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Label-free cell differentiation

Novel concentration measurements for the full blood count including 3-part white blood cell differentiation

PTB-News 2.2017
02.05.2017
Especially interesting for

manufacturers of flow cytometers

 

oncology

 

 

hematology

 

 

laboratory medicine

In the case of blood samples with pathologically modified blood cells, the hemolysis and staining methods used in routine diagnostics can lead to the wrong identification of certain cell populations or to an inaccurate determination of their concentration. In contrast to this, an AC impedance procedure, which was developed at PTB, allows whole blood samples to be analyzed without disturbing influences due to the pre-processing of the sample.

Microfluidic chip with four integrated electrodes for flow cytometric blood cell measurement.

Label-free cell differentiation in a (diluted) full blood sample by means of AC impedance measurements at two frequencies (Z1 at 2.3 MHz and Z2 at 10 MHz). The clusters correspond to the red blood cells (RBC), the platelets (Plt), and the sub-populations of white blood cells: the granulocytes (Gn), the monocytes (M), and the lymphocytes (Ly).

The blood count is a compilation of data on the quantity and the shape of cells. From this, the physician can draw conclusions concerning a patient's state of health. The corresponding measurements also include flow cytometric procedures in which optical or impedance counting devices are used. In both cases, the red blood cells are first destroyed by hemolysis to facilitate the measurement of the concentration of white blood cells which, in healthy patients, are less abundant than red blood cells by a factor of approx. 1000. In the case of certain diseases such as leukemia, the white blood cells, however, may either be modified or destroyed by hemolysis procedures. In other blood samples, e.g. from newborns or from anemic patients, hemolysis-resistant erythrocytes may be observed which impede the correct differentiation and concentration measurement of leukocytes. To ensure reliable cell differentiation, such samples require demanding microscopic investigations or staining of the DNA or of the cell membrane.

The procedure developed at PTB allows blood samples, diluted to a suitable concentration, to be analyzed by means of flow cytometry, without modifying them by hemolysis or by using staining procedures. This method is based on measuring the change which occurs in the complex resistance (impedance) when a blood cell passes through the electrode configuration of a microfluidic assembly. In order to identify the cells unequivocally, two alternating voltages are applied with different frequencies and each of the complex AC impedance signals is measured, i.e. the effective resistance, the reactance and the absolute value of the impedance are determined. By choosing suitable frequencies and measurands, it is possible to differentiate in the micro flow cytometer between the cells attributed to the full blood count including the 3-part differential white blood count, i.e. the red blood cells, the platelets, the granulocytes, the monocytes and the lymphocytes.

With this new method, it is possible to facilitate the examination of blood samples from certain groups of patients and, at the same time, to prevent systematic measurement errors. This reduces the number of subsequent microscopic differentiations and/or of cell-specific staining. Moreover, the procedure, which is based on a microfluidic assembly, offers potential for developing a point of care device utilizing a flow cytometric sensor.

Patent application

Procedure and measuring equipment for the determination of blood cells, submitted on 5 November 2015.

Contact

Jörg Neukammer
Department 8.3 Biomedical Optics
Phone: +49 (0)30 3481-7241
joerg.neukammer@ptb.de

Scientific publication

P. Simon. M. Frankowski, N. Bock, J. Neukammer: Label-free whole blood cell differentiation based on multiple frequency AC impedance and light scattering analysis in a micro flow cytometer. Lab Chip 16, 2326−2388 (2016)