RU2634262C1 - Method for determination of indications for selective adsorption of endotoxin in patients with sepsis caused by gram-negativemicroorganisms - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for determination of indications for selective adsorption of endotoxin in patients with sepsis caused by gram-negative microorganisms includes examination of body temperature, total leukocyte count, neutrophil shift, plasma procalcitonin level, determination of endotoxin activity by means of chemiluminescence analysis in the EAA test; at a body temperature less than 36°C and more than 38°C, total number of leukocytes less than 4×10⁹/l and more than 12×10⁹/l, neutrophil shift of more than 10%, procalcitonin content of more than 2 ng/ml and endotoxin activity equal to or greater than 0.6, selective sorption of endotoxin is prescribed, when the endotoxin activity of less than 0.6 is detected, neutrophils response is assessed according to the level of the "Response" index in the EAA test, at the "Response" level of less than 0.5, presepsin content in the patient's blood is further examined, at a presepsin concentration of 1500 pg/ml and above, selective adsorption of endotoxin is prescribed.

EFFECT: accuracy and objectivity increase.

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Description

The invention relates to medicine, namely to a clinical laboratory diagnosis of sepsis caused by gram-negative microorganisms in the intensive care unit and intensive care.

The use of new high-tech approaches in the treatment of patients with sepsis caused by gram-negative microorganisms is associated with the need for rapid diagnosis of endotoxinemia.

A known method for determining the indications of selective adsorption of endotoxin (Kulabukhov V.V., Chizhov A.G., Kudryavtsev A.N. Selective lipopolysaccharide hemosorption as a key link in the pathogenetically substantiated therapy of gram-negative sepsis. Medical alphabet. Emergency medicine. - 2010. No. 3. C. 60-66), which includes determining the totality of parameters: clinical signs of septic shock, bacteriological analysis, body temperature, total white blood cell count, neutrophil shift, procalcitonin concentration, LAL test values . The selective adsorption of endotoxin is prescribed for hyperthermia of more than 38 ° C, a total number of leukocytes of more than 12 × 10 ⁹ / L, a neutrophilic shift of more than 10%, a procalcitonin concentration of more than 2.0 ng / ml, and a LAL test of 1.40 UE / ml.

However, in the modern literature there are numerous data on the high frequency of false-positive results of the LAL test. Since the coagulation factors of the horseshoe crab, the cells of which are used in the LAL test, are very similar to the factors of the coagulation system of mammals, thrombin and human thromboplastin can cause gelation of the lysate. In addition to interacting with the plasma components of the patient, the conditions for setting the test contribute to the production of false positive values. Even slight temperature fluctuations (36 ° C or 38 ° C instead of the set 37 ° C) or the pH of the reagents used (6.0 or 8.0 instead of 7.0) increase the frequency of positive results of the gel thrombus LAL test in 1, 5 and 5.0 times, respectively.

Other difficulties in interpreting the results of the LAL test include the lack in clinical studies of the relationship of test values with the level of complement system activation and the expression of cytokines by mononuclear cells caused by gram-negative microflora; detection of positive test values in the presence of a fungal infection in the systemic circulation, which is associated with the activation of the coagulation cascade of the test (1 → 3) -β-D-glucans.

From the literature it is also known that to assess the endotoxin load, the level of EAA endotoxin activity (Endotoxin Activity Assay) is used. This test is based on the reaction of monoclonal antibodies to endotoxin lipid A. This antigen-antibody complex (AG-AT) activates neutrophils and causes an oxidative “explosion” in them. Active forms of oxygen produced by neutrophils are measured using a chemiluminescent reaction with luminol (Romaschin AD, Harris DM, Ribeiro M.V., Paice J., Foster DM, Walker PM, Marshall JC A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence // Journal of Immunological Methods. Elsevier; 1998; 212: 169-185).

A known method for determining the indications for selective adsorption of endotoxin in patients with sepsis (Popok Z.V. Selective sorption of endotoxin in the complex intensive therapy of sepsis in patients after cardiac surgery // Abstract of dissertation of Candidate of Medical Sciences. Moscow, 2010. - 24 p. ), at which body temperature, the total number of leukocytes, neutrophilic shift, the presence of a focus of infection and insufficiency of organs and systems, the level of endotoxin activity (EAA), the endotoxin titer according to the LAL test, and the concentration of procalcitonin are determined.

Selective endotoxin adsorption procedures are prescribed for the following values of indicators: hyperthermia more than 38 ° C, the total number of leukocytes more than 12 × 10 ⁹ / l or less than 4 × 10 ⁹ / l, neutrophilic shift more than 10%, the presence of a focus of infection and failure of more than 2 organs and systems, the level of endotoxin activity (EAA) is more than 0.6, the endotoxin titer according to the LAL test is more than 0.72 UE / ml, the concentration of procalcitonin is more than 2 ng / ml. At an EAA level of less than 0.6, selective adsorption of endotoxin is not indicated. However, the known method does not take into account the additional indicator "Response" in the EAA test, which reflects the level of ability of the patient’s neutrophils to oxidative "explosion" (an in vitro marker of phagocytic activity of neutrophils).

There is also a method of prescribing selective adsorption of endotoxin in patients with sepsis caused by gram-negative microorganisms (Chetverikov M.V. Selective adsorption of endotoxin during surgical sepsis in cancer patients // Diss.Cand. Medical Sciences. Rostov-on-Don, 2015. - 131 s.), Selected for the prototype, which takes into account the "Response", body temperature, heart rate, respiratory rate or partial pressure of carbon dioxide in arterial blood, the total number of leukocytes, neutrophil shift, The values of the test of the EAA and the concentration of procalcitonin. The appointment of selective endotoxin adsorption procedures is carried out in the presence of hyperthermia of more than 38.3 ° C or hypothermia of less than 36 ° C, tachycardia of more than 90 beats / min, tachypnea of more than 20 breaths / min or partial pressure of carbon dioxide in arterial blood (paCO ₂ ) less than 32 mm water column, the total number of leukocytes is more than 12 × 10 ⁹ / l and less than 4 × 10 ⁹ / l, neutrophilic shift is more than 10%, EAA test values are more than 0.6 or 0.4-0.6 at the level of "Response "less than 0.5, procalcitonin more than 2 ng / ml. The disadvantage of this method is that it does not take into account patients with EAA values less than 0.4 with a "Response" level of less than 0.5 and high phagocytic activity of resistant neutrophils, which also require selective endotoxin adsorption procedures.

The technical problem is the creation of a more accurate and informative way of determining the indications for selective adsorption of endotoxin in patients with sepsis caused by gram-negative microorganisms.

The technical result from the use of the method is to expand the diagnostic parameters of the express diagnostics of determining the indications for selective sorption of endotoxin in patients with sepsis caused by gram-negative microorganisms.

Measurement of endotoxin activity in the EAA test is directly related to the ability of neutrophils, opsonized by the “endotoxin-AT to endotoxin” immune complexes, to phagocytic activity followed by a respiratory burst, the intensity of which is detected by an automatic chemiluminometer. The principle of chemiluminescent assessment of phagocytic activity is based on CR1 / CR3 receptor activation of whole blood neutrophils to produce oxidants that react with luminol. In the EAA test there is an additional indicator “Response”, which reflects the level of the patient’s neutrophil ability to oxidize.

The authors found that in the group of patients with a critically low level of "Response" low EAA and a high concentration of presepsin are noteworthy. This is due to the fact that after binding to endotoxin in the systemic circulation, membrane CD14 is cleaved from the monocyte and in a soluble form enters the circulation. The lysosomal proteinases released as a result of activation of phagocytosis cleave soluble CD14 to form a fragment, which is now called prespepsin. The manufacturer positions this test as an indicator of phagocytosis activation. Thus, prespepsin levels reflect in vivo phagocytosis, while the EAA test for endotoxin activity uses the ability of neutrophils to in vitro phagocytosis.

The authors hypothesized that the patient’s neutrophils cannot react with immune complexes in the EAA test, because their CR1 / CR3 receptors are occupied or depleted by prolonged intake of massive doses of endotoxin in the bloodstream, causing high phagocytic activity, which reflects a significantly increased concentration of prespepsin (PSP )

Analysis of the Response level helps to make a more complete and reliable assessment of the clinical condition of the patient in each specific study of the level of endotoxinemia, and the use of the Response and SRP values in the methods of prescribing and monitoring the etiopathogenetic therapy of sepsis increases the information content of the whole complex of laboratory studies of the endotoxinemia level and the effectiveness of the therapy in general, which allows not to miss patients with low immune reactivity of the body who need adsorption.

The invention consists in the following.

In patients with suspected sepsis caused by gram-negative microorganisms, body temperature, total leukocyte count, neutrophil shift, procalcitonin concentration, endotoxin activity by the EAA test and the level of adequacy of the Neutrophil response “Response”, prespepsin concentration are evaluated. Selective adsorption of endotoxin is indicated at the following values: body temperature less than 36 ° C and more than 38 ° C, the total number of leukocytes less than 4 × 10 ⁹ / l and more than 12 × 10 ⁹ / l, neutrophilic shift more than 10%, the concentration of procalcitonin 2 ng / ml When the content of procalcitonin is equal to or more than 2 ng / ml, the activity of endotoxin is determined using chemoluminescent analysis in the EAA test. Assign procedures for selective adsorption of endotoxin with an activity of endotoxin equal to and more than 0.6. At the same time, when detecting endotoxin activity of less than 0.6, the neutrophil response is evaluated by the level of the “Response” indicator in the EAA test, which characterizes the functional activity of neutrophils. If the level of "Response" is less than 0.5, the prespepsin blood level of the patient is additionally examined. At a prespepsin concentration of 1500 pg / ml and above, selective endotoxin adsorption procedures are prescribed.

The method is as follows.

We examine the level of procalcitonin (PCT) in patients with suspected sepsis caused by gram-negative microorganisms, with hyperthermia greater than 38 ° C or hypothermia less than 36 ° C, the total number of leukocytes more than 12 × 10 ⁹ / l and less than 4 × 10 ⁹ / l, neutrophilic a shift of more than 10%. An analysis of the concentration of PCT is necessary to identify a group of patients with a suspected bacterial infection among patients with signs of inflammation caused by an immune response to surgical treatment or a viral infection.

Plasma PCT levels are measured on an Elecsys 2010 immunochemical analyzer (Roche) using the ECLIA quantitative electrochemiluminescent immunoassay - Elecsys BRAHMS PCT (BRAHMS Diagnostica GmbH). The method is based on the ability of antigens and antibodies to form “sandwich complexes”. Antigens (PCTs) of the test sample and monoclonal antiprocalcitonin antibodies (mice) labeled with a ruthenium complex and antibodies labeled with a biotin label are reacted.

At the next stage, we determine the level of endotoxin activity (EAA) and "Response" in patients with a PCT concentration of more than 2 ng / ml. Measurement of the level of endotoxin activity is necessary for the differential diagnosis of infection caused by gram-negative microorganisms.

The EAA method is based on the reaction of endotoxin of a patient’s whole blood with antiendotoxin antibodies contained in the reagent. Complement proteins opsonize the formed antibody / endotoxin complexes. Opsonized immune complexes interact with neutrophils through complementary receptors to complement components, which undergo respiratory explosion in the presence of zymosan.

Oxidants produced by neutrophils react with luminol. The intensity of chemiluminescence is proportional to the level of endotoxin in the test sample. Chemiluminescence is detected by counting photons in a chemiluminometer. The values measured by the device are converted by calculation into units of endotoxin, and the unit of its activity as proportional to the total possible activity (0-1.00). The EAA test has a special parameter called “Response”, which reflects the degree of adequacy of the oxidative response of neutrophils.

Assessment of the level of chemiluminescence is carried out in three test tubes attached to the kit, which differ in the composition of the reagents added to them, into which the patient's whole blood is added. Each EAA test consists of “tube 1” (control), “tube 2” (sample) and “tube 3” (maximum). In "test tube 1", basal activity is measured, which determines the nonspecific oxidative burst of the patient’s neutrophils in the absence of specific antiendotoxin antibodies. In "test tube 3" determine the level of the maximum possible oxidative response by neutrophils of the patient. In "test tube 2" carry out an analytical measurement. The EAA level is calculated by normalizing the chemiluminescence in the test sample ("tube 2") relative to the maximum chemiluminescence ("tube 3") with the correction of both results for basal chemiluminescence activity ("tube 1").

Patients with an EAA level of greater than or equal to 0.6 are prescribed selective endotoxin adsorption procedures.

At the next stage, we assess the adequacy of the neutrophil response by the "Response" level in patients with EAA less than 0.6. The "Response" values are obtained automatically when measuring the endotoxin level with the EAA test. According to the manufacturer of the EAA test, the reference range for the Response is 0.80-0.98. We evaluate the Response level so as not to exclude patients with low neutrophil reactivity and therefore falsely lowered EAA values, which also require selective endotoxin adsorption.

In patients with an EAA of less than 0.6 and a Response of less than 0.5, we examine the concentration of presepsin in the patient’s whole blood. Measurements of PSP levels are necessary to confirm excessive activation of immunity by high levels of circulating endotoxin. The concentration of presepsin is measured on a PATHFAST analyzer (Mitsubishi Chemical Medience Corp.) The test system is based on the method of chemiluminescent enzyme immunoassay (CLEIA) using a special technology for the separation of bound and free material by washing the magnetic particles in the tips of MAGTRATION (Precision System Science).

The test uses a mixture of mono- and polyclonal antibodies to presepsin: polyclonal antibodies are additionally associated with alkaline phosphatase, monoclonal antibodies with magnetic particles. When samples of test blood are added to the reaction mixture, the formation of immune complexes - PSP / labeled with the enzyme AT / AT on magnetic particles. After removal of unbound material, a chemiluminescent substrate is added to this complex, the enzymatic transformation of which initiates luminescence. The luminescence intensity is proportional to the concentration of presepsin in the sample. Calculation of the result is carried out according to the standard calibration curve.

Patients with a presepsin concentration of 1,500 pg / ml and above are prescribed selective endotoxin adsorption procedures.

We provide data confirming the possibility of realizing the declared purpose and achieving the specified technical result.

Clinical example. The postoperative period in patient M., 48 years old, after cardiac surgery on the heart valve was complicated by gram-negative sepsis, the focus of infection was ventilation-associated pneumonia; body temperature 38.5 ° C, the total number of leukocytes 3.0 × 10 ⁹ / L. By the described method, the concentration of PCT was measured in the patient’s blood, which was 5.5 ng / ml. Then, endotoxin activity was measured by the EAA test, which was less than 0.60 (0.00). Therefore, the adequacy of the neutrophil response in the EAA test was assessed by the "Response" level, which amounted to 0.01. At the last stage, the concentration of PSP was measured, the level of which was 9482 pg / ml. The patient was prescribed selective endotoxin adsorption procedures. After two procedures for selective adsorption of endotoxin, the ability of neutrophils to oxidatively explode increased to 37%, and the values of endotoxin activity reached the upper limit of the range, which can be associated with the release of CR1 / CR3 receptors of polymorphonuclear neutrophils due to a decrease in the level of endotoxin. The table shows the results of a study of patient M. before and after selective adsorption of endotoxin. Patient M. was transferred to the ward and discharged.

Claims (1)

A method for determining indications for selective adsorption of endotoxin in patients with sepsis caused by gram-negative microorganisms, including a study of body temperature, total leukocyte count, neutrophilic shift, level of procalcitonin in blood plasma, determination of endotoxin activity using chemoluminescent analysis in the EAA test; at a body temperature of less than 36 ° C and more than 38 ° C, the total number of leukocytes less than 4 × 10 ⁹ / l and more than 12 × 10 ⁹ / l, a neutrophilic shift of more than 10%, a procalcitonin content of more than 2 ng / ml and an endotoxin activity equal to or more than 0.6, selective endotoxin sorption procedures are shown, when detecting endotoxin activity of less than 0.6, the neutrophil response is evaluated by the level of the Response indicator in the EAA test, characterized in that when the level of Response is less than 0.5, the content is additionally examined in the patient’s blood of prespepsin, at a concentration of presepsin of 1500 pg / ml and the above prescribed procedures for selective adsorption of endotoxin.