A method for measuring the experimental resolution of laboratory assays (clinical biochemical, blood count, immunological, and qPCR) to evaluate analytical performance.

BACKGROUND: The measurement method for experimental resolution and related data to evaluate analytical performance is poorly explored in clinical research. We established a method to measure the experimental resolution of clinical tests, including biochemical tests, automatic hematology analyzer methods, immunoassays, chemical experiments, and qPCR, to evaluate their analytical performance. METHODS: Serially diluted samples in equal proportions were measured, and correlation analysis was performed between the relative concentration and the measured value. Results were accepted for p ≤ 0.01 of the correlation coefficient. The minimum concentration gradient (eg, 10%) was defined as the experimental resolution. For this method, the smaller the value, the higher the experimental resolution and the better the analytical performance. RESULTS: The experimental resolution of the most common biochemical indices reached 10%, with some even reaching 1%. The results of most counting experiments showed experimental resolution up to 10%, whereas the experimental resolution of the classical chemical assays reached 1%. Unexpectedly, the experimental resolution of more sensitive assays, such as immunoassays was only 25% when using the manual method and 10% for qPCR. CONCLUSION: This study established a method for measuring the experimental resolution of laboratory assays and provides a new index for evaluating the reliability of methods in clinical laboratories.

Evaluation of INSTAND e.V.'s external proficiency testing program for tetanus and diphtheria antitoxin detection: Lessons for assessing levels of immunoprotection.

OBJECTIVES: The aim of this study was to evaluate the development and status quo of the quality of high throughput in vitro diagnostic testing for tetanus and diphtheria antitoxin antibody (ATX) concentrations based on external quality assessment (EQA) data. METHODS: We analyzed manufacturer-specific data of 22 EQA surveys-each for the detection of tetanus and diphtheria ATX-to check the diagnostic strength of the corresponding in vitro diagnostic systems. RESULTS: While the results were mostly well aligned, individual surveys showed widely dispersed ATX concentrations. The medians of manufacturer collectives deviated from the overall median by up to 8.9-fold in the case of diphtheria ATX and by up to 3.5-fold in the case of tetanus ATX. Such a distribution in the results is particularly critical in the cut-off range for immunity and may lead to an incorrect assessment of vaccination status. CONCLUSION: These results were surprising as there are International Standards for both ATX; however, the results may be linked to the high ATX concentration of the reference material, which deviates considerably from clinically significant concentrations. To increase the accuracy and diagnostic strength of both assays, we recommend a recalibration of the test systems and verification of their traceability to the International Standards.

Food antigen-specific IgE in dogs with suspected food hypersensitivity.

OBJECTIVE: Knowledge of cross-reactions in food-sensitive dogs will influence the choice of elimination diets and the long-term management of those patients. The objective of this study was to evaluate food allergen-specific IgE tests of suspected allergic dogs for concurrent positive reactions as possible evidence for cross reactions between allergens. MATERIAL AND METHODS: Results of serum IgE tests from 760 suspected allergic dogs submitted to 2 laboratories were evaluated statistically. After the tested allergens were grouped by their phylogenetic relationship, odds ratios as well as a sensitivity analysis of the odds ratios were performed to evaluate if concurrent positive IgE results to 2 allergens occurred more often than expected. RESULTS: Within related allergen pairs 27% (laboratory 1) and 72% (laboratory 2) of the pairs could be considered as associated. For the unrelated allergen pairs only 6.8% and 10.6% of the analyzed pairs were considered associated respectively. Strong correlations were shown in the group of ruminant allergens, especially beef and lamb, and grain allergens. High rates of concurrent reactions were also detected in the poultry group, especially between chicken and duck, as well as between pork and ruminant allergens, and soy and grain allergens. CONCLUSION: As our results showed not only correlations within related but also between non-related allergens, the possible relevance of carbohydrate moieties as well as panallergens for canine hypersensitivities warrants further study. Further investigations are necessary to distinguish co-sensitization from cross-reactions and determine the clinical relevance of food-specific IgE reactivity. CLINICAL RELEVANCE: Due to possible cross reactivity related allergens, especially beef and lamb as well as grain allergens, should not be used for an elimination diet to avoid false results.

[Autoimmunity accreditation: training, accreditation and maintenance of skills in indirect immunofluorescence]. / Accréditation en auto-immunité : formation, habilitation et maintien des compétences en immunofluorescence indirecte.

The ISO 15189 accreditation of biological analysis needs the validation of the analytical methods allowing the evaluation of their performance including all the factors that could influence the quality of their results. The field of autoimmunity includes many analyses and methods such as the indirect immunofluorescence technique (IIF) and the performance of this technique largely depends on the competency of staff members. For each staff member, the required levels of competency have to be precisely defined and evaluated after a period of formation before the final habilitation for the IIF technique. The French group of the international group called EASI (European autoimmunity standardisation initiative) proposes two habilitation forms to be filled with criteria, evidence and maintenance of target skills for the IIF preparation of slides and reading. These forms could be used as a model for the IIF formation and habilitation and have to be adapted to the routine practice of the laboratories.

CMV-specific T-cells and CD27-CD28-CD4+ T-cells for assignment of cytomegalovirus (CMV) status in adults awaiting organ transplant.

BACKGROUND/OBJECTIVES: Determination of Cytomegalovirus (CMV) status in solid organ transplant (SOT) candidates is essential to stratify risk of post-transplant CMV disease. Passive transfusion-acquired antibodies can make serologic determination of CMV status unreliable. We evaluated 3 assays, not affected by passive antibodies (PA), in assignment of CMV status: quantification of CMV-specific CD4 + T-cells (CMV-TC) and exhausted CD27-CD28- CD4 + T-cells, and detection of CMV DNA with Nucleic Acid Amplification Testing (NAAT). STUDY DESIGN: We enrolled 50 adults awaiting SOT and 50 immunocompetent age-matched controls, and collected a throat swab, urine, saliva and blood sample on each. Using flow cytometry CD4 + T-cells were phenotypically analyzed for expression of CD27 and CD28 and CMV-specific CD4 + T-cells were identified by CD69 expression and intracellular IFN-γ quantification after stimulation with CMV-antigen lysate. CMV NAAT was performed on all specimens using real-time PCR. CMV serology (CMV IgG) was determined by enzyme immunoassay. Subjects were considered to have potential PA if they received blood products within 2 months of collection. RESULTS: The CMV-TC assay discriminated between CMV-seropositive and seronegative SOT candidates without PA well (sensitivity 79%, specificity 93%) while the CD27-CD28-CD4 + T-cell assay had good sensitivity (86%) but specificity of 74%. Detection of CMV DNA was uncommon in CMV-seropositive SOT candidates (2/21). CONCLUSIONS: Given its high specificity, the CMV-TC assay is valuable in confirming true-positive CMV status in seropositive SOT candidates with PA, while use of CD27-CD28-CD4 + T-cell analysis is limited by moderate specificity. Detection of CMV DNA is of limited value in assignment of CMV status in adults.

Major Histocompatibility Complex Binding, Eluted Ligands, and Immunogenicity: Benchmark Testing and Predictions.

Antidrug antibody (ADA) responses impact drug safety, potency, and efficacy. It is generally assumed that ADA responses are associated with human leukocyte antigen (HLA) class II-restricted CD4+ T-cell reactivity. Although this review does not address ADA responses per se, the analysis presented here is relevant to the topic, because measuring or predicting CD4+ T-cell reactivity is a common strategy to address ADA and immunogenicity concerns. Because human CD4+ T-cell reactivity relies on the recognition of peptides bound to HLA class II, prediction, or measurement of the capacity of different peptides to bind or be natural ligands of HLA class II is used as a predictor of CD4+ T-cell reactivity and ADA development. Thus, three different interconnected variables are commonly utilized in predicting T-cell reactivity: major histocompatibility complex (MHC) binding, capacity to be generated as natural HLA ligands, and T-cell immunogenicity. To provide the scientific community with guidance in the relative merit of different approaches, it is necessary to clearly define what outcomes are being considered. Thus, the accuracy of HLA binding predictions varies as a function of what the outcome predicted is, whether it is binding itself, natural processing, or T-cell immunogenicity. Furthermore, it is necessary that the accuracy of prediction is based on rigorous benchmarking, grounded by fair, objective, transparent, and experimental criteria. In this review, we provide our perspective on how different variables and methodologies predict each of the various outcomes and point out knowledge gaps and areas to be addressed by further experimental work.

Overview of antibody-mediated immunity to S. pneumoniae: pneumococcal infections, pneumococcal immunity assessment, and recommendations for IG product evaluation.

Streptococcus pneumoniae (S. pneumoniae) strains colonize the nasopharynx and can cause mucosal infections in the upper airway and middle ear, pneumonias, and invasive infections like bacteremia, sepsis, and meningitis. Over 90 serotypes, defined by the structure of their capsular polysaccharides, are known. Twenty-three of these serotypes cause most infections and several of these serotypes can develop antibiotic resistance. Susceptibility factors that increase the susceptibility to S. pneumoniae mucosal and invasive infections include all forms of primary and secondary antibody deficiencies. Many patients affected by one of these deficiencies benefit from the regular administration of human gamma globulin (IgG) preparations. Donors of plasma units used to prepare human IgG have varying concentrations of IgG antibodies against relevant S. pneumoniae serotypes. These antibodies are developed in response to colonization and common subclinical infections and by routine vaccination with S. pneumoniae polysaccharide vaccines. The presence of an adequate concentration of these protective antibodies against all prevalent serotypes needs to be determined to assure the effectiveness of human IgG. All presently available methods to assess IgG antibodies against S. pneumoniae capsular polysaccharides have advantages and pitfalls that are analyzed in this review. In vitro testing does not provide a complete or necessarily accurate measurement of the effectiveness of antibodies in vivo. For regulatory purposes, caution needs to be used in the interpretation of currently available assays that measure pneumococcal antibody levels. Monitoring S. pneumoniae infections in patients treated with IgG and tracing information about IgG lots used to treat these patients should be encouraged.

Methodological Guidelines for Accurate Detection of Viruses in Wild Plant Species.

Ecological understanding of disease risk, emergence, and dynamics and of the efficacy of control strategies relies heavily on efficient tools for microorganism identification and characterization. Misdetection, such as the misclassification of infected hosts as healthy, can strongly bias estimates of disease prevalence and lead to inaccurate conclusions. In natural plant ecosystems, interest in assessing microbial dynamics is increasing exponentially, but guidelines for detection of microorganisms in wild plants remain limited, particularly so for plant viruses. To address this gap, we explored issues and solutions associated with virus detection by serological and molecular methods in noncrop plant species as applied to the globally important Barley yellow dwarf virus PAV (Luteoviridae), which infects wild native plants as well as crops. With enzyme-linked immunosorbent assays (ELISA), we demonstrate how virus detection in a perennial wild plant species may be much greater in stems than in leaves, although leaves are most commonly sampled, and may also vary among tillers within an individual, thereby highlighting the importance of designing effective sampling strategies. With reverse transcription-PCR (RT-PCR), we demonstrate how inhibitors in tissues of perennial wild hosts can suppress virus detection but can be overcome with methods and products that improve isolation and amplification of nucleic acids. These examples demonstrate the paramount importance of testing and validating survey designs and virus detection methods for noncrop plant communities to ensure accurate ecological surveys and reliable assumptions about virus dynamics in wild hosts.

The evolution of food immune reactivity testing: why immunoglobulin G or immunoglobulin A antibody for food may not be reproducible from one lab to another.

The gold standard for identifying food reactions is the elimination-provocation diet. Embarking on this long, tedious journey takes an expert practitioner and a completely dedicated patient, with a whole lot of patience from both. In the contemporary fast lane, microwave, "give me a pill" popping, I-want-satisfaction-now society, many clinicians have turned to laboratory assessments for quick answers to food reactivity. From the introduction of cytotoxic testing for food allergies in 1947 until today, food reactivity testing has evolved and branched; it has been both pseudoimproved and scientifically improved. With multiple available options for methodology, specimen types, and clinical lab, how is a clinician expected to find the one that fits the requirements of a particular practice? How, indeed, when one self-promoting paper supports a particular methodology, while another criticizes it? In this article, with the benefit of his years of training and experience as a research scientist and test development expert, the author, who is trained in both microbiology and immunology, discusses the history of food testing, analyzes the criticisms of it, reviews the scientific literature, and tours the methodologies.

Review on the association of Group B Streptococcus capsular antibody and protection against invasive disease in infants.

A trivalent Group B streptococcus (GBS) polysaccharide-protein conjugate vaccine for vaccination of pregnant women is under development to protect their newborns against invasive GBS disease. Establishing sero-correlates of protection against invasive GBS disease in infants could expedite the licensure pathway of polysaccharide-protein conjugate vaccine. A systematic review of studies reporting on the association of capsular antibodies and invasive GBS disease in infants and colonization in women or newborns was undertaken. Most studies that described maternal and/or infant capsular antibody levels in infants with invasive GBS disease identified an association between low capsular antibody levels in invasive GBS cases compared to controls. Different assay methods and the lack of standardized reference ranges for serotype-specific antibody levels makes it difficult to select an antibody level that may be used as a reliable sero-correlate of protection. Further studies using standardized methods are warranted.

Uncertainty of measurement: an immunology laboratory perspective.

'Measurement uncertainty of measured quantity values' (ISO15189) requires that the laboratory shall determine the measurement uncertainty for procedures used to report measured quantity values on patients' samples. Where we have numeric data measurement uncertainty can be expressed as the standard deviation or as the co-efficient of variation. However, in immunology many of the assays are reported either as semi-quantitative (i.e. an antibody titre) or qualitative (positive or negative) results. In the latter context, measuring uncertainty is considerably more difficult. There are, however, strategies which can allow us to minimise uncertainty. A number of parameters can contribute to making measurements uncertain. These include bias, precision, standard uncertainty (expressed as standard deviation or coefficient of variation), sensitivity, specificity, repeatability, reproducibility and verification. Closely linked to these are traceability and standardisation. In this article we explore the challenges presented to immunology with regard to measurement uncertainty. Many of these challenges apply equally to other disciplines working with qualitative or semi-quantitative data.

Serological diagnosis of canine visceral leishmaniasis in Brazil: systematic review and meta-analysis.

OBJECTIVE: To evaluate the quality and accuracy of serological diagnosis of canine visceral leishmaniasis in the Americas. METHODS: A systematic review found original studies in the databases MEDLINE, EMBASE and LILACS up to November 2012 and in complementary sources up to February 2013. Studies were evaluated in accordance with QUADAS 2 and STARD parameters and recommended in accordance with GRADE parameters. Meta-analysis was carried out with Meta-DiSc software, using the random-effect model. RESULTS: Two hundred and eighty-four studies were identified, of which 25 met the inclusion criteria, comprising the final synthesis. All but one was conducted in Brazil, and only two were judged to be of good quality. 15 studies involving immuno-enzymatic tests with crude antigens (cELISA), 11 studies on indirect immunofluorescence tests (IFAT) and three on the immunochromatographic dual-path platform (DPP) test were meta-analysed. The combined results for sensitivity and specificity were cELISA: 0.89 (CI 95% 0.87-0.91) and 0.87 (CI 95% 0.86-0.88); IFAT: 0.88 (CI 95% 0.85-0.91) and 0.63 (CI 95% 0.61-0.65); and DPP: 0.83 (CI 95% 0.78-0.88) and 0.73 (CI 95% 0.70-0.75). CONCLUSION: Enzyme-linked immunosorbent assay with crude antigens and DPP tests have moderate accuracy for the diagnosis of canine visceral leishmaniasis, and the quality of the design, implementation and analysis of validation studies on diagnostic tests for this disease urgently require improvement. The recommendation for use of the evaluated tests is based on evidence that is scarce and restricted to Brazil.

[Expert consensus post-marketing evaluation scheme to detect immunotoxicity of Chinese medicine in clinical populations (draft version for comments)].

Through consensus, establish a post-marketing scheme and the technical processes to evaluate Chinese medicine's immunotoxicity on a population, as well as its beneficial influences on the immune system. Provide regulations on the collection, storage and transportation of serum samples. This article applies to the post-marketing scientific evaluation of the immunotoxicity of parenterally administered, and for other ways of taking Chinese medicine.

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay.

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturer's suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearson's correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989-0.99, while within laboratory it was 0.998-0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991-0.997, while within laboratory it was 0.997-0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.