Flow cytometry is the “go-to” test for diagnosing PNH. PNH happens because of a change (mutation) in the PIG-A gene of a subset of stem cells in bone marrow. This gene controls the creation of substance that helps certain proteins to stick to the surface of blood cells. Therefore, any blood cells created by this mutated bone marrow stem cell are abnormal because they can be destroyed by the complement system, which is a component of our immune system.
Using light scattering techniques and fluorescence, flow cytometry can show your doctor if any proteins are missing from the surface of blood cells. In PNH, cells are missing from some or all of two proteins (CD55 and CD59) on the surface of red blood cells, which causes the cells to break apart. This process is known as hemolysis.
Relying on CD-55 and CD-59 deficient red cells may be problematic in assessing clone size, as they can hemolyze and their percentage altered by transfusions. Therefore, it is important to identify PNH clones in either granulocytes or monocytes (certain types of white cells that are not affected by hemolysis). This is done by utilizing a special substance known as FLAER, which recognizes the missing anchor on the surface of those cells allowing the flow cytometer to identify and quantitate such cells providing an accurate estimate of the PNH clone size.
Flow cytometry is also able to quantitate the size of PNH clones to help physicians predict characteristics of the disease such as the severity of symptoms, and risk of thrombosis (blood clots). This fine level of detail provided by flow cytometry allows your healthcare provider to assess the presence and severity of PNH.
Timeline and Development of Flow Cytometry*
- 1930s: Andrew Moldavan designed a photoelectric apparatus to count individual cells flowing through narrow tubes which was mounted on a microscope stage.
- 1950s: Wallace Coulter began the development of the first instrument that could electronically calculate cell volume which became known as the Coulter Counter. Until this day, it is still used in hematological cytometers.
- 1965: Mark Fulwyler invented the forerunner to today’s type of flow cytometers, and utilized the Coulter Counter method, the cell sorter and included findings on fluidic jets and lessons learned from an inkjet printer to create the first cell sorter. After cells passed through the Coulter method, the stream of cells would be broken up into droplets. These droplets could be charged and deflected into separate containers.
- 1969: Len Harzenberg from Stanford University combined Fulwyler’s method of sorting technology with a fluorescence detector. This allowed for the detection of fluorophore-conjugated antibodies (targeting a specific antigen with a fluorescent dye) and the rapid phenotyping (physical characteristic or expression) of immune cells and fluorescence-activated cell sorting (FACS).
How is Flow Cytometry used in PNH?
Using flow cytometry, your doctor can usually divide your blood cells into 3 types.
- PNH I cells or Type I cells
These cells respond in a healthy way to the complement system (A group of proteins that move freely in the bloodstream and support (complement) the work of white blood cells by fighting infections.). They are normal cells.
- PNH II cells or Type II cells
These cells are partially sensitive to the complement system. They are missing some of the proteins that protect them from attack.
- PNH III cells or Type III cells
These cells are extremely sensitive to the complement system. Of the three groups of cells, these ones break apart (hemolyze) most easily. They are missing all the proteins that protect normal cells from attack.
4. GPI- deficient granulocytes and monocytes
Granulocytes and monocytes that are lacking the GPI anchor can be identified by using FLAER while analyzing flowcytometric data for better estimation of the PNH clone size.
Since there is a well-known association between aplastic anemia and PNH, flow cytometry is also used with aplastic anemia patients for detecting a PNH clone. All patients that are diagnosed with aplastic anemia should be tested for PNH as they are susceptible in developing PNH clones.
*Burger, Stefanie. Introduction to Flow Cytometry. Institute of Molecular Biology. https://www.imb.de/fileadmin/imb/core_facilities/Lectures/MTiLS_Lecture_Cytometry_2018_sec.pdf