Immunofluorescence pattern of Antinuclear Antibody and its Association with Autoantibody profile in Systemic lupus erythematosus

Background: Antinuclear antibody (ANA) is useful in the diagnosis of systemic lupus erythematosus (SLE). Association of specific autoantibodies with the immunofluorescence pattern of ANA in SLE as noted in Western literature has been taken as reference in all over the world. However, in Bangladesh such research work or data correlating the autoantibodies and their ANA patterns is inadequate. Objective: To identify an association between immunofluorescence patterns of antinuclear antibody on HEp-2 cell and more specific antinuclear reactivities (e.g. anti-dsDNA and anti-extractable nuclear antigen) in the serum samples of SLE patients. Methods: Serum samples of 37 SLE patients who were diagnosed by ARA (American Rheumatism Association) classification criteria and laboratory tests, attending at lupus clinic of Bangabandhu Sheikh Mujib Medical University (BSMMU) during the study period of six months were subjected for ANA testing by Indirect Immunofluorescence (IIF) on HEp-2 cell, anti-dsDNA by ELISA and antiextractable nuclear antigen (anti-ENA) by Dot Immunoblot. Dot blot strips were tested for anti-Sm, anti-RM, anti-SSAlRo, and anti-SSB/La. Results: Out of 37 SLE patients 32 (86.5%) cases were ANA positive by IIF on HEp-2 cell. ANA positive sera exhibitedthree fluorescence pattems such as speckled (43.7o/o),peipheral(34.3%) andhomogenous pattem (21.8%). Peripheral pattem (100%) was strongly associated with anti-dsDNA (p<0.05) and homogenous pattern (85.7%) was also predominantly associated with anti-dsDNA (p<0.05). Speckled pattem (85.6%) was significantly associated with anti-ENA (p<0.05). Anti-dsDNA was positive in 7 5Yo of SLE cases and majority (45.8%) of which showed peripheral pattem whereas anti-ENA was positive lm48.6% cases and maju"tty (70.5%) of which showed speckled pattem. The most commonly identified antinuclear autoreactivity was directed towards anti-RM (22.2%) then anti-Sm (16.6%), anti-SSA (16.6%) and anti-SSB (ll.l%). Multiple anti-ENA reactivities were identifiedn33.3% cases. Conclusion: Peripheral and homogenous pattern is stongly associated with anti-dsDNA therefore may be predicted that patients have active SLE and speckled pattern may predict anti-ENA (specially ribonucleoprotiens). Thus, ANA-IIF method may sufftce and probably reduce the expense of detailed immunological work-up with minimal loss in diagnostic accuracy.

autoantibodies.For that pu{pose, additional tesJing is required for the detection of specific autoantibodies o, t.
There is neither research work nor arry data available correlating the autoantibodies and their ANA patterns in Bangladesh.
In this study, samples of SLE patients were analyzed for ANA testing by IIF method and further processed for identification of specific autoantibodies by ELISA (anti- dsDNA) and Dot immunoblot (anti-ENA).The results were correlated with one another to establish any definite link between the fluorescence pattern and specific autoan- tibody reactivities.If a definite correlation is found between the ANA patterns and immune profile, one could use ANA-IIF fluorescence patterns to predict the presence of autoantibodies to diagnose SLE.This would economize on the cost of laboratory investigations.

Methods :
A total of 3 7 dragnosed SLE patients, attending at lupus clinic in BSMMU during the study period of M ay,2010 to October,2010; were enrolled in this cross sectional study.
Blood (5 ml) was drawn from SLE patient and sera were separated from the clotted blood samples of all SLE patients by centrifugation.Sera were stored at 4"c if testing was planned withinT2 hours or at -20"c for testing after three days (without freeztng and thawing).Each of the serum samples was subjected for ANA testing by Indirect Immunofluorescence on HEp-2 cell, Dot immu- noblot for anti-ENA and ELISA for anti-dsDNA.
Indirect immunofluorescence was done by a commer- cially available kit.Serum diluted Il40 in phosphate buffered saline (PBS) was overlaid onto fixed HEp-2 cell (ALPHADIA, Belgium) for 30 minutes at room tempera- ture.Slides were washed twice for five minutes each with PBS, overlaid with fluorescence labeled conjug ate, which is antihuman IgG heavy and light chain specific and incubated for an additional 30 minutes.After washing twice, a coverslip was placed over the slide, and the slides were read using a fluorescence microscope atx40 powera.
The main fluorescence patterns seen were speckled, homogeneous, and peripheral.
The Dot immunoblot method is a qualitative assay, which utilizes strips of nitrocellulose on which purified antigens are blotted at prelocated spots.Coated antigens used in this study were Sm, RNP, ssA and ssB.The antigen sources used are bovine and rabbit thymus (SSA and Sm) or calf spleen and rabbit thymus (SSB and Sm/RNp).The test procedure was performed according to directions supplied by the manufacturer (D-tek, Belgium).Test strips were incubated for 10 minutes with a diluted patient serum in a PBS-T\Meeen solution.Subsequently the test strips were washed by gentle agitation in a test tube filled with PBS-Tween for 1 min.After the excess buffer solution was removed with a filter paper, the test strips were incubated with an alkaline phosphatase-protien A conjugate for 10 min.The test strips were then washed for 1 min by gentle agitation by PBS-Tween.Again excess buffer was removed with filter paper.Finally the test strips were stained with 5-bromo-4-chloro-3-indolylphosphate for 5 min.The reaction was terminated by washing the test strips with deionized water.The strips were then air dried.
only strips on which the positive control position was stained as a clearly marked blue spot were able to be evaluated and used for this study6.
Antibody against dsDNA was detected by commercially available ELISA kit (Orgentec, Germany).Micro wells were pre-coated with calf thymus dsDNA antigen.The calibrators, controls, and diluted patient samples were added to the wells and autoantibodies recogn izing the dsDNA antigen bind during the first incubation.After washing the wells to remove all unbound proteins, purified peroxidase labeled rabbit anti-human IgG conju- gate was added.The conjugate binded to the captured human autoantibody and the excess unbound conjugate was removed by a further wash step.The bound conjugate was visualized by TMB substrate which gives a blue reaction product, the intensity of which is proportional to the concentration of autoantibody in the sample.phos- phoric acid was added to each well to stop the reaction.This produces a yellow end point colour, which was read at 450nm.Data analysis was performed using SPSS pC version 10.
A chi-square test was used to identify association." A p value <0.05 was considered signific arfi.

Results :
In this cross sectional study, 37 samples of SLE patients were analyzed who were diagnosed by ARA (American Rheumatism Association) criteria.We collected serum samples of various stages of SLE patients where autoanti- bodies were fluctuated by the disease activity.The study tried to find out the correlation of ANA fluorescence pattern with specific autoantibodies.Among these samples, 32 (86.5%) were ANA positive by IIF method.
Three patterns of nucleat fluorescence were noted: the homogenous pattern, in which the entire nucleus fluoresced (Fig. l); the peripheral pattern, in which the fluorescence is located along the rim of nucleus (Fig. 2); and the speckled pattern in which the fluorescence is localized as discrete spots in the nucleus (Fig. 3).In these fluorescence positive samples, speckled pattern was the most common pattern seen rn 43.7% cases, followed by peripheralpattern 34.3% and homogenous pattern 2I.8%.
ANA-IIF negativity was observed in 5 (13.'5yo) of the total 37 samples under study.Of these, 4(80%) exhibited positivity with anti-dsDNA.There"was a single case in the entire study that exhibited strong positivity for SSA/Ro but negative for ANA-IIF.