Rea T Count Evaluation At Canada N=51

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

BD BIOSCIENCES

ReaMetrix

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

Reference beads CD3+4+

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

Table 1

Fifty-one whole blood K2EDTA samples (31 HIV+, 20 HIV-) were prepared with 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.

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RESULTS

FIG. 2

FIG. 3

Table 1 compares the mean, median and SD based on the CD4 determinations obtained from 51 specimens .

20

+2 SD 49.8

Difference (cells/µl)

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0 M EAN -29.5

-50

+ 2 SD

5

6.82

0

MEAN -5.49

-5 -10

-100

-15

- 2 SD - 108.8

- 17.82 - 2 SD

-20

-150

-25

0

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1400

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FIG. 4 Linear Regression of Absolute CD4 values betw een the FACSCount values and the ReaT-Count values 1400 1200 Reametrix (cells/µl)

600

800

Average (cells/µl)

Average (cells/µl)

CONCLUSION The absolute CD4 T-cell counts yielded by the two kit reagents showed excellent agreement with a minimal bias. This preliminary study indicates that the results obtained using the Rea T-Count dried reagent kits are comparable to the proprietary reagent kits. The use of low-cost, dry shelf stable reagent looks promising. The new reagents can be shipped and stored at ambient temperature, a significant advantage under extreme environmental conditions. An additional multi-site validation study collecting CD4, CD8 and CD3 absolute counts is ongoing.

25

100 50

Bland-Altman plot (fig. 2) indicated a mean difference of -29.5 cells/µl (difference = RM value- BD value) against the average [(RM value + BD value)/2] with limits of agreement (defined as ± 2 Standard Deviation) between -108 and +50 cells/µl. The largest difference is observed with CD4 count range over 1000 cells/µl. Using Pollock (fig. 3) the difference is expressed as a percentage of the averages ([difference/average] *100). The % mean difference was -5.5% with limits of agreement between -17.8 and +6.8. Linear regression indicated a coefficient of correlation of 0.9933.

Pollock plot of the % difference between FACSCount values and ReaT-Count values

Bland Altman plot of the difference between FACSCount values and ReaT-Count values

Difference %

Figure 1 compares the CD3/CD4 dot plots from the FACSCount system for both reagents.

1000 800 600 400

y = 0.9469x + 2.5675 R2 = 0.987

200 0 0

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FACSCount (cells/µl)

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1400

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1400