Flow cytometry characterization of white cell-reduced blood: apoptosis markers and morphology of postfiltration elements.
OBJECTIVE
Apoptosis affects white blood cells (WBCs) contained in packed red blood cell (RBC) units. This phenomenon was recently described also in residual WBCs after filtration. The aim of this study was to better characterize the residual WBCs postfiltration by using apoptosis markers and morphology.


METHODS
Immunofluorescence, flow cytometry and cell-sorting techniques were utilized.


RESULTS
Residual leucocytes of leucodepleted packed RBC units showed increasing values of apoptotic elements in a time-course experiment. We also demonstrated that these elements are positive for APO 2.7 monoclonal antibody (mAb), poly ADP-ribose polymerase (PARP) cleavage and fluorescein isothiocyanate (FITC)-conjugated N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), all of which indicate that programmed death is a feature of this population of cells. Phenotypic analysis with CD45 side-scatter gating demonstrated also that CD15 and CD16 granulocyte-associated antigens are present on a subset of postfiltration leucocytes. Moreover, the expression of human leucocyte antigen (HLA) class I antigens is maintained. Sorting of CD45-positive cells and morphological analysis of these samples confirmed that leucocytes in postfiltration units have morphological characteristics of dying cells.


CONCLUSIONS
Our study extends previous observations regarding the morphology and function of apoptotic cells in leucodepleted blood units, which suggested the presence of apoptotic cells in postfiltration leucocytes. Cleaved PARP, APO 2.7 mAb and positivity for the FITC-conjugated Z-VAD-analogous reagent strongly suggest the activation of programmed death pathways. In addition, the maintained granulocyte-associated and HLA class I antigens might recall an immune response in multitransfused patients.
Tazzari
P L
PL
Servizio di Immunoematologia e Medicina Trasfusionale, Policlinico S. Orsola-Malpighi, Bologna, Italy.
Bontadini
A
A
Fruet
F
F
Tassi
C
C
Ricci
F
F
Manfroi
S
S
Conte
R
R
D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D017707:Erythrocyte Transfusion / Q000592:standards*; D005374:Filtration; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping; D007962:Leukocytes / Q000166:cytology / Q000276:immunology; D013997:Time Factors
D015415:Biomarkers