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Label-free flow cytometric cell differentiation by combined optical and impedance measurements in micro-fluid structures

30.12.2014

Flow cytometric counting of cells is an established procedure in laboratory medicine and serves to support the diagnosis in haematology, immunology, oncology and transfusion medicine. Besides highthroughput analysis of the samples in central laboratories, there is an increasing demand for simpler and faster point-of-care tests for different applications. These include – among other things – emergency medicine, decisions on transfusions during surgery, and the diagnosis as well as the monitoring of therapy in HIV patients in developing countries. Of particular interest are rapid tests where – usually time-consuming – cell labelling or destruction of red bloods cells by haemolysis is not necessary. To develop easy-to-handle disposable cartridges as are required for such point-of-care-tests, research as well as engineering activities in the field of microfluidic- based devices are being conducted increasingly.

Layout of the micro-fluid structure for the measurement of alternating current impedance signals of single blood cells

Differentiation of white and red blood cells and of blood platelets by simultaneous measurement of the scattered light and the impedance

At PTB, micro-fluid structures have been developed in cooperation with the TU Berlin and the RWTH Aachen. Impedance properties of single blood cells have been analysed systematically in the frequency range between 400 kHz and 100 MHz. Using the micro-fluid sensor shown in the figure, which allows simultaneous detection of side scatter of light and electric impedance, blood platelets (Plt), red blood cells (RBC) and the subpopulations of the white blood cells, the lymphocytes (Ly), granulocytes (G) and monocytes (M), were differentiated. These results demonstrate, for the first time, that combined light scatter and impedance detection allow differentiation of red and white blood cells and of platelets in a single measurement. Apart from the dilution of the sample, no additional preparations are required to measure the so-called “extended” Complete Blood Count when applying this method.

The results show that for the detection of white blood cells, haemolysis can be avoided, and that for selected applications, immunostaining is not required. Even the smallest corpuscular blood components, the thrombocytes or blood platelets, can be reliably detected in micro-fluid structures. Thus, the basis has been created for the development of specific disposable cartridges with an integrated sensor for combined optical and impedance measurements. Potential applications include, e.g., the rapid determination of concentrations of blood platelets during surgery and the differentiation of white blood cells in the whole blood of newborns or in blood samples of patients with lysis-resistant red blood cells.