ARTICLE |
Correspondence to: Zvi Malik, Microscopy Unit, Life Sciences Dept., Bar-Ilan University, Ramat-Gan 52900, Israel..
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Summary |
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Spectral morphometric characterization of typical chronic lymphocytic leukemia (B-CLL) cells vs normal small lymphocytes stained by MayGrunwaldGiemsa was carried out by multipixel spectral imaging. The light intensity (450850 nm of 104 pixels) from nuclear domains of each stained cell was recorded and represented as light transmittance spectra and optical density. Transmitted light spectra of two nuclear domains were determined, one with low-intensity light transmittance (LIT) and the other with high-intensity light transmittance (HIT). A spectral library was constructed using the four transmitted light spectra representing the HIT and LIT domains of the normal human lymphocytes and the LIT and HIT domains of the CLL cells. The spectral library served to scan CLL lymphocytes from 10 cases of CLL and the lymphocytes of 10 healthy individuals. Each spectrally similar domain in the nuclei of the lymphocytes was assigned an arbitrary color. The morphometric analysis of the spectrally classified nuclei showed specific spectral patterns for B-CLL in 92% of the cells. The specific spectral characteristics of each of the two cell populations were also observed by their optical density light absorbance spectra. We propose that spectral morphometric analysis may serve as an additional diagnostic tool for detection of CLL lymphocytes in a hematological specimen. (J Histochem Cytochem 46:11131118, 1998)
Key Words: multipixel spectroscopy, spectral similarity mapping, classified image, absorbance image, CLL
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Introduction |
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The overaccumulation of small, mature-appearing B-lymphocytes in the peripheral blood is a fundamental hallmark of chronic lymphocytic leukemia (CLL) diagnosis (
The FrenchAmericanBritish group (FAB) has proposed criteria based on cytochemical and immunological methods to establish a clear diagnosis (- or
-light chains and the monoclonality is essential to establish the diagnosis. Receptors for mouse red blood cell rosettes (MRBC-Rs) are detectable on both B-CLL cells and normal B-cells. However, the two populations express different patterns of complement receptor. Chromosomal abnormalities have been described in about 50% of CLL patients using conventional cytogenetics (
To improve CLL diagnosis, we applied spectral imaging techniques for differential analysis of peripheral blood lymphocytes. Spectral imaging enhances the information obtained with light microscopy by providing multipixel spectra that otherwise are impossible to obtain from cytological specimens (
The capability of spectral imaging to detect and identify known and unknown substances has wide biological application. In cytogenetics, spectral imaging using 24 whole chromosome probes, each one labeled and emitting a different fluorescence spectrum, enables easy detection of chromosomal aberrations in solid tumors (
Here we describe a novel spectral analysis technique, termed spectrally resolved morphometry, for the discrimination of CLL from normal lymphocytes based on multipixel spectral data. The analyzed cells were stained with MayGrunwaldGiemsa (MGG) and inspected by conventional light microscopy. The spectral morphometric measurements utilizes the specific chromatinstain complexes that enhance the shape and structure of the nuclei in each cell population.
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Materials and Methods |
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MayGrunwaldGiemsa Staining of Peripheral Blood Specimens
Peripheral blood was obtained from 10 patients with typical B-CLL, who were diagnosed according to established criteria (
Fourier Transform Multipixel Spectrometry System for Microscopy
Spectral imaging was performed using the SpectraCube SD-200 (Applied Spectral Imaging; Migdal HaEmek, Israel). The SpectraCube system consists of an interferometer situated in the parallel beam between an objective lens (infinity corrected) and a lens equivalent to an eyepiece, whose purpose is to form an image on a CCD camera. The light beam passing through the specimen is split in the interferometer in opposite directions and is united again at the exit with an optical path difference (OPD) that is a function of the angle between the incoming beam and the interferometer itself. The OPD arises because for non-zero angles the two beams undergo different optical paths in the beamsplitter. The inherent mechanical stability of this interferometer allows the Fourier technique to be successfully applied to the visible spectral region. The measurement is done by recording successive CCD frames in synchronization with the steps of the motor used to rotate the collimated beam, so that the instantaneous OPD is known for every pixel in every recorded frame and can be used in the FFT calculation (
Briefly, in spectral imaging each pixel is actually one of several tens of thousands of microspectrometers, acting simultaneously and independently. As a result, spectral imaging acquires a so-called cube whose appellate signifies the two spatial dimensions of a flat sample (x and y) and the third spectrum dimension representing light intensity at any wavelength. The calculated pixel size in a spectral image is 0.04 µm2. The spectral resolution (FWHM, full width at half maximum) is 5 nm at 400 nm (12 nm at 600 nm) and the spectral range (more than 5% response) is 4001000 nm (
Optical Density Images
The SpectraCube software provides the possibility for calculation of approximate values of optical density for each pixel in the image, based on the spectra stored in the transmitted-spectra "cube." For these calculations, the incident light was defined as a pixel outside a cell. The calculated data are stored in a new optical density cube, revealing the corrected optical density for each pixel of the cells. The optical density cube served to construct absorbance images of the cells.
Spectral Similarity Mapping
Similarity mapping is useful in a situation in which the sample is composed of a number of spatially separated components, each characterized by a known and unique spectrum, and the task is to detect and map all components. Spectral analysis of normal human lymphocytes and B-CLL cells revealed two different spectral domains composing the nucleus. The steps of this algorithm were as follows. First, the spectra of the distinct nuclear domains were stored in a "spectral library." Second, for every pixel of the cube a comparison was made between its measured spectrum and all the spectra of the library. Third, each pixel in the image was identified with the most similar spectrum in the library. Fourth, each pixel was displayed in a previously established color identifying the specific library spectrum, forming a so-called "classified image."
The comparison formula for the second step of the similarity mapping algorithm was as follows: n functions fnx,y (n is the number of spectra in the library) are defined for every pixel of spatial coordinates x and y, as follows:
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(1) |
To eliminate intensity variations, scanning was performed using normalized spectra. To eliminate the variance in dye concentration between different stained specimens, the spectra of all cubes were standardized according to the spectrum of a standard stained erythrocyte.
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Results |
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An attempt to discriminate normal human lymphocytes from B-CLL cells stained by MGG was performed by multipixel spectral analysis. We have observed two main spectral domains in the lymphocyte nuclei, representing two main condensation states of the chromatin: one domain of high condensation and low intensity light transmittance (LIT) and the other with high intensity light transmittance (HIT) from domains of low chromatin condensation. To measure the area of each domain in the two lymphocyte populations, we constructed a spectral library consisting of the four transmitted light spectra representing HIT and LIT domains of normal and CLL lymphocytes. These spectra were each assigned an arbitrary color (Figure 1C). Using the spectral library, CLL lymphocytes from 10 cases of CLL and lymphocytes from 10 healthy individuals (10 lymphocytes from every donor) were scanned and each nuclear domain was assigned the color of the library spectrum most similar to its spectral characteristics. The scan produced a classified image for each lymphocyte using a mathematical algorithm called "spectral similarity mapping" as described in Materials and Methods.
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Figure 1A and Figure 1B show the spectrally classified images obtained for the two populations of normal and B-CLL lymphocytes. Distinct nuclear domains are revealed, with each colored domain corresponding to one spectrum of the library. Spectra no. 1 (red) and 2 (blue) were uniquely associated with normal lymphocytes (Figure 1A), whereas spectrum no. 3 (green) of the library appeared to dominate the nucleus of most of the CLL cells (Figure 1B).
Morphometric measurements were performed on the spectrally classified images. Table 1 presents the number of normal and CLL lymphocytes displaying each spectral domain and the relative area of that domain in their nuclei. Most of the normal lymphocytes displayed more than one spectral domain in the nucleus. The calculations of the classified areas revealed that 82% of the normal lymphocytes displayed either spectrum no. 1 or spectrum no. 2, or both, in the nucleus. However, spectra no. 1 and 2 did not appear in the nucleus of 92% of the CLL lymphocytes, which displayed either spectrum no. 3 or 4, or both.
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Spectral distinctions between the two cell populations were made on the basis of their optical density characteristics, which reflect light absorbance properties of the stained cells. Absorbance images were constructed for the normal (Figure 1D) and B-CLL lymphocytes (Figure 1G) using the spectral information stored in the optical density cube (see Materials and Methods). Figure 2A and Figure 2B show a set of spectra revealing the chromatin optical density values for low-absorbance and high-absorbance domains. Each spectrum in Figure 2 is an average of 400 pixel spectra from four patients with CLL (Figure 2B) and 400 pixel spectra from four healthy individuals (Figure 2A). The normal lymphocytes absorbed light in the range of 450800 nm, with an absorbance peak at 520550 nm in high-absorbance domains and a peak at 510540 nm in low-absorbance domains (Figure 2A). B-CLL cells displayed absorbance peaks in the range of 480580 nm in high-absorbance domains and 500570 nm in low-absorbance domains (Figure 2B).
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The organization of the two nuclear domains in the normal and B-CLL lymphocytes was revealed by spectral similarity mapping using a single reference spectrum. The first similarity mapping was performed using a spectrum from the low-absorbance domains (Figure 1E) and the second using a spectrum from the high-absorbance domains (Figure 1F). The nucleus of normal lymphocytes was revealed as composed mainly of high-absorbance domains (Figure 1F), with a few low-absorbance domains scattered all over the nucleus (Figure 1E). In contrast, the B-CLL nucleus appeared to be composed of a single central low-absorbance domain (Figure 1H) surrounded by a single high-absorbance rim (Figure 1I).
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Discussion |
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Spectral morphometric analysis was used in the present study for the characterization of typical B-CLL and normal small lymphocytes on the basis of spectral nuclear features. The nuclear structure is a dynamic reflection of the metabolic state of the nucleus and a physical correlate of its total content of biochemical constituents (
Here we present the concept of spectral morphometry for the determination and distinction of cell populations. The spectral morphometric analysis was applied in the present study to B-CLL and normal lymphocytes. Although the Giemsa-stained CLL cells generally appear similar to normal resting lymphocytes (
The specific absorbance characteristics of the two cell populations were reflected in their optical density spectra. In normal lymphocytes an absorbance peak was seen at 520550 nm in high-absorbance domains and at 510540 nm in low-absorbance domains. B-CLL cells displayed absorbance peaks in the range of 480580 nm in low-absorbance domains and at 500570 nm in high-absorbance domains. By choosing a single optical density spectrum from each domain and performing the spectral similarity mapping process, we revealed distinct chromatin organization patterns in the nuclei of CLL and normal lymphocytes. In the normal lymphocytes, the highly condensed chromatin was scattered over the nucleus, whereas in the B-CLL the condensed chromatin concentrated in the center of the nucleus and was surrounded by a less condensed chromatin rim. We have to bear in mind that normal peripheral blood lymphocytes are mainly T-cells whereas the blood cells in the present study are B-CLLs. A situation in which normal peripheral blood displays a large population of B-lymphocytes is extremely rare. An elevation in the number of B-lymphocytes in the peripheral blood usually indicates a pathological condition. Therefore, the spectral morphological distinctions observed are of significant importance for the discrimination of peripheral blood of CLL patients from that of healthy individuals.
Micromorphometry plays an increasing role in clinical diagnostics by providing clues derived from the shape and structure determinations of cell nuclei. Image analysis of the digitized data engenders high sensitivity to subtle changes not perceived by human visual assessment and offers instant access to large image databases for comparison (
In the relatively short time that spectral morphometry has been applied to cytological research and diagnostics, it has become clear that the advantages provided by this technique include an objective tool for cytological and cytogenetic diagnosis, advancement and automation of cytological specimen examination, and the facilitation of nuclear structure and chromatin organization analysis.
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Acknowledgments |
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Supported by a grant from Applied Spectral Imaging, Migdal HaEmek, Israel.
We acknowledge the helpful remarks of Dr Judith Radnay from the laboratories of Molecular Biology and Hematology, Sapir Medical Center, Kfar Saba, Israel. We gratefully thank Ms Judith Hanania for help in editing the manuscript and Mr Jacob Langsam for skillful assistance.
Received for publication March 31, 1998; accepted June 26, 1998.
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Literature Cited |
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Andreeff M, Darzynkiewicz Z, Sharpless TK, Clarkson BD, Melamed MR (1980) Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA. Blood 55:282-293[Abstract]
AvilaCarino J, Lewin N, Tomita Y, Szeles A, Sandlund A, Mosolits S, Mellstedt H, Klein G, Klein E (1997) B-CLL cells with unusual properties. Int J Cancer 70:1-8[Medline]
Bain BJ (1993) Chronic lymphoid leukemias. In Bain J, ed. Leukemia: Diagnosis. A Guide to the FAB Classification. London, Gower Medical, 89-93
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C (1989) Proposals for the classification of chronic (mature) B and T lymphoid leukemias. J Clin Pathol 42:567-584[Abstract]
Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S, Rai KR (1996) National Cancer Institute-sponsored group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 87:4990-4997
Criel A, Verhoef G, Vlietinck R, Mecucci C, Billiet J, Michaux L, Meeus P, Louwagie A, Van Orshoven A, Van Hoof A, Boogaerts M, Van Den Berghe H, De WolfPeeters C (1997) Further characterization of morphologically defined typical and atypical CLL: a clinical, immunophenotypic, cytogenetic and prognostic study on 390 cases. Br J Haematol 97:383-391[Medline]
Freedman AS, Nadler LM (1993) Immunologic markers in B-cell chronic lymphocytic leukemia. In Cheson BD, ed. Chronic Lymphocytic Leukemia: Scientific Advances and Clinical Development. New York, Marcel Dekker, 1-12
GarciaMarco JA, Caldas C, Price CM, Wiedemann LM, Ashworth A, Catovsky D (1996) Frequent somatic deletion of the 13q12.3 locus encompassing BRCA2 in chronic lymphocytic leukemia. Blood 88:1568-1575
Garini Y, Katzir N, Cabib D, Buckwald RA, Soenksen D, Malik Z (1996) Spectral Bio-imaging. In Wang XF, Herman B, eds. Fluorescence Imaging Spectroscopy and Microscopy. New York, Wiley & Sons, 87-124
Harris NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary ML, Delsol G, De WolfPeeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, MullerHermelink HK, Pileri SA, Piris MA, Ralfkiaer E, Warnke RA (1994) A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 84:1361-1392
Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie MJ, Parker AC, Castoldi GL, Cuneo A, Knuutila S, Elonen E, Gahrton G (1990) Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med 323:720-724[Abstract]
Kipps TJ (1995) Chronic lymphocytic leukemia and related diseases. In Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams Hematology. 5th Ed. New York, McGrawHill, 1017-1033
Malik Z, Amit I, Rothmann C (1997) Subcellular localization of sulfonated tetraphenyl porphines in colon carcinoma cells by spectrally resolved imaging. J Photochem Photobiol 65:389-396
Malik Z, Cabib D, Buckwald RA, Talmi Y, Garini Y, Lipson SG (1996a) Fourier transform multipixel spectroscopy for quantitative cytology. J Microsc 182:133-140
Malik Z, Dishi M, Garini Y (1996b) Fourier transform multipixel spectroscopy and spectral imaging of protoporphyrin and photofrin in single melanoma cells. Photochem Photobiol 63:608-614[Medline]
Rothmann C, Cohen AM, Malik Z (1997) Chromatin condensation in erythropoiesis resolved by multi-pixel spectral imaging: differentiation versus apoptosis. J Histochem Cytochem 45:1097-1108
Schrock E, Du Manoir S, Veldman T, Schoell B, Wienberg J, FergusonSmith MA, Ning Y, Ledbetter DH, Bar-Am I, Soensken D, Garini Y, Ried T (1996) Multicolor spectral karyotyping of human chromosomes. Science 273:494-497[Abstract]
Silber R, Sahl R (1990) Chronic lymphocytic leukemia and related diseases. In Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. 4th Ed. New York, McGrawHill, 1011-1033
Sorensen FB (1992) Quantitive analysis of nuclear size for objective malignancy grading. A review with emphasis on new, unbiased stereologic methods. Lab Invest 66:4-23[Medline]
Veldman T, Vignon C, Schrock E, Rowley JD, Ried T (1997) Hidden chromosome abnormalities in haematological malignancies detected by multicolor spectral karyotyping. Nature Genet 15:406-410[Medline]
Wells WA, Rainer RO, Memoli VA (1992) Basic principles of image processing. Am J Clin Pathol 98:493-501[Medline]
Wied GL, Bartels PH, Bibbo M, Dytch HE (1989) Image analysis in quantitative cytopathology and histopathology. Hum Pathol 20:549-571[Medline]