title: Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event mu-targets in high-background biosamples.
abstract: A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 micros) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-microm calibration beads in environmental water dirt samples. Europium-labeled 5-microm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.
authors.affiliation: Macquarie University, Centre of MQ Photonics, New South Wales 2109, Australia. jin@ics.mq.edu.au
authors.forename: Dayong
authors.initials: D
authors.lastname: Jin
authors.forename: James A
authors.initials: JA
authors.lastname: Piper
authors.forename: Robert C
authors.initials: RC
authors.lastname: Leif
authors.forename: Sean
authors.initials: S
authors.lastname: Yang
authors.forename: Belinda C
authors.initials: BC
authors.lastname: Ferrari
authors.forename: Jingli
authors.initials: J
authors.lastname: Yuan
authors.forename: Guilan
authors.initials: G
authors.lastname: Wang
authors.forename: Lidia M
authors.initials: LM
authors.lastname: Vallarino
authors.forename: John W
authors.initials: JW
authors.lastname: Williams
mesh_terms: D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D017076:Computer-Aided Design; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D007089:Image Enhancement / Q000295:instrumentation*; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity