Ethical Review
The Cameroon National Ethics Committee approved the protocol prior to implementation with the number 2011/12/454/L/CNERH/SP. Blood samples were collected in the context of routine CD4 count testing for patients at CIRCB and other health facilities. Written informed consent is not required for receipt of routine services including CD4 testing performed at MOH facilities. Residual blood from routine testing was used for the analysis. No personally identifiable information was made available to the researchers. The institutional review boards waived the need for written informed consent.
Study Setting
This study was done in three health facilities offering CD4 + T cell enumeration: Chantal BIYA International Reference Centre with PIMA and FACSCalibur flow cytometer, Central Hospital of Yaounde with PIMA and CyFlow, and General Hospital of Yaounde with PIMA and FACSCount flow cytometer. The sites used different testing platforms (BD FACSCount™, PARTEC Cyflow™, and BD FACSCalibur™) for routine CD4 + T enumeration.
Study Participants
A total of 260 were recruited from the three study sites. Data on gender and age were available for all the patients. 56% were female while 44% were male. The median age was 36 years (range 1–65 years old) with an approximately normal distribution; 68 patients were aged 18 years or less. At least 37.7% of the patients were on antiretroviral therapy. Qualified and trained laboratory technicians conducted all tests.
Study Design
In this methods comparison study, venous blood specimens were collected consecutively from all eligible patients presenting at the health infrastructures included in the study who agreed to participate in the study through informed consent. Demographic data, CD4 + T cell count, and date of clinic visit were all recorded in a structured questionnaire and entered into a constructed database. This study was reviewed and approved by the National Ethical Review Committee. Patients were only provided with CD4 + T cell results obtained from the conventional CD4 testing platforms for further clinical management.
Laboratory Procedures
CD4 testing using each of the available devices was done according to manufacturers’ instructions. Whole blood collected in EDTA tubes was used for the BD FACSCount™, the BD FACSCalibur™, the PARTEC Cyflow™, and the Alere PIMA device. The Alere PIMA was present in all the three sites. Both internal quality assurance (IQA) were implemented for the platforms according to manufacturers’ instructions and existing individual laboratory protocols. During the study, we received samples for external quality control from QASI in Canada. Facility flow cytometers in the sites are enrolled in one External Quality Assurance Schemes. The CIRCB, was enrolled in the UKNEQAS EQA program. Control cartridges (both high and low) were run on the PIMA devices every morning before tests. PIMA devices reporting errors were not used for this work.
PIMA procedure
Twenty-five microliters of the blood sample was dispensed into a disposable anticoagulant-coated cartridge preloaded with antihuman CD3 and CD4 monoclonal antibodies conjugated with fluorescent labels (excitation at 520 nm, emission at 671 nm and 575 nm, respectively). The cartridge was capped and inserted immediately into the analyzer and the test performed. During the 20-minute analysis process, the blood sample was mixed with the freeze-dried fluorescently conjugated monoclonal antibodies present within the cartridge. A series of images of the fluorescent labeled cells in the fixed volume detection chamber were collected and the data analyzed for the absolute number of CD3 + CD4 + T-lymphocytes [14, 18, 19][13, 17, 18][14, 18, 19]
FACSCalibur procedure
Fifty microliters of whole blood was dispensed in a TruCount tube containing 20 µl of BD Multitest fluorescent conjugated monoclonal antibodies, and vortexed for 5 seconds. The Multitest consists of CD3-FITC/CD8-PE/CD45-PerCP/CD4 APC reagent. The mixture was incubated for 15 min at room temperature in the dark before adding 450 µl of FACS™ lysing solution and incubating for an additional 15 minutes in the dark prior to acquisition on the FACSCalibur. Data were analyzed using the MultiSET™ software with automated gating and analysis. In this analysis,CD3 T lymphocytes, CD3CD4 T cells and CD3CD8 T cells in absolute and relative values were determined, and the ratio CD4/CD8. Only the CD3CD4 T cells were used for comparison with PIMA[20]
FACSCount procedure
Fifty microliter of uncoagulated whole blood was added to the CD4/CD3 reagent tube (containing monoclonal antibodies and known number of microbeads) using an electronic pipette (BDB). The tube was vortexed for 5 s and incubated in the dark at room temperature for 60 min. Then, 50 µl of a fixative solution (5% of formaldehyde in PBS) provided with the reagent kit was added to the tube. The tube was vortexed, and the non lysed stained sample was analyzed in FACSCount FCM using an automated FACSCount software. After acquisition of 30,000 events for each sample, the gate was set up automatically around CD3+/CD4 + T lymphocytes [21]
CyFlow method
Twenty microliters of whole blood were added into a sample tube (on top of the lyophilized antibody spot at the bottom of the tube) gently mixed and incubated for 15 min (mix again after 5 min) in the dark at room temperature. Then, Buffer 1 was poured and mixed, and the Buffer 2 was added directly prior to the measurement on the CyFlow miniPOC. Stained blood was completely aspirated with a new syringe until the plunger reaches the position 1 ml (avoid having air bubble in the extremity of the syringe). The syringe was attached to the device, analysis started and the results (CD4 count and CD4%) were available in less than 2 min [19, 22]
Data Analysis
The results of the evaluation were analyzed using standard statistical methods. The absolute CD4 + T Cell counts derived from Alere PIMA device were compared with those derived from existing technologies by calculating the coefficient of determination (r2) and conducting regression analysis using XLSTAT. To determine interchangeability between the device and existing platforms, Bland-Altman analysis was used. For the former analysis, the bias was defined as the mean difference between two methods. Confidence intervals for bias and for limits of agreement were calculated using formulae previously described by Bland and Altman. The x axis on each Bland-Altman plot was the average value of the two methods while the y axis was the difference between the two methods.