Reviewer's report 
 Ovarian antibodies as detected by indirect immunofluorescence are unreliable in the diagnosis of Title: 
autoimmune premature ovarian failure: a controlled evaluation 
Authors: 
Judy A Novosad ( janovosad@aol.com )
Sophia N Kalantaridou ( kalantas@cc1.nichd.nih.gov )
Zhi-Bin Tong ( Tongz@cc1.nichd.nih.gov )
Dr Lawrence M Nelson ( Lawrence_Nelson@nih.gov )
 1  24 Jan 2003 Version: Date: 
 Denise Faustman Reviewer: 
 A paper whose findings are important to those with closely related research interests Level of interest: 
 Unable to decide on acceptance or rejection until the authors have responded Advice on publication: 
to the compulsory revisions 
Ovarian antibodies as detected by indirect immunofluorescence are unreliable in the diagnosis of 
autoimmune premature ovarian failure: a controlled evaluation 
J. A. Novosad, S.N. Kalantaridou, Z-B Tong, and L.M. Nelson 
Summary 
In this paper, a commercially available kit for the detection of ovarian antibodies in blood serum was 
evaluated. The kit provided sections of cynomologous monkey ovary that were incubated with blood serum from patients. Serum antibodies that bound to the zona pellucida were then visualized with a goat-anti-human second antibody tagged with FITC. The blinded sera of 26 female patients with 
premature ovarian failure were evaluated alongside two groups of controls (sera from 26 control women 
and from 26 control men). Specimens were sent out to a commercial testing laboratory and were also 
evaluated in-house. In addition, the frequency of other autoantibodies associated with ovarian autoimmunity were determined. 
Using this assay, approx. half of the women with premature ovarian failure were found to have ovarian 
antibodies. However, the sera of nearly one third of the normal control women also tested positive. The use of a different scoring method increased the number of false positives further. All male sera tested negative. 
Based on the high percentage of false positives, the authors conclude that this assay has poor 
specificity and argue that the test is not a useful diagnostic tool. Furthermore, treatment recommendations based solely on the outcome of this assay carries considerable risk to the patients. 
Recommendation 
The paper describes serious shortcomings in a commercially available test. It appears that some clinicians base their treatment recommendations on the outcome of this test, and are thus at risk of 
recommending the wrong treatment to their patients. Therefore, it is important that these findings should 
be published, but the paper requires some more work. 
According to the results, a large percentage of the control women have ovarian antibodies. This could 
be real or artefact. However, before deciding between real and artefact, the authors need to show more 
convincingly that they have tried everything they could to improve the specificity of the assay and to 
reduce the number of false positives. Currently, attempts to improve the assay were limited to using a simpler (+/-) scoring system and the testing of higher serum dilutions. Below are some points that the authors should address before the paper is publishable. 
Compulsory revisions: 
1. The subject of this paper is an indirect immunofluorescence based assay. The authors describe that 
they had difficulties with the specificity of the assay, as well as the reproduction of intermediate scoring 
data from the commercial testing laboratory. However, only numerical data is presented. The authors should include a panel of pictures that allow the reader to evaluate issues such as level of background fluorescence, amount of fluorescence in the positive and negative controls, specificity of fluorescence 
localization, as well as examples of scoring of fluorescence intensity. 
2. It is very interesting that the male serum never produced a positive outcome. This finding argues 
against artefact in the positive cases in the normal women control group (although some additional 
controls should also be performed --- see below). The authors should include this in their abstract. 
Currently this important observation goes unmentioned in the abstract. 
Discretionary revisions: 
1. It is common practice in immunocytochemistry to block aspecific binding sites with normal serum from 
the animal species in which the second antibody was produced. In this case the second antibody is 
goat-anti-human. Consequently, the assay should include a blocking step using normal goat serum. 
Does this reduce false positives? 
2. Further improvements in specificity may be obtained by including milk and/or antibody depleted 
human serum, or detergents in the block. Trying different fixatives might also improve the results. 
3. The assay uses sections of monkey ovarian tissue. The authors should show that using human 
ovarian tissue would yield the same results and specificity (or lack of it). 
Competing interests: 
None declared.