Immunology Tests

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Homogenous Pattern:
Clinical association: Common in SLE, drug induced lupus (e.g. procainamide and hydralazine), rheumatoid arthritis, juvenile chronic arthritis and systemic sclerosis. Suggest further testing for antibodies to dsDNA, ENA and RF, depending on clinical features. 
Speckled pattern:
Clinical association: A common pattern found in several diseases including SLE, Sjogren’s syndrome, subacute cutaneous lupus and scleroderma. Suggest further testing for antibodies to dsDNA, ENA. 
Centromere pattern:
Clinical association: Almost exclusively found in the CREST syndrome (calcinosis, Raynaud’s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasias) a mild variant of progressive systemic sclerosis of which approximately 60% are antibody positive.  Patients with primary Raynaud’s phenomenon who are centromere antibody positive frequently develop limited scleroderma.  Suggest further testing for antibodies to ENA.
Nucleolar pattern:
Clinical association: Systemic sclerosis and associated with skeletal muscle disease and pulmonary hypertension.  Suggest further testing for antibodies to ENA. 
Mitotic Spindle Apparatus pattern:
Clinical association: A clinically non-specific antibody found occasionally in SLE, Sjogren’s syndrome, CREST, MCTD and polyarthritis.  Suggest further testing for antibodies to dsDNA, ENA and RF. 
Peripheral or Rim pattern:
Clinical association: Found in SLE particularly when the disease is active.
Suggest further testing for antibodies to dsDNA, C3, C4 and other markers of disease activity. 
Nuclear Membrane pattern:
Clinical association: A rare pattern found in patients with unusual mixed, chronic autoimmune disorders usually including chronic hepatitis and variably associated with vasculitis, thrombocytopenia and Raynaud’s phenomenon.
Suggest further testing (eg. ANCA, dsDNA, ACA) depending on clinical features. 
Atypical Speckled pattern (PCNA):
Clinical association: May be associated with systemic vasculitis. 
Suggest further testing for antibodies to ANCA, dsDNA and ENA. 
Nuclear Dots (2-6):
Clinical association: Autoimmune and viral liver diseases such as primary biliary cirrhosis and chronic active hepatitis. Also found rarely in collagen vascular diseases and a variety of other autoimmune diseases. 
Suggest further testing for antibodies to smooth muscle, mitochondria and ENA. 
Multiple Nuclear dots:
Clinical association: A rare pattern found in 6% of primary biliary cirrhosis, often with Sjogren’s syndrome, and less frequently in SLE. Suggest further testing for antibodies to dsDNA, ENA and mitochondria. 
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 Rheumatoid Factor 

This antibody is commonly present in the serum and synovial fluid of patients with a variety of autoimmune and rheumatic diseases including rheumatoid arthritis, Sjogrens syndrome, chronic infections and healthy elderly individuals. 
Suggest ANA, ENA, serum immunoglobulins, IPG, IEPG if clinically indicated. 
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Anti-neutrophil cytoplasmic antibodies (ANCAs) recognise lysosomal enzymes of human neutrophils and monocytes. Two different staining patterns of ANCA are recognised by indirect immunofluorescence (IF). The cytoplasmic pattern (cANCA) is associated with a serine protease, proteinase 3, and is a sensitive and specific marker for Wegener's granulomatosis. The perinuclear pattern (pANCA) is associated with recognition of myeloperoxidase and has been found in patients with necrotizing and crescentic glomerulonephritis, microscopic polyangiitis, and Churg-Strauss syndrome. pANCAs have also been detected in patients with a variety of inflammatory (ulcerative colitis, chronic hepatitis, cystic fibrosis) and rheumatic diseases (e.g. Sweets syndrome). ANCAs may also have a role as prognostic markers for aggressive disease in patients with early RA. 

Drug induced ANCA (usually pANCA) observed with propylthiouracil and hydralazine therapy. Plasma ANCA titers and disease activity — Plasma ANCA titers usually parallel the course of the vasculitis, especially with C-ANCA. The disappearance of ANCA is often associated with clinical remission and patients who maintain negative or have decreasing titers after therapy are at lower risk for clinical relapse (5 to 20%). However, the relationship between ANCA titers and disease activity is not absolute, since ANCA and disease activity may be discordant in as many as one-third of patients. It has been suggested that once remission has been induced, a rise in plasma ANCA titers may be highly predictive of subsequent clinical relapse. 
An increase in ANCA titers (as determined by indirect immunofluorescence) may be a less sensitive predictor of relapse than a rise in levels using an enzyme linked immunosorbent assay (ELISA). 
Suggest: PR3 and MPO antibodies by ELISA to follow course of the disease. Consider ANA, ENA, DNA, serum immunoglobulins, EPG, IEPG and complement levels (C3, C4, CH100) if clinically indicated.
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The antiphospholipid antibody syndrome (APS) is characterised by recurrent arterial or venous thromboembolism, and/or pregnancy loss, in association with antibodies directed against either phospholipids or plasma proteins bound to anionic phospholipids. Four types of antiphospholipid antibodies (aPL) have been characterised: 
Anticardiolipin antibodies (aCL)
Anti-ß2 glycoprotein-I antibodies
False positive serologic test for syphilis (STS)
Lupus anticoagulants 

High titres of IgG ACL are associated with the cardiolipin antibody syndrome. This may be seen in patients with other autoimmune diseases (e.g. SLE), infections (e.g. HIV, syphilis) and drugs (e.g. chlorpromazine, hydralazine, procainamide, phenytoin).
Suggest: Consider ANA, ENA, DNA, serum immunoglobulins, EPG, IEPG, cryoglobulins and complement levels (C3, C4, CH100) if clinically indicated.
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The IgA anti-endomysial antibody (EMA) is the most sensitive and specific screening test for celiac disease. The antiendomysial antibody has 90 to 95% sensitivity and 90 to 95% specificity. The antigen to which EMAs react is tissue transglutaminase (tTG). Celiac sprue is associated with dermatitis herpetiformis (DH), insulin-dependent diabetes mellitus and IgA immunoglobulin deficiency. An increased incidence of both gastrointestinal and nongastrointestinal neoplasms as well as intestinal lymphoma exists in patients with celiac sprue. Suggest: serum immunoglobulins, EPG, IEPG and T cell subsets if clinically indicated. 
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Smooth Muscle Antibodies 

Low titre SMA are common and usually of no clinical significance. Autoimmune hepatitis is associated with circulating autoantibodies (nuclear, smooth muscle (directed at actin), anti-LKM antibodies, antibodies to "soluble liver antigen" (directed at a member of the glutathione S-transferase gene family), as well as antibodies to the liver-specific asialoglycoprotein receptor (or "hepatic lectin"), thyroid and rheumatoid factor), and hyperglobulinemia. Other autoimmune disorders - such as thyroiditis, rheumatoid arthritis, autoimmune haemolytic anaemia, ulcerative colitis, proliferative glomerulonephritis, juvenile diabetes mellitus and Sjögren's syndrome - occur with increased frequency in patients with autoimmune hepatitis.
Suggest (in patients with suspected autoimmune hepatitis): ANA, AMA, ENA, EPG, IEPG, Thyroid antibodies. 
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Mitochondrial Antibodies 

Antimitochondrial antibodies (AMA) are the serologic hallmark for primary biliary cirrhosis. They are 95% specific for PBC. Other non-organ specific antibodies, namely antinuclear antibody (ANA) and smooth muscle antibody (SMA), are detected in one-third of patients with AMA positive PBC and in most patients with AMA negative PBC. Serum immunoglobulin levels are elevated predominantly IgM, although there may also be a less marked elevation in serum IgG. Several other autoimmune disorders are commonly found in association with primary biliary cirrhosis, chiefly autoimmune thyroiditis, Sicca syndrome, Raynaud's disease, CREST syndrome, rheumatoid arthritis and celiac disease.
Suggest: (in patients with suspected PBC) ANA, ENA, EMA, SMA, RF, anti-thyroid anti-bodies, EPG, IEPG and serum immunoglobulins.
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Gastric Parietal Cell Antibodies 

Anti-parietal cell antibody, which is directed against the H+,K+-ATPase, occur in 90% of patients with pernicious anaemia, while 60% have antibodies to intrinsic factor. Anti-parietal cell antibody is also found in 50% of patients with gastric atrophy without pernicious anaemia, as well as in 10 to 15% of the population (anti-IF antibody is usually absent from these patients). Pernicious anaemia is associated with other autoimmune diseases, including Graves' disease, myxedema, thyroiditis, Addison’s disease, vitiligo, and hypoparathyroidism. Suggest: thyroid and adrenal autoantibodies, serum immunoglobulins, EPG, IEPG.
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Reticulin Antibodies 

These antibodies are detected in normal subjects as well as patients with a variety of immune disorders, most commonly; coeliac disease, dermatitis herpetiformis and Crohns disease. Suggest: EMA if clinically indicated.
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Reduced serum C3 results from activation of the complement cascade, either the classical (reduced C4) or alternate pathway (normal C4)) or from complement deficiency. Immune complex mediated disease lead to reduced C3 levels and this may serve as an indicator of disease activity in conditions such as SLE and cryoglobulinaemia. Increased C3 results from an acute phase response.
Suggest: C4, CH100, PH50, ANA, DNA, cryoglobulins.
Reduced serum C4 results from activation of the classical complement cascade or from complement deficiency (eg. C1 INH). Increased C4 results from an acute phase response.
Suggest: C3, C1INH, C4 allotyping.
This is a screening test for suspected inherited complement deficiency. The CH100 is reduced in association with activation of the classical complement pathway. C3 is usually also reduced.
Suggest: C3, C4 and specific complement factor determination (consult laboratory).
C3 nephritic factor is an auto-antibody that binds to C3 convertase the enzyme that metabolizes C3, and renders it resistant to inactivation. This anti-body is characteristic of Type II membranoproliferative glomerulonephritis (MPGN) and occurs in patients with partial lipodystrophy.
Suggest: C3 NeF should only be ordered when the C3 is normal and C4 is reduced (i.e. when there is alternate pathway activation). 
Altogether 3 forms of C1 inhibitor deficiency are known. Two are hereditary heterozygous deficiency states no homozygous deficiency of this protein is known one presumes the condition is fatal in utero. Type 1 C1 inhibitor deficiency: the patient only expresses from one allele and serum C1 inhibitor levels are 5-30% of normal. Type 2 C1 inhibitor deficiency: the patient expresses from both alleles but one protein is functionally inactive usually due to a mutation in or close to the C1r and C1s binding site. Serum levels appear normal but the functional activity is 5-30% of normal because of consumption of the functional protein. Acquired C1 inhibitor deficiency: patients with this disease secrete an antibody, which binds to and inactivates C1 inhibitor. Acquired C1 INH is associated with malignancy and SLE. C1 INH is unlikely to be reduced if C4 is normal.
Suggest: measure C3 and C4 before C1 INH. Level may rise with Danazol therapy.

Further Information:
A clever graphic representation of the basic assay for complement activity using red blood cell lysis, from D. Fix at University of Southern Illinois, Carbondale.

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Lymphocyte Typing

Lymphocyte numbers should be interpreted in clinical context and based on the results of FBC, lymphocyte morphology and other tests of immune function. Reduced CD4 T cells are common in HIV and other viral infections as well as in a variety of autoimmune, neoplastic and infiltrative disease, and secondary to drug therapy. Reduced CD8 T cells occurs in HIV and other viral infections as well as in a variety of autoimmune, neoplastic and infiltrative disease, and secondary to drug therapy.
Increased T cells occur in inflammatory and reactive disorders and in some infections (eg. EBV). Monoclonality suggests the presence of leukaemia or lymphoma. B cells (CD19) are absent in X-linked aggamaglobulinaemia and reduced in common variable immunodeficiency. B cells are increased in inflammatory and lymphoproliferative disorders. Monoclonality suggests the presence of leukaemia or lymphoma. NK cells (CD16) are increased in the peripheral blood in viral infections (HCV), inflammatory diseases, IV drug use and lymphomas. NK cell numbers are reduced in association with certain malignancies and in the Chediak Higashi syndrome. 

Further Information:
The SCID home page contains links to periodicals and databases with information about SCID.
Information about the global AIDS epidemic can be accessed from this site.
Up-to-date information concerning AIDS epidemiology in the United States can be obtained at this site.

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Tumour Markers 

Interpret the results of tumour marker analysis in the context of the patients clinical features. Exercise caution in comparing results using different methodologies. Note that increased serum levels of certain tumour markers might result from a large number of non-malignant conditions.
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 Immunofixation Electrophoresis (IFE), Serum and Urine EPG 

Monoclonal immunoglobulins consist of heavy and light chains. IFE identifies the presence or absence of a monoclonal protein and determines its heavy-chain and light-chain type. This test measures immune status and competence by identifying monoclonal and particle protein band immunoglobulins involved in the immune response. IFE is a follow-up test performed when a monoclonal spike is observed on EPG or when a monoclonal gammopathy is suspected on the basis of the patient’s immunoglobulin concentrations.
Clinical Implications:
1. Monoclonal protein in the serum or urine suggests a neoplastic process; a polyclonal increase in immunoglobulins is seen in chronic liver disease, connective tissue disease, and infection.
2. In multiple myeloma, 99% of patients have a monoclonal protein in the serum or urine. Waldenstrom’s macroglobulinemia is characterised by the presence of a serum monoclonal IgM protein in all cases.
3. A monoclonal light chain (K or Bence-Jones protein) is found in the urine of ~ 75% of patients with multiple myeloma. Approximately 75% of patients with Waldenstrom’s macroglobulinemia have a monoclonal light chain in the urine. Heavy-chain fragments as well as free light chains may be seen in the urine of patients with multiple myeloma or amyloidosis.
Suggest: IEPG, urine EPG, serum immunoglobulins, T and B cell analysis.
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Serum Immunoglobulins 

Polyclonal increase in serum immunoglobulins is observed in a large number of clinical conditions.
Increased IgG:
1. Chronic granulomatous infections
2. Hyperimmunization
3. Liver disease
4. Monoclonal gammopathy of uncertain origin
5. Disease associated with hypersensitivity, granulomas, dermatologic disorders, and IgG myeloma
6. Rheumatoid arthritis 
Decreased IgG:
1. Agammaglobulinemia: Brutons, Common variable immunodeficiency
2. Lymphoid aplasia
3. Selective IgG, IgA deficiency
4. IgA myeloma
5. Bence-Jones proteinemia
6. Chronic lymphoblastic leukaemia
Suggest: serum IgG subclasses, EPG, IEPG, ANA, RF, EMA, T and B cell analysis
IgG subclass deficiency:
IgG subclass deficiency is defined as the relative lack of one or more IgG subclasses, with a normal concentration of total serum IgG. If total IgG is low, the diagnosis should be common variable immunodeficiency.
There are four IgG subclasses: IgG1 (60 to 65% of total IgG), IgG2 (20 to 25%), IgG3 (5 to 10%), and IgG4 (3 to 6%). Most individuals lacking one or more IgG subclasses are asymptomatic. However, some present with recurrent sinopulmonary infections, diarrhoea, inhalant and food allergies, autoimmune disease, and epilepsy. Various IgG subclass deficiencies, occurring alone or in combination, have been described. Deficiency of the IgG2 subclass is detected most commonly in symptomatic paediatric patients, most of whom are male. It may occur in isolation, but is also frequently associated with deficiency of IgG4 and/orIgA. Selective deficiency of IgG3 alone is observed more common in adult females and is associated with the same clinical picture as previously described.
Suggest: serum Immunoglobulins, EPG, IEPG, ANA, RF, EMA, T and B cell analysis. Since most children have been immunised with tetanus toxoid and H. influenzae type B, antibody titers may be determined. If titers are low, the response to booster immunisation should be assessed.
For the rigorous assessment of polysaccharide responses, patients may also be immunised with pneumococcal or meningococcal vaccines.  However, these vaccines are unreliable in normal children under the age of two years, and negative responses should be interpreted with caution.
Increased IgM:
1. Waldenstrom’s macroglobulinemia
2. Malaria
3. Infectious mononucleosis
4. Lupus erythematosus
5. Rheumatoid arthritis
6. Dysgammaglobulinemia (certain cases) 
Decreased IgM:
1. Agammaglobulinemia
2. Lymphoproliferative disorders (certain cases)
3. Lymphoid aplasia
4. IgG and IgA myeloma
5. Dysgammaglobulinemia
6. Chronic lymphoblastic leukaemia
Suggest: serum IgG subclasses, EPG, IEPG, ANA, RF, EMA, T and B cell analysis.
IgA deficiency:
This is a common immune deficiency effecting 1:5-600 individuals. Although IgA deficiency may be seen in clinically healthy subjects it is associated with an increased risk of sino-pulmonary infections, allergy, coeliac disease, rheumatoid arthritis, SLE and lymphoma.
Decreased IgA:
1. Congenital deficit
2. Ataxia-telangiectasia
3. Late pregnancy
Suggest: IgG subclasses, ANA, EMA, anti-IgA antibodies
Increased IgA:
1. Chronic, nonalcoholic liver diseases, especially primary biliary cirrhosis (PBC)
2. Obstructive jaundice
3. Exercise
4. Alcoholism
5. Subacute and chronic infections
6. Abstinence from alcohol after a period of 1 year
7. Drugs and dextrin
8. Protein-losing enteropathy
Suggest: serum IgG subclasses, EPG, IEPG, ANA, RF, EMA, T and B cell analysis
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Bence Jones Proteins in Urine

Monoclonal immunoglobulin light chains in dimers may be detected in the urine in association with myeloma (light chain, IgG and IgA). BJ proteins may also be detected in the urine of patients with macroglobulinaemia, lympho and myelo-proliferative disorders, pyuria and renal diseases.
Suggest: serum EPG, IEPG, serum immunoglobulins, T and B cells if clinically indicated.  
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CSF oligoclonal bands 

The detection of oligoclonal bands in CSF, but not serum indicates local CNS synthesis of immunoglobulins. This is seen most frequently in patients with MS and SSPE. The test is not specific for these conditions and oligoclonal bands may also be detected in CNS infections (HIV and syphilis), Guillain Barre syndrome and after CVA. Suggest: serum immunoglobulins, EPG, IEPG. 
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Cryoglobulin Test 

Cryoglobulins are protein complexes that undergo reversible precipitationat low temperatures and redissolve on warming. This test provides additional diagnostic information about certain disorders such as malignant B-cell diseases, autoimmune disorders, acute and chronic infections, and primary cryoglobulinemia in persons with cold hypersensitivity. HCV infection is the most common cause of cryoglobulins. Detection of cryoglobulins is highly specific for immune complexes. Three different types of cryoglobulins have been described:
Type I: there is a monoclonal immunoglobulin that is most often due to underlying multiple myeloma or Waldenstrom's macroglobulinemia. In multiple myeloma, cryoglobulin-induced glomerular disease must be distinguished from primary amyloidosis and from light chain deposition disease.
Type II: or essential mixed cryoglobulinemia, in which the cryoglobulin contains both a polyclonal IgG (which may either act as an antigen or be directed against an antigen) and a monoclonal IgM rheumatoid factor directed against the IgG. It now seems clear that most cases are due to chronic infection with hepatitis C virus (HCV).
Type III: there is also a mixed cryoglobulin, but both the IgG and the rheumatoid factor IgM are polyclonal. This condition is often seen in chronic inflammatory and autoimmune disorders (such as lupus and leukocytoclastic vasculitis), lymphoproliferative malignancies, and, in as many as 50% of cases, HCV. Disorders associated with cryoglobulinemia include the following:
1. Acute and chronic infections:
2. HVC
3. Endocarditis, Infectious mononucleosis, Cytomegalovirus and syphilis
4. B-cell neoplasia (eg. multiple myeloma, Waldenstrom’s macroglobulinemia, chronic lymphocytic leukaemia)
5. Autoimmune diseases (eg. rheumatoid arthritis, Sjogren’s syndrome, SLE)
6. Sarcoidosis
7. Acute poststreptococcal glomerulonephritis
8. Cirrhosis
9. Haemolytic anaemia  
Suggest: EPG, IEPG, ANA, ENA, DNA, RF, T and B cells.
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HLA B27 

HLA B27 is a marker for genetic predisposition to a variety of inflammatory and rheumatic diseases (ankylosing spondylitis, reactive arthritis, uveitis). The antigen is found in 9% of the normal population.
Suggest: interpret the results in the clinical context. Further Information: 

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