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

Vi tackar Cassandra Bryant för att utföra IIF experimentet och Carol Buchner för hennes expert teknisk översyn.

1. Provberedning och substrat Selection <ol><li> Ta bort reagenser från förpackningar och låta varje objekt för att komma till rumstemperatur. Förbered reagenser och späda patientsera enligt riktningen insatsen. OBS: NOVA Lite diabilder är barcoded och kan enkelt integreras i automatiserade system i samband med laboratorieinformationssystem LIS. Denna procedur illustrerar manuell slide behandling; hög kapacitet laboratorier kan dock välja automatisk bildprocessutrustning. </li></ol> 2. Tillägg av kontroller och prover <ol><li> Dispensera 1 droppe av den positiva kontrollen och 1 droppe negativ kontroll till lämpliga glid brunnar. Pipettera 20-25 pl utspätt patientserum till de återstående brunnarna. Bearbeta en bild i taget. </li><li> Placera objektglaset (s) i en färgning behållare med en fuktig pappershandduk på botten. Täck behållaren och inkubera objektglaset (n) i 30 min. Anm: De fuktiga förhållanden kommerförhindra att substratet torkar ut. Torkning av brunnarna kan resultera i artefaktuella färgning. Under denna inkubationsperiod, kommer anti-nukleära antikroppar i patientens serum binder till antigener av de celler som är bundna till varje brunn. </li><li> Efter inkubationsperioden, skölj av serumet med användning av en svag ström av tvättbuffert. För att undvika skador på underlaget och förhindra cross-over av prover mellan brunnar, vinkel bilden en aning för att undvika att rikta strömmen direkt på brunnarna. Knacka bort tvättbuffert och placera objektglas i ett Coplin-kärl innehållande tvättbuffert under ca 5 min. </li></ol> 3. Tillsats av fluorescerande konjugat <ol><li> En efter en, ta bort bilderna från tvättbufferten och knacka försiktigt för att avlägsna överskottet tvättbufferten. Applicera en droppe av fluorescerande konjugat (FITC-märkt anti-humant IgG) till varje brunn. Inkubera objektglasen i 30 minuter i fuktig behållare, och se till att byta ut färgningbehållarens lock. Obs! ANA testning, är användningen av en IgG-Fc specifikt konjugat rekommenderas. Konjugatet är ljuskänslig, och locket skyddar bilderna från ljus förutom att bibehålla den fuktig miljö. Under denna inkubationsperiod, kommer konjugatet att binda till patientens anti-nukleära antikroppar, som har bundit till antigenerna cell. Detta konjugat bindande resultat i närvaro av fluorescens i brunnarna. </li><li> Skölj och tvätta sliden som beskrivits ovan. </li><li> Placera på ett täckglas på en pappershandduk och tillämpa monteringsmedium i en kontinuerlig linje till den nedre kanten av täckglas. </li><li> Ta bort varje bild från tvättbufferten och peka på skjut försiktigt för att ta bort överskottet tvättbufferten. Tryck på den nedre kanten av bilden till kanten av täckglaset. </li><li> Sänk försiktigt ned objektglaset på täckglaset så att monteringsmedium på täckflöden till den övre kanten av bilden utan luftbubbelbildning. Notera: Coverslipping, bland annat med hjälp av optimala mängden monteringsmedium, är en teknik som kräver övning för att fullända. </li></ol> 4. Identifiering av positiva och negativa resultat Manuell Tolkning <ol><li> Visa bilder med en fluorescerande mikroskop, som ligger i ett mörkt rum. Utför scanning av hela bra med en 20X eller 25X mål att bedöma distributionen och likformighet av fluorescens cell </li><li> Byt till en 40X mål att göra den slutliga tolkningen om positivitet och mönster. </li><li> Titta på de positiva och negativa kontrollerna. Grade positivitet med hjälp av en reaktivitet betygsskala från 1 + till 4 +. Obs: Den negativa kontrollen kanske inte visas helt mörkt, men kommer ofta visa låg nivå ospecifik fluorescens. Den positiva kontrollen visar ljust äppelgrön fluorescens i kärnan (fig. 1). </li></ol> Automatisk tolkning Obs: I additiom till manuell tolkning, belastning och skanna diabilder från NOVA Visa Automatiserad fluorescens mikroskop eller annan automatiserad digital bildbehandling instrument; ingen mörkrum är nödvändig. När du har skapat ett projekt genom att välja lämpligt bildtypen, förvärvar instrumentet och lagrar högupplösta digitala bilder av celler i varje brunn. Dessutom NOVA Visa mäter fluorescerande ljusintensitet, och kategoriserar resultatet som positivt eller negativt, och ger mönsterigenkänning för positiva prov. Bilderna ses av operatören på en högupplöst datorskärm, vilket möjliggör slutgiltiga tolkningen, revidering och bekräftelse av NOVA Visa resultat. Rapporter kan genereras på bekräftade resultat. Digital mikroskopi bör användas tillsammans med en erfaren laboratorietekniker som den är avsedd som hjälp vid tolkning av resultat.

<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>

Författarna Gabri Lakos, Cassandra Bryant, Carol Buchner, och Anna Eslami är anställda i 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.

Termen antinukleär antikropp (ANA) och beskriver en mångfald autoantikroppar som reagerar med beståndsdelar i cellkärnan, inklusive DNA, proteiner och ribonukleoproteiner 1, 2. HEp-2-cell, ett nativt protein array med hundratals antigener ger ett idealiskt substrat för detektion av ANA 1. Upptäckten av ANA i humant serum är en viktig screeningmetod för bindvävssjukdomar, och IIF är referensmetod för ANA testning 1. Nyligen, IIF på HEp-2 celler har ersatts i vissa laboratorier med antigenspecifika immun och multiplex metoder. På grund av oro för falskt negativa resultat och bristen på öppenhet för kliniker, American College of Rheumatology bildat en arbetsgrupp som fram till att IIF använder HEp-2-celler bör vara den &quot;gyllene standarden&quot; för ANA screening 1. På grund av den subjektiva naturen av ANA screening, är kvaliteten på HEp-2-celler itegral till korrekt och säker rapportering av resultat. Förekomsten av ett stort antal mitotiska celler optimal cellmorfologi, tillräcklig konfluens, och uttryck av relevanta antigener som är särskilt viktiga. IIF mönsterigenkänning är ett viktigt verktyg för att bistå i patientens diagnos. Att förstå betydelsen av olika mönster gör det möjligt för läkare och laboratoriepersonal att utföra lämplig uppföljning tester för att bekräfta diagnosen. Till exempel kan homogent ANA mönster inträffa i närvaro av anti-dsDNA eller kromatin-antikroppar, och kan vara associerade med systemisk lupus erythematosus (SLE) 3. Från andra sidan har det nyligen beskrivits att den så kallade täta finfläckig mönster (DFS) som ofta ses i upp till 12% av rutinprover, främst har förknippats med anti DFS70 antikroppar. Dessa autoantikroppar, då fann för sig (utan andra sjukdomsspecifika ANA) är inte förknippade med systemiska autoimmuna reumatiska sjukdomar &lt;sup&gt; 4-9. Därmed bekräftande tester för anti-DFS70 antikroppar kan bidra till att minska onödig reflex test, ger avsevärda kostnadsbesparingar, och lindra patientens ångest. Med tanke på att IIF är den första raden screeningmetod för att detektera autoantikroppar, är det av största vikt att användaren förstår hur valet av reagens och vävnads substrat kan påverka resultaten. Eftersom IIF teknik är inneboende subjektivt, är det viktigt att de reagens som används ger de högsta kvalitet. Målet med denna video protokoll är att bekanta användaren med rätt steg som behövs för att utföra IIF-metoden, de gemensamma mönster i samband med ANA, och att införa nya framsteg som kan effektivisera laboratoriearbetsflödet och standardisera resultat.

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anti-nuclear antibody screening using hep-2 cells

American College of Rheumatology ställningstagande på ANA testning föreskriver användning av IIF som guldmyntfoten metod för ANA screening 1. Även IIF är en utmärkt screeningtest i expert händer, de tekniska svårigheterna med bearbetning och läsa IIF slides - såsom arbetskrävande glid bearbetning, manuell avläsning, behovet av erfarna, utbildade tekniker och användning av mörka rummet - gör IIF-metoden svårt att passa in i arbetsflödet av moderna, automatiserade laboratorier. Den första och avgörande steg mot högkvalitativa ANA screening är noggrann glidbehandling. Detta förfarande är arbetsintensiv, och kräver fullständig förståelse för processen, såväl som uppmärksamhet på detaljer och erfarenhet. Skjut läsning utförs av fluorescensmikroskopi i mörka rum, och görs av utbildad tekniker som känner till de olika mönster, i samband med cellcykelnoch morfologi interfas och delande celler. Förutsatt att IIF är den första raden screeningmetod för SARD, förstå stegen för att korrekt utföra denna teknik är avgörande. Nyligen har digitala bildsystem utvecklats för automatisk avläsning av IIF diabilder. Dessa system, som NOVA Visa Automated Fluorescerande mikroskop, är utformade för att effektivisera rutin IIF arbetsflöde. NOVA Visa förvärvar och lagrar högupplösta digitala bilder av brunnarna och därmed separera bild förvärv från tolkning; Bilderna visade en tolkas på högupplösta datorskärmar. Den lagrar bilder för framtida referens och stödjer operatörens tolkning genom att ge fluorescerande ljusintensitet uppgifter om bilderna. Det också preliminärt kategoriserar resultat som positivt eller negativt, och ger mönsterigenkänning för positiva prov. Sammanfattningsvis eliminerar behovet av mörkrummet, och automatiserar och effektiviserar IIF reading / tolkning arbetsflöde. Viktigast av allt, det ökar överensstämmelsen mellan läsare och avläsningar. Dessutom, med hjälp av streckkodade bilder, är felskrivningar elimineras genom att prov spårbarhet och positiva patientidentifiering. Detta resulterar i ökad patientintegritet och säkerhetsuppgifter. Det övergripande målet med den här videon är att visa på IIF förfarande, där även bild bearbetning, identifiera gemensamma IIF mönster, och införandet av nya framsteg för att förenkla och harmonisera denna teknik.

Vid bedömningen av cellfärgningsresultat för Hep-2, fem stora kärnvapen mönster är vanligast rapporterade: homogen, fläckig, centromer nucleolar och nukleära prickar. Dessa mönster är resultatet av autoantikroppen att binda till specifika beståndsdelar i kärnan. Även ANA testning är specifika för kärnstrukturer, kan cytoplasmiska mönster i samband med autoantikroppar mot cytoplasmiska strukturer också observeras. I vissa fall kan flera autoantikroppar vara närvarande i ett prov, och bilden kan visas som en blandad bild. Det finns andra, mindre frekvent, och ofta cellcykelspecifika mönster som kan upptäckas av IIF-metoden; senast, har en så kallad tät finfläckig mönster med särskild klinisk betydelse har beskrivits i vissa ANA positiva sera. Homogen Mönster För att identifiera ett homogent mönster, skanna väl och identifiera mitotiska eller skiljeceller. Den kondense kromatin i de mitotiska cellerna (Figur 2, orange pil) uppvisar fast, jämn fluorescens, som ofta är mer uttalad än i vilande cellen nuclei.In den homogena mönstret, den vilande celler uppvisar enhetlig, diffus fluorescens av hela kärnan (Figur 2, vit pil). Denna karakteristiskt mönster är ofta resultatet av anti-dsDNA-antikroppar 10. Fläckigt mönster I grova fläckigt mönster de mitotiska cellerna inte visar någon färgning av de kondenserade kromosomala regioner (figur 3, orange pil). De vilande celler uppvisar granulat fluorescens i hela kärnan. Kärnkraftsfläck kan definieras som grovt eller fint. Den Grov fläckmönster är ofta resultatet av anti-Sm och anti-RNP 11. I finfläckig mönster, de vilande celler visar fina eller diffus fläck hela kärnorna, vanligen i uniform distribution (Figur 4, vit pil). Den fina spräckliga mönstret är ofta resultatet av anti-SSAand anti-SSB 12. Tät finfläckig Mönster Det är nu känt att den täta fina spräckliga mönster (DFS) är vanlig i rutintestning, och så mycket som 12% av proverna är positiva för anti-DFS70 autoantikroppar. Dessa autoantikroppar, när de finns i isolering, inte är förknippade med systemiska autoimmuna reumatiska sjukdomar 4 -6. Dessa antikroppar är vanliga hos friska individer och patienter som bär sjukdomar som saknar samband med SARD 6. Bekräftande tester för anti-DFS70 antikroppar kan bidra till att minska onödig reflex test, ger avsevärda kostnadsbesparingar, och lindra patientens ångest. Eftersom DFS mönstret är svårt att identifiera och ANA positivitet kan orsaka ångest för både patienter och läkare, är det starkt rekommenderat att utföra ett bekräftande test efter identi<html> fiera ett mönster som tyder på anti DFS70 antikroppar för att klargöra den kliniska betydelsen av ANA positivty 5,6. I detta mönster, mitotiska celler (Figur 5, orange pil) visar positiv fläckig färgning av metafas kromatin, medan vila cellkärnor (Figur 5, vit pil) uppvisar jämnt fördelade fina fläckar i hela kärnan. Centromeren Mönster För att identifiera centromer mönstret, skanna brunnarna och identifiera mitotiska eller delande celler. I mitotiska celler (Figur 6, orange pil), dessa åtskilda fläckar blir nära samband i det som ofta beskrivs som en &quot;metafas bar&quot;. I centromeren mönstret, vilande celler (Figur 6, vit pil) visar ca 40-60 åtskiljda fläckar fördelade över kärnorna. Centromeren mönstret är resultatet av anti-CENP A, B, antikroppar C 13. steg &quot;&gt; Nukleolär Mönster Vissa nukleolära mönster är associerad med diffus cytoplasmisk färgning i mitotiska celler (Figur 7, orange pil) och negativa kromosomala regionen, medan andra nucleolear mönster visar positiv färgning av den kromosomala regionen. Det nukleolära mönstret associerat med homogen eller fläckig färgning av nukleolerna (fig 7, vit pil), tillsammans med svag spräckligt eller homogen färgning av nukleoplasman. Dessa mönster är förknippade med anti-RNA-polymeras III, anti-fibrillarin, anti-Th/To och anti-PM/Scl antikroppar 14,15. Kärnbildpunktmönster Kärnpunktmönster är förknippad med negativa metafasmitotiska celler (Figur 8, orange pil) och med några diskreta fläckar i vilande cellkärnor (Figur 8, vit pil). Denna karakteristiskt mönster är ofta ett resultat av sp-100, PML, eller P80 Colin autoantikroppar. Dessa antikroppar är som<html> matiskt med primär biliär cirros och autoimmun hepatit 16. <img alt="Figur 1" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig1highres.jpg" width="500" /> . Figur 1 Negativ kontroll (vänster): Celler visar låg nivå icke-specifik fluorescens, men ingen specifik nukleär färgning Positiv kontroll (höger):. Celler visar äppelgrön nukleär fluorescens. <img alt="Figur 2" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig2highres.jpg" width="500" /> Figur 2. Homogen mönster. Mitotiska celler (orangearrow) visar fast fluorescens. Vilande celler visar även, diffus färgning (whitearrow). &quot;Width =&quot; 500 &quot;/&gt; Figur 3. Grov fläckigt mönster. Mitotiska celler (orange pil) visar negativ färgning. Vilande celler visar en tydlig spräcklig fläck (vit pil). <img alt="Figur 4" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig4highres.jpg" width="500" /> Figur 4. Fina spräckliga mönster. Mitotiska celler (orange pil) visar negativ färgning. Vilande celler visar en tydlig finfläckig färgning (vit pil). <img alt="Figur 5" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig5highres.jpg" width="500" /> Figur 5. Tät finfläckig mönster. Mitotiska celler (orange pil) visar ett finkornigt fast färgning. Vilande celler visar en mycket fin, diffus spräcklig fläck (whitearrow). Figur 6. Centromer mönster. Mitotiska celler (orangearrow) show uniform, åtskiljda fläckar. Vilande celler (whitearrow) visar 40-60 åtskiljda fläckar per cellkärna. <img alt="Figur 7" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig7highres.jpg" width="500" /> Figur 7. Nukleolär mönster. Mitotiska celler (orangearrow) visas som stora kluster av granulär färgning. Vilande cell nucleishows fluorescens i nukleolerna (whitearrow). <img alt="Figur 8" fo:content-width="5in" src="/files/ftp_upload/51211/51211fig8highres.jpg" width="500" /> Figur 8. Kärnprickmönster. Mitotiska celler (orangearrow) verkar negativt. Vilande celler visar några discrEte fläckar i kärnan (whitearrow). Dessutom kan nucelar prickmönster samexistera med cytoplasmatisk färgning i samband med autoantikroppar mot mitokondriella antigener (röd pil).

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

Indirekt immunofluorescens (IIF)-analyser har traditionellt använts för påvisande av antinukleära antikroppar (ANA) i humanserum. Förekomsten av dessa antikroppar kan vara till hjälp vid diagnos av systemiska autoimmuna reumatiska sjukdomar (SARD). Detta protokoll visar hur effektivt att utföra IIF teknik för att exakt upptäcka dessa autoantikroppar.

Anti-nukleär antikropp Screening med hjälp av HEp-2-celler

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 dissekering av Drosophila Embryon: Effektivare metoder för screening Mutant Samlingar av Antibody färgning

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.

UPPDRAG esiRNA för RNAi screening i däggdjursceller

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.

UPPDRAG LentiPlex Poolade shRNA Bibliotek Screening i däggdjursceller

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.

Konvertering av en ELISA för att en Luminex xMAP analys med hjälp av en Multiplex metod Antibody Screening

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.

Hög kapacitet Funktionell Screening med hjälp av en hemmagjord Dual-glöd luciferas analys

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.

Induktion och Analys av Epithelial till mesenkymala övergång

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.

Antikropp färgning i<em&gt; C. Elegans</em&gt; Använda &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 ...

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.

Hög kapacitet Screening för kemiska modulatorer av Post-transkription Reglerade Gener

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.

Identifiering av kinas-substrat par Använda högkapacitetsscreening


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 med hjälp av en hög kapacitet 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 ...