Autoimmune diseases (AID) are a group of diseases in which excessive and persistent autoimmune reactions lead to tissue and organ damage and cause corresponding organ lesions or clinical symptoms. Rheumatic diseases are a group of chronic diseases involving joints, cartilage, muscles and other connective tissues.
Laboratory tests, especially immunological tests, are important for the diagnosis of AID and rheumatic diseases.
Some tests can be used as one of the diagnostic indicators.
some tests can be used as indicators for monitoring the progression and prognosis of the disease
Some tests can be used as one of the evaluation indicators of drug efficacy.
With the development of immunology technology and the application of molecular biology, various autoantibody tests are increasingly used in clinical practice. As a clinician, one should be familiar with the significance of these tests and be able to use them objectively in conjunction with clinical and other examinations to assist in diagnosis, understand the condition, and guide treatment. Here we only introduce some of the commonly used clinical tests for autoantibodies.
I. Antinuclear antibody measurement
（ANA can be divided into anti-DNA antibody, anti-histone antibody, anti-nuclear antibody and antibodies against other cellular components. These antibodies together form the ANA spectrum.
Anti-nuclear antibodies (ANA) are most commonly used to screen for systemic rheumatic diseases and autoimmune disorders.ANA tests may observe autoantibodies that react with nucleic acid and protein antigens in the cell nucleus. Antibodies that react to multiple antigens may be present in the cells. IFAs can be identified only if the potency of a particular antibody is high.
homogenous
Also known as diffuse, the corresponding antigens are double-stranded DNA and histone complexes. It is commonly seen in SLE, especially in patients with renal involvement. It is also seen in other connective tissue diseases, such as rheumatoid arthritis, MCTD, dry syndrome, scleroderma, chronic active hepatitis and primary biliary hepatic sclerosis.
Peripheral (peripheral)
The corresponding antigen is double-stranded DNA, mostly seen in patients with active SLE.
Speckle (speckle type)
Antibodies mainly against ENA, including Sm, RNP, SSA, SSB, and Scl-70. It is generally believed that low potency speckle ANA is not specific to connective tissue disease and does not actually represent a clinical abnormality. Highly potent anti-Sm antibodies are mostly associated with SLE. Highly potent anti-RNP autoantibodies are seen in MCTD but also in SLE. anti-SSA and anti-SSB antibodies, seen in S.S, are also seen in SLE. when patients have anti-SSA antibodies, skin manifestations and photosensitivity are usually predominant. Scl-70 antibodies are associated with scleroderma.
Nucleoli (Nucleolar)
The target antigen is a nuclear protein associated with RNA molecules. Most commonly seen in scleroderma.
Centromere (mitotic)
Anti-centromere antibody (ACA), mostly seen in CREST syndrome of systemic scleroderma.
Commonly used detection methods are IFA, ELISA, immunoblotting, RIA, immunospot assay, colloidal gold labeled spot immuno-permeation assay, etc. Among them, IFA is one of the more widely used methods. Mouse liver sections or Hep-2 cells are generally used as substrate antigen slices, and when patient serum is added, the ANA in the patient serum can bind to the corresponding antigenic components in the cells. When fluorescently labeled anti-human IgG is added, bright green fluorescence in the cell nucleus can be seen under fluorescence microscope.
〖Reference value〗 <1_10 or negative
〖clinical significance〗
ANA positive is seen in many diseases, often used in the diagnosis of rheumatic diseases and autoimmune diseases. the positive rate of SLE patients is over 95%, and the potency is often above 1:80. In other rheumatic diseases such as RA, systemic sclerosis, dermatomyositis, dry syndrome (SS), mixed connective tissue disease (MCTD), etc. can also be positive. Other diseases such as liver disease and viral infections may also show low potency.
〖Caution〗
Low-valent non-specific antinuclear antibodies can be detected in about 1% of normal people, and the detection rate can be as high as 50% in elderly people over 80 years old. Highly potent ANAs are generally closely associated with active SLE. Different staining models can be seen using Hep-2 cells for ANA detection.
In addition, ANA can be detected in women taking oral contraceptives.
Extractable nuclear antigen (ENA) antibody
ENA is a non-histone acidic nucleoprotein particle composed of many small molecules of RNA and polypeptides in the cell. Those distributed in the cytoplasm are called small cell plasma ribonucleoproteins (scRNPs); those distributed in the nucleus are called small cell ribonucleoproteins (snRNPs). More than 20 types of anti-ENA antibodies have been identified, among which the following are commonly used.
(i) Anti-Sm antibodies
Sm is the abbreviation of patient Smith. sm antigen belongs to snRNP and consists of 5 RNAs and polypeptides with antigenic epitopes on 29KD, 28KD and 13.5KD polypeptides. anti-Sm antibodies are acidic glycoproteins and are marker antibodies for SLE.
(ii) Anti-nRNP (u1RNP) antibodies
u1RNP is composed of u1RNA and protein with antigenic epitopes on 73KD, 32KD and 17.5KD polypeptides, and is an important serum marker for mixed connective tissue disease (MCTD).
(iii) Anti-SSA antibody
SS is the abbreviation of dry syndrome. This antibody is also called anti-RO antibody, SSA antigen is RNA and protein complex, antigenic epitope on 52KD polypeptide, mainly seen in patients with primary dry syndrome and SLE.
(iv) Anti-SSB antibodies
SSB antibodies often appear together with SSA antibodies, also known as anti-La antibodies. SSB antigens belong to SnRNP, which is a complex containing RNA and 50KD protein. The antigenic epitopes are on 45KD, 47KD and 48KD polypeptides. Coexistence with SSA antibodies is specific for the diagnosis of SS.
(E) Anti-Scl-70 antibody
Scl-70 antigenic epitopes are on 86KD and 70KD fragments, which are degradation products of DNA topoisomerase I. Anti-Scl-70 antibodies are almost exclusively seen in scleroderma, with a positive rate of about 30%.
(vi) Anti-Jo-1 antibody
Jo-1 antibody is a histidyl tRNA synthetase with an antigenic epitope at 55 KD polypeptide. Anti-Jo-1 antibody is a marker antibody for polymyositis and dermatomyositis, with a 25-40% positivity rate.
(vii) Anti-ribosomal antibodies
Ribosomes are synthesized in the nucleolus and then transferred to the cytoplasm, and the antigenic epitopes are on 38KD, 16KD and 15KD polypeptides on the large subunit. Positive anti-ribosomal antibodies are mainly seen in SLE, with a positive rate of 20-30%.
The main methods to detect the above antibodies are: convective immunoelectrophoresis (CIE), biphasic immunodiffusion, immunoblotting test (IBT), immunospotting technique and ELISA. Generally, rabbit thymus or bovine thymus extracts or cell extracts are used as antigens. In recent years, kits prepared by applying molecular recombinant antigens have been put on the market.
〖Reference value〗Negative
〖clinical significance〗
1. anti-n RNP antibody: seen in 35-40% of SLE patients, the positive rate can reach 95-100% in MCTD patients, also seen in other rheumatic diseases.
2. anti-Sm antibodies: mainly seen in SLE and overlap syndrome, 75% of patients in acute stage are positive. In general, 30-40% of SLE patients are positive. However, more than 90% of those positive for Sm antibody are SLE, so it is called the marker antibody of SLE.
3.
Anti-SSA and anti-SSB antibodies: Seen in 40-45% of SS patients. Anti-SSA antibodies are also seen in 25% of SLE patients and other rheumatic diseases; anti-SSA antibodies are associated with neonatal lupus and congenital conduction block. Anti-SSA antibodies are also seen in non-rheumatic diseases such as viral infections. Anti-SSB antibodies are more specific for primary dry syndrome, but the positive rate is only about 27%.
4. anti-Scl-70 antibody: generally only seen in patients with systemic sclerosis, with a positive rate of 20-60%.
5. Anti-Jol-1 antibodies are seen in 30% of patients with polymyositis and 10% of patients with dermatomyositis. Anti-Jol-1 antibodies are associated with an increased incidence of interstitial pneumonia in patients with polymyositis.
Anti-rRNP antibody: mainly seen in SLE patients, with a positivity rate of about 20%, associated with central nervous system lesions, mostly in the active phase of the disease.
〖Caution〗
The test results should be evaluated in the context of clinical manifestations, and should also be interpreted according to the test method. Convective immunoelectrophoresis and biphasic diffusion method have low sensitivity but high specificity; immunoblotting, spot immunoassay and ELISA method have increased sensitivity but relatively reduced specificity.
III. Anti-double-stranded DNA antibody (Antidouble-stranded DNA, A-dsDNA,)
A-dsDNA
A-dsDNA is highly specific for SLE and is an autoantibody against the nucleus of the cell. The commonly used clinical detection methods are: ①Farr¡®s method: 125I-DNA and A-dsDNA in the specimen to be examined form an immune complex, add saturated ammonium sulfate to precipitate the complex, and determine the free and precipitated 125I-DNA bound in the supernatant with a gamma counter, respectively. The binding activity of A-dsDNA in serum can be calculated by measuring the content of free and bound 125I-DNA in the precipitate by γ-counter.
②ELISA method: Calf thymus DNA was encapsulated in microtiter plate, patient serum was added, and if A-dsDNA was bound to the microtiter plate, enzyme-labeled anti-human IgG was added, and the amount of A-dsDNA antibodies in serum could be known by colorimetric comparison of enzyme-labeled instrument after adding substrate.
③Immunofluorescence method: Trypanosoma equi or T. shortum was used as the substrate, and patient serum was added. The A-dsDNA in the serum binds to the natural DNA on the membrane, and fluorescently labeled anti-human IgG is added. green fluorescence is emitted from the motile substrate of Trypanosoma equi and T. shortum.
④Gold-labeled spot method: Double-stranded DNA is encapsulated on nitrocellulose membrane, patient’s serum is added, and gold-labeled anti-human IgG is added, if A-dsDNA exists, red spots will be shown.
〖Reference value〗The reference value varies according to different methods.
Farr¡®s method: ≤ 0.20
Immunofluorescence method: <1:10
ELISA method: depending on the kit, generally <70u
Gold standard method: Negative
〖clinical significance〗
A-dsDNA positivity has a high specificity for SLE. Other rheumatic diseases may also appear, but the positive rate is very low, such as RA, S.S.
〖Caution〗
False positive results are rare, but a negative result does not exclude the diagnosis of SLE.
IV. Anti-neutrophil cytoplasmic antibody (Antineutrophil cytoplasmic antibody, ANCA) assay
ANCA is an autoantibody against various components of neutrophil cytoplasm.
The main antibodies are myeloperoxidase (MPO) and Proteinase 3 (PR3). They are mainly used for the diagnosis of Wegener
granulomatosis and systemic vasculitis, especially in patients with multiple organ damage caused by concomitant renal disease, respiratory disease or other types of vasculitis. Detection methods include IFA, ELISA, IBT, etc.
〖Reference value〗 Negative.
〖clinical significance〗
Immunofluorescence method for ANCA positive is generally divided into two fluorescence types, one is cytoplasmic type, called cANCA, and one is perinuclear type, called pANCA. cANCA produces diffuse cytoplasmic staining and is an antibody against PR3
The cANCA produces diffuse cytoplasmic staining and is an antibody against PR3 , which is generally seen in patients with wegener’s granulomatosis (WG), especially those with renal and pulmonary involvement. pANCA produces perinuclear staining and is an antibody against MPO , which is mainly seen in patients with systemic vasculitis.
In patients with WG, cANCA positivity is up to 80% in the active phase and has high antibody potency. In the remission phase, the antibody positivity rate is low and the potency decreases or disappears completely.
〖Caution〗
ANCA
Positive ANCA can also be seen in patients with Good-Pastare¡®s syndrome and SLE.
The diagnosis of WG cannot be made solely on the basis of ANCA results, but must be determined in conjunction with other clinical manifestations, laboratory tests and histopathological examinations. pANCA potency does not fully reflect disease activity and response to treatment.
A positive pANCA is not a specific anti-MPO antibody, but can also be an antibody against other enzymes in the granulocyte cytoplasm, such as elastase and histone G. ANCA testing should be performed to exclude cross-reactivity of other autoantibodies, such as ANA, which can produce false positives and should be identified and tested for ANA.
Antimitochondrial antibody (AMA)
AMA is an autoantibody using mitochondria in the cell plasma as antigen, without organ and species specificity. Generally, rat kidney and stomach are used as antigenic substrates, and the positive test by IFA shows fine granular fluorescence in the cytoplasm of rat kidney and stomach.
The reactions with mitochondrial antigens can be classified into nine types from M1 to M9 using immunoblotting. Highly efficient M2 and M9 type autoantigens are associated with primary biliary hepatic sclerosis. ELISA kits for the detection of M2 are currently available.
〖Reference value〗 Negative
>1:10 is positive
〖Clinical significance〗
AMA is a marker antibody for primary biliary cirrhosis, with a positive rate of 90% and an antibody potency of 1:1280 or higher in 50% of patients. In addition, it can also be seen in chronic active hepatitis and cryptogenic cirrhosis. The AMA is often negative in patients with extrahepatic obstructive jaundice, so it can be used as an indicator for differential diagnosis. The positive rate is <10% in normal subjects and the potency is low.
VI. Antismooth muscle antibody (ASMA)
ASMA is an autoantibody against an actin in the hepatocyte membrane. This actin has cross-antigenic properties with smooth muscle and therefore reacts with smooth muscle. Generally, IFA method is used, and rat stomach is used as the antigenic substrate, and a positive test shows bright green fluorescence of smooth muscle of the stomach wall.
〖Reference value〗 Negative, >1:10 for positive
〖clinical significance〗
Mainly seen in patients with autoimmune hepatitis in apparently active stage, with positive rate above 80% and potency
above 1:80. ASMA can also be positive in the early stage of acute viral hepatitis, and it is earlier than
HBsAg appears. Other diseases such as infectious mononucleosis, SS, RA, mycoplasma pneumonia, tumors, and viral infections also have varying degrees of positivity. Only 2% of normal individuals are positive.
VII. Antithyroglobulin antibodies (ATGA) and antithyroid microsomal antibodies (ATMA)
ATGA is an autoantibody caused by thyroiditis, the antigen is a glycoprotein, ATGA is organ specific but not species specific, the antigen of ATMA is a lipoprotein in the cytoplasm of thyroid follicular epithelial cells. Commonly used detection methods are: IFA, IHA, RIA, ELISA, etc.
〖Reference value〗
IHA: serum potency ≤1:32, >1:32 for positive, ATGA and ATMA
ELISA: normal is negative, P/N < 2.1, >2.1 is positive, ATGA and ATMA
RIA: ATGA <30%; ATMA <15%
l〖clinical significance〗
It is mainly seen in patients with Hashimoto’s thyroiditis, hyperthyroidism and hypothyroidism, but also in thyroid adenoma, pernicious anemia, myasthenia gravis, Edison’s disease and liver disease, etc. SLE and other autoimmune diseases also have a certain positive rate.
Both antibodies can be detected in normal individuals, and the rate of positivity increases with age, especially in women over 40 years of age, and can reach about 18%.
It should be noted that some patients are negative for ATGA but positive for ATMA, so simultaneous testing of both antibodies may improve the detection of anti-thyroid autoantibodies.
VIII. HLA-B27 measurement
HLA-B27
The HLA-B27 antigen is the expression product of the B site of human MHC class I antigen and can be divided into several subtypes.
-B27 can be detected by various methods, complement-dependent microcytotoxic assay (CDMA); flow cytometry; roentgenographic method; ELISA; isoelectric focusing method, etc. Recently, PCR method is applied to detect HLA-B27, which is sensitive and specific, and can be used for the analysis of B27 subtypes.
l〖reference value〗
Afro-American: 3-4%.
l Caucasian: 6-8%.
Asians: 1%.
〖clinical significance〗
The HLA-B27 positivity rate in patients with ankylosing spondylitis (AS) is 90-95%. HLA-B27 is present in about 42% of young rheumatoid arthritis (JRA), and Reiter¡®s
syndrome patients with a positivity rate of approximately 79%. In addition, enteropathic arthritis and psoriatic arthritis also have a certain positive rate. the positive rate of RA patients is not high.
〖Caution〗
HLA-B27 positive results should be considered in conjunction with clinical manifestations, and the diagnosis should not be made solely on the basis of this result.
Other immunological indicators and tests
C-Reactive Protein (CRP)
CRP is an acute temporal protein, synthesized by hepatocytes, which can precipitate with pneumococcal cell wall C polysaccharide, so called CRP. serum CRP concentration increases sharply in the acute phase of various inflammatory diseases or tissue trauma.
bacteria
CRP not only binds polysaccharide substances in bacteria (bacteria), fungi (fungi) and protozoa (protozoal), but also binds phosphatidylcholine and nucleic acid in the presence of calcium ions. The bound complex has the effect of activating the complement.
Commonly used assays are
enzyme-linked immunosorbent assay (ELISA) and
Rate scattering turbidimetric method (rate nephelometry), etc.
〖Reference value〗 <8mg/L
〖clinical significance〗
Elevated serum CRP is usually seen in: acute and chronic bacterial infections; necrotic tissue injury; acute myocardial infarction; various inflammatory diseases; surgical procedures; tumor infiltration; acute rheumatic fever and active rheumatoid arthritis, as well as other inflammatory joint diseases. However, viral infections generally do not have elevated CRP, so it can be used as a differentiation indicator between viral and bacterial infections.
〖Caution〗
CRP is measured by different methods and should be taken into consideration when determining the results. In addition, estrogen and oral contraceptives can increase CRP, while corticosteroids and anti-inflammatory drugs can decrease CRP.
Rheumatoid Factor (RF)
RF is an autoantibody that reacts with denatured IgG and binds to the Fc segment of IgG.
RF can be classified into five types: IgG, IgM, IgA, IgD and IgE. RF is mainly seen in rheumatoid arthritis (rheumatoid RA) and is detected by agglutination tests.
RF is mainly seen in rheumatoid arthritis (RA), but also in other connective tissue diseases and other diseases. Detection methods include latex agglutination test, allergic sheep red blood cell agglutination method, rate scattering turbidimetric method, ELISA method, etc.
〖Reference value〗
Latex method: negative or <1:20
Rate scattering turbidimetric method: <30IU/ml
〖clinical significance〗
RF is seen in more than 90% of RA patients, the potency is often above 1:160, and the content is more than 80IU/ml. Most of the general methods detected are IgM-RF.
About RF typing, clinical application is not yet widespread. Most authors believe that: IgM-RF potency can reflect the activity of RA to some extent, but there is no clear close relationship; IgG-RF is closely related to synovitis, vasculitis and extra-articular symptoms in RA patients; IgA-RF is seen in RA, scleroderma, Felty syndrome and systemic lupus erythematosus (systemic lupus erythematosus).
lupus erythematosus (SLE), which is also an indicator of the clinical activity of RA; IgE-RF
High level IgM-RF positive patients have a poor prognosis.
In patients with early RA, patients with persistent positive IgM-RF are more likely to develop bone erosion, and IgA-RF is more prevalent in patients with SS; IgE-RF is more prevalent in patients with malignant arthritis. In RA patients, the presence of high-valent RF with severe joint function limitation often indicates a poor prognosis.
〖Caution〗
Normal people may also have a positive rate of about 4%. RF can also be negative in RA patients, and the diagnosis of RA cannot be made solely on the basis of a positive RF. RF can also be seen in a variety of other diseases, the most common of which is dry syndrome (S.S), with an incidence of over 90%.
The incidence is more than 90%, and the level is generally high. Other common RF-positive diseases include.
SLE, systemic sclerosis, hyperglobulinemia, nodular disease, syphilis, leprosy, viral infections, cirrhosis of the liver, etc. Some potencies can be above 1:160 and levels above 80 IU/ml, which should be noted when interpreting the results.
Cryoglobulin CG,)
Cryoglobulin is a kind of globulin, which precipitates at 4°C, polymerizes easily at 30°C and dissolves at 37°C. CG is divided into three types: 1.
1. Type I (monoclonal type): mostly IgM or IgG type, no anti-complement effect.
2. Type II (mixed type): two or more monoclonal Ig mixture, IgM + IgG is the most common, also visible IgA + IgG; IgG + IgM + IgA, etc.. There are anti-complement effects.
3. type III (polyclonal type): no monoclonal protein.
The detection methods are hematocrit tube method (qualitative) and spectrophotometer method (quantitative).
〖Reference value〗 Qualitative method: negative
Quantitative method: <80μg/ml.
〖Clinical significance〗
SLE
Mixed CG can be found in the serum of patients, and can also be positive in vasculitis, glomerulonephritis and lymphoproliferative diseases. It is associated with type I cryoglobulinemia in patients with macroglobulinemia or multiple myeloma with Raynaud’s phenomenon.
Type II cryoglobulinemia is associated with autoimmune diseases such as vasculitis, glomerulonephritis, SLE, RA, and SS
It is associated with autoimmune diseases such as vasculitis, glomerulonephritis, SLE, RA and SS. It can also occur in certain infections such as hepatitis, infectious mononucleosis, cytomegalovirus infection, and toxoplasmosis.
〖Caution〗1.
1. Specimens must be collected at 37°C and maintained at that temperature until the experiment is performed. 2.
2. Generally not used for screening of people without clinical manifestations of cryoglobulinemia.
3. Cold agglutinins may cause erroneous results in some automated hemocytometers.
Summary:
In conclusion, the detection of autoantibodies is of great importance for the diagnosis of autoimmune diseases. A variety of autoantibodies are present in human serum, both organ-specific and non-organ-specific. Therefore, the selection of autoantibody tests should be comprehensive and rational.
Many new autoantibodies have emerged in recent years, so we will not describe them in detail here, but we can refer to the relevant literature.
Cytoplasmic antibodies
Hep-2 cells have many cytoplasmic antigens. If specific cytoplasmic antibodies are present, strong positive cytoplasmic fluorescence can be observed. These include anti-mitochondrial antibodies (AMA), anti-ribosomal antibodies, anti-actin antibodies i.e. anti-smooth muscle antibodies (ASMA), anti-Golgi antibodies, and anti-filament antibodies (e.g. Vimentin). The most significant ones in general are AMA and ASMA, but fluorescent staining of Hep2 cells cannot identify these two antibodies, which can be further analyzed by applying murine kidney and murine stomach sections.
Evaluation of the active phase of systemic rheumatic disease
Autoantibody testing is more relevant to diagnose systemic disease than to monitor the activity of the disease. Anti-dsDNA antibody potency, however, is associated with activity. The potency of anti-dsDNA antibodies and the levels of C3 and C4 should be tested regularly. ESR and CRP are often elevated in response to inflammation, and CRP is significantly increased in exacerbations of RA. in active SLE, CRP is generally normal.
Organ-specific autoimmune diseases
Tissue antibodies
Many autoantibodies can be measured using IFA. Antibodies against mitochondria, smooth muscle, liver and kidney microsomes, and gastric lining cells can be measured using sections of murine kidney and stomach. Anti-thyroid autoantibodies can be measured in thyroid tissue sections.
Autoimmune liver disease
In liver lesions, the ANA positivity rate can be 40-80%. Anti-mitochondrial antibodies (AMA) are associated with primary biliary hepatic sclerosis. In particular, highly potent M2 and M9 mitochondrial antigens.
In addition, liver and kidney microsomal antibodies (LKMAs), anti-soluble liver antigen (SLA) antibodies, and anti-hepatocyte membrane antibodies are also used in the diagnosis and monitoring of autoimmune liver disease.
Other
Antibodies commonly measured in kidney disease include anti-dsDNA antibodies, anti-glomerular basement membrane antibodies, etc. CIC can also be measured, as well as kidney tissue biopsies for fluorescent staining.
Wegener’s granulomatosis is a necrotizing vasculitis that occurs in the upper and lower respiratory tract and also involves the kidneys; patients often die of renal and pulmonary failure. Checking for anti-neutrophil cytoplasmic antibodies (ANCAs) is one of the diagnostic indicators. Anti-myeloperoxidase antibodies, anti-proteinase 3 antibodies can also be measured by ELISA, which can help in diagnosis and differential diagnosis.
In gastrointestinal diseases, pernicious anemia is associated with anti-gastric wall autoantibodies and correlates with anti-internal factor antibodies by 75%. Anti-islet cell antibodies (ICAs), anti-glutamic acid decarboxylase antibodies (GADA) are associated with type I diabetes. Anti-skeletal muscle antibodies may be present in patients with myasthenia gravis. Anti-Pm1 and anti-Jo-1 antibodies may be present in polymyositis and dermatomyositis. Anti-cardiac antibodies are commonly seen after myocardial infarction, cardiac surgery or myocardial injury.