A technologist at Trumbull Labs fills each vial with cells for entry into the flow cytometer
A blood sample arrives at the lab where the cells are sorted, cleaned and stained with fluorescent antibodies through a series of processes that take about 90 minutes. These stained cells are being tracked to determine if a patient's lymphoma is retreating after chemotherapy. A medical technologist enters the sample into the flow cytometer, a machine that identifies the characteristics of cells using optics and lasers. Thirty seconds later the analysis cascades across the computer screen in the form of cell clusters represented by various colors like red, green and blue.
Flow cytometry can interrogate 10,000 cells per second from one sample, making determinations on various cell distinctions – specifically focused on the antibodies attached to cells.
"The number of cells that can be read by the cytometer is just phenomenal," said Bonni Hazelton, PhD, flow cytometry supervisor for Trumbull. "It not only tells you all the antibodies that are bound, it can tell you the size of the cell and if it's granular or not."
The technology behind flow cytometry is based on the principle of hydrodynamic focusing, which uses a combination of fluidics, photo multiplier tubes, lasers, prisms and filters. The fluorescently stained sample is injected into the center of a sheath flow, and the fluidics literally forces the cells into a single-file line, allowing the machine to focus on one cell at a time. Once the laser hits the cell, light scatters and the cytometer recognizes the emission wavelengths, explained Hazelton. After the optics capture the emitted fluorescent light, the signal is then switched from analog to digital and the information can be viewed on a computer workstation. All quality controls are done by the computer which produces a daily quality report.
Flow cytometry is used primarily for blood diseases like leukemia, lymphoma and multiple myeloma, and help pathologists monitor a new or recurring diagnosis. The most common specimens include single cell suspensions from bone marrow, blood, tissues and other bodily fluids.
"We can see the changes in the profile of chemo patients and see if they're getting better or if it's having no effect," noted Hazelton. "That helps the doctor control the regimen and make adjustments (if needed)."
No two patient samples are the same. The flow cytometer can apply its search to numerous applications, whatever it's told to look for – specific populations of cells, specific diseases, and specific cells.
Ultimately, flow enables scientists and physicians to peer deeper into the cell and pick up what microscopes cannot, minimal residual disease. It can even observe apoptosis.
Explained Hazelton, "There are some diseases that we can't even see by staining the surface of the cells. What we can do is permeablize the membranes and use intracellular markers."
Flow cytometry is a compliment to the other testing performed in their labs. A pathologist's interpretation accompanies each flow cytometry report and is used as additional information toward making a diagnosis. For example, if a doctor investigates a lymph node, Hazelton said, and develops a theory after looking at 1,000 cells on a slide, the sample can be sent for flow cytometry for a blood panel.
"They are relying on that information to (confirm) what they already saw," added Hazelton.
Trumbull Laboratories was founded by the Pathology Group of the Midsouth (PGM) in 1998, bringing together doctors, technologists and a wide array of lab professionals under one roof. PGM/Trumbull specializes in cytology, histology, immunohistochemistry and flow cytometry. Trumbull Labs also offers research support to technology companies by providing product testing and analysis.
The combined relationship of physicians and scientists has enhanced their ability to produce timely results in the interpretation and diagnosis of diseased tissue and fluids. About 300,000 slides are reviewed annually at PGM/Trumbull.
Another use for flow cytometry at Trumbull is stem cell identification which they do in partnership with the stem cell transplant program at Baptist Memorial Hospital-Memphis. After stem cells are collected, the flow cytometer determines the density of CD-34 positive population, which is the marker for stem cells.
"We'll run a sample here and give them a percentage or number of stem cells on the product, usually based on the patient's weight," explained Hazelton. "We can tell them how the collection went, how many stem cells are in that collection and then they can determine whether they need to make another collection."
All patients in the stem cell program collect their own cells and Trumbull provides the monitoring. Patients are seen prior to harvest to determine if they have enough stem cells to collect.
Hazelton said they are currently looking to expand flow cytometry to include fetal hemoglobin, for maternal care. Eventually, she added, flow could be used for prenatal screening of leukemia and sickle-cell disease.