Immunophenotyping using gold or silver nanoparticle-polystyrene bead conjugates with multiple light scatter.
BACKGROUND
The type of antibody-conjugated polystyrene (PS) latex beads for use as light scatter shift agents for targeted lymphocyte populations in whole blood has been expanded to include gold and silver nanoparticle-aminodextran-PS latex bead conjugates with antibodies. The linkers between antibody and colloidal metal were an aminotrithiol ligand or aminodextran polymer molecules.


METHODS
A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10-20 degrees ) and UMALS (20-65 degrees ) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead-fluorescent marker experiments.


RESULTS
Two mutually exclusive cell populations, CD4+ and CD8+ lymphocytes, have been simultaneously enumerated in blood by using a mixture of CD4-PS, CD8-Au-PS or CD4-Au-PS, CD8-PS beads, and one laser line, 633 nm, excitation. Similar measurements were made with mixtures of CD4-PS, CD8-Ag-PS or CD4-Ag-PS, CD8-PS beads. Also, simultaneous use of bead and fluorescent markers mixed with whole blood was demonstrated with CD4-PS beads and with the CD4-RD1/CD8-FITC dual marker.


CONCLUSIONS
Enumeration of CD4 and CD8 lymphocytes in whole blood by light scatter parameters only compared well with standard analyses with fluorescent markers. In simultaneous bead-fluorescent marker labeling of lymphocytes, the labeled bead had to be mixed first with cells in whole blood.
Siiman
O
O
Advanced Technology, Beckman Coulter, Miami, Florida 33196-2500, USA. olavi.siiman@coulter.com
Gordon
K
K
Burshteyn
A
A
Maples
J A
JA
Whitesell
J K
JK
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D016827:CD8 Antigens / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003102:Colloids; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006046:Gold / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007834:Lasers; D007958:Leukocyte Count; D016131:Lymphocyte Subsets / Q000276:immunology*; D008863:Microspheres; D011137:Polystyrenes / Q000378:metabolism*; D012542:Scattering, Radiation; D012834:Silver / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D016827:CD8 Antigens; D003102:Colloids; D011137:Polystyrenes; D012834:Silver; D006046:Gold; C024075:styrofoam; D016650:Fluorescein-5-isothiocyanate