title: Detection of residual donor leucocytes in leucoreduced red blood cell components using a fluorescence microplate assay.
abstract: In November 1999, universal leucoreduction of blood components was introduced in the UK to minimise the risk of variant Creutzfeldt-Jakob Disease (vCJD) transmission by blood transfusion. The UK specifications for leucodepletion processes state that 99% of leucodepleted components should contain < 5 x 10(6) leucocytes/unit, within 95% confidence limits. However, this leucocyte concentration is below the detection limits of standard haematology analysers. The development of a fluorometric immunoassay to detect the residual donor leucocytes in leucoreduced blood components is described. Monoclonal antibodies to leucocyte-specific cell surface antigens, CD45 and CD15, were adsorbed to the well surface in 96-well microplates. Red blood cell samples containing low numbers of leucocytes were added to the wells and the cells of interest captured by the monoclonal antibodies. Since leucocytes are the only nucleated cells found in significant numbers in blood components they were quantified using PicoGreen, a fluorescent stain specific for dsDNA. In comparison to flow cytometry, the method currently used to detect low numbers of leucocytes, the microplate assay demonstrated excellent sensitivity (1.00) and acceptable specificity (0.81) when standard leucodepleted samples were tested. There was no significant difference between the two methods (p < or = 0.175). In conclusion, the fluorescence microplate assay represents a simple, high throughput alternative to flow cytometry for monitoring leucodepletion compliance in blood banks.
authors.affiliation: Bristol Institute for Transfusion Sciences, National Blood Service-Bristol Centre, Southmead Road, Bristol BS10 5ND, UK. rgilbert@phls.org.uk
authors.forename: Ruth L
authors.initials: RL
authors.lastname: Gilbert
authors.forename: Janet R
authors.initials: JR
authors.lastname: Rider
authors.forename: J Richard
authors.initials: JR
authors.lastname: Turton
authors.forename: Derwood H
authors.initials: DH
authors.lastname: Pamphilon
mesh_terms: D001771:Blood Banks / Q000592:standards; D001781:Blood Component Removal; D001782:Blood Donors; D002021:Buffers; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007958:Leukocyte Count / Q000379:methods*; D009930:Organic Chemicals; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D002021:Buffers; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen