SU1093325A1 - Method of treatment of the course of inflammatory process - Google Patents

Abstract

A method for predicting the inflammatory process by posing a rosette-forming reaction with neutrophils, characterized in that, in order to reduce the morbidity of the method, blood is taken from patients, layered on a ficollurostrasse gradient with a density of 1.073 - 1.083 g / ml, then a neutrophil fraction is isolated, incubated with in equal parts with a 2.5% solution of spontaneous sheep erythrocytes and with a 2.5% solution of human complementary erythrocytes, then the number of total D-neutrophils formed is determined and their relative standards 8-19 times predict acute inflammatory process, a § with increasing D-neutrophil t is 3-7 times the norm predict chronic form during the inflammatory process.

Description

The invention relates to medicine that can be used to perturb the inflammatory process. Leukocytes play a major role in antimicrobial protection. Defects in any of the links of the antimicrobial systems of neutrocytes lead to diseases characterized by frequent repeated infections 1. The known method of differential agnosticism of acute and chronic inflammatory processes is that a method has been developed to isolate a population of precursors of the granulocyte series, allowing you to diagnose an acute and chronic inflammatory process 2. The drawbacks of the method are the duration of the study, the trauma as the bone marrow punctate is taken. The aim of the invention is to reduce the morbidity of the method, allowing to isolate a cell population reflecting the barrier function of the bone marrow and study its diagnostic value. The goal is achieved according to the method of predicting the inflammatory process by setting rosette-forming reactions with neutrophils, the patient takes blood, layer it on a ficoll-urostrash gradient with a density of 1.073-1.083 g / ml, then a neutrophil fraction is isolated, it is incubated with in equal parts with a 2.5% solution of spontaneous erythrocytes and a 2.5% solution of complementary human erythrocytes, then the number of total D-neutrophils formed is determined and with an increase in their relative norm of 8-1 An acute inflammatory process is predicted 9 times, and with an increase in D-neutrophils 3-7 times relative to the norm, a chronic form of the inflammatory process is predicted. The method is described as follows. Blood is taken from the patient, the blood cells are separated by layering whole blood on a double gradient. 1.070-1.073 and 1.080-1.083 g / ml, followed by centrifugation at 1500 rpm. Two clean cell populations are obtained: a suspension of lymphocytes between plasma and a 25 ficoll-urostras solution with a density of I, 0701.073 g / ml, and a suspension of neutrophils between two solutions with a density of 1.070-1.073 and 1.080-1.083 g / ml. 0.1 MP of neutrophils (cells to 1 ml) are drained with a 0.05 ml solution of spontaneous sheep erythrocytes and 0.05 ml of a 2.5% solution of human complementary erythrocytes. The mixture of cells incubated for 10-20 min at 0-4 ° C. Then centrifuged min. at 0–4 ° C. A second incubation is carried out for 1–2 h, centrified at 0–4 ° C and 1000–1500 rpm for 10–20 min, fixed with 3% glutaraldehyde solution in phosphate buffer pH - 7.4 for 10-20 minutes. Distilled water is added and centrifuged for 5-15 minutes at 1000-1500 rpm. The nozzles are discarded, 0.1-0.3 ml of cell suspension is left, mixed and applied on glass slides. Dry, fix with methanol for 510 minutes, paint with azure-eosin for 5-10 minutes. In the preparation, 200 cells are calculated. For the rosette-forming D-neutrophil, they consider a cell that has attached three spontaneous sheep erythrocytes and three complementary erythrocytes of chyuveka. The percentage and absolute content of rosetting cells are calculated. Example I. Patient A., a healthy donor. A siliconized tube of 3 ml of Ficoll-Urorast mixture with a density of 1.073 g / ml and 3 ml of a mixture of 1.083 g / ml density is layered with 3 ml of blood, followed by centrifugation at 4 ° C and 1500 rpm for 20 minutes. Two cell suspensions are obtained: a suspension of lymphocytes and a suspension of neutrophils. A suspension of neutrophils VU in 0.1 ml is combined with 0.05 ml of a 2.5% aqueous solution of spontaneous erythrocytes of ram and 0.05 ml of a 2.5% aqueous solution of complementary human erythrocytes. The mixture of cells is incubated for 20 min at 4c, followed by centrifugation for 15 min at 4 ° C. The second incubation is carried out for 90 minutes. at 4 ° C. Fixed for 20 minutes with a 3% solution of glutaraldehyde, centrifuged, the sediment applied to slides, dried, fixed with methanol for 10 minutes, dyed with azure-eosin for 10 minutes, counted in 6-7 different fields of view in the immersion microscope system 200 cells attached

not less than 3 DL and 3 EH. The absolute content of D-neutrophils calculated by the formula

.

N

100 100

where L is the number of neutrophils in

1 μl of peripheral blood; t - neutrophils in the blood formula,%; g. - D-rosette neutrophils,%.

100 100 indicators resulting in a volume of 1 µl. In the immersion system of the microscope in 7 different fields x 3% rosette of D-neutrophils, the leukocyte count in 1 µl of blood is 6750, the percentage of neutrophils in a blood formula is 60%. Substituting the numerical values in the formula we get:

  –6250: 3: 60, 21.5 in 1 micron

100-100.,

(norm

Example 2. Patient X., case history No. 2382, was admitted to the clinic about fibroplastic induction of the penis. Concomitant diseases - cerebral atherosclerosis with epilaptoid episodes of vascular genesis; residual effects of polymilite,

In a silicone tube of 3 MP of Ficoll-Urostras solution with a density of 1.070 g / ml and 3 ml of 1.090 g / mp, 3 ml of blood are layered, centrifuged for 10 min at 1000 rpm. As a result, erythrocytes settle to the bottom, and the lymphocytes are suspended forms a ring above the ficoll urorast solution with a density of 1.070 g / ml, and a suspension of neutrophils forms a ring above the ficoll-urotrast solution with a full surface I, 080 g / ml. Neutrophils in an amount of 310 ° in 0.1 ml are combined with 0.05 MO of a 2.5% aqueous solution of spontaneous erythrocytes of ram AND 0.05 ml of a 2.5% aqueous solution of complementary human erythrocytes. The mixture of cells was incubated for 10 min at 0 ° C, followed by centrifugation for 10 min at 0 ° C and 1000 rpm. The second incubation is carried out for one hour at 0 ° C. Fixed for 15 min with 3% glutoraldehyde solution, followed by centrifugation at room temperature for 5 min. The precipitate is placed on a glass slide, dried, fixed with 5 MVUI methanol, and painted with azure-eosin for 5 minutes. Consider in the immersion microscope system in 5-7 different fields x 200 neutrophil cells that attached at least 3 EB and 3 ECh. In this example, the content of D-ROH in peripheral blood is 2425.5 in 1 μl (35%), E-ROP ( promyelocytes) 491.4 in 1 μl (84%), E-ROM (myelocytes), 898.5 in μL (96%), E-Roy (young) 804.9 V. 1 M1SL (86%), E .-RONp (papa of neutrophils) 1467.1 per 1 micron (95%), E-RONs (segmented neutrophils) 245.7 in 1 µl (98%),

In this example, the morphological parameters of the bone marrow cell composition are within the normal range,%: promyelocytes 5.0, myelocytes 8, rod nuclear neutrophils 13.1, segmented nuclear neutrophils 21.4,

The bone marrow index of neutrophil maturation refers to the ratio of young granulocyte elements to mature neutrophils. Normally, it is equal to 0.6-0.8. In this example, it is normal and equal to 0.6. The content of neutrophilic row cells is also normal and is 55.6%. However, the comparison of the sum of rosette-forming cells of a neutrophilic row in the bone The brain and peripheral blood showed the following:

Bone marrow (ck)

 1 µl (inflammation. 5767, B in 1 µl (normal)

Late total D-neutrophils 5d.) -

-121.5 in 1 μl (normal)

I

In a practically healthy person, E-RON (in the bone

brain) 5788.5 in 1 μl,

D-RON (in peripheral blood) 121.5 in 1 μl. In a sick person: E-RON (in bone

Mogze) 6115,6 in 1 mkl,

D-RON (in peripheral blood) 2425.5 in 1 μl. Thus, during inflammation, the barrier function of the bone marrow changes, and the neutrophil population that participates in the inflammatory response is released. The number of rosette-forming late total D-neutrophils in the peripheral blood P iiop is 47 times less than the rosette precursors of the neutrophilic group in the bone margins. With the active phase of inflammation, the content of late D-ROH in peripheral blood increases 19 times. When stopping the process, these indicators are normalized. Example 3. Patient N., case history No. 2056, was admitted to the clinic for Peyronie's disease. Blood counts: leukocytes.8400 in I μl segmetoid neutrophils 4032 in 1 μl (48%), rod darn neutrophils 252 in} μl 3%), eosinophigus. 924 in 1 μl (11%), lymphocytes 2268 in I μl (27%), monocytes 924 in μl (11%). In urine analysis, protein is 0.093%, leukocytes are 20-30 in the visual field, soliaxalates and a lot of mucus.

3 ml of peripheral blood was filled with 3 ml of Ficoll-Urostast solution with a density of 1.072 g / ml and 3 mp of a solution with a density of 082 g / ml. Centrifuged for 5 min at 1000 rpm. B-result received two cell suspensions: lymphocyte. And neutrophilic. Neutrophils in the amount of 310 in 0.1 ml were combined with 0.05 ml of a 2.5% solution of EB and Oj05 ml of a 2.5% solution of EC. The mixture of cells was incubated for 15 min, followed by centrifugation for 10 min at 0 ° C and. 1000 rpm. Second incubation was carried out for 2 hours at. Fixed 20 min 3% -Hbiis solution of glutoraldehyde. :.: Centrifuged for 10 minutes at 1000 rpm, the precipitate was applied to slides, dried, fixed for 7 minutes with methanol, and painted with azure-eosin for 7 minutes. Considering from 5-7 different fields x 200 D neutrophils in the microscope's immersion system.

In this example, the content of D-ROH 378.4 in i μl (13%), E-ROP (promiyelo cytes) 14.7 in 1 went (78%), E-ROM (myelocytes) 274 in 1 μl (87), E-RO (young) 277.8 in 1 μl (63%), E-RONp. (Rod) 816.8 in 1 μl (59%) E-RONs (segmental) 578 in 1 μl (37%) . In the studied peripheral blood, the leukocyte content is 4100 in 1 μl, the segmented neutrophil count is 2911 in 1 μl (71%), the bacterial neutroma 41 in j μl (1%), the eosinophils 41 in 1 mint (l%) f lymphocytes 984 in 1 μl (24%), monocytes 123 in 1 μl (3%).

KM bone marrow

.1 2 1 1 Н 1 3 ЭУЙ) - п т

- .... .. "." "," ... "." .. ". - and J J

5767.8 in 1 μl (normal) D-ROH (PC) 378 4 1deficiency), 121.5 (normal)

In a practically healthy person, E-RON is 5788.5 in 1 μl (in the bone marrow),

D-RON - 121.5 in 1 μl (in peripheral blood).

A patient with a chronic inflammatory process:

E-ROH - 1961.3 in I μl (in the bone marrow),

 - 378.4 in 1 μl (in peripheral blood).

Thus, in the chronic inflammatory process in the bone marrow, the bone marrow neutrophil maturation index is disturbed and there is a deep deficit of rosetting neutrophils. The patient received antibacterial and vitamin therapy. After the treatment, the state is satisfactory, complaints are not normal in the morning and evening. In the analysis of urine leukocytes 2-3

in the field of vision, In the growth urine the microflo is not detected hay.

Example 4. Patient R., case history No. 645, was admitted to pain. Thus, all of the indicators except for the patient were normal. In the bone marrow, the sum of the elements of the neutrophilic series is also normal and is 56.6%. However, the bone marrow index of neutrophil maturation is well below normal and is 0.28. The sum of rosette-forming cells of the neutrophilic row in the bone marrow is 5 times less than normal: diagnosed with chronic pyelonephritis, right-sided nephroptosis. Sick for 5 years. Upon receipt of a complaint of dull pain in the lumbar region, subfibril temperature, agility. Urinalysis: weight gain 1025, sour reaction, 6-8 leukocytes in the visual field, erythrocytes 1-2 in the visual field. In the urine culture, microflora growth is not. detected. On radiographs, one hundred and one lying down, a decrease in the cleansing function of the right kidney with slowed down removal of the drug from it. The lack of total cleaning ability of the kidneys is 25%,

Claims (1)

A method for predicting the course of the inflammatory process by setting the reaction of rosette formation with neutrophils, characterized in that, in order to reduce the morbidity of the method, blood is taken from patients, layered on the ficollurotrast gradient with a density of 1.073 - 1.083 g / ml, then the neutrophil fraction is isolated, · it is incubated taken in equal parts with a 2.5% solution of spontaneous sheep erythrocytes and a 2.5% solution of complementary human erythrocytes, then determine the number of total D-neutrophils formed and with an increase from ositelno standards 8-19 times predict acute inflammatory process, while increasing the D-t neutrophils 3-7 times relative to the norm predict chronic form of inflammation.