Flow cytometry is the “gold standard” for diagnosing paroxysmal nocturnal hemoglobinuria (PNH). In other words, it’s the main test that doctors use to determine if you have this condition.

Although other diagnostic tests can provide information about whether you have various blood disorders, flow cytometry can conclusively confirm or rule out PNH. This test leads to a definitive diagnosis by looking for specific types of proteins found on your red blood cells.

What Is Flow Cytometry?

Flow cytometry is an assay (a test that analyzes a substance) that shows details about cells within a sample.

All your cells have surface proteins that help them function properly. These proteins allow each cell to learn about its environment and communicate with other cells throughout your body.

Each type of cell tends to have a slightly different mix of proteins on its surface. Flow cytometry analyzes blood cells one by one, allowing detection of PNH proteins as well as other features of a cell, such as its size. This process can help show:

• The number of cells in a sample
• The type of cells in the sample
• The main job or role of each cell
• Signs of abnormalities or faulty functioning in cells

Health care providers most often use flow cytometry to analyze cells found in various fluids, such as in your blood or bone marrow (the soft tissue in your long bones). This procedure can help diagnose medical conditions that affect these cells.

How Does Flow Cytometry Work?

Before performing flow cytometry, your doctor will collect a sample of your cells. A laboratory technician will then prepare your cells by mixing them with monoclonal antibodies (molecules that recognize certain proteins) that are attached to fluorescent molecules. This way, each antibody acts as a glue, sticking a specific fluorescent label on a specific type of protein.

Next, the lab technician will load your cells into a machine called a flow cytometer. This instrument takes up your blood cells one at a time and shines lasers onto each cell. If the cell has fluorescent molecules attached to its proteins, it will glow, and the flow cytometer detects this light. In other words, the machine “reads” which proteins are found on the outside of the cell.

The flow cytometer uses other lasers to collect additional information regarding the sample’s cell population. The way the lasers reflect off the cell reveals two characteristics: how big the cell is and the amount of proteins, particles, and other contents. These details also tell the technician what type of cell they are looking at — for example, red blood cells are very small.

During a single flow cytometric test, the machine may collect information from hundreds of thousands or even millions of cells. All of this information is compiled into graphs that show, for instance, how many big versus small cells are in a sample or how many cells contain — and how many lack — a certain protein.

How Does Flow Cytometry Help Diagnose PNH?

Flow cytometry helps detect proteins that play an important role in PNH. Whether or not you have these proteins determines if you are diagnosed with this condition.

GPI-Anchored Proteins in PNH

PNH develops when your body makes red blood cells that are missing two proteins called CD55 and CD59. These proteins belong to a category called glycosyl phosphatidylinositol (GPI)-anchored proteins or GPI-linked proteins.

People without PNH have healthy red blood cells that contain these proteins. Both CD55 and CD59 prevent the red blood cells from being attacked by the complement system — a part of your immune system that helps kill germs and keeps your body safe.

PNH is caused by a mutation in a gene called PIGA, which prevents the body from making certain GPI-anchored proteins. This leads to the production of red blood cells that have either no CD55 and CD59 or less of these proteins than they should.

Getting a Flow Cytometry Test for PNH

In the case of PNH, flow cytometry is conducted with a sample of peripheral blood (blood that circulates throughout the body), which is taken from your arm as part of a normal blood draw.

Within a lab, a technician will mix your blood cells with antibodies that recognize CD55 and CD59. If your red blood cells contain these proteins, the antibodies will label them with a fluorescent molecule.

When PNH flow cytometry is performed, the cytometer will try to detect and measure the amount of fluorescence your cells are giving off. The test may show that all your blood cells are fluorescently labeled — they all have CD55 and CD59 — or that some lack these proteins. During this process, both your red blood cells and white blood cells may be analyzed.

Looking for a PNH Clone

Flow cytometry can group your cells into different categories. The test results can show how many of your cells fall into each group:

• Type 1 cells — Normal blood cells that contain all the proteins they are supposed to
• Type 2 cells — Blood cells that have lower levels of CD55 and CD59 than usual and are somewhat vulnerable to being attacked by the complement system
• Type 3 cells — Blood cells that don’t have any CD55 and CD59 and are likely to be destroyed

If you have PNH, you will likely have both type 1 and type 2 cells, although the amounts vary quite a bit from person to person. When you get your flow cytometry results back, they may say what percentage of your blood cells are PNH cells. This group of cells is also known as the PNH clone.

You may have a very small percentage of PNH cells and not get a diagnosis of PNH. If fewer than 10 percent of your blood cells lack CD55 and CD59, you’re unlikely to ever develop PNH symptoms because you have enough normal blood cells to keep you healthy.

When Might Your Doctor Use Flow Cytometry?

A couple of situations might prompt your doctor to order a flow cytometry test. This test is used during the initial process of diagnosis but may also be repeated as you continue to live with PNH.

If Your Doctor Suspects You Have PNH

Your doctor may want to perform flow cytometry to potentially diagnose PNH if you have:

• Signs of intravascular hemolysis (destruction of red blood cells in your blood vessels), such as dark urine or high levels of an enzyme called lactate dehydrogenase
• Certain PNH symptoms, including abdominal pain, bleeding problems, or swallowing difficulties
• Thrombosis (blood clots)
• Aplastic anemia, myelodysplastic syndrome (MDS), or other bone marrow failure syndromes (conditions in which your hematopoietic stem cells — cells that make all your blood cells — can’t produce enough healthy cells)

To Monitor People With Aplastic Anemia and Myelodysplastic Syndrome

People with bone marrow failure syndromes have a greater risk of developing PNH. Many people with these conditions have a small number of PNH cells but not full-blown PNH. If you have aplastic anemia or MDS, your doctor may recommend regular flow cytometry tests to look for signs you’re developing PNH.

To Check Your PNH Treatments Are Working

If you are diagnosed with PNH, your doctor may recommend follow-up visits and additional flow cytometry tests every six to 12 months. This helps your doctor see whether your condition is worsening and if immunosuppressive therapies like eculizumab (Soliris) are working.

During these regular tests, your doctor will look at your PNH clone size and compare it with previous flow cytometry results. If the number of cells that lack CD55 and CD59 is dropping, it’s a sign that your treatment plan is working — your body is producing more healthy blood cells than PNH cells.

Talk With Others Who Understand

On myPNHteam, the social network for people with paroxysmal nocturnal hemoglobinuria and their loved ones, members come together to ask questions, give advice, and share their stories with others who understand life with PNH.

Are you living with PNH? Did you have a flow cytometry test done while getting a PNH diagnosis? Share your experience in the comments below, or start a conversation by posting on your Activities page.