Fujimoto H, Sakata T, Hamaguchi Y, Shiga S, Tohyama K, Ichiyama S, Wang F S, Houwen B
Reagent Group, Sysmex Corporation, Kobe, Japan.
Cytometry. 2000 Dec 15;42(6):371-8. doi: 10.1002/1097-0320(20001215)42:6<371::aid-cyto1004>3.0.co;2-g.
We developed a flow cytometric method for the enumeration and classification of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more mature (IG stage 2: metamyelocytes). Blood specimens from 46 patients with documented infectious or inflammatory disease and known presence of IG (by routine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated granulocytes into promyelocytes and myelocytes combined, metamyelocytes, and included band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulated blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibodies and a three-step gating procedure. The flow cytometric results were confirmed by cell sorting and microscopic evaluation of the sorted cells. A total of 10,000 events, excluding debris, were recorded per specimen and IG stage 1 (CD16-/CD11b-), IG stage 2 (CD16-/CD11b+), and mature neutrophils (CD16+/CD11b+) were categorized. Regression and correlation between flow cytometric IG and the manual differential showed y = 1.34x + 0.95, r(2) = 0.86 for IG stages 1 and 2 combined versus promyelocytes, myelocytes, and metamyelocytes. For IG stage 1 versus microscopic counts of promyelocytes and myelocytes, the results were y = 1.53x + 1.24, r(2) = 0.76; for IG stage 2 versus manual metamyelocyte count, y = 0.77x + 0.21, r(2) = 0.58. Reproducibility of the flow cytometric method showed a coefficient of variation (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manual differential IG count (based on a routine 100 WBC count). Samples were found stable at least 12 h at 25 degrees C and at least 48 h at 4 degrees C for flow cytometry. After staining and lysing, the sample was stable for at least 120 min at room temperature. We analyzed samples from patients with myelodysplastic and myeloproliferative disease separately. We found that CD16- mature neutrophils falsely elevated the flow cytometric IG count. Similar results were obtained in blood from patients treated with granulocyte-colony stimulating factor (G-CSF). Although this restricts the use of the method somewhat, we believe that this flow cytometric method is useful for enumerating reactive IG, as well as for evaluating automated methods for IG identification by hematology analyzers.
我们开发了一种用于非恶性未成熟粒细胞（IG）计数和分类的流式细胞术方法。在本研究中，IG被定义为最不成熟的（IG 1期：早幼粒细胞和中幼粒细胞）以及更成熟的（IG 2期：晚幼粒细胞）。分析了46例有记录的感染性或炎症性疾病且已知存在IG（通过常规手工显微镜检查）患者的血标本。对于参考手工分类计数，我们进行了400个白细胞（WBC）分类，并将粒细胞分为早幼粒细胞和中幼粒细胞合并、晚幼粒细胞，并将杆状核细胞纳入成熟的分叶核中性粒细胞群体。流式细胞术方法基于用CD45-PerCP、CD16-FITC和CD11b-PE标记的单克隆抗体对全血、抗凝血液进行三色染色以及三步门控程序。流式细胞术结果通过细胞分选和对分选细胞的显微镜评估得到证实。每个标本记录10,000个事件（不包括碎片），并对IG 1期（CD16-/CD11b-）、IG 2期（CD16-/CD11b+）和成熟中性粒细胞（CD16+/CD11b+）进行分类。流式细胞术IG与手工分类之间的回归和相关性显示，IG 1期和2期合并与早幼粒细胞、中幼粒细胞和晚幼粒细胞相比，y = 1.34x + 0.95，r(2) = 0.86。对于IG 1期与早幼粒细胞和中幼粒细胞的显微镜计数相比，结果为y = 1.53x + 1.24，r(2) = 0.76；对于IG 2期与手工晚幼粒细胞计数相比，y = 0.77x + 0.21，r(2) = 0.58。流式细胞术方法的重现性显示，所有IG合并的变异系数（CV）为6.8%，而手工分类IG计数（基于常规100个WBC计数）的CV为50.2%。发现样本在25℃下至少稳定12小时，在4℃下至少稳定48小时用于流式细胞术。染色和裂解后，样本在室温下至少稳定120分钟。我们分别分析了骨髓增生异常和骨髓增殖性疾病患者的样本。我们发现CD16-成熟中性粒细胞错误地提高了流式细胞术IG计数。在接受粒细胞集落刺激因子（G-CSF）治疗的患者血液中也获得了类似结果。尽管这在一定程度上限制了该方法的应用，但我们认为这种流式细胞术方法对于计数反应性IG以及评估血液学分析仪识别IG的自动化方法是有用的。
