Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples
2.2. ANA IIFA
- First incubation: Pipette an adequate volume of control samples and diluted serum samples (1:80 with sample buffer) into each well of slides. Incubate slides for 30 min ± 10 min at room temperature (20–26 °C) in the moist incubator tray.
- Washing: Remove slides from incubator tray and rinse briefly with wash buffer using a squeeze wash bottle. Wash slides 2 × 5 min with wash buffer in a slide staining dish.
- Second incubation: Return slides to incubator tray and cover each well with an adequate volume of FITC-conjugate (fluorescein labelled anti-human IgG). Incubate slides for 30 min ± 10 min at room temperature in the dark.
- Washing: Wash as described above.
- Mounting medium: Add an adequate volume of mounting medium along midline of each slide.
- Reading: Read slides at 400–800 × total magnification with a fluorescent microscope.
2.3. Solid-Phase Assay
- First incubation: Place each test strip in an empty channel. Fill each channel with 1.5 mL of the diluted serum sample (1:101 with sample buffer). Incubate for 30 min at room temperature (18–25 °C) on a rocking shaker.
- Wash: Aspirate off the liquid from each channel and wash 3 × 5 min each with 1.5 mL working-strength wash buffer on a rocking shaker.
- Second incubation: Pipette 1.5 mL diluted enzyme conjugate (alkaline phosphatase-labelled anti-human IgG) into each channel. Incubate for 30 min at room temperature on a rocking shaker.
- Wash: Wash as described above.
- Third incubation: Pipette 1.5 mL substrate solution into the channels of the incubation tray. Incubate for 10 min at room temperature on a rocking shaker.
- Stop: Aspirate off the liquid from each channel and wash each strip 3 × 1 min with distilled water.
- Evaluate: Place test strip on the evaluation protocol, air dry and evaluate.
2.4. Guidance and Re-Evaluation
2.5. Clinical Relevance
2.6. Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Anti-Nucleosome/ Histone/dsDNA (+) * | Anti-Nucleosome/ Histone/dsDNA (−) | LR+/LR− | OR (95% CI) | p-Value | |
---|---|---|---|---|---|
Total (healthy subjects + patients)—Original | |||||
AC-1 (+) | 6 | 23 | 0.89/1.10 | 0.82 (0.25–2.71) | 0.74 |
AC-1 (−) | 8 | 25 | |||
Total (healthy subjects + patients)—Excluding 1:80 | |||||
AC-1 (+) | 5 | 19 | 0.86/1.14 | 0.75 (0.20–2.78) | 0.67 |
AC-1 (−) | 7 | 20 | |||
Total (healthy subjects + patients)—Re-evaluated | |||||
AC-1 (+) | 8 | 25 | 1.10/0.89 | 1.23 (0.37–4.07) | 0.74 |
AC-1 (−) | 6 | 23 | |||
Patients—Original | |||||
AC-1 (+) | 5 | 14 | 1.18/0.87 | 1.36 (0.33–5.61) | 0.67 |
AC-1 (−) | 5 | 19 | |||
Patients—Re-evaluated | |||||
AC-1 (+) | 8 | 14 | 1.89/0.35 | 5.43 (1.00–29.61) | 0.05 |
AC-1 (−) | 2 | 19 |
Anti-DFS70 (+) | Anti-DFS70 (−) | LR+/LR− | OR (95% CI) | p-Value | |
---|---|---|---|---|---|
Total (healthy subjects + patients)—Original | |||||
AC-2 (+) | 24 | 14 | 1.71/0.41 | 4.16 (1.39–12.50) | 0.01 |
AC-2 (−) | 7 | 17 | |||
Total (healthy subjects + patients)—Excluded 1:80 | |||||
AC-2 (+) | 22 | 10 | 1.67/0.44 | 3.77 (1.14–12.46) | 0.03 |
AC-2 (−) | 7 | 12 | |||
Total (healthy subjects + patients)—Re-evaluated | |||||
AC-2 (+) | 23 | 1 | 24.53/0.24 | 101.86 (11.70–886.44) | <0.001 |
AC-2 (−) | 7 | 31 | |||
Patients—Original | |||||
AC-2 (+) | 17 | 12 | 1.23/0.65 | 1.89 (0.52–6.87) | 0.33 |
AC-2 (−) | 6 | 8 | |||
Patients—Re-evaluated | |||||
AC-2 (+) | 17 | 1 | 14.78/0.27 | 53.83 (5.87–493.61) | <0.001 |
AC-2 (−) | 6 | 19 |
n | Percentage | |
---|---|---|
Specific autoantibodies (+)/AC-2 | 13/24 | 54.2% * |
dsDNA | 4 | 16.7% |
histone | 2 | 8.3% |
Sm | 1 | 4.2% |
RNP/Sm | 2 | 8.3% |
RP155 | 4 | 16.7% |
Ku | 1 | 4.2% |
PM-Scl75 | 3 | 12.5% |
Sp100 | 1 | 4.2% |
Mi-2a | 1 | 4.2% |
Mi-2b | 1 | 4.2% |
n | ANA Pattern (Re-Evaluated) | Specific Autoantibodies | |
---|---|---|---|
SARDs (+)/patients | 10 */43 | ||
Sjögren’s syndrome | 5 | AC-1 | AMA-M2, histone, nucleosome, dsDNA, SSB, Ro52, SSA |
AC-1 & AC-4 | Ro-52, SSA, histone, DFS70 | ||
AC-1 | nil | ||
AC-1 | RP155, PM100, dsDNA | ||
AC-1 | RP155, Ro-52, SSA | ||
RA | 3 | AC-1 | nil |
AC-1 | RP11, RP155 | ||
AC-2 | PM75, Sp100, Mi-2b, DFS70 | ||
SLE | 2 | AC-1 | AMA-M2, histone, nucleosome, dsDNA, SSB, Ro52, SSA |
AC-4 | Ku, SSB, Ro-52, SSA | ||
AS | 1 | AC-2 | RP-155 |
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Yu, H.-H.; Hsieh, P.-F.; Huang, S.-W.; Chan, T.-M.; Tai, P.-L.; Yang, S.-T.; Yu, K.-H. Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging. Biomedicines 2023, 11, 3027. https://doi.org/10.3390/biomedicines11113027
Yu H-H, Hsieh P-F, Huang S-W, Chan T-M, Tai P-L, Yang S-T, Yu K-H. Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging. Biomedicines. 2023; 11(11):3027. https://doi.org/10.3390/biomedicines11113027
Chicago/Turabian StyleYu, Han-Hua, Pao-Feng Hsieh, Szu-Wei Huang, Tien-Ming Chan, Pao-Lien Tai, Shih-Ting Yang, and Kuang-Hui Yu. 2023. "Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging" Biomedicines 11, no. 11: 3027. https://doi.org/10.3390/biomedicines11113027
APA StyleYu, H.-H., Hsieh, P.-F., Huang, S.-W., Chan, T.-M., Tai, P.-L., Yang, S.-T., & Yu, K.-H. (2023). Discriminating between Homogeneous (AC-1) and Dense Fine Speckled (AC-2) Antinuclear Antibody Patterns: Re-Evaluation of Immunofluorescence Imaging. Biomedicines, 11(11), 3027. https://doi.org/10.3390/biomedicines11113027