bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in the erythrocyte chemokine receptor (Duffy) gene: the molecular basis of the Fya/Fyb antigens and identification of a deletion in the Duffy gene of an apparently healthy individual with the Fy(a – b–) phenotype
Authors: MALLINSON G. , SOO K. S. , SCHALL T. J. , PISACKA J M. , ANSTEE D. J.
Published: March 2008

Summary. The erythrocyte chemokine receptor, a receptor for Plasmodium vivax, carries the antigens of the Duffy blood group system. Sequence analysis of reticulocyte RNA from individuals of known Duffy phenotype showed that the Fya antigen differs from the Fyb antigen as a result of a single nucleotide difference (A131 or G) encoding amino acid Gly44 (Fya) or Asp (Fyb) in the N‐terminal extracellular domain of the glycoprotein. Evidence is presented for two different genetic backgrounds giving rise to the Fy(a ‐ b‐) phenotype. The most likely genetic mechanism in most individuals of the Fy(a ‐ b–) phenotype is down‐regulation of Duffy glycoprotein mRNA. However, the Duffy gene from a very rare Caucasian individual (AZ) with the Fy(a – b–) phenotype has a 14 base‐pair deletion (nucleotides 287‐301) resulting in a frameshift which introduces a stop codon and produces a putative truncated 118 amino acid protein. The occurrence of this mutation in an apparently healthy individual raises questions about the functional importance of the Duffy glycoprotein not only in normal erythrocytes but also in all human cells and tissues.
Evaluation of cerebrospinal fluid EBV‐DNA and IL‐10 as markers for in vivo diagnosis of AIDS‐related primary central nervous system lymphoma
Authors: LUCA ANDREA DE , ANTINORI ANDREA , CINGOLANI ANTONELLA , LAROCCA LUIGI MARIA , LINZALONE ANGELA , AMMASSARI ADRIANA , SCERRATI MASSIMO , ROSELLI ROMEO , TAMBURRINI ENRICA , ORTONA LUIGI
Published: March 2008

Summary. Acquired immunodeficiency syndrome (AIDS)‐related primary central nervous system lymphoma (PCNSL) is almost always associated with the Epstein‐Barr virus (EBV), and EBV‐DNA in cerebrospinal fluid (CSF) has been indicated as a useful tumour marker for this HIV‐related neoplasm. AIDS lymphomas also show an enhanced production of IL‐10 which is generally associated with the presence of EBV in lymphoma cells.

We performed a prospective study in 19 HIV seropositive patients with brain mass lesions, and in 21 other AIDS patients with or without other neurological disorders, to assess the in vivo diagnostic value of EBV‐DNA and of IL‐10 levels in the CSF for primary lymphoma of the central nervous system (CNS). EBV‐DNA was detected by a nested polymerase chain reaction (PCR) in the CSF from seven of eight patients with PCNSL, diagnosed by brain biopsy (875% sensitivity) and in none of the 11 controls with brain mass lesions (100% specificity) and of the other 21 AIDS patients with or without neurological disorders. The only patient with PCNSL without detectable EBV‐DNA in the CSF was also negative for EBV‐DNA in the lymphoma tissue, whereas the samples of the other seven brain lymphomas were all positive for EBV‐DNA by nested PCR. Therefore 100% of patients with an EBV‐positive primary CNS lymphoma had detectable EBV‐DNA in the CSF. No patient from the control group without PCNSL with EBV‐negative CSF developed a lymphoma after a mean follow‐up of 157 ± 173 d. IL‐10 levels in the CSF from the patients with PCNSL were not significantly different from those in the other groups of patients with AIDS. Due to uniformly high levels in the CSF from AIDS patients, IL‐10 is not a useful diagnostic marker for AIDS‐related brain lymphoma.

The detection of EBV‐DNA from the CSF by nested PCR is an extremely sensitive and specific diagnostic tool for AIDS‐related PCNSL and should be further evaluated as a possible alternative in patients from whom brain biopsy is not advisable.
Identification of two novel mutations in non‐Jewish factor XI deficiency
Authors: IMANAKA YASUFUMI , LAL KALPANA , NISHIMURA TAKUYA , BOLTON‐MAGGS PAULA H. B. , TITDDENHAM EDWARD G. D. , MCVEY JOHN H.
Published: March 2008

Summary. We have studied two heterozygous unrelated CRM non‐Jewish FXI‐deficient patients. Neither of the patients carries a previously described mutation. Their FXI genes were screened by SSCP analysis following PCR amplification of each exon and the flanking intronic sequences. DNA fragments showing aberrant mobility were cloned and sequenced. The following mutations were identified: in case 1, a T to G transition in exon 12 results in the substitution of Phe‐442 by Val (FXI‐F442V); in case 2 a C to A transition in exon 5 results in the substitution of Cys‐128 by a nonsense codon (FXI‐C128X). The missense mutation results in a substitution within the protease domain of FXI. Molecular modelling locates this residue in a structurally conserved region of the protease domain and the amino acid substitution may therefore interfere with either chain folding and subsequent secretion or the stability of the protein in plasma. We conclude that the mutations which we have identified are responsible for the inherited abnormality in these patients.
Mobilization of CD34‐positive tumour cells in a patient with testicular mixed germ cell tumour
Authors: VERBEEK WALTER , PIES ANDREAS , HUMPE ANDREAS , GROVE DORIS , TROFF CARMEN , KUNZE EKKEHARD , HIDDEMANN WOLFGANG , WORMANN BERNHARD
Published: March 2008

Summary. Tumour cells from a patient with recurrent testicular germ cell cancer and bone marrow infiltration were found to express CD33 and CD34 in the absence of other haemopoiesis‐associated antigens. After myelosup‐pression and treatment with G‐CSF for stem cell mobilization, CD34‐positive tumour cells were detected in the peripheral blood in addition to normal haemopoietic progenitor cells. The tumour cells were decreased in the leukapheresis product. Retrospectively, the appearance of tumour cells in the peripheral blood after stem cell mobilization was the first indication of impending relapse.
Severe congenital neutropenia unresponsive to G‐CSF
Authors: Ryan M. , Will A. M. , Testa N. , Hayworth C. , Darbyshire P. J.
Published: March 2008

Summary Severe congenital neutropenia (SCN) is an inherited disorder characterized by severe neutropenia and recurrent infections from an early age, with bone marrow showing a maturational arrest of granulopoiesis at the promyelocyte stage. Since the introduction of G‐CSF therapy the prognosis for affected children has improved dramatically. We describe two patients with SCN who were clinically unresponsive to G‐CSF therapy. The results of in‐vitro colony assays from these two patients are presented together with the results from the mother of one of these patients who also has a chronic neutropenia, and a further child with SCN who responded to treatment with G‐CSF.
Bc16/Laz3 rearrangements in post‐transplant lymphoproliferative disorders
Authors: Delecluse H. J. , Rouault J. P. , Jeammot B. , Kremmer E. , Bastard C. , Berger F.
Published: March 2008

Summary Post‐transplant lymphoproliferative disorders (PTLD) are well‐known complications of iatrogenic immune deficiency and are thought to result from the proliferation of B cells infected by the Epstein‐Barr virus (EBV). Some large cell lymphomas occurring in the general population carry a rearrangement of the Bcl6/Laz3 zinc‐finger‐encoding gene. 15 EBV‐associated PTLD were tested for the presence of Bcl6/Laz3 rearrangements by Southern blot analysis using two specific probes (F3 70, F3 72), One out of 15 cases displayed a rearranged band independent of the germline one. In contrast, 10 lymphoblastoid cell lines and one lymphoblastoid cell line passaged in an SCID mouse carried only germline alleles of Bcl6/Laz3 after Southern blot hybridization. This indicates that genetic abnormalities may also play an important role in the development of some PTLD.
Patient accrual and quality of participation in a multicentre study on myeloma: a comparison between major and minor participating centres
Authors: Hjorth Martin , Holmberg Erik , Rodjer Stig , Taube Adam , Westin Jan
Published: March 2008

Summary .The participation of minor centres in randomized trials has been questioned because of inferior quality of participation. We have studied this issue in a multicentre trial on myeloma, in which 5 74 patients were included from 99 participating centres in Sweden, Norway and Denmark from 1 June 1990 until 4 November 1992.

Two hundred and eight patients were entered from university hospitals (n= 13), 172 from major county hospitals (n= 25), denned by a population base of ± 100 000 inhabitants, and 194 from minor county hospitals (n= 61) with a population base of < 100 000 inhabitants. The accrual rate was similar for the three hospital categories, averaging 54% of all reported cases, corresponding to 38% of the expected number of newly diagnosed cases. The adherence to the study protocol from an administrative point of view was judged by the completeness of follow‐up forms and the delay in the notification of deaths, and from a clinical point of view by the dose intensity for the principal drugs of the study, melphalan and interferon. For all studied measures of quality, the values were similar for the three hospital categories.

We conclude that with due informative and educational support, minor centres can make a considerable contribution to the patient material of a large randomized trial without impairing the quality of the study.
Kostmann's disease, recombinant HuG‐CSF, monosomy 7 and MDS/AML
Authors: Smith Owen P. , Reeves Brian R. , Kempski Helena M. , Evans Jane P.
Published: March 2008

Summary Monosomy 7 (Mo7) and leukaemia predisposition are associated with Kostmann's disease (KD). The recent introduction of long‐term recombinant HuG‐CSF treatment in patients with KD, whilst showing significant reductions in infectious complications and improved quality of life, might also be associated with an increased risk of developing karyotypic abnormalities, myelodysplasia (MDS) and acute myeloid leukaemia (AML). We describe a case of an identical twin with probable autosomal dominant KD who developed anaemia, Mo7/MDS and AML at 18, 30 and 50 months respectively from starting r‐metHuG‐CSF (filgrastim) treatment. Further patient analyses are required to establish the natural history of KD and to determine what role, if any, filgrastim plays in altering the pathobiology of this disorder.
Association of changes in monocyte antigen presentation and cytokine production in haemophilic boys with treatment and blood‐borne virus infection
Authors: Pasi K. J. , Evans J. A. , Wadhwa M. , Thorpe R. , Hill F. G. H.
Published: March 2008

Summary Aspects of monocyte function (antigen presentation and cytokine production (e.g. IL‐1, IL‐6) have been studied in a normal control population and three groups of haemophilic boys: group 1 HIV and HCV seronegative and treated only with a single heat‐treated factor VIII (FVIII) concentrate; group 2 HIV seronegative but HCV seropositive; group 3 all HIV and HCV seropositive. Groups 2 and 3 have been previously treated with unheated and heated FVIII concentrate. Group 1 boys (HIV/HCV uninfected) showed no significant reduction in monocyte antigen presentation function nor IL‐1 or IL‐6 production when compared with a control population. Group 2 boys (HCV infected) showed a reduced monocyte antigen presentation activity (P< 0.05), but no significant reduction in IL‐1 or IL‐6 production. Group 3 boys (HIV and HCV infected) showed a significantly reduced monocyte antigen presentation activity (P < 0.001) and an impairment of IL‐1 and IL‐6 production (P lt 0.05). A significant reduction of IL‐1 and IL‐6 production was only seen in the HIV and HCV infected haemophilic boys, implicating HIV as an aetiological agent. In contrast, reduced monocyte antigen presentation activity was seen in haemophilic boys with both HIV and HCV infection or HCV alone. The HIV and HCV seronegative boys had normal antigen presentation. The absence of immune modulation in haemophilic boys that have not acquired HIV and HCV infection suggests that chronic blood‐borne virus infections as contributory to immune modulation seen in haemophiliacs with virus infections.
Results of long‐term deferiprone (L1) therapy: a report by the International Study Group on Oral Iron Chelators
Authors: Al‐Refaie F. N. , Hershko C. , Hoffbrand A. V. , Kosaryan M. , Olivieri N. F. , Tondury P. , Wonke B.
Published: March 2008

Summary This report updates the combined experience of four centres involved in the long‐term treatment of transfusional iron overload in 84 patients with the oral iron chelator deferiprone (L1) over 167 patient‐years. The source of L1 was variable, including two university research laboratories and three pharmaceutical firms. Compliance was rated as excellent in 48%, intermediate in 36%, and poor in 16% of patients. On a mean L1 dose of 73–81 mg/kg/d, urinary iron excretion was stable, at around 0 5 mg/kg/d, with no indication of a diminishing response with time. Serum ferritin showed a very steady decrease with time from an initial mean ±1SD of 4207 ±3118 to 1779 ± 1154μg/1 after 48 months (P< 0–001). 17 patients abandoned L1 therapy, Major complications of L1 requiring permanent discontinuation of treatment included agranulocytosis (three), severe nausea (four), arthritis (two) and persistent liver dysfunction (one). The remaining patients abandoned treatment because of low compliance (three) and conditions unrelated to L1 toxicity (four). Lesser complications permitting continued L1 treatment included transient mild neutropenia (four), zinc deficiency (12), transient increase in liver enzymes (37), moderate nausea (three) and arthropathy (17). There was no treatment‐related mortality. Although the complications associated with L1 treatment are significant and require close monitoring, they do not preclude effective long‐term therapy in the vast majority of patients. Further well‐controlled prospective studies of L1 are required in order to enable proper judgement of its suitability for general long‐term clinical use.
Fludarabine‐related autoimmune haemolytic anaemia in patients with chronic lymphocytic leukaemia
Authors: Myint H. , Copplestone J. A. , Orchard J. , Craig V. , Curtis D. , Prentice A. G. , Hamon M. D. , Oscier D. G. , Hamblin T. J.
Published: March 2008

Summary. We have treated 52 patients with chronic lymphocytic leukaemia (CLL) with fludarabine; 12 developed severe autoimmune haemolysis. Only three had a previous history of haemolytic anaemia. Six out of eight patients retreated with fludarabine after control of their haemolysis developed an exacerbation of the haemolytic anaemia. The cause of autoimmune phenomena in CLL is not known, but our findings reinforce the view that they are caused by a disturbance in immunoregulatory T cells. Fludarabine is a known suppressor of T‐cell function.
Abnormalities of 3q21 and 3q26 in myeloid malignancy: a United Kingdom Cancer Cytogenetic Group study
Authors: Secker‐Walker L. M. , Mehta A. , Bain B. ,   on behalf of the UKCCG
Published: March 2008

Summary. Cytogenetic and clinical details are presented for 66 patients with myeloid malignancy and chromosome abnormalities of 3q21 and/or 3q26 (3qabns). Bone marrow and/or peripheral blood morphology was assessed for 52 cases. 3qabns in Philadelphia negative (Ph–ve) and positive (Ph + ve) cases were inv(3)(q21q26), (21 Ph‐ve, 6Ph + ve); t(3;3)(q21;q26) (nine Ph‐ve, four Ph + ve); and t(3;21)(q26;q22) (four Ph‐ve, six Ph + ve). Ph‐ve cases also had t(l;3)(p36;q21) (three cases), and t(3;5)(q21;q31)/ (q21;q35)/(q26;q21) (five cases aged <40 years). Three cases, aged < 30 years, had t(3;12)(q26;p13) which defines a new 3qabn subgroup. Monosomy 7 and/or 5q‐ accompanied inv(3) or t(3;3) in 17/30 cases. All cases had a myeloid malignancy (predominantly AML M1, M4 or M7), frequent trilineage myelodysplasia, and markedly abnormal megakaryopoiesis with micromegakaryocytes (<30/mi). Thrombocytosis occurred in two cases only. Most Ph + ve cases were in myeloid blast crisis and in Ph + ve cases alone, micro‐megakaryocytes were uniquely small (10 μm) in 7/11 cases. There were equal numbers of males and females. Seven secondary leukaemias were found in Ph–ve cases with inv(3), t(3;3), t(3;21), t(l;3) or del(3)(q21). Three cases with t(3;21) (one Ph + ve) were de novo AML or had de novo aplastic anaemia. Survival was rarely greater than 12 months from detection of the 3qabn.
Both early and committed haemopoietic progenitors are more frequent in peripheral blood than in bone marrow during mobilization induced by high‐dose chemotherapy + G‐CSF
Authors: Tarella Corrado , Benedetti Giovanni , Caracciolo Daniele , Castellino Claudia , Cherasco Cristina , Bondesan Paola , Omede Paola , Ruggieri Daniela , Gianni Alessandro Massimo , Pileri Alessandro
Published: March 2008

Summary. Haemopoietic growth factor administration following high‐dose chemotherapy markedly amplifies progenitor cell pool in the peripheral blood (PB). Collection and reinfusion of these cells enable rapid haemopoietic recon‐stitution following autograft. Less is known on engraftment potentiality of bone marrow (BM) cells taken under analogous conditions. To investigate this tissue, PB and BM were evaluated simultaneously during maximal mobilization in a series of 14 patients undergoing the HDS chemotherapy programme. A significantly higher growth of committed progenitors was found from PB rather than from BM (663 ± 123 v 267±40CFU‐GM/105 MNC, respectively). Also, significantly more CFU‐GM could be collected by a median of three leukaphereses, compared to those harvested from BM (158 ± 31 v 16 ± 4 ± 104 CFU‐GM/kg, respectively). Most mobilized CFU‐GM were phenotypically immature (CD15−); in addition, circulating cells included primitive progenitors, as assessed by LTC‐IC assay, or by evaluation of non‐proliferating pre‐CFU‐GM, selected by an anti‐CD71 immunotoxin. The amount of pre‐CFU‐GM determined by both techniques was consistently higher in PB than in BM. Moreover, a direct correlation could be established between circulating CFU‐GM and primitive precursors. Thus, during optimally induced mobilization, PB contains many more haemopoietic progenitors, of both committed and primitive stages, than does BM. Under such conditions, PB is probably the best source of material for graft purposes.
New type of t(14; 18) in a non‐Hodgkin's lymphoma provides insight in molecular events in early B‐cell differentiation
Authors: Meijerink J. P. P. , Raemaekers J. M. M. , Mensink E. J. B. M.
Published: March 2008

Summary. In this report a follicular non‐Hodgkin's lymphoma (NHL) carrying an unusual t(14;18) is described. This translocation most likely occurred during the VH to D‐JH rearrangement process of the IgH locus. From the data combined with data from the literature we conclude that the chance for development of a t(14;18) decreases during progression of the immunoglobulin rearrangement process in the pre‐B‐cell ontogeny. This is probably due to decreased accessibility of the bcl‐2 locus by reduced transcription. We analysed the somatic mutation pattern of the productively rearranged IgH gene. Like other follicular lymphomas, somatic mutations were present in this gene and indicated positive selection, probably for an antigen. We found no evidence for ongoing induction of somatic mutations during lymphoma development. We conclude that bcl‐2 gene deregulation, but not the precise moment at which this occurs during the pre‐B‐cell stage, influences the development of follicular NHL.
High incidence of deletions but infrequent inactivation of the retinoblastoma gene in human myeloma cells
Authors: Jdge‐Morineau Nadine , Mellerin Marie‐Paule , Francois Sylvie , Rapp Marie‐José , Harousseau Jean‐Luc , Amiot Martine , Bataille Regis
Published: March 2008

Summary. We have studied the retinoblastoma (RB‐1) susceptibility gene status and pRB expression in 22 human myeloma cell lines (HMCL) and in 10 patients with advanced multiple myeloma (MM). Deletions of the RB‐1 gene were observed in 81% (17/21) of the informative HMCL, regardless of their paracrine or autocrine interleukin‐6 (IL‐6) status. Among the deleted HMCL, only one (U266) had a biallelic deletion and lacked pRB expression. Monoallelic deletions had no consequence on the RB‐1 gene activation and pRB expression. One patient of 10 presented the same biallelic deletion as U266 and six of 10 had monoallelic deletions. We conclude that monoallelic deletions of the RB‐1 gene are frequent in HMCL and MM patients but have no consequence on gene activation and pRB expression.
Decreased production of interferon gamma by anti‐CD3 monoclonal antibody and interleukin‐2 ‐stimulated peripheral blood mononuclear cells in Hodgkin's disease
Authors: Frydecka Irena , Mazur Grzegorz , Kuliczkowski Kazimierz
Published: March 2008

Summary. We have examined the production of interferon (IFN) gamma by peripheral blood mononuclear cells (PBMC) in 22 patients with Hodgkin's disease (HD) in active phase of the disease, 12 patients in clinical remission and 16 healthy subjects. The level of IFN gamma in supernatants of PBMC stimulated for 72 h with anti‐CD3 monoclonal antibody (mAb), measured by sandwich enzyme immunoassay (ELISA) was 50.4 ± 2.3 U/ml in active phase; 137.0 ±7.4 U/ml in clinical remission patients; and 520.0 ± 10.0 U/ml in the controls; the difference between the groups was statistically significant. Co‐stimulation with interleukin‐2 (rIL‐2) markedly amplified production of IFN gamma. The mean levels were 220.8 ±7.0U/ml, 590‐7 ±3‐6U/ml and 2111.1 ± 17.3 U/ml in active phase HD, clinical remission and controls, respectively, the difference between groups was statistically significant. The patients showed the same kinetic pattern as healthy individuals. Our results indicate that patients with HD have severely impaired TCR/CD3 activation pathway resulting in significantly depressed IFN gamma response to anti‐CD3 mAb and anti‐CD3 + rIL‐2 in vitro stimulation and provide support for the possible clinical use of IFN gamma as an immunopotentiating agent in patients with HD.
Persistent hepatitis C virus RNA replication in haemophiliacs: role of co‐infection with human immunodeficiency virus
Authors: Chambost H. , Gerolami V. , Halfon P. , Thuret I. , Michel G. , Sicardi F. , Rousseau S. , Perrimond H. , Cartouzou G.
Published: March 2008

Summary. In order to evaluate the evolution of transfusional hepatitis C in haemophiliacs, we performed a retrospective study of ALT levels and HCV viraemia with a RNA PCR assay in 57 patients. We found that the vast majority of HCV‐infected patients remained viraemic (43/57=75%) and higher ALT levels correlated with HCV viraemia. Although indicators of the transfusional viral load (age, severity of haemophilia) and HBV co‐infection did not correlate with HCV RNA replication, HIV seropositivity was strongly associated with persistence of HCV viraemia (23/25 = 92% in HIV‐positive versus 20/32 = 62% in HIV‐negative patients), without any correlation with CD4 counts. Genotyping of HCV in the 43 viraemic patients shows more frequent genotype 1 in the HIV‐seropositive group (14/23) than in the seronegative group (6/20). Our data emphasize that besides the role of the immunodeficiency status, the genotypes of HCV might be involved in the differences observed in terms of HCV RNA replication between the HIV‐seropositive and seronegative haemophiliacs.
Genetic diagnosis of haemophilia A of Chinese origin
Authors: Lin Shu‐Rung , Chang Shu‐Chuan , Lee Chia‐Cheng , Shen Ming‐Ching , Lin Shu‐Wha
Published: March 2008

Summary. Genetic diagnosis of haemophilia A has been studied in two aspects. One is to directly identify the mutations in the factor VIII genes of the affected probands, and the other is to examine the usefulness of several intragenic factor VIII markers for gene tracking. Direct mutational analysis by PCR‐SSCP (polymerase chain reaction‐single‐strand conformation polymorphism) has been accomplished previously in 87 haemophilia A patients, accounting for nearly 10% of cases in Taiwan. Of the 87 cases, 46% were with point mutations, short deletions or insertions, and most of the remaining were with gene inversion readily identified by Southern blotting. Further examination of 112 patients has estimated a 33% incidence for gene inversion in all the patients with haemophilia A, or 37% in severe cases. Since the direct mutational detection described above cannot be used in all Chinese families with haemophilia A, genetic markers were also investigated. The two CA repeat markers located at intron 13 (CA‐13) and intron 22 (CA‐22), respectively, were amplified and analysed simultaneously. Seven different alleles with 18‐24 CAs have been identified for CA‐13. Alleles of 20 and 21 CAs are the most common and their population frequency was 0.68 and 0.24, respectively. The CA‐22 marker contained a repetition of (GT)n(AG)n as was identified in the white European but not in the Canadian population. Alleles with 25 and 26 GT/ AGs account for 18% and 75% of this group of samples, respectively. The expected rate of heterozygosity for either CA markers was 68%, although a value of 57% was observed by haplotype analysis, indicating an association of the two repeat markers. Nevertheless, the study of 62 females showed that with the combined use of CA‐13 and CA‐22 with Bell, approximately 71% would be informative for these markers. This number may increase to 81% if Xbal polymorphism is added. We propose that a better genetic diagnosis procedure for Chinese individuals would be first to look for the inversion mutation, secondly for one of the intragenic markers, and then at the PCR‐SSCP analysis.
Heparin‐induced thrombocytopenia: cross‐reactivity between standard heparin, low molecular weight heparin, dalteparin (Fragmin) and heparinoid, danaparoid (Orgaran)
Authors: Ramakrishna R. , Manoharan A. , Kwan Y. L. , Kyle P. W.
Published: March 2008

Summary. The incidence of cross‐reactivity between unfractionated heparin and LMWH, fragmin, in patients with HIT is significantly lower (6/15, 40%) than hitherto reported in the literature. 7/9 patients with a negative cross‐reactivity test were treated with dalteparin sodium (Fragmin) without any untoward events.
Bilateral linkage between a new deletion polymorphism in intron 21 of the GP lib gene and the HPA‐3b (Bakb) determinant
Authors: Peyruchaud Olivier , Nurden Alan , Bourre Francois
Published: March 2008

Summary. Glycoproteins (GP) IIb (αIIb) and GP IIIa (β3) form heterodimeric complexes (GP IIb‐IIIa) at the platelet surface and mediate platelet aggregation by binding fibrinogen after platelet activation. The structures and DNA sequences of the GP IIb and GP IIIa genes are known. Punctual mutations resulting in alloantigen systems (HPA) have been described on both genes, as have a series of genetic defects giving rise to Glanzmann's thrombasthenia (GT), We now report a nine base pair deletion located in intron 21 of the GP lib gene. This was found both in unrelated GT patients and in normal individuals. Subsequent studies showed that the deletion polymorphism and the mutation responsible for the platelet alloantigen, HPA‐3b, were linked together. The deletion was always present when the gene carried the HPA‐3b genotype, but was never observed in association with the HPA‐3a polymorphism. Analysis of 60 independent alleles from 30 unrelated Caucasian individuals revealed no exceptions to this linkage. It is the first time that two genetic markers have been reported to be linked to each other on the GP lib gene.
The Pasteur effect in human platelets: implications for storage and metabolic control
Authors: Guppy M. , Abas L. , Arthur P. G. , Whisson M. E.
Published: March 2008

Summary. The Pasteur effect and the associated acidosis have long been considered a major cause of platelet death during storage. We have investigated this phenomenon using a defined platelet preparation and a system whereby the oxidative and glycolytic contributions to total ATP production can be measured over a range of oxygen concentrations from saturating (pO2=158mmHg) to anoxic (pO2=OmmHg). Platelets do not show a Pasteur effect until the pO2 decreases to & 2′OmmHg, whereupon lactate production increases 1‐5‐fold. The Pasteur effect is therefore not a likely cause of platelet death during storage where pO2 in a storage bag typically drops to no less than 50mmHg. The data also have implications for the role of oxygen diffusion in oxidative metabolism, and for the compensatory nature of the Pasteur effect. As platelets are relatively small cells, and the onset of the Pasteur effect occurs at a relatively low oxygen concentration, diffusion may limit the rate of oxygen consumption in most other (larger) cells. The Pasteur effect is only fully compensative if the P/O2 ratio used for the calculations is lower than the conventional one. Since recent research strongly suggests that the conventional P/O2 ratio is too high, examples of fully compensative Pasteur effects may be more common than the literature suggests.
Single amino acid mutation of FCγ receptor is associated with the development of heparin‐induced thrombocytopenia
Authors: Burgess Janette K. , Lindeman Robert , Chesterman Colin N. , Chong Beng H.
Published: March 2008

Summary. Heparin‐induced thrombocytopenia (HIT) is mediated by a heparin‐dependent antibody/platelet factor 4/heparin complex binding to platelets via the FCγ receptor (type IIA). A single base polymorphism at position 131 of FeγRIIA changes the native arginine to histidine. In the presence of murine monoclonal IgG1 the former phenotype (FcγRIIAArg131) is functionally characterized by strong platelet aggregation (high responder) and the latter (Fc7RIIAHls131) by poor aggregation (low responder). In the presence of human IgGa the opposite response is observed. It has recently been shown that the heparin‐dependent antibody is predominantly of this subclass. We hypothesize that a relationship exists between FcγRIIAHls131 and the development of HIT. We studied 24 normal individuals and 20 HIT patients using VM58, a murine monoclonal IgG1, to characterize the phenotype by platelet aggregrometry, and PCR products, amplified with primers bordering the FC7RIIA polymorphism and hybridized with oligonucleotide probes specific for the single base mutation, to determine the genotype. The distribution of phenotypes and genotypes in the two populations differed, with a greater prevalence of the FcγRIIAHis131 allele in the HIT patient population. Homozygous Fc7RIIAArg131 individuals were absent from this group. We conclude that the presence of the Fc7RIIAHis131 allele is associated with a predisposition to HIT.
Adhesion molecule expression on CD34+ progenitor cells from normal and aplastic anaemia bone marrow
Authors: Karakantza Marina , Cavenagh James D. , Gordon‐Smith Edward C. , Gibson Frances M.
Published: March 2008

Summary. Aplastic anaemia (AA) is a disease of bone marrow failure. Evidence has been produced for both a stem cell and a stromal cell defect in this disease. The contribution of deficient or defective cell adhesion molecules (CAMs) has not been determined. CAMs have been shown to be important in stem cell‐stromal cell interactions and maintenance of haemopoiesis.

In this study the expression of CAMs (LFA‐1, LFA‐3, ICAM‐1, VLA‐4, CD44, sLex and L‐selectin) on CD34+ progenitor cells from 10 normal donors and eight patients with AA was investigated using double immunofluorescence. There was no significant difference in the percentage of CD34+ cells that were CAM+ between normal and AA bone marrow, suggesting that abnormal CAM expression on AA progenitor cells is not responsible for nor contributes to the pathogenesis of the disease. However, these findings do not exclude abnormal CAM function on progenitor cells, or abnormal expression or function of CAM ligands or counter‐receptors on AA stromal cells.
Band 3 Chur: a variant associated with band 3‐deficient hereditary spherocytosis and substitution in a highly conserved position of transmembrane segment 11
Authors: Maillet Philippe , Vallier Agnès , Reinhart Walter H. , Wyss Erwin J. , Ott Peter , Texier Pascale , Baklouti Faouzi , Tanner Michael J. A. , Delaunay Jean , Alloisio Nicole
Published: March 2008

Summary. We studied a large Swiss family with dominantly inherited hereditary spherocytosis and band 3 (anion exchanger 1, AE1) deficiency. Band 3 cDNA was analysed by single‐strand conformation polymorphism analysis and nucleotide sequencing. A new point mutation was found: G771D (GGC→GAC). This change was present in all eight investigated patients but absent in four healthy members of the family. It is located at a highly conserved position in the middle of transmembrane segment 11, introducing a negative charge in a stretch of 16 apolar or neutral residues. None of the six amino‐acid substitutions already known in this region as being associated with band 3 deficiency were recorded. To rule out any major transcriptional or post‐transcriptional defect, we evaluated the amount of band 3 mRNA by RNase mapping using a band 3‐protein 4.1 chimaeric probe. Similar mRNA amounts were present in patients and controls. Our results strengthen the view that some amino‐acids, that are well conserved throughout the AE family, may be crucial for the insertion and/or the stabilization of band 3 within the lipid bilayer. At the present time, most of the mutations altering such residues are located in the C‐terminal region of band 3.
Thalassaemic erythrocytes: cellular suicide arising from iron and glutathione‐dependent oxidation reactions?
Authors: Scott Mark D. , Eaton John W.
Published: March 2008

Summary. Both β‐thalassaemic red blood cells and normal red blood cells (RBC) artificially loaded with unpaired α‐haemoglobin chains exhibit increased amounts of membrane‐bound haem and iron. In the model β‐thalassaemic RBC the amount of free haem and iron was as much as 20 times that which could have been contributed by the entrapped α‐haemoglobin chains alone. This excess haem iron arises from destabilization of haemoglobin via reactions between ferric iron (Fe3+), initially contributed by the unpaired a chains, and cytoplasmic constituents, primarily reduced glutathione (GSH). Indeed, in the presence of Fe3+ (100μM) addition of even small amounts of GSH (0‐5 mM) to dilute RBC haemolysates (0‐15 mg haemoglobin/dl) greatly accelerated methaemoglobin formation. In contrast, lysates from GSH‐depleted RBC demonstrated a significantly reduced rate of iron‐mediated haemoglobin oxidation which was reversible by addition of GSH. The initiation, and subsequent propagation, of Fe3+‐mediated haemoglobin oxidation was significantly inhibited by iron chelators. Finally, Fe3+‐driven haemoglobin oxidation was synergized by low amounts of H2O2, an oxidant spontaneously generated in thalassaemic RBC. To summarize, the release of small amounts of free iron from unpaired α‐haemoglobin chains in the β‐thalassaemic RBC can initiate self‐amplifying redox reactions which simultaneously deplete cellular reducing potential (e.g. GSH), oxidize additional haemoglobin, and accelerate the red cell destruction.
The metabolites of nitric oxide in sickle‐cell disease
Authors: Rees David C. , Cervi Paul , Grimwade David , O'Driscoll Aisling , Hamilton Malcolm , Parker Norman E. , Porter John B.
Published: March 2008

Summary. Plasma NOx concentrations were raised in 22 acute painful crises in SCD. We have measured blood concentrations of nitric oxide metabolites (NOx) in sickle‐cell disease (SCD), and shown that they are increased compared with healthy controls (P = 0.002), and haemoglobin E/β‐thalassaemic controls (P=0.05). Concentrations in steady‐state SCD were also higher than in healthy controls (P = 0.04) but not significantly different from the concentrations at the beginning of painful crises (P = 0.34). Importantly, in 12 regularly exchanged sicklers, the mean pre‐transfusion NOx concentration did not differ significantly from the control population (P=0.52), suggesting that the changes in NO metabolism can be reversed. It is unlikely that the increased concentrations of NOx in SCD result from anaemia or haemolysis as the untransfused haemoglobin E/β‐thalassaemics did not show increased levels.
Patients with multiple myeloma requiring long‐term dialysis: presenting features, response to therapy, and outcome in a series of 20 cases
Authors: Torra R. , Blade J. , Cases A. , López‐Pedret J. , Montserrat E. , Rozman C. , Revert L.
Published: March 2008

Summary. From January 1982 to December 1993, 30 patients with multiple myeloma (MM) required haemodialysis (HD) at our institution. The subgroup of 20 patients who survived more than 2 months on HD is the subject of this study. Four patients were already on HD, due to previous nephropathy, when MM was diagnosed. 13 patients presented with acute renal failure and were on dialysis from the time of diagnosis. The remaining three cases developed renal failure later in the course of the disease. The objective response rate was 40% (8/20). Only two patients could discontinue HD (one had a late partial recovery and one received a kidney graft). Mean hospitalization per year was 19.3 d. The subgroup of patients who survived < 1 year spent a mean of 38.3 d in hospital. Whereas in the subgroup with a survival > 1 year mean hospitalization days was 9.6 (P < 0.001). The median survival was 20 months and six patients survived for > 3 years. In summary, patients with MM and severe renal failure who survive the first 2 months on dialysis have an objective response rate to chemotherapy of 40% and a median survival of almost 2 years, with 30% long‐term survivors.
Isodicentric (X)(q13) in haematological malignancies: presentation of five new cases, application of fluorescence in situ hybridization (FISH) and review of the literature
Authors: Dierlamm Judith , Michaux Lucienne , Criel Arnold , Wlodarska Iwona , Zeller Wolfgang , Louwagie Andries , Michaux Jean‐Louis , Mecucci Cristina , Van Den Berghe Herman
Published: March 2008

Summary. Idic(X)(q13) represents a rare but recurrent chromosomal abnormality in haematological malignancies. We present five new cases characterized by this particular aberration and review the literature on this subject.

The patients were elderly females with a diagnosis of refractory anaemia (1/5), refractory anaemia with ringed sideroblasts (2/5), chronic myelomonocytic leukaemia (1/ 5), and Philadelphia chromosome‐negative chronic myeloid leukaemia (1/5). Three out of the five patients demonstrated an increased proportion of bone marrow ringed sideroblasts. After a follow‐up period of 30‐57 months all patients but one are alive.

Idic(X)(q13) always occurred as the sole chromosomal abnormality, either in one or in two copies. We confirmed the dicentric nature of the aberration by fluorescence in situ hybridization (FISH) on metaphases as well as interphase nuclei using an X‐chromosome‐specific alpha‐satellite probe, and performed chromosome painting to visualize possible additional chromosomal changes involving the X chromosomes.

Our findings and the data of 17 previously published cases indicate that idic(X)(q13): (1) may play a significant pathogenetic role in haematological malignancies affecting exclusively females and deriving predominantly from early progenitor cells; (2) is frequently associated with a pathological iron accumulation; (3) indicates a variable prognosis.
Absence of N‐RAS point mutations in peripheral blood cells of patients with aplastic anaemia and paroxysmal nocturnal haemoglobinurea
Authors: White Jonathan R. , Josten Klaus M. , Chopra Rima , Tooze Jennifer , Saso Radovan , Gordon‐Smith Edward C. , Rutherford Tim R.
Published: March 2008

Summary. The myelodysplastic syndromes (MDS) have a significant frequency of evolution into acute myeloid leukaemia (AML). Approximately 30% of MDS patients show activating mutations of the N‐RAS proto‐oncogene, and these patients are at increased risk of leukaemic evolution. Long‐term survivors of aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinurea (PNH) are also at significant risk of developing AML. We have screened peripheral blood DNA from 42 AA patients and 15 PNH patients for the presence of N‐RAS point mutations. No mutations were detected in these samples, indicating that the mechanisms of evolution into AML may be different from those in MDS.
t(8;21) detection by RT‐PCR: an improved primer set convenient for the routine laboratory
Authors: Lynas Caroline , Howe Denise , Sweeney Marion , Wagstaff Michael
Published: March 2008

Summary. A new set of primers to detect t(8;21) by RT‐PCR have been designed. These primers give a more specific PCR amplification than the original primers and can be used with a cycling regime that is already in use for a wide range of other PCR methods in the routine haematology laboratory. Keywords: AML, t(8;21), RT‐PCR.
BCR‐ABL rearrangement and Variant’Philadelphia chromosome in de novo acute myelogenous leukaemia FAB subtype M1
Authors: Cigudosa Juan C. , Almeida M. Teresa Acosta , Carrasco Vicente , Bello Teófilo , Carrasco José Luis , Gomez Ana Otero , Talavera Juan Garcia , Garcia Miranda José Luis
Published: March 2008

Summary. We report a case of de novo acute myelogenous leukaemia FAB subtype Ml that presents a cytogenetic complex translocation between chromosomes 7, 9 and 22, producing a ‘variant’ Philadelphia chromosome. Molecular analysis revealed a BCR‐ABL rearrangement involving exons b3 and a2 (b3a2). Haematological parameters and genetic analysis again raise the problem of the true nature of this disease, which is briefly discussed.
Induction of tissue factor expression in human monocyte/endothelium cocultures
Authors: Collins P. W. , Noble K. E. , Reittie J. R. , Hoffbrand A. V. , Pasi K. J. , Yong K. L.
Published: March 2008

Summary. Induction of tissue factor (TF) expression on monocyctes and endothelial cells is central to the development of septic coagulopathy. Serum concentrations of endotoxin in septic patients who develop disseminated intravascular coagulation (DIC) do not, however, reach the levels that would directly stimulate TF expression on either monocytes or endothelium. We show, using an in vitro coculture system, that the interaction of monocytes with endothelium induces the expression of significant levels of TF. Unstimulated cocultures of monocytes (2 × 104/well) and endothelial cells (2 × 104/well) produced 35.3± 8.5 mU of PCA/well, representing a 5‐fold increase over the combined PCA of each cell type cultured alone (7.1 ± 1.5 mU, n= 6, P < 0.001). Significant enhancement was also found in the presence of low concentrations of LPS. Induction of TF protein was confirmed by Western blotting. Fixation of monocytes with paraformaldehyde completely abolished TF induction in cocultures, whereas fixation of endothelium had no effect, suggesting that TF induction occurred in monocytes rather than endothelial cells. Induction of TF in cocultures could be further augmented by preincubating the endothelial cells with IFN‐γ. When endothelium was prestimulated with 500U/ml IFN‐γ there was 142 ±11% increase over unstimulated cocultures (n = 5, P< 0.01). TF induction was inhibited by 32 ± 6% in the presence of anti‐ICAM‐1 mAb (n= 5, P < 001). Our results suggest that monocyte interactions with vascular endothelium, regulated by inflammatory cytokines, and mediated by adhesive ligand binding, leads to the induction of functional monocyte TF protein, which may be responsible for the initiation of DIC in sepsis.
Effects of different amino‐acid substitutions in the leucine 694‐proline 708 segment of recombinant von Willebrand factor
Authors: Hubert Lysiane , Gaucher Christine , Mazurier Claudine
Published: March 2008

Summary. Type 2B von Willebrand disease (vWD) is characterized by an increased affinity of von Willebrand factor (vWF) for binding to platelet glycoprotein lb (Gplb). Most type 2B candidate mutations are clustered in the 509‐695 disulphide loop but three of them (H505D, L697V and A698V) are outside this loop. We confirm here that the A698V mutation is a type 2B mutation by its expression in Cos‐7 cells. As the L697V and A698V type 2B mutations both induce the presence of a valine residue in the 694‐708 sequence, we created and expressed different mutated recombinant vWFs (rvWFs), in substituting the other leucine and alanine residues of this sequence (at positions 694, 701 and 706) into valine residues. V694rvWF and V706rvWF displayed decreased ristocetin‐induced GpIb binding showing that it is not always the presence of a valine residue that may explain the increased affinity of type 2B vWF for GpIb. We also compared the interaction with platelets of V697rvWF and V698rvWF to those obtained with rvWFs reproducing two prevalent type 2B mutations located in the loop (R543W and V553M). We show that the two mutations located in the loop are more reactive than the two mutations identified outside the loop.
Blood platelet heterogeneity: a functional hierarchy in the platelet population
Authors: Behnke O.
Published: March 2008

Summary. The platelet population in man and rat can be divided into two classes of about equal size based on the presence/absence of a p‐nitrophenylphosphatase, which probably is a phosphotyrosine phosphatase (PTPase). Phosphorylation of tyrosines on several platelet proteins is implicated in platelet activation, and I carried out in vitro and in vivo experiments on rats to determine whether PTPase positive and negative platelets differed in their reaction time. I used adhesion to collagen in vitro and in vivo (longitudinal slits in aorta and vena portae) and platelet aggregates in clots formed in vivo. I present evidence that PTPase negative platelets react the fastest, most conspicuously seen in the arterial bleeding under high flow conditions, where the first platelets to respond and adhere are predominantly PTPase negative.
Detection of minimal residual disease in childhood acute lymphoblastic leukaemia using fluorescence in‐situ hybridization
Authors: White Denise M. , Crolla John A. , Ross Fiona M.
Published: March 2008

Summary. Using centromere‐specific probes and a fluorescence in‐situ hybridization (FISH) technique in cases of childhood hyperdiploid acute lymphoblastic leukaemia (ALL), cells with extra copies of chromosomes can be differentiated from normal cells by their extra signals in both metaphase and interphase nuclei. In this way the entire cell population, not only those cells in division, can be analysed, thereby providing a valuable technique not only for determining leukaemia cell karyotype at diagnosis but also for the detection of minimal residual disease (MRD). We have conducted 161 analyses of remission bone marrow aspirates (BMs) in 13 children with hyperdiploid ALL. Slides were analysed blind and in parallel to 35 control samples. Control BMs showed very low numbers of trisomic cells (mean ± 2 × SD = 013 ± 0.34%). MRD was detected in 5/13 cases of ALL investigated while on chemotherapy. One out of five newly diagnosed cases and all three relapse cases of ALL had significantly raised levels of hyperdiploid cells in day 28 BMs. The presence of detectable disease in day 28 BMs suggests the need for larger studies to find whether this data is of prognostic value.