Fig 1.
Typical scattergrams of the two flow cytometric platelet count channels on the XN-Series automated hematology analyzer.
The same whole-blood sample from a healthy volunteer was measured at the same time on the XN-2000 automated hematology analyzer. Each panel shows PLT-F (left) and PLT-O (right) scattergrams. FSC: forward scattered light; SFL: side fluorescent; RBC: erythrocytes; PLT: platelets; IPF: immature platelet fraction; RET: reticulocytes.
Fig 2.
Platelets and fragmented erythrocytes were distinguished with PLT-F reagents.
In each row, the left, middle, and right columns show platelets (PLT), erythrocytes (RBC), and fragmented erythrocytes (fRBC), respectively. (A) May-Giemsa-stained cells. (B) (Upper) PLT-F-stained cells. (Lower) PLT-F scattergrams. (C) (Upper) PLT-O-stained cells. (Lower) PLT-O scattergrams. Pale blue dots surrounded by yellow dashed line indicate fRBC misidentified as platelets. Bars: 5 μm.
Fig 3.
Comparison among three platelet counting methods using a fragmented RBC-PRP mixture.
Three platelet counting methods (PLT-F, PLT-O, and PLT-I) were compared using a control or fragmented RBC (fRBC) and platelet rich plasma (PRP) mixture. The mixtures were made from two donors’ blood and examined ten times per sample. (A) Typical scattergrams from the three methods. Top: PLT-F, middle: PLT-O, bottom: PLT-I. Left: control RBC and PRP mixture, right: fRBC and PRP mixture. The pale blue dots surrounded by yellow dashed line indicate fRBCs misidentified as platelets. (B) Platelet count of the mixtures. Data are expressed as mean ± standard deviation of the mean. F: PLT-F, O: PLT-O, I: PLT-I. Control: control RBC and PRP mixture, fRBC: fragmented RBC and PRP mixture. Significant code *** means p < 0.001.
Table 1.
Mean, SD, and CV of platelet counts with PLT-F, PLT-O, and PLT-I.
Fig 4.
PLT-F stain signals were overlapped with anti-Grp75 staining signals.
(A) Anti-Grp75 (red in merge channel), anti-CD63 (green in merge channel) and PLT-F (Blue in merge) triple staining. Yellow arrowhead indicates overlapped signals of anti-Grp75 and the PLT-F stain. (B) anti-calreticulin (Red in merge), CD62p (green in merge) and PLT-F signals (Blue in merge). (A and B) “with PC” channels shows merged images of immunofluorescence, PLT-F, and phase contrast images. PC stands for phase contrast. Bar = 1 μm.
Fig 5.
PLT-F reagents strongly stain CD41/CD61-positive platelets.
In each row, a mixture of platelets and fragmented erythrocytes was stained with both platelet-specific antibodies (anti-CD41 (A) or anti-CD61 (B), left) and PLT-F reagents (middle). Yellow arrowheads indicate platelets.
Fig 6.
The CD41+/CD61+ double-positive particles were separated with PLT-F reagents.
The mixture of platelets and fragmented erythrocytes that were stained with both FITC-labeled and PE-labeled antibodies were then stained with the PLT-F reagents and examined by flow cytometry. (A) The mixture was stained with both platelet-specific antibodies (anti-CD41 or anti-CD61, left) and PLT-F reagents (middle). (B) FITC-control IgG or (C) PE-control IgG was used as a negative control for fluorescence compensation.