BACKGROUND: Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker. METHODS: We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients. RESULTS: Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening. CONCLUSION: A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.
Antigens, CD15/metabolism ; Antigens, CD24/metabolism ; Antigens, CD55/metabolism ; Antigens, CD59/metabolism ; Biomarkers/metabolism ; Blood Cell Count ; Erythrocytes/cytology/metabolism ; Flow Cytometry ; Granulocytes/cytology/metabolism ; Hemoglobinuria, Paroxysmal/*diagnosis/metabolism ; Humans ; Sensitivity and Specificity

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Flow Cytometry/*methods ; Granulocytes/cytology ; Humans ; Infant ; Leukocyte Count/*methods ; Leukocytes/*cytology ; Leukopenia/*blood/diagnosis ; Male ; Middle Aged ; Reagent Kits, Diagnostic ; Software

BACKGROUND: Hematology analyzers may ineffectively recognize abnormal cells, and manual differential counts may be imprecise for leukopenic samples. We evaluated the efficacy of the Hematoflow method for determining the leukocyte differential in leukopenic samples and compared this method with the manual differential method. METHODS: We selected 249 blood samples from 167 patients with leukopenia (WBC counts, 500-2,000/microL) for analysis in this study. The EDTA-anticoagulated blood samples were analyzed using an automatic blood cell counter (DxH800; Beckman Coulter, USA) and flow cytometry (FC 500; Beckman Coulter) by using Cytodiff reagent and analysis software (Beckman Coulter). Hematoflow results were selected or calculated from DxH800 and Cytodiff results. Two trained pathologists performed a manual differential count by counting 50-100 cells. RESULTS: The precision of the Hematoflow method was superior to that of the manual method in counting 5 leukocyte subpopulations, immature granulocytes (IGs), and blasts. Blasts were detected in all 45 cases (100%) by Hematoflow. The correlation of the Cytodiff blast count to the reference count was high (r = 0.8325). For all other cell populations, the correlation of the Hematoflow results with the reference count was stronger than that of the other manual counts with the reference count. CONCLUSIONS: The Hematoflow differential counting method is more reproducible and sensitive than manual counting, and is relatively easy to perform. In particular, this method detected leukemic blasts more sensitively than manual differential counts. The Hematoflow method is a very useful supplement to automated cell counting.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Flow Cytometry/*methods ; Granulocytes/cytology ; Humans ; Infant ; Leukocyte Count/*methods ; Leukocytes/*cytology ; Leukopenia/*blood/diagnosis ; Male ; Middle Aged ; Reagent Kits, Diagnostic ; Software