Integrated On-Slide Positive Controls for Immunocytochemistry on Cytology Slides.

INTRODUCTION: Integrated on-slide positive controls are a standard quality assurance and quality control measure for immunohistochemistry on formalin-fixed paraffin-embedded tissue sections. They ensure identical analytical conditions for the control and patient samples. Our aim was to develop a procedure for preparing integrated on-slide positive controls for immunocytochemistry (ICC) on methanol-fixed cytospins. METHODS: Leftover diagnostic cytology samples with sufficient cells and confirmed expression of Calretinin, MOC31, TTF1, and hormone receptors were used as control samples. Cells from the control samples were deposited on the peripheral part of objective slides using standard cytocentrifuge equipment. Cytospins were immediately fixed in methanol for at least 30 min and then covered with polyethylene glycol (PEG). Completely dry and solid PEG was removed from the central part of the objective slides and stored at room temperature. Patient samples were subsequently added to the central part of a PEG-protected slide, with an appropriate positive control placed on the peripheral part, and then fixed in methanol. ICC was performed on the Ventana/Roche automated platform ULTRA, using optimized and validated protocols for TTF1, hormone receptors, and double immunostaining for Calretinin/MOC31. The quality of ICC reactions for both deposits on the same slide and potential cell carryover was evaluated retrospectively. RESULTS: In the period from October 2021 to December 2023, the majority of integrated positive controls (364/368, 99%) consistently exhibited unequivocally positive reactions for TTF-1 (n = 93), hormone receptors (n = 84), and double staining for Calretinin/MOC31 (n = 191), with easily interpretable ICC reactions on corresponding patient samples. No obvious carryover of cells from the control sample to the patient sample was observed during this period. CONCLUSION: A novel approach developed for preparing integrated on-slide positive controls for ICC on methanol-fixed cytospins using standard cytocentrifugation is low-cost and can be widely applied in diagnostic cytology laboratories. Simultaneous ICC procedures for the control and patient samples on the same slide ensure identical analytical conditions for both samples, providing the highest level of quality control while reducing costs. Interpreting both ICC reactions on the same slide is time-efficient and convenient.

Quantifying variation across 16S rRNA gene sequencing runs in human microbiome studies.

Recent microbiome research has incorporated a higher number of samples through more participants in a study, longitudinal studies, and metanalysis between studies. Physical limitations in a sequencing machine can result in samples spread across sequencing runs. Here we present the results of sequencing nearly 1000 16S rRNA gene sequences in fecal (stabilized and swab) and oral (swab) samples from multiple human microbiome studies and positive controls that were conducted with identical standard operating procedures. Sequencing was performed in the same center across 18 different runs. The simplified mock community showed limitations in accuracy, while precision (e.g., technical variation) was robust for the mock community and actual human positive control samples. Technical variation was the lowest for stabilized fecal samples, followed by fecal swab samples, and then oral swab samples. The order of technical variation stability was inverse of DNA concentrations (e.g., highest in stabilized fecal samples), highlighting the importance of DNA concentration in reproducibility and urging caution when analyzing low biomass samples. Coefficients of variation at the genus level also followed the same trend for lower variation with higher DNA concentrations. Technical variation across both sample types and the two human sampling locations was significantly less than the observed biological variation. Overall, this research providing comparisons between technical and biological variation, highlights the importance of using positive controls, and provides semi-quantified data to better understand variation introduced by sequencing runs. KEY POINTS: • Mock community and positive control accuracy were lower than precision. • Samples with lower DNA concentration had increased technical variation across sequencing runs. • Biological variation was significantly higher than technical variation due to sequencing runs.

Determination of five positive control drugs in hERG external solution (buffer) by LC-MS/MS to support in vitro hERG assay as recommended by ICH S7B.

ICH S7B recommends screening for hERG channel block using patch clamp recordings to assess a drug's proarrhythmic risk. Block of the hERG channel has been associated with clinical QTC prolongation as well as the rare, but potentially fatal ventricular tachyarrhythmia Torsade de Pointes (TdP). During recording, drug concentrations perfused to the cells can deviate from nominal concentrations due to molecule-specific properties (such as non-specific binding), thereby introducing error when assessing drug potency. To account for this potential source of error, both the original ICH S7B and the newly released ICH E14/S7B Q&As guidelines call for verifying drug solutions' concentrations. Dofetilide, cisapride, terfenadine, sotalol and E-4031 are hERG blockers commonly used as positive controls to illustrate hERG assay sensitivity. The first four compounds are also clinical drugs associated with high TdP risk; therefore, their safety margins may be useful comparators to better understand an investigational product's TdP risk. Having analytical methods to quantify these five compounds in the hERG external solution that will be used for patch clamp recordings is important from a regulatory science research perspective. However, a literature search revealed no analytical methods or stability information for these molecules in the high salt, serum-free matrix that constitutes the hERG external solution. This study was conducted to develop and validate LC-MS/MS methods to quantify these 5 molecules in hERG external solution. The bioanalytical methods for these positive controls were validated as per the FDA's bioanalytical method validation guidance along with various stabilities.

Assessing the effectiveness of empirical calibration under different bias scenarios.

BACKGROUND: Estimations of causal effects from observational data are subject to various sources of bias. One method for adjusting for the residual biases in the estimation of treatment effects is through the use of negative control outcomes, which are outcomes not believed to be affected by the treatment of interest. The empirical calibration procedure is a technique that uses negative control outcomes to calibrate p-values. An extension of this technique calibrates the coverage of the 95% confidence interval of a treatment effect estimate by using negative control outcomes as well as positive control outcomes, which are outcomes for which the treatment of interest has known effects. Although empirical calibration has been used in several large observational studies, there is no systematic examination of its effect under different bias scenarios. METHODS: The effect of empirical calibration of confidence intervals was analyzed using simulated datasets with known treatment effects. The simulations consisted of binary treatment and binary outcome, with biases resulting from unmeasured confounder, model misspecification, measurement error, and lack of positivity. The performance of the empirical calibration was evaluated by determining the change in the coverage of the confidence interval and the bias in the treatment effect estimate. RESULTS: Empirical calibration increased coverage of the 95% confidence interval of the treatment effect estimate under most bias scenarios but was inconsistent in adjusting the bias in the treatment effect estimate. Empirical calibration of confidence intervals was most effective when adjusting for the unmeasured confounding bias. Suitable negative controls had a large impact on the adjustment made by empirical calibration, but small improvements in the coverage of the outcome of interest were also observable when using unsuitable negative controls. CONCLUSIONS: This work adds evidence to the efficacy of empirical calibration of the confidence intervals in observational studies. Calibration of confidence intervals is most effective where there are biases due to unmeasured confounding. Further research is needed on the selection of suitable negative controls.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Toward Standards in Clinical Microbiota Studies: Comparison of Three DNA Extraction Methods and Two Bioinformatic Pipelines.

When studying the microbiome using next-generation sequencing, the DNA extraction method, sequencing procedures, and bioinformatic processing are crucial to obtain reliable data. Method choice has been demonstrated to strongly affect the final biological interpretation. We assessed the performance of three DNA extraction methods and two bioinformatic pipelines for bacterial microbiota profiling through 16S rRNA gene amplicon sequencing, using positive and negative controls for DNA extraction and sequencing and eight different types of high- or low-biomass samples. Performance was evaluated based on quality control passing, DNA yield, richness, diversity, and compositional profiles. All DNA extraction methods retrieved the theoretical relative bacterial abundance with a maximum 3-fold change, although differences were seen between methods, and library preparation and sequencing induced little variation. Bioinformatic pipelines showed different results for observed richness, but diversity and compositional profiles were comparable. DNA extraction methods were successful for feces and oral swabs, and variation induced by DNA extraction methods was lower than intersubject (biological) variation. For low-biomass samples, a mixture of genera present in negative controls and sample-specific genera, possibly representing biological signal, were observed. We conclude that the tested bioinformatic pipelines perform equally, with pipeline-specific advantages and disadvantages. Two out of three extraction methods performed equally well, while one method was less accurate regarding retrieval of compositional profiles. Lastly, we again demonstrate the importance of including negative controls when analyzing low-bacterial-biomass samples.IMPORTANCE Method choice throughout the workflow of a microbiome study, from sample collection to DNA extraction and sequencing procedures, can greatly affect results. This study evaluated three different DNA extraction methods and two bioinformatic pipelines by including positive and negative controls and various biological specimens. By identifying an optimal combination of DNA extraction method and bioinformatic pipeline use, we hope to contribute to increased methodological consistency in microbiota studies. Our methods were applied not only to commonly studied samples for microbiota analysis, e.g., feces, but also to more rarely studied, low-biomass samples. Microbiota composition profiles of low-biomass samples (e.g., urine and tumor biopsy specimens) were not always distinguishable from negative controls, or showed partial overlap, confirming the importance of including negative controls in microbiota studies, especially when low bacterial biomass is expected.

Container Closure Integrity Testing-Method Development for Freeze-Dried Products Using Laser-Based Headspace Oxygen Analysis.

The regulatory expectations introduced by the U.S. Food and Drug Administration in 1999, ICH Q5C, the revised draft of Annex 1, Eudralex volume 4 for consultation, and the recent update of USP general chapters 1207.1 and 1207.2 regarding container closure integrity (CCI) testing have created a need for further development of testing methods, although there are no universally accepted methods to test and evaluate the CCI of a biopharmaceutical drug product. Each testing method and principle has merits and demerits.This paper will present a simple approach to be used in method validation of CCI testing based on headspace oxygen analysis for freeze-dried biopharmaceutical drug products in vials, as well as a method for testing and verifying positive control vials. The model is based on Fick's law of diffusion and is empirically corrected to account for Knudsen diffusion in smaller defect sizes. The model considers storage conditions of the vials, allowing for testing of vials stored under anaerobic and near-sealing pressure conditions and different temperature conditions, as well as testing at different time points. Using this approach based on headspace oxygen analysis, the minimum timeframe for leak detection is dependent on the volume of the object tested, but standard vial sizes have shown that leak sizes of 0.2 µm can be detected within hours of vial stoppering with the model developed. CCI testing by headspace oxygen analysis using this approach can be done quickly and non-destructively. This method can prove its effectiveness in demonstration of a protective critical barrier (CCI) for a biopharmaceutical drug product during real-time situations of manufacturing, transportation, registered storage conditions, etc. (life-cycle approach).LAY ABSTRACT: The recent update of the guideline, USP general chapters 1207.1 and 1207.2, regarding container closure integrity (CCI) testing, has created a need for further development of testing methods. The new methods applied should be deterministic, i.e., describe predefined detection limits and objective quantitative data. Deterministic leak test methods are methods for which the leakage event being detected or measured is based on phenomena that follow a predictable chain of events.At present, there is no universally accepted method to test and evaluate the CCI of a biopharmaceutical drug product.This paper will present a simple model-based method based on headspace oxygen analysis for freeze-dried biopharmaceutical drug products in vials, as well as a method for testing and verifying positive control vials. Positive controls are vials with a known defect, e.g., an inserted micropipette.The model considers storage conditions of the vials, allowing for testing of vials stored under different pressure and temperature conditions, as well as testing at different time points. CCI testing by headspace oxygen analysis using the new approach can be done quickly and without destruction of the vials. This method can prove its effectiveness in demonstration of a protective critical barrier (CCI) for a biopharmaceutical drug product during real situations of manufacturing, transportation, registered storage conditions, etc.

Stabilization of RDT target antigens present in dried Plasmodium falciparum-infected samples for validating malaria rapid diagnostic tests at the point of care.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) are a great achievement in implementation of parasite based diagnosis as recommended by World Health Organization. A major drawback of RDTs is lack of positive controls to validate different batches/lots at the point of care. Dried Plasmodium falciparum-infected samples with the RDT target antigens have been suggested as possible positive control but their utility in resource limited settings is hampered by rapid loss of activity over time. METHODS: This study evaluated the effectiveness of chemical additives to improve long term storage stability of RDT target antigens (HRP2, pLDH and aldolase) in dried P. falciparum-infected samples using parasitized whole blood and culture samples. Samples were treated with ten selected chemical additives mainly sucrose, trehalose, LDH stabilizer and their combinations. After baseline activity was established, the samples were air dried in bio-safety cabinet and stored at room temperatures (~ 25 °C). Testing of the stabilized samples using SD Bioline, BinaxNOW, CareStart, and First Response was done at intervals for 53 weeks. RESULTS: Stability of HRP2 at ambient temperature was reported at 21-24 weeks while that of PAN antigens (pLDH and aldolase) was 2-18 weeks of storage at all parasite densities. The ten chemical additives increased the percentage stability of HRP2 and PAN antigens. Sucrose alone and its combinations with Alsever's solution or biostab significantly increased stability of HRP2 by 56% at 2000 p/µL (p < 0.001). Trehalose and its combinations with biostab, sucrose or glycerol significantly increased stability of HRP2 by 57% (p < 0.001). Unlike sucrose, the stability of the HRP2 was significantly retained by trehalose at lower concentrations (500, and 200 p/µL). Trehalose in combination biostab stabilizer increased the percentage stability of PAN antigens by 42, and 32% at 2000 and 500 p/µL respectively (p < 0.01). This was also the chemical combination with the shortest reconstitution time (~ < 20 min). CONCLUSIONS: These findings confirm that stabilizing RDT target antigens in dried P. falciparum-infected samples using chemical additives provides field-stable positive controls for malaria RDTs.

Method establishment for discerned immunogenicity assessment of a recombinant glycoprotein containing nonhuman sialic acid Neu5Gc residues.

AIM: Recombinant glycoprotein produced in nonhuman mammalian cell lines can be modified with the immunogenic nonhuman sialic N-glycolylneuraminic acid (Neu5Gc). We describe here a validated method for detection of antidrug antibodies against both protein and Neu5Gc-containing glycan epitopes. RESULTS: An electrochemiluminescent method was established with drug conjugates as capture and detection reagents. Rabbit antidrug polyclonal antibodies were used as the positive control for protein moiety-specific antibodies, while chicken anti-Neu5Gc polyclonal antibodies were used as the positive control for antibodies against Neu5Gc glycan epitope. Specificity to Neu5Gc was verified by signal inhibition with bovine γ-globulin that contains Neu5Gc. CONCLUSION: The assay illustrated here discerns the immunogenicity of the protein backbone and the sialic acid Neu5Gc glycan moiety of a recombinant protein containing Neu5Gc.

Quality control in diagnostic immunohistochemistry: integrated on-slide positive controls.

Standardization in immunohistochemistry is a priority in modern pathology and requires strict quality control. Cost containment has also become fundamental and auditing of all procedures must take into account both these principles. Positive controls must be routinely performed so that their positivity guarantees the appropriateness of the immunohistochemical procedure. The aim of this study is to develop a low cost (utilizing a punch biopsy-PB-tool) procedure to construct positive controls which can be integrated in the patient's tissue slide. Sixteen frequently used control blocks were selected and multiple cylindrical samples were obtained using a 5-mm diameter punch biopsy tool, separately re-embedding them in single blocks. For each diagnostic immunoreaction requiring a positive control, an integrated PB-control section (cut from the appropriate PB-control block) was added to the top right corner of the diagnostic slide before immunostaining. This integrated control technique permitted a saving of 4.75% in total direct lab costs and proved to be technically feasible and reliable. Our proposal is easy to perform and within the reach of all pathology labs, requires easily available tools, its application costs is less than using external paired controls and ensures that a specific control for each slide is always available.

Proton NMR metabolic profiling of CSF reveals distinct differentiation of meningitis from negative controls.

BACKGROUND: Cerebrospinal fluid (CSF) is an essential bio-fluid of the central nervous system (CNS), playing a vital role in the protection of CNS and performing neuronal function regulation. The chemical composition of CSF varies during onset of meningitis, neurodegenerative disorders (positive controls) and in traumatic cases (negative controls). METHODS: The study design was broadly categorized into meningitis cases, negative controls and positive controls. Further differentiation among the three groups was carried out using Principal Component Analysis (PCA) followed by supervised Partial Least Square Discriminant Analysis (PLS-DA). RESULTS: The statistical analysis of meningitis vs. negative controls using PLS-DA model resulted in R2 of 0.97 and Q2 of 0.85. There was elevation in the levels of ketone bodies, total free amino acids, glutamine, creatine, citrate and choline containing compounds (choline and GPC) in meningitis cases. Similarly, meningitis vs. positive controls resulted in R2 of 0.80 and Q2 of 0.60 and showed elevation in the levels of total free amino acids, glutamine, creatine/creatinine and citrate in the meningitis group. Four cases of HIV were identified by PLS-DA model as well as by clinical investigations. CONCLUSION: On the basis of metabolic profile it was found that negative control CSF samples are more appropriate for differentiation of meningitis than positive control CSF samples.

Potential pitfalls associated with testing of enzyme preparations in the Salmonella/microsome assay.

The effect of a sample of food enzyme preparations on S9 activity was evaluated in bacterial mutation assays with the Salmonella typhimurium strains TA98 and TA100 using benzo(a)pyrene, 2-aminoanthracene and 2-aminofluorene as model compounds. Under the experimental conditions applied, Aspergillus oryzae protease and porcine pancreas trypsin, applied at low non-toxic doses, proved to effectively inhibit the metabolic activation of benzo(a)pyrene by Aroclor induced rat liver 9, while the activation of 2-aminoanthracene and 2-aminofluorene was only marginally affected. The tolerance of metabolic activation of 2-aminoanthracene to the presence of proteolytic enzymes, compared to the strong inhibition elicited on the metabolic activation of benzo(a)pyrene, points to the involvement of different components of liver S9 in their biotransformation. Overall, data indicate that the use of 2-aminoanthracene as positive control in the Ames test can give a misleading indication of S9 proficiency, and thus it should be used with caution or in conjunction with other chemicals, especially in the testing of crude enzyme preparations in which proteases may be present as minor components.

Novel cost-effective quality control approach for the Cepheid Xpert CT/NG assay for the detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae.

The Xpert CT/NG is a rapid assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis. QC materials must be formulated to emulate human specimens, and are prohibitively expensive. A creative, cost-effective QC approach is proposed. The acceptable sample types for the Xpert CT/NG assay were extended to include eye swabs.

Botanical insecticide research: many publications, limited useful data.

Our analysis of >20000 papers on botanical insecticides from 1980 to 2012, indicates major growth in the number of papers published annually (61 in 1980 to 1207 in 2012), and their proportion among all papers on insecticides (1.43% in 1980 to 21.38% in 2012). However, only one-third of 197 random articles among the 1086 papers on botanical insecticides published in 2011 included any chemical data or characterization; and only a quarter of them included positive controls. Therefore, a substantial portion of recently published studies has design flaws that limit reproducibility and comparisons with other and/or future studies. In our opinion, much of the scientific literature on this subject is of limited use in the progress toward commercialization or advancement of knowledge, given the resources expended.

In vitro transcribed RNA molecules for the diagnosis of pandemic 2009 influenza A(H1N1) virus by real-time RT-PCR.

The 2009 influenza A(H1N1) outbreak allowed the implementation of new epidemiologic surveillance tools in several countries around the world. A new molecular protocol with appropriate sensitivity and specificity using real-time RT-PCR was developed by the Centers for Disease Control and Prevention (CDC) to identify the pandemic 2009 influenza A (H1N1) virus in human specimens. In the CDC protocol, positive controls are available only upon request and they are taken from cell cultures infected with 2009 influenza A(H1N1) virus, representing a handling risk for laboratory technicians. The poor availability of positive control materials in diagnostic laboratories may limit the public health response. The aim of the work presented in this paper was to develop positive controls for the diagnostic testing of influenza A(H1N1) virus that could be used in the CDC real-time RT-PCR protocol. A series of plasmid constructs bearing partial sequences of the viral genes were created and each construct was used as a template for in vitro transcription. RNA molecules were obtained successfully at high yield, i.e., 2×10(7) assays per microliter. Thus, the inclusion of these molecules in the influenza panel as positive controls is proposed. The in vitro transcribed RNA could also be used as quality standards in the design of international proficiency studies.