External quality assessment results.

<p>Radar graphs summarizing the results obtained with external quality assessment samples for absolute CD4 counts (left, including 10 trials, each with a low and a normal CD4 count on each respective trial) and CD4% of lymphocytes (right), both indicated by solid blue lines revealing the performance of the Aquios in relation to the respective 10 supplied retrospective trial pool means. The solid red lines indicate the acceptable 2 standard deviation range, while the interrupted black line represents the average overall performance (ideal) of the pooled results (at an SDI of zero).</p

PLG/CD4 protocol on Aquios CL cytometer.

<p>PLG/CD4 gating strategy applied for the Aquios CL cytometer platform, represented by 8 histograms (H1-8). H2 selects all CD45 positive white cells, from which all CD4 bright lymphocytes are gated (in H3) for reporting of an absolute CD4 count, while a CD4% of lymphocytes value is derived from H4. H6 and H7 indicate the bead population, currently used as an internal control measure of instrument stability.</p

Performance verification of the new fully automated Aquios flow cytometer PanLeucogate (PLG) platform for CD4-T-lymphocyte enumeration in South Africa

<div><p>Background</p><p>The National Health Laboratory Service (NHLS) offers wide-scale CD4 testing through a network of laboratories in South Africa. A new “load and go” cytometer (Aquios CL, Beckman Coulter), developed with a PLG protocol, was validated against the predicate PLG method on the Beckman Coulter FC500 MPL/CellMek platform.</p><p>Methods</p><p>Remnant routine EDTA blood CD4 reference results were compared to results from two Aquios/PLG instruments (n = 205) and a further n = 1885 samples tested to assess daily testing capacity. Reproducibility was assessed using Immunotrol^TM and patient samples with low, medium, high CD4 counts. Data was analyzed using GraphPad software for general statistics and Bland-Altman (BA) analyses. The percentage similarity (%Sim) was used to measure the level of agreement (accuracy) of the new platform versus the predicate and variance (%SimCV) reported to indicate precision of difference to predicate.</p><p>Results</p><p>205 samples were tested with a CD4 count range of 2–1228 cells/μl (median 365cells/μl). BA analysis revealed an overall -40.5±44.0cells/μl bias (LOA of 126.8 to 45.8cells/μl) and %Sim showing good agreement and tight precision to predicate results (94.83±5.39% with %SimCV = 5.69%). Workflow analysis (n = 1885) showed similar outcomes 94.9±8.9% (CV of 9.4%) and 120 samples/day capacity. Excellent intra-instrument reproducibility was noted (%Sim 98.7±2.8% and %SimCV of 2.8%). 5-day reproducibility using internal quality control material (Immunotrol™) showed tight precision (reported %CV of 4.69 and 7.62 for Normal and Low material respectively) and instrument stability.</p><p>Conclusion</p><p>The Aquios/PLG CD4 testing platform showed clinically acceptable result reporting to existing predicate results, with good system stability and reproducibility with a slight negative but precise bias. This system can replace the faded XL cytometers in low- to medium volume CD4 testing laboratories, using the standardized testing protocol, with better staff utilization especially where technical skills are lacking. Central monitoring of on-board quality assessment data facilitates proactive maintenance and networked instrument performance monitoring.</p></div

Summary of results from manufacturer provided daily quality control material (Immunotrol^TM Normal and Low) for 12 consecutive days, where target value and range refers to package insert values.

<p>Summary of results from manufacturer provided daily quality control material (Immunotrol^TM Normal and Low) for 12 consecutive days, where target value and range refers to package insert values.</p

Summary of samples tested using the predicate FC500 MPL/CellMek PLG platform and comparative statistics against the Aquios PLG platform.

<p>Summary of samples tested using the predicate FC500 MPL/CellMek PLG platform and comparative statistics against the Aquios PLG platform.</p

Summary of misclassification of absolute CD4 counts at three thresholds of importance in HIV management, indicating the number of samples misclassified and the percentage of misclassification per threshold.

<p>Summary of misclassification of absolute CD4 counts at three thresholds of importance in HIV management, indicating the number of samples misclassified and the percentage of misclassification per threshold.</p

Reproducibility data on Aquios.

<p>Reproducibility data for absolute CD4 count and CD4% of lymphocytes compiled using internal quality control material, Immunotrol™ Normal (a) and Immunotrol™ Low (b). Reproducibility data of patient samples with a low, medium and high CD4 counts are represented for absolute CD4 counts (c) and CD4% of lymphocytes (d).</p

Performance Evaluation of the Becton Dickinson FACSPresto^™ Near-Patient CD4 Instrument in a Laboratory and Typical Field Clinic Setting in South Africa

<div><p>Background</p><p>The BD-FACSPresto^™ CD4 is a new, point-of-care (POC) instrument utilising finger-stick capillary blood sampling. This study evaluated its performance against predicate CD4 testing in South Africa.</p><p>Methods</p><p>Phase-I testing: HIV+ patient samples (n = 214) were analysed on the Presto^™ under ideal laboratory conditions using venous blood. During Phase-II, 135 patients were capillary-bled for CD4 testing on FACSPresto^™, performed according to manufacturer instruction. Comparative statistical analyses against predicate PLG/CD4 method and industry standards were done using GraphPad Prism 6. It included Bland-Altman with 95% limits of agreement (LOA) and percentage similarity with coefficient of variation (%CV) analyses for absolute CD4 count (cells/μl) and CD4 percentage of lymphocytes (CD4%).</p><p>Results</p><p>In Phase-I, 179/217 samples yielded reportable results with Presto^™ using venous blood filled cartridges. Compared to predicate, a mean bias of 40.4±45.8 (LOA of -49.2 to 130.2) and %similarity (%CV) of 106.1%±7.75 (7.3%) was noted for CD4 absolute counts. In Phase-2 field study, 118/135 capillary-bled Presto^™ samples resulted CD4 parameters. Compared to predicate, a mean bias of 50.2±92.8 (LOA of -131.7 to 232) with %similarity (%CV) 105%±10.8 (10.3%), and 2.87±2.7 (LOA of -8.2 to 2.5) with similarity of 94.7±6.5% (6.83%) noted for absolute CD4 and CD4% respectively. No significant clinical differences were indicated for either parameter using two sampling methods.</p><p>Conclusion</p><p>The Presto^™ produced remarkable precision to predicate methods, irrespective of venous or capillary blood sampling. A consistent, clinically insignificant over-estimation (5–7%) of counts against PLG/CD4 and equivalency to FACSCount^™ was noted. Further field studies are awaited to confirm longer-term use.</p></div

Reproducibility of FACSPresto^™.

<p>Reproducibility of the FACSPresto^™ system was assessed for (A) absolute CD4 counts and (B) CD4% of lymphocytes. Two sets of ten samples were analysed for Beckman Coulter Immunotrol (IT) normal and low (Repro 1 and 2 IT) and for nine patient samples (PS) with low, medium and high CD4 counts (3 per group), indicated as Repro PS Set 1–3. %CV results are indicated for each data set.</p

Summary of Phase II validation results.

<p>Summary of Phase II validation results.</p