Silvia Linari

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

Summary.  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.

Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA

There is considerable interest in an autologous transplantation (AT) programme for patients with high‐risk breast cancer; however, the issue of the incidence of occult bone marrow (BM) micrometastasis at diagnosis, and the cancer contamination of peripheral blood stem cell (PBSC) collections used for haematological rescue, is still debated. The presence of BM micrometastasis was evaluated in bilateral BM biopsies obtained at diagnosis of 33 patients with stage II/IIIA breast cancer using: (i) a ‘nested’ reverse transcriptase‐polymerase chain reaction (RT‐PCR) assay for cytokeratin 19 (K19) mRNA, (ii) histology, and (iii) immunohistochemistry (IHC) analysis with a panel of three monoclonal antibodies. The RT‐PCR assay only was used to determine contamination of PBSC collections obtained after priming with recombinant human granulocyte‐colony stimulating factor (rhG‐CSF). K19 transcripts in one or both BM samples  were detected in 48% of patients at diagnosis, with an overall 85% concordance with the results of IHC analysis. On the other hand, 56% of PCR‐ and IHC‐positive BM samples were diagnosed as ‘normal’ on histological analysis. 57% of patients showed K19 mRNA in at least one PBSC collection; the possibility to have contaminated PBSC collections was significantly higher in patients with K19 positivity in BM at diagnosis. In four patients who had shown K19 positivity in BM and in PBSC collections, immunoselected CD34+ cells used for haematological rescue were K19‐negative. There was a trend towards longer relapse free survival (RFS) in patients transplanted with K19‐negative PBSC collections as compared to the others. In conclusion, a substantial proportion of patients with high‐risk non‐metastatic breast cancer present occult BM micrometastasis at diagnosis and also show cancer contamination of PBSC collections used for AT. These might represent a category of patients with poorer prognosis after AT, and possible candidates for more intensive and/or alternative therapeutic regimens, including AT with purged PBSCs.

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. J. Cell. Physiol. 180:390–401, 1999.

Clinical, instrumental, serological and histological findings suggest that hemophilia B may be less severe than hemophilia A

Recent evidence suggests that patients with severe hemophilia B may have a less severe disease compared to severe hemophilia A. To investigate clinical, radiological, laboratory and histological differences in the arthropathy of severe hemophilia A and hemophilia B, 70 patients with hemophilia A and 35 with hemophilia B with at least one joint bleeding were consecutively enrolled. Joint bleedings (<10, 10–50, >50), regimen of treatment (prophylaxis/on demand), World Federation of Hemophilia, Pettersson and ultrasound scores, serum soluble RANK ligand and osteoprotegerin were assessed in all patients. RANK, RANK ligand and osteoprotegerin expression was evaluated in synovial tissue from 18 hemophilia A and 4 hemophilia B patients. The percentage of patients with either 10–50 or more than 50 hemarthrosis was greater in hemophilia A than in hemophilia B (P<0.001 and P=0.03, respectively), while that with less than 10 hemarthrosis was higher in hemophilia B (P<0.0001). World Federation of Hemophilia (36.6 vs. 20.2; P<0.0001) and ultrasound (10.9 vs. 4.3; P<0.0001) score mean values were significantly higher in hemophilia A patients. Serum osteoprotegerin and soluble RANK ligand were decreased in hemophilia A versus hemophilia B (P<0.0001 and P=0.006, respectively). Osteoprotegerin expression was markedly reduced in synovial tissue from hemophilia A patients. In conclusion, the reduced number of hemarthrosis, the lower World Federation of Hemophilia and ultrasound scores, and higher osteoprotegerin expression in serum and synovial tissue in hemophilia B suggest that hemophilia B is a less severe disease than hemophilia A. Osteoprotegerin reduction seems to play a pivotal role in the progression of arthropathy in hemophilia A.

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 α‐ and β‐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.

RANK-RANKL-OPG in hemophilic arthropathy: from clinical and imaging diagnosis to histopathology.

Objective. Hemarthrosis triggers hemophilic arthropathy, involving the target joints. The histopathogenesis of blood-induced joint damage remains unclear. The triad of receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG; RANK-RANKL-OPG) controls bone turnover. Our aim was to evaluate RANK-RANKL-OPG expression in the synovium of hemophilic patients with severe arthropathy. Methods. Synovial biopsies were obtained from 18 patients with hemophilic arthropathy and 16 with osteoarthritis (OA) who were undergoing total knee replacement and synovectomy. The severity of hemophilic arthropathy was evaluated according to ultrasonography score, the World Federation of Hemophilia (WFH) orthopedic joint scale, and the radiographic Pettersson score. RANK-RANKL-OPG expression was examined by immunohistochemistry and Western blotting. Serum levels of soluble RANKL (sRANKL) and OPG from an extended group of 67 patients with hemophilic arthropathy and 30 healthy controls were measured by ELISA. Results. The mean ultrasonography, WFH orthopedic joint scale, and Pettersson scores in patients with hemophilic arthropathy indicated severe arthropathy. A decreased expression of OPG was found in hemophilic arthropathy synovium compared with patients with OA. RANK and RANKL immunopositivity was strong in the lining and sublining layers in hemophilic arthropathy synovial tissue. Western blotting confirmed the immunohistological findings. Serum levels of sRANKL and OPG in patients with hemophilia were lower than in healthy controls. Conclusion. In hemophilic arthropathy, the synovium highly expressed RANK and RANKL, whereas OPG immunopositivity decreased, suggesting an osteoclastic activation. Low tissue expression of OPG paralleled the serum levels of this protein and the severity of hemophilic arthropathy assessed by ultrasonography, Pettersson, and WFH orthopedic joint scale scores.

Viscosupplementation in haemophilic arthropathy: a long‐term follow‐up study

Summary.  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.

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 α‐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.

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.

Emerging Issues in Diagnosis, Biology, and Inhibitor Risk in Mild Hemophilia A.

Mild hemophilia A (MHA) is an X-linked bleeding disorder defined by factor VIII (FVIII) levels between 5 and 40 U/dL. Diagnosis occurs later in life compared with severe or moderate disease. Although bleeding episodes are especially posttraumatic, their unexpected occurrence may be potentially life threatening if diagnosis is missed or delayed. Desmopressin is the treatment of choice for MHA since it is cheap and safe, but a significant proportion of cases do not attain FVIII postinfusion greater than 50 U/dL, which is considered a safe level for major surgery. Thus, replacement therapy may be needed and is usually successful in MHA, but recent data indicate that this can be associated with the occurrence of inhibitors against FVIII, as for severe hemophilia A. However, in contrast to severe or moderate hemophilia A, patients with MHA have a lifelong risk of inhibitor formation. Inhibitors may change the clinical phenotype dramatically, as the inhibitor frequently cross-reacts with the patients endogenous FVIII, reducing the endogenous FVIII plasma levels below 1 U/dL. Specific F8 missense mutations predispose to inhibitor development. Inhibitors are frequently provoked by intensive treatment with therapeutic FVIII concentrates (more than 5 consecutive exposure days). Bleeding in inhibitor patients may be treated with desmopressin, high doses of FVIII concentrate or FVIII bypassing agents. Many inhibitors disappear over time when no FVIII concentrate is administered. However, this does not imply that a patient is tolerant and an anamnestic reaction may occur when treatment with FVIII concentrate is again necessary. To eradicate, an inhibitor different strategies may be used: watchful waiting, immunosuppression, or immune tolerance induction regimen.