SU1647404A1 - Method of differential diagnosis of idiopathic thrombocytopenic purpura and hypoplastic anemia - Google Patents

Abstract

The invention relates to medicine and can be used in the differential diagnosis of blood disorders, especially from atypical forms. The goal is to improve the accuracy of diagnosis. The immunological method is based on a specific reaction of sensitized antigen binding lymphocytes of a patient with a platelet antigen, including the isolation of lymphocytes from the patient's blood, washing them away from plasma factors, incubating the first portion of lymphocytes with the platelet antigen, washing out lymphocytes from an inappropriate anti-inflammatory body. a portion of washed lymphocytes. When the number of lymphocyte agglutinates is equal to and greater than 2.5% relative to the control, idiopathic thrombocytopenic purpura (ITP) is diagnosed, and with a result less than 2.5%, hypoplastic anemia (HA) is diagnosed. With atypical forms of ITP and HA, the diagnostic accuracy is increased to 89 -93%. (Ls

Description

The invention relates to medicine, namely to hematologists, and can be used in the diagnosis of blood diseases.

The aim of the invention is to improve the accuracy of the method.

The goal is achieved by isolating lymphocytes from the patient’s blood, washing off plasma factors, incubating the first batch of lymphocytes with a platelet antigen, then washing them from the unbound antigen, and re-incubating with the second batch of washed lymphocytes, and with 2: 2 lymphocytic agglutinates, 5% diagnose ITP, and with a result 2.5% diagnose HA.

The essence of the method lies in the fact that the patient's blood is tested, while: lymphocytes are isolated from the blood; lymphocytes are washed from plasma.

Careful plasma washing is necessary, since we found out that, with this pathology, the patient's plasma contains factors that distort the results of the study and reduce the accuracy of the method. Autoantibodies directed against the test antigen, endogenous toxins, blocking factors and

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The obtained lymphocytes are incubated with pre-prepared platelet antigen. At this stage, the antigen is specifically bound to the membranes of sensitized lymphocytes.

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Lymphocytes are washed away from unbound antigen in order to exclude its participation in the further process.

The resulting lymphocytes are reincubated with a new portion of lymphocytes washed from plasma of the same patient. In this case, the sensitized lymphocytes of the new portion through the antigenic bridge bind to the previously incubated lymphocytes, forming agglutinates.

The result is examined under a microscope by counting the percentage of agglutins to free-lying lymphocytes. When the number of lymphocyte agglutinates is equal and more than 2.5%, the diagnosis of ITP is made, and with the result less than 2.5% is diagnosed with HA.

Preparation of antigen from human platelets.

10-12 ml of donor blood is taken from the cubital vein, for heparin solution (25 units of heparin per 1 ml of blood). Centrifugation (10 min at 1500 rpm) precipitated leukocytes and erythrocytes. The supernatant fluid, which contains platelets, is neped into another siliconized tube and washed with physiological saline (0.85%) three times by centrifugation (at 3000 rpm, 20 min). Then the number of platelets is counted by the conventional method, a smear of the platelet sediment is made, dried, stained according to the Romanovsky-Giemsa method, and microscoped (90x10) by the conventional method. In this case, the presence of platelets is determined.

8 o of individual cases not only platelets are found in the smear, but also red blood cells and leukocytes of the donor. In these cases additional platelet purification is carried out. For this, the pellet is resuspended in physiological saline and centrifuged in a test tube (at 1000 rpm, 5 min). Then the supernatant containing platelets is transferred to another tube and centrifuged again (at 3000 rpm, 20 min). The supernatant is removed, and the sediment consisting of platelets is diluted in 2 ml of saline and destroyed by sequential freezing in the freezer of a domestic refrigerator and defrosting with heating to 40 ° C in warm water 3-4 times.

After that, the concentration of protein in the antigen from platelets is adjusted to a working concentration of 280 µg / ml. The antigen is poured into 0.5 ml in sterile ampoules, sealed, frozen and stored in the freezer of a refrigerated container at 4 ° C. The antigen prepared by the indicated

method suitable for 2-2.5 months. Work is carried out under aseptic conditions.

Lymphocyte isolation is carried out in a conventional manner. 2 ml of the patient's blood taken on a heparin solution (25 units of heparin per 1 ml of blood) are diluted 1: 2 with medium 199. Carefully apply blood to a ficoll solution (3 ml) with a density of 1.077 g-ml in a centrifuge

the tube and centrifuged at 1500 rpm for 30 minutes After centrifugation, the blood cells are divided into two fractions: a white layer consisting of lymphocytes is located on the surface of the ficoll, and red blood cells

and granulocytes - at the bottom of the tube.

Lymphocytes are collected with a Pasteur pipette into a conical tube and washed three times with medium 199, centrifuged at 800 rpm for 5 minutes. Lymphocyte Counting

carried out in a Gorev chamber in a conventional manner and brought to a concentration of up to 2,106 cells in 1 ml. .

Registration of antigen-binding lymphocytes in the blood of patients.

In the test sample, 0.1 ml of platelet antigen was added to 0.1 ml of lymphocytes, shaken and incubated for 30 minutes at 37 ° C. After incubation, lymphocytes are washed from excess unbound antigen in

medium 199 by centrifugation for 5 minutes at 800 rpm.

A second batch of lymphocytes (0.1 ml) is added to the cells washed from the antigen, the mixture is shaken, centrifuged for 5 minutes.

at 800 rpm Incubate for 30 minutes at 37 ° C. An equal amount of medium 199 is added to the control sample instead of antigen. Samples are prepared from the sediment, which are quickly dried and stained according to Romanovsky-Giemsa. Smears are mi- croscoped by counting 200 lymphocytes in them, noting among them agglutinates consisting of 2-3 lymphocytes, which are antigen-binding cells. The number of agglutinated lymphocytes is expressed in% of the total number of lymphocytes counted. The result is expressed by the difference between the experimental and control samples.

EXAMPLE 1. Examples of the clinical application of the method are given. Typical form of ITP. Sick L., 23, was admitted to the hospital with complaints of bruises under the skin of the extremities, gingival bleeding. Directing diagnosis: ITP. Sick in

for 3 years, I have been repeatedly treated with ie-most static preparations and corticosteroids, but the blood and its components have not been transfused. Blood test: HB-106 g / l, erythrocytes 4.85 1012 cells / l, leukocytes

6.5 -10 cells / l, lymphocytes 20%, platelets 4.3 109 cells / l. In the study of the bone marrow, the latter is rich in cellular elements, lymphocytes are 5.2%, many megakaryocytes (in two preparations, 20 megakaryocytes were counted, 12 of them do not function, 8 functions poorly). The content of antigen-binding lymphocytes was 6%, which confirmed the guiding diagnosis: itp.

Conservative treatment followed by splenectomy was performed. The patient's condition improved rapidly (the bruises were completely resolved, the number of platelets increased - 466.0 109l / l, leukocytes 12.3 109l / l.

PRI mme R 2. Atypical form ITP. Sick K., 14 years old, hospitalized with a direct diagnosis: HA. Complaints on admission to nasal, uterine and gum bleeding. For two years, unsuccessfully treated with blood transfusions and its components. Objectively: the condition of the patient upon admission is heavy, the skin is pale, multiple bruises all over the body of different sizes and old, the regional lymph nodes are not enlarged. Pulse of weak filling and voltage, accelerated to 108 beats / min, blood pressure 100/60 mm Hg. Blood test - hemoglobin 40g / l, erythrocytes 2.0 10 2 cells / l, platelets 16.0 109 cells / l, leukocytes 3.7 10 cells / l, lymphocytes 45% On the basis of the clinic - hematological data the patient was given a preliminary diagnosis: hypoplastic anemia But, since the number of antigen-binding lymphocytes, determined by the proposed method, was 7%, another diagnosis was made: a chronic relapsing form of idiopathic thrombocytopenic purpura, a complication of post-hemorrhagic anemia,

Appropriate treatment was prescribed, the condition improved, the bruises resolved, hemoglobin increased to 100 g / l, red blood cells to 3.5 1012 cells / l, white blood cells to 7.8 109 cells / l, decreased lymphocyte count to 31%, increased platelet count 38.6 10 cells / l. Subsequent observation and myelogram data confirmed the correctness of our diagnosis. Myelogram: the bone marrow is multicellular, the red poo is irritated, the lymphocytes are 17%, many megakaryocytes (of the 14 counted, 2 are functioning).

PRI me R 3. Typical form of HA. Patient M, 10 litas was admitted to the hospital about complaints of weakness, epistaxis, multiple bruising, tinnitus.

and blackout before eyes. Directing diagnosis: HA. During the year, he was repeatedly treated in various medical institutions with a small clinical effect. Objective: condition of the patient

pale skin, pale, multiple bruising, nasal and dehumous bleeding. Analysis kovi: hemoglobin 60 g / l, erythrocytes of 1.8 10 cells / l, leukocytes of 1.15 10. cells / l. lymphocytes 75%

platelets 2,24 10 cells / l. Myelogram: bone marrow is small cell, lymphocyte count increased 43.8%. Only one megakaryocyte was found in the two bone marrow preparations, and that one is not functioning. The content of antigen-binding lymphocytes according to the proposed method is 1.5%, which confirms the guiding diagnosis of hypoplastic anemia.

After conducting a focused

Conservative treatment with repeated blood transfusions of persistent improvement in clinical and hematological parameters did not occur (the bruises did not resolve, reduced hemoglobin content of 70 g / l, erythrocytes 2.0 1012 cells / l, and lymphocytes increased 63%). Taking into account the ineffectiveness of conservative treatment of the patient, surgical treatment was performed - bone marrow transplantation

from a relative, after which there was an improvement

PRI me R 4. Atypical form of HA. Patient R., 13 years old, was admitted to the hospital with a direct diagnosis: ITP. Complaints

on admission to nosebleeds and bruises, general weakness. Objectively: the state of average body weight, bruises under the skin of the lower extremities and forearms, slight nose bleeding. Blood tests hemoglobin 82 g / l, erythrocytes 3.1 -101 cells / l, leukocytes 4.3 10 cells / l, lymphocytes 32%, platelets 34.6 109 cells / l. The content of antigen-binding lymphocytes is 1%, on the basis of which

the diagnosis And GP changed to HA. After adequate therapy, the myels frame was studied: the bone marrow is moderately cellular, the lymphocyte count is increased by 39%, there are few megakaryocytes (only two megakaryocytes are found in two preparations, only one of them functions). Blood test after treatment: hemoglobin 95 g / l, erythrocytes 3.5 1012 cells / l, leukocytes 3.7 1GJ cells / l, lymphocytes 66%, platelets 92.3 10 ° cells / l, Has come

clinical condition and further operative baking, splenectomy, was performed.

PRI me R 5. Healthy bone marrow donor I., 27 years old, soon no ill. Blood test: hemoglobin 134 g / l, erythrocytes 4.3 1012 cells / l, leukocytes 7.6 -10 cells / l, lymphocytes 24%, platelets 286.0 10 cells / l. Myelogram; the bone marrow is rich in Droa-containing elements, 14.8% of lymphocytes, many megakaryocytes (14 megakaryocytes in two preparations, 12 of them are functioning), antigen-binding lymphocytes according to the proposed method 1.2%.

The implementation of the differential diagnosis of ITP and GA proposed method, compared with the known, provides;

acceleration of diagnostic research by 2-3 times in comparison with the prototype (2.5-3 hours versus 6-8 hours);

differential diagnosis of ITP and GA for blood analysis;

a decrease in the morbidity of the study / io compared with the diagnostic methods for bone marrow;

improving the accuracy of diagnosis of ITP and GA, and especially their atypical forms;

a reduction in the volume of blood and its components expended on unsuccessful treatment with an erroneous diagnosis;

as a result of the above, it is possible to save costs on the diagnosis and treatment of patients with ITP and HA

With atypical forms of ITP and HA, the diagnostic accuracy is increased to 89-93%.

Invention Formula

The method of differential diagnosis of idiopathic thrombocytopenic purpura and hypoplastic anemia by examining blood cells is also different because, in order to improve the accuracy of the method, lymphocytes were isolated from the blood, some of which are incubated with platelet antigen with a final protein concentration of 140 µg / ml in flow

30 minutes at 37 ° C, washed and combined with an equal volume of another part of the native lymphocytes, then smears are prepared, stained, the percentage of agglutinated lymphocytes is counted,

compared with a control that does not contain a platelet antigen, determine the difference in performance and, if its value is equal to or greater than 2.5%, diagnose idiopathic thrombocytopenic

purpura, and when less than 2.5%, hypoplastic anemia is diagnosed.

Claims (1)

Claim A method for differential diagnosis of idiopathic thrombocytopenic purpura and hypoplastic anemia by examining blood cells, characterized in that, in order to increase the accuracy of the method, lymphocytes are isolated from the blood, some of which are incubated with platelet antigen with a final protein concentration of 140 μg / ml for 20 30 min at 37 ° C, wash and combine with an equal volume of another part of the native lymphocytes, then smears are prepared, stained, the percentage of agglutinated lymphocytes is calculated, 25 are compared with a control that does not contain platelet antigen, the difference in indicators is determined and when its value is equal to or more than 2.5%, idiopathic thrombocytopenic 30 purpura is diagnosed, and if it is less than 2.5%, hypoplastic anemia is diagnosed.