Confocal backscattering‐based detection of leukemic cells in flowing blood samples

Abstract

The prognostic value of assessing minimal residual disease (MRD) in leukemia has been established with advancements in flow cytometry and PCR. Nonetheless, these techniques are limited by high equipment costs, complex, and costly cell processing and the need for highly trained personnel. Here, we demonstrate the potential of exploiting differences in the relative intensities of backscattered light at three wavelengths to detect the presence of leukemic cells in samples containing varying mixtures of white blood cells (WBCs) and leukemic cells flowing through microfluidic channels. Using 405, 488, and 633 nm illumination, we identify distinct light scattering intensity distributions for Nalm-6 leukemic cells, normal mononuclear (PBMC) and polymorphonuclear (PMN) white blood cells and red blood cells. We exploit these differences to develop cell classification algorithms, whose performance is evaluated based on simultaneous acquisition of light scattering and fluorescence flow cytometry data. When this algorithm is used prospectively for the analysis of samples consisting of mixtures of PBMCs and leukemic cells, we achieve an average specificity and sensitivity of leukemic cell detection of 99.6 and 45.2%, respectively. When we consider samples that include leukemic cells along with PMNs and PBMCs, which can be acquired using a simple red blood cell lysis step following venipuncture, the specificity and sensitivity of the approach decreases to 91.6 and 39.5%, respectively. On the basis of the performance of these algorithms, we estimate that 42 or 71 lL of blood would be adequate to confirm the presence of leukemia at an 80% power level in samples containing 0.01% leukemia to either PBMCs or PBMCs and PMNs, respectively. Therefore, light scattering-based flow cytometry in a microfluidic platform could provide a low cost, highly portable, minimally invasive approach for detection and monitoring of leukemic patients. This could offer significant improvements especially for pediatric patients and for patients in developing countries. ' 2011 International Society for Advancement of Cytometry

ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

“…In order to evaluate level of autofluorescence, blood was also collected from NOD/SCID mice that were not injected with any tumor cells. All blood samples were collected in heparin (100 USP units/sample), diluted and processed the same day for detection of GFP‐positive CTCs using a confocal flow cytometry system at a flow rate of 4.5 μl/min, as previously described (Greiner et al , ). Enumerated cells were identified with a previously described algorithm based on the combined detection of peaks appearing simultaneously in a fluorescence channel optimized for GFP detection and in at least one of the channels detecting scattering at either 405 or 488 nm (Hwu et al , ) (see supplementary materials).…”

Endogenous light scattering as an optical signature of circulating tumor cell clusters

“…Samples were flowed in 30x30 µm 2 microfluidic channels made of polydimethylsiloxane (PDMS) with a glass bottom microscope slide, described previously [24]. Flexible tubing on one end of the devices was submerged in a cell solution constantly being stirred by a magnetic spin bar, while tubing on the other end was connected to a syringe pump pulling at 3 µl min −1 .…”

“…The confocal backscatter and fluorescence flow cytometer has been described in greater detail by Greiner et al [24]. Briefly, light from 405nm, 488nm, and 633nm lasers was directed into a single illumination path by mirrors and dichroic filters.…”

“…The latter number was approximated based on the cell concentration assessed by haemocytometer counts, the volumetric flow rate of 3μL/min, and the total acquisition time. To calibrate for day-to-day variations in instrument throughput, the 405, 488, and 633 nm scattering intensities were normalized for each cell peak by the mean intensity of the signal for that channel reflected by a spectralon reflectance standard [24].…”

Label-free impedance detection of cancer cells from whole blood on an integrated centrifugal microfluidic platform

“…Consequently, techniques including density-gradient based centrifugation , microfiltration (Zheng et al, 2011), hydrodynamic sorting by size (Loutherback et al, 2012), separation by cell deformability (Tan et al, 2009), immunoassay based on tumour-specific surface protein expression (Alix-Panabieres and Pantel, 2014), and molecular markers (Nagrath et al, 2007) have been employed to effectively separate these cells from normal blood cells before capture. Subsequent enumeration of captured CTCs has been demonstrated with such techniques as immunofluorescence (Ignatiadis et al, 2008), confocal microscopy (Greiner et al, 2011), absorbance (VilaPlanas et al, 2011), chemiluminescence (Hun et al, 2010), interference spectroscopy (Kumeria et al, 2012), surface-enhanced Raman scattering (Lee et al, 2014b) and surface plasmon resonance (Law et al, 2011). However, a simple, sensitive and robust assay that allows routine detection and subsequent molecular characterization of CTCs in a clinical setting has not been reported.…”