Ivory K, Sarria B, Fairweather-Tait S J, Hughes D A
Gastrointestinal Biology and Health, Institute of Food Research, Colney, Norwich, East Anglia, UK.
Int J Lab Hematol. 2007 Oct;29(5):352-60. doi: 10.1111/j.1365-2257.2006.00881.x.
As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis.
作为一项铁吸收研究的一部分，在测量健康人类志愿者口服稳定铁同位素后的富集情况之前，我们需要准确计数其外周血中的网织红细胞。最近的研究表明，通过流式细胞术进行网织红细胞计数是有用的，该方法结合了差分光散射和噻唑橙（TO）与成熟红细胞内RNA的化学计量结合测量。使用这种方法，我们试图通过计数更多细胞来提高定量分析的精度，因为网织红细胞通常占红细胞总数的不到2%。为了确保排除其他细胞类型，我们分别用CD16+CD45-别藻蓝蛋白和CD61-藻红蛋白鉴定白细胞和血小板。从分析中去除CD16(+)CD45(+)TO(+)白细胞和CD61(+)TO(-)血小板后，剩余细胞为CD61(-)TO(-)红细胞、CD61(-)TO(+)网织红细胞和CD61(+)TO(+)网织血小板的组合。从分析中排除CD61(+)TO(+)细胞后，网织红细胞计数较低。当通过血型糖蛋白A表达和TO摄取阳性鉴定红细胞前体时，网织红细胞计数同样较低。我们得出结论，在流式细胞术网织红细胞分析中排除网织血小板是必要的。
