Hook Effect in Semiquantitative SARS-CoV-2 Antispike Total Antibody Assay.

OBJECTIVES: The hook effect is a common preanalytical error that results in falsely decreased analyte concentrations in immunoassays. We present here an example in a semiquantitative SARS-CoV-2 antispike total antibody assay and report the incidence of this error at our institution. METHODS: All specimens with initial results within the reportable range of the assay were diluted. Results with higher results upon dilution were determined to have the hook effect. In a subset of specimens, these results were also confirmed as elevated on an alternative SARS-CoV-2 antibody assay. RESULTS: Over 1 month, 12 (9.1%) of 132 results were within the analytical measuring range of the assay. Of these, 11 showed the hook effect and required dilution to obtain accurate results. These represented 8.3% of our total testing volume. CONCLUSIONS: The hook effect was detected in a semiquantitative SARS-CoV-2 antispike total antibody assay at a high incidence. This error results in observed concentrations much lower than is accurate. Laboratories should be aware of this issue and consider manually diluting specimens within the reportable range of the assay to detect this issue.

Elevated Lactate Dehydrogenase Concentrations in Plasma Compared to Serum.

OBJECTIVE: To evaluate the difference in lactate dehydrogenase (LDH) concentrations in plasma vs serum specimens in our patient population. MATERIALS AND METHODS: We measured LDH in 110 paired plasma and serum specimens over a 2-week period. Hemolytic indices were performed on each specimen. These paired specimens were drawn in a single setting and stored under the same conditions. For the last 14 paired specimens, cell counts were performed on the plasma/serum. RESULTS: Plasma LDH was on average 22% higher than serum LDH. There was no difference in the hemolytic indices between the plasma and the serum specimens. In the last 14 specimens, cell counts revealed increased platelets in the plasma specimens compared to the serum specimens. CONCLUSION: We propose switching back to using serum for LDH testing because there was unpredictable elevation in plasma LDH concentrations. These elevations in LDH levels may be linked to the platelets present in plasma and that may lyse or become activated with storage at refrigerated temperature.

Target specific serologic analysis of COVID-19 convalescent plasma.

This study compared the performance of four serology assays for Coronavirus Disease 2019 (COVID-19) and investigated whether COVID-19 disease history correlates with assay performance. Samples were tested at Northshore using the Elecsys Anti-SARS-CoV-2 (Roche Diagnostics), Access SARS-CoV-2 IgG anti-RBD (Beckman Coulter), and LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) as well as at Genalyte using Maverick Multi-Antigen Serology Panel. The study included one hundred clinical samples collected before December 2019 and ninety-seven samples collected from convalescent plasma donors originally diagnosed with COVID-19 by PCR. COVID-19 disease history was self-reported by the plasma donors. There was no difference in specificity between the assays tested. Clinical sensitivity of these four tests was 98% (Genalyte), 96% (Roche), 92% (DiaSorin), and 87% (Beckman). The only statistically significant differences in clinical sensitivity was between the Beckman assay and both Genalyte and Roche assays. Convalescent plasma donor characteristics and disease symptoms did not correlate with false negative results from the Beckman and DiaSorin assays. All four tests showed high specificity (100%) and varying sensitivities (89-98%). No correlations between disease history and serology results were observed. The Genalyte Multiplex assay showed as good or better sensitivity to three other previously validated assays with FDA Emergency Use Authorizations.

A New Era in Pathology Consultation: The MyPathologist Electronic Consultation Tool.

Pathologists and laboratory scientists provide valuable guidance on laboratory utilization, test ordering, interpretation, and quality control provided that clinical staff can easily access the laboratory team. To encourage consultation between clinicians with laboratory scientists and pathologists, we developed an easily accessible electronic tool termed "MyPathologist," placed on the homepage of our electronic health record system. Over its 2-year pilot, utilization of this consultation tool climbed as we continued to publicize it and incorporated education into housestaff onboarding and electronic health record training. Physician satisfaction with the tool was high. Additionally, this became the primary source of consults to our residency call service. Evaluation of MyPathologist questions received during its pilot period showed that more than half the questions were of significant educational value to the residents, often focusing on results interpretation, appropriate test ordering, and quality control. MyPathologist is a novel electronic tool for pathology consultation within our electronic health record and also represents an avenue for educating residents, improving utilization of the laboratory, and improving patient care.

Variable Potassium Concentrations: Which Is Right and Which Is Wrong?

Reverse pseudohyperkalemia is a term used to describe in vitro, falsely elevated potassium concentrations in plasma specimens that occur in association with extreme leukocytosis and are commonly associated with hematologic malignant neoplasms. Tumor lysis syndrome is an in vivo lysis of tumor cells that leads to elevated levels of potassium, uric acid, phosphate, and lactate dehydrogenase, as well as decreased calcium concentrations. Herein, we report a case of a 66-year-old Caucasian man with stage IV mantle-cell lymphoma who has elevated levels of potassium, uric acid, and phosphorus, as well as a white blood cell (WBC) count greater than 100,000 cells per mm3. The patient initially was diagnosed as having tumor lysis syndrome. His subsequent potassium concentrations in whole blood remained elevated even after hemodialysis; however, his serum potassium concentrations were decreased. The patient then was diagnosed accurately as having reverse pseudohyperkalemia, and accurate potassium measurements were obtained via serum specimens.

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

BACKGROUND: Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. CASE REPORT: To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. RESULTS: The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. CONCLUSION: The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes.

Modeling the influence of vitamin D deficiency on cigarette smoke-induced emphysema.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide. While the primary risk factor for COPD is cigarette smoke exposure, vitamin D deficiency has been epidemiologically implicated as a factor in the progressive development of COPD-associated emphysema. Because of difficulties inherent to studies involving multiple risk factors in the progression of COPD in humans, we developed a murine model in which to study the separate and combined effects of vitamin D deficiency and cigarette smoke exposure. During a 16-week period, mice were exposed to one of four conditions, control diet breathing room air (CD-NS), control diet with cigarette smoke exposure (CD-CSE), vitamin D deficient diet breathing room air (VDD-NS) or vitamin D deficient diet with cigarette smoke exposure (VDD-CSE). At the end of the exposure period, the lungs were examined by a pathologist and separately by morphometric analysis. In parallel experiments, mice were anesthetized for pulmonary function testing followed by sacrifice and analysis. Emphysema (determined by an increase in alveolar mean linear intercept length) was more severe in the VDD-CSE mice compared to control animals and animals exposed to VDD or CSE alone. The VDD-CSE and the CD-CSE mice had increased total lung capacity and increased static lung compliance. There was also a significant increase in the matrix metalloproteinase-9: tissue inhibitor of metalloproteinases-1 (TIMP-1) ratio in VDD-CSE mice compared with all controls. Alpha-1 antitrypsin (A1AT) expression was reduced in VDD-CSE mice as well. In summary, vitamin D deficiency, when combined with cigarette smoke exposure, seemed to accelerate the appearance of emphysemas, perhaps by virtue of an increased protease-antiprotease ratio in the combined VDD-CSE animals. These results support the value of our mouse model in the study of COPD.

Fludarabine, cytarabine, and attenuated-dose idarubicin (m-FLAI) combination therapy for elderly acute myeloid leukemia patients.

We performed a phase II trial to evaluate the efficacy and safety of the modified fludarabine, cytarabine, and attenuated-dose idarubicin (m-FLAI) regimen in elderly acute myeloid leukemia (AML) patients. Elderly (≥60 years) AML patients who had not previously received chemotherapy were enrolled in the study. Patients received two consecutive cycles of m-FLAI chemotherapy as an induction. The m-FLAI regimen comprised fludarabine (25 mg/m(2) , days 1-4), cytarabine (1,000 mg/m(2) , days 1-4), and attenuated-dose idarubicin (5 mg/m(2) , days 1-3). The primary end point was complete remission (CR) rate. Secondary end points were overall survival (OS), event-free survival (EFS), and treatment-related mortality (TRM). There were 108 patients (median age 68.4 years, M:F = 64:44) enrolled in the study. CR was achieved in 56.5% of patients, and the TRM rate was 21.3%. Median OS and median EFS were 10.2 and 6.6 months, respectively. The mortality at 30 and 60 days was 15 and 21%, respectively. Performance status and comorbidity did not have prognostic value in this patient cohort. Bone marrow expression of CD117 was associated with increased EFS and OS. m-FLAI is an effective induction regimen for previously untreated AML in elderly patients. In addition, bone-marrow CD117 expression is an independent favorable prognostic factor in elderly AML patients. (ClinicalTrials.gov number, NCT01247493).

The diaphragms of fenestrated endothelia: gatekeepers of vascular permeability and blood composition.

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.

Thermal stability of atomic layer deposited Ru layer on Si and TaN/Si for barrier application of Cu interconnection.

The thermal stability of thin Ru single layer and Ru/TaN bilayers grown on bare Si by plasma enhanced atomic layer deposition (PEALD) have been studied with Cu/Ru, Cu/Ru/TaN structures as a function of annealing temperature. To investigate the characteristics as a copper diffusion barrier, a 50 nm thick Cu film was sputtered on Ru and Ru/TaN layers and each samples subjected to thermal annealing under N2 ambient with varied temperature 300, 400, and 500 degrees C, respectively. It was found that the single 5 nm thick ALD Ru layer acted as an effective Cu diffusion barrier up to 400 degrees C. On the other hand ALD Ru (5 nm)/TaN (3.2 nm) showed the improved diffusion barrier characteristics even though the annealing temperature increased up to 500 degrees C. Based on the experimental results, the failure mechanism of diffusion barrier would be related to the crystallization of amorphous Ru thin film as temperature raised which implies the crystallized Ru grain boundary served as the diffusion path of Cu atoms. The combination of ALD Ru incorporated with TaN layer would be a promising barrier structure in Cu metallization.

Growth behavior of iridium on Si substrates prepared by plasma enhanced atomic layer deposition.

Growth behavior of iridium (Ir) thin film on Si substrates prepared by plasma enhanced atomic layer deposition (PEALD) was systematically studied. Ir(EtCp)(COD) and oxygen was employed as a precursor and reactant, respectively. To obtain optimal conditions for depositing nanometer scale Ir thin film, deposition temperature, cycle dependence and precursor feeding time dependence were studied. Uniform 12 nm thick Ir layer with sharp interface was grown at the temperature range of 330-360 degrees C at the fixed deposition cycles of 300. The grown Ir film showed linear properties as a function of deposition cycles which is a typical self-limiting characteristic of ALD. The XRD patterns revealed that IrOx was not formed due to relatively low partial pressure of oxygen. The optimal conditions obtained for 12 nm thick Ir thin film were 330 degrees C of deposition temperature, 300 deposition cycles, and 10 sec of precursor feeding time.

Multiple lipoprotein and electrolyte laboratory artifacts caused by lipoprotein X in obstructive biliary cholestasis secondary to pancreatic cancer.

Lipoprotein X (Lp-X) is an abnormal lipoprotein which may form in patients with intra- and extra-hepatic cholestasis. The presence of very high levels of Lp-X has been shown to be a rare cause of pseudohyponatremia. We present a patient with severe obstructive cholestasis secondary to pancreatic cancer leading to very high Lp-X concentrations resulting in pseudohyponatremia, pseudohypokalemia, pseudohypochloremia and interference with the selective micellary solubilization direct low density lipoprotein cholesterol assay. These spurious laboratory anomalies impeded the initial clinical management of the patient including the attempted correction of the electrolyte abnormalities. After relief of obstruction following biliary stent placement, the patient's lipid levels normalized. Clinicians must be wary of laboratory artifacts and remember to correlate the laboratory values with the clinical presentation of the patient. Assays employing direct ion-selective electrodes such as those in blood gas analyzers are not subject to the interference of high concentrations of lipids or proteins, and maybe useful in situations where such interference is suspected. Furthermore the Vertical Auto Profile (VAP®) ultracentrifugation assay may be useful to detect lipoprotein X and low density lipoprotein cholesterol levels when the selective micellary solubilization technique fails to detect or quantify these lipid moieties.

Warfarin genotyping using three different platforms.

Genetic testing for common variants in the CYP2C9 and VKORC1 genes may provide useful clinical information to guide dosing patients receiving oral warfarin. Specifically, the CYP2C9*2, CYP2C9*3 and either the VKORC1-1639 G>A or VKORC1 1173C>T polymorphisms can be used to help predict an approximate warfarin maintenance dose needed for a particular patient. Although clinical uptake and use of this genotyping has been slow, an increasing body of literature provides evidence of the clinical utility of supplementing traditional warfarin dosing algorithms with a pharmacogenetic approach. The availability of multiple methods for clinical genotyping provides the opportunity for molecular diagnostic laboratories to introduce genotyping assays tailored to their specific needs based on variables such as testing volumes, staffing, available instrumentation and needed turnaround times. Three assays (Invader, Verigene and TaqMan) designed to detect three genetic variations associated with warfarin dosing are evaluated and compared as potential clinical tests to assist in patient care. Identical genotypes were reported by each assay for all samples tested but the assays were found to differ in turnaround time, approval status by the U.S. Food and Drug Administration (FDA), requirements for amount of input genomic DNA and other logistical factors that might make each assay more favorable in different settings.

Pretreatment with stress cortisol enhances the human systemic inflammatory response to bacterial endotoxin.

OBJECTIVE: There is continuing controversy regarding the effect of glucocorticoids on a systemic inflammatory process. Based ona model of glucocorticoid action that includes both pro- and anti-inflammatory effects, we used the human experimental endotoxemia model to test the hypothesis that a transient elevation of plasma cortisol to stress-associated levels would enhance a subsequent (delayed) systemic inflammatory response to bacterial endotoxin. DESIGN: Prospective, randomized, double-blind, placebo-controlled clinical investigation. SETTING: Academic medical center. SUBJECTS: Thirty-six healthy human volunteers. INTERVENTIONS: Participants were randomized to receive a 6-hr intravenous infusion of saline (control), an intermediate dose of cortisol (Cort80; 6.3 mg/hr/70 kg), or a high dose of cortisol (Cort160; 12.6 mg/hr/70 kg) on day 1. On day 2, participants received an intravenous injection of 2 ng/kg Escherichia coli endotoxin followed by serial measurements of plasma cytokine concentrations. MEASUREMENTS AND MAIN RESULTS: Baseline participant characteristics and cortisol and cytokine concentrations were similar in all three groups. The plasma cortisol response to endotoxemia on day 2 was similar in all three groups. The interleukin-6 response to endotoxemia was significantly increased in the Cort80 Group compared with the control Group (p = .004), whereas the interleukin-10 response was significantly suppressed (p = .034). Corresponding results for the Cort160 Group were not significantly different from control Group values. CONCLUSIONS: In this study, transient elevation of in vivo cortisol concentrations to levels that are observed during major systemic stress enhanced a subsequent, delayed in vivo inflammatory response to endotoxin. This appeared to be a dose-dependent effect that was more prominent at intermediate concentrations of cortisol than at higher concentrations of cortisol.

Reporting variant hemoglobins discovered during hemoglobin A1c analysis - common practices in clinical laboratories.

BACKGROUND: Patients with variant hemoglobins may receive inaccurate results by some HbA(1c) methods. We examined reporting practices of clinical laboratories with respect to variant hemoglobins and limitations of methodology. METHODS: A survey of reporting practices was published in LabMedicine, and circulated to directors of Clinical Laboratory Sciences programs. Websites of reference laboratories were reviewed. RESULTS: One hundred thirty-five laboratories from 42 US states responded. 61.5% of those laboratories report only HbA(1c) value and reference interval; 5% of laboratories include methodology. 51% of laboratories use IE-HPLC, 47% use immunoassay and 2% use boronate affinity chromatography. Of laboratories using IE-HPLC, 39% routinely report the presence of hemoglobin variants, and 10% report variants only if they cause interference with the test. Of laboratories using immunoassay, only one appends the disclaimer that elevated HbF interferes with test results. All of the major reference laboratories report methodology on their websites; only 2 can detect hemoglobin variants. Six out of 7 reference laboratories state limitations of methodology on their websites. CONCLUSIONS: There is no standardized reporting format for HbA(1c) that includes methodology, test limitations or notification of variant hemoglobins. An algorithm for detection and reporting of variant hemoglobins and test methodology is proposed based on best practices.

Validation of a CYP2D6 genotyping panel on the NanoChip Molecular Biology Workstation.

BACKGROUND: CYP2D6 is a highly polymorphic phase I enzyme that metabolizes 20%-25% of clinically used drugs. The objective of this study was to validate a CYP2D6 genotyping assay with the NanoChip Molecular Biology Workstation. METHODS: We genotyped 200 anonymized human DNA samples with the Pyrosequencing platform at the Medical College of Wisconsin and with the NanoChip platform at Dartmouth Medical School. We compared CYP2D6 genotypes and resolved samples with genotypic discrepancies with the Jurilab CYP2D6 duplication/deletion assay or with traditional DNA sequencing. The Jurilab assay is a long-range PCR assay used to evaluate sequence structures 3' of the CYP2D7 and CYP2D6 coding regions. For the NanoChip platform, we performed multipad addressing and duplicate runs to test the intra- and intercartridge precision, within- and between-run precision, and reproducibility of the defined genotypes. RESULTS: We used both platforms to genotype all 200 DNA samples for CYP2D6*3, *4, *5, *6, *7, *8, and gene duplication. The 2 methods showed 99.4% concordance in the genotyping results; we found only 8 discrepant genotypes among 1400 DNA analyses. Confirmatory molecular analysis of the discrepant genotypes revealed that the NanoChip assay showed better agreement. The imprecision of the NanoChip method (CV) was 8.9%-17.7%. CONCLUSIONS: This validation study of the NanoChip electronic microarray-based CYP2D6 genotyping assay revealed a CV <20% and good concordance with the Pyrosequencing method and a confirmatory sequencing method.