Flow Rate Calibration for Absolute Cell Counting Rationale and Design

Clare Walker1, David Barnett1

1 Royal Hallamshire Hospital, Sheffield
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.24
DOI:  10.1002/0471142956.cy0624s36
Online Posting Date:  May, 2006
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Abstract

There is a need for absolute leukocyte enumeration in the clinical setting, and accurate, reliable (and affordable) technology to determine absolute leukocyte counts has been developed. Such technology includes single platform and dual platform approaches. Derivations of these counts commonly incorporate the addition of a known number of latex microsphere beads to a blood sample, although it has been suggested that the addition of beads to a sample may only be required to act as an internal quality control procedure for assessing the pipetting error. This unit provides the technical details for undertaking flow rate calibration that obviates the need to add reference beads to each sample. It is envisaged that this report will provide the basis for subsequent clinical evaluations of this novel approach.

Keywords: flow cytometry; single platform; absolute counts; quality control; CD4+ T lymphocytes

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Lysing Solution Calibration Factor
  • Basic Protocol 2: Absolute Cell Counting Using Flow Rate Calibration
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of Lysing Solution Calibration Factor

  Materials
  • Lysing solution (FacsLyse, BD Biosciences)
  • Microbeads (TruCount, BD Biosciences)
  • Blood: normal whole blood in EDTA
  • 12 × 75–mm polypropylene tubes
  • Vortex
  • Flow cytometer

Basic Protocol 2: Absolute Cell Counting Using Flow Rate Calibration

  Materials
  • FITC‐labeled CD45 and PE‐labeled CD4
  • Blood (from individual patients)
  • Lysing solution
  • 12 × 75–mm polypropylene tubes
  • Vortex
  • Flow cytometer with 488‐nm excitation and filters for collection of green (FITC) and orange (PE) fluorescence
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Figures

Videos

Literature Cited

Literature Cited
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   Bergeron, M., Phaneouf, S., Ding, T., Soucy, N., and Mandy, F. 2002. A quality control tool for single‐platform absolute counting technology: Using bead flow rate as pipetting precision verifier. Cytometry 50:281.
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