SU1589215A1 - Method of predicting recurrencies of acute lymphoblastic leukosis - Google Patents

Abstract

The invention relates to the field of medicine, in particular hematology, and concerns the early pre-morphological immunological diagnosis of recurrent acute lymphoblastic leukemia (ALL). The purpose of the invention is to improve the accuracy of early diagnosis of disease recurrence before the appearance of its clinical and morphological signs. The method is performed as follows: in the acute phase of the disease, using a panel of monoclonal antibodies (MAb), differentiation antigens characteristic of the lymphoblasts of each patient are detected, in the reaction of indirect immunofluorescence in the microwell modification, a panel of µA against antigens was developed: T6, T9, T10 , CALLA, Ia, ICO-II. Leukemia lymphoblasts are obtained from the peripheral blood or bone marrow of patients in the acute phase of the disease. Accounting for the reaction is carried out using a fluorescent microscope with an additional viewing of drugs in phase contrast. Similarly, with the same ICA panel, a study was carried out on the antigens of lymphocytes isolated from the peripheral blood of patients in remission. The studies are repeated regularly throughout the remission with an interval of 1-4 months. The appearance of lymphocytes that positively react with at least one of the used MCAs, in amounts exceeding their normal limits, is a precursor of the onset of a relapse of the disease. The positive effect of the method lies in the fact that the proposed ICA panel allows recognition of 96% of leukemic cells and early diagnosis of disease recurrence in the maximum number of cases on mature lymphocytes of any of the antigens characteristic of leukemic lymphoblasts in the acute period.

Description

The invention relates to the field of medicine, in particular, hematology, and concerns the issue of early pre-morphological immunological diagnostics.

recurrent acute lymphoblastic leukemia (ALL).

; The purpose of the invention is to improve the accuracy, early diagnosis of relapse for

diseases before the appearance of its clinical and morphological signs.

The method is carried out as follows.

In the acute phase of the disease, with the help of the developed ICA panel, the differentiation antigens characteristic of lymphoblasts of each patient in the patient, in the reaction of indirect immunofluorescence, are revealed. In the 1C remission period, the same ICA panel regularly with an interval of 1–4 months was used to study morphologically mature peripheral blood lymphocytes. In the case of lymphocytes positively reacting with at least one of the associated MCAs. in quantities that exceed their normal limits (M + 36), is a precursor of the onset of a relapse of the disease. )

The study of leukemic lymphoblasts antigens was carried out in the reaction of indirect immunofluorescence in microwell modification using six ICA preparations included in the developed panel. Leukemia lymphoblasts are obtained after lysing erythrocytes from 5-10 ml of heparinized peripheral blood or 1 ml of bone marrow taken from patients in the acute phase of the disease. The suspension is adjusted to a concentration of 4-5 IO cells in 1 ml of Hank's solution and attached to the glass surface of microwells using poly-b-lysine. Cells are first treated with the indicated preparations of MKM (20 µl) pr; incubation for 30 min at 40 ,, I and then after washing the microwells twice with Hank's solution, FITC-labeled goat anti-mouse antibodies - globulin (20 µl each) are applied to the cells. After incubation at 4 ° C for 30 min, the preparations are washed twice with a solution Hank and microwells are poured over 5 µl of 50% glycerol dilution. Prepare covered with cover glass and sealed. The reaction was recorded on a fluorescent microscope with a control view of the cells in phase contrast. Differential antigens of lymphoblasts are determined by the presence of surface luminescence on the cells. Similarly, with the same ICA panel, the antigens of lymphocytes isolated from the peripheral blood of patients in remission from

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by gradient centrifugation. The studies are repeated regularly throughout the remission with an interval of 1-4 months.

Example 1, Patient B. 1 5 years. Diagnosis: acute lymphoblastic leukemia. Blast cells isolated from 5 ml of heparinized blood in the acute period of the disease and studied with the developed ICA panel contained antigens 1a, ICO-1 1, CALLA, TY, T9 (the number of positive antigen cells was 91.55 70.90 and 50%, respectively ). The T6 antigen on the patient's lymphoblasts was not identified. At the time of ascertaining the complete clinical and hematological remission achieved after 2 months, the number of morphologically mature blood lymphocytes containing the laj, ICO-1 1, CALLA, 165-19, T10 antigens corresponded to normal values. However, after 1 month in the period of complete clinical hematological remission (in the presence of bone marrow in punctate 3% lymphoblasts) after repeated studies of blood lymphocytes with the same ICA panel, an increase in the number of cells with ICO-11 antigens (68%) T10 (18%) was detected with a normal number of lymphocytes, containing a La, CALLA, T6, T9 immunogens A relapse of the disease was detected after 1.5 months during a morphological study of bone marrow punctate (the number of lymphoblasts in it was 12.5% with a subsequent steady progression of the disease).

Example 2. Patient L „, 18 years. Diagnosis: acute lymphoblastic leukemia. Blast cells isolated from 5 ml of heparinized blood in the acute period of the disease, when examined with the developed ICA panel, contained la, PID-11 antigens (the number of antigen-positive cells was 70 and 85%, respectively). CALLA, T6, 19, T10 antigens on the lymphoblasts of the patient were not detected. One month after the statement of complete clinical and hematological remission (if 4% lymphoblasts are present in bone marrow punctate), the morphologically mature blood lymphocytes were studied with the same ICA panel along with the normal number of cells containing antigens 1a, CALLA, Tb, T9 and T10, increased populations l 1

cells containing antigen ICO-11 (46%). A recurrence of the disease was detected by clinical morphological studies after 3 months (the number of blast cells in the peripheral blood was 80%).

These examples show that using a known method would not allow predicting the recurrence of the disease. Thus, the proposed method allows to improve the accuracy of the immunological prediction of recurrence of ALL to the appearance of its clinical and hematological signs due to the use of not one, but several MCA directed to various differential antigens detected on leukemia lymphoblasts to diagnose .

The method can be applied in almost all patients with ALL, since with the help of the developed ICA panel, leukemic cells are recognized in order 89891

most cases (in 96% of patients).

Claims (1)

Invention Formula A method for predicting the recurrence of acute lymphoblastic leukemia by detecting a differentiation tigene with monoclonal antibodies on leukemic lymphoblasts in the acute period and lymphocytes during remission and upon detection of this antigen during remission in quantities of 5 wahs exceeding the limits of their normal values, a relapse of the disease is expected, differing by the fact that, in order to increase the accuracy of prediction, a complex of specific monoclonal antibodies is used to detect an20 tigens, and when detecting one second or more antigens predict relapse of disease.