Extreme γ' fibrinogen levels in COVID-19 patients.

COVID-19 disease progression can be accompanied by a "cytokine storm" that leads to secondary sequelae such as acute respiratory distress syndrome. Several inflammatory cytokines have been associated with COVID-19 disease progression, but have high daily intra-individual variability. In contrast, we have shown that the inflammatory biomarker γ' fibrinogen (GPF) has a 6-fold lower coefficient of variability compared to other inflammatory markers such as hs-CRP. The aims of the study were to measure GPF in serial blood samples from COVID-19 patients at a tertiary care medical center in order to investigate its association with clinical measures of disease progression. COVID-19 patients were retrospectively enrolled between 3/16/2020 and 8/1/2020. GPF was measured using a commercial ELISA. We found that COVID-19 patients can develop extraordinarily high levels of GPF. Our results showed that ten out of the eighteen patients with COVID-19 had the highest levels of GPF ever recorded. The previous highest GPF level of 80.3 mg/dL was found in a study of 10,601 participants in the ARIC study. GPF levels were significantly associated with the need for ECMO and mortality. These findings have potential implications regarding prophylactic anticoagulation of COVID-19 patients.

Contributions of CCLM to advances in quality control.

Abstract The discipline of laboratory medicine is relatively young when considered in the context of the history of medicine itself. The history of quality control, within the context of laboratory medicine, also enjoys a relatively brief, but rich history. Laboratory quality control continues to evolve along with advances in automation, measurement techniques and information technology. Clinical Chemistry and Laboratory Medicine (CCLM) has played a key role in helping disseminate information about the proper use and utility of quality control. Publication of important advances in quality control techniques and dissemination of guidelines concerned with laboratory quality control has undoubtedly helped readers of this journal keep up to date on the most recent developments in this field.

Prognostic utility of biochemical markers of cardiovascular risk: impact of biological variability.

BACKGROUND: Although a variety of biochemical markers are used to help predict the risk of cardiovascular disease, the prognostic utility of any marker used as a risk assessment tool is dependent on the long- and short-term biological variability that the marker shows in different individuals. METHODS: We measured total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; triglycerides; high-sensitivity C-reactive protein (hsCRP); total fibrinogen; and γ' fibrinogen in blood samples collected from 15 apparently healthy individuals over the course of 1 year. Repeated measures variation estimates were used to calculate short- and long-term intraclass correlation coefficients (ICC), within- and between-subject coefficients of variation (CVI and CVG, respectively), validity coefficients, and indices of individuality for each marker. RESULTS: HDL cholesterol demonstrated the lowest variability profile, with an ICC of 0.84 and CVI of 11.1 (95% CI: 8.3, 17.0). hsCRP showed the highest levels of short- and long-term within-subject variability [CVI (95% CI): 54.8 (32.8, 196.3) and 77.1 (53.3, 141.3), respectively]. Stated differently, it would require five separate measurements of hsCRP, performed on samples collected over multiple days, to provide the risk assessment information provided by a single measurement of HDL cholesterol. γ' Fibrinogen demonstrated an ICC of 0.79 and CVI of 14.3 (95% CI: 10.6, 21.9). CONCLUSIONS: hsCRP showed very high biological variability, such that a single measurement of hsCRP lacks sufficient clinical utility to justify routine measurement. The variability profile of γ' fibrinogen was not markedly different than HDL cholesterol, necessitating only a limited number of measurements to establish an individual's risk of cardiovascular disease.

Evaluation of Preanalytical Point-of-Care Testing Errors and Their Impact on Productivity in the Emergency Department in the United States.

BACKGROUND: Preanalytical errors due to poor sample quality or improper sample handling may occur with point-of-care testing (POCT). METHODS: A retrospective analysis was conducted using deidentified records for 15 479 i-STAT® cartridges run at the Oregon Health & Science University Emergency Department (ED) between December 2015 and August 2016. Data were collected from electronic health records and device middleware for CG4+, CHEM8+, and Troponin cartridges. The frequency of POCT errors was evaluated by cartridge type. The effect of user experience on error frequency, impact of error on hospital length of stay (LOS), and test turnaround time (TAT) were all evaluated. Direct costs incurred due to Chem8+ and Troponin cartridge waste and indirect costs as avoidable nursing staff labor were estimated over 2 years. RESULTS: A total of 935 erroneous results were identified (6.0% of all cartridges). Three hundred seventy-two (2.4%) were unusable results, and 563 (3.6%) were cartridge errors, of which 163 were classified by device error codes as poor sample quality/improper sample handling. Error rates were inversely correlated with user experience based on number of tests performed during the 9-month period. Compared to nonerroneous results, test TATs and LOS were significantly longer with erroneous results (P < 0.01). Over 2 years, direct costs incurred due to cartridge waste was $45 000, and indirect cost was estimated between 486 and 729 h in avoidable nursing labor. CONCLUSIONS: Preanalytical POCT errors were inversely correlated with user experience and significantly impacted clinical productivity in the ED based on LOS and test TAT.

Rapid Point-of-Care Test for Determination of C-Peptide Levels.

C-peptide is co-secreted with insulin and is not subject to hepatic clearance and thus reflects functional ß-cell mass. Assessment of C-peptide levels can identify individuals at risk for or with type 1 diabetes with residual ß-cell function in whom ß cell-sparing interventions can be evaluated, and can aid in distinguishing type 2 diabetes from Latent Autoimmune Diabetes in Adults and late-onset type 1 diabetes. To facilitate C-peptide testing, we describe a quantitative point-of-care C-peptide test. C-peptide levels as low as 0.2 ng/ml were measurable in a fingerstick sample, and the test was accurate over a range of 0.17 to 12.0 ng/ml. This test exhibited a correlation of r = 0.98 with a high-sensitivity commercial ELISA assay and a correlation of r = 0.90 between matched serum and fingerstick samples.

Establishment of Monoclonal Antibody Standards for Quantitative Serological Diagnosis of SARS-CoV-2 in Low-Incidence Settings.

BACKGROUND: Serological confirmation of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for understanding the dynamics of the pandemic and determining seroprevalence rates within afflicted communities. Common challenges with SARS-CoV-2 serological assays include poor analytical specificity and sensitivity and lack of a serological standard for quantitative assessment of antibody titers. METHODS: To overcome these obstacles, we developed a quantitative enzyme-linked immunosorbent assay based on an optimized 2-dimensional screening assay that utilizes SARS-CoV-2 receptor binding domain (RBD) of spike protein and SARS-CoV-2 spike S1 subunit. RESULTS: A total of 4 SARS-CoV-2-reactive monoclonal antibodies were evaluated for use as serum standards for calibrating assays performed on different days or by different laboratories. This approach provided quantitative analysis of hospitalized reverse transcription polymerase chain reaction-confirmed COVID-19 cases that in some cases reached >100 µg/mL. The assay demonstrated 72% sensitivity based on time points ranging from 2 to 52 days post-symptom onset, with 100% sensitivity at time points measured ≥13 days post-symptom onset and 100% specificity. CONCLUSIONS: Using these optimized reagents and serological standards, we believe this approach will be useful for sensitive and specific determination of seroconversion rates and quantitatively measuring the durability of antiviral antibody responses following SARS-CoV-2 infection or vaccination.

Gamma' fibrinogen: evaluation of a new assay for study of associations with cardiovascular disease.

BACKGROUND: Studies of disease associations with gamma' fibrinogen, a newly emerging risk factor for cardiovascular disease, have been hampered by the lack of a standardized and well-characterized assay. METHODS: We developed an immunometric technique to measure gamma' fibrinogen concentrations in plasma and studied the clinical utility of this test in samples from healthy individuals enrolled in the Framingham Offspring Study and in a separate case/control study of coronary artery disease (CAD). Monoclonal antibody 2.G2.H9, specific for the unique carboxyl terminal peptide of the fibrinogen gamma' chain, was used as capture antibody. Sheep antihuman fibrinogen/horseradish peroxidase conjugate was used for detection, with 3,3',5,5'-tetramethylbenzidine as substrate. We evaluated the linearity, imprecision, analytical specificity, and lower limit of quantification of the assay. We determined the reference interval for gamma' fibrinogen in healthy individuals from the Framingham Offspring Study (n = 2879) and quantified associations between gamma' fibrinogen and cardiovascular disease risk factors. The sensitivity and specificity of gamma' fibrinogen in evaluating CAD patients (n = 133) was determined with ROC curve analysis. RESULTS: The gamma' fibrinogen ELISA had within-run CVs of 13.4% at 0.127 g/L and 4.8% at 0.416 g/L. The limit of quantification at an imprecision of 20% was 0.10 g/L. The reference interval for healthy individuals was 0.088-0.551 g/L. ROC curve analysis of results from patients with CAD yielded an area under the curve of 0.76, with a diagnostic accuracy of 0.78 at a decision threshold of 0.30 g/L. CONCLUSIONS: gamma' Fibrinogen shows excellent utility for cardiovascular risk analysis.

The Impact of Undetected In Vitro Hemolysis or Sample Contamination on Patient Care and Outcomes in Point-of-Care Testing: A Retrospective Study.

BACKGROUND: Point-of-care (POC) testing is an integral diagnostic component in clinical settings like the emergency department (ED). However, most POC testing devices are unable to detect endogenous interferents such as hemolysis, which typically occurs during sample collection and handling and can falsely increase measured potassium (pseudohyperkalemia), a phenomenon we hypothesized may significantly impact patient care. METHODS: In this retrospective study, we evaluated 100 unique admissions to the Oregon Health & Science University ED, presenting with elevated potassium measured at the POC. To evaluate whether in vitro hemolysis had occurred, POC test results were compared to repeat testing of the original specimen, or other specimens tested within 90 minutes in the Core laboratory. Review of associated Electronic Health Records determined whether elevated potassium initially measured using the POC analyzer was real, or due to in vitro hemolysis or contamination, and whether pseudohyperkalemia impacted patient management or care. RESULTS: Of the 100 admissions with hyperkalemia measured using a POC analyzer, 40% were found to have pseudohyperkalemia due to hemolysis or contamination. Of these 40 patients, 6 experienced repeated testing, and an additional 5 were noted to have altered patient management, specifically due to pseudohyperkalemia. CONCLUSIONS: This study demonstrates the incidence of in vitro hemolysis, which is unknown to the POC operator, is high in patients who show an elevated potassium as measured at the POC. Furthermore, in vitro hemolysis can significantly impact patient management, suggesting that minimizing the incidence of unrecognized hemolysis will benefit hospital efficiency, decrease waste, and improve patient care.

From diagnostics to therapy: prospects of quantum dots.

Quantum dots (QDs) are among the most promising items in the nanomedicine toolbox. These nanocrystal fluorophores have several potential medical applications including nanodiagnostics, imaging, targeted drug delivery, and photodynamic therapy. The diverse potential applications of QDs are attributed to their unique optical properties including broad-range excitation, size-tunable narrow emission spectra, and high photostability. The size and composition of QDs can be varied to obtain the desired emission properties a makes them amenable for simultaneous detection of multiple targets. Also, numerous surface functionalizations can be used to adapt QDs to the needed application. Recent reports have shown successful use of QDs in various medical applications. With respect to in vivo applications, caution must be exercised with QDs due to their toxic components. Development of appropriate health and safety regulations and resolution of intellectual property issues are necessary for commercialization. In light of these obstacles however, QDs appear to be too valuable to nanomedicine to dismiss, and will eventually come into routine practical use.

Systemic Inhibition of CREB is Well-tolerated in vivo.

cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.

False-positive troponin I measured with the Abbott AxSYM attributed to fibrin interference.

BACKGROUND: Serum is often used for the measurement of cardiac troponin I (cTnI). Previous reports suggest that fibrin present in serum samples collected for cTnI analysis may interfere with measurement of this marker. We investigated the incidence and magnitude of fibrin interference in serum specimens submitted for cTnI measurement using the AxSYM analyzer by performing duplicate analysis of all specimens with increased cTnI results. METHODS: Over a 4-month period, we analyzed 3692 specimens for cTnI with the Abbott AxSYM. Of these, 307 (8.3%) showed increased cTnI. A threshold of three times the precision of the method (15%) was used to judge discrepancies between duplicate analyses of specimens; all specimens being recentrifuged between the initial and repeat cTnI analyses. RESULTS: Of 307 patient specimens with elevated cTnI concentrations, 24 (7.8%) demonstrated differences of greater than 45% between duplicate analyses. Concentrations of cTnI obtained on initial analysis of these 24 specimens ranged from 2.4 to 24.0 microg/l. Repeat analysis showed the repeat values for 20 (83%) to be within the normal reference interval, with 16 (67%) showing concentrations of less than 0.3 microg/l. CONCLUSIONS: Our finding indicates that interference should be highly suspected in serum specimens where the initially measured cTnI concentrations is in the range of 2.0-25.0 microg/l when using the Abbott AxSYM. The finding of no interference in specimens with measured troponin concentration greater than 25.0 microg/l suggests that the interference effect of fibrin is generally not sufficient to cause spurious elevations of cTnI into this range. In addition, since switching to plasma as the specimen of choice for the AxSYM, we have not observed any discrepant cTnI results following duplicate analysis of over 200 patient samples with initial measured cTnI concentrations of 2.0 microg/l or greater.

Electrical nanowell diagnostics sensors for rapid and ultrasensitive detection of prostate-specific antigen.

AIM: Alumina nanowell based disposable diagnostic biosensor for detecting and quantifying levels of prostate-specific antigen (PSA) from human serum has been designed, fabricated and tested. MATERIALS & METHODS: The biosensors were designed by integrating nanoporous alumina membranes onto printed circuit board platforms, resulting in the generation of high-density nanowell arrays with gold base electrodes. The size and density of the nanowells were leveraged toward achieving sieving action for size-based exclusion of nonspecific molecules and size-based confinement of the target PSA molecules. RESULTS & CONCLUSION: We demonstrated PSA detection between 0.01 and 1000 ng/ml and detection and quantification of PSA from a 17 patient cohort validated using the Beckman Access system with >95% correlation.

Novel Nanomonitor ultra-sensitive detection of troponin T.

BACKGROUND: Troponin is the preferred biomarker for diagnosing myocardial infarction. Point of care devices have not matched the sensitivity of laboratory-based methods for measuring troponin. The Nanomonitor is a novel point-of-care device that uses the change in electrical impedance that occurs when a biomarker binds to its antibody, which is then correlated to the concentration of the target biomarker. METHODS: Performance characteristics of the Nanomonitor were evaluated and compared to a standard laboratory-based method. RESULTS: The limit of detection of the Nanomonior for troponin T was 0.0088ng/l. Total imprecission was 2.38% and 0.85% at troponin T concentrations of 73ng/l and 1800ng/l. The functional sensitivity (10% coeffecient of variation) was 0.329ng/l. The linear regression had a slope of 0.996 (95% confidence interval, 0.991, 1.002), r=1.00, and an intercept of 15.88ng/l (95% confidence interval, -68.39ng/l, 100.15ng/l). The mean difference between the assays was -7.54ng/l, determined by Bland-Altman analysis. CONCLUSION: The Nanomonitor preliminary results have favorable performance characteristics for detecting troponin T in patient blood, provide results in 15min, and are portable. More research is needed.

Portable nanoporous electrical biosensor for ultrasensitive detection of Troponin-T.

AIM: To demonstrate the design, fabrication and testing of a portable, label-free biosensor for ultrasensitive detection of the cardiac Troponin-T (cTnT) from patient blood. MATERIALS & METHODS: The biosensor is comprised of a nanoporous membrane integrated on to a microelectrode sensor platform for nanoconfinement effects. Charge perturbations due to antigen binding are recorded as impedance changes using electrochemical impedance spectroscopy. RESULTS: The measured impedance change is used to quantitatively determine the cTnT concentration from the tested sample. We were successful in detecting and quantifying cardiac Troponin-T from a 40-patient cohort. The limit of detection was 0.01 pg/ml. CONCLUSION: This novel technology has promising preliminary results for rapid and sensitive detection of cTnT.

Comparison of hemolysis in blood samples collected using an automatic incision device and a manual lance.

OBJECTIVE: To evaluate the magnitude of hemolysis in blood specimens collected from the heels of newborns using an automated blood collection device that uses a spring-loaded lance with blood collected using a manual lance. DESIGN: A randomized controlled trial involving 134 newborns assigned to have blood collected using either an automated blood collection device or a manual lance. A single experienced individual performed all blood collections. Serum hemoglobin concentrations were measured in all samples to gauge the extent of hemolysis. SETTING: A neonatology unit in a 740-bed tertiary care teaching hospital. PATIENTS: Healthy newborns with gestational ages ranging from 33 weeks to 41 weeks. Blood samples were collected from study participants at between 7 and 126 hours postpartum. Group 1 consisted of 66 individuals who had blood collected using the manual lance. Group 2 contained 68 individuals with blood collected using a spring-loaded automatic lance. MAIN OUTCOME MEASURE: Plasma hemoglobin content as an indicator of the extent of hemolysis. RESULTS: There were no significant differences between newborns in groups 1 and 2 with respect to gestational age, birth weight, or time interval between birth and time of blood collection. We found a highly significant difference with respect to plasma hemoglobin concentrations in specimens collected with an automated lance (hemoglobin, 2.35 g/L) vs that collected using the hand-held lance (hemoglobin, 4.85 g/L). CONCLUSION: Use of an automated spring-loaded lance allows for the collection of blood specimens with smaller levels of plasma hemoglobin.

Quantum dots: heralding a brighter future for clinical diagnostics.

Quantum dots (QDs) are semiconductor nanocrystals that possess unique optical properties including broad-range excitation, size-tunable narrow emission spectra and high photostability, giving them considerable value in various biomedical applications. The size and composition of QDs can be varied to obtain the desired emission properties and make them amenable to simultaneous detection of multiple targets. Furthermore, numerous surface functionalizations can be used to adapt QDs to the needed application. The successful use of QDs has been reported in the areas of in vitro diagnostics and imaging. There is also potential for multimodal applications for simultaneous imaging. Toxicity issues are still a prime concern with regards to in vivo applications on account of the toxic constituents of QDs.

Correction factors for estimating potassium concentrations in samples with in vitro hemolysis: a detriment to patient safety.

CONTEXT: Correction factors have been proposed for estimating true potassium concentrations in blood samples with evidence of in vitro hemolysis. OBJECTIVE: We used 2 different models of true (ie, nonsimulated) in vitro hemolysis to evaluate the clinical utility of correction factors for estimating potassium concentrations in samples with evidence of in vitro hemolysis. DESIGN: Potassium correction factors were derived using 2 different models. In model 1, potassium and plasma hemoglobin were measured with the Hitachi 747 analyzer in 132 paired blood samples, with each pair consisting of 1 sample with evidence of hemolysis and 1 without, collected during the same phlebotomy procedure. The change in measured potassium concentration was plotted versus the change in plasma hemoglobin concentration for each pair of samples. In model 2, the potassium levels of 142 784 blood samples and the corresponding hemolytic index values were measured with the Beckman LX20 analyzer. Potassium concentrations at the 10th, 25th, 50th, 75th, and 90th percentiles were calculated for each hemolysis index category. RESULTS: From our 2 models, we derived correction factors expressing an increase in potassium concentration of 0.51 and 0.40 mEq/L for every increase in plasma hemoglobin concentration of 0.1 g/dL. These correction factors are much higher than those reported in studies that simulated in vitro hemolysis by freeze-thaw lysis or osmotic disruption of whole blood. CONCLUSIONS: Use of correction factors for estimating the true potassium concentration in samples with evidence of in vitro hemolysis is not recommended. Derivation of correction factors by using samples with nonsimulated in vitro hemolysis suggests that the actual increase in potassium in hemolyzed samples is much higher than that reported in previous studies that produced hemolysis with artificial means.

Application of spin label electron paramagnetic resonance in the diagnosis and prognosis of cancer and sepsis.

Diagnostic medicine has seen significant changes during the past decade. The emergence of proteomics and genomics has significantly increased our understanding of disease. These fields have also revealed the vast array of proteins that are expressed in various disease processes, such as cancer. Measurement of these unique proteins expressed in certain diseases may offer diagnostic clues or allow patient prognosis to be assessed. Another approach is to measure the effects that these ligands have on the structure and function of albumin. Albumin is known to play an important role in modulating the serum concentrations of various proteins produced by tumor cells. In this review, we introduce the reader to the technique of spin labeling followed by electron paramagnetic resonance spectroscopy. This method is a powerful tool for evaluating the structural and functional changes that can occur to albumin following the binding of various ligands. We describe the utility of this technique for the diagnosis of cancer and sepsis, as well as some other novel potential applications.