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.

Clinical decision support to improve CBC and differential ordering.

OBJECTIVES: Complete blood count and differential (CBC diff) is a common laboratory test that may be overused or misordered, particularly in an inpatient setting. We assessed the ability of a clinical decision support (CDS) alert to decrease unnecessary orders for CBC diff and analyzed its impact in the laboratory. METHODS: We designed 3 CDS alerts to provide guidance to providers ordering CBC diff on inpatients at frequencies of daily, greater than once daily, or as needed. RESULTS: The 3 alerts were highly effective in reducing orders for CBC diff at the frequencies targeted by the alert. Overall, test volume for CBC diff decreased by 32% (mean of 5257 tests per month) after implementation of the alerts, with a corresponding decrease of 22% in manual differentials performed (mean of 898 per month). Turnaround time for manual differentials decreased by a mean of 41.5 minutes, with a mean decrease of up to 90 minutes during peak morning hours. CONCLUSIONS: The 3 CDS alerts successfully decreased inpatient orders for CBC diff and improved the quality of patient care by decreasing turnaround time for manual differentials.

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.

Utility of Banff Human Organ Transplant Gene Panel in Human Kidney Transplant Biopsies.

BACKGROUND: Microarray transcript analysis of human renal transplantation biopsies has successfully identified the many patterns of graft rejection. To evaluate an alternative, this report tests whether gene expression from the Banff Human Organ Transplant (B-HOT) probe set panel, derived from validated microarrays, can identify the relevant allograft diagnoses directly from archival human renal transplant formalin-fixed paraffin-embedded biopsies. To test this hypothesis, principal components (PCs) of gene expressions were used to identify allograft diagnoses, to classify diagnoses, and to determine whether the PC data were rich enough to identify diagnostic subtypes by clustering, which are all needed if the B-HOT panel can substitute for microarrays. METHODS: RNA was isolated from routine, archival formalin-fixed paraffin-embedded tissue renal biopsy cores with both rejection and nonrejection diagnoses. The B-HOT panel expression of 770 genes was analyzed by PCs, which were then tested to determine their ability to identify diagnoses. RESULTS: PCs of microarray gene sets identified the Banff categories of renal allograft diagnoses, modeled well the aggregate diagnoses, showing a similar correspondence with the pathologic diagnoses as microarrays. Clustering of the PCs identified diagnostic subtypes including non-chronic antibody-mediated rejection with high endothelial expression. PCs of cell types and pathways identified new mechanistic patterns including differential expression of B and plasma cells. CONCLUSIONS: Using PCs of gene expression from the B-Hot panel confirms the utility of the B-HOT panel to identify allograft diagnoses and is similar to microarrays. The B-HOT panel will accelerate and expand transcript analysis and will be useful for longitudinal and outcome studies.

Banff Human Organ Transplant Transcripts Correlate with Renal Allograft Pathology and Outcome: Importance of Capillaritis and Subpathologic Rejection.

BACKGROUND: To seek insights into the pathogenesis of chronic active antibody-mediated rejection (CAMR), we performed mRNA analysis and correlated transcripts with pathologic component scores and graft outcomes. METHODS: We utilized the NanoString nCounter platform and the Banff Human Organ Transplant gene panel to quantify transcripts on 326 archived renal allograft biopsy samples. This system allowed correlation of transcripts with Banff pathology scores from the same tissue block and correlation with long-term outcomes. RESULTS: The only pathology score that correlated with AMR pathways in CAMR was peritubular capillaritis (ptc). C4d, cg, g, v, i, t, or ci scores did not correlate. DSA-negative CAMR had lower AMR pathway scores than DSA-positive CAMR. Transcript analysis in non-CAMR biopsies yielded evidence of increased risk of later CAMR. Among 108 patients without histologic CAMR, 23 developed overt biopsy-documented CAMR within 5 years and as a group had higher AMR pathway scores (P=3.4 × 10-5). Random forest analysis correlated 3-year graft loss with elevated damage, innate immunity, and macrophage pathway scores in CAMR and TCMR. Graft failure in CAMR was associated with TCMR transcripts but not with AMR transcripts, and graft failure in TCMR was associated with AMR transcripts but not with TCMR transcripts. CONCLUSIONS: Peritubular capillary inflammation and DSA are the primary drivers of AMR transcript elevation. Transcripts revealed subpathological evidence of AMR, which often preceded histologic CAMR and subpathological evidence of TCMR that predicted graft loss in CAMR.

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.

Case report: Successful simultaneous heart-kidney transplantation across a positive complement-dependent cytotoxic crossmatch.

Preformed donor-specific antibodies are associated with a higher risk of rejection and worse graft survival in organ transplantation. However, in heart transplantation, the risk and benefit balance between high mortality on the waiting list and graft survival may allow the acceptance of higher immunologic risk donors in broadly sensitized recipients. Transplanting donor-recipient pairs with a positive complement dependent cytotoxic (CDC) crossmatch carries the highest risk of hyperacute rejection and immediate graft loss and is usually avoided in kidney transplantation. Herein we report the first successful simultaneous heart-kidney transplant with a T- and B-cell CDC crossmatch positive donor using a combination of rituximab, intravenous immunoglobulin, plasmapheresis, bortezomib and rabbit anti-thymocyte globulin induction followed by eculizumab therapy for two months post-transplant. In the year following transplantation, both allografts maintained stable graft function (all echocardiographic left ventricular ejection fractions ≥ 65%, eGFR>60) and showed no histologic evidence of antibody-mediated rejection. In addition, the patient has not developed any severe infections including cytomegalovirus or BK virus infection. In conclusion, a multitarget immunosuppressive regimen can allow for combined heart/kidney transplantation across positive CDC crossmatches without evidence of antibody-mediated rejection or significant infection. Longer follow-up will be needed to further support this conclusion.

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.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Significance of the intraindividual variability of HLA IgG antibodies in renal disease patients observed with different beadsets monitored with two different secondary antibodies on a Luminex platform.

The accurate measurement of anti-HLA alloantibodies in transplant candidates is required for determining the degree of sensitization and for the listing of unacceptable antigens for organ allocation. Both the configuration of the HLA molecules coated on the beads and the nature of detection antibodies may impede assessment of the presence and strength of anti-HLA IgG- with the Luminex single-antigen-bead assay. Sera antibodies of the end-stage renal disease patients were compared using LIFECODES (LC) and LABScreen (LS) beadsets monitored with polyclonal-Fab (IgHPolyFab) and monoclonal-IgG (FcMonoIgG) second antibodies. Positive results at mean fluorescence intensity (MFI) > 500 (at serum dilution 1/10) were used to calculate panel reactive antibody (cPRA) levels. LS-beadsets are coated with monomeric variants in addition to intact HLA antigens with or without peptides, while LC-beadsets are devoid of monomeric variants and with lesser levels of peptide-free heterodimers. Consequently, IgG antibodies against both classes of HLA were reactive to more antigens with LS than with LC-beadsets. For both classes, MFIs were also frequently higher with LS than with LC. For HLA-I, MFIs were higher with IgHPolyFab than with FcMonoIgG with the exception of sera with MFIs > 5000 where they were comparable. For HLA-II, the reverse occurred, with significantly higher levels with FcMonoIgG regardless of the beadsets. The intraindividual variability observed between beadsets with two detection antibodies elucidates that antigens found as acceptable with one beadset may end up unacceptable with the other beadsets, with the possibility of denying potentially compatible transplants to candidates.

Regulation of TCRß allelic exclusion by gene segment proximity and accessibility.

Ag receptor loci are regulated to promote allelic exclusion, but the mechanisms are not well understood. Assembly of a functional TCR ß-chain gene triggers feedback inhibition of V(ß)-to-DJ(ß) recombination in double-positive (DP) thymocytes, which correlates with reduced V(ß) chromatin accessibility and a locus conformational change that separates V(ß) from DJ(ß) gene segments. We previously generated a Tcrb allele that maintained V(ß) accessibility but was still subject to feedback inhibition in DP thymocytes. We have now further analyzed the contributions of chromatin accessibility and locus conformation to feedback inhibition using two novel TCR alleles. We show that reduced V(ß) accessibility and increased distance between V(ß) and DJ(ß) gene segments both enforce feedback inhibition in DP thymocytes.

Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites.

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.

Out-of-frame T cell receptor beta transcripts are eliminated by multiple pathways in vivo.

Non-productive antigen receptor genes with frame shifts generated during the assembly of these genes are found in many mature lymphocytes. Transcripts from these genes have premature termination codons (PTCs) and could encode truncated proteins if they are not either inactivated or destroyed by nonsense-mediated decay (NMD). In mammalian cells, NMD can be activated by pathways that rely on the presence of an intron downstream of the PTC; however, NMD can also be activated by pathways that do not rely on these downstream introns, and pathways independent of NMD can inactivate PTC-containing transcripts. Here, through the generation and analysis of mice with gene-targeted modifications of the endogenous T cell receptor beta (Tcrb) locus, we demonstrate that in T cells in vivo, optimal clearance of PTC-containing Tcrb transcripts depends on the presence of an intron downstream of the PTC.

Ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases have overlapping activities during chromosomal signal joint formation.

Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.