Analytical and clinical performance of cPass neutralizing antibodies assay

Serological tests for SARS-CoV-2 are a critical component of disease control strategies. SARS-CoV-2 serology tests used in clinical diagnostic should not accurately evaluate total levels the antibodies but also closely correlate with neutralizing antibodies titers.However, only limited data is available reporting correlation of neutralization antibody assays with commercial high-throughput serological assays widely used in clinical laboratories.We performed evaluation of the GenScript cPass neutralizing antibody detection assay, to assess its value for routine clinical use to measure neutralizing titers in patients who recovered from coronavirus disease 2019 (COVID-19) or have been vaccinated. We tested its clinical performance against the commonly used Ortho Vitros IgG assay.Our combined data shows that GenScript cPass neutralizing antibody assay has satisfactory analytical and clinical performance and good correlation with Ortho Vitros IgG, supporting its use as a tool for accurate SARS-COV-2 immune surveillance of recovered or vaccinated individuals.     

Improving detection of venous thromboembolism. New technology holds promise for early, precise diagnosis

In recent years, a number of modalities have been evaluated for the diagnosis of venous thromboembolism. The role of these modalities is still evolving. While ventilation-perfusion scanning is important in the diagnosis of venous thromboembolism, spiral CT scanning, MRI, and D-dimer assays are now being used more often, either exclusively or in combination with ventilation-perfusion scanning. A number of diagnostic algorithms using these modalities are currently being evaluated. Regardless of which diagnostic approach is used, the clinician must be aware of some key limitations. Spiral CT scanning has gained popularity because it is noninvasive and can rapidly identify other cardiopulmonary diseases that mimic pulmonary embolism. Its use has been limited because of its inability to detect subsegmental pulmonary emboli. D-dimer assays offer promise as rapid, inexpensive screening tools. However, the wide variability in assay performance has limited its usefulness. We recommend that if D-dimer assays are to be used in a diagnostic algorithm, the clinician be aware of the details of the assay. At present, lack of data precludes use of MRI as a primary diagnostic tool for detection of venous thromboembolism.     

Laboratory diagnostics of Crimean hemorrhagic fever by polymerase chain reaction

Our group developed, within the present case study, two techniques' variations, i.e. a single-step RT-PCR and nested RT-PCR assays, for the purpose of detecting the Crimean-Congo hemorrhagic fever RNA virus in human samples. The above assays as well as those previously recommended by the Ministry of Health of the Russian Federation were simultaneously used in 14 clinical samples obtained from patients with Crimean hemorrhagic fever. After assessing the detection accuracy, it was found that the developed-by-us test system displayed the same or even better diagnostic values versus the previously recommended nested RT-PCR and a 1000-fold advantage over the previously recommended single-step RT-PCA. The single-step RT-PCR assay variation is always more preferable in sense of technical and economic motivations. Finally, the test system developed by us has every reason to become the method of choice in routine PCR diagnosis of Crimean hemorrhagic fever after all official trials and approvals are duly complied with.     

A fluorescence sedimentation assay for dsDNA antibodies

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, ‘Fluoro-Farr’ assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.     

Antigènes fongiques en réanimation : tests disponibles et état des lieux

Numerous serological assays try to supply the pitfalls of microbiological diagnostic methods for fungal infections. Several antigens have been assessed: the galactomannan (GM), the mannan (Mn), the β-glucan (ßG), and the capsular antigen for the diagnosis of invasive aspergillosis, yeast infections, all the fungal infections, and the Cryptococcus neoformans infection respectively. The performance characteristics of these assays are usually satisfactory when serum samples collected in well defined clinical settings are used. In contrast, the results of prospective studies are often disappointing. This underlines the difficulty in standardizing the patient populations and the definitions of fungal infections. For the GM assay, the conclusions obtained in onco-hematology can be used for immunocompromised patients hospitalized in intensive care units. For the Mn assays, the hope relies on the simultaneous detection of both antigen and antibodies. The advantage over the microbiological screening for yeasts of different anatomical sites remains to be demonstrated. For the βG assay, its best interest seems to be its negative predictive value as bacterial and fungal infections are hardly distinguished when the test is positive. Excepted for the capsular cryptococcal antigen, a single test is usually not contributive for any of the assays, which should be implemented as a screening test for patients at risk for fungal infections.     

HIV-1 RNA and viral load

Viral load monitoring has become the standard of care in clinical practice to assess risk for disease progression and to monitor treatment response. Furthermore, viral load monitoring has contributed greatly to the understanding of HIV disease pathogenesis and response to various antiretroviral regimens, and has broadened its applications to include blood bank screening. The assays that are currently available are more sensitive, precise, and robust. There is now a better understanding of their limitations and the clinical scenarios and assay performance issues that result in variations of viral load results.     

Serum markers in the emergency department diagnosis of acute myocardial infarction

No currently used cardiac-specific serum marker meets all the criteria for an "ideal" marker of AMI. No test is both highly sensitive and highly specific for acute infarction within 6 hours following the onset of chest pain, the timeframe of interest to most emergency physicians in making diagnostic and therapeutic decisions. Patients presenting to the ED with chest pain or other symptoms suggestive of acute cardiac ischemia therefore cannot make a diagnosis of AMI excluded on the basis of a single cardiac marker value obtained within a few hours after symptom onset. The total CK level is far too insensitive and nonspecific a test to be used to diagnose AMI. It retains its value, however, as a screening test, and serum of patients with abnormal total CK values should undergo a CK-MBmass assay. Elevation in CK-MB is a vital component of ultimate diagnosis of AMI, but levels of this marker are normal in one fourth to one half of patients with AMI at the time of ED presentation. The test is highly specific, however, and an abnormal value (particularly when it exceeds 5% of the total CK value) at any time in a patient with chest pain is highly suggestive of an AMI. There have been several improvements of CK-MB assay timing and subform quantification that appear highly useful for emergency physicians. Rapid serial CK-MB assessment greatly increases the diagnostic value of the assay in a timeframe suitable for ED purposes but unfortunately still misses about 10% of patients ultimately diagnosed with acute MI. Assays of CK-MB subforms have very high sensitivity, and, although unreliable within 4 hours of symptom onset, have excellent diagnostic value at 6 or more hours after chest pain begins. Automated test assays recently have become available and could prove applicable to ED settings. The cardiac troponins are highly useful as markers of acute coronary syndromes, rather than specifically of AMI, and abnormal values at any time following chest pain onset are highly predictive of an adverse cardiac event. The ED applicability of the troponins is severely limited, however, because values remain normal in most patients with acute cardiac events as long as 6 hours following symptom onset. Myoglobin appeared promising as a marker of early cardiac ischemia but appears to be only marginally more sensitive than CK-MB assays early after symptom onset and less sensitive than CK-MB at 8 hours or more after chest pain starts. Rapid serial myoglobin assessment, however, appears highly useful as an early marker of AMI. The marker has a very narrow diagnostic window. The clinician is left with several tests that are highly effective in correctly identifying patients with AMI (or at high risk for AMI), but none that can dependably exclude patients with acute coronary syndromes soon after chest pain onset. A prudent strategy when assessing ED patients with chest pain and nondiagnostic ECGs is to order CK-MB and troponin values on presentation in the hope of making an early diagnosis of AMI or unstable coronary syndrome. Although it is recognized that normal values obtained within 6 hours of symptom onset do not exclude an acute coronary syndrome, patients at low clinical risk and having normal cardiac marker tests could be provisionally admitted to low-acuity hospital settings or ED observation. After 6 to 8 hours of symptom duration has elapsed, the cardiac-specific markers are highly effective in diagnosing AMI, and such values obtained can be used more appropriately to make final disposition decisions. At no time should results of serum marker tests outweigh ECG findings or clinical assessment of the patient's risk and stability.     

Hexaplex PCR for rapid detection of virulence factors

PCR technology has emerged as a basic tool in biological research and in the detection of infectious organisms. It has the potential to provide information on a number of toxins and virulence factors, as well as allowing species identification of pathogens. Multiplex PCR assays are becoming prevalent for the simultaneous detection of toxins, virulence factors and pathogens in clinical and environmental specimens. This review will discuss the hexaplex PCR assay, its application and future directions.     

A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus

The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10^?8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2–10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples.     

High sensitivity troponin: The Sisyphean pursuit of zero percent miss rate for acute coronary syndrome in the ED

Background

The United States Food and Drug Administration recently approved a high sensitivity troponin (hsTn) assay for use. Recent literature has investigated the diagnostic accuracy of hsTn for acute coronary syndrome (ACS) in the emergency department (ED) and its use in accelerated diagnostic protocols.

Development of a novel internal positive control for Taqman based assays

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.     

Improvements in diagnosis of histoplasmosis

Understanding the uses and limitations of a battery of laboratory methods is essential to diagnose histoplasmosis. Antigen detection and serology are valuable adjuncts to histopathology and culture. Improvements incorporated into the second-generation Histoplasma antigen assay have increased its sensitivity and specificity for diagnosis of histoplasmosis. More recently, the antigen assay has been modified to provide quantitation, which improves reproducibility and facilitates monitoring antigen clearance during treatment. Furthermore, detection of antigen in bronchoalveolar lavage fluid increases the sensitivity for diagnosis of pulmonary histoplasmosis. Serological tests for antibodies are also useful, but may be falsely negative in immunosuppressed patients. In addition, elevated antibody titres persist for several years following initial infection, complicating their interpretation. Although histopathology may provide for rapid diagnosis, its sensitivity is < 50% in patients with disseminated disease and even lower in pulmonary histoplasmosis. Polymerase chain reaction has been described, but sensitivity is less than that of histopathology. Culture, although highly specific, has notable limitations, including insensitivity, a need for invasive procedures and delayed growth. This review provides the background for understanding the role of a battery of diagnostic methods in histoplasmosis. Tests facilitating a rapid diagnosis are expected to improve the outcome in patients with severe disease.     

ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non-physiological and time consuming. We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718-905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end. Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni2+-coated microtiter well plate for 2 h. The immobilized Ni2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS-13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity +/- SEM for normal plasmas diluted 1 : 50 was 40 +/- 4.2% while the value obtained for the patients was 2.4 +/- 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS-13 activity under physiological and non-denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS-13 activity in plasma.     

梅毒血清学试剂性能评估方案的优化及应用

目的制定优化的梅毒血清学试剂的评估方案,探讨新方案在发现试剂检测性能特征的价值。方法优化的评估方案首次引入最低检出限、抗干扰性、稳定性等技术参数,采用国际抗梅毒标准血清进行测量值溯源,选择国产梅毒血清学试剂及相对应的进口试剂共6种,以多中心方式进行检测性能的评价。结果 456例明确诊断样品的定性试验,梅毒免疫层析法结果与诊断100%符合;其它4种梅毒筛查试剂相互间虽然敏感性和特异性差异无统计学意义,但存在不同程度的假阳性和假阴性;最低检测限浓度由高到低分别是筛查试剂、梅毒螺旋体凝集法、免疫层析法、和酶联免疫法/化学发光法;两种促凝真空采血器96.88%干扰筛查试剂。结论国产梅毒血清学试剂的检测性能媲美进口产品。免疫层析试剂与临床诊断高度符合,且敏感性水平超过了传统的方法,为推广应用奠定了基础。新的梅毒血清学试剂的评估方案,更完整地反映以往被忽略的影响因素。     

Epitomics: serum screening for the early detection of cancer on microarrays using complex panels of tumor antigens

Efforts toward the development of early detection assays for cancers have traditionally depended on single biomarker molecules. Current technologies have been disappointing and have not resulted in diagnostic tests suitable for clinical practice. Using a high-throughput cloning method, a panel of epitopes/antigens that react with autoantibodies to tumor proteins in the serum of patients with ovarian cancer have been isolated. Discovery of biomarker panels was directed in an unbiased fashion by cloning a large panel of epitopes or tumor antigens, rather than individual biomarkers without a previous notion of their function. The binding properties of these serum antitumor antibodies on microarrays and advanced bioinformatics tools led to a panel of diagnostic antigens. The sequences that were identified using this new technology will lead to the discovery of novel disease-related proteins that have diagnostic value for the presymptomatic detection of cancer. It has been demonstrated that this approach can detect these autoantibodies in the sera of Stage I ovarian cancer patients. There are numerous advantages of employing serum antibodies as the analytes, not the least of which is the ability to rapidly adapt these assays to standard clinical platforms. This technology of global epitope/antigen profiling is referred to as 'epitomics'.     

Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus

Quantitative real-time polymerase chain reaction (qRT-PCR) is useful for diagnosis and studying virus replication. We developed positive- and negative-strand qRT-PCR assays to detect nsP3 of chikungunya virus (CHIKV), a positive-strand RNA alphavirus that causes epidemic fever, rash, and arthritis. The positive- and negative-strand qRT-PCR assays had limits of quantification of 1 and 3 log₁₀ RNA copies/reaction, respectively. Compared to a published E1 diagnostic assay using 30 laboratory-confirmed clinical samples, the positive-strand nsP3 qRT-PCR assay had higher R² and efficiency and detected more positive samples. Peak viral load of 12.9 log₁₀ RNA copies/mL was reached on day 2 of illness, and RNA was detectable up to day 9, even in the presence of anti-CHIKV IgM. There was no correlation between viral load and persistent arthralgia. The positive-strand nsP3 assay is suitable for diagnosis, while the negative-strand nsP3 assay, which uses tagged primers to increase specificity, is useful for study of active viral replication kinetics.     

Evolution and Utility of Antiplatelet Autoantibody Testing in Patients with Immune Thrombocytopenia

To this day, immune thrombocytopenia (ITP) remains a clinical diagnosis made by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis, but the lack of specificity and sensitivity of the available methods for platelet autoantibody testing limits their value in the diagnostic workup of thrombocytopenia. The introduction of methods for glycoprotein-specific autoantibody detection has improved the specificity of testing and is acceptable for ruling in ITP but not ruling it out as a diagnosis. The sensitivity of these assays varies widely, even between studies using comparable assays. A review of the relevant literature combined with our own laboratory's experience of testing large number of serum and platelet samples makes it clear that this variation can be explained by variations in the characteristics of the tests, including in the glycoprotein-specific monoclonal antibodies, the glycoproteins that are tested, the platelet numbers used in the assay and the cutoff levels for positive and negative results, as well as differences in the tested patient populations. In our opinion, further standardization and optimization of the direct autoantibody detection methods to increase sensitivity without compromising specificity seem possible but will still likely be insufficient to distinguish the often very weak specific autoantibody signals from background signals. Further developments of autoantibody detection methods will therefore be necessary to increase sensitivity to a level acceptable to provide laboratory confirmation of a diagnosis of ITP.     

Diagnostic accuracy and prognostic relevance of the measurement of cardiac natriuretic peptides: a review

BACKGROUND: The pathophysiologic and clinical relevance of cardiac natriuretic hormone (CNH) assays has been investigated in numerous experimental and clinical studies. Authors have sought to evaluate the diagnostic accuracy and prognostic relevance of the measurement of CNHs according to evidence-based laboratory medicine principles. METHODS: In June 2003, we ran a computerized literature search on National Library of Medicine using keywords "ANP" and "BNP" and found more than 12 300 and 1200 articles, respectively. A more refined search with keywords "ANP or BNP assay" extracted approximately 7000 and 800 articles, respectively. Only studies specifically designed to evaluate the diagnostic accuracy and prognostic relevance of CNH measurements were selected from this huge mass of articles to be discussed in this review. Content: Several studies suggested that CNH assays may be clinically useful for the screening and classification of patients with heart failure, as a prognostic marker in cardiovascular disease, in the follow-up of patients with heart failure, and because they may reduce the need for further cardiac investigation. However, it is difficult to compare even the best-designed studies because not only did the authors evaluate different populations, they also used different gold standards. CONCLUSIONS: CNH assays and conventional diagnostic work-ups provide complementary information for evaluation of the presence and severity of cardiac dysfunction and clinical disease. Several aspects of CNH assays are still to be elucidated, and further work is needed to carefully assess their diagnostic accuracy and prognostic value in cardiac disease.