Avaliação da incorporação de enxerto osseo homogeno fresco congelado em humanos: análise histologica e imuno-histoquímica

Allogeneic, fresh-frozen bone has been used in order to replace bone autografts. However, its osteoinduction and osteoconduction properties are not well-defined in the scientific literature. This work aimed to evaluate samples of homogenous bone grafts in humans by qualitative histological and immunohistochemical analysis. For this, ten pre-selected patients underwent surgical augmentation of bone defects. The homogenous fresh frozen block bone graft was stabilized and fixed by bicortical screws. After six months, the reopening procedure was performed for installation of osseointegrated implants. At this time surgical bone graft samples were removed by means of drill trephine. The samples were fixed in 10% formalin, processed with decalcified paraffin, and stained with hematoxylin and eosin. Immunohistochemistry was performed for the expression of Caspase 3 enzyme. The slides were brought to light microscopy for qualitative histology and immunohistochemistry. The results showed non-vital bone tissue, with few areas of deposition of new bone formation on the amorphous matrix, presence of chronic inflammatory infiltrate with areas of osteomyelitis, and expressive immunolabeling of Caspase 3. Given the methods employed and the results it was concluded that the allograft fresh-frozen block is not incorporated into the recipient bed after a healing period of six months.

Fabric-mechanical property relationships of trabecular bone allografts are altered by supercritical CO2 treatment and gamma sterilization

Tissue grafts are implanted in orthopedic surgery every day. In order to minimize infection risk, bone allografts are often delipidated with supercritical CO2 and sterilized prior to implantation. This treatment may, however, impair the mechanical behavior of the bone graft tissue. The goal of this study was to determine clinically relevant mechanical properties of treated/sterilized human trabecular bone grafts, e.g. the apparent modulus, strength, and the ability to absorb energy during compaction. They were compared with results of identical experiments performed previously on untreated/fresh frozen human trabecular bone from the same anatomical site (Charlebois, 2008). We tested the hypothesis that the morphology–mechanical property relationships of treated cancellous allografts are similar to those of fresh untreated bone. The morphology of the allografts was determined by μCT. Subsequently, cylindrical samples were tested in unconfined and confined compression. To account for various morphologies, the experimental data was fitted to phenomenological mechanical models for elasticity, strength, and dissipated energy density based on bone volume fraction (BV/TV) and the fabric tensor determined by MIL. The treatment/sterilization process does not appear to influence bone graft stiffness. However, strength and energy dissipation of the bone grafts were found to be significantly reduced by 36% to 47% and 66% to 81%, respectively, for a broad range of volume fraction (0.14 < BV/TV < 0.39) and degree of anisotropy (1.24 < DA < 2.18). Since the latter properties are strongly dominated by BV/TV, the clinical consequences of this reduction can be compensated by using grafts with lower porosity. The data of this study suggests that an increase of 5–10% in BV/TV is sufficient to compensate for the reduced post-yield mechanical properties of treated/sterilized bone in monotonic compression. In applications where graft stiffness needs to be matched and strength is not a concern, treated allograft with the same BV/TV as an appropriate fresh bone graft may be used.

Embolization for multicompartmental bleeding in patients in hemodynamically unstable condition: prognostic factors and outcome.

PURPOSE: To determine prognostic factors and evaluate outcomes of transcatheter arterial embolization in severely injured patients in hemodynamically unstable condition with multicompartmental bleeding.¦MATERIALS AND METHODS: Between June 2000 and May 2008, 36 consecutive patients treated with transcatheter arterial embolization for major retroperitoneal bleeding associated with at least one additional source of bleeding were retrospectively reviewed. Mean Injury Severity Score (ISS) was 49.4 ± 15.8. Univariate and multivariate analyses were performed to identify parameters associated with failure of embolization, need for additional surgery to control bleeding, and fatal outcome at 30 d.¦RESULTS: Embolization was technically successful in 35 of 36 patients (97.2%) and resulted in immediate and sustained (> 24 h) hemodynamic improvement in 29 (80.5%). Additional hemostatic surgery was necessary after embolization in six patients (16.6%). Fifteen patients (41.6%) died within 30 d. Failure to restore hemodynamic stability was correlated with the rate of administration of packed red blood cells (P = .014), rate of administration of fresh frozen plasma (FFP; P = .031), and systolic blood pressure (SBP) immediately before embolization (P = .002). The need for additional surgery was correlated with FFP administration rate before embolization (P = .0002) and hemodynamic success (P = .003). Death was correlated with Glasgow Coma Scale score at admission (P = .001), ISS (P = .014), New Injury Severity Score (P = .016), number of injured sites (P = .012), SBP before embolization (P = .042), need for vasopressive drugs before embolization (P = .037), and hemodynamic success (P = .0004).¦CONCLUSIONS: In patients in hemodynamically unstable condition, transcatheter arterial embolization effectively controls bleeding and improves hemodynamic stability. Immediate survival is related to hemodynamic condition before embolization, and 30-d mortality is mainly related to associated brain trauma.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Three years of haemovigilance in a general university hospital.

The aim of this study is to describe a newly implemented haemovigilance system in a general university hospital. We present a series of short cases, highlighting particular aspects of the reports, and an overview of all reported incidents between 1999 and 2001. Incidents related to transfusion of blood products were reported by the clinicians using a standard preformatted form, giving a synopsis of the incident. After analysis, we distinguished, on the one hand, transfusion reactions, that are transfusions which engendered signs or symptoms, and, on the other hand, the incidents where management errors and/or dysfunctions took place. Over 3 years, 233 incidents were reported, corresponding to 4.2 events for 1000 blood products delivered. Of the 233, 198 (85%) were acute transfusion reactions and 35 (15%) were management errors and/or dysfunctions. Platelet units gave rise to statistically (P < 0.001) more transfusion reactions (10.7 per thousand ) than red blood cells (3.5 per thousand ) and fresh frozen plasma (0.8 per thousand ), particularly febrile nonhaemolytic transfusion reactions and allergic reactions. A detailed analysis of some of the transfusion incident reports revealed complex deviations and/or failures of the procedures in place in the hospital, allowing the implementation of corrective and preventive measures. Thus, the haemovigilance system in place in the 'Centre Hospitalier Universitaire Vaudois, CHUV' appears to constitute an excellent instrument for monitoring the security of blood transfusion.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Background Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. Methods 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. Findings 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12. weeks versus 24 weeks (hazard ratio [HR] 1 98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00,0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. Interpretation While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA,exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.

Ex-vivo ultrasound control of resection margins during partial nephrectomy

Introduction: Surgery represents the treatment of choice for localized renal cell neoplasia. Partial nephrectomy (PN) has widened its indications over the past two decades and has shown oncological results equivalent to radical nephrectomy for small tumors. The role of negative surgical margins has been widely debated. Intraoperative fresh frozen section analysis is shown to be unreliable, expensive, time-consuming and not well correlated to final pathology. The goal of the present study was to assess the feasibility of intraoperative ex-vivo ultrasound (US) control of resection margins and its correlation to margin status at definitive pathology in patients undergoing PN.Material and Methods: The study was carried out in our institution from February 2008 to March 2010. Patients undergoing PN for T1-T2 renal tumors were included. Ex vivo US was performed by one single senior radiologist. Considering its availability, not all consecutive eligible patients were included. PN was undertaken in a standardized technique applying the "minimal healthy tissue margin" technique. Once resected, the specimen was kept in a saline solution and ex-vivo US was performed to evaluate the whole tumor pseudocapsule.Results: Twelve patients (five women, age (mean}SD) 65}11 years) were included. Intraoperative ex-vivo US showed negative surgical margin in all cases. US duration ranged from 1 to 4 minutes, with a median time of 1 minute. Definitive histological analysis confirmed the presence of two angiomyolipoma, eight pT1a tumors, of which seven were clear cell carcinoma and one was a type II papillary tumor, one pT1b clear cell carcinoma and one pT2 chromophobe carcinoma (size 2.9}2.3 cm). Final pathology revealed R0 margins.Conclusion: Intraoperative ex-vivo US control of resection margins in patients undergoing PN is feasible, time-efficient and well correlated to definitive pathological examination with regards to margin status.

Rôle de la MGMT et implications cliniques dans les tumeurs cérébrales [Role of MGMT and clinical applications in brain tumours]

Glioblastoma multiforme is the most common and most malignant primary brain tumour with a dismal prognosis. The advent of new chemotherapies with alkylating agents crossing the blood-brain barrier, like temozolomide, have permitted to notably ameliorate the survival of a subgroup of patients. Improved outcome was associated with epigenetic silencing of the MGMT (O6-methylguanin methyltransferase) gene by promotor methylation, thereby blocking its repair capability, thus rendering the alkylating agents more effective. This particularity can be tested by methylation specific PCR on resected tumour tissue, best on fresh frozen biopsies, and allows identification of patients more susceptible to respond favourably to the treatment.

Visceral leishmaniasis and hemophagocytic syndrome

A 11 months old female infant from Portugal, free of family history, consults for apathy, weight loss, tachycardia, tachypnea, petechiae, pallor without icterus and hepatoslenomegaly. Seven months earlier, while being in Portugal, she presented a persistent bluish pimple on her buttock. Laboratory results showed anemia (35 g/l), leucopenia (3.3 G/l), thrombocytopenia (13 G/l), impaired coagulation (INR 1.4, PTT 41 sec.), hyponatremia (124 mmol/l), elevated CRP (139 mg/l), high ferritin (34.775 μg/l) and high triglycerides (5.22 mmol/l). After correction of vital parameters, a bone marrow aspiration and biopsy (BMB) revealed both the etiological diagnosis, namely a visceral leishmaniasis (VL) as well as one of its potential complications, the hemophagocytic syndrome (HS). Transfusions of whole blood, platelets and fresh frozen plasma were immediately started. Dexamethasone (10 mg/m2) and amphotericin B (3 mg/kg/day) have also been administrated. Visceral leishmaniasis is caused by a protozoan (Leishmania donovani) transmitted by the female sandfly. It is endemic in the Mediterranean basin (including France, Italy, Spain and Portugal), South America, sub-Saharan Africa as well as in India and Bangladesh. The parasite infects macrophages and, after several weeks of incubation, the disease occurs by affection of bloodlines (anemia, leucopenia, thrombocytopenia), hepatosplenomegaly, cachexia, gastrointestinal damage. The complications of the disease may lead to death. Liposomal amphotericin B is the currently recommended treatment. HS is caused by the proliferation and activation of macrophages in the marrow in response to a cytokine storm. It may be of primary cause. When it is secondary, it may be related to infections such as leishmaniasis. Patients present with fever and laboratory diagnostic criteria include cytopenia, hypertriglyceridemia, high ferritin and hemophagocytosis in the BMB. The treatment consists among other in the administration of high doses corticosteroids and, in secondary cases, in the treatment of the underlying cause. In conclusion, the clinical and biological features of VL may mimic haematological disorders as leukemia, but an enlargement of the liver and especially of the spleen should remind in this parasitic infection and its potential fatal complication, the HS.

Biomarker analysis of stored blood products: emphasis on pre-analytical issues.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors.

PURPOSE: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. EXPERIMENTAL DESIGN: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. RESULTS: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. CONCLUSION: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.

Médecine d'urgence [Emergency medicine: updates 2013].

New evidences published this year are susceptible to change the management of several medical emergencies. Combined antiplatelet therapy might be beneficial for the management of TIA or minor stroke and rapid blood pressure lowering might improve the outcome in patients with intracerebral hemorrhage. A restrictive red cell transfusion strategy is indicated in case of upper digestive bleeding and coagulation factors concentrates are superior to fresh frozen plasma for urgent warfarin reversal. Prolonged systemic steroid therapy is not warranted in case of acute exacerbation of BPCO, and iterative physiotherapy is not beneficial after acute whiplash. Finally, family presence during cardiopulmonary resuscitation may reduce post-traumatic stress disorder among relatives.

Angiotensin converting enzyme inhibitor induced angio-oedema: a review of the pathophysiology and risk factors.

Angio-oedema (AE) is a known adverse effect of angiotensin converting enzyme inhibitor (ACE-I) therapy. Over the past several decades, evidence of failure to diagnose this important and potentially fatal reaction is commonly found in the literature. Because this reaction is often seen first in the primary care setting, a review was undertaken to analyse and document the keys to both diagnostic criteria as well as to investigate potential risk factors for ACE-I AE occurrence. A general review of published literature was conducted through Medline, EMBASE, and the Cochrane Database, targeting ACE-I-related AE pathomechanism, diagnosis, epidemiology, risk factors, and clinical decision making and treatment. The incidence and severity of AE appears to be on the rise and there is evidence of considerable delay in diagnosis contributing to significant morbidity and mortality for patients. The mechanism of AE due to ACE-I drugs is not fully understood, but some genomic and metabolomic information has been correlated. Additional epidemiologic data and clinical treatment outcome predictors have been evaluated, creating a basis for future work on the development of clinical prediction tools to aid in risk identification and diagnostic differentiation. Accurate recognition of AE by the primary care provider is essential to limit the rising morbidity associated with ACE-I treatment-related AE. Research findings on the phenotypic indicators relevant to this group of patients as well as basic research into the pathomechanism of AE are available, and should be used in the construction of better risk analysis and clinical diagnostic tools for ACE-I AE.

Médecine d'urgence [Emergency medicine: updates 2013].

New evidences published this year are susceptible to change the management of several medical emergencies. Combined antiplatelet therapy might be beneficial for the management of TIA or minor stroke and rapid blood pressure lowering might improve the outcome in patients with intracerebral hemorrhage. A restrictive red cell transfusion strategy is indicated in case of upper digestive bleeding and coagulation factors concentrates are superior to fresh frozen plasma for urgent warfarin reversal. Prolonged systemic steroid therapy is not warranted in case of acute exacerbation of BPCO, and iterative physiotherapy is not beneficial after acute whiplash. Finally, family presence during cardiopulmonary resuscitation may reduce post-traumatic stress disorder among relatives.

Epidemiological evaluation of hepatic trauma victims undergoing surgery

Objective : to evaluate the epidemiological variables and diagnostic and therapeutic modalities related to hepatic trauma patients undergoing laparotomy in a public referral hospital in the metropolitan region of Vitória-ES. Methods : we conducted a retrospective study, reviewing charts of trauma patients with liver injuries, whether isolated or in association with other organs, who underwent exploratory laparotomy, from January 2011 to December 2013. Results : We studied 392 patients, 107 of these with liver injury. The male: female ratio was 6.6 : 1 and the mean age was 30.12 years. Penetrating liver trauma occurred in 78.5% of patients, mostly with firearms. Associated injuries occurred in 86% of cases and intra-abdominal injuries were more common in penetrating trauma (p <0.01). The most commonly used operative technique was hepatorrhaphy and damage control surgery was applied in 6.5% of patients. The average amounts of blood products used were 6.07 units of packed red blood cells and 3.01 units of fresh frozen plasma. The incidence of postoperative complications was 29.9%, the most frequent being infectious, including pneumonia, peritonitis and intra-abdominal abscess. The survival rate of patients suffering from blunt trauma was 60%, and penetrating trauma, 87.5% (p <0.05). Conclusion : despite technological advances in diagnosis and treatment, mortality rates in liver trauma remain high, especially in patients suffering from blunt trauma in relation to penetrating one.