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Monitoring of Patients with Diabetes





Guidelines & Recommendations for Laboratory Analysis in the Diagnosis & Management of Diabetes Mellitus

Noel K Maclaren MD and John Marker BS
Weill College of Medicine of Cornell University
New York, NY 10021.

Auto-immune Markers

1. Use: A. 


Recommendation:  Islet cell auto-antibodies are not yet recommended for routine diagnosis or screening.  However, valuable clinical information can be gained through their identification. It is important that they be measured in a central laboratory with an established quality control program. They are comprised by cytoplasmic islet cell autoantibodies (ICA); insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), and the tyrosine phosphatase autoantibodies (IA-2A and IA-2bA). ICA are the most sensitive and specific for IMD as single tests, however the indirect immunofluorescence assay is difficult to perform reliably, while the chemical assays are more reproducible. Whereas, patients with IMD have been reported to have low frequencies of autoreactive T cells in their peripheral blood that can be demonstrated to proliferate in-vitro upon exposure to the islet cell antigens GAD and IA-2, the variability of such assays precludes their use in a clinical setting. Typing of class 2 major histocompatibility antigens or human leukocyte antigens DRB1 and DQA1 and DQB1 are not diagnostic of IMD, however certain of them are susceptible or resistant alleles. Thus HLA-DR/DQ typing can be used to increase or decrease the probability of IMD only, and thus cannot be recommended for clinical diagnosis. 

Diagnosis: When a patient has been diagnosed with diabetes, the assignment to immune mediated form of type 1 diabetes can only be made by identification of ICA. Most Caucasian children (90%) and adults (60%) with clinical type 1 diabetes have ICA, indicating that IMD accounts for most but not all of these patients. However, 15-20% of Caucasian adult patients who present without symptoms of insulin deficiency (type 2 diabetes phenotype) have ICAs. The incidence for Hispanic and African American diabetics is xq and yq, respectively. Whereas islet cell auto-antibody positive diabetic patients progress faster to absolute insulinopenia than do antibody negative patients, it should be recognized that many antibody negative adults who present with asymptomatic diabetes, also progress to become insulin dependent over time. It is believed by some that prolonged glucose mediated toxicity is responsible. Studies of gestational diabetes indicate that late post-partum progression to type 1 diabetes is greatest for patients with positive ICAs.  Young infants and children may develop transient hyperglycemia with gastro-enteritis and especially with upper respiratory tract infections. Most resolve and appear not to be at risk of diabetes in later life. The small subset with positive ICA however, is at high subsequent risk for type-1 diabetes.

Screening: There is no doubt that screening of relatives with IMD for islet cell auto-antibodies can identify the small group at high risk to also develop the disease. Relatives of patients with type 1 diabetes are at 15-fold the empirical risk of developing IMD (about 5%) than persons from the general population (1:250-300 over a life time).  ICA are detectable in about 3.5% of relatives, where they are associated with an absolute risk of progression to overt diabetes over 5-7 years of about 30%. The higher the titer of ICA and the younger the relative when ICA is first found to be positive, then the greater the risk of impending IMD. ICA comprise antigenic reactivities to a number of islet cell antigens, including the lower molecular weight isoform of GAD (GAD65), two (IA-2 and IA-2b) of the many trans-membrane tyrosine phosphatases expressed by pancreatic islets, and other islet cell antigens that have yet to be identified. When ICA occur with other defined autoantobodies (IAA, IA-2A/IA-2bA), then the risk of impending IMD rises greatly, to be almost a given. The higher titered ICA are most likely to comprise multiple component autoantibodies (GADA and/or IA-2A), explaining their associated higher risk for IMD. However, as many as 2% of normal adults have GAD reactive auto-antibodies that do not react as ICA, and such persons are at little to no risk of developing IMD unless they also have IA-2 or insulin autoantibodies (IAA) which most do not. Relatives of ___________with positive ICA can be followed by first phase of insulin release after IV glucose tolerance testing (IVGTT). HLA-DQB1*0602 containing genotypes are strongly protective of impending IMD, however such persons may develop islet cell autoantibodies where their prognostic significance is markedly reduced. When new-born infants with high risk HLA-DR/DQ genotypes are followed, those that develop islet cell autoantibodies begin to do so from about 9 months of age through 18 months of age, however some appear before and after this time. IAA and GAD65A tend to occur first, and IA-2A/IA-2bA later.  Thus the environmental inductive agent long speculated about, must thus occur most often in the infancy period of life. After age 7, few persons develop islet cell antibodies for the first time, albeit some with only a single type of autoantibody will undergo antigenic/epitopic spreading beyond this time. It is recommended that relatives being screened for islet cell antibodies be screened yearly until age 10, biannually from 10-20 years of age and if found negative beyond age 20, they need not be screened again unless for diagnosis if overt diabetes occurs beyond this age.

B.     Monitoring/Prognosis.  Recommendation:  Whereas impending IMD or risk of IMD can be identified through detection of islet cell autoantibodies, there are no therapies that are yet proven to prolong survival of islet cells once diabetes has been diagnosed, or to prevent the clinical onset of the disease in autoantibody positive subjects. Thus repeated islet cell autoantibodies to monitor the disease are not yet clinically useful, and cannot be recommended. The possible exception however, would be in islet cell or whole pancreas transplantation, where recurrence of islet cell autoimmunity often results as indicated by reappearance of or by rising titers of islet cell autoantibodies. However, diabetic patients who have islet cell autoantibodies are useful diagnostically as discussed above. Further, which antibodies are of value prognostically in adult diabetic patients particularly, to identify a subset with a high rate of failure of oral hypoglycemic agents. Although not yet formally proven in man, it appears likely to the authors that the capacity to secrete endogenous insulin from the time of diagnosis will be improved if such patients are treated by insulin replacement therapy rather than oral hypoglycemic agents, from their clinical outsets.There are a number of potential intervention therapies for IMD that are undergoing clinical trials or likely will be so tested in the near future. These include oral insulin given to patients at their diagnosis or to islet cell autoantibody positive relatives, and nasal insulin in both types of patients. The initial trials of a vaccine based upon immunization by an insulin B chain peptide are to begin soon.  Additional trials of other antigen based immunotherapies, adjuvants, cytokines and T cell accessory molecule blocking agents are likely in the future. Decreases in objective evidence of islet cell autoimmunity will be one important outcome measure, such as the quantitative decrease in titers of islet cell autoantibodies with the intervention. Metabolic outcome measures will include reductions in the rate of decline in the first phase of insulin release after IVGTT. The normal limits for this where the 1 and 3 minute levels of insulin are summed, is > 60 nU/ml for children under age 8 years, and > than 100 nU/ml for those over age 8 years. Other metabolic measures include plasma C-peptide levels after a mixed meal TT if the interventions are being tested in the post clinical onset period, where development of insulin antibodies to therapeutic insulin replacement will interfere with measurement of plasma insulin but not C-peptide levels.    

2. Rationale.

  1. Diagnosis:

Islet cell autoantibodies although not considered to be causal to the pancreatic b cell destruction of IMD, provide the diagnostic indicators of the disease. In a diabetic patient, their identification indicates that disease is IMD. This is a use that we believe should be recommended.  However it must be understood that islet cell autoantibodies in the absence of diabetes is not indicative of IMD, since most persons with such antibodies will never develop diabetes. In antibody positive individuals, impaired secretion of endogenous insulin to stimuli can be used as a surrogate for diabetes provided that the insulin or C-peptide levels are below the referance range (mean - 2SDs).

B.  Screening: The screening of relatives of patients with type 1 diabetes or persons in the general population does provide useful prognostic information, however until there is a proven intervention therapy to prevent the clinical onset of the disease available, such testing cannot be recommended outside of a research setting. In respect to the latter, it is strongly recommended that research groups get together to identify cohorts of antibody positive persons from relatives or from the general population on which to test prospective intervention strategies.

3. Analytical Issues

  1. Preanalytical

Recommendations:  Fortunately, blood samples for islet cell autoantibody determinations can be taken at any time of day and in the non fasting state, albeit lipemic serum may interfere with antibody binding in any of the assays. Serum rather than plasma is recommended, since heparin may lower the antibody titers. Hemolysis may also interfere, especially with the indirect immuno-fluorescence assay for ICA. We recommend the use of serum separator tubes to collect blood samples for the ICA assay since better quality sera give less ambiguity in the ICA test procedure. The sera may be sent to the analytical laboratory by overnight mail in an unfrozen state if freshly obtained. However when longer times than this are foreseen, the sera should be frozen and sent to the laboratory on dry ice.

Reference values: ICA are measured by end point titering, as compared to a standard sera obtained by the Immunology of Diabetes Workshop group, through plasmaphoresis of an index newly diagnosed IMD patient.  The results in comparison to this sera or its’ progeny are reported in Juvenile Diabetes Foundation (JDF) Units. Positive results depend upon the study or context that they are to be used in, but many laboratories like ours use 10 JDF units determined on two separate occasions, or a single result of 20 JDF units and higher to be significant titers which convey an increased risk of diabetes. IAA are measured quantitatively, in respect to the specific displaceable binding to the insulin ligand.  Significant results are those where the specific (antibody) binding is in excess of the mean + 3SDs levels in normal controls. For our own laboratory, this is > 107nU/ml. Significant levels of GADA and IA-2A and IA-2bA are those calculated as a ratio of a representative positive patient serum, and exceeding 99.2% of normal controls. Each laboratory at present must calculate their own reference cut-off values. In recent analyses by many laboratories worldwide on quality control sera sent out from our laboratory, there was remarkable concordance (>90%) for laboratories in their determinations of antibody positive versus antibody negative sera.B Analytical

Recommendation: ICA are best determined on human, blood group O pancreatic tail sections, where the pancreas has been obtained surgically from an organ donor and cubed and frozen within a 4 hour period, and stored at –800C until used. For IAA a radio-immunoassay method that calculates the displaceable insulin ligand binding after addition of excess “cold” insulin is recommended, albeit a micro-method has been developed recently that appears promising.  For IA-2A and GAD65A, a duel, micro-method, radio-immunoassay performed with 35S labeled human rIA-2 and tritium labeled human rGAD65 in a rabbit reticulocyte expression system is currently used by most laboratories. After labeling, these ligands are purified by a NAP-10 Sephadex column, they are mixed with the patients sera and after a overnight incubation, precipitated by protein-A sepharose and counted in a Packard top-counter and expressed as a ratio compared to a moderately high tittered standard patients serum. A similar assay system has been developed for IAA as well. Whereas these assays are not yet available commercially, they will soon be. Panels of autoantibodies must be done simultaneously, since only patients with two or more antibodies are at risk of developing diabetes, while any one of these four will identify IMD as the diagnosis when the patient is already diabetic. Such a panel will include the more specific but least sensitive IA-2b also in future.(John. This is the section that needs to be fleshed out by you a bit in the final version).Most of the islet cell autoantibodies are of the IgG type with all subtypes represented, including IgG2.  IgA and IgM antibodies are described however.  Islet cell autoantibodies react to antigens through conformational epitopes. Thus, ELISA techniques that rely on linear antigenic epitopes or denatured antigens, are not satisfactory for such autoantibody analyses, rather liquid phase reactions using native (undenatured) antigens as ligands are recommended.  In the case of the transmembrane tyrosine phosphatases IA-2 and IA-2b, the antibodies react almost exclusively with the N terminus or internal domains. These two phosphatases share more than 70% overall homologies and autoantibodies reactive to IA-2b mostly also react to I-A2, however reactivity is about twice as common to IA-2 than to IA-2b. In newly diagnosed patients with type-1 diabetes clinically, ICA are found in some 75%, GAD65A in some 60%, IA-2A in some 40% and IA-2bA in about half this number. A few patient sera react exclusively with IA-2b

  1. Interpretation
Low levels of autoantibodies of all kinds are expected even in completely normal persons with no family history of autoimmune diseases. Islet cell autoantibodies are no exception. In the context of clinical diabetes, islet cell antibodies above background can be taken as diagnostic evidence for IMD. The predictive significance of the autoantibodies based on titer should be worked out by individual groups for their assays. However for the standardized ICA, replicate titers in excess of 10 JDF units do predict an increased risk of diabetes.  Similarly, IAA above the mean + 3SDs of normal controls also predict an increased risk of diabetes, and when associated with ICA or other antibody, the risk becomes high.  In relatives of a proband with IMD, islet cell antibodies predict diabetes as mentioned above, however this is also true in the general population of children. The more of the 5 types of autoantibodies identified, then the higher the risk of diabetes. Besides those already mentioned, others such as GLIMA-38 have been identified as associated with IMD but the prognostic significance has not been established. There are other islet cell antigens that are yet to be discovered in the disease, however those available are sufficient for diagnosis and for disease prediction. In fact, trial of therapies for disease prevention could be done on relatives on the basis of autoantibodies alone. This is also true for general populations, however the positivity rates are distinctly lower than in relatives, so that more have to be screened to provide at risk subjects needed for the trials.
  1. Emerging Considerations
The above autoantibodies are now routine laboratory procedures, and will probably be reconstructed for auto-analyzers in due course.  It is also likely that other islet cell antigens will be discovered, that could lead to diagnostic and predictive tests for IMD. The need for such analyses will rise sharply, when any of a number of possible intervention strategies are finally proven in man as they have been in mice. Autoantibody screening on finger stick blood samples appears quite feasible in future, as physician room or beside tests. The whole panel of antibodies mentioned above should be done at the one time, to increase their predictive values. As destructive islet cell autoimmunity proceeds, there is co-incident spreading of the antigenic/epitopic reactivities. Multiple autoantibodies when detected give increased specificity for IMD over that for single type autoantibodies where all have be tested for, but at lower sensitivity.  Population screening of blood samples collected as blood spots on filter paper is now feasible as well. Much standardization of cellular responses to islet cell antigens needs to be done before these can be used reliably for diagnosis or prediction of IMD, however such is likely in future. In those found to have positive islet cell autoantibodies, HLA-typings and IVGTT will help define absolute risks of diabetes.