bluuebunny/crossref_metadata_2025 huggingface dataset:
Collection of Ph‐negative progenitor cells with granulocyte‐colony stimulating factor in patients with chronic myeloid leukaemia who respond to recombinant alpha‐interferon
Authors: Reiffers  , Taylor  , Gluckman  , Gorin  , Mahon  , MiclÉa  , DestrÉe  , Gautier
Published: March 2003

This study aimed to demonstrate that sufficient Ph‐negative blood progenitors could be collected following administration of glycosylated rhG‐CSF (lenograstim) to patients with Philadelphia chromosome (Ph)‐positive chronic myeloid leukaemia (CML) who responded to recombinant alpha‐interferon (α‐IFN) (Ph‐positive marrow metaphases < 35%). 23 patients received lenograstim (150 μg/m2) once daily for a median of 9 d. Peak circulating numbers of white blood cells (36.4 × 109/l), CD34+ cells (24/μl) and colony‐forming unit‐granulocyte‐macrophage (CFU‐GM; 1346.5/ml) occurred at a median of day 8, day 8 and day 7, respectively. Two to six (median three) leukaphereses (LK) were performed from days 5 to 12. The median number of mononuclear cells (MNC), CD34+ cells and CFU‐GM collected per patient was 7.35 × 108/kg, 2.72 × 106/kg and 10.23 × 104/kg, respectively. 22/23 patients had LK which contained either 104 CFU‐GM/kg and/or 106 CD34+ cells/kg; 47 LK (from 20/22 patients) were evaluable for cytogenetics. The median percentage of Ph‐positive cells was 0, and 43/47 LK (91%) contained < 35% Ph‐positive cells; 25 (53%) were entirely negative. Sixteen of 20 evaluable patients had one or more LK with < 35% Ph‐positive cells, and 12 had at least one 100% Ph‐negative LK. Mobilization and collection of Ph‐negative cells were not influenced by the dose or duration of α‐IFN administration before (or during) lenograstim administration or by the quality of cytogenetic response (complete v major) during lenograstim administration. No significant side‐effects were observed. Thus, lenograstim administration can result in satisfactory Ph‐negative blood progenitor cell collection. Autologous transplantation of such cells may be used when indicated.
Multiplex PCR for TCR delta rearrangements: a rapid and specific approach for the detection and identification of immature and mature rearrangements in ALL
Authors: Hettinger Klaudia , Fischer Susanna , Panzer Simon , Panzer‐GRÜmayer E. Renate
Published: March 2003

The preferential occurrence of immature T‐cell receptor (TCR) δ rearrangements (i.e. incomplete Dδ2‐Dδ3 and Vδ2‐Dδ3) in B‐cell precursor acute lymphoblastic leukaemia (BCP ALL) and of predominantly mature rearrangements (incomplete Dδ2‐Jδ1, complete Vδ1, Vδ2, Vδ3 to Jδ1) in T‐lineage ALL prompted us to establish two separate multiplex PCR systems for the identification of clonal TCRδ rearrangements. PCR products of the expected size for the specific rearrangements were detectable from a dilution of 100–1000 clonal cells in 150 000 polyclonal cells. Both multiplex PCR systems were used to analyse samples from 86 childhood BCP ALLs and 30 T‐lineage ALLs. The results of the multiplex PCRs were controlled by standard PCR analyses for the individual rearrangements and Southern blots, which were identical. Only immature TCRδ rearrangements were detected in BCP ALL (59%), whereas no rearrangement was found in the remaining BCP leukaemias, thus confirming the exclusive presence of immature TCRδ rearrangements in B‐lineage cells. 50% of the T‐lineage ALLs contained mature rearrangements, but no immature rearrangements were found. These two multiplex PCR techniques appear to be reliable and fast aids in the analysis of clonal TCRδ rearrangements in ALL.
Lack of association between childhood common acute lymphoblastic leukaemia and an HLA‐C locus dimorphism influencing the specificity of natural killer cells
Authors: Ghodsi  , Taylor  , Gokhale  , Dearden  , Stevens  , Birch  , Fergusson  , Eden  , Ollier
Published: March 2003

Previous serological studies documenting an association between acute lymphoblastic leukaemia (ALL) and HLA‐Cw antigens suggested that the HLA‐C locus might influence susceptibility to ALL. However, associations with more than one Cw antigen suggest that polymorphic variants shared by more than Cw allele could be involved. Recent studies have shown that the HLA‐C locus encodes two ligands (NK1 and NK2) recognized by receptors on natural killer (NK) cells. HLA‐Cw alleles encoding these ligands are dimorphic, dependent on whether they encode one or other NK ligand. To determine whether susceptibility to the common (CD10+) form of childhood ALL (c‐ALL) is associated with NK1 or NK2, we carried out a molecular analysis of 94 childhood c‐ALL patients and 136 infant controls. We found no difference in the frequency of NK1 and NK2 alleles, phenotypes or genotypes between the patients and controls, suggesting that this does not explain the role of the HLA‐C locus in susceptibility to childhood c‐ALL.
Flow cytometric analysis of platelet activation by different collagen types present in the vessel wall
Authors: Alberio Lorenzo , Dale George L.
Published: March 2003

The interaction of platelets with collagens of the vessel wall is a critical event in primary haemostasis. Although numerous studies have examined the ability of various collagen types to support platelet adhesion, little is known concerning the relative ability of different collagens to elicit specific activation markers in platelets. In this report, flow cytometric analysis has been utilized to evaluate the ability of various native collagen types to elicit secondary activation events in human platelets. Collagen types I, III, V and VI induced α‐granule secretion and up‐regulation of cell surface glycoprotein (GP) IIb/IIIa. In contrast, collagen type IV did not elicit these responses in the concentration ranges examined. Dose–response curves for α‐granule secretion induced by the various collagen types indicated that human type III and human type I collagens were less effective than human type V, human type VI and calf skin type I. In addition, the ability of these various collagens to activate GPIIb/IIIa to its ligand binding conformation was even more heterogenous with only human type VI and calf skin type I readily promoting this transition. These data demonstrate that flow cytometric analysis of collagen‐induced platelet activation is feasible and that collagen‐mediated α‐granule secretion and membrane glycoprotein redistribution in human platelets are separate events from activation of GPIIb/IIIa.
Studies of multimerin in patients with von Willebrand disease and platelet von Willebrand factor deficiency
Authors: Chen Christine I. , Federici Augusto B. , Cramer Elisabeth M. , Canciani Maria T. , Harrison Paul , Zheng Shilun , MassÉ Jean‐Marc , Mannucci Pier M. , Hayward Catherine P. M.
Published: March 2003

In normal platelet α‐granules von Willebrand factor (VWF) is stored with multimerin and factor V in an eccentric electron‐lucent zone. Because the platelet stores of VWF are deficient in ‘platelet low’ type 1 and type 3 von Willebrand disease (VWD), we investigated their electron‐lucent zone proteins. The patients with VWD had partial to complete deficiencies of plasma and platelet VWF but normal α‐granular multimerin and factor V, and normal α‐granular fibrinogen, thrombospondin‐1, fibronectin, osteonectin and P‐selectin. In type 3 VWD platelets, α‐granular electron‐lucent zones lacking VWF‐associated tubules were identified and multimerin was found in its normal α‐granular location. These findings indicate that the formation of the electron‐lucent zone and the sorting of multimerin to this region occur independent of VWF. The isolated abnormalities in VWF suggests a VWF gene mutation is the cause of ‘platelet low’ type 1 VWD.
Unusual complications after bone marrow transplantation for dyskeratosis congenita
Authors: Rocha Vanderson , Devergie Gnès , SociÉ Gerard , Ribaud Patricia , EspÉrou Hélène , Parquet Nathalie , Gluckman Eliane
Published: March 2003

Dyskeratosis congenita (DC) is a rare inherited disorder often associated with aplastic anaemia. We report the cases of five boys transplanted with an HLA‐identical related donor for severe aplastic anaemia (SAA) associated to DC; in all cases successful engraftment was observed. Three patients died 2–8 years after bone marrow transplantation (BMT) with signs of endothelial cell damage syndrome (kidney microangiopathy and liver veno‐occlusive disease). Another boy died 1 year after BMT from Evans syndrome and invasive aspergillosis. One boy currently presents anaemia, polyarthritis of unknown origin, pulmonary fibrosis and gut malabsorption 7.5 years after BMT. SAA associated with DC can be successfully treated by allogeneic BMT. However, these early and late complications observed are very unusual after BMT and probably reflect the association of transplanted‐related factors, evolution of the underlying disease, and increased sensitivity of endothelial cells. Modified conditioning approaches, advances in supportive care and surveillance of these unusual complications offer the possibility of improved outcome for these patients.
In vitro growth in acute myeloblastic leukaemia: relationship with other clinico‐biological characteristics of the disease
Authors: Del Cañizo  , Brufau  , Almeida  , Galende  , GarcÍa Marcos  , Mota  , GarcÍa  , FernÁndez Calvo  , Ramos  , Fisac  , Orfao  , San Miguel
Published: March 2003

The in vitro growth characteristics of a large series of acute myeloid leukaemia (AML) patients and their relationship with other clinical and biological disease characteristics were analysed. Patients with AML were studied, 181 with de novo AML and 45 with secondary AML (24 myelodysplastic syndrome, sAML‐MDS, 21 myeloproliferative disorder, sAML‐MPD). Leukaemic colony forming units (L‐CFU) were assayed by plating peripheral blood (PB) blast cells in methyl‐cellulose and using LCM‐PHA as stimulant. In each case parallel cultures were made with and without stimulating factors. Plating efficiency (PE) was defined as the number of clusters plus colonies/105 cells plated. Autonomous growth (AG) was the number of colonies plus clusters growing without stimulant. The autonomous proliferative index (API) was calculated as the number of clusters + colonies without stimulating factor divided by the number of clusters + colonies with stimulating factor. No significant differences in the PE between de novo and secondary AML were found. Autonomous growth was significantly higher in sAML‐MPD. The FAB subtype M3 leukaemias displayed a significantly greater PE and a significantly lower API when compared with the other FAB subgroups (P = 0.0002). Upon analysing the relationship with the immunophenotype, only CD33 expression showed a significant relationship with the in vitro growth pattern; CD33+ cases displayed a higher PE (P = 0.0002) and AG (P = 0.0003) than CD33− cases. When patients were grouped according to the level of rh123 efflux (MDR1) it was observed that cases with >30% elimination showed a higher AG and API than those with <30% (P = 0.03). Finally we found that patients with higher API (>0.05) displayed a significantly shorter overall survival as compared with patients with API < 0.05 (P = 0.04). The in vitro study properties of clonogenic cells produces relevant clinical information of leukaemic cell biology in AML patients.
Prevention of EBV‐induced B‐lymphoproliferative disorder by ex vivo marrow B‐cell depletion in HLA‐phenoidentical or non‐identical T‐depleted bone marrow transplantation
Authors: Cavazzana‐Calvo  , Bensoussan  , Jabado  , Haddad  , Yvon  , Moskwa  , Tachet Des Combes  , Buisson  , Morand  , Virion  , LE Deist  , Fischer
Published: March 2003

HLA‐mismatched bone marrow transplantation (BMT) is hampered by three major complications: graft rejection, acute graft‐versus‐host disease (aGVHD) and delayed immune reconstitution. Infusion of anti‐LFA1 plus anti‐CD2 monoclonal antibodies (MAb), combined with ex‐vivo T‐cell depletion of the graft, was efficient in preventing graft rejection and aGVHD. Nevertheless, disease‐free survival was limited by the high frequency of lethal infections, including EBV‐induced lymphoproliferative disease (BLPD), which originates mostly from donor B cells, with an incidence of 5–30%. To decrease the rate of this complication, ex‐vivo B‐cell depletion was attempted.

This study compares a group of 19 patients who received a T‐ and B‐cell‐depleted marrow from an HLA‐mismatched related donor with a retrospective control group of 19 patients, who had received T‐cell‐depleted marrow by the same method. The level of T‐cell depletion was similar in the two groups. For B‐cell depletion, two different methods were compared. The median number of B cells infused in the study group was 0.46/kg. Engraftment and aGVHD incidence were similar in the two groups. No EBV donor‐derived BPLD occurred in the study group, compared with seven in the control group, four of whom died because of EBV‐BPLD. Event‐free survival was significantly different between the two groups. We conclude that ex‐vivo B‐cell depletion of the graft may be a useful means of preventing EBV‐BPLD, and warrants further study on a larger group of patients.
A case of non‐β‐globin gene linked β thalassaemia in a Dutch family with two additional α‐gene defects: the common −α3·7 deletion and the rare IVS1–116 (A → G) acceptor splice site mutation
Authors: Giordano  , Harteveld  , Haak  , Batelaan  , Van Delft  , Plug  , Emonts  , Zanardini  , Bernini
Published: March 2003

We describe a family with β thalassaemia, apparently not linked to the β‐globin gene cluster, in combination with α thalassaemia. The propositus, an adult Dutch Caucasian male, and his son presented with microcytic hypochromic parameters. Their lysates displayed the normal adult pattern on electrophoresis. The HbA2 concentration, which is usually increased in β thalassaemia, was normal. The in vitro biosynthetic rate of the globin chains was strongly unbalanced even in the presence of a coexisting α‐thalassaemia defect. Routine analysis of the β genes, including the promoter region, was performed repeatedly by polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct sequencing. No molecular abnormalities were detected. Large β deletions were excluded by haplotype determination, using seven polymorphic markers distributed over an area of 50 kb, from 1 kb 5′ of the ε gene to 4 kb 3′ of the β gene. The haplotype analysis of the β‐gene cluster revealed that the unaffected daughter had received the same β haplotype as her β‐thalassaemic brother from their β‐thalassaemic father. These data suggest that the β‐gene cluster shared by father and son was not directly associated with a reduced β‐globin chain expression. In order to exclude the remote possibility of a β‐locus‐control region (LCR) rearrangement in the paternal haplotype of the daughter, the sequence of the HS2 element was examined in the nuclear family. We compared the haematological and clinical data of this family with the data reported in the limited number of similar cases. We discuss the possibility that the mutation of a trans‐acting erythroid factor(s), not linked to the β‐genes cluster, may impair the β‐gene expression of both alleles.
Influence of SDZ‐PSC833 on daunorubicin intracellular accumulation in bone marrow specimens from patients with acute myeloid leukaemia
Authors: Merlin Jean‐Louis , Guerci Agnès‐Paule , Marchal Sophie , Bour Corinne , Colosetti Pascal , Kataki Agapi , Guerci Oliero
Published: March 2003

The multidrug resistance (MDR) modulating activity of SDZ‐PSC833 (PSC), a non‐immunosuppressive cyclosporine analogue, was investigated and compared with cyclosporin A (CSA) in bone marrow clinical specimens from 45 patients with acute myeloid leukaemia (AML) taken at diagnosis, using double‐labelling flow cytometry with simultaneous determination of P‐glycoprotein (PGP) expression and intracellular daunorubicin fluorescence (IDF). On the basis of pre‐clinical results in multidrug‐resistant K562 leukaemic cells, concentrations leading to iso‐effective complete restoration of IDF were used: 5 and 10 μmol/l, respectively for PSC and CSA.  In the clinical specimens, PGP expression was correlated with a significant decrease in IDF. PSC was found to be significantly more potent than CSA since it was found to induce a significant increase in IDF in a higher number of cases and to a higher extent than CSA. PGP‐unrelated activity of PSC was also observed in specimens expressing no PGP but exhibiting low IDF, thus probably expressing alternative resistance mechanisms. The results confirm the potency of PSC as MDR‐modulating agent in clinical AML specimens whose resistance pattern differed from that of highly resistant cell models and suggest that the activity of PSC is not limited to P‐glycoprotein inhibition.
Heterogeneity of blood group RhE variants revealed by serological analysis and molecular alteration of the RHCE gene and transcript
Authors: Noizat‐Pirenne France , Mouro Isabelle , Gane Pierre , Okubo Yasuto , Hori Yasuto , Rouger Philippe , LE Pennec Pierre‐Yves , Cartron Jean‐Pierre
Published: March 2003

After testing red cells from 12 RhE variants with a panel of anti‐E monoclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes designated epE1, epE2, epE3 and epE4. The variants were classified into four categories (cat EI to EIV) which carried epE1 and epE2, epE1 and epE4, epE1, epE3 and epE4, and all four epitopes, respectively. Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and EIII displayed three distinct genetic alterations. Cat EI variants exhibited a point mutation (T500A) in exon 4 of the RHCE gene that resulted in a Met167Lys substitution in the third extracellular loop of the RhcE protein. Cat EII variant carried a hybrid gene structure characterized by replacement of exons 1–3 (or 2–3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant was also associated with a weak expression of the RhC antigen. In cat EIII variants there was a partial DNA exchange of exon 5 sequences (nt 697 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE‐D‐cE protein carrying the Glu233 and Val238 substitutions.

The serological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and proline at position 226 is necessary but not sufficient for the full expression of the E antigen, (ii) the lack of RhE epitope(s) is associated with heterogenous molecular alterations of the RHCE gene, and (iii) amino‐acids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.
Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells
Authors: Introna Martino , Barbui Anna Maria , Golay JosÉE , Bambacioni Federica , SchirÒ Raffaella , Bernasconi Sergio , Breviario Ferruccio , Erba Eugenio , Borleri Gianmaria , Barbui Tiziano , Biondi Andrea , Rambaldi Alessandro
Published: March 2003

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage‐dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL‐2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS‐sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram‐1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.
Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT‐PCR for cytokeratin‐19 mRNA
Authors: Vannucchi Alessandro M. , Bosi Alberto , Glinz Stephanie , Pacini Paolo , Linari Silvia , Saccardi Riccardo , Alterini Renato , Rigacci Luigi , Guidi Stefano , Lombardini Letizia , Longo Giovanni , Mariani Maria P. , Rossi‐Ferrini Pierluigi
Published: March 2003

There is considerable interest in an autologous transplantation (AT) programme for patients with high‐risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a ‘nested’ reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT‐PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte‐colony stimulating factor (rhG‐CSF). K19 transcripts in one or both BM samples  were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR‐ and IHC‐positive BM samples were diagnosed as ‘normal’ on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19‐negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19‐negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high‐risk non‐metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.
Poor response rate to a continuous schedule of Amifostine therapy for ‘low/intermediate risk’ myelodysplastic patients
Authors: Bowen  , Denzlinger  , Brugger  , Culligan  , Gelly  , Adlakha  , Groves  , Hepburn  , Kanz
Published: March 2003

Ineffective haemopoiesis leading to cytopenia presents the major clinical management problem for patients with myelodysplasia (MDS). Preliminary studies have demonstrated that the synthetic aminothiol Amifostine stimulates multilineage haemopoiesis both in vitro and in vivo in patients with MDS. We have treated 12 patients with an uninterrupted 8‐week schedule of thrice‐weekly intravenous Amifostine with a starting dose of 300 mg/m2 escalating to 450 mg/m2 in non‐responders. No patients satisfied response criteria on study but two patients showed minor responses. We conclude that therapeutic response to Amifostine in MDS may be schedule dependent.
The incidence and significance of fevers during treatment with antithymocyte globulin for aplastic anaemia
Authors: Dearden  , Foukaneli  , Lee  , Gordon‐Smith  , Marsh
Published: March 2003

Antithymocyte globulin (ATG) is a foreign protein used widely to treat aplastic anaemia (AA). Febrile reactions occurring during its administration may be impossible to distinguish clinically from fever due to sepsis, and are therefore routinely treated with intravenous antibiotics after collection of blood cultures. A statistically highly significant difference was found in positive blood cultures between 39 AA patients who developed fever during ATG therapy, and 38 febrile neutropenic patients with acute leukaemia, suggesting that most fevers developing during ATG treatment are not due to infection. It may therefore be reasonable to consider early discontinuation of intravenous antibiotics in patients who are clinically stable and have no proven sepsis.
Congenital dyserythropoiesis with intererythroblastic chromatin bridges and ultrastructurally‐normal erythroblast heterochromatin: a new disorder
Authors: Wickramasinghe  , Spearing Ruth L. , Hill
Published: March 2003

Two non‐anaemic subjects, a father and daughter, with a new form of congenital dyserythropoiesis are reported. The features of their disorder are: (1) an abnormal blood film with basophilic stippling of red cells and oval macrocytes, (2) various dysplastic changes in the erythroblasts, including internuclear chromatin bridges, (3) ultrastructurally‐normal erythroblast heterochromatin, (4) normal serum thymidine kinase activity, and (5) a probable autosomal dominant inheritance. The last three features distinguish this disorder from CDA type I.
A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an α2 globin gene variant (Hb Conakry)
Authors: Cohen‐Solal Michel , Préhu Claude , Wajcman Henri , Poyart Claude , Bardakdjian‐Michau Josiane , Kister Jean , Promé Danielle , Valentin Colette , BAchir Dora , Galactéros Frédéric
Published: March 2003

A Guinean woman, hetererozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3‐DPG concentration of twice normal levels. Sequence of the PK‐R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C → A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [α80(F1) Leu  → Val], which seemed to have a mild effect. The high intraerythrocytic 2,3‐DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3‐DPG content was modified in vitro. Hysteresis between deoxy‐ and reoxygenation curves, as well as increase in the nmax value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3‐DPG concentration.
Clonal disease of natural killer large granular lymphocytes in Taiwan
Authors: Chou Wen‐Chien , Chiang I‐Ping , Tang Jih‐Luh , Su Ih‐Jen , Huang Shang‐Yi , Chen Yao‐Chang , Liu Ming‐Chi , Lee Fenn‐Yu , Wang Chiu‐Hwa , Shen Ming‐Ching , Chuang Sou‐Ming , Tien Hwei‐Fang
Published: March 2003

Lymphoproliferative diseases of large granular lymphocytes (LDGL) may arise from either CD3+ T cells or CD3− natural killer (NK) cells. LDGL with clonal proliferation of large granular lymphoeytes (LGL) is defined as LGL leukaemia. The number of patients with NK‐LGL leukaemia reported is limited and the pathogenesis of the disease is not yet clear. From 1991 to 1998 six patients with cytogenetically proved clonal disease of NK‐LGL were identified in our institute. All were seropositive for Epstein‐Barr virus (EBV). EBV RNA or DNA could be detected in LGL from four patients by EBV in situ hybridization or Southern blot analysis. Most patients ran an aggressive clinical course and five died of the disease. Nonrandom clonal chromosomal abnormalities, including duplication of 1q, rearrangement at 3q and loss of chromosomes Y, 13 or 10, were noted in the six patients from this study and in eight from the literature. The implications of these recurrent cytogenetic aberrations in the development and progression of the disease deserve further studies.
Dyserythropoiesis and severe anaemia associated with malaria correlate with deficient interleukin‐12 production
Authors: Mohan  , Stevenson
Published: March 2003

Complex cytokine interactions occur during blood‐stage malaria which offer a unique opportunity to study their influence on the pathogenesis of malarial anaemia. Plasmodium chabaudi AS susceptible A/J mice experience severe and fatal anaemia whereas resistant C57BL/6 (B6) mice survive following moderate anaemia. In this study we analysed the role of IL‐12 in erythropoiesis and tested whether the levels of IL‐12 produced in these mice correlated with the extent of anaemia. In vitro, IL‐12 significantly enhanced the numbers of erythroid burst (BFU‐E) and colony forming units (CFU‐E) in bone marrow and spleen cells from normal and day 7 infected A/J and B6 mice. Despite the presence of IL‐12 in vitro, the level of splenic erythropoiesis in infected A/J mice was significantly lower than in B6 mice. Moreover, sera from infected B6 mice, but not A/J mice, significantly up‐regulated erythropoiesis in vitro and this enhancement correlated with several fold higher levels of IL‐12 in the sera of B6 compared to A/J mice. Furthermore, the erythropoietic potentiating effect of sera from infected B6 mice was abrogated following depletion of IL‐12. Taken together, these findings suggest that defective IL‐12 production in A/J mice during the early course of infection may result in fatal anaemia.
Erythrocyte autoantibodies in paediatric patients with sickle cell disease receiving transfusion therapy: frequency, characteristics and significance
Authors: Castellino Sharon M. , Combs Martha R. , Zimmerman Sherri A. , Issitt Peter D. , Ware Russell E.
Published: March 2003

The formation of erythrocyte autoantibodies following transfusion therapy has been described in case reports and small series. To determine the frequency, serological characteristics, and clinical significance of this phenomenon in paediatric patients with sickle cell disease, we analysed the patient database at the Duke University Pediatric Hematology Clinic. We identified children who received multiple erythrocyte transfusions, then reviewed clinical records to identify children who developed erythrocyte autoantibodies in association with transfusions. Among 184 paediatric patients who received multiple erythrocyte transfusions, 14 children (7.6%) developed warm (IgG) erythrocyte autoantibodies. Median transfusion exposure at the time of autoantibody formation was 24 erythrocyte units, range 3–341 units. The autoantibody reacted as a panagglutinin in 11 cases but had anti‐e specificity in three patients. Surface complement also was detected in five patients. Clinically significant haemolysis was documented in four patients, each of whom had both surface IgG and C3 detected. The development of erythrocyte autoantibodies was associated with the presence of erythrocyte alloantibodies. Formation of warm erythrocyte autoantibodies in association with transfusions is not rare in paediatric patients with sickle cell disease. Clinicians should be aware of this complication and recognize that the presence of surface C3 is often associated with significant haemolysis.
P‐glycoprotein, lung resistance‐related protein and multidrug resistance associated protein in de novo acute non‐lymphocytic leukaemias: biological and clinical implications
Authors: Michieli Mariagrazia , Damiani Daniela , Ermacora Anna , Masolini Paola , Raspadori Donatella , Visani Giuseppe , Scheper R. J. , Baccarani Michele
Published: March 2003

P‐glycoprotein (PGP), lung resistance‐related protein (LRP) and multidrug resistance associated protein (MRP) expression and the blast cells' intracellular daunorubicin accumulation (IDA) were evaluated in 96 previously untreated cases of de novo acute non‐lymphocytic leukaemia (ANLL). 47/96 patients (49%) were classified as PGP+, 44/96 (46%) as LRP+, and 8/96 (8%) as MRP+. The more frequent MDR clusters were PGP−/LRP−/MRP− (32/96 cases, 33%) and the PGP+/LRP+/MRP− (27/96 cases, 28%) followed by PGP+/LRP−/MRP− (15/96 cases, 16%) and PGP−/LRP+/MRP− (14/96 cases, 14%). A favourable karyotype was observed more frequently in PGP− and LRP− cases. A highly significant correlation was found between either PGP or LRP overexpression and leukaemic blast cell IDA.

All the patients received standard induction and consolidation treatments containing MDR‐related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP+ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.
Automated malaria detection by depolarization of laser light
Authors: MENDELOW BARRY V. , LYONS CHERYL , NHLANGOTHI PHILEMON , TANA MARJORIE , MUNSTER MARION , WYPKEMA EMMA , LIEBOWITZ LYNNE , MARSHALL LEONARD , SCOTT STEPHEN , COETZER THERESA L.
Published: March 2003

Anecdotal experience with full blood count (FBC) technology incorporating analysis of depolarized laser light (DLL) for the enumeration of eosinophils showed that malaria infection generated unusual distributions in the white cell channels. The objective of this study was to identify and define criteria for a diagnosis of malaria using this technology. To determine sensitivity, specificity, and positive and negative predictive values, 224 directed samples referred specifically for malaria were used; true positives were defined as those in which malaria was identified by microscopic and/or immunological methods. For the DLL method, positive was defined as one or more large mononuclear cell(s) for which the 90° depolarized signal exceeded the 90° polarized signal. To determine possible utility in a routine haematology laboratory setting, 220 random undirected FBC samples were evaluated for possible malaria infection by the DLL method. Of the 224 directed samples, 95 were malaria positive as determined by microscopic and/or immunological methods, and 129 were negative. For the DLL method, overall sensitivity was 72% (90% in the case of Black Africans), and specificity 96%. Positive and negative predictive values overall were 93% and 82% respectively. In the utility study a single positive result was identified among the 220 samples studied. This was found to be from a patient with malaria. The detection of unexpected malaria by automated screening FBC analysis could substantially lower the mortality and morbidity from unascertained infection, especially in indigenous African peoples.
Normal primitive haemopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time
Authors: FRASSONI FRANCESCO , PODESTÀ MARINA , PIAGGIO GIOVANNA , ROSTI VITTORIO , PITTO ANNA , BENVENUTO FEDERICA , FIGARI OSVALDO , VASSALLO FRANCA , CARELLA ANGELO M. , ZIKOS PANAGIOTIS , BERGAMASCHI GAETANO , FUGAZZA GIUSEPPINA , SESSAREGO MARIO , CAZZOLA MARIO
Published: March 2003

We carried out studies to quantify Ph‐negative progenitors both in steady state and during regeneration after chemotherapy and G‐CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady‐state bone marrow, Ph‐negative long‐term culture initiating cells (LTC‐IC) and Ph‐negative colony‐forming‐cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P < 0.001 and P < 0.02, respectively). The absolute number of mobilized Ph‐negative progenitors was markedly higher in group A versus group B (P < 0.02 for LTC‐IC, P < 0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph‐negative LTC‐IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph‐negative LTC‐IC in the bone marrow predicted the yield of Ph‐negative LTC‐IC mobilized into peripheral blood (P < 0.001). The bone marrow frequency of Ph‐positive LTC‐IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph‐negative circulating progenitors would appear to be soon after diagnosis.
Transferrin receptor on peripheral blood lymphocytes in iron deficiency anaemia
Authors: KINIK SIBEL TULGAR , TUNCER A. MURAT , ALTAY ÇIĞDEM
Published: March 2003

The effect of iron deficiency anaemia (IDA) on CD71 expression by peripheral blood lymphocytes was studied in 43 children with iron deficiency anaemia. 18 healthy age‐matched children were selected as the control group. 11 children with β‐thalassaemia trait were also studied. Lymphocytes bearing CD71 were enumerated by flow cytometric analysis of peripheral blood. At diagnosis, CD71+ peripheral lymphocytes (mean ± SE) was 5.90 ± 0.76% in patients with IDA and 12.60 ± 0.98% in healthy controls (P = 0.000). In β‐thalassaemia trait patients the peripheral blood CD71+ lymphocytes were 7.80 ± 1.20%. In IDA patients there was a statistically significant correlation between the levels of CD71+ peripheral lymphocytes and haemoglobin value (P = 0.000). In 19 patients studied at days 0 and 30 of oral iron therapy, the number of peripheral blood CD71+ lymphocytes was shown to be increased from 5.90 ± 0.76% to 12.11 ± 1.21%. In severe IDA presence of a limited number of CD71+ peripheral blood lymphocytes indicated that severe IDA should be borne in mind when considering conditions responsible for the suppression of lymphocyte proliferation.
Infections in adults undergoing unrelated donor bone marrow transplantation
Authors: WILLIAMSON EMMA , MILLAR MICHAEL , STEWARD COLIN , CORNISH JACQUELINE , FOOT ANNABEL , OAKHILL ANTHONY , PAMPHILON DERWOOD , REEVES BARNABY , CAUL E. , WARNOCK DAVID , MARKS DAVID
Published: March 2003

This study retrospectively reviews infections over a 7‐year period in 60 consecutive adults (median age 25 years) undergoing their first unrelated donor bone marrow transplant (UD‐BMT). T‐cell depletion was employed in 93%. More than half the patients had one or more severe, potentially life‐threatening, infections. There was a high incidence of invasive fungal infections (Aspergillus 17, Candida four), despite the use of itraconazole or amphotericin prophylaxis. Ten Aspergillus infections occurred beyond 100  d. Two patients (11%) with invasive aspergillosis survived. Clustering of infections was noted, with invasive fungal infections significantly associated with bacteraemias (OR 3.73, P = 0.06) and multiple viral infections (OR 4.25, P = 0.05). There were 21 severe viral infections in 16 patients, with CMV disease occurring in four patients only; viral pneumonitis was predominantly due to ‘community respiratory’ viruses. Most early bacteraemias (68%) were due to Gram‐positive organisms. The majority of episodes of Gram‐negative sepsis were caused by non‐fastidious non‐fermentative bacteria, such as Pseudomonas spp. and Acinetobacterspp., historically regarded as organisms of low pathogenicity. In patients with successful engraftment and minimal graft‐versus‐host disease, late infections suggestive of continued immune dysfunction (shingles, recurrent lower respiratory infections, Salmonella enteritis and extensive warts) were common.
P‐glycoprotein is an independent prognostic factor predicting relapse in childhood acute lymphoblastic leukaemia: results of a 6‐year prospective study
Authors: Dhooge C. , De Moerloose B. , Laureys G. , Kint J. , Ferster A. , De Bacquer D. , Philippe J. , Benoit Y.
Published: March 2003

P‐glycoprotein (P‐gp), a cellular drug‐efflux pump, is thought to be one of the major causes of multidrug resistance (MDR) in malignancies. Since therapeutic strategies are being developed to circumvent drug resistance by inhibiting P‐gp function, large prospective studies evaluating the clinical relevance of P‐gp in childhood acute lymphoblastic leukaemia (ALL) are warranted. P‐gp expression was evaluated over a period of 6 years in 102 consecutive patients with de novo childhood ALL and in 35 children with relapse of ALL. Bone marrow and blood smears were studied immunocytochemically with two monoclonal antibodies at initial diagnosis and at relapse. P‐gp expression was found in 14 (14%) patients at initial diagnosis. After induction treatment, complete remission was achieved in 100/102 patients (98%), of whom 19 relapsed. Cumulative event‐free survival was significantly higher in the P‐gp‐negative group compared with the P‐gp‐positive population (Logrank P = 0.02). Multivariate analysis showed the results to be independent of age, WBC count and karyotype, and concomitantly underlined the importance of MDR1 phenotype detection in childhood ALL. P‐gp expression was more frequently found at relapse (34%) than at primary diagnosis (P = 0.01). In the relapsed patient group, P‐gp‐positive patients had a 2‐fold greater risk for adverse clinical outcome than the P‐gp‐negative relapsed patients. P‐gp expression was not induced by exposure to previous chemotherapy since the majority of P‐gp‐negative patients remained negative at relapse. P‐glycoprotein expression in newly diagnosed childhood ALL is an independent adverse prognostic parameter with a predictive value for relapse.
Identification of a novel promoter mutation in the human pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia
Authors: Kugler Wilfried , Laspe Petra , Stahl Markus , Schröter Werner , Lakomek Max
Published: March 2003

Using direct sequencing we analysed the pyruvate kinase (PK) LR gene of a patient with severe haemolytic anaemia due to PK deficiency. A novel promoter mutation −249delA relative to the translation initiation site and the common 1529A mutation in exon 11 of the gene could be identified. Reverse transcription (RT)‐PCR analysis combined with restriction digestion revealed that the −249delA mutation leads to a reduction in the amount of mRNA produced from this allele to about 6% of normal. We assume that both mutations would account for the PK deficiency in the compound heterozygous patient.
A case of myelofibrosis with a t(4;13)(q25;q12): evidence for involvement of a second 13q12 locus in chronic myeloproliferative disorders
Authors: Macdonald Donald H. C. , Lahiri Diya , Chase Andrew , Sohal Jastinder , Goldman John M. , Cross Nicholas C. P.
Published: March 2003

Aberrations of 13q are frequently found in myeloproliferative disorders (MPD). In a case of primary proliferative polycythaemia which transformed to myelofibrosis, karyotype analysis at transformation revealed an acquired t(4;13)(q25;q12). FISH and Southern analysis demonstrated that the ZNF198 gene, disrupted in the t(8;13)(p11;q12) myeloproliferative syndrome, was unaffected by the t(4;13). FISH analysis mapped the 13q12 breakpoint to the genomic region flanked by the genetic markers D13S1126 and D13S1121. Physical mapping estimated this region to be <80 kbp. Our results suggest the possibility of a novel gene, distinct from ZNF198, that is located at chromosome 13q12 and involved in the pathogenesis of myelofibrosis.
Dendritic cells in chronic myelomonocytic leukaemia
Authors: Vuckovic S. , Fearnley D. B. , Gunningham S. , Spearing R. L. , Patton W. N. , Hart D. N. J.
Published: March 2003

Blood dendritic cells (DC) differentiate in vitro via two separate pathways: either directly from blood DC precursors (DCp) or from CD14+ monocytes. In chronic myelomonocytic leukaemia (CMML) abnormal bone marrow precursors contribute to blood monocyte development but DC development has not been studied previously. Monocytes comprised 60% of blood MNC in 15 CMML patients studied, compared with 20% in 16 age‐matched controls. The increase in blood monocytes was accompanied by a reciprocal decrease in mean blood DC percentage (from 0.42% of MNC in normal individuals to 0.16% of MNC in CMML patients). Absolute blood DC numbers showed a minimal (non‐significant) reduction from 9.8 × 106/l in normal individuals to 7.5 × 106/l in CMML patients. The CD14lowCD16+ monocyte subpopulation was not found in CMML patients. After culture in GM‐CSF/IL‐4, CMML CD14+ monocytes acquired the phenotype of immature monocyte derived DC (Mo‐DC) with similar yields to normal blood Mo‐DC generation. Addition of TNF‐α or LPS induced both normal and CMML Mo‐DC to express prominent dendritic processes, the CMRF44+ and CD83+ antigens and high levels of HLA‐DR, CD80 and CD86. Treatment either with TNF‐α or LPS increased the allostimulatory activity of normal Mo‐DC, but had little effect on the allostimulatory activity of CMML Mo‐DC, perhaps reflecting the underlying neoplastic changes in monocyte precursors. We conclude that the blood DC numbers are relatively unaffected in CMML, suggesting discrete regulation of monocyte and DC production.
Mechanism underlying factor VII deficiency in Jewish populations with the Ala244Val mutation
Authors: Hunault Mathilde , Arbini Arnaldo A. , Carew Josephine A. , Bauer Kenneth A.
Published: March 2003

We investigated a Sephardic Jewish patient with a mild bleeding diathesis whose plasma levels of factor VII coagulant activity and factor VII antigen were 7% and 9% of normal, respectively. Sequencing demonstrated homozygosity for the Ala244Val mutation and the Arg353Gln polymorphism, which is associated with a modest decrease in factor VII levels. To elucidate the mechanism by which Ala244Val reduced factor VII levels in this patient, transient transfections were performed in COS‐1 cells with wild type and mutant factor VII cDNAs and factor VII antigen levels in cell lysates and conditioned media were measured. The secretion of the mutant protein (FVII244V) into the media was 20% of wild type (FVIIwt), and intracellular levels of FVII244V were 60% of FVIIwt. A construct encoding Ala244Val along with the Arg353Gln polymorphism decreased the factor VII level in the media to that observed in the patient's plasma. Pulse‐chase experiments demonstrated that FVII244V did not accumulate intracellularly and that low levels of the abnormal protein were maintained throughout the chase. To test the hypothesis that FVII244V results in an unstable molecule, amino acids with smaller (Gly) or larger (Phe) side chains were substituted for Val244 by site‐directed mutagenesis. Transient transfection assays with these constructs demonstrated that the side chain of amino acid 244 is crucial in maintaining a proper conformation of the molecule. We conclude that Ala244Val results in a factor VII molecule that is unstable and is probably degraded intracellularly.