Peripheral Nucleated Red Blood Cells and Mortality in Critically Ill Children.

The present retrospective cohort study examines whether there is an association between circulating nucleated red blood cells (nRBCs) and mortality in critically ill children. nRBCs are erythropoietic progenitor cells not found in peripheral blood of healthy adults and children beyond the neonatal period. The presence of circulating nRBCs is associated with poor prognosis in adults and neonates, though little is known about their significance in children. Admissions to both the general and cardiac pediatric intensive care unit at the Stollery Children's Hospital in Edmonton, Alberta between January 1, 2015 and December 31, 2017 were examined, and logistic regression was performed to ascertain the association between the peak absolute nRBC counts and in-hospital mortality in critically ill children. A total of 2065 admissions were included. The number of admissions with detectable nRBCs was 386 (prevalence: 13.9%), and the number of deaths was 93 (mortality: 4.5%). A statistically significant association was found between the absolute value of nRBC peak and intensive care unit mortality (odds ratio=1.37; 95% confidence interval: 1.13-1.67; P=0.002) as well as hospital mortality (odds ratio=1.38; 95% confidence interval: 1.12-1.70; P=0.003) independent of the Pediatric Index of Mortality 3 score (PIM3). This result warrants more attention to nRBC values and their potential clinical use.

Proposed approach to thrombolysis in dabigatran-treated patients presenting with ischemic stroke.

BACKGROUND: Acute ischemic stroke thrombolysis in patients taking dabigatran is controversial because of a presumed increased risk of symptomatic hemorrhagic transformation. Using data from our local hematopathology laboratory, we developed a thrombolysis protocol for acute ischemic stroke patients taking dabigatran. METHODS: A local thrombin time (TT)-dabigatran concentration relationship was calculated using dabigatran calibrators. The effect of dabigatran on activated partial thromboplastin time (aPTT) and prothrombin time (PT) (international normalized ratio [INR]) was also measured. A protocol was developed, in which a dabigatran concentration less than 10 ng/mL (corresponding to a TT<38 seconds or a normal aPTT) was selected as the upper limit for thrombolysis. Consecutive patients presenting with acute stroke were then enrolled in this prospective study. RESULTS: In the 8 months after development of the protocol, 13 potential thrombolysis candidates taking dabigatran were assessed at a median (interquartile range) time of 192 (143) minutes. The median National Institutes of Health Stroke Scale score was 18 (20). The mean time from arrival to TT, aPTT, and PT (INR) results were 39±4.1 minutes, 21±6.5 minutes, and 21±6.5 minutes, respectively. Based on TT/aPTT, 4 patients were ineligible for thrombolysis. Six patients were not treated because of minor or resolving symptoms and another presented with intracerebral hemorrhage. Two patients were treated with intravenous tissue plasminogen activator (tPA), without symptomatic hemorrhagic transformation in either case. In 3 patients (42.8%), aPTT was normal, despite a prolonged TT. CONCLUSIONS: Administration of intravenous tPA in dabigatran-treated patients is feasible. Although, the relationship between dabigatran concentrations and coagulation measures varies between laboratories, individual protocols, preferably based on TT, can be developed at acute stroke treatment centers.

Hydroxocobalamin interference in routine laboratory tests: Development of a protocol for identifying samples and reporting results from patients treated with CyanokitTM.

OBJECTIVES: Hydroxocobalamin (OHCob) is an antidote for cyanide poisoning in patients rescued from house fires and is known to cause interference with certain laboratory tests. Consensus is lacking on the extent of this interference and on how to handle these samples. The objectives of this study were to characterize OHCob interference across a wide range of laboratory tests and to develop protocols for identifying and reporting these samples. DESIGNS & METHODS: Patient plasma samples (n = 5) were spiked with OHCob (1.5 mg/mL) and compared to controls without this drug. A series of analytes were measured using chemistry, urinalysis, coagulation, hematology, and blood gas instruments. Dose-response testing was performed on a subset of assays that showed interferences ≥10%. RESULTS: Of the 77 analytes evaluated, 27 (35%) showed interference from OHCob, with chemistry and coagulation analytes showing the greatest effects. Of those affected, 22 analytes had a positive interference, whereas 5 analytes had negative interference. Dose-response studies showed dose-dependent increases and/or decreases consistent with initial spiking studies. Although red in colour, plasma samples with OHCob did not trigger hemolysis index flags, necessitating a special sample identification and reporting protocol. CONCLUSION: OHCob had significant effects on several analytes across different instruments. These findings led to the development of special sample handling and reporting protocols to identify OHCob samples and ensure only accurate results are released. It is vital for emergency departments to document and notify their laboratories whenever blood samples from these patients are drawn.

Day 14 Bone Marrow Evaluation During Acute Myeloid Leukemia Induction in a Real-world Canadian Cohort.

INTRODUCTION: The 2017 National Comprehensive Cancer Network guidelines for acute myeloid leukemia have recommended performing bone marrow (BM) aspiration and BM trephine biopsy (BMTB) 14 to 21 days after starting induction therapy (commonly referred to as "day 14 [D14] marrow"). Those who do not achieve a hypoplastic marrow, with cellularity < 20% and blasts < 5%, are recommended to undergo 2-cycle induction (2CI). We performed a retrospective analysis to determine the impact of D14 BM characteristics in predicting for remission, association with overall survival (OS), and the effect of 2CI according to the D14 BM results. PATIENTS AND METHODS: Patients aged 18 to 70 years undergoing induction therapy with standard "7 + 3" regimens were included. D14 cellularity was determined from BMTB samples and the blast percentage was assessed by morphology on BM aspiration and BMTB samples. The outcomes evaluated included the rates of complete remission (CR) and OS. RESULTS: A total of 486 patients with results from D14 BM evaluation were included in the present study. On multivariate analysis, cytogenetic risk and D14 blasts < 5% were predictive of CR/CR with incomplete count recovery (P < .001). Cytogenetic risk (P < .001), age < 60 years (P = .001), and D14 blasts < 5% (P = .045) predicted for OS. 2CI was performed in 131 patients (27%). Patients with hypocellular D14 BM but residual blasts (n = 106) underwent 2CI in 46% of cases, with improved remission rates (43.9% vs. 72.0%; P = .004) but no difference in OS. CONCLUSIONS: The results from D14 BM evaluations are predictive of subsequent remission and OS. Our findings did not show a survival benefit with D14 BM-driven 2CI.

Crystals and Fatty Acid Abnormalities Are Not Present in Circulating Cells From Choroideremia Patients.

Purpose: To confirm whether choroideremia (CHM) is a systemic disease characterized by blood lipid abnormalities and crystals found in, or associated with, circulating peripheral blood cells of patients. Methods: Peripheral blood samples obtained from three subjects with confirmed mutations in the CHM gene and three age-matched normal controls were processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Fatty acids from plasma of nine male CHM subjects were analyzed and compared to reference values for a sample from a Canadian population. Results: Intracellular crystals were not observed in the cells from choroideremia-affected males. No crystals were found adherent to the external plasma membrane of red blood cells. Fatty acid profiles of patients were similar to reference values, with the exception of lower levels of nervonic acid. Conclusions: This investigation failed to observe crystals previously reported in peripheral circulating blood cells derived from CHM subjects, and showed no significant fatty acid abnormalities, not supporting the view of CHM as a systemic disease.

Persistent cytogenetic abnormalities in patients undergoing intensive chemotherapy for acute myeloid leukemia.

We evaluated the impact of bone marrow sample characteristics on the detection of persistent cytogenetic abnormalities (PCA) following induction chemotherapy for acute myeloid leukemia (AML). PCA's were identified in 20.4% of patients and were more common with complete remission without count recovery (CRi) vs. those with count recovery (CR, 45.8 vs. 13.5%, p = .001), with >2% blasts vs. ≤2% blasts (42 vs. 12%, p = .001) and with hypocellular trephine biopsies relative to those with normo/hypercellular biopsies (42.1 vs. 17.3%, p = .03), although in a multivariate analysis only CRi and blast count >2% were independently associated with a PCA. PCA's were not observed in patients with favorable risk karyotype. Amongst patients with intermediate and unfavorable risk karyotypes PCA were not associated with differences in overall or, amongst non-transplanted patients, relapse free survival. Thus, although PCAs are common post-induction it is unclear whether they provide any independent prognostic information beyond the diagnostic karyotype.

The role of bestrophin in airway epithelial ion transport.

The purpose of this study was to identify Cl- channels in the basolateral membrane of airway epithelial cells at the molecular level. We have focused on a new family of Cl- channels, bestrophins, which have previously been identified in retinal pigment epithelium. RT-PCR, Western blot and confocal microscopy studies revealed the presence of bestrophin in airway epithelial cells. Decreasing bestrophin expression using siRNA resulted in diminished 36Cl- flux. These studies also showed that bestrophin regulation is similar to that of native basolateral Cl- channels. The data indicate that the presence of a functional bestrophin may contribute to the basolateral cell conductance in airway epithelial cells.

Dabigatran assessment in patients with acute complications using routine coagulation assays.

Complications while on dabigatran therapy, particularly bleeding and thrombosis, are occurring, and require laboratory assessment. The utility of routine coagulation assays has been previously evaluated in stable patients, but not those with acute complications. The purpose of this study was to determine how to employ routine coagulation assays to assess dabigatran in patients with acute complications. Seventeen patients on dabigatran presenting with various complications were evaluated. In addition, plasma samples with various fibrinogen levels were spiked in vitro with dabigatran ranging from low trough levels to the highest supratherapeutic concentrations reported (5000 ng/ml). INR, partial thromboplastin time (PTT), thrombin time (TT, Diagnostica Stago reagent), and fibrinogen were assayed and results compared to that of the Hemoclot Thrombin Inhibitors assay. Interference in the Clauss fibrinogen assay was assessed using a variety of commercial reagents. The majority of patients on dabigatran with acute complications demonstrated a significant negative bias in PTT results compared to normal plasma. TT remained highly sensitive to the presence of dabigatran (at least 10 ng/ml) under all circumstances investigated. There was wide variation in the sensitivity of commercial fibrinogen assays to dabigatran, with some even showing interference in the therapeutic range but this could be mitigated. The PTT is unreliable as a method for assessment of dabigatran in patients with acute complications. The TT assay is a simple and reliable alternative, particularly when combined with a fibrinogen level.

The role of the basolateral outwardly rectifying chloride channel in human airway epithelial anion secretion.

The purpose of this study was to characterize basolateral anion channels in Calu-3 and normal human bronchial epithelial cells, and their role in anion secretion. Patch clamp studies identified an outwardly rectifying Cl- channel (ORCC), which could be activated by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA). Short-circuit current measurements revealed that NECA activates a basolateral, but not an apical, anion conductance sensitive to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid, and to 9-anthracenecarboxylic acid, but not to 4,4'-dinitrostilbene-2,2'-disulfonic acid. Apical membrane permeabilization studies confirmed the presence of basolateral anion channels, established their halide permeability sequence (Cl- >/= Br- >> I-), and demonstrated their outwardly rectifying nature. Experiments using H-89, forskolin, and Ht31 demonstrated that adenosine receptor dependent activation of basolateral ORCC was cAMP- and potentially A-kinase anchoring protein-dependent. Neither BAPTA-AM treatment nor basolateral Ca2+ removal had any effect on the activation of these channels. Anion replacement and 36Cl- flux studies show that Calu-3 cells primarily secrete HCO3- when stimulated with NECA, and that Cl- secretion can be stimulated by blocking basolateral ORCC, whereas normal human bronchial epithelial cells exclusively secrete Cl- under all conditions studied. We propose a novel model of anion secretion in which ORCC recycles Cl- across the basolateral membrane, allowing preferential HCO3- secretion.

Regulation of Cl- secretion by alpha2-adrenergic receptors in mouse colonic epithelium.

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.