12th Congress of the European Hematology ... - Haematologica

Myelodysplastic syndromes II
0623
MDS PATIENTS WITH ANTI ERYTHROBLAST AUTOIMMUNITY: EFFECT OF BM CULTURE
SUPERNATANTS ON CLONOGENIC ACTIVITY OF NORMAL BONE MARROW
F. Imperiali, A. Zaninoni, M Colombi, A. Zanella, W. Barcellini
Fondazione IRCCS Ospedale Maggiore, MILAN, Italy
Background. Myelodysplatic syndromes (MDS) are characterised by
ineffective and dysplastic hematopoiesis, bone marrow hypercellularity
and, paradoxically, progressive peripheral blood cytopenias. Autoimmune
phenomena, mainly directed against RBC, are thought to play a
role in bone marrow failure of early MDS, i.e. refractory anemia (RA) and
RA with ringed sideroblasts (RARS). We showed an autoimmune reactivity
against erythroblasts in roughly half patients with RA and RARS
by a method named mitogen-stimulated-direct antiglobulin test (MS-
DAT). The same patients displayed increased erythroblast counts together
with peripheral signs of hemolysis. Aims. To study the effect of BM
culture supernatants from MDS patients with anti erythroblast autoimmunity
on clonogenic activity of normal BM Methods. MS-DAT was performed
by stimulating BM cells with PMA and PHA and antibodies were
detected in supernatants by competitive solid phase ELISA. Culture
supernatants from BM MS-DAT positive and negative patients were
tested on the growth of normal BM progenitors on methylcellulose
medium. Colony forming units (CFU) count and morphological evaluation
were performed after 14 days of culture. Smears were obtained
from CFU and differential counts performed. Results. The morphology
and the number of colony forming units granulocyte-monocyte (CFU-
GM) and burst forming units erythroid (BFU-E) were comparable in cultures
performed with or without BM MS-DAT positive supernatants.
Likewise, control experiments with BM MS-DAT negative supernatants
gave negligible effect on the number of CFU-GM and BFU-E colonies.
Addition of BM MS-DAT positive supernatants increased the overall
CFU smears cellularity along with the appearance of diserythropoietic
signs (nuclear atypia i.e. multiple nuclei, nuclear inclusions, and intercellular
bridges). At variance BM MS-DAT negative supernatants had no
effect on CFU smears. Regarding the differential BM counts (percentage
of myeloid and erythroid precursors) we did not observe major differences
in the presence or absence of various supernatants. Conclusions. BM
culture supernatants from MDS patients with an autoimmune reactivity
against erythroblasts induced normal BM progenitors to an hyperplastic
and diserythropoietic growth in vitro, without a preferential destruction
of a cell subset or a blockade in red cell hematopoietic maturation.
0624
RELATIVE DISTRIBUTION OF DIFFERENT COMPARTMENTS OF CD34+ CELLS IN THE
BONE MARROW OF PATIENTS WITH MYELODYSPLASTIC SYNDROMES
S. Matarraz, 1 A. Lopez, 1 C. Salvador, 2 N. Fernandez-Mosteirin, 2
M. Giralt, 2 A. Orfao1 1 Centro de Investigación del Cáncer, SALAMANCA; 2 Hematology ServiceMiguel
Servet Hospital, ZARAGOZA, Spain
Introduction. Despite the value of the overall number of CD34 + cells in
the bone marrow (BM) of patients with myelodysplastic syndromes
(MDS), currently information about the distribution and behaviour of the
different compartments of CD34 + cells in MDS patients remains limited.
Aims. The aim of the present study is to analyze the relative distribution
of different compartments of immature, myeloid and lymphoid
CD34 + cell precursors in the BM of patients with MDS and correlate it
with the different diagnostic and prognostic subtypes of the disease.
METHODS. Overall, 73 BM samples were analyzed corresponding to 23
normal BM samples (NBM), and 50 from patients with newly diagnosed
MDS classified according to WHO and IPSS criteria into: refractory anemia
(RA) (n=11), refractory cytopenia with multilineage dysplasia
(RCMD) (n=9), RA with excess of blasts (RAEB)-1 (n=13), RAEB-2
(n=10), unclassifiable MDS (UNC) (n=2) and myelodysplastic/myeloproliferative
disorders (MDS/MPD) (n=5). Low risk (LOW) (n=12), intermediate
risk (INT)-1 (n=14), INT-2 (n=9), and high risk (HIGH) (n=4).
Analysis of CD34 + cells was performed using a standard 4-color flow
cytometry approach for the discrimination of immature precursors (ImP),
neutrophil lineage committed CD34 + cells (NP), and B-lymphoid precursors
(BCP) within CD34 + BM cells, based on their different light scatter
characteristics and expression of CD45, CD34, and cytoplasmatic
Myeloperoxidase (cyMPO). In addition, CD34 + precursors phenotypically
committed to other less represented cell lineages were also identified
12 th Congress of the European Hematology Association
using a large panel of monoclonal antibodies: CD34 + plasmacytoid dendritic
cell (pDCP), monocytic, basophyl, erythroid and mast cell precursors.
Results. Overall, the three major compartments of CD34 + cells were
identified in every NBM sample analyzed, whereas ImP and NP were
detected in 59% and 100% of low grade MDS and in 15% and 85% of
high grade MDS, respectively. As compared to NBM, an increased percentage
of ImP and a significant decreased number of BCP was detected
in RAEB-1 (p=0.003 and p