Unit 4 Flashcards
Describe flow cytometry
-automated process that analyzes cells or beads in fluid suspensions for their light- scattering characteristics
-uses fluorochromes to identify cells by size, shape, and antigenic properties
- allows for rapid and accurate detection of cells found in small numbers
What is immunophenotyping
Identifying their surface and cytoplasmic antigen expression
What are fluorochcomes
Fluorescent molecule
What do fluorochromes do?
-absorbs light across a spectrum of wavelengths and emits light of lower energy across a spectrum of longer wavelengths
What is flow cytometry used to detect?
- leukemia
- lymphoma
- AIDS
- chronic granulomatous disease
- leukocyte adhesion deficiency
- fetal RBC
- F-cell identification in maternal blood
- identification of paroxysmal nocturnal hemoglobinuria
What is flow cytometry used for?
- Enumerate hematopoietic stem cells
-detect human leukocyte antigen (HLA) antibodies in transplantation
-identify cells undergoing apoptosis
What is a significant advantage of flow cytometry?
Because the flow rate of cells is so rapid, thousands of events can be analyzed in seconds, allowing for accurate detection of cells that are present in very small numbers
What are the 4 parts of instrumentation of flow cytometry?
① fluidics
②laser light source
③optics and photodetectors
④ computer
Describe fluidics of flow cytometry
- Allows for cell transport, one at a time
-each time a cell passes in front of a laser beam, light is scattered,and the interruption o laser signal is recorded
Describe laser light source of flow cytometry
- For cell illumination and identification
-solid-state diode lasers are typically used
Describe optics and photodetectors of flow cytometry
- Signal detection
Describe computers of flow cytometry
- Data management
What colors are lasers?
- Red
-blue
-violet
-ultraviolet (UV) - yellow-green
What are the types of light scatter?
① forward scatter (FSC)
② side scatter (SSC)
Describe forward scatter
-an indicator of size
Describe side scatter
- indicative of granularity or the intracellular complexity of the cell
What are the intrinsic parameters of the light sources of flow cytometry?
- forward scatter
- side scatter
*light scattering properties
Describe extrinsic parameters in light source of flow cytometry
-require the addition of a fluorescent probe for their detection
Describe principle of hydronomically focusing
In flow cytometry
- cells pass single file through the intersection of the laser light source
What are the sample types common in flow cytometry?
- Whole blood
- bone marrow
- fluid aspirates
What WBCs can be differentiated based solely on their intrinsic parameters?
- lymphocytes
- monocytes
- neutrophils
What is flow cytometry steps?
① celli are processed into a suspension, and the cytometer draws up the cell suspension and injects the sample inside a carrier stream of isotonic saline (sheath fluid) to form a laminar flow
② The sample stream is constrained by the carrier stream and is thus hydrodynamically focused so that the cells pass in a single file through the intersection of the laser light source
③ each cell is interrogated by a light source that typically consists of one or more small air-cooled lasers
④ cells are labeled with a fluorochrome or Fluorescent molecules, that absorbs light across a
Spectrum of wavelengths and emits light of lower energy pattern of absorption (excitation) and emission
What are common speciemens tested for
Flow cytometry?
Whole blood
Bone marrow
Fluid aspirates
What is the additive in the tube that is used for whole blood collection?
EDTA
Heparin can also be used