Fluid Dynamics Analysis by CT Imaging Technique of New Sorbent Cartridges for Extracorporeal Therapies.

BACKGROUND: Recent innovations in biomaterials technology have led to the development of innovative sorbents adopted as adsorbing devices in the field of extracorporeal blood purification therapies. As removal mechanism, adsorption allows to remove specific molecules, selectively binding them to sorbent materials. In addition to the material properties, a quintessential aspect influencing device properties is blood flow distribution within the sorbent particles. OBJECTIVES: In order to adequately characterize the potential adsorbing properties for an effective blood purification therapy, an in vitro study assessing the fluid dynamics inside 3 new cartridges, HA130, HA230 and HA330 (Jafron, Zhuhai City, -China) was conducted through CT imaging technique. -Methods: The cartridges were placed in vertical position in the CT -gantry. Dye solution was circulated through the cartridges at 250 mL/min, longitudinal sections, 0.5 cm thick, were recorded for 60 s. Furthermore, an in vitro test was conducted to build pressure drop profiles. Blood was circulated at a different flow rate, 100-400 mL/min, step 50 mL/min. Pre and post cartridges pressures were acquired and pressure drop calculated. RESULTS: Sequential images demonstrated an excellent distribution of the flow inside the cartridges. Average flow velocity was 0.37 cm/s for the 3 cartridges. HA130 had a homogeneous flow profile along the entire length of the device; HA230 and HA330 showed minimal differences between central and peripheral regions. Pressure drop profiles resulted linear, increasing proportionally with blood flow rate and packing density. CONCLUSIONS: We may conclude that the structural and functional design of the studied cartridges is adequate for haemoperfusion with no channelling phenomena. This ensures maximum and optimal utilization of the sorbent contained in the devices.

Quantification of Internal Filtration in Hollow Fiber Hemodialyzers with Medium Cut-Off Membrane.

BACKGROUND: Inadequate removal of molecules between 5 and 50 KDa may cause long-term complication in chronic hemodialysis. Medium cut-off (MCO) is a new class of membranes with enhanced sieving properties and negligible albumin loss. MCO membrane makes it possible to perform expanded hemodialysis (HDx), a technique based on high internal filtration (IF).The present study is designed to quantify IF in 2 MCO dialyzers (Theranova 400 and 500, Baxter, Deerfield, USA) using a nuclear imaging technique previously validated. METHODS: Blood and dialysate compartment pressure drop along with transmembrane pressure; they were measured in a closed in vitro circuit with human blood (blood flow [QB] = 300 and 400 mL/min; dialysate flow 500 mL/min; net ultrafiltration rate 0 mL/min). A non-diffusible marker molecule (albumin macro-aggregates labeled with 99Tc metastable) was injected in the blood compartment and nuclear emission was recorded by a gamma camera. Relative variations in the concentration of the marker molecule along the length of the filter were used to calculate local cross filtration. RESULTS: Based on marker concentration profiles, IF was estimated. For Theranova 400, IF were 29.7 and 41.6 mL/min for QB of 300 and 400 mL/min. For Theranova 500, IF were 31.6 and 53.1 mL/min for QB of 300 and 400 mL/min respectively. CONCLUSIONS: MCO membrane provides significant amounts of IF due to the particular combination between hydraulic permeability of the membrane and reduced inner diameter of the fibers. High IF combined with enhanced sieving profile of MCO membrane leads to improved removal of a wider spectrum of uremia retention molecules in HDx, without requiring complex equipment.

Evans syndrome secondary to chronic lymphocytic leukaemia: presentation, treatment, and outcome.

Evans syndrome (ES) is defined by the combination (either simultaneous or sequential) of immune thrombocytopenia (ITP) and autoimmune haemolytic anaemia (AIHA). When related to secondary conditions, ES may arise in patients with chronic lymphocytic leukaemia (CLL), which is frequently associated to autoimmune cytopenias (AIC). We analysed 25 patients with ES secondary to CLL, which were identified from a large series of consecutive patients with CLL, diagnosed and followed up in two institutions. They represented 2.9 % of the whole series. Thirteen patients presented with concurrent ITP and AIHA (simultaneous ES), while others developed the two AIC sequentially. Occurrence of ES was associated with unfavourable biological prognostic factors like ZAP-70 expression, unmutated immunoglobulin heavy chain variable region gene status, 17-p13 deletion and TP53 gene mutations. Of note, the majority of patients with ES (66 %) had stereotyped B cell receptor configuration. Most patients had short-lasting remissions and required second-line treatments to control the autoimmune manifestations of ES. Patients with ES were associated with inferior survival compared to patients not developing AIC, especially when ES developed early in the course of CLL, although the reduced survival was not confirmed by multivariate analysis. In conclusion, ES secondary to CLL is a difficult-to-treat complication, characterised by adverse biological features and clinical outcome.

CVVHD treatment with CARPEDIEM: small solute clearance at different blood and dialysate flows with three different surface area filter configurations.

BACKGROUND: The CARdiorenal PEDIatric EMergency (CARPEDIEM) machine was originally designed to perform only continuous venovenous hemofiltration (CVVH) in neonatal and pediatric patients. In some cases, adequate convective clearance may not be reached because of a limited blood flow. In such conditions, the application of diffusive clearance [continuous venovenous hemodialysis (CVVHD)] would help optimize blood purification. In this study, the CARPEDIEM™ machine was modified to enable the circulation of dialysis through the filter allowing testing of the performance of CARPEDIEM™ machine in CVVHD. METHODS: Three different polyethersulfone hemodialyzers (surface area = 0.1 m(2), 0.2 m(2), and 0.35 m(2), respectively) were tested in vitro with a scheduled combination of plasma flow rates (Qp = 10-20-30 ml/min) and dialysis fluid flow rate (Qd = 5-10-15 ml/min). Three sessions were performed in co-current and one in counter-current configuration (as control) for each filter size. Clearance was measured from the blood and dialysate sides and results with mass balance error greater than 5 % were discarded. RESULTS: Urea and creatinine clearances for each plasma/dialysate combination are reported: clearance increase progressively for every filter proportionally to plasma flow rates. Similarly, clearances increase progressively with dialysate flow rates at a given plasma flow. The clearance curve tends to present a steep increase for small increases in plasma flow in the range below 10 ml/min, while the curve tends to plateau for values averaging 30 ml/min. As expected, the plateau is reached earlier with the smaller filter showing the effect of membrane surface-area limitation. At every plasma flow, the effect of dialysate flow increase is evident and well defined, showing that saturation of effluent was not achieved completely in any of the experimental conditions explored. No differences (p > 0.05 for all values) were obtained in experiments using whole blood instead of plasma or using co-current versus counter-current dialysate flow configuration. CONCLUSIONS: Although plasma flow and filter surface give an important contribution to the level of clearance urea and creatinine, it appears evident that dialysate flow plays an essential role in the blood purification process, justifying the use of CVVHD versus CVVH in case of high dialysis dose requirement and/or limited blood flow rate.

Enumeration of residual white blood cells in leukoreduced blood products: Comparing flow cytometry with a portable microscopic cell counter.

Transfusion of blood components is potentially associated to the risk of cell-mediated adverse events and current guidelines require a reduction of residual white blood cells (rWBC) below 1 × 10(6) WBC/unit. The reference method to enumerate rare events is the flow cytometry (FCM). The ADAM-rWBC microscopic cell counter has been proposed as an alternative: it measures leukocytes after their staining with propidium iodide. We have tested the Adam-rWBC for the ability to enumerate rWBC in red blood cells and concentrates. We have validated the flow cytometry (FCM) for linearity, precision accuracy and robustness and then the ADAM-rWBC results have been compared with the FCM. Our data confirm the linearity, accuracy, precision and robustness of the FCM. The ADAM-rWBC has revealed an adequate precision and accuracy. Even if the Bland-Altman analysis of the paired data has indicated that the two systems are comparable, it should be noted that the rWBC values obtained by the ADAM-rWBC were significantly higher compared to FCM. In conclusion, the Adam-rWBC cell counter could represent an alternative where FCM technology expertise is not available, even if the risk that borderline products could be misclassified exists.

Evaluation of lymphocytes inactivation by extracorporeal photopheresis using tetrazolium salt based-assay.

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP.

Clinical significance of LAIR1 (CD305) as assessed by flow cytometry in a prospective series of patients with chronic lymphocytic leukemia.

Most patients affected by chronic lymphocytic leukemia are diagnosed by flow cytometry. Several immunophenotypic markers have been identified as significant and independent prognostic variables, especially from retrospective cohorts. However, while attractive because their detection is inexpensive and feasible in most laboratories, only few have been validated by independent series. The expression of leukocyte-associated immunoglobulin-like receptor-1 (also known as LAIR1, LAIR-1 or CD305), an inhibitor of B-cell receptor-mediated signaling, has been reported to be lacking in high-risk chronic lymphocytic leukemia. However, its correlation with biological variables and its prognostic significance remain unknown. We investigated 311 consecutive patients, prospectively enrolled since 2007. Methods for studying patients were standardized and included clinical assessment, immunophenotype, fluorescence in situ hybridization, and status of immunoglobulin heavy chain variable region genes. Overall, 22.1% of patients had Binet stage B or C disease, 38.5% had unmutated immunoglobulin genes, 15.1% had high-risk cytogenetic abnormalities, 23.4% were CD38(+), 37.8% CD49d(+), and 59.8% LAIR1(+). Expression of LAIR1 was inversely related to that of CD38 (P=0.0005), but was not associated with CD49d expression (P=0.96). A significantly lower expression of LAIR1 was observed in patients with Binet stage B or C disease (P=0.023), and in the presence of high-risk cytogenetic abnormalities (P=0.048) or unmutated immunoglobulin heavy chain variable region genes (P<0.0001). At univariate analysis LAIR1(+) was significantly associated with longer time to first treatment (P=0.0002). This favorable effect of LAIR1(+) was confirmed by multivariate analysis (hazard ratio=2.1, P=0.03 for LAIR1). Our results indicate that LAIR1 expression is a reliable and inexpensive marker capable of independently predicting time to first treatment in newly diagnosed unselected patients with chronic lymphocytic leukemia.

Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: A medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed. METHODS: Platelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells. RESULTS: After 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations. CONCLUSIONS: The proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.

A flow cytometric assay for the quantification of MSC lysis by peripheral blood mononucleated cells.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy.

Testing single/combined clinical categories on 5110 Italian patients with developmental phenotypes to improve array-based detection rate.

BACKGROUND: Chromosomal microarray analysis (CMA) is nowadays widely used in the diagnostic path of patients with clinical phenotypes. However, there is no ascertained evidence to date on how to assemble single/combined clinical categories of developmental phenotypic findings to improve the array-based detection rate. METHODS: The Italian Society of Human Genetics coordinated a retrospective study which included CMA results of 5,110 Italian patients referred to 17 genetics laboratories for variable combined clinical phenotypes. RESULTS: Non-polymorphic copy number variants (CNVs) were identified in 1512 patients (30%) and 615 (32%) present in 552 patients (11%) were classified as pathogenic. CNVs were analysed according to type, size, inheritance pattern, distribution among chromosomes, and association to known syndromes. In addition, the evaluation of the detection rate of clinical subgroups of patients allowed to associate dysmorphisms and/or congenital malformations combined with any other single clinical sign to an increased detection rate, whereas non-syndromic neurodevelopmental signs and non-syndromic congenital malformations to a decreased detection rate. CONCLUSIONS: Our retrospective study resulted in confirming the high detection rate of CMA and indicated new clinical markers useful to optimize their inclusion in the diagnostic and rehabilitative path of patients with developmental phenotypes.

Prenatal detection of trisomy 8 mosaicism: Pregnancy outcome and follow up of a series of 17 consecutive cases.

OBJECTIVE: To study the outcome of a series of individuals with prenatal detection of trisomy 8 mosaicism by chorionic villus sampling (CVS) and/or amniocentesis. STUDY DESIGN: The databases of two Italian genetics units were reviewed to identify all consultations requested during pregnancy because of trisomy 8 mosaicism. To evaluate the pregnancy outcome, the regional registry of congenital malformations (including terminations of pregnancies) was consulted; additional follow-up data were collected by a telephone interview. The following outcomes were analysed: delivery, pre- and post-natal growth, psychomotor development, major malformations, other diseases/complications. RESULTS: A total of 17 consecutive cases of trisomy 8 mosaicism were identified. Fourteen cases were first detected among women undergoing prenatal diagnosis by CVS; the remaining ones were identified among women who underwent amniocentesis. In most cases diagnosed by CVS, the chromosomal anomaly was only detected in long-term cell cultures (10/14) and was not confirmed by amniocentesis (11/13). There were two terminations of pregnancy and 15 live births; no major birth defects were observed among live born infants and only a case with prenatal and postnatal growth retardation was observed (mean age at follow-up interview was 5.9 years). CONCLUSION: Our data showed an overall positive prognosis for cases with an apparent confined placental mosaicism and those with low-level mosaicism in amniotic fluid if no congenital anomalies were detected by foetal ultrasound examinations. However, larger studies are warranted to better define the associated risk of neurodevelopmental anomalies.

Genetics and Autosomal Dominant Polycystic Kidney Disease Progression.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, accounting for 10% of European patients on renal replacement therapy. In the previous years, many approaches to slow the progression of ADPKD were studied and many clinical trials published. In addition to having diagnostic role, the description of the genotype is even important to predict the progression of the disease and contributes, combined with several other factors, to a more precise patients classification. With the availability of disease-modifying drugs, "fast progression factors" are needed to early recognize those patients who would likely progress, before cyst growth reaches a critical value. ERA-EDTA working group on inherited kidney disorders included a series of recommendations resulting in a hierarchical decision algorithm to select patients who are most likely to benefit from the treatment. Beyond diagnosis, we will also discuss the important role of genetics in ADPKD progression and management.

Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use.

BACKGROUND: Increasing evidence suggests the safety and efficacy of mesenchymal stromal cells (MSC) as advanced therapy medicinal products because of their immunomodulatory properties and supportive role in hematopoiesis. Although bone marrow remains the most common source for obtaining off-the-shelf MSC, cord blood (CB) represents an alternative source, which can be collected noninvasively and without major ethical concerns. However, the low estimated frequency and inconsistency of successful isolation represent open challenges for the use of CB-derived MSC in clinical trials. This study explores whether CB may represent a suitable source of MSC for clinical use and analyzes several in vitro parameters useful to better define the quality of CB-derived MSC prior to clinical application. METHODS: CB units (n = 50) selected according to quality criteria (CB volume ≥ 20 ml, time from collection ≤ 24 h) were cultured using a standardized procedure for CB-MSC generation. MSC were analyzed for their growth potential and secondary colony-forming capacity. Immunophenotype and multilineage differentiation potential of culture-expanded CB-MSC were assessed to verify MSC identity. The immunomodulatory activity at resting conditions and after inflammatory priming (IFN-γ-1b and TNF-α for 48 hours) was explored to assess the in vitro potency of CB-MSC prior to clinical application. Molecular karyotyping was used to assess the genetic stability after prolonged MSC expansion. RESULTS: We were able to isolate MSC colonies from 44% of the processed units. Our results do not support a role of CB volume in determining the outcome of the cultures, in terms of both isolation and proliferative capacity of CB-MSC. Particularly, we have confirmed the existence of two different CB-MSC populations named short- and long-living (SL- and LL-) CBMSC, clearly diverging in their growth capacity and secondary colony-forming efficiency. Only LL-CBMSC were able to expand consistently and to survive for longer periods in vitro, while preserving genetic stability. Therefore, they may represent interesting candidates for therapeutic applications. We have also observed that LL-CBMSC were not equally immunosuppressive, particularly after inflammatory priming and despite upregulating priming-inducible markers. CONCLUSIONS: This work supports the use of CB as a potential MSC source for clinical applications, remaining more readily available compared to conventional sources. We have provided evidence that not all LL-CBMSC are equally immunosuppressive in an inflammatory environment, suggesting the need to include the assessment of potency among the release criteria for each CB-MSC batch intended for clinical use, at least for the treatment of immune disorders as GvHD.

Prospective, Randomized, Multicenter, Controlled Trial (TRIATHRON 1) on a New Antithrombogenic Hydrophilic Dialysis Membrane.

INTRODUCTION: Hemodialysis treatment requires anticoagulation to prevent thrombosis of the dialyzer. The Hydrolink® (NV series; Toray) has been designed to reduce thrombotic complications by increasing membrane hydrophilic properties. Previous studies have confirmed reduced platelet activation, improved removal of ß2-microglobulin and excellent small-solute removal. METHODS: We designed a prospective, multi-centered, randomized clinical study to compare the antithrombogenic effects (platelet count) of NV dialyzers versus conventional treatment. To compare the possibility of performing heparin-free dialysis, we carried out progressive heparin reduction tests. Patients with an average platelet count lower than 170,000 cells/mm3 using standard high flux membranes in the 6 months prior to the study were enrolled and randomized. Patients were either dialyzed for 6 months without changing the previous membrane (control group) or treated with the Hydrolink® membrane (NV group). After the third week, the heparin reduction test was conducted for 5 weeks in order to assess the minimum amount of anticoagulant needed to safely perform a 4-hour dialysis treatment. Performance and safety were evaluated measuring platelet count and activation, middle-molecule removal rate and nutritional status. RESULTS: We found no significant difference in platelet count, platelet activation factors ß-thromboglobulin and platelet factor 4 (PF-4), between the groups. More patients in the study group reached heparin-free dialysis without clotting events during the heparin reduction test. The NV dialyzers displayed anti-thrombogenic effects as compared to conventional dialyzers. CONCLUSIONS: The NV dialyzer series is safe with no adverse events reported. Further studies are required to understand the mechanisms of anti-thrombogenic effects.