1 Evaluation of two volumetric flow cytometers for the quantitation of CD4+ T-cells in Thai HIV-1 infected patients Yuwadee Phuang-ngern, Vallerut Pobkeeree, Surada Lerdwana, Egarit Noulsri, Nattawat Onlamoon, Somboon Nookhai, Kimberley Fox, Jordan Tappero, and Kovit Pattanapanyasat Office for Research and Development and Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Thailand-Ministry of Public Health-U.S. CDC Collaboration, Nonthaburi, Thailand, and Global AIDS Program, Centers for Disease Control and Prevention, Atlanta, GA, USA.

2 42 Million People Living with AIDS Nature Reviews | Immunology Vol 3, 2003

3 Prognostic Monitoring of HIV Infection Absolute CD4 T-cell counts –Flow cytometric methods (Gold Standard) Single-platform technology Dual-platform technology –Non-flow cytometric methods Viral load Genotype, phenotype of drug resistance

4 CD4 Single Platform Volumetric method (Guava, CyFlow) *Number of cells / given volume of sample determined Non-volumetric method 1. TruCount, FlowCount, FACSCount *Beads are added in known numbers *Beads and cells are counted *Results are given as cells/beads and absolute counts are calculated 2. Flow rate based method (2003)

5 Three-Color SSC/CD45 Lymphocyte Gating Method Nicholson et al. Cytometry 1993;14: Pattanapanyasat et al. Cytometry 1994;18: BD MultiSET TM Software # of events in region containing subset population # of beads per pellet Absolute CD4+ count(cells/µl) # of events in absolute sample volume (µl) of subset population count bead region x = MMWR, 2003: 52(RR02);1-13 % CD4 X % Lymphocytes X WBC (from cell counter) = Absolute CD4+ count(cells/µl) Standard Flow Cytometric CD4 Methods Dual-platform method Single-platform method

6 FACSCalibur Flow Cytometer

7 FACSCount SYSTEM

8 FACSCount: A DEDICATED FLOW CYTOMETER Simple to use compact system Gold Standard method Absolute count & T % (infants?) One tube for CD4 is available Pipetting is the only critical step No generic reagent alternative Requires sheath fluid

9 System software and analysis

10 New Developments ● CD4 Cell Enumeration - CyFlow ® (Partec) - EasyCD4 TM (Guava) - PointCARE TM (PointCare Technologies) - LabNow (LabNow) ● Comparison to FACSCount or three-color bead-based flow cytometry

11 CyFlow green 1 Parameter

12 Measuring volume 200 µl True volumetric measurements

13 Volumetric Absolute Counting: Result Display Absolute CD4+ T-cell = N * dilution factor V

14 Evaluation of a new single-platform volumetric flow cytometer for enumeration of absolute CD4 T- lymphocyte counts in HIV-1 infected Thai patients Blood samples: 200 HIV-infected blood samples CyFlow green from Partec, Germany Compare with gold standard single-platform flow cytometric methods: FACSCount and FACS three-color Quality control: use Calibrite beads, CountCheck and Calibration beads for FACSort, FACSCount and CyFlow Statistical analysis: use linear regression analysis StatView and the Bland-Altman plot for bias analysis Pattanapanyasat K, et al. Clin Diagn Lab Immunol 2005;12:

15 Methods: Quality Control Within-run variation using CD-Check Plus CD4 Low stabilized whole blood samples showed less than 6% CV % CV of between-run variation over the period of study were less than 10%

16 A R 2 = 0.96 y = 0.81x R 2 = 0.97 y = 0.86x R 2 = 0.98 y = 0.93x – 5.3 D R 2 = 0.93 y = 0.79x R 2 = 0.95 y = 0.83x R 2 = 0.97 y = 0.95x Absolute CD4+ T-LymphocytesAbsolute CD8+ T-ymphocytes CyFlow green System TruCOUNT TM System CyFlow green System FACSCount TM System TruCOUNT TM System

Absolute CD4+ T-Lymphocytes Absolute CD8+ T-Lymphocytes Difference of the Two Systems (CyFlow green –TruCOUNT TM ) Difference of the Two Systems (CyFlow green – FACSCount TM ) Difference of the Two Systems (FACSCount TM –TruCOUNT TM ) Average of the Two Systems

Absolute CD4+ T-Lymphocytes Difference of the Two Systems (CyFlow green –TruCOUNT TM ) Difference of the the Two Systems (CyFlow green – FACSCount TM ) Difference of the Two Systems (FACSCount TM –TruCOUNT TM Average of the Two Systems

19 Discussion Disadvantages:  Need some manipulation  Problems on difficult samples  TB samples  Fluorochrome-protein ratio  Dim CD8+ T-cell  Integrity of volumetric probes  Accurate dispensing precision  Waiting for international approval

20 Benchtop Microvolume Cell Analysis Application Specific Software and Data Handling Green Diode Laser 36 cm 32 cm 21 cm Capillary Flow Cell Microliter Cell Samples Waste Fluid Dell Laptop Windows 2000 Pentium III 15.9 kilos with PC Guava Personal Cell Analyzer (PCA)

21 Absolute count = No. of cell count X dilution factor (20) Total volume acquired

22 Features Smaller sample volumes and reagents Smaller numbers of cells Minimized waste & expense –Reduces biohazardous waste 500-fold %CD4 T cells –CD4 T-cells as % of total lymphs –Suitable for pediatric HIV patients

23 Patients and Methods 250 HIV-1+ blood samples Equipments used in this study –TriTEST/TruCOUNT FACSCalibur –FACSCount –Guava system Quality control Statistical analysis Submitted for publication in Clin Cytometry

24 TruCOUNT TM Correlation of Absolute CD4 Guava Bland-Altman of Absolute CD4 (TruCount-Guava) Correlation of Absolute CD8 Guava TruCOUNT TM Results: TruCOUNT TM compare to Guava y = 0.99x R 2 = 0.95 y = 0.90x R 2 = 0.92

25 Results: FACSCount TM compare to Guava FACSCount TM Guava Bland-Altman of Absolute CD4 (FACSCount-Guava) Guava FACSCount TM Correlation of Absolute CD4 Correlation of Absolute CD y = 1.04x R 2 = 0.94 y = 0.91x R 2 = 0.88

26 Results: Bland-Altman Bias for Guava measurement Mean biases were 8.9 and 16.3 cells/μL at CD4+ T-cell counts below 200 cells/μL when compared to TruCOUNT and FACSCount Mean biases were 79.5 and 52.3 cells/μL for CD8+ T-cell counts when compared to TruCOUNT and FACSCount Guava system overestimates the other two bead-based systems

27 Discussion (1) Smaller sample volumes Smaller numbers of cells Minimized waste and expense –Reduces biohazardous waste 500-fold %CD4 T cells –CD4 Tcells as % of total lymphs –Suitable for pediatric HIV patients

28 Discussion (2) Disadvantages:  Equipment requires adjustment  Precision of pipetting  Problems on difficult samples  Leucopenic samples  Low CD3 samples  Dim CD8+ T-cells  Unable to distinguish CD3 and CD4 T-cells  Integrity of microcapillary flow cells  Waiting for international approval

29 Excellent correlation when compared to the two standard bead-based methods Overall %CD4 bias is less than 1% Bias of <8% or 8-16 CD4 cells/μl in patients with CD4<200 cells/μl Reduce the CD4 testing cost of $12-25 to less than $2-3 Save up to $3,000,000/year for national HIV treatment program in Thailand Conclusion

30 Acknowledgments Global AIDS Programs, Center for Disease Control and Prevention, Atlanta, GA, USA WHO HIV/AIDS Diagnostic Support, Health Technology and Pharmaceuticals Becton Dickinson Biosciences (Thailand) AIDS 2006 International Scholarship (ID 10001) Note: The findings and conclusions in this poster are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention