The immuassay handbook parte81


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The immuassay handbook parte81

  1. 1. 783© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. The concentration of glucose in blood is closely controlled in healthy individuals, regardless of the quantity of carbo- hydrate ingested in the diet. The hormone insulin plays a central role in the regulation of blood glucose concentra- tions by influencing tissue glucose uptake and the meta- bolic pathways of gluconeogenesis, glycogenolysis, and lipolysis. It is produced by the beta cells of the Islets of Langerhans in the pancreas and secreted into the circula- tion within minutes when the concentration of glucose in the blood increases. The secretion or administration of insulin causes a fall in the concentration of blood glucose. A concentration of blood glucose below that found in healthy fasting individuals is known as hypoglycemia. Measured glucose concentrations are dependent on sam- ple type (arterial/venous/capillary, whole blood or plasma). In diabetes mellitus, the concentration of glucose in the blood is abnormally high (hyperglycemia) either due to insufficient insulin secretion or due to an inability of insu- lin to act at a cellular level or both. The hyperglycemia of diabetes may occur when the patient is fasting and/or post- prandially. Other abnormalities of intermediary metabo- lism such as lipid metabolism are found. Diabetes mellitus is treated by diet, and administering insulin or drugs that enhance insulin secretion, or drugs that increase sensitivity to insulin. The chronic hyperglycemia of diabetes can lead to long-term complications of the disease: microvascular complications involving the kidney (nephropathy) and ret- ina (retinopathy), macrovascular complications such as coronary heart disease, peripheral vascular disease, and also the neuropathies. There are two main types of diabetes mellitus, which are classified based on the pathophysiology of the disease rather than on treatment (see Table 1). Type 1 (previously insulin-dependent diabetes mellitus, IDDM) has a var- ied prevalence of 1–29 per 100,000 of the population. Those affected have a significantly reduced secretion of insulin due to autoimmune destruction of the pancreatic beta cells. Type 1 diabetes is usually diagnosed in child- hood or early adulthood, with the patient presenting acutely with polyuria (excretion of an increased volume of urine), excessive thirst, weight loss, and ketosis, which is characterized by drowsiness, headache, dehydration, and deep respiration, due to an accumulation of ketone bodies, β-hydroxybutyric acid, acetoacetic acid, and acetone in the blood. Diabetic ketoacidosis is a medical emergency. Type 1 diabetes is primarily treated with insulin. Type 2 diabetes is associated with insulin resistance and obesity. Obesity itself will also contribute to the resistance to insulin. There is some evidence to suggest insulin defi- ciency may also be a factor. Type 2 diabetes has a prevalence worldwide of about 6% (, accessed March 2011), and this can be significantly higher in certain populations such as the Pima Indians in the United States. The prevalence of Type 2 diabetes is increasing both in adults and in children around the world and has been associ- ated with the increase in obesity and changes in lifestyle (Ehtisham and Barrett, 2004). Type 2 diabetes is generally treated in the first instance by lifestyle modification (weight loss following dietary advice and increased exercise), fol- lowed sequentially with drugs that will stimulate the secre- tion of insulin (oral hypoglycemics) or drugs that improve the sensitivity to insulin. Treatment with insulin is often necessary as the disease progresses. Continued patient assessment will range from self-monitoring of blood glu- cose, to laboratory investigations for determining glucose control, and for complications such as lipid status and car- diovascular risk, and kidney involvement (nephropathy). A number of diseases can cause secondary diabetes, and there are also several rarer conditions associated with it (see Table 1). There is increasing recognition of genetic causes (mono- and polygenic) of diabetes that can have significant implications for treatment options (McCarthy and Hatters- ley, 2008), and diabetes may be associated with exocrine dis- ease of the pancreas and other endocrinopathies. Diabetes presenting in pregnancy is known as gestational diabetes. The diagnosis of diabetes has been based on the demon- stration of hyperglycemia. Nearly, all classifications have relied on blood (or plasma/serum) glucose concentrations exceeding an established glucose level in a timed sample collection. These include fasting, random relative to pran- dial status, or samples collected after a standardized stress test (such as the 75g oral glucose tolerance test, OGTT) (American Diabetes Association, 2007). The most recent criteria introduced for the diagnosis of diabetes include a random venous plasma glucose (>11.1mmol/L or 200mg/ dL) or a fasting venous plasma glucose (≥7.0mmol/L or 126mg/dL) in the presence of symptoms of diabetes mel- litus. With no symptoms, the diagnosis should not be based on a single glucose determination—a repeat mea- surement is required. Two other groups of “impaired glu- cose tolerance” and “impaired fasting glycemia” are now defined (Table 2). It is recommended that those with impaired fasting glycemia should have an OGTT (American Diabetes Association, 2007; World Health Organization, 2006). However, HbA1c has now been adopted in some countries for the diagnosis of diabetes. In the OGTT, 75g of anhydrous glucose is administered orally to a fasting patient, and the blood glucose concentra- tion is measured at timed intervals (see Table 2). The test must be standardized as a number of factors, such as the type of sample collected, the glucose dose, and the preceding diet, all have a significant effect on the results of the test. Diabetes Mellitus Penny Clark ( Tim McDonald ( C H A P T E R 9.10
  2. 2. 784 The Immunoassay Handbook Measurement of fasting plasma glucose and performing an OGTT can be difficult, and recently, it has been proposed that hemoglobin A1c with a value ≥6.5% (48mmol/mol) can be used to diagnose diabetes mellitus, though this proposal has not been universally agreed (Sacks et al. 2011). Different criteria are used for the diagnosis of gesta- tional diabetes. The measurement of plasma insulin is not necessary for the diagnosis of diabetes mellitus. An understanding of the dynamics and processes of insulin secretion and action are needed to unravel the pathophysiological mechanisms of the disease. The importance of insulin assays is underlined by the fact that the first radioimmunoassay (RIA) to be described (Yalow and Berson, 1959) was for plasma insulin. Control of blood glucose within certain limits by adjusting treatment is advocated for patients with Type 1 and Type 2 diabetes, for both immediate metabolic control and to prevent or delay the onset of diabetic complications (The Diabetic Control and Complication Trials Research Group, 1993; The United Kingdom Prospective Diabetes Study Group, 1998). Central to good control of blood glucose in clinical practice is self-monitoring of blood glucose, the measurement of markers of glycemic control such as glycated hemoglobin and markers of diabetic complications such as microalbumin. Immunoassays exist for many of these analytes. Analytes INSULIN, PROINSULIN(S), AND C-PEPTIDE Insulin, like many polypeptide hormones, is synthesized as a precursor molecule: proinsulin. Limited proteolysis of proinsulin at sites marked by pairs of basic amino acids produces insulin by way of a number of partially processed forms. The C-peptide is a fragment that connects the A and B chains of insulin in the proinsulin molecule (Fig. 1). Insulin is secreted in a pulsatile manner and is secreted in equimolar concentrations with C-peptide into the portal cir- culation. The first pass through the liver clears approximately 50% of this insulin, whereas C-peptide is excreted mainly through the kidneys. Because C-peptide has a longer bio- logical half-life than insulin, its measurement in a body fluid TABLE 1 Classification of Disorders of Glycemic Control (1) Type 1 diabetes mellitus (beta cell destruction leading to insulin deficiency) Autoimmune Idiopathic (2) Type 2 diabetes mellitus (ranges from insulin resistance/relative insulin deficiency with or without secretory defect to secretory defect with or without insulin resistance) (3) Other types Genetic defects of beta cell function, e.g., MODY Genetic defects in insulin action, e.g., Type A insulin resistance Diseases of the exocrine pancreas, e.g., cystic fibrosis, pancreatitis Endocrinopathies, e.g., acromegaly, Cushing’s syndrome Drug/chemical induced Infection, e.g., CMV, congenital rubella Uncommon immune mediated, e.g., antibodies to insulin Associated with other genetic syndromes, e.g., Turner’s, Wolfram’s (4) Gestational diabetes mellitus TABLE 2 Glucose Values (mmol/L) for the Diagnosis of Diabetes Mellitus and Hyperglycemia (American Diabetes Association, 2004) Whole Blood Whole Blood Plasma Venous Capillary Venous Diabetes mellitus Fasting or 2h post-glucose load or both ≥6.1 ≥10.0 ≥6.1 ≥11.1 ≥7.0 ≥11.1 Impaired glucose tolerance Fasting and 2h post-glucose load <6.1 ≥6.7 and <10.0 <6.1 ≥7.8 and <11.1 <7.0 ≥7.8 and <11.1 Impaired fasting glycemia (IFG) Fasting 2h post-glucose load (if measured) ≥5.6 and <6.1 <6.7 ≥5.6 and <6.1 <7.8 ≥6.1 and <7.0 <7.8 FIGURE 1 Processing of proinsulin to major intermediates and to insulin. Reproduced with permission from Temple et al Diabetic Medicine 1992 John Wiley and Sons Ltd
  3. 3. 785CHAPTER 9.10 Diabetes Mellitus may be used as an indicator of insulin secretion. Circulating concentrations of C-peptide will be elevated in renal failure. Proinsulin and its partially processed forms are thought to have little biological activity in comparison to insulin. The amino acid composition of insulin is conserved among many species except for residues 4, 8, 9, and 10 of the A chain and 1, 2, 3, 27, 29, and 30 of the B chain. There is structural homology with insulin-like growth factors I and II. Glucose is the primary stimulator of insulin secretion in mammals though secretion is also stimulated by the amino acids leucine and arginine and inhibited by the hormone somatostatin. Insulin has a number of important metabolic actions cen- tral to the control of plasma glucose. It suppresses the endogenous production of glucose. Insulin inhibits the breakdown of glycogen to glucose in the liver and stimu- lates the uptake, storage, and use of glucose in other tissues such as muscle and adipose tissue. Insulin also increases the synthesis of fatty acids, triglycerides, and proteins and sup- presses proteolysis and lipolysis. The counter-regulatory hormones to insulin, which increase the concentration of glucose in the blood, include glucagon, epinephrine (adren- aline), and to a lesser extent growth hormone and cortisol. Reference Intervals A number of factors influence the plasma concentration of these hormones, most notably the plasma glucose concen- tration, obesity (generally expressed as a body mass index, kg/m2), age, ethnic group, and the assay used. The insulin/ glucose relationship in neonates differs to that in adults. Fasting plasma insulin is increased at the time of puberty, and insulin resistance is also found in pregnancy. The upper limit of the reference range for insulin in euglycemic subjects is quoted as being between 60 and 209pmol/L and the lower limit between 0 and 20pmol/L. For C-peptide, the corresponding figures are for the upper limit 500 and 1700pmol/L and for the lower limit, 100 and 400pmol/L. These differences may reflect differences in assay sensitivity and specificity and also differences in the populations studied (Wark, 2006). Fasting plasma proinsulin concentrations are normally <7pmol/L though there is variation between assays, and the numbers of subjects studied are few. The des 64,65 split proinsulin (i.e., proinsulin cleaved between amino acids 65 and 66 with subsequent loss of the pair of basic amino acids) is usually undetectable in fasting human plasma (less than 5pmol/L), and the des 31,32 split proin- sulin may be in the range of 1–20pmol/L depending on body mass. The upper limit of the reference range for C-peptide for euglycemic subjects is quoted as being between 700 and 2300pmol/L and the lower limit between 100 and 400pmol/L. These differences may reflect differences in assay specificity and also differences in the populations studied (Wark, 2006). In the investigation of hypoglycemia and the interpre- tation of the response to the 72h fast, a range of figures are quoted as the appropriate response for plasma insu- lin, C-peptide, and proinsulin in the presence of Whip- ple’s triad (symptoms and signs of hypoglycemia, a low concentration of plasma glucose as measured by the labo- ratory and resolution of symptoms after plasma glucose is raised). In patients with symptoms and signs of hypogly- cemia and with a glucose of less than 3.0mmol/L, an insulin of at least 18pmol/L, C-peptide of at least 200pmol/L, and a proinsulin of at least 5.0pmol/L indi- cate hyperinsulinism (Cryer et al., 2009). However, these suggested cutoff values require validation with assays in current use as some insulin assays, for example, have sig- nificant bias and/or poor precision at insulin concentra- tions of less than 50pmol/L (Miller et al., 2009). Clinical Applications The concentrations of plasma insulin, proinsulin, and C-peptide are normally measured to investigate the causes of hypoglycemia (plasma glucose <3.0mmol/L, 54mg per 100mL) (see Table 3) and in particular to diagnose insuli- noma or congenital hyperinsulinemic hypoglycemia, where the plasma insulin (and/or proinsulin) is inappropri- ately high for the concentration of glucose. Blood samples may be collected after an overnight or prolonged fast and are most informative if collected at the time of hypoglyce- mia. Suppression tests are rarely used now. Clamping techniques and selective venous localization may be per- formed in specialist centers and require technical exper- tise, specialist equipment, and medical supervision. Contraindications to the performance of any test should be considered. If exogenous insulin administration is suspected, the specificity of the immunoassay for different animal and human insulins should be taken into account (Owen and Roberts, 2004), and C-peptide should be measured to TABLE 3 Causes of Hypoglycemia in Adults In the ‘well’ patient Drugs, e.g., oral hypoglycemic agents, salicylate Ethanol Insulinoma Factitious induced by insulin Ketotic hypoglycemia Intense exercise In the “ill” patient Drugs, e.g., oral hypoglycemic agents, salicylate, insulin, quinine, quinidine Liver disease Renal disease Congestive cardiac failure Non-beta cell tumors, e.g., mesenchymal Endocrine deficiency, e.g., cortisol, growth hormone Sepsis Surgical removal of pheochromocytoma Insulin antibody induced Starvation Shock Total parenteral nutrition and insulin therapy Genetic defects of beta cell function, e.g., congenital hyperinsulinemic hypoglycemia
  4. 4. 786 The Immunoassay Handbook distinguish endogenous insulin production from exogenous insulin administration. A drug screen for oral hypoglyce- mic agents should be performed. Measurement of insulin antibodies may be needed to exclude interference in the assays and to exclude autoimmune hypoglycemia (Lupsa et al., 2009). In specialized laboratories, these assays may be used in investigations into the pathophysiology or etiology of dia- betes mellitus. Standard measures of insulin and C-peptide involve blood samples collected either fasted or, most often, after a fixed stimulus (such as oral glucose, mixed meal, or IV glucagon). The mixed-meal tolerance test (MMTT) is the accepted gold standard measure of endogenous insulin secretion in Type 1 diabetes (Greenbaum et al., 2008). During the MMTT, a 90min poststimulus serum C-peptide of >200 pmol/L is considered as significant endogenous insulin secretion and is related to improved clinical outcomes. However, the MMTT is practically demanding and requires a specific lipid, protein, and car- bohydrate liquid meal and the patient to omit their morning insulin. This restricts this test to the hospital and research setting. Measurement of C-peptide 5 min following IV glucagon administration has also been advocated, but associated nausea means this test is poorly tolerated and rarely performed. The assessment of endogenous C-peptide production in diabetes can be useful in classifying the subtype of dia- betes. In a patient with young-onset diabetes, persistent C-peptide production may reflect the honeymoon period of a patient with Type 1 diabetes but also enduring C-peptide can be a feature of other types of diabetes including Type 2 diabetes where levels are typically high. There is extensive evidence that C-peptide can be used to differentiate between the classification of Type 1 diabetes and Type 2 diabetes. It has been suggested that the loss of the first-phase insulin response in the IVGTT can predict the develop- ment of Type 1 diabetes. A number of mathematical mod- els and transformations have been used to derive functions of insulin sensitivity and beta cell function, for example, homeostatic model assessment (HOMA) (Wallace et al., 2004) and the insulin/glucose ratio (Katz et al., 2000). The methods for some of these tests, and their interpretations, vary from center to center. They are applicable in research and epidemiological studies rather than to individuals. However, whichever method is used, the clinical diagnosis of diabetes mellitus is still determined from the plasma glucose concentration and clinical features. It remains to be established whether formal assessment of insulin resis- tance will become established in the assessment of those patients to be treated with drugs that improve tissue sensi- tivity to insulin. It has also been suggested that C-peptide, as a measure of beta cell function, can predict the need for insulin treat- ment in Type 2 diabetes (Hohberg, 2009). In practice, this is not used routinely, the decision on treatment pro- tocols being made on clinical grounds, and the plasma glucose profile and glycated hemoglobin results. How- ever, the C-peptide response to glucagon may be used to assess beta cell function in pancreatic/islet cell transplantation. Urine C-Peptide Measuring C-peptide excretion in urine is an alternative approach to the assessment of insulin secretion. C-peptide metabolism largely occurs in the kidneys, in contrast to insulin, which is metabolized and extracted in the liver. The total quantity of C-peptide excreted in the urine per day represents 5–10% of pancreatic secretion compared to only 0.1% of secreted insulin. Consequently, despite equi- molar secretion, C-peptide has a longer half-life of approx- imately 30 min compared with only 6min for insulin. Urinary C-peptide (UCP) is a noninvasive alternative to serum C-peptide that when collected in boric acid preser- vative is stable at room temperature for at least 3 days (McDonald et al., 2009). Previous studies suggest 24h UCP concentrations accurately assess beta cell secretory capacity and correlate with fasting and stimulated serum insulin and C-peptide. However, 24h urine collections are cumbersome, often incomplete, and impractical in a busy clinic. Serum insulin and C-peptide are correlated with single and 4h poststim- ulated UCP in both nondiabetic and insulin treated dia- betic patients. Correcting UCP for urinary creatinine (urine C-peptide creatinine ratio) adjusts for differences in urine dilution, enabling spot samples to be taken at different times of the day rather than in a 24h collection, an approach already used for routine microalbuminuria (see later). A study from Koskinen in 1986 suggested that a 2h postprandial UCP creatinine ratio (UCPCR) is as sensitive and specific as glucagon-stimulated plasma C-peptide in detecting patients who will benefit from insulin treatment. UCPCR has been shown to be a sensitive and specific alternative to 90min serum C-peptide during an MMTT, the gold standard method for detecting endogenous insu- lin secretion, in children and young adults with Type 1 diabetes and in a mixed cohort of late-onset insulin-treated diabetes. Postprandial UCPCR has also been shown to be a useful test to discriminate HNF1A- and HNF4A-MODY from long-duration Type 1 diabetes (Besser et al., 2011). Limitations G Immunoassays for insulin, proinsulin(s), and C-peptide vary in their sensitivities and specificities. These factors should be taken into account when interpreting results. A number of assays for insulin measure proinsulin(s) and when these are elevated, as in Type 2 diabetes, the immunoassay may overestimate the plasma insulin concentration. G Some assays may lack the sensitivity to detect sup- pressed insulin concentrations that are found in non- insulinoma hypoglycemia. G Hemolysis may result in a fall in the measured hormone concentration, and hemolyzed samples should not be assayed. The effects of other possible interferences should also be noted. G It is recommended that samples be collected on ice and transported to the laboratory for immediate separation of plasma/serum, which should be stored frozen. Data suggest that it is C-peptide, which is the least stable in plasma/serum, and that the degree of instability
  5. 5. 787CHAPTER 9.10 Diabetes Mellitus detected is assay dependent. Repeated freezing and thawing should be avoided. G Some patients, particularly patients with Type 1 diabetes treated with animal insulins (although now rare), those with other autoimmune disease or treated with sulfhy- dryl-containing drugs such as methimazole and penicil- lamine, may develop antibodies to the insulin. These antibodies may interfere in assays for insulin, proinsulin, and C-peptide unless the sample is pretreated, for exam- ple, with polyethylene glycol. This procedure must be validated. Insulin autoantibodies have also been described in a number of nondiabetic subjects. G Preparations of the split proinsulin(s) for use as stan- dards are not widely available. There is an international reference reagent (coded IRR 84/611) for proinsulin, for C-peptide (coded IRR 84/510), and an international reference preparation for insulin (coded IRP 66/304). Although there are commercially available quality assurance materials for insulin, these are not always suitable for assays specific for human insulin. Quality assurance materials for the proinsulins are not widely available. The external quality assurance schemes are limited to insulin and C-peptide. G There are few reported assays for the partially pro- cessed proinsulins. G Cross-reactivity with synthetic/modified insulins should be known. G Many drugs can raise or lower insulin concentrations through in vivo effects. G Immunoassays for these hormones have been compared with high-performance liquid chromatography (HPLC) and stable isotope dilution liquid chromatog- raphy mass spectrometry assays. These methods are technically demanding and use large volumes of sample and hence are unsuitable for routine use. However, they may be used to validate immunoassays. G Method comparison studies have highlighted poor comparability between C-peptide assays making trans- ferability of C-peptide values/cutoffs from research to clinical practice problematic. Assay Technology Early assays for insulin, proinsulin, and C-peptide were competitive RIAs, and care is required in reading the litera- ture to determine the specificity and calibration of these assays. Most commercial assays are immunometric assays, using monoclonal antibodies or both mono- and polyclonal antibodies in excess concentrations, which have led to improvements in sensitivity and specificity (Clark, 1999; Sapin, 2007). A number of non-isotopic assays for insulin and C-peptide are available commercially on automated immunoassay analyzers or as semiautomated/manual assays based on microtiter plate technology with either measure- ment of absorbance or luminescence. A number of com- mercial assays are available for intact or total proinsulins. Insulin and C-peptide Many commercially available assays for both insulin and C-peptide are classical RIAs though some competitive assays use horseradish peroxidase (HRP) or alkaline phos- phatase (ALP) as label. Immunometric assays for both are now widely available and have been automated to accom- modate high throughput utilizing non-isotopic signal generation systems such as chemiluminescence and fluo- rescence as labels. Proinsulin A commercial RIA is available for proinsulin, the majority of assays being immunometric assays with a microtiter plate solid phase. HRP and ALP (with enzyme amplifica- tion) have been used as the label in assays for intact and total proinsulin(s) as has chemiluminescence. Desirable Assay Performance Characteristics Insulin Competitive immunoassays for insulin tend to demon- strate significant cross-reactivity with intact proinsulin and the partially processed proinsulins while immunometric assays may show greater specificity. For the routine inves- tigation of hypoglycemia, the use of a nonspecific assay may be preferable as some insulinomas are known to secrete significant amounts of proinsulin(s) though some advocate the measurement of both insulin and proinsulin in these circumstances. In contrast, for research purposes, a more specific assay may be required in order to deter- mine more accurately insulin secretion in the presence of increased concentrations of proinsulin(s). Some commer- cially available immunoassays, whether competitive or immunometric, lack the sensitivity to determine insulin concentrations at the lower limit of the reference range. Proinsulin(s) Immunoassays for intact and partially processed proinsu- lins vary in their degree of specificity. As a minimum full cross-reactivity data should be quoted. The sensitivity of many assays is insufficient to measure concentrations at the lower limit of the reference range. C-peptide C-peptide is reported to be unstable in whole blood, on storage and on repeated freeze–thaw cycles though this view has been challenged more recently and may be over- come with the use of specific anticoagulants. Stability data should be quoted for each assay and collection conditions specified by the service provider. Types of Sample Blood should be collected and immediately taken on ice to the laboratory for separation. Plasma or serum may be used though reference ranges may differ and manufactur- ers’ instructions need to be followed. Samples should be stored frozen. There is little published information on the effects of gel tubes and clot accelerators, and this informa- tion should be sought from manufacturers. C-peptide is sometimes measured in urine and should be collected in boric acid to maximize stability. Insulin can be measured in blood spot samples though such samples should be stored frozen (Butter et al., 2001). A sample for the mea- surement of plasma glucose should be collected at the same time.
  6. 6. 788 The Immunoassay Handbook Urine C-peptide is stable for over 72h when collected in boric acid at room temperature. Urine C-peptide is sta- ble after seven freeze–thaw cycles. The stability of urine C-peptide should be validated on individual analytical platforms. Frequency of Use Mainly limited to specialized laboratories. Uncommon but an increased level of research in the clinical utility of C-peptide and insulin may see an increase in the usage of these tests in the future. GLYCATED HEMOGLOBIN (GHB) More tests are performed to monitor the health of diabetic patients than to diagnose the condition itself. This is true of both types of diabetes and careful, continuous manage- ment of the patient can lead to a full and normal life for many decades. The findings and recommendations of recent major studies (American Diabetes Association, 2010) have emphasized the importance and consequent benefits of rigorous monitoring and especially the role of glycated hemoglobin. Two types of assays are used for monitoring diabetic patients: those that check the degree of metabolic control and those that detect the appearance and progression of the complications that can accompany prolonged diabetes. In neither case are the analytes determined of primary sig- nificance to diabetes but rather they are secondary indica- tors of the short- and long-term effects of the disease. The primary indicator of metabolic control in diabetes is the mean concentration of blood glucose, which is main- tained by the patient through a combination of diet and drug therapy. Because of the rapid fluctuations in blood glu- cose concentration in response to diet and exercise, a ran- dom blood glucose determination is of limited value. Blood glucose meters do allow daily monitoring, but there are limitations to the frequency with which measurements can be made. A means of making a retrospective measurement of average glycemic control is provided by the ability of glu- cose to react directly with exposed amino groups of proteins in a nonenzymic glycation reaction. All proteins with sus- ceptible residues will react if exposed to glucose and the extent to which they are glycated reflects three parameters: their intrinsic reactivity, their half-life, and the mean glu- cose concentration. For any given protein, the first two fac- tors are normally constant and hence the extent of protein glycation is a reliable indirect measure of the time-averaged glucose concentration experienced by the protein through- out its life. Results of these measurements have traditionally been expressed as the percentage of analyte that is glycated. Many proteins, particularly plasma constituents, have been studied in this context, but the one most useful for routine monitoring is glycated hemoglobin. Hemoglobin is an α2β2 tetramer of relative molecular mass 64.5kDa, but its structure and function as an oxygen carrier are merely incidental to its use as an analyte for monitoring glycemic control in diabetes. What is important is the half- life of the molecule, which is determined in turn by the life span of the red blood cell of about 120 days. The molecule of hemoglobin is glycated at several surface lysine residues but also possesses a uniquely reactive site, the amino group of the N-terminal valine of the beta chain. Glycation at this residue sufficiently alters the pKa of the amino group to impart a relative negative charge to the molecule at neu- tral pH. It was on this basis that a glycated form of the molecule was first identified and separated by conventional biochemical techniques. The reaction between the open chain form of glucose and the beta chain valine residue is shown in Fig. 2. The reaction proceeds in two stages, the first to form an unsta- ble aldimine (or Schiff base) often termed pre-HbA1c or labile HbA1c, which reflects the prevailing glucose concen- tration, and a second, slower and effectively irreversible rearrangement that produces a stable ketoamine, HbA1c (Amadori rearrangement). Other reactive hexose phos- phates (glucose-6-phosphate and fructose-1,6-bisphos- phate) react with the same residue to form derivatives that also differ by charge and form fractions of the fast HbA1 group. A comparable reaction sequence involving the ε-amino group of lysine also occurs at a number of surface residues, to produce a heterogeneous fraction termed gly- cated hemoglobin (GHb). The analysis of GHb is complex because of the subtle chemical differences between the variously related deriva- tives of hemoglobin and the different subfractions that are measured. GHb is an unusual analyte, in that it can be determined by a multiplicity of fundamentally different methods that are not limited by considerations of sensitiv- ity, since the analyte is present in high concentrations. Methods are based on differences in the antigenicity, charge, or chemical reactivity of the glycosylated com- pared to the non-glycosylated forms. The particular method used effectively defines the exact molecular species measured and, although all methods correlate reasonably well, the differences between methods may be significant and are not always fully appreciated. Standardization In recent years, these methods have been added to, and con- siderable effort has been made to develop a consensus view and especially an absolute reference method and develop- mentofaprimaryreferencematerial.Immunoassaymethods, both enzyme immunoassays (EIAs) and immunoturbidimet- ric methods, represent only part of the methodology FIGURE 2 The formation of HbA1c from glucose and hemoglobin via the Schiff base pre-HbA1c.
  7. 7. 789CHAPTER 9.10 Diabetes Mellitus available. Older methods, such as gel electrophoresis and chemical affinity (on boronate columns), have generally been replaced and improved upon by developments such as capil- lary electrophoresis, labeled boronate ligands, and improve- ments to HPLC ion-exchange methods. Some of the chromatographic methods have now been automated in a form with a throughput of less than 5min and with instru- mentation of small enough size for use in clinics or satellite laboratories. With such a variety of methods available, it is not surprising that standardization has proved difficult. Immunoassay methods, although generally precise and free from most interferences, are not necessarily superior to other methods, and frequently, the choice is dictated by other con- siderations including cost and instrumentation. To address the issue of poor standardization the Inter- national Federation of Clinical Chemistry (IFCC) estab- lished a working group for HbA1c standardization in 1995. Since the groups’ formation, they have established an internationally accepted definition of the analyte, a pri- mary reference material, and a reference method. HbA1c was defined as Hb that is irreversibly glycated at one or both N-terminal valines of the beta chains. This does not exclude hemoglobin that is additionally glycated at other sites on the alpha or beta chains. For the calibration of the reference method, mixtures were produced of pure HbA1c and pure HbA0 characterized using capillary isoelectric focusing and electrospray ionization mass spectrometry (Finke et al., 1998). In 2001, the IFCC approved two ref- erence methods that specifically measure the glycated N-terminal residue of the beta chain. The reference method is based on enzymatic cleavage of hemoglobin fol- lowed by reverse-phase HPLC and electron-spray ioniza- tion mass spectrometry or capillary electrophoresis (Jeppsson et al., 2002). The relationship between HbA1c results from the National Glycohemoglobin Standardiza- tion Program (NGSP) network (%HbA1c) and the IFCC network (mmol/mol) has been evaluated, and a master equation has been developed with the new IFC units being consistently 1.5–2% lower than the old NGSP (%) units. Reference Intervals In 2007, the IFCC along with the American Diabetes Asso- ciation (ADA), European Association for the Study of Dia- betes (EASD), and International Diabetes Federation (IDF) recommended that newly IFCC standardized HbA1c be expressed as mmol HbA1c/mol Hb. There is also the option of reporting an “interpretation” of the HbA1c result as “esti- mated average glucose” (eAG). In the United Kingdom reporting as IFCC mmol/mol was implemented at the end of 2011 after 2 years of dual reporting of both IFCC (mmol/ mol) and DCCT (%). It was decided eAG would not be reported in the United Kingdom, but this has been adopted in many countries including the United States. National guidelines (NICE, 2009) for the treatment management of Type 2 diabetes recommend that for an individual, a target HbA1c (DCCT aligned) should be between 6.5 and 7.5% (47–58mmol/mol), dependent on the risk of micro- and macrovascular complications. The higher limit may be preferred in those at risk of iatrogenic hypoglycemia. See limitations for a discussion of HbA1c standardization and the relevance to reference intervals. Separate reference ranges may be required for preg- nancy (Mosca et al., 2006). Clinical Applications Measurements of glycated hemoglobin, whether using immunoassays or other technology, have traditionally been reserved for the monitoring of glycemic control as part of the routine management of diabetes. Hemoglobin has proved to be the most applicable analyte for this pur- pose, partly because of the relative ease with which the sample can be taken, but chiefly because the life span of the erythrocyte means that the retrospective window is about 60–120 days. Shorter time frames have not proved as use- ful except in the close monitoring of “brittle diabetics,” newly diagnosed or pregnant patients for which measure- ment of other shorter-lived glycated plasma proteins are appropriate (see later). In 2009, an international expert committee suggested HbA1c can be used as a diagnostic test for diabetes with a cutoff of HbA1c ≥6.5% (47mmol/mol) (International Expert Committee, 2009). The World Health Organiza- tion announced its support for using HbA1c as a diagnostic test for diabetes and also advocates a level of 6.5% (47mmol/mol), providing that stringent quality assurance tests are in place, and assays are standardized to criteria aligned to the international reference values (Report of a WHO Consultation, 2010). A value of less than 6.5% does not exclude diabetes diagnosed using glucose tests. The WHO concluded that there is insufficient evidence to make any formal recommendation on the interpretation of HbA1c levels below 6.5% (47mmol/mol). Limitations G Immunoassays are based on antibodies, which react directly to the glycated sequence of the beta chain of hemoglobin and have good precision and specificity. Depending on how much more of the sequence is required by the antibody for this recognition, an immu- noassay may respond to some specific hemoglobin vari- ants and not others. Of most relevance are HbS and HbC, which both have single amino acid substitutions at position 6 of the beta chain. Although glycation of these variants does not give rise to HbA1c as such, the glycated variants nevertheless still reflect the glycemic state of the patient. As long as there is no change in the percentage of the variant, and the effect of the variant in the assay system is not changed, i.e., the glycated variant is measured, it is theoretically possible to estab- lish baseline values for a given patient. However, the usual reference ranges will not apply, and this approach cannot be advocated. G Physiological (e.g., pregnancy) and pathological condi- tions (e.g., renal failure, hemolytic anemias) that lead to decreased red cell survival, result in decreased values for glycated hemoglobin, which do not reflect the degree of metabolic control. Blood glucose monitoring or measurement of glycated serum protein or glycated serum albumin may be used. The NGSP website lists specific analytical and physiological interferences in HbA1c measurement (
  8. 8. 790 The Immunoassay Handbook G The lack of agreement of GHb methods has led to international efforts to develop reference methods and standards. Clinicians should be made aware that ranges indicating good and poor glycemic control vary between assays. Assay Technology With increasing demand for the measurement of HbA1c, most assays provided by clinical laboratories will be fully automated, whether the assays are based on affinity/liquid chromatography or less commonly immunoassay. Auto- mated methods are based on immunoturbidimetry and, because they determine an effective concentration of HbA1c, also require a separate measurement of the total hemoglobin concentration, so that the result can be expressed as the conventional %HbA1c. A latex agglutina- tion method that uses a monoclonal antibody raised against a glycated peptide corresponding to the N-terminal sequence of HbA1c is widely used. The antibody aggluti- nates a suspension of latex particles that are coupled to this peptide. HbA1c in the sample competes for the antibody thus inhibiting the agglutination and produces a type of competitive immunoassay. The method also performs a simultaneous determination of the total hemoglobin con- centration of the sample, and these measurements are made, together with the necessary calibration, in a dispos- able cassette, which is read in a dedicated reader. Suitable instrumentation is available for use in diabetic clinics, using fingerprick samples and with results being available in less than 5 min. Similarly a homogeneous, competitive immunoturbidim- etry assay, together with a photometric total hemoglobin method, has been used on benchtop and larger analyzers. A polyclonal antibody recognizes the first four amino acids of the glycated beta chain of hemoglobin and forms a soluble immunocomplex with HbA1c in the analyte. Excess unreacted antibody then forms an immune complex with a poly-hapten form of the peptide coupled to a dextran carrier; aggregation is measured by increasing turbidimetry at 340 nm. There are additionally, a number of point-of-care devices that do require a degree of technical skill. It has been reported that some point-of-care instruments do not meet accepted analytical performance goals and their use is not recom- mended for diagnosing diabetes by means of an HbA1c. Desirable Assay Performance Characteristics The NGSP recommends a target precision of CV ≤3%. With respect to bias the same group recommends that the 95% confidence intervals of differences between test meth- ods and the Secondary Reference Laboratory (SRL) should be within ±1% GHb of the SRL. The IFCC and American Association of Clinical Chemistry recommend that between run coefficients of variation should be less than 5%, though CVs of less than 3% are more clinically useful. Types of Sample These assays use whole blood as the sample, typically small volumes of around 100µL. In order to liberate hemoglobin from the erythrocytes, part of the process requires the sample to be lyzed. Commonly available collection methods and anticoagulants (lithium-heparin, ethylenediaminetet- raacetic acid, fluoride-oxalate) can all be used. In addition, several attempts have been made to use hemoglobin eluted from dried blood spots, although this method is not in com- mon use. Frequency of Use The management of diabetes varies both between and within countries, but most patients in the developed world are monitored at least annually, and in some cases, as often as four times per year. The National Institute for Clinical Excellence (2009) has recommended that HbA1c should be monitored at 2–6 month intervals. The interval depends on the acceptable level of control and stability of blood glucose control and/or changes in blood glucose concen- trations and/or changes in therapy. OTHER GLYCATED PROTEINS Although a large number of proteins, both plasma and struc- tural, may be glycated in diabetes besides hemoglobin, the only other glycated protein to be used as an index of glyce- mic control is glycated albumin. This is sometimes deter- mined as a specific analyte, e.g., by immunoassay, or as a major component in the glycated proteins of plasma or serum, e.g., as fructosamine. As with hemoglobin, the struc- ture of albumin and its function as a transport protein are peripheral in this context, and the most significant property of the molecule is its half-life, which, at about 20 days, is much shorter than that of hemoglobin. Thus, albumin offers a measure of glycemic control intermediate between hemo- globin and the direct and immediate glucose determination. Glucose reacts with albumin in the same way as with hemoglobin, but no reaction has been detected at the amino group of the N-terminal asparagine. All the glycated sites are reactive lysines, and there is no fraction of albumin analogous to HbA1c that can be separated on the basis of charge. Glycated albumin is, therefore, a heterogeneous fraction of molecules modified at several sites; Lys 525 has been shown to be the most reactive with nearly half the nonenzymic glycation occurring at this residue. Reference Interval Glycated albumin measurements are not sufficiently com- mon for reference intervals to be widely reported. The suggested upper limit of the nondiabetic range can vary between 2.4 and 3.0%. Clinical Applications Immunoassays for glycated albumin may be regarded as more specific alternatives to fructosamine and can be applied where short-term monitoring is important. Limitations Glycated albumin determinations either as fructosamine or specifically by immunoassay are only of use for short- term monitoring of diabetes and are not likely to replace glycated hemoglobin as the analyte of choice for routine long-term management. Results may not reflect glycemic
  9. 9. 791CHAPTER 9.10 Diabetes Mellitus control accurately in those patients where serum albumin concentrations are low. Assay Technology As with GHb, there are a number of simple chemical tech- nologies available for the determination of glycated albu- min, and immunoassays have only recently become available. The ability of the ketoamine derivative to reduce nitro-blue tetrazolium at alkaline pH forms the basis for the fructosamine method. A number of immunoassays have been developed based both on monoclonal antibodies and on polyclonal anti- sera. The raising of these antibodies is critical to the specificity of the test. Some success has been achieved in producing antibodies specific to the glycated lysine resi- due alone, which then have broad specificities for gly- cated proteins. In a different approach, others have raised antibodies to the glucitol-lysine derivative that is formed by the reduction of the ketoamine. This produces a more immunogenic molecule, but any assay must subsequently incorporate the same reduction step into its protocol. The immunoassays have not found widespread use, though the development of an automated enzyme assay for glycated albumin may broaden its use (Kouzuma et al., 2004). Desirable Assay Performance Characteristics Though desirable and detailed performance characteristics may not have been reported, there is guidance on quality specifications (Fraser and Petersen, 1999). For long-term monitoring of glycemic control, long-term precision will be critical. Types of Sample Both serum and plasma have been used for the determina- tion of glycated albumin. Frequency of Use Rarely used. ALBUMINURIA The term “microalbuminuria,” i.e., the presence of low concentrations of albumin in the urine, is strictly a misno- mer as it suggests the presence of small molecules of albu- min, and there is a move to use the broader term of albuminuria with defined cutoff values. The determination of albuminuria is widely used as a predictor of microvascu- lar disease in diabetes but is also a risk factor for cardiovas- cular disease and end-stage renal disease. Reference Interval There has been considerable variation in the values used to define albuminuria and in the units for reporting (mg per 24h, µg per min, or µg per unit of creatinine). It is now recommended that a spot urine is collected in the early morning with repeat samples collected at the same time of day. (Table 4). Clinical Applications Classic proteinuria, of which albumin is the major compo- nent, is associated with the late and irreversible stages of renal disease, and only comparatively recently have the earlier, incipient phases been unequivocally correlated with the lower urine concentrations of albumin, previously known as microalbuminuria. As diabetic nephropathy develops, there is a loss both in the size and in the charge selectivity of the basement membrane, and the excretion of albumin increases in absolute terms and also as a propor- tion of the total excreted protein. The early detection of albuminuria, a risk factor for subsequent renal impairment and other complications, is critically important to its treatment, and once persistent proteinuria has developed, the decline of kidney func- tion seems to be irreversible. At this stage, conventional tests of renal function are used. There is much data to support the early treatment of hypertension in the dia- betic, even when mild, in order to prevent and/or delay the development of nephropathy and other complica- tions. Patients with diabetes are routinely monitored for albuminuria. Limitations G Physiological variations in excretion mean that care must be taken with the type of sample collected (see below). Transient increases have been reported, for example, during acute febrile illness, short-term hyper- glycemia, heart failure. G Contamination of the urine sample with menstrual blood or seminal fluid may cause inaccurate results. G Factors affecting urine creatinine concentrations such as ethnicity and muscle wasting will affect the albumin/ creatinine ratio (ACR). G The range of concentrations of albumin in urine will be wide, and procedures should be in place to avoid the high dose hook effect. G Differences in calibration of assays, particularly when the patient is monitored by both clinic and laboratory methods, may result in changes in measured analyte concentrations that are clinically significant, resulting in inappropriate changes to treatment. G There is a requirement for long-term stability of assays as they will be used for lifetime monitoring of patients. TABLE 4 Definitions of Urine Albumin Excretion (Sacks et al., 2011) Parameter Normal Higher Risk Urine Albumin Excretion Albuminuria Very High Albuminuria Concentration (µg/ min), ADA (2010) <20 20–200 >200 ACR (mg/mmol), ADA (2010) and NICE (2009) <3.39 3.39–33.9 >2.5 men >3.5 women >33.9 ADA, American Diabetes Association. NICE, National institute of Clinical Excellence.
  10. 10. 792 The Immunoassay Handbook G Urine albumin is stable for 7–14 days at room tempera- ture and at +4°C (in the absence of urinary tract infec- tion). Stability at −20°C has been questioned, though lower temperatures are thought to be suitable. Assay Technology Overt proteinuria does not require a sensitive assay, and simple dipstick/point-of-care tests have been based on the spectral changes caused by the binding of albumin to an organic dye. Automated chemical methods are used to quantitate overt proteinuria. Point-of-care devices should be evaluated as they may have insufficient sensitivity for the detection of albuminuria and appropriate quality assur- ance procedures put in place. To detect albumin concentrations 100 times lower, however, more sensitive immunoassay methods have been developed including RIAs, immunoturbidometric and nephelometric assays, and enzyme immunossays. The requirements for high sample throughput and fast assays mean that automation is essential. Desirable Assay Performance Characteristics Because the measurement of albuminuria is used to monitor patients over many years, long-term stability of the assays is required. Most quantitative assays for this purpose have detection limits of less than 20µg/L and given the large biological variability, achieve a total imprecision consistent with the analytical goal, of less than 15%. Types of Sample The rate of albumin excretion is subjected to several fac- tors that influence renal function, but are unconnected with any pathological state. These include posture, rest or exercise, and time of day; these considerations are reflected in the type of urine sample collected, and whether the results are expressed as a simple concentration or a rate of excretion. There are four ways in which urine is commonly collected: 1. Timed overnight. Allows the overnight albumin excretion rate (AER) to be determined; this is regarded as the most reliable index. Allows more stringent criteria to be applied and measures a lower AER because of overnight inactivity but generally is not now recommended due to the potential for errors in collection. 2. First morning “spot” urine. More convenient for the patient and retaining most of the advantages of a timed overnight sample. 3. Random. Most convenient for the patient, but single measurements of randomly taken urine are of little or no use because of the uncontrolled fac- tors already mentioned. To reduce variability, repeat samples should be collected at the same time of day. 4. 24h. Allows the average AER to be determined in a 24h period but is least convenient for the patient and subject to errors of collection. Not recommended. It is common for additional measurements to be made to eliminate variations in renal function. Creatinine is the normal choice of analyte, and albumin concentrations are then expressed as an ACR. Frequency of Use It has been suggested that urine albumin should be mea- sured at diagnosis and annually thereafter. If albuminuria or proteinuria is found, this should be repeated on at least two occasions within 1 month. More regular monitoring may be necessary to monitor treatment. AUTOIMMUNE AND OTHER ASSAYS Recent advances in the understanding of the pathogenesis of diabetes, especially of the autoimmune mechanism of Type 1 diabetes, have led to the identification of a number of immunological markers associated with this condition. These include autoantibodies to islet cell cytoplasm (ICA), endogenous insulin (IAA), two tyrosine phosphatases (IA- 2A and IA-2βA), glutamic acid decarboxylase (GAD65A), and zinc transporter 8 (ZnT8). The islet cell cytoplasmic antibody (ICA) is usually mea- sured in peripheral blood by indirect immunofluorescence methods using frozen sections of human pancreas. Results are compared to a standard serum and reported as Juvenile Diabetes Foundation (JDF) units. Generally, results of 10 JDF units on two separate occasions or a single result ≥20 JDF units are considered as significant. ICA has been detected in roughly 3% of a background population and in 15–30% of patients with Type 1 diabetes, though this rises to 70–80% in patients at the time of diagnosis. Testing for pancreatic islet cell autoantibody (ICA) is labor intensive, technically demanding and poorly standardized. Unique islet antigens contributing to the heterogeneous ICA immunofluorescence staining include GAD65, IA-2, IAA, and ZnT8. Most patients with Type 1 diabetes will have multiple islet cell autoantibodies detectable in their blood at diagnosis, and less than 5% will have only one detectable antibody when assessed using a combination of ICA and antibodies to GAD65, IA-2, IAA, and ZnT8 autoantibodies. Interestingly, all the Type 1 diabetes auto- antibodies have epitopes related to the secretory apparatus of the beta cell. Traditionally, antibodies to IA-2, IA-2 beta, and GAD65 have been assessed by radioligand-binding assays, which are technically demanding (Borg et al., 1997). Radioligand assays using 35S-labeled GAD showed high sensitivity but are technically demanding and subsequently commercial assays using 125I have become available. In recent years, robust commercial ELISA assays have been developed for both GAD65 and IA-2 antibody. The Diabetes Autoanti- body Standardization Programme, a collaboration between the US Centers for Disease Control and Prevention and the Immunology of Diabetes Society, has demonstrated that these commercial EIA methods are both as sensitive and specific as traditional radioligand techniques (Torn et al., 2008). In addition to insulin antibodies detected in patients treated with exogenous insulin (normally polyclonal IgGs), insulin autoantibodies (IAA) have been identified in
  11. 11. 793CHAPTER 9.10 Diabetes Mellitus untreated patients who are either newly diagnosed with Type 1 diabetes or shown to be at high risk of developing it. These are measured by radioisotopic methods based on the displacement of insulin radioligand after the addition of excess non-labeled insulin. Whether these antibodies are a direct causal factor or a consequence of beta cell destruc- tion is uncertain. The importance of insulin autoantibodies thus remains controversial, and there are many conflicting reports in the literature, some of which have been attrib- uted to shortcomings in the assays used (Greenham and Palmer, 1991). Insulin autoantibodies are still poorly stan- dardized and technically demanding; as a result, they are restricted to a few specialized research laboratories. The islet autoantibody assays mentioned above are not routinely regarded as diagnostic nor routinely used in screening or monitoring. They are, however, more fre- quently used in the differential diagnosis of young diabetic patients with an uncertain etiology. They are widely used as research tools, nevertheless, the hope and expectation are that there will be major advances in the prediction of those at risk of developing Type 1 diabetes that in the future may allow the development of therapeutic strategies for the prevention of this disease. ADIPOKINES Obesity and insulin resistance are clearly associated with the development of Type 2 diabetes and the metabolic syndrome. Adipose tissue is now recognized as an endo- crine tissue that plays a role in the control of energy homeostasis, lipid metabolism, angiogenesis, insulin sensi- tivity, and immune regulation. Adipose tissue secretes a number of bioactive proteins, named adipocytokines, which include leptin, adiponectin, tumor necrosis factor (TNF) alpha, resistin, visfatin, and retinol-binding protein 4 (Rasauli and Kern, 2008; Miehle et al., 2012). Leptin is a 16kDa adipocyte-derived protein that circu- lates in free and bound form. Leptin acts by binding to specific receptors in the hypothalamus to affect energy intake and expenditure stimulating the expression of pro- opiomelanocortin. Mutations in genes for both leptin and its receptor have been described in humans and are associ- ated with gross obesity and serum leptin concentrations inappropriate for the degree of obesity (Farooqi and O’Rahilly, 2009). Generally, serum concentrations are directly related to body mass index. Leptin itself may sup- press insulin secretion. A number of immunoassays are available for the measurement of circulating concentra- tions of leptin, including assays for serum free and bound leptin (Lewandoski et al., 1999). Human adiponectin is a 28kDa protein secreted by adi- pose tissue that exists in three major oligomeric forms: a low molecular weight trimer, a middle molecular weight hexamer, and a higher molecular weight multimer (Sinha et al., 2007). This complex structure has important conse- quences for the design of immunoassays for the protein. Some of the differences in reported concentrations may reflect differences in antibody specificity. A number of commercial research assays are available. Although secreted by adipocytes, paradoxically circulating concen- trations are higher in lean individuals than in obese. Adi- ponectin may have a causative role in insulin resistance, and equally, circulating concentrations may be influenced by hyperinsulinemia. Anti-inflammatory properties have also been described. Serum concentrations are influenced by age and gender. Resistin is an adipocyte-derived polypeptide that has been described in rodents with human resistin showing only 59% homology to the mouse form of the polypeptide. Study of mRNA expression, and circulating tissue concentrations in both animals and humans, has yielded conflicting data con- cerning the role of the protein in insulin resistance and obe- sity. The biological role of resistin remains to be established (Pittas et al., 2004). Both competitive and immunometric assays for resistin are available (Pfutzner et al., 2003). Measurement of adipokines is restricted chiefly to the research laboratory. Significant variability in results obtained by immunoassays can mean very different clinical interpretations, e.g., in the qualitative and quantitative assays for visfatin (Korner et al., 2007). References and Further Reading American Diabetes Association Clinical Practice Recommendations. Executive summary: standards of medical care in diabetes. Diabetes Care 33(Suppl 1), S4–S5 (2010). American Diabetes Association Position Statement. Diagnosis and classification of diabetes mellitus. Diabetes Care 27, S5–S10 (2004). American Diabetes Association Position Statement. Diagnosis and classification of diabetes mellitus. Diabetes Care 30(Suppl 1), S42–S47 (2007). Besser, R.E., Shepherd, M.H., McDonald, T.J., et al. Urinary C-peptide creatinine ratio is a practical outpatient tool for identifying hepatocyte nuclear factor 1-{alpha}/hepatocyte nuclear factor 4-{alpha} maturity-onset diabetes of the young from long-duration type 1 diabetes. Diabetes Care 34, 286–291 (2011). Borg, H., Fernlund, P. and Sundkvist, G. Measurement of antibodies against glu- tamic acid decarboxylase 65 (GADA): two new 125I assays compared with [35S] GAD 65-ligand binding assay. Clin. Chem. 43, 779–785 (1997). Butter, N.L., Hattersley, A.T. and Clark, P.M. Development of a bloodspot assay for insulin. Clin. Chim. Acta 310, 141–150 (2001). Clark, P.M. Assays for insulin, proinsulin(s) and C-peptide. Ann. Clin. Biochem. 36, 541–564 (1999). Cryer, P.E., Axelrod, L., Grossman, A.B., et al. Evaluation and management of adult hypoglycaemic disorders: an Endocrine Society clinical practice guide- line. J. Clin. Endocrinol. Metab. 94, 709–728 (2009). The Diabetic Control and Complication Trials Research Group. The effect of intensive treatment of diabetes on the development and progression of long- term complications in insulin-dependent diabetes mellitus. New Engl. J. Med. 329, 977–986 (1993). Ehtisham, S. and Barrett, T.G. Emergence of type 2 diabetes in childhood. Ann. Clin. Biochem. 41, 10–16 (2004). Farooqi, I.S. and O’Rahilly, S.O. Leptin: a pivotal regulator in human energy homeostasis. Am. J. Clin. Nutr. 89, 980S–984S (2009). Finke, A., Kobold, U., Hoelzel, W., et al. Preparation of a candidate primary refer- ence material for the international standardization of HbA1c determinations. Clin. Chem. Lab. Med. 36, 299–308 (1998). Fraser, C.G. and Petersen, P.H. Analytical performance characteristics should be judged against objective quality specifications. Clin. Chem. 45, 321–323 (1999). Greenham, C.J. and Palmer, J.P. Insulin antibodies and autoantibodies. Diab. Med. 8, 97–105 (1991). Greenbaum, C.J., Mandrup-Poulsen, T., McGee, P.F., et al. Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes. Diabetes Care 31, 1966–1971 (2008). Hohberg, C., Pfutzner, A., Forst, T., et al. Successful switch from insulin therapy to treatment with pioglitazone in type 2 diabetes patients with residual betacell func- tion: results from the PioSwitch study. Diabetes Obes. Metab. 11, 464–471 (2009). International Expert Committee report on the role of the A1C assay in the diagno- sis of diabetes. Diabetes Care 32, 1327–1334 (2009). Jeppsson, J.O., Kobold, U., Barr, J., et al. Approved IFCC reference method for the measurement of HbA1c in human blood. Clin. Chem. Lab. Med. 40, 78–89 (2002). Katz, A., Nambi, S.R., Mather, K., et al. Quantitative insulin sensitivity index: a simple, accurate method for assessing insulin sensitivity in humans. J. Clin. Endocrinol. Metabol. 85, 2402–2410 (2000). Körner, A., Garten, A., Blüher, M., et al. Molecular characteristics of serum visfatin and differential detection by immunoassays. J. Clin. Endocrinol. Metab. 92, 4783–4791 (2007).
  12. 12. 794 The Immunoassay Handbook Koskinen, P., Viikar, I.J., Irjala, K., et al. Plasma and urinary C-peptide in the clas- sification of adult diabetics. Scand. J. Clin. Lab. Invest. 46, 655–663 (1986). Kouzuma, T., Uemastu, Y., Usami, T., et al. Study of glycated amino acid elimina- tion reaction for an improved enzymatic glycated albumin measurement method. Clin. Chim. Acta 346, 135–143 (2004). Lewandoski, K., Horn, R., O’Callahan, C.J., et al. Free leptin, bound leptin and soluble leptin receptor in normal and diabetic pregnancies. J. Clin. Endocrinol. Metab. 84, 300–306 (1999). Lupsa, B.C., Chong, A.Y., Cochran, E.K., et al. Autoimmune forms of hypoglycae- mia. Medicine (Abingdon) 88, 141–153 (2009). McCarthy, M. and Hattersley, A.T. Novel insights arising from the definition of genes for monogenic and type 2 diabetes. Diabetes 57, 2889–2898 (2008). McDonald, T.J., Knight, B.A., Shields, B.M., et al. Stability and reproducibility of a single sample urinary C-peptide/creatinine ratio and its correlation with 24-h urinary C-peptide. Clin. Chem. 55, 2035–2039 (2009). Miehle, K., Stephan, H. and Fasshauer, M. Leptin, Adiponectin and other adipokines in gestational diabetes mellitus and preeclampsia. Clin. Endocrinol., 76, 2–11 (2012). Miller, W.G., Bruns, D.E., Hortin, G.L., et al. Current issues in measurement and reporting of urinary albumin excretion. Clin. Chem. 55, 24–38 (2009). Mosca, A., Paleari, R., Dalfra, M.G., et al. Reference intervals for hemoglobin A1c in pregnant women: data from an Italian multicenter study. Clin. Chem. 52, 1138–1143 (2006). National Glycohemoglobin Standardization Program: http://www.missouri. edu/~diabetes/ngsp/. National Institute of Clinical Excellence. The management of type 2 diabetes. Guideline 87 (2009). Owen, W.E. and Roberts, W.L. Cross-reactivity of three recombinant insulin ana- logs with five commercial insulin immunoassays. Clin. Chem. 50, 257–259 (2004). Pittas, A.G., Joseph, N.A. and Greenberg, A.S. Adipocytokines and insulin resis- tance. J. Clin. Endocrinol. Metab. 89, 447–452 (2004). Pfutzner, A., Langenfeld, M., Kunt, T., Lobig, M. and Forst, T. Evaluation of human resistin assays with serum from patients with type 2 diabetes and differ- ent degrees of insulin resistance. Clin. Lab. 49, 571–576 (2003). Rasouli, N. and Kern, P.A. Adipocytokines and the metabolic complications of obesity. J. Clin. Endocrinol. Metab. 93, S64–S73 (2008). Report of a WHO Consultation Use of Glycated Haemoglobin (HbA1c) in the Diagnosis of Diabetes Mellitus. World Health Organization, (2010). Sacks, D.B., Arnold, M., Bakris, G.L., et al. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin. Chem. 57, e1–e47 (2011). Sapin, R. Insulin immunoassays: fast approaching 50 years of existence and still calling for standardization. Clin. Chem. 53, 810–812 (2007). Sinha, M.K., Songer, T., Xiao, Q., et al. Analytical validation and biological evalu- ation of a high molecular-weight adiponectin ELISA. Clin. Chem. 53, 2144– 2151 (2007). Torn, C., Mueller, P.W., Schlosser, M., et al. Diabetes Antibody Standardization program: evaluation of assays for autoantibodies to glutamic acid decarboxylase and islet antigen-2. Diabetologia 51, 846–852 (2008). UK Prospective Diabetes Study (UKPDS) Group Intensive blood glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). Lancet 352, 837–853 (1998). Wallace, T.M., Levy, J.C. and Matthews, D.R. Use and abuse of HOMA Modeling. Diabetes Care 27, 1487–1495 (2004). Wark, G. UKNEQAS Guildford Peptide Hormones Annual Review. (2006). World Health Organization. Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycaemia, ISBN 92 4 159493 4 (2006). Yalow, R.S. and Berson, S.A. Assay of plasma insulin in human subjects by immu- nological methods. Nature 184, 1648–1649 (1959).