Inter-laboratory method validation of CD34+ flow-cytometry assay: the experience of Turin Metropolitan Transplant Centre.

The Turin Metropolitan Transplant Centre (CIC 305) includes four flow-cytometry laboratories assessing quality control on hematopoietic stem cells (HSC) with different instruments and operators. Therefore, the CD34+ enumeration assay should be validated on a regular basis. We describe here the validation plan to test the inter-laboratory reproducibility of CD34+ enumeration assay, based on the risk analysis. Stabilized blood samples were analysed using Stem-Kit reagent according to manufacturer's instructions and acquired using the Beckman Coulter Navios at Regina Margherita Children's' Hospital (305-1), Beckman Coulter FC500 at Candiolo Cancer Institute FPO-IRCCS (305-2), BD Biosciences FACSLyric™ at S. Luigi Hospital (305-3), and Beckman Coulter Navios EX at Mauriziano Hospital (305-4). The ISHAGE guidelines were followed for estimating % and absolute number of CD34+ cells in single-platform method. For each sample repeatability limit (r), reproducibility error, uncertainty of reproducibility error and coefficient of variation (CV) were reported. The repeated measurements from each laboratory or instrument have a variability, expressed as reproducibility error, lower than the repeatability limit for that single parameter. The corrected reproducibility error is always lower than the repeatability limit except for the percentage value of the "low" count. The analysis of inter-laboratory variance is within the maximum acceptable variance value, and the CV of all measurements for each parameter is less than 8%, indicating low measurement variability among laboratories. Evaluating the overall data, we can conclude that the four laboratories are perfectly aligned and the results are reproducible.

The dynamic sagittal balance: Definition of dynamic spino-pelvic parameters using a method based on gait analysis.

Introduction: Evaluation of sagittal balance parameters is a standard assessment before spine surgery. However, these parameters can change during walking. We aimed to describe the behavior of spino-pelvic parameters during walking in healthy subjects. Material and methods: Analyses were performed in 60 healthy subjects. Static spinal sagittal balance parameters were assessed. We performed gait analysis and we used SMART-DX 500® to analyze parameters aimed at defining dynamic sagittal balance, including pelvic tilt angle (PTA), sagittal trunk shift (STS), and trunk angle (TA). We considered rotational and obliquity movements of the pelvis, flexo-extension movements of the hip, trunk, and knees. Analyses were performed in a standing posture and during walking. Results: PTA-cycle, PTA-stance, PTA-swing, STS-cycle, STS-stance, and STS-swing showed good-to-excellent internal reliability (ICC = 0.867; ICC = 0.700; ICC = 0.817, respectively). The parameters with the lowest variability were radiographic PI (CV = 16.53%), PTA-stance (CV = 9.55%), and PTA-swing (CV = 17.22%). PT was directly related to PTA-cycle (r = 0.534, p = .027). PI was inversely correlated with trunk flexo-extension range of motion (r = -0.654, p = .004) and dynamic PT (r = -0.489, p = .047). LL and SS were directly related to knee flexo-extension (r = 0.505, p = .039; r = 0.493, p = .045, respectively). SVA was correlated with the trunk obliquity in dynamics (r = 0.529, p = .029). PTA-cycle was directly related to trunk obliquity (r = 0.538, p = .049). STS and TA in the three phases of step were related to the kinematic parameters of the pelvis. TA was related to flexo-extension of the hip and knee. Conclusions: Variations of dynamic spino-pelvic parameters occur during walking and modify sagittal balance from a static to a dynamic condition.

Evaluation of the psychometric properties of the Barthel Index in an Italian ischemic stroke population in the acute phase: a cross-sectional study.

The objective of this study was to assess and validate the psychometric properties of the Italian culturally adapted Barthel Index (IcaBI) in a cohort of people with ischemic stroke. The validation process was conducted in an Italian cohort of 99 stroke inpatients to whom the IcaBI was administered in order to test its structural validity, and inter-and intrarater reliability. The internal consistency (Cronbach's alpha) was 0.901. Factor analysis revealed a two-factor structure. The interclass correlation coefficient 3,1 (ICC) for intra-rater reliability was estimated at 0.987 (95% CI: 0.975-0.993), while the ICC for inter-rater reliability was 0.909 (95% CI: 0.852-0.948). This study demonstrates the psychometric properties of the IcaBI in an Italian stroke population, and therefore shows that the scale can be considered a valid and reliable assessment tool for measuring functional disability in Italian acute ischemic stroke survivors.

The culturally adapted Italian version of the Barthel Index (IcaBI): assessment of structural validity, inter-rater reliability and responsiveness to clinically relevant improvements in patients admitted to inpatient rehabilitation centers.

The Barthel Index (BI) is widely used to determine eligibility criteria for inpatient rehabilitation and to monitor patients' recovery, irrespective of the illnesses that affect them. The culturally adapted Italian version of the Barthel Index (IcaBI) was recently validated. This paper reports the structural validity and inter-rater reliability of the IcaBI and its responsiveness to the results of inpatient rehabilitation. The IcaBI was administered to a cohort of 264 patients hospitalized in two rehabilitation centers in Rome, Italy. Factor analysis using principal component analysis revealed a monofactorial structure for neurological patients and, after removal of item 1 "feeding", also for orthopedic patients. Substantial to optimal inter-rater reliability was found (0.74 > intraclass correlation coefficient < 0.96). The IcaBI was found to be accurate (area under the curve= 0.72) with a minimal clinically important change score of 35 points. This work confirms that IcaBI is a useful tool for measuring disability in health and social care settings along the continuum of care. Further studies are needed to assess its criterion validity, interpretability and responsiveness in other specific disease conditions.

Un estudio completo sobre la obtención de 99mTc-UBI 29-41 y su comportamiento in vitro e in vivo: su potencial uso en imágenes de infecciones / A complete study about the procurement of 99mTc-UBI 29-41 and its in vitro and in vivo behaviour: potential use in infection imaging

El objetivo del presente trabajo es investigar como se modifica el comportamiento in vitro e in vivo (localización de sitios de infección experimental con Staphilococcus aureus (S.a.)) del UBI 29-41 cuando es marcado con 99mTc por cuatro métodos distintos: a) con borohidruro de potasio y pirofosfato de estaño, b) con hidróxido de sodio y cloruro estañoso (métodos directos), c) con NHS-MAG3 y d) con NHS-HYNIC (métodos indirectos). En segundo lugar comparar los valores PI/PN (Pata Infectada / Pata Normal) del 99mTc-UBI 29-41 (método a), con el 99mTc-scrambled (Sc) UBI 29-41 (péptido control) y con 99mTc-IgG (radiofármaco no específico para infecciones) en ratones infectados con S.a. viables; y los valores pata inflamada/ PN del 99mTc-UBI 29-41 99mTc (método a) con 99mTc-IgG en ratones con inflamación estéril. Cuando el 99mTc-UBI 29-41 fue obtenido por el método a, se obtuvieron los mejores resultados in vitro y las biodistribuciones mostraron la máxima acumulación en sitios con S.a. viables y muy baja acumulación en sitios con inflamación estéril, demostrando su potencial uso en el diagnóstico de infecciones.

Biodistribution of p-borophenylalanine (BPA) in dogs with spontaneous undifferentiated thyroid carcinoma (UTC).

Human undifferentiated thyroid carcinoma (UTC) is a very aggressive tumor which lacks an adequate treatment. The UTC human cell line ARO has a selective uptake of BPA in vitro and after transplanting into nude mice. Applications of boron neutron capture therapy (BNCT) to mice showed a 100% control of growth and a 50% histological cure of tumors with an initial volume of 50 mm(3) or less. As a further step towards the potential application in humans we have performed the present studies. Four dogs with diagnosis of spontaneous UTC were studied. A BPA-fructose solution was infused during 60 min and dogs were submitted to thyroidectomy. Samples of blood and from different areas of the tumors (and in one dog from normal thyroid) were obtained and the boron was determined by ICP-OES. Selective BPA uptake by the tumor was found in all animals, the tumor/blood ratios ranged between 2.02 and 3.76, while the tumor/normal thyroid ratio was 6.78. Individual samples had tumor/blood ratios between 8.36 and 0.33. These ratios were related to the two histological patterns observed: homogeneous and heterogeneous tumors. We confirm the selective uptake of BPA by spontaneous UTC in dogs and plan to apply BNCT in the future.

Biodistribution studies of boronophenylalanine-fructose in melanoma and brain tumor patients in Argentina.

A study of the (10)B-enriched p-boronophenylalanine-fructose complex ((10)BPA-F) infusion procedure in potential BNCT patients, including four melanoma of extremities and two high-grade gliomas (glioblastoma and ganglioglioma) was performed. T/B and S/B ratios for (10)B concentrations in tumor (T), blood (B) and skin (S) were determined. The T/B ratio for the glioblastoma was in the 1.8-3.4 range. The ganglioglioma did not show any significant boron uptake. For the nodular metastasic melanoma T/B values were between 1.5 and 2.6 (average 2.1+/-0.4), corresponding to the lower limit of the mean values reported for different melanoma categories. This result might suggest a lower boron uptake for nodular metastasic melanomas. S/B was 1.5+/-0.4. An open two-compartment pharmacokinetic model was applied to predict the boron concentration during the course and at the end of a BNCT irradiation.

First BNCT treatment of a skin melanoma in Argentina: dosimetric analysis and clinical outcome.

A Phase I/II protocol for treating cutaneuos melanomas with BNCT was designed in Argentina by the Comisión Nacional de Energía Atómica and the medical center Instituto Roffo. The first of a cohort of thirty planned patients was treated on October 9, 2003. This article depicts the protocol-based procedure and describes the first clinical case, treatment regime and planning, patient irradiation, retrospective dosimetric analysis and clinical outcome. Considering the low acute skin toxicity and the complete response in 21 of the 25 subcutaneous melanoma nodules treated, a second irradiation was performed in a different location of the extremity of the same patient. The corresponding clinical outcome is still under evaluation.

99mTc-UBI 29-41: biodistribución y autoradiografía digital en ratones con S.a. viables, S.a. no viables obtenidos por irradiación gamma y S.a. muertos por calor / 99mTc-UBI 29-41: biodistribution and digital autoradiography in mices with viable S.a., non viable S.a. obtained from gamma irradiation and heat killed S.a

El objetivo del presente trabajo es evaluar la capacidad del 99mTc-UBI 29-41 marcado por un método directo en la localización de sitios de infección experimental con Staphilococcus aureus (S.a.). En segundo lugar comparar los valores PI/PN (Pata Infectada / Pata Normal) de las biodistribuciones en ratones portadores de S.a. viables, S.a. no viables obtenidos por irradiación gamma y por calentamiento y en sitios de inflamación estéril y cuantificar las relaciones sitio infectado / sitio normal (SI / SN) através de autorradiografía digital. El UBI 29-41 es un fragmento sintético de la ubiquicidina y el scrambled (Sc) UBI 29-41 es el péptido control. 99mTc-IgG fue el control positivo para inflamaciones estériles. Cuando el 99mTc-UBI 29-41 fue obtenido por este método rápido y reproducible, se visualizaron sitios de infección a 2h p.i.. Las biodistribuciones mostraron mayor acumulación en sitios con S.a. viables que con S.a. irradiados y no hubo acumulación en inflamaciones estériles.

Análogos de somatostatina marcados con 99m-TC / Somatostatine analogs marked with 99m-TC

El objetivo del presente trabajo es el estudio biológico y radioquímico de dos análogos de la somatostatina, el péptido RC-160 y el Tyr3-Octreotide (TOC), marcados con 99mTc por método directo e indirecto usando como quelantes mercapto acetil triglicina (MAG3) e hidrazinonicotinamida (HYNIC) respectivamente. Se realizó la síntesis del benzoil MAG3-RC-160 y del HYNIC-Tyr3-Octreotide los cuales se marcaron con 99mTc obteniéndose purezas radioquímicas mayor que 70 por ciento y mayor que 95 por ciento respectivamente. Se realizaron biodistribuciones en ratas Wistar normales y los resultados se correlacionaron con los datos cromatográficos y de unión a proteinas, encontrándose que a menor lipofilicidad de los complejos se incrementaba la excreción renal. Se realizaron ensayos de unión a receptores utilizando como fuente de los mismos membranas de corteza de cerebro de rata. Los mejores resultados se obtuvieron con el conjugado de HYNIC-TOC marcando con tricina como coligando

In vivo evaluation of three different 99mTc-labelled radiopharmaceuticals for sentinel lymph node identification.

This work was designed to compare sentinel lymph node (SLN) uptake of 99mTc-labelled human serum albumin colloid (99mTc-HSAC), 99mTc-labelled antimony sulphur colloid (99mTc-SC) and a 99mTc-labelled dextran 70 solution (99mTc-Dx) and their selectivity in the identification of this node in the right rear footpad (RRF) of normal mice and tumour bearing mice. Radiopharmaceutical uptake in the SLN (popliteal lymph node) and the lumbar lymph node (LLN), the second lymphatic node station from RRF, were measured at different time points post-intradermal or intratumoural injection into the RRF of NIH normal mice and of Balb/c mice harbouring the murine mammary tumour M2. 99mTc-HSAC uptake in the SLN was significantly higher than LLN uptake. The 99mTc-SC demonstrated high uptake in SLN, but accumulation in LLN was also high. 99mTc-Dx showed low uptakes in both SLN and LLN. The intradermal injection resulted in a more effective radiopharmaceutical accumulation in SLN than did the intratumoural inoculation. Data also show that increments in tumour volume reduced radiopharmaceutical uptake in the SLN. Our results show that 99mTc-HSAC exhibits the highest uptake in the SLN combined with the smallest amounts of radiopharmaceutical passing through to the LLN. Therefore, 99mTc-HSAC appears to be the best radiopharmaceutical for sentinel node detection.

Marcación de un anticuerpo monoclonal con 99mTc por medio del grupo 99cTcN: evaluación de un modelo biológico / 99mTC labeling of a monoclonal antibody via a 99mTcN group: a biological model evaluation

El anticuerpo monoclonal (AcMo) anti-CEA B2C114 fue marcado con 99mTc utilizando uno de los métodos directos de marcación, en el cual la proteína (AcMo IgG1) se reuce utilizando una solución de Ditiotreitol (DTT) para generar grupos sulfhidrilos, uniendo el 99mTc a los mismos a través del anión [99mTcNCl4]- preparado por calentamiento a reflujo de pertecneciato en presencia de azida sódica y HCl conc. El exceso de DTT se eliminó pasando el AcMo reducido por Sephadex G-25 y del mismo modo se realizó la purificación del anticuerpo marcado. Las cromatografías en TLC y Whatman Nº1 usando Metanol 85 por ciento y solución fisiológica como solventes, mostraron una actividad unida a proteínas entre 55-60 por ciento, obteniéndose luego de la purificación por Sephadex G-25 un producto con 90-95 por ciento de pureza radioquímica. Se realizó la biodistribución en ratones Balb/c a las 21 h post inyección obteniéndose una relación de 2:1 de la dosis inyectada por gramo de tejido, con respecto al tumor reactivo:no reactivo. El ensayo de unión realizado demostró que el AcMo conservó su actividad biológica después de marcado

Marcación de un anticuerpo monoclonal con 99mTc por medio del grupo 99cTcN: evaluación de un modelo biológico / 99mTC labeling of a monoclonal antibody via a 99mTcN group: a biological model evaluation

El anticuerpo monoclonal (AcMo) anti-CEA B2C114 fue marcado con 99mTc utilizando uno de los métodos directos de marcación, en el cual la proteína (AcMo IgG1) se reuce utilizando una solución de Ditiotreitol (DTT) para generar grupos sulfhidrilos, uniendo el 99mTc a los mismos a través del anión [99mTcNCl4]- preparado por calentamiento a reflujo de pertecneciato en presencia de azida sódica y HCl conc. El exceso de DTT se eliminó pasando el AcMo reducido por Sephadex G-25 y del mismo modo se realizó la purificación del anticuerpo marcado. Las cromatografías en TLC y Whatman Nº1 usando Metanol 85 por ciento y solución fisiológica como solventes, mostraron una actividad unida a proteínas entre 55-60 por ciento, obteniéndose luego de la purificación por Sephadex G-25 un producto con 90-95 por ciento de pureza radioquímica. Se rea

Utilización de inmunoglobina humana marcada con 99mTc en localización de abscesos / Abscess localization using 99mTc labeled human immunoglobulin

Se formuló un kit liofilizado de IgG, se marcó con 99mTc y se utilizó en ratones Balb/c portadores de un sitio de inflamación. La marcación se basó en la reducción de los puentes disulfuro (S-S) usando 2-mercaptoetanol (2-ME) y metilendifosfonato como ligando la transferencia. Las especies marcadas fueron analizadas comparativamente por cromatografía instantánea en placa delgada (ITLC Gelman SG), cromatografía líquida de alta presión (HPLC) y electroforesis en gel de poliacrilamida (PAGE). Los ensayos de biodistribución y las imágenes fueron realizadas en ratones Balb/c con un absceso de 48 h inducido por la inyección de trementina (pata derecha) y mostraron una clara diferencia entre la proteína marcada y el 99mTcO4Na. Las relaciones pata normal/pata inflamada de 24 h no mostraron diferencias con el 67Ga, pero fueron mayores para la 99mTc-IgG a las 4 h. En conclusión la 99mTc-IgG puede ser usada en las detecciones tempranas de sitios de inflamación/infección con todas las ventajas sobre el 67Ga debido a las propiedades físicas ideales del 99mTc, fácil preparación, bajo costo y mayor disponibilidad

Utilización de inmunoglobina humana marcada con 99mTc en localización de abscesos / Abscess localization using 99mTc labeled human immunoglobulin

Se formuló un kit liofilizado de IgG, se marcó con 99mTc y se utilizó en ratones Balb/c portadores de un sitio de inflamación. La marcación se basó en la reducción de los puentes disulfuro (S-S) usando 2-mercaptoetanol (2-ME) y metilendifosfonato como ligando la transferencia. Las especies marcadas fueron analizadas comparativamente por cromatografía instantánea en placa delgada (ITLC Gelman SG), cromatografía líquida de alta presión (HPLC) y electroforesis en gel de poliacrilamida (PAGE). Los ensayos de biodistribución y las imágenes fueron realizadas en ratones Balb/c con un absceso de 48 h inducido por la inyección de trementina (pata derecha) y mostraron una clara diferencia entre la proteína marcada y el 99mTcO4Na. Las relaciones pata normal/pata inflamada de 24 h no mostraron diferencias con el 67Ga, pero fueron mayores para la 99mTc-IgG a las 4 h. En conclusión la 99mTc-IgG puede ser usada en las detecciones tempranas de sitios de inflamación/infección con todas las ventajas sobre el 67Ga debido a las propiedades físicas ideales del 99mTc, fácil preparación, bajo costo y mayor disponibilidad (AU)

99mTc direct labeling of anti-CEA monoclonal antibodies: quality control and preclinical studies.

The anti-carcinoembryonic B2C114 monoclonal antibody was radiolabeled with 99mTc by a direct method and quality control tested in vitro by instant thin layer chromatography, gel column scanning and cellulose acetate electrophoresis and assessed in vivo for radioimmunodetection on a murine spontaneous mammary carcinoma. The optimal results of percent 99mTc bound to protein were obtained at a dithiothreitol:antibody molar ratio ranging from 800:1 to 1000:1 and at a methylene diphosphonate:stannous fluoride weight ratio of 4.3:1. Although cysteine removed up to 18% of the label during the first 4 h, the stability of the tracer appeared to be excellent in human serum at 37 degrees C and when challenged with DTPA. 99mTc-labeled B2C114 demonstrated good and specific in vivo tumor targeting.

Direct labeling of monoclonal antibodies with 99mTc and radioimmunodetection of a murine mammary carcinoma with 99mTc-B2C114.

Two anti-CEA antibodies, B2C114 and IORCEA1, were radiolabeled with 99mTc by two direct methods (mercaptoethanol and ascorbic acid reduction), and the radio-immunoimaging properties of B2C114 were assessed in mice bearing an M3-reactive tumor. The labeling efficiency was greater than 90% as measured by ITLC in saline, methylethylketone and with serum albumin impregnated sheets using ethanol: water: NH4OH (2:5:1). The label was stable to challenge with excess DTPA, and in the case of ascorbic acid reduction, serum analysis showed that 10-15% of the radioactivity was lost during incubation. In vitro studies demonstrated that the radiolabeled antibodies retained their immunoreactivity. Biodistribution studies in normal Balb/c mice showed that the pattern of uptake was quite similar for both antibodies. Biodistribution of the 99mTc-B2C114 and image studies in the animal model showed that the tumor was clearly visualized and that B2C114 labeled with 99mTc is a possible candidate for human radioimmunodetection of CEA-expressing tumors.

Marcación del anticuerpo monoclonal B2C114 con 111In usando un quelante funcional / 111In labelling of B2C114 monoclonal antibody with a bifunctional chelating agent

El anticuerpo monoclonal (AcMo) B2C114, dirigido contra el antígeno carcinoembrionario (CEA) fue conjugado con el anhídrido bicíclico del DTPA (CA-DTPA) usando diferentes relaciones molares CA-DTPA: AcMo desde 1:1 hasta 30:1. Se determinó para cada caso la eficiencia de acoplamiento y el número de moléculas de DTPA por molécula de AcMo. Se realizó la marcación con 111In para todas las relaciones molares CA-DTPA: AcMo y se determinó la pureza radioquímica por cromatografía instantánea en placa delgada (ITLC Gelman SG) y cromatografía en gel (Sephadex G-25). La biodistribución del AcMo marcado en ratones normales Balb/c y en portadores de tumor reactivo (M3), a diferentes horas post-inyección, mostró una acumulación creciente en el tumor al cabo de 72 h, con captación en hígado y riñón. Se observó también que al aumentar la relación molar CA-DTPA se incrementó el porcentaje de radiactividad asociada al riñón, lo cual indicaría una mayor inestabilidad del radiofármaco