bluuebunny/crossref_metadata_2025 huggingface dataset:
The epitope specificity of heparin‐induced thrombocytopenia
Authors: HORSEWOOD PETER , WARKENTIN THEODORE E. , HAYWARD CATHERINE P. M. , KELTON JOHN G.
Published: November 2003

Heparin‐induced thrombocytopenia (HIT) is caused by antibodies (HIT‐Abs) that bind to a complex of heparin and platelet factor 4. We investigated the epitope specificity of the HIT‐Abs, and found that the HIT‐Abs recognized solid‐phase immobilized complexes with an optimum ratio of four to eight molecules of PF4 per molecule of heparin. To try to define the epitopes within the PF4 molecule, intact and reduced (linearized) PF4 was tested against 29 different sera from patients with HIT. In addition, eight different peptides that spanned the PF4 molecule were studied for their ability to bind to the HIT‐Abs either alone or in the presence of heparin. With the exception of a subpopulation of patient samples (5/29, 17%), we found that reduced PF4 and the peptides were uniformly nonreactive with the HIT‐Abs in the presence of heparin. Reduced PF4 and PF4 carboxy‐terminal peptides with a minimum size of 19 amino acids were recognized by a minority (5/29) of HIT‐Abs samples but only when heparin was present. The specificity of this subgroup of samples from patients with HIT was highly restricted and the loss of one amino acid (peptide reduced in length from 19 to 18 amino acids) rendered the peptides non‐reactive. The clinical characteristics of these patients were similar to the other HIT patients.

These studies demonstrate that the majority of HIT‐Abs recognize a noncontiguous conformational epitope on the PF4 molecule that is produced when four to eight PF4 molecules are bound together by heparin.
Thymoma and agranulocytosis: two case reports and literature review
Authors: Yip Desmond , Rasko John E. J. , Lee Choon , Kronenberg Harry , O'Neill Barry
Published: November 2003

Thymoma with agranulocytosis is a rare association. We describe two cases of agranulocytosis presenting with sepsis which were both found to have coincident benign spindle cell thymomas. One case, associated with promyelocyte arrest and hypogammaglobulinaemia, was treated successfully with granulocyte colony‐stimulating factor (G‐CSF). Thymectomy had no effect. The other case, associated with complete myeloid aplasia, proceeded to a fatal outcome after failure of treatment with granulocyte‐macrophage colony stimulating factor (GM‐CSF), plasmapheresis, thymectomy, intravenous immunoglobulin, cyclophosphamide and methylprednisolone. We also review the literature of thymoma in association with agranulocytosis.
Translocation t(3;17)(q23;q21): a new translocation in acute lymphoblastic leukaemia
Authors: Raanani Pia , Berkowicz Miriam , Rosner Esther , Rosenthal Ester , Berman Sara , Rechavi Gideon , Ben‐Bassat Isaac
Published: November 2003

We report on two adult patients with CD10+ positive acute lymphoblastic leukaemia (ALL) who presented with similar clinical and laboratory features and with a new chromosomal translocation: t(3;17)(q23;q21). This translocation may be involved in the formation of a new chimaeric transcription factor. Both patients shared several poor prognostic factors at presentation and an adverse clinical course. The t(3;17)(q23;q21) translocation may therefore predict a poor outcome in ALL.
Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines
Authors: Ernofsson Mats , Tenno Taavo , Siegbahn Agneta
Published: November 2003

Interleukin (IL)‐4, IL‐10, IL‐13 and transforming growth factor beta (TGF‐β) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT‐PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)‐induced tissue factor (TF) expression in human monocytes. The effects of IL‐4 and IL‐10 on monocyte chemoattractant protein‐1 (MCP‐1)‐ and C‐reactive protein (CRP)‐induced TF expression were also studied. A direct comparison revealed that IL‐4, IL‐10 and IL‐13 all down‐regulated LPS‐induced TF expression in a concentration‐dependent manner without the need for priming. In contrast, TGF‐β required 4 h of priming to inhibit TF expression induced by LPS. IL‐10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL‐4 and IL‐13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL‐4 and IL‐10 showed similar concentration‐dependent inhibition of MCP‐1‐ and CRP‐induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL‐10, in particular, may be a possible candidate as a TF‐preventing drug.
Sea‐blue histiocyte syndrome in bone marrow secondary to total parenteral nutrition including fat‐emulsion sources: a clinicopathologic study of seven cases
Authors: BIGORGNE C. , LE TOURNEAU A. , MESSING B. , RIO B. , GIRAUD V. , MOLINA T. , AUDOUIN J. , DIEBOLD J.
Published: November 2003

Bone marrow examination revealed a lipid‐laden histiocytosis in seven patients undergoing long‐term total parenteral nutrition necessitated by extensive short‐bowel surgical resection. Clinical abnormalities occurred during this treatment which required bone marrow examination. These included hepatosplenomegaly and peripheral blood cytopenia; the median time to the detection of these abnormalities was 64 months.  The most striking change within the bone marrow was the presence of many pigment‐laden histiocytes which had the typical morphology of sea‐blue histiocytes seen in the so‐called idiopathic sea‐blue histiocyte syndrome. The occurrence of sea‐blue histiocytosis in the bone marrow in association with long‐term parenteral nutrition for short‐bowel syndrome has not, to our knowledge, been reported previously and should now be considered in the differential diagnosis of bone marrow sea‐blue histiocytosis.
Clonal identification of Burkitt's Lymphoma Arising from Lymphocyte‐Predominant Hodgkin's Disease
Authors: Yoshinaga Haruhiko , Ohashi Kazuteru , Yamamoto Koh , Miki Tohru , Miura Osamu , Saito Takako , Koyama Takatoshi , Kanazawa Akira , Nemoto Tetsuo , Miyasaka Nobuyuki , Kamiyama Ryuichi , Hirosawa Shinsaku
Published: November 2003

The occurrence of large cell lymphomas subsequent to, or concurrent with, lymphocyte predominant Hodgkin's disease (LPHD) is a well‐documented phenomenon. We present a case of Burkitt's lymphoma of the bladder, occurring after the successful treatment of LPHD of a cervical lymph node. To evaluate the clonal relationship of the two tumours, we amplified the complementarity‐determining‐region 3 of two samples from paraffin‐embedded slides, using the polymerase chain reaction (PCR). The sequences of the PCR products showed 96% homology to each other. These results indicate that the malignant clone of Burkitt's lymphoma arose from the corresponding LPHD.
Amplification of mitochondrial DNA in acute myeloid leukaemia
Authors: Boultwood J. , Fidler C. , Mills K. I. , Frodsham P. M. , Kusec R. , Gaiger A. , Gale R. E. , Linch D. C. , Littlewood T. J. , Moss P. A. H. , Wainscoat J. S.
Published: November 2003

There is a long‐standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML).

We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2–50 were observed in 25 AML samples and 13 of these showed 8‐fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3–10‐fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.
Specificities of anti‐platelet antibodies in multitransfused patients with haemato‐oncological disorders
Authors: Kurz Martin , Greinix Hildegard , HO¨cker Paul , Kalhs Peter , Knöbl Paul , Mayr Wolfgang Richard , Pober Michael , Panzer Simon
Published: November 2003

The clinical condition and the formation of platelet‐reactive antibodies influence the post‐transfusion platelet increment. We analysed the specificities of platelet‐reactive antibodies in 81 multitransfused patients with haemato‐oncological diseases refractory to platelet transfusions, or prior to a scheduled stem cell transplantation. In 17 additional patients we prospectively determined the development of platelet‐reactive antibodies at the time of chemotherapy in weekly intervals. Sera were tested by the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA)‐technique for antiplatelet antibodies against HLA class I antigens, the human platelet‐specific alloantigens (HPA)‐1, ‐2, ‐3, ‐5, and the platelet membrane glycoproteins (GP) Ia/IIa, Ib/IX, IIb/IIIa (panreactive).

Platelet reactive antibodies were found in 54% of blood samples. They were frequent in patients with a history of possible previous immunization, particularly if patients were refractory to platelet transfusions. Platelet‐reactive HLA antibodies were the most common antibodies. Even patients without a known risk of primary immunization who received exclusively leucocyte‐depleted blood products formed antibodies. By the MAIPA technique, even LCT‐negative sera were found to contain platelet‐reactive antibodies.
Fluorescence in situ hybridization in combination with morphology detects minimal residual disease in remission and heralds relapse in acute leukaemia
Authors: Bernell Per , Arvidsson Ingrid , Jacobsson Björn , Hast Robert
Published: November 2003

Fluorescence in situ hybridization in combination with morphology (MGG/FISH) was used to detect minimal residual disease (MRD) in complete remission (CR) in 12 cases of acute leukaemia (six MDS‐AML, five de novo AML, one pre‐B ALL) with numerical chromosomal aberrations at diagnosis. Residual leukaemic cells could be detected in the remission bone marrows by MGG/FISH in five patients, whereas the other seven showed no abnormalities. All five patients with signs of MRD at CR relapsed in the bone marrow within 2–9 months, in contrast to two of seven with a normal finding by MGG/FISH at CR. In both these patients a second MGG/FISH analysis showed that a subpopulation of leukaemic blasts had reappeared, 4 and 5 months prior to the leukaemia becoming clinically overt. One patient suffered a CNS relapse, but without any evidence of bone marrow involvement. The remaining four patients with no evidence of MRD at CR were still in haematological remission at follow‐up after 4, 11, 12 and 13 months, respectively. We conclude that MGG/FISH seems to be a clinically useful method to detect MRD in acute leukaemia and to predict relapses, particularly when repeat studies are performed during CR.
Probable regulation of factor VIIa–tissue factor and prothrombinase by factor Xa–TFPI and TFPI in vivo
Authors: Gouin‐Thibault Isabelle , Dewar Lori , Craven Sharon , Kulczycky Myron , Wun Tze‐Chein , Ofosu Frederick A.
Published: November 2003

Given that factor VIIa–tissue factor (TF) probably initiates coagulation in vivo, this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1+2 in plasma (the latter as an index of prothrombinase activity in vivo). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa–TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa–TFPI inactivates factor VIIa–TF and prothrombinase. Established enzyme‐linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1+2, whereas we developed an ELISA to quantify factor Xa–TFPI using affinity purified rabbit (anti‐human TFPI)‐IgG and chicken anti‐(human factor Xa–TFPI)‐IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1+2 increased in parallel in the plasmas of up to 145 healthy adults assayed (P = 0.007), as did the concentrations of factor VIIa and TFPI (P = 0.0039), and prothrombin fragment 1+2 and TFPI (P = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa–TFPI and factor VIIa (P <0.0001) and free factor Xa–TFPI and prothrombin fragment 1+2 (P=0.0095). These results are consistent with factor Xa–TFPI regulating factor VIIa–tissue factor and prothrombinase in vivo.
Serum thrombopoietin levels in patients with aplastic anaemia
Authors: Marsh J. C. W. , Gibson F. M. , Prue R. L. , Bowen A. , Dunn V. T. , Hornkohl A. C. , Nichol J. L. , Gordon‐Smith E. C.
Published: November 2003

Endogenous serum thrombopoietin (TPO) levels were measured in 31 patients with aplastic anaemia (AA) using an enzyme immunoassay with a sensitivity of 20 pg/ml. The median platelet count for all AA patients was 30 ± 29 × 109/l (range 5–102) compared with a median of 284 ± 59 × 109/l (range 148–538) for normal controls. Serum TPO levels were significantly elevated in all patients compared with normals (1706 ± 1114.2, range 375–5000 v 78 ± 54, range 16.5–312.9, P < 0.0001). There was no correlation between serum TPO levels and the degree of thrombocytopenia in AA patients, but TPO levels were significantly higher in patients who were platelet transfusion dependent than in patients who were transfusion independent (P < 0.01). There was a trend for higher TPO levels in patients with severe AA compared with non‐severe AA patients. Clinical trials of TPO and a related truncated, pegylated molecule, megakaryocyte growth and development factor (PEG‐rHuMGDF), are awaited to determine whether treatment with these drugs will result in increased platelet counts in patients with AA.
Flow cytometric analysis of platelets from childrenwith the Wiskott‐Aldrich syndrome reveals defects in platelet development, activation and structure
Authors: Semple John W. , Siminovitch Katherine A. , Mody Meera , Milev Youli , Lazarus Alan H. , Wright J. Fraser , Freedman John
Published: November 2003

The pathophysiology of platelet dysfunction in the Wiskott‐Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet‐associated IgG and reduced platelet‐dense granule content. Levels of reticulated ‘young’ platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar‐sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin‐ and ADP‐induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F‐actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality.
Immunomagnetic selection of CD34+peripheral blood stem cells for autografting in patients with breast cancer
Authors: Hohaus Stefan , Pförsich Margit , Murea Simona , Abdallah Alhossain , Lin Yung‐Sheng , Funk Liane , Voso Maria Teresa , Kaul Sepp , Schmid Hans , Wallwiener Diethelm , Haas Rainer
Published: November 2003

Contamination of transplants with tumour cells may contribute to relapse after peripheral blood stem cell transplantation (PBSCT). We studied the feasibility of CD34+ cell selection from blood‐derived autografts obtained following G‐CSF‐supported cytotoxic chemotherapy in a group of 25 patients with breast cancer (10 with high‐risk stage II/III and 15 with stage IV without bone or bone marrow involvement).

Using immunomagnetic beads (Isolex 300 SA, Baxter) CD34+ cells were enriched and released by chymopapain resulting in a median purity of 95% (range 82–99%) and a median recovery of 80% (range 27–132%). The enrichment procedure did not change the proportion of CD34+ subsets coexpressing HLA‐DR, CD38 and Thy‐1, while L‐selectin was removed from the cell surface following selection. Using a sensitive immunocytological technique with a cocktail of epithelial‐specific antibodies (anti‐cytokeratin 8, 18 and 19; HEA125; BM7 and BM8), five leukaphereses products contained epithelial cells, whereas the selected CD34+ cell fraction was free of tumour cells. A neutrophil count of 0.5×109/l and a platelet count of 20×109/l was reached after a median time of 14 and 10 d following 40 high‐dose chemotherapy (HDC) cycles. Our results indicate that immunomagnetic selection of CD34+ cells yields highly purified autografts devoid of tumour cells whereas the engraftment ability of the progenitor and stem cells is fully retained.
Immunoglobulin heavy chain Diversity genes rearrangement pattern indicates that MALT‐type gastric lymphoma B cells have undergone an antigen selection process
Authors: Bertoni Francesco , Cazzaniga Giovanni , Bosshard Giovanna , Roggero Enrico , Barbazza Renzo , De Boni Michele , Capella Carlo , Pedrinis Ennio , Cavalli Franco , Biondi Andrea , Zucca Emanuele
Published: November 2003

Gastric MALT lymphoma usually develops from chronic gastritis, the vast majority of which (>90%) is associated with Helicobacter pylori infection. We sequenced the third complementarity determining region (CDR3) of immunoglobulin heavy chain genes in 19 gastric MALT lymphoma clones to determine the pattern of variable (V), diversity (D) and joining (J) gene utilization during immunoglobulin gene rearrangement.

DNA was extracted from paraffin‐embedded sections and the rearranged CDR3 regions were amplified using a semi‐nested polymerase chain reaction (with primers complementary to the conserved framework‐three segment of the variable region [FR3A] and J regions). The DNA used for cloning and sequencing was obtained after purification of monoclonal bands excised from polyacrylamide gels. The N‐D‐N region specific to each clone was compared with known germline D sequences.

Similarly to that observed in normal and leukaemic B cells, our series of gastric MALT lymphomas showed apparent preferential utilization of genes from the DXP family. In two cases no similarity between the CDR3 nucleotide sequences of the neoplastic clones and the known germline D sequences could be found. In 10/19 analysed alleles the lymphoma B‐cell clones appeared to contain two D gene segments (D‐D recombination), a rare occurrence in normal individuals but one which has been described as a significant event in the determination of idiotype expression and antigen‐binding affinity. Remarkably, despite the use of different D and J segments, the resultant amino acid sequences matched in two patients, suggesting the presence of a common selecting antigen.

The observed pattern of D gene rearrangement suggests that MALT lymphoma B‐cell clones have undergone antigen selection, which seems to indicate that the antigen stimulation plays a pivotal role in the development of the lymphoma.
IMMUNOGLOBULIN CLASS SWITCH FROM IgA1 TO IgG2 AND SIMULTANEOUS ASSOCIATION WITH BENCE JONES PROTEINURIA IN THE ESCAPE PHASE IN A MYELOMA PATIENT TREATED WITH INTERFERON ALPHA
Authors: Kozuru M. , Uike N. , Takahira H. , Yufu Y. , Goto T. , Muta K.
Published: November 2003

Immunoglobulin (Ig) class switch from α1 to γ2 associated with κ‐type Bence Jones proteinuria was evident in the escape phase of an IgA1 myeloma patient treated with interferon α (IFNα). The additional M‐protein, IgG2‐κ, level rapidly increased and was associated with Bence Jones proteinuria, whereas monoclonal IgA1‐κ progressively declined. The N‐terminal 21 amino acid sequences of the κ‐chains of monoclonal IgA1, IgG2 and the Bence Jones protein were the same. The N‐terminal 15 amino acid sequence of the γ2‐chain was identical to that of the α1‐chain. Based on these findings, the IgA1 myeloma cells underwent a class switch in CH gene expression from α1 to γ2 with cell differentiation in vivo. The mechanism of the Ig class switching is discussed from three points of view: (1) Increase in immature and plasmablastic myeloma cells in the escape phase is susceptible to Ig class switching by the T‐cell‐ derived cytokines. (2) We presumed that administered IFNα increased the amounts of secreted IFNγ from the Th1 cells. (3) Due to a large quantity of IFNγ, an inducer of Cγ2 germ‐line transcript, Ig class switching occurred stepwise from the α1 constant region gene to the next 3′CH γ2 gene.
Childhood acute lymphoblastic leukaemia with ider(21)(q10)t(12;21)(p12;q22): a new recurrent abnormality showing ETV6/CBFA2 fusion
Authors: ANDREASSON PATRIK , JOHANSSON BERTIL , STRÖMBECK BODIL , DONNÉR MIKAEL , MITELMAN FELIX , HÖGLUND MATTIAS
Published: November 2003

The cytogenetically unidentifiable t(12;21) (p12;q22), resulting in ETV6/CBFA2 fusion, is the most frequent chromosomal aberration in childhood acute lymphoblastic leukaemia (ALL). We report a variant, ider(21) (q10)t(12;21)(p12;q22), which was shown to contain double ETV6/CBFA2 fusions by fluorescence in situ hybridization. This is the second case of such an ider(21) in childhood ALL, suggesting that it is a new recurrent abnormality. Since the ider(21) is cytogenetically indistinguishable from i(21)(q10) and idic(21)(p11), changes associated with similar clinical features as the t(12;21), i.e. pre‐B‐cell ALL and age 1–10 years, we suggest that all ALL displaying these changes should be tested for ETV6/CBFA2 fusion transcript.
Microsatellite instability and other molecular abnormalities in childhood acute lymphoblastic leukaemia
Authors: Takeuchi Seisho , Seriu Taku , Tasaka Taizo , Koike Michiaki , Cho Steve K. , Park Susan , Slater James , Mufti Iqbal , Hatta Yoshihiro , Miyoshi Isao , Bartram Claus R. , Koeffler H. Phillip
Published: November 2003

Microsatellite instability (MSI) has been considered to represent the defect of DNA mismatch repair systems and has been implicated in the tumourigenesis of several human malignancies. To investigate the possible presence of microsatellite instability in childhood acute lymphoblastic leukaemia (ALL), we examined 48 primary ALL samples. Instability was determined at 85 different microsatellite loci localized to 12 different chromosome arms.  Microsatellite instability was detected in five (10%) samples. Interestingly, the instability was found at chromosomal regions associated with frequent alterations. Two samples had instability at the microsatellite marker within the TEL gene on chromosome arm 12p. Two other samples had instability at a microsatellite marker close to CDKN2/p16 on 9p; one of these samples had a homozygous deletion at 9p21. The fifth sample had instability at the microsatellite marker on 6q, which we have found is a frequent region of loss of heterozygosity in childhood ALL.

Taken together, instability was rare in childhood ALL, but was localized to the three most frequently deleted chromosome regions in childhood ALL, suggesting that localized microsatellite instability may identify a fragile chromosomal region which could result in alteration of surrounding target genes and lead to leukaemia.
Stem cell mobilization in resistant or relapsed lymphoma: superior yield of progenitor cells following a salvage regimen comprising ifosphamide, etoposide and epirubicin compared to intermediate‐dose cyclophosphamide
Authors: McQuaker I. G. , Haynes A. P. , Stainer C. , Anderson S. , Russell N. H.
Published: November 2003

We analysed the factors influencing the efficacy of peripheral blood stem cell (PBSC) collection in patients with lymphoma. Sixty‐six patients underwent initial PBSC collection following mobilization with chemotherapy plus recombinant granulocyte colony‐stimulating factor (300 μg/d). Patients were mobilized with one of two chemotherapy regimens, either cyclophophamide (3 g/m2 or 4 g/m2) (n=50) or ifosphamide, etoposide and epirubicin (IVE; n=16). The target of collecting >2.0×106 CD34+ cells/kg was achieved in 43/66 (65%) patients with a median of two apheresis procedures. The IVE plus G‐CSF mobilization regimen gave a significantly higher median yield of CD34+ cells (8.62 × 106/kg) compared with cyclophosphamide plus G‐CSF (3.59 × 106/kg) (P=0.045). The median yield of CD34+ cells per leukapheresis was almost twice as high in patients receiving IVE (1.94 × 106/kg) compared to cyclophosphamide (1.03 ×106/kg) (P= 0.035). In a univariate analysis of the factors affecting mobilization, the subtype of lymphoma (high‐grade NHL) and the mobilization regimen were the only factors associated with high CD34+ cell yield. However, in a multivariate analysis of factors affecting mobilization including age, lymphoma subtype, previous chemotherapy and radiotherapy, only the use of the IVE protocol was predictive of a high yield of CD34+ cells. In 13 patients undergoing a second mobilization procedure the use of IVE was associated with a significantly higher yield of CD34+ cells compared to cyclophosphamide; three patients who failed cyclophosphamide plus G‐CSF mobilization were able to proceed to transplantation following success‐ful mobilization with IVE + G‐CSF. These results demon‐strate that IVE is a highly effective mobilization regimen which is superior to cyclophophamide and has the benefit of being effective salvage therapy for lymphoma patients.
Re‐induction of complete remission with a new synthetic retinoid, Am‐80, for relapse of acute promyelocytic leukaemia previously treated with all‐trans retinoic acid
Authors: Takeuchi Makoto , Yano Tomofumi , Omoto Eijiro , Takahashi Kiyoshi , Kibata Masayoshi , Shudo Koichi , Ueda Ryuzo , Ohno Ryuzo , Harada Mine
Published: November 2003

Two patients with relapsed acute promyelocytic leukaemia previously treated with all‐trans retinoic acid (ATRA), were treated with a new synthetic retinoid, Am‐80. In both patients pancytopenia gradually resolved without an increase in leukaemic cells, and differentiation of leukaemic cells was observed morphologically in bone marrow. Without the use of anti‐leukaemic agents, both cases achieved complete remission (CR) on days 52 and 38 of treatment, respectively. On the day of CR, PML gene rearrangement and the t(15;17) translocation disappeared, though PML‐RAR α chimaeric messenger RNA was still detected by reverse transcriptase polymerase chain reaction. Both patients then received conventional chemotherapy for consolidation of CR. These clinical experiences suggest that Am‐80 may be an active agent for APL patients who have relapsed from ATRA‐induced remission.
Immunophenotypic and ultrastructural study in peripheral blood neutrophil granulocytes following bone marrow transplantation
Authors: MASAT T. , FELIU E. , VILLAMOR N. , CASTELLSAGUÉ J. , ORDI J. , FABREGUES M. , ROZMAN C.
Published: November 2003

Neutrophil studies after bone marrow transplantation (BMT) describe chemotactic and phagocytotic alterations and dyshaemopoiesis. Neutrophil granulocytes (NG) in peripheral blood after BMT were analysed in 28 patients. 14 patients (six receiving GM‐CSF) underwent autologous BMT and 14 underwent allogeneic BMT. Immunophenotypic and electron microscopic studies were performed during post‐BMT granulopoietic regeneration. Results were compared with NG from 15 healthy bone marrow donors (control group A) and from six patients receiving intensive chemotherapy before autologous BMT (control group B).

A significant increase in CD15 and a decrease in 8C7 antigen expression was observed in peripheral blood NG from BMT patients compared with controls A. MPO‐7 in NG after BMT did not differ from control group A. Autologous BMT patients showed a lower percentage of NG expressing 13F6, 31D8 and CD16 (Leu 11a) than allogeneic BMT patients, and a significant decrease in 8C7 antigen expression compared with patients receiving intensive chemotherapy. Ultrastructurally, a marked decrease of azurophilic granules was observed in NG from BMT patients compared with control groups A and B. These data indicate that repopulation after BMT was made by phenotypically less mature NG with dysgranulopoietic features. Differences between autologous and allogeneic BMT patients may be partly related to GM‐CSF usage. In conclusion, NG present immunophenotypic and ultrastructural changes after BMT which may be involved in abnormal NG response against bacterial infections, although further investigation is needed.
Clonal haemopoiesis in normal elderly women: implications for the myeloproliferative disorders and myelodysplastic syndromes
Authors: Champion Kim M. , Gilbert James G. R. , Asimakopoulos Fotios A. , Hinshelwood Stephen , Green Anthony R.
Published: November 2003

Studies of X chromosome inactivation patterns are central to many aspects of our understanding of the pathogenesis of haematological malignancies. In patients with myeloproliferative disorders and myelodysplastic syndromes the demonstration of skewed X inactivation patterns in multiple haemopoietic lineages has been taken to indicate a stem cell origin for these groups of diseases. However, stem cell depletion or selection pressures can also produce skewed X inactivation patterns and might increase with age. We have therefore used the HUMARA assay to study X inactivation patterns of elderly patients with myeloproliferative disorders together with an age‐matched control group of normal elderly women.  A clonal pattern (clonal granulocytes and polyclonal T cells) was observed in 23.1% of normal women and 63.4% of patients with myeloproliferative disorders. This is the first report of X inactivation patterns in purified subpopulations of blood cells in normal elderly women. These results have three significant implications. Firstly, the finding of clonal granulocytes and polyclonal T cells in normal elderly women is likely to reflect age‐related stem cell depletion or selection pressures. Secondly, the demonstration of clonal granulocytes and polyclonal T cells is not a useful diagnostic marker for myeloproliferative disorders or myelodysplastic syndromes in elderly women. Thirdly, our data raise the possibility that clonal blood cell patterns may precede rather than follow mutations which subsequently give rise to myelodysplastic or myeloproliferative phenotypes.
A human monoclonal anti‐D antibody which detects a nonconformation‐dependent epitope on the RhD protein by immunoblot
Authors: Apoil P. A. , Reid M. E. , Halverson G. , Mouro I. , Colin Y. , Roubinet F. , Cartron J‐P. , Blancher A.
Published: November 2003

We describe the first human monoclonal anti‐D (LOR‐15C9) which reacts with a D‐specific motif exposed either on a native form on intact D‐positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR‐15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription‐translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR‐15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314–358). These findings correlate well with the reactivity of LOR‐15C9 with erythrocytes of some nonhuman primates (Dgor‐positive gorillas), but not of chimpanzee and Old or New World monkeys.

In membrane proteins from partial DVI red cells, LOR‐15C9 detected two proteins of molecular weight 33 and 21 kD; the presence of the latter was specific for category DVI and presumably represented the product of an alternatively spliced RHDVI transcript in these cells. This is consistent with the finding that LOR‐15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription‐translation and lacking amino‐acids 163–313 encoded by exons 4–6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D−−, using a rabbit anti‐Rh polypeptide antibody (MPC8) raised against the C‐terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript.

This study demonstrates that LOR‐15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.
Thrombopoietin‐independent effect of interferon‐γ on the proliferation of human megakaryocyte progenitors
Authors: Muraoka Kenji , Tsuji Kohichiro , Yoshida Makoto , Ebihara Yasuhiro , Yamada Kaoru , Sui Xingwei , Tanaka Ryuhei , Nakahata Tatsutoshi
Published: November 2003

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon‐γ receptor (IFN‐γR). To clarify the precise functional roles of IFN‐γR in human CD34+ cells, we examined the effect of IFN‐γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum‐free clonal culture. Surprisingly, IFN‐γ alone supported only megakaryocyte (MK) colonies in a dose‐dependent manner with a plateau level at 1000 U/ml of IFN‐γ. IFN‐γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 103 CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)‐3, stem cell factor (SCF) or IL‐6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti‐IFN‐γ monoclonal antibody (mAb) to the IFN‐γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO‐dependent production of MK colonies. In contrast, although anti‐TPO polyclonal Ab almost completely blocked TPO‐dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN‐γ, suggesting that the observed effect of IFN‐γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN‐γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL‐3‐dependent MK colony formation. Additionally, IFN‐γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa‐positive megakaryocytes. These results suggest that IFN‐γ may have regulatory roles in human megakaryocytopoiesis.
Acquired skewing of X‐chromosome inactivation patterns in myeloid cells of the elderly suggests stochastic clonal loss with age
Authors: Gale Rosemary E. , Fielding Adele K. , Harrison Claire N. , Linch David C.
Published: November 2003

More frequent skewing of X‐chromosome inactivation patterns (XCIPs) occurs in the white blood cells of elderly females; this study was performed to determine whether this occurs in myeloid or lymphoid lineages. XCIPs were analysed in purified neutrophils and T cells from 80 females  > 75 years and the results were compared with 23 cord blood and 94 younger adult blood samples. The degree of XCIP skewing in cord blood and younger adult blood cells was similar, with 3–4% having  > 90% expression of one allele. Skewing was markedly increased in the neutrophils of elderly females, with 33% having  > 90% expression of one allele (P < 0.0001). Extreme skewing was present in only 9% of the elderly T‐cell samples and no evidence of T‐cell clonality was found by PCR analysis of the TCRγ gene. The high level of acquired skewing of the XCIPs in myeloid cells of the elderly suggests that with time there is a change in stem cell usage with stochastic loss of some of the original stem cells. This has major implications for the use of XCIP analysis in the diagnosis of myeloid malignancies in the elderly and for gene therapy into haemopoietic stem cells.
Danaparoid for cardiopulmonary bypass in patients with previous heparin‐induced thrombocytopenia
Authors: Gillis Shmuel , Merin Gideon , Zahger Doron , Deeb Maher , Drenger Benjamin , Hyam Esti , Eldor Amiram
Published: November 2003

Anticoagulation for cardiopulmonary bypass in patients with heparin‐induced thrombocytopenia requires the use of other anticoagulants. We report a case in whom this was achieved using the heparinoid danaparoid (Orgaran). Based on our experience and a review of the literature, we provide guidelines for managing these rare patients. A danaparoid dose substantially lower than that recommended by the manufacturer may minimize bleeding complications.
Allogeneic blood or marrow transplantation for chronic lymphocytic leukaemia: timing of transplantation and potential effect of fludarabine on acute graft‐versus‐host disease
Authors: Khouri Issa F. , Przepiorka Donna , Van Besien Koen , O'Brien Susan , Palmer Judy L. , Lerner Susan , Mehra Rakesh C. , Vriesendorp Huibert M. , Andersson Borje S. , Giralt Sergio , Körbling Martin , Keating Michael J. , Champlin Richard E.
Published: November 2003

The outcome of allogeneic haemopoietic transplants including the rate of immune complications for patients with chronic lymphocytic leukaemia (CLL) refractory to or relapsing after chemotherapy with fludarabine was analysed.

Fifteen patients with advanced CLL who received allogeneic transplantation were prospectively analysed. All patients had previously received chemotherapy with fludarabine for 3–15 courses; 12 were refractory. The median number of circulating CD4^plus; and CD8^plus; lymphocytes at the time of transplant was 0.49 ×109 l and 0.23 ×109 l, respectively. One patient was transplanted from a one HLA‐antigen mismatched unrelated donor. Three others received a one or two antigen mismatched graft and 11 had HLA‐identical sibling donors. Patients received cyclosporine or tacrolimus in addition to methotrexate or methylprednisolone for prophylaxis of acute graft‐versus‐host disease (aGVHD).

Fourteen patients engrafted; one patient had graft failure, but recovered after therapy with intravenous immunoglobulin. 13 (87%) achieved complete remission (CR). Nine (53%) remain alive and in CR with a median follow‐up of 36 (range 3–60) months. None developed visceral graft‐versus‐host disease. These data compared favourably to published reports in other leukaemia patients and for patients with CLL who received a comparable immunosuppressive therapy but without prior fludarabine exposure.

This data indicates that allogeneic haemopoietic transplantation can induce durable remission in patients with CLL refractory to fludarabine and that it is reasonable to delay transplantation until failure of fludarabine therapy. It also suggests that prior exposure to fludarabine may decrease the incidence of severe aGVHD, possibly through its immunosuppressive effects. Further studies are warranted to evaluate this observation.
Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system
Authors: Drake M. , Ranaghan L. , Morris T. C. M. , Nolan L. , Desai Z. R. , Irvine A. E. , Jordan A. , Magill K. , Price S.
Published: November 2003

A quantitative analysis of peripheral blood stem cell (PBSC) yield, measuring absolute numbers of CD34+ cells × 106/kg and CFU‐C × 104/kg was performed in 74 consecutive patients. The interval or ‘gap’ from the end of previous chemotherapy to the date of priming was recorded in weeks.

Geometric mean CD34 and CFU‐C values were significantly higher in patients with a score of ≤ 60 compared to those with score > 60 (P = 0.003 and 0.02, respectively) and a significant difference in CD34 values was also found when scores of ≤ 38 were compared with scores > 38 (P = 0.003), with the difference in CFU‐C values approaching significance (P = 0.08). Patients exposed to toxicity factor 4 drugs had significant lowering of both CD34 and CFU‐C values (P < 0.001 and P = 0.038) and this emerged as the only independent factor when analysed using linear regression. No significant difference in the geometric mean CD34 or CFU‐C values of patients was found in any of the gap categories analysed. Prior exposure to toxicity factor 4 drugs had a significant adverse effect on stem cell yield and should be avoided or minimized prior to stem cell harvesting.
Beta‐thalassaemia in the immigrant and non‐immigrant German populations
Authors: Vetter B. , Schwarz C. , Kohne E. , Kulozik A. E.
Published: November 2003

In Germany homozygous β‐thalassaemia mainly occurs in the immigrant population from endemic regions. In non‐immigrants β‐thalassaemia is rare. Heterozygous β‐thalassaemia minor, however, is more common and must be considered in the differential diagnosis of hypochromic anaemia. The clinical and molecular data of 221 homozygous patients and 256 non‐immigrant German heterozygous individuals are presented. Clinically, 87% (n =192) of the homozygotes are classified as thalassaemia major (TM) and the other 13% as thalassaemia intermedia (TI). There is a wide spectrum of 39 thalassaemia mutations which occur with relatively low frequencies in individual cases. In 17/29 TI patients ‘mild’ mutations have been found and in 16/29 there are mutations that are associated with increased γ‐globin gene activity. α‐Thalassaemia is rare and found only in 3/29.

In the 256 Germans with heterozygous β‐thalassaemia there are 27 different thalassaemia mutations. The three most common are Mediterranean, together accounting for 61%. Also relatively common (5%) is an otherwise rare frameshift mutation of codon 83 (FS83 ΔG). The other mutations occur in < 10 individuals. Two mutations described here are novel. One of them affects position −2 of the intron 1 splice acceptor site (IVSI‐129 A‐G) and the other is a deletion of a single G in codon 15/16 (FS 15/16 ΔG). These data suggest that β‐thalassaemia in Germans was introduced from the Mediterranean in about two‐thirds of cases and that the remaining third has probably originated locally.
Elevated levels of shed syndecan‐1 correlate with tumour mass and decreased matrix metalloproteinase‐9 activity in the serum of patients with multiple myeloma
Authors: Dhodapkar Madhav V. , Kelly Thomas , Theus Allison , Athota Anupama B. , Barlogie Barthel , Sanderson Ralph D
Published: November 2003

Sera from 20 myeloma patients and 12 normal controls were analysed for the presence of syndecan‐1 and matrix metalloproteinase‐9 (MMP‐9). The level of syndecan‐1 in the serum was elevated in 7/20 (35%) myeloma patients whilst 6/19 patients (31%) had decreased serum MMP‐9 activity. The presence of increased syndecan‐1 was associated with decreased serum MMP‐9. Both elevated syndecan‐1 and decreased MMP‐9 were associated with higher marrow plasmacytosis, serum beta‐2 microglobulin and paraprotein levels. These data provide evidence that the syndecan‐1 ectodomain is shed in vivo. Quantitation of serum syndecan‐1 may be a useful measure of tumour mass and may have important implications for myeloma biology.
The venous thrombosis risk factor 20210 A allele of the prothrombin gene is not a major risk factor for arterial thrombotic disease
Authors: Corral Javier , Gonzalez‐Conejero Rocio , Lozano Maria L. , Rivera Jose , Heras Inmaculada , Vicente Vicente
Published: November 2003

A nucleotide change (G to A transition) at position 20210 has recently been demonstrated to be a risk factor for venous thrombosis. The relevance of this polymorphism to thrombotic disease was investigated by genotypic identification in three prospective case–control studies: 101 case patients with acute coronary heart disease (CHD), 104 patients with acute cerebrovascular disease (CVD), 82 patients with a confirmed diagnosis of deep venous thrombosis (DVT), and one control age‐ and sex‐matched for each patient. The prevalence of the genetic variation was significantly associated with the occurrence of DVT, but did not differ in patients with CHD or CVD from that in controls, suggesting that this allele should not be considered a major risk factor for arterial thrombotic disease.
De novo methylation of tumour suppressor genes CDKN2A and CDKN2B is a rare finding in B‐cell chronic lymphocytic leukaemia
Authors: Martel Véronique , Guerci Agnès , Humbert Jean‐Claude , Gregoire Marie‐José , Chery Michèle , Lederlin Pierre , Jonveaux Philippe
Published: November 2003

De novo methylation of the 5′CpG island has been recently reported as an alternative mechanism of inactivation for the tumour suppressor genes CDKN2A and CDKN2B. We examined CDKN2A methylation status at diagnosis in 42 B‐cell chronic lymphocytic leukaemia (CLL) patients, in 19 cases the CDKN2B methylation status was also analysed. No homozygous CDKN2A/2B deletion was detected, but four patients (9%) displayed an aberrant CDKN2A methylation status and only one had hypermethylated CDKN2B. De novo methylation was associated with silencing of gene expression. These results confirm that CDKN2A/2B inactivation by deletion is a rare event in CLL and suggest that aberrant methylation could be an alternative way of inactivation very rarely involved in the development of some CLL.
Mucormycosis in patients with haematological malignancies: a retrospective clinical study of 37 cases
Authors: Pagano Livio , Ricci Paolo , Tonso Anna , Nosari Annamaria , Cudillo Laura , Montillo Marco , Cenacchi Annarita , Pacilli Leonardo , Fabbiano Francesco , Del Favero Albano ,   GIMEMA Infection Program
Published: November 2003

A retrospective study of 37 patients with haematological malignancy (21 acute myeloid leukaemia, 11 acute lymphoid leukaemia, two lymphoma, two hairy cell leukaemia, one Hodgkin's disease) and histologically documented mucormycosis was conducted to evaluate the clinical characteristics and ascertain the factors which influenced the outcome from mycotic infection. Patients were admitted to 18 haematology divisions in tertiary care or university hospitals in Italy between 1987 and 1995.

Fever, thoracic pain, dyspnoea and cough were the most frequent presenting symptoms. At the onset, 89% patients were neutropenic (neutrophil counts < 0.5 × 109/l) with a median duration of previous neutropenia of 14 d (range 6–60). The most frequent sites of infection were lungs (81%), CNS (27%), sinus (16%), liver (16%) and orbital space (10%). Only three patients were asymptomatic. A correct in vivo diagnosis was made in only 13 (35%) patients. When performed, thoracic and cranial CT scan were the most useful diagnostic investigations. Despite the fact that 26 febrile patients were treated with empirical antifungal treatment, 28 of the 37 patients (76%) died from fungal infection at a median time of 17 d from the onset of clinical symptoms. Nine patients were cured by antifungal therapy plus, in five cases, radical surgery procedures.

An analysis of factors influencing outcome demonstrated that the resolution of chemotherapy‐induced neutropenia and prolonged treatment with amphotericin B and, if feasible, radical surgical debridement treatment, were significantly correlated with recovery from infection.

Mucormycosis, a rare filamentous fungal infection that occurs most frequently in neutropenic acute leukaemia patients, is characterized by a high mortality rate. Extensive and aggressive diagnostic and therapeutic procedures are essential to improve the prognosis in these patients.