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    • 专利摘要:
    In vitro method and kit to evaluate cellular immunity. A procedure for directly assessing the cellular immunity of an animal or human being is described, from a sample of biological fluid comprising mononuclear cells, such as whole blood or saliva, by using a panel of biomarkers related to oxidative metabolism and/or inflammation, obtained by subjecting the mononuclear cells to cellular stress. Likewise, a kit is described that allows to carry out said method of evaluation of cellular immunity. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2725373A1
    申请号:ES201830280
    申请日:2018-03-21
    公开日:2019-09-24
    发明作者:Madrigal José Joaquín Cerón；Marín Luis Pardo；Vicente Fernando Tecles；Subiela Silvia Martínez；Gambín Luis Jesús Bernal；Asta Tvarijonaviciute；Tortosa Damián Escribano；Rubio Camila Peres
    申请人:Universidad de Murcia；
    IPC主号:
    专利说明:

    [0001]
    [0002]
    [0003]
    [0004] OBJECT OF THE INVENTION
    [0005]
    [0006] The objective of the present invention is to develop an in vitro method to directly assess cellular immunity, in a human being or in an animal, by using a panel of biomarkers related to oxidative metabolism and inflammation, obtained from a sample of Mononuclear cells subjected to cell stress by freezing. Likewise, the invention also relates to a kit that allows carrying out the in vitro method of evaluation of cellular immunity described in the present invention.
    [0007]
    [0008] BACKGROUND OF THE INVENTION
    [0009]
    [0010] Cellular immunity is an acquired and specific response that is fundamentally mediated by T lymphocytes. This cellular immunity is essential for the defense of the organism and is responsible for destroying pathogens or cells infected by viruses, bacteria or parasites and neoplastic cells.
    [0011]
    [0012] Therefore, this immunity defends the body from suffering various diseases caused by living agents. For example, in human and canine leishmaniasis the correct functioning and activation of cellular immunity has been associated with disease control and parasite destruction (Brelaz de Castro et al., 2012). And today numerous vaccines seek to activate said cellular immunity.
    [0013]
    [0014] So far, two techniques are used to assess cellular immunity:
    [0015]
    [0016] (a) Tests based on lymphocyte proliferation. These assays are based on the incubation of mononuclear cells (lymphocytes and monocytes) with different substances for the growth of said mononuclear cells. These substances are usually mitogens (which induce cell division) or antigens (of the disease to be studied).
    [0017]
    [0018] Subsequently, the growth of said mononuclear cells is measured, as an increase in their populations and / or the production of cytokines such as gamma interferon, IL-2 or IL-4 (Arce Fonseca et al., 2013). Within this system there are:
    [0019] a1. trials described as the "cytotoxic T lymphocyte assay" (Burleson et al., 2010) where the response of lymphocytes to T-dependent antigens is evaluated; a2. commercially available trials based on ELISPOT assays.
    [0020]
    [0021] These tests (a1, a2) usually consist of:
    [0022]
    [0023] i. Add a specific capture antibody to the analyte to be determined (usually cytokines) attached to an ELISA plate.
    [0024] ii. Block the plate normally with serum
    [0025] iii. Add to cells that want to study a cell proliferation stimulant
    [0026] iv. Incubate the plates so that the secreted cytokines are captured by the antibody bound to the ELISA plate.
    [0027] v. Wash and add a biotinylated detection antibody to allow detection of cytokines bound to the plate antibody.
    [0028] saw. Visualize the cytokines using avidin conjugates and a colored substrate. vii. Each colored point represents a cytokine secretory cell that can be counted visually or automatically.
    [0029]
    [0030] The cytokines that are usually determined are IL-2, IL-4, IL-17, IFN- and or TNF-a.
    [0031] (b) Flow cytometric counts of CD4 and CD8 lymphocytes (Brelaz de Castro et al., 2012). In this type of assay, lymphocytes are labeled with specific antibodies bound to a molecule that is capable of emitting a quantifiable signal and the number of lymphocytes labeled in a flow cytometer is measured.
    [0032]
    [0033] Therefore, assays to assess the cellular immunity currently used require, in general, the incubation of the patient's cells and / or the use of flow cytometry.
    [0034]
    [0035] That is why there is a need to have tests that can assess the state of cellular immunity of an individual (human or animal) that can be carried out directly, without the need for incubation or proliferation tests and thus obtain the results of said test in short periods of time On the other hand, it is also of interest that such tests do not require specialized laboratory or personal material (as is the case when the use and handling of flow cytometers is required).
    [0036] DESCRIPTION
    [0037]
    [0038] The present invention describes an in vitro method for directly evaluating cellular immunity from a panel of biomarkers (analytes) related to oxidative metabolism and inflammation, obtained from a sample of mononuclear cells, separated from a sample of biological fluid, and subjected to cell stress by freezing.
    [0039]
    [0040] This method:
    [0041]
    [0042] - does not require the use of a flow cytometer, so the costs are lower;
    [0043]
    [0044] - it does not require incubation, or cell culture, or the addition of antigens, so the time to obtain a result is shorter;
    [0045]
    [0046] - does not require specialized personnel.
    [0047]
    [0048] Another feature of the method of the invention is that the evaluation of these biomarkers or analytes can be determined, both absolutely and in a relative way. Therefore, the values of each biomarker per mononuclear cell of the sample, in particular the values of each biomarker per lymphocyte and the absolute values of said biomarker, are estimated in the whole sample. This makes it a more sensitive test than if it were only expressed by absolute values, since relative values measure the cellular immune response capacity of each cell.
    [0049]
    [0050] The present invention thus relates to an in vitro method for evaluating cellular immunity from a sample of biological fluid comprising mononuclear cells, from an animal or from a human being, wherein said method comprises:
    [0051]
    [0052] i. separate the mononuclear cells from the biological fluid sample;
    [0053] ii. suspend the separated mononuclear cells in a saline solution;
    [0054] iii. quantify the mononuclear cells present in the saline solution of mononuclear cells obtained in step (ii);
    [0055] iv. freeze the saline solution of mononuclear cells;
    [0056] v. thaw the mononuclear cell saline solution and then perform a centrifugation;
    [0057] saw. collect the supernatant liquid resulting from the centrifugation of step (v); and vii. determine the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid collected in step (vi) viii compare the content of at least one analyte indicator of inflammation and / or oxidative stress indicator determined the step (viii) with the average reference content of the same analyte obtained by reproducing said method under the same conditions from at least two samples of the same type of biological fluid that comprise mononuclear cells from animals or healthy humans.
    [0058]
    [0059] In one embodiment, the in vitro method of the invention comprises:
    [0060]
    [0061] i. separating the mononuclear cells from the biological fluid sample by adding said sample onto a mononuclear cell separating solution and subjecting it to centrifugation;
    [0062] ii. collect the phase that contains the mononuclear cells of the sample, comprised between the upper phase and the lower phase formed in the centrifugation, and suspend it in a saline solution;
    [0063] iii. quantify the mononuclear cells present in the saline solution of mononuclear cells obtained in step (ii);
    [0064] iv. freeze the saline solution of mononuclear cells;
    [0065] v. thaw the mononuclear cell saline solution by letting it stand at room temperature and then perform centrifugation;
    [0066] saw. collect the supernatant liquid resulting from the centrifugation of step (v); and vii. determine the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid collected in step (vi) viii. compare the content of at least one analyte indicator of inflammation and / or oxidative stress indicator determined the step (viii) with the average reference content of the same analyte obtained by reproducing said method under the same conditions from at least two samples of the same type of biological fluid comprising mononuclear cells from animals or healthy humans.
    [0067]
    [0068] In an embodiment of the in vitro method of the invention, the biological fluid sample comprising mononuclear cells is a saliva sample.
    [0069]
    [0070] In an embodiment of the in vitro method of the invention, the biological fluid sample comprising mononuclear cells is a whole blood sample.
    [0071]
    [0072] In an embodiment of the in vitro method of the invention, the whole blood sample is pretreated with an anticoagulant. For the purposes of the present invention, the term Anticoagulant refers to any substance suitable for inhibiting the natural coagulation of blood where, if it is not contacted with said substance, the blood loses its liquidity, becoming a gel and forming clots.
    [0073]
    [0074] For the purposes of the present invention, the whole blood sample can be pretreated with any anticoagulant used in routine hematological and biochemical determinations.
    [0075]
    [0076] In an embodiment of the in vitro method of the invention said anticoagulant is ethylenediamine tetraacetic acid (EDTA). In another embodiment of the in vitro method of the invention said anticoagulant is heparin.
    [0077]
    [0078] For the purposes of the present invention, anticoagulants are preferably employed at the concentrations described in the state of the art for routine hematological and biochemical determinations.
    [0079]
    [0080] In another embodiment of the in vitro method of the invention, said anticoagulant is a citrate solution, and the whole blood sample is diluted in a 1:10 volume ratio with said citrate solution. In an embodiment of the in vitro method of the invention, when a citrate solution is used as an anticoagulant and the whole blood sample is diluted in relation to 1:10 with said citrate solution, the analyte values determined in step (vii ) of the method of the invention must be multiplied by 10.
    [0081]
    [0082] In an embodiment of the in vitro method of the invention, the citrate solution is preferably used at the concentrations described in the state of the art for routine hematological and biochemical determinations.
    [0083]
    [0084] In an embodiment of the in vitro method of the invention, step (i) comprises separating the mononuclear cells from the biological fluid sample by adding said sample onto a mononuclear cell separating solution and subjecting it to centrifugation.
    [0085]
    [0086] In an embodiment of the in vitro method of the invention, step (i) comprises separating the mononuclear cells from the biological fluid sample by any means or separating process that allows a separation of the mononuclear cells and subjecting them to centrifugation.
    [0087]
    [0088] In an embodiment of the in vitro method of the invention, step (i) comprises separating the mononuclear cells from the biological fluid sample by density gradient and subjecting it to centrifugation.
    [0089] In one embodiment of the in vitro method of the invention, step (i) comprises separating the mononuclear cells from the biological fluid sample by adding said biological fluid sample onto a mononuclear cell separating medium and subjecting it to centrifugation.
    [0090] In one embodiment of the in vitro method of the invention, the mononuclear cell separating medium has a density of 1077 mg / ml.
    [0091]
    [0092] By way of indication and not limitation, separating means suitable for carrying out the in vitro method of the invention include means prepared in the laboratory itself or commercial means such as Histopaque® or Ficoll®.
    [0093]
    [0094] In an embodiment of the in vitro method of the invention, step (i) comprises separating the mononuclear cells by adding the biological fluid sample onto a mononuclear cell separating medium and subjecting it to a centrifugation of between 1000 to 2000 rpm. In a more preferred embodiment of the in vitro method of the invention, said centrifugation is carried out for at least 15 minutes. In an even more preferred embodiment of the in vitro method of the invention, said centrifugation is performed for at least 30 minutes.
    [0095]
    [0096] In an embodiment of the in vitro method of the invention, step (ii) comprises collecting the phase containing the mononuclear cells of the biological fluid sample, comprised between the upper phase and the lower phase formed in the centrifugation, and suspending it in a saline solution with a salt concentration equivalent to the salt concentration inside the cells. Said saline solution containing a salt concentration equivalent to the saline concentration inside the cells is also called normal saline solution or physiological serum. In a more preferred embodiment of the in vitro method of the invention, said saline solution comprises distilled water and 0.9% (w / v) sodium chloride.
    [0097]
    [0098] In an embodiment of the in vitro method of the invention, when the biological fluid sample comprising mononuclear cells is a whole blood sample, the saline solution is of a volume equal to the initial volume of blood sample after the addition of the anticoagulant.
    [0099]
    [0100] In an embodiment of the in vitro method of the invention, when the biological fluid sample comprising mononuclear cells is a whole blood sample, the relationship between the volume of saline solution and the volume of whole blood sample after the addition of the anticoagulant It is a 1: 1 ratio.
    [0101] In an embodiment of the in vitro method of the invention, when the biological fluid sample comprising mononuclear cells is a whole blood sample, the relationship between the volume of saline solution and the volume of whole blood sample after the addition of the anticoagulant It is different from a 1: 1 volume ratio.
    [0102]
    [0103] In general, in the in vitro method of the invention, the volume ratio between the biological fluid sample comprising mononuclear cells and the saline solution must be in a range that allows the detection of the analytes by the methods used for their quantification. In one embodiment of the invention, the total volume of the biological fluid sample and the saline solution must be at least 0.5 ml. In another embodiment of the in vitro method of the invention, the total volume of the biological fluid sample and the saline solution is 0.5 ml or even smaller, depending on whether the analyte to be measured and the measurement technique thereof. allow.
    [0104]
    [0105] In an embodiment of the in vitro method of the invention, step (iii) comprises quantifying the mononuclear cells present in the mononuclear cell saline solution obtained in step (ii) in any hematology analyzer that allows mononuclear cell counts to be performed. Examples of such analyzers comprise, but are not limited to electrical impedance analyzers, centrifuges or based on laser technology. In an embodiment of the in vitro method of the invention, step (iii) comprises quantifying the mononuclear cells present in the mononuclear cell saline solution obtained in step (ii) in a hematology analyzer that is independently selected from an impedance analyzer electrical, a centrifugal analyzer or an analyzer based on laser technology.
    [0106]
    [0107] Therefore, performing said step (iii), according to the in vitro method of the invention, does not require the use of a hematology analyzer based on the flow cytometry technique.
    [0108]
    [0109] In one embodiment of the in vitro method of the invention, step (iv) comprises freezing the saline solution of mononuclear cells obtained in step (ii) at -20 ° C. In one embodiment of the in vitro method of the invention, step (iv) comprises freezing the mononuclear cells for at least 1 hour.
    [0110]
    [0111] This allows the in vitro method of the invention to be performed in a laboratory or clinic without the need for a freezer of -80 ° C and quickly.
    [0112]
    [0113] In other embodiments of the in vitro method of the invention step (iv) comprises freezing the saline solution of mononuclear cells obtained in step (ii) at -20 ° C for at least 1 hour, at least 2 hours, at least 5 hours, at least 8 hours, at least 24 hours, or more than 24 hours; at -80 ° C for at least 1 hour, at least 2 hours, at least 5 hours, at least 8 hours, at least 24 hours, or more than 24 hours; or during any time that allows to freeze the saline solution of mononuclear cells obtained in step (ii).
    [0114]
    [0115] The in vitro method of the invention can also be carried out by performing step (iv) at -15 ° C, -15 ° C, -20 ° C, -30 ° C, -45 ° C , at -65 ° C or at any temperature at which the freezing of the saline solution of mononuclear cells obtained in step (ii) occurs.
    [0116]
    [0117] In an embodiment of the in vitro method of the invention, step (v) comprises defrosting the mononuclear cell saline solution obtained in step (ii) by allowing it to stand at room temperature and then perform centrifugation.
    [0118]
    [0119] For the purposes of the present invention, room temperature is defined as a temperature between 10 ° C and 30 ° C.
    [0120]
    [0121] In an embodiment of the in vitro method of the invention, step (v) comprises thawing the saline solution of mononuclear cells obtained in step (ii) by allowing it to stand at room temperature for half an hour, and then performing a centrifugation. In another embodiment of the in vitro method of the invention, step (v) comprises thawing the saline solution of mononuclear cells obtained in step (ii) by allowing it to stand at room temperature for at least 20 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour; or during the time necessary for defrosting, and then perform a centrifugation.
    [0122]
    [0123] In an embodiment of the in vitro method of the invention, step (v) comprises thawing the saline solution of mononuclear cells obtained in step (ii) and then performing a centrifugation at least at 1700 rpm. In an embodiment of the in vitro method of the invention, step (v) comprises then performing a centrifugation between 1500 to 15000 rpm. In an embodiment of the in vitro method of the invention, step (v) comprises then performing a centrifugation between 1500 and 10,000 rpm. In an embodiment of the in vitro method of the invention, step (v) comprises performing a centrifugation between 1500 and 3000 rpm.
    [0124]
    [0125] In an embodiment of the in vitro method of the invention, step (v) comprises thawing the saline solution of mononuclear cells obtained in step (ii) by allowing it to stand at room temperature and perform a centrifugation for at least 5 minutes at least 1700 rpm
    [0126] In an embodiment of the in vitro method of the invention, step (vi) comprises collecting the supernatant liquid resulting from the centrifugation of step (v).
    [0127]
    [0128] In an embodiment of the in vitro method of the invention, step (vii) comprises determining the content of at least one inflammatory indicator and / or oxidative stress indicator in the supernatant liquid collected in step (vi) with the use of methods previously described in the state of the art for said analyte. Such methods may be spectrophotometric or immunological. For example, in order to determine ferritin, in the in vitro method of the invention, an immunological method having an antibody that allows the detection of ferritin can be used as reagent. Or to determine total oxidants (TOS), in the in vitro method of the invention, a spectrophotometric method can be used in which one of the reagents carries o-dianisidine that reacts with the oxidants in the sample and produces color variation. Other reagents that react with the oxidants in the sample can also be used in the in vitro method of the invention. These methods can be carried out in the laboratory itself or commercially available.
    [0129]
    [0130] Biochemistry analyzers carry out different types of evaluations, including spectrophotometric, immunoturbity, selective ion determination, among others.
    [0131]
    [0132] Non-limiting examples of biochemistry analyzers are automated biochemistry analyzers (eg Olympus A400, Olympus 600), manual biochemistry analyzers (any conventional spectrophotometer) or spectrophotometric microplate readers (eg Biotech, Tecan).
    [0133]
    [0134] In an embodiment of the in vitro method of the invention, the determination of at least one analyte of the passage (vii) is carried out in manual or automated spectrophotometers.
    [0135]
    [0136] In an embodiment of the in vitro method of the invention, in step (vii) the content, total and / or content in relation to the number of mononuclear cells quantified in step (iii), of at least one indicator analyte is determined of inflammation, and / or oxidative stress.
    [0137]
    [0138] In one embodiment of the in vitro method of the invention, the mononuclear cells are lymphocytes.
    [0139]
    [0140] For the purposes of the present invention, the term "inflammation indicator analyte" refers to an analyte existing in said mononuclear cells that varies in concentration when In these cells inflammation occurs. An example of such an analyte indicator of inflammation is, for example, ferritin.
    [0141]
    [0142] For the purposes of the present invention, the term "oxidative stress indicator analyte" refers to an analyte existing in said mononuclear cells that varies in concentration when oxidative stress occurs in said cells. An example of said oxidative stress indicator analyte is , for example, the concentration of total antioxidants (TOS).
    [0143]
    [0144] In an embodiment of the in vitro method of the invention, in step (vii) at least one indicator analyte for inflammation and / or oxidative stress is determined which is independently selected from the group consisting of ferritin, C-reactive protein, haptoglobin, myeloperoxidase , total oxidants (TOS), cupric ion reducing antioxidant capacity (CUPRAC), paraoxonase-1 (PONI), interleukins, total antioxidants (TAC), iron antioxidant status (FRAP), total esterase (TEA), catalase, nitrite, thiol or nitric oxide; or combinations thereof. In one embodiment the interleukins are selected from IL-2, IL-4, IL-17 IFN- and or TNF-a. In general, any analyte related to inflammation, oxidative stress or interleukins can be determined.
    [0145]
    [0146] In general, the in vitro method of the invention includes the determination of at least one analyte indicator of inflammation and / or oxidative stress, variations in the response of individual analytes may exist depending on the disease or clinical situation and some analytes may be more sensitive than others in said disease or said clinical situation. For example, in situations of acute inflammation the determination of C-reactive protein is more appropriate than that of haptoglobin, while in chronic inflammation the determination of haptoglobin provides a more sensitive result. Depending on the possible disease of the animal or human being from which the whole blood sample comes, the appropriate analytes will be chosen for the determination of their immune status.
    [0147]
    [0148] In another embodiment of the in vitro method of the invention, in step (vii) an analyte related to inflammation is selected independently selected from ferritin, an acute phase protein such as C-reactive protein or one or more interleukins; or combinations thereof. In one embodiment, the interleukins are selected from IL-2, IL-4, IL-17 IFN- and or TNF-a, although any interleukin for which a method is available can be used.
    [0149] In another embodiment of the in vitro method of the invention, in step (vii) an analyte related to oxidative stress independently selected from total oxidants (TOS), total antioxidants (TAC), myeloperoxidase, iron antioxidant status (FRAP) is determined , antioxidant capacity of cupric ion (CUPRAC), paraoxonase-1 (PON1), total esterase (TEA), catalase, nitrite, thiol or nitric oxide; or combinations thereof or any other oxidative state marker for which a method is available.
    [0150]
    [0151] In another embodiment of the in vitro method of the invention, in step (vii) the content of an analyte related to oxidative stress independently selected from myeloperoxidase, iron antioxidant status (FRAP), cupric ion reducing antioxidant capacity (CUPRAC) is determined ) and paraoxonase-1 (PON1).
    [0152]
    [0153] In another embodiment of the in vitro method of the invention, in step (vii) the ferritin content, total oxidants (TOS), myeloperoxidase, iron antioxidant status (FRAP), cupric ion reducing antioxidant capacity (CUPRAC) are determined and paraoxonase-1 (PON1).
    [0154]
    [0155] In an embodiment of the in vitro method of the invention, in step (vii) the content of at least ferritin and total oxidants (TOS) is determined.
    [0156]
    [0157] In step (viii) of the in vitro method of the invention, the content of at least one inflammatory indicator analyzer and / or oxidative stress indicator determined is compared to step (viii) with the average reference content of the same analyte obtained. when reproducing said method under the same conditions from at least two samples of biological fluid from animals or healthy humans.
    [0158]
    [0159] As seen in Example 1, the method of the invention is carried out under the same conditions in a sample of whole blood, both from healthy animals (dogs), and from sick animals (leishmaniosis). The results show that diseased animals obtain ferritin and TOS values much higher (per mononuclear cell) than healthy animals (Table 1), therefore being an increase in these analytes an indicator of the immune status of said animals. Specifically, in the group of dogs analyzed in Example 1, it can be determined, in general, that values greater than 25 ^ g / l of ferritin in 103 cells / ^ l and greater than 60 ^ g / l of TOS in 103 cells / ^ l are indicative of sick animals with leishmaniasis.
    [0160]
    [0161] The results of tables 2 and 3 show that the determination of the levels of CUPRA, FRAP, ASD, catalase and myeloperoxidase, also show higher values in the sick animals, and in many cases the increase in these values, in relation to the number of mononuclear cells in the sample, is of the order of 10 times more than in healthy animals.
    [0162]
    [0163] On the other hand, the particular freezing conditions, in the temperature and time ranges described in the present description, cause the mononuclear cells to release analytes to the medium, as observed in Example 2, so that they can be used to establish the cellular immune status of the subject.
    [0164]
    [0165] As can be seen in Example 3, at higher freezing times, higher values of the measured analytes are obtained.
    [0166]
    [0167] On the other hand, as observed in Example 4, the samples of supernatant liquid obtained, once the mononuclear cells have been thawed and centrifuged, are stable for at least 24 hours, both frozen and at room temperature, making the method flexible. when determining a more or less extensive set of analytes, and allows the analytes to be determined in the supernatants collected in step (vi) of the in vitro method of the invention just after performing said step (vi) or later, and its transport is also possible so that the determination of the analytes is carried out in other laboratories.
    [0168]
    [0169] Another embodiment of the invention relates to a cell immunity evaluation kit from a biological fluid sample comprising mononuclear cells of an animal or a human being, comprising:
    [0170]
    [0171] to. a first support comprising a mononuclear cell separating solution by density gradient;
    [0172]
    [0173] b. a second support comprising a saline solution;
    [0174]
    [0175] C. a third suitable support for quantifying an analyte in a biochemistry analyzer or in a spectrophotometer or in a microplate reader;
    [0176]
    [0177] wherein said kit further comprises instructions, wherein said instructions comprise:
    [0178]
    [0179] i. add without mixing the biological fluid sample comprising mononuclear cells on the mononuclear cell separator solution of the first support and subject it to centrifugation;
    [0180] ii. collect the mononuclear cells of the biological fluid sample from the interface between the supernatant and the pellet formed in the centrifugation, and suspend them in the saline solution of the second support;
    [0181] iii. quantify the mononuclear cells present in the saline solution of mononuclear cells of the second support obtained in step (ii);
    [0182] iv. freeze the mononuclear cell saline solution from the second support; v. thaw the mononuclear cell saline solution from the second support by allowing it to stand at room temperature and then perform a centrifugation;
    [0183] saw. collect the supernatant liquid resulting from the centrifugation of step (v) and deposit it on the third support; Y
    [0184] vii. determine the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid collected in step (vi) viii. compare the content of at least one analyte indicator of inflammation and / or oxidative stress indicator determined the step (viii) with the average reference content of the same analyte obtained by reproducing said method under the same conditions from at least two samples of the same type of biological fluid from animals or healthy humans.
    [0185]
    [0186] In an embodiment of the kit of the invention, the instructions comprise that the biological fluid sample comprising mononuclear cells is a saliva sample.
    [0187]
    [0188] In an embodiment of the kit of the invention, the instructions comprise that the biological fluid sample comprising mononuclear cells is a whole blood sample.
    [0189] In one embodiment of the kit of the invention, the instructions in step (i) comprise adding a whole blood sample, previously treated with an anticoagulant, to the mononuclear cell separator solution of the first support without mixing and subject to centrifugation.
    [0190]
    [0191] For the purposes of the present invention, the instructions in step (i) comprise that the whole blood sample can be previously treated with any anticoagulant used in routine hematological and biochemical determinations.
    [0192]
    [0193] In an embodiment of the kit of the invention, the instructions comprise that said anticoagulant is ethylenediamine tetraacetic acid (EDTA). In another embodiment of the kit of the invention, the instructions comprise that said anticoagulant is heparin.
    [0194] In another embodiment of the kit of the invention, the instructions comprise that said anticoagulant is a citrate solution, and the whole blood sample is diluted 1:10 with said citrate solution. In another embodiment of the kit of the invention, the instructions comprise that when a citrate solution is used as an anticoagulant and the whole blood sample is diluted in relation to 1:10 with said citrate solution, the analyte values determined in the step (vii) of the instructions of the invention kit must be multiplied by 10.
    [0195]
    [0196] In an embodiment of the kit of the invention, the instructions in step (i) comprise that the first support separating solution has a density of 1077 mg / ml.
    [0197]
    [0198] In general, the first support of the kit of the invention can comprise any separating solution that allows a correct separation of the mononuclear cells. By way of indication and not limitation, the instructions of the kit of the invention may include that any separating solution is suitable for separating mononuclear cells, including media prepared in the laboratory itself or commercial media such as Histopaque® or Ficoll®.
    [0199]
    [0200] In one embodiment of the kit of the invention, the instructions in step (i) comprise that the volume of the biological fluid sample is equal to the volume of the mononuclear cell separating solution. That is, the biological fluid sample and the mononuclear cell separating solution are in a 1: 1 volume ratio.
    [0201]
    [0202] In an embodiment of the kit of the invention, the instructions of step (i), when the biological fluid sample is a whole blood sample, comprise that the volume of the whole blood sample after the addition of the anticoagulant is equal to the volume the mononuclear cell separating solution.
    [0203]
    [0204] In an embodiment of the kit of the invention, the instructions of step (i) comprise adding a biological fluid sample without mixing the mononuclear cell separator solution of the first support and subjecting it to centrifugation.
    [0205]
    [0206] In an embodiment of the kit of the invention, the instructions of step (i) comprise adding a biological fluid sample without mixing the mononuclear cell separator solution of the first support and subjecting it to a centrifugation at between 1000 to 2000 rpm. In an embodiment of the kit of the invention, the instructions comprise that said centrifugation is carried out for at least 15 minutes. In another embodiment of the kit of the invention, the instructions comprise that said centrifugation is carried out for at least 30 minutes.
    [0207] In an embodiment of the kit of the invention, the instructions of step (ii) comprise. collect the mononuclear cells from the biological fluid sample from the interface between the supernatant and the pellet formed in the centrifugation, and suspend them in the saline solution of the second support.
    [0208]
    [0209] In an embodiment of the kit of the invention, the salt solution of the second support comprises a salt concentration equivalent to the salt concentration inside the cells. Said saline solution containing a salt concentration equivalent to the saline concentration inside the cells is also called normal saline solution or physiological serum. In a more preferred embodiment of the kit of the invention said saline solution comprises distilled water and 0.9% (w / v) sodium chloride.
    [0210]
    [0211] In an embodiment of the kit of the invention, the instructions of step (ii) comprise collecting the mononuclear cells from the biological fluid sample from the interface between the supernatant and the pellet formed in the centrifugation, and suspending them in a volume saline solution equal to the initial sample volume
    [0212]
    [0213] In an embodiment of the kit of the invention, the instructions of step (ii), when the biological fluid sample is a whole blood sample, comprise collecting the mononuclear cells from the whole blood sample from the interface between the supernatant and the pellets formed in centrifugation, and suspend them in a saline solution of volume equal to the initial volume of blood sample after the addition of the anticoagulant.
    [0214]
    [0215] In an embodiment of the kit of the invention, the instructions of step (ii), when the biological fluid sample is a whole blood sample, comprise that the relationship between the volume of saline solution and the volume of whole blood sample after The addition of the anticoagulant is 1: 1.
    [0216]
    [0217] In an embodiment of the kit of the invention, the instructions of step (ii), when the biological fluid sample is a whole blood sample, comprise that the relationship between the volume of saline solution and the volume of whole blood sample after The addition of the anticoagulant is different than a 1: 1 volume ratio.
    [0218]
    [0219] In an embodiment of the kit of the invention, the instructions of step (ii) comprise that the volume ratio between the biological fluid sample comprising mononuclear cells and the saline solution must be in a range that allows the detection of the analytes by the methods used for quantification. In an embodiment of the kit of the invention, the instructions in step (ii) comprise that, the volume Total biological fluid sample and saline solution should be at least 0.5 ml. In another embodiment, the instructions in step (ii) of the kit of the invention comprise that the total volume of the biological fluid sample and the saline solution is 0.5 ml or even less, depending on whether the analyte to be measured and the Measurement technique allows it.
    [0220]
    [0221] In an embodiment of the kit of the invention, the instructions of step (iii) comprise quantifying the mononuclear cells present in the mononuclear cell saline solution of the second support obtained in step (ii) in any hematology analyzer that allows cell counts to be performed mononuclear
    [0222]
    [0223] In one embodiment of the kit of the invention, the instructions in step (iv) comprise freezing the mononuclear cell saline solution of the second support at -20 ° C. In one embodiment, the instructions in step (iv) comprise freezing the mononuclear cells in the second support for at least 1 hour.
    [0224]
    [0225] In one embodiment of the kit of the invention, the instructions in step (iv) comprise freezing the mononuclear cell saline solution of the second support at -20 ° C for at least 1 hour, at least 2 hours, at least 5 hours, at least 8 hours, at least 24 hours, or more than 24 hours; at -80 ° C for at least 1 hour, at least 2 hours, at least 5 hours, at least 8 hours, for at least 24 hours, or for more than 24 hours; or during any time that allows to freeze the saline solution of mononuclear cells of the second support.
    [0226]
    [0227] In other embodiments of the kit of the invention, the instructions in step (iv) comprise freezing the mononuclear cell saline solution of the second support at -15 ° C, -20 ° C, -30 ° C, -45 ° C , at -65 ° C, or at any temperature at which the freezing of the mononuclear cell salt solution of the second support occurs.
    [0228]
    [0229] In an embodiment of the kit of the invention, the instructions in step (v) comprise defrosting the mononuclear cell saline solution from the second support by allowing it to stand at room temperature and then perform centrifugation.
    [0230]
    [0231] In one embodiment of the kit of the invention, the instructions in step (v) comprise defrosting the mononuclear cell saline solution from the second support by allowing it to stand at room temperature for half an hour and then perform centrifugation. In other embodiments of the kit of the invention, the instructions in step (v), the instructions comprise defrosting the mononuclear cell saline solution from the second support by allowing it to stand at room temperature for at least 20 minutes, at at least 40 minutes, at least 50 minutes, or at least 1 hour; or during any time that allows defrosting of the mononuclear cell saline solution of the second support, and then performing a centrifugation.
    [0232]
    [0233] In an embodiment of the kit of the invention, the instructions in step (v) comprise defrosting the mononuclear cell saline solution from the second support by allowing it to stand at room temperature and then performing a centrifugation at at least 1700 rpm. In another embodiment of the kit of the invention, the instructions comprise, in relation to the centrifugation of step (v), that it is carried out at 1500 to 15000 rpm. In a further embodiment of the kit of the invention, the instructions comprise, in relation to the centrifugation of step (v), that it is performed at between 1500 and 10,000 rpm. In one embodiment said centrifugation of step (v) is performed at between 1500 and 3000 rpm.
    [0234]
    [0235] In a further embodiment of the kit of the invention, the instructions comprise, in relation to the centrifugation of step (v), that it is performed for at least 5 minutes at at least 1700 rpm.
    [0236]
    [0237] In an embodiment of the kit of the invention, the instructions of step (vi) comprise collecting the supernatant liquid resulting from the centrifugation of step (v).
    [0238]
    [0239] In an embodiment of the kit of the invention, the instructions in step (vii) comprise determining the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in said supernatant liquid.
    [0240]
    [0241] In an embodiment of the kit of the invention, the instructions of step (vii) comprise determining the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid resulting from the centrifugation of step (vi) with the use of methods previously described in the state of the art for said analyte. Such methods may be spectrophotometric or immunological.
    [0242]
    [0243] The methods for determining the content of at least one inflammation indicator analyte and / or oxidative stress indicator included in the instructions of the kit of the invention in relation to step (vii), can be carried out using any biochemistry analyzer. Automated biochemistry analyzers (eg Olympus A400, Olympus 600), or spectrophotometric microplate readers (eg Biotech, Tecan) can be used.
    [0244]
    [0245] In an embodiment of the kit of the invention, the instructions in step (vii) comprise determining at least one analyte in manual or automated spectrophotometers.
    [0246] In an embodiment of the kit of the invention, the instructions in step (vii) comprise determining the content, total and / or content in relation to the number of mononuclear cells quantified in step (iii), of at least one analyte indicator of inflammation, and / or oxidative stress.
    [0247]
    [0248] In one embodiment of the kit of the invention, the instructions indicate that the mononuclear cells separated in step (i) are lymphocytes.
    [0249]
    [0250] In one embodiment of the kit of the invention, the instructions in step (vii) comprise determining at least one inflammatory and / or oxidative stress indicator analyte that is independently selected from the group consisting of ferritin, C-reactive protein, haptoglobin, myeloperoxidase, oxidants Total (TOS), cupric ion reducing antioxidant capacity (CUPRAC), paraoxonase-1 (PON1), interleukins, total antioxidants (TAC), iron antioxidant status (FRAP), total esterase (TEA), catalase, nitrite, thiol or Nitric oxide; or combinations thereof. In one embodiment the interleukins are selected from IL-2, IL-4, IL-17 IFN-y or TNF-a.
    [0251]
    [0252] In an embodiment of the kit of the invention, the instructions in step (vii) comprise determining an inflammation-related analyte independently selected from ferritin, an acute phase protein such as C-reactive protein or one or more interleukins; or combinations thereof. In one embodiment, the interleukins are selected from IL-2, IL-4, IL-17 IFN-y or TNF-a.
    [0253]
    [0254] In one embodiment of the kit of the invention, the instructions in step (vii) comprise determining an analyte related to oxidative stress independently selected from total oxidants (TOS), total antioxidants (TAC), iron antioxidant status (FRAP), total esterase (TEA), catalase, nitrite, thiol or nitric oxide; or combinations thereof.
    [0255]
    [0256] In one embodiment of the kit of the invention, the instructions in step (vii) comprise determining an analyte related to oxidative stress independently selected from myeloperoxidase, iron antioxidant status (FRAP), antioxidant capacity of cupric ion reducer (CUPRAC), paraoxonase. 1 (PONI).
    [0257]
    [0258] In an embodiment of the kit of the invention, the instructions in step (vii) comprise determining the content of at least ferritin and TOS.
    [0259]
    [0260] In an embodiment of the kit of the invention, the instructions in step (viii) comprise comparing the content of at least one analyte indicator of inflammation and / or indicator of Oxidative stress determined the step (viii) with the average reference content of the same analyte that is obtained by reproducing said method under the same conditions from at least two samples of the same type of biological fluid from animals or healthy humans.
    [0261]
    [0262] EXAMPLES
    [0263]
    [0264] The examples described below are illustrative and are not intended to limit the scope of the present invention.
    [0265]
    [0266] Example 1: Use of the in vitro method of the invention to evaluate the cellular immunity of healthy dogs and patients with leishmaniasis.
    [0267]
    [0268] Samples of whole blood were taken from 4 sick dogs with leishmaniosis (a disease that causes a decrease in cellular immunity) and 4 healthy dogs and the in vitro method of the invention was carried out to establish their cellular immune status.
    [0269]
    [0270] The sick dogs came from the Dinos clinic and the Dr. Bernal clinic in Murcia (Spain) and were diagnosed with leishmania by serology and PCRs. They were in clinical stage 3 according to the guide of the leishvet group. Healthy dogs were dogs that came to the clinic for routine periodic checkups, showed no external clinical signs and were negative for leishmania due to serology and PCR. In no case had they received treatment in the last 6 months.
    [0271]
    [0272] Both the total content of an inflammation marker (ferritin) and an oxidative stress marker (TOS) were determined, as well as the content of said markers relative to the number of lymphocytes in the sample. Ferritin was determined by an immunoturbidimetric method, which uses a specific antibody against ferritin for quantification, using a commercial kit from the Roche house (Tina-quant Ferritin Gen 4. Roche Diagnostics Gmbh), although it can be determined by any capable method of quantifying ferritin. The TOS was determined using a substrate that reacts with the oxidants of the sample produces color, following the method described by Erel (Erel's TOS, 2005) although it can be determined by any method capable of quantifying the TOS.
    [0273] The results are shown in Tables 1A and 1B :
    [0274]
    [0275]
    [0276]
    [0277] fable 1A
    [0278]
    [0279]
    [0280]
    [0281] Standard deviation (SD) indicated in brackets for each value * p <0.05
    [0282]
    [0283] Table 1B
    [0284]
    [0285] Tables 1A and 1B show significantly higher values of ferritin / lymphocytes and TOS / lymphocytes in the analyzed samples of sick dogs compared to the analyzed samples of healthy dogs. In addition, it can be seen that, in the case of ferritin, only when the value is corrected with the number of mononuclear cells in the sample, significant differences appear between healthy and diseased animals.
    [0286]
    [0287] Healthy dogs had undetectable antibody levels, while patients had antibody levels greater than 1: 360, which would indicate a predominance of humoral immunity capable of producing very high levels of antibodies and a decrease in cellular immunity. Thus the levels of ferritin / lymphocytes and TOS / high lymphocytes can be associated with (1) an alteration of the immune system in which there are a predominance of humoral immunity with antibody production, and also to (2) appearance of clinical signs in animals.
    [0288]
    [0289] The values obtained for the CUPRAC, FRAP, TEA, catalase and myeloperoxidase analytes in two healthy dogs and two with leishmania were also compared, following the in vitro method of the invention.
    [0290]
    [0291] In this case, the values of cupric ion reducing antioxidant capacity (CUPRAC), iron antioxidant status (FRAP) and total esterase (TEA) were obtained by spectrophotometric methods previously described in Serum antioxidant capacity and oxidative damage in clinical and subclinical canine ehrlichiosis. Rubio CP, Yilmaz Z, Martínez-Subiela S, Kocaturk M, Hernández-Ruiz J, Yalcin E, Tvarijonaviciute A, Escribano D, Ceron JJ.Res Vet Sci. 2017 Dec; 115: 301-306.
    [0292]
    [0293] On the other hand, the catalase values were also obtained in this case by a spectrophotometric method previously described in Serum markers of lipid peroxidation, antioxidant enzymatic defense, and collagen degradation in an experimental (Pond-Nuki) canine model of osteoarthritis. Goranov NV. Vet Clin Pathol. 2007 Jun; 36 (2): 192-5.
    [0294]
    [0295] Finally, myeloperoxidase values were also obtained by a spectrophotometric method previously described in Evaluation of serum myeloperoxidase concentration in dogs with heart failure due to chronic mitral valvular insufficiency. Park JI, Suh SI, Hyun C. Can J Vet Res. 2017 Jan; 81 (1): 37-40.
    [0296]
    [0297] The results are shown in Tables 2 and 3:
    [0298]
    [0299]
    [0300]
    [0301] Table 2
    [0302]
    [0303]
    [0304] Table 3
    [0305]
    [0306] You can see in both tables higher values of these analytes in sick dogs compared to the values obtained in healthy dogs, in many cases the increase is of the order of 10 times more, when comparing the values obtained relative to the number of mononuclear cells of the sample analyzed, in sick dogs compared to healthy dogs.
    [0307]
    [0308] Example 2: Influence of freezing on the release of analytes in mononuclear cells.
    [0309]
    [0310] Samples of two leishmania dogs diagnosed with Example 1 were used and the in vitro method of the invention was carried out. Of all the samples 2 aliquots were made:
    [0311]
    [0312] -in an aliquot the ferritin values were measured using the separated mononuclear cells without freezing and after 1 hour after their separation.
    [0313]
    [0314] - in the other the ferritin values were measured after freezing the separated mononuclear cells according to the in vitro method described in the invention, 1 hour at -20 ° C.
    [0315]
    [0316] Ferritin values were determined as previously indicated in example 1.
    [0317] The results are shown in Table 4:
    [0318]
    [0319]
    [0320]
    [0321] Table 4
    [0322]
    [0323] It can be seen from the values shown in Table 4 that freezing at -20 ° C produces a much more important release of ferritin than when the mononuclear cells do not freeze.
    [0324]
    [0325] Example 3: Effect of different freezing times at -20 ° C on the values of the analytes obtained.
    [0326]
    [0327] In this example, 2 leishmaniasis sick dogs from Example 1 were used. Following the in vitro method of cell immunity evaluation of the present invention, both the total content of ferritin and TOS were determined as described in example 1, as well as content of said markers relative to the number of lymphocytes in the sample, using 30, 60 and 90 min freezing.
    [0328]
    [0329] The results are shown in Table 5 :
    [0330]
    [0331]
    [0332]
    [0333] Table 5
    [0334] It can be seen that, in general, there is an increase in analyte values when freezing times are increased.
    [0335]
    [0336] Example 4: Stability test.
    [0337]
    [0338] Ferritin and TOS were analyzed by the methods described above in the samples of two of the healthy animals and two of the sick animals directly after the in vitro method of the invention was performed, and subsequently after leaving the samples of the supernatant fluid from the passage (v ), 24 hours at room temperature. The results are shown in Table 6 :
    [0339]
    [0340]
    [0341]
    [0342] Table 6
    [0343]
    [0344] It is appreciated that both the values obtained from ferritin and those obtained from TOS are practically stable both if they are measured directly after isolating the supernatant in step (v) of the method of the invention, and if they are measured in the supernatant 24 hours later by keeping it at room temperature.
    [0345] Example 5: comparison between centrifugation speeds of step (iv).
    [0346]
    [0347] A whole blood sample was taken from a sick dog with leishmaniosis and the method of the invention was carried out, determining in that sample the ferritin (FRR) and TOS values by the methods described in example 1.
    [0348]
    [0349] The results are shown in Table 7 :
    [0350]
    [0351]
    [0352]
    [0353] Table 7
    [0354]
    [0355] It is appreciated that there are no differences in analyte values when the centrifugation rate changes.
    [0356]
    [0357] BIBLIOGRAPHY:
    [0358]
    [0359] - Brelaz de Castro et al., Cellular Immunology 279 (2012), 180-186
    [0360] - Arce Fonseca et al., Veterinary Research (2013), 44:15
    [0361] - Burleson et al., Immunology Testing, p. 195-205, Methods and Protocols, Methods in Molecular Biology, vol. 58, Chapter 14, Ed. Human Press (2010)
    [0362] - Erel et al., Clin Biochem 38 (2005), 1103-1111
    [0363] - Rubio CP, Yilmaz Z, et al., Res Vet Sci. 2017 Dec; 115: 301-306.
    [0364] - Goranov NV et al. Vet Clin Pathol. 2007 Jun; 36 (2): 192-5.
    [0365] - Park JI et al. Can J Vet Res. 2017 Jan; 81 (1): 37-40.
    权利要求:
    Claims (15)
    [1]
    1- In vitro method for evaluating cellular immunity from a biological fluid sample comprising mononuclear cells of an animal or a human being, wherein said method comprises:
    i. separate the mononuclear cells from the biological fluid sample;
    ii. suspend the separated mononuclear cells in a saline solution;
    iii. quantify the mononuclear cells present in the saline solution of mononuclear cells obtained in step (ii);
    iv. freeze the saline solution of mononuclear cells;
    v. thaw the mononuclear cell saline solution and then perform a centrifugation;
    saw. collect the supernatant liquid resulting from the centrifugation of step (v); and vii. determine the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid collected in step (vi)
    viii compare the content of at least one analyte indicator of inflammation and / or oxidative stress indicator determined the step (viii) with the average reference content of the same analyte obtained by reproducing said method under the same conditions from at least two samples of the same type of biological fluid that comprise mononuclear cells from animals or healthy humans.
    [2]
    2- In vitro method according to claim 1, wherein step (iv) is performed at a temperature of -20 ° C.
    [3]
    3- In vitro method according to any of claims 1 or 2, wherein step (iv) is carried out for at least 1 hour.
    [4]
    4- In vitro method according to any of claims 1 to 3, wherein in step (vii) the total content and / or the content is determined in relation to the number of mononuclear cells quantified in step (iii), of At least one analyte indicator of inflammation and / or oxidative stress.
    [5]
    5- In vitro method according to any one of claims 1 to 4, wherein the at least one analyte indicator of inflammation and / or oxidative stress is independently selected from the group consisting of ferritin, total oxidants (TOS), myeloperoxidase, protein C reactive, haptoglobin, antioxidant reducing capacity of cupric ion (CUPRAC), paraoxonase-1 (PONI), interleukins, total antioxidants (TAC), antioxidant iron status (FRAP), total esterase (TEA), catalase, nitrite, thiol or nitric oxide; or combinations thereof.
    [6]
    6- In vitro method according to any one of claims 1 to 5 wherein in step (vii) the content of at least ferritin and total oxidants (TOS) is determined.
    [7]
    7- In vitro method according to any one of claims 1 to 6, wherein step (iii) is performed in a hematology analyzer not based on flow cytometry technology.
    [8]
    8- In vitro method according to any of claims 1 to 7, wherein step (i) is performed by a density gradient separation.
    [9]
    9- Cellular immunity evaluation kit from a sample of biological fluid comprising mononuclear cells of an animal or a human being, comprising:
    to. a first support comprising a mononuclear cell separating solution by density gradient;
    b. a second support comprising a saline solution;
    C. a third suitable support for quantifying an analyte in a biochemistry analyzer;
    wherein said kit further comprises instructions, wherein said instructions comprise:
    i. add without mixing the biological fluid sample on the mononuclear cell separating solution of the first support and subject it to centrifugation; ii. collect the mononuclear cells of the biological fluid sample from the interface between the supernatant and the pellet formed in the centrifugation, and suspend them in the saline solution of the second support; iii. quantify the mononuclear cells present in the mononuclear cell saline solution of the second support obtained in step (ii); iv. freeze the mononuclear cell saline solution from the second support; v. thaw the mononuclear cell saline solution of the second support by allowing said support to stand at room temperature and then perform centrifugation;
    saw. collect the supernatant liquid resulting from the centrifugation of step (v) and deposit it on the third support; Y
    vii. determine the content of at least one analyte indicator of inflammation and / or indicator of oxidative stress in the supernatant liquid collected in step (vi) viii. compare the content of at least one analyte indicator of inflammation and / or oxidative stress indicator determined the step (viii) with the average reference content of the same analyte that is obtained by reproducing said method from at least two samples of biological fluid which comprise mononuclear cells of animals or healthy humans.
    [10]
    10. Kit according to claim 9 wherein the instructions comprise that the freezing in step (iv) is carried out at a temperature of -20 ° C.
    [11]
    11. Kit according to any of claims 9 or 10, wherein the instructions comprise that the freezing in step (iv) is carried out for at least 1 hour.
    [12]
    12. Kit according to any of claims 9 to 11, wherein the instructions comprise that step (vii) comprises determining the total content and / or the content in relation to the number of mononuclear cells quantified in step (iii), of at least one analyte indicator of inflammation and / or oxidative stress.
    [13]
    13. Kit according to any of claims 9 to 12, wherein the instructions comprise that the at least one analyte indicator of inflammation and / or oxidative stress is independently selected from the group consisting of ferritin, total oxidants (TOS), Myeloperoxidase, C-reactive protein, haptoglobin, cupric ion reducing antioxidant capacity (CUPRAC), paraoxonase-1 (PON1), interleukins, total antioxidants (TAC), iron antioxidant status (FRAP), total esterase (TEA), catalase, nitrite , thiol or nitric oxide.
    [14]
    14. Kit according to any of claims 9 to 13, wherein the instructions comprise that in step (vii) the content of at least ferritin and total oxidants (TOS) is determined.
    [15]
    15. Kit according to any of claims 9 to 14, wherein the instructions comprise that step (iii) is performed in a hematology analyzer not based on flow cytometry technology.
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