Giuseppe d'Onofrio

Autologous stem cell transplantation: evaluation of erythropoietic reconstitution by highly fluorescent reticulocyte counts, erythropoietin, soluble transferrin receptors, ferritin, TIBC and iron dosages

The plasma concentrations of erythropoietin (Ep), soluble transferrin receptors (sTfRs), iron, total iron binding capacity (TIBC) and ferritin were monitored in five leukaemia patients undergoing autologous bone marrow stem cell transplantation (BMSCT) and in 10 lymphoma and 21 ovarian cancer patients undergoing autologous peripheral blood SCT (PBSCT); 9/21 ovarian cancer patients received recombinant human G‐CSF and Ep and six recombinant human GM‐CSF and Ep following SCT. All parameters were evaluated in relation to the kinetics of erythroid reconstitution as evaluated by haemoglobin (Hb) and reticulocyte levels [including the fraction of immature reticulocytes, also called highly fluorescent reticulocytes (HFR)].

The combination of erythropoietin and granulocyte colony-stimulating factor increases the rate of haemopoietic recovery with clinical benefit after peripheral blood progenitor cell transplantation

In order to investigate the effects of erythropoietin (EPO) plus granulocyte colony‐stimulating factor (G‐CSF) administration after peripheral blood progenitor cell transplantation (PBPCT) we performed a phase I/II study in patients with high‐risk cancer. 15 consecutive patients were treated with recombinant human G‐CSF (rhG‐CSF) at the dose of 5 μg/kg subcutaneously (s.c.) every 24 h until day +12 and with recombinant human EPO (rhEPO) at the dose of 150 IU/kg s.c. every 48 h until day +11 following PBPCT. Their haemopoietic recovery was compared to that obtained in eight historic control patients who did not receive any cytokines after PBPCT. No side‐effects were observed during EPO plus G‐CSF treatment and the treatment was not discontinued in any of the patients before completion of the treatment plan. The administration of EPO plus G‐CSF after PBPCT produced a significant increase in the rate of white blood cell (WBC) (P = 0.0005), polymorphonuclear leucocyte (PMN) (P = 0.0005) and platelet (PLT) (P = 0.0105) recovery compared to the control group. The acceleration in haemopoietic recovery observed in the EPO plus G‐CSF‐treated patients produced a significant reduction of the days with WBC < 1×109/l (P = 0.0009), PMN  < 0.2×109/l (P = 0.0030) and PMN < 0.5×109/l (P = 0.0006). EPO plus G‐CSF‐treated patients required a significantly lower number of single donor PLT transfusions (P = 0.0142) and did not experience neutropenic fever, but historic control patients experienced fever > 38°C for a median period of 4 d (0–12) with a median period of parenteral antibiotic administration of 7.5 d (0–17). The length of the hospital stay was significantly shorter in the study group than in the historic control group (P = 0.0264). In conclusion, we can confirm that EPO plus G‐CSF treatment is feasible and potentiates the haemopoietic recovery after PBPCT, thus simplifying the clinical management of cancer patients who undergo high‐dose chemotherapy.

A European consensus report on blood cell identification: terminology utilized and morphological diagnosis concordance among 28 experts from 17 countries within the European LeukemiaNet network WP10, on behalf of the ELN Morphology Faculty

This paper describes the methodology used to develop a consensual glossary for haematopoietic cells within Diagnostics‐WP10 of European‐LeukemiaNet EU‐project. This highly interactive work was made possible through the use of the net, requiring only a single two‐day meeting of actual confrontation and debate. It resulted in the production of a freely accessible tool that could be useful for training as well as harmonization of morphological reports in onco‐haematology especially, without geographic limitation, not limited to European countries. Moreover, this collective work resulted in the production of a consensus statement, taking into account individual practices, collegial agreement and literature data.

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors.

Circulating CD34+ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34− contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3+ T‐lymphocytes (range 2–12%), CD3−/CD16+/CD56+ natural killer cells (range 2–11%) and rare mature CD15+/CD11b+ granulocytes (range 1–2%). CD34+ cells were cultured in liquid medium in the presence of interleukin‐3, granulocyte‐macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate‐resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34+ cells until day 35.

Flow Cytometric Detection of CD44 (H-CAM) in Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a rare clonal B-cell disorder characterized by a distinctive pattern of infiltration of hairy cells (HCs) in the bone marrow (BM), hepatic sinusoids and splenic red pulp. HCs express a wide spectrum of matrix-binding integrins, which influence their migratory behaviour and subsequent tissue localization. CD44 distribution and staining intensity on HCs from the BM and peripheral blood (PB) were investigated using dual-color flow cytometry. HCs from all cases expressed CD44, but the staining intensity of the positive population was significantly higher than normal residual lymphocytes (p < 0.0005) and medullary HCs in those patients exhibiting additional hairy cell dissemination in the peripheral blood. It is of interest to speculate that the BM microenvironmental may deliver signals which down-regulate CD44 expression on resident HCs compared to HCs recirculating in peripheral blood. CD44 expression on HCs may help clarify their homing mechanisms in the bone marrow and to define phenotypic subsets with different clinical and pathological behaviours.

Lower hemoglobin levels in human immunodeficiency virus–infected patients with a positive direct antiglobulin test (DAT): relationship with DAT strength and clinical stages

BACKGROUND: There are conflicting opinions regarding the effect of positive direct antiglobulin test (DAT) on hemoglobin (Hb) levels in human immunodeficiency virus–infected (HIV+) patients.

Purified unfractionated G-CSF/chemotherapy mobilized CD34+ peripheral blood progenitors and not bone marrow CD34+ progenitors undergo selective erythroid differentiation in liquid culture in the presence of erythropoietin and stem cell factor

A combination of erythropoietin (EPO) plus stem cell factor (SCF) drove purified unfractionated granulocyte colony stimulating factor (G‐CSF)/chemotherapy mobilized peripheral blood CD34+ cells to selective erythroid differentiation in liquid culture with an average 28‐fold increase in the total cell number after 21 d. From day 6 of culture, cytologic and cytofluorimetric characterization revealed that cultured cells belonged to the erythroid lineage with a gradual wave of maturation along the erythroid pathway to terminal cells. A similar pattern of erythroid differentiation was observed when the same peripheral blood CD34+ cells were cultured with EPO plus SCF in serum‐free medium. This cytokine combination produced selective erythroid differentiation with the complete exhaustion of the clonogenic potential on day 21. In parallel experiments the same circulating CD34+ cells underwent granulocytic/monocytic differentiation in liquid culture in response to granulocyte‐macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and SCF, demonstrating that these CD34+ progenitors had intact pluripotent differentiating potential. Conversely, bone marrow CD34+ cells isolated from bone marrow allografts were unable to selectively differentiate along the erythroid pathway when they were exposed to EPO plus SCF combination. However, these cells maintained a greater number of colony forming cells on day 21 of culture compared to mobilized peripheral blood CD34+ cells. This model is a simple and reliable way to obtain selective erythroid differentiation of peripheral blood G‐CSF/chemotherapy mobilized CD34+ progenitor cells in liquid culture. The absence of cytokines such as GM‐CSF and IL‐3 in the culture medium permits studies on in vitro erythropoiesis without disturbance of prevalent myelopoiesis.

Characterization of anti-D monoclonal antibody reagents based on their reactivity with the weak D phenotype

BACKGROUND: Anti‐D monoclonal antibody (MoAb) reagents have improved D typing in routine tests. However, they exhibit a wide range of reactivity with the weak D phenotype depending on the characteristics of the different MoAbs used. We analyzed the reactivity of immunoglobulin (IgM) anti‐D by cluster analysis to characterize MoAb that have similar reactivities with the weak D phenotype.

Reticulocyte population data in different erythropoietic states

Aims To assess changes in reticulocyte impedance volume, conductivity and light scatter (reticulocyte population data or RPD) obtained with the volume, conductivity and laser light scatter (VCS) technology in healthy subjects and in different erythropoietic states. Methods Blood samples were analysed with the Beckman–Coulter LH750 system, using the VCS method, from a group of 40 healthy subjects and three groups of patients with different types of untreated or treated anaemia: 24 cases of iron deficiency at the time of diagnosis, 16 patients with iron deficiency anaemia during intravenous iron administration, and 57 patients with chronic kidney disease undergoing dialysis and administration of rHu-erythropoietin and intravenous iron. Results RPD data were reproducible. Average mean channels for volume were 50.9 for controls and 49.2, 55.7 and 64.0 for the three patient groups, respectively. Average mean channels for conductivity were 52.0 for controls and 59.8, 55.7 and 56.1 for the three patient groups. Average mean channels for light scatter were 108.4 for controls and 113.3, 117.3 and 128.2 for the three patient groups. SD data indicated increased dispersion in the patient groups. Conclusion When values in the patient groups are compared with reference values obtained in healthy controls, the main differences are found in impedance volume and light scatter, which were both increased in patients with stimulated erythropoiesis. These data indicate the opportunity to further evaluate the clinical usefulness of RPD in haematological diseases.

Clinical Applications of Automated Reticulocyte Indices.

Automated analysis of reticulocytes provides pathologists and clinicians with several new parameters, which need to be evaluated for their role in the diagnosis and management of diseases. We review here the current knowledge on reticulocyte cell volume, hemoglobin concentration and content. Several studies have provided reference values for reticulocyte cell volume (MCVr), cell hemoglobin concentration (CHCMr) and cell hemoglobin content (CHr). Data are available on the changes of these indices in iron deficiency and megaloblastic anemias and their response to therapy. CHr has been shown to be an early indicator of functional irondeficiency in subjects treated with recombinant human erythropoietin (r-HuEPO). Reticulocyte changes have also been described in the early phases of hydroxyurea therapy for sickle cell disease and in bone marrow transplantation. The real-time information provided by reticulocyte indices on the functional state of the erythroid marrow is an important tool in the diagnosis and management of several hematological disorders and in the use of r-HuEPO.