Flow cytometry and Cytometry Flashcards
Most common clinical application of flow cytometry
Diagnosis of leukemias and lymphomas
Flow cytometry’s original purpose
To evaluate physical properties of cells based on their ability to deflect light
Discovery that improved flow cytometry
Development of monoclonal antibodies
Particles studied in flow cytometry
Cells, chromosomes, microorganisms, proteins
Properties measured by flow cytometry
Physical, antigenic, and functional properties of particles suspended in a fluid
Components of a flow cytometer
Fluidics, light source (laser), multiple detectors, computer
Main advantage of flow cytometry
Ability to quickly and simultaneously analyze multiple parameters in a large number of cells
Specimens commonly analyzed by flow cytometry
Bone marrow, peripheral blood, lymphoid tissues, body cavity fluids, solid tissues
Processing time for peripheral blood and bone marrow specimens
Must be processed within 24 to 48 hours from time of collection
Preferred anticoagulant for specimens in flow cytometry
Heparin
Flow cytometry specimen transportation
Transported at room temperature
Meaning of LASER
Light Amplified by Stimulated Emission of Radiation (Photon is the basic unit of radiation)
Flow cytometry antigen detection
Can detect 17 antigens on an individual cell simultaneously using monoclonal antibodies conjugated to fluorochromes
Flow cytometry analysis method
Cells must pass separately through the illumination and detection system
Method for individual cell analysis
Hydrodynamic focusing (cells aligned in a core surrounded by sheath fluid)
Fluorescence detection in flow cytometry
Particles emit fluorescent signals detected by detectors, converted to digital output
Additional signals recorded in flow cytometry
Forward scatter (FS), Side scatter (SS), and fluorescence
Forward scatter (FS) detection
Measures particle volume or size, detected by a photodetector aligned with the laser beam
Side scatter (SS) detection
Measures surface complexity and internal structures (granules and vacuoles), detected by a photodetector positioned to the side
Display and registration of flow cytometry signals
FS, SS, and fluorescence displayed simultaneously on the instrument screen and registered by the computer system
Markers for Immature lineage
CD 34, CD 117, Terminal deoxynucleotidyl transferase
Markers for Granulocytic/Monocytic lineage
CD 33, CD 13, CD 15, CD 14
Markers for Erythroid lineage
CD 71, Glycophorin A
Markers for Megakaryocytic lineage
CD 41, CD 42, CD 61
Markers for B lymphocytes
CD 19, CD 20, CD 22, κ Light chain, λ Light chain
Markers for T lymphocytes
CD 2, CD 3, CD 4, CD 5, CD 7, CD 8
Study of chemical constituents of cells
“Cytochemistry definition
Enzymatic technique specimen requirement
Fresh smears
Nonenzymatic technique specimen stability
Stable for months at room temperature (e.g., PAS/SBB)
Enzymes seen in primary granules of neutrophils
eosinophils
Use of MYELOPEROXIDASE (MPO)
Used to differentiate blasts of AML from ALL
Positive MPO stain interpretation
Rules out ALL
Positive MPO results
Neutrophilic granulocytes (except normal blasts), Auer rods, leukemic blasts in FAB M1, M2, M3, eosinophils
Weakly positive or negative MPO results
Monocytes
Negative MPO results
Myeloblasts, basophils, lymphocytic cell series, erythrocytic cell series
Stain reactions parallel those of MPO’s in most cases
“SUDAN BLACK B (SBB)”
SUDAN BLACK B (SBB) stains
Sterols, neutral fats, phospholipids (found in primary and secondary granules of neutrophils and lysosomal granules of monocytes)
Positive SBB results
Promyelocyte, myelocyte, metamyelocytes, bands, segmented neutrophils (STRONGLY POSITIVE), leukemic blasts, Auer rods, eosinophils
Weakly positive or negative SBB results
Myeloblasts, monocytic cells
Negative SBB results
Lymphocytes and its precursors, megakaryocytes and platelets, erythrocytes
Stain to differentiate acute granulocytic leukemias from monocytic leukemias
“ESTERASES”
Esters substrates
α-naphthyl acetate, α-naphthyl butyrate (nonspecific), Naphthol AS-D Chloroacetate (specific)
α-naphthyl acetate esterase (NSE) positive results
Monocytes (strong positive reaction), positive for certain cell types
α-naphthyl acetate esterase (NSE) weakly positive or negative results
Granulocytes, lymphoid cells
α-naphthyl acetate esterase (NSE) negative results
Monocytes (with NaF inhibition)
α-naphthyl butyrate esterase (NSE) positive results
Monocytes, positive for certain cell types
α-naphthyl butyrate esterase (NSE) weakly positive or negative results
Granulocytes, lymphoid cells
α-naphthyl butyrate esterase (NSE) negative results
Monocytes (with NaF inhibition)
Naphthol AS-D Chloroacetate esterase positive results
Promyelocyte, myelocyte, metamyelocyte, bands, segmented neutrophils, leukemic myeloblasts, Auer rods
Naphthol AS-D Chloroacetate esterase weakly positive or negative results
Monocytic cells
Naphthol AS-D Chloroacetate esterase negative results
Myeloblasts (variable), monoblasts, promonocytes, monocytes
Stain for glycogen
“PERIODIC ACID-SCHIFF (PAS)”
PERIODIC ACID-SCHIFF (PAS) is useful in identifying
“FAB M6 leukemia”
Strongly positive Leukemia using PERIODIC ACID-SCHIFF (PAS) REACTION
“RBCs in erythroleukemia (FAB M6)”
Normal erythroid precursors reaction in PAS
“negative”
Enzyme marker for primitive lymphoid cells
“TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT)”
Strong TdT activity – observed in approximately 90% of patients with
“ALL”
Helpful in the recognition of the ‘lymphoblastic transformation’ of CML
“TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT)”
