SUPPLEMENTARY INFORMATION In format provided by Stevens et al. (NOVEMBER 2008)

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1 S1 Table CD4 enumeration technologies Manual bead-based technologi es Co m p a ny As s ay Beckman Coulter (Fullerton, California, USA) Invitrogen (San Diego, California, USA) Coulter Manual CD4 Count Kit 1 4 Dynal T4 Quant Kit 5 7 Key features Latex bead-based labelling Counting using light microscope Manual count of rosetted cells Magnetic bead cell isolation P ara m eters meas ured CD4 absolute count CD4 absolute count Rea g ent s u s ed Anti-CD4- and anti-cd14-coated latex beads Anti-CD14 and anti-cd4 Au to m a ti o n No, manually performed No, manually performed S pec im en type Whole blood Whole blood S pec im en vo lu m e (µ l) 100 µl 125 µl Co ntrol s Di s a dv a n ta g es None known Immunotrol tested inadequate (DKG, unpublished data) Manual preparation is labour-intensive and cumbersome Training crucial to ensure reliability 3 Needs insight and skills applicable to use of a Neubauer counting chamber Time consuming a single technician can perform up to 12 Counting using fluorescent microscope, but can be counted using light microscope Dynabead-based systems have been automated and applied on haematology instrumentation by Sysmex, Japan on pochi and KXpert systems None known CDChex efficacy tested but reported as poor 8 Immunotrol tested inadequate (DKG, unpublished data) Manual preparation is labour-intensive and cumbersome Needs insight and skills applicable to use of a Neubauer counting chamber Time consuming a single technician can perform a maximum of 15 tests per day 5 NATURE REVIEWS MICROBIOLOGY 1

2 Adv a nta g es tests per day 4 Precision poor 9 Very low capital costs, requires simple light microscope (estimated cost of microscope US$6000 2,4 ) Co s t per tes t, kit o nly (US D) $10 1 $ Precision less than flow cytometry Very low capital costs, requires fluorescence microscopy but can also be adapted for use on a simple light microscope Flow cytometry systems that require minim al technical input Co m p a ny Becton Dickinson (Franklin Lakes, New Jersey, USA ) PointCare Technologies (Marlborough, Massachussetts,USA) As s ay nam e FACSCount 10,11 PointCare NOW Key features P ara m eters meas ured Rea g ent s u s ed Dedicated (duplicate bead analysis as SP) Two-colour Newly released FACSCount CD4 for CD4% lymphocyte values 12 CD4 (CD3+) absolute count CD8 (CD3+) absolute count CD3 absolute count CD4/CD8 ratio New FACSCount system CD4% lymphocytes 12 Specific bead calibrators Control tubes Specific reagent tubes SP Volumetric Single parameter colloidal gold based, no fluorescence CD4 absolute count CD4 percentage Haematology white blood cells; lymphocyte count and percentage; monocyte count and percentage; neutrophil count and percentage; eosinophil count and percentage; haemoglobin Specific CD4 (gold labelled) Disposal tubes Lysis buffer if CD4% required NATURE REVIEWS MICROBIOLOGY 2

3 Au to m a ti o n Pre-dispensed beads Pre-dispensed CD4/CD3 and CD8/CD3 reagents Requires accurately calibrated pipette and vortex mixer Can be automated, operator semi-independent; may require manual pipetting of blood Automatic pipettes can also be used Manual completely operator independent S pec im en type Whole EDTA blood only Whole EDTA blood only S pec im en vo lu m e (µ l per tes t) Co ntrol s Prep ara ti o n and a naly sis tim e Tec h nic a l skill s La b orat ory equ ipment 50 µl (requires 3 5 ml EDTA whole blood sample for accurate cell counting) As per supplier Calibration and use of T lineage gating built into analysis ensures robust CD3 count; bead reproducibility ensures precision Immuntrol (BC) and Streck products can be used as process controls Preparation: 60 minutes (30 minutes for new BD FACSCount) minutes (but CD4 12 samples with lower cell counts take longer to analyse Low technical skills needed Use of automated precision pipette can ensure accuracy and precision, otherwise manual pipetting skills mandatory to ensure quality control 10 FACSCount analyzer Automatic pipette Vortex mixer 100 µl (requires 3 5 ml EDTA whole blood sample for accurate cell counting) Daily quality control: bead volumetric calibration Immuntrol (BC) and Streck products not applicable Analysis time: ~8 minutes No sample preparation Low technical skills needed Computer literacy (minimal) PointCare NOW analyzer NATURE REVIEWS MICROBIOLOGY 3

4 T hrou g h p u t (s a m p l es per run) Adv a nta g es Di s a dv a n ta g es Low Maximum capacity 50 per day (if doing both CD4 and CD8); 100 per day (CD4 only 12 ) Automated analysis but with individual manual sample preparation and sample loading May need manual pipetting of blood if automatic pipetting not available Operator-independent analysis (automatic gating) No red cell lysis step Little flow cytometric experience required CD4 percentage available with paediatric reagents and software update Designed for small laboratory Footprint 38.1 x 43.2 x 55.9 cm In original format no CD4 percentage (*NB for infants) in original format model Dedicated no menu expansion Specific reagents in specific kit Long processing time (up to 2 hours) Requires accurate reverse pipetting skill Low 7 samples per hour Maximum capacity 50 per day Daily quality control: bead volumetric calibration Additional options of white cell count, 4-part white blood count differential and haemoglobin Reagents insensitive to light and do not require cold-chain shipment Touch screen No computer literacy or knowledge of flow cytometry needed Small footprint (35.4 x 25 x 33.7 cm) Weight under 12 kg Long-lasting LED, no alignment needed Storage capacity for 8000 data sets (has USB connection for downloading data) Generates Levey Jennings plots automatically Closed system, no blood handling No sample preparation Designed for near patient care CD4 percentage available Waste disposal of more tubes required Low-throughput No peer-reviewed validation study publications at this time NATURE REVIEWS MICROBIOLOGY 4

5 High cost per test Separate reagents for CD4 count and percentage 12 Co s t per tes t, kit o nly (US D) Variable, volume dependent; from $3 5 per test Variable, volume dependent $10 12 FDA a p proved Yes Yes Flow-cytometry-based systems that require operator input/flow cytometry skill Co m p a ny Becton Dickinson Beckman Coulter Beckman Coulter Guava Technologies (Hayward, California, USA) As s ay nam e/ ins tru ment ty pe Key features Multitest CD3/8/45/4 or variations with Trucount /and analysis using Multiset algorithm driven software 13 FACSCalibur Standard one or two laser flow cytometry Option for fully automated analysis/trucount/ Multiset SP TetraONE CD45/4/8/3 Flow Count 14 XL or FC500 Standard one or two laser flow cytometry Gating analysis typically based on international guidelines FlowCare PLG CD4 CD45/4 Typically XL Can be applied with standard one laser flow cytometry Bead-based SP 20 DP option 16,21 23 equals ogies.com EasyCD4 PCA CD3/4 Dedicated SP Volumetric Two-colour Partec (Munster, Germany) CyFlow CyFlow Counter CyFlow SL Green CyFlow SL Blue 31 1 or 2 parameter single CD4 6,30,32,35 CD45/3/4 or CD4/SS Dedicated SP Volumetric Single colour in simplest CyFlow Counter or multicolour in more NATURE REVIEWS MICROBIOLOGY 5

6 Gating analysis typically based on international guidelines SP precision 15,19,39 Simpler gating strategy Instrument independent 15 17,19 23,39 41 sophisticated models, some with side scatter measurement Advocated in recent guidelines 36,37 P ara m eters m eas ured Depends on the monoclonal antibody combinations used Absolute CD3/CD8/CD45/CD4 counts CD4% Ly Some variations do not include CD45, i.e. CD4/CD8/CD3 Can be modified to include full international guidelines, i.e.cd16/cd56, CD19 and CD45 to above CD45/CD4/CD8/CD3 counts CD4% Ly Can be modified to include full international guidelines, i.e.cd16/cd56, CD19 and CD45 to above Absolute CD4 count CD4% Ly DP variation requires white cell count from haematology analyser to calculate absolute CD4 counts CD4% of lymphocytes possible using CD45 bright gating BCR 20,42,43 ensure withinsample quality control Absolute CD4/CD3 and/or CD8/CD3 counts Not suitable for paediatric counting CD4% Ly EasyCD4 % assay available Absolute CD4 or CD8 count Depends on choice of reagents Not suitable for pediatric counting no CD4% Ly on low end models (CyFlow) Rea g ent s u s ed Depends on variation employed CD45/CD4/CD8/CD3 or variation Typically FACSlyse used for red cell lysis CD45/CD4/CD8/CD3 or variation Typically Immunoprep used for red cell lysis CD45/CD4 Typically Immunoprep used for red cell lysis 16,20 or FACSLyse 15,21,22,39 41, depending on manufacturer system used CD4 and/or CD8 and CD3 Calibration beads Lysing reagent (FACSLyse or new GuavaLyse) CD4 only or CD4 and CD8, or CD3/CD4/CD45 dependent on module and model of flow cytometer Lysing reagent for percentages only NATURE REVIEWS MICROBIOLOGY 6

7 Au to m a ti o n Analysis can be automated BD FACS Sample Prep Assistant (manual pipetting of blood) MultiSet software Carousel driven/closed system architecture Analysis can be automated PrepPLUS automated pipetting (beads and blood) Multi-TQ Prep automated sample preparation Carousel driven/closed system architecture Semi automated at this time, requires some input of manual gating but simple strategy ensures that gates do not vary much between analyses PrepPLUS automated pipetting can be employed Carousel driven/ closed system architecture Manual analysis and pipetting Open architecture single tube by tube analysis Manual analysis and pipetting Open architecture single tube by tube analysis S pec im en type Whole blood (EDTA) 36,37 Whole blood (EDTA) 36,37 Whole blood (EDTA) 36,37 Whole blood (EDTA) 36,37 Whole blood (EDTA) 36,37 S pec im en vo lu m e Co ntrol s 100 µl µl µl 10 µl (but still requires 3 5 ml EDTA whole blood sample for accurate cell counting, so reduced testing volume only relevant for reduced reagent cost) Process control: stabilised blood products can be used; Immunotrol (BCI) Streck (CDChex) option Flow cytometry control: daily laser alignment and Process control: stabilised blood products can be used; Immunotrol and Immunotrol LOW (BCI) Streck (CDChex) option Process control: stabilised blood products can be used; preferably Immunotrol and Immunotrol LOW (BCI) Problematic for leukopenic samples 25 Flow cytometry daily quality control: bead volumetric calibration and laser/pmt 50 µl Volumetric precision: none advised by company. However, commercially available beads have been recommended for use in quality control volumetric NATURE REVIEWS MICROBIOLOGY 7

8 T ime t o resu l t linearity check imperative; Calibrite beads 15 minutes with full blood count required for DP result or beads for SP result Flow cytometry control: daily laser alignment and linearity check imperative; FlowCheck 16 minutes with full blood count or beads Streck (CDChex) option Flow cytometry control: daily laser alignment and linearity check imperative; FlowCheck 15 minutes with full blood count or beads Tec h nic a l skill Skilled laboratory worker Skilled laboratory worker Semi-skilled or skilled laboratory worker La b orat ory equ ipment Flow cytometer: footprint: 200 x 60 cm Accurately calibrated pipette necessary for use with pre-pipetted beads Flow cytometer XL or FC500: footprint: 150 x 60 cm Does not absolutely require accurately calibrated pipettes as long as same pipette is used for both beads and blood for SP analyses 45 Typically utilizes XL Footprint: 150 x 60 cm Does not absolutely require accurately calibrated pipettes as long as same pipette is used for both beads and blood for SP analyses 45 precision 44 >30 minutes described minutes including two required pipetting steps (company lists 15 minutes incubation). Skilled laboratory worker Computer literacy Guava PCA Vortex mixer Small footprint: 23 x 39 x 46 cm Weight: 22.7 kg Requires the same sophisticated infrastructure, facilities and technical support as standard flow cytometry 29 For percentage, requires 3 pipetting steps (company lists 20 minutes). Suggested technical input required for adequate operation 31,32 Accurately calibrated pipette, vortex mixer; Variable footprint Counter: 32.5 x 33 x 26.5 cm Weight: 9.7 kg SL: 43 x 16 x 47 cm NATURE REVIEWS MICROBIOLOGY 8

9 T hrou g h p u t High throughput, 36 samples per hour High throughput, 36 samples per hour Very high throughput (~200 or more samples per day) or 36 per hour 20 Low, 20 samples per day; ~6 samples per hour with manual loading Manufacturer claims: in excess of 30; turnaround 15 minutes per sample Adv a nta g es CDC guidelines NIH guidelines Single platform capability Accuracy and precision validated Industry reference Multiple reagent choices Additional applications CDC guidelines NIH guidelines Single platform capability Accuracy and precision validated Industry reference Multiple reagent choices Additional applications CD4 absolute and percentages Can be applied with good reproducibility SP and DP FCR 20,42 monitoring allows for individual sample quality control Stable on aged blood up to 5 days good for centralized facility Simple and easy gating strategy Additional applications applied in LEGO building block fashion 20,46,47 Additional applications Small footprint Small test volume Minimal waste disposal Small reagent volume Generates automated clinical results report User replaceable flow cell Self-aligning Fixed optical and fluidics system Easy to learn, run, and maintain (1 day training) CD4 percentage reagent available No lyse system, No beads (although beads required for calibration) Expandable menu dependent on antibodies chosen Can give percentage CD4 if lyse no wash system used Also operable on 12V DC Colour touch-screen to operate Counter built-in printer for results Dis a dvantages High cost of instruments High cost of maintenance Significant skill required to operate Need good technical support Need carefully controlled High cost of instruments High cost of maintenance Significant skill required to operate Need good technical support Need carefully controlled No CD8 (although can be added as third antibody and incorporated as PLG CD8 (gated on CD8+++) Awaits automation of software still requires data input for centralized result Accurate reverse pipetting Knowledge of flow cytometry and computer literacy Requires red cell lysis and specific antibody reagents Daily bead calibration recommended 44 Accurate reverse pipetting Requires knowledge of flow cytometry and computer literacy NATURE REVIEWS MICROBIOLOGY 9

10 Per tes t c o s t f or rea gent s (US D) environment Reverse pipetting required Relatively high cost of instruments; Clinton Foundation has negotiated good reagent/instrument prices, maintenance support, etc environment Relatively high cost of instruments; Clinton Foundation has negotiated good reagent/instrument prices, maintenance support, etc generation Relatively high cost of instruments; Clinton Foundation has negotiated good reagent/instrument prices, maintenance support, etc Needs good technical support Need carefully controlled environment Low throughput, ~8 samples per hour Needs technical support and controlled environment 44 Separate reagents for CD4 count and percentage Separate laptop computer Lower cost of reagents No multisite independent validation of performance available Automated software currently not available Needs technical support and controlled environment 44 Separate reagents for CD4 count and percentage For SL models, separate laptop computer required Negotiable Negotiable Less than $2 3 CD4 count ~$4 5 25,29 CD4 count ~ $2.7; CD4 percentage ~ $3.9 (priced in Euros) FDA a p proved Yes Yes Yes No Instruments actively marketed for resourceconstrained countries No Instruments actively marketed for resourceconstrained countries Refe ren ces 1. Balakrishnan, P. et al. An inexpensive, simple, and manual method of CD4 T-cell quantitation in HIV-infected individuals for use in developing countries. J. Acquir. Immune Defic. Syndr. 3 6, (2004). 2. Carella, A. V., Moss, M. W., Provost, V. & Quinn, T. C. A manual bead assay for the determination of absolute CD4+ and CD8+ lymphocyte counts in human immunodeficiency virus-infected individuals. Clin. Diagn. Lab. Immunol. 2, (1995). 3. Didier, J. M. et al. Comparative assessment of five alternative methods for CD4+ T-lymphocyte enumeration for implementation in developing countries. J. Acquir. Immune Defic. Syndr. 26, (2001). NATURE REVIEWS MICROBIOLOGY 10

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13 Lab. Immunol. 12, (2005). 33. Shapiro, H. M., Mandy, F. F., Rinke de Wit, T.F. & Sandstrom, P. Dried blood spot technology for CD4+ T- cell counting. Lancet 363, 164; author reply (2004). 34. Zijenah, L. S. et al. Affordable flow cytometry for enumeration of absolute CD4+ T-lymphocytes to identify subtype C HIV-1 infected adults requiring antiretroviral therapy (ART) and monitoring response to ART in a resourcelimited setting. J. Transl. Med. 4, 33 (2006). 35. Fryland, M. et al. The Partec CyFlow Counter could provide an option for CD4+ T-cell monitoring in the context of scaling-up antiretroviral treatment at the district level in Malawi. Trans. R. Soc. Trop. Med. Hyg. 100, (2006). 36. Clinical and Laboratory Standards Institute. [online], < (Wayne, Pennysylvania, USA, 2007). 37. Mandy, F. F., Nicholson, J. K. & McDougal, J. S. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. MMWR Recomm. Rep. 52, 1 13 (2003). 38. Schnizlein-Bick, C.T. et al. Use of CD45 gating in three and four-color flow cytometric immunophenotyping: guideline from the National Institute of Allergy and Infectious Diseases, Division of AIDS. Cytometry 50, (2002). 39. Storie, I. et al. Flow rate calibration II: a clinical evaluation study using PanLeucoGating as a single-platform protocol. Cytometry B Clin. Cytom. 55B, 8 13 (2003). 40. Walker, C. L. et al. Flow rate calibration. III. The use of stabilized biostandards to calibrate the flow rate and calculate absolute CD4+ T- cell counts. Cytometry B Clin. Cytom. 70B, (2006). 41. Pattanapanyasat, K. et al. Low cost CD4 enumeration using generic monoclonal antibody reagents and a two-color user-defined MultiSET protocol. Cytometry B Clin. Cytom. 70B, (2006). 42. Scott, L. E. & Glencross, D. K. Monitoring reproducibility of single analysis, single platform CD4 cell counts in a high volume, low resource laboratory setting using sequential bead count rates. Cytometry B Clin. Cytom. 67B, (2005). 43. Bergeron, M. et al. Stability of currently used cytometers facilitates the identification of pipetting errors and their volumetric operation: time can tell all. Cytometry B Clin. Cytom. 52B, (2003). 44. Dieye, T. N. et al. Evaluation of an affordable instrument for absolute CD4 counting in resource-poor settings against two reference clinical flow cytometers. Abstract The 2nd IAS Conference on HIV pathogenesis and treatment (Paris, France, 2003). 45. Brando, B. et al. Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. Cytometry 4 2, (2000). 46. Glencross, D. K. et al. CD8/CD38 activation yields important clinical information of effective antiretroviral therapy: Findings from the first year of the CIPRA-SA cohort. Cytometry B Clin. Cytom. 74B (Suppl. 1), S (2008). 47. Jani, I. V., Janossy, G., Brown, D. W. & Mandy, F. Multiplexed immunoassays by flow cytometry for diagnosis and surveillance of infectious diseases in resource-poor settings. Lancet Infect. Dis. 2, (2002). NATURE REVIEWS MICROBIOLOGY 13