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Flow Cytometry-Clinical Applications

Flow Cytometry-Clinical Applications

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Beckman Coulter Portfolio
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Published by: candiddreams on Jul 03, 2014
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Flow Cytometry
low cytometry is a technique of quantitative single cell analysis. The flow cytometer was developed in the 1970’s and rapidly became an essential instrument for the biologic sciences. purred by the !"# pandemic and a plethora of discoveries in hematology$ speciali%ed flow cytometers for use in the clinical laboratory were de&veloped by several manufacturers. The ma'or clinical application of flow cytometry is diagnosis of hematologic malignancy$ but a wide variety of other applications e(ist$ such as reticulocyte enumeration and cell function analysis. )resently$ more than *0$000 'ournal articles referencing flow cytometry have been published. This brief review of the principles and ma'or clinical applications of flow cytometry may be supplemented by several recent review articles and boo+s.The technique of analy%ing individual cells in a fluidic channel was first described by ,allace -oulter in the 190s$ and applied to automated blood cell counting. ubsequent developments in the fields of computer science$ laser technology$ monoclonal antibody production$ cytochemistry$ and fluorochrome chemistry led to the development of the flow cytometer two decades later. /ecause the first commercial flow cytometers were large$ comple($ e(pensive$ and difficult to operate and maintain$ they were primarily used in the research laboratory. !owever$ the enormous value of the flow cytometer in the medical and biologic sciences was quic+ly appreciated$ and its cost and comple(ity gradually decreased as its analytic capability increased.
The present state&of&the art2 flow cytometers are capable of analy%ing up to 13 parameters 4forward scatter$ side scatter$ 11 colors of immuno&fluorescence5 per cell at rates up to 100$000 cells per second. 6utomation and robotics is increasingly being applied to flow cytometry to reduce analytic cost and improve efficiency.
asic Principles of Flow Cytometry
)repared single cell or particle suspensions are necessary for flow cytometric analysis. #arious immunoflurescent dyes or antibodies can be attached to the antigen or protein of interest. The suspension of cells or particles is aspirated into a flow cell where$ surrounded by a narrow fluid stream$ they pass one at a time through a focused laser beam. The light is either scattered or absorbed when it stri+es a cell. 6bsorbed light of the appropriate wavelength may be re&emitted as fluorescence if the cell contains a naturally fluorescent substance or one or more fluorochrome&labeled antibodies are attached to surface or internal cell structures. ight scatter is dependent on the internal structure of the cell and its si%e and shape. Fluorescent substances absorb light of an appropriate wavelength and reemit light of a different wavelength. Fluorescein isothiocyanate 4F"T-5$ Te(as red$ and
phycoerythrin 4)85 are the most common fluorescent dyes used in the biomedical sciences. ight andor fluorescence scatter signals are detected by a series of photodiodes and amplified. :ptical filters are essential to bloc+ unwanted light and permit light of the desired wavelength to reach the photodetector. The resulting electrical pulses are digiti%ed$ and the data is stored$ analy%ed$ and displayed through a computer system.
7$ ;
The end result is quantitative information about every cell analy%ed 4Fig. 15. ince large numbers of cells are analy%ed in a short period of time 4<1$000sec5$ statistically valid information about cell populations is quic+ly obtained.
Flow Cytometry And Its Clinical Applications
Flow cytometry measures optical and fluorescence characteristics of single cells (or any other particle, including nuclei, microorganisms, chromosome preparations, and latex beads). Physical properties, such as size represented by forward angle light scatter) and internal complexity(represented by right-angle scatter) can resolve certain cell populations. Fluorescent dyes may bind or intercalate with different cellular components such as !" or #!".  "dditionally, antibodies con$ugated to fluorescent dyes can bind specific proteins on cell membranes or inside cells. %hen labeled cells are passed by a light source, thefluorescent molecules are excited to a higher energy state. &pon returning to their resting states, the fluorochromes emit light energy at higher wavelengths. 'he use of multiple fluorochromes, each with similar excitation wavelengths and different emission wavelengths (or colors), allows several cell properties to be measured simultaneously.
Immunophenotyping of Leukemias and Lymphomas
*mmunophenotyping by flow cytometry is an important tool in the diagnosis and staging of patients with a haematological neoplasm. *t is used in con$unction with classical morphology.*n the bone marrow, normal blood cells develop from stem cells in a progressive series of differentiations, branching off to give different lineages of cells (for example, myeloid or lymphoid, ' cell or + cell), each of which has a distinct maturation pathway. "t each stage, the cells carry a distinctive set of marers classified by the  (cluster of differentiation) nomenclature.'he panel selected will depend on initial clinical diagnosis. 'he results from the initial screen may indicate the need for further classification with an extended panel that is more specific for a particular disease. 'he marers include+ cell /, 01, 02, 31, 4/, 5appa, 6ambda7 ' cell 3, 8, 4, /, 9, :, 4/, /;7 <yeloid cells 9, 00b, 08, 04, 0/, 0;, 88, 84, 4/, /;, 009, =6"-#, <P>.Plasma cell 02, 8:, 4/, /;, 08:.
Detection of minimal residual disease
<inimal residual disease (<#) was defined as disease beyond the limit of morphological detection using conventional microscopy. Patients with acute leuaemia were considered to be in remission when bone marrow samples contained ?/@ neoplastic cells. Flow cytometric methods can detect far lower levels of disease, which can be important in the clinical management of leuaemia. 'he residual tumour cells are detected using immunofluorescence of surface marers.
Stem cell enumeration
=aemopoietic stem cells in the bone marrow can be identified by their expression of 84. !ormally, the number of such cells in bone marrow is low and is negligible in peripheral blood. =owever, if mobilisation of 84 positive cells from the bone marrow is stimulated, stem cells can be harvested from the peripheral blood as well as the marrow. 'hese cells can be used to repopulate a depleted bone marrow after, for example, high dose chemotherapy.'o assist in the identification of a small percentage of 84 positive cells, a double stain of 84 and 4/ is used. 'he method gives the percentage of 84 Ave cells.
Solid organ transplantationT cell cross-match
Flow cytometry can be used to crossmatch a recipientBs serum with donor lymphocytes to detect antibodies that could interfere with engraftment. Prior to organ transplantation, the organ donorBs lymphocytes are incubated with serum from the potential recipient of the graft. "fter washing, bound immunoglobulins are detected using an F*'-con$ugated anti-human *gC antibody. 'he ' cells are identified using a PD-8 con$ugate.
Postoperatie monitoring
 "fter the organ transplant, analysis of the peripheral blood lymphocytes may help to indicate early re$ection and bone marrow toxicity during immunosuppressive therapies, and to help in the differentiation of infections from transplant re$ection. " variety of cell surface marers and activation antigens can be used depending on the clinical condition and the organ transplanted. Peripheral blood testing may also be used to monitor the effectiveness of anti-re$ection immunosuppressive therapy, which may include, for example, antibodies designed to destroy ' cells. 'he number of circulating '-cells is usually determined by measuring the cell surface marer 8.
Detection of autoanti!odies
 "utoantibodies to leucocytes, platelets and erythrocytes may be found in a variety of autoimmune conditions and can cause anaemia, leuopenia, or thrombocytopenia. 'hey are detected by immunofluorescence in either a direct or an indirect assay. *n the former, anti-human *g antibodies are used to detect *g on the surface of the patientEs cells. *n the indirect assay, the reaction of antibodies in the patientEs serum with cells from a normal person is observed. 'he procedures are similar to those used for a ' cell crossmatch.
"I# infection$
etermination of the numbers of 4 Ave lymphocytes in the peripheral blood is used to monitor patients with =* infections.'he percentage of 4 Ave cells can be obtained in a single tube by staining for 4/G8G4. " cytogram of HH versus 4/ is used to identify the lymphocytes and a cytogram of 4 versus 8 to enumerate the 4Ave ' cells. "n extended panel is used to obtain a more complete picture of the peripheral blood lymphocytes.
HLA B27 antigen determination
 "nylosis spondylarthritis is strongly associated with =6" +39 phenotypes. Facing a borderline symptomatology, the detection of this phenotype can help the diagnosis. lassically =6"

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