Heart transplant in a factor VIII-deficient patient with a high-titre inhibitor: perioperative management using high-dose continuous infusion factor VIII and recombinant factor VIIa.

Four years prior to transplantation, a 14-year-old boy with severe haemophilia A and a high-responding factor VIII (FVIII) inhibitor developed an anteroseptal myocardial infarct while receiving high doses of an activated prothrombin complex concentrate (PCC). Cardiac transplantation was required for survival because of the ensuing cardiomyopathy. At surgery, the patient's inhibitor titre was 1.8 Bethesda units (BU). High-dose bolus therapy, followed by a continuous infusion of FVIII provided excellent operative and initial postoperative haemostasis without additional blood-product support. Once anamnaesis developed on day 6 postoperatively, recombinant factor VIIa (rFVIIa) therapy was initiated. Haemostasis remained excellent, except for the transient increase in chest-tube bleeding that was noted on day 7. epsilon-Aminocaproic acid was added and haemostasis was re-established. On day 15, rFVIIa was replaced with alternate day infusions of prothrombin complex concentrates (PCCs). On day 21 following the transplant, the patient was discharged, remaining on daily FVIII immune tolerance and thrice-weekly PCC prophylaxis. He remains well 24 months after transplant with an inhibitor titre of 39 BU. This paper describes the second case of cardiac transplantation complicated by haemophilia and an inhibitor, and discusses preoperative planning and operative and postsurgical haemostasis management.

CT imaging of splenic sequestration in sickle cell disease.

Pooling of blood in the spleen is a frequent occurrence in children with sickle cell diseases, particularly in the first few years of life, resulting in what is termed "splenic sequestration crisis." The spectrum of severity in this syndrome is wide, ranging from mild splenomegaly to massive enlargement, circulatory collapse, and even death. The diagnosis is usually clinical, based on the enlargement of the spleen with a drop in hemoglobin level by > 2 g/dl, and it is rare that imaging studies are ordered. However, in the patient who presents to the emergency department with non-specific findings of an acute abdomen, it is important to recognize the appearance of sequestration on imaging studies. We studied seven patients utilizing contrast-enhanced CT scans and found two distinct patterns--multiple, peripheral, non-enhancing low-density areas or large, diffuse areas of low density in the majority of the splenic tissue. Although radiological imaging is not always necessary to diagnose splenic sequestration, in those situations where this diagnosis is not immediately obvious, it makes an important clarifying contribution.

Human HKR isozyme: organization of the hexokinase I gene, the erythroid-specific promoter, and transcription initiation site.

We previously described a cDNA for the human HKR isozyme, whose sequence is identical to that of the ubiquitous HKI isozyme, except for a unique 5' end sequence. Screening a human genomic library with a DNA fragment containing an erythroid-specific sequence we found one clone including 5' ends for both HKR and HKI genes. The first HKR exon was located 3 kb 5' of the first HKI exon. These results confirmed that HKR is produced from the HKI gene by alternate promoter and splicing. The HKI gene consisted of 19 exons. All exon-intron boundaries are conserved among the genes for hexokinase and glucokinase. The HKI gene length was estimated at over 67 kb. The initiation site for the HKR was identified by primer extension. Its promoter did not have a canonical TATA box, but an inverted GATA at nt -177 (i.e., 36 nt 5' to the transcription initiation site). In the HKR promoter a DNA fragment spanning nt -275 to nt -107 exhibited erythroid-specific activity. However, this was absent in shorter promoter fragments (nt -206 to -107 or nt -229 to -107). The sequence nt -275 to -229, which appeared critical for the erythroid-specific expression of the HKR gene, contained a consensus motif for Sp-1 and GATA, CCAAT, and GGAA motifs. The electrophoretic mobility shift assay (EMSA) suggested erythroid-specific cooperative protein-protein interaction in this region. Deletion of the GATA sequence as well as reaction with a specific antibody identified GATA-1 as one of the interacting proteins.

Serum transferrin receptor as a marker of erythropoiesis suppression in patients on chronic transfusion.

In the management of patients requiring chronic transfusion, various parameters may be used to evaluate the degree of erythroid marrow suppression. The aim of our study was to assess which of these parameters provide the most useful assessment of erythropoiesis. We studied 27 chronically transfused patients, 19 with sickle cell disease (SS patients) and 8 with thalassemia. Thirty-one nonchronically transfused SS patients and 74 healthy children served as controls. We measured serum transferrin receptor levels, reticulocyte counts, hemoglobin (Hb) concentrations and erythropoietin levels. The serum transferrin receptor levels were very elevated in control SS patients and remained significantly elevated in those on transfusion therapy, but were normal in thalassemia patients, indicating a more complete suppression of erythropoiesis. The reticulocyte counts were elevated in all SS patients, even when on chronic transfusion, but were in the normal range in patients with thalassemia. Erythropoietin levels were elevated in patients with thalassemia and in all the SS patients. Hb levels negatively correlated with serum transferrin receptor and erythropoietin in all SS patients. In the transfused SS patients, a higher HbS level correlated with higher reticulocyte counts, transferrin receptor, and erythropoietin levels. In thalassemia patients, erythropoiesis was more completely suppressed, as reflected both by normal reticulocyte counts and near-normal transferrin receptor levels. Though the reticulocyte counts were not significantly different in the transfused SS patients, the serum transferrin receptor levels were less elevated than in SS patients not on transfusion. The serum transferrin receptor level appears to be the most useful marker of marrow erythropoietic activity in chronically transfused SS patients. We recommend that reticulocyte counts be integrated with periodic measurements of serum transferrin receptor levels.

Erythropoietic protoporphyria: four novel frameshift mutations in the ferrochelatase gene.

Human erythropoietic protoporphyria is an inherited disorder of the heme metabolic pathway caused by defects in the gene for ferrochelatase, the terminal enzyme of the pathway that catalyzes chelation of ferrous iron into protoporphyrin IX to form heme. Mutation analysis was performed for families with erythropoietic protoporphyria and four novel frameshift mutations were identified. Two of the mutations, 205insA and 215insT in exon 3 of the ferrochelatase gene, are single bp insertions. The other two, 400delA in exon 4 and 678delG in exon 6, are single bp deletions. All of the mutations result in premature termination codons downstream shortly after the mutation sites, and in one case the premature termination codon caused by 400delA was also shown to reduce mRNA level via nonsense-mediated mRNA decay.

Identification of the cDNA for human red blood cell-specific hexokinase isozyme.

A unique cDNA for hexokinase (HK) was identified from poly(A)+ RNA of human reticulocytes by anchored polymerase chain reaction. This appeared to represent the cDNA for the red blood cell (RBC)-specific HK isozyme (HKR) described in our previous study (Murakami et al: Blood 75:770, 1990). Its nucleotide sequence was identical to HKI cDNA except for the 5' extreme end. It lacked the first 62 nucleotides of the HKI coding region: instead, it contained a unique sequence of 60 nucleotides at the beginning of the coding sequence as well as another unique sequence upstream of the putative translation initiation site. It lacked the porin-binding domain which facilitates binding to the mitochondria, thus explaining the exclusive cytoplasmic localization of HKR. It was the major cDNA derived from reticulocytes, consistent with the observation that HKR activity is predominant in reticulocytes. Northern blot analysis showed that it was expressed in the reticulocytes and in the K562 erythroleukemic cell line, but not in a lymphocytic cell line. In the extract of K562 cells, HKR activity co-eluted with the HKR of human RBCs on a MonoQ column (Pharmacia, Piscataway, NJ) chromatography, using a salt gradient elution. The separate genetic control of the RBC-specific HK isozyme explains the clinical reports of two types of HK deficiency, one in which the HK activity was reduced exclusively in the RBC (HKR defect) and another with general decrease of HK activity in several tissues (HKI defect).

Pulmonary embolism developing in patients with sickle cell disease on hypertransfusion and IV deferoxamine chelation therapy.

Pulmonary disease, including thromboembolic problems, accounts for a large portion of the morbidity of sickle cell disease. Chronic transfusion therapy is now a part of long-term treatment of sickle cell patients with stroke and chest syndrome. The resultant iron overload must be treated with chelation therapy using deferoxamine. Poor compliance with subcutaneous chelation therapy has necessitated intravenous deferoxamine treatment. We describe two patients with sickle cell disease on such a regimen, who became hypoxic as a result of pulmonary thromboembolism, secondary to venous thrombophlebitis. The thrombophlebitis and subsequent pulmonary embolism probably reflect the hypercoagulable state seen in sickle cell and are not due to the deferoxamine therapy.

Deferoxamine-induced platyspondyly in hypertransfused thalassemic patients.

Deferoxamine chelation therapy (widely used to reduce iron overload in hypertransfused thalassemic patients) has been implicated in causing skeletal growth abnormalities (rachitic-like changes in the long bones and vertebral body flattening), particularly when used in early infancy and at high dose levels. Radiographs of seven hypertransfused and well-chelated patients with thalassemia were reviewed. For two patients, serial films of the spine from the early 1970s to the present revealed a sequence of changes in the vertebral bodies, beginning with normal bodies that became bulbous and subsequently flattened. These two patients had begun deferoxamine chelation therapy early in infancy. The bone changes, though slightly reminiscent of post-radiation changes, are milder and result in a final Scheuermann-like picture.

Haplotypes of the human beta-like globin gene cluster: analyzing the 3' Hpa I polymorphic site by long distance PCR.

Haplotypes of the beta-like globin gene cluster in individuals with sickle cell disease appear to be important as prognostic factors for the severity of the disease. The Hpa I polymorphic site 3' of the beta-globin gene is very often involved in haplotyping. However, this restriction site cannot be ascertained by conventional PCR, as it is in the middle of a long repetitive sequence. Long distance PCR was employed to amplify this Hpa I-containing region. By positioning either one or both primers outside the repetitive sequence, specific amplification was achieved. Moreover, sufficient amplification can be obtained by using a white cell lysate instead of purified DNA, thereby significantly reducing the amount of blood needed, and the total procedure time.

Systematic screening for RNA with skipped exons--splicing mutations of the ferrochelatase gene.

A systematic method was designed to screen a large population of patients with erythropoietic protoporphyria (EPP) for aberrant ferrochelatase RNA with skipped exons. The method utilizes the new junction sequence created by exon skipping as the probe to detect such RNA species. In 7 of 17 EPP families, an aberrant ferrochelatase RNA with one exon missing was observed. Two previously unreported splicing mutations were also identified in 2 EPP families. One was a G >> T transversion at the +1 position of the acceptor site of intron 8, causing exon 9 to be skipped during RNA splicing. Both the patient and her father were found to be heterozygous for this mutation. In another family, an A >> G transition at the +3 position of the donor site of intron 10 was identified, associated with exon 10 skipping during RNA splicing. Both the patient and her father were heterozygous for this mutation.

Recent advances in the management of thalassemia.

Although the current treatment of thalassemia with regular transfusions and assiduous chelation leads to a good quality of life and long survival, it is cumbersome and expensive. Various treatments have recently been explored. Bone marrow transplantation can cure thalassemia, but there was severe mortality in initial trials. It is safely successful only in patients in good clinical condition and with a compatible donor. Certain drugs, including azacytidine, butyrate, hydroxyurea, and erythropoietin may increase the production of fetal hemoglobin; their practical value is being explored. New potential oral iron chelators are under investigation. L1, the best evaluated, appears effective, but its potential toxicity remains undefined.

Human protoporphyria: genetic heterogeneity at the ferrochelatase locus.

Inherited deficiency of ferrochelatase results in erythropoietic protoporphyria (EPP). Genetic heterogeneity at the locus for human ferrochelatase was investigated. Analysis of genomic DNA of patients with EPP and of control subjects by restriction endonuclease techniques using ten different enzymes detected polymorphisms only at sites recognized by EcoRI, HincII, PstI and TaqI. None of these polymorphisms alone was specific for expression of the disease since each was observed in control subjects as well. Three of these polymorphisms (at EcoRI, HincII and PstI sites) were always associated, indicating linkage. These and other studies demonstrate that the ferrochelatase gene is markedly heterogeneous. It is not yet clear whether some of the mutations associated with these polymorphisms contribute to expression of EPP.

MRI marrow observations in thalassemia: the effects of the primary disease, transfusional therapy, and chelation.

The magnetic resonance bone marrow patterns in thalassemia were evaluated to determine changes produced by transfusion and chelation therapy. Thirteen patients had T1- and T2-weighted images of the spine, pelvis and femurs. Three received no therapy (age range 2.5-3 years). Three were hypertransfused (transfused to maintain a hemoglobin greater than 10 g/dl) and not chelated because of age (age range 6 months-8 years). Seven were hypertransfused and chelated (age range 12-35 years). Signal characteristics of marrow were compared with those of surrounding muscle and fat. Fatty marrow (isointense with subcutaneous fat) was compared with red marrow (hypointense to fat and slightly hyperintense to muscle). Marrow hypointense to muscle was identified as iron deposition within red marrow. The untreated group demonstrated signal consistent with red marrow throughout the central and peripheral skeleton. Hypertransfused but not chelated patients demonstrated marked iron deposition in the central and peripheral skeleton. Hypertransfused and chelated patients demonstrated iron deposition in the central skeleton and a mixed appearance of marrow in the peripheral skeleton. The MR appearance of marrow in thalassemia is a reflection of the patient s transfusion and chelation therapy. Iron deposition occurs despite chelation therapy in sites of active red marrow. As red marrow retreats centrally with age, so does the pattern of iron deposition. The long-term biological effects of this iron deposition are unknown.

MR marrow signs of iron overload in transfusion-dependent patients with sickle cell disease.

Magnetic resonance (MR) marrow signal in the axial and appendicular skeleton of 13 transfusion-dependent and chelated pediatric patients with sickle cell anemia (SSD) was compared with marrow signal in six non-transfusion-dependent patients with SSD. Hepatic, pancreatic, and renal MR signal were also evaluated. Indication for hypertransfusion therapy was primarily prior history of stroke. Transfusion-dependent patients had evidence of iron deposition throughout the imaged marrow and the liver, despite deferoxamine chelation therapy. Non-transfusion-dependent patients did not demonstrate grossly apparent signs of iron overload. Red marrow restoration was present in the spine, pelvis, and long bones and, in some patients, within the epiphyses. Marrow edema secondary to vaso-occlusive crises was evident in the metaphyses and diaphyses of long bones in areas of both red and fatty marrow and was best seen using fat-saturated T2-weighted imaging techniques.

Middle cerebral artery velocity changes during transfusion in sickle cell anemia.

BACKGROUND AND PURPOSE: Sickle cell disease is associated with cerebral hyperemia, which is therapeutically reduced by transfusion; however, the process of transfusion-induced cerebral perfusion changes has heretofore not been observed. METHODS: We document the acute changes of intracranial arterial velocity in 10 patients (7 with strokes, 3 without) undergoing transfusion therapy using transcranial Doppler ultrasonography. Middle cerebral artery velocities were bilaterally measured every 30 minutes for the duration of transfusion (4 to 5 hours). Regional cerebral blood flow was quantified in 5 of these patients before the transfusion and 24 hours later by the 133Xe technique. RESULTS: Velocities in stroke-associated vessels (64.33 +/- 18.65 cm/s; n = 6) were significantly lower than in uninfarcted territories (99.54 +/- 27.39 cm/s; n = 13), and both types of vessels showed a robust reduction of blood flow velocities during transfusion. The rates of reduction were not significantly different as a function of prior stroke but did correlate with pretransfusion velocities and with the rise in hematocrit (multiple r = .887, P < .001). These reductions occurred rapidly within the first 3 hours of transfusion. Velocities attained at the end of transfusion were maintained in the hour after transfusion and the next day. CONCLUSIONS: We conclude that transfusion induces rapid changes in cerebral hemodynamics that are related to pretransfusion velocities and a rise in hematocrit. Transcranial Doppler provides a safe, simple, and noninvasive technique of monitoring these changes and may provide a means of making therapeutic decisions regarding transfusion therapy in patients with sickle cell anemia.

A novel splicing mutation in the ferrochelatase gene responsible for erythropoietic protoporphyria.

An aberrant ferrochelatase mRNA lacking exon 7 was found in a patient with erythropoietic protoporphyria (EPP). The exon 7 skipping appears to result from a G >> A transition at position +5 of the donor site of intron 7 of the ferrochelatase gene. The patient is heterozygous for the mutation. Since the patient's paternal half-brother (not available for testing) also has clinically obvious EPP, their father appeared to be the source of the mutant allele. The father was in fact found to be a carrier of the same mutation and his ferrochelatase activity was 35% of normal; however, he is asymptomatic, with only a slightly elevated erythrocyte protoporphyrin level. These findings confirm that the observed mutation is responsible for the defect. The variability in clinical expression of EPP probably reflects the great heterogeneity of the ferrochelatase gene defects and the contribution of additional exogenous and endogenous inducers of latent disease.

Experience with 500 prenatal diagnoses of sickle cell diseases: the effect of gestational age on affected pregnancy outcome.

Prenatal diagnosis of sickle cell diseases is obtained rapidly and precisely by polymerase chain reaction (PCR) with Ddel restriction analysis and dot-blotting with allele-specific oligonucleotides (ASO). Prenatal diagnosis of HgbSS and HgbSC was performed in 500 pregnancies, 196 by Southern blot and 304 by PCR. PCR drastically shortened the interval from sampling to reporting, allowing acceptance even of samples with unknown paternal phenotype, and resulted in an overall four-fold increase in diagnoses. In 108 pregnancies, the diagnosis was an affected fetus; 25 were HgbSC: 3 (12 per cent) were terminated; 83 were HgbSS: four ended in miscarriage; 40/79 (51 per cent) were terminated. The gestational age at the time of report to the mother appeared to be a major outcome determinant when the fetal diagnosis was HgbSS. The change-point in the maternal decision was found at 20 weeks of gestation. Before the 20th week, most mothers (64 per cent) chose termination; thereafter, the majority (72 per cent) chose continuation. The odds ratio of termination in earlier relative to later reporting was 4.7. In order to offer a choice to the mothers at risk of delivering a fetus affected by sickle cell disease, the diagnosis should be reported before the 20th week of gestation.

Effects of total hemoglobin and hemoglobin S concentration on cerebral blood flow during transfusion therapy to prevent stroke in sickle cell disease.

BACKGROUND: The standard treatment of stroke in sickle cell disease is chronic transfusion to maintain the fraction of abnormal hemoglobin (hemoglobin S [HbS]) below 20%. Risks associated with such transfusion can be reduced by allowing higher HbS levels, but the physiological consequences of this modification are unknown. Cerebral blood flow is elevated in sickle cell disease proportionate to the degree of anemia and is reduced by transfusion. We tested the effects of various HbS levels on cerebral blood flow during the course of transfusion therapy. CASE DESCRIPTIONS: We monitored cerebral blood flow (by the 133Xe inhalation method) in three patients whose chronic transfusion program was changed from a traditional regimen (HbS < 20%) to a moderate one, allowing HbS to rise to 45% to 50% between treatments. As expected, cerebral blood flow was higher with lower hemoglobin and higher HbS concentration. However, the HbS fraction appeared to exert a separate influence on the hyperemia, independent of total hemoglobin concentration. Furthermore, cerebral blood flow was higher during the modified regimen, despite equivalent anemia. CONCLUSIONS: These results suggest caution in adapting the modified transfusion regimen. Although HbS concentrations of 50% did not cause any frank neurological sequelae, the possible consequences of the associated hyperemia over time are unknown. We conclude that larger clinical and physiological studies of moderate transfusion regimens (allowing higher concentration of HbS) are necessary before it can become standard therapy.