EP4320437A1

EP4320437A1 - Method for analyzing a blood sample for a disease

Info

Publication number: EP4320437A1
Application number: EP22718983.4A
Authority: EP
Inventors: Andrej MANTEI; Christian Meisel; Tim Meyer; Alexander Scheffold
Original assignee: Labor Berlin Charite Vivantes Services GmbH
Current assignee: Labor Berlin Charite Vivantes Services GmbH
Priority date: 2021-04-08
Filing date: 2022-03-30
Publication date: 2024-02-14
Also published as: DE102021203480A1; WO2022214371A1

Abstract

The present invention relates to a method for analyzing a blood sample from a human for a disease, especially a bacteria-based disease, comprising: - stimulating provided cells from the blood sample with bacterial antigens to induce at least one presence marker on T cells of the provided cells, - labeling at least one of the induced presence markers on T cells in the provided cells, which presence marker is specifically formed on T cells which recognized bacterial antigen during the stimulation, - labeling a first status marker and a further status marker on T cells in the provided cells that are specific for the activation status thereof, said status markers differing from one another and from the presence marker, - evaluating a first labeling event of the labeling steps by analysis of the frequency of T cells having a labeled first status marker and a labeled presence marker based on a reference value and making a comparison with a first combination limit, - evaluating at least one further labeling event of the labeling steps by analysis of the frequency of T cells having a labeled further status marker and a labeled presence marker based on a reference value and making a comparison with a further combination limit, - forming an overall result from the evaluated labeling events and comparing the overall result with an overall limit.

Description

PROCEDURE FOR ANALYZING A BLOOD SAMPLE FOR DISEASE

The present invention relates to a method for analyzing a blood sample from a human for a disease, caused in particular by bacteria.

There are bacterial diseases, such as tuberculosis, which can be divided into a latent disease and an active disease. Tuberculosis is a chronic infection caused by mycobacteria of the Mycobacterium tuberculosis complex (MTBC). In most cases, tuberculosis infections in humans are caused by M. tuberculosis sensu stricto and M. africanum. In humans, an infection with tuberculosis bacteria leads to a tuberculosis disease or active tuberculosis infection in only about 10% of cases. In 90% of the cases there is no disease, one speaks of a latent tuberculosis infection (LTBI). In this case too, tuberculosis bacteria remain in humans. A transition from latent to active tuberculosis infection and thus disease is possible. In order to select a tuberculosis therapy or to verify the success of the therapy, the clearest possible proof of a tuberculosis disease is necessary. In the case of any known diagnostic method, 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.

Culture or nucleic acid amplification tests, and thus clear evidence of tuberculosis bacteria from patient material, have been required for clear evidence of an active tuberculosis disease. However, these methods have significant disadvantages. They are often associated with an invasive procedure to obtain the appropriate tissue sample. In addition, the detection of an active tuberculosis disease by culture of tuberculosis bacteria can take several weeks and thus entails disadvantages in terms of time and costs, as well as a delayed start of treatment. One The decision as to whether tuberculosis therapy is carried out is therefore often based on clinical experience or monitoring of the patient, or therapy must be carried out without there being any possibility of differentiation.

It is the object of the present invention to at least partially eliminate the disadvantages described above. In particular, it is the object of the present inventions to make it possible to distinguish between latent and active disease in a cost-effective and simple manner with minimal invasiveness by analyzing a blood sample.

The above object is achieved by methods for analyzing a blood sample from a human having the features of claim 1 and claim 2. Further features and details of the invention result from the dependent claims, the description and the drawings. Features and details that are described in connection with the methods according to claims 1 and 2 also apply, of course, in connection with the method according to the subclaims and vice versa, so that the disclosure of the individual aspects of the invention is always referred to or mutually referenced . can be.

According to the invention, the method is used to analyze a blood sample from a human for a disease, in particular based on bacteria, having the following steps:

- providing cells from the blood sample,

- stimulation of the cells provided with bacterial antigens to induce at least one presence marker on T cells of the cells provided,

- Labeling of at least one of the induced presence markers on T cells in the cells provided, which is formed specifically on T cells that have recognized the bacterial antigen during the stimulation, - marking of a first status marker on T-cells in the cells provided, which is specific to their activation status and differs from at least one presence marker,

- Marking of at least one further status marker on T-cells in the cells provided, which is specific to their activation status and differs from the at least one presence marker and the first status marker,

- Evaluation of a first marking result of the marking steps by analyzing the frequency of T-cells with marked first status marker and marked presence marker based on a reference variable and comparison with a first combination limit value,

- Evaluation of at least one further marking result of the marking steps by analyzing the frequency of T cells with marked further status markers and marked presence markers based on a reference value and comparison with a further combination limit value,

- Forming an overall result from the evaluated marker results and comparing the overall result with an overall limit value.

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:

- providing cells from the blood sample,

- stimulation of the cells provided with bacterial antigens to induce at least one presence marker on T cells of the cells provided, - Labeling of at least one of the induced presence markers on T cells in the cells provided, which is formed specifically on T cells that have recognized the bacterial antigen during the stimulation,

- marking of a first status marker on T-cells in the cells provided, which is specific to their activation status and differs from at least one presence marker,

- Evaluation of a first marking result of the marking steps by analyzing the frequency of T cells with marked first status marker and marked presence marker based on a reference variable,

- Evaluating at least one further marking result (ME2, ME3) of the marking steps by analyzing the frequency of T-cells (112) with marked further status markers (SM2, SM3) and marked presence markers (AM) based on a reference value,

- Forming an overall result from the evaluated marker results by means of a weighting relationship, having weighting factors for each marking result, and

- Comparison of the overall result with an overall limit value.

Based on the known methods, an infection, in particular a bacterial infection, in a blood sample from a person is to be detected and classified here as well. In principle, however, a method according to the invention can also be used for other diseases caused by pathogens. These include, for example, pathogens in the form of viruses, fungi or parasites, which have a pathogen-specific immune response. For this purpose, cells from a blood sample (e.g. in the form of isolated mononuclear cells or whole blood) are used in a first step provided. 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.

A core idea of the present invention is based on the analysis of T-cells (e.g. CD4+ T-cells), which are part of the cells provided. For this purpose, the cells provided are stimulated with 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.

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.

In 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.

In these two steps, the presence marker can be used to draw conclusions about the presence of bacteria, and 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. In a further step, in order to be able to make the actual statement as to whether an active bacterial disease is present, 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. In other words, marked cells that carry a combination of at least one presence marker and at least one status marker can now be identified and evaluated in the set of cells made available. 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. This means that the second step of marking status markers is carried out for at least two different status markers. This means that in a first marking step, the presence markers are marked on the cells. In a second step, all T cells are marked with a presence marker and a first status marker, and in a third marking step, T cells are marked with a presence marker and a further status marker. In other words, 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.

Within the blood sample and the cells provided are 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. In the same way, it can be assumed that 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.

In summary, specific marking steps are carried out for different status markers, so that a marking result can also be made available for each status marker. An evaluation is possible for each marking result by normalizing the marking result using a reference value. The result of this evaluation is a comparison with a specific combination limit, which is specific to the respective status marker and the reference value. These specific combination limit values are therefore preferably designed differently and are specific to the respective marking result and the reference variable from a qualitative and/or quantitative point of view.

In a final step according to the invention, the individual marking results and their evaluations are now summarized in 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. In the simplest case, 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. This is how this example goes using three different status markers. Accordingly, three marking steps are carried out specifically for each of these different status markers. In this 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, while 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. In accordance with the invention, an overall result is now generated on the basis of these individual results. This can be done in the simplest way by adding the three qualitative individual marking results, so that the overall result is 0 + 1 + 1 = 2. In this case, for example, 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.

In the context of the present invention, 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. In addition, 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) can be used for the stimulation.

In order to be able to distinguish between latent and active disease, information is also required as to how many cells that carry the presence marker (and here are specific for tuberculosis antigen) also carry one of the status markers or to what extent these in the cells are present. During a latent tuberculosis infection, 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). After transition to an active tuberculosis infection, characterized among other things by intensive replication of the tuberculosis bacteria, there is intensive contact and activation of the patient's immune system, in particular the tuberculosis-specific 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.

As can be seen from the above explanations, 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. With the avoidance of an invasive procedure, 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.

For the variant of the method according to claim 2, the overall result is formed in a slightly modified manner. There is no comparison with 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. In particular, it can be assumed that high frequency values for all marking results show the same direction, for example for an active disease. In addition, 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.

It is also advantageous if 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.

It is also advantageous if at least the amount of all T cells, in particular all CD4+ T cells, in the blood sample is used as a reference value in a method according to the invention for the evaluation of the labeling results. The 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. In this way, it is possible to relate 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.

In addition or as an alternative to the embodiment of the previous paragraph, there can be advantages if, in a method according to the invention for the evaluation of the marking results, at least the amount of all T cells, in particular all CD4+ T cells, with presence markers in the blood sample is used as a reference value becomes. Of all the T cells present in the blood sample, only a portion will also carry the presence marker. Here, too, it is now possible to relate the marker results, i.e. the set of all cells that carry a presence marker and specifically the respective status marker, to a reduced reference set as a reference value, namely in this case as a reference value all T cells, in particular all CD4+ T cells that also have a presence marker (e.g., CD154). It should be explicitly pointed out that 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 . As a result, 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. It can bring further advantages if, in a method according to the invention, 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. This is a particularly advantageous solution to the method, in which a choice can be made between two different reference values. A combination of different reference values is also possible here, as explained in the two previous paragraphs.

It can also bring advantages if, in a method according to the invention, this is carried out with at least two different bacterial antigens in separate stimulation steps. For example, 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. Thus, a further doubling, tripling or corresponding multiplication of the number of marking results can be achieved by using different bacterial antigens for stimulation. Here, too, it is possible to increase the total number of marking results, so that the significance of the summary in the overall result for the number of marking results increased in this way can be increased accordingly.

It is also advantageous if, in a method according to the invention, the combination limit values used are specific to the respective status marker and/or specific to the reference variable used. For example, 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. For a method according to the invention, 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.

In addition, it is advantageous if 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.

There are further advantages if, in a method according to the invention, the overall result is formed quantitatively on the basis of a quantitative evaluation of the marker results. For example, 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. Of course, weightings can also be included here, as explained in the previous paragraph.

It is also advantageous if 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.

Further advantages can be achieved if, in a method according to the invention, if a marking result is found to be unusable, it is excluded from the formation of the overall result and the overall limit value is adapted to this exclusion. As has already been explained in the previous paragraph, this takes place either with reference to the maximum qualitative and/or the maximum quantitative evaluation of the excluded marking result. From a qualitative and/or quantitative point of view, the overall limit value can also be reduced by the amount that is no longer available as an overall result for the overall score as a result of the exclusion.

It is also advantageous if, in a method according to the invention, the at least one presence marker specifically indicates the contact of the cells with at least one tuberculosis bacteria antigen in humans. This means in particular that, after in vitro stimulation, the presence marker on the T cells that is specific against tuberculosis antigens is expressed. As has already been explained, 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. What is decisive in this embodiment is that 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.

It is also advantageous if, in a method according to the invention, the at least one status marker has at least one activity parameter for the activation status of the T cells in the blood sample. Depending on 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.

It can also be advantageous if the status marker has at least one of the following configurations in a method according to the invention:

- quantitative correlation with activation status,

- the at least one status marker is changed by in vivo activation of the specific T cells,

- the change caused by infection in vivo is not changed for the duration of the test and by the stimulation conditions in vitro,

- Independence from the step of marking and/or the step of evaluating the marking result of the presence marker.

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. In particular, 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.

In addition, there are advantages if, in a 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. In other words, it is possible in this way to specify a preferably exact limit value, which distinguishes between a positive and a negative result in the method according to the invention. 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. It is also advantageous if, in a method according to the invention, the combination limit value has at least two limit values with different probabilities for the detection of an active tuberculosis disease. Not only can a distinction be made between a negative and a positive test result, but different probabilities can be assumed, particularly in the case of a positive result. For example, if the test result is between the two limit values, the probability of contracting tuberculosis is lower than if both limit values are exceeded. A quantitative statement is thus possible, at least in stages, with regard to the probability of the test procedure.

It can be a further advantage if, in a method according to the invention, the at least one presence marker and the at least one status marker are marked at the same time, at least in sections. Preferably 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.

In addition, it can be advantageous if, in a method according to the invention, 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.

It is also advantageous if, in a method according to the invention, 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. This means that 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. Furthermore, it is advantageous if, in a method according to the invention, at least some of the cells provided from the blood sample are stimulated with a control stimulant. This means that in particular the one with the Control stimulant-stimulated part of the blood sample remains as a positive control in order to be available later as an analysis to verify the test result. 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. In particular, 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.

It is also advantageous if at least one of the following is used as a presence marker in a method according to the invention:

- Surface markers, especially CD154

It is also advantageous if at least one of the following is used as a status marker in a method according to the invention:

- CD38

- HLA-DR

- Ki-67

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. Further advantages, features and details of the invention emerge from the following description, in which exemplary embodiments of the invention are described in detail with reference to the drawings. The features mentioned in the claims and in the description can each be essential to the invention individually or in any combination. They show schematically:

1 shows an embodiment of a method according to the invention,

2 shows a further embodiment of a method according to the invention,

3 shows an embodiment in the evaluation of a presence marker and

4 shows a further embodiment in the evaluation of a status marker according to the invention,

5 shows a schematic representation of cells with different markers,

6 shows a schematic representation of a set of T cells,

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

status marker,

10 shows an overview matrix of the result of a method according to the invention,

11 shows an overview of individual results and an overall result, and

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. Finally, 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.

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. In the right sample in FIG. 3, 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.

Three analysis results can now be seen in FIG. 4 . In the presence of a tuberculosis disease, ie in the representation according to FIG. 3 in the right sample, 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. In such a case, for example, this result could be excluded from the further course of the procedure as not usable. 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. In FIG. 5, starting from the top left, 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. Of course, 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.

If a method according to the invention is carried out, a presence marker AM is induced in T cells 112 by stimulation with bacterial antigens BA. In cells such as are shown in the middle of the upper line of FIG. 5, if they are 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. The activity is then evaluated in the form of status markers SM1, SM2 or SM3 being marked, as shown in FIG.

The results of the method can now be presented as follows. 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.

Starting from the set of FIG. 6, FIG. 7 shows a base set as a reference variable BG, which here represents the marked T cells 112 with the presence marker AM. If 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. For the evaluation of the marking result ME2, this quantity must now be related to a reference variable BG, which enables the quantitative evaluation. In the embodiment of FIG. 8, two different reference values BG are shown. The recognized and marked set of all T-cells 112 with status marker SM2 and presence marker AM in Fig. 8 represents a proportion of the total set of all T-cells 112 as a second reference value BG2. At the same time, 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.

9 shows the similar evaluation, but with reference to the status marker SM1. Here, too, 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.

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.

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. In this example, 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.

11 shows an overview of the individual results as an evaluation and summary in an overall result GE. By simply listing the individual results, 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. For example, in this embodiment 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.

In the above explanation of the embodiments, the present invention is described only by way of examples. It goes without saying that individual features of the embodiments can be freely combined with one another, provided this makes technical sense, without departing from the scope of the present invention. Reference List

100 blood sample 110 cells 112 T cells 200 evaluation unit

BA bacterial antigen BA1 first bacterial antigen BA2 second bacterial antigen FM marker

AM presence marker SM1 first status marker SM2 second status marker SM3 third status marker

ME1 first marking result ME2 second marking result ME3 third marking result GE overall result

BG reference variable BG1 first reference variable BG2 second reference variable

KG1 first combination limit value KG2 second combination limit value KG3 third combination limit value GG total limit value

Claims

patent claims

1. A method for analyzing a blood sample (100) from a human for a disease, in particular based on bacteria, comprising the following steps:

- providing cells (110) from the blood sample (100),

- stimulation of the cells (110) provided with bacterial antigens (BA) to induce at least one presence marker (AM) on T cells (112) of the cells (110) provided,

- Marking of at least one of the induced presence markers (AM) on T-cells (112) in the provided cells (110), which is specifically formed on T-cells (112), the bacterial antigen (BA) during have recognized the stimulation

- Marking a first status marker (SM 1) on T-cells (112) in the provided cells (110), which is specific to their activation status and differs from at least one presence marker (AM),

- Marking of at least one further status marker (SM2, SM3) on T-cells (112) in the provided cells (110), which is specific to their activation status and differs from the at least one presence marker (AM) and the first status marker ( SM1) distinguishes

- Evaluation of a first marking result (ME1) of the marking steps by analyzing the frequency of T-cells (112) with marked first status marker (SM1) and marked presence marker (AM) related to a reference variable (BG) and comparison with a first combination limit value ( KG1),

- Evaluation of at least one further marking result (ME2, ME3) of the marking steps by analyzing the frequency of T-cells (112) with marked further status markers (SM2, SM3) and marked Presence marker (AM) based on a reference variable (BG) and comparison with another combination limit value (KG2, KG3),

- Forming an overall result (GE) from the evaluated marking results (ME1, ME2, ME3) and comparing the overall result ses (GE) with an overall limit value (GG).

2. Method for analyzing a blood sample (100) from a human for a disease, in particular based on bacteria, comprising the following steps:

- providing cells (110) from the blood sample (100),

- Evaluation of a first marking result (ME1) of the marking steps by analyzing the frequency of T-cells (112) with a marked first Status marker (SM1) and marked presence marker (AM) related to a reference value (BG),

- Evaluating at least one further marking result (ME2, ME3) of the marking steps by analyzing the frequency of T-cells (112) with marked further status markers (SM2, SM3) and marked presence markers (AM) based on a reference variable (BG),

- forming an overall result (GE) from the evaluated marking results (ME1, ME2, ME3) by means of a weighting relationship, having weighting factors for each marking result, and

- Comparison of the overall result (GE) with an overall limit value (GG).

3. The method as claimed in one of the preceding claims, characterized in that it is designed for the analysis of a tuberculosis disease based on tuberculosis bacteria, in that antigens of the tuberculosis pathogen are used as the bacterial antigen (BA).

4. The method according to any one of the preceding claims, characterized in that for the evaluation of the marking results (ME1, ME2, ME3) at least the amount of all T cells (112) in the blood sample (100), in particular all CD4+ T cells ( 112) is used as a reference variable (BG).

5. The method according to any one of the preceding claims, characterized in that for the evaluation of the marking results (ME1, ME2, ME3) at least the amount of all T cells (112), in particular all CD4 + T cells (112), with presence marker ( AM) in the blood sample (100) is used as a reference variable (BG).

6. Method according to one of the preceding claims, characterized in that the marking results (ME1, ME2, ME3) of the marking steps are generated by analyzing the frequency of T-cells (112) in the available cells (110) provided with marked status markers (SM1, SM2, SM3) and marked presence markers (AM) or by analyzing the marking strength of the status markers (SM1, SM2, SM3) in the cells (110) provided with marked presence markers (AM ) and in particular comparison with a respective combination limit value (KG1, KG2, KG3).

7. The method according to any one of the preceding claims, characterized in that it is carried out with at least two different bacterial antigens (BA) in separate stimulation steps.

8. Method according to one of the preceding claims, characterized in that the combination limit values (KG1, KG2, KG3) used are specific to the respective status marker (SM1, SM2, SM3) and/or specific to the reference variable (BZ) used.

9. Method according to one of the preceding claims, characterized in that the individual marking results (ME1, ME2, ME3) are weighted when the overall result (GE) is formed.

10. The method as claimed in one of the preceding claims, characterized in that the overall result (GE) is formed quantitatively on the basis of a quantitative evaluation of the marking results (ME1, ME2, ME3).

11. Method according to one of the preceding claims, characterized in that the marking results (ME1, ME2, ME3) are checked for usability.

12. The method according to claim 11, characterized in that if a marking result (ME1, ME2, ME3) is found to be unusable, it is excluded from the formation of the overall result (GE), with the overall limit value (GG) being adjusted to this exclusion.

13. The method according to any one of the preceding claims, characterized in that the at least one presence marker (AM) during the in vitro Stimulation with bacterial antigen (BA) is only formed by T cells (112) that have specifically recognized the antigen during stimulation

14. The method according to any one of the preceding claims, characterized in that the at least one status marker (SM) correlates with the activation status of the T cells (112) as a measure of the infection status.

15. The method according to claim 14, characterized in that the status marker (SM1, SM2, SM3) has at least one of the following configurations:

- The at least one status marker (SM1, SM2, SM3) is changed by in vivo activation of the specific T cells (112).

- the change induced in vivo by infection is not altered for the duration of the test and by the in vitro stimulation conditions

- Independence from the step of marking and/or the step of evaluating the marking result (ME1, ME2, ME3) of the presence marker (AM)

16. The method as claimed in one of the preceding claims, characterized in that a limit value is defined for the combination limit value (KG1, KG2, KG3) and an active disease is recognized when this limit is exceeded.

17. The method according to claim 16, characterized in that the combination limit value (KG1, KG2, KG3) has at least two limit values with different probabilities for the detection of an active disease.

18. The method as claimed in one of the preceding claims, characterized in that the at least one presence marker (AM) and the at least one status marker (SM1, SM2, SM3) are marked at least in sections at the same time.

19. The method according to any one of the preceding claims, characterized in that the marking of the at least one presence marker (AM) and the at least one status marker (SM1, SM2, SM3) takes place with the same marking mixture.

20. The method according to any one of the preceding claims, characterized in that at least some of the cells (110) provided from the blood sample (100) are stimulated and/or at least some of the cells (110) provided from the blood sample are stimulated (100) remain unstimulated.

21. The method according to any one of the preceding claims, characterized in that the cells (110) are made available from a blood sample (100) with peripheral blood.

22. The method according to any one of the preceding claims, characterized in that at least one of the following is used as a presence marker (AM):

- Surface markers, especially CD154

23. The method according to any one of the preceding claims, characterized in that at least one of the following is used as a status marker (SM1, SM2, SM3):

- CD38

- HLA-DR

- Ki-67

24. The method according to any one of the preceding claims, characterized in that the steps of marking are carried out without permeabilization on the cells (110) provided.