bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of thrombosis and haemorrhage, relationship with anti‐Xa, anti‐IIa activities, and D‐dimer plasma levels in patients receiving a low molecular weight heparin, enoxaparin or tinzaparin, to prevent deep vein thrombosis after hip surgery
Authors: Bara Lucienne , Planes André , Samama Meyer‐Michel
Published: March 2003

Studies in experimental animal models and in patients receiving low molecular weight heparin (LMWH) to prevent thromboembolic events after surgery have not demonstrated a clear relationship between anti‐Xa and anti‐IIa activities in plasma and either bleeding or prevention of thrombosis. The relationship between these clinical outcomes and ex vivo anti‐Xa and anti‐IIa activities, activated partial thromboplastin time (APTT) and D‐dimers were evaluated in 440 patients undergoing total hip replacement and given prophylaxis once daily with a LMWH (tinzaparin or enoxaparin) in a multicentre double‐blind randomized study. 221 patients received 4500 anti‐Xa IU of tinzaparin; 219 patients received 40 mg (4000 anti‐Xa IU) of enoxaparin. Both regimens were administered subcutaneously once daily. Blood samples for anti‐IIa, anti‐Xa, D‐dimers levels and APTT were taken at baseline, on day 1, day 5 and on the day of discharge (days 8–14) and clinical assessments were performed daily until day 14. All patients had bilateral venography between days 8 and 14. All coagulation tests were performed in central laboratories. A significant correlation was observed between anti‐IIa activity and anti‐Xa activity and the dose of each LMWH injected. The anti‐Xa activity was significantly higher with enoxaparin and the anti‐IIa activity was significantly higher with tinzaparin. No clear relationship between these two activities and the clinical outcomes was observed. This was also true with regards to APTT. Before and after surgery, D‐dimers were significantly higher in patients with deep vein thrombosis (DVT) than in those without DVT but had no predictive value. Interestingly, a significant post‐operative increase of D‐dimers persisted in both groups of patients during the whole observation period, possibly suggesting that a longer duration of prophylactic treatment may be appropriate.
Periodic chronic myelogenous leukaemia: spectral analysis of blood cell counts and aetiological implications
Authors: Fortin Pascal , Mackey Michael C.
Published: March 2003

Of 24 published clinical reports of periodic chronic myelogenous leukaemia (PCML), 21 had sufficient data to analyse for periodicity, and 12 showed significant periodicity ( p  0.05) using the Lomb periodogram. Leucocyte oscillations had periods T ranging from 37 to 83 d. When data were also reported for platelets and reticulocytes there was no significant difference between their periods and those of the leucocytes. These data and their analysis provide strong circumstantial evidence for a haemopoietic stem cell origin of PCML. Namely, the known chromosomal changes in CML patients may, on occasion, also be accompanied by a destabilization resulting in an oscillatory efflux into the leucocyte, platelet and erythrocytic pathways.
Combination therapy with fludarabine and cyclophosphamide as salvage treatment in lymphoproliferative disorders
Authors: FREWIN REBECCA , TURNER DEBORAH , TIGHE MARY , DAVIES SIMON , RULE SIMON , JOHNSON STEPHEN
Published: March 2003

Seventeen patients (aged 50–85 years) with relapsed or refractory non‐Hodgkin's lymphoma (NHL, 10 patients) or chronic lymphocytic leukaemia (CLL, seven patients) were treated with a combination of fludarabine 25 mg/m2/d and cyclophosphamide 250 mg/m2/d for 3 d repeated every 4 weeks. 12 patients had previously received purine analogue therapy of which four had progressive disease during treatment. The overall response rate of patients with CLL was 71% (28% CR, 43% PR) and for NHL was 50% (0% CR, 50% PR). Toxicity consisted of nausea and vomiting which was maximal in the 3 d after therapy, infections and haematological suppression which was prolonged in some patients. This combination, which is based on a rational prediction of synergistic activity, is highly effective but associated with significant problems with tolerance.
Mapping the prevalence of sickle cell and beta thalassaemia in England: estimating and validating ethnic‐specific rates
Authors: Hickman M. , Modell B. , Greengross P. , Chapman C. , Layton M. , Falconer S. , Davies S. C.
Published: March 2003

A range of estimates for sickle cell and β thalassaemia have been derived for the different ethnic groups living in the U.K., reflecting uncertainty over the true population value in certain countries and the heterogeneity within and between countries of origin comprising the same ethnic group. These were validated against six community screening programmes, with the estimated range correctly predicting the number of affected births observed by the programmes.

In England approximately 3000 affected babies (0.47%) carry sickle cell trait and 2800 (0.44%) carry β thalassaemia trait annually; with approximately 178 (0.28 per 1000 conceptions) affected by sickle cell disease (SCD) and 43 (0.07 per 1000) by β thalassaemia major/intermedia. Allowing for termination, about 140–175 (0.22–0.28 per 1000) affected infants are born annually with SCD and from 10 to 25 (0.02–0.04 per 1000) with β thalassaemia major/intermedia.

These are the first evidence‐based rates for sickle cell and β thalassaemia for use in the U.K., and should underpin the future planning of services. The long‐term solution to monitoring changes in the rates of trait and disease in the population is to introduce a standardized instrument for collecting ethnicity for all community screening programmes.
PK‐LR gene mutations in pyruvate kinase deficient Portuguese patients
Authors: Manco Licínio , Ribeiro M. Letícia , Almeida Helena , Freitas Orquídea , Abade Augusto , Tamagnini Gabriel
Published: March 2003

In nine unrelated Portuguese patients with pyruvate kinase (PK) deficient anaemia, whose symptoms ranged from a mild chronic haemolytic anaemia to a severe anaemia presenting at birth and requiring multiple transfusions, the PK‐LR gene mutations were identified and correlated with their phenotypes. Five different mutations were identified, three of them for the first time: a missense mutation 1670G → C on exon 12 and two 5′ splice donor site (GT) mutations on intron 8 [IVS8(+2)T → G] and intron 10 [IVS10(+1)G → C]. Two previously described missense mutations, 1456C → T and 993C → A, were also found. The genotype/phenotype correlation showed that patients with two missense mutations or with a missense mutation and a splicing mutation had a mild haemolytic anaemia. The three patients with severe anaemia, who were transfusion dependent until splenectomy, were homozygous for the splicing site mutations IVS10(+1)G → C or IVS8(+2)T → G.
Semiquantitative analysis of residual disease in patients treated for adult T‐cell leukaemia/lymphoma (ATLL)
Authors: Leclercq India , Mortreux Franck , Morschhauser Franck , Duthilleul Patrick , Desgranges Claude , Gessain Antoine , Cavrois Marielle , Vernant Jean‐Paul , Hermine Olivier , Wattel Eric
Published: March 2003

Many adult T‐cell leukaemia/lymphoma (ATLL) patients who respond to induction treatment, then relapse. Knowing the clonality pattern of residual tumourous clones during treatment could help understand disease evolution and aid therapeutic decisions. We developed a sensitive and semi‐quantitative molecular analysis of these clones in ATLL patients. DNA samples from PBMCs derived from eight ATLL patients were studied over time by quadruplicate linker mediated PCR (LMPCR) amplification of HTLV‐1 integration sites. Patients were treated with combination chemotherapy, zidovudine–interferon‐alpha and/or by peripheral stem cell transplantation or allogeneic bone marrow transplantation. Persistence of tumourous clones at a high frequency (>1/300 PBMCs) was frequently observed, even in complete responders, and was invariably correlated with relapse and/or poor outcome. Fluctuation in the frequency of some tumourous clones was observed with evidence for clonal change under treatment in one patient, indicating that treatment of ATLL can result in the selection of resistant clones. Finally, allogeneic bone marrow transplantation (BMT) using an HTLV‐1 infected sibling as donor was found to be associated with long‐lasting disappearance of tumourous clones and a possible cure of the disease. Long‐term persistent clonal expansion of circulating HTLV‐1 bearing T cells which derived from the donor bone marrow was evidenced in this patient. In conclusion, variable success in treatment of ATLL is probably due to the clonal heterogeneity which results in the selection of resistant clones. Semi‐quantitative assessment of residual disease (RD) through LMPCR may predict treatment failure. Accordingly, additional therapy may be tailored to the clonality pattern observed after first‐line therapy.
Thrombosis and bleeding in myeloproliferative disorders: identification of at‐risk patients with whole blood platelet aggregation studies
Authors: Manoharan A. , Gemmell R. , Brighton T. , Dunkley S. , Lopez K. , Kyle P.
Published: March 2003

Seventy‐five patients with chronic myeloproliferative disorders were studied to investigate platelet function by simultaneous measurement of platelet aggregation by the impedance method and ATP dense granule release using a whole blood platelet lumi‐aggregometer, in an attempt to identify patients at risk for thrombosis and bleeding.

Thirty‐nine patients had at least one abnormal result indicating platelet hyperactivity (i.e. impedance or release with one agonist being above the reference range); 16 patients had platelet hypoactivity (i.e. at least one result was below the reference range), whilst 14 had co‐existence of hyper‐ and hypoactivity. Six patients had normal results. 20/53 patients with platelet hyperactivity (alone or mixed) had a positive history of venous and/or arterial thrombosis; in comparison, only two of the other 22 patients had a positive history. During a median follow‐up of 33 months, nine patients with and one patient without platelet hyperactivity respectively developed new thrombotic events before the addition of specific therapy. A total of 50 patients with and eight patients without platelet hyperactivity respectively received specific treatment including aspirin and/or cytotoxic therapy. All but one elderly patient with platelet hyperactivity have remained free of new thrombotic events on specific therapy. Two of the 17 patients with platelet hypoactivity had major clinical bleeding.

These observations highlight the need to test platelets for hyper‐ as well as hypo‐function and suggest a useful role for routine whole blood platelet aggregation studies to identify the patients at risk for thrombosis or bleeding.
Low prevalence of flower cells in U.S.A. blood donors infected with human T‐lymphotrophic virus types I and II
Authors: Sacher Ronald A. , Luban Naomi L. C. , Ameti Dannie I. , Friend Shirley , Schreiber George B. , Murphy Edward L. ,   for the Retrovirus Epidemiology Donor Study Group (REDS)
Published: March 2003

Large lymphocytes with basophilic cytoplasm and cleaved/cerebriform nuclei called flower cells have been described in human T‐lymphotrophic virus type I (HTLV‐I) seropositive individuals and may be precursors of adult T‐cell leukaemia (ATL). A cohort of 546 HTLV‐seropositive former blood donors, 32 HTLV‐positive sexual partners of these donors and 799 HTLV‐seronegative controls has been followed as part of the Retrovirus Epidemiology Donor Study. A novel methodology was developed to systematically review peripheral blood slides from these subjects for HTLV‐related lymphocyte abnormalities, using an algorithm based on morphologic features to objectively identify flower cells. The algorithm included: absence of azurophil granules; nuclear chromatin condensation; cell size >1.5 small lymphocytes; nuclear to cytoplasmic ratio >80%; and presence of nuclear folding/lobulation. Peripheral slides from subjects were screened by a medical technologist blinded to HTLV status. 6.8% of HTLV‐I subjects (P = 0.0001 versus seronegatives), 0.9% of HTLV‐II subjects and 1.1% of seronegatives were confirmed to have cells classified as flower cells by two haematologists using objective criteria, and blinded to serostatus. Despite the higher prevalence of flower cells in HTLV‐I positives, no clinical correlations were found. Longitudinal follow‐up may yield higher rates of cellular abnormalities as the sequelae of HTLV infection develop.
Fatal immune haemolysis due to a degradation product of ceftriaxone
Authors: Meyer Oliver , Hackstein Holger , Hoppe Berthold , Göbel Franz‐Josef , Bein Gregor , Salama Abdulgabar
Published: March 2003

A 16‐year‐old girl who was treated with ceftriaxone developed two intravascular haemolytic attacks that led to acute renal failure and death. The direct antiglobulin test was positive only for C3d, and no ceftriaxone‐dependent antibodies were detectable. Her serum reacted strongly positive with red blood cells in the presence of ex vivo antigen related to ceftriaxone (urine samples from patients receiving the drug). This is the first reported case in which the causative antibodies appeared to be stimulated solely by a degradation product of ceftriaxone.
Liposome‐encapsulated daunorubicin for PGP‐related multidrug resistance
Authors: Michieli Mariagrazia , Damiani Daniela , Ermacora Anna , Masolini Paola , Michelutti Angela , Michelutti Teresa , Russo Domenico , Pea Federico , Baccarani Michele
Published: March 2003

The possibility that Daunoxome (DNX), a combination of daunorubicin (DNR) with a liposomal targeting system, escapes PGP was tested. Two pairs of leukaemic cell lines, each consisting of the parental non‐multidrug resistance (MDR) line and of a MDR variant, were studied for cytotoxicity (MTT test) and for cellular DNR kinetic and accumulation (flow cytometry). DNX and free DNR were equally toxic against non‐MDR cells, whereas the liposomal anthracycline was more toxic than the free drug against the MDR variant. Non‐MDR cells accumulated DNR more rapidly when they were exposed to free DNR than to DNX, but MDR cells accumulated more DNR when they were exposed to DNX. The kinetics of DNX and free DNR were also studied in the blast cells of 41 cases of acute leukaemia and they were found to be related to blast cell PGP expression. In 15 cases with a low PGP expression intracellular DNR accumulation was faster and higher with free DNR than with DNX. In 26 cases with a high PGP expression the area under the curve was similar with DNX and free DNR, but the kinetics of intracellular DNR accumulation showed an early low plateau with free DNR and a slow and continuous increase with DNX. In MDR cell lines the ratio was more favourable to DNX than to free DNR. We conclude that liposome encapsulated DNR is partially protected from PGP and that it is worth testing for the treatment of PGP‐positive acute leukaemia.
Selective effect of cyclosporine monotherapy for pure red cell aplasia not associated with granular lymphocyte‐proliferative disorders
Authors: Yamada Osamu , Motoji Toshiko , Mizoguchi Hideaki
Published: March 2003

Morphological characteristics of lymphocytes and the response to cyclosporine treatment have revealed some unique patients with pure red cell aplasia. Lymphocytes from these patients consisted mainly of non‐granulated lymphocytes. All of the patients were successfully managed by cyclosporine monotherapy irrespective of prior treatment. A reduction in lymphocyte mass was not a prerequisite for the remission of pure red cell aplasia, and responses occurred within 1 month from the start of therapy. Clonal T‐cell proliferation was detected in four patients, which raised the possibility of idiopathic pure red cell aplasia being associated with a clonal proliferation of T cells. An examination of the lymphocytes in patients with pure red cell aplasia could potentially be used to plan better therapeutic modalities and assess prognosis.
Interaction of thrombopoietin with the platelet c‐mpl receptor in plasma: binding, internalization, stability and pharmacokinetics
Authors: Li Junzhi , Xia Yuping , Kuter David J.
Published: March 2003

Thrombopoietin (TPO) is the primary regulator of platelet production and acts through binding its receptor, c‐mpl, found on megakaryocyte progenitor cells, megakaryocytes and platelets. Circulating levels of TPO are regulated primarily by the clearance of TPO after it binds to c‐mpl receptors on circulating platelets. In this study the interaction of TPO with the platelet c‐mpl receptor has been analysed under physiological conditions using radiochemical and pharmacokinetic approaches. 125I‐rHuTPO was prepared using a novel method of gentle iodination that preserved its biological activity and used to demonstrate that platelets, but not endothelial cells, have a single class of binding sites (56 ± 17 binding sites/platelet) with high affinity (Kd = 163 ± 31 pM). Cross‐linking experiments confirmed that TPO, but not erythropoietin (EPO), specifically associated with the 95 kD platelet c‐mpl receptor. Upon addition of TPO to platelets, 80% of the TPO binding sites were internalized within an hour and were not recycled. TPO that was not bound by platelets was stable for up to 6 d in both platelet‐poor and platelet‐rich plasma. Using unlabelled recombinant human TPO (rHuTPO), standard pharmacokinetic analysis demonstrated that platelets have an average TPO clearance of 1.24 ± 0.38 ml/h/109 platelets and that TPO clearance was reduced by low temperature but not by a number of drugs or metabolic inhibitors. The maximal amount of TPO removed by platelets in vitro was identical to that predicted by the total number of TPO binding sites. These results provide a biochemical and pharmacokinetic basis for the clinical use of TPO and for understanding possible disorders of platelet production.
Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias
Authors: Auner Holger W. , Olipitz Werner , Hoefler Gerald , Bodner Claudia , Konrad Dagmar , Crevenna Richard , Linkesch Werner , Sill Heinz
Published: March 2003

Mutations of the DNA mismatch repair (MMR) gene hMLH1 have recently been linked to the development of some hereditary and sporadic cancers which frequently display widespread microsatellite instability (MSI). Conflicting results regarding the extent of MSI in myeloid leukaemias prompted us to perform mutational analysis of all 19 exons of the hMLH1 gene by polymerase chain reaction–single‐stranded conformation polymorphism (PCR‐SSCP) and sequence analysis in a total of 133 patients with acute and chronic myeloid leukaemia. Apart from one exonic and one intronic polymorphism, no mutations were detected in any of the samples indicating that the major MMR gene hMLH1 is not involved in the pathogenesis or progression of myeloid malignancies.
Therapeutic monitoring of von Willebrand disease: interest and limits of a platelet function analyser at high shear rates
Authors: Fressinaud Edith , Veyradier Agnès , Sigaud Marianne , Boyer‐Neumann Catherine , Le Boterff Cécile , Meyer Dominique
Published: March 2003

We have evaluated the position of the Platelet Function Analyzer PFA‐100TM in the management of 41 patients with von Willebrand disease (VWD) receiving either desmopressin (23 patients with type 1, five with type 2M, three with type 2A and three with type 2B) or von Willebrand factor (VWF) concentrates (four patients with type 3, two with type 2M ‘type B’, two with type 2A and one type 1 ‘platelet low’). In all patients the following were studied before and 30 min after infusion of desmopressin and/or VWF concentrates: VWF ristocetin cofactor activity (VWFRCo), bleeding time (BT) and closure time with the PFA‐100 using ADP (CT‐ADP) as well as epinephrine (CT‐Epi) cartridges. After the infusion of desmopressin, the CT was modified in the same way as the VWFRCo levels, being always normalized in patients with type 1 and not constantly corrected in those with type 2. Thus, our results indicated that the measurement of the CT enabled a quick and accurate evaluation of the response to desmopressin which, in fact, measured the releasable VWF cellular compartment containing the highly multimerized forms of VWF. For patients with type 2 or 3 VWD who were non‐responsive to desmopressin, VWF concentrates corrected the VWFRCo defect but not the CT as none of these patients had a normal platelet VWF content and the VWF concentrates did not contain the ultralarge VWF multimers. In conclusion, the very high shear conditions in the PFA‐100 make it very sensitive to the contribution of platelet VWF and to the ultralarge VWF multimers, indicating that the evaluation of the CT is a very simple and rapid tool to discriminate between good and non‐responders to desmopressin.
Standardization of the PFA‐100® platelet function test in 105 mmol/l buffered citrate: effect of gender, smoking, and oral contraceptives
Authors: Böck  , De Haan  , Beck  , Gutensohn  , Hertfelder  , Karger  , Heim  , Beeser  , Weber  , Kretschmer
Published: March 2003

The PFA‐100® (PFA) diagnostic system for the detection of platelet dysfunction was evaluated to determine reference ranges in a normal population. The PFA determines the primary haemostasis capacity (PHC) of anticoagulated whole blood, expressed by the system's closure time (CT). In this study the CT reference ranges were determined for blood samples collected in 105 mmol/l (3.2%) buffered citrate and the effect of gender, smoking, and use of oral contraceptives on reference ranges was assessed. Each of the 309 healthy blood donors from five blood centres was confirmed to have normal platelet function before inclusion in the study. Blood samples were tested in duplicate with both the collagen/epinephrine (Col/Epi) and collagen/ADP (Col/ADP) test cartridges.

PFA reference ranges (90% central intervals of measured closure times) for both cartridge types were similar for all groups. Subgroup analysis showed that neither gender nor oral contraceptive usage had any effect on PHC. The 95% cut‐off value for the Col/Epi CT was slightly higher for smokers than for non‐smokers, an effect more pronounced in female than in male donors. However, the small difference did not justify establishment of specific reference ranges for smokers. Data from all included subjects were pooled to calculate the CT reference ranges for blood samples collected in 105 mmol/l buffered citrate (Col/Epi 82–150 s; Col/ADP 62–100 s). Normal levels of fibrinogen, as well as normal platelet counts and normal haematocrit levels, appeared not to influence the PHC. Because slight but significant differences of the reference ranges were observed between some of the participating sites, in‐house confirmation of these reference range guidelines is recommended.
VH gene usage by family members affected with chronic lymphocytic leukaemia
Authors: Pritsch Otto , Troussard Xavier , Magnac Christian , Mauro Francesca R. , Davi Frederic , Payelle‐Brogard Beatrice , Dumas Gerard , Pulik Marc , Clerget Françoise , Mandelli Franco , Chiorazzi Nicholas , Schroeder Jr Harry W. , Leporrier Michel , Dighiero Guillaume
Published: March 2003

The excess risk of chronic lymphocytic leukaemia (CLL) in the first‐degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non‐random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B‐cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members.

Among familial cases, VH gene segment utilization proved non‐random and diverged from the frequencies previously reported among unrelated patients with CLL. Specifically, although the 4‐34 and 5‐51 gene segments were found repeatedly, the 1‐69 and 4‐39 gene segments were used sparingly and the 3‐23 gene segment presented with increased frequency. Following the pattern detected in studies of unrelated patients, the single 1‐69 expressing CLL contained an unmutated H chain sequence and included a long HCDR3 interval. In contrast, 3‐23 containing H chains all used JH4, retained at most 93% homology with germline sequence, and included only short HCDR3 intervals. The vast majority of the CLL variable domains contained a high degree of somatic mutation and exhibited an excess of replacement mutations in the CDR intervals. These findings suggest that familial CLL cases may preferentially derive from B‐cell progenitors that have responded to antigen.
Discrepant expression of membrane and soluble isoforms of Fas (CD95/APO‐1) in adult T‐cell leukaemia: soluble Fas isoform is an independent risk factor for prognosis
Authors: Kamihira Shimeru , Yamada Yasuaki , Tomonaga Masao , Sugahara Kazuyuki , Tsuruda Kazuto
Published: March 2003

The Fas signalling system probably plays a critical role in the natural and chemotherapeutic cell death machinery, suggesting that aberrant Fas expression is involved in growth control of tumours. The membrane isoform (mFas) is a 45 kD cell surface protein containing a single transmembrane region, and induces apoptosis in normal or tumour cells, whereas the soluble isoform (sFas) lacks the transmembrane domain due to alternative splicing of the transcript and is thought to block Fas‐mediated apoptosis. To clarify the clinical roles of expression of these two Fas isoforms in adult T‐cell leukaemia (ATL), we investigated the levels of the Fas isoforms in 81 patients with ATL. The expression patterns of the Fas isoforms were heterogenous, and there was no significant correlation between mFas and sFas levels: 10/81 cases were negative for mFas and had high serum sFas levels, whereas the remaining 71 cases were positive for mFas and had various levels of expression of the two Fas isoforms. Irrespective of the status of mFas expression in leukaemic cells, the mRNAs encoding these isoforms were always detectable, indicating the potential for protein translation. Although mFas expressed on freshly isolated ATL cells could iduce apoptosis in vitro, positive versus negative mFas status was not associated with any clinical aspects of ATL, whereas the sFas level was strongly correlated with clinical parameters such as serum LDH activity, tumour burden, serum soluble IL‐2R level, hypercalcaemia and prognosis. These results suggest that the ratio of Fas isoforms varies, and high expression of the sFas protein and message reflects the malignant behaviour of ATL and is an independent risk factor for the prognosis.
Transmission of symptomatic parvovirus B19 infection by fibrin sealant used during surgery
Authors: Hino Masayuki , Ishiko Osamu , Honda Ken‐ichi , Yamane Takahisa , Ohta Kensuke , Takubo Takayuki , Tatsumi Noriyuki
Published: March 2003

Human parvovirus B19 infection has been shown to be transmissible by blood and blood products and to result in transient aplastic crisis in patients with rapid red cell turnover. We report three cases of iatrogenic parvovirus B19 infection resulting from the use of the same batch of fibrin sealant under operation. Fibrin sealant, which is a typical haemostatic agent produced from blood, has been used during surgery. Human parvovirus is resistant to existing virus‐inactivating techniques, suggesting that infection may occur from blood products contaminated with it. Use of recombinant products for these proteins may thus be necessary.
Liver biopsy in Irish hepatitis C‐infected patients with inherited bleeding disorders
Authors: McMahon C. , Pilkington R. , Shea E. O. , Kelleher D. , Smith O. P.
Published: March 2003

The majority of patients receiving plasma‐derived clotting factor concentrates between 1970s and the mid‐1980s are now hepatitis C positive. The progression of hepatitis C is extremely variable and there is frequently a poor correlation among liver biochemistry, viral load and the stage of liver disease. Liver biopsy remains the only definitive way of staging fibrosis and grading necroinflammatory activity. Concerns have been expressed about the safety of the procedure; however, with modern regimes for the correction of coagulopathy in patients with inherited bleeding disorders, normal haemostasis may be maintained during the peribiopsy period. We performed 21 liver biopsies between 1984 and 1997 on patients with factor VIII (FVIII) or IX (FIX) deficiency and von Willebrand's Disease (VWD). Four had concomitant human immunodeficiency virus (HIV) infection, five were thrombocytopenic and one had a prolonged prothrombin time (PT). Haemostasis was achieved using an intermittent bolus of factor concentrate or continuous infusion regimens. One patient with VWD received Desmopressin (DDAVP). There were no bleeding episodes associated with biopsy. We suggest that liver biopsy is a safe procedure in patients with inherited bleeding disorders when the coagulopathy is fully corrected. It is the only definitive method of staging the extent of fibrosis associated with hepatitis C infection, and it is this that defines prognosis.
Asparagine synthetase activity in paediatric acute leukaemias: AML‐M5 subtype shows lowest activity
Authors: Dübbers A. , Würthwein G. , Müller H. J. , Schulze‐Westhoff P. , Winkelhorst M. , Kurzknabe E. , Lanvers C. , Pieters R. , Kaspers G. J. L. , Creutzig U. , Ritter J. , Boos J.
Published: March 2003

Lack of sufficient cellular activity of asparagine synthetase (AS) in blast cells compared with normal tissue is thought to be the basis of the antileukaemic effect of l‐asparaginase in acute lymphoblastic leukaemia (ALL). Although
l‐asparaginase is routinely used in ALL, its role and value in the treatment of acute myelogenous leukaemia (AML) is still being discussed. To evaluate the pharmacological basis for
l‐asparaginase treatment, we established pretreatment monitoring of the intracellular AS activity in blast cells of patients with AML and ALL. There was no general difference in AS activity between ALL and AML samples. Significantly lower AS activity, however, was found in the B‐lineage ALL subgroups as well as AML‐M5.
A combination of anti‐interleukin 6 murine monoclonal antibody with dexamethasone and high‐dose melphalan induces high complete response rates in advanced multiple myeloma
Authors: Moreau Philippe , Harousseau Jean‐Luc , Wijdenes John , Morineau Nadine , Milpied Noël , Bataille Régis
Published: March 2003

To improve the complete response (CR) rate in advanced multiple myeloma (MM) without increasing the toxicity of high‐dose therapy, we have used a new conditioning regimen. A combination of BE‐8 [an anti‐interleukin 6 (IL‐6) murine monoclonal antibody] and dexamethasone followed by high‐dose melphalan (220 mg/m2) and autologous stem cell transplantation was used to treat a series of 16 patients with advanced multiple myeloma. A strong inhibition of IL‐6 activity evaluated by quantification of C‐reactive protein was observed in all patients and was correlated with the high CR rate achieved with this combination therapy.
Induction therapy consisting of alternating cycles of ranimustine, vincristine, melphalan, dexamethasone and interferon α (ROAD‐IN) and a randomized comparison of interferon α maintenance in multiple myeloma: a co‐operative study in Japan
Authors: Wada Makio , Mizoguchi Hideaki , Kuriya Shin‐ichiro , Taguchi Hirokuni , Kawamura Tsugumichi , Maekawa Isao , Shimazaki Chihiro , Sato Yutaka , Niho Yoshiyuki , Miyazaki Tamotsu , Shibata Akira , Kitani Teruo , Hamajima Nobuyuki , Ohno Ryuzo ,   the Japanese Co‐operative Study Group for the Treatment of Multiple Myeloma
Published: March 2003

This pilot study evaluated the efficacy of a new combination chemotherapy with a newly developed nitrosourea derivative ranimustine and evaluated the efficacy of interferon α (IFN‐α) maintenance in previously untreated patients with multiple myeloma (MM). The induction therapy (ROAD‐IN) was a 6‐week regimen consisting of chemotherapy with ranimustine, vincristine (Oncovin), melphalan (Alkeran) and dexamethasone starting on day 1 and IFN‐α, which was administered three times weekly for 3 weeks starting on day 22. This was repeated for three cycles. The responders were subsequently randomized into two groups that received or did not receive IFN‐α as maintenance therapy. Of the 164 patients registered, 161 were evaluated. An objective response to induction therapy was seen in 75% of patients; complete remission (CR) in 38 (24%) and partial remission (PR) in 82 (51%). The median survival for all patients was 3·6 years from registration. The survival of responders (CR + PR) was significantly better than that of non‐responders (median survival 4·3 years vs. 1·4 years; 7‐year survival rate 32% vs. 9%; P < 0·0001). The IFN‐α maintenance did not show any advantage for either response duration or survival. This pilot study demonstrated that a comparatively short period of induction therapy with the ROAD‐IN regimen produced a rather high response rate and a similar survival rate to those achieved with other longer induction regimens, and that good responders to the initial therapy survived significantly longer than non‐responders.
Hereditary thrombocythaemia is a genetically heterogeneous disorder: exclusion of TPO and MPL in two families with hereditary thrombocythaemia
Authors: Wiestner A. , Padosch S. A. , Ghilardi N. , Cesar J. M. , Odriozola J. , Shapiro A. , Skoda R. C.
Published: March 2003

Hereditary thrombocythaemia (HT) is an autosomal dominant disorder with clinical presentation and complications resembling sporadic essential thrombocythaemia (ET). Mutations in the thrombopoietin (TPO) gene causing overproduction of TPO and elevated TPO serum levels have been found previously in three families with HT. Here, we present evidence for genetic heterogeneity by demonstrating that HT in a Spanish and a US family is caused by genes other than TPO. Affected family members in both families had normal TPO serum levels. Genetic linkage analysis with TPO microsatellite markers excluded TPO as the disease gene in the Spanish HT family, and sequencing of the TPO gene revealed no mutations in the propositus of the US family. To test a role for MPL, the gene for the TPO receptor, we identified three single nucleotide polymorphisms (SNP) and a novel polymorphic CA microsatellite marker. By linkage analysis, we excluded MPL as the cause of HT in the Spanish family. Interestingly, mapping of the CA microsatellite marker to a region 40·5 kb upstream of MPL revealed the presence of sequences from the TIE gene, which encodes a tyrosine kinase receptor expressed on megakaryocytes and endothelial cells. Thus, MPL and TIE are in close physical proximity, and the CA microsatellite described here will be a useful genetic marker for both genes.
A case of purpura fulminans is caused by homozygous Δ8857 mutation (protein C‐Nagoya) and successfully treated with activated protein C concentrate
Authors: Nakayama Takayuki , Matsushita Tadashi , Hidano Hiroshi , Suzuki Chizuko , Hamaguchi Motohiro , Kojima Tetsuhito , Saito Hidehiko
Published: March 2003

We report a Japanese patient who developed purpura fulminans and disseminated intravascular coagulation (DIC) shortly after birth. The patient was diagnosed to be homozygous for protein C deficiency and was treated with an activated protein C (APC) concentrate. Intravenous infusions of APC markedly improved the necrotic skin lesions and the anticoagulation by APC enabled successful DIC control. The identified mutation (Δ8857) results in impaired intracellular transport and protein maturation and would be the cause of the complete protein C deficiency. This is the seventh case of the mutation that has been exclusively reported in Japan, but is the first report of a homozygous case. Our findings propose new therapeutic and diagnostic tools for the management of this fatal thrombotic disease.
A quantitative study of peripheral blood stem cell contamination in diffuse large‐cell non‐Hodgkin's lymphoma: one‐half of patients significantly mobilize malignant cells
Authors: Jacquy C. , Sorée A. , Lambert F. , Bosly A. , Ferrant A. , André M. , Parma J. , Kentos A. , Martiat P.
Published: March 2003

Autologous transplantation using peripheral blood stem cells (PBSCs) collected after chemotherapy, followed by growth factor administration (ASCT), is increasingly used in the treatment of non‐Hodgkin's lymphoma (NHL). However, quantitative data regarding contaminating malignant cells in the harvests are still scarce. We prospectively investigated 37 diffuse large‐cell lymphomas (DLCLs) in complete remission (CR) that were treated according to multicentric protocols at our centre. DNA was extracted from the diagnostic lymph node. The complementarity‐determining region (CDR) III was sequenced and a patient‐specific oligomer synthesized. Contamination was evaluated semiquantitatively by polymerase chain reaction (PCR) and was confirmed by a limiting dilution analysis. PBSCs collected at regeneration after administration of granulocyte colony‐stimulating factor (G‐CSF), steady‐state bone marrow (BM) and peripheral blood samples at CR were compared. DNA was available in 37 patients, from which 22 rearrangements could be sequenced. Patients (n = 15) who had both the required follow‐up samples and a suitable clonal marker were investigated. In two cases, the patient‐specific PCR assay set up at diagnosis later gave false‐negative results in samples in which clonal DNA was still detectable by other sets of primers. PBSC contamination was highly variable: 7 out of 15 patients showed a PBSC/BM ratio of NHL cells greater than 1 log, whereas 8 out of 15 patients showed no difference and could vary from one apheresis to another. Eight ASCTs were performed, five of which used highly contaminated PBSCs: four patients relapsed early, three with disseminated lymphoma. Thus, 50% of DLCLs in CR seem to mobilize significantly malignant cells at regeneration under G‐CSF. Considering the higher numbers of cells reinfused, this translates into a much higher number of lymphoma cells reinfused when compared with autologous bone marrow transplantation (ABMT). However, their clonogenic potential remains unknown and, despite concerning observations in certain cases, it is still unclear whether this has an impact upon the outcome of ASCT.
Nephrotic syndrome caused by protein thrombi in glomerulocapillary lumen in Waldenström's macroglobulinaemia
Authors: Harada Yoshimi , Ido Nobuhiro , Okada Tomoya , Otani Masako , Shirota Tsunemichi , Nakao Toshiyuki , Hayashi Tohru
Published: March 2003

Waldenström's macroglobulinaemia (WM) is described as a disorder of plasmacytoid lymphocytes. The renal complications of WM are less common and severe than those of multiple myeloma. We present a case of WM complicated by nephrotic syndrome. A biopsy specimen of the kidney revealed the intraglomerular thrombi of immunoglobulin M paraprotein. Corticosteroid pulse therapy and plasmapheresis were effective in improving proteinuria and reducing protein thrombi. The nephrotic syndrome caused by protein thrombi in WM may be reversible, at least in its early stage.
Relationship between bleeding time, aspirin and the PlA1/A2 polymorphism of platelet glycoprotein IIIa
Authors: Szczeklik Andrzej , Undas Anetta , Sanak Marek , Frołow Marzena , Węgrzyn Wojciech
Published: March 2003

A single nucleotide T to C transition of the gene encoding glycoprotein IIIa leads to a common diallelic polymorphism Leu‐33→Pro (PLA1/A2). We studied the relationship between the PlA1/A2 polymorphism and platelet function in 80 healthy men, aged 20–25 years. Before aspirin ingestion, bleeding time (BT) was shorter in carriers of the PlA2 than in carriers of the PlA1/A1 allele. At 4 h after ingestion of 300 mg of aspirin, BT became prolonged, and the intergroup difference was enhanced. In seven out of 26 PLA2 allele carriers, aspirin shortened BT on average by 30 s, compared with only one among 54 subjects with the PlA1/A1 genotype. Thus, BT both at baseline and after aspirin depends on the PlA1/A2 polymorphism of glycoprotein IIIa. Carriers of the PlA2 allele appear to be more resistant to the antithrombotic action of aspirin.
Generation of human osteoclasts in stromal cell‐free and stromal cell‐rich cultures: differences in osteoclast CD11c/CD18 integrin expression
Authors: Lader Charlotte S. , Scopes John , Horton Michael A. , Flanagan Adrienne M.
Published: March 2003

Osteoclasts form in the presence of macrophage colony‐stimulating factor (M‐CSF) and receptor activator of Nfκb ligand (RANKL), a membrane‐bound differentiation factor that is now available as a soluble recombinant molecule. Acquisition of the osteoclast phenotype [the αvβ3 subunit of the vitronectin receptor (VNR)‐, calcitonin receptor (CTR)‐ and F‐actin ring‐positive cells] is associated with loss of monocyte/macrophage‐associated integrins, specifically CD11b, CD11c and CD18. We hypothesized that differences in the osteoclast integrin adhesion molecule profile may exist in osteoclasts generated in stromal cell‐rich and in stromal‐free conditions. Unlike osteoclasts generated in vivo, F‐actin ring‐positive (resorbing) osteoclasts formed in soluble RANKL in vitro, in the absence of stromal cells, and co‐expressed CD11c and CD18. However, when osteoclasts were generated from peripheral blood mononuclear cells (PBMNCs) in co‐cultures with the murine bone marrow stromal cell line 218 (which does not produce membrane‐bound RANKL) in the presence of soluble RANKL, CD11c and CD18 were not expressed by osteoclasts. These findings indicate that the persistent expression of CD11c and CD18 is not accounted for by RANKL being presented in a soluble form and that membrane‐bound RANKL is not required for the normal integrin expression in resorbing osteoclasts. This study demonstrates that potentially misleading information may arise by using data obtained from osteoclasts generated in the absence of stromal cells as they do not completely reflect the situation in vivo.
Von Willebrand factor collagen binding activity in the diagnosis of von Willebrand disease: an alternative to ristocetin co‐factor activity?
Authors: Casonato A. , Pontara E. , Bertomoro A. , Sartorello F. , Cattini M. G. , Girolami A.
Published: March 2003

The capability of von Willebrand factor (VWF) to bind platelet glycoprotein Ib (GPIb) and promote platelet plug formation is currently evaluated in vitro using the ristocetin co‐factor activity (VWF:RCo) assay. The replacement of this cumbersome and not always reproducible test with the collagen binding activity of VWF (VWF:CBA) has been attempted with controversial results. To evaluate the capacity of VWF:CBA to identify classic and variant von Willebrand disease (VWD) compared with VWF:RCo, we studied 10 type 2A and 12 type 2B VWD patients, together with 30 type 1 VWD patients with reduced platelet VWF content. In both 2A and 2B VWD, VWF:CBA and VWF:RCo were decreased, but that of VWF:CBA was more consistent. The difference was more evident when values were expressed as a ratio, obtained by normalizing VWF:CBA and VWF:RCo with the VWF antigen value; the ratio for VWF:CBA was always below 0·2, while that for VWF:RCo was greater than 0·4, and in no patient was the VWF:CBA value higher than VWF:RCo. In contrast, in type 1 VWD, the decrease in VWF:CBA was similar to that seen in VWF:RCo with the ratios always within the normal range. To better investigate the relationship between VWF:CBA and VWF:RCo, and the representation of large/intermediate VWF multimers, to which both tests are sensitive, 1‐deamino‐cys‐8‐D‐arginine‐vasopressin (DDAVP) was infused in type 2A and 2B VWD patients. The differences between the two tests were even more evident after DDAVP, and in type 2A, even though large multimers were persistently decreased, VWF:RCo was normalized, while VWF:CBA remained defective. These findings clearly indicate that VWF:CBA detects the absence of large and intermediate VWF multimers better than VWF:RCo. Hence, we suggest adding VWF:CBA to the panel of tests employed in the diagnosis of VWD. Moreover, owing to the difficulty in performing VWF:RCo and its low reproducibility, we suggest that, when necessary, VWF:CBA may be substituted for VWF:RCo.
Prognostic significance of tumour‐infiltrating T lymphocytes and T‐cell subsets in de novo diffuse large B‐cell lymphoma: a multiparameter flow cytometry study
Authors: Xu Yin , Kroft Steven H. , McKenna Robert W. , Aquino Deborah B.
Published: March 2003

Tumour‐infiltrating T lymphocytes (TIL‐T) have been implicated in playing a role in controlling tumour growth. We evaluated TIL‐T in 55 cases of de novo diffuse large B‐cell lymphoma (DLBCL) using three‐ or four‐colour flow cytometric immunophenotyping (FCI). The percentage of TIL‐T varied from 3% to 72% of total viable cellular events (mean 32 ± 20%). The CD4:CD8 ratio varied from 0·17 to 13 (mean 2·3 ± 2·2). Cases with ≥ 20% T cells and those with CD4:CD8 ratios ≥ 2·0 showed a significantly better overall survival (P = 0·017 and P = 0·034 respectively). These findings were independent of clinical stage at diagnosis. The T‐cell percentage and CD4:CD8 ratio were moderately correlated (Spearman correlation coefficient = 0·47, P = 0·001) and multivariate analysis revealed that the association of the two factors with prognosis was mutually dependent. The T cells in 23 cases were studied for CD45RO. The mean percentage of total T cells expressing CD45RO was 86 ± 10%. There was a trend towards better survival for those patients with a higher percentage of CD45RO+ T cells (P = 0·06). These results suggest that TIL‐T, particularly CD4+ T cells, may play a role in the control of DLBCL, and measurement of T‐cell percentage and T‐cell subsets using FCI may be useful in predicting the clinical behaviour of DLBCL.
Long‐term follow‐up in patients treated with Mini‐BEAM as salvage therapy for relapsed or refractory Hodgkin's disease
Authors: Martín A. , Fernández‐Jiménez M. C. , Caballero M. D. , Canales M. A. , Pérez‐Simón J. A. , García de Bustos J. , Vázquez L. , Hernández‐Navarro F. , San Miguel J. F.
Published: March 2003

Several studies have focused on investigation of the optimal salvage regimen to induce maximum response before autologous stem cell transplantation (ASCT) in patients with relapsed or refractory Hodgkin's disease (HD). However, in most of these studies, the follow‐up is relatively short. In the present study, we report on long‐term results of 55 consecutive patients with HD who received Mini‐BEAM [BCNU (carmustine), etoposide, cytarabine, melphalan] as salvage therapy before ASCT. Eleven patients were refractory to front‐line therapy, 17 were partial responders, and 27 patients had relapsed from HD. Twenty‐eight patients achieved complete response, and 18 achieved partial response with a median of two cycles of Mini‐BEAM, giving a total response rate of 84%. Significant factors predicting poor response (P < 0·05) were: initial treatment with MOPP (mechloroethamine, oncovin, procarbazine, prednisolone), ≥ two previous chemotherapy regimens and three disease characteristics at Mini‐BEAM treatment: presence of B symptoms, extranodal involvement or low serum albumin. However, only the last two factors retained independent influence on multivariate analysis. In total, 45/55 patients have been transplanted. Median follow‐up after Mini‐BEAM administration for living patients is 68 months. At the time of reporting, 31 out of 55 patients (56·4%) are still alive, 21 patients (38%) have relapsed, three (5·4%) have developed secondary neoplasias, and five have died of other complications not related to disease progression. The actuarial 7‐year overall survival (OS) was 52%, the progression‐free survival (PFS) 54% and the event‐free survival (EFS) 36%. The response to Mini‐BEAM was the most important prognostic factor for predicting the long‐term probability of surviving the disease: none of the eight patients who did not respond to Mini‐BEAM were alive at 3 years. On multivariate analysis, response to Mini‐BEAM and extranodal involvement before Mini‐BEAM had a significant influence on OS. Our results show the safety and efficacy of Mini‐BEAM before ASCT for refractory or relapsed HD patients.