Cytoplasmic Islet Cell Autoantibodies Research Articles

Practical Clinical Applications of Islet Autoantibody Testing in Type 1 Diabetes.

The distinction between type 1 diabetes (T1D) and type 2 diabetes (T2D) is extremely important for the choice of therapy, body weight and dietary management, screening for coexistent autoimmune diseases and comorbidities, anticipated prognosis, and risk assessment in relatives. Not uncommonly, the presentation of the patient may not allow an unambiguous discrimination between T1D and T2D. To help resolve this challenge, the detection of islet autoantibodies can support the diagnosis of T1D. The presence of islet autoantibodies in a person with diabetes indicates an autoimmune etiology therefore establishing the diagnosis of T1D. Presently 5 islet autoantibodies are available for routine clinical use: islet cell cytoplasmic autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), insulinoma associated-2 autoantibodies (IA-2A), and zinc transporter-8 autoantibodies (ZnT8A). There are caveats to the selection of which islet autoantibodies should be measured. Islet autoantibodies can also predict the development of T1D. Therefore, once safe and effective therapies are available to prevent T1D, islet autoantibody testing is expected to become a routine part of medical practice. A very rare cause of autoimmune diabetes is the type B insulin resistance syndrome resulting from antagonistic autoantibodies to the insulin receptor. Rarely hypoglycemia can result from agonistic insulin receptor autoantibodies, or high-titer IAA causing the autoimmune insulin syndrome (i.e., Hirata disease). In summary, autoimmune causes of dysglycemia are increasing in clinical importance requiring the scrutiny of laboratorians. The determination of islet autoantibodies can greatly aid in the diagnosis and the prediction of T1D.

Glutamic Acid Decarboxylase and Islet Cell Cytoplasmic Autoantibodies Positivity in Latent Autoimmune Diabetes of Adults: Need for Early Insulinisation

Introduction: Latent Autoimmune Diabetes of Adults (LADA) remains a poorly understood disease at both the clinical and research level and usually misdiagnosed as Type 2 Diabetes Mellitus (T2DM) and treated with Oral Hypoglycaemic Agents (OHA). This might lead to accelerated b-cell destruction leading to early insulin-dependency in these patients. Screening of T2DM and determination of C-peptide and insulin antibodies helps to differentiate this disease from LADA. Early insulinisation of these patients prevents b-cell destruction and preserves residual b cell function. Aim: To screen type 2 diabetic patients for potential LADA subjects based on LADA clinical risk score and to confirm them as LADA based on Glutamic Acid Decarboxylase (GADA) positivity and Islet Cell Cytoplasmic Autoantibodies (ICA) positivity. Materials and Methods: The hospital based cross-sectional study was done on 945 T2DM patients attending Out Patient Department (OPD) of Rajarajeswari Medical College and Hospital, Bengaluru, Karnataka, India from March 2017 to May 2019. Patients were screened using LADA clinical risk score and potential LADA subjects were identified and tested for C-peptide, GAD and IC Antibodies to confirm LADA. C-Peptide and GAD antibodies were measured by ELISA method. IC antibodies were observed using immunoflorescence microscopy. Inferential Statistics (Chi-square test, Z test, t-test) by using SPSS V20 and MS Excel was used. Results: Out of 945 T2DM patients, 188 patients (20%) were considered as potential LADA subjects based on LADA clinical risk score. All 188 subjects were tested for C-peptide, GAD and IC antibodies. 36% (68 subjects) of them had low C-peptide levels. Among them four subjects (6%) had high GADA titer confirming them as LADA subjects. Three (4%) of the subjects had low GADA titer, remaining 61 subjects did not show antibody positivity. Only one of them had both IC antibody positivity out of four subjects with high GADA titer. Remaining 64% (120 subjects) had normal C-peptide levels and antibody negative. Conclusion: Using 5-point LADA clinical risk score 20% were identified as potential LADA subjects out of 945 T2DM subjects. 6% of the potential LADA subjects had high GADA titer confirming them as LADA, who may be benefited by early insulinisation.

Autoantibodies in Diabetes Mellitus

Based on American Diabetes Association (ADA), diabetes can be classified into the following general categories: type 1 diabetes (T1D), type 2 diabetes (T2D), gestational diabetes mellitus (GDM) and specific types of diabetes due to other cause. Obesity is by far the main underlying factor causing T2D and its pathological potential lies in obesity-associated insulin resistance, activation of innate immunity and chronic low-grade inflammation. When tissue inflammation induced, tissue destruction occurs, 'self' antigens, which are generally not accessible to T cells, can be released from the affected tissues and promote autoimmune activation. The 4 major autoantibodies are islet-cell cytoplasmic autoantibodies (ICA), glutamid acid decarboxylase antibody (GADA), islet antigen-2 antibody (IA-2A) and insulin autoantibodies (IAA). In addition, ZnT8A has recently been found to predict T1D. ZnT8 is contained in the islets of Langerhans, with the highest expression is in β cells of the pancreas. ZnT8A measurements simultaneously with GADA, IA-2A and IAA achieve rates of 98% detection for onset level of autoimmune diabetes. Presence of antibodies in T2D also shows the potential serious complications compared with T2D without antibodies. The combination of GADA, IA-2A and ZnT8A can be suggested as the most powerful and cost-effective diagnostic approach in patients with T1D.Keywords: autoantibody, autoimmune, diabetes mellitus, ICA, GADA, IA-2A, IAA, ZnT8A

A novel mutation in the hepatocyte nuclear factor-1β gene in maturity onset diabetes of the young 5 with multiple renal cysts and pancreas hypogenesis: A case report.

A 17-year-old Chinese male was hospitalized exhibiting hyperglycemia and increased serum urea nitrogen and creatinine levels in addition to weight loss. The patient was treated with gliclazide. The patient was 150 cm tall, weighed 35 kg and had no family history of diabetes or kidney disease. Physical examination revealed cephalus quadratus, rachitic rosary and a visible toe-out gait. Laboratory examinations revealed that the patient's fasting plasma glucose and glycosylated hemoglobin levels were markedly increased, fasting plasma C-peptide level was slightly increased and no peak 2 h postprandial was observed. Diabetic autoimmune antibodies [islet cell cytoplasmic autoantibodies (ICA), glutamic acid decarboxylase autoantibodies (GADA), isulinoma-2-associated autoantibodies (IA2A) and insulin autoantibodies (IAA)] were negative. Levels of serum electrolytes decreased, uric acid and parathyroid hormone increased, mild albuminuria was detected and there was a low proportion of urine. The patient also presented with low bone mass and cataracts. Abdominal computed tomography (CT) revealed a bilateral atrophic kidney with multiple renal cysts, primarily located at the junction of renal cortex and medulla, with a diameter of 0.3-0.7 cm. CT also revealed hypogenesis of the body and tail of the pancreas. In an oral glucose tolerance test, the mother and paternal uncle of the patient were diagnosed with type II diabetes and the patient's sister, maternal uncle and paternal grandpa were diagnosed with glucose tolerance impairment. Genetic testing revealed an unreported amino acid mutation in exon 2 of hepatocyte nuclear factor 1β (c.391C>T), a nonsense mutation of CAA to TAA at codon 131. This mutation was identified in the proband but not in any other family members.

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Enhancing the Understanding of Pre-Type 1 Diabetes in the General Population

Type 1 diabetes occurs in ∼90% of patients with no family history of the disease. The majority of studies seeking to identify at-risk subjects have been conducted in first-degree relatives who comprise just 10% of all cases of type 1 diabetes but may have a 20-fold increased risk for type 1 diabetes (1). Although the peak incidence occurs at adolescence, there are data to suggest that 5–10% of all adults diagnosed with type 2 diabetes may actually have type 1 diabetes (latent autoimmune diabetes in adults [LADA]). If type 1 diabetes is to be prevented, significantly enhanced understanding of both the mechanisms leading to type 1 diabetes and the natural history of the pre-diabetic period is essential. Current evidence suggests that type 1 diabetes results from a complex interaction between type 1 diabetes genetic susceptibility (predominantly HLA Class II associations) and environmental exposure(s) leading to a breakdown of tolerance culminating in β-cell autoimmunity and destruction (2). In the process leading to overt type 1 diabetes, both T and B lymphocytes are activated. B-lymphocyte activation is characterized by the emergence of cytoplasmic islet cell autoantibodies (ICAs) and one or more autoantibodies against the β-cell–specific autoantigen insulin, glutamate decarboxylase, the IA-2 protein tyrosine phosphatase, or the zinc transporter ZnT8 (IAA, GADA, IA-2A, and ZnT8A, respectively). For the most part, IAA, GADA, IA-2A, and ZnT8A have replaced ICA, which is measured using indirect immunofluorescence that requires a subjective evaluation of the fluorescence intensity. The IAA, GADA, IA-2A, and ZnT8A analyses require only small amounts of serum (5–20 μl in most assays) and are thus well suited for large-scale studies comprising both infants and small children. Serum samples can be safely stored at −20°C for years before the measurement of the autoantibodies is undertaken, and the samples usually also tolerate repeated freeze-thaw cycles without …

Insulin autoantibody could help to screen latent autoimmune diabetes in adults in phenotypic type 2 diabetes mellitus in Chinese

Latent autoimmune diabetes in adults (LADA) is characterized by a relatively mild diabetes onset, autoantibody positivity, and eventual requirement for insulin therapy. Glutamic acid decarboxylase autoantibodies (GADA) or cytoplasmic islet cell autoantibodies (ICA) play a key role in distinguishing LADA from type 2 diabetes mellitus (T2DM) in clinical practice. The aim of our research was to determine whether insulin autoantibody (IAA) has some additional value in diagnosing LADA. We analyzed IAA, GADA, and IA-2A (antibodies to insulinoma-associated antigen-2) in 1,003 newly diagnosed phenotypic T2DM patients, 110 type 1 diabetes mellitus (T1DM) patients, and 317 normal controls to survey the prevalence of IAA in phenotypic T2DM patients and the overlapping positivity of IAA with other autoantibodies. Sera were drawn within 7 days from the start of insulin therapy. Results showed that 3.39% of the newly diagnosed phenotypic T2DM, 0.95% of normal control (χ(2) = 5.3, P < 0.05), and 21.82% of T1DM (χ(2) = 68.2, P < 0.001) were positive for IAA at diagnosis. The combination frequency of three antibodies was 10.47%, which was higher than any single antibody testing. Combination testing of IAA with GADA and IA-2A could improve LADA diagnose rate by 2.39% than that of GADA and IA-2A. IAA-positive subjects had diabetes family history more common compared to its matched group (67.6% vs. 14.7%, P = 0.000). Postprandial C-peptide in IAA-positive group tended to be lower, but the difference was not statistically significant (P = 0.084). We concluded that IAA can be used to screen LADA in phenotypic T2DM in the Chinese population.

Diabetes classification: grey zones, sound and smoke: Action LADA 1

Diseases gain identity from clinical phenotype as well as genetic and environmental aetiology. The definition of type 1 diabetes is clinically exclusive, comprising patients who are considered insulin dependent at diagnosis, whilst the definition of type 2 diabetes is inclusive, only excluding those who are initially insulin dependent. Ketosis-prone diabetes (KPD) and latent autoimmune diabetes in adults (LADA) are each exclusive forms of diabetes which are, at least initially, clinically distinct from type 2 diabetes and type 1 diabetes, and each have a different natural history from these major types of diabetes.KPD can be diagnosed unequivocally as diabetes presenting with the categorical clinical feature, ketoacidosis. In contrast, LADA can be diagnosed by the co-occurrence of three traits, not one of which is categorical or exclusive to the condition: adult-onset non-insulin-requiring diabetes, an islet autoantibody such as glutamic acid decarboxylase autoantibodies (GADA) or cytoplasmic islet cell autoantibodies (ICA), and no need for insulin treatment for several months post-diagnosis. But while some would split diabetes into distinct subtypes, there is a strong case that these subtypes form a continuum of varying severity of immune and metabolic dysfunction modified by genetic and non-genetic factors. This article discusses the nature of disease classification in general, and KPD and LADA in particular, emphasizing the potential value and pitfalls in classifying diabetes and suggesting a need for more research in this area.

Oral Insulin Therapy to Prevent Progression of Immune‐Mediated (Type 1) Diabetes

Repeated ingestion of insulin has been suggested as an immune tolerization therapy to prevent immune-mediated (type 1) diabetes. We performed a placebo-controlled, two-dose, oral insulin tolerance trial in newly diagnosed (< 2 years) diabetic patients who had required insulin replacement for less than 4 weeks and were found to have cytoplasmic islet cell autoantibodies (ICAs). No oral hypoglycemic agents were permitted during the trial. Endogenous insulin reserves were estimated at six-month intervals by plasma C-peptide responses to a mixed meal. Positive ICAs were found in 262 (31%) of the 846 patients screened. Of the 197 who agreed to participate, 187 could be followed for 6 to 36 months. Endogenous insulin retention was dependent upon initial stimulated C-peptide response, age at diabetes onset, and numbers of specific islet cell autoantibodies found. Oral insulin improved plasma C-peptide responses in patients diagnosed at ages greater than 20 years, best seen at the low (1 mg/day) over the high (10 mg/day) insulin dose (P = .003 and P = .01, respectively). In patients diagnosed before age 20 years, the 1 mg dose was ineffective, whereas the 10 mg dose actually accelerated C-peptide loss (P = .003). There were no adverse effects. If confirmed, these findings suggest that diabetic patients over age 20 years with ICA evidence of late-onset immune-mediated diabetes should be considered for oral insulin at 1 mg/day to better retain endogenous insulin secretion.

Type 1 diabetes islet autoantibody markers.

The diagnosis of type 1 diabetes versus other forms of diabetes such as type 2 diabetes is paramount to guiding proper therapy. Several islet autoantibodies have been identified that serve to diagnose immune-mediated, type 1a diabetes in clinically ambiguous cases. These autoantibodies also serve to predict type 1 diabetes in nondiabetic individuals. The most useful islet autoantibodies include islet cell cytoplasmic autoantibodies, insulin autoantibodies, glutamic acid decarboxylase autoantibodies, and insulinoma-associated-2 autoantibodies. Once type 1 diabetes can be safely and reliably prevented, large-scale islet autoantibody screening programs of the general pediatric population may be warranted. It is controversial whether islet autoantibodies influence the course of type 1 diabetes following diagnosis.

No alteration in T lymphocyte expression of CD40 ligand (CD154) in individuals with or at increased risk for insulin-dependent diabetes mellitus.

CD40 ligand (CD40L) regulates multiple phases of the humoral and cellular immune response through binding to CD40. Previous investigations have suggested that insulin-dependent diabetes (IDDM) in both humans and nonobese diabetic mice may be strongly influenced by similar immunoregulatory molecules. As persons with or at increased risk for the disease are characterized by a number of immunological abnormalities, including that of self-reactive autoantibody production (e.g. islet cell cytoplasmic autoantibodies), we analyzed the expression of CD40L on T lymphocytes (CD3+ cells) in a series of individuals with newly diagnosed IDDM (n = 11), nondiabetic relatives of IDDM probands at increased risk for the disease (n = 21; islet cell cytoplasmic autoantibodies positive; Juvenile Diabetes Foundation titer, > or = 20), and healthy controls (n = 13). Both phorbol myristate acetate (PMA)-stimulated and unstimulated peripheral blood mononuclear cells from study subjects were analyzed by flow cytometry with a series of phenotypic antibody markers (CD3, CD40L, and isotype controls). The kinetics of CD3 and CD40L expression on peripheral blood mononuclear cells under PMA-stimulated and unstimulated conditions were similar in the three study groups (6, 24, and 48 h; all P = NS). Similarly, unstimulated and PMA stimulated CD40L expressions (percentage of positive cells and level) on CD3+ cells from newly diagnosed IDDM patients and persons at increased risk for the disease were similar to those in healthy controls (6, 24, and 48 h; all P = NS). These findings do not support abnormal CD40L expression as the mechanism underlying the functional defect(s) in communication between T lymphocytes and antigen-presenting cells that allows for autoantibody production or the inability of individuals to regulate antiself immunity in IDDM.

Sexual dimorphism in transmission of expression of islet autoantibodies to offspring.

To help elucidate the mode of inheritance of insulin-dependent diabetes mellitus (IDDM), we measured GAD (glutamic acid decarboxylase) autoantibodies (GAD65Ab), insulin autoantibodies (IAA), and cytoplasmic islet cell autoantibodies (ICA) in 292 sequentially screened non-diabetic offspring of patients with IDDM. The prevalence of these islet autoantibodies was higher in offspring of diabetic fathers than in offspring of diabetic mothers. The prevalences of GAD65Ab, IAA, and ICA in the offspring of diabetic fathers were 11.5%, 10.8%, and 8.1% vs 2.1%, 1.4%, and 2.8%, respectively in the offspring of diabetic mothers (p < 0.002, p < 0.001, and p = 0.06 NS). Amongst autoantibody-positive relatives the IAA and ICA levels were significantly higher in offspring of diabetic fathers than of diabetic mothers (p < 0.002 and p < 0.01, respectively). The frequencies of these autoantibodies were equal in male and female offspring. We conclude that IDDM mothers transmitted islet autoimmunity less frequently to their offspring than IDDM fathers. Given the markedly lower frequency of autoantibodies in offspring of mothers, larger sample sizes will be required to determine whether islet autoantibodies are influenced by age of IDDM onset of mothers, maternal age of pregnancy, and presence of diabetes in these mothers prior to conception.

Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the target antigen.

The autoimmune phenomena associated with destruction of the beta cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Prognostically significant heterogeneity of cytoplasmic islet cell antibodies in relatives of patients with type I diabetes.

A significant proportion of relatives of patients with insulin-dependent (type I) diabetes with high titers of cytoplasmic islet cell autoantibodies (ICAs) do not progress to overt diabetes with up to 8 yr of follow-up. This may reflect that follow-up of such relatives has not been long enough to observe diabetes, that despite expression of identical ICAs, some relatives will not progress to diabetes; or that there is heterogeneity in what is identified as ICA. We identified a subset of ICA that was restricted in its species (not reacting with mouse islets) and cell-type reactivity within islets (beta-cell specific). Only one of eight relatives whose sera had the restricted pattern of reactivity progressed to overt diabetes, and on sequential evaluation, all but the one relative who progressed to diabetes have maintained normal first-phase insulin secretion to intravenous glucose. In contrast, by life-table analysis, 70% of relatives expressing nonrestricted ICA became diabetic within 5 yr of follow-up (1 of 8 vs. 16 of 25 diabetic at last follow-up, P less than 0.02). Moreover, preliminary data suggest a significant association of the human leukocyte antigen DQB1*0602 allele of DR2 haplotypes with the restricted ICA pattern (4 of 5 DQB1*0602 restricted vs. 0 nonrestricted ICA, P = 0.006). We propose that expression of a genetically determined restricted ICA pattern confers a markedly lower risk for progression to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenetic heterogeneity in type I (insulin-dependent) diabetes among the Tunisian population. HLA-DR antigens and organ-specific autoantibodies (family study)

The frequency of HLA-DR antigens, as well as the prevelence of islet cell insulin autoantibodies and other autoimmunity disorders, were investigated in Tunisian patients with insulin-dependent diabetes mellitus (IDDM) and were compared with family members (sibs) and healthy control subjects. Cytoplasmic islet cell autoantibodies (ICA) were found in 79 of 175 (45.1%) patients with IDDM, in 23 of 126 (18.25%) unaffected first degree relatives of type I diabetes patients and in only two of 146 (1.3%) control subjects. In 79 ICA positive patients with IDDM, 46.8% presented other evidence of autoimmunity by testing for specific autoantibodies. Insulin autoantibodies were found in 86.9% of healthy ICA-positive sibs. A good correlation between HLA-DR3/DR4 heterozygous phenotypes and the presence of ICA in patients with IDDM and their unaffected sibs was observed in the Tunisian population. In fact, this heterozygous phenotype is found in 63.3% of ICA-positive diabetic patients and in 44.4% of ICA-positive unaffected sibs, whereas, HLA-DR3/DR4 antigens were noted in only 22.9% of ICA-negative diabetic patients and in no ICA-negative unaffected sibs. In these studies, we also summarized the distribution of HLA-DR antigens in patients with IDDM who presented autoimmune disorders other than ICA.

64 000 M r autoantibodies as predictors of insulin-dependent diabetes

The occurrence of autoantibodies to an islet-cell protein of 64 000 M r (64KA) was examined in relation to development of insulin-dependent diabetes. 64KA were absent in 26 normal controls and present in only 1 of 41 first-degree relatives who lacked islet-cell cytoplasmic autoantibodies (ICA) or insulin autoantibodies (IAA). Among first-degree relatives at high risk for IDD, 64KA were identified in 23 of 28 persons positive for ICA and 4 of 5 with IAA but no ICA. Among 31 patients with newly diagnosed IDD, 64KA were found in 26.64KA were identified in 23 of 28 persons studied up to 75 months before clinical onset of IDD. Of these 23 64KA-positive prediabetic subjects, 5 were ICA-negative and 10 lacked IAA. 64KA were most predictive in those who became diabetic before age 34 (22/24). In several individuals, 64KA were detected before the other autoantibodies appeared. These findings suggest that 64KA may be an early and useful predictive marker for IDD.

Heterogeneity of islet cell autoantibodies in terms of insulin release from rat islets and insulinoma cells

The clinical significance of cytoplasmic islet cell autoantibodies (ICA) has been studied since their discovery by Bottazzo et al. in 1974 [1]. Some ICAs destroy pancreatic B cells in the presence of complement [2], whereas others take no part in this destruction [3]. This suggests that islet function varies with the amount of ICA produced. In the present investigation we report the heterogeneity of monoclonal islet cell antibodies produced by one of us in terms of insulin release from isolated rat islets as well as from rat insulinoma cells (RINr).

Cytoplasmic islet cell autoantibodies: prevalence and pathognomic significance.

For many decades insulin-dependent diabetes mellitus (IDDM = Type I diabetes) has been regarded as a metabolic disorder with a typical acute clinical onset. In the past 15 years, however, there have been substantial changes in previously established concepts of the etiopathogenesis of Type I diabetes (1,2). Based on immunologic, genetic and epidemiologic studies it is apparent that the majority of patients with Type I diabetes have a long, clinically silent prediabetic period. This latency period, which can antedate clinically overt manifestation of insulin-deficiency for up to several years, is featured by the presence of various autoimmune phenomena (2,3). Currently chronic autoimmune reactions are widely believed to play a key role in the progressive destruction of the insulin-producing beta-cells of the Langerhans islets, although a direct autoimmune attack has still to be proved.KeywordsIslet Cell AntibodyIDDM PatientPrecision ScoreJuvenile Diabetes MellitusOvert ManifestationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Progress Toward Standardization of Cytoplasmic Islet Cell–Antibody Assay

Cytoplasmic islet cell autoantibodies (ICAs) of 13 coded sera were determined by 26 laboratories. ICAs were determined by 24 laboratories according to a standard protocol based on the conventional indirect-immunofluorescence technique on cryostat sections of human pancreas. In addition, these 24 laboratories had performed any of 8 modifications of the assay. Test samples were titrated to end point, and the results obtained with the different methods were compared with those obtained by the standard protocol. The remaining 2 laboratories used either exclusively pancreatic sections of monkey instead of human as substrate (lab 23) or applied immunohistochemical staining (lab 22). By following the standard protocol, interlaboratory concordance was greater than 90% for the presence or absence of islet cell antibodies in 7 of the 13 samples circulated. However, a wide variability of titers was recorded, ranging from negative to 128. Of the modifications, prolonged incubation in the presence of aprotinin was performed by 10 laboratories and was found to improve the sensitivity in 57 of 70 (81%) determinations with samples that had been ICA positive by the standard protocol. Improved sensitivity was also noted by 2 laboratories with sections of monkey pancreas. Acetone-fixed sections, used by 6 laboratories, or a two-color immunofluorescence method, applied by 3 laboratories, did not change the titers in 27 of 35 (77%) and 14 of 25 (56%) determinations with samples that had been ICA positive by the standard protocol. In contrast, heat inactivation of the samples before testing, performed by 5 laboratories, resulted in a decrease in titers in 25 of 39 (64%) determinations.(ABSTRACT TRUNCATED AT 250 WORDS)