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Immunophenotyping with flow cytometry


Medical editor Bjørn Risberg MD
Pathologist
Oslo University Hospital

General

Flow cytometry immunophenotyping is an important link in the evaluation and treatment assessment of lymphoma and leukemia. The test provides a quick and quantitative analysis of a cell population, cell line origin, stage of differentiation, maturition, and biological activity.   

Flow cytometry immunophenotying is a method for measuring physical and chemical properties of cells and particles in a stream of fluid. Within immunology, flow cytometry is more commonly used, since it can be used to classify different blood cells. DNA measurements are also used. Evaluation of results requires special knowledge and expertise.

Indications

  • Suspicion of lymphoma
  • Suspicion of leukemia
  • Response evaluation of treatment for lymphoma or leukemia

Goal

  • Confirm or exclude malignant disease
  • Be a contributing factor for determining which treatment plan the patient should follow. 
  • Diagnose disease recurrence

Definitions

Flow cytometry

The cells are in a suspension, which are led individually through a nozzle where they are illuminated with a laser. The fluorochromes (substances which fluoresce) are excited by the laser and emit light. This emitted light is transmitted by a lens and filter system to a detector which amplifies and translates the electrical pulses to numerical values.

Knowledge of how the cells spread light after illumination in the flow cytometer allows us to differentiate the cells from each other. Multiple parameters are measured simultaneously: unique fluorescence as well as degree and direction of light spreading. Low angle (FSC) and wide angle (SSC) light spreading indicate the size and complexity of the cell.

The computer stores data of DNA content for every cell passing the laser, so that diagrams are made which provide a precise overview over the number of cells with a certain amount of DNA. In this way, it is possible to see how many cells are found in different phases of the cell cycle.

Immunophenotyping

Blood contains many cell types with different functions. These cells originate from multi-potent stem cells from bone marrow. Classification of leukemia and lymphomas are determined to a large extent by the antigen expression of the cell surface.  

The cells are labeled with fluorochromes attached to monoclonal antibodies which are targeted for antigens on cell surfaces or in cytoplasm. By utilizing fluorescent monoclonal antibodies against the surface antigens, it is possible to identify immunophenotypes expressed on leukemic cells but not on normal cells.

Minimal Residual Disease (MRD)

Flow cytometry is a sensitive method used to identify one leukemic cell among 10,000 normal cells. It is used for surveillance of treatment effect when the risk for recurrence of leukemia is under assessment. If the blast cells are under 5%, the disease is considered to be in morphological remission. 

Due to chemotherapy-resistant blast cells which remain in very small amounts in bone marrow, some patients will have recurrence of the disease. The probability of this is associated with the number of blast cells during and after treatment.

These blast cells are called MRD. Patients having an MRD level < 0.01% after treatment have a better prognosis than those with a level of ≥ 0.01 %. Flow cytometry is a useful examination for assessing the risk for recurrence of the disease.

Cancer cells usually have an abnormally high DNA content. Having information about this is of prognostic importance. It appears that tumors and leukemia cells with abnormal DNA content have a poorer prognostic impact than normal DNA content.

DNA flow cytometry

DNA flow cytometry can be used to measure DNA of cell nuclei, which has a prognostic value.


Preparation

An aspirate is taken of: 

  • peripheral blood
  • bone marrow 
  • cell suspension from lymph nodes

Implementation

  • A smear of the test material is made and stained for a microscopic examination.
  • The specimen is washed with physiological saline to remove plasma proteins which can interfere with antibody-antigen binding.  

From the microscopy assessment, clinical information and patient history, it is determined which combinations of antibodies will be used for staining in each case.

  • The red blood cells are hemolysed leaving a concentrate consisting of only white blood cells.
  • The specimen is analyzed by the flow cytometer.
  • The raw data are analyzed using dedicated software.

Follow-up

  • The result from the analysis is assessed by the treating doctor.
  • The result is available after a few hours up to a few days, depending on the complexity of the analysis.

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