For a long time I wanted to write about antinuclear factor, but something always stopped me. Why? Well, because writing about this is trying to grasp the immensity, and brevity is clearly not the sister of my talent)))
Determination of antinuclear factor (ANF) is the main method for detecting antinuclear antibodies, which allows identifying most types of antinuclear antibodies. The result of determining ANF is the very fact of the presence of autoantibodies, the final titer of serum dilution and the type of luminescence of the cell nucleus.
ANF is not just one antibody!
The method for detecting ANF was developed by Tan (1982), who used the human continuous epithelioid cell line HEp-2 (you may have seen this name on the analysis form) as a substrate. This cell line is derived from a human laryngeal tumor (adenocarcinoma). The comparative unpretentiousness of this line, large nuclei and the presence of all human antigens have made the method of indirect immunofluorescence (indirect immunofluorescence) using the continuous cell line HEp-2 the main method for detecting ANF.
Other cell lines can also be used as substrates (see carousel), however, due to their good morphology and ease of cultivation, it is the Hep-2 line that has become a generally accepted substrate for indirect immunofluorescence. In the English-language literature, this method is sometimes referred to as FANA (Fluorescent AntiNuclear Antibody detection).
Sometimes the term “antinuclear antibodies” is used by immunological laboratories to refer to tests based on enzyme immunoassay and immunochemical analysis methods, as well as the immunofluorescence method.
Antinuclear factor (ANF) is an antibody detected by immunofluorescence.
It is necessary to identify ANF of the IgG class, since the detection of ANF represented by other classes of immunoglobulins has no independent significance.
The ANF titer is defined as the last dilution of the patient's serum, which has clear fluorescence (glow) of cell nuclei. When using the HEp-2 cell line, it is recommended to use an initial titer of 1:80; in this case, the normal ANF titers are less than 1:160. To determine the final titer, titration in steps of x2 is usually used (1:160-1:320 - 1:640 - 1:1280 - 1:2560 - 1:5120, etc.).
During an exacerbation of the disease, ANF titers of more than 1:640 are usually observed, and during remission, titers can decrease to 1:160-1:320.
Scoring of detection results (“in crosses” +++) allows laboratories to save reagents and reduce labor costs for performing research. In this case, determining the end titer is not possible❌
It is advisable to determine the final titer in all positive patients, which makes it possible to clarify the presence of autoantibodies in the serum, which are more closely related to the activity of the process.
TYPES OF CORE GLOW
The type of core glow is one of key characteristics when detecting ANF. Each type of antinuclear antibody (ANA) has specific targets in the cell and reflects the interaction of the patient's serum with structures within the cell. The type of glow depends on the presence of specific autoantibodies in the blood, on the basis of which a preliminary conclusion can be made regarding the types of antibodies that are present in a given serum. The type of luminescence of the cell nucleus significantly increases the information content of ANF detection.
The use of HEp-2 cells makes it possible to characterize more than 20 various options staining the nucleus, however, for clinical practice It is enough to highlight the main ones. Each type of glow has very characteristic features, which allow you to distinguish one option from another.
At homogeneous type of glow autoantibodies react with those antigens that are distributed diffusely in the nucleus, i.e. are part of chromatin.
Due to what? Antibodies against nucleosomes, DNA, histones.
When? In patients with SLE and drug-induced lupus, as well as in patients with scleroderma.
Often, the detection of a high titer of ANF with a homogeneous type of luminescence indicates a diagnosis of SLE.
Peripheral type of glow can be isolated separately, although it is a type of homogeneous. The peripheral type of glow is found in patients with antibodies to double-stranded DNA and is detected mainly in SLE and liver diseases.
Granular type (mottled, mesh) is the most common and, at the same time, the most nonspecific.
Due to what? Sm, U1-RNP, SS-A, SS-B antigens and PCNA.
When? SLE, Sjogren's disease/syndrome, scleroderma, dermatomyositis/polymyositis, RA and a number of other diseases. Low titers (1:160-1:320) of ANF with a granular type of luminescence can be present in the blood serum of clinically healthy individuals WITHOUT signs of systemic disease.
Detection of very high ANP titers (1:2560-1:10000) with a coarse-granular type of nuclear luminescence usually indicates a diagnosis and requires further examination to identify the RNP antigen.
Nucleolar antigens can act as targets of ANA, leading to the detection nucleolar type of fluorescence.
Due to what? RNA polymerase 1, NOR, U3RNP, PM/Scl.
When? Scleroderma and its varieties.
Centromeric type noted when antibodies appear to the centromeres of chromosomes, and is found only in dividing cells. Its presence is characteristic of CREST syndrome (a variant of scleroderma).
Cytoplasmic type of luminescence.
Due to what? Antibodies to tRNA synthetases, in particular Jo-1, which are observed in polymyositis. In addition, it is detected in patients with ANA directed against other components of the cell cytoplasm: in autoimmune hepatitis, in primary biliary cirrhosis.
A number of other types of glow can be characterized. Antibodies to Scl-70 give fine granular staining of the nucleus and nucleoli, anti-p80 - luminous spots in the nucleus that are absent in mitotic cells.
A combination of several types of luminescence can often occur, for example, fine granular and nucleolar, which is typical for antibodies to Scl-70.
Sometimes in low dilutions one type of fluorescence predominates, for example granular, and with further dilutions homogeneous or centromeric types of fluorescence are revealed, which indicates the presence of various types ANA.
General information about the study
Determination of antinuclear factor (ANF) is the “gold standard” for detection of antinuclear antibodies (ANA) and diagnosis autoimmune diseases.
Pathogenesis systemic diseases connective tissue(CTD) is closely associated with disorders of the immune system and increased production antibodies to the structures of one's own cells. Autoantibodies to the components of the cell nucleus - antinuclear antibodies - interact with nucleic acids and nuclear proteins, cytoplasmic antigens, which is manifested by inflammatory changes in tissues and organs, pain in joints and muscles, severe fatigue, weight loss, and skin changes. ANAs are found in many autoimmune diseases, but are most common in systemic lupus erythematosus (SLE). ANAs occur in more than 90% of patients with systemic connective tissue diseases; currently about 200 varieties have been described, which are united by a single name - antinuclear factor.
When determining ANF by indirect fluorescence, the continuous line of epithelial cells of human laryngeal adenocarcinoma HEp-2 is most often used. HEp-2 cells are a very convenient substrate for laboratory research, since they have large nuclei and grow in one layer on glass. ANAs are detected by binding to intracellular antigens of HEp-2 cells.
During the study, HEp-2 epithelial cells are grown on glass slides, fixed and incubated with diluted patient serum. After removing excess serum, the cells are incubated with fluorescein-labeled antibodies, then washed again and examined under a fluorescence microscope. In this case, the antibody titer and the type of glow are determined. A titer of more than 1:160 is considered diagnostically significant. During exacerbation of rheumatic diseases, it exceeds 1:640, and during remission it decreases to 1:160-1:320. The more antibodies, the higher the titer. Based on the type of glow, one can identify the targets of antinuclear antibodies, which is important clinical significance and determines the tactics for further examination of the patient. The main ones are peripheral, granular (small/large), nucleolar, centromeric and cytoplasmic types of nuclear staining. Each type of glow has very characteristic features that make it possible to distinguish one option from another.
Homogeneous (diffuse) staining is associated with the presence of antibodies to double-stranded DNA, histones and is characteristic of SLE and drug-induced lupus.
Peripheral luminescence is caused by the peripheral distribution of chromatin in the nucleus, is associated with antibodies to DNA and is specific for SLE. It is important to differentiate this type of luminescence from staining of the nuclear membrane, which occurs in autoimmune liver diseases.
Granular (mottled, reticular) staining is the most common and least specific; it is detected in many autoimmune diseases. Autoantigens in in this case nucleoprotein complexes appear in the nucleus (Sm, U1-RNP, SS-A, SS-B antigens and PCNA).
Very high titers of ANF with a coarse-granular type of luminescence often indicate a mixed connective tissue disease.
Nuclear (nucleolar) staining is caused by antibody formation to the components of the nucleolus (RNA polymerase-1, NOR, U 3 RNP, PM/Scl), detected in scleroderma, Sjögren's disease. ANA sometimes increases with endocrine diseases(diabetes mellitus type 1, thyroiditis, thyrotoxicosis, polyendocrine syndrome), skin diseases(psoriasis, pemphigus), during pregnancy, after organ and tissue transplantation, in patients on hemodialysis.
Centromere luminescence appears in the presence of antibodies to the centromeres of chromosomes and is characteristic of a form of scleroderma - CREST syndrome.
The cytoplasmic type of luminescence is associated with antibodies to tRNA synthetase, in particular to Jo-1, which is characteristic of dermatomyositis and polymyositis. This type of staining is also determined in the presence of antibodies to other components of the cytoplasm in autoimmune hepatitis and primary biliary cirrhosis.
Simultaneous detection of different types of glow indicates the presence different types antibodies.
In healthy individuals, fine-grained luminescence may be detected at low, moderate, or high titers of ANA, but normally coarse-grained or homogeneous types of luminescence should not be detected.
Depending on the results of assessing the type of glow, further treatment tactics for the patient are developed and additional studies are prescribed to clarify the spectrum of ANA.
What is the research used for?
When is the study scheduled?
There are a number of diseases in which the defense system mistakes the cells of one’s own body for foreign ones, after which it begins to mistakenly attack them. Majority autoimmune pathologies have chronic form and pose a serious health risk. In order to identify these ailments on the most early stage their development, doctors prescribe an ANF analysis. This abbreviation stands for “antinuclear factor.” Some reports label the study as AHA. The meaning stands for “antinuclear antibody test.” ANF is clinical significant indicator, helping the doctor draw up the maximum effective scheme treatment and prevent the development of complications.
The biological material for research is blood. When any pathogenic microorganism enters the body immune system begins to produce specific antibodies, the task of which is to destroy foreign antigens. The essence of the method is to detect and quantification of these substances in liquid connective tissue.
Doctors say that the ANF blood test is a laboratory test that has high degree information content. With its help, it is possible to identify any autoimmune pathologies even at an early stage of their development.
Specific antibodies are also often detected in people suffering from severe forms of hepatitis, oncology and some infectious diseases. These substances can also be detected in healthy people. A situation like this requires integrated approach to identify the cause.
ANF analysis sometimes involves assessing the quantitative content of immunoglobulins. Their presence may indicate the development of collagenosis and rheumatic diseases.
It is important to know that an ANF blood test is a study that is prescribed to confirm or exclude the presence of autoimmune pathologies, as well as to evaluate the effectiveness of the treatment.
A doctor can suspect an illness by the following symptoms:
In addition, ANF analysis is prescribed if the presence of rheumatic disease. The study is carried out after receiving the results laboratory diagnostics, in which ESR, CEC and C-reactive protein are increased.
ANF blood test allows you to identify pathologies of an autoimmune nature. The study is informative regarding the following diseases:
This is an incomplete list of diseases. It is important that the analysis of ANF shows the development of autoimmune pathologies at the earliest stage of their course. This allows the doctor to decide on treatment tactics and subsequently evaluate its effectiveness.
The collection of biomaterial is carried out in the morning. It is necessary to donate blood on an empty stomach. The last meal should be taken at least 4 hours before. At the same time, drinking water is acceptable at any time and in any quantity. Alcohol consumption is prohibited.
Rest is indicated 1 day before the study. Physical and psycho-emotional stress often lead to false results. Smoking is prohibited half an hour before donating blood.
It is necessary to inform the doctor about the medications you are taking when prescribing an ANF test. This is due to the fact that active ingredients Some drugs provoke the production of antibodies and can also cause drug-induced lupus. False Negatives are often a consequence of taking glucocorticosteroids.
If the patient has been prescribed physical therapy or instrumental examinations, they must be carried out only after donating blood.
It is carried out in the morning. The biological material is venous blood. Its collection is carried out according to the standard algorithm. As a rule, blood is taken from a vein located on the elbow.
Once the liquid connective tissue is obtained, serum is extracted from it. This is what is needed to carry out the analysis.
Currently, it is possible to detect antibodies in biomaterial in several ways:
The ANF analysis should be deciphered by the attending physician. Subject to availability positive results additional tests are prescribed. Final diagnosis is not assigned based on the conclusion of one analysis.
Best result is one in which antinuclear antibodies are absent. However, it is worth remembering that they can also be found in completely healthy people. In this case, a repeat study is indicated.
The standard for ANF analysis is titer no more than 1:160. At the same time this indicator relevant for both adults and children.
When deciphering the ANF blood test, it is important to consider the following information:
ANF analysis performed using HEp-2 cells is considered normal if the titer is not higher than 1:160. A result of more than 1:640 indicates an exacerbation of rheumatic pathologies. During the period of remission of the disease, the titer decreases to 1:320. In this case, only a doctor will be able to identify the fact that such a person is talking about low rate, based on medical history and individual health characteristics of the patient.
By binding to antigens, they form an immune complex. The latter, in turn, is a trigger for the development inflammatory process in the walls of blood vessels. As a result, a person develops the first alarming symptoms systemic diseases. The analysis shows high titers.
In this case, pathology can be identified by determining the type of glow. Interpretation of results:
Reducing the level of antinuclear antibodies is clinically important exclusively in predicting and monitoring existing and previously identified systemic diseases.
The ANF indicator directly depends on the intensity pathological process. In this regard, its decrease is a favorable sign, indicating that the treatment was successful and the disease has entered the remission stage.
Each autoimmune pathology requires a specific therapeutic approach. The purpose of a blood test for ANF is to identify antibodies in liquid connective tissue and evaluate the nature of their interaction with specific antigens. Based on the diagnostic results, the doctor can make a preliminary diagnosis. Additional research is needed to confirm this. And only after this the doctor draws up a treatment regimen. Choice medications directly depends on what pathology is detected in a person.
You can take an ANF test in an independent laboratory, private clinic or a government medical facility. It is important to know that not all budget clinics provide such a service. It is necessary to check with the reception regarding its availability.
Research is paid even in government medical institutions. The cost of the analysis directly depends on the clinic’s pricing policy, which consists of many factors. The minimum price is 1000 rubles, the maximum does not exceed 1700 rubles. In addition, you must additionally pay for blood sampling. The cost of this service, as a rule, does not exceed 200 rubles.
The abbreviation ANF stands for antinuclear factor. Normally it should not be present in the blood healthy person or its concentration should be less than 1:160. An analysis for ANF is prescribed in order to identify autoimmune pathologies in a patient at the earliest stage of their development.
The essence of the method: when provoking agents penetrate the body, the defense system begins to produce antibodies. Their job is to attack antigens and destroy them. To identify this reaction, a sample is taken from the patient. venous blood followed by separation of the whey. Specific antigens are added to the latter and further reactions are assessed.
Systemic rheumatic diseases
Antinuclear factor (ANF) and type of luminescence
Antinuclear factor (ANF) determination is the main method for detecting antinuclear antibodies, which allows the detection of most types of antinuclear antibodies, including autoantibodies to nucleic acids (dsDNA, ssDNA, RNA), conformational and insoluble antigens. The result of determining ANF is the presence of autoantibodies in the diagnostic titer, the final titer of serum dilution, reflecting the affinity and concentration of autoantibodies, as well as the type of luminescence of the cell nucleus (test 01.02.15.005). The characteristics of a particular technique depend primarily on the choice of substrate, which can vary from cryosections (rat liver or kidney) to cultured proliferating cell lines (Hep2, Kb, HeLa), as well as the method of tissue fixation, which can affect the preservation of a number of nuclear antigens ANA.
History of the development of tests for the detection of antinuclear antibodies:
Beginning of the twentieth century - “hyaline bodies” in the cytoplasm of leukocytes
1948 Hargraves, Richmond, Morton - method for in vitro detection of LE cells during long-term incubation of leukemia from SLE patients
1951 Lee, Michael, Vural - discovered a serum factor that stimulates phagocytosis of lymphocyte nuclei by peripheral blood leukocytes
1957 Holoborow, Weir, Johnson used the indirect immunofluorescence method developed by Coons in 1950 to detect ANF in cryosections of human liver
1961 Beck - used tissue from laboratory animals
1982 Tan - used the human continuous cell line HEp-2, this test became the gold standard for detecting ANA
1958 Jones - discovered in the blood of patients with Sjögren's syndrome a factor capable of precipitating extracts of cell nuclei, which served as an impetus for the description of most varieties of antinuclear antibodies
Indirect immunofluorescence on the continuous cell line Hep2
A method for detecting ANF was developed by Tan (1982), who used the human continuous epithelioid cell line HEp-2 (ATCC ref. no. CCL-23) as a substrate. This cell line, derived from human laryngeal adenocarcinoma, is a large polyploid non-keratinizing squamous epithelial cell that forms a monolayer on plastic and glass.
Figure: cells of the HEp-2 cell line, stained with hematological dye.
The comparative unpretentiousness of this line, large nuclei and the presence of all human antigens made the method of indirect immunofluorescence using the continuous cell line HEp-2 the main method for detecting ANF. In the English-language literature there is no single name for this method; sometimes this method is referred to as FANA (Fluorescent AntiNuclear Antibody detection) for brevity. The lack of unity of nomenclature has created confusion in the names of laboratory tests. Sometimes the term “antinuclear antibodies” is used by immunological laboratories to refer to tests based on enzyme immunoassay and immunochemical analysis methods, as well as the immunofluorescence method. In the Russian-language literature, we consider it necessary to preserve the domestic name for this indicator - “antinuclear factor” (ANF) in order to distinguish the immunofluorescent method from other tests for detecting ANA.
It is necessary to identify ANF of the IgG class, since the detection of ANF represented by other classes of immunoglobulins has no independent diagnostic value. Standard dilutions of the patient's serum are incubated with tissue substrate, allowing antinuclear antibodies from the patient's serum to bind to appropriate nuclear targets. The autoantibody binding sites are then detected using fluorescein-labeled anti-human antiserum.
Although other human cell lines can be used as substrates, due to their good morphology and ease of cultivation, Hep-2 has become a generally accepted substrate for indirect immunofluorescence. Its use improves the sensitivity of the test due to bright fluorescence even with significant dilutions of the patient’s serum, and the large, euchromatin-rich nucleus allows an accurate description of the type of fluorescence. In addition, the use of the Hep-2 cell line facilitates the detection of Ro/SS-A antibodies, as well as antibodies to nucleolar antigens, which are poor when using cryosections of laboratory animal tissue. Other benefits of HEp-2 include high frequency cell division, which makes it possible to detect antibodies to antigens expressed only during cell division, and the absence of a tissue cellular matrix, which makes it difficult to visualize specific luminescence when compared with histological sections.
Antinuclear factor titers
The ANF titer is defined as the last dilution of the patient's serum, which has clear fluorescence of cell nuclei. When using laboratory animal tissues, the diagnostic ANF titer is 1:8-1:10, while when using the HEp-2 cell line, it is recommended to use an initial titer of 1:80. In this case, the normal ANF titers are less than 1:160.
When using titers less than 1:160, the frequency of weakly positive results in clinically healthy individuals will be no more than 5%, and at the same time will not allow significant ANA titers to be missed in patients diffuse diseases connective tissue.
To determine the final titer, titration in x2 steps is usually used (1:160-1:320 - 1:640 - 1:1280 - 1:2560 - 1:5120, etc. Rougher titration can also be used. Against the background of exacerbation of rheumatic diseases ANF titers of more than 1:640 are usually observed, and during remission the titers decrease to 1:160-1:320.
There are recommendations for scoring the results of identifying ANF, indicating the content “in the crosses”. This allows laboratories to save reagents and reduce labor costs for performing research. In this case, determination of the final titer is not possible. It must be taken into account that the final titer is more important than the amount of autoantibodies bound to the substrate, since it is directly related to the affinity of their interaction with the antigen. It is advisable to determine the final titer in all positive patients, which makes it possible to clarify the presence in the serum of high-affinity autoantibodies, which are more closely related to the activity of the process.
Type of luminescence of the antinuclear factor nucleus
Already in the first experience clinical application Indirect immunofluorescence to detect ANF using cryosections of laboratory tissues, it was noted that the serum of patients with autoimmune diseases “stained” the cell nuclei differently, which led to selective luminescence of some nuclear structures. Clarification of this phenomenon led to the description of the so-called “types of luminescence” of the nucleus in the immunofluorescence test. This is due to the fact that each type of ANA has specific cellular targets, as a result of which the type of luminescence of the nucleus, nucleolus and cytoplasm of the cell reflects the interaction of ANA in the patient’s serum with antigen-containing structures inside the cell. The type of luminescence depends on the presence of specific autoantibodies in the patient’s blood serum, on the basis of which a preliminary conclusion can be made regarding the types of ANA that are present in this serum. However, the use of histological sections of tissue from laboratory animals does not allow one to reliably establish the type of luminescence. At the same time, the use of continuous cell lines, such as Hep-2, makes it possible to describe a significant number of variants of nuclear and cytoplasmic staining. There are three main groups of antigens, the presence of antibodies to which determines various types glow:
The type of luminescence of the cell nucleus significantly increases the information content of identifying ANF, and therefore should be routinely determined when determining this indicator. The use of HEp-2 cells makes it possible to characterize more than 20 different variants of nuclear staining, which depend on the spectrum of ANAs present in the serum under study. However, for a practical laboratory it is enough to distinguish between 6 main options for the binding of autoantibodies to antigen-containing cell structures.
There are homogeneous, peripheral, granular (small/large), nucleolar, centromeric and cytoplasmic types of nuclear luminescence. Each type of luminescence has very characteristic features that make it possible to distinguish one option from another, as well as a set of antigens with which autoantibodies react in the sera of patients. Description of glow types is valuable clinical information in itself, in addition, the type of glow may indicate the need for certain laboratory tests in the future.
At homogeneous type glow autoantibodies react with those antigens that are distributed diffusely in the nucleus, i.e. are part of chromatin. Typically, when a homogeneous type of luminescence is detected, condensed chromosomes are brightly colored in dividing cells. The main structural units of chromatin are nucleosomes - complexes of DNA and histones. Thus, the homogeneous type of luminescence suggests the presence of antibodies against nucleosomes, dsDNA and antibodies to histones. It occurs in patients with SLE and drug-induced lupus, as well as in patients with scleroderma. Typically, the detection of a high titer of ANF with a homogeneous type of luminescence indicates a diagnosis of SLE.
Peripheral type glow is often isolated separately, although it is a type of homogeneous glow. Its detection is an artifact of cell fixation, which leads to redistribution of chromatin in the nucleus to the periphery. It is important to distinguish the peripheral type of luminescence from the staining of the nuclear membrane, which is observed in autoimmune liver diseases. The peripheral type of luminescence is found in patients with antibodies to double-stranded DNA and is detected mainly in patients with SLE.
Granular type is the most common and, at the same time, the most nonspecific. Sometimes this type of glow in Russian literature is called “speckled” or “mesh”. The name “granular” more accurately reflects this phenomenon, since in this case the autoantibodies react with granules in the nucleus, which are supramolecular nucleoprotein complexes. Such complexes of proteins and nucleic acids carry out in the nucleus a number of functions necessary for the normal functioning of the cell. Such complexes, in particular, include spliceosomes, which carry out post-transcriptional rearrangement of mRNA necessary for protein synthesis on ribosomes. The composition contains many different nucleoproteins, which determines the diversity of antigenic targets when detecting a granular type of luminescence. The main autoantigens, antibodies to which lead to visualization of the granular type of luminescence, include Sm, U1-RNP, SS-A, SS-B antigens and PCNA. During the process of cell division, cells lose most of the formed nucleoprotein complexes; therefore, mitotic figures in a cell line with a granular type of luminescence are not stained. The granular type of nuclear luminescence is observed in patients with SLE, SS, SS, DM/PM, RA and a number of other autoimmune diseases. Low titers of granular type ANF predominate in the blood sera of clinically healthy individuals with ANF without signs of systemic disease.
Detection of very high ANF titers (1:2560-1:10000) with coarse granular type nuclear luminescence usually indicates a diagnosis of mixed connective tissue disease and requires further examination to identify the RNP antigen, which is the main serological marker of this disease.
Nucleolar antigens can act as targets of ANA, leading to the detection nucleolar type fluorescence. Identification of the nucleolar type of glow is characteristic of scleroderma and its varieties. Nucleolar type fluorescence determined in patients with antibodies to components of the nucleolus, such as RNA polymerase 1, NOR, U 3 RNP, PM/Scl.
Centromeric type fluorescence is noted when antibodies appear to the centromeres of chromosomes, and is found only in dividing cells. Its presence is characteristic of the CREST variant of scleroderma.
Cytoplasmic type glow indicates antibodies to tRNA synthetases, in particular Jo-1, which are observed in polymyositis. In addition, it is detected in patients with ANA directed against other components of the cell cytoplasm: antibodies to actin in autoimmune hepatitis, antibodies to mitochondria in primary biliary cirrhosis.
A number of other types of luminescence can be characterized, and these types of staining of the cell nucleus may be characteristic of a particular type of ANA. Thus, antibodies to Scl-70 in an immunofluorescent test on HEp-20 cells give fine-grained staining of the nucleus and nucleoli, anti-p80 - luminous dots in the nucleus, which are absent in mitotic cells. However, it is necessary to point out the rarity and diversity of such phenomena, which makes their identification during routine research unnecessary.
A combination of several types of luminescence can often occur, for example, fine granular and nucleolar, which is typical for antibodies to Scl-70. Moreover, often in low dilutions one type of fluorescence predominates, for example granular, and with further dilutions homogeneous or centromeric types of fluorescence are revealed, which indicates the presence of various types of ANA in the patient’s serum. Although the type of glow provides the doctor with certain data in favor of a particular diagnosis, it is necessary to take into account its relatively low specificity and the variety of phenomena encountered, which requires further laboratory examination.
The detection of ANA is the basis for the use of other laboratory tests that clarify the spectrum of ANA in the blood of patients. Given the extreme information content of this test, further testing and analysis of the results of a subsequent examination should be interpreted depending on the results of assessing the type of luminescence and ANF titer.
Although the absence of significant ANA titers almost always excludes the diagnosis of active systemic rheumatic disease, when using the HEp-2 cell line, in 2-4% of patients meeting the criteria for the diagnosis of SLE, ANA are not detected or titers are low. These patients are sometimes classified as " ANF-negative SLE“To clarify the diagnosis in such patients, further examination is required, primarily the identification of antibodies to SS-A antigens. These antigens are highly soluble and can be lost from cell nuclei. To reduce this category of patients, comprehensive testing is used, including the determination of antibodies to extracted nuclear antigens, the so-called “ screening for connective tissue diseases», test 000723. A negative result of such an examination allows, with a very high probability, to exclude the diagnosis of SLE and other systemic rheumatic diseases.
Systemic rheumatic diseases
Antinuclear factor (ANF) and type of luminescence
Antinuclear factor (ANF) determination is the main method for detecting antinuclear antibodies, which allows the detection of most types of antinuclear antibodies, including autoantibodies to nucleic acids (dsDNA, ssDNA, RNA), conformational and insoluble antigens. The result of determining ANF is the presence of autoantibodies in the diagnostic titer, the final titer of serum dilution, reflecting the affinity and concentration of autoantibodies, as well as the type of luminescence of the cell nucleus (test 01.02.15.005). The characteristics of a particular technique depend primarily on the choice of substrate, which can vary from cryosections (rat liver or kidney) to cultured proliferating cell lines (Hep2, Kb, HeLa), as well as the method of tissue fixation, which can affect the preservation of a number of nuclear antigens ANA.
History of the development of tests for the detection of antinuclear antibodies:
Beginning of the twentieth century - “hyaline bodies” in the cytoplasm of leukocytes
1948 Hargraves, Richmond, Morton - method for in vitro detection of LE cells during long-term incubation of leukemia from SLE patients
1951 Lee, Michael, Vural - discovered a serum factor that stimulates phagocytosis of lymphocyte nuclei by peripheral blood leukocytes
1957 Holoborow, Weir, Johnson used the indirect immunofluorescence method developed by Coons in 1950 to detect ANF in cryosections of human liver
1961 Beck - used tissue from laboratory animals
1982 Tan - used the human continuous cell line HEp-2, this test became the gold standard for detecting ANA
1958 Jones - discovered in the blood of patients with Sjögren's syndrome a factor capable of precipitating extracts of cell nuclei, which served as an impetus for the description of most varieties of antinuclear antibodies
Indirect immunofluorescence on the continuous cell line Hep2
A method for detecting ANF was developed by Tan (1982), who used the human continuous epithelioid cell line HEp-2 (ATCC ref. no. CCL-23) as a substrate. This cell line, derived from human laryngeal adenocarcinoma, is a large polyploid non-keratinizing squamous epithelial cell that forms a monolayer on plastic and glass.
Figure: cells of the HEp-2 cell line, stained with hematological dye.
The comparative unpretentiousness of this line, large nuclei and the presence of all human antigens made the method of indirect immunofluorescence using the continuous cell line HEp-2 the main method for detecting ANF. In the English-language literature there is no single name for this method; sometimes this method is referred to as FANA (Fluorescent AntiNuclear Antibody detection) for brevity. The lack of unity of nomenclature has created confusion in the names of laboratory tests. Sometimes the term “antinuclear antibodies” is used by immunological laboratories to refer to tests based on enzyme immunoassay and immunochemical analysis methods, as well as the immunofluorescence method. In the Russian-language literature, we consider it necessary to preserve the domestic name for this indicator - “antinuclear factor” (ANF) in order to distinguish the immunofluorescent method from other tests for detecting ANA.
It is necessary to identify ANF of the IgG class, since the detection of ANF represented by other classes of immunoglobulins does not have independent diagnostic value. Standard dilutions of the patient's serum are incubated with tissue substrate, allowing antinuclear antibodies from the patient's serum to bind to appropriate nuclear targets. The autoantibody binding sites are then detected using fluorescein-labeled anti-human antiserum.
Although other human cell lines can be used as substrates, due to their good morphology and ease of cultivation, Hep-2 has become a generally accepted substrate for indirect immunofluorescence. Its use improves the sensitivity of the test due to bright fluorescence even with significant dilutions of the patient’s serum, and the large, euchromatin-rich nucleus allows an accurate description of the type of fluorescence. In addition, the use of the Hep-2 cell line facilitates the detection of Ro/SS-A antibodies, as well as antibodies to nucleolar antigens, which are poor when using cryosections of laboratory animal tissue. Other advantages of HEp-2 include a high frequency of cell division, which makes it possible to detect antibodies to antigens expressed only during cell division, and the absence of a tissue cellular matrix, which makes it difficult to visualize specific luminescence when compared with histological sections.
Antinuclear factor titers
The ANF titer is defined as the last dilution of the patient's serum, which has clear fluorescence of cell nuclei. When using laboratory animal tissues, the diagnostic ANF titer is 1:8-1:10, while when using the HEp-2 cell line, it is recommended to use an initial titer of 1:80. In this case, the normal ANF titers are less than 1:160.
When using titers less than 1:160, the frequency of weakly positive results in clinically healthy individuals will be no more than 5%, and at the same time will not allow significant ANA titers to be missed in patients with diffuse connective tissue diseases.
To determine the final titer, titration in x2 steps is usually used (1:160-1:320 - 1:640 - 1:1280 - 1:2560 - 1:5120, etc. Rougher titration can also be used. Against the background of exacerbation of rheumatic diseases ANF titers of more than 1:640 are usually observed, and during remission the titers decrease to 1:160-1:320.
There are recommendations for scoring the results of identifying ANF, indicating the content “in the crosses”. This allows laboratories to save reagents and reduce labor costs for performing research. In this case, determination of the final titer is not possible. It must be taken into account that the final titer is more important than the amount of autoantibodies bound to the substrate, since it is directly related to the affinity of their interaction with the antigen. It is advisable to determine the final titer in all positive patients, which makes it possible to clarify the presence in the serum of high-affinity autoantibodies, which are more closely related to the activity of the process.
Type of luminescence of the antinuclear factor nucleus
Already in the first experience of the clinical use of indirect immunofluorescence to detect ANF using cryosections of laboratory tissues, it was noted that the serum of patients with autoimmune diseases “stained” the cell nuclei differently, which led to selective luminescence of some nuclear structures. Clarification of this phenomenon led to the description of the so-called “types of luminescence” of the nucleus in the immunofluorescence test. This is due to the fact that each type of ANA has specific cellular targets, as a result of which the type of luminescence of the nucleus, nucleolus and cytoplasm of the cell reflects the interaction of ANA in the patient’s serum with antigen-containing structures inside the cell. The type of luminescence depends on the presence of specific autoantibodies in the patient’s blood serum, on the basis of which a preliminary conclusion can be made regarding the types of ANA that are present in this serum. However, the use of histological sections of tissue from laboratory animals does not allow us to reliably establish the type of luminescence. At the same time, the use of continuous cell lines, such as Hep-2, makes it possible to describe a significant number of variants of nuclear and cytoplasmic staining. There are three main groups of antigens, the presence of antibodies to which determines different types of luminescence:
The type of luminescence of the cell nucleus significantly increases the information content of identifying ANF, and therefore should be routinely determined when determining this indicator. The use of HEp-2 cells makes it possible to characterize more than 20 different variants of nuclear staining, which depend on the spectrum of ANAs present in the serum under study. However, for a practical laboratory it is enough to distinguish between 6 main options for the binding of autoantibodies to antigen-containing cell structures.
There are homogeneous, peripheral, granular (small/large), nucleolar, centromeric and cytoplasmic types of nuclear luminescence. Each type of luminescence has very characteristic features that make it possible to distinguish one option from another, as well as a set of antigens with which autoantibodies react in the sera of patients. The description of the types of luminescence provides valuable clinical information in itself, in addition, the type of luminescence may indicate the need for certain laboratory tests in the future.
At homogeneous type glow autoantibodies react with those antigens that are distributed diffusely in the nucleus, i.e. are part of chromatin. Typically, when a homogeneous type of luminescence is detected, condensed chromosomes are brightly colored in dividing cells. The main structural units of chromatin are nucleosomes - complexes of DNA and histones. Thus, the homogeneous type of luminescence suggests the presence of antibodies against nucleosomes, dsDNA and antibodies to histones. It occurs in patients with SLE and drug-induced lupus, as well as in patients with scleroderma. Typically, the detection of a high titer of ANF with a homogeneous type of luminescence indicates a diagnosis of SLE.
Peripheral type glow is often isolated separately, although it is a type of homogeneous glow. Its detection is an artifact of cell fixation, which leads to redistribution of chromatin in the nucleus to the periphery. It is important to distinguish the peripheral type of luminescence from the staining of the nuclear membrane, which is observed in autoimmune liver diseases. The peripheral type of luminescence is found in patients with antibodies to double-stranded DNA and is detected mainly in patients with SLE.
Granular type is the most common and, at the same time, the most nonspecific. Sometimes this type of glow in Russian literature is called “speckled” or “mesh”. The name “granular” more accurately reflects this phenomenon, since in this case the autoantibodies react with granules in the nucleus, which are supramolecular nucleoprotein complexes. Such complexes of proteins and nucleic acids perform a number of functions in the nucleus that are necessary for the normal functioning of the cell. Such complexes, in particular, include spliceosomes, which carry out post-transcriptional rearrangement of mRNA necessary for protein synthesis on ribosomes. The composition contains many different nucleoproteins, which determines the diversity of antigenic targets when detecting a granular type of luminescence. The main autoantigens, antibodies to which lead to visualization of the granular type of luminescence, include Sm, U1-RNP, SS-A, SS-B antigens and PCNA. During the process of cell division, cells lose most of the formed nucleoprotein complexes; therefore, mitotic figures in a cell line with a granular type of luminescence are not stained. The granular type of nuclear luminescence is observed in patients with SLE, SS, SS, DM/PM, RA and a number of other autoimmune diseases. Low titers of granular type ANF predominate in the blood sera of clinically healthy individuals with ANF without signs of systemic disease.
Detection of very high ANF titers (1:2560-1:10000) with coarse granular type nuclear luminescence usually indicates a diagnosis of mixed connective tissue disease and requires further examination to identify the RNP antigen, which is the main serological marker of this disease.
Nucleolar antigens can act as targets of ANA, leading to the detection nucleolar type fluorescence. Identification of the nucleolar type of glow is characteristic of scleroderma and its varieties. Nucleolar type fluorescence determined in patients with antibodies to components of the nucleolus, such as RNA polymerase 1, NOR, U 3 RNP, PM/Scl.
Centromeric type fluorescence is noted when antibodies appear to the centromeres of chromosomes, and is found only in dividing cells. Its presence is characteristic of the CREST variant of scleroderma.
Cytoplasmic type glow indicates antibodies to tRNA synthetases, in particular Jo-1, which are observed in polymyositis. In addition, it is detected in patients with ANA directed against other components of the cell cytoplasm: antibodies to actin in autoimmune hepatitis, antibodies to mitochondria in primary biliary cirrhosis.
A number of other types of luminescence can be characterized, and these types of staining of the cell nucleus may be characteristic of a particular type of ANA. Thus, antibodies to Scl-70 in an immunofluorescent test on HEp-20 cells give fine-grained staining of the nucleus and nucleoli, anti-p80 - luminous dots in the nucleus, which are absent in mitotic cells. However, it is necessary to point out the rarity and diversity of such phenomena, which makes their identification during routine research unnecessary.
A combination of several types of luminescence can often occur, for example, fine granular and nucleolar, which is typical for antibodies to Scl-70. Moreover, often in low dilutions one type of fluorescence predominates, for example granular, and with further dilutions homogeneous or centromeric types of fluorescence are revealed, which indicates the presence of various types of ANA in the patient’s serum. Although the type of glow provides the doctor with certain data in favor of a particular diagnosis, it is necessary to take into account its relatively low specificity and the variety of phenomena encountered, which requires further laboratory examination.
The detection of ANA is the basis for the use of other laboratory tests that clarify the spectrum of ANA in the blood of patients. Given the extreme information content of this test, further testing and analysis of the results of a subsequent examination should be interpreted depending on the results of assessing the type of luminescence and ANF titer.
Although the absence of significant ANA titers almost always excludes the diagnosis of active systemic rheumatic disease, when using the HEp-2 cell line, in 2-4% of patients meeting the criteria for the diagnosis of SLE, ANA are not detected or titers are low. These patients are sometimes classified as " ANF-negative SLE“To clarify the diagnosis in such patients, further examination is required, primarily the identification of antibodies to SS-A antigens. These antigens are highly soluble and can be lost from cell nuclei. To reduce this category of patients, comprehensive testing is used, including the determination of antibodies to extracted nuclear antigens, the so-called “ screening for connective tissue diseases», test 000723. A negative result of such an examination allows, with a very high probability, to exclude the diagnosis of SLE and other systemic rheumatic diseases.
