High-throughput Analyzers Research Articles

Development and validation of an LC-MSMS method to quantify creatinine from dried blood spots

IntroductionScreening for chronic kidney disease relies on accurate and precise creatinine measurements. Traditionally, creatinine is measured in serum or plasma using high-throughput chemistry analyzers. However, dried blood spots (DBS) can also be utilized to improve testing access. MethodsSamples were obtained from a 6 mm DBS punch, which was reconstituted in water before undergoing an acetonitrile crash. The resulting supernatant was diluted using an 80:20 acetonitrile: water before injection. Creatinine was identified using an isocratic gradient, and detected using an API 4000 triple quadrupole mass analyzer. Quantification relied on matrix-matched calibrators, with values harmonized to the Roche Cobas enzymatic assay. Validation studies assessing method performance included precision, linearity, accuracy, method comparison, stability, interference, and matrix effects. ResultsThe LC-MSMS assay was linear from 0.3 to 20 mg/dL (y = 1.02x-0.11; R2 = 0.996). Precision ranged from 5.2 to 8.1 % using matrix-matched controls (n = 4) that spanned the analytical measurement range. LC-MSMS results corresponded to the enzymatic assay (Roche) with a fitted line equation of y = 0.956x–0.07 (R2 = 0.995; n = 173). The Siemens and Roche enzymatic assays demonstrated higher accuracy in correlating to the DBS creatinine concentration (n = 40 paired venous/DBS collections) compared to the Beckman Jaffe assay (-2.5 % and −0.8 % versus −6.3 % and −4.1 %, respectively) or the iSTAT (-28.4 % and −27.1 %, respectively). Accuracy was unaffected by hematocrit, blood spot volume, excess IgG or IgA, or hypertriglyceridemia. No matrix effects were observed, and both extraction and processing efficiency were robust.Ambient stability extended to at least 10 days, and exposure to extreme temperature did not affect the creatinine results. ConclusionWe successfully developed an accurate and precise LC-MSMS method for quantifying creatinine in DBS.

Phage Display's Prospects for Early Diagnosis of Prostate Cancer.

Prostate cancer (PC) is the second most diagnosed cancer among men. It was observed that early diagnosis of disease is highly beneficial for the survival of cancer patients. Therefore, the extension and increasing quality of life of PC patients can be achieved by broadening the cancer screening programs that are aimed at the identification of cancer manifestation in patients at earlier stages, before they demonstrate well-understood signs of the disease. Therefore, there is an urgent need for standard, sensitive, robust, and commonly available screening and diagnosis tools for the identification of early signs of cancer pathologies. In this respect, the "Holy Grail" of cancer researchers and bioengineers for decades has been molecular sensing probes that would allow for the diagnosis, prognosis, and monitoring of cancer diseases via their interaction with cell-secreted and cell-associated PC biomarkers, e.g., PSA and PSMA, respectively. At present, most PSA tests are performed at centralized laboratories using high-throughput total PSA immune analyzers, which are suitable for dedicated laboratories and are not readily available for broad health screenings. Therefore, the current trend in the detection of PC is the development of portable biosensors for mobile laboratories and individual use. Phage display, since its conception by George Smith in 1985, has emerged as a premier tool in molecular biology with widespread application. This review describes the role of the molecular evolution and phage display paradigm in revolutionizing the methods for the early diagnosis and monitoring of PC.

B-203 Development and Validation of an LC/MS/MS Method to Quantify Creatinine in Dried Blood Spots

Abstract Background Health disparities in diagnosis and treatment of chronic kidney disease (CKD) are apparent with Black, Brown, and socially disadvantaged people experiencing worse outcomes. Improving access to CKD screening may decrease disparities. Blood creatinine concentrations are a mainstay in CKD screening, as it is commonly used to estimate glomerular filtration rate. Creatinine is classically measured in serum/plasma using high-throughput chemistry analyzers. Dried blood spots (DBS) could offer a solution to under-screening, but require an alternative method since extraction from DBS retrieves minimal sample volume and is difficult to scale using conventional automation. The purpose of this study was to develop an LC/MS/MS DBS method to quantify creatinine and ultimately to increase screening access for underprivileged communities. Methods Creatinine was measured using a 100 × 2.1 mm Hypersil Silica column with an ABSciex API4000 MS/MS detector. A 5-point calibration curve was made using creatinine certified reference material (Cerilliant) and blank whole blood (UTAK) (target concentrations: 0.575, 0.975, 4.575, 10.575, 20.575 mg/dL). Calibrators were spotted onto Whatman903 cards and allowed to dry for at least 24-hours. A 6 mm punch was used for analysis of calibration points and patient samples. Once punched, water (50 ul) was added and the samples were incubated at 60°C for 15 min before shaking at 800 RPM. Acetonitrile with 2H-creatinine internal standard (IS; .06 mg/dL) was added and the mixture was shaken and centrifuged. The resulting supernatant was diluted to contain 20% water before injecting 10 uL. Creatinine was separated using an isocratic mobile phase (80% Acetonitrile, 20% DI water, 10 mM Ammonium Formate) and detected using MRM mode (Q1 114.0 m/z; Q3 86.8 m/z (quantifier) and 44.5 m/z (qualifier)). For the IS a Q1 of 117.0 m/z and Q3 of 89.9 m/z was applied and the extraction ratio of endogenous creatinine to IS was calculated to determine the concentration. Linearity was confirmed by running five levels of DBS calibration standards for five days. Imprecision was established by running 3 control levels five times for five consecutive days. Control material was purchased from BioRad and run as liquid. Accuracy was assessed using residual liquid CAP serum creatinine proficiency testing (PT) material in triplicate. DBS creatinine concentration determined by LC/MS/MS was compared to paired venous serum creatinine concentration determined by the Roche Cobas enzymatic creatinine assay (n = 63). Results Creatinine concentrations from 0.2–20 mg/dL were within the allowable nonlinearity of 0.075 mg/dL or 5.0% (y = 1.00x−0.010;R = 0.999). Intraday and interday precision was <2%and <3%, respectively for all levels (mean concentrations 1.00, 2.02, and 7.05 mg/dL). Residual CAP PT samples (n = 4) were within 2.5% of the peer group mean. Compared to venous results, DBS had a bias of −0.03 mg/dL (−3.573%) and a linear equation of (y = .904x +0.049; R = 0.83). Conclusion Preliminary results indicate that DBS are a viable sample source for measuring creatinine. Creatinine concentrations measured by DBS compared favorably with CAP PT. A clinically insignificant negative bias was seen when DBS creatinine concentration was compared to venous blood.

A universal CRISPR/Cas9-based electrochemiluminescence probe for sensitive and single-base-specific DNA detection

Electrochemiluminescence (ECL) is a sensitive detection technology. Commercial ECL analyzer democratizes ECL as a convenient tool for clinical immunoassay. Still, commercial DNA ECL detection kits are absent, because oligonucleotide-based ECL probes often lack double-stranded DNA (dsDNA) recognition capability and specificity. In this study, we harnessed CRISPR/dCas9 as a universal electrochemiluminescence (ECL) probe for sensitive and single-base-specific DNA detection. Benefiting from the unique characteristics of Cas9, including flexible programmability, dsDNA binding ability and high specificity, the dCas9-ECL probe can directly recognize dsDNA amplification products under constant and mild conditions, and provide higher signal-to-background ratio. Experimental results demonstrated our method can detect 38 cfu‧mL-1 genome of Listeria monocytogenes. Only changing the targeting region of the sgRNA, the probe can be employed to detect 0.1 pg‧μL-1 EGFR L858R mutant with a mutation discrimination selectivity factor of 0.01%. Moreover, the dCas9-ECL probe can be used for DNA detection in complex samples (e.g. milk & cell extracts). In conclusion, the dCas9-ECL probe could be formed into a universal DNA detection kit and be compatible with automated and high-throughput ECL immunoassay analyzers, thus holds a certain application potential in pathogen detection and gene typing.

Antibody profiling and prevalence in US patients during the SARS-CoV2 pandemic.

BackgroundAntibody diagnostics play an important role in disease detection and can potentially aid in monitoring of the immune responses to see if an individual has developed immunity. Developing high throughput diagnostics which does not involve handling of infectious material becomes imperative in the case of pandemics such as the recent outbreak of SARS-CoV2.MethodsA protein microarray technology was used to detect the plurality of antibody response to four novel antigens namely S1 glycoprotein, Receptor binding domain (RBD), S2 glycoprotein and Nucleoprotein of the novel coronavirus named SARS-CoV2 using serum samples. A DBS card was additionally used to compare its performance with a venipuncture-based serum separator tube (SST) draw.ResultsThe three main subclasses of antibodies IgM, IgA and IgG were analyzed to see the variations in immune responses in the affected population and compared to their microbial RT-PCR based NP swab results. The clinical sensitivity and specificity were determined to be 99.67% and 99.77%. In the matrix comparison study, which would enable patients to test without risk of transmitting the virus, DBS (Dried Blood Spot) matched with higher than 98% accuracy to a venipuncture-based SST collection.ConclusionMultiplex testing enables higher sensitivity and specificity which is essential while establishing exposure on a population scale. This flexible platform along with a discrete collection methodology would be crucial and broadly useful to scale up testing in current and future pandemics. Minimum sample volume that can be collected using DBS cards can be processed in this multiplex pillar plate format enabling the capacity to provide the reliability of high throughput analyzers while having the ease of collection similar to rapid tests.

Validation of a novel and accurate ApoE4 assay for automated chemistry analyzers

The allele ε4 of the apolipoprotein E gene (APOE ε4) is the major genetic risk factor for non-dominantly inherited Alzheimer’s Disease (AD). Current techniques for APOE ε4 carriers identification show good accuracy but have several disadvantages that limit its implementation in a clinical laboratory. These include the need for sample preprocessing, poor automation, low throughput, requirement of additional equipment, and high cost. We followed ISO 13485 guidelines to validate the e4Risk test, a new latex-enhanced immunoturbidimetric blood assay for apolipoprotein E4 (ApoE4) determination in human plasma samples. The test showed high performance in terms of lot to lot variability, precision, interferences, reagents stability, prozone, and detectability. Furthermore, diagnostic accuracy is almost equal (99%) to the gold standard, APOE ε4 genotyping by polymerase chain reaction (PCR). Furthermore, we demonstrated that the e4Risk test can be adapted to any clinical chemistry analyzer, including the high throughput analyzers present in most hospitals and clinical laboratories. The e4Risk test versatility, low cost, and easiness provides an excellent solution for APOE ε4 carriers identification using the same blood sample drawn for biochemical diagnostic work-up of AD patients, which can have important advantages for patient stratification in clinical trials, preventative strategies for AD, and clinical assessment of risk for brain amyloidosis.

The diagnostic potential of the iron-regulatory hormone hepcidin.

The diagnostic potential of the iron-regulatory hormone hepcidin.

Advanced combinational microfluidic multiplexer using multiple levels of control pressures

We propose an advanced combinational microfluidic multiplexing method capable of increasing the number of addressable fluidic channels dramatically. Using only 4 control lines, the proposed advanced combinational multiplexer, utilizing two different levels of control pressure, could address up to 19 fluidic channels, which is at least two times larger than previous multiplexers. The difference between the maximum addressable channels in the present and previous methods increases dramatically when the control lines and control pressure levels increase. The present multiplexer, with its high control efficiency and simple structure for channel addressing, could be used in the application areas of integrated microfluidic systems such as high-throughput analyzers and dynamic pressure generators.

Hybrid modeling framework for process analytical technology: Application toBordetella pertussiscultures

In the process analytical technology (PAT) initiative, the application of sensors technology and modeling methods is promoted. The emphasis is on Quality by Design, online monitoring, and closed-loop control with the general aim of building in product quality into manufacturing operations. As a result, online high-throughput process analyzers find increasing application and therewith high amounts of highly correlated data become available online. In this study, an hybrid chemometric/mathematical modeling method is adopted for data analysis, which is shown to be advantageous over the commonly used chemometric techniques in PAT applications. This methodology was applied to the analysis of process data of Bordetella pertussis cultivations, namely online data of near-infrared, (NIR), pH, temperature and dissolved oxygen, and off-line data of biomass, glutamate, and lactate concentrations. The hybrid model structure consisted of macroscopic material balance equations in which the specific reactions rates are modeled by nonlinear partial least square (PLS). This methodology revealed a significant higher statistical confidence in comparison to PLSs, translated in a reduction of mean squared prediction errors (e.g., individual root mean squared prediction errors calibration/validation obtained through the hybrid model for the concentrations of lactate: 0.8699/0.7190 mmol/L; glutamate: 0.6057/0.2917 mmol/L; and biomass: 0.0520/0.0283 OD; and obtained through the PLS model for the concentrations of lactate: 1.3549/1.0087 mmol/L; glutamate: 0.7628/0.3504 mmol/L; and biomass: 0.0949/0.0412 OD). Moreover, the analysis of loadings and scores in the hybrid approach revealed that process features can, as for PLS, be extracted by the hybrid method.

Pseudohyponatremia revisited: a modern-day pitfall.

Abstract Factitiously low sodium estimations are a hazard in most modern clinical laboratories. Most modern high-throughput analyzers use indirect ion-selective electrodes to estimate electrolyte concentrations in serum samples. This analysis is preceded by a dilution step of the sample. If the water concentration is altered by the presence of increased lipid or protein, the dilution step and the subsequent calculation of concentration by the analyzer results in a falsely low sodium value. This places patients at risk, particularly if the factitious result is acted upon by the physician. In this short review, we highlight this problem and review the methodology and situations where this artifact can occur and discuss strategies to circumvent this problem. When factitious results are suspected, whole blood sodium can be assessed using a direct ion-selective electrode, by measurement of osmolality, or by calculation of the serum water fraction and applying a correction to the reported value.

Pseudohyponatremia Revisited: A Modern-Day Pitfall

Factitiously low sodium estimations are a hazard in most modern clinical laboratories. Most modern high-throughput analyzers use indirect ion-selective electrodes to estimate electrolyte concentrations in serum samples. This analysis is preceded by a dilution step of the sample. If the water concentration is altered by the presence of increased lipid or protein, the dilution step and the subsequent calculation of concentration by the analyzer results in a falsely low sodium value. This places patients at risk, particularly if the factitious result is acted upon by the physician. In this short review, we highlight this problem and review the methodology and situations where this artifact can occur and discuss strategies to circumvent this problem. When factitious results are suspected, whole blood sodium can be assessed using a direct ion-selective electrode, by measurement of osmolality, or by calculation of the serum water fraction and applying a correction to the reported value.

Microfluidic Devices for the Analysis of Single Cells: Leaving No Protein Uncounted

Microfluidic devices are revolutionizing bioanalysis, and designs capable of detecting single protein molecules are now available. Two recently described microfluidic devices provide information on the number of beta(2)-adrenergic receptors in individual cultured insect cells and measure the degradation of phycobilisomes in individual cyanobacteria, respectively. This latter experiment, which included the analysis of three single cells in parallel, heralds a bright future for high-throughput single-cell analyzers. These devices could greatly advance research in signal transduction and studies of the effects of environmental stimuli or xenobiotics on cellular responses.

High Throughput Coagulation Analyzers Review

The increasing no. of ordered coagulation screening tests has led to a remarkable improvement in the efficiency of automated coagulation testing, and appearance of a new generation of high throughput analyzers ensuring accuracy and precision, and reducing the strain and human error. The earliest analyzers operated mechanically, using the hook to detect the clot in the cuvette are now replaced with more sophisticated analyzer that simultaneously uses the clotting, chromogenic, and immunological principles, to provide a versatile test menu covering antigenic and functional aspects of coagulation. The armamentarium of coagulation testing is complimented with tools like; automated platelet function analyzer, flowcytometer, and molecular techniques including the polymerase chain reaction, and lately the microarrary (biochip) technology. This review addresses the principle of operation and distinctive features of various automated high throughput coagulation analyzers, their impact on improving efficiency, eliminating preanalytical and postanalytical handling thus maximizing productivity and return on investment.

Evaluation of the automated coagulation analyzer Sysmex® CA-7000

Introduction Centralization of laboratory diagnostics and an increasing number of urgent requests and nonstandard samples raise the need for short turn-around times and high-throughput analyzers for coagulation tests. The aim of the present study was to evaluate the analytical and technical performance of the Sysmex® CA-7000 coagulation analyzer under routine laboratory conditions. Materials and methods We evaluated the Sysmex® CA-7000 in comparison to the Sysmex® CA-6000 analyzer for PT, INR, aPTT, Clauss, and derived fibrinogen. We also compared antithrombin (AT) measured on the Sysmex® CA-7000 and Dimension® RxL. Imprecision studies were performed, and throughput, online, and STAT functions and the handling of routine samples were evaluated. Results The Sysmex® CA-7000 showed very low intra-assay and interassay variability for all parameters. The method comparison study showed good comparability to the Sysmex® CA-6000 and Dimension® RxL. The throughput of the Sysmex® CA-7000 was 2.5–3 times faster than that of Sysmex® CA-6000. No interference was seen for total bilirubin up to 710 μmol/L. Triglyceride levels >11.4 mmol/L resulted in invalid PT measurements and levels >3.42 mmol/L gave invalid results for Clauss fibrinogen on the CA-7000 analyzer. Conclusions This is the first published evaluation of the analytical and technical performance of the coagulation analyzer Sysmex® CA-7000. We conclude that the CA-7000 is well suited for coagulation laboratories with high sample throughput and a high number of nonstandard samples.

Influence of practicable virus inactivation procedures on tests for frequently measured analytes in plasma.

Clinical specimens from patients carrying highly infectious agents such as corona, Lassa, Ebola, or Marburg viruses may present a biohazard to laboratory workers. Although specialized medical microbiology laboratories amplify and analyze such viruses under the required biosafety measures, few general clinical laboratories have the equipment to perform their analyses without putting their personnel at risk. The recent spread of severe acute respiratory syndrome (SARS) has heightened concern about personnel safety. Although the WHO (1) and the CDC(2) recommend that blood samples from patients in whom SARS is suspected should be analyzed under biosafety level II conditions, most high-throughput analyzers in the clinical chemistry laboratory use open tubes and therefore do not meet the biosafety level II standards. In such cases, the CDC recommends administrative measures and/or additional personal protective equipment to reduce risk. Some manufacturers announced that their analyzers should not be used for samples from patients in whom SARS is suspected, citing possible aerosol production during analysis. Hence, laboratory directors and biosafety officers are in the dilemma of how to offer urgent diagnostic and surveillance tests to clinicians and at the same time protect their coworkers from potential biohazards. Gamma irradiation and heat inactivation procedures have been investigated for the mentioned lipid-enveloped viruses (3)(4). Gamma …

Observations on Centrifugation: Application to Centrifuge Development

This report outlines the background to the development of an automated, serial, discrete centrifuge, reporting on the criteria considered essential in such an instrument. We established the criteria by examining the detailed logistics of centrifuge operation in a hospital laboratory. The mean sample load per run, using six centrifuges, was 13.6 samples, and the user-selectable cycle time ranged from 00:01:10 to 00:12:33 (hours:minutes:seconds) with a fixed g value of 1050. During the laboratory working window, (0900-1700), only 50% of the centrifuge capacity was utilized and more than one-third of the sample workload was delayed for >5 min because the centrifuges were not emptied promptly. In addition, 35% of the sample workload was centrifuged for less than the time prescribed in the operational specifications. Based on these findings, we designed a new continuous, serial centrifuge to overcome some of the deficiencies noted in the logistics study. The centrifuge operates continuously, nominally treating 150 samples/h, with a cycle time of 5 min at 1,000 g. The cycle time and g value are variable between limits, and their selection governs the throughput rate. Each sample is centrifuged separately in individual rotors mounted in a sturdy carousel with a periphery that traverses a load/unload station. There is no sample delay because of operator absence, and the capacity is fully utilized. The centrifuge can operate in a stand-alone capacity or has the capability of being integrated into a sample preparation system or as a direct front end for high-throughput analyzers.

Human genome sequencing to be fast and free

A new joint-venture genomics company plans to overtake the federal government's Human Genome Project by sequencing the entire human genome in only three years at its own expense. announcement by the Institute for Genomic Research (TIGR), Rockville, Md., and Perkin-Elmer Corp. (PE), Norwalk, Conn., surprised those associated with the 15-year, $3 billion federal effort. Researchers have sequenced 4% of the human genome and were planning to sequence the rest over the project's remaining seven years. as-yet-unnamed company is relying on a new generation of high-throughput DNA sequence analyzers developed by PE's Applied Biosystems Division. project will be sited at a single center to be built in Rockville, a Washington, D.C., suburb, and will use an efficient but controversial shotgun sequencing technique. The numbers for this are really quite staggering, TIGR President and Director J. Craig Venter said last week at a press conference at the National Institutes of Health. facility ...

The role of microchemistry in laboratory medicine

During the last 30 years, automation of chemical analysis in clinical chemistry has reduced the quantity of blood serum consumed per analysis from approximately one milliliter to a few microliters. Particularly during the last 15 years, this has been the result of improved designs of automated, high-throughput serum analyzers. One consideration in the design changes has been a need to reduce costs by minimizing reagent consumption. In hematology laboratories, automated microchemical analysis of individual blood cells is replacing manual methods of counting white blood cells. Refined methods of cell analysis are coming into use, which give clinically important information that has not heretofore been available to physicians.