Performance evaluation of platelet counting by novel fluorescent dye staining in the XN-series automated hematology analyzers

Abstract

Background: Conventional automated hematology analyzers have limitations in platelet measurements such as poor accuracy and precision in the low count range and interference by nonplatelet particles. In order to improve it, the newly developed XN-Series automated hematology analyzers (Sysmex Corporation, Kobe, Japan) have been installed with a new dedicated channel for platelet analysis (PLT-F), which is based on a fluorescence flow cytometry method with uses of a novel fluorescent dye specifically staining platelets. We evaluated the basic performance of this new PLT-F channel.

Methods: Basic performance of the PLT-F channel in within-run reproducibility and assay linearity was studied using standard methods. Correlation was studied between PLT-F and a conventional automated hematology analyzer (XE-2100) and immunoplatelet analysis using anti-CD61 monoclonal antibody (Cell-Dyn Sapphire; Abbott Laboratories). The assay interference by nonplatelet particles such as fragmented red and white blood cells was evaluated by using clinical samples, respectively, from burn injury and acute leukemia.

Results: Basic performance of the PLT-F platelet counting was satisfactory in within-run reproducibility, linearity and correlation with the conventional analyzer. The correlation was satisfactory also with the immunoplatelet analysis, even for samples from a patient with burn injury, and those with white blood cell fragments displayed, platelet abnormal flag and low platelet counts (<50 × 10(9)/l).

Conclusion: The platelet counting performance of the PLT-F channel of the XN Series had improved accuracy and precision in the low range and in abnormal samples, avoiding the interference by nonplatelet particles.

Keywords: PLT-F channel; automated hematology analyzer; nonplatelet particle; platelet counting; thrombocytopenia.

Figure 1

Principle of PLT‐F channel. (A) Platelets stained with the dedicated reagent of PLT‐F (fluorescent microscopy image). The staining pattern of platelets by the fluorescent dye is localized, reflecting its specific binding to nucleic acid‐rich organelles. (B) PLT‐F scattergram. After platelets are stained with fluorescence dye (A), they are differentiated using information from the forward scattered light and side fluorescence intensity (B).* IPF, immature platelet fraction.

Figure 2

Correlation of platelet counts between CD61 and XN. (A) Samples that did not display the “PLT abnormal distribution” flag and had a platelet count below 50 × 10⁹/l. (B) Samples that displayed the “PLT abnormal distribution” flag. (C) Samples with a platelet count below 50 × 10⁹/l among samples that displayed the “PLT abnormal distribution” flag. White dots show results with PLT‐I that differed from the CD61 counts by more than 20 × 10⁹/l.

Figure 3

Histograms (PLT‐I) and scattergrams (PLT‐O and PLT‐F) of samples appeared white blood cell fragments. Dashed line in the PLT‐I histogram shows platelet volume 15 fL. In PLT‐F scattergram, the boundary line between the PLT and WBC areas is in the zone between them with few dots of particles. The part circled with the red line is believed to be where white blood cell fragments are plotted.

Figure 4

Correlation with CD61 platelet counts and monitoring of platelet counts in samples from burn injury patients. (A) Correlation of platelet counts between CD61 and XN using samples from burn injury patients. (B) Monitoring of platelet counts in burn injury samples.