FAQ - Frequently Asked Questions

Submit your question to ICAP. Only confirmed registered users can submit a question. If you are not registered, click here to start. Submitted questions will be routed to ICAP Coordinators Edward K. L. Chan (echan@ufl.edu) and Luis E. C. Andrade (luis.andrade@unifesp.br). Simple questions will be answered within 24 hours. Complex questions will be routed to several ICAP members either for a consensus or to provide different viewpoints and it may take 72 hours to 2 weeks for an answer. Note that some questions/answers may be edited and may appear in the FAQ section of ICAP website. Please note that we do not offer clinical advice on individual patients as it is unethical for us to provide medical advice. Patients may consider referring their doctors to the website. When submitting a question, if applicable please provide information, such as 1) serum dilution and 2) manufacturer information of HEp-2 substrate used. It is now feasible to include up to three IFA images together with your question. When images are submitted, please first consider providing high quality images (e.g. iPhone images capturing a IFA pattern on a computer screen is not appropriate as resolution is much reduced for any useful interpretation). Please also indicate 1) how many images are submitted with your question and 2) if possible, indicate whether the IFA pattern in question changes with serial dilutions. Please note by submitting your images, there is an implied agreement for ICAP to include them as part of a FAQ in the ICAP website. Only images in JPEG extension will be accepted.

Anti-PCNA positive by blot and yet negative in HEp-2 IFA?
Can you explain when anti-PCNA antibodies is positive in immunoblot and yet negative in HEp-2 IFA?

How best to recognize the metaphase plate?
Question: As a new person learning HEp-2 IFA, is there any good tip to identify whether the metaphase plate is positive or negative?

When anti-SS-A/Ro was lowly positive and changed to negative?
A sample was weakly positive for ANA and anti-SS-A/Ro test last year and now it is negative. Is it possible? Is anti-SS-A/Ro indicative of Sjögren’s syndrome?
How to deal with just a “nuclear speckled” IFA report?
In my practice I have followed patients with ANA findings, with a nuclear speckled pattern (without specifying whether fine/dense/coarse), in patients with very heterogeneous phenotypes, some with a clinical picture that suggests further investigation of systemic autoimmune disease (one patient with proximal muscle weakness and skin thickening) and others who represent only non-specific findings. In such situations, as a precaution, I request more specific autoantibodies. However, this pattern (nuclear speckled pattern) is not described by the "ICAP" and I am in doubt about which antigenic association it represents, even to guide which autoantibody may be present and which ones to look after. How to interpret this pattern? Does the lab describe it when it is not possible to "refine" such a conclusion? Could this be associated with deficiency in the methodology, sample, interpretation?

Tips to identify AC-4a pattern
Question: Any good tip to observe AC-4a pattern?

What is the magnification needed for assigning HEp-2 IFA patterns?
Question: Has ICAP made any clear statement about the magnification at which HEp-2 IFA staining patterns in should be evaluated before assigning a pattern?

Differences between multiple, mixed, and composite HEp-2 IFA patterns?
Question: Can you provide more information on how ICAP defines the differences between multiple patterns, mixed patterns, and composite patterns in HEp-2 IFA?

Reporting AC-18 - does it change with the 2021 classification chart revision?
Question: I have one question for the new classification tree. As AC-18 was removed from the category of cytoplasmic speckled and assigned still at the expert level. If the lab only reports competent-level patterns, how should AC-18 be reported? Should they just report the first branch of the classification tree as "cytoplasmic"?
True IFA staining masked at low dilution? 
Question: In the IFA testing of a 9-year-old female lupus patient, initial dilution at 1/40 produced a bright fuzzy cytoplasmic staining with no well-defined pattern. Surprisingly nuclear fine speckled AC-4 was observed at 1/160. Is it common that the cytoplasmic staining completely masked the nuclear staining at low dilution?
Low titer anti-dsDNA serum negative by HEp-2 IFA?
Question: Can I have a negative HEp-2 IFA result in a sample with positive Crithidia assay at 1/20? The negative HEp-2 IFA was confirmed with slides from different commercial brands.
IFA pattern changed from coarse granular to homogeneous 
Question: The HEp-2 IFA pattern of a patient has changed from coarse granular to homogenous after 6 months. Is it possible?
Mitotic patterns are reported as ANA-positive or -negative?
Question: Should I report positive mitotic patterns as ANA-positive or ANA-negative?
Discrepancy in HEp-2 IFA and western blot data. How do you explain the detection of antibodies by western blot (WB) that are not related to the pattern observed in HEp-2 cells by indirect immunofluorescence (IFA)? 
Anti-Ro52 antibodies with an AC pattern? Do anti-Ro52 antibodies show any staining pattern matching with known ICAP AC designation? I have a patient with exclusive anti-Ro52 +++ (strong) in immunoblot and I do not know which AC pattern it should correspond to. AC-4? AC-XX?
The pseudo-DFS pattern? Some samples yield a nuclear speckled pattern with similar staining at the mitotic chromatin (metaphase and anaphase), very similar to AC-2 (nuclear dense fine speckled pattern), but do not yield a positive result in immunoassays specific for anti-DFS70 antibodies. How should I report such pattern since it is not exactly the AC-2 pattern and there is no anti-DFS70 reactivity? Is this pattern defined by ICAP?
Cutting corners in ANA titer reporting. I want your comment about my way of ANA titration and reporting. Typically I perform only 2 dilutions at 1/40 and 1/160. However, I report titers from negative at 1/40, 1/40, 1/80, 1/160, and further report estimated titers of 1/320, 1/640, 1/1280 and over 1/1280 based on signal intensity. I think I can accurately estimate the titers just from the above 2 dilutions.
Issue with increasing UV light intensity. I want to clarify when increasing the intensity of UV light, ANA-negative samples may become visible or positive. How to deal with this issue?

Nuclear periphery positive and yet center negative? Some time we observed periphery of the nucleus is stained like positive and center of the nucleus is negative.what is the reason for that ?
Double IFA protocol. What is the protocol for double IFA that can help to identify, for example, a subcellular compartment such as the nucleolus?

Cytoplasmic positive alone is ANA positive or negative? Hello, I would appreciate if you can share with me how to report cytoplasmic staining (without nuclear staining) as ANA positive or negative?

Ro/SS-A and La/SS-B negative in HEp-2 IFA. Can I get positive Ro and La with negative ANA IFA? How to explain if yes?

ANA titer reporting. We would also like recommendations on the report of ANA results. For example, when report such as 1:80 dilution AC-1 homogeneous 4+ are made, is it correct to report in crosses (e.g. +++) or only the dilution and the pattern?
Fine Art with AC-29. Is it critical that all five elements of the subcellular domains associated with the AC-29 pattern be evaluated in order to correctly classify an individual serum as AC-29? In other words, can it be classified as AC-29 with staining of only some of the five elements?
Online since 19 May 2015