Accurate and Precise Performance of the BD FACSVia™ System

for Determination of CD4 Cell Count and %CD4 Cell concentration

Charlene Bush-Donovan, Julie Nguyen, Maya Kalyan, Anna Lin, Tom Frey

BD Life Sciences, 2350 Qume Drive, San Jose, CA 95131

Introduction HIV/AIDS affects approximately 37 million people globally including 2.6 million children generating severe health and

development issues in the world, particularly in resource poor countries. A steady decrease of CD3+CD4+ lymphocyte

counts typically exhibits in HIV+ individuals as the infection progresses resulting in a dysfunctional immune system with

increased opportunistic infections and complications which often can lead to death. The full access to the highly effec-

tive ART therapy by the HIV+ community has dramatically reduced mortality and morbidity by allowing sustained control

of viral replication with improved life quality and expectancy. Determination of CD4 absolute count and percentages

plays a critical role in the diagnosis of HIV/AIDS, initiation of ART, treatment monitoring and patient risk management

rendering CD4 count as an important clinical laboratory test. Providing easy access to flow cytometric technology for

HIV/AIDS diagnosis and monitoring is part of the global effort to decrease the rate of HIV infections and to end the AIDS

epidemic by 2030.

To meet global health needs, BD has developed the new and affordable BD FACSVia™ System. It is a small and

easy-to-use flow cytometer that features a compact optical design with fixed alignment. The BD FACSVia System

contains two lasers and four fluorescence detectors for use with various biological assays. The BD FACSVia instru-

ment provides pre-optimized detector settings so that users do not have to adjust PMT voltages and compensation

during daily use and operation. Daily one-step BD™ Cytometer Setup and Tracking (CS&T) procedure for instru-

ment QC on the BD FACSVia System can be accomplished in less than five minutes, which provides quality control

for electronics, fluidics, and optical performance as well as automatic optimization of instrument compensation. QC

results are tracked by BD FACSVia™ clinical software for long-term monitoring of instrument performance. New

design features, coupled with an intuitive user interface (UI) on the BD FACSVia system, significantly reduce the

number of clicks users have to make when running clinical assays. Improved workflow on the BD FACSVia Sys-

tem, such as simultaneous data analysis during sample acquisition or system cleaning, saves time and labor for

routine laboratory operation. The BD FACSVia™ Loader option is available to increase sample throughput in clini-

cal settings.

The BD Tritest™ CD4/CD8/CD3, and BD Tritest CD3/CD4/CD45, and BD Multitest IMK assays available on the BD

FACSVia system in Europe is designed to automatically enumerate absolute count and percentages of T, B and NK

lymphocytes using the BD Testest™ , BD Multitest IMK reagents with BD Trucount™ Tubes. To validate the BD

FACSVia System using these assays, we evaluated multiple BD FACSVia systems to demonstrate robust perfor-

mance using HIV+ patients and normal donors.

Methods

Instrument setup and QC

The BD FACSVia systems were set up using BD™ CS&T Beads and BD FACSVia clinical software. The CS&T Beads

were prepared by adding two drops of beads into 500 µL of DI water, then run on the BD FACSVia instrument to gener-

ate a passing Instrument QC report.

On the predicate system, the BD FACSCalibur™, BD Calibrite™ beads were prepared per the BD instructions for use

and run on the BD FACSCalibur instrument using BD FACSComp™ software to set up the BD FACSCalibur instrument.

A Pass on the BD FACSComp QC report was obtained on each study day.

The BD FACSVia and BD FACSCalibur instrument systems were also qualified by running process controls. BD Mul-

tiCheck control cells were prepared using the BD Tritest and Multitest reagents and run on both BD FACSVia and BD

FACSCalibur systems. The absolute count of lymphocyte subsets of the BD Multicheck control cells must pass the

specification range provided by the manufacturer on both the BD FACSVia and FACSCalibur instruments.

Method comparison

Method comparison was performed at BD Life Sciences with a total of 208 HIV+ and normal donor samples in EDTA

tubes. Each whole blood specimen was stained with the BD Tritest CD4/CD8/CD3, and BD Tritest CD3/CD4/CD45, and

BD Multitest IMK (CD3/CD8/CD45/CD4 and CD3/CD16+56/CD45/CD19) reagents in BD Trucount tubes and

analyzed on the BD FACSVia System (Test) and BD FACSCalibur cytometer (Predicate). Acquisition was carried out on

a sample loader or using manual tube loading method. Lymphocyte subset absolute count and percentages were gen-

erated and compared between the predicate (reference) and test systems. Deming regression and Bland-Altman analy-

sis and Concordance Analysis were performed for lymphocyte subsets for the cell absolute count and percentages.

Precision

A 21-day study was conducted at BD Life Sciences, to assess within-site precision. Estimates of precision for the enu-

meration of lymphocyte subset percentages and absolute counts were determined across three BD FACSVia flow cy-

tometers and three operators by acquiring two concentrations of analyte, BD Multi-Check CD4 Low control and BD Mul-

ti-Check control, stained in duplicate with three lots of the BD Multitest IMK (CD3/CD8/CD45/CD4 and CD3/ CD16+56/

CD45/CD19) reagents. Two separate runs were analyzed separately during each of the 21 tested days.

Linearity

Eleven concentration levels of CD4 cells were prepared from normal donor whole blood. Triplicate stained samples us-

ing each of the reagents were measured on one BD FACSVia system to determine lymphocyte subset absolute counts

in the 11 linearity samples. Statistical analysis based on the CLSI guidance document EP6-A was performed to deter-

mine system linearity.

PRESENTED AT THE 21ST

INTERNATIONAL AIDS CONFERENCE - DURBAN,

SOUTH AFRICA

BD Tritest CD4/CD8/CD3

Method Comparison

BD Tritest CD3/CD4/CD45

Method Comparison

BD Multitest CD3/CD8/CD45/CD4

Method Comparison

BD Multitest CD3/CD16+56/CD45/CD19

Method Comparison

T –Lymphocyte

Subset

Linear range

(cells/µL)

CD3+CD4+ 5—6,500

CD3+CD8+ 5—3,500

CD3+ 5—10,000

T –Lymphocyte

Subset

(cells/µL)

control mean %CV

(repeatability)

%CV

(within-site

precision)

CD3+CD4+ MC

Low

148.9 5.8 6.6

CD3+CD8+ 533.5 5.1 5.5

CD3+ 754.4 5 5.5

CD3+CD4+ 754.6 4.5 5.2 MC

normal CD3+CD8+ 369.0 4.6 5.3

CD3+ 1180.2 4.1 5.1

T –Lymphocyte

Subset (% of T

Lymphocyte)

control mean SD

(repeatability)

SD

(within-site

precision)

CD3+CD4+ MC

Low

19.7 0.6 0.7

CD3+CD8+ 70.7 0.9 1.1

CD3+CD4+ 63.9 0.9 1 MC

normal CD3+CD8+ 31.1 0.8 0.9

Linearity

Precision

T –Lymphocyte

Subset

Linear range

(cells/µL)

CD3+CD4+ 5—6,500

CD3+ 5—10,000

Linearity

Precision T –Lymphocyte

Subset

(cells/µL)

control mean %CV

(repeatability)

%CV

(within-site

precision)

CD3+CD4+ MC

Low

146.2 7.5 8.3

CD3+ 791.8 5.2 6.1

CD3+CD4+ 779.1 5.3 6.4 MC

normal CD3+ 1304.0 5.3 6.3

T –Lymphocyte Subset

(% of total Lympho-

cyte)

control mean SD

(repeatability)

SD

(within-site

precision)

CD3+CD4+ MC

Low

10.2 0.5 0.6

CD3+ 55.0 1 1.1

CD3+CD4+ 43.2 1 1 MC

normal CD3+ 72.3 1.1 1.2

T –Lymphocyte

Subset

Linear range

(cells/uL)

CD3+CD4+ 5—6500

CD3+CD8+ 5—3500

CD3+ 10—14,000

T –Lymphocyte

Subset

Linear range

(cells/uL)

CD3-CD19+ 5—1800

CD3-(CD16+56)+ 5—1300

CD3+ 10—14,000

T –

Lymphocyte

Subset

(cells/µL)

control mean %CV

(repeatability)

%CV

(within-site

precision)

CD3+CD4+ MC

Low

136.8 9.3 10.0

CD3+CD8+ 587.1 5.0 6.7

CD3+ 773.0 4.5 6.5

CD3+CD4+ 752.9 5.7 6.2 MC

normal CD3+CD8+ 374.4 5.3 6.3

CD3+ 1227.3 5.4 6.1

T –Lymphocyte Sub-

set (% of total Lym-

phocyte)

control mean SD

(repeatability)

SD

(within-site

precision)

CD3+CD4+ MC

Low

9.5 0.7 0.7

CD3+CD8+ 37.3 1.2 1.5

CD3+ 53.6 1.2 1.2

CD3+CD4+ 42.1 1.0 1.2 MC

normal CD3+CD8+ 20.9 0.8 1.1

CD3+ 68.6 1.4 1.7

T –Lymphocyte

Subset

(cells/µL)

control mean %CV

(repeatability)

%CV

(within-site pre-

cision)

CD3-CD19+ MC

Low

337.0 6.5 10.1

CD3-(CD16+CD56)

+

282.2 5.8 7.0

CD3+ 771.9 4.0 5.6

CD3-CD19+ 259.6 7.6 11.5 MC

normal CD3-(CD16+56)+ 218.7 7.5 7.5

CD3+ 1237.2 4.6 5.4

T –Lymphocyte Sub-

set (% of total Lym-

phocyte)

control mean SD

(repeatability)

SD

(within-site

precision)

CD3-CD19+ MC

Low

23.4 0.9 1.5

CD3-(CD16+CD56)+ 19.6 0.7 1.1

CD3+ 53.6 1.0 1.3

CD3-CD19+ 14.3 0.9 1.3 MC

normal CD3-(CD16+CD56)+ 12.1 0.6 0.7

CD3+ 68.5 1.5 1.5

Conclusion The BD FACSVia system demonstrated equivalent results of lymphocyte subset absolute count and percentages running the two BD Trit-

est (CD3/CD8/CD4 and CD3/CD4/CD45) and BD Multitest IMK (CD3/CD8/CD45/CD4) and (CD3/CD16+56/CD45/CD19) assays compared to

its predicate BD FACSCalibur system using FACSVia loader or manual tube loading method. Other system performance characteristics

such as reproducibility and within-site precision, system linearity, showed robust results and met study criteria. Analysis results of sam-

ples with CD4 < 500 cells/µL showed the mean absolute bias between BD FACSVia System and FACSCalibur for the absolute CD4 T cell

counts are 1.7 for the BD Multitest CD3/CD8/CD45/CD4, -9.8 for the BD Tritest CD3/CD8/CD4 and –6.6 for the BD Tritest CD3/CD4/CD45.

The BD FACSVia system provides an easy, reliable, and affordable solution to enumerate T, B and NK lymphocyte subsets for immune

monitoring for adults and pediatric populations, particularly for the HIV/AIDS diagnosis and monitoring.

Linearity Linearity

Precision Precision

Class 1 Laser Product. The BD FACSVia System is not yet available for sale in the US. © 2016 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company.

Parameters Range Intercept

Slope

(95% CI)

R2 Mean relative Bias %

(95% CI)

Mean absolute Bias

(95% CI)

T –Lymphocyte

Subset

(cells/µL)

CD3+CD4+ 14, 2608 2.63 0.99 (0.97,1.00) 0.99 -0.90 (-2.2, 0.5) -8.6 (-16.7, -0.5)

CD3+CD8+ 150, 2848 14.11 0.97 (0.95,0.98) 0.97 -1.7 (-2.8, -0.7)

*CD3+ 306, 4683 39.63 0.96 (0.94,0.98) 0.98 -1.4 (-2.1, -0.6)

T –Lymphocyte

Subset (% of total

Lymphocyte)

CD3+CD4+ 0.29 1.00 (0.99,1.01) 0.99 0.31 (0.14, 0.47)

CD3+CD8+ 0.62 0.99 (0.97, 1.00) 0.99 0.068

(-0.15, 0.28)

*CD3+ 0.97 1.00 (0.98,1.02) 0.98 0.69 (0.50, 0.88)

Parameters Range Intercept

Slope

(95% CI)

R2 Mean relative Bias %

(95% CI)

Mean absolute Bias

(95% CI)

T –Lymphocyte

Subset

(cells/µL)

CD3-CD19+ 28, 1088 -4.98 0.96 (0.93, 0.99) 0.95 -8.2 (-10.5, -5.8)

CD3-(CD16+CD56)+ 22, 1497 12.01 0.90 (0.87, 0.93) 0.95 -4.3 (-6.6, -1.9)

T –Lymphocyte

Subset (% of total

Lymphocyte)

CD3-CD19+ -0.80 1.03 (1.00, 1.06) 0.95 -0.39 (-0.57, -0.20)

CD3-(CD16+CD56)+ 0.20 0.96 (0.94, 0.99) 0.96 -0.27 (-0.45, -0.077)

*: CD3+ is from average results of 2 IMK reagent tubes.

Range Intercept

Slope

(95% CI)

R2 Mean Relative Bias %

(95% CI)

Mean Absolute Bias

(95% CI)

Parameter

T –Lymphocyte

Subset

(cells/µL)

CD3+CD4+ 19, 2686 -1.57 0.98 (0.97, 1.00) 0.98 -2.7 (-4.0, -1.4) -13.5 (-22.1, -5.0)

CD3+CD8+ 149, 3252 7.88 0.98 (0.96, 1.00) 0.98 -10 (-20, -0.1)

CD3+ 303. 5061 28.52 0.97 (0.96, 0.99) 0.97 -0.8 (-1.7, 0.0)

T –Lymphocyte

Subset (% of T

Lymphocyte)

CD3+CD4+ -0.56 1.01 (1.00, 1.02) 1.00 -0.23 (-0.41, -0.052)

CD3+CD8+ -0.34 1.01 (1.00, 1.02) 0.99 -0.052 (-0.25, 0.15)

Range Intercept

Slope

(95% CI)

R2 Mean relative Bias %

(95% CI)

Mean absolute Bias

(95% CI)

Parameter

T –Lymphocyte

Subset

(cells/µL)

CD3+CD4+ 10, 2575 3.32 0.97 (0.96, 0.99) 0.98 -1.8 (-3.2, -0.5) -14.5 (-22.9, -6.0)

CD3+ 314, 4858 36.78 0.96 (0.94, 0.98) 0.97 -1.5 (-2.4, -0.7)

T –Lymphocyte

Subset (% of total

Lymphocyte)

CD3+CD4+ -0.022 1.00 (0.99, 1.02) 0.99 0.091 (-0.085, 0.27)

CD3+ 1.72 0.98 (0.96, 1.01) 0.96 0.53 (0.28, 0.78)

CD4 Bin CD4 Absolute Count

(cells/µL)

Number of

samples

<200 34

200—500 51

500—1000 83

1000—4500 41

CD4 Bin CD4 Absolute

Count

(cells/uL)

Number of

samples

<200 35

200—500 48

500—1000 84

1000—4500 41

CD4 Bin CD4 Absolute Count

(cells/µL)

Number of

samples

<200 33

200 —500 51

500 —1000 83

1000 — 4500 42

Absolute mean

bias (95% CI)

Cells/µL

95% Limits of

agreement

Cells/µL

n

-9.8 (-13.8, -5.8) -46.3, 26.6 85

CD4 < 500 cells/µL CD4 < 500 cells/µL Absolute mean

bias (95% CI)

Cells/µL

95% Limits of

agreement

Cells/µL

n

-6.6 (-12.5, -0.6) -59.9, 46.8 84

Absolute mean

bias (95% CI)

Cells/µL

95% Limits of

agreement

Cells/µL

n

1.7 (-3.2, 6.6) -42.4, 45.8 83

CD4 < 500 cells/µL