RU-2861290-C1 - METHOD FOR QUANTITATIVE AND QUALITATIVE ASSESSMENT OF NEUTROPHIL EXTRACELLULAR TRAPS FOR PATIENTS WITH ESSENTIAL THROMBOCYTHAEMIA

Abstract

FIELD: medicine. SUBSTANCE: invention relates to clinical laboratory diagnostics, can be used in studying the risk of developing immunothrombosis. When implementing a method for quantitative and qualitative assessment of neutrophil extracellular DNA traps for patients with essential thrombocythaemia, a sample of venous blood taken from a patient in a plastic tube with K3-EDTA anticoagulant is allowed to settle, the plasma is completely taken into a separate tube and mixed by rocking. Plasma is applied to the edge of a glass slide, spread over the surface of the slide, after drying, the smear is fixed in 1% buffered formalin solution and stained with primary antibodies specific to proteins of neutrophil extracellular DNA traps and secondary antibodies. The stained smears are examined using a confocal fluorescence microscope and a quantitative and qualitative assessment of neutrophil extracellular traps is carried out. EFFECT: method provides unambiguous identification in samples and quantitative assessment of neutrophil extracellular DNA traps, includes their qualitative assessment, observation of both canonical suicidal types of extracellular DNA traps and vital ones, and minimisation of mechanical impact on cells. 1 cl, 2 dwg

Inventors

• Adamanskaia Ekaterina Aleksandrovna
• Pshonkin Aleksei Vadimovich
• Smetanina Nataliia Sergeevna
• Sveshnikova Anastasiia Nikitichna

Dates

Publication Date

20260504

Application Date

20250228

Claims (1)

1. The method of quantitative and qualitative assessment of neutrophil extracellular DNA traps for patients with essential thrombocythemia is carried out as follows: a sample of venous blood taken from a patient in a plastic tube with the anticoagulant K3-EDTA, settled for 45 minutes at a temperature of 37 ° C, after which the plasma is completely collected in a separate tube and mixed by shaking, then 4 μl of plasma is applied to the edge of a glass slide and spread on the surface of the glass, after drying, the smear is fixed in a 1% solution of buffered formalin and stained with primary antibodies specific to proteins of extracellular DNA traps of neutrophils and secondary antibodies, then the stained smears are examined on a confocal fluorescence microscope and a quantitative and qualitative assessment of neutrophil extracellular traps is carried out, while: if the cell membrane is destroyed, has strong breaks and chromatin threads with antimicrobial peptides distributed in the extracellular space, the objects are characterized as a suicidal type; if the plasma membrane is intact, the detected cell lacks chromatin or the DNA is in vesicles at the cell periphery, and myeloperoxidase and neutrophil elastase are detected in the cytoplasm, such a manifestation is considered a vital type of release of extracellular DNA traps.

Description

The invention relates to the field of medicine, in particular to clinical laboratory diagnostics, and can be used in studying the risk of developing immunothrombosis. The formation of neutrophil extracellular traps (NETs) is a key mechanism of innate immunity, characterized by the release of DNA strands enriched with antibacterial proteins to combat pathogens. NETs are also known to activate the intrinsic coagulation pathway. Patients with myeloproliferative neoplasms (MPN) are thought to be at higher risk of thrombosis, in part due to elevated levels of NETs [5], but quantitative data on this rare class of diseases in children are limited. Therefore, the development of a method for quantifying neutrophil extracellular DNA traps is highly relevant. There are several studies devoted to assessing the number of NETs in patients or the level of NETosis in a patient. However, all studies pre-isolate neutrophils, which is known [3] to activate segmented leukocytes and can lead to incorrect results. For example, the article "Quantitative Method for Characterizing NETosis" describes an optimized high-speed imaging method followed by semi-automated image analysis to identify features characteristic of neutrophils undergoing "suicidal" and "vital" NETosis. Also, a comparison of neutrophils obtained from the peripheral blood of healthy individuals and individual neutrophil subsets isolated from the blood of patients with systemic lupus erythematosus [6] is made. The disadvantage of this method is that it provides only a comparative analysis of the obtained data, without recommendations for further treatment of patients. Moreover, the results obtained in the described work are applicable only to patients with systemic lupus erythematosus. A well-known method for assessing neutrophils is the technique used in the work of Fedorov et al. [1]. Or, based on this work, in the patent application "Identification of neutrophil extracellular traps in biological samples" (patent application No. WO2021022165A1). This method consists of a computer-based method for identifying neutrophil extracellular traps in the blood, including: obtaining images of the biological sample and analyzing its morphological characteristics. A drawback of this patent is that its application to standard hematoxylin and eosin-stained blood smears may yield false-positive results due to the nonspecificity of the staining. Applying a single marker to NETosis may mislead the identification of the cell type: smeared cells or manifestations of NETosis. Furthermore, the method is not designed to obtain healthy norms and does not characterize the type of neutrophil extracellular traps. Furthermore, this method does not provide recommendations for clinical use, taking into account the minimization of impact on neutrophil status. An analog of the claimed method for assessing neutrophil extracellular DNA traps is the invention "Method for Detecting Neutrophil Extracellular Traps in a Supravitally Stained Blood Preparation" (RU Patent No. 2768152). The invention involves identifying neutrophils in a suspension obtained using a double density gradient of a Ficoll-Vergografin solution. Cell staining occurs supravitally, i.e., without cell fixation, using propidium iodide dye and monoclonal antibodies to CD15. The main drawback of this invention compared to the claimed method is the use of a Ficoll-verografin solution to obtain a neutrophil suspension. This solution, as demonstrated, for example, in the work of TaináMosca and Wilma C. N. Forte "Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficolland, and Spontaneous Sedimentation Methods" [3], additionally activates neutrophils, which affects the reliability of determining the baseline level of extracellular DNA traps. Furthermore, this method does not provide unambiguous identification of neutrophil DNA traps from other extracellular DNA, which, as a result, does not allow the detection of vital NETosis. Staining with propidium iodide and antibodies to CD15 does not provide sufficient specificity for NETosis. Another drawback of this method is that it uses hepanized peripheral venous blood. The anticoagulant heparin affects leukocyte activity [2], particularly neutrophils, which also influences the formation of extracellular DNA traps and the determination of baseline NETosis levels at rest. This method is also not suitable for clinical use with high patient sample throughput and potential repeat testing, as the samples are not fixed, preventing storage for repeat testing. The use of unfixed cells also limits the time required to process the resulting blood sample, as blood is considered suitable for analysis for up to 3-4 hours. In terms of subject matter, this result of intellectual activity (RIA) corresponds to the RIA "Quantitation of NETosis using image analysis" [4]. However, this RIA is a computer program for image processing and is methodologically incompatible wit