Corrigendum to: Comparison of Five Common Analyzers in the Measurement of Chemistry Analytes in an Authentic Cohort of Body Fluid Specimens

Corrigendum to "Comparison of Five Common Analyzers in the Measurement of Chemistry Analytes in an Authentic Cohort of Body Fluid Specimens

Comparison of Five Common Analyzers in the Measurement of Chemistry Analytes in an Authentic Cohort of Body Fluid Specimens

OBJECTIVES: Interpretation of body fluid (BF) results is based on published studies and clinical guidelines. The aim of this study is to determine whether the assays from five common commercial vendors produce similar results in BFs for 12 analytes in a BF cohort. METHODS: BFs (n = 25) and serum (n = 5) were analyzed on five instruments (Roche cobas c501, Ortho 5600, Beckman AU5800 and DXI800, Siemens Vista 1500, and Abbott Architect c8000) to measure albumin, amylase, total bilirubin, cholesterol, creatinine, glucose, lactate dehydrogenase (LDH), lipase, total protein, triglycerides, urea nitrogen, and carcinoembryonic antigen. Deming regression and Bland-Altman analysis were used for method comparison to Roche. RESULTS: Results were significantly different from Roche for LDH and lipase on Ortho and lipase on Siemens but similar for both BFs and serum. BF differences were larger than serum differences when measuring creatinine, glucose, and urea nitrogen on Ortho and glucose on Siemens. CONCLUSIONS: Five instruments used to perform BF testing produce results that are not significantly different except for lipase and LDH measurements. Bias of similar magnitude observed in both BF and serum should not affect interpretation. Further investigations into Ortho and Siemens measuring glucose and Ortho measuring creatinine and urea nitrogen are warranted

Comparison of Five Common Analyzers in the Measurement of Chemistry Analytes in an Authentic Cohort of Body Fluid Specimens

Abstract Objectives Interpretation of body fluid (BF) results is based on published studies and clinical guidelines. The aim of this study is to determine whether the assays from five common commercial vendors produce similar results in BFs for 12 analytes in a BF cohort. Methods BFs (n = 25) and serum (n = 5) were analyzed on five instruments (Roche cobas c501, Ortho 5600, Beckman AU5800 and DXI800, Siemens Vista 1500, and Abbott Architect c8000) to measure albumin, amylase, total bilirubin, cholesterol, creatinine, glucose, lactate dehydrogenase (LDH), lipase, total protein, triglycerides, urea nitrogen, and carcinoembryonic antigen. Deming regression and Bland-Altman analysis were used for method comparison to Roche. Results Results were significantly different from Roche for LDH and lipase on Ortho and lipase on Siemens but similar for both BFs and serum. BF differences were larger than serum differences when measuring creatinine, glucose, and urea nitrogen on Ortho and glucose on Siemens. Conclusions Five instruments used to perform BF testing produce results that are not significantly different except for lipase and LDH measurements. Bias of similar magnitude observed in both BF and serum should not affect interpretation. Further investigations into Ortho and Siemens measuring glucose and Ortho measuring creatinine and urea nitrogen are warranted. </jats:sec

Substrate-Mediated Oxygen Activation by Homoprotocatechuate 2,3-Dioxygenase: Intermediates Formed by a Tyrosine 257 Variant

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O₂ bind in adjacent ligand sites of the active site Fe^II of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe^II to O₂ gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases <i>k</i>_cat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F_Int1^HPCA and Y257F_Int2^HPCA, accumulate during the Y257F-HPCA + O₂ reaction prior to formation of the ring-cleaved product. Y257F_Int1^HPCA is colorless and is formed as O₂ binds reversibly to the HPCA–enzyme complex. Y257F_Int2^HPCA forms spontaneously from Y257F_Int1^HPCA and displays a chromophore at 425 nm (ε₄₂₅ = 10 500 M^–1 cm^–1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe^II. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe^II, and the low O₂ affinity suggest that Y257F_Int1^HPCA is an HPCA-Fe^II-O₂ complex with little electron delocalization onto the O₂. In contrast, the intense spectrum of Y257F_Int2^HPCA suggests the intermediate is most likely an HPCA quinone-Fe^II-(hydro)­peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O₂

Volumetric Microsampling of Capillary Blood Spot vs Whole Blood Sampling for Therapeutic Drug Monitoring of Tacrolimus and Cyclosporin A: Accuracy and Patient Satisfaction

Abstract Background Immunosuppressant therapeutic drug monitoring (TDM) usually requires outpatient travel to hospitals or phlebotomy sites for venous blood collection; however Mitra® Microsampling Device (MSD) sampling could allow self-collection and shipping of samples to a laboratory for analysis. This study examined the feasibility of using volumetric microsampling by MSD for TDM of tacrolimus (TaC) and cyclosporin A (CsA) in transplant patients, along with their feedback on the process. Methods MSD was used to collect TaC and CsA from venous (VB) or capillary (CB) blood. The MSDs were rehydrated, extracted, and analyzed using on-line solid phase extraction coupled to tandem mass spectrometry (SPE-MS/MS). We report an abbreviated method validation of the MSD including: accuracy, precision, linearity, carry-over, and stability using residual venous whole blood (VB) samples. Subsequent clinical validation compared serially collected MSD + CB against VB (200 µL) from transplant patients. Results Accuracy comparing VB vs. MSD+VB showed high clinical concordance (TaC = 89% and CsA = 98%). Inter- and intra-precision was ≤11.5 %CV for TaC and CsA. Samples were stable for up to 7 days at room temperature with an average difference of &amp;lt;10%. Clinical validation with MSD+CB correlated well with VB for CsA (slope = 0.95, r2 = 0.88, n = 47) and TaC (slope = 0.98, r2 = 0.82, n = 49). CB vs. VB gave concordance of 94% for CsA and 79% for TaC. A satisfaction survey showed 82% of patients preferred having the capillary collection option. Conclusion Transplant patients favored having the ability to collect capillary samples at home for TaC/CsA monitoring. Our results demonstrate good concordance between MSD+CB and VB for TaC and CsA TDM, but additional studies are warranted. </jats:sec

Substrate-Mediated Oxygen Activation by Homoprotocatechuate 2,3-Dioxygenase: Intermediates Formed by a Tyrosine 257 Variant

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O(2) bind in adjacent ligand sites of the active site Fe(II) of Homoprotocatechuate 2,3-Dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe(II) to O(2) gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases k(cat) by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed [Formula: see text] and [Formula: see text] , accumulate during the Y257F-HPCA + O(2) reaction prior to formation of the ring-cleaved product. [Formula: see text] is colorless and is formed as O(2) binds reversibly to the HPCA-enzyme complex. [Formula: see text] forms spontaneously from [Formula: see text] and displays a chromophore at 425 nm (ε(425) = 10,500 M-1 cm(−1)). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe(II). The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe(II), and the low O(2) affinity suggests that [Formula: see text] is an HPCA-Fe(II)-O(2) complex with little electron delocalization onto the O(2). In contrast, the intense spectrum of [Formula: see text] suggests the intermediate is most likely an HPCA quinone-Fe(II)-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O(2)