bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum levels of cytokines after bone marrow transplantation: increased IL‐8 levels during severe veno‐occlusive disease of the liver
Authors: Remberger M. , Ringdén O.
Published: July 2010

Abstract: We investigated the cytokine profile and peak levels of interleukin (IL) ‐6, IL‐8, IL‐10 and tumour necrosis factor (TNF) ‐α levels in 42 patients after allogeneic bone marrow transplantation (BMT). Eleven of them developed veno‐occlusive disease (VOD) of the liver. Fourteen patients had moderate‐to‐severe acute graft‐versus‐host disease (aGvHD), 10 isolated bacteraemia and 7 had no major complication. Those who developed severe VOD (n = 6) showed a short, very high IL‐8 peak (median: 6632 pg/ml, range: 5546 – 10,000 vs. 280 pg/ml, 0 – 2042 in controls, p < 0.01) 1 – 4 d after diagnosis of the liver disease. Five of these patients had high peak levels of IL‐6. Five patients with mild VOD showed a lower increase in the cytokines tested. Bilirubin levels, at day of IL‐8 peak, did not differ statistically between mild and severe VOD. The highest levels of IL‐10 were found in those with aGvHD. IL‐8 levels were also increased, but not to the same extent as in patients with severe VOD (p = 0.01 vs. VOD). In patients with bacteraemia, very high levels of IL‐6 were seen. In patients without major complications, the levels of cytokines were low. In conclusion, high levels of IL‐8 occurred in severe VOD of the liver, which may be of value to determine prognosis.
Relationship between the severity of β‐thalassaemia syndromes and the number of alleviating mutations
Authors: Ratip S. , Petrou M. , Old J. M. , Wonke B. , Porter J. B. , Modell B.
Published: July 2010

Abstract:  Thalassaemia intermedia, defined as homozygous β‐thalassaemia in which patients are not transfusion‐dependent, covers a wide range of clinical severity. It may arise because one or more genetic factors ameliorate the otherwise severe phenotype of thalassaemia major. Exactly which and how many such mutations are necessary to produce a thalassaemia intermedia phenotype is incompletely understood, although such information would be useful both clinically and for prenatal diagnosis. We examined DNA from 28 patients with thalassaemia intermedia resident in London and 28 matched patients with thalassaemia major, for 3 types of genetic modifying factors, namely; mild β‐thalassaemia mutations, the upstream XmnI G‐gamma globin gene polymorphism, and α‐globin gene deletions. The results show that the number of alleviating mutations present has a large influence on the phenotype of patients with homozygous β‐thalassaemias. A single alleviating mutation was present in 56% of thalassaemia intermedia subjects compared with 26% of thalassaemia major subjects. Two alleviating mutations were present in 33% of thalassaemia intermedia subjects compared with 1 thalassaemia major subject. No patients with thalassaemia major had 3 alleviating mutations, in contrast to 11% of those with thalassaemia intermedia. Although the findings did not account for the full range of phenotypic variation, such information is of potential value both in the clinical management and the prenatal diagnosis of homozygous β‐thalassaemia.
Chronic isolated macrothrombocytopenia with autosomal dominant transmission: a morphological and qualitative platelet disorder
Authors: Fabris F. , Cordiano I. , Salvan F. , Ramon R. , Valente M. , Luzzatto G. , Girolami A.
Published: July 2010

Abstract:  We studied 47 subjects belonging to 13 unrelated families with a history of mild haemorrhagic diathesis and chronic thrombocytopenia. 36 patients presented some degree of thrombocytopenia: 7/36 (19%) had slight thrombocytopenia (100–150×109/L); 26/36 (72%) had mild thrombocytopenia (50–100×109/L) and 3/36 (8%) had severe thrombocytopenia (<50×109/L). No correlation was observed between platelet count and the degree of haemorrhagic diathesis, which was mild in the majority of patients. Transmission was autosomal dominant. Platelet anisocytosis, increased percentage of large platelets and absence of leukocyte inclusions were observed in 26/30 (87%) of the examined blood smears. The ultrastructural appearance of platelets was normal. Megakaryocytes appeared normal in number in 10/10 patients, but showed asynchronous nuclear‐cytoplasm maturation and mainly nonlobulated nuclei. Platelet aggregation was studied in 26 patients and either increased or decreased curves were variably observed in response to different aggregating agents. Platelet‐associated IgG (PAIgG) was increased in 18/31 (58%) patients, while serum autoantibodies against platelet glycoproteins Ib/IX or IIb/IIIa were demonstrable in only 1 case. An increased expression of platelet surface glycoproteins Ib and IIb/IIIa, as studied by murine monoclonal antibodies binding in 17 cases, was observed. Platelet survival performed by 111In‐oxine‐labelled autologous platelets was normal in the 3 studied patients. Congenital macrothrombocytopenia confirms to be a distinct clinical disorder for which the name of “chronic isolated hereditary macrothrombocytopenia” is proposed.
Quantification of loss of haemoglobin components from the circulating red blood cell in vivo
Authors: Willekens F. L. A. , Bosch F. H. , Roerdinkholder‐Stoelwinder B. , Groenen‐Döpp Y. A. M. , Werre J. M.
Published: July 2010

Abstract:  Previous studies have shown that a considerable amount of haemoglobin is lost from the intact red cell during its lifespan. The aim of this study was to determine the relative contribution of all the haemoglobin components to this process. Therefore, the relative amount of haemoglobins A0, A2, F and the glycated haemoglobins were determined in 24 fractions of different cell age. These fractions were obtained by the combination of counterflow and density centrifugation. When the absolute amount of all haemoglobin components were calculated using the MCH‐values of each fraction, it appeared that the mean red cell loss of haemoglobins A0, A2, F, an unknown X and “rest” comprised, respectively, 440, 23, 1, 4 and 1 amol per cell, while the mean gain of the glycated haemoglobins was 84 amol per cell. This resulted in a net loss of 385 amol of haemoglobin per cell. One of the glycated haemoglobins (HbA1e2) turned out to be the product of further carbamylation. It was concluded that in the first half of the red cell lifespan HbA0 and HbA2 decreased by glycation and carbamylation and that in the second half some of the HbA0 and HbA2 but also some of the glycated and carbamylated haemoglobin components leave the red cell. The total loss amounted to about 20%.
Decreased erythropoietin response in Plasmodium falciparum malaria‐associated anaemia
Authors: Hassan A. M. A. El , Saeed A. M. , Fandrey J. , Jelkmann W.
Published: July 2010

Abstract:  One of the most serious manifestations of Plasmodium falciparum malaria is anaemia. Its established causes are increased red cell destruction and ineffective erythropoiesis. Since proinflammatory cytokines have been shown to suppress the in vitro synthesis of erythropoietin (Epo), we measured serum immunoreactive Epo in 90 Sudanese patients suffering from malaria. Even in severe cases of anaemia (blood haemoglobin <80 g/l), serum Epo levels rarely exceeded 300 U/l. For comparison, serum Epo was increased up to 12,000 U/l in a reference group of Caucasian patients with anaemia not associated with infection. Moreover, the slope of the log Epo/haemoglobin regression line was less steep in malarial anaemia. Thus, as documented for other chronic inflammatory disorders, there is a relative lack of Epo in malaria‐associated anaemia. Treatment with the antimalarial drug chloroquine may aggravate the defect in Epo production, because chloroquine inhibited Epo synthesis when tested in cell culture.
Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture
Authors: Gross Sherilyn , Helm Karen , Gruntmeir Jennifer J. , Stillman Wayne S. , Pyatt David W. , Irons Richard D.
Published: July 2010

Abstract:  Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6‐d period using 3‐colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine‐, drug‐ or chemical‐induced alterations in haematopoietic progenitor cell differentiation in vitro.
Non‐inpatient parenteral antibiotic therapy in febrile adults
Authors: Newland A. C. , Ketley N. J.
Published: July 2010

Abstract:  Non‐inpatient care is becoming an increasingly attractive option in many therapeutic areas, including the treatment of infections in patients with haematological malignancies. The choice of antibiotics for this care is governed by infection patterns and experience within particular institutions. There is an increase in infections caused by Gram‐positive pathogens due to the long‐term use of tunnelled catheters and intensive, punishing chemotherapy regimens. Patients suitable for non‐inpatient care include both long‐ and short‐term patients who fulfil specific clinical and social criteria. Haematological malignancy patients are often suitable for this type of care. Benefits for patients include improved quality of life, while benefits for the clinician include effective, safe care as well as reduced costs and greater bed availability. The glycopeptide teicoplanin has been assessed for use in non‐inpatient care, and is particularly suitable due to its long half‐life, no need for monitoring and its activity against Gram‐positive pathogens. A comparative study of a teicoplanin–ciprofloxacin combination was conducted in inpatients, followed by a cohort study in non‐inpatients. This combination was found to be clinically and microbiologically effective, suitable for non‐inpatient administration and generated cost savings due to early discharge. The organization of a non‐inpatient service demands dedicated team members, with well‐defined roles and a designated treatment area. The day ward is the focus of care, which can then take place in the day ward or in the patient's home. Good communication between immediate and wider team members, and patient education are cornerstones of a successful service.
Impact of clinical and morphological variables in classification and regression tree‐based survival (CART) analysis of CML with special emphasis on dynamic features
Authors: Thiele J. , Kvasnicka H. M. , Zirbes T. K. , Flucke U. , Niederle N. , Leder L. D. , Diehl V. , Fischer R.
Published: July 2010

Abstract: To determine parameters of predictive value in CML, a retrospective clinico‐pathological study was performed. This included laboratory data and (pretreatment) bone marrow biopsies of 120 patients with a monotherapy by busulfan (BU) and 50 patients with interferon‐alpha 2b (IFN)treatment. Median survival in the BU group was 39 months and in the IFN‐treated patients 65 months. Morphological features (CD61‐positive megakaryocytes, argyrophilic fibres, pseudo‐Gaucher cells) were evaluated by morphometry. Additionally, we measured the incidence of apoptosis (in situ end‐labelling technique) and the expression of the proliferating cell nuclear antigen (PCNA). The ratio between the proliferative and apoptotic cell fraction was coined leukaemia turnover index (LTI). In order to estimate the impact of clinical and various morphological as well as dynamic features of prognostic significance, a multivariate analysis was carried out using the classification and regression tree approach (CART). Discrimination of single disease parameters revealed that fibrosis remained the most significant variable for survival in both therapeutic groups. Indicators of myeloid metaplasia such as occurrence of erythro‐normoblasts and/or splenomegaly were important clinical parameters for prognosis. Inclusion of morphological as well as dynamic disease features in risk classification resulted in a substantial improvement of prognostic efficiency compared to other predictive scores which could be demonstrated by means of ROC‐analysis.
The changing profile of idiopathic myelofibrosis: a comparison of the presenting features of patients diagnosed in two different decades
Authors: Cervantes Francisco , Pereira Arturo , Esteve Jordi , Cobo Francesc , Rozman Ciril , Montserrat Emilio
Published: July 2010

Abstract: In an attempt to ascertain whether the presenting features of idiopathic myelofibrosis (IM) have changed in recent years, 2 groups of patients diagnosed with IM in a single institution in different time periods were compared. The first group included 53 patients diagnosed from 1975 to 1986, and the second included 56 patients diagnosed from 1987 to 1997. No significant differences were observed between the two groups with regard to age, gender, delay from first symptoms to disease diagnosis, peripheral blood hematological values and serum biochemical parameters. Patients diagnosed prior to 1987 presented more often with constitutional symptoms (fever, night sweats, weight loss), but the difference did not reach statistical significance. These latter patients had, however, a higher frequency of splenomegaly (91% vs. 73%, p=0.01) and hepatomegaly (79% vs. 48%, p=0.002), and were more often in the osteosclerotic phase at diagnosis (p=0.05) than patients more recently diagnosed. Finally, no significant differences were found between both groups in either the distribution by prognostic scores or survival. The above results seem to indicate a trend towards a less florid clinical picture of IM at presentation in recent years. This, however, does not result in a longer patient survival.
Plasma levels of D‐dimer and circulating endothelial adhesion molecules in veno‐occlusive disease of the liver following allogeneic bone marrow transplantation
Authors: Südhoff Thomas , Heins Michael , Söhngen Dietmar , Lenz Viola , Wehmeier Artur , Meckenstock Gerald , Schneider Wolfgang , Reinauer Hans , Heyll Axel , Aul Carlo
Published: July 2010

Abstract: Veno‐occlusive disease (VOD) of the liver is a frequent and life‐threatening complication of BMT. Recently, successful treatment by t‐PA has been reported but has been compromised by fatal bleeding events. Therefore, t‐PA application should be restricted to patients with severe VOD. However, moderate and severe forms of VOD are difficult to distinguish in early stages. We analyzed plasma levels of cross‐linked fibrin degradation products (D‐dimer) and soluble endothelial adhesion molecules such as sE‐selectin, sVCAM‐1 and sICAM‐1 in 10 consecutive patients undergoing allogeneic BMT to evaluate their use in identifying severe forms of VOD. During the observation period, 4 episodes of VOD occurred, 2 of which were fatal due to early onset of multiorgan failure. Concentrations of D‐dimer generally increased after transplantation. However, there was an additional significant increase in D‐dimer levels during severe VOD. Thus, D‐dimer levels above 1000 μg/1 were only found in 2 cases with severe VOD and fatal outcome. When compared with bilirubin concentrations substantial increases of D‐dimers appeared earlier during the course of severe VOD. In contrast, VOD episodes were not accompanied by significant increases in sE‐selectin, sVCAM‐1 and sICAM‐1 levels. It is concluded that measurement of D‐dimer concentrations may aid accuracy to the early diagnosis of severe VOD.
Diffuse large B‐cell lymphoma: is morphologic subdivision useful in clinical management?
Authors: Salar A. , De Sevilla A. Fernández , Romagosa V. , Domingo‐Claros A. , González‐Barca E. , Pera J. , Climent J. , Grañena A.
Published: July 2010

Abstract: The diffuse large B‐cell lymphoma category of the REAL classification encompasses different morphologic lymphoma subtypes in a single entity. The aim of this study is to determine the influence of the morphologic subdivision within this category with respect to clinical features and response to treatment. From January 1993 to October 1996, 132 patients were diagnosed de novo with diffuse large B‐cell lymphoma in our institution. All cases were classified according to the REAL and the Updated Kiel classifications, and immunohistochemical study was performed in all of them. Sixty‐three per cent of patients received chemotherapy with a curative approach. Of the 105 assessable patients, 80 cases (74%) were classified as centroblastic (CB) and 25 cases (26%) as immunoblastic (IB), according to the updated Kiel classification. These 2 subsets of lymphomas did not differ with respect to major clinical features and laboratory parameters. Both groups had a similar complete response rate with a uniform therapeutic approach and the overall 2‐yr survival did not show statistical differences (49% in CB vs. 45% in IB). In conclusion, for clinicians, morphologic subdivision of the diffuse large B‐cell lymphoma category into CB and IB subtypes has little clinical and prognostic significance.
Combined use of erythrocyte zinc protoporphyrin and mean corpuscular volume in differentiation of thalassemia from iron deficiency anemia
Authors: Harthoorn‐Lasthuizen Else J. , Lindemans Jan , Langenhuijsen Mart M. A. C.
Published: July 2010

Abstract: In a retrospective study the diagnostic value of erythrocyte zinc protoporphyrin (ZPP) measurement as a means of distinguishing iron deficiency anemia from thalassemia syndromes in patients with microcytosis was explored. ZPP values were increased in all patients with iron deficiency and in part of the patients with thalassemia. The combined measurement of erythrocyte mean corpuscular volume (MCV) and ZPP resulted in a correct classification of patients with iron deficiency and with thalassemia in more than 95%. The predictive value of this method is better than the results obtained by using formulae derived from red cell indices. In population screening programs for thalassemia syndromes, in which MCV determination is used as the initial test, the ZPP test is recommended as a second test, in order to discriminate between patients with microcytosis due to iron deficiency and patients with microcytosis due to thalassemia syndromes.
Evaluation of marrow and blood haemopoietic progenitors in chronic lymphocytic leukaemia before and after chemotherapy
Authors: Sala Roberta , Mauro Francesca R. , Bellucci Roberto , De Propris Maria Stefania , Cordone Iole , Lisci Alessandro , Foà Robin , De Fabritiis Paolo
Published: July 2010

Abstract: We have evaluated the number and differentiation pattern of CD34+ cells, as well as the CFU–GM, BFU–E and CFU–GEMM progenitors from the blood (PB) and marrow (BM) of 53 chronic lymphocytic leukaemia (CLL) patients. Twenty‐four patients were untreated and 29 were studied at 2 months from the last course of fludarabine or chlorambucil; 6 patients, studied after fludarabine therapy, were further evaluated after mobilization with cyclophosphamide and G–CSF. PB of untreated patients showed a median number of CD34+ cells, CFU–GM, BFU–E and CFU–GEMM/105 seeded cells and per litre of PB similar to those of normal controls. No differences were also found in the number of clonogenic progenitors/105 cells in patients studied before and after therapy, while significantly fewer BFU–E/l of PB were found after fludarabine. The number of circulating CD34+ cells/l of PB was significantly lower in patients treated with fludarabine or chlorambucil compared to untreated patients. BM growth was significantly reduced in untreated CLL patients compared to healthy donors. Treatment with fludarabine or chlorambucil restored BM progenitors at levels similar to those of normal controls; this effect did not occur for CFU–GM in patients treated with fludarabine. Three‐colour fluorescence analysis demonstrated a differentiation pattern of CD34+ cells, with a greater expression of CD13 and CD33 after treatment with fludarabine compared to untreated patients and normal controls. In 4 patients previously treated with fludarabine who underwent a successful cyclophosphamide and G‐CSF mobilization therapy, 4 × 106 CD34+ cells/kg were collected. These 4 patients showed a notable increase of CD34+ cells and of clonogenic cells in the PB, but a marked decrease of BM progenitor cells. The 2 patients who failed CD34+ cell mobilization had a reduced CFU‐GM growth both in the PB and in the BM. Taken together, these studies indicate that residual haemopoietic progenitors are present in untreated CLL patients and that stem cell mobilization and collection can be carried out following fludarabine treatment.
Engraftment of human myelodysplastic syndrome derived cell line in transgenic severe combined immunodeficient (TG‐SCID) mice expressing human GM–CSF and IL‐3
Authors: Kim Dong Ku , Kojima Masaharu , Fukushima Takashi , Miyasaka Masayuki , Nakauchi Hiromitsu
Published: July 2010

Abstract: A transgenic SCID (TG‐SCID) mouse expressing the human cytokines interleukin‐3 (IL‐3) and granulocyte‐macrophage colony‐stimulating factor (GM–CSF) has been generated with the aim of making a model system allowing the in vivo proliferation of human hematopoietic cells. Using TG‐SCID mice expressing high levels (30–35 ng/ml in the serum) of human GM–CSF and IL‐3, we attempted to engraft a human myeloid leukemia cell line, F‐36P, derived from a myelodysplastic syndrome (MDS) patient. When F‐36P cells were transferred intravenously into sublethally irradiated TG‐SCID mice, extensive proliferation of F‐36P cells was found 4–6 wk later. Successful engraftment, however, required the preadministration of a monoclonal antibody to mouse interleukin‐2 receptor (IL‐2R) β chain, neutralizing NK activity. Surprisingly, all the transplanted TG‐SCID mice engrafted with F‐36P cells developed hind leg paralysis approximately 6 wk after transfer. Histological analysis demonstrated extensive invasion and formation of osteolytic lesions by the F‐36P cells in the vertebrae. These data indicate that transgenic SCID mice expressing human IL‐3 and GM–CSF provide a useful system for the study of human leukemia. In addition, NK cells appear to play an important role in rejection of human cells.
Outcome of secondary myeloid malignancy in Hodgkin's disease: the BNLI experience
Authors: Harrison Claire N. , Hudson Gillian Vaughan , Devereux Stephen , Linch David C.
Published: July 2010

Abstract: In Hodgkin's disease where the majority of patients are long‐term survivors secondary myeloid malignancies are a well‐documented complication. The survival of those who develop secondary myelodysplasia/ acute myeloid leukaemia (MDS/AML) is historically said to be extremely poor. This study from the BNLI database of over 4900 patients with Hodgkin's disease reports long‐term follow‐up of 30 patients with secondary MDS/AML. Five patients have survived at least 5 yr (1>12 yr) from the time of diagnosis of AML. These patients were significantly younger (p=0.03) than those who succumbed to this complication and each also had standard or favourable risk cytogenetics. The actuarial 5‐ and 10‐yr survival rates are 17.4% (7.7–34.9%, 95% CI) and 8.7% (1.9–31.7%, 95% CI), respectively. There is therefore a subgroup of patients who will achieve long‐term survival despite the development of secondary myeloid malignancy.
The risk of bacteremia in patients with monoclonal gammopathy of undetermined significance
Authors: Gregersen Henrik , Madsen Kreesten Meldgaard , Sørensen Henrik Toft , Schønheyder Henrik Carl , Ibsen Jenna Sailing , Dahlerup Jens Frederik
Published: July 2010

Abstract: We estimated the risk of bacteremia in patients with monoclonal gammopathy of undetermined significance (MGUS) compared with the general population; 1237 cases of MGUS were identified by linking information on detected monoclonal components in the North Jutland County with the Danish Cancer Registry. We evaluated the risk of bacteremia in the MGUS cohort during the 13‐yr period from 1981 to 1993 by linkage to the Bacteremia Registry in the County. Follow‐up for the occurrence of bacteremia started 30 d after detection of the M‐component and continued until malignant transformation, death or until 31 December 1993, whichever came first. The expected numbers of bacteremia was based upon county, age, sex and period‐specific incidence rates. The median follow‐up period was 3.8 yr. Forty episodes of bacteremia occurred during 5500 person‐years versus 18 expected. The crude standardized incidence ratio of bacteremia was 2.2 (95% confidence interval, 1.6–3.0). There was no distinct pattern of bacterial etiology in the MGUS cohort. Although we found an association between MGUS and risk of bacteremia, the overall risk is small and this finding hardly affects the clinical handling of MGUS patients.
Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol
Authors: Cortesi R. , Gui V. , Osti F. , Nastruzzi C. , Gambari R.
Published: July 2010

Abstract: Human leukemia K562 cells can be induced to erythroid differentiation when treated with a variety of compounds, including hemin, cytosine arabinoside and 5‐azacytidine. Following erythroid induction, K562 cells express at high level γ‐globin and accumulate both Hb Portland and Hb Gower 1. In this paper we determined whether a combination treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids lead to full expression of differentiated functions. Cell growth kinetics studies, intracellular detection of hemoglobin by benzidine staining and hemoglobin analysis by cellulose acetate were performed. The results obtained show that (a) retinoic acid and retinol are not able to induce differentiaton of K562 cells and (b) cytosine arabinoside induces differentiation only when used at 100–300 nmol/l concentrations. In addition, our data demonstrate that erythroid differentiation of K562 occurs when 40 μmol/l of retinoic acid or retinol are added together with 75 nmol/l cytosine arabinoside.
Platelet activation in the Dutch haemocytometry quality assessment programme: correlation between P‐selectin expression and variation in platelet count
Authors: Pouwels J. G. J. , Van Maastricht B. M. N. , van de Leur J. J. C. M. , Kuiper‐Kramer P. A.
Published: July 2010

Abstract: Since 1990, our laboratory has prepared a set of 8 fresh whole blood samples for use in a countrywide quality assessment (QA) programme. The samples are intended as external controls for haemocytometry analysers. These samples are of 8 different haematocrit levels and each one is prepared from a single donor. About 210 laboratories participate in this QA programme. From the start of this programme large interlaboratory variations in platelet counts were encountered in some of the samples. This variability was much higher than would be expected and was independent of the platelet count of the samples. The main cause was thought to be formation of platelet aggregates. The aim of the present study was to find a parameter that can predict a high interlaboratory variation in the QA programme. Therefore we investigated the initial platelet activation status in the donors and the activation status of platelets in the prepared QA blood. As a marker for platelet activation P‐selectin expression on the platelets was measured using flow cytometry. During 5 rounds of the QA programme we found a good correlation of r = 0.53 (p < 0.001) between P‐selectin expression on platelets in the reconstituted QA blood and the interlaboratory platelet count variability. We conclude that P‐selectin expression in the prepared QA blood is an important parameter to exclude samples that lead to high CVs of platelet counts in the QA programme.
Downregulation of the CD95 receptor and defect CD40‐mediated signal transduction in B‐chronic lymphocytic leukemia cells
Authors: Laytragoon‐Lewin N. , Duhony E. , Bai X.‐F. , Mellstedt H.
Published: July 2010

Cross‐linking of the CD40 receptors has been shown to induce protein tyrosine kinases (PTK) phosphorylation and prevent apoptosis in Bcl‐2 negative germinal center B cells. The expression of CD40 on B chronic lymphocytic leukemia (B‐CLL) cells was found to be similar to that of normal B cells. Activation of normal B cells with soluble anti‐CD40 monoclonal antibody (mAb) induced tyrosine phosphorylation, prolonged survival and prevented apoptosis. However, activation of CD40 on B‐CLL cells using soluble anti‐CD40 mAb does not influence survival or apoptosis. Normal B cells entered apoptosis when cultured in the presence of soluble anti‐CD95 mAb. This process was independent of PTK activity. On B‐CLL cells, the CD95 molecules were downregulated and a transient PTK signal was observed when cross‐linking of the receptor by soluble anti‐CD95 mAb occurred. Interestingly, B‐CLL cells did not enter apoptosis in the presence of anti‐CD95 mAb. Our study indicates that survival signals mediated through the CD40 molecule and death signals mediated through the CD95 molecule used different intracellular pathways in control donor B cells. In contrast, B‐CLL cells do not respond to these signals. The leukemic B cells showed a defective CD40‐mediated signal transduction and downregulated CD95 receptor expression. As a consequence, no apoptosis could be induced in B‐CLL cells by a soluble anti‐CD95 mAb. The abnormalities of these receptors may contribute to the long‐lived status of B‐CLL cells.
Retrospective study of candidemia in patients with hematological malignancies. Clinical features, risk factors and outcome of 76 episodes
Authors: Pagano Livio , Antinori Andrea , Ammassari Adriana , Mele Luca , Nosari Annamaria , Melillo Lorella , Martino Bruno , Sanguinetti Maurizio , Equitani Francesco , Nobile Francesco , Carotenuto Mario , Morra Enrica , Morace Giulia , Leone Giuseppe
Published: July 2010

A retrospective study of 76 episodes of candidemia in 73 patients with underlying hematological malignancy, from 1988 until 1997, has been conducted to evaluate the clinical characteristics and to ascertain the variables related to the onset and the outcome of candidemia. The most frequent malignancy was acute myeloid leukemia (29 episodes). Candidemia developed mainly during aplasia in patients refractory to chemotherapy (42%). In 65 episodes (86%) the patients were neutropenic (ANC < 1×109/l) before the candidemia diagnosis for a median time of 13 d, and in 53 episodes (70%) at microbiological diagnosis of candidemia ANC was <1×109/l. Candida albicans was the most frequently isolated etiologic agent (31 episodes), but C. non‐albicans species sustained the majority of candidemia. Seventeen candidemias developed during azoles prophylaxis. One month after the diagnosis of candidemia, 26 patients died. In 19 cases, death was attributable to candidemia. The case‐control study demonstrated, at univariate analysis, that the colonization with Candida, spp. (p=0.004), antimycotic prophylaxis (p=0.01), presence of central venous catheter (p=0.01), neutropenia (p=0.002), and the use of glycopeptide (p=0.0001) increased the risk of candidemia. Using multivariate regression analysis only colonization with Candida spp. and the previous therapy with glycopeptide were associated with a significantly increased risk. Acute mortality, expressed by a cumulative probability of survival at 30 d from diagnosis of candidemia, was 0.67 (95% C.I. 0.55–0.77) and was significantly reduced in patients with neutrophils <1×109/l when compared to those with neutrophils > 1×109/l (p at Mantel‐Cox=0.029). Overall cumulative probability of survival at 1 yr was 0.38 (95% C.I. 0.27–0.49) and only the treatment with Amfotericin B significantly reduced the risk of death.
Membrane protein pattern in hereditary spherocytosis in five subjects from north‐east Italy obtained by SDS‐PAGE using N, N‘‐diallyltartardiamide
Authors: Giuliani Anna Lisa , Bigoni Beatrice , Veronesi Massimo , Manservigi Roberto , Mischiati Carlo , Berti Gilberto , Zavagli Giorgio , Ricci Giorgio
Published: July 2010

Abstract: In the present study we examined five subjects affected by hereditary spherocytosis (three unsplenectomized and two splenectomized), coming from an area in the north‐east of Italy where hereditary spherocytosis is an anaemic disease with very low incidence. All patients showed a low degree of spectrin deficiency (14%), detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, when this analysis was performed with N, N'‐diallyltartardiamide as cross‐linking agent instead of N, N'‐methylenbisacrylamide, some unusual bands appeared in the region between proteins 4.2 and 5, the three unsplenectomized and two splenectomized patients showing different patterns. We hypothesise that some alterations of proteins in this region (e.g. the 4.5 or 4.9 bands), possibly due to proteolysis, must have occurred in relation to the disease.
Apoptosis, proliferation and NF‐κB activation induced by agonistic Fas antibodies in the human myeloma cell line OH‐2: amplification of Fas‐mediated apoptosis by tumor necrosis factor
Authors: Børset Magne , Hjorth‐Hansen Henrik , Johnsen Ann‐Charlotte , Seidel Carina , Waage Anders , Espevik Terje , Sundan Anders
Published: July 2010

Abstract: Tumor necrosis factor (TNF) is known to be a growth factor for several myeloma cell lines. However, in the presence of the agonistic Fas antibody CHI 1, TNF enhanced the level of apoptosis in cultures of the human myeloma cell line OH‐2. This pro‐apoptotic effect of TNF was explained at least in part by a TNF‐mediated enhancement of Fas expression. TNF induces proliferation of OH‐2 by activating nuclear transcription factor kappa‐B (NF‐κB). The proliferative effect of TNF on OH‐2 cells was abrogated by CH11, but this was not caused by an inhibition of the translocation of NF‐κB. On the contrary, CH11 could by itself activate NF‐κB in OH‐2 cells, and in the presence of an inhibitor of caspase‐1 induce proliferation of the cells. The relationship between stimulation of TNF receptors and Fas and the level of NF‐κB activation was also examined in three other myeloma cell lines. RPMI‐8226 cells showed NF‐κB activation by TNF, but contrary to OH‐2, not by CHI 1. Unstimulated U‐266 and JJN‐3 cells had high levels of activated NF‐κB. This shows that NFκ‐B is either constitutively activated or inducible in myeloma cells. Modulation of Fas expression and inhibition of NF‐κB activation can potentially be of therapeutic importance in multiple myeloma.
Cryopreservation of the bone marrow from patients with acute myeloid leukaemia leads to functional abnormalities
Authors: Novitzky Nicolas , Kruger William , Mohamed Rygana
Published: July 2010

Abstract:  We compared the performance of the stroma (SL) and haematopoietic progenitors before (group A) or following cryopreservation from patients in complete remission (CR) of acute myeloid leukaemia before autologous transplantation (AML) (group B), to similarly treated normal cells (groups A and B). From each group, fibroblastic (F) colony‐forming units (CFU) and SL were established and their growth quantitated. Upon confluence of SL, 1 ×104 selected CD34+ cells from patient or control samples (of group A and B) were seeded on pre‐formed SL from AML or control group A and B layers and adhesive precursors were scored for the formation of blastic (‐bl) CFU (>20 cells). Lastly, CD34+ cells from the 4 progenitor sources were cultured in the presence of growth factors and scored for the production of granulocyte‐macrophage (GM) CFU, erythroid burst forming units (BFU‐E) and mixed colonies (CFU‐MIX). In the patients, CFU‐F numbers were decreased and by 5 wk SL failed to reach confluence. Group A AML CD34+ progenitors gave a mean of 119.2 (SD 80.4) CFU‐bl/104 CD34+ cells, but following cryopreservation these numbers declined (83.2, SD 72.71; p = 0.009). Although AML SL prior to cryopreservation supported control CFU‐bl adequately, contrary to the norm, this property decreased in group B stroma (mean 50.9/104CD34+ cells, SD 49.33; p = 0.02). Group B AML samples gave significantly fewer CFU‐MIX and BFU‐E than pre‐freezing cultures and than control samples at the first 2‐growth factor combination but numbers improved following further addition of cytokines. We conclude that cryopreservation of the bone marrow of patients in CR of AML results in an inhibition of the proliferation of the CD34+ precursors. It also modifies the stroma, leading to reductions in the support of normal clones.
Biological characteristics of myelodysplastic syndrome patients who demonstrated high versus no intramedullary apoptosis
Authors: Dar Saleem , Mundle Suneel , Andric Tanja , Qawi Huma , Shetty Vilasini , Reza Samina , Mativi B. Yifwayimare , Allampallam Krishnan , Ali Ambereen , Venugopal Parameswaren , Gezer Sefer , Broady‐Robinson LaTanya , Cartlidge John , Showel Margaret , Hussaini Seema , Ragasa Deborah , Ali Irfan , Chaudhry Ambreen , Waggoner Samina , Lisak Laurie , Huang Ray‐Win , Raza Azra
Published: July 2010

Abstract:  Spontaneous intramedullary apoptosis was measured in bone marrow (BM) biopsies of 175 patients with myelodysplastic syndromes (MDS) using in situ end‐labeling (ISEL) of fragmented DNA. Two groups of high (n = 71) versus low (n = 43) levels of apoptosis were identified while 61 patients were ISEL‐negative. Semiquantitative assessment of 3 cytokines, the number of macrophages and in vivo labeling indices (LI) were also determined from consecutive sections of the biopsy. Patients with high apoptosis levels tended to have a high LI (p = 0.013), more macrophages in their BM biopsies (p = 0.006) and higher tumor necrosis factor alpha (TNF‐α) levels (not significant) compared to patients with no apoptosis. In addition, low risk MDS patients had significantly lower rates of apoptosis (p = 0.047) and lower levels of TNF‐α (p = 0.055) compared to high‐risk MDS patients. We conclude that the genesis of cytopenias in MDS is of multifactorial origin and that cytokine‐associated apoptosis clearly identifies a distinct biological subgroup of patients who may benefit selectively by use of anti‐cytokine therapies.
VH gene family utilization in different B‐cell lymphoma subgroups
Authors: Rosenquist Richard , Lindström Anita , Holmberg Dan , Lindh Jack , Roos Göran
Published: July 2010

Abstract:  VH gene family specific polymerase chain reaction (PCR) amplification was performed in 87 B‐cell lymphoma samples from 4 different subgroups. No apparent restriction in the VH gene usage was found in follicular lymphomas, lymphoplasmacytoid lymphomas or large B‐cell lymphomas, whereas a biased Vh1 utilization was shown in patients with chronic lymphocytic leukemia. Eleven of 18 chronic lymphocytic leukemia cases utilized the VH1 gene family, and nucleotide sequencing of the VH1 gene rearrangements revealed that a majority utilized the DP10 (51p1) germline gene, which has been reported to be strongly associated with autoimmune disease. No VH5 or VH6 rearrangements were amplified in the chronic lymphocytic leukemia subgroup, 2 gene families which previously have been found to be over‐represented in these patients. In a high proportion (40%) of large B‐cell lymphomas, VH gene family‐specific PCR failed to amplify any rearrangement. Using primers hybridizing to the framework regions 2 and 3 and Southern blot analysis of the immunoglobulin heavy chain locus, clonal rearrangements were displayed in two‐thirds of these PCR negative cases. However, the rearrangement status could not be elucidated in 5 of 35 patients with large B‐cell lymphoma.
Natriuretic peptides as markers of cardiotoxicity during doxorubicin treatment for non‐Hodgkin's lymphoma
Authors: Nousiainen T. , Jantunen E. , Vanninen E. , Remes J. , Vuolteenaho O. , Hartikainen J.
Published: July 2010

Abstract:  Thirty adult patients with non‐Hodgkin's lymphoma who were planned to receive up to 8–10 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) to a cumulative doxorubicin dose of 400–500 mg/m2 were studied to evaluate the value of serial plasma atrial natriuretic peptide (ANP), N‐terminal pro‐ANP (NT‐proANP) and brain natriuretic peptide (BNP) measurements in the early detection of doxorubicin‐induced left ventricular dysfunction. Plasma levels of natriuretic peptides were measured before every treatment course and 4 wk after the last one. Cardiac function was monitored with serial radionuclide ventriculography. Twenty‐eight patients were evaluable for cardiotoxicity. Clinical heart failure developed in 2 patients (7%). Left ventricular ejection fraction (LVEF) decreased from 58.0 ± 1.3% to 49.6 ± 1.7% (p<0.001). Plasma levels of ANP increased from 16.4 ± 1.3 pmol/l to 22.7 ± 2.4 pmol/l (p = 0.002), NT‐proANP from 288 ± 22 to 380 ± 42 pmol/l (p = 0.019) and BNP from 3.3 ±0.4 to 8.5 ± 2.0 pmol/l (p =0.020). There was a significant correlation between the increase in plasma ANP and the decrease in LVEF (r = ‐0.447, p = 0.029), and a trend towards significance between the increase in NT‐proANP and the decrease in LVEF (r=‐0.390, p = 0.059). The decrease in LVEF started very early and could already be seen after the cumulative doxorubicin dose of 200 mg/m2, whereas the increase in plasma natriuretic peptides was not evident until the cumulative doxorubicin dose of 400 mg/m2. Our results show that neuroendocrine activation — increased concentrations of plasma natriuretic peptides — occurs when left ventricular function has reduced substantially and its compensatory capacity has been exceeded resulting in atrial and ventricular overload. Thus, serial natriuretic peptide measurements cannot be used in predicting the impairment of left ventricular function. On the other hand, our study suggests that natriuretic peptides are useful in the detection of subclinical left ventricular dysfunction in patients receiving doxorubicin therapy.
Clonal evolution as judged by immunoglobulin heavy chain gene rearrangements in relapsing precursor‐B acute lymphoblastic leukemia
Authors: Rosenquist R. , Thunberg U. , Li A.‐H. , Forestier E. , Lönnerholm G. , Lindh J. , Sundström C. , Sällström J. , Holmberg D. , Roos G.
Published: July 2010

Abstract:  Oligoclonality and ongoing clonal evolution are common features in patients with precursor‐B (pre‐B) acute lymphoblastic leukemia (ALL), as judged by immunoglobulin heavy chain (IgH) gene rearrangement analysis. These features are considered to be results of secondary rearrangements after malignant transformation or emergence of new tumor clones. In the present study we analyzed the IgH gene rearrangement status in 18 cases with relapsing pre‐B ALL using variable heavy chain (VH) gene family specific polymerase chain reaction (PCR) amplification and single stranded conformation polymorphism (SSCP) analysis. Clonal IgH rearrangements were displayed in all leukemias but one, and altered rearrangement patterns occurred in five cases (29%), which were selected for detailed nucleotide sequence analysis. In one case, multiple subclones at diagnosis were suggested to be derived from a progenitor clone through joining of different VH germline gene segments to a pre‐existing D–JH complex (VH to D–JH joining). Evidence for VH gene replacement with identical N‐sequences at the VH‐D junction and a common D–JH region was observed in one case. Diversification at the VH–D junction consisting of heterogeneous N‐sequences were observed in one case. This molecular modification of the VH‐D region could fit a hypothesized “open‐and‐shut” mechanism. Nevertheless, despite these ongoing events at least one IgH rearrangement remained unchanged throughout the disease in most patients, indicating that the immunoglobulin heavy chain locus can be a suitable marker for detection of minimal residual disease (MRD).
Clonal rearrangements in childhood and adult precursor B acute lymphoblastic leukemia: a comparative polymerase chain reaction study using multiple sets of primers
Authors: Li Ai‐Hong , Rosenquist Richard , Forestier Erik , Holmberg Dan , Lindh Jack , Löfvenberg Eva , Roos Göran
Published: July 2010

Abstract: Ig heavy chain (IgH) and T‐cell receptor (TCR) gene rearrangements were investigated by polymerase chain reaction (PCR) amplification of diagnostic tumour samples from 91 patients (57 children and 34 adults, with cut‐off at age 16) with precursor B acute lymphoblastic leukemia (ALL). Using primers directed to the framework regions (FR) 1, 2 and 3 of the IgH gene, clonal IgH rearrangements were observed in 82, 58 and 58%, respectively, whereas clonality was presented in 45 and 27% using primers hybridising to the TCR δ and γ genes. A combination of all five primer sets used resulted in 96% positive cases (children 100%, adults 88%). The frequency of clonal IgH rearrangements correlated to patient age with a significantly lower fraction of positive cases in the adult group. The concomitant usage of more than one VH family gene was similar for childhood and adult ALL, and an over‐representation of VH6 rearrangements was found in childhood ALL. Twenty‐five out of 91 cases (27%) displayed an oligoclonal pattern for either IgH or TCR gene rearrangements (children 37%, adults 12%). A comparative analysis of samples from different compartments was performed in 23 patients, and differences between two or three compartments were observed in seven cases. Unexpectedly large, clonally appearing PCR products of 540–715 bp were found in three leukemias and sequence analysis verified their clonal nature. In summary, using multiple sets of primers clonal rearrangements of IgH and TCR genes can be detected in a very high frequency, including previously neglected large size PCR products. A common heterogeneity was demonstrated in different compartments reflecting ongoing clonal evolution, which can make detection of minimal residual disease (MRD) in ALL troublesome. Therefore, we suggest that a minimum of three targets should be used to minimise false‐negative results.