bluuebunny/crossref_metadata_2025 huggingface dataset:
Human herpesvirus type 7 in Hodgkin's disease
Authors: Secchiero Paola , Davico Bonino Laura , Lusso Paolo , Abele Maria Cristina , Reato Gigliola , Kerim Simonetta , Palestro Giorgio , Zauli Giorgio , Valente Guido
Published: March 2003

Several lines of evidence have pointed to the involvement of a viral agent in the pathogenesis of Hodgkin's disease (HD). Therefore we investigated the presence of human herpesvirus type 7 (HHV‐7) in 53 cases of HD by polymerase chain reaction (PCR), DNA in situ hybridization (ISH) and immunohistochemistry. HHV‐7 DNA was frequently detected (68% of the cases) in HD biopsies by PCR independently of the histological type, whereas only 32% (P < 0.05) of positive cases were found in 19 reactive lymph nodes. However, by applying the quantitative PCR technique, the majority of the samples showed a low level of viral load. Moreover, ISH for HHV‐7 DNA was positive in a low number of small T lymphocytes and consistently negative in Hodgkin and Reed‐Sternberg (HRS) cells, which appeared negative for HHV‐7 also at immunohistochemistry.  These results indicate that the high frequency of HHV‐7 infection in HD: (i) is probably non‐productive, (ii) mainly involves small lymphocytes belonging to the T‐lineage, and (iii) is probably due to the recruitment of non‐malignant reactive cells in HD tissue.
Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse
Authors: Kurata Hitoshi , Mancini Gian Carlo , Alespeiti Gabriel , Migliaccio Anna Rita , Migliaccio Giovanni
Published: March 2003

We have investigated the kinetics of the amplification of the progenitor cell compartments (CFC) in haemopoietic organs during murine ontogenesis and compared the growth requirements of fetal and adult CFC. Two haemopoietic phases were recognized in the fetal liver (FL): an exponential growth phase, from 11.5 to 15.5 d post conception (p.c.), during which the mean number of nucleated cells and of CFC in the FL increased from 4.9 × 105 to 7.0 × 107 and from 4.5 × 103 to 2.7 × 105, respectively, and a recessive phase after 15.5 d p.c., during which the CFC number in the FL gradually decreased, although some CFC were still detectable in the liver after birth. In serum‐deprived cultures, FL and adult marrow (AM) CFC had similar responses to GM‐CSF, and did not respond to G‐CSF or IL‐3. In contrast, FL, but not AM, erythroid colonies grew Epo‐independently whereas SCF alone induced formation of maximal numbers of erythroid bursts from FL, but not from AM cells. The proliferative and differentiative effect of SCF alone on fetal cells was confirmed in serum‐deprived cultures of purified early progenitor cells isolated by cell sorting on the basis of multiple parameters from FL and AM light‐density cells. In culture of purified FL cells, SCF alone induced a similar amplification of total cells (maximal amplification at day 12: 800–300‐fold) and total CFC (11–38‐fold of maximal amplification at day 6) to the combination of SCF plus IL‐3 (1300–800‐fold amplification of total cells and 31–88‐fold amplification of CFC). In contrast, SCF alone allowed only survival of purified AM early progenitor cells. Therefore FL early progenitor cells have an intrinsic higher potential than their adult counterpart to respond to SCF, confirming the potent role of this growth factor in the development of the murine haemopoietic system.
Molecular abnormality observed in a patient with coagulation factor X (FX) deficiency: a novel three‐base‐pair (CTT) deletion within the polypyrimidine tract of the FX intron D
Authors: Hayashi Tomohiro , Yahagi Akito , Suzuki Keijiroh , Sato Shinji , Sasaki Hideo
Published: March 2003

We investigated the molecular defect underlying congenital factor X (FX) deficiency in a Japanese patient. A novel three‐base‐pair deletion was identified within intron D of the FX gene. An allele‐specific restriction analysis showed the propositus was homozygous and her two daughters were heterozygous for the deletion. It was not detected in 53 unrelated Japanese (106 alleles), indicating a probable cause of the FX deficiency. The deletion resides within a polypyrimidine tract of the acceptor splicing site where U2 snRNP binds to form spliceosomes. The defect could alter the formation of spliceosomes, resulting in incorrect splicing and decreased FX production.
Quinine improves the results of intensive chemotherapy in myelodysplastic syndromes expressing P glycoprotein: results of a randomized study
Authors: Wattel  , Solary  , Hecquet  , Caillot  , Ifrah  , Brion  , MahÉ  , Milpied  , Janvier  , Guerci  , Rochant  , Cordonnier  , Dreyfus  , Buzyn  , Hoang‐Ngoc  , Stoppa  , Gratecos  , Sadoun  , Stamatoulas  , Tilly  , Brice  , Maloisel  , Lioure  , Desablens  , Pignon  , Abgrall  , Leporrier  , Dupriez  , Guyotat  , Lepelley  , Fenaux
Published: March 2003

Intensive chemotherapy produces a lower complete remission (CR) rate in the myelodysplastic syndromes (MDS) than in de novo acute myeloid leukaemia (AML), possibly due in part to a higher incidence of P glycoprotein (PGP) expression in MDS blast cells. We designed a randomized trial of intensive chemotherapy with or without quinine, an agent capable of reverting the multidrug resistance (mdr) phenotype, in patients aged ⩽ 65 years with high‐risk MDS. Patients were randomized to receive mitoxantrone 12 mg/m2/d days 2–5 + AraC 1 g/m2/12 h days 1–5, with (Q+) or without (Q−) quinine (30 mg/kg/d). 131 patients were included. PGP expression analysis was successful in 91 patients. In the 42 PGP‐positive cases, 13/25 (52%) patients in the Q+ group achieved CR, compared to 3/17 (18%) patients in the Q− group (P = 0.02) and median Kaplan‐Meier survival was 13 months in the Q+ group, and 8 months in the Q− group (P = 0.01). No life‐threatening toxicity was observed with quinine. In conclusion, the results of this randomized study show that quinine increases the CR rate and survival in PGP‐positive MDS cases treated with intensive chemotherapy.
Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line
Authors: Daibata Masanori , Taguchi Takahiro , Taguchi Hirokuni , Miyoshi Isao
Published: March 2003

Human herpesvirus 6 (HHV‐6) genome has been found in several human lymphoid malignancies, but configuration of the HHV‐6 genome has not been well delineated. We established the HHV‐6‐positive, Epstein‐Barr virus‐negative Burkitt's lymphoma cell line Katata. In this study we investigated the status of the HHV‐6 genome in Katata cells. Neither linear nor circular HHV‐6 DNA was detected by Gardella gel analysis. The fluorescence in situ hybridization technique enabled us to directly visualize the integrated HHV‐6 DNA at the single‐cell level. Only one integrated site of viral DNA was detected in metaphase chromosomes and it was preferentially located at the long arm of chromosome 22 (22q13). Treatment of the cells with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) or with calcium ionophore A23187 led to induction of the HHV‐6 immediate‐early gene as well as the late gene. Sodium n‐butyrate also gave rise to expression of the HHV‐6 genes. The TPA inducibility was synergistically enhanced when combined with A23187 or n‐butyrate. Our study provides, for the first time, an in vitro model system of latent HHV‐6 infection whose genome is integrated into host DNA of lymphoma cells.
Liposomal amphotericin B compared with amphotericin B deoxycholate in the treatment of documented and suspected neutropenia‐associated invasive fungal infections
Authors: Leenders Alexander C. A. P. , Daenen Simon , Jansen Rob L. H. , Hop Wim C. J. , Lowenberg Bob , Wijermans Pierre W. , Cornelissen Jan , Herbrecht Raoul , Lelie Hans Van Der , Hoogsteden Henk C. , Verbrugh Henri A. , Marie Siem De
Published: March 2003

It has been suggested that a better outcome of neutropenia‐associated invasive fungal infections can be achieved when high doses of lipid formulations of amphotericin B are used. We now report a randomized multicentre study comparing liposomal amphotericin B (AmBisome, 5 mg/kg/d) to amphotericin B deoxycholate (AmB, 1 mg/kg/d) in the treatment of these infections. Of 106 possible patients, 66 were enrolled and analysed for efficacy: nine had documented fungaemia, 17 had other invasive mould infections and 40 had suspected pulmonary aspergillosis. After completion of the course medication, in the AmBisome group (n = 32) 14 patients had achieved complete response, seven a partial response and 11 were failures as compared to 6, 13 and 15 patients (n = 34) treated with AmB (P = 0.09);P = 0.03 for complete responders. A favourable trend for AmBisome was found at day 14, in patients with documented infections and in patients with pulmonary aspergillosis (P = 0.05 andP = 0.096 respectively). Mortality rates were lower in patients treated with AmBisome (adjusted for malignancy status,P = 0.03). More patients on AmB had a >100% increase of their baseline serum creatinine (P < 0.001).

The results indicate that, in neutropenic patients with documented or suspected invasive fungal infections AmBisome 5 mg/kg/d was superior to AmB 1 mg/kg/d with respect to efficacy and safety.
A polymorphism in exon b2 of the major breakpoint cluster region (M‐bcr) identified in chronic myeloid leukaemia patients
Authors: Meissner Rosely De V. , Dias Paula M. B. , Covas Dimas T. , Job Fani , Leite Márcia , Nardi Nance B.
Published: March 2003

The BCR/ABL junctional region and the b3 exon from chronic myeloid leukaemia (CML) patients were sequenced. In all 21 samples analysed the junctional region, as well as the b3 exon of 8 b3a2 mRNA molecules, presented no differences to the already described sequences. However, we identified a polymorphic base in the b2 exon in two out of seven b3a2 samples, four out of 10 b2a2 samples and all four b3a2/b2a2 samples analysed. In the eighth position before the junctional region of BCR/ABL cDNA, a cytosine replaces thymine in these cases. The polymorphism described here could be a useful marker for the differentiation of normal and rearranged BCR alleles in heterozygotes.
Transplantation for Fanconi's anaemia: long‐term follow‐up of fifty patients transplanted from a sibling donor after low‐dose cyclophosphamide and thoraco‐abdominal irradiation for conditioning
Authors: SociÉ Gérard , Devergie AgnÈs , Girinski Théodore , Piel Gaelle , Ribaud Patricia , Esperou Hélène , Parquet Nathalie , Maarek Odile , Noguera Maria‐Helena , Richard Patrice , Brison Olivier , Gluckman Eliane
Published: March 2003

We describe the long‐term follow‐up of 50 Fanconi's anaemia patients who were transplanted from a related donor with a median follow‐up of >6 years. The survival estimate was 74.4% at 54 months and 58.5% at 100 months. All patients were conditioned with low‐dose cyclophosphamide and thoraco‐abdominal irradiation. Acute graft‐versus‐host disease (GvHD) of grade II or more developed in 26 patients and chronic GvHD developed in 30/43 (69.9%) patients. The survival of patients without chronic GvHD (n = 13) was 100%. In addition to chronic GvHD, 20 pre‐transplant transfusions was shown to have an adverse impact on survival by multivariate analysis (relative risk = 7.08, P = 0.0003). Prospective follow‐up of growth and endocrine function could be performed in 31 patients. Of 20 boys, six have already reached normal puberty within the expected time. Among the 11 girls, three were at the pubertal age at the time of analysis. Growth retardation was common, whereas late complications (e.g. peripheral hypothyroidism, cataract) were rare. However, the most important long‐term complication was the occurrence of cancer in seven patients (8‐year projected incidence 24%).

Among the 32 survivors, 27 (84.5%) had a normal and four a moderately reduced performance status, and all achieved complete engraftment with donor cells. Therefore transplantation was able to cure these patients who remain at high risk for developing late complications. Clearly, a genetic predisposition and chronic GvHD could have led to the development of these cancers. However, we cannot completely rule out irradiation as a cofactor in the genesis of these cancers, and therefore no longer use irradiation for the conditioning of Fanconi's anaemia patients.
Oncogenesis in utero: fetal death due to acute myelogenous leukaemia with an MLL translocation
Authors: Hunger Stephen P. , Mcgavran Loris , Meltesen Lynne , Parker Natalie B. , Kassenbrock C. Kenneth , Bitter Mitchell A.
Published: March 2003

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants < 1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero. In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin‐embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero.
Restarting anticoagulation in prosthetic heart valve patients after intracranial haemorrhage: a 2‐year follow‐up
Authors: Butler  , Tait
Published: March 2003

35 patients with oral anticoagulant (OAC) related intracranial or intraspinal haemorrhage were studied to determine treatment received, outcome and rate of recurrent bleeding and thromboembolism after restarting OAC. All patients underwent active anticoagulant reversal and in 14 patients with prosthetic heart valves (PHV) the INR remained below 2.0 for 0–19 d (median 7) with no thromboembolic events. 10 patients received heparin, although a therapeutic level was rarely achieved. 13 patients with PHV were restarted on OAC and followed for a median 23.5 months. One patient had recurrent intracranial bleeding. 3/13 patients had cerebral embolic events despite anticoagulation. We conclude that in PHV patients temporary cessation of OAC is safe and the risk of recurrent bleeding after restarting OAC is low.
Measurement of apoptosis and proliferation of bone marrow plasma cells in patients with plasma cell proliferative disorders
Authors: Witzig Thomas , Timm Michael , Larson Dirk , Therneau Terry , Greipp Philip
Published: March 2003

The proliferative rate of malignant plasma cells, as measured by the plasma cell labelling index (PCLI), is an important prognostic factor in multiple myeloma (MM); however, the PCLI alone is probably inadequate to describe tumour growth because it ignores the idea that myeloma cells may have a reduced rate of apoptosis. The aims of this study were to develop a flow cytometric method to measure the apoptosis index of fresh marrow plasma cells and develop a plasma cell growth index (PCGI) that related both proliferation and apoptosis to disease activity. Marrow aspirates were obtained from 91 patients with plasma cell disorders and the plasma cells in apoptosis were identified by either 7‐amino actinomycin‐D (7‐AAD) or annexin V–FITC three‐colour flow cytometry. The median plasma cell apoptotic index (PCAI) for patients with monoclonal gammopathy of undetermined significance (MGUS), smouldering or indolent myeloma (SMM/IMM), and new multiple myeloma (MM) was 5.2, 3.4 and 2.4, respectively (P=0.03, MGUS v MM). The median PCLI for these same patient groups was 0.0, 0.2 and 0.6, respectively (P<0.001, MGUS v MM). The paired PCLI and PCAI for each sample were used to derive the PCGI = 2 + [PCLI − (0.1)(PCAI)]. The median PCGI for patients with inactive disease (MGUS, SMM/IMM or amyloidosis) was 1.8 compared to 2.4 for those with active disease (new or relapsed MM) (P < 0.001). These results suggest that a decrease in the PCAI may be a factor in the progression from MGUS to SMM to overt MM.
Variable pathogenicity of exon 43del (FAA) in four Fanconi anaemia patients within a consanguineous family
Authors: Koc Ahmet , Pronk Jan C. , Alikasifoglu Mehmet , Joenje Hans , Altay Çigdem
Published: March 2003

Four Fanconi anaemia group A (FAA) patients within two related consanguineous families are presented: the propositus (male, 13 years, transplanted at age 10), and his three cousins (one male, 8 years, and two female newborns). Assignment of the patients to FAA was based on the functional complementation analysis by somatic cell hybridization and confirmed by mutation screening showing a homozygous deletion of exon 43 (4267–4404del) in the FAA gene to be present in all four patients. The newborn patients had been diagnosed prenatally by DNA analysis. In spite of identical molecular pathology and close familial relationship the clinical phenotypes of the four patients were not concordant. Discordant symptoms included birthweight, pigmentation abnormalities, skeletal, renal and genital abnormalities, whereas microcephaly and possibly the haematological course were concordant. Differences in environmental conditions and/or genetic make‐up along with chance effects during development may explain discordant phenotypes despite identical molecular pathology in these patients. However, our results do not rule out the possibility that the exon 43del mutation may have prognostic value for the haematological course of the disease.
Protein disulphide isomerase mediates platelet aggregation and secretion
Authors: ESSEX DAVID W. , LI MENGRU
Published: March 2003

Platelet surface thiols and disulphides play an important role in platelet responses. Agents that reduce disulphide bonds expose the fibrinogen receptor in platelets and activate the purified glycoprotein (GP) IIbIIIa receptor. Protein disulphide isomerase (PDI), an enzyme that rearranges disulphides bonds, is found on the platelet surface where it is catalytically active. We investigated the role of PDI in platelet responses using (1) rabbit anti‐PDI IgG specific for PDI, (2) a competing substrate (scrambled ribonuclease A), and (3) the PDI inhibitor, bacitracin. Fab fragments of the rabbit anti‐PDI IgG inhibited platelet responses to the agonists tested (ADP and collagen), whereas Fab fragments prepared identically from normal rabbit IgG had no inhibitory effect. Scrambled ribonuclease A blocked platelet aggregation and secretion, but native ribonuclease A did not. When biphasic platelet aggregation was examined using platelets in citrated plasma, the principle effect of bacitracin was on second phase or irreversible aggregation responses and the accompanying secretion. Using flow cytometry and an antibody specific for activated GPIIbIIIa (PAC‐1), the rabbit anti‐PDI Fab fragments substantially inhibited activation of GPIIbIIIa when added before, but not after, platelet activation. In summary, we have demonstrated that protein disulphide isomerase mediates platelet aggregation and secretion, and that it activates GPIIbIIIa, suggesting this receptor as the target of the enzyme.
Detection of abnormalities in B‐cell differentiation pattern is a useful tool to predict relapse in precursor‐B‐ALL
Authors: Ciudad J. , San Miguel J. F. , López‐Berges M. C. , García Marcos M. A. , González M. , Vázquez L. , Del Cañizo M. C. , López A. , Van Dongen J. J. M. , Orfao A.
Published: March 2003

Immunophenotypic investigation of minimal residual disease (MRD) has traditionally been based on the investigation of phenotypic aberrants at diagnosis to be used later as a target for MRD detection. This approach has several shortcomings (it is only applicable to patients with aberrant phenotypes, requires a diagnostic sample, and is patient‐specific) and therefore a search for simpler alternatives is warranted. The present study is based on the hypothesis that in precursor‐B‐ALL patients the persistence of residual leukaemic cells may induce abnormalities in the precursor‐B‐cell compartment in bone marrow (BM) and these could be used as a criteria to predict relapse.  These abnormalities may include: (1) the presence of an increase in the frequencies of immature B cells (CD34+/CD19+ or CD20−/CD19+) or (2) the existence of an altered B‐cell differentiation pathway due to a blockade or to the presence of B cells outside the normal pathway. A total of 180 BM samples from 45 consecutive precursor‐B‐ALL patients who achieved morphological complete remission (CR) were analysed by multiparametric flow cytometry. Our results show that a significant increase in immature B‐cell subsets or an altered B‐cell differentiation predicts a high relapse rate (P < 0.01) and a shorter disease‐free survival (P < 0.01). Moreover, abnormalities in either of these two criteria detected at specific time points during follow‐up (end of induction, maintenance, or after treatment) were associated with a significantly shorter disease‐free survival (P < 0.01).  In summary, the investigation of abnormalities in B‐cell differentiation is a relatively simple and cheap approach for predicting relapse in precursor‐B‐ALL patients.
Chronic lymphocytic leukaemia with symptomatic diffuse central nervous system infiltration responding to therapy with systemic fludarabine
Authors: Elliott Michelle A. , Letendre Louis , Li Chin‐Yang , Hoyer James D. , Hammack Julie E.
Published: March 2003

B‐cell chronic lymphocytic leukaemia is an indolent disease characterized by the insidious accumulation of small mature‐appearing lymphocytes in the peripheral blood, bone marrow and lymphoid tissues. Direct symptomatic invasion of the central nervous system is exceedingly rare and, to our knowledge, only three cases histologically confirmed as true chronic lymphocytic leukaemia have been reported in the literature. We describe the first case of early Rai stage B‐cell chronic lymphocytic leukaemia presenting with symptomatic infiltration of the brain and spinal cord which could be demonstrated radiographically by magnetic resonance imaging. The diagnosis was confirmed by examination of peripheral blood, cerebrospinal fluid, brain and bone marrow biopsies, both morphologically and immunophenotypically by means of flow cytometric analysis. The patient demonstrated a complete response to therapy with standard‐dose systemic fludarabine and remains in complete remission 6 months after completion of therapy.
Red cell deformability in oral contraceptive pill users with sickle cell anaemia
Authors: Yoong W. C. , Tuck S. M. , Yardumian A.
Published: March 2003

The use of the combined oral contraceptive pill (COCP) in women with sickle cell anaemia (SCA) is controversial, as contraceptive steroids are thought to adversely affect erythrocyte deformability. This observational study was performed to investigate whether hormonal contraception influenced erythrocyte deformability in women with SCA. 30 women with SCA using various contraceptive modalities: COCP (n = 10); progestogen only (PO) contraception (n = 10) and non‐hormonal contraception (n = 10) were recruited. Erythrocyte deformability was assessed using the clogging rate (CR) and red cell transit time (RCTT). There was no statistical difference in the mean CR and RCTT between the three groups of women (one‐way ANOVA). Current contraceptive steroids do not appear to impair red cell deformability in women with SCA.
The sequential analysis of trisomy 12 in B‐cell chronic lymphocytic leukaemia
Authors: Auer R. L. , Bienz N. , Neilson J. , Cal M. , Waters J. J. , Milligan D. W. , Fegan C. D.
Published: March 2003

In this longitudinal study we evaluated the clinical significance of screening for trisomy 12 by fluorescence in situ hybridization (FISH) and correlated these findings with survival, disease progression and need for treatment. Interphase FISH was performed on 85 patients in 1993/94 and repeated on 41 patients in 1996/97. Over the 4‐year period there was no significant change in the percentage of trisomic cells, even in patients with disease progression. Overall survival and requirement for treatment were similar for patients with and without FISH‐defined trisomy 12.
Expression of Concern: Effect of insulin‐like growth factor 1 on PHA‐stimulated cord blood mononuclear cell telomerase activity
Authors: Tu Wenwei , Zhang De‐Kai , Cheung Pik‐To , Tsao Sai‐Wah , Lau Yu‐Lung
Published: March 2003

Telomerase may contribute to the capacity for cell replication by compensating for the loss of telomere length. Exploring the use of biological modifiers in increasing cellular replicative potential through telomerase activity may be useful for in vitro expansion of haemopoietic stem cells for transplantation or lymphoid cells for adoptive immunotherapy. In this study we showed for the first time that insulin‐like growth factor 1 (IGF‐1) modulated telomerase activity in human cord blood mononuclear cells (MNC) and some of the known functional determinants of telomerase activity. We found that cord blood MNC expressed constitutively a low level of telomerase activity and human telomerase reverse transcriptase (hTRT) mRNA, and a high level of human telomerase RNA component (hTR) and telomerase‐associated protein‐1 (TP1) mRNA. Interestingly, IGF‐I alone did not increase the telomerase activity of cord blood MNC but could enhance the PHA‐induced increase in telomerase activity. These alterations in telomerase activity were not completely in phase with those of proliferation response. On the other hand, IGF‐I did not alter hTRT mRNA expression but enhanced the PHA‐induced increase in hTRT whereas TP1 mRNA expression was stimulated by either IGF‐I or PHA but showed no additive increase when stimulated by both IGF‐1 and PHA. Neither IGF‐1 nor PHA altered hTR expression. Finally, the temporal dynamics of hTRT mRNA expression and telomerase activity in cord blood MNC over 5 d in culture were not totally concordant, suggesting that key factors other than hTRT were involved in regulating telomerase activity in cord blood MNC. The modulatory effect of IGF‐1 on telomerase activity supports its potential role in increasing replicative potential of cord blood lymphoid cells or haemopoietic stem cells.
An ELISA specific for murine erythropoietin
Authors: Noé Gudrun , Riedel Waltraut , Kubanek Bernhard , Rich Ivan
Published: March 2003

Murine recombinant erythropoietin (EPO) was purified from an EPO‐producing cell line and used for the production of polyclonal monospecific anti‐murine EPO antibodies in rabbits. The anti‐mouse EPO antibodies were purified by two affinity chromatography procedures. In order to obtain the most sensitive ELISA, different antibody combinations were tested in the ELISA sandwich assay. The best combination was achieved with an anti‐human EPO antibody as coating and the biotinylated anti‐murine EPO antibody as detecting antibody. With this sandwich‐ELISA a sensitive standard curve in the range of 0.6–30 mU/ml could be established. The assay provides a sensitive and reliable measure of murine EPO in serum and cell culture supernatants ranging from normal to highly elevated EPO levels.
Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia
Authors: McLeod Howard L. , Coulthard Sally , Thomas Angela E. , Pritchard Stuart C. , King Derek J. , Richards Susan M. , Eden O. B. , Hall Andrew G. , Gibson Brenda E. S.
Published: March 2003

The role of 6‐mercaptopurine (6MP) in the treatment of childhood acute lymphoblastic leukaemia (ALL) is well established. However, the efficacy of 6MP is significantly influenced by inactivation by the polymorphic enzyme thiopurine methyltransferase (TPMT). In the general population 89–94% have high TPMT activity, 6–11% have intermediate activity, and approximately 0.3% have low activity. Individuals with low‐activity experience severe or fatal toxicity with standard 6MP doses. Prospective identification of this group of patients might prevent this problem.

Recent identification of the molecular basis for low TPMT activity enabled rapid assessment of altered 6MP metabolism by PCR methods. This study evaluated the frequency of mutant TPMT alleles in 147 children with ALL. One patient was homozygous mutant (0.7%), and 16 patients were heterozygous for variant TPMT alleles (10.9%). The majority of mutant alleles were TPMT*3A. Both the allele frequency and the pattern of TPMT mutations were similar to that observed in an adult British population.

The number of weeks when 6MP therapy was administered at full dose was determined in patients on MRC UKALL X and XI. The 94 patients spent a median 11% of the maintenance period receiving no therapy as a result of haematological toxicity. There was no significant difference in the number of weeks when no therapy could be administered among patients with a wild‐type or heterozygous genotype. However, the one patient with a homozygous mutant genotype had severe haematological toxicity and no therapy could be administered for 53% of the maintenance period.

This study demonstrates that 11.6% of the children had variant TPMT alleles. Prospective identification of TPMT genotype may be a promising tool for decreasing excessive haematological toxicity in individuals with low activity.
The response to high‐dose intravenous immunoglobulin or steroids is not predictive of outcome after splenectomy in adults with autoimmune thrombocytopenic purpura
Authors: Ruivard M. , Caulier M. T. , Vantelon J. M. , Tournilhac O. , Schaeffer A. , Godeau B. , Bierling P.
Published: March 2003

The response to high‐dose intravenous immunoglobulin (IVIg) was recently reported to be predictive of outcome after splenectomy in patients with autoimmune thrombocytopenic purpura (AITP). We analysed the records of 75 adults with chronic AITP who received IVIg and subsequently underwent splenectomy. There was no significant difference in the response rate to splenectomy according to whether or not patients had responded to IVIg (81% v 67%, P = 0.36). Age, the time from diagnosis to splenectomy, and the response to steroids were also not significantly associated with outcome after splenectomy. These results indicate that the response to IVIg or steroids is not predictive of the efficacy of splenectomy.
High inducibility of heat shock protein 72 (hsp72) in peripheral blood mononuclear cells of aplastic anaemia patients: a reliable marker of immune‐mediated aplastic anaemia responsive to cyclosporine therapy
Authors: Takami Akiyoshi , Nakao Shinji , Tatsumi Yasuaki , Wang Hongbo , Weihua Zeng , Yamazaki Hirohito , Yasue Shizuka , Shiobara Shintaro , Matsuda Tamotsu , Mizoguchi Hideaki
Published: March 2003

To better characterize immunologic aberrations in aplastic anaemia (AA), we examined peripheral blood mononuclear cells (PBMC) of 67 patients with AA and other patients with various haematological diseases for the expression of heat shock protein 72 (hsp72), which is inducible in lymphocytes by various stressors including antigenic stimulation. When freshly obtained PBMC were examined using flow cytometry, the proportion of cells expressing hsp72 in cytoplasm was significantly higher in allogeneic marrow transplant recipients (22 ± 15%, mean ±standard deviation (SD)) and AA patients (17 ± 21%) than in normal individuals (6 ± 3%). When PBMC were tested after heat treatment, only the proportion of hsp72+ cells of AA patients (37 ± 30%) was significantly higher than that of the normal control (17 ± 11%). Dual fluorescence analysis of the PBMC revealed that the majority of hsp72+ cells was CD3+. For 28 untreated AA patients, the proportion of hsp72+ cells in those who later responded to cyclosporine (CyA) (62 ± 24%) was higher than that in non‐responders (19 ± 13%). Immunoblotting analysis revealed predominant expression of hsp72 in T cells. These findings indicate that high inducibility of hsp72 in PBMC by heat treatment is an immunologic aberration characteristic of CyA‐responsive AA and that this simple test may be useful for identifying a subset of AA patients responsive to immunosuppressive therapy.
Multiplex PCR reaction for the detection and identification of immunoglobulin kappa deleting element rearrangements in B‐lineage leukaemias
Authors: Stolz Frank , Panzer Simon , Renate Panzer‐Grümayer E.
Published: March 2003

Immunoglobulin kappa (Igκ) gene recombinations can be used — similarly to IgH rearrangements — as clonal markers in B‐lineage leukaemias. Based on the extensive junctional diversity, these rearrangements represent valuable targets for the analysis of minimal residual disease (MRD). In order to provide a simple method for the rapid detection of leukaemia clone‐specific kappa deleting element (Kde) mediated rearrangements, we developed a multiplex PCR reaction that is able to amplify the five most frequent rearrangements in one tube. Position of the amplimers were chosen to enable identification of the involved segments according to the size of the PCR product. This method was tested on 101 B‐lineage leukaemias (71 childhood B‐cell precursor acute lymphoblastic leukaemias (BCP ALL) and 30 chronic lymphocytic leukaemias (CLL)). 39 and 22 Kde rearrangements could be readily detected in 30 (44%) BCP ALL and 22 (56%) CLL, respectively. 36% of the Kde rearrangements in BCP ALL and 45% in CLL were intron recombination signal sequence (RSS)‐Kde rearrangements. The other Kde rearrangements involved the Vκ families: VκI in 36% and 50%, VκII in 32% and 16.7%, VκIII in 24% and 25%, and VκIV in 8% and 8.3% in BCP ALL and CLL, respectively. The sensitivity of the multiplex system was 10−2–10−3. We compared this multiplex PCR assay with multiple single PCR reactions using different sets of primer combinations. Thereby the number and types of rearrangements were confirmed in all cases. Clonality of rearrangements was proven by sequence analysis. Our data show that by this method clonal Kde rearrangements were rapidly detected and precisely identified.
Lipid composition of seven APTT reagents in relation to heparin sensitivity
Authors: Kitchen S. , Cartwright I. , Woods T. A. L. , Jennings I. , Preston F. E.
Published: March 2003

The phospholipid content of different activated partial thromboplastin time (APTT) reagents was determined and compared to heparin sensitivity. The seven reagents included were those most widely used amongst participants of the U.K. National External Quality Assessment Scheme (NEQAS) at the time of study. Heparin sensitivity was assessed using the APTT ratios obtained by more than 300 NEQAS participants on five plasmas prepared from patients receiving unfractionated heparin. The concentrations of three neutral lipids and six phospholipids present in the seven APTT reagents were determined by high‐performance thin‐layer chromatography (HPTLC) and densitometry. Both the concentrations and the relative percentages of individual phospholipid components varied markedly between reagents. The total phospholipid concentration included a 12‐fold range from 16 to 205 μg/ml. Phosphatidylserine (PS) was completely lacking from one reagent prepared from vegetable material and ranged from 3 to 22 μg/ml in the other six reagents containing extracts from animal tissue. The concentration of phosphatidylcholine ranged from 3 to 109 μg/ml. There was no demonstrable relationship between the concentration of any individual lipid components and heparin sensitivity. However, the relative percentage phospholipid composition was important since a lower % of PS or phosphatidylinositol (PI) correlated with increasing heparin sensitivity.
Kaposi's sarcoma‐associated herpesvirus (KSHV/HHV‐8) DNA sequences are absent in leukapheresis products and ex vivo expanded CD34+ cells from multiple myeloma patients
Authors: De Greef Catherine , Van De Voorde Wendy , Bakkus Marleen , Corthals Jurgen , Heirman Carlo , Schots Rik , Lacor Patrick , Van Camp Ben , Van Riet Ivan
Published: March 2003

Recently it was reported that Kaposi's sarcoma‐associated herpesvirus (KSHV/HHV‐8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development. Because of the use of myeloid growth factors like GM‐CSF and G‐CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts. Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed. KSHV DNA could not be amplified in any of them.
Absolute CD4+ T‐lymphocyte and CD34+ stem cell counts by single‐platform flow cytometry: the way forward
Authors: Barnett  , Granger  , Whitby  , Storie  , Reilly
Published: March 2003

To determine the potential advantage of single‐platform technology in the enumeration of CD4+ T lymphocyte and CD34+ stem cells, data has been analysed from the UK NEQAS for Leucocyte Immunophenotyping schemes. The inter‐laboratory CVs for CD4+ T lymphocyte counts were consistently lower for single‐platform (mean 13.7%, range 10–18.3%) compared to dual‐platform methodology (mean 23.4%, range 14.5–43.7%). Subgroup analysis of single‐platform users demonstrated mean overall inter‐laboratory CVs of 17.2%, 13% and 7.1% for the FlowCount, TruCount and volumetric approach respectively. The lowest inter‐laboratory CVs obtained for a single sample by each single platform approach were 4% (TruCount), 4.4% (volumetric), 4.6% (FACSCount) and 12.7% (FlowCount). Similarly, the mean inter‐laboratory CV for CD34+ stem cell enumeration using non‐standardized single‐platform approaches was 18.6% (range 3.1–36.9%) compared to 28.6% (range 19–44.2%) for the dual‐platform technology. Our results suggest absolute cell subset enumeration should be performed by single‐platform technology and that such an approach should improve the quality control of multi‐centre clinical trial data for CD4+ T lymphocyte and CD34+ stem cells.
Persistent leucocyte abnormalities in children years after previous long‐term low‐dose radiation exposure
Authors: Chang Wushou P. , Lin Yi‐ping , Hwang Pai‐tsang , Tang Jih‐Luh , Chen Jer‐yuan D. , Lee Shy‐Dye
Published: March 2003

Two hundred and eighty‐nine children who had been educated in a kindergarten during 1983–92 and received continuous low‐dose whole body gamma‐irradiation from 60Co‐contaminated steel window frames in their classrooms were investigated for residual effects on their haematological tissues. Another 751 children, sex and age‐matched, received similar but much lower exposure in an elementary school with classrooms built with contaminated steel rebars. The peripheral leucocytes of these children were examined 5–7 years after they had stopped using these irradiated classrooms. Children who received higher exposure in the kindergarten were shown to have a significant decrease in total leucocytes and neutrophils and an increase in eosinophils. Moreover, they were shown to be at significantly higher risk of developing relative leucopenia and neutropenia, but not lymphocytopenia, than those who received a lower exposure at the elementary school. Children from the kindergarten who had much higher exposure were shown to have a significant lowering of total leucocytes and neutrophils, and an increase in eosinophils years after exposure. Residual adverse haematological effects on the exposed children are strongly suspected.
Increased serum levels of β2m‐free HLA class I heavy chain in multiple myeloma
Authors: Perosa Federico , Luccarelli Grazia , Prete Marcella , Ferrone Soldano , Dammacco Franco
Published: March 2003

Serum levels of β2‐microglobulin (β2m)‐free HLA class I heavy chain (FHC) in 94 patients with multiple myeloma (MM) were higher than in 29 patients with monoclonal gammopathy of undetermined significance (MGUS) (P = 0.023) and in 97 sex‐ and age‐matched healthy controls (P < 0.0001). Spearman correlation analysis indicated that in MM, FHC correlated with β2m (r = 0.31, P = 0.003) and the percentage of bone marrow plasma cells (BMPC%) (r = 0.36, P = 0.002), whereas β2m, in addition to BMPC% (r = 0.43, P = 0.0003), also correlated with creatinine levels (r = 0.63, P < 0.0001), haemoglobin levels (r = −0.35, P = 0.0007) and patient age (r = 0.34, P < 0.0011). Furthermore, MM patients with poor prognosis (β2m  6 mg/l) displayed higher FHC levels than those with a better prognosis (β2m < 6mg/l) (P < 0.021). At variance from β2m, these levels were not influenced by renal failure, as indicated by the lack of Spearman correlation of FHC with creatinine concentration and of statistical significance between the median FHC concentration of MM patients with creatinine < 176.6 μmol/l and those with creatinine  176.6 μmol/l (P = 0.3). Stratification of patients according to disease activity and stage showed that FHC levels were only statistically different (P = 0.04) for disease activity, whereas β2m and C‐reactive protein were not. Taken together, our data indicate that serum FHC may be a useful disease marker in MM.
TGF‐β is not the principal immunosuppressive component in coagulation factor concentrates
Authors: Pearson Helen J. L. , Stirling David , Ludlam Christopher A. , Steel C. Michael
Published: March 2003

Coagulation factor concentrates are known to inhibit a variety of immune reactions when assessed in vitro. This study assessed the immunomodulatory activity of a wide range of coagulation factor concentrates by measuring their inhibition of PHA‐stimulated lymphocyte proliferation and reduction in IL‐2 secretion. The hypothesis that TGF‐β is responsible for most of these effects was tested by measuring biologically active TGF‐β and immunoreactive TGF‐β1 in the concentrates and comparing the levels recorded with immunosuppressive activity. In addition, the coagulation factors were compared directly with a standard preparation of TGF‐β in a TGF‐β‐specific bioassay and in lymphocyte proliferation assays. Although there was a broad correlation between levels of total or active TGF‐β and immunosuppressive activity across all of the coagulation factors tested, individual data sets showed clear discrepancies. Implying that TGF‐β probably serves as a surrogate marker for other immunomodulatory contaminants and that neither TGF‐β nor any other single substance could account for all of the immunosuppressive activity observed. Furthermore, there was a difference of more than 100‐fold in the relative potencies of coagulation factors and pure TGF‐β, when compared in immunosuppression assays, indicating that the different assays did not measure the same substance. Whereas anti‐TGF‐β antibody almost completely blocked the activity of coagulation factor concentrates (TGF‐β‐specific bioassay) and abrogated the effect of authentic TGF‐β (immunosuppression assays) at high concentrations it achieved <50% reversal of the immunosuppressive effects of coagulation factors in immunosuppression assays. These findings indicated that TGF‐β accounted for only a minor proportion of the immunosuppressive activity in most coagulation factor concentrates.
Age‐related changes in thrombopoietin in children: reference interval for serum thrombopoietin levels
Authors: Ishiguro Akira , Nakahata Tatsutoshi , Matsubara Kousaku , Hayashi Yasuhide , Kato Takashi , Suzuki Yoko , Shimbo Toshikazu
Published: March 2003

We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease‐free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73–5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r = 0.27) or in children alone (r = 0.12). In children > 1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = −0.257 + 4.039 × plasma TPO values (r = 0.951, P < 0.001, n = 22). This study is the first to report age‐dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.
Revision of the diagnosis of T‐zone lymphoma in the father of a patient with autoimmune lymphoproliferative syndrome type II
Authors: Bosch Jutte van der Werff ten , Delabie Jan , Böhler Thomas , Verschuere Johan , Thielemans Kris
Published: March 2003

Autoimmune lymphoproliferative syndrome (ALPS) is a disease of childhood characterized by typical clinical and laboratory findings. Here we describe an adult patient presenting with lymph node enlargement and splenomegaly. Pathological examination of an adenopathy supported the diagnosis of malignant T‐zone lymphoma. The patient was treated accordingly. 3 years later his child was diagnosed with ALPS. Therefore the diagnosis of the father's disease was reconsidered. Review of the slides and functional tests led to the diagnosis of ALPS in both father and son. ALPS should be considered as a possible differential diagnosis in adult patients presenting with rare types of T‐cell lymphomas.
Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow
Authors: Imai Kiyotoshi , Kobayashi Masanobu , Wang Jingxin , Shinobu Noriaki , Yoshida Hideaki , Hamada Jun‐ichi , Shindo Masanobu , Higashino Fumihiro , Tanaka Junji , Asaka Masahiro , Hosokawa Masuo
Published: March 2003

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP‐2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility‐stimulating activity. The bone marrow endothelial cells expressed stromal‐cell‐derived factor‐1 (SDF‐1) mRNA and produced SDF‐1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP‐2 cells to the bone marrow endothelial cells was partially inhibited by anti‐SDF‐1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF‐1 and random motility‐stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.
Laparoscopic splenectomy: single‐centre experience of a district general hospital
Authors: Bernard  , Rhodes  , Turner  , Wimperis  , Deane
Published: March 2003

We report the results of 20 consecutive laparoscopic splenectomies performed on haematology patients for a number of indications. Our series includes patients up to 77 years of age at the time of surgery and removal of spleens weighing up to 3530 g. The most significant benefit is the early rate of discharge post‐operatively (median 2 d); however, there is a risk of conversion to open laparotomy (in this series 3/20, 15%). We show that laparoscopic splenectomy can be offered as a therapeutic option to patients unfit for conventional laparotomy and that even large and bulky spleens can be removed safely using this approach.