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A sensitive quantitative single-platform flow cytometry protocol to measure human platelets in mouse peripheral blood

Authors

  • Laurus F. Schipper,

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    • *

      LFS and YVH contributed equally to this manuscript.

  • Yvette Van Hensbergen,

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    • *

      LFS and YVH contributed equally to this manuscript.

  • Willem E. Fibbe,

    1. From the Sanquin Blood Bank, Southwest Region, Leiden; and the Department of Immunohematology & Blood Transfusion and Department of Hematology, Laboratory of Experimental Hematology, Leiden University Medical Center, Leiden, the Netherlands.
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  • Anneke Brand

    1. From the Sanquin Blood Bank, Southwest Region, Leiden; and the Department of Immunohematology & Blood Transfusion and Department of Hematology, Laboratory of Experimental Hematology, Leiden University Medical Center, Leiden, the Netherlands.
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  • Financial support by Grant PPOC-01-003 from the Sanquin Blood Bank.

Anneke Brand, Department of Research and Education, Sanquin Blood Bank, Southwest Region, Plesmanlaan 1A, 23333 BZ, Leiden, the Netherlands; e-mail: a.brand@sanquin.nl.

Abstract

BACKGROUND: The NOD/SCID mouse is a widely used model for human cord blood (CB) transplantation. Engraftment is generally estimated with semiquantitative methods, measuring the percentage of human cells among mouse cells. To compare protocols aiming to improve hematopoietic recovery, quantitative methods to enumerate human cells would be preferred. This study describes a single-platform protocol to count human platelets (hPLTs) after transfusion and CB transplantation in the peripheral blood (PB) of the mouse.

METHODS: With an anti-human CD41 antibody against hPLTs and counting beads, the sensitivity to detect hPLTs in mouse blood by flow cytometry was validated. PLT recovery after hPLT transfusions and PLT kinetics after transplantation with CB CD34+ cells was followed in time in NOD/SCID mice.

RESULTS: hPLTs could be reliably detected to a level as low as 1 PLT per μL with this single-platform protocol, what appeared to be at least 10 times more sensitive than detection with the dual-platform protocol. To verify the applicability for mouse studies, hPLTs were measured serially in transfusion and transplantation studies in NOD/SCID mice. The results showed that earlier detection of PLT recovery was feasible with the single-platform protocol.

CONCLUSION: A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.

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