Flow cytometry and fluorescence activated cell sorting (FACS)

Definition, Principles involved, Fluoroscence activated cell sorting (FACS), Interpretation of histogram, Applications of flow cytometry

1. FLOW CYTOMETRY Abu sufiyan chhipa m. pharm (pharmacology) I sem.

2. Flow cytometry  is a technology that is used to analyse the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Flow cytometry  is a laser- or impedance-based, biophysical technology employed in cell counting, cell sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them through an electronic detection apparatus.

3. Principle of Flow cytometry The basic principle of flow cytometry is the passage of cells in single line in front of a laser so that they can be detected, counted and sorted. A flow cytometer is generally composed of 3 components: The flow system(fluidics) The optical system(light sensing) The electronic system(signal processing)

4. The flow system The fluidics system includes a flow cell, where the sample fluid is injected. The flow cell requires sheath fluid to carry and align the cells or particles so that they pass through a narrow channel and into the laser intercept (light beam) in a single file. This hydrodynamic focusing allows the analysis of one cell at a time by laser interrogation .

5. The optical system  The optics system consists of various filters, light detectors, and the light source, which is usually a laser line producing a single wavelength of light at a specific frequency. This is where the particles are passed through at least one laser beam. Lasers are available at different wavelengths ranging from ultraviolet to far red. Interrogation by the laser beam excites any compatible fluorescent probes that are conjugated to antibodies, causing the probes to emit light (or fluoresce) at specified wavelengths.

6. The electronic system These light signals are converted by the electronics system to data that can be visualized and interpreted by software.

7. Fluorescence Activated Cell Sorting (FACS) Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry . It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

8. Procedure

9. Interpretation of histogram

10. Forward Scatter(FSC) Scattering of light by the cells in forward direction. Intensity of forward scattering is directly proportional to cell size.

11. Side Scatter(SSC) Light scattered at degrees to the axis of laser path is referred as side scatter. The intensity of side scatter is directly proportional to the amount of cytosolic structures(organelles ).

12. Applications of flow cytometry

13. Leukocyte Analysis As HIV disease progresses, CD4-positive T lymphocytes decrease in total number while CD38 is a protein whose expression increases upon activation in HIV infected individuals. The absolute CD4 count provides a powerful laboratory measurement for predicting and monitoring disease progression and response to treatment in HIV infected individuals.

14. DNA Analysis Flow Cytometry is used to differentiate malignant cells from their normal counterparts. The distinction between normal and leukemic bone marrow precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia. DNA aneuploidy generally is associated with malignancy; often correlates with many types of cancer such as multiple myeloma, and childhood acute lymphoblastic leukemia (ALL).

15. Enzymatic Deficiencies Flow cytometry is a tool for measuring Beta glucocerebrosidase activity in Gaucher’s disease and to study the abnormalities RBCs shape . Gaucher disease is caused by a deficiency of the enzyme glucocerebrosidase . Macrophages transform into pathogenic Gaucher cells following the phagocytosis of red blood cells (RBCs) and subsequentaccumulation of glucosylceramide .

16. Detection of minimal residual disease Minimal residual disease (MRD) is defined as disease beyond the limit of morphological detection using conventional microscopy. Patients with acute leukaemia were considered to be in remission when bone marrow samples contained <5% neoplastic cells. Flow cytometric methods can detect far lower levels of disease, which can be important in the clinical management of leukaemia .

17. Procedure Mononucleated cells are separated on a density gradient and labelled with three antibodies associated with the phenotype of the leukaemia  - CD19/CD34/CD13. One sample contains an isotype control in place of the CD13 antibody. The CD13 versus CD34 plot is gated on light scatter and the CD19, CD34 + ve cluster. 110,000 events were recorded; there were 62 cells in the region set on the isotype control and 2021 cells in the same region set on the positive sample.

18. Detection of Autoantibodies Autoantibodies to leucocytes, platelets and erythrocytes may be found in a variety of autoimmune conditions and can cause anaemia , leukopenia , or thrombocytopenia . Detected by immunofluorescence in either a direct or an indirect assay. In direct assay, anti-human Ig antibodies are used to detect Ig on the surface of the patient’s cells. In the indirect assay, the reaction of antibodies in the patient’s serum with cells from a normal person is observed.

19. Foeto -maternal haemorrhage Foeto -maternal bleeding can sensitise a Rhesus blood group D- ve mother to D+ve blood cells from the foetus . Haemolytic disease of the new born child can be caused by the destruction of Rhesus D + ve   blood cells of the foetus by maternal anti-D antibodies. Prophylactic anti-D given to the Rhesus D - ve mother shortly after delivery of a Rhesus D + ve  child significantly reduces the incidence of anti-D sensitisation in the mother. Since the dose of anti-D given is related to the size of the foeto -maternal haemorrhage , quantitation of foetal -maternal haemorrhage is therefore important.

20. Quantitation is achieved by labelling the erythrocytes in a sample of maternal blood with FITC-conjugated, non-agglutinating anti-D antibodies. A population of as few as 0.1% foetal cells is sufficient to sensitise the parent so at least 500,000 cells should be analysed to obtain a statistically significant estimation.  

21. Reticulocyte analysis Reticulocytes can be distinguished from erythrocytes by their high content of RNA. There are several stains that can be used for RNA. Ex. Thaizole orange