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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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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Basic Protocol 1: Determination of Lysing Solution Calibration Factor

  • 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

  • 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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Literature Cited

Literature Cited
   Barnett, D., Granger, V., Whitby, L., Storie, I., and Reilly, J.T. 1999. Absolute CD4+ T‐lymphocyte and CD34+ stem cell counts by single platform flow cytometry: The way forward. Br. J. Haematol. 106:1059‐1062.
   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.
   Bergeron, M., Lustyik, G., Ding, T., Nicholson, J., Janossy, G., Shapiro, H., Barnett, D., and Mandy, F. 2003. When do non‐volumetric flow cytometers become volumetric? Time can tell, how absolute your instrument is about absolute cell counts. Cytometry 52B:37‐39.
   Brand, A., van de Watering, L.M., and Claas, F.H. 2000. Clinical significance of leukoreduktion of blood components. Vox Sang. 78:227‐229.
   Brando, B., Barnett, D., Janossy, G., Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J.L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S., and Gratama, JW. 2000. Cytometric methods for assessing absolute numbers of cell subsets in blood. Cytometry 42:327‐346.
   Cutnell, J.D. and Johnson, K.W. 2001. Physics (5th Edition). John Wiley & Sons, Hoboken, N.J.
   Glencross, D., Scott, L.E., Jani, I.V., Barnett, D., and Janossy, G. 2002. CD45 assisted PanLeucoGating for accurate, cost effective dual platform CD4+ T cell enumeration. Cytometry 50:69‐77.
   Janossy, G., Jani, I.V., and Goehde, W. 2000. Affordable CD4+ T cell counts on “single platform” flow cytometers I. Primary CD4 gating. Br. J. Haematol. 111:1198‐1208.
   Knudsen, L.M., Gaarsdal, E., Jensen, L., Nikolaisen, K., and Johnsen, H.E. 1996. Improved priming for mobilization of and optimal timing for harvest of peripheral blood stem cells. J. Hematother. 5:399‐406.
   Lowenthal, R.M., Faberes, C., Marit, G., Boiron, J.M., Cony‐Makhoul, P., Pigneux, A., Agape, P., Vezon, G., Bouzgarou, R., Dazey, B., Fizet, D., Bernard, P., Lacombe, F., and Reiffers, J. 1998. Factors influencing haemopoietic recovery following chemotherapy‐mobilised autologous peripheral blood progenitor cell transplantation for haematological malignancies: A retrospective analysis of a 10‐year single institution experience. Bone Marrow Transplant. 22:763‐770.
   Mandy, F., Nicholson, J., Autran, B., and Janossy, G. 2002. T‐Cell subset counting and the fight against AIDS: Reflection over a 20‐year struggle. Cytometry 50:39‐45.
   Poiseuille, J.L.M. 1846. Recherches expérimentales sur le mouvementes liquides dans les tubes de très‐petits diameters. Mém. Acad. Roy. Sci. 9:433‐544.
   Schnizlein‐Blick, C.T., Spritzler, J., Wilkening, C.L., Nicholson, J.K., and O'Gorman, M.R. 2000. Evaluation of TruCount absolute‐count tubes for determining CD4 and CD8 cell numbers in human immunodeficiency virus positive adults. Site Investigators and The NIAID DAIDS New Technologies Evaluation Group. Clin. Diagn. Lab. Immunol. 7:336‐343.
   Storie, I., Sawle, A., Whitby, L., Goodfellow, K., Granger, V., Reilly, J.T., and Barnett, D. 2003. Flow rate calibration II: Clinical evaluation study using a single‐platform PanLeucoGating protocol. Cytometry 55B:8‐13.
   Strauss, K., Hannet, I., Engel, S., Becker, R., Shiba, A., Jinguji, M.G., Valinsky, J., Barnett, D., Orfao, A., and Kestens, L. 1996. Performance evaluation of FACSCount: A dedicated system for clinical cellular analysis. Cytometry 26:52‐59.
   Walker, C.L., Whitby, L., Granger, V., Storie, I., Reilly, J.T., and Barnett, D. Flow rate calibration III: The use of stabilised biostandards to calibrate the flow rate and calculate absolute CD4+ T‐Cell counts. Cytometry (Clinical Cytometry) In press.
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