Application In Clinical Laboratories Research Articles

Development of an automated immunologic mass spectrometry (iMS) method to overcome matrix effect for quantification: Steroid hormones as the example

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) shows great promise in clinical application for its high specificity, high sensitivity and wide linear range for the determination of small molecules. However, its application in clinical laboratory is hampered by matrix effect of clinical samples which could greatly affect quantification accuracy and the difficulty to be automated for the traditional sample preparation procedures. Thus, new techniques which could achieve selective enrichment to minimize matrix effect and automatic sample preparation of mass spectrometry are needed. We developed an immunologic mass spectrometry (iMS) method to overcome matrix effect and its clinical application was demonstrated for automatic analysis of testosterone (T), progesterone (P) and estradiol (E2) in human serum simultaneously. Firstly, three monoclonal antibodies were coupled to magnetic beads for selective enrichment of target hormones from serum. The immunomagnetic beads were separated, washed and eluted automatically for LC-MS/MS analysis. Analytical performance of the iMS method was validated and compared with traditional LC-MS/MS and chemiluminescence immunoassay (CLIA). Hormone levels were measured for 160 pregnancy women at different gestational weeks. Results showed that target hormones could be selectively captured with absolute recoveries of 93.9%–110.8 %. Relative responses for high, medium and low concentrations of the hormones between serum and methanol solution were 98.0%–109.7 %, 92.2%–105.3 % and 91.7%–96.0 % for T, P and E2, respectively. Calibration curves prepared in methanol solution, BSA solution and blank serum showed good consistency for the iMS method. The automated iMS method could overcome matrix effect of LC-MS/MS and cross-reaction of CLIA. Matrix effect of the iMS method was negligible as high specificity of target hormone enrichment before LC-MS/MS analysis. Matrix-matched calibration standards were no longer necessary for accurate quantification, which was of great benefit for the clinical application of mass spetrometry.

Development of two ultra-sensitive UHPLC-QqQ-MS/MS methods for the simultaneous determination of hydroxyzine and its active metabolite (cetirizine) in human blood: applications to real cases of forensic toxicology.

Both postmortem toxicological and medical-forensic examinations are very important in the case of analyzing various types of chemical substances. Hydroxyzine (HZ) is a first-generation antihistamine drug with a sedative effect that disrupts cognitive function and affects the ability to drive motor vehicles. Enzymatic oxidation of the hydroxy-methyl group to the carboxyl group leads to the formation of its main metabolite-cetirizine (CZ). CZ is the active substance of antiallergic drugs. Because it does not cross the BBB (blood-brain barrier) easily, it is less likely to cause drowsiness or affect memory and impair cognitive function. Therefore, in criminal studies, it is often important what medication had been taken by a person involved, e.g., in a car accident, HZ or CZ. The analysis of both antihistamine drugs is challenging, as usually very low concentrations of the compound of interest need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of HZ and CZ in biological fluid samples. The lower limit of quantification (LOQ) for HZ and CZ was calculated as 0.345 and 0.3696ng/mL, respectively. Together with a reduced sample volume to 200 μL, it makes the developed method suitable for a sensitive multidrug forensic toxicological analysis. Samples were extracted with simple and fast liquid-liquid extraction (ethyl acetate, pH 9). The present method for the determination of HZ and CZ in human blood proved to be simple, fast, selective, and sensitive. The quantification by LC-MS/MS was successfully applied to the samples coming from 28 authentic biological fluids (blood, urine, vitreous humor, bile and stomach content), both antemortem and postmortem. The performed studies confirm that the developed method is characterized by a high extraction efficiency. Its accuracy, reproducibility, simplicity, and selectivity suggest its application in clinical, toxicological, and forensic laboratories.

Quantification of Urinary Exosomal Prostate-Specific Antigen for the Diagnosis of Prostate Cancer Using Clinical Laboratory-Based Techniques: Protocol for a Case-Control Study.

Prostate cancer is the most common cancer in men and represents a major public health problem. The current method for the diagnosis or screening of prostate cancer is invasive and costly. There have been renewed and innovative studies searching for urinary biomarkers to aid in the diagnosis of prostate cancer, especially with technologies based on urinary exosomes. However, technologies based on urine exosomes usually need expensive machines such as an ultracentrifuge and they are difficult to standardize, which hinder their application in clinical laboratories. We have optimized and standardized the isolation of urinary exosomes with the precipitation method. We have found that urinary exosomal prostate-specific antigen (PSA) can be quantified by automatic Elecsys total PSA technique. In this study, our objective is to utilize urinary exosomes from prostate cancer for the development of a test to aid in its diagnosis. Exosomes from the prostate cancer cell line LNCaP was used to set up the technique. To analyze urine samples from patients, the methods include the collection of first-void urine using the Colli-Pee device, the isolation of urine exosomes using the optimized precipitation method, and the quantification of exosomal PSA by Elecsys total PSA. This will be a 2-year study. We will start including patients and controls in the last quarter of 2024. We expect the results to be published in the second quarter of 2027. This is the first study to quantify urinary exosomal PSA using the Elecsys total PSA technique for the diagnosis of prostate cancer. This study emphasizes techniques that are suitable for implementation in clinical laboratories, which will facilitate the application of urinary exosomes to simplify and improve the diagnosis and screening of prostate cancer. PRR1-10.2196/63551.

Detecting M-Protein via Mass Spectrometry and Affinity Beads: Enrichment With Mixed Kappa-Lambda Beads Enables Prompt Application in Clinical Laboratories.

Detecting monoclonal protein (M-protein), a hallmark of plasma cell disorders, traditionally relies on methods such as protein electrophoresis, immune-electrophoresis, and immunofixation electrophoresis (IFE). Mass spectrometry (MS)-based methods, such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-quadrupole time-of-flight (ESI-qTOF) MS, have emerged as sensitive methods. We explored the M-protein-detection efficacies of different MS techniques. To isolate immunoglobulin and light chain proteins, six types of beads (IgG, IgA, IgM, kappa, lambda, and mixed kappa and lambda) were used to prepare samples along with CaptureSelect nanobody affinity beads (NBs). After purification, both MALDI-TOF MS and liquid chromatography coupled with Synapt G2 ESI-qTOF high-resolution MS analysis were performed. We purified 25 normal and 25 abnormal IFE samples using NBs and MALDI-TOF MS (NB-MALDI-TOF). Abnormal samples showed monoclonal peaks, whereas normal samples showed polyclonal peaks. The IgG and mixed kappa and lambda beads showed monoclonal peaks following the use of daratumumab (an IgG/kappa type of monoclonal antibody) with both MALDI-TOF and ESI-qTOF MS analysis. The limits of detection for MALDI-TOF MS and ESI-qTOF MS were established as 0.1 g/dL and 0.025 g/dL, respectively. NB-MALDI-TOF and IFE exhibited comparable sensitivity and specificity (92% and 92%, respectively). NBs for M-protein detection, particularly with mixed kappa-lambda beads, identified monoclonal peaks with both MALDI-TOF and ESI-qTOF analyses. Qualitative analysis using MALDI-TOF yielded results comparable with that of IFE. NB-MALDI-TOF might be used as an alternative method to replace conventional tests (such as IFE) to detect M-protein with high sensitivity.

A preliminary study on the reference intervals of serum tumor marker in apparently healthy elderly population in southwestern China using real-world data

BackgroundThe aim is to establish and verify reference intervals (RIs) for serum tumor markers for an apparently healthy elderly population in Southwestern China using an indirect method.MethodsData from 35,635 apparently healthy elderly individuals aged 60 years and above were obtained in West China Hospital from April 2020 to December 2021. We utilized the Box-Cox conversion combined with the Tukey method to normalize the data and eliminate outliers. Subgroups are divided according to gender and age to examine the division of RIs. The Z-test was used to compare differences between groups, and 95% distribution RIs were calculated using a nonparametric method.ResultsIn the study, we observed that the RIs for serum ferritin and Des-γ-carboxy prothrombin (DCP) were wider for men, ranging from 64.18 to 865.80 ng/ml and 14.00 to 33.00 mAU/ml, respectively, compared to women, whose ranges were 52.58 to 585.88 ng/ml and 13.00 to 29.00 mAU/ml. For other biomarkers, the overall RIs were established as follows: alpha-fetoprotein (AFP) 0–6.75 ng/ml, carcinoembryonic antigen (CEA) 0–4.85 ng/ml, carbohydrate antigen15-3 (CA15-3) for females 0–22.00 U/ml, carbohydrate antigen19-9 (CA19-9) 0–28.10 U/ml, carbohydrate antigen125 (CA125) 0–20.96 U/ml, cytokeratin 19 fragment (CYFRA21-1) 0–4.66 U/ml, neuron-specific enolase (NSE) 0–19.41 ng/ml, total and free prostate-specific antigens (tPSA and fPSA) for males 0–5.26 ng/ml and 0–1.09 ng/ml. The RIs for all these biomarkers have been validated through our rigorous processes.ConclusionThis study preliminarily established 95% RIs for an apparently healthy elderly population in Southwestern China. Using real-world data and an indirect method, simple and reliable RIs for an elderly population can be both established and verified, which are suitable for application in various clinical laboratories.

Design of an Insulin Tracer Protein-Based Biosensor for Insulin Determination

Aim: The regulation of blood glucose levels is controlled by insulin, which is produced by the pancreatic beta system. Inadequate synthesis of beta insulin, results in elevated glucose levels, a condition known as diabetes, which can lead to various chronic health issues. In recent times, the diagnosis of diabetes, particularly type 1, has shifted towards the direct measurement of insulin levels. To facilitate this, an immunosensor was created to enable rapid and sensitive examination of insulin levels, with the goal of improving the quality for life for diabetic patients. Material and Method: For this purpose, an insulin tracer protein based biosensor was designed for the determination of insulin at all solutions. For determination of insulin, electrobiochemical analyses were performed. Optimisation and characterisation studies were performed using differential pulse voltammetry. The performance of bioelectrochemical system was analysed by Receiver Operating Characteristic method. Results: The insulin biosensor cyclic woltammogram was obtained between -0,1 and 0,6 V potantial. At 0,45 V was found as the anodic peak side for determination the insulin. Optimisation and characterisation studies performed at 0,45 V with differential pulse voltammetry. Conclusion: The study successfully identified stable and easy-to-use insulin concentrations, indicating the potential of the newly developed immunosensor for applications in clinical biochemistry laboratories.

Immune-monitoring of myelodysplastic neoplasms: Recommendations from the i4MDS consortium.

Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing "immune classes" among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune "risk factors." By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Review of the potential benefits and challenges of artificial intelligence in clinical laboratory

Over the past few years, medical artificial intelligence (AI) has been extensively utilized within the healthcare industry. However, the deployment of AI raises complicated social and ethical issues related to security, privacy, and human rights. While the use of artificial intelligence (AI) has the potential to improve healthcare outcomes and operational efficiency, this article gives a detailed assessment of current cutting-edge AI breakthroughs in clinical laboratories. It focuses on the potential benefits of AI and its application in clinical laboratory. The use of AI in clinical laboratory is rapidly growing, with the potential to alter patient care in the near future. Furthermore, it has the potential to democratize modern laboratory services, making them available to people all around the world.

Development and Validation of a Bioanalytical Method for Estimating Ellagic Acid in Wistar Rat Plasma by RP-HPLC Using a Gradient Elution Technique

Abstract Background: Ellagic acid (EA) is a natural polyphenolic compound recognized for bioactive and pharmacologic properties that is found abundantly in various plant groups, particularly eudicotyledons. In the current study a novel, reliable, and cost-effective bioanalytical method was developed for quantifying EA in Wistar rat plasma using RP-HPLC. Methods: Zorbax SB C18 (5μ, 4.6150 mm) and Ascentis C18 (5μ, 1004.6 mm) columns were used in the stationary phase, while the mobile phase consisted of water with 0.1 % formic acid (A) and acetonitrile (ACN) with 0.08% formic acid (B). Results: Optimized parameters were as follows: temperature, 30°C; flow rate, 1.0 ml/min; and PDA detectors, 254 nm. The method exhibited high linearity (r2 = 0.9993) between 5 and 300 μg/ml. Precision, both intra- and inter-day, was within acceptable limits (relative standard deviation <2%), and the mean recovery was 99.73%. The LOD and LOQ were 1.564 ± 0.026 μg/ml and 5.015 ± 0.025 μg/ml, respectively. Stability tests, including short- and long-term evaluations, demonstrated stability under various conditions. Conclusion: The developed method met the necessary criteria and holds promise for application in clinical laboratories for assessing EA levels, either alone or with analytes.

Dual fluorescence enhancement of loratidine by photoinduced electron transfer blocking and micellization: Application to the development of novel highly sensitive microwell spectrofluorimetric assay for analysis of dosage forms and urine samples

This study describes the enhancement of the weak native fluorescence of loratadine (LOR) by dual strategy via photoinduced electron transfer (PET) blocking followed by micellization into sodium dodecyl sulfate micelles. The enhanced fluorescence was employed as a basis for the development of a novel microwell spectrofluorimetric assay (MW-SFA) for the determination of LOR in its pharmaceutical dosage forms and urine samples. The assay was conducted in 96-microwell assay plates, and the enhanced fluorescence signals were measured by a microplate reader at 290 and 435 nm for excitation and emission, respectively. The optimum conditions of the assay were established, calibration curve was generated, and the linear regression equation was computed. The relation between the fluorescence signals and LOR concentrations was linear with good determination coefficients (0.9992) in the range of 10 – 2000 ng mL−1. The assay limits of detection and quantitation were 4.1 and 12.5 ng mL−1, respectively. The precision was satisfactory, with values of relative standard deviation not exceeding 1.68%, and the assay’s accuracy was ≥ 99.1%. The proposed was successfully applied to the analysis of LOR in its pharmaceutical dosage forms with acceptable accuracy and precision. The label claims were 99.3 – 100.5% (±0.95 – 1.59%). Statistical analysis comparing the results of the proposed assay with those obtained by a reported pre-validated assay revealed no significant difference between both methods in terms of the accuracy and precision at the 95% confidence level. The assay was also applied to the analysis of urine samples containing LOR with accuracy ≥ 98.24%. The greenness of the proposed assay was confirmed by three efficient metric tools. In overall conclusion, the proposed assay is characterized by high sensitivity, procedure simplicity, and high throughput, enabling the simultaneous analysis of many samples in a short time. Therefore, it is a valuable tool for rapid routine application in pharmaceutical quality control units and clinical laboratories for the determination of LOR.

Application of high-throughput next-generation sequencing technology in clinical laboratory diagnosis of infectious diseases

With advances in sequencing technologies, high-throughput next-generation sequencing (NGS) has triggered increased attention on its application in clinical laboratories and facilitates the molecular diagnosis and treatment of infectious diseases. Compared with conventional microbiology laboratory methods, NGS has greatly increased the sensitivity and accuracy of diagnosis, and reduced detection time for infectious pathogens, especially for diagnosis of complex and mixed infections. However, there are still some problems that hamper the NGS application in infections diagnosis, including lack of standardization, cost, and variation in data interpretation, etc. In recent years, with the developing of policies and legislation, and guidance and supports from Chinese government, the sequencing industry has gained continuously healthy development and sequencing application market gradually becomes mature. Meanwhile, worldwide microbiology experts are striving to develop the standards and reach the consensus, there are more and more clinical laboratories equipped with sequencing instruments and personnel with expertise. All of these measures would certainly promote the clinical application of NGS, and making full use of high-throughput NGS could contribute to the accurate clinical diagnosis and appropriate treatment. The current article describes the application of high-throughput next-generation sequencing technology in the laboratory diagnosis of clinical microbial infectious diseases, as well as the policy system support and development direction.

Detection and analysis of rotavirus in clinical stool samples using silver nanoparticle functionalized paper as SERS substrate

Among the different analytical techniques, surface-enhanced Raman scattering (SERS) approach is a widely used technique for the detection and analysis of various chemicals and biological samples. Present study reports a low-cost, sensitive SERS substrate that has an ability to detect rotavirus in clinical stool samples. The proposed SERS substrate has been fabricated through drop-casting of silver nanoparticles (AgNPs) on a printing-grade paper. Rotavirus particles were extracted from clinical stool samples. The presence of rotavirus antigen in stool samples was confirmed using enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and sequencing. The characteristic Raman peaks of rotavirus (RV) particles in solution were found to be significantly enhanced when Raman signals were recorded from the paper-based SERS substrates. Using the proposed SERS substrate, rotavirus samples with concentration as low as 1% could be reliably recorded by the Raman spectrometer. The paper SERS substrate reported herein is an extremely cost-efficient platform and may find applications in other research and clinical laboratories as well.

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice.

Proteomics continues to forge significant strides in the discovery of essential biological processes, uncovering valuable information on the identity, global protein abundance, protein modifications, proteoform levels, and signal transduction pathways. Cancer is a complicated and heterogeneous disease, and the onset and progression involve multiple dysregulated proteoforms and their downstream signaling pathways. These are modulated by various factors such as molecular, genetic, tissue, cellular, ethnic/racial, socioeconomic status, environmental, and demographic differences that vary with time. The knowledge of cancer has improved the treatment and clinical management; however, the survival rates have not increased significantly, and cancer remains a major cause of mortality. Oncoproteomics studies help to develop and validate proteomics technologies for routine application in clinical laboratories for (1) diagnostic and prognostic categorization of cancer, (2) real-time monitoring of treatment, (3) assessing drug efficacy and toxicity, (4) therapeutic modulations based on the changes with prognosis and drug resistance, and (5) personalized medication. Investigation of tumor-specific proteomic profiles in conjunction with healthy controls provides crucial information in mechanistic studies on tumorigenesis, metastasis, and drug resistance. This review provides an overview of proteomics technologies that assist the discovery of novel drug targets, biomarkers for early detection, surveillance, prognosis, drug monitoring, and tailoring therapy to the cancer patient. The information gained from such technologies has drastically improved cancer research. We further provide exemplars from recent oncoproteomics applications in the discovery of biomarkers in various cancers, drug discovery, and clinical treatment. Overall, the future of oncoproteomics holds enormous potential for translating technologies from the bench to the bedside.

Robust and high-throughput lipidomic quantitation of human blood samples using flow injection analysis with tandem mass spectrometry for clinical use

Direct infusion of lipid extracts into the ion source of a mass spectrometer is a well-established method for lipid analysis. In most cases, nanofluidic devices are used for sample introduction. However, flow injection analysis (FIA) based on sample infusion from a chromatographic pump can offer a simple alternative to shotgun-based approaches. Here, we describe important modification of a method based on FIA and tandem mass spectrometry (MS/MS). We focus on minimizing contamination of the FIA/MS both to render the lipidomic platform more robust and to increase its capacity and applicability for long-sequence measurements required in clinical applications. Robust validation of the developed method confirms its suitability for lipid quantitation in human plasma analysis. Measurements of standard human plasma reference material (NIST SRM 1950) and a set of plasma samples collected from kidney cancer patients and from healthy volunteers yielded highly similar results between FIA-MS/MS and ultra-high-performance supercritical fluid chromatography (UHPSFC)/MS, thereby demonstrating that all modifications have practically no effect on the statistical output. Newly modified FIA-MS/MS allows for the quantitation of 141 lipid species in plasma (11 major lipid classes) within 5.7min. Finally, we tested the method in a clinical laboratory of the General University Hospital in Prague. In the clinical setting, the method capacity reached 257 samples/day. We also show similar performance of the classification models trained based on the results obtained in clinical settings and the analytical laboratory at the University of Pardubice. Together, these findings demonstrate the high potential of the modified FIA-MS/MS for application in clinical laboratories to measure plasma and serum lipid profiles.

Petri-plate, bacteria, and laser optical scattering sensor.

Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibility testing , and for characterizing industrially beneficial traits. An optical scattering sensor designated BARDOT (bacterial rapid detection using optical scattering technology) that uses a red-diode laser, developed at the beginning of the 21st century at Purdue University, some 220 years after the Petri-plate discovery can identify and study bacteria directly on the plate as a diagnostic tool akin to Raman scattering and hyperspectral imaging systems for application in clinical and food microbiology laboratories.

Development of a Real-Time Recombinase-Aided Amplification Method to Rapidly Detect Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.

An accurate genetic assay to identify human neutrophil antigen 2 deficiency.

We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.

A Simple, Robust, and Cost-effective Method for Genotyping Small-scale Mutations.

Background and objectives:Genotyping is an important tool for studying gene functions in animals or detecting genetic variants in humans. Various methods using low to high concentrations of agarose or polyacrylamide gel electrophoresis have been developed for genotyping. These methods rely on the detection of large-size differences (20–2,000 bp) of targeted PCR products between a wild-type gene and a mutant gene. Endonuclease digestion was introduced to identify heterozygous mutations, but it was not possible to differentiate the wild-type from the homozygous mutants with the same or similar size. This study thus developed a novel, simple, and reliable test for genotyping animals or cells following genetic modifications.Methods:We developed an improved and simple method that used 2% agarose gel electrophoresis following T7E1 or Surveyor endonuclease digestion to firstly separate the heterozygous mutations from the wild-type or homozygous mutations. By adding a wild-type PCR product to a potentially homozygous product, which would form heteroduplexes, we could then separate the wild-type from a homozygous mutation with a nearly identical size or only a single base pair substitution without Sanger sequencing.Results:We verified this method in genotyping zebrafish mutants with a 2-8-bp deletion or insertion and mouse mutants with a 1- or 8-bp substitution. The wild-type, heterozygous, and homozygous mutations ranged 1–8 bp were clearly differentiated on agarose gel. Sanger sequencing also confirmed our genotyping results.Conclusions:This novel and improved genotyping method may have a broad application in many clinical and research laboratories for rapid and economical genotyping of patients and animals with a small area deletion or single base pair substitution, particularly in the era of gene editing or in those with naturally occurring mutations.

Gold nanoparticle decorated blu-ray digital versatile disc as a highly reproducible surface-enhanced Raman scattering substrate for detection and analysis of rotavirus RNA in laboratory environment.

Detection and estimation of various biomolecular samples are often required in research and clinical laboratory applications. Present work demonstrates the functioning of a surface-enhanced Raman scattering (SERS) substrate that has been obtained by drop-casting of citrate-reduced gold nanoparticles (AuNPs) of average dimension of 23 nm on a bare blu-ray digital versatile disc (BR-DVD) substrate. The performance of the proposed SERS substrate has been initially evaluated with standard Raman active samples, namely malachite green (MG) and 1,2-bis(4-pyridyl)ethylene (BPE). The designed SERS substrate yields an average enhancement factor of 3.2 × 106 while maintaining reproducibility characteristics as good as 94% over the sensing region of the substrate. The usability of the designed SERS substrate has been demonstrated through the detection and analysis of purified rotavirus double-stranded RNA (dsRNA) samples in the laboratory environment condition. Rotavirus RNA concentrations as low as 10ng/μL could be detected with the proposed sensing scheme.

Обзор практик применения бережливого производства в клинических лабораториях

The main role of clinical laboratory diagnostics is to obtain reliable, reproducible and timely results that help to make the right clinical decisions. As the literature review shows, there are a number of common problems for many clinical laboratories, such as: long turnaround times, high laboratory costs for consumables, insufficient level of satisfaction with quality of services received by patients and high number of complaints. The use of lean manufacturing can be one way of solving these problems. Purpose of the study is to summarize and analyze the literature regarding the application of lean manufacturing in clinical laboratories. Materials and methods. A literature review was performed using the following abstract databases: Web of Science, Scopus, PubMed, ScienceDirect, Mendeley, and NCBI. The depth of the search according to the time parameter had no limitations. The following keywords were used as search markers: “lean management”, “lean thinking”, “lean six sigma”, “lean tools”, “healthcare”, “health services”, “clinical laboratory”. More than 100 publications were analyzed, from which 28 most representative studies were selected. Results. The implementation of lean production methods provides a reduction in turnaround time, costs and errors in the clinical laboratory, reduces the contamination of biological material at the preanalytical stage and reduces waiting time for services and, as a consequence, reduces the number of patient complaints. Conclusion. The above results indicate that lean production methods can be used in laboratories to solve frequently occurring problems and demonstrate the effectiveness of their application in clinical laboratories.