bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of a normal Hb A2β‐thalassaemia determinant in a Sardinian family
Authors: Oggiano L. , Pirastu M. , Moi P. , Longinotti M. , Perseu L. , Cao† A.
Published: March 2008

In this study we have carried out haplotype analysis at the β‐globin gene cluster and defined the β‐thalassaemia mutations in a large Sardinian family, ascertained through a proband with thalassaemia major, in which several members were carriers of a β‐thalassaemia allele characterized by microcytosis, hypochromia and normal Hb A2 levels (type 2 normal Hb A2 heterozygous β‐thalassemia). The proband was a compound heterozygote for the β°‐39 and the β+ IVS‐2, nt 745 mutations and all the β‐thalassaemia heterozygotes with normal Hb A2 showed the β+ IVS‐2, nt 745 mutation, always associated with haplotype VII. Because of the consistent association of a specific β‐thalassaemia mutation and normal Hb A2 levels, we postulate that this β‐thalassaemia chromosome carries a δ gene (δ‐thalassaemia) which is unable to increase the δ‐globin output in response to β‐thalassaemia.
Disappearance of HLA and platelet‐specific antibodies in acute leukaemia patients alloimmunized by multiple transfusions
Authors: Murphy M. F. , Metcalfe P. , Ord J. , Lister T. A. , Waters A. H.
Published: March 2008

Alloimmunization by platelet transfusions was studied in 154 patients with acute leukaemia. 17 patients had HLA antibodies at initial presentation induced by previous transfusions or pregnancies; one of these also had platelet‐specific antibodies and one other patient had platelet‐specific antibodies alone. A further 38 patients developed HLA antibodies during therapy; three also had platelet‐specific antibodies and two patients developed platelet‐specific antibodies alone. Of these, 37 patients with HLA antibodies including three with platelet‐specific antibodies and one patient with platelet‐specific antibodies alone survived their initial therapy and formed the basis of this study.

Antibodies once detected persisted throughout the study in seven of the 37 patients with HLA antibodies including one patient with platelet‐specific antibodies and in the patient with platelet‐specific antibodies alone. HLA antibodies disappeared after discontinuation of transfusions in six patients, and after switching to HLA matched platelet transfusions and leucocyte‐poor blood in eight patients: two of the latter patients also had platelet‐specific antibodies which disappeared. The other 16 patients with HLA antibodies lost their antibodies despite continued transfusions.
Nuclear c‐myc protein, maturation, and cell‐cycle status of human haemopoietic cells
Authors: Bains M. A. , Hoy T. G. , Baines P. , Jacobs A.
Published: March 2008

Human c‐myc protein, p62c‐myc, has been quantitated by flow cytometry in the nuclei of normal marrow and peripheral blood cells, and the HL60 cell line. Marrow and peripheral blood cells exhibit nuclear c‐myc protein throughout the cell‐cycle, at an average level 2–3–fold lower than HL60 cells. In no cells did p62c‐myc vary more than 2‐fold throughout the cell cycle. A small subset of marrow Go/G1 cells, enriched in early myeloid and blast cell fractions, contained p62c‐myc at levels equal to or even exceeding those of HL60.

Overall c‐myc protein content was higher in myeloid, compared to erythroid and lymphoid marrow fractions. Within the myeloid lineage, the highest average p62c‐myc level was present in cells of intermediate maturation, i.e. myelocytes and metamyelocytes. In the erythroid lineage, c‐myc protein level was highest in the most immature cells and declined with maturation. Significant amounts of p62c‐myc were present in post‐mitotic, end‐stage neutrophils, but were barely detectable in cycling late erythroblasts or in quiescent lymphocytes and monocytes. HL60 cells, despite c‐myc gene amplification and increased gene expression, contain c‐myc protein at a level corresponding to promyelocytes in normal human marrow. The virtual absence of p62c‐myc in cycling late erythroblasts, and its presence in post‐mitotic end‐stage granulocytes, suggests that c‐myc protein may have functions unrelated to cell proliferation.
Familial lupus anticoagulants
Authors: Mackie I. J. , Colaco C. B. , Machin S. J.
Published: March 2008

Three families having more than one affected member with SLE or lupus‐like disease were investigated by global coagulation tests as well as methods based on dilute thromboplastin, Russell's viper venom and thermal stability/absorption, and by RIA for anticardiolipin (CL) antibodies. Of the 19 persons, 11 had SLE or lupus‐like disease. Eight of these 11 had a prolonged KPTT and other evidence of LA, while only 5/11 had high anticardiolipin titres. Four healthy spouses of affected females, and three asymptomatic siblings also had prolonged non‐correctable KPTTs. These persons had no bleeding or thrombotic history and normal clotting factor levels. Further clotting tests were negative, although one had raised anti‐CL antibody. Such cases may account for some of the patients one finds during routine haemostatic screening with unexplained prolonged KPTT. Although anticardiolipin levels are raised in subjects with LA, there was no close correlation between length of KPTT and anticardiolipin titre. These findings would support a hypothesis of transmissible agents or other environmental factors being involved in lupus‐like disorders.
Analysis of leucocyte differentiation antigens in blood and bone marrow from patients with Waldenström's macroglobulinaemia
Authors: Kucharska‐Pulczynska Malgorzata , Ellegaard Jørgen , Hokland Peter
Published: March 2008

Summary. Peripheral blood and bone marrow mononuclear cells from 10 patients with newly diagnosed Waldenström's macroglobulinaemia were analysed for the presence of leucocyte differentiation antigens using flow cytometry. Most patients had circulating intracytoplasmic‐μ cells that were positive for the B‐cell specific antigens B1, B2 and B4 and for the plasma cell antigen PCA‐1. When such monoclonal cyto‐μ+ cells were purified by immune rosette depletion they were shown to express B1 to a much lesser extent than B2, B4 and PCA‐1. In bone marrow samples cells with a similar phenotype, but with higher B1 expression were found in all patients.

When the serum IgM paraprotein concentrations were compared to surface markers longitudinally from presentation and during the first courses of chemotherapy, a decreased serum IgM level was accompanied by a decrease in B1 cells in peripheral blood. These data demonstrate that WM cells possess a unique phenotype with coexpression of B‐cell and plasma cell associated antigens.
Adult acute lymphoblastic leukaemia: is cell proliferation related to other clinical and biological features?
Authors: Ffrench Martine , Manel Anne Marie , Magaud Jean Pierre , Fiere Denis , Adeleine Patrice , Guyotat Denis , Bryon Paul André
Published: March 2008

Summary. Flow cytometry with propidium iodide and fluorescein isothiocyanate was used to study 46 cases of adult acute lymphoid leukaemia (ALL) before any form of chemotherapy. Cell proliferation was related to the other clinical and biological characteristics and its prognostic significance was evaluated. The following cell‐cycle variables were determined: S. G2+ M, and the Low Protein Content fraction of G1 (LPC fraction). The L3 group, corresponding to B‐ALL, had significantly higher proliferation than LI and L2 (P < 0·01). The proliferation rate was not significantly higher for T‐ALL than for the other phenotypes. Complete remission was succesfully induced significantly more often in cases with the LPC fraction under 50% (P < 0·05). Failure was mainly related to resistance to chemotherapy. Of the four patients who died during aplasia, three had an LPC fraction below 25%. Duration of complete remission and survival were significantly shorter for L3 which is the most proliferative ALL (P < 0·01). Survival was also found to be longer (P < 0·05) when G2+ M was between 3·8% and 5·1%. This finding and the negative correlation between S and G2+ M (P < 0·01) are discussed.
Haem arginate as a treatment for myelodysplastic syndromes
Authors: Ruutu Tapani , Volin Liisa , Tenhunen Raimo
Published: March 2008

Summary. We report on two patients with a myelodysplastic syndrome in whom the blood cell counts markedly improved during treatment with haem arginate. One patient received haem arginate only, the other haem arginate in combination with low dose androgen. The drug was given as weekly infusions of 2‐3 mg/kg body weight for 8 ‐ 12 weeks. In one patient the percentage of ringed and other abnormal sideroblasts in the bone marrow was clearly reduced as a result of the treatment. In both patients the effect on blood cell counts lasted for several months after the cessation of the haem arginate treatment. Eleven other patients showed no clear response. No adverse effects of the infusions were observed. Further studies on the possible therapeutic role of haem arginate are indicated.
Identification and characterization of a new antithrombin III familial variant (AT Dublin) with possible increased frequency in children with cancer
Authors: Daly M. , O'Meara A. , Hallinan F. M.
Published: March 2008

Summary. The antithrombin III (ATIII) isoform pattern of a number of serum and plasma samples was analysed by isoelectric focusing and immuno‐blotting. A novel ATIII isoform pattern which was observed in 4/80 children with acute lymphatic leukaemia (ALL) and in 1/4 children with Ewing's sarcoma, has been shown by family studies to be due to a mutant form of ATIII (AT Dublin) in the heterozygous state. The coagulation properties of AT Dublin heterozygotes were normal. In addition the immunological and activity levels of their ATIII were normal. The effects of thrombin and heparin on the mutant ATIII were similar to controls. Neuraminidase treatment reduced the ATIII isoforms to one in controls and two in the mutant. Two‐dimensional gel analysis showed the mutant ATIII to have an identical molecular size distribution to the normal form. This mutant is, thus, most likely due to an amino acid substitution giving a more basic molecule that is clinically silent (at the coagulation level). It may be of interest that the frequency of AT Dublin in the ALL group is significantly higher than in the control group (3/430) studied (P < 0·001).
Studies on the effect of heat treatment on the thrombogenicity of factor IX concentrates in dogs
Authors: Littlewood Janet D. , Dawes Joan , Smith James K. , Feldman Peter A. , Haddon Michael E. , Mcquillan Thomas A. , Foster Peter R. , Ferguson Joyce , Prowse Christopher V.
Published: March 2008

Summary. This study examines the effects of heat treatment for 72 h at 80°C on the potential thrombogenicity of lyophilized human coagulation factor IX concentrates. Since heating generated minor amounts of thrombin, concentrate was prepared with antithrombin III addition prior to heat treatment. Changes in coagulation parameters were followed prior to and after infusion of 100 iu/kg of heated and unheated concentrates to dogs. All batches produced a transient fall in platelet count during infusion and a delayed rise in plasma fibrinopeptide A, accompanied by a minor prolongation of the activated partial thromboplastin time. Such changes were less marked for heated batches.

Control infusion of a‘failed’ factor IX concentrate showed an additional fall in fibrinogen, rise in fibrin degradation products and a more rapid rise in fibrinopeptide A, while thrombin infusion caused an even more dramatic intravascular coagulation.

These studies indicated no increase in the potential thrombogenicity of freeze dried factor IX concentrates as a result of heat treatment.
Characteristics of a ferritin‐binding protein present in human serum
Authors: Bellotti Vittorio , Arosio Paolo , Cazzola Mario , Cozzi Anna , Levi Sonia , Meloni Federica , Zappone Elisabetta
Published: March 2008

Summary. The ferritin present in human serum differs from the ferritins found in tissues and other body fluids in having negligible proportions of H subunits. This has been related to the possible presence of binding factors which would form complexes with H‐subunit containing ferritins and thereby determine their rapid clearance and/or interference with immunoassays ('serum inhibition’). In this work we have tried to identify and characterize these binding factors. Dotting and blotting experiments demonstrated an interaction between tissue ferritins and human serum. This was stronger with human heart and recombinant H‐type ferritin obtained from E. coli than with human liver ferritin. The serum binder appeared to be a glycoprotein migrating in the beta‐2 region and with a molecular weight of about 200 000 and pi between 4 and 5. Two different approaches to purification of the ferritin‐binding protein yielded enriched fractions containing also the complement proteins C3 and C4, the plasma protease inhibitor alpha‐2‐macroglobulin, and immunoglobulins. These in vitro findings may have physiological relevance.
Utility of the FAB classification for myelodysplastic syndromes: investigation of prognostic factors in 237 cases
Authors: Kerkhofs H. , Hermans J. , Haak H. L. , Leeksma C. H. W.
Published: March 2008

The utility and prognostic significance of the FAB classification was studied in 237 patients with a myelodysplastic syndrome. No significant differences in actuarial survival and probability of transformation to acute leukaemia were found in patients with RA, AISA or CMML. The median survival time for the RA group was 50 months, for the AISA and CMML subclasses more than 60 months. The probability of transformation for the RA, AISA and CMML subgroups showed a linear trend with a probability of 25% for the RA, 16% for the AISA and of 18% for the CMML groups after a 5 year observation period. A uniformly poor prognosis was found for the RAEB and RAEB/t subgroups with median survival times of respectively 9 and 6 months.

Chromosomal abnormalities were found in 68 out of 155 patients (44%). Patients with only normal metaphases or with abnormal metaphases together with karyotypic normal cells had a longer median survival time and a lower probability for transformation as compared to those with only abnormal metaphases.

The most important factor in prognosis is the number of blast cells in blood and bone marrow. Age and sex, and certain quantitative and qualitative abnormalities in the peripheral blood appear of limited prognostic value for patients with RA, AISA and CMML.

The longer life expectancy of 35 patients with CMML as compared to other series seems to be related to the percentage of blast cells at the time of diagnosis.
Familial splenomegaly syndrome with reduced circulating T helper cells and splenic germinal centre hypoplasia
Authors: McKinley R. A. , Kwan Y. L. , Lam‐po‐Tang P. R. L.
Published: March 2008

Summary. Familial splenomegaly is a rare occurrence and is occasionally associated with immune abnormalities. We report three members of a family with massive splenomegaly associated with a reduction in circulating T helper cells, a reversed T4/T8 ratio and cutaneous anergy. The spleen and lymph nodes were shown in one family member to have germinal centre hypoplasia with T helper cells being present in normal numbers and distribution in these tissues. Various abnormalities of immunoglobulins were also noted. Despite the demonstrated immune abnormalities, the affected subjects showed few serious consequences. The pathogenesis of this disorder is unclear but we postulate it may involve a functional defect of the T helper cell. This defect probably is responsible both for the diminished circulating capacity of T helper cells as well as a reduced ability to aid in the formation of germinal centres.
Elimination of T cells from human peripheral blood and bone marrow using a cocktail of three anti‐T cell immunotoxins
Authors: Katz Fay E. , Janossy George , Cumber Alan , Ross Walter , Blacklock Hilary A. , Tax Wil , Thorpe Philip E.
Published: March 2008

Summary. Four anti‐T cell monoclonal antibodies were coupled to ricin‐A and tested for their ability to kill T cells in peripheral blood and bone marrow using a clonogenic assay to quantify T cell survival. The immunotoxins (IT) prepared from RFT11 (CD2) and WT1 (CD7) antibodies were the most toxic to peripheral blood T cells. The immunotoxin prepared from RFT1 (CD5) was the next most efficient toxin and the immunotoxin prepared from RFT8 (CD8) was the least toxic. When these reagents were applied to peripheral blood cells at 3×10–8 M the number of T cell colonies was reduced by an average of 95%, 94%, 84% and 50%, respectively. Peripheral blood T cells from different donors showed marked variability in their sensitivity to ITs. However, a cocktail of three ITs prepared from RFT11, WT1 and RFT1 gave superior and consistent killing (mean 99.9%; range 99.8–100%) of peripheral blood T cells from six donors. When this cocktail was applied to bone marrow cells from six donors, an average of 99.6% (range 99.5–99.8%) of the T cells were killed. Under the same conditions there was little or no reduction in the number of normal haematopoietic progenitors (CFU‐GM, CFU‐GEMM, CFU‐Meg and BFU‐E).
Reduced erythrocyte survival following clearance of malarial parasitaemia in Thai patients
Authors: Looareesuwan Sornchai , Merry A. H. , Phillips R. E. , Pleehachinda R. , Wattanagoon Yupaporn , Ho May , Charoenlarp Pricha , Warrell D. A. , Weatherall D. J.
Published: March 2008

Summary. Erythrocyte survival times were measured in healthy Thai controls and in patients following clearance of asexual P. falciparum or P. vivax parasitaemia. In five controls the mean cell life (MCL) of compatible donor erythrocytes was 89.6 d (mean range 73–101 d) compared with a mean MCL of 56.8 d (range 30–66 d) for autologous erythrocytes in 12 falciparum patients. In one of these patients the survival curve was biphasic with a rapid loss of some labelled cells. The survival of compatible donor erythrocytes was also studied in 10 patients and two types of survival curve could be distinguished. In five patients the cells had a mean MCL of 64.4 d (range 42–90 d). In the others survival curves were curvilinear, suggesting a complex mechanism of cell clearance or the presence of more than one cell population. There was initially a more rapid rate of destruction. In P. vivax malaria the MCL of autologous erythrocytes in seven patients was a mean of 67.2 d (range 34.74 d) and that of compatible donor cells in six patients was 66.8 d (range 54–76 d). In all except one of these patients both autologous and donor cell survival curves could be fitted to straight lines. No increase in cell‐bound IgG or C3 was evident in 12 patients tested. The differences between the mean MCL in all the groups of patients and the controls were statistically significant at the 5% level. This indicates an increased rate of erythrocyte destruction following clearance of P. falciparum or P. vivax parasites which is not antibody or complement mediated. The mechanism is unknown, but appears to be extrinsic to the erythrocytes themselves and may result from nonspecific activation of the reticuloendothelial function associated with the parasitic infection.
A simple index for initiating transfusion treatment in thalassaemia intermedia
Authors: Sbyrakis S. , Karagiorga‐Lagana M. , Voskaki I. , Efthimiou H. , Karaklis A.
Published: March 2008

Summary. In order to develop an objective test for discriminating between patients with thalassaemia intermedia requiring blood transfusion, and those not likely to require transfusion the medullary width (MW) in the midpoint of the second left metacarpal and the bone mass were measured in 34 normal children and in 37 patients. In patients, blood haemoglobin (Hb) and serum ferritin concentration were measured and cephalofacial deformities (CFD) were scored. The mean values of MW were 0.37±0.06 and 0.44±0.1 cm (P<0.01) and the bone mass 0.091 ±0.012 and 0.078±0.02 (P<0.005) in normal children and patients, respectively. In 13 of these patients who had MW more than 2 SD above the mean of the controls, i.e. more than 0.5 cm, regular blood transfusions were instituted. Measurements of MW 12 and 1 month before and 12 months after the initiation of transfusions showed an increase from 0.50 to 0.60 and a decrease to 0.49, respectively. Bone mass measured at the same times decreased from 0.083 to 0.045 and increased to 0.071, respectively. These changes were visible radiologically. It is concluded, therefore, that the measurement of MW seems to be an objective, simple test for discriminating between patients requiring or not blood transfusions, and that bone deformities will be reversible if transfusions are instituted using as criterion the MW (> 0.5 cm) regardless of age or haemoglobin concentration. This test may help clinicians to decide about the optimal time for institution of regular transfusions in patients with thalassaemia intermedia.
Plasma fibrinolysis during and after normal childbirth
Authors: Mackinnon S. , Walker I. D. , Davidson J. F. , Walker J. J.
Published: March 2008

A detailed sequential study of plasma fibrinolytic components in 42 healthy women during and after childbirth showed that striking changes occurred within minutes of placental separation. Significant increases in overall fibrinolytic activity were detected 1 5 min after delivery. During this period statistically significant increases in tissue plasminogen activator levels were noted but were overshadowed by a dramatic decrease in the level of plasminogen activator inhibitor (PAI). A 10‐fold fall in PAI was noted by days 3‐5 post‐partum, suggesting that the placenta is the major source of PAI detectable at the end of pregnancy and during labour. Persisting slightly elevated levels of PAI were detected for the first few days post partum. This may be due to increased production of normal‘endothelial’PAI in response to the vascular trauma incurred by placental separation, and in response to uterine vascular changes immediately post‐partum.
Characterization of the blast cell population in two neonates with Down's syndrome and transient myeloproliferative disorder
Authors: Coulombel L. , Derycke M. , Villeval J. L. , Leonard C. , Breton‐Gorius J. , Vial M. , Bourgeois P. , Tchernia G.
Published: March 2008

The phenotype and in vitro growth properties of blood and marrow blast cells detected in two neonates with Down's syndrome and a transient leukaemic picture are presented. In both patients, blast cells at diagnosis were heterogeneous and expressed predominantly megakaryocyte and erythroid markers identified by membrane fluorescence using monoclonal antibodies or ultrastructural detection of platelet peroxidase and ferritin. An additional trisomy involving chromosome 22 was detected in blast cells from one patient. Blood and marrow cells colony‐assays performed at diagnosis revealed precursors with an abnormal differentiation capacity similar to those found in acute myelogenous leukaemia colony assays. However, an unusual feature was the persistence of high numbers of precursor cells (namely erythroid) following a normal differentiation pathway. Phenotypically and cytogenetically abnormal cells spontaneously disappeared by week 4‐6, but overt relapse occurred in one patient 20 months later. These results bring strong arguments in favour of the neoplastic nature of the transient leukaemic picture observed in some neonates with Down's syndrome. Furthermore, we suggest that this disorder can be individualized as a separate entity with specific phenotypic and biological properties.
Ph‐negative chronic myeloid leukaemia
Authors: Fitzgerald P. H. , Beard M. E. J. , Morris C. M. , Heaton D. C. , Reeve A. E.
Published: March 2008

An analysis of five patients with Philadelphia chromosome (Ph) negative chronic myeloid leukaemia (CML) revealed that two were clinically and haematologically identical to Ph‐positive CML whereas three should be reclassified as chronic myelomonocytic leukaemia (CMML). At a molecular level the first two patients showed the same juxtaposition of c‐abl and bcr genes as is seen in Ph‐positive CML. These genomic changes were not seen in the other three patients. Observations on these five patients suggest that the clinical course and prognosis of the rare patient who carries the Ph ‘molecular defect’but lacks the Philadelphia chromosome is no different from Ph‐positive CML.
A new specific chromosomal rearrangement, t(8;16) (p11;p13), in acute monocytic leukaemia
Authors: Heim Sverre , Avanzi Gian‐Carlo , Billstroum Rolf , Kristoffersson Ulf , Mandahl Nils , Bekassy Albert N. , Garwicz Stanislaw , Wiebe Thomas , Pegoraro Loigi , Falda Michele , Resegotti Luigi , Mitelman Felix
Published: March 2008

The translocation t(8;16) (p11;p13) was found as the sole deviation from the normal karyotype in three patients with acute monocytic leukaemia. The bone marrow morphology was strikingly similar in the two cases where smears were available for re‐evaluation: the leukaemic cells showed signs of differentiation, and active erythrophagocytosis was a particularly conspicuous feature. We suggest that t(8;16) (p11;p13) represents a new consistent abnormality in acute monocytic leukaemia, specifically associated with the differentiated subtype (M5b) and with pronounced phagocytic activity by the leukaemic monocytes.
Allogeneic bone marrow transplantation for acute leukaemia: comparative outcomes for adults and children
Authors: Weisdorf Daniel J. , McGlave Philip B. , Ramsay Norma K. C. , Miller Wesley J. , Nesbit Mark E. , Woods William G. , Goldman Anne I. , Kim Tae H. , Kersey John H.
Published: March 2008

Advances in both allogeneic marrow transplantation and conventional therapy for acute leukaemia have complicated the choice between bone marrow transplantation (BMT) and other remission treatment options. Because older patients may be more susceptible to BMT‐related complications, this study analysed the effect of age on clinical outcome for 149 patients with acute leukaemia in remission receiving allogeneic BMT. Overall projected relapse‐free survival at 3 years post‐transplant is equivalent for 48 adults (18 years or older) and 101 children (less than 18) at 45.4% (31.1–59.6; 95% confidence interval) and 39.9% (30.1–49.7; 95% C.I.), respectively. Among 73 patients with acute lymphocytic leukaemia (ALL) 35.3% of adults and 30.1% of children survive relapse‐free at 3 years. Cox multiple regression analysis demonstrated that higher diagnostic white count, but not pre‐transplant extramedullary leukaemia, remission number, or age, was important as an independent adverse clinical prognostic factor for patients with ALL. Overall outcome was better for 76 patients with acute myeloid leukaemia (AML) with 51.6% of adults and 52.9% of children surviving relapse‐free at 3 years post‐transplant. Cox multivariate regression analysis identified first remission status and lower white cell count, but not patient age, as independent predictors of improved relapse‐free survival for AML patients. Adults had greater transplant morbidity, predominantly related to a higher incidence of acute graft‐versus‐host disease (GVHD), resulting in longer hospital stay. Survival at 100 d, long‐term survival and clinical performance status were similar in both age groups. These data suggest that results of allogeneic BMT for adults with acute leukaemia compare favourably with those found in children and are superior to most reports of conventional chemotherapy. Allogeneic BMT remains a reasonable option for remission acute leukaemia patients up to the age of 45.
Interleukin 2 stimulates the T‐cells from patients with eosinophilia to produce CFU‐Eo growth stimulating factor
Authors: Enokihara Hideo , Furusawa Shimpei , Kajitani Hiroshi , Hamaguchi Hiroyuki , Saito Kenji , Fukuda Takeshi , Shishido Hideo
Published: March 2008

To explore the mechanism of eosinophilopoiesis in patients with reactive eosinophilia, we studied the effect of interleukin 2 (IL‐2) on the production of CSFs, especially CFU‐eosinophil growth stimulating factor (CFU‐Eo GSF) from T‐lymphocytes in patients with reactive eosinophilia. Conditioned media (CM) prepared from patients’E rosette forming cells (ERFC) with or without IL‐2 was assayed for CFU‐Eo, CFU‐monocyte, macrophage (CFU‐M) and CFU‐neutrophil (CFU‐N) GSF. The addition of IL‐2 to the ERFC significantly stimulated the production of CFU‐Eo and CFU‐M GSF while only CFU‐M GSF increased in normals. Serial testing of the CFU‐Eo GSF in ERFC‐CM demonstrated that the ability of ERFC to produce CFU‐Eo GSF with IL‐2 stimulation was retained even when the eosinophilia had disappeared. These results suggest that CFU‐Eo GSF is produced from T‐cells with IL‐2 stimulation and that the T‐cells from patients produce CFU‐Eo GSF with IL‐2 stimulation after the disappearance of eosinophilia.
Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults
Authors: Planque Mariet M. de , Kluin‐Nelemans Hanneke C. , Krieken Han J. M. van , Kluin Philip M. , Brand Anneke , Beverstock Geoffrey C. , Willemze Roel , Rood Jon J. van
Published: March 2008

Myelodysplasia (MDS) and leukaemia following acquired aplastic anaemia has been reported as a rare event occurring in about 5% of patients. Improved results in survival of patients with severe aplastic anaemia (SAA) and subsequent prolonged follow‐up created the possibility of evaluating the occurrence of MDS and leukaemia in 38 adult patients with acquired SAA surviving two or more years without bone marrow transplantation. Five patients, age 22, 35, 47, 56, 72 years, two females, three males, all with idiopathic SAA and normal cytogenetic analysis developed a refractory anaemia (RA) 7, 30, 48, 56, 142 months after diagnosis of SAA. In 3/5 RA evolved into an acute myeloid leukaemia (AML) either via a chronic myelomonocytic leukaemia (CMML) (2/3) or via RA with excess of blasts (RAEB) (1/3). Three patients revealed a monosomy 7 during MDS and/or leukaemic phase. One patient died during RA phase without cytogenetic abnormalities. A pattern of evolution could be identified in these patients revealing well‐documented SAA ‐ improvement of bone marrow haematopoiesis ‐ dyshaematopoietic features of one or more cell lines with predominance of dyserythropoiesis ‐ RA ‐ RAEB or CMML ‐ AML. These five patients represent more than 10% of all patients surviving at least 2 years. This implies that the risk of developing MDS and leukaemia in SAA patients surviving with autologous marrow, might increase with longer follow‐up.
Carrier detection in 50 haemophilia A kindred by means of three intragenic and two extragenic restriction fragment length polymorphisms
Authors: Moodie P. , Liddell M. B. , Peake I. R. , Bloom A. L.
Published: March 2008

Carrier detection was attempted in 50 haemophilia A kindred by means of restriction fragment length polymorphism (RFLP) analysis using two linked and three intragenic probes. The sample comprised 330 individuals including 70 haemophiliacs, 45 obligate carriers and 100 possible carriers. Non‐related haplotypes contained within the sample group were used to tabulate the intragenic RFLP allele frequencies. The linkage disequilibrium existing between the XbaI and Bc 1I RFLP was shown to be less than previously reported, such that 69% of women are informative for at least one of these two polymorphisms. 36 women were subsequently diagnosed as carriers and 37 as being normal: in 52 of these, diagnosis was based either on intragenic RFLPs or linked RFLP plus strong phenotypic data, and was considered to be unequivocal; in the remaining 21, diagnosis was based on linked RFLPs alone and was considered to be probabilistic, with a 5‐10% possible error rate. 27 of the possible carriers, either from families with sporadic haemophilia or from families where missing members precluded haplotype analysis, remained unassigned. 72% of the obligate carriers and firmly diagnosed carriers were heterozygous and phase known for at least one intragenic RFLP, whereas 6% were uninformative for the RFLPs for which they were examined. In 54 informative meioses, three recombinations between the factor VIII locus and the DX13 and/or ST14 loci were observed, giving a recombination rale of 5.5%. 15 prenatal diagnoses were carried out. Of the 11 male fetuses, six were shown to be affected and five to be normal. In three of four prenatal diagnoses where only a linked RFLP was informative, the result was confirmed by fetoscopy, fetal blood sampling, and factor VIII assay.
Use of a hypervariable minisatellite DNA probe (33.15) for evaluating engraftment two or more years after bone marrow transplantation for aplastic anaemia
Authors: Weitzel Jeffrey N. , Hows Jill M. , Jeffreys Alec J. , Min Gao Ling , Goldman John M.
Published: March 2008

We have studied long‐term engraftment in 24 multiply transfused patients transplanted for severe aplastic anaemia (SAA) 2‐7 years previously from HLA identical sibling donors. All 24 patients had engrafted initially; nine (38%) developed grade II‐IV a‐GVHD, but only 5 (21%) developed chronic GVHD, which was mild, localized and transient. In 22 cases DNA ‘fingerprint’analysis using a hypervariable minisatellite DNA probe (33.15) confirmed the donor/recipient origin of patient peripheral blood (PB) nucleated cells. Red cell antigens and PB lymphocyte chromosomes were also analysed in informative cases. In 19 patients (79%) PB cells were of donor origin confirming sustained engraftment, whereas five (21%) had PB cells of recipient origin. In four of these five cases complete autologous reconstitution was demonstrated. In one case DNA fingerprinting revealed mixed haemopoietic chimaerism. In three of the four cases of autologous reconstitution there had been a previous episode of late graft failure. The low incidence of chronic GVHD in the study group was not explained by autologous reconstitution or mixed chimaerism. We conclude that the hypervariable minisatellite probes are valuable in the study of engraftment after BMT, especially when patient and donor are HLA identical, of the same sex, and have the same ABO‐Rh blood type. Pre‐transplant specimens from the patient are not necessary for interpretation of the results provided that DNA from the donor is available.
Interleukin 3 enhances the cytotoxic activity of 1‐β‐d‐arabinofuranosylcytosine (ara‐C) on acute myeloblastic leukaemia (AML) cells
Authors: Lista Patrizia , Porcu Pierluigi , Avanzi Gian Carlo , Pegoraro Luigi
Published: March 2008

We have evaluated the possibility of enhancing the cell killing effect of ara‐C on AML blasts by increasing their proliferative activity with haemopoietic growth factors. Leukaemic cells from 10 AML patients were incubated for 3 d in liquid culture in the presence or in the absence of the human recombinant growth factors IL‐1β (5 U/ml) and IL‐3 (3 U/ml), and subsequently exposed to ara‐C (3 μ/ml) for the last 24 h. The number of residual leukaemic stem cells was evaluated by a clonogenic assay in semisolid medium. The results showed that ara‐C exposure inhibits the proliferation of a higher proportion of clonogenic cells in cultures pretreated with growth factors than in the controls (mean inhibitory values: in the absence of growth factors = 49.8%: with IL‐1β=58.3%; with IL‐3 78.9%) The effect was statistically significant only when IL‐3 was used as a growth factor.

The results suggest that haemopoietic growth factors may help to improve the therapeutic index of cytostatic agents.
Effect of recombinant and purified human haematopoietic growth factors on in vitro colony formation by enriched populations of human megakaryocyte progenitor cells
Authors: Lu Li , Briddell Robert A. , Graham Cathy D. , Brandt John E. , Bruno Edward , Hoffman Ronald
Published: March 2008

Nonadherent low density T‐lymphocyte depleted (NALT‐) marrow cells from normal donors were sorted on a Coulter Epics 753 Dye Laser System using Texas Red labelled My 10 and phycoerythrin conjugated anti HLA‐DR monoclonal antibodies in order to obtain enriched populations of colony forming unit‐megakaryocyte (CFU‐MK). The CFU‐MK cloning efficiency (CE) was 1.1 ± 0.5% for cells expressing both high densities of My 10 and low densities of HLA‐DR (My 10+++DR+). This procedure resulted in an 18‐fold increase in CE over NALT‐ cells. The effect of purified or recombinant human haematopoietic growth factors including erythropoietin (Epo), thrombocytopoiesis stimulating factor (TSF), interleukin 1α (IL‐1α), granulocyte colony stimulating factor (G‐CSF), granulocyte‐macrophage colony stimulating factor (GM‐CSF), macrophage colony stimulating factor (M‐CSF or CSF‐1) and interleukin‐3 (IL‐3) on MK colony formation by My10+++DR+ cells was determined utilizing a serum depleted assay system. Neither Epo, TSF, CSF‐1, IL‐lα nor G‐CSF alone augmented MK colony formation above baseline (2.5 ± 0.8/5 × 103 My 10+++DR+ cells plated). In contrast, the addition of GM‐CSF and IL‐3 each increased both CFU‐MK colony formation and the size of colonies with maximal stimulation occurring following the addition of 200 units/ml of IL‐3 and 25 units/ml of GM‐CSF. At maximal concentration, IL‐3 had a greater ability to promote megakaryocyte colony formation than GM‐CSF. The stimulatory effects of GM‐CSF and IL‐3 were also additive in that the effects of a combination of the two factors approximated the sum of colony formation in the presence of each factor alone. The CFU‐MK appears, therefore, to express HPCA‐1 and HLA‐DR antigens. These studies also indicate that GM‐CSF and IL‐3 are important in vitro regulators of megakaryocytopoiesis, and that these growth factors are not dependent on the presence of large numbers of macrophages or T cells for their activity since the My 10+++DR+ cells are largely devoid of these accessory cells.
Terminal deoxynucleotidyl transferase and HLA‐DR expression appear unrelated to prognosis of acute myeloid leukaemia
Authors: Swirsky D. M. , Greaves M. F. , Gray R. G. , Rees J. K. H.
Published: March 2008

Mononuclear cells from peripheral blood or bone marrow from 314 patients with acute myeloid leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (304 patients), surface membrane expression of HLA‐DR (314 patients) and the common acute lymphoblastic leukaemia antigen (281 patients). All patients were treated with identical remission induction chemotherapy, and morphological diagnosis was carried out in a central laboratory. The overall complete remission rate was 70%. There were no significant correlations between the immunological markers and complete remission rate, duration of remission, or survival.
Histidine‐rich glycoprotein binding to activated human platelets
Authors: Lerch P. G. , Nydegger U. E. , Kuyas C. , Haeberli A.
Published: March 2008

Specific binding of purified histidine rich glycoprotein (HRGP) to human platelets stimulated with either bisdiazoniumbenzidine‐crosslinked immunoglobulin G (BDB‐IgG), with thrombin or with collagen was dose‐ and divalent cation dependent. A 5–10‐fold increase of platelet bound 125I‐HRGP was obtained when 0.5–0.8 × 109 platelets/ml were activated with 100 μg BDB‐IgG/ml, 0.1 U thrombin/ml or 15 μg collagen/ml. At maximal binding tested 16000 molecules of HRGP became bound per platelet, but saturation was not achieved. Such platelet inhibitors as acetylsalicylic acid, prostaglandin E1 and cytochalasin B reduced the capacity of platelets to bind ligand, and by kinetic experiments involving enzymatic digestion of radiolabelled bound HRGP the ligand revealed to remain surface bound rather than being taken up to inner parts of the cell.
Bone marrow lymphoid nodules in myeloproliferative disorders: association with the nonmyelosclerotic phases of idiopathic myelofibrosis and immunological significance
Authors: Cervantes Francisco , Pereira Arturo , Marti Josep‐Maria , Feliu Evaristo , Rozman Ciril
Published: March 2008

The presence of lymphoid nodules in bone marrow biopsy was investigated at diagnosis in 200 patients with chronic myeloproliferative disorders (MPD). Twelve out of 51 patients with idiopathic myelofibrosis (IM) showed such a feature (23.5%), versus two out of 100 with Ph1‐positive chronic myeloid leukaemia, two of 32 with polycythaemia vera, and one of 17 with essential thrombocythaemia, the difference between IM and the remaining MPD being statistically significant (P < 0.0001). When IM patients were compared for their initial characteristics according to the presence or not of bone marrow lymphoid nodules, patients with such a histological finding showed significantly lower values for either WBC counts, number of primitive cells in the blood, and serum lactic dehydrogenase levels. Moreover, it was observed that virtually all patients with lymphoid nodules were in the nonmyelosclerotic phases of IM. Finally, among the 14 of 32 IM patients (44%) investigated for circulating immune complexes who gave a positive test, a significant association between this immunological abnormality and bone marrow lymphoid nodules was found. The above results reinforce the immunological significance of the finding of bone marrow lymphoid nodules in IM and give support to the hypothesis of an immune component in the pathogenesis of the disorder.
Plasminogen activator inhibitor (PAI‐1) in plasma and platelets
Authors: Booth N. A. , Simpson A. J. , Croll A. , Bennett B. , MacGregor I. R.
Published: March 2008

The distribution of PAI‐1 in the plasma and platelets of normal individuals and of patients with platelet abnormalities was studied. An ELISA, capable of measuring PAI‐1 in plasma at 1.5 ng/ml, and a functional assay of t‐PA inhibition were used to assay platelet‐free plasma (PFP), platelet‐rich plasma in which the platelets were lysed (PRP) and serum. The PAI‐1 concentration of normal PFP was 21.0 ± 7.2 ng/ml (mean ± SD) and those of PRP and serum were 282.6 ± 68.0 and 270.3 ± 71.9 ng/ml. The concentration of PAI‐1 in PRP was proportional to the platelet count with 0.67 ± 0.18 ng/106 platelets. Patients with thrombocy‐topenia had approximately normal PAI‐1 concentrations in PFP; the extremely low concentrations in serum or PRP reflected the platelet count. A patient with grey platelet syndrome showed a comparable pattern, confirming that PAI‐1 occurs in the platelet α‐granules and indicating that the plasma concentration of PAI‐1 is independent of the platelet pool of PAI‐1. The median inhibitory activities towards t‐PA were 1.6, 8.7 and 8.3 units/ml in normal PFP. PRP and serum respectively. PAI‐1 in PFP had a median specific activity (units/mg PAI‐1) about 5‐fold higher than platelet PAI‐1. Plasma and platelets represent two distinct pools of PAI‐1, both of which should be considered in studies on the relationship between circulating PAI‐1 and thrombotic disease.
Molecular characterization of β‐globin gene mutations in patients with β‐thalassaemia intermedia in South China
Authors: Antonarakis Stylianos E. , Kang Julie , Lam V. M. S. , Tam Joseph W. O. , Li Anita M. C.
Published: March 2008

We have studied the spectrum of mutations producting β‐thalassaemia intermedia in South China. The methods of mutation detection include oligonucleotide analysis, polymerase chain reaction amplification of the β‐globin gene and direct genomic sequencing. The mutations have been identified in 22 β‐globin genes from the patients in 11 unrelated families. Seven different mutations have been identified and the A to G substitution in the TATA box of the β‐globin gene accounts for 42% of these mutant β‐globin genes. Most patients have a β+ thalassaemia and one copy of the TATA box mutation. In two patients with β) thalassaemia intermedia the mild phenotype may be explained in one by the presence of the ‐ + ‐ + + 5’β‐globin gene cluster haplotype which contains the Xmn I site ‐158 nt to the Gγ‐globin gene or in the other by the number of α‐globin genes present.
Replacement therapy for a homozygous protein C deficiency‐state using a concentrate of human protein C and S
Authors: Vukovich Thomas , Auberger Karin , Weil Jochen , Engelmann Hartmut , Knöbl Paul , Hadorn Hans Beat
Published: March 2008

A severe congenital deficiency of protein C was diagnosed in a 10‐month‐old girl who had been suffering from skin necrosis since the age of 7 months. The patient was treated initially with fresh frozen plasma, 10 ml per kg body weight, every 24 h. Following treatment, the mean plasma level of protein C was 0.1 U/ml after 30 min and less than 0.02 U/ml after 24 h. The child was then treated with a concentrate of human protein C and S, 100 U protein C per kg body weight, given every 48 h for a period of 9 months. The mean plasma level of protein C was 0.93 U/ml 30 min after administration of the concentrate and 0.13 and 0.08 U/ml after 24 and 48 h, respectively. The mean post‐transfusional in vivo recovery of protein C was 44% and the half life was 8.3 h. The mean plasma level of ‘free’protein S increased from 1.1 to 2.2 U/ml after administration of the concentrate. There was no increase in ‘bound’protein S. The in vivo recovery of ‘free’protein S was 49% and the half life was about 17 h. Since the start of this replacement therapy using a human protein C and S concentrate, the patient has not developed any thromboembolic complications. These results indicate the therapeutic value of human protein C and S concentrate in the treatment of severe protein C deficiency.
Rapid and non‐radioactive prenatal diagnosis of β thalassaemia and sickle cell disease: application of the polymerase chain reaction (PCR)
Authors: Kulozik Andreas E. , Lyons John , Kohne Elisabeth , Bartram Claus R. , Kleihauer Enno
Published: March 2008

The standard method for the prenatal diagnosis of the haemoglobinopathies is by restriction enzyme mapping of chorionic villus DNA using Southern blotting and radioactively labelled gene probes. An improvement of the procedure which involves the selective amplification of DNA fragments by the polymerase chain reaction allows one to visualize restriction fragments directly without the use of radioactivity and within 2 d after obtaining the sample.

We report here the prenatal diagnosis of two pregnancies at risk for homozygous β thalassaemia and homozygous sickle cell disease using this novel approach.
Characterization of a newly discovered α‐thalassaemia‐1 in two Spanish patients with Hb H disease
Authors: Gonzalez‐Redondo J. M. , Diaz‐Chico J. C. , Malcorra‐Azpiazu J. J. , Balda‐Aguirre M. I. , Huisman T. H. J.
Published: March 2008

A new deletion of more than 27 kb, removing the Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 globin genes has been found in four members of a Spanish family, including two patients with Hb H disease. The 5’end point of the deletion is located between the ζ and Ψζ genes, and the 3’end of the deletion is downstream of the 3’hypervariable region.
Characterization of a monoclonal antibody ITI‐Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes
Authors: Thorsen L. I. , Gaudernack G. , Hack N. , Wilkinson J. M. , Brosstad F. , Solum N. O. , Crawford N.
Published: March 2008

Summary. A monoclonal antibody (mAb) termed ITI‐Pl 1 has been prepared by the hybridoma procedure. Using immuno‐absorption and crossed immunoelectrophoresis of Triton X‐100 extracts of untreated and EDTA‐treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP‐stimulation and the presence of Ca2+‐ions. It saturates at around 870 ng/108 cells corresponding to approximately 35 800 molecules/platelet. ITI‐Pl 1 did not significantly inhibit GP IIb‐IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI‐Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno‐electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I‐labelled ITI‐Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb‐IIIa complex. The epitope on GP IIb detected by ITI‐Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase‐treated GP IIb‐IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb‐IIIa from untreated internal membranes isolated by continuous flow electrophoresis.