This slide is an illustration of a HEp-2 or HEp-2000® cell with several nuclear and cytoplasmic structures indicated. Antibodies to DNA, histones, centromere, nuclear RNP, cytoplasmic RNP, mitochondria, ribosomes, lysosomes, golgi apparatus, as well as a variety of cytoskeletal proteins such as microfilaments, intermediate filaments, and microtubules, can be detected using the HEp-2 or HEp-2000® cell lines.(Ref10) It is important to note that RNA constitutes the bulk of the nucleic acid in the cells, being 5-10 times more abundant than DNA.(Ref11) Generally we consider 2 compartments in the cell when detecting autoantibodies in systemic rheumatic disease: 1) nucleus, and 2) cytoplasm. Staining of the nucleus includes staining inside the nucleoli.
|Fluorescent ANA Testing|
The most common method of ANA testing is indirect fluorescent assay (IFA) utilizing fluorescein isothiocyanate (FITC) as the marker on the secondary antibody.The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein. When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized with the aid of a fluorescent microscope. In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence. The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)
A similar procedure that is also widely used is called Colorzyme®.(Ref7) This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope. The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANA's are present, a stable antigen-antibody complex is formed. After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody reagent conjugated to horseradish peroxidase. When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme specific substrate. The reaction between the enzyme labeled antibody and enzyme specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker staining. The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.)
Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA
|For the ANA test to be positive what must be present in the nucleus of the interphase cell?||View Page|
This is an example of a centromere ANA pattern.This pattern is characterized by staining of discrete speckles in the nucleus of the interphase cells (a). This is staining of the centromere. There are usually 46 speckles, one for each set of chromosomes. Notice the discrete speckling is also seen in the chromosomal area of the metaphase mitotics (b). The presence of anti-centromere antibodies can be determined by the staining pattern alone. No specific follow-up testing is necessary. Anti-centromere antibodies are most often seen in patients with the limited form of systemic sclerosis called the CREST syndrome. CREST is an acronym for the clinical features:Calcinosis cutisRaynaud's phenomenonEsophageal dysfunctionSclerodactylyTelangiectasiaAnti-centromere antibodies have been detected years prior to the onset of symptoms. Finding these antibodies in patients with Raynaud's phenomenon or other risk factors is an important indicator for the potential development of systemic sclerosis.This pattern is reported as ANA positive Centromere; titering is necessary.Centromere:Interphase cells Usually 46 discrete speckled Evenly distributed in the nucleusMetaphase mitotic cells Discrete speckles align in the chromosomal region of the mitotics No staining outside of the chromosomal area
|The image on the right represents the result of a fluorescent antinuclear antibody (ANA) test. What pattern should be reported?Note: (a) points to the nuclei of several interphase cells, the primary consideration for discerning the ANA pattern and (b) indicates a metaphase mitotic cell. Observing the chromosomal area and cytoplasm of the metaphase cell may assist in identification of the ANA pattern.||View Page|
|NSp I or Multiple Nuclear Dots (MND)|
This is an example of a Multiple Nuclear Dots (MND) ANA pattern.This pattern is characterized by several (15-30) discrete speckles, or dots, in the nucleus of the interphase cells (a). There is no staining in the nucleoli and the chromosome area of the metaphase mitotics is negative (b). This pattern is distinguished from centromere by having fewer speckles and negative mitotics.Follow-up testing for antibodies to Sp-100 is available. These antibodies are seen inpatients with primary biliary cirrhosis.This pattern is reported as ANA positive; unusual or atypicial speckled; titering is optional.NSp I or MND:Interphase cells 15-30 discrete speckles Evenly distributed in the nucleusMetaphase mitotic cells No staining in chromosomal region Region outside of the chromosome area will stain with variable intensity
|NSp II or CENP-F|
This is an example of the ANA pattern seen with antibodies to CENP-F or NSp II.This pattern is characterized by the absence of staining in the interphase cells (a). In late interphase speckling begins to appear (b). The metaphase mitotics demonstrate staining similar to centromere pattern but the speckling is often in two rows (c). No follow-up testing is available. These antibodies are seen inpatients with various cancers.This pattern is reported as ANA positive, NSp II or CENP-F; titering is optional.CENP-F or NSp II:Interphase cells Speckled staining appears in late interphaseProphase cells Look like CentromereMetaphase mitotic cells Look somewhat like Centromere
This is an example of a Midbody ANA pattern.In this pattern there is no staining in the early interphase cells (a). By late interphase fine speckling is present (b). In metaphase the speckling is located in chromosomal area and is very similar to centromere staining (c). As the cells progress into anaphase and telophase staining is seen in the intercellular bridge (d).No follow-up testing is necessary.This pattern is reported as ANA positive, Midbody; titering is optional.Midbody:Interphase cells• Speckled staining appears in late interphaseProphase cells• Discrete speckling resembles CentromereMetaphase mitotic cells• Discrete speckling in chromosomal region resembles CentromereAnaphase, Telophase and Cytokenisis cells• As cells separate it becomes apparent the staining is in the cleavage furrow between the dividing cells
In addition to the usual speckled patterns seen previously, there is a group of unusual (or atypical) speckled patterns. These patterns can demonstrate some characteristics of the usual speckled patterns but also have unique features, do not normally require follow-up testing, and can be seen in various diseases.Some of these patterns have also been referred to as the "pseudo centromere" patterns because some have features that can resemble centromere staining . By considering the number of speckles and location of the the speckles it is easy to differentiate these patterns from centromere.
|When identifying the Midbody pattern only cells in interphase and metaphase are useful.||View Page|
|Homogeneous and Centromere|
This is an example of a mix of homogeneous and centromere ANA patterns.In this sample notice the smooth homogeneous pattern of the interphase cells and smooth staining of the chromosomal area of the mitotics (a). Also notice the discrete speckling of the centromere pattern present in the nucleus of the interphase cells as well as in the chromosomal area of the metaphase mitotic cells (b). In cases of mixed homogeneous and centromere ANAs, follow-up testing for anti-dsDNA is necessary.When mixed patterns are titered, the endpoint for each pattern is reported.This pattern is reported as ANA positive, Homogeneous and Centromere; titering is necessary.Homogeneous and Centromere:Interphase cells Smooth staining of entire nucleus Usually 46 discrete speckledMetaphase mitotic cells Smooth staining of the chromosomal region Discrete speckles align in the chromosomal region of the mitotics Region outside of the chromosome area will stain with variable intensity
|Other Mixed Patterns|
Mixed patterns are not limited just to the ones viewed previously. Virtually any combination is possible.Other Combinations: Centromere and SS-A/Ro (HEp-2000®) Homogeneous, speckled, and SS-A/Ro (HEp-2000®) Homogeneous and nucleolar Both patterns may be caused by antibodies to one antigen Remember: any combination is possible!
|What is the most common combination of ANA patterns?||View Page|
Anti-mitotic spindles antibodies refers to several different patterns which, in a broad sense, includes CENP-F, Centromere and Midbody. However, in the case of the cytoplasmic patterns it only applies to the staining of the spindle fibers. Mitotic Spindle Antibodies:Several antigens NuMA, HsEg5, othersCytoplasmic pattern Stain spindle fibers in metaphase mitoticReport as Suspect mitotic spindleClinical significance Various autoimmune disease Mycoplasma pneumoniae infections