Title - PROCEDURE FOR DETERMINING THE CONCENTRATION OF EPITHELIAL CELLS IN A BLOOD SAMPLE OR ASPIRATE SAMPLE

Abstract

A procedure for determining the concentration of epithelial cells in a blood sample or aspirate from a human or mammal, mixed with an anticoagulant. In this procedure, the sample is incubated after the addition of antibodies, antibody fragments, or antibody mimetics, each directed against a specific epithelial cell antigen, until a decreasing rate of binding of the antibodies, antibody fragments, or antibody mimetics to the cells is achieved. Only then is the number of labeled cells and their original concentration in the blood sample or aspirate determined.

Description

PROCEDURE FOR DETERMINING THE CONCENTRATION OF EPITHELIAL CELLS IN A BLOOD SAMPLE OR SAMPLE VACUUMING The invention relates to a method for determining the concentration of epithelial cells in a blood sample or aspirate sample from a human or mammal, mixed with an anticoagulant. In the case of the aspirate sample, this may be a bone marrow aspirate, a pleural aspirate, or a peritoneal aspirate. A procedure of this type is described in US patent 7,615,358 B2. In this procedure, epithelial tumor cells contained in a body fluid are labeled by the addition of antibodies or antibody fragments, where the antibodies or antibody fragments are directed against the human epithelial antigen, which is recognized by the monoclonal antibody HEA 125. The sample is then placed on a support and incubated to allow the tumor cells to adhere to the support. The surface of the support may be coated with an agent that promotes nonspecific cell adhesion, such as poly-Lysine. Cell adhesion usually lasts 10 to 15 minutes. Viable cells are then identified and quantified among the adherent epithelial tumor cells by their morphology, and the concentration of vital epithelial tumor cells in the body fluid is calculated.In this case, the morphology of adherent epithelial tumor cells is analyzed by means of t. Laser scanning cytometry, detecting live tumor cells by their unique surface staining and excluding dead cells due to intracellular staining. WO 2014 / 047285 A procedure is known for the detection and / or treatment of a subgroup of prostate cancer patients who may benefit from taxane therapy. In this case, a sample taken from the patient is tested for the presence of an androgen receptor splicing variant. This can be achieved by capturing circulating tumor cells from the sample and testing them for the presence of a specific splicing variant. Capture can be performed using immobilized antibodies. The assay involves contacting the sample with an antibody directed against the splicing variant and detecting the binding of the antibody to the splicing variant. A procedure for determining the IGF-1 and VEGF-2 receptors in circulating epithelial tumor cells in blood samples from breast cancer patients is known from Pizón, M. et al., PLOS ONE, February 2013, Volume 8, Issue 2, e56836, Pages 1-6. In this procedure, blood samples were prepared using EDTA as an anticoagulant within 48 hours of collection. Erythrocytes in the blood sample were lysed. The remaining cells were incubated overnight at 4°C with either PE-labeled mouse monoclonal antibodies directed against the VEGF-2 receptor, FITC-conjugated mouse antibodies directed against EpCAM, or PE-labeled mouse monoclonal antibodies directed against the IGF-1 receptor.A defined volume of the resulting cell suspensions was transferred into the cavities of the ELISA plates and measured using a laser scanning cytometer. De Hekimian, K. et al., Clin. Chem. Lab. Med. 2012; 50(4), pages 701-708, describes the unmasking of epithelial cell adhesion molecules (EpCAMs) in circulating epithelial tumor cells from breast cancer patients treated with Tween®2 0. The publication speculates that EpCAMs are masked by membrane glycoproteins or lipoproteins, thus preventing binding with antibodies. In one experiment, 1 ml of coagulation-inhibited peripheral blood was incubated for 5 minutes on the day of collection and on the first and second days post-collection with 20 μA of Tween®2 0 or without the detergent. The erythrocytes contained within were then used with an erythrocyte lysis buffer. The epithelial cells were subsequently detected with an antibody directed against EpCAMs.In this case, it was shown that with treatment using Tween®20 on the day of collection, and on days 1 and 2 post-collection, a similarly high number of cells could be detected within the margin of error. Without Tween®20, no cells could be detected on the day of collection, slightly fewer on the first day, and on the second day, as many cells as with the Tween®20 treatment. This demonstrates that storing blood samples before incubation with antibodies also increases EpCAM's accessibility to the cells for the antibodies. The object of the present invention is to indicate an alternative procedure for determining a concentration of epithelial tumor cells in a blood sample or aspirate sample from a human or mammal and mixing it with an anticoagulant agent. The object is solved by the features of patent claim 1. The suitable configurations result from the features of patent claims 2 to 15. The invention relates to a method for determining the concentration of epithelial cells in a blood sample or aspirate sample from a human or mammal and mixed with an anticoagulant. In this case, the sample is stored after the addition of antibodies, antibody fragments, or antibody mimetics, each directed against a specific antigen for epithelial cells, until the increase in the amount of antibodies bound to the cells, antibody fragments, or antibody mimetics slows down over time; that is, until the binding rate decreases or until a binding curve obtained by plotting binding over time begins to flatten and passes the saturation area.Only then is the number of labeled cells determined, and from this, the original concentration of these cells in the blood or aspirate sample. It was surprisingly shown that reaching the point of leveling off on the saturation curve takes much longer than would be expected for a given number of binding pairs. It is assumed that many of the antigens specific to epithelial cells are initially present in a masked state and only become accessible to binding by antibodies, antibody fragments, or antibody mimetics over time. Only then is a reliable determination of the cell count possible. Specifically, the procedure according to the invention for determining the concentration of epithelial cells in a blood sample or aspirate sample from a human or mammal and mixed with an anticoagulant comprises the following steps: a) lysation of the erythrocytes contained in the blood sample or aspirate sample by adding a buffer that causes lysation of the erythrocytes, separation of the unlysed cells in this case and suspension of the separated cells in a buffer, b) adding the antibodies, antibody fragments or antibody mimetics each bearing at least one tag, each directed against the epithelial cell adhesion molecule (EpCAM) and / or at least one epithelial cell-specific antigen, to a cell suspension obtained in step a) or a partial quantity obtained by separating this cell suspension, mixing the antibodies, antibody fragments or antibody mimetics and the cell suspension or the partial quantity and incubating a mixture thus obtained for at least a time necessary to achieve a decreasing rate of binding of the antibodies, antibody fragments or antibody mimetics to the cells, wherein the incubation is carried out for at least 12 hours, c) determination of the number of cells labeled by a binding of antibodies, antibody fragments or antibody mimetics in the mixture obtained in step d) and d) Calculation of the concentration of the amount of labeled cells determined in step c) in the blood sample or aspirate sample. The entire procedure is performed outside the human and animal body. The mixing of the antibodies, antibody fragments, or antibody mimetics, and the partial quantity according to step b), can be carried out directly after adding the antibodies, antibody fragments, or antibody mimetics to the partial quantity. Incubation according to step b), can be carried out at a predetermined temperature, for example, 0 to 20 °C, and especially 2 to 15 °C. In calculating the concentration of the amount of labeled cells determined in step c) in the blood sample or aspirate sample according to step d), a dilution possibly carried out previously and / or an increase in the concentration of the cells with respect to the blood sample or aspirate sample must be considered. Normally, epithelial cells are not found in circulating blood. However, it has been shown that circulating epithelial tumor cells are present in the blood of patients with malignant epithelial tumors, such as lung, breast, bowel, or prostate tumors. Furthermore, epithelial cells of epithelial tissue have been found in the peripheral blood of patients with chronic inflammatory conditions affecting epithelial tissue, such as rheumatoid arthritis, asthma, COPD, Crohn's disease, or ulcerative colitis. Beyond this, the cells Epithelial cells enter the peripheral bloodstream through events that damage epithelial tissue, such as surgery or trauma. Therefore, for diagnostic purposes in epithelial tumors or chronic inflammatory conditions of epithelial tissue, to monitor the course of a disease or healing process, and for predictive or mass screening, it is useful when the concentration of epithelial cells can be determined as precisely as possible in a blood sample or aspirate from a human or mammal. These cells may be from an epithelial tumor, epithelial cells from inflamed tissue, or other epithelial cells that entered the bloodstream through an event that damaged epithelial tissue. In the case of an anticoagulant agent, it could be, for example, ethylenediaminetetraacetate (EDTA), citrate or heparin. In the case of antibody fragments, these could be, for example, Fab', F(ab')2, or Fab. However, they could also be recombinant antibody fragments, such as scFv, di-scFv, sdAb (single-domain antibody), or chemically linked antibody fragments like F(ab')2. Antibody mimetics, on the other hand, are compounds that, like antibodies, can bind to antigens, but are not antibodies or antibody fragments themselves. These are mostly synthetic peptides, proteins, or lectins. The determination of the quantity of antibodies, antibody fragments or bound antibody mimetics according to step c) occurs as a function of time from the mixing according to step b) . The expert in the area of ​​research on the binding of antibodies, antibody fragments or antibody mimetics to substrates knows how to determine a binding rate.The inventors of the present invention recognized that the binding of antibodies, antibody fragments, or antibody mimetics labeled with a specific epithelial cell antigen to the surface of circulating epithelial cells, as expected, achieves saturation within a few minutes after their addition and mixing. However, saturation is only achieved after several hours. Since this cannot be based solely on the antigen-antibody reaction, it is assumed that there is greater accessibility of previously masked epitopes. Accessibility is already increased by storage prior to incubation with the antibodies, as known from Hekimian, K. et al. However, it is further increased by prolonged incubation in the presence of the antibodies, antibody fragments, or antibody mimetics. The reason for this could be that their binding results in the unmasking of the antigens.This effect is completely surprising to the expert. Between stages a) and b), the following subsequent stages can be carried out: a) separation of at least a partial quantity or other partial quantity of the cell suspension obtained in step a), a2) addition of antibodies, antibody fragments or antibody mimetics each bearing at least one label, each of which is directed against the epithelial cell adhesion molecule (EpCAM) and / or at least one epithelial cell-specific antigen, to another partial quantity in such quantity that its concentration in the other partial quantity is identical to the concentration of antibodies, antibody fragments or antibody mimetics in the cell suspension or the partial quantity according to step b), mixing the antibodies, antibody fragments or antibody mimetics and the further partial quantity and incubating a mixture thus obtained at a predetermined temperature and a3) single or multiple determination of a quantity of antibodies, antibody fragments or antibody mimetics, which are bound to cells contained in the other partial quantity as a function of time from the mixture according to step a2), calculating a binding rate therefrom and determining a time,which is necessary to achieve a decreasing binding rate of the binding of antibodies, antibody fragments or antibody mimetics to these cells, wherein the incubation in step b) occurs at the predetermined temperature, wherein the time according to step b) is selected such that it is at least as long as the time determined in step a3). In the case of the quantity of antibodies, antibody fragments, or antibody mimetics to be determined in step a3), this can be a relative or absolute quantity. A relative quantity is sufficient for determining a decreasing binding rate. When steps a)ac) or a)ad) are performed several times under identical conditions, it is sufficient for steps a1) to a3) to be carried out only once for the The determination of the time according to step a3) and the time according to step b) is selected in each case in such a way that it is at least as long as the time determined once in step a3). As is usual in binding assays, the antibodies, antibody fragments or antibody mimetics are added in steps a2) and b) in such quantity in each case that their concentration in the cell suspension, partial amount or further partial amount far exceeds the concentration of increasing EpCAM or other specific antigen for epithelial cells, but at least in factor 2. In steps b) and a2), after the addition of the antibodies, antibody fragments or antibody mimetics and an incubation for 5 to 60 minutes, for example, after 15 minutes or between steps b) and c) or a2) and a3), a dilution of the suspension can be produced, for example, with PBS, in order to obtain in the determination according to steps c) and a3) better measurement results, for example, because a background signal resulting from the unbound antibodies, antibody fragments and antibody mimetics is reduced. By determining the appropriate method according to step a3), the incubation time of the cells required for the reliable determination of a concentration of epithelial cells with antibodies, antibody fragments, or antibody mimetics is established. This time can range from 8 to 25 hours. By selecting a relatively high temperature, a decreasing binding rate can be achieved sooner, for example, after 6 to 8 hours. Conversely, with a relatively low temperature, i.e., a temperature of 0 to 8 °C, especially 2 to 8 °C, achieving a decreasing binding rate may require more than 25 hours, and, for example, only after 25 to 30 hours. The default temperature can be a temperature in the range of 0 to 30 °C, especially 0 to 20 °C, especially 2 to 15 °C, especially 8 to 15 °C. Incubation according to step b) can be carried out, for example, for at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, or at least 30 hours Labeling can be fluorescent using a fluorochrome such as fluorescein isothiocyanate (FITC) or phycoerythrin (PE), colorimetric using a chromophore, or indirectly detectable using biotin, avidin, streptavidin, or another affinity label, epitope label, or protein label. Direct antibody labeling has the advantage over indirect labeling, for example, using a secondary antibody, that a washing step, which usually results in the loss of some epithelial cells, can be omitted. The determination of the quantity of antibodies, antibody fragments, or bound antibody mimetics according to step a3) and / or the determination of the quantity of cells labeled by binding antibodies, antibody fragments, or antibody mimetics according to step c) can be performed by laser scanning cytometry, fluorescence microscopy, especially quantitative fluorescence microscopy, or light microscopy, especially quantitative light microscopy. When determining the quantity of antibodies, antibody fragments, or bound antibody mimetics and / or the quantity of labeled cells by fluorescence microscopy or light microscopy, image recognition software can be used for cell recognition. This software can distinguish labeled cells from labeled cell debris or other non-specifically labeled structures. When fluorescent labeling is used, in the case of laser scanning cytometry or fluorescence microscopy, a general fluorescence measurement can be performed for each cell, and a background fluorescence can be dynamically determined for that cell. This allows for equivalent fluorescence values ​​to be obtained for each cell as the difference between the general fluorescence calculated for each cell and the background fluorescence calculated for that cell. In the case of dynamic background fluorescence determination, the background fluorescence in the cell's area is measured at the same time as the general fluorescence measurement at the time of that measurement; that is, at a distance of up to approximately half a cell diameter from the cell membrane.In this way, the accuracy of the corresponding measurement and the validity of the measurement results can be increased. The separation of unused cells according to step a) can be achieved by centrifugation, Mere sedimentation or filtration. The suspension of the separated cells in a buffer according to step a) occurs directly after separation, i.e., in such a way that the cells do not dry out and become damaged by drying. In the case of buffering, PBS, i.e., phosphate-buffered saline, can be used. In one embodiment of the procedure, a blocking reagent to block nonspecific binding sites and / or Fe receptors is added to the cell suspension, the partial amount, or the further partial amount of the cell suspension in each case before the addition of the antibodies, antibody fragments, or antibody mimetics, each bearing at least one label according to steps b) and a2), and is mixed and incubated with the cell suspension, the partial amount, or the further partial amount of the cell suspension. Incubation can be carried out in this case, for example, for approximately 15 minutes at 8 °C. In one formulation of the procedure, no detergent is used at any stage and / or no cell fixation occurs at any stage. This prevents damage to the epithelial cells and, consequently, avoids influencing the measurement result. This is particularly important when the procedure requires determining the concentration of cells with intact cell membranes or even the concentration of live epithelial cells. All stages of the procedure, or at least stages b) and e), or at least stages b), a2), a3), and c), can be carried out using an automated system designed for this purpose. This allows for greater objectivity and improved reproducibility of the measurement results. In one embodiment of the procedure, several partial quantities are separated in step a1), wherein in step a2) a different quantity of antibodies, antibody fragments or antibody mimetics is added to each of the partial quantities, specifically immediately mixed with the corresponding partial quantities, wherein in step a3) the time required to achieve the decreasing binding rate is determined for each of the partial quantities, wherein for the performance of step b) the quantity of antibodies, antibody fragments or antibody mimetics with which the decreasing binding rate is achieved in a desired time is selected. The special antigen specific to epithelial cells can be selected, for example, from the following group: androgen receptor, epithelial growth factor receptor (EGF receptor), estrogen receptor, progesterone receptor, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), programmed death ligand 1 (PDL-1), Melan A, B7-H3, vascular endothelial growth factor receptor (VEGF receptor), Ki67, insulin-like growth factor receptor (IGF receptor), and Her2neu. The blood sample or aspirate sample can be stored for at least 14 hours, especially for at least 16 hours, especially for at least 18 hours, especially for at least 20 hours, especially for at least 22 hours, especially for at least 24 hours, at a temperature between 0 and 40 °C, especially at a temperature between 15 and 25 °C, before lysing the erythrocytes. This can further improve the accessibility of specific antigens to epithelial cells. The invention is then explained by means of an embodiment example. A sample of EDTA-anticoagulated blood is allowed to stand for 24 hours at room temperature. The sample is then brought up to 15 mL with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM Na2-EDTA) and stored for 15 minutes at 8 °C. The sample is then centrifuged at 780 g for 7 minutes. The supernatant is discarded. The pellet is resuspended in 500 μL of PBS-EDTA (137 mM NaCl, 2.8 mM KCl, 8.1 mM NaHPO4, 1.4–7 mM KH2PO4, 2 mM EDTA, pH 7.4). From the sample, 50 µA are poured into a reaction vessel and mixed with 15 µA of FcR blocking reagent (Miltenyi Biotec GmbH) and incubated for 15 minutes at 8 °C. Then, 5 µA of a monoclonal antibody directed against EpCAM (HEA-125, Miltenyi Biotec GmbH) are added and mixed with the suspension. A further 15-minute incubation is performed under light protection at 8 °C. Then, 430 µA of PBS-EDTA are added and mixed with the suspension. The resulting suspension is incubated in the dark at 8 °C. After remixing, 100 µA are removed from the suspension at different intervals and measured using an iCys laser scanning microscope (CompuCyte, Beckman Coulter GmbH). The measurement can also be carried out using another microscope suitable for fluorescence calculation.Based on the measured binding, at different times, the time from the mixture with the antibody needed to achieve a decreasing binding rate can be determined. Next, 50 µA of the cell pellets resuspended in PBS-EDTA are mixed with 15 µA of FcR blocking reagent and incubated for 15 minutes at 8 °C. Then, 5 µA of the aforementioned EpCAM-directed antibody is added and mixed with the cell suspension. After a 15-minute incubation protected from light at 8 °C, 430 µA of PBS-EDTA is added and mixed with the suspension. The suspension is then incubated for the time required to reach a falling rate of binding, for example, 14 or 16 hours, at 8 °C in the dark. After further thorough mixing of the suspension, 100 µA is extracted and fluorescence is measured using a laser scanning microscope or other appropriate microscope. In this case, the quantity of cells marked by a binding of antibodies is determined, and from this, the concentration of these cells in the original blood sample.

Claims

1. An in vitro procedure for determining the concentration of epithelial cells in a blood sample or aspirate sample from a human or mammal, which has been dosed with an anticoagulant, wherein the erythrocytes contained in the blood sample or aspirate sample, the antibodies, antibody fragments, and antigens specific to the procedure are excluded from protection, characterized in that it comprises the following steps: a) lysing the erythrocytes contained in the blood sample or aspirate sample by adding a buffer that causes lysis of the erythrocytes, separating the unlysed cells, and suspending the separated cells in a buffer, wherein the blood sample or aspirate sample is stored at a temperature between 0 and 40°C for at least 24 hours before the erythrocytes are lysed, b) adding antibodies,antibody fragments or antibody mimetics carrying at least one label, and each directed against epithelial cell adhesion molecule (EpCAM) and / or at least one epithelial cell-specific antigen, to a cell suspension obtained in step a) or to a partial quantity obtained by separating this cell suspension, mixing the antibodies, antibody fragments or antibody mimetics and the cell suspension or the partial quantity and incubating a mixture so obtained for at least a time necessary to achieve a decreasing rate of binding of the antibodies, antibody fragments or antibody mimetics to the cells, wherein the incubation is carried out for at least 6 hours, c) determination of the quantity of cells labeled by binding of the antibodies,antibody fragments or antibody mimetics in the mixture obtained in step b) and d) calculation of the concentration of labeled cells of the amount determined in step c) in the blood sample or aspirate sample. 13 Claims follow,