Clinical Impact of New Reference Intervals for the Roche Prolactin II Immunoassay.

Context: The Roche prolactin immunoassay is used throughout the world. It reports higher values than the Siemens immunoassay but the manufacturer-defined reference intervals are similar. Patient results are often above the Roche upper limit but within the Siemens interval, causing diagnostic confusion. Objective: Establish new reference intervals for the Roche and Siemens prolactin immunoassays. Methods: We established new reference intervals for the Roche and Siemens immunoassays using 374 specimens from healthy outpatients. We performed chart review for unnecessary testing and treatment for 298 patients in a 6-month period with at least 1 Roche prolactin value above the manufacturer-defined upper limit and below our new upper limit. Results: The new upper limit for the Roche assay was 37.8 ng/mL (females) and 22.8 ng/mL (males). The manufacturer-defined limits were 23.3 ng/mL and 15.2 ng/mL, respectively. New intervals for the Siemens assay matched the manufacturer. No cases of clinically significant pathophysiologic prolactin excess were identified in patients with values between the manufacturer-defined upper reference limit and our new Roche upper limit. Unnecessary further evaluation in these patients included 459 repeat prolactin measurements, 57 macroprolactin measurements, 39 magnetic resonance imaging studies, and 28 endocrine referrals. Eleven patients received dopamine agonists. The minimum cost of excess care using Medicare reimbursement rates was $34 134, with substantially higher amounts billed to patients and their insurance providers. Conclusion: Adoption of new upper reference limits for the Roche prolactin assay of 37.8 ng/mL (females) and 22.8 ng/mL (males) would not delay diagnosis or necessary intervention in patients with clinically significant pituitary tumors but would reduce unnecessary evaluation in patients without pathophysiologic prolactin excess.

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products.

Potential Impact of Pharmacogenomic Single Nucleotide Variants in a Rural Caucasian Population.

BACKGROUND: In the US adverse drug reactions (ADRs) are estimated to cause 100 000 fatalities and cost over $136 billion annually. A patient's genes play a significant role in their response to a drug. Pharmacogenomics aims to optimize drug choice and dose for individual patients by characterizing patients' pharmacologically relevant genes to identify variants of known impact. METHODS: DNA was extracted from randomly selected remnant whole blood samples from Caucasian patients with previously performed complete blood counts. Samples were genotyped by mass spectrometry using a customized pharmacogenomics panel. A third-party result interpretation service used genotypic results to predict likely individual responses to frequently prescribed drugs. RESULTS: Complete genotypic and phenotypic calls for all tested Cytochrome P450 isoenzymes and other genes were obtained from 152 DNA samples. Of these 152 unique genomic DNA samples, 140 had genetic variants suggesting dose adjustment for at least one drug. Cardiovascular and psychiatry drugs had the highest number of recommendations, which included United States Food and Drug Administration warnings for highly prescribed drugs metabolized by CYP2C19, CYP2C9, CYP2D6, HLA-A, and VKORC1. CONCLUSIONS: Risk for each drug:gene pairing primarily depends upon the degree of predicted enzyme impairment or activation, width of the therapeutic window, and whether parent compound or metabolite is pharmacologically active. The resulting metabolic variations range from risk of toxicity to therapeutic failure. Pharmacogenomic profiling likely reduces ADR potential by allowing up front drug/dose selection to fit a patient's unique drug-response profile.

AACC Guidance Document on the Use of Point-of-Care Testing in Fertility and Reproduction.

BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.

An Opioid Hiding in Plain Sight: Loperamide-Induced False-Positive Fentanyl and Buprenorphine Immunoassay Results.

BACKGROUND: Loperamide (Imodium®), a commonly used anti-diarrheal, is a mu opioid receptor agonist that, like all opioids, reduces gastrointestinal tract peristalsis. Loperamide is considered to have low abuse potential as it does not produce an analgesic or euphoric effect due to low bioavailability and first-pass metabolism. However, reports of individuals misusing loperamide through the use of super-therapeutic doses, alone or in combination with P-glycoprotein and/or CYP450 enzyme inhibitors, is increasing. We hypothesized that loperamide could potentially cross-react with laboratory immunoassay drug screens. METHODS: Drug-free urine was spiked with loperamide or its principal metabolite, N-desmethyl loperamide (dLop), and assayed on multiple fentanyl and buprenorphine assays. Fentanyl immunoassay screen-positive results at one institution were examined by high-resolution mass spectrometry (MS) for the presence of loperamide and quantified by liquid chromatography- tandem MS when positive. RESULTS: Loperamide produced positive results on the Thermo DRI Fentanyl and Immunalysis Fentanyl assays at concentrations greater than 5.72 mg/L and 23.7 mg/L. dLop generated positive results for the Thermo DRI and Immunalysis fentanyl assays at concentrations exceeding 6.9 mg/L and 35.7 mg/L. dLop also produced positive buprenorphine results on the Thermo CEDIA buprenorphine assay at concentrations exceeding 12.2 mg/L. High-resolution MS analysis of 225 fentanyl immunoassay positives (Thermo DRI) yielded 5 specimens containing loperamide and/or dLop, 4 of which contained measurable quantities of fentanyl in addition to loperamide/dLop. CONCLUSIONS: Laboratories using these assays should be aware of the potential for false-positive screening results due to the presence of high concentrations of loperamide and its metabolite dLop.

Reevaluating the Icterus Index Cutoff of a Jaffé Creatinine Method.

BACKGROUND: The aim of this study was to redefine the icterus index cutoff for the Roche Jaffé creatinine method using both conjugated and unconjugated bilirubin on 3 Roche cobas modules (c311, c501, and c701/c702) at laboratories across our hospital network. METHODS: Interference was evaluated by adding conjugated bilirubin (as bilirubin conjugate, ditaurate) and unconjugated bilirubin to pooled remnant plasma. The effects of conjugated and unconjugated bilirubin were tested separately to assess the contribution of each species. The magnitude of interference was calculated as both absolute and percentage error with total allowable error limits set at 0.1 mg/dL (8 µmol/L or 8%). RESULTS: Analysis of interference data across the 3 Roche modules did not show bias exceeding our total allowable error limits for plasma creatinine up to a conjugated bilirubin icterus index of 16.2 (approximately 16.2 mg/dL or 277 µmol/L) or an unconjugated bilirubin icterus index of 18.5 (approximately 18.5 mg/dL or 316 µmol/L), the highest concentrations tested. CONCLUSIONS: Our results demonstrate that the Roche Jaffé method exhibits acceptable performance in the presence of icterus at icterus indexes above the manufacturer's current recommendations of 5 (approximately 5 mg/dL or 86 µmol/L) and 10 (approximately 10 mg/dL or 171 µmol/L) for conjugated and unconjugated bilirubin, respectively. We have updated the icterus index in our hospital system to 16 for conjugated bilirubin and 18 for unconjugated bilirubin.

Wastewater-Based SARS-CoV-2 Surveillance in Northern New England.

SARS-CoV-2 viral RNA is shed in the stool of 55-70% of infected individuals and can be detected in community wastewater up to 7 days before people present with COVID-19 symptoms. The detection of SARS-CoV-2 RNA in wastewater may serve as a lead indicator of increased community transmission. Here, we monitored viral concentrations in samples collected from nine municipal wastewater facilities in New Hampshire (NH) and Vermont (VT).Twenty-four-h composite primary influent wastewater samples were collected from nine municipal wastewater treatment facilities twice per week for 5 months (late September 2020 to early February 2021). Wastewater was centrifuged for 30 min at 4600 × g, then the supernatant was frozen until further analysis. Once thawed, samples were concentrated, extracted, and tested for SARS-CoV-2 RNA using reverse transcriptase-quantitative PCR (RT-qPCR) and reverse transcriptase-droplet digital PCR (RT-ddPCR) detection methods. Active case counts for each municipality were tracked from the NH and VT state COVID-19 dashboards. We received a total of 283 wastewater samples from all sites during the study period. Viral RNA was detected in 175/283 (61.8%) samples using RT-qPCR and in 195/283 (68.9%) samples using RT-ddPCR. All nine sites showed positivity in the wastewater, with 8/9 (88.8%) sites having over 50% of their samples test positive over the course of the study. Larger municipalities, such as Nashua, Concord, and Lebanon, NH, showed that SARS-CoV-2 positivity in the wastewater can precede spikes in active COVID-19 case counts by as much as 7 days. Smaller municipalities, such as Woodsville, NH and Hartford, VT, showed sporadic SARS-COV-2 detection and did not always precede a rise in active case counts. We detected SARS-CoV-2 RNA in samples from all 9 municipalities tested, including cities and small towns within this region, and showed wastewater positivity as an early indicator of active case count increases in some regions. Some of the smaller rural municipalities with low case counts may require more frequent sampling to detect SARS-CoV-2 in wastewater before a case surge. With timely collection and analysis of wastewater samples, a community could potentially respond to results by increasing public health initiatives, such as tightening mask mandates and banning large indoor gatherings, to mitigate community transmission of SARS-CoV-2. IMPORTANCE Despite vaccination efforts, the delta and omicron variants of SARS-CoV-2 have caused global surges of COVID-19. As the COVID-19 pandemic continues, it is important to find new ways of tracking early signs of SARS-CoV-2 outbreaks. The manuscript outlines how to collect wastewater from treatment facilities, concentrate the virus in a dilute wastewater sample, and detect it using two sensitive PCR-based methods. It also describes important trends in SARS-CoV-2 concentration in wastewater of a rural region of the United States from Fall 2020 - Winter 2021 and demonstrates the utility of wastewater monitoring as a leading indicator of active SARS-CoV-2 cases. Monitoring changes in concentration of SARS-CoV-2 virus in wastewater may offer an early indicator of increased case counts and enable appropriate public health actions to be taken.

Evaluating thyroid function in pregnant women.

Thyroid hormones are primarily responsible for regulating the basal metabolic rate but also make important contributions to reproductive function and fetal development. Both hyper- and hypothyroidism in pregnancy have been associated with increased risks of complications that include preeclampsia and low birth weight, among others. Furthermore, thyroid hormone deficiency in the developing fetus results in neurodevelopmental delay. As the fetus is exclusively reliant on maternal thyroid hormone for most of the first trimester and requires continued maternal supply until birth, identifying maternal thyroid dysfunction is critically important. However, evaluating thyroid function in pregnancy is challenging because of the many physiological changes that affect concentrations of thyroid-related analytes. Increasing plasma human chorionic gonadotropin (hCG) concentrations in the second half of the first trimester elicit a corresponding transient decrease in thyroid-stimulating hormone (TSH), and continually increasing estradiol concentrations throughout pregnancy cause substantial increases in thyroxine-binding globulin (TBG) and total thyroxine (T4) relative to the nonpregnant state. Lastly, free T4 concentrations gradually decrease with increasing gestational age. For these reasons, it is essential to interpret thyroid function test results in the context of trimester-specific reference intervals to avoid misclassification of thyroid status. This review summarizes the effects of thyroid dysfunction prior to conception and during pregnancy and describes considerations for the laboratory assessment of thyroid function in pregnant women.

Comparison of Symptoms and Antibody Response Following Administration of Moderna or Pfizer SARS-CoV-2 Vaccines.

CONTEXT.­: Moderna (mRNA-1272) and Pfizer (BNT162b2) SARS-CoV-2 vaccines demonstrate favorable safety and efficacy profiles, but direct comparison data are lacking. OBJECTIVE.­: To determine the vaccines' side effect profiles and expected antibody responses. These data may help personalize vaccine selection and identify individuals with a suboptimal vaccine response. DESIGN.­: One hundred forty-nine healthy, largely seronegative adults were assigned Moderna (n = 79) or Pfizer (n = 70). Following the second dose, participants completed a survey documenting their side effects. Serum was collected 0 to 4 days prior to dose 2, and 14 ± 4 days, 30 ± 4 days, 90 ± 10 days, and 180 ± 20 days after dose 2. Convalescent serum specimens were collected 32 to 54 days from donors after a polymerase chain reaction-confirmed SARS-CoV-2 infection (n = 20). Anti-spike antibodies were measured using the Roche Diagnostics Elecys Anti-SARS-CoV-2 S assay on a Roche cobas e801 instrument. RESULTS.­: Participants receiving the Moderna vaccine experienced side effects with greater frequency and severity. Both vaccines elicited a robust antibody response, but median signal was higher in Moderna recipients. Symptom severity decreased with age. Antibody response in Pfizer recipients negatively correlated with age. Antibody response decreased after 6 months (84% reduction in Moderna, 79% Pfizer), but values remained greater than for convalescent donors. Antibody response did not correlate with gender or symptom severity. CONCLUSIONS.­: Moderna may be preferred in individuals in need of greater immune stimulation (eg, older individuals), whereas Pfizer may be preferred in those concerned about vaccine reactions. Anti-spike antibody signal varies by vaccine, so specific reference intervals will be needed to identify individuals with a suboptimal response.

Longitudinal Systemic and Mucosal Immune Responses to SARS-CoV-2 Infection.

BACKGROUND: A longitudinal study was performed to determine the breadth, kinetics, and correlations of systemic and mucosal antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Twenty-six unvaccinated adults with confirmed coronavirus disease 2019 (COVID-19) were followed for 6 months with 3 collections of blood, nasal secretions, and stool. Control samples were obtained from 16 unvaccinated uninfected individuals. SARS-CoV-2 neutralizing and binding antibody responses were respectively evaluated by pseudovirus assays and multiplex bead arrays. RESULTS: Neutralizing antibody responses to SARS-CoV-2 were detected in serum and respiratory samples for 96% (25/26) and 54% (14/26), respectively, of infected participants. Robust binding antibody responses against SARS-CoV-2 spike protein and S1, S2, and receptor binding (RBD) domains occurred in serum and respiratory nasal secretions, but not in stool samples. Serum neutralization correlated with RBD-specific immunoglobulin (Ig)G, IgM, and IgA in serum (Spearman ρ = 0.74, 0.66, and 0.57, respectively), RBD-specific IgG in respiratory secretions (ρ = 0.52), disease severity (ρ = 0.59), and age (ρ = 0.40). Respiratory mucosal neutralization correlated with RBD-specific IgM (ρ = 0.42) and IgA (ρ = 0.63). CONCLUSIONS: Sustained antibody responses occurred after SARS-CoV-2 infection. Notably, there was independent induction of IgM and IgA binding antibody and neutralizing responses in systemic and respiratory compartments. These observations have implications for current vaccine strategies and understanding SARS-CoV-2 reinfection and transmission.

Retrospective Evaluation of the Antibody Prevalence in Epilepsy and Encephalopathy (APE2) Score.

BACKGROUND: Autoimmune encephalitis (AE) is a rare collection of disorders that present with a diverse and often nebulous set of clinical symptoms. Indiscriminate use of multi-antibody panels decreases their overall utility and predictive value. Application of a standardized scoring system may help reduce the number of specimens that generate misleading or uninformative results. METHODS: The results of autoimmune encephalopathy, epilepsy, or dementia autoantibody panels performed on serum (n = 251) or cerebrospinal fluid (CSF) (n = 235) specimens from October 9th, 2016 to October 11th, 2019 were collected. Retrospective chart review was performed to calculate the Antibody Prevalence in Epilepsy and Encephalopathy (APE2) score for patients with an antibody above the assay-specific reference interval and to classify results as true or false positive. RESULTS: Of the 486 specimens, 60 (12.3%) generated positive results for any AE antibody (6 CSF and 54 serum). After removing 2 duplicate specimens collected from a single patient, 10 of the remaining 58 were determined to be true positives and 8 contained neural-specific antibodies. Application of the APE2 score revealed that 89% of all true positives and 86% of specimens with neural-specific antibodies had a score ≥4. In contrast, 76% of false positives, 74% of clinically nonspecific antibodies, and 85% of the negative specimens had an APE2 score <4. CONCLUSION: The APE2 score can improve the diagnostic utility of autoimmune encephalopathy evaluation panels.

Gamma Glutamyl Transferase Activity Has Limited Utility in Assessment of Alkaline Phosphatase Elevations.

INTRODUCTION: As part of an ongoing effort to improve healthcare value for patients, laboratories increasingly implement test utilization review. Alkaline phosphatase (ALP) isoenzymes (hereafter: isoenzymes) testing distinguishes the various ALP isoforms to explain elevations in total serum ALP. Gamma glutamyl transferase activity (GGT) has served as a proxy for total ALP elevations attributable to the hepatic isoform given that both are membrane-bound proteins with a shared mechanism of release. We assessed the utility of GGT in evaluating isoenzymes requests. METHODS: We obtained 8 years of isoenzymes results and identified same-patient GGT measurements obtained within 7 days. We assessed the ability of GGT to predict elevations in hepatic, bone, intestinal, and nonhepatic ALP isoforms overall. We generated ROC curves and calculated sensitivity and specificity using our in-house reference limits for GGT. RESULTS: GGT as a predictor of hepatic isoform elevation had an area under the ROC curve (AUC) of 0.68, and GGT activity above the upper reference limit was 46.6% sensitive and 85.0% specific for hepatic ALP elevation. GGT activity as a predictor of nonhepatic isoform elevation had an AUC of 0.52, and GGT within reference limits was 59.8% sensitive and 46.4% specific for elevation in a nonhepatic ALP isoform. In 133 individuals with hepatic isoform elevations, 93 had a concurrent elevation in a nonhepatic ALP isoform. CONCLUSION: GGT was reasonably specific but insensitive for hepatic ALP isoform elevation and was a poor predictor of ALP isoform elevation overall, suggesting that its usefulness in evaluating isoenzymes orders is limited.

A comparison of SARS-CoV-2 nucleocapsid and spike antibody detection using three commercially available automated immunoassays.

INTRODUCTION: Commercially available serological assays for SARS-CoV-2 detect antibodies to either the nucleocapsid or spike protein. Here we compare the performance of the Beckman-Coulter SARS-CoV-2 spike IgG assay to that of the Abbott SARS-CoV-2 nucleocapsid IgG and Roche Anti-SARS-CoV-2 nucleocapsid total antibody assays. In addition, we document the trend in nucleocapsid and spike antibodies in sequential samples collected from convalescent plasma donors. METHODS: Plasma or serum samples from 20 individual SARS-CoV-2 RT-PCR-positive inpatients (n = 172), 20 individual convalescent donors with a previous RT-PCR-confirmed SARS-CoV-2 infection (n = 20), were deemed positive SARS-CoV-2 samples. RT-PCR-negative inpatients (n = 24), and 109 pre-SARS-CoV-2 samples were determined to be SARS-CoV-2 negative. Samples were assayed by the Abbott, Roche, and Beckman assays. RESULTS: All three assays demonstrated 100% specificity. Abbott, Beckman, and Roche platforms had sensitivities of 98%, 93%, and 90% respectively, with the difference in sensitivity attributed primarily to samples from immunocompromised patients. After the exclusion of samples immunocompromised patients, all assays exhibited ≥ 95% sensitivity. In sequential samples collected from the same individuals, the Roche nucleocapsid antibody assay demonstrated continually increasing signal intensity, with maximal values observed at the last time point examined. In contrast, the Beckman spike IgG antibody signal peaked between 14 and 28 days post positive SARS-CoV-2 PCR and steadily declined in subsequent samples. Subsequent collections 51-200 days (median of 139 days) post positive SARS-CoV-2 RT-PCR from five inpatients and five convalescent donors revealed that spike and nucleocapsid antibodies remained detectable for several months after confirmed infection. CONCLUSIONS: The three assays are sensitive and specific for SARS-CoV-2 antibodies. Nucleocapsid and spike antibodies were detectable for up to 200 days post-positive SARS-CoV-2 PCR but demonstrated markedly different trends in signal intensity.