Specifičnosti hemostaznog mehanizma kod obolelih od SARS-CoV-2 virusne infekcije

Abstract

Uvod: Obolljenje Covid-19, izazvano korona virusom teškog akutnog respiratornog sidroma 2 (SARS-CoV-2) u 2020. i 2021. godini nametnulo se kao značajan uzrok morbiditeta i mortaliteta u svetu. Već na samom početku pandemije uočen je značajan broj slučajeva infekcije koji su bili praćeni poremećajima funkcionalnosti hemostaznog mehanizma, koji se ogledao u izrazito povišenom nivou stimiulacije trombinske aktivnosti, menifestovanim u vidu diseminovane intravaskularne koagulacije kao i razvojem areterijskih i venskih tromboza različitih lokalizacija. Precizan mehanizam razvoja koagulopatija pri infekciji SARS-CoV-2 virusnom infekcijom nije u potpunosti razjašnjen . Uključenost sve tri komponente Virhivljeve trijade u sadejstvu sa inflamatornim promenama nastalim kao posledica infekcije ali i samog imunskog odgovora organizma mofuće je objašnjenje patofiziološkog mehanizma koagulopatije. Materijal i metode: Ispitivanje je koncipirano kao u vidu prospektivne studje tokom čije izrade je izvršeno regrutovanje 60 pacijenata sa RT-PCR potvrđenom infekcijom SARS-CoV-2, hospitalizovanih inicijalno u COVID jedinicama Kliničkog centra Vojvodine, izuzimajući pacijente koji su se nalazili u jedinicama intenzivne nege. Za potrebe istraživanja korišteni su osnovni anamnestički podaci kao i podaci laboratorijskih analiza uzoraka pacijenata dobijenih flebotomijom i analiziranjem uzoraka pune geparinizirane krvi kao i analizom EDTA krvi i citratne plazme. Određivanje parametara krvne slike, uključujući određivanje broja trombocia i trombocitnih indeksa izvršnjno je na automatizovanom hematološkom brojaču. Ispitivanje funkcionalnosti primarne hemostaze, pored određivanja broja trombocita i trombocitnih indeksa, obukvatilo je agregometrijska merenja metodom impredansne agregometrije određivanjem TRAP testa kao pokazatelja bazalne funkcionalnosti trombocita iz uzorka pune krvi sa litijum-heparinom kao antikoagulansom. Procena koagulacionog mehanizma izvršena je određivanjem aktivisanog parcijalnog tromboplasninskog vremena (aPTT), protrombinskog vremena (PT), fibrinogena i d-dimera iz uzoraka citratne plazema na automatizovanim koagulometrima.Euglobulinsko vreme lize koaguluma kao test procene funkcionalnosti fibrinoliznog mehanizma je određivano metodom prema Makfarlanu i Pilinguiz uzoraka citratne plazme. Za unos i obradu podataka korišćen je program SPSS, verzija 24.0.U okviru komparativne statistike u culju određivanja ratlika imeđu ispitivanih grupa korišćen je Studentov t-test za nezavisne uzorke, ili odgovarajuća neparametrijska zamena Man-Vitnijev test. Kako bi se utvrdila povezanost između različitih posmatranih kliničkih parametara primenjen je Spirmanov koeficijent rang korelacije i Pirsonov koeficijent linearne korelacije ako su u pitanju kontinuirane varijavle sa normalnom distribucijom. Za procenu uticaja pojedinačnig ispitivanih parametara na rizik od nastanka smrtnog ishoda ili tromboznih komplikacija korišćena je nekondicionalna logistička regresija. Rezultati: U grupi ispitanika je bilo 36 muškaraca (60%) i 24 žene (40%) ispitanika. Prosečna starost iznosila je 65 godina, prilikom čega je najmlađi ipitanik imao 32 godine, dok je najstariji imao 96 godina. Od ukupnog broja ispitanika, kod četiri (6,7%) je dijagnostikovana tromboza dubokih vena ili plućna tromboembolija dok je smrtni ishod nastupio kod 12 pacijenata što iznosi 20% ukupnog broja ispitanika. Grupisanjem ispitanika u pogledu ishoda lečenja uočena su 44 pozitivna ishoda (73,3%). dok je negativan ishod, koji obuhvata tromboembolijske komplikacije i smrtni ishod bio prisutan kod 16 ispitanika (26,7%). Ispitivanje statističke značajnosti razlika u parametrima primarne hemostaze, koagulacionog i fibrinoliznog mehanizma pokazalo je postojanje statistički značajne razlike (p<0,001) za sve ispitivane parametre poredeći grupu ispitanika sa negativnim ishodom, kao i poredeći grupu ispitanika sa smrtnim ishodom sa grupom ispitanika sa pozitivnim ishodom. Primenom nekondicionalne logističke regresije izvršena je procena relativnog rizika za nastanak negativnog ishoda kod pacijenata u odnosu na pojedinačne laboratorijske parametre hemostaznog mehanizma ispitivane u trenutku prijema na bolničko lečennje. Kako svi primenjivani tesotvi za procenu primarne hemostaze, koagulacionog i fibrinoliznog mehanizma imaju uniformne referentne opsege koji su jednaki za oba pola, logistička regresija prilagođena je za godine ispitanika. Rezultati procene rizika za nastanak negativnog ishoda bolesti u odnosu na ispitivane pojedinačne parametre primarne hemostaze pokazali su da ispitivani parametri imaju uticaj na porast rizika na negativan ishod pri čemu njihov pojedinačni uticaj iznosi 7%. Nekondicionalna logistika regresija primenjena je i na parametre koagulacionog mehanizma čiji rezultati su ukazali na blag uticaj svakog od ispitivanih parametara. Test euglobulinskog vremena lize koaguluma kojime je vršena procena funkcionalnosti fibrinoliznog mehanizma, testiran nekondicionalnom logističkom regredijom pokazao je blag uticaj (OR 1.084 95%CI 1.023-1.160) ovog parametra na ishod samog oboljenja. Multivarijantni regresioni model pokazao je da, uzimajući u obzir doprinos svakog pojedinačnog izmerenog parametra primarne hemostaze, koagulacionog i fibrinoliznog mehanizma, njihovu međusobnu interakciju i efekat na ishod oboljenja euglobulinsko vreme lize koaguluma se izdvojilo kao statistički signifikantan prediktor ishoda, sa statističkom značajnošću na nivou p<0.001. Zaključak: Rezultati ove studije još jednom su pokazali značaj fibrinoliznog mehanizma u regulaciji celokupnog hemostaznog mehanizma i pokazali potencijalnu korist od određivanja euglobulinskog vremena lize koaguluma kao pokazatelja globalne procene fibrinoliznog mehanizma kod osoba obolelih od SARS-CoV-2 virusne infekcije u trenutku prijema na bolničko lečenje kod kojih je došlo do razvoja tromboznih komplikacija i negativnog ishoda.

Introduction: Covid-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 and 2021, has emerged as a significant cause of morbidity and mortality worldwide. At the very beginning of the pandemic, a significant number of cases of infection were observed, which were accompanied by dysfunctions of the hemostatic mechanism, which was reflected in markedly elevated levels of thrombin activity, manifested as disseminated intravascular coagulation and development of arterial and venous thrombosis. The exact mechanism of coagulopathy development in SARS-CoV-2 virus infection has not been fully elucidated. The involvement of all three components of the Virchow' triad in cooperation with inflammatory changes caused by infection, but also by the immune response of the organism itself, may be an explanation of the pathophysiological mechanism of coagulopathy. Material and methods: The study was conducted as a prospective study during which 60 patients with RT-PCR confirmed SARS-CoV-2 infection were recruited, initially hospitalized in COVID units of the Clinical Center of Vojvodina, except for patients who were in intensive care units. For the purposes of the research, the basic anamnestic data and laboratory data of patient samples obtained by phlebotomy and analysis of whole heparinized blood as well as EDTA and citrate plasma were used. Determination of blood count parameters, including determination of platelet count and platelet indices, was performed on an automated hematology analyzer. Examination of the functionality of primary hemostasis, in addition to determining the number of platelets and platelet indices, included aggregometric measurements by the method of impedance aggregometry by determining the TRAP test as an indicator of basal platelet function from a whole blood sample with lithium heparin as an anticoagulant. Evaluation of coagulation mechanism was performed by determination of activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and d-dimer from citrate plasma samples on automated coagulometers. Euglobulin clot lysis time as an assessment of the functionality of the fibrinolytic mechanism was determined by the method according to McFarlane and Peeling from citrate plasma samples SPSS program, version 24.0, was used for data entry and processing. In order to determine the correlation between the different observed clinical parameters, Spearman 's correlation rank coefficient and Pearson' s coefficient of linear correlation were applied in the case of continuous variables with normal distribution. Non - conditional logistic regression was used to assess the influence of individual examined parameters on the risk of death or thrombotic complications. Results: In the group of respondents there were 36 male (60%) and 24 female (40%). The average age was 65, during which the youngest respondent was 32 years old, while the oldest was 96 years old. Of the total number of subjects, four (6.7%) were diagnosed with deep vein thrombosis or pulmonary thromboembolism, while death occurred in 12 patients, which is 20% of the total number of subjects. By grouping the respondents in terms of treatment outcomes, 44 positive outcomes were observed (73.3%). while a negative outcome, which includes thromboembolic complications and death, was present in 16 subjects (26.7%). Examination of statistical significance of differences in the parameters of primary hemostasis, coagulation and fibrinolytic mechanism showed the existence of statistically significant differences (p <0.001) for all examined parameters comparing the group of subjects with negative outcome and the group of subjects with fatal outcome with the group of subjects with positive outcome. Using non - conditional logistic regression, the assessment of the relative risk for the occurrence of a negative outcome in patients in relation to individual laboratory parameters of the hemostastic mechanism examined at the time of admission to hospital treatment was performed. As all applied tests for the assessment of primary hemostasis, coagulation and fibrinolytic mechanism have uniform reference ranges that are equal for both sexes, logistic regression was adjusted for the age of the subjects. The results of the risk assessment for the occurrence of a negative outcome of the disease in relation to the examined individual parameters of primary hemostasis showed that the examined parameters have an impact on the increase in risk of a negative outcome with their individual impact of 7%. Non-conventional regression logistics was also applied to the parameters of the coagulation mechanism, the results of which indicated a slight influence of each of the examined parameters. The euglobulin clot lysis time, which assessed the functionality of the fibrinolytic mechanism, tested by non-conditional logistic regression, showed a mild effect (OR 1,084 95% CI 1,023-1,160) of this parameter on the outcome of the disease. Multivariate regression model showed that, taking into account the contribution of each individual measured parameter of primary hemostasis, coagulation and fibrinolytic mechanism, their interaction and effect on disease outcome, euglobulin clot lysis time was isolated as a statistically significant predictor of outcome p<0.001. Conclusion: The results of this study once again showed the importance of the fibrinolytic mechanism in the regulation of the overall hemostatic mechanism and showed the potential benefit of determining the euglobulin clot lysis time as an indicator of global assessment of fibrinolytic mechanism in persons with SARS-CoV-2 viral infection at hospital admission. in whom thrombotic complications and a negative outcome have developed.