Analysis of red cell and platelet morphology using an imaging-combined flow cytometer

Laser-based flow cytometry and aperture-impedance methods are still the dominant technologies used for cell analysis in haematology, but both are limited to areas such as morphological analysis of red cell shape and high-sensitivity detection of platelet agglutinates and aggregates. Flow cytometry alone does not provide precise measurement of red cell volume without chemical pretreatment before detection and aperture-impedance is still considered the gold standard in the field of particle volume analysis. In the present study, an experimental prototype instrument called the imaging-combined flow cytometer (IFC) was evaluated. The IFC is equipped with an imaging device consisting of a pulse laser, lens units and a charge-coupled device (CCD) camera in addition to the flow-cytometric optical set-up. A personal computer was attached to the instrument to handle images derived from the imaging device. Laser illumination was triggered so that the image of an object was captured for each exposure of the CCD camera. Objects in the images were used to calculate size and shape information and to compute fractal texture features by image processing after each measurement. The advantage of the IFC is that it can capture images of selected cells of interest at the same time as flow-cytometric detection. Estimation of red cell volume, discrimination of red cells and platelets, and detection of platelet agglutinates and aggregates were attempted using the IFC in combination with image processing, It was found that image analysis on the IFC could provide a substitutional function for mean corpuscular volume (MCV) estimation and detection of platelet agglutinates and aggregates. The additional information generated by the IFC may be useful in diagnostic haematology.