We thank Cassandra Bryant for performing the IIF experiment and Carol Buchner for her expert technical review.

1. Sample Preparation and Substrate Selection
<ol>
	<li>Remove reagents from packaging and allow each item to come to room temperature. Prepare reagents and dilute patient sera according to the direction insert. Note: NOVA Lite slides are barcoded and can be easily integrated into automated systems in conjunction with the Laboratory Information System LIS. This procedure illustrates manual slide processing; high throughput laboratories, however, may opt for automated slide processing equipment.</li>
</ol>
2. Addition of Controls and Samples
<ol>
	<li>Dispense 1 drop of positive control and 1 drop of negative control to the appropriate slide wells.&#160;Pipette 20-25 &#956;l of diluted patient serum to the remaining wells. Process one slide at a time.</li>
	<li>Place the slide(s) in a staining container with a damp paper towel on the bottom.&#160;Cover the container and incubate the slide(s) for 30 min. Note: The humid conditions will prevent the substrate from drying out. Drying of the wells could result in artifactual staining. During this incubation period, the anti-nuclear antibodies in the patient&#8217;s serum will bind to antigens of the cells that are fixed onto each well.</li>
	<li>After the incubation period, rinse off the serum using a gentle stream of wash buffer.&#160;In order to avoid damaging the substrate and prevent cross-over of samples between wells, angle the slide slightly to avoid directing the stream directly onto the wells. Tap off excess wash buffer and place the slide into a Coplin jar containing wash buffer for approximately 5 min.</li>
</ol>
3. Addition of Fluorescent Conjugate
<ol>
	<li>One by one, remove the slides from the wash buffer and gently tap to remove the excess wash buffer. Apply 1 drop of fluorescent conjugate (FITC labeled anti-human IgG) onto each well. Incubate the slides for 30 min in the humidified container, and be sure to replace the staining container cover. Note: For ANA testing, the use of an IgG Fc specific conjugate is recommended. The conjugate is light sensitive, and the cover will protect the slides from exposure to light in addition to maintaining the humidified environment. During this incubation period, the conjugate will bind to the patient&#8217;s anti-nuclear antibodies that have bound to the cell antigens. This conjugate binding results in the presence of fluorescence in the wells.</li>
	<li>Rinse and wash the slide as described above.</li>
	<li>Place a coverslip on a paper towel and apply mounting medium in a continuous line to the bottom edge of the coverslip.</li>
	<li>Remove each slide from the wash buffer and tap the slide gently to remove the excess wash buffer. Touch the lower edge of the slide to the edge of the coverslip.</li>
	<li>Gently lower the slide onto the coverslip in such a way that the mounting medium on the coverslip flows to the top edge of the slide without air bubble formation. Note: Coverslipping, including using the optimal amount of mounting medium, is a technique that takes practice to perfect.</li>
</ol>
4. Identification of Positive and Negative Results
Manual Interpretation
<ol>
	<li>View slides with a fluorescent microscope, located in a dark room. Perform scanning of the entire well with a 20X or 25X objective to assess cell distribution and uniformity of fluorescence</li>
	<li>Switch to a 40X objective to make the final interpretation regarding positivity and pattern.</li>
	<li>Look at the positive and negative controls. Grade positivity using a reactivity grading scale from 1+ to 4+. Note: The negative control may not appear completely dark, but will often display low level nonspecific fluorescence. The positive control will display bright apple green fluorescence in the nucleus (Figure 1).</li>
</ol>
Automated Interpretation
Note: In addition to manual interpretation, load and scan slides by NOVA View Automated Fluorescence Microscope or other automated digital imaging instrument; no darkroom is necessary. After creating a project by selecting the appropriate slide type, the instrument acquires and stores high resolution digital images of cells in each well. Additionally, NOVA View measures fluorescent light intensity, and categorizes results as positive or negative, and provides pattern recognition for positive samples. Images are viewed by the operator on a high resolution computer monitor, allowing for final interpretation, revision and confirmation of NOVA View result. Reports can be generated on confirmed results. Digital microscopy should be used in conjunction with an experienced laboratory technician as it is intended to aid in interpretation of results.

<ol>
	<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>Tan, E. M. <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+nuclear+antigens+(ANA):+Their+immunobiology+and+medicine.">Autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine.</a> Advances in immunology. 33, 167-240 (1982).</li><li>Sack, U., 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=Autoantibody+detection+using+indirect+immunofluorescence+on+HEp-2+cells.">Autoantibody detection using indirect immunofluorescence on HEp-2 cells.</a> Ann NY Acad Sci. 1173, 166-173 (2009).</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>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=Importance+of+the+dense+fine+speckled+pattern+on+HEp-2+cells+and+anti-DFS70+antibodies+for+the+diagnosis+of+systemic+autoimmune+diseases.">Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases.</a> Autoimmun Revi. 11 (9), 642-645 (2012).</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).</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).</li><li>Mariz, 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=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>Muro, Y., 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=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>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>Craft, 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=The+U2+small+nuclear+ribonucleoprotein+particle+as+an+autoantigen.">The U2 small nuclear ribonucleoprotein particle as an autoantigen.</a> J Clin Invest. 81 (6), 1716-1724 (1998).</li><li>Salomonsson, 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=A+serologic+marker+for+fetal+risk+of+congenital+heart+block.">A serologic marker for fetal risk of congenital heart block.</a> Arthritis Rheum. 46 (5), 1233-1241 (2002).</li><li>Miyawaki, 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=Clinical+and+serological+heterogeneity+in+patients+with+anticentromere+antibodies.">Clinical and serological heterogeneity in patients with anticentromere antibodies.</a> J Rheumatology. 32 (8), 1488-1494 (2005).</li><li>Raijamkers, R., 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=PM-Scl-75+is+the+main+autoantigen+in+patients+with+the+polymyositis/scleroderma+overlap+syndrome.">PM-Scl-75 is the main autoantigen in patients with the polymyositis/scleroderma overlap syndrome.</a> Arthritis Rheum. 50 (2), 565-569 (2004).</li><li>Yang, J. 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=Human+scleroderma+sera+contain+autoantibodies+to+protein+components+specific+to+the+UC+small+nucleolar+RNP+complex.">Human scleroderma sera contain autoantibodies to protein components specific to the UC small nucleolar RNP complex.</a> Arthritis Rheum. 48 (1), 210-217 (2003).</li><li>Granito, 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=Antinuclear+antibodies+as+ancillary+markers+in+primary+biliary+cirrhosis.">Antinuclear antibodies as ancillary markers in primary biliary cirrhosis.</a> Expert Rev Mol Diagn. 12 (1), 65-74 (2012).</li></ol>

The authors Gabriella Lakos, Cassandra Bryant, Carol Buchner, and Anna Eslami are employees of INOVA Diagnostics.

Screening by HEp-2 cells is a critical first step in the diagnosis of patients with SARD. However, IIF methods lack harmonization. Sources of variability include preparation of slides, conjugate specificity, and efficiency of the fluorescent microscope and experience of the reader. Despite these concerns, IIF remains the &#8220;gold standard&#8221; for ANA testing. HEp-2 cells contain a large variety of autoantigens, and provide the ideal substrate for the detection of these autoantibodies. In some laboratories, IIF has been replaced with solid phase screening methods such as multiplex assay or ELISA. The shortcoming of such tests is that they do not display the full range of antigens to be sufficiently sensitive. As a consequence, true positive patients may be missed which can have deleterious consequences In addition to the issues of treatment delay and wrong diagnosis, additional healthcare costs may occur due to the repetition of confirmatory tests or unnecessary diagnostic investigations. Given the inherent challenges to IIF, it is paramount to perform this technique properly to avoid subjectivity in results.
To ensure accurate interpretation and reporting of results for ANA screening, it is vital to use the highest quality substrate. When selecting a HEp-2 substrate, it is critical that the cells be optimized to express clinically relevant epitopes in their native protein state. Transfected or otherwise modified HEp-2 cell lines may not allow proper antibody-antigen binding. In addition, when several different autoantibodies are present simultaneously, recombinant cells may mask one or more patterns. High number of mitotic cells should also be present in the substrate. Adequate number of mitotic cells allows quick and accurate identification of patterns. 
In addition to the attributes of the substrate, the objective of this video protocol is to describe the IIF technique by showing critical steps such as the addition of sample, slide washing, addition of conjugate, cover slipping, and determination of positive and negative results. Results can be compromised if proper technique is not used for each of these steps. Proper washing is important to remove all unbound antibody. Some patients display very high amounts of autoantibodies and in these cases it is important to wash the serum from the wells such that it does not contaminate other wells. 
Although IIF has traditionally been a very labor intensive and subjective laboratory method, new technologies such as slide processors, barcoded slides and automated digital microscopy can greatly simplify the workflow, increase the consistency and reduce the sources of variability of interpretation.

The term antinuclear antibody (ANA) describes a variety of autoantibodies that react with constituents of cell nuclei including DNA, proteins and ribonucleoproteins1,2. The HEp-2 cell, a native protein array with hundreds of antigens, provides an ideal substrate for the detection of ANA1. The detection of ANA in human serum is an important screening tool for connective tissue diseases, and IIF is the reference method for ANA testing1. Recently, IIF on HEp-2 cells has been replaced in some laboratories with antigen-specific immunoassays and multiplex methods. Due to concerns over false negative results and the lack of transparency to clinicians, the American College of Rheumatology formed a Task Force that concluded that IIF using HEp-2 cells should be the &#8220;gold standard&#8221; for ANA screening1.
Due to the subjective nature of ANA screening, the quality of HEp-2 cells is integral to accurate and confident reporting of results. The presence of a high number of mitotic cells, optimal cell morphology, sufficient confluency, and expression of relevant antigens are particularly important. IIF pattern recognition serves as an important tool to aid in patient diagnosis. Understanding the significance of various patterns enables the clinicians and laboratory personnel to perform the appropriate follow-up testing to confirm diagnosis. For example, homogeneous ANA pattern can occur in the presence of anti-dsDNA or chromatin antibodies, and may be associated with systemic lupus erythematosus (SLE)3. From the other side, it has recently been described that the so called dense fine speckled pattern (DFS) that is commonly seen in up to 12% of routine samples, has mostly been associated with anti-DFS70 antibodies. These autoantibodies, when found in isolation (without other disease-specific ANA) are not associated with systemic autoimmune rheumatic diseases4-9. Thereby confirmatory testing for anti-DFS70 antibodies can help reduce unnecessary reflex testing, offer considerable cost savings, and ease patient anxiety.
Given that IIF is the first line screening methodology to detect autoantibodies, it is paramount that the user understands how the selection of reagents and tissue substrates can affect results. Since the IIF technique is inherently subjective, it is important that the reagents used provide the highest quality results.
This goal of this video protocol is to familiarize the user with the correct steps needed to perform the IIF method, the common patterns associated with ANA, and to introduce new advancements which can streamline the laboratory workflow and standardize results.

<table><tbody><tr><td>NOVA Lite HEp-2 IgG (DAPI conjugate)</td><td>INOVA Diagnostics</td><td>708102</td><td></td></tr><tr><td>NOVA View Instrument</td><td>INOVA Diagnostics</td><td>NV2000</td><td></td></tr><tr><td>QUANTA Link Workstation</td><td>INOVA Diagnostics</td><td>LINK010</td><td></td></tr><tr><td>QUANTA Link Workstation License</td><td>INOVA Diagnostics</td><td>LINK001</td><td></td></tr><tr><td>Barcode Scanner</td><td>INOVA Diagnostics</td><td>LINK019</td><td></td></tr></tbody></table>

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.

When evaluating cell staining results for HEp-2, five major nuclear patterns are most commonly reported: homogenous, speckled, centromere, nucleolar, and nuclear dots. These patterns are the result of autoantibody binding to specific constituents of the nucleus. Although ANA testing is specific to nuclear structures, cytoplasmic patterns associated with autoantibodies against cytoplasmic structures may also be observed. In some cases, several autoantibodies may be present in a sample, and the image may appear as a mixed pattern.
There are other, less frequently observed, and often cell cycle specific patterns that can be detected by the IIF method; most recently, a so called dense fine speckled pattern with special clinical significance has been described in some ANA positive sera.
Homogeneous Pattern
To identify a homogeneous pattern, scan the well and identify mitotic or dividing cells. The condensed chromatin of the mitotic cells (Figure 2, orange arrow) exhibits solid, uniform fluorescence which is often more pronounced than in the resting cell nuclei.In the homogeneous pattern, the resting cells exhibit uniform, diffuse fluorescence of the entire nucleus (Figure 2, white arrow). This characteristic pattern is often the result of anti-dsDNA antibodies10.
Speckled Pattern
In the coarse speckled pattern, the mitotic cells show no staining of the condensed chromosomal regions (Figure 3, orange arrow). The resting cells exhibit granular fluorescence throughout the entire nucleus. The nuclear speckling can be defined as coarse or fine. The Coarse speckling pattern is often the result of anti-Sm and anti-RNP11.
In the fine speckled pattern, the resting cells show fine or diffuse speckling throughout the nuclei, typically in uniform distribution (Figure 4, white arrow). The fine speckled pattern is often the result of anti-SSAand anti-SSB12.
Dense Fine Speckled Pattern
It is now recognized that the dense fine speckled pattern (DFS) is common in routine testing, and as much as 12% of samples are positive for anti-DFS70 autoantibodies. However, these autoantibodies, when found in isolation, are not associated with systemic autoimmune rheumatic diseases4-6. These antibodies are prevalent in healthy individuals and patients bearing diseases unrelated to SARD6. Confirmatory testing for anti-DFS70 antibodies can help reduce unnecessary reflex testing, offer considerable cost savings, and ease patient anxiety.
Since the DFS pattern is difficult to identify and ANA positivity can cause anxiety to both patients and physicians, it is highly recommended to perform a confirmatory test after identifying a pattern suggestive of anti-DFS70 antibodies to clarify the clinical significance of ANA positivty5,6.
In this pattern, mitotic cells (Figure 5, orange arrow) show positive speckled staining of the metaphase chromatin, while resting cell nuclei (Figure 5, white arrow) exhibit uniformly distributed fine speckles throughout the nucleus.

Centromere Pattern
To identify the centromere pattern, scan the wells and identify mitotic or dividing cells. In mitotic cells (Figure 6, orange arrow), these discrete speckles become closely associated in what is often described as a “metaphase bar”. In the centromere pattern, resting cells (Figure 6, white arrow) show approximately 40-60 discrete speckles distributed throughout the nuclei. The centromere pattern is the result of anti-CENP A,B,C antibodies13.
Nucleolar Pattern
Some nucleolar patterns are associated with diffuse cytoplasmic staining in mitotic cells (Figure 7, orange arrow) and negative chromosomal region, while other nucleolear patterns display positive staining of the chromosomal region. The nucleolar pattern is associated with homogenous or speckled staining of the nucleoli (Figure 7, white arrow), along with weak speckled or homogenous staining of the nucleoplasm. These patterns are associated with anti-RNA polymerase III, anti-fibrillarin, anti-Th/To and anti-PM/Scl antibodies14,15.
Nuclear Dot Pattern
The nuclear dot pattern is associated with negative metaphase mitotic cells (Figure 8, orange arrow)and with few discrete speckles in the resting cell nuclei (Figure 8, white arrow). This characteristic pattern is often the result of sp-100, PML, or p80 colin autoantibodies. These antibodies are associated with primary biliary cirrhosis and autoimmune hepatitis16.
<img alt="Fluorescent microscopy comparing low/high intensity; cell nuclei." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Negative control (left): Cells display low level non-specific fluorescence, but no specific nuclear staining. Positive control (right): Cells display apple green nuclear fluorescence.
<img alt="Fluorescent microscopy image highlighting cell nuclei and mitotic chromosomes in green, arrow notation." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Homogenous pattern. Mitotic cells (orangearrow) show solid fluorescence. Resting cells show even, diffuse staining (whitearrow).
<img alt="Fluorescence microscopy image of cell nuclei, green staining, nuclear structure analysis." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig3highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Coarse speckled pattern. Mitotic cells (orange arrow) show negative staining. Resting cells show a distinct speckled stain (white arrow).
<img alt="Confocal microscopy image; fluorescent-stained cells with DNA, protein markers for cell analysis." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Fine speckled pattern. Mitotic cells (orange arrow) show negative staining. Resting cells show a distinct fine speckled staining (white arrow).
<img alt="Fluorescent microscopy image showing cellular nuclei, DNA staining, and protein localization." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Dense fine speckled pattern. Mitotic cells (orange arrow) show a fine granular solid staining. Resting cells show a very fine, diffuse speckled stain (whitearrow).
<img alt="Fluorescent microscopy image; cell nuclei stained for DNA analysis; highlights mitosis process." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig6highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Centromere pattern. Mitotic cells (orangearrow) show uniform, discrete speckles. Resting cells (whitearrow) shows 40-60 discrete speckles per cell nucleus.
<img alt="Fluorescent microscopy image showing cell structures with marked features for analysis." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7. Nucleolar pattern. Mitotic cells (orangearrow) appear as large clusters of granular staining. Resting cell nucleishows fluorescence in the nucleoli(whitearrow).
<img alt="Fluorescent microscopy image; cell structure analysis; GFP-tagged protein localization." fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8. Nuclear dot pattern. Mitotic cells (orangearrow) appear negative. Resting cells show a few discrete speckles in the nucleus (whitearrow).Additionally, the nucelar dot pattern can coexist with cytoplasmic staining associated with autoantibodies to mitochondrial antigens (red arrow).

Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Anti-Nuclear Antibody Screening Using HEp-2 Cells

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 ...

anti-nuclear-antibody-screening-using-hep-2-cells

Research

JoVE Journal

Biology

134.4K Views.  INOVA Diagnostics, Inc..  Indirecte immunofluorescentie (IIF) assays traditioneel gebruikt voor de detectie van antinucleaire antilichamen (ANA) in menselijk serum. De aanwezigheid van deze antilichamen kunnen helpen bij de diagnose van systemische auto reumatische aandoeningen (SARD). Dit protocol laat zien hoe effectief uit te voeren de IIF techniek om deze autoantilichamen nauwkeurig te detecteren. 

Video: Anti-Nuclear Antibody Screening Using HEp-2 Cells

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Cancer Research

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

We present a protocol to screen anti-hepatitis B virus (HBV) compounds targeting pre- and post-viral entry lifecycle stages, using isothermal titration calorimetry to measure binding affinity (KD) with host sodium taurocholate cotransporting polypeptide. Antiviral efficacy was determined through the suppression of viral lifecycle markers (cccDNA formation, transcription, and viral assembly).

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load ...

Biochemistry

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.

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 ...

Immunology and Infection

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug&#39;s pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.
In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-na&#239;ve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Here we present a cell-based flow cytometry method to detect neutralizing antibodies or other factors that interfere with the cellular uptake of enzyme replacement therapies in a human matrix, such as cerebral spinal fluid (CSF) or human serum.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Medicine

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

Anti-Nuclear Antibody Screening Using HEp-2 Cells

Samenvatting

Meer video's verkennen