GB2493965A - Hematoxylin/eosin staining of circulating tumour cells in a microfluidic device - Google Patents
Hematoxylin/eosin staining of circulating tumour cells in a microfluidic device Download PDFInfo
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- GB2493965A GB2493965A GB1114752.7A GB201114752A GB2493965A GB 2493965 A GB2493965 A GB 2493965A GB 201114752 A GB201114752 A GB 201114752A GB 2493965 A GB2493965 A GB 2493965A
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- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 210000005266 circulating tumour cell Anatomy 0.000 title claims abstract description 25
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 238000010186 staining Methods 0.000 title claims description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 38
- 210000004027 cell Anatomy 0.000 claims abstract description 33
- 210000004369 blood Anatomy 0.000 claims abstract description 26
- 239000008280 blood Substances 0.000 claims abstract description 26
- 238000002955 isolation Methods 0.000 claims abstract description 26
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 claims abstract description 11
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 10
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 10
- 239000000470 constituent Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 64
- 239000000834 fixative Substances 0.000 claims description 21
- 230000037452 priming Effects 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 18
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 13
- 239000002953 phosphate buffered saline Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 5
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 9
- 201000011510 cancer Diseases 0.000 abstract description 8
- 239000008367 deionised water Substances 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 7
- 239000002699 waste material Substances 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000011045 prefiltration Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- HGAZMNJKRQFZKS-UHFFFAOYSA-N chloroethene;ethenyl acetate Chemical compound ClC=C.CC(=O)OC=C HGAZMNJKRQFZKS-UHFFFAOYSA-N 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5002—Partitioning blood components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- Immunology (AREA)
- Urology & Nephrology (AREA)
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- Biomedical Technology (AREA)
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Abstract
The invention relates to a process for carrying out hematoxylin and eosin staining of circulating tumour cells in a microfluidic device. The device comprises an inlet and outlet connected by a microfluidic channel, wherein the channel has a plurality of cell isolation wells positioned therein that can trap a circulating tumour cells whilst allowing passage of other blood constituents. The blood sample is passed through the channel, and the circulating tumour cells are trapped in the isolation wells. The cancer cells are then fixed using paraformaldehyde and methanol, and then stained with hematoxylin and eosin.
Description
CELL STAINING PROCESS
FIELD
This invention relates to process for carrying out hematoxylin and eosin staining in a microfluidic device.
BACKGROUND
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. eta!, Biosensors and Bioelectronics 26 (2010)1701-1705 and Tan, S. J. etal, BioniedMicrodevices 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 pm. The size of this gap ensures the flow through of blood constituents due to their defonnability 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 -ye, DM1 +ve), yet this is not how cancer is defined by World Health Organisation criteria. In clinical practice, cancer is defined by hematoxylin arid eosin appearance, supplemented when necessary with antibody markers.
1-lematoxylin arid 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 S 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 microfluidie device as described above without inducing undesired precipitation in the device.
SUMMARY
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 (a) flowing a blood sample through the channel and allowing trapping of circulating tumour cells by the isolation structures to occur; (b) washing by flowing a liquid through the channel; (c) flowing a fixative solution through the channel, wherein the fixative solution comprises paraformaldehyde and methanol; (d) washing by flowing a liquid through the channel to remove excess fixative; (e) staining with hematoxylin solution; (0 washing by flowing a liquid through the channel; and (g) staining with eosin solution.
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: (a') priming the microfluidic device by flowing a priming solution through the channel; (a) flowing a blood sample through the channel and allowing trapping of circulating tumour cells by the isolation structure to occur; (b) washing by flowing a liquid through the channel; (c) flowing a fixative solution through the channel, wherein the fixative solution comprises paraformaldehyde and methanol; (d) washing by flowing a liquid through the channel to remove excess fixative; (e) staining with hematoxylin solution; (f) washing by flowing a liquid through the channel; and (g) staining with eosin solution.
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 hematoxytin and eosin staining to be successfully carried out in a microfluidie 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 8-12mM EDTA solution in phosphate buffered saline (PBS). Preferably, the priming solution is an EDTA/PBS solution at a concentration of about 10mM.
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-l00. 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.
BRIEF DESCRIPTION OF THE DRAWINGS
Specific embodiments of the invention are described below by way of example only and with reference to the accompanying drawings, in which: Figure 1 shows a schematic view of a microfluidic device in which a process of the invention may be performed, wherein (a) is a photograph of the microfluidic device; (b) illustrates the isolation well dimensions and (c) is a plan view showing a detailed layout of the microfluidic device.
DETAILED DESCRIPTION
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 1mm.
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 chatmel 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 isotation 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 m, preferably about 5 pm between the structures forming the well. Preferably, the cell isolations wells are positioned with a spacing of about 40-60 pm, preferably about 50 pm.
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 andlor to stain varying cell types. Accordingly, in a further aspect the invention provides a process of carrying out heniatoxylin 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 Figures 1(a), (b) and (c). The device comprises a sample inlet 100, first and second waste outlets 101, 105 linked in fluid communication by a microfluidie channel system within which are located a pre-filter 102 and a plurality of cell isolation wells 103. Details of this device are disclosed in Tan, S. J. et al, Biosensors and Bioelectronics 26 (2010) 1701-1 705 and Tan, S. J. et a!, Biotned Microdevices 11 (4):883-892, 2009, the disclosures of which are incorporated herein by reference. In operation, a blood sample is flowed through the device from the sample inlet and through the array of cell isolation wells 103, where trapping of CTCs, separating them from the remainder of blood constituents, occurs. Separation of CTCs in the device utilizes the fact that the size and stiffness of cancer cells are larger than that of blood cells. Each of the plurality of cell isolations wells 103 is made up of an array of three projections (isolation structures) in a crescent shaped configuration and separated from each other by a gap of 5 pm (Fig 1(b)). 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. The cell isolation wells are positioned with a spacing of 50 urn apart, which prevents cells clogging in the device. The pre filter 102 has gaps of 20 urn, prevents debris within a blood sample from clogging the device and, by connection to the waste outlet 101, enables removal of debris. A cell collection point 104 is also present.
Once CTCs are trapped within the device, staining of the cells can be performed in-situ using the staining protocol described herein, to identif' 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 jim) before injection into the CTCh1p to minimize the introduction of debris into system, which adversely affects CTC isolation and staining.
I. CTChip priming 1. Three Tygon tubings are cut and attached to a tubing connection assembly such as a Luer Lock connector.
2. Each tubing is carefully inserted into one of three ports in the CTChip.
3. The tubings are then attached, via the tubing connection, to three reservoir barrels set-up on a stand. Each of the three ports in the CTChip now corresponds to an individual barrel.
4. 2m1 of lOniM EDTAIPBS is filtered and injected into the barrels connected to both the blood and secondary ports.
5. The manifold, which is connected to a syringe pump that controls the pressure through the CTChip, is set to the priming' configuration.
6. The CTChip is primed once it is entirely filled with 10mM EDTAJPBS and no air bubbles are present in the traps or ports. This is determined visually, via a camera.
II. CTC Isolation 7. Fresh blood is collected in EDTA-containing blood collection tubes, kept at 4°C and processed within 24 hours of collection.
8. The manifold and pump are set to the isolation' configuration. Negative pressure through the waste port causes fluid to flow from the blood and secondary port, through the chip and out through the waste port.
9. Flow through the secondary port is stopped by closing the secondary tubing with a tubing clamp.
10. The blood is filtered through a 40tm cell strainer to remove clots and imi is injected into the blood port.
11. Once the blood enters the chip, it takes about 2 hours for I in! of blood to be filtered through the traps and end up in the waste barrel.
IlL Washing and fixing 12. After lml of blood has been processed, the secondary tubing is released and the blood tubing is closed, also via a tubing clamp. This stops the flow of blood through the chip and washes 10mM EDTAJPBS through the traps.
13. After 15 minutes, any 10mM EDTAIPBS remaining in the secondary barrel is removed.
14. A 4% paraformaldehyde fixative containing 20% methanol is prepared. SOOpI of this solution is filtered and injected into the secondary barrel.
15. After 20 minutes, any remaining fixative is removed from the secondary barrel and replaced with SOOpfl of deionised water.
IV. Staining 16. After 15 minutes of washing, the deionised water is replaced by 500 j.xl of Harris's hematoxylin (e.g. from Leica Microsystems).
17. Hematoxylin staining carries on for 3 minutes and is followed by another 15 minute wash with deionised water.
18. Subsequently, the water is removed and replaced by a 0.5% aqueous eosin-Y solution (diluted 1:5).
19. After 15 minutes, a final wash with deionised water is carried out.
20. The CTChip and its trapped cells are then ready to be observed under higher magnification.
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 4g PFA in lOOmi 0.OIM PBS, heat and stir mixture on a stirring block until all the PFA is dissolved; 2) To make imI of 20% methanol in 4% PFA, add 200pl of 100% methanol to 800p1 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 10mM EDTAJPBS priming solution is utilised and gives good results.
However, decreasing this concentration to 5mM 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 heniatoxylin 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.
Claims (1)
- <claim-text>Claims 1. 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: (a) flowing a blood sample through the channel and allowing trapping of circulating tumour cells by the isolation structure to occur; (b) washing by flowing a liquid through the channel; (c) flowing a fixative solution through the channel, wherein the fixative solution comprises paraformaldehyde and methanol; (d) washing by flowing a liquid through the channel to remove excess fixative; and (e) staining with hematoxylin solution; (0 washing by flowing a liquid through the channel and (g) staining with eosin solution.</claim-text> <claim-text>2. The process of claim 1, comprising step (a'), prior to step (a), of priming the microfluidic device by flowing a priming solution through the channel.</claim-text> <claim-text>3. The process of claim 2, wherein the priming solution is an EDTA solution.</claim-text> <claim-text>4. The process of claim 3, wherein the priming solution is an 8-12 mM EDTA solution.</claim-text> <claim-text>5. The process of claim 4, wherein the priming solution is an 8-12mM EDTA solution in PBS.</claim-text> <claim-text>6. The process of any one of claims 3 to 5, wherein the EDTA priming solution has an EDTA concentration of about 10mM.</claim-text> <claim-text>7. The process of any preceding claim, wherein the fixative solution is a solution of paraformaldehyde and methanol in a solvent.</claim-text> <claim-text>8. The process of claim 7, wherein the fixative solution is a solution in phosphate buffered saline (PBS).</claim-text> <claim-text>9. The process of claim 7 or 8, wherein the fixative solution is a 3-5 w/v% parafornialdehyde solution, containing 15-25 v/v % methanol.</claim-text> <claim-text>10. The process of claim 9, wherein the fixative solution is a 4 w/v% paraformaldehyde solution, containing 20 v/v% methanol.</claim-text> <claim-text>11. The process of any preceding claim, wherein step (0 comprises staining with hematoxylin for 1-5 minutes.</claim-text> <claim-text>12. The process of any preceding claim, wherein the eosin solution used in step (h) is an aqueous eosin-Y solution with a concentration of about 0.1% (w/v).</claim-text> <claim-text>13. The process of any one of claims 2 to 12, wherein the liquid used in washing step (c) is the same as the priming solution.</claim-text> <claim-text>14. A process of carrying out hematoxylin and eosin staining of cells trapped in a microfluidic device, the process comprising process steps (a) to (g), and optionally (a'), as defined in any one of claims Ito 13.</claim-text> <claim-text>15. A process substantially as described herein with reference to the accompanying drawings.</claim-text>
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| Application Number | Priority Date | Filing Date | Title |
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| GB1114752.7A GB2493965A (en) | 2011-08-25 | 2011-08-25 | Hematoxylin/eosin staining of circulating tumour cells in a microfluidic device |
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| GB1114752.7A GB2493965A (en) | 2011-08-25 | 2011-08-25 | Hematoxylin/eosin staining of circulating tumour cells in a microfluidic device |
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| GB2493965A true GB2493965A (en) | 2013-02-27 |
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| CN114295452A (en) * | 2021-12-31 | 2022-04-08 | 力因精准医疗产品（上海）有限公司 | Human sperm morphological staining reagent and staining method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060228772A1 (en) * | 2005-04-11 | 2006-10-12 | Donndelinger Thomas M | Compositions and methods for preparing specimens for microscopic analysis |
| WO2009051734A1 (en) * | 2007-10-17 | 2009-04-23 | The General Hospital Corporation | Microchip-based devices for capturing circulating tumor cells and methods of their use |
| WO2011025976A2 (en) * | 2009-08-28 | 2011-03-03 | On-Q-Ity Inc. | Methods of detaching and collecting cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060228772A1 (en) * | 2005-04-11 | 2006-10-12 | Donndelinger Thomas M | Compositions and methods for preparing specimens for microscopic analysis |
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| Publication number | Publication date |
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| GB201114752D0 (en) | 2011-10-12 |
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