WO2017202729A1

WO2017202729A1 - Microfluidic device and method for providing a sample of paraffin-embedded tissue

Info

Publication number: WO2017202729A1
Application number: PCT/EP2017/062156
Authority: WO
Inventors: Jochen Hoffmann
Original assignee: Robert Bosch Gmbh
Priority date: 2016-05-24
Filing date: 2017-05-19
Publication date: 2017-11-30
Also published as: DE102016208936A1

Abstract

The invention relates to a method (500) for providing a sample of paraffin-embedded tissue using a microfluidic device (100), having the following steps: • introducing a paraffin-embedded tissue sample (1) into a first chamber (110) of the microfluidic device (100), • introducing oil into the first chamber (110), in particular by pumping the oil out of a first reservoir (120) of the microfluidic device (100) into the first chamber (110), and • extracting paraffin out of the paraffin-embedded tissue sample (1) by contacting the tissue sample (1) with the oil, wherein the oil is brought to a first temperature by a heating device (130) of the microfluidic device (100) and preferably held at the first temperature.

Description

description

title

Microfluidic device and method for sample preparation of paraffin-embedded tissue

State of the art

In the field of tumor diagnostics, tissue of a carcinoma taken by a surgeon is usually initially treated with formalin for later analysis in order to inactivate cellular enzymes and to fix the DNA. For preservation, the tissue is then embedded in paraffin.

In order to detect the genetic status of a carcinoma by means of molecular diagnostic methods, for example a polymerase chain reaction, both the formalin and the paraffin must be removed before the analysis. This is usually done by a solution of the paraffin in xylene, followed by removal of the xylene with ethanol, and heating of the tissue sample to inactivate the formalin fixation. Such a method is known for example from WO 2011/150316 AI.

Disclosure of the invention

Advantages of the invention

The invention relates to a method for sample preparation of paraffin-embedded tissue with a microfluidic device. The method comprises, in a first step, introducing a paraffin-embedded tissue sample into a first chamber of the microfluidic device. In a second step, oil is introduced into the first chamber, in particular by a Pumping the oil from a first reservoir of the microfluidic device into the first chamber. The method further comprises a step of extracting paraffin from the paraffin-embedded tissue sample by contacting the tissue sample with the oil, wherein the oil is brought to a first temperature by a heater of the microfluidic device and

is preferably maintained at the first temperature. In this case, the oil can be brought to the first temperature either before it is introduced into the first chamber or only after it has been introduced into the first chamber.

A paraffin-embedded tissue sample may, in particular, be understood as meaning a part of a human or animal tissue which has been embedded in paraffin. In addition, the tissue may have been treated with formalin prior to embedding in paraffin, and such prepared tissue is also referred to as formalin-fixed paraffin-embedded tissue, FFPE tissue for short. In particular, a device in which microfluidic processes can run at least partially automated can be understood as a microfluidic device, for example a lab-on-a-chip device. Extraction of paraffin from the paraffin-embedded tissue sample may, in particular, be understood to mean a separation of at least a portion of the paraffin in which the tissue sample is embedded from the tissue sample.

The invention is based on the recognition that extracting paraffin from a paraffin-embedded tissue sample using oil in a microfluidic device is particularly advantageous. Oil, such as a mineral oil, has the advantage that it can be stored in microfluidic devices, which usually comprise at least a partial structure of polymers, long-term stable. The use of oil makes it possible in particular to dispense with the customary use of xylene for extracting the paraffin from the paraffin-embedded tissue sample. Unlike oil, xylene can not readily exist in microfluidic

Prevented devices based on plastic. Furthermore, the use of oil can be dispensed with a customary rinsing of the sample to remove xylene residues. Since it can be dispensed with the introduction or pre-storage of a rinsing liquid, the microfluidic In addition, the microfluidic device can be disposed of in a simple manner after use, while with use of xylene must be made separate disposal.

In a particularly advantageous embodiment of the invention, the oil having the first temperature via a loop-shaped channel of

microfluidic device several times for the extraction of paraffin through the first chamber. This makes the tissue sample extra

be effectively mixed with the oil.

According to an advantageous embodiment of the invention, a lysis buffer for lysis of cells of the tissue sample is added after extraction of the paraffin. This can be done in a simple way DNA from cells of the

Tissue sample to be extracted. The lysis buffer may in particular be a buffer suitable for chemical, thermal or ultrasound lysis.

According to a particularly advantageous embodiment of the invention, the oil mixed with extracted paraffin is conveyed with an aqueous solution, in particular with a lysis buffer, after a lysis of cells of the tissue sample, in a phase separation channel of the microfluidic device. In this case, at least part of the oil is cut off by the hydrophobic filter, in particular a hydrophobic membrane filter, from the aqueous solution in a section of the phase separation channel bounded by the filter. Thus, advantageously, the two-phase mixture is divided by the filter into a non-polar phase containing the oil and a polar phase containing the aqueous components of the aqueous solution. The two phases can then advantageously be further processed separately.

Preferably, after extracting the paraffin, the tissue sample is supplemented with an aqueous reagent for enrichment of the DNA from the tissue sample in the aqueous reagent. Thus, advantageously, especially after the lysis has been carried out, the DNA can be separated from the tissue sample in a simple manner for a further analysis of the DNA. In an advantageous embodiment of the invention, the tissue sample for removing a Formalinfixierung is brought to a second temperature, wherein the second temperature is preferably between 60 and 110 degrees Celsius, preferably between 70 and 90 degrees Celsius. This has the advantage that fixation of formalin of the tissue by heating can be easily and effectively reversed.

The invention also relates to a microfluidic device for

Sample preparation of paraffin-embedded tissue. The microfluidic device comprises a first chamber for receiving a paraffin-embedded tissue sample and a heating device. The first chamber is fluidly connected to a reservoir for oil and preferably filled with oil, especially mineral oil. The heater is configured to bring oil from the reservoir for extracting paraffin from a sample received in the first chamber to a first temperature by contacting the oil with the tissue sample. The microfluidic device according to the invention advantageously makes it possible, by providing a

Reservoirs for oil and a heater to extract paraffin from the paraffin-embedded tissue. By using oil instead of the xylene commonly used, the oil can easily be stored in the microfluidic device in a long-term stable manner and the device can be disposed of after use without any special post-processing.

In an advantageous development of the microfluidic device, an outlet of the first chamber is connected via a loop-shaped channel to an inlet in the first chamber. This allows repeated transport of the oil for extracting the paraffin through the first chamber.

In a further advantageous embodiment of the invention, the first chamber is fluidly connected to a phase separation channel, wherein the

Phase separation channel by a hydrophobic filter, in particular a hydrophobic membrane filter, limited portion. As a result, when passing through an aqueous solution which comprises oil mixed with paraffin extracted from the tissue sample, it is advantageously carried out by the Phase separation channel at least a portion of the oil through the hydrophobic filter from the aqueous solution deposited in the section.

According to an advantageous development of the microfluidic device, the heating device is also set up. The tissue sample for a removal of one

Formalinfixierung bring to a second temperature, wherein a second temperature is preferably between 50 and 110 degrees Celsius, preferably between 70 and 90 degrees Celsius. The invention further relates to a computer program that is set up all

Perform steps of the method according to the invention, as well as a machine-readable storage medium with a computer program stored thereon according to the invention. Brief description of the drawings

Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. Show it:

Figure 1 is a schematic representation of an embodiment of the

Invention and Figure 2 is a flowchart of an embodiment of the invention

Process.

EMBODIMENTS OF THE INVENTION FIG. 1 shows a schematic representation of a part of a microfluidic one

Device 100, which has a first chamber 110 for receiving a

Tissue sample 1, for example via an opening 115 of the first chamber 110 includes. The first chamber 110 is fluidly connected to a reservoir 120 for oil, wherein the fluidic connection 160 through a first valve 116 at an inlet opening 111 to the first chamber 110 and through a second Valve 117 can be interrupted near the reservoir 120. The oil may be a mineral oil, which is pre-stored in the reservoir 120 in an amount of 50 to 2000 microliters, preferably 200 to 800 microliters. The microfluidic device 100 further comprises a heater 130, which is adapted to bring oil from the reservoir 120 to a first temperature and preferably to hold there. For this, the heater 130 may be thermally connected to the reservoir 120 or the first chamber 110. The oil from the reservoir 120 can be passed via the fluidic connection 160 into the first chamber 110, wherein the oil can be brought to the first temperature by the heater 130 before being introduced into the first chamber 110 or only after being introduced into the first chamber 110 is brought to the first temperature. To aid in the introduction of the oil from the reservoir 120 into the first chamber 110, the microfluidic

Device preferably a pump 165, which preferably in the fluidic connection 160 between the first chamber 110 and the reservoir

120 is arranged.

The microfluidic device 110 in this embodiment comprises a loop-shaped channel 140, so that an outlet 112 of the first chamber 110 via the loop-shaped channel 140 with the input 111 in the first

Chamber 110 is connected. This allows for repeated transport of the oil through the first chamber 110, preferably assisted by the pump 160. To fluidically decouple the looped channel 140, the looped channel 140 includes a third at an entrance to the channel 140 and an exit from the channel 140 Valve 118 or a fourth

Valve 119.

The output 112 of the first chamber 110 is fluidly connected to a phase separation channel 150 via a fifth valve 151. The phase separation channel 150 has a hydrophobic filter 155 which has a portion 156 in the

Phase separation channel 150 limited. The hydrophobic filter 155 may be a fluorinated filter, such as a fluorinated one

Membrane filter act, which includes polytetrafluoroethylene, and for example pores having a size between 5 nanometers and 500 microns, preferably between 0.1 and 10 microns. Due to the hydrophobic Characteristics of the filter 155 is a non-polar constituent in an aqueous solution, for example, oil in water, as the aqueous solution is passed through the phase separation channel of polar constituents of the solution into section 156, so as to separate a polar from a non-polar phase. The non-polar phase can be forwarded via a first output 152 of the section 156 and the polar phase via a second output 153 of the phase separation channel 150.

FIG. 2 shows a flowchart of an embodiment of the invention

A method according to the invention for the sample preparation of paraffin-embedded tissue with a microfluidic device, for example with a microfluidic device 100 from FIG. 1.

In a first step 501, a paraffin-embedded tissue sample 1 is taken up in a first chamber 110 of the microfluidic device 100. In a second step 502, oil is introduced from the reservoir 120 into the first chamber 110 with the assistance of the pump 160. In a third 503, at least a portion of the paraffin is extracted from the paraffin-embedded tissue sample 1 by contacting the tissue sample 1 with the oil, the oil being heated to a first temperature by the heater 130, for example at a temperature between 70 and 90 degrees Celsius. In order to increase the effectiveness of the extraction of the paraffin, the oil is pumped through the first chamber 110 via the loop-shaped channel 140 of the microfluidic device 100. Due to the first temperature of the oil, the paraffin melts and combines with the oil. If the paraffin-embedded tissue additionally has a formalin fixation, then in a fourth step 504 the formalin fixation may be achieved by heating the

Tissue sample 1 can be inactivated to a temperature between 70 and 95 degrees Celsius for 5 to 60 minutes.

In a fifth step 505 of the embodiment, a lysis of cells of the recorded tissue sample 1. The lysis can be done either via a chemical lysis, a thermal lysis or ultrasound lysis. In the case of chemical, enzymatic lysis or thermal lysis, a lysis buffer is added to the first chamber 110. This can either also take place via the opening 115 in the first chamber 110 or, as shown in Figure 1, via a feed channel 170, which can be fluidly connected to the first chamber via a closable sixth valve 171 with the first chamber done. In the case of ultrasonic lysis, ultrasonic energy is coupled into the first chamber 110.

After lysis has taken place, in a sixth step 506, the lysis buffer, which contains both the paraffin-blended oil and the dissolved DNA from the tissue, via the outlet 112 of the first chamber 110

Phase separation channel 150 forwarded, where due to the hydrophobic

Filter 155 of the phase separation channel 150 a separation of the non-polar oil is separated together with the paraffin from the polar components of the buffer, in particular the DNA. The paraffin-separated oil can be disposed of via the first exit 152 of the phase separation channel while the polar components, in particular the DNA, via the second

Output 153 of the phase separation channel can be conveyed out of the phase separation channel 150 for further analysis. The sixth step can be supplemented by DNA extraction methods known from the prior art.

Claims

claims

A method (500) for paraffin-embedded tissue sample preparation with a microfluidic device (100), comprising the steps of:

Introducing a paraffin-embedded tissue sample (1) into a first chamber (110) of the microfluidic device (100)

Introducing oil into the first chamber (110), in particular by pumping the oil from a first reservoir (120) of the microfluidic device (100) into the first chamber (110)

Extracting paraffin from the paraffin-embedded tissue sample (1) by contacting the tissue sample (1) with the oil, the oil being brought to a first temperature by a heater (130) of the microfluidic device (100), and preferably at the first temperature is held.

2. The method (500) of claim 1, wherein the first temperature having oil via a loop-shaped channel (140) of the microfluidic

Device (100) is repeatedly transported for the extraction of the paraffin through the first chamber (110).

3. The method (500) according to claim 1 or 2, wherein after the extraction of the paraffin, a lysis buffer for lysis of cells of the tissue sample (1) is added.

A method (500) according to any one of the preceding claims, wherein the extracted paraffin-blended oil is delivered to a phase separation channel (150) of the microfluidic device (100) with an aqueous solution, in particular a lysis buffer, after lysis of cells of the tissue sample wherein at least a portion of the oil passes through a hydrophobic filter (155) from the aqueous solution into a portion (156) of the aqueous solution defined by the filter (155) Phase separation channel (155) is deposited.

The method (500) of any of the preceding claims, wherein after extraction of the paraffin, the tissue sample (1) is supplemented with an aqueous reagent for enrichment of DNA from the tissue sample (1) in the aqueous reagent.

A method (500) according to any one of the preceding claims, wherein the tissue sample (1) is for removing a formalin fixation to a second

Temperature is brought, wherein the second temperature is preferably between 60 and 110 ° C, preferably between 70 and 90 ° C.

A microfluidic apparatus (100) for sample conditioning of paraffin-embedded tissue comprising a first chamber (110) for receiving a paraffin-embedded tissue sample (1) and a heater (130), characterized in that the first chamber is fluidically connected to a reservoir for oil and wherein the heater (130) is configured to bring oil from the reservoir (120) for extracting paraffin from a tissue sample received in the first chamber to a first temperature by contacting the oil with the tissue sample (1).

The microfluidic device (100) of claim 7, wherein an exit (112) of the first chamber (110) via a looped channel (140) having an inlet (111) into the first chamber (110) for repeated transport of the oil the extraction of the paraffin is connected.

The microfluidic device (100) of claim 7 or 8, wherein the first chamber (110) is fluidly connected to a phase separation channel (150), the phase separation channel (150) having a portion (156) defined by a hydrophobic filter (155) such that when passing an aqueous solution comprising oil mixed with paraffin extracted from the tissue sample (1), at least a portion of the oil passes through the phase separation channel (150) through the hydrophobic filter (155) from the aqueous solution to the portion (Fig. 156) is deposited.

The microfluidic device (100) of any one of claims 7 to 9, wherein the heater (130) is further adapted to place the tissue sample (1) for a

Removal of a Formalinfixierung to bring a second temperature, wherein the second temperature is preferably between 50 and 110 ° C, preferably between 70 and 90 ° C.

A computer program adapted to perform all the steps of a method (500) according to any one of claims 1 to 6.

12. A machine-readable storage medium with a computer program stored thereon according to claim 11.