Liquid biopsy detection of leukemia using closed-loop microfluidics

Patent metadata

Field	Value
Publication number	US-10697964-B2
Application number	US-201715726710-A
Country	US
Kind code	B2
Filing date	Oct 6, 2017
Priority date	Nov 15, 2016
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

Abstract

This invention describes a one-step technique for the simultaneous label-free detection and concentration of blast cells from a blood sample. Enrichment of blast cells is achieved using a closed loop microfluidics system, allowing continuous removal of waste and non-target cells to generate concentrated samples of high purity without the need for specific targeting of proteins by antibodies. The technique is highly effective for samples which cannot be purified in a single run. The application of detecting rare blast cells for monitoring minimal residual disease in leukemia patients is demonstrated. The sensitivity of the invention promotes the detection of blast cells in blood samples of early-stage patients.

First claim

What is claimed is:

1. A method of detecting blast cells in a blood sample comprising the steps of:
a. introducing a blood sample into an inlet reservoir of a microfluidic system comprising:
i. At least one inlet reservoir;
ii. At least one output reservoir;
iii. A first curvilinear microchannel comprising a first inlet in fluid communication with an inlet reservoir, a first outlet in fluid connection with the inlet reservoir, and a second outlet in fluid communication with an output reservoir; wherein said curvilinear microchannel is configured to separate particles from a fluid comprising a mixture of particles and wherein the microfluidic system is configured to provide a closed-loop recirculation of the fluid through the first curvilinear microchannel;

b. directing the blood sample from the inlet reservoir into the first inlet of the first curvilinear microchannel, bifurcating the blood sample into a first stream containing blast cells and at least one additional stream, wherein the at least one additional stream contains waste;
c. directing the first stream to the inlet reservoir and the second stream to the output reservoir, wherein the first stream comprises blast cells; and
d. detecting blast cells in the first stream.

2. The method of claim 1 , wherein the curvilinear microchannel is a spiral microchannel or a serpentine microchannel.

3. The method of claim 2 , wherein the microchannel is a spiral microchannel comprising at least 4 loops.

4. The method of claim 1 wherein the curvilinear microchannel has a trapezoidal cross section defined by a radially inner side, a radially outer side, a bottom side, and a top side, the cross section having a) the radially inner side and the radially outer side unequal in height, or b) the radially inner side equal in height to the radially outer side, and wherein the top side has at least two continuous straight sections, each unequal in width to the bottom side.

5. The method of claim 4 wherein the curvilinear microchannel cross section has (a) the height of the radially inner side larger than the height of the radially outer side, or (b) the height of the radially inner side is smaller than the height of the radially outer side, or (c) the top side includes at least one step forming a stepped profile, or (d) the top side includes at least one shallow region in between the radially inner side and the radially outer side.

6. The method of claim 4 , wherein the microchannel has a right trapezoidal cross section.

7. The method of claim 4 , wherein the radially inner side of at least one microchannel cross section has a height in a range of between about 20 microns and about 200 microns.

8. The method of claim 4 , wherein the radially outer side of at least one microchannel cross section has a height in a range of between about 20 microns and about 200 microns.

9. The method of claim 4 , wherein the bottom side of at least one microchannel cross section has a width in a range of between about 100 microns and about 2000 microns.

10. The method of claim 4 , wherein the top side of at least one microchannel cross section has a width in a range of between about 100 microns and about 2000 microns.

11. The method of claim 4 , wherein at least one curvilinear microchannel has a radius of curvature in a range of between about 2.5 mm and about 25 mm.

12. The method of claim 4 , wherein at least one curvilinear microchannel has a length in a range of between about 4 cm and about 100 cm.

13. The method of claim 1 , wherein the first inlet of the first curvilinear microchannel is the only inlet of the first curvilinear microchannel.

14. The method of claim 13 , wherein the first inlet is on the interior of a spiral microchannel.

15. The method of claim 14 , wherein the outlets are on the circumference of a spiral microchannel.

16. The method of claim 1 , wherein the first outlet is located on the radially outer side of the microchannel.

17. The method of claim 16 , wherein the system comprises at least one additional outlet and wherein the first outlet is located between the second outlet and the at least one additional outlet.

18. The method of claim 17 , wherein the at least one additional outlet is in fluid communication with a second output reservoir or is in fluid communication with the same output reservoir as the second outlet.

19. The method of claim 1 , wherein the first curvilinear microchannel comprises a third outlet.

20. The method of claim 19 , wherein the third outlet is in fluid communication with a second output reservoir or is in fluid communication with the same output reservoir as the second outlet.

21. The method of claim 19 , wherein at least one of the second and third outlets are located on the radially inner side of the microchannel.

22. The method of claim 19 , wherein at least one of the second and third outlets are located on the radially outer side of the microchannel.

23. The method of claim 19 , wherein the second outlet is located on the radially inner side of the microchannel, wherein the third outlet is located on the radially outer side or the microchannel, and wherein the first outlet is located on the microchannel between the second and third outlets.

24. The method of claim 1 , further comprising a pump configured to pump fluid from the first reservoir to the inlet of the first curvilinear microchannel.

25. The method of claim 1 , further comprising the step of lysing red blood cells in the blood sample prior to introducing the sample into the device.

26. The method of claim 1 , wherein the step of detecting blast cells comprises immunostaining.

27. A method for separating blast cells from a blood sample comprising the steps of:
a. introducing a blood sample into an inlet reservoir of a microfluidic system comprising:
i. At least one inlet reservoir;
ii. At least one output reservoir;
iii. A first curvilinear microchannel comprising a first inlet in fluid communication with an inlet reservoir, a first outlet in fluid connection with the inlet reservoir, and a second outlet in fluid communication with an output reservoir; wherein said curvilinear microchannel is configured to separate particles from a fluid comprising a mixture of particles and wherein the microfluidic system is configured to provide a closed-loop recirculation of the fluid through the first curvilinear microchannel;

b. directing the blood sample from the inlet reservoir into the first inlet of the first curvilinear microchannel, bifurcating the blood sample into a first stream containing blast cells and at least one additional stream, wherein the at least one additional stream contains waste;
c. directing the first stream to the inlet reservoir and the second stream to the output reservoir, wherein the first stream comprises blast cells; and
d. collecting the first stream from the inlet reservoir.

Assignees

• Massachusetts Inst Technology

Inventors

• Khoo Bee Luan
• Han Jongyoon
• Choi Kyungyong
• Ryu Hyunryul

Classifications

• G01N33/57585

  involving compounds identifiable in body fluids · CPC title

• G01N33/575 Primary

  for cancer · CPC title

• B01L3/502715 Primary

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title

• B01L2300/088

  Channel loops · CPC title

• B01L2400/0409

  centrifugal forces · CPC title