Evaluation of the Blood Stabilizers TransFix™ and Cyto-Chex^®BCT for Low-Cost CD4 T-Cell Methodologies

Abstract

TransFix(TM) and Cyto-Chex((R)) BCT (blood collection tube) reagents have been shown to maintain whole blood integrity for delayed immunophenotyping by flow cytometry. We evaluated the ability of these blood-stabilizing reagents to preserve HIV-seropositive blood for delayed CD4(+) T-cell quantification utilizing the Dynal((R)) Biotech T4 Quant Kit. TransFix was added to EDTA-anticoagulated whole blood and tested at a 1:10 dilution over 7 d using the Dynal (n = 21) manual method. Compared to baseline analysis, a significant decrease in mean CD4(+) counts was observed over time. Cyto-Chex BCT-preserved samples (n = 20) were tested for CD4(+) counts by Dynal over 7 d, with storage at varying temperatures: room temperature (21 degrees C), 37 degrees C, and 37 degrees C with intermittent storage at 42 degrees C. A significant decline in mean CD4(+) counts was observed in samples at all temperatures compared to baseline (p < 0.05). Increases in temperature to and above 37 degrees C resulted in a greater decline in mean CD4(+) counts over time. Our findings indicated that neither TransFix or Cyto-Chex BCT was a suitable blood stabilizer when used for delayed CD4(+) quantification with a low-cost manual CD4(+) bead-based method.

Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials

“…markers for the white blood cells in whole blood sample. These studies were in contrast to work done by Plate et al (2009), which illustrated that neither Transfix® nor Cyto-Chex® BCT was useful in preserving CD4+ quantification. In all of these studies, less than 5-color fluorophore staining was employed.…”

Interlaboratory comparison of the TransFix®/EDTA Vacuum Blood Collection tube with the 5 mL Cyto‐Chex® BCT

“…to this one (12,13,15), the authors believe that this study is the first assessment of the performance of the 3 mL TVT device with the 5 mL BCT device with regard to their shared intended use of stabilizing leukocytes for 14 days where statistically relevant donor numbers have been evaluated. BCT samples exhibit the statistical equivalent (P > 0.05) level of autofluorescence as the control tube on only two occasions: CD4 MFI within Cytotoxic T-cells for both time points.…”

“…However, similarly to the HIV cohort, BCT samples exhibit a marked increase in levels of auto-fluorescence compared to the control. to this one (12,13,15), the authors believe that this study is the first assessment of the performance of the 3 mL TVT device with the 5 mL BCT device with regard to their shared intended use of stabilizing leukocytes for 14 days where statistically relevant donor numbers have been evaluated. It was also designed to demonstrate that the devices performed consistently for donor samples of differing disease status, hence the inclusion of healthy and HIV+ samples.…”

Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development

“…Stability of light scatter and some surface markers have been reported for up to 14 days post-collection (Warrino et al, 2005;Plate et al, 2009;Davis et al, 2011;Ng et al, 2012) These products contain fixatives which render the samples unacceptable for functional studies. Furthermore, the fixatives may denature certain epitopes resulting in decreased binding by certain mAb.…”