8/14/2019 Rea T Count Evaluation at Canada n=51

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EVALUATION OF AFFORDABLE ALTERNATIVE DRIEDREAGENTS FOR CD4 T-CELL ENUMERATION USING THE

FACSCOUNT SYSTEMN. Soucy1, F. Mandy1, T. Ding1, M. Bergeron1

1 National HIV Immunology Laboratory, Public Health Agency of Canada, Ottawa

ACKGROUND

forts to develop affordable alternative CD4 T-cell counting technologies are

itical to accelerate the implementation of HIV antiretroviral therapy in resources

mited regions. Substituting proprietary reagents for generic dried reagents is a

ost effective solution. ReaMetrix (RM) has developed an alternative reagent kit in

dry format, compatible with the FACSCount system, a widely used dedicated

ow cytometer. This preliminary study compares CD4 counts determined by both

e new generic and standard proprietary kit.

ETHOD

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

aT-Count (ReaMetrix) and FACSCount (BD Biosciences) reagents. 50 l of

mple was pipetted into the CD3/CD4 reagent tube and incubated for 60 minutes

the dark at room temperature. Following the calibration of the instrument using

e FACSCount control kit, the samples were fixed and analyzed within 2 hours of

eparation. To measure the agreement between methods, the differences

tween CD4 T-cell results were estimated using Bland Altman and Pollock

atistical analysis. Linear regression equation was used to calculate the

efficient of correlation.

ESULTS

gure 1 compares the CD3/CD4 dot plots from the FACSCount system for both

agents.

ble 1 compares the mean, median and SD based on the CD4 determinations

tained from 51 specimens .

and-Altman plot (fig. 2) indicated a mean difference of -29.5 cells/l (difference =

M value- BD value) against the average [(RM value + BD value)/2] with limits of

reement (defined as 2 Standard Deviation) between -108 and +50 cells/l. The

gest difference is observed with CD4 count range over 1000 cells/l. Using

llock (fig. 3) the difference is expressed as a percentage of the averages

ifference/average] *100). The % mean difference was -5.5% with limits of

reement between -17.8 and +6.8. Linear regression indicated a coefficient of

rrelation of 0.9933.

ONCLUSION

he absolute CD4 T-cell counts yielded by the two kit reagents showed excellent

reement with a minimal bias. This preliminary study indicates that the results

btained using the Rea T-Count dried reagent kits are comparable to the

oprietary reagent kits. The use of low-cost, dry shelf stable reagent looks

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

gnificant advantage under extreme environmental conditions. An additional

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

ngoing.

OBJECTIVE

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

easured with both ReaMetrix and BD BioSciences reagents.

Pollock plot of the % difference between FACSCount

values and ReaT-Count values

-25

-20

-15

-10

-5

0

5

10

15

20

25

0 200 400 600 800 1000 1200

Average (cells/l)

Difference%

- 2 SD

6.82

MEAN -5

- 17.82

+ 2 SD

Linear Regression of Absolute CD4 values between the

FACSCount values and the ReaT-Count values

y = 0.9469x + 2.5675

R2

= 0.9870

200

400

600

800

1000

1200

1400

0 200 400 600 800 1000 1200 1400

FACSCount (cells/l)

Reametrix(cells/l)

Bland Altman plot of the difference between

FACSCount values and ReaT-Count values

-150

-100

-50

0

50

100

0 200 400 600 800 1000 1200 1400

Average (cells/l)

Difference(cells/l)

+2 SD

MEAN -29.5

- 108.8

9.8

- 2 SD

FACSCount Reagents

BD BIOSCIENCES

ReaT-Count Reagents

ReaMetrix

Referencebeads

CD3+4+

CD3+CD4-

FIG. 1 FACSCOUNT FLOW CYTOMETRIC ANALYSIS

!

Table 1

FIG. 2 FIG. 3

FIG. 4