zi Experimental data were fitted with simulations of light scattering from homogeneous spheres with fixed refractive indexes. The morphologic pattern of CLL lymphocytes at diagnosis has been associated with likelihood of different prognoses, while its prognostic significance at the time of disease progression is uncertain. and refractive index of 1.606. C ∼7.6μm . with a step of This is partly due to the large nucleus diameter used However, the five-layer model of mononuclear cells has a few disadvantages. Mean diameter ratios Microscopically, in a Wright's stained peripheral blood smear, a normal lymphocyte has a large, dark-staining nucleus with little to no eosinophilic cytoplasm. Dn∕Dc=0.903 α0 Many textbooks on nonlinear regression deal with errors of parameter estimates (see e.g., Refs. mn You currently do not have any folders to save your paper to! Concepts. : The advantage of this procedure is the automatic decrease of importance of measurements with large errors. The correlation due to angular resolution (about was varied from Dc=6.38μm axis of the figure, is the same as shown in Fig. In this work, we calculate the expectation value As we discussed in Sec. Author information: (1)Medical Institute of Novosibirsk, Institute of Clinical and Experimental Lymphology, Siberian Section of RAMS, Novosibirsk. ) through a hole in the mirror (mirror 4). 3.2). Dot plots of these two integral parameters were verified to be similar to and display the same population percentages as ones obtained by the standard flow cytometer Coulter Epics (Beckman Coulter, Fullerton, California) (data not shown). Results of global optimization for four simulated LSPs corresponding to the enhanced optical model of a lymphocyte. The main difference in morphology of T- and B-lymphocytes is found to be the larger mean diameters of the latter. Such a plot for mean cell diameter over sample including errors of mean is shown in Fig. One can see that the coated sphere is an adequate first-order model, and this model was used for the solution of the inverse light-scattering problem. .35 Its confidence level is. and A Rigorous methods to deal with correlated residuals exist,35 but they are quite cumbersome. The essential feature of the SFC is the ability to measure the absolute differential light-scattering cross section of single particles of any shape and structure. Complete cases of common blood disorders (peripheral blood, bone marrow, and diagnostic studies). 4 χ2 . 49 using the distribution of lymphocytes over the side scattering signal. . Preparation of lymphocyte samples was carried out by a density-gradient separation procedure.26 This procedure consists of diluting The bias increase with relative weight of the cut tail, so we define a confidence level However, we have shown and analyzed only the diagonal elements of the matrices (for 12 donors),32 and from Lymphocytes are a heterogeneous population of blood cells that can be distinguished from other leukocytes by their characteristic morphology and structural features. If in high numbers, neoplastic lymphocytes are morphologically similar, forming a uniform population. Lymphocyte morphology #00061292. Dn∕Dc∊[0.7,0.95] In particular, blood platelet volume and refractive index, red blood cell volume, and hemoglobin concentration are the only physical characteristics that can be measured reliably with automatic hematology analyzers. map. 420nm and , whereas the remainder can live from hundreds of days to many years. μi There seems to be no reason for our processing algorithm to decrease the natural variability of mean T-cell sizes between the donors, e.g., by somehow concentrating it to This study is an attempt to evaluate the significance of morphological evaluation of lymphocytes on peripheral smear examination in adult σ(⟨.⟩) The ratio of LSPs that passed the confidence level threshold varied from 10 to 20% from sample to sample. The above described neutrophil morphological changes had almost completely disappeared and the lymphocyte population showed wide morphological heterogeneity with large atypical lymphocytes , lymphoplasmacytoid cells and increased proportion of large granular lymphocytes. than in previous studies, or the microscopic method together with corresponding sample preparation is susceptible to some measurement errors that increases the visible variability between the donors. of staining, the sample was washed again and diluted to a concentration of about 12to50deg Cytoplasmic vacuoles and/or azurophilic granules may also be present. α0=0.8 , refractive indices of cytoplasm ; filters—BP This work was supported by grants from the Russian Foundation for Basic Research, numbers 07-04-00356-a and 08-02-91954-NHИO̱a; integration grants of the Siberian Branch of the Russian Academy of Science, numbers 2009-37 and 2009-7; a grant from the program of the Presidium of the Russian Academy of Science, number 2009-276; and a grant from the Novosibirsk City Hall for young scientists. and detected by PMT 1. This is an important goal for future research. . α0 We use these confidence regions to assess the influence of parameter space boundaries on the results. preceding parturition. is to assess biases of They all have relatively mature clumped nuclear chromatin and no visible nucleoli. α0=0 A lymphocyte has a nearly spherical shape and a single nucleus.32 3-D images of a few cells were obtained from confocal microscopy, and one slice of them is presented in the left part of Fig. φ B You have requested a machine translation of selected content from our databases. The angular range, where experimental weighted LSP Results for other characteristics and donor 1 are similar (data not shown). But currently we can only conclude that the difference in mean diameter at least partly explains the difference in averaged LSPs. We characterized samples of B-cells only for two donors because of the small amount of B-lymphocytes above the threshold for the other donors. mc σ(.) Plasmacytoid lymphocytes: a clue to dengue diagnosis, Transformed lymphocytes mimicking blasts/atypical cells. Currently, processing one LSP with the proposed method takes approximately These cells are often large with abundant cytoplasm. α0=0.8 This pattern is rather sensitive to cell morphology, in particular to the cell size and nucleus shape and displacement for lymphocytes.15 This sensitivity offers the opportunity to determine individual cell characteristics from a solution of the inverse light-scattering problem. The light scattered in the forward direction is collected by objective 1 All simulations were run on the Dutch compute cluster Lisa.31. is caused by a significant fraction of cells having α<0.35 Our method is based on fitting experimental light-scattering patterns (LSPs) by a coated sphere model using the global optimization algorithm DIRECT. It is important to note that we estimate a full covariance matrix of the sample of individual lymphocytes as well, including correlations between different characteristics. θ inside each sample. This fact disagrees with recent microscopic measurements,32, 33 and currently we cannot explain this. This functionality is provided solely for your convenience and is in no way intended to replace human translation. The global optimization algorithm described in Secs. ⟨.⟩, They vary in morphologic detail as well as surface marker characteristics, indicating that they comprise a heterogeneous mixture of cell types. Introduction csw lsuhsc 2002 . We describes a method for the solution of the inverse light-scattering problem for individual mononuclear cells. The platelet counts were totally normal otherwise in this case. In general, features include: Size - 15 - 20 μ Nucleus - large, reticular chromatin pattern. and both The graphs correspond to a different reliability level of the fit It occurs when the lymphocyte is activated by an antigen (from antigen-presenting cells) and increased in volume by nucleus and cytoplasm growth as well as new mRNA and protein synthesis. with steps of Although the difference in This is usually seen as enlarged nuclei, a moderately loose, coarse chromatin structure, and a marked widening of the basophilic cytoplasmic layer. However, one may try to estimate the relative count of T- to B-lymphocytes in a sample, processing the distribution of all lymphocytes over morphological characteristics as a bimodal distribution (e.g., a sum of two multidimensional normal distributions). Results for these two donors comparing B- and T-lymphocytes are shown in Table 2 for characterization of the samples. 60mW k=3 BACKGROUND AND OBJECTIVES: Chronic lymphocytic leukemia (CLL) is characterized by clinical, immunophenotypic and morphologic heterogeneity. Dependence of estimated mean cell diameter and fraction of processed cells on confidence level threshold for a sample of T-lymphocytes of donor 6. Although of the particle (more precisely, of associated probability distribution over Analogously, in SFC we used absolute values of the integrated whole light-scattering trace instead of the side scattering [Fig. ,42, 43 by electron microscopy were Your use of this feature and the translations is subject to all use restrictions contained in the Terms and Conditions of Use of the SPIE website. value±standard Second, we tested the enhanced model including nucleus inhomogeneity (see Sec. is different from the rest of the interval, because it contains almost no oscillations. given a specific experimental LSP is then , which is why it may seem to contradict earlier simulations using smaller nucleus diameters, e.g., Ref. Therefore, we can only hypothesize that either donors studied in this work are more uniform (in age, health condition, etc.) Reactive lymphocytes show a range of morphologic features. A similar method was proposed by Terstappen 1.077g∕ml α 560–615nm To study the effect of such nuclear inhomogeneity on the light scattering, we enhanced the optical model by introducing an oblate spheroid into the nucleus (Fig. [Morphological and functional characteristics of lymphocytes of mothers of children with Down syndrome]. This optical setup of the SFC (PMT 2 in Fig. Cattle have small lymphocytes (as described), as well as large lymphocytes with dark areas in the nucleus (chromocenters) and abundant pale blue cytoplasm. The typical parameters of the microscopy used in this work were as follows: objective—Plan-Apochromat β 5.4μm . The beam stop prevents illumination of PMT 1 by the incoming laser beam. . 0to90deg • Causative agent maybe viral or non-viral. Comparison of model characteristics of T- and B-lymphocytes of two donors. . Dogs and cats have mostly small circulating lymphocytes (Figure 22.3a). Observe the differences in the cytoplasm between the two cell types. The theory of light scattering by a sphere consisting of two layers is well known,24 and light-scattering profiles are easily and quickly computed. 150sec and detected by PMT 3. These lymphocytes are activated lymphocytes with varying morphology and size. This can be explained by the acceptance of values close to the parameter space boundary and larger sample size, respectively. However, as discussed in Sec. μ However, this difference is much smaller than the natural variability of LSPs inside each sample (see Fig. Most lymphocytes in newborn children look somewhat blastoid with finer chromatin and clefted nuclei often worrisome for acute leukemia. Another way of looking at differences between T- and B-lymphocytes is studying average LSPs. However, one may try to estimate the relative count of T- to B-lymphocytes in a sample, processing the distribution of all lymphocytes over morphological characteristics as a bimodal distribution (e.g., a sum of two multidimensional normal distributions). Then it stabilizes, however, since new accepted measurements have relatively large errors and hence contribute very little new information. The size, shape and staining characteristics of lymphocytes vary among and within animal species. This model was used to mimic the effect that nuclear inhomogeneity may have on the LSPs and to verify the results of global optimization algorithms in the presence of model errors. The solution of the inverse problem is performed by a least-squares method, i.e., by minimizing the weighted sum of squares: We use the following cellular characteristics: cell diameter , Knowledge of this morphologic variation is essential to avoid performing unnecessary flow cytometry on peripheral blood specimens of these patients. Therefore, we consider them less reliable and stick to results obtained with confidence level thresholds in further discussions. The correlation is caused by: 1. finite angular resolution of the SFC, causing dependence between nearby σ(Dc) Hence, at present we cannot reliably classify a single lymphocyte as being T- or B-, using only its morphological characteristics determined from LSP. For illustration, sample parameters related to cell diameter are shown in Table 3 Absolute Lymphocytosis with Normal Lymphocyte Morphology • Acute Infectious Lymphocytosis • Usually found in children between ages of 1 to 10 years, and occasionally up to 14 years of ages. Next, we apply a Bayesian inference method with standard noninformative (or homogeneous) prior The mean values of lymphocyte diameter reported in the literature for normal donors as determined by the Coulter principle were Reactive lymphocytes have an increased amount of RNA in the cytoplasm, which is reflected by an associated increase in cytoplasmic basophilia. pleomorphic lymphocytosis). Results of processing the simulated LSPs with the global optimization are shown in Fig. .35 A typical example of this function for a lymphocyte is shown in Fig. 3. Then the average (effective medium) refractive index of the nucleus equals the one specified before. The discrete dipole approximation (DDA) is a general method to compute scattering and absorption of electromagnetic waves by particles of arbitrary geometry and composition.28 The ADDA computer code is an efficient DDA implementation on a cluster of computers, parallelizing a single DDA computation;29 ADDA’s source code and documentation is freely available.30 Simulations of light scattering by the nonsymmetric optical model of a lymphocyte (see Sec. 8, right). into the capillary of the optical cell. Polyribosomes are a prominent feature in the lymphocytes and can be viewed with an ele… , where The measured light-scattering trace of the same microsphere is shown in the inset. 11a ⟨Dc⟩ . , Dc However, most of the theory is worked out for the case of normally distributed and independent experimental errors. and their total amount was less than 0.7% of all cells in each sample. Hence, at present we cannot reliably classify a single lymphocyte as being T- or B-, using only its morphological characteristics determined from LSP. csw lsuhsc 2002 menu This study module is designed for LSUHSC L2 students enrolled in Clinical Pathology 201. As a compromise, we chose a round value Therefore, we can seamlessly include all measurements into the estimation of cell characteristics, even those that seem unreliable due to large sums of squares and/or standard deviations of the characteristics. The hydrofocusing head (not shown) produces two concentric fluid streams: a sheath stream without particles and a probe stream that carries the particles. We also compared morphological characteristics of T- and B-lymphocytes of the same donors. Clusters containing all lymphocytes were gated by light-scattering signals. Plots for other parameters and samples support this choice (data not shown). Typical autocorrelation function of residuals, resulting from fitting of a lymphocyte LSP by LSPs calculated from the Mie theory for coated spheres. 11b. 2.5). Only parameters related to cell diameter are shown. . Relative goodness of fit is better assessed by the matrix is a normalization constant obtained as, Since we know the complete probability distribution of model parameters for any measured particle, we can infer any statistical characteristics of this distribution. Lymphatic cells show wide variability and transform easily. . 47. A The inverse light-scattering problem was solved with an optimization procedure based on a multistart Levenberg-Marquardt algorithm. . 30deg Z deviation) are also shown. 2ml The region . 0.4to1.2μm Opt. The discontinuity at and the covariance matrix The SFC was assembled on the bench of a FACStar Plus BD flow cytometer (BD, Franklin, Lakes, New Jersey). Their nuclear shapes vary quite markedly. It is important to note that in 4-D parameter space, P(β|Iexp)=κ[S(β)]−n∕2 Knowledge of this morphologic variation is essential to avoid performing unnecessary flow cytometry on peripheral blood specimens of these patients. While plasma cells have their origins in the bone marrow as B-cells, they usually leave the bone marrow to develop and mature in the lymph nodes or spleen. and 0.85. For that we develop a method to characterize the sample based on individual measurements with different errors. We use an estimator proposed by Fuller,38 whose first step is based on means of Second, and more important, that method did not provide (statistically reliable) errors of estimates of characteristics of individual cells, which is crucial to assess the overall adequacy of the fitting procedure. 0.6μm A Carl Zeiss LSM 510 META confocal microscope was used to develop our optical model of lymphocytes. B , using steps of ⟨.⟩, The main purpose of Disorders – characteristic morphology . Over the last few decades substantial progress has been achieved in flow cytometry immunophenotyping procedures.2 Nevertheless, morphological analysis and automatic cell sizing remain one of the key points in studying, diagnosing, and classifying lymphoproliferative disorders.3, 4 It is well known that the average size of lymphocytes decreases and density changes (generally increasing) in leukemia.5, 6 The cytoplasmic refractive index of circulating lymphocytes remains invariant among normal human individuals and is elevated for patients bearing malignant tumors.7 This refractive index also increases significantly in active rheumatoid arthritis, in the immune reaction to a kidney transplant, and pregnancy, especially during the and However, doing so is not the best option because of the bias due to the region boundaries, which are especially important for particles with small confidence level value±standard The structure of these LSPs in the angular range from It can also be used as a measure of goodness of fit but only approximately, because it depends on the position of In normal situations, the coarse, dense nucleus of a lymphocyte is approximately the size of a red blood cell (about 7 μm in diameter). 0.5deg The fluorescence of specific dye molecules linked at the single cell is collected by objective 3 distribution is. Results of characterization of all lymphocyte samples using no confidence level threshold. Some lymphocytes show a clear perinuclear zone (or halo) around the nucleus or could exhibit a small clear zone to one side of the nucleus. Slice from the confocal image (left) and (improved) optical model (right) of a lymphocyte. is much larger then the weight of the tail cut by the boundary. The prepared samples were analyzed with a scanning flow cytometer (SFC) measuring the LSPs and fluorescence signals from each cell. α 6.4μm , ratio of nucleus and cell diameters Dmitry I. Strokotov, Maxim A. Yurkin, Konstantin V. Gilev, Dirk R. Van Bockstaele, Alfons G. Hoekstra, Nikolay Rubtsov, Valerii Pavlovich Maltsev, "Is there a difference between T- and B-lymphocyte morphology?," J. Biomed. 0.5to10μm is defined by their boundary values: 3, where the inset shows the measured light-scattering trace of a single microsphere. To perform global optimization, we use the algorithm DIRECT (an acronym for DIviding RECTangles)25 and propose a method to compute errors of the estimated cellular characteristics. Over the last decade we have, however, developed an advanced flow cytometer that is capable of acquiring more physical characteristics of individual particles13 by measuring detailed light scattering profiles and inverting them by relying on dedicated optical models.14 In this work we demonstrate the applicability of this scanning flow cytometry to mononuclear cells and apply it to a detailed analysis of the morphology and optical properties of human B- and T-lymphocytes. Unfortunately, there is significant dependence between residuals 20min The structured and hierarchical ontology is composed of all the significant morphological characteristics, considered in an individual way. , , and Such LSPs are successfully processed, with the global optimization resulting in a confidence level above 0.95 and accurate estimation of the model parameters (data not shown). Comparing it with Tables 1, 2, one can see that for all samples the standard deviation is about 1.5 times larger and the standard error of mean is about two times smaller. To better understand these errors, we performed a limited set of DDA simulations. CD3×CD19 This content is available for download via your institution's subscription. Second, we simulated LSPs of lymphocytes specified by estimates of the mean values of model characteristics, resulting in Fig. α Here we propose to use an approximate but much simpler approach. diam into the optical cell (Fig. 6.5μm equal to The results suggest that deviations of real lymphocyte LSPs from the best-fit LSP of the coated sphere model may be due to nuclear inhomogeneity. of whole anticoagulated blood in . implies a small bias of Having determined characteristics of each lymphocyte with certain errors (i.e., 2.1. Renal lymphoma in dogs is rare and has a poor prognosis. α0 (12 donors),32 and from 1] for (six donors),33 showing no significant difference between different methods. We also note that B-cells are generally better fitted by the coated sphere model than T-cells. μ The distribution of model characteristics over sample (its mean and covariance matrix) is estimated, accounting for different reliability of parameter estimates for different particles. Differential light-scattering cross section of T-lymphocytes and a polymer microsphere at a wavelength of You will have access to both the presentation and article (if available). 22 used a flow cytometer together with an intensified charge-coupled device (ICCD) camera to record 2-D patterns of the light scattered by single lymphocytes. The lymphocytes in this field have a more regular cytoplasmic border without the cytoplasmic blebbing and pseudopods that are present in monocytes. mn The platelet counts were totally normal otherwise in this case. 35, 36). leaves only cells with a relatively homogeneous nucleus (but possibly shifted from the center of the cell). of phosphate buffered saline (PBS) and centrifuging at This will count as one of your downloads. ; scaling— To choose the threshold To access this item, please sign in to your personal account. Neoplastic lymphocytes, as discussed in Section V: Hematopoietic Neoplasia, may be small, intermediate sized, and blastic. In this work we construct a solution of the inverse light-scattering problem for mononuclear cells, modeled as a coated sphere. Morphologically, lymphocytes are classified as small, medium and large depending on their size. 41 and Please enable JavaScript or upgrade to a JavaScript-capable browser to use the ASH Image Bank. M onocytic cells seen in body fluids have variable morphology as they have the ability to transform into phagocytic macrophages. Dn∕Dc (1.8GHz) . −0.8 . However, much more detailed study is required to make any definite conclusion about the exact shape of nuclear inhomogeneity of lymphocytes and its effect on their LSPs. Cellular and nuclear size of mononuclear leucocytes and densities of their cytoplasm and nucleus are still out of scope of routine hematological analysis. . . From the obtained confidence levels, we can suggest that using the threshold Images of peripheral blood and/or bone marrow of blood disorders and normal hematopoiesis. The lymphocytes are typically … . . After 2.5, this is mostly due to model errors, i.e., the fact that real lymphocytes are not perfect coated spheres. There are also certain errors associated with imperfect alignment of SFC and noncentral positions of the particle in the flow.13 However, we consider these errors as part of model errors, since the exact structure and cause of the latter is not important for our characterization method, nor can they be distinguished from real model errors. • It is contagious, benign, and self-limited. 7 1b]. κ Results for T-lymphocytes duplicate corresponding columns of Table 1. Comparing the results of the global optimization for characterization of T- and B-lymphocytes (Table 2), one can see that the main difference in T- and B-cell morphology is the mean cell size This feature is realized by measurement of a mixture of unknown particles and polymer microspheres.39 The LSP of the polymer microsphere, measured with the SFC, gives a high accuracy agreement with the Mie theory.40 This allows calibration in absolute light-scattering units. and a sample covariance matrix based on T-lymphocytes and B-lymphocytes usually cannot be differentiated on routine blood … Absolute Lymphocytosis with Normal Lymphocyte Morphology 19. Index of lymphocytes based on morphological appearances From www.haematologyetc.co.uk. in our case, which can be characterized by a sample autocorrelation function , and Dc With decreasing ) was used for generation of the scattering pattern, for excitation of fluorescence, and for triggering the electronic unit. (SSC×FSC) First, we averaged 100 experimental LSPs from the samples of T- and B-lymphocytes for donor 6, resulting in Fig. One can see that there are a number of cells, for which does not significantly change the results. Distribution of standard deviations of estimated cell diameter for samples of T- and B-lymphocytes of donor 6. For now we concentrated on the robustness of the algorithm (reaching global minima and sufficiently detailed description of the least-square surface), and did not pursue its numerical efficiency.

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