EP4320437A1 - Procédé d'analyse d'un échantillon de sang pour détecter une maladie - Google Patents

Procédé d'analyse d'un échantillon de sang pour détecter une maladie

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Publication number
EP4320437A1
EP4320437A1 EP22718983.4A EP22718983A EP4320437A1 EP 4320437 A1 EP4320437 A1 EP 4320437A1 EP 22718983 A EP22718983 A EP 22718983A EP 4320437 A1 EP4320437 A1 EP 4320437A1
Authority
EP
European Patent Office
Prior art keywords
cells
marker
marking
status
markers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22718983.4A
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German (de)
English (en)
Inventor
Andrej MANTEI
Christian Meisel
Tim Meyer
Alexander Scheffold
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Labor Berlin Charite Vivantes Services GmbH
Original Assignee
Labor Berlin Charite Vivantes Services GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Labor Berlin Charite Vivantes Services GmbH filed Critical Labor Berlin Charite Vivantes Services GmbH
Publication of EP4320437A1 publication Critical patent/EP4320437A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to a method for analyzing a blood sample from a human for a disease, caused in particular by bacteria.
  • Tuberculosis is a chronic infection caused by mycobacteria of the Mycobacterium tuberculosis complex (MTBC).
  • MTBC Mycobacterium tuberculosis complex
  • M. tuberculosis sensu stricto and M. africanum M. tuberculosis sensu stricto and M. africanum.
  • an infection with tuberculosis bacteria leads to a tuberculosis disease or active tuberculosis infection in only about 10% of cases.
  • LTBI latent tuberculosis infection
  • tuberculosis bacteria remain in humans.
  • a transition from latent to active tuberculosis infection and thus disease is possible.
  • the clearest possible proof of a tuberculosis disease is necessary.
  • a tuberculosis disease is detected in a very wide variety of ways. In principle, it is possible to detect a tuberculosis infection on the basis of blood samples. The reaction of the immune system to tuberculosis antigens is also being examined. If there is a reaction, it can be concluded that the patient in question is infected with tuberculosis bacteria. However, the blood tests currently used cannot be used to determine whether the person concerned is only latently infected with tuberculosis or whether the tuberculosis disease is active.
  • the method is used to analyze a blood sample from a human for a disease, in particular based on bacteria, having the following steps:
  • the present invention also relates to a method for analyzing a blood sample (100) from a human for a disease, in particular based on bacteria, having the following steps:
  • an infection in particular a bacterial infection
  • a method according to the invention can also be used for other diseases caused by pathogens.
  • pathogens include, for example, pathogens in the form of viruses, fungi or parasites, which have a pathogen-specific immune response.
  • cells from a blood sample e.g. in the form of isolated mononuclear cells or whole blood
  • the starting material can therefore be obtained in a minimally invasive manner in the form of a peripheral venipuncture. Biopsies and time-consuming cultivation of bacteria are no longer necessary.
  • T-cells e.g. CD4+ T-cells
  • the cells provided are stimulated with bacterial antigens, such as tuberculosis antigens.
  • bacterial antigens such as tuberculosis antigens.
  • Structures that are part of bacteria of the Mycobacterium tuberculosis complex (MTBC), e.g. ESAT-6/CFP-10 peptides or purified protein derivatives (PPD) can be used as tuberculosis antigens.
  • MTBC Mycobacterium tuberculosis complex
  • PPD purified protein derivatives
  • the T cells that respond to stimulation with bacterial antigens are labeled and characterized. Labeling is made possible by inducing the at least one presence marker on these T cells.
  • the method can be presented as a two-stage process. In the first step, tuberculosis antigen-specific T cells are identified using the at least one presence marker. These cells are found in both latently and actively infected patients with tuberculosis.
  • the second step it is examined whether the cells that carry the at least one presence marker also carry one of the status markers.
  • This at least one status marker allows conclusions to be drawn about the activation status of the bacterial antigen-specific cells, as cells with a presence marker.
  • the presence marker can be used to draw conclusions about the presence of bacteria
  • the status marker can be used to draw conclusions about the status, i.e. the distinction between latent and active expression of the bacterial infection.
  • the frequency of the T cells carrying presence markers and status markers or the labeling strength of the status markers on the T cells carrying presence markers is determined and compared with a combination limit value.
  • Combination limits can be defined as a clear limit or as a transition range, as explained later.
  • the number of such cells with a combined marking or the marking strength of a status marker as a measure of its frequency on the cells that also carry the at least one status marker can now be normalized to a total cell number in the form of a reference value and compared to a combination limit value. If the number of cells marked in combination exceeds the combination limit value, an active bacterial infection, for example active tuberculosis, can be assumed, particularly with a certain probability. As will be explained later, the informative value of this individual result is further improved by combining it into an overall result and comparing it with an overall limit value.
  • a further core idea according to the invention is based on the fact that different status markers are used and combined in the analysis.
  • the presence markers are marked on the cells.
  • all T cells are marked with a presence marker and a first status marker
  • T cells are marked with a presence marker and a further status marker.
  • different marked sets arise. This is particularly relevant with regard to the reference value, which will be explained later, and should be used in the further evaluation, which is why the individual result sets will be briefly discussed below.
  • T cells which, particularly after stimulation, may or may not have a presence marker.
  • These represent two possible reference values, namely either all T cells in the blood sample, in particular the CD4+ T cells, or as a second reference value only those T cells, in particular the CD4+ T cells, which also have a presence marker in marked form exhibit. More cell sets will be formed by the marked status markers. However, status markers are only considered on cells on which a presence marker has also been marked. It can therefore be assumed that there is a first set as the first marker result, which contains T cells with a marked presence marker and a marked first status marker.
  • a further marking result correspondingly includes all T cells which have a marked presence marker and the marked at least one further status marker. If, for example, a third status marker is also used in a method according to the invention, this means that a third marking result can be made available accordingly.
  • an overall result which can also be referred to as a score, overall score or disease score.
  • This overall result is compared with an overall limit value in order to be able to make a statement about an activity or a latency of the bacterial disease.
  • the overall result is generated by adding the individual results. Both a qualitative addition and a quantitative addition can be carried out. Of course, more complex relationships can also be taken into account when forming the overall result, as will be explained later.
  • the improvement by a method according to the invention will be briefly described below with the aid of a simple example.
  • the three different marking results are now evaluated and the following picture emerges, for example.
  • the first labeling result is indicative of a latent infection
  • the second and third labeling results are indicative of an active bacterial infection. From a qualitative point of view, this means that the first marking result can be interpreted as "0" and the other two marking results as "1".
  • the individual marking results are therefore not unequivocal, since they lead to different individual interpretations.
  • an overall result is now generated on the basis of these individual results.
  • the value 2 can be set as the overall limit. In the present case, this means that an overall result of 2 or higher indicates an active infection, while an overall result of ⁇ 2, ie 0 or 1, indicates a latent infection. It now makes it possible to summarize the individual marking results and to arrive at a superordinate analysis result, which contains a clear statement about the activity status of the infection. With the known solutions, the use of the first status marker would have led to an interpretation of a latent infection, while the individual evaluation of the second or the third status marker would have led to the interpretation of an active infection. Only by combining the different status markers in the manner according to the invention can the certainty in distinguishing between latent and active forms of the infection be significantly increased.
  • a presence marker and a status marker are to be understood as meaning proteins on a subgroup of the cells provided which have specific correlations with the parameters to be analyzed.
  • the frequency of the cells that carry the presence marker after stimulation with tuberculosis antigens correlates with the frequency of tuberculosis-specific T cells in the patient's blood.
  • a presence marker must fulfill two conditions in particular: After in vitro stimulation, it should only be formed by cells that specifically produce the stimulant (here: tuberculosis antigen). recognize.
  • the duration for which the presence marker can be found on the cells after stimulation should be limited so that the method does not erroneously identify cells that have already been activated in vivo before in vitro stimulation.
  • the labeling of the presence marker thus allows the analysis of tuberculosis-specific T-cells in the cells provided with appropriate stimulation with tuberculosis antigens.
  • the stimulation takes place over a defined period of 1 to 72 hours, in particular 1 to 48 hours, in particular 1 to 24 hours, in particular 1 to 12 hours, in particular 1 to 7 hours, in particular 4 to 6 hours.
  • the cells which are specific for tuberculosis antigens can therefore be recognized and distinguished from other cells by the stimulation and the associated induction of the at least one presence marker.
  • Live bacteria, dead bacteria or, in particular, fragments or synthesized structures that resemble the structures of tuberculosis bacteria e.g. lysates, protein extracts, purified proteins, protein mixtures, recombinantly produced proteins or protein fragments, peptides or nucleic acid sequences
  • tuberculosis bacteria are often locked away in an encapsulated area of the body and in this state have only limited interaction with the patient's adaptive immune system (including T cells).
  • T cells including T cells
  • the activation of the T-cells through this contact causes them to start forming activation markers, some of which can be used as status markers.
  • the analysis of the marking of the at least one status marker in a quantitative and/or qualitative manner allows a statement to be made for the purpose of distinguishing between latent and active tuberculosis disease. There is still to point out that the status markers on the T cells are already generated in the body and no separate stimulation is necessary for this.
  • the status markers differ from the at least one presence marker.
  • a method can now be made available with the aid of a simple, inexpensive and, above all, minimally invasive venipuncture, which can be analyzed in a two-stage or parallel process with regard to the status of a bacterial infection.
  • a precise analysis with the ability to distinguish, for example, between latent and active tuberculosis disease is possible. This leads to significantly cheaper and faster results, which can also represent a detailed and meaningful source of information for subsequent therapy requests or for monitoring the success of therapy.
  • the overall result is formed in a slightly modified manner.
  • a combination limit value but rather an algorithmic weighting by means of a weighting context. This makes it possible to dispense with an individual evaluation of the marking results, since the statement of the respective marking result is reflected in the respective weighting.
  • the weighting can also take into account the accuracy of the statement of the respective status marker.
  • the final step of the comparison with an overall limit value is again identical for both variants of the procedure.
  • a method according to the invention is designed for the analysis of a tuberculosis disease based on tuberculosis bacteria, in which tuberculosis antigens are used as bacterial antigens.
  • a particularly advantageous embodiment of the method can be achieved because, as has already been explained, a tuberculosis infection is very often divided into latent and active tuberculosis infections.
  • the combination according to the invention of several status markers in a common overall result means that a Distinguish between latent tuberculosis infection and active tuberculosis infection.
  • T cells considered in the blood sample can in particular be CD4+ T cells.
  • Such T-cells thus form a first total quantity or reference quantity as a reference quantity, in particular if the combination limit value specific for this reference quantity is set accordingly.
  • the individual marking results i.e. the respective quantity of T cells with the respective status marker and presence marker, to the total quantity of all T cells and accordingly an initial statement about the distinction between latent and active infection to obtain.
  • the two different reference values can also be used in parallel in the evaluation of the marker results, so that different results can also be generated in the evaluations with different reference values for one and the same status marker by using a different reference value in each case .
  • the method according to the invention can be based on a doubled or even greater number of marking results, as a result of which the security and in particular the specificity of the evaluation in the overall result can be increased even further.
  • the marking results of the marking steps are determined by analyzing the frequency of T cells in the cells provided with marked status markers and marked presence markers or by analyzing the marking strength of the status markers in the Have the cells made available with the marked presence marker evaluated and compared with a respective combination limit value.
  • a sub-sample can be diverted from a blood sample, for which sub-sample the method is carried out with a first stimulation means, while at least one further sub-sample is stimulated with a different stimulation means.
  • a further doubling, tripling or corresponding multiplication of the number of marking results can be achieved by using different bacterial antigens for stimulation.
  • the combination limit values used are specific to the respective status marker and/or specific to the reference variable used.
  • test series or therapy series can be used to evaluate which patients with a latent infection and which patients with an active infection are part of the therapy. If a method according to the invention is used in such patients in whom the knowledge of an activity status of the infection is available, a large number of patient data can be used to statistically evaluate which combination limit values for the respective status marker and/or the respective reference value used more appropriate security apply.
  • combination limit values determined in this way are stored and used in a specific way for the respective status marker and/or the respective reference variable during implementation.
  • the individual marking results are weighted in a method according to the invention when the overall result is formed. While in principle a purely qualitative transfer of the individual marking results is carried out in the overall result, the correspondingly different informative value of individual marking results can be taken into account by weighting. For example, an influence can be exerted on whether a marking result is very slightly above the respective combination limit value or whether there is a clear distance from the respective combination limit value. The clearer or more clearly the respective combination limit value is exceeded and/or fallen below, a weighting can be taken into account in the summary in the overall result. It is also possible that when the combination limit values are created, different status markers bring with them different strengths of information about the status of the respective infection situation. In this way, particularly meaningful marking results can be weighted more heavily than is the case for less meaningful status markers.
  • the overall result is formed quantitatively on the basis of a quantitative evaluation of the marker results.
  • a quantitative evaluation it is possible for a quantitative evaluation to be determined for each marking result. This is conceivable between 0 and 1, for example as a percentage parameter.
  • weightings can also be included here, as explained in the previous paragraph.
  • the marking results are checked for usability in a method according to the invention. It can happen that individual marking results lead to an implausible interpretation or the usability is questionable for other reasons. Since there is now more than one marking result, such unusable marking results can be excluded from the evaluation in the overall result. Is this the If this is the case, the overall limit value is advantageously adapted to the type and/or the number of the excluded marking results.
  • the at least one presence marker specifically indicates the contact of the cells with at least one tuberculosis bacteria antigen in humans.
  • the presence marker on the T cells that is specific against tuberculosis antigens is expressed.
  • tuberculosis bacteria produce corresponding biological, chemical and/or biochemical reactions in the body. This is based in particular on a corresponding reaction of the human immune system.
  • the presence marker is stimulated specifically and without a second indication only in T cells, which in turn are specific for a tuberculosis infection. This is possible with a stimulation duration of 1 to 72 hours, of which in particular 1 to 48 hours, of which in particular 1 to 24 hours, of which in particular 1 to 12 hours, of which in particular 1 to 7 hours, of which in particular 4-6 hours.
  • the at least one status marker has at least one activity parameter for the activation status of the T cells in the blood sample.
  • the activation status of the T cells in humans they have corresponding status markers on the surface.
  • Activated T cells can be distinguished from other T cells.
  • the activation and thus also the presence of a status marker is not included limited to tuberculosis infection. In combination with the at least one presence marker, however, the restriction to a tuberculosis infection can be made by means of the combination limit value.
  • the at least one status marker is changed by in vivo activation of the specific T cells
  • the above list is a non-exhaustive list.
  • the quantitative correlation allows an activity parameter to provide a quantitative and thus a probability statement as to whether the tuberculosis is active or latent.
  • the activity parameter is mapped into the combination limit. If the activity parameter is stable for the duration of the test, a sample that is easier to handle can be made available and, in particular, transport between the sampling location and the analysis location can also be made available. Since the status marker is not generated by its own stimulation in the method, but is already in it when the blood sample is taken, this stability over time ensures greater flexibility in the application of the method according to the invention.
  • a limit value is defined for the combination limit value, and if this limit is exceeded, an active tuberculosis disease is detected.
  • This limit value is preferably provided with a safety margin in order to have the corresponding safety for the subsequent treatment in the event of a positive test result.
  • the combination limit value has at least two limit values with different probabilities for the detection of an active tuberculosis disease.
  • the at least one presence marker and the at least one status marker are marked at the same time, at least in sections.
  • the marking takes place simultaneously or substantially simultaneously. It is irrelevant whether a joint marking mixture is to be used for marking both markers or separate marking agents are to be used.
  • the parallel configuration of this marking step, at least in sections, further reduces the time required for a method according to the invention and the corresponding costs.
  • the at least one presence marker and the at least one status marker are stained with the same marker mixture. This is the case in particular in combination with the embodiment according to the previous paragraph.
  • At least some of the cells made available from the blood sample are stimulated and/or at least some of the cells made available from the blood sample remain unstimulated.
  • the stimulation-free part of the blood sample in particular remains as a negative control in order to be available later as an analysis to verify the test result.
  • at least some of the cells provided from the blood sample are stimulated with a control stimulant.
  • the stimulation of the blood sample is in particular biological, chemical and/or biochemical. In a qualitative and/or quantitative manner, it can provide reinforcement or simplification in the analysis according to the invention.
  • Such a negative control is in particular combined with such a positive control.
  • a further advantage can be achieved if, in a method according to the invention, the cells are made available from a blood sample with peripheral blood. This further reduces the invasiveness of the necessary preliminary procedure for preparing the blood sample.
  • the blood sample can be made available in a quick and simple manner and can serve as the starting point for a method according to the invention.
  • a further advantage can be achieved if, in a method according to the invention, the steps of labeling are carried out on the cells provided without permeabilization, insofar as this is possible. It is therefore not necessary to loosen or open the cell walls of the cells provided, so that the preparation effort when carrying out a method according to the invention can be reduced. The time required for the analysis can also be reduced. In this way, a further simplification and reduction in complexity for a method according to the invention can be made available.
  • FIG. 7 shows the number of T-cells according to FIG. 6 with a reference value
  • Fig. 8 shows the set of T-cells of Figs. 6 and 7 with different reference values
  • Fig. 9 shows the set according to Fig. 8 with an evaluation with respect to another
  • FIG. 12 shows a higher-level comparison step.
  • 1 shows schematically how cells (110) provided from a peripheral blood sample 100 are stimulated with bacterial antigens BA.
  • Marking means FM either in the form of a mixture of marking means or different marking means FM, can be used to mark presence markers AM and status markers SM1, SM2, SM3 on/in the cells 110 provided.
  • this marking is evaluated in an evaluation unit 200, so that the combination limit value KG1, KG2, KG3 can be analyzed qualitatively and/or quantitatively.
  • FIG. 3 and 4 shows schematically how the corresponding limits for the presence marker AM and the respective status marker SM1, SM2, SM3 can be formed.
  • Two blood sample analysis results can be seen in FIG. While the analysis on the left is a combination limit value KG with a single limit value, FIG. 4 shows a combination limit value KG with two individual partial limit values.
  • the combination of the two markers AM and SM1 is above the combination limit value KG, so that the presence of an active tuberculosis disease can be affirmed here. Accordingly, the sample on the left designates a negative test, since the combination of the two markers AM and SM1 is below the combination limit KG.
  • FIG. 4 Three analysis results can now be seen in FIG. 4 .
  • additional information can be made available above the combination limit value KG. If the combination of the two markers AM and SM1 is below a lower limit of the combination limit KG, latent tuberculosis can be detected. If the value is above the upper limit value of the combination limit value KG, an active tuberculosis disease can be assumed. If the value for the combination of the two markers AM and SM1 is within the two specified limit values, then a further detailed analysis is necessary or a correspondingly reduced probability is linked to the statement.
  • FIG. 2 shows a further embodiment of a method according to the invention, in which the cells 110 made available are divided up. While for further processing and marking with the marker FM here a stimulation is carried out using a stimulating agent BA, a negative sample is diverted beforehand from the cells 110 provided. It is also possible to subject such a split sample to the method, but with a different bacterial antigen for stimulation or a control stimulant.
  • FIG. 5 shows schematically how different cell types are characterized in the following figures.
  • a cell is shown as a circular shape. If the cell is a T cell 112, in particular a CD4+ T cell 112, a corresponding protein represented as a diamond can be seen in the middle of the top line of FIG.
  • a rectangular protein is found on the surface of the cell as the presence marker AM at the right-hand end of the top line in FIG. 5, which schematically represents a presence marker AM.
  • the bottom line of FIG. 5 now shows possible combinations of status markers SM1, SM2.
  • a triangle is shown here as the first status marker SM1 and a circle is shown as the second status marker SM2.
  • these two status markers SM1 and SM2 can also occur in combination with one another in a T-cell 112 in the bottom line on the far right.
  • a presence marker AM is induced in T cells 112 by stimulation with bacterial antigens BA.
  • T cells 112 in particular CD4+ T cells 112, which have already been exposed to the respective disease bacterium, Presence marker AM, ie cells of the structures in the top row on the far right in FIG. 5 develop.
  • Presence marker AM ie cells of the structures in the top row on the far right in FIG. 5 develop.
  • the activity is then evaluated in the form of status markers SM1, SM2 or SM3 being marked, as shown in FIG.
  • T-cells 112 The set of all cells which are defined as T-cells 112 is shown schematically in FIG. 6 . These are presence markers AM-free T-cells 112, T-cells 12 which have a presence marker AM and T-cells 112 with presence marker AM and at least one status marker SM1, SM2 or SM3.
  • FIG. 7 shows a base set as a reference variable BG, which here represents the marked T cells 112 with the presence marker AM.
  • BG represents the marked T cells 112 with the presence marker AM.
  • the second status marker SM2 is evaluated according to FIG. 8, the result is the set of cells 110 shown in FIG. 8, which have both the presence marker AM in the form of a rectangle and the second status marker SM2 in the form of a circle.
  • this quantity must now be related to a reference variable BG, which enables the quantitative evaluation.
  • two different reference values BG are shown.
  • the said set of T-cells 112 with second status marker SM2 and presence marker AM represents a subset of an intermediate set of all T-cells 112 with presence marker AM as first reference variable BG1.
  • Two marking results ME2 can therefore be distinguished from one another in this evaluation, depending on which quantitative reference variable BG is referred to.
  • FIG. 9 shows the similar evaluation, but with reference to the status marker SM1.
  • a division into two different reference values BG is selected as the first marking result ME1, namely the large reference value BG2 as all T-cells 112 and the smaller intermediate set as reference value BG1 for all T-cells 112 with presence marker AM.
  • FIG. 10 shows the result of a method according to the invention as an overview matrix. Based on the explanation for FIGS. 6 to 9, an evaluation of the upper left half of the matrix would be provided here. So who the two different reference values BG1 and BG2 combined with two different status markers SM1 and SM2. If a further, third status marker SM3 is also evaluated, then the matrix expands to the three columns, as shown in FIG. In addition, Fig. 10 is down again by two more Rows are doubled by stimulating in different ways with two different bacterial antigens BA1 and BA2. The same allocations to the three different status markers SM1, SM2 and SM3 and the two different reference variables BZ1 and BZ2 are again selected for both bacterial antigens BA1 and BA2. In total, there is a matrix of 4x3 individual analyses, so that a total of twelve individual marking results ME1, ME2, ME3... can be evaluated.
  • FIG. 10 also shows that a specific limit can be used as a combination limit value KG1, KG2 and/or KG3 for each individual marking result ME1, ME2, ME3. This applies to all reference values BG1, BG2, ... and all bacterial antigens BA1, BA2, ... . It can be seen in FIG. 10 that in the case of one and the same blood sample, a division into a total of twelve different marker results ME is made available, which in total can have a different individual characteristic.
  • a qualitative evaluation is selected for the evaluation in such a way that if the respective combination limit value KG1, KG2 or KG3 is exceeded, the respective measurement is evaluated as positive, while if the respective combination limit value KG1, KG2 and KG3 is not reached, the measurement result is considered negative.
  • Positive marker results ME1 , ME2 and ME3 receive a 1 and negative ones a 0.
  • FIG. 11 shows an overview of the individual results as an evaluation and summary in an overall result GE.
  • the overall result GE and thus also the overall score here is 7.
  • This can now be compared with an overall limit value GG in a higher-level comparison step, as shown in FIG.
  • the overall limit value GG is now 5, so that as an overall result GE of 7 exceeding this overall limit value GG of 5 leads to the detection of an active bacterial infection.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'analyse d'un échantillon de sang issu d'un être humain pour détecter une maladie, en particulier une maladie causée par des bactéries, comportant les étapes consistant à: - stimuler des cellules fournies à partir de l'échantillon de sang avec des antigènes bactériens pour induire au moins un marqueur de présence sur des lymphocytes T des cellules fournies, - étiqueter au moins un des marqueurs de présence induits sur des lymphocytes T dans les cellules fournies, ledit marqueur de présence étant spécifiquement formé sur des lymphocytes T qui ont reconnu l'antigène bactérien pendant la stimulation, - étiqueter un premier marqueur d'état et un marqueur d'état supplémentaire sur des lymphocytes T dans les cellules fournies qui sont spécifiques à leur état d'activation, lesdits marqueurs d'état différant les uns des autres et du marqueur de présence, - évaluer un premier événement d'étiquetage des étapes d'étiquetage par une analyse de la fréquence de lymphocytes T présentant un premier marqueur d'état étiqueté et un marqueur de présence étiqueté d'après une valeur de référence et effectuer une comparaison avec une première limite de combinaison, - évaluer au moins un événement d'étiquetage supplémentaire des étapes d'étiquetage par une analyse de la fréquence de lymphocytes T présentant un marqueur d'état supplémentaire étiqueté et un marqueur de présence étiqueté d'après une valeur de référence et effectuer une comparaison avec une limite supplémentaire de combinaison, - former un résultat global à partir des événements d'étiquetage évalués et comparer le résultat global avec une limite globale.
EP22718983.4A 2021-04-08 2022-03-30 Procédé d'analyse d'un échantillon de sang pour détecter une maladie Pending EP4320437A1 (fr)

Applications Claiming Priority (2)

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DE102021203480.2A DE102021203480A1 (de) 2021-04-08 2021-04-08 Verfahren für die Analyse einer Blutprobe auf eine Erkrankung
PCT/EP2022/058377 WO2022214371A1 (fr) 2021-04-08 2022-03-30 Procédé d'analyse d'un échantillon de sang pour détecter une maladie

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EP4320437A1 true EP4320437A1 (fr) 2024-02-14

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EP (1) EP4320437A1 (fr)
DE (1) DE102021203480A1 (fr)
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ZA (1) ZA202309330B (fr)

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Publication number Priority date Publication date Assignee Title
EP3139922B1 (fr) 2014-05-05 2019-08-14 Emory University Méthodes de diagnostic et de traitement de la tuberculose
EP3555630B1 (fr) * 2016-12-14 2023-05-31 Becton, Dickinson and Company Procédés et compositions permettant d'obtenir une évaluation de la tuberculose chez un sujet
US20200408771A1 (en) * 2018-02-22 2020-12-31 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Cd153 and/or cd30 in infection
DE102018131696B4 (de) * 2018-12-11 2020-09-17 Labor Berlin - Charité Vivantes Services GmbH Verfahren für die Analyse einer Blutprobe eines Menschen auf eine Tuberkuloseerkrankung

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ZA202309330B (en) 2024-05-30
DE102021203480A1 (de) 2022-10-13
WO2022214371A1 (fr) 2022-10-13

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