Features, reason for testing, and changes with time of 583 paroxysmal nocturnal hemoglobinuria clones from 529 patients: a multicenter Italian study.

In this study, we aimed at disclosing the main features of paroxysmal nocturnal hemoglobinuria (PNH) clones, their association with presentation syndromes, and their changes during follow-up. A large-scale, cooperative collection (583 clones from 529 patients) of flow cytometric and clinical data was entered into a national repository. Reason for testing guidelines were provided to the 41 participating laboratories, which followed the 2010 technical recommendations for PNH testing by Borowitz. Subsequently, the 30 second-level laboratories adopted the 2012 guidelines for high-resolution PNH testing, both upon order by the local clinicians and as an independent laboratory initiative in selected cases. Type3 and Type2 PNH clones (total and partial absence of glycosyl-phosphatidyl-inositol-anchor, respectively) were simultaneously present in 54 patients. In these patients, Type3 component was sevenfold larger than Type2 (p < 0.001). Frequency distribution analysis of solitary Type3 clone size (N = 442) evidenced two discrete patterns: small (20% of peripheral neutrophils) and large (> 70%) clones. The first pattern was significantly associated with bone marrow failure and myelodysplastic syndromes, the second one with hemolysis, hemoglobinuria, and thrombosis. Pediatric patients (N = 34) showed significant preponderance of small clones and bone marrow failure. The majority of PNH clones involved neutrophils, monocytes, and erythrocytes. Nevertheless, we found clones made exclusively by white cells (N = 13) or erythrocytes (N = 3). Rare cases showed clonal white cells restricted only to monocytes (6 cases) or neutrophils (3 cases). Retesting over 1-year follow-up in 151 cases showed a marked clone size increase in 4 cases and a decrease in 13, demonstrating that early breaking-down of PNH clones is not a rare event (8.6% of cases). This collaborative nationwide study demonstrates a clear-cut difference in size between Type2 and Type3 clones, emphasizes the existence of just two classes of PNH presentations based on Type3 clone size, depicts an asymmetric cellular composition of PNH clones, and documents the possible occurrence of changes in clone size during the follow-up.

Morphologic and immunophenotypic features of a case of acute monoblastic leukemia with unusual positivity for Glycophorin-A.

Acute monoblastic leukemia (AMoL) is characterized by cells with highly undifferentiated morphology. Cytochemistry with non-specific esterases is negative in up to 20% of cases. Immunophenotyping by flow cytometry has an essential role in diagnosing such a subtype of leukemia and a multiparametric approach with a wide monoclonal antibody panel is necessary. We describe a case of AMoL with morphology resembling either plasma blasts or very immature erythroblasts. Diagnosis was made by alpha-naphtyl-acetate esterase staining and with immunophenotyping, which was made with a wide monoclonal antibody panel. Blasts were positive for monocytic markers. Most of leukemic cells, however, were positive for Glycophorin-A. The presence of Glycophorin-A, which is considered as a specific marker of the erythroid lineage, has never been reported previously in cases of AMoL. This peculiar immunophenotype might be interpreted as deriving from a common myelo-erythroid precursor undergone leukemic transformation.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Modifications in B-Lymphocyte Number and Phenotype in the Course of Pregnancy in a Woman with Persistent Polyclonal B-Cell Lymphocytosis: A Flow Cytometric Study.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare clinical condition, characterized by a persistent, generally moderate lymphocytosis, generally due to stimulation of central memory B-lymphocytes, and by a moderate increase of polyclonal IgM. In some patients, slight or moderate splenomegaly is observed. A variable percentage of circulating, bone marrow and splenic lymphocytes display an abnormal nucleus (generally bilobated) or are binucleated. The clinical course is benign in most cases and transformation into splenic B-cell lymphoma occurs in few cases. In the current paper we report the first case of pregnancy in PPBL. Our patient became pregnant 18 months after diagnosis. In the course of pregnancy, a marked down-regulation of lymphocytosis (from 6 × 10(9)/L to 2.1 × 10(9)/L) and a decrease in B-lymphocyte number was observed (from 3.6 × 10(9)/L to 1 × 10(9)/L), mainly due to a marked reduction in the percentage and absolute number of central memory B-cells. Such modifications were similar to those described in normal pregnant women. One year after the delivery of a healthy female baby, the number of total lymphocytes and B-lymphocytes showed an inverse behavior, with a new expansion of central memory B-cells. Our case shows that a normal pregnancy can occur in patients with PPBL and that pregnancy can induce marked modifications in B-lymphocyte kinetics and phenotype.

Kinetics of hematogones in bone marrow samples from patients with non-Hodgkin lymphomas treated with rituximab-containing regimens: a flow cytometric study.

Treatment with rituximab, either alone or in combination with antiblastic drugs, causes significant depletion of circulating B-lymphocytes and modifications of B cell maturation in the bone marrow. In the present study, we analyzed the kinetics of hematogones in bone marrow samples from 55 patients suffering from non-Hodgkin lymphomas and treated with rituximab-containing regimens. Maturation arrest at the level of stage 2 hematogones, along with complete depletion of naïve, mature B-lymphocytes, was observed as short-term effects (2 months after completion of chemo-immunotherapy). Further bone marrow samples, obtained 12 months after the last rituximab infusion in 21 patients undergoing long-term follow-up and treated with rituximab maintenance therapy, showed complete normalization of B-lymphocyte ontogeny. Hypogammaglobulinemia developed in 26 patients, and was still observed in nine of the 21 patients undergoing long-term follow-up. Our study provides novel data on hematogone kinetics in the setting of patients with non-Hodgkin lymphomas treated with chemo-immunotherapy containing rituximab and with rituximab maintenance. Our observations show that hypogammaglobulinemia can persist in a significant percentage of patients, despite complete recovery of B-lymphocyte ontogeny.

Myelomatous meningitis evaluated by multiparameter flow cytometry : report of a case and review of the literature.

Central nervous system (CNS) involvement in multiple myeloma (MM) is uncommon. Among its possible presentations, leptomeningeal involvement of MM, also termed central nervous system myelomatosis (CNS-MM) is rare and is characterized by the presence of neoplastic plasma cells in the cerebrospinal fluid (CSF). So far, 187 cases of CNS-MM have been reported : the great majority of them were diagnosed by cytological assays and flow cytometry was used in only eight cases. We describe a case of CNS-MM in a 62-year-old woman, previously treated with chemotherapy (VTD) and autologous peripheral blood hematopoietic stem cell transplantation for stage IIIB IgG-λ MM. After achieving a very good partial response, the patient showed progression of disease, with an extramedullary localization. During administration of second-line therapy, the patient showed severe neurological symptoms. MRI resulted negative. Diagnosis of CNS-MM was made by multiparameter flow cytometry, which showed the presence of CD56(+) plasma cells in a CSF sample, in the absence of plasma cell leukemia. In this paper we also present a review of the eight previous cases of CNS-MM diagnosed by flow cytometry. We found that the application of flow cytometry in cases of MM with neurological symptoms allows a rapid diagnosis of CNS-MM and provides useful information about plasma cell phenotype (including CD56 expression). Some cases of CNS-MM are characterized by normal MRI. In addition, some evidences deriving from the review of literature suggest that CSF monitoring by flow cytometry in such cases might be used to evaluate the efficacy of drugs capable of crossing the blood-brain barrier.

Multiparameter Flow Cytometry to Detect Hematogones and to Assess B-lymphocyte clonality in Bone Marrow Samples from Patients with Non-Hodgkin Lymphomas.

Hematogones are precursors of B-lymphocytes detected in small numbers in the bone marrow. Flow cytometry is the most useful tool to identify hematogones and, so far, 4-color methods have been published. In addition, flow cytometry is used in the diagnosis and follow-up of lymphomas. We developed a flow cytometric 7-color method to enumerate hematogones and to assess B-lymphocyte clonality for routine purposes. We evaluated 171 cases of B-cell non-Hodgkin lymphomas, either at diagnosis or in the course of follow-up. By our diagnostic method, which was carried out by the combination K/λ/CD20/CD19/CD10/CD45/CD5, we were able to detect hematogones in 97.6% of samples and to distinguish normal B-lymphocytes, neoplastic lymphocytes and hematogones in a single step. The percentage of hematogones showed a significant inverse correlation with the degree of neoplastic infiltration and, when bone marrow samples not involved by disease were taken into consideration, resulted higher in patients during follow-up than in patients evaluated at diagnosis.

Simultaneous presentation of Waldenström macroglobulinemia and multiple myeloma: multidisciplinary diagnosis, treatment and 30-month follow-up.

Waldenström macroglobulinemia and multiple myeloma are mature B-cell neoplasms deriving from post-germinal cells at different stages of differentiation. The simultaneous presentation of Waldenström macroglobulinemia and multiple myeloma in the same patient is a very rare phenomenon and, so far, only two cases have been described. We report the case of a 75-year Caucasian female patient, with a silent clinical history, who presented with anemia and two different monoclonal proteins (IgMκ and IgGκ). The trephine biopsy showed the presence of a dual population, represented by small lymphoplasmacytoid cells and by plasma cells, which infiltrated the bone marrow with a clearly different pattern. Both immunohistochemistry and flow cytometry demonstrated the biclonal origin such neoplastic cells, since lymphoplasmacytoid cells resulted IgMκ while plasma cells were IgGκ. This biclonal pattern was further confirmed by the demonstration of a different IgH gene rearrangement of the two neoplasms. The patient was treated with bortezomib, dexamethasone and rituximab, achieving partial remission of both Waldenström macroglobulinemia and multiple myeloma. After a 30-month follow-up, she is in stable disease. Multiple myeloma has been described in association with other indolent B-cell neoplasms, mostly chronic lymphocytic leukemia, while Waldenström macroglobulinemia can be followed by diffuse large B-cell lymphoma in some instances, after chemotherapy. The association of Waldenström macroglobulinemia and multiple myeloma seems to be very rare. Our study shows that an integrated diagnostic work-up is very useful in such cases, with an interesting role for flow cytometry. [J Clin Exp Hematop 53(1): 29-36, 2013].

Regional evidence of modulation of cardiac adiponectin level in dilated cardiomyopathy: pilot study in a porcine animal model.

BACKGROUND: The role of systemic and myocardial adiponectin (ADN) in dilated cardiomyopathy is still debated. We tested the regulation of both systemic and myocardial ADN and the relationship with AMP-activated protein kinase (AMPK) activity in a swine model of non-ischemic dilated cardiomyopathy. METHODS AND RESULTS: Cardiac tissue was collected from seven instrumented adult male minipigs by pacing the left ventricular (LV) free wall (180 beats/min, 3 weeks), both from pacing (PS) and opposite sites (OS), and from five controls. Circulating ADN levels were inversely related to global and regional cardiac function. Myocardial ADN in PS was down-regulated compared to control (p < 0.05), yet ADN receptor 1 was significantly up-regulated (p < 0.05). No modifications of AMPK were observed in either region of the failing heart. Similarly, myocardial mRNA levels of PPARγ, PPARα, TNFα, iNOS were unchanged compared to controls. CONCLUSIONS: Paradoxically, circulating ADN did not show any cardioprotective effect, confirming its role as negative prognostic biomarker of heart failure. Myocardial ADN was reduced in PS compared to control in an AMPK-independent fashion, suggesting the occurrence of novel mechanisms by which reduced cardiac ADN levels may regionally mediate the decline of cardiac function.

B-cell acute lymphoblastic leukemia with t(4;11)(q21;q23) in a young woman: evolution into mixed phenotype acute leukemia with additional chromosomal aberrations in the course of therapy.

About 5% of adult B-cell acute lymphoblastic leukemias (B-ALL) are characterized by t(4;11)(q21;q23), which confers peculiar features to this B-ALL subtype, including a very immature immunophenotype and poor prognosis. We describe the case of a 21-year-old female who presented with B-ALL carrying the t(4;11)(q21;q23) and blasts positive for CD19, TdT, CD79a, CD38, HLA-DR. Before completing the Hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) therapy regimen, the B-cell leukemic clone still was detected, but an additional leukemic clone appeared, with morphology and immunophenotype (CD13, CD33, CD64, CD38, CD56, CD15, CD4(dim)) compatible with derivation from the myeloid/monocytic lineage. Karyotype showed the co-existence of three cell lines, with persistence of t(4;11)(q21;q23) and appearance of +8,+12,+13 and two der(4). The patient died because of disseminated intravascular coagulation. Our report describes a rare, possible evolution of such a subtype of B-ALL, with transformation into mixed phenotype acute leukemia in the course of therapy. This finding suggests a blast cell derivation from a common lymphoid/monocytic precursor leading to a final bilineal acute leukemia.

The role of CD19 and CD27 in the diagnosis of multiple myeloma by flow cytometry: a new statistical model.

We have developed a new statistical diagnostic model that examines the correlation between immunophenotype and clonality as detected by flow cytometry (FC) and histology, defining the diagnostic role of FC in multiple myeloma (MM). The 192 bone marrow samples from patients and control subjects were studied for routine diagnostic analysis of MM; a minimum of 100 plasma cells (PCs) were analyzed for each patient sample. A direct 7- or 8-color method was applied to study the immunophenotype of PCs, utilizing a FACSCanto II (BD Biosciences, San Jose, CA). Samples were labeled with fluorochrome-conjugated monoclonal antibodies (AmCyan, Pac Blue, fluorescein isothiocyanate, phycoerythrin [PE], PECy7, peridinin-chlorophyll protein, allophycocyanin [APC], and APC-Cy7) to the following antigens: CD138, CD81, CD200, CD221, CD45, CD38, CD28, CD19, CD27, CD117, CD38, CD33, CD20, CD56, CD10, and immunoglobulin κ and λ light chains. Among all antigens tested, CD19 and CD27, when applied to our model, resulted in optimal concordance with histology. This model defines the effective diagnostic role FC could have in MM and in the detection of minimal residual disease.

Discordant lymphoma consisting of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood: a case report.

INTRODUCTION: Discordant lymphomas are rare entities characterized by the simultaneous presence of two distinct types of lymphomas in different anatomic sites. We describe a very rare case of simultaneous occurrence of splenic mantle cell lymphoma and marginal zone lymphoma involving the bone marrow and peripheral blood. CASE PRESENTATION: We report the case of a 60-year-old asymptomatic Caucasian woman in whom discordant lymphomas were discovered when a slight lymphocytosis and a conspicuous splenomegaly were observed. The different morphological, immunophenotypical and immunohistochemical features found in the different pathologic samples obtained from peripheral blood, bone marrow and spleen sections made it possible to differentiate two types of non-Hodgkin B-cell lymphomas: a mantle cell lymphoma infiltrating the spleen and a marginal zone lymphoma involving both the bone marrow and peripheral blood. Since a similar IgH gene rearrangement was found both in the bone marrow and in the spleen, the hypothesis of a common origin, followed by a different clonal selection of the neoplastic lymphocytes may be taken into consideration. CONCLUSION: Our case emphasizes the usefulness of investigating simultaneous specimens from different anatomic sites from the same patient and the relevant diagnostic role of splenectomy.

Mitochondrial pathway mediates the antileukemic effects of Hemidesmus indicus, a promising botanical drug.

BACKGROUND: Although cancers are characterized by the deregulation of multiple signalling pathways, most current anticancer therapies involve the modulation of a single target. Because of the enormous biological diversity of cancer, strategic combination of agents targeted against the most critical of those alterations is needed. Due to their complex nature, plant products interact with numerous targets and influence several biochemical and molecular cascades. The interest in further development of botanical drugs has been increasing steadily and the FDA recently approved the first new botanical prescription drug. The present study is designed to explore the potential antileukemic properties of Hemidesmus indicus with a view to contributing to further development of botanical drugs. Hemidesmus was submitted to an extensive in vitro preclinical evaluation. METHODOLOGY/PRINCIPAL FINDINGS: A variety of cellular assays and flow cytometry, as well as a phytochemical screening, were performed on different leukemic cell lines. We have demonstrated that Hemidesmus modulated many components of intracellular signaling pathways involved in cell viability and proliferation and altered the protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential and increased Bax/Bcl-2 ratio. ADP, adenine nucleotide translocator and mitochondrial permeability transition pore inhibitors did not reverse Hemidesmus-induced mitochondrial depolarization. Hemidesmus induced a significant [Ca(2+)](i) raise through the mobilization of intracellular Ca(2+) stores. Moreover, Hemidesmus significantly enhanced the antitumor activity of three commonly used chemotherapeutic drugs (methotrexate, 6-thioguanine, cytarabine). A clinically relevant observation is that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemic patients. CONCLUSIONS/SIGNIFICANCE: These results indicate the molecular basis of the antileukemic effects of Hemidesmus and identify the mitochondrial pathways and [Ca(2+)](i) as crucial actors in its anticancer activity. On these bases, we conclude that Hemidesmus can represent a valuable tool in the anticancer pharmacology, and should be considered for further investigations.

Cytochrome P-450 3A13 and endothelin jointly mediate ductus arteriosus constriction to oxygen in mice.

The fetal ductus arteriosus (DA) contracts to oxygen, and this feature, maturing through gestation, is considered important for its closure at birth. We have previously obtained evidence of the involvement of cytochrome P-450, possibly of the 3A subfamily (CYP3A), in oxygen sensing and have also identified endothelin (ET)-1 as the attendant effector for the contraction. Here, we examined comparatively wild-type (WT) and CYP3A-null (Cyp3a(-/-)) mice for direct validation of this concept. We found that the CYP3A subfamily is represented only by CYP3A13 in the WT DA. CYP3A13 was also detected in the DA by immunofluorescence microscopy, being primarily colocalized with the endoplasmic reticulum in both endothelial and muscle cells. However, a distinct signal was also evident in the plasma membrane. Isolated DAs from term WT animals developed a sustained contraction to oxygen with transient contractions superimposed. Conversely, no tonic response occurred in Cyp3a(-/-) DAs, whereas the phasic response persisted unabated. Oxygen did not contract the preterm WT DA but caused a full-fledged contraction after retinoic acid (RA) treatment. RA also promoted an oxygen contraction in the Cyp3a(-/-) DA. However, responses of RA-treated WT and Cyp3a(-/-) mice differed in that only the former abated with ET-1 suppression. This implies the existence of an alternative target for RA responsible for the oxygen-induced contraction in the absence of CYP3A13. In vivo, the DA was constricted in WT and Cyp3a(-/-) newborns, although with a tendency to be less narrowed in the mutant. We conclude that oxygen acts primarily through the complex CYP3A13 (sensor)/ET-1 (effector) and, in an accessory way, directly onto ET-1. However, even in the absence of CYP3A13, the DA may close postnatally thanks to the contribution of ET-1 and the likely involvement of compensating mechanism(s) identifiable with an alternative oxygen-sensing system and/or the withdrawal of relaxing influence(s) operating prenatally.

Severity of regional myocardial dysfunction is not affected by cardiomyocyte apoptosis in non-ischemic heart failure.

The role of myocardial apoptosis during the development of heart failure (HF), in the absence of coronary artery stenosis, is still debated. The aim of the study was to evaluate whether (similar to functional impairment) the activation of myocardial apoptosis follows a regional pattern in an established model of pacing-induced HF. HF was induced in adult male minipigs by rapid and sustained left ventricular (LV) epicardial pacing (n=8; n=5 healthy controls). Progressive regional derangement of the contractile function and perfusion was assessed by magnetic resonance imaging as LV end-systolic wall thickening (LVESWT) and relative upslope of signal intensity (LVRUSI, %) in the anterior/anterior-lateral (pacing site, PS) and infero-septal LV region (opposite site, OS). LV tissue from PS and OS was analyzed for biomarkers of cell apoptosis and injury. After 21 days of LV pacing, LVESWT was lower in the PS compared to OS (7.6±3.7 vs 24.16±3.6%, p<0.05), and LV ejection fraction was 24.0±3.7 (p<0.05 vs control). The mRNA expression of caspase (Casp)-3 was significantly higher in the PS of HF hearts than in controls (1.28±0.125 vs 0.82±0.10), but not Casp-9. Bcl-2 and Hsp72 expression was significantly increased in PS compared to control (0.90±0.60 vs 0.63±0.033; 0.72±0.10 vs 0.28±0.098), in the presence of a TNF-α level increased by 50.7%. The regional myocardial apoptotic index, assessed by TUNEL, was unchanged in HF. In conclusion, the activators and inhibitors of cell apoptosis are equally expressed without affecting the survival of cardiomyocytes and the magnitude of regional myocardial dysfunction during development of non-ischemic HF.

Prethymic cytoplasmic CD3 negative acute lymphoblastic leukemia or acute undifferentiated leukemia: a case report.

Acute undiffentiated leukemia (AUL) is an acute leukemia with no more than one membrane marker of any given lineage. Blasts often express HLA-DR, CD34, and/or CD38 and may be positive for terminal deoxynucleotidyl transferase (TdT). The expression of CD34, HLA-DR, and CD38 has been shown in pro-T-ALL, although in this case, blasts should also express CD7 and cyCD3. However, some cases of T-ALL without CD3 in the cytoplasm and all TCR chain genes in germ line configuration are reported, features that fit well with a very early hematopoietic cell. We report a case of acute leukemia CD34+/-HLADR+CD7+CD38+cyCD3- in which a diagnosis of AUL was considered. However the blasts were also positive for CD99 and TCR delta gene rearrangement which was found on molecular studies. Therefore a differential diagnosis between AUL and an early cyCD3 negative T-ALL was debated.