroxidation than polyunsaturated fatty acids (PUFAs). It has been observed that cholesterol is peroxidized much slower than PUFAs in plasma but that, contrary to expectations from chemical reactivity toward peroxyl radicals, cholesterol appears to be more readily autoxidized than linoleates in cell membranes. The levels of oxidation products of cholesterol and linoleates observed in humans support this notion. It is speculated that this discrepancy is ascribed to the fact that cholesterol and phospholipids bearing PUFAs are localized apart in raft and non-raft domains of cell membranes respectively and that the antioxidant vitamin E distributed predominantly in the non-raft domains cannot suppress the oxidation of cholesterol lying in raft domains which are relatively deficient in antioxidant.

Impact of microsampling on toxicological evaluation in rodent safety studies.

Recently, animal welfare has been attracting worldwide attention, and implementation of 3Rs (replacement, reduction, and refinement) is prioritized in every way possible in the drug development. Microsampling, in which small amounts of blood are collected, is attracting attention in this context. ICH S3A Q&A focused on microsampling was published in November 2017 to help accelerate the application of microsampling for toxicokinetic assessment. The increased sensitivity of drug measurement apparatuses such as mass spectrometers has made it possible to measure drug concentrations with small amounts of blood samples. In this review, we summarized the reports on toxicological influence of microsampling in rodents (rats and mice) with or without drug administration or recovery period after blood collection and influences that may arise from differences in the blood sampling site or blood sampling volume. We also summarized some perspectives on further implementation of microsampling in toxicology studies. The use of microsampling in regulatory toxicology studies has gradually increased, although at a lower rate than in discovery studies. Since more animals are used in GLP toxicology studies than in discovery studies, the effect of reducing the number of animals by microsampling is expected to be greater in the toxicology studies. This report aims to promote the application of microsampling to nonclinical studies, as it is beneficial for improving animal welfare and can contribute to the 3Rs.

Effects of microsampling on toxicity evaluation of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance, in a mouse toxicity study.

ICH S3A Q&A focused on microsampling (MS) was published to help accelerate the use of MS and states that MS is useful because toxicokinetic (TK) evaluation with conventional blood sampling volume requires many animals for TK satellite groups; however, there are few reports of MS application in mice. We investigated the influence of MS on toxicity evaluation in mice by comparing the toxicity parameters with and without MS after a single oral administration of 1-naphthylisothiocyanate (ANIT), a hepatotoxic substance. Blood samples (50 µL/point) were collected from the tail vein of 3 mice per group at 2 or 3 time points during a 24-hr period, and toxicity was evaluated 2 days after administration. ANIT-related changes suggesting liver or gallbladder injury were noted in blood chemistry and histopathology. Some of these changes such as increases in focal hepatocyte necrosis and inflammatory cell infiltration in the liver as well as mucosal epithelium necrosis in the gallbladder were apparently influenced by MS. A tendency to anemia was noted in animals with MS but not without MS, which was also noted in the vehicle-treated controls, suggesting influence of blood loss. The current results indicate that ANIT hepatotoxicity could be evaluated in mice in which blood samples were collected by MS for most parameters; however, parameters in anemia and pathology in the liver and gallbladder were influenced by MS in this study condition with ANIT. Therefore, MS application in mice should be carefully considered.

Sulforaphane decreases serum selenoprotein P levels through enhancement of lysosomal degradation independent of Nrf2.

Selenoprotein P (SeP) is a major selenoprotein in serum predominantly produced in the liver. Excess SeP impairs insulin secretion from the pancreas and insulin sensitivity in skeletal muscle, thus inhibition of SeP could be a therapeutic strategy for type 2 diabetes. In this study, we examine the effect of sulforaphane (SFN), a phytochemical of broccoli sprouts and an Nrf2 activator, on SeP expression in vitro and in vivo. Treatment of HepG2 cells with SFN decreases inter- and intra-cellular SeP levels. SFN enhances lysosomal acidification and expression of V-ATPase, and inhibition of this process cancels the decrease of SeP by SFN. SFN activates Nrf2 in the cells, while Nrf2 siRNA does not affect the decrease of SeP by SFN or lysosomal acidification. These results indicate that SFN decreases SeP by enhancing lysosomal degradation, independent of Nrf2. Injection of SFN to mice results in induction of cathepsin and a decrease of SeP in serum. The findings from this study are expected to contribute to developing SeP inhibitors in the future, thereby contributing to treating and preventing diseases related to increased SeP.

Pharmacokinetic Models Scaled-up from Humanized-liver Mouse Data Can Account for Drug Monitoring Results of Atomoxetine and Its 4-Hydroxylated and N-Demethylated Metabolites i n Pediatric Patients Genotyped for Cytochrome P450 2D6.

Atomoxetine is a cytochrome P450 (P450) 2D6 probe substrate and an approved medicine for attention-deficit/hyperactivity disorder. In this humanized-liver mouse study, interactions between atomoxetine and the P450 2D6 probe drug paroxetine were observed. Human physiologically based pharmacokinetic (PBPK) models were established by scaling up humanized-liver mouse data obtained in the absence or presence of paroxetine. These models could explain the drug monitoring results of atomoxetine and its primary 4-hydroxylated and N-demethylated metabolites in Japanese children aged 8-14 years and could be used to help establish the correct dosage and for the evaluation of clinical outcomes. The results of simple PBPK models (using input parameters that reflected the subjects' small body size and normal or reduced P450 2D6-dependent clearance) were in general agreement with one-point measured plasma concentrations of atomoxetine and its 4-hydroxylated and N-demethylated metabolites in 13 pediatric participants. Unexpectedly high hepatic exposure, possibly in intermediate-metabolizer patients harboring CYP2D6*10 or 2D6*36 alleles, might in part explain the adverse effects of atomoxetine prescribed alone recorded in a Japanese adverse-event database. The steady-state, one-point drug monitoring data from the participants indicated extensive biotransformation of atomoxetine to 4-hydroxyatomoxetine under individually prescribed doses of atomoxetine. These results also suggest that a relatively narrow range of 4-hydroxyatomoxetine and N-desmethylatomoxetine concentration ratios in spot urine and/or plasma samples from pediatric patients could be a simple semiquantitative determinant factor for P450 2D6 intermediate metabolizers, compared with the wide range of concentrations of the two primary metabolites and substrate in extensive metabolizers. Significance Statement Validated simple pharmacokinetic models are able to predict steady-state plasma concentrations of the approved medicine atomoxetine and its primary metabolites in the majority of pediatric patients. The package insert advises careful dose escalation, especially for poor metabolizers; however, no simple way exists to determine P450 2D6 phenotypes. A relatively narrow range ratio of 4-hydroxyatomoxetine and N-desmethylatomoxetine in spot urine/plasma samples could be a simple semi-quantitative determinant factor for P450 2D6 intermediate metabolizers to optimize or confirm the correct dosage.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.