Physics of a rapid CD4 lymphocyte count with colloidal gold

Abstract

The inherent surface charges and small diameters that confer colloidal stability to gold particle conjugates (immunogold) are detrimental to rapid cell surface labeling and distinct cluster definition in flow cytometric light scatter assays. Although the inherent immunogold surface charge prevents self aggregation when stored in liquid suspension, it also slows binding to cells to timeframes of hours and inhibits cell surface coverage. Although the small diameter of immunogold particles prevents settling when in liquid suspension, small particles have small light scattering cross sections and weak light scatter signals. We report a new, small particle lyophilized immunogold reagent that maintains activity after 428C storage for a year and can be rapidly dissolved into stable liquid suspension for use in labelling cells with larger particle aggregates that have enhanced scattering cross section. Labeling requires less than 1 min at 208C, which is $30 times faster than customary fluorescent antibody labeling. The labeling step involves neutralizing the surface charge of immunogold and creating specifically bound aggregates of gold on the cell surface. This process provides distinct side-scatter cluster separation with blue laser light at 488 nm, which is further improved by using red laser light at 640 nm. Similar comparisons using LED light sources showed less improvement with red light, thereby indicating that coherent light scatter is of significance in enhancing side-scatter cluster separation. The physical principles elucidated here for this technique are compatible with most flow cytometers; however, future studies of its clinical efficacy should be of primary interest in point-of-care applications where robust reagents and rapid results are important. ' 2011 International Society for Advancement of Cytometry Key terms CD4 lymphocytes; nanoparticles; point of care; light scatter; cytometry FLOW cytometry is the recognized clinical standard by which human T-cell subclasses are enumerated in HIV-infected patients. Bringing this standard to resource poor environments requires simplified instrumentation, robust reagents, and rapid test results. This report investigates light scatter as a simplification for flow cytometry instrumentation, immunogold as a robust reagent, and a novel cationic surfactant to speed immunogold binding to cell surface antigens, thus potentially providing CD4 T-cell results to patients in minutes without the need for a return visit.The potential for simplification offered by light scatter detection of immunogold conjugates of monoclonal antibodies has attracted attention for more than 2 decades. In 1984, Bohmer and King (1) reported immunogold labelling of mouse spleen lymphocytes. They were unable to achieve useful side scatter cluster separation with an argon laser operating at several wavelengths from 451.7 nm to 528.9 nm but were able to achieve side scatter cluster separation with a HeNe laser at 632.8 nm. The impractical feature of their assay was that to overcome very long...

On-Chip Cytometry using Plasmonic Nanoparticle Enhanced Lensfree Holography

“…Each specimen is imaged sequentially at seven different illumination wavelengths (λ) ranging from 480 nm to 950 nm, each with a bandwidth of ~10 nm. 100-nm diameter Au NPs (plasmon-resonance peak wavelength: ~554 nm, see Figure S2a) are conjugated with antiCD4 antibodies (denoted as Au-antiCD4) and are used to specifically label CD4 cells (see Methods)40. In our initial experiments, CD4 cells with and without Au NP labeling exhibited strong contrast differences under a conventional darkfield scattering microscope (see Figures 1c,f).…”

“…Labeling CD4 cells with Au-antiCD4 conjugates followed a similar procedure described by Hansen et al40. CD4 cells were maintained in RPMI 1640 medium under normal cell culture conditions (37°C, 5% CO 2 ).…”

“…Polybrene was added to CD4 cells with a final concentration of 10 μg/mL. Polybrene served as a charge neutralization agent to increase the affinity of negatively charged antibody to the cell membrane during the incubation40. CD4 cells were then mixed with 4, 8, 15, and 60 μL of Au-antiCD4 conjugates (OD ~ 10), and incubated at 37°C with continuous mixing (100 rpm) overnight.…”

Plasmonic Nanoparticle-Based Digital Cytometry to Quantify MUC16 Binding on the Surface of Leukocytes in Ovarian Cancer

“…This limitation arises from a combination of factors including cellular autofluorescence, interference and attenuation of fluorescence emission due to the nucleus and organelles, and cross-talk between fluorescent labels. Although several studies have proposed to utilize plasmonic gold nanoparticles to enhance sensitivity and to avoid photobleaching and spectral overlap of fluorescent labels in multiparametric flow cytometry, − these studies mainly investigated cell surface receptors at a density of 20,000 to 100,000 per cell (e.g., CD4 and CD8 receptors). Here, the challenge was to detect low levels of receptor-bound antigens/ligands on a single cell.…”