• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTANA6YR - Six-year SSANA subsample weights
  • SSCYT - total cytoplasm intensity
  • SSNUC - total nucleus intensity
  • SSTOMIC - total mitotic intensity
  • SSMSI - maxim signal intensity
  • SSTSUM - titer summary
  • SSNUCPOS - nucleus nucleolar positive
  • SSNUCSIG - nucleus nucleolar signal
  • SSNUHOMP - nucleus homogenous positive
  • SSNUHOMS - nucleus homogenous signal
  • SSNDFSPS - nucleus dense fine speckles positive
  • SSNDSPSG - nucleus dense fine speckles signal
  • SSNFSPOS - nucleus fine speckles positive
  • SSNFSPSG - nucleus fine speckles signal
  • SSNUCSPP - nucleus coarse speckles positive
  • SSNUCSPS - nucleus coarse speckles signal
  • SSNUCENP - nucleus centromere positive
  • SSNUCENS - nuclear centomere signal
  • SSNUDOTP - nuclear dots positive
  • SSNUDOTS - nuclear dots signal
  • SSNUENVP - nuclear envelope positive
  • SSNUENVS - nuclear envelope signal
  • SSNPSPOS - nucleus PCNA-Like positive
  • SSNPSSIG - nucleus PCNP-Like signal
  • SSCENPOS - nucleus CEN-F-like positive
  • SSCENSIG - nucleus CEC-F like signal
  • SSCYHOMP - cytoplasm homogeneous positive
  • SSCYHOMS - cytoplasm homogeneous signal
  • SSCYSPS - cytoplasm speckled positive
  • SSCYTSG - cytoplasm speckled signal
  • SSCYMIP - cytoplasm mitochondria-like positive
  • SSCYMIS - cytoplasm mitochondria-like signal
  • SSCYGOP - cytoplasm golgi complex positive
  • SSCYGOS - cytoplasm golgi complex signal
  • SSCYGWP - cytoplasm GW body positive
  • SSCYGWS - cytoplasm GW body signal
  • SSCYFLPS - cytoplasm cytofilament positive
  • SSCYFLSG - cytoplasm cytofilament signal
  • SSCYRRPS - cytoplasm rods/rings positive
  • SSCYRRSG - cytoplasm rods/rings signal
  • SSMTCNPS - mitotic centrosome positive
  • SSMTCNSG - mitotic centrosome signal
  • SSMISFPS - mitotic spindle fibers positive
  • SSMISFSG - mitotic spindle fibers signal
  • SSMTNMPS - mitotic NuMa positive
  • SSMTNMSG - mitotic NuMa signal
  • SSMTBRPS - mitotic intracellular bridge positive
  • SSMTBRSG - mitotic intracellular bridge signal
  • SSMICHP - mitotic chromosomes positive
  • SSMICHS - mitotic chromosomes signal

National Health and Nutrition Examination Survey

1999-2004 Data Documentation, Codebook, and Frequencies

Autoantibodies - Immunofluorescence Analyses (Surplus) (SSANA2_A)

Data File: SSANA2_A.xpt

First Published: August 2019

Last Revised: July 2022

Note: The NHANES Biospecimen Program processes were reevaluated in 2021 and 2022 to monitor quality control after a procedural error was identified. This error did not pose any risk of participant disclosure. Addressing this error resulted in the removal of some records from various stored biospecimen data files between 1999 and 2018 that did not meet program standards. After a comprehensive review of all stored specimen datasets, this data file was modified to remove records (<5% of records) that were initially included in error. No data values were altered. The sample weights and overall estimates were compared between the original and revised files. The sample weights provided with the original data release are appropriate to use with this updated data file; therefore, no new survey weights were created. The overall estimates from these files were examined and are consistent with the previous file. However, not all possible analyses were performed.

Component Description

IgG autoantibody testing of a subsample of stored serum from 1999-2004 participants was conducted to estimate the prevalence and specificities of selected autoantibodies in the US population.

Eligible Sample

One third subsample of participants aged 12 years and older from 1999-2004.

Description of Laboratory Methodology

Immunofluorescence analysis
Testing of IgG autoantibodies to human cellular antigens was performed by the HEp-2 cell immunofluorescence assay using slides from INOVA Diagnostics, San Diego, CA (Cat # 508100) following the manufacturer’s instructions and evaluated primarily using their NOVA View system. The cellular pattern of staining and staining intensity from 0 to 4+ was described using a reference gallery per below and description of patterns are consistent with those described by the International Consensus on ANA Pattern (ICAP) committee [ref. Chan EKL, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PL, Fritzler MJ, Garcia-De La Torre I, Herold M, Mimori T, Satoh M, von Muhlen CA, Andrade LE. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015;6:412. PMC:4542633]. The determination of autoantibody titers from 1:80 to 1:1280 was performed by serial dilution on samples that showed a 3+ or greater nuclear and/or cytoplasmic immunofluorescence pattern.

Laboratory Quality Assurance and Monitoring

Immunofluorescence analysis
Serum samples were tested using established protocols outlined by manufacturer. A set of CDC autoimmune reference sera (12 different specificities) (Chan EKL et al., 2007) and 2 healthy control sera were coded and tested in a blinded fashion two times to verify consistent results throughout screening tests: towards the beginning and when approximately half of the samples were tested.

Samples that showed discrepant results were repeated. Repeats were performed whenever there was any doubt for accuracy or technical issues.

Data Processing and Editing

Data Access: All data are publicly available.

Analytic Notes

The NHANES Survey Design:

The analysis of NHANES laboratory data should be conducted using the key survey design variables. The NHANES Demographic data files for each 2-year survey cycle contain core information on the survey and the survey design variables for strata and PSU (SDMVSTRA and SDMVPSU, respectively) which are recommended to be used in variance estimation. This laboratory data files may be linked to other NHANES data files using the unique survey participant identifier SEQN.

Weighting:

Special subsample sample weights WTANA6YR in the file are required to analyze the NHANES 1999-2004 ANA data. This is because the ANA data were analyzed using stored serum samples obtained from survey participants who had been selected from NHANES 1999-2004 Dioxin subsamples. The NHANES 1999-2004 ANA sample and the subsample weights were created for 6 years analysis and should be used to analyze all six years together.

Immunofluorescence analysis 

A standard gallery of images showing the intensity of staining and the cellular patterns of reactivity is shown below and the following patterns were reported:

Nuclear patterns
1) homogeneous (AC-1); 2) dense fine speckled (AC-2); 3) centromere (AC-3); 4) fine speckled (AC-4); 5) Coarse speckled (AC-5); 6) nuclear dots (AC-6/AC-7); 7) nucleolar (AC-8/AC-9/AC-10); 8) nuclear envelope (AC-11/AC-12); 9) PCNA-like (AC-13); 10) CENP-F-like (AC-14)

Cytoplasmic patterns
1) cytofilament (AC-15/AC-16/AC-17); 2) GW Body (AC-18); 3) homogeneous (AC-19); 4) speckled (AC-20); 5) mitochondria-like (AC-21); 6) Golgi complex (AC-22); 7) rods/rings (AC-23)

Mitotic cell patterns
1) centrosome (AC-24); 2) spindle apparatus (AC-25); 3) NuMA-like (AC-26); 4) mid-body (AC-27); 5) mitotic chromosomal (AC-28)

Nuclear staining	Images	Description of staining patterns
Homogeneous (AC-1)		Homogeneous and regular fluorescence across all nucleoplasm
Nuclear dense fine speckled (AC-2)		Speckled pattern distributed throughout the interphase nucleus with characteristic heterogeneity in the size, brightness and distribution of the speckles. The metaphase plate depicts strong speckled pattern with some coarse speckles standing out.
Centromere (AC-3)		Discrete coarse speckles (40-80/cell) scattered in interphase cells and aligned at the chromatin mass on mitotic cells
Nuclear fine Speckled (AC-4)		Fine tiny speckles across all nucleoplasm
Nuclear coarse speckled (AC-5)		Coarse speckles across all nucleoplasm.
Nuclear dots (AC-6/AC-7)		Countable discrete speckles PML bodies, Cajal bodies (coiled bodies)
Nucleolar (AC-8/AC-9/AC-10)		Homogeneous or speckled staining of the nucleolus
Nuclear envelope (AC-11/AC-12)		Homogeneous nuclear staining with greater intensity at its outer rim
PCNA-like (AC-13)		Pleomorphic speckled nucleoplasmic staining, with variability in size and brightness of the speckles. In interphase, some cells are negative (G1 phase), some are intensely stained (S-phase) and some present rare and scattered speckles with occasional nucleolar staining.
CENP-F-like (AC-14)		Nuclear speckled pattern with striking variability in intensity with the strongest staining in G2 phase and weakest/negative staining in G1. The centromeres are positive only in prometaphase and metaphase, revealing multiple aligned small and faint dots.
Cytofilament (AC-15/AC-16/AC-17)		Cytoskeleton staining with variable intensity in interphase cells
Cytoplasmic discrete dots/GW body-like (AC-18)		Staining of GW bodies in the cytoplasm of interphase cells with high numbers in late S/G2 cells
Cytoplasmic dense fine speckled (or homogeneous,AC-19)		Uniform diffuse staining of the cytoplasm in interphase cells
Speckled (AC-20)		Scattered small speckles in the cytoplasm
Mitochondria-like (AC-21)		Coarse granular filamentous staining
Golgi complex (AC-22)		Discontinuous speckled or granular perinuclear staining
Rods/Rings (AC-23)		Distinct rod and ring structures throughout the cytoplasm
Centrosome (AC-24)		Distinct dots (2-4/cell) in cytoplasm and at the poles of mitotic spindle
Spindle fibers (AC-25)		The spindle fibers between the poles are stained in mitotic cells, associated with cone-shaped decoration of the mitotic poles. Spindle fibers cover both NuMA-like and non-NuMA patterns. NuMA-like pattern has associated distinct nuclear speckles. Only non-NuMA patterns are shown here while NuMA-like patterns are shown in the NuMA-like group
NuMA-like (AC-26)		Nuclear speckled staining with spindle fibers
Intercellular bridge (AC-27)		Staining of the intercellular bridge that connects daughter cells by the end of cell division, but before cell separation
Mitotic Chromosomal (AC-28)		Diffuse staining of condensed chromatin in mitotic cells

 

 

References

• Chan EKL, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PL, Fritzler MJ, Garcia-De La Torre I, Herold M, Mimori T, Satoh M, von Muhlen CA, Andrade LE. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015;6:412.
• Chan EKL, Fritzler MJ, Wiik A, Andrade LE, Reeves WH, Tincani A, Meroni PL. AutoAbSC.Org - Autoantibody Standardization Committee in 2006. Autoimmun.Rev. 2007;6(8):577-80.
• Reeves WH, Satoh M, Lyons R, Nichols C, Narain S: Detection of autoantibodies against proteins and ribonucleoproteins by double immunodiffusion, immunoprecipitation, and western blotting. In: Manual of Molecular and Clinical Laboratory Immunology, 7th edition. Edited by Rose NR, Hamilton RG, Detrick B, Reeves WH. Washington, D.C.: American Society of Microbiology Press; 2006: 1007-1018.

Codebook and Frequencies