Fentanyl in the labor epidural impacts the results of intrapartum and postpartum maternal and neonatal toxicology tests.

BACKGROUND: A positive urine fentanyl toxicology test may have considerable consequences for peripartum individuals, yet the extent to which fentanyl administration in a labor epidural may lead to such a positive test is poorly characterized. OBJECTIVE: This study aimed to quantify the extent to which neuraxial fentanyl in labor neuraxial analgesia can lead to a positive peripartum maternal or neonatal urine toxicology test. STUDY DESIGN: We performed a prospective cohort study of pregnant participants planning a vaginal delivery with neuraxial analgesia. Participants with a history of substance use disorder, hypertension, or renal or liver disease were excluded. A urine sample was collected before initiation of neuraxial analgesia, each time the bladder was emptied during labor, and up to 4 times postpartum. Neonatal urine was collected once. Urine fentanyl testing was performed using 2 common toxicology testing methods, namely immunoassay and liquid chromatography with tandem mass spectrometric detection. RESULTS: A total of 33 maternal-infant dyads yielded a total of 178 urine specimens. All maternal specimens were negative for fentanyl using liquid chromatography with tandem mass spectrometric analysis and immunoassay before initiation of neuraxial analgesia. Intrapartum, 26 of 30 (76.7%) participants had positive liquid chromatography with tandem mass spectrometry results for fentanyl or its metabolites, and 12 of 30 (40%) participants had positive immunoassay results. Postpartum, 19 of 21 (90.5%) participants had positive liquid chromatograph with tandem mass spectrometric results, and 13 of 21 (61.9%) had a positive immunoassay result. Of the 13 neonatal specimens collected, 10 (76.9%) were positive on liquid chromatography with tandem mass spectrometry analysis, the last of which remained positive 29 hours and 50 minutes after delivery. CONCLUSION: Neuraxial fentanyl for labor analgesia may lead to positive maternal and neonatal toxicology tests at various times after epidural initiation and cessation and at different rates depending on the testing method used. Caution should be used in interpreting toxicology test results of individuals who received neuraxial analgesia to avoid false assumptions about nonprescribed use.

Despite the improved clinical sensitivity of the Roche benzodiazepines II assay it cannot replace mass spectrometry in all patient populations.

Introduction: Benzodiazepines are frequently prescribed and misused therefore urine drug screening (UDS) is performed in many patient populations. Most current benzodiazepine immunoassays have poor sensitivity, particularly for detecting the metabolites of newer benzodiazepines such as lorazepam in urine. Objectives: We aimed to verify the clinical performance of the new qualitative Roche Benzodiazepines II (BNZ2) immunoassay, as well as compare its performance to the Roche Benzodiazepines Plus (BENZ) assay in two patient populations: UDS in the emergency department (ED) and compliance monitoring. Methods: An initial verification study was performed, selecting for samples containing clonazepam and lorazepam metabolites. Performance of the BNZ2 and BENZ assays was compared to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the reference method. Sensitivity, specificity, false positive rate (FPR) and false negative rate (FNR) were determined. Results: We verified the performance claims in the initial verification and demonstrated similar precision, with coefficient of variations (CVs) of 12.8% and 7.7% for negative and positive controls, respectively. Furthermore, we observed higher clinical sensitivity and lower FNR with the BNZ2 assay in both the ED and compliance monitoring populations due to improved cross-reactivity for lorazepam and clonazepam metabolites. Despite these improvements, the BNZ2 assay was unable to detect 27% of specimens positive by LC-MS/MS, including specimens from patients using benzodiazepines without prescription. Discussion: Due to its improved performance and rapid turnaround time, the BNZ2 assay should be implemented for UDS in the ED. However, the assay should not replace LC-MS/MS testing for compliance monitoring, as unsuspected benzodiazepine use may go undetected.

Performance of a Norfentanyl Immunoassay in Specimens with Low Concentrations of Fentanyl and/or Norfentanyl.

BACKGROUND: Many fentanyl immunoassays are limited in their ability to detect norfentanyl. Urine specimens collected from individuals who have been exposed to fentanyl frequently have detectable concentrations of norfentanyl (≥2 ng/mL) but low concentrations of fentanyl (<2 ng/mL) by LC-MS/MS. The Lin-Zhi Fentanyl II Immunoassay (Lin-Zhi) claims 100% cross-reactivity with norfentanyl and therefore may detect exposure missed by other assays. METHODS: In addition to verifying the manufacturer's analytical sensitivity claims, we selected 92 urine specimens with low-positive Lin-Zhi results (1-99 absorbance units, lowest 10%) for analysis by the Immunalysis Health Equity Impact Assessment and ARK II fentanyl methods. The accuracy of the 3 immunoassays was compared to LC-MS/MS as the reference method. RESULTS: Spiking studies using purified fentanyl and norfentanyl and a set of 100 consecutive specimens confirmed the manufacturer's claims of limit of detection for fentanyl (3.8 ng/mL) and norfentanyl (5.0 ng/mL). However, the 92 low-positive patient specimens demonstrated concentrations of norfentanyl and fentanyl below 2.0 ng/mL by LC-MS/MS, with 47 (51%) having only norfentanyl detected. When comparing Lin-Zhi to the Immunalysis and ARK II immunoassays, only 27 (29%) of the 92 specimens were concordant. Fifty-two (57%) of the specimens were positive by LC-MS/MS and Lin-Zhi but false negative by one or both other immunoassays. Seven specimens (8%) were positive by Lin-Zhi but negative by the other immunoassays and had undetectable concentrations (<2 ng/mL) of fentanyl and norfentanyl by LC-MS/MS. CONCLUSIONS: The clinical sensitivity of the Lin-Zhi exceeds the manufacturer's claims, providing results comparable to LC-MS/MS methods.

Advances in fentanyl testing.

Fentanyl is a synthetic opioid that was approved by the FDA in the late 1960s. In the decades since, non-prescription use of fentanyl, its analogs, and structurally unrelated novel synthetic opioids (NSO) has become a worsening public health crisis. There is a clear need for accessible testing for these substances in biological specimens and in apprehended drugs. Immunoassays for fentanyl in urine are available but their performance is restricted to facilities that hold moderate complexity laboratory licenses. Immunoassays for other matrices such as oral fluid (OF), blood, and meconium have been developed but are not widely available. Point of care tests (POCT), such as lateral flow immunoassays or fentanyl test strips (FTS), are widely available but not approved by the FDA for clinical use. All immunoassays are vulnerable to false positive and false negative results. Immunoassays may or may not be able to detect fentanyl analogs and NSOs. Mass spectrometry (MS) can accurately and reliably measure fentanyl and its major metabolite norfentanyl in urine and oral fluid. MS is available at reference laboratories and large hospitals. Liquid chromatography paired with tandem mass spectrometry (LC-MS/MS) is the most widely used method and has outstanding specificity and sensitivity for fentanyl and norfentanyl. When compared to immunoassays, MS is more expensive, requires more technical skill, and takes longer to result. Newer mass spectrometry methods can measure fentanyl analogs and NSO. Both mass spectrometry assays and immunoassays [in the form of fentanyl test strips (FTS)] have potential use in harm reduction programs.

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.

Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding.

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for Trib2-induced AML. Deletion or mutation of the COP1-binding site abrogated the ability of Trib2 to degrade CCAAT/enhancer-binding protein-α (C/EBP-α), block granulocytic differentiation, and to induce AML in vivo. Furthermore, COP1 knockdown inhibited the ability of Trib2 to degrade C/EBP-α, showing that it is important for mediating Trib2 activity. We also show that the Trib2 kinase domain is essential for its function. Trib2 contains variant catalytic loop sequences, compared with conventional kinases, that we show are necessary for Trib2 activity. The kinase domain mutants bind, but cannot efficiently degrade, C/EBP-α. Together, our data demonstrate that Trib2 can bind both COP1 and C/EBP-α, leading to degradation of C/EBP-α. Identification of the functional regions of Trib2 that are essential to its oncogenic role provides the basis for developing inhibitors that will block Trib functions in cancer.

Differential ability of Tribbles family members to promote degradation of C/EBPalpha and induce acute myelogenous leukemia.

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha, which account for their differential ability to induce leukemia.

Bupivacaine Metabolite Can Interfere with Norfentanyl Measurement by LC-MS/MS.

BACKGROUND: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard for the measurement of fentanyl and norfentanyl (NF) in urine and is favored over immunoassays due to its superior specificity. NF is the principal metabolite of fentanyl found in the urine and is typically present in higher abundance than fentanyl. Thus, the sensitivity and specificity of LC-MS/MS relies largely on the ability to identify and quantitate NF. METHODS: We analyzed urine specimens from women who had received bupivacaine and fentanyl for epidural analgesia during labor. We analyzed the contents of the epidural bag itself and purified bupivacaine metabolite N-desbutyl bupivacaine [or N-(2,6-dimethylphenyl)piperidine-2-carboxamide (NDB)] by LC-MS/MS. RESULTS: NDB interferes with the LC-MS/MS assay for NF. NDB passes through the Q1 mass selection filter because it is isobaric with the NF precursor ion (233 m/z). Further, it shares product ions with NF (84 m/z and 150 m/z), used as quantifier and qualifier ions, respectively, in our urine NF detection method. Baseline resolution of NDB and NF using these quantifier and qualifier ions could not be achieved. A unique product ion of NF (177 m/z) was useful for distinguishing NDB from NF. CONCLUSION: Bupivacaine is a commonly used drug. Recognition of this interference by laboratories is critical for preventing the misidentification of NF, which can have profound effects on patient care.

Impact of marijuana legalization on cannabis-related visits to the emergency department.

BACKGROUND: Cannabis is widely used in the United States despite federal laws. In US states that have progressed toward legalization, there have been various reported impacts on cannabis-related emergency department (ED) visits. However, studies on the impact of legalization in Massachusetts (MA) EDs are lacking. METHODS: Cannabinoid immunoassay (THC IA) results and cannabis-related ICD-10 codes were obtained for consecutive patient ED visits at two academic medical centers in Boston, MA over the following legalization periods (January 2012-December 2019): decriminalized (DEC), before medical dispensaries (MED BD), medical dispensaries available (MED DISP), before recreational dispensaries (REC BD) and recreational dispensaries available (REC DISP). Trends and monthly positivity rates for THC IA and ICD-10 codes were determined for these legalization periods. RESULTS: There was an increase in both THC IA (p < .0001) and cannabis-related ICD-10 codes (p < .0001) in the ED as legalization progressed at both institutions. Positivity rates significantly increased by 7% for THC IA and 0.4% for ICD-10 codes. Increases in THC IA positivity were seen in females, patients aged 30-39, older adults (>59 years), and those in the highest income tertile. There was an increasing trend in amphetamine positivity and decreasing trend in opiate positivity in patients with positive THC IA. Unlike THC IA, significant trends per patient demographics were not seen with ICD-10 codes. CONCLUSIONS: Legalization of marijuana in MA has led to an increase in cannabis use as indicated by both increasing rates of positive THC IA results, in older adults, as well as increasing cannabis-related ICD-10 codes. Data suggest a steady increase in THC use associated with legalization that was not associated with an increase in opiate, fentanyl, or cocaine use. We recommend using ED THC IA positivity, an objective laboratory measure, to monitor THC use and the impact of state-specific progression in cannabis legalization.

Comparison of Oral Fluid and Urine for Detection of Fentanyl Use Using Liquid Chromatography with Tandem Mass Spectrometry.

BACKGROUND: We compared oral fluid (OF) and urine (UR) for detection of fentanyl (FEN) use in addiction medicine-psychiatry (AMP) clinics. METHODS: We measured FEN and norfentanyl (NRFEN) in UR with a limit of detection (LOD) of 2.0 µg/L and FEN in OF with an LOD of 0.5 µg/L by LC-MS/MS in 311 paired samples and compared the 2 matrices when higher OF and UR LODs were used. RESULTS: Urine (UR) detected more FEN use than OF using a LOD of 2.0 µg/L and 0.5 µg/L, respectively. FEN and/or NRFEN were detected in 44 and 59 UR specimens, respectively, and FEN in 46 OF specimens (43 OF+UR+, 3 OF+UR-, 16 OF-UR+, and 249 OF-UR-). In UR there were no instances with FEN positive and NORFEN negative. UR creatinine was <20 mg/dL in the 3 OF+UR- specimen pairs. The median OF/UR analyte concentration ratios in positive sample pairs were 0.23 for OF FEN/UR FEN and 0.02 for OF FEN/UR NRFEN. CONCLUSIONS: We demonstrate that UR detects more FEN use than OF in an AMP setting when UR FEN and UR NORFEN LODs of 2.0 µg/L are used. OF is less sensitive than UR in detecting FEN use, but is still valuable for cases with low UR creatinine and/or suspected adulteration or substitution of UR. The UR vs OF comparison statistics are greatly impacted by even minimal adjustments of the LOD.

Validation and Implementation of an Ordering Alert to Improve the Efficiency of Monoclonal Gammopathy Evaluation.

OBJECTIVES: To evaluate the use of a provider ordering alert to improve laboratory efficiency and reduce costs. METHODS: We conducted a retrospective study to assess the use of an institutional reflex panel for monoclonal gammopathy evaluation. We then created a clinical decision support (CDS) alert to educate and encourage providers to change their less-efficient orders to the reflex panel. RESULTS: Our retrospective analysis demonstrated that an institutional reflex panel could be safely substituted for a less-efficient and higher-cost panel. The implemented CDS alert resulted in 79% of providers changing their high-cost order panel to an order panel based on the reflex algorithm. CONCLUSIONS: The validated decision support alert demonstrated high levels of provider acceptance and directly led to operational and cost savings within the laboratory. Furthermore, these studies highlight the value of laboratory involvement with CDS efforts to provide agile and targeted provider ordering assistance.

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.

Vortioxetine use may cause false positive immunoassay results for urine methadone.

BACKGROUND: Urine immunoassays are frequently employed for methadone screening because they are relatively inexpensive and widely available. However, immunoassays are notoriously prone to false positives. We report that the use of vortioxetine (Trintellix® in the USA and Canada, Brintellix® worldwide) could cause false positives in the Roche KIMS Methadone II Urine immunoassay (MDN2). METHODS: We performed a spiking study using a parent drug vortioxetine concentration of 7500 ng/ml. RESULTS: Urine specimens from seven patients on typical vortioxetine doses tested positive for methadone in the Roche assay but negative for methadone in a confirmatory (GC/MS) assay and two other immunoassay platforms. Because of the pharmacokinetics of vortioxetine and the high cross-reactivity of a metabolite in the MDN2 assay, routine use of the drug could cause false positives even without detectible parent drug in the urine. CONCLUSIONS: Vortioxetine is commonly prescribed for mood disorders, which have high prevalence in patients treated for opioid addiction. For that reason, it is important that clinicians are aware of this interference.

STK40 Is a Pseudokinase that Binds the E3 Ubiquitin Ligase COP1.

Serine/threonine kinase 40 (STK40) was originally identified as a distant homolog of Tribbles-family proteins. Despite accumulating data attesting to the importance of STK40 in a variety of different physiologic processes, little is known about its biological activity or mechanism of action. Here, we show that STK40 interacts with Constitutive Photomorphogenic Protein 1 (COP1), relying primarily on a C-terminal sequence analogous to the motif found in Tribbles proteins. In order to further elucidate structure-function relationships in STK40, we determined the crystal structure of the STK40 kinase homology domain at 2.5 Å resolution. The structure, together with ATP-binding assay results, show that STK40 is a pseudokinase, in which substitutions of conserved residues within the kinase domain prevent ATP binding. Although the structure of the kinase homology domain diverges from the analogous region of Trib1, the results reported here suggest functional parallels between STK40 and Tribbles-family proteins as COP1 adaptors.

Crystal structure of the catalytic core of human DNA polymerase kappa.

We present the crystal structure of the catalytic core of human DNA polymerase kappa (hPolkappa), the first structure of a human Y-family polymerase. hPolkappa is implicated in the proficient extension of mispaired primer termini on undamaged DNAs, and in the extension step of lesion bypass. The structure reveals a stubby "fingers" subdomain, which despite its small size appears to be tightly restrained with respect to a putative templating base. The structure also reveals a novel "thumb" subdomain that provides a basis for the importance of the N-terminal extension unique to hPolkappa. And, most surprisingly, the structure reveals the polymerase-associated domain (PAD) juxtaposed on the dorsal side of the "palm" subdomain, as opposed to the fingers subdomain. Together, these properties suggest that the hPolkappa active site is constrained at the site of the templating base and incoming nucleotide, but the polymerase is less constrained following translocation of the lesion.