Introduction
Autoimmune diseases
Autoantibodies - Introduction
Autoantibodies - Determination
 
Autoantibodies
Rheumatoid Factor
Antinuclear Antibodies (ANA)
Specific Antibodies
Anti-neutrophil Cytoplasmic Antibodies
(ANCA)
Anti-phospholipid Antibodies
Anti-mitochondrial Antibodies (AMA)
Anti-endothelial Cell Antibodies (AECA)
Anti CCP antibodies
Antibodies against DNases
 
Quality Assurance
 
Reference ranges
 
Algorithm
ANA and incidence of diseases
Proposed stepwise diagnosis scheme
Positive Immunoflourescence -
Nucleoplasmic
Positive Immunoflourescence - Nucleolar
Positive Immunoflourescence -
Cytoplasmic
Type of autoimmune diseases
Conditions associated with antinuclear
antibodies (ANA)
 
Slide show
 
References
 
Collaborators
Antinuclear Antibodies (ANA) - Determination

Table 8 Assays for detection of ANA

Antinuclear antibodies

Indirect immunofluorescence assay

Immunodiffusion

Radioimmunoprecipitation

Western blotting

Enzyme-linked immunosorbent assays

Anti-DNA-antibodies

Farr binding assay

Filter binding assay

Crithidia luciliae immunofluorescence assay
(for the detection of native or double-stranded DNA)

Enzyme-linked immunosorbent assays (ELISA)

Over the past 30 years, the identification of new autoantibody systems was advanced by the initiation or adaptation of novel techniques such as double immunodiffusion to detect antibodies to saline-soluble nuclear antigens, immunoblotting, immunoprecipitation and ELISA techniques to detect a wide range of antibodies directed against naturally occurring and recombinant proteins. These techniques have been made possible by advances in cellular and molecular biology and in turn, the sera from index patients have been important reagents to identify novel intracellular macromolecules.

Serum
Specimen stable at 2-8°C for 1 week.

The immunofluorescence techniques used in autoimmune disease diagnosis can be divided into direct detection methods and indirect fluorescent antibody (IFA) assays, in the IFA methods a substrate (antigen) is immobilized on a solid phase (often a slide) and incubated with the patient's serum (containing the antibodies in question). After a washing step, specifically bound antibodies (usually IgG class; Fig B 4.5.5) are detected using labeled anti-IgG antibodies. Fluorescein isothiocyanate (FITC) is generally used as the conjugate, the characteristic apple-green fluo­rescence then being observed under the microscope.

Early indirect immunofluorescence assays (IFA) used a fluoresceinated anti-immunglobulin to detect human ANA bound to Kidney Stomach Liver (KSL) tissue of mouse or rat (29). Since these first assays, which are nowadays as first line tool for the ANA diagnostics obsolete, several significant improvements have been made in the ANA assay. The single most important change was the replacement of mouse tissue substrate with HEP-2 cells for ANA screening. (30). The HEP-2 cells show greater sensitivity, have large nuclei, lower background staining, and can be manipulated to ensure the presence of a significant number of dividing cells. The utilization of cells with mitotic figures has improved the ability of the assay to detect antibodies to antigens present in higher amounts in dividing cells (31, 32). Nevertheless, in certain cases it is necessary to use KSL tissue for the confirmation of the pattern found using HEP-2 cells (lamin, nuclear pores, AMA, ASMA).

The most common IFA variant uses HEp2 (human epithelioma cell, type 2) monolayers previously cultured in vitro directly on slides. This cell line, which was originally isolated from a laryngeal carcinoma, is distinguished by the presence of a large number of actively dividing cells (mitotic cell stages) and readily visible nuclei. This meets the prerequisites for a differential assessment of fluo­rescence patterns, since both interphase stages and all 4 mitotic phases (prophase, metaphase, anaphase and telophase) can be analyzed at the same time.
An IFA test is positive if the pattern of interest in the test serum fluoresces more intensely than in the negative control. IFA results are often expressed in dilution stages (endpoint titers) at which the fluorescence intensity just permits differentiation be­tween positive and negative sera. In adults, antibody titers from > 1:80 to > 1:160 (depending on laboratory) are regarded as a diagnostically significant elevations. A positive IFA result (Section C 1) is a key criterion for steering the diagnosis towards autoimmune disease, but it is also found in the normal popula­tion with a frequency that increases with advancing age (Table C1.1).

Nowadays IFA using HEP-2 cells is the assay widely used for ANA screening and for the detection of several of these autoantibodies. In this assay, patient sera in increasing dilutions are incubated with microscope slides to which HEP-2 cells are attached. The HEP-2 cells in each well consists of a mixture of stationary cells and dividing cells. During mitosis the chromatin consolidates, making the chromosomes visible. Interpretation should include observation of the staining intensity for both the stationary and dividing cells.
Bound antibodies then are revealed by a fluorescent-labeled anti-immunoglobulin reagent, such as an antiserum raised in goat or rabbit. The final step is inspection of the slide by fluorescence microscopy. Results are given as titer, whereby a titer of 1:40 or higher is nowadays generally accepted as cutoff point with a high diagnostic sensitivity but a low specificity (33). In children the cutoff point for detecting autoimmune diseases is reported to be at least 1:160 (34). The IFA-method requires personal ”hands-on” manipulation by the laboratorians. The interpretation depends on the experience and the knowledge of the examinator. Both of these factors can lead to varying and non-reproducible results. Interlaboratory coefficients of variation (CV) between 36% and 51% are reported even for the examination of healthy individuals. Pathological findings should be confirmed by specific ELISA (Table 9).

Table 10 Releationship between the pattern on HEP-2 cells and the specificity for ANA and cytoplasmatic antibodies

Pattern

Possible Specificity for

Centromere (nuclear staining)

anti-CENP-B

Cytoplasmatic

anti-desmin
anti-Golgi’s apparatus
anti-Jo-1
anti-keratin
anti-mitochondria
anti-smooth-muscle-cells (anti-Actin)
anti-vimentin
anti-vinculin

Homogenous or peripheral
(nuclear staining)

anti-dsDNA anti-histone
lamin nuclear pores

Nucleolar
(nuclear staining)

anti-fibrillarin
anti-Pm-Scl anti-Scl-70
NOR 90
RNA polymerase

Speckled
(nuclear staining)

anti-Jo-1
ant-Ku
anti-RNP anti-SS-A/Ro anti-SS-B/La anti-Sm

 

Centromere or discrete speckled:
Unlike the numerous, scattered uniformly-fluorescing particles in the speckled pattern, discrete larger fluorescent particles are seen in a centromere pattern. In dividing cells, the centromeres (kinetochores), which are attached to the spindle fibers, are stained. This pattern is associated with the CREST syndrome variant of progressive systemic sclerosis (35).

Homogeneous or tissue:
The entire nucleus is evenly stained. In dividing cells (mitotic figures), the chromosome is solidly stained. A homogeneous pattern may indicate autoantibody to dsDNA and/or histone or nucleosome. This pattern is generally associated with SLE, but may also be associated with other connective tissue diseases (36).

Nucleolar:
Nucleoli, which typically remain unstained in the homogeneous pattern, stain brightly. This staining pattern manifests as very large, individual round-shaped objects (35, 37, 38) within the nucleus. Dividing cells do not stain. This pattern may be associated with scleroderma and Sjögren's syndrome (39). These are different nucleolar decorations which should be differentiated from each other.

Morphology

 

Clumpy

Fibrillarin

Speckled

Scl-70

Homogenous

PM-Scl

Speckled with 2 – 4 dots in the chromosomes

RNA polymerase I (NOR-90)

 

Speckled:
A fine to coarse uniform staining of specks or grains of nuclear material exists throughout the nucleus. The mitotic figures do not stain. The mitotic cell cytoplasm may also show a fine speckled staining pattern. This pattern may be associated with autoantibodies to Sm, ribonucleoprotein (RNP), SS-B and other antigens and may be associated with SLE, scleroderma, MCTD, Sjögren's syndrome, and other connective tissue diseases (40).

Peripheral:
These are antibodies against the nuclear lamina (lamin) or nulear pores and are generally associated with autoimmune liver diseases.

HEP-2 cells may also aid in the identification of other patterns (described below), but are usually not considered the substrate of choice for verification of these autoantibodies. The following types of patterns should be reported as negative for ANA, but may be suggestive of the presence of other autoantibodies. Fluorescence may occasionally be seen in the cytoplasm: however, the clinical significance of theses cytoplasmatic antibodies should be carefully interpreted.

Spindle:
A fine, thread-like fluorescence in dividing cells suggests autoantibodies to the spindle apparatus.
Mitochondrial:
Fine-speckled, perinulclear condensed staining throughout the cytoplasm, especially in dividing cells, may be indicative of the mitochondria pattern.
Golgi apparatus:
Speckled staining restricted to one side of the cytoplasm, in the perinuclear region, may be specific for the Golgi apparatus.
Ribosomal:
Speckled staining throughout the cytoplasm, which is generally finer than that observed in mitochondrial staining, may be suggestive of antibodies to ribosomes.
Smooth muscle:
Uniform, fibrous-like staining across the cytoplasm which often appears to stain the nucleus may indicate the presence of antibodies to smooth muscle. Mitotic cells may show a speckled pattern.

 

Screening for ANA is performed for various systemic autoimmune diseases with various different techniques (43, 44). Recently, the first fully automated assay was developed to measure human antibodies to HEP-2 cells by means of an indirect non-competitive solid phase immunoassay. This assay is only a screening method and does not allow any differentiation between the patterns. The obtained results can be interpreted as negative, grey zone or positive. When comparing this EIA to IFA, the results corresponded well in healthy subjects, SLE, MCTD and RA (43-45). In the case of Sjögren's syndrome and scleroderma patients the EIA yielded a lower rate of positive results compared to IFA (45). Routine practicability and usefulness of this screening method have to be further investigated.

Table 11

Methods

Concentration

Ouchterlony

20 – 40 µg

RID

5 – 10 µg

Immunoelectrophoresis

20 – 80 µg

“Rocket” immunoelectrophoresis

2 – 5 µg

Immunonefelometry

20 – 50 µg

Hemaglutination

10 – 1000 ng

Complement fixation

100 – 1000ng

Immunofluorescence

1 –100 ng

Immunoblot

0.2 – 100 pg

ELISA

0.1 – 100 pg

Luminoescence

0.1 – 100 pg

RIA

0.1 – 100 pg

In the classic Ouchterlony assay, a serum and antigen source are placed in opposing wells in a semisolid support medium. If an antibody is present, a precipitation line forms, and the formation of a line of immunologic identity with a reference serum proves specificity. Separation of antigens by electrophoresis also can be used to determine specificity by a technique called counterimmunoelectrophoresis. The immunodiffusion assay is convenient and inexpensive, although the results are not quantitative. Extractable nuclear antigen (ENA) commonly has been used for Ouchterlony assays and is derived from thymic tissue of animals (46, 47).

Immunoprecipitation assays were a major advance in ANA testing and the key to the molecular definition of the Sm and RNP antigens. In these assays, the antigen source is the soluble extract of cells grown in vitro with radiolabelled precursors for incorporation into RNA or protein. Serum is incubated with the antigen and resulting immune complexes purified by protein-A immunosorbents. The radiolabelled RNA or proteins then are separated by gel electrophoresis, using radioautography to detect the antigens at the characteristic molecular weight positions (48, 49).
Although many nuclear antigens are complexes comprising multiple protein and RNA species, ANA usually bind a single protein species. Nevertheless, the immunoprecipitation pattern shows the whole array of molecules in the complex, whether or not they are antigenic and directly bound by antibody (50).

In this technique, an antigen preparation means the separation by gel electrophoresis following dissociation and denaturation by a detergent and a reducing agent. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Recently, Tween 20 was recommended as the preferable detergent having a more pronounced renaturating effect on proteins than other detergents and thereby improving antigen-antibody binding (51). The protein components are transferred to nitrocellulose paper and then assessed for binding to serum antibodies. Antibody detection is accomplished by radioautography, using a radiolablled anti-immunoglobulin reagent. Alternatively, the developing anti-immunoglobulin reagent may be conjugated to an enzyme - e.g., horseradish peroxidase-that can convert a substrate into an insoluble product for visual identification. A Western blot reveals only antigenic proteins and produces a much simpler pattern than a radioimmunoprecipitation assay (52, 53).
In routine diagnostics Western blots are more and more replaced by dot blots or line blots.


The antigen (Ag) is bound via a spacer (SP) to polystyrene (Ps). The immobilised antigen reacts with the patients antibodies (Ab) (5-25 \il serum in 250 jil diluent) for 20 min at 37°C. The un­bound antibodies are removed in a washing step.

2. Step:



Murine-anti-human-lgG-peroxidase (POD) is added. Incuba­tion for 15-30 min. at 37°C, followed by a washing step.

3. Step:


Addition of substrate (H2O2) and chromogen (TMB). Oxi­dized TMB produces a blue color change.

ELISA can be performed with nuclear antigens isolated from tissue or cell sources, although most preparations now are cloned recombinant products produced in bacteria. In an ELISA, an antigen is adhered to wells of a plastic microtiter plate and exposed sequentially to a serum, enzyme conjugated anti-immunoglobulin reagent, and a substrate, the breakdown product of which can be measured colorimetrically (54, 55).
Immunoblot and ELISA are less time consuming than immunodiffusion and show good interassay sensitivity without loss of specificity. The combination of immunoblot and enzyme immunoassay are reported to yield excellent assay sensitivity (100%) and specificity (99%) for detection of autoantibodies (56).