A highly diluted suspension of red blood cells (hematocrit 0.01) was illuminated with an Ar or a dye laser in the wavelength range of 458-660 nm. The extinction and the angle-resolved intensity of scattered light were measured and compared with the predictions of Mie theory, the Rayleigh-Gans approximation, and the anomalous diffraction approximation. Furthermore, empirical phase functions were fitted to the measurements. The measurements were in satisfactory agreement with the predictions of Mie theory. However, better agreement was found with the anomalous diffraction model. In the Rayleigh-Gans approximation, only small-angle scattering is described appropriately. The scattering phase function of erythrocytes may be represented by the Gegenbauer kernel phase function.

Single scattering by red blood cells

Red blood cell (RBC) parameters such as morphology, volume, refractive index, and hemoglobin content are of great importance for diagnostic purposes. Existing approaches require complicated calibration procedures and robust cell perturbation. As a result, reference values for normal RBC differ depending on the method used. We present a way for measuring parameters of intact individual RBCs by using digital holographic microscopy (DHM), a new interferometric and label-free technique with nanometric axial sensitivity. The results are compared with values achieved by conventional techniques for RBC of the same donor and previously published figures. A DHM equipped with a laser diode (lambda = 663 nm) was used to record holograms in an off-axis geometry. Measurements of both RBC refractive indices and volumes were achieved via monitoring the quantitative phase map of RBC by means of a sequential perfusion of two isotonic solutions with different refractive indices obtained by the use of Nycodenz (decoupling procedure). Volume of RBCs labeled by membrane dye Dil was analyzed by confocal microscopy. The mean cell volume (MCV), red blood cell distribution width (RDW), and mean cell hemoglobin concentration (MCHC) were also measured with an impedance volume analyzer. DHM yielded RBC refractive index n = 1.418 +/- 0.012, volume 83 +/- 14 fl, MCH = 29.9 pg, and MCHC 362 +/- 40 g/l. Erythrocyte MCV, MCH, and MCHC achieved by an impedance volume analyzer were 82 fl, 28.6 pg, and 349 g/l, respectively. Confocal microscopy yielded 91 +/- 17 fl for RBC volume. In conclusion, DHM in combination with a decoupling procedure allows measuring noninvasively volume, refractive index, and hemoglobin content of single-living RBCs with a high accuracy.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/cyto.a.20605

Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer

3 – Basic Haematological Techniques

Simultaneous measurement of reticulocyte and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia

Summary
The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method. However, it is time-consuming and results in high levels of imprecision. The introduction of automated full blood counters using impedance technology resulted in a dramatic improvement in precision. However, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelet counting, but there are still occasional cases where an accurate platelet count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting. This method utilizes monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore. This permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples. In future, analysers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1751-553X.2007.00909.x

Continuing developments with the automated platelet count.

A light scattering technique for simultaneously determining the volume (V) and hemoglobin concentration (HC) of individual sphered red blood cells (RBCs) is described. Light scattered into two angular intervals yields measurements S1 and S2, respectively. Since a sphered RBC is essentially a homogeneous dielectric sphere having a complex refractive index that is linear in HC, with a proper choice of detector acceptance angles, tables relating V and HC to S1 and S2 can be computed via Mie theory. Absolute calibration is possible using droplets of water-immisible oils of accurately known refractive index. Results of experimental tests of the method are compared with those obtained from hematological reference measurements.

D. H. Tycko

Papers

Flow-cytometric light scattering measurement of red blood cell volume and hemoglobin concentration.