WO2018218341A1 - System and method for collecting and preserving blood samples for subsequent antibody-independent imaging - Google Patents

Abstract

A method is provided for preserving blood samples for subsequent antibody-independent imaging, which includes transferring a blood sample to a fixative and red blood cell (RBC) lysis buffer. The method can further include transferring a mixture of the blood sample and the buffer to a refrigeration device for storage. The mixture can be incubated at room temperature and comprises a dilution buffer added thereto to achieve a target volume. The method can also include having a working concentration of the fixative and RBC lysis buffer is prepared by adding an amount of water that reduces a concentration of the buffer to produce the working concentration. The blood sample can be collected using a lancing technique.

Description

SYSTEM AND METHOD FOR COLLECTING AND PRESERVING BLOOD SAMPLES FOR SUBSEQUENT ANTIBODY-INDEPENDENT IMAGING

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of priority to U.S. Provisional Patent

Application No. 62/512,141 filed on May 29, 2017, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

[0002] The following relates to systems and methods for collecting and preserving bodily fluids samples, particularly blood samples, for subsequent antibody-independent imaging such as cytometry.

DESCRIPTION OF THE RELATED ART

[0003] The ability to monitor and visualize an individual's immune system homeostasis over time would be valuable in systems biology and for guiding personalized medicine and wellbeing. However, current mechanisms for studying immune cell subpopulations from peripheral blood typically require venipuncture, separation or lysis of red blood cells (RBC), and immunolabeling of fresh white blood cells (WBC) using fluorescently-tagged antibodies, followed by sample fixation. Current methods also typically require one or several rounds of centrifugation followed by near-term data acquisition and analysis by flow cytometry or other means. The time, cost of reagents, and requirement for laboratory equipment to perform this procedure precludes the use of such methods to efficiently and reproducibly monitor the immune system status of individuals repeatedly and/or longitudinally outside of a hospital, clinic, or testing laboratory setting. Recent advances in computer-based image analysis, and developments in machine-learning and deep learning tools have made possible classification of blood cell subsets by analysis of digital images acquired by microscopy and imaging flow cytometry. However, expensive and specialized laboratory equipment is still required to image sufficient numbers of blood cells at sufficient magnification for classification, precluding point-of-care diagnostics.

[0004] It is an object of the following to address at least one of the above disadvantages. SUMMARY

[0005] In order to enable monitoring of individual's immune system status over time, a system and method are described that allows simple and reproducible collection and preservation of WBC from small volumes of blood, which can be stored for long periods (at least several months to one year) without loss of cell integrity or morphometric quality. [0006] The method described herein can also be performed by non-experts/non- healthcare professionals at convenient locations, such as, but not limited to, the subject's home. This method can be used alongside recent advances in antibody-independent single cell classification, for example by digital image analysis software, machine-learning-based image analysis and nascent deep learning computing tools. The method thereby allows for reproducible collection and storage of peripheral blood leukocytes at virtually any location that can be subsequently classified and analyzed without the need for staining with fluorochrome-tagged antibodies.

[0007] In one aspect, there is provided a method for a method of preserving blood samples for subsequent antibody-independent imaging, the method comprising transferring a blood sample to a fixative and red blood cell (RBC) lysis buffer.

[0008] In another aspect, there is provided a kit comprising suitable items, materials and instruments for performing the method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Embodiments will now be described by way of example only with reference to the appended drawings wherein:

[0010] FIG. 1 is a flow chart illustrating operations performed in collecting and preserving bodily fluids samples, particularly blood samples, for subsequent antibody- independent imaging such as cytometry;

[0011] FIG. 2A illustrates a preparation of working concentration of fixation and RBC lysis buffer according to one preparation method;

[0012] FIG. 2B illustrates a preparation of working concentration of fixation and RBC lysis buffer according to an alternative preparation method;

[0013] FIG. 2C illustrates a transfer of working concentration of fixation and RBC lysis buffer to an empty vessel;

[0014] FIG. 3A is an illustrative view showing blood being drawn from a subject;

[0015] FIG. 3B is a schematic view of a blood sample transferred to a microcentrifuge tube containing the working concentration of fixation and RBC buffer;

[0016] FIG. 3C is a schematic view of a sample with dilution buffer added prior to storage;

[0017] FIG. 4 provides example Brightfield images of WBC collected one day (left) and four months (right) prior to imaging an image analysis, for heterogeneous WBC populations; [0018] FIG. 5 provides example nuclear stained images of WBC collected one day (left) and four months (right) prior to imaging an image analysis, for heterogeneous WBC populations;

[0019] FIG. 6 provides example darkfield images of WBC collected one day (left) and four months (right) prior to imaging an image analysis, for heterogeneous WBC populations;

[0020] FIG. 7A provides example image analysis metrics applied to samples collected six weeks prior to image analysis and illustrates Nuclear Symmetry_2 versus Ratio of Nuclear Area to Cytoplasmic Area for each cell in the sample (left), Nuclear Intensity- weighted Aspect Ratio versus Darkfield Intensity-weighted Aspect Ratio (middle), and Nuclear Compactness to Darkfield Intensity (right);

[0021] FIG. 7B provides example image analysis metrics applied to samples collected four months prior to image analysis and illustrates Nuclear Symmetry_2 versus Ratio of Nuclear Area to Cytoplasmic Area for each cell in the sample (left), Nuclear Intensity- weighted Aspect Ratio versus Darkfield Intensity-weighted Aspect Ratio (middle), and Nuclear Compactness to Darkfield Intensity (right);

[0022] FIG. 7C provides example image analysis metrics applied to samples collected one day prior to image analysis but underfixed due to low fixation buffer concentration, and illustrates Nuclear Symmetry_2 versus Ratio of Nuclear Area to Cytoplasmic Area for each cell in the sample (left), Nuclear Intensity-weighted Aspect Ratio versus Darkfield Intensity- weighted Aspect Ratio (middle), and Nuclear Compactness to Darkfield Intensity (right);

[0023] FIG. 7D shows that image analysis metrics can be conserved with cells held at room temperature for a period of time prior to imaging;

[0024] FIG. 8A shows that samples of individual subject's blood leukocytes can be used to train a classifier to recognize main blood cell subsets using supervised machine-learning algorithms, wherein antibody-labelled cells are used to determine features in Brightfield and Darkfield imagery that identify the cell type independent of the antibody labeling;

[0025] FIG 8B shows that the classifier developed in FIG. 8A can then be used to classify blood cells collected by the current method with the absence of antibody staining; and

[0026] FIG 8C shows that the classification of blood leukocyte subsets identified by the machine-learning algorithms used in FIG 8A results in population percentages similar to those known to be present in human blood (published averages). DETAILED DESCRIPTION

[0027] Development of a method designed and optimized for collection, preparation, and preservation of cell populations such as WBC subsets for subsequent characterization, without the need for fluorescently-labeled antibodies, would be highly advantageous for systems biology and personalized medicine. This approach would obviate the current need for blood to be drawn, processed, antibody labelled, fixed, and washed immediately within a hospital, clinic or testing laboratory, and could therefore greatly reduce costs and

simultaneously allow for longitudinal study of the immune system within individuals undergoing treatment, changing lifestyle habits, or ageing, as examples .

[0028] Described herein is a method for collecting blood or bodily fluids at any location that can be performed by non-experts, in the absence of specialized lab equipment, and that is compatible with short- or long-term storage prior to subsequent image data acquisition and analysis. The method described herein is particularly advantageous for the collection and preparation of WBC from a finger stick blood sample, however the method would apply similarly to blood collected by other methods, such as venipuncture. The ability to obtain blood by finger stick exemplified in this method is particularly advantageous as it allows for use by non-professionals in a safe, repeatable, reproducible, and minimally invasive manner.

[0029] The method described herein can be applied in several applications, for example the collection and preservation of cells for research or clinical applications with the goal of monitoring an individual's personal immune system homeostasis, or in response to medical treatments, prescriptions, procedures such as cancer chemotherapy, radiation therapy, immunotherapy, anti-microbial agents, vaccinations, or lifestyle changes including but not limited to changes in diet, activity level, tobacco or drug use, environment. Potential applications of the method include cancer, heart disease, autoimmune disorders, pathogenic infections, neurological disorders, etc. Also described herein is a simple, safe, and affordable kit with the necessary components for use by non-experts.

[0030] This method described herein can be used along with recent advances in antibody-independent single cell analysis, for example by machine-learning-based image analysis. An example of such machine-learning bases image analysis can be found in WO 2015/168026 A2. Generally, the fixation of cells prior to antibody labeling destroys or modifies antibody-ligand binding epitopes, resulting in reduced antibody binding (Burel et al, Journal of Immunology 2017, doi 10.4049/jimmunol.1601750). Therefore, this method, which allows for immune cell classification and quantification independent of antibody labeling represents a significant advance in technology. It may be noted that this does not exclude the possibility that in some cases the method could be used in conjunction with antibody labeling, as certain epitopes may be stable to the fixation procedure described herein and thus be targeted by antibody-dependent labeling and analysis if desired.

[0031] Turning now to the figures, FIGS. 1 -3 illustrate a method for blood collection for WBC fixation/preservation/permeabiliization, RBC lysis, and storage and antibody-free imaging and classification.

[0032] As shown in FIG. 1 , the method begins at steps 50 and 52 with preparing a working concentration of fixation and RBC buffer (step 50) and collecting a blood sample (step 52).

[0033] In step 50, as shown in FIGS. 2A, 2B, and 2C, to an empty, sterile vessel 12a such as a 1 .5ml microcentrifuge tube is added one drop of a mixture of 10x concentrated buffer for WBC fixation and 10x RBC lysis 14 along with 9 drops of water 16, e.g., using a plastic transfer pipette 10 (FIGS. 2A or 2B). 3 drops or 45 - 90 μΙ of the resulting working concentration of fixation / RBC lysis buffer is added to an empty, sterile vessel 12b as shown in FIG. 2C.

[0034] Fixation in this example, is performed with 1 % formaldehyde, however alternative concentrations of formaldehyde, or other fixatives could be used, such as paraformaldehyde (PFA) or formalin. RBC lysis is typically performed using diethylene glycol buffer 14 but could be achieved by alternative means such as hypotonic lysis buffer or water, or ammonium chloride potassium (ACK). Transfer of liquids in this example is performed with a disposable plastic transfer pipette 10 but other means of liquid transfer could be used. The exemplary technique used here includes a commercially available one- step fix/lyse buffer to minimize variability / allow consistency, however other reagents for fixation and RBC lysis or removal may be used. The buffer 14 contains both 10x formaldehyde for fixation and 10X diethylene glycol for RBC lysis. This allows the white blood cells to be fixed and the red blood cells lysed (or otherwise removed). Herein described is one particular method to fix with formaldehyde and lyse with diethylene glycol in a single step (i.e. using a commercially available reagent designed to do this). However, it can be appreciated that one could fix with another fixative and lyse with another reagent and produce similar results. As such, the buffer 14 in Figure 2A is one volume of 10x formaldehyde and 10x diethylene glycol. The addition of 9 volumes (in this case each drop is one volume) of water produces the working concentration of both formaldehyde and RBC lysis buffer. It has been found that this working concentration is less effective after one month of storage and is thus ideally freshly prepared. Therefore, the alternative of having the 10x reagents separated from water in a single tube as in Figure 2B can allow a simpler preparation of fresh working concentration, make the procedure more reproducible, and pose less risk to the user (e.g., exposure to formaldehyde, which is an irritant and can be hazardous) by not requiring pipetting by the user.

[0035] In an alternative method, one could use an ACK lysis or hypotonic lysis of RBC. To address the preservation of cell morphology and light scattering properties, one could optimize conditions with other RBC lysis approaches and integrate such an approach within the method described herein. Similarly, while this procedure uses formaldehyde fixation, the use of alternative fixation solutions such as paraformaldehyde (PFA) or formalin, or methanol are possible in other embodiments. Importantly, according to the principles described herein, adding a blood sample directly into a fixative with the intent to characterize and classify WBC subpopulations by image analysis provides the particular advantages discussed herein. In contrast, with current methodologies, blood would be collected and stained with antibodies and then fixed and the RBCs lysed. That is, blood is added immediately and directly to fixative and RBC lysis buffer rather than collected into anticoagulant-containing tubes and then labeled with antibodies before fixation. An advantage is that the technique can be performed easily, anywhere, by anyone. In one embodiment, this may include 1x formaldehyde and diethylene glycol-mediated RBC lysis in a commercial one-step solution. In another embodiment, paraformaldehyde (PFA) or formalin or methanol for fixation can be used, or a hypotonic RBC lysis buffer or ACK instead of diethylene glycol.

[0036] Here, the exemplary method for dilution of concentrated fixation / RBC lysis solution uses one drop of the 10x buffer from a plastic transfer pipette added to 9 drops of water to produce 1x (working concentration) buffer. However, it can be appreciated that alternative methods could be used to prepare the working concentration of fixation/RBC buffer. For example, having a defined volume of 10x or other stock concentration of fixation / RBC lysis buffer separated from a defined volume of dilution buffer by a breakable or removeable septum or other barrier 18 as shown in FIG. 2B could be used. In this case, the user could break away the barrier by applying pressure to the external surface of the vessel or could otherwise remove the barrier and mix to dilute to the working concentration. Such design would minimize risk of exposure of the subject to the formaldehyde.

[0037] At step 52, the area of skin where stick is to be performed should be wiped with a towelette soaked in 70% isopropanol or other disinfectant. A small volume (typically 5μΙ - 25μΙ but as much as 200 μΙ) of blood is drawn by finger stick as illustrated in FIG. 3A. The skin is punctured using a lancing device 22 outfitted with a single use, sterile lancet 20.

[0038] Next, at step 54, one or two drops of blood, totalling 5μΙ to 20μΙ is wiped from skin at the lancing site to the tube 12 containing the 45μΙ - 90μΙ of working concentration buffer for WBC cell fixation and RBC lysis. It can be appreciated that the method described herein should scale linearly. As such, if an application requires collection of a greater number of cells, increasing the volume of blood drawn, along with increasing the volumes of buffer proportionally, can be performed to provide the same result. Larger containment vessels would be required, but the method performed would be the same. For example, it is possible to draw up to 200 μΙ of blood from a finger stick, and thus as much as 10 times as many cells could be collected if required, for example for rare cell analyses.

[0039] Alternative lancing techniques and blood sample collection may be used. Blood droplets or a defined volume of blood between 5μΙ and 200μΙ could be drawn into buffer- containing tubes by microfluidics, capillary tubes or vacuum or other available techniques.

[0040] In step 54, the microcentrifuge tube is recapped and the blood sample is mixed within the fixation / RBC lysis buffer by gentle agitation, for example shaking or flicking the tube, and the sample 24 is set aside for 15 minutes at room temperature in step 56, and shown in FIG. 3B. The incubation period of 15 minutes is exemplary for this method but certain variations in incubation times and temperatures may be acceptable and provide similar results. It has been observed that overly short incubation times, overly dilute fixation buffer, or use of working concentration of fixation buffer that has been stored for too long can have an adverse effect on image quality and reproducibility as shown in the subsequently described figures.

[0041] At step 58, phosphate buffered saline (PBS), which has been stored at 4-10 degrees Celsius in a refrigerator, is then added to the blood sample, e.g., by plastic transfer pipette until a total volume 26 of at least 1 ml is achieved. Next, the tube is recapped and inverted several times to mix the volume 26, and terminate the fixation reaction as shown in FIG. 3C.

[0042] It can be appreciated that buffer addition by transfer pipette is exemplary and other methods for transfer may be used. Similarly, buffer and reagent storage temperatures given herein are exemplary and other storage conditions may be used. It can also be appreciated that buffer volumes and final volume given are exemplary and other volumes may be used. [0043] At step 60, the sample tube is transferred to a refrigerator and maintained at 4- 10 degrees Celsius for between one day and 6 months or longer, before data acquisition by imaging cytometry or other use at step 62. It can be appreciated that sample storage times and temperatures given are exemplary and other storage conditions may be used.

[0044] It has also been found that a kit can be created to enable the above method to be readily performed by non-experts, e.g., at home or elsewhere in the absence of specialized lab equipment. Such a kit can include the following elements:

[0045] (a) Bottle containing stock solution for cell fixation and permeabilization as well as RBC lysis.

[0046] (b) Bottle containing phosphate-buffered saline, or other dilution buffer.

[0047] (c) Cardboard or plastic box containing sterile, empty microcentrifuge tubes for collection of samples, and for storage of tubes after sampling and before analysis.

[0048] (d) Labels and markers for noting date of collection.

[0049] (e) Second vessel containing diluent or optionally, collapsible or destructible septum for dilution of stock solution with dilution buffer to produce working solution.

[0050] (f) Spring-loaded lancing device and disposable lancets.

[0051] (g) Sharps collection container for used lancet storage and disposal.

[0052] (h) Sealed sterile alcohol wipes, bandages.

[0053] (i) Instruction manual including link to web-based video or mobile application video showing how to perform the sampling.

[0054] FIGS. 4, 5 and 6 provide example images of WBC collected one day (left panels) or 4 months (right panels) prior to imaging and image analysis. Exemplary montages of Brightfield (FIG. 4), nuclear stained (FIG. 5), and darkfield images (FIG. 6) of heterogeneous WBC populations are shown. The image quality, and size, shape, and contrast features of the WBC as observed by Brightfield, darkfield, and nuclear dye staining are preserved during at least 4 months of storage in a household refrigerator. This preservation of WBC is important to allow sample collection at any location, to obviate the need for immediate cryopreservation or immediate cell preparation and analysis typical of existing classification methodologies (Burel et al 2017, J Immunol). This enables the samples to be stable for periods of time longer than what is the current standard, since the method is not being performed in a laboratory by trained personnel. The images shown in FIGS. 4-6, and importantly the analysis of those images (not shown) demonstrates that the cells can be well preserved and stable for several months. This would allow, for example, subjects to collect samples, e.g., once per month for six or twelve months, then submit them for analysis. In such a scenario, it is important that samples be stable for long periods. In some applications, results may be desired immediately and long-term stability is not required. However, for purposes of long-term monitoring, and cost-effectiveness of a kit, for example, sample stability is considered to be important.

[0055] FIG. 7A, 7B, and 7C provide dotplots that illustrate the successful preservation of WBC population for six weeks and twenty-four weeks after blood collection, versus the poor preservation of WBC when fixation is improperly performed. The diagrams shown in FIGS. 7A-C demonstrate the clustering of WBC populations using certain image analysis features that can be useful in classifying WBC types without the need for fluorescently- labelled antibodies. FIG. 7A shows cell populations imaged four weeks after initial blood collection by finger stick using this method. FIG. 7B shows cell populations imaged twenty- four weeks after initial blood collection by finger stick using this method. FIG. 7C illustrates a poorly preserved blood sample where, although the blood sample was collected one day before imaging, the fixation buffer used had expired and was no longer capable of optimally preserving cell morphology and integrity, as evidenced by the spreading out of the cell populations by the ratio of Nuclear Area to Cytoplasmic Area (left plot), the Intensity- weighted Aspect Ratio of the Darkfield signal (middle plot) and the loss of distinct population clusters by Darkfield Intensity (right plot).

[0056] FIGS. 7A-7C therefore show the image analysis metrics that demonstrate what heterogeneous cells look like after 6 or 24 weeks of storage using the presently described method versus what they may look like when appropriate fixation conditions are not met.

[0057] FIG. 7D shows that image analysis metrics can be conserved even with cells that were held at room temperature for 24 hours prior to imaging. As such, in at least some implementations, embodiments, or scenarios, refrigeration may not be required.

[0058] FIGS. 8A, 8B, and 8C provide bar graphs that establish that, in the absence of antibody staining, machine learning algorithms are able to classify many of the normal blood leukocyte subpopulations with high fidelity.

[0059] FIG. 8A shows that, when machine learning algorithms, such as Gradient Boosting, are applied to images cells acquired in the absence of antibody, high degrees of accuracy in cell type determination can be achieved for some of the major blood cell subsets. In this case, antibody-labelled cells are used as 'ground truth' for training the analysis software. ~ 100,000 images of cells from blood samples taken from 13 subjects were used as ground truth in the training process. Features from Brightfield and Darkfield (Side Scatter) images are identified by the software that classify the cell subsets without the use of antibodies. In this case, high (above 90% accuracy) is attained for Monocytes, T lymphocytes, Neutrophils, and Eosinophils, with lower accuracy results obtained for B lymphocytes.

[0060] FIG. 8B demonstrates that the same classifier used above, trained on samples collected separately from the current method described herein, can be used to classify cells collected using the method described herein, with similar accuracy, in the absence of any antibody-dependent labeling or biomarker staining. In this example, blood collected several weeks or months prior to digital image acquisition was analyzed using the classifier developed and describe in FIG 8A. High accuracy of classification was obtained for most subsets, despite the widely differing collection times and procedures.

[0061] Figure 8C demonstrates that the frequency of cell populations identified using the classifier to predict cell classes using the method described herein is similar to the published average cell frequency of populations in human subjects sampled by traditional blood drawing and antibody-dependent cytometric analysis.

[0062] It can be appreciated that while the examples discussed herein refer to the collecting and preserving of blood samples, the techniques described herein can also be adapted to processing other bodily fluids that contain immune cells, such as tears, saliva, etc.

[0063] For simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the examples described herein. However, it will be understood by those of ordinary skill in the art that the examples described herein may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the examples described herein. Also, the description is not to be considered as limiting the scope of the examples described herein.

[0064] It will be appreciated that the examples and corresponding diagrams used herein are for illustrative purposes only. Different configurations and terminology can be used without departing from the principles expressed herein. For instance, components and modules can be added, deleted, modified, or arranged with differing connections without departing from these principles. [0065] The steps or operations in the flow charts and diagrams described herein are just for example. There may be many variations to these steps or operations without departing from the principles discussed above. For instance, the steps may be performed in a differing order, or steps may be added, deleted, or modified.

[0066] Although the above principles have been described with reference to certain specific examples, various modifications thereof will be apparent to those skilled in the art as outlined in the appended claims.

Claims

Claims:

1. A method of preserving blood samples for subsequent antibody-independent imaging, the method comprising:

transferring a blood sample to a fixative and red blood cell (RBC) lysis buffer.

2. The method of claim 1 , further comprising transferring a mixture of the blood sample and the buffer to a refrigeration device for storage.

3. The method of claim 1 , wherein the mixture is incubated at room temperature and comprises a dilution buffer added thereto to achieve a target volume.

4. The method of claim 3, wherein the mixture is incubated for fifteen minutes.

5. The method of claim 1 , wherein the fixative comprises formaldehyde.

6. The method of claim 1 , wherein the RBC lysis buffer comprises diethylene glycol.

7. The method of claim 1 , wherein a working concentration of the fixative and RBC lysis buffer is prepared by adding an amount of water that reduces a concentration of the buffer to produce the working concentration.

8. The method of claim 3, wherein the dilution buffer comprises phosphate buffered saline (PBS).

9. The method of claim 1 , further comprising collecting the blood sample using a lancing technique.

10. The method of claim 9, wherein the lancing technique utilizes a single use sterile lancet.

1 1. The method of claim 2, further comprising extracting the sample from the refrigeration device for subsequent image analysis.

12. The method of claim 9, wherein the blood sample volume is up to 200μΙ.

13. The method of claim 12, wherein the blood sample volume is between 5μΙ and 25μΙ.

14. The method of claim 2, wherein the mixture is stored for between one day and 6 months or longer.