- Review Article
- Open Access
The immunofluorescence techniques in the diagnosis of endocrine autoimmune diseases
© Springer-Verlag 2012
- Received: 3 April 2012
- Accepted: 5 May 2012
- Published: 6 June 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.
- Immunofluorescence techniques
- Autoimmune 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.
Analytical techniques used in the autoimmunity laboratory
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).
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.
Criteria for defining a disease as autoimmune (modified from Witebsky )
Presence of autoantibodies and/or cell-mediated events in the serum
Presence of lympho-monocyte infiltration in the target organs
Possibility of identification and isolation of autoantigens
Possibility of experimentally induction of the disease in animals by immunization with autoantigens and the transfer the disease passively by serum or lymphocytes
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” .
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 .
Autoimmune endocrine diseases
Type I diabetes mellitus
Polyendocrine syndromes Type I, II, III, IV
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).
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
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 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
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
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
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 .
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.
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.
Conflict of interest
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