Counting reticulated platelets by flow cytometry

Summary

Platelet RNA levels correlate with thrombopoietic status and can distinguish thrombocytopenia due to myelosuppression or increased peripheral blood destruction. Reticulocyte platelets can be used to monitor megakaryocyte and thrombopoietic status after radiation therapy or transplantation. This experiment is based on the "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Counting reticulated platelets by flow cytometry

Principle

Platelet RNA levels correlate with thrombopoietic status and can distinguish thrombocytopenia due to myelosuppression or increased peripheral blood destruction. Reticulocyte platelets can be used to monitor megakaryocyte and thrombopoietic status after radiation therapy or after transplantation.

Materials and Instruments

EDTA Formaldehyde Tyrode Buffer Thiazole Orange Stain
Flow Cytometry

Move

1. Preparation of platelet-rich plasma: centrifuge EDTA-anticoagulated whole blood at 500 r/min for 8 min, take out the upper layer of platelet-containing plasma, centrifuge it at 2000 r/min for 10 min, discard the supernatant, and then re-suspend the platelets in Sodium Citrate Dextrose EDTA buffer (pH 7.4), wash it once, and then wash it twice in Tyrode's buffer (containing bovine serum albumin and EDTA). Wash twice with Tyrode buffer (containing bovine serum albumin and EDTA) and resuspend in Tyrode buffer, and adjust the platelet concentration to 100 x ¹⁰⁹ /L as the sample.

2. Aldehyde fixation: Add 1% formaldehyde to the specimen, and place the specimen in 4℃ for more than 2 h to fix the platelets. Wash with Tyrode buffer once before detection.

3. Staining and counting: Add 450 ul of thiazole orange stain to 50 ul of aldehyde-fixed platelet suspension and place it in the dark at room temperature for 1~2 h. Determine the fluorescence intensity at 525 nm wavelength by flow cytometry, which represents the number of RPs. At the same time, the total number of platelets was counted and the ratio of RP was calculated. This is the semi-automatic counting method of RP. Because this method uses thiazole orange staining,
stained platelets are also called thiazole orange positive platelets ( ^TO+ platelets).

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