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Related Experiment Videos

Reticulated platelets interfere with flow cytometric reticulocyte counts.

K Ivory1, B Sarria, S J Fairweather-Tait

  • 1Gastrointestinal Biology and Health, Institute of Food Research, Colney, Norwich, East Anglia, UK. kamal.ivory@bbsrc.ac.uk

International Journal of Laboratory Hematology
|September 11, 2007
PubMed
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Accurate reticulocyte counting using flow cytometry requires excluding reticulated platelets. This ensures precise measurement of red blood cell precursors in iron absorption studies.

Area of Science:

  • Hematology
  • Cell Biology
  • Biotechnology

Background:

  • Accurate reticulocyte quantification is crucial for iron absorption studies.
  • Flow cytometry offers a precise method for counting reticulocytes via thiazole orange (TO) RNA binding.
  • Reticulocytes, immature red blood cells, constitute a small fraction (<2%) of the peripheral blood cell population.

Purpose of the Study:

  • To enhance the precision of reticulocyte counting in human peripheral blood using flow cytometry.
  • To refine flow cytometry protocols for distinguishing reticulocytes from other blood components.
  • To validate a method for accurate reticulocyte enumeration in the context of iron metabolism research.

Main Methods:

  • Flow cytometry was employed for cell analysis.

Related Experiment Videos

  • Thiazole orange (TO) was used to stain RNA in maturing erythrocytes (reticulocytes).
  • Antibodies (CD16, CD45, CD61, glycophorin A) were used for cell identification and exclusion of white blood cells (WBCs) and platelets.

    • Main Results:

    • Exclusion of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets was performed.
    • Analysis revealed that CD61(+) TO(+) reticulated platelets were present alongside reticulocytes.
    • Reticulocyte counts were significantly lower and more accurate after the exclusion of reticulated platelets.

    Conclusions:

    • Reticulated platelets can be misidentified as reticulocytes during flow cytometry.
    • Excluding CD61(+) TO(+) reticulated platelets is essential for accurate reticulocyte counting.
    • This refined flow cytometry approach improves the reliability of reticulocyte measurements in hematological studies.