bluuebunny/crossref_metadata_2025 huggingface dataset:
Splenic irradiation before bone marrow transplantation for chronic myeloid leukaemia
Authors: Gratwohl A. , Hermans J. , V. Biezen A. , Arcese W. , Debusscher L. , Ernst P. , Ferrant A. , Frassoni F. , Gahrton G. , Iriondo A. , Kolb H. J. , Link H. , Niederwieser D. , Ruutu T. , Schattenberg A. , Schmitz N. , Siegert W. , Torres‐Gomez A. , Vernant J. P. , Zwaan F. E. , Apperley J.
Published: November 2003

A total of 229 patients with chronic myeloid leukaemia (CML) in chronic phase were randomized between 1986 and 1990 to receive or not receive additional splenic irradiation as part of their conditioning prior to bone marrow transplantation (BMT). Both groups, 115 patients with and 114 patients without splenic irradiation, were very similar regarding distribution of age, sex, donor/recipient sex combination, conditioning, graft‐versus‐host disease (GvHD) prevention method and blood counts at diagnosis or prior to transplant. 135 patients (59%) are alive as of October 1995 with a minimum follow‐up of 5 years. 52 patients have relapsed (23%), 26 patients in the irradiated, 26 patients in the non‐irradiated group (n.s.) with a relapse incidence at 6 years of 28%. The main risk factor for relapse was T‐cell depletion as the method for GvHD prevention, and an elevated basophil count in the peripheral blood prior to transplant. Relapse incidence between patients with or without splenic irradiation was no different in patients at high risk for relapse, e.g. patients transplanted with T‐cell‐ depleted marrows (P = n.s.) and in patients with low risk for relapse, e.g. patients transplanted with non‐T‐cell‐depleted transplants and basophil counts <3% prior to transplant (P = n.s.). However, relapse incidence differed significantly in patients with non‐T‐cell‐depleted transplants and high basophil counts (> 3% basophils in peripheral blood). In this patient group, relapse incidence was 11% at 6 years with splenic irradiation but 32% in the non‐irradiated group (P = 0.05). Transplant‐related mortality was similar whether patients received splenic irradiation or not. This study suggests an advantage in splenic irradiation prior to transplantation for CML in this subgroup of patients and illustrates the need for tailored therapy.
Eosinophilia and granulocytic dysplasia terminating in acute myeloid leukaemia after 24 years
Authors: Doorduijn J. K. , van Lom K. , Löwenberg B.
Published: November 2003

Eosinophilia of variable duration, and subsequent progression to granulocytic sarcoma and acute myeloid leukaemia, has been infrequently reported in the literature. We report a patient with eosinophilia and normal cytogenetics who, after 24 years, showed transformation to a granulocytic sarcoma of the brain. Haematological counts were normal but the marrow revealed the cytogenetic abnormality trisomy 8 in 25% of mitoses. Subsequently an AML‐M2 developed, showing a complex karyotype including the trisomy 8 in all metaphases. FISH analysis combined with cytological examination identified the trisomy 8 in blasts, eosinophils and dysplastic granulocytes only. Thus progressive leukaemic transformation selectively involved the myeloid compartment.
A novel approach to investigating the erythroid lineage, using both receptor analysis and haemoglobin detection
Authors: McGuckin Colin P. , Liu Wai M. , Gordon‐Smith Edward C. , Uhr Mario R.
Published: November 2003

Progenitor cell failure in the erythroid lineage is a particular problem in bone marrow failure. To provide insight into early erythopoietic development we used sensitive techniques to examine the effects of SCF, IL‐3 and MIP‐1α on two developmentally arrested progenitor cell lines, HEL and K562. Quantitative flowcytometric analysis showed that both expressed receptors (SCF > MIP‐1α >IL‐3). Qualitative analysis revealed HEL cells expressed more receptors than K562 cells. Clonogenic assays with sensitive haemoglobin detection showed that SCF and IL‐3 did not support HEL development and reduced haemoglobin production. MIP‐1α reduced partially developed HEL colonies and haemoglobin in developed colonies. SCF increased development, but not haemoglobin in K562 cells, with IL‐3 being more effective in both. MIP‐1α increased the proportion of well‐developed K562 colonies but not haemoglobin. This suggests SCF, IL‐3 and MIP‐1α all have a role to play in early erythroid cellular development, with differing actions depending on the stage of development.
Quantitative PCR of the immunoglobulin heavy chain gene using genomic DNA
Authors: ELSWORTH A. M. , EVANS P. A. S. , MORGAN G. J. , KINSEY S. E. , SHIACH C. R.
Published: November 2003

Techniques currently available enable the detection of clonal rearrangements of the immunoglobulin heavy chain gene using fluorescent PCR technology. It is possible to use this technique to analyse minimal residual disease throughout patient treatment; however, without the development of a quantitative assay, only the presence or absence of a clonal population can be determined. We describe here the development of a quantitative competitive PCR technique using genomic DNA which enables the rate of clearance of disease to be measured. In future, the ability to detect and also quantitate minimal residual disease may enhance the application of molecular investigations in the clinical management of patients.
A non‐thrombocytopenic bleeding disorder due to an IgG4‐kappa anti‐GPIIb/IIIa autoantibody
Authors: McMillan R. , Bowditch R. D. , Tani P. , Anderson H. , Goodnight S.
Published: November 2003

Autoantibodies in chronic immune thrombo‐cytopenic purpura occasionally interfere with platelet function. We describe a patient with a normal platelet count who had clinically significant mucosal bleeding, a prolonged bleeding time and abnormal platelet aggregation. The patient had high titres of an IgG4 kappa autoantibody, directed to a cation‐dependent epitope on platelet glycoprotein IIb/IIIa, which blocked the binding of fibrinogen and fibronectin to this complex. Corticosteroid treatment resulted in clinical improvement and a marked drop in autoantibody concentration. The lack of thrombocytopenia in this patient, despite high autoantibody levels, is best explained by the poor recognition of IgG4 antibodies by phagocytic cells.
Clonal involvement of eosinophils in therapy‐related myelodysplastic syndrome with eosinophilia, translocation t(1;7) and lung cancer
Authors: Imai Yasufumi , YASUHARA SHINGO , Hanafusa Norio , Ohsaka Akimichi , Enokihara Hideo , Tomizuka Hiroshi , Sonoyama Masayuki , Miura Yasusada , Tohda Syuji , Nara Nobuo , Takahashi Atsushi
Published: November 2003

We report a therapy‐related MDS (RAEB) patient with eosinophilia, unbalanced translocation der(7)t(1;7) (q12;q22) and lung cancer. We observed no increase in cytokine levels in serum or in the conditioned medium (CM) of peripheral T cells cultured with or without IL‐2. When bone marrow (BM) cells were cultured with GM‐CSF, IL‐3 and SCF in a semisolid system, the colonies were exclusively eosinophilic. Cytogenetic analysis of the colony cells identified the same chromosome abnormality in all metaphases to that of BM cells. Suspension and clonogenic colony assay of BM cells cultured with various cytokines showed predominant eosinophilic growth and differentiation with GM‐CSF, but not with the other cytokines examined. These findings, together with mild morphological abnormalities of eosinophils, indicate clonal involvement of eosinophils in the myelodysplastic syndrome (MDS) clone, and that the eosinophilia was derived from the neoplastic clone with the translocation and was not associated with the patient’s lung cancer.
Pitfalls in the immunophenotyping of leukaemia and leukaemic lymphomas: survey of 9 years of quality control in The Netherlands
Authors: Kluin‐Nelemans J. C. , Van Wering E. R. , Van 't Veer M. B. , Van Der Schoot C. E. , Adriaansen H. J. , Van Der Burgh F. J. , Gratama J. W.
Published: November 2003

During the last decade, biannual quality controls were performed in The Netherlands focusing on the immunophenotyping of leukaemic haematological malignancies. All results on 48 specimens obtained by 18–34 laboratories were analysed. The interlaboratory variability and percentages of discordant results from 30 markers were measured by assessing false positive or negative (cut‐off 10%) results in comparison with median results of the group. The quality of the immunophenotypic diagnoses obtained from the interpretation of these markers in relation to clinical data was evaluated by scoring them as ‘correct’, ‘minor fault’, ‘major fault’, ‘not based upon the markers used’, and ‘no diagnosis’. CD3, CD8, CD19, CD61 and Smλ had the lowest percentage discordancy (sum of total negative and positive discordant values 5–7.5% of assays); CD13, CD15, cyCD22, CD33 and TdT scored worst with 14–20% cumulative discordancy. The analysis of each diagnosis yielded 78% acceptable immunophenotypic conclusions (correct 54% and minor fault 24%). It appeared that the major faults in immunophenotyping were caused by suboptimal antibody selection and erroneous interpretation of the results obtained, rather than by technical errors. Large differences per diagnostic category were observed, with the best scores for mature B‐cell leukaemias, AMLs and common‐ALL, and the poorest scores for T‐cell malignancies which were correctly diagnosed in only 24–60% of specimens. Mature T‐NHL and T‐PLL were mistakenly diagnosed as T‐ALL by 40% of the centres. Misinterpretation of TdT immunofluorescence or omitting this marker contributed significantly to these wrong diagnoses. A median of 4% of immunophenotypic diagnoses were not based on a correct panel of antibodies, but upon the morphology of the accompanying blood smear, and was often flawed by overinterpretation.

In conclusion, both the technical performance of immunophenotyping of haematological malignancies in The Netherlands and the procedure by which a final diagnosis is obtained needs improvement, especially for T‐cell malignancies.
Oligoclonal accumulation of T cells in peripheral blood from patients with idiopathic thrombocytopenic purpura
Authors: Shimomura Takeshi , Fujimura Kingo , Takafuta Toshiro , Fujii Teruhisa , Katsutani Shinya , Noda Masaaki , Fujimoto Tetsuro , Kuramoto Atsushi
Published: November 2003

To determine whether clonal T cells accumulate in idiopathic thrombocytopenic purpura (ITP), we performed single‐strand conformation polymorphism (SSCP) analysis to detect T‐cell receptor (TCR) β‐chain usage of peripheral T cells. We detected significantly more oligoclonal T cells (15.5 ± 8.9 bands representative for clonal T‐cell expansions) in peripheral blood from ITP patients than from healthy donors (2.8 ± 2.6 bands). Frequently used Vβ genes in these accumulated T cells in ITP were Vβ 3, 6, 10, 13.1 and 14. To determine whether these bands were derived from clonal T cells, presumably in a preactivated state, we established some T‐cell clones (expressing CD4 and TCR Vβ 6, 13.1, or 14) by nonspecific stimulation from patients’ peripheral mononuclear cells, and examined their clonotypes. Clonal identities for three out of seven clones tested were confirmed using SSCP analyses to compare the migration of their β‐chain complementarity determining region 3 (CDR3) cDNAs, expanded by polymerase chain reaction (PCR) with those from peripheral blood. Therefore, distinctive T‐cell clones accumulated in the periphery in ITP and they may be related to the autoimmune‐mediated destruction of platelets.
Clearance of marrow infiltration after 1 week of therapy for childhood lymphoblastic leukaemia: clinical importance and the effect of daunorubicin
Authors: Lilleyman J. S. , Gibson B. E. S. , Stevens R. F. , Will A. M. , Hann I. M. , Richards S. M. , Hill F. G. H.
Published: November 2003

At the commencement of UKALL XI, a national MRC trial for childhood lymphoblastic leukaemia (ALL), the therapy included a bolus of daunorubicin (DR) on the first 2 d of the protocol. This component of treatment was subsequently withdrawn because of concern about long‐term cardiotoxicity. All children both before and after this change of policy had their marrow status at the end of the first week assessed by central review as part of the trial to examine the clinical importance of the rate of disease clearance. This also afforded an opportunity to observe the effect of DR on gross residual disease at an early stage of therapy.

1419 children were studied: 342 received DR (‘recipients’), 1077 did not. 44% of the recipients completely cleared their marrow of blast cells after 8 d compared with 13% of the non‐recipients (χ2=158.2, P<0.0001). The difference in the proportion with massive residual disease (>80% blasts) was less impressive but there was still a difference in favour of DR recipients (DR 9%, no DR 15%; χ2=7.7, P=0.006). The rate of disease clearance correlated with disease‐free survival for both recipients and non‐recipients, but there was no significant difference in outcome when comparing the two groups with each other, either in terms of disease‐free or relapse‐free survival.

DR accelerated the rate of blast cell disappearance from the marrow but the difference this made to disease free survival is small or non‐existent. It appears to be the relative speed of response to a given therapeutic regimen that is prognostically important rather than the absolute rate of response when comparing one treatment with another.
Myelomonoblastic leukaemia cells carrying the PEBP2β/MYH11 fusion gene are CD34+, c‐KIT+ immature cells
Authors: Osato Motomi , Asou Norio , Okubo Toshiya , Nishimura Shintaro , Yamasaki Hiroshi , Era Takumi , Suzushima Hitoshi , Kawano Fumio , Matsuoka Rumiko , Oka Hiroya , Bae Suk‐Chul , Ito Yoshiaki , Takatsuki Kiyoshi
Published: November 2003

To clarify the aspects affected by the PEBP2β/MYH11 fusion gene involved in the inv(16), we analysed immunophenotypes in myelomonoblastic leukaemias. We found high expressions of CD34 and c‐KIT antigens in myelomonoblastic cells from all patients carrying this fusion gene, including two with M4 and one CML blastic phase, in contrast to those with M4 without the fusion gene. These findings indicate that immunophenotyping is useful for detecting a leukaemia with the fusion gene in myelomonoblastic leukaemias and that the PEBP2β/MYH11 gene is involved in immature cells expressing CD34 and c‐KIT antigens.
Common clonal origin of lymphocytes and plasma cells in splenic lymphoma with villous lymphocytes
Authors: KATAYAMA YOSHIO , KOJIMA KENSUKE , YOSHINO TADASHI , MATSUO YOSHINOBU , ISOKAWA MASAFUMI , YANO TOMOFUMI , OKA HIDEYUKI , YAMAGUCHI MIKA , DEGUCHI SEIGO , TSUCHIYAMA JUNJIRO , HAYASHI KYOICHI , TESHIMA TAKANORI , SHINAGAWA KATSUJI , ISHIMARU FUMIHIKO , OMOTO EIJIRO , HARADA MINE
Published: November 2003

In two‐thirds of patients with splenic lymphoma with villous lymphocytes (SLVL) a small amount of M‐protein can be detected in association with the presence of plasma cells in the peripheral blood (PB) and/or bone marrow (BM). However, it is not known whether lymphoma cells and plasma cells originate from the same clone. In this report we describe a case of SLVL which was characterized by the presence of marked monoclonal gammopathy (IgG‐κ 90 g/l) and increased plasma cells in the BM. In an attempt to elucidate the origin of lymphoma cells and plasma cells, we performed morphological, cytogenetic and molecular studies on PB mononuclear cells (PBMNC) without plasma cells and BMMNC containing 10% plasma cells from this patient.

Immunofluorescence showed that lymphoma cells and plasma cells were positive for cytoplasmic γ heavy and κ light chains. Well‐developed endoplasmic reticulum was observed in the cytoplasmic organelles of PBMNC using an electron microscope. The mean IgG concentration in the 3 d supernatant cultures of PBMNC was 374±24μg/l. More than 50% PBMNC differentiated into plasmacytoid cells in 6 d of liquid culture with IL‐3 and IL‐6. Analysis by two‐colour FISH revealed that karyotypic abnormalities of monosomy X and trisomy 17 existed simultaneously in both lymphoma cells and plasma cells. JH gene rearranged bands from PBMNC and BMMNC by Southern blot hybridization were identical, whereas DNAs from PBMNC failed to hybridize with the Cμ probe.

These observations strongly suggest that lymphoma cells and plasma cells originate from the same clone, and that plasma cells, as well as lymphoma cells, which have undergone class switch recombination, could produce IgG type M‐protein in this case.
In vivo‘Purging’ of residual disease in CLL with Campath‐1H
Authors: DYER M. J. S. , KELSEY S. M. , MACKAY H. J. , EMMETT E. , THORNTON P. , HALE G. , WALDMANN H. , NEWLAND A. C. , CATOVSKY D.
Published: November 2003

We assessed the role of human CD52 antibody (Campath‐1H) in six patients with chronic lymphocytic leukaemia (CLL) treated to maximal response with purine analogues (fludarabine/deoxycoformycin) in whom persistent leukaemic infiltration of blood and bone marrow had precluded autologous stem cell transplantation. Five patients achieved haematological and histological complete remission following Campath‐1H and one had minimal focal residual CLL in a trephine biopsy. Autologous transplantation was performed in two patients without complications and with rapid haemopoietic engraftment. Treatment with Campath‐1H may be of value in eradicating residual disease in CLL and may facilitate high‐dose therapy in young patients.
Novel and diagnostically applicable information from optical waveform analysis of blood coagulation in disseminated intravascular coagulation
Authors: DOWNEY COLIN , KAZMI RASHID , TOH CHENG HOCK
Published: November 2003

Transmittance waveform is the term applied to the optical profile generated from the process of clot formation on standard coagulation tests run on the MDA‐180, a new‐generation automated coagulation analyser. In patients with disseminated intravascular coagulation, a characteristically abnormal ‘biphasic change’ is seen on both the activated partial thromboplastin time and thrombin time waveforms. Increasing steepness of the initial slope on the waveform correlates with clinical deterioration and fulminant progression. Although the mechanism underlying the biphasic appearance remains to be elucidated, its identification provides the diagnostic laboratory with a simple, rapid and robust assay for disseminated intravascular coagulation that can help the clinician with urgent and appropriate therapeutic interventions.
Treatment of adults with acute lymphoblastic leukaemia in first bone marrow relapse: results of the ALL R‐87 protocol
Authors: Giona Fiorina , Annino Luciana , Rondelli Roberto , Arcese William , Meloni Giovanna , Testi Anna Maria , Moleti Maria Luisa , Amadori Sergio , Resegotti Luigi , Tabilio Antonio , Ladogana Saverio , Fioritoni Giuseppe , Camera Andrea , Liso Vincenzo , Leoni Pietro , Mandelli Franco
Published: November 2003

Sixty‐one adults aged <55 years with acute lymphoblastic leukaemia (ALL) in first bone marrow relapse were enrolled in an Italian cooperative study (ALL R‐87 protocol) from 12 GIMEMA Institutions. The treatment programme consisted of: (1) an induction phase with intermediate‐dose cytarabine (IDARA‐C 1 g/m2, 6 h daily infusion ×6 d) plus idarubicin (IDA; 5 mg/m2/d × 6 d) and prednisone (40 mg/m2/d × 21 d), (2) a consolidation phase followed by (3) bone marrow transplant (BMT). Median first complete remission (CR) duration was 8.5 months (range 1–54 months). 34/61 patients achieved CR (56%); 24 (39%) failed to respond and three (5%) died during induction. Most responders (24 patients) could not enter the BMT programme; 15 relapsed early (median time to relapse 2 months); nine were withdrawn due to toxicity and one died in CR of infection. Nine of the 34 CRs underwent BMT (five autologous and four allogeneic). Three of the four allotransplanted patients are alive in continuous CR at 22, 43 and 63 months; only one of the five who underwent an autologous BMT is alive in CR at 46 months.

The estimated disease‐free survival (DFS ± SE) at 36 months was 0.16 ± 0.08 for all responders. Univariate analysis showed that previous therapy was the only prognostic factor influencing DFS. The estimated probabilities of event‐free survival (EFS ± SE) and survival ± SE at 37 months were 0.09 ± 0.04 and 0.10 ± 0.04, respectively. The EFS was significantly better in patients with a preceding CR >24 months, compared to those with a shorter first remission.

Our results confirm the tolerance and efficacy of IDARA‐C plus IDA in inducing CR in poor‐risk adult ALL. Even though the number of transplanted patients was small, allogeneic BMT seems to give a real opportunity of cure in this category of patients.
IBMTR Severity INDEX FOR GRADING ACUTE GRAFT‐VERSUS‐HOST DISEASE: RETROSPECTIVE COMPARISON WITH GLUCKSBERG GRADE
Authors: Rowlings Philip A. , Przepiorka Donna , Klein John P. , Gale Robert Peter , Passweg Jakob R. , Jean Henslee‐Downey P. , Cahn Jean‐Yves , Calderwood Stan , Gratwohl Alois , Socié Gérard , Abecasis M. M. , Sobocinski Kathleen A. , Zhang Mei‐Jie , Horowitz Mary M.
Published: November 2003

Acute graft‐versus‐host disease (GVHD) severity is graded by pattern of organ involvement and clinical performance status using a system introduced by Glucksberg and colleagues 21 years ago. We examined how well Glucksberg grade predicted transplant outcome and constructed a Severity Index not requiring subjective assessment of performance in 2881 adults receiving an HLA‐identical sibling T‐cell‐depleted (n = 752) or non‐T‐cell‐depleted (n = 2129) bone marrow transplant for leukaemia between 1986 and 1992. Relative risks (RR) of relapse, treatment‐related mortality (TRM) and treatment failure (TF) (relapse or death) were calculated for patients with Glucksberg Grade I, II or III/IV acute GVHD versus those without acute GVHD and for patients with distinct patterns of organ involvement regardless of Glucksberg grade. Using data for non‐T‐cell‐depleted transplants, a Severity Index was developed grouping patients with patterns of organ involvement associated with similar risks of TRM and TF. Higher Glucksberg grade predicted poorer outcome; however, patients with the same grade but different patterns of skin, liver or gut involvement often had significantly different outcomes. The revised Severity Index groups patients in four categories, A–D. Compared to patients without acute GVHD, RRs (95% confidence interval) of TF were 0.85 (0.69, 1.05) for patients with Index A, 1.21 (1.02, 1.43) with B, 2.19 (1.78, 2.71) with C, and 5.69 (4.57, 7.08) with D. Prognostic utility of the Index was tested in patients receiving T‐cell‐depleted transplants; similar RRs of TF were observed. An acute GVHD Severity Index is proposed to enhance design and interpretation of clinical trials in the current era of allogeneic blood and bone marrow transplantation.
Multilineage mobilization of peripheral blood progenitor cells in humans following administration of PEG‐rHuMGDF
Authors: RASKO JOHN E. J. , BASSER RUSSELL L. , BOYD JAN , MANSFIELD RACHEL , O'MALLEY CINDY J. , HUSSEIN SONAY , BERNDT MICHAEL C. , CLARKE KERRIE , O'Byrne JOAN , Sheridan William P. , Grigg Andrew P. , Begley C. Glenn
Published: November 2003

The most important physiological regulator of megakaryocytopoiesis is the ligand for the c‐mpl receptor (thrombopoietin/megakaryocyte growth and development factor, MGDF). We examined the effect of pegylated‐recombinant human MGDF (PEG‐rHuMGDF): patients received PEG‐rHuMGDF at doses of 0.03, 0.1, 0.3 or 1.0 μg/kg/d or placebo for 10 d maximum in a double‐blinded randomized study. There was a dose‐dependent elevation in circulating platelet counts but no alteration in erythrocyte or total leucocyte counts. The number of bone marrow megakaryocytes was increased approximately 2‐fold. The frequency of bone marrow progenitor cells was not altered. In contrast, both to the bone marrow results and to published pre‐clinical data, there was a dose‐dependent mobilization into the blood of progenitor cells of multiple cell lineages. Increased levels of Meg‐CFC (maximum increase 30‐fold), day 7 and day 14 GM‐CFC and BFU‐E were demonstrated at doses of 0.3 and 1.0 μg/kg/d PEG‐rHuMGDF. At 0.1 μg/kg/d, mobilization of Meg‐CFC alone occurred in two‐thirds of patients. Maximum blood levels of progenitor cells occurred at day 12. Thus, administration of PEG‐rHuMGDF to humans resulted in mobilization of progenitor cells of multiple lineages despite its ‘lineage‐specific’ activity on mature cell development.
PROTEOLYTIC DEGRADATION OF HIGH MOLECULAR WEIGHT KININOGEN IN ACUTE THROMBOTIC THROMBOCYTOPENIC PURPURA
Authors: Pavord Susannah , Kelton John G. , Warner Margaret N. , Moore Jane C. , Warkentin Theodore E. , Hayward Catherine P. M.
Published: November 2003

Thrombotic thrombocytopenic purpura (TTP) is a disorder characterized by thrombocytopenia and schistocytic haemolytic anaemia. The majority of patients have normal coagulation parameters including the activated partial thromboplastin time (APTT). An intracellular cysteine protease, calpain, has been found in the plasma of many patients with acute TTP, and we hypothesize that it participates in the pathogenesis of the disorder. However, certain aspects of the disorder remain unresolved. For example, high molecular weight kininogen (HK) is one of the primary plasma inhibitors of calpain, and it also can act as a substrate for calpain. Consequently, one might anticipate that the HK could be defective or altered in TTP. In this report we describe the analysis of HK in plasma from five patients with acute TTP and following recovery. The HK was studied immunogenically and functionally. We observed that the HK in plasma samples from patients with acute TTP was proteolysed. This degradation was not observed in remission samples from the same patients. However, both acute and remission TTP samples had normal HK coagulant activity (acute samples, x =0.84 ±0.26 U/ml; remission samples, x = 0.89 ± 0.21 U/ml; control samples, x = 0.87 ± 0.05 U/ml). Although the TTP plasmas were able to inhibit calpain activity, less inhibition activity was found in the acute samples (acute: mean inhibition 71 ±2.4%; remission: mean 92 ± 2.1%; control samples: mean 93 ± 5.4%: P<0.001). Normal HK treated with calpain also had reduced calpain inhibition capacity (mean 58%). This study suggests that although HK is proteolysed during acute TTP, the proteolysis occurs without a major loss in the coagulation function or depletion of the protease inhibitory activity of HK.
Bone mineralization and turnover in children with congenital neutropenia, and its relationship to treatment with recombinant human granulocyte‐colony stimulating factor
Authors: Fewtrell M. S. , Kinsey S. E. , Williams D. M. , Bishop N. J.
Published: November 2003

Bone mineral content (BMC) of the radius was measured using single photon absorptiometry (SPA) in nine children with congenital neutropenia. Five had normal values. Two children with severe congenital neutropenia (SCN) had low BMC, and two boys with Schwachman syndrome had biochemistry suggestive of rickets.
The VITA Project: C677T mutation in the methylene‐tetrahydrofolate reductase gene and risk of venous thromboembolism
Authors: TOSETTO ALBERTO , MISSIAGLIA EDOARDO , FREZZATO MAURIZIO , RODEGHIERO FRANCESCO
Published: November 2003

We evaluated the hypothesis that a common polymorphism of the methylenetetrahydrofolate reductase gene (C677T), which results in increased levels of plasma homocysteine, may be a putative risk factor for venous thromboembolism (VT). Sixty‐five cases of VT and 130 controls, both identified within the framework of an epidemiologic survey on thrombophilia, the Vicenza Thrombophilia and Arteriosclerosis (VITA) Project, were genotyped for the mutation. No increased risk of VT was found in carriers of the mutation. We conclude that screening for the C677T mutation of the methylenetetrahydrofolate reductase gene should not be recommended in unselected patients with VT.
Types and sources of fuels for platelets in a medium containing minimal added fuels and a low carryover plasma
Authors: Niu X. , Whisson M. E. , Guppy M.
Published: November 2003

The storage of platelets in synthetic media can result in plasma savings and reduced transfusion reactions. Accordingly, a wide range of storage formulations have been developed with the aim of replacing at least a proportion of the plasma in the storage medium. However, the concentrations and types of fuels in the carryover plasma, and the utilization of these fuels by platelets in storage, has not been investigated. We have developed a system which can measure total ATP turnover, and the contribution to total ATP turnover by the oxidation of various fuels and by lactate production, in a bag of partially purified platelets in a buffered saline with minimal carryover citrate phosphate double dextrose (CP2D) plasma.

Carryover plasma was about 1% and the final platelet suspension contained, on average, 0.62 mM glucose, 9.6 mg/l free fatty acids, 32 mg/l triglycerides and 0.23 mM total amino acids. The oxidation of carbohydrate (glucose, glycogen and lactate) accounted for 60% of total ATP turnover. The platelets also produced lactate (<6% of total ATP turnover) and consumed free fatty acids and amino acids/proteins (15.2% of total ATP turnover). Therefore we have identified the fuels that account for about 80% of oxygen consumption and ATP turnover by platelets in a medium with low carryover plasma. The implications of these data for storage strategies are discussed.
Heterogenous expression of bcr‐abl fusion mRNA in a patient with Philadelphia‐chromosome‐positive acute lymphoblastic leukaemia
Authors: Inokuchi Koiti , Futaki Makoto , Hanawa Hideki , Tanosaki Sakae , Yamaguchi Hiroki , Iwakiri Rika , Nomura Takeo , Dan Kazuo
Published: November 2003

We performed molecular and cytogenetic analysis on a 56‐year‐old woman with Philadelphia chromosome (Ph1)‐positive acute lymphoblastic leukaemia (ALL) having two types of major and minor bcr‐abl (M‐bcr‐abl, m‐bcr‐abl) fusion mRNA at diagnosis. In the course of her disease, unexpected heterogenous bcr‐abl fusion mRNA was detected by sequential analysis using the reverse transcription and polymerase chain reaction (RT‐PCR).

The RT‐PCR analysis showed both M‐bcr‐abl (b2‐a2 type) and m‐bcr‐abl at diagnosis. Although b2‐a2 type M‐bcr‐abl disappeared after complete remission (CR) was achieved with intensive induction chemotherapy, expression of both m‐bcr‐abl and a new type of M‐bcr‐abl mRNA (b1‐a2 type), which may have been produced through splicing out of exon b2, was detected in the early stage of CR. The leukaemic cells contained these heterogenous bcr‐abl mRNAs in both the CR and relapsed state, and showed more stable expression of the m‐bcr‐abl type mRNA than that of the b2‐a2 type. These findings of heterogenous bcr‐abl mRNA due to alternative or missplicing during the disease course in the present ALL case may provide important evidence of disease progression.
CD56+ NK lymphomas:clinicopathological features and prognosis
Authors: KWONG Y. L. , CHAN  , LIANG RAYMOND , CHIANG ALAN K. S. , CHIM C. S. , CHAN T. K. , TODD DAVID , HO FAITH C. S.
Published: November 2003

The surface molecule CD56 marks a category of malignant lymphoma of putative natural killer (NK) cell origin. We conducted a retrospective analysis of 24 cases of CD56+ NK lymphoma/leukaemia to define the clinicopathologic and prognostic features of this specific group of lymphomas. 56 cases of nasal lymphomas and 204 cases with an initial diagnosis of peripheral T‐cell lymphoma were retrospectively analysed. To specifically examine lymphomas of putative NK origin, only those that were negative for surface expression of CD3 but positive for CD56 were analysed. 24 cases were identified. The initial predominant sites of involvement were nasal (n =18), palate (n = 1), nodal (n = 1) and multi‐organ (n =4). Clinically, in patients with disease localized to one anatomical site (n = 20), most had symptoms confined to the nose, with a high percentage in early stage (I: 91%; IV: 9%). The marrow was not involved in any of these cases. However, patients with multi‐organ involvement at presentation (n = 4) behaved differently. All presented acutely with pancytopenia, hepatosplenomegaly, and marrow infiltration with haemophagocytosis. A leukaemic phase was observed in one case. Anthracycline containing combination chemotherapy resulted in complete remission in 75% of patients with localized disease, but only in 25% with multi‐organ involvement. The median survival of patients with localized disease was 12 months, compared with 2 months in the multi‐organ group (P =0.06); the disease‐free survival was significantly better in the former (P <0.01). The overall median survival of all patients was still poor at 11 months.

We conclude that CD56+ NK lymphomas could be divided into two main patterns of disease presentations: localized (predominantly nasal), and multi‐organ involvement. Each has different clinicopathologic and prognostic features. Conventional chemotherapy appeared ineffective for the majority of patients, and innovative treatment modalities are needed to improve outcome.
DISTRIBUTION OF T‐CELL SIGNALLING MOLECULES IN HUMAN MYELOMA
Authors: Bianchi Alberto , Mariani Sara , Beggiato Eloise , Borrione Paolo , Peola Silvia , Boccadoro Mario , Pileri Alessandro , Massaia Massimo
Published: November 2003

It is controversial whether altered levels of TCR/CD3‐associated signalling molecules play a role in the T‐cell dysfunction of cancer patients. In multiple myeloma (MM), peripheral blood T (PBT) lymphocytes are functionally impaired by prolonged exposure to tumour cells, and so we investigated the organization of the TCR/CD3‐associated signal transduction machinery. The aim of this study was two‐fold: first, to investigate the levels of CD3ζ, p56lck, p59fyn, ZAP‐70, protein kinase C‐α (PKC‐α) and phospholipase C‐γ (PLC‐γ) in MM PBT cells; second, to determine whether levels of expression were correlated with clinical or prognostic factors. Forty‐four MM patients were studied and 25 age‐matched normal donors served as controls. On average, PKC‐α was the only significantly decreased (P<0.001) signalling molecule, whereas levels of CD3ζ, p56lck, p59fyn, PLC‐γ and ZAP‐70 were not statistically different. However, there was wide variation between individual patients, and levels for each single protein also varied. A 75% or greater decrease in protein expression was observed, ranging from 8% (p59fyn) to 68% (PCK‐α) of MM patients. When patients were grouped according to the cut‐off values of prognostic factors such as the serum levels of C reactive protein (CRP), β2‐microglobulin (β2M), neopterin (NPT) and the labelling index (LI%) of bone marrow (BM) plasma cells, the only difference observed was the lower PKC‐α expression in patients with high serum NPT values. None of the T‐cell signalling molecule levels was affected by the duration of tumour exposure, calculated on the number of years and/or months that had elapsed since diagnosis, or by disease status. In conclusion, there was a significant decrease of PCK‐α in MM T cells; however, neither this decrease nor the heterogenous levels of the other T‐cell signalling molecules were clearly correlated with prognosis, duration of tumour exposure, and disease status.
Exon/intron structure of the human AF‐4 gene, a member of the AF‐4/LAF‐4/FMR‐2 gene family coding for a nuclear protein with structural alterations in acute leukaemia
Authors: Nilson I. , Reichel M. , Ennas M. G. , Greim R. , Knörr C. , Siegler G. , Greil J. , Fey G. H. , Marschalek R.
Published: November 2003

The AF‐4 gene on human chromosome 4q21 is involved in reciprocal translocations to the ALL‐1 gene on chromosome 11q23, which are associated with acute lymphoblastic leukaemias. A set of recombinant phage carrying genomic fragments for the coding region and flanking sequences of the AF‐4 gene were isolated. Phage inserts were assembled into four contigs with 21 exons, and an intron phase map was produced enabling the interpretation of translocation‐generated fusion proteins. The gene contains two alternative first exons, 1a and 1b, both including a translation initiation codon. The translocation breakpoint cluster region is flanked by exons 3 and 6 and two different polyadenylation signals were identified. Polyclonal antisera directed against three different portions of the AF‐4 protein were produced and used to detect a 116 kD protein in cellular extracts of human B‐lymphoblastoid and proB cell lines. In mitogen‐stimulated human peripheral blood mononuclear cells the AF‐4 antigen was predominantly located in the nucleus. The AF‐4 gene is a member of the AF‐4, LAF‐4 and FMR‐2 gene family. The members of this family encode serine‐proline‐rich proteins with properties of nuclear transcription factors. Comparison of AF‐4 protein coding sequences with the LAF‐4 and FMR‐2 sequences revealed five highly conserved domains of potential functional relevance.
Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia
Authors: Callera Fernando , Falcão Roberto P.
Published: November 2003

In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in paraffin‐embedded bone marrow biopsies from patients with aplastic anaemia using an in situ TdT‐catalysed DNA nick end labelling (TUNEL) staining method. A significant increase in the proportion of mononuclear apoptotic cells was demonstrated in biopsies from patients with aplastic anaemia (8.19 ± 1.45%) when compared with controls (2.07 ± 0.86%). These data support the view that apoptosis may play a role in the pathophysiology of bone marrow failure.
International Normalized Ratio determination using calibrated reference plasmas
Authors: Hubbard Anthony R. , Margetts Sarah M. L. , Barrowcliffe Trevor W.
Published: November 2003

We have compared the conventional method of International Normalized Ratio (INR) determination with an alternative method involving extrapolation from a calibration curve using freeze‐dried ‘reference’ plasmas. The latter approach does not require the determination of a mean normal prothrombin time (MNPT) or local system International Sensitivity Index (ISI). Calibration curves were constructed by plotting local prothrombin time (PT) against assigned INR values for a normal plasma and either two plasma pools from patients on oral anticoagulants or two artificially depleted plasmas. Six laboratories determined the INR of a freeze‐dried test plasma and frozen patient plasma samples using the conventional method and by extrapolation. Similarities in the results with the freeze‐dried test plasma and the frozen plasmas were encouraging for the projected use with fresh plasma samples. INR values by the conventional method for the test plasma gave an overall mean of 2.73 and inter‐laboratory variability (gcv%) of 8.92%, whereas estimates by extrapolation against the normal and patient plasmas or the normal and artificially depleted plasmas gave identical overall mean INR values of 2.70 with inter‐laboratory variability (gcv%) of 3.44% and 4.92% respectively. The results indicate that INR determination by extrapolation is associated with reduced inter‐laboratory variability.
Successive transformation of chronic myelomonocytic leukaemia into acute myeloblastic then lymphoblastic leukaemia, both with minor‐bcr rearrangement
Authors: DAUTEL Marie Michèle , FRANÇOIS Sylvie , BERTHEAS Marie‐FranÇoise , BARANGER Laurence , GARDAIS Jacques , BOASSON Marc , IFRAH Norbert , BLANCHET Odile
Published: November 2003

We report a case of chronic myelomonocytic leukaemia (CMML), which transformed first into acute myeloblastic leukaemia (AML) and then into acute lymphoblastic leukaemia (ALL). In the AML and ALL phases, chromosome analysis showed a classic Philadelphia chromosome (Ph) t(9;22)(q34;q11). Molecular studies showed breakpoint cluster region rearrangement between exons e1 and a2 compatible with a p190bcr/abl breakpoint as observed in Ph‐positive lymphoblastic acute leukaemia. The minor (m‐bcr) rearrangement was also detected during complete remission.

This observation supports a multistep pathogenesis of leukaemias, and that the p190bcr/abl breakpoint may influence the course of the disease.
Robertsonian translocation as an acquired karyotypic abnormality in leukaemia
Authors: Ma S. K. , Chow E. Y. D. , Wan T. S. K. , Chan L. C.
Published: November 2003

Robertsonian translocations, although relatively common as a constitutional genetic aberration, are rarely encountered in leukaemia. We report a case of acute myeloid leukaemia which showed an acquired Robertsonian translocation in the form of der(14;21) by cytogenetic analysis of leukaemic cells. This was confirmed by the PHA‐stimulated culture of peripheral blood lymphocytes. A review of the literature identifies only eight reported cases of acquired Robertsonian translocations in leukaemia. In the majority of cases the Robertsonian translocation occurs as a secondary change in a complex abnormal clone, whereas in two out of nine patients reported, including ours, it is found as a sole karyotypic abnormality.
Do HbSS erythrocytes lose KCl in physiological conditions?
Authors: GODART H. , DORMANDY A. , ELLORY J. C.
Published: November 2003

KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in ‘simple’ physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both Vmax and Km for K+ of the transporter. The plasma factor(s) responsible was heat‐stable and dialysable (i.e. size &60; 10 kD).

Adding magnesium, calcium, inorganic phosphate or bicarbonate to ‘simple’ saline to mimic the effect of plasma revealed that Mg2+ and Ca2+ had no significant effect at physiological concentrations. Pi was not effective at 1.1 mM , but did inhibit significantly (42±2%) at 5.6 mM . HCO−3   had a major inhibitory effect on K+ influx when added to saline, and was identified as the principal candidate for the plasma effect.

We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo. It acts by altering the set point of the transporter via the signalling systems involved in its regulation.
Bernard‐Soulier syndrome Karlstad: Trp 498→Stop mutation resulting in a truncated glycoprotein Ibα that contains part of the transmembranous domain
Authors: Holmberg Lars , Karpman Diana , Nilsson Ingmar , Olofsson Tor
Published: November 2003

In Bernard‐Soulier syndrome, a hereditary bleeding disorder, the platelets are deficient in the glycoprotein (GP) Ib–IX–V complex, a major receptor for the von Willebrand factor. The components of the complex are encoded by separate genes. Patients with this syndrome have a variable expression level of the receptor protein on platelets depending on the specific genetic abnormality. We describe a patient with life‐long bleeding symptoms, who is homozygous for a unique stop mutation, Trp 498→ Stop in the GPIbα gene, resulting in a truncated GPIbα polypeptide chain. In contrast to previously reported truncated forms of GPIbα, this form contains a portion of the transmembranous domain as well as the juxtamembranous cysteines engaged in a disulphide bond with GPIbβ. Flow cytometry with GPIbα antibodies demonstrated the presence of GPIb on the patient’s platelets, although in reduced amounts compared to normal controls. GPIX was similarly detectable. Immunoblotting demonstrated that the patient synthesized a truncated GPIbα of the expected size of 130 K, which was, however, sensitive to proteolysis. These studies show that GPIbα lacking the intracytoplasmic tail can be expressed at the platelet surface provided elements of the transmembranous domain are present.
A prospective study of minimal residual disease in childhood B‐lineage acute lymphoblastic leukaemia: MRD level at the end of induction is a strong predictive factor of relapse
Authors: Jacquy C. , Delepaut B. , Van Daele S. , Vaerman J. L. , Zenebergh A. , Brichard B. , Vermylen C. , Cornu G. , Martiat P.
Published: November 2003

We prospectively investigated minimal residual disease (MRD) in 51 children with B‐lineage acute lymphoblastic leukaemia (ALL) treated according to the Fralle 93 protocol. PCR follow‐up was performed in children in morphological and cytogenetic complete remission, provided an immunoglobulin (IgH) gene rearrangement could be detected using FR3/JH amplimers. MRD was studied according to our previously described methodology, with a few modifications including the use of a consensus JH probe to control for PCR efficiency variations.

Out of the initial 51 patients, 34 were assessable for MRD at the end of induction at the time of analysis. MRD levels were as follows: >1/103 in 26%, 1/103 to 1/104 in 50% and <1/104 or not detectable in 24%. With a median follow‐up of 20 months there were five relapses, all of which occurred in the group of patients with MRD >1/103. To date, none of the patients with MRD 1/103 (good molecular responder) has relapsed. Classification according to molecular response at the end of induction did not correlate with the conventional risks groups: there were no statistically significant differences between good and bad molecular responders. Of particular interest is the absence of correlation between WBC at diagnosis and MRD level at the end of induction. We conclude that classification of patients into good and bad molecular responders using PCR seems to be a better prognostic indicator than conventional risk factors in childhood B‐lineage ALL. Patients with MRD level >1/103 have a particularly poor outcome and should always be considered for alternative therapeutic strategies in the future, whereas in good molecular responders belonging to poor or intermediate risk categories, treatment de‐escalation might be contemplated.
Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis
Authors: Ratajczak Mariusz Z. , Ratajczak Janina , Marlicz Woytek , Pletcher, JR Charles H. , Machalinski Bogdan , Moore Jonni , Hung Hsiao‐ling , Gewirtz Alan M.
Published: November 2003

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum‐free cell‐culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum‐free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL‐3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7 d in serum‐free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre‐cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit‐erythroid (BFU‐E) colonies as cells which were pre‐cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3–7 d under serum‐free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.