Autoimmune Diseases and Antinuclear Antibody Testing: Methods and Staining Patterns

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Robert R. Boyes and David J. Moffa, PhD, BCLD
Reviewer: Nadine Lerret, PhD, MLS(ASCP)CM

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Continuing Education Credits

  • P.A.C.E.® Contact Hours: 2.5 hour(s)
  • Florida Board of Clinical Laboratory Science CE - General (Serology/Immunology): 2.5 hour(s)


  • Understand the autoimmune reaction and describe the various types of autoimmune diseases.
  • Describe the signs, symptoms, and causes of autoimmune diseases.
  • Describe the laboratory tests and exams used to diagnose autoimmune diseases.
  • Define the antinuclear antibody test (ANA) and explain the differences in ANA methods.
  • Distinguish positive from negative samples.
  • Identify and classify ANA patterns.
  • Discuss the clinical significance of ANAs.

Course Outline

  • Copyright and Disclosure
      • Copyright
      • Disclosure
  • Overview of Autoimmune Diseases
      • Autoimmune Disease Definition and Overview
      • Autoimmune Disease Overview (continued)
      • Autoimmune Disease (continued)
      • Which of the following statements are TRUE with regard to autoimmune diseases? (Choose all that apply.)
      • A gender and genetic predisposition is usually the first step toward the development of an autoimmune reaction. The second step is the occurrence of a...
      • Common Autoimmune Diseases
      • Systemic Autoimmune Rheumatic Diseases (SARDs)
      • Systemic Autoimmune Rheumatic Diseases (SARDs), continued
      • Which of these is NOT considered one of the systemic autoimmune rheumatic diseases (SARDs)?
      • In which autoimmune disorder (and a form of SARDs) does the body's immune system mistakenly attack tissue in glands that produce moisture, such as tea...
      • Signs, Symptoms, Causes, and Treatment of Autoimmune Diseases
      • Diagnosis of Autoimmune Diseases: Laboratory Tests
      • Laboratory Tests (Continued): Other Inflammatory Biomarkers
      • The initial laboratory evaluation for autoimmune diseases often involves which tests?
  • Introduction to ANA testing
      • Antinuclear Antibody Test
      • The Antinuclear Antibody Test: Background, History, and Overview
      • Cell Morphology (The HEp-2 or HEp-2000 cell)
      • Stages of Mitosis
      • Which statement is FALSE when describing the ANA test?
      • Which two stages of cell division are the most important for reading ANAs?
  • ANA Testing Methods
      • Indirect Immunofluorescence Assay (IFA)
      • Fluorescent ANA Testing
      • Colorzyme®
      • Which of the following statements is FALSE when describing indirect immunofluorescence assays (IFAs)?
      • Which statement accurately describes the Colorzyme® system?
      • Enzyme-Linked Immunosorbent Assay (ELISA)
      • True or False: Depending on the laboratory and specific method used, an ANA titer is typically considered negative when the titer is equal to or less ...
      • Other Methods for ANA Testing
      • Conditions Associated with a Positive ANA Test
      • About 95% of those individuals with a particular condition have a positive ANA test. Therefore, the most common condition associated with a positive A...
  • ANA Pattern Interpretation
      • Reading ANA Patterns Using a HEp-2 or HEp-2000® Substrate
      • For the ANA test to be positive, what must be present in the nucleus of the interphase cell?
      • ANA Testing Scheme
      • Why are ANA positive samples titered? (Select all that apply.)
      • Follow-up Testing
      • Antigen Chart
      • What type of follow-up testing is recommended for a speckled ANA pattern?
  • ANA Patterns
      • ANA patterns
      • Negative
      • Homogeneous (AC-1)
      • Speckled (AC-5)
      • Nucleolar (AC9)
      • Centromere (AC-3)
      • SSA/Ro Pattern
      • Proliferating Cell Nuclear Antigen (PCNA) (AC-13)
      • Nuclear Membrane (AC-11)
      • The image on the right represents the result of a fluorescent antinuclear antibody (ANA) test. What pattern should be reported?Note: (a) points to the...
    • Unusual or Atypical Speckled Patterns
      • Unusual or Atypical Speckled Patterns: Overview
      • Description of Unusual or Atypical Speckled Patterns
      • Description of Unusual or Atypical Speckled Patterns (continued)
      • When identifying the Midbody pattern, only cells in interphase and metaphase are useful.
    • Mixed ANA Pattterns
      • Mixed ANA Patterns: Overview
      • Homogeneous and Speckled
      • Homogeneous and Centromere
      • Homogeneous and SSA/Ro (on HEp-2000®)
      • Speckled and SSA/Ro Pattern
      • Scl-70 Pattern
      • Other Mixed Patterns
      • What is the most common combination of ANA patterns?
    • Cytoplasmic Patterns
      • Cytoplasmic ANA Patterns: Overview
      • Common Cytoplasmic Staining Patterns
      • Common Cytoplasmic Staining Patterns (continued)
      • Cytoplasmic patterns are interesting to look at but have no clinical significance and should NOT be reported.
  • References
      • References

Additional Information

Level of instruction: Intermediate

Intended Audience: Clinical laboratory professionals including medical laboratory scientists, medical technologists, and technicians. This course is also appropriate for clinical laboratory science and MLT students, pathologists, clinicians, and rheumatologists.

Author Information: Robert R. Boyes was the former Sr. Vice President and General Manager for Immuno Concepts located in Sacramento, California. His degree is in Medical Technology. In his career as a clinical laboratory professional, he has worked in hospital laboratories, specialty clinics, and in the corporate setting. Mr. Boyes has conducted numerous research presentations and has authored or co-authored several publications related to antinuclear antibodies.

David J. Moffa, PhD, BCLD, has over 30 years of experience in the healthcare industry as an executive manager, clinical laboratory director, and medical laboratory scientist. He is currently a technical consultant for Kentmere Healthcare, Wilmington, DE, and until his retirement, was the Regional Director for LabCorp, Inc. He holds a PhD in medical biochemistry from the School of Medicine, West Virginia University.

Reviewer Information:  Nadine Lerret, PhD, MLS(ASCP)CM ​is an assistant professor in the Department of Medical Laboratory Science at Rush University in Chicago, Illinois. She is currently the Clinical Chemistry and Clinical Immunology Course Director and performs research focused on the interplay between hyperglycemia and the immune system. She received a PhD in immunology from Rush University and then was a Postdoctoral fellow in the Division of Nephrology and Hypertension at Northwestern University in Chicago, Illinois.

Course Description: This course concentrates and expands upon both the diagnosis and laboratory testing for autoimmune diseases, as well as on the interpretation of antinuclear antibody (ANA) staining patterns. Laboratory methods for detection and identification of ANAs are complex. Proper training is required to correctly interpret results at both the laboratory and clinical level. This educational program provides a comprehensive review of the methods, interpretation, and clinical significance of ANA testing. The material presented will describe various autoimmune diseases with emphasis on diagnosis using laboratory&a

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