Van Hove L, Goossens W, Van Duppen V, Verwilghen R L
University Hospital Leuven, Department of Haematology, Belgium.
Clin Lab Haematol. 1990;12(3):287-99. doi: 10.1111/j.1365-2257.1990.tb00039.x.
Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.
最近，流式细胞术技术已得到发展，以取代显微镜网织红细胞计数。我们使用噻唑橙，一种RNA结合荧光染料，来区分网织红细胞和成熟红细胞。对噻唑橙和Retic-COUNT软件包在不同流式细胞仪上进行常规分析的性能进行了评估。所应用的方法以一种易于操作的方式半自动分析10⁴个细胞。使用乙二胺四乙酸二钾抗凝血液（采血后30小时内稳定），在25℃下于噻唑橙溶液中孵育2至7小时，可获得一致的结果。这使得样本采集、储存和检测性能具有灵活性，且结果无变化。高达30℃的孵育温度变化没有可测量的影响。对于正常、低和高网织红细胞计数，所获得的值显示出良好的线性、精密度和准确性。然而，观察到存在干扰：红细胞自发荧光、有核红细胞、豪-焦小体、高白细胞计数、高血小板计数和巨大血小板，所有这些都会错误地增加网织红细胞数量。通过软件门控校正消除了这些假象，因此只需不到5%的样本通过显微镜方法重新分析。噻唑橙流式细胞术方法被确定为一种用于网织红细胞常规临床定量的快速、可靠方法。
