Reviews/Commentaries/ADA Statements POSITION STATEMENT

Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Mellitus

1 6 DAVID B. SACKS M. SUE KIRKMAN mellitus, formerly known as - 2 7 MARK ARNOLD AKE LERNMARK 3 8 dependent diabetes mellitus (IDDM) or GEORGE L. BAKRIS BOYD E. METZGER 4 9 juvenile-onset diabetes mellitus, is usu- DAVID E. BRUNS DAVID M. NATHAN 5 ally caused by autoimmune destruction ANDREA RITA HORVATH of the pancreatic islet b-cells, rendering the unable to synthesize and se- crete insulin (2). mellitus, BACKGROUND—Multiple laboratory tests are used to diagnose and manage patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these tests varies formerly known as non-IDDM or adult- substantially. onset diabetes, is caused by a combina- tion of and inadequate APPROACH—An expert committee compiled evidence-based recommendations for the use of insulin secretion (3,4). Gestational diabe- laboratory testing for patients with diabetes. A new system was developed to grade the overall quality tes mellitus (GDM), which resembles type of the evidence and the strength of the recommendations. Draft guidelines were posted on the fi 2diabetesmorethantype1,develops Internet and presented at the 2007 Arnold O. Beckman Conference. The document was modi ed in during approximately 7% (range, 5%– response to oral and written comments, and a revised draft was posted in 2010 and again modified in response to written comments. The National Academy of Clinical Biochemistry and the Evidence- 15%) of pregnancies, usually remits after Based Laboratory Medicine Committee of the American Association for Clinical Chemistry jointly delivery, and constitutes a major risk fac- reviewed the guidelines, which were accepted after revisions by the Professional Practice Committee tor for the development of type 2 diabetes and subsequently approved by the Executive Committee of the American Diabetes Association. later in life. Other types of diabetes are rare. Type 2 is the most common form, — CONTENT In addition to long-standing criteria based on measurement of plasma , accounting for 85%–95% of diabetes in diabetes can be diagnosed by demonstrating increased blood hemoglobin A1c (HbA1c) concen- developed countries. Some patients can- trations. Monitoring of glycemic control is performed by self-monitoring of plasma or blood not be clearly classified as type 1 or type 2 glucose with meters and by laboratory analysis of HbA1c. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, diabetes (5). proinsulin, C-peptide, and other analytes are addressed. Diabetes is a common disease. The current worldwide prevalence is esti- SUMMARY—The guidelines provide specific recommendations that are based on published mated to be approximately 250 x 106, data or derived from expert consensus. Several analytes have minimal clinical value at present, and it is expected to reach 380 x 106 by and their measurement is not recommended. 2025 (6). The prevalence of diabetes Diabetes Care 34:e61–e99, 2011 [based on fasting plasma glucose (FPG) results] in U.S. adults in 1999–2002 was 9.3%, of which 30% of the cases were un- fi iabetes mellitus is a group of met- . The disease is classi ed diagnosed (7). The most recent data, abolic disorders of carbohydrate into several categories. The revised clas- – D fi which were derived from the 2005 metabolism in which glucose is si cation, published in 1997 (1), is pre- 2006 National Health and Nutrition Ex- underutilized and overproduced, causing sented in Table 1. amination Survey (NHANES) with both ccccccccccccccccccccccccccccccccccccccccccccccccc FPG and 2-h oral (OGTT) results, show a prevalence of di- From the 1Department of Laboratory Medicine, National Institutes of Health, Bethesda, Maryland; the 2Department 3 abetes in U.S. persons $20 years old of of Chemistry, University of Iowa, Iowa City, Iowa; the Department of Medicine, Hypertensive Disease Unit, 6 Section of , Diabetes and Metabolism, University of Chicago, Chicago, Illinois; the 4Department 12.9% (approximately 40 x 10 )(8).Of of Pathology, University of Virginia Medical School, Charlottesville, Virginia; the 5Screening and Test Evaluation these individuals, 40% (approximately 16 Program, School of Public Health, University of Sydney, SEALS Department of Clinical Chemistry, Prince million) are undiagnosed. The prevalence of Wales Hospital, Sydney, Australia; the 6American Diabetes Association, Alexandria, Virginia; the 7Department of Clinical Sciences, Lund University/CRC, Skane University Hospital Malmö, Malmö, Sweden; the 8Division of of diabetes has also increased in other Endocrinology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois; and 9Massachusetts parts of the world. For example, recent General Hospital and Harvard Medical School, Diabetes Center, Boston, Massachusetts. estimates suggest 110 x 106 diabetic indi- Corresponding author: David B. Sacks, [email protected]. viduals in Asia in 2007 (9), but the true Received 30 December 2010 and accepted 28 February 2011. DOI: 10.2337/dc11-9998 number is likely to be substantially This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/suppl/doi:10. greater, because China alone was thought 6 2337/dc11-9998/-/DC1. to have 92.4 x 10 adults with diabetes in This article is simultaneously published in Clinical Chemistry and Diabetes Care, under joint copyright, and by 2008 (10). the National Academy of Clinical Biochemistry. The worldwide costs of diabetes were © 2011 by the American Diabetes Association and the American Association for Clinical Chemistry. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the approximately $232 billion in 2007 and work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. are likely to be $302 billion by 2025 (6). See accompanying article, p. 1419. In 2007, the costs of diabetes in the U.S. care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e61 Position Statement

Table 1—Classification of diabetes quality of the evidence (Table 2) and the several headings and subheadings (in mellitusa strength of recommendations (Table 3). parentheses), which are as follows: use This guideline focuses primarily on (diagnosis, screening, monitoring, and I. Type 1 diabetes the laboratory aspects of testing in di- prognosis); rationale (diagnosis and A. Immune-mediated abetes. It does not address any issues screening); analytical considerations (pre- B. Idiopathic related to the clinical management of analytical, including reference intervals; II. Type 2 diabetes diabetes, which are already covered in and analytical, such as methods); inter- III. Other specifictypes the American Diabetes Association (ADA) pretation (including frequency of mea- A. Genetic defects of b-cell function guidelines. The NACB guideline intends surement and turnaround time); and, B. Genetic defects in insulin action to supplement the ADA guidelines in where applicable, emerging considera- C. Diseases of the exocrine pancreas order to avoid duplication or repetition tions, which alert the reader to ongoing D. Endocrinopathies of information. Therefore, it focuses on studies and potential future aspects rele- E. Drug- or chemical-induced practical aspects of care to assist with vant to that analyte. F. Infections decisions related to the use or interpreta- G. Uncommon forms of immune-mediated tion of laboratory tests while screening, diabetes GLUCOSE diagnosing, or monitoring patients with H. Other genetic syndromes sometimes diabetes. Additional details concerning 1. Use associated with diabetes the scope, purpose, key topics, and tar- IV. GDM a gets of this guideline are described in the From the ADA (378). accompanying Supplementary Data. RECOMMENDATION: WHEN GLUCOSE IS To facilitate comprehension and as- USED TO ESTABLISH THE DIAGNOSIS were estimated to be $174 billion (11). sist the reader, we divide each analyte into OF DIABETES, IT SHOULD BE MEASURED IN The mean annual per capita healthcare VENOUS PLASMA costs for an individual with diabetes are A(high). approximately 2.3-fold higher than those Table 2—Rating scale for the quality of for individuals who do not have diabetes evidence RECOMMENDATION: WHEN GLUCOSE IS (11). Similarly, diabetes in the U.K. ac- USED FOR SCREENING OF HIGH-RISK counts for roughly 10% of the National High: Further research is very unlikely to INDIVIDUALS, IT SHOULD BE MEASURED Health Service budget (equivalent in change our confidence in the estimate of effect. The body of evidence comes from IN VENOUS PLASMA 2008 to £9 billion/year). The high costs B (moderate). of diabetes are attributable to care for both high-level individual studies that are acute conditions (such as sufficiently powered and provide precise, consistent, and directly applicable results in and ketoacidosis) and debilitating compli- RECOMMENDATION: PLASMA GLUCOSE a relevant population. cations (12). The latter include both micro- SHOULD BE MEASURED IN AN — Moderate: Further research is likely to have an vascular complications predominantly ACCREDITED LABORATORY WHEN USED — important impact on our confidence in the retinopathy, nephropathy, and neuropathy FOR DIAGNOSIS OF OR SCREENING FOR estimate of effect and may change the and macrovascular complications, partic- DIABETES estimate and the recommendation. The ularly stroke and coronary artery disease. Good Practice Point (GPP). Together, they make diabetes the fourth body of evidence comes from high-/ most common cause of death in the de- moderate-level individual studies that are 6 sufficient to determine effects, but the veloped world (13). About 3.8 x 10 peo- RECOMMENDATION: OUTCOME STUDIES strength of the evidence is limited by the ple worldwide were estimated to have ARE NEEDED TO DETERMINE THE number, quality, or consistency of the died from diabetes-related causes in 2007 EFFECTIVENESS OF SCREENING included studies; generalizability of results (6). C (moderate). The National Academy of Clinical to routine practice; or indirect nature of the Biochemistry (NACB) issued its “Guide- evidence. lines and Recommendations for Labora- Low: Further research is very likely to have an A. Diagnosis/screening. The diagnosis tory Analysis in the Diagnosis and important impact on our confidence in the of diabetes is established by identifying Management of Diabetes Mellitus” in estimate of effect and is likely to change the the presence of hyperglycemia. For many 2002 (14). These recommendations estimate and the recommendation. The years the only method recommended for werereviewedandupdatedwithan body of evidence is of low level and comes diagnosis was a direct demonstration of evidence-based approach, especially in key from studies with serious design flaws, or hyperglycemia by measuring increased areas in which new evidence has emerged evidence is indirect. glucose concentrations in the plasma since the 2002 publication. The process of Very low: Any estimate of effect is very (15,16). In 1979, a set of criteria based updating guideline recommendations fol- uncertain. Recommendation may change on the distribution of glucose concentra- lowed the standard operating procedures when higher-quality evidence becomes tions in high-risk populations was estab- for preparing, publishing, and editing available. Evidence is insufficient to assess lished to standardize the diagnosis (15). NACB laboratory medicine practice the effects on health outcomes because of These recommendations were endorsed guidelines, and the key steps are detailed limited number or power of studies, by the WHO (16). In 1997, the diagnostic in the Supplementary Data that accom- important flaws in their design or conduct, criteria were modified (1) to better iden- panies this special report. A new system gaps in the chain of evidence, or lack of tify individuals at risk of retinopathy and was developed to grade both the overall information. nephropathy (17,18). The revised criteria e62 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates

Table 3—Grading the strength of recommendations Table 4—Criteria for the diagnosis of diabetesa A. The NACB strongly recommends adoption Strong recommendations for adoption are made when c There is high-quality evidence and strong or very strong agreement of experts that the Any one of the following is diagnostic: $ b intervention improves important health outcomes and that benefits substantially 1. HbA1c 6.5% (48 mmol/mol) outweigh harms; or OR c c There is moderate-quality evidence and strong or very strong agreement of experts that 2. FPG $7.0 mmol/L (126 mg/dL) the intervention improves important health outcomes and that benefits substantially OR outweigh harms. 3. 2-h Plasma glucose $11.1 mmol/L Strong recommendations against adoption are made when (200 mg/dL) during an OGTTd c There is high-quality evidence and strong or very strong agreement of experts that the OR intervention is ineffective or that benefits are closely balanced with harms, or that harms 4. Symptoms of hyperglycemia and casual clearly outweigh benefits; or plasma glucose $11.1 mmol/L e c There is moderate-quality evidence and strong or very strong agreement of experts that (200 mg/dL) the intervention is ineffective or that benefits are closely balanced with harms, or that aIn the absence of unequivocal hyperglycemia, these fi criteria should be confirmed by repeat testing. From harms outweigh bene ts. b B. The NACB recommends adoption the ADA (378). The test should be performed in a laboratory that is NGSP certified and standardized Recommendations for adoption are made when to the DCCT assay. Point-of-care assays should not c There is moderate-quality evidence and level of agreement of experts that the be used for diagnosis. cFasting is defined as no ca- intervention improves important health outcomes and that benefits outweigh harms; or loric intake for at least 8 h. dThe OGTT should be c There is low-quality evidence but strong or very strong agreement and high level of performed as described by the WHO, with a glucose fi load containing the equivalent of 75 g of anhydrous con dence of experts that the intervention improves important health outcomes and that glucose dissolved in water. e“Casual” is defined as benefits outweigh harms; or any time of day without regard to time since previous c There is very low–quality evidence but very strong agreement and very high level of meal. The classic symptoms of hyperglycemia in- confidence of experts that the intervention improves important health outcomes and that clude polyuria, polydipsia, and unexplained weight benefits outweigh harms. loss. Recommendations against adoption are made when who have unequivocal hyperglycemia, c There is moderate-quality evidence and level of agreement of experts that the intervention is . ineffective or that benefits are closely balanced with harms, or that harms outweigh benefits; or i.e., 11.1 mmol/L (200 mg/dL) with symptoms consistent with hyperglyce- c There is low-quality evidence but strong or very strong agreement and high level of confidence of experts that the intervention is ineffective or that benefits are closely mia]. The WHO and the International Di- balanced with harms, or that harms outweigh benefits; or abetes Federation (IDF) recommend either an FPG test or a 2-h postload glu- c There is very low–quality evidence but very strong agreement and very high levels of confidence of experts that the intervention is ineffective or that benefits are closely cose test that uses the same cutoffs as the balanced with harms, or that harms outweigh benefits. ADA (19) (Table 5). In 2009, the Interna- C. The NACB concludes that there is insufficient information to make a recommendation tional Expert Committee (20), which Grade C is applied in the following circumstances: comprised members appointed by the ADA, the European Association for the c Evidence is lacking or scarce or of very low quality, the balance of benefits and harms cannot be determined, and there is no or very low level of agreement of experts for or Study of Diabetes, and the IDF, recom- against adoption of the recommendation. mended that diabetes be diagnosed — by measurement of hemoglobin A1c c At any level of evidence particularly if the evidence is heterogeneous or inconsistent, fl indirect, or inconclusive—if there is no agreement of experts for or against adoption of (HbA1c), which re ects long-term blood the recommendation. glucose concentrations (see HbA1c section GPP. The NACB recommends it as a good practice point below). The ADA (21) and the WHO have GPPs are recommendations mostly driven by expert consensus and professional agreement endorsed the use of HbA1c for diagnosis of and are based on the information listed below and/or professional experience, or widely diabetes. accepted standards of best practice. This category applies predominantly to technical (e.g., Testing to detect type 2 diabetes in preanalytical, analytical, postanalytical), organizational, economic, or quality-management asymptomatic people, previously contro- aspects of laboratory practice. In these cases, evidence often comes from observational studies, versial, is now recommended for those at audit reports, case series or case studies, nonsystematic reviews, guidance or technical risk of developing the disease (21,22). – The ADA proposes that all asymptomatic documents, non evidence-based guidelines, personal opinions, expert consensus, or position $ statements. Recommendations are often based on empirical data, usual practice, quality people 45 years of age be screened in a requirements, and standards set by professional or legislative authorities or accreditation healthcare setting. An HbA1c,FPG,or2-h bodies, and so forth. OGTT evaluation is appropriate for screening (21). The IDF recommends that the health service in each country de- comprised: 1)anFPGvalue$7.0 mmol/L glucose concentration $11.1 mmol/L cide whether to implement screening for (126 mg/dL); 2) a 2-h postload glucose (200 mg/dL) (Table 4) (1). If any one of diabetes (23). FPG is the suggested test. In concentration $11.1 mmol/L (200 mg/dL) these three criteria is met, confirmation by contrast, the International Expert Com- during an OGTT; or 3) symptoms of di- repeat testing on a subsequent day is nec- mittee and the ADA have recommended abetes and a casual (i.e., regardless of the essary to establish the diagnosis [note that that HbA1c can be used for screening for time of the preceding meal) plasma repeat testing is not required for patients diabetes (20,21,24) (see section on HbA1c care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e63 Position Statement

Table 5—WHO criteria for interpreting 2-h OGTTa B. Monitoring/prognosis. There is a di- rect relationship between the degree of 2-h OGTT result, mmol/L (mg/dL) chronic plasma glucose control and the risk of late renal, retinal, and neurologic 0h 2h complications. This correlation has been Impaired fasting glucoseb .6.1 (110) to ,7.0 (126) ,7.8 (140) documented in epidemiologic studies Impaired glucose tolerancec ,7.0 (126) .7.8 (140) to ,11.1 (200) and clinical trials for both type 1 (42) Diabetesd .7.0 (126) .11.1 (200) and type 2 (43) diabetes. The important aValues are for venous plasma glucose using a 75-g oral glucose load. From the WHO (19). bIf 2-h glucose is causal role of hyperglycemia in the devel- not measured, status is uncertain as diabetes or impaired glucose tolerance cannot be excluded. cBoth fasting opment and progression of complications and 2-h values need to meet criteria. dEither fasting or 2-h measurement can be used. Any single positive has been documented in clinical trials. result should be repeated on a separate day. Persons with type 1 diabetes who maintain lower mean plasma glucose concentrations below). If an FPG result is ,5.6 mmol/L $34,375, with ages between 55 and 75 exhibit a significantly lower incidence of (100 mg/dL) and/or a 2-h plasma glucose years being the most cost-effective (33). microvascular complications—namely, concentration is ,7.8 mmol/L (140 Modeling run on 1 x 106 individuals sug- diabetic retinopathy, nephropathy, and mg/dL), testing should be repeated at 3- gests considerable uncertainty as to neuropathy (44). Although intensive insu- year intervals. Screening should be consid- whether screening for diabetes would be lin therapy reduced hypercholesterolemia ered at a younger age or be carried out more cost-effective (34). By contrast, the results by 34%, the risk of macrovascular disease frequently in individuals who are over- of a more recent modeling study imply that was not significantly decreased in the weight (BMI $25 kg/m2)orobeseand screening commencing at 30 or 45 years is original analysis (44). Longer follow-up who have a least one additional risk factor highly cost-effective (,$11,000 per QALY documented a significant reduction in car- for diabetes [see (21) for conditions asso- gained) (35). Long-term outcome studies diovascular disease in patients with type 1 ciated with increased risk]. Because of the are necessary to provide evidence to resolve diabetes treated with intensive glycemic increasing prevalence of type 2 diabetes in the question of the efficacy of diabetes control (45). The effects of tight glycemic children, screening of children is now ad- screening (36). control on microvascular complications in vocated (25). Starting at age 10 years (or at In 2003, the ADA lowered the thresh- patients with type 2 diabetes (46) are sim- the onset of puberty if puberty occurs at a old for “normal” FPG from ,6.1 mmol/L ilar to those with type 1 diabetes, given the younger age), testing should be performed (110 mg/dL) to ,5.6 mmol/L (100 mg/dL) differences in glycemia achieved between every 3 years in overweight individuals (37). This change has been contentious and the active-intervention and control groups who have two other risk factors—namely, has not been accepted by all organizations in the various trials. Intensive plasma glu- family history, a race/ethnicity recognized (19,38). The rationale is based on data that cose control significantly reduced micro- to increase risk, signs of insulin resistance, individuals with FPG values between 5.6 vascular complications in patients with and a maternal history of diabetes or GDM mmol/L (100 mg/dL) and 6.05 mmol/L type 2 diabetes. Although meta-analyses during the child’s gestation (25). Despite (109 mg/dL) are at increased risk for devel- have suggested that intensive glycemic these recommendations and the demon- oping type 2 diabetes (39,40). More-recent control reduces cardiovascular disease in stration that interventions can delay and evidence indicates that FPG concentrations individuals with type 2 diabetes (47,48), sometimes prevent the onset of type 2 di- even lower than 5.6 mmol/L (100 mg/dL) clinical trials have not consistently dem- abetes in individuals with impaired glu- are associated with a graded risk for type 2 onstrated a reduction in macrovascular cose tolerance (26,27), there is as yet no diabetes (41). Data were obtained from disease (myocardial infarction or stroke) published evidence that treatment based 13,163 men between 26 and 45 years of with intensive therapy aimed at lowering on screening has an effect on long-term age who had FPG values ,5.55 mmol/L glucose concentrations in type 2 diabetes. complications. In addition, the published (100 mg/dL) and were followed for a Long-term follow-up of the United King- literature lacks consensus as to which mean of 5.7 years. Men with FPG values dom Prospective Diabetes Study (UKPDS) screening procedure (FPG, OGTT, and/ of 4.83–5.05 mmol/L (87–91 mg/dL) population supported a benefit of inten- or HbA1c)isthemostappropriate have a significantly increased risk of type sive therapy on macrovascular disease (20,28–30). On the basis of an evaluation 2 diabetes, compared with men with FPG (49), but three other recent trials failed of NHANES III data, a strategy has been values ,4.5 mmol/L (81 mg/dL). Although to demonstrate a significant difference in proposed to use FPG to screen whites $40 the prevalence of diabetes is low at these macrovascular disease outcomes between years and other populations $30 years of glucose concentrations, the data support very intensive treatment strategies, which age (31). The cost-effectiveness of screen- the concept of a continuum between FPG achieved HbA1c concentrations of approx- ing for type 2 diabetes has been estimated. and the risk of diabetes. imately 6.5% (48 mmol/mol), and the The incremental cost of screening all per- control groups, which had HbA1c concen- sons $25 years of age has been estimated trations 0.8%–1.1% higher (50–52). One to be $236,449 per life-year gained RECOMMENDATION: ROUTINE study even observed higher cardiovascular and $56,649 per quality-adjusted life- MEASUREMENT OF PLASMA GLUCOSE mortality in the intensive-treatment arm year (QALY) gained (32). Interestingly, CONCENTRATIONS IN AN ACCREDITED (50). In both the Diabetes Control and screening was more cost-effective at ages LABORATORY IS NOT RECOMMENDED AS Complications Trial (DCCT) and the younger than the 45 years currently THE PRIMARY MEANS OF MONITORING OR UKPDS, patients in the intensive-treatment recommended. In contrast, screening tar- EVALUATING THERAPY IN INDIVIDUALS group maintained lower median plasma geted to individuals with hypertension WITH DIABETES glucose concentrations; however, analyses B(low). reduces the QALY from $360,966 to of the outcomes were linked to HbA1c, e64 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates which was used to evaluate glycemic (59) (leukocytosis will increase RECOMMENDATION: TO MINIMIZE control, rather than glucose concentration. eveninthepresenceoffluoride if the leu- GLYCOLYSIS, ONE SHOULD PLACE THE Moreover, most clinicians use the recom- kocyte count is very high). SAMPLE TUBE IMMEDIATELY IN AN mendations of the ADA and other organi- Few effective and practical methods ICE–WATER SLURRY, AND THE PLASMA zations, which define a target HbA are available for prompt stabilization of 1c SHOULD BE SEPARATED FROM THE CELLS concentration as the goal for optimum gly- glucose in whole-blood samples. Loss of WITHIN 30 MIN. IF THAT CANNOT BE cemic control (21,53). glucose can be minimized in two classic ACHIEVED, A TUBE CONTAINING A Neither random nor fasting glucose ways: 1) immediate separation of plasma RAPIDLY EFFECTIVE GLYCOLYSIS concentrations should be measured in from blood cells after blood collection INHIBITOR, SUCH AS CITRATE BUFFER, an accredited laboratory as the primary [the glucose concentration is stable for SHOULD BE USED FOR COLLECTING THE means of routine outpatient monitoring 8 h at 25°C and 72 h at 4°C in separated, SAMPLE. TUBES WITH ONLY ENOLASE of patients with diabetes. Laboratory nonhemolyzed, sterile serum without INHIBITORS, SUCH AS SODIUM FLUORIDE, plasma glucose testing can be used to sup- fluoride (61)]; and 2) placing the blood SHOULD NOT BE RELIED ON TO PREVENT plement information from other testing, tube in an ice–water slurry immediately GLYCOLYSIS to test the accuracy of self-monitoring (see after blood collection and separating the B (moderate). below), or to adjust the dosage of oral plasma from the cells within 30 min hypoglycemic agents (22,54). In addi- (19,62). These methods are not always tion, individuals with well-controlled A. Preanalytical. Blood should be drawn practical and are not widely used. type 2 diabetes who are not on insulin in the morning after an overnight fast (no A recent study showed that acidifica- therapy can be monitored with periodic caloric intake for at least 8 h), during tion of blood with citrate buffer inhibits in measurement of the FPG concentration, which time the individual may consume vitro glycolysis far more effectively than although analysis need not be done in water ad libitum (1). Published evidence fluoride (62). The mean glucose concen- an accredited laboratory (54,55). reveals diurnal variation in FPG, with the tration in samples stored at 37°C de- mean FPG being higher in the morning creased by only 0.3% at 2 h and 1.2% at 2. Rationale than in the afternoon, indicating that 24 h when blood was drawn into tubes A. Diagnosis. The disordered carbohy- many diabetes cases would be missed in containing citrate buffer, sodium fluo- drate metabolism that underlies diabetes patients seen in the afternoon (57). ride, and EDTA. The use of these blood- manifests as hyperglycemia. Therefore, Loss of glucose from sample contain- collection tubes, where they are available, measurement of either plasma glucose ers is a serious and underappreciated appears to offer a practical solution to the or HbA1c is the diagnostic criterion. This problem (58). Decreases in glucose con- glycolysis problem. strategy is indirect, because hypergly- centrations in whole blood ex vivo are due Glucose can be measured in whole cemia reflects the consequence of the to glycolysis. The rate of glycolysis— blood, serum, or plasma, but plasma is metabolic derangement, not the cause; reported to average 5%–7%/h [ap- recommended for diagnosis [note that however, until the underlying molecular proximately 0.6 mmol/L (10 mg/dL)] although both the ADA and WHO rec- pathophysiology of the disease is identi- (59)—varies with the glucose concentra- ommend venous plasma, the WHO also fied, measurement of glycemia is likely to tion, temperature, leukocyte count, and accepts measurement of glucose in capil- remain an essential diagnostic modality. other factors (60). Such decreases in glu- lary blood (19,21)]. The molality of glu- B. Screening. Screening is recommended cose concentration will lead to missed di- cose (i.e., the amount of glucose per unit for several reasons. The onset of type 2 abetes diagnoses in the large proportion water mass) in whole blood is identical diabetes is estimated to occur approxi- of the population who have glucose con- to that in plasma. Although erythrocytes mately 4–7 years (or more) before clinical centrations near the cut points for diag- are essentially freely permeable to glu- diagnosis (56), and epidemiologic evi- nosis of diabetes. cose (glucose is taken up by facilitated dence indicates that complications may The commonly used glycolysis inhib- transport), the concentration of water (in begin several years before clinical diagno- itors are unable to prevent short-term kilograms per liter) in plasma is approxi- sis. Furthermore, it is estimated that 40% glycolysis. Glycolysis can be attenuated mately 11% higher than in whole blood. of people in the U.S. with type 2 diabetes by inhibiting enolase with sodium fluo- Therefore, glucose concentrations are ap- are undiagnosed (8). Notwithstanding ride (2.5 mg/mL of blood) or, less com- proximately 11% higher in plasma than in this recommendation, there is no pub- monly, lithium iodoacetate (0.5 mg/mL of whole blood if the hematocrit is normal. lished evidence that population screening blood). These reagents can be used alone Glucose concentrations in heparinized for hyperglycemia provides any long- or, more commonly, with such anti- plasma were reported in 1974 to be 5% term benefit. Outcome studies examining coagulants as potassium oxalate, EDTA, lower than in serum (63). The reasons for the potential long-term benefits of screen- citrate, or lithium heparin. Unfortunately, the difference are not apparent but have ing are ongoing. although fluoride helps to maintain long- been attributed to the shift in fluid from term glucose stability, the rates of decline erythrocytes to plasma caused by anti- 3. Analytical considerations in the glucose concentration in the first coagulants. In contrast, some more recent hour after sample collection are virtually studies found that glucose concentrations identical for tubes with and without are slightly higher in plasma than in se- RECOMMENDATION: BLOOD FOR FPG fluoride, and glycolysis continues for up rum. The observed differences were ap- ANALYSIS SHOULD BE DRAWN IN THE to 4 h in samples containing fluoride (59). proximately 0.2 mmol/L (3.6 mg/dL) MORNING AFTER THE INDIVIDUAL HAS After 4 h, the concentration of glucose (64), or approximately 2% (65), or 0.9% FASTED OVERNIGHT (AT LEAST 8h) in whole blood in the presence of fluoride (62). Other studies have found that glu- B(low). remains stable for 72 h at room temperature cose values measured in serum and care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e65 Position Statement plasma are essentially the same (66,67). B. Analytical. Glucose is measured al- be developed to assist in achieving this Given these findings, it is unlikely that val- most exclusively by enzymatic methods. objective. ues for plasma and serum glucose will be An analysis of proficiency surveys con- substantially different when glucose is as- ducted by the College of American Pa- 4. Interpretation sayed with current instruments, and any thologists (CAP) reveals that hexokinase Despite the low analytical imprecision at differences will be small compared with or glucose oxidase is used in virtually all the diagnostic decision limits of 7.0 the day-to-day biological variation of glu- analyses performed in the U.S. (70). A mmol/L (126 mg/dL) and 11.1 mmol/L cose. Clinical organizations do not recom- very few laboratories (,1%) use glucose (200 mg/dL), classification errors may mend the measurement of glucose in dehydrogenase. Enzymatic methods for occur. Knowledge of intraindividual serum (rather than plasma) for the diag- glucose analysis are relatively well stan- (within-person) variation in FPG concen- nosis of diabetes (19,21). Use of plasma dardized. At a plasma glucose concentra- trations is essential for meaningful inter- allows samples to be centrifuged promptly tion of approximately 7.5 mmol/L (135 pretation of patient values (although total to prevent glycolysis without waiting for mg/dL), the imprecision (CV) among lab- biological variation includes within-person the blood to clot. The glucose concentra- oratories that used the same method was and between-person variation, most dis- tions in capillary blood obtained during an #2.6% (70). Similar findings have been cussions focus on the within-person var- OGTT are significantly higher than those reported for glucose analyses of samples iation). An early study, which repeated the in venous blood [mean, 1.7 mmol/L (30 from patients. The method of glucose OGTT in 31 nondiabetic adults at a 48-h mg/dL), which is equivalent to 20%–25% measurement does not influence the re- interval, revealed that the FPG concentra- higher (68)], probably owing to glucose sult. A comparison of results from ap- tion varied between the 2 values by ,10% consumption in the tissues. In contrast, proximately 6,000 clinical laboratories in 22 participants (77%) and by ,20% in the mean difference in fasting samples is reveals that the mean glucose concentra- 30 participants (97%) (80). A careful eval- only 0.1 mmol/L (2 mg/dL) (68,69). tions measured in serum samples by the uation of healthy individuals over several Reference intervals. Glucose concentrations hexokinase and glucose oxidase methods consecutive days revealed that the biolog- vary with age in healthy individuals. The are essentially the same (76). Compared ical variation in FPG [mean glucose, 4.9 reference interval for children is 3.3–5.6 with a reference measurement procedure, mmol/L (88 mg/dL)] exhibited within- mmol/L (60–100 mg/dL), which is similar significant bias (P , 0.001) was observed and between-individual CVs of 4.8%– to the adult interval of 4.1–6.1 mmol/L for 40.6% of the peer groups (76). If sim- 6.1% and 7.5%–7.8%, respectively (74–110 mg/dL) (70). Note that the ADA ilar biases occur with plasma, patients (81–83). Larger studies have revealed and WHO criteria (19,21), not the refer- near the diagnostic threshold could be intraindividual CVs of 4.8% and 7.1% ence intervals, are used for the diagnosis misclassified. for FPG in 246 healthy individuals and of diabetes. Moreover, the threshold for No consensus has been achieved on 80 previously undiagnosed individuals the diagnosis of hypoglycemia is variable. the goals for glucose analysis. Numerous with diabetes, respectively (83). Similar Reference intervals are not useful for diag- criteria have been proposed to establish findings were obtained from an analysis nosing these conditions. In adults, the analytical goals. These criteria include of 685 adults from NHANES III, in which mean FPG concentration increases with expert opinion (consensus conferences), the mean within-person variation in FPG increasing age from the third to the sixth the opinion of clinicians, regulation, the measured 2–4 weeks apart was 5.7% decade (71) but does not increase signifi- state of the art, and biological variation (95% CI, 5.3%–6.1%) (84). An analysis cantly after 60 years of age (72,73). By con- (77). A rational and realistic recommen- of larger numbers of individuals from trast, glucose concentrations after a glucose dation that has received some support thesameNHANESIIIdatabaseyielded challenge are substantially higher in older is to use biological criteria as the basis within- and between-person CVs of individuals (72,73). The evidence for an for analytical goals. It has been suggested 8.3% and 12.5%, respectively, at a glucose association between increasing insulin re- that imprecision should not exceed one- concentration of approximately 5.1 sistance and age is inconsistent (74). Aging half of the within-individual biological mmol/L (92 mg/dL) (85). If a within- appears to influence glucose homeostasis, CV (78,79). For plasma glucose, a CV person biological CV of 5.7% is applied and visceral obesity seems to be responsible #2.2% has been suggested as a target to a true glucose concentration of 7.0 for the reported continuous decrease in for imprecision, with a 0% bias (79). Al- mmol/L (126 mg/dL), the 95% CI would glucose tolerance that begins in middle though this recommendation was pro- encompass glucose concentrations of 6.2– age (75). posed for within-laboratory error, it 7.8 mmol/L (112–140 mg/dL). If the would be desirable to achieve this goal analytical CV of the glucose assay (ap- for interlaboratory imprecision to mini- proximately 3%) is included, the 95% CI mize differences among laboratories in is approximately 612.88%. Thus, the RECOMMENDATION: ON THE BASIS OF the diagnosis of diabetes in individuals 95% CI for a fasting glucose concentration BIOLOGICAL VARIATION, GLUCOSE with glucose concentrations close to the of 7.0 mmol/L (126 mg/dL) would be 7.0 MEASUREMENT SHOULD HAVE AN threshold value. Therefore, the goal for mmol/L 6 6.4% (126 mg/dL 6 6.4%), ANALYTICAL IMPRECISION £2.9%, ABIAS glucose analysis should be to minimize i.e., 6.1–7.9 mmol/L (110–142 mg/dL). £2.2%, AND A TOTAL ERROR £6.9%. TO total analytical error, and methods should Use of an assay CV of 3% only (excluding AVOID MISCLASSIFICATION OF PATIENTS, be without measurable bias. A national or biological variation) would yield a 95% CI THE GOAL FOR GLUCOSE ANALYSIS international program that uses commut- of 6.6–7.4 mmol/L (118–134 mg/dL) SHOULD BE TO MINIMIZE TOTAL able samples (e.g., fresh frozen plasma) to among laboratories, for a true glucose con- ANALYTICAL ERROR, AND METHODS eliminate matrix effects and has accuracy- centration of 7.0 mmol/L (126 mg/dL). SHOULD BE WITHOUT MEASURABLE BIAS based grading with values derived with a Performing the same calculations at the B(low). reference measurement procedure should cutoff for yields a e66 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates

95% CI of 5.6 mmol/L 6 6.4% (100 GLUCOSE METERS—Portable me- below) precludes their use from the di- mg/dL 6 6.4%), i.e., 4.9–6.3 mmol/L ters for the measurement of blood glucose agnosis of diabetes. Similarly, screening (87–113 mg/dL). One should bear in concentrations are used in three major with portable meters—although attrac- mind that these intervals include 95% of settings: 1)inacute-andchronic-carefacil- tive because of convenience, ease, and the results and that the remaining 5% will ities, including intensive care units (ICUs); accessibility—would generate many be outside this interval. Thus, the biological 2) in physicians’ offices; and 3) by patients false positives and false negatives. variation is substantially greater than the at home, work, and school. Measurement analytical variation. Using biological varia- in the last setting, self-monitoring of blood tion as the basis for deriving analytical per- glucose (SMBG), was performed at least RECOMMENDATION: SMBG formance characteristics (77), Westgard once per day by 40% and 26% of individ- IS RECOMMENDED FOR ALL proposed the following desirable specifi- uals with type 1 and type 2 diabetes, re- INSULIN-TREATED PATIENTS cations for glucose (86): analytical impre- spectively, in the U.S. in 1993 (90). The WITH DIABETES cision, #2.9%; bias, #2.2%; and total overall rate of daily SMBG among adults A(high). error, #6.9%. with diabetes in the U.S. increased to A. Turnaround time. A short turnaround 40.6% in 1997 and to 63.4% in 2006 time for glucose analysis is not usually (91). The ADA summarized the uses of necessary for diagnosis of diabetes. In some SMBG as early as 1987 [see (92) and refer- RECOMMENDATION: IN PATIENTS WITH clinical situations, such as acute hyper- or ences therein] and currently recommends TYPE 2 DIABETES TREATED WITH DIET hypoglycemic episodes in the emergency that SMBG be carried out $3 times daily by AND ORAL AGENTS, SMBG MAY HELP department or treatment of diabetic keto- patients who use multiple insulin injec- ACHIEVE BETTER CONTROL, acidosis (DKA), rapid analysis is desirable. tions or insulin pump therapy (92,93). It PARTICULARLY WHEN THERAPY IS A turnaround time of 30 min has been is recommended that most individuals with INITIATED OR CHANGED. DATA ARE proposed (87). This value is based on the diabetes attempt to achieve and maintain INSUFFICIENT, HOWEVER, TO CLAIM AN suggestions of clinicians, however, and no blood glucose concentrations as close to ASSOCIATED IMPROVEMENT OF HEALTH outcome data that validate this time interval those in nondiabetic individuals as is safely OUTCOMES. THE ROLE OF SMBG IN have been published. Inpatient manage- possible. PATIENTS WITH STABLE TYPE 2 DIABETES ment of diabetic patients on occasion may CONTROLLED BY DIET ALONE IS require a rapid turnaround time (minutes, 1. Use NOT KNOWN not hours). Similarly, for protocols with in- C(high). tensive glucose control in critically ill pa- RECOMMENDATION: THERE ARE tients (88), rapid glucose results are required in order to calculate the insulin INSUFFICIENT PUBLISHED DATA B. Monitoring/prognosis. SMBG is rec- dose. Bedside monitoring with glucose me- OUTCOME TO SUPPORT A ROLE FOR ommended for all insulin-treated patients PORTABLE METERS AND SKIN-PRICK ters (see below) has been adopted by many with diabetes. Intensive glycemic control (FINGER-STICK) BLOOD SAMPLES IN THE as a practical solution. can decrease microvascular complica- B. Frequency of measurement. The fre- DIAGNOSIS OF DIABETES OR FOR tions in individuals with type 1 (44) or quency of measurement of plasma glucose POPULATION SCREENING type 2 (46) diabetes. In the DCCT, pa- C (moderate). is dictated by the clinical situation. The tients with type 1 diabetes achieved inten- ADA, WHO, and IDF recommend that an sive glycemic control by performing increased FPG or an abnormal OGTT result SMBG at least 4 times per day (44). Ther- must be confirmed to establish the diag- RECOMMENDATION: THE IMPRECISION OF apy in patients with type 2 diabetes in the nosis of diabetes (19,89). Screening by FPG THE RESULTS, COUPLED WITH THE UKPDS (46) was adjusted according to is recommended every 3 years, beginning SUBSTANTIAL DIFFERENCES AMONG FPG concentration; SMBG was not evalu- at 45 years of age and more frequently in METERS, PRECLUDES THE USE OF ated. high-risk individuals; however, the fre- GLUCOSE METERS FROM THE DIAGNOSIS The role of SMBG in individuals with quency of analysis has not been specified OF DIABETES AND LIMITS THEIR type 2 diabetes has generated consider- for the latter group. Monitoring is per- USEFULNESS IN SCREENING FOR DIABETES able controversy (94,95). Faas et al. (96) formed by patients who measure their glu- A(moderate). reviewed 11 studies published between cose themselves with meters and by 1976 and 1996 that evaluated SMBG in assessment of HbA1c in an accredited labo- patients with type 2 diabetes. Only one of ratory (see below). The appropriate interval A. Diagnosis/screening. The glucose- the published studies reported that SMBG between glucose measurements in acute based criteria for the diagnosis of di- produced a significant improvement in clinical situations (e.g., patients admitted abetes are based on outcome data (the glycated Hb (GHb). The review’sauthors to a hospital, patients with DKA, neonatal risk of micro- and macrovascular dis- concluded that the efficacy of SMBG in hypoglycemia, and so forth) is highly vari- ease) correlated with plasma glucose type 2 diabetes is questionable (96). Sim- able and may range from 30 min to 24 h or concentrations—both fasting and 2 h ilar conclusions were drawn in an early more. after a glucose load—assayedinanac- (2000) meta-analysis (97) of a sample credited laboratory (1). Whole blood is of patients with type 2 diabetes in 5. Emerging considerations used in portable meters. Although most the NHANES (98) and the Freemantle Di- Continuous minimally invasive and non- portable meters have been programmed abetes Study (99). Two early randomized invasive analysis of glucose is addressed to report a plasma glucose concentration, trials assessed the use of glucose meters below. the imprecision of the current meters (see in individuals with type 2 diabetes care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e67 Position Statement

(100,101). One of these trials (100) had Two recent systematic reviews of normally precede neuroglycopenia statistical power to detect a 0.5% reduc- randomized controlled studies of SMBG (“hypoglycemic unawareness”) (113), tion in HbA1c but reported only a modest in people with type 2 diabetes not treated increasing the risk of hypoglycemia. decrease (0.3%) in HbA1c among poorly with insulin reported small but signifi- SMBG can be useful for detecting asymp- controlled patients treated with oral cantly greater decreases in HbA1c among tomatic hypoglycemia and allowing pa- agents. The second study (101) failed to patients using SMBG than in controls tients to avoid major hypoglycemic demonstrate a significant difference in (110,111). In the first review (110), episodes. HbA1c in patients who were assigned to SMBG was associated with a larger reduc- use meters, compared with those who tion in HbA1c compared with non-SMBG were not. (weighted mean difference, 20.31%; 3. Analytical considerations For individuals with type 2 diabetes, 95% CI, 20.44 to 20.17). In the second cross-sectional and longitudinal obser- study (111), the relative decrease in vational studies in several countries have HbA1c was 20.24% (95% CI, 20.34% RECOMMENDATION: PATIENTS SHOULD failed to demonstrate an improvement in to 20.14%). The effect of SMBG was BE INSTRUCTED IN THE CORRECT USE OF glycemic control (as measured by mean limited to patients with HbA1c values GLUCOSE METERS, INCLUDING QUALITY HbA1c concentration) associated with $8% (64 mmol/mol). CONTROL. COMPARISON BETWEEN SMBG the use of SMBG (102–104). This lack A 2009 review of studies of patients AND CONCURRENT LABORATORY of effect was seen in individuals treated with type 2 diabetes (112) addressed re- GLUCOSE ANALYSIS SHOULD BE with insulin, oral agents, or both. Fre- cent large randomized trials of tight gly- PERFORMED AT REGULAR INTERVALS TO quency of meter use did not predict cemic control, a major rationale for SMBG EVALUATE THE PERFORMANCE OF THE ’ HbA1c. use in these patients. It concluded that METERS IN THE PATIENT SHANDS A 2005 Cochrane review (105,106) “tight glycemic control burdens patients B (moderate). of self-monitoring in individuals with with complex treatment programs, hypo- type 2 diabetes not using insulin con- glycemia, weight gain, and costs and of- cluded that SMBG might be effective in fers uncertain benefits in return,” thus A. Preanalytical. Numerous factors improving glucose control. There was in- raising additional uncertainty about can interfere with glucose analysis with sufficient evidence to evaluate whether it the use of SMBG in people with type 2 portable meters. Several of these factors, was beneficial in improving quality of life, diabetes. such as improper application, timing, improving well-being or patient satisfac- and removal of excess blood (61), have tion, or decreasing the number of hypo- been mitigated or eliminated by advan- glycemic episodes. 2. Rationale ces in technology. Important variables The randomized controlled Diabetes Knowledge of ambient plasma or blood that may influence the results of bedside Glycaemic Education and Monitoring glucose concentrations is used by insulin- glucose monitoring include changes in (DiGEM) trial (107) studied people with requiring patients, particularly those with hematocrit (114), altitude, environmen- type 2 diabetes, a third of whom were type 1 diabetes, as an aid in determining tal temperature or humidity, hypoten- treated with diet alone. In 2007, the in- appropriate insulin doses at different sion, hypoxia and high triglyceride vestigators reported, “Evidence is not times of the day (92). Patients adjust the concentrations (115), and various convincing of an effect of self monitoring amount of insulin according to their drugs. Furthermore, most meters are in- blood glucose ...in improving glycaemic plasma or blood glucose concentration. accurate at very high or very low glucose control [as assessed by HbA1c]compared Frequent SMBG is particularly impor- concentrations. Another important fac- with usual care in reasonably well con- tant for tight glycemic control in type 1 tor is variation in results among different trolled non-insulin treated patients with diabetes. glucose meters. Different assay methods type 2 diabetes.” A cost-effectiveness Hypoglycemia is a major, potentially and architectures lead to a lack of corre- analysis of data from the DiGEM trial life-threatening complication of the lation among meters, even from a single concluded, “Self monitoring of blood glu- treatment of diabetes. The risk of hy- manufacturer. In fact, two meters of the cose with or without additional training poglycemia is seen primarily in patients same brand have been observed to differ in incorporating the results into self care treated with insulin or insulin secre- substantially in accuracy (116,117). was associated with higher costs and tagogues, and it increases substantially Patient factors are also important, partic- lower quality of life in patients with when pharmacologic therapy is directed ularly adequate training. Recurrent edu- non-insulin treated type 2 diabetes. In towards maintaining the glycemic con- cation at clinic visits and comparison of light of this, and no clinically significant centrations as close to those found in SMBG with concurrent laboratory glu- differences in other outcomes, self mon- nondiabetic individuals as is safely possi- cose analysis improved the accuracy itoring of blood glucose is unlikely to be ble (44,46). The incidence of major hypo- of patients’ blood glucose readings cost effective in addition to standardised glycemic episodes—requiring third-party (118). Thus, it is important to evaluate usual care” (108). help or medical intervention—was 2- to the patient’s technique at regular inter- The later ESMON study (109), a ran- 3-fold higher in the intensive-treatment vals (21). In addition to these technical domized controlled trial of SMBG in group than in the conventional group issues, the anatomic site where skin- newly diagnosed people with diabetes in clinical trials of patients with type 1 puncture samples are obtained influ- not treated with insulin, found no benefit and type 2 diabetes (44,46). Fur- ences results. Testing blood from so-called of SMBG on glycemic control but did thermore, many patients with diabetes, alternative sites may introduce a tempo- find higher scores on a depression sub- particularly those with type 1, lose ral lag in changes in measured blood scale. the autonomic warning symptoms that glucose. e68 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates

error signal if the sample volume is in- (30 mg/dL) or 3.3 mmol/L (60 mg/dL) RECOMMENDATION: MULTIPLE adequate, “lock out” if controls are not and be considered acceptable. Such errors PERFORMANCE GOALS FOR PORTABLE assayed, and bar code readers to identify do not appear to be acceptable for reliably GLUCOSE METERS HAVE BEEN PROPOSED. the lot of the strips. Moreover, meters detecting hypoglycemia. Similarly, errors THESE TARGETS VARY WIDELY AND ARE store up to several hundred results that of 20% can lead to errors in insulin HIGHLY CONTROVERSIAL. can subsequently be downloaded for dosing, which, when combined with MANUFACTURERS SHOULD WORK TO analysis. Together, these improvements other factors, can lead to hypoglycemia. IMPROVE THE IMPRECISION OF CURRENT have improved the performance of new Others have proposed different ap- METERS, WITH AN INTERMEDIATE GOAL meters (122,123). Nonetheless, meter proaches to establishing quality require- OF LIMITING TOTAL ERROR FOR 95% OF performance in the hands of patients ments. Clarke et al. (128) developed an SAMPLES TO £15% AT GLUCOSE does not equal potential performance as error grid that attempts to define clinically CONCENTRATIONS ‡5.6 mmol/L judged by performance in the hands of important errors by identifying fairly (100 mg/dL) AND TO ,0.8 mmol/L skilled medical technologists (124). broad target ranges. In another approach, (15 mg/dL) AT GLUCOSE Numerous analytical goals have been 201 patients with long-standing type 1 CONCENTRATIONS ,5.6 mmol/L proposed for the performance of glucose diabetes were questioned to estimate (100 mg/dL). LOWER TOTAL ERROR meters. The rationale for these goals is not quality expectations for glucose meters WOULD BE DESIRABLE AND MAY PROVE always clear. In 1987, the ADA recommen- (129). On the basis of patients’ percep- NECESSARY IN TIGHT GLUCOSE-CONTROL ded a goal of total error (user plus analyt- tions of their needs and their reported PROTOCOLS AND FOR AVOIDING ical) of ,10% at glucose concentrations of actions in response to changes in mea- HYPOGLYCEMIA IN ALL SETTINGS 1.7–22.2 mmol/L (30–400 mg/dL) 100% sured glucose concentrations, a goal for C(low). of the time (125). In addition, the ADA analytical quality at hypoglycemic con- proposed that values should differ by centrations was a CV of 3.1%. With hy- #15% from those obtained by a laboratory poglycemia excluded, the analytical CV to RECOMMENDATION: METERS SHOULD reference method. The recommendation meet the expectations of 75% of the pa- MEASURE AND REPORT PLASMA GLUCOSE was modified in response to the significant tients was 6.4% to 9.7%. The authors rec- CONCENTRATIONS TO FACILITATE reduction in complications obtained by ommended an analytical CV of 5% with a COMPARISON WITH ASSAYS PERFORMED tight glucose control in the DCCT. A re- bias #5% (129). A third approach used IN ACCREDITED LABORATORIES GPP. vised performance goal, published in simulation modeling of errors in insulin 1996 (92), was for a total analytical error dose (130). The results revealed that meters of ,5%. To our knowledge, there are no that achieve both a CV and a bias ,5% B. Analytical. Virtually all glucose meters published studies of diabetic patients rarely lead to major errors in insulin dose. use strips that contain enzymes, such as achieving the goal of an analytical error of To provide the intended insulin dosage glucose oxidase or glucose dehydroge- ,5% with any glucose meters. 95% of the time, however, the bias and nase. A drop of whole blood is applied to a The less stringent CLSI (formerly CV needed to be ,1%–2%, depending strip that contains all the reagents neces- NCCLS) recommendations are that, for on the dosing schedule for insulin and sary for the assay. Some meters have a 95% of the samples, the difference be- the intervals of glucose concentrations for porous membrane that separates erythro- tween meter and laboratory measure- the individual patient (130). No meters cytes, and analysis is performed on the ments of glucose be 1) ,20% when the have been shown to achieve CVs of 1%– resultant plasma. Meters can be calibrated laboratory glucose value is .5.5 mmol/L 2% in routine use in the hands of patients. to report plasma glucose values, even (100 mg/dL) and 2) ,0.83 mmol/L (15 The lack of consensus on quality when the sample is whole blood. An mg/dL) of the laboratory glucose value goals for glucose meters reflects the ab- IFCC working group recommended that when the glucose concentration is #5.5 sence of agreed objective criteria. With glucose meters report the plasma glucose mmol/L (100 mg/dL) (126). The 2003 the same biological-variation criteria de- concentration, irrespective of the sample International Organization for Standard- scribed above for glucose analysis in type or technology (119,120). This ap- ization (ISO) recommendations (127) accredited laboratories (section 4, Inter- proach can improve harmonization propose that for test readings .4.2 pretation), a biological goal would be a and allow comparison with laboratory- mmol/L (75 mg/dL), the discrepancy be- total error #6.9% with an imprecision (as generated results (121). The meters use tween meters and an accredited labora- the CV of measurements over several days reflectance photometry or electrochemis- tory should be ,20%; for glucose or weeks) #2.9% and a bias #2.2% (86). try to measure the rate of the reaction or readings #4.2 mmol/L (75 mg/dL), the Additional studies, however, are neces- the final concentration of the products, discrepancy should not exceed 0.83 sary to define a goal that is related to med- and they provide digital readouts of glu- mmol/L (15 mg/dL) in 95% of the sam- ical needs. cose concentration. Manufacturers claim ples. In both the CLSI and ISO guidelines, Current meters exhibit performance reportable concentration ranges as large as 5% of these results can be substantially superior to prior generations of meters 33.3 mmol/L (600 mg/dL), e.g., 0–33.3 outsidetheselimits.Atthetimeofwrit- (122,123). A variety of studies of newer mmol/L (0–600 mg/dL). ing, both the CLSI and ISO recommenda- analyzers have documented CVs of about Several important technological ad- tions were undergoing revision. 2% in the hands of trained workers. vances decrease operator error. These These criteria serve as de facto min- Nonetheless, there is room for improve- improvements include automatic com- imal quality requirements for manufac- ment. In a study conducted under care- mencement of timing when both the turers wishing to sell meters. With these fully controlled conditions in which a sample and the strip are in the meter, criteria, a concentration of 2.5 mmol/L single medical technologist performed smaller sample-volume requirements, an (45 mg/dL) may be read as 1.7 mmol/L all of the assays, about 50% of the analyses care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e69 Position Statement met the 1996 ADA criterion of ,5% de- (138). Moreover, the error of glucose me- 4. Interpretation viation from reference intervals (122). ters may compound the problem and A. Frequency of measurement. SMBG Another study that evaluated meter per- compromise the ability to control blood should be performed at least 3 times per formance in 226 hospitals with split sam- glucose and avoid hypoglycemia. Simula- day in patients with type 1 diabetes. ples analyzed simultaneously on meters tion modeling studies have demonstrated Monitoring less frequently than 3 times and laboratory glucose analyzers revealed that errors in glucose measurement per day leads to deterioration in glycemic that 45.6%, 25%, and 14% of the split (which include errors related to sample control (92,144,145). Patients perform samplesdifferedfromeachotherby type and sample collection) lead to self-monitoring much less frequently .10%, .15%, and .20%, respectively marked degradation of glycemic control than recommended. Data from NHANES (131). In another study, none of the me- in tight glucose-control protocols (139). III collected between 1988 and 1994 re- ters met the 1996 ADA criterion (132). In In this study, frequencies of both hy- veal that SMBG was performed at least an evaluation in which “all testing was perglycemia and hypoglycemia were once a day by 39% of patients taking in- performed by trained study staff in an in- increased with increasing assay impreci- sulin and by 5%–6% of patients treated patient Clinical Research Center setting,” sion. In a 2005 study of ICU patients with oral agents or diet alone (98). More- only 81% of results with a meter that (140), the agreement of meter results over, 29% and 65% of patients treated used a hexokinase method were within with accredited laboratory results was with insulin and oral agents, respectively, 10% of results obtained from an accred- poor:Among767pairedresults,the monitored their blood glucose less than ited laboratory (133). We are aware of no 95% limits of agreement were 12.4 to once per month; however, no evaluation studies that document patient-generated 21.5 mmol/L (143.1 to 227.2 mg/dL). has been performed to verify that 3 times results that meet the 1996 ADA criteria. Hoedemaekers et al. (141), in a study of per day is ideal or whether a different fre- Moreover, an analysis of published stud- 197 arterial blood samples from ICU pa- quency would improve glycemic control. ies of glucose meters demonstrated that tients, reported that the evaluated meter For example, adjustment of insulin ther- the studies suffered from deficiencies in did not meet the ISO total-error criteria. apy in women with GDM according to the study design, methodology, and report- They also demonstrated that the total results of postprandial, rather than pre- ing (134), raising the possibility that the error of meters used in ICU patients was prandial, plasma glucose concentrations reported total error underestimates the greater than in non-ICU patients. A later improved glycemic control and reduced true total error of the meters. A standard- report, which also studied arterial blood the risk of neonatal complications (146). ized method for evaluating meters has from ICU patients, measured glucose in The optimal frequency of SMBG for pa- been developed in Norway (134), and 239 samples by a portable meter and by a tients with type 2 diabetes is unknown. the Norwegian health authorities have laboratory method and found that the The ADA recommends that patients decided that all SMBG instruments mar- meter results did not meet the CLSI/ISO treated with multiple daily injections of ketedinNorwayshouldbeexaminedbya criteria (142). Similarly, a 2005 study of insulin perform SMBG $3timesperday similar procedure (135). Results of evalu- arterial, venous, and capillary samples (21) and states that “SMBG is useful in ations of nine brands of meters according from a mixed medical/surgical ICU of a ter- achieving glycemic goals” in other pa- to this method showed that three of nine tiary care hospital in Canada found that tients. The last statement is based on ex- meters did not meet the ISO criteria, and meters did not meet proposed CLSI goals pert opinion. none met the 1996 ADA criteria in the but that a blood gas analyzer did (143). hands of patients (135). CONTINUOUS MINIMALLY Glucose meters are also used to sup- RECOMMENDATION: STUDIES ARE INVASIVE GLUCOSE port tight control of glucose in patients in ANALYSES ICU settings. A 2001 report of a seminal NEEDED TO DETERMINE THE ANALYTICAL GOALS (QUALITY SPECIFICATIONS) FOR randomized controlled trial by van den 1. Use Berghe et al. described a 34% reduction in GLUCOSE METERS IN SMBG AND IN ICUS C (moderate). mortality in surgical ICU patients man- aged according to a tight glucose-control RECOMMENDATION: REAL-TIME protocol (88). A meta-analysis of multiple CONTINUOUS GLUCOSE MONITORING randomized controlled trials of tight glu- RECOMMENDATIONS FOR FUTURE (CGM) IN CONJUNCTION WITH INTENSIVE cose control conducted 7 years later failed RESEARCH: IMPORTANT END POINTS IN INSULIN REGIMENS CAN BE A USEFUL STUDIES OF SMBG SHOULD INCLUDE, AT A to identify any improved outcomes but TOOL TO LOWER HbA1c IN SELECTED > did find an increased incidence of hypo- MINIMUM, HbA1c AND FREQUENCY OF ADULTS (AGE 25 YEARS) WITH TYPE 1 glycemia (136). A Clinical Chemistry Per- HYPOGLYCEMIC EPISODES TO ASCERTAIN DIABETES spective article (137) pointed out that the WHETHER IMPROVED METERS ENABLE A(high). study of van den Berghe et al. used a pre- PATIENTS TO ACHIEVE BETTER GLUCOSE cise and accurate glucose analyzer and CONTROL. FOR STUDIES OF METER USE IN collected arterial blood samples, whereas INTENSIVE OR CRITICAL CARE, RECOMMENDATION: ALTHOUGH THE subsequent studies often used glucose IMPORTANT END POINTS INCLUDE MEAN EVIDENCE FOR LOWERING HbA1c IS NOT meters and capillary blood samples ob- BLOOD GLUCOSE, FREQUENCY OF AS STRONG FOR CHILDREN, TEENS, AND tained by finger stick. The integrity of re- HYPOGLYCEMIA, AND VARIATION OF YOUNGER ADULTS, REAL-TIME CGM MAY sults obtained with finger-stick samples GLUCOSE CONTROL. IDEALLY, OUTCOMES BE HELPFUL IN THESE GROUPS. SUCCESS can be compromised by such factors as (e.g., LONG-TERM COMPLICATIONS) CORRELATES WITH ADHERENCE TO shock, hypoxia, and low hematocrit, SHOULD ALSO BE EXAMINED ONGOING USE OF THE DEVICE which are common in these settings GPP. B (moderate). e70 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates

type 1 diabetic patients showed that on binding glucose, or the use of com- RECOMMENDATION: REAL-TIME CGM MAY adults $25 years of age who used inten- petitive-binding assays that use two fluo- BE A SUPPLEMENTAL TOOL TO SMBG IN sive insulin therapy and real-time CGM rescent molecules in the fluorescent INDIVIDUALS WITH HYPOGLYCEMIA experienced a 0.5% reduction in HbA , resonance energy transfer technique UNAWARENESS AND/OR FREQUENT 1c from approximately 7.6% to 7.1% (ap- (153–157). EPISODES OF HYPOGLYCEMIA proximately 60 to 54 mmol/mol), com- B(low). pared with the usual intensive insulin therapy with SMBG (150). Sensor use in 4. Interpretation children, teens, and adults to 24 years of The subcutaneous sensors are generally RECOMMENDATION: PATIENTS REQUIRE age did not lower HbA significantly, and worn for a number of days and require EXTENSIVE TRAINING IN USING THE 1c there was no significant difference in hypo- calibration with SMBG readings several DEVICE. AVAILABLE DEVICES MUST BE glycemia for any group. The greatest pre- times per day. A few small studies have CALIBRATED WITH SMBG READINGS, AND dictor of HbA reduction in this study examined their accuracy compared with THE LATTER ARE RECOMMENDED FOR 1c among all age-groups was frequency of sen- SMBG and/or plasma glucose assays. For MAKING TREATMENT CHANGES GPP. sor use, which was lower in younger age- the Medtronic CGMS System Gold de- groups. Although CGM is an evolving vice, the mean (SD) absolute difference technology, the emerging data suggest between sensor readings and blood glu- The development of a device for “contin- that it may offer benefit in appropriately cose readings was 15.0% (12.2%) for 735 uous” in vivo monitoring of glucose con- selected patients who are motivated to paired samples, whereas the GlucoDay centrations in blood has become a very wear it most of the time. CGM may be microdialysis device (Menarini) had a high priority as patients are required to particularly useful for patients with hypo- mean absolute difference of 13.6% control their plasma glucose more closely glycemia unawareness and/or frequent (10.2%) for 1,156 paired samples (158). (21,44,147). The first device approved by episodes of hypoglycemia; studies in For both devices, accuracy was lowest in the U.S. Food and Drug Administration this area are ongoing. the hypoglycemic ranges. Approximately (FDA) for minimally invasive interstitial 97% of the values for both devices were fluid glucose sensing, the transcutaneous within zones A and B of a Clarke error GlucoWatch Biographer, is no longer on 2. Rationale grid, with none falling in zone E (158). the market. Several implanted-catheter The first goal for developing a reliable in A study of 91 insulin-treated patients systems have subsequently been ap- vivo continuous glucose sensor is to detect using the DexCom device showed that proved. The initial device in the latter unsuspected hypoglycemia. The impor- 95% of 6,767 paired glucose values fell category is the Continuous Glucose Mon- tance of this goal has been increasingly within Clarke error grid zones A and B, itoring System (CGMS) (Medtronic), a appreciated with the recognition that strict with a mean absolute difference of 21.2% system that does not provide real-time glucose control is accompanied by a (148). data to the patient, but rather one the pa- marked increase in the risk of hypoglyce- Currently, there are no analytical goals tient wears for 3 days and then returns to mia (44,147). Therefore, a sensor designed for noninvasive and minimally invasive the provider’soffice for its data to be to detect severe hypoglycemia alone would glucose analyses. Such standards will downloaded for trend analyses. More be of value. In contrast, a full-range, reliable clearly need to be different for different recently, a number of real-time devices continuous in vivo glucose monitor is a proposed uses. For example, the reliability, that allow patients to read both current prerequisite for the development of a precision, and accuracy requirements for a glucose concentrations and trends have closed-loop pump or “artificial pancreas” glucose sensor that is linked to a system that become commercially available. In the that would measure blood glucose concen- automatically adjusts insulin doses will be U.S., these devices include the Guardian trations and automatically adjust insulin much more stringent than those for a sensor Real-Time (Medtronic Diabetes), the administration. designed to trigger an alarm in cases of Seven Plus System (DexCom), and the apparent extreme hyper- or hypoglycemia. Freestyle Navigator (Abbott Laborato- It seems intuitively obvious that a larger ries). CGM devices require calibration 3. Analytical considerations imprecision can be tolerated in instruments and confirmation of accuracy with con- The methods to sample biological fluids that make frequent readings during each ventional SMBG, and the FDA advises us- in a continuous and minimally invasive hour than in an instrument used only 2 or 3 ing the latter for treatment decisions, such way vary among test systems. The under- times per day to adjust a major portion of a as calculating premeal insulin doses. lying fundamental concept is that the person’s daily insulin dose. The clinical studies of these devices, concentration of glucose in the interstitial generally in highly selected populations, fluid correlates with blood glucose. The had primarily been limited to assessments implanted sensors use multiple detection 5. Emerging considerations of their accuracy or to short-term trials systems, including enzyme- (usually glu- With FDA approval of several self- demonstrating reductions in the time cose oxidase), electrode-, and fluorescence- monitoring continuous glucose sensors, patients spend within hypo- and hyper- based techniques. Alternatives to enzymes, it is anticipated that there will be renewed glycemic intervals (148). A systematic re- including artificial glucose “receptors,” as efforts to bring other technologies for- view of trials of the non–real-time CGM glucose-recognition molecules are being ward into clinical studies. Ultimately, we system device suggests that it does not developed (151,152). Fluorescence tech- shall see improved methods for noninva- lead to significantly lower HbA1c values nologies include the use of engineered sive or minimally invasive glucose mea- compared with SMBG (149). In 2008, a molecules that exhibit altered fluores- surements that will complement current large 26-week randomized trial of 322 cence intensity or spectral characteristics glucose self-monitoring techniques. care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e71 Position Statement

NONINVASIVE GLUCOSE point-of-care monitoring, screening for di- considers all noninvasive sensing tech- ANALYSIS abetes, and control of hyperglycemia in crit- nologies to be high-risk medical devices, ically ill patients. To date, none of these and premarket approval documentation applications has been realized. will be required for commercialization RECOMMENDATION: NO NONINVASIVE in the U.S. (165). SENSING TECHNOLOGY IS CURRENTLY 2. Rationale Many reports of attempts to measure APPROVED FOR CLINICAL GLUCOSE Indirect and direct methods are being glucose noninvasively lack sufficient in- MEASUREMENTS OF ANY KIND. MAJOR developed for noninvasive glucose sens- formation to judge the likelihood that TECHNOLOGICAL HURDLES MUST BE ing. Indirect methods rely on the effect of glucose is actually being measured. The OVERCOME BEFORE NONINVASIVE in vivo glucose concentrations on a mea- interpretation of such clinical data is SENSING TECHNOLOGY WILL BE surable parameter. The classic example of complicated by the common use of mul- SUFFICIENTLY RELIABLE TO REPLACE this approach is the effect of blood glu- tivariate statistical methods, such as par- EXISTING PORTABLE METERS, cose concentrations on the scattering tial least squares regression and artificial IMPLANTABLE BIOSENSORS, OR properties of skin (163). Changes in neural networks. These multivariate MINIMALLY INVASIVE TECHNOLOGIES blood glucose substantially affect the dif- methods are prone to spurious correla- C (very low). ference in refractive index between skin tions that can generate apparently func- cells and the surrounding interstitial fluid tional glucose measurements in the 1. Use and thereby alter the scattering coefficient complete absence of glucose-specifican- Noninvasive glucose-sensing technolo- of skin. This parameter can be measured alytical information (166,167). Given this gies represent a group of potential ana- in a number of ways, including ocular co- known limitation of these multivariate lytical methods for measuring blood herent tomography. Skin impedance and methods, care must be used in their im- glucose concentrations without implant- the aggregation properties of erythrocytes plementation. Tests for spurious correla- ing a probe or collecting a sample of are other indirect approaches. tions (168–170) must be developed and any type. The most commonly explored Direct methods measure a property implemented with all future clinical data methods involve passing a selected band of the glucose molecule itself. Vibrational to avoid reports of false success. of nonionizing electromagnetic radiation spectroscopy is the primary direct method Despite the limitations noted above, (light) through a vascular region of the and generally involves mid-infrared, near- real progress is being made to further the body and then determining the in vivo infrared, photoacoustic, or Raman scat- development of noninvasive glucose- glucose concentration from an analysis of tering spectroscopy. The basis of these sensing technologies (171,172). Rigorous the resulting light or spectrum. The dis- measurements is the unique spectral sig- testing of noninvasive technologies must tinguishing feature of this approach is a nature of glucose relative to the back- be continued in concert with efforts to lack of physical contact between the ground tissue matrix. understand the underlying chemical basis sample matrix and a measurement probe. Selectivity is the primary factor that of selectivity. Issues of calibration stability The only functional interaction is the light must be addressed for either indirect or must also be investigated. Overall prog- passing through the sample. direct approaches. The lack of an isolated ress demands advances in both instru- A truly noninvasive method would be sample precludes the use of physical mentation and methods of data analysis. painless in operation and capable of con- separations or chemical reactions to en- For each, meaningful benchmarks must tinuous readings over time. In addition, hance measurement selectivity. All of the be established to allow rigorous inter- noninvasive sensing technology may be less analytical information must originate and intralaboratory comparisons. expensive to implement than existing tech- from the noninvasive signal. Ultimately, nologies that demand either a fresh test strip the success of any approach demands a for each measurement or a new implantable full understanding of the fundamental MELLITUS probe that requires multiple daily calibra- basis of selectivity. To this end, basic tion measurements with fresh test strips. research efforts are paramount to estab- 1. Use Furthermore, most noninvasive strategies lish such a level of understanding. offer the potential for measuring multiple RECOMMENDATION: ALL PREGNANT analytes from a single noninvasive mea- 3. Analytical considerations WOMEN NOT PREVIOUSLY KNOWN TO surement. The development of this tech- It should no longer be acceptable to HAVE DIABETES SHOULD UNDERGO nology is driven by the features of both low publish results that simply demonstrate TESTING FOR GDM AT 24–28 WEEKS OF cost and painless, continuous operation the ability to follow glucose transients GESTATION with no reagents or waste for disposal. during simple glucose tolerance tests A(high). Reports in the peer-reviewed litera- (164). This ability is well established in ture describe noninvasive measurements the literature for numerous approaches, GDM has been defined as any degree of based on a variety of techniques, such as both indirect and direct. In fact, it is rather glucose intolerance with onset or first absorption spectroscopy, photoacoustic easy to monitor optical changes that cor- recognition occurring during pregnancy spectroscopy, Raman scattering, static relate with in vivo glucose concentrations (1). After recent discussions, the Interna- light scattering, polarimetry, and optical during glucose tolerance tests. It is con- tional Association of the Diabetes and coherent tomography (159–162). Po- siderably more difficult, however, to Pregnancy Study Groups (IADPSG) rec- tential applications include discrete home demonstrate that such measurements are ommended that high-risk women who glucose testing, continuous home glucose reliable and selective. Reliability and se- have diabetes established according to monitoring, nocturnal hypoglycemia lectivity must be the focus of the next gen- standard criteria (Table 4) at their initial alarm, measurements in a physician’soffice, eration of research. Indeed, the FDA prenatal visit receive a diagnosis of overt, e72 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates not gestational, diabetes (21). The Table 6—Screening for and diagnosis of performed and that patients at average or IADPSG recommendations are not iden- GDM high risk receive a glucose-challenge test. tical to the criteria for nonpregnant indi- Several diagnostic strategies could be viduals, in that an OGTT result with an used. They were a “1-step” approach, in , Glucose FPG value 7.0 mmol/L (126 mg/dL) and concentration Percentage which an OGTT was performed initially, 2-h value .11.1 mmol/L (200 mg/dL) is . or a “2-step” approach, in which an ad- “ ” Glucose threshold, threshold not called overt diabetes. As the preva- measure mmol/L (mg/dL)a (cumulative)b ministered 50-g oral glucose load (regard- lence of obesity and type 2 diabetes has less of whether the patient was fasting) was increased, the number of women with un- FPG 5.1 (92) 8.3% followed by a plasma glucose measure- diagnosed diabetes has risen (173). There- 1-h PG 10.0 (180) 14.0% ment at 1 h. A plasma glucose value fore, the ADA now recommends that 2-h PG 8.5 (153) 16.1%c $7.8 mmol/L (140 mg/dL) indicates the women with risk factors for type 2 diabetes aOne or more of these values from a 75-g OGTT need for definitive testing with an OGTT; must be equaled or exceeded for the diagnosis of be screened for diabetes according to stan- b however, a consensus was lacking as to dard diagnostic criteria (Table 4) at the first GDM. Cumulative proportion of HAPO cohort whether a 100-g or 75-g OGTT should equaling or exceeding those thresholds. cIn addition, prenatal visit (93). Women with diabetes 1.7% of participants in the initial cohort were un- be performed and what cutoff values diagnosed with this approach should blinded because of an FPG value .5.8 mmol/L (105 should be used for diagnosis. receive a diagnosis of overt diabetes. mg/dL) or a 2-h OGTT value .11.1 mmol/L (200 Some GDM cases may represent pre- Two randomized clinical trials have mg/dL), bringing the total to 17.8%. existing, but undiagnosed, type 2 diabe- now demonstrated a benefitfromthe tes. Therefore, women with GDM should “ ” – treatment of mild GDM. Both studies These recommendations were adopted by be screened for diabetes 6 12 weeks post- found that treatment of GDM can reduce the ADA in 2011 (93) and are currently un- partum according to the OGTT criteria both serious adverse outcomes and the der consideration by the American College for nonpregnant women (Table 5) (93). frequency of large babies (macrosomia) of Obstetrics and Gynecology in the U.S. In addition, because women with GDM (174,175). and by corresponding groups in other are at a considerably increased risk of de- countries. Using the new criteria substan- veloping diabetes later (179), lifelong 2. Rationale tially increases the incidence of GDM, screening for diabetes should be per- The ADA states that because of the risks of mainly because only one increased glucose formed at least every 3 years according GDM to the mother and the neonate, value is required to diagnose GDM (prior to standard criteria for nonpregnant screening and diagnosis are warranted recommendations required two increased women (Table 4) (93). (21). The screening and diagnostic criteria fi glucose concentrations). Treatment will re- for GDM have recently been modi ed ex- quire additional resources, and outcome URINARY GLUCOSE tensively. The Hyperglycemia and Adverse studies will be necessary to ascertain Pregnancy Outcome (HAPO) study was a whether therapy is beneficial for GDM di- RECOMMENDATION: SEMIQUANTITATIVE large (approximately 25,000 pregnant agnosed with the new criteria; however, the URINE GLUCOSE TESTING IS NOT women) prospective, multinational epide- two trials that focused on the treatment of RECOMMENDED FOR ROUTINE CARE OF miologic study to assess adverse outcomes “mild GDM” (identified with the old crite- PATIENTS WITH DIABETES MELLITUS as a function of maternal glycemia (176). ria) achieved an improvement in outcomes, B(low). The study revealed strong, graded, predom- with only 10%–20% of the patients requir- inantly linear associations between mater- ing pharmacologic treatment in addition to 1. Use nal glycemia and primary study outcomes, medical nutritional therapy (174,175). i.e., birth weight .90th percentile, delivery Semiquantitative urine glucose testing, by cesarean section, clinical neonatal hypo- once the hallmark of diabetes care in the 3. Analytical considerations glycemia, and cord serum insulin (C- home setting, has now been replaced by These considerations have been ad- peptide) concentrations .90th percentile SMBG (see above). Semiquantitative dressed earlier in the Glucose sections. of values in the HAPO study population. urine glucose monitoring should be con- Given the strict cutoffs, it is very impor- The associations remain strong after adjust- sidered only for patients who are unable tant that close attention be paid to strin- ments for multiple potentially confounding or refuse to perform SMBG, because the gent sample-handling procedures to factors. Strong associations were also found urine glucose concentration does not minimize glycolysis after phlebotomy. with infant adiposity (177), with some sec- accurately reflect the plasma glucose con- ondary outcomes (including risks of shoul- centration (147,180). Notwithstanding der dystocia and/or birth injury), and with 4. Interpretation these limitations, urine glucose monitoring preeclampsia (176). On the strength of is supported by the IDF in those situations in which blood glucose monitoring is not these results, an expert consensus panel ap- RECOMMENDATION: GDM SHOULD BE accessible or affordable, particularly in pointed by the IADPSG recommended DIAGNOSED BY A 75-G OGTT ACCORDING “ ” fi resource-poor settings (23). outcome based criteria for the classi ca- TO THE IADPSG CRITERIA DERIVED FROM tion of glucose concentrations in pregnancy THE HAPO STUDY (178). All pregnant women not previously A(moderate). 2. Rationale known to have diabetes should be evaluated Although urine glucose is detectable in by a 75-g OGTT for GDM at 24–28 weeks patients with grossly increased blood of gestation (178). Diagnostic cut points for The ADA previously recommended glucose concentrations, it provides no fasting, 1-h, and 2-h plasma glucose con- that a “risk assessment” (based on age, information about blood glucose concen- centrations have been established (Table 6). weight, past history, and so on) be trations below the variable renal glucose care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e73 Position Statement threshold [approximately 10 mmol/L Increased ketone concentrations detected 2. Analytical. Several assay principles have (180 mg/dL)]. This fact alone limits its in patients with known diabetes or in pre- been described. Most commonly used is usefulness for monitoring diabetes under viously undiagnosed patients presenting the colorimetric reaction that occurs be- modern care recommendations. Semi- with hyperglycemia suggest impending tween AcAc and nitroprusside (sodium quantitative urine glucose tests also cannot or established DKA, a medical emergency. nitroferricyanide) to produce a purple distinguish between euglycemia and hypo- The two major mechanisms for high ke- color (181). This method is widely avail- glycemia. Furthermore, the extent to which tone concentrations in patients with dia- able in the form of dipsticks and tablets the kidney concentrates the urine will affect betes are increased production from and is used to measure ketones in both urine glucose concentrations, and only triglycerides and decreased utilization in the urine and blood (either serum or mean glucose values between voidings are the liver—both of which are due to an plasma). Several manufacturers offer dip- reflected. These facts further minimize the absolute or relative insulin deficiency sticks for measuring glucose and ketones. value of urine glucose measurements. and increased counter-regulatory hor- A combination dipstick is necessary only mones, including cortisol, epinephrine, if the patient monitors urine glucose in- 3. Analytical considerations glucagon, and growth hormone (183). stead of or in addition to blood glucose. Semiquantitative test-strip methods that The principal ketone bodies bHBA The nitroprusside method measures only use reactions specific for glucose are and AcAc are typically present in approx- AcAc unless the reagent contains glycine, recommended. Commercially available imately equimolar amounts. Acetone, in which case acetone is also measured. strips use the glucose oxidase reaction usually present in only small quantities, The nitroprusside-containing reagent is (181). Test methods that detect reducing is derived from spontaneous decarboxyl- much more sensitive to AcAc than ace- substances are not recommended because ation of AcAc. The equilibrium between tone with respect to color generation. Im- they are subject to numerous interfer- AcAc and bHBA is shifted towards bHBA portantly, this reagent cannot be used to ences, including numerous drugs and non- formation in any condition that alters the measure bHBA (181). glucose sugars. When used, single voided redox state of hepatic mitochondria to in- B. Blood ketones urine samples are recommended (147). crease NADH concentrations, such as 1. Preanalytical. Serum/plasma ketones 4. Interpretation hypoxia, fasting, metabolic disorders (in- can be measured with the tablets or dip- Because of the limited use of urine glucose cluding DKA), and alcoholic ketoacidosis sticks routinely used for urine ketone measurements, semiquantitative specific (184–186). Thus, assay methods for ke- measurements. Although samples can be reaction–based test-strip methods are ad- tones that do not include bHBA measure- diluted with saline to “titer” theketonecon- equate. ment may provide misleading clinical centration (results are typically reported as information by underestimating total ke- “positive at a 1/x dilution”), bHBA, the pre- KETONE TESTING tone body concentration (187). dominant ketone body in DKA, is not de- tected, as with urine ketone testing. 1. Use 3. Analytical considerations For specific bHBA measurements, A. Urine ketones sample requirements differ among meth- RECOMMENDATION: KETONES MEASURED 1. Preanalytical. The concentrations of ods, as is described below. In general, ketones in the urine of healthy individuals blood samples can be collected into tubes IN URINE OR BLOOD IN THE HOME fl SETTING BY PATIENTS WITH DIABETES are below the detection limits of commer- containing heparin, EDTA, uoride, cit- AND IN THE CLINIC/HOSPITAL SETTING cially available testing materials. False- rate, or oxalate. Ascorbic acid interferes SHOULD BE CONSIDERED ONLY AN positive results have been reported with with some assay methods. AcAc interferes ADJUNCT TO THE DIAGNOSIS OF DKA highly colored urine and in the presence with some assay methods unless the sam- GPP. of several sulfhydryl-containing drugs, ples are highly dilute. Sample stability dif- including angiotensin-converting en- fers among methods, but whole-blood The ketone bodies acetoacetate (AcAc), zyme inhibitors (188). Urine test reagents samples are generally stable at 4°C for acetone, and b-hydroxybutyric acid deteriorate with exposure to air, giving up to 24 h. Serum/plasma samples are (bHBA) are catabolic products of free fatty false-negative readings; therefore, testing stable for up to 1 week at 4°C and for at acids. Measurements of ketones in urine and material should be stored in tightly sealed least several weeks at 220°C (long-term blood are widely used in the management of containers and discarded after the expira- stability data are not available for most patients with diabetes as adjuncts for both tion date on the manufacturer’s label assay methods). diagnosis and ongoing monitoring of DKA. (189). False-negative readings have also 2. Analytical. Although several different Measurements of ketone bodies are rou- been reported with highly acidic urine assay methods (e.g., colorimetric, gas tinely performed, both in an office/hospital samples, such as after large intakes of chromatography, capillary electrophore- setting and by patients at home. The ADA ascorbic acid. Loss of ketones from urine sis, and enzymatic) have been described recommends that ketosis-prone patients attributable to microbial action can also for blood ketones, including specific mea- with diabetes check urine or blood ketones cause false-negative readings. Because ac- surement of bHBA, enzymatic methods in situations characterized by deterioration etone is a highly volatile substance, sam- appear to be the most widely used for in glycemic control in order to detect and ples should be kept in a closed container. the quantification of bHBA for routine preempt the development of DKA (21,182). For point-of-care analyses in medical fa- clinical management (190–192). The cilities and for patients in the home set- principle of the enzymatic methods is 2. Rationale ting, control materials (that give both that b-hydroxybutyrate dehydrogenase 1 Ketone bodies are usually present in urine negative and positive readings) are not in the presence of NAD converts bHBA and blood, but in very low concentrations commercially available but would be de- to AcAc and NADH. Under alkaline con- (e.g., total serum ketones, ,0.5 mmol/L). sirable to ensure accuracy of test results. ditions (pH 8.5–9.5), the reaction favors e74 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates the formation of AcAc from bHBA. The the results do not quantify bHBA, the pre- equivalent HbA1c andbereportedas fi NADH produced can be quanti ed spec- dominant ketone in DKA. The test should HbA1c for purposes of harmonization of trophotometrically (usually kinetically) not be used to monitor the course of results. HbA1 is composed of HbA1a, with the use of a peroxidase reagent. therapy, because AcAc and acetone may HbA1b, and HbA1c and should not be mea- Most methods permit the use of whole- increase as bHBA decreases during suc- sured or reported. The term “A1C test” is blood, plasma, or serum samples (re- cessful therapy (147,183–187). Blood used by the ADA in place of HbA1c to fa- quired volumes are generally #200 mL). ketone measurements that measure bHBA cilitate communication with patients. As Some methods permit the analysis of mul- specifically are useful for both the diag- describedinthetext,mostoftheclini- tiple analytes; these methods are designed nosis and ongoing monitoring of DKA cal-outcome data that are available for for point-of-care testing. Several methods (194–196). Reference intervals for bHBA the effects of metabolic control on compli- are available as handheld meters, which differ among assay methods, but concen- cations (at least for the DCCT and UKPDS) have been FDA cleared for both laboratory trations in healthy individuals who have involved the use of assay methods that use and home use by patients. These meth- fasted overnight are generally ,0.5 quantified HbA1c. In this report, we use ods use dry-chemistry test strips to which a mmol/L. Patients with well-documented the abbreviation GHb to include all forms , drop of whole blood, serum, or plasma is DKA [serum CO2 17 mmol/L, arterial of glycated hemoglobin.] HbA1c is used added. Results are displayed on the instru- pH ,7.3, plasma glucose .14.9 mmol/L both as an index of mean glycemia and ments within approximately 2 min. (250 mg/dL)] generally have bHBA con- as a measure of risk for the development centrations .2 mmol/L. of diabetes complications (147,197). 4. Interpretation HbA1c testing and maintenance of speci- 5. Emerging considerations fied concentrations during pregnancy in RECOMMENDATION: URINE KETONE Further studies are needed to determine patients with preexisting type 1 or type 2 MEASUREMENTS SHOULD NOT BE USED whether blood ketone measurements by diabetes are important for maximizing TO DIAGNOSE OR MONITOR THE COURSE patients with diabetes are preferable (e.g., the health of the newborn and decreasing OF DKA better accepted by patients, more prompt perinatal risks for the mother. Specifi- GPP. diagnosis of DKA) to urine ketone meas- cally, stringent control of HbA1c values urements. Studies are necessary to evalu- during pregnancy decreases the risk of A. Urine ketone measurements. The ate whether the test offers any clinical congenital malformations, large-for-date presence of positive urine ketone readings advantage over more traditional manage- infants, and the complications of preg- in a patient with known diabetes or a ment approaches (e.g., measurements of nancy and delivery that can otherwise oc- patient not previously diagnosed with serum CO2, anion gap, or pH). cur when glycemic control is not carefully diabetes but who presents with typical managed (198). A recent consensus state- symptoms of diabetes and hyperglycemia HbA1c ment (198) recommends an HbA1c value suggests the possibility of impending or of ,6% (42 mmol/mol) in these patients established DKA. Although DKA is most 1. Use if it can be achieved without excessive commonly associated with type 1 diabe- hypoglycemia. HbA1c is also being used tes, it may occur rarely in type 2 diabetic RECOMMENDATION: HbA1c SHOULD BE increasingly by quality-assurance pro- patients (193). Patients with alcoholic MEASURED ROUTINELY IN ALL PATIENTS gramstoassessthequalityofdiabetes ketoacidosis will have positive urine WITH DIABETES MELLITUS TO DOCUMENT care (e.g., requiring that healthcare pro- ketone readings, but hyperglycemia is THEIR DEGREE OF GLYCEMIC CONTROL viders document the frequency of HbA1c not usually present. Positive urine ketone A(moderate). testing in patients with diabetes and the readings are found in up to 30% of first proportion of patients with HbA1c values morning urine samples from pregnant Measurement of glycated proteins, pri- below a specified value) (199,200). women (with or without diabetes), dur- marily HbA1c, is widely used for routine The ADA and other organizations that ing starvation, and after hypoglycemia monitoring of long-term glycemic status have addressed this issue recommend (187). in patients with diabetes. [The terms “gly- HbA1c measurement in both type 1 and cated hemoglobin,”“glycohemoglobin,” type 2 diabetic patients to document the “glycosylated” (which should not be degree of glycemic control and to assess RECOMMENDATION: BLOOD KETONE used), “glucosylated hemoglobin,”“HbA1,” response to therapy (21,93,201). The DETERMINATIONS THAT RELY ON THE “ ” fi and HbA1c have all been used to refer to ADA has recommended speci c treat- NITROPRUSSIDE REACTION SHOULD BE hemoglobin that has been modified by the ment goals for HbA1c on the basis of re- USED ONLY AS AN ADJUNCT TO DIAGNOSE nonenzymatic addition of glucose. These sults from prospective randomized DKA AND SHOULD NOT BE USED TO terms are not interchangeable, however. clinical trials, most notably the DCCT MONITOR DKA TREATMENT. SPECIFIC The current acceptable term for glycation for type 1 diabetes (44,197) and the MEASUREMENT OF bHBA IN BLOOD CAN of hemoglobin in general is “glycated he- UKPDS for type 2 diabetes (46). These BE USED FOR DIAGNOSIS AND moglobin” (GHb). HbA1c is the specific trials have documented the relationship MONITORING OF DKA glycated species that is modified by glu- between glycemic control (as quantified B (moderate). cose on the N terminus of the hemoglobin by longitudinal HbA1c measurements) b chain. “HbA1c” is also the internation- and the risks for the development and B. Blood ketone measurements. Blood ally accepted term for reporting all GHb progression of chronic complications of ketone measurements that rely on the results. Assay methods that measure to- diabetes. Because different GHb assays nitroprusside reaction should be used tal GHbs (e.g., boronate affinity meth- can produce different GHb values, the with caution for DKA diagnosis, because ods) should be calibrated to report an ADA recommends that laboratories use care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e75 Position Statement only assay methods that have been certi- that relates their concentrations to the type or analyte is clinically superior to any fied as traceable to the DCCT GHb refer- chronic complications of diabetes other. The GHb results reported for the ence (21,187); these results are reported (147,187). same blood sample could differ consider- as HbA1c. The ADA recommends that in ably among methods, however, unless , general an HbA1c target of 7% (53 3. Analytical considerations they have been standardized to a common mmol/mol) is desirable for nonpregnant reference [e.g., without standardization, adults, with higher values recommended the same blood sample could be read as RECOMMENDATION: LABORATORIES for children and adolescents (21). HbA 7% (42 mmol/mol) in one laboratory and 1c SHOULD USE ONLY HbA ASSAY METHODS goals should be individualized according 1c 9% (75 mmol/mol) in another] (53,147, THAT ARE CERTIFIED BY THE NATIONAL to the potential for benefit with regard to 204,212–215). GLYCOHEMOGLOBIN STANDARDIZATION long-term complications and be bal- In 1996, the NGSP was initiated to PROGRAM (NGSP) AS TRACEABLE TO THE anced against the increased risk for the standardize GHb test results among labo- DCCT REFERENCE. THE MANUFACTURERS hypoglycemia that attends intensive ratories to DCCT-equivalent values (215). OF HbA ASSAYS SHOULD ALSO SHOW therapy. For selected individual pa- 1c The rationale for standardizing GHb test TRACEABILITY TO THE IFCC tients, more-stringent targets could be results to DCCT values was that the REFERENCE METHOD suggested, provided that this goal can GPP. DCCT had determined the relationship be- be achieved without substantial hypo- tween the results obtained for a specific glycemia or other adverse effects of GHb test (HbA1c) and long-term complica- treatment. Such patients might include RECOMMENDATION: LABORATORIES THAT tions in patients with type 1 diabetes thosewithashortdurationofdiabetes, (44,147,187). The NGSP was developed fi MEASURE HbA1c SHOULD PARTICIPATE a long life expectancy, and no signi cant IN A PROFICIENCY-TESTING PROGRAM, under the auspices of the American Associ- cardiovascular disease (93). Conversely, SUCH AS THE COLLEGE OF AMERICAN ation for Clinical Chemistry (AACC) and is higher HbA1c goals should be chosen endorsed by the ADA, which recommends PATHOLOGISTS (CAP) HbA1c SURVEY, for patients with a history of severe that laboratories use only GHb methods THAT USES FRESH BLOOD SAMPLES fi hypoglycemia, a limited life expectancy, WITH TARGETS SET BY THE NGSP that have passed certi cation testing by advanced microvascular or macrovascular LABORATORY NETWORK the NGSP (21,147). In addition, the ADA complications, or extensive comorbid con- GPP. recommends that all laboratories perform- ditions. Other clinical organizations recom- ing GHb testing participate in the CAP mend similar HbA1c targets, which range proficiency-testing survey for HbA1c,which from 6.5% to 7% (48 to 53 mmol/mol) Approximately 100 different GHb assay uses fresh whole-blood samples (216). (53,202). methods are in current use. They range The NGSP Laboratory Network from low-throughput research laboratory includes a variety of certified assay meth- 2. Rationale component systems and manual mini- ods, each calibrated to the DCCT refer- Glycated proteins are formed posttransla- column methods to high-throughput ence. The DCCT reference is an HPLC fi tionally from the slow, nonenzymatic automated systems dedicated to HbA1c cation-exchange method that quanti es reaction between glucose and free amino measurements. Most methods can be HbA1c; this method is a CLSI-designated groups on proteins (203). For Hb, the rate classified into one of two groups ac- comparison method (217). The assay of GHb synthesis is principally a function cording to assay principle (147,181, method has been used since 1978 and of the glucose concentration to which the 204). The first group includes methods has demonstrated good long-term preci- erythrocytes are exposed, integrated over that quantify GHb on the basis of charge sion (between-run CVs are consistently the time of exposure. GHb is a clinically differences between glycated and nongly- ,3%) (216). Secondary reference labora- useful index of mean glycemia during the cated components. Examples include tories in the Network interact with man- preceding 120 days, the average life span cation-exchange chromatography and ufacturers of GHb methods to assist them, of erythrocytes (147,203–206). Several agar-gel electrophoresis. The second first in calibrating their methods and then studies have demonstrated a close math- group includes methods that separate in providing comparison data for certifi- ematical relationship between HbA1c con- components on the basis of structural cation of traceability to the DCCT. Certi- centration and mean glycemia, which differences between glycated and nongly- fication is valid for 1 year. An important should allow the expression of HbA1c as cated components. Examples include adjunct to the program is the HbA1c an estimated average glucose (eAG) con- boronate affinity chromatography and proficiency-testing survey administered centration (205,207–209). Analogous to immunoassay. Most charge-based and by CAP. Since 1996 (starting with a pilot Hb (in erythrocytes), serum proteins be- immunoassay methods quantify HbA1c, project including 500 laboratories and ex- come glycated. Commercial assays are which is defined as HbA with glucose at- panded to all laboratories in 1998), the available that measure total glycated pro- tached to the N-terminal valine of one or survey has used fresh whole-blood sam- tein (termed fructosamine) or glycated al- both b chains. Other methods quantify ples with NGSP-assigned target values. bumin in the serum. The concentrations “total glycated hemoglobin,” which in- Since initiation of the NGSP in 1996, fl of these glycated proteins also re ect cludes both HbA1c and other Hb–glucose the survey has documented a steady im- mean glycemia, but over a much shorter adducts (e.g., glucose–lysine adducts and provement in comparability of GHb val- time (15–30 days) than GHb (60–120 glucose–a-chain N-terminal valine ad- ues among laboratories, both within and days) (147,203–206,210,211). The clini- ducts). Generally, the results of methods between methods (216,218). In 2007, cal utility of glycated proteins other than that use different assay principles show CAP initiated “accuracy-based” grading Hb has not been clearly established, how- excellent correlation, and there are no with the value of each sample assigned ever, and there is no convincing evidence convincing data to show that any method bytheNGSPNetwork.Theobjectiveis e76 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates to reduce bias and imprecision among as- recommended, but the decision to report however, race did not modify the associ- says. The NGSP Web site (http://www. eAG was left to the discretion of individ- ation between HbA1c concentration and ngsp.org) provides detailed information ual countries. Notwithstanding the agree- adverse cardiovascular outcomes or death on the certification process and maintains ment, it appears unlikely that universal (229). The ADAG study, which included fi a listing of certi ed assay methods (up- reporting of HbA1c will be adopted; how- frequent glucose measurements, did not dated monthly) and factors that are ever, the master equation allows conver- show a significantly different relationship known to interfere with specific methods. sion between IFCC and NGSP numbers. between the calculated mean glucose con- In 1997, the IFCC formed a commit- centration during 3 months and the teetodevelopahigher-orderreference A. Preanalytical HbA1c value at the end of the 3 months method and reference materials for HbA1c for Africans/African Americans and Cau- analysis; the method was approved in casians. The relatively small size of the 2001 (219,220). The analysis is per- RECOMMENDATION: LABORATORIES African/African American population, formed by cleaving Hb with endopro- SHOULD BE AWARE OF POTENTIAL however, limits the interpretation of this teinase Glu-C and separating the resulting INTERFERENCES, INCLUDING finding (209). glycated and nonglycated N-terminal HEMOGLOBINOPATHIES, THAT MAY Any condition that shortens erythro- b-chain hexapeptides by HPLC (220). AFFECT HbA1c TEST RESULTS, cyte survival or decreases mean erythro- The hexapeptides are quantified with elec- DEPENDING ON THE METHOD USED. cyte age (e.g., recovery from acute blood trospray ionization mass spectrometry IN SELECTING ASSAY METHODS, loss, hemolytic anemia) falsely lowers LABORATORIES SHOULD CONSIDER THE or capillary electrophoresis. The two HbA1c test results, regardless of the assay methods use the same primary reference POTENTIAL FOR INTERFERENCES IN THEIR method (147). Vitamins C and E are re- materials, and the results are essentially PARTICULAR PATIENT POPULATION. ported to falsely lower test results, possi- IN ADDITION, DISORDERS THAT AFFECT identical. HbA1c is measured as the ratio bly by inhibiting Hb glycation (230,231). of the glycated N-terminal peptide to the ERYTHROCYTE TURNOVER MAY CAUSE Iron deficiency anemia increases test re- nonglycated N-terminal peptide and is re- SPURIOUS RESULTS, REGARDLESS OF sults (232). Food intake has no significant ported in millimoles of deoxyfructosyl Hb THE METHOD USED effect on test results. Hypertriglyceride- per mole of Hb. Of note, preparing and GPP. mia, hyperbilirubinemia, uremia, chronic measuring samples with this method is alcoholism, chronic ingestion of salicy- laborious, very expensive, and time- 1. Patient variables. HbA1c results are not lates, and opiate addiction reportedly in- consuming. The method was never envi- affected significantly by acute fluctuations terfere with some assay methods, falsely sioned as a practical means of assaying in blood glucose concentrations, such as increasing results (204,233). clinical samples. It will only be used by those occurring with illness or after meals; Several Hb variants (e.g., Hbs S, C, D, manufacturers to standardize the assays. however, age and race reportedly influ- and E) and chemically modified Hb de- Like the NGSP, the IFCC has established ence HbA1c. Published data show age- rivatives interfere with some assay meth- a network of laboratories (221) (11 at the related increases in HbA1c values of ods [independently of any effects due to time of writing). The IFCC offers manu- approximately 0.1% per decade after age shortened erythrocyte survival (234– facturers calibrators and controls as well 30 years (224,225). Careful phenotyping 236); for a review, see (233)]. Depending as a monitoring program (221). Unlike of individuals with OGTT supports an in- on the particular hemoglobinopathy and the NGSP, the IFCC network does not crease in HbA1c with age, even after re- assay method, results can be either falsely have a certification program. moving from the study population increased or falsely decreased. Some AcomparisonofHbA1c results patients with otherwise undiagnosed di- methods may give a value in the reference obtained with pooled blood samples in abetes and persons with impaired glucose interval for a nondiabetic individual with the IFCC and NGSP (DCCT-aligned) tolerance (224). The clinical implications an Hb variant, but that is no assurance networks has revealed a linear relation- of the small, but statistically significant, pro- that no interference is present. The inter- “ ” ship (termed the master equation ): gressive increase in “normal” HbA1c levels ference may be subtle in the reference in- NGSP% 5 (0.915 x IFCC%) 1 2.15 with aging remain to be determined (226). terval but may increase steadily with fi (220). Although the clinical values ob- The effects of race on HbA1c values increasing HbA1c. Boronate af nity chro- tained with assays standardized with the are controversial. Several studies have matography assay methods are generally new IFCC method correlate tightly with suggested a relatively higher HbA1c in Af- considered to be less affected by Hb variants NGSP values, the absolute HbA1c values rican American and Hispanic populations than other methods. In some instances, reported differ by 1.5%–2.0% HbA1c. than in Caucasian populations at the same such as with most cation-exchange HPLC Concern regarding the clinical impact of level of glycemia (225,227,228). The ac- methods, manual inspection of chromato- changing patients’ HbA1c values led in cumulated evidence suggests that there grams or an automated report by the device 2007 to an agreement between the IFCC are differences in HbA1c among racial can alert the laboratory to the presence of and the major diabetes organizations to groups; however, the measurement of either a variant or a possible interference. report IFCC HbA1c results (in millimoles chronic glucose concentrations in these If an appropriate method is used, HbA1c per mole) as the equivalent NGSP DCCT- studies has not been sufficiently frequent can be measured accurately in the vast ma- aligned result (a percentage based on the to capture adequately the actual mean gly- jority of individuals heterozygous for Hb master equation) and as a calculated eAG cemia. Moreover, it is not clear that the variants (for a summary of published stud- based on the A1c-Derived Average Glu- differences in HbA1c have clinical signifi- ies, see http://www.ngsp.org). If altered cose (ADAG) study (209,222). In the re- cance. A recent analysis of 11,092 adults erythrocyte turnover interferes with the re- vised agreement, published in 2010 showed that blacks had mean HbA1c val- lationship between mean blood glucose and (223), both NGSP and IFCC units were ues 0.4% higher than whites (229); HbA1c values, or if a suitable assay method care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e77 Position Statement is not available for interfering Hb variants, Early reports recommended that the organizations, not reference intervals, alternative non–Hb-based methods for as- interassay CV be ,5% at normal and di- are used to evaluate metabolic control in sessing long-term glycemic control (such as abetic GHb concentrations (244). Subse- patients. fructosamine assay) may be useful (233). quent reports have suggested lower CVs Given that interferences are method [e.g., intralaboratory CVs ,3% (245) or fi , RECOMMENDATION: SAMPLES WITH HbA1c speci c, product instructions from the 2% (246), and interlaboratory CVs RESULTS BELOW THE LOWER LIMIT OF manufacturer should be reviewed before ,5% (245)]. Intraindividual CVs for > , THE REFERENCE INTERVAL OR 15% the HbA1c assay method is used. A list healthy persons are very small ( 2%), fi HbA1c SHOULD BE VERIFIED BY REPEAT of interfering factors for speci c assays is and many current assay methods can TESTING maintained on the NGSP Web site (http:// achieve intralaboratory and interlabora- B(low). www.ngsp.org). In selecting an assay tory CVs of ,2% and ,3%, respectively method, a laboratory should consider char- (247). A recent statistical analysis calcu- acteristics of the patient population served lated appropriate goals for HbA1c assay RECOMMENDATION: HbA1c VALUES THAT (e.g., a high prevalence of Hb variants). performance (218). If the reference ARE INCONSISTENT WITH THE CLINICAL 2. Sample collection, handling, and storage. change value (also termed “critical differ- PRESENTATION SHOULD BE Blood can be obtained by venipuncture ence”) is used, an analytical CV #2% will INVESTIGATED FURTHER or by finger-stick capillary sampling produce a 95% probability that a differ- GPP. $ (237,238). Blood tubes should contain ence of 0.5% HbA1c between successive the anticoagulant specified by the manu- patient samples is due to a significant 3. Out-of-range samples. A laboratory facturer of the HbA1c assay method change in glycemic control [when HbA1c should repeat testing for all sample results (EDTA can be used unless the manufac- is 7% (53 mmol/mol)]. In addition, if a below the lower limit of the reference in- turer specifies otherwise). Sample stabil- method has no bias, a CV of 3.5% is nec- terval, and if these results are confirmed, ity is assay method specific (239,240). In essary to have 95% confidence that the the physician should be informed to de- “ ” general, whole-blood samples are stable for HbA1c result for a patient with a true termine whether the patient has a variant up to 1 week at 4°C (240). For most meth- HbA1c of 7% (53 mmol/mol) will be be- Hb or shows evidence of erythrocyte de- 2 ods, whole-blood samples stored at 70°C tween 6.5% and 7.5% (between 48 and struction. If possible, the repeat HbA1c or colder are stable over the long term (at 58 mmol/mol) (218). We recommend measurement should be performed least 1 year), but samples are not as stable at an intralaboratory CV ,2% and an in- with a method based on an analytical 220°C. Improper handling of samples, terlaboratory CV ,3.5%. For a single principle that is different from the initial such as storage at high temperatures, can method, the goal should be an interlabor- assay. In addition, samples with results , introduce large artifacts that may not be de- atory CV 3%. .15% HbA1c (140 mmol/mol) should tectable, depending on the assay method. A laboratory should include two be assayed a second time; if the results are Manufacturers have introduced a num- control materials with different mean confirmed, the possibility of an Hb variant ber of convenient blood-collection systems, values (high and low) at both the begin- should be considered (233). Any result that including filter paper and small vials con- ning and the end of each day’s run. Fro- does not correlate with the clinical impres- taining stabilizing/lysing reagent (241– zen whole-blood controls stored in sion should also be investigated. 243). These systems are designed for field single-use aliquots at 270°C or colder 4. Removal of labile GHb. The formation collection of samples and routine mailing to are ideal and are stable for months or of HbA1c involves an intermediate Schiff the laboratory and are generally matched even years, depending on the assay base, which is called “pre-A1c” or “labile fi ” with speci c assay methods. They should method. Lyophilized controls are com- A1c (248). This Schiff base is formed rap- be used only if studies have been performed mercially available but, depending on idly with hyperglycemia and can interfere to establish the comparability of test results the assay method, may show matrix ef- with some HbA1c assay methods if it is not for these collection systems with standard fects when new reagents or columns are completely removed or separated. Most sample-collection and handling methods introduced. We recommend that a labo- currently available automated assays ei- fi for the speci c assay method used. ratory consider using both commercial ther remove the labile pre-HbA1c during and in-house controls to optimize per- the assay process or do not measure the B. Analytical formance monitoring. labile product. 2. Reference intervals. A laboratory should RECOMMENDATION: DESIRABLE determine its own reference interval 4. Interpretation – SPECIFICATIONS FOR HbA1c according to CLSI guidelines (CLSI A. Laboratory physician interactions. MEASUREMENT ARE AN Document C28A), even if the manufac- A laboratory should work closely with INTRALABORATORY CV ,2% AND AN turer has provided one. Nondiabetic test physicians who order HbA1c testing. INTERLABORATORY CV ,3.5%. AT LEAST individuals should be nonobese, have an Proper interpretation of test results re- TWO CONTROL MATERIALS WITH FPG concentration ,5.6 mmol/L (100 quires an understanding of the assay DIFFERENT MEAN VALUES SHOULD BE mg/dL), and, ideally, have a 2-h post- method, including its known interfer- ANALYZED AS AN INDEPENDENT MEASURE OGTT plasma glucose value of ,11.1 ences. For example, if the assay method OF ASSAY PERFORMANCE mmol/L (200 mg/dL). For NGSP-certified is affected by hemoglobinopathies (inde- B(low). assay methods, reference intervals should pendently of any shortened erythrocyte not deviate substantially (e.g., .0.5%) survival) or uremia, the physician should 1. Performance goals and quality control. from 4%–6% (20–42 mmol/mol). Note be made aware of this interference. Several expert groups have presented rec- that treatment target values recom- An important advantage of using ommendations for assay performance. mended by the ADA and other clinical an NGSP-certified method is that the e78 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates laboratory can provide specificinfor- results, the ADA has recommended that a either type 1 or type 2 diabetes. In addition, mation relating HbA1c test results to primary goal of therapy be an HbA1c value all patients with diabetes who are admitted both mean glycemia and outcome risks ,7% (53 mmol/mol) (21). Lower targets to a hospital should have HbA1c measured as definedintheDCCTandUKPDS may be considered for individual patients, if the results of testing in the previous 2–3 (44,147,187). This information is avail- e.g., in diet-treated type 2 diabetes. Other months are not available (21). Diabetes able on the NGSP Web site. For example, major clinical organizations have recom- quality-assurance programs [e.g., Provider each 1% (approximately 11 mmol/mol) mended similar targets (53); however, Recognition Program and HEDIS (Health- change in HbA1c is related to a change in recent studies that used multiple medica- care Effectiveness Data and Information the mean plasma glucose concentration of tions to treat type 2 diabetes and aimed Set) (199,200)] have generally required approximately 1.6 mmol/L (29 mg/dL). Re- for HbA1c concentrations ,6.5% (48 documentation of the percentage of dia- porting HbA1c results with a calculated mmol/mol) have not demonstrated con- betic patients who have had at least one eAG will eliminate the need for healthcare sistent benefits and failed to observe any HbA1c measurement during the preceding providers or patients to perform these benefit with regard to macrovascular dis- year. Studies have established that serial calculations themselves. The equation ease, compared with interventions that HbA1c measurements (quarterly for 1 year) generated by the ADAG study is the achieved HbA1c values 0.8% to 1.1% produce large improvements in HbA1c val- mostreliabletodate(209). higher (50–52). The ACCORD (Action ues in patients with type 1 diabetes (252). Some evidence suggests that imme- to Control Cardiovascular Risk in Dia- 3. Interpretation. HbA1c values in patients diate feedback of HbA1c test results to pa- betes) study demonstrated increased with diabetes constitute a continuum. tients at the time of the clinic visit leads to mortality with very intensive diabetes They range from within the reference in- an improvement in their long-term glyce- therapy [HbA1c, 6.4% vs. 7.5% (46 vs. terval in a small percentage of patients mic control (249,250). Not all publica- 58 mmol/mol)]. These HbA1c values ap- whose mean plasma glucose concentra- tions have supported this observation ply only to assay methods that have been tions are close to those of nondiabetic in- (251), however, and additional studies certified as traceable to the DCCT refer- dividuals, to markedly increased values are needed to confirm these findings be- ence, with a reference interval of approxi- (e.g., two- to threefold increases in some – – fl fore this strategy can be generally recom- mately 4% 6% HbA1c (20 42 mmol/mol). patients) that re ect an extreme degree of mended. It is possible to achieve the goal In the DCCT, each 10% reduction in hyperglycemia. A proper interpretation of of having HbA1c test results available at HbA1c (e.g., 12% vs. 10.8% or 8% vs. HbA1c test results requires that physi- the time of the clinic visit by either having 7.2%) was associated with an approxi- cians understand the relationship be- the patient send in a blood sample shortly mately 45% lower risk for the progression tween HbA1c values and mean plasma before the scheduled clinic visit or of diabetic retinopathy (42). Comparable glucose, the kinetics of HbA1c,andspe- having a rapid-assay system convenient risk reductions were found in the UKPDS cific assay limitations/interferences (147). to the clinic. (197). Also of note is that decreases in Small changes in HbA1c (e.g., 60.3% HbA1c were associated in the DCCT and HbA1c) over time may reflect assay impre- B. Clinical application UKPDS with an increased risk for severe cision rather than a true change in glycemic hypoglycemia. status (218).

RECOMMENDATION: TREATMENT GOALS 5. Emerging considerations SHOULD BE BASED ON ADA RECOMMENDATION: HbA1c TESTING RECOMMENDATIONS, WHICH INCLUDE SHOULD BE PERFORMED AT LEAST GENERALLY MAINTAINING HbA1c BIANNUALLY IN ALL PATIENTS AND RECOMMENDATION: HbA1c MAY BE USED CONCENTRATIONS AT ,7% AND QUARTERLY FOR PATIENTS WHOSE FOR THE DIAGNOSIS OF DIABETES, WITH MORE-STRINGENT GOALS IN SELECTED THERAPY HAS CHANGED OR WHO ARE NOT VALUES ‡6.5% BEING DIAGNOSTIC. AN INDIVIDUAL PATIENTS IF THEY CAN BE MEETING TREATMENT GOALS NGSP-CERTIFIED METHOD SHOULD BE ACHIEVED WITHOUT SIGNIFICANT B(low). PERFORMED IN AN ACCREDITED HYPOGLYCEMIA OR OTHER ADVERSE LABORATORY. ANALOGOUS TO ITS USE IN TREATMENT EFFECTS. SOMEWHAT 2. Testing frequency. There is no consensus THE MANAGEMENT OF DIABETES, HIGHER INTERVALS ARE RECOMMENDED on the optimal frequency of HbA1c test- FACTORS THAT INTERFERE WITH OR FOR CHILDREN AND ADOLESCENTS AND ing. The ADA recommends (21), “For ADVERSELY AFFECT THE HbA1c ASSAY MAY BE APPROPRIATE FOR PATIENTS WITH any individual patient, the frequency of WILL PRECLUDE ITS USE IN DIAGNOSIS A LIMITED LIFE EXPECTANCY, EXTENSIVE A1C testing should be dependent on the A(moderate). COMORBID ILLNESSES, A HISTORY OF clinical situation, the treatment regimen SEVERE HYPOGLYCEMIA, OR ADVANCED used, and the judgment of the clinician.” COMPLICATIONS (NOTE THAT THESE In the absence of well-controlled studies VALUESAREAPPLICABLEONLYIFTHE that suggest a definite testing protocol, RECOMMENDATION: POINT-OF-CARE NGSP HAS CERTIFIED THE ASSAY METHOD HbA1c ASSAYS ARE NOT SUFFICIENTLY expert opinion recommends HbA1c test- AS TRACEABLE TO THE DCCT REFERENCE) ACCURATE TO USE FOR THE DIAGNOSIS ing “at least two times a year in patients A (high). OF DIABETES who are meeting treatment goals (and who have stable glycemic control) ... B (moderate). 1. Treatment goals. HbA1c measurements and quarterly in patients whose therapy are now a routine component of the clin- has changed or who are not meeting glyce- A. Use of HbA1c for diabetes screening/ ical management of patients with diabe- mic goals” (21). These testing recommen- diagnosis. The role of HbA1c in the diag- tes. Principally on the basis of the DCCT dations are for nonpregnant patients with nosis of diabetes has been considered for care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e79 Position Statement several years (19,24,37,253). In the past, the IDF, are considering HbA1c testing GENETIC MARKERS the lack of standardization has been a ma- for diabetes diagnosis and screening. jor barrier. With improved standardiza- Note that glucose-based testing for diag- 1. Use tion through the NGSP and the IFCC, nosis remains valid. Analogous to the A. Diagnosis/screening and new data demonstrating the associa- concept of impaired fasting glucose tion between HbA1c concentrations and and impaired glucose tolerance, individ- the risk for retinopathy, the International RECOMMENDATION: ROUTINE uals with HbA1c values between 5.7% Expert Committee recommended the use and 6.4% (39 and 46 mmol/mol) should MEASUREMENT OF GENETIC MARKERS IS NOT OF VALUE AT THIS TIME FOR THE of HbA1c in the diagnosis of diabetes (20). be considered at high risk for future di- In making its recommendation, the Com- DIAGNOSIS OR MANAGEMENT OF abetes and should be counseled about mittee also considered several technical PATIENTS WITH TYPE 1 DIABETES. FOR effective measures to reduce their risk advantages of HbA1c testing compared SELECTED DIABETIC SYNDROMES, with glucose testing, such as its preana- (93). INCLUDING NEONATAL DIABETES, lytical stability and decreased biological B. Use of other glycated proteins, in- VALUABLE INFORMATION CAN BE variation. Finally, the clinical conve- cluding advanced glycation end prod- OBTAINED WITH DEFINITION OF ucts, for routine management of nience of the HbA1c assay, which requires DIABETES-ASSOCIATED MUTATIONS no patient fasting or tolerance tests, com- diabetes. Further studies are needed to de- A(moderate). pared with glucose-based diagnosis, con- termine whether other glycated proteins, vinced the Committee to recommend such as fructosamine or glycated serum al- HbA1c testing for diagnosis. A value bumin, are clinically useful for routine mon- 1. Type 1 diabetes. Genetic markers are $6.5% (48 mmol/mol) was considered itoring of patients’ glycemic status. Further currently of limited clinical value in eval- diagnostic on the basis of the observed studies are also needed to determine if meas- uating and managing patients with diabe- relationship with retinopathy. For urements of advanced glycation end prod- tes; however, mutational analysis is diagnosis, a positive test result [$6.5% ucts are clinically useful as predictors of risk rapidly emerging for classifying diabetes (48 mmol/mol)] should be confirmed for chronic diabetes complications (256). in the neonate (258–260) and in young with a repeat assay. The ADA indicates Only one study of a subset of DCCT patients patients with a dominant family history of “ that although either an HbA1c assay or a evaluated advanced glycation end products diabetes, often referred to as maturity- glucose assay (FPG or OGTT) can be used onset diabetes of the young” (MODY) fi in dermal collagen obtained with skin biop- as the con rmatory test, repeating the sies. Interestingly, the concentration of ad- (261). Type 1 or autoimmune diabetes same test is preferred (93). The frequency vanced glycation end products in dermal is strongly associated with HLA-DR (ma- of HbA testing for diagnosis has not jor histocompatibility complex, class II, 1c collagen correlated more strongly with the been established, but guidelines similar DR) and HLA-DQ (major histocompatibility presence of complications than the mean to those for glucose-based testing seem complex, class II, DQ) genes. HLA-DQA1 fi HbA1c values (257). The clinical role of appropriate. Only NGSP-certi ed HbA1c fi and HLA-DQB1 genotyping can be useful methods should be used to diagnose (or such measurements remains unde ned. to indicate the absolute risk of diabetes. screen for) diabetes. The ADA cautions Similarly, the role of noninvasive methods The HLA DQA1*0301–DQB1*0302 and that point-of-care devices for measuring that use light to measure glycation transder- DQA1*0501–DQB1*0201 haplotypes, fi HbA1c should not be used for diagnosis mally is unde ned. alone or in combination, may account (93). Although several point-of-care C. Global harmonization of HbA1c for up to 90% of children and young HbA1c assays are NGSP certified, the test testing and uniform reporting of re- adults with type 1 diabetes (262). These is waived in the U.S., and proficiency test- sults. As noted above, the NGSP has two haplotypes may be present in 30%– ing is not necessary. Therefore, no objec- largely succeeded in standardizing the 40% of a Caucasian population, and HLA tive information is available concerning GHb assay across methods and laborato- is therefore necessary but not sufficient their performance in the hands of those ries. Furthermore, the IFCC standardi- for disease. The HLA-DQ and HLA-DR ge- who measure HbA1c in patient samples. A zation, which provides a chemically netic factors are by far the most important recent evaluation revealed that few point- discrete standard, is being implemented determinants of type 1 diabetes risk of-care devices that measure HbA1c met worldwide. The reporting recommenda- (263). HLA typing may be used in com- acceptable analytical performance criteria tions (223) need to be implemented with bination with islet autoantibody analyses — — (254). Absent objective and ongoing the education of healthcare providers to exclude type 1 diabetes in assisting documentation of performance with ac- and patients. Some believe that report- in the diagnosis of genetic forms of dia- curacy-based proficiency testing that ing eAG should complement the current betes. uses whole blood (or other suitable mate- As indicated below, HLA-DR/DQ typ- reporting in NGSP/DCCT-aligned units rial that is free from matrix effects), point- ing can be useful to indicate a modified (percentages) and the new IFCC results of-care HbA1c devices should not be used risk of type 1 diabetes in persons positive for diabetes diagnosis or screening. (millimoles per mole), because the eAG for islet cell autoantibodies, because pro- results will be in the same units (millimoles The ADA has endorsed the use of HbA1c tective alleles do not prevent the appear- for the diagnosis of diabetes (Table 4) per liter or milligrams per deciliter) as ance of islet cell autoantibodies (most (21), as have The Endocrine Society patients’ self-monitoring. Educational often as single autoantibodies) but may (255) and the WHO. The American Asso- campaigns will be necessary, however, delay the onset of clinical diabetes. Typ- ciation of Clinical Endocrinologists sup- to ensure clear understanding of this ing of the class II major histocompati- ports it in a more limited fashion. Other assay, which is central to diabetes man- bility antigens or HLA-DRB1, -DQA1, international organizations, including agement. and -DQB1 is not diagnostic for type 1 e80 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates diabetes. Some haplotypes induce suscep- (odds ratios of 1.1 to 1.3), however, and also autoantibodies, which are markers tibility, however, whereas others provide do not significantly enhance our ability to of the disease’s pathogenesis, are often as- significant delay or even protection. Thus, predict the risk of type 2 diabetes (274). sociated with HLA-DRB1, HLA-DQA1, and HLA-DR/DQ typing can be used only to 3. MODY. Detecting mutations in MODY HLA-DQB1, indicating that self-peptides increase or decrease the probability of type patients and their relatives is technically may also be presented to T cells (262). 1 diabetes presentation and cannot be feasible. The reduced costs of sequencing Genetic testing for syndromic forms recommended for routine clinical diagno- and emerging new technologies make it of diabetes is the same as that for the sis or classification (264). possible to identify mutations and to underlying syndrome itself (1). Such The precision in the genetic character- properly classify MODY patients on the formsofdiabetesmaybesecondaryto ization of type 1 diabetes may be extended basis of specific mutations. As direct au- the obesity associated with Prader–Willi by typing for polymorphisms in several tomated sequencing of genes becomes syndrome, which maps to chromosome genetic factors identified in genome-wide standard, it is likely that the detection of 15q, or to the absence of adipose tissue association studies (265). Non-HLA ge- specific diabetes mutations will become inherent to the recessive Seip–Berardinelli netic factors include the INS (insulin), routine. syndrome of generalized lipodystrophy, PTPN22 [protein tyrosine phosphatase, B. Monitoring/prognosis. Although ge- which maps to chromosome 9q34 (1,276). non-receptor type 22 (lymphoid)], and netic screening may provide information More than 60 distinct genetic disorders are CTLA4 (cytotoxic T-lymphocyte–associated about prognosis and could be useful for associated with glucose intolerance or protein 4) genes and several others genetic counseling, genotype may not frank diabetes. Many forms of type 2 (263,265). These additional genetic fac- correlate with the phenotype. In addition diabetes (which are usually strongly famil- tors may assist in assigning a probability to environmental factors, interactions ial) will probably be understood in de- for a diagnosis of type 1 diabetes of uncer- among multiple loci for the expression fined genetic terms. The complexity of tain etiology (266). of quantitative traits may be involved. Ge- the genetic factors that contribute to type It is possible to screen newborn chil- netic identification of a defined MODY 2 diabetes risk is substantial (272,273). dren to identify those at increased risk for will have value for anticipating the prog- Several genetic factors for MODY have developing type 1 diabetes (267–269). This nosis. Infants with neonatal diabetes due been identified, and there are large num- strategy cannot be recommended until a to a mutation in the KCNJ11 (potassium bers of individual mutants. Persons at risk proven intervention is available to delay inwardly-rectifying channel, subfamily J, within MODY pedigrees can be identified or prevent the disease (270). There is member 11; also known as KIR6.2)gene through genetic means. Depending on the some evidence that early diagnosis may may be treated with sulfonylurea rather specific MODY mutation, the disease can prevent hospitalization for ketoacidosis than with insulin (258,259). be mild (e.g., mutation) and and preserve residual b-cells (271). The ra- not usually associated with long-term tionale for the approach is thus discussed 2. Rationale complications of diabetes, or it can be as below under Emerging Considerations. The HLA system, which has a fundamen- severe as typical type 1 diabetes [e.g., he- tal role in the adaptive immune response, patocyte nuclear factor (HNF) mutations] exhibits considerable genetic complexity. (277). RECOMMENDATION: THERE IS NO ROLE The HLA complex on chromosome 6 Eight different MODYs have been FOR ROUTINE GENETIC TESTING IN contains class I and class II genes that identified. MODY-1, -3, -4, -5, -6, and PATIENTS WITH TYPE 2 DIABETES. THESE code for several polypeptide chains (275). -7 are all caused by mutations in the genes STUDIES SHOULD BE CONFINED TO THE The major (classic) class I genes are HLA- encoding transcription factors that regu- RESEARCH SETTING AND EVALUATION OF A (major histocompatibility complex, late the expression of genes in pancreatic SPECIFIC SYNDROMES classI,A),HLA-B (major histocompatibil- b-cells. These genes are HNF4A (hepato- A(moderate). ity complex, class I, B), and HLA-C (major cyte nuclear factor 4, alpha) in MODY-1, histocompatibility complex, class I, C). HNF1A (HNF1 homeobox A) in MODY-3, 2. Type 2 diabetes. Fewer than 5% of pa- The loci of class II genes are designated HNF1B (HNF1 homeobox B) in MODY-5, tients with type 2 diabetes have been re- by three letters: the first (D) indicates the PDX1 (pancreatic and duodenal homeobox solved on a molecular genetic basis, and, class, the second (M, O, P, Q, or R) indi- 1; formerly known as IPF1)inMODY-4, not surprisingly, most of these patients cates the family, and the third (A or B) NEUROD1 (neurogenic differentiation 1; have an autosomal dominant form of the indicates the chain. Both classes of the also known as NeuroD and BETA2)in disease or very high degrees of insulin encoded molecules are heterodimers. MODY-6, and KLF1 [Kruppel-like factor 1 resistance. Type 2 diabetes is a heteroge- Class I molecules consist of an a chain (erythroid)] in MODY-7. Homozygous mu- b neous polygenic disease with both resis- and 2-microglobulin, and class II mole- tations of the PDX1 gene have been shown tance to the action of insulin and cules have a and b chains. The function of to lead to pancreatic agenesis, and hetero- defective insulin secretion (3,4). Multiple theHLAmoleculesistopresentshort zygous PDX1 mutations have been shown genetic factors interact with exogenous in- peptides derived from pathogens or auto- to cause MODY-4 (276). The modes of ac- fluences (e.g., environmental factors such antigens to T cells to initiate the adaptive tion of the HNF lesions in MODY are still as obesity) to produce the phenotype. immune response (275). Genetic studies not clear. It is likely that mutations in Identification of the affected genes is there- have revealed an association between cer- HNF1A, HNF1B,andHNF4A cause diabetes fore highly complex. Recent genome-wide tain HLA alleles and autoimmune dis- because they impair insulin secretion. association studies have identified .30 eases. These diseases include, but are MODY-2 is caused by mutations in the genetic factors that increase the risk for not confined to, ankylosing spondylitis, GCK [glucokinase (hexokinase 4)] gene. type 2 diabetes (272,273). The risk alleles celiac disease, Addison disease, and type The product of the gene is an essential en- in these loci all have relatively small effects 1 diabetes (275). Not only the disease but zyme in the glucose-sensing mechanism of care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e81 Position Statement b-cells, and mutations in this gene lead to persons heterozygous for HLA DRB1*04– advances in the identification of the genetic partial deficiencies of insulin secretion. DQA1*0301–DQB1*0302 and DRB1* bases for both type 1 and type 2 diabetes. MODY-8 is due to mutations in the CEL 03–DQA1*0501–DQB1*0201 are the This progress should ultimately lead to [carboxyl ester lipase (bile salt-stimulated most susceptible, with an absolute lifetime family counseling, prognostic information, lipase)] gene. risk of type 1 diabetes in the general pop- and the selection of optimal treatments ulation of about 1 in 12. Persons who are (276,284). 3. Analytical considerations protected from developing type 1 diabetes A detailed review of analytical issues will atayoungagearethosewithHLA not be attempted here, because genetic DRB1*15–DQA1*0201–DQB1*0602 AUTOIMMUNE MARKERS testing for diabetes outside of a research haplotypes in particular (281). Individu- 1. Use setting is currently not recommended for als with DRB1*11 or 04 who also have clinical care. Serologic HLA typing should DQB1*0301 are not likely to develop be replaced by molecular methods, be- type 1 diabetes at a young age. HLA-DR cause antibodies with a mixture of spe- is also involved in susceptibility to type 1 RECOMMENDATION: ISLET CELL cificities and cross-reactivities have been diabetes, in that the B1*0401 and 0405 AUTOANTIBODIES ARE RECOMMENDED estimated to give inaccurate results in subtypes of DRB1*04 are susceptible, FOR SCREENING NONDIABETIC FAMILY approximately 15% of typings. whereas the 0403 and 0406 subtypes MEMBERS WHO WISH TO DONATE PART OF A. Preanalytical. Mutations are detected are negatively associated with the disease, THEIR PANCREAS FOR TRANSPLANTATION by using genomic DNA extracted from pe- even when found in HLA genotypes with INTO A RELATIVE WITH END-STAGE ripheral blood leukocytes. Blood samples the susceptible DQA1*0301–DQB1*0302. TYPE 1 DIABETES should be drawn into test tubes containing DR molecules are heterodimers also; how- B(low). EDTA, and the DNA should be extracted ever, the DRa chain is invariant in all per- within 3 days; longer periods both lower sons. Additional DRb chains (B3, B4, and the yield and degrade the quality of the B5) are not important. DNA obtained. Genomic DNA can be iso- Class II MHC molecules are in- RECOMMENDATION: ISLET CELL lated from fresh or frozen whole blood by volved in antigen presentation to CD4 AUTOANTIBODIES ARE NOT lysis, digestion with proteinase K, extrac- helper cells, and the associations out- RECOMMENDED FOR ROUTINE DIAGNOSIS tion with phenol, and then dialysis. The lined above are likely to be explained by OF DIABETES, BUT STANDARDIZED ISLET average yield is 100–200 mg DNA from defective affinities to islet cell antigenic CELL AUTOANTIBODY TESTS MAY BE USED 10 mL of whole blood. DNA samples are peptides, leading to persistence of FOR CLASSIFICATION OF DIABETES IN best kept at 280°C in Tris-EDTA solution. T-helper cells that escape thymic abla- ADULTS AND IN PROSPECTIVE STUDIES OF These conditions maintain DNA sample in- tion. Class I HLA molecules are also CHILDREN AT GENETIC RISK FOR TYPE 1 tegrity virtually indefinitely. implicated in type 1 diabetes. Multiple DIABETES AFTER HLA TYPING AT BIRTH B. Analytical. Methods for the detection non-HLA loci also contribute to suscep- B(low). of mutations vary with the type of muta- tibility to type 1 diabetes (279). For ex- tion. MODY mutations have substitution, ample, the variable nucleotide tandem deletion, or insertion of nucleotides in the repeat (VNTR) upstream from the INS No therapeutic intervention that will pre- coding regions of the genes. These muta- gene on chromosome 11q is useful for vent diabetes has been identified (279,280). tions are detected by the PCR. Detailed predicting the development of type 1 di- Therefore, although several islet cell protocols for detecting specificmutations abetes, with alleles with the longest autoantibodies have been detected in in- are beyond the scope of this review. VNTR having protective effects. Typing dividuals with type 1 diabetes, their mea- newborn infants for both HLA-DR and surement has limited use outside of 4. Interpretation HLA-DQ—and to a lesser degree the INS clinical studies. Currently, islet cell auto- For screening for the propensity for type 1 gene—allows prediction of type 1 diabe- antibodies are not used in routine man- diabetes in general populations, HLA-D tes to better than 1 in 10 in the general agement of patients with diabetes. This genes are the most important, contribut- population. The risk of type 1 diabetes in section focuses on the pragmatic aspects ing as much as 50% of familial suscepti- HLA-identical siblings of a proband with of clinical laboratory testing for islet cell bility (278). HLA-DQ genes appear to be type 1 diabetes is 1 in 4, whereas siblings autoantibodies. central to the HLA-associated risk of type who have HLA haplotype identity have a 1 A. Diagnosis/screening 1 diabetes, albeit HLA-DR genes may be in 12 risk and those with no shared haplo- 1. Diagnosis. In type 1 diabetes, the pan- independently involved [for reviews, see type have a 1 in 100 risk (280). Genome- creatic islet b-cells are destroyed and lost. (279,280)]. The heterodimeric proteins wide association studies have confirmed In the vast majority of these patients, the that are expressed on antigen-presenting that the following non-HLA genetic factors destruction is mediated by an autoimmune cells, B lymphocytes, platelets, and acti- increase the risk for type 1 diabetes, both in attack (285). This disease is termed “type vated T cells—but not other somatic first-degree relatives of type 1 diabetic pa- 1A” or “immune-mediated diabetes” (Table cells—are composed of cis- and trans- tients and in the general population: INS, 1). Islet cell autoantibodies comprise auto- complementated a-andb-chain hetero- VNTR, CTLA4, PTPN22, and others antibodies to islet cell cytoplasm (ICA), to dimers. Thus, in any individual, four (263,265,282,283). native insulin [referred to as “insulin auto- possible DQ dimers are encoded. Persons antibodies” (IAA) (286)], to the 65-kDa at the highest genetic risk for type 1 di- 5. Emerging considerations isoform of glutamic acid decarboxyl- abetes are those in whom all four DQ com- The sequencing of the human genome and ase (GAD65A) (287–289), to two insu- binations meet this criterion. Thus, the formation of consortia have produced linoma antigen 2 proteins [IA-2A (290) e82 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates and IA-2bA (also known as phogrin) 2. Screening. Only about 15% of patients RECOMMENDATION: THERE IS (291)], and to three variants of zinc with newly diagnosed type 1 diabetes CURRENTLY NO ROLE FOR MEASUREMENT transporter 8 (ZnT8A) (292,293). Auto- have a first-degree relative with the dis- OF ISLET CELL AUTOANTIBODIES IN THE antibody markers of immune destruc- ease (294). The risk of developing type 1 MONITORING OF PATIENTS IN CLINICAL tion are usually present in 85% to diabetes in relatives of patients with the PRACTICE. ISLET CELL AUTOANTIBODIES 90% of individuals with type 1 diabetes disease is approximately 5%, which is 15- ARE MEASURED IN RESEARCH PROTOCOLS when fasting hyperglycemia is initially fold higher than the risk in the general AND IN SOME CLINICAL TRIALS AS detected (1). Autoimmune destruction population (1 in 250–300 lifetime risk). SURROGATE END POINTS of b-cells has multiple genetic predispo- Screening relatives of type 1 diabetic pa- B(low). sitions and is modulated by undefined tients for islet cell autoantibodies can environmental influences. The autoim- identify those at high risk for the dis- munity may be present for months or ease; however, as many as 1%–2% of B. Monitoring/prognosis. No acceptable years before the onset of hyperglycemia healthy individuals have a single auto- therapy has been demonstrated to pro- and subsequent symptoms of diabetes. antibody against insulin, IA-2, GAD65, long the survival of islet cells once di- After years of type 1 diabetes, some anti- or ZnT8 and are at low risk of develop- abetes has been diagnosed or to prevent bodies fall below detection limits, but ing type 1 diabetes (295). Because of the clinical onset of diabetes in islet cell GAD65A usually remains increased. the low prevalence of type 1 diabetes autoantibody–positive individuals (279). Patients with type 1A diabetes have a (approximately 0.3% in the general pop- Thus, the use of repeated testing for islet significantly increased risk of other ulation), the positive predictive value cell autoantibodies to monitor islet cell autoimmune disorders, including celiac of a single islet cell autoantibody will autoimmunity is not clinically useful at disease, Graves disease, thyroiditis, Ad- be low (280). The presence of multiple present. In islet cell or pancreas trans- dison disease, and pernicious anemia islet cell autoantibodies (IAA, GAD65A, plantation, the presence or absence of (128). As many as 1 in 4 females with IA-2A/IA-2bA, or ZnT8A) is associated islet cell autoantibodies may clarify type 1 diabetes have autoimmune thy- with a .90% risk of type 1 diabetes whether subsequent failure of the trans- roid disease, whereas 1 in 280 patients (292,295,296); however, until cost- planted islets is due to recurrent auto- develop adrenal autoantibodies and ad- effective screening strategies can be immune disease or to rejection (302). renal insufficiency. A minority of pa- developed for young children and until When a partial pancreas has been trans- tients with type 1 diabetes (type 1B, effective intervention therapy to prevent planted from an identical twin or other idiopathic) have no known etiology or delay the onset of the disease becomes HLA-identical sibling, the appearance and no evidence of autoimmunity. available, such testing cannot be recom- of islet cell autoantibodies may raise con- Many of these patients are of African or mended outside of a research setting. sideration regarding the use of immuno- Asian origin. Children with certain HLA-DR and/ suppressive agents to try to halt the or HLA-DQB1 chains (*0602/*0603/ recurrence of diabetes. Notwithstanding *0301) are mostly protected from type these theoretical advantages, the value of 1 diabetes, but not from developing islet this therapeutic strategy has not been es- cell autoantibodies (297). Because islet tablished. RECOMMENDATION: SCREENING cell autoantibodies in these individuals Some experts have proposed that test- have substantially reduced predictive ing for islet cell autoantibodies may be PATIENTS WITH TYPE 2 DIABETES FOR fi ISLET CELL AUTOANTIBODIES IS NOT signi cance, they are often excluded useful in the following situations: 1)to from prevention trials. identify a subset of adults initially thought RECOMMENDED AT PRESENT. – STANDARDIZED ISLET CELL Approximately 5% 10% of adult to have type 2 diabetes but who have islet AUTOANTIBODIES ARE TESTED IN Caucasian patients who present with a cell autoantibody markers of type 1 diabe- PROSPECTIVE CLINICAL STUDIES OF TYPE type 2 diabetes phenotype also have islet tes and who progress to insulin depen- 2 DIABETIC PATIENTS TO IDENTIFY cell autoantibodies (298), particularly dency (303); 2) to screen nondiabetic POSSIBLE MECHANISMS OF SECONDARY GAD65A, which predict insulin depen- family members who wish to donate a kid- FAILURES OF TREATMENT OF TYPE 2 dency. This condition has been termed ney or part of their pancreas for transplan- “latent autoimmune diabetes of adult- tation; 3) to screen women with GDM to DIABETES ” “ ” B(low). hood (LADA) (299), type 1.5 diabetes identify those at high risk of progression to (300), or “slowly progressive IDDM” type 1 diabetes; and 4) to distinguish type 1 (301). Although GAD65A-positive dia- from type 2 diabetes in children to institute betic patients progress faster to absolute insulin therapy at the time of diagnosis insulinopenia than do antibody-negative (304,305). For example, some pediatric RECOMMENDATION: SCREENING FOR patients, many antibody-negative (type diabetologists now treat children thought ISLET CELL AUTOANTIBODIES IN 2) diabetic adults also progress (albeit to have type 2 diabetes with oral medica- RELATIVES OF PATIENTS WITH TYPE 1 more slowly) to insulin dependency tions but treat autoantibody-positive DIABETES OR IN PERSONS FROM THE with time. Some of these patients may children immediately with insulin. It is GENERAL POPULATION IS NOT show T-cell reactivity to islet cell com- possible, however, to follow patients RECOMMENDED AT PRESENT. ponents (300). Islet cell autoantibody who are islet cell autoantibody positive STANDARDIZED ISLET CELL testing in patients with type 2 diabetes to the point of metabolic decompensa- AUTOANTIBODIES ARE TESTED IN has limited utility, because the institu- tion and then institute insulin therapy. PROSPECTIVE CLINICAL STUDIES tionofinsulintherapyisbasedonglu- The Diabetes Prevention Trial of Type 1 B(low). cose control. Diabetes (DPT-1) study failed to show a care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e83 Position Statement protective effect of parenteral insulin develop after insulin therapy, even in per- that still carry out the ICA assay has de- (306). sons who use human insulin. Data from creased markedly, and the test is no lon- the Diabetes Autoantibody Standardiza- ger included in the DASP program. 2. Rationale tion Program (DASP) demonstrate that The presence of islet cell autoantibodies the interlaboratory imprecision for IAA 4. Interpretation suggests that insulin therapy is the most is inappropriately large (309). GAD65A may be present in approximately appropriate therapeutic option, especially GAD65A and IA-2A are measured with 60%–80% of patients with newly diag- in a young person. Conversely, in children standardized radiobinding assays, which nosed type 1 diabetes, but the frequency or young people without islet cell autoanti- are performed with 35S-labeled recombi- varies with sex and age. GAD65A is asso- bodies, consideration may be given to a nant human GAD65 or IA-2 generated by ciated with HLA DR3–DQA1*0501– trial of oral agents and lifestyle changes. coupled in vitro transcription translation DQB1*0201 in both patients and healthy There is no unanimity of opinion, but the with [35S]methionine or other 35S- or individuals. IA-2As may be present presence of islet cell autoantibodies may 3H-labeled amino acids (310). Commer- in 40%–50% of patients with newly diag- alter therapy for subsets of patients, in- cially available methods for GAD65A and nosed type 1 diabetes, but the frequency cluding Hispanic and African American IA-2A are available as a radioimmunoassay is highest in the young. The frequency children with a potential diagnosis of non- with 125I-labeled GAD65 (truncated at the decreases with increasing age. IA-2As are autoimmune diabetes, adults with islet cell N-terminal end to promote solubility) and associated with HLA DR4–DQA1*0301– autoantibodies but clinically classified as IA-2, respectively. In addition, immunoas- DQB1*0302. IAA positivity occurs in type 2 diabetic, and children with transient says without radiolabel are commercially .70%–80% of children who develop hyperglycemia. The majority of nondia- available for both GAD65A and IA-2A. type 1 diabetes before 5 years of age but betic individuals who have only one auto- Major efforts have been made to standard- occurs in ,40% of individuals who de- antibody may never develop diabetes. ize GAD65A and IA-2A measurements velop diabetes after the age of 12 years. Although the production of multiple islet (309,311). A WHO standard for both IAAs are associated with HLA DR4– cell autoantibodies is associated with con- GAD65A and IA-2A has been established, DQA1*0301–DQB1*0302 and with INS siderably increased diabetes risk (295,296), and GAD65A and IA-2A amounts are ex- VNTR (262). ICA is found in about approximately 20% of individuals present- pressed in international units (312). The 75%–85% of new-onset patients. ing with new-onset diabetes produce only a binding of labeled autoantigen to autoanti- The ICA assay is labor-intensive and single autoantibody. Prospective studies of bodies is normally distributed. Cutoff val- difficult to standardize, and marked in- children reveal that islet cell autoantibodies ues should be determined from 100–200 terlaboratory variation in sensitivity and may be transient, indicating that an islet serum samples obtained from healthy indi- specificity has been demonstrated in autoantibody may have disappeared prior viduals. GAD65A and IA-2A results should workshops (284,314). Few clinical labo- to the onset of hyperglycemia or diabetes be reported as positive when the signal ratories are likely to implement this test. symptoms (307). exceeds the 99th percentile. Comparison Theimmunoassaysaremorereproduc- of multiple laboratories worldwide is car- ible and are amenable to standardization fi 3. Analytical considerations ried out in the DASP, a pro ciency-testing (309). Measurement of T-cell reactivity in program organized by the CDC under the peripheral blood is theoretically appeal- auspices of the Immunology of Diabetes So- ing, but the imprecision of such assays RECOMMENDATION: IT IS IMPORTANT ciety. That commercially available GAD65A precludes their use from a clinical setting THAT ISLET CELL AUTOANTIBODIES BE and IA-2A methods are also participating in (315,316). Autoantibody positivity (by MEASURED ONLY IN AN ACCREDITED the DASP program demonstrates that it definition) occurs in healthy individuals LABORATORY WITH AN ESTABLISHED should be possible not only to harmonize despite an absence of a family history of QUALITY-CONTROL PROGRAM participating laboratories but also eventu- autoimmune diseases. Islet cell autoanti- AND PARTICIPATION IN A ally to standardize GAD65A and IA-2A bodies are no exception. If one autoanti- PROFICIENCY-TESTING PROGRAM (311). body is found, the others should be GPP. ICAs are measured by indirect immu- assayed, because the risk of type 1 diabe- nofluorescence of frozen sections of hu- tes increases if an individual tests positive man pancreas (313). ICA assays measure for two or more autoantibodies (306). For IAAs, a radioisotopic method that the degree of immunoglobulin binding to The following suggestions (279) have calculates the displaceable insulin radio- islets, and results are compared with a been proposed as a rational approach to ligand binding after the addition of excess WHO standard serum available from the the use of autoantibodies in diabetes: 1) nonradiolabeled insulin (308) is recom- National Institute of Biological Standards antibody assays should have a specificity mended. Results are reported as positive and Control (312). The results are re- .99%; 2)proficiency testing should be when specific antibody binding exceeds ported in Juvenile Diabetes Foundation documented; 3) multiple autoantibodies the 99th percentile or possibly exceeds (JDF) units. Positive results depend on should be assayed; and 4) sequential mea- themeanplus2(or3)SDsforhealthy the study or context in which they are surement should be performed. These persons. Insulin autoantibody binding used, but many laboratories use 10 JDF strategies will reduce false-positive and has been noted not to be normally distrib- units measured on two separate occasions false-negative results. uted. Each laboratory needs to assay at or a single result $20 JDF units as titers least 100–200 healthy individuals to de- that may indicate a significantly increased 5. Emerging considerations termine the distribution of binding. An risk of type 1 diabetes. The method is Immunoassays for IAA, GAD65A, IA-2A/ important caveat concerning IAA mea- cumbersome and has proved difficult to IA-2bA, and ZnT8A are now available, surement is that insulin antibodies standardize. The number of laboratories and a panel of these autoantibodies is e84 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates currently used in screening studies (317). semiquantitative or qualitative screening Disease: Improving Global Outcomes Because ICA assays are difficult to standard- tests must be shown to be positive in Committee (21,325–327) as excretion ize, their use has declined substantially. .95% of patients with albuminuria. Pos- of 30–300 mg of albumin/24 h, 20–200 It is likely that other islet cell antigens itive results of such tests must be confirmed mg/min, or 30–300 mg/mg creatinine will be discovered, and such discoveries by quantitative testing in an accredited (Table 8) on two of three urine collec- could lead to additional diagnostic and laboratory. tions. Recent data, however, suggest predictive tests for type 1 diabetes. Auto- that risk extends below the lower limit antibody screening of dried spots obtained of 20 mg/min (328–330), reinforcing from finger-stick blood samples appears 1. Use the notion that this factor is a continuous quitefeasibleinthefuture.Forindividuals variable for cardiovascular risk (331–333). who are positive for islet cell autoantibodies, The JNC 7, the National Kidney RECOMMENDATION: ANNUAL TESTING HLA-DR/HLA-DQ genotyping will help de- Foundation (NKF), and the ADA all finetheabsoluteriskoftype1diabetes. FOR ALBUMINURIA IN PATIENTS WITHOUT recommend the use of morning spot Several clinical trials to prevent or CLINICAL PROTEINURIA SHOULD BEGIN IN albumin/creatinine measurement for an- intervene with type 1 diabetes are being PUBERTAL OR POSTPUBERTAL nual quantitative testing for urine albu- actively pursued (317). Such trials can INDIVIDUALS 5 YEARS AFTER DIAGNOSIS min in adults with diabetes (21,326,327). now be done with relatives of patients OF TYPE 1 DIABETES AND AT THE TIME OF Individuals should be fasting. The opti- DIAGNOSIS OF TYPE 2 DIABETES, with type 1 diabetes or in the general pop- mal time for spot urine collection is the ulation on the basis of the islet cell auto- REGARDLESS OF TREATMENT early morning, but for minimizing varia- B (moderate). antibody and HLA-DR/HLA-DQ genotype tion, all collections should be at the same status. Risk can be assessed by islet cell time of day; the individual preferably autoantibodies alone, without the need should not have ingested food for at least for evaluating endogenous insulin re- RECOMMENDATION: URINE ALBUMIN AT 2 h (334). “ serves, as was done for the U.S. DPT-1 CONCENTRATIONS ‡30 mg/g CREATININE Positive test results represent albu- ” trial (306). Rates of islet cell autoantibody SHOULD BE CONSIDERED A CONTINUOUS minuria in these guidelines, correspond- . positivity are distinctly lower in the RISK MARKER FOR CARDIOVASCULAR ing to protein excretion of 300 mg/24 h, . m . general population than in relatives of EVENTS 200 g/min, or 300 mg/g creatinine individuals with type 1 diabetes; conse- B (moderate). (Table 8). In these patients, quantitative quently, trials with the latter group are measurement of urine albumin excretion more economical. Potential interven- is used in assessing the severity of albu- tional therapies (for type 1 diabetes) un- A. Diagnosis/screening. Diabetes is as- minuria and its progression, in planning dergoing clinical trials include oral insulin sociated with a very high rate of cardio- treatment, and in determining the impact (317) or nasal insulin (318) given to non- vascular events and is the leading cause of of therapy. To properly assess the stage of diabetic (but islet cell autoantibody– end-stage renal disease in the Western kidney disease, the estimated glomerular positive) relatives of individuals with world (321). Early detection of risk mark- filtration rate (eGFR) can be calculated type 1 diabetes or to children with islet ers, such as albumin in the urine (for- from the serum creatinine value, age, cell autoantibodies and HLA genotypes merly termed “microalbuminuria”), sex, and race of the patient (335). An conferring increased risk. Phase II clinical relies on tests for urinary excretion of al- eGFR of ,60 mL/min, regardless of the trials with alum-formulated GAD65 have bumin. Conventional qualitative tests presence of low levels of albuminuria, is reported no adverse events and some (chemical strips or “dipsticks”) for albu- an independent cardiovascular risk factor preservation of endogenous insulin pro- minuria do not detect the small increases (325,327). A urine albumin value of ,30 duction in GAD65A-positive diabetic pa- of urinary albumin excretion. For this mg/g creatinine, although considered tients (319,320). Additional trials of purpose, tests to detect albumin concen- “normal,” should be reassessed annually, other antigen-based immunotherapies, trationsareused(Table7)(322–324). because values as low as 10 mg/g creati- adjuvants, cytokines, and T-cell acces- Low levels of albuminuria have been de- nine have been associated in some studies sory molecule–blocking agents are likely fined by the Joint National Committee with an increased cardiovascular risk. If in the future (270). Decreased islet cell (JNC) 7 and the ADA and have more re- the value is $30 mg/g creatinine, changes autoimmunity will be one important out- cently been redefined by the Kidney should be reassessed after 6 to 12 months come measure of these therapies. — ALBUMINURIA (FORMERLY Table 7 Review of assays to assess albuminuria MICROALBUMINURIA)—Albu- minuria (formerly microalbuminuria) Method Interassay CV Detection limit are a well-established cardiovascular risk marker, in which increases over time to Immunonephelometry (Beckman 4.2% at 12.1 mg/L 2mg/L macroalbuminuria (.300 mg/day) are Coulter Array analyzer) 5.3% at 45 mg/L associated with kidney disease and an Immunoturbidimetry (Dade 4.1% at 10.6 mg/L 6mg/L increased risk for progression to end- Behring turbimeter) 2.2% at 77.9 mg/L stage renal disease. Annual testing for Hemocue (point of care) 2.2% at 77.9 mg/L 5mg/L albuminuria is recommended by all 4.3% at 82 mg/L m major guidelines for patients with diabe- Radioimmunoassay 9.2% at 12.2 mg/dL 16 g/L tes and/or kidney disease. To be useful, 4.8% at 33 mg/L care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e85 Position Statement

Table 8—Definitions of albuminuriaa cardiovascular risk and delaying the onset of overt diabetic nephropathy. Thus, it is an Unit of measure indicator of the need for more intensive efforts to reduce cardiovascular risk factors. mg/24 h mg/min mg/mg creatinine Albuminuria (stage A2) rarely occurs Normal ,30 ,20 ,30 with a short duration of type 1 diabetes or High albuminuria (formerly microalbuminuria) 30–300 20–200 30–300 before puberty. Thus, testing is less ur- Very high albuminuriab .300 .200 .300 gent in these situations. Nevertheless, the fi aFrom the ADA (21). bAlso called “overt nephropathy.” dif culty in precisely dating the onset of type 2 diabetes warrants initiation of annual testing at the time of diagnosis of if antihypertensve therapy is required or patients with type 1 diabetes and low levels diabetes. Although older patients (age annually in those who are normotensive of albuminuria, urinary albumin excretion .75 years or a life expectancy ,20 years) (326). For children with type 1 diabetes, can increase by as much as 10%–20%/year, may not be at risk for clinically significant testing for low levels of albuminuria is with the development of clinical protein- nephropathy because of a short projected recommended to begin after puberty uria (.300 mg albumin/day) in 10–15 life span, they will be at higher cardiovas- and after a diabetes duration of 5 years. years in more than half the patients. After cular risk. In such patients, the role of Of note is that most longitudinal cohort clinical-grade proteinuria occurs, .90% treating albuminuria is far from clear. studies have reported significant increases of patients develop a decreased GFR and, Published studies have demonstrated in the prevalence of low levels of albumin- ultimately, end-stage renal disease. In type that it is cost-effective to screen all pa- uria only after diabetes has been present for 2 diabetes, 20%–40% of patients with tients with diabetes and/or kidney disease 5 years (326,336). stage A2 albuminuria (Table 8) progress for albuminuria (346,347). In the algorithms of both the NKF and to overt nephropathy, but by 20 years after the ADA for urine protein testing (321), overt nephropathy, approximately 20% 3. Analytical considerations the diagnosis of low levels of albuminuria develop end-stage renal disease. In addi- requires both the demonstration of in- tion, patients with diabetes (type 1 or type fi RECOMMENDATION: THE ANALYTICAL CV creased albumin excretion (as de ned 2) and stage A2 albuminuria are at in- OF METHODS TO MEASURE LOW LEVELS above) on two of three tests repeated at creased risk for cardiovascular disease. OF ALBUMINURIA SHOULD BE ,15% intervals of 3 to 6 months and the exclu- Of note is that low levels of albuminuria B (moderate). sion of conditions that “invalidate” the alone indicate neither an increased risk for test (Fig. 1). progression to end-stage kidney disease B. Prognosis. Albuminuria values .30 nor kidney disease per se; hypertension A. Analytical. Analytical goals can be mg/g creatinine [and lower values if the needs to be present for the risk of progres- related to the degree of biological varia- eGFR is ,60 mL/min (Table 8)] have sion (339,340). Moreover, about 20% of tion, with less precision required for prognostic significance. Multiple epide- people progress to end-stage kidney dis- analytes that vary widely. Detection limits miologic studies have shown it to be an ease without an increase in low levels of and imprecision data are summarized in independent risk marker for cardiovas- albuminuria (341). Another factor that in- Table 7. Commercially available quantita- cular death (325,337,338). In 80% of dicates progression is an increase in albu- tive methods for low levels of albuminuria minuria from stage A2 to A3 over time have documented detection limits of ap- despite achievement of blood pressure proximately 20 mg/L or less. Within-run goals (342). imprecision and day-to-day (total) impre- C. Monitoring. The roles of routine cision are well within the analytical goal of urinalysis and albumin measurements approximately 15% and are often consid- are less clear in patients with stage A2 erably less. Most, but not all, methods albuminuria. Some experts have advo- agree well and support a reference interval cated urine protein testing to monitor of 2–20 mg albumin/mg creatinine (348). treatment, which may include improved The within-person variation in albu- glycemic control, more assiduous control min excretion is large in people without of hypertension, dietary protein restric- diabetes and is even higher in patients tion, and therapy with blockers of the with diabetes. Howey et al. (349) studied renin angiotensin system (321). Several day-to-day variation, over 3–4 weeks, in factors are known to slow the rate of uri- the 24-h albumin excretion, the concen- nary albumin excretion or to prevent its tration of albumin, and the albumin– development. They include reducing creatinine ratio. The last two variables blood pressure (with a blocker of the renin were measured in the 24-h urine sample, angiotensin system as part of the regimen), the first morning void, and random un- glycemic control, and lipid-lowering ther- timed urine collections. In healthy volun- apy (45,343–345). teers, the lowest within-person CVs were obtained for the concentration of albumin 2. Rationale in the first morning void (36%) and for Early detection of albuminuria allows early the albumin–creatinine ratio in that sam- Figure 1—Algorithm for urine protein testing. intervention with the goal of reducing ple (31%) (349). Multiple studies have e86 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates evaluated the best procedure to assess al- In two subsequent studies (353,354), the Genzyme Diagnostics) (322). Screening buminuria. Most studies have found that sensitivities were 67%–86%. False-positive 182 patient samples with this method the spot urine albumin–creatinine con- results also appear to be common, with with an albumin–creatinine ratio of $30 centration in the first morning void, rates as high as 15% (352). Thus, it appears mg/mg as positive yielded a sensitivity of rather than the 24-h urinary excretion of that at least some of the tests, especially as 96%, a specificity of 80%, a positive pre- albuminorthetimedcollection,isthe used in practice, have the wrong character- dictive value of 66%, and a negative pre- most practical and reliable technique istics for screening because of low sensitivity dictive value of 98%. In a separate study, (346,350,351). (high false-negative rates), and positive re- 165 patients had the HemoCue point-of- To keep the analytical CV less than sults must be confirmed by a laboratory care system for albumin compared with half the biological CV, an analytical goal method. Of the available methods, the im- the Clinitek Microalbumin (Siemens) and of an 18% CV has been proposed (349). munoturbidimetric assay is the most reli- Chemstrip Micral (Roche Diagnostics) Alternatively, if the albumin–creatinine able and should be considered the standard tests, as well as with an HPLC assay, for ratio is to be used, one may calculate the for comparison, because it has .95% sen- spot albumin–creatinine ratio measure- need for a somewhat lower imprecision sitivity and specificity to detect very low ment (324). Further studies are needed (that is, a better precision) to accommo- levels of albuminuria. Semiquantitative or before the dipstick tests for low levels of date the lower biological CV for the ratio qualitative screening tests should be posi- albuminuria can be recommended as re- and the imprecision contributed by the tive in .95% of patients for the detection placements for the quantitative tests. The creatinine measurement. Assuming a CV of albuminuria to be useful for assessment use of qualitative tests at the point of care of 5% for creatinine measurement, we of cardiovascular risk and progression of is reasonable only when it can be shown calculate a goal of 14.7% for the analytical kidney disease. Positive results obtained that this approach eliminates quantitative CV for albumin when it is used to estimate with such methodologies must be con- testing in a sizeable proportion of patients the albumin–creatinine ratio. A goal of firmed by an immunoturbidimetric assay and detects those patients who have early 15% appears reasonable to accommodate in an accredited laboratory (355). renal disease. use of the measured albumin concentra- tion for calculating either the timed excre- RECOMMENDATION: CURRENTLY tion rate or the albumin–creatinine ratio. RECOMMENDATION: ACCEPTABLE AVAILABLE DIPSTICK TESTS DO NOT HAVE SAMPLES TO TEST FOR INCREASED ADEQUATE ANALYTICAL SENSITIVITY TO URINARY ALBUMIN EXCRETION ARE TIMED DETECT LOW LEVELS OF ALBUMINURIA RECOMMENDATION: SEMIQUANTITATIVE COLLECTIONS (e.g., 12 OR 24 h) FOR B (moderate). OR QUALITATIVE SCREENING TESTS MEASUREMENT OF THE ALBUMIN SHOULD BE POSITIVE IN >95% OF CONCENTRATION AND TIMED OR PATIENTS WITH LOW LEVELS OF Chemical-strip methods are not sen- UNTIMED SAMPLES FOR MEASUREMENT ALBUMINURIA TO BE USEFUL FOR sitive when the albumin concentration in OF THE ALBUMIN–CREATININE RATIO SCREENING. POSITIVE RESULTS MUST BE the urine is in the interval of 20–50 mg/L. B (moderate). CONFIRMED BY ANALYSIS IN AN Thus, no recommendation can be made ACCREDITED LABORATORY for the use of any specific screening test. GPP. Dipstick tests for low levels of albumin- uria cannot be recommended as a replace- RECOMMENDATION: THE OPTIMAL TIME ment for the quantitative tests. FOR SPOT URINE COLLECTION IS THE Qualitative (or semiquantitative) as- The available dipstick methods to de- EARLY MORNING. ALL COLLECTIONS says have been proposed as screening tect low levels of albuminuria do not SHOULD BE AT THE SAME TIME OF DAY TO tests for low levels of albuminuria. To be appear to lend themselves to viable screen- MINIMIZE VARIATION. THE PATIENT useful, screening tests must have high ing strategies, either in the physician’soffice SHOULD NOT HAVE INGESTED FOOD detection rates, i.e., a high clinical sensitivity. or for home testing. Usual screening tests WITHIN THE PRECEDING 2 hBUTSHOULD Although many studies have assessed (e.g., for phenylketonuria) have low false- BE WELL HYDRATED (i.e., NOT VOLUME the ability of reagent strips (“dipstick” negative rates, and thus only positive re- DEPLETED) methods) to detect increased albumin con- sults require confirmation by a quantitative GPP. centrations in urine, the important question method. If a screening test has low sensi- is whether the method can detect low levels tivity, negative results also must be con- of albuminuria, that is, an increased albu- firmed, a completely untenable approach. B. Preanalytical. Collection of 24-h min excretion rate or its surrogate, an in- With semiquantitative tests, it may be pos- samples has disadvantages, specifically creased albumin–creatinine ratio. We can sible (or indeed necessary) to use a cutoff because many samples are collected find no documentation of any test in which ,20 mg/L to ensure the detection of sam- inadequately and because total creatinine the sensitivity for detection of an increased ples with albumin values .20 mg/L as is not routinely checked to evaluate the albumin excretion rate consistently reached measured by laboratory methods. adequacy of collection. The albumin– 95% in .1 study. For example, in a large Recent studies have compared se- creatinine ratio is the superior method study (352), the sensitivity for detection of lected dipstick methods to laboratory to predict renal events in patients with an albumin excretion rate .30 mg/24 h assays. One dipstick was found to have type 2 diabetes (356). The ratio has a was 91% when the test was performed .95% sensitivity (322,324). One such within-person biological variation similar by a single laboratory technician, 86% study evaluated an office-screening test to that of the excretion rate and correlates when performed by nurses, and 66% that uses a monoclonal antibody against well with both timed excretion and the when performed by general practitioners. human serum albumin (ImmunoDip; albumin concentration in a first morning care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e87 Position Statement void of urine (349). For the ratio, a first with albumin–creatinine ratios ,30 A. Diagnosis. In the last several years, morning void sample is preferable because mg/mg. After the documentation of stage interest has increased in the possibility this sample has a lower within-person var- A2 albuminuria (i.e., with results as de- that measurements of the concentrations iation than the ratio for a random urine fined above on two of three tests per- of plasma insulin and its precursors might sample taken during the day (349). Al- formed within 3 to 6 months), repeated be of clinical benefit. In particular, pub- though the ratio appears entirely accept- testing is reasonable to determine lished evidence reveals that increased able for screening, limited data are whether a chosen therapy is effective. It concentrations of insulin and/or proinsu- available on its use in monitoring the re- may also be useful in determining the lin in nondiabetic individuals predict the sponse to therapy. Recent post hoc analy- rate of disease progression and thus development of coronary artery disease ses of clinical trials, however, have found may support planning for care of end- (362). Although this possibility may be that the albumin–creatinine ratio is a rea- stage renal disease. Although the ADA scientifically valid, its clinical value is sonable method to assess change over time recommendations suggest that such test- questionable. An increased insulin con- (357). For screening, an untimed sample ing is not generally needed before pu- centration is a surrogate marker that can for albumin measurement (without creat- berty, testing may be considered on an be used to estimate resistance to insulin- inine) may be considered if one uses a individual basis if it appears appropriate mediated glucose disposal, and it can concentration cutoff that allows high sen- becauseofanearlyonsetofdiabetes, identify individuals at risk for developing sitivity for detecting an increased albumin poor control, or a family history of dia- syndrome X, also known as the insulin excretion rate. betic nephropathy. The duration of dia- resistance syndrome or the metabolic syn- Albumin is stable in untreated urine betes prior to puberty is reportedly an drome (363). Accurate measurement stored at 4°C or 20°C for at least a week important risk factor in this age-group of insulin sensitivity requires the use of com- (358). Neither centrifugation nor filtra- and thus can be used to support such plex methods, such as the hyperinsulinemic tion appears necessary before storage at testing in individual patients (361). euglycemic clamp technique, which are 220°C or 280°C (359). Whether a urine generally confined to research laboratories sample is centrifuged, filtered, or not (364,365). Because of the critical role of treated, the albumin concentration de- insulin resistance in the pathogenesis of MISCELLANEOUS creases by 0.27%/day at 220°C but type 2 diabetes, hyperinsulinemia would POTENTIALLY IMPORTANT shows no decreases over 160 days at also appear to be a logical risk predictor ANALYTES. I. INSULIN AND 280°C (359). The urinary albumin excre- for incident type 2 diabetes. PRECURSORS tion rate does not show marked diurnal Earlier studies may not have con- variation in diabetes but does so in essen- trolled well for glycemic status and other 1. Use tial hypertension (360). confounders. More-recent analyses sug- gest that insulin values do not add signif- icantly to diabetes risk prediction carried 4. Interpretation RECOMMENDATION: THERE IS NO ROLE out with more traditional clinical and A. Nonanalytical sources of variation. FOR ROUTINE TESTING FOR INSULIN, laboratory measurements (366) and that Transient increases in urinary albumin C-PEPTIDE, OR PROINSULIN IN MOST measures of insulin resistance (that in- excretion have been reported with short- PATIENTS WITH DIABETES. clude insulin measurements) predict the term hyperglycemia, exercise, urinary DIFFERENTIATION BETWEEN TYPE 1 AND risk of diabetes or coronary artery disease tract infections, marked hypertension, TYPE 2 DIABETES MAY BE MADE IN MOST only moderately well, with no threshold heart failure, acute febrile illness, and CASES ON THE BASIS OF THE CLINICAL effects (367). Consequently, it seems of hyperlipidemia (321). PRESENTATION AND THE SUBSEQUENT greater clinical importance to quantify COURSE. THESE ASSAYS ARE USEFUL the consequences of the insulin resistance PRIMARILY FOR RESEARCH PURPOSES RECOMMENDATION: LOW URINE . and hyperinsulinemia (or hyperproinsu- OCCASIONALLY C PEPTIDE ALBUMIN CONCENTRATIONS (i.e., ,30 , - linemia) rather than the hormone values MEASUREMENTS MAY HELP DISTINGUISH mg/g CREATININE) ARE NOT themselves, i.e., by measuring blood pres- TYPE 1 FROM TYPE 2 DIABETES IN ASSOCIATED WITH HIGH sure, the degree of glucose tolerance, and AMBIGUOUS CASES SUCH AS PATIENTS CARDIOVASCULAR RISK IF THE eGFR IS , plasma lipid/lipoprotein concentrations. 21 2 21 WHO HAVE A TYPE 2 PHENOTYPE BUT >60 mL · min · (1.73 m ) AND THE It is these variables that are the focus of PRESENT IN KETOACIDOSIS PATIENT IS NORMOTENSIVE. IF THE eGFR clinical interventions, not plasma insulin 21 2 IS ,60 mL · min · (1.73 m2) 1 B (moderate). or proinsulin concentrations (366,367). AND/OR THE LEVEL OF ALBUMINURIA The clinical utility of measuring IS ‡30 mg/g CREATININE ON A SPOT insulin, C-peptide, or proinsulin concen- URINE SAMPLE, A REPEAT MEASUREMENT trations to help select the best antihyper- glycemic agent for initial therapy in SHOULD BE TAKEN WITHIN THE YEAR RECOMMENDATION: THERE IS NO ROLE patients with type 2 diabetes is a question TO ASSESS CHANGE AMONG PEOPLE FOR MEASUREMENT OF INSULIN WITH HYPERTENSION that arises from consideration of the CONCENTRATION IN THE ASSESSMENT OF pathophysiology of type 2 diabetes. In A(moderate). CARDIOMETABOLIC RISK, BECAUSE theory, the lower the pretreatment insulin KNOWLEDGE OF THIS VALUE DOES NOT concentration, the more appropriate might B. Frequency of measurement. The ALTER THE MANAGEMENT OF THESE be insulin, or an insulin secretagogue, as NKF, ADA, and JNC 7 recommend an- PATIENTS the drug of choice to initiate treatment. B (moderate). nual measurement in diabetic patients Although this line of reasoning may have e88 DIABETES CARE, VOLUME 34, JUNE 2011 care.diabetesjournals.org Sacks and Associates some intellectual appeal, there is no evi- 2. Analytical considerations C-peptide concentration be #0.5 ng/mL; dence that measurement of plasma insulin however, because of the noncomparability or proinsulin concentrations will lead to of results from different assays, which led to more efficacious treatment of patients with RECOMMENDATION: BECAUSE CURRENT denial of payment for some patients with type 2 diabetes. MEASURES OF INSULIN ARE POORLY values .0.5 ng/mL, the requirement now In contrast to the above considera- HARMONIZED, ASTANDARDIZEDINSULIN states that the C-peptide concentration tions, measurement of plasma insulin and ASSAY SHOULD BE DEVELOPED TO should be #110% of the lower limit of proinsulin concentrations is necessary to ENCOURAGE THE DEVELOPMENT OF the reference interval of the laboratory’s establish the pathogenesis of fasting hy- MEASURES OF INSULIN SENSITIVITY THAT measurement method (376). poglycemia (368). The diagnosis of an is- WILL BE PRACTICAL FOR CLINICAL CARE let cell tumor is based on the persistence GPP. of inappropriately increased plasma insu- MISCELLANEOUS lin concentrations in the face of a low Although it has been assayed for .40 POTENTIALLY IMPORTANT glucose concentration. In addition, an in- years, there is no standardized method ANALYTES. II. crease in the ratio of fasting proinsulin to available to measure serum insulin INSULIN ANTIBODIES insulin in patients with hypoglycemia (371). Attempts to harmonize insulin as- strongly suggests the presence of an islet says with commercial insulin reagent cell tumor. The absence of these associ- sets have produced greatly discordant RECOMMENDATION: THERE IS NO ated changes in glucose, insulin, and pro- results (372). Recently, an insulin stan- PUBLISHED EVIDENCE TO SUPPORT THE insulin concentrations in an individual dardization workgroup of the ADA, in USE OF INSULIN ANTIBODY TESTING FOR with fasting hypoglycemia makes the diag- conjunction with the National Institute ROUTINE CARE OF PATIENTS WITH nosis of an islet cell tumor most unlikely, of Diabetes and Digestive and Kidney DIABETES and alternative explanations should be Diseases, the CDC, and the European C (very low). sought for the inability to maintain fasting Association for the Study of Diabetes, euglycemia. called for harmonization of insulin assay Measurement of the C-peptide re- results through traceability to an isotope- Given sufficiently sensitive techniques, sponse to intravenous glucagon can aid dilution liquid chromatography–tandom insulin antibodies can be detected in any in instances in which it is difficult to mass spectrometry reference (373). The In- patient being treated with exogenous in- differentiate between the diagnosis of sulin Standardization Workgroup called sulin (371). In the vast majority of pa- type 1 and type 2 diabetes (5). Even in for harmonization of the insulin assay to tients, the titer of insulin antibodies is this clinical situation, however, the re- encourage the development of measures low, and their presence is of no clinical sponse to drug therapy will provide use- of insulin sensitivity and secretion that significance. Very low values are seen in ful information, and measurement of will be practical for clinical care (374). patients treated exclusively with human C-peptide may not be clinically necessary. Analogous to insulin, considerable impre- recombinant insulin (377). On occasion, Measurement of C-peptide is essential in cision among laboratories has also been however, the titer of insulin antibodies in the investigation of possible factitious hy- observed for measurement of C-peptide. the circulation can be quite high and as- poglycemia due to surreptitious insulin A comparison of 15 laboratories that sociated with a dramatic resistance to the administration (369). used nine different routine C-peptide as- ability of exogenous insulin to lower In the past, some advocated insulin say methods, found within- and between- plasma glucose concentrations. This clin- assays in the evaluation and management run CVs as high as .10% and 18%, ical situation is quite rare, it usually oc- of patients with the polycystic ovary syn- respectively (375). A committee has been curs in insulin-treated patients with type drome. Women with this syndrome man- established under the auspices of the CDC 2 diabetes, and the cause-and-effect rela- ifest insulin resistance by androgen excess, to harmonize C-peptide analysis. tionships between the magnitude of the as well as by abnormalities of carbohydrate Measurements of proinsulin and C- increase in insulin antibodies and the de- metabolism; both abnormalities may re- peptide are accomplished by immuno- gree of insulin resistance are unclear. spond to treatment with metformin or metric methods. Proinsulin reference There are several therapeutic approaches thiazolidinediones. Although clinical tri- intervals are dependent on methodology, for treating these patients, and a quantita- als have generally evaluated insulin re- and each laboratory should establish its tive estimate of the concentration of cir- sistance by using the hyperinsulinemic own reference interval. Although it has culating insulin antibodies does not euglycemic clamp, ratios of fasting glu- been suggested by some, insulin mea- appear to be of significant benefit. cose to insulin, and other modalities, the surement should not be used in an OGTT The prior version of these guidelines optimal laboratory evaluation of these to diagnose diabetes. In the case of C- (14) contained short sections on amylin patients in routine clinical care has not peptide, there is a discrepancy in reliabil- and leptin, both of which were the focus been clearly defined. It is unclear whether ity because of variable specificity among of active clinical studies. The evidence assessing insulin resistance through insulin antisera, lack of standardization of C- that has accumulated in the last 7 to 8 measurement has any advantage over peptide calibration, and variable cross- years has failed to identify any clinical assessment of physical signs of insulin reactivity with proinsulin. Of note is the value in measuring these analytes in pa- resistance (BMI, presence of acanthosis requirement of the U.S. Centers for Medi- tients with diabetes. Similarly, although nigricans), and routine measurements care and Medicaid Services that Medicare cardiovascular disease is the major cause of insulin are not recommended by the patients have C-peptide measured in order of mortality for persons with diabetes, no American College of Obstetrics and Gy- to be eligible for coverage of insulin pumps. evidence supports the measurement of necology (370). Initially, the requirement was that the nontraditional cardiovascular risk factors care.diabetesjournals.org DIABETES CARE, VOLUME 34, JUNE 2011 e89 Position Statement for routine assessment of risk in patients Maldonado MR. Accuracy and predictive 20. The International Expert Committee. with diabetes. These sections have, there- value of classification schemes for ketosis- International Expert Committee report fore, been removed. prone diabetes. Diabetes Care 2006;29: on the role of the A1c assay in the di- 2575–9 agnosis of diabetes. Diabetes Care 2009; 6. IDF. Diabetes Atlas. 3rd ed. Brussels: 32:1327–34 Acknowledgments—Upon manuscript sub- IDF; 2008 21. ADA. Standards of medical care in di- mission, all authors completed the Disclosures 7. Cowie CC, Rust KF, Byrd-Holt DD, abetes—2010. Diabetes Care 2010;33 of Potential Conflict of Interest form. D.B.S. and Eberhardt MS, Flegal KM, Engelgau MM, (Suppl. 1):S11–61 D.E.B. reported employment or leadership po- et al. Prevalence of diabetes and impaired 22. ADA. Tests of glycemia in diabetes. Di- sitions with Clinical Chemistry, AACC. M.A. re- fasting glucose in adults in the U.S. abetes Care 2001;24(Suppl. 1):S80–2 ported employment or leadership positions population: National Health and Nutri- 23. IDF. Task Force. Global guideline for with and stock ownership in ASL Analytical, tion Examination Survey 1999–2002. type 2 diabetes. Brussels: IDF; 2005. Inc. G.L.B. reported employment or leadership Diabetes Care 2006;29:1263–8 p1–11 positions with American Society of Hyperten- 8. Cowie CC, Rust KF, Ford ES, Eberhardt 24. Saudek CD, Herman WH, Sacks DB, sion and National Kidney Foundation; reported MS, Byrd-Holt DD, Li C, et al. Full ac- Bergenstal RM, Edelman D, Davidson consultant or advisory role for Novartis, Merck, counting of diabetes and pre-diabetes MB. A new look at screening and di- Walgreen Co., and Takeda; and received re- in the U.S. population in 1988–1994 agnosing diabetes mellitus. J Clin En- search funding from GlaxoSmithKline, Forest and 2005–2006. Diabetes Care 2009;32: docrinol Metab 2008;93:2447–53 Laboratories, Novartis, and PepsiCo. D.E.B. re- 287–94 25. ADA. Type 2 diabetes in children ceived research funding from Abbott Diag- 9. 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Tuomilehto J, Lindstrom J, Eriksson JG, the design of the study. U.S. in 2007. Diabetes Care 2008;31: Valle TT, Hamalainen H, Ilanne-Parikka P, All authors confirmed they have contrib- 596–615 et al. Prevention of type 2 diabetes uted to the intellectual content of this article 12. Nathan DM. Long-term complications of mellitus by changes in lifestyle among and have met the following three require- diabetes mellitus. N Engl J Med 1993; subjects with impaired glucose toler- ments: 1) significant contributions to the 328:1676–85 ance. N Engl J Med 2001;344:1343– conception and design, acquisition of data, or 13. Roglic G, Unwin N, Bennett PH, Mathers 50 analysis and interpretation of data; 2)drafting C, Tuomilehto J, Nag S, et al. The burden 28. Icks A, Rathmann W, Haastert B, or revising the article for intellectual content; of mortality attributable to diabetes: re- John J, Löwel H, Holle R, Giani G. 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