Peter J. Larson

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 (F.IX) 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 (2×1011 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 F.IX 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.

Delayed hemolytic transfusion reaction due to anti-Goa, an antibody against the low-prevalence Gonzales antigen

Goa (DCor) is a low‐frequency antigen in the Rh system found on red cells lacking part of the D mosaic (category IVa). Anti‐Goa has not been previously reported to cause hemolytic transfusion reactions. A 27‐year‐old African American male with sickle‐cell disease, maintained on chronic transfusion, was noted to have dark plasma during an erythrocytapheresis, procedure, and the pretransfusion hemoglobin was noted to be 1 g/dl lower than 4 weeks before (with hyperbilirubinemia and a significantly increased LDH). Polyspecific direct antiglobulin test (DAT) was weakly positive (C3‐weak, IgG‐weak), and indirect antiglobulin tests (IATs) performed on the serum (pre‐ and posttransfusion reaction) and a red blood cell (RBC) eluate from the postreaction sample were negative. A segment from one of the four implicated units from the prior months transfusion was strongly reactive at 37°C and using anti‐human globulin (AHG) when crossmatched with the postreaction serum and the eluate. The postreaction serum, screened with a panel of red cells positive for low‐prevalence antigens, reacted with three Go(a+) cells. The implicated unit was reactive with a previously identified anti‐Goa serum.

Gene therapy for hemophilia B: AAV-mediated transfer of the gene for coagulation factor IX to human muscle.

Hemophilia B is an X-linked bleeding disorder caused by a deficiency of coagulation factor IX. In its severe form, the disease results in spontaneous bleeding as well as bleeding following surgery or trauma, and is clinically indistinguishable from hemophilia A (factor VIII deficiency). The major morbidity of the disease is the chronic arthropathy of hemophilia that develops following spontaneous hemorrhage into the joint spaces. Blood in the joint space serves as an irritant causing proliferation of the vascular synovial membranes. Hypertrophied synovium is more susceptible to injury resulting in a vicious cycle of rebleeding that furthers the destruction of the joint. The major bleeding-related mortality of hemophilia is intracranial hemorrhage that occurs in between 3 and 14% of patients (1, 2, 3). The mortality rate from intracranial hemorrhage ranges from 20–50%, and survivors are often left with severe neurological sequelae such as seizures, paresis or mental retardation (1). Current treatment of hemophilia is based on the episodic intravenous infusion of highly purified plasma-derived or recombinant clotting factor concentrates in response to bleeding episodes (on-demand therapy).

The presurgical management with erythrocytapheresis of a patient with a high-oxygen-affinity, unstable Hb variant (Hb Bryn Mawr)

BACKGROUND: Hemoglobin (Hb) Bryn Mawr is an unstable Hb variant resulting in congenital hemolytic anemia. This variant Hb also has an increased affinity for oxygen. The perioperative transfusion management of this disorder is described, and the first genomic analysis of this Hb variant is given. CASE REPORT: An 11‐year‐old boy, heterozygous for Hb Bryn Mawr, was referred for cholecystectomy. Sequence analysis of genomic DNA confirmed that the patients was heterozygous for a T–>C transition in the codon for amino acid 85, causing a substitution of serine for phenylalanine in the beta‐globin chain. On the basis of whole‐blood O2 dissociation studies, projected tissue O2 delivery would have been suboptimal during general anesthesia; therefore, a partial red cell exchange transfusion was performed to lower variant Hb and prevent tissue hypoxia during surgery. The red cell mass to be exchanged (50%) was determined from the calculated increase in O2 delivery capacity required to maintain an O2 extraction of 4 to 5 mL of O2 per dL of whole blood. The p50 of whole blood from the patients immediately after the exchange transfusion was 16.0 torr. At the time of surgery, the p50 was normal (25.9 torr). The patients whole blood 2,3 DPG levels were 4.70 mmol per mL of red cells (before transfusion) (normal range=4.8 +/− 0.3 mmol/mL red cells), 4.07 mmol per mL of red cells (immediately after transfusion), and 4.55 mmol per mL of red cells (48 hours after transfusion). CONCLUSION: This patient with Hb Bryn Mawr was prepared for surgery with a partial exchange transfusion to prevent tissue hypoxia during anesthesia. Decreased 2,3 DPG levels immediately after transfusion resulted in increased O2 affinity of whole blood; however, 48 hours after exchange transfusion, a normal p50 (due to both removal of variant Hb and regeneration of 2,3, DPG) was observed. Partial exchange transfusion is useful in the preoperative management of patients with Hb variants characterized by increased O2 affinity.

Other Noninfectious Complications of Transfusion

Publisher Summary The transfusionists must be familiar with many potential noninfectious complications of the infusion of blood or blood products, including circulatory overload, transfusion related acute lung injury (TRALI), and nonimmune-mediated hemolysis. Some of these may be because of human errors and are therefore preventable. Specific therapies are indicated for the treatment of many of these events. Circulatory overload is defined as the expansion of a patients intravascular volume because of the infusion of blood components and/or other fluids that results in cardiac decompensation. When signs of circulatory overload develop, if at all possible, the transfusion should be terminated. Therapy for congestive heart failure (CHF) should be instituted, including fluid restriction, supplemental O2, administration of diuretics, and morphine. TRALI is a syndrome of acute hypoxia because of noncardiogenic pulmonary edema that develops within 16 hours following transfusion. If symptoms develop during infusion of a blood product, the transfusion should be terminated. Supportive care includes supplemental O2 and mechanical ventilation, if indicated. Vasopressors may be necessary for prolonged hypotension. Non-immune-mediated hemolysis involves the destruction of red blood cells (RBCs) within the circulation because of physical injury to the RBC membrane. This may be because of osmotic, thermal, chemical, or mechanical damage to the RBCs. As opposed to immune-mediated intravascular hemolysis, these reactions are rarely life-threatening. Management involves terminating the transfusion, ruling out an immune-mediated mechanism, and determining the cause of RBC injury prior to continuing RBC transfusion. Intravenous access is maintained to provide adequate hydration for good urine output.

Efficacy and inhibitor development in previously treated patients with haemophilia A switched to a B domain‐deleted recombinant factor VIII – clarification of Kogenate inhibitor data

I have read with interest the paper of Gringeri et al (2004). The investigators described both a prospective study of B domaindeleted (BDD) recombinant factor VIII (rFVIII) use in previously treated patients (PTPs), and a retrospective analysis of this product used by Italian haemophilia patients (PTPs) who were switched from another replacement factor to BDDrFVIII. In the presentation of the data, the authors make reference to other studies in haemophilia patients treated with rFVIII products, including several Kogenate /Kogenate -FS studies. We respectfully wish to provide clarification regarding several aspects of the Kogenate studies, particularly the numbers reported in Table IV of the Gringeri et al (2004) paper. Two of the publications (Schwartz et al, 1990; Seremetis et al, 1999) appear to be separate Kogenate studies, while in fact they are reports of the same PTP population at different time points in the study. Seremetis et al (1999) reported the final results of the worldwide study. Similarly, Abshire et al (2000) and Rothschild et al (2002), listed in Table IV of Gringeri et al (2004), are papers on the same study. The original reports (Abshire et al, 2000; Rothschild et al, 2002) clearly specified that there were no de novo inhibitors in the patients studied. It should also be noted that Giles et al (1998) reported a three time-point prevalence (not incidence) study and the population included PTPs/minimally treated patients and previously untreated patients, not representative of PTPs with greater than 100–150 exposure days only, therefore should not be part of the analysis of inhibitor incidence in PTPs. In the small postmarketing surveillance study by AygörenPürsün and Scharrer (1997), of patients who had previously been treated with products containing factor VIII, the age of the patients in the cohort ranged from 2 to 62 years, and of the 39 patients, 13 had moderate disease and 4 had mild disease. Seven of the patients in the total cohort were reported to have a single inhibitor occurrence by non-modified Bethesda assay at the study site; however, as detailed in the Methods section, repeat testing at a central reference laboratory in Frankfurt (using the Nijmegen-modified Bethesda assay) failed to confirm the presence of inhibitors in five of these patients (Aygören-Pürsün & Scharrer, 1997). Samples of two patients were not retested at the reference laboratory and are therefore unconfirmed. I hereby provide a revised table (Table I) reflecting the above clarifications pertaining to Kogenate .