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Review of CD4 Technologies

Suzanne Crowe, Burnet Institute On behalf of Members of the CD4 Working Group, Forum for Collaborative HIV Research

Laboratory monitoring & the 3x5 program
Cost of ARV Rx in developing countries is potentially exceeded by the cost of laboratory monitoring. Sophisticated assays often established as part of clinical trial infrastructure, paid for by international grants HIV+ individuals outside trials must find funds to pay for their own monitoring Monitoring HIV treatment is complex
– – – – CD4 and VL Drug toxicity Adherence Drug resistance

New CD4 monitoring tests and technologies
Flow based assays
Guava EasyCD4 System Partec CyFlow PointCARE Panleukogating technology

Manual assays
Dynal immune bead-based assay (microscopy) Coulter immune bead-based assay (microscopy)

In the pipeline
LabNow microchip technology SemiBio slide test (microscopy) suPAR (soluble urokinase plasminogen activator receptor) CD4 dipstick for remote care

Dual Platform Flow Cytometry

Slide courtesy of Roland Gohde

Single Platform Flow Cytometry

Slide courtesy of Roland Gohde

Pan-leukogate or PLG CD4 Methodology Slides courtesy of Angela Vernon and Meryl Foreman. Beckman Coulter .

count not reliable beyond 24 hours. Beckman Coulter . • Hematology lymph % is affected by EDTA.PLG CD4 Methodology Ungated • Identifies CD4+ lymphocytes based on a pan-leucocyte count • WBC count (cells/ul) X CD4 events from region B CD45 events from region A = Absolute CD4 Gated on WBC • The WBC gate is not affected by EDTA changes that occur with older specimens. Slide courtesy of Angela Vernon and Meryl Foreman.

2002 .990) Glencross et al.PLG CD4 Count (r2 = 0. 50:2. Clinical Cytometry.

Aged Specimen Performance – Limitations of Scatter Gating Forward Scatter cellular structure lost over time. Beckman Coulter . results in inability to define appropriate gates using scatter alone WBC Gate Day 1 Lymphs CD4 T cells Day 5 Slides courtesy of Angela Vernon and Meryl Foreman.

Median CD4 count = 371 cells/μL   Slide courtesy of Ank Gowans.8919 CD4 Count Range: 7 – 1579 cells/Μl. p=0. China n = 39 HIV+ volunteers CD4 Absolute Count 1800 1600 1400 1200 1000 800 600 400 200 0 0 Mean Specimens Linear (Mean) 1 2 3 4 5 6 Days Mixed Model ANOVA for trend over time.PLG: Aged Specimen Performance Beijing. Beckman Coulter and CDC Beijing .

South Africa High capacity: good for high volume centralized labs .Summary: PLG CD4 New flow cytometry-based method Based on a pan-leukocyte marker uses a 2-color pre-optimized reagent provides both CD4% and absolute counts extends sample age beyond 24 hrs to up to 5 days good correlation with “gold standard” flow compatible with most flow cytometers – with 2 color capability & 488 nm laser line <$6 per test Licensed by Beckman Coulter from NHLS.

Leonard Buchner . Tina Baumgartner.Guava EasyCD4 Slides courtesy of Jeff Harvey.

data acquisition and analysis .Guava EasyCD4 Measures absolute CD4 (can measure CD8) Sample volume: 10 µL of whole blood (EDTA) Reagents 10 µL of antibody cocktail – Anti-CD3-PE-Cy5 – Anti-CD4-PE 180 µL of Lyse-Fix solution Components/Software Dell LapTop computer included Software includes instrument set-up.

9 kilos with PC .The Guava EasyCD4 System: Dell Laptop Windows 2000 Pentium IV EasyCD4 Software and Data Handling Capillary Flow Cell 21 cm Green Diode Laser Microliter Cell Samples 36 cm Waste Fluid 32 cm 15.

turn on power and allow 10 minute warm-up Run Guava Check QC procedure (5 min) Adjust (or recall) instrument settings Acquire samples. vortex.Guava EasyCD4 Protocol Add 10uL of antibody cocktail to each tube Add 10uL EDTA whole blood to each tube. incubate 15min During sample incubation. Analyze results . incubate 15min Add 180uL of Lyse/Fix solution.

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EasyCD4 vs MultitestCD4 2000 1800 1600 1400 1200 1000 800 R1 600 400 200 0 0 200 400 600 800 1000 1200 1400 1600 1800 R2 R3 Linear (x =y) Linear (R1) Linear (R2) Linear (R3) EasyCD4 Triplicates R=0.97 R2 = 0.9725 TM MultiTest TM CD4 UCSF-GCRC/GIVI-CFAR Core Immunology Laboratory .

Tina Baumgartner.North America – California 3 Site Trial 200 Residual Difference 150 100 50 0 -50 -100 -150 -200 0 500 1000 1500 Site1 Site2 Site3 Mean +2SD -2SD N = 75 Average Guava & BD MultiTest CD4 Slides courtesy of Jeff Harvey. Leonard Buchner .

Guava EasyCD4 at YRG CARE .

Summary Easy CD4 (Guava) Quick assay Reagent cost is low ($3/test) Capital costs high ($45.000) Existing QA panels not compatible with this technology Measures percentage CD4 as %CD3+ T cells Still undergoing evaluation .

Partec Cy-Flow eg CyFlow Counter1P CyFlow SLGreen 2P CyFlow SL Blue 5P Slides courtesy of Roland Gohde .

Cy-Flow no lyse .no wash CD4 protocol 1   2  3 3-Step Protocol 50µl blood from the patient into a sample tube add 10µl of CD4-PE and incubate for 10 minutes at RT in the dark add 850µl of the no lyse dilution buffer .

Slides courtesy of Roland Gohde .

9899 CD4 count (FACSCount) 1000 500 0 0 500 1000 1500 CD4 count (CyFlow) Douala and Marua.Cameroon: CD4 Counting .CyFlow vs. FACSCount 1500 r = 0. Cameroon .

Cameroon: CD4 . Cameroon . FACSCount Bland-Altman Plot 600 bias 8 CD4/µl difference between CyFlow and FACSCount (CD4/µl) 400 200 mean +2sd mean of difference (bias) zero bias mean -2sd 0 -200 -400 -600 0 500 1000 1500 mean of methods CyFlow and FACSCount (CD4/µl) Douala and Marua.CyFlow vs.

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000 Absolute CD4 only .Summary Partec Cyflow Simple and portable flow cytometric assay No formal comparisons with samples provided by external QA programs available at this time Reagent costs $2-3/test Capital costs approx $20.

PointCARE Slides courtesy of Cecil Sherrer .

LY% and count Mobile. battery backup Room temperature reagent storage and operation .PointCARE System %CD4 and Absolute CD4 WBC.

•Ideal for low-volume.tube operation – biohazard containment via cap piercing 4.Closed. Patient Whole Blood Sample Tube •Patient sample and reagents bar-code are tracked in the instrument. decentralized labs . Rinse Tube 2. Lysing Reagent Tube or Cleaning Solution Tube 3. CD4 Reagent Tube 1.

cost of patient result is under US$10 •Cost of patient result includes: All reagents and disposables Operator time CD4. LY. CD4%. WBC.Automated Patient Results •Both CD4% & Absolute CD4 •without beads •critical for pediatrics •Depending on test volume. LY% Service .

PointCARE comparison with DP Flow CD4 Count.0718x + 30.98 800 400 0 0 500 1000 1500 2000 CD4 Count.Method Comparison N = 68 PointCARE CD4 Counts 2000 1600 1200 y = 1.REF) / REF 150 100 50 0 -50 -100 150 170 190 210 230 250 270 290 310 330 350 REF Dual Platform CD4 Counts .29 R = 0.REF 150-350/uL Bias Plot N = 23 REF Dual Platform CD4 Counts % Bias = (FC .

Summary PointCARE Recently received FDA approval %CD4 (as percentage of CD3+T cells) Cost approximately $10/test Capital cost approx $15.000 .000-20.

Manual low cost assays for monitoring CD4 Vidal. Cecile Goddard . Omah Mooleedhar. Burnet Institute Melb and Dr Balakrishnan’s lab. Shahir Ali. CAREC Data from Crowe lab. YRG Care Chennai Arlene Darmanie.

CID 2003: 37 (suppl 1) S25-35 . et al.CD4 manual methods Dynal assay CD14 Dynabeads® Monocyte-depleted blood removed to new tube CD4 Dynabeads® Lysis and staining of nuclei Count stained nuclei + - + - + - Coulter assay Monocyte blocking agent CD4 cytospheres® Add blood to staining solution Count cells with beads attached Crowe.

1 mm deep Manual counter Tubes Pipettes Plus rotating wheel and magnet for Dynal assay .What equipment is needed for these manual CD4 assays? Microscope with 40x objective Hemocytometer 0.

200 Difference 100 0 -100 -200 -300 0 200 400 600 800 1000 1200 Mean Balakrishnan Pachamuthu et al .Bland-Altman plot for difference against mean for CD4+ T-lymphocyte counts by flow cytometry and Coulter cyto-sphere assay 300 (n=122).

n=54 1000 1200 Dynal assay shows excellent association with flow cytometry Flow cytometry (cells/ul) Difference in CD4 count Bland-Altman Plot 350 300 250 200 150 100 50 0 -50 -100 0 -150 n=54 200 400 600 800 1000 1200 Average Flow cytometry result is 65 cells/µl higher than Dynal assay Crowe et al 2003 (cells/ul) Average CD4 count by Flow cytometry and Dynal assay (cells/ul) .97.Correlation of Flow SP and Dynal assay Dynal assay 1500 (cells/ul) 1000 500 0 0 200 400 600 800 R= 0.

et al CAREC .Comparison of CD4 Count between DYNAL and FACSCount Series1 (n=85) Mean 200 Mean + 2STD Mean .2STD Residual (Dynal-FACSCount) 150 100 50 0 -50 -100 -150 -200 0 200 400 600 800 1000 1200 1400 1600 Average of Dynal IFA and FACSCount Arlene Darmanie.

0 7 Flow Cytometry Reference site CV n 8.3 20 Site 6 9.0 24 Site 5 5.4 130 CV n 14.8 14 All Sites 8.6 16 7.Coefficient of variation Dynal assay vs flow cytometry in West Africa DYNABEADSR Sites Site 1 Site 2 Site 3 Ref site 9.3 20 Sites Distant from Reference site 19.7 45 Diagbouga S et al AIDS 2004 .7 49 Site 4 6.

4) Diagbouga S et al AIDS 2004 . 36.9 (6. 69.7 (2.Impact of the delay in sample handling on DynabeadsR Technique at room temperature samples exhibiting ≥ 20% decrease in CD4 cell counts Time Hour 4 Hour 8 Hour 12 Hour 24 n 0 / 28 3 / 28 5 / 28 14 / 28 % (95% CI) 0 (0.1.9) 50.3) 10. 28. 12.0 (30.2) 17.7.3.

NEQAS UK and data from Amanda Lindholm Streck Laboratories .Blood stabilizers Slides courtesy of Viv Granger and Dave Barnett.

CD4+ T cell analysis: must be done within 18 hrs  haematology analysers: difficulty producing a differential after 24 hrs  CytoChexTM (Streck laboratories)  Member of family of non cross-linking fixatives  Orig.Reagents for stabilizing blood samples  Guidelines. Barnett et al Cytometry 1996 26:216 Jani et al J Imm M eth 2001 257:145 . designed to preserve WBCs in whole blood (1:1)  Cyto-Chex BCT contains 57ul preservative/anticoagulant  Samples stable 7 days at ambient temps  NEQAS (UK)  TransFixTM lasts >10days. (1:10). <250 C  Transfix: allows transportation of fixed samples  Both compatible with flow technology  No data on stabilized blood and manual CD4 counts Turpen & Collins Amer Clin Lab 1996 15:30.

Flow Cytometric Analysis Fresh Day 7 .

Preservation of Lymphocyte Subsets with TransFIX 80 70 CD 3 Normal s Unfixed TRANSFix Specimens % Lymphocytes 60 50 40 30 20 10 0 Day 1 CD 4 CD 8 CD 16/56 CD 19 Day 2 Day 3 Day 4 Day 6 Time after collection .

Stability of CD3/4+ TCells post addition of TransFIX Stability of CD3/4+ T Cells post addition of TransFIX CD3/4+ T Cell count (cells/uL) 1000 800 600 400 200 0 0 1 10 Days post addition of TransFIX CD4 <100 >100 but <300 >300 but <500 >500 .

reader and digital camera Individual biochips Absolute CD4 Tentatively planned for availability late 2005 Semi-Bio manual slide technology – Slide with an antiCD4 coated chamber that traps CD4+ cells during incubation – Count CD4+T cells after staining Remote point-of-care technology – No technical skill required – For estimation of CD4+ T cells – Early stages of development .In the pipeline LabNow – – – – Microchip.

Microchip technology with a reader device and digital camera .

Data/Slide from Bill Rodriguez .

Which low-cost CD4 assay to introduce? .

Depends on Number of samples per day – Low throughput. opt for manual or ship samples or ensure local engineers/technicians trained Cost . flow method most cost effective (and definitely more practical) Sophistication of lab – Current methods require varying degree of technical skill – Remote point of care methods under development Availability of technical support & equipment maintenance training – A key issue for flow cytometers – Remote area. manual may be most cost-effective – High throughput.

Word of caution when considering cost Hidden costs (ie non-kit costs) greatly influence the final cost to a testing laboratory in a resourceconstrained country        Labour (often less expensive) Disposables (if available often more expensive) Shipping costs Importation costs Infrastructure Repeat assay runs Instrument repair  Pricing may be best negotiated at an international or country level with bulk procurement schemes Elbeik et al Expert Rev PharmacoEconomics Outcomes Res 2003 3:383-407 .

including clinical trial evaluation and independent statistical analysis.Where are we up to? All assays/methods continuing to undergo incountry analyses Rigorous independent evaluation. is absolutely essential Some technologies /assays recently licensed in USA (PLG. PointCARE) However all are still emerging technologies QA participation essential and establishing this should be part of the deal Countries should not purchase technologies that have not been inadequately validated .

Tina Baumgarten. Debbie K. YRGCare. Glencross and the NHLS of South Africa • Dr Balakrishnan. NEQAS UK • Cecil Sherrer (PointCARE) • Vicki Greengrass. Cameroon • Viv Granger and Dave Barnett.Final thanks to  Forum for Collaborative Research Ben Cheng Houtan Movafagh Ben Collins Veronica Miller Alan Landay (Chair) All those who provided slides for this presentation. especially • Rolande Gohde (Partec) • Jeff Harvey. (Beckman Coulter) • Ank Gowans. Megan Plate. Beckman Coulter and CDC Beijing • Dr. Leonard Buchner (Guava) • Angela Vernon and Meryl Foreman. Chennai • Douala and Marua. Mandy Dunne. Pauline Steele (Burnet Institute) • Roche (SC support to attend CROI) .