Name: anti-nuclear antibody screening using hep-2 cells
Uploaded: 2014-06-23T18:15:00
Duration: 13 min 1 s
Description: Watch this Scientific Journal Video about Anti-Nuclear Antibody Screening Using HEp-2 Cells at JoVE.com

Wij danken Cassandra Bryant voor het uitvoeren van de IIF experiment en Carol Buchner voor haar deskundige technische beoordeling.

1. Monstervoorbereiding en de gekozen ondergrond <ol><li> Verwijder reagentia uit de verpakking en laat elk item om op kamertemperatuur te komen. Bereid reagentia en verdun patiëntensera volgens de richting insert. Opmerking: NOVA Lite slides barcode en kan gemakkelijk in combinatie met het Laboratorium Informatie Systeem LIS worden geïntegreerd in geautomatiseerde systemen. Deze procedure illustreert handleiding dia verwerking; hoge doorvoer laboratoria evenwel kiezen voor geautomatiseerde dia apparatuur. </li></ol> 2. Toevoeging van controles en-monsters <ol><li> Breng 1 druppel positieve controle en 1 druppel negatieve controle aan de juiste putjes dia. Pipetteer 20-25 gl verdunde patiëntenserum de resterende putjes. Verwerk een dia per keer. </li><li> Plaats de schuif (s) in een kleuring container met een vochtige papieren handdoek op de bodem. Bedek de houder en incubeer de schuif (s) gedurende 30 minuten. Opmerking: De vochtige omstandigheden zalvoorkomen dat de ondergrond uitdroogt. Drogen van de putten kan leiden tot kunstmatig kleuring. Tijdens deze incubatietijd wordt de antinucleaire antilichamen in het serum van de patiënt binden aan antigenen van de cellen die zich binden aan elk putje. </li><li> Na de incubatieperiode, spoelen het serum met behulp van een zachte stroom van wasbuffer. Om te voorkomen dat schade aan de ondergrond en het voorkomen van cross-over van de monsters tussen de putten, de hoek van de glijbaan iets om te voorkomen dat de leiding van de stroom direct op de putten. Tik overtollige wasbuffer en plaats de dia in een Coplin pot met wasbuffer gedurende ongeveer 5 minuten. </li></ol> 3. Toevoeging van TL-conjugaat <ol><li> Een voor een, verwijdert u de dia&#39;s van de wasbuffer en tik zachtjes om het overtollige wasbuffer verwijderen. Breng 1 druppel fluorescente conjugaat (FITC gemerkt anti-humaan IgG) op elk putje. Incubeer de glaasjes gedurende 30 min in de bevochtigde container, en zorg ervoor dat de vlekken te vervangencontainer deksel. Opmerking: ANA test is het gebruik van een IgG Fc specifiek conjugaat aanbevolen. Het conjugaat is lichtgevoelig en het deksel zal de dia tegen blootstelling aan licht naast het handhaven van de bevochtigde omgeving. Tijdens deze incubatieperiode wordt het conjugaat binden aan anti-nucleaire antilichamen van de patiënt die zijn gebonden aan de cel antigenen. Dit conjugaat binding resulteert in de aanwezigheid van fluorescentie in de putjes. </li><li> Spoel en was de dia zoals hierboven beschreven. </li><li> Plaats een dekglaasje op keukenpapier en fixeermiddel toepassing in een continue lijn met de onderkant van het dekglaasje. </li><li> Verwijder elke dia uit de wasbuffer en tik op de schuif zachtjes om het overtollige wasbuffer verwijderen. Raak de onderrand van de schuif naar de rand van het dekglaasje. </li><li> Sluit voorzichtig de schuif op het dekglaasje zodanig dat het fixeermiddel op het dekglaasje stroomt naar de bovenrand van de schuif zonder vorming luchtbel. Opmerking: Coverslipping, inclusief het gebruik van de optimale hoeveelheid van de montage medium, is een techniek die praktijk neemt om te perfectioneren. </li></ol> 4. Identificatie van positieve en negatieve resultaten Handmatig Interpretatie <ol><li> Bekijk dia&#39;s met een fluorescentiemicroscoop, gelegen in een donkere kamer. Voer scannen van het gehele goed met een 20X en 25X objectief celverdeling en uniformiteit van fluorescentie beoordelen </li><li> Schakel over naar een 40x doelstelling om de uiteindelijke interpretatie met betrekking tot positiviteit en patroon te maken. </li><li> Kijk naar de positieve en negatieve controles. Grade positiviteit met een reactiviteit beoordelingsschaal van 1 + tot 4 +. Opmerking: De negatieve controle mogelijk niet helemaal donker, maar zal vertonen vaak een laag niveau niet-specifieke fluorescentie. De positieve controle zal helder appel groene fluorescentie in de kern (figuur 1) weer te geven. </li></ol> Automatische interpretatie Opmerking: In additiop handmatige interpretatie, belasting en scan dia&#39;s door NOVA Bekijk Automated fluorescentiemicroscoop of andere geautomatiseerde digitale imaging-instrument; geen donkere kamer nodig. Na het aanmaken van een project door het selecteren van het juiste type glijbaan, het instrument verwerft en slaat digitale beelden van cellen van hoge resolutie in elk putje. Daarnaast meet NOVA Bekijk fluorescerend licht-intensiteit, en categoriseert de resultaten als positief of negatief, en biedt patroonherkenning voor positieve monsters. De beelden worden bekeken door de exploitant op een hoge resolutie computer monitor, waardoor uiteindelijke interpretatie, revisie en bevestiging van NOVA View resultaat. Rapporten kunnen worden gegenereerd op bevestigd resultaten. Digitale microscopie moet in combinatie met een ervaren laborant worden gebruikt zoals het is bedoeld om te helpen bij de interpretatie van de resultaten.

<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>

De auteurs Gabriella Lakos, Cassandra Bryant, Carol Buchner, en Anna Eslami zijn medewerkers van 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.

Het antinucleaire antilichaam term (ANA) beschrijft verschillende auto-antilichamen die reageren met bestanddelen van celkernen waaronder DNA, eiwitten en ribonucleoproteins 1, 2. De HEp-2 cellen, een natief eiwit array met honderden antigenen, een ideale substraat voor de detectie van ANA 1. De detectie van ANA in menselijk serum is een belangrijk onderzoekshulpmiddel voor bindweefselziekten en IIF is de referentiemethode ANA test 1. Onlangs heeft IIF op HEp-2-cellen vervangen in sommige laboratoria antigeenspecifieke multiplex immunoassays en werkwijzen. Als gevolg van bezorgdheid over vals negatieve resultaten en het gebrek aan transparantie aan werkers, de American College of Rheumatology vormden een Task Force concludeerde dat IIF gebruik HEp-2-cellen de &quot;gouden standaard&quot; voor ANA screening 1 zou moeten zijn. Door de subjectieve aard van ANA screening, de kwaliteit van HEp-2-cellen inTEGRAL om nauwkeurige en vol vertrouwen rapportage van de resultaten. De aanwezigheid van een groot aantal mitotische cellen optimaal celmorfologie voldoende confluentie en expressie van relevante antigenen zijn bijzonder belangrijk. IIF patroonherkenning fungeert als een belangrijk instrument om te helpen bij de patiënt de diagnose. Inzicht in de betekenis van de verschillende patronen kan de artsen en laboratoriumpersoneel om de passende follow-up testen uit te voeren om de diagnose te bevestigen. Zo kunnen homogeen ANA patroon voorkomen in de aanwezigheid van anti-dsDNA antilichamen of chromatine, en kan gepaard gaan met systemische lupus erythematosus (SLE) 3. Van de andere kant, is onlangs beschreven dat de zogenaamde dichte fijn gespikkelde patroon (DFS), die vaak wordt gezien in maximaal 12% van de routine monsters, is meestal in verband gebracht met anti-DFS70 antilichamen. Deze auto-antilichamen, indien in isolatie (zonder andere ziektespecifieke ANA) zijn niet geassocieerd met systemische auto reumatische aandoeningen &lt;sup&gt; 4-9. Daarbij bevestigend onderzoek naar anti-DFS70 antilichamen kunnen helpen onnodige reflex tests te beperken, bieden aanzienlijke kostenbesparingen en gemak patiënt angst. Gezien het feit dat IIF is de eerste lijn screening methodologie om autoantilichamen sporen, is het van het grootste belang dat de gebruiker begrijpt hoe de selectie van reagentia en weefsel substraten resultaten kunnen beïnvloeden. Aangezien de IIF techniek subjectief, is het belangrijk dat de gebruikte reagentia bieden de hoogste kwaliteit. Dit doel van deze video-protocol is om de gebruiker vertrouwd te maken met de juiste stappen die nodig zijn om de IIF-methode, de gemeenschappelijke patronen geassocieerd met ANA voeren, en om nieuwe ontwikkelingen waarvoor het laboratorium workflow kunnen stroomlijnen en de resultaten te standaardiseren introduceren.

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:252px;">NOVA Lite HEp-2 IgG (DAPI conjugate)</td>
			<td style="width:189px;">INOVA Diagnostics</td>
			<td style="width:135px;">708102</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NOVA View Instrument</td>
			<td>INOVA Diagnostics</td>
			<td>NV2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation</td>
			<td>INOVA Diagnostics</td>
			<td>LINK010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QUANTA Link Workstation License</td>
			<td>INOVA Diagnostics</td>
			<td>LINK001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barcode Scanner</td>
			<td>INOVA Diagnostics</td>
			<td>LINK019</td>
			<td></td>
		</tr>
	</tbody>
</table>

anti-nuclear antibody screening using hep-2 cells

De American College of Rheumatology positie verklaring over ANA testen, wordt het gebruik van IIF als de gouden standaard methode voor het ANA screening 1. Hoewel IIF is een uitstekende screeningstest in deskundige handen, de technische problemen van de verwerking en het lezen IIF dia&#39;s - zoals de arbeidsintensieve verwerking glijbaan, handleiding lezen, de behoefte aan ervaren, opgeleide technologen en het gebruik van donkere kamer - maken het IIF methode moeilijk in te passen in de workflow van de moderne, geautomatiseerde laboratoria. De eerste en cruciale stap op weg naar een hoge kwaliteit ANA screening is voorzichtig glijbaan verwerking. Deze procedure is arbeidsintensief en volledig begrip van de werkwijze en aandacht voor details en ervaring vereist. Dia lezing wordt uitgevoerd door fluorescentie microscopie in donkere kamers, en wordt gedaan door opgeleide technologen die vertrouwd zijn met de verschillende patronen, in het kader van de celcyclusen de morfologie van interfase en delende cellen. Mits IIF is de eerste lijn screening tool voor SARD, het begrijpen van de stappen om deze techniek correct uit te voeren is van cruciaal belang. Recent zijn digitale beeldvormingssystemen ontwikkeld voor het automatisch lezen van IIF dia. Deze systemen, zoals het NOVA View Automated fluorescentiemicroscoop, zijn ontworpen om de routine IIF stroomlijnen. NOVA Bekijk verwerft en slaat digitale beelden van de putten hoge resolutie, waardoor beeldaanwinst scheiden van interpretatie; beelden worden bekeken een geïnterpreteerde op hoge resolutie beeldschermen. Het slaat beelden voor toekomstige referentie en ondersteunt de interpretatie van de exploitant door middel van fluorescerende lichtintensiteit gegevens op de afbeeldingen. Ook voorlopig categoriseert resultaten als positief of negatief, en biedt patroonherkenning voor positieve monsters. Samengevat, het elimineert de behoefte aan donkere kamer, en automatiseert en stroomlijnt het IIF reading / interpretatie workflow. Belangrijker nog, het verhoogt de consistentie tussen lezers en lezingen. Bovendien, met het gebruik van barcodes glijbanen, transcriptie fouten worden voorkomen door te bepalen monster traceerbaarheid en positieve patiënt identificatie. Dit resulteert in een verhoogde patiëntgegevens integriteit en veiligheid. Het algemene doel van deze video is om de IIF-procedure aan te tonen, waaronder dia verwerking, de identificatie van gemeenschappelijke IIF patronen, en de introductie van nieuwe ontwikkelingen te vereenvoudigen en harmoniseren van deze techniek.

Bij het evalueren van celkleuring resultaten voor HEp-2, vijf grote nucleaire patronen zijn meest gemelde: homogeen, gespikkeld, centromeer, nucleolar, en nucleaire stippen. Deze patronen zijn het gevolg van auto-antilichaam binden aan specifieke bestanddelen van de kern. Hoewel ANA test specifiek nucleaire structuren kunnen cytoplasmatische patronen geassocieerd met autoantilichamen tegen cytoplasmatische structuren worden waargenomen. In sommige gevallen kunnen verschillende auto-antilichamen aanwezig in een monster, en kan het beeld als gemengd patroon. Er zijn andere, minder vaak waargenomen en vaak celcyclus specifieke patronen die kunnen worden gedetecteerd door de IIF werkwijze; meest recent is een zogenaamde dichte fijn gespikkelde patroon met speciale klinische betekenis is beschreven in een aantal ANA positieve sera. Homogene Patroon Om een ​​homogeen patroon te identificeren, scan de put en identificeren mitotische of scheidingslijnencellen. De gecondenseerde chromatine van de mitotische cellen (Figuur 2, oranje pijl) vertoont solide, uniforme fluorescentie die vaak meer uitgesproken dan in de rustende cel nuclei.In het homogeen patroon, de rustende cellen vertonen uniform, diffuse fluorescentie van de gehele kern (Figuur 2, witte pijl). Deze karakteristiek patroon is vaak het resultaat van anti-dsDNA-antilichamen 10. Gespikkelde Patroon In de grove gespikkeld patroon, de mitotische cellen vertonen geen kleuring van het gecondenseerde chromosomale regio&#39;s (figuur 3, oranje pijl). De rustende cellen vertonen korrelvormige fluorescentie gedurende de gehele kern. De nucleaire spikkels kan worden gedefinieerd als grof of fijn. De grove spikkels patroon is vaak het resultaat van anti-Sm en anti-RNP 11. In het fijn gespikkelde patroon, de rustende cellen vertonen boete of diffuse speckling de gehele kernen, meestal in uniform ditkering (Figuur 4, witte pijl). De fijne gespikkelde patroon is vaak het resultaat van anti-SSAand anti-SSB 12. Dichte Fine gespikkelde Patroon Het wordt nu erkend dat de dichte fijne gespikkelde patroon (DFS) is gemeenschappelijk in routine testen, en maar liefst 12% van de monsters positief zijn voor anti-DFS70 autoantilichamen. Echter, deze auto-antilichamen, wanneer gevonden op zichzelf niet geassocieerd met systemische auto reumatische aandoeningen 4 -6. Deze antilichamen zijn overwegend in gezonde individuen en patiënten dragen ziekten die niets met SARD 6. Bevestigend onderzoek naar anti-DFS70 antilichamen kunnen helpen onnodige reflex tests te beperken, bieden een aanzienlijke kostenbesparing en gemak patiënt angst. Sinds de DFS patroon is moeilijk te identificeren en ANA positiviteit kan leiden tot angst voor zowel patiënten als artsen, is het sterk aanbevolen om een ​​bevestigende test uit te voeren na identificatie<html> fying een patroon suggestief van anti-DFS70 antilichamen tegen de klinische betekenis van ANA verduidelijken positivty 5,6. In dit patroon, mitotische cellen (Figuur 5, oranje pijl) laten positieve gespikkelde kleuring van de metafase chromatine, terwijl rust celkernen (Figuur 5, witte pijl) vertonen uniform verdeeld fijne spikkels in de hele kern. Centromeer Patroon Om de centromeer patroon te identificeren, scan de putten en identificeren mitotische of delende cellen. In mitotische cellen (Figuur 6, oranje pijl), die discrete spikkels worden nauw betrokken bij wat vaak omschreven als een &quot;metafase bar&quot;. In de centromeer patroon, rustende cellen (Figuur 6, witte pijl) laten ongeveer 40-60 discrete spikkels verspreid over de kernen. Het centromeer patroon is het resultaat van anti-CENP A, B, C antilichamen 13. stap &quot;&gt; Hergroepering van nucleoli Patroon Sommige nucleolair patronen worden geassocieerd met diffuse cytoplasmatische kleuring in mitotische cellen (figuur 7, oranje pijl) en negatieve chromosomaal gebied, terwijl andere nucleolear patronen tonen positieve kleuring van het chromosomale gebied. De nucleolair patroon wordt geassocieerd met homogene of gespikkeld kleuring van de nucleoli (figuur 7, witte pijl), samen met zwakke gespikkelde of homogene kleuring van het nucleoplasma. Deze patronen worden geassocieerd met anti-RNA polymerase III, anti-fibrillarin, anti-Th/To en anti-PM/Scl antilichamen 14,15. Nucleaire Dot Pattern De nucleaire puntpatroon geassocieerd met negatieve metafase mitotische cellen (Figuur 8, oranje pijl) en met weinig discrete spikkels in rustpositie celkernen (Figuur 8, witte pijl). Deze karakteristiek patroon is vaak het gevolg van SP-100, PML of p80 Colin autoantilichamen. Deze antilichamen zijn<html> samenhingen met primaire biliaire cirrose en auto-immune hepatitis 16. <img alt="Figuur 1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> . Figuur 1 Negatieve controle (links): Cellen tonen lage niveau niet-specifieke fluorescentie, maar geen specifieke kernkleuring Positieve controle (rechts):. Cellen geven appelgroen nucleaire fluorescentie. <img alt="Figuur 2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> Figuur 2. Homogene patroon. Mitotische cellen (orangearrow) Solide fluorescentie. Rustende cellen vertonen zelfs, diffuse kleuring (whitearrow). &quot;Width =&quot; 500 &quot;/&gt; Figuur 3. Grof gespikkeld patroon. Mitotische cellen (oranje pijl) geven negatieve kleuring. Rustende cellen vertonen een duidelijke gespikkelde vlek (witte pijl). <img alt="Figuur 4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> Figuur 4. Fine gespikkelde patroon. Mitotische cellen (oranje pijl) geven negatieve kleuring. Rustende cellen vertonen een duidelijke fijn gespikkelde kleuring (witte pijl). <img alt="Figuur 5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> Figuur 5. Dichte fijn gespikkelde patroon. Mitotische cellen (oranje pijl) laten een fijne korrelige vaste stof kleuring. Rustende cellen vertonen een zeer fijne, diffuse gespikkelde vlek (whitearrow). Figuur 6. Centromeer patroon. Mitotische cellen (orangearrow) toon uniform, discrete spikkels. Rustende cellen (whitearrow) toont 40-60 discrete spikkels per celkern. <img alt="Figuur 7" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" /> Figuur 7. Hergroepering van nucleoli patroon. Mitotische cellen (orangearrow) verschijnen als grote clusters van granulaire kleuring. Rusten cel nucleishows fluorescentie in de nucleoli (whitearrow). <img alt="Figuur 8" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" /> Figuur 8. Nuclear stippenpatroon. Mitotische cellen (orangearrow) lijken negatief. Rustende cellen vertonen een paar discrete spikkels in de kern (whitearrow). Daarnaast is de Nucelar dot patroon kan samengaan met cytoplasmakleuring geassocieerd met auto-antilichamen tegen mitochondriale antigenen (rode pijl).

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

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.

Anti-nucleaire antilichaam screening met HEp-2 cellen

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

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Live-dissectie van Drosophila Embryo&#39;s: Gestroomlijnde methoden voor het screenen Mutant Collections door antilichaam kleuring

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISSIE esiRNA voor RNAi Screening in zoogdiercellen

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

MISSIE LentiPlex Gepoolde shRNA Library Screening in zoogdiercellen

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

Omzetting van een Capture ELISA aan een Luminex xMAP test met behulp van een Multiplex Antilichaam screeningsmethode

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

High-throughput Functionele Screening met behulp van een zelfgemaakte Dual-gloed Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inductie en analyse van epitheliale naar mesenchymale transitie

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Antilichaamkleuring in<em&gt; C. Elegans</em&gt; Een &#39;Freeze-Cracking &quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

High-throughput screening voor Chemische Modulators van Post-transcriptioneel gereguleerde genen

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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.

Identificatie van de kinase-substraat Paren Met behulp van 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 ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Multi-enzym screening met een hoge verwerkingscapaciteit Genetische screening Enzyme System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...