Določevanje trombocitnih protiteles pri zdravih darovalcih plazme in pri prebolevnikih covid-19

Bohinc, Klara (author), Rožman, Primož (mentor)

Med pandemijo koronavirusne bolezni 2019 so za zdravljenje ljudi s težjim potekom priporočili uporabo kovidne prebolevniške plazme, ki so jo zbrali od posameznikov po preboleli okužbi z virusom SARS-CoV-2. Nekatere študije so pokazale, da lahko zmanjša resnost bolezni in izboljša stopnjo preživetja, medtem ko druge niso odkrile pomembne koristi. Kljub temu da so danes na voljo druge oblike zdravljenja in preventive, kovidno prebolevniško plazmo še vedno uporabljamo, njeno učinkovitost pa posledično nadalje preučujemo. Eden izmed razlogov, ki lahko prispeva k njenemu relativno šibkemu terapevtskemu učinku, je prisotnost nezaželenih snovi, ki v telesu nastanejo med okužbo in po zdravljenju povzročijo iatrogene zaplete. Sem sodijo številne molekule, ki so posledica akutnega vnetja ter drugih imunskih, presnovnih in koagulacijskih motenj, mednje pa spadajo tudi trombocitna avtoprotitelesa. V okviru magistrske naloge smo s štirimi metodami določili trombocitna avtoprotitelesa pri 115 darovalcih kovidne prebolevniške plazme ter pri 130 zdravih darovalcih plazme, ki okužbe niso preboleli. Med skupinama smo najprej preverili morebitno prisotnost razlik v demografskih značilnostih in ugotovili, da so bili prebolevniki v povprečju starejši za približno 5 let. V prvem delu magistrske naloge smo izvedli presejalni indirektni imunofluorescenčni test, potem pa še potrditveni fluorescenčno pretočni citometrični test Pak Lx® za specifikacijo protiteles. Razlik v prisotnosti in specifičnosti trombocitnih protiteles med skupinama nismo dokazali. V drugem delu smo z encimskim imunoadsorpcijskim testom dodatno poskušali dokazati še protitelesa proti trombocitnemu faktorju 4. Na vzorcih s pozitivnim rezultatom smo v nadaljevanju izvedli še funkcijski test za preverjanje sposobnosti aktivacije trombocitov. Med skupinama nismo odkrili statistično značilne razlike v prisotnosti protiteles proti trombocitnemu faktorju 4, vzorci, ki smo jih testirali s funkcijskim testom, pa sposobnosti aktivacije trombocitov niso izkazali. S tem smo dokazali, da trombocitna avtoprotitelesa verjetno ne prispevajo k slabši terapevtski učinkovitosti enot kovidne prebolevniške plazme. Predlagamo, da se zbrane enote testira še na morebitno prisotnost drugih patoloških molekul, ki se pojavijo med okužbo.

• Language: Slovenian
• Keywords: kovidna prebolevniška plazma, trombocitna avtoprotitelesa, trombociti, SARS-CoV-2, covid-19
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-146743
• Publication date in RUL: 10.06.2023

Secondary language

• Language: English
• Title: Determination of platelet antibodies in healthy plasma donors and COVID-19 convalescents

Abstract

During the pandemic of coronavirus disease 2019, the use of covid convalescent plasma collected from individuals after their recovery from the infection was recommended for the treatment of patients with severe course of the disease. Some studies have shown that convalescent plasma can reduce disease severity and improve survival rates, while others have found no significant benefit. Despite the availability of other forms of treatment and prevention, covid plasma is still used and its effectiveness is being further studied. One of the reasons that may contribute to its relatively weak therapeutic effect is the presence of unwanted substances that are produced in the body during infection and cause iatrogenic complications after treatment. These include many molecules that result from acute inflammation and other immune, metabolic and coagulation disorders, including platelet autoantibodies. In the scope of Master's thesis, we used four methods to determine the presence of platelet autoantibodies in 115 covid convalescent plasma donors and in 130 healthy plasma donors that have not been infected. We first checked for any differences in demographic characteristics between the two groups and found that on average the convalescents were about 5 years older. In the first part of the study, we performed screening using an indirect immunofluorescence test, followed by the confirmatory Pak Lx® fluorescence flow cytometric test for antibody specification. We found no differences in the presence and specificity of platelet antibodies between the two groups. In the second part of the study, we additionally tried to detect antibodies against platelet factor 4 using an enzyme-linked immunosorbent assay. The positive samples were further subjected to a functional assay to verify platelet activation capacity. We found no statistically significant difference in the presence of antibodies against platelet factor 4 between the two groups, and the samples tested in the functional test did not show any platelet activation capacity. This demonstrates that platelet antibodies are unlikely to contribute to weaker therapeutic efficacy in the covid convalescent plasma. We suggest that the collected units are also tested for the presence of other pathological molecules that may be present during the infection.

• Keywords: covid convalescent plasma, platelet autoantibodies, platelets, SARS-CoV-2, COVID-19