A practical approach for gmp-compliant validation of real-time PCR method for mycoplasma detection in human mesenchymal stromal cells as advanced therapy medicinal product.

BACKGROUND: Manufacturing of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP) for clinical use involves an ex vivo expansion, which leads to a risk of contamination by microbiological agents. Even if manufacturing under Good Manufacturing Practice (GMP) license minimizes this risk, contamination of cell cultures by mycoplasmas still represents a widespread problem. Furthermore, the absence of mycoplasma contamination represents one of ATMPs release criteria. Since July 2007, European Pharmacopoeia (EuPh) offers the possibility to replace official mycoplasma detection methods with Nucleic Acid Amplification techniques, after suitable validation. As an Italian authorized Cell Factory, we developed an in-house GMP-compliant validation of real-time PCR method for mycoplasma detection in human Mesenchymal Stromal Cells, according to EuPh sec. 2.6.7 and International Conference on Harmonization Q2. MATERIALS AND METHODS: The study was performed in compliance with GMP international requirements with MycoSEQ™ Mycoplasma Detection Assay (Thermofisher) on QuantStudio5 real-Time PCR (Applied Biosystems). Assay validation was developed to evaluate sensitivity, interferences matrix-related, specificity and robustness. RESULTS: MycoSEQ™ Mycoplasma Detection Assay has been successfully validated on human Mesenchymal Stromal Cells as results comply with validation protocol acceptance criteria. CONCLUSIONS: MycoSEQ™ Mycoplasma Detection Assay is a fast, sensitive and specific PCR-based Nucleic Acid Test assay that can be used as an alternative to official mycoplasma test methods for lot release of human Mesenchymal Stromal Cells as advanced therapy medicinal product (ATMP). Moreover, our study underlines the presence of interference on real-time PCR reaction due to matrix composition, pointing out a practical approach for method validation (i.e interference removal).

Extracorporeal photopheresis as an immunomodulatory agent: Haematocrit-dependent effects on natural killer cells.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Leukapheresis for autologous stem cell transplantation: Comparative study of two different thawing methods WSCFD(®) Stem Cell Fast Thawer KW versus 37 °C thermostatic bath.

BACKGROUND: Leukapheresis for autologous stem cell transplantation represents an efficient technique for the reconstitution of haematopoietic system in patients subjected to a high-dose chemotherapy for the treatment of haematological malignancies. The current regulations emphasise first steps of leukapheresis procedure but do not recommend methods for thawing, only suggesting that it must be performed as soon as possible in a 37 °C thermostatic bath. AIM OF THE STUDY: We compared the classic method of thawing with an innovative and fully traceable method that uses WSCFD(®) Stem Cell Fast Thawer KW. MATERIALS AND METHODS: The first part of the study was focused on the thermodynamic process of the two methods, thawing 6 "simulated" leukapheresis (buffy coats of healthy donors cryopreserved with saline solution, 5% HSA and 5% DMSO) and analysing the thawing curve obtained, by using an inside probe. In the second part, we focused on the recovery of viable CD34+ cells and leukocytes, thawing 20 real leukapheresis from paediatric patients. In this phase we also analyse final core bag temperature, time of procedure, cellular recovery with ISHAGE single platform flow cytometry assay and clonogenic potential performing a CFU assay. RESULTS: We found no significant differences between the two methods, both for thermodynamic aspect and cellular recovery. Thawing curves were similar and the paired Student's-t test used for statistical analysis showed a CD34+ cells recovery of 92.2% ± 11.4 using WSCFD(®) versus 90% ± 11.1 of thermostatic bath. Data were similar even for leukocytes recovery (80.8% ± 9.5 with WSCFD(®) and 79.2% ± 14.4 with thermostatic bath). All thawed products never exceeded the core temperature of 30 °C and no differences were found about the post-thaw clonogenic potential (614 × 10(4) ± 98.3 total CFU using WSCFD(®) versus 592 × 10(4) ± 78.5 using thermostatic bath). The only difference observed was about the thawing time: WSCFD method requires a slightly longer time but, on the other hand, it correlates with reduced mean increase in temperature per minute, as a result of a more linear thawing curve. CONCLUSIONS: WSCFD(®) can replace the 37 °C thermostatic bath thawing procedure for leukapheresis, providing more security and fully traceable process data.

[Flat, depressed and polypoid colorectal lesions: a comparative study using a index of histological advance]. / Lesiones planas, deprimidas y polipoides colorrectales: estudio comparativo utilizando un índice de avance histológico.

BACKGROUND: It has been described that the histological changes in flat and depressed colon lesions are more advanced than the ones in polypoid lesions. OBJECTIVES: To compare the histological findings in flat or depressed (non polypoid) and elevated (polypoid) colon lesions. To validate the use of a newly developed Histological Advance Index to compare results. MATERIALS & METHODS: Prospective observational study. Population in study consisted of adult patients programmed for an elective colonoscopy at a private endoscopy center in Lima- Perú. Two groups: 417 found to have non polypoid lesions (which included flat, depressed and lateral spreading tumors or LST), and 405 with polypoid lesions. RESULTS: Total of 8,248 patients, with 417(5%) in the non polypoid group; 368(4.5%) in the flat lesion group, 27(0.32%) in the depressed and 22 (0.26%) LSTs. According to our index, flat and polypoid lesions showed no difference in histological findings. LSTs had a more advanced histology and depressed lesions reached the highest index scores. Flat lesions were found more often in right colon compared with polypoid ones (31% vs 22%, p<0.01), with a higher percentage of serrated lesions (9% vs 2%, p<0.01) and high grade dysplasia (5% vs 3%, NS). In contrast, depressed lesions, showed high grade dysplasia in 3.7% (NS) but cancer in 18% (p<0.01) LSTs were found mainly in right colon and rectum, showing villous component in 23%(p< 0.01) and high grade dysplasia in 32%(p <0.01), but no cancer was found. CONCLUSIONS: Flat and polypoid lesions showed similar histological findings, but LSTs were found to have a higher prevalence of villous lesions and high grade dysplasia. Depressed lesions were found to have a higher prevalence of malignancy. Histological Advance Index proved to be a useful tool to compare groups and quantify differences. .

Lesiones planas, deprimidas y polipoides colorrectales: estudio comparativo utilizando un índice de avance histológico / Flat lesions, and polypoid colorectal depressed: a comparative study using a histologic progress index

ANTECEDENTES: Se ha descrito que las lesiones planas y deprimidas colorrectales, llamadas también no polipoides (LNP) tienen una histología más avanzada que las polipoides o protruidas (LP). OBJETIVOS: Comparar el grado de avance histológico de las LNP con el de las LP, a nivel del colon y recto. Validar el uso de un Índice de Avance Histológico (IAH) para objetivar estas diferencias. METODOLOGÍA: Estudio observacional, prospectivo, realizado en adultos programados a colonoscopía en un Centro Endoscópico privado de Lima- Perú. Dos grupos: 417 pacientes con LNP (planas, deprimidas y de crecimiento lateral o LST) y 405 pacientes con solo LP. RESULTADOS: 417/ 8,248 pacientes (5%) tuvieron LNP; 368 (4.5%) planas, 27(0.32%) deprimidas y 22(0.26%) LST. Según nuestro IAH, las lesiones planas y polipoides mostraron similar avance histológico. Las LST tuvieron una histología más avanzada y las deprimidas alcanzaron los valores más altos. Las lesiones planas tuvieron mayor tendencia que las polipoides a situarse en colon derecho (31% vs 22% p< 0.01), a presentar histología aserrada (9% vs 2% p< 0.01) y displasia de alto grado (5% vs 3% NS), pero menor tendencia al cáncer (0.2% vs 1% NS). Comparadas con las polipoides, las deprimidas tuvieron similar displasia de alto grado (3.7% /NS) pero una alta proporción de cáncer (18 % p < 0.01), mientras que las LST se localizaron sobre todo en colon derecho y recto, con componente velloso en 23% (p< 0.01) y displasia de alto grado en 32% (p< 0.01), pero no cáncer. CONCLUSIONES: Las lesiones planas mostraron un grado de avance histológico similar a las polipoides, pero las de crecimiento lateral si tuvieron una histología más avanzada y las deprimidas desarrollaron cáncer en una elevada proporción. El Índice de Avance Histológico fue una herramienta útil para comparar los grupos y resaltar sus diferencias.

BACKGROUND: It has been described that the histological changes in flat and depressed colon lesions are more advanced than the ones in polypoid lesions. Objectives: To compare the histological findings in flat or depressed (non polypoid) and elevated (polypoid) colon lesions. To validate the use of a newly developed Histological Advance Index to compare results. MATERIALS & METHODS: Prospective observational study. Population in study consisted of adult patients programmed for an elective colonoscopy at a private endoscopy center in Lima- Perú. Two groups: 417 found to have non polypoid lesions (which included flat, depressed and lateral spreading tumors or LST), and 405 with polypoid lesions. RESULTS: Total of 8,248 patients, with 417(5%) in the non polypoid group; 368(4.5%) in the flat lesion group, 27(0.32%) in the depressed and 22 (0.26%) LSTs. According to our index, flat and polypoid lesions showed no difference in histological findings. LSTs had a more advanced histology and depressed lesions reached the highest index scores. Flat lesions were found more often in right colon compared with polypoid ones (31% vs 22%, p<0.01), with a higher percentage of serrated lesions (9% vs 2%, p<0.01) and high grade dysplasia (5% vs 3%, NS). In contrast, depressed lesions, showed high grade dysplasia in 3.7% (NS) but cancer in 18% (p<0.01) LSTs were found mainly in right colon and rectum, showing villous component in 23%(p< 0.01) and high grade dysplasia in 32%(p <0.01), but no cancer was found. CONCLUSIONS: Flat and polypoid lesions showed similar histological findings, but LSTs were found to have a higher prevalence of villous lesions and high grade dysplasia. Depressed lesions were found to have a higher prevalence of malignancy. Histological Advance Index proved to be a useful tool to compare groups and quantify differences.

Telefetalcare: a first prototype of a wearable fetal electrocardiograph.

Fetal heart rate monitoring is fundamental to infer information about fetal health state during pregnancy. The cardiotocography (CTG) is the most common antepartum monitoring technique. Abdominal ECG recording represents the most valuable alternative to cardiotocography, as it allows passive, non invasive and long term fetal monitoring. Unluckily fetal ECG has low SNR and needs to be extracted from abdominal recordings using ad hoc algorithms. This work describes a prototype of a wearable fetal ECG electrocardiograph. The system has flat band frequency response between 1-60 Hz and guarantees good signal quality. It was tested on pregnant women between the 30(th) and 34(th) gestational week. Several electrodes configurations were tested, in order to identify the best solution. Implementation of a simple algorithm for FECG extraction permitted the reliable detection of maternal and fetal QRS complexes. The system will allow continuative and deep screening of fetal heart rate, introducing the possibility of home fetal monitoring.

A four command BCI system based on the SSVEP protocol.

This paper discusses the development of a four command BCI system. This system is composed of a wearable electroencephalogram acquisition unit interfaced to a computer by a wireless Bluetooth (BT) connection. The implemented system relies on the steady-state visual evoked potential (SSVEP) protocol applied to a four selection system. In order to achieve the maximum reliability against false positives a five class classifier was used considering the idle state as an independent class. In order to maximize the usability of the system a two channel solution was tested and adopted. The BCI algorithm was based on a supervised multi-class classifier implemented by combining different binary regularized linear discriminant analysis (RLDA) classifiers. The biofeedback was evaluated by combining the resultant time signed distance with quality index related to the number of coherent identification obtained with the one-vs-all approach.

A wearable device for continuous monitoring of heart mechanical function based on impedance cardiography.

In this study we explored the possibility to realize a low power device for Cardiac Output continuous monitoring based on impedance cardiography technique. We assessed the possibility to develop a system able to record data allow an intra-subjective analysis based on the daily variations of this measure. The device was able to acquire and to send signals using a wireless Bluetooth transmission. The electronic circuit was designed in order to minimize power consumption, dimension and weight. The reported results were interesting for what concerns the power consumption and then noise level. In this way was obtained a wearable device that will permit to define specific clinical protocols based on continuous monitoring of the Cardiac Output signal.

Spasticity management in the child with spastic quadriplegia.

In children with spastic quadriplegia, also described as 'whole body involvement', spasticity can interfere with motor function, contributes to the development of deformities and adversely impacts on care, positioning, and comfort. In this population, spasticity interventions address goals such as improving comfort, reducing pain, easing the burden of carers, slowing the progression of musculoskeletal deformities and perhaps improving function. Children with severe diplegia are distinguished from those with quadriplegia by their ability to ambulate, as well as by a greater emphasis being placed on functional motor goals even though similar treatment modalities are often employed to manage spasticity. The many treatment options currently available include, but are not limited to, botulinum toxin type A, phenol neurolysis, oral medications, intrathecal baclofen, selective dorsal rhizotomy, and orthopaedic surgery. The integration of these treatment modalities can help to optimize the overall care and function for a child with spastic quadriplegia or severe diplegia. However, the development of a management programme is complex and needs to take into account many factors, including age, weight and nutritional status, rate of progression of musculoskeletal deformities, developmental potential, comorbid conditions, current functional status and prognosis, and family and patient treatment goals. Children with marked spasticity are likely to benefit from a combination of interventions, rather than a single treatment modality. Because of these complexities, management should be planned and coordinated by a multidisciplinary team of medical and allied health professionals which recognizes the central role of the family in all decisions. Once the special characteristics of the child with spastic quadriplegia and the various treatment options are understood, outcomes can be maximized.

[Digoxin-like immunoreactive factors. Review of the literature]. / Digoxin-like immunoreactive factor. Revisione della letteratura.

It is confirmed by several studies that in normal subjects a substance recognized by antibodies anti digoxin exists. Such a substance can be found at increased concentration in pregnant women, neonates, in liver or kidney diseases. A limited increase in concentration has been also registered in patients with essential hypertension and in normotensive patients with a family history of hypertension. Serum or urines rich in such a substance show an increased capacity of inhibiting in vitro the sodium-potassium pump and therefore in reducing also in vivo the capacity of reabsorption of sodium and with it, of water. The investigators interest for this substance has two main reasons: 1) the interference that such a substance has in dosages of digitalis in therapeutic monitorizing; 2) the possibility that such a substance has an important physiological role in hydroelectrolytic metabolism.

Lingual thyroid and hypothyroidism. Report of a case in a middle aged woman.

There are approximately 400 reported cases of lingual thyroid (LT) with a clear cut prevalence in paediatric age. These glands are probably quantitatively deficient and function at marginal levels. Thyro-stimulating hormone (TSH) causes hypertrophy producing local symptoms and in younger patients may induce dangerous obstruction. The presumptive diagnosis is usually made by indirect laryngoscopy and thyroscintigraphy. The authors describe a case of LT in a middle aged patient in whom the anomaly remained undiscovered, being without local symptoms, and permitting a normal life. The discovery was made possible by the development in the last four years of a progressive, frank hypothyroidism, probably induced by silent thyroiditis into the ectopic gland.

[Changes in the cardio-thoracic index during the treatment with ibopamine]. / Modificazioni del rapporto cardio-toracico in corso di trattamento con ibopamina.

In order to confirm the efficacy of ibopamine for the management of congestive heart failure, as shown by the disappearance of clinical symptoms, the authors have assessed the cardiothoracic index before and after drug administration. In the 17 female patients examined the index was markedly improved, i.e. from an average of 0.561 +/- 0.058 to an average of 0.538 +/- 0.052, (p less than 0.01). This finding, together with the disappearance of dyspnea and edema, confirms that there was a change in disease pattern and that treatment with this drug was effective.

[Behavior of ionized calcium in hypertensive and normotensive patients]. / Comportamento del calcio ionizzato in soggetti ipertesi e normotesi.

Serum concentrations of ionized calcium have been measured in a group of 38 patients with essential hypertension (26 women and 12 men with mean age 61 +/- 12 years) and in a group of 19 normotensives (14 women and 5 men with mean age 70 +/- 12 years). Serum Ca++ levels were significantly lower in hypertensives than in normotensives (4.16 +/- 0.43 mg/dl and 4.46 +/- 0.38 mg/dl respectively, with p less than 0.05). The lower serum concentrations found in hypertensives may be due ions in arterial smooth muscle cells may cause an increase of vascular tone and peripheral resistances. Our results support, in agreement with other Authors, the possible role of ionized calcium in the pathogenesis of essential hypertension.