EVALUATION OF AFFORDABLE ALTERNATIVE DRIED

REAGENTS FOR CD4 T-CELL ENUMERATION USING THE

FACSCOUNT SYSTEM
N. Soucy1, F. Mandy1, T. Ding1, M. Bergeron1
1 National HIV Immunology Laboratory, Public Health Agency of Canada, Ottawa

BACKGROUND FIG. 1 FACSCOUNT FLOW CYTOMETRIC ANALYSIS

FACSCount Reagents ReaT-Count Reagents

Efforts to develop affordable alternative CD4 T-cell counting technologies are BD BIOSCIENCES ReaMetrix
critical to accelerate the implementation of HIV antiretroviral therapy in resources
limited regions. Substituting proprietary reagents for generic dried reagents is a Reference
cost effective solution. ReaMetrix (RM) has developed an alternative reagent kit in beads
a dry format, compatible with the FACSCount system, a widely used dedicated
flow cytometer. This preliminary study compares CD4 counts determined by both
CD3+4+
the new generic and standard proprietary kit.

OBJECTIVE CD3+CD4-

The objective is to assess the intra-laboratory variability of CD4 T-cell counts

measured with both ReaMetrix and BD BioSciences reagents.

METHOD

Fifty-one whole blood K2EDTA samples (31 HIV+, 20 HIV-) were prepared with
Table 1
ReaT-Count (ReaMetrix) and FACSCount (BD Biosciences) reagents. 50 µl of
sample was pipetted into the CD3/CD4 reagent tube and incubated for 60 minutes !
in the dark at room temperature. Following the calibration of the instrument using
the FACSCount control kit, the samples were fixed and analyzed within 2 hours of
preparation. To measure the agreement between methods, the differences
between CD4 T-cell results were estimated using Bland Altman and Pollock
statistical analysis. Linear regression equation was used to calculate the
coefficient of correlation.

RESULTS

Figure 1 compares the CD3/CD4 dot plots from the FACSCount system for both
reagents. FIG. 2 FIG. 3
Bland Altman plot of the difference between Pollock plot of the % difference between FACSCount
Table 1 compares the mean, median and SD based on the CD4 determinations FACSCount values and ReaT-Count values values and ReaT-Count values
25
obtained from 51 specimens . 100
20
+2 SD 15
50 49.8
10
Bland-Altman plot (fig. 2) indicated a mean difference of -29.5 cells/µl (difference =
Difference (cells/µl)

+ 2 SD
Difference %

5 6.82
0
RM value- BD value) against the average [(RM value + BD value)/2] with limits of M EAN -29.5 0
MEAN -5.49

agreement (defined as ± 2 Standard Deviation) between -108 and +50 cells/µl. The -50 -5

-10
largest difference is observed with CD4 count range over 1000 cells/µl. Using -100 - 2 SD -15 - 17.82
- 108.8
Pollock (fig. 3) the difference is expressed as a percentage of the averages -20 - 2 SD

-150 -25
([difference/average] *100). The % mean difference was -5.5% with limits of 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400

Average (cells/µl) Average (cells/µl)

agreement between -17.8 and +6.8. Linear regression indicated a coefficient of
correlation of 0.9933.

CONCLUSION FIG. 4
The absolute CD4 T-cell counts yielded by the two kit reagents showed excellent Linear Regression of Absolute CD4 values betw een the
agreement with a minimal bias. This preliminary study indicates that the results FACSCount values and the ReaT-Count values
1400
obtained using the Rea T-Count dried reagent kits are comparable to the
1200
proprietary reagent kits. The use of low-cost, dry shelf stable reagent looks
1000
Reametrix (cells/µl)

promising. The new reagents can be shipped and stored at ambient temperature, a 800

significant advantage under extreme environmental conditions. An additional 600

multi-site validation study collecting CD4, CD8 and CD3 absolute counts is 400

ongoing. 200
y = 0.9469x + 2.5675
R2 = 0.987
0
0 200 400 600 800 1000 1200 1400
FACSCount (cells/µl)