METHOD FOR DETECTING INFLAMMATIONS

Description

FIELD OF THE INVENTION

[0001] This invention relates to methods of detecting early stage inflammation by measuring levels of amyloid in a subject's blood.BACKGROUND TO THE INVENTION

[0002] The global disease burden is continuing to shift away from communicable diseases to non-communicable diseases such as diabetes, atherosclerosis, Alzheimer's disease, cardiovascular disease and cancer - all of which are linked to chronic low-grade inflammation. Furthermore, about 80% of people dying from these diseases now live in the developing world, which holds a particular danger for health systems of developing countries which are already under-resourced and over-stretched. It is thus essential to investigate possible markers which link inflammation to these diseases and to develop low cost methods of early detection.

[0003] Amyloids are one class of markers that have been implicated in a number of inflammatory conditions. These conditions, referred to as amyloidogenic diseases, are characterised by the formation of amyloid plaques or amyloid deposits in the body. The term amyloid refers to a highly ordered and insoluble type of protein that forms as the result of a normally soluble protein aggregating via a self-association process. In the body, this process is linked to a breakdown in the systems that ensure efficient protein synthesis and folding. Amyloid deposits can be cerebral (e.g. Alzheimer's disease and Huntington's disease), or peripheral (e.g. light chain amyloidosis and type 2 diabetes). Amyloids can affect any organ or tissue but the kidneys, pancreas, liver, spleen, nervous tissue and heart constitute the major sites of deposition in patients with peripheral amyloidosis.

[0004] Several pro-inflammatory gene products have been identified as inducers of amyloid formation, one example being serum amyloid A (SAA). SAA is a generic term for a family of acute phase proteins synthesised by the liver which are mainly regulated by inflammation associated cytokine-peptide hormone signals. Inflammation resulting from cancer, cardiovascular disease, rheumatoid arthritis, bacterial infection, and tissue damage, may cause SAA levels to rise 1000-fold. It may therefore be advantageous to measure levels of SAA in order to determine a degree of inflammation in a subject. Currently, SAA levels can be detected using enzyme-linked immunosorbent assays (ELISA) and mass spectrometry (MS). However, these methods are poorly sensitive, extremely expensive and time-consuming, which may limit their application, particularly in under resourced clinical contexts. Furthermore, due to their insensitivity, these methods may only be capable of detecting SAA levels in advanced disease states where SAA levels are considerably elevated. There is therefore room for new methods of detecting SAA levels in subjects at a higher degree of sensitivity than previously available methods.

[0005] A problem that is encountered in the field of therapy of amyloid-related and other inflammatory disorders is an inability to detect or quantify amyloid formation at an early stage of disease progression. To date, no satisfactory method has been developed by which it is possible to obtain rapid, quantitative, detection of low levels of amyloid in patients suffering from amyloid-associated conditions. Consequently, it is difficult to accurately monitor patient responses to therapy. A need exists for a method by which low levels of inflammation may be detected in such patients.

[0006] The preceding discussion of the background to the invention is intended only to facilitate an understanding of the present invention. It should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was part of the common general knowledge in the art as at the priority date of the application.RELEVANT BACKGROUND ART

[0007] Pretorius Etheresia et al. "Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide", JOURNAL OF THE ROYAL SOCIETY. INTERFACE, vol. 13, no. 122, 1 September 2016, page 20160539, DOI: 10.1098 / rsif.2016.0539 discloses how the addition of tiny concentrations (0.2 ngl -1< ) of bacterial lipopolysaccharide (LPS) to both whole blood and platelet-poor plasma of normal, healthy donors leads to marked changes in the nature of the fibrin fibres so formed, as observed by ultrastructural and fluorescence microscopy. They resemble those seen in a number of inflammatory (and also amyloid) diseases, consistent with the involvement of LPS. Further, it is disclosed that general inflammation may cause hypercoagulation.

[0008] Kell Douglas B et al. "Proteins behaving badly. Substoichiometric molecular control and amplification of the initiation and nature of amyloid fibril formation: lessons from and for blood clotting", PROGRESS IN BIOPHYSICS AND MOLECULAR BIOLOGY, vol. 123, pages 16-41, DOI: 10.1016 / J.PBIOMOLBIO.2016.08.006 discloses that when fibrin is formed in samples from patients harbouring different diseases it can have widely varying diameters and morphologies, and that in many cases the anomalous fibrin fibre formation seen in such diseases actually amounts to amyloidogenesis. Further, serum amyloid A is described to be a heterogeneous family of apolipoproteins (and variants) that form amyloid fibrils in the blood, typically in response to inflammation or infection.

[0009] Etheresia Pretorius et al. "Lipopolysaccharide-binding protein (LBP) reverses the amyloid state of fibrin seen in plasma of type 2 diabetics with cardiovascular co-morbidities", SCIENTIFIC REPORTS, vol. 7, no. 1, 29 August 2017, DOI: 10.1038 / s41598-017-09860-4 relates to a method of analysing a blood sample for signs of type 2 diabetes (T2D) wherein platelet poor plasma is allowed to clot by addition of thrombin and are analysed for hypercoagulability and amyloid formation. The thus formed fibrils are analysed with the amyloid-binding dye thioflavin T. This paper concludes that clots formed in samples from T2D patients are amyloid in nature.

[0010] Etheresia Pretorius et al. "Substantial fibrin amyloidogenesis in type 2 diabetes assessed using amyloid-selective fluorescent stains", CARDIOVASCULAR DIABETOLOGY, vol. 16, no. 1, 2 November 2017, DOI: 10.1186 / s12933-017-0624-5 relates to a method of analysing a blood sample for signs of type 2 diabetes (T2D) wherein platelet poor plasma (PPP) is allowed to clot by addition of thrombin and are analysed for hypercoagulability and amyloid formation. The thus formed fibrils are analysed with the amyloid-binding dyes thioflavin T (ThT), amytracker 480, and amytracker 680. This paper concludes that PPP from T2D display a substantial potential for amyloidogenesis and that this can be prevented with LPB (lipopolysaccharide binding protein).SUMMARY OF THE INVENTION

[0011] This invention provides a method of detecting early stage inflammation in a subject having no symptoms of inflammation or disease, wherein the early stage inflammation indicates a disease, wherein the disease is cancer, the method comprising contacting a blood sample obtained from the subject with thrombin to form fibrin clots, determining a quantity of amyloid present in the fibrin clots, associating the quantity of amyloid with a concentration of serum amyloid A (SAA), and making a diagnosis of early-stage inflammation as an indicator of stage 0 or stage 1 cancer in the subject if the concentration of SAA is greater than 30 µg / ml and less than 10 000 µg / ml.

[0012] The amount of amyloid may be measured by contacting the blood sample with an amyloid-binding fluorescent marker, allowing amyloid in the sample to bind to the marker, measuring fluorescence emitted upon binding of the amyloid to the marker, and assigning an amount of amyloid based on the measured fluorescence.

[0013] The quantity of SAA may be determined by comparing the amount of amyloid to a reference. The reference may be one or more predetermined values, a chart or a graph.

[0014] The subject is diagnosed as having early-stage inflammation when the level of SAA in the blood sample is greater than a threshold level. The threshold level is any value between 30 and 10,000 micrograms / ml, such as 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 micrograms / ml. The threshold level is provided as a range between 30 and 10,000 micrograms / ml.

[0015] The fluorescence marker may be selected from Thioflavin T, NIAD-4, a luminescent conjugated oligothiophene (LCO) and Congo red.

[0016] The fluorescence may be measured by a spectrophotometer, a diode array detector or a fluorescence detector. The fluorescence may be measured by confocal microscopy and quantified by image processing software. The detected fluorescence may be compared to a reference fluorescence value in order to determine a level of amyloid in the sample.

[0017] The blood sample may be whole blood or blood plasma.

[0018] The detected fluorescence may be compared to a standard curve of predetermined fluorescence values having associated SAA levels in order to determine a level of SAA in the sample.BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1is a series of confocal images of clotted platelet poor plasma (PPP) in the presence of Thioflavin T (ThT), Amytracker ®< 480 and Amytracker ®< 680: First column - Amytracker ®< 480; second column - Amytracker ®< 680; third column - ThT. A to C) healthy blood; D to F) inflamed blood. Figure 2is a boxplot of the distribution of the coefficients of variation (CV) for the pixel intensities in the confocal clot images from Amytracker ®< 480 (median coefficients of variation and STDs are reported above the plot). Data of the inflamed amyloidogenic blood differed significantly from that of the healthy control (P < 0.0001). Figure 3is a boxplot of the distribution of the coefficients of variation (CV) for the pixel intensities in the confocal clot images from Amytracker ®< 680 (median coefficients of variation and STDs are reported above the plot). Data of the inflamed amyloidogenic blood differed significantly from that of the healthy control (P < 0.0001). Figure 4is a boxplot of the distribution of the coefficients of variation (CV) for the pixel intensities in the confocal clot images from ThT (median coefficients of variation and STDs are reported above the plot). Data of the inflamed amyloidogenic blood differed significantly from that of the healthy control (P < 0.0001). Figure 5shows representative confocal micrographs of plasma from a healthy individual with a BMI of 23 before and after exposure to cancer cells using 3 amyloid markers. A) Naïve healthy whole blood. B) Plasma from the same individual after exposure to cultured cancer cells. Figure 6shows representative confocal micrographs of plasma from a healthy individual with a BMI of 27.1 before and after exposure to cancer cells with 3 amyloid markers. A) Naïve healthy whole blood. B) Plasma from the same individual after exposure to cultured cancer cells. Figure 7is a graph of the coefficient of variation (CV) data obtained with Amytracker ®< 480 for naïve cells (NC) and cells contacted with cancer cells (Cells). Vertical bars indicate standard deviation. Figure 8is a graph of the coefficient of variation (CV) data obtained with Amytracker ®< 680 for naïve cells (NC) and cells contacted with cancer cells (Cells). Vertical bars indicate standard deviation. Figure 9is a graph of the coefficient of variation (CV) data obtained with ThT for naïve cells (NC) and cells contacted with cancer cells (Cells). Vertical bars indicate standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a convenient, non-invasive method of detecting early stage inflammation in a subject having no symptoms of inflammation or disease.

[0021] The method comprises determining a quantity of amyloid present in fibrin clots in a blood sample obtained from the subject and contacted with thrombin, and associating the quantity of amyloid with a concentration of serum amyloid A (SAA), and making a diagnosis of early-stage inflammation as an indicator of stage 0 or stage 1 cancer in the subject if the concentration of SAA is greater than 30 µg / ml and less than 10 000 µg / ml. Amyloid fibrin structure is significantly upregulated in all inflammatory conditions, when compared to fibrin structure in healthy individuals. The fibrin amyloid comprises fibrin protein which has incorrectly folded or is otherwise in an anomalous state. The proportion of fibrin(ogen) present as amyloid can be correlated with a degree of inflammation in the subject. High levels of inflammation correspond with high levels of amyloid and low levels of inflammation with low levels of amyloid.

[0022] The present method can aid in the early detection of the presence of inflammation associated with cancer, assist in determining the prognosis, and define the therapeutic options.

[0023] The quantity of fibrin amyloid can be determined by contacting the blood sample with an amyloid-binding fluorescent marker, allowing amyloid in the sample to bind to the marker, measuring fluorescence emitted upon binding of the amyloid to the marker, and assigning a quantity of amyloid based on the measured fluorescence. Thrombin is added to the sample to induce fibrin formation and blood clotting.

[0024] The quantity of amyloid can be determined by comparing the amount of fluorescence to a reference. The reference may be one or more predetermined values, such as a value of fluorescence previously obtained from the subject or from other subjects. Each predetermined value can be associated with a quantity of amyloid, from which the quantity of amyloid determined by the method can be derived. Alternatively the reference can be a chart or a graph comprising levels of fluorescence or inflammation (or values derived therefrom) and associated amyloid or fluorescence quantities from which the quantity of amyloid determined by the method can be derived. In an exemplary embodiment, the reference can be a predetermined value on a standard curve which correlates amount of fluorescence with quantity of amyloid.

[0025] The amount of fibrin amyloid is associated with a level of serum amyloid A (SAA). By associating a quantity of amyloid with a level of SAA, a diagnosis of early stage inflammation as an indicator of stage 0 or stage 1 cancer is possible if the concentration of SAA is greater than 30 µg / ml and less than 10 000 µg / ml. Equally, a quantity of SAA in the sample may be determinable from the amount of amyloid measured, for example, by comparing the amount of amyloid to a reference. The reference may be a predetermined value, a chart or a graph.

[0026] The subject is diagnosed as having early-stage inflammation when the quantity of SAA in the blood sample is greater than a threshold level. A blood level of SAA in a healthy subject is typically from 5 to 30 micrograms / ml and the threshold level is greater than 30 µg / ml and less than 10 000 µg / , such as 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 micrograms / ml of SAA. The threshold level can be provided as a range of values between 30 and 10,000 µg / ml, such as 30 - 1000 µg / ml, 50 - 1000 µg / ml, 100 - 1000 µg / ml, 500 - 1000 µg / ml, 1000 - 2000 µg / ml, 2000 - 3000 µg / ml, 3000 - 4000 µg / ml, 4000 - 5000 µg / ml, 5000 - 6000 µg / ml, 6000 - 7000 µg / ml, 7000 - 8000 µg / ml, 8000 - 9000 µg / ml, 9000 - 10000 µg / ml.

[0027] The early stage inflammation in a subject having no symptoms of inflammation or disease, indicates a disease state or a pre-disease state in the subject. The disease is cancer and the cancer may be pre-cancer or early stage cancer.

[0028] Association of a quantity of amyloid in the sample with a concentration of SAA is used to diagnose early stage inflammation in a subject having no symptoms of inflammatory disease. Levels of SAA greater than 30 µg / ml but less than 10,000 µg / ml indicate early stage inflammation. The early stage inflammation is indicative of cancer, which can be pre-cancer or early stage cancer. A level of SAA between 5,000 and 10,000 µg / ml can indicate early stage cancer, and a level of SAA more than 30 and less than 5000 µg / ml can indicate pre-cancer. The stages of cancer is determined by comparing the quantity of amyloid to a reference indicating a stage of cancer.

[0029] Pre-cancer refers to a state of disordered morphology of cells that is associated with an increased risk of cancer. It is also referred to as stage 0 cancer or cancer in situ, which is a non-invasive cancer that has not progressed to an aggressive, invasive stage. These types of cancers are located in the place in which they originated and have not spread to other tissues. Early stage cancer refers to stage 1 cancer, which is associated with a small tumour that has not grown deeply into nearby tissues. It has typically not spread to the lymph nodes or other areas on the body. Other stages of cancer such as stage 2 and 3, are characterised by larger cancers or tumours that have grown deeply into nearby tissue and which may have spread to lymph nodes but not to other parts of the body, and stage 4 in which the cancer has spread to other organs or parts of the body. Stage 4 is also referred to as advanced or metastatic cancer.

[0030] The method is for detecting early stage inflammation in a subject having no symptoms of inflammation or disease. In particular, in the case of cancer, the subject may have no detectable tumours.

[0031] The term "cancer" refers to disease of skin tissues, organs, blood, and vessels, including, but not limited to, cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, and uterus. Specific cancers include, but are not limited to, advanced malignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastase, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressive, hormone refractory prostate cancer, resected high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen independent prostate cancer, androgen dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma. The term "cancer" includes both solid tumours and blood born tumours.

[0032] The fluorescence marker used in the method can be selected from Thioflavin T, NIAD-4 [chemical name: 2-((5'-(4-hydroxyphenyl)-2,2'-bithiophen-5-yl)methylene)propanedinitrile], luminescent conjugated oligothiophenes (LCOs) and Congo red [chemical name: disodium 4-amino-3-[4-[4-(1-amino-4-sulfonato-naphthalen-2-yl)diazenylphenyl]phenyl]diazenyl-naphthalene-1-sulfonate]. The LCOs can be selected from the structures provided in Table 1 below, or a salt or free acid thereof.

[0033] The LCO can be an Amytracker ®< 480 having an excitation range of 405-458 nm and a detection range of 470-550 nm, an Amytracker ®< 515 having excitation at 458 or 488 nm and a detection range of 500-650 nm, an Amytracker ®< 545 having excitation at 458 or 488 nm and a detection range of 500-650 nm, or an Amytracker ®< 680 having an excitation range of 530-565 nm and a detection range of 600-800 nm.

[0034] The fluorescence may be measured by a spectrophotometer, a diode array detector or a fluorescence detector. In some embodiments, the fluorescence can be measured using a confocal microscope and quantified by image processing software. In a typical embodiment, a blood sample is contacted with a fluorescence marker and the marker allowed to bind and form a conjugate with amyloid in the sample. The sample is exposed to laser excitation at a suitable wavelength to induce the marker-amyloid conjugate to fluoresce. An image is captured and fluorescence in the image quantified by suitable software, such as Zeiss ZEN or ImageJ (Fiji) software.

[0035] The blood sample can be whole blood or blood plasma. In some embodiments, whole blood can be clarified by centrifugation and the resulting platelet poor plasma used in the method. In other embodiments, whole blood can be used without clarification.

[0036] Importantly, the method of the present invention makes use of a blood test rather than a biopsy to determine early-stage inflammation. This permits the risks, discomfort and inconvenience associated with biopsies to be circumvented. The present invention is also less expensive, faster and has a higher degree of sensitivity than existing methods used to detect early-stage inflammation associated with cancer. Furthermore, the use of fibrin amyloid as a biomarker to detect early stage inflammation enables sensitive, early diagnosis of a number of potentially life threatening inflammatory diseases, thereby increasing the prospects of successful treatment.

[0037] The invention will now be described in further detail by the following non-limiting examples.Examples Example 1

[0038] Twenty healthy whole blood (WB) samples and twenty amyloidogenic blood samples with low grade inflammation were prepared in citrate tubes. Platelet poor plasma (PPP) was used for confocal and super-resolution analysis. Platelet poor plasma (PPP) was prepared by centrifuging WB for 15 minutes at 3000 x g. One of three fluorescent markers, Thioflavin T (ThT), Amytracker ®< 480 or Amytracker ®< 680, was added to the PPP to detect amyloid formation. The tubes were incubated for 30 minutes with ThT at a final concentration of 5 µM and Amytracker ®< 480 and 680 (0.1 µL into 100 µL PPP).

[0039] Clots were allowed to form and were viewed under confocal microscope. Before viewing clots on the confocal microscope, thrombin was added in the ratio 1:2, (5 µL thrombin: 10 µL PPP) and extensive fibrin networks, created. Thrombin was provided by the South African National Blood Service, and the thrombin solution was at a concentration of 20 U / ml and made up in a biological buffer containing 0.2% human serum albumin. A coverslip was placed over the prepared clot, and samples were viewed using a Zeiss LSM 510 META confocal microscope with a Plan-Apochromat 63x / 1.4 Oil DIC objective. For ThT, the excitation laser used was 488 nm and emission measured at 508 to 570 nm, for Amytracker ®< 480 the 405 nm laser was used with emission measured at 478 to 539 nm, and for Amytracker ®< 680 the 561 nm laser was used for excitation with emission measured at 597 to 695 nm. A selection of micrographs of the prepared clots was captured. Fluorescent signals of each of the three fluorescent markers were captured as a composite.czi file in the Zeiss ZEN software, and ImageJ (FIJI) was used to split the channels. Variance between (black) background and fluorescent pixels (binary comparison) for each of the three fluorescent markers was assessed using the histogram function in ImageJ(FIJI). The coefficient of variation (CV) (as SD / mean) was calculated as a metric to quantify and discriminate between clots of healthy naïve PPP and clots of the amyloidogenic blood. Sample analysis was performed with the Mann-Whitney U test, using the STATSDIRECT (version 2.8.0) software.

[0040] Confocal analysis of healthy clotted PPP, in the presence of ThT, Amytracker ™< 480 and 680, showed occasional small patches of fluorescence (see Figure 1A to C). However, when amyloidogenic blood was treated with thrombin, fluorescence was considerably increased for all three markers (Figure 1D to F). This demonstrated binding of the markers to fibrin amyloid and showed that amyloid formation could be detected.

[0041] Statistical (coefficient of variation, CV) data are plotted in Figures 2-4. These data provide quantified measures of fibrin amyloid for the amyloidogenic blood having low grade inflammation. Data from the three different markers - Amytracker ®< 480 (Figure 2), Amytracker ®< 680 (Figure 3) and ThT (Figure 4) - are shown.

[0042] A quantity of amyloid can be calculated by plotting the quantity of fluorescence on a standard curve and reading off the concentration of amyloid that corresponds to the quantity of fluorescence. The amyloid level can in turn be positively correlated to a level of inflammation by comparing the level of amyloid with a plurality of predetermined concentration ranges in which each concentration range has an associated level of inflammation. Furthermore, since SAA is an inducer of amyloid formation, a level of SAA in the blood can be determined based on the amount of amyloid present. A level of inflammation can then be assigned based on the level of SAA present in the blood.Example 2

[0043] In order to demonstrate the detectability of cancer in a subject by measuring amyloid in a blood sample from the subject, naïve healthy cells from twenty-one individuals were contacted with metastatic human breast adenocarcinoma cell line MDA-MB-231 cells. The induction of healthy cells into a state of hypercoagulation, or amyloidogenesis, by cancer cells occurs in early stage cancer as the cancerous cells metastasize in the subject's blood and release elevated levels of SAA and other inflammogens. SAA is induces amyloid formation, which can be measured. By treating the blood with amyloid-binding fluorescent markers (and thrombin to induce fibrin formation) and measuring fluorescence, a quantity of amyloid structures present in fibrin protein in the blood can be determined. A level of SAA present in the blood can be determined based on the quantity of amyloid present. A level of inflammation in the subject can be assigned based on the quantity of amyloid or SAA present, and cancer detected.

[0044] Amyloid formation was measured using confocal microscopy according to the following protocol: Naïve whole blood (WB), as well as WB exposed to human breast adenocarcinoma cell line MDA-MB-231 cells, were centrifuged to obtain platelet poor plasma (PPP). The PPP was incubated with a fluorescent marker, either ThT at a final concentration of 5 µM, or Amytracker ™< 480 or Amytracker ™< 680 (0.1 µL into 100 µL PPP) for 30-minutes. Before viewing clots on the confocal microscope, thrombin was added in the ratio 1:2, (5 µL thrombin: 10 µL PPP) to create extensive fibrin networks. Thrombin solution was at a concentration of 20 U.mL -1< and made up in PBS containing 0.2% human serum albumin. A coverslip was placed over the prepared clot, and samples were viewed using a Zeiss LSM 780 with ELYRA PS1 confocal microscope with a Plan-Apochromat 63x / 1.4 Oil DIC objective. For ThT, the 488 nm excitation laser was used, with emission measured at 508 to 570 nm; for Amytracker ™< 480 the 405 nm excitation laser was used, with emission measured at 478 to 539 nm; and for Amytracker ™< 680, the 561 nm excitation laser was used, with emission measured at 597 to 695 nm. A selection of micrographs of the prepared clots were captured. Gain settings were kept the same during all data capture and used for statistical analyses; however, brightness and contrast were slightly adjusted for figure preparation. The fluorescent signal of each of the three fluorescent markers was captured as a composite LSM file in the Zeiss ZEN software and then ImageJ (FIJI) was used to split and analyse the RGB channels.

[0045] The fluorescence of each stained clot was quantified by assessing the variance between the black background and the fluorescent pixels (binary comparison) for each of the three fluorescent markers in the clots. The histogram function in ImageJ (FIJI) was used to calculate the coefficient of variation (CV) (as SD / mean) of the histogram of different pixel intensities as a metric to quantify and discriminate between clots of healthy (age-controlled) naïve PPP and clots of cells induced into a state of hypercoagulation through contact with cancer cells. CVs were calculated from the data shown in each histogram. GraphPad Prism with Kruskal-Wallis posthoc test and Dunn's Multiple Comparison Test were used for the statistical analysis.

[0046] Figures 5 and 6 show confocal micrographs of amyloid formation in plasma from representative normal body mass index (BMI) and high BMI individuals, before and after exposure to cancer cells. There were no significant differences between normal and high BMI individuals, although the high BMI individuals did show slightly increased amyloid formation when the micrographs were visually inspected. This is likely due to underlying systemic inflammation due to an increased body weight. The addition of cancer cells to plasma significantly increased the amyloid areas in the fibrin clot (this was noted from statistical analysis of all 3 fluorescent markers of amyloid structure). This was seen irrespective of the BMI of the individual.

[0047] Coefficient of variation (CV) data are provided in Figures 7-9 and were used to obtain the statistical data represented in Tables 2-4. Higher CV values indicate higher levels of amyloid formation. These data provide quantified measures of amyloidogenesis for the blood having low grade inflammation resulting from contact with cancer cells. Data from the three different markers - Amytracker ®< 480 (Table 2, Figure 7), Amytracker ®< 680 (Table 3, Figure 8) and ThT (Table 4, Figure 9) - are shown. Table 2: Amytracker 480 results of confocal parameters of naïve blood with and without treatment: NC = healthy naive control blood; Cells = NC blood with cancer cells addedAmytracker 480 = Blue Kruskal-Wallis test P valueP<0.0001Exact or approximate P value?Gaussian ApproximationP value summary***Do the medians vary signif. (P < 0.05)YesNumber of groups4Kruskal-Wallis statistic46,33Dunn's Multiple Comparison Test Difference in rank sum Significant? P < 0.05? Summary NC vs Cells-78,60Yes*** Table 3: Amytracker 680 results of confocal parameters of naïve blood with and without treatment: NC = healthy naive control blood; Cells = NC blood with cancer cells added Amytracker 680 = Red Kruskal-Wallis test P valueP<0.0001Exact or approximate P value?Gaussian ApproximationP value summary***Do the medians vary signif. (P < 0.05)YesNumber of groups4Kruskal-Wallis statistic34,77 Dunn's Multiple Comparison Test Difference in rank sum Significant? P < 0.05? Summary NC vs Cells-64,72Yes*** Table 4: ThT results of confocal parameters of naïve blood with and without treatment: NC = healthy naive control blood; Cells = NC blood with cancer cells added ThT = Green Kruskal-Wallis test P value0,0002Exact or approximate P value?Gaussian ApproximationP value summary***Do the medians vary signif. (P < 0.05)YesNumber of groups4Kruskal-Wallis statistic20,02 Dunn's Multiple Comparison Test Difference in rank sum Significant? P < 0.05? Summary NC vs Cells-53,08Yes**

[0048] The quantified fibrin amyloid structures can be positively correlated to a level of inflammation by comparing the level of amyloid with a plurality of predetermined concentration ranges in which each concentration range has an associated level of inflammation. Where the level of inflammation falls within a predetermined range for cancer, cancer can be detected.

[0049] Throughout the specification unless the content requires otherwise the word 'comprise' or variations such as 'comprises' or 'comprising' will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Claims

1. A method of detecting early stage inflammation in a subject having no symptoms of inflammation or disease, wherein the early stage inflammation indicates a disease, wherein the disease is cancer, the method comprising contacting a blood sample obtained from the subject with thrombin to form fibrin clots, determining a quantity of amyloid present in the fibrin clots, associating the quantity of amyloid with a concentration of serum amyloid A (SAA), and making a diagnosis of early-stage inflammation as an indicator of stage 0 or stage 1 cancer in the subject if the concentration of SAA is greater than 30 µg / ml and less than 10 000 µg / ml.

2. A method as claimed in claim 1, wherein the quantity of amyloid is determined by contacting the blood sample with an amyloid-binding fluorescent marker, allowing amyloid in the sample to bind to the marker, measuring fluorescence emitted upon binding of the amyloid to the marker, and assigning a quantity of amyloid based on the measured fluorescence.

3. A method as claimed in claim 1 or claim 2, wherein the quantity of amyloid is determined by comparing the measured fluorescence to a reference.

4. A method as claimed in any one of claims 1 to 3, wherein the reference is selected from one or more predetermined values, a chart and a graph.