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Presented at the 4th INTEREST Workshop25-28 May 2010, Maputo Mozambique

Point‐of Care CD4 Testing• Bulk CD4 testing is done at accredited centralized National

Laboratories (NHLS SA) equipped to handle high volume sample throughput– TAT <24 hours

– Time delay from sampling to result‐in‐hand vary• Transport

• Reporting of results (electronic vs. manual)

• Patient return to clinics


Point‐of Care CD4 Testing• Decentralizing low volume CD4 testing to clinic level

– “same‐day‐results”

– “one‐stop” service of testing, counseling, initiation of therapy

– Get people onto therapy on 1st clinic visit if they qualify

– Not restricted to rural areas as urban clinics experience high % loss of patients

– POC systems


POC CD4 Technologies1. Rapid CD4 tests = laminar flow strips

BC ZyomyxTM

2. Low volume Fluorescent Cartridge Analyzers

PIMATM LabNowTM

3. Low volume Flow Cytometer Analyzers

FACSCountTM PointCareTM

ScreeningPositive/Negative answerCut-off 350cells/µlAccuracy questionable

Absolute CD4 count (no CD4%)Semi-Quantitative10-15 samples/instrument/dayBiohazard waste-disposalNo cold-chain

Absolute CD4 count and CD4%Quantitative20-40 samples/instrument/day“mini”-laboratory requiredCold chain required for some


(1) CD4 Test Strips

• Preliminary results with FirstSign strips– 1st generation done on plasma and serum

– 2nd generation done on whole blood

– Current cut‐off 250 cells/µl

– 95% false positive/negative results


(2) PIMATM

PHASE I: Laboratory evaluation

• Evaluated in the NHLS CD4 Reference Laboratory in Johannesburg

• 4 Pima Analyzers in parallel

• Testing done on whole blood drawn in EDTA tubes

• Manually pipetted 30µl of venous EDTA blood into cartridge capillary

• Results compared to reference CD4 method (PLG/CD4 by flow cytometry)

Closed cartridge systemFluorescent detection of CD4+ cells20min/day internal QC procedureSample analyses 20 minutes10 sample/instrument/8 hour day


Performance Comparison• In total 100 samples tested across 4 instruments

• Intra‐instrument variability negligible (50 samples run on 3 or 4 instruments in parallel) (1‐way ANOVA, p=0.95)

• Overall performance showed no significant difference in CD4 values (p>0.05)

• Error rate (NO READ) 1.3% across 4 instruments


Bias and Overall Agreement• Bland‐Altman analyses revealed a bias of ‐16±36 (95%LOA ‐88 to 55.2)

– At higher CD4 counts = bigger differences merely a function of the larger number

– %Similarity indicated tight reproducibility with overall agreement of 98% (slight under‐estimation of <2%) and precision of similarity (CV) of 4.6%

• Good correlation confirmed by linear regression analyses



2 PIMA analyzers moved to HIV clinic in CM Hospital– Nursing staff trained to operate and use special finger prick lancet

– Dual testing of fingerprick/ capillary blood fill (CLINIC) versus EDTA venous blood pipette filled (LAB)

– PLG/CD4 reference method in the LAB

Preliminary data of 43 patients showed• Laboratory comparison good with %SIM CV<7% with outliers removed (differences not

significant)

• Finger prick derived data revealed 4 outliers with clinical impact and confirmed slight under‐estimation of absolute counts

• 13% NO READ in clinic = cartridge not filled correctly/closed properly (no results generated and patient already had left the clinic)

PIMATM PHASE IIClinic validation using finger prick derived capillary blood



• Internal instrument QC • Manufacturer QC data confirmed reproducibility

(daily bead control) of system over time (CV<2%) in both laboratory and clinic analyzers

Control Cartridges

Pima 1

NPim

a 2 N

Pim

a 3 N

Pima 4

NPim

a 1L

Pima 2

LPim

a 3 L

Pima 4

L

0

500

1000

1500

Instrument PIMA 1 PIMA 2 PIMA 3 PIMA 4Low Bead ControlNumber of values 32 32 32 22Mean 152 220.9 150.1 148.595% CI of mean 151.3‐152.8 219.4‐222.4 149‐151.1 147.3‐149.8%CV 1.38 1.88 1.91 2.09High Bead ControlNumber of values 32 32 32 26Mean 994.6 828.4 960.6 988.595% CI of mean 998.8‐1000 825.2‐831.7 958.7‐962.5 985.4‐991.7%CV 1.60 1.09 0.55 0.72

Instrument and Control

Abso

lute

Bea

d C

ount


• External Quality Assessment– Retrospective AFREQAS EQA samples (n=10) showed system can process

stabilized blood products enabling external quality assessment on AFREQAS or equivalent EQAS

– Values between 1 to ‐2SDI with overall negative bias (NCS)



(3) Low volume Flow Cytometers• FACSCount comparison to PLG/CD4

– Both in laboratory and clinic

– Data summarized on poster no P‐28• Good overall agreement

• %similarity 98% with CV of <5%

• PointCare comparison to PLG/CD4– Laboratory evaluation

– Data presented on poster no P‐28• Overall agreement not acceptable

• %similarity 122% with CV of >10%


Inter-instrument comparisonsPIMA™, PointCARE™, FACSCount™

Pima vs. PointCARE Pima vs. FACSCount™• Significant differences No significant differences• PCare vs. PLG or PIMA• Bias 125±137 (%sim of 122%, CV 18%) Bias 3.5±38 cells/µl; (%sim 98%, CV<5%)


SUMMARY• POC systems should be evaluated for purpose

• Choice of system depend on– Screening or monitoring

– Patient group (Adult vs. Pediatrics; #CD4 vs CD4%)

– Volume of samples/day

• Results within acceptable limit of error rate– Not adversely affect patient care

Acknowledgements

• RHRU, Hillbrow– Dr Regina Osih from RHRU for organizing clinic

– Prof Francois Venter

• Ms Dinah Ramasegane for operating the PIMA analyzer in clinic

• NHLS Johannesburg– Ms Sithembile Mojalefa for analyzing samples in the laboratory

– Professor Glencross, Stevens and Denise Lawrie, NHLS Johannesburg Hospital

• Alere for supply of PIMA and reagents.• Lynton Scorgie for supply of PointCARE

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