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Keywords:

  • light scattering;
  • spectroscopy;
  • leukemia;
  • blood cells

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Leukemia is the most common pediatric cancer and leading cause of cancer related deaths in children. Improvements in the assessment of leukemic cells have the potential to influence not only the diagnosis of leukemia, but also the risk assessment of patients during the course of the treatment, both of which are important for improving the cure rate for this disease. In this study, we report on the design and performance of a confocal laser based system built to collect backscattered light over a range of 26° at 405, 488, and 633 nm to discriminate leukemic cells from normal red blood cells (RBC) and white blood cells (WBC). The design of the system is based on the spectral differences observed from spectroscopy measurements with a similar system designed with a white light source. Significant differences are observed in the intensity and wavelength dependence of leukemic cells from normal RBC and WBC. Specifically, the distinct light scattering of RBC is due to hemoglobin absorption, allowing for its discrimination from leukemic cells, mononuclear, and polymorphonuclear WBC particularly at certain wavelengths. Meanwhile, the high scattering intensities of polymorphonuclear WBC reflect the intracellular complexity of these cells in comparison to the leukemic or normal lymphocytes. Additionally, the detected light scattering spectra for leukemic cells are consistently steeper in comparison to normal WBC, which we attributed to differences in the fractal organization of intracellular scatterers. Based on our findings, the system has potential applications in the detection and quantification of leukemic cells in blood either in vivo or in vitro, using microfluidic-based systems, for disease monitoring. © 2011 International Society for Advancement of Cytometry

Hematopoiesis, which describes the production of mature blood cells from stem cells in the bone marrow, is a highly regulated and precise process. Disruption in normal hematopoiesis can result in serious conditions such as leukemia, the most common blood cancer. Leukemia is characterized by the overproduction of malignant and immature leukocytes that are unable to carry out normal hematopoietic functions, resulting in clinical manifestations related to infectious or hemorrhagic complications (1). It is expected to affect more than 44,000 adults and ∼3,500 children under the age of 15 in the United States (2). Although leukemia affects more adults than children, it is the most common pediatric cancer, accounting for 25% of cancer cases, and is the leading cause of disease related death in children under 20 years of age (3, 4). Acute lymphoblastic leukemia (ALL), the most common form of leukemia in children, involves the increased proliferation of lymphocytes arrested in the early stage of development, resulting in overcrowding in the bone marrow and their eventual presence in the circulation, where they are not commonly found.

Blood analysis has been found to be of clinical importance in the prognostic assessment of patients after therapy, with patients showing a persistent number of leukemic blasts in the bone marrow or peripheral blood associated with slow therapeutic response and a high risk for relapse (5–7). Morphological analysis of cell samples, which is the traditional technique for assessing the number of residual leukemic cells that represent minimal residual disease (MRD), is subjective and limited in sensitivity (8). Thus, a considerable proportion of patients who are considered to be in remission, defined by a morphological criterion of having less than 5% blast cells in the bone marrow, may in fact harbor as many as 1010 residual leukemic cells and eventually experience a relapse (8, 9). To improve the assessment of MRD for monitoring therapeutic response, methods, such as flow cytometric detection of aberrant immunophenotypes and polymerase chain reaction (PCR) analysis for gene rearrangement, have been developed (10–12). Nonetheless, the applicability of flow cytometry or PCR is limited and depends on the type of ALL and MRD assay used (9, 13–16). Moreover, false positives may result not only from immunophenotypic changes or clonal gene rearrangements, but also from practical differences in laboratory protocols, including sample handling, flow cytometry gating or choice of PCR reference genes (10, 14, 17). Therefore, improvements in the detection of leukemic cells in blood or bone marrow aspirates can have a significant clinical impact, by improving MRD assessment and allowing effective therapeutic intervention.

Optical techniques have the potential to yield improved or novel approaches to detect leukemia and monitor MRD. For example, in vivo flow cytometry, which combines the principles of confocal detection and flow cytometry, has been used for the detection of fluorescently labeled cancer cells directly in the circulation of animals and has provided insights in terms of the kinetics of cancer cells and metastasis (18–25). However, since few dyes are approved for use in humans, techniques that are based on natural sources of signal contrast may have more clinical applicability. Specifically, intrinsic light scattering is sensitive to cellular and subcellular morphology and organization and has been used for the discrimination of different cell types. The angular dependence of light scattering has been shown to differentiate between sub-populations of white blood cells (WBC) in a flow cytometry set-up (26–29). In the context of cancer, light scattering spectroscopy has identified differences in the wavelength dependence of backscattered light between normal and diseased cells due to cellular transformations in early stages of cancer development (30–36). Despite these studies, little work has explored the potential of light scattering for the identification of leukemic cells from blood cells for leukemia diagnosis or MRD assessment. With this in mind, we developed light scattering based systems to characterize the backscattering of leukemic and normal white blood cells (WBC) and red blood cells (RBC). These systems provide the basis for the design of in vitro microfluidic-based and potentially in vivo light scattering flow cytometry devices.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

Optical System Design

Spectroscopy measurements of non-flowing Nalm-6, WBC, and RBC, were performed using a white light arc-lamp backscattering spectroscopy system (ABSS), which was based on the design of previously developed fluorescence in-vivo flow cytometry and angular light scattering spectroscopy systems (18, 37). As shown in the optical schematic of this system, Figure 1a, light from a 100 W Xenon lamp was spatially filtered, collimated, and linearly polarized before filling the back aperture of a long working distance 20×, 0.4 NA microscope objective (Olympus). The same objective collected the backscattered light from the cells, a portion of which was directed to an imaging CCD to verify sample placement. The remaining scattered light was directed towards the detection path with an analyzer oriented either parallel or perpendicular to the input polarization and an achromatic lens that focused the scattered light into a pinhole. This detection pinhole was placed confocal to the front focal plane of the objective to reduce the signal contribution from out-of-focus objects and to optimize the signal detection of light scattered by the cells at the front focal plane of the objective. To further reduce the contribution from specular reflections from various optical surfaces that can degrade the backscattered signal from the cells, a beam block was placed covering over half of the microscope objective back aperture (see inset in Fig. 1a). Backscattered light that made it through the pinhole was then spectrally filtered by a series of bandpass filters in a filter wheel prior to being detected by a photomultiplier tube, PMT1 (Hamamatsu), that integrated the intensity of scattered light at specific wavelengths. Each bandpass filter had a bandwidth of 20 nm with center wavelengths from 450 to 730 nm. Integrated backscattered intensity from the PMT was converted into voltage, sampled by a data acquisition card, and recorded for analysis into a computer. With the beam block at the back of the objective, the system detected light backscattered at angles less than 26° (setting 0° as the exact backscattering angle). The lateral resolution was determined by laterally scanning across the focal plane a single 4.5 μm polystyrene bead suspended in 10% agarose solution (w/v). The axial resolution was measured by axially scanning a mirror. Based on the full-width half max of the resulting intensity profiles, the lateral and axial resolution of the ABSS were 20 and 60 μm, respectively, for a 500 μm pinhole.

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Figure 1. (a) Schematic of the ABSS. (b) Schematic of the LBSS. Insert drawing shows how specular reflections are blocked in both the ABSS and LBSS by blocking the back aperture of the sample objective (×20). Legend: D = dichroic, L = lens, M = mirror, P = pinhole, BS = beam splitter, Pol = polarizer, and Diff = diffuser.

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From the spectroscopy results with the white light system, we selected laser sources emitting light at 405 (56IC425, Melles-Griot), 488 (PCI13589, Spectra-Physics), and 633 (1144P, JDS Uniphase) nm for the construction of a laser-based backscattering system (LBSS) that could be ultimately used for rapid data acquisition from flowing cell populations. As shown in the optical schematic for this system, Figure 1b, the beams from the three lasers were combined into a single illumination path by dichroic filters and passed through a diffuser to reduce laser speckle. The beam was then focused by a 40× objective (0.60 NA, Olympus) into a 50 μm core diameter multimode fiber (0.20 NA, Thorlabs). The beam exiting the fiber was re-imaged at the sample plane at the front focal plane of the 20× sample objective (0.40 NA, Olympus) via a 10× objective (0.30 NA, Olympus). As in the ABSS, the scattered light from the sample was collected by the objective, directed to an analyzer, and spatially filtered by the confocal pinhole in the detection path. We also used a beam block at the back aperture of the 20× objective to reduce signal contributions from specular reflections. Scattered light that passed through the pinhole was spectrally separated by dichroic filters and directed to three PMTs, to detect light scattered at each wavelength. The optical fiber, 10× and 40× objectives in the illumination path were selected for the design of LBSS to achieve comparable lateral and axial resolutions to the ABSS. Accordingly, the lateral and axial resolutions of the LBSS were 15 and 80 μm, respectively, for a 300 μm confocal pinhole.

Cell Preparations

Nalm-6, a B-cell precursor cell line established from the peripheral blood of a 19-year-old man with ALL, was used to compare the light scattering of leukemic cells to normal blood cells (38). Nalm-6 cells were purchased from DSMZ and cultured in RPMI-1640 with 10% fetal bovine serum (FBS) plus 1% penicillin/streptomycin (PSF). RBC were acquired from the Massachusetts General Hospital blood bank. Peripheral blood mononuclear white blood cells (PBMC) and polymorphonuclear white blood cells (PMN) were isolated from fresh blood acquired by venipuncture from healthy donors and processed within 2 hours of the blood draw. This procedure was approved by the Tufts Institutional Review Board. For PBMC isolation, 1 part whole blood was layered on 1 part density gradient, Histopaque-1077 (Sigma-Aldrich), and centrifuged at 400 × g for 30 minutes at room temperature. The PBMC fraction, found between the density gradient and the plasma, was collected and washed twice in phosphate-buffered saline (PBS) without Ca2+ and Mg2+. For PMN isolation, a rosette sedimentation technique was used, as previously described (39). Briefly, 50 μL of RosetteSep antibody cocktail for granulocyte enrichment (Stemcell Technologies) was added to every 1 mL of whole blood for 20 minutes. The sample was then diluted with an equal volume of PBS with 2% FBS, layered on top of the density gradient, Histopaque 1119 (Sigma Adrich), in 1:1 ratio, and centrifuged at 1200 × g for 20 minutes. The PMN enriched layer was collected and washed twice in RPMI supplemented with 2% FBS. After collecting the cells, the cell concentration was determined using a hemacytometer.

Light Scattering Measurements

For initial measurements on the full spectral dependence of scattering from each cell type, measurements were obtained for different wavelengths using the ABSS. Pure cell samples of Nalm-6, PBMC, PMN, or RBC, at a concentration of more than 7 × 106 cells/mL, were suspended in 300 mL of RPMI-1640 with 2% FBS and 1% PSF and placed in a 10.6 × 6.4 mm well, attached to a 1 mm microscope slide. The cells were allowed to settle for at least 30 minutes prior to measurements, forming several cell layers that were <120 μm thick. This ensured that more than one cell was within the confocal measurement volume to enhance the level of detected backscattered signal. Light scattering measurements were acquired along the parallel (I||) and perpendicular (I) polarization relative to the incident light from 3 to 5 different areas at one particular axial position, 45 μm above the microscope slide. To account for background scattering (Imedia), cell media (RPMI + 2% FBS) used to suspend the cells was measured using similar sample conditions. Lastly, a 99% white light reflectance standard (LabSphere) was measured from 10 different areas and the scattering intensities were averaged (Ispectralon) to correct for the angular and spectral dependence of the system. Light scattering measurements were obtained with the LBSS to acquire the backscattering at 405, 488, and 633 nm using a similar protocol. Independent measurements of each cell type were obtained at least four times to assess signal reproducibility.

The scattering intensity from the cells at each polarization was normalized at every wavelength as follows:

  • equation image

where Pfactor is the ratio of the illumination power used during cell measurements to the power used during spectralon measurements. Light scattering was obtained with the analyzer either parallel or perpendicular to the polarizer to determine the type of scattering from cells associated with focused illumination and confocal detection. Specifically, multiply scattered light depolarizes the input beam contributing to scattering intensities in both polarization directions, that is, I|| and I. However, light that has been scattered once or a few times maintains the original polarization contributing mainly to detected light along the same polarization as that of the incident light (I||), particularly for scatterers significantly smaller than the wavelength of the scattered light. Therefore, the differential scattering, Idiff =I||I, is mainly attributed to single scattering.

To quantify the differences observed in the detected light scattering spectra, we described them using a model with an inverse power-law wavelength dependence, that is, I(λ) ∝ λγ. Such a model has been used previously to describe light scattering spectra acquired from different types of cells, and is consistent with a fractal organization of subcellular scatterers, even though the precise nature of the subcellular fractality is not thoroughly defined yet (30, 32, 39–41).

Discriminant Analysis

To assess the strength of cell discrimination by light scattering, we used SPSS software to perform discriminant analysis of the normalized light scattering intensities from all areas measured from independent samples. For the ABSS data, a number of areas were measured from PBMC (n = 19 from 4 independent samples), PMN (n = 19 from 4 independent samples), Nalm-6 (n = 18 from 4 independent samples), and RBC (n = 16 from 4 independent samples) and used in the analysis (ntotal = 72). For the LBSS, we acquired light scattering from 33 areas within 5 PBMC samples, 30 areas within 4 PMN samples, 48 areas within 10 Nalm-6 samples, and 48 areas within 9 RBC samples (ntotal = 159).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

ABSS Measurements

To assess the feasibility of using backscattered light to discriminate leukemic cells from normal peripheral blood cells, we determined the scattering characteristics of cells at different wavelengths using the ABSS. Light that has been scattered once or a few times from submicron spherical scatterers largely retains the polarization of the incident light. The average backscattered intensities, I||, I, and Idiff, from each cell type are shown in Figures 2a–2c, respectively. The error bars represent standard deviations indicating the variability in backscattering intensities from biological samples. We note that most of the scattering from cells is singly or minimally scattered light, since I is on average <6%, of I||. Moreover, I is found to be spectrally flat, therefore I|| and Idiff were spectrally similar.

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Figure 2. Mean backscattering spectra from ABSS measurements of leukemic Nalm-6 (○), PBMC (⋄), PMN (▵), and RBC (x) samples. (a) Spectra with analyzer (a) parallel to the input polarization (I||) and (b) perpendicular to input polarization (I). (c) Spectra of the differential scattering (Idiff). (d) Inverse power-law fits (dashed lines) to I|| spectra for Nalm-6, PBMC, and PMN.

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Comparison of minimally or singly scattered light between different cell types reveals systematic differences in both the spectral dependence as well as the intensity. Specifically, the backscattering spectrum of RBC is dominated by the characteristic hemoglobin features, as expected. Interestingly, while both normal WBC populations, that is, the granulocytes and mononuclear cells, exhibit a fairly flat wavelength dependence, the leukemic cells are characterized by a decrease in intensity as a function of wavelength that can be described well by an expression with an inverse power-law wavelength dependence, I(λ) ∝ λγ, as shown in Figure 2d. When we employ the same expression to describe the wavelength dependence of the PBMC and PMN spectra, we find that while the exponents for these two populations are similar (γ = 0.04 ± 0.31 for PBMC and γ = 0.08 ± 0.18 for PMN), they are both statistically different (P < 0.05) from the exponent characterizing the leukemic cells (γ = 1.31 ± 0.23 for Nalm-6).

To establish wavelength regimes with the highest potential of identifying the different blood cell populations, we use discriminant analysis on normalized scattering intensity measurements acquired with the ABSS system. The results from the discriminant analysis at each scattering wavelength are summarized in Figures 3a and 3b. Figure 3a compares the degree of correctly classified cells as normal blood or leukemic cells. We find that more than 77.8% of normal and cancerous cells are classified accurately in the wavelength regions corresponding to 440–520, 540–580, and 620–640 nm. Consequently, both sensitivity and specificity are high at these wavelength regions, as shown in Figure 4b. Specifically, the sensitivity is at least 77.8, 88.9, and 83.3% in wavelength regions 440–520, 540–580, and 620–640 nm, respectively. The specificity is better than 92.6, 90.7, and 79.6% at 440–520, 540–580, and 620–640 nm, respectively. In addition, we note that cell classification, sensitivity and specificity are best at the shortest wavelengths measured, that is, 440–500 nm, attributed to large differences in absorption by hemoglobin, and the steeper wavelength dependence of scattering exhibited by the leukemic cells. For this reason, we expect wavelengths in the 400–440 nm to allow additional improvements in classification both between RBC and non-RBC and between normal and leukemic WBCs.

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Figure 3. Results from discriminant analysis of normalized scattering intensities from ABSS measurements. (a) A comparison of the percentage of cancer cells and normal cells correctly classified and (b) a comparison of specificity and sensitivity at each scattering wavelength.

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Figure 4. Mean backscattering spectra at 405, 488, and 633 nm from LBSS measurements with the analyzer oriented parallel to the input polarizer (I||). Inverse power-law fits to I|| spectra are shown in dashed lines for Nalm-6 (○), PBMC (⋄), and PMN (▵). Scattering for RBC (x) are also shown but without a power-law fit.

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LBSS Measurements

Based on spectral differences noted from the data acquired with the ABSS system, we designed the LBSS to deliver light at 405, 488, and 633 nm to enable simultaneous acquisition of all wavelength information and the delivery of enough power to acquire backscattering signal ultimately from a single cell flowing either in a microfluidic device or a small blood vessel. Since the contribution of multiply scattered light is minimal and the spectra for I|| are similar to those for Idiff, scattering measurements with the LBSS are obtained only with the analyzer parallel to the polarizer, reducing the total measurement time and/or complexity of the system. Data acquired with the LBSS are consistent with the dominant light scattering features of the cell populations characterized by the ABSS system (Figure 4). Specifically, the backscattering signals acquired from RBC exhibit an increasing trend as a function of wavelength, with 405 nm yielding the highest difference in intensity between RBC and non-RBC. In agreement with observations from ABSS measurements, RBC scattering intensity is higher than that of the Nalm-6 and PBMC cells at 633 nm. Similarly, the intensity of PMN scattering is highest for all scattering wavelengths compared to all other cell types. No statistical differences are observed in the calibrated scattering intensity values at 488 and 633 nm from LBSS compared with 490 and 630 nm from ABSS measurements for all cell types based on a two-tailed t-test with P = 0.05.

As in the white light spectroscopy measurements, we note a steeper wavelength dependence of the scattering from leukemic cells in comparison to normal WBCs. When the inverse power-law wavelength dependent model is used to characterize the detected wavelength dependence of the scattered light using this system, the exponent values are γ = 1.57 ± 0.38 for Nalm-6, γ = 0.11 ± 0.56 for PBMC, and γ = 0.04 ± 0.40 for PMN. We find that these values are not statistically different from those obtained from ABSS measurements based on a 2-tailed t-test and P = 0.05. Similarly, we determined the potential for cell classification by light scattering by performing discriminant analysis on the backscattered light at 405, 488, and 633 nm acquired with the LBSS. Results show that with the light scattering from all three wavelengths, more than 80% of our Nalm-6 samples can be correctly classified, as shown in Table 1. Additionally, normal PBMC and PMN can be discriminated from RBC.

Table 1. Classification Results Based on Discriminant Analysis of Calibrated Scattering Intensities at 405, 488, and 633 nm from LBSS Measurements of Leukemic and Normal Blood Cell Samples
Original Group
 Predicted Group Membership (%)
Cell TypeRBCPBMCPMNNALM
  1. Cells correctly classified (%) 94.3.

  2. Normal WBCs were isolated from two donors.

RBC100000
PBMC010000
PMN001000
NALM018.8081.3

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED

In our study, we characterized the backscattering of leukemic cells, WBCS, and RBC at various wavelengths. This approach takes advantage of the light scattering spectral differences of these populations that are attributed to hemoglobin (for the differentiation of RBC from non-RBC) as well as differences in the morphology and organization of the cells, especially in the context of separating leukemic from normal WBCs.

Hemoglobin absorption effects were observed in both ABSS and LBSS measurements of RBC. Notably, hemoglobin absorption resulted in the lowest detected intensities for RBC for wavelengths up to 600 nm. At longer wavelengths, higher scattering was observed for RBC than for normal PBMC and cancerous Nalm-6 cells. These results suggest that the choice of wavelength can be critical in cell discrimination. Indeed, this was noted in a flow cytometry study that showed improved discrimination of WBC sub-populations from RBC at a shorter wavelength, namely 413 nm, compared with 488 nm (29). By selecting wavelengths associated with low, medium, and high hemoglobin absorption, as in our LBSS design, RBC may readily be identified from both normal and cancerous WBCs.

Meanwhile, morphological scale invariance is evident by the inverse power-law spectral dependence of scattering, likely reflecting some type of fractal subcellular morphology especially in diseased WBCs. Although the exact form of the fractal nature (e.g., self-affine vs. mass fractal geometry) cannot be uniquely inferred by our current light scattering experiments, we note that changes in the light scattering power exponent, γ, have been correlated to scale invariant subcellular inhomogeneities observed by differential interference contrast (DIC) microscopy on these cells (42). Similar power-law spectral dependence has been observed for leukemic and normal WBCs in a previous study by our group that characterized the scattering at narrower angles, (0° ± 4° from the exact backscattering angle), but with γ values that are higher in comparison to our study (42). Specifically, the wavelength dependent exponent values for fits to spectra formed by light scattered at 1° relative to exact backscattering angle from the previous study are γ = 1.83 ± 0.07, γ = 0.86 ± 0.10, and γ = 0.7 ± 0.04 for Nalm-6, PBMC, and PMN, respectively. Since our spectra represent the scattered light over a large range of angles (0–26°) as opposed to a specific angle (i.e., 1°), the differences in the exponents suggest that light scattering at higher angles exhibits a more shallow wavelength dependence, resulting in the overall flatter wavelength profiles observed in our study. Nevertheless, we should note that in both studies, the leukemic cells exhibit significantly steeper wavelength dependence than the two normal leukocyte populations, which in turn are characterized by similar wavelength decays. Therefore, our results show significant promise for the light scattering for the non-invasive or minimally invasive diagnosis of leukemic conditions, as well as for providing insight into the subcellular morphological transformations associated that give rise to the observed differences.

In addition to spectral differences, consistent and significant differences are observed in the overall intensity of the detected light in both ABSS and LBSS measurements. Specifically, the scattering intensity from PMN is highest at all wavelengths and lowest for PBMC compared with leukemic cells. The high total scattering intensity from PMN maybe due to the large number of granules found in the cytoplasm of these cells, which are not present in PBMC or Nalm-6 cells (38, 43–45). These differences in the scattering intensity of spectra from normal and leukemic WBCs are also consistent with our previous study using a collimated illumination system (42).

In summary, our main findings indicate that leukemic cell discrimination from cells most commonly found in blood is possible with light scattering spectroscopy, based on a system design employing focused illumination, confocal detection, and collection of light over a broad range of backscattering angles, at a small number of excitation wavelengths spanning the visible range. Therefore, light scattering based approaches may result in the development of novel, minimally or noninvasive techniques, such as microfluidic-based lab-on-chip or in vivo flow cytometry platforms with confocal detection, to improve monitoring of leukemic cells for disease diagnosis or MRD assessment particularly in children, for whom blood withdrawal maybe difficult and traumatic.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITERATURE CITED
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