RU2790833C1 - Method for determining antierythrocytic alloantibodies in patients with multiple myeloma - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular, to haematology, namely to treating patients with multiple myeloma administered treatment with the medical product Daratumumab. Method for determining antierythrocytic alloantibodies in patients with multiple myeloma in an indirect antiglobulin test using pretreatment of test erythrocytes includes pretreatment conducted using monoclonal antibodies anti-CD38 (F(ab′)2) taken in a volume ratio of 1:5 to the sample.

EFFECT: higher reliability of alloantibody screening assessment.

1 cl, 2 ex

Description

The claimed invention relates to medicine, and in particular to hematology, namely to the treatment of patients with multiple myeloma (MM) receiving treatment with the drug Daratumumab. It can find application in hematological practice.

Daratumumab (international non-proprietary name) is a human monoclonal antibody to the CD38 protein of the IgG class. In patients with MM, plasma cells (malignant clones) express a large amount of CD38 molecules, and treatment is aimed at eliminating these malignant cells.

Drugs based on monoclonal anti-CD38 antibodies have been introduced into the clinical practice of MM therapy, which has significantly improved the results of treatment of patients with multiple myeloma. However, the use of this drug introduced a number of previously unknown problems when conducting pre-transfusion tests.

In the complex treatment of a patient with MM, one of the types of therapy is the transfusion of erythrocyte suspension (hemocomponent therapy). In this regard, a mandatory study is the screening of anti-erythrocyte alloantibodies, and its conduct is regulated by guidelines. Screening of alloantibodies allows you to identify alloantibodies of clinical significance in the recipient (patient). Transfusion of donor erythrocytes with an antigen to which the recipient has alloantibodies to the recipient will lead to the development of a post-transfusion complication of the hemolytic type in the recipient. The severity of a post-transfusion complication will depend on the specificity and titer of antibodies and the timely medical care provided. These complications can lead to disability and even death.

Therapy with anti-CD38 drugs is accompanied by binding not only to pathological cells, but also to other cells expressing CD38. However, drugs based on anti-CD38 monoclonal antibodies make it difficult to interpret the screening and identification of alloantibodies in an indirect antiglobulin test (NAGT), because they cause in vitro panreactivity, which leads to false positive results that prevent further treatment of the patient (hemocomponent therapy).

A false positive result leads to the fact that the donor hemocomponents are incompatible with the recipient. False-positive results in NAHT, which make it difficult to interpret the results of alloantibody screening in such patients, can be obtained within 6 months after discontinuation of treatment with these drugs.

A known method for determining anti-erythrocyte alloantibodies in patients with multiple myeloma receiving treatment with Daratumumab [Chapui CI., Nicholson RT, Aguad MD et al. Transfusion 2015; 55(6(pt)2); 1545-1554; Chapui CI, Aguad MD, Nicholson RT et al. Blood 2015; 126 (Suppl): Abstract 3567], which includes the treatment of test erythrocytes used for screening and identification of antibodies with dithiothreitol, which denatures proteins and fragments of CD38 receptors located on the surface of erythrocytes. The preparation of control samples consists in the treatment of donor K ⁺ erythrocytes with dithiothreitol, incubation and centrifugation. If the antigens are not completely denatured, then the processing procedure is repeated until the antigen is completely denatured.

Dithiothreitol is toxic and requires special precautions when handling it. The known method is time-consuming and lengthy (determination takes 3-3.5 hours). However, the treatment of test erythrocytes with dithiothreitol, in addition to laboriousness and duration, has a significant drawback - it violates the integrity of erythrocytes, causing denaturation of antigens of the Kell, Dombrock, Yt and Lutheran systems, which makes it impossible to determine the presence of alloantibodies to these antigens.

The purpose of the claimed invention, in addition to reducing the duration of the process, is to increase the reliability of the assessment of screening of alloantibodies and the establishment of the specificity of alloantibodies in patients with MM receiving therapy with the drug Daratumumab.

This result is achieved by the fact that in the method for determining alloimmune antierythrocyte antibodies in patients with multiple myeloma in an indirect antiglobulin test using pretreatment of test erythrocytes, pretreatment is carried out using anti-CD38 monoclonal antibodies (F(ab')2), taken in a volume ratio to a sample equal to 1:5. Treatment of monoclonal antibodies anti-CD38 (F(ab')2) does not violate the integrity of erythrocyte antigens, allowing the detection of clinically significant antibodies to antigens of all erythrocyte blood group systems in NAG, which is an undoubted advantage in comparison with the generally accepted technology of treatment with dithiothreitol.

It is known to use a diagnostic reagent - monoclonal antibodies CD38(F(ab')2) - for typing antigens on leukocytes [Kudryavtseva IV, Borisov AG and other Ross. Journal of Immunology, 2015, 9(18), No. 2, pp. 42-47]. RBCs do not carry CD38 molecules, and we are not aware of the use of CD38(F(ab')2) monoclonal antibodies for typing RBC antigens.

The specified processing is carried out directly in the test tube (microtube) - 50 μl of test erythrocytes (tests I-II-III of the panel for screening anti-erythrocyte antibodies) and 10 μl of CD38 monoclonal antibodies (F(ab')2) are placed in each microtube. Incubation is carried out for 10 minutes at a temperature of +37°C. At this stage, proteins and fragments of CD38 receptors are neutralized.

Further, in the same microtubes, an indirect antiglobulin test is performed in the classical formulation [Mineeva N.V. Blood groups. Fundamentals of immunohematology, St. Petersburg, A-Print, 2007, 186 pp.]: 25 μl of the patient's serum is added to each microtube with processed test erythrocytes, incubated for 15 minutes at a temperature of +37 ° C, followed by centrifugation for 10 minutes ( 1000g). The result is evaluated by the presence or absence of agglutination.

When a positive screening result is obtained (antibodies are detected), it is necessary to establish the specificity of the antibodies, that is, to determine which antigen the antibodies are directed against (antibody identification). Identification of anti-erythrocyte alloantibodies is carried out using NAHT using an extended panel of test erythrocytes, represented by at least eleven samples of erythrocytes with different phenotypes. This treatment is carried out directly in the test tube (microtube): 50 μl of test erythrocytes (1-11 panel for the identification of anti-erythrocyte antibodies) and 10 μl of CD38 monoclonal antibodies (F(ab')2) are placed in each microtube. Incubation is carried out for 10 minutes at a temperature of +37°C, followed by centrifugation and interpretation of the result.

Further, the method is illustrated by examples of a specific embodiment.

Example 1

Patient G. with a diagnosis of multiple myeloma is receiving therapy with the drug Daratumumab. According to clinical indications, the patient was prescribed a transfusion of erythrocyte mass.

When screening anti-erythrocyte alloantibodies in NAGT, agglutination of different reaction strengths (from 1+ to 3+) is observed. It is impossible to make a conclusion about the presence or absence of antibodies of clinical significance.

The test erythrocytes used for screening were treated with monoclonal antibodies CD38(F(ab')2). Treatment with monoclonal antibodies CD38(F(ab')2) eliminated non-specific binding of test erythrocytes. Repeated screening showed the absence of alloimmune antibodies in the patient. The transfusion of erythrocyte mass was successfully carried out.

There were no reactions or complications during and after transfusions.

Example 2

Patient V. diagnosed with multiple myeloma is receiving therapy with the drug Daratumumab. According to clinical indications, the patient was prescribed a transfusion of erythrocyte mass.

When screening anti-erythrocyte alloantibodies in NAGT, agglutination of different reaction strengths (from 1+ to 3+) is observed. It was not possible to conclude on the presence or absence of antibodies of clinical significance.

Treatment of test erythrocytes for screening with CD38(F(ab')2) monoclonal antibodies eliminated non-specific binding, and when screening was repeated, agglutination was observed in tubes I and II, which made it possible to conclude the presence of clinically significant antibodies. As a result of the identification of antibodies with the processed extended panel of test erythrocytes, the specificity of anti-D alloantibodies was established. For such a patient, an individual selection of a donor erythrocyte mass is necessary.

3 doses of erythrocytes that do not have antigens, to which the recipient has alloantibodies, were delivered for selection. With two of the three unprocessed donor RBC samples, the recipient's plasma samples were incompatible. After treatment with anti-CD38 monoclonal antibodies, all donor erythrocyte samples were compatible with the recipient's plasma.

There were no reactions or complications during and after transfusions.

As can be seen from the above data, the treatment of test erythrocytes for screening and identification of antibodies with the diagnostic reagent "monoclonal antibody anti-CD38 (F(ab')2)" makes it possible to neutralize nonspecific agglutination in plasma samples containing the drug Daratumumab, which makes it possible to reliably determine specificity of clinically significant anti-erythrocyte alloimmune antibodies. Moreover, this processing method preserves the integrity of erythrocyte antigens, making it possible to detect clinically significant antibodies to antigens of all erythrocyte blood group systems in NAHT, which is of great clinical importance and is an undoubted advantage of the proposed method.

Claims (1)

A method for determining anti-erythrocyte alloantibodies in patients with multiple myeloma in an indirect antiglobulin test using pre-treatment of test erythrocytes, characterized in that pre-treatment is carried out using anti-CD38 monoclonal antibodies (F(ab')2), taken in a volume ratio to the sample, equal to 1:5.