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

OBJECTIVETo confirm the malignant phenotype of hepatocarcinoma cell (HCC) lines at various stages of differentiation (MHCC97L, MHCC97H and HCCLM3) and to explore their expression levels of cancer stem cell (CSC) markers.

METHODSThe invasive and proliferative properties of each HCC line were assessed by transwell assay and the Cell Counting Kit-8 (CCK-8) colorimetric assay. Sensitivity to chemotherapy was assessed by treatment with oxaliplatin and determination of the half inhibitory concentration (IC50). The expression of CD90, EpCAM and CD24 was measured by flow cytometry.

RESULTSThe number of cells that migrated through the invasion assay membrane were significantly different between the three HCC lines: HCCLM3 (30.57 +/- 8.95) more than MHCC97H (21.33 +/- 4.17) more than HCC97L (9.33 +/- 3.85), P less than 0.01. The IC50 was significantly different between the three HCC lines: HCCLM3 (36.57 +/- 6.95) mumol/L more than MHCC97H (26.35+/-3.88) mumol/L more than MHCC97L (17.68 +/- 3.25) mumol/L. The CSC marker with the highest expression on all three HCC lines was CD90 (HCCLM3: 0.92% +/- 0.21%, MHCC97H: 1.98% +/- 0.23%, and MHCC97L: 2.55% +/- 0.34%), followed by EpCAM (2.11% +/- 0.32%, 3.23% +/- 0.18%, and 4.38% +/-0.49%, respectively), and CD24 as the lowest (0.68% +/- 0.37%, 1.22% +/- 0.26%, and 1.36% +/- 0.24%, respectively).

CONCLUSIONHigher expression of CSC markers on HCC lines is associated with a stronger invasive ability and higher sensitivity to chemotherapy.

Antigens, Neoplasm ; metabolism ; CD24 Antigen ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Adhesion Molecules ; metabolism ; Cell Differentiation ; Cell Line, Tumor ; Epithelial Cell Adhesion Molecule ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplastic Stem Cells ; cytology ; metabolism ; Signal Transduction ; Thy-1 Antigens ; metabolism

BACKGROUNDRecent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell.

METHODSTissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1).

RESULTSMore than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Biparametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24.

CONCLUSIONSNon-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Adult ; Antigens, CD ; metabolism ; CD24 Antigen ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Endoglin ; Female ; Flow Cytometry ; Humans ; Hyaluronan Receptors ; metabolism ; Integrin beta1 ; metabolism ; Intervertebral Disc ; cytology ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, Cell Surface ; metabolism ; Young Adult

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; CD24 Antigen ; metabolism ; CD55 Antigens ; metabolism ; Female ; Gangliosides ; metabolism ; Glycoproteins ; metabolism ; Hedgehog Proteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Isoenzymes ; metabolism ; Neoplastic Stem Cells ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; Peptides ; metabolism ; Receptors, Notch ; metabolism ; Retinal Dehydrogenase ; metabolism ; Signal Transduction ; Wnt Signaling Pathway