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Evaluation of assay improvement options for an existing clinical vitamin K assay
1100
Abstracts
Standards calibration verifiers. Correlation with the current ARCHITECT Magnesium assay (List #7D70) was analyzed using patient samples (n = 60). Potential interferences (hemoglobin, lipemia, bilirubin, and ascorbate) were examined in patient pools at high and low magnesium concentrations. Reference interval verification was performed with samples from healthy volunteers. Results: The ARCHITECT Magnesium assay displayed imprecision of 1.7% at 0.72 mmol/L and 1.4% at 1.8 mmol/L (20 days, one calibration, one reagent lot). The linear measurable range was verified between 0.18 and 2.70 mmol/L. Results of the new assay (x) compared well with the predicate assay (y) with the relationship y = 0.951x − 0.023; R = 0.9747. This method showed b10% error for interference by hemoglobin (3 g/L as hemolysate), lipemia (20 g/L Intralipid), unconjugated bilirubin (1026 μmol/L), and ascorbate (680 μmol/L). The observed reference interval in adults was consistent with the manufacturer's stated values (0.66–1.07 mmol/L). Conclusions: The next generation ARCHITECT Magnesium assay provides acceptable analytical characteristics for the measurement of magnesium in patient samples. In comparison to the current assay, notable improvements are seen in assay precision, 30-day calibration stability, and minimal interference in hemolyzed and lipemic samples. doi:10.1016/j.clinbiochem.2012.07.006
P504 Evaluation of assay improvement options for an existing clinical vitamin K assay V.E. Barakauskasa, E.L. Franka,b a Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA b ARUP Laboratories, Salt Lake City, UT, USA Objectives: This study sought to identify options for increasing assay throughput, reducing sample volume requirements and improving calibrator matrix of a high-volume reference test for serum/plasma vitamin K (vitK). Methods: Measurement involves liquid–liquid and normal-phase solid-phase extraction (NP-SPE), reverse-phase high-pressure liquid chromatography (RP-HPLC) and fluorescent detection. Calibrators are made in delipidated plasma, which can be contaminated with organic solvents. Light-based depletion of vitK was evaluated. To increase sample throughput, different sample extraction and chromatographic conditions were investigated. Volume reduction was tested using the existing NP-SPE method, smaller capacity NP-SPE columns (200 mg/ 3 mL, 100 mg/1 mL) and RP-SPE methods. Results: UV-light exposure (3 h) depleted plasma of vitK. Recovery was lower from spiked UV-depleted plasma compared to delipidated plasma (3.6–11.3% less). Two smaller dimension RP-HPLC columns were evaluated — Phenomenex Luna C18(2) (50 × 3.0 mm, 2.5 μm) and Agilent Poroshell-120 EC-C18 (50 × 3mm, 2.7 μm). Both performed similarly to the existing column (average %bias: −3.4%, 2.3% respectively) with good peak resolution (R = 2.8). VitK eluted 4.4 min sooner with 4-fold less solvent used. Optimal PMT settings (12), data collection frequency (1 s), and signal linearity (100-fold) were determined. Using NP-SPE with reduced sample volume (0.5 mL), vitK recovery was 105–115% and independent of SPE column size. Of the RP-SPE methods evaluated, only C18-SPE performed adequately with shorter sample processing times. Conclusions: UV-depleted plasma improves matrix matching of calibrators. A smaller HPLC column reduces sample analysis time and reagent usage. Reduced sample volume and smaller scale SPE appear viable. doi:10.1016/j.clinbiochem.2012.07.007
P505 Urine arsenic speciation in clinical toxicology: Usefulness and interpretation Patrick Bélanger, Normand Fleury Laboratoire de Toxicologie, Direction de la santé environnementale et de la Toxicology, Nstitut National de Santé Publique du Québec, 945, Avenue Wolfe, 4e étage, Sainte-Foy (Québec), Canada G1V 5B3 Objectives: Analysis of the five main arsenic species in urine allows us to get important information about potential source of exposition, real level of toxicity and if the sample has been altered post-micturition. Toxicity of arsenic has been widely demonstrated. Analysis of arsenic species in urine of exposed people (worker usually) is necessary to establish the true toxicity level. Inorganic arsenic species, Arsenite (As(III)) and Arsenate (As(V)), are the main naturally-occurring arsenic species in the environment and are identified as having the highest level of toxicity. Seafood usually contains less toxic arsenic compounds such of arsenobetaine (AsB) and arsenosugars. These compounds are the end of the arsenic metabolic pathway (seafood), and their polarity and inertia allows for fast elimination. There are many proposed metabolic elimination pathways. In human urine, di-methylated arsenic species (DMA) are more abundant, which demonstrates the terminal metabolism pathway of inorganic arsenic. Results: Analytical method used (Atom. Spectrosc., 2010;31(6): 175-181) allows us to get the level of the five main arsenic species in urine in less than 4 min per sample.
Conclusions: After some years of routine clinical analysis of arsenic speciation, we observed some trend. These observations can help in the diagnostic or source of exposition in arsenic contaminated people. doi:10.1016/j.clinbiochem.2012.07.008
P506 Rehaussement du programme de dépistage néonatal sanguin par MSMS au Québec: Validation De La Trousse Néobase De Perkin-Elmer Marie-Thérèse Berthier, Jean-Guy Girard, Mohamed Chouaibou, Yves Giguère Programme québécois de Dépistage Néonatal Sanguin, Service de biochimie, Département de Biologie Médicale, CHUQ-Hôpital pavillon St François d'Assise, Québec, Canada Objectifs: Le CHUQ effectue le dépistage sanguin de tous les nouveau-nés de la province pour l'hypothyroïdie congénitale, la phénylcétonurie et la tyrosinémie type I. En septembre 2011, la déficience en MCAD (medium chain acyl carnitines dehydrogenase) a été ajoutée. Cette maladie, souvent mortelle si non dépistée, est traitable en présence d'une intervention précoce. Nous avons rehaussé le Programme via l'acquisition de deux UPLC-MSMS. Nous avons validé la méthode afin de pouvoir dépister jusqu'à 43 maladies métaboliques héréditaires en utilisant la nouvelle trousse de dépistage Neobase sans dérivation de Perkin Elmer, en vue des possibles futurs mandats ministériels.
Abstracts
Standards calibration verifiers. Correlation with the current ARCHITECT Magnesium assay (List #7D70) was analyzed using patient samples (n = 60). Potential interferences (hemoglobin, lipemia, bilirubin, and ascorbate) were examined in patient pools at high and low magnesium concentrations. Reference interval verification was performed with samples from healthy volunteers. Results: The ARCHITECT Magnesium assay displayed imprecision of 1.7% at 0.72 mmol/L and 1.4% at 1.8 mmol/L (20 days, one calibration, one reagent lot). The linear measurable range was verified between 0.18 and 2.70 mmol/L. Results of the new assay (x) compared well with the predicate assay (y) with the relationship y = 0.951x − 0.023; R = 0.9747. This method showed b10% error for interference by hemoglobin (3 g/L as hemolysate), lipemia (20 g/L Intralipid), unconjugated bilirubin (1026 μmol/L), and ascorbate (680 μmol/L). The observed reference interval in adults was consistent with the manufacturer's stated values (0.66–1.07 mmol/L). Conclusions: The next generation ARCHITECT Magnesium assay provides acceptable analytical characteristics for the measurement of magnesium in patient samples. In comparison to the current assay, notable improvements are seen in assay precision, 30-day calibration stability, and minimal interference in hemolyzed and lipemic samples. doi:10.1016/j.clinbiochem.2012.07.006
P504 Evaluation of assay improvement options for an existing clinical vitamin K assay V.E. Barakauskasa, E.L. Franka,b a Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA b ARUP Laboratories, Salt Lake City, UT, USA Objectives: This study sought to identify options for increasing assay throughput, reducing sample volume requirements and improving calibrator matrix of a high-volume reference test for serum/plasma vitamin K (vitK). Methods: Measurement involves liquid–liquid and normal-phase solid-phase extraction (NP-SPE), reverse-phase high-pressure liquid chromatography (RP-HPLC) and fluorescent detection. Calibrators are made in delipidated plasma, which can be contaminated with organic solvents. Light-based depletion of vitK was evaluated. To increase sample throughput, different sample extraction and chromatographic conditions were investigated. Volume reduction was tested using the existing NP-SPE method, smaller capacity NP-SPE columns (200 mg/ 3 mL, 100 mg/1 mL) and RP-SPE methods. Results: UV-light exposure (3 h) depleted plasma of vitK. Recovery was lower from spiked UV-depleted plasma compared to delipidated plasma (3.6–11.3% less). Two smaller dimension RP-HPLC columns were evaluated — Phenomenex Luna C18(2) (50 × 3.0 mm, 2.5 μm) and Agilent Poroshell-120 EC-C18 (50 × 3mm, 2.7 μm). Both performed similarly to the existing column (average %bias: −3.4%, 2.3% respectively) with good peak resolution (R = 2.8). VitK eluted 4.4 min sooner with 4-fold less solvent used. Optimal PMT settings (12), data collection frequency (1 s), and signal linearity (100-fold) were determined. Using NP-SPE with reduced sample volume (0.5 mL), vitK recovery was 105–115% and independent of SPE column size. Of the RP-SPE methods evaluated, only C18-SPE performed adequately with shorter sample processing times. Conclusions: UV-depleted plasma improves matrix matching of calibrators. A smaller HPLC column reduces sample analysis time and reagent usage. Reduced sample volume and smaller scale SPE appear viable. doi:10.1016/j.clinbiochem.2012.07.007
P505 Urine arsenic speciation in clinical toxicology: Usefulness and interpretation Patrick Bélanger, Normand Fleury Laboratoire de Toxicologie, Direction de la santé environnementale et de la Toxicology, Nstitut National de Santé Publique du Québec, 945, Avenue Wolfe, 4e étage, Sainte-Foy (Québec), Canada G1V 5B3 Objectives: Analysis of the five main arsenic species in urine allows us to get important information about potential source of exposition, real level of toxicity and if the sample has been altered post-micturition. Toxicity of arsenic has been widely demonstrated. Analysis of arsenic species in urine of exposed people (worker usually) is necessary to establish the true toxicity level. Inorganic arsenic species, Arsenite (As(III)) and Arsenate (As(V)), are the main naturally-occurring arsenic species in the environment and are identified as having the highest level of toxicity. Seafood usually contains less toxic arsenic compounds such of arsenobetaine (AsB) and arsenosugars. These compounds are the end of the arsenic metabolic pathway (seafood), and their polarity and inertia allows for fast elimination. There are many proposed metabolic elimination pathways. In human urine, di-methylated arsenic species (DMA) are more abundant, which demonstrates the terminal metabolism pathway of inorganic arsenic. Results: Analytical method used (Atom. Spectrosc., 2010;31(6): 175-181) allows us to get the level of the five main arsenic species in urine in less than 4 min per sample.
Conclusions: After some years of routine clinical analysis of arsenic speciation, we observed some trend. These observations can help in the diagnostic or source of exposition in arsenic contaminated people. doi:10.1016/j.clinbiochem.2012.07.008
P506 Rehaussement du programme de dépistage néonatal sanguin par MSMS au Québec: Validation De La Trousse Néobase De Perkin-Elmer Marie-Thérèse Berthier, Jean-Guy Girard, Mohamed Chouaibou, Yves Giguère Programme québécois de Dépistage Néonatal Sanguin, Service de biochimie, Département de Biologie Médicale, CHUQ-Hôpital pavillon St François d'Assise, Québec, Canada Objectifs: Le CHUQ effectue le dépistage sanguin de tous les nouveau-nés de la province pour l'hypothyroïdie congénitale, la phénylcétonurie et la tyrosinémie type I. En septembre 2011, la déficience en MCAD (medium chain acyl carnitines dehydrogenase) a été ajoutée. Cette maladie, souvent mortelle si non dépistée, est traitable en présence d'une intervention précoce. Nous avons rehaussé le Programme via l'acquisition de deux UPLC-MSMS. Nous avons validé la méthode afin de pouvoir dépister jusqu'à 43 maladies métaboliques héréditaires en utilisant la nouvelle trousse de dépistage Neobase sans dérivation de Perkin Elmer, en vue des possibles futurs mandats ministériels.