bluuebunny/crossref_metadata_2025 huggingface dataset:
Late‐appearing Philadelphia chromosome in a patient with acute nonlymphocytic leukaemia derived from myelodysplastic syndrome: detection of P210‐ and P190‐type bcr/abl fusion gene transcripts at the leukaemic stage
Authors: Katsuno Makoto , Yamashita Sayuri , Sadamura Shingo , Umemura Tsukuru , Hirata Jouji , Nishimura Junji , Nawata Hajime
Published: March 2008

Summary. We describe a patient with acute nonlymphocytic leukaemia (ANLL) derived from myelodysplastic syndrome in whom the Philadelphia chromosome (Ph1) first emerged at the late stage of ANLL transformation. Cytogenetically, the Ph1 chromosome was not detected until the late stage of ANLL transformation, 14 months after the transformation following a 3‐month history of refractory anaemia with excess of blasts. The cells with and without the Ph1 chromosome had a common abnormal chromosome, t(3;3) (q21;q26). The reverse transcription‐polymerase chain reaction analysis showed no bcr/abl message at diagnosis. However, the mRNA encoding P210bcr/abl was detected in the early stage of ANLL transformation. Furthermore, the mRNAs encoding both P210bcr/abl and P190bcr/abl were detected in the late stage of ANLL transformation when the Ph1 chromosome was detected by cytogenetic analysis. These evidences support a multistep pathogenesis of leukaemias, and the product of bcr/abl fusion gene may influence the course of disease.
Optimization of the dilute prothrombin time for the detection of the lupus anticoagulant by use of a recombinant tissue thromboplastin
Authors: Arnout J. , Vanrusselt M. , Huybrechts E. , Vermylen J.
Published: March 2008

The dilute prothrombin time (dPT) is a widely accepted sensitive screening test for the lupus anticoagulant (LA). In general, Simplastin (Organon Teknika), a rabbit brain thromboplastin, diluted 1/500, is used in this test. Recently, human tissue thromboplastin obtained by recombinant DNA technology has become available and we have evaluated the usefulness of one such preparation, Innovin (Dade), for the detection of the LA. dPTs, using several dilutions of Innovin were determined on plasmas from 18 normal individuals and 15 patients with a well‐documented LA. The dPT ratios, calculated as the individual result divided by the mean normal, were statistically compared. Innovin in dilutions from 1/100 onwards was found to be significantly more responsive to the LA than Simplastin (P < 0.05). Intraassay coefficients of variation (CVs) ranged from 1.05% to 14.8% with Innovin, versus 2.7%‐24.3% with Simplastin (P < 0.05); inter‐assay CVs ranged from 5.6%‐11.8% with Innovin, versus 4.2%‐33.8% with Simplastin (P < 0.001). Analysis of 316 consecutive plasma samples from nonanticoagulated patients, on which a LA determination was requested, showed a 100% sensitivity and a 96% specificity of the dPT performed with Innovin, as compared to 81% and 93% respectively for the dPT using Simplastin.
Immunochemical estimation of haemoglobin types in red blood cells by FACS analysis
Authors: Thorpe Susan J. , Thein Swee Lay , Sampietro M. , Craig J. E. , Mahon B. , Huehns E. R.
Published: March 2008

Summary. A fixation and permeabilization procedure using formaldehyde and acetone has been developed which allows immunostaining of intracellular haemoglobin for fluorescence activated cell sorter (FACS) analysis of erythrocytes. The treatment preserves antigenicity and light‐scattering properties. Validation of the method was given by the correlation of F cell number in adults determined by FACS analysis with that assessed by microscopic examination of cell smears, and by the direct relationship between β chain synthesis and intensity of β chain/Hb A immunofluorescence within fetal erythrocyte samples known to vary in their β chain/Hb A content. The procedure is rapid, non‐subjective and sensitive, and makes analysis of haemoglobin content, type and distribution amongst red cell populations possible.
Diagnosis of thalassaemia by non‐isotope detection of α/β and ζ/α mRNA ratios
Authors: Lin Sheng‐Fung , Liu Ta‐Chiu , Chen Tyen‐Po , Chiou Shyh‐Shin , Liu Hong‐Wen , Chang Jan‐Gowth
Published: March 2008

Summary. The α/β and ζ/α messenger RNA (mRNA) ratios in the thalassaemia syndromes were investigated by polymerase chain reaction (PCR) with silver staining of the PCR products. In this study we used the PCR to amplify cDNA copies of circulating erythroid cell mRNA in order to measure the relative amounts of α‐, β‐ and ζ‐globin contained within. Quantitation was performed by scanning the silver stain of specific globin cDNA bands. We found that there were significant differences of α/β‐mRNA and ζ/α‐mRNA in patients with Hb H disease and α‐thalassaemia‐1 compared to normal subjects. There was a marked increase in the α/β‐mRNA ratio but not in the ζ/α‐mRNA ratio in patients with β‐thalassaemia. In two β‐thalassaemia cases abnormal increases of ζ‐globin bands were noted and they were confirmed through DNA analysis to be combined with α‐thalassaemia‐1. This method provides a simple, rapid and non‐radioactive approach to detect thalassaemia syndromes, and can help to screen cases of β‐thalassaemia with α‐thalassaemia‐1.
Glucose 6‐phosphate dehydrogenase deficiency and red cell membrane defects: additive or synergistic interaction in producing chronic haemolytic anaemia
Authors: Alfinito F. , Calabro V. , Cappellini M. D. , Fiorelli G. , Filosa S. , Iolascon A. , Giudice E. Miraglia del , Perrotta S. , Migliorati R. , Vallone D. , Rotoli B. , Luzzatto L.
Published: March 2008

Summary. We have investigated two unrelated patients with congenital haemolytic anaemia in both of whom we found a combination of hereditary spherocytosis (HS) and glucose‐6‐phosphate dehydrogenase (G6PD) deficiency. Segregation of the two defects was documented in both families, who had different molecular abnormalities for both HS and G6PD deficiency. In one family the propositus had a reduced level of spectrin and G6PD Seattle (282Asp His). In the other family the propositus had a band 3 abnormality and was heterozygous for G6PD Mediterranean (188Ser Phe). From a comparison of clinical and haematological findings in family members with either or both abnormalities we conclude that in one case the two defects exhibited a synergistic effect, resulting in a severe chronic haemolytic anaemia; whereas in the other the association was simply additive.
Rolling and stationary cytoadhesion of red blood cells parasitized by Plasmodium falciparum: separate roles for ICAM‐1, CD36 and thrombospondin
Authors: Cooke Brian M. , Berendt Anthony R. , Craig Alister G. , MacGregor John , Newbold Christopher I. , Nash Gerard B.
Published: March 2008

Summary. Adhesion of parasitized erythrocytes to microvascular endothelium is a central event in the pathogenesis of severe falciparum malaria. We have characterized the adhesion of flowing parasitized red blood cells to three of the known endothelial receptors coated on plastic surfaces (CD36, intercellular adhesion molecule‐1 (ICAM‐1) and thrombospondin (TSP)), and also to cells bearing these receptors (human umbilical vein endothelial cells (HUVEC) and platelets). All of the surfaces could mediate adhesion at wall shear stress within the physiological range. The great majority of adherent parasitized cells formed rolling rather than static attachments to HUVEC and ICAM‐1, whereas static attachments predominated for platelets, CD36 and TSP. Studies with monoclonal antibodies verified that binding the HUVEC was mainly via ICAM‐1, and to platelets via CD36. Adhesion via ICAM‐1 was least sensitive to increasing wall shear stress, but absolute efficiency of adhesion was greatest for CD36, followed by ICAM‐1, and least for TSP. TSP did not give long‐lasting adhesion under flow, whereas cells remained adherent to CD36 or ICAM‐1. We propose that the different receptors may have complementary roles in modulating adhesion in microvessels. Initial interaction at high wall shear stress may be of a rolling type, mediated by ICAM‐1 or other receptors, with immobilization and stabilization occurring via CD36 and/or TSP.
Non‐invasive assessment of tissue iron overload in the liver by magnetic resonance imaging
Authors: Jensen P. D. , Jensen F. T. , Christensen T. , Ellegaard J.
Published: March 2008

Summary. We investigated the clinical usefulness of a standard magnetic resonance imaging (MRI) system for non‐invasive determination of the liver iron concentration in 38 patients with iron overload and 15 normal controls by measurement of the signal intensity ratio between liver and skeletal muscle (SIR). However, SIR was found dependent on the applied repetition time (TR) of the MRI system, which led us to investigate this relationship in autopsy material of liver and muscle tissue specimens with various iron content. Based on these results, adjustment of SIR measurements to a constant value of TR was achieved. By use of this technique we found a close correlation between MRI and chemically determined liver iron concentration (r2= 0.98) as well as the serum ferritin concentration (r2= 0.86). The reproducibility was sufficiently good for the use of MRI in the follow‐up of iron reductive treatment. The use of iron store parameters in serum was found insufficient as indicators of endpoint for venesection therapy, if 20 μmol Fe/g dry weight was applied as the upper reference limit of the liver iron concentration.

It is concluded that MRI based on SIR measurements offers a precise and reproducible non‐invasive method for the determination and follow‐up of iron overload within a wide range of liver iron concentrations. Our findings may increase the clinical use of MRI in haematological patients with iron overload.
Androgen therapy in myelodysplastic syndromes with thrombocytopenia: a report on 20 cases
Authors: Wattel Eric , Cambier Nathalie , Caulier Marie‐Therese , Sautiere Daniele , Bauters Francis , Fenaux Pierre
Published: March 2008

Summary. Twenty patients with myelodysplastic syndromes (MDS) and (i) platelets <50 × 109/1 and (ii) bone marrow blasts 10% were treated with androgen therapy (fluoxymesterone at 1 mg/kg/d: seven patients: danazol at 600 mg/d: 13 patients) for at least 3 months. 11 of them (55%) had an increase in platelet counts by at least 30 × 109/1 and a disappearance of bleeding symptoms was seen in 6/6 patients with initial bleeding. A response with neutrophil counts (six cases) or haemoglobin levels (five cases) was less often seen. Treatment was continued for 3+ to 27 months in responders (the dose being reduced by 50% after 6 months). Seven patients on maintenance treatment were still responding. Another patient died while he was still responding, and the remaining three patients relapsed after discontinuation (two cases) and dose reduction to 50% (one case) of the androgen used. Side‐effects of treatment were moderate. In our experience, androgen therapy can be useful in patients with ‘low risks’MDS (i.e. with marrow blasts 10%) and severe thrombocytopenia, especially because no growth factor regularly active on platelets is currently available.
Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure
Authors: Maciejewski Jaroslaw P. , Anderson Stacie , Katevas Prokopis , Young Neal S.
Published: March 2008

SUMMARY. Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colonyforming cells, and AA progenitors fail to proliferate in longterm assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34+ and CD33+ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34+ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34+ cell reduction. A decrease in BM CD33+ cells was a common finding in MDS patients, but reduction in CD34+ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purged CD34+ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c‐kit receptor expression on CD34+ cells, we found that the mitotically active CD34+ cells bearing the c‐kit antigen were reduced in AA. With clinical improvement, CD34+ and CD33+ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34+ cells from recovered patents showed improved plating efficiency. In patients with aplastic anaemia, use of CD34 antigen as a phenotypic marker of progenitor cells may be helpful for the analysis of the early haemopoietic cell compartment and BM recovery.
Detection of peripheral blood plasma cells as a predictor of disease course in patients with smouldering multiple myeloma
Authors: Witzig Thomas E. , Kyle Robert A. , O'Fallon William M. , Greipp Philip R.
Published: March 2008

SUMMARY. We evaluated the number and labelling index of circulating peripheral blood monoclonal plasma cells (PBPC) in 57 patients with newly diagnosed smouldering multiple myeloma (SMM) to determine if these measurements could distinguish SMM from active multiple myeloma (MM). The PBPC were detected by a sensitive slide‐based immunofluorescence procedure that identified PC by their morphology and monoclonal light chain staining. The presence of abnormal PBPC (defined as an increase in number or proliferative rate of circulating monoclonal plasma cells) was correlated with patient status at 6 and 12 months after testing. 16 of the 57 patients progressed within 12 months and 63% of these had abnormal PBPC. In contrast, only 10% (4/41) of those patients who remained stable had abnormal PBPC. The median time to progression for patients with abnormal PBPC was 0.75 years, compared to 2.5 years for those patients without abnormal PBPC (P < 0.01). The detection of PBPC is important and helps to identify patients with active MM when other parameters suggest SMM. The lack of abnormal PBPC suggests SMM, and these patients may have a stable course without the need for immediate chemotherapy.
Further characterization of platelet‐aggregating cysteine proteinase activity in thrombotic thrombocytopenic purpura
Authors: Consonni R. , Falanga A. , Barbui T.
Published: March 2008

SUMMARY. Circulating platelet‐aggregating activities have been described by several authors. In a previous study we have confirmed the presence in serum and plasma from patients with thrombotic thrombocytopenic purpura (TTP) of a platelet aggregating cysteine proteinase (PACP). This activity differed in that, among the class of thiol enzymes, it showed characteristics of a lysosomal cathepsin rather than of a cytoplasmic calpain.

To further investigate the enzymatic properties of this PACP we have designed a study to evaluate the dependence of the activity on Ca++. Calcium‐dependence is a property of calpains but not of cathepsins. A proteolytic assay was set up and conducted with and without Ca++. The release of acid‐soluble peptide from denatured human globin promoted by TTP samples and standards was fluorimetrically measured. The results show that TTP samples were equally active with and without Ca++ similar to standard palpain (the cathepsin B‐like proteinase), whereas standard calpain only acted with Ca++.

An inhibition study performed by the proteolytic assay confirmed the same pattern of sensitivity of PACP to a series of specific cystein proteinase inhibitiors previously shown by the proaggregating assay. The enzymatic behaviour of PACP of TTP resembled a lysosomal (cathepsins) rather than a cytoplalsmic (calpains) cysteine proteinase.
Characterization of platelet aggregation induced by human colon adenocarcinoma cells and its inhibition by snake venom peptides, trigramin and rhodostomin
Authors: Chiang Huei‐Shien , Swaim Mark W. , Huang Tur‐Fu
Published: March 2008

SUMMARY. SW‐480 cells, derived from a primary human colon adenocarcinoma, caused dose‐dependent platelet aggregation in heparinized human platelet‐rich plasma. SW‐480 tumour cell‐induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E‐64 (10 μM) but was limited by cell pretreatment with phospholipase A2. SW‐480 cell suspension caused marked dose‐dependent decreases in plasma recalcification times using normal, factor VIII‐deficient and factor IX‐deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW‐480 cell suspension did not affect the recalcification time of factor VII‐deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW‐480 TCIPA. Taken together, these data suggest that SW‐480 TCIPA arises from SW‐480 tissue factor activity expression. Trigramin and rhodostomin, RGD‐containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW‐480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotien IIb/IIIa and Ib prevent SW‐480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50 0.09 μM) and rhodostomin (IC50 0.03 μM) were about 6000 and 18000 times, respectively, more potent than GRGDS (IC50 0.56mM).
The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes
Authors: Brinkman Herm‐Jan M. , Mertens Koen , Holthuis Joost , Zwart‐Huinink Lineke A. , Grijm Karin , Mourik Jan A. Van
Published: March 2008

SUMMARY. Rates of factor X activation on endothelial cells were compared with activation rates on other vascular cells, platelets, monocytes and negatively charged phospholipid vesicles. Factor VIIa‐mediated factor X activation was observed on smooth muscle cells and fibroblasts in the absence of cell‐perturbing agents, whereas endothelial cells required activation in order to allow extrinsic activation of factor X. On the other hand, unperturbed endothelial cells did promote intrinsic, factor VIII/IXa‐dependent activation of factor X. The rate of factor X activation on these cells was about one‐sixth of that on ionophore A23187‐stimulated platelets. Also, smooth muscle cells and fibroblasts were able to activate factor X through the intrinsic pathway, altough to a lesser extent than endothelial cells. Monocytes were ineffective in this respect. Prothrombin fragment 1, the prothrombin fragment containing the γ‐carboxyglutamic acid domain known to mediate binding of vitamin K‐dependent coagulation factors to phospholipid surfaces, inhibited factor VIII/IXa‐dependent factor X activation on endothelial cells (IC50 3.2 μM) to a lesser extent than on phospholipid vesicles (IC50 0.2 μM). Therefore, besides negatively charged phospholipids, other membrane constituents seem to be involved in endothelial cell mediated, intrinsic activation of factor X. Perturbation of endothelial cells with phorbol myristate acetate (PMA) or lipopoly‐saccharide (LPS) was without effect on intrinsic activation of factor X. This observation indicates that membrane constituents of endothelial cells involved in factor VIII/IXa‐dependent activation of factor‐X are constitutively expressed.
Determination of plasma tissue factor antigen and its clinical significance
Authors: Koyama Takatoshi , Nishida Kenji , Ohdama Shinichi , Sawada Megumi , Murakami Naomi , Hirosawa Shinsaku , Kuriyama Renjiro , Matsuzawa Kimihiko , Hasegawa Ryoichi , Aoki Nobuo
Published: March 2008

SUMMARY. To investigate the clinical significance of determination of plasma tissue factor (TF) antigen, we have developed a highly sensitive enzyme‐linked immunosorbent assay (ELISA) for plasma TF, using two different monoclonal antibodies against TF apoprotein, 6B4 (catching antibody) and 5G9 (detecting antibody), and tetramethyl benzidine/H2O2 as substrates. Titration curves of recombinant human TF in buffer containing Triton X‐100 were linear within the range from 50 to 2000pg/ml. The total assay time was 3h. Ultracentrifugation and immunoblot analysis indicated that human plasma and urine contained 50 000g sedimentable and non‐sedimentable forms of TF, both of which were detected by our ELISA method.

Plasma and urine concentrations of TF in healthy subjects and patients with various diseases were measured by the ELISA method. In healthy subjects, plasma and urinary TF levels were found to be 149± 72pg/ml (n = 30) and 175±60pg TF/urine creatinine mg (n = 95). respectively. TF was increased in plasma of patients with disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, vasculitis associated with collagen diseases, diabetic microangiopathy and chronic renal failure receiving haemodialysis, but not in the plasma of endotoxaemic patients without DIC. The plasma TF/serum creatinine ratio did not show a positive correlation. Measurement of TF antigen in plasma may be useful for evaluating the endothelial damage and cell destruction in TF‐containing tissues.
Effects of 3‐5 log10 pre‐storage leucocyte depletion on red cell storage and metabolism
Authors: Heaton W. A. L. , Holme S. , Smith K. , Brecher M. E. , Pineda A. , AuBuchon J. P. , Nelson E.
Published: March 2008

SUMMARY. A new, in‐line high‐efficiency 3‐5 log10 leucodepletion filter system (Leukotrap° RC system) was used to investigate the effect of pre‐storage white cell removal on the quality of AS‐3 red cell concentrates stored for 42 d at 4°. Median residual white cell content was 4 × 105 when filtration was performed at 22° within 8 h of phlebotomy (n= 20) and 3.2 × 104 when filtration was performed at 4° 12‐24 h after phlebotomy (n= 24). None exceeded 1 × 106 WBC per red cell product. Filtration was rapid (median 28 min), and red cell loss averaged (mean ± 1 SD) 6.4 ± 0.7%. In a paired study design, post‐transfusion recoveries of 42 d stored red cells in the filtered units averaged 84 ± 6% v 82 ± 8% for unfiltered units (P < 0.05) and post‐storage haemolysis, ATP, osmotic fragility, K+ and pH were significantly (P < 0.05) better in the filtered units. Reduced glycolytic activity was also observed in the filtered units, and there was a correlation between osmotic fragility, glucose consumption, and lactate produced in standard units that was not present in leucodepleted units. In conclusion, this study suggests that leucodepletion of AS‐3 red cell concentrates prior to storage results in better maintenance of the integrity of the red cell membrane with reduced glycolytic activity. There was a modest improvement in post‐infusion viability sufficient to offset the filtration‐induced loss and to result in an equivalent red cell product.
Philadelphia‐negative chronic myeloid leukaemia: detection by FISH of BCR‐ABL fusion gene localized either to chromosome 9 or chromosome 22
Authors: Nacheva E. , Holloway T. , Brown K. , Bloxham D. , Green A. R.
Published: March 2008

SUMMARY. Dual‐colour FISH has been used to study two patients with chronic myeloid leukaemia (CML) associated with a normal karyotype. Co‐localization of signals from BCR and ABL cosmids was observed in interphase nuclei from both patients. In one patient, analysis of metaphase spreads showed that the 3’region of the ABL gene was deleted from one chromosome 9 and inserted into chromosome 22. In a second patient 5’BCR sequences were missing from one copy of chromosome 22, and co‐localized with 3’ABL sequences on chromosome 9. These results demonstrate the molecular heterogeneity of Ph‐negative CML. In addition, they illustrate the potential usefulness of dualcolour FISH on interphase nuclei for monitoring the response to treatment of patients with Ph‐negative CML.
Cytokine gene expression in the mixed lymphocyte culture in allogeneic bone marrow transplants as a predictive method for transplantation‐related complications
Authors: Tanaka Junji , Imamura Masahiro , Kasai Masaharu , Kobayashi Sumiko , Hashino Satoshi , Kobayashi Hajime , Sakurada Keisuke , Miyazaki Tamotsu
Published: March 2008

SUMMARY. We have investigated cytokine gene expression in the two‐way mixed lymphocyte cultures (MLC) enhanced by concanavalin A to assess whether this is a useful predictive method for severe graft‐versus‐host disease (GVHD) and graft failure in nine allogeneic bone marrow transplantation (allo‐BMT) patients. Our present study revealed that increased IL‐2, IL‐5 and IFN‐γ mRNA expression and IL‐2 and IFN‐γ production in the MLC in two cases with severe lethal transplantation‐related complications (graft failure and grade III acute GVHD). These findings suggest that increased cytokine mRNA expression and cytokine products in this assay may be predictive of the development of transplantation‐related complications.
Reducing erythropoietin in cultures of human erythroid precursors elevates the proportion of fetal haemoglobin
Authors: Fibach Eitan , Schechter Alan N. , Noguchi Constance T. , Rodgers Griffen P.
Published: March 2008

Summary. In order to clarify the mechanism of the effect of erythropoietin (Epo) on the fetal haemoglobin (HbF) phenotype of peripheral erythrocytes, we studiesd the doseresponse effect of Epo on HbF production by erythroid precursors derived from the peripheral blood of normal adult individuals and grown in a two‐phase liquid culture system. The proportion of HbF out of the total haemoglobin (Hb) content (%HbF) was dependent on the duration of exposure to Epo; on day 6 it comprised up to 15%, but dropped to < 2% on day 14. Both cell yield and cellular Hb content were markedly increased by high (1 U/ml) Epo, compared to normal physiological (20–50 mU/ml) levels, but neither the initial nor final %HbF were dependent on the increased Epo dose. However, when cells grown with high Epo were transferred on day 7 to low Epo, their progeny contained by day 14 a higher %HbF as compared to cells that were continuously exposed to high Epo. This was accompanied by acceleration and synchronization of their maturation process, as evidenced by their morphology, density and size, and restriction on cell multiplication, as indicated by the lower cell yield. These results are consistent with the following model. As early erythroid precursors, with relatively high HbF, mature under steady‐state levels of Epo, HbA production predominates and HbF is diluted. However, when such precursors are switched from high to low levels of Epo they undergo a synchronized, accelerated maturation which shortens the period of HbA production, leading to a decreased Hb content and a relatively high proportion of HbF. This mechanism may contribute to the elevated HbF observed following Epo administration (due to short half‐life of Epo in vivo). and might also explain the HbF‐augmenting effect of Epo administered together with hydroxyurea observed in patients with sickle cell anaemia.
The effect of iron overload and iron reductive treatment on the serum concentration of carbohydrate‐deficient transferrin
Authors: Jensen P.D. , Peterslund N.A. , Poulsen J. Hjelm , Jensen F. T. , Christensen T. , Ellegaard J.
Published: March 2008

Summary. The concentration of carbohydrate‐deficient transferrin in serum (CDT) has been used as a reliable indicator of recent alcohol consumption. We have investigated the utility of this laboratory test in 20 patients with hereditary haemochromatosis (HH) by simultaneous evaluation of serum concentrations of liver transaminases, γ‐glutamyl transpeptidase, iron, transferrin and asessment of the liver iron concentration by magnetic resonance imaging. 11 patients were re‐examined during iron depletion with phelebotomies. In all 11 patients intensive but not maintenance iron removal was associated with an increase in serum CDT, in three patients even to levels above the reference range. The mean serum CDT increased from 8.5 (SD 2.2) U/1 to 16.6 (SD 7.2) U/1 (P < 0.001). Iron mobilization from the liver was found particularly responsible for the increase in serum CDT. Independent of this finding we found a significant semi‐logarithmic correlation (r =−0.77. P = 0.009) between the MRI determined liver iron depletion. Our findings indicate that the utility of serum CDT as a measure of alcohol consumption in patients with HH may be compromised, especially during intensive iron depletion.
Influence of donor lymphocytes on the incidence of primary graft failure after allogeneic bone marrow transplatition in a murine model
Authors: Uharek Ltz , Glass Bertram , Gassmann Winfried , Loeffler Helmut , MuellerRchholtz Wolfgang
Published: March 2008

Summary. We used a murine mode to determine the impact of donor lymphocyte subsets on the incidence of primary marror graft fallure after transplantation of lymphoctytedepleted bone marroe. After lethal irradiaton with 7.5Gy. Balb/c mice receivd 1 * 105 to 4 * 107 GvH‐nonreactive (C57 * Balb)F1 or GvH‐reactive C57Bl/6 marrow cells. Pretdreatment with anti‐Thy‐1,2, anti‐CD4/CD8, anti‐asialoGMl or L‐leucyl‐L‐Leucine methyld ester (Leu‐Leu‐OMe) was employed to eliminate T lymphocytes and/or natural Killer cells. Primary graft failure was defined as death with neurtophild < 0.5 * 109/1. To assess longterm chimaerism, the precentage of H‐bb ‐positive spleen cells was determined. Pretreatment with anti‐Thy‐1.2, anti‐CD4/CD8 or Leu‐Leu‐OMe successfully eliminated GvHR‐induced mortality. Graft failure rates gradually declined from 88% after transplantation of 1 * 105 cells to 0% after transplantation of 4 * 107 C57B/6 cells. The incidence of graft failure, however, was not altered by T‐cell depletion, provided that the unspecific loss of marrow cells was compensated for. After transplantation of GvH‐nonreactive (C57 * Balb)Fl bone marroe, neigher ex‐viro treatment with anti‐asialo GMl nor addition of 1 * 107 donor thymocytes to the allograft significantly influenced engraftment. The data obtained in our animal model suggest that the total number of marrow cells in of critical importance for successful marrow engraftment and not the presence of absence to T celld, NK cells or GvHR.
Unique immunophenotype of acute promyelocytic leukaemia as defined by CD9 and CD68 antibodies
Authors: Erber Wendy N. , Asbahr Heinke , Rule Simon A. , Scott Colin Stephen
Published: March 2008

Summary. Twenty‐one cases of acute promyelocytic leukaemia (FAB M3) demonstrating t(15,17) chromosomal translocation were studied in detail by immunocytochemical techniques using a panel of monoclonal antibodies. A characteristic myeloid phenotype of the leukaemic cells, co‐expression of CD9 and CD68 antigens and absence of HLA‐DR, was noted in all cases. Although the cellular morphology of acute promyelocytic leukaemia provides the most rapid means for diagnosis, this unique leukaemic cell phenotype provides confirmatory diagnostic evidence. In view of the new therapeutic options and prognosis in acute promyelocytic leukaemia, the detection of cases with atypical morphology based on this unique phenotype would be of value. In addition, the phenotype of acute promyelocytic leukaemia is similar to that of basophils and mast cells and raises the possibility that the leukaemic cells may have undergone a degree of basophilic differentiation.
Molecular detection of clonally rearranged cells in peripheral blood progenitor cell harvests from multiple myeloma patients
Authors: Bird J. M. , Bloxham D. , Samson Diana , Marcus R. E. , Russell N. H. , Kelsey S. M. , Newland A. C. , Apperley J. F.
Published: March 2008

Summary. Peripheral blood progenitor cells (PBPC) are increasingly used for autologous reconstitution following high‐dose chemotherapy in multiple myeloma but it is unclear whether these cells are less likely to be contaminated with malignant cells than bone marrow (BM). We have investigated this using immunoglobulin heavy‐chain (IgH) gene fingerprinting, a polymerase chain reaction based technique with a sensitivity of 0.1–0.01% (10‐3 ‐ 10‐4). We have looked for patient‐specific IgH rearrangements in leukapheresis samples from eight myeloma patients undergoing PBPC harvest. Seven were in first remission (six partial, one complete) and one in second complete remission. Mobilization of PBPC was accomplished using cyclophosphamide (4 or 7 mg/m2) and rhG‐ or GM‐CSF. Between two and five leukaphereses were performed in each patient. Patient‐specific IgH rearrangements were identified in diagnostic BM in all patients and bands of identical size were found in one or more leukaphereses from 6/8 patients. Overall, 14/32 leukaphereses were shown to be contaminated. Two patients who showed contamination of at least one PBPC harvest had BM harvests in which contaminating cells were not detectable, suggesting that PBPC are not necessarily less likely to be contaminated than marrow stem cells.

These results indicate that PBPC harvests from the majority of myeloma patients are likely to contain contaminating cells. Further studies are needed to determine whether these cells are clonogenic and whether they contribute to relapse.
A haemorrhagic platelet disorder associated with altered stimulus‐response coupling and abnormal membrane phospholipid composition
Authors: Cartwright I. J. , Hampton K. K. , Macneil S. , Colvin B. T. , Preston F. E.
Published: March 2008

Summary. Haemorrhagic diatheses due to platelet function defects are a heterogenous and poorly understood group of conditions. We report the investigation of a female with a lifelong history of epistaxes, haemarthroses, menorrhagia and persistent iron‐deficiency anaemia. Although platelet numbers and morphology were normal, platelet function was abnormal both in vivo and in vitro. Skin bleeding time was prolonged and aggregation thresholds in platelet‐rich plasma to a variety of weak and strong agonists were increased. Platelet granule contents were normal and membrane glycoproteins GpIb and GpIIIa were present in normal amounts. Polyphosphoinositide metabolism and phosphatidic acid generation were diminished in thrombinstimulated platelets, as was phosphorylation of the 47 kD substrate for protein kinase C and the 20 kD protein myosin light chain kinase, indicating impaired generation of the intracellular second messengers diacylglycerol and inositol trisphosphate due to diminished stimulated phospholipase C activity. Although intracellular free calcium, calmodulin activity and basal cAMP concentrations were normal, washed platelets showed increased cAMP accumulation following stimulation with prostaglandin E1 and forskolin. Platelet membrane lipid analysis revealed a reduction in plasmalogen phosphatidylethanolamine content. It is suggested that the membrane phospholipid abnormalities cause the abnormal platelet reactivity by interfering with signal transduction from platelet receptor, via intermediary G proteins, to phospholipase C and adenylate cylase. The bleeding tendency is likely to be a consequence of the altered stimulus‐response coupling.
Characterization of human blood group scFv antibodies derived from a V gene phage‐display library
Authors: Hughes‐Jones N. C. , Gorick B. D. , Bye J. M. , Finnern R. , Scott M. L. , Voak D. , Marks J. D. , Ouwehand W. H.
Published: March 2008

Summary. We previously reported the initial characterization of five human single‐chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phageantibody library constructed from the variable region genes of two non‐immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti‐B and anti‐HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross‐reactivity with other red cell antigens, with the exception of the anti‐E which reacted weakly with E‐negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti‐D scFv recognized 17 600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 × 107m1 and a rate constant for dissociation (koff) of 1.9 × 102 s‐1. The anti‐E scFv recognized 29 800 and 39 800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 × 106 and 4.4 × 107m‐1. The koff for this antibody was 2.7 × 10‐2s‐1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affnities typical of the primary immune response can be obtained from a phage‐display library.
Predicting the severity of rhesus alloimmunization: monocyte‐mediated chemiluminescence versus maternal anti‐D antibody estimation
Authors: Buggins A. G. S. , Thilaganathan B. , Hambley H. , Nicolaides† K. H.
Published: March 2008

Summary. Anti‐D haemolytic antibody concentration and chemiluminescence (CLT) opsonic index was measured in maternal blood obtained from 20 alloimmunized pregnancies at 17–28 weeks undergoing intrauterine fetal blood sampling for the estimation of fetal haemoglobin concentration. The fetal haemoglobin concentration. The fetal haemoglobin concentration was significantly associated with the maternal serum CLT opsonic index (r =−0.566, P < 0.01) but not with the maternal anti‐D concentration (r =−0.329). The data of this study indicate that measurement of maternal serum CLT opsonic index may be more accurate than anti‐D quantification in providing non‐invasive prediction of the degree of fetal anaemia.
High‐dose gamma‐globulin therapy in the reactive haemophagocytic syndrome
Authors: Gill D. S. , Spencer A. , Cobcroft R. G.
Published: March 2008

Summary. Reactive haemophagocytic syndrome (RHS) is a disorder characterized by systemic proliferation of non‐malignant histiocytes occuring most commonly in patients with pre‐existing immunological abnormalities or neoplasma. Patients, particularly those with immunosuppression, often have a rapidly progressive fatal course. Treatment is directed at the underlying disorder. In the absence of identifiable cause, the therapy is less satisfactory. We report here three cases of RHS successfully treated with high‐dose gamma‐globulin therapy. Two of the three patients were immunocompromised and the third occurred during pregnancy. The improvement occurred within 24–72 h and all patients recovered. High‐dose i.v. gamma‐globulin therapy may be beneficial in RHS.
Cyclic haemopoiesis at 7‐ or 8‐day intervals
Authors: Sawamura Morio , Yamaguchi Sayuri , Murakami Hirokazu , Kitahara Tokuyuki , Itoh Katsuhiko , Maehara Tadashi , Kawada Etsuo , Matsushima Takafumi , Tamura Jun'ichi , Naruse Takuji , Tsuchiya Jun
Published: March 2008

Summary. We report a patient with severe anaemia and cyclic oscillations of reticulocyte and leucocyte counts, as well as serum iron (Fe), unsaturated iron‐binding capacity (UIBC), ferritin, C‐reactive protein (CRP) levels and temperature, at regular intervals of 7 or 8 d. After treatment with prednisolone, anaemia was corrected and the cyclic oscillations of these parameters ceased; whereas treatment with indomethacin, recombinant granulocyte‐colony stimulating factor (G‐CSF) and erythropoietin (Epo) were unsuccessful.
Serum levels of granulocyte‐colony stimulating factor (G‐CSF) in bacterial and viral infections, and in atypical pneumonia
Authors: Pauksen Karlis , Elfman Lena , Ulfgren Ann‐Kristin , Venge Per
Published: March 2008

Summary. Serum granulocyte‐colony stimulating factor (G‐CSF) was measured with an ELISA method in patients with acute bacterial and viral infections, or with and atypical pneumonia. Before initiation of antibiotic treatment, G‐CSF was found to be significantly increased (799 ± 1501 ng/1) in sera from 34 patients with and acute bacterial infection compared with the 27 patients with the 27 patients with viral infection (58 ± 34 ng/1; P < 0.001) and with the eight patients with an atypical pneumonia (60 ± 33) ng/1; P < 0.001). No significant difference in G‐CSF levels was seen between gram‐positive and gram‐negative bacterial infections. In septic shock, increased G‐CSF levels were seen both in patients with leucocytosis and leucopenia. In uncomplicated bacterial infections, both G‐CSF and IL‐6 were increased on day 0, and decreased rapidly after initiation of antibacterial therapy and before the patients became afebrile. In bacterial infections on day 0, G‐CSF levels correlated with mononuclear cells (rs=−0.62, p < 0.001), IL‐6 (rs= 0.40, P < 0.05 and S‐MPO (rs=−0.5, P < 0.01). In viral infections, G‐CSF was correlated with mononuclear cells (rs= 0.041, P < 0.05), White blood cell counts (rs= 0.56, P < 0.01), neutorphils (rs= 0.41, P < 0.05) and CRP (rs= 0.47, P < 0.05). We conclude that G‐CSF is rapidly rised in the blood in acute baterica infections but not in acute viral infections or in infections with Mycoplasma pneumonia. Our results also support the theory that G‐CSF is involved in the mechanisms of mobilization of neutrophils into the peripheral circulation.
Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia
Authors: Schrezenmeier Hubert , Noe Gudrun , Raghavachar Aruna , Rich Ivan N. , Heimpel Hermann , Kubanek Bernhard
Published: March 2008

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron‐deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron‐deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti‐thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre‐treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.

Results of this study argue against the model of a simple feedback regulation of EPO via hypoxic anaemia. Our data support the hypothesis that cytokines and the erythropoietic progenitor pool are involved in the regulation of EPO production. The results illustrate that serial measurements of EPO along with therapeutic interventions are necessary to identify patients who might benefit from treatment with exogenous recombmant human EPO.
Red cell membrane protein abnormalities in hereditary spherocytosis in Brazil
Authors: Saad Sara T. O. , Costa Fernando F. , L Dilmara , Tereza Vicentim , Salles S. I. , Pranke Patricia H. L.
Published: March 2008

Summary. We studied 14 kindred and nine unrelated patients from southeastern Brazil with the typical form of hereditary spherocytosis. Diagnosis was made on the basis of clinical features, presence of spherocytes on the peripheral blood smears and an abnormal osmotic fragility test. By densitometric tracing of SDS‐PAGE stained by Coomassie blue, we detected isolated deficiency of spectrin in 39% of our patients, combined spectrin and ankyrion deficiency in 13% and deficiency of band 3 in 13%. One of our patients presented ankyrin deficiency without spectrin reduction. Our data suggest that, despite ethnic differences among the Brazilian and European or North‐American populations, these biochemical abnormalities in HS patients may be similar.
Translocation t(5;12)(q31‐q33;p12‐p13): a non‐random translocation associated with a myeloid disorder with eosinophilia
Authors: Baranger Laurence , Szapiro Nathalie , Gardais Jacques , Hillion Josette , Derre Josette , Francois Sylvie , Blanchet Odile , Boasson Marc , Berger Roland
Published: March 2008

Summary. A t(5;12)(q33;p13) translocation has been detected in two patients with myeloid disorder and eosinophilia. Six other patients with haematological disease with eosinophilia with similar translocation have been published previously. The existence of a new entity, a myeloproliferative disorder with eosinophilia and t(5;12) (q31‐q33;p12‐p13), is suggested by the results of the present study.
CD30‐positive lymphoma in human T‐cell leukaemia virus type 1 carrier without monoclonal integration of HTLV‐1
Authors: Hori Mitsuo , Mitsuhashi Shouichi , Kobayashi Toshitaka , Nagasawa Toshiro , Mori Naoyoshi , Miwa Masanao , Abe Tsukasa
Published: March 2008

Summary.CD30, Ki‐1 antigen, an activated T‐cell antigen, is a member of the nerve growth factor receptor family. This antigen is expressed on the lymphoma cells of some adult T‐cell leukaemia/lymphoma (ATL/L) patients and some patients with Epstein‐Barr virus infection. CD30‐positive large cell cutaneous T‐cell lymphomas occasionally integrate a defective HTLV‐1 provirus. We describe here an HTLV‐1 carrier who developed Ki‐1 lymphoma with no evidence of monoclonal integration of the HTLV‐1 proviral sequence.
PML‐RARα PCR positivity in the bone marrow of patients with APL precedes haematological relapse by 2–3 months
Authors: Korninger L. , Knöbl P. , Mustafa S. , Quehenberger P. , Schwarzinger I. , Lechner K. , Jaeger U. , Mannhalter C.
Published: March 2008

Summary. We report the results of serial tests in three patients with hyperleucocytotic AML M3 by haematological methods, coagulation assays and PCR analyses following treatment with ATRA and chemotherapy. All three patients became PCR negative in bone marrow and peripheral blood (sensitivity level 1 in 105 cells) after one cycle of ATRA+chemotherapy. However, they relapsed haematologically 6–9 months after achieving complete haematological remission. The haematological relapse was precded by PCR positivity in the bone marrow by 3 months. Platelet counts decreased already during CHR, but dropped below normal levels only at the time of relapse. Coagulation parameters were not helpful for early prediction of relapse. Our results demonstrate that PCR analysis in the bone marrow is the best way to monitor patients with APL. However, in hyperleucocytotic patients, PCR negativity does not seem to indicate long‐term remission.