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Objectives Monitoring quality indicators (QIs) is an important part of laboratory quality assurance (QA). Here, the Canadian Society of Clinical Chemists (CSCC) Point of Care Testing (POCT) and QI Special Interest Groups describe a process for establishing and monitoring QIs for POCT glucose testing. Methods Key, error prone steps in the POCT glucose testing process were collaboratively mapped out, followed by risk assessment for each step. Steps with the highest risk and ability to detect a non-conformance were chosen for follow-up. These were positive patient identification (PPID) and repeat of critically high glucose measurements. Participating sites were asked to submit aggregate data for these indicators from their site(s) for a one-month period. The PPID QI was also included as part of a national QI monitoring program for which fifty-seven sites submitted data. Results The percentage of POCT glucose tests performed without valid PPID ranged from 0–87%. Sites without Admission-Discharge-Transfer (ADT) connectivity to POCT meters were among those with the highest percentage of POCT glucose tests performed without valid PPID. The percentage repeated critically high glucose measurements ranged from 0–50%, indicating low compliance with this recommendation. A high rate of discordance was also noted when critically high POCT glucose measurements were repeated, demonstrating the importance of repeat testing prior to insulin administration. Conclusions Here, a process for establishing these QIs is described, with preliminary data for two QIs chosen from this process. The findings demonstrate the importance of QIs for identification and comparative performance monitoring of non-conformances to improve POCT quality.

In a CODE-MI sub-study, six troponin assays were evaluated over 12 months using 3-samples (normal, female and male 99th-percentile levels; n=2,142 results from 67 instruments). Mean differences varied per assay with maximum values being ±4, ±8 and ±9 ng/L at the normal and sex-specific 99th-percentiles, respectively. A pragmatic approach, combining all assays, established maximum differences of ±3 ng/L or ±30%.

Introduction: Hypervitaminosis D is a relatively uncommon etiology of hypercalcemia. Toxicity is usually caused by very high doses, mostly secondary to erroneous prescription or administration of Vitamin D, and less commonly, contaminated foods or manufacturing errors of vitamin D-containing supplements. Case description: 16 year old male, previously healthy, presented with 2-week history of non specific symptoms (fatigue, gastrointestinal complaints). Investigations showed acute kidney injury and hypercalcemia (total calcium 3.81 mmol/L). Further diagnostic workup revealed markedly elevated 25-hydroxy-vitamin D levels (1910 nmol/L). He denied taking any vitamin D supplements; however, he reported consumption of creatine and protein supplements. Mass spectrometry analysis of the creatine supplement estimated a vitamin D content of 425,000 IU per serving (100 times the upper tolerable daily dose). A few months later, another previously healthy adolescent presented with severe hypercalcemia and acute kidney injury secondary to hypervitaminosis D. He was also using a creatine supplement from the same manufacturer brand and lot. Both patients were treated with IV hydration, calcitonin and pamidronate. They maintained normocalcemia after their initial presentation but required low-calcium diets and laboratory testing for months after this exposure. Discussion: We present 2 cases of hypervitaminosis D caused by a manufacturing error of a natural health product which did not claim to contain vitamin D. The use of dietary supplements is highly prevalent; their use should be incorporated in anamnesis and considered a potential source of toxicity when an alternative source cannot be found, regardless of the product label.

Point of Care Testing (POCT) refers to clinical laboratory testing performed outside the central laboratory, nearer to the patient and sometimes at the patient bedside. The testing is usually performed by clinical staff, such as physicians or nurses, who are not laboratory trained. This document was developed by the POCT Interest group of the Canadian Society of Clinical Chemists (CSCC) as practical guidance for quality assurance practices related to POCT performed in hospital and outside hospital environments. The aspects of quality assurance addressed in this document include: (1) device selection, (2) initial device verification, (3) ongoing device verification, (4) ongoing quality assurance including reagent and quality control (QC) lot changes, and (5) quality management including operator and document management.

Background: Serum IgG4 is typically measured to investigate for Immunoglobulin G4-related Disease (IgG4-RD), a fibroinflammatory condition associated with polyclonal increase in serum IgG4. However, increased IgG4 can also be monoclonal, and little is known about IgG4 myeloma. Methods: We describe two cases of IgG4 myeloma without clinical, radiologic, or laboratory features of IgG4-related disease. Results: An 84 year old man presented with anemia and compression fractures and a 77 year old man presented with anemia, hypercalcemia and renal failure. Both had markedly elevated monoclonal serum IgG4, 34 g/L and 48 g/L in the beta region, and increased IgG positive bone marrow plasma cells, 50% and 80%, respectively. Neither had clinical or radiological manifestations of IgG4-related disease (IgG4-RD) such as salivary or lacrimal gland swelling, autoimmune pancreatitis , or retroperitoneal fibrosis. Both cases responded well to standard myeloma therapy. The IgG4 paraprotein caused spuriously elevated beta-2 microglobulin of 45.2 mg/L in case two due to interference with the assay. Conclusion: These cases illustrate the importance of performing serum protein electrophoresis in tandem with IgG subclasses to distinguish between polyclonal and monoclonal increases in serum IgG4. The lack of typical IgG4-RD features in these two patients suggests that monoclonal elevation in serum IgG4 alone is insufficient to cause the organ damage characteristic of IgG4-RD. Larger studies of IgG myeloma subtypes are warranted to explore whether IgG1, IgG2, IgG3 and IgG4 myeloma differ in natural history and whether the interference with beta-2 microglobulin is specific to IgG4 monoclonal proteins.

A 71-year-old man presented with increasing confusion after dialysis. He was admitted for progressive decline in functional status over a 1-month period including delirium, a fall, and difficulty with pain, speaking, and walking. Four years ago, he was diagnosed with stage IIIB multiple myeloma with IgAκ M-protein and corresponding κ free light chain (FLC).3 He was treated with combination chemotherapy of cyclophosphamide, bortezomib, and dexamethasone, and he achieved partial remission. Medical history was significant for myeloma-related end-stage renal failure, hypertension, osteonecrosis of the jaw secondary to bisphosphonate, and recent onset of squamous cell carcinoma. At presentation, physical examination was unremarkable. The patient was alert and oriented, with a Glasgow coma scale of 15. Laboratory findings included hemoglobin concentration of 10.4 g/dL [reference interval (RI), 13.5–17.0 g/dL] and mean red cell volume of 114 fL (RI, 82–98 fL). Additional test results included a plasma total protein concentration of 6.5 g/dL (RI, 6.0–8.0 g/dL), albumin concentration of 4.2 g/dL (RI, 3.5–4.8 g/dL), total calcium concentration of 12.4 mg/dL (RI, 8.7–10.3 mg/dL; 3.11 mmol/L; RI, 2.18–2.58 mmol/L), ionized calcium concentration of 6.12 mg/dL (RI, 4.68–5.16 mg/dL; 1.53 mmol/L; RI, 1.17–1.29 mmol/L), phosphorus concentration of 7.3 mg/dL (RI, 2.5–5.0 mg/dL; 2.36 mmol/L; RI, 0.80–1.60 mmol/L), alkaline phosphatase concentration of 75 U/L (RI, 30–105 U/L), creatinine concentration of 10.2 mg/dL (RI, 0.68–1.13 mg/dL; 903 μmol/L; RI, 60–100 μmol/L), and blood urea nitrogen concentration of 69 mg/dL (RI, 7–22 mg/dL; 24.6 mmol/L; RI, 2.5–8.0 mmol/L). ### QUESTIONS TO CONSIDER 1. What are causes of discordance between total protein and serum FLC quantification? 2. What are limitations of serum FLC assays? 3. What strategies can be used to clarify suspected inaccurate FLC results? Serum protein electrophoresis (SPE) (Fig. 1, A and C) showed hypogammaglobulinemia, with a β region of 1.0 g/dL. Immunofixation electrophoresis identified the presence of IgAκ M-protein in the β …

Key Messages • Consider safety precautions and infection control processes, particularly in remote testing locations when using rapid SARS-CoV-2 devices. • Seek oversight and partnership with an accredited clinical laboratory for guidance on setting up a quality assurance framework. • Rapid-testing for SARS-CoV-2 requires method verification prior to clinical implementation.

The StatSensor is a point-of-care device which measures creatinine in capillary whole blood. Previous studies reported an underestimation of the creatinine measurements at high creatinine concentrations and were performed in the prestandardization era for creatinine.This accuracy-based study evaluates the use of this device in kidney-transplanted patients and those with chronic kidney disease (CKD).Cross-sectional diagnostic accuracy study.Nephrology outpatient clinic in an urban tertiary center.Adults with CKD or a functioning kidney transplant.Duplicate StatSensor creatinine measurements were performed on capillary whole blood samples collected by direct fingerstick and SAFE-T-FILL collection device. Results were compared with simultaneous venous blood sampling for serum and plasma creatinine measured by an enzymatic method on the Roche Integra 400 mainframe analyzer with traceability to the ID-GC-MS (isotope dilution gas chromatography mass spectrometry) reference method.Deming regression, Pearson correlation coefficient, and Bland-Altman analysis were used to assess accuracy and comparability between capillary whole blood measured by StatSensor and plasma creatinine measured by routine analyzer with traceability to the reference method. Estimated glomerular filtration (eGFR) rates were calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation and concordance with Kidney Disease Improving Global Outcomes (KDIGO) CKD stage classification was evaluated.There were 60 participants (mean age = 61.9 ± 15.0 years, 55% men, 33% transplant, mean plasma creatinine = 137 ± 59 µmol/L). Bland-Altman analysis indicated a positive mean bias of 12.7 µmol/L between StatSensor fingerstick creatinine measurement and plasma creatinine. Comparison of eGFR (CKD-EPI) calculated from the StatSensor fingerstick creatinine versus plasma creatinine showed misclassification across all KDIGO CKD stages. Postanalytical correction of the bias did not improve misclassifications. The use of mean of duplicate StatSensor creatinine results did not improve performance compared with the use of singlet results.Single center, limited participant numbers.The results of our study suggest that the limiting characteristics of the StatSensor device are not only bias, but also imprecision. The level of imprecision observed may influence clinical decision-making and limit the usefulness of StatSensor as a CKD screening tool. If choosing to utilize it for either screening for or monitoring CKD, it is essential that clinicians understand the limitations of point-of-care devices and apply this knowledge to test interpretation.Le StatSensor est un appareil portatif conçu pour mesurer le taux de créatinine dans le sang capillaire total. Des études antérieures, réalisées avant la standardisation des mesures de la créatinine, ont rapporté une sous-estimation des mesures à des concentrations élevées.Cette étude centrée sur la précision a examiné l’utilisation de cet appareil chez des patients transplantés d’un rein et des patients atteints d’insuffisance rénale chronique (IRC).Étude transversale centrée sur la précision du diagnostic.La clinique ambulatoire de néphrologie d’un centre de soins tertiaires en milieu urbain.Des adultes atteints d’IRC ou transplantés avec un rein fonctionnel.Les mesures de créatinine par StatSensor ont été effectuées en double sur des échantillons de sang capillaire total prélevés par ponction digitale directe et à l’aide du dispositif de prélèvement SAFE-T-FILL. Ces résultats ont été comparés à un prélèvement veineux simultané pour la mesure des taux de créatinine sérique et plasmatique par la méthode enzymatique avec l’analyseur Integra 400 de Roche avec traçabilité à la méthode de référence ID-GC-MS.La régression de Deming, le coefficient de corrélation de Pearson et l’analyse de Bland-Altman ont été utilisés pour évaluer la précision et la comparabilité entre les mesures du sang capillaire total par StatSensor et la mesure de créatinine plasmatique obtenue par l’analyseur de routine avec traçabilité à la méthode de référence. Le débit de filtration glomérulaire estimé (DFGe) a été calculé avec l’équation CKD-EPI, puis la concordance avec la classification des stades KDIGO pour l’IRC a été évaluée.L’étude a inclus 60 patients (55 % d’hommes; âge moyen 61,9 ± 15,0 ans) dont 33 % étaient transplantés. Le taux moyen de créatinine plasmatique s’établissait à 137 ± 59 µmol/L. L’analyse de Bland-Altman indique un biais positif moyen de 12,7 µmol/L entre la mesure de créatinine obtenue avec StatSensor par ponction digitale et le taux de créatinine plasmatique. La comparaison entre le DFGe (CKD-EPI) calculé à partir des mesures obtenues par ponction digitale avec StatSensor et de la mesure de créatinine plasmatique a montré une classification erronée à tous les stades KDIGO pour l’IRC. La correction du biais après l’analyse n’a pas amélioré les erreurs de classification. L’utilisation de la moyenne des résultats obtenus par StatSensor sur les échantillons prélevés en double n’a pas amélioré les performances par rapport à l’utilisation de singulets.Étude monocentrique, nombre de participants limité.Nos résultats suggèrent que les caractéristiques de limitation du StatSensor ne constituent pas qu’un biais, mais également une imprécision. Ce degré d’imprécision peut influencer la prise de décision clinique et limiter l’utilité du StatSensor comme outil de dépistage de l’IRC. Il est essentiel que les cliniciens soient conscients des limites de ces dispositifs et qu’ils appliquent ces connaissances à l’interprétation des résultats s’ils choisissent de les utiliser pour dépister ou surveiller l’IRC.Sans objet, il ne s’agissait pas d’un essai clinique.

This article will review the structure and function of IgG4, methods of measuring serum IgG4 concentrations, clinical conditions associated with increased and decreased serum IgG4, and the test characteristics of serum IgG4 in the diagnosis and management of Immunoglobulin G4-Related Disease (IgG4-RD). The four subclasses of IgG were discovered in 1964 through experiments on monoclonal IgG in patients with myeloma. Since 2001, interest in measuring serum IgG subclasses has increased dramatically due to the emergence of IgG4-RD, a multisystem fibroinflammatory condition wherein polyclonal serum IgG4 concentration is increased in approximately 70% of cases. Increased serum IgG4 typically manifests as a restriction in the anodal gamma region on serum protein electrophoresis, often with beta-gamma bridging, and can be mistaken as a monoclonal protein or polyclonal increase in IgA. Limitations of current clinical methods used in quantitation of serum IgG4 concentrations will be discussed, including the common immunonephelometric assays and LC-MS/MS based assays. Polyclonal IgG4 elevation is not specific for IgG4-RD, and may also occur in conditions such as eosinophilic granulomatosis with polyangiitis (EGPA), lymphoma, and multicentric Castleman disease (MCD). Race and gender differences also affect interpretation of serum IgG4 concentrations, for instance Asians have a higher serum IgG4 concentration than Whites and males have a higher concentration than females.

Background: 1,25-dihydroxyvitamin D (1,25(OH)2D), the biologically active vitamin D metabolite, plays a critical role in calcium and phosphate homeostasis. 1,25(OH)2D is measured to assess calcium and phosphate metabolism, particularly during periods of profound growth and development. Despite its importance, no reliable pediatric reference interval exists, with those available developed using adult populations or out-dated methodologies. Using the fully automated chemiluminescence immunoassay by DiaSorin, we established 1,25(OH)2D pediatric reference intervals using healthy children and adolescents from the CALIPER cohort. Methods: Serum samples from healthy subjects (0 to 2D using the DiaSorin LIAISON XL assay and age-specific reference intervals were established. The Mann-Whitney U-test was used to determine seasonal differences. Pooled neonatal and infantile samples were quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine if elevated concentrations during the first year of life may be attributed to cross-reacting moieties. Results: Three reference interval age partitions were required with highest levels in subjects 0 to 2D concentration was not significantly affected by seasonal variation or sex. Elevated 1,25(OH)2D concentrations in neonatal and infantile samples may be the result of an interfering substance. The absence of 3-epi-1,25-dihydroxyvitamin D in the pooled samples makes it unlikely to be the interfering moiety. Conclusions: Pediatric reference intervals for 1,25(OH)2D were established to improve test result interpretation in children and adolescents. 1,25(OH)2D is elevated in a proportion of neonates and infants, which may be the result of a cross-reacting moiety.

Background: Accurate reference intervals (RIs) based on a healthy pediatric population are essential for pediatric test result interpretation. The CALIPER project has recruited a large healthy cohort and completed a series of a priori studies to address gaps in pediatric RIs. As immunoassays from different manufacturers for endocrine and special chemistry markers are not standardized and show marked intermethod differences, direct RI studies are needed for each major analytical platform. Here, we report age- and sex-specific pediatric RIs for 29 immunoassays on the Ortho Clinical Diagnostics (Ortho) VITROS® 5600 analyzer. Methods: Health information and blood samples were collected from healthy pediatric subjects. Using the Ortho VITROS 5600 Integrated System MicroWell Technology, 29 biomarkers were measured. Analyte concentrations were partitioned by age and sex according to the Harris and Boyd method. After removing outliers, age- and sex-specific RIs and corresponding 90% confidence intervals were calculated according to CLSI guidelines. Results: All analytes required age partitioning except β-human chorionic gonadotropin (β-hCG), cancer antigen 15-3 (CA15-3), rubella immunoglobulin G (rubella IgG), and vitamin D. Several analytes including estradiol, progesterone, testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), free triiodothyronine (FT3), total triiodothyronine (TT3), total thyroxine (TT4), thyroid uptake, ferritin, intact parathyroid hormone (iPTH), total prostate-specific antigen (tPSA), free prostate-specific antigen (fPSA), cancer antigen 125 (CA125), creatine kinase MB (CK-MB), and myoglobin showed sex differences, observed mostly with the onset of puberty. Conclusions: Complex reference value trends were observed across the pediatric age range for several biomarkers examined on Ortho VITROS immunoassays. The availability of VITROS immunoassay RIs will enable accurate laboratory test interpretation and diagnosis for the pediatric population. As recommended by the CLSI EP28-A3c guidelines, implementation of these RIs should be validated for each laboratory’s local pediatric population.

Mass spectrometry-based assays have been increasingly implemented in various disciplines in clinical diagnostic laboratories for their combined advantages in multiplexing capacity and high analytical specificity and sensitivity. It is now routinely used in areas including reference methods development, therapeutic drug monitoring, toxicology, endocrinology, paediatrics, immunology and microbiology to identify and quantify biomolecules in a variety of biological specimens. As new ionisation methods, instrumentation and techniques are continuously being improved and developed, novel mass spectrometry-based clinical applications will emerge for areas such as proteomics, metabolomics, haematology and anatomical pathology. This review will summarise the general principles of mass spectrometry and specifically highlight current and future clinical applications in anatomical pathology.

The N-end rule pathway is a conserved targeted proteolytic process observed in organisms ranging from eubacteria to mammals. The N-end rule relates the metabolic stability of a protein to its N-terminal amino acid residue. The identity of the N-terminal amino acid residue is a primary degradation signal, often referred to as an N-degron, which is recognized by the components of the N-end rule when it is a destabilizing N-terminus. N-degrons may be exposed by non-processive proteolytic cleavages or by post-translational modifications. One modification is the post-translational addition of amino acids to the N-termini of proteins, a reaction catalyzed by aminoacyl-tRNA protein transferases. The aminoacyl-tRNA protein transferase in eubacteria like Escherichia coli is L/F transferase. Recent investigations have reported unexpected observations regarding the L/F transferase catalytic mechanism and its mechanisms of substrate recognition. Additionally, recent proteome-wide identification of putative in vivo substrates facilitates hypothesis into the yet elusive biological functions of the prokaryotic N-end rule pathway. Here we summarize the recent findings on the molecular mechanisms of catalysis and substrate recognition by the E. coli L/F transferase in the prokaryotic N-end rule pathway.

Background: Therapeutic drug monitoring of immunosuppressant drugs are used to monitor drug efficacy and toxicity and to prevent organ transplantation 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 identify method suitability and limitations.Methods:Residual whole blood samples from patients undergoing immunosuppressant therapy were used for evaluation. Experiments included imprecision, linearity, functional sensitivity, method comparisons and lot-to-lot assessments.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 rof 0.985 for CSA, 0.938 (95%CI: 0.895-0.981) androf 0.974 for TAC, and 0.842 (0.810-1.110) androf 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) androf 0.969 for CSA, 0.924 (95%CI: 0.843-1.005) androf 0.984 for TAC, and 0.971 (95%CI: 0.913-1.030) androf 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.

Aminoacyl-tRNA protein transferases catalyze the transfer of amino acids from aminoacyl-tRNAs to polypeptide substrates. Different forms of these enzymes are found in the different kingdoms of life and have been identified to be central to a wide variety of cellular processes. L/F-transferase is the sole member of this class of enzyme found in Escherichia coli and catalyzes the transfer of leucine to the N-termini of proteins which result in the targeted degradation of the modified protein. Recent investigations on the tRNA specificity of L/F-transferase have revealed the unique recognition nucleotides for a preferred Leu-tRNA(Leu) isoacceptor substrate. In addition to discussing this tRNA selectivity by L/F-transferase, we present and discuss a hypothesis and its implications regarding the apparent competition for this aminoacyl-tRNA between L/F-transferase and the translational machinery. Our discussion reveals a hypothetical involvement of the bacterial stringent response that occurs upon amino acid limitation as a potential cellular event that may reduce this competition and provide the opportunity for L/F-transferase to readily increase its access to the pool of aminoacylated tRNA substrates.

Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3′ aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNALeu (anticodon 5′-CAG-3′) isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNALeu (CAG)—a G3:C70 base pair and a set of 4 nt (C72, A4:U69, C68)—that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNA(Leu) and phenylalanyl-tRNA(Phe) as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.