Enumeration of hematopoietic stem cells in venous blood and apheresis products\u27 samples using hematology analyzer

V procesu presaditve krvotvornih matičnih celic (KMC) iz venske krvi običajno določamo količino KMC pred zbiranjem celic (aferezo) v venski krvi in po zbiranju v koncentratu. Določamo jih s pretočnim citometrom po priporočilih International Society of Hematotherapy and Graft Engineering, v raziskovalne namene pa tudi z nekaterimi hematološkimi analizatorji, ki so opremljeni s kanalom za določanje nezrelih celic. Z nalogo smo želeli opredeliti, kako dobro se metodi ujemata in v katerih primerih bi analize na pretočnem citometru lahko zamenjali ali dopolnili z analizami na hematološkem analizatorju. Predlagati smo želeli spremembe odločitvenih poti za pravilnejše in hitrejše sprejemanje kliničnih odločitev. V eksperimentalnem delu smo z obema metodama – s pretočnim citometrom in s hematološkim analizatorjem – analizirali vzorce venske krvi pred aferezo, vzorce svežih koncentratov in vzorce zamrznjenih koncentratov. Zbrane rezultate smo statistično analizirali s programom MedCalc. Ločeno smo obravnavali vse tri skupine analiziranih vzorcev in vzorce venske krvi z zelo nizkimi koncentracijami levkocitov (? 3 × 109/L), pri katerih po sedaj uveljavljenih odločitvenih kriterijih ne določamo koncentracije KMC. Korelacijo med metodama smo ovrednotili s pomočjo Spearmanovega koeficienta tako za deleže kot za koncentracije KMC. Korelacija je zelo dobra pri vseh vzorcih skupaj (N = 179) ter posebej pri vzorcih venske krvi (N = 103) in svežih koncentratov (N = 56), slaba pa pri vzorcih venske krvi z zelo nizkimi koncentracijami levkocitov (N = 12) in vzorcih zamrznjenih koncentratov (N = 20). Tudi Bland-Altmanova analiza je potrdila zelo dobro ujemanje med metodama pri vzorcih venske krvi in svežih koncentratov ter slabo ujemanje pri vzorcih venske krvi z zelo nizkimi koncentracijami levkocitov in zamrznjenih koncentratov. Analize s pretočnim citometrom bi pri vzorcih venske krvi v večini primerov lahko nadomestili z analizami na hematološkem analizatorju in na osnovi rezultatov sprejemali ustrezne klinične odločitve o začetku afereze. Pri vzorcih venske krvi z zelo nizkimi koncentracijami levkocitov bi lahko uvedli analize s hematološkim analizatorjem in v primeru določitve visokih vrednosti KMC rezultate potrdili s pretočnim citometrom. Vzorce svežih koncentratov kljub zelo dobremu ujemanju med metodama vedno analiziramo s pretočnim citometrom, saj količina KMC v koncentratu pomembno vpliva na uspešnost presaditve.During the process of transplantation of hematopoietic stem cells (HSCs) from venous blood cell count is usually performed before stem cell collection (apheresis) in venous blood and after apheresis procedure in apheresis products. Enumeration of HSCs is performed by flow cytometry according to the protocol suggested by International Society of Hematotherapy and Graft Engineering. For research purposes, hematology analyzers upgraded with a channel for the determination of immature cells can also be used. The purpose of this dissertation was to determine the agreement between the two methods of measurement and to determine in which cases analyses performed by flow cytometry could be replaced by analyses on hematology analyzer. We wanted to suggest changes in decision-making scheme that could help make clinical decisions more accurate and faster. In the experimental part, venous blood samples, fresh and frozen apheresis products\u27 samples were analysed by both methods using flow cytometer and hematology analyzer. Statistical analyses of the results were performed by MedCalc statistical software. All three groups of the analysed samples were treated separately, as well as venous blood samples with very low leucocyte concentration (? 3 × 109/L) in which HSC concentration is not determined according to the established decision criteria. Correlation between the two methods was assessed using Spearman\u27s correlation coefficient both for percentages and concentrations of HSCs. We observed a very strong correlation in the group of all analysed samples (N = 179), in venous blood samples (N = 103) and in fresh apheresis products\u27 samples (N = 56), and a weak correlation in venous blood samples with very low leucocyte concentration (N = 12) and in frozen apheresis products\u27 samples (N = 20). Bland-Altman analysis confirmed that the results of analyses with both methods agreed very well in venous blood and fresh apheresis products, but there was a poor agreement between methods in venous blood with low leucocyte concentration and in frozen apheresis products. Hematology analyzer could be used to substitute most of the analyses of venous blood samples performed with flow cytometer to determine the optimal starting point of apheresis. In venous blood samples with very low leucocyte concentration analyses by hematology analyzer could be introduced and the results, in cases of high levels of HSCs, confirmed with flow cytometer. In spite of a good agreement between the methods, fresh apheresis products are always analysed by flow cytometry because the amount of HSCs in a transplant is crucial for a successful transplantation