Absolute TCD4+ counting by a minimalist dual-platform flow cytometric method.
The aim of this work was to compare the performance of an absolute TCD4+ counting method based on total WBC gating versus the standard lymphocyte (Ly) gating method, in order to develop a flow cytometric (FCM) minimalist strategy for TCD4+ enumeration.


METHODS
132 routine peripheral blood samples, mainly from HIV infected patients, were labelled with CD3-FITC/CD4-PE/CD45-PECy5 and analyzed by two gating methods: a) standard method based on Ly immunological gating (CD45++SSClow), followed by the determination of CD3+CD4+ percentage and absolute number (# calculation using Ly # from hematological analyser (HA); b) total WBC immunological gate on biparametric scatter CD45/CD4, followed by CD4++SSClow percentage determination and absolute number calculation using WBC absolute number from hematological counter without using the WBC differential. Moreover on 63 samples Ly # based on Ly % from FCM and WBC counting from HA was compared with Ly # from HA.


RESULTS
The TCD4+/microL ranged from 3 to 3277 and the statistical analysis results showed: a) linear regression: r2 = 0.9847; b) Bland & Altman analysis: difference mean = -56.22; agreement range = +95.68 / -208.12; c) the mean of result difference/mean value*100 between two methods was -9.06%; d) comparison between regression line and the boundaries for acceptable residual values based on regressed confidence limits found by A. Kunkl et al showed regression line within boundaries near the upper limits. The Ly/microL count ranged from 635 to 8752. The statistical analysis results showed: a) linear regression: r2 = 0.9764; b) Bland & Altman analysis: difference mean = -362.93; agreement range = +134.51 / -860.37; c) the mean of result difference/mean value*100 between two methods was -16.12%.


CONCLUSIONS
Our results suggest a fair agreement between the two gating methods, but the one based on total WBC gate gives TCD4+/microL counts systematically higher than the standard method. This finding can be attributed to a systematic lower estimation of Ly% by HA.
Chianese
R
R
Department of Hematology and Blood Transfusion, Hospital of Legnano, Milano, Italy. chianese.r@libero.it
Nebuloni
E
E
De Paschale
M
M
Gatti
A
A
Mena
M
M
D000911:Antibodies, Monoclonal / Q000737:chemistry; D017252:CD3 Complex / Q000096:biosynthesis; D015704:CD4 Antigens / Q000096:biosynthesis; D018791:CD4 Lymphocyte Count; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D006679:HIV Seropositivity / Q000097:blood; D006801:Humans; D016014:Linear Models; D008214:Lymphocytes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D015704:CD4 Antigens