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- MATERIALS AND METHODS
- LITERATURE CITED
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 μL 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
Leukemia is characterized by the overproduction of immature and malignant white blood cells (WBCs) that are unable to carry out normal hematopoietic functions (1). This disease is expected to affect more than 44,000 adults and ∼3,500 children under the age of 15 in the United States (2). Although more adults are affected by this disease compared to children, leukemia is the most common pediatric cancer, accounting for 25% of cancer cases, and the leading cause of death in children under 20 years of age (3, 4). Of the different types of leukemia, acute lymphoblastic leukemia (ALL) is the most common form in children affecting ∼80% of patients. Progress in the development of effective treatment and supportive care has improved the cure rate of ALL, particularly in children (5, 6). However, clinical and biological parameters, such as age and WBC count, may not be sufficient for individualized treatment stratification or risk assessment, resulting in the over-treatment or insufficient treatment of some patients (7, 8). Studies of minimal residual disease (MRD) have aimed to estimate leukemic burden after initial therapy, providing clinicians with an indication of the aggressiveness of the disease and the efficacy of treatment (9–11). Such information could be used to optimize treatment strategy, minimizing patients' risk for relapse and ultimately improving cure rates.
At diagnosis, patients with ALL may have more than 1012 leukemic cells (12). Patients are considered to be in complete remission, after therapy, if fewer than 5% of the cells in the bone marrow are identified as blasts according to morphological standards. However at this level, patients may still harbor as many as 1010 leukemic cells (12). As a result, more than 25% of ALL patients may eventually experience a relapse after induction therapy (13). Advances in flow cytometry and PCR techniques have been able to improve the detection of these residual leukemic cells, which represent MRD, at neoplastic cell proportions of <1% relative to normal nucleated blood cells. A number of flow cytometry and PCR studies have shown that patients with MRD levels of 0.01–0.1% (i.e., 1 leukemic cell to 1,000–10,000 normal nucleated cells) during or after therapy have higher chances of relapse (9, 14–16). However, neither technique is applicable to all patients (17, 18). Specific immnophenotypic or molecular probes may not be found in all patients and the sensitivity and specificity of the technique may vary with the choice of marker (6, 17–19). Furthermore, phenotypic switches or changes in antigen- receptor gene rearrangements that can occur during the course of the disease cause false-negative results (6, 18, 20, 21). Although most MRD assessments are performed on bone marrow aspirates or biopsies, few MRD studies showed that peripheral blood MRD monitoring in patients with ALL is possible and may even provide strong prognostic value in comparison to bone marrow MRD assessment (18, 22). For example, peripheral blood-based approaches can improve the frequency with which residual leukemia can be monitored, especially in children, for whom bone marrow aspirations are particularly difficult and painful. Therefore, improved methods in the detection of leukemic cells within bone marrow aspirates or peripheral blood could significantly impact the treatment of this disease.
Light scattering techniques in conjunction with microfluidic-based devices may be a novel approach suitable for monitoring MRD. Microfluidic-based platforms have the advantage of allowing for single cell analysis and enumeration, as conventional bench top flow systems, but at a lower cost, better portability and higher throughput. Additionally, since light scattering is a natural source of signal contrast, it obviates the need for cell labeling, minimizing the perturbation of biological samples as well as the need for complicated and time consuming processing, as in PCR and standard flow cytometry. Though light scattering in the forward and perpendicular direction are routinely used in standard flow cytometry, light scattering in the backward direction has not been applied thus far. Studies of light scattered in the backwards direction from cell monolayers and tissue samples have shown the sensitivity of backscattering to morphological differences that occur during neoplastic transformation, thus allowing for the discrimination of normal from cancerous cells (23–25). In addition, we have previously shown characteristic differences between the back-scattered light from ALL cells (Nalm-6) and that from red blood cells (RBCs), peripheral blood mononuclear (PBMCs) and polymorphonuclear (PMNs) white blood cells normally found in the blood (Ref. 26; Greiner et al. in press, Cytometry A). Here, we describe the work extending our light scattering spectroscopy studies on static, layered cell samples to flowing leukemic and normal blood cell samples, further assessing the potential applicability of this approach for MRD assessment of ALL. Specifically, leukemic cells, in various concentrations from 0.01% to >20%, are combined with normal blood cells and flowed in single microfluidic channels for light scattering measurements. Classification equations, based on discriminant analysis, are applied to identify leukemic cells from normal blood cells based on differences in their backscattering characteristics. Finally, statistical analyses are performed to prospectively assess the diagnostic performance of our approach.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
Flow cytometry and PCR have improved the assessment of MRD of ALL compared to morphological analysis. Nonetheless, these techniques are limited by the large volume of blood that needs to be sampled and extensive sample processing that not only increases cost, but also can result in the loss of already rare cells. Microfluidics is a fast growing technology that can potentially overcome some of these limitations. The reduction in scale leads to significant reductions in the volume of needed reagents and sample and often allows for a degree of automation. In combining microfluidics with backscattering, sample processing can be minimized and reliability maybe improved. This unique combination of capabilities makes for an appealing candidate for the development of a portable, simple, and cost-effective alternative for MRD assessment in leukemia patients, particularly appealing in developing countries where material resources and qualified personnel are limited and cure rates are lower (34). In resource-rich countries, it can additionally result in more continuous monitoring of patients, allowing for more effective therapeutic interventions. Further benefits may also be realized in the simultaneous monitoring of WBCs and leukemia cells. For example, absolute lymphocyte counts during chemotherapy have recently been shown to be an independent predictor of ALL patient relapse and survival (34). But most importantly, a diagnostic or monitoring test for leukemia that would be based on the use of a small volume of blood would be beneficial to pediatric patients throughout the world.
In this study, we demonstrate that, using the backscattered light at 405, 488, and 633 nm, we can detect characteristic light scattering distributions from flowing RBCs, PBMCs, PMNs, and leukemic cells. These results are in agreement with previous light scattering studies performed with similar, but static cell samples (Ref. 26; Greiner et al., manuscript submitted). Specifically, the scattering intensity from PMNs is highest in comparison to Nalm-6 and PBMCs. RBC scattering is the lowest at 405 nm, but higher than that of PBMCs and leukemic cells at 633 nm. Thus, measurements performed at 405 nm and 633 nm are particularly sensitive to hemoglobin absorption changes, allowing for discrimination of RBCs from the other cell types. This was also noted by Ost et al. in their flow cytometric measurements of forward and perpendicular scattering of diluted blood samples with a 413 nm laser source (35). A steeper decrease in the wavelength dependence of the backscattered light from Nalm-6 cells relative to that from the normal PBMCs and PMNs, as well as differences in the scattering intensities, forms the basis for separating leukemic from non-leukemic WBCs.
To assess the diagnostic potential of our approach, we evaluated prospectively blood samples with mixtures of RBCs, WBCs, and varying concentrations of leukemic cells in the range of 0.01–35% (leukemic:WBC). We used calcein fluorescence labeling of the leukemic cells, to assess the validity of the classification results acquired based on using the backscattered light alone. Even though this is not a perfect gold standard, it provides a good measure of performance.
Our results indicate that the proposed light scattering based diagnostic would perform better for the analysis of blood samples that undergo an initial density gradient isolation step to collect mostly the mononuclear cells of the sample. This is still a much simpler sample preparation requirement in comparison to multi-labeling for flow cytometry or to RNA/DNA extraction and multiple gene amplification for PCR. Even though a portion of cells could be lost during this step, with one study as an example indicating ∼67% cell recovery rate, we could ascertain the presence of leukemia at a power level of 80% using less then 63 μL of whole blood (for 0.01% leukemia cells relative to PBMCs) (36). This is a smaller blood volume in comparison to the volume needed for other MRD assessment techniques, which can range from 2 to 5 mL of bone marrow aspirates or 10 mL of peripheral blood samples with leukemic cell proportions of 0.01% or more relative to normal mononuclear cells (37).
When only a simple RBC lysis processing step is expected prior to light scattering assessment, which would result in the presence of both PBMCs and PMNs along with leukemic cells, the blood volume required to have a positive test for the presence of leukemic cells with a 0.80 power level increases to 71 μL. Even if 10% of leukemic cells are lost during this initial processing, we would still be able to perform the test using very small volumes of blood that could be isolated with a simple finger prick (38). Since in this case, both the RBC lysis and the light scattering based measurements could be performed using microfluidics, the potential for developing a truly minimally invasive, cheap and highly portable lab-on-a-chip type device would be highly feasible (38).
Additionally, we should note that the classification algorithm developed in this study is simple and can be implemented for real-time data analysis. A decrease in the sensitivity in our validation data compared to our training data suggests that further improvements in classification performance may be possible by considering larger data sets or other, slightly more complex algorithms, such as discriminant analysis based on quadratic instead of linear classification optimization or discriminant analysis with kernels (39). Nonetheless, the application of a standard classification algorithm presents improvements over the sophisticated data analysis software and personnel training needed to correlate the various parameters in multidimensional space for proper cell classification, when performing multiparameter flow cytometry (9, 40). Finally, automated data analysis in combination with the capability for parallel processing in a microfluidic device offers the potential for processing times that are significantly lower than the 24-h time frame typically offered by standard PCR and flow cytometry analysis (9, 41).
To the best of our knowledge, our backscattering microfluidic-based platform is the first to be applied for MRD assessment. Nonetheless, we expect to easily apply our microfluidic chip to other forms of cancer, since spectroscopy studies have noted characteristic backscattering from other types cancer cells (23–25). As the number of studies increasingly supports the clinical value of enumerating circulating tumor cells in blood, researchers are developing microfluidic-based platforms to capture circulating epithelial cancer cells (42, 43). One example is a microfluidic chip developed to capture epithelial tumor cells, without blood pre-processing, using antibody coated microposts (42). This device can process a large volume of blood (1–2.5 mL h−1) compared to most microfluidic chips, but the capture rate varies from 20 to 60% depending on the flow rate (42). Moreover, the average purity of the captured cells is ∼50% and therefore, it requires further cell characterization procedures via cell staining and fluorescence imaging (42). In comparison, our microfluidic platform technique is simpler and may be easier to apply to different types of cancer.
In conclusion, we have demonstrated and assessed the potential of back scattering in combination with a microfluidic chip platform for MRD assessment of ALL. We demonstrate that this approach could lead to a simple, cost-effective, minimally invasive, portable alternative that allows for automated and real-time diagnosis compared to conventional MRD assessment techniques.