Routine Laboratory Setting Research Articles

P1.07-010 Peripheral Blood Biomarkers Associated with Clinical Outcome in Non–Small Cell Lung Cancer Patients Treated with Nivolumab

Targeting of the immune system has been found to confer clinical benefit for patients with some types of advanced solid tumor. Given that only a limited number of patients experience a durable response, whereas all those treated are at risk for specific immune side effects, the identification of individuals who are most likely to benefit from nivolumab and similar agents is an important clinical goal. We have now examined the possible impact of clinical parameters determined in the routine laboratory setting—including peripheral blood cell counts such as ANC, absolute lymphocyte count (ALC), absolute monocyte count (AMC), and absolute eosinophil count (AEC)—on outcome in patients with advanced or recurrent NSCLC treated with nivolumab.

DMSO Assisted Electrospray Ionization for the Detection of Small Peptide Hormones in Urine by Dilute-and-Shoot-Liquid-Chromatography-High Resolution Mass Spectrometry

The mobile phase additive (DMSO) has been described as a useful tool to enhance electrospray ionization (ESI) of peptides and proteins. So far, this technique has mainly been used in proteomic/peptide research, and its applicability in a routine clinical laboratory setting (i.e., doping control analysis) has not been described yet. This work provides a simple, easy to implement screening method for the detection of doping relevant small peptides (GHRPs, GnRHs, GHS, and vasopressin-analogues) with molecular weight less than 2 kDa applying DMSO in the mobile phase. The gain in sensitivity was sufficient to inject the urine samples after a 2-fold dilution step omitting a time consuming sample preparation. The employed analytical procedure was validated for the qualitative determination of 36 compounds, including 13 metabolites. The detection limits (LODs) ranged between 50 and 1000 pg/mL and were compliant with the 2 ng/mL minimum detection level required by the World Anti-Doping Agency (WADA) for all the target peptides. To demonstrate the feasibility of the work, urine samples obtained from patients who have been treated with desmopressin or leuprolide and urine samples that have been declared as adverse analytical findings were analyzed. Graphical Abstract ᅟ.

Intake of carp meat from two aquaculture production systems aimed at secondary prevention of ischemic heart disease - a follow-up study.

Our previous study showed that a diet enriched with 400 g of carp per week improved plasma lipids in subjects after aortocoronary bypass (CABG). The aim of the present study is to determine whether the different carp farming systems have an impact on the effects of carp meat in secondary cardiovascular prevention. We examined 3 groups of patients after CABG over a 4-week period of spa treatment (108 persons, 73 males, 35 females, age over 60 years). We found no differences in baseline values of blood pressure or plasma lipids. The patients were given a standard spa diet (controls; N=36) or a diet enriched of 400 g of carp meat per week, enriched omega 3 (N=37) or cereal carp (N=35). Plasma lipid parameters were examined at start and after 4 weeks in a routine laboratory setting. Group consuming omega-3 carp showed the largest decline in total cholesterol, LDL cholesterol, triglycerides and an increase in HDL cholesterol (all p<0.01). We found that carp meat from the two production systems showed significantly different effects on plasma lipids. Further trials should be performed to clarify the exact causes of the differences.

Detection of drugs of abuse in urine using the Bruker Toxtyper™: Experiences in a routine clinical laboratory setting

Urine screening can be used to detect misuse of illicit drugs and validate opioid replacement therapy compliance. It is common that immunochemical assays are combined with GC-MS for these applications. Bruker has recently released an ion trap mass spectrometer, called Toxtyper™, with the potential to replace current screening algorithms to detect drug misuse.Here, we compare our current strategy of urine screening for misuse of cannabis, amphetamines, cocaine, opiates, benzodiazepine, methadone, sufentanil, and pregabalin to the Toxtyper protocols provided by the manufacturer.The analytical performance of the instrument was determined on a selected drug panel and with 188 urine samples being compared to establish concordance between our currently established approach and the Toxtyper.The lower limits of detection and identification for acetylcodeine, amphetamine, benzoylecgonine, methadone, and nordiazepam were below the common cut-offs for immunological screening assays and comparable to GC-MS. Imprecision and accuracy, both within- and between-series, were consistently <25%. Toxtyper screening for pregabalin and sufentail was less sensitive than a targeted LC-MS/MS assay. Concordance met the predefined criterion of >90% for all drugs, except for pregabalin. Cannabis misuse could not be detected due to the limited sensitivity of the Toxtyper assay protocols used and the inherent imprecision of the assay.Our study has revealed that a considerable portion of our current time-consuming protocol for screening drugs of abuse in urine, based on the combination of multiple analytical methods, could be consolidated by the Toxtyper for a majority of the most-relevant substances in our patient population.

Evaluation of the Xpert Norovirus assay for the rapid detection of norovirus genogroups I and II in faecal specimens within a routine laboratory setting

Norovirus infection accounts for over 90% of all viral gastroenteritis cases and approximately 50% of all global outbreaks [1]. It is estimated that norovirus infects over 267 million people and ma...

Highlights of This Issue

To explore a novel molecularly targeted strategy that may abrogate or delay the emergence of antiestrogen resistance in hormone-dependent breast cancer, Mayer and colleagues conducted a phase Ib clinical trial of the PI3Kα-specific inhibitor, alpelisib, and the aromatase inhibitor, letrozole, in patients with ER+ metastatic breast cancers refractory to endocrine therapies. Study results show that the above combination was safe, tolerable, and active, particularly in those patients whose tumors harbored an activating mutation in PIK3CA. Phase II and III trials of alpelisib and endocrine therapy in patients with ER+ breast cancer are ongoing.Clinical management of early-stage lung cancer is becoming crucial due to recent activation of cancer prevention and screening programs. The availability of prognostic biomarkers for patients with early-stage lung cancer (stage I) is paramount for therapy optimization. Dama and colleagues developed a molecular test easily implementable in a routine laboratory setting to diagnose an aggressive subtype of stage I lung adenocarcinoma. Remarkably, a comprehensive analysis of genetic, epigenetic, and proteomic profiles identified molecular features in this subtype that are peculiar in metastatic lung cancer. These findings also suggest new possibilities for tailored therapies in patients with early-stage lung cancer.Human Ether-à-go-go-related gene (hERG) encodes a voltage-gated potassium channel that is overexpressed in glioblastoma (GBM) cell lines. Pointer and colleagues discovered that high hERG expression in GBM is correlated with poor patient survival. Furthermore, patients with high hERG-expressing tumors who also received drugs with incidental hERG inhibitory activity experienced significantly improved median survival compared with those who did not receive a hERG blocker. This study establishes hERG as a new GBM biomarker and the rationale for targeting clinical trials of hERG inhibitors for the subset of high hERG-expressing GBM patients.The presence of stem-like cells and the intratumor heterogeneity are key features for resistance to anticancer treatments, including targeted therapy. In colorectal cancer (CRC) EphA2 overexpression has been linked to stem-like properties of cells and tumor malignancy. De Robertis and colleagues characterized murine homogeneous EphA2high CRC cell subpopulation, unveiling a novel potential molecular signature related to EphA2/EGFR pathway. These preclinical findings strongly correlated with clinical outcome and cetuximab responsiveness. Because anti-EGFR drug resistance remains a critical issue in treating CRC, this study may provide new potential biomarkers and therapeutically actionable kinase targets in the EphA2/Efna1/EGFR/mir-26b/mir-200a linked pathways.

Prospective evaluation of Cepheid Xpert HIV-1 Viral Load assay as a supplemental confirmatory HIV-1 test in the routine clinical laboratory setting

Prospective evaluation of Cepheid Xpert HIV-1 Viral Load assay as a supplemental confirmatory HIV-1 test in the routine clinical laboratory setting

Recent "omics" advances in Helicobacter pylori.

The development of high-throughput whole genome sequencing (WGS) technologies is changing the face of microbiology, facilitating the comparison of large numbers of genomes from different lineages of a same organism. Our aim was to review the main advances on Helicobacter pylori "omics" and to understand how this is improving our knowledge of the biology, diversity and pathogenesis of H.pylori. Since the first H.pylori isolate was sequenced in 1997, 510 genomes have been deposited in the NCBI archive, providing a basis for improved understanding of the epidemiology and evolution of this important pathogen. This review focuses on works published between April 2015 and March 2016. Helicobacter "omics" is already making an impact and is a growing research field. Ultimately these advances will be translated into a routine clinical laboratory setting in order to improve public health.

VEGF-A mRNA measurement in meningiomas using a new simplified approach: branched DNA and chemiluminescence

For decades, the preferred and almost sole method for measurement of gene expression has been RT-qPCR. The method is robust, inexpensive, and well-studied; however, PCR is also quite laborious and vulnerable to contamination. As part of an investigation of VEGF-A gene expression in meningiomas, an alternative and less laborious method for gene expression analysis based on branched DNA hybridization and chemiluminescence (Lumistar) was tested. Albeit the two methods differ, in principle, cellular mRNA-concentration is measured with both. Because they both determine gene expression via the measurement of mRNA-concentration, they were expected to be comparable. The aim of the present study was to compare Lumistar to the traditional RT-qPCR approach in a routine laboratory setting, where there is emphasis on rapid analysis response. Meningioma (nÂ=Â10) and control brain tissue (nÂ=Â5) samples were collected and VEGF-A and GAPDH mRNA were quantified using both RT-qPCR and Lumistar. Furthermore, two dilution series of two of the meningioma samples were prepared in order to make quantitative analyses. Both Lumistar and RT-qPCR-results were found to follow concentration dependent linear paths when diluted (p<0.0001 and p<0.01). Finally, Lumistar and RT-qPCR analyses were performed with the inclusion of a reference gene (GAPDH), where similar results were obtained with the two methods (R2=0.48; p=0.01). It is intriguing that in spite of the vast difference in handling and assay principles, gene expression results are similar. The preferred method depends on the variability of the samples, budget, and time. Lumistar was less time consuming, while RT-qPCR was less expensive and best suited for data sets with large sample variability.

Aluminium foil as a potential substrate for ATR-FTIR, transflection FTIR or Raman spectrochemical analysis of biological specimens

Aluminium foil is a potential low-cost alternative substrate for spectrochemical analyses: facilitating translation to a routine clinical or laboratory setting.

Development of a novel multiplex type-specific quantitative real-time PCR for detection and differentiation of infections with human papillomavirus types HPV2, HPV27, and HPV57

The present study describes the development and evaluation of the first multiplex type-specific quantitative real-time PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction. The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR. The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.

Remarkable magnitude of the self-disproportionation of enantiomers (SDE) via achiral chromatography: application to the practical-scale enantiopurification of β-amino acid esters

We report the best performance yet for the self-disproportionation of enantiomers (SDE) via achiral chromatography as typically used in laboratories for the isolated yield of the excess enantiomer using N-acetyl β-amino acid ethyl esters. The results are the most convincing ever demonstration of the capability of the SDE for practical-scale enantiopurification as comparable, or even superior for some systems, to that of recrystallization. For example, from a sample of 94.4% ee, a yield of 71% of enantiopure material was isolated in a single chromatographic run. Moreover, the lack of an esoteric structural entity, e.g. strongly polarizing groups, such as, for instance CF3, highlights the fact that the phenomenon is not dependent on the presence of such and thus the process is relevant to any usual-type structure. In contrast to recrystallization, the procedure is predictable, general, and dependable, boding well for its widespread application in routine laboratory settings.

Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections.

Group B Streptococcus (GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in the cyl operon, encoding the β-h/c pigment biosynthetic pathway and, in the abx1 gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.

Robust, compact implementation of an off-axis digital holographic microscope.

Recent advances in digital technologies, such as high-speed computers and large-format digital imagers, have led to a burgeoning interest in the science and engineering of digital holographic microscopy (DHM). Here we report on a novel off-axis DHM, based on a twin-beam optical design, which avoids the limitations of prior systems, and provides many advantages, including compactness, intrinsic stability, robustness against misalignment, ease of use, and cost. These advantages are traded for a physically constrained sample volume, as well as a fixed fringe spacing. The first trade is not overly restrictive for most applications, and the latter provides for a pre-set assembly alignment that optimizes the spatial frequency sampling. Moreover, our new design supports use in both routine laboratory settings as well as extreme environments without any sacrifice in performance, enabling ready observation of microbial species in the field. The instrument design is presented in detail here, along with a demonstration of bacterial video imaging at sub-micrometer resolution at temperatures down to -15 °C.

Improved sensitivity to venom specific-immunoglobulin E by spiking with the allergen component in Japanese patients suspected of Hymenoptera venom allergy

BackgroundVes v 5 and Pol d 5, which constitute antigen 5, are recognized as the major, most potent allergens of family Vespidae. Several studies have reported the diagnostic sensitivity of the novel recombinant (r)Ves v 5 and rPol d 5 allergens in routine clinical laboratory settings by analyzing a group of Vespula and Polistes venom-allergic patients. In this study, we analyzed the sensitivity to venom specific (s)IgE by spiking with rVes v 5 and rPol d 5 in Japanese patients suspected of Hymenoptera venom allergy. MethodsSubjects were 41 patients who had experienced systemic reactions to hornet and/or paper wasp stings. Levels of serum sIgE against hornet and paper wasp venom by spiking with rVes v 5 and rPold d 5, respectively, as improvement testing, compared with hornet and paper wasp venom, as conventional testing, were measured by ImmunoCAP. ResultsOf the 41 patients, 33 (80.5%) were positive (≥0.35 UA/ml) for hornet and/or paper wasp venom in conventional sIgE testing. sIgE levels correlated significantly (P < 0.01) between hornet (R = 0.92) or paper wasp venom (R = 0.78) in improvement testing and conventional testing. To determine specificity, 20 volunteers who had never experienced a Hymenoptera sting were all negative for sIgE against these venoms in both improvement and conventional testing. Improved sensitivity was seen in 8 patients negative for sIgE against both venoms in conventional testing, while improvement testing revealed sIgE against hornet or paper wasp venom in 5 (total 38 (92.7%)) patients. ConclusionsThe measurement of sIgE following spiking of rVes v 5 and rPol d 5 by conventional testing in Japanese subjects with sIgE against hornet and paper wasp venom, respectively, improved the sensitivity for detecting Hymenoptera venom allergy. Improvement testing for measuring sIgE levels against hornet and paper wasp venom has potential for serologically elucidating Hymenoptera allergy in Japan.

Comparison of MALDI-TOF mass spectra with microsatellite length polymorphisms in Candida albicans.

Candida albicans is the most frequent yeast involved in human infections. Its population structure can be divided into several genetic clades, some of which have been associated with antifungal susceptibility. Therefore, detecting and monitoring fungal clones in a routine laboratory setting would be a major epidemiological advance. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra results are now widely used as bar codes to identify microorganisms in clinical microbiology laboratories. This study aimed at testing MALDI-TOF mass spectra bar codes to identify clades among a set of C. albicans isolates. Accordingly, 102 clinical strains were genotyped using 10 microsatellite markers and analyzed via MALDI-TOF mass spectrometry. The mass spectra were compared with a reference spectral library including 33 well-characterized collection strains, using a Microflex(TM) system and Biotyper(TM) software, to test the capacity of the spectrum of a given isolate to match with the reference mass spectrum of an isolate from the same genetic clade. Despite high confidence species identification, the spectra failed to significantly match with the corresponding clade (p = 0.74). This was confirmed with the MALDI-TOF spectra similarity dendrogram, in which the strains were dispersed irrespective of their genetic clade. Various attempts to improve intra-clade spectra recognition were unsuccessful. In conclusion, MALDI-TOF mass spectra bar code analysis failed to reliably recognize genetically related C. albicans isolates. Further studies are warranted to develop alternative MALDI-TOF mass spectra analytical approaches to identify and monitor C. albicans clades in the routine clinical laboratory.

Prospective study comparing enzyme‐linked immunosorbent assay and glycoarray assay to detect antiglycolipid antibodies in a routine diagnostic neuroimmunology laboratory setting

AbstractObjectiveEnzyme‐linked immunosorbent assay (ELISA) is the conventional technique for antiglycolipid antibody testing in inflammatory neuropathy sera. Miniaturized array‐based assays (glycoarrays) have also been used to detect these antibodies. As previous studies have focused on specific disease categories, such as Guillain–Barré syndrome, the array has never been tested on an unselected population in a routine diagnostic laboratory setting.MethodsIn the present prospective study, we compared the results of the glycoarray with data obtained with a standardized inflammatory neuropathy cause and treatment‐ELISA. A total of 300 sera sent to the Glasgow Neuroimmunology Laboratory for routine antiglycolipid antibody testing during a 6‐month period were tested both with ELISA and glycoarray.ResultsThe two techniques were significantly correlated and showed good agreement. By ELISA, six sera were positive for immunoglobulin G antibodies against GM1 or GD1a, 11 for immunoglobulin G anti‐GQ1b antibodies, five for immunoglobulin M anti‐GM1 antibodies and three for immunoglobulin M antibodies against disialosyl gangliosides. The glycoarray had a sensitivity of 92% to detect ELISA‐positive sera with a specificity above 92% for all the different ELISA patterns.ConclusionsThe glycoarray allows testing of large panels of antibodies against single glycolipids and complexes of glycolipids on a very small scale. Its technical characteristics make it suitable as a diagnostic screening test. As data provided by the glycoarray and ELISA were reliably correlated in the present study, the glycoarray can be used in a routine setting to detect antiglycolipid antibodies. Further studies, including more positive samples, are required to clarify the future position of the array in the biological investigation of inflammatory neuropathies.

Comparative Analysis of Disc Diffusion and E-test with Broth Micro-dilution for Susceptibility Testing of Clinical Candida Isolates Against Amphotericin B, Fluconazole, Voriconazole and Caspofungin.

Antifungal susceptibility testing remains an area of intense interest because of the increasing number of clinical isolates resistant to antifungal therapy. Clinical and Laboratory Standards Institute has proposed reference broth micro dilution (BMD) method for susceptibility testing. The reference method is time-consuming and poorly suited for the routine clinical laboratory setting. Agar-based susceptibility testing methods, disk diffusion (DD) method and the E-test method can be an easier, reliable and less time consuming alternative for the BMD method. To compare the results of Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility testing by DD, and the E-test method with the CLSI reference method for clinical Candida isolates. Broth Microdilution (BMD), E-test and Disk diffusion testing of the various clinical Candida isolates was performed in accordance with CLSI documents. The results obtained were analysed and compared. The categorical agreement for Amphotericin B, fluconazole, voriconazole, and Caspofungin susceptibility results by E-test and DD method was 65.2%, 67.4%; 100%, 82.6%; 100%, 100%; 100%, 97.8% respectively. The agar-based E-test and disk diffusion methods are reliable alternatives to the BMD method for Candida isolates when test susceptible to fluconazole, voriconazole, and Caspofungin, however the susceptibility testing results must be interpreted with caution in case of Amphotericin B.

Availability and use of molecular microbiological and immunological tests for the diagnosis of tuberculosis in europe.

IntroductionCurrently only limited data exist regarding the availability and clinical use of molecular and immunological tests for tuberculosis (TB) in the European setting.MethodsWeb-based survey of Paediatric-Tuberculosis-Network-European-Trialsgroup (ptbnet) and Tuberculosis-Network-European-Trialsgroup (TBnet) members conducted June to December 2013. Both networks comprise clinicians, microbiologists, epidemiologists and researchers predominately based in Europe.Results191 healthcare professionals from 31 European countries participated. Overall, 26.8% of respondents did not have access to the Xpert MTB/RIF assay; only 44.6% had access to the assay in-house. However, a substantial proportion had access to other commercial and/or non-commercial PCR-based assays for TB (68.8% and 31.8%, respectively). Only 6.4% did not have access to any PCR-based assays for TB. A large proportion of participants with access to the Xpert MTB/RIF assay had used it for the analysis of non-respiratory samples [pleural fluid: 36.5%, gastric aspirates: 34.7%, cerebrospinal fluid: 34.7%, stool samples: 4.3%, blood/serum: 2.6%, ‘other samples’ (which included biopsy/tissue samples, lymph node aspirates, joint aspirates and urine samples): 16.5%]. Regarding interferon-gamma release assays, a greater proportion of respondents had access to the QuantiFERON-TB Gold assay (84.7%) than to the T-SPOT.TB assay (52.2%).ConclusionsBoth immunological and molecular TB tests are widely available across Europe. The QuantiFERON-TB Gold assay is more widely used than the T-SPOT.TB assay, which may reflect the difficulties of integrating an ELISPOT assay into the routine laboratory setting. Although Xpert MTB/RIF assays are optimised and solely licensed for the analysis of sputum samples, in clinical practice they are commonly used for non-respiratory samples. Further research is needed to establish how current molecular TB tests impact on patient care and outcome in the routine clinical setting.

MxA mRNA quantification and disability progression in interferon beta-treated multiple sclerosis patients.

Even though anti-interferon beta (IFNβ) antibodies are the main determinants of IFNβ bioactivity loss and Myxovirus-resistance protein A (MxA) is the most established marker of IFNβ biological activity in IFNβ-treated multiple sclerosis patients, their usefulness in the routine clinical practice is still debated. Therefore, 118 multiple sclerosis patients naïve for treatment were enrolled for a 3-year longitudinal observational study mimicking the conditions of a real-world setting. In order to evaluate the kinetics of bioactivity loss in blood samples obtained every 6 months after therapy initiation, MxA and interferon receptor isoform/subunit mRNA were quantified by real-time PCR, anti-IFNβ binding antibodies were detected by radioimmunoprecipitation, and neutralizing antibodies by cytopathic effect inhibition assay. Clinical measures of disease activity and disability progression were also obtained at all time points. We found that, at the individual-patient level, the response to IFNβ therapy was extremely heterogeneous, including patients with stable or transitory, early or late loss of IFNβ bioactivity, and patients with samples lacking MxA mRNA induction in spite of absence of antibodies. No interferon receptor isoform alterations that could explain these findings were found. At the group level, none of these biological features correlated with the measures of clinical disease activity or progression. However, when MxA mRNA was evaluated not at the single time point as a dichotomic marker (induced vs. non-induced), but as the mean of its values measured over the 6-to-24 month period, the increasing average MxA predicted a decreasing risk of short-term disability progression, independently from the presence of relapses. Therefore, a more bioactive treatment, even if unable to suppress relapses, reduces their severity by an amount that is proportional to MxA levels. Together with its feasibility in the routine laboratory setting, these data warrant the quantification of MxA mRNA as a primary tool for a routine monitoring of IFNβ therapy.