Lessons learned: A look back at the performance of nine COVID-19 serologic assays and their proposed utility.

BACKGROUND: Serologic assays for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been proposed to assist with the acute diagnosis of infection, support epidemiological studies, identify convalescent plasma donors, and evaluate vaccine response. METHODS: We report an evaluation of nine serologic assays: Abbott (AB) and Epitope (EP) IgG and IgM, EUROIMMUN (EU) IgG and IgA, Roche anti-N (RN TOT) and anti-S (RS TOT) total antibody, and DiaSorin (DS) IgG. We evaluated 291 negative controls (NEG CTRL), 91 PCR positive (PCR POS) patients (179 samples), 126 convalescent plasma donors (CPD), 27 healthy vaccinated donors (VD), and 20 allogeneic hematopoietic stem cell transplant (HSCT) recipients (45 samples). RESULTS: We observed good agreement with the method performance claims for specificity (93-100%) in NEG CTRL but only 85% for EU IgA. The sensitivity claims in the first 2 weeks of symptom onset was lower (26-61%) than performance claims based on > 2 weeks since PCR positivity. We observed high sensitivities (94-100%) in CPD except for AB IgM (77%), EP IgM (0%). Significantly higher RS TOT was observed for Moderna vaccine recipients then Pfizer (p-values < 0.0001). A sustained RS TOT response was observed for the five months following vaccination. HSCT recipients demonstrated significantly lower RS TOT than healthy VD (p < 0.0001) at dose 2 and 4 weeks after. CONCLUSIONS: Our data suggests against the use of anti-SARS-CoV-2 assays to aid in acute diagnosis. RN TOT and RS TOT can readily identify past-resolved infection and vaccine response in the absence of native infection. We provide an estimate of expected antibody response in healthy VD over the time course of vaccination for which to compare antibody responses in immunosuppressed patients.

Patient misidentifications caused by errors in standard bar code technology.

BACKGROUND: Bar code technology has decreased transcription errors in many healthcare applications. However, we have found that linear bar code identification methods are not failsafe. In this study, we sought to identify the sources of bar code decoding errors that generated incorrect patient identifiers when bar codes were scanned for point-of-care glucose testing and to develop solutions to prevent their occurrence. METHODS: We identified misread wristband bar codes, removed them from service, and rescanned them by using 5 different scanner models. Bar codes were reprinted in pristine condition for use as controls. We determined error rates for each bar code-scanner pair and manually calculated internal bar code data integrity checks. RESULTS: As many as 3 incorrect patient identifiers were generated from a single bar code. Minor bar code imperfections, failure to control for bar code scanner resolution requirements, and less than optimal printed bar code orientation were confirmed as sources of these errors. Of the scanner models tested, the Roche ACCU-CHEK® glucometer had the highest error rate. The internal data integrity check system did not detect these errors. CONCLUSIONS: Bar code-related patient misidentifications can occur. In the worst case, misidentified patient results could have been transmitted to the incorrect patient medical record. This report has profound implications not only for point-of-care testing but also for bar coded medication administration, transfusion recipient certification systems, and other areas where patient misidentifications can be life-threatening. Careful control of bar code scanning and printing equipment specifications will minimize this threat to patient safety. Ultimately, healthcare device manufacturers should adopt more robust and higher fidelity alternatives to linear bar code symbologies.

Surface-enhanced Raman spectroscopy for in situ measurements of signaling molecules (autoinducers) relevant to bacteria quorum sensing.

Autoinducer (AI) molecules are used by quorum sensing (QS) bacteria to communicate information about their environment and are critical to their ability to coordinate certain physiological activities. Studying how these organisms react to environmental stresses could provide insight into methods to control these activities. To this end, we are investigating spectroscopic methods of analysis that allow in situ measurements of these AI molecules under different environmental conditions. We found that for one class of AIs, N-acyl-homoserine lactones (AHLs), surface-enhanced Raman spectroscopy (SERS) is a method capable of performing such measurements in situ. SERS spectra of seven different AHLs with acyl chain lengths from 4 to 12 carbons were collected for the first time using Ag colloidal nanoparticles synthesized via both citrate and borohydride reduction methods. Strong SERS spectra were obtained in as little as 10 seconds for 80 microM solutions of AI that exhibited the strongest SERS response, whereas 20 seconds was typical for most AI SERS spectra collected during this study. Although all spectra were similar, significant differences were detected in the SERS spectra of C4-AHL and 3-oxo-C6-AHL and more subtle differences were noted between all AHLs. Initial results indicate a detection limit of approximately 10(-6)M for C6-AHL, which is within the limits of biologically relevant concentrations of AI molecules (nM-microM). Based on these results, the SERS method shows promise for monitoring AI molecule concentrations in situ, within biofilms containing QS bacteria. This new capability offers the possibility to "listen in" on chemical communications between bacteria in their natural environment as that environment is stressed.

Sequential-pulse laser-induced breakdown spectroscopy of high-pressure bulk aqueous solutions.

Sequential-pulse (or dual-pulse) laser-induced breakdown spectroscopy (DP-LIBS) with an orthogonal spark orientation is described for elemental analysis of bulk aqueous solutions at pressures up to approximately 138 x 10(5) Pa (138 bar). The use of sequential laser pulses for excitation, when compared to single-pulse LIBS excitation (SP-LIBS), provides significant emission intensity enhancements for a wide range of elements in bulk solution and allows additional elements to be measured using LIBS. Our current investigations of high-pressure solutions reveal that increasing solution pressure leads to a significant decrease in DP-LIBS emission enhancements for all elements examined, such that we see little or no emission enhancements for pressures above 100 bar. Observed pressure effects on DP-LIBS enhancements are thought to result from pressure effects on the laser-induced bubble formed by the first laser pulse. These results provide insight into the feasibility and limitations of DP-LIBS for in situ multi-elemental detection in high-pressure aqueous environments like the deep ocean.

Immunoassay-Based Drug Tests Are Inadequately Sensitive for Medication Compliance Monitoring in Patients Treated for Chronic Pain.

BACKGROUND: Enzyme immunoassays (EIA) have notable limitations for monitoring therapeutic compliance in pain management. Chromatography coupled with mass spectrometry provides definitive results and superior sensitivity and specificity over traditional EIA testing. OBJECTIVE: To analyze and compare the sensitivity of EIA results together with known prescriptions to liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring drug use (and abuse) in patients treated for chronic pain. STUDY DESIGN: A total of 530 urine samples from patients being treated for chronic pain were studied. SETTING: Pain management clinic in the United States. METHODS: The samples were tested for a profile of chronic pain medications and illicit drugs with commercially available EIA kits followed by analysis with Agilent LC-MS/MS system. RESULTS: The EIAs exhibited poor sensitivity and high rates of false negative results in the pain management setting. For example, 21% of EIA for opiates show false negative results. Mass spectrometry methods were more sensitive, detected a broader range of drugs and metabolites, and could detect non-prescribed drug use and simulations in compliance. LIMITATIONS: Patients do not always accurately report drug use information, and some drugs do not have EIA methods available for comparative purposes. CONCLUSIONS: Mass spectrometry is a more robust and reliable method for detection of drugs used in the pain management setting. Due to the extent of undisclosed use and abuse of medications and illicit drugs, LC-MS/MS testing is necessary for adequate and accurate drug detection. In addition, LC-MS/MS methods are superior in terms of sensitivity and number of compounds that can be screened, making this a better method for use in pain management. Key words: Pain management, enzyme immunoassays, mass spectrometry, urine drug testing, prescription status, compliance.

Laser-induced breakdown spectroscopy of high-pressure bulk aqueous solutions.

Laser-induced breakdown spectroscopy (LIBS) is presented for detection of several Group I and II elements (e.g., Na, Ca, Li, and K), as well as Mn and CaOH, in bulk aqueous solution at pressures exceeding 2.76 x 10(7) Pa (276 bar). Preliminary investigations reveal only minor pressure effects on the emission intensity and line width for all elements examined. These effects are found to depend on detector timing and laser pulse energy. The results of these investigations have implications for potential applications of LIBS for in situ multi-elemental detection in deep-ocean environments.

Standoff detection of high explosive materials at 50 meters in ambient light conditions using a small Raman instrument.

We have designed and demonstrated a standoff Raman system for detecting high explosive materials at distances up to 50 meters in ambient light conditions. In the system, light is collected using an 8-in. Schmidt-Cassegrain telescope fiber-coupled to an f/1.8 spectrograph with a gated intensified charge-coupled device (ICCD) detector. A frequency-doubled Nd : YAG (532 nm) pulsed (10 Hz) laser is used as the excitation source for measuring remote spectra of samples containing up to 8% explosive materials. The explosives RDX, TNT, and PETN as well as nitrate- and chlorate-containing materials were used to evaluate the performance of the system with samples placed at distances of 27 and 50 meters. Laser power studies were performed to determine the effects of laser heating and photodegradation on the samples. Raman signal levels were found to increase linearly with increasing laser energy up to approximately 3 x 10(6) W/cm2 for all samples except TNT, which showed some evidence of photo- or thermal degradation at higher laser power densities. Detector gate width studies showed that Raman spectra could be acquired in high levels of ambient light using a 10 microsecond gate width.

KIMS, CEDIA, and HS-CEDIA immunoassays are inadequately sensitive for detection of benzodiazepines in urine from patients treated for chronic pain.

BACKGROUND: Patients treated for chronic pain may frequently undergo urine drug testing to monitor medication compliance and detect undisclosed prescribed or illicit drug use. Due to the increasing use and abuse of benzodiazepines, this class of medications is often included in drug screening panels. However, immunoassay-based methods lack the requisite sensitivity for detecting benzodiazepine use in this population primarily due to their poor cross-reactivity with several major urinary benzodiazepine metabolites. A High Sensitivity Cloned Enzyme Donor Immunoassay (HS-CEDIA), in which beta-glucuronidase is added to the reagent, has been shown to perform better than traditional assays, but its performance in patients treated for chronic pain is not well characterized. OBJECTIVES: To determine the diagnostic accuracy of HS-CEDIA, as compared to the Cloned Enzyme Donor Immunoassay (CEDIA) and Kinetic Interaction of Microparticles in Solution (KIMS) screening immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS), for monitoring benzodiazepine use in patients treated for chronic pain. STUDY DESIGN: A study of the diagnostic accuracy of urine benzodiazepine immunoassays. SETTING: The study was conducted at an academic tertiary care hospital with a clinical laboratory that performs urine drug testing for monitoring medication compliance in pain management. METHODS: A total of 299 urine specimens from patients treated for chronic pain were screened for the presence of benzodiazepines using the HS-CEDIA, CEDIA, and KIMS assays. The sensitivity and specificity of the screening assays were determined using the LC-MS/MS results as the reference method. RESULTS: Of the 299 urine specimens tested, 141 (47%) confirmed positive for one or more of the benzodiazepines/metabolites by LC-MS/MS. All 3 screens were 100% specific with no false-positive results. The CEDIA and KIMS sensitivities were 55% (78/141) and 47% (66/141), respectively. Despite the relatively higher sensitivity of the HS-CEDIA screening assay (78%; 110/141), primarily due to increased detection of lorazepam, it still missed 22% (31/141) of benzodiazepine-positive urines. The KIMS, CEDIA, and HS-CEDIA assays yielded accuracies of 75%, 79%, and 90%, respectively, in comparison with LC-MS/MS. LIMITATIONS: This study was limited by its single-site location and the modest size of the urine samples utilized. CONCLUSIONS: While the HS-CEDIA provides higher sensitivity than the KIMS and CEDIA assays, it still missed an unacceptably high percentage of benzodiazepine-positive samples from patients treated for chronic pain. LC-MS/MS quantification with enzymatic sample pretreatment offers superior sensitivity and specificity for monitoring benzodiazepines in patients treated for chronic pain.

Improved buprenorphine immunoassay performance after urine treatment with ß-glucuronidase.

Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with ß-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods.

Significant cost savings achieved by in-sourcing urine drug testing for monitoring medication compliance in pain management.

INTRODUCTION: Reference laboratory testing can represent a significant component of the laboratory budget. Therefore, most laboratories continually reassess the feasibility of in-sourcing various tests. We describe the transfer of urine drug testing performed for monitoring medication compliance in pain management from a reference laboratory into an academic clinical laboratory. METHODS: The process of implementing of both screening immunoassays and confirmatory LC-MS/MS testing and the associated cost savings is outlined. RESULTS: The initial proposal for in-sourcing this testing, which included the tests to be in-sourced, resources required, estimated cost savings and timeline for implementation, was approved in January 2009. All proposed testing was implemented by March 2011. CONCLUSIONS: Keys to the successful implementation included budgeting adequate resources and developing a realistic timeline, incorporating the changes with the highest budget impact first. We were able to in-source testing in 27 months and save the laboratory approximately $1 million in the first 3 year.

A new highly specific buprenorphine immunoassay for monitoring buprenorphine compliance and abuse.

Urine buprenorphine screening is utilized to assess buprenorphine compliance and to detect illicit use. Robust screening assays should be specific for buprenorphine without cross-reactivity with other opioids, which are frequently present in patients treated for opioid addiction and chronic pain. We evaluated the new Lin-Zhi urine buprenorphine enzyme immunoassay (EIA) as a potentially more specific alternative to the Microgenics cloned enzyme donor immunoassay (CEDIA) by using 149 urines originating from patients treated for chronic pain and opioid addiction. The EIA methodology offered specific detection of buprenorphine use (100%) (106/106) and provided superior overall agreement with liquid chromatography-tandem mass spectrometry, 95% (142/149) and 91% (135/149) using 5 ng/mL (EIA[5]) and 10 ng/mL (EIA[10]) cutoffs, respectively, compared to CEDIA, 79% (117/149). CEDIA generated 27 false positives, most of which were observed in patients positive for other opioids, providing an overall specificity of 75% (79/106). CEDIA also demonstrated interference from structurally unrelated drugs, chloroquine and hydroxychloroquine. CEDIA and EIA[5] yielded similar sensitivities, both detecting 96% (22/23) of positive samples from patients prescribed buprenorphine, and 88% (38/43) and 81% (35/43), respectively, of all positive samples (illicit and prescribed users). The EIA methodology provides highly specific and sensitive detection of buprenorphine use, without the potential for opioid cross-reactivity.

Development of a fast and simple liquid chromatography-tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC-MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100 µL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY TQD mass spectrometer using a UPLC C18 BEH 1.7 µm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC-MS/MS method was linear over the concentration range of 0.003-3.0 µg/mL, with a lower limit of quantitation for argatroban of 0.003 µg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC-MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC-MS/MS method was unaffected. UPLC-MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.

A new automated urine fentanyl immunoassay: technical performance and clinical utility for monitoring fentanyl compliance.

BACKGROUND: Opiate and other drugs of abuse screening assays have been available for many years, and successfully utilized for monitoring patient medication compliance and for detecting misuse. However, immunoassays designed to rapidly detect the highly potent synthetic opioid, fentanyl, have not been available. METHODS: We evaluated a new fentanyl homogeneous enzyme immunoassay (HEIA) (Immunalysis Corporation) for its ability to accurately detect fentanyl in 307 urine samples from patients prescribed chronic opioid therapy. Samples were screened by HEIA and confirmed by LC-MS/MS and ELISA for diagnostic comparison. HEIA precision and interference studies were performed. RESULTS: HEIA diagnostic sensitivity, specificity and accuracy were 97%, 99%, and 99%, respectively, in comparison with LC-MS/MS and ELISA. HEIA detected 37 of the 38 LC-MS/MS positive samples identified in our initial evaluation, including some that contained very low levels of fentanyl, <1 ng/ml (<3 fmol/l). HEIA showed minimal cross-reactivity with other opioid analgesics and commonly encountered drugs. Interferences by common urine contaminants were negligible, but we observed considerable signal suppression in acidic samples (pH<4.0). CONCLUSIONS: The HEIA urine fentanyl assay allows for rapid and accurate fentanyl detection, illustrating its utility in monitoring fentanyl compliance and abuse.

Quantification of busulfan in plasma using liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS).

Busulfan is a chemotherapy drug widely used as part of conditioning regimens for patients undergoing bone marrow transplantation (BMT). Challenges of busulfan treatment include a narrow therapeutic window and wide inter- and intra-patient variability. Inappropriately low drug levels lead to relapse and even graft rejection, while higher doses frequently have toxic and sometimes fatal consequences. Maintenance of plasma busulfan concentrations using repeated measurements and proper adjustment of dosage can reduce busulfan-related toxicity and improve treatment outcomes. We describe a rapid (2-minute total analysis time per sample) and simple method for accurate and precise busulfan concentration determination in plasma samples (100 microL) using high performance liquid chromatography combined with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Busulfan is isolated from plasma after internal standard (busulfan-D(8))-methanol extraction, dilution with mobile phase (ammonium acetate-formic acid-water), and centrifugation. The supernatant plasma is injected onto the HPLC-ESI-MS/MS and quantified using a six-point standard curve. The assay is linear from 0.025 microg/mL (approximately 0.1 micromol/L) to at least 6.2 microg/mL (approximately 25 micromol/L) with precisions of <5% over the entire range.

Laser-induced breakdown spectroscopy of bulk aqueous solutions at oceanic pressures: evaluation of key measurement parameters.

The development of in situ chemical sensors is critical for present-day expeditionary oceanography and the new mode of ocean observing systems that we are entering. New sensors take a significant amount of time to develop; therefore, validation of techniques in the laboratory for use in the ocean environment is necessary. Laser-induced breakdown spectroscopy (LIBS) is a promising in situ technique for oceanography. Laboratory investigations on the feasibility of using LIBS to detect analytes in bulk liquids at oceanic pressures were carried out. LIBS was successfully used to detect dissolved Na, Mn, Ca, K, and Li at pressures up to 2.76 x 10(7) Pa. The effects of pressure, laser-pulse energy, interpulse delay, gate delay, temperature, and NaCl concentration on the LIBS signal were examined. An optimal range of laser-pulse energies was found to exist for analyte detection in bulk aqueous solutions at both low and high pressures. No pressure effect was seen on the emission intensity for Ca and Na, and an increase in emission intensity with increased pressure was seen for Mn. Using the dual-pulse technique for several analytes, a very short interpulse delay resulted in the greatest emission intensity. The presence of NaCl enhanced the emission intensity for Ca, but had no effect on peak intensity of Mn or K. Overall, increased pressure, the addition of NaCl to a solution, and temperature did not inhibit detection of analytes in solution and sometimes even enhanced the ability to detect the analytes. The results suggest that LIBS is a viable chemical sensing method for in situ analyte detection in high-pressure environments such as the deep ocean.