Two Years of Genomic Surveillance in Belgium during the SARS-CoV-2 Pandemic to Attain Country-Wide Coverage and Monitor the Introduction and Spread of Emerging Variants.

An adequate SARS-CoV-2 genomic surveillance strategy has proven to be essential for countries to obtain a thorough understanding of the variants and lineages being imported and successfully established within their borders. During 2020, genomic surveillance in Belgium was not structurally implemented but performed by individual research laboratories that had to acquire the necessary funds themselves to perform this important task. At the start of 2021, a nationwide genomic surveillance consortium was established in Belgium to markedly increase the country's genomic sequencing efforts (both in terms of intensity and representativeness), to perform quality control among participating laboratories, and to enable coordination and collaboration of research projects and publications. We here discuss the genomic surveillance efforts in Belgium before and after the establishment of its genomic sequencing consortium, provide an overview of the specifics of the consortium, and explore more details regarding the scientific studies that have been published as a result of the increased number of Belgian SARS-CoV-2 genomes that have become available.

Evaluation of a Capillary Electrophoresis System for the Separation of Proteins.

BACKGROUND: Serum protein electrophoresis is one of the core investigations for screening for monoclonal proteins. Among the available capillary systems, the Helena V8 system has been evaluated in a limited number of studies. METHODS: In total, 310 sera samples were assessed on the Helena V8 system and compared with the Sebia Capillarys instrument. Abnormalities suggestive of monoclonal proteins were confirmed by immunofixation. Imprecision studies and reference intervals were determined. RESULTS: The imprecision of the Helena V8 was inferior or equal to 5.8%. The mean bias of Helena V8 vs Sebia Capillarys was about -0.9 g/L for albumin; -0.2 g/L for alpha-1; 1.1 g/L for alpha-2; -0.2 g/L for beta; 0.3 g/L for gamma; -0.5 g/L for monoclonal protein in beta; and 0.3 g/L for monoclonal protein in gamma. Among the 56 samples with monoclonal proteins confirmed by immunofixation, all were seen on both methods, with only 1 discordant result at a cutoff of 5.0 g/L. Reference intervals were statistically different between the 2 analyzers, except for the beta fraction. CONCLUSIONS: Our evaluation confirms the good analytical performance of the Helena V8 analyzer as a suitable alternative to the Sebia Capillarys instrument.

Liver transplantation in children with fulminant hepatic failure: The UCL experience.

The outcome of pediatric LT for FHF was shown to be poor in our center. To better understand such results, recipient and transplant parameters with a putative impact on post-transplant outcome were analyzed in LT for FHF. Between March 1984 and June 2002, 33 children with FHF received a primary liver allograft. The overall results in this series were studied with respect to pre-operative demographic and metabolic variables, peri-operative events, and outcome. Five-yr patient and graft survivals were 71% and 66%, respectively, with a retransplantation rate at 18%. Incidences of perioperative hemorrhage, of HAT and PVT were 14%, 8%, and 4%, respectively. Five-yr acute rejection-free survival rate was 55%. These data confirm the worse outcome following LT for FHF when compared with LT in elective, non-malignant indications such as BA; results in FHF could not be related to surgical or immunological complications in the post-transplant period and it is hypothesized that the MOF associated with FHF contributes to early post-transplant mortality which would justify special management, including aggressive renal and hepatic support.

Steroid-free, tacrolimus-basiliximab immunosuppression in pediatric liver transplantation: clinical and pharmacoeconomic study in 50 children.

Corticosteroid-free immunosuppression (IS) may be potentially beneficial for transplanted patients, particularly children. The purpose of this study was to evaluate the efficacy and cost of such strategy in primary pediatric liver transplantation (LT). Fifty pediatric LT recipients were prospectively treated with a steroid-free, tacrolimus-basiliximab-based IS (group TB). A group of 34 children transplanted under a conventional tacrolimus-steroids regimen served as control series (group TS). Groups TB and TS were compared regarding patient and graft survival, rejection incidence, infectious complications, and growth, as well as cost of the transplant procedure. Patient and graft survivals at 3 years were 96% and 94% in group TB, versus 91% and 88% in group TS (P = 0.380 and P = 0.370, respectively). Rejection-free graft survival at 3 years was 72% in group TB, versus 41% in group TS (P = 0.007). Patients in group TB had significantly less viral infections than patients in group TS (P = 0.045). Height standard deviation score was significantly enhanced in children from group TB, when compared to group TS. Medical care costs were similar in both groups. Steroid avoidance together with basiliximab immunoprophylaxis was not harmful in terms of allograft acceptance, and even seemed to be beneficial in the long term.

Monitoring tolerance after human liver transplantation.

The validation of reliable, non-invasive immunological assays evaluating anti-donor responsiveness in allograft recipients would provide a clinically relevant tool for the early detection of ongoing rejection process as well as for the identification of operational tolerance in the long term. A sequential approach towards immunological monitoring of allografts is proposed in this review: (i) investigations exploring the initial donor-recipient alloresponses, including the analysis of the cytokine network; (ii) investigations regarding graft acceptance and operational tolerance in long-term transplant patients, consisting in the analysis of regulatory T cells and of circulating precursors of dendritic cells, in the measurement of T cell alloreactivity as well as in the study of T cell receptor repertoires. Beside the conventional in vivo and in vitro immunological techniques, the potential applications of molecular imaging in transplantation also deserve further exploration, with particular respect to allograft immune monitoring. Enforced collaboration between transplant clinicians and immunologists will be required to develop the translational research protocols required for the development of immunological monitoring, within an international multicentric network.

Immunological monitoring after organ transplantation: potential role of soluble CD30 blood level measurement.

Analysing the relevance of soluble CD30 (sCD30) in the bloodstream before and after transplantation may be important for the monitoring of transplant recipients. In this study, 27 patients (15 pediatric liver and 12 adult kidney graft recipients) were investigated. In the liver graft group, the patients who developed acute rejection during the first month (n=9) had a slightly higher sCD30 value on pre-transplantation baseline (day 0) and post-transplantation day 7, when compared to patients with normal graft function (n=6) (day 0: 102(1.6) U/ml versus 118(1.5) U/ml, p=0.52) and (day 7: 69(1.5) U/ml versus 83(1.6) U/ml, p=0.47). Increased serum sCD30 was shown to correlate with increased interleukin-10 circulating levels between day 0 and day 7 (r=0.53; p=0.04), whereas, no correlation could be evidenced between interferon-gamma (IFN-gamma) and sCD30 (r=0.02; p=0.47). Similarly, in the kidney transplantation group, no significant difference was found in sCD30 levels at day 0 in both groups with graft rejection or normal graft function (n=6) (85(1.3) U/ml versus 77(1.6) U/ml, p=0.66), but sCD30 decreased significantly at day 7 post-transplantation from baseline value in the rejection group (n=6) (77(1.6) versus 35(1.4); p=0.02). We conclude that increased serum sCD30 was correlated with increased IL-10 (interleukin-10) circulating levels, but not with IFN-gamma levels in the post-transplantation period. Neither pre-transplantation sCD30 nor sCD30 at day 7 post-transplantation could be correlated with acute rejection in liver graft recipient. The monitoring of sCD30 might constitute a tool to assess the risk of acute rejection in renal transplant but did not appear as a valuable mean for early immunological monitoring in the small group of liver allograft recipients patients analysed in this study.

Assessment of FUS-200 Performance: Comparison with Quantitative Urine Culture Shows that Identifying the Right Cutoff for Optimizing Analyzer Performance Is Challenging.

BACKGROUND: Urine sediment analysis is a frequently ordered test, since it permits screening for many clinical conditions. Here, the technical and bacteriuria diagnostic performance of FUS-200, a new sediment analyzer, was assessed. METHODS: Carry-over, imprecision, and linearity were measured according to CLSI protocols. FUS-200 was compared to sediMAX™, our current laboratory analyzer, in terms of particle recognition/counting in 382 fresh urine samples, and bacteriuria diagnosis was based on white blood cell (WBC) and bacteria counts in a subgroup of the same samples. In the diagnostic study, quantitative bacterial cultures served to classify the samples as bacteria-positive or bacteria-negative. RESULTS: FUS-200 did not show carryover for the particles tested. Total imprecision for red blood cells (RBCs) and WBCs in positive controls was 3.6%-10.5% and complied with European guidelines. RBC and WBC recovery was linear. When FUS-200 particle counts were edited by a reviewer, concordance with sediMAX improved for epithelial cells, yeast, and crystals, and recognition of casts and crystals improved. FUS-200 concordance with sediMAX varied between 97% for yeast and 58% for bacteria and was satisfactory. FUS-200 detected bacteriuria better than sediMAX (P = 0.004). FUS-200 WBC and bacteria cutoff values based on the Youden index detected bacteriuria better than manufacturer cutoffs. The best sensitivity with which FUS-200 detected bacteriuria was 79%. CONCLUSIONS: Although casts and crystal recognition should be improved, the overall technical performance of FUS-200 was acceptable to good. FUS-200 exhibited good screening accuracy for bacteria and WBC. Editing only mildly influenced FUS-200 outcomes.

Steroid-free liver transplantation in children.

Although corticosteroids have been part of immunosuppressive regimens since the early days of transplantation, steroid avoidance could be beneficial. To test this hypothesis in paediatric liver transplantation, we compared liver-transplantation under steroid-free immunosuppression in 20 children, who received combined tacrolimus and basiliximab, with that under tacrolimus and steroids in 20 matched historical recipients as a historical control group. 12-month rejection-free survival was 75% in the tacrolimus-basiliximab group compared with 50% in the steroid group (p=0.05). Growth in the first year after transplantation was significantly better in the tacrolimus-basiliximab group than in the steroid group. Steroid avoidance was, therefore, not harmful to our patients, and combining tacrolimus with basiliximab as a steroid substitution seems a safe alternative to tacrolimus and steroid immunosuppression.

PELD score and posttransplant outcome in pediatric liver transplantation: a retrospective study of 100 recipients.

BACKGROUND: Pediatric End-stage Liver Disease (PELD) score is proposed as an objective tool to prioritize children awaiting liver transplantation (LT), higher PELD being associated with increased pre-LT mortality. This study investigated whether PELD may also impact on post-LT results. METHODS: PELD was retrospectively analyzed in 100 pediatric recipients of a primary LT from living-related (n = 49) or postmortem donors (PMD, n = 51). The main pre-LT diagnosis was biliary atresia (n = 64), hepatic malignancy and fulminant hepatitis cases being excluded. PELD was calculated in all patients at the time of pre-LT assessment. Considering the median delay of 117 days between listing and LT in the PMD subgroup, a second PELD was calculated at the time of LT, allowing the determination of a delta PELD during the waiting period. PMD grafts were allocated using an allocation system taking into account waiting times as well as medical urgency, operative at EuroTransplant. RESULTS: Overall 5-year actuarial patient and graft survivals were 96% and 91%, respectively. PELD at listing (13.3 +/- 9.7) showed a normal statistical distribution. PELD scores at listing and at LT were not found to significantly impact on post-LT outcome (NS). In contrast, higher delta PELD might be associated with lower posttransplant patient survival (p = 0.094). CONCLUSIONS: The results of this retrospective analysis suggest that giving priority to high PELD recipients may not result in worsening post-LT outcome. Accordingly, these data support such "sickest children first" allocation policy, which should contribute to reduce pre-LT mortality without worsening post-LT results and increasing organ waste.

Iron storage is significantly increased in peritoneal macrophages of endometriosis patients and correlates with iron overload in peritoneal fluid.

OBJECTIVE: To further investigate peritoneal iron disruption in endometriosis by studying iron storage in peritoneal macrophages of patients with endometriosis compared with controls. DESIGN: Cross-sectional study. SETTING: Academic gynecology research unit in a university hospital. PATIENT(S): Fifty patients undergoing laparoscopy. INTERVENTION(S): Collection of peritoneal fluid samples (N = 50) from patients with (n = 27) and without (n = 23) endometriosis undergoing laparoscopy. MAIN OUTCOME MEASURE(S): Quantification of peritoneal macrophage ferritin by immunocytochemical staining and immunodensitometry and measurement of peritoneal iron, transferrin, ferritin, and prohepcidin concentrations. RESULT(S): The optical density of peritoneal macrophage ferritin staining was statistically significantly higher in endometriosis patients than in controls. Higher iron concentrations, transferrin saturations, and ferritin concentrations were also detected in case of endometriosis. A statistically significant positive correlation was found between the optical density of macrophage ferritin staining and peritoneal iron concentrations in endometriosis and control patients. CONCLUSION(S): Iron storage is statistically significantly increased in peritoneal macrophages of patients with endometriosis and correlates with iron overload in peritoneal fluid. The potential implications of iron accumulation in peritoneal macrophages in case of endometriosis are discussed.

Application of the Six Sigma concept in clinical laboratories: a review.

Six Sigma is a global management strategy introduced to the industrial world in the 1980s. This methodology has been widely implemented in companies such as Motorola, General Electric, Allied Signal and many others, with tremendous success in terms of customer satisfaction and global profitability. To achieve similar benefits in the healthcare field, Six Sigma is currently being deployed in several laboratories around the world. Despite this situation, few articles have been published in the peer-reviewed literature on this subject. The aim of this article is to clarify the different aspects of Six Sigma and their potential applications in clinical laboratories, as well as to systematically review articles and books discussing Six Sigma strategy implementation in the laboratory field.

Percutaneous and surgical radiofrequency ablation of liver malignancies: a single institutional experience.

BACKGROUND: the purpose of this study was to report a single academic institution's experience with radiofrequency ablation (RFA) of liver malignancies METHODS: Sixty-five patients underwent RFA technique through a percutaneous (Group I: 33 patients) or a surgical approach (Group II: 32 patients). The two groups were different according to type of disease selection (more hepatocellular carcinoma in Group I and liver metastases in Group II) and tumour features (smaller size but greater number of lesions in Group II). In Group II, RFA was associated to liver resection in 23 patients (72%). RESULTS: The 2-month postoperative mortality and complication rates were low in both groups. The postoperative hospital stay was longer in Group II. During a median follow-up of 24 months in Group I and 21 months in Group II, the local "in-situ" recurrence rate was 41.4% and 9.1%, respectively. For RFA-treated tumours < 30 mm in size, the local "in-situ" recurrence rate was 40.5% in Group I and 0% in Group II. Multivariate statistical analysis demonstrated that larger tumour and a percutaneous approach for RFA were independent predictive factors of local "in-situ" liver tumour recurrence. CONCLUSIONS: RFA appears to be a safe technique for treating liver malignancies by both approaches. Tumour size and type of RFA approach are predictive factors of in-situ liver tumour recurrence.

Early immunological monitoring after pediatric liver transplantation: cytokine immune deviation and graft acceptance in 40 recipients.

Cytokine deviation may be a factor contributing to graft acceptance. We analyze, in the context of liver transplantation, circulating cytokine levels and their mRNA precursors in liver biopsy samples to study a putative correlation with early immunologic outcome. Forty primary pediatric liver recipients were submitted to a prospective immune monitoring protocol, including 8 of 40 patients with an early, biopsy-proven acute rejection episode. The 32 patients with graft acceptance showed markedly increased interleukin (IL)-10 blood levels at 2 hours after reperfusion on days 1 and 4 after transplantation as compared with baseline, whereas patients with graft rejection only exhibited increased IL-10 levels at 2 hours. A good correlation was observed between IL-10 peripheral levels and levels ascertained by IL-10 reverse transcriptase-polymerase chain reaction at 2 hours and on day 7. Patients with graft acceptance also showed a decrease in interferon gamma (IFN-gamma) at 1 and 2 hours after reperfusion on days 1, 4, 7, 14, and 28 after transplantation. One patient with graft tolerance who had subsequent immunosuppression withdrawal after posttransplantation lymphoproliferative disease showed a similar intraoperative IL-10 pattern, whereas posttransplantation tumor necrosis factor alpha and IFN-gamma levels greatly decreased. The occurrence of cytokine immune deviation may therefore be related to early graft acceptance in children who receive liver transplants.

The immunological monitoring of alloreactive responses in liver transplant recipients: a review.

The aim of this work is to review the current knowledge in the field of immunological monitoring of allogenic responsiveness in clinical liver transplantation. When compared to other solid-organ transplants, liver allografts are considered as immunologically privileged, and, accordingly, constitute a favorable setting to develop experimental as well as clinical strategies for minimization of immunosuppression and even induction of operational tolerance. The validation of simple, reliable, noninvasive assays exploring antidonor alloreactivity will constitute a crucial step toward implementing such approaches in the clinic. In contrast to research in rodents claiming the development of donor-specific tolerance in case of graft survivals of over 100 days without immunosuppression, it is impractical to confirm tolerance induction in this way in humans. Promising candidate assays include the detection of post-transplant immune deviation, of circulating precursors of dendritic cells subtypes, and of regulatory T cells. A conceptual framework for the development of tolerance assays in clinical liver transplantation is also proposed.

Stepwise minimization of the immunosuppressive therapy in pediatric liver transplantation. A conceptual approach towards operational tolerance.

The evolution of immunosuppression in pediatric liver transplantation has been characterized by a steady reduction of the immunosuppressive load, including removal of anti-lymphocyte antibodies, with the aim to reduce the incidence of EBV-related post-transplant lymphoproliferative disorders. Acute rejection rates were studied retrospectively over two decades of pediatric liver transplantation, according to the successive immunoprophylactic regimens. 318 primary pediatric liver transplant recipients, included between 1984 and 2004 in successive prospective trials, were analyzed, with respect to the impact of the immunosuppressive protocol on acute rejection occurrence. A progressive decrease of rejection incidences was observed, which corresponded to reduced immunosuppressive load and to transplant eras. Such trend might be related to changing approaches towards acute rejection histology and therapy by transplant clinicians, but also to the stepwise minimization of immunosuppressive protocols, putatively enhancing graft acceptance. We hypothesize that the recent population of liver transplant recipients with low immunosuppression might be more suitable for progressive immunosuppression withdrawal trial, with the aim to reach ultimately operational tolerance.