We thank Andrew Zenger for performing the IIF experiments and collecting images.

The protocol follows the guidelines of Trinity Biotech institutional human research ethics committee.
1. Sample and Substrate Preparation
<ol>
	<li>Allow pouches containing substrate slides (glass slides with 12 wells on each slide containing either HEp2 or HEp-2+HEp2 KO cell mixture) to equilibrate to room temperature for optimal performance and prevent condensation of moisture on slide surface prior to the addition of sample. This should take approximately between 10 - 15 min.</li>
	<li>Once equilibrated to room temperature, carefully remove the substrate slides using fingers without them touching the sides of the pouch.</li>
	<li>Label the slides and place them in an incubation chamber (at room temperature) lined with paper towels moistened with water to prevent drying of slides during sample and conjugate incubation. 
	NOTE: Non-specific staining can result from dry wells; a wet paper towel inside the incubation chamber provides humidity and prevents well dryness.</li>
</ol>
2. Addition of Controls and Samples
<ol>
	<li>Dispense approximately 50 &#181;L of the negative/positive controls, combinations of DFS70-ANA positive sera as described in this study, or patient serum samples onto individual slide wells.</li>
	<li>Dispense control(s) and samples into each well on the slide.</li>
	<li>To prevent well cross-contamination, avoid overfilling the wells. While not overfilling wells, ensure complete coverage of well surface and avoid air bubbles. 
	NOTE: The recommended volume is 50 &#181;L as per step 2.1.</li>
	<li>Place the lid on the incubation chamber and incubate slides for 30 min at room temperature. If there is any ANA present in the serum of the patient, it will bind to the cells present in each well during this incubation period.</li>
	<li>Once the incubation period is over, remove the slide from the incubation chamber. Hold the slide at tab end and rinse gently with approximately 10 mL phosphate-buffered saline (PBS) wash buffer provided in the kit, using a pipette or in a beaker filled with PBS for 10 - 15 s. Transfer the slide immediately into a Coplin jar with PBS wash buffer and wash (soak) for 10 min. Repeat the process with all remaining slides. 
	NOTE: When placing multiple slides in a Coplin jar, ensure that the cell side of the slide does not contact another slide, as this will result in damaging the cells adhered on the glass slide.</li>
</ol>
3. Addition of Fluorescent Conjugate
<ol>
	<li>Remove only one slide at a time from the Coplin jar. To remove the excess PBS wash buffer from the slide, gently tap or blot the slide on the paper towel. Each kit is provided with FITC labeled anti-human IgG fluorescent conjugate. On to each well, gently apply 1 drop (approximately 50 &#181;L) of the provided conjugate.</li>
	<li>Incubate the slides in the closed staining incubation chamber for 30 min. 
	NOTE: The use of Fc specific IgG conjugate is recommended. If there is any ANA present in the serum of the patient, the conjugate will bind to the cells present in each well during this incubation period, which results in the specific fluorescence of the cells present in the wells. The conjugate is sensitive to light. The closed incubation chamber will help to prevent light interference with the conjugate reaction of the cells present in each well.</li>
	<li>Once the incubation period is over, remove the slide from the incubation chamber. Hold the slide at the tab end and rinse gently with approximately 10 mL PBS wash buffer provided in the kit, using a pipette or in a beaker filled with PBS. Transfer the slide immediately into a Coplin jar with PBS wash buffer and wash (soak) for 10 min. Repeat the process with all remaining slides. 
	NOTE: When placing multiple slides in a Coplin jar, ensure that the cell side of the slide does not contact another slide, as this will result in damaging the cells adhered on the glass slide.</li>
	<li>Place the coverslips that are provided in the kit on a dry paper towel. The mounting media is provided with the kit. To the bottom edge of the coverslip, apply between 3 - 4 drops of the mounting media in a&#160;line.</li>
	<li>Take out one slide at a time from the Coplin jar containing the wash buffer. To remove the excess wash buffer, gently tap or blot the slide on the paper towel.</li>
	<li>Lower the slide gently on to the coverslip by placing the lower edge of the slide to the edge of the coverslip. This allows the mounting media present on the lower edge coverslip to flow to the top edge of the slide, and minimizes the formation of air bubbles, which may interfere with the reading of the each well of a slide. 
	NOTE: Minimal pressure should be used to mount the cover slide on to the slide. Any excess pressure can cause lateral movement of the cover slips. This can cause damage to the monolayer preparation of the cells and result in distortion.</li>
	<li>Store the slides in the dark at 2 - 8 &#176;C. Examine the slides as soon as they are mounted or within 48 h. 
	NOTE: Longer storage may result in deterioration of the pattern and fluorescent signal.</li>
</ol>
4. Identification of Positive and Negative Results
<ol>
	<li>View the slides with a fluorescent microscope using a FITC filter set, located in a dark room.</li>
	<li>Examine for specific bright green fluorescence under a fluorescence microscope at a magnification of 200X or greater to make the final interpretation regarding positivity and pattern. Observe the positive and negative controls. 
	NOTE: The positive control will display bright green fluorescence in the nucleus (Figure 2A, Homogeneous). The negative control will display no specific green fluorescence under a fluorescence microscope.</li>
	<li>Record the positive/negative result for each well and include the ANA pattern for positive cases.</li>
</ol>

<ol>
	<li>. Position Statement. Methodology of testing for antinuclear antibodies Available from: <a target="_blank" href="https://www.rheumatology.org/Portals/0/Files/Methodology%20of%20Testing%20Antinuclear%20Antibodies%20Position%20Statement.pdf">https://www.rheumatology.org/Portals/0/Files/Methodology%20of%20Testing%20Antinuclear%20Antibodies%20Position%20Statement.pdf</a> (2011)</li><li>Meroni, P. L., Schur, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ANA+screening:+an+old+test+with+new+recommendations.">ANA screening: an old test with new recommendations.</a> Ann Rheum Dis. 69 (8), 1420-1422 (2010).</li><li>Agmon-Levin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+recommendations+for+the+assessment+of+autoantibodies+to+cellular+antigens+referred+to+as+anti-nuclear+antibodies.">International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies.</a> Ann Rheum Dis. 73 (1), 17-23 (2014).</li><li>Chan, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+of+the+First+International+Consensus+on+Standardized+Nomenclature+of+Antinuclear+Antibody+HEp-2+Cell+Patterns+2014-2015.">Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015.</a> Front Immunol. 6, 412 (2015).</li><li>Malyavantham, K., Suresh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+DFS70+pattern+and+impact+on+ANA+screening+using+a+novel+HEp-2+ELITE/DFS70+knockout+substrate.">Analysis of DFS70 pattern and impact on ANA screening using a novel HEp-2 ELITE/DFS70 knockout substrate.</a> Auto Immun Highlights. 8 (1), 3 (2017).</li><li>Sperotto, F., Seguso, M., Gallo, N., Plebani, M., Zulian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-DFS70+antibodies+in+healthy+schoolchildren:+A+follow-up+analysis.">Anti-DFS70 antibodies in healthy schoolchildren: A follow-up analysis.</a> Autoimmun Rev. , (2017).</li><li>Ayaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+cytotoxic+anti-LEDGF+autoantibodies+in+atopic+dermatitis.">Detection of cytotoxic anti-LEDGF autoantibodies in atopic dermatitis.</a> Autoimmunity. 35 (5), 319-327 (2002).</li><li>Ochs, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+DFS+70+kd/transcription+coactivator+p75+in+atopic+dermatitis+and+other+conditions.">Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.</a> J Allergy Clin Immunol. 105 (6 Pt 1), 1211-1220 (2000).</li><li>Watanabe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-DFS70+antibodies+in+597+healthy+hospital+workers.">Anti-DFS70 antibodies in 597 healthy hospital workers.</a> Arthritis Rheum. 50 (3), 892-900 (2004).</li><li>Yamada, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humoral+immune+response+directed+against+LEDGF+in+patients+with+VKH.">Humoral immune response directed against LEDGF in patients with VKH.</a> Immunol Lett. 78 (3), 161-168 (2001).</li><li>Seelig, C. A., Bauer, O., Seelig, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+Against+DFS70/LEDGF+Exclusion+Markers+for+Systemic+Autoimmune+Rheumatic+Diseases+(SARD).">Autoantibodies Against DFS70/LEDGF Exclusion Markers for Systemic Autoimmune Rheumatic Diseases (SARD).</a> Clin Lab. 62 (4), 499-517 (2016).</li><li>Ochs, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+autoantibodies+to+DFS70/LEDGFp75+in+health+and+disease:+integrating+basic+science+with+clinical+understanding.">The significance of autoantibodies to DFS70/LEDGFp75 in health and disease: integrating basic science with clinical understanding.</a> Clin Exp Med. 16 (3), 273-293 (2016).</li><li>Bizzaro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+chemoluminescence+and+immunoasdorption+tests+for+anti-DFS70+antibodies+avoid+false+positive+results+by+indirect+immunofluorescence.">Specific chemoluminescence and immunoasdorption tests for anti-DFS70 antibodies avoid false positive results by indirect immunofluorescence.</a> Clin Chim Acta. 451 (Pt B), 271-277 (2015).</li><li>Bizzaro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+the+lens+and+cornea+in+anti-DFS70-positive+subjects.">Antibodies to the lens and cornea in anti-DFS70-positive subjects.</a> Ann N Y Acad Sci. 1107, 174-183 (2007).</li><li>Daniels, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antinuclear+autoantibodies+in+prostate+cancer:+immunity+to+LEDGF/p75,+a+survival+protein+highly+expressed+in+prostate+tumors+and+cleaved+during+apoptosis.">Antinuclear autoantibodies in prostate cancer: immunity to LEDGF/p75, a survival protein highly expressed in prostate tumors and cleaved during apoptosis.</a> Prostate. 62 (1), 14-26 (2005).</li><li>Dellavance, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+spectrum+of+antinuclear+antibodies+associated+with+the+nuclear+dense+fine+speckled+immunofluorescence+pattern.">The clinical spectrum of antinuclear antibodies associated with the nuclear dense fine speckled immunofluorescence pattern.</a> J Rheumatol. 32 (11), 2144-2149 (2005).</li><li>Gundin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+anti-DFS70+antibodies+in+patients+with+ANA-associated+autoimmune+rheumatic+diseases+suspicion+is+cost-effective.">Measurement of anti-DFS70 antibodies in patients with ANA-associated autoimmune rheumatic diseases suspicion is cost-effective.</a> Auto Immun Highlights. 7 (1), 10 (2016).</li><li>Kang, S. Y., Lee, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+significance+of+dense+fine+speckled+pattern+in+anti-nuclear+antibody+test+using+indirect+immunofluorescence+method.">Clinical significance of dense fine speckled pattern in anti-nuclear antibody test using indirect immunofluorescence method.</a> Korean J Lab Med. 29 (2), 145-151 (2009).</li><li>Lee, H., Kim, Y., Han, K., Oh, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+anti-DFS70+antibody+and+specific+autoantibody+test+algorithms+to+patients+with+the+dense+fine+speckled+pattern+on+HEp-2+cells.">Application of anti-DFS70 antibody and specific autoantibody test algorithms to patients with the dense fine speckled pattern on HEp-2 cells.</a> Scand J Rheumatol. 45 (2), 122-128 (2016).</li><li>Mariz, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+on+the+antinuclear+antibody-HEp-2+test+is+a+critical+parameter+for+discriminating+antinuclear+antibody-positive+healthy+individuals+and+patients+with+autoimmune+rheumatic+diseases.">Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases.</a> Arthritis Rheum. 63 (1), 191-200 (2011).</li><li>Marlet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombophilia+Associated+with+Anti-DFS70+Autoantibodies.">Thrombophilia Associated with Anti-DFS70 Autoantibodies.</a> PLoS One. 10 (9), e0138671 (2015).</li><li>Mercado, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+autoantibodies+to+DSF70/LEDGFp75+in+Mexican+Hispanics+using+multiple+complementary+assay+platforms.">Detection of autoantibodies to DSF70/LEDGFp75 in Mexican Hispanics using multiple complementary assay platforms.</a> Auto Immun Highlights. 8 (1), 1 (2017).</li><li>Muro, Y., Sugiura, K., Morita, Y., Tomita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concomitance+of+disease+marker+autoantibodies+in+anti-DFS70/LEDGF+autoantibody-positive+patients+with+autoimmune+rheumatic+disease.">High concomitance of disease marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive patients with autoimmune rheumatic disease.</a> Lupus. 17 (3), 171-176 (2008).</li><li>Muro, Y., Sugiura, K., Nakashima, R., Mimori, T., Akiyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+prevalence+of+anti-DFS70/LEDGF+antibodies+in+patients+with+dermatomyositis+and+other+systemic+autoimmune+rheumatic+diseases.">Low prevalence of anti-DFS70/LEDGF antibodies in patients with dermatomyositis and other systemic autoimmune rheumatic diseases.</a> J Rheumatol. 40 (1), 92-93 (2013).</li><li>Mutlu, E., Eyigor, M., Mutlu, D., Gultekin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+anti-DFS70+antibodies+is+needed+in+routine+clinical+samples+with+DFS+staining+pattern.">Confirmation of anti-DFS70 antibodies is needed in routine clinical samples with DFS staining pattern.</a> Cent Eur J Immunol. 41 (1), 6-11 (2016).</li><li>Okamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+DFS70/LEDGF+are+increased+in+alopecia+areata+patients.">Autoantibodies to DFS70/LEDGF are increased in alopecia areata patients.</a> J Autoimmun. 23 (3), 257-266 (2004).</li><li>Pazini, A. M., Fleck, J., dos Santos, R. S., Beck, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+relevance+and+frequency+of+cytoplasmic+and+nuclear+dense+fine+speckled+patterns+observed+in+ANA-HEp-2.">Clinical relevance and frequency of cytoplasmic and nuclear dense fine speckled patterns observed in ANA-HEp-2.</a> Rev Bras Reumatol. 50 (6), 655-660 (2010).</li><li>Schmeling, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+Dense+Fine+Speckles+in+Pediatric+Diseases+and+Controls.">Autoantibodies to Dense Fine Speckles in Pediatric Diseases and Controls.</a> J Rheumatol. 42 (12), 2419-2426 (2015).</li><li>Wiik, A. S., Hoier-Madsen, M., Forslid, J., Charles, P., Meyrowitsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antinuclear+antibodies:+a+contemporary+nomenclature+using+HEp-2+cells.">Antinuclear antibodies: a contemporary nomenclature using HEp-2 cells.</a> J Autoimmun. 35 (3), 276-290 (2010).</li><li>Mahler, M., Fritzler, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Clinical+Significance+of+the+Dense+Fine+Speckled+Immunofluorescence+Pattern+on+HEp-2+Cells+for+the+Diagnosis+of+Systemic+Autoimmune+Diseases.">The Clinical Significance of the Dense Fine Speckled Immunofluorescence Pattern on HEp-2 Cells for the Diagnosis of Systemic Autoimmune Diseases.</a> Clin Dev Immunol. 2012, 494356 (2012).</li><li>Mahler, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-DFS70/LEDGF+Antibodies+Are+More+Prevalent+in+Healthy+Individuals+Compared+to+Patients+with+Systemic+Autoimmune+Rheumatic+Diseases.">Anti-DFS70/LEDGF Antibodies Are More Prevalent in Healthy Individuals Compared to Patients with Systemic Autoimmune Rheumatic Diseases.</a> J Rheumatol. 39 (11), 2104-2110 (2012).</li><li>Conrad, K., Rober, N., Andrade, L. E., Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Clinical+Relevance+of+Anti-DFS70+Autoantibodies.">The Clinical Relevance of Anti-DFS70 Autoantibodies.</a> Clin Rev Allergy Immunol. , (2016).</li><li>Miyara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Phenotypes+of+Patients+with+Anti-DFS70/LEDGF+Antibodies+in+a+Routine+ANA+Referral+Cohort.">Clinical Phenotypes of Patients with Anti-DFS70/LEDGF Antibodies in a Routine ANA Referral Cohort.</a> Clin Dev Immunol. 2013, 703759 (2013).</li><li>Bizzaro, N., Tonutti, E., Villalta, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognizing+the+dense+fine+speckled/lens+epithelium-derived+growth+factor/p75+pattern+on+HEP-2+cells:+not+an+easy+task!+Comment+on+the+article+by+Mariz+et+al.">Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! 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The authors Kishore Malyavantham and Lakshmanan Suresh are employees of Trinity Biotech, USA.

Autoantibodies to nuclear and cytoplasmic antigens are highly prevalent in systemic autoimmune diseases. Although no single assay provides the best possible sensitivity and specificity, IIF by HEp-2 is widely regarded as a highly sensitive and cost-effective screening method for systemic autoimmune diseases2,3,4. Despite the prevalence of IIF protocol for more than 50 years, accuracy of IIF assay is highly dependent on the skill of the technician, careful execution of the individual steps of the protocol, and accurate interpretation of results using a well-maintained fluorescence microscope. Diagnosis of systemic autoimmune disease should not be based on the results of a single serological test such as IIF by HEp-2.
Reconstitution of reagents using high quality water (distilled/deionized), use of standardized kit components (slides with HEp-2 cell monolayers, sample diluent, positive and negative controls, pre-diluted optimized FITC-conjugate, and mounting medium) have a positive impact on the results. Skipping the addition of controls or samples on wells can encourage false negative interpretations. Drying of slides at any point after the addition of sample or control can result in artifactual false positive reactions and/or improper patterns. Too much wash buffer left on slide or drying of slide wells prior to dispensation of mounting medium can result in artifacts. Adding insufficient quantity of mounting medium or generating bubbles on the slide surface prior to placing a coverslip can result in reactions that look blurry or out of focus when observed under the microscope. Mounted slides must be stored at 2 - 8 &#176;C and analyzed within the recommended window of time to minimize false negative or artifactual results.
Using a well-maintained epi-fluorescent microscope which houses an appropriate LED/Hg/Xenon light source and clean optical components including excitation and emission filters that are not damaged is critical to obtain accurate IIF results. End user training for discriminating positive/negative reaction intensities and interpretation of patterns is critical. HEp-2 IIF assays are vulnerable to lack of standardization in procedures, microscope configuration, and end-user training or skill. The consensus nomenclature and representative patterns and examples of images acquired using various manufacturers are made available by ICAP (&#60;www.ANApatterns.org&#62;) for educational purposes4. A HEp-2 cell pattern classification tree provided by ICAP for various nuclear, cytoplasmic, and mitotic patterns is a valuable resource to learn the subtle differences between various patterns that may look similar to the untrained eyes4. One of the primary examples of a challenging pattern is the DFS70 which also lacks a distinct association with an autoimmune condition, and as discussed in previous sections this pattern is highly prevalent in healthy populations5.
Depending on the study, the prevalence of anti-DFS70 autoantibodies vary between healthy cohorts, ANA screening populations, and ANA positive individuals with no evidence of systemic autoimmune disease9,16,20,30,31. DFS70 autoantibodies have been reported to occur in isolation as well as with other disease-associated ANA patterns. Isolated or monospecific DFS70 pattern is reported in 2 - 22% of healthy controls but in less than 1% of the cases with autoimmune rheumatic disease32. Based on these trends, DFS70 mono-positive pattern was proposed as a criterion to exclude in autoimmune rheumatic diseases by ICAP committee3,31,32. The ICAP committee, established to promote harmonization of autoantibody test nomenclature and interpretation of the HEp-2 IIF results, recommends reporting DFS70 positivity while reporting ANA screening results4.
Prior to ruling out a systemic autoimmune disease based on DFS70 mono-positivity, it is vital to review the state of current screening and confirmation methods (particularly those specific for DFS70). Agreement between DFS70 positivity by HEp-2 IIF and solid phase confirmatory assays namely ELISA, CLIA, and line immunoassay/dot blot methods) varied widely between various studies13,22,25,33. IIF interpretation and confirmatory assay parameters that contribute to this disagreement have been discussed previously5,13,34. A recently proposed immunoadsorption approach for anti-DFS70 antibody confirmation addresses some of the deficiencies of current solid phase assays (for DFS70) but requires the labs to perform an additional step in IIF procedure on suspected samples, which increases the cost and turnaround time for reporting results13. This additional step is not required when HEp-2 ELITE/DFS70 KO cells are used for routine ANA screening. This reduces the overall cost and moreover there is no impact on turnaround time as DFS70 pattern is discerned in the initial screening step27. An additional disadvantage of using conventional HEp-2 IIF method is that it is unable to distinguish DFS70 pattern co-occurring with other ANA patterns at the screening stage. However, this disadvantage is overcome by the use of HEp-2 ELITE/DFS70 KO cells27.
Comparative evaluation of conventional HEp-2 and the new engineered HEp-2 (HEp-2 ELITE/DFS70 KO) using ANA positive controls and their combinations with a mono-positive DFS70 control, demonstrate the utility of the new substrate&#39;s ability to simultaneously screen for ANAs and confirm DFS70 pattern (Figure 1). The IIF protocol for the new substrate is identical to the conventional HEp-2 IIF method. Interpretation scheme for all disease-associated ANA patterns is identical with the exception of the DFS70 pattern (Table 1). Interpretation of both mono-positive and mixed DFS70 patterns was relatively easier using the new method and it does not warrant additional assays (Figure 1B). Implementation of this new method in clinical lab simplifies the challenge associated with the interpretation of DFS70 pattern and improves the overall accuracy of the ANA screening by further revealing disease-associated ANA patterns previously concealed by DFS70 (mixed patterns).

ANAs are a hallmark of autoimmune mediated systemic connective tissue diseases1. Several methods are employed for screening and confirmation of ANAs. IIF technique using HEp-2 substrate remains the most widely used and frequently recommended method for screening ANAs1,2. Despite advances in solid phase diagnostic assay methodologies such as enzyme linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), chemiluminescence immunoassay (CLIA), and bead-based multiplex assays, IIF by HEp-2 is the most prevalent screening method due to its diagnostic usefulness and cost effectiveness1,2,3. The above-mentioned assay technologies rely on a limited set of purified native or recombinant autoantigens whereas the HEp-2 cell monolayer preparations on glass slide wells present an extensive array of autoantigens in their natural form, distributed across the nucleus and cytoplasm, that react with the autoantibodies from patient sera or positive controls, resulting in a multitude of patterns that are associated with various autoimmune conditions. These patterns are classified into nuclear, cytoplasmic, and mitotic patterns based on the location of the antigen in the cells. Each pattern and the associated antigen/antigens and disease states are well characterized4. In the IIF method, patient and control sera are incubated on glass slide wells that present a monolayer culture of HEp-2 cells suitably fixed to preserve a multitude of autoantigens in their natural configuration. The autoantibodies in the patient sera or positive controls are allowed to bind to the autoantigens presented by HEp-2 cells on the substrate (glass slide well) during incubation. Post incubation, unbound antibodies are washed off and bound antibodies of the IgG class are detected with fluorescein/fluorescein isothiocyanate (FITC) conjugated anti-human IgG reagent. Unbound reported-conjugate is washed away and the glass coverslips are mounted on top of the slides using a mounting medium that preserves the reactions and facilitates observation under a fluorescent microscope. Reactions are observed under a fluorescence microscope equipped with appropriate excitation-emission filter combination that is configured as per the reporter used (for FITC reporter, a diagnostic microscope typically uses filters with excitation range of 467 - 498 nm and an emission range of 513&#160;- 556 nm). The presence of ANA is demonstrated by a bright green fluorescence of specific structures in the cells. Unlike automated assay platforms, IIF methodology relies on the laborious process of serial diluting the sera and the visual positive determination of the staining patterns which requires significant user expertise5,6. Despite these limitations, IIF by HEp-2 remains the most widely used and recommended method for screening of ANAs due to the standardization efforts towards pattern interpretation and nomenclature by the International consensus on ANA patterns (ICAP) committee, increased availability of automation solutions for IIF slide processing, and result interpretation3.
Among the multitude of patterns detected by the HEp-2 IIF method, autoantibodies targeting the psip1 gene product (also referred to as LEDGF/p75/DFS70), have gained significant interest in recent years for several reasons4,5,6,7,8,9,10. DFS70 autoantibodies are present in high titers in healthy controls, and studies so far have failed to demonstrate a distinct clinical association for the presence of DFS70 autoantibodies7,8,9,10,11,12,13. The rates of reported positive results for DFS70 autoantibodies in various disease states and healthy cohorts varied widely between studies6,8,9,10,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28. The monospecific DFS70 pattern is well differentiated from a homogeneous or speckled pattern but interpretation of mixed homogeneous-speckled patterns and anti-DFS70 antibodies co-occurring with other ANAs is challenging even for expert readers. The current generation of IIF screening methods and DFS70 specific solid phase assays are not capable of differentiating a monospecific DFS70 reaction from mixed patterns (Figure 1A, yellow shaded area of the algorithm). Misinterpretation of DFS70 pattern as homogeneous, speckled, or mixed pattern, or vice versa, may have serious implications on patient care. The current commonly used protocol is as follows: the clinical labs employ a number of solid phase confirmatory tests for disease-associated ANAs and/or an immunoadsorption method for DFS70/LEDGF when dense fine speckled (AC-2) pattern is suspected (Figure 1A)4,5.
The goal of this protocol is to demonstrate the utility of a novel engineered HEp-2 IIF substrate (HEp-2 ELITE/DFS70 knockout (KO), will be referred to as HEp-2 ELITE) that retains all the advantages of conventional HEp-2 for screening ANAs while simultaneously distinguishing DFS70 pattern with high confidence in both monospecific and mixed ANA positive cases (Figure 1B, yellow shaded area of the algorithm)5. HEp-2 ELITE substrate is composed of an approximate mixture of unmodified conventional HEp-2 cells and engineered cells devoid of LEDGF/p75/DFS70 antigen in 1:9 ratio5. The IIF procedure for HEp-2 ELITE is identical to the conventional HEp-2 method. The unique configuration of HEp-2 ELITE substrate not only retains all the advantages of conventional HEp2 but can differentiate homogeneous, speckled, and mixed reaction patterns from DFS70 pattern at the initial screening or testing of a sample (Figure 1B)5. Patient sera to be reacted on slides with HEp-2 IIF substrate should be diluted 1:40 with screening dilution or as per the individual laboratory criterion. A specific reaction at 1:40 or higher titer is considered positive.
In this study, positive sera for homogeneous, speckled, centromere, nucleolar, cytoplasmic reticular/AMA, and DFS70 patterns that produced a medium positive signal (relative to the negative control and positive ANA control provided by the kit) at screening dilution of 1:40 were selected for simulating the mono-specific and mixed patterns on HEp-2 substrates (conventional and HEp-2 ELITE). The 1:40 diluted DFS70 positive sera were mixed with 1:40 dilutions of individual disease-associated ANA pattern controls (homogeneous, speckled, centromere, nucleolar, or cytoplasmic reticular-AMA) in various ratios (control:DFS70 in 100:0, 80:20, 50:50, 20:80, and 0:100) and evaluated on conventional and HEp-2 ELITE substrates following standard IIF procedure outlined below.

<table><tbody><tr><td>HEp-2 ELITE / DFS70-KO 12 Well Slide sealed individually in pouch</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240</td><td></td></tr><tr><td>ANA positive control</td><td>Trinity Biotech&amp;nbsp;</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>DFS70 antibody positive control</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240</td><td></td></tr><tr><td>Negative control</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Anti-human IgG FITC conjugate containing Evan&#039;s blue counterstain</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Buffer diluent for serum</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Mounting medium</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>Coverslips&amp;nbsp;</td><td>Trinity Biotech</td><td>part of kit# 1108, 1108-120, 1108-240, 1103, 1103-240</td><td></td></tr><tr><td>ImmuGlo ANA HEp-2 Cells 12 well slide sealed individually in pouch</td><td>Trinity Biotech</td><td>part of kit# 1103, 1103-240</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials used in the study but not provided by the kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Diagnostic fluorescent microscope</td><td>Nikon Eclipse 50i</td><td></td><td>Equipped with a spot dignostic camera, model 2.2.1</td></tr><tr><td>Incubation chamber</td><td>Trinity Biotech</td><td></td><td>provided for customers (no catalog #)</td></tr><tr><td>Coplin jar</td><td>n/a</td><td></td><td>General labware</td></tr><tr><td>Paper towels</td><td>n/a</td><td></td><td>General lab supplies</td></tr><tr><td>distilled/deionized water</td><td>n/a</td><td></td><td>General lab supplies</td></tr></tbody></table>

simultaneous distinction of monospecific and mixed dfs70 patterns during ana screening with a novel hep-2 elite/dfs70 knockout substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

HEp-2 ELITE/DFS70 KO Substrate Preserves Classic ANA Patterns and Facilitates Clear Distinction of DFS70 Pattern:
Conventional and engineered cells on HEp-2 ELITE/DFS70 KO substrate are identical, preserving all autoantigens except for the LEDGF/p75. The negative control provided by the kit establishes the baseline fluorescent signal. Positive controls for homogeneous, speckled, centromere, nucleolar, and mitochondrial patterns on HEp-2 ELITE produce a pattern identical to expected patterns on conventional HEp-2 IIF substrate (Figure 2A). These reference patterns have been described in detail along with each pattern's antigen and disease association previously4,29. A brief description of patterns observed in Figure 2A are provided in Table 1.
DFS70 pattern on HEp-2 ELITE:
Approximately 10% of the cells in each well show a dense fine speckled pattern (ICAP assigned nomenclature: AC-2) that can be observed on the interphase nucleus, and chromatin associated staining is seen on mitotic nuclei (Figure 2B). On a conventional HEp-2 substrate, all the cells will have this pattern when reacted with a DFS70 positive sample. The remaining ~90% of HEp-2 cells have the psip1 gene encoding the LEDGF antigen knocked out and therefore will not produce a signal or pattern when reacted with a monospecific DFS70 positive serum (Figure 2B). If 10% of the HEp-2 cells have brighter fluorescence corresponding to the DFS70 pattern and rest of the cells present additional patterns (e.g., fine speckled or nucleolar), then this indicates the presence of mixed patterns. Closer examination of such patterns is essential to confirm all the autoantibody specificities that are present in addition to the DFS70 pattern. To demonstrate the capability of the HEp-2 ELITE substrate, a panel of sera samples was created with various concentrations of DFS70 and ANA positive control sera and tested on both conventional and HEp-2 ELITE substrates using a standard IIF protocol (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7).
Interpretation of DFS70 Monospecific and Mixed Reactions on Conventional and HEp-2 ELITE Substrates:
For each specific disease-associated ANA pattern (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7), a monospecific ANA pattern on left most column and a monospecific DFS70 pattern on right most column can be observed. The three columns in the middle represent various ratios of disease-associated and DFS70 pattern positive controls. The resulting mixed patterns are challenging to discern on conventional HEp-2 substrate (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, top row). However, with the novel Hep-2 ELITE substrate, in most of the samples, the difference in intensities between unmodified and engineered cells is distinct which not only confirms the presence of DFS70 autoantibodies (when the ratio of brighter to less bright cells is 1:9) but also reveals a secondary ANA pattern. In the case of homogeneous-DFS70 or speckled-DFS70 mixed reactions, it is impossible to confidently predict the correct pattern which leads to the labs opting to run a number of additional assays (Figure 1A). In the case of centromere-DFS70 and nucleolar-DFS70 mixed reactions, suspecting DFS70 positivity is very challenging (Figure 5, Figure 6). With nucleolar and cytoplasmic reticular/AMA reactions, the DFS70 pattern is not dominant until the sera ratio reaches 50%. In all the examples, the HEp-2 ELITE substrate can present the DFS70 pattern and the underlying secondary pattern over a wider range of intensity levels and thus facilitates a more accurate ANA interpretation.
To closely observe the mixed patterns, results for various 50:50 combinations of ANA and DFS70 positive controls were enlarged (Figure 8) for both conventional and HEp-2 ELITE substrates. It is encouraged to observe both the interphase and mitotic staining for all ANA patterns including DFS70 on HEp-2 substrates to improve interpretation and accuracy of the reported pattern. However, for mixed patterns, the differential pattern presented by dividing cell nuclei may not be sufficient to facilitate accurate interpretation. Red arrows (Figure 8) indicate the mitotic nuclei in both conventional and engineered cell. It can be observed that mixed DFS70 reactions on dividing cells do not comply with the established set of rules for interpretation of disease-associated ANA patterns. Yellow and blue arrows indicate unmodified and engineered (DFS70 KO) HEp-2 cell nuclei, respectively. In all the combinations (Figure 8), a clear intensity difference between the unmodified cell nuclear staining (~10%) and engineered cell nuclear (~90%) pattern in the same microscopic field of view is observed, which enables simultaneous screening and confirmation of the DFS70 pattern.
<img alt="ANA screening algorithms flowchart; HEp-2 IIF and DFS70 methods; diagnostic process illustration." class="xfigimg" src="/files/ftp_upload/56722/56722fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: ANA screening algorithms using conventional HEp-2 and HEp-2 ELITE/DFS70 KO IIF methods. (A) Schematic unravels the deficiency of conventional HEp-2 IIF in identifying monospecific and mixed DFS70 positive patterns at the screening stage (yellow shaded area) and its inability to rule out a secondary disease-associated ANA pattern that may be concealed by DFS70 pattern. Further, the current generation of DFS70 specific solid phase confirmatory assays are not able to confirm monospecific DFS70 positivity which leads to additional confirmatory testing for ANAs. (B) Schematic reveals the capabilities of HEp-2 ELITE for screening of ANAs, simultaneous distinction of monospecific and mixed DFS70 reactions, and eliminating the need for solid phase DFS70 confirmation assays. Algorithm using HEp-2 ELITE significantly simplifies the ANA screening and reduces the required number of confirmation assays. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="HEp-2 ELITE/DFS70 KO substrate diagram showing classic ANA patterns: homogeneous, speckled, centromere, nucleolar, cytoplasmic reticular/AMA with DFS70 positive reaction for diagnostic interpretation." class="xfigimg" src="/files/ftp_upload/56722/56722fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Classic ANA and DFS70 pattern on HEp-2 ELITE/DFS70 KO substrate. (A) HEp-2 ELITE/DFS70 KO substrate preserves classic ANA patterns. Classic homogeneous reaction and DFS70 positive reactions were produced using the positive controls provided by the kit. Other reactions were produced using previously established positive control sera for speckled, centromere, nucleolar, and cytoplasmic reticular/AMA reactions5. (B) HEp-2 ELITE/DFS70 KO substrate provides both conventional and engineered cells, which facilitates easy distinction of DFS70 pattern. Schematic shows the resulting DFS70 monospecific reaction and the staining pattern on interphase and dividing cell nuclei for both conventional and engineered (DFS70-KO) cells. Engineered HEp-2 cells are devoid of LEDGF/p75/DFS70 antigen which enables the simultaneous detection of secondary patterns that are usually concealed by DFS70 autoantibody reaction on conventional HEp-2 substrates. Arrows (yellow) indicate examples of mitotic cell nuclei. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, HEp-2 cells, antibody pattern comparison, DFS70 labeling, immunofluorescence." class="xfigimg" src="/files/ftp_upload/56722/56722fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: DFS70 and homogeneous sera in combination. DFS70 monospecific sera were combined with a positive homogeneous pattern control in various ratios (100:0, 80:20, 50:50, 20:80, and 0:100) and tested on conventional and engineered (DFS70-KO) HEp-2 substrates. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="HEp-2 cell fluorescence microscopy; DFS70 intensity variation; comparative analysis." class="xfigimg" src="/files/ftp_upload/56722/56722fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: DFS70 and speckled sera in combination. DFS70 monospecific sera were combined with a positive speckled pattern control in various ratios (100:0, 80:20, 50:50, 20:80, and 0:100) and tested on conventional and engineered (DFS70-KO) HEp-2 substrates. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope images of Hep-2 cells with varying centromere:DFS70 ratios showing fluorescence patterns." class="xfigimg" src="/files/ftp_upload/56722/56722fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: DFS70 and centromere sera in combination. DFS 70 monospecific sera were combined with a positive centromere pattern control in various ratios (100:0, 80:20, 50:50, 20:80, and 0:100) and tested on conventional and engineered (DFS70-KO) HEp-2 substrates. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, protein expression, nucleolar DF570 ratios, HEP-2 cell comparison chart." class="xfigimg" src="/files/ftp_upload/56722/56722fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: DFS70 and nucleolar sera in combination. DFS 70 monospecific sera were combined with a positive nucleolar pattern control in various ratios (100:0, 80:20, 50:50, 20:80, and 0:100) and tested on conventional and engineered (DFS70-KO) HEp-2 substrates. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cytoplasmic fluorescence, DFS70 analysis, HEP-2 cells, immunofluorescence microscopy results." class="xfigimg" src="/files/ftp_upload/56722/56722fig7.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: DFS70 and cytoplasmic reticular/AMA sera in combination. DFS70 monospecific sera were combined with a positive mitochondrial pattern control in various ratios (100:0, 80:20, 50:50, 20:80, and 0:100) and tested on conventional and engineered (DFS70-KO) HEp-2 substrates. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy comparison of HEp-2 cells with DFS70-KO; protein expression patterns." class="xfigimg" src="/files/ftp_upload/56722/56722fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Mixed patterns (50:50 ratio) produced on conventional and HEp-2 ELITE substrates. Differences between the two substrates are revealed. Red arrows indicate the mitotic nuclei in both conventional and engineered cells on both substrates. For these combinations, the differential pattern presented by dividing cell nuclei on conventional HEp-2 substrate is not sufficient for accurate interpretation of mixed patterns. It can be observed that mixed DFS70 reactions on dividing cells can perturb the established set of rules for interpretation of disease-associated ANA patterns. Yellow and blue arrows indicate unmodified and engineered (DFS70 KO) HEp-2 cell nuclei, respectively. Results obtained for all the combinations tested on the engineered substrate: a clear intensity difference between the interphase nuclear pattern belonging to unmodified cells (~10%) and engineered cells (~90%) was observed, which enabled simultaneous detection and confirmation of DFS70 pattern. Scale bars represent 20 µm. <a href="https://cloudflare.jove.com/files/ftp_upload/56722/56722fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ANA pattern</td>
			<td colspan="2">Description</td>
		</tr>
		<tr>
			<td>Homogeneous</td>
			<td colspan="2">The entire nucleus fluoresces evenly with a diffuse staining pattern.</td>
		</tr>
		<tr>
			<td>Speckled</td>
			<td colspan="2">Discrete, coarse to fine speckles of fluoresce throughout the nucleus.  Fine speckled and coarse speckled patterns have been described.</td>
		</tr>
		<tr>
			<td>Nucleolar</td>
			<td colspan="2">The nucleoli stain as multiple solid or speckled bodies within the nucleus. Subtypes have been described previously.</td>
		</tr>
		<tr>
			<td>Centromere</td>
			<td colspan="2">Large speckles of finite number. Reactive antigen segregates with condensed chromosomes in cells undergoing mitosis.</td>
		</tr>
		<tr>
			<td>Cytoplasmic reticular/AMA</td>
			<td colspan="2">The cytoplasm appears granular with positive staining of the mitochondria.</td>
		</tr>
		<tr>
			<td rowspan="2">DFS70</td>
			<td colspan="2">•Conventional HEp-2 cells: A dense fine speckled pattern is observed on the interphase nucleus and chromatin associated staining is seen on mitotic nuclei.</td>
		</tr>
		<tr>
			<td colspan="2">•Engineered DFS70 KO cells: Anti-DFS70 antibodies produce negative staining</td>
		</tr>
		<tr>
			<td colspan="3">HEp-2 Elite/ DFS70 KO has conventional and engineered (DFS70 KO) cells in approximately 1:9 ratio. Conventional cells produce typical DFS70 pattern and engineered cells produce a negative pattern when reacted with a DFS70 monospecific/isolated positive reactions.</td>
		</tr>
	</tbody>
</table>
Table 1: Description of ANA Patterns.

Watch this Scientific Journal Video about Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate at JoVE.com

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

DFS70 autoantibodies mimic common disease-associated antinuclear antibody patterns making accurate interpretation challenging when using conventional HEp-2 substrates. The protocol describes advantages of novel engineered HEp-2 substrate over conventional HEp-2 in ANA screening and distinguishing DFS70 patterns with high confidence in both monospecific and mixed ANA positive cases.

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...

simultaneous-distinction-monospecific-mixed-dfs70-patterns-during-ana

Research

JoVE Journal

Immunology and Infection

35.6K Views.  A Trinity Biotech Company. DFS70 autoantilichamen nabootsen gemeenschappelijk ziekte-geassocieerde antinuclear antilichaam patronen maken nauwkeurige interpretatie uitdaging bij het gebruik van conventionele HEp-2 substraten. Het protocol beschrijft de voordelen van roman gemanipuleerde HEp-2 substraat ten opzichte van conventionele HEp-2 in ANA screening en het registratienummer van de DFS70 patronen met hoge vertrouwen in zowel monospecifieke als gemengde ANA positieve gevallen.

Video: Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Cancer Research

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Genetics

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Bioengineering

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a cost-effective in vivo method to identify heterochromatin-promoting compounds by using Drosophila. The Drosophila&#39;s DX1 strain, having a variegated eye color phenotype that reflects the extents of heterochromatin formation, thereby providing a tool for a heterochromatin-promoting drug screen. In this screening method, eye variegation is quantified based on the surface area of red pigmentation occupying parts of the eye and is scored on a scale from 1 to 5. The screening method is straightforward and sensitive and allows for testing compounds in vivo. Drug screening using this method provides a fast and inexpensive way for identifying heterochromatin-promoting drugs that could have beneficial effects in cancer therapeutics. Identifying compounds that promote the formation of heterochromatin could also lead to the discovery of epigenetic mechanisms of cancer development.

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

Drosophila is an excellent model organism that can be used to screen compounds that might be useful for cancer therapy. The method described here is a ...

Single Nucleotide Polymorphism-sensitive FISH Detection of Locus-specific Ribosomal RNA Transcription in Drosophila melanogaster

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences that encode rRNAs, composing regions called ribosomal DNA (rDNA) loci. Many organisms' genomes contain more than one rDNA locus distributed across different chromosomes, and rRNA may be transcribed from multiple rDNA loci or only a single locus. Changes in rRNA transcription sources often indicate ribosomal distress. However, the homogeneity of rRNA obstructs distinguishing if rRNA is transcribed from multiple or a single rDNA locus. Here, we describe a method that uses single nucleotide polymorphism-sensitive fluorescence in situ hybridization (SNP-FISH) to distinguish rRNA transcription between Drosophila melanogaster rDNA loci on the X and Y chromosomes. This method leverages locus-specific rRNA variants to enable easy detection of the source of rRNA transcription with single-cell resolution. This assay can be applied to any Drosophila cell type and may be adapted for use in other systems.

The protocol describes single nucleotide polymorphisms-sensitive fluorescence in situ hybridization (SNP-FISH) method to distinguish ribosomal RNA transcripts derived from the X or Y chromosome ribosomal DNA locus in Drosophila melanogaster.

In order to ensure sufficient ribosomal RNA (rRNA) is transcribed for ribosome function, genomes contain hundreds of tandem duplications of the sequences ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Biochemistry

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Medicine

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biology

Anti-Nuclear Antibody Screening Using HEp-2 Cells

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although IIF is an excellent screening test in expert hands, the technical difficulties of processing and reading IIF slides &#8211; such as the labor intensive slide processing, manual reading, the need for experienced, trained technologists and the use of dark room &#8211; make the IIF method difficult to fit in the workflow of modern, automated laboratories.
The first and crucial step towards high quality ANA screening is careful slide processing. This procedure is labor intensive, and requires full understanding of the process, as well as attention to details and experience.
Slide reading is performed by fluorescent microscopy in dark rooms, and is done by trained technologists who are familiar with the various patterns, in the context of cell cycle and the morphology of interphase and dividing cells. Provided that IIF is the first line screening tool for SARD, understanding the steps to correctly perform this technique is critical.
Recently, digital imaging systems have been developed for the automated reading of IIF slides. These systems, such as the NOVA View Automated Fluorescent Microscope, are designed to streamline the routine IIF workflow. NOVA View acquires and stores high resolution digital images of the wells, thereby separating image acquisition from interpretation; images are viewed an interpreted on high resolution computer monitors. It stores images for future reference and supports the operator&#8217;s interpretation by providing fluorescent light intensity data on the images. It also preliminarily categorizes results as positive or negative, and provides pattern recognition for positive samples. In summary, it eliminates the need for darkroom, and automates and streamlines the IIF reading/interpretation workflow. Most importantly, it increases consistency between readers and readings. Moreover, with the use of barcoded slides, transcription errors are eliminated by providing sample traceability and positive patient identification. This results in increased patient data integrity and safety.
The overall goal of this video is to demonstrate the IIF procedure, including slide processing, identification of common IIF patterns, and the introduction of new advancements to simplify and harmonize this technique.

Indirect immunofluorescent (IIF) assays have traditionally been used for the detection of antinuclear antibodies (ANA) in human serum. The presence of these antibodies can aid in the diagnosis of systemic autoimmune rheumatic diseases (SARD). This protocol demonstrates how to effectively perform the IIF technique to accurately detect these autoantibodies.

The American College of Rheumatology position statement on ANA testing stipulates the use of IIF as the gold standard method for ANA screening1. Although ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

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