FACSCount CD4 and Pima CD4 T-Cell Count Systems
FACSCount CD4 and Pima CD4 T-Cell Count Systems
The study was approved by the Institutional Review Board of the Institute of Tropical Medicine (ITM) and the University of Antwerp, Belgium, and by the Senate Research and Publications Committee of Muhimbili University of Health and Allied Sciences in Dar es Salam, Tanzania. Study participants were recruited among HIV-infected patients presenting for routine CD4 T-cell counting at the HIV outpatient clinic of the ITM Antwerp and at the Infectious Diseases Clinic in Dar es Salam. At least 200 participants were enrolled per site, targeting 50 patients with CD4 T cells <200 per microliter, 100 with CD4 T cells between 200 and 500 per microliter, and 50 with CD4 T cells >500 per microliter. The recruitment was initially done consecutively and was then focused on patients with low CD4 count looking at their previous CD4 T-cell count. CD4 enumeration was part of the routine follow-up, and study participants signed an informed consent before enrollment to provide an additional capillary blood sample. Capillary blood was collected from a finger stick directly onto Pima cartridges and venous blood from venepuncture in K3 EDTA vacutainer tubes.
The new BD FACSCount CD4 assay is an updated version of the BD FACSCount with an ability to provide percentage of CD4 T cells (CD4%) in addition to absolute CD4 T cells.
The Alere Pima CD4 is an automated image-based immune hematology POC test intended for rapid in vitro quantitative measurement of absolute CD3CD4 T cells from capillary or venous blood samples.
Precision of FACSCount CD4 included instrument precision, intra-assay variation, inter-assay variation, and carry over. Precision of Pima CD4 was evaluated on venous blood only and included intra-assay, inter-assay, intra-instrument, and inter-instrument variabilities. Instrument precision, not applicable on blood samples (single-use cartridges), was determined using the Pima control beads. Carry over was not applicable on Pima CD4.
For the FACSCount CD4, the instrument precision (run to run) was assessed on 15 different blood specimens containing 100–300 cells per microliter. Each stained sample was run 10 times or as many times as possible if less than 10 because of volume shortage.
The intra-assay variability assesses the tube-to-tube variability and includes the variation induced by pipetting errors made by the operator. The intra-assay variability was determined on 10 different blood samples with CD4 T cells ranging from 100 to 300 per microliter. For each blood sample, the entire CD4 staining procedure and sample acquisition were repeated 10 times. In Antwerp, for Pima CD4, each of the 10 venous blood samples was run twice on each of the 5 Pima analyzers, for a total of 10 readings per blood sample. In Dar es Salam, each blood sample was read 10 times on the same device, and 10 different blood samples were run using 6 different Pima analyzers.
The inter-assay variation, which assesses the day-to-day variation, was determined on 10 consecutive blood samples (7 with 100–300 cells per microliter and 3 with 301–550 cells per microliter). An aliquot from the blood samples was stained 3 times: at 6, 24, and 48 hours after specimen collection, with storage at room temperature.
The carry over assessment determines if the result of a high–CD4 count sample has an influence on the result of a subsequent low–CD4 count sample. This effect was studied by analyzing 5 batches of 2 different blood samples, one with a high CD4 count and the other with low CD4 count. The high–CD4 count sample (>600 per microliter) was read in duplicate (recorded as a1 and a2) followed by the duplicate reading of the low–CD4 count (100–300 cells per microliter) sample (recorded as b1 and b2). The carry over (k) is defined by k = (b1 − b2) × 100/(a2 − b2).
Additionally, in Antwerp, we assessed the true intra-assay variability by running 1 fresh venous blood sample 10 times on each of the 5 Pima devices. By this, we also calculated the inter-instrument variability (device to device), comparing the 5 Pima devices used in this study.
An acceptable assay should have the percent coefficient of variation (%CV) less than 15% (or 30 cells) for CD4 counts ≤200 cells per microliter and less than 10% for CD4 counts >200 cells per microliter as agreed in WHO prequalification protocol. The acceptable carry over must be less than 2%.
Routine venous blood samples brought into the laboratory and capillary blood samples collected by finger stick from the same patients were used to determine agreement between methods. Capillary blood samples were run on 1 of the 5 different Pima CD4 analyzers within 5 minutes after finger-prick collection. Venous blood samples were collected and stored at ambient temperature (17–25° C) in the outpatient clinic before being transported to the laboratory for analysis within 6 hours after venipuncture on FACSCalibur using Trucount tubes, FACSCount, FACSCount CD4, and Pima CD4 analyzers. For each capillary sample, the corresponding venous sample was run on the same Pima analyzer to avoid inter-instrument variability. All tests were performed according to the manufacturer's instructions. Before running samples, specific control beads were successfully tested daily on FACSCalibur and Pima analyzers and weekly on FACSCount. In addition, the Multicheck controls (BD Biosciences) were run daily before samples to ensure the accuracy and reliability of the FACSCalibur reference system. CD4 T-cell measurements on FACSCalibur reference and on the evaluated instruments were performed by different operators to allow a blind reading.
The reference method for CD4 counting used in this study was the FACSCalibur in combination with Trucount (BD Biosciences). Trucount is an established flow cytometric CD4 cell counting assay, which allows SP measurements of both absolute CD4 T-cell counts and CD4% and has excellent repeatability and quality assurance scores. Briefly, 50 μL of whole venous blood and 20 μL of Multitest monoclonal antibody (mAb) reagents (CD3-FITC/CD4-PE/CD8-PerCP/CD45-APC) were pipetted into a Trucount tube, mixed, and incubated for 15 minutes. Subsequently, 450 μL of lysis solution (BD Biosciences) was added to each tube and incubated for 15 minutes before reading the samples on the FACSCalibur.
The FACSCount CD4 reagent kit consists of 50 single tubes containing each a mixture of 3 mAbs (CD4-PE/CD14-PE-Cy5/CD15-PE-Cy5), a fluorescent nuclear dye, and fluorescent beads. The FACSCount reagent kit consists of 50 twin tubes containing each a mixture of mAbs (CD3-PE-Cy5 and CD4-PE or CD8-PE) and fluorescent beads. Fifty microliters of venous blood was added into single tube and into each of the twin tubes. The tubes were capped, vortexed, and incubated for 30 minutes (single tubes) or 60 minutes (twin tubes). After incubation, 50 μL of fixative solution was added into each tube, and samples were run on the FACSCount instrument with the respective software. All incubation steps were done in the dark at room temperature.
The Pima CD4 uses disposable anticoagulant-coated cartridges preloaded with antihuman CD3-dye1 and CD4-dye2 mAbs. Capillary blood was directly collected from the finger into a cartridge according to the manufacturer's instructions. Twenty-five microliters of venous blood was loaded into a disposable cartridge. Once the control window was filled, the blood collector was removed and the cartridge capped and immediately inserted into a Pima analyzer for 20 minutes incubation followed by automatic analysis of the test sample.
Data were analyzed using MedCalc version 10.0.2.0 (MedCalc Software, Mariakerke, Belgium). Precision expressed as the CV was determined by dividing the SD of the measurements by the mean (CV = SD × 100/mean). We calculated the CV for instrument precision and intra-assay, inter-assay, and inter-instrument variations. Measurement of linear regression was determined using Passing–Bablok regression analysis. Pollock and Bland–Altman analyses were used to determine the mean biases and the limits of agreement (LOA = mean ± 1.96 SD) on CD4 count and on CD4%, respectively. Percentage similarity was calculated for each sample as Similarity = Average of methods A and B × 100/Method A (with A = reference and B = evaluated). For each group, the mean percent similarity and the CV were determined. We first performed comparisons between each alternative method (FACSCount CD4 or Pima CD4) and the FACSCalibur (Trucount) reference system in the overall data and then within each of the 3 CD4 T-cell count categories. Second, comparisons were done between FACSCount CD4 and FACSCount and between CD4 counts from capillary blood and those from venous blood on the Pima CD4.
Misclassification probabilities were calculated at ART initiation thresholds of 200, 350, and 500 cells per microliter for CD4 counts and 25% for CD4% setting of the FACSCalibur as the reference method to determine eligible patients on ART.
Materials and Methods
Study Population
The study was approved by the Institutional Review Board of the Institute of Tropical Medicine (ITM) and the University of Antwerp, Belgium, and by the Senate Research and Publications Committee of Muhimbili University of Health and Allied Sciences in Dar es Salam, Tanzania. Study participants were recruited among HIV-infected patients presenting for routine CD4 T-cell counting at the HIV outpatient clinic of the ITM Antwerp and at the Infectious Diseases Clinic in Dar es Salam. At least 200 participants were enrolled per site, targeting 50 patients with CD4 T cells <200 per microliter, 100 with CD4 T cells between 200 and 500 per microliter, and 50 with CD4 T cells >500 per microliter. The recruitment was initially done consecutively and was then focused on patients with low CD4 count looking at their previous CD4 T-cell count. CD4 enumeration was part of the routine follow-up, and study participants signed an informed consent before enrollment to provide an additional capillary blood sample. Capillary blood was collected from a finger stick directly onto Pima cartridges and venous blood from venepuncture in K3 EDTA vacutainer tubes.
Description of the Technologies
The new BD FACSCount CD4 assay is an updated version of the BD FACSCount with an ability to provide percentage of CD4 T cells (CD4%) in addition to absolute CD4 T cells.
The Alere Pima CD4 is an automated image-based immune hematology POC test intended for rapid in vitro quantitative measurement of absolute CD3CD4 T cells from capillary or venous blood samples.
Precision Assessment
Precision of FACSCount CD4 included instrument precision, intra-assay variation, inter-assay variation, and carry over. Precision of Pima CD4 was evaluated on venous blood only and included intra-assay, inter-assay, intra-instrument, and inter-instrument variabilities. Instrument precision, not applicable on blood samples (single-use cartridges), was determined using the Pima control beads. Carry over was not applicable on Pima CD4.
For the FACSCount CD4, the instrument precision (run to run) was assessed on 15 different blood specimens containing 100–300 cells per microliter. Each stained sample was run 10 times or as many times as possible if less than 10 because of volume shortage.
The intra-assay variability assesses the tube-to-tube variability and includes the variation induced by pipetting errors made by the operator. The intra-assay variability was determined on 10 different blood samples with CD4 T cells ranging from 100 to 300 per microliter. For each blood sample, the entire CD4 staining procedure and sample acquisition were repeated 10 times. In Antwerp, for Pima CD4, each of the 10 venous blood samples was run twice on each of the 5 Pima analyzers, for a total of 10 readings per blood sample. In Dar es Salam, each blood sample was read 10 times on the same device, and 10 different blood samples were run using 6 different Pima analyzers.
The inter-assay variation, which assesses the day-to-day variation, was determined on 10 consecutive blood samples (7 with 100–300 cells per microliter and 3 with 301–550 cells per microliter). An aliquot from the blood samples was stained 3 times: at 6, 24, and 48 hours after specimen collection, with storage at room temperature.
The carry over assessment determines if the result of a high–CD4 count sample has an influence on the result of a subsequent low–CD4 count sample. This effect was studied by analyzing 5 batches of 2 different blood samples, one with a high CD4 count and the other with low CD4 count. The high–CD4 count sample (>600 per microliter) was read in duplicate (recorded as a1 and a2) followed by the duplicate reading of the low–CD4 count (100–300 cells per microliter) sample (recorded as b1 and b2). The carry over (k) is defined by k = (b1 − b2) × 100/(a2 − b2).
Additionally, in Antwerp, we assessed the true intra-assay variability by running 1 fresh venous blood sample 10 times on each of the 5 Pima devices. By this, we also calculated the inter-instrument variability (device to device), comparing the 5 Pima devices used in this study.
An acceptable assay should have the percent coefficient of variation (%CV) less than 15% (or 30 cells) for CD4 counts ≤200 cells per microliter and less than 10% for CD4 counts >200 cells per microliter as agreed in WHO prequalification protocol. The acceptable carry over must be less than 2%.
Agreement Between Methods
Routine venous blood samples brought into the laboratory and capillary blood samples collected by finger stick from the same patients were used to determine agreement between methods. Capillary blood samples were run on 1 of the 5 different Pima CD4 analyzers within 5 minutes after finger-prick collection. Venous blood samples were collected and stored at ambient temperature (17–25° C) in the outpatient clinic before being transported to the laboratory for analysis within 6 hours after venipuncture on FACSCalibur using Trucount tubes, FACSCount, FACSCount CD4, and Pima CD4 analyzers. For each capillary sample, the corresponding venous sample was run on the same Pima analyzer to avoid inter-instrument variability. All tests were performed according to the manufacturer's instructions. Before running samples, specific control beads were successfully tested daily on FACSCalibur and Pima analyzers and weekly on FACSCount. In addition, the Multicheck controls (BD Biosciences) were run daily before samples to ensure the accuracy and reliability of the FACSCalibur reference system. CD4 T-cell measurements on FACSCalibur reference and on the evaluated instruments were performed by different operators to allow a blind reading.
The reference method for CD4 counting used in this study was the FACSCalibur in combination with Trucount (BD Biosciences). Trucount is an established flow cytometric CD4 cell counting assay, which allows SP measurements of both absolute CD4 T-cell counts and CD4% and has excellent repeatability and quality assurance scores. Briefly, 50 μL of whole venous blood and 20 μL of Multitest monoclonal antibody (mAb) reagents (CD3-FITC/CD4-PE/CD8-PerCP/CD45-APC) were pipetted into a Trucount tube, mixed, and incubated for 15 minutes. Subsequently, 450 μL of lysis solution (BD Biosciences) was added to each tube and incubated for 15 minutes before reading the samples on the FACSCalibur.
The FACSCount CD4 reagent kit consists of 50 single tubes containing each a mixture of 3 mAbs (CD4-PE/CD14-PE-Cy5/CD15-PE-Cy5), a fluorescent nuclear dye, and fluorescent beads. The FACSCount reagent kit consists of 50 twin tubes containing each a mixture of mAbs (CD3-PE-Cy5 and CD4-PE or CD8-PE) and fluorescent beads. Fifty microliters of venous blood was added into single tube and into each of the twin tubes. The tubes were capped, vortexed, and incubated for 30 minutes (single tubes) or 60 minutes (twin tubes). After incubation, 50 μL of fixative solution was added into each tube, and samples were run on the FACSCount instrument with the respective software. All incubation steps were done in the dark at room temperature.
The Pima CD4 uses disposable anticoagulant-coated cartridges preloaded with antihuman CD3-dye1 and CD4-dye2 mAbs. Capillary blood was directly collected from the finger into a cartridge according to the manufacturer's instructions. Twenty-five microliters of venous blood was loaded into a disposable cartridge. Once the control window was filled, the blood collector was removed and the cartridge capped and immediately inserted into a Pima analyzer for 20 minutes incubation followed by automatic analysis of the test sample.
Statistical Analyses
Data were analyzed using MedCalc version 10.0.2.0 (MedCalc Software, Mariakerke, Belgium). Precision expressed as the CV was determined by dividing the SD of the measurements by the mean (CV = SD × 100/mean). We calculated the CV for instrument precision and intra-assay, inter-assay, and inter-instrument variations. Measurement of linear regression was determined using Passing–Bablok regression analysis. Pollock and Bland–Altman analyses were used to determine the mean biases and the limits of agreement (LOA = mean ± 1.96 SD) on CD4 count and on CD4%, respectively. Percentage similarity was calculated for each sample as Similarity = Average of methods A and B × 100/Method A (with A = reference and B = evaluated). For each group, the mean percent similarity and the CV were determined. We first performed comparisons between each alternative method (FACSCount CD4 or Pima CD4) and the FACSCalibur (Trucount) reference system in the overall data and then within each of the 3 CD4 T-cell count categories. Second, comparisons were done between FACSCount CD4 and FACSCount and between CD4 counts from capillary blood and those from venous blood on the Pima CD4.
Misclassification probabilities were calculated at ART initiation thresholds of 200, 350, and 500 cells per microliter for CD4 counts and 25% for CD4% setting of the FACSCalibur as the reference method to determine eligible patients on ART.