The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

Stability and Analytical Characterization of Voriconazole as Measured by Immunoassay.

BACKGROUND: Voriconazole is a broad-spectrum triazole antifungal agent recommended for invasive fungal diseases, including invasive aspergillosis. Therapeutic drug monitoring via voriconazole target trough concentration is important to ensure efficacy while preventing toxicity. Our aim was to determine the stability of voriconazole as adapted and measured by an immunoassay. METHODS: Plasma from patient samples (n = 45) evaluated by a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was compared against an ARK immunoassay method, adapted and optimized on the Abbott Alinity c analyzer. Stability of voriconazole and analytical performance of ARK immunoassay was assessed, including functional sensitivity, limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ), linearity, and precision. RESULTS: ARK voriconazole immunoassay was highly correlated (Pearson R = 0.988) to the LC-MS/MS method, with an average bias of 0.09 mg/L (2%). CV at LoQ of 0.5 mg/L was 3.7% while the functional sensitivity was established at 0.05 mg/L. Overall imprecision with liquid quality control material obtained from ARK was 5.0%, 6.3%, and 5.9% at 1 mg/L, 5 mg/L, and 10 mg/L, respectively. Limit of blank and LoD were 0.02 mg/L and 0.05 mg/L, respectively. Voriconazole in lithium heparin plasma separator tube declines over time, with a decrease that is more evident near or above toxic concentrations. CONCLUSION: Voriconazole collected in gel separation tubes declines over time, possibly due to absorptive properties. Voriconazole measurements by immunoassay and LC-MS/MS demonstrated acceptable comparability with sufficient level of sensitivity and precision.

Factors to consider with estimated low-density lipoprotein cholesterol using the new Sampson/NIH equation.

INTRODUCTION: The most commonly utilized method for determining low-density lipoprotein cholesterol (LDLc) is by Friedewald estimation (FeLDLc). A new approach to better estimate LDLc has been proposed by Sampson et al. 2020, known as the Sampson/National Institutes of Health (NIH) estimation of LDLc (NeLDLc), to overcome the limitations of FeLDLc. Non-high-density lipoprotein-cholesterol (Non-HDLc), has equivalent cut-offs to LDLc, established by the 2021 Canadian Cardiovascular Society (CCS) guideline. We hypothesized that NeLDLc remains an inadequate substitute at high triglyceride levels when compared to Non-HDLc. METHODS: A retrospective analysis of 120,959 lipid profiles (47085 patients) spanning five years across a large academic medical center was utilized for comparison of NeLDLc and FeLDLc relative to Non-HDLc as a function of triglyceride content. Regression and concordance between calculated methods were determined at various triglyceride levels to determine optimal utilization of NeLDLc. RESULTS: NeLDLc is generally more correlated and has greater concordance than FeLDLc with Non-HDLc. NeLDLc with increasing triglycerides can produce negatively erroneous results, even with triglycerides < 4.52 mmol/L (400 mg/dL). The largest variation of NeLDLc results is notable at < 0.5 mmol/L (19 mg/dL). Currently, the 2021 CCS guideline recommends reliance on Non-HDLc when triglycerides are > 1.5 mmol/L (133 mg/dL). With the use of NeLDLc, this triglyceride cut-off can be increased to 1.7 mmol/L(150 mg/dL), making it consistent with the hypertriglyceridemia flagging limit. CONCLUSION: NeLDLc offers increased concordance and correlation to Non-HDLc when compared to FeLDLc. However, caution is warranted when triglycerides are > 4.5 mmol/L and when NeLDLc results are < 0.5 mmol/L. Adopting NeLDLc enables flagging at 1.7 mmol/L (vs. 1.5 mmol/L) of triglycerides to suggest reliance on Non-HDLc while simultaneoulsly indicating hypertriglyceridemia.

Evaluation of hemolysis, lipemia, and icterus interference with common clinical immunoassays.

OBJECTIVES: Hemolysis, icterus, and lipemia (HIL) are common sources of endogenous interference in clinical laboratory testing. Defining the threshold of interference for immunoassays enables appropriate reporting of their results when they are affected by HIL. METHODS: Pools of residual patient serum samples were spiked with a known amount of interferent to create samples with varying concentrations of hemolysate, bilirubin, and Intralipid that mimicked the effects of endogenous HIL. Samples were analysed on the Alinity i analyser (Abbott Diagnostics) for more than 25 immunoassays. The average recovery relative to the non-spiked sample was calculated for each interference level and was compared to a predefined allowable bias. RESULTS: C-peptide, estradiol, serum folate, free T4, homocysteine, insulin, and vitamin B12 were found to be affected by hemolysis, at hemoglobin concentrations between 0.3 to 20 g/L. Immunoassays for BNP, estradiol, free T3, and homocysteine were affected by icterus at conjugated bilirubin concentrations between 50 to 1,044 µmol/L. BNP, serum folate, and homocysteine were affected by Intralipid with measured triglyceride concentrations between 0.8 to 10 mmol/L. Lastly, serological immunoassays for HIV and hepatitis A, B and C were also affected by interferences. CONCLUSIONS: Immunoassays are impacted by varying degrees of HIL interference. Some measurands, in the presence of interference, are affected in a manner not previously indicated. The data presented herein provide an independent evaluation of HIL thresholds and will be of aid to resource-limited clinical laboratories that are unable to internally verify endogenous interferences when implementing the Alinity i analyser.

Identification of core allosteric sites through temperature- and nucleus-invariant chemical shift covariance.

Allosteric regulation is essential to control biological function. In addition, allosteric sites offer a promising venue for selective drug targeting. However, accurate mapping of allosteric sites remains challenging since allostery relies on often subtle, yet functionally relevant, structural and dynamical changes. A viable approach proposed to overcome such challenge is chemical shift covariance analysis (CHESCA). Although CHESCA offers an exhaustive map of allosteric networks, it is critical to define the core allosteric sites to be prioritized in subsequent functional studies or in the design of allosteric drugs. Here, we propose two new CHESCA-based methodologies, called temperature CHESCA (T-CHESCA) and CLASS-CHESCA, aimed at narrowing down allosteric maps to the core allosteric residues. Both T- and CLASS-CHESCAs rely on the invariance of core inter-residue correlations to changes in the chemical shifts of the active and inactive conformations interconverting in fast exchange. In T-CHESCA the chemical shifts of such states are modulated through temperature changes, while in CLASS-CHESCA through variations in the spin-active nuclei involved in pairwise correlations. T- and CLASS-CHESCAs, as well as complete-linkage CHESCA, were applied to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC), which serves as a prototypical allosteric switch. Residues consistently identified by the three CHESCA methods were found in previously identified EPAC allosteric core sites. Hence, T-, CLASS-, and CL-CHESCA provide a toolset to establish allosteric site hierarchy and triage allosteric sites to be further analyzed by mutations and functional assays. Furthermore, the core allosteric networks selectively revealed through T- and CLASS-CHESCA are expected to facilitate the mechanistic understanding of disease-related mutations and the design of selective allosteric modulators.

Alerting to acute kidney injury - Challenges, benefits, and strategies.

Acute Kidney Injury (AKI) is a complex heterogeneous syndrome that often can go unrecognized and is encountered in multiple clinical settings. One strategy for proactive identification of AKI has been through electronic alerts (e-alerts) to improve clinical outcomes. The two traditional criteria for AKI diagnosis and staging have been urinary output and serum creatinine. The latter has dominated in aiding identification and prediction of AKI by alert models. While creatinine can provide information to estimate glomerular filtration rate, the utility to depict real-time change in rapidly declining kidney function is paradoxical. Alerts for AKI have recently been popularized by several studies in the UK showcasing the various use cases for detection and management by simply relying on creatinine changes. Predictive models for real-time alerting to AKI have also gone beyond simple delta checks of creatinine as reviewed here, and hold promise to leverage data contained beyond the laboratory domain. However, laboratory data still remains vital to e-alerts in AKI. Here, we highlight a select number of approaches for real-time alerting to AKI built on traditional consensus definitions, evaluate impact on clinical outcomes from e-alerts, and offer critiques on new and expanded definitions of AKI.

Measuring magnesium - Physiological, clinical and analytical perspectives.

Magnesium is the fourth most abundant cation in the human body, essential for physiological processes and is the electrolyte with levels commonly deranged in critically ill patients. These derangements of magnesium imbalance can go unnoticed and result in poor clinical outcomes, requiring both worthy attention to abnormal values and accurate tools and methods to measure magnesium reliably. At present, clinical laboratories employ various methodologies for measuring magnesium in blood and urine. This review aims to address the role of magnesium from not only physiological and pathophysiological perspectives, but importantly to review the methods for measuring magnesium with relevant analytical considerations. Given the role of magnesium and drugs for various treatments, measuring magnesium has become more relevant as drugs can lead to magnesium imbalances. Clinical manifestations and etiology of magnesium imbalance as divided into hypomagnesemia and hypermagnesemia are also reviewed.

Optimizing Measurement and Interpretation of the G6PD/Hb Ratio.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD)/hemoglobin (Hb) ratio helps detect G6PD deficiency, an X-linked disorder that can be asymptomatic or cause acute hemolytic anemia and chronic hemolysis. We investigated preanalytical, analytical, and postanalytical aspects to optimize G6PD/Hb measurement and interpretation. METHODS: G6PD was measured with the Pointe Scientific assay and Hb with Drabkin's reagent on Alinity c® (Abbott Diagnostics). Stability of G6PD/Hb was assessed after 7 and 14 days while stored at 2-8 °C. Stability of hemolysate prepared for G6PD analysis was assessed using QC and patient samples up to 4 h at room temperature or 2-8 °C. Analytical performance specifications including precision, method comparison, linearity, LOQ, and carry-over were established for the enzymatic reaction of G6PD and spectrophotometric reading of Hb. G6PD/Hb reference interval and cut-offs were established indirectly using truncated maximum likelihood method (TML) using retrospective data (n = 4715 patient data points). RESULTS: Samples were stable after 7 days at 2-8°C, unless grossly hemolyzed. Hemolysate prepared for G6PD measurement remained stable for up to 4 h for QC at room temperature and 2-8°C, but up to 30 min-1 h at room temperature and 1-2 h at 2-8 °C for patient samples. Precision, linearity, LOQ, and carryover were acceptable. G6PD/Hb cut-offs were <3.3, ≥3.3, 3.3-8.9, and ≥8.9 U/g Hb for deficient males/females, normal males, intermediate females, and normal females, respectively. CONCLUSIONS: In vitro hemolysis and delayed hemolysate analysis significantly reduce G6PD/Hb stability. QC material cannot detect the impact of delayed hemolysate analysis. These findings were foundational for optimizing G6PD/Hb protocols for a new platform and establishing laboratory-specific G6PD/Hb cut-offs.

Mechanisms of Specific versus Nonspecific Interactions of Aggregation-Prone Inhibitors and Attenuators.

A common source of false positives in drug discovery is ligand self-association into large colloidal assemblies that nonspecifically inhibit target proteins. However, the mechanisms of aggregation-based inhibition (ABI) and ABI-attenuation by additives, such as Triton X-100 (TX) and human serum albumin (HSA), are not fully understood. Here, we investigate the molecular basis of ABI and ABI-attenuation through the lens of NMR and coupled thermodynamic cycles. We unexpectedly discover a new class of aggregating ligands that exhibit negligible interactions with proteins but act as competitive sinks for the free inhibitor, resulting in bell-shaped dose-response curves. TX attenuates ABI by converting inhibitory, protein-binding aggregates into nonbinding coaggregates, whereas HSA minimizes nonspecific ligand interactions by functioning as a reservoir for free inhibitor and preventing self-association. Hence, both TX and HSA are useful tools to minimize false positives arising from nonspecific binding but at the cost of potentially introducing false negatives due to suppression of specific interactions.

Mechanism of Selective Enzyme Inhibition through Uncompetitive Regulation of an Allosteric Agonist.

Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4R's action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBD's hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.

What's a Lab to Do During and After a Hurricane?

Although laboratories may be able to rely on a comprehensive Hurricane Plan during a hurricane, alarming and unanticipated events frequently occur. To minimize disruption of lab operations, it is important to try to mitigate the impact of these unexpected events as quickly as possible, in the quest to minimize negative outcomes. In this article, we discuss approaches to dealing with unanticipated events during and after hurricanes, based on our personal experiences.

Hurricanes: Are You Prepared?

Severe weather events such as hurricanes have the potential to cause significant disruption of laboratory operations. Comprehensive planning is essential to mitigate the impact of such events. The essential elements of a Hurricane Plan, based on our personal experiences, are detailed in this article.

Implementation of the NMR CHEmical Shift Covariance Analysis (CHESCA): A Chemical Biologist's Approach to Allostery.

Mapping allosteric sites is emerging as one of the central challenges in physiology, pathology, and pharmacology. Nuclear Magnetic Resonance (NMR) spectroscopy is ideally suited to map allosteric sites, given its ability to sense at atomic resolution the dynamics underlying allostery. Here, we focus specifically on the NMR CHEmical Shift Covariance Analysis (CHESCA), in which allosteric systems are interrogated through a targeted library of perturbations (e.g., mutations and/or analogs of the allosteric effector ligand). The atomic resolution readout for the response to such perturbation library is provided by NMR chemical shifts. These are then subject to statistical correlation and covariance analyses resulting in clusters of allosterically coupled residues that exhibit concerted responses to the common set of perturbations. This chapter provides a description of how each step in the CHESCA is implemented, starting from the selection of the perturbation library and ending with an overview of different clustering options.

Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG).

Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state.