Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with [89]zirconium.

Intrabone (IB) hematopoietic cell transplantation (HCT) of umbilical cord blood in humans remains experimental and the technique has not been optimized. It is unknown whether hematopoietic progenitor cells (HPCs) injected IB are initially retained in the marrow or rapidly enter into the venous circulation before homing to the marrow. To develop an IB-injection technique that maximizes HPC marrow-retention, we tracked radiolabeled human HPCs following IB-injection into swine. We developed a method to radionuclide-label HPCs using a long-lived positron emitter (89) Zr and protamine sulfate that resulted in cellular-retention of low-dose radioactivity. This approach achieved radioactivity levels sufficient for detection by positron emission tomography with both high sensitivity and spatial resolution when fused with computed tomography. We found that conditions utilized in pilot IB-HCT clinical trials conducted by others led to both rapid drainage into the central venous circulation and cellular extravasation into surrounding muscle and soft tissues. By optimizing the needle design, using continuous real-time intra-marrow pressure monitoring, and by reducing the infusion-volume and infusion-rate, we overcame this limitation and achieved high retention of HPCs in the marrow. This method of IB cellular delivery is readily applicable in the clinic and could be utilized in future investigational IB-HCT trials aimed at maximizing marrow retention of HPCs.

Validation of upper extremity post-thrombotic syndrome outcome measurement in children.

Using the Manco-Johnson instrument in a derivation cohort of 107 children with or without a central venous catheter, upper extremity physical findings of post-thrombotic syndrome were absent, and the pain score was 0 in all but one child. Interrater reliability in an independent validation cohort (n = 38) of children with or without upper extremity deep venous thrombosis was 97%-100%.

A thrombolytic regimen for high-risk deep venous thrombosis may substantially reduce the risk of postthrombotic syndrome in children.

Important predictors of adverse outcomes of thrombosis in children, including postthrombotic syndrome (PTS), have recently been identified. Given this knowledge and the encouraging preliminary pediatric experience with systemic thrombolysis, we sought to retrospectively analyze our institutional experience with a thrombolytic regimen versus standard anticoagulation for acute, occlusive deep venous thrombosis (DVT) of the proximal lower extremities in children in whom plasma factor VIII activity and/or D-dimer concentration were elevated at diagnosis, from within a longitudinal pediatric cohort. Nine children who underwent the thrombolytic regimen and 13 who received standard anticoagulation alone were followed from time of diagnosis with serial clinical evaluation and standardized PTS outcome assessments conducted in uniform fashion. The thrombolytic regimen was associated with a markedly decreased odds of PTS at 18 to 24 months compared with standard anticoagulation alone, which persisted after adjustment for significant covariates of age and lag time to therapy (odds ratio [OR] = 0.018, 95% confidence interval [CI] = < 0.001-0.483; P = .02). Major bleeding developed in 1 child, clinically judged as not directly related to thrombolysis for DVT. These findings suggest that the use of a thrombolysis regimen may safely and substantially reduce the risk of PTS in children with occlusive lower-extremity acute DVT, providing the basis for a future clinical trial.

Lemierre's and Lemierre's-like syndromes in children: survival and thromboembolic outcomes.

OBJECTIVE: Lemierre's syndrome, or jugular vein thrombosis (JVT) associated with anaerobic infection of the head and neck and frequently complicated by septic pulmonary embolism (PE), has historically been described as a disease of young adults. In recent years, an increasing number of case reports of childhood Lemierre's syndrome have been published, focusing mostly on the clinical and laboratory findings at disease presentation and the outcomes of infection. Given the potentially life-threatening thromboembolic complications of this disorder, we reviewed our single-institutional experience with pediatric Lemierre's and Lemierre's-like syndromes (LALLS) from within the context of a larger cohort study of thrombosis in children. METHODS: Children who were aged from birth to 21 years and had received a diagnosis of JVT and Lemierre's syndrome at the Children's Hospital (Denver, CO) between 2001 and 2005 were identified for inclusion. Case designation of LALLS required all the following: (1) radiologic confirmation of JVT, (2) clinical diagnosis of pharyngitis or other febrile illness, and (3) intraoperative evidence of loculated infection in the head and neck region or radiologic demonstration of bilateral pulmonary infiltrates. Isolation of a causative organism by microbiologic culture of blood, tissue, or purulent fluid was also a necessary diagnostic criterion among patients who had not been treated with antibiotics before culture. A designation of classic Lemierre's syndrome was reserved for documented cases of anaerobic infection. Children in whom JVT was associated with the presence of an ipsilateral central venous catheter were excluded from the analysis. Analysis included information on underlying medical conditions, microbiologic and radiologic findings, and comprehensive hypercoagulability testing results from the time of diagnosis, as well as antimicrobial and anticoagulant therapies administered. In addition, clinical outcomes were evaluated via serial follow-up and included bleeding complications, thrombus resolution on serial radiologic studies, symptomatic recurrent venous thromboembolism (VTE), and mortality. RESULTS: From January 2001 to January 2005, 9 children with LALLS were identified. Median age was 15 years (range: 2.5-20 years). Clinical presentation was consistent with septic PE in 5 cases and septic shock in 2. Thrombophilia was present in 100% (7 of 7) of children tested, consisting principally of antiphospholipid antibodies and elevated factor VIII activity. Anticoagulation was given in 89% (8 of 9), for a median duration of 3 months (range: 7 weeks-1 year). After a median follow-up time of 1 year, all children had survived without recurrent VTE or anticoagulant-associated major hemorrhage. JVT failed to resolve at 3 to 6 months in 38% of anticoagulated children. CONCLUSIONS: Our experience suggests that LALLS is an emerging pediatric concern with serious acute (eg, septic PE) and chronic (eg, persistent vascular occlusion) complications. Septic JVT may not be uniquely anaerobic, and the inflammatory prothrombotic state is often characterized by antiphospholipid antibodies and elevated factor VIII levels. Early diagnosis and aggressive antimicrobial and antithrombotic therapies in LALLS may be necessary for optimal long-term outcomes.

Elevated plasma factor VIII and D-dimer levels as predictors of poor outcomes of thrombosis in children.

BACKGROUND: Elevated levels of plasma factor VIII and D-dimer predict recurrent venous thromboembolism in adults. We sought to determine whether an elevation of factor VIII, D-dimer, or both at diagnosis and persistence of the laboratory abnormality after three to six months of anticoagulant therapy correlate with poor outcomes of thrombosis in children. METHODS: We evaluated levels of factor VIII and D-dimer and additional components of an extensive laboratory thrombophilia (i.e., hypercoagulability) panel at the time of diagnosis in 144 children with a radiologically confirmed acute thrombotic event. All patients were treated initially with heparin and then with either warfarin or low-molecular-weight heparin for at least three to six months, according to the current standard of care. Patients were examined at follow-up visits 3, 6, and 12 months after diagnosis and then annually, at which times testing was repeated in children with previously abnormal factor VIII and D-dimer test results and a uniform evaluation for the post-thrombotic syndrome was performed. RESULTS: Among 82 children for whom complete data were available regarding laboratory test results at diagnosis and thrombotic outcomes during follow-up, 67 percent had factor VIII levels above the cutoff value of 150 IU per deciliter, D-dimer levels above 500 ng per milliliter, or both at diagnosis, and at least one of the two laboratory values was persistently elevated in 43 percent of the 75 patients in whom testing was performed after three to six months of anticoagulant therapy. Fifty-one percent of the 82 patients had a poor outcome (i.e., a lack of thrombus resolution, recurrent thrombosis, or the post-thrombotic syndrome) during a median follow-up of 12 months (range, 3 months to 5 years). Elevated levels of factor VIII, D-dimer, or both at diagnosis were highly predictive of a poor outcome (odds ratio, 6.1; P=0.008), as was the persistence of at least one laboratory abnormality at three to six months (odds ratio, 4.7; P=0.002). The combination of a factor VIII level above 150 IU per deciliter and a D-dimer level above 500 ng per milliliter at diagnosis was 91 percent specific for a poor outcome, and after three to six months of standard anticoagulation, the combination was 88 percent specific. CONCLUSIONS: Elevated levels of plasma factor VIII, D-dimer, or both at diagnosis and a persistent elevation of at least one of these factors after standard-duration anticoagulant therapy predict a poor outcome in children with thrombosis.

Introduction to microsurgery: an emerging discipline in biomedical research.

Using microsurgical techniques, biomedical researchers are able to perform procedures that would otherwise be impossible on small laboratory animals. The authors provide a primer on learning microsurgical technique, from correct posture and hand position, to understanding lenses and proper handling of surgical needles and suture material.

Microsurgical instrumentation and suture material.

Microsurgery requires specialized instruments and very fine suture material. The authors describe microsurgical instruments and suturing materials available for small animal microsurgery.

Manometric changes during retrograde biliary infusion in mice.

The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [(14)C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 A in diameter to specific histological compartments.

Paragordius varius.

We describe a 30-year-old man who presented with a tangled knot of worms that he "passed per rectum." The worms were identified as Paragordius varius. This is an uncommon pseudoparasite that is ingested from contaminated food or water. Identifying characteristics and the life cycle of Gordian worms are briefly discussed. Identification of this worm is important to distinguish it from true parasitic worms.

Nosocomial nasal myiasis.

Sixty-five fly maggots were retrieved from the nasal cavity of an unconscious 64-year-old man who had been admitted 18 days earlier with diabetic hyperosmolar coma. The larvae were identified as Cochliomyia macellaria, an organism commonly associated with myiasis in the United States. The clinical time sequence indicates that this infection was acquired in the hospital. This incident provides further evidence that immobile and debilitated patients are at risk to acquire myiasis.