bluuebunny/crossref_metadata_2025 huggingface dataset:
Random X chromosome inactivation in a female with a variant of Wiskott‐Aldrich syndrome
Authors: RDSSELL SUSAN J. , NISEN PERRY D.
Published: March 2008

A 15‐month‐old female presented with eczema, thrombocytopenia, recurrent infections and failure to thrive. She had low serum IgM and IgG subclasses and an abnormal lymphocyte proliferative response to periodate in vitro. Molecular X chromosome inactivation analysis, using the polymorphic HUMARA DNA probe, showed that the infant has random X chromosome inactivation. We conclude that she has an atypical form of Wiskott‐Aldrich syndrome which may be inherited in an autosomal recessive manner.
Trisomy 11: an association with stem/progenitor cell immunophenotype
Authors: SLOVAK MARILYN L. , TRAWEEK S. THOMAS , WILLMAN CHERYL L. , HEAD DAVID R. , KOPECKY KENNETH J. , MAGENIS R. ELLEN , APPELBAUM FREDERICK R. , FORMAN STEPHEN J.
Published: March 2008

Summary. The clinicopathological features and the prognostic significance of acute myeloid leukaemia (AML) with trisomy 11 are currently unknown. In this study we describe 15 adult AML cases with trisomy 11. Trisomy 11 was the sole chromosomal anomaly in eight cases; the remaining seven cases were characterized by +11 in association with other karyotypic aberrations. Patient ages ranged from 34 to 79 years. 12 patients were male; three were female. Although there was no correlation of trisomy 11 with any specific FAB subgroup [M2 (n=7). Ml (n=5), M4/5 (n=2), M3 (n=l)] less mature forms predominated. Immunologically, the leukaemic blasts showed a strikingly consistent stem cell phenotype with expression of HLA‐DR, CD34 and the myeloid antigens (CD15, CD33 and/or CD13). In addition, two cases expressed the B‐cell associated antigen CD19. The presence of trilineage dysplasia, suggesting the presence of an underlying myelodysplasia (MDS), was observed at presentation in five cases; in another case MDS was evident at relapse only. Unexpectedly, MLL gene rearrangements were observed in two of four cases characterized by trisomy 11 as the sole karyotypic abnormality; however, MLL aberrations were not identified in three cases with trisomy 11 accompanied by other karyotypic anomalies. The majority of patients in each subgroup (i.e. those with and without additional cytogenetic abnormalities) achieved a short first complete remission (CR) (mean 8 months) and failed to obtain a second CR. Only one patient in each trisomy 11 subgroup is in a continuous CR for >34 months. These findings suggest that trisomy 11 leukaemia is characterized by a stem/progenitor cell immunophenotype with poor response to standard chemo‐therapeutic regimens and an unfavourable prognosis.
Antenatal screening for fetal alloimmune thrombocytopenia: the results of a pilot study
Authors: DOUGHTY H. A. , MURPHY M. F. , METCALFE P. , WATERS A. H.
Published: March 2008

Summary. Feto‐maternal incompatibility for the human platelet antigen HPA‐la is an important cause of severe fetal thrombocytopenia. The incidence is 1 in 1000‐2000 pregnancies, which is more common than other conditions for which screening is presently carried out. Antenatal diagnosis and management are now available, but only for subsequent siblings following diagnosis of a previously affected infant. This study describes a pilot prospective screening programme for the antenatal detection of feto‐maternal alloimmune thrombocytopenia (FMAIT) due to HPA‐la incompatibility. 3473 women were typed for HPA‐la using a method designed for large‐scale typing. 71 women found to be HPA‐la negative were further tested for HLA‐DR52a as a risk factor for alloimmunization. All women were monitored for the development of anti‐HPA‐la throughout pregnancy and a cord full blood count was taken at delivery. Two affected pregnancies were found and treated: a singleton pregnancy was treated antenatally and a twin pregnancy after delivery. The study showed that screening for FMAIT could be established within the pre‐existing antenatal red cell serology programme. It was concluded that screening should be based on platelet typing and offered regardless of parity. Further stratification, combining DR52a typing and HPA‐la antibody screening, although focusing on the group of women at greater risk, may not identify all affected pregnancies. Confirmation of the diagnosis and severity of FMAIT continues to depend on fetal blood sampling during pregnancy or cord blood samples after birth.
Philadelphia chromosome‐negative chronic myeloid leukaemia: a report of 14 new cases
Authors: COSTELLO R. , LAFAGE M. , TOIRON Y. , BRUNEL V. , SAINTY D. , ARNOULET C. , MOZZICONACCI M.‐J. , BOUABDALLAH R. , GASTAUT J.‐A. , MARANINCHI D. , GABERT J.
Published: March 2008

Summary. The Philadelphia chromosome (Ph) is the cytogenetic hallmark of chronic myeloid leukaemia (CML) and is used to confirm the diagnosis of CML based on clinical and morphological criteria. We investigated 14 patients with features of CML but without detectable Ph chromosome. In seven patients, referred to as BCR+, M‐bcr/abl rearrangement was detected by polymerase chain reaction (PCR). The seven remaining patients did not have M‐bcr/abl rearrangement and are described as BCR. BCR patients were younger, had lower white blood cell counts (WBC) and lower basophilia. Four BCR‐ and four BCR+ patients underwent blastic transformation (BT), Response to therapy was fairly similar in both populations. According to French‐American‐British (FAB) Cooperative Leukaemia Group guidelines, all BCR˜ patients were classified as having classic form CML or‘chronic granulocytic leukaemia’(CGL) when based only on morphological data. This study further confirms the existence of true CML cases without Ph chromosome or M‐bcr/abl rearrangement and shows that this entity differs only slightly from classic form Ph+ CML. The Ph‐BCR‐ subgroup raises two problems. First, the differential diagnosis with atypical CML or CMML, based on morphological data, and secondly, the therapeutic follow‐up in the absence of a specific marker. In contrast, the residual disease of Ph˜BCR˜ patients can be monitored by PCR. More advanced molecular and biochemical techniques will be required to understand which molecular mechanisms underlie Ph‐BCR‐ CML, resulting in phenotypes sometimes indistinguishable from Ph+ CML.
Circumvention of P‐glycoprotein‐mediated drug resistance in human leukaemic cells by non‐immunosuppressive cyclosporin D analogue, SDZ PSC 833
Authors: JIANG XU‐RONG , KELSEY STEPHEN M. , Wn YU‐LING , NEWLAND ADRIAN C.
Published: March 2008

Summary. Cyclosporin A (CSA) exhibits greater multidrug resistance (MDR) modulating activity in vitro than other MDR modulators such as verapamil and quinidine. However, the immunosuppressive and nephrotoxic effects of CSA may limit its clinical use. PSC 833, a new cyclosporin D derivative, exerts a higher MDR reversal activity but lacks toxic or immunosuppressive effects. The drug‐resistant sublines K/DAV10.,, K/DAU200. K/DAU30o. K/DAU40o, K/ DAU500 and K/DAU60o have been derived from the drug‐sensitive parental cell line, K562 cl.6 and CEM/VLB100 is a drug‐resistant derivative of CCRF‐CEM. We report a comparison of the effects of PSC 833 and CSA on daunorubicin (DAU) transport kinetics and chemosensitivity in these cell lines. Both CEM/VBL100 and KS62 cl.6 DAU‐resistant cells displayed high levels of P‐glycoprotein (PGP), decreased DAU accumulation and increased DAU efflux when compared to their parental cells. PSC 833 was 1‐6‐, 3‐4‐, 4‐9‐ and 4‐6‐fold more effective than CSA in reversing DAU resistance in higher resistance CEM/VLB10o, K/DAU40o, K/DAU500 and K/DAU60o cells respectively. DAU transport kinetics showed that PSC 833 was more effective than CSA in increasing cellular DAU accumulation and decreasing DAU efflux in higher resistant leukaemia subclones. PSC 833 could restore DAU retention at lower doses and was more active than CSA in all the resistant cells. A 89‐100% restoration of intracellular DAU retention were gained by PSC 833 at 1‐0ftM in K562 cl.6 DAU‐resistant sublines, whereas a 73‐100% restoration of DAU retention was obtained by CSA only at 30‐0 β, M in the same resistant sublines. PSC 833 at 3‐0/XM is sufficient to restore full DAU retention in all resistant cells. CSA, however, even at 30‐0 pM, cannot confer full restoration of DAU retention in higher resistance K562 cl.6/DAU sublines. By measuring MDR modulator‐mediated short‐term inhibition of PGP function, PSC 833 was found to be at least 10‐30 times more active than CSA. As no effect on DAU retention and sensitivity has been found in sensitive parental cells with PSC 833, it is suggested that PSC 833 may act by blocking the effluxing function of PGP in the resistant leukaemia cells.
An ELISA for factor × activation peptide: application to the investigation of thrombogenesis in cardiopulmonary bypass
Authors: PHILIPPOU HELEN , ADAMI ANTONELLA , BOISCLAIR MICHAEL D. , LANE DAVID A.
Published: March 2008

Summary. An ELISA for measurement of factor × activation peptide (FXAP) in plasma has been developed. The capture antibody was generated by immunization with a carrier‐coupled synthetic peptide based on the amino acid sequence of the C terminal region of native human FXAP: the tag antibody was a commercial polyclonal antibody to factor X. Because of limited specificity of the capture antibody to FXAP compared with factor X, a plasma processing step precipitated plasma factor × and also permitted a concentration step, enabling detection of FXAP below the lower limit of the normal range in plasma. The overall intra‐ and inter‐assay coefficients of variation were ˜5% and ˜11%, respectively. 18 normal laboratory control subjects had FXAP levels of 2‐12 – 0‐82 ng/ml (mean –SEM). Eight patients under‐ going surgery and cardiopulmonary bypass progressively generated FXAP throughout the surgery with mean FXAP rising to 11‐73 – 4′66ng/ml, and this resulted in increased generation of thrombin detected by measurement of plasma levels of Fl + 2. Levels of FXAP rose significantly ahead of those of factor IX activation peptide (FLXAP), supporting a suggestion that contact system activation can not be the primary stimulus to coagulation in bypass. The ELISA to FXAP will be useful in the study of mechanisms of thrombogenesis in clinical situations where the coagulation system is activated.
Severe hypophosphataemia during stem cell harvesting in chronic myeloid leukaemia
Authors: CLARK RICHARD E. , LEE EDWIN S.
Published: March 2008

Summary. Peripheral blood stem cell autografts for the treatment of chronic myeloid leukaemia (CML) are currently under evaluation. A patient with CML received intensive chemotherapy followed by granulocyte colony‐stimulating factor prior to the collection of peripheral blood derived stem cells. He developed unusually severe, and fatal, hypophosphataemia and this coincided with the rapid rise of his peripheral blood white cell count. The hypophosphataemia was considered to be due to a combination of severe anorexia, sepsis and the rapid growth factor‐stimulated myeloid regeneration in CML.
Pearson's marrow/pancreas syndrome: haematological features associated with deletion and duplication of mitochondrial DNA
Authors: SMITH O. P. , HANN I. M. , WOODWARD C. E. , BROCKINGTON M.
Published: March 2008

Summary. Pearson's marrow/pancreas syndrome (PS) is a rare, often fatal, disorder of infancy that is characterized by impaired bone marrow, exocrine pancreatic, hepatic and renal function. Large‐scale rearrangements of mitochondrial DNA (mtDNA) are present in blood. We report a patient with PS who has predominantly impaired haemopoiesis manifesting as sideroblastic anaemia, vacuolization of bone marrow precursors, and neutropenia. 77% of bone marrow mtDNA was rearranged (64% deleted an 13% duplicated). We suggest that rearrangements of mtDNA should be sought in all infants presenting with sideroblastic anaemia.
C‐myc amplification, double minutes and homogenous staining regions in a case of AML
Authors: FEGAN C. D. , WHITE D. , SWEENEY M.
Published: March 2008

Summary. c‐myc amplification is usually associated with lymphoid malignancies and Burkitt's lymphoma in particular. We present a case of AML with c‐myc amplification which was associated with homogenous staining regions (hsr) and double minutes (dmin). The administration of GCSF following induction chemotherapy resulted in a marked increase in blast numbers. The GCSF was stopped and further courses of chemotherapy given, which resulted in complete remission. The patient relapsed 7 months after diagnosis and failed to go into a second remission with reinduction therapy.

We conclude that c‐myc amplification is a rare event in AML, but may be associated with chemotherapy resistance and a poor prognosis, are as dmins and hsr. Growth factors should be used with caution in these patients.
Hereditary xerocytosis: a report of six unrelated Spanish families with leaky red cell syndrome and increased heat stability of the erythrocyte membrane
Authors: CORRONS J. LL. VIVES , BESSON I. , AYMERICH M. , AYALA S. , ALLOISIO N. , DELAUNAY J. , GONZALEZ I. , MANRUBIA E.
Published: March 2008

Summary. Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na++K+ fluxes with low total cation (Na++K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60min) and two different temperature values (46°C and 49°C). Under these conditions control RBCs (50 normal subjects) exhibited at 49°C and 30min a maximum of 30% fragmented RBCs. This value increased to 80% after 60min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality.

In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.
Heterogeneity in cell proliferation and expression of p53 and bcl‐2 during the indolent phase of germinal centre cell lymphoma: an explanation for clinical variability
Authors: BARRANS S. , RANDERSON J. , EVANS P. , BLYTHE D. , SHIACH C. , CHILD J. A. , MORGAN G. , JACK A. S.
Published: March 2008

Summary. Germinal centre cell lymphomas (GCCL) show a wide range of clinical outcomes from persistent indolent disease to large cell transformation. To investigate possible mechanisms of this heterogeneity, a combined morpho‐metric and immunohistological study of p53, bcl‐2 and cell proliferation was carried out. There was wide variation in p53 expression between biopsies and between individual follicles in the same tumour. A similar pattern of variation was seen using the cell‐cycle marker MIB1, but this did not correlate with p53 expression. Even in cases in which a t(14:18) was demonstrated by PCR, variation occurred in the number of cells expressing bcl‐2.

On the basis of these results, we suggest that the probability of the clonal expansion of GCCL tumour cells carrying additional genetic abnormalities depends on a complex interaction of cell proliferation with p5 3 and bcl‐2 expression, and that this may account for variation seen in the clinical behaviour seen in this group of tumours.
A chromogenic assay for activated protein C resistance
Authors: VARADI K. , MORITZ B. , LANG H. , BAUER K. , PRESTON E. , PEAKE I. , RIVARD G. E. , KEIL B. , SCHWARZ H. P.
Published: March 2008

Summary. Resistance to activated protein C (APC) diagnosed on the basis of prolongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophilia. The majority of patients with APC resistance carry a factor V molecule with a point mutation at one APC cleavage site (Arg506Gln) which prevents the optimal inactivation of activated factor V by APC. To overcome the limitations of aPTT‐based assays in the diagnosis of APC resistance, we have developed a chromogenic assay which is based on the capacity of APC to limit the generation of factor Xa by inactivating factor Villa in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62‐2.06. APC response ratios as measured by the chromogenic assay correlated with ratios measured by the aPTT assay and were below the normal range in 23.24 individuals with Arg506Gln mutant factor V from three different families with familial thrombosis and from 11 unrelated asymptomatic individuals. In reconstitu‐tion experiments, purified factor V corrected the decreased APC response in plasma samples from patients with the Arg506Gln mutation as well as with factor V deficiency, and increased the APC response in normal plasma, whereas the addition of activated factor V had no enhancing effect.
Major basic protein binding to thrombomodulin potentially contributes to the thrombosis in patients with eosinophilia
Authors: MUKAI HARUMI YAMAMOTO , NINOMIYA HARUHIKO , OHTANI KOJI , NAGASAWA TOSHIRO , ABE TSUKASA
Published: March 2008

Summary. The contribution of an eosinophil granule protein, major basic protein (MBP), to the pathogenesis of thrombosis seen in patients with eosinophilia was investigated. The sera from eosinophilic patients containing elevated levels of MBP inhibited thrombomodulin (TM) function as a cofactor for the thrombin‐catalysed activation of protein C more significantly than those from normal individuals (means 48.5%v 17.4%, respectively). It was suggested that the binding of mature MBP in the sera to TM was electrostatic, because mature MBP (pi 10.9) bound to TM, whereas pro‐MBP (pi 6.2) did not. The inhibition of TM cofactor activity by eosinophil granule proteins was mainly attributed to the mature MBP, because MBP‐depleted eosinophil granule proteins did not inhibit TM cofactor activity significantly. This inhibition seemed to be due to the specific thrombin‐binding to TM being blocked. We concluded that eosinophil granule proteins, particularly MBP, potentially contribute to the hypercoagulation seen in some conditions of eosinophilia, at least because of the inhibition of TM function as a cofactor of the anticoagulation system.
The effect of interleukin‐12 in ex‐vivo expansion of human haemopoietic progenitors
Authors: BERTOLINI FRANCESCO , SOLIGO DAVIDE , LAZZARI LORENZA , CORSINI CHIARA , SERVIDA FEDERICA , SIRCHIA GIROLAMO
Published: March 2008

Summary. We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL‐12. In cultures of low density cells, cytokine combinations including IL‐12 were associated to a greater proliferation (up to 6.7 ± 2.5 CFU‐GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 ± 49 CFU‐GM fold expansion) was SCF, IL‐3, IL‐11 and MlP‐la, and the addition of IL‐12 did not further enhance expansion of progenitors.

These results indicate that accessory cells, lost in CD34+ cell purification, could be in part responsible for IL‐12 effect on progenitor cell proliferation. In CD34+ cell cultures the addition of IL‐12 led to CD19 mRNA generation, suggesting that IL‐12 acts on haemopoietic cells with both myeloid and lymphoid potential.
Quantitation of soluble and membrane‐bound FC7RIIA (CD32A) mRNA in platelets and megakaryoblastic cell line (Meg‐01)
Authors: Markovic Boban , Wu Zhanhe , Chesterman Colin N. , Chong Beng H.
Published: March 2008

Summary .FCγ receptors (FCγRS) are glycoproteins on platelet surface that bind IgG‐containing immune complexes. However, excessive binding of immune complexes leads to platelet activation and thrombosis or increased platelet clearance and thrombocytopenia. In this study, FC7R transcripts in platelets and megakaryoblastic cell line (Meg‐01) were investigated using specifically designed oligonucleotides and a new quantitative in situ hybridization assay. Platelets and Meg‐01 cells were found to express only FCγRII transcripts. Of FCγRIIA mRNA isoforms (FCγRIIA, B and C), FCgMRIIA mRNA predominates in these cells. Platelets and Meg‐01 cells contain both alternative spliced forms of FCγRIIA mRNA, those with and without the transmembrane (TM) exon and both forms were present in near equal amounts. In contrast, FCγRIIA transcript with the TM exon predominates in neutrophils and monocytes, suggesting that the splicing of the TM exon is under lineage‐specific control.
A simple and sensitive method for determining plasma cell isotype and monoclonality in bone marrow using flowcytometry
Authors: van Zaanen H. C. T. , Vet R. J. W. M. , de Jong C. M. , von dem Borne A. E. G. Kr. , van Oers M. H. J.
Published: March 2008

Summary. In this paper we describe a new, rapid and sensitive method to determine plasma cell isotype and clonality in bone marrow using flowcytometry. With the use of a new fixation and permeabilization reagent (Permeafix®), which preserves cell structure and morphology, and a monoclonal antibody (Mab) specific for plasma cells (B‐B4), it has become possible to specifically select plasma cells and to determine the cytoplasmatic immunoglobulins by flowcytometry.

Thirty successive bone marrow aspirates from multiple myeloma patients and patients with MGUS were studied as well as 10 bone marrow samples from patients with reactive plasmacytosis. Each sample was analysed both by immunofluorescence on cytospin smears and FACS analysis. There were no discrepancies between plasma cell isotype as determined by FACS and cytospin. Moreover, FACS analysis was shown to allow detection of very low numbers of plasma cells and to determine whether these plasma cells are mono‐or polyclonal. Possible applications are discussed.
An association of aplastic anaemia in Thailand with low socioeconomic status
Authors: Issaragrisil Surapol , Kaufman David W. , Anderson Theresa E. , Chansung Kanchana , Thamprasit Tharatorn , Sirijirachai Jittima , Piankijagum Anong , Porapakham Yaowarat , Vannasaeng Sathit , Leaverton Paul E. , Shapiro Samuel , Young Neal S.
Published: March 2008

summary. The relationship of socioeconomic status to the risk of aplastic anaemia was evaluated in a case‐control study conducted in Bangkok and two rural regions of Thailand (Khonkaen and Songkla). Among 152 cases and 921 controls there were significant trends of increasing risk with decreasing years of education (P = O01) and total household income (P = 0–0001), after control for confounding. The relative risk estimate for those with monthly incomes of >1500 baht (about 60 U.S.) was 3–9 (95% confidence interval 2–1–7–3) compared to those with monthly incomes of at least 5000 baht (about 200). The pattern of increasing risk with decreasing income was observed in all three regions, with significant trends in Bangkok (P= 0–004) and Khonkaen (P = 0–003). This finding may partly explain the high incidence of aplastic anaemia in Thailand. Low socioeconomic status may be a surrogate for one or more environmental factors that could cause aplastic anaemia, such as infectious pathogens or toxic exposures.
Clinical significance of elevated soluble interleukin‐6 receptor levels in the sera of patients with plasma cell dyscrasias
Authors: Ohtani Koji , Ninomiya Haruhiko , Hasegawa Yuichi , Kobayashi Toshitaka , Kojima Hiroshi , Nagasawa Toshiro , Abe Tsukasa
Published: March 2008

Summary .We investigated the clinical significance of the serum soluble interleukin‐6 receptor (sIL‐6R) in 42 patients with plasma cell dyscrasias (27 with multiple myeloma (MM), 13 with monoclonal gammopathy of undetermined significance (MGUS), and two with plasma cell leukaemia (PCL)). Serum levels of sIL‐6R in normal individuals were 77 ± 21 ng/ml (mean ± SD, n= 18); those in patients with MGUS and with MM were elevated (102 ± 33 ng/ml, mean ± SD, P < 0–05 and 126 ± 60ng/ml, mean ± SD, P < 0–01, respectively). Significant correlations were not found between the serum levels of sIL‐6R and known prognostic factors (C‐reactive protein, haemoglobin levels, calcium, creatinine, β2‐microglobulin, amounts of M‐protein, or percentages of plasma cells in bone marrow). Elevated serum sIL‐6R did not affect the survival of the patients with MM. Serial measurements of sIL‐6R together with the clinical course of patients with plasma cell neoplasias revealed a good correlation between the sIL‐6R level and disease activity. We conclude that sIL‐6R can be used as a clinical factor correlated with the disease activity, at least in some patients with plasma cell neoplasias.
Variable numbers of BCR‐ABL transcripts persist in CML patients who achieve complete cytogenetic remission with interferon‐α
Authors: Hochhaus A. , Lin F. , Reiter A. , Skladny H. , Rhee F. van , Shepherd P. C. A. , Allan N. C. , Hehlmann R. , Goldman J. M. , Cross N. C. P.
Published: March 2008

Summary .A substantial minority of patients with chronic myeloid leukaemia (CML) achieve a complete response to treatment with interferon‐α (IFN‐α), defined as the disappearance of Philadelphia chromosome positive metaphases or, for patients who are Philadelphia chromosome negative but BCR‐ABL positive, the disappearance of the leukaemic clone as assayed by Southern blot. We have measured the levels of BCR‐ABL transcripts in 20 such patients by quantitative PCR. Results were standardized for both quality and quantity of cDNA by quantification of ABL as an internal control. All 20 patients had evidence of residual disease; the median number of transcripts was 750/μg RNA (range 10–22 000) and the median BCR‐ABL/ABL ratio was 0–17% (range 0–0008–3–6%). Our findings show that CML has not been eradicated in any patient and that the quantity of residual disease in complete responders may vary by as much as four orders of magnitude.
Hairy cell leukaemia, occupation, and smoking
Authors: Clavel J. , Mandereau L. , Cordier S. , Goaster C. Le , Heamon D. , Conso F. , Flandrin G.
Published: March 2008

Summary The roles of farm practices, occupational exposures to organic solvents, and ionizing radiation in the risk of hairy cell leukaemia (HCL) were examined in a French hospital‐based multicentre case‐control study including 291 cases (229 men and 62 women) and 541 controls (425 men and 116 women). No positive association was observed with occupations involving exposure to organic solvents or with self‐declared exposures to solvents, but a significant association with self‐reported exposure to petrol or diesel was found for men (OR =1–5 CI95% [1–0–2–1]). No association with ionizing radiation was detected. Agriculture employment gave an odds ratio of 1–7 (CI95% [11–2–4]) for men and 2–7 (CI95% [1–1–6–7]) for women. Among men, the association seems to affect farmers rather than agricultural workers. Self‐declared exposure to pesticides or bovine cattle breeding was related to HCL risk in both genders. Finally, a significant negative association with smoking was observed in men, with an inverse exposure‐risk relationship odds ratios of 0–6, 0–5 and 0–2, respectively, for cumulative consumptions of <10, 10–23 and 24 pack‐years), contrasting with an odds ratios clearly <1 in women.
Expression of interleukin‐5 receptors on acute myeloid leukaemia cells: association with immunophenotype and karyotype
Authors: Nishii Kazuhiro , Kita Kenkichi , Nadim Mahumd , Miwa Hiroshi , Ohoishi Koushi , Yamaguchi Motoko , Shirakawa Shigeru
Published: March 2008

Summary Eosinophilia sometimes occurs in acute myeloid leukaemia (AML) and several laboratories have reported that cells with the t(8;21) or structural abnormalities of chromosome 16 can proliferate and differentiate to eosinophils in the presence of IL‐5 in vitro. However, cases without these chromosomal abnormalities can also have eosinophilia. We investigated the association between the expression of IL‐5 receptor (IL‐5R) gene, karyotype and phenotype in 35 patients with AML. All cases expressed the IL‐3R but the IL‐5R gene was expressed predominantly in leukaemic cells with either t(8;21) or CD4‐positive immunophenotype and was associated with the presence of eosinophilia.
Use of central venous catheters in children with severe congenital coagulopathy
Authors: Liesner R. J. , Vora A. J. , Hann I. M. , Lilleymann J. S.
Published: March 2008

Summary From two U.K. centres 23 children with severe congenital coagulopathy had a total of 27 port‐a‐cath devices inserted to facilitate factor VIII or IX prophylaxis (eight patients), domiciliary therapy (seven patients), immunotolerance (four patients), or a combination thereof (four patients). Six children had a factor VIII inhibitor at the time of insertion. The mean age at operation was 30 months, with a range of 9–76 months. The cumulative length of follow‐up is 639 months with a mean of 27–8 months and a range of 5–79 months.

Haemostasis was achieved peri‐ and post‐operatively with high‐purity concentrate in the majority of patients without an inhibitor. All those with an inhibitor had porcine factor VIII, except one who had recombinant factor Vila.

The post‐operative complication rate was 27% (6/23): three had a port‐site haematoma (one required removal and replacement), two had post‐operative infection, and one had swelling caused by extravasation.

To date there have been 13 documented infections in 10/ 23 patients (five with inhibitor): a rate of 0–24 per follow‐up year or 0–67 per 1000 patient‐days. Six were caused by Gram‐positive and seven by Gram‐negative organisms. Six infections could not be eradicated by antibiotics and the port‐a‐cath system had to be removed; in three it was replaced by a second port‐a‐cath.

Although there are risks involved in the use of port‐a‐caths in this population, both clinicians and parents involved in the care of these children believe that the benefits are considerable and the potential hazards are acceptable.
In vitro platelet ageing at 22°C is reduced compared to in vivo ageing at 3 7°C
Authors: Holme S. , Heaton A.
Published: March 2008

Summary .In these studies, platelet ageing during in vitro at 22°C was compared with in vivo ageing using isotope labelling. Paired fresh and 5‐d‐stored platelets had a mean residual life‐span (MRL) of 4–8 ± 0–7d and 3–2 ± 0–9 d, respectively. After 2–1 ± 0–4 d in vivo circulation, the MRL of the fresh platelets was equivalent to that of the 5‐d‐stored in vitro platelets. This suggests that platelet ageing for 5 d in vitro at 22°C corresponds to 2–1 d in vivo ageing at 37°C. Thus, the relative ageing at 22°CC in vitro was (2.1 d/ 5d) = 0–42 of that at 37°C in vivo. A similar ageing ratio (0–44) was obtained by measurement of the decrease in MRL during storage at 22°C of platelets stored for 1, 5, 7, 10 and 14 d relative to the decrease in MRL of fresh platelets in vivo.

ATP turnover rate at 22°C was compared to the rate of 37°C by measurement of the rates of platelet oxygen consumption and lactate production in vitro. In vitro ATP turnover at 22°C versus 37°C, was found to be 10–5 ± 10 versus 21–6 ± l.4/μmol/1012plts/min, respectively. Thus, the ATP turnover ratio (0–48) at these two temperatures suggests that the relative decrease in ageing at 22°C compared to 37°C is similar to the relative decrease in metabolic rate at this temperature.
Interleukin‐3 priming in acute myeloid leukaemia patients
Authors: Tafuri Agostino , Felice Lidia de , Goodacre Angela , Fenu Susanna , Petrucci Maria Teresa , Valentini Tiziana , Alimena Giuliana , Petti Maria Concetta , Meloni Giovanna , Mandelli Franco , Andreeff Michael
Published: March 2008

Summary Several studies have demonstrated that G‐CSF, GM‐CSF and, in particular, IL‐3 can effectively recruit acute myeloid leukaemia (AML) blasts into the cell cycle, resulting in a significant increase in cytosine‐arabinoside (Ara‐C) mediated cytotoxicityin vitro. Since IL‐3 has shown biological and clinical activity, we investigated the cell kinetic effects of rIL‐3 and high‐dose Ara‐C/idarubicin in three patients with refractory AML selected for the presence of chromosome 7 monosomy; this enabled differentiation between the effects of IL‐3 on leukaemic and on normal cells. Thein vivo administration of rhIL‐3 (250μ/m2 d s.c. for 6–10 d) recruited AML blasts into the cell cycle in two of the three patients, and this effect resulted in an increase inin vitro growth of clonogenic cells (CFU‐L) and of their S‐phase fraction. The percentage of leukaemic cells with monosomy 7 increased only in the two cases who showed a proliferative response. Normal cells were not recruited, even when rhIL‐3 was administered for up to 10 d.In vitro studies showed an increased Ara‐C cytotoxicity on clonogenic AML cells, in particular with LL‐3 plus GM‐CSF, thus confirming the priming effects of IL‐3 in the two responding cases.

The results of this study suggest that rhIL‐3 can selectively recruit leukaemic cells into the cell cycle. Although leukaemic blasts can be sensitized to Ara‐C, other mechanisms of primary blast resistance may limit the clinical benefit of kinetic‐based approaches.
Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia
Authors: Maciejewski Jaroslaw P. , Selleri Carmine , Sato Tadatsugu , Anderson Stacie , Young Neal S.
Published: March 2008

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T‐cell‐mediated killing of malignant, virus‐infected or allogeneic target cells. Interferon‐γ (IFN‐γ) and tumour necrosis factored (TNF‐α), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34+ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas‐mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN‐γ, increased TNF‐β expression, and elevated numbers of activated cytotoxic T‐cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34+ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas‐mediated inhibition of colony formation. In contrast, in early AA, BM CD34+ cells showed markedly increased percentages of Fas receptor‐expressing CD34+ cells, which correlated with increased sensitivity of AA marrow cells to anti‐Fas antibody‐mediated inhibition of colony formation. The proportion of Fas antigen‐bearing cells was lower in recovered patients’BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34+ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN‐γ and/or TNF‐α and are suitable targets for T‐cell‐mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.
VLA‐4 and VCAM‐1 are the principal adhesion molecules involved in the interaction between blast colony‐forming cells and bone marrow stromal cells
Authors: Oostendorp Robert A. J. , Reisbach Gilbert , Spitzer Elisabeth , Thalmeier Karin , Dienemann Hendrik , Mergenthaler Hans‐Günther , Dörmer Peter
Published: March 2008

Summary. The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony‐forming capacity of stroma‐adherent Bl‐CFC, BFU‐E and GM‐CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow‐derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against β1‐, α4‐integrin (both chains of the very late activation antigen‐4 (VLA‐4)) and vascular cell adhesion molecule (VCAM‐1) inhibited colony formation from stroma‐adherent Bl‐CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the α5‐integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA‐4 on progenitors interacted with stromal VCAM‐1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU‐E and GM‐CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as Bl‐CFC, remains to be determined.
The CD4 receptor plays essential but distinct roles in HIV‐1 infection and induction of apoptosis in primary bone marrow GPIIb/IIIa+ megakaryocytes and the HEL cell line
Authors: Zauli Giorgio , Catani Lucia , Gibellini Davide , Re Maria Carla , Milani Daniela , Borgatti Paola , Bassini Alessandra , Placa Michele La , Capitani Silvano
Published: March 2008

Summary. We investigated whether cells belonging to the megakaryocyte lineage could be infected in vitro with human immunodeficiency virus type‐1 (HIV‐1). Primary GPIIb/IIIa+ bone marrow (BM) cells and HEL continuous cell line were first phenotypically characterized for the presence of megakaryocytic markers and CD4 antigen, then challenged in vitro with the laboratory strain IIIB of HIV‐1. Both GPIIb/IIIa+ BM and HEL cells expressed significant levels of CD4 receptor (<50%) and were efficiently infected with HIV‐1, as judged by the presence of proviral DNA after polymerase chain reaction analysis and by quantitative evaluation of gag p24 antigen in the culture supernatants. Of note, infection with HIV‐1 in both primary BM megakaryocytes and HEL cells was specifically blocked by soluble recombinant CD4. To ascertain whether the CD4 receptor was essential for infection of megakaryocytic cells, HEL were subcloned into CD4+ and CD4 cells. Although unfractionated and CD4+ HEL cells were productively infected with HIV‐1, CD4 HEL cells could not be infected. Infection of HEL cells did not induce gross cytotoxic effects or a significant increase of apoptosis. On the other hand, treatment of unfractionated or CD4+ HEL cells with cross‐linked recombinant env gp120 or Leu3a anti‐CD4 monoclonal antibody markedly (P< 0.01) increased the degree of apoptosis with respect to HEL cells infected with HIV‐1 or treated with cross‐linked gag p24 or anti‐GPIIb/IIIa antibody. Taken together, these data indicate that the CD4 receptor represents the main route of infection in cells belonging to the megakaryocytic lineage. Moreover, an inappropriate engagement of CD4 by either free env gpl20 or anti‐CD4 monoclonal antibody could be more relevant than a direct infection with HIV‐1 in the induction of the frequent BM megakaryocyte abnormalities found in HIV‐1 seropositive thrombocytopenic patients.
The role of haemopoietic growth factors in the pathogenesis of the early anaemia of premature infants
Authors: Han P. , Stacy D. , Story C. , Owens P. C.
Published: March 2008

Summary. The anaemia of prematurity has been attributed to an insufficient erythropoietin (Epo) level. However, haemopoiesis is known to be regulated by a cohort of growth factors including interleukin‐3 (IL‐3), IL‐6, stem cell factor (SCF), granulocyte monocyte‐colony stimulating factor (GM‐CSF) and insulin‐like growth factors‐I and ‐II (IGF‐1, IGF‐II). Circulating levels of these growth factors were measured in cord blood at the following gestational ages: 25‐28 weeks, 29‐32 weeks, 33‐36 weeks and >37 weeks. This study indicates that low concentrations of IGFs as well as a low Epo level in early gestational ages may play a role in anaemia of prematurity.
Long survival after splenic immunoblastic transformation of Waldenström's macroglobulinaemia
Authors: Winter A. J. , Obeid D. , Jones E. L.
Published: March 2008

Summary. Immunoblastic transformation of Waldenström's macroglobulinaemia is normally a preterminal event. We report a case in which the immunoblastic transformation appeared to be limited to the spleen. Splenectomy was more effective than cytotoxic chemotherapy in controlling the disease, and the patient remains free of disease 45 months later.
Characterization of mutations within the factor VIII gene of 73 unrelated mild and moderate haemophiliacs
Authors: Schwaab R. , Oldenburg J. , Schwaab U. , Johnson D. J. D. , Schmidt W. , Olek K. , Brackman H.‐H. , Tdddenham E. G. D.
Published: March 2008

Summary.To Screen for mutations within the factor VIII gene of 101 patients (85 unrelated), we used denaturing gradient gel electrophoresis (DGGE) after DNA amplification of target regions, including all coding regions except for the middle part (amino acid 757 to amino acid 1649) of the B domain. With this method, missense mutations were identified in 86% of unrelated patients. 41 different mutations were identified; 25 of them have not been described previously. Five of the genotypes are associated with CRM+and 26 with CRMredstatus. Patients who are definitely related to each other showed no differences in DNA sequence. One patient showed two different base pair alterations, the first at amino acid 469 [ala(GCA→gly(GGA)] and the second at position 473 [tyr(TAT)→cys(TGT)]. One patient with an amino acid change at position 1689 [arg(CGC)→his(CAC)] has developed an inhibitor against factor VIII.
Haemopoietic growth factor production by normal and aplastic anaemia stroma in long‐term bone marrow culture
Authors: Gibson F. M. , Scopes J. , Daly S. , Ball S. , Gordon‐Smith E. C.
Published: March 2008

Summary. Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive‐ or negative‐acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma‐stem cell interactions.

We have used a sensitive bioassay to investigate production of granulocyte‐colony stimulating factor (G‐CSF), granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), interleukin (IL)‐3, IL‐6 and stem cell growth factor (SCF), by normal and AA stroma In long‐term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34+ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony‐stimulating activity (CSA) and burst‐promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G‐CSF and GM‐CSF, 93% produced IL‐3, 80% produced IL‐6, and 70% produced SCF. Similarly, all AA stroma produced G‐CSF and GM‐CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL‐3 and 36% produced IL‐6. Target cell stimulation was not dependent on direct stroma‐target cell contact, suggesting production of soluble cytokines. However, although both IL‐6 and G‐CSF were detected in LTBMC supernatants by enzyme‐linked immunoassay (ELISA), IL‐3 and GM‐CSF were undetectable, perhaps indicating low‐level local production of these factors.
Quantitative flow cytometry can distinguish between normal and leukaemic B‐cell precursors
Authors: Farahat Nahla , Lens Daniela , Zomas Athanasios , Morilla Ricardo , Matutes Esteixa , Catovsky Daniel
Published: March 2008

Summary. The immunological detection of minimal residual disease in B‐lineage acute lymphoblastic leukaemia (ALL) has been hampered by the fact that the leukaemic cells represent the malignant counterparts of normal haemo‐poietic precursors expressing terminal deoxynucleotidyl transferase (TdT), CD10 and CD19. We have used quantitative double‐labelling flow cytometry with standard fluorescent beads to convert the mean fluorescence to the number of antigen molecules per cell. The number of TdT, CD10 and CD19 molecules per cell was determined in normal B‐cell precursors from 22 healthy donors and eight regenerating marrows from patients with various malignancies and in 20 cases of B‐lineage ALL. In normal bone marrow we characterized two different B‐cell populations: TdT+/CD10+/ CD19+ and TdT/CD10+/CD19+. We demonstrated a major difference in the level of expression of TdT, CD 10 and CD 19 between normal bone marrow and B‐lineage ALL blasts. Normal TdT+ precursors have significantly higher number of TdT (>100×l03) and lower number of CD10 (<50×l03) and CD19 (<10×103) molecules per cell than B‐lineage ALL blasts (<100, >50, >10×l03 molecules per cell respectively); these differences were statistically highly significant. Furthermore, regenerating marrows had a significantly higher percentage of B‐cell precursors than healthy donors. This increase was at the expense of the TdT−/ CD10+/CD19+ population which, in the context of B‐lineage ALL, could be wrongly interpreted as evidence of relapse if TdT is not included in the analysis. Therefore the quantitative analysis of TdT combined with CD10 and CD19 may allow a clear distinction between normal precursors and minimal residual leukaemia in B‐lineage ALL and avoid the pitfall of misinterpreting regenerating B‐cells as evidence of relapse.
2 ‐Chloro‐deoxyadenosine therapy for giant lymph node hyperplasia
Authors: Bordeleau Louise , Bredeson Christopher , Markman Saul
Published: March 2008

Summary. Giant lymph node hyperplasia (GLNH) or Castle‐ man disease is a heterogenous group of atypical lympho‐ proliferative disorders. Two main histologic variants, the hyaline vascular variant and the plasma cell variant, have been recognized. Although localized GLNH can often be managed successfully with surgery, optimal therapy for multifocal disease has yet to be identified. We report two cases of GLNH treated with 2‐chloro‐deoxyadenosine (2‐ CDA), a synthetic purine analogue. 2‐CDA was utilized based on its relative lymphocytic toxicity and the putative pathophysiologic process in GLNH being either hamarto‐ matous overgrowth (hyaline‐vascular variant) or immune dysfunction and lymphoproliferation (plasma cell variant). One patient with unresectable localid hyaline‐vascular GLNH has had a 9‐month continuous complete remission following two courses of 2‐CDA therapy followed by radiation therapy. The second patient with disseminated plasma cell type had a partial response to two cycles of 2‐ CDA therapy; however, further cycles were not given due to development of possible early neurotoxicity. Although the optimal management of non‐resectable GLNH is yet to be determined, 2‐CDA appears to be a viable therapeutic option for patients with this disease process.