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.

Multi-laboratory evaluation of immunoaffinity LC-MS-based glucagon-like peptide-1 assay.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Highlights of the 13th Japan Bioanalysis Forum Symposium.

Approximately 280 people from pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 13th Japan Bioanalysis Forum Symposium. The symposium was held via web to prevent the spread of COVID-19 from the 28 February to 2 March 2022. The theme of the symposium was 'All for One Goal', and the event has provided an opportunity for open discussion among researchers with different backgrounds but who share a common goal: "to deliver more effective and safe pharmaceuticals to patients as quickly as possible". The speakers focused on hot topics in bioanalysis, including chromatography, biomarker analysis, cell and gene therapy, COVID-19 and antidrug antibody. This symposium provided a great opportunity for the participants to have meaningful discussions, even though 'on the web' was a limited space.

Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Age and composition of the thrombus retrieved by mechanical thrombectomy from patients with acute ischemic stroke are associated with revascularization and clinical outcomes.

INTRODUCTION: Understanding the composition of stroke thrombi retrieved by mechanical thrombectomy is essential to clarify the pathogenesis of stroke. However, it is difficult to evaluate thrombus composition precisely and objectively. Immunohistochemical staining was used to evaluate thrombus composition and age. MATERIALS AND METHODS: Consecutive thrombi (n = 108) retrieved from patients who underwent mechanical thrombectomy for acute large-vessel ischemic stroke were retrospectively analyzed. Lytic features of granulocytes and CD163 were estimated as indicators of the age of the cardioembolic (CE) thrombus. RESULTS: The stroke subtypes were as follows: CE, 74 cases; large artery atherosclerosis, 11; undetermined etiology, 12; and other determined etiology, 11. There were no statistical differences in thrombi composition according to stroke subtypes. The fibrin area was positively correlated with the red blood cell (RBC) and platelet areas. The following analysis was performed using CE only. Regarding age, the thrombus was judged as fresh in 30.0 % and older in 70.0 % based on the lytic features. The RBC areas of older thrombi were smaller than those of fresh thrombi. The puncture-to-reperfusion time of older thrombi was longer than that of fresh thrombi. Platelet-rich thrombi were associated with a greater number of maneuvers, a smaller prevalence of TICI 3, and unfavorable functional outcomes compared to platelet-poor thrombi. The number of CD163 positive cells in thrombi with anticoagulants was higher than in those without anticoagulants. CONCLUSION: Thrombus composition correlated with revascularization and clinical outcomes. The composition of an acute ischemic thrombus may reflect the pathophysiology of stroke and influence treatment efficacy.

Highlights of the 12th Japan Bioanalysis Forum Symposium.

Approximately 300 people associated with pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 12th Japan Bioanalysis Forum Symposium. The webinar was conducted from 9 to 11 March 2021. The theme of the symposium was 'for the next generation', and the event provided 'an opportunity for young researchers in bioanalysis (including students)' and 'an opportunity to discuss new frontiers of bioanalysis'. The speakers focused on hot topics of bioanalysis, including biomarker analysis, patient centric sampling, virtual clinical trials, gene therapy, cancer genome medicine and therapeutic middle molecules. The symposium presented a platform for the discussion of the prospects and challenges facing bioanalysts working in the field of pharmacokinetics. This report presents the key issues discussed.

Quantitative application of flow cytometry for the analysis of circulating human T cells: A preclinical pharmacokinetic study.

As the application of flow cytometry to a quantitative pharmacokinetic study with adoptive T cell therapy is new, we aimed to investigate the quantitativity of flow cytometry-based analysis for the pharmacokinetic assessment of circulating human T cells in a preclinical study. We evaluated the selectivity, linearity, accuracy, precision, and sensitivity of flow cytometry-based analysis for human CD8+ T cells in immunodeficient mouse blood. The CD3/8/45-positive cell population was successfully distinguished from the negative population. Linear regression analysis for the calibration curve showed good linearity and recovery was approximately 100%. Acceptable inter- and intra-day precision and accuracy were observed and the lower limit of quantification (30 cells/50 µL) was validated with acceptable precision and accuracy. Blood concentrations of human CD8+ T cells in immunodeficient mice were then evaluated after administration using this method and the time-concentration profile of human T cells in mice was successfully assessed. The present study is the first to clarify the quantitativity of flow cytometry-based analysis for circulating human T cells in animals. The concept of the present study would be applicable to quantitative pharmacokinetics/efficacy or safety analysis of adoptive T cell therapy.

Development of a bioanalytical method for circulating human T cells in animals using Arthrobacter luteus-based quantitative polymerase chain reaction and its application in preclinical biodistribution studies.

INTRODUCTION: In the development of cell therapy products for human use, studies on the biodistribution of transplanted cells in animals are important for assessing the safety and efficacy of these products. Although a few reports have described the biodistribution of human cells in animals using Arthrobacter luteus-based-polymerase chain reaction (Alu-PCR), most have used genomic DNA or synthetic oligonucleotide as calibrators, as opposed to actual cells. In addition, bioanalytical variability in the quantification of cells with respect to specificity, selectivity, accuracy, and precision, has not been evaluated. Accordingly, in this study, we validated the utility of this bioanalytical method for human T cells in mice to establish assay performance using cells as a calibrator. METHODS: A standard curve was constructed for the addition of cell lysates to mouse tissues and blood, and DNA was extracted. Alu-PCR was applied for the quantification of human peripheral blood CD8+ T cells in mice. To determine assay performance, we evaluated accuracy, precision, selectivity, specificity, and stability. In vivo cell kinetics and biodistribution were investigated based on intravenous administration of human T cells to mice. RESULTS: Alu-PCR enabled us to specifically detect human T cells in mouse blood and tissues. The lower detection limit of Alu-PCR was 10 cells/15 mg tissue (7.5 mg for spleen and lung) or cells/50 µL blood. Given that PCR threshold cycle (Cq) values among mouse samples (blood, liver spleen, lung, heart, and kidney) show slight variation, calibration curves should be generated using the same tissue as used for the assay. Most coefficients of variation in the assay were within 30%. The cell kinetics of administered human T cells in mice were successfully evaluated using the established Alu-qPCR. CONCLUSIONS: The Alu-PCR technique developed in this study showed sufficient specificity and sensitivity in detecting human peripheral blood CD8+ T cells in mice. This technique, which targets the primate-specific Alu gene, is applicable for quantifying transplanted human cells in animals without the necessity of cell labeling. The data presented herein will be useful for standardizing bioanalytical approaches in biodistribution studies of cell therapy products.

Anti-sortilin1 Antibody Up-Regulates Progranulin via Sortilin1 Down-Regulation.

Progranulin (PGRN) haploinsufficiency associated with loss-of-function mutations in the granulin gene causes frontotemporal dementia (FTD). This suggests that increasing PGRN levels could have promising therapeutic implications for patients carrying GRN mutations. In this study, we explored the therapeutic potential of sortilin1 (SORT1), a clearance receptor of PGRN, by generating and characterizing monoclonal antibodies against SORT1. Anti-SORT1 monoclonal antibodies were generated by immunizing Sort1 knockout mice with SORT1 protein. The antibodies were classified into 7 epitope bins based on their competitive binding to the SORT1 protein and further defined by epitope bin-dependent characteristics, including SORT1-PGRN blocking, SORT1 down-regulation, and binding to human and mouse SORT1. We identified a positive correlation between PGRN up-regulation and SORT1 down-regulation. Furthermore, we also characterized K1-67 antibody via SORT1 down-regulation and binding to mouse SORT1 in vivo and confirmed that K1-67 significantly up-regulated PGRN levels in plasma and brain interstitial fluid of mice. These data indicate that SORT1 down-regulation is a key mechanism in increasing PGRN levels via anti-SORT1 antibodies and suggest that SORT1 is a potential target to correct PGRN reduction, such as that in patients with FTD caused by GRN mutation.

When and How Should Chinese Pregnant Women Exercise? A Longitudinal Study in China.

This study aimed to examine when and how physical activity (PA) influences gestational weight gain (GWG) and infant birthweight (BW) by considering the PA's total volume, timing, intensity, and type, controlling for the influence of energy intake. A total of 1272 participants in different stages of pregnancy were recruited from hospital. The associations between PA and GWG or BW in the latter half of pregnancy were significant. Women with the highest PA volume in the third trimester had significantly lower risks of inadequate and excessive GWG by 69% (OR = 0.31, 95%CI: 0.10-0.91) and 67% (OR = 0.33, 95% CI: 0.12-0.91), respectively, compared to women in the lowest quartile. Women who achieved the recommended moderate intensity of PA during their second and third trimesters, independent of total volume of PA, had infants with significantly lower BWs compared to those who did not (ß = -0.15, SE = 66.33, p = 0.04; ß = -0.20, SE = 64.54, p = 0.01, respectively). Therefore, the effects of total volume and intensity of PA on GWG and BW were different. Interventions to prevent inappropriate GWG and macrosomia may need to set different priorities and timing regarding total volume or intensity of PA.

Physical activity and dietary intake among Chinese pregnant women: an observational study.

BACKGROUND: Physical activity (PA) and dietary intake are important modifiable factors associated with health outcomes. However, Chinese pregnant women's PA and dietary intake are only vaguely understood. The aim of this study was to reveal the characteristics of PA and dietary intake of Chinese women in different trimesters as well as the associations between PA and dietary intake. METHODS: This is a cross-sectional observational study. PA, dietary intake, and demographics of 1077 Chinese pregnant women were measured. The Chi-square test, Kruskal-Wallis test, multiple logistic regression, and multiple linear regression were used for data analysis. RESULTS: About 57.1% of the participants met the international guideline for PA. Household activity and occupational activity contributed the most to the total PA, while sports/exercise contributed little. The mean energy intake of the participants was 2008 ± 748.0 kcal. Most participants had normal energy intake, but they obtained excessive energy from fat (mean = 41.7 ± 8.7%). PA was not found to be significantly associated with dietary intake. Further, the participants who were unemployed during pregnancy (OR = 0.72, 95% CI: 0.55-0.95; p < 0.05) or had no exercise habits before pregnancy (OR = 0.62, 95% CI: 0.47-0.80; p < 0.01) were less likely to meet the PA guideline. The participants in the third trimester (OR = 1.43, 95% CI: 1.03-1.99; p < 0.05) were more likely to meet the PA guideline compared to those in the first trimester. The older participants (> 30 years) showed higher dietary intake than the younger (< 25 years) participants (p < 0.01). CONCLUSIONS: The total PA of Chinese women during pregnancy mostly consists of household and occupational activities, but little sports/exercise. Starting exercise before pregnancy may help women achieve adequate PA during pregnancy. Moreover, these women consumed an excessive amount of fat and their diet intake varies by age.

The effect of elevated α1-acid glycoprotein on the pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, in rats.

The pharmacokinetics of TAK-272 (SCO-272), an orally active renin inhibitor, was investigated in rats with subcutaneously injected turpentine oil, which was an inflammation animal model. Following intravenous administration of TAK-272 to the turpentine-treated rats, the systemic clearance and volume of distribution decreased with the elevated plasma α1-acid glycoprotein (AGP) levels. The elevated plasma AGP levels were negatively correlated with the plasma unbound fraction of TAK-272 in the rats. Although the AUCs of total TAK-272 in the turpentine-treated rats were higher than those in the control rats after intravenous and oral administration, those of unbound TAK-272, which seem to directly contribute to the pharmacological effect and safety, were nearly equal between the turpentine-treated and control rats in the respective dose routes. TAK-272 has been shown to primarily bind to AGP in the human plasma. These results strongly suggested that the pharmacokinetic of TAK-272 in humans would also be affected by the variation in the plasma AGP levels and should be discussed with not only the total concentrations but also the unbound concentrations in the clinical trial for patients with elevated plasma AGP levels.

A dural metastatic small cell carcinoma of the gallbladder as the first manifestation: a case report.

BACKGROUND: A dural metastasis is one of the essential differential diagnoses of meningioma. In general, carcinomas of the breast and lung in females and prostate in males have been the most commonly reported primary lesions of dural metastases. However, dural metastasis of gallbladder carcinoma is extremely rare. Here, we report a unique case of a dural matter metastasis of gallbladder carcinoma as the first manifestation, which was autopsy-defined as small cell carcinoma. CASE PRESENTATION: A 78-year-old man came to our hospital complaining of left hemianopia. Brain computed tomography (CT) revealed a sizeable parasagittal dural-based extra-axial tumor. However, the findings for meningioma were atypical by magnetic resonance imaging, suggesting a meningioma mimic. A contrast-enhanced CT scan of the abdomen revealed a large gallbladder carcinoma. The patient opted for the best supportive care and died 2 months later. The post-mortem examination revealed small cell carcinoma in gallbladder carcinoma. Moreover, an immunologically similar carcinoma was detected in the dural metastasis. CONCLUSIONS: To the best of our knowledge, this is the first case of a dural metastasis of gallbladder small cell carcinoma. A systemic examination is essential for clinicians when atypical findings of meningioma are observed, suggesting a meningioma mimic. We present this rare case with a review of the literature.

Pathologic Findings of Symptomatic Carotid Artery Stenosis Several Decades after Radiation Therapy: A Case Report.

Improved long-term survival of malignancy has drawn increased attention to late cerebrovascular toxicity after neck radiotherapy. Recently, neck radiotherapy has been found as a significant risk factor of carotid artery stenosis and ischemic stroke; however, long-term adverse effects of radiation in large arteries remain unknown. Here, we described an autopsied case with recurrent ischemic stroke associated with ipsilateral carotid artery stenosis several decades after neck radiation therapy. Pathologically, there were intima-media fibrosis, endothelial cell loss, and decreased expression of thrombomodulin in irradiated carotid artery stenosis. Our findings support the hypothesis that long-term radiation-induced vascular injury in large arteries is morphologically different from atherosclerotic change. Furthermore, endothelial cell injury may promote fibrin thrombus formation through decreased expression of thrombomodulin, which may cause ischemic stroke associated with radiation-induced carotid artery stenosis.

Takotsubo-like Myocardial Dysfunction in a Patient with Botulism.

Botulinum toxin A (BTXA) can disrupt the neuromuscular and autonomic functions. We herein report a case of autonomic system dysfunction that manifested as Takotsubo-like myocardial dysfunction in a patient with botulism. Takotsubo syndrome results in acute cardiac insufficiency, another fatal complication of botulism in addition to respiratory muscle paralysis, particularly in patients with cardiovascular disease.

A case of hemifacial paresis in a patient with Lyme neuroborreliosis treated with antibiotics in whom Borrelia meningitis developed.

A 38-year-old man visited our hospital because of hemifacial paresis that developed 2 months after being bit by a tick. We diagnosed idiopathic peripheral facial palsy and gave the patient oral prednisolone and valacyclovir. Although the symptoms completely resolved in about 2 weeks, there was a risk of Lyme neuroborreliosis. The patient therefore received doxycycline (100 mg twice daily) and amoxicillin (1,000 mg 3 times daily) for 14 days. Two months later, he had symptoms of meningitis such as headache and fever accompanied by lymphocytic cerebrospinal fluid pleocytosis. Viral meningitis was diagnosed and treated with parenteral acyclovir. The symptoms of meningitis improved. Tests for serum IgG antibodies against borrelia were positive. We gave the patient a diagnosis of Lyme neuroborreliosis. The patient received intravenous ceftriaxone and had no relapse. It is a rare for meningitis to develop in a patient with cranial neuropathy who received doxycycline. Lyme neuroborreliosis is a rare disease in Japan. Care should therefore be exercised in the diagnosis of Lyme neuroborreliosis and evaluation of the response to treatment.

Acquired hemophilia A associated with internal border-zone infarction triggered by major hemorrhage of thigh and leg: a case report.

It is unusual to be complicated with cerebral infarction for bleeding disorders. We describe a first case of internal border-zone infarction (I-BZI) complicated with acquired hemophilia A. A 79-year-old man was introduced from other hospital by cerebral infarction and severe anemia. His left thigh and leg were swollen with subcutaneous bleeding. Activated partial thromboplastin time was 99.4 seconds. Factor VIII activity was less than 1% and Factor VIII inhibitor concentration was 85 BU, respectively. The platelet count and prothrombin time were normal. These results were consistent with the diagnosis of acquired hemophilia A. Magnetic resonance imaging of the brain showed multiple small infarction in bilateral internal border zone. To our knowledge, this is the first case of I-BZI complicated with acquired hemophilia A. This case suggested that major hemorrhage is one of the sole causes of I-BZI.

[A case of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in which lomerizine hydrochloride was suggested to prevent recurrent stroke].

A 60-year-old man visited our hospital because of left hemiparesis in September 2006. Magnetic resonance imaging (MRI) revealed a high-intensity lesions in the right corona radiata on diffusion-weighted images and a high-intensity lesions in the basal ganglia and deep white matter on T2-weighted images. He recovered with no sequelae. Antithrombotic agents such as aspirin were given to prevent stroke, but stroke recurred three times over the course of 3 years. In February 2009, neurological examination revealed right hemiparalysis and dysarthria. Dysphagia and cognitive decline had been progressing gradually. We suspected cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) on the basis of the clinical and family history. An Arg75Pro mutation in the Notch3 gene was found, but did not involve a cysteine residue. Antithrombotic agents were ineffective. We tried lomerizine hydrochloride, which was reported to prevent stroke in a patient with CADASIL. In Japan, lomerizine hydrochloride is used to prevent migraine and to selectively inhibit cerebral artery contraction. During treatment with lomerizine hydrochloride (5 mg/day) for more than 3 years, there was no recurrence of cerebral infarction and no further deterioration of cognitive function or MRI findings. There is no evidence supporting the efficacy of antithrombotic agents in CADASIL patients. Moreover, antithrombotic agents have been reported to increase the frequency of clinically silent microbleeds on MRI in CADASIL. Lomerizine hydrochloride might therefore be one option for the treatment of CADASIL.