scholarly journals Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues

2016 ◽  
Vol 54 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Yen-Rei A. Yu ◽  
Danielle F. Hotten ◽  
Yuryi Malakhau ◽  
Ellen Volker ◽  
Andrew J. Ghio ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage ◽  
Human Blood ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Flow Cytometric

scholarly journals bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1291-1298 ◽  
Author(s):  
D Delia ◽  
A Aiello ◽  
D Soligo ◽  
E Fontanella ◽  
C Melani ◽  
...  
Keyword(s):  
Cell Lines ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Leukemic Cell ◽  
Refractory Anemia ◽  
Polymorphonuclear Cells ◽  
Color Flow ◽  
Western Blots ◽  
Flow Cytometric ◽  
Myeloid Leukemias

The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) to the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33+ and CD33+/CD34+ myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KG1, GM-1, and K562 were bcl-2 positive together with 11 of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti- bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KG1 cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a cross- reactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto- oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner.

scholarly journals Two-Color flow cytometric analysis of monocyte depleted human blood lymphocyte subsets

Cytometry ◽  
1988 ◽  
Vol 9 (4) ◽  
pp. 309-315 ◽  
Author(s):  
Thomas A. Fleisher ◽  
Claire Hagengruber ◽  
Gerald E. Marti
Keyword(s):  
Human Blood ◽  
Blood Lymphocyte ◽  
Lymphocyte Subsets ◽  
Flow Cytometric Analysis ◽  
Color Flow ◽  
Flow Cytometric ◽  
Human Blood Lymphocyte

Correcting Flow Cytometric Blast Counts for Blood Contamination in Bone Marrow Aspirates.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4613-4613
Author(s):  
Michael R. Loken ◽  
Sung-Chao Chu ◽  
Wayne K. Fritschle ◽  
Dian-Kun Li ◽  
Denise A. Wells
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Light Scatter ◽  
Blood Contamination ◽  
Simple Method ◽  
Flow Cytometric ◽  
Biopsy Specimens ◽  
Blast Count

Abstract An accurate blast count is pivotal in the diagnosis, classification and prognosis of patients with myelodysplasia. Blast counts in all previous classification schemes are based on morphologic assessment of marrow aspirates with a poor correlation to blast counts determined by flow cytometry. A significant problem in blast enumeration by flow cytometry is the variable hemodilution of the marrow during collection for flow cytometric analysis. Blast counts can vary depending on which aspirate tube is used for flow analysis, e.g., 2.4%, 1st 5ml tube; 0.62%, 2nd 5ml tube; 0.58% 3rd 5ml tube. Morphologists circumvent this problem by selecting a region for assessment close to a spicule with minimal blood dilution. Cell surface antigens can be used to distinguish mature cells found in blood as distinct from immature cells identified in marrow. CD16 intensity on neutrophils reaches a maximum at the band/segmented stage of development with a low coefficient of variation, thereby becoming a marker for mature myeloid forms. A simple method to distinguish immature from mature myeloid cells was developed to assess extent of blood contamination in marrow aspirates using a combination of CD16, CD13, and CD45. The average mature neutrophil content of a marrow was determined from phenotypically normal bone marrow biopsy specimens, assumed to have minimal blood contamination. The proportion of dimCD16 cells gated on the myeloid cells based on CD45 and right angle light scatter in 31 biopsy specimens was 82% (range 69–93, SD=6.2) (Figure 1). A value of 80% (rather than 82%) was used for the subsequent calculations to correct for the excess mature neutrophils found in an aspirate as compared to the biopsies (Corrected Blasts = [80 / % dim CD16 myeloid] x determined blast count). To test this hypothesis bone marrow aspirates were diluted with blood at different ratios to mimic blood marrow hemodilution. Blasts (defined as CD45 dim, low right angle light scatter, HLA-DR positive, CD11b negative) were determined for the various dilutions, then corrected based solely on the proportion of dim CD16 myeloid cells (Figure 2). A marrow from an MDS case was also diluted (1:5 v/v) with blood for comparison. The original marrow contained 80% dim CD16 myeloid cells with a blast count of 9.2%. After dilution, only 12% dim CD16 cells were detected with 1.1% blasts, however upon correction (6.67), the blast count was 7.3%, close to the original determination. This approach may provide for more standardization and consistency in the determination of blast counts in MDS marrow specimens using flow cytometric analysis. Figure 1, CD16 of marrow myeloid cells. Figure 1,. CD16 of marrow myeloid cells. Figure 2, Uncorrected/Corrected Blast Count Figure 2,. Uncorrected/Corrected Blast Count

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