Anca Roxana Lupu

Abnormalities of platelet aggregation in chronic myeloproliferative disorders

A large variety of platelet dysfunctions has been described in chronic myeloproliferative disorders. These abnormalities may be due to deficiency of platelet granules, arahidonic acid metabolism defects or platelet membrane glycoproteins abnormalities. In this study we intend to detect the incidence of platelet function defects in 76 patients with various types of chronic myeloproliferative disorders. The platelet activity was studied in vitro by measuring platelet aggregation in response to ADP, epinephrine, collagen, arachidonic acid and ristocetin. These results were subsequently correlated with bleeding time and clinical aspects (bleeding or thrombosis). We found complex changes in platelet response with all agonists, in varied proportions. These abnormalities include absent, decreased or abnormal platelet aggregation response. In a few cases we found a markedly decreased, almost absent platelet response to all agonists while in some patients a normal platelet aggregation was noted. The correlation between these results and template bleeding time, thrombotic or hemorrhagic events and the type of diseases was difficult to establish and sometimes conflictual. Despite this fact, we consider that investigating platelet aggregation may be useful not only for the assesment of the hemostatic balance in chronic myeloproliferative disorders but also for a better insight into cell abnormalities occuring in these pathologic conditions.

Relationship between chromosomal changes complexity and disease aggressiveness in myeloid and lymphoid disorders.

In this paper are presented four cases, with unusual chromosomal abnormalities, identified at the first presentation, among over 100 patients with myeloid and lymphoid acute and chronic leukemias cytogenetically investigated. The complexity and nature of cytogenetic abnormalities was in direct relationship with the disease evolution. The first case, a 22 years old man with acute lymphoblastic leukemia type L3, exhibited many structural changes in bone marrow cells with diploid number of chromosomes: del(3)(q26); del (5)(p13); t(8;14) (q24;q32); del(9)(p11q11);inv(15)(p12qter). The second case, a 62 years old woman, diagnosed as poorly differentiated acute leukemia, refractory to treatment, showed hiperdiploidy (48–54 chromosomes) and 3–4 markers derived from chromosomes 5 and 12. The third case, a young man of 27 years old, diagnosed as acute myeloid leukemia, apart of Philadelphia chromosome, presented trisomy 16, both in diploid and aneuploid cells. None of these three patients did respond to any medical therapy. Their rapid death was a powerful proof of the correlation between the complexity of genome changes and disease aggressiveness. In the fourth case, a constitutional translocation t(3;5)(q26.3;q21) identified in a 72 years old woman with essential thrombocythemia, appeared not to be involved in the etiology of the disease. In this case, the treatment with hydroxyurea was successful and the disease evolution was favourable. In conclusion, we appreciate that in the three cases of myeloid and lymphoid leukemias it was a direct relationship between the complexity of genomic changes and the aggressiveness of the disease.

What about microparticles? Perspectives and practical aspects

Abstract The first description of microparticles dates back to 1967, when Wolf reported platelet membrane fragments in human plasma and called them “platelet dust”. These vesicles were later called microparticles and the knowledge about their characterization and function has advanced since then. The generation of microparticles represents a mechanism of intercellular communication, playing various roles in both physiological and pathological conditions. Besides other multiple roles in pathology such as inflammation, atherogenesis and cancer spreading, platelet-derived microparticles are involved in thrombogenesis. Tissue factor and phosphatidylserine are both exposed on the outer membrane of platelet-derived microparticles, providing catalytic procoagulant surfaces. The evaluation of microparticles may represent a possible investigation and diagnostic tool. Their enumeration and characterization is challenging and flow cytometry remains the most widely used method for the analysis of microparticles. The aim of the authors is to review the most relevant information on the main properties, mechanisms of generation, and clinical relevance of platelet-derived microparticles, since their evaluation is increasingly considered as a diagnostic biomarker. Rezumat Prima descriere a mieroporticulelor datează din 1967, când Wolf a comunicat prezenţa fragmentelor membranare trombocitare în plasma umană, numindu-le “praf placlietar”. Aceste vezicule au fost ulterior denumite microparticule, iar cunoştinţele despre caracterizarea şi funcţia lor au avansat de atunci. Generarea de microparticule reprezintă un mecanism de comunicare intercelulară, având diferite roluri, atât în statusul fiziologic, cât şi în diverse afecţiuni. în afară de participarea lor în patogenia unor procese precum inflamaţia, aterogeneza sau metastazarea neoplaziilor, microparticulele trombocitare sunt implicate în trombogeneză. Factorul tisular şi fosfatidilserina sunt ambele expuse pe membrana externă a microparticulelor trombocitare, oferind suprafeţe catalitice procoagulante. Evaluarea microparticulelor constituie un potenţial instrument de investigare şi diagnostic. Enumerarea şi caracterizarea lor reprezintă o provocare, iar metoda de analiză cea mai utilizată este citometria în flux. Având în vedere tendinţa actuală de a considera microparticulele ca fiind un adevărat rnarker de diagnostic, autorii îşi propun să prezinte o imagine de ansamblu asupra celor mai relevante informaţii cu privire la proprietăţile principale, mecanismele de producţie şi importanţa clinică a microparticulelor trombocitare

Therapy-related myelodysplastic syndrome after successful treatment of acute promyelocytic leukemia: case report and literature review

Abstract In the 2016 revision of the World Health Organization classification the term therapy-related myeloid neoplasia (t-MN) defines a subgroup of acute myeloid leukemia (AML) comprising patients who develop myelodysplastic syndrome (MDS-t) or acute myeloid leukemia (AML-t) after treatment with cytotoxic and/or radiation therapy for various malignancies or autoimmune disorders. We report the case of a 36 year old patient with t-MN (t-MDS) after achieving complete remission (CR) of a PML-RARA positive acute promyelocytic leukemia (APL) at 32 months after diagnosis. Initially classified as low risk APL and treated according to the AIDA protocol - induction and 3 consolidation cycles - the patient achieved a complete molecular response in September 2013 and started maintenance therapy. On follow-up PML-RARA transcript remained negative. In January 2016 leukopenia and thrombocytopenia developed and a peripheral blood smear revealed hypogranular and agranular neutrophils. Immunophenotyping in the bone marrow aspirate identified undifferentiated blast cells that did not express cytoplasmic myeloperoxidase. The cytogenetic study showed normal karyotype. The molecular biology tests not identified PMLRARA transcript. A diagnosis of t-MDS (AREB-2 - WHO 2008) was established. Treatment of AML was started with 2 “3+7” regimens and 1 MEC cycle. Two months from diagnosis, while in CR, an allogeneic HSCT from an unrelated HLA compatible donor was performed after myeloablative regimen. An unfavorable clinical evolution was followed by death on day 9 after transplantation. The occurrence of t-MNs during CR of APL represents a particular problem in terms of follow-up and differential diagnosis of relapse and constitutes a dramatic complication for a disease with a favorable prognosis. This work was supported by the grants PN 41-087 /PN2-099 from the Romanian Ministry of Research and Technology

Acquired von Willebrand disease: from theory to practice. A single center experience - three case reports

Abstract Acquired von Willebrand disease (AvWD) represents a rare, potentially severe and most likely underdiagnosed category of hemorrhagic syndromes determined by quantitative, qualitative or functional, nonhereditary, alterations of von Willebrand factor (vWF) that occur in the context of various underlying diseases. It is diagnosed mainly in adults, without any personal or familial history of bleeding. The etiopathogeny of AvWD is complex, marked by the intervention of multiple mechanisms, occuring in the evolution of neoplasia, autoimmune disorders, cardiovascular diseases and other conditions. The clinical and laboratory manifestations are similar to the congenital form with mucocutaneous hemorrhage in patients without bleeding history and demonstration of quantitative and/or functional anomalies of vWF. Treatment has two major objectives: control of bleeding and therapy of the underlying condition. As a practical illustration of the theoretical aspects we present 3 clinical cases of AvWD diagnosed in the Colţea Hospital Department of Hematology during the last 10 years.

Evaluation of platelet aggregation and platelet derived microparticles in acute leukemia patients

Abstract Patients with acute leukemia develop abnormalities of haemostasis, leading not only to bleeding, but also to thrombotic complications. The pathogenesis of these complications is complex and multifactorial. Because platelets and platelet derived microparticles are key players in haemostasis and thrombosis, we presumed their roles in the prediction of bleeding and thrombotic complications. Our study groups included 24 patients with acute leukemia and 16 healthy volunteers. Platelet aggregation evaluation was performed by impedance whole blood aggregometry and the ennumeration of platelet derived microparticles was done by means of flow cytometry. Eight patints developed hemorrhagic complications associated with reduced platelet aggregation response at hospital admission. Major thrombotic events occurred in 5 patients, being preceded by increased platelet aggregation in 3 cases and high level of platelet derived microparticles in 2 cases. Our findings reveal that whole blood platelet aggregometry could be a valuable tool especially in the detection of platelet hyperreactivity and in the prediction of thrombotic events. A high level of platelet derived microparticles could also predict thrombosis. These hypotheses need further evaluation and confirmation on larger number of patients. Rezumat Pacienţii cu leucemie acută dezvoltă anomalii ale hemostazei care determină apariţia de complicaţii hemoragipare şi chiar trombotice. Patogeneza acestor complicaţii este complexă si multifactorială. Deoarece trombocitele şi microparticulele trombocitare reprezintă elemente cheie în cadrul proceselor de hemostază si tromboză, am încercat să evidenţiem rolul lor în predicţia complicaţiilor hemoragice şi trombotice. Grupurile studiate au inclus 24 de pacienţi cu leucemie acută şi 16 voluntari sănătoşi. Evaluarea agregării plachetare s-a realizat prin agregometrie prin impedanţa în sânge integral, iar pentru determinarea microparticulelor trombocitare s-a utilizat citometria în flux. Opt pacienţi au dezvoltat complicaţii hemoragipare, care au fost asociate cu agregabilitate trombocitară diminuată la internare. Evenimentele trombotice majore au complicat evoluţia a 5 pacienţi şi au fost precedate de agregare trombocitară crescută la 3 cazuri şi nivel crescut de microparticule trombocitare la 2 cazuri. Rezultatele noastre evidenţiaza faptul că agregometria plachetară în sânge integral poate fi un instrument valoros în special în identificarea hiperreactivitaţii plachetare şi în predicţia evenimentelor trombotice. Un nivel crescut de microparticule trombocitare poate, de asemenea, să anticipeze apariţia trombozei. Aceste ipoteze necesită evaluare si confirmare pe un număr mai mare de pacienţi.

The importance of the new prognostic scoring system for evaluating patients with lower-risk myelodysplastic syndrome at diagnosis

Abstract Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell disorders characterized by ineffective hematopoiesis with dysplastic changes in one or more myeloid cell lines and increased risk of progression to acute leukemia. The current diagnosis criteria include the morphology of peripheral blood (PB) and bone marrow (BM), bone marrow biopsy and cytogenetic exam. Material and method. For this study, we have analyzed 33 patients diagnosed with lower-risk MDS (IPSS 0 and intermediate-1) according to the World Health Organization (WHO) classification (2001) between 2008 and 2012. The diagnosis was confirmed by blood cell counts, bone marrow (aspirate and biopsy) exam and cytogenetic exam. Other causes of cytopenia or dysplastic changes were excluded. Results. The types of MDS according to the WHO classification were: nine patients with refractory anemia (RA) (27.27%), sixteen patients with refractory anemia with ringed sideroblasts (RARS) (48.48%), and eight patients with refractory cytopenia with multilineage dysplasia (RCMD) (24.24%) out of which two with refractory cytopenia with multilineage dysplasia with ringed sideroblasts (RCMD-RS). Cytogenetic exam was performed in all patients, but analyzable metaphases for cytogenetic exam were obtained only from twenty five patients. The patients who did not have analyzable metaphases on cytogenetic exam were considered low risk if: they had only one cytopenia and the percent of bone marrow blasts was less than 5%. For all patients who had analyzable metaphases at cytogenetic exam, the International Prognostic Scoring System (IPSS) and Revised International Prognostic Scoring System (R-IPSS) scores were determined, and their survival and the death leading events were observed. According to IPPS (1997), the cytogenetic exam was good in 17 cases, intermediate in 1 case and poor in 7 cases. The IPSS score was low in 13 cases and intermediate-1 in 12 cases. According to R-IPSS, cytogenetic exams had been very good and good in 17 cases, intermediate in 1 case, poor in 6 cases and very poor in 1 case. R-IPSS was very low and low in 17 cases and intermediate and high in 8 cases. Conclusions. This new R-IPSS score at diagnosis allows a more accurate classification of patients into risk groups and thus enables risk adapted therapy Rezumat Sindroamele mielodisplazice (SMD) reprezintă un grup heterogen de hemopatii clonale ale celulei stem caracterizate prin hematopoieză ineficientă cu afectarea uneia sau mai multor linii mieloide şi risc crescut de transformare în leucemie acută. Diagnosticul curent include morfologia sângelui periferic şi a măduvei, examenul citogenetic şi biopsia osteomedulară. Material și metodă. În acest studiu am analizat un lot de 33 de pacienţi diagnosticaţi cu SMD cu risc scăzut (IPSS 0 şi intermediar-1) conform clasificării OMS 2001 în perioada 2008-2012. Diagnosticul s-a stabilit pe baza hemogramei, a examenului medular şi a examenului citogenetic. Au fost excluse alte cauze de citopenie sau modificări displazice secundare. Rezultate. Tipurile de SMD conform clasificării OMS pe lotul nostru de pacienţi au fost: 9 cazuri de AR, 16 cazuri de ARSI, şi 8 cazuri de CRDM din care 2 cazuri CRDM-SI. Examenul citogenetic a fost efectuat la toţi pacienţii, dar numai la 25 de pacienţi au fost obţinute metafaze analizabile. Pacienţii la care nu s-au obţinut metafaze la examenul citogenetic au fost consideraţi cu risc scăzut dacă au avut o singură citopenie şi procentul de blaşti în măduvă a fost sub 5%. Scorurile IPSS şi IPSS-R au fost calculate la pacienţii la care s-au obţinut metafaze analizabile la examenul cytogenetic şi s-au urmărit supravieţuirea şi evenimentele care au dus la deces. Conform scorului IPPS din 1997, examenul citogenetic a fost favorabil în 17 cazuri, intermediary întrun caz şi nefavorabil în 7 cazuri. Scorul IPSS a fost favorabil în 13 cazuri şi intermediar-1 în 12 cazuri. Conform IPSSR, examenul citogenetic a fost foarte favorabil şi favorabil în 17 cazuri, intermediar într-un caz, nefavorabil în 6 cazuri şi foarte nefavorabil în 1 caz. IPSS-R a fost cu risc foarte scăzut şi scăzut în 17 cazuri şi intermediar şi crescut în 8 cazuri. Concluzii. Aplicarea acestui nou scor IPSS-R tuturor pacienţilor la diagnostic ajută la aprecierea prognosticului, a riscului de transformare în leucemie acută şi a tratamentului. Singura terapie cu intenţie curativă este allotransplantul de celule stem hematopoietice

Lymphoma immunophenotyping: “borderline” lymphomas

Immunophenotyping of B‐cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(‐) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as “borderline” CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.