Blood cell counting device and method

• US 9,914,120 B2
• Filed: 03/12/2013
• Issued: 03/13/2018
• Est. Priority Date: 03/12/2012
• Status: Expired due to Fees

• Current Assignee: Biosurfit, S.A.
• Original Assignee: Biosurfit, S.A.
• Inventors: De Oliveira Garcia Da Fonseca, Joo Manuel, Cabea, Ricardo
• Primary Examiner(s): Lee, Jonathan S
• Application Number:
  US14/384,200
• Publication Number:
  US 20150024426A1
• Time in Patent Office: 1,827 Days
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0803 Disc shape
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0688 surface tension valves, capillary stop, capillary break
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: G01N 15/042 by centrifuging and investigating centrifugates centrifuges per se B04B
• CPC Class Codes: G01N 15/05 in blood
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1484 microstructural devices
• CPC Class Codes: G01N 2015/045 by optical analysis
• CPC Class Codes: G01N 2015/055 for hematocrite determination
• CPC Class Codes: G01N 21/07 Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 33/491 by separating the blood components G01N15/05 takes precedence

Abstract

A device for use in imaging a liquid sample comprises an inlet for accepting the sample, a connection conduit and a detection chamber for detection of the sample, preferably optical detection of the sample. The connection conduit connects the inlet to the detection chamber and contains one or more dry reagents for reaction with the sample as the sample passes through the connection conduit. Specific embodiments include devices arranged for treating a blood sample, in particular lysing and staining the sample. The liquid flow may be driven by capillary effect. The device may further include liquid handling structures arranged for centrifugally driven liquid flow, for example to meter a volume of sample and separate the sample into phases by centrifugation.

Claims

1. A device for use in imaging a liquid sample, the device comprising:

an inlet for accepting a sample into the device;

a connection conduit;

a detection chamber for optical detection of the sample;

a metering chamber arranged to hold a predefined volume of the sample, wherein the metering chamber is in fluidic connection with the inlet and the connection conduit;

a split feature arranged between the metering chamber and the connection conduit to split the sample between the metering chamber and the connection conduit; and

a downstream chamber in fluidic connection with the metering, chamber and arranged to receive the predefined volume of the sample;

wherein the connection conduit connects the inlet to the detection chamber,

wherein the connection conduit contains one or more dry reagents for reaction with the sample as the sample passes through the connection conduit,

wherein at least one dimension of the connection conduit is less than the smallest dimension of the inlet and at least one dimension of the detection chamber is less than the smallest dimension of the connection conduit,

wherein the device is arranged for rotation about an axis of rotation to drive liquid flow to the downstream chamber, and

wherein the split feature is arranged to separate a volume of the sample in the metering chamber from a volume of the sample in the connection conduit in response to the centrifugal force.

2. The device of claim 1, wherein the sample is a blood sample, the one or more dry reagents include a haemolysing agent for selective lysis of erythrocytes in the blood sample and a staining agent for selectively staining leukocytes in the blood sample.

3. The device of claim 1, wherein the connection conduit comprises a main conduit portion and one or more protrusions extending from the main conduit portion wherein the dry reagents are stored in the one or more protrusions and respective junction regions between the one or more protrusions and the main conduit portion provide a reaction region in which gradual resuspension of the dry reagents can occur.

4. The device of claim 3, wherein the protrusions comprise a main portion and a neck portion in the region of the junction, the neck portion having a smaller cross-sectional area along the main conduit portion than the main portion.

5. The device of claim 1, wherein the connection conduit is of a meandering configuration.

6. The device of claim 1, wherein the connection conduit further contains a stabilizer agent in dry form.

7. The device of claim 1, wherein the connection conduit further contains a surfactant in dry form.

8. The device of claim 1, further comprising an overflow chamber in fluidic connection with the downstream chamber and further comprising a siphon in fluidic connection with the downstream chamber, wherein an inlet of the siphon is arranged radially inwards from a portion of the downstream chamber such that when the device is subjected to a centrifugal force and the siphon is primed, a predetermined volume of the sample is siphoned from the sample in the downstream chamber.

9. The device of claim 1, wherein dry anticoagulant is stored in the metering chamber.

10. The device of claim 1, wherein the metering chamber comprises a surface tension barrier, the surface tension barrier being arranged to stop capillary driven flow between the metering chamber and the downstream chamber.

11. The device of claim 1, further comprising a waste chamber wherein the waste chamber is in fluidic connection with the detection chamber, and arranged radially inward from the detection chamber to receive centrifugally driven overflow from the detection chamber.

12. A system for imaging a sample comprising the device of claim 1, the system further comprising:

an imager for acquiring at least one image of the sample in the detection chamber, the downstream chamber or both.

13. The system of claim 12, further comprising a drive for rotating the device.

14. The system of claim 12, further comprising a processor configured to process the at least one image of the sample in the detection chamber, when the sample is a blood sample, to segment stained cells in the image, the processor being further configured to determine a haematocrit fraction of the blood sample in the downstream chamber and, in the overflow chamber.

15. A method for imaging a liquid sample, the method comprising the steps of:

inserting a sample into a first chamber of a cartridge;

causing the sample to flow by capillary action from the first chamber through a connection conduit into a detection chamber;

while the sample flows through the connection conduit gradually re-suspending one or more dry reagents in the sample

filling a metering chamber with the sample by capillary action;

rotating the cartridge to separate cellular material from blood plasma in the downstream chamber and imaging the sample in the downstream chamber once cellular material has been separated; and

capturing at least one image of the sample in the detection chamber.

16. The method of claim 15, wherein the sample is a blood sample, the one or more dry reagents include a haemolysing agent for selective lysis of erythrocytes in the blood sample and a staining agent for selective stain of leukocytes in the blood sample.

17. The method of claim 16, further comprising performing image cell segmentation and classification of leukocytes by comparison of obtained images of the lysed and stained blood in the detection chamber with pre-defined image property thresholds.

18. The method of claim 15, wherein the first chamber and the metering chamber are filled from a common inlet.

19. The method of claim 15, further comprising rotating the cartridge to separate cellular material from blood plasma in the downstream chamber and imaging the sample in the downstream chamber once cellular material has been separated.