Important Serological Tests in Auto Immune Diseases
NATIONAL JOURNAL OF HOMOEOPATHY 1999 Mar / Apr VOL VIII NO 2.
Dr Camilia Rodriquez
The use and interpretation of serological tests in Rheumatological disorders are dependent on how the results relate to the clinical status. Simply put, a positive test is not an illness in the absence of clinical disease.
Important tests include reactants of Acute phase like:
- Erythrocyte Sedimentation Rate (ESR)
- C-reactive Protein (CRP)
- Rheumatoid factor/ antibody (RF/RA)
- Anti Nuclear Antibody (ANA)
- Inflammatory mediators as Ceruloplasmin, Complement etc.
- Inflammatory inhibitors as antitryspsin
- Scavengers as C-Reactive Protein (CRP)
- Proteins in repair and resolution as fibrinogen.
The Erythrocyte Sedimentation Rate (ESR) is a simple method for evaluating this Acute phase plasma protein response. Red Blood Cells (RBC) suspended in plasma normally repel each other because of their surrounding net negative charge. Fibrinogen bridges these charges and as the concentration of such molecules increase red blood cells aggregate more readily to form rouleaux structures that fall through plasma at an accelerated rate.
The Wintrobe method is preferred to the Westergreen as it is not affected by packed volume of RBC's.
Normal values of ESR are slightly higher in women than in men and aging is associated with a moderate increase in ESR. Normal values at age 50 have been taken to be 20mm at the end of 1 hour for men and 30mm at the end of 1 hour for women. An unusually low ESR < 1mm at the end of 1 hour is occasionally noted and maybe associated with Abnormal Immunoglobulin.
In the absence of erythrocyte and immunoglobulin abnormalities ESR increase reflects increase in the plasma fibrinogen which takes several days to peak. Changes in RBC morphology as anisocytosis, poikilocytosis or polycythemia etc affect the ESR and can give normal values even in the presence of an Acute phase reaction.
ESR helps to differentiate inflammatory Vs non-inflammatory joint disease (Rheumatoid arthritis versus Osteoarthritis).
Marked elevations of ESR (>100mm/hr are associated with severe deep tissue infections as Tuberculosis or Osteomyelitis, Systemic Lupus Erythematosus (SLE), Polymyalgia Rheumatica, metastatic carcinoma, myeloma etc. Increase in ESR is seen in Monoclonal gammopathy, increasing age and increasing dietary lipid intake. ESR maybe also be spuriously elevated in patients on heparin.
| Table 1 Factors That Affect The ESR | |
| Low ESR | High ESR |
| Male sex | Female sex |
| Young age | Old age |
| Sample standing before estimation | Heparinisation |
| Polycythemia | Anemia |
| Changes of red cell shape | Anisocytosis |
| Spherocytosis | Thyroid disorders |
| Anti inflammatory drugs | Oral contraceptives |
| Hypofibrinogenemia | IM injections as Penicillin |
CRP: Studies on relationship between ESR and CRP have suggested that CRP levels correlate better with degree of activity than ESR (Table 2) in certain diseases such as Rheumatoid Arthritis (RA) and Ankylosing Spondylitis (AS). CRP is used in medical practice to differentiate cystitis from pyelonephritis; Sarcoid from Tuberculosis, Crohn's disease from ulcerative colitis and viral meningitis (<10mg/dl) from Bacterial meningitis. It is also useful in MONITORING INFECTION in neonatal sepsis, post operative patients, malignancy and SLE.
In Rheumatic diseases CRP accurately reflects disease activity and is used to assess this in RA, gout, psoriatic arthritis, AS etc.
CRP levels are elevated above the normal range in most patients with active SLE and tend to fall as disease improves. Therefore it is a good index for improvement.
CRP elevation in lupus patients is an indication of infection and must be expressed quantitatively.
Rheumatoid Factors & Antibody/IES
These are autoantibodies that are directed against epitopes on the Fc portion of Immunoglobulin molecules- they occur within the IgM, IgG and IgA classes. Classical RF is the 19S IgM molecule.
In standard clinical assays, tests sera is combined with RBC's or latex particles coated with Rabbit/or human IgG and the degree of agglutination is observed. Other methods for detecting RF include ELISA and rate limited nephelometry. Rheumatoid factors are non-specific and may occur in a broad range of diseases and are found in TB, Leprosy, Chronic active Hepatitis. They are found in 40% of patients with infective endocarditis, 50%of patients with pulmonary fibrosis and virtually all patients with essential mixed cryoglobulinemia.
A critical question concerns the significance of a positive RA in a healthy person. These persons have increased susceptibility to RA.
Tests for RF are used as screening tests for Rheumatoid Arthritis as the RF is present in at least 75% of such patients using a cut off level for positivity, which excludes 95% of the normal population.
The presence of RF in Rheumatoid Arthritis indicates a poorer prognosis and high frequency of systemic and extra articular manifestation.
Anti Nuclear Antibodies (ANA)
ANA is the common denominator that unites nearly all patients with SLE. Hargreaves and colleagues initially discovered it in 1940 as the Lupus Erythematosus (LE) cell phenomenon. Indirect immunoflouresence has taken over from the LE cell test because it is more sensitive, simpler to perform and easier to interpret.
A large number of substrates are used for ANA as mouse, rat liver or kidney. Hep-2 cells (derived from laryngeal carcinoma cell line) are used with the advantage that these cells have larger nuclei to visualize. The antinuclear antibodies are visualized by adding a fluoresce in-conjugated antibody to human gamma globulin and examining the slide under UV light using fluorescence microscopy. In SLE patients ANA is positive in about 95%.
Different patterns of fluorescence can be observed depending on the types and relative amounts of autoantibodies present in the sample. (Table 2)
| Table 2 - The Relative Merits of ESR And CRP in Patients with Rheumatic Disease. | ||
| ESR | CRP | |
| Sample | Must be fresh | can be stored |
| Method | Takes at least 1 hr | can be automated |
| Variable | affected by age, sex, anemia etc | Independent of age & anemia |
| Half life | rises in 24-48 hrs and subsides with half life of 6 days | rises within 6 hrs and subsides with life of 48 hrs |
| Useful for monitoring | Chronic inflammation | Acute inflammation |
The following types can be observed:
- Homogenous: solid staining of Nucleus.
Nuclear Antigens: dsDNA, ssDNA, histories
Disease Association: SLE, lower titers in other connective tissue disorders. - Peripheral solid staining primarily: around the outer region of the nucleus.
Nuclear Antigen: dsDNA, ss DNA, histories and DNP.
Disease Association: SLE - Speckled: A fine grainy appearing staining of the nucleus.
Nuclear Antigen: Sm, RNP, SSA (Ro) SS-B (LA) Scl.70
Disease Association: High titers in SLE (Sm)
Mixed connective tissue disease
Sjogrens syndrome (SSA /SSB)
Scleroderma (Scl 70%) - Nucleolar: large coarse speckled staining of the nucleoli in the nucleus.
Nuclear Antigen: 4-6 S RNA
Disease Association: Scleroderma and Sjogrens Syndrome.
A low positive ANA (+) maybe seen in-patients on certain drugs (Isoniazide, Chlorpromazine). In chronic infection and in aging 60-75% of elderly persons without apparent disease may show a low titer positively.
