Louis D. Wadsworth

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in β thalassemia patients.

A population-based study of childhood myelodysplastic syndrome in British Columbia, Canada

Myelodysplastic syndrome (MDS) is considered to be very rare in children. However, the only two published population‐based studies reported widely divergent incidence figures. To further explore the epidemiology of childhood MDS and to evaluate the accuracy of cancer registry and treatment trial data, we conducted a population‐based study of children aged 0–14 years in British Columbia (BC), Canada, between 1982 and 1996. MDS was diagnosed in 31 cases corresponding to an annual incidence of 3.2 per million children or 6% of all leukaemias, compared with an incidence of 6.0/million for acute myeloid leukaemia (AML), and of 0.5/million for chronic myeloid leukaemia. There was a non‐significant (P = 0.19) trend toward an increase in MDS incidence with time, the increase was partly explained by an increasing number of patients with Down syndrome. Associated abnormalities were found in 48% of the MDS cases with Down syndrome as the most common (seven cases). Only one third of the MDS cases were correctly registered in the Cancer Registry and less than half of the eligible MDS patients were enrolled on a cooperative group study. Data on MDS from treatment‐based studies and cancer registries were inaccurate and seemed to significantly underestimate the incidence of MDS in children.

Utilization of red blood cell transfusion in an obstetric setting

The transfusion experience for a 1-year period (September 1985 to August 1986) at a tertiary referral obstetric hospital was reviewed retrospectively. During the review period 7731 mothers were delivered and 6003 patients (83%) underwent type-and-screen procedures. A total of 1057 units of red blood cells were crossmatched, and 362 of these 1057 units were transfused to 100 parturient women so that the overall crossmatch/transfusion ratio was 2.9:1. Five percent of transfused patients received 1 unit; 52% of patients received 2 units, 19% received 3 units and 24% received greater than or equal to 4 units of packed red blood cells. Major indications for transfusion were uterine atony, 27%; retained placenta, 17%; trauma, 17%, placenta previa, 7%; and abruptio placentae, 5%. In 12% of patients transfusions were done because of anemia. This study shows the value of audit and confirms that the type-and-screen procedure is an effective way of reducing the crossmatch/transfusion ratio without compromising patient care, even in high-risk patients.

Transcobalamin II deficiency: Case report and review of the literature

A male Caucasian infant presented at 6 weeks of age with failure to thrive, diarrhoea, macrocytic anaemia, and decreased IgG. He had normal serum B12 and folate levels. Serum cobalamin binding capacity showed no detectable transcobalamin II. Both parents showed levels consistent with a heterozygous state. The literature is extensively reviewed, and the importance of early diagnosis to prevent neurological dysfunction is stressed.

An assessment of published pediatric dosage guidelines for enoxaparin: a retrospective review.

Objective To evaluate the ability of published dosage guidelines for enoxaparin to achieve therapeutic anticoagulation and to determine whether the routine monitoring of anti-Xa levels is still necessary at a tertiary care pediatric institution. Methods Consecutive charts and laboratory records were reviewed for all patients receiving treatment doses of enoxaparin for thrombosis in the authors’ institution over a 4-year period (1998–2002). Results Sixty-six percent (25/38) of the anti-Xa levels were within the recommended therapeutic range (0.5–1.0 [± 10%] U/mL) after two doses. The success rates of achieving therapeutic levels were 1/6, 2/3, 6/9, 10/11, and 6/9, for patients 2 months or younger, more than 2 months to 1 year, more than 1 year to 6 years, more than 6 years to 12 years, and more than 12 years of age, respectively. Patients with cardiac or renal disease were more likely to achieve high anti-Xa levels. Thirty-seven percent of patients reported adverse effects. The most common effects were injection site-related bruising and minor bleeding. One patient experienced a major bleed that was not life-threatening. Conclusions Most patients achieved therapeutic anticoagulation when dosed according to the published guidelines. Children with cardiac conditions or renal insufficiency or those younger than 2 months were more likely to require dosage adjustments to achieve the therapeutic range. Routine monitoring of anti-Xa levels is still necessary in these patient populations, particularly when the early establishment of therapeutic anticoagulation may be critical. Enoxaparin appears to be well tolerated in the authors’ patient population.

The use of acute hemodilution in parturients undergoing cesarean section

OBJECTIVE Concern over transmissible disease has increased interest in methods of minimizing homologous blood transfusion during elective surgery. One method is acute hemodilution, a technique previously unreported in parturients. This study was designed to determine its feasibility and safety in women at risk of hemorrhage during cesarean section. STUDY DESIGN This technique was performed on 38 parturients. Collected blood was retransfused at the end of surgery or earlier, if required. Hemoglobin was measured before hemodilution, after hemodilution, before transfusion, after transfusion, and 24 hours postoperatively. Neonatal assessment included umbilical blood gases and Apgar scores. RESULTS All patients were hemodynamically stable and no fetal heart rate abnormalities were observed during the procedure. One patient received homologous blood and 14 received previously donated autologous blood. Umbilical blood gases were normal and 5-minute Apgar scores were > or = 7. CONCLUSION This study suggests that acute hemodilution is well tolerated in parturients undergoing cesarean section. This may limit exposure to homologous blood transfusion.

Importance of the day 7 bone marrow biopsy as a prognostic measure of the outcome in children with acute lymphoblastic leukemia.

The presence of > or = 25% blasts in a marrow aspirate obtained on day 7 of induction followed by a remission at day 28 has been associated with a poor prognosis in children with acute lymphoblastic leukemia (ALL). We evaluated whether a day 7 marrow biopsy may be used to more accurately assess therapeutic reduction of leukemia tumor burden. Studied were 76 children with ALL enrolled on CCG protocols at B.Cs Childrens Hospital who received both a day 7 aspirate and biopsy and were in remission by day 28. Evaluation for the correlation of the percentage aspirate blasts on day 7 with the biopsy demonstrated a moderate correlation with the percentage biopsy blasts (R = 61), but not correlation with the biopsy cellularity. We saw a similar prediction of outcome by the percentage blasts on day 7 marrow aspirate in the study as reported previously although it was not significant. Outcome analysis was done using leukemia burden as measured by the day 7 absolute blast index-aspirate (ABI-aspirate) calculated as the product of the biopsy cellularity with the percentage blasts on the aspirate. The ABI-aspirate significantly predicted patient outcome with 83% survival in those with an ABI-aspirate of < .06 compared to 51% in those > or = .06 (P = .01) and was highly significant when analyzed as a continuous predictor (P = .004). This is the first study to demonstrate that information gained from the day 7 marrow biopsy can improve prediction of outcome in children with ALL. Based on this preliminary study, we recommend that large population ALL therapy trials evaluate the role of the day 7 marrow biopsy for outcome prediction in children with ALL.

Diamond-blackfan Anemia and Cyclosporine Therapy Revisited

A girl with Diamond-Blackfan anemia diagnosed in infancy started cyclosporine A (CSA) therapy at 9 years and 8 months of age after experiencing unacceptable side effects while receiving prednisone. Since then, she has been followed-up for more than 4 years. She exhibited a dramatic response to CSA, with weaning and then cessation of steroid therapy after 5 months. She has remained transfusion-independent. Attempts to discontinue CSA therapy have been unsuccessful. Relapse of the anemia has occurred in the context of viral infections with missed CSA doses. The major clinical problem during treatment has been recurrent oral aphthous ulceration, which responds to topical therapy. She is currently maintained on CSA 100 mg twice daily with a hemoglobin of 10.2 g/dL and a reticulocyte count of 1.6%. A trial of CSA therapy should be considered in patients with Diamond-Blackfan anemia in whom steroid therapy has failed before a transfusion program is instituted or alternative donor stem cell transplantation is entertained.

Pleural relapse during hematopoietic remission in childhood acute lymphoblastic leukemia.

Purpose This report describes an isolated pleural relapse during hematopoietic remission in a child previously treated for acute lymphoblastic leukemia (ALL). Patient and Methods: An 11-year-old boy had a cough and exertional dyspnea 34 months after an initial diagnosis of ALL and 10 months after completion of therapy. Imaging studies revealed a large left pleural effusion. Bone marrow and cerebrospinal fluid studies were negative for disease at this time. Results: Histopathologic examination of biopsy samples revealed cells with morphologic features of acute lymphoblastic leukemia blasts. Immunophenotyping, cytogenetic, and gene rearrangement studies confirmed the presence of a leukemic blast cell population similar to that detected at initial diagnosis. An isolated extramedullary relapse in the pleura was diagnosed. The patient underwent successful reinduction therapy and subsequently a matched unrelated donor bone marrow transplant; he died of disseminated infection in the posttransplant period. Conclusions: Unusual extramedullary sites of relapse are recognized with increasing frequency as long-term survival in childhood ALL improves. The length of the disease-free interval before relapse is felt to be of prognostic significance. Isolated relapse to the pleural space has not previously been described. The mechanism for persistence of leukemic clones in patients who appear to be in hematopoietic remission is unknown.

Transient erythroporphyria of infancy

We describe a neonate with hemolytic disease of the newborn in whom a photosensitivity eruption developed during phototherapy for treatment of hyperbilirubinemia. Free erythrocyte protoporphyrin and zinc protoporphyrin levels were markedly elevated during the neonatal period. Porphyrin levels were normal at 19 weeks of age. The infant had residual skin atrophy and showed clinical and radiologic evidence of kernicterus. The pathogenesis of transient porphyrinemia associated with hemolytic disease of the newborn is unclear.