bluuebunny/crossref_metadata_2025 huggingface dataset:
EBV infection patterns in Hodgkin's disease and normal lymphoid tissue: expression and cellular localization of EBV gene products
Authors: Hummel Michael , Anagnostopoulos Ioannis , Dallenbach Friederike , Korbjuhn Petra , Dimmler Christiane , Stein Harald
Published: March 2008

The present study was performed to clarify the reported inconsistencies regarding the frequency of the association of Epstein‐Barr virus (EBV) and Hodgkin's disease (HD). Biopsies from 102 patients with HD were screened for the presence of EBV‐encoded small nuclear RNA (EBER) and latent membrane protein (LMP) by using a non‐isotopic in situ hybridization (ISH) and immunohistology (IH), respectively. The results were additionally compared with those obtained by polymerase chain reaction (PCR) for EBV‐DNA detection. EBV was detected by EBER‐ISH in 67% of the HD cases and in 25% of the control group cases consisting of normal lymph nodes. The results of PCR performed on cases with amplifiable DNA were overall congruent with those obtained by EBER‐ISH. With respect to the cellular localization of EBV. four categories of HD could be established: (a) cases with EBV‐infected tumour cells (42/102). (b) cases with additional infection of bystander cells (4/102): (c) cases with EBV infection restricted to non‐malignant bystander cells (23/102); and (d) cases with neither EBV‐infected tumour cells nor bystander cells (33/102). LMP expression was detectable only in the neoplastic cell population of those cases with EBER‐positive tumour cells, suggesting a frequent involvement of EBV in the pathogenesis of HD.
Detection of minimal residual disease by polymerase chain reaction of bcr/abl transcripts in chronic myelogenous leukaemia following allogeneic bone marrow transplantation
Authors: Lee M. , Khouri I. , Champlin R. , Kantarjian H. , Talpaz M. , Trujillo J. , Freireich E. , Deisseroth A. , Stass S.
Published: March 2008

The prognostic significance of detecting minimal residual disease by polymerase chain reaction (PCR) amplification of bcr/abl mRNA transcripts was investigated in 27 bone marrow samples from 20 patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukaemia (CML) in complete cytogenetic remission following allogeneic bone marrow transplantation. Sixteen were transplanted in first chronic phase, two were in second chronic phase, one was in accelerated phase and one was in blast crisis. All 20 achieved complete cytogenetic remission post transplant and 15 patients had detectable bcriabl mRNA by PCR from 2 to 22 months following the procedure. One of these patients had graft failure and one died from graft‐versus‐host‐disease at 7 months. Of the remaining 13 PCR‐positive patients, only one (8%) relapsed after 23 months: the other 12 were alive and still in remission after a median follow‐up of 16 + months (ranging 5+ to 29+ months). Five patients were PCR negative: all are alive in complete clinical and cytogenetic remission at lo +, 11 +, 19 +, 25+ and 25+ months post transplant. In this study, detection of subclinical Phl‐positive cells by PCR was not associated with imminent clinical or cytogenetic relapse. Since late recurrence may potentially occur, long‐term follow‐up is required to definitely determine the prognostic value of the PCR assay.
Adjusted dose continuous infusion of factor VIII in patients with haemophilia A
Authors: Martinowitz U. , Schulman S. , Gitel S. , Horozowski H. , Heim M. , Taron D.
Published: March 2008

Surgical interventions in patients suffering from haemophilia A require infusions of large doses of factor VIII (F VIII) concentrates. These are expensive and may pose a burden on the immune system, which is already compromised in many haemophiliacs. F VIII is usually given as bolus injections, although there are reports on fixed dose continuous infusion. We have developed a continuous infusion programme with dosage adjusted according to daily calculation of the clearance of F VIII.

Twenty‐four haemophiliacs received a total of 205 d of continuous infusion with F VIII (168 d in hospital, 37 d home therapy). Eighteen patients underwent surgeries (11 elective, seven emergency) and six were treated for serious haemor rhages. We observed a significant, progressive decrease of the clearance of F VIII over the first 5 d from 3.2 (2.8–3.5) to 1.7 (1.3–1.9) ml/kg/h (median and interquartile range). The decrease of the clearance together with the fact that a continuous infusion requires less drug than bolus injections to keep the same minimal concentration, contributed to much lower doses of F VIII than reported in the literature or than required by historical controls, matched for the type of operation. Other advantages of our method are improved safety with stable activities of F VIII, lack of hazardous troughs below the haemostatic range, and the convenience, which permits ambulant therapy even when high doses of F VIII are required.
Incompletely processed N‐glycans of serum glycoproteins in congenital dyserythropoietic anaemia type II (HEMPAS)
Authors: Fukuda Michiko N. , Gaetani Gian F. , Izzo Pietro , Scartezzini Paolo , Dell Anne
Published: March 2008

Congenital dyserythropoietic anaemia type II, or HEMPAS (hereditary erythroblastic multinuclearity with positive acidifed serum lysis test) is a genetic disease caused by membrane disorganization of erythroid cells. The primary defect of this disease lies in the gene encoding enzyme(s) which is responsible for the biosnythesis of Asn‐linked oligosacharides chains of glycoproteins (Fukuda et al. 1990). In order to know whether this gene defect affects the glycosylation in the cells other than the erythroid cells, the carbohydrate structures of the transferrin isolated from the sera of HEMPAS patients were analysed. Fast atom bombardment mass spectrometry analysis showed the presence of high mannose type and hybrid type oligosaccharides in the HEMPAS transferrin which is in contrast to the complex‐type oligosac charides found in the normal transferrin. The results strongly suggest that biosynthesis of Asn‐linked oligosaccharide chains in HEMPAS hepatocytes is disturbed. As a result, the serum glycoproteins with incompletely processed carbohydrates are circulating in the plasma in HEMPAS patients. but they must have been absorbed by the cells in the liver and the reticuloendothelial cells. Upon intravenous infusion into rats, as much as 30% of the HEMPAS transferrin was cleared from the plasma circulation. The majority of the HEMPAS transferrins was taken up by the liver, and transferrin was distributed both in the hepatocytes and the Kupffer cells. The presence of enormous amounts of aberrantly glycosylated serum glycoproteins may lead to the liver cirrhosis and secondary tissue siderosis seen in HEMPAS patients.
Red cell autoantibody production by colonic mononuclear cells from a patient with ulcerative colitis and autoimmune haemolytic anaemia
Authors: Yates P. , Macht L. M. , Williams N. A. , Elson C. J.
Published: March 2008

We have studied the ability of mononuclear cells extracted from the colon (CMC), draining lymph nodes (LNMC), peripheral blood (PBMC) and spleen (SMC) of a patient with ulcerative colitis and severe autoimmune haemolytic anaemia to produce red cell autoantibodies. The LNMC, PBMC and SMC secreted immunoglobulin in vitro in pokeweed mitogen culture but not spontaneously. They also produced immunoglobulin when transferred to severe combined immunodeficient (SCID) mice but no anti‐red cell activity was demonstrable. In contrast, in vitro cultures of CMC produced immunoglobulin spontaneously, showing the presence of activated B‐cells and plasma cells, but anti‐red cell activity could not be demonstrated. However, CMC transferred to SCID mice were able to produce IgG with anti‐red cell activity. These results concur with clinical observations suggesting that the colon is the source of red cell autoantibodies in these patients.
A sensitive sandwich ELISA for measuring erythropoietin in human serum
Authors: Noea Gudrun , Riedel Waltraut , Kubanek Bernhard , Rich Ivan N.
Published: March 2008

A sandwich, non‐competitive enzyme‐linked immunosorbant assay (ELBA) for erythropoietin (EPO) is described. The ELISA utilizes a monospecific, polyclonal antibody raised in rabbits against human recombinant EPO (rhu EPO) and purified over a rhu EPO affinity chromatography column. The ELISA procedure can be summarized as follows: Anti‐EPO is coated onto 96‐well ELISA microtitre plates; standard EPO or sample is added and left to bind to this catching antibody: this is followed by the addition of the same antibody which has been biotinylated; finally, anti‐biotin conjugated to alkaline phosphatase is added and the enzyme reaction developed and read at 405 nm. All parameters of the assay have been optimized. Recombinant human EPO was standardized against the World Health Organization 2nd International Reference Preparation for erythropoietin. The minimal detectable concentration of rhu EPO was 0.3–0.5 mil/ml, which corresponded to 1.2–2 mU/ml of EPO in serum (serum diluted 1:4). No reaction was obtained with a variety of blood components and cytokines, indicating that the anti‐EPO antibody did not cross‐react with those substances to produce false‐positive results. The intra‐assay variation ranged from 3% to 10%, while the inter‐assay variation ranged from 8.5% to 24%. Serum dose‐response curves were parallel to the standard dose‐response curve. The assay is easy to use, rapid, reproducible, but above all quantitative, specific and sensitive to measure the EPO content in all serum samples.
Granulocytic differentiation of the human myelomonocytic leukaemia cell line ME‐1 in serum‐free culture
Authors: Yanagisawa Kohsuke , Hasegawa Hitoshi , Fujita Shigeru
Published: March 2008

We have recently established ME‐1, a human myelomonocytic leukaemia cell line derived from acute myelomonocytic leukaemia with eosinophilia (M4E0). When ME‐1 cells were cultured in serum‐free medium, they stopped proliferating and began to differentiate morphologically, functionally and phenotypically to mature granulocyte‐like cells. The protein kinase inhibitor, 1‐(5‐isoquinolinyl‐sulphonyl)‐2‐methylpiperazine (H‐7) enhanced this differentiation dose‐dependently. Upon addition of fetal calf serum (FCS) to the serum‐free medium, the differentiation of ME‐1 cells into granulocyte‐like cells was inhibited and they resumed cell growth. We have recently reported that the differentiation of ME‐1 cells into macrophage‐like cells induced by IL‐3 and GM‐CSF involved the activation of protein kinase C. The present results indicate that ME‐1 is a bipotential cell line that can differentiate into granulocyte‐like cells or macrophage‐like cells, and that protein kinase C is closely related to each form of differentiation.
Diagnostic and clinical importance of interleukin‐2 receptor alpha chain expression on non‐T‐cell acute leukaemia cells
Authors: Nakase Kazunori , Kita Kenkichi , Otsuji Akira , Anazawa Hiroshi , Hoshino Kenichiro , Sekine Takao , Shirakawa Shigeru , Tanaka Isao , Nasu Kaori , Tsutani Hiroshi , Dohy Hiroo , Tsudo Mitsuru
Published: March 2008

The expression of interleukin‐2 receptors (IL‐2R) was examined in 328 adult patients with non‐T‐cell (non‐T) acute leukaemia and blast crisis of chronic myelocytic leukaemia (CML.BC) using two monoclonal antibodies, anti‐Tac for IL‐2R α chain (IL‐2Rα) and Mikβ1 for IL‐2R β chain (IL‐2Rβ). Leukaemic cells in the following cases were positive for anti‐Tac; 28/192 of acute myelocytic leukaemia (AML), 24/44 CML‐BC, 4/28 CD19(+)CD10(‐) acute lympho‐blastic leukaemia (ALL), and 20/64 common ALL (c‐ALL). IL‐2Rβ was not detected on leukaemic cells of any case examined. Eleven of IL‐2Rα(+) AML were derived from myelodysplastic syndrome. None of the IL‐2Rα positive leukaemic cells responded to exogenous recombinant human IL‐2 (rhIL‐2) in culture. In addition, IL‐2Rα expression on non‐T leukaemic cells was closely correlated with coexpressing different lineage markers and the presence of the Philadelphia abnormality. Marked increase of serum soluble IL‐2Rα was demonstrated in the IL‐2Rα(+) patients examined. Clinically, the IL‐2Rα(+) patients showed significantly lower response to chemotherapy and poorer prognosis than IL‐2Rα(‐) patients. Our results clearly indicate the diagnostic importance of IL‐2Rα expression in non‐T acute leukaemia with a close relation to the particular cellular characteristics and the prognosis.
Possible evidence for genomic imprinting in childhood acute myeloblastic leukaemia associated with monosomy for chromosome 7
Authors: Katz Fay , Webb David , Gibbons Barbara , Reeves Brian , McMahon Cathy , Chessells Judith , Mitchell Christopher
Published: March 2008

Monosomy or deletion of chromosome 7 is a frequent finding in both de novo and secondary acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS). Based on analysis of deletions of chromosome 7 in such patients, it has been suggested that there is a critical region of the chromosome lying within bands q21‐q31. We have examined bone marrow and peripheral blood samples from 10 patients with MDS, AML and biphenotypic acute leukaemia who had monosomy for or rearrangement of chromosome 7, seeking evidence of non‐random allele loss that might suggest the presence of imprinted genes on the chromosome.

Bone marrow cells from one patient with the infant monosomy 7 syndrome had loss of maternal alleles as did two patients with biphenotypic leukaemia. Five out of five patients with MDS and both patients with de novo AML had loss of paternal alleles. One of the latter patients had a del(7) (q31q36) rather than monosomy 7. These findings suggest that imprinting of a gene(s) on chromosome 7, within the bands q31‐q36, may be of importance in MDS and AML.

Despite the reported increased incidence of AML amongst relatives of patients with cystic fibrosis (CF) the gene for which lies in chromosome region 7q31, none of the patients nor parents studied here appeared to be carriers of the most common gene mutation seen in patients with CF, the delta F508.
Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: effect of conditioning on the specific immune response of the graft recipient
Authors: Labadie J. , van Tol M.J. D. , Dijkstra N.H. , Zwaan F. E. , Vossen J. M.
Published: March 2008

Transfer of specific immunity was investigated in a group of 28 paediatric and adult leukaemia patients during the first 100 d after allogeneic bone marrow transplantation (BMT). These patients and/or their donors were immunized 7–13 d before transplantation with the recall antigen tetanus toxoid (TT) and the neo‐antigen Helix pomatia haemocyanin (HPH). The recipients were booster immunized with both antigens at day 42 after transplantation. Transfer of a primary IgM and IgG response to HPH was successful in most paediatric and adult patients, but transfer of a secondary response to TT was established in only a few paediatric recipients. After booster immunization at day 42 most paediatric recipients responded with a rise in serum antibody titre to HPH as opposed to only two of 18 adult recipients. This incapability of the adult recipients to mount a secondary immune response may be related to their conditioning regimen which included Campath‐IG in vivo. The results from this study indicate that transfer of immunity against recall‐and neo‐antigens is possible. However, the establishment of long‐term memory may be affected by the regimen used to condition the graft recipient.
Surface marker expression in acute myeloid leukaemia at first relapse
Authors: Thomas Xavier , Campos Lydia , Archimbaud Eric , Shi Zhi‐Hua , Treille‐Ritouet Danièle , Anglaret Bruno , Fiere Denis
Published: March 2008

Surface markers were studied at first relapse in 66 cases of acute myeloid leukaemia (AML), using a panel of five monoclonal antibodies directed to CD13, CD14, CD15, CD33 and CD34 antigens. At time of relapse, there was increased expression of CD33 (P=0·002) and CD34 (P=0·0001), and decreased expression of CD13 (P=0·004) and CD15 (P=0·0001) antigens by comparison to initial diagnosis. There was no strict correlation with the FAB classification. However, CD13 and CD33 expression changes preferentially affected granulocytic leukaemias. At relapse, CD14 and CD34 were significantly more expressed in monocytic than in granulocytic AML (P=001 and 0·003 respectively). In a multivariate analysis, CD34 expression was associated with a low CR rate (P=0·001) and short survival (P=0·05), whereas CD15 expression was associated with long survival (P=0·0004). These results suggest that AML tends to relapse with a less differentiated phenotype than observed at diagnosis and that AML with less differentiated phenotype is of poor prognosis after first relapse, as also observed at diagnosis.
Immunophenotypic subclassification of chronic lymphocytic leukaemia (CLL)
Authors: Kurec A. S. , Threatte G. A. , Gottlieb A. J. , Smith J. R. , Anderson J. , Davey F. R.
Published: March 2008

To determine the significance of the immunophenotypic heterogeneity of B‐cell chronic lymphocytic leukaemia (CLL), surface immunoglobulins (SIgs), mouse rosette assays (MR), and a panel of monoclonal antibodies for B cells, T cells and myeloid cells were performed on peripheral blood samples from 61 newly diagnosed cases. Four groups were observed: group I (SIg+, MR+, CD19/20+, CD5+, T antigen (Ag)‐; 27 cases); group II (SIg+, MR+, CD19/20+, CD5+, T Ag+; 17 cases); group III (SIg+, MR+ CD19/20+, CD5‐, T AG‐; 12 cases); and group IV (SIg‐, MR+, CD19/20+, Cd5+, T Ag‐; 5 cases). Groups were compared according to French–American–British Cooperative Group subtypes, clinical and laboratory features, Rai staging, and survival. Typical CLL morphology (>90% small lymphocytes) was present in 20/20 (100%) of group I cases and 23/27 (85%) group II, III and IV cases (P=0·09). Expression of a myeloid antigen was seen in 5/27 group I cases (18%) and 1/16 group II cases (6%), but was not predictive of survival (P=0·36). The CD5‐ group III had a lower haemoglobin level (P<0·0001), higher Rai stage (P<0·002), and poorer survival at 5 years (P<0·02) than the other groups. We conclude that at least four distinct immunophenotypic subgroups of B‐cell CLL can be determined. Expression of myeloid or T‐cell antigens does not appear to predict for patient survival: however, lack of CD5 antigen may be associated with more advanced stage of disease and poor patient survival.
A family with haemolytic anaemia and three β‐globins: the deletion in haemoglobin Atlanta‐Coventry (β75 Leu→Pro, 141 Leu deleted) is not present at the nucleotide level
Authors: George Peter M. , Myles Timothy , Williamson David , Higuchi Russell , Symmans W. Ashley , Brennan Stephen O.
Published: March 2008

Analyses of haemoglobin from a family with an unstable haemoglobin haemolytic anaemia demonstrated that the affected individuals had three β‐globins, namely, normal (βA), Atlanta (βAt) with a mutation of β75 Leu→Pro, and β‐Atlanta‐Coventry (βAt‐Co) with mutation of β75 Leu→Pro and β141 Leu deleted. These were present in the ratio 66:23:11 respectively. The structure of the β‐globin cluster, however, was found to be normal by Southern blotting: also cytogenetic analysis failed to show any abnormality. DNA sequence analyses demonstrated the presence of the βAt mutation in genomic DNA isolated from leucocytes but the Coventry deletion of 141 Leu in βAt‐Co was not present in genomic DNA. PCR amplification of the β‐globin cDNA and direct sequencing of the product also failed to demonstrate the Coventry deletion. Thus, it appears that the absence of 141 Leu in the βAt‐Co globin is a consequence of the βAt mutation in these patients and that both βAt and βAt‐Co are the product of a single gene. This unusual conclusion is paralleled in the bizarre case of Hb Vicksburg where the deletion of a leucine at β75 is not coded for in genomic DNA.
Chromosome 11q rearrangements in B non Hodgkin's lymphoma
Authors: Vandenberghe E. , Peeters C. De Wolf , Wlodarska I. , Stul M. , Louwagie A. , Verhoef G. , Thomas J. , Criel A. , Cassiman J. J. , Mecucci C. , Van den Berghe H.
Published: March 2008

Summary. The clinical features, morphology and immunophenotype of 20 cases of B non Hodgkin's lymphoma (B‐NHL) with chromosome abnormalities involving 11q13–14 were studied, to determine if this abnormality was closely associated with a specific sub‐type of β‐NHL. A t(11:14)(q13;q32) was found in 11 cases of intermediately differentiated lymphocytic lymphoma (IDLL). A breakpoint in the major translocation cluster of the BCL‐.1 locus was found in six of these cases. These patients were male with lymphomatous involvement of the bone marrow, marked splenomegaly and frequently had mucosa associated lymphoid tissue involvement. One patient with IDLL had a t(8;11)(p21;q13) and a rearranged BCL‐1 locus, suggesting that this may be a variant of t(11;14)(q13;q32). Diagnoses of IDLL, chronic lymphocytic leukaemia, lymphoplasmacytic lymphoma and monocytoid B cell lymphoma were made in all but one of the remaining cases. These cases had either a translocation involving 1 Iq13–14 and various partner chromosomes or an 11q13 deletion. This study demonstrates that 11q abnormalities occur mainly in a group of low‐grade B‐NHL of non follicle centre cell lineage.
Studies on macromolecular activators of phagocytosis from platelets (MAPPs) using anti MAPP murine monoclonal antibodies
Authors: Yokoya Yukihiro , Sakamoto Haruhiko , Oryu Makoto , Uda Hirotsugu
Published: March 2008

1‐MAPP(290 kD) and s‐MAPP(150 kD) are glycoproteins which have been shown to be involved in released products from platelets (PRPr) and to activate monocyte and neutrophil phagocytosis via the Fc receptors. Three IgG 2 a (κ) murine monoclonal antibodies against 1‐MAPP and nine IgG1 (κ) antibodies against s‐MAPP have been raised.

By affinity chromatography using these monoclonal antibodies, active MAPPs were obtained from the supernatant of outdated platelet concentrates in the presence of 60% saturated ammonium sulphate and from PRPr‐rich medium, and substantial amounts of non‐functioning immunoreactive MAPPs were obtained from the precipitate.

Fab fragments of one of the two anti 1‐MAPP and of two of the four anti s‐MAPP monoclonal antibodies inhibited the function of their corresponding MAPPs, but none of them could inhibit the other MAPPs.

1‐MAPP and s‐MAPP in the platelets were visualized by indirect fluorescent antibody technique using Fab fragments of the antibodies.
Heparin‐associated thrombocytopenia in a patient treated with polysulphated chondroitin sulphate: evidence for immunological crossreactivity between heparin and polysulphated glycosaminoglycan
Authors: Greinacher A. , Michels I. , Schäfer M. , Kiefel V. , Mueller‐Eckhardt C.
Published: March 2008

Heparin‐associated thrombocytopenia (HAT) type II, a severe side effect of heparin therapy, is thought to be induced by an immunological mechanism. By crossreactivity studies we have demonstrated that sera of patients with HAT type II activate platelets in vitro not only after the addition of heparin but also after addition of a chemically polysulphated chondroitin‐like substance, Arteparon®, used for treatment of degenerative joint disease. In addition here, we describe a patient who developed deep venous thrombosis and pulmonary embolism following administration of Arteparon® and typical HAT type II with thrombocytopenia, 36 h after the first administration of heparin. This patient had never received heparin, but had repeatedly been treated with Arteparon® for degenerative joint disease. We conclude that this patient had been presensitized by Arteparon®, as indicated by his clinical course. In vitro studies again confirm crossreactivity between heparin and Arteparon®.
Ratios of anti‐factor Xa to antithrombin activities of heparins as determined in recalcified human plasma
Authors: Schoen Pieter , Lindhoux Theo , Hemker H. Coenraad
Published: March 2008

Anti‐factor Xa and anti‐thrombin activities of unfractionated (UF) and low molecular weight (LMW) heparins have been measured in human plasma and with purified human antithrombin III (ATIII) in the absence and presence of 1.5 mM calcium. The anti‐factor Xa and antithrombin activities were measured directly, by assessing the heparin‐dependent pseudo‐first order rate constants of inactivation of human factor Xa or thrombin. These activities were studied with the 4th International Standard for UF heparin, the 1st International Standard for LMW heparin, CY216, enoxaparin, CY222, and the synthetic pentasaccharide. In plasma, calcium equally well increased the specific anti‐factor Xa catalytic activities as compared to purified ATIII. That is. 1.5 mM calcium stimulated the UF standard heparincatalysed inactivation of factor Xa 2.1–2.4 times. In the presence of the LMW heparins the effect of calcium was smaller (1.3–1.7 times), and in plasma there was no effect of calcium on the pentasaccharide‐catalysed inactivation of factor Xa. Thus, the largest effects of calcium in the inactivation reaction of factor Xa is seen with UF standard heparin. Calcium reduced the anti‐thrombin activities of all the heparin preparations studied about 1 × 5 times when purified ATIII was used, although in plasma this effect was less clear. Consequently, in the presence of 1.5 mM calcium the ratio of the anti‐factor Xa to the anti‐thrombin activities of UF standard heparin approximated those of the LMW heparins. The only exception was CY222, which under all conditions retained anti‐factor Xa/anti‐thrombin ratios significantly higher than those of UF standard heparin.
The effect of macrophage colony‐stimulating factor on haemopoietic recovery after autologous bone marrow transplantation
Authors: Khwaja Asim , Yong Kwee , Jones H. Mark , Chopra Rajesh , McMillan Andrew K. , Goldstone Anthony H. , Patterson Keith G. , Matheson Catherine , Ruthven Karen , Abramson Stephan B. , Linch David C.
Published: March 2008

Macrophage colony‐stimulating factor (M‐CSF) is active in the late stages of monocyte maturation, activates mature monocyte‐macrophages and enhances their production of various other cytokines. We have examined the effects of a 21 d course of escalating doses of M‐CSF purified from human urine (hM‐CSF) on recovery following autologous bone marrow transplantation (ABMT) in 20 patients with malignant lymphomas. Four patients were treated at each dose level of 4, 8, 16, 32 and 64.1099 U/m2/d and results compared to 46 concurrent controls. There was no significant difference in recovery to an absolute neutrophil count (ANC) of 0.5×109/1 (median 20 d in hM‐CSF group versus 22 in controls) or in recovery of platelets to 50.109/1 (32 d versus 39 d, 0.05< P <0.1); hM‐CSF patients received a median of 81 platelet units following ABMT (controls 112 units, P = NS). hM‐CSF patients had a median of 5.5 d with fever >37.5°C (control 8, P=NS), received parenteral antibiotics for 14.5 d (control 17, P=NS) and had a 50% incidence of bacteraemia (control 48%). hM‐CSF treated patients were discharged by a median of day 29 following transplantation (control 33, P<0.05). Platelet and neutrophil recovery correlated significantly with the number of marrow mononuclear cells (MNC) reinfused in the hM‐CSF group (P=0.05 and P=0.014 respectively) but not in controls. Subgroup analysis showed that hM‐CSF patients receiving > 2.108 MNC/kg body weight reached an ANC of 0.5×109/1 by a median of day 16.5 (control 18.5, NS), became platelet transfusion independent by day 17 (control 29, P<0.05) and reached a platelet count of 50.109/1 by day 21 (control 40, P<0.05). No significant toxicity attributable to hM‐CSF treatment was seen. These results suggest that hM‐CSF accelerates platelet recovery following ABMT and that relatively large marrow innocula are required to see this effect.
Trisomy 8 detection in granulomonocytic, erythrocytic and megakaryocytic lineages by chromosomal in situ suppression hybridization in a case of refractory anaemia with ringed sideroblasts complicating the course of paroxysmal nocturnal haemoglobinuria
Authors: PArlier V. , Tiainen M. , Beris Ph. , Miescher P. A. , Kimuutila S. , Bellomo M. Jotterand
Published: March 2008

Paroxysmal nocturnal haemoglobinuria (PNH) was diagnosed in a 20‐year‐old male patient who suffered from anaemia since the age of 11. Eighteen years after diagnosis, PNH transformed into refractory anaemia with ringed sideroblasts (RARS). Trisomy 8 was observed in 27%, 45% and 53% of the bone marrow metaphase cells analysed in 1987, 1988 and 1990 respectively. In order to determine which bone marrow cell lineages were affected by trisomy 8 and at which stage of stem cell differentiation, MAC (Morphology, Antibody, Chromosomes) and CISS (Chromosomal in Situ Suppression) hybridization techniques were combined. The MAC technique enables karyotypic analysis of morphologically and immunologically classified mitotic cells. CISS hybridization makes it possible to detect individual chromosomes and chromosome aberrations using recombinant DNA libraries from sorted human chromosomes. Trisomy 8 was detected in granulomonocytic (50.6%), erythrocytic (67.2%) and megakaryocytic (one megakaryocyte with trisomy 8, one normal) lineages, providing evidence for the occurrence of trisomy 8 in early haematopoietic cell precursors, at the GEMM or pluripotent level. Cytogenetic and clinical data suggest that the sideroblastic clone originated from a mutation affecting a cell of the PNH clone, progressively replaced by the PNH/RARS clone, due to proliferative advantage.
Detection of latent subclones with abnormal karyotypes by long‐term bone marrow cultures in cases of myelodysplastic syndrome
Authors: Tamura Shu , Kanamaru Akihisa , Kakishita Eizo , Nagai Kiyoyasu
Published: March 2008

Cytogenetic studies on non‐adherent cells from long‐term bone marrow cultures (LTBMC) were done for 23 patients with myelodysplastic syndrome (MDS) and compared with the karyotypes detected during their clinical courses. Of 14 cases with normal karyotype before culture, abnormal karyotypes were detected first in LTBMC from seven. Novel abnormal karyotypes were observed after LTBMC in two of four cases which had had both normal and abnormal karyotypes before culture. Abnormal karyotypes were also newly detected in four of five cases with only abnormal karyotypes before culture. Among 13 cases in which abnormal karyotypes were observed during the cultures, three patients showed the same karyotypes 1–11 months later in their clinical courses. These findings suggest that our LTBMC might be useful for evaluating the prognosis and choice of treatment for MDS patients.
Relapse into blast crisis following bone marrow transplantation for chronic phase chronic myeloid leukaemia: a report of five cases
Authors: Cullis J. O. , Marks D. I. , Schwarer A. P. , Barrett A. J. , Hows J. M. , Swirsky D. M. , Goldman J. M.
Published: March 2008

A proportion of patients receiving allogeneic bone marrow transplants (BMT) for chronic myeloid leukaemia (CML) in first chronic phase relapse; most of these relapses show features of chronic phase disease. We report here a series of five patients seen at a single institution over a 10 year period who developed blast crisis as the first sign of relapse after BMT for CML in chronic phase. The blast cells were myeloid in three cases and lymphoid in two. In one case the relapse may have occurred in cells of donor origin. The possible explanations for this unusual sequence of events include incipient transformation that was not detected before BMT, undetected relapse into chronic phase proceeding into transformation post‐BMT, and transformation occurring de novo post‐BMT in small numbers of residual leukaemic stem cells.
Identification of CD71 (transferrin receptor) expressing erythrocytes by multiparameter‐flow‐cytometry (MP‐FCM): correlation to the quantitation of reticulocytes as determined by conventional microscopy and by MP‐FCM using a RNA‐staining dye
Authors: Serke Stefan , Huhn Dieter
Published: March 2008

Reticulocytes express the CD71‐defined antigen, the transferrin receptor. This report describes how by means of dual‐colour immunofluorescence using MP‐FCM (multiparameter‐flow‐cytometry) CD71+ erythrocytes can be detected regularly in blood of healthy adults. Percentages of these CD71+ erythrocytes were compared to the percentages of reticulocytes as determined by conventional microscopy using brilliant cressyl blue, and to the percentages of erythrocytes with high RNA content, as detected by MP‐FCM using a RNA‐staining dye (thiazole‐orange). Only about two‐thirds of the percentages determined by the two latter methods were detected by MP‐FCM using the CD71 expression for definition of reticulocytes. Studying clinical samples, however, including both specimens with very low and very high numbers of reticulocytes. almost identical percentages were determined by all the three methods described.

Studying reticulocytes in vitro, a rapid decline of the expression of the transferrin receptor was observed on reticulocytes. Due to the differential expression of the transferrin receptor on reticulocytes, different subsets of reticulocytes could be identified.

The dual‐colour MP‐FCM method described allows for the characterization and enumeration of immature erythrocytes, representing the major subset of reticulocytes as determined by conventional methods. Furthermore, it allows for subset dissection of reticulocytes.
Prospective follow‐up of monoclonal gammopathies in HIV‐infected individuals
Authors: Lefrère Jean‐Jacques , Debbia Martine , Lambin Patrick
Published: March 2008

Summary. A cohort of 341 symptomless anti‐human immunodeficiency virus (HIV)‐1 positive individuals was screened over a 6‐year period to detect a serum monoclonal gammopathy (MG) and to approach the prognostic significance of such anomaly in HIV infection. Eleven individuals with a MG were followed‐up over a mean period of 50 months from the date of discovery of the MG. At the end of this period, the MG had disappeared in seven individuals, was still present in the four others. The appearance of a second MG was noticed in two cases, and of a third in one case. Immunoglobulin (Ig) typing identified eleven IgG kappa and three IgG lambda. Mean serum concentration of MG of individuals with persistent MG (14 · 3 g/l) and of individuals without (4 · 2 g/l) was significantly different (P < 0 · 05). The discovery of a MG was without prognosis value on the disease progression and did not appear as a primary event in the subsequent development of lymphoma.
PCR‐based immunogenotyping at the Ig heavy chain CDR3 locus: improvements in resolution
Authors: Pollard Peter , Owen Gwyneth , Worwood Mark
Published: March 2008

Summary. An improved electrophoretic separation of products after PCR employing consensus primers to amplify the CDR3 of the immunoglobulin heavy chain variable region allows visualization of a ladder of fragments in polyclonal samples of peripheral blood as well as a single band where there is clonal proliferation.
A new method to study the efficiency of third generation blood filters
Authors: Sivakumaran M. , Norfolk D. R. , Major K. E. , Revill J. A. , Hutchinson R. M. , Wood J. K.
Published: March 2008

Summary. In order to study the efficiency of third generation blood filters we have devised a new method using 3 μm pore size polycarbonate filter membranes and a filtration chamber to trap leucocytes passing through the blood filter. The method has enabled us to make two important observations: (1) The filters function very efficiently at the start but the efficiency diminishes progressively with time. (2) When the blood flow through the filter is retarded, due to defective priming and/or filter malfunction, the filters fail even at the outset of the transfusion.
Impairment of GM‐CSF production in myelodysplastic syndromes
Authors: Visani G. , Zauli G. , Tosi P. , Fogli M. , Cenacchi A. , Finelli C. , Gamberi B. , Ottaviani E. , Manfoi S. , Tura S.
Published: March 2008

Summary. In this study we evaluated the production of granulocyte/macrophage‐colony stimulating factor (GM‐CSF), interleukin‐6 (IL‐6) and tumour necrosis factor‐α (TNF‐α) by enriched bone marrow (BM) macrophages in 15 patients affected by myelodysplasia and 20 normal BM donors.

The presence of GM‐CSF, IL‐6 and TNF‐α in the culture supernatants of BM macrophages was detectable only after stimulation with lipopolysaccharide (LPS), whereas no differences were present in the amount of IL‐6 and TNF‐α between myelodysplastic patients and normal controls, GM‐CSF production appeared eight‐fold reduced in BM macrophage culture supernatants from myelodysplastic patients with respect to normal controls. After further experiments, we concluded that the impaired release of GM‐CSF by BM macrophages could not be due to a different production kinetic in myelodysplastic patients.

Moreover, the number of multipotent (CFU‐GEMM), granulocyte/macrophage (CFU‐GM) and erythroid (BFU‐E) progenitors was significantly impaired in myelodysplastic patients.

In conclusion, we demonstrated that the production of GM‐CSF by purified adherent cells from MDS patients is markedly impaired in spite of the peripheral blood cytopenia. This selective defect in GM‐CSF production, along with an intrinsic defect of haematopoietic progenitor cells, might contribute to the impairment of haematopoiesis always observed in myelodysplastic patients.
Serum thymidine kinase as a prognostic indicator for patients with multiple myeloma: results from the MRC (UK) V Trial
Authors: Brown R. D. , Joshua D. E. , Nelson M. , Gibson J. , Dunn J. , MacLennan I. C. M.
Published: March 2008

Summary. The significance of serum thymidine kinase (STK) as a prognostic indicator for patients with myeloma has been evaluated by determining the pre‐treatment level of STK in a retrospective study of 547 patients from the Medical Research Council (MRC) V Myeloma Trial. Overall, STK was a highly significant prognostic factor (Chi2= 7.91; P= 0.005). At the time of censor, 23.4% of patients with a presentation STK of < 6 U/l but only 7.9% of the patients with an STK of > 11 U/l were still alive. STK proved to be a highly significant prognostic indicator for the 270 melphalan‐treated patients (Chi2= 12·37; P, = 0.0004) but had no prognostic significance for the 277 ABCM (adriamycin, BCNU, cyclophosphamide and melphalan) treated patients (Chi2= 0.29; P= 0.59). When the STK data was stratified for serum B‐2‐microglobulin (SB2M) it was demonstrated that STK was independent of SB2M and provided additional prognostic information for patients who were treated with melphalan. Thus patients with both a low STK and SB2M had the best prognosis (median survival 1677 d) and those patients with a high STK and SB2M had the worst prognosis (median survival 519 d). STK is a good prognostic marker for patients treated with melphalan but the prognostic significance of STK may disappear with the introduction of new chemotherapy regimens.
Quantitation of follicular non‐Hodgkin's lymphoma cells carrying t(14;18) by competitive polymerase chain reaction
Authors: Meijerink J. P. P. , Smetsers T. F. C. M. , Raemaekers J. M. M. , Bogman M. J. J. T. , de Witte T. , Mensink E. J. B. M.
Published: March 2008

Summary. A competitive polymerase chain reaction (PCR) technique was developed to quantify residual malignant cells in the peripheral blood and bone marrow of patients with low‐grade follicular non‐Hodgkin's lymphoma carrying a translocation between chromosomes 14 and 18. Artificial segments were constructed imitating a translocation between chromosome 14 and 18. These artificial translocation segments were used as competitor molecules in the quantitative PCR. Serial dilutions of a known amount of patient‐derived translocation segments were coamplified with a fixed number of competitor molecules, and a patient specific calibration curve was constructed. Several patient samples were coamplified with an equal number of competitor molecules and the number of t(14;18) translocations within the samples was calculated by comparison with the calibration curve.

The method was demonstrated on samples of four follicular non‐Hodgkin's lymphoma (NHL) patients. In a patient transplanted with allogeneic bone marrow declining numbers of residual lymphoma cells were observed.

We conclude that the method is accurate, relatively fast and the general principle of this method can be applied to all malignancies with characteristic abnormalities on DNA or RNA level that are detectable by PCR.
Confirmation of Rb gene defects in B‐CLL clones and evidence for variable predominance of the Rb defective cells within the CLL clone
Authors: Kay Neil E. , Suen Rosa , Ranheim Erik , Peterson LoAnn C.
Published: March 2008

Summary. In 70 B‐CLL patients, deletion or translocation, at or near the retinoblastoma (Rb) site, was detected in 20 by cytogenetic analysis. Purified B cell clones from 13 of these B‐CLL patients were isolated and studied for Rb gene status, Rb mRNA and the Rb protein product. Southern blot analysis of the Rb site detected internal deletions (N= 1) or a single allele loss (N= 2) in five patients. Northern blots detected reduced Rb mRNA in four patients. Immunoblot of whole cell lysate revealed reduced levels of unphosphorylated Rb protein in six CLL patients. No CLL B cell clone contained phosphorylated Rb species. These molecular studies have confirmed the cytogenetic alteration of 13q12–14 sites in B‐CLL cells. In addition, cytogenetic and molecular biologic analysis suggest heterogeneity in the B cell clone for Rb gene abnormality. B‐CLL patients with abnormalities in both cytogenetic and Rb DNA/RNA analysis will have a dominance of B cells with an Rb abnormality (N= 5). In patients whose Rb defective CLL cells constitute only a minor subpopulation of the total B cell clone, only cytogenetic defects would likely be detected (N= 7).
The behaviour of different factor VIII concentrates in a chromogenic factor X‐activating system
Authors: Kemball‐Cook G. , Tubbs J. E. , Dawson N. J. , Barrowcliffe T. W.
Published: March 2008

Summary. A chromogenic factor Xa generation method has been developed for comparing the co‐factor activity of factor VIII concentrates at physiological factor VIII concentrations (1 iu/ml). In the presence of thrombin all concentrates gave similar rapid rates of factor Xa generation, but in the absence of thrombin there were major differences between the rates of Xa generation between different products. High purity products, particularly those prepared by monoclonal antibody purification from plasma and recombinant sources, gave more rapid Xa generation than most intermediate‐purity products. There was a very strong correlation between the rate of Xa generation and the difference in factor VIII potency by one‐stage and two‐stage assays. These results suggest the possible presence of small amounts of activated factor VIII in some concentrates, but differences in von Willebrand factor content could also contribute towards the different rates of factor Xa generation observed.
Reduced coagulation activation following infusion of a highly purified factor IX concentrate compared to a prothrombin complex concentrate
Authors: Hampton K. K. , Preston F. E. , Lowe G. D. O. , Walker I. D. , Sampson B.
Published: March 2008

Summary. We have looked for evidence of coagulation activation in six subjects with haemophilia B by performing a single‐blind active control cross‐over study comparing a recently developed factor IX concentrate with a conventional prothrombin complex concentrate (PCC). Samples were obtained before infusion and at 0·25, 0·5, 1, 2, 4, 6, 12, 24, 36 and 48 h for assay of factor IX, prothrombin time, fibrinopeptide A (FPA), prothrombin fragment F1 + 2, D‐dimer, thrombin–antithrombin complexes (TAT) and antithrombin III (ATIII). Following administration of the PCC there was evidence of coagulation activation in five of the six recipients for up to 6 h after the infusion. The factor IX concentrate induced a moderate degree of coagulation activation in one subject. There was no significant difference between the two products in respect of either recovery or half‐life. This study provides further evidence that the new high purity preparations of factor IX concentrates produce significantly less coagulation activation than currently available PCCs. It remains to be established whether this will result in a corresponding reduction in thromboembolic complications in clinical use.
The effect of methyldopa and procainamide on suppressor cell activity in relation to red cell autoantibody production
Authors: Garratty George , Arndt Patricia , Prince Harry E. , Shulman Ira A.
Published: March 2008

Summary. Kirtland et al (1980) suggested that methyldopa caused the production of red cell (RBC) autoantibodies by causing a persistent increase in lymphocyte cyclic AMP, which inhibited suppressor T cell function, leading to unregulated autoantibody production in some patients. They showed that significantly higher lymphocyte cyclic AMP concentrations were generated by lymphocytes from healthy donors after adding methyldopa, and by lymphocytes from patients who were receiving methyldopa compared to lymphocytes from healthy donors without methyldopa present. They also showed that methyldopa affected suppressor cell activity. We measured the effect of methyldopa and procainamide on suppressor cell activity, using a similar approach to Kirtland et al (1980). Suppressor cell activity was measured by measuring the amount of IgG, produced in vitro, by B cells following mitogen stimulation preceded by a 24 h incubation period. We found no significant increase in the amount of IgG generated by normal donor lymphocytes, when methyldopa or procainamide was present during the preincubation period. This is in contrast to the findings of Kirtland et al (1980). We also measured the amount of IgG generated in vitro by mitogen‐stimulated lymphocytes from patients (with and without positive direct antiglobulin tests) taking methyldopa and compared this to the amount of IgG generated by lymphocytes from normal donors and patients (with and without positive direct antiglobulin tests). The results were similar for each group. This does not agree with the findings of Kirtland et al (1980) who found that lymphocytes from patients taking methyldopa produced more IgG in vitro than lymphocytes from normal donors.

Our results do not support the hypothesis that methyldopa and procainamide induce autoantibodies by affecting suppressor cell function.
BCR‐ABL rearrangement in a child with acute myelogeneous leukaemia without a Philadelphia chromosome
Authors: Borkhardt A. , Repp R. , Harbott J. , Lakomek M. , Janssen J. W. G. , Schlieben S. , Bartram C. R. , Lampert F.
Published: March 2008

Summary. We describe a BCR/ABL rearrangement positive but Philadelphia chromosome negative status in a 9‐year‐old boy suffering from an acute myelogeneous leukaemia (AML). This case was detected in a prospective PCR screening procedure including 21 children with newly diagnosed AML and 150 children with acute lymphoblastic leukaemia (ALL). We found a 5·4% incidence of BCR/ABL rearrangement positive cases in pre‐B and c‐ALL in childhood.
Lymphoepithelioid cell lymphoma (Lennert's lymphoma): clinical features derived from analysis of 108 cases
Authors: Patsouris E. , Engelhard M. , Zwingers Th. , Lennert K.
Published: March 2008

Summary. In a retrospective analysis of 108 patients with lymphoepithelioid cell lymphoma (LeL) (median age 60, range 21–87 years) initial extent of disease was Ann Arbor stage I in 9%, II in 19%, III in 35%, and IV in 37% of the patients with a high incidence of generalized lymphadenopathy (71%) but seldom extralymphatic disease (13%). Unstable spontaneous remissions were observed occasionally. Complete response rates were 75% in the patients treated with radio‐ and/or chemotherapy but relapse occurred in 65%. Median overall survival was 16 months with a significantly better prognosis for stage I/II than for stage III/IV disease (P= 0·014). LeL thus seems to possess an unfavourable prognosis possibly to be improved by standardized treatment approaches.