Influence of sex on chronic steroid-induced glaucoma: 24-Weeks follow-up study in rats.

The objective was to evaluate ocular changes based on sex in steroid-induced glaucoma models in rats comparing healthy controls, over 24 weeks follow-up. Eighty-nine Long-Evans rats (38 males and 51 females) with steroid-induced glaucoma were analysed. Two steroid-induced glaucoma models were generated by injecting poly-co-lactic-glycolic acid microspheres loaded with dexamethasone (MMDEX model) and dexamethasone-fibronectin (MMDEXAFIBRO model) into the ocular anterior chamber. Intraocular pressure was measured by rebound tonometer Tonolab®. Neuroretinal function was analysed using dark- and light-adapted electroretinography (Roland consult® RETIanimal ERG), and structure was analysed using optical coherence tomography (OCT Spectralis, Heidelberg® Engineering) using Retina Posterior Pole, Retinal Nerve Fibre Layer and Ganglion Cell Layer protocols over 24 weeks. Males showed statistically (p < 0.05) higher intraocular pressure measurements. In both sexes and models neuroretinal thickness tended to decrease over time. In the MMDEX model, males showed higher IOP values and greatest percentage thickness loss in the Ganglion Cell Layer (p = 0.015). Females receiving MMDEXAFIBRO experienced large fluctuations in thickness, a higher percentage loss (on average) in Retina Posterior Pole (p = 0.035), Retinal Nerve Fibre Layer and Ganglion Cell Layer than aged-matched males, and the highest thickness loss rate by mmHg. Although no difference was found by sex in dark- and light-adapted electroretinography, increased amplitude in photopic negative response was found in MMDEX males and MMDEXAFIBRO females at 12 weeks. Although both glaucoma models used dexamethasone, different intraocular pressure and neuroretinal changes were observed depending on sex and other influential cofactors (fibronectin). Both sex and the induced glaucoma model influenced neuroretinal degeneration.

Performance of the SolWat system operating in static mode vs. dynamic for wastewater treatment: Power generation and obtaining reclaimed water.

Two hybrid SolWat systems operating in static (without flow circulation) versus dynamic (with flow circulation) mode were simultaneously compared. This work aims to demonstrate the viability of SolWat in dynamic mode to: a) adapt to the operation of the WWTP with a continuous flow, in which the wastewater flows continuously for treatment, b) treat a larger volume of water in the system and c) increase the cooling of the PV modules thanks to the cooling of the temperature of the water sample to improve the energy efficiency in the system. Real secondary wastewater effluents from wastewater treatment plants were used, using solar energy for water disinfection and photovoltaic energy production, in order to use the SolWat systems and implement it as a tertiary treatment. A total of five experiments were performed during autumn, winter, spring and summer. Solar disinfection of Escherichia coli, Enterococcus faecalis and Clostridium perfringens was assessed, and physicochemical parameters were also analysed. The UV dose received by the SolWat systems was the same, but not for the microorganisms in the water sample. The static SolWat irradiated a particle (microorganism) for 4 h, while the dynamic SolWat irradiated intermittently, so the latter system received a shorter UV exposure time, and therefore a lower UV dose. Results indicated that, although the microorganisms did not obtain the absolute bacterial inactivation during the SODIS treatment in any SolWat system, adequate inactivation levels were achieved to allow the reuse of the water for various uses (Royal Decree 1620/2007, Regulation (EU) 2020/741), although to a lesser extent for SolWat in dynamic mode, which treated twice the volume of water and reached cooler temperatures. C. perfringens proved to be the most resistant bacterium tested. The total photovoltaic energy production in the dynamic mode system was more energy efficient than the static mode, being even more efficient than the single PV reference system during the spring (3.5%) and summer (2.7%) test, due to the compensating effect by the cooling of the water on the photovoltaic module against the losses caused by radiation.

Deactivating environmental strains of Escherichia coli, Enterococcus faecalis and Clostridium perfringens from a real wastewater effluent using UV-LEDs.

Environmental bacteria strains are known to be more resistant but studies on UV-LEDs are scarce, especially for Clostridium perfringens and Enterococcus faecalis. UV-LEDs of different wavelengths (268 nm, 279 nm and 307 nm) have been used for treating real wastewater from the effluent of the municipal plant in Linares (Spain), with real organic matter content, for E. coli, Enterococcus faecalis and Clostridium perfringens disinfection. Experimental results demonstrate that 268 nm was the most effective wavelength for inactivation of the three different bacteria strains: E. coli showed an inactivation rate of 0.561 at 268 nm vs. 0.245 at 279 nm and 0.0029 for 307 nm; E. faecalis inactivation rate was 0.313 at 268 nm, 0.231 at 279 nm and 0.0023 at 307 nm; and C. perfringens inactivation rate was 0.084 at 268 nm, 0.033 at 279 nm and 6.9e-4 at 307 nm. In general, 307 nm wavelength showed a significantly lower inactivation rate so it would not be recommended for practical applications. C. Perfringens required higher UV doses and longer times to achieve complete inactivation.

Diagnosis of multiple sclerosis using multifocal ERG data feature fusion.

The purpose of this paper is to implement a computer-aided diagnosis (CAD) system for multiple sclerosis (MS) based on analysing the outer retina as assessed by multifocal electroretinograms (mfERGs). MfERG recordings taken with the RETI-port/scan 21 (Roland Consult) device from 15 eyes of patients diagnosed with incipient relapsing-remitting MS and without prior optic neuritis, and from 6 eyes of control subjects, are selected. The mfERG recordings are grouped (whole macular visual field, five rings, and four quadrants). For each group, the correlation with a normative database of adaptively filtered signals, based on empirical model decomposition (EMD) and three features from the continuous wavelet transform (CWT) domain, are obtained. Of the initial 40 features, the 4 most relevant are selected in two stages: a) using a filter method and b) using a wrapper-feature selection method. The Support Vector Machine (SVM) is used as a classifier. With the optimal CAD configuration, a Matthews correlation coefficient value of 0.89 (accuracy = 0.95, specificity = 1.0 and sensitivity = 0.93) is obtained. This study identified an outer retina dysfunction in patients with recent MS by analysing the outer retina responses in the mfERG and employing an SVM as a classifier. In conclusion, a promising new electrophysiological-biomarker method based on feature fusion for MS diagnosis was identified.

Long-term corticosteroid-induced chronic glaucoma model produced by intracameral injection of dexamethasone-loaded PLGA microspheres.

PURPOSE: To evaluate a new chronic glaucoma model produced by intracameral injection of dexamethasone-loaded poly lactic-co-glycolic acid microspheres (Dex-PLGA-Ms) over six months. METHODS: Healthy rats received two injections (at baseline and Week 4) of Dex-PLGA-Ms into the anterior chamber of the right eye. Clinical signs and intraocular pressure (IOP) were weekly recorded. The structure of the retina and optic nerve was in vivo evaluated using optical coherence tomography (OCT) every two weeks and functionally using dark- and light-adapted electroretinography at 0-12-24 weeks. Histological studies were also performed. RESULTS: IOP progressively increased up to hypertension (23.22 ± 3.63 mmHg) in both eyes but did so later in left eyes. OCT quantified a decrease in full-thickness retina posterior pole (R), retinal-nerve-fiber layer (RNFL), and ganglion-cell layer (GCL) thickness up to 24 weeks. Right eyes showed higher neuroretinal thickness loss up to week 8. RNFL experienced the highest percentage thickness loss at the inferior-superior axis, while in GCL the inner sectors of the horizontal axis (Nasal-Temporal) suffered the greatest decrease in thickness. Retinal ganglion cell, photoreceptor, and intermediate cell functionality decreased over time. Increased deposition of collagen IV was also found in zonular fibers and the ciliary body. CONCLUSIONS: This work shows the usefulness of drug delivery systems, not to treat pathology but to induce it. Only two injections of Dex-PLGA-Ms in the anterior chamber of rat eyes were enough to progressively create ocular hypertension and subsequent functional and structural neuroretinal degeneration, at least over 6 months.

Early diagnosis of multiple sclerosis by OCT analysis using Cohen's d method and a neural network as classifier.

BACKGROUND: The consequences of inflammation, demyelination, axonal degeneration and neuronal loss in the central nervous system, typical of the development of multiple sclerosis (MS), are manifested in thinning of the retina and optic nerve. The purpose of this work is to diagnose early-stage MS patients based on analysis of retinal layer thickness obtained by swept-source optical coherence tomography (SS-OCT). METHOD: OCT (Triton® SS-OCT device -Topcon, Tokyo, Japan-) recordings were obtained from 48 control subjects and 48 recently diagnosed MS patients. The following thicknesses were measured on a 45 × 60 grid: retinal nerve fibre layer (RNFL), ganglion cell layer (GCL+), GCL++, retinal thickness and choroid. Using Cohen's d effect size, it was determined the regions and layers with greatest capacity to discriminate between control subjects and patients. Points exceeding the threshold set were used as inputs for an automatic classifier: support vector machine and feed-forward neural network. RESULTS: In MS at clinical onset the layer with greatest discriminant capacity is GCL++ [AUC = 0.83] which exhibits a horseshoe-like macular topographic distribution. It is followed by retina, GCL+ and RNFL; choroidal thicknesses do not provide discriminatory capacity. Using a neural network as a classifier between controls and MS patients, obtains sensitivity of 0.98 and specificity of 0.98. CONCLUSIONS: This work suggest that OCT may serve as an important complementary role to other clinical tests, particularly regarding neurodegeneration. It is possible to characterise structural alterations in retina and diagnose early-stage MS with high degree of accuracy using OCT and artificial neural networks.

Brimonidine-LAPONITE® intravitreal formulation has an ocular hypotensive and neuroprotective effect throughout 6 months of follow-up in a glaucoma animal model.

Intravitreal administration is widely used in ophthalmological practice to maintain therapeutic drug levels near the neuroretina and because drug delivery systems are necessary to avoid reinjections and sight-threatening side effects. However, currently there is no intravitreal treatment for glaucoma. The brimonidine-LAPONITE® formulation was created with the aim of treating glaucoma for extended periods with a single intravitreal injection. Glaucoma was induced by producing ocular hypertension in two rat cohorts: [BRI-LAP] and [non-bri], with and without treatment, respectively. Eyes treated with brimonidine-LAPONITE® showed lower ocular pressure levels up to week 8 (p < 0.001), functional neuroprotection explored by scotopic and photopic negative response electroretinography (p = 0.042), and structural protection of the retina, retinal nerve fibre layer and ganglion cell layer (p = 0.038), especially on the superior-inferior axis explored by optical coherence tomography, which was corroborated by a higher retinal ganglion cell count (p = 0.040) using immunohistochemistry (Brn3a antibody) up to the end of the study (week 24). Furthermore, delayed neuroprotection was detected in the contralateral eye. Brimonidine was detected in treated rat eyes for up to 6 months. Brimonidine-LAPONITE® seems to be a potential sustained-delivery intravitreal drug for glaucoma treatment.

Photovoltaic and solar disinfection technology meeting the needs of water and electricity of a typical household in developing countries: From a Solar Home System to a full-functional hybrid system.

A novel SolWat system designed exclusively as a Solar Home System that also meets the drinking water access in a family of a rural community in a developing country has been designed, manufactured and tested outdoors. The system is composed of 5 photovoltaic modules of monocrystalline silicon solar cells technology, each 20 Wp, parallel-interconnected, adding up to a 100 Wp system. The modules have a water reactor on top with the capacity of providing a minimum of 37.5 L per day for a family of 5 members, guaranteeing the minimum daily needs. Experimental campaign run tests of SODIS of 3 h each, running the system 3 times per day (with a total of 9 h of experimentation per day). Results show that the water treatment of 3 h should be increased at certain periods of the day when the UV dose is not sufficient (late in the afternoon). E. coli and Enterococcus spp achieved total inactivation or almost total disinfection. Regarding electrical production, although energy losses of 5.6-10% were observed in comparison with a single PV module, it was sufficient to fully meet the load demand of the solar home system. The system could be used in a household of a developing country, using only solar energy to meet the electricity and drinking water demand.

Identification of clusters in multifocal electrophysiology recordings to maximize discriminant capacity (patients vs. control subjects).

PURPOSE: To propose a new method of identifying clusters in multifocal electrophysiology (multifocal electroretinogram: mfERG; multifocal visual-evoked potential: mfVEP) that conserve the maximum capacity to discriminate between patients and control subjects. METHODS: The theoretical framework proposed creates arbitrary N-size clusters of sectors. The capacity to discriminate between patients and control subjects is assessed by analysing the area under the receiver operator characteristic curve (AUC). As proof of concept, the method is validated using mfERG recordings taken from both eyes of control subjects (n = 6) and from patients with multiple sclerosis (n = 15). RESULTS: Considering the amplitude of wave P1 as the analysis parameter, the maximum value of AUC = 0.7042 is obtained with N = 9 sectors. Taking into account the AUC of the amplitudes and latencies of waves N1 and P1, the maximum value of the AUC = 0.6917 with N = 8 clustered sectors. The greatest discriminant capacity is obtained by analysing the latency of wave P1: AUC = 0.8854 with a cluster of N = 12 sectors. CONCLUSION: This paper demonstrates the effectiveness of a method able to determine the arbitrary clustering of multifocal responses that possesses the greatest capacity to discriminate between control subjects and patients when applied to the visual field of mfERG or mfVEP recordings. The method may prove helpful in diagnosing any disease that is identifiable in patients' mfERG or mfVEP recordings and is extensible to other clinical tests, such as optical coherence tomography.

Functional Evaluation of the Visual Pathway in Patients with Multiple Sclerosis Using a Multifunction Stimulator Monitor.

OBJECTIVES: To assess the capability of the vision monitor unit Monpack One of detecting visual function alterations in patients with multiple sclerosis (MS) and to evaluate the correlation between structural retinal parameters and functional measurements obtained with this device. METHODS: Forty-eight patients with MS and 46 healthy controls were included in a cross-sectional study. All participants underwent a complete functional evaluation of the visual pathway, which included low-contrast visual acuity (LCVA), contrast sensitivity vision (CSV), automated perimetry, multifocal visual evoked potentials (mfVEPs), and pattern electroretinogram (ERG). All tests were performed using the vision monitor unit Monpack One (Metrovision, France), a multifunction stimulator device. Retinal structural measurements were obtained in all subjects using Triton swept source optical coherence tomography (Topcon, Japan). RESULTS: Patients with MS presented reduced low-contrast VA (p < 0.001) and reduced CSV at medium (p=0.001, p=0.013) and low (p=0.001, p=0.002) spatial frequencies. All visual field parameters were found to be altered in MS patients compared with controls (≤0.001). Patients with MS presented lower amplitude of the P100 waveform of the mfVEP in areas corresponding to central (p < 0.001), inferonasal (p=0.001), and inferotemporal (p=0.003) retina. The pattern ERG did not show significant differences. Significant correlations were observed between structural retinal measurements and functional parameters, especially between the inner macular areas and measurements corresponding to contrast sensitivity and perimetry indexes. CONCLUSIONS: Patients with MS present visual dysfunction detectable with the vision monitor unit Monpack One. This device may be a fast and useful tool to provide a full evaluation of axonal damage in patients with multiple sclerosis.

Visual dysfunction and its correlation with retinal changes in patients with Alzheimer's disease.

AimTo evaluate visual dysfunction and its correlation with structural changes in the retina in patients with Alzheimer's disease (AD).MethodsPatients with AD (n=24) and controls (n=24) underwent evaluation of visual acuity (VA), color vision (using the Farnsworth and L'Anthony desaturated (D) 15 color tests), and contrast sensitivity vision (CSV; using the Pelli-Robson chart and CSV-1000E test) to measure visual dysfunction. Structural measurements of the retinal nerve fiber layer (RNFL) and macular thickness were obtained using spectral domain-optical coherence tomography (SD-OCT).ResultsCSV at three of the four spatial frequencies was significantly worse in AD patients than in controls. Color vision was significantly affected in AD patients based on the Farnsworth color test. Compared with controls, macular thinning was detected in all sectors except the fovea, and the RNFL exhibited significant thinning in the superior quadrant and lower average thickness (P<0.05). CSV was the functional parameter most strongly correlated with structural measurements in patients with AD. Color vision was strongly associated with macular volume (r>0.70, P<0.05). VA at different levels of contrast was associated with macular and RNFL thickness.ConclusionsPatients with AD had visual dysfunction that correlated with structural changes evaluated by SD-OCT. Macular measurements may be reliable indicators of visual impairment in AD patients.

Ethylene biosynthesis and perception during ripening of loquat fruit (Eriobotrya japonica Lindl.).

In order to gain insights into the controversial ripening behavior of loquat fruits, in the present study we have analyzed the expression of three genes related to ethylene biosynthesis (ACS1, ACO1 and ACO2), two ethylene receptors (ERS1a and ERS1b), one signal transduction component (CTR1) and one transcription factor (EIL1) in peel and pulp of loquat fruit during natural ripening and also in fruits treated with ethylene (10µLL-1) and 1-MCP (10µLL-1), an ethylene action inhibitor. In fruits attached to or detached from the tree, a slight increase in ethylene production was detected at the yellow stage, but the respiration rate declined progressively during ripening. Accumulation of transcripts of ethylene biosynthetic genes did not correlate with changes in ethylene production, since the maximum accumulation of ACS1 and ACO1 mRNA was detected in fully coloured fruits. Expression of ethylene receptor and signaling genes followed a different pattern in peel and pulp tissues. After fruit detachment and incubation at 20°C for up to 6days, ACS1 mRNA slightly increased, ACO1 experienced a substantial increment and ACO2 declined. In the peel, these changes were advanced by exogenous ethylene and partially inhibited by 1-MCP. In the pulp, 1-MCP repressed most of the changes in the expression of biosynthetic genes, while ethylene had almost no effects. Expression of ethylene perception and signaling genes was barely affected by ethylene or 1-MCP. Collectively, a differential transcriptional regulation of ethylene biosynthetic genes operates in peel and pulp, and support the notion of non-climacteric ripening in loquat fruits. Ethylene action, however, appears to be required to sustain or maintain the expression of specific genes.

Visual dysfunction and its correlation with retinal changes in patients with Parkinson's disease: an observational cross-sectional study.

OBJECTIVES: To evaluate visual dysfunction and its correlation with structural changes in the retina in patients with Parkinson's disease (PD). METHODS: Patients with PD (n=37) and controls (n=37) were included in an observational cross-sectional study, and underwent visual acuity (VA), colour vision (using the Farnsworth and Lanthony desaturated D15 colour tests) and contrast sensitivity vision (CSV; using the Pelli-Robson chart and CSV 1000E test) evaluation to measure visual dysfunction. Structural measurements of the retinal nerve fibre layer (RNFL), and macular and ganglion cell layer (GCL) thicknesses, were obtained using spectral domain optical coherence tomography (SD-OCT). Comparison of obtained data, and correlation analysis between functional and structural results were performed. RESULTS: VA (in all different contrast levels) and all CSV spatial frequencies were significantly worse in patients with PD than in controls. Colour vision was significantly affected based on the Lanthony colour test. Significant GCL loss was observed in the minimum GCL+inner plexiform layer. A clear tendency towards a reduction in several macular sectors (central, outer inferior, outer temporal and superior (inner and outer)) and in the temporal quadrant of the RNFL thickness was observed, although the difference was not significant. CSV was the functional parameter most strongly correlated with structural measurements in PD. Colour vision was associated with most GCL measurements. Macular thickness was strongly correlated with macular volume and functional parameters (r>0.70, p<0.05). CONCLUSIONS: Patients with PD had visual dysfunction that correlated with structural changes evaluated by SD-OCT. GCL measurements may be reliable indicators of visual impairment in patients with PD.

Patterns of individual change in a parenting program for child maltreatment and their relation to family and professional environments.

In the area of child maltreatment prevention, little is known about the typology of changes that individuals undergo in response to parent-training interventions. In this study, we examined the patterns of change observed in parents immediately after their completion of the Apoyo Personal y Familiar (APF, Personal and Family Support) parenting program. We identified five clusters and classified 496 parents according to two criteria: (a) the amount of pre-post changes (total or partial) as reflected in their self-reports on implicit theories, parental agency and childrearing practices, and (b) the positive, negative or mixed character of these changes. The study also included a follow-up of a subset of 95 participants intended to examine the extent to which the patterns of change identified in the first part of the study might predict the quality of the childrearing environment at home and the successful integration of the APF program into social services structures one year on. In this follow-up study, external evaluators observed families' home environments and collected the parenting program facilitators' self-reports on changes to their work environment. The evaluators found higher-quality childrearing environments and more positive appraisals of the changes to the teams' work with families in those cases where participants had experienced partial or total positive changes as a result of the APF. This approach offers insights into processes of individual change that have practical implications for the successful implementation of parenting programs in child maltreatment prevention services.

Infiltrados pulmonares en paciente en hemodiálisis y tratamiento inmunosupresor / No disponible

No disponible

Immunoquantitative measurement of soybean aeroallergen emissions at industrial sites.

BACKGROUND: Asthma attacks and mortality due to inhalation of soybean antigens in Barcelona, Spain have been well documented. A new control scheme was adopted in the city to avoid the emission and dispersion of soybean dust into the atmosphere during unloading. We studied soybean allergen emission during unloading and at 3 industrial sites and compared the results obtained. METHODS: Over a period of 31 months, 628 paired air samples from 3 plants (A, B, C) involved in soybean manipulation in Barcelona harbor were collected. Samples were analyzed by a radiometric competitive inhibition assay (RCIA) and the enzyme-linked immunosorbent assay (ELISA). A Bland-Altman plot was used to compare the soybean concentrations measured by each assay. RESULTS: The median values for the 628 samples were 5535 U/m3 (range, 370-18,416,751) for the RCIA and 9955 U/m3 (range, 400-22,349,059) for the ELISA. Plant A had the lowest emission levels and the lowest Spearman rank correlation coefficient (0.409). The correlation coefficients were 0.747 and 0.794 for plants B and C. Soybean aeroallergen concentrations differed by plant. The highest variability in values was seen for plant A, which had the lowest allergen concentrations. CONCLUSIONS: The competitive assays described are useful tools for the measurement of soybean allergen emission levels at industrial sites. These methods may be used to monitor unloading and the impact of environmental interventions.

Assessment of soy aeroallergen levels in different work environments.

BACKGROUND: Airborne soybean hull proteins are known causes of asthma epidemics around harbours and soy processing plants. Soy flour dust proteins may cause occupational allergy in food and feed industries. OBJECTIVE: To compare enzyme immunoassays (EIAs) for soy hull and soy flour aeroallergens, exposure assessment in various work environments. METHODS: Airborne dust samples (n=324) from soy unloading and/or processing plants, the animal feed industry and pig stables were analysed by two soy flour assays: one assay for measuring complete soy hull proteins and two assays for measuring the purified low-molecular-weight (LMW) soy hull allergens. RESULTS: Immunoblotting confirmed strong differences between antibody specificities and soy preparations. The results of the two soy flour assays and the assay for measuring complete soy hull proteins were highly correlated (r>0.85). The two LMW soy hull assays also showed a strong mutual correlation (r=0.91), but much less correlation with assays for measuring soy flour and complete soy hull. The levels of LMW soy hull proteins were the highest at sites of soybean unloading or processing, while soy flour levels were particularly high in the soy and animal feed industry. CONCLUSIONS: The optimal EIA procedure for soy aeroallergen exposure assessment depends on the type of work environment and the local soy dust composition. Thus, the type of work environment should always be taken into account in future soy allergy studies in order to prevent a possible underestimation of the workers' actual risk of developing soy allergy.

Optimized methods for fungal alpha-amylase airborne exposure assessment in bakeries and mills.

BACKGROUND: In order to enable reproducible and comparable exposure measurements of fungal alpha-amylase (alpha-amylase) in different laboratories and countries, the entire procedure from sampling of airborne dust to measuring extracted samples (including standards and the used enzyme) immunoassays must be standardized. The aim of this study was to establish optimal elution and assay conditions. METHODS: A parallel sampler was used for simultaneous collection of 10 samples of inhalable dust in bakeries and mills in Germany, England, the Netherlands and Spain. Three enzyme-immunoassays (EIAs) for detection of fungal alpha-amylase based on monoclonal antibodies or polyclonal antibodies were used for the measurement of the parallel-sampled filters (n=432) extracted using several methods. The results were analysed by regression analysis of variance. Additional filters (n=54) were extracted and analysed using two EIAs to investigate the storage stability of the extracts. RESULTS: Although alpha-amylase concentrations correlated well (r> or =0.88), differences were found between the EIAs in the sensitivity and nominal values (up to a mean factor 5.8). The best elution medium for airborne filters (phosphate-buffered saline 'PBS' with 0.05% Tween-20) led to 1.2 to two times higher alpha-amylase allergen yields than extraction in PBS only, while higher Tween-20 concentrations decreased the extracted alpha-amylase yield. During storage of frozen dust/filter extracts for 3-4 months at -20 degrees C, a loss of approximately 40% of measurable alpha-amylase was observed, which could be partially prevented by addition of 0.1% casein to the medium directly after extraction. CONCLUSION: Although the effects of only a few of many possible causes of variation were investigated, for these factors a clear choice could be made with regard to optimal elution conditions and the use of validated EIAs with calibrated standards, thus making significant progress towards a completely standardized procedure for airborne alpha-amylase measurements.