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Chapter 120 : Endocrinopathies

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Abstract:

This chapter describes the various procedures currently used to detect circulating autoantibodies in patients with endocrine disease, i.e., thryoiditis, Graves’ disease, insulin-dependent diabetes mellitus, Addison’s disease, and pernicious anemia. The general method of indirect immunofluorescence (IF) is the screening test used most frequently to detect tissue- or organ-specific autoantibodies. Commercial hemagglutination kits for thyroglobulin antibodies are less sensitive than the tanned-cell hemagglutination (TCH) test described. Indirect IF performed on sections of human or monkey thyroid can demonstrate antibodies to thyroglobulin, CA2, and microsomes of thyroid epithelial cells, and antinuclear antibodies. The most commonly used tests for determining thyroid peroxidase (TPO) antibodies are indirect IF and commercially available hemagglutination assays or ELISAs. Separate diabetes mellitus categories consist of gestational diabetes and ‘’specific types of diabetes’’ usually with defined mutations or accompanying pathology (e.g., pancreatic abnormalities, endocrinopathies, or drug induced). The current insulin autoantibodies (IAA) assay is not able to distinguish between natural IAA and induced insulin antibodies. The current major format for determination of islet autoantibodies uses fluid-phase radioimmunobinding assays, applied to all three major islet autoantibody tests, including glutamic acid decarboxylase (GAD) autoantibodies (GAA), ICA512AA, and IAA. The majority of sera from type 1 diabetes patients target more than two epitopes on ICA512 molecules, while non-disease-related antibodies usually react with only one epitope. Anti-islet autoantibodies find their primary application in the differential diagnosis of the forms of diabetes and in predicting risk of progression to overt diabetes.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120

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Image of FIGURE 1
FIGURE 1

Overall correlation between the CCH test and ELISA. Each dot represents the mean ELISA value for the group of sera with the individual CCH titers.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 2
FIGURE 2

Indirect IF for antibodies to thyroid microsomes with an unfixed, air-dried section of monkey thyroid. Only the cytoplasm of the thyroid epithelial cells is stained. (Magnification, ×80.)

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 3
FIGURE 3

Indirect IF for antibodies to adrenal cortex, with an unfixed, air-dried section of monkey adrenal cortex. All layers of the adrenal cortex are stained. (Magnification, × 32.)

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 4
FIGURE 4

Same as Fig. 3 but at a higher magnification. Only the cytoplasm of the adrenal cells is stained. (Magnification, ×80.)

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 5
FIGURE 5

Indirect IF for antibodies to parietal cells of the gastric mucosa, with an unfixed, air-dried section of rat stomach. The cytoplasm of most cells is stained. (Magnification, ×100.)

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 6
FIGURE 6

TSAb-induced cAMP accumulation in FRTL5 and CHO-TSH-R cells incubated in isotonic medium. Cells were incubated for 2 h in the absence (control) or presence of the indicated concentrations of TSAb (purified IgG), and cAMP was measured in cell extracts.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 7
FIGURE 7

TBI assay with a commercial kit (Kronus, Inc., Boise, Idaho). Dilutions were carried out with pooled serum from healthy humans. A, B, C, and D refer to sera from mothers, all on replacement therapy, who had hypothyroid neonates. TSAb was from a mother who was treated with I for hyper-thyroidism of Graves’ disease and subsequently had two hyper-thyroid neonates. (Reprinted, with permission of The Endocrine Society, from M. Zakarija, J. M. McKenzie, and M. S. Eidson, Transient neonatal hypothyroidism: characterization of maternal antibodies to the thyrotropin receptor, 1239-1246, 1990.)

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 8
FIGURE 8

Assays with CHO-TSH-R cells in a low-salt medium. (A) Inhibitory effect of TBAb on TSAb- and TSH-induced accumulation of cAMP. (B) Biphasic effect of a patient’s IgG (IgG-Z). Cells were incubated for 2 h with the test substances (TRAb as purified IgG) and concentrations indicated, and cAMP in the medium was measured.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 9
FIGURE 9

Progression to overt type 1A diabetes of first-degree relatives of patients with type 1A diabetes (DM) relative to number of anti-islet autoantibodies expressed (autoantibod-ies to GAD65, ICA512, and insulin). ♦, three antibodies; ▪, two antibodies; ▴, one antibody. (From http://www.barabaradavis-center.org,

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 10
FIGURE 10

The current well-identified anti-islet autoantibody assays. (From http://www.barbaradaviscenter.org, Islet autoimmunity is defined as one or more autoanti-bodies for at least 3 to 6 months.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 11
FIGURE 11

General outline of fluid-phase 96-well plate assays for autoantibodies. In this example I-insulin is utilized, but the format is identical for GAD65 and ICA512 assays.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Image of FIGURE 12
FIGURE 12

Standard curve for calculation of WHO units. The axis shows the counts per minute from the assay after removing the background counts per minute, and the axis shows known log2 WHO units.

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120
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Tables

Generic image for table
TABLE 1

Biochemically characterized autoantigens

Citation: Burek C, Bigazzi P, Rose N, Zakarija M, McKenzie J, Yu L, Wang J, Eisenbarth G. 2006. Endocrinopathies, p 1062-1077. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch120

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