BACKGROUND: Implementation of WBC reduction of the blood supply increases the importance of measurement of residual WBC subtypes responsible for immunologic and infectious complications of transfusion. STUDY DESIGN AND METHODS: Real-time RT-PCR assays were developed to detect mRNA encoding lineage-specific WBC markers. Primers and fluorescent probes were designed for CD45 (pan-WBC), CD3 (T-lymphocyte), CD19 (B-lymphocyte), CD14 (monocyte), and CD66 (granulocyte), and the specificity was assessed by comparison with flow cytometric analysis of enriched cell populations. WBC subsets were examined in WBC-reduced whole blood prepared with filters (WBF2, Pall; and RZ2000, Baxter) and in platelet concentrates prepared with other filters (Autostop, Pall; and PLX-5, Baxter) and apheresis (COBE Spectra LRS, Gambro). RESULTS: All real-time RT-PCR assays were linear over >5 log concentration range, allowing pre-WBC-reduction and post-WBC-reduction comparisons. Sensitivity limits ranged from 10 cells per mL (CD45) to 200 cells per mL (CD19). Assay specificity was confirmed by the close correlation of real-time RT-PCR and immunophenotyping results by flow cytometry. For all subsets, >3.8 log and >3.1 log reduction was obtained during WBC reduction of whole blood and platelets, respectively. CONCLUSION: Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.
BACKGROUND: Implementation of WBC reduction of the blood supply increases the importance of measurement of residual WBC subtypes responsible for immunologic and infectious complications of transfusion. STUDY DESIGN AND METHODS: Real-time RT-PCR assays were developed to detect mRNA encoding lineage-specific WBC markers. Primers and fluorescent probes were designed for CD45 (pan-WBC), CD3 (T-lymphocyte), CD19 (B-lymphocyte), CD14 (monocyte), and CD66 (granulocyte), and the specificity was assessed by comparison with flow cytometric analysis of enriched cell populations. WBC subsets were examined in WBC-reduced whole blood prepared with filters (WBF2, Pall; and RZ2000, Baxter) and in platelet concentrates prepared with other filters (Autostop, Pall; and PLX-5, Baxter) and apheresis (COBE Spectra LRS, Gambro). RESULTS: All real-time RT-PCR assays were linear over >5 log concentration range, allowing pre-WBC-reduction and post-WBC-reduction comparisons. Sensitivity limits ranged from 10 cells per mL (CD45) to 200 cells per mL (CD19). Assay specificity was confirmed by the close correlation of real-time RT-PCR and immunophenotyping results by flow cytometry. For all subsets, >3.8 log and >3.1 log reduction was obtained during WBC reduction of whole blood and platelets, respectively. CONCLUSION: Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.
Authors: Tamarah D de Jong; Joyce Lübbers; Samina Turk; Saskia Vosslamber; Elise Mantel; Hetty J Bontkes; Conny J van der Laken; Johannes W Bijlsma; Dirkjan van Schaardenburg; Cornelis L Verweij Journal: Arthritis Res Ther Date: 2016-07-13 Impact factor: 5.156