Flow Cytometric Analysis of Reticulated Platelets

Goran B. Matic1, Gregor Rothe1, Gerd Schmitz1

1 University of Regensburg, Regensburg, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.10
DOI:  10.1002/0471142956.cy0710s17
Online Posting Date:  August, 2001
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Flow Cytometric Analysis of Reticulated Platelets (G. Matic, G. Rothe, and G. Schmitz, University of Regensburg, Regensburg, Germany). In the last several years, flow cytometry has emerged as one of the tools of choice in evaluation of platelets. In particular, the distinction between reticulated platelets and mature platelets based on RNA fluorescence has proved to be a vital tool in the clinical hematology laboratory. Further, as is now well understood, it is important to evaluate platelets in whole blood rather than in isolated populations. Platelet quantification in a dual‐color whole‐blood method is of interest for the characterization of thrombocytopoiesis in (immune)‐thrombocytopenia or in the regenerating bone marrow.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Normal and patient whole blood samples anticoagulated with di‐ or tripotassium EDTA (1.5 mg/ml final EDTA concentration)
  • Phycoerythrin‐conjugated platelet‐specific monoclonal antibody—e.g., anti‐GPIb (CD42b) or anti‐GPIIb/GPIIIa (CD41/CD61)—at concentration such that required quantity (determined by titration) can be delivered in 5 µl
  • 1 µg/ml thiazole orange (TO) working solution (see recipe)
  • Formaldehyde solution (see recipe)
  • 12 × 75–mm polypropylene or polystyrene tubes as required by flow cytometer
  • Flow cytometer with 488‐nm argon laser and band‐pass filters centered around 520 nm and 585 nm
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Literature Cited

Literature Cited
   Ault, K.A. and Knowles, C. 1995. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp. Hematol. 23:996‐1001.
   Ault, K.A., Rinder, H.M., Mitchell, J., Carmody, M.B., Vary, C.P., and Hillman, R.S. 1992. The significance of platelets with increased RNA content (reticulated platelets): A measure of the rate of thrombopoiesis. Am. J. Clin. Pathol. 98:637‐646.
   Bonan, J.L., Rinder, H.M., and Smith, B.R. 1993. Determination of the percentage of thiazole orange (TO)‐positive, “reticulated” platelets using autologous erythrocyte TO fluorescence as an internal standard. Cytometry 14:690‐694.
   Dale, G.L., Friese, P., Hynes, L.A., and Burstein, S.A. 1995. Demonstration that thiazole‐orange‐positive platelets in the dog are less than 24 hours old. Blood 85:1822‐1825.
   Ingram, M. and Coopersmith, S. 1969. Reticulated platelets following acute blood loss. Br. J. Haematol. 17:225‐228.
   Kienast, J. and Schmitz, G. 1990. Flow cytometric analysis of thiazole orange uptake by platelets: A diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 75:116‐121.
   Lee, L.G., Chen, C.H., and Chiu, L.A. 1986. Thiazole orange: A new dye for reticulocyte analysis. Cytometry 7:508‐517.
   Matic, G.B., Chapman, E.S., Zaiss, M., Rothe, G., and Schmitz, G. 1998. Whole blood analysis of reticulated platelets: Improvements of detection and assay stability. Cytometry In press.
   Richards, E.M. and Baglin, T.P. 1995. Quantitation of reticulated platelets: Methodology and clinical application. Br. J. Haematol. 91:445‐451.
   Rinder, H.M., Munz, U.J., Ault, K.A., Bonan, J.L., and Smith, B.R. 1993. Reticulated platelets in the evaluation of thrombopoietic disorders. Arch. Pathol. Lab. Med. 117:606‐610.
   Robinson, M.C., Mackie, I.J., Khair, K., Liesner, R., Goodall, A.H., Savidge, G.F., Machin, S.J., and Harrisson, P. 1998. Flow cytometric analysis of reticulated platelets: Evidence for a large proportion of non‐specific labelling of dense granules by fluorescent dyes. Br. J. Haematol. 100:351‐357.
   Romp, K.G., Peters, W.P., and Hoffman, M. 1994. Reticulated platelet counts in patients undergoing autologous bone marrow transplantation: An aid in assessing marrow recovery. Am. J. Hematol. 46:319‐324.
   Schmitz, G., Rothe, G., Ruf, A., Barlage, S., Tschöpe, D., Clemetson, K.J., Goodall, A.H., Michelson, A.D., Nurden, A.T., and Shankey, T.V. 1998. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb. Haemost. 79:885‐896.
   Watanabe, K., Takeuchi, K., Kawai, Y., Ikeda, Y., Kubota, F., and Nakamoto, H. 1995. Automated measurement of reticulated platelets in estimating thrombopoiesis. Eur. J. Haematol. 54:163‐171.
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