lewis-x-antigen and Hemoglobinuria--Paroxysmal

The flow cytometry analysis of GPI-linked proteins on red blood cells and leukocytes is crucial for paroxysmal nocturnal hemoglobinuria (PNH) diagnostics. However, the commonly used multicolor panels cannot be implemented in low-resourced hematology laboratories. In order to develop a simple prediagnostic test for PNH screening, we analyzed the diagnostic accuracy of the two-color (FLAER/CD15) detection of GPI-deficient neutrophils.. We reanalyzed multicolor data set of 1594 peripheral blood samples of patients screened for PNH applying only two markers (FLAER/CD15). The quantitative positivity/negativity was reported. Then, these results were compared in a blinded manner with previously obtained multicolor data from the same samples.. Among the 1594 samples included in the study, 507 samples were PNH-positive by the multicolor assay. The two-color method revealed 510 PNH-positive samples. The detailed examination of this discrepancy revealed 12 false-positives and 9 false-negatives. Therefore, FLAER/CD15 screening method displayed 98.90% of the diagnostic specificity and 98.22% of the sensitivity.. This simple two-color evaluation of FLAER-negative neutrophils is a highly effective screening test for PNH. Although this approach is not intended to replace the multicolor diagnostic procedure, it could minimize the number of patients requiring a conventional multicolor flow cytometric assay.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers; Child; Child, Preschool; Female; Flow Cytometry; GPI-Linked Proteins; Hemoglobinuria, Paroxysmal; Humans; Lewis X Antigen; Male; Middle Aged; Neutrophils; Young Adult

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.. 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.. 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.. 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.

Topics: Biomarkers; Blood Cell Count; CD24 Antigen; CD55 Antigens; CD59 Antigens; Erythrocytes; Flow Cytometry; Granulocytes; Hemoglobinuria, Paroxysmal; Humans; Lewis X Antigen; Sensitivity and Specificity

Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.. CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages.. Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples.. While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.

Topics: ADP-ribosyl Cyclase; Antigens, CD; Bacterial Toxins; CD24 Antigen; Flow Cytometry; Fucosyltransferases; GPI-Linked Proteins; Granulocytes; Hemoglobinuria, Paroxysmal; Humans; Leukocyte Common Antigens; Lewis X Antigen; Lipopolysaccharide Receptors; Monocytes; Pore Forming Cytotoxic Proteins; Receptors, IgG

Identification of paroxysmal nocturnal hemoglobinuria (PNH) by detecting a glycophosphatidylinositol-anchored defect by flow cytometry is presently the standard method of choice for diagnosing PNH. However, the selection of suitable markers will be critical and significantly affect the determination and quantification of PNH clones in various cell lineages.. In this study, we investigated the performance of various immunophenotypic markers including CD59, GPHA (a clustered antigen, CD235a), CD33, CD15 and fluorescent aerolysin (FLAER) combined with CD16, CD24 and CD14 in a PNH panel using six-color flow cytometry.. The results strongly indicate that these markers can collectively and effectively identify and quantify PNH clones in erythrocyte, granulocyte and monocyte populations derived from peripheral blood and bone marrow (BM). A sensitivity threshold as low as 0.01% in identifying PNH clones in erythrocyte and granulocyte populations from peripheral blood is achieved by this panel in a series dilution assay. In addition, a direct side-by-side comparison between BM and peripheral blood from the same patients suggests that the FLAER PNH test is capable of identifying to PNH clones in BM specimens.. The data support the premise that a six-color flow cytometry PNH panel using the combination of CD59, CD235a, CD33, CD15, FLAER, CD16, CD24 and CD14 can enhance and improve the current methods used in diagnosis and management of PNH by specifically identifying PNH clones in the erythrocyte, granulocyte and monocyte population.

Topics: Adult; Aged; Bacterial Toxins; Blood Cell Count; Bone Marrow; CD24 Antigen; CD59 Antigens; Cell Lineage; Erythrocytes; Female; Flow Cytometry; GPI-Linked Proteins; Granulocytes; Hemoglobinuria, Paroxysmal; Humans; Immunophenotyping; Lewis X Antigen; Lipopolysaccharide Receptors; Male; Middle Aged; Monocytes; Pore Forming Cytotoxic Proteins; Receptors, IgG; Sialic Acid Binding Ig-like Lectin 3