Flow Cytometric Analysis of Human Bone Marrow
Adrian P. Gee in BONE MARROW PROCESSING and PURGING, 2020
The advent of flow cytometry provides an objective method for analyzing thousands of cells per second and the use of multiparameter analysis permits the evaluation of multiple characteristics of each cell. This chapter presents approaches for using flow cytometry to evaluate bone marrow preparations during processing or tumor cell purging. In the area of clinical flow cytometry, progress is being made in the standardization of the flow cytometric analysis of lymphocyte subsets. To optimize the light scatter for the collection of nucleated cells, an aliquot of the sample can be stained with LDS-751, a DNA-binding dye which will resolve the nucleated cells from the contaminating RBCs and debris. Because mature cells represent the major cell population in bone marrow, a cocktail of antibodies to mature cells has been used to identify this component, and exclude it from the analysis, thereby permitting a detailed evaluation, by multicolor analysis, of antigen expression on the minor immature component.
Effects of Folic Acid Deficiency on Tumor Cell Biology
Maryce M. Jacobs in Vitamins and Minerals in the Prevention and Treatment of Cancer, 2018
Folic acid is a vitamin which is found most abundantly in green leafy vegetables, liver, navy beans, nuts and whole wheat products. While replacement therapy with folic acid is usually indicated when deficiency is detected, treatment of folic acid deficiency in cancer patients is controversial because of concerns about possible promotion of tumor growth. Subsequent studies in rodents indicated that nutritional deficiency of folic acid retarded the growth of ascitic lymphocytic neoplasms in mice and Walker carcinosarcoma 256 in rats. Tumor cell size increases coincident with the development of folate deficiency. Murine melanoma cells incubated in low-folate medium demonstrated a progressive increase in the number of cells in S phase as folate deficiency developed, when studied by flow cytometry. The onset of folate deficiency in tumor cells is accompanied by changes in the adhesive properties of the cells. The mechanism or mechanisms by which nutritional folate deficiency enhances tumor cell metastatic potential is unclear.
Pancreas
Victor A. Bernstam in Pocket Guide to GENE LEVEL DIAGNOSTICS in Clinical Practice, 2019
Flow cytometry (FCM) allows the separatation of endocrine pancreas cells into individual cell populations. A tentative and weak correlation of aneuploidy of pancreatic neuroendocrine tumors with a poor prognosis has been observed by FCM. The fraction of cells with aneuploid Deoxyribonucleic acid (DNA) content in ductal adenocarcinomas of the pancreas and adenocarcinomas of the ampulla of Vater, although being relatively low, was significantly higher in carcinomas than in nonneoplastic tissue. Studies in cultured pancreatic endocrine tumor cells have identified the genes encoding various DNA-binding proteins specifically interacting with DNA regulatory elements in controlling the function of islet cell hormones. In the case of insulinoma, for example, half of the tumors studied displayed aneuploidy, despite that most of the patients were alive and disease free 2 to 5 years postresection. Numerical or structural abnormalities of chromosome 7 have been found in human pancreatic carcinoma cell lines.
Agreement between blood draw techniques for assessing platelet activation by flow cytometry
Published in Platelets, 2019
Emma L. Welch, Michael G. Crooks, Simon P. Hart
It is widely believed that assays of platelet activation are susceptible to preanalytical variables related to blood draw technique. We assessed platelet activation by whole blood flow cytometry and investigated the effects of: (1) drawing blood into vacuum tubes or manually aspirated syringes, and (2) discarding the first drawn blood sample (discard tube). Platelet P-selectin expression and platelet-monocyte complexes were measured by flow cytometry under both basal conditions and following stimulation with 0.1, 1, or 10 µM ADP. Bland-Altman plots demonstrated agreement between results for vacuum tube and syringe-aspirated samples with an a priori-defined clinically relevant agreement limit of 5%. Agreement of results was also observed between discard tube and second draw samples for both vacuum-driven and manually aspirated blood. We conclude that a vacuum tube or a manually-aspirated syringe can be used when assessing platelet activation by flow cytometry and that there is no need for a discard tube.
Neutrophil CD64 expression – comparison of two different flow cytometry protocols on EPICs MCL and the Leuko64
Published in Scandinavian Journal of Clinical and Laboratory Investigation, 2015
Oskar Eriksson, Lena Douhan Håkansson, Malgorzata Karawajczyk, Daniel Garwicz
Objective. To evaluate the Trillium Diagnostics Leuko64™ assay on Abbott Celldyn Sapphire haematology analyser compared to two flow cytometry protocols on Beckman Coulter EPICS MCL flow cytometer. Materials and methods. CD64 expression on neutrophils was determined by two flow cytometry protocols and by a commercial assay on an automatic haematology analyser. The inclusion of study subjects was based on elevated procalcitonin (PCT) values, identifying patients where a systemic infection was suspected. Healthy blood donors were used as a reference group. Results. Statistically significant correlations between the Trillium Diagnostics Leuko64™ assay and the flow cytometry methods were found when measuring neutrophil CD64 expression. Conclusions. The good correlation between a reference method and an automated haematology analyser method for CD64 expression on neutrophils supports introduction of the latter assay for routine use as an independent biomarker of bacterial infection and inflammation.
High Throughput, Parallel Imaging and Biomarker Quantification of Human Spermatozoa by ImageStream Flow Cytometry
Published in Systems Biology in Reproductive Medicine, 2009
Clayton Buckman, Thaddeus C. George, Sherree Friend, Miriam Sutovsky, Antonio Miranda-Vizuete, Christophe Ozanon, Phil Morrissey, Peter Sutovsky
Spermatid specific thioredoxin-3 protein (SPTRX-3) accumulates in the superfluous cytoplasm of defective human spermatozoa. Novel ImageStream technology combining flow cytometry with cell imaging was used for parallel quantification and visualization of SPTRX-3 protein in defective spermatozoa of five men from infertile couples. The majority of the SPTRX-3 containing cells were overwhelmingly spermatozoa with a variety of morphological defects, detectable in the ImageStream recorded images. Quantitative parameters of relative SPTRX-3 induced fluorescence measured by ImageStream correlated closely with conventional flow cytometric measurements of the same sample set and reflected the results of clinical semen evaluation. Image Stream quantification of SPTRX-3 combines and surpasses the informative value of both conventional flow cytometry and light microscopic semen evaluation. The observed patterns of the retention of SPTRX-3 in the sperm samples from infertility patients support the view that SPTRX3 is a biomarker of male infertility.
Related Knowledge Centers
- Clinical Trial
- Cytometry
- DNA
- Hematology
- Cell Separation
- Cytophotometry
- Blood Cancer