CLASSIFICATION, SCREENING AND DIAGNOSTIC METHODS AND APPARATUS

A method of measuring the morphological profile of a sample of red blood cells including measuring the projected area distribution of the cells and identifying characteristic peak areas from the distribution. The spectrum of peak areas may be compared with those from known blood disorders, and provides a classification, screening and diagnostic methods for a range of blood disorders, especially anaemias. Apparatus for carrying out the methods is also provided, which in some embodiments includes the use of microfluidic devices.

FIELD OF THE INVENTION

The invention relates to methods and apparatus for monitoring the physiological state of red blood cells (RBCs). This provides methods for detecting abnormalities in such cells, giving the ability to pre-screen patients for such abnormalities (such as those produced by anaemias, especially rare anaemias). Methods are also presented for diagnosing such diseases. Apparatus for carrying out such methods are also disclosed.

Background and Prior Art

It is known that red blood cells can adopt a number of morphological forms, and that the presence of some of these forms can be indicative of a disease state.

Automated blood cell analysers are able to provide counts of red and white blood cells, and of platelets. Centrifugal segregation of red blood cells in a density gradient gives an indication of the presence and relative abundance of the various morphological forms of the red blood cells but requires 0.5-1.0 ml of blood, limiting the use of this technique to adult patients. However, accurate identification of the distribution of morphological forms of RBCs typically requires time-consuming and expensive analysis by direct visual inspection of cells by a skilled technician. Blood films (smears) that are currently used for assessing RBC morphology do not always adequately reflect true morphological appearance of cells in suspension.

Some recent advances (Deb, N. and Chakroborty, S., “A Noble Technique for Detecting Anemia through Classification of Red Blood Cells in a Blood Smear”, IEEE International Conference on Recent Advances and Innovations in Engineering (ICRAIE-2014), May 9, 2011, Jaipur, India. FIG. 3 of that paper shows that a lot of shape artefacts may be introduced by this technique: cells are “angular” and artificially elongated in one direction, also “echinocytes” may appear as a result of film drying. The authors show that analysis of images of RBCs involving extraction of colour data from images, measurement of cell aspect ratios and the calculation of Fourier descriptors for blood cells (involving the calculation of the spatial frequency content of the boundary of the cells) can be used to provide at least a degree of differentiation between different RBC morphologies and differentiation between white blood cells and nucleated RBCs.

Despite these advances, automatic morphological analysis of RBCs for use in diagnostic tests, pre-screening and patient monitoring is not yet available.

For many anaemias, especially the rare anaemias, genetic analysis to identify individual mutations or groups of mutations is required to reach even a putative diagnosis.

It is among the objects of the present invention to attempt a solution to these and other problems.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method of measuring the morphological profile of a sample of red blood cells (RBCs) comprising the steps of: (a) measuring the greatest projected area of each RBC in a population of said RBCs to create a projected area distribution of the RBCs; and (b) identifying characteristic peak areas from said projected area distribution;

each relative peak area indicating the proportion of phenotypic cell types within the RBC sample.

The invention also provides a method of pre-screening for a disorder affecting red blood cells (RBCs) in a subject comprising the steps of: (a) providing a sample of red blood cells previously obtained from a subject; (b) measuring the greatest projected area of each RBC in a population of said RBCs to create a projected area distribution of the RBCs; (c) providing a database storing like projected area distributions of RBCs obtained from subjects including those with a range of such disorders; and (d) comparing said measured projected area distribution to those stored in said database to find a closest match, the potential RBC disorder being that associated with the matched distribution.

Preferably said disorder is a red blood cell disorder, and more preferably, an anaemia.

Said RBC disorder can also be a conditions associated with stress erythropoiesis.

More preferably, said anaemia is selected from the group consisting of: Hereditary Spherocytosis; and Hereditary Xerocytosis.

The invention also provides a method of identifying a subject likely to have a mutation associated with a red blood cell (RBC) disorder comprising the steps of: (a) providing a sample of red blood cells previously obtained from a subject; (b) measuring the greatest projected area of each RBC in a population of said RBCs to create a projected area distribution of the RBCs; (c) providing a database storing like projected area distributions of RBCs obtained from subjects including those with a range of such mutations; and (d) comparing said measured projected area distribution to those stored in said database to find a closest match, the potential mutation being that associated with the matched distribution.

Preferably, said mutation is associated with a gene selected from the group consisting of: ANK1 (coding for erythrocytic Ankyrin 1 protein); SPTB (coding for erythrocytic spectrin beta chain protein); SCL4A1 (coding for erythrocytic spectrin alpha chain protein); and EPB42 (coding for erythrocyte membrane protein band 4.2).

In any such method, it is preferred that the greatest projected area of each RBC is measured by: (a) allowing said RBCs to sediment onto a surface; and (b) measuring the projected surface area of each RBC in a direction substantially normal to the plane of said surface.

Also in any such method, it is preferred that said sample of red blood cells is diluted before measurement, preferably with a diluent containing an albumin.

Also in any such method, it is preferred that said cells are exposed to a stressor before measurement.

Preferably, said stressor is selected from the group consisting of: osmotic stress; pH stress; temperature stress; and mechanical stress.

In any method of the invention it is also preferred that said projected area is measured by superimposing an ellipse over an image of each RBC, said ellipse having major and minor radii r1and r2, and said projected area being measured as π, r1, r2.

Also in any such method using a database, it is preferred that said database of like projected area distributions contains averaged projected area distributions taken from a plurality of subjects.

The invention further provides apparatus for carrying out a method according to a method described herein, said apparatus comprising: (a) a chamber for accepting a sample of RBCs; (b) imaging apparatus to image cells within said sedimentation chamber; (c) a processor configured to measure the distribution of greatest projected areas of said cells; (d) a database storing projected area distributions of RBCs obtained from subjects including those with a range of disorders affecting RBC morphology; and (e) a processor configured to compare said measured distribution of projected areas to those stored in said database to find a closest match between said measured and stored projected area distributions.

Preferably, said chamber is a sedimentation chamber, and more preferably said sedimentation chamber comprises a microfluidic chamber.

Along with raw diagnosis, these methods and apparatus may be used for assessment of disease severity for individual patients. Furthermore they may also be used for monitoring of therapeutic efficacy of treatment comparing the changes in projected areas distribution density for individual patient over time.

Among the advantages of the inventions is that the techniques exclude handling- and drying-related deformations of morphology and allows visualisation of shapes even for cells with unstable membrane such as those for rare hereditary hemolytic anemia.

DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors have surprisingly found that when the projected surface areas of members of a population of red blood cells are measured, the areas do not form a smooth distribution, but are clustered around characteristic peaks. These distributions resemble a projected area spectrum, characteristic of the state of the blood sample and reflect several morphologically defined populations of RBCs corresponding to well-known classes of cells (e.g. discocytes, disco-stomatocytes, stomatocytes, spherocytes, echinocytes).FIG. 1shows the average projected surface area distribution (or “spectrum”) from eleven samples of blood from healthy subjects. Heparinized blood samples were suspended in a dilution medium at a dilution of 1:1000. The cells were allowed to sediment on a microscope slide, and were imaged using a Zeiss Axiovert 200M microscope with a 100× objective. When the cells sediment, they lie with their largest faces substantially parallel to the plane of the slide (rather than e.g. landing on their edges), and so the image observed is in effect the largest projected surface area of the RBC.

In this experiment, the dilution medium was a blood plasma analogue of the following composition:

ConcentrationComponent(mM)NaCl140KCl4MgCl20.75CaCl22ZnCl20.015alanine0.2glutamate-Na0.2glycine0.2arginine0.1glutamine0.6glucose10HEPES-imidazole20
In addition, the medium contained 0.1% (w/v) BSA (bovine serum albumin), and was adjusted to pH 7.4. The procedure was carried out at room temperature (22-27° C., typically 25° C.).

The projected cell areas were measured by image analysis, approximating the cell borders as an ellipse having major and minor radii r1and r2, and the projected area being measured as π, r1, r2.FIG. 2illustrates this measurement showing the ellipse1and the major and minor radii r1and r2.

Returning toFIG. 1, eight characteristic peaks can be seen, each one centred approximately around the projected cell areas indicated. The distribution of projected areas is presented as a probability distribution, the area under the curve between any two projected areas being the probability of finding a cell within that area range.

The inventors were able to associate each of these peaks with a particular cell morphology, by visual inspection. The peaks corresponded to morphological forms as follows:Peak 1: terminal echinocytosisPeak 2: echinocytosisPeak 3: stomato-RBCPeak 4: stomato-biconcavePeak 5: tense biconcavePeak 6: common biconcavePeak 7: wavy relaxed biconcavePeak 8: relaxed biconcave

The inventors obtained blood samples from subjects that had been diagnosed with a range of rare anaemias, and performed the same analysis.

FIG. 3shows the projected area distributions of RBCs from four subjects (identified as P005, P007, P053 and P059) diagnosed as having a mutation in the gene coding for the Band 3 anion exchanger protein (also known as anion exchanger 1 [AE1], band 3, or solute carrier family 4 member 1 [SLC4A1). It can be seen that, although there is some intra-subject variability, each of the morphological spectra (i.e. the area distributions) show very similar characteristic features.

FIG. 4shows the average projected area density distributions of RBCs for subjects having the Band 3 mutation vs. average plot for 11 healthy control subjects. The differences in the spectra are evident, for example, and most notably, the difference in relative sizes of peaks 5 and 6 (located at 46 μm2and 53 μm2respectively).

FIG. 5shows the projected area distributions of RBCs from four subjects (identified as P033, P084, P088, and P090) diagnosed as having a mutation in the SPTA gene, coding for erythrocytic spectrin alpha chain protein, causing blood disorders including spherocytic haemolytic anemia (hereditary spherocytosis)). Again, although there is some intra-subject variability, each of the morphological spectra (i.e. the area distributions) show very similar characteristic features.

FIG. 6shows the average projected area distributions of red blood cells for subjects having the SPTA mutation vs. healthy controls. Characteristic differences include a decrease in the relative abundance of cells having larger projected areas and a marked reduction in the proportion of cells in peak 6, located around 53 μm2.

FIG. 7shows the projected area distributions of RBCs from two subjects (identified as P2 and P9) diagnosed as having a mutation in the SPTB gene, coding for erythrocytic spectrin beta chain protein. Again, although some variability is present the two morphological spectra show similar characteristics.

FIG. 8shows the average projected area distributions of red blood cells for subjects having the SPTB mutation vs. healthy controls. Marked characteristic differences include a much higher presence of smaller projected area cells in the SPTB subjects compared to the controls.

FIG. 9compares the projected area spectra for the three above pathologies vs. spectra from healthy control subjects.

The differences in these projected area spectrum therefor provide a method of differentiating between the different pathologies, or at least pre-screening subjects to select those who might benefit from a more detailed diagnostic study such as genetic sequencing.

A variety of heuristics may be used to assign a putative pathology to a blood sample.FIG. 10illustrates, as a flow diagram, a method for pre-screening subjects suspected of having an RBC disorder, or diagnosing such a disorder.

A sample of RBCs is provided 2, previously taken from a subject. A projected area distribution of the RBCs is measured 3. In order to take the measurement, the sample is preferably diluted. If sedimentation onto a surface is to be used to determine the maximum projected surface area, the sample is preferably diluted by approximately a factor of 1:1000, but this dilution could also by 1:5000, 1:2000, 1:500 or even 1:200.

The dilution medium should be selected so as not to change the morphological spectrum of the RBCs prior to measurement. The inventors have found that this may be achieved by using a medium that mimics blood plasma. In this regard, it is convenient to use a substitute such as the dilution medium described above. Alternative isotonic media should contain an albumin, preferably an albumin such as BSA (bovine serum albumin) or FCS (foetal calf serum). For example, PBS (phosphate buffered saline) or 0.9% NaCl solution each supplemented with 0.1% albumin, such as BSA would also be suitable.

Whilst the maximum projected area of each RBC may be conveniently measured by allowing the cells to sediment onto a surface, thereby orienting themselves with their largest surface area parallel to the plane of that surface, other methods of achieving this are also envisaged. For example, if the projected surface area of an RBC in free suspension is measured over time, as the cell is tumbling, a measure of the largest such projected area over time will give equivalent results. Similarly, if the RBCs are randomly oriented and not moving, then imaging the projected areas of the cells from multiple directions, and choosing the largest such projected area for each cell would also similarly produce equivalent results. Allowing the cells to sediment before imaging is, however, a simple and preferred method of achieving the projected surface area distribution. Such a method also aids in focussing the imaging apparatus, as all of the cells are essentially lying in the same plane.

It will also be appreciated that, although the present analysis is presented using a continuous distribution of projected cell areas, a similar analysis may be carried out by assigning each cell to a “bin” depending on its projected area. For example, by knowing the position of the peaks in the distribution, bins for each peak (and therefore each RBC morphology) may be constructed e.g. as follows:

PeakLowerUpperPositionBoundBoundBin(μm2)(μm2)(μm2)Morphology12520*27.5terminal echinocytosis23027.533echinocytosis3363339stomato-RBC4423944stomato-biconcave5464449.5tense biconcave65349.555common biconcave7575560wavy relaxedbiconcave8636065*relaxed biconcave*The choice of these values may be chosen to exclude artefacts from imaging cell fragments, or platelets (for the lower bound), or larger cells such as white blood cells (for the upper bound).

A database4is also provided having representative RBC area distributions (either continuous or in bins, e.g. as above) taken from subjects with known pathologies, as well as healthy controls.

The measured projected area distribution of RBCs from the sample can then be compared 5 with those stored in the database to find a closest match6.

Various methods of matching the measured distribution to the stored distributions will be evident to the skilled person. For example, among non-linear methods, a neural network may be trained to identify a measured peak as being most representative of a particular pathology.

Similarly, genetic algorithms could also be used to good effect. A linear approach could also be taken, such as the use of cross-correlation between measured and stored spectra, or a simple approach such as singular value decomposition may be used.

In order to demonstrate this, on the relatively small dataset obtained by the inventors thus far, a singular value decomposition analysis using a least squares approach was applied to the data.

Mean values for the projected area distributions for each of the pathologies and for control subjects were calculated. These data are those presented graphically inFIG. 9. These correspond to the database4of projected area number distributions.

Each of the patient and control samples was then compared against the database and a least squares difference calculated between each sample and the reference distributions. The least square difference (LSD) was calculated as follows:

Where:LSDjis the least square difference for reference distribution j;Siis the population density of the sample at projected area i;

Ri,jis the population density of the reference distributions at projected area i for reference distribution j; andwhere the continuous distribution is approximated by n data points.

The reference distribution having the lowest LSD gives an indication of the pathology represented by the sample. Table 1 shows the results of this analysis

It can be seen that, even with the small amount of data used in this analysis, and with a simple least squares discriminator, the technique correctly identified the pathology associated with a sample for over 80% of all samples. It is expected that with more data, and a more sophisticated matching algorithm, better predictive power will ensue.

FIG. 11illustrates, schematically, an embodiment of an apparatus for carrying out a method of pre-screening, diagnosis or patient monitoring as described herein, generally indicated by7. The apparatus comprises a chamber8for accepting and holding a sample of red blood cells9. In this embodiment, the cells9are suspended within the fluid in the chamber. They may be allowed to sediment, into a configuration illustrated schematically inFIG. 12where they lie on a bottom surface of the chamber8. Imaging apparatus10is also provided, e.g. in the form of a camera and microscope arrangement that can pass images to a processor11to produce a measured distribution12. A database13is also provided, having projected cell area distributions from known pathologies and healthy subjects. The same11or a different processor14is also configured to compare the measured distribution12with those stored in the database13, in order to find a closest match15, and thereby indicate a putative assessment of the blood sample, e.g. by producing a diagnosis, or a pre-screening indicator.

Typically around 2000 RBCs are measured by automatic image analysis, but smaller numbers could also be counted, say 200, 500, or 1000 RBCs. As the field of view of most imaging systems is not able to image such numbers, the apparatus is preferably arranged to allow the field of view of the imaging apparatus and chamber to move relative to one another so that sufficient numbers of cells may be measured. In particularly preferred embodiments, a microfluidic cell may be employed, to allow samples to be moved into and out of an imaging chamber, which itself may be long enough to allow sufficient cells to sediment on its base such that they can be scanned with the imaging system.

Whilst this application has been described primarily in the context of identifying pathological conditions of red blood cells, the measurement of a morphological profile of a sample of red blood cells also has other applications.

Other diseases, such as diabetes, are known to affect the morphology of red blood cells in a subject, even though diabetes is not per se a pathology of RBCs. The techniques could also equally be used to identify disorders of RBC ion transport, and metabolic structure disorders of RBCs.

Furthermore, it is also known that as donated blood is stored, the morphological pattern of the red blood cells changes over time. The methods of measuring the morphological profile of a sample of red blood cells can therefore be applied to samples taken from stored, donated blood to assess potentially unwanted changes during storage.

The techniques described here could also be used to study osmotically induced red blood cell shrinkage or swelling and compensatory responses by the cells.

FIG. 14illustrates apparatus for carrying out a method of RBC analysis as described herein, generally indicated by24. The microfluidic test apparatus24comprises a microfluidic device25, first and second pumps26and27, respectively, and a controller28that operates the pumps26,27.

The microfluidic device25comprises a first reservoir29and a second reservoir30for receiving a first fluid and a second fluid, respectively, and a microfluidic test region31that may serve as the sedimentation chamber for analysis of the RBCs. A first microfluidic pathway32is provided between the first reservoir29and the microfluidic test region31. A second microfluidic pathway33is provided between the second reservoir30and the microfluidic test region31. In the microfluidic device25illustrated inFIG. 14, a further microfluidic pathway34is provided between the microfluidic test region31and a port35.

The first pump26is connected to the port34via a valve36. The first pump26and valve36are arranged to pump a priming fluid into the port34when operated. The first pump26may be a syringe pump, in which the syringe filled with the priming fluid. The priming fluid may contain a wetting agent to reduce air being trapped in the microfluidic device25.

The second pump27is connected to the port35via a valve37. The second pump27and valve37are arranged to apply suction at the port35when operated and draw fluid therefrom.

The valves36and37may take any suitable form, including a one-way valve, non-return valve, or an activated valve. In some embodiments the valves36,37may be omitted.

The controller28is configured to control operation of the first and second pumps26and27, and the valves36,376where the valves are activated. The controller28may be any suitable device such as a microcontroller, embedded controller, programmable logic controller (PLC), microprocessor, portable computing device or computer and may include a control program. The controller28is configured to operate the first pump26to prime the microfluidic device12. The first pump14preferably has a pump rate in the order of mL/second; this relatively high flow rate aids priming the microfluidic device25and reduces air entrapment. The controller28operates the first pump26to pump priming fluid into the microfluidic device25such that priming fluid enters the reservoirs29,30.

After priming, the first and second fluids are then added to the reservoirs29and30, respectively. Where priming fluid has entered the reservoirs29,30, in some embodiments the priming fluid may be removed before the first and second fluids are added. The first fluid comprises a sample of red blood cells, which may be suitable diluted with a dilution medium as described above, typically at a dilution of approximately 1:1000. The second fluid is chosen according to the test requirements and may for example include a label and/or a stressor to the cells that cause a distinctive change in cells which may include cell lysis, aggregation, swelling, shrinkage, and/or shape change. In some embodiments, a series of second fluids may be added one by one to the second reservoir30as a test is performed, each second fluid having a different stressors, stressor concentration, and/or different labels.

If no stressor or other second reagent is required, the second reservoir30and its microfluidic passage33may be omitted.

The controller28is configured to operate the second pump27to draw a test volume of first fluid from the first reservoir29into the microfluidic test region31. If used, a volume of second fluid will also be drawn from the second reservoir30, according to the dimensions of the microfluidic pathways32,33. Since the second pump27applies suction to the port35, pressure on the cells in the first fluid is limited. Using a pump to ‘push’ the first fluid through the microfluidic device25can result in higher pressure on the cells and cause cell ruptures, which may affect testing. It is preferred that the second pump27has a pump rate in the order of μL/second.

In the microfluidic device25shown inFIG. 14, the microfluidic test region31comprises a microfluidic channel into which the first and (if used) second fluids flow. Other forms of microfluidic test region31may be employed; for instance, the microfluidic test region31may comprise a microfluidic channel formed into a spiral. Forming the microfluidic test region31in a spiral may permit the imaging device10to capture fluid flow at several locations along the microfluidic test region31in a small area covered by a single image.

In order to perform an analysis as described herein a suitable protocol can include configuring the pump controller28to draw a sample of diluted RBCs into the microfluidic test region31, to stop the flow to allow the RBCs to sediment in the microfluidic test region (i.e. to use it as a sedimentation chamber) and send a signal to the imaging system10,11to cause it to capture an image of the sedimented cells. The pump controller can then restart the pump27to allow a further sample of diluted RBCs to be drawn into the microfluidic test region31, and again stop the pump27and signal the imaging system10,11to capture a further image of a second aliquot of diluted, sedimented RBCs. This can be repeated as often as required until a required number of cells have been imaged. The imaging system10,11may be configured to send instructions to the pump controller28indicating whether sufficient images have been obtained.

It will be appreciated that the imaging apparatus10may be arranged to image the sedimented RBCs through either the top or bottom of the microfluidic device25. Additionally, the microfluidic device25and imaging apparatus10may be configured such that the two are moved relative to each other after the cells have sedimented, to enable a larger field of view to be captured.

FIG. 15illustrates, schematically, an alternative microfluidic device25that may be used as part of the apparatus. This device25differs from that illustrated inFIG. 14in that a single reservoir29is provided, with a single microfluidic channel32connecting it to the microfluidic test region31. This configuration is preferred where no second fluid (e.g. a stressor fluid or stain) is required in the methodology.

The device further comprises a microfluidic waste region38provided between the microfluidic test region31and the port35. The microfluidic waste region38may comprise a circuitous microfluidic pathway39. Whilst shown in two dimensions in the drawings for clarity it will be appreciated that the pathway39may be formed in three dimensions. The microfluidic waste region38defines a microfluidic volume commensurate with the test volume to prevent the test fluid from reaching the port30. The microfluidic waste region38prevents the test fluid from leaving the microfluidic device25, thereby avoiding cross-contamination that would result if some of the first or second fluids were to leave the microfluidic device25and then subsequently be pumped into another microfluidic device during the priming thereof. It will be appreciated that such a microfluidic waste region28may also be employed in an analogous fashion to the test device25ofFIG. 13.