Che Maraina, Che Hussin
(2013)
Distribution of anti-nuclear antibodies (ANA),
anti-dsdna and extractable nuclear antigens
(ENA) antibodies among apparently healthy
malay women and determination of ana cutofftitre.
Other.
Pusat Pengajian Sains Perubatan.
(Submitted)
Abstract
Distribution of anti-nuclear antibodies (ANA), anti-dsDNA and extractable nuclear
antigens (ENA) antibodies among apparently healthy Malay women and determination of
ANA cut-off titre.
Previous studies discovered that autoantibodies were present in healthy people at various
dilutions. Some autoantibodies play a very important role in the pathogenesis, diagnosis
and prognosis of autoimmune diseases including antinuclear antibodies (ANA), antidsDNA
and ENA antibodies. In diagnostic procedures, establishing a cut-off value that
can discriminate between healthy and diseased people is very important to reduce false
positive results. Therefore, this study aimed to determine the cut-off titre for ANA test,
the distributions of ANA, anti-dsDNA and ENA antibodies among apparently healthy
Malay women and the seroconversion of ANA positivity after 6, 12 and 18 months in this
cohort.
This study was divided into two phases. Phase I was designed as a case-control study that
involved a group of 59 SLE patients as cases and 101 healthy women as controls at childbearing
age. Medical records review was used to obtain ANA results of the patients.
Healthy women were recruited from HUSM based on the inclusion and exclusion criteria.
Phase 2 was designed as a prospective cohort study and was a continued evaluation of
healthy women for their ANA, anti-dsDNA and ENA antibodies levels. The blood was
tested for ANA and anti-dsDNA antibodies using immunofluorescence method that used
HEp-2 cells and Crithidia luciliae as the substrate, respectively. The current cut-off titres
for ANA and anti-dsDNA antibodies tests are :0:1:40 and <:I :10, respectively. ENA
antibodies were determined based on the principle of an enzyme immunoassay. Twelve
antigens coated on a strip were tested. A sample was considered positive for a specilic
antibody if the colour intensity of the corresponding antigen dot was higher than the
intensity of the negative control dot. Data entry and analysis were done using Predictive
Analytics SoftWare (PASW) Statistics 18. Receiver operating characteristics (ROC)
curve was plotted in StataSE 9 for determination of ANA cut-offtitre.
In healthy women aged 20 to 40 years, the prevalence of ANA positive and ENA positive
were 3.0% (0, 6.0) and 5.0% (0.6, 9.4) respectively. Three subjects had positive ANA
composed of one positive subject at the highest dilution I :40 and two subjects at 1:80. All
0 Division of Research & Innovation- Universiti Sains Malaysia -20130118
positive subjects displayed speckled pattern. No positivity of anti-dsDNA antibodies was
found in all the subjects. For ENA antibodies test, one subject was positive for RNP
antigen, three subjects for SSA antigen and one subject for PM-Scl antigen. However,
these five positive subjects were found to be ANA-negative. The follow-up on ANA
seroconversion at 6-month, 12-month and IS-month period found three subjects with
circulating ANA in their bloodstream which occurred at fluctuating dilutions. Eleven
subjects had detectable ANA at least once. The highest dilution of ANA was detected at
l :80. Based on the ROC curve, the cut-off titre for ANA was chosen at I :80 with 97.4%
sensitivity and 83.5% specificity.
In conclusion, the prevalence of ANA in healthy Malay women at this population was
comparatively low as compared to other studies. Anti-dsDNA antibodies were not
detected, which explained the significance of these antibodies as a specific diagnostic
marker for SLE. This study also discovered the positivity of ENA antibodies in five
negative-ANA subjects. There was insufficient information to conclude the significance
of this event in non-symptomatic subjects. The fluctuations of ANA positivity in healthy
women over 2-year period occurred mainly below the level of the current cut-off titre,
l :40. The cut-off titre for ANA was determined at I :80.
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