Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2.

Laboratories faced many challenges throughout the COVID-19 pandemic. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments-waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory-trained individuals. However, these methods must be validated to ensure the manufacturer's performance specifications are met and they are found to be fit-for-purpose in the clinical workflows they are implemented.

National Kidney Foundation Laboratory Engagement Working Group Recommendations for Implementing the CKD-EPI 2021 Race-Free Equations for Estimated Glomerular Filtration Rate: Practical Guidance for Clinical Laboratories.

Recognizing that race is a social and not a biological construct, healthcare professionals and the public have called for removal of race in clinical algorithms. In response, the National Kidney Foundation and the American Society of Nephrology created the Task Force on Reassessing the Inclusion of Race in Diagnosing Kidney Diseases to examine the issue and provide recommendations. The final report from the Task Force recommends calculating estimated glomerular filtration rate (eGFR) without a race coefficient using the recently published CKD-EPI 2021 creatinine (cr) and creatinine-cystatin C (cr-cys) equations. The Task Force recommends immediately replacing older eGFRcr equations (MDRD Study and CKD-EPI 2009) with the new CKD-EPI 2021 equation. In a 2019 survey by the College of American Pathologists, 23% of 6200 laboratories reporting eGFRcr used an incorrect equation that is not suitable for use with standardized creatinine measurements, 34% used the CKD-EPI 2009 equation and 43% used the MDRD Study 2006 equation re-expressed for standardized creatinine measurement. Rapid transition to using the CKD-EPI 2021 equation is an opportunity for laboratories to standardize to a single equation to eliminate differences in eGFRcr due to different equations used by different laboratories, and to report eGFR without use of race. We provide guidance to laboratories for implementing the CKD-EPI 2021 equations for both eGFRcr and eGFRcr-cys.

Rapid detection and identification of bacteria directly from whole blood with light scattering spectroscopy based biosensor.

Bacterial infections are one of the major causes of death worldwide. The identification of a bacterial species that is the source of an infection generally takes a long time, and often exceeds the treatment window for seriously ill patients. Many of these deaths are preventable if the bacterial species can be identified quickly. Here we present an optical spectroscopic method for rapid detection and identification of bacteria directly from whole blood using a light scattering spectroscopy technique. This technique was originally developed to detect pre-cancerous changes in epithelial tissues, characterize changes in tissue on the cellular scale, and characterize biological structures comparable to or smaller than a single wavelength. We demonstrate here that not only can an inexpensive light scattering spectroscopy-based biosensor rapidly detect and identify four bacteria species in the blood, responsible for the majority of death causing infections, but that species-level identification can potentially be made based on approximately one thousand bacterial cells per milliliter of blood. Observing entire colonies or performing susceptibility testing is therefore not required.

Analytic Sensitivity of 3 Nucleic Acid Detection Assays in Diagnosis of SARS-CoV-2 Infection.

BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse transcription PCR is the primary method to diagnose coronavirus disease 2019 (COVID-19). However, the analytic sensitivity required is not well defined and it is unclear how available assays compare. METHODS: For the Abbott RealTime SARS-CoV-2 assay (m2000; Abbott Molecular), we determined that it could detect viral concentrations as low as 26 copies/mL, we defined the relationship between cycle number and viral concentrations, and we tested naso- and oropharyngeal swab specimens from 8538 consecutive individuals. Using the m2000 as a reference assay method, we described the distribution of viral concentrations in these patients. We then used selected clinical specimens to determine the positive percent agreement of 2 other assays with more rapid turnaround times [Cepheid Xpert Xpress (GeneXpert; Cepheid); n = 27] and a laboratory developed test on the Luminex ARIES system [ARIES LDT (Luminex); n = 50] as a function of virus concentrations, from which we projected their false-negative rates in our patient population. RESULTS: SARS-CoV-2 was detected in 27% (95% CI: 26%-28%) of all specimens. Estimated viral concentrations were widely distributed, and 17% (95% CI: 16%-19%) of positive individuals had viral concentrations <845 copies/mL. Positive percent agreement was strongly related to viral concentration, and reliable detection (i.e., ≥95%) was observed at concentrations >100 copies/mL for the GeneXpert but not the ARIES LDT, corresponding to projected false-negative rates of 4% (95% CI: 0%-21%) and 27% (95% CI: 11%-46%), respectively. CONCLUSIONS: Substantial proportions of clinical specimens have low to moderate viral concentrations and may be missed by methods with less analytic sensitivity.

Picoanalysis of Drugs in Biofluids with Quantitative Label-Free Surface-Enhanced Raman Spectroscopy.

The enormous increase of Raman signal in the vicinity of metal nanoparticles allows surface-enhanced Raman spectroscopy (SERS) to be employed for label-free detection of substances at extremely low concentrations. However, the ultimate potential of label-free SERS to identify pharmaceutical compounds at low concentrations, especially in relation to biofluid sensing, is far from being fully realized. Opioids are a particular challenge for rapid clinical identification because their molecular structural similarities prevent their differentiation with immunolabeling approaches. In this paper, a new method called quantitative label-free SERS (QLF-SERS) which involves the formation of halide-conjugated gold nanoclusters trapping the analyte of interest near the SERS hot spots is reported, and it is demonstrated that it yields a 105 fold improvement in the detection limit over previously reported results for the entire class of clinically relevant opioids and their metabolites. Measurements of opioid concentrations in multicomponent mixtures are also demonstrated. QLF-SERS has comparable detection limits as currently existing laboratory urine drug testing techniques but is significantly faster and inexpensive and, therefore, can be easily adapted as part of a rapid clinical laboratory routine.

Label-free spectrochemical probe for determination of hemoglobin glycation in clinical blood samples.

Glycated hemoglobin, HbA1c, is an important biomarker that reveals the average value of blood glucose over the preceding 3 months. While significant recent attention has been focused on the use of optical and direct molecular spectroscopic methods for determination of HbA1c, a facile test that minimizes sample preparation needs and turnaround time still remains elusive. Here, we report a label-free approach for identifying low, mid and high-HbA1c groups in hemolysate and in whole blood samples featuring resonance Raman (RR) spectroscopy and support vector machine (SVM)-based classification of spectral patterns. The diagnostic power of RR measurements stems from its selective enhancement of hemoglobin-specific features, which simultaneously minimizes the blood matrix spectral interference and permits detection in the native solution. In this pilot study, our spectroscopic observations reveal that glycation of hemoglobin results in subtle but reproducible changes even when detected in the whole blood matrix. Leveraging SVM analysis of the principal component scores determined from the RR spectra, we show high degree of accuracy in classifying clinical specimen. We envisage that the promising findings will pave the way for more extensive clinical specimen investigations with the ultimate goal of translating molecular spectroscopy for routine point-of-care testing.

Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results.

BACKGROUND: With a 10-year sustained increase in 25-hydroxyvitamin D [25(OH)D] testing, laboratories have swapped their LC-MS/MS methods for high-throughput automated immunoassays. Although it is generally well-known that immunoassays have poor recoveries for 25-hydroxyvitamin D2 [25(OH)D2], the frequency and extent to which this impacts total 25(OH)D have not been previously demonstrated. We evaluated 3 automated immunoassays against the first FDA-cleared CDC/NIST-traceable LC-MS/MS method. METHODS: Method comparison was performed for the Siemens ADVIA Centaur, Roche Elecsys Cobas, and Abbott Architect 25(OH)D immunoassay methods in real patient samples (n = 105). We calculated the mean bias in samples containing >20 ng/mL 25(OH)D2 and estimated the percent 25(OH)D2 cross-reactivities. We determined the prevalence of appreciable concentrations of 25(OH)D2 in our patient population through random sampling (n = 120) and projected the frequency of inaccurate 25(OH)D immunoassay results. RESULTS: Linear regression for 25(OH)D was y = 1.09x - 4.44 (Centaur), y = 0.84 + 0.43 (Cobas), and y = 0.83x - 0.48 (Architect). The mean biases of 25(OH)D concentrations were 5.6 (11.0) ng/mL (Centaur), -17.5 (7.2) ng/mL (Cobas), and -20.3 (9.8) ng/mL (Architect) in samples containing >20 ng/mL 25(OH)D2. The observed percent cross-reactivities for 25(OH)D2 were 115% (Centaur), 52% (Cobas), and 44% (Architect). We estimate that 8% of our population has >20 ng/mL 25(OH)D2, thereby compromising the accuracy of 25(OH)D results in >3000 samples annually. CONCLUSIONS: We demonstrate that immunoassay manufacturer package inserts indicate much better recoveries of 25(OH)D2 than what is observed in unadulterated real patient samples. We estimate the frequency of inaccurate total 25(OH)D determination by these immunoassay methods to be largely dependent on the concentration of 25(OH)D2 in each sample.

Assessment of Intensive Care Unit Laboratory Values That Differ From Reference Ranges and Association With Patient Mortality and Length of Stay.

Importance: Laboratory data are frequently collected throughout the care of critically ill patients. Currently, these data are interpreted by comparison with values from healthy outpatient volunteers. Whether this is the most useful comparison has yet to be demonstrated. Objectives: To understand how the distribution of intensive care unit (ICU) laboratory values differs from the reference range, and how these distributions are related to patient outcomes. Design, Setting, and Participants: Cross-sectional study of a large critical care database, the Medical Information Mart for Intensive Care database, from January 1, 2001, to October 31, 2012. The database is collected from ICU data from a large tertiary medical center in Boston, Massachusetts. The data are collected from medical, cardiac, neurologic, and surgical ICUs. All patients in the database from all ICUs for 2001 to 2012 were included. Common laboratory measurements over the time window of interest were sampled. The analysis was conducted from March to June 2017. Main Outcomes and Measures: The overlapping coefficient and Cohen standardized mean difference between distributions were calculated, and kernel density estimate visualizations for the association between laboratory values and the probability of death or quartile of ICU length of stay were created. Results: Among 38 605 patients in the ICU (21 852 [56.6%] male; mean [SD] age, 74.5 [55.1] years), 8878 (23%) had the best outcome (ICU survival, shortest quartile length of stay) and 3090 (8%) had the worst outcome (ICU nonsurvival). Distribution curves based on ICU data differed significantly from the hospital standard range (mean [SD] overlapping coefficient, 0.51 [0.32-0.69]). All laboratory values for the best outcome group differed significantly from those in the worst outcome group. Both the best and worst outcome group curves revealed little overlap with and marked divergence from the reference range. Conclusions and Relevance: The standard reference ranges obtained from healthy volunteers differ from the analogous range generated from data from patients in intensive care. Laboratory data interpretation may benefit from greater consideration of clinically contextual and outcomes-related factors.

Practices for Identifying and Rejecting Hemolyzed Specimens Are Highly Variable in Clinical Laboratories.

CONTEXT: Hemolysis is an important clinical laboratory quality attribute that influences result reliability. OBJECTIVE: To determine hemolysis identification and rejection practices occurring in clinical laboratories. DESIGN: We used the College of American Pathologists Survey program to distribute a Q-Probes-type questionnaire about hemolysis practices to Chemistry Survey participants. RESULTS: Of 3495 participants sent the questionnaire, 846 (24%) responded. In 71% of 772 laboratories, the hemolysis rate was less than 3.0%, whereas in 5%, it was 6.0% or greater. A visual scale, an instrument scale, and combination of visual and instrument scales were used to identify hemolysis in 48%, 11%, and 41% of laboratories, respectively. A picture of the hemolysis level was used as an aid to technologists' visual interpretation of hemolysis levels in 40% of laboratories. In 7.0% of laboratories, all hemolyzed specimens were rejected; in 4% of laboratories, no hemolyzed specimens were rejected; and in 88% of laboratories, some specimens were rejected depending on hemolysis levels. Participants used 69 different terms to describe hemolysis scales, with 21 terms used in more than 10 laboratories. Slight and moderate were the terms used most commonly. Of 16 different cutoffs used to reject hemolyzed specimens, moderate was the most common, occurring in 30% of laboratories. For whole blood electrolyte measurements performed in 86 laboratories, 57% did not evaluate the presence of hemolysis, but for those that did, the most common practice in 21 laboratories (24%) was centrifuging and visually determining the presence of hemolysis in all specimens. CONCLUSIONS: Hemolysis practices vary widely. Standard assessment and consistent reporting are the first steps in reducing interlaboratory variability among results.

Clinical Laboratory Quality Practices When Hemolysis Occurs.

CONTEXT: Hemolyzed specimens delay clinical laboratory results, proliferate unnecessary testing, complicate physician decisions, injure patients indirectly, and increase health care costs. OBJECTIVE: To determine quality improvement practices when hemolysis occurs. DESIGN: We used the College of American Pathologists (CAP) Survey Program to distribute a Q-Probes-type questionnaire about hemolysis practices to CAP Chemistry Survey participants. RESULTS: Of 3495 participants sent the questionnaire, 846 (24%) responded. Although 85%, 69%, and 55% of participants had written hemolysis policies for potassium, lactate dehydrogenase, and glucose, respectively, only a few (46%, 40%, and 40%) had standardized hemolysis reports between their primary and secondary chemistry analyzers for these 3 analytes. Most participants (70%) had not attempted to validate the manufacturers' hemolysis data for these 3 analytes; however, essentially all who tried, succeeded. Forty-nine percent of participants had taken corrective action to reduce hemolysis during the past year and used, on average, 2.4 different actions, with collection and distribution of hemolysis data to administrative leadership (57%), troubleshooting outliers (55%), retraining phlebotomist (53%), and establishment of quality improvement teams among the laboratory and at problem locations (37%) being the most common actions. When asked to assess their progress in reducing hemolysis, 70% noted slow to no progress, and 2% gave up on improvement. Upon measuring potassium, lactate dehydrogenase, and glucose, approximately 60% of participants used the same specimen flag for hemolysis as for lipemia and icterus. CONCLUSIONS: Hemolysis decreases the quality and increases the cost of health care. Practices for measuring, reporting, and decreasing hemolysis rates need improvement.

Emerging trends in optical sensing of glycemic markers for diabetes monitoring.

In the past decade, considerable attention has been focused on the measurement of glycemic markers, such as glycated hemoglobin and glycated albumin, that provide retrospective indices of average glucose levels in the bloodstream. While these biomarkers have been regularly used to monitor long-term glucose control in established diabetics, they have also gained traction in diabetic screening. Detection of such glycemic markers is challenging, especially in a point-of-care setting, due to the stringent requirements for sensitivity and robustness. A number of non-separation based measurement strategies were recently proposed, including photonic tools that are well suited to reagent-free marker quantitation. Here, we critically review these methods while focusing on vibrational spectroscopic methods, which offer highly specific molecular fingerprinting capability. We examine the underlying principles and the utility of these approaches as reagentless assays capable of multiplexed detection of glycemic markers and also the challenges in their eventual use in the clinic.

Long-term evaluation of analytical methods used in sirolimus therapeutic drug monitoring.

Results of therapeutic monitoring of sirolimus blood concentrations are assay and laboratory dependent. This study compared performance over time of the IMx microparticle enzyme immunoassay (MEIA), Architect chemiluminescent microparticle immunoassay (CMIA), and liquid chromatography with mass spectrometric detection (LC/MS/MS) as part of a proficiency testing scheme. Pooled samples from sirolimus-treated patients and whole-blood samples spiked with known quantities of sirolimus were assayed monthly between 2004 and 2012. When results of pooled patient samples were compared with LC/MS/MS, the MEIA assay showed an overall mean percent bias of -2.3% ± 11.2% that, although initially positive, became increasingly negative from 2007 through 2009. The CMIA, which replaced the MEIA assay, had a mean percent bias of 21.9% ± 12.3%, remaining stable from 2007 through 2012. Similarly, for spiked samples, the MEIA showed an increasingly negative bias over time vs. LC/MS/MS, whereas CMIA maintained a stable positive bias. Based on comparison of immunoassay measurements on individual patient samples, CMIA values were more than 25% higher than MEIA values. These results highlight the importance of continued proficiency testing and regular monitoring of sirolimus assay performance. Clinicians must be aware of the methodology used and adjust target levels accordingly to avoid potential effects on efficacy and toxicity.