WO2014177700A1 - Procédé d'immunofluorescence indirecte pur détecter des auto-anticorps anti-nucléaires - Google Patents

Procédé d'immunofluorescence indirecte pur détecter des auto-anticorps anti-nucléaires Download PDF

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WO2014177700A1
WO2014177700A1 PCT/EP2014/058999 EP2014058999W WO2014177700A1 WO 2014177700 A1 WO2014177700 A1 WO 2014177700A1 EP 2014058999 W EP2014058999 W EP 2014058999W WO 2014177700 A1 WO2014177700 A1 WO 2014177700A1
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image
subject
ana
cells
anyone
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Daniel Bertin
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Universite D'aix-Marseille
Assistance Publique Hôpitaux De Marseille
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts
    • G06V20/695—Preprocessing, e.g. image segmentation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10064—Fluorescence image
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro

Definitions

  • the invention relates to a method for detecting the presence of antinuclear autoantibodies (ANA) in a biological sample of a subject, typically of a human being, and, when present, determining the titer of the tested sample.
  • ANA antinuclear autoantibodies
  • Such a method can be used in particular to diagnose a disease and to monitor the efficiency of a treatment of said disease in a subject.
  • IIF Indirect ImmunoFluorescence
  • ANA antinuclear antibodies
  • Antinuclear antibodies are biological markers that contribute to the diagnostic of several autoimmune diseases such as for example systemic lupus erythematosus (SLE) [1 ] or autoimmune liver diseases [2] . Moreover, they also provide information for sub-classification and disease activity monitoring [3] . Given this central role in autoimmune diagnosis, ANA detection and identification should be accurate and reproducible. For several decades, indirect immunofluorescence (IIF) on Hep-2 cells has been the reference technique for ANA testing. Although, new available techniques [4, 5] such as ELISA or multiplexing solid phase technologies have been proposed to replace IIF, the recommendations of the American College of Rheumatology (ACR) still point IIF as the gold standard method for ANA detection [6] .
  • IIF indirect immunofluorescence
  • a high throughput process allowing accurate and reproducible ANA detection would be most helpful for autoimmunity laboratories.
  • Commercial systems for automated analysis of ANA by IIF are described in the literature [7-1 1 ].
  • Egerer et al. [7] describes the AKLIDES® interpretation system which is controlled by a specific software consisting of modules for device and autofocus control, image analysis, and pattern recognition algorithms.
  • a reactivity index (RI) is calculated by combining absolute image intensity, contrast, and number of grey-scale levels of the total image for the assessment of image data.
  • the RI is influenced by the exposure time that depends in turn on the cell density and on the highest signal occurring in the image after exclusion of artifacts.
  • Inventors now provide an accurate and reproducible method allowing the IIF ANA detection in a biological sample, even if the ANA's concentration in the biological sample is very low or weak.
  • This method presents advantageous performance when compared to available methods and processes of the art, in that i) it is an automatable or computerizable method, ii) it can be adapted to the analyses of any HEp-2 cell preparations, in particular to any commercially available HEp-2 cell preparations, and it is not limited by the cells' density or distribution in the sample/preparation, and in that iii) this method is able to more efficiently discriminate ANA positive/negative biological samples, even ANA weak- (or very low-) positive/negative biological samples.
  • the herein described method further advantageously allows determining the correct titer of the sample without the time-consuming step of serial dilution.
  • FI fluorescence index
  • COFRAC French Accreditation Committee
  • ANAs antinuclear antibodies
  • step b) contacting Hep-2 cells of step a) with at least one secondary antibody labeled with a fluorochrome A, said at least one secondary antibody recognizing, when present, at least one anti- nuclear antibody (ANA) of the subject,
  • step b) labeling the nuclei of Hep-2 cells of step b) with a fluorochrome B distinct from fluorochrome
  • step f) superimposing either the selection of step e) to the first image of step d), or the first image of step d) to the selection of step e), in order to get a third image
  • FI fluorescence index
  • step f) comparing the fluorescence index (FI) of step f) to a predetermined cut-off or titer range, an index i) at least equal to or above said cut-off, or in or above the titer range, revealing the presence of antinuclear antibodies in the biological sample of the subject and an index ii) below said cut-off, or below said titer range, revealing the absence of antinuclear antibodies in the biological sample of the subject.
  • FI fluorescence index
  • kit comprising typically i) at least one slide with fixed cells used as substrat, typically HEp-2 cells or cells derived from Hep-2 cells such as recombinant Hep-2 cells, ii) at least one computer program allowing the implementation of the herein described method, and optionally iii) at least one motorized material, preferably a computer, a camera, a microscope, and/or a slide handling system, allowing implementation of at least one, preferably several, or each of steps d) to g) of the herein described method.
  • Anti-nuclear antibodies are autoantibodies that bind to contents of the cell nucleus.
  • the immune system produces antibodies to foreign proteins (antigens) but not to human proteins (autoantigens).
  • antibodies to human antigens are produced.
  • ANAs there are many subtypes of ANAs such as anti-Ro antibodies (also called anti-SSA), anti-La antibodies (also called anti-SSB), anti-Sm antibodies, anti-ribonucleoprotein antibodies (anti-RNP), anti-Scl-70 antibodies (anti-topoisomerase I antibodies), anti-dsDNA antibodies, anti-histone antibodies, antibodies to nuclear pore complexes (anti-gp210 antibodies), anti- centromere antibodies, anti-splOO antibodies, anti-RNA polymerase III antibody.
  • anti-Mi-2 antibodies anti-Ku antibodies, anti-fibrillarine antibodies and anti-PM-Scl antibodies.
  • Each of these antibody subtypes bind to different proteins or protein complexes within the nucleus. They are found in many disorders including autoimmunity, cancer and infection, with different prevalences of antibodies depending on the condition. This allows the use of ANAs in the diagnosis of some autoimmune diseases or disorders.
  • Autoimmune diseases arise from an overactive immune response of the body against substances and tissues normally present in the body. In other words, the body actually attacks its own cells. The immune system mistakes some part of the body as a pathogen and attacks it. This may be restricted to certain organs or involve a particular tissue in different places. A substantial minority of the population suffers from these diseases, which are often chronic, debilitating, and life-threatening. There are more than eighty illnesses caused by autoimmunity. Autoimmune diseases strike women three times more than men. It has been estimated that autoimmune diseases are among the ten leading causes of death among US women age 65 and younger (Walsh SJ, Rau LM. Autoimmune diseases: a leading cause of death among young and middle-aged women in the United States. Am J Public Health. 2000 Sep;90(9):1463-6)
  • ANAs may be used to diagnose autoimmune diseases or disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, limited or diffuse systemic scleroderma, mixed connective tissue disease, polymyositis, dermatomyositis, autoimmune hepatitis, primary biliary cirrhosis, drug induced lupus, Raynaud's phenomenon, multiple sclerosis, discoid lupus, thyroid disease, fibromyalgia, antiphospholipid syndrome, juvenile idiopathic arthritis, psoriatic arthritis, juvenile dermatomyositis, idiopathic thrombocytopaenic purpura, infection and cancer.
  • SLE systemic lupus erythematosus
  • Sjogren's syndrome limited or diffuse systemic scleroderma
  • mixed connective tissue disease polymyositis, dermatomyositis,
  • ANAs are found in many disorders as explained previously but also in some healthy individuals. These antibodies can be subdivided or classified according to their specificity, and each subset has different propensities for specific disorders such as those identified herein above. For example, anti- double stranded DNA (anti ds DNA) antibodies are highly specific (near 100%) for active SLE and are therefore used in the diagnosis and monitoring of this disease.
  • anti ds DNA anti- double stranded DNA
  • Enzyme-linked immunosorbent assay uses antigen-coated microtitre plates for the detection of ANAs. Each well of a microtitre plate is coated with either a single antigen or multiple antigens to detect specific antibodies or to screen for ANAs, respectively. The antigens are either from cell extracts or recombinant proteins. Blood serum is incubated in the wells of the plate and is washed out. If antibodies that bind to antigens are present then they will remain in the coated well after washing. Then, the well is contacted with a secondary anti-human antibody conjugated to an enzyme such as horseradish peroxidase. The enzyme reaction will with its substrate produce a change in colour of the solution that is proportional to the amount of antibody bound to the antigen,
  • IIF is the gold standard method for ANA detection.
  • HEp-2 cells from human epidermoid carcinoma cell line
  • Microscope slides are coated with HEp-2 cells and the serum is incubated with the cells. If antibodies are present then they will bind to the antigens on the cells; in the case of ANAs, the antibodies will bind to the nucleus.
  • a fluorophore tagged usually Fluorescein isothiocyanate (FITC) or rhodopsin B
  • FITC Fluorescein isothiocyanate
  • rhodopsin B anti-human antibody that binds to the autoantibodies.
  • the fluorophore becomes fluorescent when it is excited by a specific wavelength of light. Then, the emitted fluorescence can be seen under the microscope equipped with an appropriate filter, as well known by the skilled person.
  • both ANA detection and identification may vary depending on the skill of the human observers.
  • titer also identified as "titre"
  • the titer or titre is the highest dilution of the serum at which autoantibodies are still detectable.
  • Positive autoantibody titers at a dilution equal to or greater than 1 : 160 are usually considered as clinically significant.
  • Positive titers of less than 1 : 160 are present in up to 20% of the healthy population, especially the elderly.
  • Positive titers of 1 : 160 or higher, in particular higher than 1 :200, are strongly associated with autoimmune disorders.
  • Autoantibody screening is useful in the diagnosis of autoimmune disorders and monitoring levels help to predict the progression of disease.
  • ANAs first have to be detected, in particular when present at very low levels, i.e. below 1 : 160, preferably between 1 : 160 and 1 :80, typically at or around 1 :80 or 1 : 100, in what are herein identified as the "very low positive ANA titers' samples". Positive titers of 1 : 160 or higher are also inadequately found in 5% of healthy individuals (Tan EM et al. Range of antinuclear antibodies in "healthy” individuals. Arthritis Rheum. 1997 Sep;40(9) : 1601 - 1 1 ).
  • Inventors now herein describe an original IIF method for accurately and reproducibly detecting ANAs (also identified by inventors as the "algorithm”, “algorithm approach” or “algorithm method”) comprising the determination of a fluorescence index (FI) that objectively discriminates between positive and negative samples by minimizing any artefacts' influence, and in particular advantageously allows the detection of very low- (or weak-) positive biological samples as defined herein above.
  • FI fluorescence index
  • the herein described fluorescence index does not depend from the time-exposure of the tested sample to a particular material, typically to the camera capturing fluorescence images.
  • a first object of the invention is a method for detecting, in vitro or ex vivo, anti-nuclear antibodies (ANAs) in a biological sample of a subject, wherein the process comprises the following steps of: a) contacting cells, typically Hep-2 cells, with a biological sample from a subject, in particular that of a subject suspected of containing antinuclear antibodies (ANAs),
  • step b) contacting cells of step a) with at least one secondary antibody labeled with a fluorochrome A, said at least one secondary antibody recognizing, when present, at least one anti-nuclear antibody (ANA) of the subject (said being considered as the primary antibody),
  • ANA anti-nuclear antibody
  • step b) labeling the nuclei of cells of step b) with a fluorochrome B distinct from fluorochrome A, the measure of a fluorochrome B emission confirming in particular that the tested sample has been concretely contacted with the cells of step b),
  • step f) superimposing either the selection of step e) to the first image of step d) or the first image of step d) to the selection of step e) in order to get a third image
  • FI fluorescence index
  • FI Ibackgroimd/Imicieus
  • the fluorescence index (FI) of step f) to a predetermined cut-off or titer range, preferably to a predetermined cut-off, an index i) at least equal to or above said cut-off, or in or above the titer range, revealing the presence of antinuclear antibodies (ANAs) in the biological sample of the subject and an index ii) below said cut-off, or below said titer range, revealing the absence of antinuclear antibodies in the biological sample of the subject.
  • ANAs antinuclear antibodies
  • the term "cut-off can refer to the mean of values, typically of titers, obtained with the biological samples of a reference population, typically of a population or cohort of subjects who suffer from an autoimmune disease, disorder or anomaly as herein defined.
  • the control value can also be a statistic or discriminating value, i.e., a value which has been determined by measuring the parameter in both a healthy control population and a population with a known disease or disorder as herein defined.
  • the discriminating value identifies the diseased population with a predetermined specificity and/or a predetermined sensitivity based on an analysis of the relation between the parameter values and the known clinical data of the healthy control population and of the diseased patient population
  • the discriminating value may also be expressed as a concentration of the biomarker in the biological sample of the tested subject for a particular specificity and/or sensitivity, or may be a normalized cutoff value expressed as a ratio for a particular specificity and/or sensitivity.
  • the cut-off can be further easily adapted by the skilled person to the particular biological sample, typically Hep-2 cell preparation, to be studied.
  • the cut-off is determined or predetermined through a Receiver Operating Characteristic (ROC) curve analysis (cf. Metz, 1978).
  • a ROC curve is a graphical plot of the sensitivity (or true positive rate), vs. false positive rate (1 - specificity or 1 - true negative rate), for a binary classifier system. Each point on the ROC plot represents a sensitivity/specificity pair corresponding to a particular decision threshold. The area under the ROC curve is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normal).
  • sensitivity also called the true positive rate
  • sensitivity measures the proportion of positives which are correctly identified as such.
  • sensitivity designates the probability of a positive test given the patient is ill.
  • the positive predictive value is the proportion of positive test results that are true positives (such as correct diagnoses). It is a critical measure of the performance of a diagnostic method, as it reflects the probability of having the disease when the test result is positive. Its value does however depends on the prevalence of the outcome of interest, which may be unknown for a particular target population.
  • the negative predictive value is a summary statistic used to describe the performance of a diagnostic testing procedure.
  • NPV reflects the probability of not having the disease when the test result is negative. It is defined as the proportion of subjects with a negative test result who are correctly diagnosed.
  • a high NPV for a given test means that when the test yields a negative result, it is most likely correct in its assessment.
  • a high NPV means that the test only rarely misclassifies a sick person as being healthy. Note that, contrary to the specificity, this says nothing about the tendency of the test to mistakenly classify a healthy person as being sick.
  • the ROC curve analysis allowed inventors to precisely determine a cut-off for FI that optimize accuracy between the herein described method and the standard visual IIF method by comparing their respective ability to discriminate diseased cases from normal cases in terms of both specificity and sensitivity.
  • the sensitivity of the method has been determined by inventors as of at least 94% and the specificity of the method as of at least 98% for positive/negative discrimination, i.e. for a fluorescence index above the cut-off, the sensitivity being in a particularly advantageous manner of at least 86%o and the specificity of at least 98%> for weak positive/negative discrimination, i.e. when the fluorescence index is at or around the predetermined cut-off.
  • the positive predictive value of the method has been determined by inventors as of at least 98 %> and the negative predictive value of the method as of at least 93 %> for positive/negative discrimination, i.e. for a fluorescence index above the cut-off, the positive predictive value being in a particularly advantageous manner of at least 95 %> and the negative predictive value being of at least 93 %> for weak positive/negative discrimination, i.e. when the fluorescence index is at or around the predetermined cut-off.
  • the method of the invention can advantageously further comprise an additional step (step i) of determining the titer of the detected ANAs in the biological sample by comparing the fluorescence index of the biological sample to the fluorescence index of a reference sample of defined titer or to a standard curve of fluorescence indexes.
  • An antibody titer is a measurement of how much antibody an organism has produced that recognizes a particular epitope, expressed as the greatest dilution that still gives a positive result. Titers expressed as 1/X (1 part biological sample to X-1 parts diluents) are also herein expressed by the denominator only, i.e. "X" in this particular example. ELISA and Coombs tests are common means of determining antibody titers.
  • the indirect Coombs test detects the presence of anti-Rh antibodies in a pregnant woman's blood serum.
  • a patient might be reported to have an "indirect Coombs titer" of 16. This means that the patient's serum gives a positive indirect Coombs test at any dilution down to 1/16 (1 part serum to 15 parts diluent). At greater dilutions the indirect Coombs test is negative. If a few weeks later the same patient had an indirect Coombs titer of 32 (1/32 dilution which is 1 part serum to 31 parts diluent), this would mean that she was making more anti-Rh antibody, since it took a greater dilution to abolish the positive test.
  • the method of the invention comprises no step of dilution of the tested biological sample, in particular no step of serial dilution thereof.
  • the fluorescence index (FI) may also be compared during step h) to a reference titer (or "control value"), said titer being typically comprised in a range (herein identified as the "titer range”), instead of being compared to a cut-off.
  • Such a comparison may occur for example for samples suspected of having, or recognized as having, a very low positive ANA titer.
  • a typical very low positive ANA titer range is [80-180], preferably [80- 160], for example [80-120] or [90-110].
  • the term "around" used in the herein used expression "when the fluorescence index is at or around the predetermined cut-off is typically associated to the [80- 160] titer range, a typical cut-off being the cut-off usually considered in standard visual IIF, i.e. 100 (or 1 :100).
  • the term "biological sample” includes any biological sample from a subject, in particular a mammalian subject, typically a human being.
  • the biological sample may be a tissue biopsy, for example a skin biopsy.
  • the biological sample is typically a biological fluid sample, preferably a bodily fluid.
  • Typical examples of biological samples usable in the context of the present invention may be selected from plasma, blood, serum, urine, cerebrospinal fluid, synovial fluid, and saliva.
  • the biological sample is a blood, a plasma or a serum sample, even more preferably a serum sample.
  • subject herein refers to any testable subject and typically designates a patient.
  • the subject is a mammal, even more preferably a human being.
  • the invention may be used both for an individual and for an entire population.
  • the subject may be tested whatever his/her age or sex.
  • the subject can be a subject at risk of developing any disease or disorder leading to the expression, in particular to an overexpression, of ANAs, typically an autoimmune disease or disorder, in particular a disease or disorder leading to a variation of the concentration of ANAs in serum sample.
  • any disease or disorder leading to the expression, in particular to an overexpression, of ANAs typically an autoimmune disease or disorder, in particular a disease or disorder leading to a variation of the concentration of ANAs in serum sample.
  • the subject can be a subject at risk, or suspected to be at risk, of developing a specific disease, disorder or anomaly as herein described.
  • the patient can be a subject predisposed to (or suspected to be predisposed to) develop a lupus, for example a systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • the subject can be asymptomatic, or present early or advanced signs of such a disease, disorder or anomaly.
  • the subject may be selected for example from patient presenting at least one symptom of a known autoimmune disease, in particular of a disease known to induce the expression of ANA by a subject suffering of such a disease.
  • the symptom may be for example a symptom of lupus, in particular a symptom of SLE, or a symptom of rheumatoid arthritis, a symptom of vasculitis, etc.
  • HEp-2 cells are currently one of the most common substrates for ANA detection by immunofluorescence. They are superior to the previously used animal tissues because of their large size and the high rate of mitosis (cell division) in the cell line. This allows the detection of antibodies to mitosis-specific antigens, such as centromere antibodies. They also allow identification of anti-Ro antibodies, because acetone can be used for fixation of the cells (other fixatives can wash the antigen away).
  • the terms "HEp-2 cells” herein designates HEp-2 cells as well as cells derived from HEp-2 cells such as recombinant Hep-2 cells. Epithelial- like cell lines such as HEp-2000® can thus also be used in the herein described methods.
  • HEp-2000® is a HEp-2 cell line that has been transfected for overexpression of the SSA/Ro antigen. This result in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen.
  • human epithelial tumor cell lines such as Hela cells could also be used as a substrate in the herein described method.
  • the detection may be performed on a solid support, for example on a glass slide on which a substrate, such as Hep-2 cells, is fixed, in addition to the subject's ANAs to be detected, or on solid particles, microplaques, beads, test tubes, etc.
  • the claimed method can advantageously be adapted to any commercial support of the art (whatever the selected reagents, antibodies, etc.) and in particular to any HEp-2 cells preparations such as HEp-2 cells fixed on glass slides.
  • cell density does not influence the fluorescence index thanks to step c-g) of the herein claimed algorithm, and in particular to the selection of the regions corresponding to the nuclei of cells on the second image in order to get a selection comprising i) a first compartment corresponding to the nucleus region of the second image and ii) a second compartment corresponding to the non-nucleus background region of the second image.
  • image herein designates the total image which has been captured.
  • the claimed algorithm in particular does not require to distinguish between grey-scale levels.
  • Fluorescent dye with blue, green, or red emission are respectively 4,6-diamidino-2-phenylindol (DAPI) (blue emission), fluorescein isothiocyanate (FITC) or rhodamine (green emission), and Cy5 and/or allophycocyanin (APC) (red emission).
  • DAPI 4,6-diamidino-2-phenylindol
  • FITC fluorescein isothiocyanate
  • rhodamine green emission
  • APC allophycocyanin
  • fluorochrome A and fluorochrome B are distinct and both selected froms fluorescein isothiocyanate (FITC) and 4,6-diamidino-2-phenylindol (DAPI).
  • fluorochrome A is fluorescein isothiocyanate (FITC) and fluorochrome B is 4,6-diamidino-2- phenylindol (DAPI).
  • the image capturing of step d), the selection of step e), the superimposition of step f), the fluorescence intensity measures performed during step g), and the comparison of step h) are carried out using a computer and/or an adapted computer program (allowing implementation of said step(s)).
  • the fluorescence intensity used as a possible threshold for the selection/segmentation in step e) of the regions corresponding to the nuclei of Hep-2 cells is a multiple of, for example twice, the fluorescence intensity of the background part of the second image obtained or captured in step d).
  • the superimposition of step f) is a superimposition of the selection of step e) to the first image obtained or captured in step d), or is a superimposition of the first image obtained or captured in step d) to the selection of step e). It is typically a correct superimposition and preferably a perfect superimposition.
  • the correct or perfect superimposition will typically vary depending on the selected material (in particular on the selected microscope and/or camera) used to perform said superimposition and on its ability to capture images of a single microscopic field at different wavelengths, or in other words on its ability to differentiate the fluorescence emissions of distinct fluorochromes.
  • This superimposition can be performed using any material know by the skilled person such as conventional optical microscope or fully motorized microscope.
  • the correct or perfect superimposition can further vary if a material-dependant overlap exists between captured images of the same microscopic field.
  • the correct or perfect superimposition is controlled during an additional step f ), said step f ) being performed before step g).
  • Such a control can be performed with the help of a correlation coefficient, typically calculated in the following way:
  • r p is the correlation coefficient, x ; (also herein identified as Pi) is the fluorescence intensity of pixels of a first compartment, and y; (also herein identified as P 2 ) is the fluorescence intensity of pixels of the second compartment (relative to the first).
  • superimposition is not correct or imperfect.
  • Superimposition will be from correct when r is distinct from 0 to perfect when r is about
  • the superimposition can be improved for example by translating/shifting one image relative to the other, typically the first image relative to the second image, or the second image relative to the first image, for example with the help of an appropriate computer program.
  • the method is automated (implemented with the help of a computer and of an adapted program) only pixels located within a rectangular window obtained after removing strips of 20 pixel width along each border are considered. The calculation is repeated after shifting of the second image with respect to the first image by 1 to 10 pixels along both the height and width of the image.
  • the fluorescence intensities of each of the first and second compartments of the third image are measured in step g) using a material classically used by the skilled person, typically a computer, possibly with the help of an adapted computer program performing such measures of pixel fluorescence intensity and preferably further calculating a mean fluorescence intensity for each region (compartment).
  • the fluorescence intensity as determined for each region (compartment) is a mean intensity.
  • the intensities of each compartment are then used to determine the fluorescence index (FI).
  • the fluorescence index can be the ratio of I nU cieus Iback g round.
  • a method wherein the fluorescence index corresponds to the ratio of Ibackground/Imicieus ( ⁇ 1 ) is nothing else that a method equivalent to the herein described method wherein the fluorescence index corresponds to the ratio Inucieus Ibackground ( ⁇ 1 ).
  • the (mean) fluorescence intensity of one compartment, for example of the second compartment, of the third image is measured in step g) following inversion of the other compartment selection, in this example of the first compartment selection, using a computer program.
  • the (mean) fluorescence intensity of a second compartment of the third image is measured in step g) following inversion of a first compartment selection using preferably a computer program.
  • the invention also relates to in vitro or ex vivo methods comprising the determination of the presence of ANA in a biological sample from a subject (i) for the detection of an autoimmune disease (diagnostic), (ii) for the determination of a predisposition to an autoimmune disease, (iii) for the determination of the prognosis or monitoring of the course of an autoimmune disease, or (iv) for assessing or monitoring the efficiency of a treatment of an autoimmune disease.
  • diagnosis refers to the detection or identification of an autoimmune disease or disorder as herein defined, or to the evaluation of the severity or of the progression of such a disease or disorder in a subject as herein defined.
  • a diagnostic method of the invention comprises the determination of the presence of ANA in a biological sample of a subject, and preferably the determination of an ANA titer.
  • Prognostic refers to the assessment or “monitoring” of the progression (course) of an autoimmune disease or disorder (as herein defined) in a subject (as herein defined), treated or not, typically the prediction of the worsening of such a disease or disorder and associated harmful effects or, on the contrary, the prediction of an improvement of the subject's health.
  • a prognostic method of the invention can comprise one or several steps of monitoring the ANA's titer at various stages, including early, pre-symptomatic stages, and late stages, in a biological sample or in biological samples from the subject.
  • Prognosis typically includes the assessment (prediction) of the progression of an autoimmune disease and the characterization of a subject to define the most appropriate treatment.
  • a particular matter herein described thus relates to the use of the herein described method of the invention for the diagnostic and/or monitoring of a disease responsible for the production of ANA in a subject suffering of said disease.
  • the present description provides, in an embodiment, an in vitro or ex vivo method for the detection of an autoimmune disease in a subject.
  • the method comprises the determination, as herein explained, of the presence of ANAs in a biological sample of the subject.
  • the presence of ANA in the subject is typically indicative of the presence of an autoimmune disease.
  • the presence of ANAs in the subject can further be indicative of a predisposition or risk to develop such an autoimmune disease.
  • the invention provides an in vitro or ex vivo method of determining the prognosis of, or of monitoring the course of an autoimmune disease in a subject.
  • the method comprises the determination of the presence of ANA in a biological sample of the subject, at different times, the presence (following the absence) or a titer variation (increase or decrease), typically a titer increase (for example from 1/X to 1/(Y>X)) during time being indicative of a worsening of the autoimmune disease.
  • a further herein described matter relates to the use of the herein described method for monitoring the efficiency of a therapeutic treatment of a subject suffering of a disease responsible for the production of ANA.
  • the invention relates to an in vitro or ex vivo method of assessing the efficiency of a treatment of an autoimmune disease in a subject.
  • the method comprises the determination, as herein explained, of the presence of ANA in a biological sample of the subject, at different times before, during and/or after the treatment, the absence (following the presence) or a variation (increase or decrease), typically a decrease (for example from 1/X to 1/(Y ⁇ X)), in the ANA titer during time being indicative of an improvement of the health's subject regarding the autoimmune disease.
  • a variation typically a decrease (for example from 1/X to 1/(Y ⁇ X)
  • Such an absence or variation may be indicative of a decreased risk that the autoimmune disease (as herein described), occurs.
  • kits comprising any one or more of the herein-described products.
  • the kit is a kit for detecting ANA in a bodily fluid of a subject, preferably of a mammal, even more preferably of a human being.
  • This kit comprises i) at least one slide with fixed cells used as substrat (substrate), typically HEp-2 cells, preferably several slides with such fixed cells, ii) at least one computer program allowing the implementation of the herein described method (also herein identified as the "algorithm"), and optionally iii) at least one motorized material (for example a computer, camera, microscope, and/or slide handling system) or at least one non motorized material allowing IIF detection, image acquisition and/or image analysis (i.e. allowing implementation of at least one, preferably several, or each of steps d) to h), of the herein described method of the invention).
  • a preferred program allows typically the implementation of the herein described process and facilitates data communication with the informatics system used in a given laboratory.
  • Such a program preferably comprises data (images, results) storing means.
  • the program is able to pilot a motorized microscope, typically a motorized microscope comprising a camera and preferably also a slide handling system.
  • a preferred kit comprises i) at least one slide with fixed cells used as substrate, typically HEp-2 cells, preferably several slide, ii) at least one computer program allowing the implementation of the herein described method, and optionally iii) at least one motorized material, preferably a computer, a camera, a microscope, and/or a slide handling system, allowing implementation of at least one, preferably several, or each of steps d) to g) of the herein described method.
  • the kit can also comprise one or more containers filled with one or more of the herein described products, in particular fluorochrome(s), antibody(ies) recognizing at least one ANA of the subject, reagent(s), standard(s), calibrant(s), wash buffer(s), etc.
  • a labelling notice providing instructions for using the products in the context of a method according to the present invention can further be added to the kit.
  • Figure 1 Examples of captured images. Examples of images obtained by Indirect ImmunoFluorescence (IIF) on Human Epithelial cell line type 2 (HEp-2) cells from 2 different serum samples: one (AntiNuclear Antibody) ANA negative (a,b) and one ANA positive (c,d) for both DAPI (a,c) and FITC (b,d) stainings. Objective: x20.
  • IIF Indirect ImmunoFluorescence
  • HEp-2 Human Epithelial cell line type 2
  • A Receiver Operating Characteristic (ROC) of ANA detection using the method of the invention.
  • Sens sensitivity
  • Spec Specificity
  • PV+ positive predictive value
  • PV- Negative predictive value.
  • B Accuracy as a function of fluorescence index cut-off. Accuracy is the ratio between the number of correct prediction allowed by the method of the invention and the total number of patients. Here the determined cut-off is equal to 1.246.
  • Figure 4 Study of fluorescence index as a function of dilution.
  • Inventors collected serum samples from a total number of 237 patients. The samples were submitted to the clinical laboratory of immunology for analysis of antinuclear antibodies. All patients were outpatients or hospitalized patients from Assistance Good - Hopitaux de Marseille (AP-HM) University hospital. The most prescribing hospital departments were internal medicine, cardiology, dermatology and rheumatology.
  • ds-DNA Anti-Double Stranded DNA antibodies levels were measured in sera with fluorescence-enzyme immunoassay (EliATM dsDNA; Phadia, Uppsala, Sweden; now part of Thermo Fisher Scientific).
  • ANA AntiNuclear Antibody
  • IIF indirect immunofluorescence
  • FITC fluorescein isothiocyanate
  • DAPI 4,6-diamidino-2-phenylindol
  • DAPI image was used to determine/localize nucleus position. This was performed using a thresholding method based on image histogram analysis. Inventors defined, as an example, the background intensity of DAPI image as the first peak of DAPI histogram. A threshold defined as twice this background intensity allowed an appropriate segmentation and selection of nucleus region of DAPI image. This nucleus region selection was then superimposed on FITC image which allowed mean fluorescence intensity measurement of nucleus region of FITC image (MFI n). Then, an inversion of selection leading to the selection of the non- nucleus background region, allowed mean fluorescence intensity measurement of said non-nucleus background region of FITC image (MFI b).
  • MFI n mean fluorescence intensity measurement of nucleus region of FITC image
  • FI fluorescence index
  • Inventors also evaluated the effect of time-exposure of the camera on FI by studying the FI as a function of the time-exposure (50-300 ms) for positive ANA patients (data not shown). No variation was observed attesting that FI values were time exposure independent.
  • Performance of the method was evaluated by calculating sensitivity (Se), specificity (Spe), positive predictive value (PPV) and negative predictive value (NPV). Accuracy was defined as the proportion of the total number of correct method predictions. Student's t-test was used for comparing the means of indexes and Spearman's rank correlation coefficient for studying the correlation between the indexes and fluorescence titers. The agreement between visual (standard) interpretation and method discrimination was evaluated using Cohen's Kappa coefficient which takes on the value i) zero if there is no more agreement between two tests as can be expected on the basis of chance, ii) 1 if there is perfect agreement.
  • the main studied population was composed of 237 patients with male/female ratio 93: 144,
  • the positive ANA group Among the positive ANA group, several single and mixed fluorescence patterns were represented (91 speckled, 10 centromeric, 6 nucleolar, 4 homogenous, 5 nuclear dots, 3 mitotic spindle apparatus, 11 homogenous-speckled, and 2 homogenous-nucleolar).
  • the fluorescence titers range from 100 to more than 800.
  • Cut-off determination of FI was performed using ROC analysis (Figure 3A) and accuracy curve (Figure 3B), showing an FI cut-off value equal to 1,246.
  • Area under curve (AUC) was 0,991 attesting the very good performance of the method.
  • sensitivity and specificity for positive(i) / negative(ii) discrimination were respectively i) of at least 94% or 94,5%, preferably at least 95%, and ii )of at least 98%.
  • the agreement between visual evaluation and method discrimination (“algorithmic method”), was excellent (Kappa 0.923) (cf. Table 3).
  • Table 3 Method performance for ANA screening.
  • PPV Positive predictive value
  • NPV Negative predictive value
  • the herein described method now allows the efficient automation of IIF ANA detection with the calculation of a fluorescence index that objectively discriminates positive and negative samples and further allows a quantitative analysis.
  • Inventors validated it on a large cohort of 237 routinely investigated samples by comparing the results obtained with their method and with those obtained by standard visual evaluation.
  • the present approach is based on a quantitative strategy.
  • the first step is the positive/negative screening that eliminates 60-70% of requests because they are negative and do not require further investigation.

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Abstract

La présente invention concerne un procédé de détection de la présence d'auto-anticorps anti-nucléaires (ANA) dans un échantillon biologique d'un sujet, typiquement d'un être humain et, le cas échéant, de détermination du titre de l'échantillon testé. Un tel procédé peut être utilisé en particulier pour diagnostiquer une maladie et surveiller l'efficacité d'un traitement de ladite maladie chez un sujet.
PCT/EP2014/058999 2013-05-02 2014-05-02 Procédé d'immunofluorescence indirecte pur détecter des auto-anticorps anti-nucléaires WO2014177700A1 (fr)

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CN108507849A (zh) * 2018-04-08 2018-09-07 华中农业大学 一种适用于免疫荧光分析的小麦根部细胞核提取方法
CN110443105A (zh) * 2018-05-03 2019-11-12 长庚医疗财团法人林口长庚纪念医院 自体免疫抗体的免疫荧光影像型态识别方法
CN110543806A (zh) * 2018-05-29 2019-12-06 长庚医疗财团法人林口长庚纪念医院 一种自体免疫抗体的免疫荧光影像分类系统及其分类方法
CN109858428A (zh) * 2019-01-28 2019-06-07 四川大学 基于机器学习和深度学习的ana荧光片自动识别方法
CN109858428B (zh) * 2019-01-28 2021-08-17 四川大学 基于机器学习和深度学习的ana荧光片自动识别方法
CN112240878A (zh) * 2019-07-19 2021-01-19 欧蒙医学实验诊断股份公司 用于检测不同抗核抗体荧光图案类型的存在的方法和装置
CN112240878B (zh) * 2019-07-19 2023-05-26 欧蒙医学实验诊断股份公司 用于检测不同抗核抗体荧光图案类型的存在的方法和装置
CN111403004A (zh) * 2020-02-26 2020-07-10 广州和硕信息技术有限公司 一种人工智能ana检测图文报告系统

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