Evaluation of the ARK Diagnostics immunoassay for qualitative detection of xylazine in urine.

Xylazine exposure is common in some US cities, but a commercial assay for routine laboratory testing for xylazine is not currently available. We evaluated a pre-release version of the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxyxylazine in urine. Studies were conducted using either the semi-quantitative assay application (A. Roche Cobas 503 analyzer) or the qualitative assay application (B. Beckman Coulter AU480 analyzer). Study specimens consisted of deidentified patient urine samples submitted for routine drugs-of-abuse testing. Measurements of xylazine (X) were performed by LC-MS-MS to obtain X-NEGATIVE (X <10 ng/mL) and X-POSITIVE (X ≥10 ng/mL). The semi-quantitative ARK assay was calibrated with a 10 ng/mL cutoff for ARK-POSITVE. For (A): among 74 X-POSITIVE samples, there was 1 ARK-NEGATIVE result (false-negative rate = 1.4%); among 78 X-NEGATIVE samples by LC-MS-MS, there were 0% ARK-POSITIVE results (false-positive rate = 0%). For (B), among 74 X-POSITIVE samples, there were 0 ARK-NEGATIVE results (false-negative rate = 0%); among 78 X-NEGATIVE samples there was 1 ARK-POSITIVE sample (false-positive rate = 1.3%). Common sources of interferences were investigated without evidence of interference. The ARK xylazine/4-OH-xylazine immunoassay was found to be suitable for routine use in screening patient urine samples for presence of xylazine >10 ng/mL.

High prevalence of xylazine among fentanyl screen-positive urines from hospitalized patients, Philadelphia, 2021.

BACKGROUND: Xylazine is an α-2 adrenoreceptor agonist used as a sedative/analgesic in veterinary medicine. Xylazine is known to be present within the street supply of opiates in urban Philadelphia. Medical staff at our hospital asked if we could test for xylazine in fentanyl screen-positive urine samples. We developed an LC-MS/MS assay for this purpose, and determined prevalence of xylazine among fentanyl screen-positive urine samples at our hospital. METHODS: The LC-MS/MS assay utilized d5-norfentanyl as internal standard (IS). One hundred microliter samples were extracted with 200 µl of MeOH/IS. LC was performed using a Phenomenex Kinetix C18 column (100 A, 5 µm, 50 × 4.6 mm) at 40 °C. Time-variable mobile phases (A = H2O, 0.1% formic acid; B = MeOH, 0.1% formic acid) were used at a fixed flow rate of 0.5 ml/min. MS/MS used positive electrospray ionization, monitoring m/z transitions of 221 > 164 for xylazine (primary), 221 > 90 for xylazine (qualifier), and 238 > 84 for d5-norfentanyl (IS). Retention time was 3.9 min for both xylazine and IS. RESULTS: Calibration curve was linear (0-500 ng/ml; r > 0.99). Inter-assay CVs (n = 20) were 5.2% (18 ng/ml) and 6.6% (95 ng/ml). Lower limit of detection was set at 10 ng/ml (CV = 15%). Among 81 urine samples that were screen-positive for fentanyl (Ark Diagnostics immunoassay), 63 (78%) were positive for xylazine (>10 ng/ml). CONCLUSIONS: By LC-MS/MS, there was high prevalence (78%) of xylazine in fentanyl screen-positive urine samples submitted to the laboratory. Because α-2 adrenoreceptor agonists may be used in treatment of opioid addiction, knowledge of xylazine exposure may be clinically useful to guide patient management.

MicroRNA-Related DNA Repair/Cell-Cycle Genes Independently Associated With Relapse After Radiation Therapy for Early Breast Cancer.

PURPOSE: Local recurrence and distant failure after adjuvant radiation therapy for breast cancer remain significant clinical problems, incompletely predicted by conventional clinicopathologic markers. We had previously identified microRNA-139-5p and microRNA-1274a as key regulators of breast cancer radiation response in vitro. The purpose of this study was to investigate standard clinicopathologic markers of local recurrence in a contemporary series and to establish whether putative target genes of microRNAs involved in DNA repair and cell cycle control could better predict radiation therapy response in vivo. METHODS AND MATERIALS: With institutional ethics board approval, local recurrence was measured in a contemporary, prospectively collected series of 458 patients treated with radiation therapy after breast-conserving surgery. Additionally, independent publicly available mRNA/microRNA microarray expression datasets totaling >1000 early-stage breast cancer patients, treated with adjuvant radiation therapy, with >10 years of follow-up, were analyzed. The expression of putative microRNA target biomarkers--TOP2A, POLQ, RAD54L, SKP2, PLK2, and RAG1--were correlated with standard clinicopathologic variables using 2-sided nonparametric tests, and to local/distant relapse and survival using Kaplan-Meier and Cox regression analysis. RESULTS: We found a low rate of isolated local recurrence (1.95%) in our modern series, and that few clinicopathologic variables (such as lymphovascular invasion) were significantly predictive. In multiple independent datasets (n>1000), however, high expression of RAD54L, TOP2A, POLQ, and SKP2 significantly correlated with local recurrence, survival, or both in univariate and multivariate analyses (P<.001). Low RAG1 expression significantly correlated with local recurrence (multivariate, P=.008). Additionally, RAD54L, SKP2, and PLK2 may be predictive, being prognostic in radiation therapy-treated patients but not in untreated matched control individuals (n=107; P<.05). CONCLUSIONS: Biomarkers of DNA repair and cell cycle control can identify patients at high risk of treatment failure in those receiving radiation therapy for early breast cancer in independent cohorts. These should be further investigated prospectively, especially TOP2A and SKP2, for which targeted therapies are available.