Laboratory Studies in Autoimmune Diseases

Abstract

Autoimmune diseases (AID) are a great percentage of the patients needing to be seen by Rheumatologists. Their initial work-up by their primary care physician (PCP) can be helpful in determining who needs to be seen in consultation. If interpreted properly, initial laboratory studies can help the PCP know when to initiate a consultation. The following laboratory studies as outlined will provide the PCP with the necessary information to determine if a further AID work-up is indicated.

Introduction and General Studies

There are general laboratory studies which can indicate the possibility of an underlying AID. These include the complete blood count (CBC), urine analysis (UA), sedimentation rate (ESR), C-reactive protein (CRP), comprehensive metabolic panel (CMP), and muscle enzymes. In the CBC, we are looking for an anemia of inflammatory disease which can develop. In active inflammatory diseases, absorption of iron is limited and not distributed to the bone marrow resulting in a chronic anemia unless the inflammation is controlled. We also look for acute falls in hemoglobin and hematocrit, mainly seen in patients with systemic lupus erythematosus (SLE) or Sjogren’s Syndrome (SS). They have developed autoantibodies to red cells. The most common cause of leukopenia and thrombocytopenia is due to an autoimmune disease with the development of anti-white cell and anti-platelet antibodies in these patients causing peripheral destruction.

Urinalysis as a screen is self-evident. Acidic pH, proteinuria, red cells, white cells, and red cell casts are all evidence of possible autoimmune renal damage. If the UA is abnormal, a 24-hour urine for creatinine clearance and total protein is ordered. If abnormal, a renal biopsy is indicated to determine etiology and possible therapy.

General inflammation is monitored by ESR and should be less than 15 mm/hr. CRP should be <0.3 mg/dl and ideally <0.1 mg/dl. Values out of this range may indicate a possible inflammatory process and need for further work up.¹

The CMP findings are again fairly self-evident. Increased creatinine indicates renal disease, increased liver enzymes, a possible hepatitis or drug reaction, and increased muscle enzymes, a possible myositis. It is emphasized that all patients with possible myositis should have all three muscle enzymes (creatinine phosphokinase (CPK), aldolase, lactic dehydrogenase (LDH)) evaluated. One thing we emphasize is in different genetic populations normals can be different. African-Americans have a higher normal CPK, between 200–400 IU. Caucasians and Asians should be less than 200 IU, but, CPK is a very sensitive assay. Exercise can elevate CPK in many patients. Aldolase and LDH are not affected and can be relied on to complete an evaluation of myositis. Neopterin and von Willebrand’s Factor Antigen can, also, be measured. One thing that is most misunderstood in the CMP is the albumin/globulin ratio. Albumin should be around 4 g/dl and globulin 2.5 g/dl, so the ratio should be around 1.6 normally. If that ratio is closer to 1 or less than 1 then you have a reversal of the albumin/globulin ratio and possible hypergammaglobulinemia. This is usually the first evidence that there may be an underlying inflammatory process or a patient with an autoimmune disease. These are the screening tests and thought processes that should be entertained to determine a more definitive work up.²

A more definitive work up for AID includes rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibodies, antinuclear antibodies (ANA), complement levels, antiphospholipid (APLS) antibodies, anti-neutrophilic cytoplasmic antibodies (ANCA), and genetic studies.

Rheumatoid Factor

The RF that is measured by most assays is a 19S IgM molecule; however, RFs are found in all immunoglobulin classes. It is found in a higher percentage of rheumatoid arthritis (RA) patients, about 80–90%, but can be seen in all connective tissue diseases. By definition, it is an anti-IgG globulin that binds to the Fc fragment of IgG forming an immune complex. By enzyme-linked immunoabsorbent assay (ELISA), an abnormal level is > than 20 IU, but normally RA patients are > 40 IU. About 10–30% by ELISA depending on the sensitivity are positive in juvenile idiopathic arthritis (JIA) patients, and about one-third of patients with SLE and scleroderma (SSc) are positive for RF; however, about 80% of patients with SS are positive. There are a multiplicity of viral infections that cause transient RF positivity including the Epstein-Barr virus (EBV), cytomegalovirus (CMV), parvovirus, and hepatitis B and C. These are usually negative for αCCP antibodies. RF also can be found in extravatory fluids. In fact, when you look at extravatory fluids, synovial, pleural, and pericardial fluid, these fluids have a high propensity for immune complex formation, so you are going to find autoantibodies in the extravatory fluids in higher concentrations than in the peripheral blood.³

Anti-Cyclic Citrullinated Peptide Antibodies

The second antibody that we assay for in RA seems to correlate better with disease and is called αCCP antibodies. These are antibodies that are reactive with synthetic peptides containing the amino acid citrulline, the most translationally modified arginine residue found in approximately 75–80% of adult RA patients. Testing in most assays is only for the IgG antibody to CCP. Studies in Sweden have shown that they may appear many years before actual disease.³

eTa Protein

Recently, a possible new inflammatory marker has been identified. They are called 14-3-3 eTa proteins, ubiquitously expressed intracellular isoforms. These isoforms are expressed in inflammatory processes and are detected mainly in RA and in psoriatic arthritis.

Antinuclear Antibodies

ANA positivity is a common cause for referral to Rheumatologists. ANA are a diverse collection of antibodies directed against numerous macromolecules which are normal constituents of the cell nucleus. ANA presence are a hallmark of AID, but not diagnostic. About 30% of the healthy female population will be ANA positive. If positive, then an ANA profile is indicated to determine the exact antibody causing the ANA to be positive. Again, immunogenic viruses such as EBV, CMV, and parvovirus can cause a transiently or permanently positive ANA. Also, each year we see probably five to ten patients referred to us with a positive ANA that have autoimmune thyroiditis with antibodies to thyroperoxidase (TPO) or thyroglobulin. ANA are a screening test. It can define a specific AID, but titers, rising or falling, are not associated with disease activity. However, some autoantibodies may define rises and falls in disease activity.⁴

The most widely used technique for the detection of ANA is immunofluorescence (IF) using human epithelial cells (Hep-2). These cells have abundance of double (ds) and single-stranded (ss) DNA, histones, Sm, RNP, Scl-70, and SS-B substrate, but a very low concentration of antibodies to SSA or cytoplasmic antigens. Therefore, even if the ANA is negative, if SS is suspected, antibodies to SSA should be run. By IF, five patterns are generally described. The homogenous pattern which is very nonspecific, the speckled pattern which is produced by a multitude of antigens, the rim pattern, the nucleolar pattern which is usually found in patients with scleroderma and scleroderma dermatomyocitis overlap, and the fifth pattern is discreet speckled which is what we see with antibodies to centromere. If the IF ANA is positive, an ELISA profile for the specific antibodies is needed. Most profiles will contain antibodies to dsDNA, SSA, SSB, Sm, RNP, and chromatin. They may not contain antibodies to Scl70, histones, Jo-1, RNA polymerase III, centromere, and lesser antibodies. You may have to ask for a specific assay or ELISA for those.⁵

Autoantibodies in SLE

ANA should be positive in 100% of SLE patients by one method. Antibodies to dsDNA occur in about 75% of patients. These are the patients that have high incidence of renal disease, central nervous system (CNS) disease, vasculitis, and low complement levels. In most patients, these levels will rise and fall with active disease. There are two assays for antibodies to dsDNA. There is the ELISA assay which is sensitive but it can give positive results for ss DNA. It can also cause false positivity with certain drugs, hepatitis B and C, etc.. High titers are very specific for SLE. The most specific assay is IF with Crithidia luciliae substrate. It is completely specific for dsDNA antibodies. High titers are very specific for SLE and correlate with very active disease.⁵

Laboratory studies as outlined in this article will provide the PCP with the necessary information to determine if a further autoimmune diseases work-up is indicated.

Other autoantibodies include antibody to chromatin, antibodies to nucleosomes, which occur in about 75% of patients. These are also patients who usually have renal disease. Antibodies to chromatin may occur with antibodies to histones in drug-induced syndromes, especially associated with oral contraceptives of high estrogen content, propylthiouracil, and some of the hypertensive medications. A more specific antibody that is part of the criteria for SLE, are antibodies to Sm and occurs in about 30–40% of patients. This antibody correlates with pleural or pericardial disease, so if antibody to Sm is positive, screening with baseline pulmonary function tests (PFTs) to look for restrictive lung disease and an echocardiogram to look for pulmonary hypertension (PAH) is indicated and repeated in two year intervals. The other antibody with the same association is antibodies to RNP. This is the antibody by itself that we define mixed connective tissue disease (MCTD). This antibody alone may correlate with milder disease or less renal disease. These are the patients with high incidence of Raynaud’s, myositis, and can have restrictive lung disease and PAH. Antibodies to SSA or Ro and antibodies to SSB or La are the antibodies we find in 30 to 60% of SLE patients. These are the antibodies seen with subcutaneous lupus and are the antibodies that cross the placenta and bind to the fetal conduction system. The fetus can have neonatal lupus. These are the patients who have dry eyes and dry mouth (sicca complaints) and have a high incidence of partial C2 and C4 deficiencies. Lastly, there is a cytoplasmic antibody called anti-ribosomal P which does correlate in certain cases with CNS vasculitis.⁶

Autoantibodies in SS

The majority of patients (90%) with SS have antibodies to SSA and/or SSB. They again correlate with the presence of vasculitis, leukopenia, thrombocytopenia, and positive RF. These are the antibodies associated with anti-white cell and anti-platelet antibodies and hypergammaglobulinemia. Seven to ten percent of these patients may have an underlying B cell hyperproliferative syndrome and so at baseline, measure IgG levels. IgG levels over 2,000 mg/dl at baseline may indicate a propensity for developing a B cell lymphoma. Anti-SSB antibodies are usually associated with anti-SSA antibodies. Anti-SSB antibody by itself may indicate an underlying lymphoproliferative disease.^{5, 6}

Autoantibodies in Polymyositis (PM)/Dermatomyositis (DM)

There are multiple antibodies in PM/DM. These are anti-synthetase antibodies. The most common is anti-histidyl tRNA synthetase antibody (anti-Jo-1). It occurs in about 40% of patients with PM/DM. It is also associated again with restrictive lung disease, so these patients need PF Ts and followed very carefully. There are a multiplicity of other autoantibodies which can be defined by a myositis panel which include antibodies to PL-7, PL-12, OJ, EJ, SRP, Zo, KS, Mi2, SAE, TIF1-¥, NXP2, and Ha. A small percentage of patients have each of these, but their presence is very specific for an underlying autoimmune myositis. Myositis-associated interstitial pneumonitis can be found in 95% of patients that have antibodies to PL-12, KS, and OJ. Arthritis, Raynaud’s, fever, mechanic hands, Gottron’s papules, and heliotropic rash have a high association with the presence of anti-Jo-1 and PL-12 antibodies. There is also an anti-HMGCoA antibody which we mainly see in patients with statin induced myositis.⁷

Autoantibodies in SSc

There are three more common antibodies found in SSc, anti-centromere, anti-topoisomerase (Scl70), and anti-RNA polymerase III antibody. Anti-centromere antibody is usually found in limited scleroderma (CREST). They form the discreet speckled pattern ANA. This form is not benign with a good percentage of these patients developing PAH. Therefore, these patients with anti-centromere antibody should also be followed closely with PFTs, echocardiogram, and possibly right heart catheterization if there is evidence that the pulmonary artery pressure is greater than 35 mmHg on estimate from echocardiogram. Antibodies to Scl70 and RNA polymerase III are associated with a, sclerodactyly and diffuse skin involvement. A high incidence of these patients develops restrictive lung disease. So, PF Ts and echocardiogram need to be followed. The importance of the antibody to RNA polymerase III to clinicians is there is a high incidence of the patient having an underlying cancer. Therefore, cancer screening has to be emphasized. Chest x-ray, mammograms, pelvic, and pap smear, and colonoscopy have to be up to date on any of these patients with antibody to RNA polymerase III. The other antibodies to PM-Scl, fibrillarin, high nuclear RNP1, etc., are found in a small percentage of patients.⁸

Overall, specificity of ANA presence in AID in SLE are antibodies to dsDNA, Sm, RNP, SS-A, and SS-B, antibodies in MCTD, are specifically antibodies to RNP alone, antibodies in SSc to centromere, topoisomerase I (Scl70), and RNA-polymerase III, antibodies in PM/DM are antibodies to Jo-1, etc., and in SS antibodies to SS-A and SS-B. One needs to look at these and then make a specific diagnosis. Many times it’s more important to actually know what antibody is present than specifically making a specific disease diagnosis because many of these antibodies define what organ systems are involved. These include antibodies to dsDNA in renal disease and vasculitis, antibodies to Sm/RNP/centromere/Jo1/RNA polymerase III, and Scl70 for pulmonary and pleural involvement, and antibodies to SS-A and SS-B for sicca complaints and hypergammaglobulanemia.⁵

Complement Measurement

Measuring disease activity directly is difficult, because none of the assays give direct disease measurement, just indications. These are immune complex diseases, but the immune complex assays available are not very helpful. We do use complement protein measurements to aid in defining activity of disease. Immune complexes fix complement; and, therefore, we may see a fall in complement proteins. Three complement pathways are present, the classical, mannose binding lectin, and the alternative pathway. Most of the time in our AID, we are dealing with the classical pathway. We mainly assay for CH50, C4, and C3. C4 is the rate limiting step of the classical pathway and therefore it falls quicker and is more sensitive to immune complex activation. However, you need to know the genetics. There are four alleles coding for C4, two for C4A and two alleles for C4B. Knowing the genetic make-up will determine if low C4 levels are significant. Measuring C3 can give you information on all three pathways. Measuring CH50 gives you a picture of the whole pathway to screen to rule out complement deficiencies. If you have a low CH50 with a normal C3 and C4 then you have evidence of a complement deficiency. You then measure each component separately to determine the deficiency. About 1.9% of the population has a partial C2 deficiency and about 0.7% of the population has a partial C4 deficiency. Partial C4 deficiencies are present in a high number of SLE patients.^9,10

Anti-Phospholoipid Antibodies

Any patient evaluated with AID, we want to define whether there is any underlying anti-phospholipid syndrome (APLS). Many patients with SLE may also have associated APLS, but APLS can also be primary. Measuring anti-cardiolipin antibodies, lupus anti-coagulant, and anti-beta 2 glycoproteins on a fasting specimen is required. The IgG antibody of the three is associated with more clotting abnormalities and symptoms of migraine headaches and miscarriages in the family. The presence of these antibodies can cause problems with pregnancy, so you want to counsel any young female about these antibodies before they become pregnant.¹¹

Anti-Nuclear Cytopasmic Antibodies (ANCA)

We now have assays (IF and ELISA) that can screen for vasculitidies, such as granulomatosis with polyangiitis (GPA) and microscopic polyarteritis (mPA). There are two types of ANCAs by IF; cANCA which is cytoplasmic ANCA and pANCA which is perinuclear ANCA. cANCA are antibodies to proteinase 3 (PR3) which is seen in about 90% of patients with GPA. In most patients with GPA, it can be correlated with disease activity. These antibody levels will rise and fall with disease activity. pANCA is antibody to myeloperoxidase (MPO). These are antibodies seen in eosinophilic GPA, microscopic polyarteritis, a small percentage in inflammatory bowel patients, and in drug-induced syndromes, especially in patients on minocycline or thyroid patients on propylthiouracil.¹²

Genetic Studies

These studies can be helpful in evaluating young adults for seronegative spondyloarthritides. In ankylosing spondylitis about 90% of those patients will be positive for HLAB27. Those with enthesitis-related arthritis will be positive in about 85% of cases. A smaller percentage of inflammatory bowel disease and those with psoriatic arthritis will be positive, especially those with spinal involvement. HLAB51 positivity can be found in about 60% of patients with Behcet’s.¹³

Additional Studies

What other studies can be helpful? In patients that have peripheral vasculitis, you may want to measure cold precipitable proteins or cryoglobulins. The problem is that many labs do not perform this assay correctly. The blood has to be drawn in a tube that is at 37 degrees or body temperature. After drawing the blood, it has to be placed in a 37 degree water bath to clot, the serum aspirated, and then placed at 4 degrees and observed. If a precipitate forms, a cryoglobulin is present and is very specific for immune complex presence. There are three types of cryoglobulins, monoclonal, mixed, and polyclonal. What we look for in AID disease are the mixed or polyclonal. Also, about 20 % of patients with hepatitis B and C will be positive for cryoglobulins.

Organ Evaluating Studies

Skin biopsies may help define an immune complex disease, a 24-hour urine for creatinine clearance and total protein spillage may determine if a renal biopsy is needed, PFTs for restrictive lung disease, echocardiogram for PAH, and barium swallow for esophageal problems may be helpful to evaluate specific organ disease. The skin biopsy may show a lympho-proliferative response by H&E staining. IF studies for immunoglobulin and complement deposition are more specific for immune complex deposition. Immune complex renal disease in SLE is defined by IF studies showing immunoglobulin and complement deposition on the biopsy, which will determine severity and the future medical regimen.

Synovial Fluid

A normal synovial fluid is straw colored, transparent, very viscous with <200 WBCs/mm. In inflammatory arthritis, such as RA, you are going to see cloudy fluid, less viscous because of the inflammation, and the white count is going to be anywhere from 2,000 to 100,000/mm. In trauma, you may see straw to bloody-grayish fluid, transparent, and viscosity is high because of not many inflammatory cells, mainly red cells. Septic fluid is going to be bloody and it’s going to be turbulent, very low viscosity, and the white count usually will be greater than 20,000/mm, but gram staining the fluid and cultures are indicated to define further. Pseudogout and gout are going to produce an inflammatory fluid with low viscosity and crystals are to be evaluated by polarizing microscope. ¹⁴

Overall, these outlined studies will provide an excellent evaluation of a patient for AID, and provide information to provide a correct diagnosis and help in determining a therapeutic regimen for the patient.

Biography

Terry L. Moore, MD, FACP, FAAP, MACR, is the Director, Division of Adult and Pediatric Rheumatology, Professor of Internal Medicine, Pediatrics, and Molecular Biology and Immunology at Saint Louis University Medical Center and SSM Cardinal Glennon Children’s Hospital. Austin M. Dalrymple, DO, is Senior Fellow, Pediatric Rheumatology, at Saint Louis University Medical Center and SSM Cardinal Glennon Children’s Hospital.

Contact: mooretl@slu.edu