Detection of macroenzymes: establishing upper reference limits for eight enzymes after polyethylene glycol precipitation

IntroductionThe presence of macroenzymes in blood can cause diagnostic confusion. Therefore, confirming the presence of macroenzymes is important to reduce unnecessary (non-)invasive investigations. Polyethylene glycol (PEG) precipitation is a simple and fast first-line method for the detection of macroenzymes. However, there is no consensus on the upper reference limit for the PEG-precipitable activity (%PPA) of monomeric enzymes. The aim of this study was to verify a PEG precipitation protocol for the detection of macroenzymes in our laboratory by establishing upper reference limits (URLs) and determining imprecision for eight enzymes after PEG precipitation. In addition, we aimed to clinically verify the URLs using samples containing macroenzymes as identified by electrophoresis. Materials and methodsPer enzyme, at least 40 leftover blood samples from adult patients with either normal or increased enzyme activities were diluted 1:1 with 25% PEG 6000 and 1:1 with 0.9% NaCl. Mixtures were incubated for 10 min at 37°C and centrifuged. Supernatant enzyme activity was measured on Cobas c702 and the %PPA was calculated. ResultsThe following URLs were obtained: 26% PPA for amylase, 29% PPA for alkaline phosphatase (ALP), 61% PPA for alanine aminotransferase, 48% PPA for aspartate aminotransferase, 24% PPA for creatine kinase (CK), 55% PPA for gamma-glutamyltransferase, 65% PPA for lactate dehydrogenase, and 56% PPA for lipase. The within-lab imprecision was < 15%. Regarding the clinical verification, the two historical samples with proven macroCK showed a %PPA of 69% and 43%, respectively, and a sample with proven macroALP had a %PPA of 52%. ConclusionIn this study, URLs for monomeric enzyme activities after PEG precipitation for eight different enzymes were established. The URLs are suitable for clinical use, but are only partially in line with other studies. Therefore, our data highlight the importance of establishing laboratory-specific upper reference limits for %PPA to allow a correct interpretation

A case of severe pseudohyperkalaemia due to muscle contraction

Introduction: Severe hyperkalaemia is a serious medical condition requiring immediate medical attention. Before medical treatment is started, pseudohyperkalaemia has to be ruled out. Case description: A 10-month old infant presented to the emergency department with fever and coughing since 1 week. Routine venous blood testing revealed a severe hyperkalaemia of 6.9 mmol/L without any indication of haemolysis. Reanalysis of the plasma sample confirmed the hyperkalaemia (7.1 mmol/L). Based on these results, the clinical pathologist suggested to perform a venous blood gas analysis and electrocardiogram (ECG) which revealed a normal potassium of 3.7 mmol/L and normal ECG, ruling out a potentially life-treating hyperkalaemia. The child was diagnosed with pneumonia. The paediatrician had difficulty to perform the first venous blood collection due to excessive movement of the infant during venipuncture. The muscle contractions of the child in combination with venous stasis most probably led to a local increase of potassium in the sampled limbs. The second sample collected under optimal preanalytical circumstances had a normal potassium. Since muscle contraction typically does not cause severe hyperkalaemia, other causes of pseudohyperkalaemia were excluded. K3-EDTA contamination and familial hyperkalaemia were ruled out and the patient did not have extreme leucocytosis or thrombocytosis. By exclusion a diagnosis of pseudohyperkalaemia due to intense muscle movement and venous stasis was made. Conclusion: This case suggests that intense muscle contraction and venous stasis can cause severe pseudohyperkalemia without hemolysis. Once true hyperkalemia has been ruled out, a laboratory work-up can help identify the cause of pseudohyperkalaemia

Delayed diagnosis and treatment of extreme hypertriglyceridemia due to rejection of a lipemic sample

Most laboratories routinely determine haemolysis, icterus and lipemia indices to identify lipemic samples and reject potentially affected results. Hypertriglyceridemia is the most common cause of lipemia and severe hypertriglyceridemia (≥ 11.3 mmol/L) is a major risk factor of acute pancreatitis. A 56-year-old woman attended the outpatient clinic for a follow-up visit 1 month after a kidney transplantation. Her immunosuppressive therapy consisted of corticosteroids, cyclosporine, and mycophenolic acid. The routine clinical chemistry sample was rejected due to extreme lipemia. The comment “extreme lipemic sample” was added on the report, but the requesting physician could not be reached. The Cobas 8000 gave a technical error (absorption > 3.3) for the HIL-indices (L-index: 38.6 mmol/L) which persisted after high-speed centrifugation. The patient was given a new appointment 2 days later. The new sample was also grossly lipemic and gave the same technical error (L-index: 35.9 mmol/L). The second sample was manually diluted 20-fold after centrifugation to obtain a result for triglycerides within the measuring range (0.10–50.0 mmol/L). Triglycerides were 169.1 mmol/L, corresponding to very severe hypertriglyceridemia. This result was communicated to the nephrologist and the patient immediately recalled to the hospital. She received therapeutic plasma exchange the next day and did not develop acute pancreatitis. This case illustrates the delicate balance between avoiding the release of unreliable results due to lipemia and the risk of delayed diagnosis when results are rejected. Providing an estimate of the degree of hypertriglyceridemia might be preferable to rejecting the result

Case report: Myocarditis in congenital STAT1 gain-of function

Autosomal dominant Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in an inborn error of immunity characterized by chronic mucocutaneous candidiasis, recurrent viral and bacterial infections, and diverse autoimmune manifestations. Current treatment consists of chronic antifungal therapy, antibiotics for concomitant infections, and immunosuppressive therapy in case of autoimmune diseases. More recently, treatment with Janus kinases 1 and 2 (JAK1/2) inhibitors have shown promising yet variable results. We describe a STAT1 GOF patient with an incidental finding of elevated cardiac troponins, leading to a diagnosis of a longstanding, slowly progressive idiopathic myocarditis, attributed to STAT1 GOF. Treatment with a JAK-inhibitor (baricitinib) mitigated cardiac inflammation on MRI but was unable to alter fibrosis, possibly due to the diagnostic and therapeutic delay, which finally led to fatal arrhythmia. Our case illustrates that myocarditis could be part of the heterogeneous disease spectrum of STAT1 GOF. Given the insidious presentation in our case, a low threshold for cardiac evaluation in STAT1 GOF patients seems warranted

Unraveling defects in the human innate immune system: Diagnosis of inherited disorders in the NF-κB pathway

Primary immunodeficiencies are inherited diseases that predispose children and adults to recurrent bacterial, viral, and/or fungal infections. The awareness for primary immunodeficiencies is still limited in routine clinical practice and many patients are diagnosed too late to prevent life-threatening infections by appropriate therapy. Toll-like/IL-1R (TIR) receptors and the associated NF-κB signaling pathway are important mediators of the innate immune response. Understanding their function in human cells is necessary as defects lead to invasive pyogenic bacterial infections, herpes simplex encephalitis, common variable immunodeficiency, chronic mucocutaneous candidiasis, and combined immunodeficiency. In the first part of the study, we determined the value of TLR/NF-κB-specific functional assays to diagnose these disorders. In Chapter 1, we studied induction of IL-6 by PBMCs after 24 hour stimulation with various TIR ligands in 770 patients presenting with increased susceptibility to infection. We established cut-off values for IL-6 induction by PBMCs after stimulation with various TIR ligands by applying the Bhattacharya algorithm. Application of the established cut-off values resulted in the identification of 22 patients with impaired induction of IL-6 after TIR stimulation, including one IRAK-4 deficient and two NEMO-ID patients. The other 19 patients presented with a phenotype reminiscent of a mild IRAK-4 or MyD88 deficiency, but without mutations in these genes. Chapter 2 focused on the development of a rapid flow cytometric assay that can screen for genetic defects in the canonical NF-κB pathway. In terms of validation, this assay based on the evaluation of IκB-α degradation can already be used to screen for IRAK-4, MyD88-, and CARD11-deficiencies. It remains to be determined how patients with other defects in the canonical NF-κB pathway respond (study still ongoing in collaboration with Hôpital Necker, Paris). Despite the usefulness of functional assays, genetic analysis is still the only way to accurately establish a diagnosis of NF-κB immunodeficiencies. In the second part, we explored the currently available genetic methods that can be used to identify disease-causing mutations in the innate immune system. In Chapter 3, we set up a highly reliable parallel resequencing technique to sequence IKBKG, IRAK4, and MYD88 for several patients in one run by comparing Single Molecule Real Time (SMRT) sequencing on the PacBio RSII (Pacific Biosciences) with Sequencing-by-Synthesis on the Hiseq 2500 (Illumina). We targeted IKBKG specifically as it is one of the genes with insufficient coverage due to the presence of the highly homologous pseudogene IKBKGP1. Chapter 4 describes three patients who obtained a genetic diagnosis through Sanger sequencing. We describe two patients in whom an IKBKG mutation was found. In a third patient, carrying no IKBKG variants, a gain-of-function STAT1 mutation was identified. We studied pathologic impact of the identified mutations and their contribution to the immunological phenotype in all three cases. Chapter 5 describes the next-generation sequencing application of an array-based panel covering 174 PID genes that has been implemented in our center since 2014. This technique was used to diagnose IRAK-4 deficiency in an adult woman who was originally thought to suffer from autosomal dominant hyper-IgE syndrome. In Chapter 6, patients in whom an innate immune defect was suspected, were subjected to whole exome sequencing (WES) to discover yet unappreciated disease-causing gene candidates. A total of 25 patients with recurrent pyogenic bacterial infections were evaluated with WES. In five of them, mutations were found in the CVID-associated TNFRSF13B gene. Two patients with an unknown syndromic immunodeficiency were also subjected to whole exome sequencing. The first patient suffered from X-linked Kabuki syndrome caused by a novel hemizygous de novo mutation in KDM6A. A homozygous mutation in TRNT1 was identified in the second patient leading to a severe immunodeficiency characterized by gastro-intestinal inflammation and B-cell deficiency. In conclusion, we evaluated diagnostic strategies that can be used in the clinical and genetics laboratory for a specific subset of primary immunodeficiencies affecting the innate immune system.status: publishe

Mild humoral immunodeficiency in a patient with X-linked Kabuki syndrome

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Migrating a lab-developed MERS-CoV real-time PCR to 3 "Sample to Result" systems: experiences on optimization and validation

The goal of the study was to adapt our Middle East respiratory syndrome coronavirus (MERS-CoV) lab-developed test (LDT) to 3 "Sample to Result" (S2R) systems: BD MAX (BD), ELITe InGenius (ELITechGroup), and ARIES (Luminex). The BD MAX and InGenius system allowed use of lab-developed primers and TaqMan probes, while ARIES required conversion to MultiCode primers for melting curve analysis. Each device required ≤1 day of training and assay optimization. No discordant results were noted after analysis of 32 External Quality Control (EQC) samples. On a 10-fold dilution series of a MERS-CoV-positive EQC sample, InGenius obtained the highest detection rate. Laboratory technicians rated the ARIES as the user-friendliest. It also required the least hands-on time. BD MAX had the lowest turnaround time and highest throughput. While each device had distinguishing system properties with associated (dis)advantages, the 3 S2R systems were comparable in terms of assay development and validation.status: publishe

How to meet ISO15189:2012 pre-analytical requirements in clinical laboratories? A consensus document by the EFLM WG-PRE

The International Organization for Standardization (ISO) 15189:2012 standard aims to improve quality in medical laboratories through standardization of all key elements in the total testing process, including the pre-analytical phase. It is hence essential that accreditation bodies, assessing laboratories against ISO15189:2012, pay sufficient attention to auditing pre-analytical activities. However, there are significant differences in how technical auditors interpret the pre-analytical requirements described in ISO15189:2012. In this consensus document, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Pre-analytical Phase (WG-PRE) sets out to review pre-analytical requirements contained in ISO15189:2012 and provide guidance for laboratories on how to meet these requirements. The target audience for this consensus document is laboratory professionals who wish to improve the quality of the pre-analytical phase in their laboratory. For each of the ISO requirements described in ISO15189:2012, members of EFLM WG-PRE agreed by consensus on minimal recommendations and best-in-class solutions. The minimal consensus recommendation was defined as the minimal specification which laboratories should implement in their quality management system to adequately address the pre-analytical requirement described in ISO15189:2012. The best-in-class solution describes the current state-of-the-art in fulfilling a particular pre-analytical requirement in ISO15189:2012. We fully acknowledge that not every laboratory has the means to implement these best-in-class solutions, but we hope to challenge laboratories in critically evaluating and improving their current procedures by providing this expanded guidance

Identification of two new gain-of-function STAT1 mutations in the DNA-binding domain

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