Method for Treating a Biological Sample, and Device for Isolating Cells from a Transport Medium

Abstract

The present disclosure relates to a method for treating a biological sample contained in a transport medium. At least one complexing agent is added to the transport medium, which complexing agent forms complexes with alkaline-earth metal ions. In the method, a device can be used which comprises, in succession, a porous volume filter and a membrane filter having a pore size in the range of 0.2 μm to 2.0 μm.

Claims

1. A method for treating a biological sample present in a transport medium, comprising: adding at least one complexing agent that forms complexes with alkaline earth metal ions to the transport medium.

2. The method as claimed in claim 1, wherein the transport medium is an Amies medium.

3. The method as claimed in claim 1, wherein the alkaline earth metal ions are at least one of calcium ions and/or magnesium ions.

4. The method as claimed in claim 1, wherein the at least one complexing agent comprises [NR.sup.1R.sup.2R.sup.3R.sup.4].sup.+ ions, where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently selected from H and alkyl groups.

5. The method as claimed in claim 4, wherein the at least one complexing agent comprises at least one salt selected from a group consisting of ammonium hydrogencitrate, ammonium citrate, ammonium thioglycolate, ammonium chloride, ammonium acetate, tetramethylammonium citrate, and tetramethylammonium thioglycolate.

6. The method as claimed in claim 1, further comprising: adjusting the transport medium to a pH in the range of 5 to 6.

7. The method as claimed in claim 6, further comprising: adding at least one of a citric acid/citrate buffer, an acetic acid/acetate buffer, and/or a thioglycolic acid/thioglycolate buffer to the transport medium.

8. The method as claimed in claim 1, further comprising: filtering, after adding the complexing agent, the transport medium through a porous volume filter; and filtering the filtered transport medium through a membrane filter with a pore size in the range of 0.2 μm to 2.0 μm.

9. The method as claimed in claim 8, characterized in that further comprising: freeing cells that remain on the membrane filter of alkaline earth metal hydrogenphosphates, alkaline earth metal dihydrogenphosphates and impurities from the transport medium.

10. The method as claimed in claim 8, further comprising: lyzing cells that remain on the membrane filter.

11. The method as claimed in claim 10, further comprising: eluting nucleic acids released during the lysis from the membrane filter.

12. A device for isolating cells from a transport medium, comprising: a porous volume filter; and a membrane filter with a pore size in the range of 0.2 μm to 2.0 μm located downstream of the porous filter.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows a flow diagram of an exemplary embodiment of the method according to the invention.

[0015] FIG. 2 shows a schematic representation of an exemplary embodiment of the device according to the invention.

EXEMPLARY EMBODIMENTS OF THE INVENTION

[0016] In one exemplary embodiment of the invention, a biological sample, which is a bacterial sample in the form of a patient swab, is introduced into an Amies medium as transport medium. In the present exemplary embodiment, the Amies medium comprises [0017] 0.2 g/l potassium chloride [0018] 0.2 g/l potassium dihydrogenphosphate [0019] 0.1 g/l calcium chloride [0020] 3.0 g/l sodium chloride [0021] 1.15 g/l sodium hydrogenphosphate [0022] 1.0 g/l sodium thioglycolate [0023] 0.1 g/l magnesium chloride [0024] 6.5 g/l agar

[0025] It has a pH of around 7.3. Once the Amies medium with the biological sample has been transported to a diagnostic laboratory, it is treated there by means of an exemplary embodiment of the method according to the invention. As shown in FIG. 1, after the method has started 10, the Amies medium with the biological sample is inserted 11 into a lab-on-chip system. In this, a complex-forming buffer mixture is added 12 to the Amies medium. In the present exemplary embodiment, the complex-forming buffer mixture comprises ammonium hydrogencitrate and citric acid in a mixing ratio which buffers the Amies medium to a pH in the range of 5 to 6. Calcium dihydrogenphosphate, calcium hydrogenphosphate, magnesium dihydrogenphosphate and magnesium hydrogenphosphate, formed from the chlorides of the two alkaline earth metal elements along with the potassium dihydrogenphosphate and the sodium hydrogenphosphate, are suspended in the Amies medium and go back into solution with formation of diamminecalcium complexes and diamminemagnesium complexes. The Amies medium thus treated is then introduced into a device 20 for isolating cells that is arranged in the lab-on-chip and is shown in FIG. 2.

[0026] The device 20 has a porous volume filter 21. Arranged immediately downstream of this is a porous membrane filter 22 that has a pore size of 0.9 μm in the present exemplary embodiment. The Amies medium is introduced into the device 20 at an introduction position 23 on the volume filter 21. The Amies medium is first filtered 13 through the volume filter 21 as a result. This holds back agar and relatively large particles or sample impurities in the volume filter 21, while bacterial cells easily pass through this volume filter and thus reach the membrane filter 22. The Amies medium is then further filtered 14 through the membrane filter 22. Here, the bacterial cells from the biological sample remain on the membrane filter 22, while all soluble components of the Amies medium leave the device 20 through the membrane filter 22 and a subsequent exit position 24 as an aqueous solution.

[0027] The cells are then washed 15 on the membrane filter 22 by introducing a Tween®/water mixture (polysorbate/water mixture) into the device 20 through the inlet point 23. This washes remaining soluble constituents of the Amies medium and soluble constituents of the biological sample still adhering to the surface of the cells out of the device 20 through the membrane filter 22 and through the outlet point 24. Afterward, the cells are lyzed 16 by introducing a 0.1 wt % solution of Triton® X-100 (octoxynol-9) into the device 20 through the introduction position 23. This functions as a lysis medium and simultaneously elutes the free nucleic acids from the bacteria, obtained by way of the lysis, out of the device through the outlet opening 24. The eluate is mixed with a PCR master mix in the form of a lyophilized PCR bead and a PCR detection reaction is carried out 17. The method is then ended 18.