{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T08:06:49Z","timestamp":1788854809589,"version":"build-2803163510"},"reference-count":22,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytometry Part B Clinical"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Receptor occupancy (RO) assessments are a critical component of elucidating PK\/PD\/safety relationships during early clinical development. As an initial measure of target engagement, RO can inform the pharmacological activity, and RO data from nonclinical studies are often translated to support human dose projection when the therapeutic target is a membrane\u2010bound protein. Currently, across the industry, RO measurements are typically performed using freshly collected blood samples that require real\u2010time bioanalysis, often resulting in highly variable data, missed time points, substantial operational costs, and logistical challenges. Here, we describe a novel RO method that utilizes cryopreserved whole blood, representing a substantial process improvement and enhanced robustness of RO endpoints. Instead of measuring cell surface target engagement in freshly collected whole blood, this method utilizes whole blood that is cryopreserved at the time of collection without subsequent peripheral blood mononuclear cell (PBMC) isolation. This approach extends the sample stability window from 2 to 3\u2009days to 12\u2009months post\u2010collection. Internal method development demonstrated that a 1:1 dilution of whole blood with freezing media followed by cryopreservation produced RO results in T cells comparable to those obtained from fresh blood. Follow\u2010up studies confirmed that this observation extended to multiple target binding arms of multispecific T\u2010cell engagers, as well as RO measurements in B\u2010cell and monocyte targets. Additional studies evaluated the feasibility of implementing this method in clinical settings by assessing assay robustness, sample freezing conditions, and shipping methods.<\/jats:p>","DOI":"10.1002\/cyto.b.70069","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T07:10:21Z","timestamp":1788851421000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Improved sample stability for clinical whole blood receptor occupancy assays with cryopreservation"],"prefix":"10.1002","author":[{"given":"Chunli","family":"Huang","sequence":"first","affiliation":[{"name":"Bioanalytical Discovery &amp; Development Sciences Johnson &amp; Johnson  Spring House Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen\u2010I","family":"Tsou","sequence":"additional","affiliation":[{"name":"Bioanalytical Discovery &amp; Development Sciences Johnson &amp; Johnson  Spring House Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pu","family":"Fang","sequence":"additional","affiliation":[{"name":"Bioanalytical Discovery &amp; Development Sciences Johnson &amp; Johnson  Spring House Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katharine D.","family":"Grugan","sequence":"additional","affiliation":[{"name":"Bioanalytical Discovery &amp; Development Sciences Johnson &amp; Johnson  Spring House Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1002\/ajpa.24251","article-title":"A field method for cryopreservation of whole blood from a finger prick for later analysis with flow cytometry","volume":"174","author":"Blackwell A.","year":"2021","journal-title":"American Journal of Physical Anthropology"},{"key":"e_1_2_7_3_1","doi-asserted-by":"crossref","first-page":"652","DOI":"10.1002\/cyto.b.21994","article-title":"An easy and reliable whole blood freezing method for flow cytometry immune\u2010phenotyping and functional analyses","volume":"100","author":"Braudeau C.","year":"2021","journal-title":"Cytometry"},{"key":"e_1_2_7_4_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jim.2015.01.008","article-title":"Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development","volume":"418","author":"Brown L.","year":"2015","journal-title":"Journal of Immunological Methods"},{"key":"e_1_2_7_5_1","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/S0304-3835(01)00400-1","article-title":"Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies","volume":"166","author":"Cheng L.","year":"2001","journal-title":"Cancer Letters"},{"issue":"9","key":"e_1_2_7_6_1","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.4252\/wjsc.v13.i9.1197","article-title":"Effects of storage media, supplements and cryopreservation methods on quality of stem cells","volume":"13","author":"Erol O.","year":"2021","journal-title":"World Journal of Stem Cells"},{"key":"e_1_2_7_7_1","first-page":"176","volume-title":"Immuno\u2010oncology cellular and translational approaches","author":"Flye\u2010Blakemore L.","year":"2020"},{"key":"e_1_2_7_8_1","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1136\/jcp.30.8.758","article-title":"Cryopreservation of human granulocytes in liquid nitrogen","volume":"30","author":"Graham\u2010Pole J.","year":"1977","journal-title":"Journal of Clinical Pathology"},{"key":"e_1_2_7_9_1","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1002\/cyto.b.21339","article-title":"Recommendations for the development and validation of flow cytometry\u2010based receptor occupancy assays","volume":"90","author":"Green C.","year":"2016","journal-title":"Cytometry. Part B, Clinical Cytometry"},{"key":"e_1_2_7_10_1","first-page":"1492","article-title":"Whole blood cryopreservation in epidemiological studies","volume":"11","author":"Hayes R.","year":"2002","journal-title":"Cancer Epidemiology, Biomarkers & Prevention"},{"key":"e_1_2_7_11_1","doi-asserted-by":"crossref","DOI":"10.1186\/s12967-024-06010-z","article-title":"CryoSCAPE: Scalable immune profiling using cryopreserved whole blood for multi\u2010omic single cell and functional assays","volume":"23","author":"Heubeck A.","year":"2025","journal-title":"Journal of Translational Medicine"},{"issue":"1","key":"e_1_2_7_12_1","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1002\/cyto.b.21970","article-title":"Best practices for optimization and validation of flow cytometry\u2010based receptor occupancy assays","volume":"100","author":"Hilt E.","year":"2021","journal-title":"Cytometry. Part B, Clinical Cytometry"},{"issue":"10","key":"e_1_2_7_13_1","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0205520","article-title":"Modification of cellular membranes conveys cryoprotection to cells during rapid, non\u2010equilibrium cryopreservation","volume":"13","author":"Huebinger J.","year":"2018","journal-title":"PLoS One"},{"key":"e_1_2_7_14_1","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.jim.2010.01.014","article-title":"Dimethyl sulfoxide (DMSO) exposure to human peripheral blood mononuclear cells (PBMCs) abolish T cell responses only in higher concentrations and following coincubation for more than two hours","volume":"356","author":"Kloverpris H.","year":"2010","journal-title":"Journal of Immunological Methods"},{"key":"e_1_2_7_15_1","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.cryobiol.2019.04.004","article-title":"Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents","volume":"88","author":"Kojayan G.","year":"2019","journal-title":"Cryobiology"},{"key":"e_1_2_7_16_1","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1002\/cyto.a.24700","article-title":"A comprehensive assessment of four whole blood stabilizers for flow\u2010cytometric analysis of leukocyte populations","volume":"103","author":"Nguyen N.","year":"2023","journal-title":"Cytometry"},{"issue":"4","key":"e_1_2_7_17_1","first-page":"210","article-title":"Cryopreservation modified flow\u2010cytometric analysis of hemopoietic cells","volume":"68","author":"Rosillo M.","year":"1995","journal-title":"Vox Sanguinis"},{"issue":"9","key":"e_1_2_7_18_1","doi-asserted-by":"crossref","DOI":"10.3390\/cells11091527","article-title":"Comparison of whole blood cryopreservation methods for extensive flow cytometry immunophenotyping","volume":"11","author":"Serra V.","year":"2022","journal-title":"Cells"},{"issue":"10","key":"e_1_2_7_19_1","doi-asserted-by":"crossref","first-page":"2160","DOI":"10.1158\/1055-9965.EPI-07-0604","article-title":"Cryopreservation of whole blood samples collected in the field for a large epidemiologic study","volume":"16","author":"Stevens V.","year":"2007","journal-title":"Cancer Epidemiology, Biomarkers & Prevention"},{"key":"e_1_2_7_20_1","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1002\/cyto.b.21355","article-title":"Role of receptor occupancy assays for flow cytometery in drug development","volume":"90","author":"Stewart J.","year":"2015","journal-title":"Cytometry. 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Part B, Clinical Cytometry"},{"key":"e_1_2_7_22_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1177\/0963689721999617","article-title":"Cryopreservation: An overview of principles and cell\u2010specific considerations","volume":"30","author":"Whaley D.","year":"2021","journal-title":"Cell Transplantation"},{"issue":"10","key":"e_1_2_7_23_1","doi-asserted-by":"crossref","first-page":"655","DOI":"10.4155\/bio-2020-0080","article-title":"Extending flow cytometry sample stability by freezing lysed whole blood for clinical monitoring of Treg","volume":"12","author":"Yu J.","year":"2020","journal-title":"Bioanalysis"}],"container-title":["Cytometry Part B: Clinical Cytometry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.b.70069","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cyto.b.70069","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.b.70069","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T07:10:24Z","timestamp":1788851424000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.b.70069"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,8]]},"references-count":22,"alternative-id":["10.1002\/cyto.b.70069"],"URL":"https:\/\/doi.org\/10.1002\/cyto.b.70069","archive":["Portico"],"relation":{},"ISSN":["1552-4949","1552-4957"],"issn-type":[{"value":"1552-4949","type":"print"},{"value":"1552-4957","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,8]]},"assertion":[{"value":"2026-03-16","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-28","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-08","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"cyto.b.70069"}}