Tests for Ana Detection
Screening tests are used for detection of ANAs in patient serum. The presence of ANAs in blood represents an important criterion for the diagnosis of connective tissue diseases (CTDs). Also, identifying ANA subtypes may be useful in determining a specific CTD. Despite that there are many tests available for detection of ANAs, the ones most commonly used in daily practice are the indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA).[4,8,9]
The ELISA test is based on the interaction between antibodies existing in the blood specimen, a preprepared antigen, and special antibodies that are able to stick to the previous 2 components of the final complex, which leads to a color change in the final solution that is proportional to the quantity of ANAs. The results can be positive, equivocal, or negative, based on the determination of the optical density value of the solution.[4,8,10,11]
IFA is the standard ANA testing method because it is easy to perform, inexpensive, and has high specificity and sensitivity. The presence of ANAs in patient blood specimens is detected using a cell-line substrate from human laryngeal carcinoma (HEp-2 cells). Microscope slides are coated with a monolayer of Hep-2 cells because those cells are a sensitive substrate for ANAs. Those cells confer the following advantages: they are the most sensitive substrate used at the moment, they offer the possibility to identify many patterns, they have better specificity than other methods due to their human origin, they have large nuclei so more details can be seen, all nuclei can be seen, antigens that are produced only in cell division can be more easily identified due to high cell-division rates, and the distribution of antigens is uniform.
These slides are incubated with blood specimens, and if the antibodies are present, they will bind to the nuclear antigens. The antigen-antibody complex can be visualized as a distinct pattern in fluorescence microscopy, after adding a fluorescent-labeled antibody that is able to adhere to the complex. The amount of ANAs in the serum specimen is established by determination of antibody titers. ANA titer is determined by diluting the specimens in a buffered solution. Screening tests for ANAs use 1:40 and 1:160 dilutions. If the pattern can be seen in both dilutions, the specimen will continue to be diluted until staining can no longer be seen. The end dilution corresponds to the antibody titer. It is generally considered that a titer of 1:160 is significant for the diagnosis of CTDs. The fluorescence patterns visualized under fluorescence microscope can be correlated with certain autoimmune diseases.[2,4,8,11–14]
Hep-2 Nuclear Patterns
These staining patterns appear as a result of the adhesion of autoantibodies to certain nuclear antigens. Although ANAs are specific to nuclear antigens, in some cases, cytoplasmic patterns correlated with autoantibodies against cytoplasmic antigens can also be visualized. Sometimes, a mixed pattern can be observed. Cases in which mitosis-associated patterns may be observed are less common.
Smooth nuclear membrane pattern. This pattern is characterized by an intense linear fluorescence represented by the nuclear membrane and a less-intense homogeneous fluorescent appearance of the nucleus. In anaphase and metaphase, the chromatin plate result is negative, and greater fluorescent intensity can be seen at the contact between cells. In this pattern, the nucleoli cannot be seen. A fine membranous fluorescence surrounds newly formed nuclei in telophase. The antigens associated with this pattern are lamins A, B1, B2, and C, as well as lamin-associated proteins such as LAP 1A and LAP 2. This pattern has been observed in SLE (Image 1), SS, chronic active hepatitis, seronegative arthritis, and chronic fatigue syndrome.[14–16]
Punctate nuclear membrane pattern. In this pattern, also known as nuclear membrane pores, the nuclear membrane presents granular staining in interphase cells. In anaphase and metaphase, no chromosomal staining can be visualized. The contact surfaces of adjacent nuclear membranes emit more powerful fluorescent signals. Frequently, this aspect is associated with mitochondrial antibodies. This pattern can be observed in polymyositis (Image 2) and PBC. In patients with PBC, if testing for anti-Gp210 antibodies yields a positive result, the disease is more aggressive. The most frequent antigen is Gp210, which is a glycoprotein of 210 kDa from nuclear pore complexes. These pores play an important role in substances movement between the cytoplasm and the nucleus. Another antigen identified in this pattern is nucleoporin p62.[14,17–19]
Homogeneous nuclear pattern. Uniform staining across all nucleoplasm is seen in this pattern. The intensity of fluorescence depends on the titer of antibodies in the serum. Sometimes, the nuclear rim can be visualized as a more intense fluorescent emission of the nucleus inner edge. In mitotic cells, chromatin mass is often more intensely stained. Nucleolar staining may yield a positive or negative result and, in some cases, a peripheal nucleolar emission can be observed. Autoantibodies are directed against antigens from dsDNA, histones, or nucleosomes. Although anti-Ku antibodies are more commonly associated with a speckled pattern, in some cases, they are able to show a homogeneous aspect. This pattern is common in SLE (Image 3), RA, lupus induced by hydralazine and procainamide, juvenile chronic arthritis, and SS.[14,20–22]
Nuclear matrix/large speckled pattern. A network of large speckles with a sponge-like aspect characterizes this pattern. Chromatin is not stained in mitotic cells (anaphase, telophase, or metaphase). Nucleoli can be stained or unstained. The nuclear matrix represents a structure made from insoluble proteins resistant to RNAase, DNAase, and high salt treatment. In this pattern, the antigens are heterogeneous ribonuclear proteins. In patients with SLE (Image 4), MCTD, and RA, the autoantigens are represented by hnRNP-A1, hnRNP-A2, hnRNP-B1, and hnRNP-B2. In SS, autoantigens hnRNP-C1, hnRNP-C2, and hnRNP-I have been detected. Usually, nuclear matrix pattern is reported in MCTD but it may also occur in RA, SS, and SLE.[14,23,24]
Nuclear matrix/large speckled pattern in serum of patient with systemic lupus erythematosus.
Coarse speckled. This pattern is characterized by the presence of dense, intermediate-sized speckles and large speckles. The nucleoli are unstained. Cells in mitosis are without staining of the chromatin mass. The antigens identified in this pattern are ribonucleoproteins Sm (U2, U4, U5, U6) and U1RNP. Sm autoantibodies and U1-snRNP (small nuclear ribonucleoprotein) are markers for SLE and MCTD. U2-RNPs are presented in SS-PM overlap, MCTD, SLE, psoriasis (Image 5), and Raynaud phenomenon. In SS and SjS, researchers have identified U4-snRNP and U6-snRNP autoantibodies.[14,25,26]
Fine speckled. Also known as fine granular, this pattern is characterized by fine, tiny speckles emission of interphase nuclei, with a uniform distribution. Nucleoli are mainly unstained; however, in cases of positive anti–SS-B or anti-Ku, some nucleoli may be visualized. Chromatin is not observed in mitotic cells. Antigens identified in this pattern are nuclear proteins such as SSA, SSB, Ku, Ki, RNA polymerases II and III, and Mi-2. The diseases associated with this pattern are SLE, SjS, myositis, MCTD, and SS. As many as one-fourth of patients with dermatomyositis (Image 6) present autoantibodies against Mi-2.[14,27–29]
Dense fine speckled. In interphase, characteristic for this pattern are speckles of various size, distribution, and brightness (Image 7). Nucleoli present no staining. Another highly characteristic aspect is the presence of some denser and looser areas of speckles. Mitotic cells present intense speckled fluorescence of the chromosomes. Associated antigens may be LEDGF-p75 (lens epithelium–derived growth factor 75). The exclusive presence of LEDGF-p75 antibodies in the specimen may be useful to identify patients who have no rheumatologic disease, even if the IFA test result is positive.[14,30]
Few nuclear dots pattern. An average of 2 (1–6) discrete dots are visualized in interphase. Frequently, these dots are presented near the nucleolus and are known as coiled bodies or Cajal bodies. Those dots represent a distinct pattern from the multiple nuclear dots pattern, despite being found in similar clinical conditions. Antigens identified in this fluorescent aspect are proteins (80 kDa) designated p80-coilin. Particles are associated with fibrillarin and are presumed to have a role in snRNP maturation and transport. Clinical associations: chronic active hepatitis, PBC and, in rare occasions, collagen vascular diseases (Image 8).[14,31,32]
Multiple nuclear dots. This pattern is similar to the few-nuclear-dots pattern, but the number of dots is variable, namely, from 6 to 20 per cell (an average of 10/cell). Also, the dots are of variable size. In mitotic cells, the emission is visualized in the cell periphery. More than 30% of patients with PBC are associated with this pattern. Other pathological conditions associated are SjS (Image 9) and, in rare occasions, SLE. Autoantibodies are directed against nuclear multiprotein complexes that include promyelocytic leukemia protein (PML), Sp-100 protein, and NDP53.[33,34]
Centromere pattern. In interphase, discrete coarse speckles (40–60/cell) spread over the entire nucleus are observed. A block of closely associated speckles is found in the chromatin mass of mitotic cells. Antigens targeted by autoantibodies found in the centromere pattern include CENP-A, -B, -C, -D, -E, -G, and -H. This pattern can be present in limited cutaneous SS-CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; Image 10), PBC and, in rare instances, SjS.[35,36]
Centromere pattern in serum of patient with limited cutaneous SS-CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia).
Hep-2 Nucleolar Patterns
Nucleolar homogeneous. This pattern involves diffuse, homogeneous fluorescence of the entire nucleolus, with weak, fine granular staining of the nucleoplasm. In mitotic cells, chromatin mass is unstained; however, diffuse cytoplasmic emission is seen. PM-Scl 75 and PM-Scl 100 represent antigens associated with this pattern and are found in patients with myositis-scleroderma overlap syndrome (25%–50% of patients). Another antigen identified is Rpp25, which is a part of the Th/To complex and is associated with SS (Image 11), (4%–10% of patients with limited cutaneous form). Rpp25 have also been reported in SLE, RA, and polymyositis.[14,35,37,38]
Nucleolar clumpiness. Clustered granules are observed in the nucleoli. Nucleoplasm is not stained; however, coiled bodies may be seen as nuclear dots. In mitotic cells, chromatin mass is stained. This pattern has a high specificity (5% of patients) for SS (Image 12) and may be detected in pulmonary hypertension. It has been also associated with hepatocellular carcinoma. The identified antigen is fibrillarin, which is a component of snoRNP (small nucleolar ribonucleoprotein) that includes U3snoRNP.[39,40]
Nucleolar speckled/punctuated. Distinct grains with dense distribution are visualized in the nucleoli and are frequently associated with fine speckled nucleoplasmic emission. In metaphase cells, bright spots corresponding to nucleolar organizing regions (NORs) are usually seen within the chromatin body. Regarding antigens (RNAP I/NOR), RNAP I (RNA polymerase I) complex is located inside the nucleoli, and RNAP II and III are located in the nucleoplasm. Due to certain common polypeptides, a cross-reaction between RNAP I, II, and III may by possible. The nucleolar speckled pattern has high specificity for SS (4%–20%), which is often associated with kidney and heart involvement. Other clinical associations are SLE (Image 13), MCTD, and RA.[35,41,42]
Nucleolar speckled/punctated pattern in serum of patient with systemic lupus erythematosus.
As many as 100 autoantibodies are incriminated in the determination of overt 35 IFA patterns. In addition to nuclear and nucleolar patterns, cytoplasmic patterns have also been described. In several cases, these patterns are mixed due to similar antigenic structures.
Lab Med. 2018;49(3):e62-e73. © 2018 American Society for Clinical Pathology