An appraisal of the practice of duplicate testing for the detection of irregular analytical errors.

OBJECTIVES: Our study aimed to determine the usefulness of duplicate testing in identifying irregular analytical errors and subsequent prevention of patient mismanagement. METHODS: In our laboratory, all requests for Na+, Ca2+, alkaline phosphatase (ALP), and high-sensitivity cardiac-troponin-I (hs-cTnI) are run in duplicate. Data from four separate weeks for Na+ (n=21,649), Ca2+ (n=14,803) and ALP (n=19,698); and a full year for hs-cTnI (n=17,036) were gathered. For each test, pre-defined limits for differences between duplicates were used to identify erroneous results (Fliers). We further characterised a subset of such fliers as "critical errors", where duplicates fell on opposing sides of a reference/decision making threshold. The costs/benefits of running these tests in duplicate were then considered in light of increased number of tests analysed by this approach. RESULTS: For Na+, 0.03 % of duplicates met our flier defining criteria, and 0.01 % of specimens were considered critical errors. For Ca2+ requests, 4.58 % of results met our flier defining criteria and 0.84 % were critical errors. For ALP, 0.22 % of results were fliers, and 0.01 % were critical errors. For hs-cTnI, 1.58 % of results were classified as fliers, whilst 0.14 % were classified as a critical error. Depending on the test in question, running all analyses in duplicate increased annual costs by as little as €1,100 (for sodium), and as much as €48,000 (for hs-cTnI). CONCLUSIONS: Duplicate testing is effective at identifying and mitigating irregular laboratory errors, and is best suited for assays predisposed to such error, where costs are minimal, and clinical significance of an incorrect result can justify the practice.

Adverse effects of cannabidiol: a systematic review and meta-analysis of randomized clinical trials.

Cannabidiol (CBD) is being investigated as a treatment for several medical disorders but there is uncertainty about its safety. We conducted the first systematic review and meta-analysis of the adverse effects of CBD across all medical indications. Double-blind randomized placebo-controlled clinical trials lasting ≥7 days were included. Twelve trials contributed data from 803 participants to the meta-analysis. Compared with placebo, CBD was associated with an increased likelihood of withdrawal for any reason (OR 2.61, 95% CI: 1.38-4.96) or due to adverse events (OR 2.65, 95% CI: 1.04-6.80), any serious adverse event (OR 2.30, 95% CI: 1.18-4.48), serious adverse events related to abnormal liver function tests (OR 11.19, 95% CI: 2.09-60.02) or pneumonia (OR 5.37, 95% CI: 1.17-24.65), any adverse event (OR 1.55, 95% CI: 1.03-2.33), adverse events due to decreased appetite (OR 3.56, 95% CI: 1.94-6.53), diarrhoea (OR 2.61, 95% CI: 1.46-4.67), somnolence (OR 2.23, 95% CI: 1.07-4.64) and sedation (OR 4.21, 95% CI: 1.18-15.01). Associations with abnormal liver function tests, somnolence, sedation and pneumonia were limited to childhood epilepsy studies, where CBD may have interacted with other medications such as clobazam and/or sodium valproate. After excluding studies in childhood epilepsy, the only adverse outcome associated with CBD treatment was diarrhoea (OR 5.03, 95% CI: 1.44-17.61). In summary, the available data from clinical trials suggest that CBD is well tolerated and has relatively few serious adverse effects, however interactions with other medications should be monitored carefully. Additional safety data from clinical trials outside of childhood epilepsy syndromes and from studies of over-the-counter CBD products are needed to assess whether the conclusions drawn from clinical trials can be applied more broadly.

Investigation of the long-term yield of auditing for conformity with the ISO 15189:2012 quality standard in a hospital pathology laboratory.

INTRODUCTION: Pathology laboratories are increasingly seeking accreditation to quality standards to assure Quality of Service (QoS). However, there is little data available regarding the value of this in laboratories with well-established Quality Management Systems (QMS). Moreover, critics of accreditation claim it redirects resources toward trivial issues. Our objective was to investigate the value of auditing for conformity with the ISO 15189:2012 standard in such laboratories. DESIGN: and Methods: In total, 483 Audit-Identified Non-Conformities (AINCs) were documented within our department since transitioning to an ISO 15189:2012 compliant QMS. The potential consequences of these were assessed by three clinical laboratorians who assigned them into categories based on their likely impact. These were: Unlikely (no clear consequences); Possible (potential for poor QoS/harm); and Probable (Likely to cause poor QoS/harm). Additionally, total numbers/severity of Real-Time Non-Conformities (RTNCs) detected outside of auditing were examined to provide additional insight into the effects of accreditation on QoS. RESULTS: According to majority decision: 395 (81.8%) of AINCs were classified Unlikely, 88 (18.2%) were Possible, and none were Probable. The relative proportion of Unlikely AINCs also rose over time. Total numbers and severity of RTNCs dropped in the short-term following transition to an ISO 15189:2012 QMS, but steadily rose thereafter. CONCLUSIONS: Our data suggest auditing for conformity with ISO 15189:2012 standards may be effective in attaining accreditation, but may have diminishing returns in the long-term once the QMS is established, unless there is continual improvement in the audit process to promote better use of resources.

Closure beyond clozapine: successfully averting rebound symptoms in a patient with schizoaffective disorder and agranulocytosis.

SUMMARY: 'Rebound' or 'withdrawal' symptoms are frequently observed after a sudden discontinuation of clozapine. We describe a patient with treatment-resistant schizoaffective disorder who developed agranulocytosis on clozapine but was successfully switched to treatment with olanzapine with no deterioration in her condition. We put forward three possible theories which may have accounted for the lack of rebound symptoms in this patient: the pharmacological profile of olanzapine, the anticholinergic effects of hyoscine hydrobromide, and the possibility that this patient may not be treatment-resistant and so have a reduced risk of rebound psychosis due to displaying a different pathophysiology. DECLARATION OF INTEREST: None.

Implanting 1.1B4 human ß-cell pseudoislets improves glycaemic control in diabetic severe combined immune deficient mice.

AIM: To investigate the potential of implanting pseudoislets formed from human insulin-releasing ß-cell lines as an alternative to islet transplantation. METHODS: In this study, the anti-diabetic potential of novel human insulin releasing 1.1B4 ß-cells was evaluated by implanting the cells, either as free cell suspensions, or as three-dimensional pseudoislets, into the subscapular region of severe combined immune deficient mice rendered diabetic by single high-dose administration of streptozotocin. Metabolic parameters including food and fluid intake, bodyweight and blood glucose were monitored throughout the study. At the end of the study animals were given an intraperitoneal glucose tolerance test. Animals were then culled and blood and tissues were collected for analysis. Insulin and glucagon contents of plasma and tissues were measured by insulin radioimmunoassay and chemiluminescent enzyme-linked immunosorbance assay respectively. Histological analyses of pancreatic islets were carried out by quantitative fluorescence immunohistochemistry staining. RESULTS: Both pseudoislet and cell suspension implants yielded well vascularised ß-cell masses of similar insulin content. This was associated with progressive amelioration of hyperphagia (P < 0.05), polydipsia (P < 0.05), body weight loss (P < 0.05), hypoinsulinaemia (P < 0.05), hyperglycaemia (P < 0.05 - P < 0.001) and glucose tolerance (P < 0.01). Islet morphology was also significantly improved in both groups of transplanted mice, with increased ß-cell (P < 0.05 - P < 0.001) and decreased alpha cell (P < 0.05 - P < 0.001) areas. Whereas mice receiving 1.1B4 cell suspensions eventually exhibited hypoglycaemic complications, pseudoislet recipients displayed a more gradual amelioration of diabetes, and achieved stable blood glucose control similar to non-diabetic mice at the end of the study. CONCLUSION: Although further work is needed to address safety issues, these results provide proof of concept for possible therapeutic applicability of human ß-cell line pseudoislets in diabetes.

Functionality and antidiabetic utility of ß- and L-cell containing pseudoislets.

Unavailability of tissue and poor engraftment remain significant obstacles to clinical islet transplantation. Here, the therapeutic potential of pseudoislets generated from the insulin and GLP-1 releasing cell-lines MIN6 and GLUTag was investigated. Glucose and other secretagogues evoked 1.3-5.7 fold increases in insulin secretion from both pseudoislet types. Secretion expressed in relation to basal values did not greatly differ between configurations. Exposure of both types of pseudoislets to ninhydrin, H2O2, streptozotocin or cytokine cocktails decreased viability and increased apoptosis. However, combined pseudoislets exhibited enhanced resistance (1.2-1.7 fold increased LD50, 1.2-1.4 fold decreased apoptosis). Implantation of pseudoislets into streptozotocin-diabetic SCID mice precipitated cell masses containing immunoreactive insulin and GLP-1. Implantation of both pseudoislet types was associated with significant reductions in blood glucose, increased plasma insulin, greater bodyweight, decreased polydipsia and improved glucose tolerance. These changes greatly exaggerated in MIN6 pseudoislet recipients, with mice becoming severely hypoglycaemic. In contract, combined pseudoislet recipients achieved tempered restoration of normoglycaemia and exhibited increased plasma GLP-1, decreased plasma and pancreatic glucagon, increased pancreatic insulin and enhancements in islet ß:α cells and the ratio of Ki67: TUNEL positive ß-cells. MIN6 pseudoislet implantation increased islet ß:α cell ratio but did not affect ß-cell proliferation or hormone content. Our observations highlight the potential of combining insulin and GLP-1 cell therapy using heterotypic pseudoislets.

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors.

Pseudoislet formation enhances gene expression, insulin secretion and cytoprotective mechanisms of clonal human insulin-secreting 1.1B4 cells.

We have studied the effects of cell communication on human beta cell function and resistance to cytotoxicity using the novel human insulin-secreting cell line 1.1B4 configured as monolayers and pseudoislets. Incubation with the incretin gut hormones GLP-1 and GIP caused dose-dependent stimulation of insulin secretion from 1.1B4 cell monolayers and pseudoislets. The secretory responses were 1.5-2.7-fold greater than monolayers. Cell viability (MTT), DNA damage (comet assay) and apoptosis (acridine orange/ethidium bromide staining) were investigated following 2-h exposure of 1.1B4 monolayers and pseudoislets to ninhydrin, H2O2, streptozotocin, glucose, palmitate or cocktails of proinflammatory cytokines. All agents tested decreased viability and increased DNA damage and apoptosis in both 1.1B4 monolayers and pseudoislets. However, pseudoislets exhibited significantly greater resistance to cytotoxicity (1.5-2.7-fold increases in LD50) and lower levels of DNA damage (1.3-3.4-fold differences in percentage tail DNA and olive tail moment) and apoptosis (1.3-1.5-fold difference) compared to monolayers. Measurement of gene expression by reverse-transcription, real-time PCR showed that genes involved with insulin secretion (INS, PDX1, PCSK1, PCSK2, GLP1R and GIPR), cell-cell communication (GJD2, GJA1 and CDH1) and antioxidant defence (SOD1, SOD2, GPX1 and CAT) were significantly upregulated in pseudoislets compared to monolayers, whilst the expression of proapoptotic genes (NOS2, MAPK8, MAPK10 and NFKB1) showed no significant differences. In summary, these data indicate cell-communication associated with three-dimensional islet architecture is important both for effective insulin secretion and for protection of human beta cells against cytotoxicity.