wileyonlinelibrary.com/journal/ijlh Int J Lab Hem. 2018;40:152–158.
© 2017 John Wiley & Sons Ltd
Received: 2 March 2017
Accepted: 4 September 2017
Relevance of sample preparation for flow cytometry
V. E. Muccio | E. Saraci | M. Gilestro | D. Oddolo | M. Ruggeri | S. Caltagirone |
B. Bruno | M. Boccadoro | P. Omedè
Divisione Universitaria di Ematologia, A.O.U.
Città della Salute e della Scienza di Torino,
Vittorio Emanuele Muccio, Divisione
Universitaria di Ematologia, Torino, Italy.
Introduction: Flow cytometry is a useful tool for diagnosis and minimal residual dis-
ease (MRD) study of hematological diseases. Standard sample preparation protocols
are characterized by stain- lyse- wash (SLW). To prevent nonspecific bindings and
achieve high sensitivity in MRD studies, lyse- wash- stain- wash (LWSW) is required. To
our knowledge, no comparison between the two methods has been performed.
Methods: We compared mean fluorescence intensity (MFI), stain index, signal- to-
noise ratio, and percentage of positive cells of 104 antibodies and of 13 selected anti-
bodies tested in 10 samples simultaneously prepared with the two methods.
Results: MFI and percentages of positive cells obtained by the two methods did not
show significant differences and showed a very high correlation. Stain index and
signal- to- noise ratio presented higher values for kappa and lambda surface chains in
LWSW samples and a trend of higher values for the other antibodies in SLW
Conclusions: We suggest to use LWSW method also at diagnosis to obtain more
comparable antibody intensity expressions when samples from the same patient are
processed for MRD evaluation after bulk lysis. Moreover, LWSW can prevent
nonspecific bindings, shows no differences in the identification and quantitation of the
populations of interest, and reduces acquisition of cell debris.
antibody binding, flow cytometry, lyse-wash-stain, sample preparation, stain-lyse-wash
1 | INTRODUCTION
Over the past decades, multiparameter flow cytometry has become
the method of choice for the differential diagnosis of several hema-
tological diseases, for the definition of prognostic factors, and for the
identification of rare cell populations in peripheral blood (PB) and bone
marrow samples (BM).
Flow cytometry, through the presence or absence of specific sur-
face antigens, detects abnormal cells and helps in identifying their
lineage and maturation stage, detects abnormal cells through identifi-
cation of antigen expression that differs from the normal. The wider ap-
plication of flow cytometry in diagnostic and research fields increases
the need to distinguish between real and artifact positive signals given
that antigen- antibody interactions, characterized by weak bindings,
may involve unexpected molecules. The presence of dead cells, cell
doublets, and the nonspecific antibody binding can compete to gen-
erate artifacts in the phenotype. In some cases, an antibody can bind
similar antigens not of interest or an epitope can be shared by different
antigens or the Fc of many antibodies can be bound by Fc- receptors
expressed by different cells of the immune system.
body’s binding depends not only on the antibody’s specificity but also
on a wide variety of other interactions, as well as the physical and bi-
ological conditions that the target cell population encountered during
The ability to reliably distinguish between positive and negative
populations of cells is an essential aspect of clinical flow cytometry.