Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method
Background Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking. Methods Replicates of 10 or 20 measure...
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| Veröffentlicht in: | Cytometry. Part B, Clinical cytometry Clinical cytometry, 2013-09, Vol.84 (5), p.329-337 |
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| creator | Davis, Bruce H. McLaren, Christine E. Carcio, Anthony J. Wong, Linda Hedley, Benjamin D. Keeney, Mike Curtis, Adam Culp, Naomi B. |
| description | Background
Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking.
Methods
Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined.
Results
For all assays and most instrument platforms, |
| doi_str_mv | 10.1002/cyto.b.21116 |
| format | Article |
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Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking.
Methods
Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined.
Results
For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5–10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data‐driven means for determining optimal replicate numbers.
Conclusions
The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes. © 2013 International Clinical Cytometry Society</description><identifier>ISSN: 1552-4949</identifier><identifier>EISSN: 1552-4957</identifier><identifier>DOI: 10.1002/cyto.b.21116</identifier><identifier>PMID: 24022856</identifier><language>eng</language><publisher>United States: Wiley Subscription Services, Inc</publisher><subject>Antigens, CD34 - isolation & purification ; assay validation ; Female ; Fetal Blood ; fetomaternal hemorrhage test ; flow cytometry ; Flow Cytometry - methods ; Flow Cytometry - standards ; Fluorescent Dyes ; Hematopoietic Stem Cells - pathology ; hENT1 assay ; Humans ; IVD clearance ; laboratory‐developed test ; Pregnancy ; quality assessment ; regulatory science ; Sepsis - blood ; sepsis test ; stem cell assay</subject><ispartof>Cytometry. Part B, Clinical cytometry, 2013-09, Vol.84 (5), p.329-337</ispartof><rights>2013 International Clinical Cytometry Society</rights><rights>2013 International Clinical Cytometry Society.</rights><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2426-cf1b24de33a3dd891e621d3c47cc6795fc5dbbc8777f06c24fe49b44421fd31a3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fcyto.b.21116$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fcyto.b.21116$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,1427,27901,27902,45550,45551,46384,46808</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24022856$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Davis, Bruce H.</creatorcontrib><creatorcontrib>McLaren, Christine E.</creatorcontrib><creatorcontrib>Carcio, Anthony J.</creatorcontrib><creatorcontrib>Wong, Linda</creatorcontrib><creatorcontrib>Hedley, Benjamin D.</creatorcontrib><creatorcontrib>Keeney, Mike</creatorcontrib><creatorcontrib>Curtis, Adam</creatorcontrib><creatorcontrib>Culp, Naomi B.</creatorcontrib><title>Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method</title><title>Cytometry. Part B, Clinical cytometry</title><addtitle>Cytometry B Clin Cytom</addtitle><description>Background
Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking.
Methods
Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined.
Results
For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5–10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data‐driven means for determining optimal replicate numbers.
Conclusions
The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes. © 2013 International Clinical Cytometry Society</description><subject>Antigens, CD34 - isolation & purification</subject><subject>assay validation</subject><subject>Female</subject><subject>Fetal Blood</subject><subject>fetomaternal hemorrhage test</subject><subject>flow cytometry</subject><subject>Flow Cytometry - methods</subject><subject>Flow Cytometry - standards</subject><subject>Fluorescent Dyes</subject><subject>Hematopoietic Stem Cells - pathology</subject><subject>hENT1 assay</subject><subject>Humans</subject><subject>IVD clearance</subject><subject>laboratory‐developed test</subject><subject>Pregnancy</subject><subject>quality assessment</subject><subject>regulatory science</subject><subject>Sepsis - blood</subject><subject>sepsis test</subject><subject>stem cell assay</subject><issn>1552-4949</issn><issn>1552-4957</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkTlvFDEUx60IlIQkXerIEg3NLr5nJx0spxQpKUJBZfl4Bkee8WDPgLajpeMz8kmYScIWVO_6vUPvj9A5JWtKCHvpdmNe2zWjlKoDdEylZCvRyubJ3hftEXpW6x0hXArVHKIjJghjG6mO0a83MELpYm_GmHucA87DGDuTcIEhRWdGwP3UWSg45IK_mxT9Ho3dUMDFuoRTjf0XHNKUC1QHvQPsIKU_P39bU8FjU6vZ1Ut8U_KQl8RQjBvnBQl3MH7N_hQ9DSZVOHu0J-jTu7e32w-rq-v3H7evrlaOCaZWLlDLhAfODfd-01JQjHruROOcaloZnPTWuk3TNIGouSeAaK0QgtHgOTX8BL14mDuU_G2COuou1uVU00OeqqaCM9LIDeMz-vw_9C5PpZ-vmymlFJMtJzN18UhNtgOvhzL_r-z0vyfPgHgAfsQEu32dEr0IqBcBtdX3Aurt59vr1_cu_wvFaZLC</recordid><startdate>201309</startdate><enddate>201309</enddate><creator>Davis, Bruce H.</creator><creator>McLaren, Christine E.</creator><creator>Carcio, Anthony J.</creator><creator>Wong, Linda</creator><creator>Hedley, Benjamin D.</creator><creator>Keeney, Mike</creator><creator>Curtis, Adam</creator><creator>Culp, Naomi B.</creator><general>Wiley Subscription Services, Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QO</scope><scope>7T5</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>201309</creationdate><title>Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method</title><author>Davis, Bruce H. ; McLaren, Christine E. ; Carcio, Anthony J. ; Wong, Linda ; Hedley, Benjamin D. ; Keeney, Mike ; Curtis, Adam ; Culp, Naomi B.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2426-cf1b24de33a3dd891e621d3c47cc6795fc5dbbc8777f06c24fe49b44421fd31a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Antigens, CD34 - isolation & purification</topic><topic>assay validation</topic><topic>Female</topic><topic>Fetal Blood</topic><topic>fetomaternal hemorrhage test</topic><topic>flow cytometry</topic><topic>Flow Cytometry - methods</topic><topic>Flow Cytometry - standards</topic><topic>Fluorescent Dyes</topic><topic>Hematopoietic Stem Cells - pathology</topic><topic>hENT1 assay</topic><topic>Humans</topic><topic>IVD clearance</topic><topic>laboratory‐developed test</topic><topic>Pregnancy</topic><topic>quality assessment</topic><topic>regulatory science</topic><topic>Sepsis - blood</topic><topic>sepsis test</topic><topic>stem cell assay</topic><toplevel>online_resources</toplevel><creatorcontrib>Davis, Bruce H.</creatorcontrib><creatorcontrib>McLaren, Christine E.</creatorcontrib><creatorcontrib>Carcio, Anthony J.</creatorcontrib><creatorcontrib>Wong, Linda</creatorcontrib><creatorcontrib>Hedley, Benjamin D.</creatorcontrib><creatorcontrib>Keeney, Mike</creatorcontrib><creatorcontrib>Curtis, Adam</creatorcontrib><creatorcontrib>Culp, Naomi B.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Biotechnology Research Abstracts</collection><collection>Immunology Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Cytometry. Part B, Clinical cytometry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Davis, Bruce H.</au><au>McLaren, Christine E.</au><au>Carcio, Anthony J.</au><au>Wong, Linda</au><au>Hedley, Benjamin D.</au><au>Keeney, Mike</au><au>Curtis, Adam</au><au>Culp, Naomi B.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method</atitle><jtitle>Cytometry. Part B, Clinical cytometry</jtitle><addtitle>Cytometry B Clin Cytom</addtitle><date>2013-09</date><risdate>2013</risdate><volume>84</volume><issue>5</issue><spage>329</spage><epage>337</epage><pages>329-337</pages><issn>1552-4949</issn><eissn>1552-4957</eissn><abstract>Background
Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking.
Methods
Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical in vitro diagnostic use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals (95% CIs) of the mean, standard deviation, and coefficient of variation (CV) of sequential replicates were determined.
Results
For all assays and most instrument platforms, <5 replicates were found adequate to validate assay imprecision levels below the 5–10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95% CI and the cumulative mean for replicates, termed variance factor (VF), provide a data‐driven means for determining optimal replicate numbers.
Conclusions
The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate that three to four replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes. © 2013 International Clinical Cytometry Society</abstract><cop>United States</cop><pub>Wiley Subscription Services, Inc</pub><pmid>24022856</pmid><doi>10.1002/cyto.b.21116</doi><tpages>9</tpages></addata></record> |
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| subjects | Antigens, CD34 - isolation & purification assay validation Female Fetal Blood fetomaternal hemorrhage test flow cytometry Flow Cytometry - methods Flow Cytometry - standards Fluorescent Dyes Hematopoietic Stem Cells - pathology hENT1 assay Humans IVD clearance laboratory‐developed test Pregnancy quality assessment regulatory science Sepsis - blood sepsis test stem cell assay |
| title | Determination of optimal replicate number for validation of imprecision using fluorescence cell‐based assays: Proposed practical method |
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