Commercial insulin immunoassays fail to detect commonly prescribed insulin analogues.

OBJECTIVES: Blood insulin and C-peptide are key investigations in the differential diagnosis of hypoglycaemia. Analogues of insulin have modified primary-sequences compared to native human insulin, as such may not cross react with insulin assays. This has important implications in detecting surreptitious or malicious insulin administration. The aim of this study is to assess the cross-reactivity of all insulins currently listed in the British National Formulary (BNF65, 2013) in clinical insulin assays currently used in UK clinical laboratories. DESIGN AND METHODS: Sample sets were prepared for all 15 exogenous insulin classes listed in the BNF, at concentrations of 1000 pmol/L and 300 pmol/L, using pooled human serum. Samples were sent blinded to 5 participating analytical laboratories to cover analysis on the 10 major clinical insulin assays used in the UK. RESULTS: The ability of insulin assays to detect exogenous insulin preparations was highly variable and ranged from 0% to >140% for a single exogenous insulin. Four assays were highly specific for the human insulin sequence and had no cross-reactivity with any synthetic analogue insulin. Two detected all insulin types (human sequence, animal and synthetic analogue), with the remaining having variable cross-reactivity. CONCLUSION: The cross-reactivity of the 15 exogenous insulin preparations is highly variable in the assays used in clinical laboratories around the UK. It is important that laboratories and clinicians are aware of the limitations of their local assays to avoid missing the important diagnosis of hypoglycaemia secondary to excessive exogenous insulin. Where necessary, samples should be referred to specialist centres for insulin analysis and ideally by a validated and fully-quantitative mass spectrometry-based method.

Analytical and clinical challenges in a patient with concurrent type 1 diabetes, subcutaneous insulin resistance and insulin autoimmune syndrome.

UNLABELLED: A lean 15-year-old girl was diagnosed with type 1 diabetes based on symptomatic hyperglycaemia and positive anti-islet cell antibodies. Glycaemia was initially stabilised on twice-daily mixed insulin. After 11 months from the time of diagnosis, she complained of hyperglycaemia and ketosis alternating with hypoglycaemia. This progressively worsened until prolonged hospital admission was required for treatment of refractory hypoglycaemia. A high titre of anti-insulin antibodies was detected associated with a very low recovery of immunoreactive (free) insulin from plasma after precipitation with polyethylene glycol, suggesting the presence of insulin in bound complexes. Insulin autoimmune syndrome was diagnosed and metabolic fluctuations were initially managed supportively. However, due to poor glucose control, immunosuppressive therapy was initiated first with steroids and plasmapheresis and later with anti-CD20 antibody therapy (Rituximab). This treatment was associated with a gradual disappearance of anti-insulin antibodies and her underlying type 1 diabetes has subsequently been successfully managed with an insulin pump. LEARNING POINTS: Anti-insulin antibodies may result in low levels of free insulin.Polyclonal anti-insulin antibodies can interfere with the pharmacological action of administered insulin, resulting in hypoglycaemia and insulin resistance, due to varying affinities and capacities.In this patient, rituximab administration was associated with a gradual disappearance of anti-insulin antibodies.It is hypothesised that this patient had subcutaneous insulin resistance (SIR) caused by insulin capture at the tissue level, either by antibodies or by sequestration.A prolonged tissue resistance protocol may be more appropriate in patients with immune-mediated SIR syndrome.

Hypoglycemia due to an insulin binding antibody in a patient with an IgA-kappa myeloma.

CONTEXT: Autoantibodies to insulin have been described to cause spontaneous hypoglycemia in nondiabetic subjects. There have been occasional reports of spontaneous hypoglycemia due to monoclonal anti-insulin antibodies. We present the first report of a patient with an IgA-kappa myeloma in whom frequent hypoglycemia resulted from the ability of the monoclonal IgA-kappa to bind insulin. OBJECTIVES: The aim of this study was to describe the occurrence of profound hypoglycemia in a patient with IgA-kappa myeloma, characterize biochemically the nature of the IgA:insulin complex present, and place this case in the context of the published literature on hypoglycemia resulting from autoantibodies to insulin. DESIGN: A case study was performed. PATIENTS: A single case of profound hypoglycemia associated with IgA-kappa myeloma was studied. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURES: A case study was performed. RESULTS: Polyethylene glycol precipitation and gel filtration chromatography were used to demonstrate high-molecular weight insulin immunoreactivity in the patient's plasma. This was characterized as an insulin binding IgA-kappa paraprotein present at 4200 mg/dl (42 g/liter) with a relatively high insulin dissociation constant of 0.32 microm/liter using radiolabelled insulin binding studies. CONCLUSIONS: We present the first case of hypoglycemia due to IgA binding insulin antibodies in a patient with an IgA-kappa paraprotein myeloma. The hypoglycemia was associated with high-plasma insulin levels and relatively low C-peptide levels. A plausible mechanism for the hypoglycemia is the delayed clearance of insulin. This case broadens the spectrum of monoclonal gammopathies that have been associated with anti-insulin reactivity and spontaneous hypoglycemia.

Variation in GH and IGF-I assays limits the applicability of international consensus criteria to local practice.

BACKGROUND: There is increasing reliance on consensus criteria for decision making. Recent criteria state that acromegaly is excluded by a nadir GH during an oral glucose tolerance test (OGTT) of < 1 microg/l and a normal level of IGF-I. OBJECTIVE: To study GH and IGF-I assay performance close to cut-off values for active acromegaly. DESIGN AND METHODS: Two serum samples known to give borderline results were sent to all centres participating in the UK National External Quality Assessment Service (NEQAS). Sample A was assigned to be a nadir during an OGTT and sent for GH assessment to 104 centres. Sample B, with a clinical scenario, was sent to 23 centres that measure IGF-I, and these centres were asked to measure IGF-I, interpret the result and provide the source of their reference ranges (RRs). RESULTS: For sample A, the median GH was 2.6 mU/l (range 1.04-3.5 mU/l). Applying a conversion factor (CF) of 2.0 (1 microg/l = 2 mU/l), the most negatively biased method classified 10% of the values consistent with acromegaly, while the most positively biased method classified all values as consistent with the diagnosis. Applying a CF of 3.0 (1 microg/l = 3 mU/l), only 11% of results were consistent with acromegaly. For sample B, the median IGF-I was 50.8 nmol/l (range 24.3-60.9 nmol/l). All centres used age-related RRs. There was a 50% variation in the upper limit of the RRs between centres. Overall, 30% of the IGF-I results were against the diagnosis. There was little agreement in the RRs quoted by centres using the same method. CONCLUSION: Variability in assay performance, coupled with use of inappropriate CFs and RRs, undermines the applicability of international consensus criteria to local practice.

Most commercial insulin assays fail to detect recombinant insulin analogues.

Insulin assays are utilized in various clinical scenarios, including the assessment of insulin therapy compliance or of suspected insulin overdose. In an interpretative exercise carried out by UK National External Quality Assessment Service (NEQAS), serum sent to the participating laboratories was spiked with 30 pmol/L of the short-acting insulin analogue Human Actrapid. Only two out of 24 participant laboratories had sufficient assay cross-reactivity with Actrapid to interpret the results as suggestive of insulin administration. The development of specific insulin assays has led to deterioration in the ability to detect non-compliance or overdose with recombinant insulin treatment. Clinicians should be aware of this significant limitation, which could lead to misdiagnosis.

The effectiveness of different treatment options for non-islet cell tumour hypoglycaemia.

OBJECTIVE: To compare the outcome of different treatment options used in several cases of non-islet cell tumour hypoglycaemia (NICTH). PATIENTS: Eight cases of NICTH were referred for diagnosis and monitoring following either surgical or medical treatment. METHODS: Serum samples collected throughout the time-course of each case were analysed for glucose, insulin, C-peptide, IGF-I, total IGF-II, total IGF-II to IGF-I ratio and, in most of the cases, big IGF-II. RESULTS: Surgical excision was successful in the relief of symptoms and normalization of the biochemical parameters. Therapeutic treatment with glucocorticoids confirmed previous studies showing the suppressive effect on tumour (big) IGF-II production. The present data show that the effect was dose-dependent and reversible if doses were below a critical level. CONCLUSIONS: Within the limits of the cases studied, and the time-scales involved, moderate- to high-dose glucocorticoid therapy had immediate beneficial influence on symptomatic hypoglycaemia and, if tolerated in the long term, was effective in correcting the underlying biochemical dysfunction, unlike other therapeutic regimens. This effectiveness was only achieved when the dose exceeded a threshold level specific to the patient. In addition, reduction of the dose or withdrawal of the drug caused a return of the abnormal biochemical profile. Surgical removal of the malignancy, where this was an option, was successful within the periods studied.

The biochemical investigation of cases of hypoglycaemia: an assessment of the clinical effectiveness of analytical services.

AIM: To assess the extent to which biochemical analytical services contribute to the diagnosis and management of clinical cases of hypoglycaemia. METHODS: All cases of confirmed hypoglycaemia, referred during a six month period, were included in the survey. Questionnaires were sent to each referring laboratory requesting information on the clinical progress and current status of the patient. RESULTS: The level of influence exerted by analytical data was assigned in each case and those with similar outcomes combined. Identifiable case groups were: (1) Results not recorded in the patients' notes (15.7%). (2) Inappropriate requesting of insulin and C peptide measurements in cases of diabetes (11.4%). (3) Patient died soon after investigation (20.0%). (4) Patient recovered spontaneously (17.1%). (5) Patient received effective medical or surgical treatment (12.9%). (6) Patient awaiting or not requiring pathology based treatment (31.4%). (7) Inconclusive outcome prompting further investigation (5.7%). CONCLUSIONS: Within the timescale of the survey (approximately 12 months), positive progress had been made towards diagnosis and subsequent treatment in only 10% of cases. Another 30% were either awaiting some form of treatment or further diagnostic tests. The remaining 60% did not appear to benefit in any way from the biochemical investigations.

An activated protein kinase C alpha gives a differentiation signal for hematopoietic progenitor cells and mimicks macrophage colony-stimulating factor-stimulated signaling events.

Highly enriched, bipotent, hematopoietic granulocyte macrophage colony-forming cells (GM-CFC) require cytokines for their survival, proliferation, and development. GM-CFC will form neutrophils in the presence of the cytokines stem cell factor and granulocyte colony-stimulating factor, whereas macrophage colony-stimulating factor leads to macrophage formation. Previously, we have shown that the commitment to the macrophage lineage is associated with lipid hydrolysis and translocation of protein kinase C alpha (PKCalpha) to the nucleus. Here we have transfected freshly prepared GM-CFC with a constitutively activated form of PKCalpha, namely PKAC, in which the regulatory domain has been truncated. Greater than 95% of the transfected cells showed over a twofold increase in PKCalpha expression with the protein being located primarily within the nucleus. The expression of PKAC caused macrophage development even in the presence of stimuli that normally promote only neutrophilic development. Thus, M-CSF-stimulated translocation of PKCalpha to the nucleus is a signal associated with macrophage development in primary mammalian hematopoietic progenitor cells, and this signal can be mimicked by ectopic PKAC, which is also expressed in the nucleus.

Abl protein kinase abrogates the response of multipotent haemopoietic cells to the growth inhibitor macrophage inflammatory protein-1 alpha.

The clonogenic cells of chronic myeloid leukaemia (CML), unlike normal haemopoietic progenitor cells, are resistant to the growth inhibitory effects of the chemokine macrophage inflammatory protein-1 alpha (MIP-1alpha). CML is also relatively resistant to chemotherapy and the disease is difficult to cure using conventional therapeutic routes. CML is associated with increased abl oncogene protein tyrosine kinase (PTK) activity. Here, we have tested the hypothesis that these aberrant responses to MIP-1alpha and the relative resistance to chemotherapy are directly related to this increased abl PTK activity in primitive haemopoietic cells. To do this we have expressed a temperature sensitive abl PTK in a growth factor dependent, multipotent stem cell line (FDCP-Mix) in which growth is normally suppressed by MIP-1alpha. In FDCP-Mix cells expressing the ts v-abl PTK and grown at the restrictive temperature for PTK activity the cells were relatively sensitive to cytotoxic agents such as cytosine arabinoside and 5-fluorouracil but MIP-1alpha could induce growth inhibition and confer some degree of protection from these agents. At the permissive temperature for abl PTK, the cells were relatively resistant to cytotoxic drugs and MIP-1alpha treatment neither induced growth inhibition nor protected the cells from cytotoxic drug induced cell death. This lack of response to MIP-1alpha was not due to receptor down modulation as neither the affinity nor the number of 125I-MIP-1alpha binding sites was altered by activating Abl PTK. However, MIP-1alpha mediated increases in cytosolic Ca2+ levels were abrogated by switching cells to the permissive temperature for Abl PTK activity. These data suggest that the relative resistance of CML progenitor cells to therapeutic drugs and the lack of response to MIP-1alpha occurs as a direct consequence of abl PTK activity and involves desensitisation of signal transduction events stimulated by MIP-1alpha receptors. Thus one contributory mechanism to transformation of primitive haemopoietic cells is abrogation of response to a growth inhibitor.

Macrophage inflammatory protein-1 alpha mediated growth inhibition in a haemopoietic stem cell line is associated with inositol 1,4,5 triphosphate generation.

Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.

Effect of trypsin on a Ca(2+)-activated K+ channel reconstituted into planar phospholipid bilayers.

Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.