Resolving Interference of Direct Oral Anticoagulants (DOACs) on Routine Coagulation, Thrombophilia, and Lupus Anticoagulant Testing Using Activated Carbon

Background: Direct (anti-IIa and anti-Xa) oral anticoagulants (DOACs) have a significant impact on various coagulation test results. As the correct interpretation of these tests is mandatory to prevent misclassification and subsequent clinical consequences, withholding treatment could be necessary, with the associated risk of thrombosis. Aims: To evaluate the performance of the activated carbon DOAC-Remove (5-Diagnostics, Basel, Switzerland) in extracting DOACs from plasma samples and its effect on various routine and esoteric coagulation test results. Patients, Materials and Methods: Left-over plasmas from patients treated or not with DOAC obtained in the routine laboratory workload were evaluated. Briefly, 0.8 mL of plasma sample was incubated with 1 tablet of the activated carbon for 10 min at room temperature using a rotating shaker. After a 2 min-centrifugation at 2000x g and room temperature, the supernatant was pipetted before being analyzed. Tests were performed before and after such incubation. DOACs were measured using either a specific direct thrombin inhibitor or an anti-Xa assay with specific calibrations. Routine coagulation tests (PT, aPTT, and factor(F)V, FVIII:C, FIX, fibrinogen, and D-dimer) were performed, as well as thrombophilia panel [antithrombin, protein C (PC, chromogenic and clotting assays), PS (free PS antigen and clotting assays)], and lupus anticoagulant (LA) panel [silica clotting time (SCT), and dilute Russell venom clotting time (dRVVT)]. All assays were performed using reagents from Werfen (Bedford, MA, USA) on the ACL TOP 700 analyzer. As the distribution of data was not normal, test results were compared using non-parametric tests. Results: We evaluated a total of 756 plasma samples, obtained from patients treated with dabigatran (n=139, median concentration 129 ng/mL [range:18-905]), rivaroxaban (n=157, median concentration 159 ng/mL [range:19-815]), or apixaban (n=155, median concentration=154 ng/mL (range:11-510), and from 305 patients with various disease states and not on DOAC including 35 patients on coumadin and 32 patients on heparin (UFH: n=18 ; LMWH: n=14). In untreated patients, the DOAC-Remove had no significant impact on test results (n>30 for each parameter), except for D-dimer, free PS and FVIII:C. However, the mean biases, evaluated according to Bland-Altman, were below the accepted limits, and changes would not have had any clinical relevance. In the plasma from patients treated on DOAC, the DOAC-Remove eliminated all the three DOACs, with levels far below the detection limit of the techniques after a 10 min-incubation, leading to a dramatic correction of the DOAC-induced prolongations of PT and APTT, as well as of their lowering effect on FV, FVIII and FIX activities. The same applied to the DOAC-induced elevation of PC and PS anticoagulant activities (clotting assays), and antithrombin activity in the plasma from patients on rivaroxaban and apixaban. DRVVT screen/confirm ratios, which were above the normal ranges in 45% of the patients on dabigatran (n=31), and 78% of the patients on rivaroxaban (n=32), were normalized in most samples after a 10 min-incubation with the carbon and remained positive in only 10% of the patients on dabigatran and 12% of the patients on rivaroxaban. The impact of the DOACs on the SCT screen/confirm ratio was less noticeable with baseline positive test results in only one of the samples from patients on dabigatran (n=31), and in 2 patients on rivaroxaban (n=32). Treatment with the carbon leaded to a correction in the positive sample from patient on dabigatran and in one of the two positive samples from patients on rivaroxaban. Both dRVVT and SCT test results were within the normal range in the plasma from 32 patients on apixaban before and after treatment with the activated carbon. As expected, fibrinogen, PC activity evaluated using a chromogenic assay, free PS antigen concentration, VWF:RCo and VWF:Ag were not affected by any of the 3 DOACs, and test results were not significantly different before and after incubation with the activated carbon. Conclusion: The DOAC-Remove had no effect on test results obtained in the plasma from untreated patients and from patients on traditional anticoagulants. It effectively removed all 3 tested DOACs from the plasma of treated patients, allowing an accurate measurement of routine and esoteric coagulation tests in DOAC treated patients without withholding the treatment. Disclosures No relevant conflicts of interest to declare.

Analysing cord blood levels of TNF inhibitors to validate the EULAR points to consider for TNF inhibitor use during pregnancy

BackgroundTo minimise placental transfer of tumour necrosis factor inhibitors (TNFi), the European League Against Rheumatism (EULAR) created points to consider (PtC) for the use of TNFi during pregnancy. We are the first to validate the EULAR-PtC by analysing TNFi concentrations in cord blood.MethodsPatients were derived from the Preconceptional Counselling in Active Rheumatoid Arthritis Study. TNFi was stopped at the time points recommended by the EULAR. Maternal blood and cord blood were collected and analysed for the concentration of TNFi.Results111 patients were eligible for the analysis. Median stop time points were gestational age (GA) 37.0 weeks for certolizumab pegol, GA 25.0 weeks for etanercept, GA 19.0 weeks for adalimumab and GA 18.4 weeks for infliximab. Certolizumab pegol (n=68) was detectable in 5.9% of cord blood samples, with a median concentration of 0.3 µg/mL (IQR: 0.2–1.3) and a median cord/maternal concentration ratio of 0.010. Etanercept (n=30) was not detected in any cord blood samples. Adalimumab (n=25) was detectable in 48.0% of cord blood samples, with a median concentration of 0.5 µg/mL (IQR: 0.2–0.7) and a median concentration ratio of 0.062 (IQR: 0.018–0.15). Infliximab (n=14) was detectable in 57.1% of cord blood samples, with a median concentration of 0.4 µg/mL (IQR: 0.1–1.2) and a median concentration ratio of 0.012 (IQR: 0.006–0.081).ConclusionCompliance with the EULAR-PtC results in absence or low levels of TNFi in cord blood.

Pharmacokinetics of direct oral anticoagulants in emergency situations – Results of the prospective observational RADOA-registry

Background Direct oral anticoagulants (DOAC) are increasingly used worldwide. Little is known so far about their pharmacokinetics in emergency situations. Methods A prospective, observational registry was performed to determine the clinical course in consecutive patients with major bleeding or urgent surgery treated with DOACs. In back-up blood samples from routine care DOAC drug concentrations were measured using UPLC-MS/MS. Anticoagulant intensity at first presentation and drug half-life (t1/2), tested in repeat samples, were evaluated. Results 140 patients were prospectively included. Pharmacokinetic data were available in 94% (132/140) of patients. 67% (89/132) experienced life-threatening bleeding and 33% (43/132) needed an urgent surgery. For pharmacokinetic analysis a total of 605 blood samples was available. Median concentration on admission was 205 ng/mL for rivaroxaban and 108 ng/mL for apixaban. All treatment groups showed a high variation of drug concentrations at baseline. In rivaroxaban treated patients t½ was 17.3 hours (95% CI: 15.4 – 19.7) without significant difference in both groups (major bleeding: t½ 16.7 hours, 95% CI: 14.7 - 19.3; urgent surgery: t½ 19.7 hours, 95% CI 15.2 - 27.9; p=0.292). In apixaban treated patients t½ was 25.0 hours (95% CI: 22.9 – 27.6) with a longer t½ after urgent surgery (t1/2: 30.8 hours; 95% CI: 26.9 – 36.4) compared to severe bleeding (t1/2: 20.8 hours; 95% CI: 18.8 – 23.2; p<0.001). Conclusions Emergency patients under DOAC treatment show a high variation of anticoagulant concentrations at baseline. Compared with rivaroxaban, apixaban showed a lower median concentration on admission and a longer t½.

An Unrecognized Hazard in E-Cigarette Vapor: Preliminary Quantification of Methylglyoxal Formation from Propylene Glycol in E-Cigarettes

Up to 95% of the liquid volume in an e-cigarette consists of propylene glycol. Previous research has shown that propylene glycol can generate diacetyl and formaldehyde when heated. New research shows that propylene glycol can also generate methylglyoxal, an alpha di-carbonyl compound recently shown to cause epithelial necrosis at even lower concentrations than diacetyl, the flavoring chemical associated with bronchiolitis obliterans (“Popcorn Lung”). We analyzed chemical emissions from 13 JUUL pod flavors. Diacetyl and methylglyoxal was detected in 100% of samples with median concentration (range) of 20 µg/m3 (less than limit of quantification: 54 µg/m3) and 4219 µg/m3 (677–15,342 µg/m3), respectively. We also detected acetaldehyde (median concentration: 341 µg/m3) and propionaldehyde (median concentration: 87 µg/m3) in all samples. The recent evidence that methylglyoxal is more cytotoxic to airway epithelial cells than diacetyl makes this an urgent public health concern. Current smokers considering e-cigarettes as a smoking cessation tool, and never users, who may be under the impression that e-cigarettes are harmless, need information on emissions and potential risks to make informed decisions.

Concentration of Penicillin G in Jawbone Affected by Antiresorptive Agent-Related Osteonecrosis Following a Single Preoperative Dose

The aim of this study was to evaluate the concentration of penicillin G in bone affected by antiresorptive agent-related osteonecrosis of the jaw (ARONJ) following a single preoperative dose of 10 million international units (6000 mg). ARONJ is a major concern in patients administered antiresorptive agents for conditions associated with pathologically increased bone resorption. Antibiotic therapy is a key component of most treatment approaches for ARONJ and penicillin based regimens, providing a cost effective therapy option with a favorable side effect profile, are administered most frequently. In this study, high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was applied to evaluate penicillin G concentration in serum and bone samples of 19 patients suffering from ARONJ and undergoing surgical treatment under perioperative intravenous (IV) antibiotic therapy. Penicillin G bone concentrations were above the limit of detection (0.1 μg/g bone tissue) in 16 out of 19 samples, with a median concentration of 2.7 μg/g (range 0.1–8.8 μg/g). Penicillin G concentrations in intraoperative serum samples were above the limit of detection in all serum samples, with a median concentration of 116 μg/mL (range 1–232 μg/mL). Thus, considering bacteria frequently found in ARONJ lesions, penicillin G at levels providing adequate antimicrobial activity was detected in the serum and 16 out of 19 osteonecrotic lesions of patients suffering from ARONJ.

Urine neutrophil gelatinase-associated lipocalin concentration in healthy newborns during the first three postnatal days

Introduction: Urine neutrophil gelatinase-associated lipocalin (uNGAL) is a biochemical marker significant for early prediction of acute kidney injury in adults. However, it has not been examined sufficiently among the infant population, particularly newborns in terms of reference values. The aim of our study was to determine the concentration of uNGAL in healthy term newborns and to determine if there was a difference in uNGAL concentration according to gender, postnatal age and birth weight. Materials and methods: Our study involved 81 healthy term newborns birth (≥ 37 weeks, Apgar score ≥ 8 in the first minute after birth, CRP < 5 mg/L). Urine NGAL was measured using chemiluminescent microparticle immunoassay (CMIA) within 72 hours after birth, on Architect plus ci8200 analyser (Abbott, Chicago, USA). Data were analysed using Statistica software. Results: The median concentration of uNGAL in the whole study group of healthy term newborns was 27.1 ng/mL (16.5-56.0 ng/mL) (newborn girls, 27.1 ng/mL (15.8-47.9 ng/mL); newborn boys, 27.9 ng/mL (16.5-61.0 ng/mL), P = 0.941). Median uNGAL concentration according to postnatal age expressed in days was 28.2 ng/mL (11.7-57.2 ng/mL) 1st day, 28.9 ng/mL (16.5-64.2 ng/mL ) 2nd day and 23.9 ng/mL (20.2-46.6) 3rd day, P = 0.863. Regarding birth weight for newborns < 3500 g, median concentration was 25.0 ng/mL (16.5-45.4 ng/mL ) and for weight ≥ 3500 g 30.6 ng/mL (16.5-64.2 ng/mL), P = 0.455. Conclusions: There were no significant difference in uNGAL concentration in relation to gender, postnatal age and birth weight.

Association between ELABELA Serum Concentrations in First Trimester and Pregnancy-Induced Hypertension

ELABELA (ELA) is considered to be implicated in the pathophysiology of preeclampsia (PE), since ELA-deficient mice exhibited PE-like symptoms and infusion of exogenous ELA normalized the gestational hypertension (GH) and proteinuria. However, no evidence show that circulating ELA is deficient in early placental development among women who destined to develop GH/PE. This nested case-control study was conducted to investigate the association between serum ELA concentration in early pregnancy and the risk of later GH/PE. Participants were recruited and sampled in 10-14+6 weeks of gestation. Definite GH/PE cases were matched 1 : 3 to controls with respect to age and gestational age. Serum concentration of ELA was measured using enzyme immunoassay. Women with later GH ( N = 28 ) had a slightly lower median concentration of ELA (46.72 ng/mL versus 53.54 ng/mL), while those with later PE ( N = 16 ) had a slightly higher median concentration of ELA (74.8 ng/mL versus 66.30 ng/mL) compared to the controls. Yet, both the increments did not reach statistically significant difference (GH: P = 0.380 , PE: P = 0.799 ). ELA serum concentrations were unchanged in first trimester in women with GH/PE. Further studies are still needed to identify the dynamic changes in serum ELA concentrations during the whole pregnancy, especially in those with pregnancy-induced hypertensive disorders.

Effects of Lactobacillus reuteri DSM 17938 and vitamin D combination on the level of 25(OH)D in blood serum of young children

Background. The search for optimal vitamin D levels and supplementation approaches to provide a wide range of preventive effects on the human body remains the focus of the world’s specialists. One of the promising directions is to improve the absorption of vitamin D in the gastrointestinal tract by using probiotics. Objective: to study the effect of using a combination of Lactobacillus reuteri DSM 17938 with vitamin D on the serum level of 25(OH)D in young children. Materials and methods. A total of 56 children from 1 to 36 months of age with the signs of functional gastrointestinal disorders were examined; 45 of them were randomized to participate in the comparative efficacy study of using a combination of 400 IU of cholecalciferol and 108 CFU of viable Lactobacillus reuteri DSM 17938 bacteria and an oily vitamin D solution at a dose of 500 IU for 12 weeks. At the start and the end of the study, the indicators of calcium and phosphorus metabolism (25(OH)D, total and ionized calcium, inorganic phosphorus, alkaline phosphatase) and the serum levels of total IgE were determined in all children, and the dynamics of the functional status of the gastrointestinal tract was assessed. Results. At baseline, the median serum concentration of 25(OH)D in children was 30.3 ng/mL (23.2–41.2); this indicator exceeded 30 ng/mL only in 53.3±7.4% (24/45) of cases. The use of a combination of vitamin D3 with Lactobacillus reuteri DSM 17938 made it possible to reach a statistical difference in the median concentration of 25(OH)D after 12 weeks of use compared to the same indicator at baseline: 37.4 ng/mL (30.2–52.3) versus 28.3 ng/mL (20.8–42.9), p=0.000. The level of 25(OH)D in all these children exceeded 20 ng/mL; the concentration level of 25(OH)D exceeded 30 ng/mL in 75.0±8.8% (18/24) of these. The use of 400 IU of vitamin D supplements in combination with L. reuteri for 12 weeks made it possible to reduce the median concentration of total IgE by almost half — from 64.1 IU/mL (9.2–120.0) to 31.9 IU/mL (6.4–143.6) but with significant individual variability of data. The use of vitamin D supplements in combination with Lactobacillus reuteri DSM 17938 had a positive effect on the functional activity of the gastrointestinal tract in young children and significantly reduced the frequency of parents’ complaints of stool retention and anxiety during defecation in their children. Conclusions. The use of vitamin D3 supplements at a dose of 400 IU in combination with the probiotic strain Lactobacillus reuteri DSM 17938 has a positive effect on the functional status of the gastrointestinal tract in young children and increases the serum level of 25(OH)D. The effects of vitamin D in combination with L. reuteri in children with a burdened history of allergies require further study. Key words: vitamin D; Lactobacillus reuteri DSM 17938; young children; supplementation.

Flualprazolam Blood Concentrations in 197 Forensic Investigation Cases

Flualprazolam is a designer benzodiazepine and novel psychoactive substance that is increasing in prevalence and appearing in forensic investigations. Flualprazolam was quantitatively confirmed in 197 blood samples from medicolegal death investigations and human performance cases reported between August 2019 and February 2020. Drug screening was performed using liquid chromatography–time-of-flight mass spectrometry and quantitative confirmation was performed using liquid chromatography–tandem mass spectrometry. A three-point standard addition protocol was implemented for quantitation in the absence of an available traditionally validated assay. In postmortem cases with quantitative results (n = 167), the mean (±standard deviation [SD]) flualprazolam concentration was 20 (±63) ng/mL, the median concentration was 8.2 ng/mL and the range of concentrations was 2.0–620 ng/mL. Four additional postmortem cases were reported positive (&lt;2.0 ng/mL). In drug impaired driving cases (n = 22), the mean (±SD) flualprazolam concentration was 22 (±18) ng/mL, the median concentration was 14 ng/mL and the range of concentrations was 4.4 to 68 ng/mL. The four remaining cases were of unknown circumstances. This report details the most extensive dataset of flualprazolam intoxication cases reported to date. There was significant overlap in concentrations of flualprazolam between postmortem and DUID cases. Flualprazolam was commonly (83% of the time) found in combination with opioids (e.g. fentanyl). Toxicologists should consider quantitative flualprazolam results in the context of case history, observations, and/or other toxicological findings. Addition of flualprazolam to the scope of drug testing should be considered by all laboratories.

Time-Dependent Changes in THC Concentrations in Deceased Persons

Changes in the concentrations of Δ9-tetrahydrocannabinol (THC) in the postmortem period were investigated in a series of cases by comparing concentrations in blood taken on receipt of the body in the mortuary (admission specimen, AD) with the concentrations obtained in blood taken at autopsy some time later and also from blood specimens taken antemortem. Overall, the median THC concentration in AD blood was 13.7 ng/mL (n = 239, range LOQ–220), while the median concentration at autopsy was 13.8 ng/mL (n = 106, range LOQ–810) and 1.9 ng/mL (n = 147, range LOQ–48) antemortem. Fourteen cases had all three specimens taken from the same decedent. The corresponding AM, AD and PM median concentrations were 4.0 (range LOQ–48), 15.5 (range 4.0–176) and 4.4 ng/mL (LOQ–56), respectively. The median elapsed times from AM to AD and AD to PM were 33 and 97.5 h, respectively. In contrast, acetaminophen showed no change in blood concentration from AM to AD (6.8 and 6.0 mg/L, respectively). These data show large increases in THC concentration in the early postmortem period, followed by a decline, although the median blood concentrations at autopsy were similar to that obtained antemortem. In contrast, when blood was taken from the femoral region, subclavian and heart ventricles sites, in the same case, the THC concentrations, while variable, showed overall no significant difference. These dynamic changes reflect complex phenomenon occurring in deceased persons and will further serve to increase the uncertainty over any interpretation of postmortem THC concentrations.