Confounding effects of platelets on flow cytometric analysis and cell-sorting experiments using blood-derived cells

Area of Science:

• Immunology
• Cell Biology
• Hematology

Background:

• Flow cytometric analysis and cell sorting of peripheral blood leukocytes are standard laboratory techniques.
• Platelet contamination in these analyses is often overlooked, potentially affecting results.

Purpose of the Study:

• To investigate the extent of platelet contamination during flow cytometric analysis and cell sorting of rat blood leukocytes.
• To evaluate the impact of different sample preparation methods on platelet contamination.

Main Methods:

• Sorting of red blood cells, platelets, lymphocytes, monocytes, and granulocytes from rat blood.
• Presort enrichment using differential centrifugation.
• Leukocyte sample preparation with ammonium chloride lysis of red blood cells.

Main Results:

• Significant platelet contamination occurred unless proper precautions were taken during cell sorting.
• The level of platelet contamination varied between experiments, with neutrophils/granulocytes showing the highest association in samples prepared with ammonium chloride lysis.

Conclusions:

• Incorporating an immunophenotypic marker for platelet identification is a simple and effective measure to assess contamination.
• This practice should be routine for flow cytometric analysis and cell sorting of blood-derived cells.
• Both free and cell-associated platelets contribute to contamination and require specific attention.