Whole Blood Flow Cytometry: Principles and Applications

Whole blood flow cytometry is an analytical method that examines the physical and chemical properties of individual cells within a blood sample. The technique analyzes thousands of cells per second, allowing for the rapid identification and quantification of diverse cell populations. This provides a quantitative assessment of a heterogeneous mixture of cells directly from the sample.

Understanding Whole Blood Flow Cytometry Principles

The instrument’s fluidics system uses hydrodynamic focusing, where a central stream of the sample fluid is injected into a faster-moving outer stream of sheath fluid. This narrows the sample stream, forcing cells to align in a single file before they intersect the laser. This precise alignment ensures each cell is individually illuminated and measured.

When a cell crosses the laser beam, it scatters the light in multiple directions. Light scattered in the forward direction, known as forward scatter (FSC), is primarily related to the cell’s size. Light scattered at a 90-degree angle, called side scatter (SSC), is influenced by the cell’s internal complexity or granularity. For example, a lymphocyte is relatively small with a non-granular cytoplasm, resulting in low FSC and low SSC, while a neutrophil is larger and contains many granules, leading to higher FSC and much higher SSC.

In addition to light scatter, the technique measures fluorescence. Cells can be labeled with fluorescent dyes or antibodies chemically linked to fluorescent molecules called fluorochromes. These antibodies bind to specific proteins, or markers, on or inside the cell. When a labeled cell passes through the laser, the fluorochromes are excited and emit light at a specific wavelength, which is then detected, allowing for identification based on protein expression.

Using whole blood for this analysis examines cells in a state closer to their natural physiological condition, minimizing changes from extensive sample processing. The instrument’s fluidics, optics, and electronics work together to convert light signals from each cell into digital data for a detailed, multi-parameter analysis.

Preparing Whole Blood Samples for Cytometric Analysis

The process of preparing a sample begins with the collection of blood into tubes containing an anticoagulant, such as EDTA or heparin, to prevent clotting. The choice of anticoagulant can influence the results, so consistency is important for longitudinal studies or when comparing different samples.

The abundance of red blood cells (RBCs), which can outnumber leukocytes by about 600 to 1, can interfere with the analysis of white blood cells. To address this, most protocols employ a lysis step to selectively destroy RBCs while leaving leukocytes intact. A common method uses an ammonium chloride-based lysis buffer, often performed after antibody staining in a “lyse/no-wash” protocol to simplify the procedure and reduce cell loss.

For some applications, particularly those focused on platelets or certain rare cell types, a “no-lyse” protocol might be preferred to avoid any potential impact of the lysis buffer on the cells of interest. In these cases, smaller sample volumes are used, or specific triggers are set on the instrument to focus only on the target cell populations. The decision between lysis and no-lyse methods depends on the specific cells being investigated and the experimental question.

Staining cells with fluorescently-labeled antibodies involves adding an antibody cocktail to the whole blood sample and incubating it in the dark. Each antibody must be titrated to determine the optimal concentration for a bright signal with minimal background staining. A blocking step is also frequently included to prevent antibodies from binding non-specifically to certain immune cells.

Key Applications in Diagnostics and Research

One of the most common uses for flow cytometry is in hematology-oncology for the immunophenotyping of leukemia and lymphoma. By analyzing the unique combination of cell surface markers on cancerous cells, clinicians can accurately diagnose the specific type of blood cancer, determine prognosis, and monitor the patient’s response to therapy.

Another diagnostic application is monitoring patients with Human Immunodeficiency Virus (HIV). The progression of HIV is tracked by measuring the number of CD4+ T-cells, a type of lymphocyte that is the primary target of the virus. Regular counting of these cells helps guide treatment decisions, and the technique is also used to monitor immune system recovery in patients after hematopoietic stem cell transplantation.

The technique can be used for the detection of fetal red blood cells in a maternal blood sample to assess the risk of hemolytic disease of the newborn. It is also employed in platelet function tests to evaluate bleeding disorders or monitor the effects of anti-platelet medications.

Immunologists use it to study the immune system, such as analyzing the activation states of different immune cells in response to infection or vaccination. Researchers in infectious diseases can use it to detect parasites within blood cells or to characterize the host’s immune response. In drug discovery, it serves to validate that a new drug is engaging its intended cellular target and to measure its downstream effects on various cell populations, known as pharmacodynamic studies.

Interpreting Data from Whole Blood Flow Cytometry

After data acquisition, the information is displayed in plots for interpretation through a process called gating. Gating involves selecting a cell population on a data plot based on its light scatter or fluorescence characteristics. This isolates the selected population for further analysis.

Analysis begins with a dot plot of forward scatter (FSC) versus side scatter (SSC) to identify the main leukocyte populations. Based on their scatter properties, lymphocytes (low FSC/SSC), monocytes (intermediate FSC/SSC), and granulocytes (high FSC/SSC) can be distinguished as distinct populations.

Once these primary populations are identified, further sub-gating can be performed using fluorescence data. For example, after gating on the lymphocyte population based on scatter, a new plot can be created to look at the fluorescence of markers that distinguish different lymphocyte subtypes. A plot of CD3 fluorescence versus CD19 fluorescence would separate T-cells (CD3 positive) from B-cells (CD19 positive). This hierarchical gating strategy allows for the quantification of very specific subpopulations.

When using multiple fluorochromes, spectral overlap can occur where fluorescence from one dye spills into another’s detector; this is corrected through a process called compensation. Interpreting data also requires excluding artifacts, like debris or platelet clumps, through careful gating. Data is presented as dot plots (two parameters) or histograms (one parameter).