bluuebunny/crossref_metadata_2025 huggingface dataset:
Red cell glycolytic intermediates in normal, anaemic and transfused human fetuses
Authors: Lestas A. N. , Bellingham A. J. , Nicolaides K. H.
Published: March 2008

Normal fetal ranges for red cell glycolytic intermediates at 18–24 weeks gestation, which are useful as reference values for the prenatal diagnosis of erythroenzymopathies, were established for the first time. Characteristic increases in glucose‐6‐phosphate (G6P), fructose‐6‐phosphate (F6P) and particularly fructose‐ 1,6‐diphosphate (FDP) suggest that there is no metabolic block at the phospho‐fructokinase (PFK) step of glycolysis as previously suggested by others for premature infants on the first day of life.

Neither reticulocytosis nor anaemia consistently led to further increases in the early metabolites as occurs in adults. However, very large increases in G6P, F6P and particularly FDP may occur independently of anaemia and reticulocytosis. This suggests that activation of hexokinase (HK) and/or PFK can take place as in adults but the stimulus is probably different to adults.

The 2,3‐DPG in normal fetuses is higher than in adults and increases still further in anaemic fetuses with or without transfusion of adult blood. The pattern of intermediates found in the fetus suggests that the controlling mechanism for the increased 2,3‐DPG may be an in vivo relative preponderance of PFK activity over that of pyruvate kinase (PK) rather than regulation of HK as proposed for adult subjects with anaemia or high altitude hypoxia.
Secretion of neutrophil secondary granules occurs during granulocyte‐macrophage colony stimulating factor induced margination
Authors: Devereux S. , Porter J. B. , Hoyes K. P. , Abeysinghe R. D. , Saib R. , Linch D. C.
Published: March 2008

The effects of recombinant human granulocytemacrophage colony‐stimulating factor (rhGM‐CSF) on neutrophil lactoferrin (LF) and transcobalamin (TC) 1 and 3 secretion were determined in vitro and during in vivo administration in humans. In whole blood, in vitro incubation with GM‐CSF reproducibly produced a rise in plasma LF concentration (P<0.05) whereas in purified neutrophils the results were variable. Exposure of whole blood to GM‐CSF also resulted in a significant rise in plasma TC 1 and 3 (190.60%, P<0.05). The response was dose dependent with maximal effect at GM‐CSF concentrations of 10 ng/ml and above. rhGM‐CSF was administered on seven occasions to six patients with malignant disease prior to chemotherapy. Plasma LF and unsaturated TC 1 and 3 levels rose significantly in each patient studied and the rise coincided with the initial neutropenia due to margination that occurs during infusions of rhGM‐CSF. Patients receiving rhGM‐CSF may therefore have hypofunctional neutrophils due to secondary granule depletion.
Two types of acquired idiopathic sideroblastic anaemia (AISA)
Authors: Gattermann Norbert , Aul Carlo , Schneider Wolfgang
Published: March 2008

On cytological bone marrow examination we distinguished between pure sideroblastic anaemia (PSA), which is confined to dyserythropoiesis, and refractory anaemia with ring sideroblasts (RARS), which is characterized by additional dysplastic features of granulopoiesis and/or megakaryopoiesis. In a follow‐up study of 94 patients with AISA diagnosed according to FAB criteria for myelodysplastic syndromes we found a striking difference in the risk of leukaemic transformation between PSA and RARS (5 year cumulative rate 1.9%v. 48%). Overall survival was much better in PSA than in RARS (5 year cumulative chance 69%v. 19%). Infections and haemorrhages were frequent causes of death in RARS but not in PSA.

Bone marrow culture studies (CFU‐GM) were performed on 10 consecutive patients with PSA and RARS, respectively. RARS patients showed grossly impaired colony growth, typical of the myelodysplastic syndromes. Patients with PSA had persisting colony formation, even if moderately decreased in frequency, with numbers of CFU‐GM being inversely correlated with the degree of erythroid hyperplasia in the bone marrow.

We conclude that on cytomorphological grounds AISA can be divided into pure (dyserythropoietic) sideroblastic anaemia (PSA) and a true myelodysplastic form (RARS), with both types differing considerably in terms of survival, risk of leukaemic transformation and findings on bone marrow culture (CFU‐GM).
A new human plasma cell line, Karpas 620, with translocations involving chromosomes 1, 11 and 14
Authors: Nacheva Elisabeth , Fischer Patricia E. , Sherrington Paul D. , Labastide Wayne , Lawlor Emer , Conneally Eibhlin , Blaney Clare , Hayhoe Frank G. J. , Karpas Abraham
Published: March 2008

We report here the establishment of a new cell line, Karpas 620 (K620), from the peripheral blood of an elderly woman with an IgG‐kappa plasma cell leukaemia (PCL). The line has the same hypotetraploid karyotype as the fresh cells from the patient. The cultured cells have the ultrastructural appearance of plasma cells with abundant rough endoplasmic reticulum (RER) and secrete kappa light chain. They are positive for surface antigens HLA DR, and WR17 (CD 37) and negative for CD1, CD3, CD4 and CD8. Using high resolution (HR) cytogenetic analysis it has been possible to identify all the marker chromosomes including several rearrangements commonly seen in malignancies of B cell lineage. These are a 14q + marker with a typical ‘Burkitt’morphology der(14)(pter→q32.3::8q24.1→qter) but with no reciprocal 8q–, and three translocations involving chromosome 11 at q 13 with partners other than chromosome 14, namely 1q32.1, 8q24.22 and 13q14.3. An earlier report of molecular studies on the DNA of K620 has shown a rearrangement near the region on 11q13 designated BCL‐1 (Rabbitts et al, 1988). This is the first report of a rearrangement in the region of 11q 13 in a cell line originating from a case of plasma cell leukaemia.
Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack
Authors: Yonemura Yuji , Kawakita Makoto , Koito Atsushi , Kawaguchi Tatsuya , Nakakuma Hideki , Kagimoto Tadashi , Shichishima Tsutomu , Terasawa Takashi , Akagaki Yohji , Inai Shinya , Takatsuki Kiyoshi
Published: March 2008

A 47‐year‐old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement‐sensitivity lysis tests, 80% of her erythrocytes were markedly complement‐sensitive (PNH‐III). Laser cytofluorimetry with a monoclonal antibody against decay‐accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF‐negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusions of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patient's erythrocytes. When the patient's serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.
Monoclonal antibodies against myeloperoxidase are valuable immunological reagents for the diagnosis of acute myeloid leukaemia
Authors: Van Der Schoot C. Ellen , Daams G. Marjolein , Pinkster Jantina , Vet Raymond , Kr.vondem Borne Albert E. G.
Published: March 2008

Whereas the diagnosis of acute lymphoid leukaemia greatly depends on immunophenotyping on the leukae‐mic cells, the diagnosis of acute myeloid leukaemia (AML) is still only based on morphological and cytochemical criteria. Here we describe that with a monoclonal antibody, directed against myeloperoxidase (MPO), the immunological diagnosis of AML is possible in most cases. A monoclonal antibody against lactoferrin (LF) was used to detect more mature myeloperoxidase‐containing cells. Of the cell samples tested from 206 different patients with AML, 95% were found to express myeloperoxidase in more than 15% of lactoferrin negative cells. Compared with other myeloid‐reactive monoclonal antibodies (VIM2, anti‐CD13, anti‐CD14, anti‐CD15 and anti‐CD33), a higher diagnostic sensitivity and specificity for AML was found. No significant correlation with the FAB classification was found. In most patients, more MPO‐positive cells were detected by the monoclonal antibody than by the cytochemical staining. This could be due to the recognition of enzymatically inactive precursor forms of myeloperoxidase by the antibody. The use of anti‐myeloperoxidase monoclonal antibodies for the diagnosis of AML has the advantage that objective quantification is possible.
Myelodysplastic syndrome (MDS)‐associated inhibitory activity on haemopoietic progenitor cells
Authors: Ohmori Masami , Ohmori Seiichi , Ueda Yasunori , Tohyama Kaoru , Yoshida Yataro , Uchino Haruto
Published: March 2008

We studied MDS‐associated inhibitory activity, which inhibited colony formation in vitro of granulocyte‐macrophage progenitors (CFU‐GM). Macrophages obtained from MDS bone marrow mononuclear cells (BM‐MNC) when pretreated with granulocyte‐macrophage colony stimulating factor (GM‐CSF) suppressed the growth of normal CFU‐GM. These macrophages were designated as ‘MDS‐derived inhibitory macrophages’. Media conditioned by MDS‐derived inhibitory macrophages (MDS‐CM) also suppressed the growth of normal CFU‐GM. In the MDS‐CM, high levels of prostaglandin E2 (PGE2) and ferritin were found. However, MDS‐CM did not contain detectable levels of tumour necrosis factor (TNF) or gamma‐interferon (γ‐IFN). Antiserum against human placental ferritin and/or against PGE2 blocked the haemopoietic inhibitory activity to some extent.

These results suggest that inhibitory macrophages may be responsible for the suppression of granulopoiesis in patients with MDS and that the suppression may be mediated by soluble factors including PGE2 and ferritin.
P47 phosphoprotein of blood platelets (pleckstrin) is a major target for phorbol ester‐induced protein phosphorylation in intact platelets, granulocytes, lymphocytes, monocytes and cultured leukaemic cells: absence of P47 in non‐haematopoietic cells
Authors: Gailani David , Fisher Thomas C. , Mills David C. B. , Macfarlane Donald E.
Published: March 2008

Aggregating agents including phorbol esters which activate protein kinase C induce the rapid phosphorylation of a Mr= 47 000 cytosolic protein in blood platelets (P47 or pleckstrin). This protein is well resolved by analytical 16‐BAC‐SDS two‐dimensional PAGE and was purified from platelets by preparative 16‐BAC‐SDS PAGE. Polyclonal antibodies were raised to the protein in mice and rabbits. These antisera detected a single protein with the migration of P47 on Western blots of platelet extracts, and the rabbit antisera immunoprecipitated 32P‐labelled P47 from platelet cytosol. The presence of P47 in other haematopoietic cells was determined by prelabelling them with 32P and observing increased 32P incorporation into the location of P47 on autoradiographs of 16‐BAC‐SDS analytical PAGE of cells exposed to phorbol ester. The identity of the phosphoprotein found in this location was further established by probing Western blots of SDS PAGE gels of cultured cell lines with the P47 antisera. P47 was detected in peripheral blood lymphocytes, monocytes and granulocytes (including the granulocytes of two unrelated patients with X‐linked chronic granulomatous disease). P47 was also found in HL‐60 promyelocytes (especially after differentiation with retinoic acid), U937 histiocytes, HEL leukaemia cells, and Raji ‘B’lymphoblasts. It was not detected in normal erythrocytes. K562 leukaemic cells, MOLT‐3 ‘T’lymphoblasts, or in wide range of non‐haematopoietic cell lines. We conclude that P47 is a major target for the action of phorbol ester induced phosphorylation in platelets, normal leucocytes and some haematopoietic cell lines. These cells have as their common feature the ability when stimulated to develop adhesive functions on their plasma membranes.
Re‐evaluation of the role of azathioprine in the treatment of adult chronic idiopathic thrombocytopenic purpura: a report on 53 cases
Authors: Quiquandon Isabelle , Fenaux Pierre , Caulier Marie Thérèse , Pagniez Dominique , Huart Jean Jacques , Bauters Francis
Published: March 2008

We treated 53 adults (mean age 54 years, range 17‐89; 37 females and 16 males) with chronic idiopathic thrombocytopenic purpura (ITP) by azathioprine. All patients had received at least one form of therapy (including splenectomy in 40 patients) and had less than 50 x 109/1 platelets. The duration of ITP before azathioprine was started ranged from 6 to 350 months (median 19). All patients initially received 150 mg/d of azathioprine. This was associated with a short initial course of prednisone (0.3–0.5 mg/kg d) in 10 of them, who were refractory to prednisone alone. 34 (64%) patients responded, including 24 (45%) complete remissions (CR), three (6%) partial remissions (PR) and seven (13%) minor responses (MR). Median time to achieve response was 4 months. 17 of the CR persisted after 7–182 months, 10 of them after discontinuation of azathioprine. Seven patients relapsed after 4–26 months, five of them after azathioprine was stopped or its dose was reduced. PR were short and the median duration of MR was 8 months. Overall, 21 patients (40%) had responses lasting 1 year or more and 17 (32%) lasting 2 years or more. Median duration of treatment was 18 months (range 3–84). Five patients died of bleeding while on treatment. No prognostic factors for response to azathioprine were found. Mild leucopenia was seen in seven patients and a moderate (x3) increase in transaminases in two patients. No opportunistic infections were seen and no malignancy has occurred since the onset of azathioprine. We conclude that azathioprine gives a relatively high incidence of durable responses and very limited side effects in chronic ITP, when splenectomy has failed or is contraindicated. This efficacy, in our experience, is superior to that obtained with other therapeutic approaches. As responses may be delayed, a course of azathioprine of 4 months is required before one can infer a failure to respond. In responding patients, however, the optimum duration of treatment remains to be established.
Increased platelet sensitivity to ristocetin is predicted by the binding characteristics of a GPIb/IX determinant
Authors: Miller Jonathan L. , Hustad Kent O. , Kupinski John M. , Lyle Vicki A. , Kunicki Thomas J.
Published: March 2008

Platelets from patients with platelet‐type von Willebrand disease (vWD) were used as immunogens for the production of murine monoclonal antibodies (MoAbs). One such MoAb, C‐34, inhibited ristocetin‐induced aggregation of patient or normal platelets. but not aggregation induced by other aggregating agents. As demonstrated by crossedimmunoelectrophoresis, C‐34 recognized an epitope within the GPIb/IX complex. In indirect immunofluorescence studies on fresh platelets, the ratio of any of four different anti‐GPIb MoAbs to one another was near unity (0.88‐1.14) both for normals and for patients. In contrast, the ratio of the binding of C‐34 to such a MoAb (AP‐1) was 0.31 ± 0.02 (x± SE) for normal platelets and significantly increased to 0.54 ± 0.01 for patient platelets (P < 0.001). In NP‐40 lysates of 3H‐labelled platelets, saturating concentrations of C‐34 produced much fainter bands than did AS‐2 or other anti‐GPIb MoAbs in the GPIb and GPIX regions. In contrast to the other anti‐GPIb MoAbs. C‐34 did not bind to the purified 125I‐Iabelled glycocalicin fragment of GPIb, to the glycocalicin derivative identified by crossed‐immunoelectrophoresis, or to the amino‐terminal ˜ 40 kDa portion of GPIbα cleaved from 3H‐labelled platelets by trypsin. C‐34 appears to be the first MoAb that is quantitatively informative in identifying the abnormal platelets in platelet‐type vWD. The observed differences between the patient and normal platelets may reflect an abnormality in the primary structure of the GPIb/ IX complex. Alternatively, patient platelets may have an abnormality of other structures near this region that impose less of a steric hindrance upon binding of antibody to the C‐34 epitope.
A genetic defect of erythrocyte band 4.2 protein associated with hereditary spherocytosis
Authors: Ideguchi Hiroshi , Nishimura Junji , Nawata Hajime , Hamasaki Naotaka
Published: March 2008

We report two patients with hereditary spherocytosis associated with band 4.2 protein deficiency from a Japanese family. The defect of band 4.2 protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) not only in freshly prepared white ghosts but also in washed whole erythrocytes. The finding was quite reproducible and was also recognized postsplenectomy. The interaction of ankyrin with band 3 in the patients’ghosts was stable both at low ionic strength and at acidic pH. Our results suggested that band 4.2 protein might not be essential for the structural stability of band 3‐ankyrin interaction. On the other hand, membrane protein phosphorylation studies revealed an increased phosphorylation of spectrin/ankyrin, band 3 and band 4.1 in the patients’erythrocytes as compared with normal cells. The finding might be related to a dysregulation of protein phosphorylation which could result in membrane instability in affected cells. Band 4.2 deficiency is an inherited disorder in association with hereditary haemolytic anaemias and seems to be relatively prevalent in the Japanese population.
CRM+ haemophilia A due to a missense mutation (372→Cys) at the internal heavy chain thrombin cleavage site
Authors: Pattinson J. K. , McVey J. H. , Boon M. , Ajani A. , Tuddenham E. G. D.
Published: March 2008

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non‐conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non‐carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.
An infrequent DNA polymorphism associated with severe von Willebrand's disease
Authors: Caekebeke‐Peerlinck Kathelijne M. J. , Bakker Egbert , Briet Ernest
Published: March 2008

Genomic DNA of six unrelated Dutch patients with severe von Willebrand's disease (vWD) was submitted to restriction fragment length polymorphism analysis. We observed a strong association between a 36 kb allele detected by a partial complementary DNA probe (pvWF 1100) and the restriction enzyme XbaI with severe von Willebrand's disease. This 36 kb allele is rare (allele frequency of 7%) both in the general population and in patients with autosomal dominant types of von Willebrand's disease. Three of our six patients were found to be homozygous for this allele while two others were heterozygous. The association of this rare XbaI allele with severe vWD enables carrier detection and prenatal diagnosis in these families. The high frequency (67%) of the 36 kb allele observed in this patient group raises the possibility that a subgroup of patients with severe vWD has a genetic defect with a common origin.
Development of a highly sensitive assay, based on the polymerase chain reaction, for rare B‐lymphocyte clones in a polyclonal population
Authors: Brisco M. J. , Tan L. W. , Orsborn A. M. , Morley A. A.
Published: March 2008

A method has been developed to use the polymerase chain reaction to amplify and sequence the chain determining region 3 (CDR3) of the human immunoglobulin heavy‐chain gene, and to use the sequence as a marker for rare neoplastic B lymphocytes. Consensus primers for the Variable and Joining regions of the gene were constructed and shown to enable efficient amplification, directed cloning, and sequencing of CDR3. Using leukaemic cell line PFMC as a test system, CDR3 was sequenced, specific primers synthesized, and PFMC DNA was detected down to a dilution of 1:1300 in DNA from normal lymphocytes. This strategy should be useful for monitoring therapy and detecting early disease relapse in B lymphoproliferative disease.
Stem cell recovery from cyclophosphamide‐induced myelosuppression requires the presence of CD4+ cells
Authors: Pantel Klaus , And Zora Djuric , Nakeff Alexander
Published: March 2008

Recently, we have reviewed studies regarding the growth‐stimulating effect of CD4+ cells on haematopoietic cells in culture (Pantel & Nakeff, 1989a). In the present study we have tested the physiologic relevance of this interaction using a drug‐perturbed mouse model. The long‐term application of cyclophosphamide (CY, 30 mg/kg/d, five i.p. injections per week over 7 weeks) in B6D2F1 mice resulted in initial CY‐induced cytotoxicity to CFU‐S followed by the re‐establishment to pretreatment values of the femoral content of CFU‐S within 2–3 weeks of CY‐treatment. An examination of CY‐metabolism in these treated mice excluded a pharmacological explanation for the compensation of CY‐cytotoxicity. However, a three‐fold increase in the cycling fraction of CFU‐S (determined by in vivo hydroxyurea suicide) was observed concomitant with a two‐fold increase in the femoral content of L3T4+ cells (the murine equivalent to human CD4+ cells), as compared to the corresponding values in untreated mice. Ablating these L3T4+ cells in vivo by means of a cytotoxic monoclonal antibody (MoAb) to the L3T4 determinant resulted in a decrease in the cycling fraction of CFU‐S from 56.8% to essentially zero and a decrease in the femoral content of CFU‐S when comparing mice receiving either CY alone or CY plus MoAb, respectively. It would appear that the CY‐induced increase in the proliferative activity of CFU‐S requires the presence of L3T4+ cells. These data constitute the first in situ evidence for the physiologic relevance of immunocompetent L3T4+ cells as regulators involved in the recovery of stem cells from drug‐induced myelosuppression.
Exercise‐induced CD8 lymphocytosis: a phenomenon associated with large granular lymphocyte leukaemia
Authors: Mulligan S. P. , Wills E. J. , Young G. A. R.
Published: March 2008

This report describes a patient with a large granular lymphocyte leukaemia (CD8 + lymphoproliferative disease) and severe neutropenia (<0.5×109/l) in whom exercise resulted in a marked lymphocytosis, a phenomenon which has not previously been recorded. The lymphocyte count at rest was within normal limits (2.2 × 109/l), rose to a peak with strenuous exercise (38 x 109/l), then fell to the resting level within 15 min of cessation of exercise. The peripheral blood mononuclear cells showed the morphology of large granular lymphocytes (LGL) by light and electron microscopy both at rest (30%) and to a much greater extent during exercise (70%). Immunophenotyping of these lymphocytes during exercise demonstrated that the predominant cell was CD3+, CD8+, CD57+ (Leu7)/CD4‐, CD16‐, CD25‐. In the resting state, despite a total lymphocyte count within the normal range, surface marker studies indicated an excess of cells with the CD8+/CD57 + T cell phenotype (26%; cf. normal range ≦10%). Functional assays revealed a minimal increase in natural killer (NK) activity during exercise. T cell receptor β chain gene rearrangement was demonstrable in the peripheral blood at rest and during exercise. Although severe neutropenia was present, the growth of normal colony forming units, granulocyte‐macrophage (CFU‐GM) was not inhibited by patient lymphocytes and no anti‐neutrophil antibodies were demonstrated. Finally, hyposplenism has developed and the relationship of this to the LGL leukaemia is discussed. In summary, the findings demonstrated large granular lymphocyte leukaemia as the primary disorder for which the primary manifestation, apart from the neutropenia, was a marked exercise‐induced lymphocytosis.
A mutation adjacent to the beta cleavage site of factor IX (valine 182 to leucine) results in mild haemophilia Bm
Authors: Taylor S. A. M. , Liddell M. B. , Peake I. R. , Bloom A. L. , Lillicrap D. P.
Published: March 2008

The genetic basis of a mild form of haemophilia Bm has been investigated. The patient under investigation has a mild bleeding disorder and has never experienced spontaneous bleeds. Factor IX coagulant activity (FIX:C) was 0.15 units/ml and factor IX antigen (FIX:Ag) 1.32 units/ml. The prothrombin time performed with an ox brain thromboplastin was 65 s (normal plasma 31 s). Studies of the abnormal factor IX protein in this patient showed a normal molecular weight and normal calcium binding properties. Activation of the mutant factor IX with factor XIa showed normal proteolytic cleavage. DNA sequence from the eight factor IX exons and flanking introns was amplified from this patient using the polymerase chain reaction. The amplified material was subjected to direct chain termination nucleotide sequencing. The only nucleotide sequence alteration found was a G→C transversion at nucleotide 20.524, changing the amino acid encoded at residue 182 from valine to leucine. This residue is one amino acid removed from the beta cleavage site of factor IX. This residue is highly conserved in other vitamin K dependent serine proteases and we propose that its alteration in this patient is responsible for his mild haemophilic phenotype, and for the abnormal interaction of this factor IX protein with the extrinsic system of coagulation.
Clinical and laboratory evaluation of the treatment of von Willebrand's disease patients with heat‐treated factor VIII concentrate (BPL 8Y)
Authors: Pasi K. J. , Williams M. D. , Enayat M. S. , Hill F. G. H.
Published: March 2008

Adequate rises of vWF:activity and satisfactory haemostasis have been obtained in six von Willebrand patients treated with heated 8Y factor VIII concentrate, despite multimeric analysis showing the concentrate to have an alteration in the proportions of the different vWF:Ag multimers including slight loss of the highest molecular weight multimers and an abnormal triplet. As heat‐treated concentrates reduce the risk of transmitting blood borne viruses 8Y concentrate should be considered as a possible first line treatment of vWD patients in the U.K. if they do not have mild forms of type I von Willebrand's disease which can be treated with desmopressin (DDAVP) infusions.
Two human monoclonal antiplatelet autoantibodies established from patients with chronic idiopathic thrombocytopenic purpura
Authors: Honda Shigenori , Tsubakio Tadahiro , Tomiyama Yoshiaki , Take Hironori , Furubayashi Takayasu , Mizutani Hajime , Kurata Yoshiyuki , Yonezawa Takeshi , Tarui Seiichiro
Published: March 2008

Two human hybridomas secreting antiplatelet autoantibodies were established by somatic cell fusion using splenocytes from two patients with chronic idiopathic thrombocytopenic purpura (ITP). These monoclonal antibodies, HT7F and HT8C, were of the IgM isotype and reacted with autologous and allogeneic platelets fixed with paraformaldehyde (PFA). They also bound to fresh platelets. An elution study showed that eluates from allogeneic platelets reacted with autologous platelets. These results indicated that HT7F and HT8C were autoantibodies recognizing a site on the platelet surface. Both monoclonal antibodies were able to induce complement activation in vitro. HT7F was demonstrated to bind to a platelet protein having a molecular mass of 105 kDa under both nonreducing and reducing conditions. No human hybridoma synthesizing antibody against 105 kDa platelet protein has been reported to date. These antibodies may play a role in the pathogenesis of thrombocytopenia in some ITP patients.
Studies of an oral iron chelator: 1,2 ‐dimethyl‐ 3 ‐hydroxy‐pyrid‐4‐one I. IRON EXCRETION IN RATS: DEVELOPMENT OF A NEW RAPID MICROWAVE METHOD FOR IRON ANALYSIS IN FAECES
Authors: Venkataram S. , Rahman Y. E.
Published: March 2008

We have developed a simple, rapid method for analysing faecal iron using a microwave oven for digestion followed by atomic absorption spectrometry. The chelating effect of l,2‐dimethyl‐3‐hydroxy‐pyrid‐4‐one (DMHP) has been tested in rats with experimental iron overloading and three routes of DHMP administration, oral, subcutaneous, and intraperitoneal, have been compared. Regardless of the route of administration, we have found that DMHP promotes iron excretion via the urine. We have not observed a difference in the amount of iron excreted in the faeces before and after DMHP administration by any route. The subcutaneous route of administration is the most effective in promoting iron excretion, followed by the intraperitoneal route. Although most convenient for clinical use, oral administration promotes the excretion of only a small fraction of that by the subcutaneous route.
Inhibition of lymphocyte IL2‐receptor expression by factor VIII concentrate: a possible cause of immunosuppression in haemophiliacs
Authors: Hay C. R. M. , McEvoy P. , Duggan‐Keen M.
Published: March 2008

Factor VIII concentrate inhibits T‐cell function in vitro and in vivo. The mechanisms underlying this phenomenon were investigated. Factor VIII concentrate has a direct effect on lymphocytes, uninfluenced by haemophilic mono‐cyte dysfunction, since it inhibited lymphocyte transformation with phorbol myriscate acetate, a reaction unaffected by monocyte depletion. Inhibition of lymphocyte transformation by factor VIII concentrate is not corrected by the addition of exogenous IL2, suggesting that it does not inhibit lymphocyte function by suppression of IL2 secretion alone. Factor VIII concentrate causes profound inhibition of IL2‐receptor expression (CD25); with an 89% reduction in CD25‐positive CD4 cells and a 50% reduction in CD25‐antigen molecules per cell. CDS lymphocytes are similarly affected. Smaller reductions in CD71 and HLA‐DR expression are also observed. Down modulation of CD25‐antigen may explain the reduced IL2 secretion observed by others, and may be an important cause of immunodeficiency in HIV‐seronegative haemophiliacs.
Tissue sources of murine megakaryocyte potentiator: biochemical and immunological studies
Authors: Banu Naheed , Fawcett Jenny , Williams Neil , De Giorgio Toni , Withy Raymond
Published: March 2008

Summary. The immunological and biochemical characteristics of murine megakaryocyte potentiator from lung and bone marrow were examined and compared with thrombopoietic stimulatory factor. Biological activity was not neutralized by anti‐erythropoietin, but megakaryocyte potentiator activity from all three sources was abolished or reduced when the preparations were treated with anti‐thrombopoietic stimulatory factor or anti‐interleukin‐6. Megakaryocyte potentiator levels in lung conditioned medium were not found to be enhanced from mice treated with lipopolysaccharide, in contrast to granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) levels. The biochemical properties of murine megakaryocyte potentiator from lung and bone marrow were compared and found to be similar in the elution profiles from anion exchange, gel filtration and reversed phase liquid chromatography. It is concluded that the activities in lung and bone marrow are very similar if not identical, to interleukin‐6.
Colony stimulating activity in the serum of patients with multiple myeloma is enhanced by interleukin 3: a possible role for interleukin 3 after high dose melphalan and autologous bone marrow transplantation for multiple myeloma
Authors: Millar B. C. , Bell J. B. G. , Montes A. , Millar J. L. , Maitland J. A. , Treleaven J. , Viner C. , Gore M. , McElwain T. J.
Published: March 2008

Summary. Sera from 36/37 multiple myeloma patients and 19/21 sera from patients with other solid or liquid tumours had granulocyte‐macrophage colony stimulating activity (CSA) towards normal human donor bone marrow whereas 1/16 sera from normal donors had this activity. Unlike human rhGM‐CSF and GM‐CSF from 5637 (human bladder cell line) conditioned medium which is heat stable, CSA from serum is heat labile (56°C/30 min). In multiple myeloma patients, CSA was detectable more than 2 years after treatment with high dose melphalan. Although multiple myeloma patients, at relapse, have sufficient CSA in their serum to produce maximal stimulation of GM‐CFUc from normal donor bone marrow in vitro, their own GM population responds poorly. The results suggest that the failure of patients own bone marrow to respond to endogenous CSA may be due to damage to the stem cells of the marrow or the failure of precursor cells to respond to CSA. Addition of rhIL‐3 to myelomatous serum increased the number of GM‐CFUc from both normal and myelomatous bone marrow but did not stimulate the growth of MY‐CFU, significantly. The results suggest that rhIL‐3 may assist bone marrow recovery in multiple myeloma patients after intensive chemotherapy.
Development of a combined storage medium for 7‐day storage of platelet concentrates and 42‐day storage of red cell concentrates
Authors: Heaton W. A. L. , Holme S. , Keegan T.
Published: March 2008

Summary. Studies were carried out to examine whether a single additive solution could support both platelet and red cell storage. An ionically balanced electrolyte solution fortified with citrate, glucose and bicarbonate was used. This solution has previously been shown to provide good platelet viability with storage for up to 7 d. Optimization studies demonstrated that with adenine added to this solution (CSM), it also allowed for satisfactory preservation of in vitro red cell parameters for up to 49 d of storage. Confirmatory paired in vivo post‐transfusion studies were carried out in which platelets and red cells obtained from the same donor were processed and stored in CSM on one occasion, and in CPD‐plasma (platelets) and AS‐1 (red cells) on another occasion. Five paired studies were conducted with platelets stored for 5 d and red cells for 42 d; another five paired studies with platelets stored for 7 d and red cells for 49 d. Except for a slight decrease in platelet survival (P<0.05) with storage in CSM, there were no statistically significant differences in post‐transfusion recoveries and survivals between test and control media, demonstrating that both platelets and red cells may be satisfactorily stored in a combined single additive solution.
Co‐expression of haemopoietic antigens on vascular endothelial cells: a detailed phenotypic analysis
Authors: Favaloro Emmanuel J. , Moraitis Nick , Bradstock Kenneth , Koutts Jerry
Published: March 2008

Over 1000 monoclonal antibodies (MAB), previously characterized in terms of reactivity with various haemopoietic cells and including representatives from each of the currently known Clusters of Differentiation (CD), have now been evaluated by us for reactivity against human endothelial cells. In the current study, screening was performed on both fixed, and on non‐fixed, endothelial cells derived and cultured from umbilical cord veins. Results indicate that endothelial cells co‐express a wide variety of antigens that are otherwise characterized by expression on haemopoietic cells. The most intense and consistent degree of reactivity was seen using MAB to CD‐9, CD‐13, CD‐29, CD‐31, CD‐44, CD‐46, CD‐51, CD‐54 and CD‐55, although reactivity was also observed using MAB belonging to CD‐41, CD‐47, CD‐49, CD‐61 and CD‐71, in addition to a number of non‐clustered MAB.
Negative regulation of human megakaryocytopoiesis by human platelet factor 4 and beta thromboglobulin: comparative analysis in bone marrow cultures from normal individuals and patients with essential thrombocythaemia and immune thrombocytopenic purpura
Authors: Han Z. C. , Bellucci S. , Tenza D. , Caen J. P.
Published: March 2008

The effect of human platelet factor 4 (PF4) and beta‐thromboglobulin (BTG) on megakaryocyte colony formation in normal subjects as well as in essential thrombocythaemia (ET) and in immune thrombocytopenic purpura (ITP) was studied. Both PF4 and BTG were found to be capable of inhibiting the development of isolated megakaryo‐cytes and their colonies in normal marrow cultures in a dose‐dependent fashion. A significant 50% inhibition was seen at a PF4 or BTG concentration of 1–2·5 μg/ml, and complete inhibition in the range of 5–10 μg PF4 or BTG/ml. The two platelet proteins had similar effects on megakaryocyte development. A combination of PF4 and BTG resulted in an additive effect. Antibodies against PF4 or BTG could effectively neutralize the inhibitory effect of PF4 or BTG respectively. In ET and ITP, in vitro megakaryocyte development was also inhibited by PF4 and BTG in a similar way to that seen in normal subjects, suggesting that the responsiveness of megakaryocyte progenitors to PF4 and BTG is normal in these two disorders. PF4 and BTG did not affect the growth of colony forming units granulocyte‐macrophage (CFU‐GM) except at very high concentration (≥ 10 γg/ml) but they did inhibit erythroid colony formation by normal and ET burst forming units erythroid (BFU‐E). However, the inhibition of BFU‐E by PF4 and BTG was dose‐related, and a 50% inhibition required a PF4 or BTG dose ranging from 5 to 10 μg/ml. These results indicate that PF4 and BTG are involved in negative regulation of normal and pathologic megakaryocytopoiesis and that their inhibition acts predominantly on the megakaryocytic lineage.
Ras activation in myelodysplastic syndromes: clinical and molecular study of the chronic phase of the disease
Authors: Melani Cecilia , Haliassos Alexandre , Chomel Jean Claude , Miglino Maurizio , Ferraris Anna Maria , Gaetani Gian Franco , Kaplan Jean Claude , Kitzis Alain
Published: March 2008

We studied N‐ras and Ki‐ras point mutations respectively at codons 12–13 and 12 in 15 patients with myelodysplastic syndromes (MDS) using the polymerase chain reaction (PCR) method for DNA amplification, and slot blot hybridization to allele specific oligonucleotide (ASO) probes. We analysed peripheral blood and bone marrow samples collected at diagnosis and repeatedly during the chronic phase of the disease to define when the activation occurred and in which haemopoietic cell populations, in order to establish possible relationships between clinical and molecular features. In three cases the N‐ras oncogene was mutated at codon 12 in every cell population, both at diagnosis and throughout the chronic phase. Point mutations were not seen at the 12 codon of the Ki‐ras oncogene. In patients lacking activated ras oncogene at diagnosis, mutations were not discovered during the entire period of observation. Therefore in our cases disease progression and leukaemic transformation did not correlate with the presence of the activated N‐ras. Our data suggest that ras activation occurs early in the pathogenesis of MDS and involves a haemopoietic progenitor with multiple differentiative capacity, without however conferring an apparent proliferative advantage on its progeny.
A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin–antithrombin complex, D‐dimer and fibrinogen/fibrin fragment E antigen
Authors: Boisclair M. D. , Lane D. A. , Wilde J. T. , Ireland H. , Preston F. E. , Ofosu F. A.
Published: March 2008

Measurements were made of levels of D‐dimer in plasma and serum, thrombin–antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r>0·9) were established between plasma and serum levels of D‐dimer, between plasma levels of D‐dimer and serum levels of FgE, and between serum levels of D‐dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97–100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D‐dimer assay, whilst both the FgE and D‐dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad‐spectrum FgE assay was better than the more specific D‐dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT.
Previous donor pregnancy as a risk factor for acute graft‐versus‐host disease in patients with aplastic anaemia treated by allogeneic marrow transplantation
Authors: Flowers Mary E. D. , Pepe Margaret Sullivan , Longton Gary , Doney Kristine C. , Monroe Deborah , Witherspoon Robert P. , Sullivan Keith M. , Storb Rainer
Published: March 2008

To determine if previous donor pregnancies influence the development of acute graft‐versus‐host disease (GVHD) we evaluated data from 136 patients with aplastic anaemia greater than 15 years of age and given marrow grafts from HLA‐identical sibling donors. Of the 136 marrow donors, 30 were parous females (previous history of pregnancy), 30 were nulliparous females (no history of pregnancy or abortions), and 76 were males. The cumulative incidence of grade II‐IV GVHD was 57%, 21% and 46% for patients with parous, nulliparous and male donors, respectively. A multivariate analysis of the data confirmed that the risk of grade II‐IV acute GVHD was significantly increased among patients receiving marrow from parous females as compared to those from nulliparous females (relative risk = 2·5,P= 0·02). There was no statistically significant difference in the incidence of acute GVHD, however, between patients with parous donors and male donors (relative risk = 1·3,P= 0·26). Male patients given grafts from parous donors showed a higher incidence of acute GVHD (63%) than female patients (45%), though this difference was not statistically significant. The 5‐year probability of survival was 47% for patients with parous donors, 68% for patients with nulliparous donors and 70% for those with male donors. We confirm that prior donor pregnancy represents an important factor in selecting marrow donors or designing clinical protocols for GVHD prophylaxis.
Deficiency of glycosyl‐phosphatidylinositol anchored proteins on paroxysmal nocturnal haemoglobinuria (PNH) neutrophils and monocytes: heterogeneous deficiency of decay‐accelerating factor (DAF) and CD16 on PNH neutrophils
Authors: Kawakami Zenji , Ninomiya Haruhiko , Tomiyama Junji , Abe Tsukasa
Published: March 2008

Glycosyl‐phosphatidylinositol (GPI) anchored membrane proteins have been reported to be deficient on affected paroxysmal nocturnal haemoglobinuria (PNH) blood cells. In the present study we investigated the deficiency of several GPI anchored membrane proteins on PNH neutrophils (PMN) and monocytes from 10 patients with PNH. Decay‐accelerating factor (DAF) and FcyR‐III (CD 16) on PMN, DAF and CD14 on monocytes, were investigated by two‐colour immunofluorocytometry. Neutrophil alkaline phosphatase activity was also assayed on PNH neutrophils. Normal human PMN were always shown phenotypically to be DAF+/CD16+. A DAF−/CD16− subpopulation of PMN was demonstrated in all the patients studied. In six out of the 10 patients, deficiencies of DAF and CD16 were found simultaneously on affected PNH PMN. The percentage of DAF− PMN showed a positive correlation with the neutrophil alkaline phosphatase (NAP) score. However, it should be noted that, in four out of the 10 patients with PNH, a DAF+/CD16− subpopulation of PMN was also clearly found. This may indicate that the deficiencies of DAF and CD16 on PNH PMN are heterogeneous. Normal human monocytes were demonstrated to be DAF+/CD14+, whereas PNH monocytes consisted of subpopulations of DAF+/CD14+ and DAF−/CD14−. In the same patients with PNH, the deficiencies of DAF on PMN and monocytes correlated well with each other. These results suggest that, at least in some patients with PNH, the mechanisms which induce the membrane defects of PNH blood cells are heterogeneous.
Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem‐cell glycoprotein CD34 (My 10)
Authors: Geller Robert B. , Zahurak Marianna , Hurwitz Craig A. , Burke Philip J. , Karp Judith E. , Piantadosi Steven , Civin Curt I.
Published: March 2008

A series of 23 monoclonal antibodies reactive with normal lymphoid and myeloid cells at various stages of differentiation were used to characterize 96 adult patients with acute myelocytic leukaemia (AML), concentrating on the possible role the expression of these antigens may have in predicting response to intensive chemotherapy. Only the expression of CD34 (P= 0·008) and HLA‐DR (P= 0·035) was significant in predicting response to therapy; patients with leukaemic cells expressing CD34 (My10) had a complete remission (CR) rate of 59% compared to 87% for those with blasts not expressing the antigen. In a multivariate analysis predicting for CR, the expression of CD34, the disease category (de novoAML versus secondary AML [SAML] or a history of antecedent haematological disorder [AHD]), and WBC were significant covariates. Adjusting for disease category and WBC, patients with CD34‐positive AML were one‐third as likely to enter CR as with those with disease not expressing the antigen (P= 0·066). Comparison of clinical characteristics between the 58 patients whose leukaemia expressed CD34 and the 33 which were CD34‐negative found that patients with CD34‐positive AML had a higher incidence of SAML and AHD, a lower WBC at diagnosis, and a more frequent incidence of chromosomal abnormalities involving chromosomes 5 and/or 7. Twenty‐eight of these patients also had immunophenotyping performed at relapse. Patients who presented with CD34‐positive AML, and entered remission, and then relapsed all recurred with CD34‐positive leukaemia; there was no case of CD34‐positive AML at diagnosis relapsing with CD34‐negative disease. In addition, there were patients presenting with CD34‐negative AML and then relapsing with CD34‐positive AML. These results suggest that intensive cytoreductive therapy is ineffective against CD34‐positive AML. Patients who present with CD34‐positive AML may require different therapeutic approaches to completely eradicate their disease.