FLOW FLOW CYTOMETRYCYTOMETRY

PRINCIPLE OF A FLOW CYTOMETRY (FCM)

technique for counting and examining microscopic particles such as cell, chromosome Uses the principle of

light scattering light excitation emission of fluorochrome molecules

generate specific multi parameter data from particles. FCM that it measure fluorescence per cell or particle this process is performed at rates of thousands of cells per second.

FCM combines laser-induced fluorometry and particles light scattering analysis FCM incorporated with two or more fluorescence emission detection system FCM is able to measure multiple parameters

cell sizetotal protein cell receptors membrane potential calcium ion concentration granularity

Figure 1

Based on figure 1 use the principle of hydrodynamic focusing sample injected into the center of the sheath flow combined flow reduced the diameter forcing the cell into the center of stream

COMPONENTS OF A FLOW

CYTOMETRY

A FLOW CELL

A flow cell consists of a liquid stream which it wills carries and arrange the cells properly.

The arrangement processes are important so that the liquid stream can pass as a single file through the light for a purpose of sensing.

A MEASURING SYSTEM

An optical system and measurement of conductivity are the most commonly used measuring system.

A used of lamps such as xenon or mercury, a high-power of cooled water lasers like argon, krypton, dye laser, low-power air-cooled lasers like argon will resulting in light signals.

DETECTOR

Detector are able to measure the brief flashes of light emitted as cells flow past the laser beam as well as fluorescence signals from light into electrical signals that can later be processed by the read-out device.

READ-OUT DEVICE

The results are determine by measuring the visible and fluorescent light emissions, allowing cell sorting based on physical, biochemical and antigenic trait. 

Flow CytometryHow is it used in

diagnosing a disease?

Immunophenotyping Applications

in Hematology

Enzymatic deficiencies

Genetic disease and carriage

DNA analysis (Malignancy)

Leukocyte Analysis

Clinical use of Flow

Cytometry

1. 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.

2. DNA analysis (Malignancy)

DNA aneuploidy generally is associated with malignancy; often correlates with many types of cancer such as multiple myeloma, and childhood acute lymphoblastic leukemia (ALL).

Flow Cytometry 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.

Although conventional cytogenetics can detect smaller DNA content differences, flow cytometry allows more rapid analysis of a larger number of cells.

4. Enzymatic deficiencies

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 subsequent accumulation of glucosylceramide.

Flow cytometry is a tool for measuring B-glucocerebrosidase activity in Gauchers disease and to study the abnormalities RBCs shape.

5. Immunophenotyping Applications in Hematology

Flow cytometry is used in “Immunophenotyping”. Immunophenotyping is a technique used to study the protein expressed by cells such as erythrocytes, leukocytes, and platelets.

Example:Immunophenotyping for HLA-B27.HLA-B27 is one of the multiple major histocompatibility complex (MHC) class I-specific antigens. Detection of HLA-B27 (positive) may associate the patient with several other disorders, such as Reiter’s syndrome, psoriatic arthritis, and inflammatory bowel disease.Immunophenotyping of leukemias and lymphomas by differentiating their antigen profiles. Flow cytometry can also be used to identify leukemias that may be resistant to therapy .

Flow cytometry not only is can detect the presence or absence of antigens but also the strength of antigen expression which can also aid in diagnosis.

6. Genetic disease and carriage

Flow cytometry can be useful in discriminating heterozygous gene mutations from normal phenotypes.

It may also identify heterozygous family members who are carriers of a mutation for the purpose of genetic counseling.

Flow Cytometry In The Clinical Laboratory

WHICH LAB ARE USING FLOW CYTOMETRY

The clinical and research areas that use flow cytometry include : Hematology lab Immunology Tumor cell analysis Urinalysis microbiology lab

Flow cytometry in Hematology lab

• Blood cell routinely counted manually. Now, blood cell routinely counted by machine

• This instrument combine the principle of light excitation and emission of the fluorescent signals

• Flow cytometer is combination of cyt means cell , meter means measure

• This instrument is widely use for count RBC, WBC, Hb, platelet, and other blood component

• This instrument is the basic cell- counting technologies used in hematology lab

Hematology analyzer

Flow cytometry in Immunology lab

• In this lab, flow cytometry also a primary instrument used for diagnosis disease

• Instrument capable of cell sorting can separated cell by category and keep them alive for later used

• This instrument useful in separating blood cell of lymphocytes

Flow Cytometer in Urinalysis

• This instrument perform the physical, and microscopic part of routine urinalysis

• In microscopy analyzer, urine sediment can be identify and categories based on their size, shape, and texture.

• Lab technologist reported the result using a video screen

Urine analyzer

SUMMARYSUMMARY Flow cytometry is a powerful technique for correlating multiple characteristics on single cells.

This qualitative and quantitative technique has made the transition from a research tool to standard clinical testing. 

Smaller, less expensive instruments and an increasing number of clinically useful antibodies are creating more opportunities for routine clinical laboratories to use flow cytometry in the diagnosis and management of disease.

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