The demand for antinuclear antibody (ANA) testing has risen sharply over the past decade due to the difficulty in diagnosing connective tissue disease (CTD) and the increasingly common practice of using the ANA test to rule out an autoimmune disease. Traditionally, indirect immunofluorescence (IFA), using HEp-2 cells, has been the method used to test for ANA. IFA is a labor-intensive, subjective method requiring highly trained medical technologists to interpret results that are always subject to reader bias. In addition, IFA has variables that can cause inconsistent results between laboratories. To meet the increased demand for ANA testing, alternative methods have been developed. These include enzyme-linked immunosorbent assay (ELISA) and multiplex microsphere immunofluorescence antibody detection (MFAD) patented by Luminex Corp.
ARUP Laboratories perform a large volume of ANA tests-more than 15,000 every month. To keep current with what is commercially available and how these assays perform, we have been involved in multiple studies over the past few years investigating MFAD, ELISA and HEp-2 IFA assays for ANA. Using clinically defined disease sera, which included but was not limited to rheumatoid arthritis (RA), systemic lupus erythematosis (SLE), Sjögren syndrome, polymyositis and scleroderma, along with healthy donor sera, we examined multiple vendor assays utilizing each of these methodologies.
The MFAD method utilizes 5.4 micron polystyrene microspheres impregnated with a unique ratio of red and infra-red fluorescent dyes. This patented process creates 100 unique ratios of the two fluorophores, allowing detection of multiple antibodies utilizing flow cytometry. We investigated commercially available MFAD systems and found two compared well with HEp-2 IFA. A limitation of MFAD is the limited number of antigens present and often the lack of HEp-2 substrate.
ELISA utilizes a 96-well polystyrene microtiter plate read by a spectrophotometer at the end of the reaction. Most ANA ELISA assays contain these antigens: dsDNA, SSA 60 kDa, SSA 52 kDa, SSB, Scl-70, U1 RNP, Smith, Jo-1, ribonucleo protein, Pm/Scl, histone, mitochondria and chromatin; most contain a disrupted HEp-2 substrate, which contributes to the assay's high sensitivity. Over the past 12 months, we reviewed seven ANA ELISA assays that are FDA cleared and commercially available and determined that the current ANA ELISA assays containing the disrupted HEp-2 substrate are at least, if not more, sensitive than the ANA IFA method for screening ANA. The sensitivity among the seven assays ranged from 90-97%, compared to as low as 80% using the HEp-2 IFA method. However, the ELISA assays may miss the rare speckled pattern and antibody to fibrilarin.
IFA is labor intensive and requires an experienced medical technologist. With the current medical technologist shortage, individuals with experience in reading IFA are often hard to find. At the ARUP Autoimmune Laboratory, we have eight medical technologists with two to 10 years of experience reading IFA daily. Recently, we compared HEp-2 IFA assays from five vendors for standardization. Using the sera mentioned above, we found the five assays agreed from 44 to 92% of the time depending on the disease group or healthy donor group being examined. Agreement between the five assays was established when the serum being tested gave the same titer, plus or minus one doubling dilution. The agreement for the clinically defined SLE sera was only 74% among the five assays. Thus, the five HEp-2 assays did not appear to be well standardized. The five assays contained different conjugates, f/p ratios and fixatives, which affected the final result.
In 1996 the National Committee for Clinical Laboratory Standards (NCCLS) recommended the use of an IgG-specific conjugate. Polyconjugates secondary antibodies detect IgM antibodies due to rheumatoid arthritis, medications, age and other conditions usually not of diagnostic significance. When reviewing CAP ANA surveys, results for the same sample vary from a titer of 1:40 to 1:1,280 between laboratories. Sensitivity of the HEp-2 IFA assay was as low as 80% when testing clinically defined SLE sera. In addition, HEp-2 IFA is prone to missing some SSA speckled patterns.
Which is Best?
Which assay is best for screening ANA has been hotly debated over the past few years, with many papers published on this topic. Moreover, with the increased number of ANA tests being performed, some reports are finding titers as high as 1:320 in healthy individuals. If there was one test that was 100% sensitive and specific, we would likely all be using it. There is, however, not a perfect test for ANA as yet. CTD is diagnosed predominately by the patient's symptoms and medical history. To add more to the issue, standardization does not exist within or between the ELISA, IFA or MFAD methodologies.
For example, the ribonucleoprotein (RNP) antigen used in one assay is not the same RNP antigen used in other assays. The conjugate being used in the assay is also important to understand which antibody isotypes the test detects. As more vendors add autoimmune testing to their menu, standardization is further compromised. It is, therefore, up to the laboratory to stay abreast of the assay(s) they are using. It is important to test different patient populations and understand the limitations of the assay being used to screen ANA to assure quality results are obtained and what those results mean in the diagnosis of CTD. Understanding the differences between the methods and the differences between assays provided by the numerous vendors is essential to quality testing.
Strategies at ARUP
At ARUP, we use a combination of two methods. An ANA ELISA with a high sensitivity (over 95%) is used to screen the numerous sera submitted for ANA tests each month. The ANA ELISA assay has high sensitivity but low specificity. Therefore, all samples detected by ANA ELISA are then run on HEp-2 IFA using an IgG specific conjugate for confirmation that the sample is positive and to report titer and pattern. This allows the vast majority of the samples, which are negative, to be reported out the same day and at a low cost to the client. If the HEp-2 IFA is positive, it is recommended that extractable nuclear antigens (ENA) tests be run using MFAD to determine specificity of the antibody.
As laboratorians, we need to investigate and understand the advantages and limitations of the specific assays we use in each step to provide the highest quality results for clinicians and patients.
Susan S. Copple is Autoimmune Technical Supervisor, ARUP Laboratories.
• Copple SS, et al. Enzyme linked immunoassay (ELISA) screening then indirect immunofluorescence (IFA) confirmation of antinuclear antibodies: A statistical analysis; AJCP in process.
• Qin X, et al. Comparison of indirect immunofluorescence assay and ELISA for detecting antinuclear antibodies and anti-double-stranded DNA antibodies. Nan Fang Yi Ke Da Zue Xue Bao. 2009;29(3):472-5.
• Copple SS, et al. Comparison of three multiplex immunoassays for detection of antibodies to extractable nuclear antigens using clinically defined sera. Ann N Y Acad Sci 2007;70(7):1663-9.
• Tonuttia E, et al. Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunofluorescence for the detection of antinuclear antibodies. Evaluation of five commercial kits. Autoimmunity 2004;37(2):171-6.
• Hahm D, Anderer U. Establishiment of HEp-2 cell preparation for automated analysis of ANA fluorescence pattern. Cytometry A 2006; 69(3):178-81.