The present invention relates generally to methods and systems for performing flow cytometry, and more particularly to such methods and systems that allow performing in-vivo flow cytometry in a live subject without extracting blood.
Many current techniques for detecting and quantifying various cell types circulating within a subject require extracting a blood sample from the subject followed by analysis of the extracted blood. For example, one such conventional method includes withdrawing a blood sample, fluorescently labeling specific cell populations ex vivo with antibody-targeted fluorescent markers, and passing those cells through a flow stream to be interrogated by a light source (typically a laser). Fluorescence and light scattering signals emitted, or reemitted, by the cells in response to the interrogating light can be employed to determine the types and the number of cells. Automated hematology analyzers can separate and count blood cell populations based on their size/volume characteristics in addition to their light scattering properties. Another blood analysis method, known as hemocytometry, is based on counting cells against a grid while being viewed with a microscope.
These techniques, though useful, require invasive blood withdrawal and hence are not particularly suited for continuous, real-time monitoring of the circulation. Further, blood withdrawal can be difficult in certain patient populations, e.g., children and psychiatric patients. In addition, such blood withdrawal can expose patients to various risks, such as infection and/or anemia. For example, in patients with low blood volume (e.g., newborns and premature infants), blood loss due to phlebotomy for common tests can equal as much as 45% of the total blood volume. Hence, diagnostic blood withdrawal is among the leading causes of anemia in neonates, which can lead to the need for frequent transfusion or erythropoietin treatment. Further, for monitoring patients for potential infection or in diseases such as leukemia and other types of cancer, frequent monitoring of the white blood cell count (WBC) can be beneficial but is currently not practical due to the frequency of blood withdrawal required. Additionally, blood withdrawal can be dangerous in immune-compromised patients, such as AIDS patients, transplant patients, or patients undergoing chemotherapy, as the skin penetration for extracting blood carries an inherent risk of infection.
Some methods for performing in-vivo flow cytometry have also been proposed previously. These methods typically utilize fluorescent antibodies or fluorescent proteins, such as green and red fluorescent proteins (e.g., GFP and DsRed), for labeling cells. However, fluorescent antibodies and fluorescent proteins are not approved for human use and require either genetic manipulation or intravenous injection. As such, the applicability of such methods can be limited.
Accordingly, there is a need for improved methods and systems for performing flow cytometry in vivo, and particularly for such methods and systems that would allow non-invasive, real-time, continuous monitoring of cell populations in circulating blood.