Izboljšave laboratorijske diagnostike s heparinom izzvane trombocitopenije v Sloveniji

Železnik, Klara (author), Maličev, Elvira (mentor)

Diagnozo s heparinom izzvane trombocitopenije (HIT) postavimo na podlagi klinične slike in laboratorijskih preiskav. Najprej smo analizirali demografske podatke in rezultate testiranja bolnikov s sumom na HIT, ki smo jih obravnavali med letoma 2015 in 2020. Opazovani parametri so primerljivi z zapisi v tuji literaturi. V nadaljevanju smo se osredotočili na možnosti izboljšave lastnega funkcijskega testa aktivacije trombocitov s pretočnim citometrom, pri katerem uporabljamo nativni serum bolnika in testne trombocite 4 dajalcev v trombocitni plazmi (PRP). Preverjali smo različne spremenljivke pri izvedbi testa: število dajalcev testnih trombocitov, pripravo testnih trombocitov in pripravo bolnikovega seruma. Pri preverjanju odziva testnih trombocitov na protitelesa HIT smo z razširjenim panelom 10 testnih trombocitov dokazali, da so vzorci z višjo izmerjeno vrednostjo optične gostote pri presejalnem testu (ELISA OD) ali višjo klinično oceno verjetnosti za HIT (test 4T) v povprečju aktivirali višje število testnih trombocitov. Na podlagi pridobljenih rezultatov in dosedanjih izkušenj s funkcijskim testom zaključujemo, da je izvedba testa s trombociti 4 dajalcev primerna. Dodatni predanalitični spremenljivki, ki smo ju preverjali, sta spiranje testnih trombocitov z različnimi pufri ter toplotna inaktivacija bolnikovega seruma. Vpliv dodatnih postopkov na rezultat testa smo preverjali s sočasnim vzporednim testiranjem vzorcev po standardnem postopku s trombociti istih dajalcev, česar v literaturi pri tovrstnem testu nismo zasledili. Izkazalo se je, da spiranje testnih trombocitov v primerjavi s PRP in toplotna inaktivacija bolnikovega seruma v primerjavi z nativnim serumom ne predstavljata izboljšave postopka. Toplotna inaktivacija seruma bi pri naših pogojih testiranja potencialno lahko predstavljala doprinos pri nejasnih primerih kot vzporedna metoda standardnemu postopku po predhodni prilagoditvi algoritma za interpretacijo. Kot pomoč pri interpretaciji rezultatov in za oceno aktivacije testnih trombocitov smo zasnovali indeks aktivacije trombocitov, ki ima zelo dobro negativno napovedno vrednost pri vrednosti 1,1 ali manj ter zelo dobro pozitivno napovedno vrednost pri vrednosti 1,6 ali več.

• Language: Slovenian
• Keywords: s heparinom izzvana trombocitopenija, trombociti, laboratorijska diagnostika, funkcijski test, pretočni citometer
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2023
• PID: 20.500.12556/RUL-143925
• COBISS.SI-ID: 146849283
• Publication date in RUL: 20.01.2023

Secondary language

• Language: English
• Title: Laboratory diagnostics improvements of heparin-induced thrombocytopenia in Slovenia

Abstract

Heparin-induced thrombocytopenia (HIT) diagnosis requires an integrated diagnostic approach combining clinical information with concomitant screening and confirmatory laboratory tests. First, we analyzed the test results and demographic data of patients with suspected HIT tested between 2015 and 2020. The observed parameters are comparable with descriptions in the foreign literature. Further, we have focused on the potential to improve our in-house confirmatory flow cytometric platelet-activating test, which tests native patient serum and 4 donor platelets in platelet-rich plasma (PRP). We tested different variables: number of platelet donors, preparation of donor platelets, and patient serum. The response of donor platelets to HIT antibodies in a patient serum was evaluated with an expanded panel of 10 donor platelets. We demonstrated that samples with a higher optical density value measured in the screening test (ELISA OD) or with a higher clinical probability of HIT (4T score) activated a higher number of donor platelets on average. Based on the results obtained and our experience with the functional test to date, we concluded that the use of 4 donor platelets is appropriate. The observed pre-analytical variables were also platelet washing with different buffers and heat inactivation of patient serum. The influence of these additional procedures on the test result was evaluated by testing patient samples in parallel according to the standard procedure with platelets obtained from the same donors, which has not been reported in the literature for this type of test. It seems that washed platelets vs. PRP and heat inactivation vs. native serum are not an improvement of the procedure. In our testing settings, heat inactivation could potentially resolve unclear cases as a parallel method to the standard protocol after adjusting the algorithm for result interpretation. We have designed a platelet activation index to help with result interpretation and assess platelet activation. Index has a very good negative predictive value at 1.1 or less and a good positive predictive value at 1.6 or more.

• Keywords: heparin-induced thrombocytopenia, platelets, laboratory diagnostics, functional assay, flow cytometer