What to know about CLL flow cytometry

According to the World Health Organization (WHO), the diagnosis of CLL largely depends on identifying leukemic cells under a microscope by looking for specific toxins on the cell’s surface.

A 2021 paper states that CLL typically displays white blood cell markers or antigens on the surface of cells. The presence and absence of such antigens — which experts sometimes call clusters of differentiation (CD) markers — allows for a definitive CLL diagnosis.

However, relying on a cell’s appearance alone may prove difficult to differentiate CLL from other diseases, such as mantle cell lymphoma, a disease with similar microscopic properties.

Flow cytometers are instruments that determine the properties of cells or particles, one cell at a time. It provides information about:

• cell size
• cell structure
• the type and amount of specific surface receptors
• total DNA
• intracellular proteins
• DNA gene expression
• newly-synthesized DNA
• transient signaling

Each cell has specific groupings of antigens that determine the cell it came from — the cell lineage — and how far it is along the process of changing from one cell type to another, which healthcare professionals refer to as cellular differentiation. Flow cytometry detects the presence of these antigens on a cell’s surface.

Before testing, a medical practitioner obtains a sample of a person’s blood or bone marrow. They treat samples with biological stains, known as fluorochromes, highlighting specific cellular markers when “excited” by lasers. After preparation, the sample dilutes in a salt-based solution that resists pH changes, and lab technicians place the diluted sample on a narrow tube. They then run the sample through a laser or multiple lasers one at a time.

Detectors read the scattered and fluorescent light that appears once cells pass through laser beams and convert the signals into data for a doctor or healthcare professional to interpret.

Regular cells display a pattern of antigens or molecules on their surface that determine their type and maturity. These antigens have CD numbers for naming purposes.

Results from the flow cytometry show the detected CD numbers, which doctors use to compare to regular and irregular cells, allowing them to form a diagnosis.

Based on a 2018 study, the minimum marker sets that doctors look for in CLL diagnosis include CD5, CD19, CD23, CD20, Kappa, and Lambda.

Results can also determine a person’s prognosis. A 2020 study found that CD38 and D49d are prognostic markers that doctors can find in almost half of the individuals with CLL, while in a 2021 study, expression of ZAP-70 has associations with a more aggressive form of CLL.

A doctor will first perform a thorough physical examination and look at a person’s medical history if they have symptoms of CLL. They will typically check for swollen lymph nodes, tenderness in the abdomen, and other symptoms, including:

• chills
• feeling tired
• fever
• night sweats
• weight loss
• weakness

While signs and symptoms may already suggest CLL, doctors will usually request more than flow cytometry. Other tests may include:

• CBC with differential: Measures the number of blood cells, including red blood cells, platelets, and various white blood cells.
• Peripheral blood smear: Looks for the remains of abnormal white blood cells typically found in the lymphatic system, called smudge cells or basket cells, which have no clear cell boundaries. Numerous smudge cells have links to leukemia.
• Morphology: Specialists, such as pathologists, hematologists, and oncologists, may look at a person’s bone marrow sample under a microscope to check cell size and structure, which can aid in classifying leukemias.

There are other relevant tests that healthcare professionals perform for CLL monitoring and treatment purposes. They include the below.

• Bone marrow test: Helps rule out other diseases during the diagnostic stage and checks the progression of the CLL. Doctors also use this test during treatment to monitor its effectiveness.
• Beta-2 microglobulin blood levels: High levels of this blood protein indicate a more advanced CLL. A 2020 study found that beta-2 microglobulin, along with interleukin-4 and interleukin-6, suggests a less favorable CLL prognosis.
• Blood immunoglobulin levels: Measures the antibodies in a person’s blood. Since CLL cells affect B-cells that create antibodies, an individual with CLL often has low immunoglobulin, putting them at a higher risk of acquiring an infection.
• Cytogenetics: Requires growing a cell — whether blood, bone marrow, or another tissue — in a lab for study under a microscope to detect chromosomal changes.
• Fluorescent in situ hybridization: This chromosome test uses bone marrow or blood samples to look for specific genes or chromosomal changes. It uses specialized fluorescent dyes that attach to particular parts of certain chromosomes.

A doctor will consider the results of all tests along with a person’s medical history and symptoms to make a diagnosis.

The results help healthcare professionals determine CLL, its stage, and the progression of the cancer. These findings can also indicate which cancer treatment is the most suitable and how it might be effective.

Flow cytometry is one of the tools that doctors use to diagnose CLL in individuals. It uses technology to find antigens in cells that characterize the condition to determine the type of CLL a person has.

Medical practitioners use flow cytometry results in conjunction with other blood tests, an individual’s medical history, and other signs and symptoms to determine treatment options and prognosis.