Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.
BACKGROUND
Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM.


OBJECTIVE
The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results.


METHODS
PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining.


RESULTS
Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD.


CONCLUSIONS
LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.
Gerstner
Andreas O H
AO
Department of Otorhinolaryngology/Plastic Surgery, Sigmund-Freud-Str. 25, D-53127, University of Bonn, Germany. gerstaoh@web.de
Mittag
Anja
A
Laffers
Wiebke
W
Dähnert
Ingo
I
Lenz
Dominik
D
Bootz
Friedrich
F
Bocsi
Jozsef
J
Tárnok
Attila
A
D003609:Dactinomycin / Q000031:analogs & derivatives / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D047569:Laser Scanning Cytometry / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D016014:Linear Models
D003609:Dactinomycin; C025942:7-aminoactinomycin D; D017493:Leukocyte Common Antigens