Tissue microarrays - brief history, techniques and clinical future.

INTRODUCTION AND AIM: There is a growing need for better, cheaper and faster histopathological diagnostic. The authors reviewed the main steps of the efforts towards the improvement of the pre-analytical phase of tissue processing for histological examination. RESULTS: Since their introduction decades ago tissue microarrays (TMAs) proved their value by increasing efficiency, standardization and accuracy of many histological techniques, such as histochemistry, histoenzymology, immunohistochemistry, in situ hybridization, etc. By allowing the simultaneous analysis and comparison of multiple different tissues on a single histology slide (up to 1000 individual samples), TMAs are also having a significant economic advantage (consumables and labor). From its first description until recent years, the TMA techniques have evolved steadily but slowly despite many attempts to adapt it for clinical diagnostics. In this paper, we are reviewing the main techniques of obtaining TMA blocks from the beginning to the present day, as well as recent developments that are expanding their scope into high accuracy/efficiency clinical diagnostics. CONCLUSIONS: Considering recent developments, we believe that the prospect of high-throughput histology might be achievable in the not-so-distant future.

The Impact of Socioeconomic Status and Nutritional Biochemical Markers on Quality of Life of Chronic Dialysis Patients?

Aim: This cross-sectional study aimed to determine the relationship between socio-economic status and laboratory values with mental and physical components of QoL (quality of life) in chronic dialysis patients. Material and Methods: 254 patients were included in the study and divided into two groups: 243 hemodialysis (HD) patients and 11 peritoneal dialysis (PD) patients. QoL was measured by the Short Form 36(SF-36). Scores for all dimensions are expressed on a 0-100 scale. Higher score indicates better health status. Two more questions related to monthly food expenditure and the presence/or lack of family support were added. Laboratory investigations included the following nutritional parameters: serum albumin and creatinine. Results: Our study results showed that compared to PD group in HD patients QoL was compromised in all SF-36 subscale except physical functioning, role limitations due to physical functioning and role limitations due to emotional functioning. Also, the presence of supportive family members/ caregivers showed a significant improvement of all QoL scores. HD patients who spend at least 90 euros monthly on groceries/food had higher values of SF-36 items. Better QoL is associated with high pre-dialysis serum creatinine and serum albumin levels. Conclusions: Future work should incorporate larger and more balanced sample sizes and patient recruitment from multiple dialysis centers to truly capture the variability in patient characteristics.

Changes in the histological spectrum of glomerular diseases in the past 16 years in the North-Eastern region of Romania.

BACKGROUND: The aim of this study was to describe the findings of renal biopsies from a large nephrology center in Iasi, Romania, performed between 2005 and 2010. We compared these findings with our previous ones, from 1995 to 2004, as well as with similar reports. METHODS: We studied retrospectively 239 renal biopsies. The indications for renal biopsy were categorized into: nephrotic syndrome, acute nephritic syndrome, asymptomatic urinary abnormalities, acute kidney injury, and chronic kidney disease of unknown etiology. RESULTS: During the past 16 years, a gradual increase in the annual number of renal biopsies/per million population (p.m.p.)/year was observed, although this incidence remained lower than in other European countries. Nephrotic syndrome was the indication for renal biopsy in over 50% of cases. Glomerulonephritis (GN) was the main histological diagnosis in 91% of cases, of which 56% were primary GN and 35% were secondary GN. The frequency of various types of primary GN was: membranoproliferative GN (MPGN) - 29.3%, membranous nephropathy (MN) -27.5%, focal segmental glomerulosclerosis (FSGS) - 17.2%, mesangial GN (including IgAN) -13.7%, crescentic GN - 9.4%, and minimal change disease (MCD) - 2.5%. Compared to the previously reported period (1994-2004), we observed a significant decrease in the frequency of MPGN and significant increases in the frequency of FSGS and, particularly MN - which more than doubled. CONCLUSION: We report significant changes in the histological spectrum of GN in North-Eastern Romania in 2005-2010, compared to the previously reported 10-yrs. These changes seem to be following a trend that has also been observed in Western countries a few decades ago, and which may have a socioeconomic explanation.

The prognostic value of electrocardiographic estimation of left ventricular hypertrophy in dialysis patients.

BACKGROUND: Left ventricular hypertrophy (LVH) is associated with poor cardiovascular outcome in CKD. Electrocardiogram (ECG) is low-cost but infrequently used to assess presence of LVH in dialysis patients. The aim of this study was to establish which ECG-determined LVH method is most sensitive in dialysis patients, and also most predictive of death. METHODS: This was a longitudinal observational study in dialysis patients from a single center, undergoing interval ECGs. Fourteen methods of ECG LVH assessment were compared. Survival was also compared between four LVH evolutionary categories: persistent LVH; new LVH; LVH regression; and no LVH. RESULTS: The study included 418 dialysis patients (46.3% women, mean age 51 years, mean follow up 67 months, 76 deaths, 37 cardiovascular deaths). LVH prevalence varied according to method (range 13.4-41.9%). No measurement predicted all-cause mortality. After Cox regression, there was an independent association between LVH and cardiovascular mortality using Novacode (HR = 3.04; 95% [CI] = 1.11-8.28, P < 0.05), but not with other methods. Patients with persistent ECG changes of LVH had increased risk of cardiovascular mortality compared to other LVH evolutionary categories (P < 0.044). CONCLUSIONS: ECG scoring of LVH can be predictive of cardiovascular mortality. The Novacode method, based on repolarization abnormalities, is a better predictor than standard ECG techniques that are based on voltage criteria. Novacode LVH estimation at dialysis initiation may prove to be a noninvasive and cost-effective bedside tool for cardiovascular risk stratification in patients receiving dialysis.

Visualization of membrane rafts using a perylene monoimide derivative and fluorescence lifetime imaging.

A new membrane probe, based on the perylene imide chromophore, with excellent photophysical properties (high absorption coefficient, quantum yield (QY) approximately 1, high photostability) and excited in the visible domain is proposed for the study of membrane rafts. Visualization of separation between the liquid-ordered (Lo) and the liquid-disordered (Ld) phases can be achieved in artificial membranes by fluorescence lifetime imaging due to the different decay times of the membrane probe in the two phases. Rafts on micrometer-scale in cell membranes due to cellular activation can also be observed by this method. The decay time of the dye in the Lo phase is higher than in organic solvents where its QY is 1. This allows proposing a (possible general) mechanism for the decay time increase in the Lo phase, based on the local field effects of the surrounding molecules. For other fluorophores with QY<1, the suggested mechanism could also contribute, in addition to effects reducing the nonradiative decay pathways, to an increase of the fluorescence decay time in the Lo phase.

Non-conjugated, phenyl assisted coupling in through bond electron transfer in a perylenemonoimide-triphenylamine system.

Two donor-bridge-acceptor compounds containing triphenylamine (TPA) donors and perylenemonoimide (PMI) acceptors have been studied by spectroscopic techniques and quantum chemical computation. Both systems have been observed to emit prompt and delayed fluorescence under certain conditions indicating that forward and reverse electron transfer (ET) processes can occur between the locally excited and the charge separated states. The experimental and computational results show that the TPA and PMI chromophores are better coupled by almost 50% in the meta isomers which undergo ET more readily than the para isomers. Quantum chemical calculations indicate that this unexpected situation is the result of a phenyl group on the side of the bridge being advantageously positioned in the meta isomers. This leads to more extensive delocalisation of the TPA HOMO into the bridge enhancing the total through bond electronic coupling between the TPA and PMI chromophores. The calculations also indicate a strong angle dependence of the total coupling in both isomers. The experimental results are discussed in the context of the high temperature limit of Marcus's theory of non-adiabatic ET.

Photophysical properties of BODIPY-derived hydroxyaryl fluorescent pH probes in solution.

The photophysical properties of five fluorescent pH probes derived from 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene with phenolic or naphtholic subunits at position 8 and with substituents having different electron driving forces at positions 3 and 5 have been investigated in several organic solvents, by means of absorption, steady-state, and time-resolved fluorimetry. For each compound, the fluorescence quantum yield and lifetime are lower in solvents with higher polarity, owing to an increase in the rate of nonradiative deactivation. The rate constants for radiative deactivation, k(f), are nearly constant for all dyes in all solvents studied [k(f)=(1.7+/-0.2)x10(8) s(-1)]. In aqueous solution, these probes undergo a reversible protonation-deprotonation in the near-neutral to basic pH range, producing intensity increases with lower pH. The pK(a) values of the indicators are between 7.5 and 9.3, depending on the substitution pattern on positions 3, 5, and 8. The difference between the absorption and excitation spectra as a function of pH is indicative of the presence of two species in aqueous solution: the phenol- or naphthol-based indicator and its conjugate base.

Electron transfer at the single-molecule level in a triphenylamine-perylene imide molecule.

Photoinduced electron transfer (ET) processes in a donor-acceptor system based on triphenylamine and perylene imide have been studied at the single-molecule (SM) and ensemble levels. The system exists as two isomers, one of which undergoes forward and reverse ET in toluene with decay constants of 3.0 and 2.2x10(9) s(-1), respectively, resulting in the dual emission of quenched and delayed fluorescence while the other isomer remains ET-inactive. The fluorescence of both isomers is heavily quenched in the more polar solvent, diethyl ether, by ET. A broad range of ET dynamics is seen at the SM level in polystryene with the two isomers nonresolvable indicating that the local nanoenvironment of the SMs varies considerably throughout the polymer matrix. Both the electronic coupling and the driving force for ET are shown to influence the ET dynamics. Many fluorescence trajectories of SMs show long periods (tens of milliseconds to seconds) where the count rate is attenuated either partly (a "dim" state) or to the background level (an "off-time"). During these periods, the reduction or interruption of emission is attributed to cycles of rapid charge separation followed by charge recombination to the ground state reducing the fluorescence quantum yield of the SM.

Dendronized perylenetetracarboxdiimides with peripheral triphenylamines for intramolecular energy and electron transfer.

Novel perylene-3,4,9,10-tetracarboxdiimides (PDI) dyes functionalized with polyphenylene dendrimers attached at the bay region are reported. Derivatives of PDI bearing polyphenylene dendrimers up to the second generation, substituted with an increasing number of triphenylamine (TPA) moieties at the periphery, as well as a related nondendronized model compound were prepared. Intramolecular energy transfer was demonstrated by the observation of PDI emission on excitation of the triphenylamines, and electron transfer was detected by comparing photoluminescence quenching in solvents of different polarity.

Intramolecular directional förster resonance energy transfer at the single-molecule level in a dendritic system.

We report on the directional Förster resonance energy transfer (FRET) process taking place in single molecules of a first (T1P4) and a second (T2P8) generation of a perylenemonoimide (P)-terrylenediimide (T)-based dendrimer in which the chromophores are separated by rigid polyphenylene arms. At low excitation powers, single-molecule detection and spectroscopy of T1P4 and T2P8 dendrimers point to a highly efficient directional FRET from P donors to the central T acceptor, optical excitation at 488 nm resulting in exclusively acceptor emission in the beginning of the detected fluorescence intensity. Donor emission is seen only upon the bleaching of the acceptor. High-resolution time-resolved single-molecule fluorescence data measured with a microchannel plate photomultiplier reveal, for T2P8, a broad range of FRET rates as a result of a broad range of distances and orientations experienced by the donor-acceptor dendrimers when immobilized in a polymer matrix. Single-molecule data from T2P8 on 488 nm excitation are indicative for the presence, after terrylenediimide bleaching, of a P-P excited dimer characterized by a broad emission spectrum peaking around 600 nm and by fluctuating fluorescence decay times. At high excitation powers, single T1P4 and T2P8 molecules display simultaneous emission from both donor and acceptor chromophores. The effect, called "exciton blockade", occurs due to the presence of multiple excitations in a single molecule.