Validation Of Qualitative Methods Research Articles

Method Validation: Evaluation of the Analytical Performance of Standard Hematoxylin-Eosin Staining

Introduction The optimization of diagnostic quality is a major issue in Pathological Anatomy and Cytology (ACP) to secure analytical circuits and protect the operational flow of histological analyses and molecular biology examinations. The objective of this work is to validate the analytical performance of standard hematoxylin-eosin (H.E) staining according to the evaluation criteria of slides in the laboratory.  Materials and Methods Method validation involves numerous steps requiring close collaboration between pathologists and technicians. For our study, the validation covered performances such as inter-technician variability, inter- pathologist variability, and inter-block contamination. The study included 4 paraffin-embedded blocks from 2 surgical specimens, each block being sectioned by 4 different technicians on 4 different microtomes. The 16 obtained sections were stained and read by 2 expert pathologists.  Results Across the 7 evaluation criteria, a preliminary concordance of 98 % was generally found, with no differences impacting the diagnosis.  Conclusion The process of qualitative method validation in ACP is complex due to the multiplicity of stakeholders, the presence of several sub-processes, the difficulty in managing interfaces, and the desire to align with current regulations as a reference site (ISO 20166-4/2021 standard).4. Nevertheless, it is necessary within a quality assurance approach.

A proper and systematic qualitative method validation procedure and its application to gas chromatography-mass spectrometry analysis of Chemical Weapons Convention related chemicals

Performing a detailed qualitative validation, which is carried out by many laboratories in the forensic community, has been the main goal of this study. In this study, a proper and systematic qualitative method validation procedure was proposed, and its application was shown on the analysis of Chemical Weapon Convention (CWC) related compounds in organic samples. All validation steps were described in detail. The study was carried out in pump oil and dichloromethane (DCM). The limit of detection values were determined for each compound and were found in the range of 0.5–2.0 µg mL−1 in pump oil and 0.08–1.5 µg mL−1 in DCM. The validation parameters were calculated, such as the rates of sensitivity, selectivity, false-negative, false-positive, also accordance and concordance. The predicted and obtained results were compared by using Cohen's Kappa Coefficient Test, and the compatibility of the results was found as "very good". After the validation procedure, all of the validation results were evaluated, and the proposed method was confirmed as appropriate for the analysis of CWC-related compounds in organic samples. The applicability of the validated method was proved by determining the CWC-related compounds in organic samples provided by the Organization for the Prohibition of Chemical Weapons during proficiency tests.

Study of \u03949-tetrahydrocannabinol (THC) and cannabidiol (CBD) extraction FROM dried oral fluid spots (DOFS) and LC\u2013MS/MS detection

BackgroundOral fluid is a widely studied matrix able to isolate the primary Cannabis constituent THC, facilitating its detection via mass spectrometry, and in most cases link these findings to recent drug use. As an alternative to liquid oral fluid, dried oral fluid spots (DOFS) is a simple and a low-cost sampling technique. It has shown improved stability compared to liquid samples, allowing for the possibility to preserve the specimens under various temperature and humidity conditions. The sampling strategy is straightforward and involves the application of a small quantity of oral fluid aliquot to a paper substrate that is set to air dry allowing for on-site collection at a large-scale demand. The goal of this study is to study THC and CBD extraction from DOFS, applying a previous established protocol for a LC–MS/MS qualitative method validation. Although other drugs of abuse have been included in DOFS methods, this is the first method validation including cannabinoids. An alternative oral fluid extraction method (WAX-S tips) is demonstrated to improve the recovery of the analytes.MethodsA pool of blank oral fluid was used to prepare THC and CBD spiked DOFS samples for method validation and application. Spiked oral fluid was used to demonstrate WAX-S tips THC and CBD extraction. All samples were analyzed on a LC–MS/MS instrument.ResultsThe qualitative method validation for THC and CBD confirmation in DOFS included method selectivity, matrix effects (< 20%), recovery (average of 25%), process efficiency (average of 21%), LOD (2 ng/mL for THC and 4 ng/mL for CBD), absence of carryover, and DOFS stability (70% in 35 days) as figures of merit. The method application in blindly prepared samples demonstrated the method capability to identify THC and CBD. WAX-S tips extraction showed an average of 91% recovery of THC and CBD from liquid oral fluid.ConclusionsTHC and CBD extraction from DOFS showed low recoveries. However, the LC–MS/MS qualitative confirmation of THC and CBD in DOFS could improve cannabinoids screening in oral fluid, as it shows adequate LOD and stability over time. This method has potential for assisting the screening of drivers under possible drug influence by facilitating sample transportation and temporary storage in dried spot form. Additional research is suggested for WAX-S tips extraction and quantitative method validation.

First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS.

The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.

Validation of qualitative method for the detection of Staphylococcus aureus and Streptococcus agalactiae in milk samples for the diagnosis of bovine mastitis

Validation of qualitative method for the detection of Staphylococcus aureus and Streptococcus agalactiae in milk samples for the diagnosis of bovine mastitis

Qualitative Methods to Advance Care, Diagnosis, and Therapy in Rheumatic Diseases.

This article provides an overview of the basis, usefulness, and validity of qualitative methods in research. It is aimed to enhance the understanding of a broad spectrum of readers, ranging from those mystified by such approaches, to those wanting a better critical knowledge to apply to literature review, and for health care providers considering developing an interest in the field. Qualitative research is crucial in augmentation of disease knowledge as well as the development of incremental care strategies and operational aspects of care that improves health outcomes.

Efficient estimation of the limit of detection and the relative limit of detection along with their reproducibility in the validation of qualitative microbiological methods by means of generalized linear mixed models

The reproducibility of measurement results is a core performance characteristic for quantitative methods. However, in the validation of qualitative methods it is not clear how to characterize a method’s reproducibility. One approach for determining a qualitative method’s reproducibility is presented for microbiological methods, where the distribution of colony forming units (CFU) follows a Poisson distribution. The method’s reproducibility is defined in terms of the variability of the limit of detection (LOD) values. For a better estimation of reproducibility precision, our proposed approach is using an orthogonal factorial plan. Since an exact determination of absolute contamination levels is often not possible, following the ISO 16140-2:2016 [Microbiology of food and animal feed—method validation—part 2: protocol for the validation of alternative (proprietary) methods against a reference method, 2016], an approach is proposed which is based on the ratio of the LOD values of a reference and an alternative method. This approach is illustrated on the basis of an example.

Single-laboratory validation of a method for detection of Roundup Ready soy in soybeans: application of new strategies for qualitative validation

Considering expansion of genetically modified organisms and the basic principle of consumers’ right to access information about products, legislations of several countries establish a limit for labelling transgenic food. Qualitative tests based on the polymerase chain reaction (PCR) have been recommended. However, validation of qualitative methods is still a critical point in the quality management of food analysis laboratories. A screening method for the detection of Roundup Ready (RR) soy in soybeans by nested PCR was validated by a novel qualitative approach, in a single-laboratory, considering all fundamental parameters for an adequate evaluation of fitness for purpose. Blank samples of soybeans and formulations containing 0.001 to 1% of RR soy were analysed. Agarose gel electrophoresis and fluorimetry techniques were compared in terms of the quantification of extracted DNA. False-positive rate obtained was 0%, with selectivity and reliability rates of 100.0% for both techniques. Sensitivity and relia...

Inter-laboratory studies for the validation of two singleplex (tE9 and pea lectin) and one duplex (pat/bar) real-time PCR methods for GMO detection

This paper presents the inter-laboratory studies for the successful validation of qualitative real-time Polymerase Chain Reaction methods for the detection of Genetically Modified Organisms in food and feed. Two singleplex TaqMan methods were developed for the detection of the tE9 terminator present in several genetically modified events and the detection of the endogenous lectin gene from pea; the tE9 terminator donor organism. In addition, a TaqMan duplex method was validated, which allows the simultaneous detection of the pat and bar elements.The paper explains the different acceptance parameters used during the in-house validation, the method transferability study and finally the inter-laboratory validation, how this validation was conducted and the problems encountered. Statistical methods for the validation of the qualitative methods are also presented. This information can be used by laboratories for the verification of these methods during their implementation and for the validation of new methods.

An appropriate and systematized procedure for validating qualitative methods: Its application in the detection of sulfonamide residues in raw milk

The lack of well-established references for the validation of qualitative analyses and the increasing demand for reliable binary responses were the main motivating factors for this study. A detailed procedure for single-laboratory validation of qualitative methods is proposed. The experimental design and the tools for data analysis were based on the theoretical background, as well as the aspects of efficiency, convenience and simplicity. Four experimental steps were defined, as follows: (i) preliminary tests for the determination of the concentration range, (ii) a study of the rates, unreliability region, detection limit, and the accordance and concordance values, (iii) a study of the selectivity in the presence of known interferences, and (iv) a study of robustness. The applicability of the procedure was demonstrated by the validation of a qualitative commercial kit for detecting sulfonamide residues in raw milk using both the visual and instrumental reading techniques. Reliability rates of 100% were obtained for the blank samples. For the samples spiked with sulfamethazine at 10.8 and 108μgL−1 and with sulfadimethoxine or sulfathiazole at 10 and 100μgL−1, the reliability rates ranged from 93.3 to 100%. Selectivity was demonstrated using trimethoprim as a potential interferent. The method was considered robust for the factors of the temperature (54 and 58°C) and time (6 and 10min) for incubating the test strips. The estimated detection limits and unreliability regions confirmed the suitability of the kit for this purpose, based on the legislated residue limits.

Probability of identification: a statistical model for the validation of qualitative botanical identification methods.

A qualitative botanical identification method (BIM) is an analytical procedure that returns a binary result (1 = Identified, 0 = Not Identified). A BIM may be used by a buyer, manufacturer, or regulator to determine whether a botanical material being tested is the same as the target (desired) material, or whether it contains excessive nontarget (undesirable) material. The report describes the development and validation of studies for a BIM based on the proportion of replicates identified, or probability of identification (POI), as the basic observed statistic. The statistical procedures proposed for data analysis follow closely those of the probability of detection, and harmonize the statistical concepts and parameters between quantitative and qualitative method validation. Use of POI statistics also harmonizes statistical concepts for botanical, microbiological, toxin, and other analyte identification methods that produce binary results. The POI statistical model provides a tool for graphical representation of response curves for qualitative methods, reporting of descriptive statistics, and application of performance requirements. Single collaborator and multicollaborative study examples are given.

A protocol for the validation of qualitative methods of detection

This paper presents a draft protocol for analyzing the results of validation studies for qualitative methods of detection which is designed to meet three competing goals: (1) to give correct answers, (2) have a broad scope of application, and (3) be accessible to a wide range of users. The draft protocol can be applied to the validation of methods by collaborative trial or to single-laboratory studies. The protocol produces an estimate of the probability of a positive response with a prediction interval within which 95% of laboratories (or analytical runs) are expected to fall when the method is applied in practice. The interval is calculated from the observed reproducibility (or within-laboratory reproducibility) standard deviation. Then a simple plot of prediction intervals for the probability of detection against the concentration of analyte, where the concentration is known, is used to provide an estimate of the range of limits of detection and false positive probability that we can expect to see when the validated method is applied in practice. The use of the draft protocol is demonstrated using results produced by three collaborative trials. A simulation study showed that a conclusion that a method is fit for purpose that is generated by the draft protocol is likely to be safe.

Validity of method for analysing critical traceability points

The requirements for documenting food products are even increasing. Better documentation can be achieved by using traceability. Several studies mapping Critical Traceability Points (CTP) in food supply chains have been carried out.The purpose of this paper was to discuss the validity of qualitative methods for detecting CTPs in a seafood supply chain. Data from a single case study is interesting, because it provides real industry data, which can be used to develop knowledge of and theories on traceability to improve food process systems. The discussion of validity can in addition be used as input for studies aiming to identify the CTPs of other food supply chains.

Probability of Detection (POD) as a Statistical Model for the Validation of Qualitative Methods

A statistical model is presented for use in validation of qualitative methods. This model, termed Probability of Detection (POD), harmonizes the statistical concepts and parameters between quantitative and qualitative method validation. POD characterizes method response with respect to concentration as a continuous variable. The POD model provides a tool for graphical representation of response curves for qualitative methods. In addition, the model allows comparisons between candidate and reference methods, and provides calculations of repeatability, reproducibility, and laboratory effects from collaborative study data. Single laboratory study and collaborative study examples are given.

Identification and confirmation of chemical residues in food by chromatography-mass spectrometry and other techniques

A quantitative answer cannot exist in analysis without a qualitative component to give enough confidence that the result meets the analytical needs (i.e. the result relates to the analyte and not something else). Just as a quantitative method must typically undergo an empirical validation process to demonstrate that it is fit for purpose, qualitative methods should also empirically demonstrate that they are suitable to meet the analytical needs. However, thorough qualitative method validation requires analysis of a great number of samples (possibly more than can be reasonably done), which is generally avoided due to the time and the effort involved. Instead, mass spectrometry (MS) is generally assumed to be the gold standard for qualitative methods, and its results are typically unquestioned. For example, a system was developed by European regulators of veterinary drug residues in food animals (2002/657/EC), in which the number of identification points given in MS analyses depends on the general degree of selectivity of the MS technique used. This well-defined approach gives a definite answer for decision-makers, so it has grown in popularity. However, the identification-points system is not scientific. The reality is that each situation requires information gathering and careful deductive thinking on the part of the analyst to make MS identifications. Rather than devise arbitrary requirements that need to be met by an unthinking analyst, we remind the analytical community that confirmation can be given only if two or more independent analyses are in agreement, preferably using orthogonally selective (independent) chemical mechanisms. In this article, we discuss the proper use of terminology, highlight the identification power of various MS techniques, demonstrate how MS identifications can fail if precautions are not taken, and re-assert the value of basic confirmation practices, qualitative method validation, information checklists, routine quality-control procedures, and blind proficiency-test analyses.

Validation of qualitative methods of analysis that use control samples

Qualitative methods are frequently used for screening. In some applications, the resulting positive samples are subsequently analyzed by a suitable quantitative confirmatory method, so it is important that the qualitative assay provides reliable results. Although some validation procedures have been reported in this area, much work is still required because there are many different qualitative methods with many different characteristics. In this report, we examine the different types of control samples used in qualitative analysis that provide instrumental responses, we review the most important quality parameter in the validation process, we propose a procedure for estimating the selected quality parameters – traceability, the unreliability region, sensitivity and specificity rates, and false positive and negative rates – and we show how their values can be calculated in a case study: an ELISA method used in a clinical context.

Semiqualitative Method for Detection of Pesticide Residues Over Established Limits in Vegetables by Use of GC–μECD and GC–(EI)MS

A new method is presented for the rapid classification of vegetable samples into negatives and non-negatives depending on their pesticide residue content. The method uses intervals developed during qualitative method validation to accurately distinguish between both types of sample, minimizing the possibility of releasing false positive and false negative results. It also enables a large number of samples to be processed simply and reliably. The method, which includes twenty pesticides including pyrethroids, and organophosphorus and organochlorine compounds, has been applied to two different vegetables (peppers and green beans). Recoveries are acceptable in all instances and limits of detection (LOD) are always below regulatory limits. Full-scan GC–(EI)MS is used to change the non-negative conclusion into either positive or negative by following typical confirmation procedures found in the literature.

Validation of qualitative analytical methods

This article reviews the state of the art in validating qualitative analytical methods. After introducing the scope of these qualitative methods, their main characteristics and how they differ from quantitative analysis methods, we propose a classification according to the detection system. We discuss the institutions, programs and documents dealing with the validation of qualitative methods, and we present the performance parameters- false positive and negative, sensitivity and specificity rate, cut-off, unreliability region, ruggedness and cross-reactivity. We also briefly describe the various strategies used to validate qualitative analytical methods — Contingency Tables, Bayes’ Theorem, Statistical Hypothesis Tests and Performance Characteristic Curves.

Validation of qualitative chromatographic methods: strategy in antidoping control laboratories

An experimental approach for the validation of chromatographic qualitative methods and its application in an antidoping control laboratory is described. The proposed strategy for validation of qualitative methods consists of the verification of selectivity/specificity, limit of detection (LOD), extraction recovery and repeatability (intra-assay precision). A one-day assay protocol, based on the analysis of five blank samples obtained from different sources and four replicates of control samples at two different concentrations of the analytes, has been defined to evaluate the validation parameters. The following evaluation criteria have been applied: absence of interfering substances at the retention time of the analytes in the blank samples to check the selectivity/specificity of the method, the LOD recommended by international sports authorities has to be attained, and for repeatability, the relative standard deviation should be <25% for the low concentration control sample and <15% for the high concentration control sample. Qualitative screening procedures are able to detect a great number of analytes so that extraction and analysis conditions are always a compromise for the different analytes. For this reason, no minimum acceptance criteria have been defined for data of extraction recoveries. The proposed protocol has been used for the validation of the screening and confirmation qualitative methods included in the scope of the accreditation of an antidoping control laboratory according to ISO quality standards.