Resolution of PTHrP-Mediated Hypercalcemia Following Treatment with Dual BRAF/MEK Inhibition for BRAFV600E-Positive Metastatic Ameloblastoma.

Ameloblastoma is a rare odontogenic tumor which may be complicated by hypercalcemia in advanced disease. Tumoral parathyroid hormone-related peptide (PTHrP) production and local osteolysis from paracrine factors have been proposed as mechanisms. Mitogen-activated protein kinase (MAPK) inhibitors have been successfully used in ameloblastomas with BRAF V600E mutation to reduce symptoms and decrease tumor burden. Serum calcium has been observed to normalize following treatment with MAPK inhibitors; however, the response of PTHrP and markers of bone turnover has not been reported. We describe a case of a 55-year-old female with PTHrP-mediated hypercalcemia secondary to BRAF V600E-positive ameloblastoma with pulmonary metastases. Following treatment with dabrafenib and trametinib, the patient experienced the regression of pulmonary lesions and normalization of serum calcium, PTHrP, and markers of bone turnover. Tissue samples of ameloblastoma carrying BRAF V600E mutation are more likely to express PTHrP than tissue samples carrying wild-type BRAF. In our case, resolution of PTHrP-mediated hypercalcemia following initiation of BRAF/MEK inhibition provides additional evidence that the MAPK pathway contributes to PTHrP synthesis. It also raises the question of whether MAPK inhibitors would be effective in treating PTHrP-mediated hypercalcemia associated with other malignancies harboring BRAF V600E mutation.

A pragmatic randomized controlled trial of rapid on-site influenza and respiratory syncytial virus PCR testing in paediatric and adult populations.

BACKGROUND: Rapid/point-of-care respiratory virus nucleic acid tests (NAT) may improve oseltamivir, antibiotic, diagnostic test, and hospital bed utilization. Previous randomized controlled trials (RCT) on this topic have not used standard procedures of an accredited healthcare and laboratory system. METHODS: We conducted a parallel RCT at two hospitals [paediatric = Alberta Children's Hospital (ACH); primarily adult = Peter Lougheed Centre (PLC)]. Patients with a respiratory viral testing order were randomized to testing at either a central accredited laboratory (standard arm) or with a rapid polymerase chain reaction test at an on-site accredited laboratory followed by standard testing [rapid on-site test (ROST) arm] based on day of specimen receipt at the laboratory. Patients and clinicians were blinded to assignment. The primary outcome for ACH was inpatient length of stay (LOS) and for PLC was the proportion of inpatients prescribed oseltamivir. RESULTS: 706 patient encounters were included at ACH; 322 assigned to ROST (181 inpatients) and 384 to the standard arm (194 inpatients). 422 patient encounters were included at PLC; 200 assigned to ROST (157 inpatients) and 222 to the standard arm (175 inpatients). The rate of oseltamivir prescription and number of doses given was reduced in PLC inpatients negative for influenza in the ROST arm compared to standard arm [mean 14.9% (95% CI 9.87-21.9) vs. 27.5% (21.0-35.2), p = 0.0135; mean 2.85 doses (SEM 2.39-3.32) vs. 4.17 doses (3.85-4.49) p = 0.022, respectively]. ROST also significantly reduced oseltamivir use at ACH, reduced chest radiographs (ACH), and laboratory test ordering (PLC), but not antibiotic prescriptions. ROST also reduced the median turnaround time by > 24 h (ACH and PLC). The LOS at ACH was not significantly different between the ROST and standard arms [median 4.05 days (SEM 1.79-18.2) vs 4.89 days (2.07-22.9), p = 0.062, respectively]. No adverse events were reported. CONCLUSIONS: In a RCT representing implementation of ROST in an accredited laboratory system, we found that a ROST improved oseltamivir utilization and is the first RCT to show reduced ancillary testing in both paediatric and adult populations. A larger study is required to assess reduction in paediatric LOS as ACH was underpowered. These findings help justify the implementation of rapid on-site respiratory virus testing for inpatients. Trial registration ISRCTN, number 10110119, Retrospectively Registered, 01/12/2021.

Filling in the GAPS: validation of anion gap (AGAP) measurement uncertainty estimates for use in clinical decision making.

OBJECTIVES: We compare measurement uncertainty (MU) calculations to real patient result variation observed by physicians using as our model anion gap (AGAP) sequentially measured on two different instrument types. An approach for discretely quantifying the pre-analytical contributions and validating AGAP MU estimates for interpretation of patient results is proposed. METHODS: AGAP was calculated from sodium, chloride, and bicarbonate reported from chemistry or blood gas analyzers which employ different methodologies and specimen types. AGAP MU was calculated using a top-down approach both assuming no correlation between measurands and alternatively, including consideration of measurand correlation. MU-derived reference change values (RCV) were calculated between chemistry and blood gas analyzers results. Observational paired AGAP data (n=39,626 subjects) was obtained from retrospectively analyzed specimens from five urban tertiary care hospitals in Calgary, Alberta, Canada. RESULTS: The MU derived AGAP RCV for paired specimen data by the two platforms was 5.2-6.1 mmol/L assuming no correlation and 2.6-3.1 mmol/L assuming correlation. From the paired chemistry and blood gas data, total observed variation on a reported AGAP has a 95% confidence interval of ±6.0 mmol/L. When the MU-derived RCV assuming correlation is directly compared against the observed distribution of patient results, we obtained a pre-analytical variation contribution of 2.9-3.5 mmol/L to the AGAP observed variation. In contrast, assuming no correlation leads to a negligible pre-analytical contribution (<1.0 mmol/L). CONCLUSIONS: MU estimates assuming no correlation are more representative of the total variation seen in real patient data. We present a pragmatic approach for validating an MU calculation to inform clinical decisions and determine the pre-analytical contribution to MU in this system.

Whole Blood Cardiac Troponin "Triaging" to Improve Early Detection of Myocardial Injury at a Pediatric Hospital.

BACKGROUND: The importance of offering on-site cardiac troponin (cTn) testing at pediatric hospitals may be underappreciated. We developed a rapid rule-in process for myocardial injury at a pediatric hospital experiencing delays in off-site high-sensitivity cardiac troponin T (hs-cTnT) testing. METHODS: Collect-to-verify turnaround times (TATs) for off-site testing were reviewed. Pre-analytic changes to improve TATs were devised, implemented and evaluated, after which a new analyzer was selected and evaluated for on-site cTn testing. Performance of the new analyzer's assay was compared to the off-site hs-cTnT assay, and post go-live TATs for on-site testing were assessed. RESULTS: Median collect-to-verify TAT for short turnaround-time (STAT) priority off-site plasma hs-cTnT testing was 104 min, with 35% of orders having a TAT >120 min. Eliminating serum separator tubes and requiring a separate plasma separator tube did not significantly reduce TATs. A QuidelOrtho Triage® MeterPro whole blood cardiac troponin I (cTnI) assay was implemented to "triage" time-critical and STAT priority specimens collected for off-site hs-cTnT testing. Elevated cTnI (≥0.02 µg/L) had a sensitivity of 91% for clear elevations in hs-cTnT (≥53 ng/L) but a 0% sensitivity for modest elevations (5 to 13 ng/L, 14 to 52 ng/L). An interpretive comment was auto-appended to cTnI results indicating that clinicians should wait for the hs-cTnT result if cTnI was normal. Median collect-to-verify TAT for on-site cTnI testing was <50% the TAT for off-site hs-cTnT testing. CONCLUSIONS: On-site point-of-care whole blood cTn testing can rapidly confirm significant or late-presenting myocardial injury. Combined with simultaneous off-site high-sensitivity cardiac troponin (hs-cTn) testing, this workflow is a viable interim solution for pediatric hospitals without on-site hs-cTn testing.

Not T too! False elevations in high-sensitivity cardiac troponin T (hs-TnT) following specimen transport.

Though false elevations attributed to preanalytical specimen handling have been widely reported for Troponin I (TnI), Troponin T (TnT) has appeared more robust to falsely elevated Tn. We describe reproducible false elevations in high sensitivity TnT (hs-TnT) in specimens after courier transport in plasma separator tubes (PST) off-site for testing. Hs-TnT was measured under 5 different conditions: 1) at collection location (N = 24); 2) after transport upright in racks (N = 66); 3) after transport with no control over tube agitation (N = 69); 4) on transported aliquots (N = 84); or 5) immediately after transport with no control over tube agitation (N = 16), followed by keeping the specimen upright and re-measuring at 1hr, 2hr, 4hr, and 20-24hrs (N = 6). To assess the degree of discrepancy, plasma from the original PST was aliquotted, re-centrifuged, potential debris removed, and hs-TnT re-measured. 43% of PST specimens collected offsite and transported with no control over tube agitation had clinically significant false elevations of hs-TnT which subsequently decreased following aliquotting and re-centrifugation (median decrease = 9.9 ng/L). Onsite testing or transported aliquots demonstrated no discrepancy. After being kept upright, discrepant specimens were not different from re-centrifuged aliquots by 4hrs (p = 0.6141, repeated measures ANOVA with Dunn's multiple comparisons). Clinically significant false elevations of hs-TnT occurred in approximately 40% of separated PSTs that were transported in containers where specimens are transported with no control over tube agitation. This interference does not occur if plasma is aliquoted or if hs-TnT is tested at the collection site. In order to prevent these false elevations, and their potential patient impact on the diagnosis of acute myocardial infarction, specimens for hs-TnT measurement should be aliquoted at the collection location prior to transport.

Drug testing in support of the diagnosis of neonatal abstinence syndrome: The current situation.

Illicit drug use during pregnancy is a concern worldwide, with many international studies describing attempted strategies to mitigate this problem. Drug misuse during pregnancy is associated with significant maternal as well as perinatal complications, which include a high incidence of stillbirths, fetal distress, neonatal abstinence syndrome (NAS) and increased neonatal mortality. Unfortunately, the identification of a drug-exposed mother or neonate is challenging. Maternal disclosure of drug use is often inaccurate, principally due to psychosocial factors including behavioral denial or the fear of the consequences resulting from such admissions. Likewise, many infants who have been exposed to drugs in utero may appear normal at birth and initially show no overt manifestations of drug effects. Thus, the identification of the drug-exposed infant requires a high index of clinical suspicion. Conversely, analytical testing is an objective means of determining drug exposure when it may be necessary to document proof of the infant's exposure to illicit drugs. The review will discuss the different matrices that are most commonly used for testing (e.g., maternal urine, neonatal urine, meconium, and umbilical cord), the strengths and limitations for each matrix, which drugs and metabolites are appropriate for testing, the various testing methods, and the advantages and disadvantages of each method.

Low partial pressure of oxygen causes significant and unrecognized under-recovery of glucose on blood gas analyzers.

BACKGROUND: Blood gas analyzers employing glucose-oxidase biosensors under-recover glucose when pO2 is low. The manufacturer of the GEM®Premier™ series of analyzers introduced an algorithm to detect specimens at risk of low pO2 interference. We investigated the reliability of this algorithm. METHODS: Whole blood specimens were tested by GEM®Premier™ 4000 (GEM 4000) and 5000 (GEM 5000). Specimens with an incalculable ("incalc") error code for glucose result or that had a glucose ≥ 20 mmol/L were retested on a second analyzer of the same type within 5 min over the course of 30 months in 5 hospitals in Calgary, Alberta. Discordant retests were defined as either: 1) paired numeric results with a difference >10 %, or 2) an "incalc" code that yielded a numeric result upon retesting. Glucose recovery in relation to pO2 level was assessed by comparing specimens experimentally depleted of pO2 between GEM 5000 and a laboratory analyzer (Siemens Vista®). RESULTS: Of 1,776 glucose tests repeated on the GEM 5000 or 1,544 on GEM 4000, 10% were discordant. GEM 5000 produced twice as many discordant numeric retests versus the GEM 4000 [5.9% (98/1,651) vs 2.7% (38/1,391)]. The majority of "incalc" error codes repeated with a numeric glucose result on both GEM analyzers [(79.7% (122/153) vs 75.2% (94/125)]. Among specimens experimentally depleted of pO2, the GEM 5000 under-recovered glucose by up to 30% compared to the Siemens Vista and were not flagged by an "incalc" code. CONCLUSIONS: The algorithm in the GEM®PremierTM series of analyzers that flags specimens at risk for glucose under-recovery due to low pO2 does not reliably detect specimens at risk for glucose under-recovery.

Distribution of videos demonstrating best practices in preventing hemolysis is associated with reduced hemolysis among nurse-collected specimens in hospitals.

BACKGROUND: Minimizing hemolysis during phlebotomy ensures accurate chemistry results and reduces test cancellations and specimen recollections. We developed videos demonstrating best practices to reduce hemolysis and tested whether distribution to clinical nurse educators (CNEs) for provision to nursing staff affected the degree of specimen hemolysis in hospital inpatient units and outpatient clinics. METHODS: Videos of common blood collections demonstrating best practices to reduce hemolysis were filmed and then distributed via email link to all hospital-based CNEs in Calgary, Alberta, Canada. (https://vimeo.com/user18866730/review/159869683/a0cec9827f). Roche Cobas hemolysis index (H-index) results from specimens collected +/- 12 months from the date of video distribution were extracted from Roche Cobas IT middleware (cITM) and linked to collection location. An interrupted time series (ITS) analysis with collection location as the unit of anlaysis was used to quantify impact of video distribution on the trajectory of weekly mean log-H-index weighted by inverse variance. RESULTS: In +/- 3 months of data flanking video distribution (n = 137 241 collections), where overall impact was strongest, H-index trajectory (change in units per week) decreased immediately following video distribution (-5.7% / week, p < 0.01). This was accompanied by a 22% drop in overall H-index from the week before to the week after video distribution (y-intercept change, or gap). There was also a small but significant overall decrease in the proportion of hemolyzed specimens (-0.3%, p < 0.01). These changes were not observed at all collection locations, and in fact increases occured at some locations. CONCLUSIONS: We developed a novel and convenient educational aid that, when distributed, was associated with beneficial changes in specimen hemolysis at hospital inpatient units and outpatient clinics. Including it in ongoing nursing education will fill a knowledge gap that may help to reduce specimen hemolysis.

Prospective comparative evaluation of the European Society of Cardiology (ESC) 1-hour and a 2-hour rapid diagnostic algorithm for myocardial infarction using high-sensitivity troponin-T.

OBJECTIVE: Both 1- and 2-hour rapid diagnostic algorithms using high-sensitivity troponin (hs-cTn) have been validated to diagnose acute myocardial infarction (MI), leaving physicians uncertain which algorithm is preferable. The objective of this study was to prospectively evaluate the diagnostic performance of 1- and 2-hour algorithms in clinical practice in a Canadian emergency department (ED). METHODS: ED patients with chest pain had high-sensitivity cardiac troponin-T (hs-cTnT) collected on presentation and 1- and 2-hours later at a single academic centre over a 2-year period. The primary outcome was index MI, and the secondary outcome was 30-day major adverse cardiac events (MACE). All outcomes were adjudicated. RESULTS: We enrolled 608 patients undergoing serial hs-cTnT sampling. Of these, 350 had a valid 1-hour and 550 had a 2-hour hs-cTnT sample. Index MI and 30-day MACE prevalence was ~12% and 14%. Sensitivity of the 1- and 2-hour algorithms was similar for index MI 97.3% (95% CI: 85.8-99.9%) and 100% (95% CI: 91.6-100%) and 30-day MACE: 80.9% (95% CI: 66.7-90.9%) and 83.3% (95% CI: 73.2-90.8%), respectively. Both algorithms accurately identified about 10% of patients as high risk. CONCLUSIONS: Both algorithms were able to classify almost two-thirds of patients as low risk, effectively ruling out MI and conferring a low risk of 30-day MACE for this group, while reliably identifying high-risk patients. While both algorithms had equivalent diagnostic performance, the 2-hour algorithm offers several practical advantages, which may make it preferable to implement. Broad implementation of similar algorithms across Canada can expedite patient disposition and lead to resource savings.

Urine protein detection by dipstick: No interference from alkalinity or specific gravity.

Tetrabromphenol blue dye based methods are used to detect proteinuria using urinalysis dipsticks. Manufacturers have claimed that alkalinity leads to false positive proteinuria, and that high specific gravity leads to false negative protein results. However, published reports describing this phenomenon remain equivocal. This study aimed to determine whether pH and/or specific gravity affect protein detection in patient urine using three different tetrabromophenol blue dye-based dipsticks. Patient urine pools were divided into individual aliquots with varied pH or specific gravity, and measured for protein in triplicate using iChem 10SG, iChem Velocity, and Multistix 8SG dipsticks. The pH experiment involved progressive alkalinization of urine aliquots with either 1M NaOH, Na2CO3, or NaHCO3; pH was recorded by electrode. The specific gravity experiment involved mixing aliquots with NaCl and spiking with human albumin. Urine electrolytes and total CO2 were measured (Roche cobas 8000). Fresh patient urines (N = 35) were analyzed for physiological urine pH and total CO2. Urine protein results were not affected by NaOH alkalinization up to pH 10.9. False positive protein occurred at pH 9.9 and >97 mmol/L total CO2 (Na2CO3 alkalization; P < .05). Moreover, false positive protein occurred at pH 7.6 when total CO2 exceeded 137 mmol/L (NaHCO3 alkalization; P < .05). Fresh patient urines did not exceed pH 8.5 or 86 mmol/L total CO2. NaCl elevated specific gravity and caused false negative protein detection when urine ionic strength was >1100 mmol/L (P < .05). Tetrabromphenol blue dipsticks provide robust detection of proteinuria when human urine is within physiological pH, total CO2 and ionic strength.

Rising above helium: A hydrogen carrier gas chromatography flame ionization detection (GC-FID) method for the simultaneous quantification of toxic alcohols and ethylene glycol in human plasma specimens.

Here we validate a GC, Flame Ionization Detection (GC-FID), liquid injection method using hydrogen as a carrier gas combining analysis of toxic volatile alcohols (VA): methanol, ethanol, isopropanol, acetone, as well as glycols, ethylene glycol (EG) and propylene glycol (PG), in a single method. METHODOLOGY: 200 µL of calibrator, QC, or patient specimen were deproteinized with 400 µL of acetonitrile containing internal standards (10 mmol/L N-propyl alcohol for VA and 2.5 mmol/L 1,2-butanediol for glycols). GC-FID analysis using hydrogen carrier gas and nitrogen makeup gas utilized an Agilent 7890 system equipped with Agilent 7683 liquid autosampler on a 30 m × 530 µm RTX-200 fused silica column. Method validation included repeatability, recovery, carryover, linearity, lower limit of quantification (LLOQ), accuracy, selectivity and measurement uncertainty. RESULTS: The 8.3 min from injection to injection reduced time of analysis by 45% over a previously reported method using Helium carrier gas with no loss in resolution. Within-run and Between-run variability were ≤1.4% and ≤6.8% respectively. Recovery was 100% within a 95% confidence interval. Carryover was negligible for all but EG. LLOQ was <1 mmol/L for all analytes. The upper range of linearity was 120 mmol/L for methanol, ethanol and isopropanol, 100 mmol/L for acetone and 50 mmol/L for EG. Analytes demonstrated acceptable accuracy and measurement uncertainty using College of American Pathologists (CAP) criteria. Toluene can cause a false positive EG, while benzene, xylene and 1,3 butanediol can cause false negative EG. CONCLUSIONS: Converting from Helium to Hydrogen carrier gas benefits patient care through a reduction in turnaround time and provides a cost savings to the laboratory.

Low High-Sensitivity Troponin Thresholds Identify Low-Risk Patients With Chest Pain Unlikely to Benefit From Further Risk Stratification.

BACKGROUND: Very low high-sensitivity cardiac troponin T (hs-cTnT) thresholds on presentation can rule out acute myocardial infarction (AMI), but the ability to identify patients at low risk of 30-day major adverse cardiac events (MACE) is less clear. This study examines the sensitivity of low concentrations of hs-cTnT on presentation to rule out 30-day MACE. METHODS: This prospective cohort study enrolled patients with chest pain presenting to the emergency department with nonischemic electrocardiograms who underwent AMI rule-out with an hs-cTnT assay. The primary outcome was 30-day MACE; secondary outcomes were individual MACE components. Because guidelines recommend using a single hs-cTnT strategy only for patients with more than 3 hours since symptom onset, a subgroup analysis was performed for this population. Outcomes were adjudicated on the basis of review of medical records and telephone follow-up. RESULTS: Of 1167 patients enrolled, 125 (10.7%) experienced 30-day MACE and 97 (8.3%) had AMI on the index visit. More than one-third of patients (35.6%) had presenting hs-cTnT concentrations below the limit of detection (5 ng/L), which was 94.4% (95% confidence interval [CI], 88.8-97.7) sensitive for 30-day MACE and 99.0% (95% CI, 94.5-100) sensitive for index AMI. Of 292 patients (25.0%) with hs-cTnT < 5 ng/L and at least 3 hours since symptom onset, only 3 experienced 30-day MACE (sensitivity 97.6%; 95% CI, 93.2-100) and none had AMI within 30 days (sensitivity 100%; 95% CI, 96.3-100). CONCLUSIONS: Among patients with nonischemic electrocardiograms and > 3 hours since symptom onset, low hs-cTnT thresholds on presentation confer a very low risk of 30-day MACE. In the absence of a high-risk clinical presentation, further risk stratification is likely to be low yield.

CONTEXTE: Un seuil de troponine T cardiaque hypersensible (TnTc-hs) très bas au moment de la consultation permet d'écarter le diagnostic d'infarctus aigu du myocarde (IAM), mais l'utilité de ce paramètre pour reconnaître les patients exposés à un faible risque d'événement cardiaque indésirable majeur (ECIM) à 30 jours est moins bien établie. Les auteurs examinent la sensibilité de la présence d'une faible concentration de TnTc-hs à la consultation comme critère pour écarter la possibilité d'un ECIM à 30 jours. MÉTHODE: Ont été admis dans cette étude de cohorte prospective les patients qui se sont présentés à l'urgence en raison d'une douleur à la poitrine, dont l'électrocardiogramme n'a pas révélé d'ischémie et chez qui le diagnostic d'IAM a été écarté au moyen d'un dosage de la TnTc-hs. Le critère d'évaluation principal était la survenue d'un ECIM à 30 jours; les critères d'évaluation secondaires étaient les composantes individuelles de l'ECIM. Comme les lignes directrices recommandent le recours à un simple dosage de la TnTc-hs seulement pour les patients présentant des symptômes depuis plus de 3 heures, une analyse a été réalisée dans ce sous-groupe de la population à l'étude. Les critères d'évaluation ont été confirmés par un examen des dossiers médicaux et par un suivi téléphonique. RÉSULTATS: Des 1167 patients retenus, 125 (10,7 %) ont présenté un ECIM à 30 jours et 97 (8,3 %) avaient reçu un diagnostic d'IAM à la visite de référence. Au moment de la consultation, plus du tiers des patients (35,6 %) présentaient une concentration de TnTc-hs sous le seuil de détection (5 ng/l), ce qui représente une sensibilité de 94,4 % (intervalle de confiance [IC] à 95 % : de 88,8 à 97,7) dans le cas de l'ECIM à 30 jours et de 99,0 % (IC à 95 % : de 94,5 à 100) dans le cas de l'IAM de référence. Des 292 patients (25,0 %) présentant un taux de TnTc-hs < 5 ng/l et des symptômes apparus depuis au moins 3 heures, seulement 3 ont subi un ECIM à 30 jours (sensibilité de 97,6 %; IC à 95 % : de 93,2 à 100) et aucun n'a subi d'IAM dans les 30 jours (sensibilité de 100 %; IC à 95 % : de 96,3 à 100). CONCLUSIONS: Chez les patients dont l'électrocardiogramme ne révèle pas d'ischémie et qui présentent des symptômes depuis au moins 3 heures, un seuil de TnTc-hs faible au moment de la consultation est associé à un très faible risque d'ECIM à 30 jours. En l'absence d'un tableau clinique associé à un risque élevé, il est peu probable qu'une stratification du risque plus poussée soit utile.

Sex-specific, high-sensitivity cardiac troponin T cut-off concentrations for ruling out acute myocardial infarction with a single measurement.

OBJECTIVE: Sex-specific diagnostic cut-offs may improve the test characteristics of high-sensitivity troponin assays for the diagnosis of myocardial infarction (MI). The objective of this study was to quantify test characteristics of sex-specific cut-offs of a single, high-sensitivity cardiac troponin T (hs-cTnT) assay for 7-day MI in patients with chest pain. METHODS: This observational cohort study included consecutive emergency department (ED) patients with suspected cardiac chest pain from four Canadian EDs who had an hs-cTnT assay performed within 60 minutes of ED arrival. The primary outcome was MI at 7 days. We quantified test characteristics (sensitivity, negative predictive value [NPV], likelihood ratios and proportion of patients ruled out) for multiple combinations of sex-specific, rule-out cut-offs. We calculated the net reclassification index compared to universal rule-out cut-offs. RESULTS: In 7,130 patients (3,931 men and 3,199 women), the 7-day MI incidence was 7.38% among men and 3.78% among women. Optimal sex-specific cut-offs (<8 ng/L for men and <7 ng/L for women) had a 98.5% sensitivity for MI and ruled out MI in 55.8% of patients. This would enable an absolute increase in the proportion of patients who were able to be ruled out with a single hs-cTnT of 13.2% to 22.2%, depending on the universal rule-out concentration used as a comparator. CONCLUSIONS: Sex-specific hs-cTnT cut-offs for ruling out MI at ED arrival may improve classification performance, enabling more patients to be safely ruled out at ED arrival. However, differences between sex-specific and universal cut-off concentrations are within the variation of the assay, limiting the clinical utility of this approach. These findings should be confirmed in other data sets.

Gab1 but not Grb2 mediates tumor progression in Met overexpressing colorectal cancer cells.

Hepatocyte growth factor receptor (Met) plays an important role in the progression of multiple cancer types. The overexpression of Met in DLD-1 colon carcinoma cells with kirsten rat sarcoma oncogene homolog (KRAS) oncogene activation resulted in enhanced subcutaneous and orthotopic tumor growth rate and increased metastatic potential. To elucidate the mechanism of this effect, we stably expressed kinase-inactive Met(K1110A), Src homology 2 (SH2)-binding domain-inactive Met(Y1349/1356F), growth factor receptor-bound protein 2 (Grb2) non-binding Met(N1358H) and mutant receptors with ability to selectively recruit signaling proteins Grb2, src homology domain c-terminal adaptor homolog (Shc), phospholipase c-gamma (PLCgamma) and p85 phosphatidyl inositol 3 kinase. As subcutaneous implants, DLD-1 cells that expressed the majority of these receptor constructs failed to recapitulate the tumor growth-enhancing effect of the wild-type Met receptor. The Grb2- and Shc-recruiting Met mutants demonstrated slight but consistent tumor-suppressive activity, whereas the expression of N1358H mutant stimulated tumor growth rate comparable with the wild-type receptor. This suggests that direct Grb2/Shc binding does not contribute to the tumor progression activity of Met receptor. The tumors expressing Grb2- and Shc-recruiting Met receptors demonstrated a marked loss in Grb2-associated adaptor protein 1 (Gab1) protein levels, which was not observed in the cell lines, consistent with a post-translationally regulated process. Moreover, a moderate level of Gab1 overexpression stimulated tumor growth. The findings suggest a delicate balance for intact Y1349/1356 SH2-binding domain to mediate the tumor progression activity of the coactivated Met-rat sarcoma oncogene homolog (RAS) pathways. Selectivity for specific adaptor protein involvement may be the key that determines the tissue- and cell-type specificity of Met-mediated tumorigenicity in human cancers.

Stabilizing specimens for routine ammonia testing in the clinical laboratory.

BACKGROUND: In vitro deamination generates ammonia in freshly collected blood specimens. To prevent this, samples for ammonia testing are usually collected on ice and run rapidly (e.g., within 1h). We developed a method to stabilize specimens for ammonia analysis. METHODS: Following plasma separation, 500µmol/l cycloserine or a combination of 2mmol/l sodium borate with 5mmol/l l-serine were added to sample pools with normal or increased concentrations of ALT and/or GGT to inhibit deamination; and/or residual platelets were removed via centrifugation. Sample pools were then incubated at room temperature or 4°C. Untreated sample pools were also incubated at -80°C. Ammonia was measured at 0, 1, 2, 4, 8, 16, and 24h. RESULTS: When incubated at 4°C without treatment, sample pools with enzymes within their reference limit had an increase of 0.5µmol/l/h, whereas sample pools with ALT and/or GGT activity above their upper reference limit had an increase of 3.6µmol/l/h (p<0.001). When sample pools were incubated at 4°C with sodium borate/l-serine, the rate of ammonia increase was significantly reduced in samples with normal (0.3µmol/l/h, p<0.001 vs. untreated controls) or high enzyme activity (0.1µmol/l/h, p<0.001 vs. untreated controls). Independent of the ALT and/or GGT concentrations, storing the sample at -80°C also preserved the specimens for ammonia analysis (0.2µmol/l/h, p<0.001 vs. untreated controls). CONCLUSIONS: By combining sodium borate/l-serine with refrigeration, plasma ammonia specimens can be stabilized for >12h.

Comparative Evaluation of 2-Hour Rapid Diagnostic Algorithms for Acute Myocardial Infarction Using High-Sensitivity Cardiac Troponin T.

BACKGROUND: Symptoms of acute coronary syndrome account for a large proportion of emergency department (ED) visits and hospitalizations. High-sensitivity troponin can rapidly rule out or rule in acute myocardial infarction (AMI) within a short time of ED arrival. We sought to validate test characteristics and classification performance of 2-hour high-sensitivity troponin T (hsTnT) algorithms for the rapid diagnosis of AMI. METHODS: We included consecutive patients from 4 academic EDs with suspected cardiac chest pain who had hsTnT assays performed 2 hours apart (± 30 minutes) as part of routine care. The primary outcome was AMI at 7 days. Secondary outcomes included major adverse cardiac events (mortality, AMI, and revascularization). Test characteristics and classification performance for multiple 2-hour algorithms were quantified. RESULTS: Seven hundred twenty-two patients met inclusion criteria. Seven-day AMI incidence was 10.9% and major adverse cardiac event incidence was 13.7%. A 2-hour rule-out algorithm proposed by Reichlin and colleagues ruled out AMI in 59.4% of patients with 98.7% sensitivity and 99.8% negative predictive value (NPV). The 2-hour rule-out algorithm proposed by the United Kingdom National Institute for Health and Care Excellence ruled out AMI in 50.3% of patients with similar sensitivity and NPV. Other exploratory algorithms had similar sensitivity but marginally better classification performance. According to Reichlin et al., the 2-hour rule-in algorithm ruled in AMI in 16.5% of patients with 92.4% specificity and 58.5% positive predictive value. CONCLUSIONS: Two-hour hsTnT algorithms can rule out AMI with very high sensitivity and NPV. The algorithm developed by Reichlin et al. had superior classification performance. Reichlin and colleagues' 2-hour rule-in algorithm had poor positive predictive value and might not be suitable for early rule-in decision-making.

Undetectable Concentrations of a Food and Drug Administration-approved High-sensitivity Cardiac Troponin T Assay to Rule Out Acute Myocardial Infarction at Emergency Department Arrival.

BACKGROUND: The objective of this study was to quantify the sensitivity of very low concentrations of high-sensitivity cardiac troponin T (hsTnT) at ED arrival for acute myocardial infarction (AMI) in a large cohort of chest pain patients evaluated in real-world clinical practice. METHODS: This retrospective study included consecutive ED patients with suspected cardiac chest pain evaluated in four urban EDs, excluding those with ST-elevation AMI, cardiac arrest or abnormal kidney function. The primary outcomes were AMI at 7, 30, and 90 days. Secondary outcomes included major adverse cardiac events (MACE; all-cause mortality, AMI, and revascularization) and the individual MACE components. Test characteristics were calculated for hsTnT values from 3 to 200 ng/L . RESULTS: A total of 7,130 patients met inclusion criteria. AMI incidences at 7, 30, and 90 days were 5.8, 6.0, and 6.2%. When the hsTnT assay was performed at ED arrival, the limit of blank of the assay (3 ng/L) ruled out 7-day AMI in 15.5% of patients with 100% sensitivity and negative predictive value (NPV). The limit of detection of the assay (5 ng/L) ruled out AMI in 33.6% of patients with 99.8% sensitivity and 99.95% NPV for 7-day AMI. The limit of quantification (the Food and Drug Administration [FDA]-approved cutoff for lower the reportable limit) of 6 ng/L ruled out AMI in 42.2% of patients with 99.8% sensitivity and 99.95% NPV. The sensitivities of the cutoffs of <3, <5, and <6 ng/L for 7-day MACE were 99.6, 97.4, and 96.6%, respectively. The NPVs of the cutoffs of <3, <5, and <6 ng/L for 7-day MACE were 99.8, 99.5, and 99.4%, respectively. A secondary analysis was performed in a subgroup of 3,549 higher-risk patients who underwent serial troponin testing. In this subgroup, a cutoff of 3 ng/L ruled out 7-day AMI in 9.6% of patients with 100% sensitivity and NPV, a cutoff of 5 ng/L ruled out 7-day AMI in 23.3% of patients with 99.7% sensitivity and 99.9% NPV, and a cutoff of 6 ng/L ruled out 7-day AMI in 29.8% of patients with 99.7 and 99.9% NPV. In the higher-risk subgroup, the sensitivities of cutoffs of <3, <5, and <6 ng/L for 7-day MACE were 99.8, 97.4, and 96.6%, respectively. In this higher-risk subgroup, the NPV of cutoffs of <3, <5, and <6 ng/L for 7-day MACE were 99.7, 98.5, and 98.4%, respectively. CONCLUSIONS: When used in real-world clinical practice conditions, hsTnT concentrations < 6 ng/L (below the lower reportable limit for an FDA-approved assay) at the time of ED arrival can rule out AMI with very high sensitivity and NPV. The sensitivity for MACE is unacceptably low, and thus a single-troponin rule-out strategy should only be used in the context of a structured risk evaluation.

One-step extraction and quantitation of toxic alcohols and ethylene glycol in plasma by capillary gas chromatography (GC) with flame ionization detection (FID).

OBJECTIVES: Clinical analysis of volatile alcohols (i.e. methanol, ethanol, isopropanol, and metabolite acetone) and ethylene glycol (EG) generally employs separate gas chromatography (GC) methods for analysis. Here, a method for combined analysis of volatile alcohols and EG is described. DESIGN AND METHODS: Volatile alcohols and EG were extracted with 2:1 (v:v) acetonitrile containing internal standards (IS) 1,2 butanediol (for EG) and n-propanol (for alcohols). Samples were analyzed on an Agilent 6890 GC FID. The method was evaluated for precision, accuracy, reproducibility, linearity, selectivity and limit of quantitation (LOQ), followed by correlation to existing GC methods using patient samples, Bio-Rad QC, and in-house prepared QC material. RESULTS: Inter-day precision was from 6.5-11.3% CV, and linearity was verified from down to 0.6mmol/L up to 150mmol/L for each analyte. The method showed good recovery (~100%) and the LOQ was calculated to be between 0.25 and 0.44mmol/L. Patient correlation against current GC methods showed good agreement (slopes from 1.03-1.12, and y-intercepts from 0 to 0.85mmol/L; R(2)>0.98; N=35). Carryover was negligible for volatile alcohols in the measuring range, and of the potential interferences tested, only toluene and 1,3 propanediol interfered. The method was able to resolve 2,3 butanediol, diethylene glycol, and propylene glycol in addition to the peaks quantified. CONCLUSIONS: Here we describe a simple procedure for simultaneous analysis of EG and volatile alcohols that comes at low cost and with a simple liquid-liquid extraction requiring no derivitization to obtain adequate sensitivity for clinical specimens.