Patent Application
20130122534
This invention relates to process for carrying out hematoxylin and eosin staining in a microfluidic device.
The detection of circulating tumour cells (CTC) present in the bloodstream of patients with cancer provides a means for characterisation and monitoring of cancers. A microfluidic device has been developed which allows the isolation of CTCs by taking advantage of the fact that CTCs have different physical properties to other blood constituents (Tan, S. J. et al, Biosensors and Bioelectronics 26 (2010) 1701-1705 and Tan, S. J. et al, Biomed Microdevices 11(4):883-892, 2009).
This microfluidic device (also referred to as a CTChip) is able to isolate CTCs in a label-free manner, with no need for biomarkers. Separation of CTCs in the device utilizes the fact that the size and stiffness of cancer cells are larger than that of blood cells. The device comprises a microfluidic channel through which a blood sample is flowed. Within the channel is an array of multiple cell isolation wells, each well being made up of a three projections (isolation structures) in a crescent shaped configuration and separated from each other by a gap of 5 μm. The size of this gap ensures the flow through of blood constituents due to their deformability and ability to traverse small constrictions. CTCs which are less deformable are trapped in the cell isolation wells.
A problem with CTC work is that cancer cells are identified by a combination of antibody markers (e.g. Ep CAM+ve, CD 45−e, DAPI+ve), yet this is not how cancer is defined by World Health Organisation criteria. In clinical practice, cancer is defined by hematoxylin and eosin appearance, supplemented when necessary with antibody markers.
Hematoxylin and eosin staining is the most widely used cell staining technique in medical diagnosis. Standard hematoxylin and eosin staining protocols are carried out by fixing cells on a slide using a formalin fixative and then sequentially applying hematoxylin and eosin stain solutions.
It has been observed that it is not possible to successfully carry out hematoxylin and eosin staining on a microfluidic device as described above using conventional staining protocols because undesired precipitation occurs during the staining process. It is therefore desirable to provide an optimised hematoxylin and eosin staining protocol that can be used to stain cells in a microfluidic device as described above without inducing undesired precipitation in the device.
Accordingly, in a first aspect of the invention there is provided a process for carrying out hematoxylin and eosin staining of circulating tumour cells trapped in a microfluidic device, the device comprising a sample inlet and at least one outlet connected in fluid communication by a microfluidic channel, the microfluidic channel having a plurality of cell isolation wells positioned therein, each well comprising an array of isolation structures arranged to trap a circulating tumour cell, but allow passage of other blood constituents, wherein the process comprises the steps of:
The process may further comprise the step (a′), prior to step (a), of priming the microfluidic device by flowing a priming solution through the channel. Accordingly, the process may comprise the steps of:
It has been determined that the combination of fixative reagents used in the above process, preferably in combination with the priming reagents described herein, enables hematoxylin and eosin staining to be successfully carried out in a microfluidic device without stain precipitation occurring.
The priming solution may be an aqueous EDTA solution, preferably at a concentration of 8-12 mM. The priming solution may be an 8-12 mM EDTA solution in phosphate buffered saline (PBS). Preferably, the priming solution is an EDTA/PBS solution at a concentration of about 10 mM.
The fixative solution may be an aqueous solution of paraformaldehyde and methanol. The solution may be in water or an aqueous buffer solution as a solvent. The solvent is preferably phosphate buffered saline (PBS). The fixative solution may be a 3-5 w/v % paraformaldehyde solution (e.g. in PBS), containing 15-25 v/v % methanol. Preferably, the fixative solution is a 4 w/v % paraformaldehyde solution in PBS, containing 20 v/v % methanol. Methanol is incorporated for membrane permeabilisation and avoids the need for treatment with a surfactant such as Triton X-100. Accordingly, steps (c) and (d), and preferably the entire process, of the invention may be carried out in the absence of surfactants such as Triton X-100.
Staining is carried out by exposing fixed cells to hematoxylin solution, washing, then subsequently exposing the cells to eosin solution. The hematoxylin staining may be carried out for up to 15 minutes. Preferably, hematoxylin staining is carried out for 1-5 minutes, more preferably for about 3 minutes. The hematoxylin solution is preferably a Harris's hematoxylin solution.
The eosin solution is preferably a 0.5% (w/v) eosin-Y solution (preferably an aqueous solution) diluted to a ratio of about 1:5 in deionised water to give a working concentration of 0.1%.
A final washing step (step (h)) may be carried out subsequent to eosin staining, by flowing a liquid through the channel. In general, washing steps may be carried out using water or an aqueous solution. In the process described above, washing steps (d), (f) and (h) are preferably carried out by washing with water or a buffered aqueous solution (such as PBS), preferably deionised water. Washing step (b) may preferably be carried out using the priming solution, as described above.
Specific embodiments of the invention are described below by way of example only and with reference to the accompanying drawings, in which:
In the context of this disclosure, a microfluidic device is a device comprising at least one channel having at least one dimension of less than 1 mm.
In the context used herein, ‘about’ is taken to encompass a variation of ±10% of any value to which it refers.
Hematoxylin and eosin staining steps are carried out by flowing hematoxylin and eosin stain solutions, respectively, into the channel thereby exposing trapped cells to stain solutions in situ.
As mentioned above, in a device in which the process of the first aspect of the invention is performed, each cell isolation well comprises an array of isolation structures. These structures are projections extending into the channel, preferably from the channel floor. Each well preferably comprises 3 or more (preferably 3) isolation structures arranged in a curved array (preferably crescent-shaped), with a gap of about 4-6 μm, preferably about 5 μm between the structures forming the well. Preferably, the cell isolations wells are positioned with a spacing of about 40-60 μm, preferably about 50 μm.
It will be appreciated that the process of the invention could also be used to perform hematoxylin and eosin staining of cells in a microfluidic device of varying structure and/or to stain varying cell types. Accordingly, in a further aspect the invention provides a process of carrying out hematoxylin and eosin staining of cells trapped in a microfluidic device, the process comprising the process steps as described above in respect of the first aspect of the invention. The microfluidic device may optionally have the structure defined in respect of the first aspect of the invention.
A microfluidic device within which the staining protocol of the invention can be carried out is illustrated in
Once CTCs are trapped within the device, staining of the cells can be performed in-situ using the staining protocol described herein, to identify the different cancer cells present.
An exemplary staining protocol which has been carried out on a device as described above (referred to as the CTChip) and has successfully achieved hematoxylin and eosin staining without precipitation is as follows:
The set-up is housed in a laminar flow hood and all fluids are filtered through a membrane (pore size=0.22 μm) before injection into the CTChip to minimize the introduction of debris into system, which adversely affects CTC isolation and staining
The protocol set out above was successful in enabling hematoxylin and eosin staining to be carried out without undesired precipitation.
The fixative solution can be prepared according to the following process: 1) Dissolve 4 g PFA in 100 ml 0.01M PBS, heat and stir mixture on a stirring block until all the PFA is dissolved; 2) To make 1 ml of 20% methanol in 4% PFA, add 200 μl of 100% methanol to 800 μl of the 4% PFA solution made in step 1; 3) Filter before use.
Experimental work was carried out to determine the effect of varying a number of the process steps in the protocol described above. In this work, the following was determined.
The concentration of the priming solution was found to be important. In the protocol above, a 10 mM EDTA/PBS priming solution is utilised and gives good results. However, decreasing this concentration to 5 mM EDTA/PBS produced undesirable streaky precipitation in the device prior to introduction of blood, as did use of an acid-citrate-dextrose solution.
The timing of hematoxylin staining in step 17 was also found to be important. A lower duration of hematoxylin staining, was found to give the best results, avoiding overstaining which can obscure cellular content.
In addition, a dilution of the eosin solution to a ratio of about 1:5 in deionised water, to give a working concentration of 0.1%, was found to be optimal in avoiding stain precipitation.
Whilst specific components have been described above as making up the embodiments described above, it is envisaged that, even when not explicitly stated above, alternative components may be substituted therefore, where those alternative components are substantially functionally equivalent to those described above. In the embodiments set forth below, and of the recited embodiments can be combined with any of the previously described embodiments. That is, where a particular claim is dependent upon another claim, it should be understood that the present disclosure includes such claim also being dependent upon any other previously recited claim.
| Number | Date | Country | |
|---|---|---|---|
| 61554004 | Nov 2011 | US |
