The only clinically validated markers of pancreatic autoimmune disease, islet autoantibodies (iAb), recognize antigens found in insulin-producing pancreatic beta cells. The breakthrough discoveries of islet cell antibodies (ICA) from a section of frozen pancreas by indirect immunofluorescence (1970) and ICA in the serum of people with type 1 diabetes (T1D) (1974) ushered in the era of iAb testing.

However, even though ICA are highly specific for beta-cell injury, they have many target molecules and therefore lack sensitivity. Consequently, clinical laboratories no longer use ICA in routine diagnostic testing.

Currently, laboratories measure four well-characterized iAb: glutamic acid decarboxylase antibody (GADA), insulinoma-associated-2 autoantibody (IA-2A), zinc transporter 8 auto­antibody (ZnT8A), and insulin autoantibodies (IAA). These are directed against the autoantigens glutamic decarboxylase (GAD65), insulinoma-like tyrosine ­phosphatase-2 (IA-2), islet beta-cell zinc cation efflux transporter 8 (ZnT8), and insulin, respectively. IA-2 and ZnT8 reside on the surface of secretory granules in beta cells, insulin lies within the secretory granules, and GAD65 inhabits the cytoplasm of synaptic-like microvesicles.

Clinicians use commercially available measurements of all four iAb to confirm a diagnosis of T1D. Laboratories have evaluated and harmonized these assays through standardization efforts such as the Islet Autoantibody Standardization Program (IASP).

Islet Autoantibody Measurement in Practice

Clinically, iAb are best known for their role in confirming the diagnosis of T1D, a chronic autoimmune disease in which the immune system erroneously targets and destroys insulin-producing beta cells in the pancreas, eventually leading to absolute insulin deficiency. In patients with clinical symptoms of T1D, the presence of one or more iAb indicates pancreatic autoimmunity and is consistent with a diagnosis of this disease (1).

The American Diabetes Association (ADA), the Juvenile Diabetes Research Foundation, and the Endocrine Society published a joint statement in 2015 that describes distinct stages of T1D in which the measurement of islet autoantibodies plays a major role (2) (See Figure 1). In Stage 1 of T1D, a person is euglycemic with no symptoms but positive for multiple islet autoantibodies.

Stage 2 of T1D occurs when a person with multiple autoantibodies begins to have metabolic abnormalities (dysglycemia) but remains clinically asymptomatic. In Stage 3 of T1D, a patient has classical diabetes symptoms in the presence of dysglycemia and is therefore diagnosed with T1D based on standard clinical diagnostic criteria.

The most recent Standards of Medical Care in Diabetes, published annually by the ADA, supports the use of islet autoantibodies to make an early diagnosis of T1D (1). Clinically, an early diagnosis based on multiple autoantibodies may help reduce the rate of diabetic ketoacidosis and the associated morbidity and mortality that can occur at onset of T1D. According to ADA, diagnosing early stages of T1D is a necessary framework for research and regulatory decisions related to T1D.

However, despite the clinical utility of iAb measurement in diagnosing T1D, approximately 10% of patients with the disease do not have measureable iAb at onset. These patients are classified diagnostically as having type 1b diabetes and likely have an immune response to self-antigens that have yet to be identified. Of all T1D patients, approximately 50%80% are GADA-positive at the time of diagnosis, 30%70% are IA-2A-positive, 50%70% are ZnT8-positive, and 50%90% are IAA-positive (3). IAA are more prevalent in younger children.

Of note, IAA must be measured within 2 weeks of beginning treatment with insulin: After that point, antibodies may be produced in response to exogenously administered insulin, rendering their presence unhelpful in confirming a diagnosis of T1D. As noted above, the autoimmune markers of T1D are islet cell autoantibodies (ICA, GADA, IA-2A, ZnT8A) and autoantibodies to insulin (IAA), and T1D is defined by the presence of one or more of these autoimmune markers (1).

Therefore, we recommend measuring all four iAb (GADA, IA-2A, ZnT8A, IAA) when possible to accurately diagnose the disease and maximize sensitivity for detecting autoimmune diabetes.

Beyond confirming their diagnosis of T1D, clinicians often order iAb testing to help differentiate T1D from type 2 diabetes (T2D), as well as to detect rarer forms of diabetes such as maturity onset diabetes of the young (MODY). Clinicians also must rely on iAb when clinical and metabolic markers such as age, pubertal status, sex, body mass index, HbA1c, and family history do not readily help with the differential diagnosis.

One of these rarer forms of diabetes is latent autoimmune diabetes in adults (LADA), also known as type 1.5 diabetes. In newly diagnosed cases of T2D in adults, approximately 10% of patients are positive for a single iAb, predominately GADA. These patients, who also progress slowly to needing insulin, often receive a LADA diagnosis. Given the phenotypic variability of LADA-diagnosed patients in combination with the presence of iAb, LADA most likely is not a distinct entity but rather a slowly progressive form of autoimmune T1D.

By the same token, close to 30% of adolescents with newly diagnosed T2D will show evidence of autoimmunity when tested for iAb, with GADA or IA-2A being most common. These young patients also likely have a slowly progressive form of autoimmune T1D or have been misdiagnosed based on clinical features such as obesity—an increasingly prevalent condition. iAb typically are not detectible in cases of diabetes caused by defects in beta-cell function (e.g., MODY), genetic defects in insulin action, drug- or chemical-induced beta-cell damage, diseases of the exocrine pancreas (e.g., cystic fibrosis-related diabetes), or gestational diabetes.

Comparing Islet Autoantibody Assays

Although commercial assays measure all four iAb, the sensitivity and specificity of iAb for beta-cell injury varies greatly by the method of measurement (Table 1). A commercial enzyme-linked immunosorbent assay (ELISA) for ZnT8A became available only in recent years. ELISA is highly specific but has poor sensitivity compared to gold standard methods of iAb measurement—such as fluid-phase radioimmunoassay.

A major reason for poor ELISA sensitivity is that antibody binding occurs at the interface of a solid surface coated with antigen instead of in the fluid phase, in which there is a greater capacity for binding. In addition to fluid-phase radioimmunoassay, bridge-ELISA and electrochemiluminescence detection (ECL) are acceptable due to their high sensitivity and specificity.

iAb assays have improved significantly in the last decade due to efforts of the IASP, previously known as the Diabetes Antibody Standardization Program (DASP) (3). IASP aims to improve testing of iAb globally and meets biannually at the Immunology of Diabetes Society meeting. The TrialNet Islet Cell Autoantibody Core Laboratory at the University of Florida runs IASP with support from the National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases. TrialNet (www.diabetestrialnet.org) is a clinical prevention trial network sponsored by NIH that started in the early 1990s as the Diabetes Prevention Trial-Type 1 (DPT-1).

Notably, in IASP workshops the IAA assay has shown relatively wide discrepancies between laboratories, and has not yet achieved satisfactory levels of sensitivity and specificity.

The ADAs Standards of Medical Care in Diabetes now recommend that relatives of individuals with T1D have yearly monitoring for iAb through a clinical research trial. Monitoring relatives for iAb positivity primarily occurs through TrialNet, and iAb-positive individuals are eligible for clinical research trials available through TrialNet aimed at preventing the onset of T1D and preserving residual beta-cell mass.

New Research and Practice Developments

By measuring iAb, laboratories have the ability to identify T1D years before patients develop clinical symptoms. In 2015, the Juvenile Diabetes Research Foundation, Endocrine Society, and ADA published a joint scientific statement proposing that T1D be identified prior to any manifestations of the disease (4). In the groupsproposed staging, Stage 1 of T1D occurs when a euglycemic, asymptomatic person has two or more iAb. In Stage 2 of T1D, an individual with multiple iAb begins to have blood glucose abnormalities. Those with iAb and dysglycemia enter Stage 3 of T1D when they develop clinical symptoms of diabetes.

In fact, several prospective, longitudinal studies following individuals at high genetic risk for or with a family history of T1D have shown that T1D progresses through several distinct stages prior to the onset of clinical symptoms (Figure 1). Importantly, the presence of two or more iAb confers an approximate 70% risk of T1D developing within 10 years and nearly 100% over time.

The rate of progression to T1D from seroconversion varies greatly, from a few months to more than a decade. In children with a single iAb, 15% progress to clinical T1D within 10 years. About 70% of these children who go on to develop multiple iAb will do so within 2 years from the time the first iAb appears in their serum. Progression to clinical T1D occurs in nearly 75% of people with three or more iAb within 10 years from seroconversion to iAb positivity. Factors involved in the rate of progression are poorly understood, although younger age at seroconversion, number of positive autoantibodies, and higher levels of IAA and IA-2A have been associated with faster progression to T1D (5, 6).

Identifying T1D at an early stage is critical. The incidence of T1D has increased over time, and there is significant risk of morbidity and mortality from the time of diagnosis and beyond. In countries with either a high or low prevalence of T1D, the incidence of T1D has increased approximately 3%5% in the last decade, with about 1 in 300 children expected to develop the disease.

Nearly 90% of people diagnosed with T1D do not have a family member with T1D and do not recognize the diseases symptoms until theyre critically ill. In Colorado, 40% of children at the time of T1D onset are hospitalized with diabetic ketoacidosis (DKA), which sometimes causes life-threatening cerebral edema.

Prospective research studies have shown that monitoring at-risk children for iAb over time greatly reduces the incidence of DKA at diagnosis; in the DPT-1 that followed high-risk individuals who were iAb positive, the incidence of DKA at T1D onset was only 3.67%. In addition, children diagnosed through prospective studies such as The Environmental Determinants of Diabetes in the Young (TEDDY) have higher C-peptide levels, lower HbA1c, and lower insulin requirements at diagnosis and throughout the first year post-diagnosis.

Improving Care Through Screening

Now that iAb testing offers the ability to identify T1D at a preclinical stage and avoid DKA, the laboratory medicine community must build strategies to screen the general population for islet autoimmunity. General population screening for iAb is currently underway in two large clinical research projects that have a goal of changing the standard of care to include routine iAb testing in the pediatric population. While clinical research on general population screening for iAb has been ongoing since 1985, studies have been relatively small in scope, performed mostly in school settings, and have not included all four major iAb.

In 2015, the German state of Bavaria launched project Fr1Da that aims to screen 100,000 children age 25 years for iAb. The study uses capillary blood samples and a multiplex ELISA assay for GAD, IA-2A, and IAA. If results are greater than the 97.5th percentile, the lab performs a confirmatory test by screening for all four major iAb using gold-standard radioimmunoassay. In the first 10 months, the study screened an average of 2,676 children per month, demonstrating the feasibility of screening for iAb in a general population of young children through collaboration with primary care pediatricians (7).

Another study launched in 2016, Autoimmunity Screening for Kids, has a goal of screening 70,000 children in the Denver metropolitan area for both T1D and celiac disease by measuring the four major iAb and tissue transglutaminase antibody, respectively. The researchers will measure iAb primarily from serum obtained by venipuncture but also from capillary blood using gold-standard radioimmunoassay and ECL assay.

With the possibility of the standard of care changing to include routine measurement of iAb in children, iAb measurement methodology needs to be optimized for widespread screening. Continuing to increase the sensitivity and specificity of assays through the IASP remains important. Ideally, commercial laboratories would adopt the most sensitive and specific iAb assays available. In addition, it must be a priority to validate newer, highly sensitive and specific iAb assays such as ECL that do not use radioactivity and have the ability to measure all four iAb from a small volume of serum in a single well.

ECL assays are better at identifying high-affinity iAb, which are more likely to predict progression to T1D, possibly even as a single iAb (8). ECL assays also identify IAA much earlier than any other iAb measured by the current gold-standard radioimmunoassay (9). This is important as IAA is often the first iAb to develop in young children who are at highest risk for DKA.

Sample collection optimization also will be important for successful widespread iAb measurement. Capillary blood screening offers a feasible alternative to venous sampling, with the potential to facilitate autoantibody screening for T1D risk; the pilot TrialNet study showed that relatives of patients with T1D prefer initial capillary blood screening over venipuncture.

Capillary blood can be collected in microcentrifuge tubes or as dried blood spots, which are familiar to pediatric offices due to routine newborn screens completed at well-child visits. Dried blood spots are simple to collect, cost-effective, stable over time before extraction, and easily mailed to central laboratories with expertise in measuring iAb (10).

Conclusion

The identification of iAb established T1D as an autoimmune disease nearly 50 years ago and has enabled the diagnosis and appropriate treatment of scores of individuals with T1D globally. T1D can now be predicted by measuring iAb, which hopefully will reduce the morbidity and mortality associated with the diseases onset as well as give patients the opportunity to enroll in available T1D prevention trials.

With routine iAb measurement in the pediatric population looming on the horizon, now is the time to continue optimizing sample collection and iAb assays to make them reliable and cost-effective.

References

1.       American Diabetes A. (2) classifi­ca­tion and diagnosis of diabetes. Diabetes Care 2015;38 Suppl:S8–S16.

2.       Hummel K, McFann KK, Realsen J, et al. The increasing onset of type 1 diabetes in children. J Pediatr 2012;161:652–7 e1.

3.       Schlosser M, Mueller PW, Torn C, et al. Participating L. Diabetes antibody standardization program: Evaluation of assays for insulin autoantibodies. Diabetologia 2010;53:2611–20.

4.       Insel RA, Dunne JL, Atkinson MA, et al. Staging presymptomatic type 1 diabetes: A scientific statement of jdrf, the Endocrine Society, and the American Diabetes Association. Diabetes Care 2015;38:1964–74.

5.       Ziegler AG, Rewers M, Simell O, et al. Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in children. JAMA 2013;309:2473–9.

6.       Steck AK, Johnson K, Barriga KJ, et al. Age of islet autoantibody appearance and mean levels of insulin, but not GAD or IA-2 autoantibodies, predict age of diagnosis of type 1 diabetes: Diabetes autoimmunity study in the young. Diabetes Care 2011;34:1397–9.

7.       Raab J, Haupt F, Scholz M, et al. Capillary blood islet autoantibody screening for identifying pre-type 1 diabetes in the general population: Design and initial results of the Fr1Da study. BMJ Open 2016;6:e011144.

8.       Miao D, Guyer KM, Dong F, et al. Gad65 autoantibodies detected by electrochemiluminescence assay identify high risk for type 1 diabetes. Diabetes 2013;62:4174–8.

9.       Yu L, Dong F, Miao D, et al. Proinsulin/insulin autoantibodies measured with electrochemiluminescent assay are the earliest indicator of prediabetic islet autoimmunity. Diabetes Care 2013;36:2266–70.

10.       Bingley PJ, Rafkin LE, Matheson D, et al. Use of dried capillary blood sampling for islet autoantibody screening in relatives: A feasibility study. Diabetes Technol Ther 2015;17:867–71.

Kimber Simmons, MD, is an assistant professor of pediatric diabetes and endocrinology at the University of Colorado Barbara Davis Center for Diabetes in Denver. +Email: kimber.simmons@ucdenver.edu

Andrea Steck, MD, is an associate professor of pediatrics at the University of Colorado Barbara Davis Center for Diabetes in Denver. +Email: andrea.steck@ucdenver.edu