Scattered Light And Fluorescent Emissions From A Single Cell Biology

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The development of biotechnology has become widespread in clinical laboratories for several purposes and one example of its progress is flow cytometry, which has the ability of analysing numerous features of single cell in a short period of time, thus accelerating the diagnosis of diseases and treatment monitoring.

There are two types of scattered light: forward scattered (FSC), which is detected by the forward detector at a slight angle in the forward direction and thus is proportional to the cell size and surface, and side scattered (SSC), which is reflected or refracted by the intracellular components when passed through the laser beam, which provides information about the cell contents (Macey 2007). Flow cytometry can also measure fluorescent molecules either by labelling cells with fluorescent antibodies to bind specific proteins or by staining cellular contents with fluorescent dyes. This method allows us to recognize cell types, specific receptors and cell components (OrmerodĀ 2008).

The method of Flow cytometry starts by suspending cells in a stream of fluid and directing them one by one to the integration point. At this point, a laser beam is focused on one particular cell, scattering light in all directions. These light emissions are then collected by lenses, followed by routeing them to specific filters and dichroic mirrors, which direct them to the appropriate detectors, according to the particular wavelengths. Photodetectors translate these light signals into electronic signals and computers are then used to exhibit the results in histogram or dot plot formats (Brown and Wittwer 2000). Figure.1. illustrate the typical principle of flow cytometry.

Figure 1: Schematic overview of a typical flow cytometer setup

Flow cytometry can be designed with a sorting future which has the ability to separate target cells after identifying them by the electronic gate. This isolation is based on the electronic system which is preselected to capture those cells and by applying electronically charged droplets before moving them to the positive and the negative charged plates. These plates deflect the desired cells into appropriate collection tubes for further analysis, whereas unwanted cells move to the waste container (Introduction to Flow Cytometry, 2000).


There are many applications of flow cytometry in clinical laboratories. For example, In cell biology it is used to measure DNA content by staining the cell with fluorescent dyes, such as propidium iodide (PI), which bind the DNA so that it produces a strong fluorescent signal when excited by the laser beam at a suitable wavelength. As a result, the amount of DNA is proportional to the fluorescent signal. In addition, flow cytometry can analyse the distribution of cell cycle phases and DNA ploidy (OrmerodĀ 2008), and can also distinguish apoptotic cells from necrotic cells, resulting in an increase of FSC light in the necrosis and a decreases in the apoptosis (Vermes, Haanen and Reutelingsperger 2000).

Another interesting application of flow cytometry is related to clinical microbiology. In this field, flow cytometry can facilitate the identification of microorganisms, the determination of antibiotics and the performance of microbial growth (Alvarez-Barrientos et al. 2000). Besides this, flow cytometry has great potential for use in the transplantation of organs; for instance, the detection of anti-HLA antibodies and the monitoring of patients after organ transplantation (Zou et al. 2006) .

Immunophonotyping is the largest application of flow cytometry in clinical laboratories. It is dependent on the ability of specific monoclonal antibodies labelled with fluorochrome, to attach specific antigens onto the cell surface. This binding can be detected by flow cytometry to facilitate the diagnosis and the classification of various diseases such as leukemia and lymphoma. Moreover, it can facilitate the prognosis of the disease and observe the progress of patients, who are treated with chemotherapy or bone marrow transplantation (Brown and Wittwer 2000)

The most significant use of flow cytometry for immunophenotyping is associated with the area of hematology. In this field, flow cytometry has been used to analyse several hematological disorders such as leukaemia, lymphoma, paroxysmal nocturnal hemoglobinuria (PNH), HIV infection, foeto-maternal haemorrhage, enumeration of stem cell and reticulocytes (Bakke, 2000).

The beneficial effect of using flow cytometry in clinical hematology is that it has the ability to identify acute lymphoid leukemia and classify it into subgroups according to CD markers. For instance, CD3 and CD7 are positive in T-ALL, while CD79 and CD19 are positive in B -ALL (Orfao et al.1995). Moreover, the classification of AML is awkward by using phenotyping only, while the differentiation between AML is useful when using flowcytometry (Brown and Wittwer, 2000).

Another of its features is related to the deficiency of glycosylphosphatidylinositol linked proteins which lead to PNH disease. This disease has an abnormal deficiency in CD55 and CD59 markers which can be detected by flow cytometry and thus identify the disease. What is more, flow cytometry can monitor immunocompromised patients by counting their CD4 T-cells and comparing them with known samples (Tkachuk and Hirschmann, 2007), and it also provides the accurate amount of fetal blood cells found in a maternal blood sample, in contrast with the kleihauer betke test (KBT) (Porra et al. 2007).

Flow cytometry has great potential to enumerate stem cells in peripheral blood. Normally, stem cells do not appear in the blood, but after stem cell transplantation the production of CD34 stem cells increase and are released in the peripheral blood. Consequently, flow cytometry can identify CD34, stain it and obtain the percentage of CD34 cells. Moreover, it can estimate the percentage of reticulocytes in the blood by detecting the fluorescent dye, which binds to the residual RNA in the reticulocytes. In addition to this, it has the ability to distinguish platelets from other small particles by labelling them with a phycoerythrin- conjugated CD41 antibody (Bakke, 2000).

Despite the benefits of flow cytometry in the diagnoses of various haematological diseases, it is unable to diagnose Hodgkin's disease. This is due to the presence of malignant cells in the fibrous tissue which are difficult to separate them into a single cell and so the immunohistochemistry technique is used to detect this disease instead (Stetler-Stevenson 2003).


Flow cytometry is one of the most significant techniques to have been used in clinical laboratories to analyse multiparameters of single cells in a short period of time and therefore has been applied in many clinical laboratories for diagnoses, prognoses and for monitoring therapeutic responses. (1110 words)


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