Author/Editor		Ihan, A
Title		Primerjava eno-, dvo- in tribarvne fluorescentne metode za ugotavljanje koncentracije celic T pomagalk in zaviralk v krvi
Translated title		A comparison of one, two and three color T cell subset analysis with single laser flow cytometer for the immune status monitoring
Type		članek
Source		Zdrav Vestn
Vol. and No.		Letnik 64, št. Suppl 3
Publication year		1995
Volume		str. III-81-3
Language		slo
Abstract		Background. Although a data collection and analysis with modern, electronically controlled systems seems to be very simple and accurate, the results, if unproperly managed, can be far from reality. In the article the flow cytometric analysis of peripheral blood lymphocyte populations is described. The author desired to investigate the accuracy of one-, two-, and three-colour flow cytometric methods for the analysis of peripheral blood lymphocyte populations. Methods. Blood specimens of 29 blood donors were analysed. Blood donors were divided into three groups. The group A consisted of 12 healthy blood donors with normal white blood picture. The group B consisted of 8 patients with lymphopenia (more th. 1000 lymph/mm3). The group C consisted of 9 patients with leucocytosis (WBC count between 15000 and 20000). The immune status was reported as:1) the percentages of CD4 and CD8 positive lymphocytes, obtained by one colour flow cytometry, 2) the percentages of CD4 T lymphocytes and CD8 T lymphocytes, obtained by two colour flow cytometry, and 3) the percentages of CD4 T lymphocytes, CD8 T lymphocytes, obtained by three colour flow cytometry. Results. Our results demonstrate, that the most economic one colour flow cytometry may give relevant results when monitoring patients with normal T lymphocyte counts. When lymphopenia develops, the relevant results can be obtained only by combining CD3 and CD4 antibodies for detection of Th cells, and by combining CD3 and CD8 antibodies for detection of Ts cells. Three colour examination with CD3, CD4 and CD8 antibodies is able to obtain so me additional data, but most importantly, it is able to obtain more precise data, it the lymphocyte gate is contaminated with unlysed erythrocytes. In that case, cells can be gated on the CD3 positive cells and the number of CD4 and CD8 T lymphocytes precisely evaluated. Conclusions. The choice of one-, two-, three-colour flow cytometric analysis influence the accuracy of results.(trunc.)
Summary		Izhodišča. čeprav se zdi pridobivanje in analiza podatkov z modernimi elektronsko vodenimi instrumenti enostavna in točna, pa rezultati mnogokrat ob neprimernem ravnanju niso odraz resničnega stanja. Prispevek opisuje analizo limfocitnih populacij v krvi z metodo pretočne citometrije. Avtor je želel raziskati, kako na rezultate vplivajo posamezni načini analize vzorca (enobarvna, dvobarvna, tribarvna analiza). Metode. na treh skupinah preiskovancev (zdravi, bolniki z limfopenijo, bolniki z levkocitozo) smo primerjali tri metode pretočne citometrije za ugotavljanje koncentracije celic T zaviralk v krvi. Z metodo enojnega barvanja smo s protitelesi CD4 ugotavljali koncentracijo celic T pomagalk, s protitelesi CD8 pa koncentracijo celic T zaviralk. Pri metodi dvojnega barvanja smo celice T pomagalke prepoznali s kombinacijo protiteles CD3 in CD4, celice T zaviralke pa s kombinacijo protiteles CD3 in CD8. Pri metodi trojnega barvanja smo kri hkrati barvali s kombinacijo protiteles CD3, CD4 in CD8. Rezultati. Pri skupini zdravih preiskovancev so vse tri barvilne metode dale primerljive rezultate. Pri skupini limfopeničnih bolnikov metoda enojnega barvanja ne daje zadovoljivih rezultatov, medtem ko smo z metodo dvojnega in trojnega barvanja dobili primerljive in zanesljive rezultate. Pri bolnikih z levkocitozo omogoča metoda trojnega barvanja najnatančnejšo določitev celic T pomagalk in celic T zaviralk v krvi. Zaključki. Rezultati analize limfocitnih populacij v krvi so odvisni tudi od izbire metode za analizo vzorca, pri izbiri metode pa je treba upoštevati diferencialno krvno sliko preiskovanca. Pri večini primerov omogoča zadovoljive rezultate dvobarvna analiza, pri bolnikih z levkocitozo pa je primernejša tribarvna analiza.
Descriptors		LYMPHOPENIA LEUKOCYTOSIS T-LYMPHOCYTES, HELPER-INDUCER T-LYMPHOCYTES, SUPPRESSOR-EFFECTOR FLOW CYTOMETRY T-LYMPHOCYTE SUBSETS CD4-CD8 RATIO ANTIBODIES, MONOCLONAL