Cell population heterogeneity consistently emerges as an interesting and biologically relevant feature of biological systems. Nevertheless, many experimental tools are informative only about entire populations. While this level of inquiry is fundamental to our understanding of many phenomena governing the living world, only a limited number of approaches are available to dissect the differential behavior of selected individual cells within a group.
Among the techniques able to characterize cells from heterogeneous populations based on discrete properties, flow cytometry has experienced a significant expansion in recent years. At CHI’s flow cytometry meeting held recently in Boston, Peter O. Krutzik, Ph.D., senior research scientist in the department of microbiology and immunology at the Baxter laboratory of genetic pharmacology, Stanford University, talked about a new tool that he and collaborators recently developed.
“The phospho-specific flow cytometry platform enables us to analyze not only the surface markers of different cells but, at the same time, the phosphorylation level of intracellular proteins,” said Dr. Krutzik. “We can now perform complex biochemistry at the single-cell level in primary cell populations.”
Intracellular signaling pathways are perturbed in specific ways during disease states such as malignant tumors, viral infections, or autoimmune conditions, and surveying phosphorylation changes at the single-cell level by flow cytometry provides diagnostic, therapeutic, and prognostic benefits. “Not only can we stratify patients but, for the same patient, or for a particular tumor sample, we can identify heterogeneous responses that might point to different progenitor cells leading to that tumor,” he explained.
In addition, this approach could help characterize different signaling responses such as the ones correlating to more aggressive behaviors. “This also enables us to identify patients who could have a subset of cells that is more resistant to classical treatment and who might immediately require more robust or second-stage therapy.”
During high-throughput flow cytometry experiments, the time needed for data acquisition and the high cost of antibodies represent two of the major limiting factors.
One way to address these limitations is by combining several samples into one tube, a technique known as multiplexing. Dr. Krutzik and collaborators recently developed a multiplexing technique, known as fluorescent cell barcoding, in which each individual sample is treated with different concentrations and combinations of fluorescent dyes that covalently attach to cellular proteins and establish specific fluorescence signatures, conferring thus an additional dimension.
For example, 64–216 different signatures can be generated with only three fluorophores. When used for a 96-well plate, fluorescent cell barcoding decreased antibody costs 100-fold and reduced the acquisition time to 5–15 minutes, while providing excellent resolution.
“The cytometer is no longer a bottleneck,” emphasized Dr. Krutzik. “Fluorescent cell barcoding enables us to perform larger screens that we typically would not have done before. In addition, the data is quantitatively more robust because we can include both positive and negative controls in the same tube with the drug-treated samples.”
At the same meeting, Jonathan R. Fromm, M.D., Ph.D., assistant professor of laboratory medicine and associate director of the hematopathology laboratory at the University of Washington, talked about recent advances in using flow cytometry to detect classical Hodgkin lymphoma. Traditionally, the diagnosis of this condition has relied on the use of immunocytochemistry, a technique in which paraffin-embedded tissue sections are stained and examined microscopically for the presence of specific cellular proteins.
A few years ago, Dr. Fromm and collaborators developed the first flow cytometry-based approach and were able to reliably identify Hodgkin and Reed-Sternberg cells, the anatomopathological hallmark of this hematopoietic malignancy. Guided by previous observations, which reported that Hodgkin and Reed-Sternberg cells appear surrounded by T cells in paraffin sections, the authors hypothesized that this phenomenon, known as “rosetting”, could explain the difficulties accompanying their flow cytometric detection.
When mixtures of Hodgkin lymphoma cell lines and T lymphocytes were examined by flow cytometry, the investigators noticed that, although the cells looked like the original cell lines, they were also expressing T cell-specific markers such as CD3 and CD45, suggesting that they were binding to surrounding T cells.
Adhesion molecules were another antigen class observed on the cell surface, and by preincubating the cells with monoclonal antibodies directed against them, the authors were able to disrupt these interactions. When used on clinical samples, this approach allowed the identification of malignant cells with a sensitivity of 89% and a specificity of 100%, Dr. Fromm said. “In no case did we see a Hodgkin lymphoma cell population that was not Hodgkin lymphoma, suggesting that this could potentially be a clinically useful assay.”
More recently, Dr. Fromm and collaborators developed a single-tube, nine-color flow cytometry assay. “We looked at 420 tissues that were independently evaluated by morphology and examined the data without knowing what the morphology shows.” The assay sensitivity and specificity were similar to the ones reported in the previous study, and in October 2007 the authors started routinely using this approach as a diagnostic tool for classical Hodgkin lymphoma in the clinical laboratory.