Bifunctional molecular probe targeting tumor PD-L1 enhances anti-tumor efficacy by promoting ferroptosis in lung cancer mouse model.

PURPOSE: Immune checkpoint inhibitors (ICIs) targeting tumor-specific PD-1/PD-L1 significantly improve the overall survival rate of patients with advanced cancer by reactivating the immune system to attack cancer cells. To explore their tumor killing effect, we used the radionuclide iodine-131 (131I) to label the anti-PD-L1 antibody Atezolizumab (131I-PD-L1 mAb). METHOD: We prepared the radioimmunoassay molecular probe 131I-PD-L1 mAb by the chloramine-T method and evaluated its affinity using Lewis lung cancer (LLC) cells. The uptake of 131I-PD-L1 mAb by transplanted tumors was examined through SPECT and its in vivo distribution. We then compared the in vitro and in vivo anti-tumor efficacy of groups treated with control, PD-L1 mAb, 131I-PD-L1 mAb, and 131I-PD-L1 mAb + PD-L1 mAb combined treatment. We performed H&E staining to examine the changes in tumor, as well as the damage in major tissues and organs caused by potential side effects. The anti-tumor mechanism of 131I-PD-L1 mAb was analyzed by Western blot, RT-qPCR and immunohistochemistry (IHC). RESULT: 131I-PD-L1 mAb was highly stable and specific, and easily penetrated into tumor. 131I-PD-L1 mAb suppressed cancer cell proliferation in vitro, and inhibited tumor growth in vivo by inducing ferroptosis, thus prolonging the survival of experimental animals while demonstrating biological safety. CONCLUSION: Therefore, our study suggested that 131I-PD-L1 mAb affected the expression of tumor-related factors through ß-rays and thus promoted ferroptosis in tumor. Combined treatment showed better anti-tumor effect compared to single ICI treatment.

Investigating the cut-off values of captopril challenge test for primary aldosteronism using the novel chemiluminescent enzyme immunoassay method: a retrospective cohort study.

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Adrenal venous sampling criteria for chemiluminescent enzyme immunoassay as a preferable alternative to radioimmunoassay in primary aldosteronism.

Plasma aldosterone concentration (PAC) was routinely measured using radioimmunoassay (RIA); however, the RIA kit was discontinued in March 2021 in Japan. This study examined PAC conversion in adrenal venous sampling (AVS) and AVS criteria when measured using chemiluminescent enzyme immunoassay (CLEIA). PAC of 415 adrenal venous blood samples from AVS (including segmental AVS) of 63 patients with primary aldosteronism was measured using RIA (Spac-S aldosterone kit; Fujirebio Inc.) and CLEIA (Lumipulse Presto Aldosterone; Fujirebio Inc.). PAC of 70 AVS samples was also measured using liquid chromatography-mass spectrometry (LC-MS/MS, ASKA Pharma Medical Co., Ltd.). PAC conversion formulas were determined for each AVS sample assay. PAC measured using CLEIA was significantly correlated with that measured using RIA (correlation coefficient = 0.971). The PAC conversion formula was PAC (CLEIA) = PAC (RIA) × 0.772 - 1,199 pg/mL. The PAC of 14,000 pg/mL in RIA was equivalent to 9,613 pg/mL in CLEIA. PAC measured using CLEIA was also correlated with that measured using LC-MS/MS, and the PAC conversion formula was PAC (CLEIA, pg/mL) = 0.97 × PAC (LC-MS/MS, pg/mL) + 211. The inter-assay coefficient of variability (CV) was 1.1-1.3% and intra-assay CV was 1.0-1.7%, measured using CLEIA. The PAC conversion formula for AVS samples was obtained using CLEIA and RIA, and the conversion formula was different from that for peripheral blood. PAC values measured by CLEIA showed preferable accuracy and high concordance with those measured by LC-MS/MS, even in AVS samples. The study outcomes are useful for interpreting AVS results using non-RIA measurement methods.

An independent comparison of the Novolytix salivary melatonin radioimmunoassay with the new Novolytix salivary melatonin enzyme-linked immunosorbent assay.

The dim light melatonin onset (DLMO) is the current gold standard biomarker of the timing of the central circadian clock in humans and is often assessed from saliva samples. To date, only one commercially available salivary melatonin assay is considered accurate at the low daytime levels required to accurately detect the DLMO (Novolytix RIA RK-DSM2). The aim of this study was to conduct the first independent evaluation of a newly improved enzyme-linked immunosorbent assay (ELISA; Novolytix MLTN-96) and compare it with the recommended radioimmunoassay (RIA)-both in terms of melatonin concentrations and derived DLMOs. Twenty participants (15 females, 18-59 years old) provided saliva samples every 30 min in dim light starting 6 h before their habitual bedtime, yielding a total of 260 saliva samples. Both the RIA and ELISA yielded daytime melatonin concentrations <2 pg/mL, indicating adequate accuracy to detect the DLMO. The melatonin concentrations from the two assays were highly correlated (r = .94, p < .001), although the RIA yielded lower levels of melatonin concentration than the ELISA, on average by 0.70 pg/mL (p = .006). Seventeen DLMOs were calculated from the melatonin profiles and the DLMOs from both assays were not statistically different (p = .36) and were highly correlated (r = .97, p < .001). Two DLMOs derived from the RIA occurred more than 30 min earlier than the DLMO derived from the ELISA. These results indicate that the new Novolytix ELISA is an appropriate assay to use if the Novolytix RIA is not feasible or available.

An optimized radioimmunoassay for quantification of total serum thyroxine (T4) in fetal, neonatal, and pregnant rats.

Identifying xenobiotics that interrupt the thyroid axis has significant public health implications, given that thyroid hormones are required for brain development. As such, some developmental and reproductive toxicology (DART) studies now require or recommend serum total thyroxine (T4) measurements in pregnant, lactating, and developing rats. However, serum T4 concentrations are normally low in the fetus and pup which makes quantification difficult. These challenges can be circumvented by technologies like mass spectrometry, but these approaches are expensive and not always widely available. To demonstrate the feasibility of measuring T4 using a commercially available assay, we examine technical replicates of rat serum samples measured both by liquid chromatography mass spectrometry (LC/MS/MS) and radioimmunoassay (RIA). These samples were obtained from rats on gestational day 20 (dams and fetuses) or postnatal day 5 (pups), following maternal exposure to the goitrogen propylthiouracil (0-3 ppm) to incrementally decrease T4. We show that with assay modification, it is possible to measure serum T4 using low sample volumes (25-50 µL) by an RIA, including in the GD20 fetus exposed to propylthiouracil. This proof-of-concept study demonstrates the technical feasibility of measuring serum T4 in DART studies.

Thyroglobulin immunoassay with a fully automated pretreatment process provides accurate thyroglobulin values in anti-thyroglobulin antibody positive specimens.

OBJECTIVES: Human thyroglobulin (Tg) is widely used as a tumor marker for recurrence and metastasis of differentiated thyroid cancer (DTC). Currently, serum Tg values are measured using second-generation sandwich immunoassays (2nd-IMA). However, interference by endogenous autoantibodies to thyroglobulin (TgAbs) can lead to false-negative results or falsely low Tg values. Here, we describe a new Tg assay using the immunoassay for total antigen including complex via pretreatment (iTACT) method to prevent TgAb interference and compare it with 2nd-IMA. METHODS: Tg values were evaluated by three assays: iTACT Tg, Elecsys Tg-II, which is a 2nd-IMA, and LC-MS/MS (Liquid chromatography tandem-mass spectrometry). The ratio of Tg values between each assay was then compared to the Tg value by LC-MS/MS and TgAb titer. Tg immunoreactivity was analyzed by size-exclusion chromatography. RESULTS: Correlation between iTACT Tg and LC-MS/MS using TgAb-positive specimens was good: Passing-Bablok regression with iTACT Tg = 1.084 × LC-MS/MS + 0.831. Correlation between 2nd-IMA and LC-MS/MS showed a relatively lower slope: 2nd-IMA = 0.747 × LC-MS/MS - 0.518. Thus, Tg values determined by iTACT Tg are equivalent to those of LC-MS/MS regardless of TgAb titer, whereas 2nd-IMA gave lower Tg values due to TgAb interference. Tg-TgAb complexes of various molecular weights were verified by size-exclusion chromatography. Tg values measured by 2nd-IMA fluctuated depending on the molecular weight of the Tg-TgAb complexes, whereas iTACT Tg accurately quantified Tg values regardless of the size of the Tg-TgAb complexes. CONCLUSION: Tg values in TgAb-positive specimens were accurately determined by iTACT Tg. TgAb-positive specimens contain Tg-TgAb complexes of various molecular weights that interfere with Tg value determination by 2nd-IMA, whereas iTACT Tg is unaffected by the presence of Tg-TgAb complexes.

Evaluation of a rapid immunoassay for bacteriuria in dogs.

BACKGROUND: The ability to detect bacteriuria in dogs with a point-of-care test might improve medical care and antimicrobial stewardship. HYPOTHESIS AND OBJECTIVE: A rapid immunoassay (RIA; RapidBac) will provide acceptable sensitivity and specificity for diagnosis of bacteriuria. ANIMALS: Forty-four client-owned dogs with a clinical indication for urinalysis and aerobic bacterial urine culture. METHODS: Prospective study. Urine, collected by cystocentesis, was submitted for urinalysis and culture at a diagnostic laboratory. Owners completed an enrollment questionnaire regarding their dogs' clinical signs. The RIA was performed according to the manufacturer's guidelines. Results were compared to culture. RESULTS: Forty-four urine specimens were evaluated from 44 dogs. The sensitivity and specificity of the RIA test to detect bacteriuria compared to urine culture were 81.8% (95% CI, 65.7%-97.9%) and 95.5% (95% CI, 86.8%-99.9%), respectively. For cultures yielding ≥103  CFU/mL, sensitivity increased to 90.0% (95% CI, 76.9%-100%) and specificity was similar at 95.2% (95% CI, 86.1%-99.9%). Malodorous urine, bacteriuria, and pyuria were more likely to be present in dogs with positive RIA or urine culture results compared to dogs with negative results. CONCLUSIONS AND CLINICAL IMPORTANCE: The RIA was easy to perform and had good sensitivity and excellent specificity in this group of dogs. The RIA might be a useful screening test for decision-making regarding antimicrobial therapy in dogs with a clinical indication for urine culture. Consideration could be given to amending the International Society for Companion Animal Infectious Disease definition of bacterial cystitis as the presence of signs of lower urinary tract disease together with positive culture or a positive RIA.

Assessment of the Androstenedione Assay on a Roche Analytical Platform and Comparison with Different Methods.

BACKGROUND: D4-androstenedione (D4ASD) is an intermediate hormone of androgen biosynthesis by the gonads and the adrenal glands. The interest in D4ASD concentration assessment resides in diagnostics of androgenic hyperproduction pathologies. Currently, many D4ASD quantification methods are available on the market including immunological methods that remain problematic due to the possible cross-reactivity with endogenous or exogenous steroids. METHODS: Recently Roche® launched a new fully automated instrument for the measurement of D4ASD concentration. In this paper, the criteria of analytical performance (repeatability and intermediate precision) of the D4ASD Roche® assay were assessed and compared with 2 different methods including a radioimmunoassay (RIA) as well as a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. RESULTS: Repeatability and intermediate precision of the D4ASD Roche® were acceptable according to the prede-fined RICOS standard (CV ≤ 7.9%) and the assay showed a good correlation with other assays considering the 95% CI obtained for the slope and the y-intercept. CONCLUSIONS: This method demonstrates acceptable criteria of analytical performance with an intermediate imprecision and a trueness within the fixed acceptance limits.

Determination of parathyroid hormone: from radioimmunoassay to LCMS/MS.

Parathyroid hormone (PTH) determination is of paramount importance for the exploration of diseases related with calcium metabolism and for the follow-up of patients suffering from bone and mineral disorders associated with chronic kidney diseases (CKD-MBD). Unfortunately, the biologically active form of PTH, i.e. 1-84 PTH, circulates in the blood stream with many fragments and post-translationally modified forms, which decreases the specificity of immunoassays. The assays used to measure PTH, either from 2nd or 3rd generation, are not standardised, which may lead to interpretation errors and clinical consequences. Reference ranges for PTH have neither been always correctly established and the stability of the peptide is also a matter of concern. Fortunately, these last years, newer techniques using mass spectrometry (either high resolution or triple quadripole) coupled with liquid chromatography have been developed, which will help to standardise the different assays. Indeed, PTH assays standardisation is one of the task of the IFCC Committee for Bone Metabolism. Such standardisation will allow a better consistency in the interpretation of the results and will promote studies aiming at the establishment of correct reference ranges.

Validation and method comparison for a point-of-care lateral flow assay measuring equine whole blood insulin concentrations.

The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from <139 to >695 pmol/L (<20 to >100 µIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 µIU/mL), intermediate (278-417 pmol/L; 40-60 µIU/mL), and high (>417 pmol/L; >60 µIU/mL) concentrations, respectively. For 10 replicates at each level (3 assay lots), inter-assay CVs were 15.9%, 11.0%, and 11.7%, respectively. In the weighted linear regression of 5 measured insulin concentrations against expected concentrations, R2 = 0.98, slope = 1.02, and y-intercept = 14.4 pmol/L (2.08 µIU/mL). The Spearman correlation coefficient (rs) was 0.90 (95% CI: 0.85-0.94) between the WRT and RIA; the WRT = f(RIA) Passing-Bablok regression yielded the fit, y = 1.005x + 24.3 pmol/L (3.50 µIU/mL). The WRT result averaged 10.4% higher than the RIA result, with targeted bias of 25.9, 26.1, and 26.7 pmol/L (3.74, 3.76, and 3.84 µIU/mL) for cutoffs used to diagnose insulin dysregulation of 312, 347, and 451 pmol/L (45, 50, and 65 µIU/mL). Assay clinical sensitivities, specificities, and accuracies determined at the 3 selected clinical cutoffs and using the RIA as gold standard were 87-95%, 92-96%, and 91-95%, respectively (n = 99 samples). Observed total error was 28.4-30.4%. The WRT had acceptable precision, excellent linearity, and good association with the RIA.

Decreased Testosterone Levels Precede a Myocardial Infarction in Both Men and Women.

The potential role of the hormone testosterone in the risk for myocardial infarction is investigated in this study of middle-aged men and women compared with a large random control sample from the general population. Radioimmunoassay was used to measure testosterone levels in hair, approximately 1 month and 3 months before an ST-elevation or non-ST-elevation acute myocardial infarction. Mean testosterone levels were measured for middle-aged men and women (n = 168) with diagnosed myocardial infarction (the acute myocardial infarction [AMI] cases). As controls, n = 3,150 randomly selected subjects from the general population of similar age were measured at 1 time point. No significant difference in testosterone levels in hair was found 3 months before AMI for men and women compared with the controls. However, 1 month before AMI, the testosterone levels were decreasing (p <0.001) for both men (from 2.84 to 2.10 pg/mg) and women (from 1.43 to 1.10 pg/mg), indicating that a decrease in testosterone concentrations precedes a severe cardiac event. Conventional cardiovascular risk factors were tested as confounders but did not alter this tendency. The AMI cases were also compared with a randomly selected second control group from the general population (n = 205), for whom comparable segmental hair analyses were conducted. A tendency of some decreasing testosterone levels, also in the small control group, was only significant for men. This control group was a small sample, and there might be some natural biologic variation in testosterone levels over time. This study indicates that decreased testosterone levels may be among the pathophysiological processes preceding myocardial infarction and merits further investigation.

Comparing the clinical significance and antigen specificity of insulinoma-associated antigen-2 autoantibodies between radioimmunoassay and enzyme-linked immunosorbent assay in Japanese patients with type 1 diabetes.

AIMS/INTRODUCTION: This study aimed to investigate the clinical significance and antigen specificity of autoantibodies to insulinoma-associated antigen-2 (IA-2A) by radioimmunoassay (RIA; IA-2A-RIA) and enzyme-linked immunosorbent assay (ELISA; IA-2A-ELISA) in Japanese patients with type 1 diabetes. MATERIALS AND METHODS: A total of 338 type 1 diabetic patients were enrolled, including 38 fulminant type 1 diabetes, 168 acute-onset type 1 diabetes and 137 slowly-progressive type 1 diabetes (SPIDDM). The concordance, correlation of autoantibody titer, and the relationship between IA-2A and progression to the insulin-deficient state were examined. Also, competitive assay was used to examine the antigen specificity. RESULTS: The prevalence of IA-2A-ELISA was 4-5% lower than that of IA-2A-RIA in both the acute-onset type 1 diabetes and SPIDDM, but the diagnostic sensitivities of both subtypes, when measured in combination with glutamic acid decarboxylase autoantibody, were comparable. The diagnosis of type 1 diabetes using either the RIA or ELISA methods showed substantial agreement with the exponential correlation of autoantibody titers detected by RIA and ELISA. Among the SPIDDM patients, the fasting C-peptide for IA-2A-positive cases by ELISA, but not the RIA method, was significantly lower than in the negative cases (P < 0.05). Furthermore, IA-2A-ELISA proved superior to the RIA method in predicting the progression to insulin deficiency in SPIDDM. Competitive analysis showed that even sera with discrepant results by RIA and ELISA have IA-2-specific autoantibodies. CONCLUSION: These results suggest that IA-2A-ELISA is a reliable marker not only for the diagnosis of type 1 diabetes, but also for the prediction of future insulin dependency; that is, detection of IA-2A-ELISA helps identify a subtype of SPIDDM patients who would likely progress onto insulin-deficient state.

Monitoring physiological and biochemical indices in the systematic training of sprinters / Monitoramento de índices fisiológicos e bioquímicos no treino sistemático de velocistas / Monitoreo de los índices fisiológicos y bioquímicos en el entrenamiento sistemático de velocistas

ABSTRACT Introduction This paper studies physiological and biochemical indicators in the systematic training of sprinters. This paper analyzes the data measured during the athletes' training process and studies the detailed data of their physical functions. Objective This study aimed to find a link between exercise data and biochemical indicator data in sprinter athletes. By analyzing the data from this article, the researchers were able to find the optimal training program for the athletes. Methods High-intensity aerobic training tests were performed with statistical analysis of various physiological and biochemical indicators. Results Hemoglobin data were shown to be highly sensitive to intensity. The researchers found that long-term high-load training in athletes can lead to physical fatigue. This fatigue production is positively correlated with the intensity of the training load. Conclusion There is a strong positive correlation between biochemical and physiological indicators on performance levels in sprinter athletes. Evidence Level II; Therapeutic Studies - Investigating the results.

RESUMO Introdução Este artigo estuda o monitoramento de indicadores fisiológicos e bioquímicos no treino sistemático de velocistas. Este documento analisa os dados medidos durante o processo de treino das atletas e estuda os dados detalhados de suas funções físicas. Objetivo O objetivo deste estudo foi encontrar uma ligação entre os dados de exercício e os dados de indicadores bioquímicos nas atletas velocistas. Ao analisar as informações deste artigo, os pesquisadores conseguiram encontrar um programa de treino ideal para as atletas. Métodos Foram empegadas experiências de treino aeróbico de alta intensidade, com análise estatística de vários indicadores fisiológicos e bioquímicos. Resultados Os dados de hemoglobina mostraram-se altamente sensíveis à intensidade. Os pesquisadores descobriram que o treino a longo prazo de alta carga em atletas pode acarretar numa fadiga física. Essa produção de fadiga está positivamente correlacionada com a intensidade da carga de treino. Conclusão Há uma forte correlação positiva entre indicadores bioquímicos e fisiológicos nos níveis de desempenho em atletas velocistas. Nível de evidência II; Estudos Terapêuticos - Investigação de Resultados.

RESUMEN Introducción Este trabajo estudia el seguimiento de los indicadores fisiológicos y bioquímicos en el entrenamiento sistemático de los velocistas. Este artículo analiza los datos medidos durante el proceso de entrenamiento de los atletas y estudia los datos detallados de sus funciones físicas. Objetivo El objetivo de este estudio fue encontrar una relación entre los datos del ejercicio y los datos de los indicadores bioquímicos en los atletas velocistas. Al analizar las informaciones de este artículo, los investigadores pudieron encontrar un programa de entrenamiento óptimo para los atletas. Métodos Se realizaron pruebas de entrenamiento aeróbico de alta intensidad con análisis estadístico de varios indicadores fisiológicos y bioquímicos. Resultados Los datos de la hemoglobina se mostraron muy sensibles a la intensidad. Los investigadores descubrieron que el entrenamiento de alta carga a largo plazo en los atletas puede conducir a la fatiga física. Esta producción de fatiga está positivamente correlacionada con la intensidad de la carga de entrenamiento. Conclusión Existe una fuerte correlación positiva entre los indicadores bioquímicos y fisiológicos en los niveles de rendimiento de los atletas velocistas. Nivel de evidencia II; Estudios terapéuticos - Investigación de resultados.

Effect of Radioimmunoassay on Accuracy of Thyroid Hormone Detection.

At present, the most important method for the detection of thyroid hormones in hospitals in China is radioimmunoassay. Besides radioimmunoassay, there are blood test and antibody test methods for thyroid hormone detection. However, after long-term clinical investigations, the accuracy of the results of thyroid hormone detection by radioimmunoassay has been affected by many factors. Possible influencing factors include inaccurate thyroid hormone test results due to improper way of blood collection by nurses and improper way of keeping and transporting blood samples by nurses. Therefore, this paper analyzes and discusses the influencing factors of the accuracy of thyroid hormone (T4) detection by radioimmunoassay technology. In this paper, we conducted research using statistical analysis of clinical data, improved separation methods for quality control in the laboratory, and blood specimen collection methods. Radioimmunoassay occurs with antibodies. Of the 10 batches of 964 cases in the improved separation methods for quality control in the laboratory, 154 mismatched items accounted for 16%, and the error of method and operation only accounted for 5.8% of unmatched specimens, most of which were the biochemical characteristics and clinical manifestations of thyroid hormones. The blood sample collection method research found that mild hemolysis had no significant effect on the measurement results, severe hemolysis had a tendency to affect the results, and blood collection tubes had no effect on the test results. Mild hemolysis refers to the increase in the rate of red blood cell destruction due to various internal and external factors in the body. The symptoms when mild hemolysis occurs are generally not obvious. Severe hemolysis refers to a disease caused by blood group incompatibility, mainly referring to immune hemolysis caused by blood group incompatibility between mother and baby, as well as severe jaundice or severe anemia. The statistical analysis of clinical data found that, among 160 patients, the reasons for the inaccuracy of T4 results using radioimmunoassay technology were as follows: 104 patients were inaccurate due to personal factors, and the results were due to hospital factors. A total of 56 patients had inaccurate results. During the measurement of thyroid hormone, it will be affected by many factors. For this reason, the influencing factors of the accuracy of radioimmunoassay should be clarified, and appropriate measures should be taken to deal with it, so as to give full play to the role of radioimmunoassay and improve the detection.

Method Performance Verification of Anti-GAD65 and Anti-Insulin antibody Assays.

BACKGROUND: The aim of this study was to evaluate the performance of chemiluminescence immunoassays for anti-GAD65 and anti-insulin antibodies following user verification guidelines. METHODS: The analytical performance of anti-GAD65 and anti-insulin antibodies using a MAGLUMI 2000 analyzer was verified following user verification guidelines by the Clinical and Laboratory Standards Institute. RESULTS: Performance specifications including precision, linearity, carry-over, cutoffs for positive results, reference intervals, and comparability with pre-existing commercially available radioimmunoassays using patient specimens and certified reference material were verified (coefficients of variation for precision of anti-GAD65 and anti-insulin antibodies were 2.6% and 3.4%, respectively). Comparability assessed using clinical serum specimens showed overall agreement with radioimmunoassay of 87.2% (95% confidence interval 74.8% - 94.0%) for the anti-GAD65 antibody assay and 85.4% (95% confidence interval 71.6% - 93.1%) for the anti-insulin antibody assay. CONCLUSIONS: The results of this study verified the analytical performance of MAGLUMI anti-GAD65 and anti-insulin antibody assays for clinical use.

Variability and quantification of serum medroxyprogesterone acetate levels.

Quantification of serum progestin levels in clinical contraceptive studies is now routinely performed to understand progestin pharmacokinetics and to correct for unreliable self-reporting of contraceptive use by study participants. Many such studies are focussed on the three-monthly progestin-only intramuscular (IM) injectable contraceptive depot medroxyprogesterone acetate (DMPA-IM). Methods commonly used to measure serum MPA levels include liquid chromatography coupled to mass spectrometry (LC/MS) and radioimmunoassay (RIA); however, RIA methods have not been used in recent years. We review the available literature and find that these methods vary widely in terms of use of organic solvent extraction, use of derivitization and choice of organic solvent and chromatography columns. There is a lack of standardization of LC/MS methodology, including a lack of detailed extraction protocols. Limited evidence suggests that RIA, without organic solvent extraction, likely over-estimates progestin levels. Maximum MPA concentrations in the first two weeks post-injection show wide inter-individual and inter-study variation, regardless of quantification method used. Standardization of quantification methods and sampling time post-injection is required to improve interpretation of clinical data, in particular the side effects arising at different times depending on the pharmacokinetic profile unique to injectable contraceptives.

Effect of iodinated radiographic contrast media on radioimmunoassay for measuring thyroid hormones.

Radioimmunoassay (RIA) is one of the most routine laboratory tests for diagnosing thyroid disease. Patients might receive iodine in the form of intravenous iodinated radiographic contrast media (IRCM) before testing of serum thyroxin (T4) or triiodothyronine (T3) concentration by RIA. The objective was to determine the effect of IRCM on T4 and T3 hormone tests in normal, hypothyroid, and hyperthyroid hormone conditions by RIA. IRCMs (0, 2.5, 5 and 10 mgI/mL) used in this study were iopromide and iodixanol. RIA was determined by commercial T4 RIA kit and T3 RIA kits. The method suggested by the manufacturer was followed. Normal, hypothyroid, and hyperthyroid hormones condition were 1.2 ng/mL, 0.2 ng/mL and 2.2 ng/mL for T3 hormone concentration and 70 ng/mL, 30 ng/mL and 140 ng/mL for T4 hormone concentration, respectively. %Bound values were compared between IRCM-incubated groups and non-incubated group. The data showed that iopromide-incubated groups did not statistically significant change %bound values of T3 and T4 hormone tests in normal, hypothyroid, and hyperthyroid conditions, compared to the non-incubated group. In the same way, %bound values of T3 and T4 hormone tests in iodixanol-incubated groups did not change at all conditions when compared to the non-incubated group. This finding suggested that iodinated radiographic contrast media was unlikely to result in significant problems with radioimmunoassay for measuring T3 and T4 thyroid hormones.

Hypocretin-1 measurements in cerebrospinal fluid using radioimmunoassay: within and between assay reliability and limit of quantification.

STUDY OBJECTIVES: The most sensitive and specific investigative method for the diagnosis of narcolepsy type 1 (NT1) is the determination of hypocretin-1 (orexin-A) deficiency (≤110 pg/mL) in cerebrospinal fluid using a radioimmunoassay (RIA). We aimed to assess the reliability of the Phoenix Pharmaceuticals hypocretin-1 RIA, by determining the lower limit of quantification (LLOQ), the variability around the cutoff of 110 pg/mL, and the inter- and intra-assay variability. METHODS: Raw data of 80 consecutive hypocretin-1 RIAs were used to estimate the intra- and inter-assay coefficient of variation (CV). The LLOQ was established and defined as the lowest converted concentration with a CV <25%; the conversion is performed using a harmonization sample which is internationally used to minimize variation between RIAs. RESULTS: The mean intra-assay CV was 4.7%, while the unconverted inter-assay CV was 28.3% (18.5% excluding 2 outliers) and 7.5% when converted to international values. The LLOQ was determined as 27.9 pg/mL. The intra-assay CV of RIAs with lower specific radioactive activity showed a median of 5.6% (n = 41, range 1.6%-17.0%), which was significantly higher than in RIAs with higher specific activity (n = 36; median 3.2%, range 0.4%-11.6%, p = .013). The CV around the 110 pg/mL cutoff was <7%. CONCLUSIONS: Hypocretin-1 RIAs should always be harmonized using standard reference material. The specific activity of an RIA has a significant impact on its reliability, because of the decay of 125I radioactivity. Values around the hypocretin-1 cut-off can reliably be measured. Hypocretin-1 concentrations below 28 pg/mL should be reported as "undetectable" when measured with the Phoenix Pharmaceuticals RIA. CLINICAL TRIAL INFORMATION: This study is not registered in a clinical trial register, as it has a retrospective database design.

Variability in testosterone measurement between radioimmunoassay (RIA), chemiluminescence assay (CLIA) and liquid chromatography-tandem mass spectrometry (MS) among prostate cancer patients on androgen deprivation therapy (ADT).

INTRODUCTION & AIMS: Monitoring testosterone (T) levels is recommended to assess the effectiveness of androgen deprivation therapy (ADT) in advanced prostate cancer. T levels below 20 ng/dL have been associated with better outcomes. Three main measures for T exist including radioimmunoassay (RIA), chemiluminescence assay (CLIA) and mass spectrometry (MS). While CLIA and RIA are ubiquitous, MS is regarded as the reference standard. We set out to determine the discordance of T measurements amongst men on ADT. METHODS: A retrospective review of men with prostate cancer on ADT for ≥3 months was conducted. Serum samples were split in triplicate. Observational data was reported and T measurements were compared analyzing for variability looking for categorical concordance. Over and under-estimation rates were calculated. RESULTS: Ninety-five patients were included with a mean age of 70 (50-92) years. Mean ADT duration was 24.1 (3-144) months. Ninety-five percent of patients had T ≤20ng/dL by MS and CLIA as compared to only 80% by RIA. After subdividing into T categories of ≤20, 20 to 50 and ≥50 ng/dL concordance analysis showed that 4.3% and 18.9% of T measured by MS would have a different category result when remeasured by CLIA (Kappa 0.84) or RIA (Kappa 0.50) respectively. CLIA and RIA overestimated T in 66.7% of patients with T <20 ng/dL measured by MS. Conversely CLIA and RIA underestimated T in only 4.4% of cases with T >20 ng/dL measured by MS. CONCLUSIONS: There is significant variability in T measured with RIA, CLIA and MS. CLIA and RIA overestimated T levels in majority of patients leaving a concern of misdiagnosing truly castrate patients as being inadequately treated.

Quantification of thyroid hormones and analogs by liquid chromatography coupled to mass spectrometry. Preliminary results in athletes and non-athletes serum samples.

This paper aimed to assess a method to measure eight thyroid-related compounds in serum by liquid chromatography-mass spectrometry (LC-MS/MS), to verify the correlation with radioimmunoassay (RIA), to evaluate the possible cross-reactivity, and to observe differences between athletes declaring the consumption of sodium levothyroxine and nonathletes serum samples. Validation was carried out to assess carryover, working range and linearity, limit of detection and limit of quantification, precision, matrix influence, recovery, accuracy, and uncertainty. Comparison between RIA and LC-MS/MS results was done. The assay was applied to serum samples, and comparison with RIA was done for T3 and T4 levels supported by RIA Thyroid-stimulating hormone (TSH) measurements. Validation parameters showed satisfactory results. Correlation between RIA and LC-MS/MS for T3 and T4 showed good results, but a cross-reactivity between T3 and T3AA was observed. Although no significant differences were proved, preliminary comparison between athletes and nonathletes serum samples showed a shift towards high values of TSH and lower for T4 values in the athletes' group. Differences between thyronine and T4AA concentrations and ratios were observed. The trend of T4 values supported by TSH measures might indicate subclinical hypothyroidism in athletes. This represents one of the most controversial thyroid statuses as different criteria about its treatment are described, especially since one of the exogenous causes is inadequate levothyroxine therapy. Because the variation of thyroid hormones and TSH has been extensively studied in high-performance sports, it is worth considering the need to set an adequate reference interval to accurately assess the thyroid status in athletes.