Optimization of Automated Sample Preparation for Vitamin D Determination on a Biomek i7 Workstation

2021 ◽  
pp. 247263032110302
Author(s):  
Anna Bach ◽  
Heidi Fleischer ◽  
Bhagya Wijayawardena ◽  
Kerstin Thurow
Keyword(s):  
Vitamin D ◽  
Sample Preparation ◽  
Reference Method ◽  
Positive Pressure ◽  
Additional Risk Factor ◽  
Serum Samples ◽  
Recovery Rates ◽  
Preparation Methods ◽  
Sample Throughput ◽  
Automated Sample Preparation

Vitamin D belongs to the fat-soluble vitamins and is an integral part of bone metabolism. In the human body, a decreased vitamin D level can be an additional risk factor for diseases like cancer, diabetes, and mental diseases. As a result, an enormous increase in the demand for vitamin D testing has been observed in recent years, increasing the demand for powerful methods for vitamin D determination at the same time. Automation is the key factor in increasing sample throughput. This study compares three fully automated sample preparation methods for the determination of 25(OH)D2 and 25(OH)D3 in plasma and serum samples. Starting from a semiautomated reference method, the method is tested manually and subsequently fully automated on the Biomek i7 Workstation by integrating a centrifuge and a positive pressure extractor into the workstation. Alternatively, the centrifugation for the separation of protein aggregates and supernatant is replaced by a filter plate. Finally, the sample throughput is further increased by using phospholipid removal cartridges. The results show that phospholipid removal significantly increases the recovery rates in liquid chromatography–mass spectrometry. With the phospholipid removal cartridges, recovery rates of 97.36% for 25(OH)D2 and 102.5% for 25(OH)D3 were achieved, whereas with the automated classic automated preparation method, the recovery rates were 83.31% for 25(OH)D2 and 86.54% for 25(OH)D3. In addition to the technical evaluation, the different methods were also examined with regard to their economic efficiency. Finally, the qualitative and quantitative performance of the developed methods is benchmarked with a selected semiautomatic reference method.

scholarly journals Looking beyond linear regression and Bland-Altman plots: a comparison of the clinical performance of 25-hydroxyvitamin D tests

2017 ◽  
Vol 55 (3) ◽  
Author(s):  
Gellert Karvaly ◽  
Katalin Mészáros ◽  
Krisztián Kovács ◽  
Attila Patócs ◽  
Zoltán Sipák ◽  
...  
Keyword(s):  
Vitamin D ◽  
Clinical Performance ◽  
Hypothesis Test ◽  
Reference Method ◽  
Systematic Evaluation ◽  
Assay Method ◽  
Percentage Agreement ◽  
Serum Samples ◽  
Hydroxyvitamin D ◽  
25 Hydroxyvitamin D

AbstractBackground:The systematic evaluation of the clinical concordance of various 25-hydroxyvitamin D (25OHD) testing methods is presented. The need for this approach is raised by the discrepancies in the analytical performance of the available assays.Methods:The analytical and clinical performance of six automated 25OHD assays and an in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was investigated. Leftover serum samples (n=162, SA: n=114) were analyzed and all 21 assay combinations were evaluated. The utility of Cohen’s κ values was assessed by transforming them into minimum percentage agreement (MPA). McNemar’s hypothesis test was employed for testing the symmetry of the disagreeing classification outcomes within each method pair.Results:Depending on the assay method, the ratio of results classified as positive (<20 ng/mL) was 13.5%–40.0%. The percentage agreement (PA) was 74.1%–92.6%. Compared to other methods, significantly more hypovitaminosis cases were delivered by DiaSorin Liaison® 25 OH vitamin D Total (DL) and significantly fewer by IDS-iSYS 25-Hydroxy Vitamin DS (II). The strongest clinical concordance was exerted by II vs. LC-MS/MS. The κ-derived MPA showed close similarity to the PA scores. McNemar’s tests confirmed the asymmetry of the disagreement in the classification in 14 method combinations.Conclusions:The presented approach allows the prediction of the clinical consequences of a 25OHD method transfer. Differences in the clinical classification of assay results are likely encountered when transferring to a new method, even between assays standardized according to the Vitamin D Standardization Program (VDSP) Reference Method Procedure (RMP).

scholarly journals Accuracy-Based Vitamin D Survey: Six Years of Quality Improvement Guided by Proficiency Testing

2019 ◽  
Vol 143 (12) ◽  
pp. 1531-1538 ◽  
Author(s):  
Patrick Erdman ◽  
Darryl E. Palmer-Toy ◽  
Gary Horowitz ◽  
Andrew Hoofnagle
Keyword(s):  
Vitamin D ◽  
Proficiency Testing ◽  
Clinical Laboratory ◽  
Matrix Effects ◽  
Pearson Correlation ◽  
Correlation Coefficients ◽  
Reference Method ◽  
Serum Samples ◽  
Testing Program ◽  
Target Values

Context.— The goal of the College of American Pathologists Accuracy-Based Proficiency Testing Program is to promote the quality, standardization, and harmonization of clinical laboratory results through proficiency testing specimens that are free from matrix effects, have target values that are traceable to reference methods, and that probe the limitations of current methods. Objective.— To summarize the first 6 years of the Accuracy-Based Vitamin D Survey and highlight key insights from the data generated as it relates to assay performance. Design.— Accuracy-based challenges were created by using pooled human serum samples. Certain samples were derived from participants in an institutional review board–approved protocol in which vitamin D–deficient participants were treated with ergocalciferol (vitamin D2). Reference targets for the survey were set by the Centers for Disease Control and Prevention using isotope-dilution liquid chromatography–tandem mass spectrometry. Each method was compared with the reference method procedure over the course of the program (n = 43 proficiency testing samples). Results.— Linear regression versus the reference method procedure revealed proportional biases across the methods, ranging from 0.0% to 16.7%. Pearson correlation coefficients (r2) ranged from 0.902 to 0.996. Results were influenced by the concentration of 25-hydroxyvitamin D2 as well as the C-3 epimer of 25-hydroxyvitamin D3. During the 6 years, 2 manufacturers altered their assays to match the reference method procedure more closely. Conclusions.— There is considerable bias, both proportional bias and sample-specific matrix effects, affecting many assays. This ongoing accuracy-based proficiency testing program for vitamin D will provide the data needed for laboratories and manufacturers to improve their assays and thereby patient care.

scholarly journals Evaluation of Applied Biosystems MicroSEQ® Real-Time PCR System for Detection of Listeria monocytogenes in Food

2011 ◽  
Vol 94 (5) ◽  
pp. 1481-1489 ◽  
Author(s):  
Robert S Tebbs ◽  
Priya Balachandran ◽  
Lily Y Wong ◽  
Patrick Zoder ◽  
Manohar R Furtado ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Sample Preparation ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Cow’S Milk ◽  
Preparation Methods ◽  
Cow's Milk ◽  
Smoked Salmon ◽  
Sample Preparation Methods

Abstract Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. this report describes the validation of a new Real-time PCR method to detect Listeria monocytogenes in nine different food matrixes. the complete system consists of the MicroSEQ®L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-time PCR instrument, and RapidFinder™ Express software. two sample preparation methods were validated: the PrepSEQ® Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. the test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. the MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. the MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.

scholarly journals Multicenter Comparison of Seven 25Oh Vitamin D Automated Immunoassays / Multicentrično Poređenje Sedam Automatizovanih Imunoeseja Za 25Oh Vitamin D

2015 ◽  
Vol 34 (3) ◽  
pp. 344-350 ◽  
Author(s):  
Giuseppe Lippi ◽  
Gian Luca Salvagno ◽  
Antonio Fortunato ◽  
Mariella Dipalo ◽  
Rosalia Aloe ◽  
...  
Keyword(s):  
Vitamin D ◽  
Reference Method ◽  
Excellent Correlation ◽  
Serum Samples ◽  
Performance Goal ◽  
Clinical Laboratories ◽  
Hplc Technique ◽  
Routine Assessment ◽  
Minimum Performance ◽  
Abbott Architect

SummaryBackground: The measurement of 25OH vitamin D continues to grow in clinical laboratories. The aim of this multicenter study was to compare the results of seven automated commercial immunoassays with a reference HPLC technique.Methods: One hundred and twenty consecutive outpatient serum samples were centrifuged, divided in aliquots, frozen and shipped to the participating laboratories. 25OH Vita - min D was measured with a reference HPLC system and with seven automated commercial immunoassays (Roche Cobas E601, Beckman Coulter Unicel DXI 800, Ortho Vitros ES, DiaSorin Liaison, Siemens Advia Centaur, Abbott Architect i System and IDS iSYS).Results: Compared to the reference method, the regression coefficients ranged from 0.923 to 0.961 (all p<0.001). The slope of Deming fit ranged from 0.95 to 1.06, whereas the intercept was comprised between -15.2 and 9.2 nmol/L. The bias from the reference HPLC technique varied from 14.5 to 8.7 nmol/L. The minimum performance goal for bias was slightly exceeded by only one immunoassay. The agreement between HPLC and the different immunoassays at 50 nmol/L 25OH Vitamin D varied between 0.61 and 0.85 (all p<0.001). The percentage of samples below this cut-off was significantly different with only one immunoassay.Conclusions:The excellent correlation with the reference HPLC technique attests that all seven automated immuno - assays may be reliably used for routine assessment of 25OH-D in clinical laboratories. The significant bias among the different methods seems mostly attributable to the lack of standardization and calls for additional efforts for improving harmonization of 25OH-D immunoassays.

scholarly journals Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents

2008 ◽  
Vol 54 (4) ◽  
pp. 665-672 ◽  
Author(s):  
Rita Hannisdal ◽  
Asbjørn Svardal ◽  
Per Magne Ueland
Keyword(s):  
Sample Preparation ◽  
Acid Hydrolysis ◽  
Degradation Products ◽  
Serum Folate ◽  
Serum Samples ◽  
Limit Of Quantification ◽  
Fresh Serum ◽  
Liquid Chromatography Tandem Mass ◽  
Automated Sample Preparation ◽  
Good Agreement

Abstract Background: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis. Methods: Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Results: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25–96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was ≤5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay. Conclusions: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.

Validation of Solus One Salmonella from Select Food Matrixes and Stainless-Steel and Plastic Environmental Surfaces

2019 ◽  
Vol 102 (4) ◽  
pp. 1145-1161
Author(s):  
David Higgins ◽  
Holly Urquhart ◽  
Siobhan Kelly ◽  
Simon Illingworth ◽  
Nevin Perera ◽  
...  
Keyword(s):  
Stainless Steel ◽  
Sample Preparation ◽  
Preparation Method ◽  
Comparison Study ◽  
Sample Preparation Method ◽  
Preparation Methods ◽  
Environmental Surfaces ◽  
Automated Sample Preparation ◽  
The U.S ◽  
Sample Preparation Methods

Abstract Background: Solus One Salmonella is designed to accurately detect Salmonella species (Salmonella enterica subspecies enterica, salamae, arizonae, diarizonae, houtenae, indica, and Salmonella bongori) from select food matrixes and stainless-steel and plastic environmental surfaces. Solus One Salmonella uses an antibody-based technology test system that is paired with media and our proprietary media supplement, the Solus One Salmonella supplement combined with a manual or automated sample preparation method. Objective: Solus One Salmonella was evaluated for inclusivity and exclusivity, and a matrix comparison study was done for six food matrixes (raw beef trim, pasteurized liquid egg, raw salmon, cheddar cheese, Romaine lettuce, nonfat dry milk) and two environmental surfaces (stainless steel and polystyrene). Methods: Solus One Salmonella was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5: Salmonella (July 2018) and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Manual, 4.09 (January 2017) in the matrix study. Both the manual and automated sample preparation methods were performed for cheddar cheese and stainless-steel environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Salmonella correctly detected all 108 target organism isolates and correctly excluded all 35 nontarget strains that were analyzed. Conclusions: In the method comparison study, both Solus One Salmonella manual and automated sample preparation methods demonstrated no significant differences based on probability of detection (POD) statistical analysis between presumptive and confirmed results or between candidate and reference method results for the six food matrixes after 20–22 h and two environmental surfaces after 16–20 h of enrichment time. POD analysis of Solus One Salmonella method robustness, product consistency, and stability studies using the automated sample preparation method demonstrated no statistically significant differences.

Improving Sample Preparation Methods For Cannabis-Infused Edible And Topical Products

Planta Medica ◽  
2016 ◽  
Vol 82 (05) ◽  
Author(s):  
M Wilcox ◽  
M Jacyno ◽  
J Marcu ◽  
J Neal-Kababick
Keyword(s):  
Sample Preparation ◽  
Preparation Methods ◽  
Sample Preparation Methods

Methodology for TEM Analysis of Barrier Profiles

2002 ◽  
Author(s):  
Tan-Chen Lee ◽  
Jui-Yen Huang ◽  
Li-Chien Chen ◽  
Ruey-Lian Hwang ◽  
David Su
Keyword(s):  
Sample Preparation ◽  
Profile Analysis ◽  
Image Interpretation ◽  
Sample Thickness ◽  
Image Resolution ◽  
Specimen Preparation ◽  
Tem Analysis ◽  
Preparation Methods ◽  
High Image Resolution ◽  
Common Technique

Abstract Device shrinkage has resulted in thinner barriers and smaller vias. Transmission Electron Microscopy (TEM) has become a common technique for barrier profile analysis because of its high image resolution. TEM sample preparation and image interpretation becomes difficult when the size of the small cylindrical via is close to the TEM sample thickness. Effects of different sample thickness and specimen preparation methods, therefore, have been investigated. An automatic FIB program has been shown to be useful in via sample preparation. Techniques for imaging a TEM specimen will be discussed in the paper. Conventional TEM bright field (BF) image is adequate to examine the barrieronly via; however, other techniques are more suitable for a Cu filled via.

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