Adie Viljoen

Analytical error and interference in immunoassay: minimizing risk

Although generally robust, immunoassays remain vulnerable to occasional analytical errors that may have serious implications for patient care. Sporadic errors that occur as a result of properties of the specimen are particularly difficult to detect. They may be due to the presence of cross-reacting substances, antianalyte antibodies or antireagent antibodies, all of which may lead to erroneously high or low results. Low results may be observed for tumour markers due to high-dose hooking in the presence of very high analyte concentrations. Erroneous results can occur unexpectedly with any specimen and there is no practical means of identifying specimens likely to cause problems in immunoassays. The possibility of interference should always be considered when results do not appear to be in accord with the clinical picture. Errors can occur in even the best-managed laboratories and their early investigation is always desirable. If there is any doubt whatsoever about a result, clinical staff should be encouraged to contact the laboratory. Investigations for possible interference that can be undertaken in most laboratories include testing for linearity on dilution, recovery experiments, treatment with heterophilic blocking tubes and confirmation using a different method. It may be desirable to consult specialist laboratories if more complex studies are necessary. Informing clinical and laboratory staff of the ever-present possibility of unexpected interference, ensuring brief clinical details are available to laboratory staff, and above all facilitating excellent communication between laboratory and clinical staff are key to minimizing the risk of clinical mismanagement due to unsuspected interference.

New lipid-lowering drugs: an update

Lipid lowering is established as a proven intervention to reduce atherosclerosis and its complications. Statins form the basis of care but are not able to treat all aspects of dyslipidaemia. Many novel therapeutic compounds are being developed. These include additional therapeutics for low‐density lipoprotein cholesterol, for example, thyroid mimetics (thyroid receptor beta‐agonists), antisense oligonucleotides or microsomal transfer protein inhibitors (MTPI); triglycerides, for example, novel peroxosimal proliferator activating receptors agonists, MTPIs, diacylglycerol acyl transferase‐1 inhibitors and high‐density lipoprotein cholesterol (HDL‐C), for example, mimetic peptides; HDL delipidation strategies and cholesterol ester transfer protein inhibitors and modulators of inflammation, for example, phospholipase inhibitors. Gene therapy for specific rare disorders, for example, lipoprotein lipase deficiency using alipogene tiparvovec is also in clinical trials. Lipid‐lowering drugs are likely to prove a fast‐developing area for novel treatments as possible synergies exist between new and established compounds for the treatment of atherosclerosis.

Inhibition of pre-protein convertase serine kexin-9 (PCSK-9) as a treatment for hyperlipidaemia

Introduction: Pre-protein convertase subtilisin kexin (PCSK)-9 is a newly discovered protein involved in intracellular and extracellular regulation of low-density lipoprotein receptor (LDLR) expression. Autosomal dominant activating mutations in PCSK-9 cause familial hypercholesterolaemia whereas inactivating mutations in man reduce LDL cholesterol (LDL-C) and are associated with a decreased lifetime risk of cardiovascular events. Areas covered: As PCSK-9 binds to the LDLR, a number of approaches involving small molecule or peptide inhibition of binding, antibody-mediated inactivation of binding and the use of antisense oligonucleotides are being investigated as therapeutic approaches to lower LDL-C in man. This article reviews the biochemistry and physiology of PCSK-9 and details the efforts made to design novel molecules with the ability to inhibit PCSK-9 activity. Work in animal models has confirmed that reducing PCSK-9 expression can reduce atherosclerosis in mice, rats and primates. Monoclonal antibodies such as REGN-727 and AMG-145 have been shown to reduce LDL-C in patients with familial hypercholesterolaemia already treated with statins or healthy normocholesterolaemic controls. Expert opinion: PCSK-9 inhibition is a potentially interesting novel addition to the armamentarium of LDL-C reducing drugs. Its effects in reducing LDL-C will need to be confirmed to reduce CVD events in large-scale clinical trials.

Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes (SUSTAIN 7): a randomised, open-label, phase 3b trial

BACKGROUND Despite common mechanisms of actions, glucagon-like peptide-1 receptor agonists differ in structure, pharmacokinetic profile, and clinical effects. This head-to-head trial compared semaglutide with dulaglutide in patients with inadequately controlled type 2 diabetes. METHODS This was an open-label, parallel-group, phase 3b trial done at 194 hospitals, clinical institutions or private practices in 16 countries. Eligible patients were aged 18 years or older and had type 2 diabetes with HbA1c 7·0-10·5% (53·0-91·0 mmol/mol) on metformin monotherapy. Patients were randomly assigned (1:1:1:1) by use of an interactive web-response system to once a week treatment with either semaglutide 0·5 mg, dulaglutide 0·75 mg, semaglutide 1·0 mg, or dulaglutide 1·5 mg subcutaneously. The primary endpoint was change from baseline in percentage HbA1c; the confirmatory secondary endpoint was change in bodyweight, both at week 40. The primary analysis population included all randomly assigned patients exposed to at least one dose of trial product obtained while on treatment and before the onset of rescue medication. The safety population included all randomly assigned patients exposed to at least one dose of trial product obtained while on treatment. The trial was powered for HbA1c non-inferiority (margin 0·4%) and bodyweight superiority. This trial is registered with ClinicalTrials.gov, number NCT02648204. FINDINGS Between Jan 6, 2016, and June 22, 2016, 1201 patients were randomly assigned to treatment; of these, 301 were exposed to semaglutide 0·5 mg, 299 to dulaglutide 0·75 mg, 300 to semaglutide 1·0 mg, and 299 to dulaglutide 1·5 mg. 72 (6%) patients withdrew from the trial (22 receiving semaglutide 0·5 mg, 13 receiving dulaglutide 0·75 mg, 21 receiving semaglutide 1·0 mg, and 16 receiving dulaglutide 1·5 mg). From overall baseline mean, mean percentage HbA1c was reduced by 1·5 (SE 0·06) percentage points with semaglutide 0·5 mg versus 1·1 (0·05) percentage points with dulaglutide 0·75 mg (estimated treatment difference [ETD] -0·40 percentage points [95% CI -0·55 to -0·25]; p<0·0001) and by 1·8 (0·06) percentage points with semaglutide 1·0 mg versus 1·4 (0·06) percentage points with dulaglutide 1·5 mg (ETD -0·41 percentage points [-0·57 to -0·25]; p<0·0001). From overall baseline mean, mean bodyweight was reduced by 4·6 kg (SE 0·28) with semaglutide 0·5 mg compared with 2·3 kg (0·27) with dulaglutide 0·75 mg (ETD -2·26 kg [-3·02 to -1·51]; p<0·0001) and by 6·5 kg (0·28) with semaglutide 1·0 mg compared with 3·0 kg (0·27) with dulaglutide 1·5 mg (ETD -3·55 kg [-4·32 to -2·78]; p<0·0001). Gastrointestinal disorders were the most frequently reported adverse event, occurring in 129 (43%) of 301 patients receiving semaglutide 0·5 mg, 133 (44%) of 300 patients receiving semaglutide 1·0 mg, 100 (33%) of 299 patients receiving dulaglutide 0·75 mg, and in 143 (48%) of 299 patients receiving dulaglutide 1·5 mg. Gastrointestinal disorders were also the most common reason for discontinuing treatment with semaglutide and dulaglutide. There were six fatalities: one in each semaglutide group and two in each dulaglutide group. INTERPRETATION At low and high doses, semaglutide was superior to dulaglutide in improving glycaemic control and reducing bodyweight, enabling a significantly greater number of patients with type 2 diabetes to achieve clinically meaningful glycaemic targets and weight loss, with a similar safety profile. FUNDING Novo Nordisk.

Triglycerides: a case for treatment?

Purpose of review To discuss the relevance of triglycerides to cardiovascular disease (CVD) risk. Recent findings Triglycerides are a commonly measured component of lipid profiles. Raised triglycerides are a component of the metabolic syndrome and are strongly associated with future risk of diabetes as well as cardiovascular disease. Triglyceride-rich particles form a component of cardiovascular risk above that delineated by low density lipoprotein (LDL) cholesterol. Elevated triglycerides are a marker of atherogenic small dense LDL, excess baseline and residual CVD risk even after statin therapy. Additional methods to lower triglycerides include niacin, fibrates and omega-3 fatty acids. Trials in monotherapy with both niacin and fibrates suggest some benefit in reducing CVD events based on evidence mostly derived from older studies. However, endpoint trials of adding either niacin or fenofibrate to statins have not shown any benefit, except possibly in patients with an increased atherogenic index (triglyceride : HDL-C ratio), or have been underpowered. Trials of omega-3 fatty acids have been performed at doses insufficient to affect lipid profiles in populations with inadequate control of LDL-C but did reduce CVD events. Summary Further trials of lipid-lowering agents beyond statins will be required in patients with LDL-C adequately controlled on statin therapy.

Alipogene tiparvovec: gene therapy for lipoprotein lipase deficiency.

Homozygous lipoprotein lipase (LPL) deficiency is an ultra-orphan disease associated with increased rates of pancreatitis. Current treatments based on acute plasmapheresis allied with ultra-low fat diets are inadequate as responses to fibrates or other triglyceride-lowering therapies tend to be poor. Alipogene tiparvovec is an adeno-associated virus type I (AAV1) gene therapy using a hyper-functional LPL serine447-stop (S447X) insert administered intramuscularly under general anaesthetic with allied immunosuppression. Treatment results in histological muscle expression of LPL allied with a transient 40% reduction in triglycerides and improvements in postprandial chylomicron triglyceride content. Alipogene tiparvovec is the first possibly curative treatment for LPL deficiency.

Daily and intermittent rosuvastatin 5 mg therapy in statin intolerant patients: an observational study

Abstract Objective: To examine the efficacy and tolerability of rosuvastatin 5 mg at daily and non-daily dosing regimens. Research design and methods: A retrospective survey was conducted at nine primary, secondary and tertiary healthcare centres in the United Kingdom. Main outcome measures: Changes in lipid fractions from baseline values after more than 3 months’ treatment. Results: A total of 325 patients were identified. These patients were aged 63 ± 10 years, 50% male and prescription was mostly for primary prevention of cardiovascular disease (CVD) (59%). Co-morbidities included: established CVD present in 41%, type 2 diabetes mellitus (15%), hypertension (74%) and smoking (9%). Adverse effects had been documented to simvastatin (75%) or atorvastatin (63%). A total of 289 patients (89%) tolerated rosuvastatin well and were still adherent after a median follow-up of 14.9 (3–79) months. The remainder (n = 36; 11%) discontinued the medication after median 5 months’ treatment due to adverse effects. Efficacy was assessed in 224 patients who had adequate data. Baseline lipids were total cholesterol (TC) 7.41 ± 1.50 mmol/L, triglycerides (TG) 2.26 (range 0.36–18.4) mmol/L; high density lipoprotein cholesterol (HDL-C) 1.43 ± 0.47 mmol/L and low density lipoprotein cholesterol (LDL-C) 4.76 ± 1.38 mmol/L. Daily rosuvastatin (n = 134) reduced mean TC by 31%, TG 15% and LDL-C 43% (p < 0.001). Rosuvastatin 5 mg 2–3 times weekly (n = 79) reduced TC 26%, TG 16% and LDL-C 32% (p < 0.001). Weekly rosuvastatin (n = 11) reduced TC 17%, LDL-C by 23% (p < 0.001) but had no effect on TGs. Targets were attained in 17% of CHD-risk equivalent patients and 41% of primary prevention patients by National Cholesterol Education Program criteria and 27% and 68% using UK targets. No myositis or rhabdomyolysis was observed and alanine aminotransferase (ALT) and creatine kinase (CK) were similar to baseline. Conclusions: In this retrospective observational multicentre study, rosuvastatin 5 mg was found to be safe and biochemically effective either as daily or intermittent therapy in patients intolerant to other conventional statin regimens.

Analytical quality goals for 25-vitamin D based on biological variation.

Background: Measurement of 25‐hydroxyvitamin D, (25D) is central in the investigation of pathologies of bone and mineral ion metabolism and in determining a patients vitamin D status. More recently much research interest has lead to investigating the role it can play in decreasing the risk of many chronic illnesses, including common cancers, autoimmune diseases, infectious diseases, and cardiovascular disease. Knowledge of the biological variation of an analyte forms an essential part of evaluating a new analyte enabling the objective assessment of the changes in serial results, the utility of reference intervals as well as establishing laboratory quality specifications. Methods: This study determined the biological variation of 25D in 20 healthy individuals that was calculated according to the familiar methods outlined by Fraser and Harris. Results: The within‐subject variation was 12.1% and the between subject variation was 40.3%. The critical difference for sequential values significant at P<0.05 was calculated as 38.4%. The within‐subject variation forms a relatively small part of the reference interval shown by the low index of individuality of 0.3. Objective analytical quality goals have also been established which have shown achievable minimum performance for imprecision of ∼6%. The desirable analytical bias goal was ∼10%. Conclusion: This study has objectively shown that the analytical precision of current instruments is being achieved contrary to the known problems surrounding the analytical bias for 25D assays. The limitations of using reference intervals for 25D, both in diagnoses and monitoring are shown. J. Clin. Lab. Anal. 25:130–133, 2011.

Copper:caeruloplasmin ratio

Investigation of copper status can be a diagnostic challenge. The non-caeruloplasmin-bound copper (NCC) has deficiencies; accordingly, the copper:caeruloplasmin ratio has been suggested as an alternative index of copper status. A reference interval for this index was derived. In addition to making the interpretation of copper easier, the copper:caeruloplasmin ratio should also enable adjustment for relatively high caeruloplasmin concentrations without recourse to producing gender- and age-derived intervals. The copper:caeruloplasmin ratio has weaknesses similar to those identified for NCC in that immunological methods used for caeruloplasmin can cross react with apocaeruloplasmin and there is no standardised method for caeruloplasmin. Caeruloplasmin assays also have uncertainty from precision, bias and specificity and, accordingly, method-related differences may have a large effect on the copper:caeruloplasmin ratio in a manner similar to the NCC.

Diagnosis and treatment of severe hypertriglyceridemia.

Severe hypertriglyceridemia is associated with acute pancreatitis and can be a manifestation of lipoprotein lipase (LPL) deficiency. It is associated with a spectrum of disorders, ranging from heterozygous LPL deficiency allied with environmental factors to rare severe cases of homozygous LPL deficiency. The genes associated with reduced LPL activity include LPL, its cofactor apoC-2, a controlling protein apoA-5 and the LPL receptor GPI-HBP1. The effects of mutations are exacerbated by environmental factors such as diet, pregnancy and insulin resistance. Treatment of clinical LPL deficiency is by ultra-low-fat diet along with the use of fibrates, omega-3 fatty acids, niacin, statins and insulin-sensitizing therapies, depending on the extent of residual LPL activity. Novel therapies that target lipoprotein particle assembly through the antisense oligonucleotides or by interference with triglyceride-loading microsomal transport protein inhibitors offer new potential options for treating hypertriglyceridemia.