MICROFLUIDIC DEVICE AND LEUCOCYTE ANTIGEN MEDIATED MICROFLUIDIC ASSAY

• US 20090011451A1
• Filed: 05/13/2005
• Published: 01/08/2009
• Est. Priority Date: 05/13/2004
• Status: Active Grant

• Current Assignee: Advanced Animal Diagnostics, Inc.
• Original Assignee: Advanced Animal Diagnostics, Inc.
• Inventors: Rodriguez, Rodolfo R., Darr, Douglas J.
• Granted Patent: US 9,023,641 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/28 disposable or single use
• CPC Class Codes: C12M 23/58 Reaction vessels connected in series or in parallel combinations of bioreactors with other apparatus, C12M43/00
• CPC Class Codes: C12M 29/00 Means for introduction, extraction or recirculation of materials, e.g. pumps pumps per se F04B
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2333/8107 Endopeptidase (E.C. 3.4.21-99) inhibitors
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/56972 White blood cells

Abstract

The present invention relates to an leucocyte antigen mediated microfluidic assay and a microfluidic device for analyzing a subjects'"'"' body fluids containing leucocytes to determine if the subject has been previously exposed to a predetermined antigen.

Claims

1. A leucocyte antigen mediated microfluidic assay for testing a leucocyte containing fluid from a subject for prior exposure to a predetermined antigen which would have caused the leucocyte to become presensitized to the antigen comprising:

performing a first test comprising the steps of;

a) placing a microvolume first sample of the subjects leucocyte containing fluid in a first sample microchamber;

b) transporting the first sample to a first reaction microchamber containing the predetermined antigen and an antigen accelerator and allowing them to react together for a predetermined time;

c) transporting the reacted first sample to a first observation microchamber;

d) mixing one or more leucocyte observation colorants with the reacted first sample during transportation to the observation microchamber or in the observation microchamber;

e) optically performing an optical scan of the reacted sample in the first observation microchamber under conditions which allow the measurement of at least 1 measurement factor of the leucocytes in the reacted first sample;

performing a second test comprising the steps of;

a) placing a microvolume second sample of the subjects leucocyte containing fluid in a second sample microchamber;

b) transporting the second sample to an antigen free second reaction microchamber containing the same antigen accelerator as the first test and allowing them to react for the predetermined time;

c) transporting the second sample to a second observation microchamber;

d) mixing the same leucocyte observation colorants as the first test with the reacted second sample during transport to the second observation microchamber or in the second observation microchamber;

e) optically performing an optical scan of the reacted second sample in the second observation microchamber under conditions that allow the measurement of the same leucocyte measurement factors measured in the first test;

comparing the results of the first test scan to the second test scan to determine if any differences between the two scans are due to a leucocyte-antigen mediated response.

2. A method according to claim 1 wherein the leucocyte are T-lymphocytes or memory T lymphocytes or neutrophils.

3. A method according to claim 1 wherein transportation is accomplished by capillary action.

4. A method according to claim 1 wherein the optical scan is accomplished by multi-wavelength fluorescence image analysis.

5. A method according to claim 1 wherein the entire method is performed on a single disposable device.

6. A method according to claim 1 wherein the measurement factors that are measured are morphological factors, spectral factors or both.

7. A method according to claim 1 wherein a plurality of predetermined antigens are placed in the first reaction micro chamber.

8. A method according to claim 1 wherein the quantitative antigen leucocyte reactivity is determined by either measuring the number, relative intensity or both of the subject'"'"'s reactive leucocytes while comparing the results of the first scan to the second.

9. A method according to claim 1 wherein the first reaction microchamber and the first observation microchamber are the same microchamber and the second reaction microchamber and the second observation microchamber are the same microchamber

10. A microfluidic device for use in performing leucocyte-antigen mediated assay on a leucocyte containing fluid sample from a subject comprising:

a) a first fluid sample addition microchamber in fluid communication by means of a first fluid channel with a first sample reaction microchamber containing a predetermined antigen and an antigen accelerator;

b) a first sample observation microchamber in fluid communication by means of a second fluid channel with the first sample reaction microchamber;

c) one or more leucocyte observation colorants positioned either in the second fluid channel, the first sample observation microchamber or both;

d) a second fluid sample addition microchamber in fluid communication by means of a third fluid channel with an antigen free second sample reaction microchamber containing the same antigen accelerator as the first sample reaction chamber;

e) a second sample observation microchamber in fluid communication by means of a fourth fluid channel with the second sample reaction microchamber;

f) the same leucocyte observation colorants as the first test positioned either in the fourth fluid channel, the second sample observation microchamber or both;

g) a means for moving at least a portion of each of the fluid samples through each of their respective fluid channels into successive microchambers.

11. A device according to claim 10 wherein at least one of the channels has a valve positioned therein for controlling the fluid flow between microchambers.

12. A device according to claim 10 wherein the means for moving at least a portion of the fluid samples through the fluid channels is capillary action.

13. A device according to claim 10 wherein the means for moving at least a portion of the fluid samples through the fluid channels is by use of a micropump.

14. A device according to claim 10 wherein the antigen accelerator is α

₂-macroglobulin.

15. A device according to claim 10 wherein the leucocyte observation colorant is a dry meta-chromatic stain.

16. A device according to claim 10 wherein the first and second sample addition microchambers are the same microchamber.

17. A device according to claim 10 wherein the first reaction and observation microchambers are the same microchambers and the second reaction and observation microchambers are the same microchamber.

18. A device according to claim 10 wherein the antigen is a plurality of antigens.

19. A device according to claim 10 wherein the device is disposable.

20. A device according to claim 10 wherein the first and second observation microchambers are designed to be able to be read by multi-wavelength fluorescence image analysis.

21. A device according to claim 10 wherein both the first and second fluid sample addition microchambers contain a blood anticoagulant.

22. A kit of parts comprising a microfluidic device according to claim 10 and an apparatus capable of performing an optical multifield scan on the observation microchambers in the microfluidic device.