Flow cytometric method for enumeration and classification of reactive immature granulocyte populations

We developed a flow cytometric method for tbe enumeration and classificatio n of nonmalignant immature granulocytes (IG). In this study, IG are defined as most immature (IG stage 1: promyelocytes and myelocytes) and as more ma ture (IG stage 2: metamyelocytes). Blood specimens from 46 patients with do cumented infectious or inflammatory disease and known presence of IG (by ro utine manual microscopy) were analyzed. For a reference manual differential count, we used a 400 white blood cell (WBC) differential and separated gra nulocytes into promyelocytes and myelocytes combined, metamyelocytes, and i ncluded band cells in the mature, segmented neutrophil population. The flow cytometric method is based on three-color staining of whole, anticoagulate d blood with CD45-PerCP, CD16-FITC, and CD11b-PE-labeled monoclonal antibod ies and a three-step gating procedure. The flow cytometric results were con firmed by cell sorting and microscopic evaluation of the sorted cells. A to tal of 10,000 events, excluding debris, were recorded per specimen and IG s tage 1 (CD16(-)/CD11b(-)), IG stage 2 (CD16(-)/CD11b(+)), and mature neutro phils (CD16(+)/CD11b(+)) were categorized. Regression and correlation betwe en 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 promyeloc ytes 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 varia tion (CV) of 6.8% for all IG combined compared with a CV of 50.2% for manua l differential IG count (based on a routine 100 WBC count). Samples were fo und stable at least 12 h at 25 degreesC and at least 48 h at 4 degreesC for flow cytometry. After staining and lysing, the sample was stable for at le ast 120 min at room temperature. We analyzed samples from patients with mye lodysplastic and myeloproliferative disease separately. We found that CD16( -) mature neutrophils falsely elevated the flow cytometric IG count. Simila r results were obtained in blood from patients treated with granulocyte-col ony stimulating factor (G-CSF). Although this restricts the use of the meth od somewhat, we believe that this flow cytometric method is useful for enum erating reactive IG, as well as for evaluating automated methods for IG ide ntification by hematology analyzers. Cytometry (Comm. Clin. Cytometry) 42:3 71-378, 2000. (C) 2000 Wiley-Liss, Inc.