Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Use of recombinant factor VIIa for refractory hemorrhage during extracorporeal membrane oxygenation.

OBJECTIVE: To describe the outcome and treatment of two patients with recombinant factor VIIa (rFVIIa) for severe hemorrhage associated with extracorporeal membrane oxygenation (ECMO). DESIGN: Case report. SETTING: A 38-bed pediatric intensive care unit and 20-bed pediatric cardiac intensive care unit at a tertiary care children's hospital. PATIENT: Two patients with life-threatening hemorrhagic complications associated with ECMO requiring massive transfusion of blood products. INTERVENTIONS: Administration of repeated doses of rFVIIa at 90 microg/kg/dose. MEASUREMENT AND MAIN RESULTS: PATIENT 1 was an 11-yr-old male with a dilated cardiomyopathy who had undergone an orthotopic heart transplant treated with venoarterial ECMO postoperatively for right ventricular dysfunction. PATIENT 2 was a 13-yr-old male treated with venoarterial ECMO for cardiopulmonary failure from necrotizing staphylococcal pneumonia. Both patients had severe hemorrhage from the cannulation sites and thoracostomy tubes requiring massive transfusion to maintain intravascular blood volume and replace clotting factors. Both patients were treated with rFVIIa every 2-4 hrs and attained hemostasis. PATIENT 1 was administered three doses and PATIENT 2 was administered ten doses. No evidence of abnormal thrombus formation was noted in their respective ECMO circuits. CONCLUSIONS: The efficacy of rFVIIa in reducing intractable bleeding postcardiac surgery and in other coagulopathic states is being investigated. Despite theoretical concerns of thrombosis, these cases illustrate that there may be a role for the cautious use of rFVIIa in treating severe and intractable hemorrhage associated with ECMO.

Biventricular function in sickle-cell anemia: radionuclide angiographic and thallium-201 scintigraphic evaluation.

Left ventricular (LV) and right ventricular (RV) function were evaluated at rest and during exercise using radionuclide ventriculography in 10 patients, aged 19-53 years, with sickle-cell anemia (SCA). Seven patients were in New York Heart Association functional class I and 3 were in class II. The resting LV ejection fraction (EF) was normal in 9 patients and the resting RVEF was normal in 4. LV dilation and high cardiac output were observed in 6 patients at rest. The LVEF during exercise was normal in all 10 patients, whereas only 2 patients had normal RVEF at rest and during exercise. The LVEF was lower in patients with SCA at rest (54 +/- 4% versus 61 +/- 6%, p less than 0.001) and exercise (66 +/- 4% versus 74 +/- 6%, p less than 0.001) than in 42 age-matched normal subjects. Rest thallium-201 images from 9 patients showed abnormal RV uptake in 8 and normal LV uptake in 8. Thus, in adult patients with SCA, LV function was normal during exercise in all patients and at rest in all but 1 patient. The LVEF, however, was lower than that in age-matched normal subjects. RV function was abnormal in most patients at rest and during exercise. RV thallium-201 uptake suggested pressure or volume overload (or both), most likely due to pulmonary vaso-occlusive complications of the disease.

Desmopressin does not decrease bleeding after cardiac operation in young children.

Young children undergoing complex cardiac operation lose more blood after cardiopulmonary bypass than do older patients. This study was designed to investigate the effect of desmopressin on blood loss during the first 24 hours after cardiac operation in children undergoing principally complex surgical procedures. The study consisted of a randomized, blinded comparison of 112 pediatric patients who received either desmopressin 0.3 microgram/kg or saline solution placebo after cardiopulmonary bypass. A coagulation profile including bleeding time, quantitation of von Willebrand factor, and qualitative analysis of the factor VII:von Willebrand factor complex was performed before, 30 minutes after, and 3 hours after the operation. Blood loss and blood replacement were recorded for the first 24 hours after the operation. The surgeon classified the technical difficulty of each procedure as simple or complex. Statistical analysis was performed with Student's unpaired t test and chi 2 analysis. Significance was defined as p < 0.05. Results are listed as mean +/- standard deviation. Data collection was completed for 95 patients. The mean age of all patients was 26 +/- 40 months, and the mean weight was 10 +/- 11 kg, with 84% undergoing complex procedures. There were no differences between the desmopressin and placebo groups with respect to age, weight, or surgical complexity. Twenty-four-hour blood loss and replacement between the desmopressin and placebo groups were not different (blood loss: desmopressin 30 +/- 33 ml/kg, placebo 35 +/- 36; blood replacement: desmopressin 65 +/- 43 ml/kg, placebo 64 +/- 46 ml/kg). Coagulation profiles between the desmopressin and placebo groups were not different at any time. We conclude that desmopressin does not reduce blood loss or blood replacement in young children after cardiopulmonary bypass for either simple or complex cardiac surgical procedures.

Diagnosis and management of iron-induced heart disease in Cooley's anemia.

Patients with homozygous beta-thalassemia are chronically transfused and, if not assiduously chelated, are at risk for cardiac dysfunction. Available data suggest that even in optimally chelated patients, cardiac pathology is abnormal secondary to iron deposition, fibrosis, hypertrophy, and the structural effects of chronic anemia. Evidence of myopericarditis may also be found. Cardiac performance is usually only subtly affected, primarily with diastolic abnormalities not routinely detected on echocardiograms or nuclear scan. In poorly chelated patients, severe heart failure occurs and is easily predictable but invariably fatal, despite treatment with diuretics, vasodilators, inotropes, and antiarrhythmics. Based on successful prevention of heart failure with ACE inhibitors in other forms of cardiomyopathy, we suggest multicenter trials to explore methods to stabilize cardiac function in patients at risk for iron-induced heart disease. Long-term adverse effects of iron deposition, diastolic dysfunction, and abnormal hormone regulation need to be quantitated in patients reaching their third and fourth decades when the potential for ischemic cardiac disease could compound cardiac dysfunction.

Difficult pediatric diagnoses. Bruising and bleeding.

An approach to the evaluation of a child who presents to the pediatrician with bruising is outlined. Important clues in the history and physical examination that suggest disorders of platelet number and function or coagulation abnormalities are presented. A scheme for using screening tests of coagulation and platelet function is followed by a brief summary of currently available therapies appropriate for bleeding episodes in children.

What's new in transfusion medicine?

The safety of the blood supply has increased tremendously in the past decade. Donor screening and improved infectious disease testing of units for transfusion have contributed to the decreased risk of transfusion-transmitted diseases. Reduction of the number of passenger leukocytes from RBC and platelet transfusions decreases the rate of febrile transfusion reactions and alloimmunization. Irradiation of cellular products helps prevent TA-GVHD. The practice of obtaining an informed consent prior to transfusion helps patients and families understand the risks and benefits of and alternatives to transfusion therapy.

Use of acetyl chloride/methanol for assumed selective methylation of plasma nonesterified fatty acids results in significant methylation of esterified fatty acids.

The albumin-bound nonesterified fatty acid pool in plasma, which represents a very small percentage of total plasma fatty acids, has previously been quantitated by a variety of methods. In the present study we determined that the nonesterified fatty acid concentrations in the plasma, quantitated by a popular method using acetyl chloride and methanol which is reported to be specific for methylation of nonesterified fatty acids in the presence of esterified fatty acids (i.e., without prior isolation of the plasma nonesterified fatty acids), were significantly overestimated due to cleavage and methylation of esterified fatty acids. Quantitation of the contaminating fatty acid from the esterified pool demonstrated that the amount of fatty acid cleaved from the esterified pool was enough to exceed the entire mass of nonesterified fatty acids. As an established method for comparison, we isolated nonesterified fatty acids from the plasma by thin-layer chromatography prior to methylation, using a number of simple precautions to limit oxidation. By performing all thin-layer chromatography steps in an atmosphere of nitrogen and by including fatty acid standards in the plasma with 0, 1, 2 or 4 double bonds, we were able to accurately and reproducibly determine the concentration of nonesterified fatty acids in the plasma, including arachidonate. We demonstrated that no oxidation occurred in the thin-layer chromatographic isolation of nonesterified fatty acids and that the coefficients of variation for repeat measurements of the same sample were < 11% using our reference method. Our data indicate that the use of acetyl chloride and methanol for assumed selective methylation of plasma nonesterified fatty acids results in significant methylation of esterified fatty acids.

Utility of computed tomography of the head following head trauma in boys with haemophilia.

The most serious site of bleeding for patients with haemophilia is the central nervous system. Intracranial haemorrhage (ICH) in patients with haemophilia can occur spontaneously or following mild head trauma however no guidelines exist for the approach to these patients. The goal of this review was to determine the utility of screening computed tomography (CT) of the head for patients with haemophilia who experience head trauma and to determine if the use of clinical criteria could allow a selective approach to radiographic imaging. In a retrospective study we reviewed the management of head trauma in a cohort of paediatric patients with haemophilia in a single institution. The cohort included males, ages birth to 18 years with haemophilia A or B who were followed at the haemophilia treatment center at The Children's Hospital of Philadelphia from 1994 to 2005. Between the years of 1994 and 2005, 97 patients were evaluated for head trauma for a total of 374 emergency department visits. There were 295 head CT scans performed to identify 9 (3%) episodes of intracranial bleeding. Fifty-six per cent of the patients with intracranial bleeding had no clinical signs or symptoms. The clinical outcome was excellent in all cases with no deaths or reported morbidity. In this cohort, a lack of symptoms and a normal neurological exam did not exclude ICH, especially in patients with severe haemophilia who were evaluated soon after a mild head trauma event suggesting the utility of early head CT imaging.

Treatment options for bleeding episodes in patients undergoing immune tolerance therapy.

Inhibitors to factor VIII develop in 4-20% of haemophilia A patients, with the percentage rising to 52% in certain subpopulations. The management of inhibitor patients is directed toward stopping acute haemorrhages, providing short-term haemostasis before and after surgery, and inducing immune tolerance to factor VIII (immune tolerance therapy or ITT). Several different protocols have been used for ITT, but they are all centred around ongoing exposure to high doses of factor VIII. High responders (those patients with a large increase in inhibitor level after exposure to factor VIII) are the prime candidates for ITT, but low responders may also benefit from this treatment. It is often necessary to treat bleeding episodes during ITT, because elimination of inhibitors may require many years of therapy. Treatment of haemorrhages in inhibitor patients is reviewed for both low and high responders during ITT and in the absence of ITT. The choice of clotting agent for inhibitor patients who have not yet responded to ITT depends on current and past inhibitor levels, the severity of the haemorrhage, the site of the haemorrhage or the setting in which it occurs (e.g. surgical), and the extent of inhibitor cross-reactivity with porcine factor VIII. Patients with high-titre inhibitors experiencing a critical haemorrhage are generally best managed with bypassing agents (AUTOPLEX T or FEIBA VH), porcine factor VIII or rFVIIa.

Rapid excretion of gallium-67 isotope in an iron-overloaded patient receiving high-dose intravenous deferoxamine.

A 23 year-old black male with homozygous sickle cell disease (Hb SS disease) and transfusional iron overload was admitted for evaluation of response to intravenous deferoxamine (DFO) therapy. Soon after admission, the patient suffered an intraventricular hemorrhage and during his subsequent hospitalization developed a persistent fever of undetermined origin (f.u.o.). Included in the diagnostic evaluation of fever was a gallium 67 scan (Ga-67), which was initially nondiagnostic because of Ga-67 citrate's preferential chelation by DFO. After DFO was discontinued, a repeat scan demonstrated a lesion above the left kidney. To our knowledge the unusual interaction in vivo of DFO with Ga-67 citrate has not been reported in the clinical literature. With the anticipated increased use of chelation therapy for patients with transfusional iron overload, this interaction may be encountered more frequently. DFO should be discontinued before the use of Ga-67 scanning in this clinical situation, or an alternative isotopic scan, such as indium-labelled white cells, should be considered.

Blood transfusion support in pediatric cardiovascular surgery.

The majority of children who undergo open-heart surgery with cardiopulmonary bypass (CPB) require perioperative blood transfusion. Blood product requirements are affected by factors such as patient age, underlying cardiac disease, complexity of the surgical procedure, and hemostatic alterations induced by CPB. Transfusion support may include the use of whole blood and/or individual blood components with transfusion practices varying widely based on individual preferences and blood product availability. Approaches to limit allogeneic blood exposure include the use of modified ultrafiltration and smaller bypass circuits, preoperative autologous blood donation and intraoperative blood salvage, and adjunctive antifibrinolytic agents. Potential advantages and disadvantages of the different blood products and pharmacological agents must be considered in managing the pediatric cardiac surgery patient.

Splenectomy in mild hereditary spherocytosis: is it worth the risk?

Patients with mild hereditary spherocytosis (HS) often undergo splenectomy for the sole purpose of preventing gallstone formation. Splenectomy carries a surgical risk as well as the risk of postsplenectomy sepsis. Gallstones develop in less than half of mild HS patients and do not always cause symptomatic biliary tract disease. Using decision analysis, a quantitative approach to problem solving under conditions of uncertainty, we have compared the likelihood of surviving the complications of gallstones with the likelihood of surviving routine splenectomy. Probability figures for critical events were obtained from the medical literature; final outcome is recovery (utility = 1.0) or death (utility = 0.0). Our analysis shows that expectant management of gallstones is the preferred choice, being associated with a higher utility than is routine splenectomy (0.9980 vs 0.9755, respectively). The utility values for the two choices become equivalent only when the risk of postsplenectomy sepsis is lowered from 0.022 to 0.0001.

Treatment of refractory Evans syndrome with alternate-day cyclosporine and prednisone.

PURPOSE: We report that the use of alternate-day cyclosporine and prednisone improved the clinical course of a 6-year-old child with severe Evans syndrome. Before the use of cyclosporine the child had experienced life-threatening episodes of hemolytic anemia despite the use of multiple therapeutic modalities. METHODS: Cyclosporine was given at a dose of 10 mg/kg/day divided into two doses on alternate days. RESULTS: The use of cyclosporine resulted in increased hemoglobin levels, increased platelet counts, and the reduction of the patient's prednisone dose from 2 mg/kg/day to as low as 1 mg/kg every other day. With this regimen, the patient had less severe hemolytic anemia, was less thrombocytopenic, and had fewer hospitalizations. No major toxic effects were associated with cyclosporine therapy. CONCLUSION: The regimen of alternate-day cyclosporine and prednisone may prove to be useful in the treatment of other patients with refractory Evans syndrome.

Neonatal lupus erythematosus with microvascular hemolysis.

A female, term newborn born to a mother with a history of idiopathic thrombocytopenic purpura and antinuclear antibodies, single-stranded A antibody, and IgM anticardiolipin antibodies presented with immune thrombocytopenia, disseminated intravascular coagulation (DIC), microangiopathic hemolytic anemia, and a characteristic lupus rash in the periorbital areas. She responded to combined treatment with dexamethasone and intravenous immunoglobulin (IVIG). At age 9 months, she was readmitted with severe thrombocytopenia, DIC, and microangiopathic hemolytic anemia. She again responded to IVIG. This suggests that microangiopathic hemolysis can be a presenting symptom in neonatal lupus erythematosus and that reoccurrence of the microangiopathic hemolysis may occur even after the disappearance of lupus antibodies.

Emerging and receding risks of therapeutic regimens for haemophilia.

During the past two decades, the improvement of therapeutic agents for the management of haemophilia has created the opportunity for individuals with haemophilia to live normal lives. However, in some instances, the progress made has been accompanied by emergence of unexpected risks and other new complications. A number of viruses have either emerged, or become greater risks to people with haemophilia. In addition, the drive of many countries towards self-sufficiency in blood products may in fact be endangering people with haemophilia by restricting blood donation to a pool of donors with high infection risk, discouraging commercial interests from developing safer products, and discouraging use of 'foreign' products even where that may be the safer option. Gene therapy for haemophilia, although an encouraging new treatment, has brought with it a number of adverse events, including risk of virus infection and development of carcinomas. The risk of inhibitors is still the most important problem for people with haemophilia, and a recent report showed that the type of factor concentrate does not impact significantly on this risk. Despite the advent of new and promising treatments for haemophilia, heathcare providers must be alert to new risks posed by them.

Low recovery in vivo of highly purified factor VIII in patients with hemophilia.

To assess whether the response and recovery of highly purified factor VIII products approach the levels predicted by use of manufacturers' suggestions, we studied response in vivo and percentage of recovery in boys and men with hemophilia after infusion of 50 IU of an immunoaffinity-purified factor VIII preparation (Hemofil M) per kilogram of body weight. We also studied dose response and percentage of recovery in the same boys after infusion of a factor VII concentrate prepared by solvent detergent treatment and gel filtration (Koate-HP). For boys the difference between mean peak factor VIII response with Hemofil M (1.02% +/- 0.07%) and the response with Koate-HP (1.21% +/- 0.10%) was not significant (p = 0.10), but the response of both products was considerably less than the predicted response of 2.0%. The response to Hemofil M in men (1.38% +/- 0.12%) was significantly better than the response in boys (p = 0.004) but, again, fell short of the anticipated response of 2.0%. In boys the percentage of recovery of Hemofil M was 38.4% +/- 2.1%, compared with Koate-HP recovery, which was 47.0% +/- 3.6% (p = 0.034). The percentage of recovery in men was 47.5% +/- 2.8%. The better response observed in men appears to be, in part, a function of larger body size. We conclude that peak factor VIII response and recovery in boys and men who receive highly purified factor VIII concentrates are lower than those predicted by use of current prescribing recommendations.