RU2416795C1 - Method for prediction of clinical course of acute leukemia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for prediction of clinical course of acute leukemia in children, characterised by the fact that peripheral blood is analysed for cell ploidy and kinetics by flow cytometry, and if observing aneuploidy and DNA index less than 1.19, unfavourable outcome is predicted.

EFFECT: method provides more accurate and reliable prediction of the clinical outcome.

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Description

The present invention relates to medicine, namely to immunology, and can be used to predict the course in the complex diagnosis of acute leukemia (OL) in children.

The high relevance of the problem of acute leukemia in children is due to both the increase in the number of patients suffering from neoplasms, and the characteristics of the course of the disease, and a high mortality rate.

In the structure of infant mortality in the Russian Federation, neoplasms, diseases of the blood and blood-forming organs take 6th place after accidents, certain conditions of the perinatal period, congenital malformations, respiratory diseases and infectious diseases. According to statistics from the Ministry of Health of the Republic of Bashkortostan, the incidence of acute leukemia is an average of 3.7 per 100,000 children.

At the present stage of development of oncohematology, close attention is paid to the problems associated with the diagnosis of AL; the main direction of modern research is the diagnosis and verification of acute leukemia with in-depth biological characteristics of cells by flow cytometry. Not all issues have been resolved regarding the prognostic value of the expression of certain antigens in acute leukemia and the development of treatment tactics. In this regard, a comprehensive study of the characteristics of leukemia in children, the determination of diagnostic and prognostic criteria for the disease using the latest technologies seems to be very relevant.

A known method for predicting the nature of the course of acute lymphoblastic leukemia in children, which consists in the fact that the number of autosockets and the intensity of exocytic lysis of red blood cells are determined in smears of peripheral blood of patients. With the initial absolute number of autosockets with exocytic lysis of erythrocytes in the active phase of acute lymphoblastic leukemia of more than 13.0 • 10 ⁹ / L, the onset of early relapse of leukemia and (or) the development of extra-cerebral proliferates is predicted, and with the initial content of the number of autosockets with exocytic lysis of erythrocytes 3.0 • 10 ⁹ / l predict long-term remission (patent RU 2152028, 2000).

The prototype of the invention is a method for predicting the nature of the course of acute lymphoblastic leukemia in children, proposed by Chernov, characterized in that the ploidy and DNA index of bone marrow cells are determined by flow cytometry and, if hyperploidy and a DNA index of more than 1.16 are detected, a positive treatment outcome is predicted (Khamatdinova Z. R. Immunophenotypic variants of acute leukemia in children in the Republic of Bashkortostan. Thesis for the degree of candidate of biological sciences. Ufa. 2005, p.5). The method is time consuming and invasive, as it involves the collection of bone marrow cells.

The task of the invention is to develop an effective and easy to use method for predicting the course of acute leukemia, expanding the arsenal of diagnostic methods.

EFFECT: obtaining criteria for the course of the disease based on indicators of ploidy and kinetics of peripheral blood cells, reducing invasiveness during diagnosis.

The proposed method is as follows. Peripheral blood sampling is performed in an amount of 5 ml in the morning on an empty stomach. The ploidy and kinetics of peripheral blood cells are determined by flow cytometry. If aneuploidy and a DNA index of <1.19 are detected (a marker characterizing the aggressiveness of the disease), an unfavorable outcome is predicted. The diploid type of histograms is prognostically favorable.

The proposed method is safe, non-invasive, technically feasible.

Research method

DNA analysis in tissues and cell suspension.

To characterize the proliferative activity of cells, a method is used based on the binding of DNA to a fluorescent probe - propidium iodide, which allows you to determine the proportion of cells in various phases of the mitotic cycle: G0 / G1, S and G2 + M.

Reagents The CyclTEST PLUS DNA kit consists of four components.

- Trypsin in a solution of detergent (spermine tetrachloride) for enzymatic disaggregation of tissue sites, destruction of the cell membrane and cytoskeleton.

- Inhibitor of trypsin and ribonuclease A in citrate buffer with spermine tetrachloride to suppress the effects of trypsin and RNA cleavage.

- Propidium iodide (PI) and spermine tetrachloride in citrate buffer. PI binds to DNA at a concentration of 125 µg / ml.

- A buffer solution containing sodium citrate, sucrose and dimethyl sulfoxide (DMSO) to collect and / or freeze the cell suspension.

Staining procedure. Trypsin buffer 250 μl, trypsin inhibitor with RNAse 200 μl and a solution of propidium iodide 200 μl with intermediate stirring and 10-minute incubations are successively added to a test tube with 100 μl of blood, then the sample is filtered through nylon tissue with 50 μm cells. Samples within 3 hours after the addition of PI (Vindelov L., 1989) (at least 20,000 cells) were analyzed on a FACSCalibur flow cytometer, equipped with a duplet discrimination module in the ModFit program.

The invention is illustrated by the following drawings: figure 1 shows a DNA histogram of peripheral blood cells of patient L. according to example 1; figure 2 is a DNA histogram of peripheral blood cells of a patient M. according to example 2; figure 3 - DNA histogram of peripheral blood cells of a patient R. according to example 3.

The diagnosis of acute leukemia is established on the basis of a cytomorphological study and immunophenotyping of bone marrow blast cells, complaints, medical history, and the clinical picture of the disease. The paper analyzes indicators of the immune status and DNA cytometry of peripheral blood in 16 children with B-ALL.

Depending on the ploidy of peripheral blood cells, children with B-ALL were divided into two groups (with or without aneuploid cells).

The obtained results became the basis for the recommendation to include DNA cytometry of peripheral blood cells in immunological monitoring before starting chemotherapy.

Having analyzed these two groups of patients, we can say that significant differences relate only to the ploidy and kinetics of the cell cycle in peripheral blood. But, analyzing patients with aneuploidy, we can say that, although morphological remission was achieved by the 15th day of chemotherapy, these children (unlike children with a diploid set of chromosomes) had a significantly worse quality of life (frequent infections, cardiac complications development of secondary tumors).

In 3 children with aneuploidy during the course of polychemotherapy, aplasia of hematopoiesis was accompanied by a septic process. Against the background of the therapy, mortality occurred. The DNA index in these cases was <1.19.

The proposed method is illustrated by the following examples.

Example 1. Patient L., upon admission on the basis of bone marrow immunophenotyping, was diagnosed with B-2 ALL. When conducting DNA cytometry of peripheral blood, aneuploidy was - 5% (figure 1). The DNA index is 1.16. Polychemotherapy was started. Aplasia of blood formation was accompanied by a septic process. Against the background of the therapy, death has occurred.

Example 2. Patient M., upon admission on the basis of bone marrow immunophenotyping, was diagnosed with B-2 ALL. In bone marrow, blasts were 100%. When conducting DNA cytometry of peripheral blood, the histogram was characterized by a diploid type (figure 2). After chemotherapy, the child was discharged home in remission.

Example 3. Patient R. upon admission on the basis of bone marrow immunophenotyping was diagnosed with ALL-2. In bone marrow, blasts were 100%. When conducting DNA cytometry of peripheral blood, aneuploidy was 7% (figure 3). The DNA index is 1.19. Polychemotherapy was started. On the 33rd day, no blast cells were found in the bone marrow; 100% diploidy was detected with peripheral blood DNA cytometry. After programmed chemotherapy, the child was discharged home in remission.

Claims (1)

A method for predicting the course of acute leukemia in children, characterized in that the peripheral blood determines the ploidy and kinetics of the cells by flow cytometry and, when aneuploidy and a DNA index of less than 1.19 are detected, an unfavorable outcome is predicted.

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