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.

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.

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.

Raman spectroscopy-based sensitive and specific detection of glycated hemoglobin.

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the nonenzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and nonglycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision--with a limit of detection that is a factor of ~15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multicomponent mixtures and potentially in hemolysate and whole blood lysate samples.

Should creatine kinase-MB index be eliminated in patients with indeterminate troponins in the ED?

OBJECTIVES: The objective of this study is to determine whether creatine kinase-MB (CK-MB) index (CK-MBi) is useful in the evaluation of acute myocardial infarction (AMI) in patients with indeterminate troponin (Tn) in the emergency department (ED). METHODS: A retrospective cohort study was conducted of patients at an urban academic ED with over 55 000 annual visits who underwent Tn T (Roche, Indianapolis, IN) and CK-MB testing. One year of ED patients who had Tn testing were identified, and their corresponding CK-MBi was examined to find patients with indeterminate Tn (0.01-0.09) and positive CK-MBi (>6.0). Subsequent cardiac enzymes and hospital course were reviewed to identify patients diagnosed with AMI. A 95% confidence interval around point estimates were used in data analysis. RESULTS: Over 1 year, 11 718 initial Tn were identified. Indeterminate Tn was seen in 2512 cases. Of these, 28 had positive CK-MBi. Of the 28, 5 were judged by treating physicians to be having AMI and underwent cardiac catheterization. Of the 5 patients, 4 had subsequent positive Tns on serial enzyme testing. One of the patients thought to be having AMI had no coronary artery disease on catheterization. The rate of true positive CK-MBi with indeterminate Tn was 0.16% (95% confidence interval, 0.04%-0.41%). CONCLUSION: Initial results identify rare cases of AMI where CK-MBi is positive in the setting of indeterminate Tn. However, most patients with indeterminate Tn and positive CK-MBi were not judged to be having AMI. In most cases, CK-MBi is not positive with indeterminate Tn and when positive more commonly confuses the picture. This suggests CK-MBi could be eliminated in patients with indeterminate Tns.

Creatine kinase-MB does not add additional benefit to a negative troponin in the evaluation of chest pain.

OBJECTIVE: The aim of this study was to determine whether current troponin assay alone can be used for initial screening for acute myocardial infarction (AMI) and whether creatine kinase-MB (CK-MB) can safely be eliminated from this evaluation in the emergency department (ED). METHODS: A retrospective cohort study of patients who had cardiac troponin T (Roche, Basel, Switzerland) and CK-MB ordered at an urban academic level 1 trauma center with more than 55,000 annual visits. Patients with troponin testing in the ED were identified over a period of 12 months, and corresponding CK-MB indexes were examined identifying patients with negative troponins (<0.01) and positive CK-MB indexes (>6.0). In these patients, further cardiac markers, hospital course, and 30-day mortality were then evaluated. A 99% confidence interval around point estimate was used in data analysis. RESULTS: During the study period, there were 11,092 separate ED patient encounters where a patient had at least one troponin resulted. Most (97.9%) of the samples had an associated CK-MB ordered. There were 7545 initial negative troponins representing 68% of all initial samples. Seven of these had an associated positive MB index. When subsequent troponins were evaluated, an additional 4910 negative troponins were identified, with 4 patients having a positive MB. None of these 11 patients were judged to have ruled in for AMI by the treating physicians. The rate of true-positive CK-MB index with negative troponin was 0% (99% confidence interval, 0-0.04%). CONCLUSION: Our results suggest that CK-MB is not necessary in the initial screening for AMI and may safely be omitted in patients with negative troponins.

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.

Proficiency testing/external quality assessment: current challenges and future directions.

BACKGROUND: Proficiency testing (PT), or external quality assessment (EQA), is intended to verify on a recurring basis that laboratory results conform to expectations for the quality required for patient care. CONTENT: Key factors for interpreting PT/EQA results are knowledge of the commutability of the samples used and the process used for target value assignment. A commutable PT/EQA sample demonstrates the same numeric relationship between different measurement procedures as that expected for patients' samples. Noncommutable PT/EQA samples frequently have a matrix-related bias of unknown magnitude that limits interpretation of results. PT/EQA results for commutable samples can be used to assess accuracy against a reference measurement procedure or a designated comparison method. In addition, the agreement of the results between different measurement procedures for commutable samples reflects that which would be seen for patients' samples. PT/EQA results for noncommutable samples must be compared to a peer group mean/median of results from participants who use measurement procedures that are expected to have the same or very similar matrix-related bias. Peer group evaluation is used to asses whether a laboratory is using a measurement procedure in conformance to the manufacturer's specifications and/or in conformance to other laboratories using the same technology. A noncommutable PT/EQA sample does not give meaningful information about the relationship of results for patients' samples between different measurement procedures. SUMMARY: PT/EQA provides substantial value to the practice of laboratory medicine by assessing the performance of individual laboratories and, when commutable samples are used, the status of standardization or harmonization among different measurement procedures.

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.