bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting
Authors: Davies S. V. , Cavill I. , Bentley N. , Fegan C. D. , Poynton C. H. , Whittaker J. A.
Published: March 2008

Erythroid regeneration is an important and separate element in the engraftment process in allogeneic and autologous bone marrow transplantation (alloBMT, autoBMT). Qualitative visual reticulocyte counting has proved inadequate in the evaluation of erythropoiesis after BMT but automated flow cytometry now allows the reliable quantitation of reticulocytes even to very low levels. Reticulocyte counts and highly fluorescent reticulocyte (HFR) counts (very early reticulocytes) were estimated daily in recipients of 22 autoBMT and 14 alloBMT using a Sysmex R‐1000 automated reticulocyte counter. Marrow ablation caused an immediate and rapid fall in both the reticulocyte count and the HFR. Measurable numbers of reticulocytes persisted throughout the hypoplastic period, but HFR fell to zero in the majority of both the autoBMT and alloBMT. HFR rose significantly after a median time of 14 d post‐autoBMT, and 12 d post‐alloBMT. Attainment of 15 × 109/l reticulocytes and 0·5 × 109/l HFR at day 21 post‐transplant was associated with ultimate engraftment in 100% cases. Inadequate engraftment was seen in the majority of patients whose responses fell below these levels. Graft‐versus‐host disease was associated with a transient slight reduction in reticulocyte count. Neither episodes of infection nor blood transfusions had any significant impact on trends of reticulocytes or HFR.

Automated flow cytometric reticulocyte counting has been shown to provide an accessible measure of erythroid activity which may be of predictive value in the management of patients following bone marrow transplantation.
Ultraviolet irradiation modulates MHC‐alloreactive cytotoxic T‐cell precursors involved in the onset of graft‐versus‐host disease
Authors: van Prooijen H. C. , Aarts‐Riemens M. I. , Grijzenhout M. A. , Weelden H. van
Published: March 2008

Ultraviolet B (UVB) irradiation of cellular blood components has been proposed as a new technology to prevent HLA sensitization in recipients. Earlier studies have shown that a dose of 2 J/cm2 abrogates the ability of lymphocytes to serve as stimulators in mixed lymphocyte cultures (MLC). In this study we have evaluated the effect of UV energy on T‐lymphocytes for the prevention of transfusion‐associated graft‐versus‐host disease (TA‐GvHD). The response of cytotoxic T‐lymphocyte precursors against host alloantigens was almost undetectable at a dose of 0·5 J/cm2. T‐cell proliferation in MLC or in response to phytohaemagglutinin was inhibited by more than 95% at doses of 1 J/cm2 or higher. The data suggest that UV irradiation can be used to prevent both HLA sensitization and TA‐GvHD in recipients.
Book Review
Authors: Worwood M.
Published: March 2008

Book Reviewed in this article

Inorganic Biochemistry of Iron Metabolism.
Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells
Authors: Marie Jean‐Pierre , Brophy Nathalie A. , Ehsan Mohamed N. , Aihara Yukoh , Mohamed Named A. , Cornbleet Joanne , Chao Nelson J. , Sikic Branimir I.
Published: March 2008

The multidrug resistance gene mdr1, encoding P‐glycoprotein (P‐gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene. In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.). To detect cells expressing mdr1 gene in normal and post‐chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection. The presence of P‐gp was evaluated by immunocytochemistry with MRK16. Fifteen b.m. (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.). With in situ mRNA hybridization, a subset (7.7%×3.1%) of b.m. cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m. and post chemotherapy b.m. 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA. By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m. expressed mdr1. MRK16 was unable to detect a significant number of cells expressing P‐gp either by immunocytochemistry in the 12 b.m. tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m. (0–1.4% positive cells).
Abnormal CD45R expression in patients with common variable immunodeficiency and X‐linked agammaglobulinaemia
Authors: Richards Stephen J. , Scott Colin Stephen , Cole June C. , Gooi H. C.
Published: March 2008

This study has investigated the patterns of membrane 2H4 (CD45RA) andUCHL1 (CD45RO) expression by CD4+ and CD8 + lymphocyte subpopulations in 10 adults and seven children with common variable immunodeficiency (CVI) and X‐linked hypogammaglobulinaemia (XLA). Of the 10 adults (CVI, n= 8; XLA, n= 2), only one with a diagnosis of CVI showed normal CD45R expression. For the remaining seven adult CVI patients the abnormal CD45R profiles were primarily associated with CD4+ lymphocytes in three and CD8+ lymphocytes in four. Specific increases in CD45RA‐CD45RO+ fractions were found in four CVI patients and in all of these there was a concomitant reduction in circulating CD19+ B‐cell numbers. Two of the CVI cases were identical twin sisters and both had the same CD45R abnormality. The two adults with XLA also showed abnormal CD45R expression (increased CD45RA+CD45RO∼ components) but of particular note, in view of the fact that these were brothers, one was associated with the CD4+ subpopulation and the other with the CD8+ fraction. Similar analyses for the paediatric group revealed, in distinct contrast to the adult patients, no significant abnormalities of CD45R expression suggesting that these defects may not become apparent until a later age. Further investigations of in vitro lymphocyte proliferation (PHA and PWM) in the eight adults with CVI showed a significant correlation between abnormal responses and disordered CD45R expression. It is proposed that these abnormal patterns of CD45R expression by lymphocyte subpopulations in CVI and XLA may be of fundamental importance with respect to the pathogenesis of these particular immunodeficiencies.
The value of detecting surface and cytoplasmic antigens in acute myeloid leukaemia
Authors: Urbano‐Ispizua Alvaro , Matutes Estela , Villamor Neus , Sierra Jorge , Pujades Asumpció , Reverter Joan‐Carles , Feliu Evarist , Cervantes Francisco , Vives‐Corrons Joan‐Lluis , Montserrat Emilio , Rozman Ciril
Published: March 2008

The immunophenotype of leukaemia cells from 60 patients with acute myeloid leukaemia (AML) was analysed with the APAAP technique using a panel of anti‐myeloid and lymphoid associated monoclonal antibodies (McAb). Cells from all cases, including three with negative cytochemical features, were labelled by at least one of the anti‐myeloid McAb CD13, anti‐myeloperoxidase (anti‐Mpo), and/or CD14. The most sensitive marker was GD13, since it was positive in 90% of cases. In two out of three AML cases defined as MO‐AML, CD13 was expressed in the cytoplasm but not on the membrane; in these three cases peroxidase (Mpo) was not detected by conventional cytochemistry, but could be demonstrated in all of them using the McAb anti‐Mpo. The simultaneous expression of GD14 and CD68 McAb was often confined to the M4 and MS FAB AML subtypes (92% cases) as compared to the others: Ml, M2, M3 (18% cases). Lymphoid antigens were rarely positive (TdT +: 13%, CD7 +: 15%, CD19 +: 5%) and none of the AML cases were CD3+ or CD10 +. By contrast, CD4 was expressed in blasts from 44% of cases and this was not restricted to AML with a monocytic component (M4, M5) but also found in other subtypes. There were no significant differences in the clinical or prognostic features according to the positivity or negativity with TdT and CD4. By contrast, expression of CD7 was associated with refractoriness to the treatment or short complete remission duration, although the number of patients is too small to draw firm conclusions. Our findings support the clinical and diagnostic relevance of immunophenotypic studies in AML.
Monitoring circulating B cells in patients with multiple myeloma at diagnosis or in plateau phase: how prevalent is light chain isotype suppression?
Authors: King Malcolm A. , Radicchi Monica A.
Published: March 2008

Peripheral blood lymphocytes from 25 patients with multiple myeloma at diagnosis or in plateau phase, and from 18 healthy subjects, were analysed for the surface expression of CD19 and immunoglobulin (Ig) light chain isotypes by dual parameter flow cytometry. The aim was to test for the existence of light chain isotype suppression (LCIS). LCIS is a hypothetical suppressor mechanism which supposedly operates in myeloma patients with stable disease, causing a drop in the number of circulating B cells bearing the same Ig light chain isotype as the myeloma paraprotein. Our experiments emphasized that the use of a non‐Ig, pan‐B cell marker, combined with dual antigen analysis, is necessary to distinguish Ig on B cells from (cytophilic) Ig on some non‐B cells. We found good correlation between results obtained using polyclonal versus monoclonal anti‐light chain antibodies, and washed blood versus peripheral blood mononuclear cells. There was no evidence of an abnormal ratio of CD19+, K+ to CD19+, λ+ cells in any of the patients. Thus, these patients did not manifest LCIS, and these experiments raise doubts about the existence of this phenomenon. The results also illustrate the problem that may arise in the interpretation of one parameter flow cytometry.
Measurement of free kappa and lambda chains in serum and the significance of their ratio in patients with multiple myeloma
Authors: Nelson M. , Brown R. D. , Gibson J. , Joshua D. E.
Published: March 2008

An inhibition enzyme‐linked immunoassay technique using commercially available antibodies has been developed for the quantitation of both kappa and lambda light chains in the serum of patients with B‐cell malignancies. Assay conditions were selected to enable measurement of free light chains in the concentration range between 0.1 and 20 mg/1. The normal range for free lambda chains in serum was found to be 0.4–4.2 mg/1 and for free kappa chains it was 1.6–15.2 mg/1. At diagnosis the serum of most patients with multiple myeloma contained increased levels of the malignant free light chain and in some cases there was also elevation of the non‐malignant light chain. The absolute level of the malignant light chain at diagnosis did not correlate with survival nor with laboratory parameters such as IgM or creatinine. A correlation with β2M and serum paraprotein levels was evident only in cases of IgA myeloma. Although the absolute level of free serum light chain had no value as a prognostic indicator, the ratio of kappa: lambda chains closely followed the clinical assessment of disease status, being near the normal range (1.2–9.1) in plateau phase or stable disease. During periods of progressive disease this ratio ranged from 19 to 460 (n=14) in patients with kappa myeloma, and 0.0013–0.14 (n= 9) in patients with lambda myeloma. Determination of the ratio of free light chains in the serum may allow effective monitoring and earlier warning of disease progression in patients with multiple myeloma.
Rapid detection of a protein C gene mutation present in the asymptomatic and not in the thrombosis‐prone lineage
Authors: Bernardi F. , Patracchini P. , Gemmati D. , Boninsegna S. , Guerra S. , Legnani C. , Ballerini G. , Marchetti G.
Published: March 2008

The presence of mutations in the serine protease domain of protein C was investigated by temperature gradient gel electrophoresis of PCR products in five patients with protein C deficiency and thrombosis. Molecules with an altered melting behaviour were detected in one subject with a history of venous and arterial thrombosis. Direct sequencing showed that a G deletion, present in the heterozygous state, caused a reading frame shift at Trp 300 and subsequently a premature termination at the codon 335. The resulting suppression of the protein C catalytic function explains the reduction of protease activity to half. In addition the mutation caused a reduction of the antigen level in plasma. Temperature gradient gel electrophoresis enabled the rapid detection of the gene alteration in the family of the propositus. Several members of the paternal lineage had had severe thrombotic episodes. Unexpectedly the mutation was found to be inherited from the clinically asymptomatic maternal lineage, thus suggesting that an additional unknown defect from the paternal lineage is present in the thrombosis‐prone propositus.
A β‐thalassaemia phenotype not linked to the β‐globin cluster in an Italian family
Authors: Murru S. , Loudianos G. , Porcu S. , Sciarratta G. V. , Agosti S. , Parodi M. I. , Cao A. , Pirastu M.
Published: March 2008

Summary. This paper describes a family of Central Italian origin in which three patients in two generations had either thalassaemia intermedia or a late presenting form of thalassaemia major. Sequence analysis of the patients’ UNA revealed that only one of the β‐globin genes was affected by a β‐thalassaemia mutation (the codon 39 nonsense mutation), the other being completely normal, apart from the complex rearrangement (‐T + ATA) at position −5305’ to the CAP site of the β‐globin gene, which has uncertain clinical significance. Haematologically, all these patients were characterized by unusually low HbF levels (1.8–7.3%) for a β‐thalassaemia major or intermedia phenotype. The mother of the two patients with thalassaemia intermedia was heterozygous for β‐thalassaemia (codon 39 nonsense mutation), while the father had thalassaemia‐like red cell indices, an increased α/nonα chain synthesis ratio, a slight increase of HbF and a low HbA2 level, but showed entirely normal β‐globin gene sequences, apart from the complex rearrangement (‐T +ATA) at position −530 5’ to the CAP site. One of the thalassaemia intermedia patients married a normal woman and they had a child with thalassaemia major who inherited only the codon 39 nonsense mutation but not the complex rearrangement at position −530. The clinical phenotype of thalassaemia‐intermedia or major in the patients from this family may be explained by postulating the inheritance of the double heterozygous state for β‐thalassaemia and for a mutation in a gene coding for an erythroid‐specific DNA binding protein which may impair the function of the normal β‐globin gene. Heterozygosity for this postulated mutation (father of the patients with thalassaemia intermedia) may result in the production of a β‐thalassaemia carrier state with normal HbA2 level.
The in vitro effect of high‐dose recombinant human erythropoietin on granulocyte‐macrophage colony production in premature infants using a defined serum deprived cell culture system
Authors: Ward C. S. , Westwood N. B. , Emmerson A. J. B. , Pearson T. C.
Published: March 2008

Recent reports of neutropenia associated with the use of recombinant human erythropoietin (r‐HuEpo) in preterm infants with the anaemia of prematurity have raised concern over the clinical use of this hormone. The present studies were undertaken to determine whether high‐dose r‐HuEpo has an effect on granulocyte production in vitro. The studies used a serum deprived, optimized semi‐solid cell culture system to investigate the effect of lineage specific and non‐specific granulocyte and erythroid colony stimulating factors on circulating peripheral blood granulocyte‐macrophage colony forming units (CFU‐GM), erythroid burst forming units (BFU‐E) and multilineage colonies (CFU‐Mix) from nine premature infants and seven healthy adults. CFU‐GM were grown in the presence of interleukin 3 (IL3) 8 ng/ml, granulocyte‐macrophage colony stimulating factor (GM‐CSF) 20 ng/ml and granulocyte colony stimulating factor (G‐CSF) 15 ng/ml alone and combinations of G‐CSF with GM‐CSF or IL3. The number, size and differentiation of CFU‐GM colonies were then analysed in the presence and absence of high dose r‐HuEpo (4 U/ml).

High‐dose r‐HuEpo did not exert any significant modulatory effects on the number of CFU‐GM colonies produced in the presence of IL3, GM‐CSF and G‐CSF alone or in combination. The number of cells within each CFU‐GM colony did not change significantly, nor was there a significant change in the degree of differentiation. The combined number of BFU‐E. CFU‐GM and CFU‐Mix colonies increased with r‐HuEpo in both adults (1·8x) and preterm infants (1·4x), almost exclusively due to an increase in BFU‐E derived colonies. Thus, no evidence was found for an r‐HuEpo mediated redirection of multipotential haemopoietic stem cells into committed erythroid precursors at the expense of myeloid precursors.
Bone marrow involvement in non‐Hodgkin's lymphoma: increased diagnostic sensitivity by combination of immunocytology, cytomorphology and trephine histology
Authors: Schwonzen M. , Pohl C. , Steinmetz T. , Wickramanayake P. D. , Thiele J. , Diehl V.
Published: March 2008

Diagnostic results from cytomorphology and immunocytology of aspirated bone marrow (BM) were compared with the findings from standard trephine histology of 100 adult patients with non‐leukaemic non‐Hodgkin's lymphomas (NHL) in a retrospective study. Immunocytological investigations were performed by the immunoenzymatic APAAP‐technique on BM smears monoclonal antibodies against CD19, CD3, CD10 or TdT antigens and determination of positive cells in relation to total BM leucocytes. Corresponding results were obtained for trephine histology and for the combination of cytomorphology and immunocytology in 93/100 cases. Four cases with BM involvement by trephine histology were missed by the combination of immunocytology and cytomorphology. In turn, three cases negative by trephine histology, were found to be positive by the combination of immunocytology and cytomorphology. Immunocyto‐chemistry considerably increased the number of true positive detected BM‐infiltrations by cytomorphology in low grade B‐cell lymphoma from 58% to 97%. For the diagnosis of BM involvement in high‐grade NHL cytomorphology of the aspirate was of equal sensitivity to the biopsy and was always confirmed by immunocytology. The high diagnostic sensitivity of immunocytology was mainly due to high B‐cell counts in BM involved by B‐cell lymphoma (x=38%, s= 23) in contrast to low B‐cell counts in BM not involved by NHL (x=4·5%, s= 3·8). We conclude from our data that immunocytology in addition to standard cytomorphology improves diagnostic sensitivity in the detection of BM involvement by NHL.
Platelet alloimmunization after multiple transfusions: a prospective study of 50 patients
Authors: Godeau Bertrand , Fromont Patricia , Seror Thérese , Duedari Najib , Bierling Philippe
Published: March 2008

Fifty polytransfused patients were prospectively studied to determine the frequency of post‐transfusion alloimmunization and its influence on the response to platelet transfusion. Platelet‐ and HLA‐specific antibodies were detected by means of the standard and antiglobulin‐augmented lymphocytotoxicity techniques (LCT), the platelet suspension indirect immunofluorescence test (PSIIFT), and monoclonal antibody immobilization of platelet antigens (MAIPA). HLA antibodies were detected in 13 patients (26%) (IgM = 6; IgG = 6; IgM + IgG = 1). The standard LCT was positive in 12 of these 13 patients. Complement‐independent HLA antibodies, only detectable in the PSIIFT and the antiglobulin‐augmented LCT, were documented in two patients and were associated with poor post‐transfusion platelet recovery in the patient who could be evaluated. All the HLA antibodies were detected in the PSIIFT, while only four were detected in the MAIPA. Platelet‐specific alloantibodies were found in two patients by means of PSIIFT or MAIPA and may have led to poor post‐transfusion platelet recovery in one patient. Platelet autoantibodies were detected in two patients but were not associated with platelet refractoriness. Paraformaldehyde‐dependent platelet antibodies were detected in 11 patients but were not associated with platelet refractoriness.
Human parvovirus B19 in clotting factor concentrates: B19 DNA detection by the nested polymerase chain reaction
Authors: Zakrzewska K. , Azzi A. , Patou G. , Morfini M. , Rafanelli D. , Pattison J. R.
Published: March 2008

The presence of B19 parvovirus in plasma from blood donors is seldom demonstrable, but clotting factor concentrates, prepared from large plasma pools, may be able to transmit B19 virus infection, and the effectiveness of different chemical and physical treatment to inactivate this virus is not yet known.

In this study we report on the detection of B19 DNA in 25 clotting factor concentrates, prepared by a variety of procedures of purification and inactivation; dot blot hybridization and Southern blot hybridization assays, as well as a ‘nested’polymerase chain reaction (PCR) have been employed. Nine out of 25 products were B19 DNA positive by PCR, whereas only two gave positive results by hybridization techniques. B19 DNA positive concentrates have been found in ‘untreated’products but also in some solvent/detergent or steam‐treated products and even in monoclonal purified concentrates.

PCR may be useful for the screening of blood products to be used in immunocompromised haemophiliacs, particularly in HIV positive subjects, at risk of severe chronic anaemia following B19 infection.
Substituted benzaldehydes (12C79 and 589C80) that stabilize oxyhaemoglobin also protect sickle cells against calcium‐mediated dehydration
Authors: Stone P. C. W. , Nash G. B. , Stuart J.
Published: March 2008

Reversibly sickled cells from patients with homozygous sickle‐cell disease were prepared by Percoll–Isopaque density gradient separation and subjected to 15 h of cyclical deoxygenation–reoxygenation in the presence of Ca. After 15 h the sickle cells became dehydrated, losing volume secondary to K efflux via the Ca‐activated (Gardos) channel, and showed impaired filterability through 5 μm diameter pores. The substituted benzaldehydes 12C79 and 589C80, which stabilize the oxy‐conformation of sickle haemoglobin, showed an additional protective effect at pharmacological concentration by maintaining the K concentration, mean cell volume, and deformability of sickle cells. Drugs that increase the oxygen affinity of sickle haemoglobin may be more effective than specific inhibitors of Ca entry or K efflux in preserving the cation homeostasis and deformability of sickle cells during sickling in vivo.
Desferrithiocin is an effective iron chelator in vivo and in vitro but ferrithiocin is toxic
Authors: Baker E. , Wong A. , Peter H. , Jacobs A.
Published: March 2008

The efficacy and toxicity of the siderophore desferrithiocin (DFT), which has shown potential application in iron chelation therapy, were assessed in vivo and in vitro. DFT was evaluated in vivo in two ways: firstly, by measuring the effect of a single dose of DFT (10–100 mg/kg) on 59Fe excretion in iron‐loaded rats labelled with 59Fe; and secondly, by examining the effect of the daily oral administration for 2 weeks of DFT (10–25 mg/kg/d) on the growing rat. DFT and its ferric complex, ferrithiocin (FT), were assessed in vitro from their effects on transferrin and iron uptake and mobilization from rat hepatocytes in culture using transferrin doubly labelled with 125I and 59Fe. Both oral and subcutaneous DFT were highly effective in promoting iron excretion in vivo, but showed evidence of toxicity after oral administration for 2 weeks at 25 mg/kg/d. In addition, DFT was much more effective than desferrioxamine or pyridoxal isonicotinyl hydrazone in reducing hepatocyte iron in vitro. However, FT was cytotoxic, causing membrane disruption and release of intracellular aspartate aminotransferase. It was concluded that DFT should not be considered for chronic iron chelation therapy without extensive further evaluation.
Rare occurrence of P53 gene mutations in multiple myeloma
Authors: Preudhomme Claude , Facon Thierry , Zandecki Marc , Vanrumbeke Mickael , Laï Jean Luc , Nataf Eric , Loucheux‐Lefebvre Marie Henriette , Kerckaert Jean Pierre , Fenaux Pierre
Published: March 2008

We looked for mutations of exons 5–8 of the P53 gene in bone marrow cell from 37 cases of multiple myeloma, using polymerase chain reaction–single strand conformation polymorphism (PCR‐SSCP) analysis and DNA sequencing. 25 patients also had cytogenetic analysis. A point mutation, leading to an amino acid change in the P53 protein was found in only one case, involving exon 5. These findings suggest that P53 mutations are very rare in multiple myeloma, and that this disease may be categorized among the few neoplasms where P53 abnormalities have very limited role, if any.
Effect of recombinant human interleukin‐11 on rat megakaryopoiesis and thrombopoiesis in vivo: comparative study with interleukin‐6
Authors: Yonemura Yuji , Kawakita Makoto , Masuda Tetsuya , Fujimoto Kouji , Takatsuki Kiyoshi
Published: March 2008

Summary. The ability of recombinant human interleukin‐11 (IL‐11) to stimulate rat megakaryopoiesis and thrombopoiesis in vivo was investigated. Once daily subcutaneous injections of IL‐11 at doses of 2, 8 and 20 μg/rat for 5 d caused dose‐dependent increases in platelet counts. The chronic administration of 20 μg/rat/d for 14 d resulted in biphasic increases in platelet counts with peaks at days 8 and 15 of up to 30% over the control, continuing for more than 5 d after cessation of IL‐11 injections. Moreover, a striking increase in megakaryocytic size and ploidy in bone marrow in response to IL‐11 was elicited. IL‐11 induced a dose‐dependent elevation in bone marrow cell numbers but not in splenic weight and cell numbers. Modifications of these parameters were noted as soon as 24 h after the first IL‐11 injections. IL‐11 had a same potency of thrombopoietic effect in rats as compared with IL‐6. However, elevation of acute phase protein such as immunosuppressive acidic protein was 2–2‐fold in rats given 20 μg/d of IL‐6 over those receiving a same dose of IL‐11 (470 v 210 μg/ml). In addition, the rate of body‐weight increase in rats receiving IL‐11 for 5 d as well as 14 d did not differ from that in control animals. In IL‐6 treated rats, the increase in body weight was significantly slower than the controls, which was observed even in the group given 8 μg/d of IL‐6. These results suggest that IL‐11 may be an effective strategy for the treatment of thrombocytopenia.
Kinetics of circulating haematopoietic progenitors during chemotherapy‐induced mobilization with or without granulocyte colony‐stimulating factor
Authors: Liu Kai‐Yan , Akashi Koichi , Harada Mine , Takamatsu Yasushi , Niho Yoshiyuki
Published: March 2008

Summary. Kinetics of circulating haematopoietic progenitors was analysed during chemotherapy‐ or chemotherapy plus granulocyte colony‐stimulating factor (G‐CSF)‐induced mobilization of peripheral blood stem cells. Circulating progenitors including colony‐forming unit granulocyte/macrophage (CFU‐GM), burst forming‐unit erythroid (BFU‐E) and multilineage colony forming unit (CFU‐Mix) were studied serially on alternate days during a recovery phase from chemotherapy for consolidation of complete remission. In 18 patients with acute leukaemia, 27 courses of consolidation chemotherapy were performed with a combination of an intermediate‐dose cytosine arabinoside with etoposide (Ara‐C/Etop) or mitoxantron (Ara‐C/Mit). G‐CSF (5 μg/kg) was administered during the recovery phase in 6/14 courses with Ara‐C/Etop and in 4/13 courses with Ara‐C/Mit. G‐CSF induced a significant and synchronized increase of circulating CFU‐GM, BFU‐E and CFU‐Mix by more than 4‐fold at their peaks. The peak of CFU‐GM was significantly correlated with that of both BFU‐E and CFU‐Mix, irrespective of additional G‐CSF mobilization. G‐CSF also produced a significant increase of monocytes in a synchronized fashion with an increase of circulating CFU‐GM. Interestingly, peripheral blood monocytes spontaneously produced high concentrations of IL‐6; a significant correlation was observed between absolute monocyte counts and plasma levels of IL‐6 or peak levels of CFU‐GM.

These observations indicate that the addition of G‐CSF to chemotherapy‐induced mobilization can facilitate further expansion of a blood progenitor pool during the haematopoietic recovery, probably through the stimulation of monocytes to proliferate and to induce their monokine production such as IL‐6. The data also suggest that absolute monocyte counts may be a useful indicator to predict the peak of circulating progenitors for collecting autologous blood stem cells.
Acute biphenotypic leukaemia: immunophenotypic and cytogenetic analysis
Authors: Hanson Curtis A. , Abaza Mohamed , Sheldon Susan , Ross Charles W. , Schnitzer Bertram , Stoolman Lloyd M.
Published: March 2008

Summary. The incidence of acute biphenotypic leukaemia has ranged from less than 1% to almost 50% in various reports in the literature. This wide variability may be attributed to a number of reasons including lack of consistent diagnostic criteria, use of various panels of antibodies, and the failure to recognize the lack of lineage specificity of some of the antibodies used. The morphology, cytochemistry, immunophenotype and cytogenetics of acute biphenotypic leukaemias from our institution were studied. The diagnostic criteria took into consideration the morphology of the analysed cells, light scatter characteristics, and evaluation of antibody fluorescence histograms in determining whether the aberrant marker expression was arising from leukaemic blasts or differentiated bone marrow elements. Fifty‐two of 746 cases (7%) fulfilled our criteria for acute biphenotypic leukaemias. These included 30 cases of acute lymphoblastic leukaemia (ALL) expressing myeloid antigens, 21 cases of acute myelogenous leukaemia (AML) expressing lymphoid markers, and one case of ALL expressing both B‐ and T‐cell associated antigens. The acute biphenotypic leukaemia cases consisted of four major immunophenotypic subgroups: CD2± AML (11), CD19± AML (8), CD13 and/or CD33± ALL (24), CD11b± ALL (5) and others (4). Chromosomal analysis was carried out in 42/52 of the acute biphenotypic leukaemia cases; a clonal abnormality was found in 31 of these 42 cases. This study highlights the problems encountered in the diagnosis of acute biphenotypic leukaemia, some of which may be reponsible for the wide variation in the reported incidence of this leukaemia. We suggest that the use of strict, uniform diagnostic criteria may help in establishing a more consistent approach towards diagnosis of this leukaemic entity. We also suggest that biphenotypic leukaemia is comprised of biologically different groups of leukaemia based on immunophenotypic and cytogenetic findings.
Differentiation and multidrug resistance in response to drug treatment in the K562 human leukaemia cell line
Authors: Marks Denese C. , Davey Mary W. , Davey Ross A. , Kidman Antony D.
Published: March 2008

Summary. The relationship between differentiation and P‐glycoprotein expression in response to chemotherapeutic drugs was studied in the K562 human leukaemia cell line by treatment with low, but clinically achievable levels of vinblastine and epirubicin. Resistant sublines were easily generated with the multidrug resistant phenotype being expressed in response to drug treatment as low as 1 ng/ml vinblastine and 10 ng/ml epirubicin. These sublines showed stable but heterogeneous expression of P‐glycoprotein as revealed by immunocytochemistry, and confirmed by cloning. This heterogeneity was maintained over 18 months with intermittent drug treatment. While selection for resistance induced erythroid and myeloid differentiation, expression of P‐glycoprotein was not correlated with the stem cell antigen CD34 or with specific markers of erythroid or myeloid differentiation.
The molecular basis of HPFH in a British family identified by heteroduplex formation
Authors: Craig J. E. , Sheerin S. M. , Barnetson R. , Thein S. L.
Published: March 2008

Summary. Single‐base substitutions in the immediate 5′‐flanking region of the fetal Gγ and Aγ globin genes have been associated with non‐deletional forms of hereditary persistence of fetal haemoglobin (HPFH). Previously, the sole promoter mutation associated with HPFH in British individuals has been the T to C substitution at position – 198 relative to the Aγ globin gene CAP site.

We have investigated a British family with Gγ HPFH and found a T to C substitution at position – 175 of the Gγ globin gene. The mutation was first detected by examining the amplified 5’regions of both the Gγ and Aγ globin genes for heteroduplex formation after electrophoresis in a Hydrolink gel. The potential of such a system for the study of sequence variations in the γ gene promoter regions associated with elevated HbF expression has been evaluated.

Previously reported cases of an identical mutation in an American‐Black and a Sardinian family display haematological phenotypes remarkably similar to that of the British family described here.
Erythrocyte autoantibodies, the monocyte monolayer assay and in vivo haemolysis
Authors: Zupanska B. , Sokol R. J. , Booker D. J. , Stamps R.
Published: March 2008

Summary. Monocyte monolayer assays (MMA) were performed using red cells (RBCs) from 159 patients with a positive direct antiglobulin test and monocytes from healthy individuals. The results of the MMA reflected, to a great extent, in vivo haemolysis; 56/63 patients with a positive result, but only 10/96 with a negative one, had evidence of in vivo RBC destruction. Positive MMAs were observed with autoantibodies of all types: warm (42 cases), cold (14 cases), mixed (seven cases), but never in patients with only C3d on their RBCs and no detectable autoantibodies. With warm autoantibodies, the MMA results depended, in part, on the level of IgG sensitization; additional C3d coating acted synergistically to lower the amount of IgG necessary for monocyte interaction to occur. The presence of IgG3 as well as IgG 1 on the RBCs was usually associated with a high MMA result; most of these cases, however, also exhibited larger amounts of cell‐bound IgG. When the MMA results were analysed in respect of the various types of autoantibodies, differences in the phagocytosis: adherence ratio were observed; in patients with warm autoantibodies, there was a preponderance of phagocytosis, whereas in those with cold and mixed types, adherent RBCs predominated or were found alone.
Normal individuals with high Hb A2 levels
Authors: Gasperini D. , Cao A. , Paderi L. , Barella S. , Paglietti E. , Perseu L. , Loi D. , Galanello R.
Published: March 2008

Summary. Increased haemoglobin (Hb) A2 levels associated with reduced mean corpuscular volume (MCV) and Hb content per cell (MCH) are the most typical features of heterozygous β thalassaemia. However, double heterozygotes for α and β thalassaemia may have normal MCV and MCH but Hb A2 always in the carrier range. In this report we describe two Sardinian families who have increased Hb A2 levels, normal red blood cell indices and normal globin chain synthesis and in whom DNA sequence analysis of β and δ globin genes did not reveal any abnormality. Our findings demonstrate the existence of a genetic trait not resulting from a defect of the β globin gene cluster, transmitted in a dominant manner and manifested as isolated increase of Hb A2.
Identification of IgG Fc receptor type II on human megakaryoblastic cell lines (MEG‐01 and UT‐7)
Authors: Wu Zhanhe , Chesterman Colin N. , Chong Beng H.
Published: March 2008

Summary. In this study, Fcγ receptors (FcγR) on the cells of human megakaryoblastic leukaemia cell lines (Meg‐01 and UT‐7) were investigated. Binding of the anti‐FcγRII monoclonal antibody (MoAb) IV. 3 but not anti‐FcγRI MoAb (32.2), anti‐FcγRIII MoAb (3G8) nor control murine IgGs to Meg‐01 and UT‐7 cells was demonstrated by immunocytochemical staining and flow cytometric analysis. There was specific and saturable binding of 125I‐labelled IV.3 Fab fragments to the megakaryoblasts but no binding of 125I‐labelled 32.2 Fab nor 3G8 Fab fragments. MoAb IV.3 immunoprecipitated a 40 kD protein (FcγRII) from detergent cell lysates of surface radio‐labelled Meg‐01 and UT‐7 cells. More importantly, IV.3 Fab almost completely inhibited the binding of 125I‐labelled human aggregated IgG to the Meg‐01 cells. In contrast, neither 32.2 Fab, 3G8 Fab nor Fab fragments of the control murine IgGs were inhibitory. These results indicate that only FcγR Type II is expressed on megakaryoblasts in these two cell lines.
Demonstration of a ubiquitin binding site on murine haemopoietic progenitor cells: implication of ubiquitin in homing and adhesion
Authors: Parakh K. A. , Kannan K.
Published: March 2008

Summary. Investigations into the nature of the molecular interactions mediating the recognition of the haemopoietic progenitor cells by the haemopoietic stroma, indicate that ubiquitin mediates the binding between murine haemopoietic progenitors and the haemopoietic stroma. The adhesion of haemopoietic progenitors to anti‐ubiquitin antibody treated bone marrow stromal cultures, shows inhibition of binding by approximately 78%. Affinity purification of the 1% Triton X‐100 soluble stromal membrane fraction, on anti‐ubiquitin‐sepharose revealed a ubiquitinated 55 kD subunit. Progenitor cells treated with ubiquitin show approximately 58% inhibition in their ability to home into spleen, indicating the direct involvement of ubiquitin in homing. Histochemical staining of bone marrow cells using ubiquitin as a probe further delineates a population of cells possessing specific binding sites for ubiquitin. We demonstrate here the presence of a ubiquitin binding site on the haemopoietic progenitor cells, which may play a major role in the targeting of such progenitors to their ‘niche’ within the haemopoietic tissue. Such ubiquitin‐mediated recognition may thus constitute a common molecular mechanism for homing and adhesion to both bone marrow and spleen, and may be implicated in the homing of more primitive, less differentiated haemopoietic progenitors. The results also indicate that the homing of haemopoietic progenitors within the haemopoietic micro‐environment may be mediated by both a ubiquitin dependent and another ubiquitin independent mechanism.
Clonal evolutions during long‐term cultures of bone marrow from de novo acute myeloid leukaemia with trilineage myelodysplasia and with myelodysplastic remission marrow
Authors: Tamura Shu , Kanamaru Akihisa , Takemoto Yoshinobu , Kakishita Eizo , Nagai Kiyoyasu
Published: March 2008

Summary. We previously established a long‐term bone marrow culture (LTBMC) system in which novel abnormal karyotypes could emerge in vitro prior to the appearance of the same karyotypes in vivo in patients with myelodysplastic syndrome (MDS). We extended our study to examine whether acute myeloid leukaemia (AML) transformed from MDS (MDS/AML) and de novo AML with trilineage myelodysplasia (AML/TMDS) show clonal evolution in LTBMC similar to that of typical AML or MDS. We also analysed the cytogenetic changes in cultures with bone marrows from AML with myelodysplastic remission marrow (AML/MRM) as well as chronic myeloid leukaemia (CML) to compare them with typical AML with respect to the liability of clonal evolution. Among the 34 AML cases, abnormal karyotypes were newly detected in four of seven MDS/AML, three of six AML/TMDS and three of three AML/MRM. Novel abnormal karyotypes were also observed in nine out of 13 CML cases after culture. In contrast, no other abnormal karyotypes were found after culture in 18 typical AML without myelodysplasia. These findings suggest that AML/TMDS and AML/MRM are different from typical AML and are similar to MDS/AML and CML in view of their potential for disease progression from latent multiple clones. Typical AML may develop from a single abnormal clone without any subclones.
Confirmation of viral safety of dry heated factor VIII concentrate (8Y) prepared by Bio Products Laboratory (BPL): a report on behalf of U.K. Haemophilia Centre Directors*
Authors: Rizza C. R. , Fletcher M. L. , Kernoff P. B. A.
Published: March 2008

Summary. Twenty‐seven factor VIII deficient patients who had previously not been treated with blood or blood products were studied after infusion of a total of 24 batches of NHS factor VIII (8Y) concentrate produced by Bio‐Products Laboratory, Elstree. Follow‐up was carried out according to guidelines laid down by the International Society for Thrombosis and Haemostasis.

Serial estimations of amino transferase level carried out over a 26‐week period revealed no elevation of these enzymes attributable to hepatitis. Studies of various virological markers found no evidence of infection with hepatitis C, hepatitis B or HIV following transfusion. This confirms a previous finding that severe dry heating of factor VIII at 80°C for 72 h seems to reduce the risk of transmitting hepatitis C from approximately 90% to a rate of 0–11%.
Pulmonary fibrosis with megakaryocytoid cell infiltration in accelerated phase of chronic myelogenous leukaemia
Authors: Yamauchi Kunihiko , Oda Koji , Shimamura Kazuo , Arimori Shigeru , Nagao Tadami
Published: March 2008

Summary. We report a patient in the accelerated phase of Philadelphia‐chromosome‐positive chronic myelogenous leukaemia who developed fibrosis in lungs, spleen and bone marrow. In the lungs, fibrosis was demonstrated in the alveolar septa which had been infiltrated by giant, bizarreshaped cells resembling the megakaryocytes in the bone marrow. The relationship between the fibrosis and megakaryocytoid cell infiltration in the lungs is discussed.
Gastro‐intestinal bleeding in acquired von Willebrand's disease: efficacy of high‐dose immuno‐globulin where substitution treatments failed
Authors: White Lucy A. , Chisholm Morag
Published: March 2008

Summary. We report a case of acquired von Willebrand's disease with severe and persistent gastro‐intestinal bleeding from multiple bleeding points. He received maximum substitution treatment including factor VIII concentrates, cryoprecipitate and platelets. He also received DDAVP. There was no change in the rate of blood loss.

He was then treated with high‐dose intravenous gammaglobulin and the blood loss stopped abruptly. He remained well for several months when bleeding recommenced and he again responded promptly to gamma‐globulin. This is a useful maintenance treatment for patients with acquired von Willebrand's disease.
Heparin‐induced skin reaction due to two different preparations of low molecular weight heparin (LMWH)
Authors: Phillips J. K. , Majumdar G. , Hunt B. J. , Savidge G. F.
Published: March 2008

Summary. Heparin‐induced skin reaction is a recognized complication of subcutaneously administered, unfractionated heparin, and has recently been described in association with the use of two LMWH preparations. Fragmin and Fraxiparin. We describe the case of a 25‐year‐old woman in whom characteristic skin lesions were induced on separate occasions by unfractionated heparin, Fragmin and a third preparation of LMWH, Clexane. This case demonstates that substitution of one preparation of heparin for another does not prevent recurrence of skin reaction in susceptible patients. Alternative anticoagulants should be considered in these cases.
Alpha interferon raises serum beta‐2‐microglobulin in patients treated for thrombocythaemia
Authors: Remes K. , Tienhaara A. , Pelliniemi T‐T.
Published: March 2008

Summary. Serum beta‐2‐microglobulin (b2m) levels were studied in 10 patients treated with alpha interferon (IFN) for thrombocythaemia associated with chronic myeloproliferative diseases. In eight patients the IFN treatment decreased the blood platelet level to <600 × 109/1. Serum b2m levels rose significantly and dose‐dependently immediately after the onset of treatment, by a median maximal increase of 72% with IFN 3 MU every other day, and 95% with 3 MU daily. The b2m rise caused by IFN abolishes the value of serum b2m in states where b2m is used as an indicator of disease activity.
Sweet's syndrome associated with myelodysplasia: possible role of cytokines in the pathogenesis of the disease
Authors: Reuss‐Borst M. A. , Pawelec G. , Saal J. G. , Horny H. P. , Müller C. A. , Waller H. D.
Published: March 2008

Summary. The clinical course of a 56‐year‐old female patient with Sweet's syndrome (SS) preceded by a myelodysplastic syndrome (MDS) is described. During the acute phase of the disease with high remittent fever, painful skin lesions and maximal leucocytosis IL‐6 and G‐CSF serum levels were extremely high, while TNF‐alpha was only slightly elevated and gamma‐interferon and IL1‐β were not increased. On clinical improvement IL‐6 serum levels rapidly fell, whereas G‐CSF values already slightly elevated before the manifestation of the disease slowly declined.

High G‐CSF levels triggered by a yet unknown factor could explain the leucocytosis, neutrophilic dermatosis and skin lesions in SS, while IL‐6 probably induced the associated clinical symptoms of fever and pain.
Analysis of rearranged immunoglobulin genes indicating a process of clonal evolution in chronic lymphocytic leukaemia
Authors: Hakim Itzchak , Rechavi Gideon , Brok‐Simoni Frida , Grossman Zehava , Amariglio Ninetta , Mandel Mathilda , Ramot Bracha , Ben‐Bassat Isaac , Katzir Nurit
Published: March 2008

Summary. Chronic lymphocytic leukaemia (CLL) is known to be a stable monoclonal neoplasm. In contrast to early studies demonstrating no more than two hybridizing immunoglobulin heavy chain bands corresponding to the two expected alleles, we have demonstrated an unexpected multiband pattern when the HindIII‐digested DNA samples from 38 CLL patients were analysed by Southern blot hybridization using JH and Cμ gene probes. In order to characterize the genetic basis for the multiband pattern, we molecularly cloned the immunoglobulin heavy chain genes of one of the patients whose leukaemic DNA sample demonstrated three hybridizing JH bands and a loss of the germline band. The cloned rearranged immunoglobulin genes could be divided, based on the restriction mapping and the hybridization with the various probes, into two basic patterns representing two alleles. In one of the cloned rearranged immunoglobulin genes a secondary rearrangement occurred that resulted in the addition of 300 base‐pair long sequence into the switch region, and the creation of a HindIII restriction site.

The results of the study suggest that clonal evolution occurs in some CLL, and that many of these neoplasms are indeed oligoclonal due to the accumulation of secondary genetic changes.
Neonatal thrombocytopenia and hidden maternal autoimmunity
Authors: Tchernia G. , Morel‐Kopp M. C. , Yvart J. , Kaplan C.
Published: March 2008

Summary. Severe transient thrombocytopenia was observed in 17 newborns delivered by 11 mothers who persistently exhibited normal platelet counts and had no previous immunological or platelet disorder. Maternofetal alloimmunization as well as other causes for fetal thrombocytopenia were ruled out. A specific circulating autoantibody directed against the platelet glycoprotein Ib/IX complex was found in 10/11 mothers and in 3/4 offspring. After delivery, maternal compensated thrombocytolysis and/or hypersplenism were demonstrated by 111Indium labelled platelet studies in 10/11 women. The association of a compensated thrombocytolysis and of a specific maternal circulating antiplatelet autoantibody makes likely the diagnosis of mild maternal chronic autoimmune thrombocytopenic purpura (AITP) only expressed by the fetal or neonatal thrombocytopenia. The finding of anti‐IbIX autoantibody in control women who gave birth to neonates without thrombocytopenia raises the question of the incidence and clinical significance of such antibodies which could be natural autoantibodies as well as associated with compensated thrombocytolysis or with overt immune thrombocytopenia. The cause of the discrepancy between the fetal and maternal platelet status, which could be explained by a different conformational state of fetal as compared to adult GP Ib/IX, deserves further investigation.