Kit containing solutions that eliminate the cd38 antigen

Abstract

The present invention relates to a kit that is used as a therapeutic agent in cases of multiple myeloma, and that is used to prevent false positivity in conformity tests performed (cross match, indirect agglutination, and antibody screening tests) before blood transfusion by causing panagglutination, and the application method thereof. The solutions contained in said test kit directly eliminate the CD38 antigen in the anti-CD38 monoclonal antibody, which affects the conformity tests, instead of blocking them, and prevent the monoclonal antibody from binding to the CD38 antigen present on the cell membrane of red blood cells, thereby preventing a false positive result.

Description

[0001] KIT CONTAINING SOLUTIONS THAT ELIMINATE THE CD38 ANTIGEN

[0002] Technical Field of the Invention

[0003] The present invention relates to a kit that is used as a therapeutic agent in cases of multiple myeloma, and that is used to prevent false positivity in compatibility tests performed (cross match, indirect agglutination, and antibody screening tests) before blood transfusion by causing panagglutination, and the application method thereof. The solutions contained in said kit directly eliminate the CD38 antigen on the erythrocyte surface instead of blocking them, and prevent the anti-CD38 monoclonal antibody, which affects the compatibility tests, from binding to the CD38 antigen on the cell membrane of red blood cells, thereby preventing false positive results.

[0004] State of the Art

[0005] Blood transfusion is the transfer of blood or substances of the blood, such as thrombocyte, plasma, or erythrocyte, to people who require it, through a needle. Blood is defined as a tissue. Therefore, a blood and blood product transfusion is a tissue transfusion, not a simple fluid infusion. Blood transfusion, which is defined as the introduction of blood or blood products into the circulation for therapeutic purposes, is a very common form of complex treatment. Blood transfusion reactions can have serious and dangerous consequences that may lead to death. For this reason, some compatibility tests should be performed between the donor and the recipient before the transfusion. Blood group determination, cross match, indirect agglutination, and antibody screening tests are some of the compatibility tests used before blood transfusion.

[0006] In the state of the art, anti-CD38 monoclonal antibody-based drugs (daratumumab, isatuximab, MOR202 etc.) used in cases of multiple myeloma causes false positivity by causing panagglutination in conformity tests applied before blood transfusion. This poses a problem in blood transfusion applications for immunohematology laboratories. There are test kits developed in the state of the art in order to prevent anti-CD38 monoclonal antibody-based drugs used in multiple myeloma cases from causing false positives by causing panagglutination in conformity tests performed prior to blood transfusion. However, dithiothreitol (DTT) chemical, which is included in test kits used in current techniques and which is a water-soluble and reducing reagent and quantitatively dissolves the disulfide bonds between cysteine amino acids, is a chemical that is very sensitive to temperature and light, and therefore it can decompose rapidly. Therefore, the difficulty of keeping the test kits containing DTT in blood banking conditions creates a problem during the preparation of the test solution. In addition, in order to dissolve disulfide bonds between cysteine amino acids, test protocols in which only DTT chemical is used are carried out for as long as 2 hours. Considering the urgency and importance of conformity tests and blood transfusion prior to blood transfusion to the patient, 2 hours spent in the blood banking is too long to ignore.

[0007] In another state of the art, in order to prevent anti-CD38 monoclonal antibody-based drugs used in cases of multiple myeloma in a study conducted by Ye Zhou et al. from causing false positives by causing panagglutination in existing tests performed prior to blood transfusion, 2-mercaptoethanol was used instead of DTT chemical to quantitatively dissolve the disulfide bonds between cysteine amino acids in test kits developed in the state of the art. However, the application time of the test protocols developed as a result of the use of the chemical 2-mercaptoethanol extends up to 2 hours.

[0001]

[0008] Although existing test kits developed to prevent anti-CD38 monoclonal antibody-based drugs from causing false positives by causing panagglutination in existing tests applied before blood transfusion are known to reduce the severity of panagglutination reactions seen in patients' conformity tests, said panagglutination reaction intensities cannot be completely eliminated.

[0009] In the state of the art, there are no studies conducted to prevent the test kits developed to prevent anti-CD38 monoclonal antibody-based drugs used in multiple myeloma cases from causing false positives by causing panagglutination in existing tests applied before blood transfusion from deteriorating and thereby ensuring their long-term storage under blood banking conditions. In addition, there is no study conducted to reduce the test protocols in existing techniques to a shorter period of time than a vital time such as 2 hours. In the state of the art, although test kits developed to prevent anti-CD38 monoclonal antibody-based drugs from causing false positivities by causing panagglutination in conformity tests applied before blood transfusion reduce the panagglutination reaction severity seen in the conformity tests of the patients, they cannot completely eliminate the panagglutination reaction severity.

[0010] It is necessitated to develop a test kit that completely eliminates the severity of panagglutination reaction that causes false positives in conformity tests performed before blood transfusion, that can be stored for a long time in blood banking and has a short application time due to reasons such as the limitations and inadequacies of the solutions in the state of the art, the absence of any development in the test kits developed to prevent anti-CD38 monoclonal antibody-based drugs from causing false positivities by causing panagglutination in conformity tests applied before blood transfusion to prevent their deterioration and thereby ensuring their long-term storage in blood banking conditions, the absence of any study performed to reduce the test protocols in the current techniques to a shorter period of time that cannot be ignored and is of vital importance, such as 2 hours, and the inability of test kits developed to prevent false positives by causing panagglutination in conformity tests applied before blood transfusion, to completely eliminate the panagglutination reaction intensities seen in the conformity tests of the patients.

[0011] Brief Description and Objects of the Invention

[0012] In the present invention, it is disclosed a kit that is used as a therapeutic agent in cases of multiple myeloma, and that is used to prevent false positivity in conformity tests performed (cross match, indirect agglutination, and antibody screening tests) before blood transfusion by causing panagglutination, and the application method thereof. The solutions contained in said test kit directly eliminate the CD38 antigen in the anti- CD38 monoclonal antibody, which affects the conformity tests, instead of blocking them, and prevent the monoclonal antibody from binding to the CD38 antigen present on the cell membrane of red blood cells, thereby preventing a false positive result.

[0013] An object of the present invention is to eliminate the panagglutination observed in conformity tests performed prior to blood transfusion in multiple myeloma patients treated with anti-CD38 antibody-derived drugs. In the present invention, Elimination of panagglutination observed in conformity tests performed before blood transfusion, in other words, prevention of false positivity is provided by blocking the CD38 molecule on the surface of the erythrocyte. When the kit of the present invention is applied to erythrocytes to be used in blood transfusion, false positivity is prevented by giving the expected results as if the patient's blood sample or anti-CD38 antibody-based drugs were not present.

[0014] Another object of the present invention is to directly eliminate the integrity of the CD38 antigen, which is found in anti-CD38 antibody-based drugs, which causes adverse effects in patients' conformity tests. In the present invention, directly eliminating the integrity of the CD38 antigen is achieved by the degradation of the molecular structure of the CD38 antigen after the use of DTT and 2-mercaptoethanol in specific ratios in the content of the test kit of the present invention.

[0015] By means of the invention, a test kit with a long shelf life is provided under blood banking conditions to be used in conformity tests before blood transfusion. In the present invention, test kit with long shelf life in blood banking conditions to be used in conformity tests before blood transfusion is provided by stabilizing the DTT chemical, which is included in the test kits and which is a temperature and light sensitive, and rapidly degrading agent.

[0016] By means of the kit, which is the subject of the invention, the seventy of panagglutination, which causes false positivity in conformity tests applied before blood transfusion, is completely eliminated. In the present invention, complete elimination of the seventy of panagglutination that causes false positives is achieved by using specific ratios of DTT and 2-mercaptoethanol chemicals together.

[0017] Yet another object of the present invention is to shorten the duration of conformity tests performed before blood transfusion. In the present invention, eliminating the panagglutination observed in the conformity tests performed before the blood transfusion in a short time such as 30-45 minutes and completing the test is achieved by DTT and 2-mercaptoethanol chemicals used in specific ratios. Detailed Description of the Invention

[0018] In the present invention, it is disclosed a kit that is used as a therapeutic agent in cases of multiple myeloma, and that is used to prevent false positivity in conformity tests performed (cross match, indirect agglutination, and antibody screening tests) before blood transfusion by causing panagglutination, and the application method thereof. The solutions contained in said test kit directly eliminate the CD38 antigen in the anti- CD38 monoclonal antibody, which affects the conformity tests, instead of blocking them, and prevent the monoclonal antibody from binding to the CD38 antigen present on the cell membrane of red blood cells, thereby preventing a false positive result.

[0019] The test kit of the present invention comprises 1 mL primary solution containing 2.25- 2.7 g sodium chloride (NaCI), 0.08-0.15 g potassium phosphate (KH2PO4), 2-2.5 g sodium phosphate dibasic dihydrate (Na2HPO4.2H2O), 0.8-1 .2 g sodium nitrogenide (NaNs), 0.005-0.02 mL Tween-20, and 0.05-0.15 mL bovine serum albumin (BSA), per liter, and a second solution containing 140-160 mg of DTT (dithiotreitol), 0,05-0,15 mL BSA, 0.0020-0.0026 g KH2PO4, and 0.01 -0.03 mL 2-mercaptoethanol in 10 milliliters

[0020] Application method of the test kit of the present invention, which prevents false positive reaction in conformity tests by eliminating anti-CD38 antibody effect of anti-CD38 monoclonal antibody and anti-CD38 monoclonal antibody-based drugs that are used as therapeutic agents in cases of multiple myeloma and that cause panagglutination and affect conformity tests prior to blood transfusion, in other words inhibiting the agglutination effect, comprises the process steps of; i. preparing 1 mL primary solution containing 2.25-2.7 g NaCI, 0.08-0.15 g KH2PO4, 2-2.5 g Na2HPO4.2H2O, 0.8- 1.2 g NaN3, 0.005-0.02 mL Tween-20, and 0.05-0.15 mL BSA in 1 liter, ii. preparing the second solution containing 140-160 mg DTT, 0.05-0.15 mL BSA, 0.0020-0.0026 g KH2PO4, and 0.01 -0.03 mL 2-mercaptoethanol in 10 milliliters, iii. Diluting the second solution with the primary solution at a ratio of 1 / 5, iv. adding 40-50 pl of erythrocyte suspension prepared as 1 %-0.8% and 25- 35 pl of second solution diluted 1 / 5 into a well of the antihuman globulin (AHG) card used when performing cross-match, indirect agglutination, and antibody screening tests, v. pipetting 5 times inside the anti-human globulin card microtube, and then incubating the anti-human globulin card at 35-38°C for 15-30 minutes for mixing the erythrocytes and the 1 / 5 diluted second solution, vi. adding 25-35 pl of patient serum into the well of the anti-human globulin card, which has finished incubation, containing the erythrocytes treated with the second solution diluted 1 / 5, and mixing the patient serum added to the anti-human globulin card by pipetting 5 times, and incubating said card at 35-38°C for 15 minutes, vii. centrifugating the incubated anti-human globulin card at 990 rpm, viii. examining the anti-human globulin card result as a result of centrifugation.

[0021] Said test kit should be kept away from light and stored at 2-8°C.

[0022] In multiple myeloma patients treated with anti-CD38 antibody-derived drugs with the test kit of the present invention, the panagglutination problem observed in the conformity tests performed before the blood transfusion and the preventing false positivity as a result of this problem are provided by blocking the CD38 molecule on the surface of the erythrocyte. Eliminating the negative effects of anti-CD38 antibodybased drugs in patients' conformity tests by completely eliminating the seventy of panagglutination is achieved by inactivating the integrity of the CD38 antigen by complete elimination with the use of DTT and 2-mercaptoethanol at specific rates. Said specific ratios are 150 mg DTT and 0.02 mL 2-mercaptoethanol contained in the 10 mL second solution. The technical effect of 150 mg DTT and 0.02 mL 2- mercaptoethanol used has been proven as a result of the studies. As a result of the use of specific ratios of 150 mg DTT and 0.02 mL 2-mercaptoethanol, it has been proven that the rate of reduction in the seventy of the panagglutination reaction reaches 100% while the rate of decrease in the severity of the panagglutination reaction is 50% when only DTT is used in the test kit, which is the subject of the invention, and the reduction rate of the panagglutination reaction is 83% when only 2-mercaptoethanol is used, In other words, as a result of using 150 mg DTT and 0.02 mL 2-mercaptoethanol, panagglutination and therefore false positives caused by panagglutination are completely eliminated. Table 1. The results of the effectiveness of the use of 150 mg DTT and 0.02 mL 2- mercaptoethanol included in the test kit, which is the subject of the invention, on the cross-match test applied before blood transfusion. The storage problem in blood banking conditions is prevented by means of the the stabilization of the DTT chemical, which is a temperature-sensitive and rapidly degrading agent, contained in the test kit of the present invention developed to prevent anti-CD38 monoclonal antibody-based drugs used in cases of multiple myeloma from causing false positives by causing panagglutination. Stabilization of DTT chemical, which is a light sensitive and rapidly degrading agent, is provided by using the primary solution containing 2.5 g NaCI, 0.12 g KH2PO4, 2,25 g Na2HPO4.2H2O, 1 g NaNs, 0.01 mL Tween-20, and 0.1 mL BSA, which has the specific ratios used in the content of the test kit, which is the subject of the invention, and thus, the problem of preservation in blood banking conditions is eliminated. By means of the application method of the test kit of the present invention developed in order to prevent anti-CD38 monoclonal antibody-based drugs used in multiple myeloma cases from causing false positives by causing panagglutination, panagglutination observed in conformity tests performed before blood transfusion is eliminated and the test result, in which false positivity is prevented, can be obtained in a short time such as 30-45 minutes. Considering the importance of blood transfusion on human life, this period has been shortened to a degree that cannot be ignored. While applying the test kit of the present invention developed in order to prevent anti- CD38 monoclonal antibody-based drugs used in multiple myeloma cases from causing false positives by causing panagglutination, no other equipment is required other than the equipment in the blood centers. The equipments used are listed as simple centrifuge device, pipette, and AHG card.

[0023] REFERENCES

[0024] 1. 2-mercaptoethanol (2-me)-based IATS or polybrene ... (n.d.). Retrieved

[0025] November 30, 2021 , from ME-based_IATs_or_polybrene_method_mitigates_the_interference_of_anti-

[0026] CD38_immunotherapy_on_blood_compatibility_tests.

Claims

CLAIMS A test kit for use in cross-matching, indirect agglutination, and antibody screening tests prior to blood transfusion in order to prevent false positive reaction by eliminating the anti-CD38 antibody effect caused by anti-CD38 monoclonal antibody-based drugs, characterized by comprising; 1 mL primary solution containing 2.25-2.7 g sodium chloride (NaCI), 0.08-0.15 g potassium phosphate (KH2PO4), 2-2.5 g sodium phosphate dibasic dihydrate (Na2HPO4.2H2O), 0.8-1 .2 g sodium nitrogenide (NaNs), 0.005-0.02 mL Tween- 20, and 0.05-0.15 mL bovine serum albumin (BSA), in 1 liter, and a second solution containing 140-160 mg of dithiotreitol ( DTT), 0.05-0.15 mL bovine serum albumin (BSA), 0.0020-0.0026 g potassium phosphate (KH2PO4), and 0.01 -0.03 mL 2-mercaptoethanol in 10 milliliters Application method of a test kit that prevents false positive reaction in conformity tests by eliminating the anti-CD38 antibody effect of anti-CD38 monoclonal antibody-based drugs, characterized by comprising the process steps of; i. preparing 1 mL primary solution containing 2.25-2.7 g sodium chloride (NaCI), 0.08-0.15 g potassium phosphate (KH2PO4), 2-2.5 g sodium phosphate dibasic dihydrate (Na2HPO4.2H2O), 0.8-1 .2 g sodium nitrogenide (NaNs), 0.005-0.02 mL Tween-20, and 0.05-0.15 mL bovine serum albumin (BSA), in 1 litre, ii. preparing the second solution containing 140-160 mg d ith iothreitol (DTT), 0.05-0.15 mL bovine serum albumin (BSA), 0.0020-0.0026 g potassium phosphate (KH2PO4), and 0.01 -0.03 mL 2-mercaptoethanol, in 10 milliliters, iii. Diluting the second solution with the primary solution at a ratio of 1 / 5, iv. adding 40-50 pl of erythrocyte suspension prepared as 1 %- 0.8% and 25- 35 pl of second solution diluted 1 / 5 into a well of the antihuman globulin card used when performing cross-match, indirect agglutination, and antibody screening tests,V. pipetting 5 times inside the anti-human globulin card microtube, and then incubating the anti-human globulin card at 35-38°C for 15-30 minutes for mixing the erythrocytes and the 1 / 5 diluted second solution, vi. adding 25-35 pl of patient serum into the well of the anti-human globulin card, which has finished incubation, containing the erythrocytes treated with the second solution diluted 1 / 5, and mixing the patient serum added to the anti-human globulin card by pipetting 5 times, and incubating said card at 35-38°C for 15 minutes, vii. centrifugating the incubated anti-human globulin card at 990 rpm, viii. examining the anti-human globulin card result as a result of centrifugation. A test kit according to Claim 1 , characterized in that, said anti-CD38 monoclonal antibody-based drugs are daratumumab, isatuximab, or MOR202. Application method of a test kit according to Claim 2, characterized in that, said anti-CD38 monoclonal antibody-based drugs are daratumumab, isatuximab, or MOR202. A test kit according to Claim 1 , characterized in that, said test kit is used in conformity tests. Application method of a test kit according to Claim 2, characterized in that, it is used in conformity tests.

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