How I approach hereditary hemolytic anemia and splenectomy.

Hereditary hemolytic anemias (HHA) are a heterogeneous group of anemias associated with decreased red cell survival. While there can be clinical benefit of splenectomy in many cases, splenectomy is not appropriate for all types of HHA. Additionally, there are significant risks during and following splenectomy including surgical risks, postsplenectomy sepsis, and thrombotic complications. This review discusses the diagnostic approach to HHA as well as the role of splenectomy in the management. Surgical approaches and outcomes for total and partial splenectomy are discussed.

Intra-amniotic delivery of amniotic-derived neural stem cells in a syngeneic model of spina bifida.

OBJECTIVE: Neural stem cells (NSCs) may promote spinal cord repair in fetuses with experimental spina bifida. We sought to determine the fate of amniotic-derived NSCs (aNSCs) after simple intra-amniotic injection in a syngeneic model of spina bifida. METHODS: Fetal neural tube defects were induced on 20 pregnant Lewis dams by prenatal administration of retinoic acid. Ten dams served as amniotic fluid donors for epigenetic isolation of aNSCs, which were expanded and labeled with 5-bromo-2'-deoxyuridine. The remaining 10 dams received intra-amniotic injections of the processed aNSCs, blindly in all their fetuses (n = 37) on gestational day 17 (term = E21-22). Fetuses with spina bifida underwent screening for the presence of donor aNSCs in the spinal cord at term. RESULTS: Donor cells were identified in 93.3% of the animals with spina bifida, selectively populating the neural placode, typically in clusters, retaining an undifferentiated morphology, and predominantly on exposed neural surfaces, though some were detected deeper in neighboring neural tissue. CONCLUSIONS: The amniotic cavity can serve as a route of administration of NSCs in experimental spina bifida. Simple intra-amniotic delivery of NSCs may be a practical adjuvant to regenerative strategies for the treatment of spina bifida.

Craniofacial repair with fetal bone grafts engineered from amniotic mesenchymal stem cells.

BACKGROUND: Ethically acceptable applications of fetal tissue engineering as a perinatal therapy can be expanded beyond life-threatening anomalies by amniotic fluid cell-based methods, in which cell procurement poses no additional risk to the mother. We sought to start to determine whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be an adjunct to craniofacial repair. METHODS: New Zealand rabbits (n = 12) underwent creation of a full-thickness diploic nasal bone defect. We then equally divided animals into two groups based on how the defect was repaired: namely, size-matched implants of electrospun biodegradable nanofibers with or without nuclear labeled, allogeneic aMSCs maintained in osteogenic medium. We killed animals 8 wk post-implantation for multiple analyses. Statistical analysis included analysis of variance, post-hoc Bonferroni adjusted comparisons, and Levene's F-test, as appropriate (P < 0.05), with significance set at P < 0.05. RESULTS: Micro-computed tomography scanning (two- and three-dimensional) showed no significant differences in defect radiodensity between groups. However, extracellular calcium levels were significantly higher in engineered grafts than in acellular implants (P = 0.003). There was significantly greater variability in mineralization in acellular implants than in engineered grafts by both direct calcium (P = 0.008) and micro-computed tomography measurements (P = 0.032). There were no significant differences in alkaline phosphatase activity or variance between groups. We documented labeled cells in the engineered grafts. CONCLUSIONS: Craniofacial repair with osseous grafts engineered from aMSCs lead to enhanced and more consistent mineralization compared with an equivalent acellular prosthetic repair. Amniotic fluid-derived engineered bone may become a practical adjunct to perinatal craniofacial reconstruction.

Limb reconstruction with decellularized, non-demineralized bone in a young leporine model.

Limb salvage from a variety of pathological processes in children is often limited by the unavailability of optimal allograft bone, or an appropriate structural bone substitute. In this study, we sought to examine a practical alternative for pediatric limb repair, based on decellularized, non-demineralized bone grafts, and to determine whether controlled recellularization prior to implantation has any impact on outcome. Growing New Zealand rabbits (n = 12) with a complete, critical-size defect on the left tibiofibula were equally divided into two groups. One group received a decellularized, non-demineralized leporine tibiofibula graft. The other group received an equivalent graft seeded with mesenchymal stem cells labeled with green fluorescent protein (GFP), at a fixed density. Animals were euthanized at comparable time points 3-8 weeks post-implantation. Statistical analysis was by the Student t-test and Fisher's exact test (P < 0.05). There was no significant difference in the rate of non-union between the two groups, including on 3D micro-CT. Incorporated grafts achieved adequate axial bending rigidity, torsional rigidity, union yield and flexural strength, with no significant differences or unequal variances between the groups. Correspondingly, there were no significant differences in extracellular calcium levels, or alkaline phosphatase activity. Histology confirmed the presence of neobone in both groups, with GFP-positive cells in the recellularized grafts. It was shown that osseous grafts derived from decellularized, non-demineralized bone undergo adequate remodeling in vivo after the repair of critical-size limb defects in a growing leporine model, irrespective of subsequent recellularization. This methodology may become a practical alternative for pediatric limb reconstruction.

Targeted quantitative amniotic cell profiling: a potential diagnostic tool in the prenatal management of neural tube defects.

PURPOSE: We sought to determine whether amniotic cell profiles correlate quantitatively with neural tube defect (NTD) type and/or size. METHODS: Sprague-Dawley fetuses exposed to retinoic acid (n=61) underwent amniotic fluid sample procurement before term. Samples were analyzed by flow cytometry for the presence of cells concomitantly expressing Nestin and Sox-2 (neural stem cells, aNSCs), and cells concomitantly expressing CD29 and CD44 (mesenchymal stem cells, aMSCs). Statistical analysis included ANOVA and post-hoc Bonferroni adjusted comparisons (P<0.05). RESULTS: There was a statistically significant increase in the proportion of aNSCs in fetuses with spina bifida (6.78%± 1.87%) when compared to those with exencephaly (0.64%± 0.23%) or with both spina bifida and exencephaly (0.22%± 0.09%). Conversely, there was a statistically significant decrease in the proportion of aMSCs in fetuses with exencephaly, either isolated (1.09%± 0.42%) or in combination defects (2.37%± 0.63%) when compared with normal fetuses (8.83%± 1.38%). In fetuses with isolated exencephaly, there was a statistically significant inverse correlation between the proportion of aNSCs and defect size. CONCLUSIONS: The proportions of neural and mesenchymal stem cells in the amniotic fluid correlate with the type and size of experimental NTDs. Targeted quantitative amniotic cell profiling may become a useful diagnostic tool in the prenatal evaluation of these anomalies.

Predictive value of the Pediatric Ulcerative Colitis Activity Index in the surgical management of ulcerative colitis.

PURPOSE: The primary purpose of this study was to investigate the relationship between Pediatric Ulcerative Colitis Activity Index (PUCAI) and operative management. We also specifically evaluated those patients receiving tacrolimus for their disease. METHODS: A retrospective review (1/06-1/11) identified ulcerative colitis patients (≤21 years old) undergoing restorative proctocolectomy with rectal mucosectomy and ileal pouch-anal anastomosis. Main outcomes included pre-operative PUCAI, combined versus staged procedure, and postoperative complications. Patients receiving tacrolimus within 3 months of surgical intervention were identified. PUCAI at tacrolimus induction and medication side effects were also noted. RESULTS: Sixty patients were identified. Forty-two (70%) underwent combined and 18 (30%) had staged procedures. Pre-operative PUCAI was lower for combined versus staged patients (p = < 0.001). Furthermore, a higher pre-operative PUCAI strongly correlated with the likelihood of undergoing a staged procedure (p < 0.001). Forty-four patients (73%) received tacrolimus. Significant improvement in their PUCAI was noted from induction to pre-operative evaluation (p < 0.001). Minor and reversible side effects occurred in 46% of patients receiving tacrolimus, but complication rates were not significantly different. CONCLUSIONS: There is a very strong correlation between the PUCAI and the likelihood of undergoing a staged procedure. A significant improvement in PUCAI occurs following preoperative tacrolimus therapy.

Prenatal tracheal reconstruction with a hybrid amniotic mesenchymal stem cells-engineered construct derived from decellularized airway.

PURPOSE: This study was aimed at examining an airway construct engineered from autologous amniotic mesenchymal stem cells (aMSCs) and a xenologous decellularized airway scaffold as a means for tracheal repair. METHODS: Fetal lambs (N = 13) with a tracheal defect were divided into 2 groups. One group (acellular, n = 6) was repaired with a decellularized leporine tracheal segment. The other group (engineered, n = 7) received an identical graft seeded with expanded/labeled autologous aMSCs. Newborns were euthanized for multiple analyses. RESULTS: Eleven lambs survived to term, 10 of which could breathe at birth. Engineered grafts showed a significant increase in diameter in vivo (P = .04) unlike acellular grafts (P = .62), although variable stenosis was present in all implants. Engineered constructs exhibited full epithelialization, compared with none of the acellular grafts (P = .002). Engineered grafts had a significantly greater degree of increase in elastin levels after implantation than acellular implants (P = .04). No such differences were noted in collagen and glycosaminoglycan contents. Donor cells were detected in engineered grafts, which displayed a pseudostratified columnar epithelium. CONCLUSIONS: Constructs engineered from aMSCs and decellularized airway undergo enhanced remodeling and epithelialization in vivo when compared with equivalent acellular implants. Amniotic mesenchymal stem cell-engineered airways may become an alternative for perinatal airway repair.

Adrenal cortical tumors in children: factors associated with poor outcome.

PURPOSE: The purpose of this study was to evaluate recurrence and survival outcomes in pediatric adrenal cortical neoplasms. METHODS: A 90-year retrospective review of children with adrenal cortical neoplasms was performed using multivariate Cox regression analysis to identify factors associated with recurrence and tumor-related mortality. RESULTS: The evaluable cohort included 29 patients. Twenty-seven underwent resection. Twenty-two (81%) had localized disease, and 5 (19%) had locally advanced disease (all received chemotherapy and 2 of 5 were cured). Two patients presenting with metastatic disease died despite treatment. There were 4 recurrences; all patients died. Tumor-related mortality was 24% (7/29). Kaplan-Meier freedom from recurrence was 85% at 1 year (95% confidence interval, 75%-95%). Multivariate Cox regression revealed that older age (P = .01), higher mitotic rate (P = .005), and necrosis (P < .001) were independent predictors of tumor-related death. Higher mitotic rate (P = .007) and larger tumor size (P = .03) were significant predictors of tumor recurrence. CONCLUSION: Risk factors for poor outcomes in patients with adrenocortical tumors include older age, higher mitotic rate, higher percent necrosis, and larger tumor size. Therefore, the presence of these factors may warrant consideration of adjuvant chemotherapy, even in the absence of advanced disease.