bluuebunny/crossref_metadata_2025 huggingface dataset:
Standardization of Factor VIII II. A BRITISH STANDARD FOR FACTOR VIII RELATED ANTIGEN
Authors: Kirkwood T. B. L. , Barrowcliffe T. W.
Published: July 2010

Summary.

A collaborative study on factor VIII related antigen (VIII R: Ag) has been carried out, involving 11 laboratories in the U.K. Samples of two different freeze‐dried plasmas were assayed against participants' own local standards by the Laurell electroimmunoassay method. There was reasonably good agreement on the relative potencies of the two freeze‐dried plasmas, but there were considerable differences in the VIII R: Ag content of the plasma pools used as local standards, with values ranging from 83% to 129% of the mean. All participants agreed on the need for a standard for VIII R: Ag, and that the unit be defined by the mean of the local standards. Accordingly, freeze‐dried plasma 66/355 was established as the 1st British Standard for factor VIII related antigen, with an assigned potency of 1.05 units per ampoule.
The British Comparative Thromboplastin: the Relationship between Lipid Class Composition and Procoagulant Activity
Authors: Stevenson K. J. , Slater P. J. , Puller L.
Published: July 2010

Summary. Twenty‐eight consecutive batches of the reference reagent British Comparative Thromboplastin (BCT) were produced in the National (UK) Reference Laboratory for Anticoagulant Reagents and Control (NRLARC) between 1969 and 1977. The relationship between procoagulant activity and lipid class composition in these batches at various stages of age deterioration on storage has been studied by a modification of the method of high pressure chromatography which allows better definition of the individual lipids. The free fatty acid concentration rose markedly while concentrations of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine were reduced. An oxidative process is suggested and supported by the increase in malonaldehyde levels. The method of production of the BCT involves maceration of human brain which causes the breakdown of cell membranes and the release of many potentially oxidative materials from subcellular particles. The loss of procoagulant activity of BCT on storage may be due to the loss of phospholipid or to the increase of inhibitory degradation products, i.e. free fatty acid and malonaldehyde. The examination of the lipid class composition of tissue thromboplastin extracts appears useful, therefore, not only in determining the phospholipids necessary for procoagulant activity, but also in monitoring the deterioration of tissue thromboplastins on storage.
Citrate Induced Hypocalcaemia during Cell Separation
Authors: Watson D. K. , Penny A. F. , Marshall R. W. , Robinson E. Angela E.
Published: July 2010

Summary. The value of calcium addition during cell separation by the Haemonetics Model 30 has been investigated in two patient groups. Where citrated plasma was used as the replacement fluid the addition of calcium abolished clinical symptoms and reduced the degree of citrate induced hypocalcaemia. When Plasma Protein Fraction was used as the replacement fluid, calcium addition was not necessary as clinical symptoms and significant hypocalcaemia did not occur.
Enzyme Linked Immunoassay for the Detection of Platelet Associated IgG
Authors: Hegde U. M. , Powell D. K. , Bowes A. , Gordon‐Smith E. C.
Published: July 2010

Summary. An enzyme linked immunoassay incorporating antihuman globulin coupled with alkaline phosphatase has been developed to measure platelet associated IgG (PAIgG). Using a method in which platelet IgG is extracted into the fluid phase after appropriate procedures, we were able to bind the ‘solubilized’ PAIgG to commercially obtained antihuman IgG (AHG) which had previously been coated onto polystyrene. The amount of PAIgG thus bound was subsequently measured by the addition of the enzyme reagent using p‐nitro phenyl phosphate as substrate. With this technique platelets from normal donors were found to have 2·6–17·4 ng/106 platelets (mean ± 2 SD). These values are higher than those obtained when assay systems using intact platelets are employed. Platelets from patients with immune thrombocytopenia had PAIgG values of 8·2–98·0 ng/106 platelets. In a few patients with disorders other than autoimmune thrombocytopenia (AITP) increased levels of PAIgG were also demonstrated. The assumption that increased PAIgG always represents platelet autoantibody may not be valid. The relevance of PAIgG as a parameter in the diagnosis and clinical management of patients with AITP is discussed.
Waldenström‐like Lymphoma with Monoclonal IgG1kappa: Evidence of Common Clonal Origin
Authors: Waele Marc De , Coulie Roland , Camp Benjamin Van
Published: July 2010

Summary. A patient with a pleomorphic lymphoplasmacytic bone marrow infiltration, a peripheral lymphocytosis and a monoclonal IgG1‐κ gammopathy is described. An antiidiotype serum, raised against the monoclonal serum protein showed a common clonal origin of the pleomorphic cell population. In the light of recent reports on B‐cell neoplasms associated with M‐components, a hypothetical scheme of the cellular clonal event is proposed.
Surface IgMκ of Prolymphocytic Leukaemia Cells with Antibody Activity to Surface Antigens of Sheep and Guinea‐Pig Red Blood Cells
Authors: Bergmann Lothar , Mitrou Paris S. , Rogge Helmut
Published: July 2010

Summary. The immunological findings of a B‐cell prolymphocytic leukaemia forming spontaneous rosettes with sheep (SRBC) and guinea‐pig red blood cells (GPRBC) are reported. The B‐cell lineage was revealed by Ia‐like antigen, surface IgMK and lack of cytotoxicity of anti‐human T‐cell serum. Despite B‐cell characteristics, prolymphocytes formed spontaneous rosettes with SRBC. Inhibition procedures demonstrated rosette formation to be the result of antibody activity of surface IGM to SRBC and GPRBC. Pretreatment of prolymphocytes with pronase or antibodies to μ or K‐chains inhibited rosette formation. Furthermore, pre‐treatment of SRBC with the patient's serum or IgM decreased rosette formation demonstrating circulating antibodies to antigen(s) of SRBC. Antigen(s) on SRBC and GPRBC are not related to Forssman or Paul Bunnel antigen.
Simultaneous or Spontaneous Occurrence of Lympho‐ and Myeloproliferative Disorders: a Report of Four Cases
Authors: Manoharan A. , Catovsky D. , Clein P. , Traub N. E. , Costello C. , O'Brien M. , Boralessa H. , Galton D. A. G.
Published: July 2010

Summary. We describe four patients with mixed lympho‐ and myeloproliferative disorders. One patient had hairy cell leukaemia and acute myelomonocytic leukaemia, another lymphocytic lymphoma in leukaemic phase and chronic myelomonocytic leukaemia and the third patient had chronic lymphocytic leukaemia and polycythaemia rubra vera; none of these patients had received any prior therapy, and in two the diagnosis of the two malignancies was simultaneous. The fourth patient developed acute monocytic leukaemia 4 years after the diagnosis of chronic lymphocytic leukaemia after only 2 weeks of therapy with chlorambucil. The number of cases with concurrent or sequential but spontaneous occurrence of lympho‐ and myeloproliferative disorders reported so far is now 38. The questions relating to the pathogenesis of the two malignancies are discussed.
DNA Synthesis in Thymic‐Acute Lymphoblastic Leukaemia
Authors: Piga A. , Ganeshaguru K. , Lee N. , Breatnach F. , Prentice H. G. , Hoffbrand A. V.
Published: July 2010

Summary.

Deoxyribonucleoside triphosphate (dNTP) concentrations were measured in bone marrow and peripheral blood leucocytes from seven patients with acute Thy‐lymphoblastic leukaemia (Thy‐ALL), 12 patients with acute myeloblastic leukaemia and 15 patients with acute non‐T, non‐B lymphoblastic leukaemia (c‐ALL), and in thymocytes from patients with myasthenia gravis. Labelled thymidine and deoxycytidine incorporation into DNA was also studied.

In Thy‐ALL, dNTP concentrations were markedly increased compared with those in the other acute leukaemias. The dNTP concentrations in thymocytes were, however, similar to those in Thy‐ALL. 3H‐nucleoside incorporation studies showed a marked difference in labelled deoxycytidine incorporation and particularly in the deoxycytidine/thymidine DNA labelling ratio between Thy‐ALL and the other cell types.

We conclude that the pathways of DNA synthesis in Thy‐ALL blasts are different from those in the cells from other acute leukaemias and some but not all these differences may correspond to differences between normal cortical thymocytes and bone marrow cells.
Membrane‐active agents and heat‐induced erythrocyte fragmentation
Authors: Zarkowsky Harold S.
Published: July 2010

Morphological observations of heated erythrocytes were made after preincubation with lysolecithin, diamide, p‐chloromercuribenzoate, or N‐ethyl maleimide and after ATP‐depletion. Diamide (1.0 mM) reduced the critical temperature of fragmentation from 49°C to 47°C, and at higher concentrations the critical temperature was further reduced. The other sulphydryl reacting agents had little or no effect. Drug‐induced spherocytes and ATP‐depleted cells did not fragment. Membrane‐active agents can effect the response of membranes to heat and the effect is dose‐dependent.
Comparison of purine degradative enzymes and terminal deoxynucleotidyl transferase in T cell leukaemias and in normal thymic and post‐thymic T cells
Authors: Ma D. D. F. , Massaia M. , Sylwestrowicz T. A. , Price G. , Hoffbrand A. V.
Published: July 2010

Summary. Adenosine deaminase (ADA), ecto 5’ nucleotidase (5′NT), purine nucleoside phosphorylase (PNP) and terminal deoxynucleotidyl transferase (TdT) were measured in the cells of patients with acute or chronic T cell leukaemia and compared with normal putative prothymocytes (large, blast‐like cortical thymocytes), cortical and medullary thymocytes and peripheral blood T lymphocytes. Distinct patterns of enzyme activities were found in the individual types of T cell leukaemia. Mean ADA, TdT and 5′NT activities in thymic acute lymphoblastic leukaemia (Thy‐ALL) were 41·9 u/108 cells, 31·1 u/108 cells and 4·7 u/106 cells respectively; in chronic T cell leukaemia they were 7·1 u/108 cells, 0·6 u/108 cells and 18·1 u/106 cells respectively. Mean PNP activity was similar between these two groups of leukaemia (68·6 u/106 cells in Thy‐ALL and 77·9 u/106 cells in chronic T cell leukaemia). The activities of these four enzymes in OKT4+ chronic T cell leukaemia did not differ significantly from those in the OKT8+ chronic T cell leukaemia cases. The activities of TdT, ADA, PNP and 5′NT in Thy‐ALL closely resembled those in normal immature thymocytes, and in the chronic T cell leukaemias showed a similar pattern of enzyme activities to that of mature T lymphocytes. These findings are consistent with surface phenotypic studies of T cell malignancies which suggest that different T cell leukaemias represent malignant proliferation of T cell clones arrested at different stages of T cell differentiation. They also demonstrate the value of biochemical markers in defining the different subtypes of acute and chronic leukaemia.
Von Willebrand factor multimer patterns in von Willebrand's disease
Authors: Hoyer Leon W. , Rizza Charles R. , Tuddenham Edward G. D. , Carta Carl A. , Armitage Helen , Rotblat Francis
Published: July 2010

Summary. The von Willebrand factor antigen (factor VIII‐related antigen, VIIIR:Ag) multimer pattern has been analysed by SDS‐agarose electrophoresis of plasmas from 116 patients (47 families) with von Willebrand's disease. In addition to previously recognized patterns, a subclassification was established between plasmas that had a type Ia pattern (VIIIR:Ag multimer pattern like that of normal plasma) and those that had a type Ib pattern in which there was a relative reduction in the concentration of the larger VIIIR:Ag multimers even though all multimeric forms were present. The different patterns were consistent within families and were inherited by autosomal dominant transmission. Von Willebrand's disease heterogeneity was apparent in the distribution of these plasmas: type Ia, 43 patients in 18 families; type Ib, 39 patients in 15 families; type II, 22 patients in 10 families, one of which was further classified as type IIB, one of which was type IIC, and three were IIA. Seven patients with severe von Willebrand's disease were also studied. In general, the interpretation of SDS‐agarose multimer patterns corresponded to those previously obtained by crossed immunoelectrophoresis, but the former technique was more sensitive and could identify differences that were not apparent by crossed immunoelectrophoresis.
Red cell alloimmunization in sickle cell disease
Authors: Davies Sally C. , Mcwilliam A. C. , Hewitt Patricia E. , Devenish A. , Brozovic Milica
Published: July 2010

Summary. Alloimmunization to red cell antigens contributes to morbidity in transfused patients. It has been recommended that blood for sickle cell patients need not be matched for antigens other than ABO and Rh(D), as there is no greater incidence of antibody production than in other multitransfused patient populations. Post transfusion alloimmunization was studied in a group of 34 sickle cell disease patients attending a U.K. haemoglobinopathy clinic. Red cell antibodies were formed in 17‐6% of the transfused patients and Rhesus and Kell antibodies accounted for 66% of this total. In order to reduce alloimmunization, a policy of performing extended red cell phenotyping on the patients, and providing blood matched for Kell, and in certain circumstances the Rhesus antigens other than Rh(D), is recommened.
HTLV‐III antibody and T‐cell subset ratios in haemophiliacs and their spouses
Authors: Mcverry B. A. , Machin S. J. , Galloway M. J. , Mccarthy K. , Evans P. , Barnes R. M. R. , Turner G. C. , CHEINGSONG‐POPOV R. , Tedder R. S. , Winter M.
Published: July 2010

Summary. 44% of 63 British patients with either haemophilia A or B were HTLV‐III antibody positive (HTLV‐VIII +). HTLV‐III+ was more frequent in high factor VIII concentrate users and 75% of severely affected haemophilia A patients were HTLV‐III+. All eight patients who were exposed to factor IX concentrate were HTLV‐III+. 17 haemophilia A patients who received only British made factor VIII concentrate (average 12 000 units/year) were HTLV‐III−. Two of 63 patients had evidence of a pre‐AIDS type symptom complex and both were HTLV‐III+. Information from a cohort of 21 Liverpool haemophiliacs suggests that HTLV‐III was first introduced into this country in 19 81. OKT4/T8 ratios were abnormal in 52% of 21 patients studied but this finding was not confined to either HTLV‐III+ or HTLV‐III− individuals. The spouses of 14 HTLV‐III+ haemophiliacs were all HTLV‐III−.
Familial elevation of plasma histidine‐rich glycoprotein in a family with thrombophilia
Authors: Engesser L. , Kluft C. , Briët E. , Brommer E. J. P.
Published: July 2010

A patient with spontaneous venous thrombotic events, myocardial infarction and a positive family history of thrombosis was investigated for underlying disorders of haemostasis. Abnormally high levels of histidine‐rich glycoprotein were found. No other abnormality known or suspected of increasing the risk for thrombosis was present.

Elevated levels of histidine‐rich glycoprotein appeared to be present in four other members of the family over two generations suggesting a hereditary disorder. Further study on the relation of elevated histidine‐rich glycoprotein levels and thrombosis is indicated.
The 12.6 kilobase DNA deletion in Dutch β°‐thalassaemia
Authors: Gilman John G.
Published: July 2010

The Dutch β°‐thalassaemia has few clinical symptoms in homozygotes, elevated fetal haemoglobin (4–11%) in heterozygotes, and has a DNA deletion previously estimated as 10 kb which removes the β‐globin gene (Gilman et al, 1984). A DNA fragment containing the breakpoints of the Dutch β°‐thalassaemia deletion has now been cloned. Sequencing across the deletion junction region showed the 3’endpoint to be about 3 kb further 3’than originally thought, so that the deletion covers 12.6 kb. The 3’endpoint lies in a region of Kpn I (LI) repeated sequences, which is also the case for several other deletions. A six bp region of homology (AAATTT) between the 5’and 3’normal sequences at the breakpoint may have contributed to the non‐homologous recombination event that led to the Dutch β°‐thalassaemia deletion. The 12.6 kb Dutch β°‐thalassaemia deletion is now seen to be a member of a 12–13 kb size category of deletions which also includes two δβ‐thalassaemias.
Lymphoid cells and tissue mast cells of bone marrow lesions in systemic mastocytosis: a histological and immunohistological study
Authors: Horny Hans‐Peter , Kaiserling Edwin
Published: July 2010

Systemic mastocytosis (SM) can be regarded as a tumorous proliferation of tissue mast cells (TMC) involving various organs, particularly the bone marrow. The infiltrates, however, are by no means composed exclusively of TMC, but also contain eosinophils and lymphocytes. The varying composition of the TMC infiltrates and the immunohistological characteristics of the lymphatic cells were the main subjects of investigation in this study.

Three different types of bone marrow infiltrates could be identified: (1) A pure mastocytic infiltrate. (2) A mixed mastocytic/lymphocytic infiltrate. (3) A predominantly lymphocytic infiltrate containing loosely‐scattered TMC.

The mixed mastocytic/lymphocytic infiltrate seems to be a unique finding confined to the bone marrow in cases of SM, and is not detected in this conformation in other tissue sites normally involved in SM (spleen, liver, lymph nodes and skin), nor in cases of malignant mastocytosis. The lymphoid cells could be identified immunohistologically as being an admixture of T lymphocytes and B lymphocytes, while NK cells were virtually absent from the composite nodules. The TMC reacted strongly with antibodies (monoclonal or polyclonal) against vimentin, common leucocyte antigen, lysozyme, α1antichymotrypsin and α1‐antitrypsin, but were negative with a variety of other antibodies tested (UCHL1, MB1, Ki‐B3, Leu‐7, KL1, desmin, S‐100 protein, F VIII‐related antigen and chromogranin A).
Spontaneous programmed death (apoptosis) of B‐chronic lymphocytic leukaemia cells following their culture in vitro
Authors: Collins Russell J. , Verschuer Louise A. , Harmon Brian V. , Prentice Roger L. , Pope John H. , Kerr John F. R.
Published: July 2010

Summary When B‐cell lymphocytic leukaemia (B‐CLL) cells derived from peripheral blood were cultured in vitro, a substantial proportion of them spontaneously died by apoptosis. This type of cell death is morphologically and biochemically distinct from necrosis and has previously been found to occur under physiologic and certain pathologic conditions where cell deletion appears controlled and biologically meaningful. By 30 h of culture, approximately 20% of the unfractionated B‐CLL cells were affected. There was no significant difference in the incidence of apoptosis in T‐cell depleted and undepleted cultures or when either autologous or normal human serum was used. Furthermore, seeding densities of 2 × 106 and 5 × 105 cells/ml resulted in a similar incidence of apoptosis, indicating that cell density was unlikely to be a contributing factor in producing the death. The finding that B‐CLL cells spontaneously die in vitro has at least two important implications. Firstly, previous work relating to some of the functions of B‐CLL cells and their interactions with T cells may require re‐evaluation. Secondly, an understanding of the mechanisms involved in the induction of apoptosis in this disease may have therapeutic consequences.
A base substitution (T→C) in codon 29 of the α2‐globin gene causes α thalassaemia
Authors: Hall Georgina W. , Thein Swee Lay , Newland Adrian C. , Chisholm Morag , Traeger‐Synodinos Jan , Kanavakis E. , Kattamis C. , Higgs Douglas R.
Published: July 2010

Summary. We have identified three individuals of Greek or Greek Cypriot origin with an atypical form of HbH disease characterized by a severe hypochromic microcytic anaemia associated with relatively small amounts of HbH in the peripheral blood. Molecular analysis has shown that each is a compound heterozygote for a previously described mutation affecting the poly A addition signal (AATAAA→ÁTAAG) and a previously undescribed mutation involving a T→C transition in codon 29 of the α2 gene causing a leucine→pro‐line substitution. Although this mutation would be expected to produce an unstable haemoglobin and hence a haemolytic anaemia, simple heterozygotes for the α29Leu→Pro mutation have the phenotype of α‐thalassaemia trait.
Cytomegalovirus infection in leucocytes after bone marrow transplantation demonstrated by mRNA in situ hybridization
Authors: Link Hartmut , Battmer Karin , Stumme Carsten
Published: July 2010

Summary. Cytomegalovirus (CMV) infection in leucocytes after bone marrow transplantation (BMT) was identified using a 35S‐labelled antisense RNA probe specific for CMV immediate early (IE) gene mRNA. 54 patients were examined regularly up to 14 weeks after BMT, 36 after allogeneic BMT and 18 after autologous BMT. Only mononuclear cells with monocyte morphology were CMV IE mRNA positive. The number of CMV positive leucocytes was higher after allogeneic BMT than after autologous BMT (P=0.006), and in patients with acute graft‐versus‐host disease (GvHD) II‐IV than with GvHD 0‐I (P=0.06). In patients who later developed chronic GvHD, the mean value of CMV infected leucocytes during the first week after BMT was higher than in patients without chronic GvHD (P=0.034). Patients with symptomatic CMV infection had greater numbers of CMV infected leucocytes during the fourth and fifth week after BMT than patients without CMV disease, but the difference did not reach statistical significance.

CMV infection of monocytes may be an important factor in the early onset of CMV infection and of GvHD.
High‐dose vincristine, fractionated total‐body irradiation and cyclophosphamide as conditioning regimen in allogeneic and autologous bone marrow transplantation for childhood acute lymphoblastic leukaemia in second remission: a 7‐year Italian multicentre study
Authors: Uderzo Cornelio , Rondelli Roberto , Dini Giorgio , Dallorso Sandro , Messina Chiara , Miniero Roberto , Locatelli Franco , Manzini Andrea De , Pession Andrea , Balduzzi Adriana , Locasciulli Anna , Masera Giuseppe
Published: July 2010

We investigated the feasibility and efficacy of high‐dose vincristine (4 mg/m2 over 4 d) combined with fractionated total body irradiation (F‐TBI) (200 cGyx2 over 3 d) and cyclophosphamide (60 mg/kg for 2 d) as a preparative regimen in allogeneic (AlloBMT) and autologous (ABMT) bone marrow transplantation for 75 consecutive children (median age at transplant 8‐5 years) with acute lymphoblastic leukaemia in second complete remission (CR). Median duration of first CR was 26 and 25 months in the AlloBMT and ABMT group, respectively. Of the 46 patients who underwent AlloBMT, 33 had isolated or combined marrow relapse and 13 isolated extramedullary relapse. Of the 29 patients given ABMT, 23 had preBMT isolated or combined marrow relapse and six isolated extramedullary relapse. 44/75 patients are alive and in CR at a median follow‐up of 35 months (range 10‐90 months). Seven children given AlloBMT (15.8%) and two given ABMT (7%) died from transplant‐related causes. No major early organ toxicity, including vincristine‐related toxicity, was recorded. The overall 3‐year EFS estimate (95% CL) was 53.8% (42‐66%): in particular, 58.2% (40‐76%) for AlloBMT and 27.6% (9‐46%) for ABMT patients who experienced a marrow relapse before transplant. The overall 3‐year relapse rate estimate (95% CL) was 39.2% (27‐51%): in particular, 30.1% (12‐49%) in the AlloBMT group and 72% (54‐91%) in the ABMT group (P < 0.01) who presented a preBMT isolated or combined marrow relapse. We conclude that the conditioning regimen with high‐dose vincristine combined with cyclophosphamide and F‐TBI is feasible and promising, although its therapeutic advantage should be tested in larger series of patients enrolled in randomized studies.
Platelet activation induced by a murine monoclonal antibody directed against a novel tetra‐span antigen
Authors: Roberts J. J. , Rodgers S. E. , Drury J. , Ashman L. K. , Lloyd J. V.
Published: July 2010

MAb 14A2.H1 identifies a novel low‐abundance platelet surface antigen, PETA‐3, which is a member of the tetra‐span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E1, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, FcγRII, and the specific antigen as it was prevented by either a blocking MAb to FcγRII (IV.3) or F(ab')2fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of FcγRII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb rv.3, or F(ab')2fragments of 14A2.H1. The similar low copy number of PETA‐3 and FcγRII in the platelet membrane (approximately 1000/platelet). together with the dependence on FcγRII for activation by MAb 14A2.H1, suggests that PETA‐3 may be a component of the FcγRII signal transducing complex in platelets.
Soluble c‐kit levels in acute GVHD after allogeneic bone marrow transplantation
Authors: Hashino Santoshi , Imamura Masahiro , Kobayashi Sumiko , Tanaka Junji , Kasai Masaharu , Sakurada Keisuke , Asaka Masahiro
Published: July 2010

Serum soluble c‐kit concentrations were measured in 11 patients with or without acute graft‐versus‐host disease (GVHD) after allogeneic bone marrow transplantation. The serum soluble c‐kit levels in the patients with moderate to severe acute GVHD (grade II‐IV) were significantly lower than those in the patients with no or mild acute GVHD (grade 0‐I) following the onset. The data from this study indicate that measurement of serum soluble c‐kit concentration is a useful indicator of severe acute GVHD.
Alpha‐interferon therapy for congenital dyserythropoiesis type I
Authors: Lavabre‐Bertrand Thierry , Blanc Pierre , Navarro Robert , Saghroun Mezziana , Vannereau Henri , Braun Michel , Wagner André , Taiub Jacques , Lavabre‐Bertrand Christine , Navarro Maurice
Published: July 2010

We report the case of a 28‐year‐old female followed for congenital dyserythropoiesis type I which required repeated transfusions. Alpha‐2a interferon treatment was started because of post‐transfusion chronic viral hepatitis type C. Following this treatment, haemoglobin level increased and reached normal value during the 24 weeks of interferon treatment. When interferon therapy was stopped, haemoglobin level returned to previous values, requiring more transfusions. Resumption of interferon therapy resulted again in a complete normalization of haemoglobin level. Erythrokinetic studies demonstrated a striking reduction of the ineffective erythropolesis, and electron microscopy study a reduction in nuclear structure abnormalities. To our knowledge, this is the first report of the efficacy of interferon in congenital dyserythropoiesis.
A novel (288delC) mutation in exon 2 of GPIIb associated with type I Glanzmann's thrombasthenia
Authors: Tao Jianming , Arias‐Salgado Elena García , González‐Manchón Consuelo , Díaz‐Cremades Juan , Ayuso Matilde S. , Parrilla Roberto
Published: July 2010

This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb–IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site, causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb–IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb–IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co‐transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb–IIIa requires the integrity of exons 2 and 3 of GPIIb.
Stroma‐supported progenitor production as a prognostic tool for graft failure following autologous stem cell transplantation
Authors: Van Hennik Paula B. , Breems Dimitri A. , Kusadasi Nuray , Slaper‐Cortenbach Ineke C. M. , Van Den Berg Henk , Van Der Lelie Hans J. , Schipperus Martin R. , Cornelissen JaN. J. , Ploemacher Rob E.
Published: July 2010

To analyse the involvement of a possible numerical or qualitative stem cell defect in the development of sustained graft failure after autologous transplantation, we have determined the graft content of CD34+ nucleated cells, colony‐forming cells and cobblestone area‐forming cell subsets, as well as transplant ability to produce progenitors using the long‐term culture colony‐forming cell (LTC‐CFC) assay. We evaluated material from the graft reference ampoules of 13 graft failure patients after bone marrow transplantation (BMT), four graft failure patients and four isolated thrombocytopenia patients after peripheral blood stem cell transplantation (PBSCT). We compared these data with those from six successfully engrafted BMT patients and 20 engrafted PBSCT patients respectively. In the BMT setting, the LTC‐CFC 6‐week assay represented a highly significant graft failure predictor. In the PBSCT setting, the total number of 2‐week and 6‐week LTC‐CFCs transplanted per kg bodyweight (BW) showed the highest significant difference between the engrafted and the graft failure patients, as well as between the engrafted patients and the patients suffering from isolated thrombocytopenia after transplantation. These data show that the ability of a graft to generate progenitors in vitro rather than the number of primitive progenitors transplanted can have prognostic value for post‐transplant haematological reconstitution.
The 8p12 myeloproliferative disorder. t(8;19)(p12;q13.3): a novel translocation involving the FGFR1 gene
Authors: Mugneret Francine , Chaffanet Max , Maynadié Marc , Guasch Géraldine , Favre Bernardine , Casasnovas Olivier , Birnbaum Daniel , Pébusque Marie‐Josèphe
Published: July 2010

Translocations affecting the chromosomal locus 8p12 are hallmarks of an atypical stem cell myeloproliferative disorder. These events disrupt the fibroblast growth factor receptor 1 (FGFR1) gene and fuse the FGFR1 C‐terminus catalytic domain with unrelated proteins. Here, we report on the characterization of the 19q13.3 locus as the fifth FGFR1 chromosomal partner.
Effective lysis of model thrombi by a t‐PA mutant (A473S) that is resistant to α2‐antiplasmin
Authors: Robbie L. A. , Bennett B. , Keyt B. A. , Booth N. A.
Published: July 2010

This study used two mutants of tissue‐type plasminogen activator (t‐PA) with resistance to inhibitors of fibrinolysis to define the contribution of plasminogen activator inhibitor (PAI)‐1 and α2‐antiplasmin (α2‐AP) to the control of fibrin lysis. Wild‐type t‐PA was compared with KHRR296–299AAAA, which is resistant to PAI‐1, and with A473S, which is resistant to α2‐AP. We examined these forms of t‐PA in model systems that are physiologically relevant. Neutralization of α2‐AP was essential for lysis of plasma clots, irrespective of their platelet content, by either wild‐type t‐PA or KHRR296–299AAAA. In marked contrast, A473S lysed plasma clots without neutralization of α2‐AP. Model thrombi, with structures similar to in vivo thrombi, were lysed slowly by wild‐type t‐PA; the rate and extent of lysis were enhanced by the addition of antibodies to α2‐AP or PAI‐1. A473S was more effective than wild‐type t‐PA without the addition of antibodies by virtue of its resistance to α2‐AP. This resistance was remarkable, in that no complex formed between A473S t‐PA and α2‐AP, even after extended incubation, when 50% of wild‐type t‐PA could be converted to complex. Comparison of A473S and KHRR296–299AAAA mutants showed their similar effectiveness in lysis of platelet‐rich model thrombi. Thus, PAI‐1 and α2‐AP contribute approximately equally to the inhibition of thrombus lysis. This study underlines the functional significance of α2‐AP as a direct inhibitor of t‐PA and further explains the basis of the accepted role of α2‐AP as a regulator of fibrin persistence and thrombus resistance to lysis.
Differential cellular expression of the human MSH2 protein in normal and myelodysplastic haematopoiesis
Authors: Maeck Lienhard , Kohaus Petra , Haase Detlef , Hiddemann Wolfgang , Alves Frauke
Published: July 2010

Loss of human MSH2 (hMSH2) protein might be involved in the multistep pathogenesis of haematological malignancies associated with genetic instability. Here, we examine cellular hMSH2 expression in bone marrow samples from 10 haematopoietically normal individuals in comparison with nine patients with myelodysplastic syndrome (MDS) [one refractory anaemia (RA), two RA with ringed sideroblasts (RARS), four RA with excess blasts (RAEB)  and two RAEB in transformation (RAEB‐T)]. HMSH2 protein was predominantly expressed in myeloblasts and promyelocytes. Blast cells from three patients with RAEB and one with RAEB‐T displayed absent or very low hMSH2 expression. As no correlation between hMSH2 expression and chromosomal aberrations was observed, further genetic events seem to be required to induce karyotype instability.
The relationship between idiopathic thrombocytopenic purpura and pernicious anaemia
Authors: Juncà Jordi , Flores Alonso , Granada Maria Luisa , Jiménez Orlando , Sancho Juan M.
Published: July 2010

Some reports on the simultaneous presence of chronic idiopathic thrombocytopenic purpura (ITP) and pernicious anaemia (PA) may be found in the literature. However, little is known about the coexistence of these autoimmune disorders. For this reason, we studied the prevalence of PA in a series of patients with a diagnosis of chronic ITP by means of the analysis of the concentration of the most sensitive marker of type A atrophic (autoimmune) gastritis, serum pepsinogen I. Serum pepsinogen I was low in 20·3% of the 133 patients studied. Gastrin was elevated in 15·2% of patients, but the coexistence of both abnormalities was rather low (7·6% of patients). However, the progressive decrease in serum cobalamin as biochemical abnormalities related with atrophic gastritis appeared was noticeable. The time to progression to frank PA from type A atrophic gastritis may span some years.
Factor IX gene sequencing by a simple and sensitive 15‐hour procedure for haemophilia B diagnosis: identification of two novel mutations
Authors: Vidal Francisco , Farssac Elisenda , Altisent Carme , Puig Lluís , Gallardo Dominique
Published: July 2010

We have developed a simple, sensitive and cost‐effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA. Using this approach, we have found mutations in 10 out of 10 haemophilia B patients, two of which are novel (P287T and S123C). Very suitable for individual studies, this procedure can be easily scaled up for higher throughput.
Expression of tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) receptors and sensitivity to TRAIL‐induced apoptosis in primary B‐cell acute lymphoblastic leukaemia cells
Authors: Clodi Katharina , Wimmer Doris , Li Yang , Goodwin Raymond , Jaeger Ulrich , Mann Georg , Gadner Helmut , Younes Anas
Published: July 2010

Because tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) (Apo2 ligand) preferentially kills malignant cells while sparing normal cells, it may be therapeutically useful against cancers, including those of haematopoietic origin. Although the activity of TRAIL has been studied in tumour cell lines and in a limited number of different primary tumours, its overall activity in a large number of uniform cases of primary tumours is not known. We therefore studied the activity of TRAIL in 29 primary precursor B‐cell acute lymphoblastic leukaemia (ALL) samples. TRAIL was found to have a modest activity as it killed a maximum of 29% of ALL cells within 18 h compared with killing 75% of Jurkat cells. The sensitivity to TRAIL did not correlate with the pattern of TRAIL receptor expression or FLIP expression, as determined by Western blot analysis. The CD40 receptor, which can transduce survival signals in mature malignant B cells, was less frequently expressed on ALL cells, but incubation with an exogenous soluble CD40 ligand trimer did not rescue them from spontaneous apoptosis and did not mediate their resistance to TRAIL. Further, although ALL cells expressed TRAIL protein, they failed to kill target Jurkat cells in a TRAIL‐dependent manner. Our data delineate major biological differences between mature and precursor malignant B cells and suggest a limited therapeutic role for TRAIL as a single agent in primary B‐cell ALL.
Vincristine‐induced apoptosis in vivo in peripheral blood mononuclear cells of children with acute lymphoblastic leukaemia (ALL)
Authors: Groninger E. , de Graaf S. S. N. , Meeuwsen‐de Boer G. J. , Sluiter W. J. , Poppema S.
Published: July 2010

We conducted a study to demonstrate vincristine‐induced apoptosis in vivo in peripheral blood mononuclear cells of children with newly diagnosed acute lymphoblastic leukaemia (ALL). In five children, apoptosis was detected by terminal deoxynucleotide transferase‐mediated dUTP‐digoxigenin nick‐end labelling (TUNEL) assay in blood samples collected during an up‐front window study of vincristine monotherapy. In one patient, we found a striking increase of apoptotic cells from 2% (SEM 0·6) before to 40·7% (SEM 2·9) 3 h after vincristine injection. In the other patients, the maximum increase ranged from 0·8% to 4·3%. Wilcoxon matched pairs analysis of all patients confirmed that vincristine induces apoptosis in vivo in peripheral blood mononuclear cells in childhood ALL (P = 0·043).
Cytokine requirement for the development of T‐lymphoid lineage potential in clonal lymphohaematopoietic progenitors in vitro
Authors: Ma Feng , Yang Feng‐Chun , Kaneko Azusa , Manabe Atsushi , Tanaka Ryuhei , Asano Shigetaka , Nakahata Tatsutoshi , Tsuji Kohichiro
Published: July 2010

The early process of T‐cell development prior to thymic colonization has been poorly investigated because of the lack of a sensitive assay. We have developed a two‐step in vitro culture system by combining a clonal culture with a fetal thymus organ culture (FTOC) and analysed the early development of T cells from lymphohaematopoietic progenitors. Cells of immature colonies derived from bone marrow cells of 5‐fluorouracil (5FU)‐treated mice using various combinations of early acting cytokines were transferred into a FTOC. All the combinations of stem cell factor (SCF), interleukin (IL)‐3 and IL‐6 capable of inducing colony formation supported T‐cell generation. IL‐11 and the Flt3 ligand possessed T‐lineage promotional effects similar to IL‐6 and SCF respectively. However, there were some quantitative differences in the final T‐cell yield among cytokine combinations. Thus, the commitment towards T lineage in lymphohaematopoietic progenitors may be an event determined intrinsically rather than induced by specific stimuli, but there may be a hierarchy between the activity of cytokines in further development. Furthermore, we examined the T‐lineage potential of individual colonies derived from Lin−c‐Kit+Sca‐1+ cells clone‐sorted from post‐5FU marrow cells. No colonies that contained only myelocytic progenitors showed T‐lineage potential, but 23·3% of colonies with a haematopoietic multipotentiality did. Therefore, the divergence of the T lineage from other lineages such as myeloid potential may occur at an early stage of the hierarchy of haematopoiesis. The proposed method should prove valuable for exploring the molecular and cellular changes that occur during early T‐cell development before thymic colonization.
Haemochromatosis in patients with β‐thalassaemia trait
Authors: Piperno Alberto , Mariani Raffaella , Arosio Cristina , Vergani Anna , Bosio Sandra , Fargion Silvia , Sampietro Maurizio , Girelli Domenico , Fraquelli Mirella , Conte Dario , Fiorelli Gemino , Camaschella Clara
Published: July 2010

Severe iron overload has been reported in patients with the β‐thalassaemia trait. Studies performed before the discovery of the haemochromatosis gene (HFE) have yielded conflicting results: some suggest that iron overload might arise from the interaction of the β‐thalassaemia trait with heterozygosity for haemochromatosis, some with homozygosity for haemochromatosis and others that it was unrelated to haemochromatosis. We have studied the clinical phenotype, iron indices and HFE genotypes of 22 unrelated patients with the β‐thalassaemia trait and haemochromatosis, the inheritance of chromosome 6p and 1q haplotypes in families of non‐homozygous C282Y probands and serum measures of iron status in relatives heterozygous for C282Y with or without the β‐thalassaemia trait. We demonstrate that the β‐thalassaemia trait aggravates the clinical picture of C282Y homozygotes, favouring higher rates of iron accumulation and the development of severe iron‐related complications. We suggest that the coexistence of the β‐thalassaemia trait might also increase the risk of iron overload in patients with HFE genotypes at a mild risk of haemochromatosis. Our findings do not support the hypothesis that the association of the β‐thalassaemia trait with a single C282Y or H63D allele might lead to iron overload and suggest that other non‐HFE‐related inherited factors are present in haemochromatosis patients with incomplete HFE genotypes.
The phenotypic profile of CD34‐positive peripheral blood stem cells in different mobilization regimens
Authors: De Boer F. , Dräger A. M. , Van Haperen M. J. A. M. , Van Der Wall E. , Kessler F. , Huijgens P. C. , Pinedo H. M. , Schuurhuis G. J.
Published: July 2010

The type of regimen used might result in mobilization of phenotypically and functionally different CD34+ cells. We compared the phenotype of CD34+ cells in leukapheresis products of three homogeneous groups: I, healthy individuals treated with granulocyte colony‐stimulating factor (G‐CSF) alone (n = 13); II, patients mobilized with G‐CSF following chemotherapy (n = 16); and III, patients mobilized with G‐CSF after high‐dose chemotherapeutic pretreatment (n = 24). Multiparameter flow cytometry was performed for CD34+ subpopulation analysis and focused on adhesion molecules, differentiation markers and megakaryocytic markers relevant for stem cell homing, with special reference to the importance of L‐selectin expression. Regimens I and II led to higher numbers of mobilized CD34+ cells (mean 468 × 106 and 491 × 106 CD34+ cells per leukapheresis procedure respectively) than regimen III (mean 41 × 106 CD34+ cells per leukapheresis procedure). Both the expression of L‐selectin and CD54 on CD34+ cells was significantly lower in group III, as was the percentage of megakaryocytic (CD41+) progenitors. A higher percentage of primitive (CD38− and/or HLA−DR−) CD34+ cells was found in group III, correlating with a higher clonogenicity of the CD34+ cells. However, when comparing the CD34+ subpopulations that were also positive for L‐selectin, there was no significant difference between the three regimens. A similar approach for the megakaryocytic CD34+ population resulted in an even worse quality of regimen III: 5·1% of CD34+ being CD41+/L‐selectin+ compared with 9·2% and 8·9% in regimens I and II respectively. We concluded that the phenotypes of the CD34+ cells in the G‐CSF (group I) and G‐CSF–chemotherapy (group II) regimens are similar, whereas the phenotype of the CD34+ cells mobilized in the high‐dose regimen (group III) displayed features that might negatively influence homing of the cells. Future studies will be directed towards regimens that will lead to the mobilization of a higher amount of CD34+ cells with a phenotypically favourable phenotype.
A recurrent frameshift mutation of the ankyrin gene associated with severe hereditary spherocytosis
Authors: Gallagher Patrick G. , Ferreira Jovino D. S. , Costa Fernando F. , Saad Sara T. O. , Forget Bernard G.
Published: July 2010

During a survey of typical, dominant hereditary spherocytosis (HS) patients, we identified a frameshift mutation of the ankyrin gene in three unrelated probands. All three probands, who were from different ethnic backgrounds, suffered from severe HS requiring splenectomy. Analysis of both intragenic and flanking polymorphisms revealed that these probands did not share a common ankyrin allele, providing evidence that these mutations arose independently on separate chromosomal backgrounds. This frameshift mutation associated with severe HS, Ankyrin Florianópolis, is the first description of a recurrent mutation in the ankyrin gene.
Combined defect in membrane expression and activation of platelet GPIIb–IIIa complex without primary sequence abnormalities in myeloproliferative disease
Authors: Kaplan Robert , Gabbeta Jagadeesh , Sun Ling , Mao Guang Fen , Rao A. Koneti
Published: July 2010

Defects in glycoprotein (GP)IIb–IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67‐year‐old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex‐specific anti‐GPIIb–IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was ∼50% of normal. An enzyme‐linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb–IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb–IIIa, monitored with mAb PAC‐1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet‐activating factor (PAF); expression of ligand‐induced binding sites (LIBS) was ≤ 30% of normal. Signal transduction‐independent LIBS expression, induced by echistatin, was ∼60% of normal, suggesting that the integrin present had intact ligand‐binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post‐translational processing of the complex) and inside‐out signalling‐dependent activation of the GPIIb–IIIa complex.
BCL10 deletions in haematological malignancies: a cloning artefact?
Authors: Duke Veronique , Kapoor Aarti , Gricks Clair , Melo Junia , Foroni Letizia
Published: July 2010

Mutations within the BCL10 gene, a gene involved in apoptosis, have recently been found to occur in a variety of cancers and it was suggested that BCL10 was involved in the pathogenesis of human malignancies. Our investigations of the involvement of this gene in various malignancies, however, do not uphold this suggestion. We would like to propose that, in fact, the original results were obtained as a result of a rare cloning artefact.