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Revista Brasileira de Reumatologia

Print version ISSN 0482-5004On-line version ISSN 1809-4570

Rev. Bras. Reumatol. vol.49 no.2 São Paulo Mar./Apr. 2009 



Third Brazilian Consensus for autoantibodies screening in HEp-2 cells (ANA). Recommendations for standardization of autoantibodies screening trial in HEp-2 cells, quality control and clinical associations



Alessandra DellavanceI; Alexandre Gabriel JúniorII; Barbara NuccitelliIII; Ben Hur TalibertiIV; Carlos Alberto von MühlenV; Carlos David Araújo BicharaVI; Cláudio Henrique Ramos dos SantosVII, Cleonice BuenoVIIIS; Cristiane Martinez YanoIX; Cristóvão Luis Pitangueira MangueiraX; Darlene Gonçalves CarvalhoXI; Elizângela CardosoXII; Eloísa BonfáXIII; Flávia Ikeda e AraújoXIC; Gustavo Gabriel RassiXV; Hugo Mendonça MundimXVI; Izidro BendetXVII; Jozélia RegoXVIII; Lisiane Maria Enriconi dos Anjos VieiraXIX; Luis Eduardo Coelho AndradeXX; Maria Ordália Ferro BarbosaXXI; Mitiko SugiyamaXXII; Mittermayer Barreto SantiagoXXXIII; Natasha SlhessarenkoXXIV; Nilzio Antônio da SilvaXXV; Paulo Luiz Carvalho FrancescantonioXXVI; Renata JarachXXVII; Roberto SudaXXVIII; Roger Abramino LevyXXIX; Silvia Oliveira SampaioXXX; Suzane Pretti Figueiredo NevesXXI; Wilson de Melo CruvinelXXXII; Wilton Silva dos SantosXXXIII; Yanna K. de M. NóbregaXXXIV

IDoctorate student in Rheumatology at Universidade Federal de São Paulo (UNIFESP); Scientific advisor of the Immunology Department at Fleury Medicina Diagnóstica
IIPostdoctorate at Scripps Medical Center La Jolla La USA; Medical Coordinator of AFIP - Medicina Laboratorial e Centro Imuno-Reumatológico de São Paulo
IIIBiomedical researcher at Padrão Laboratório Clínico
IVPostdoctorate at Reumaklinik Aachen-Germany; Full Professor of Clinical Medicine at Universidade Federal de Uberlândia; Coordinator of the Rheumatology Department at HCUFU
VProfessor of Rheumatology at PUCRS Medical School; Doctorate in Medicine at Rheinisch-Westfalische Technische Hochschule/Aachen Medical School, Germany; Postdoctorate at The Scripps Research Institute, La Jolla, USA; Technical Director of Metanalysis Centro de Diagnósticos Médicos, Porto Alegre
VIDoctor and Biomedical Researcher; Expert in Clinical Pathology; Expert in Public Health; Master's student in Biology of Infectious and Parasite Agents/UFPA; Technical Supervisor at Laboratório Amaral Costa; Professor of Clinical Pathology at CESEP/PA
VIIScientific adviser at NewLife Comércio de Produtos Laboratoriais
VIIIMedical Research Laboratories at Hospital das Clínicas/FMUSP
IXMaster in Tropical Medicine at Universidade Federal de Goiás; Assistant Professor of Immunology at Universidade Católica de Goiás
XClinical Pathologist and Rheumatologist, Doctorate in Pathology at Universidade de São Paulo Medical School (FMUSP); Doctor at the Immunology Section at the Central Laboratory Division (LIM 03) at Hospital das Clínicas, FMUSP; Medical Manager of the Department of Clinical Pathology (Preventive and Diagnostic Medicine) at Hospital Israelita Albert Einstein
XIClinical Pathologist - Immunology at Instituto Hermes Pardini
XIICellular and Molecular Biology, Scientific and Technical Support at Biometrix Diagnóstica
XIIIProfessor of Rheumatology Medical School, University of São Paulo, Universidade Católica de Goiás; Coordenadora do Laboratório de Apoio Didático do Departamento de Biomedicina UCG
XIVMaster in Environmental Science and Health at Universidade Católica de Goiás (UCG); Assistant Professor I of Immunology in the Biomedicine course at Universidade Católica de Goiás; Coordinator of Laboratório de Apoio Didático (Educational Support Laboratory) at the Department of Biomedicine (UCG)
XVClinical Pathologist at Laboratório Atalaia
XVIExame Medicina Laboratorial, Distrito Federal
XVIIClinical Pathologist and Virologist; Postgraduate in Parasitary Biology at FIOCRUZ; Scientific Consultant of Immunoassay Sector at Sérgio Franco Medicina Diagnóstica
XVIIIRheumatologist and Coordinator of the Immunorheumatology Laboratory at Hospital das Clínicas, Universidade Federal de Goiás Medical School
XIXMaster in Medical Sciences at Universidade Federal de Santa Catarina; Professor at Universidade do Sul de Santa Catarina; Professor at Universidade do Vale do Itajaí; Clinical Pathologist at Laboratório Médico Santa Luzia
XXAssociate Professor of Rheumatology at UNIFESP, Coordinator of Postgraduate Course of Rheumatology of Universidade Federal de São Paulo; Medical Adviser of the Immunology Section at Fleury Medicina e Saúde
XXIMaster in Biomedical Science at San Francisco State University, California; Biochemist at Saúde and Hospital and Maternidade and Jardim América laboratories
XXIIScientific Adviser at Hemagen Diagnósticos
XXIIIPostdoctorate at University of Calgary; Rheumatologist at Universidade Federal da Bahia; Associate Professor at Fundação Bahiana para Desenvolvimento das Ciências (Science Development Foundation) and Head of the Rheumatology Department at Hospital Santa Izabel
XXIVClinical Pathologist, Master in Medicine (Pediatrics) at University of São Paulo; Professor at Universidade Federal de Mato Grosso
XXVDoctor in Medicine (Rheumatology) at Universidade de São Paulo, Full Professor of Rheumatology at Universidade Federal de Goiás Medical School
XXVIMaster in Environmental Science and Health at Universidade Católica de Goiás (UCG); Assistant Professor I of Immunology in the Biomedicine Course at Universidade Católica de Goiás; Coordinator of the 3rd Brazilian Consensus for autoantibodies screening
XXVIIMaster in Tropical Medicine at Universidade Federal de Goiás; Biomedicine Researcher at Padrão Laboratório Clínico
XXVIIIScientific Adviser at Alka
XXIXDoctor in Biological Sciences (Biophysics) of Universidade Federal do Rio de Janeiro; Employee at Universidade do Estado do Rio de Janeiro; Assistant Professor of Rheumatology at UERJ; Coordinator of Autoimmunity Center at Hospital Pró-cardíaco RJ and scientific consultant at Diagnósticos da América SA
XXXDoctorate in Immunology at Institute of Biomedical Science at Universidade de São Paulo, Graduate at Universidade de São Paulo Medical School (FMUSP), Specialist in Rheumatology (FMUSP); Doctor at the Immunology Section at Laboratório Diagnósticos da América SA
XXXIClinical Pathologist; Doctor in Science at Fundação Oswaldo Cruz; Associate Professor of Universidade Federal de Minas Gerais Medical School; Coordinator of the Serum Immunology Section of the Laboratory Medicine Department at Hospital das Clínicas - UFMG
XXXIIDoctorate student in Rheumatology at Universidade Federal de São Paulo (UNIFESP); Assistant Professor I of Immunology in the Biomedicine and Medicine Courses at Universidade Católica de Goiás; Secretary of 3rd Brazilian Consensus for autoantibodies screening
XXXIIIRheumatologist, Coordinator of the Rheumatology Laboratory at Hospital Universitário de Brasília; Doctorate in Rheumatology (UNIFESP)
XXXIVMaster in Molecular Pathology/Immunology at Universidade de Brasília (UnB); Professor at União Educacional do Planalto Central and Centro Universitário Unieuro; Scientific Adviser at Imunotech Sistemas Diagnósticos Importação e Exportação

Correspondence to




OBJECTIVE: The Third Brazilian Consensus for autoantibodies Screening in HEp-2 cells had as purpose the evaluation of difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, the discussion of strategies for quality control of the assay and the promotion of an update of the clinical associations of the several immunofluorescent patterns.
METHODS:Several ANA experts from university centers and private laboratories in different areas in Brazil joined the workshop in Goiânia on 2008 April 13 and 14 with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to the workshop.
RESULTS AND CONCLUSIONS: The 3rd Consensus emphasized the need for quality control in indirect immunofluorescent since there is a considerable heterogeneity of available microscopes and reagents. It also promoted adaptations in the previously approved terminology used to classify the different patterns and finally updated the clinical associations of the several patterns with the purpose of providing guidance for interpretation of the assay by clinical pathologists and assistant physicians.

Keywords: autoantibodies, HEp-2 cells, Antinuclear antibodies, immunofluorescent.




Historic outlook of autoantibodies screening using HEp-2 in Brazil

Technological and scientific development has promoted changes in laboratory tests that affect important parameters for clinical interpretation, such as positive and negative predictive values, sensibility and specificity. An excellent example of this situation is the antinuclear antibody (ANA) screening using the indirect immunofluorescent (IIF) technique, also known as antinuclear factor, today called "screening for antibody against cell antigens" in serum of patients under autoimmune disease suspicion. It is an excellent test to track autoantibodies that, along the last decades, has been technically changed to provide a progressively higher sensitivity. As a consequence, the antibody screening against cell antigens also started to present lower specificity.1

However, the increment of ANA-IIF test sensitivity using HEp-2 cells also brought up a loss in specificity, as some individuals without clinical or laboratory evidence of autoimmune disease also presented positive results in HEp-2 cells, requiring a strict interpretation of serological findings.2 The high frequency of positive results in healthy individuals or individuals with vague clinical manifestations has revealed a situation called "syndrome of idiopathic antinuclear antibody".1 Loss of specificity in this test has also been aggravated due to the fact that a wide range of expert physicians started to use it. Initially, rheumatologists and nephrologists were the main users of this exam and, due to a familiarity with it and their patients' characteristics, they had more chances to request this test for those who really had an autoimmune history.

Today, ANA-IIF using HEp-2 is a test requested with fewer criteria by a wide range of specialists, who obviously see to different patients, in which autoimmune rheumatic disease diagnosis is less prevalent. Thus, the chance of positive results in healthy individuals, or with low expressive clinical presentations, became higher. Some elements are important to suitable value the ANA-IIF in HEp-2 test. Firstly, this test should be requested only under convincing suspicion of autoimmune disease. Requesting this test for a patient presenting uncertain complaints frequently will bring more confusion to the clinical judgment, as a positive result does not necessary mean autoimmunity. A second point to be considered is ANA-IIF in HEp-2 titer: in general, autoimmune patients tend to present moderate (1/160 and 1/320) and high (> 1/640) titers, while healthy individuals tend to present low titers (1/80).3 However, both situations may present exceptions.4 Another important point is the fluorescence pattern that shows the identity of autoantibody(ies) in consideration;5 it should be carefully analyzed taking in account the experience and expertise of observer, as well as the capacity to reproduce the pattern using kits by different manufacturers.

Autoantibodies against some antigens have specific association with certain autoimmune diseases or to autoimmunity state while others occur indiscriminately in autoimmune and non-autoimmune individuals. So, certain fluorescence patterns are more specific to autoimmune disease while others happen frequently in healthy individuals or in patients with other non-autoimmune diseases.1

Another point to be considered is that the physiological autoimmunity level, or basal level, may fluctuate depending on the overcharges the immune system is exposed to. The autoantibody presence broken out by infections, drugs or neoplasia is well demonstrated. High prevalence of autoantibodies in patients with HIV (human immunodeficiency virus) and other lymphotropic virus6 has been clearly demonstrated. Therefore, an important point to take into consideration when evaluating a positive finding of ANA-IIF using HEp-2 cells refers to the possibility of recent viral infections, drug use and neoplasic processes.

Several evidences demonstrate that autoantibodies frequently precede clinical diagnosis of autoimmune diseases.7 A positive ANA-IIF using HEp-2 test may precede clinical systemic lupus erythematosus (SLE) in up to nine years. Almost 80% of patients with SLE present positive ANA-IIF in HEp-2 before the first symptoms. Although in a lower percentage, it is also valid for several autoantibodies specific for this disease, such as double stranded anti-DNA and anti-Sm. Consequently, another possibility to be considered facing a clinically inconsistent finding of positive ANA-IIF in HEp-2 is that the patient may develop an autoimmune disease in the future years. However, some individuals may continue for decades with circulating autoantibodies without developing any signal of autoimmune disease.8

It is essential to better characterize a positive ANA-IIF in HEp-2 cell by searching for hallmark antibodies proper to autoimmune pathologies by specific techniques. This evaluation should be supported by clinical or laboratorial evidence of a systemic autoimmune disease. Additionally to clinical examination, it is important to check possible changes in blood count, urine, C-reactive protein and erythrocyte sedimentation rate (VHS), which may be considered extensions of the clinical exam. In some cases, analyzing hepatic and muscular enzymes may be valid. Vague symptoms, like arthralgias and asthenia, with normal general laboratory tests are not sufficient to provide support for a laboratorial finding of ANA-IIF in HEp-2 in low titer and with low specific fluorescence pattern. In such cases, common sense and follow-up of the patients with regular visits may be the best decision.

The first two Consensuses contributed to improve readings and interpretation of ANA-IIF using HEp-2 patterns by the definition of morphological criteria to be followed during the reading of test, and by the establishment of a combination criteria including the main groups (nucleus, nucleolus, cytoplasm, mitotic apparatus and mixed). Information on main clinical associations of different patterns was approached and a new terminology was suggested so that the test would express its diagnostic dimension.

After the second Consensus, some questions arose requiring more discussion and better orientation. One of the problems observed concerns the classification of homogeneous nuclear pattern regarding the nucleolus reactivity and the mixed pattern classification, approaching multiple reactivities in the same group, e.g., a pattern with two or more autoantibodies against nuclear antigens. Another relevant aspect was the need to advice about substract heterogeneity, beginning with conjugate titer. Since some laboratories are not familiarized with titering the conjugate against an absolute or consensual pattern, the same test carried out in kits by different brands may present different titers. These problems, as a whole, are responsible for heterogeneity in results among several laboratories and should be deeply discussed.

Finally, along these two years, there was a need to review clinical associations of different patterns established in the 2nd Consensus. Thus, the Third Brazilian Consensus for autoantibodies Screening in HEp-2 Cells (ANA) intended to evaluate the difficulties in the accomplishment of the 2nd Consensus recommendations that took place in the year of 2002, to discuss strategies for quality control of the assay and to promote an update for the clinical associations of the several immunofluorescent patterns.



Several ANA experts from university centers and private laboratories from different areas in Brazil joined the workshop in Goiânia on April 13 and 14, 2008, with the purpose of discussing and approving the recommendations for standardization, interpretation and use of the test by physicians. Commercial representatives of different ANA slide brands were also invited as listeners to ANA-IIF in HEp-2 test.

The group approached problems like the need for test quality control, definition of some controversial aspects in the classification proposed in the 2nd Consensus, report of new fluorescence patterns and review of clinical associations. These problems were presented to members and widely discussed in order to get a consensus among several participants. Discussions were based on previous review of the literature concerning different subjects, as well as presentation of their own data.



Nucleolus classification according to homogeneous nuclear pattern becomes negative

The 3rd Consensus reaffirms the current classification of fluorescence patterns in four cell structures (cytoplasm, nucleus, nucleolus and mitotic apparatus). Additionally, some definitions were done for some possible ambiguous or vague situations. In cases where nucleus is uniformly stained and nucleolus region is not highlighted, the Consensus members understand that there is no essential reactivity against nucleolus; therefore, it should be described as "negative". Obviously, nucleolus will also be described as "negative" in cases where it is not stained. When there is a nuclear pattern, nucleolus will only be described as "positive" when its stain becomes visible over the nucleus stain. The example below showing homogeneous nuclear pattern report is recorded:

  • Patient: F.C.O.F.
  • Assay: antibody screening against cell antigens (ANA)
  • Nucleus: Positive.
  • Nucleolus: Negative.
  • Cytoplasm: Negative.
  • Mitotic apparatus: Negative.
  • Chromosomal metaphase plate: Positive
  • Pattern: Homogeneous nuclear

Mixed patterns

Mixed Patterns definition was corrected. Every case where different staining for cellular structures (nucleus, nucleolus, cytoplasm or mitotic apparatus) or different fluorescence patterns in one cell structure was classified as mixed pattern. Thus, for example, NuMA-1 pattern is considered a mixed pattern, because the nucleus and mitotic apparatus are stained. Another example is represented by a serum with an autoantibody mixture that simultaneously stain the nucleus with a fine speckled pattern and centromere speckled pattern.

Speckled nuclear pattern with separate dots

The 3rd Consensus changed the decision of the 2nd Consensus about the need for subclassification of the number of dots in speckled nuclear pattern with isolate dots in >10 and <10. Pattern terminology has been established as speckled with isolated dots. This change happens because the number of nuclear stained bodies by anti-p80-coilin and anti-sp-100 antibodies varies according cell substrate in use. Although, in most cases, expert observer may safely suggest the most probable autoantibody, the number of dots per nucleus is not an absolute criterion.

Non-characterized patterns or patterns with new features

Consensus members acknowledge that there are non-characterized patterns or patterns with no defined characteristics in the existing classification. In these cases, recommendation is to describe morphologically the pattern observed and to add a note specifying that it is not part of the Consensus terminology and that its immunologic and clinical associations have not been defined yet. In such cases, it is essential that the laboratory tests this new proposed pattern in commercial kit under a different brand than the one where the pattern was originally observed, avoiding human conditions that could result a false interpretation.

Two new fluorescence patterns were communicated by few Consensus members. Many other members declared they had already observed such patterns.

The first one is a nuclear fine speckled immunofluorescent pattern, with an almost homogeneous texture and with homogeneously stained metaphase plate. Its clinical association and immunological identity are not defined. Its importance comes from the fact that it may be easily confused with a dense fine speckled pattern and a homogeneous pattern (Figure 1).



The second is a cytoplasmatic rod- and ring-shaped pattern which is apparently associated to HCV infection. There are ongoing studies to establish its immunological identity (Figure 2).



As they are not definitely characterized, these two new patterns have been considered preliminary and the 3rd Consensus recommended that both patterns have to be completely characterized and presented in the next meeting.

Genetically modified cell-based substracts

The 3rd Consensus did not discuss any systematic study with genetically modified cell substracts; this could be a possible topic for the next meetings.



The Third Consensus promotes and recommends strategies for quality control. This recommendation aims at facing difficulties to assure test quality due to specialized trained professionals needs, heterogeneity of commercial kits and non-standardization of optical equipment at laboratories. Among institutional quality programs, the following programs were mentioned: College of American Pathologists (CAP) and educational program for ANA-IIF in HEp-2 at Controlab. Other programs and reagents for quality control were also mentioned, such as PCQAUTO by GMK Diagnósticos, Conexão HEp-2 by Hemagen Diagnósticos and the blade FITC-QC® by ALKA Tecnologia em Diagnósticos.

It was also recommended the titration of conjugate as a fundamental measure to adjust the amount of fluorochrome according to the lamp power, matching the different departments. This procedure should also be performed for every new kit in a different lot. This titration maintenance in kits from the same lot may be performed using low intensity controls. In this item, it is useful to use commercial slides with pre-calibrated microsphere for different fluorescence intensity and that may be used for training and internal calibration of reading.

In order to stimulate the achievement of authentic results, the 3rd Consensus recommends the laboratories to take part at quality control programs and systematically perform technical quality control. It is fundamental to remember that IIF reaction depends on five factors: Optical system (microscope), light power (20, 50 or 100W), conjugate concentration, control serum of minimum reactivity (1/80) and observer.

Conjugate concentration allows matching different optical systems, different power lights and observer's reading. For example, if the light power is low, a more concentrate conjugate use is recommended in order to get the same fluorescence of a light with more power. In order to establish the ideal concentration of conjugate (conjugate's titer), one should use titration technique in blocks where several dilutions of conjugate are crossed with several concentrations of a reference serum, searching the higher dilution of conjugate that is capable to reproduce nominal titer of reference serum.

This reference serum may be included with the kit and the laboratory will begin to be based on the optical, light and reader system according to patterns established by the manufacturer. This reference serum may be commercially acquired from quality control institutions or the laboratory will be able to send its serum to a laboratory of its reference and, then, with the results, start to consider this laboratory as reference. Once the first reference is done, the laboratory may store weekly aliquots of serum of titers established in this laboratory.

The laboratory should keep in its serum bank samples for controlling with minimum reactivity titer (1/80), to be diluted in 1/40, 1/80, 1/160 and 1/320. At each daily set, the laboratory should process the low control and consider this set valid if titer variation is more or less one dilution. If any control inconformity is seen, for example, if a sample with average titer of 1/80 is negative, this set should be considered invalid. Using the minimum reactivity control (1/80), a set of tests will only be considered valid after reading one titer at more or less 80. In case of a decrease over 1 titer, the system is probably unstable. It is recommended to first verify if there was any problem in the aliquot stored; in this case, repeat the test with the aliquot stored the week before. If in the end of repetition, the titer found was the expected, we may conclude that the control aliquot used was deteriorated. If the new aliquot also presented a titer decrease of more than one dilution, we should check the optical system (UV filter deterioration, buffered glycerin in the objective lens and lamp performance). If no change is found in these components and in the number of hours of the lamp use, the most probable cause is conjugate degradation; if so, conjugate should go through a new titration process, as described before.


  1. It is recommended to perform conjugate control every 15 days.
  2. When a new kit is opened, a new titration of conjugate should be performed.
  3. Microscope's objective lens and filters interfere in the definition of conjugate titer and should be periodically checked.
  4. Additionally to checking the time of the lamp, it is necessary to check if the lamp is in the center. It can be performed by placing a white paper sheet on the microscope table and using the objective lens 10 to see if the lamp is in the center. If you notice any dark part of the field, adjust it by using the following bottoms: left-right center, up and down center, and lamp focus.



Members of the 3rd Consensus proceeded to a wide discussion for validation and reevaluation of clinical and immunological associations about ANA-IIF patterns in HEp-2. Recommendations are listed in Table 1.

Most of the associations above had been accepted by general agreement by the Consensus members. In rare non-unanimous cases, the majority opinion prevailed.

Such initiative shows the intense activity in auto antibodies research, and particularly, in antinuclear antibody, in Brazil. We should emphasize that this activity has been providing a progressive and remarkable development in our scientific and professional community, establishing an independent national position about the way to perform and interpret assay to screen autoantibodies using HEp-2 cells. The physicians and consequently their patients has also benefited of this laboratory test. Finally, it is necessary to explain that this is a continuous and progressive process and it depends on the interaction of the whole community involved in several steps of this process.



  • Brazilian Society of Rheumatology (BSR)
  • Brazilian Society of Clinical Patology and Laboratorial Medicine (BSCP)
  • Brazilian Society of Clinical Analysis (BSCA)
  • Local Advisory Board of Biomedicine 3th Region



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Correspondence to:
Laboratório de Apoio Didático
Departamento de Biomedicina - Universidade Católica de Goiás
Avenida Universitária, 1069. Setor Universitário
Zip Code:74605-010, Goiânia - Goiás
Phone.: 55 62 3946-1393

Received on 11/17/2008
Approved 01/14/09.



Third Brazilian Consensus received grant from Rheumatology Aid Fund, of Brazilian Society of Rheumatology.

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