European retrospective study of real-life haemophilia treatment.

INTRODUCTION: Haemophilia treatment varies significantly between individuals, countries and regions and details of bleed rates, factor consumption and injection frequency are often not available. AIM: To provide an overview of the FVIII/FIX treatment practice and outcome for patients with haemophilia A (HA) or haemophilia B (HB) across Europe. METHODS: Non-interventional, 12-month retrospective study where anonymized data were retrieved from haemophilia centres/registers in Belgium, France, Germany, Italy, Spain, Sweden and the United Kingdom. Male patients (all ages) receiving coagulation factor treatment 24 months prior to the study, with basal FVIII/FIX levels ≤5 IU dL-1 , without inhibitors, were included. Data were summarized descriptively. RESULTS: In total, 1346 patients with HA and 312 with HB were included in the analysis; 75% and 57% had severe disease (FVIII/FIX < 1 IU dL-1 ) respectively. Prophylaxis was most common for severe haemophilia, especially for children, whereas on-demand treatment was more common for moderate haemophilia in most countries. The mean (SD) prescribed prophylactic treatment ranged from 67.9 (30.4) to 108.4 (78.1) (HA) and 32.3 (10.2) to 97.7 (32.1) (HB) IU kg-1 per week, across countries. Most patients on prophylaxis were treated ≥3 times/week (HA) or two times/week (HB). The median annual bleeding rate (ABR) for patients on prophylaxis ranged from 1.0 to 4.0 for severe HA, and from 1.0 to 6.0 for severe HB, while those with moderate haemophilia generally had slightly higher ABRs. Median ABRs for on-demand-treated severe HA ranged from 4.5 to 18.0, and for HB, 1.5 to 14.0. CONCLUSION: Treatment practice varied greatly between centres and countries and patients treated on-demand and prophylactically both experienced bleeds, emphasizing the need for further optimization of care.

Agonistic anti-human Fas monoclonal antibody induces fibroblast-like synoviocyte apoptosis in haemophilic arthropathy: potential therapeutic implications.

Haemophilic arthropathy (HA) is characterized by chronic proliferative synovitis leading to cartilage destruction and shares some pathological features with rheumatoid arthritis (RA). Apoptosis has been implicated in RA pathogenesis, and an agonistic anti-Fas monoclonal antibody (mAb) was found to induce RA fibroblast-like synoviocyte (FLS) apoptosis and suppress synovial hyperplasia in animal models of RA. The aim of this study was to evaluate the effect of anti-Fas mAb on HA-FLS. FLS were isolated from knee synovial biopsies from six HA patients, six RA patients and six healthy subjects. The expression of Fas in synovial biopsies was investigated by immunohistochemistry. FLS were stimulated with anti-Fas mAb at different concentrations, alone or in combination with tumour necrosis factor-α (TNF-α) and basic fibroblast growth factor (bFGF). Fas expression in FLS was assessed by Western blot. Cell viability was studied with the WST-1 assay. Active caspase-3 levels were measured using ELISA and Western blot. A strong Fas-immunoreactivity was observed in different cells of HA synovium, including FLS, inflammatory cells and endothelial cells. Fas antigen was constitutively overexpressed in cultured HA-FLS. Anti-Fas mAb had a significant cytotoxicity on HA-FLS in a dose-dependent manner, either alone or in combination with TNF-α and bFGF. These cytotoxic effects were due to the ability of anti-Fas to induce HA-FLS apoptosis, as shown by the increased active caspase-3 levels. Anti-Fas mAb exhibited a more pronounced pro-apoptotic effect on HA-FLS than RA-FLS. Fas antigen is highly expressed on HA-FLS and its stimulation by anti-Fas mAb may be an effective strategy to induce HA-FLS apoptosis.

Hypovitaminosis D and osteopenia/osteoporosis in a haemophilia population: a study in HCV/HIV or HCV infected patients.

Recent reports show a correlation between haemophilia and osteoporosis. HIV, HCV and their treatments are independently associated with an increased risk of osteoporosis. Vitamin D plays a pivotal role in bone mineralization. The aim of our study was to compare Vitamin D levels, bone metabolism markers and bone mineral density (BMD) in patients with haemophilia with or without co-infections. Seventy-eight adult patients with severe or moderate haemophilia A or B were subdivided into three groups of 26 patients each (HIV-HCV co-infected, HCV mono-infected and uninfected). The BMD was measured by dual energy X-ray absorptiometry (DXA) at both the femoral area (F) and lumbar spine (L). This was correlated to laboratory values and haemophilic arthropathy was assessed using validated clinical and radiological scores. The DXA showed a homogeneous F-BMD reduction in all the three groups, whereas L-BMD was significantly lower in co-infected patients (P < 0.05). The clinical score was higher in co-infected (P < 0.002) and mono-infected (P < 0.006). The radiological score was higher in mono-infected than in the other two groups (P < 0.001). Overall 25-hydroxyvitamin D (25-OH Vit D) was reduced (87%). Bone-specific alkaline phosphatase (b-ALP) and telopeptide were increased in co-infected (P < 0.001 and P < 0.01) and mono-infected (P < 0.001 and P < 0.02). The result of the homogeneous F-BMD reduction in all groups could be explained by the pivotal role of arthropathy; the lower L-BMD in co-infected and the increase of b-ALP and telopeptide in co-infected and mono-infected groups suggest faster bone metabolism in case of infections.

Viscosupplementation in haemophilic arthropathy: a long-term follow-up study.

Haemophilic arthropathy is the most common clinical manifestation of haemophilia, secondary to recurrent haemarthroses and chronic synovitis. Modern bleeding-preventing drugs have limited significantly the incidence of severe arthropathy, and primary approach is usually conservative. Use of intra-articular injections of hyaluronan acid is considered one of the most efficient treatments for early stages of articular degenerative diseases. Assessment of long-term effectiveness of intra-articular administration of hyaluronic acid (HA) in knees, ankles and elbows of patients affected by haemophilic arthropathy was done for 46 patients (10 elbows, 24 knees and 25 ankles) affected by haemophilic arthropathy. They received injections of HA and were evaluated with Visual Analogue Scale, Short Form-36, World Federation of Haemophilia score and Petterson score with a 6-year mean follow-up. Most of the patients showed improvement in pain relief and functional recovery without any complications: only a limited number of patients (8.6%) found poor results, undergoing surgery or other further treatments in the follow-up period for persistent pain or limitation. Viscosupplementation is an effective therapeutic strategy in early stages of haemophilic arthropathy, with no complications and long-term good clinical results.

Ultrasound detects joint damage and bleeding in haemophilic arthropathy: a proposal of a score.

Haemarthrosis triggers haemophilic arthropathy (HA) because bleeding starts synovitis immediately, damages cartilage and leads to loss of function and disability. The aim of our study was to investigate the capacity of ultrasonography (US) in detecting bleeding and joint damage in HA. The joints of 62 patients (pts) with haemophilia A or haemophilia B were consecutively evaluated and scored (score ranging from 0 to 21) for effusion (E), bone remodelling (BR), cartilage damage (CD), synovial hypertrophy (SH), haemosiderin (H), osteophytes (O), haemarthrosis (Hae), erosion (Er) and fibrotic septa (FS) with US. X-rays [Pettersson Score (PXS)] were performed in 61 patients and clinical evaluation [World Federation Haemophiliac orthopaedic score (WFHO)] was performed in all patients. A total of 20 healthy subjects and 20 patients affected by Rheumatoid Arthritis (RA) were used as controls. Power Doppler US (PDUS) was performed in all patients on the knee, ankle and elbow joints. A total of 83 joints were studied (50 knees; 12 elbows and 21 ankles). US showed effusion in 57 joint, bone remodelling in 62, cartilage damage in 64, synovial hypertrophy in 45, haemosiderin in 39, osteophytes in 30, haemarthrosis in 24, erosion in 5 and fibrotic septa in 3. The X-rays score showed remodelling in 47 joints, narrowing joint space in 44, displacement/angulation in 39, osteoporosis in 42, subchondral irregularity in 44, subchondral cyst formation in 37, osteophytes in 36 and erosions in 25. The US score in healthy subjects was always ≤ 5 (range 0 to 4). In haemophiliacs, 34 of 83 joints showed US score ≤ 5, and 49 US score > 5. Joints with US score ≤ 5 had a low PXS (SRCC = 0.375, P < 0.01) and joints with US score > 5 showed a high PXS (SRCC = 0.440, P < 0.01). A significant correlation between US score and PXS for bone remodelling [Spearman's rho Correlation Coefficient (SRCC) = 0.429, P < 0.01] and for osteophytes (SRCC = 0.308, P < 0.05) was found. The correlation between the US score and number of bleedings in 83 joints was very significant (SRCC = 0.375, P < 0.01). A total of 24 bleeding joints were identified and verified with aspiration of haematic fluid. US may detect bone and cartilage alterations and synovitis. Indeed, PDUS identified bleeding also in asymptomatic joints and was able to show different entity of haemarthrosis. US may be a feasible and reliable tool to evaluate joint modifications in HA.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

A modular total knee arthroplasty in haemophilic arthropathy.

The results of 24 modular Genesis II (Smith & Nephew, Memphis, TN, USA) total knee arthroplasties, performed in 20 patients with haemophilia were prospectively reviewed. The hypothesis that the use of a new-generation implant and advanced, more aggressive hematological care, will result in a decrease rate of complications and better functional results for the haemophilic patients with TKA was tested. The mean age at the time of surgery was 36 years (25 to 44). All the patients were reviewed clinically and radiologically with an average follow-up of 4.4 years (2 to 7 years). At the final follow-up the knee score improved from an average of 23 points (11 to 45) to 86 points (62 to 100; p<0.001). The mean knee flexion contracture improved from 22 degrees (0 degrees to 45 degrees ) to 3 degrees (0 degrees to 10 degrees ; p<0.0001). The mean total flexion arc improved from 69 degrees (5 degrees to 130 degrees ) to 92 degrees (80 degrees to 145 degrees p<0.001). The results of our study confirm that the introduction of modular design may improve the functional results of TKA in haemophilic arthropathy which results in a better range of motion and lower flexion contracture.

Hemorrhagic symptoms and bleeding risk in obligatory carriers of type 3 von Willebrand disease: an international, multicenter study.

OBJECTIVES: We undertook an international, multicenter study to describe the clinical picture and to estimate the bleeding risk in a group of obligatory carriers of type 3 von Willebrand disease (VWD). PATIENTS AND METHODS: Obligatory carriers (OC) of type 3 VWD were identified by the presence of offspring with type 3 VWD or by being an offspring of a type 3 patient. Normal controls were age- and sex-matched with the obligatory carriers. A physician-administered standardized questionnaire was used to evaluate hemorrhagic symptoms at presentation. A score system ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion) was used to quantitate bleeding manifestations. Odds ratios were computed for each symptom. RESULTS: Ten centers participated to the study, enrolling a total of 35 type 3 VWD families, with 70 OC. A total of 215 normal controls and 42 OC for type 1 VWD were also included. About 40% of type 3 OC had at least one bleeding symptom compared to 23% of normal controls and 81.8% of type 1 OC (P < 0.0001 by chi-squared test), showing that type 3 OC clearly represent a distinct population from type 1 OC. The clinical situations associated with an increase of bleeding risk in type 3 OC were epistaxis [odds ratio 3.6; 90% confidence intervals (CI) 1.84-21.5], cutaneous bleeding (odds ratio 5.5; 90% CI 2.5-14.1) and postsurgical bleeding (odds ratio 16.3; 90% CI 4.5-59). The severity of bleeding score correlated with the degree of factor (F) VIII reduction in plasma. CONCLUSIONS: OC for type 3 VWD represent a distinctive population from type 1 OC. These patients, however, present with more frequent bleeding symptoms in comparison to normal controls, especially in case of significantly low FVIII. Desmopressin and/or tranexamic acid might be useful to prevent or treat bleeding in these cases.

The discriminant power of bleeding history for the diagnosis of type 1 von Willebrand disease: an international, multicenter study.

OBJECTIVE: The aim of this study was the validation of the criteria defining a significant mucocutaneous-bleeding history in type 1 von Willebrand disease (VWD). SUBJECTS AND METHODS: To avoid selection bias, 42 obligatory carriers (OC) of type 1 VWD were identified from a panel of 42 families with type 1 VWD enrolled by 10 expert centers. OC were identified by the presence of an offspring and another first degree relative with type 1 VWD (affected subjects, AFF). A standardized questionnaire was administered to evaluate hemorrhagic symptoms at the time of first examination, using a bleeding score ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion). Sensitivity, specificity, diagnostic likelihood ratios, positive and negative predictive values for the diagnosis of type 1 VWD were calculated from the data collected in OC and in 215 controls. RESULTS: Having at least three hemorrhagic symptoms or a bleeding score of 3 in males and 5 in females was very specific (98.6%) for the bleeding history of type 1 VWD, although less sensitive (69.1%). None of the misclassified OC had life-threatening bleeding episodes after diagnosis. CONCLUSIONS: We suggest that the use of a standardized questionnaire and bleeding score may be useful for the identification of subjects requiring laboratory evaluation for VWD.

Monitoring the bioavailability of FEIBA with a thrombin generation assay.

BACKGROUND: Hemophilia A patients with inhibitors are generally treated with preparations containing activated coagulation factors to achieve hemostasis by bypassing factor (F)VIII. OBJECTIVES: We developed an assay for monitoring the kinetic of thrombin generation in human FVIII inhibitor plasma reconstituted in vitro with activated prothrombin complex concentrate, FEIBA, and in plasma samples from hemophilia A patients taken after FEIBA treatment. PATIENTS AND METHODS: For pharmacokinetic studies three patients with severe hemophilia A and with a high-titer inhibitor received a single dose of FEIBA. Repeated FEIBA treatment was monitored in one patient with acquired hemophilia A. Coagulation was triggered in citrated plasma by adding a low concentration of tissue factor/phospholipid complex and CaCl2 in the presence of a fluorogenic thrombin substrate. The intensity of the fluorescence signal (FU) was continuously monitored, and the rate of increase in the fluorescence signal for every time point, which reflects the actual thrombin concentrations, was calculated. RESULTS: The maximum rate of substrate conversion, which indicates the highest thrombin concentration, was approximately 1900 FU min(-1) in a normal plasma pool. Practically no thrombin generation was observed in the FVIII inhibitor plasma, but when it was spiked with FEIBA, the rate and the peak of thrombin generation increased dose-dependently to close to normal. Plasma samples from FVIII inhibitor patients treated with a single dose of FEIBA had an improved thrombin maximum within an hour after treatment, which gradually returned to baseline values with a half-life of 4-7 h. Changes in the characteristic parameters of thrombin generation coincided with the repeated administration of FEIBA in a patient with acquired hemophilia A. CONCLUSIONS: This assay enables the pharmacodynamic and pharmacokinetic properties of bypassing therapies to be monitored, thus helping to optimize treatment.

Combination therapy with G-CSF and erythropoietin after autologous bone marrow transplantation for lymphoid malignancies: a randomized trial.

Previous studies have shown that, unlike in patients submitted to allogeneic BMT, administration of recombinant erythropoietin (Epo) after autologous BMT (ABMT) had no significant effect on erythroid recovery and transfusional requirements. On the other hand, it has also been shown that combining Epo with recombinant granulocyte colony-stimulating factor (G-CSF) in patients with the acquired immunodeficiency syndrome (AIDS) and with myelodysplastic syndromes resulted in additive effects on erythropoiesis. To test the effects of combined G-CSF + Epo therapy on erythroid recovery after autologous bone marrow transplantation a pilot randomized, three-arm trial was designed. Thirty patients suffering from lymphoid malignancies submitted to ABMT were randomly assigned to receive G-CSF alone (5 micrograms/kg, from day + 1 up to reaching an ANC > or = 10(9)/1), G-CSF + Epo (150 U/kg, from day +1 to +21), or neither of these (controls). Patients receiving G-CSF + Epo had significantly more reticulocytes on day +21 and reached 30 x 10(9)/1 reticulocytes earlier when compared to both G-CSF and control patients; however, the number of red blood cell (RBC) transfusions was not modified by the addition of Epo to G-CSF, although both groups had significantly fewer units transfused than controls. No effect on platelet recovery or platelet transfusional requirements was observed. Myeloid recovery was comparable in the G-CSF and G-CSF+Epo groups, and significantly accelerated as compared to controls. We conclude that the addition of Epo to G-CSF causes a slight acceleration of erythroid recovery after ABMT, but is not associated with transfusional benefits. Therefore, the present data do not support the use of Epo in association with G-CSF to hasten erythroid recovery after ABMT.

Prevalence of hemostatic disorders in adolescents with abnormal uterine bleeding.

STUDY OBJECTIVE: To evaluate the incidence of hemostatic disorders in a population of adolescents with various patterns of abnormal uterine bleeding (AUB). DESIGN: Retrospective observational study. SETTING: University hospital. PARTICIPANTS: One hundred thirteen adolescents with AUB; mean age at menarche and mean age at the onset of symptoms 12 ± 1.2 years and 13.5 ± 2.8 years, respectively. MAIN OUTCOME MEASURES: Data on menstrual history, bleeding symptoms, co-existing medical conditions, and medical therapies were assessed. All patients were screened for hemostatic disorders with laboratory testing. The incidence of the disorders was calculated. Subjects were further divided in 2 groups based on whether the AUB started in the first 2 years from menarche (group 1) or later (group 2). A statistical analysis was performed using a chi-square test to compare incidence of hemostatic disorders between the groups. RESULTS: One hundred thirteen adolescents with AUB were identified. Overall, 54 (47.8%) patients had some underlying hemostatic disorder, of which a platelet dysfunction was the most common (17.7%). Von Willebrand disease was detected in 13.3% of cases and a deficiency of a coagulation factor in 12.4%. In 7.1% of patients an isolated increase of bleeding time was observed. When divided in 2 groups, 44.2% of patients in group 1 and 59.2% in group 2 had a coagulation disorders, with no statistically significant difference between the 2 groups (P = .17). CONCLUSION: AUB in adolescents is frequently associated with an underlying disorder of hemostasis, most commonly a platelet function disorder. The results highlight the importance of screening for coagulation disorders in adolescents with AUB, independently from the gynecologic age at onset.

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells.

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT-PCR for the constitutive and inducible expression of megakaryocyte-specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor-4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid alpha- and beta-globin genes were not transcribed appreciably. However, MELC exposed to 5 mM hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA. and simultaneous induction of the globin genes. Megakaryocyte-related genes were expressed. in the absence of globin gene transcription, by MELC treated with either phorbol-12-myristate acetate (PMA; 100 ng/ml) or colcemid (40 nM), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c-mpl. PMA and colcemid did not affect high basal c-myb mRNA levels which, in turn, were down-regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo-R and IL-3R mRNAs, whereas no expression of granulocyte/macrophage-related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro-megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

Multivariate analysis of factors influencing quality of life and utility in patients with haemophilia.

BACKGROUND AND OBJECTIVES: Although several studies have determined quality of life (QOL) in patients with hemophilia, generic questionnaires have rarely been used. The objectives of our study were; 1) to measure QOL and utility in patients with hemophilia using the Short Form 36 (SF-36) and the EuroQOL questionnaires; 2) to evaluate the influence of some clinical variables on QOL and utility; 3) to assess the correlation between the two questionnaires. DESIGN AND METHODS: All consecutive patients with hemophilia were asked to complete the SF-36 and the EuroQOL questionnaires. The following information was recorded from each patient: age, type of hemophilia, severity of disease, HCV and HIV infection, number of bleeding episodes and cumulative dose of coagulation factors over the previous year. These items were entered into a multivariate analysis to assess their effect on QOL. Correlation analyses were conducted to evaluate the relationship between the EuroQOL and SF-36. RESULTS: Fifty-six patients completed the SF-36 and the EuroQOL questionnaires. The mean scores of the SF-36 ranged from 55.2 (general health) to 74.7 (social functioning). The EuroQOLself-classifier and the EuroQOLvas showed a mean score of 0.67 (SD=0.26) and 0.66 (SD=0.17), respectively. Among the clinical variables, age significantly influenced both the EuroQOL and the SF-36 scores. The EuroQOL indices showed a statistically significant correlation with each dimension of the SF-36. INTERPRETATION AND CONCLUSIONS: Our study quantified the degree to which QOL is impaired in patients with hemophilia by using both a generic questionnaire and a utility-based approach.

Increased expression of the distal, but not of the proximal, Gata1 transcripts during differentiation of primary erythroid cells.

Gata1 is expressed from either one of two alternative promoters, the erythroid (proximal to the AUG) and the testis (distal to the AUG) promoter, both used by hemopoietic cells. To clarify the role of the distal and proximal Gata1 transcripts in erythroid differentiation, we determined by specific reverse transcriptase-polymerase chain reactions their relative levels of expression during the differentiation of erythroid precursors purified from the spleen of mice treated with phenylhydrazine (PHZ) or infected with the anemia-inducing strain of the Friend virus (FVA cells). PHZ cells are erythroid precursors that progress in vivo to erythroblasts in 3 days. Both PHZ and FVA cells synchronously proliferate and differentiate in vitro in the presence of erythropoietin (EPO). The levels of total and of distal, but not of proximal, Gata1 transcripts increased by five- to eightfold during in vivo and in vitro differentiation of FVA and PHZ cells. The increase in expression was temporally associated with an increase in the expression of Eklf, Scl, and Nfe2, three genes required for erythroid differentiation, and preceded by 24 h the repression of Gata2 and Myb expression. The day 1 PHZ cells that survived 18 h in the absence of EPO do not express globin genes and express detectable levels of distal but not of proximal Gata1 transcripts. These cells activate the expression of the globin genes within 2 h when exposed to EPO. Therefore, during erythroid differentiation of primary cells, increased expression of distal Gata1 transcripts underlies the increase in the expression of total Gata1 associated with the establishment of the erythroid differentiation program.

High doses of recombinant human erythropoietin fail to accelerate platelet reconstitution in allogeneic bone marrow transplantation. Results of a pilot study.

BACKGROUND AND OBJECTIVE: The effectiveness of recombinant human erythropoietin (rhEpo) in accelerating erythroid engraftment in patients undergoing allogeneic bone marrow transplantation (BMT) has been demonstrated in previous studies. On the other hand, there are experimental data suggesting that high doses of rhEpo might also exert a stimulatory effect on thrombopoiesis. METHODS: We carried out a pilot study on the use of high doses of rhEpo (500 U/kg/day for 30 days after transplant) in ten patients (HD-Epo group) receiving BMT to evaluate the effects on both erythroid and platelet (Plt) engrafment. This group was compared to ten BMT patients who had not received the hormone (Placebo group). RESULTS: The HD-Epo group patients showed signs of accelerated erythropoietic recovery; in fact, the time required to reach a reticulocyte count higher than 30 x 10(9)/L was significantly shorter than in the Placebo group, while the number of high RNA content reticulocytes (HFR) was about three times greater. Circulating transferrin receptor (TfR) levels 30 days after BMT were also significantly higher in the HD-Epo group than in the other. Finally, the number of red blood cell (RBC) transfusions in the first 30 days following BMT was about twofold lower in the HD-Epo group; moreover, 4/10 patients who were treated with HD-Epo did not require any RBC units. No significant effects on the engraftment of platelets or on the number of Plt transfusions were observed in the HD-Epo as compared to the Placebo group. No adverse effect was noted on granulocytopoiesis, nor were any adverse clinical experiences found in patients who had been treated with erythropoietin at high dosages. INTERPRETATION AND CONCLUSIONS: These data confirm that rhEpo may stimulate erythroid reconstitution after BMT, while its effects on Plt engraftment and on Plt transfusion requirements are minimal.

Identification and characterization of a bipotent (erythroid and megakaryocytic) cell precursor from the spleen of phenylhydrazine-treated mice.

We have identified a cell population expressing erythroid (TER-119) and megakaryocyte (4A5) markers in the bone marrow of normal mice. This population is present at high frequency in the marrows and in the spleens involved in the erythroid expansion that occurs in mice recovering from phenylhydrazine (PHZ)-induced hemolytic anemia. TER-119(+)/4A5(+) cells were isolated from the spleen of PHZ-treated animals and were found to be blast-like benzidine-negative cells that generate erythroid and megakaryocytic cells within 24-48 hours of culture in the presence of erythropoietin (EPO) or thrombopoietin (TPO). TER-119(+)/4A5(+) cells represent a late bipotent erythroid and megakaryocytic cell precursors that may exert an important role in the recovery from PHZ-induced anemia. (Blood. 2000;95:2559-2568)

Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias.

There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT-PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1-M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for alpha-globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo-R) and thrombopoietin receptor (c-mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB-subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.

Factor VIII inhibitor-bypassing agents act by inducing thrombin generation and can be monitored by a thrombin generation assay.

Factor VIII (FVIII)-bypassing agents have complex modes of action but all control bleeding in inhibitor patients by triggering the generation of thrombin. No routine test is available for monitoring this therapy in patients with inhibitors against FVIII. We present an assay that records FEIBA- or FVIIa-mediated changes in thrombin generation (TG) in FVIII inhibitor plasma samples. In plasma samples spiked with FEIBA TG was normalized above 0.4 U/ml, while for recombinant FVIIa (rFVIIa) more than 12.5 microg/ml were required to induce TG in the absence of tissue factor (TF). Addition of TF increased the TG potential of rFVIIa in vitro. This assay seems suitable for monitoring the pharmacokinetics of inhibitor bypassing agents during treatment and possibly for predicting responses to treatment.