The insulin autoimmune syndrome (IAS) as a cause of hypoglycaemia: an update on the pathophysiology, biochemical investigations and diagnosis.

Insulin autoimmune syndrome (IAS) is considered to be very rare in Caucasians. Understanding its pathophysiology is paramount in (a) appreciating its potential impact on analyses of pancreatic hormones and (b) explaining its highly variable clinical manifestations in non-diabetic, non-acutely ill patients with indeterminate hypoglycaemia. The underlying aetiology of IAS is the presence of variable affinity/avidity endogenous insulin antibodies in significant amounts. The two types of insulin antibodies namely antibodies which bind insulin and/or proinsulin(s) and receptor antibodies (insulin mimetic) will be discussed. Their biochemical and immunological roles in causing hypoglycaemia will be highlighted. Clinical manifestations of IAS can vary from mild and transient to spontaneous, severe and protracted hypoglycaemia necessitating in extreme cases plasmapheresis for glycaemic control. Antibodies of IAS can interfere in pancreatic immunoassay tests causing erroneous and potentially misleading results. Thorough testing for endogenous insulin antibodies must be considered in the investigations of non-diabetic, non-acutely ill patients with indeterminate and/or unexplained hypoglycaemia.

Identifying and reducing potentially wrong immunoassay results even when plausible and "not-unreasonable".

The primary role of the clinical laboratory is to report accurate results for diagnosis of disease and management of illnesses. This goal has, to a large extent been achieved for routine biochemical tests, but not for immunoassays which remained susceptible to interference from endogenous immunoglobulin antibodies, causing false, and clinically misleading results. Clinicians regard all abnormal results including false ones as "pathological" necessitating further investigations, or concluding iniquitous diagnosis. Even more seriously, "false-negative" results may wrongly exclude pathology, thus denying patients' necessary treatment. Analytical error rate in immunoassays is relatively high, ranging from 0.4% to 4.0%. Because analytical interference from endogenous antibodies is confined to individuals' sera, it can be inconspicuous, pernicious, sporadic, and insidious because it cannot be detected by internal or external quality assessment procedures. An approach based on Bayesian reasoning can enhance the robustness of clinical validation in highlighting potentially erroneous immunoassay results. When this rational clinical/statistical approach is followed by analytical affirmative follow-up tests, it can help identifying inaccurate and clinically misleading immunoassay data even when they appear plausible and "not-unreasonable." This chapter is largely based on peer reviewed articles associated with and related to this approach. The first section underlines (without mathematical equations) the dominance and misuse of conventional statistics and the underuse of Bayesian paradigm and shows that laboratorians are intuitively (albeit unwittingly) practicing Bayesians. Secondly, because interference from endogenous antibodies is method's dependent (with numerous formats and different reagents), it is almost impossible to accurately assess its incidence in all differently formulated immunoassays and for each analytes/biomarkers. However, reiterating the basic concepts underpinning interference from endogenous antibodies can highlight why interference will remain analytically pernicious, sporadic, and an inveterate problem. The following section discuses various stratagems to reduce this source of inaccuracy in current immunoassay results including the role of Bayesian reasoning. Finally, the role of three commonly used follow-up affirmative tests and their interpretation in confirming analytical interference is discussed.

Deceptive potassium and magnesium measurements.

Potassium and magnesium are important circulating cations and are predominantly intracellular elements. Only a small fraction of these elements is present in extracellular fluids including blood (∼1%). Measurement of the concentration of such small fractions in blood is commonly used to assess and reflect their body content levels. However, some of these measurements can be flawed and a failure to recognise the limitations of these tests may result in misdiagnosis and/or unnecessary follow-up investigations and/or expensive hospital admissions. The focus of this note is three-fold (a) to highlight and discuss separately the less appreciated pitfalls of potassium and magnesium measurements per se, (b) suggestions to identify and rectify these snags and to improve their clinical interpretation, and finally

Probabilistic Bayesian reasoning can help identifying potentially wrong immunoassays results in clinical practice: even when they appear 'not-unreasonable'.

BACKGROUND: Immunoassays are susceptible to analytical interferences including from endogenous immunoglobulin antibodies at a rate of ∼0.4% to 4%. Hundreds of millions of immunoassay tests (>10 millions in the UK alone) are performed yearly worldwide for measurements of an array of large and small moieties such as proteins, hormones, tumour markers, rheumatoid factor, troponin, small peptides, steroids and drugs. METHODS: Interference in these tests can lead to false results which when suspected, or surmised, can be analytically confirmed in most cases. Suspecting false laboratory data in the first place is not difficult when results are gross and without clinical correlates. However, when false results are subtle and/or plausible, it can be difficult to suspect with adverse clinical sequelae. This problem can be ameliorated by using a probabilistic Bayesian reasoning to flag up potentially suspect results even when laboratory data appear "not-unreasonable". RESULTS: Essentially, in disorders with low prevalence, the majority of positive results caused by analytical interference are likely to be false positives. On the other hand, when the disease prevalence is high, false negative results increase and become more significant. To illustrate the scope and utility of this approach, six different examples covering wide range of analytes are given, each highlighting specific aspect/nature of interference and suggested options to reduce it. CONCLUSION: Bayesian reasoning would allow laboratorians and/or clinicians to extract information about potentially false results, thus seeking follow-up confirmatory tests prior to the initiation of more expensive/invasive procedures or concluding a potentially wrong diagnosis.

The underestimated problem of using serum magnesium measurements to exclude magnesium deficiency in adults; a health warning is needed for "normal" results.

BACKGROUND: A major use of serum magnesium measurements in clinical practice is to identify patients with deficiency. However, numerous studies have shown that magnesium deficiency is common and may be present in over 10% of hospitalized patients, as well as in the general population. An important cause for under diagnosis of deficiency is that serum magnesium, the most commonly used test, can be normal despite negative body stores. This article focuses on the limitations of "normal" magnesium results and highlights the importance of lifestyle or "modus vivendi" as a pragmatic means of identifying those individuals potentially at risk for negative body magnesium stores. METHODS: Researched peer reviewed articles on magnesium published between 1990 and 2008 in MEDLINE and EMBASE, using database keywords "magnesium, deficiency, diagnosis, treatment and hypomagnesaemia". Bibliographies of retrieved articles have been searched and followed. We have also performed a manual search of each individual issue in which most of these reports have appeared. RESULTS: In 183 peer reviewed studies published from 1990 to 2008, magnesium deficiency was associated with increased prevalence and risk in 11 major conditions. Similarly, in 68 studies performed over the same period, magnesium deficiency was found to predict adverse events and a decreased risk of pathology was noted when supplementation or treatment was instituted. CONCLUSIONS: The perception that "normal" serum magnesium excludes deficiency is common among clinicians. This perception is probably enforced by the common laboratory practice of highlighting only abnormal results. A health warning is therefore warranted regarding potential misuse of "normal" serum magnesium because restoration of magnesium stores in deficient patients is simple, tolerable, inexpensive and can be clinically beneficial.

The double whammy of endogenous insulin antibodies in non-diabetic subjects.

The presence of high affinity/avidity endogenous insulin antibodies in significant amounts in non-diabetic individuals could uniquely be a double whammy. Clinically it could trigger pathology, namely hypoglycaemia, and analytically it could cause erroneous and potentially misleading results (i.e., falsely high or falsely low) in the key biochemical parameters essential in the differential diagnosis of this pathology, namely serum insulin, proinsulin and even C-peptide. The purpose of this paper is to highlight the clinical and analytical sequelae of endogenous insulin antibodies in (a) delaying or even confusing the differential diagnosis of unexpected/unexplained hypoglycaemia, and (b) interfering in some immunoassays of pancreatic hormones (commercial or in-house).

Erroneous laboratory results: what clinicians need to know.

Laboratory tests such as 'conventional biochemistry' are analytically robust and trusted, however, some common tests performed by immunoassays, eg thyroid function tests, are inherently more prone to analytical interference, giving rise to incorrect results. Interfering antibodies capable of causing potentially misleading results in immunoassay varied from about 0.4% to 4%. Furthermore, this form of interference cannot be predicted a priori and cannot be detected even by most stringent laboratory quality control assurance schemes because it is unique to an individual sample. Since more than 10 million immunoassay tests are carried out yearly in the UK alone, the impact of this problem on delivering appropriate patient care can no longer be ignored. Clinicians tend to perceive all laboratory data in the same light. Because of this, increased awareness of the inherent limitations of these laboratory tests should trigger a more measured and thoughtful approach, thus ensuring patients receive appropriate investigations and treatment.

On detecting interference from endogenous antibodies in immunoassays by doubling dilutions test.

The clinical laboratory may perform additional test or tests to ascertain the integrity of immunoassay analyses when the results are in doubt. Doubling serial dilutions is one of these confirmatory tests. Doubling dilutions is a useful and informative test for detecting inaccuracy and potential interference from endogenous antibodies in immunoassays. Despite its technical simplicity, the science underpinning this test is highly complex. Appreciating such complexities could help in understanding the various outcomes of such tests from misleadingly inaccurate but virtually linear/parallel results, despite the presence of interfering antibodies in some immunoassays, to an array of non-linearity/non-parallelism synonymous with interference in others. A lack of parallelism/linearity could identify some 60% of samples with inaccurate and potentially misleading results caused by endogenous interfering antibodies. Assessing real-time binding kinetics using techniques such as non-linear regression analysis or linearised transformations such as the Scatchard plot could help in identifying more samples in which the doubling dilutions test remains linear and parallel.

On the interpretation of affirmative follow-up tests in immunoassays: what must not be done?

The integrity of analyses and their limitations fall firmly within the laboratory domain. The shortcomings of analyses performed by immunoassays are well recognized. When a result is unexpected or in doubt, the laboratory may perform additional tests to substantiate the integrity of that analysis, such as repeat analysis by an alternative method, serial dilutions, or antibody blocking regents. Though commendable practice, these extra tests could in some cases lead to a false sense of assurance, misleading clinicians and wasting resources. I would like to highlight the use and misuse of these tests.

Limitations of 17-hydroxyprogesterone in investigating neonatal hyponatraemia.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a possible cause of hyponatraemia in the neonate. Elevated 17-hydroxyprogesterone (17-OHP) is considered diagnostic of the condition, although there have been reports of anomalous high concentrations, up to 110 nmol/L, in premature, sick infants subsequently shown to have normal adrenocortical function. We describe a case of a 6-week-old girl with a chest infection and hyponatraemia whose plasma 17-OHP concentration was 300 nmol/L, well within the range associated with 21-hydroxylase deficiency. However, there was no genital ambiguity and plasma cortisol was also significantly elevated, raising the possibility of generalized adrenal hyperstimulation rather than CAH. The patient was treated with antibiotic and saline infusions but no steroids. CAH was subsequently excluded by normal 17-OHP and cortisol responses to Synacthen stimulation. In sickness, an increased plasma 17-OHP concentration may not be synonymous with 21-hydroxylase deficiency, even when grossly raised. Simultaneous measurement of plasma cortisol could aid interpretation and avoid potential misdiagnosis, especially in male infants.

Wrong biochemistry results: two case reports and observational study in 5310 patients on potentially misleading thyroid-stimulating hormone and gonadotropin immunoassay results.

BACKGROUND: Immunoassays are used in almost all medical and surgical specialties, but they suffer from interference from proteins such as antibodies in some patients' sera. Such interferences are usually reported in the literature only as case reports after the introduction of a new assay. METHODS: We undertook a prospective observational study on 5310 patients for whom the common immunoassay tests for thyroid-stimulating hormone (TSH) and/or gonadotropins were requested. All TSH and gonadotropin results were critically assessed for a mismatch between the clinical details and analytical results to identify samples suspected of analytical unreliability. These were tested further by three approaches to screen for interference. RESULTS: From the 5310 sets of results, 59 patients' samples were identified as suspect and were tested further. Analytically incorrect results were found in 28 (0.53% of the total studied). The magnitude of interference varied, but in 23 of 28 patients (82%), it was considered large enough to have a potentially adverse effect on cost and/or the clinical care of these patients. Two cases, described in detail, illustrate the adverse effect of error on patient care and cost, and the second highlights the difficulties and limitations of current approaches for identifying interference and inaccuracy in immunoassays. CONCLUSIONS: Because millions of TSH/gonadotropin tests are carried out in UK hospital laboratories alone, our data suggest that thousands of patients could be adversely affected by errors from interferences. Early identification of interference in cases with unusual results could be valuable.