Fig. 1 a, Orthogonal polarization spectral imaging probe. b, Optical schematic of the OPS imaging probe. A typical magnification of ×10 is maintained between the target and its image. This results in a resolution of approximately 1 µm/pixel, which is limited by the dimension of the CCD pixel. The probe can be focused from the target surface to 1.0-mm depth, depending on the type of target and the optics used. In vivo, the typical depth of focus is approximately 0.2 mm. c, Optical density of a graduated gray scale (catalog number 152-7662; Kodak) was measured in the presence of the polarization analyzer (b) or with a 0.5 OD neutral density filter (ć) used in place of the analyzer. Average light intensity for each gray level was converted to OD by the following formula: OD = log 10 ((I m -I d )/(I max -I d )) where I m = measured light intensity, I d = dark light intensity (obtained using a black velvet target), I max = intensity of white target. NEW TECHNOLOGYDifferent disease states, including diabetes, hypertension and coronary heart disease, produce distinctive microvascular pathologies. So far, imaging of the human microcirculation has been limited to vascular beds in which the vessels are visible and close to the surface (for example, nailfold, conjunctiva). We report here on orthogonal polarization spectral (OPS) imaging, a new method for imaging the microcirculation using reflected light that allows imaging of the microcirculation noninvasively through mucus membranes and on the surface of solid organs. In OPS imaging, the tissue is illuminated with linearly polarized light and imaged through a polarizer oriented orthogonal to the plane of the illuminating light. Only depolarized photons scattered in the tissue contribute to the image. The optical response of OPS imaging is linear and can be used for reflection spectrophotometry over the wide range of optical density typically achieved by transmission spectrophotometry. A comparison of fluorescence intravital microscopy with OPS imaging in the hamster demonstrated equivalence in measured physiological parameters under control conditions and after ischemic injury. OPS imaging produced high-contrast microvascular images in people from sublingual sites and the brain surface that appear as in transillumination. The technology can be implemented in a small optical probe, providing a convenient method for intravital microscopy on otherwise inaccessible sites and organs in the awake subject or during surgery for research and for clinical diagnostic applications.At present, the use of microvascular imaging in diagnosis and treatment of human disease is limited. Use has been made of nailfold capillaroscopy in the diagnosis and treatment of peripheral vascular diseases, diabetes and hematological disorders 1-3 . Problems with movement have restricted the use of the bulbar conjunctiva for clinical applications in opthalmology 4-6 . Other locations observed by intravital microscopy include the microcirculation of the skin, lip, gingival tissue and tongue 4 . Laser-sca...

Cell volume (MCV) and hemoglobin concentration (MCHC) are the red cell indices used to characterize the blood of patients with anemia. Since the introduction of flow cytometric methods for the measurement of these indices, it has generally been assumed that the values derived by these instruments are accurate. However, it has recently been shown that a number of cellular factors, including alterations in cellular deformability, can lead to inaccurate measurement of cell volume by these automated instruments. Because cell hemoglobin concentration and hematocrit are computed from the measured values of cell volume, accuracy of these indices is also compromised by inaccurate determination of cell volume. A recently developed experimental flow cytometric method based on laser light scattering, which can independently measure volume and hemoglobin concentration, has been used in the present study to measure MCV and MCHC of density- fractionated normal and sickle red cells, hydrated and dehydrated normal red cells, and various pathologic cells. We found that the new method accurately measures both volume and hemoglobin concentrations over a wide range of MCV (30 to 120 fL) and MCHC (27 to 45 g/dL) values. This is in contrast to currently available methods in which hemoglobin concentration values are accurately measured over a more limited range (27 to 35 g/dL). In addition, as the experimental method independently measures volume and hemoglobin concentration of individual red cells, it allowed us to generate histograms of volume and hemoglobin concentration distribution and derive coefficient of variation for volume distribution and standard deviation of hemoglobin concentration distribution. We have been able to document that volume and hemoglobin concentration distributions can vary independently of each other in pathologic red cell samples.

The laser light scattered by erythrocytes subjected to a well-defined shear stess can be analyzed with the ektacytometer to obtain information regarding the changes in cell shape due to fluid shear. We describe an optical technique whereby an observed quantitative output derived from a mesurement of light intensity through a spatial filter is related to the change in cellular dimensions that were previously observed under similar fluid-shear conditions by use of microscopy and a cone-plate viscometer (rheoscope). We also present the predictions of a theoretical model (of the ektacytometer) based on approximations of light-scattering theory developed for nonspherical particles, and give preliminary results for the accuracy and sensitivity of this measurement of erythrocyte deformability. With this optical technique the instrumentation (ektacytometer) is made quite simple and suitable for use in the typical laboratory. This would allow a regular, quantitative assessment of this important blood cell quality, to supplement the data obtained from the complete blood count.

There is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)-infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets.We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*l, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built-in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples.Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory.

The Hemalog D system performs automated differential white cell counts utilizing principles of cytochemistry, electro-optical measurement and signal logic processing. Using 0.4 ml of anticoagulated whole blood and an automated continuous flow staining technique, the system is designed to process a new sample each minute. The decision logic classifies individual cells by size and intensity of staining as they flow through detectors designed to measure light loss and light scattering simultaneously. With this system, 10,000 cells/ sample are classified in less than 1 min. Alcian Blue identifies basophils when used in the presence of quaternary ammonium salts and other counterions. Monocytes are the only cells stained in an esterase method using α-naphthol butyrate at pH 6.1 with hexazonium pararosanilin as coupler. This unstable diazonium is produced on stream continuously from stable components. Size and peroxidase staining serve to classify the remaining cells. Lymphocytes and large mononuclear cells are unstained, neutrophils have moderate peroxidase activity, while eosinophils stain very strongly. Threshold detection logic for light scattering and absorption differentiate the neutrophils, eosinophils and lymphocytes. The distribution of peroxidase activity in neutrophils serves as an index of maturity of these cells. The clinical evaluation of this index is reported elsewhere. Results are reported as percentage and as cells per cubic millimeter. A total white blood cell count is also reported and the pattern of cell measurements for 10,000 cells is provided as a picture with diagnostic information content. Advantages of this system in rapid screening result from improved precision of automation, the processing of large numbers of cells and the saving of labor. Precision also makes the acquisition of sequential data on single patients more meaningful and diagnostically valuable.