Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.
BACKGROUND
Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing.


METHODS
Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets.


RESULTS
In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets.


CONCLUSIONS
With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.
Mittag
Anja
A
Interdisciplinary Center for Clinical Research Leipzig, University of Leipzig, Leipzig, Germany.
Lenz
Dominik
D
Gerstner
Andreas O H
AO
Sack
Ulrich
U
Steinbrecher
Michael
M
Koksch
Mario
M
Raffael
Alexander
A
Bocsi
Jozsef
J
Tárnok
Attila
A
D017252:CD3 Complex / Q000096:biosynthesis; D015704:CD4 Antigens / Q000096:biosynthesis; D019002:CD56 Antigen / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D002232:Carbocyanines / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007694:Killer Cells, Natural / Q000166:cytology*; D007962:Leukocytes / Q000166:cytology*; D016131:Lymphocyte Subsets / Q000166:cytology; D008214:Lymphocytes / Q000166:cytology; D009504:Neutrophils / Q000166:cytology; D010641:Phenotype; D017452:Receptors, IgG / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000166:cytology*
D017252:CD3 Complex; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D017452:Receptors, IgG; C501744:indotricarbocyanine