Frederick G. Strathmann

Blood-based biomarkers for traumatic brain injury: evaluation of research approaches, available methods and potential utility from the clinician and clinical laboratory perspectives

Blood-based biomarkers for traumatic brain injury (TBI) have been investigated and proposed for decades, yet the current clinical assessment of TBI is largely based on clinical symptoms that can vary widely amongst patients, and have significant overlap with unrelated disease states. A careful review of current treatment guidelines for TBI further highlights the potential utility of a blood-based TBI biomarker panel in augmenting clinical decision making. Numerous expert reviews on blood-based TBI biomarkers have been published but a close look at the methods used and the astonishing paucity of validation and quality control data has not been undertaken from the vantage point of the clinical laboratory. Further, the field of blood-based TBI biomarker research has failed to adequately examine sex and gender differences between men and women with respect to the clinical care settings, as well as differences in physiological outcomes of TBI biomarker studies. Discussions of tried-and-true laboratory techniques in addition to a few new ones already operating in the clinical laboratory are summarized with a consideration of their utility in TBI biomarker assessment. In the context of TBI biomarkers, the central concerns discussed in this review are the readiness of the clinical laboratory, the willingness of the research environment and the inherent ability of each to radically affect patient outcomes in TBI.

Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND Immunoassays for 1α,25-dihydroxyvitamin D [1α,25(OH)(2)D] lack analytical specificity. We characterized the cross-reactivity of an anti-1α,25(OH)(2)D antibody with purified vitamin D metabolites and used these data to map the chemical features of 1α,25(OH)(2)D that are important for antibody binding. Additionally, we hypothesized that when combined with isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibody cross-reactivity could be used to semiselectively enrich for structurally similar metabolites of vitamin D in a multiplexed assay. METHODS Sample preparation consisted of immunoaffinity enrichment with a solid-phase anti-1α,25(OH)(2)D antibody and derivatization. Analytes were quantified with LC-MS/MS. Supplementation and recovery studies were performed for 11 vitamin D metabolites. We developed a method for simultaneously quantifying 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25(OH)(2)D(3) that included deuterated internal standards for each analyte. RESULTS The important chemical features of vitamin D metabolites for binding to the antibody were (a) native orientation of the hydroxyl group on carbon C3 in the A ring, (b) the lack of substitution at carbon C4 in the A ring, and (c) the overall polarity of the vitamin D metabolite. The multiplexed method had lower limits of quantification (20% CV) of 0.2 ng/mL, 1.0 ng/mL, 0.06 ng/mL, 3.4 pg/mL, and 2.8 pg/mL for 25(OH)D(2), 25(OH)D(3), 24,25(OH)(2)D(3), 1α,25(OH)(2)D(2), and 1α,25(OH)(2)D(3), respectively. Method comparisons to 3 other LC-MS/MS methods yielded an r(2) value >0.9, an intercept less than the lower limit of quantification, and a slope statistically indistinguishable from 1.0. CONCLUSIONS LC-MS/MS can be used to characterize antibody cross-reactivity, a conclusion supported by our multiplexed assay for 5 vitamin D metabolites with immunoenrichment in a targeted metabolomic assay.

A Systematic Review of the Biomarker S100B: Implications for Sport-Related Concussion Management

OBJECTIVE Elevated levels of the astroglial protein S100B have been shown to predict sport-related concussion. However, S100B levels within an athlete can vary depending on the type of physical activity (PA) engaged in and the methodologic approach used to measure them. Thus, appropriate reference values in the diagnosis of concussed athletes remain undefined. The purpose of our systematic literature review was to provide an overview of the current literature examining S100B measurement in the context of PA. The overall goal is to improve the use of the biomarker S100B in the context of sport-related concussion management. DATA SOURCES PubMed, SciVerse Scopus, SPORTDiscus, CINAHL, and Cochrane. STUDY SELECTION We selected articles that contained (1) research studies focusing exclusively on humans in which (2) either PA was used as an intervention or the test participants or athletes were involved in PA and (3) S100B was measured as a dependent variable. DATA EXTRACTION We identified 24 articles. Study variations included the mode of PA used as an intervention, sample types, sample-processing procedures, and analytic techniques. DATA SYNTHESIS Given the nonuniformity of the analytical methods used and the data samples collected, as well as differences in the types of PA investigated, we were not able to determine a single consistent reference value of S100B in the context of PA. Thus, a clear distinction between a concussed athlete and a healthy athlete based solely on the existing S100B cutoff value of 0.1 μg/L remains unclear. However, because of its high sensitivity and excellent negative predictive value, S100B measurement seems to have the potential to be a diagnostic adjunct for concussion in sports settings. We recommend that the interpretation of S100B values be based on congruent study designs to ensure measurement reliability and validity.

Detection of neonatal drug exposure using umbilical cord tissue and liquid chromatography time-of-flight mass spectrometry.

Background: A method for qualitative detection of 57 drugs and metabolites in umbilical cord tissue using liquid chromatography time-of-flight (TOF) mass spectrometry is described. Methods: Results from 32 deidentified positive specimens analyzed by an outside laboratory using “screen with reflex to confirmation” testing were compared with TOF results. In addition, 57 umbilical cord tissue specimens paired with corresponding chart review data and 37 with meconium test results were analyzed by TOF. Urine drug test results from mother (n = 18) and neonate (n = 30) were included if available. Cutoff concentrations, recovery, and matrix effects were determined by analyzing fortified drug-free cord tissue and negative specimens. Cutoffs (in nanograms per gram) ranged from 1 to 10 for opioids and opioid antagonists, 5–10 for benzodiazepines and nonbenzodiazepine hypnotics, 20–40 for barbiturates, 8 for stimulants, and 4 for phencyclidine. Adequate sensitivity for the detection of cannabis exposure could not be realized with this method. Conclusions: Liquid chromatography time-of-flight mass spectrometry can provide accurate and sensitive detection of in utero drug exposure using umbilical cord tissue.

Patterns of Drugs and Drug Metabolites Observed in Meconium: What Do They Mean?

Background: Meconium drug testing is performed to detect potentially harmful drug exposures in a newborn. Interpretation of meconium drug testing results can be complicated based on the patterns and proportional concentrations of the drug(s) and/or drug metabolite(s) detected. Methods: The objective of this study was to analyze meconium drug testing patterns in a de-identified dataset from a national reference laboratory (n = 76,631) and in a subset of the data, wherein specimens originated at a single academic medical center for which detailed chart review was possible (n = 3635). Meconium testing was performed using 11 immunoassay-based drug screens. Specimens that were positive for one or more drug screens were reflexed to corresponding confirmation tests performed by gas chromatography or liquid chromatography with mass spectrometric detection, targeted to identify and quantitate specific parent drug(s) and metabolite(s). Results: The positivity rate was the highest for the cannabis metabolite 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (25.2%, n = 18,643), followed by opiates/oxycodone (23.2%, n = 17,778), amphetamine/methamphetamine (6.7%, n = 5134), cocaine metabolites (5.5%, n = 4205), methadone (5.3%, n = 4093), benzodiazepines (3.4%, n = 2603), barbiturates (1.1%, n = 834), propoxyphene (1.0%, n = 749), and phencyclidine (0.1%, n = 44). Based on documented pharmacy history, drugs administered to either the mother or newborn during the birth hospitalization were detected in meconium, providing evidence that drugs can be incorporated into meconium rapidly. Drugs administered directly to the newborn after birth were recovered in meconium as both parent drug and metabolites, providing evidence of neonatal metabolism. Overall, patterns observed in meconium exhibited many similarities to those patterns commonly reported with urine drug testing. Conclusions: Interpretation of meconium drug testing results requires comparison of results with clinical and analytical expectations, including maternal admissions to drug use, pharmacy history, recognized metabolic patterns for drugs of interest, cutoff concentrations, and other performance characteristics of the test. Concentrations of drug(s) and drug metabolites(s) may not reliably predict timing of drug use, extent of drug use, or frequency of drug exposures.

Retrospective analysis of the diagnostic yield of newborn drug testing

BackgroundThe objective of this study was to identify high-yield screening risk factors for detecting maternal non-medical drug use during pregnancy.MethodsA four year retrospective analysis was conducted at an academic medical center. Detailed chart review of both the newborn and mother’s medical record was performed on all cases for which one or more drug(s) or metabolite(s) were identified and confirmed in meconium or urine.Results229 (9.2%) of 2,497 meconium samples out of 7,749 live births confirmed positive for one or more non-medical drugs. History of maternal non-medical drug and/or tobacco use in pregnancy was present in 90.8% of non-medical drug use cases. Addition of social risk factors and inadequate prenatal care increased the yield to 96.9%.ConclusionsUse of focused screening criteria based on specific maternal and social risk factors may detect many prenatal non-medical drug exposures.

Use of synthetic urine as a matrix substitute for standard and quality control materials in the clinical assessment of iodine by inductively coupled plasma mass spectrometry.

OBJECTIVES We developed and validated the use of synthetic urine as a matrix substitute for standard and quality control material preparation in the clinical assessment of iodine status in urine. DESIGN AND METHODS Measurement of iodine in urine was conducted using inductively coupled-plasma mass spectrometry. Analytical and clinical recoveries were assessed to investigate comparability between synthetic urine and pooled patient urine. Method performance characteristics were determined in accordance with clinical laboratory standards. RESULTS Established assay performance characteristics included inter- and intra-assay imprecision <10%, carryover of <0.2%, analytical measurement range of 5 to 1000μg/L, limit of quantification of 5μg/L (coefficient of variation <10%), proportional bias of 0.92 and constant bias of 8.8 in comparison to an outside reference laboratory. CONCLUSIONS Synthetic urine is an appropriate alternative matrix for standard and quality control material preparation for the measurement of iodine in urine.

Meconium Drug Testing in Multiple Births in the USA

Little is published about newborn drug testing in multiple gestations. The objective of this study was to review the results of meconium drug screening in multiple births to compare drug(s) and/or drug metabolite(s) detected. A retrospective analysis was conducted using data from a national reference laboratory and an academic medical center. The data were de-identified for the reference laboratory dataset. For the academic center data, a detailed chart review of the newborn and mothers medical record was performed on cases for which one or more drug(s) and/or metabolites(s) were identified and confirmed in meconium. Meconium was analyzed for amphetamine, methamphetamine, barbiturates, benzodiazepines, cannabinoid metabolites, cocaine metabolites, methadone, opiates, oxycodone, phencyclidine and propoxyphene. One hundred and forty-two of 1,084 sets of twins and 2 of 20 sets of triplets had mismatched results. The incidence of mismatched results among the individual drug or drug classes tested was 0.9% (208 of 23,848 total results). For the panel of drug testing performed, mismatches were seen in 13% (142 of 1,084) sets of twins and 10% (2 of 20) sets of triplets. Barbiturates (33%), opiates (30%) and benzodiazepines (28%) were the most common mismatches in the national reference laboratory dataset. Benzodiazepines (89%) and opiates (51%) were most common in the academic medical center dataset with most explained by iatrogenic medications administered to one infant but not the other. Mismatches for cannabinoids most often occurred when tetrahydrocannabinol metabolites were present at a low concentration (near lower reporting limit) in one infant but not the other. Mismatched results of meconium drug testing in multiples not explainable by differences in prescribed medications are uncommon and most often occur when an analyte is barely above reporting cutoff in only one infant. Administration of iatrogenic medications to one infant but not the other(s) is another frequent cause of such mismatches.

Pathology Consultation on Urine Compliance Testing and Drug Abuse Screening

OBJECTIVES Compliance testing in pain management requires a distinct approach compared with classic clinical toxicology testing. Differences in the patient populations and clinical expectations require modifications to established reporting cutoffs, assay performance expectations, and critical review of how best to apply the available testing methods. Although other approaches to testing are emerging, immunoassay screening followed by mass spectrometry confirmation remains the most common testing workflow for pain management compliance and drug abuse testing. METHODS A case-based approach was used to illustrate the complexities inherent to and uniqueness of pain management compliance testing for both clinicians and laboratories. RESULTS A basic understanding of the inherent strengths and weaknesses of immunoassays and mass spectrometry provides the clinician a better understanding of how best to approach pain management compliance testing. CONCLUSIONS Pain management compliance testing is a textbook example of an emerging field requiring open communication between physician and performing laboratory to fully optimize patient care.

Ordering of the Serum Angiotensin-Converting Enzyme Test in Patients Receiving Angiotensin-Converting Enzyme Inhibitor Therapy: An Avoidable but Common Error

BACKGROUND Serum angiotensin-converting enzyme (ACE) levels may be decreased by use of ACE inhibitor (ACEI) medication. In this study, we determined how often ACE levels were measured in patients receiving ACEI therapy. METHODS ACE levels analyzed over a 54-month preintervention time period at an academic medical center were reviewed retrospectively for tests performed during ACEI therapy. These data were compared with a large, deidentified dataset of ACE levels measured at a national reference laboratory; in vitro studies of ACEI inhibition; and liquid chromatography time-of-flight mass spectrometry detection of lisinopril in a subset of clinical specimens. RESULTS Over a 54-month period, 1,292 patients had ACE levels measured, with 108 patients (8.4%) receiving ACEI therapy at the time of testing. ACE levels measured for patients receiving ACEI therapy were substantially lower. In general, clinical teams did not recognize a medication effect on ACE levels. Introduction of a warning prompt in the electronic health record reduced the ordering of ACE levels in patients receiving ACEIs by > 60% in a 17-month postintervention time period. The deidentified dataset of ACE levels at a reference laboratory showed a bimodal distribution, with a peak of very low ACE levels. Using liquid chromatography time-of-flight mass spectrometry, the presence of lisinopril was confirmed in a subset of specimens with low ACE activity. In vitro studies of two different ACE assays showed significant inhibition of activity at clinically relevant concentrations. CONCLUSIONS Assessment of ACE activity is often measured for patients receiving ACEIs, potentially leading to low ACE concentrations and inaccurate interpretations.