Exertional chest pain is sometimes more than just coronary atherosclerosis.

CLINICAL CASE: A 64-year-old male, with cardiovascular risk factors and previous history of bilateral carpal tunnel syndrome, presented with exertional retrosternal pain. The resting echocardiogram was unremarkable. A stress echocardiogram with dobutamine revealed hypokinesis of the inferior wall, associated with angina, followed by ventricular tachycardia. The coronary angiography revealed slow flow, a dominant right coronary artery with non-obstructive atherosclerosis and a left anterior descending artery with intermediate lesions in mid and distal segments. The invasive functional evaluation, including fractional flow reserve, thermodilution coronary flow reserve and index of microvascular resistance, led to the diagnosis of microvascular angina, treated with calcium channel blockers and transdermal nitrate, giving symptom relief. EVOLUTION: Three years later he developed complete atrioventricular block and a dual chamber pacemaker was implanted. Shortly after, the patient developed progressive symmetrical tetraparesis, associated with marked muscle atrophy, hand numbness, orthostatic hypotension and dysphagia. The neurology workup led to the diagnosis of familial amyloidotic polyneuropathy, with the Val30Met mutation in the transthyretin gene. The following year he developed congestive heart failure. The echocardiogram showed moderate concentric left ventricular hypertrophy with preserved ejection fraction. A 99mTc-DPD Scintigraphy showed significant myocardial tracer uptake, leading to a diagnosis of TTR amyloid infiltration. DISCUSSION: Patients with exertional angina and microvascular disease should be kept under close surveillance, as they may have systemic disease with cardiac involvement. Carpal tunnel syndrome, in the context of undiagnosed cardiac disease, should trigger suspicion of cardiac amyloidosis.

Assessment of the Absorbed Dose in the Kidney of Nuclear Nephrology Paediatric Patients using ICRP Biokinetic Data and Monte Carlo Simulations with Mass-Scaled Paediatric Voxel Phantoms.

The aim of this work is to use Monte Carlo simulations and VOXEL phantoms to estimate the absorbed dose in paediatric patients (aged from 2 weeks to 16 y), with normal renal function, to whom technetium-99m-dimercaptosuccinic acid (99mTc-DMSA) was administered, for diagnostic renal scintigraphy purposes; and compare them with values obtained using the International Commission on Radiological Protection (ICRP) methodology. In the ICRP methodology, the cumulated absorbed dose in the kidneys is estimated by multiplying the administered activity with the corresponding given dose coefficients. The other methods were based on Monte Carlo simulations performed on two paediatric voxel phantoms (CHILD and BABY), and another three phantoms, which were modified to suit the mass of the patients' kidneys, and other anatomical factors. Different S-values were estimated using this methodology, which together with solving the ICRP biokinetic model to determine the cumulated activities, allowed for the estimation of absorbed doses different from those obtained with the ICRP method, together with new dose coefficients. The obtained values were then compared. The deviations suggest that the S-values are strongly dependent on the patient's total body weight, which could be in contrast with the ICRP data, which is provided by age, regardless of other anatomical parameters.

Pulmonary ventilation/perfusion single photon emission tomography--Initial experience of a Nuclear Medicine Department.

INTRODUCTION: Lung ventilation/perfusion scintigraphy with planar images (V/QS-planar) is very useful for the diagnosis and follow-up of pulmonary thromboembolism (PTE). Acquiring tomographic images (V/QS-SPECT) is a recent development with potential to increase the technique's accuracy. The purpose of this work is to evaluate the added benefits of V/QS-SPECT studies as opposed to traditional planar imaging. PATIENTS AND METHODS: We prospectively revised 53 V/QS-planar and V/QS-SPECT exams, performed according to the European Association of Nuclear Medicine guidelines. We evaluated the exams independently, by consensus of two Nuclear Medicine physicians. For both methods, we gave each lung a score expressing the dimension and extension of perfusion defects with normal ventilation. For each lung, we compared the scores with the paired Wilcoxon test, estimating the 95% confidence interval (95 CI) for the respective difference. RESULTS: We performed V/QS-SPECT exams without technical difficulties. The paired Wilcoxon test estimated the score difference to be -0.75 (95 CI of -1.0 to -0.5; p-value=9.6 × 10(-7)), expressing a statistically significant difference of about 1 subsegmental defect between both methods, with V/QS-SPECT detecting more defects. DISCUSSION: The results demonstrate that V/QS-SPECT identifies a slightly larger number of perfusion defects than V/QS-planar, suggesting a higher sensitivity of this technique. However, more studies are necessary to evaluate the clinical meaning of this fact. CONCLUSION: V/QS-SPECT demonstrates a higher capability to identify perfusion defects. This method looks promising, allowing for a greater role of this exam in pulmonary thromboembolism diagnosis and follow-up.

Estimation of the collective ionizing dose in the Portuguese population for the years 2011 and 2012, due to nuclear medicine exams.

OBJECTIVES: In 2009-2010 a Portuguese consortium was created to implement the methodologies proposed by the Dose Datamed II (DDM2) project, aiming to collect data from diagnostic X-ray and nuclear medicine (NM) procedures, in order to determine the most frequently prescribed exams and the associated ionizing radiation doses for the Portuguese population. The current study is the continuation of this work, although it focuses only on NM exams for the years 2011 and 2012. MATERIAL AND METHODS: The annual frequency of each of the 28 selected NM exams and the average administered activity per procedure was obtained by means of a nationwide survey sent to the 35 NM centres in Portugal. RESULTS: The results show a reduction of the number of cardiac exams performed in the last two years compared with 2010, leading to a reduction of the annual average effective dose of Portuguese population due to NM exams from 0.08 mSv ± 0.017 mSv/caput to 0.059 ± 0.011 mSv/caput in 2011 and 0.054 ± 0.011 mSv/caput in 2012. Portuguese total annual average collective effective dose due to medical procedures was estimated to be 625.6 ± 110.9 manSv in 2011 and 565.1 ± 117.3 manSv in 2012, a reduction in comparison with 2010 (840.3 ± 183.8 manSv). CONCLUSIONS: The most frequent exams and the ones that contributed the most for total population dose were the cardiac and bone exams, although a decrease observed in 2011 and in 2012 was verified. The authors intend to perform this study periodically to identify trends in the annual Portuguese average effective dose and to help to raise awareness about the potential dose optimization.

S-nitrosation and neuronal plasticity.

Nitric oxide (NO) has long been recognized as a multifaceted participant in brain physiology. Despite the knowledge that was gathered over many years regarding the contribution of NO to neuronal plasticity, for example the ability of the brain to change in response to new stimuli, only in recent years have we begun to understand how NO acts on the molecular and cellular level to orchestrate such important phenomena as synaptic plasticity (modification of the strength of existing synapses) or the formation of new synapses (synaptogenesis) and new neurons (neurogenesis). Post-translational modification of proteins by NO derivatives or reactive nitrogen species is a non-classical mechanism for signalling by NO. S-nitrosation is a reversible post-translational modification of thiol groups (mainly on cysteines) that may result in a change of function of the modified protein. S-nitrosation of key target proteins has emerged as a main regulatory mechanism by which NO can influence several levels of brain plasticity, which are reviewed in this work. Understanding how S-nitrosation contributes to neural plasticity can help us to better understand the physiology of these processes, and to better address pathological changes in plasticity that are involved in the pathophysiology of several neurological diseases.

Renal radionuclide imaging, an evergreen forty years old.

Urinary tract congenital abnormalities (UCA) and febrile infections (UTI) are, respectively, 2 of the commonest congenital and acquired health problems in childhood. In both, radionuclide imaging still represent a cornerstone of diagnostic imaging, although the involved techniques are more or less the same from the early '80 s. During the last 2 decades, published papers focused on a deep revision about the optimal use and usefulness of such imaging tools in affected children, with the aim of reducing invasiveness, radiation burden and costs without losing efficacy. This approach leads to different results. In UCA, no consensus for a diagnostic algorithm was up to now reached, whilst, about febrile UTIs, guidelines were published in 2007 by the UK's National Institute for Clinical Excellence (NICE) and by the European Society of Paediatric Radiology (ESPR), in 2011 by the American Academy of Paediatrics (AAP), and in 2012 by the Italian Society of Paediatric Nephrology (SINP). Nevertheless, new data continuously arise and the scientific debate always revives. Every imaging tool now available has its own strengths and weaknesses, and so all published guidelines. All this body of knowledge must be critically analysed for obtaining a complete, up-to-date and flexible overview about these "always hot" topics.

Lung ventilation/perfusion scintigraphy in pulmonary capillary hemangiomatosis: a pattern to consider.

Pulmonary capillary hemangiomatosis is a rare disease, characterized by small capillaries proliferation, leading to pulmonary hypertension. The authors report a case of pulmonary capillary hemangiomatosis, and discuss its diagnostic difficulties. Special attention is presented to ventilation/perfusion scintigraphy, given both its importance to the evaluation of pulmonary hypertension, and its referred limited usefulness in pulmonary capillary hemangiomatosis. The few published cases of ventilation/perfusion scintigraphy on this entity have showed different patterns, which are discussed. This case presents a pattern with augmented perfusion on lung bases and normal ventilation, which has been described by other authors as typical for pulmonary capillary hemangiomatosis. The authors consider important to retain this pattern, when evaluating pulmonary hypertensive patients, given not only its possible ability to help on pulmonary capillary hemangiomatosis diagnosis, but also mainly its risk of misinterpretation as a decreased perfusion on upper lung lobes, leading to erroneous diagnostic hypotheses.

Pregnancy or stress decrease complexity of CA3 pyramidal neurons in the hippocampus of adult female rats.

Pregnancy is a time of distinct neural, physiological and behavioral plasticity in the female. It is also a time when a growing number of women are vulnerable to stress and experience stress-related diseases, such as depression and anxiety. However, the impact of stress during gestation on the neurobiology of the mother has yet to be determined, particularly with regard to changes in the hippocampus; a brain area that plays an important role in stress-related diseases. Therefore, the aim of the present study was to understand how stress and reproductive state may alter dendritic morphology of CA1 and CA3 pyramidal neurons in the hippocampus. To do this, adult age-matched pregnant and virgin female Wistar rats were divided into two conditions: (1) control and (2) stress. Females in the stress condition were restrained for 1h/day for the last 2 weeks of gestation and at matched time-points in virgin females. Females were sacrificed the day after the last restraint session and brains were processed for Golgi impregnation. Dendritic length and number of branch points were quantified for apical and basal regions of CA1 and CA3 pyramidal neurons. Results show that regardless of reproductive state, stressed females had significantly shorter apical dendrites and fewer apical branch points in CA3 pyramidal cells. In addition, pregnant females, regardless of stress exposure, had less complex CA3 pyramidal neurons, as measured by Sholl analysis. No differences between conditions were seen in morphology of CA1 pyramidal neurons. This work shows that both repeated restraint stress and pregnancy affect dendritic morphology by decreasing complexity of CA3, but not CA1, neurons in the hippocampus.

Study of nuclear medicine practices in Portugal from an internal dosimetry perspective.

Nuclear medicine practices involve the handling of a wide range of pharmaceuticals labelled with different radionuclides, for diagnostic and therapeutic purposes. This work intends to evaluate the potential risks of internal contamination of nuclear medicine staff in several Portuguese nuclear medicine services and to conclude about the requirement of a routine internal monitoring. A methodology proposed by the International Atomic Energy Agency (IAEA), providing a set of criteria to determine the need, or not, for an internal monitoring programme, was applied. The evaluation of the risk of internal contaminations in a given set of working conditions is based on the type and amount of radionuclides being handled, as well as the safety conditions with which they are manipulated. The application of the IAEA criteria showed that 73.1% of all the workers included in this study should be integrated in a routine monitoring programme for internal contaminations; more specifically, 100% of workers performing radioimmunoassay techniques should be monitored. This study suggests that a routine monitoring programme for internal exposures should be implemented in Portugal for most nuclear medicine workers.

Tributyltin-induced imposex in marine gastropods involves tissue-specific modulation of the retinoid X receptor.

Despite the large number of studies on the phenomenon of imposex, the mechanism underlying the abnormal growth of male sexual characters onto females in numerous gastropod species is yet to be fully elucidated. Although several hypotheses have been raised over the years, a convincing body of evidence indicates that tributyltin-induced imposex involves the abnormal modulation of the retinoid X receptor (RXR). Here, we investigate the RXR gene transcription at different timings and tissues upon exposure to environmentally relevant concentrations of tributyltin (TBT) (100 ng Sn/L TBT) in both genders of the imposex susceptible gastropod Nucella lapillus. RXR gene transcription was determined at two time-points (i.e., before and after imposex initiation) by quantitative Real Time PCR in potential target tissues: the central nervous system (CNS), penis/penis forming area (PFA), gonads and digestive gland. TBT-exposure altered transcription of RXR gene in a tissue and sex specific manner. In the CNS, a significant down-regulation was observed in females both before and after imposex initiation (P≤0.01 and P≤0.05, respectively). A similar trend was observed in male CNS at the first time-point, although differences between control and the TBT-exposed group were just above significance (P=0.059). The penis/PFA showed no differences in transcription of RXR gene between control and TBT exposed female snails before imposex induction, or before and after imposex initiation for males. However, male penis showed higher transcription of RXR gene in comparison to the PFA of females. After imposex has been induced, a significant (P≤0.001) increase in transcription of RXR gene was observed in penis of females with vas deference sequence index (VDS) levels of 3-4 in comparison with the PFA of both control and imposex females with VDS 1-2. At advanced stages of imposex, females displayed RXR transcription patterns in penis identical to those of males, which points to a functional role of RXR in the penis of both genders. In the other tissues, gonads and digestive gland, RXR gene transcription was not affected by TBT, at any of the analysed time-points. These patterns of RXR gene transcription upon TBT exposure highlight the pivotal involvement of the CNS in the mechanism of imposex induction. We integrate the results in a conceptual model, and discuss the central role of RXR and the retinoic acid signalling pathways in imposex and male genitalia formation in gastropods.

Assessment of salivary gland function in Sjögren's syndrome: the role of salivary gland scintigraphy.

Salivary gland scintigraphy (SGS) is a non invasive method of salivary gland function assessment. This technique is easy to perform, reproducible and well tolerated by patients. Additionally, an abnormal salivary gland scintigraphy result is accepted by the American-European consensus group as a criterion for the diagnosis of Sjögren's syndrome. Scintigraphic evaluation of salivary gland function also plays an important role in therapeutic decision and patient follow-up. Schall's categorical classification is usually considered the standard method for salivary scintigraphy interpretation, though subjective and with limited capacity to discriminate borderline results. In order to improve the diagnostic accuracy of SGS, there has been an increasing interest in the quantification of glandular function. However, the debate on the most reliable and suitable parameters for the diagnosis of SS persists.

Clear-PEM: a dedicated PET camera for improved breast cancer detection.

Positron emission mammography (PEM) can offer a non-invasive method for the diagnosis of breast cancer. Metabolic images from PEM using 18F-fluoro-deoxy-glucose, contain unique information not available from conventional morphologic imaging techniques like X-ray radiography. In this work, the concept of Clear-PEM, the system presently developed in the frame of the Crystal Clear Collaboration at CERN, is described. Clear-PEM will be a dedicated scanner, offering better perspectives in terms of position resolution and detection sensitivity.

Sodium channel currents in maturing acutely isolated rat hippocampal CA1 neurones.

Sodium channel currents were recorded in excised inside-out patches from immature (P(4-10)) and older (P(20-46)) rat CA1 neurones. Channel conductance was 16.6+/-0.013 pS (P(20-46)) and 19.0+/-0.031 pS (P(4-10)). Opening patterns varied with step voltage and with age. In some patches bursting was apparent at voltages positive to -30 mV. Non-bursting behaviour was more dominant in patches from younger animals. In older animals mean open time (m.o.t.) was best described by two exponentials especially in the older cells; in the immature, there were fewer cases with two exponentials. The time constant of inactivation (tau(h)) estimated in ensemble averages was best described by two exponentials (tau(hf) and tau(hs)) in most patches from older cells. tau(hf) decreased with depolarization; tau(hs) increased in the range -30 to 0 mV. The voltage dependence of tau(hf) in the older cells is identical to that of the single tau(h) found in the younger; the results indicate a dominance of tau(hf) in the younger. Patches from younger cells more often showed one apparent active channel; in such cases, m.o.t. was described by a single exponential. However, in two cases, channels showed bursting behaviour with one of these channels showing a shift between bursting and non-bursting modes. Our findings are consistent with a heterogeneous channel population and with changes in the population in the course of maturation.

Molecular genetic analysis of factor XI deficiency: identification of five novel gene alterations and the origin of type II mutation in Portuguese families.

Coagulation factor XI (FXI) deficiency is an inherited autosomal recessive mild bleeding disorder. In this study, we report the molecular genetic analysis of FXI deficiency in six unrelated families of Portuguese origin. The Jewish type II mutation was found in two families, of seemingly Portuguese origin. Haplotype analysis in these families demonstrated that this mutation is of Jewish origin. In the remaining families, five novel FXI mutations have been identified. Two of these mutations (FXI IVS K -10T-->A and FXI 1026G-->T, cd 324) affect the FXI pre-mRNA splicing. A further two (FXI 307 ins AAGCAAT, cd 85 and FXI 1072 del A, cd 340) introduce frameshifts leading to premature termination codons. The FXI splicing mutation, 1026G-->T cd 324, was found in compound heterozygosity with missense mutation FXI K518N. Analysis of the FXI mRNA from the latter genotype demonstrated new donor splice site usage. All reported mutations most likely result in functional null-alleles. In addition, three novel polymorphisms have been identified: at nt -138 in intron A, at codon D125 in exon 5 and at codon T249 in exon 8.

Multiple endocrine neoplasia type 2A. Study of a family.

INTRODUCTION: Pheochromocytomas (Pheo) can occur sporadically, isolated or in association with other neuroendocrine lesions. In multiple endocrine neoplasia type 2A (MEN-2A), Pheo is associated to medullary thyroid carcinoma (MTC) or its precursor, C-cell hyperplasia (CCH) and parathyroid hyperplasia. Genetic screening provides early diagnosis and preventive treatment. In order to validate DNA analysis as a reliable method of early identification of gene carriers, we compared the results of genetic screening with clinical, biochemical, imaging and pathological findings in the members of an affected family. POPULATION AND METHODS: The diagnosis of a bilateral necrotic Pheo in a female patient led to the study of a family with four generations, aged 3 to 78 years (mean = 30.3 yrs). The study included a clinical examination; basal and pentagastrin stimulated calcitonin values; urinary catecholamines and their metabolites; serum calcium and a genetic study (direct sequence of PCR products from genomic DNA isolated from leucocytes using specific primers in exon 11 of the RET protooncogene of chromosome 10). The radiologic study, gammagraphic study (131I-MIBG) and magnetic resonance study were performed in members with clinical suspicion of Pheo. RESULTS: Seven out of nine patients had a mutation on codon 634 of exon 11 of RET (TGC-CGC), leading to cysteine arginine substitution in the codified protein; all gene carriers had biochemical markers of MTC/CCH and four of Pheo. The Pheo patients underwent adrenalectomy (bilateral in three) and all the gene carriers underwent prophylactic thyroidectomy. The pathologic findings were: MTC in four (metastasized in one); CCH in three and parathyroid hyperplasia in one. CONCLUSIONS: Phenotypic penetration of RET mutation was 100% for MTC/CCH, but only 57% of the gene carriers had Pheo. Genetic screening allowed early prophylactic treatment in four out of seven patients; pathologic findings revealed several evolutionary stages of the disease. Patients not yet showing Pheo are under close clinical and laboratory surveillance.

Sustained potassium currents in maturing CA1 hippocampal neurones.

Whole-cell voltage clamp techniques were used to characterize sustained outward currents in maturing (P4 to P48) acutely isolated rat CA1 hippocampal neurones. Sodium removal and signal subtraction were used to isolate a sodium dependent sustained potassium current (IKNa). Calcium blockade (Co2+), sensitivity to a low TEA dose (0.5 mM) and sensitivity to Charibdotoxin (CTX 25 nM) and Iberiotoxin (IbTX 25 nM), in conjunction with signal subtraction, were used to isolate a sustained current with the characteristics of IC (IKCa). IKNa was found in both immature (P4-5) and older (P > 21) cells; this corresponded, respectively, to 56 +/- 5% and 36 +/- 6% of the outward current in younger and older cells. In the course of maturation, the voltage dependence of activation of IKNa shifted to more hyperpolarized values by approximately 20 mV. In the younger cells (P5-18) there was no evidence for sensitivity to CTX or IbTX. In 55 out of 77 older cells we found a component sensitive to CTX, IbTX, 0.5 mM TEA and Co2+.

Technetium-99m labelling of the IOR CEA 1 monoclonal antibody: evaluation of different methods.

AIM: The aim of this study was to investigate the in vivo and in vitro properties of 99mTc labelled monoclonal antibody, IOR CEA 1 when radiolabelled by different methods. METHODS: To achieve that purpose IOR CEA was directly radiolabelled via 2-mercaptoethanol (2-Me) and stannous ion (SnCl2) reduction and indirectly via the 2-iminothiolane (2-Im) conjugation. The resulting 99mTc-MoAbs were analysed for number of free sulfhydryl groups, chemical and radiochemical purity (checked by HPLC and SDS PAGE), immunoreactivity and biological distribution in mice. RESULTS: Experimental results indicated a similar radiochemical purity and immunoreactivity for direct labelling methods and a decrease of both for 2-Im method. 2-Me antibody reduction led to a high antibody fragmentation as indicated by non-denaturing SDS PAGE analysis. Nevertheless SnCl2 and 2-Im labels revealed lower in vivo stability. CONCLUSION: 99mTc-(2-Me) IOR CEA presented favorable in vitro and in vivo properties. Therefore this label was compared to 99mTc-monoclonal antibody BW 431/26. Similar characteristics were found. Clinical studies also revealed identical biodistribution profile.

[Anti-melanoma immunoscintigraphy]. / Imunocintigrafia anti-melanoma.

Nuclear Medicine has always contributed to the study of oncologic diseases. Immunoscintigraphy, one of its more recent developments, consists of the evaluation of the biodistribution of antibodies, directed against tumoral antigens and labelled with radionuclides. This technique, which has proven of special interest in some neoplasias, was used for the first time in 1983 in malignant melanoma of the skin. The antibody that has been more frequently used and which is used in the Instituto de Medicina Nuclear (IMN) of the Faculdade de Medicina de Lisboa, is antibody 225.28S, an IgG2a directed against a high molecular weight antigen present in the melanoma cell. In the IMN, we started immunoscintigraphy anti-melanoma in February 1992. During these two years, we have performed 67 exams, 44 on patients with malignant melanoma of the skin and 23 on uveal melanoma. We have obtained true positive rates and true negative ones, respectively, of 87.5% and 90% in melanoma of the skin, and 94% and 83% in uveal melanoma. It has been shown that the main clinical contribution of immunoscintigraphy for malignant melanoma of the skin is the study of loco-regional and distant metastases, namely those clinically unsuspected, as well as in the differential diagnosis of a lesion already known. In uveal melanoma, it is accepted that immunoscintigraphy may be useful in the evaluation of the primary lesion, namely in the differential diagnosis with other intra-ocular lesions.

Potassium currents in acutely isolated maturing rat hippocampal CA1 neurones.

Whole-cell voltage clamp techniques were used to characterize the postnatal development of current amplitude and inactivation and activation kinetics of two potassium currents in acutely isolated CA1 cells from rats P4 to P52: the A-current (IA) and a slow-rising, slow inactivating current (IK). In the course of maturation, changes in the relative proportion of IA and IK currents were observed, the latter becoming a dominant current in older cells. The half-maximal point (Vh) of steady-state inactivation and activation of IA and IK shifted in the course of the first and second postnatal weeks. The shifts were hyperpolarizing in the case of IK, whereas IA shifted to less negative values. The shifts in steady-state inactivation Vh were accompanied by a change in the slope factor (Vs), which is an indication of a modification in the voltage sensitivity of the steady-state inactivation. The kinetics of IK evolve after birth in a fashion that matches the changes in action potential parameters previously reported.

Yeast exo-beta-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae.

Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.