RU2800821C1 - Method for early diagnosis of sepsis using determination of absolute number of neutrophilic extracellular traps - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: clinical laboratory diagnostics, used for early diagnosis of sepsis. On the 1st day of the disease, the absolute number of extracellular traps formed by neutrophilic granulocytes in peripheral blood is determined. When the level of the absolute number of extracellular traps is more than 0.354× 10⁹/l sepsis is diagnosed.

EFFECT: method provides quick, accessible and informative diagnostics of sepsis in the early stages by determining the absolute number of extracellular traps formed by neutrophilic granulocytes in the peripheral blood on the 1st day of the disease.

1 cl, 1 ex

Description

The invention relates to medicine, in particular to clinical laboratory diagnostics, and can be used for early diagnosis of sepsis in an inflammatory process by determining extracellular DNA in peripheral blood formed by granulocytes.

Sepsis is a life-threatening organ dysfunction caused by dysregulation of the body's response to infection [1]. If sepsis is not recognized early and treated promptly, it can cause septic shock, multiple organ failure, and death. According to calculations, the incidence of sepsis worldwide annually is more than 30 million people and, according to recent studies, annually claims the lives of 11 million people [2]. The danger continues to threaten the surviving patients, of whom only half recover completely, and the rest either die within 1 year or live with the burden of chronic disability [3]. Therefore, the search for methods of early and accurate diagnosis and prognosis of sepsis, as well as specific treatment, is still relevant [4].

Currently, the following methods of laboratory diagnosis of sepsis are known.

Microbiological, which are based on the isolation and identification of a microbial agent from the blood on artificial nutrient media. The microbiological method has its drawbacks: the process is laborious, taking into account the deadlines (24-48 hours or more), cannot be used as an early criterion for sepsis, and bacteremia is determined only in 25-45% of cases.

One of the laboratory criteria for a generalized infection is the methods for determining bacterial endotoxins (BE) (LAL test-Limulus Amebocyte Lysate Test, EAA - Endotoxin activity assay, etc.). The disadvantages of the methods are: insufficient specificity in the analysis of the content of BE in such multicomponent solutions as blood and plasma; the need for special expensive equipment, which makes it difficult to use them as express methods in a hospital, difficulties with sample preparation and reproducibility of results due to interfering with the determination of glucans and other molecules, the content of which in the blood of patients varies significantly. Particularly ambiguous is the determination of the content of BE in the blood as a diagnostic criterion for septic complications of critical conditions [5].

"Classic" markers of inflammation, such as the total number of leukocytes, platelets, leukocyte formula, erythrocyte sedimentation rate, as well as proteins of the acute phase of inflammation (C-reactive protein, ceruloplasmin, ferritin, albumin, etc.) have low specificity and, accordingly, for early and accurate diagnosis of sepsis is unreliable [6].

Determination of the so-called "sepsis biomarkers". An “ideal” sepsis biomarker should have the following advantages: high sensitivity and specificity; accessibility for practice; fast results; high reproducibility; correlation with condition severity and outcome; coincidence of the dynamics of the content with the clinical response to the ongoing therapy [7]. Today, under experimental conditions and in the clinic, such markers as procalcitonin, C-reactive protein, proadrenomedullin, TREM-1, TNF, IL-6, IL-8, LPS-binding protein, endotoxin, presepsin are considered. However, according to the position of experts, none of them can currently claim to be a generally recognized sepsis biomarker that meets all the stated criteria. Among the shortcomings of the above markers, one can single out: insufficiently high sensitivity and specificity, the duration of the study, the need for expensive specialized equipment and reagents, a limited number of clinical observations, and specially trained personnel.

The invention is based on the task of developing a fast, affordable and informative method for the early diagnosis of sepsis. This problem is solved by the fact that in patients admitted to the intensive care unit or intensive care unit, the absolute number of extracellular traps formed by peripheral blood granulocytes is determined in a smear stained according to Romanovsky-Giemsa. The claimed method allows for more accurate early diagnosis of sepsis, which will allow the attending physician to optimize treatment tactics to reduce the likelihood of developing severe forms of sepsis and death.

In the proposed method, for the first time, the determination of the absolute number of extracellular traps formed by peripheral blood granulocytes on day 1 in patients admitted to the intensive care unit or intensive care unit can serve as an additional marker for the early diagnosis of sepsis. When the level of the absolute number of extracellular traps formed by peripheral blood granulocytes is more than 0.354×10 ⁹ /l, sepsis is diagnosed with 93% sensitivity and 90% specificity.

The method is carried out as follows. Upon admission to the intensive care unit, patients are taken whole peripheral blood, and neutrophil extracellular traps are counted. To do this, a smear is prepared from whole peripheral blood on a defatted glass slide, the fixed preparation is stained according to the Romanovsky-Giemsa method, the count is carried out using a light microscope (extracellular DNA is represented by thin violet-red threads occupying a space 2-3 times greater than the diameter of an unchanged leukocyte [8].

Next, the total number of leukocytes is counted. To count the total number of leukocytes, a Goryaev camera is used. Before the study, 5 µl of whole blood is mixed with 100 µl of 5% acetic acid to destroy the leukocyte membrane. Further, in 100 large squares, according to Egorov's rule, leukocytes are counted at low magnification (eyepiece ×10, lens ×8). The calculation is made according to the formula: L \u003d B * 250 * 20 * 10 ^ 6 / 100 (where L is the number of leukocytes in 1 liter of blood; B is the sum of leukocytes in 100 large squares; 250 is a multiplier leading to a blood volume of 1 μl (volume large square 1/250 µl); 20 - blood dilution; 100 - number of large squares; 10^6 - number of µl in one liter).

Next, the absolute number of neutrophilic extracellular traps is calculated, and sepsis is diagnosed at the NVL level of more than 0.354×10 ⁹ /l. The study involved 60 patients after surgery, 49 of them diagnosed with sepsis.

Diagnostic efficiency was assessed in terms of sensitivity, specificity, and the area under the characteristic curve (ROC curve). AUC (Area Under Curve) for diagnosis by determining the absolute number of NIL on the 1st day of the disease was 0.988 (p<0.0001), the sensitivity of the test was 93%, the specificity of the test was 90%, all this indicates a high quality of the model.

Example 1. Patient A., 62 years old, was treated after surgery for cholecystectomy. The patient's body temperature increased to 38-39°C, the pulse increased to 101 beats per 1 minute, the respiratory rate was 22 per 1 minute. To determine extracellular traps, blood was taken from the cubital vein for a complete blood count. When calculating the blood formula, the number of extracellular networks formed by granulocytes was also estimated. In the general blood test, the number of leukocytes was increased to 15×10 ⁹ /l. The absolute number of extracellular traps formed by peripheral blood granulocytes was 0.434×10 ⁹ /L. Based on this indicator, sepsis was diagnosed and treatment was prescribed.

Also, the blood taken from the patient was sent for bacteriological examination to confirm the diagnosis. Blood samples were processed using the BacT/Alert system. A positive blood culture was obtained only after 24 hours of incubation. Microorganisms were identified using typical Gram stain morphology and standard clinical microbiology techniques. Isolate identification was confirmed using special cards processed with a VITEK 2 instrument (BioMerieux, France). The result of a bacteriological blood test was obtained, the growth of Staphylococcus aureus was detected.

Thus, the proposed method for diagnosing sepsis in comparison with existing ones has the following advantages: the measurement does not require a long time, the method refers to express diagnostics (execution time is not more than 1 hour), which also allows you to track the effectiveness of the therapy, is not laborious, does not require expensive equipment and test systems, the method allows to diagnose sepsis on the 1st day of the disease.

Bibliography

1. Singer, M. The third international consensus definitions for sepsis and septic shock (Sepsis-3) / M. Singer, C.S. Deutschman, C.W. Seymour [et al.] // JAMA. - 2016. - Vol. 315, no. 8. - P. 801-810.

2. Fleischmann, C. Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations / C. Fleischmann, A. Scherag, N.K. Adhikari [et al.] // American journal of respiratory and critical care medicine. - 2016. - Vol. 193, no. 3. - P. 259-272.

3. Prescott, H.C. Enhancing recovery from sepsis: a review / H.C. Prescott, D.C. Angus // JAMA. - 2018. - Vol. 319, no. 1. - P. 62-75.

4. Seventieth World Health Assembly. - WHA70.7. - May 29, 2017. - P. 5.

5. Methods for determination of bacterial endotoxin in critical care medicine (review). M.N. Kopitsyna, A.S. Morozov, I.V. Bessonov, V.M. Pisarev / General resuscitation, 2017, 13, 5. P. 109-120

6. Belkov, V.V. Complex laboratory diagnostics of systemic infections and sepsis: C-reactive protein, procalcitonin, presepsin / V.V. Belkov. - Moscow: CJSC "Deacon", 2015. - 117 p.

7. Marshall J.C, Vincent J-L., Fink M. et al. Measurs, markers and mediators: towards a staging system for clinical sepsis. A report of the fifth Toronto sepsis roundtable // Ctrit. Care Med. - 2003. - Vol. 31, no. 5. - P.1560-1567.

8. RF patent No. 271555 Savochkina A.Yu., Pykhova L.R., Abramovskikh O.S., Chetvernina E.A., Poltorak A.E. Method for detecting extracellular DNA in whole peripheral blood. Application No. 2019119629, date of receipt 06/24/2019, priority 06/24/2019, registered in the State Register on 03/02/2020.

Claims (1)

A method for early diagnosis of sepsis, characterized in that on the 1st day of the disease, the absolute number of extracellular traps formed by neutrophilic granulocytes in peripheral blood is determined, and at the level of the absolute number of extracellular traps more than 0.354×10 ⁹ /l, sepsis is diagnosed.