- Review Article
The immunofluorescence techniques in the diagnosis of endocrine autoimmune diseases
Autoimmunity Highlights volume 3, pages 67–78 (2012)
In the study of autoimmune diseases, the laboratory plays a very important role. We describe the immunofluorescence techniques (direct, indirect, complement-fixing, double) for determining the presence of autoantibodies and their role in the autoimmune endocrine diseases.
In the study of autoimmune diseases, the laboratory plays a very important role in predicting or detecting, by immunological studies, the reactions between antigens (Ags) and antibodies (Abs).
Many researchers applied these techniques, year after year more sophisticated and accurate as reported by Betterle , for detecting the presence of autoantibodies in patients affected by the autoimmune diseases.
The main analytical techniques used in clinical immunology’s laboratories may be summarized in Table 1.
Immuno-histochemical and immuno-enzymatic techniques
The key reactions of immunology and immune defense are the interaction of Abs and Ags forming large irreversible macromolecule complexes. These interactions, analogous to those observed in enzyme-substrate interactions, involve non-covalent binding of antigenic determinants.
In research, purified antibodies (monoclonal or polyclonal) are most commonly used to identify and locate intracellular and extracellular proteins or autoantibodies present in body fluids or attached to some cellular or tissue antigens. Antibodies are used in flow cytometry, immunoprecipitation , Western blot analyses  and in immunohistochemistry or immunofluorescence (IF)  to examine the protein expression in tissue sections or to locate proteins within cells.
Some general characteristics of the laboratory’s techniques (accuracy, precision, linearity, sensitivity, specificity and plausibility) must be applied also to the immunofluorescence techniques. This is an important point, because sometimes it is not easy to find all these parameters simultaneously present in the same test because for e.g., the sensitivity is not always in accord with the specificity. It is certainly difficult to find equilibrium between these parameters but this must be the purpose both the implementation of the various tests and international standardization studies.
Some other parameters (preparation of substrates, solutions buffers, fixation and others)  may play an important role in the immunofluorescence studies: in the past, the laboratory’s techniques were entrusted to the skill and dexterity of the technicians, today there are many companies specialized in the preparation of substrates for immunohistochemistry.
Today, the technical procedures have changed significantly because the previous manual techniques have been replaced by automated systems. But today only the visual check by microscope is left to the experience of the technician who reads the reaction.
The immunofluorescence is a histochemical laboratory staining technique that uses the specificity of Abs to their antigen. It is a widely used in immunohistochemistry based on the use of some fluorochromes  to visualize the location of the Abs.
The immunofluorescence technique was firstly described in 1942 and refined by Coons in 1950, which used a fluorescence microscope able to read the specific immunological reaction on tissue or cellular prepared on slides. Subsequently, in 1963, granular deposits of IgG and C3 were described along the dermo-epidermal junction in lesions of lupus erythematosus. In 1964, Beutner  used the indirect immunofluorescence (IIF) technique to demonstrate antibodies in the sera of patients affected by pemphigus and, in the same years, this technique was used to detect the most important autoantibodies in endocrine autoimmune diseases (see below).
There are four major IF techniques:
Direct immunofluorescence technique: it is a one-step histological staining procedure for identifying in vivo antibodies that are bound to tissue antigens, using a single antibody labeled with a fluorophore  for staining the tissues or cells. The antibody recognizes the target molecule and binds to it.
Indirect immunofluorescence technique: it is a two-step serological technique for the detection of circulating antibodies in body fluids, using two antibodies. The unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognizes the primary antibody and binds to it.
Indirect immunofluorescence complement-fixation (IIF-CF) technique: Ags and Abs bind to one another to generate many molecules of C3. This amplification principle is used in complement IIF, which is more sensitive than IIF.
Double immunofluorescence technique: this technique allows identifying the presence of two different antigens on a cell or for the identification of specific cell in a tissue by two antibodies labeled with different fluorophores (for es. FITC or rhodamine). Double staining can be used as a direct/indirect method. The indirect method has very high sensitivity.
Historical features in autoimmune diseases of the endocrine glands
The ante litteram description of the autoimmune nature of the endocrine glands goes back to 1855, when Addison  described a case with adrenal insufficiency due to an idiopathic (lymphocytic) infiltration of the adrenals; and to 1912, when Hashimoto  described mononuclear leukocyte infiltration with an enlarged thyroid gland in patients, defining it as “lymphomatous goiter” and to 1940, when Von Mayerburg  described a lymphocytic infiltration of pancreatic islets (insulitis) in patients with type 1 diabetes mellitus. In 1956, three important discoveries were reported permitting the start of the era of autoimmune diseases: (a) the demonstration of autoantibodies against thyroglobulin in chronic thyroiditis , (b) the presence of an agent stimulating the thyroid in patients with Graves’ disease  and (c) the demonstration that chronic thyroiditis was reproducible in rabbits by immunization with homogenates of autologous thyroid tissue . In 1957, Witebsky  established the criteria for defining an autoimmune disease summarized in Table 2.
In the 1957, it was discovered that “idiopathic” Addison’s disease (AD) had complement fixing autoantibodies against adrenal cortex extracts . On 1962, antibodies to parietal cells were identified by complement fixation test in patients with pernicious anemia . In the 1968, antibodies to steroid-producing cells were described by IIF technique in patients with gonadal failure . On 1974, the autoimmune nature of type 1 diabetes mellitus was firstly described by IIF when islet cells autoantibodies were demonstrated .
On the basis of these discoveries from 1956 to 2006, a progressively increased number of diseases previously considered as idiopathic, entered in this new group of disorders, so that in 2006 in the preface of the book that celebrated the 50th Anniversary of the discovery of autoimmunity, Rose and Mackey affirmed that “more than 80 diseases are attributable to autoimmunity and one or another affect some 7 % of the population” .
Natural history of endocrine autoimmune diseases
The autoimmune endocrine diseases are chronic disorders characterized by genetic predisposition, presence of circulating autoantibodies and lymphocytic infiltration in the target organs and the natural history of these diseases develops in three separate phases: (a) potential, (b) subclinical or latent, and (c) clinical. Autoantibodies are the circulating markers that encompassed all the three phases. On the basis of their ability to induce or not a damage of target organ, the autoantibodies can be subdivided as pathogenetic and non-pathogenetic .
Autoantibodies in autoimmune endocrine diseases
The endocrine system is one of the systems more affected by autoimmunity. The main autoimmune diseases of the endocrine system are summarized in the Table 3.
Thyroid autoimmune diseases
Thyroid autoimmune diseases (TAD) are summarized in Table 3. They are marked by the presence of thyroglobulin autoantibodies (TGAbs) and thyroid microsomal autoantibodies (TMAbs).
TGAb were initially detected by immunoprecipitation in agar  and then by IIF technique on thyroid sections fixed in methanol . On 1961, they were detected by passive hemoagglutination using sheep red cells  and on 1978 using turkey-nucleated red cells . In 1974, TGAbs were tested by RIA [23, 24] and, in 1980, by ELISA [25, 26], nevertheless for a long period TGAbs were tested using IIF on unfixed thyroid tissue (Fig. 1).
TMAbs were detected, since 1967, by IIF on unfixed thyroid tissue , and from 1973 also by passive hemoagglutination using sheep red cells , and from 1976 using or turkey-nucleated red cells . They were tested by RIA or ELISA using microsomal antigens [29, 30].
These methods are a slightly more sensitive and specific than the previous tests , nevertheless, the IIF test on unfixed thyroid tissue was used for many years after.
TGAbs and/or TMAbs/TPOAbs were also present in 50 % of first-degree relatives of patients with autoimmune thyroid diseases, in 15–40 % of patients with other autoimmune diseases and they can also be found in 20 % of patients with thyroid carcinoma . These auto-Abs can be found in 12–26 % of euthyroid females and in 2.8–14.4 % of euthyroid males . The presence of these autoAbs was considered a marker of future thyroid dysfunction [37, 38].
The IIF test on thyroid tissue in addition to the detection of TMAbs and TGAbs permitted also to individuate others autoantibodies (such as mitrochondria antibodies AMA and anti-nuclear antibodies ANA) (Fig. 1e, f).
Gastric autoimmune diseases
The gastric autoimmune diseases are summarized in Table 3. The diseases are marked by the presence of parietal cells autoantibodies (PCA). Initially, PCA were detected by complement fixation test using homogenates of stomach [39, 40] and were found in 62 % of patients with pernicious anemia and in 4 % of normal controls. Subsequently they were detected by IIF using human or animal gastric mucosa; with this technique, PCA were found in 86 % of patients with pernicious anemia and in 11 % of controls . PCA are organ, but not strictly species-specific auto-Abs of IgG class, reacting with the microsomal fraction of parietal cells  (Fig. 2). On 1984, a RIA was introduced using microsomal gastric antigen in order to detect PCA .
On 1987, ATPasi H+/K+ was identified as the target antigen detected by PCA and an ELISA was produced to detect ATPasiH+/K+Abs and with this method were found in 88 % of patients with chronic gastritis . PCA can be found in normal population with a frequency that increases with the age, in fact they are present in 4.8 % in subjects under 50 years but reached 9.6 % in those with more than 80 years . PCA are also present in 5–25 % of the patients with other autoimmune diseases, particularly in those with thyroid autoimmune diseases, Addison’s disease, type 1 diabetes or vitiligo [42, 44]. PCA can be found in asymptomatic patients and they can be considered markers of latent autoimmune gastritis  or predictors of future autoimmune gastritis . Until today, the IIF test on gastric tissue is already the most used to detect PCA.
The IIF test on gastric mucosa is able to detect also other auto-Abs such as ANA, AMA, ribosomal antibodies (ARA) and smooth muscle antibodies (SMA) (Fig. 2d–f).
Autoimmune diseases of the endocrine pancreas
The autoimmune diseases of the endocrine pancreas are summarized in Table 3. Islet-cell autoantibodies (ICA) were discovered on 1974 by IIF test using normal human group 0 pancreas . They react with an unidentified antigen common to all the cells of the pancreatic islets (Fig. 3b). They are of IgG class, able to fix the complement, organ- but not strictly species-specific because they can react also with primate pancreas. On 1985 under the aegis of the Juvenile Diabetes Foundation, a standard positive serum with a value a 80 U/JDF was identified and the distribution worldwide of this standard serum permitted to improve the sensitivity and specificity of the various laboratories detecting ICA by IIF [47–49]. Subsequently, other pancreatic autoantibodies were identified against insulin (IAA) , GAD (GADAb) , second islet autoantigen (IA2Ab) also called ICA 512 . The IIF test identified ICA in 48-60 % of newly onset type 1 DM [53, 54]. ICA can be also present in about 9 % of the patients with type 2 DM  and they are able to identify, in combination with GADAb, the latent autoimmune diabetes of the adults (LADA) . ICA associated to other pancreatic Abs can also be identified in individuals without diabetes (first-degree relatives of patients with type 1 DM, schoolchildren, or patients with other autoimmune diseases) and they can be markers of future disease with an increased risk directly correlated with the number of pancreatic auto-Abs [56, 57].
Using IIF technique on human pancreas, other reactivities against different cells of the islets were also discovered (Fig. 3c) and identified by the double IF technique as autoantibodies to glucagon-producing cells (GPCAb) (Fig. 4) or to somatostatin-producing cells (SPCAb) (Fig. 5) . But, so far these antibodies were not correlated with a clinical deficiency of these hormones .
The determination of ICA in combination with other pancreatic Abs remains, today, the best method for the identification of patients with autoimmune type 1 DM, LADA or the subjects at risk of type 1 DM.
The IIF test on pancreas identified other reactivities such as AMA, ANA, ARA (Fig. 3d, e) and Abs against cytokeratin.
Autoimmune diseases of the adrenal
The adrenal cortex autoimmune diseases are summarized in Table 3. Adrenal cortex autoantibodies (ACA) are markers of autoimmune adrenal insufficiency and were discovered on 1957 by complement fixation using adrenal cortex extracts . From 1963, they were tested by IIF technique using human or animal tissues . ACA react with the cytoplasm of all the three cells layers of adrenal cortex (Fig. 6b), they are of IgG class, organ, but not strictly species, specific and fix the complement. Using the IIF technique on human or animal adrenal tissues, many patients affected by autoimmune and tuberculous AD were studied, and ACA were found positive in 61 and 6.7 %, respectively .
In 1992, it was discovered that the enzyme 21-hydroxylase (21-OH) is the major autoantigen recognized by ACA [62, 63]. After this discovery, a RIA test using 35S-21-OH  was used to detect 21-OHAbs. Subsequently, a more convenient assay to measure 21-OHAbs has been developed using 125I-21-OH expressed in yeast .
ACA by IIF on human adrenal cortex or 21-OHAbs by IPA are present in 89 and 91 %, respectively, of patients at the onset of autoimmune Addison’s disease, but absent in patients with non-autoimmune forms. There is a strict correlation between the two methods even if 21-OHAbs are able to detect very low titers of antibodies and ACA can detect some autoantibodies different from 21-OHAbs .
They can be present also in individuals without hypoadrenalism and they can predict the future adrenal insufficiency . On 2012, under the aegis of Euradrenal Committee, a program for the standardization of 21-OH Abs involving multiple international laboratories has been initiated and the results of this study will be published (Betterle personal observation).
Adrenal medulla autoimmunity
Autoimmune diseases of the adrenal medulla are summarized in Table 3. On 1984, it was described an adrenal-medullary-cell antibody (AMCA) of IgG class, reacting to an unidentified cytoplasmic antigen of adrenal medulla . The AMCA can produce a “diffuse” (Fig. 6) or a “granular” pattern. AMCA are detected in 30 % of ICA-positive and 4 % of ICA-negative patients with type 1 DM, and in 32 % of ICA-positive patients without type 1 DM .
The clinical significance of this Abs is not clear. But it is important to remember that: (a) a deficit of catecholamines has been described in type 1 DM patients with AMCA , (b) it has been demonstrated an adrenal medulla fibrosis in type 1 DM with longstanding disease , (c) some patients with type 1 DM revealed an insufficient adrenergic response to insulin-induced hypoglycaemia . On the basis of all these data it has been hypothesized that these Abs mark, in type 1 DM, a lymphocytic medullitis inducing a deficit of catecholamines [69, 71].
Autoimmune diseases of the gonads
Autoimmune diseases of the gonads are summarized in Table 3. On 1968, an antibody to steroid-producing cells (StCA) was detected by IIF test , reacting to the cells of the ovarian theca, to Leydig cells of the testis, syncytiotrophoblast of the placenta and to adrenal cortex cells (Fig. 7).
In females affected by Addison’s disease, they are markers of premature ovarian failure (POF)  characterized by a lymphocytic oophoritis . After the discovery that StCA reacted with steroidogenic enzymes 17α-hydroxylase (17α-OH) or side chain cleavage (SCC) , autoantibodies to 17α-OH (17α-OHAbs) or to SCC (SCCAbs) were tested by a RIA technique, using recombinant antigens . These Abs are present with high frequency (92 %) of Addisonian patients with POF but they are also present in female patients affected by Addison’s disease with normal menses and can be predictive markers of future POF .
Despite the StCA were initially identified in two male patients affected by Addison’s disease , it is not clear if in those patients they can be markers of autoimmune testicular insufficiency.
Autoimmune parathyroid diseases
Parathyroid autoimmune diseases are shown in Table 3. The detection of parathyroid autoantibodies represents one the most problematic and not so far defined cases in autoimmunity. Initially, they were detected in 1966 by IIF using unspecified parathyroid tissue , subsequently confirmed with the same method . These autoantibodies reacted to the cytoplasm of the cells of parathyroid tissue (Fig. 8), but they were present with low frequency in chronic hypoparathyroidism (CH), in patients with other autoimmune diseases and also in normal controls . Subsequently, by IIF, autoantibodies reacting with the surface of human parathyroid cells and able to inhibit the PTH secretion or to mediate a complement-dependent cytotoxicity in cultured bovine parathyroid cells were reported in patients with CH [79, 80] but different studies did not confirm this report . Other studies reported that the parathyroid cytoplasmic antibodies, initially detected by IIF, were reactive only with oxyphil parathyroid cells  recognizing a human mitochondrial antigen of 46 kDa molecular weight . Subsequently, autoantibodies against a human parathyroid membrane-antigen of 120–40 kDa, identified as the calcium sensing receptor (Ca-SR) by immunoblot and Western blotting analysis were identified in 20 % of the patients with CH (isolated or in the context of autoimmune polyendocrine syndrome type 1) and in 0 % of controls . Subsequently, Ca-SRAbs detected by Western blot, were found in 49 % of the patients with CH but also in 13 % of the normal controls . In contrast, by immunoprecipitation assay using 35S labelled recombinant Ca-SR antigen, Ca-SRAbs have not been detected in any of 90 patients with CH .
Recently, an antibody to a parathyroid antigen defined NACHT leucine-rich-repeat protein 5 (NALP5Abs) was identified in 48 % of the patients with CH in the context of autoimmune polyendocrine syndrome type 1 and not in the other forms of CH or in other autoimmune diseases .
Autoimmune pituitary diseases
Autoimmune pituitary diseases are summarized in Table 3. Using human pituitary, the autoantibodies against some pituitary cells permitted to demonstrate, by a double IF technique, a reaction to prolactin-secreting cells . These Abs were described in patients with autoimmune endocrine diseases but in none affected by hypopituitarism .
Subsequently, pituitary antibodies were investigated in patients with lymphocytic adenohypophysitis (LAH) by indirect IIF (Fig. 9). Using as substrates human, primate or rodent pituitary specimens, human fetuses, or animal pituitary cell lines, pituitary Abs were found in only 36 % of patients with documented LAH as reviewed by Caturegli . The specificity of pituitary Abs is poor and they were found in various other non-autoimmune diseases as Cushing’s syndrome, pituitary adenomas, empty sella syndrome and Sheehan syndrome, as in other autoimmune diseases such as type 1 diabetes, Hashimoto’s thyroiditis, and Graves’ disease as reviewed by Caturegli . Autoantibodies to a not specified anterior pituitary components were also described in patients with autoimmune diseases and able to predict future hypopituitarism . The biggest problem about the evaluation of these autoantibodies is that do not exist an international standardization of this procedure, furthermore it was documented that the Fc fragment of immunoglobulins bind to a receptor of ACTH producing cells of the pituitary .
As regard to Abs to diencephalon or hypothalamus, initially they were tested by IIF using baboon diencephalon, and antibodies to vasopressin-producing cells were described in 37 % of patients with idiopathic diabetes insipidus  (Fig. 10). The specificity of these antibodies is poor being described also in 54 % of patients with DI secondary to histiocytosis X and in other autoimmune disorders .
Concluding our study and considering that, in these recent years, IF was considered as obsolete and outdate, we think important to report these Mackay’s considerations in a recent review on the history of autoimmunity : “in the early 1960s’, the immunofluorescence led to the confident nomination of diverse diseases as autoimmune and the technique soon became the laboratory “workhorse” for routine laboratory diagnosis”. These words may be valid also today because this technique may be considered very useful in the diagnosis of various organ- and non-organ specific autoimmune diseases.
Betterle C (1997) Gli Autoanticorpi: Manuale-Atlante a colori di diagnostica. In: Piccin (ed), Padova
Williams N (2000) Immunoprecipitation procedures. Methods Cell Biol 62:449–453
Kurien B, Scofield R (2006) Western blotting. Methods 38:283–293
Scanziani E (1998) Immunohistochemical staining of fixed tissues. Methods Mol Biol 104:133–140
Brehm-Stecher B, Johnson E (2004) Single-cell microbiology: tools, technologies, and applications. Microbiol Mol Biol Rev 68:538–559
Beutner EH, Jordon R (1964) Demonstration of skin antibodies in sera of pemphigus vulgaris patients by indirect immunofluorescent staining. Proc Soc Exp Biol Med 117:505–510
Addison T (1937) Physician to Guy’s Hospital. London: New Sydenham Society 1868. Reprinted in Medical Classics 2:244–293
Hashimoto H (1912) Zur kenntnis der lymphomatosen veranderung der schilddruse (struma lymphomatosa). Acta Klin Chir 97:219–248
Von Mayenburg H (1940) Uber “insulitis” bei diabetes. Schweitz Med Wochenschr 71:554–557
Campbell PN, Doniach D, Hudson RV, Roitt IM (1956) Autoantibodies in Hashimoto’s disease (lymphadenoid goiter). Lancet 20:820–821
Adams DD, Purves HD (1956) Abnormal responses in the assay of thyrotropin. Proc Univ Otago Med School 34:11–12
Rose NR, Witebsky E (1956) Studies in organ specificity. V. Changes in the thyroid glands of rabbit following active immunization with rabbit thyroid extracts. J Immunol 76:417–427
Witebsky E, Rose NR, Terplan K, Paine JR, Egan RW (1957) Chronic thyroiditis and autoimmunization. JAMA 164:1439–1447
Anderson JR, Goudie RB, Gray KG, Timbury GC (1957) Autoantibodies in Addison’s disease. Lancet 1:1123–1124
Irvine WJ, Davies SH, Delamore IW, Williams AW (1962) Immunological relationship between pernicious anemia and thyroid diseases. Brit Med J 2:454–456
Anderson JR, Goudie RB, Gray KG, Stuart-Smith DA (1968) Immunological features of idiopathic Addison’s disease: an antibody to cells producing steroid hormones. Clin Exp Immunol 3:107–117
Bottazzo GF, Florin-Christensen A, Doniach D (1974) Islet cell antibodies in diabetes mellitus with autoimmune polyendocrine syndromes. Lancet 2:1279–1282
The autoimmune diseases (2006) In: Rose N, MacKay IR (eds) Elsevier, Academic Press, San Diego
Betterle C, Presotto F, Pedini B, Greggio N, Accolla R, Zanchetta R (1997) Generalità sulle malattie autoimmuni. In: Betterle C (ed) Gli Autoanticorpi Manuale-Atlante a colori di diagnostica. Piccin, Padova, 1–40
Doniach D (1967) Thyroid autoimmune diseases: symposium on thyroid gland. J Clin Pathol 20:385–390
Fulthorpe AJ, Roitt IM, Doniach D, Couchman K (1961) A stable sheep cell preparation for detecting thyroglobulin autoantibodies and its clinical application. J Clin Pathol 14:654–660
Cayzer I, Chalmers SR, Doniach D, Swana G (1978) An evolution of two new haemoagglutination tests for the rapid diagnosis of autoimmune thyroid diseases. J Clin Pathol 31:1147–1151
Salabè GB, Salabe H, Dominici D, Davoli C, Andreoli M (1974) Radioimmunoassay for human antithyroglobulin antibodies. II. Determination of antigen binding capacity. J Clin Endocrinol Metab 39:1125–1132
Peake RL, Wills DB, Asimakis GK, Deiss W (1974) Radioimmunological assay for antithyroglobulin antibodies. J Lab Clin Med 84:907–919
Voller A, Bidwell DE, Burek CL (1980) An enzyme-linked immunosorbent assay (ELISA) for antibodies to thyroglobulin. Proc Soc Exp Biol Med 163:402–405
Endo YE, Junichi N, Horinuchi K, Sachiya O, Motomori I, Ishikawa E (1980) An enzyme immunoassay for the measurement of antithyroglobulin autoantibodies in human serum. Clin Chim Acta 103:67–77
Bird T, Stephenson J (1973) Evaluation of a tanned red cell technique for thyroid microsomal antibodies. J Clin Pathol 26:623–627
Amino N, Hagen SR, Yamada N, Refetoff S (1976) Measurement of circulating thyroid microsomal antibodies by tanned red cell haemoagglutination technique: its usefulness in the diagnosis of autoimmune thyroid diseases. Clin Endocrinol 5:115–125
Mori T, Fisher JP (1971) Measurement by competitive binding radioassay of serum anti-microsomal and anti-thyroglobulin antibodies in Graves’ disease and other thyroid disorders. J Clin Endocrinol Metab 33:688–698
Scardt CW, McLachlan SM, Matheson J, Rees Smth B (1982) An enzyme linked immunoassay for thyroid microsomal antibodies. J Immunol Meth 55:155–168
Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitzky S (1985) Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. FEBS Lett 190:147–152
Mariotti S, Anelli S, Ruf J, Bechi R, Czarnocka B, Lombardi A, Carayon P, Pinchera A (1987) Comparison of serum thyroid microsomal and thyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab 65:987–993
Ruf J, Czarnocka B, Ferrand M, Doullais F, Carayon P (1988) Novel routine assay of thyroperoxidase autoantibodies. Clin Chem 34:2231–2234
Kaufman KD, Filetti S, Seto P, Rapaport B (1990) Recombinant human thyroid peroxidase generated in eukaryotic cells: a source of specific antigen for the immunological assay of antimicrosomal antibodies in the sera of patients with autoimmune thyroid disease. J Clin Endocrinol Metab 70:724–728
Mariotti S, Caturegli P, Piccolo P, Barbesino G, Pinchera A (1990) Antithyroid peroxidase autoantibodies in thyroid diseases. J Clin Endocrinol Metab 71:661–669
Prummel MF (2005) Thyroid peroxidase autoantibodies in euthyroid subjects. Best Practice Res Clin Endocrinol Metab 19:1–15
Vanderpump MPJ, Tunbridge WMG, French JM, Appleton D, Batest D, Clark F, Grimley Evans J, Hasan DM, Rodgers H, Tunbridge F, Young ET (1995) The incidence of thyroid disorders in the community: a twenty-year follow up of the Whickham survey. Clin Endocrinol 43:55–698
Walsh JP, Bremner AP, Feddema P, Leedman PJ, Brown SJ, O’Leary P (2010) Thyrotropin and thyroid antibodies as predictors of hypothyroidism: a 13-year, longitudinal study of a community-based cohort using immunoassay techniques. J Clin Endocrinol Metab 95:1095–1104
Irvine WJ, Davies SH, Delamore IW, Williams AW (1962) Immunological relationship between pernicious anemia and thyroid diseases. Brit Med J 2:454–456
Taylor KB, Roitt IM, Doniach D, Couchman KG, Shapland C (1962) Autoimmune phenomena in pernicious anemia: gastric antibodies. Br Med J 2:1347–1352
Mariotti S, Pisani S, Russova A, Bechi R, Giacomelli M, Passaleva A, Massai G, Baschieri L, Pinchera A (1984) A solid phase immunoradiometric assay for gastric parietal cell antibodies. Clin Exp Immunol 58:745–753
Karlsson FA, Burman P, Loof L, Olsson M, Scheynius A, Mardh S (1987) Enzyme-linked immunosorbent assay of H+, K+,-ATPase, the parietal cell antigen. Clin Exp Immunol 70:604–610
Strickland RG, Hooper B (1972) The parietal cell heteroantibody in human sera: prevalence in a normal population and relationship to parietal cell antibody. Pathology 4:259–263
Betterle C, Mazzi PA, Pedini B, Accordi F, Cecchetto A, Presotto F (1988) Complement-fixing gastric parietal cell autoantibodies: a good marker for the identification of type A chronic atrophic gastritis. Autoimmunity 1:267–274
Segni M, Borrelli O, Pucarelli I, Delle Fave G, Pasquino AM, Annibale B (2004) Early manifestations of gastric autoimmunity in patients with juvenile thyroid autoimmune diseases. J Clin Endocrinol Metab 89:4944–4948
Tozzoli R, Kodermaz G, Perosa AR, Tampoia M, Zucano A, Antico A, Bizzaro N (2010) Autoantibodies to parietal cells as predictors of atrophic body gastritis: a five-year prospective study in patients with autoimmune thyroid diseases. Autoim Rev 10:80–83
Bonifacio E, Lernmark Å, Dawkins Rl et al (1988) Serum exchange and use of dilutions have improved precision of measurement of islet cell antibodies. J Immunol Methods 106:83–88
Bonifacio E, Boitard C, Gleichmann H, Shattock MA, Molenaar J, Bottazzo GF, Betterle C (1990) Assessment of precision, concordance, specificity, and sensitivity of islet cell antibodies measurement in 41 assays. Diabetologia 33:731–736
Greenbaum CJ, Palmer JP, Nagataki S, Yamaguchi Y, Molenaar JL, Wam Van Beers, Maclaren NK, Lernmak A (1992) Improved specificity of ICA assays in the Fourth International Immunology of Diabetes Serum Exchange Workshop. Diabetes 41:1570–1574
Palmer JP, Asplin CM, Clemons P, Lyen K, Tatpati O, Raghu PK, Paquette TL (1983) Insulin antibodies in insulin-dependent diabetics before insulin treatment. Science 222:1337–1339
Solimena M, Folli F, Denis-Donini S, Comi GC, Pozza G, De Camilli P, Vicari AM (1988) Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and Type 1 diabetes mellitus. N Engl J Med 318:1012–1020
Rabin DU, Pleasic SM, Shapiro JA, Yoon-Warren H, Oles J, Hicks JM, Goldstein DE, Rae PMM (1994) Islet cell antigen 512 is a diabetic-specific islet autoantigen related to protein tyrosine phosphatases. J Immunol 152:3183–3188
Lendrum R, Walker G, Gamble DR (1975) Islet-cell antibodies in juvenile diabetes mellitus of recent onset. Lancet 880–882
Del Prete GF, Betterle C, Padovan D, Erle G, Toffolo A, Bersani G (1977) Incidence and significance of islet-cell autoantibodies in different types of diabetes mellitus. Diabetes 26:909–915
Turner R, Stratton I, Horton V, Manley S, Zimmet P, Mackay IR, Shattock M, Bottazzo GF, Holman R (1997) UKPDS 25: autoantibodies to islet-cell cytoplasm and glutamic acid decarboxylase for prediction of insulin requirement in type 2 diabetes. UK Prospective Diabetes Study Group. Lancet 1:1288–1293
Slower RH, Eisenbarth G (1997) Prevention of type 1 diabetes and recurrent beta-cell destruction of transplanted Islets. Endocrine Rev 18:241–258
Betterle C, Spadaccino AC, Presotto F, Zanchetta R, Pedini B, Lai M, Greggio NA, Bottazzo GF (2002) The number of markers of pancreatic autoimmunity is proportional to the risk for type 1 diabetes mellitus (DM) in Italian and English patients with organ-specific autoimmune diseases (OSAD). Ann N Y Acad Sci 958:276–280
Bottazzo GF, Lendrum R (1976) Separate autoantibodies to human pancreatic glucagon and somatostatin cells. Lancet 2:873–876
Betterle C, Zanette F, Presotto F, Pedini B, Tessari P, Valerio A, Tiengo A (1986) Alpha-cells autoantibodies: immunological and metabolic follow-up study. Horm Met Res 5:327–330
Blizzard RM, Kyle M (1963) Studies of the adrenal antigens and antibodies in Addison’s disease. J Clin Invest 42:1653–1660
Betterle C, Coco G, Zanchetta R (2005) Adrenal cortex autoantibodies in subjects with normal adrenal function. Best Practice Res Clin Endocrinol Metab 19:85–99
Baumann-Antczak A, Wedlock N, Bednarek J, Kiso Y, Krishnan H, Fowler S, Rees Smith B, Furmaniak J (1992) Autoimmune Addison’s disease and 21-hydroxylase. Lancet 340:429–430
Winqvist O, Karlsson FA, Kampe O (1992) 21-hydroxylase, a major autoantigen in idiopathic Addison’s disease. Lancet 339:1559–1562
Colls J, Betterle C, Volpato M, Rees Smith B, Furmaniak J (1995) A new immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in Addison’s disease. Clin Chem 41:375–380
Tanaka H, Perez MS, Powell M, Sanders JF, Sawicka J, Chen S, Prentice L, Asawa T, Betterle C, Volpato M, Rees Smith B, Furmaniak J (1997) Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipitation assay. J Clin Endocrinol Metab 82:1440–1446
Betterle C, Volpato M, Pedini B, Chen S, Rees Smith B, Furmaniak J (1999) Adrenal-cortex autoantibodies and steroid producing cells autoantibodies in patients with Addison’s disease: comparison of immunofluorescence and immunoprecipitation assays. J Clin Endocrinol Metab 84(2):618–622
Coco G, Dal Pra C, Presotto F, Albergoni MP, Canova C, Pedini B, Zanchetta R, Chen S, Furmaniak J, Rees Smith B, Mantero F, Betterle C (2006) Estimated risk for developing autoimmune Addison’s disease in patients with adrenal cortex autoantibodies. J Clin Endocrinol Metab 91:1637–1645
Schopfer K, Matter L, Tenschert R, Bauer S, Zuppinger K (1984) Anti-glucagon-cell and anti-adrenal-medullary-cell antibodies in islet-cell-autoantibody positive diabetic children. N Engl J Med 310:1536–1537
Brown F, Smith AM, Longway S, Rabinowe SL (1990) Adrenal medullitis in Type 1 diabetes. J Clin Endocrinol Metab 71:1491–1495
Brown FM, Brink JS, Freeman R, Rabinowe SL (1989) Anti-sympathetic nervous system autoantibodies. Diminished catecholamines with orthostasis. Diabetes 38:938–941
Rabinowe SL (1990) Immunology of diabetic and polyglandular neuropathy. Diabetes/Metabolism Rev 6:169–188
Irvine WJ, Chan MMW, Scarth L (1968) Immunological aspects of premature ovarian failure associated with idiopathic Addison’s disease. Lancet 2:886–887
Hoek A, Schoemaker J, Drexhage H (1997) Premature ovarian failure and ovarian autoimmunity. Endocrine Rev 18:107–134
Winqvist O, Gebre-Medhin G, Gustafsson J, Martin Ritzén E, Lundkvist O, Anders Karlsson F, Kämpe O (1995) Identification of the main gonadal autoantigens in patients with adrenal insufficiency and associated ovarian failure. J Clin Endocrinol Metab 80:1717–1723
Chen S, Sawicka J, Betterle C, Powell M, Prentice L, Volpato M, Rees Smith B, Furmaniak J (1996) Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison’s disease, and premature ovarian failure. J Clin Endocrinol Metab 83:2977–2986
Reato G, Morlin L, Chen S, Furmaniak J, Rees-Smith B, Masiero S, Albergoni MP, Cervato S, Zanchetta R, Betterle C (2011) Premature ovarian failure in patients with autoimmune Addison’s disease: clinical, genetic, and immunological evaluation. J Clin Endocrinol Metab 96:E1255-E1261
Blizzard RM, Chee D, Davis W (1966) The incidence of parathyroid and other antibodies in the sera of patients with idiopathic hypoparathyroidism. Clin Exp Immunol 1:119–128
Irvine WJ, Scarth L (1969) Antibody to the oxyphil cells of the human parathyroid in idiopathic hypoparathyroidism. Clin Exp Immunol 4:505–510
Posillico JT, Wortsman J, Sikanta S, Eisembarth GS, Mallette JW, Brown EM (1986) Parathyroid cell surface antibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells. J Bone Miner Res 1:475–483
Brandi ML, Aurbach GD, Fattorossi A, Quarto R, Marx SJ, Fitzpatrick LA (1986) Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism. Proc Natl Acad Sci (USA) 83:8366–8369
Fattorossi A, Aurbach GD, Sakaguchi K (1988) Anti-endothelial cell antibodies: detection and characterization in sera from patients with autoimmune hypoparathyroidism. Proc Natl Acad Sci (USA) 85:4015–4019
Swana GT, Swana MR, Bottazzo GF, Doniach D (1977) A human specific mitochondria antibody: its importance in the identification of organ-specific reactions. Clin Exp Immunol 28:517–525
Betterle C, Caretto A, Zeviani M, Pedini B, Salviati G (1985) Demonstration and characterization of anti-human mitochondria autoantibodies in idiopathic hypoparathyroidism and in other conditions. Clin Exp Immunol 62:353–360
Li Y, Song YH, Rais N, Connor E, Schatz D, Muir A, Maclaren N (1996) Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. J Clin Invest 97:910–914
Goswami R, Brown EM, Kochupillai N, Gupta N, Rani R, Kifor O, Chattopadhyay N (2004) Prevalence of calcium sensing receptor autoantibodies in patients with sporadic idiopathic hypoparathyroidism. Eur J Endocrinol 150:9–18
Soderberg A, Myhre AG, Gebre-Medhin G, Hedstrand H, Landgren E, Miettinen A, Eskelin P, Halonen M, Tuomi T, Gustafsson J, Husebye ES, Perheentupa J, Gylling M, Manns MP, Rorsman F, Kampe O, Nilsson T (2004) Prevalence and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 89:557–562
Alimohammadi M, Björklund P, Hallgren A, Pöntynen N, Szinnai G, Shikama N, Keller MP, Ekwall O, Kinkel SA, Husebye ES, Gustafsson J, Rorsman F, Peltonen L, Betterle C, Perheentupa J, Akerström G, Westin G, Scott HS, Holländer GA, Kämpe O (2008) Autoimmune polyendocrine syndrome type 1 and NALP5, a parathyroid autoantigen. N Engl J Med 358:1018–1028
Bottazzo GF, Pouplard A, Florin-Christensen A, Doniach D (1975) Autoantibodies to prolactin-secreting cells of human pituitary. Lancet 2:97–101
Caturegli P, Newschaffer C, Olivi A, Pomper MG, Burger PC, Rose NR (2005) Autoimmune hypophysitis. Endocrine Rev 26:599–614
Bellastella G, Rotondi M, Pane E, Dello Iacovo A, Pirali B, Dalla Mora L, Falorni A, Sinisi AG, Bizzarro A, Colao A, Chiovato L, De Bellis AM (2010) Predictive role of the immunostaining pattern of immunofluorescence and the titers of antipituitary antibodies at presentation for the occurrence of autoimmune hypopituitarism in patients with autoimmune polyendocrine syndromes over a five-year follow-up. J Clin Endocrinol Metab 95:3750–3757
Pouplard A, Bottazzo GF, Doniach D, Roitt IM (1976) Binding of human immunoglobulins to pituitary ACTH cells. Nature 13(261):142–144
Scherbaum WA, Bottazzo GF (1983) Autoantibodies to vasopressin cells in idiopathic diabetes insipidus: evidence for an autoimmune variant. Lancet 1:897–901
Scherbaum WA, Wass JAH, Besser GM, Bottazzo GF, Doniach D (1986) Autoimmune cranial diabetes insipidus: its association with other endocrine diseases and with histiocytosis X. Clin Endocrinol 25:411–420
Mackay IR (2010) Travels and travails of autoimmunity: a historical journey from discovery to rediscovery. Autoim Rev 9:A251–A258
This study was supported in part by a grant from the European Union Seventh Framework Programme, the Euradrenal project: Pathophysiology and Natural Course of Autoimmune Adrenal Failure in Europe. Grant Agreement No. 2008-201167 and from Grant ex 60 % of University of Padova. We like to thank Mr. Stefano Masiero for his technical assistance.
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Betterle, C., Zanchetta, R. The immunofluorescence techniques in the diagnosis of endocrine autoimmune diseases. Autoimmun Highlights 3, 67–78 (2012). https://doi.org/10.1007/s13317-012-0034-3
- Immunofluorescence techniques
- Autoimmune diseases