Canadian guidance for diagnosis and management of acute hepatic porphyrias.

Acute hepatic porphyrias (AHP) comprise four rare monogenic autosomal conditions. Each is linked to a deficiency of heme metabolizing enzymes. Common manifestations include severe abdominal pain, nausea, confusion, hyponatremia, hypertension, tachycardia, and neuropathy. Diagnosis is challenging due to a non-specific, variable presentation with symptoms mimicking other common conditions. Initial diagnosis of AHP can be made with a test for urinary porphobilinogen, δ-aminolevulinic acid and porphyrins using a single random (spot) sample. However, many patients have complications due to delays in diagnosis and management. A novel small interfering RNA-based agent, givosiran, has demonstrated efficacy in reducing acute attacks in a recent Phase III trial, leading to its approval for the management of AHP. Early diagnosis is crucial for the timely introduction of disease-modifying treatments that reduce impairments, enhance quality of life, and extend survival. In this guidance, we aim to improve awareness and outcomes of AHP by making recommendations about diagnosis, monitoring, and treatment in Canada.

Effective interventions to improve the quality of critically high point-of-care glucose meter results.

OBJECTIVES: Point-of-care testing (POCT) is testing performed outside the traditional laboratory, often at the patient bedside. In hospital settings, blood glucose is the most common POCT. Staff performing POCT are not usually laboratory trained; they are clinical staff with a primary focus on treating patients. Clinical staff find POCT quality assurance (QA) practices burdensome and are often non-compliant. In hospitals within EORLA (Eastern Ontario Regional Laboratories Association), all critically high POCT glucose results must be repeated prior to acting, according to policy. Compliance with this policy is audited regularly. DESIGN: and methods: All POCT glucose tests performed in participating sites between January and June 2018 and June and December 2019 were audited for compliance with the critical repeat policy. The discordant repeat rate was also determined for each audit period. Between January and May 2019, there were interventions aimed at improving compliance with the repeat policy. RESULTS: Compliance with the critical repeat policy increased from 30 to 57% in 2019 compared to 2018, following nursing education and implementation of notifications on the glucose meters themselves. The rate of discordant repeat results (>20% different from initial) also improved at most sites in 2019 compared to 2018. Nurses cited insufficient cleaning of patient hands prior to initial testing as the primary reason for discordant repeats. CONCLUSIONS: Operator compliance with POCT QA policies is an ongoing challenge requiring continual audit, feedback and education. A strong POCT multi-disciplinary committee with supports from senior and clinical leadership in an organization are key to improving compliance.

Predicting Escitalopram Exposure to Breastfeeding Infants: Integrating Analytical and In Silico Techniques.

BACKGROUND: Escitalopram is used for post-partum depression; however, there are limited pharmacokinetic data of escitalopram in milk and plasma of infants breastfed by women taking the drug. OBJECTIVE: The objective of this study was to apply physiologically-based pharmacokinetic (PBPK) modelling to predict infant drug exposure (plasma area under the curve from time zero to infinity [AUC∞]) based on drug monitoring data of escitalopram in breast milk. METHODS: Using a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, we quantified escitalopram concentrations in milk samples of 18 breastfeeding women with escitalopram therapy at steady state, collected at three to five time points. The escitalopram concentrations in breast milk were used with infant feeding parameters from the literature to simulate infant daily dose. We used PK-Sim® to develop an adult PBPK model for escitalopram and extrapolated it to a population of 1600 infants up to 12 months of age. An integration of the simulated infant daily dose and the virtual infants with variable physiological-pharmacological parameters was used to predict drug exposure (plasma AUC∞) distribution in the population of infants breastfed by women receiving escitalopram 20 mg/day. RESULTS: Escitalopram concentrations in milk were 50 ± 17 ng/mL (mean ± standard deviation). The simulated infant plasma AUC∞ following escitalopram exposure through breast milk was low, with a median of 1.7% (range 0.5-5.9%) of the corresponding maternal plasma AUC∞, indicating no substantial exposure. CONCLUSIONS: Infant exposure levels to escitalopram in breast milk are low. A PBPK modeling approach can be used to translate data on drug monitoring in milk into a population distribution of infant plasma levels for drug safety assessment.

Evaluation of electrochemiluminescence immunoassays for immunosuppressive drugs on the Roche cobas e411 analyzer.

Background:  Therapeutic drug monitoring of immunosuppressant drugs are used to monitor drug efficacy and toxicity and to prevent organ transplant rejection. This study evaluates the analytical performance of semi-automated electrochemiluminescence immunoassays (ECLIA) for cyclosporine (CSA), tacrolimus (TAC) and sirolimus (SRL) on the Roche cobas e 411 analyzer at a major transplant hospital to assess method suitability and limitations. Methods: Residual whole blood samples from patients undergoing immunosuppressant therapy were used for evaluation. Imprecision, linearity, functional sensitivity, method comparisons and lot-to-lot comparisons were assessed. Results: Total imprecision ranged from 3.3 to 7.1% for CSA, 3.9 to 9.4% for TAC, and 4.6 to 8.2% for SRL. Linearity was verified from 30.0 to 960.9 µg/L for CSA, from 1.1 to 27.1 µg/L for TAC, and from 0.5 to 32.3 µg/L for SRL. The functional sensitivity met the manufacturer's claims and was determined to be <6.5 µg/L for CSA, 1.1 µg/L for TAC, and <0.1 µg/L for SRL (CV≤20%). Deming regression analysis of method comparisons with the ARCHITECT immunoassay yielded slopes of 0.917 (95%CI: 0.885-0.949) and r of 0.985 for CSA, 0.938 (95%CI: 0.895-0.981) and r of 0.974 for TAC, and 0.842 (0.810-1.110) and r of 0.982 for SRL. Deming regression analysis of comparisons with the LC-MS/MS method yielded slopes of 1.331 (95%CI: 1.167-1.496) and r of 0.969 for CSA, 0.924 (95%CI: 0.843-1.005) and r of 0.984 for TAC, and 0.971 (95%CI: 0.913-1.030) and r of 0.993 for SRL. Conclusions: The cobas e 411 ECLIA for CSA, TAC, and SRL have acceptable precision, linearity, and functional sensitivity. The method comparisons correlated well with the ARCHITECT immunoassay and LC-MS/MS and is fit for therapeutic drug monitoring.

Milrinone therapeutic drug monitoring in a pediatric population: Development and validation of a quantitative liquid chromatography-tandem mass spectrometry method.

BACKGROUND: Milrinone is a potent selective phosphodiesterase type III inhibitor which stimulates myocardial function and improves myocardial relaxation. Although therapeutic monitoring is crucial to maintain therapeutic outcome, little data is available. A proof-of-principle study has been initiated in our institution to evaluate the clinical impact of optimizing milrinone dosing through therapeutic drug monitoring (TDM) in children following cardiac surgery. We developed a robust LC-MS/MS method to quantify milrinone in serum from pediatric patients in real-time. METHODS: A liquid-liquid extraction procedure was used to prepare samples for analysis prior to measurement by LC-MS/MS. Performance characteristics, such as linearity, limit of quantitation (LOQ) and precision, were assessed. Patient samples were acquired post-surgery and analyzed to determine the concentration-time profile of the drug as well as to track turn-around-times. RESULTS: Within day precision was <8.3% across 3 levels of QC. Between-day precision was <12%. The method was linear from 50 to 800µg/l; the lower limit of quantification was 22µg/l. Comparison with another LC-MS/MS method showed good agreement. Using this simplified method, turnaround times within 3-6h were achievable, and patient drug profiles demonstrated that some milrinone levels were either sub-therapeutic or in the toxic range, highlighting the importance for milrinone TDM. CONCLUSIONS: This simplified and quick method proved to be analytically robust and able to provide therapeutic monitoring of milrinone in real-time in patients post-cardiac surgery.

Implications of serum creatinine measurements on GFR estimation and vancomycin dosing in children.

BACKGROUND: Different serum creatinine (sCr) assays may obtain different values in the same patient, causing discrepancies in estimated glomerular filtration rate (eGFR) and sCr-based vancomycin dosing calculations. OBJECTIVE: To identify potential discrepancies in sCr concentrations obtained by different assays, the compensated Jaffe (sCr-Jaffe) and the enzymatic (sCr-enz), and to compare between the eGFR and vancomycin daily dose, based on these sCr values. METHOD: sCr-Jaffe and, sCr-enz concentrations of 890 healthy children, aged 1-18 years, were available from the Canadian Laboratory Initiative in Pediatric Reference Intervals study in Ontario. For each subject, eGFR (eGFR-Jaffe, eGFR-enz) was calculated using the revised Schwartz equation, and vancomycin daily dose (Vdose-Jaffe, Vdose-enz) was calculated using a sCr-based pharmacokinetic model. RESULT: Significant, age-related differences were found in sCr concentrations, and in subsequent eGFR and Vdose, between the two assays. In children aged 1-5 years, mean sCr-Jaffe was higher than sCr-enz (44.0 ± 5.0 vs. 27.7 ± 7.3 µmol/L, P < 0.001), leading to lower eGFR-Jaffe (83.2 ± 9.0 vs. 137.9 ± 27.1 mL/min/1.73m2, P < 0.001) and lower Vdose-Jaffe (44.7 ± 2.5 vs. 53.5 ± 5.1 mg/kg/24 h, P < 0.001). CONCLUSION: Based on these findings, young children may be at risk for vancomycin under-treatment. Further research is needed to define the more accurate sCr assay in young children treated with renally excreted drugs.

Validity of establishing pediatric reference intervals based on hospital patient data: a comparison of the modified Hoffmann approach to CALIPER reference intervals obtained in healthy children.

OBJECTIVES: To compare pediatric reference intervals calculated using hospital-based patient data with those calculated using samples collected from healthy children in the community as part of the CALIPER study. METHODS: Hospital-based data for 13 analytes (calcium, phosphate, iron, ALP, cholesterol, triglycerides, creatinine, direct bilirubin, total bilirubin, ALT, AST, albumin and magnesium), measured on the Vitros 5600, collected between 2007 and 2011 were obtained. The data for each analyte were partitioned by age and gender as previously defined by the CALIPER study. Outliers in each partition were removed using the Tukey method. The cumulative distribution function (cdf) was then determined for each analyte value following which, the inverse cdf values of a standard Gaussian distribution were calculated. The analyte values were plotted against the inverse cdf of the standard Gaussian distribution. Piece-wise regression determined the linear portion of the resulting graph using the statistical software R. Linear regression determined an equation for the linear portion in each partition and reference intervals were calculated by extrapolating to identify the 2.5th and 97.5th centiles in each partition based on the inverse cdf values (which would correspond to the values -1.96 and 1.96 of the Gaussian distribution). Using the 90% confidence intervals for the reference intervals defined by CALIPER and the Reference Change Value (RCV) as the criteria, these calculated reference intervals were compared to those reported previously by CALIPER. Reference samples were also measured on the Vitros 5600 analyzer in an attempt to validate the calculated reference intervals. RESULTS: In general, the reference intervals calculated from hospital-based data were generally wider than those calculated by CALIPER. None of the reference intervals calculated using the Hoffmann approach fell completely within the 90% confidence intervals calculated by CALIPER. CONCLUSIONS: These results suggest that calculating pediatric reference intervals from hospital-based data may be useful, as a guide, in some cases but will likely not replace the need to establish reference intervals in healthy pediatric populations.

Pediatric within-day biological variation and quality specifications for 38 biochemical markers in the CALIPER cohort.

BACKGROUND: Studies of biological variation provide insight into the physiological changes that occur within and between study participants. Values obtained from such investigations are important for patient monitoring and for establishing quality specifications. In this study we evaluated the short-term biological variation of 38 chemistry, lipid, enzyme, and protein analytes in a pediatric population, assessed the effect of age partitions on interindividual variation, and compared the findings to adult values. METHODS: Four plasma samples each were obtained within 8 h from 29 healthy children (45% males), age 4-18 years. Samples were stored at -80 °C and analyzed in 3 batches, with samples from 9-10 study participants per batch. Within-person and between-person biological variation values were established using nested ANOVA after exclusion of outliers by use of the Tukey outlier test. Analytical quality specifications were established with the Fraser method. RESULTS: Biological variation coefficients and analytical goals were established for 38 analytes. Age partitioning was required for 6 analytes. Biological variation characteristics of 14 assays (37%) were distinct from adult values found in the Westgard database on biological variation. Biological variation characteristics were established for 2 previously unreported analytes, unconjugated bilirubin and soluble transferrin receptor. CONCLUSIONS: This study is the first to examine biological variation and to establish analytical quality specifications on the basis of biological variation for common assays in a pediatric population. These results provide insight into pediatric physiology, are of use for reference change value calculations, clarify the appropriateness of reference interval use, and aid in the development of quality management strategies specific to pediatric laboratories.

CLSI-based transference of the CALIPER database of pediatric reference intervals from Abbott to Beckman, Ortho, Roche and Siemens Clinical Chemistry Assays: direct validation using reference samples from the CALIPER cohort.

OBJECTIVES: The CALIPER program recently established a comprehensive database of age- and sex-stratified pediatric reference intervals for 40 biochemical markers. However, this database was only directly applicable for Abbott ARCHITECT assays. We therefore sought to expand the scope of this database to biochemical assays from other major manufacturers, allowing for a much wider application of the CALIPER database. DESIGN AND METHODS: Based on CLSI C28-A3 and EP9-A2 guidelines, CALIPER reference intervals were transferred (using specific statistical criteria) to assays performed on four other commonly used clinical chemistry platforms including Beckman Coulter DxC800, Ortho Vitros 5600, Roche Cobas 6000, and Siemens Vista 1500. The resulting reference intervals were subjected to a thorough validation using 100 reference specimens (healthy community children and adolescents) from the CALIPER bio-bank, and all testing centers participated in an external quality assessment (EQA) evaluation. RESULTS: In general, the transferred pediatric reference intervals were similar to those established in our previous study. However, assay-specific differences in reference limits were observed for many analytes, and in some instances were considerable. The results of the EQA evaluation generally mimicked the similarities and differences in reference limits among the five manufacturers' assays. In addition, the majority of transferred reference intervals were validated through the analysis of CALIPER reference samples. CONCLUSIONS: This study greatly extends the utility of the CALIPER reference interval database which is now directly applicable for assays performed on five major analytical platforms in clinical use, and should permit the worldwide application of CALIPER pediatric reference intervals.

Closing the gaps in pediatric laboratory reference intervals: a CALIPER database of 40 biochemical markers in a healthy and multiethnic population of children.

BACKGROUND: Pediatric healthcare is critically dependent on the availability of accurate and precise laboratory biomarkers of pediatric disease, and on the availability of reference intervals to allow appropriate clinical interpretation. The development and growth of children profoundly influence normal circulating concentrations of biochemical markers and thus the respective reference intervals. There are currently substantial gaps in our knowledge of the influences of age, sex, and ethnicity on reference intervals. We report a comprehensive covariate-stratified reference interval database established from a healthy, nonhospitalized, and multiethnic pediatric population. METHODS: Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer. RESULTS: Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers. CONCLUSIONS: The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

Pediatric reference intervals for 28 chemistries and immunoassays on the Roche cobas 6000 analyzer--a CALIPER pilot study.

OBJECTIVE: The aim of this study was to determine age- and sex-specific pediatric reference intervals for 28 analytes on the Roche cobas 6000 analyzer. DESIGN AND METHODS: The study was conducted at the Hospital for Sick Children in Toronto, Canada. Approximately 600 outpatient samples from a pediatric population deemed to be metabolically stable were subdivided into five age classes ranging from 0 to 20 years of age and further partitioned by gender. Reference intervals were established, after removal of samples significantly affected by hemolysis, icterus and lipemia and outlier exclusion, using the Robust statistical method to obtain the 2.5th and 97.5th percentiles. RESULTS: Age (birth to 20 years of age) and gender-appropriate pediatric reference intervals for 28 analytes are reported. CONCLUSIONS: These reference intervals provide the basis for clinical interpretation of laboratory results using the Roche cobas 6000 analyzer or related instrumentation/methods, provided adequate reference interval verification studies are performed.

Comparison of the CEDIA and MEIA assays for the measurement of sirolimus in organ transplant recipients.

OBJECTIVES: This study evaluated two immunoassays, the CEDIA assay and the MEIA assay, used for the measurement of whole blood levels of sirolimus in organ transplant recipients. DESIGN AND METHODS: We report on the performance characteristics (total precision, limit of quantitation (functional sensitivity), limit of detection (analytical sensitivity), linearity, accuracy) for each assay. Patient correlation studies were performed, and the results were analyzed using Bland-Altman plots and Passing-Bablok analysis. RESULTS: Total precision for the MEIA assay, corresponding to three mean concentrations of 5.0, 10.6 and 20.2 ng/mL, was 10.5, 8.5, and 6.7%, respectively. The limit of detection was determined to be 1.1 ng/mL and the limit of quantitation was 1.5 ng/mL. The mean recovery for CEDIA was 105.4%, and analysis of proficiency material demonstrated a large negative bias with respect to the mass spectrometry peer mean-later determined to be due to matrix interference. Results for the CEDIA assay showed a total precision, corresponding to a mean concentration of 5.4, 10.5 and 20.7 ng/mL, of 13.5, 5.6, and 4.1%, respectively. The limit of detection was found to be 4.8 ng/mL, with a limit of quantitation of 5.2 ng/mL. The mean recovery for MEIA was 110.1%, and analysis of proficiency material demonstrated good agreement with the mass spectrometry peer mean with a slight positive bias. Both assays were acceptably linear over the reportable range of the assay. Patient correlation studies demonstrated a positive average bias for both assays versus results from LC-MS measurement (0.9 ng/mL for MEIA, 2.1 ng/mL for CEDIA). CONCLUSION: Based on this evaluation, the MEIA demonstrated acceptable performance for use in clinical monitoring of sirolimus. However, based on a higher limit of quantitation that falls within the therapeutic interval, the CEDIA is not recommended for clinical monitoring of sirolimus.

Effective removal of albumin from serum.

The protein constituents of serum can range from grams to picograms per liter, making it technically difficult to achieve in-depth proteomic analysis. Removal of highly abundant proteins, such as albumin, coupled to powerful protein separation methods is required for increased sample load, thus facilitating detection and identification of low-abundant proteins. We report here a chemical-based extraction method for the effective and specific removal of albumin from serum.

The clinical application of proteomics.

BACKGROUND: Proteomics is defined as a scientific approach used to elucidate all protein species within a cell or tissue, and many researchers are taking advantage of proteomic technology to elucidate protein changes between healthy and diseased states. METHODS: The application of proteomic techniques and strategies to the field of medicine is slowly transforming the way biomarker discovery is conducted. However, the complexity of serum is the source of both its promise to clinical applications and its challenge to proteomic analysis. Like any new technology when it is first introduced, proteomics has been touted with much hope and promise. RESULTS AND CONCLUSIONS: We provide a review of the clinical application of proteomics with the emphasis on current practical issues and challenges facing proteomic research.

Stunned peri-infarct canine myocardium is characterized by degradation of troponin T, not troponin I.

OBJECTIVE: Degradation of cardiac troponin I (cTnI) has been proposed to represent the underlying molecular mechanism responsible for post-ischemic contractile dysfunction of viable but 'stunned' myocardium. However, this concept is largely derived from models of brief, sublethal ischemia essentially devoid of necrosis, and there is speculation that defects in cTnI may be model-dependent. Accordingly, our primary aim was to evaluate the integrity of cardiac troponins-i.e., cTnI, as well as cTnT and cTnC-in viable but stunned peri-infarct tissue. In addition, we addressed the as-yet unexplored issue of whether the profound reduction of infarct size evoked by brief preconditioning ischemia (PC) was accompanied by a favorable attenuation in ischemia/reperfusion-induced degradation of cTnI, cTnT or cTnC in the remaining viable subepicardium. METHODS: Anesthetized open-chest dogs received 10 min of PC ischemia or a comparable control period, followed by 1 h of sustained coronary occlusion and 3 h of reperfusion. Subepicardial biopsies from the center of the soon-to-be ischemic territory were obtained at baseline and at 30 min and 3 h post-reflow, and myofilament protein integrity (intact cTnI, cTnT and cTnC, as well as degradation bands and covalent complexes) were assessed by Western immunoblotting. In addition, in all dogs, wall thickening was measured by echocardiography, collateral blood flow was assessed during sustained occlusion by injection of radiolabeled microspheres, and infarct size was delineated by tetrazolium staining. RESULTS: Although PC was, as expected, cardioprotective (infarct size of 2 +/- 1% of the risk region vs. 17 +/- 6% in controls; p < 0.05), both control and PC groups exhibited profound and comparable contractile dysfunction following reflow (mean wall thickening reduced to 20-22% of baseline values). There was, however, no significant degradation of cTnI in the viable but stunned, peri-infarct tissue. We did observe degradation of cTnT in the stunned subepicardium, an effect that was attenuated in dogs that received antecedent PC ischemia. However, there was no correlation between post-ischemic wall thickening and the immunoreactivity of the intact cTnT band, or wall thickening and the intensity of the cTnT degradation products. CONCLUSIONS: Our results suggest cTnI degradation is not a universal determinant of post-ischemic myocardial stunning. Moreover, the dissociation between cTnT degradation and wall thickening argue against a direct 'cause-and-effect' relationship between proteolysis of cTnT and acute, post-ischemic contractile dysfunction of stunned peri-infarct myocardium.