MICROFLUIDIC DEVICE FOR FULL BLOOD COUNT

Abstract

A microfluidic device (10) for full blood count includes a first measurement channel (11) and a second measurement channel (12) separated from the first measurement channel (11). The microfluidic device (10) furthermore includes a first inlet (13) for providing a whole blood sample to the first and second measurement channel (11, 12), a second inlet (14) for providing a lysis agent for white blood cell count in to the first channel (11), a third inlet (15) for providing a quench solution to the first channel (11), and a fourth inlet (16) for providing a lysis agent for hemoglobin measurement to the second channel (12). A method for forming such a microfluidic device (10) and a method for performing a full blood count test using such a microfluidic device (10) are described.

Claims

1. A method for manufacturing a microfluidic device for full blood count, the method comprising: providing a first measurement channel, providing a second measurement channel the second measurement channel separated from the first measurement channel, providing a first inlet for providing a whole blood sample to the first and second measurement channel, providing a second inlet at the first measurement channel for providing a lysis agent for white blood cell count in to the first channel, providing a third inlet at the first measurement channel for providing a quench solution to the first channel, and providing a fourth inlet at the second measurement channel for providing a lysis agent for hemoglobin measurement to the second channel.

2. A method for performing full blood count, the method comprising: providing a blood sample to a first and a second measurement channel of a microfluidic device, the first and second measurement channel being separated from each other, providing a lysis agent suitable for white blood cells to the blood sample in the first channel, providing a quench solution to the blood sample in the first channel, providing a lysis agent for hemoglobin to the blood sample in the second channel, at the end of the first channel performing measurements for determining white blood cell counts, and at the end of the second channel performing measurements for determining properties of red blood cells.

3. The method according to claim 2, wherein performing measurements for determining white blood cell counts is performed by impedance measurements.

4. The method according to claim 2, wherein performing measurements for determining properties of red blood cells is performed by optical measurements.

5. The method according to claim 2, wherein providing a lysis agent suitable for white blood cells to the blood sample in the first channel is performed by providing a mixture of formic acid and saponin.

6. The method according to claim 2, wherein providing a quench solution to the blood sample in the first channel is performed by providing a solution of sodium chloride and sodium bicarbonate.

7. The method according to claim 2, wherein providing a lysis agent for hemoglobin to the blood sample in the second channel is performed by providing a solution of SLS in phosphate buffered saline.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGS. 1A-1C illustrate different measurements required for a full blood count.

[0057] FIGS. 2A and 2B illustrate flow through impedance spectroscopy.

[0058] FIG. 3 schematically illustrates a microfluidic device 10 according to an embodiment of the invention.

[0059] FIG. 4A illustrates an implementation of a first measurement channel in a microfluidic device according to an embodiment of the present invention, and FIG. 4B illustrates sheat flow interfaces in the microfluidic device both for lysis and for quench.

[0060] FIGS. 5A and 5B illustrate the concept of hemoglobin measurement in a microfluidic device according to an embodiment of the present invention.

[0061] FIGS. 6A and 6B show results of optical measurements of hemoglobin detection according to embodiments of the invention.

[0062] FIG. 7 shows an exemplary embodiment of flow rates through the microfluidic device 10.

[0063] FIG. 8A shows 3 part impedance spectroscopy using standard bench bulk preparation techniques.

[0064] FIG. 8B shows impedance spectroscopy of the blood after microfluidic lysis and quench.

[0065] FIG. 8C shows micrographs of the microfluidic lysis and quench steps in process.

[0066] FIG. 9 shows an embodiment of a microfluidic diluter 100.

[0067] FIGS. 10A-10C show microscopic images of the working diluter

[0068] FIG. 11A illustrates an embodiment of the integrated microfluidic lysis, dilution and haemoglobin measurement device, while FIGS. 11B-11F show enlargements of various portions of FIG. 11A.

[0069] FIGS. 12A and 12B show experimental cell counts obtained with the microfluidic device.

[0070] In the different figures, the same reference signs refer to the same or analogous elements.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0071] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and the relative dimensions do not correspond to actual reductions to practice of the invention.

[0072] Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0073] The present invention provides a microfluidic device for full blood count (FBC), a method for forming such a microfluidic device and a method for performing a full blood count test (FBC test) using such a microfluidic device.

[0074] Several factors prohibit the FBC test from being performed in a point of care setting. First, the cost of purchasing and servicing the hematology analyzer instrument is prohibitive. Skilled technicians are also required to conduct quality control assays to ensure the test gives a result with an acceptable degree of accuracy and precision. Large (4 ml) venous blood samples are required for the test and central lab containment and disinfection facilities are required for handling these potentially infectious samples. Hematology analyzers are very bulky and often contain complex precision optics, meaning that they have a very limited portability. One known type of analyzer, i.e., the Chempaq analyser, is capable of measuring a WBC (white blood cell) differential count, platelet count and hemoglobin (Hb), but is, however, not capable of measuring the RBC (red blood cell) indices.

[0075] A microfluidic device and methods according to embodiments of the invention are capable of measuring all parameters that are required for an FBC device at the point of care from a finger-prick of blood, i.e., it is capable of measuring WBC differential count, platelet count, RBC count and Hb.

[0076] A key obstacle to a point of care device has been that no one has been able to achieve a microfluidic-based integrated Hb, RBC count, platelet count and white blood cell differential device that is able to process blood from a finger prick, i.e., from a volume of about 10 m to 50 l. The primary reason for this is that it is difficult to combine sample preparation steps required for WBC differentiation and for labeling and detecting Hb in a microfluidic format. The reasons for this are:

[0077] The need for dry reagents in case of azide measurement of Hb is not compatible with a wet microfluidic system that is required for the calibration and measurement with a microfluidic impedance/coulter counter.

[0078] The need for large dilutions/long optical path lengths in current sodium dodecyl sulphate (SDS) and/or cyanmethemoglobin (HbCN) conversion measurements mean that they are not compatible with a simple microfluidic system. The requirement for very shallow channels; e.g., channels with a diameter of about 50 m, means that very inaccurate Hb concentrations will be obtained if attempts are made to measure the absorbance of one of these strongly diluted solutions in a microfluidic channel.

[0079] For chemical solutions aimed at treating the whole blood to measure a WBC count and Hb from a same sample, there is a danger of erroneous Hb measurements being obtained due to optical scattering by the WBCs.

[0080] Many Hb labeling solutions, including those which seek to label with azide or imidazole, are incapable of labeling certain species of Hb, including sulfhemoglobin and carboxyhemoglobin. This leads to errors in the Hb reading for patient samples with high levels of these Hb species.

[0081] Problems with turbidity also lead to falsely high Hb readings.

[0082] A microfluidic device and method according to embodiments of the invention solve all of the above described problems (see further).

[0083] In a first aspect, the present invention provides a microfluidic device for full blood count. As shown in FIG. 3, the microfluidic device 10 comprises:

[0084] a first measurement channel 11,

[0085] a second measurement channel 12 different and separated from the first measurement channel 11,

[0086] a first inlet 13 for providing a whole blood sample to the first and second measurement channel,

[0087] a second inlet 14 for providing a lysis agent for white blood cell count to the first channel,

[0088] a third inlet 15 for providing a quench solution to the first channel, and

[0089] a fourth inlet 16 for providing a lysis agent for hemoglobin measurement to the second channel.

[0090] The microfluidic device according to embodiments of the invention comprises a combination of two microfluidic sample preparation protocols. The first protocol carries out a carefully controlled red blood cell lysis designed to preserve the white blood cells, before delivering the sample to an impedance measurement means (see further), e.g., impedance spectroscope, for a WBC differential measurement. The second protocol lyses the red blood cells and labels the Hb using a SLS (sodium lauryl sulphate) method that requires low dilution factors and therefore short path length absorption spectroscopy (see further).

[0091] The use of two separate measurement channels has as an advantage that it allows each lysis solution to be specifically tailored to either a WBC differential count or a Hb measurement. This separation means that problems previously associated with integrated Hb measurement in a microfluidic format, specifically WBC scattering, turbidity and the conflicting requirements of short path lengths/high dilution, are eliminated.

[0092] FIG. 2 shows the impedance measurement means as described in Impedance spectroscopy flow cytometry: on-chip label-free cell differentiation, Cheung, K., S. Gawad, and P. Renaud, Cytometry A, 2005. 65(2): p. 124-132. FIG. 2 shows a side view of the microfluidic channel and a sample cell passing between the measurement and reference electrodes. The Cell Signal is the output of a Lock-In Amplifier measuring the current difference between both electrode pairs. In this way impedance spectroscopy can be performed for different cells.

[0093] FIG. 3 schematically illustrates a microfluidic device 10 according to an embodiment of the invention. The microfluidic device 10 comprise a first microfluidic channel 11 and a second microfluidic channel 12. The second microfluidic channel 12 is different and separated from the first microfluidic channel 11. The microfluidic device 10 comprises a first inlet 13 for providing a whole blood sample to the first and second measurement channels 11, 12. The blood sample may be taken from a patient by a finger prick, because only a limited amount of blood, i.e. a volume of about 10 m to 50 l, is required for performing the FBC test with a microfluidic device according to embodiments of the present invention. A second and third inlet 14, 15 are provided at the first channel 11. The second inlet 14 is for providing a lysis agent for WBC differential count to the first channel 11 while the third inlet 15 is for providing a quench solution to the first channel 11. At the end of the first channel 11, means for determining WBC differential count, e.g., an impedance measurement means 17 may be provided, while at the end of the second channel 12, means for determining properties of red blood cells, i.e., RBC count, HB and platelet count, e.g., an optical measurement means 18 may be provided.

[0094] In a second aspect, the present invention also provides a method for performing full blood count. The method comprises:

[0095] providing a blood sample to a first and a second measurement channel 11, 12 of a microfluidic device 10, the first and second measurement channels 11, 12 being separated from each other,

[0096] providing a lysis agent suitable for white blood cells to the blood sample in the first channel 11,

[0097] providing a quench solution to the blood sample in the first channel 11, providing a lysis agent for hemoglobin to the blood sample in the second channel 12,

[0098] at the end of the first channel 11, performing measurements for determining white blood cell count, and

[0099] at the end of the second channel 12, performing measurements for determining properties of red blood cells, i.e., for determining RBC count, platelets count and Hb.

[0100] FIGS. 4A and 4B schematically illustrate what happens in the first measurement channel 11. A whole blood sample is delivered to the first channel 11 through the first inlet 13 (indicated by reference number 20 in FIGS. 4A and 4B). The whole blood sample may be taken from a patient by a finger prick, because only a limited amount of blood, i.e., a volume of about 10 m to 50 l, is required for performing the FBC test with a microfluidic device according to embodiments of the present invention. Providing the whole blood sample to the first channel 11 may be done at a known flow rate, e.g., 3 l/min. A lysis agent, e.g., a mixture of formic acid and saponin, may then be provided to the blood sample in the first measurement channel 11 through a second inlet 14 (indicated by reference number 21 in FIGS. 4A and 4B). This may be performed at a defined flow rate of e.g., 37 l/min. The lysis agent mixes by diffusion with the whole blood sample over the length of a microfluidic snake stage 19 and lyses all the red blood cells which are present in the whole blood sample. The length and channel dimensions of the snake stage 19 are chosen so that the time of contact between the lysis agent and the blood is between 5 and 7 seconds, for example, 6 seconds. At the end of the snake stage 19, the quench solution, e.g., a solution comprising sodium chloride and sodium bicarbonate, is provided to the blood sample in the first channel 11 at a defined flow rate of e.g., 16.3 l/min (indicated by reference number 22 in FIGS. 4A and 4B). The blood sample is then transferred to the measurement means 17 to measure the WBC differential. According to embodiments of the invention, the measurement means 17 to measure the WBC differential may be an impedance measurement means.

[0101] FIGS. 5A and 5B schematically illustrate what happens in the second measurement channel 12. A whole blood sample is delivered to the second measurement channel 12 through the first inlet 13 (indicated by reference number 20 in FIG. 5B). As providing the whole blood sample to the second measurement channel 12 is also performed through the first inlet 13, according to embodiments of the invention, the blood sample may simultaneously be provided to the first and second measurement channel 11, 12. Providing the blood sample to the second measurement channel 12 may be done at a flow rate, e.g., 1

[0102] The flow rate in different microfluidic channels can be different. When designing a system that slows the flow in one channel relative to the other(s) it is common to introduce some form of hydrodynamic resistance. This is achieved either by altering the length of the tube (longer tube, more resistance, slower flow) or by reducing one of the other dimensions of the channel (the method of channel fabrication generally dictates that it is the channel width that is adjusted).

[0103] A lysis agent for Hb measurement is provided to the blood sample in the second measurement channel 12 through a fourth inlet 16, e.g., a solution of 150 mM SLS in phosphate buffered saline (PBS), also referred to as SLS reagent (indicated by reference number 23 in FIG. 5B). This may be performed at a constant flow rate of e.g., 4 l/min. The lysis agent mixes by diffusion with the whole blood sample over the length of a microfluidic snake stage 24 and lyses all the red blood cells, platelets and white blood cells. The use of PBS is necessary to prevent the precipitation of the Hb in the second measurement channel 12. The primary lysis agent used in the present example is SLS, which also labels the Hb. At the end of the second measurement channel 12, properties of the red blood cells, i.e., RBC count, platelets count and Hb, are determined by means of measurement means 18. According to embodiments of the invention, measurement means 18 may be an optical measurement means 18. In the case of an optical measurement means 18, according to embodiments of the invention, at the end of the microfluidic snake stage 24, the second measurement channel 12 may open into a microfluidic chamber 25. A possible implementation of an optical measurement means 18 that can be used with embodiments of the present invention is illustrated in FIG. 5A. Collimated light 26 at a wavelength of 535 nm may from a light source 27, e.g., a pulsed LED source, be directed at right angles to the microfluidic chamber 25, where it passes through the lysed blood sample. The collimated light 26 is therefore first sent through a filter 28. By means of an aperture 29, it may then be directed towards the microfluidic chamber 25 of the microfluidic device 10. Light 30 transmitted through the device 10 is sent through a lens 31 and is then measured using an amplified photodiode 32.

[0104] The depth of the microfluidic chamber 25 may be between 50 m and 200 m, and consequently, the optical path length of the light going through the microfluidic chamber 25 may also be between 50 m and 200 m. Larger depths for the microfluidic chamber 25 may be used if the dilution factor required is higher.

[0105] Because the WBC measurement and the RBC measurements are separated from each other, a reliable FBC test can be performed which at the end gives a result for all parameters of the FBC test in once.

[0106] With the microfluidic device 10 and method according to embodiments of the invention, turbidity correction is not required as the high concentration of SLS will dissociate any cell fragments that would otherwise cause the light to scatter. Similarly, since all the WBCs are destroyed, no scattering losses occur due to the presence of white cells.

[0107] FIGS. 6A and 6B show typical data obtained from an integrated Hb measurement as discussed above. FIG. 6A shows that microfluidic sample preparation and optical measurement show good linear behavior on a Beer-Lambert plot over a clinically relevant range. These data are from a whole blood sample. FIG. 6B shows an UV/VIS spectrum of the lysate from the microfluidic device which confirms that all Hb has successfully been converted into a SLS-coordinated species, with an absorption peak at 535 nm. The zero baseline at 800 nm proves that there is no contribution from scattering by WBCs.

[0108] It is desirable, for the microfluidic device for cost reasons, that lysis be achieved not only microfluidically but also using as few hydrodynamic pumps as possible. Thus, in an advantageous embodiment of FIG. 7, the microfluidic device has been designed to use only a single hydrodynamic pump 200 sucking on the waste outlet of the microfluidic device. The three solutions involved in the reaction (blood, lysis and quench reagents) are stored under atmospheric pressure in reservoirs within the microfluidic device. The flow rates of the reagents (in l/min) from these reservoirs as they progress towards the detection chip are dictated by:

[0109] The optimum flow rate of cells for detection at the chip and

[0110] The ratios in which the different reagents should be mixed (see FIG. 7).

[0111] Where reagents must be in contact with each other for a defined time, this is incorporated into the design through combination of flow rate and channel dimension, thus in the design shown in FIG. 7, a six second contact time following addition of the lysis solution to the blood (flow=17.76 l/min) is obtained through use of a 8.8 cm section of 200100 m channel.

[0112] Where reagents are mixed (for example, where the lysis reagent is introduced to the blood, or the quench reagent introduced to the lysate) the correct mixing ratios are achieved by adjusting the flow rates of the reagents in the incoming fluidic channels. This adjustment was made by tuning the fluidic resistance of the channels, through variation of the channel height, width or length according to appropriate microfluidic formulae. In FIG. 7, an example of the flow rates through the micro fluidic device is indicated, where the desired flow rate for detection through the detection chip is 25 l/min. Other flow rates are defined by the desired mixing ratios of the reagents involved relative to 25 l/min.

[0113] In the microfluidic device shown in FIG. 7, lysis and quench reagents are added from both sides of the reaction channel. This is to aid mixing of the blood and the added reagent. It may be desirable to introduce reagent from only one side or to have multiple introductions of the same reagent along the length of the reaction channel. Where this is necessary a similar design approach to the one described would be applicable.

[0114] Microfluidic devices made using the design rationale described could be manufactured in any of the materials commonly used for micro fabricated devices.

[0115] In FIG. 8A, 3 Part Impedance spectroscopy using standard bench bulk preparation techniques is shown. FIG. 8B shows impedance spectroscopy of the blood after microfluidic lysis and quench. FIG. 8C shows micrographs of the microfluidic lysis and quench steps of the microfluidic device in operation. Impedance spectroscopy of the blood, post lysis reveals populations of cells in good agreement with those obtained when the lysis was performed using standard laboratory procedures (Compare FIG. 8B and FIG. 8A) and in proportions consistent with those expected in human blood. These results confirm that microfluidic lysis can be achieved within a microfluidic system using a system of balanced fluidic resistances and under the control of only a single syringe pump.

[0116] The microfluidic device according to the invention enables large dilutions to be performed. In standard laboratory procedure large dilutions are performed in a serial fashion where dilution is achieved by performing several smaller dilutions (such that for a 1:10,000 dilution four sequential 1:10 dilutions of the sample might be performed). Such a procedure requires a skilled individual to perform the many pipetting steps, often using large amounts of reagent and time. In moving such a dilution protocol to a microfluidic format, for example for use in a medical device to be used by an unskilled individual, it is desirable to reduce the amount of reagent used (lowering the cost of the overall device) and to minimise the time needed to run the device (fast start-up). In addition, as with the lysis device described above, it would be preferable in terms of cost if such a device used as few hydrodynamic pumps as possible. Such a device would have application in a point of care haematology analyser where the quantity of red blood cells within the blood makes large dilution necessary.

[0117] As with the standard laboratory technique, dilution on the microfluidic platform is by a sequence of smaller dilutions (this can be by any combination, such that a 1:10,000 dilution can be achieved by four 1:10 dilutions, two 1:100 dilutions or any other combination that achieves a 1;10,000 dilution). Fast start up and minimal reagent usage are achieved by discarding the majority of the sample prior to each dilution step (such that at each dilution step only a small amount of the already dilute sample gets further diluted). As with the lysis device the two reagents (blood and diluent) are stored under atmospheric pressure in a reservoir on the fluidic block. Detection of the diluted blood is by flow through impedance spectroscopy (the detection chip is again integrated on the microfluidic block).

[0118] In this case, flow rates through the microfluidic block are dictated by the desired rate for detection at the impedance chip and by the required dilution ratios. FIG. 9 shows an example of a microfluidic diluter 100. Thus for a 1:10,000 dilution by four sequential 1:10 dilutions with a desired flow rate of 40 l/min at the detection chip, the flow rates through the fluidic block are as shown in FIG. 9. In this example, the blood enters the device at 4 l/min and is diluted 1:10. In the device shown in FIG. 9, following each dilution step, there is a channel with residence time of 30 seconds to facilitate mixing of the blood with diluent (this could be substituted by active mixing structures built into the device which might reduce the time needed in these sections). Before undergoing further dilution steps, 9/10ths of the, now diluted, blood is channelled to waste with 1/10.sup.th continuing along the device where it undergoes a further 1:10 dilution. This process is repeated along the fluidic block until the blood is diluted 10,000 times where it is channelled through the impedance chip and detected. The waste is combined at each stage and is channelled straight to the waste syringe (bypassing the chip).

[0119] Within the diluter device relative flow rates of the fluidic channels are again controlled by modification of the fluidic resistances by adjusting the length, width and height of the fluidic channels according to the equation described above. The more complicated network of fluidic resistances found in this structure necessitates use of a number of possible design tools, for instance a circuit simulator.

[0120] FIGS. 10A-10C show microscope images of the diluter working. The Stages 1-3 (FIGS. 10A-10C, respectively) correspond to different positions in FIG. 9.

[0121] The individual elements described above (microfluidic red blood cell lysis, haemoglobin detection and 1:10,000 dilution) can be combined using the same design rationale described for the above devices.

[0122] FIGS. 11A-11F show the microfluidic block and blood/reagent flow in micrographs of the relevant parts operating.

[0123] FIGS. 12A and 12B show experimental cell counts obtained with this system.

[0124] It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for devices according to the present invention, various changes or modifications in form and detail may be made without departing from the scope of this invention as defined by the appended claims.