{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,3,19]],"date-time":"2024-03-19T10:24:40Z","timestamp":1710843880851},"reference-count":15,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2006,8,11]],"date-time":"2006-08-11T00:00:00Z","timestamp":1155254400000},"content-version":"vor","delay-in-days":27,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Part B Clinical"],"published-print":{"date-parts":[[2006,7,15]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background:<\/jats:title><jats:p>In this study we developed a method to measure the amount of ZAP\u201070 [zeta accessory protein] in B\u2010CLL cells without relying on the ZAP\u201070 expression of patient B or T cells to normalize fluorescence intensity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods:<\/jats:title><jats:p>B\u2010CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP\u201070 antigen was blocked in one tube with unlabeled antibody to ZAP\u201070 [clone 1E7.2]. Zap\u201070\u2010PE was then added to this tube. ZAP\u201070\u2010PE was added to a second tube without unlabeled antibody to ZAP\u201070. The mean fluorescence intensity of the ZAP\u201070 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP\u201070 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non\u2010specific ZAP\u201070 fluorescence in the B\u2010CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP\u201070 fluorescence intensity in the tube with unlabeled antibody to ZAP\u201070. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP\u201070 cells in the tube without unlabeled antibody to ZAP\u201070. The brighter the ZAP\u201070 fluorescence above the non\u2010specific background, the higher the %POS.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results:<\/jats:title><jats:p>Due to the varying amount of non\u2010specific staining between patient B\u2010CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP\u201070 in B\u2010CLL cells than other methods, which use the ratio of B\u2010CLL fluorescence to normal B or T\u2010cell fluorescence. Using this improved method, ZAP\u201070 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP\u201070 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut\u2010off value lower than previous values published, due to exclusion of non\u2010specific staining. Both cut\u2010offs were based upon patient specimen distribution profiling.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions:<\/jats:title><jats:p>Use of a blocking antibody resulted in a robust, reproducible clinical B\u2010CLL assay that is not influenced by the need to measure the amount of ZAP\u201070 in other cells. ZAP\u201070 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. \u00a9 2006 International Society for Analytical Cytology<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.b.20125","type":"journal-article","created":{"date-parts":[[2006,8,12]],"date-time":"2006-08-12T00:17:19Z","timestamp":1155341839000},"page":"251-258","source":"Crossref","is-referenced-by-count":9,"title":["Use of a blocking antibody method for the flow cytometric measurement of ZAP\u201070 in B\u2010CLL"],"prefix":"10.1002","volume":"70B","author":[{"given":"Mark","family":"Shenkin","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Russell","family":"Maiese","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2006,8,11]]},"reference":[{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa023143"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1309\/4AGUT3LKEURD7T7K"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1309\/7Q1J-1AA8-DU4Q-PVLQ"},{"key":"e_1_2_6_5_2","first-page":"1","article-title":"Immunohistochemical detection of ZAP\u201070 in 341 cases of non\u2010Hodgkin and Hodgkin lymphoma","author":"Admirand J","year":"2004","journal-title":"Modern Pathology"},{"key":"e_1_2_6_6_2","unstructured":"ShankeyTV FormanM CobbJ ScibelliP SmithC MillsRAn Optimized Whole Blood Assay for Flow Cytometric Measurement of ZAP\u201070 Protein Expression CCS ZAP70 Workshop2005 20th Annual Meeting of the Clinical Cytometry Society Savannah GA."},{"key":"e_1_2_6_7_2","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa040857"},{"key":"e_1_2_6_8_2","doi-asserted-by":"publisher","DOI":"10.1002\/1097-0320(20000615)42:3<188::AID-CYTO5>3.0.CO;2-Q"},{"key":"e_1_2_6_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/sj.leu.2403762"},{"key":"e_1_2_6_10_2","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V94.6.1840"},{"key":"e_1_2_6_11_2","doi-asserted-by":"publisher","DOI":"10.1182\/blood.V94.6.1848"},{"key":"e_1_2_6_12_2","doi-asserted-by":"publisher","DOI":"10.1080\/10428190410001714016"},{"key":"e_1_2_6_13_2","doi-asserted-by":"publisher","DOI":"10.1080\/09638280500260079"},{"key":"e_1_2_6_14_2","unstructured":"FrederickL KatzmannJ MoriceW.Storage of B\u2010CLL Patient Peripheral Blood at 4oC Stabilizes the ZAP\u201070 Protein for up to 4 Days. Poster B8 2005 20th Annual Meeting of the Clinical Cytometry Society Savannah GA."},{"key":"e_1_2_6_15_2","doi-asserted-by":"publisher","DOI":"10.1080\/10428190410001714016"},{"key":"e_1_2_6_16_2","doi-asserted-by":"publisher","DOI":"10.1309\/WVQPVDF8UF7AV21X"}],"container-title":["Cytometry Part B: Clinical Cytometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fcyto.b.20125","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.b.20125","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,9,12]],"date-time":"2023-09-12T05:20:06Z","timestamp":1694496006000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.b.20125"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2006,7,15]]},"references-count":15,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2006,7,15]]}},"alternative-id":["10.1002\/cyto.b.20125"],"URL":"https:\/\/doi.org\/10.1002\/cyto.b.20125","archive":["Portico"],"relation":{},"ISSN":["1552-4949","1552-4957"],"issn-type":[{"value":"1552-4949","type":"print"},{"value":"1552-4957","type":"electronic"}],"subject":[],"published":{"date-parts":[[2006,7,15]]}}}