Light-scattering polarization measurements as a new parameter in flow cytometry

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argon ion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation.     

Increased light scattering resolution facilitates multidimensional flow cytometric analysis

Multidimensional flow cytometry identifies cell populations as clusters in a space created by the analysis of multiple parameters simultaneously. Optimal use of this multidimensional space requires each of the individual parameters to provide additional information for cell population discrimination as well as maximum utilization of the dynamic range available for each parameter. In this study we improve the visualization of the information present in light scattering signals from leukocytes to facilitate multidimensional flow cytometric analysis. Optimization of cell preparation techniques are essential to obtain high resolution light scattering signals that give complete separation of the granulocytes, monocytes, and granular and nongranular lymphocytes. The angle at which the forward scattered light was collected was modified to enhance the separation between leukocyte populations. Although orthogonal light scattering signals separate granular and nongranular lymphocytes, the resolution and dynamic range could not be displayed using linear or logarithmic functions. By applying a polynomial function to the orthogonal light scattering signals, all leukocyte populations could be displayed while maintaining high resolution. The combination of high resolution light scattering with a nonlinear display resulted in an equally spaced distribution of the cell populations distinguished by correlating forward and orthogonal light scattering signals. Using this approach, peripheral blood neutrophils, eosinophils, basophils, monocytes, and granular and nongranular lymphocytes were shown to occupy distinct locations in the correlation of orthogonal and forward light scattering. Surprisingly, the basophilic granulocytes were located close to granular lymphocytes and monocytes rather than near neutrophils and eosinophils.     

Relative frequency of malaria pigment-carrying monocytes of nonimmune and semi-immune patients from flow cytometric depolarized side scatter

BACKGROUND: Recently, it was observed that malaria can be detected by performing automated complete blood count analysis including depolarization measurement of scattered laser light. To explain large discrepancies in sensitivity and specificity observed in semi-immune and nonimmune malaria patients, we determined the relative frequencies of malaria pigment-carrying monocytes (PCM) by flow cytometric measurements combined with rare event analysis. METHODS: An experimental cell-sorting unit utilizing argon, krypton, and helium-neon lasers measured the relative frequencies of leukocytes of malaria patients. Single white blood cells showing high intensity in their depolarized side scatter were sorted for subsequent microscopic analysis. RESULTS: From microscopic inspection of sorted cells, we identified malaria PCM as a distinct cluster in scatter diagrams that is well separated from normal leukocytes. For nonimmune patients, the average relative frequency of PCM is 1.5 x 10(-4) (median), for semi-immune patients 8.8 x 10(-4), and for malaria-negative persons 4.4 x 10(-6). Results derived from depolarized side scatter at 488, 633, or 647 nm agree well. Furthermore, malaria pigment-carrying neutrophilic granulocytes were identified microscopically after sorting. We discuss briefly how pigment-carrying neutrophils might be differentiated from normal leukocytes and PCM by using flow cytometry and measuring depolarized side scatter at two wavelengths. CONCLUSION: Our results confirm the feasibility of malaria detection by flow cytometry for semi-immune patients and extend malaria detection to nonimmune patients with low frequencies of PCM. High sensitivity and specificity for malaria detection were obtained.     

Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.     

Cytochemical identification of the leukocytes of torpedoes (Torpedo marmorata Risso and Torpedo ocellata Rafinisque)

The authors subjected peripheral blood smears of Torpedoes to cytochemical analysis of lipids, protein, neutral and acid polysaccahrides and of some enzymatic activities, i.e. adenosine triphosphatase (ATP-ase), acid and alkaline phosphatase, aliesterase and peroxidase. It was found that neutrophilic granulocytes are intensely PAS and aliesterase positive and weakly ATP-ase positive. Eosinophilic granulocytes show the presence of neutral polysaccharides in the matrix (which is PAS positive) and strong ATP-ase and acid phosphatase activities in the granules. Lymphocytes sometimes contain weakly PAS and aliesterase positive granules. Monocytes show some small PAS positive granules and weak acid phosphatase and aliesterase activities. Thrombocytes contain some peripheral granules which are PAS positive and slightly ATP-ase positive. There are no transitional forms between the various cellular types. The results confirm the classification of leukocytes of Torpedoes into neutrophilic granulocytes, eosinophilic granulocytes, lymphocytes, monocytes and thrombocytes and contribute some informations about the histoenzymatic content of Elasmobranch leukocytes.     

The cytochemistry of rabbit peripheral blood leukocytes.

The authors report on their investigations of the differentiation between neutrophilic and eosinophilic granulocytes of the peripheral blood in healthy rabbits. By varying the pH-value and the incubation time it is possible to achieve a reliable differentiation between neutrophilic and eosinophilic granulocytes of the rabbit by means of 6 different cytochemical methods which could only be made incompletely with the routine methods used up till now (Eosin-, Giemsa-, Wright's-colouring etc.). Moreover, monocytes and lymphocytes can also be identified reliably.     

Separation of leucocytes in the dogfish (Scyliorhinus canicula) using density gradient centrifugation and differential adhesion to glass coverslips

Summary The blood of the dogfish, S. canicula, contains several types of leucocytes, namely thrombocytes, monocytes, lymphocytes and four populations of granulocytes. Three of these granulocyte types, G1, G3 and G4, are eosinophilic while G2 is heterophilic/neutrophilic. All of the leucocyte types, with the exception of G2 granulocytes and monocytes, can be separated by means of their differential adherent properties to glass and by density gradient centrifugation. Thrombocytes, G3 and G4 granulocytes can be separated in good purity by single-step methods while G1 granulocytes and lymphocytes require a combination of density gradient centrifugation followed by adherence to glass to remove contaminating thrombocytes. Depending on the cell type, between 11–45% of cells with consistently high viability can be recovered after separation. Separated populations of the thrombocytes and granulocytes will be especially useful for studies on the role of such cell types in inflammation.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies

BACKGROUND: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.     

Simultaneous separate detection of low angle and large angle light scattering in an arc lamp-based flow cytometer

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp-based flow cytometer with epi-illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2 degrees and 15 degrees, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser-based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25-micron diameter. A potential application may be differentiation of bacteria.     

Leukocyte differential analysis in multiple laboratory species by a laser multi-angle polarized light scattering separation method.

Leukocyte differential analysis was performed in various species, particularly laboratory animals, by the laser multi-angle polarized light scattering separation method. Venous blood specimens were drawn from the following subjects: healthy adult men and women ("humans"); cynomolgus monkeys ("monkeys"); common marmosets ("marmosets"); beagle dogs ("dogs"); miniature potbelly pigs ("swine"); Japanese white rabbits ("rabbits"); Hartley guinea pigs ("guinea pigs"); and Sprague-Dawley rats ("rats"). 90 degrees/10 degrees scatter plot: Basophils and mononuclear-polymorphonuclear cells were separated in all subjects, but individual 10 degrees and 90 degrees scatter plots overlapped in dogs and guinea pigs, respectively. 90 degrees depolarized /90 degrees scatter plot: Neutrophils and eosinophils were clearly separated in human, monkey, guinea pig, swine and rat subjects. The eosinophil cluster was not clearly plotted in marmoset, dog, or rabbit. 0 degree/10 degrees scatter plot: Regarding this plot for monocytes and lymphocytes, cells were plotted in the following order in all subjects: lymphocytes < basophils < or = monocytes in the 0 degree (size) scatter; and lymphocytes [symbol: see text] monocytes < or = basophils in the 10 degrees (complexity) scatter. Compared to other species, the rat scatter showed a tendency to overlapping plots in both the 0 degree and 10 degrees scatters in the monocyte and lymphocyte clusters. In both dog and guinea pig, the monocyte and neutrophil plots overlapped in the 0 degree and 10 degrees scatters. Basobox: In the human and rabbit subjects, the basophil cluster was plotted within the established basobox, but no clear cell cluster was plotted in the other subjects. As a result of comparing the percentage values for leukocytes in various species obtained by using the CD3500 apparatus versus the corresponding values obtained manually, good correspondence was found in the monkey, and eosinophils in the marmoset were lower with CD3500 than manually. In the rabbit, the mean measured value for basophils matched in the manual and CD3500 findings. In the guinea pig, the CD3500 values were lower than the manual values for lymphocytes, but higher for monocytes and neutrophils. The above findings suggest that the laser multi-angle polarized light scattering separation method is indeed capable of analyzing leukocytes from various species based on cell size and cell complexity, i.e., the presence or absence of nuclei, granules and cell enclosures.     

Flow cytometer based on triggered supercontinuum laser illumination

Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source. In this study, we first wished to validate the use of a specific triggered supercontinuum laser in a flow cytometer based on white light scattering and electric sizing on human blood cells. Subsequently, to show the various advantages of this attractive system, using scattering effect, electrical detections, and fluorescence analysis, we realized cells sorting based on DNA/RNA stained by thiazole orange. Discrimination of white blood cells is efficiently demonstrated by using a triggered supercontinuum-based flow cytometer operating in a "one cell-one shot" configuration. The discriminated leukocyte populations are monocytes, lymphocytes, granulocytes, immature granulocytes, and cells having a high RNA content (monoblasts, lymphoblasts, and plasma cells). To the best of our knowledge, these results constitute the first practical demonstration of flow cytometry based on triggered supercontinuum illumination. This study is the starting point of a series of new experiments fully exploiting the spectral features of such a laser source. For example, the large flexibility in the choice of the excitation wavelength allows to use a larger number of fluorochromes and to excite them more efficiently. Moreover, this work opens up new research directions in the biophotonics field, such as the combination of coherent Raman spectroscopy and flow cytometry techniques.     

Differential of light-scattering detection in an arc-lamp-based epi-illumination flow cytometer

Light-scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc-lamp-based flow cytometer (AL-FCM). The results were compared with those obtained with a conventional, laser-based flow cytometer (L-FCM) with forward scattering (2-20 degrees) and scattering at right angles. Measuring with the AL-FCM in the angle range upward from 13 degrees, the relative light-scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right-angle scattering in the L-FCM. With a range of scattering angles upward from 2 degrees the AL-FCM yielded histograms similar although not identical to that of the forward-scattering detector in the L-FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.     

Asialogangliosides as differentiation markers of rat erythropoietic and granulopoietic cells in rat bone marrow. Flow cytometric analysis of rat bone marrow cells using anti-asialoganglioside antibodies

Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (IV2 alpha Fuc-Gg4Cer) and IV3 alpha Gal-fucogangliotetraosylceramide (IV3 alpha GalIV2 alpha Fuc-Gg4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grünwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glycolipids on their cell surfaces; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulocytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.     

Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations

A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.     

A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma mansoni.

Centrifugation of human white blood cells over either Ficoll-Hypaque or slightly hypertonic Metrizamide discontinuous gradients reliably produces separate fractions that are enriched for either neutrophilic or eosinophilic granulocytes. This single step purification routinely yields 90 to 100% pure neutrophils and 85 to 100% pure eosinophils. Metrizamide gradients, in particular, reproducibly provide high yields of 90 to 100% pure eosinophils from normal subjects with 2 to 3% eosinophils in their peripheral blood. The method does not damage cells as judged by morphologic or functional criteria. The purified cell populations were tested for their ability to damage antibody-coated schistosomula either by the measurement of 51Cr release from labeled organisms, or by direct morphologic assessment. Neutrophils were superior in their ability to release 51Cr from labeled organisms, but eosinophils adhered to the organisms to a greater extent and induced microscopically detectable damage.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Resolving leukocytes using axial light loss

Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward-angle and right-angle light scatter, they are clearly resolved in right-angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin-labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T-cells. B-cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T-cell subset is resolved from the CD3+ CD4- subset comprising mainly the CD3+ CD8+ T-cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformaldehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.     

Cell analysis in cerebrospinal fluid (CSF) using Sysmex? hematology analyzers XT-4000i and XE-5000: evaluation with CSF controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine (DGKL)

In cerebrospinal fluid (CSF) analysis, hematology analyzers (HAs) Sysmex? XT-4000i and XE-5000, equipped with flow cytometry (FCM), were used to count cells and differentiate leukocytes into mononuclear and polymorphonuclear cells (MNCs, PMCs) applying body fluid mode. FCM was evaluated with 20 DGKL CSF controls containing viable human leukocytes and erythrocytes. HA values were compared with reference values by Passing/Bablok regression analysis to reveal conformity. Conformity of white blood cells (WBCs) was obtained with native leukocytes, counted in calibrated Fuchs-Rosenthal chamber as reference; red blood cell counts proved inaccurate. CV <40% with WBC counts <20 per μL impairs accuracy. Reference WBC differentiation was assayed using FACS Canto II? and FC-500 SN with anti-CD45, anti-CD14, anti-CD16, anti-CD16/56 [Becton Dickinson (BD); Beckman Coulter (BC)]. BD FACS lysing solution?-no-wash-procedure was applied. BC pretreatment with Versalyse lysing solution was not recommended. MNCs (lymphocytes + monocytes) were significantly lower (～14%) on both HAs; PMCs (granulocytes or sum of neutrophils + eosinophils + basophils: range 1-86 M/L) were significantly higher (～2.2-fold). WBC HA differentiation is not reliable because MNC/PMC differentiation yielded lower and higher values than FACS-FCM references, respectively. This is attributed to incorrect discrimination of leukocytes with rounded/nonrounded nuclei; adding leukocytes with nonrounded nuclei to too low HA MNCs (about 40% not-activated) yielded P/B conformity; subtraction of leukocytes with nonrounded nuclei from elevated HA PMCs showed conformity (about 85% activated). Nucleus/activation state of leukocytes was assessed using microhistology. Sysmex XT-4000i and XE-5000 HAs systems are inappropriate for complete CSF cell analysis.