Compliance with hand sanitizer quality during the SARS-CoV-2 pandemic: Assessing the impurities in an ethanol plant.

Using alcohol-based disinfectants is an effective method for preventing the spread of COVID-19. However, non-traditional manufacturers of alcohol-based disinfectants, such as ethanol plants, need to undergo additional treatment to curb their impurities to limits set by the Food and Drug Association (FDA) to produce alcohol-based disinfectants. To transform them to disinfectant-grade alcohol, 17 process streams in a dry-mill ethanol plant were analyzed to determine the quality parameters for acetaldehyde, acetal, propanol, methanol, and water, including chemical oxygen demand, total suspended solids, and nutrients. Results suggest that the process stream generated by the distillation column requires further treatment because the acetaldehyde and acetal concentrations are significantly higher than the impurity limit set by the FDA. The addition of a second distillation column could be a potential method for addressing impurities and it will have minimal influence on hazardous air pollutant generation and water use.

An automated sample preparation approach for routine liquid chromatography tandem-mass spectrometry measurement of the alcohol biomarkers phosphatidylethanol 16:0/18:1, 16:0/16:0 and 18:1/18:1.

BACKGROUND: Phosphatidylethanols (PEths) are currently under investigation as highly sensitive and specific direct biomarkers of long-term alcohol abuse. PEths belong to a group of aberrant phospholipids formed in erythrocyte membranes in presence of ethanol by the catalytic action of the enzyme phospholipase D on phosphatidylcholine. Compared to other alcohol biomarkers, a higher sensitivity (94.5-100%) and specificity (100%) characterizes PEth species. METHOD: Prior to detection, an important practical aspect in the work-flow of PEths analysis is the sample preparation step. To date, traditional techniques such as liquid-liquid extraction (LLE) and solid phase extraction (SPE) require multiple steps to remove blood interferences. Due to the simplicity of use and the possibility of automation, sample filtration is also a widespread technique in biomedical laboratories. In this work, a reliable sample preparation method based on an automated filtration with Phree™ Phospholipid Removal Plates (Phenomenex, California, USA) was developed to extract PEths from human whole blood. Surface characteristics of Phospholipids Removal material allow phospholipids retention on the filter and a suitable PEths recovery after elution. The blood samples were added with internal standard (IS) and purified in acetonitrile (1 mL). After centrifugation, supernatants were applied to the Phospholipids Removal Plates in an automated workstation. After washing, the phospholipids retained on the filter were eluted with 1-mL 2-propanol 1% ammonia. PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 were extracted using the proposed method and detected by LC-MS/MS operated in electron spray ionization (ESI). The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. This method was validated for the quantitative profiling of PEth molecular species in human blood collected from heavy and social drinkers. RESULTS: The method was validated according to Food and Drug Administration (FDA) guidelines. Linearity was observed in the 25-1250 (PEth 16:0/18:1) and 5-250 (PEth 16:0/16:0 and PEth 18:1/18:1) ng/mL range with a correlation coefficient (r²) between 0.997 and 0.999 for all three compounds. Moreover, the nominal concentrations of non-zero calibrators were ±15%. Variation coefficient (%CV) was < 10% for all the analytes, while lowest limit of quantitation (LLOQ) was found to be 1.25 ng/mL for PEth 16:0/18:1, 0.50 ng/mL for PEth 16:0/16:0 and 0.50 ng/mL for PEth 18:1/18:1. Intra- and inter-day precision and accuracy were always lower than 14% and 11%, respectively. Analytical recovery was higher than 68.8% for all analytes. Sample stability at 4 °C and -20 °C showed a concentration drop lower than 20% up to 4 weeks. Extracts were stable for 7 days in the autosampler and 30 days at -20 °C and 4 °C in a closed vial. The procedure was successfully applied to blood samples collected from heavy drinkers (n = 8), social drinkers (n = 5), and teetotalers (n = 7). CONCLUSIONS: Due to the simplicity of application and the possibility of automation, sample filtration is well suited for a clinical and forensic laboratory. To monitor alcohol consumption, an analytical method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) with novel and automated sample preparation was developed and validated for the simultaneous quantification of PEth 16:0/18:1, PEth 16:0/16:0 and PEth 18:1/18:1 in whole blood samples, characterized by a fast sample preparation and lower pre-analysis costs than other extraction procedures.

Green and fast determination of the alcoholic content of wines using thermal infrared enthalpimetry.

An innovative use of thermal infrared enthalpimetry (TIE) is proposed for the determination of alcoholic content of red and white wines. Notwithstanding the presence of ethanol in beverages, absolute ethanol was added directly to wines, and the temperature rise caused by the heat of dilution was monitored using an infrared camera. Analytical signals were obtained in only 10 s for four samples simultaneously, and a calibration curve was constructed with hydroalcoholic reference solutions. A linear calibration curve was obtained from 3.0 to 18.0% (v/v) ethanol (R2 = 0.9987). The results showed agreement ranging from 98.2 to 104.0% with 942.06 and 969.12 methods of AOAC. Organic compounds (e.g., sugar) did not interfere in the determinations. The proposed method provided fast results, with a throughput of 480 samples per hour and negligible energy consumption (0.001 kWh). In addition, the consumption of reagents was reduced when compared with conventional method fulfilling green analytical chemistry requirements.

Measurement uncertainty of blood ethanol concentration in drink-driving cases in an emergency laboratory.

INTRODUCTION: The quality of blood ethanol concentration (BEC) determination is important because of its legal ramifications. Measurement uncertainty provides quantitative information about the quality and reliability of test results. In this study, we aim to calculate the measurement uncertainty for the ethanol test in our laboratory measured with a Synchron Systems Ethanol assay kit by employing an enzymatic rate method on the Beckman-Coulter Olympus AU400 auto analyzer (Beckman Coulter Inc, Melville, USA). MATERIALS AND METHODS: The measurement uncertainty values were calculated in accordance to the Nordtest guidelines. All vehicle drivers involved in a traffic accident were retrospectively inspected for the BEC test conducted during July to December 2016 in our emergency laboratory. RESULTS: A 1034 vehicle drivers had their BEC tested. The results for 181 drivers were > 0.50 g/L and reported as positive. The serum ethanol concentration in those showing a positive result was 2.04 ± 1.01 g/L, over four times the legal limit. The median BEC in those showing a negative result was 0.03 (IQR: 0.03) g/L. The expanded uncertainty obtained was 19.74%. When measurement uncertainty values were added to the results of the 1034 drivers who were retrospectively screened, eight vehicle drivers had results with 95% confidence intervals that exceeded the legal limit 0.50 g/L. CONCLUSIONS: The BEC test results for vehicle drivers with values close to legal limits should be reported as the obtained ethanol concentration with corresponding measurement uncertainty.

Chemical data as markers of the geographical origins of sugarcane spirits.

In an attempt to classify sugarcane spirits according to their geographic region of origin, chemical data for 24 analytes were evaluated in 50 cachaças produced using a similar procedure in selected regions of Brazil: São Paulo - SP (15), Minas Gerais - MG (11), Rio de Janeiro - RJ (11), Paraiba -PB (9), and Ceará - CE (4). Multivariate analysis was applied to the analytical results, and the predictive abilities of different classification methods were evaluated. Principal component analysis identified five groups, and chemical similarities were observed between MG and SP samples and between RJ and PB samples. CE samples presented a distinct chemical profile. Among the samples, partial linear square discriminant analysis (PLS-DA) classified 50.2% of the samples correctly, K-nearest neighbor (KNN) 86%, and soft independent modeling of class analogy (SIMCA) 56.2%. Therefore, in this proof of concept demonstration, the proposed approach based on chemical data satisfactorily predicted the cachaças' geographic origins.

A simple visual ethanol biosensor based on alcohol oxidase immobilized onto polyaniline film for halal verification of fermented beverage samples.

A simple visual ethanol biosensor based on alcohol oxidase (AOX) immobilised onto polyaniline (PANI) film for halal verification of fermented beverage samples is described. This biosensor responds to ethanol via a colour change from green to blue, due to the enzymatic reaction of ethanol that produces acetaldehyde and hydrogen peroxide, when the latter oxidizes the PANI film. The procedure to obtain this biosensor consists of the immobilization of AOX onto PANI film by adsorption. For the immobilisation, an AOX solution is deposited on the PANI film and left at room temperature until dried (30 min). The biosensor was constructed as a dip stick for visual and simple use. The colour changes of the films have been scanned and analysed using image analysis software (i.e., ImageJ) to study the characteristics of the biosensor's response toward ethanol. The biosensor has a linear response in an ethanol concentration range of 0.01%-0.8%, with a correlation coefficient (r) of 0.996. The limit detection of the biosensor was 0.001%, with reproducibility (RSD) of 1.6% and a life time up to seven weeks when stored at 4 °C. The biosensor provides accurate results for ethanol determination in fermented drinks and was in good agreement with the standard method (gas chromatography) results. Thus, the biosensor could be used as a simple visual method for ethanol determination in fermented beverage samples that can be useful for Muslim community for halal verification.

Álcool

O Etanol (ch3 ch2oh), também chamado álcool etílico e, na linguagem corrente, simplesmente álcool, é uma substância orgânica obtida da fermentação de açucáres, hidratação do

Characterization of certified reference material for the quantification of water in bioethanol.

The National Metrology Institute of Japan has issued a certified reference material of bioethanol (NMIJ CRM 8301-a) for the quantification of water, methanol, sulfur, and copper. This paper presents technical details for the characterization of the water in NMIJ CRM 8301-a. The characterization was performed using coulometric and volumetric Karl-Fischer (KF) titrations. To reduce moisture absorption, sample handling and KF titration were performed in a glove box under a dried nitrogen atmosphere. In addition, a rubber cap with a three-way valve was attached to the ampoule immediately after opening so as to minimize the influence of moisture. Sample aliquots were obtained using a gas-tight microsyringe through the valve, and injected into the KF cell as soon as possible. The certified value of water obtained from coulometric and volumetric KF titrations was 1.688 mg g(-1), and the expanded uncertainty (coverage factor, k = 2) was 0.028 mg g(-1). This CRM would be suitable for the monitoring of water in bioethanol and similar matrices.

Moção de apoio nº 007, de 28 de junho de 2012: vem a público manifestar apoio para aprovação do Substitutivo da Comissão de Defesa do Consumidor da Câmara dos Deputados para o Projeto de Lei nº 692/2007 e seus apensados, cujo substitutivo dispõe sobre restrições do acesso e venda do álcool líquido de uso doméstico e se encontra em discussão na Comissão de Constituição e Justiça e de Cidadania da Câmara dos Deputados, sob a relatoria da deputada federal Sandra Rosado / Supporting motion No. 007, dated June 28, 2012: public support for approval of the Substitute for the Consumer Protection Committee of the Chamber of Deputies for Bill 692/2007 and its members, whose substitute provides for restrictions access and sale of liquid alcohol for domestic use and is being discussed by the Constitution and Justice and Citizenship Commission of the Chamber of Deputies, un

Vem a público manifestar apoio para aprovação do Substitutivo da Comissão de Defesa do Consumidor da Câmara dos Deputados para o Projeto de Lei nº 692/2007 e seus apensados, cujo substitutivo dispõe sobre restrições do acesso e venda do álcool líquido de uso doméstico e se encontra em discussão na Comissão de Constituição e Justiça e de Cidadania da Câmara dos Deputados, sob a relatoria da deputada federal Sandra Rosado.

[The effectiveness of hand hygiene products on MRSA colonization of health care workers by using CHROMagar MRSA]. / El Hijyen Ürünlerinin Saglik Çalisanlarinda MRSA Kolonizasyonuna Etkilerinin CHROMagar MRSA ile Arastirilmasi

The aims of this study were; to investigate the hand hygiene compliance of the health care workers (HCWs) during their routine patient care, to determine the methicillin-resistant Staphylococcus aureus (MRSA) hand colonization of the HCWs, to investigate the effect of different hand hygiene products on MRSA colonization and to evaluate the effectiveness of chromogenic agar for detecting MRSA. HCWs were investigated during their routine patient care and hand cultures were taken before and after hand wash/hygiene. Two different techniques were used to obtain the hand cultures: fingertip method (CHROMagar MRSA containing HygiSlide); and direct swab method and then inoculation to CHROMagar MRSA media. MRSA strains grown on those cultures were confirmed with conventional methods. A total of 100 HCWs (of them 61 were female; mean age: 32.7 ± 5.2 years; age range: 25-51 years) involving physicians (n= 33), nurses (n= 38) and health care assistants (n= 29), were included in the study. MRSA was detected in 39% and 11% before hand hygiene and in 13% and 6% after hand hygiene, with HygiSlide CHROMagar media and with CHROMagar in plate media, respectively. No difference were found regarding clinics, occupations, or the type of patient handling in those HCWs who were positive (n= 13) for MRSA colonization following hand hygiene, and those who were negative (n= 26). However, the type of the hand hygiene product used exhibited a statistical difference. None of the seven HCWs who used alcohol based hand rub revealed growth in the second culture while 10 of 19 (53%) HCWs who used soap and three of 13 (23%) HCWs who used chlorhexidine were still colonized with MRSA. In terms of reduction in the MRSA counts, the most effective one was the alcohol based hand rub while the soap was the least, since seven of 19 (37%) HCWs who used soap showed no reduction at all in the MRSA counts. A high ratio of hand colonization with MRSA was detected in our hospital staff (39%). It was shown that the colonization could be reduced significantly (with a rate of 66%) with hand hygiene. Alcohol based hand rub was found to be the most effective method in hand hygiene. The fingertip technique was found to be superior to inoculation to plate media for obtaining hand cultures and CHROMagar MRSA media was found to be rapid, effective and practical for detecting the MRSA hand colonization.

Benefits of ß-xylanase for wheat biomass conversion to bioethanol.

BACKGROUND: The efficiency of bioethanol production from wheat biomass is related to the quality of end products as well as to safety criteria of co-products such as distiller's dried grains with solubles (DDGS). The inclusion of a new biocatalyst for non-starch polysaccharide degradation in fermentation processes could be one of the solutions. The objective of this study was to evaluate the influence of ß-xylanases in combination with traditional amylolytic enzymes on the efficiency of bioethanol production and DON detoxification during fermentation of Fusarium-contaminated wheat biomass with high concentration of deoxynivalenol (DON; 3.95 mg kg(-1)). RESULTS: The results showed that the negative effect of Fusarium spp. on yield and quality of bioethanol could be eliminated by the application of Trichoderma reesei xylanase in combination with amylolytic enzymes. This technological solution allowed to increase the concentration of ethanol in the fermented wort by 35.3% and to improve the quality of bioethanol by decreasing the concentrations of methanol, methyl acetate, isoamyl and isobutyl alcohols. Mass balance calculations showed that DDGS was the main source of DON contamination, comprising 74% of toxin found in wheat biomass. By using new enzyme combination for wheat biomass saccharification, a higher level of detoxification (41%) of DON was achieved during the fermentation process. CONCLUSION: The addition of Trichoderma reesei xylanase played a positive role in bioethanol production from Fusarium-contaminated wheat biomass, indicating that the yeast-growing medium was enriched during the enzymatic treatment.

Efficacy of alcohol-based hand sanitizer on hands soiled with dirt and cooking oil.

Handwashing education and promotion are well established as effective strategies to reduce diarrhea and respiratory illness in countries around the world. However, access to reliable water supplies has been identified as an important barrier to regular handwashing in low-income countries. Alcohol-based hand sanitizer (ABHS) is an effective hand hygiene method that does not require water, but its use is not currently recommended when hands are visibly soiled. This study evaluated the efficacy of ABHS on volunteers' hands artificially contaminated with Escherichia coli in the presence of dirt (soil from Tanzania) and cooking oil. ABHS reduced levels of E. coli by a mean of 2.33 log colony forming units (CFU) per clean hand, 2.32 log CFU per dirt-covered hand, and 2.13 log CFU per oil-coated hand. No significant difference in efficacy was detected between hands that were clean versus dirty or oily. ABHS may be an appropriate hand hygiene method for hands that are moderately soiled, and an attractive option for field settings in which access to water and soap is limited.

[Correction of the alkylnitrite method for the detection of ethanol in blood taking into account toxico-kinetic and anthropometric data].

The correction coefficient for the systemic inaccuracy arising during determination of blood ethanol by alkylnitrite gas chromatography and concomitant calibration of aqueous solutions was estimated to equal 0.82; this finding was confirmed by the results of the toxico-kinetic assay for the measurement of total body water (TBW) from the kinetic curve characterizing the time dependence of ethanol concentration in the exhaled air, saliva, capillary and venous blood in combination with 4 anthropometric methods and (in several cases) direct physical detection of TBW. When detecting the blood ethanol level with a correction coefficient of 0.82, the mutual position of the kinetic curves for ethanol concentrations in the blood and the exhaled air (recalculated for the blood level with a coefficient of 2100) as well as in the blood and saliva agreed with that reported in the available literature; it significantly differed from the position of the curves obtained with a correction coefficient of 0.95. The causes accounting for the systematic inaccuracy and erroneous values of the correction coefficients in earlier studies are discussed.

Safety and efficacy of high-dose fomepizole compared with ethanol as therapy for ethylene glycol intoxication in cats.

OBJECTIVE: To determine the safety and efficacy of high-dose fomepizole compared with ethanol (EtOH) in cats with ethylene glycol (EG) toxicosis. DESIGN: Prospective study. SETTING: University veterinary research laboratory. ANIMALS: Thirteen cats. INTERVENTIONS: Two cats received injections of high-dose fomepizole (Study 1). Three cats received lethal doses of EG and fomepizole treatment was initiated 1, 2, or 3 hours later (Study 2). Eight cats received a lethal dose of EG and were treated with fomepizole or EtOH (Study 3). Cats treated with fomepizole received 125 mg/kg IV initially, then 31.25 mg/kg at 12, 24, and 36 hours. Cats treated with EtOH received 5 mL of 20% EtOH/kg IV initially, then every 6 hours for 5 treatments, then every 8 hours for 4 treatments. Cats also received fluids and supportive therapy as needed. MEASUREMENTS AND MAIN RESULTS: Clinical signs were monitored and serial blood analyses performed. Cats receiving fomepizole experienced mild sedation but no biochemical evidence of toxicity. Cats receiving fomepizole for EG intoxication survived if therapy was initiated within 3 hours of EG ingestion. One of the 6 developed acute renal failure (ARF) but survived. Only 1 of the 3 cats treated with EtOH 3 hours following EG ingestion survived; 2 developed ARF and were euthanized. Cats treated 4 hours following EG ingestion developed ARF, whether treated with EtOH or fomepizole. CONCLUSIONS: Fomepizole is safe when administered to cats in high doses, prevents EG-induced fatal ARF when therapy is instituted within 3 hours of EG ingestion, and is more effective than treatment with EtOH.

Estimating alcohol content of traditional brew in Western Kenya using culturally relevant methods: the case for cost over volume.

Traditional homemade brew is believed to represent the highest proportion of alcohol use in sub-Saharan Africa. In Eldoret, Kenya, two types of brew are common: chang'aa, spirits, and busaa, maize beer. Local residents refer to the amount of brew consumed by the amount of money spent, suggesting a culturally relevant estimation method. The purposes of this study were to analyze ethanol content of chang'aa and busaa; and to compare two methods of alcohol estimation: use by cost, and use by volume, the latter the current international standard. Laboratory results showed mean ethanol content was 34% (SD = 14%) for chang'aa and 4% (SD = 1%) for busaa. Standard drink unit equivalents for chang'aa and busaa, respectively, were 2 and 1.3 (US) and 3.5 and 2.3 (Great Britain). Using a computational approach, both methods demonstrated comparable results. We conclude that cost estimation of alcohol content is more culturally relevant and does not differ in accuracy from the international standard.

Novel method for identification and quantification of methanol and ethanol in alcoholic beverages by gas chromatography-Fourier transform infrared spectroscopy and horizontal attenuated total reflectance-Fourier transform infrared spectroscopy.

Numerous methods are being used to identify and quantify methanol and ethanol in alcoholic beverages, including country liquors. Some of the known methods are density and refractive index measurements, and spectrophotometric measurements using Schiff's reagent or chromatropic acid. Other advanced techniques involve head space gas chromatography (GC), GC-flame ionization detection, high-performance liquid chromatography, enzymatic reactions, and biosensors. However, identification and quantification of methanol and ethanol in beverages can be accurately done using GC-Fourier transform infrared spectroscopy (FTIR) and horizontal attenuated total reflectance (HATR)-FTIR. Identification of alcohols is possible from library matching of the IR spectra obtained from GC-FTIR. In water, methanol and ethanol show a very strong peak for C-O, stretching at 1015.3 and 1044.2 cm(-1), respectively. The strong absorption of vibrational stretching frequency of C-O present in alcohols was used for quantification purposes. The absorptions of C-O group frequency of alcohols in water mixtures were measured using HATR-FTIR with a zinc-selenide crystal. Samples were placed directly on the HATR crystal, with alcohol concentrations ranging from 0.2 to 50.0% (v/v). The plot of absorptions against concentrations of methanol and ethanol obeyed Beer's law (r2 = 0.9998 and 0.9987, respectively), from which alcohol in the mixtures was quantified. Propan-2-ol and n-butanol showed no interference. The method is validated from absorption measurements of known mixtures of standard ethanol in water. This is a simple, specific, rapid, accurate, and nondestructive method of identification and quantification of methanol and ethanol in mixtures. It can be used to ascertain methanol contamination in alcoholic beverages that can lead to death or methanol poisoning by alcohol consumption.

Quality of alcohol-based hand disinfectants and their regulatory status. Production, sales and post-marketing surveillance.

A high standard of hand hygiene is achieved by developing, producing and distributing hand disinfectants compliant with the German law for medicinal products. This ensures optimal protection of patients and staff from infections. In addition all local requirements are automatically fulfilled independent of the place within which the product is being used. It is shown that continuous improvement can be stimulated by intensive cooperation between the customer and supplier to ensure that customer expectations are met.

Evaluation of the value of frozen tissue section used as "gold standard" for immunohistochemistry.

To examine the use of acetone- or ethanol-fixed frozen tissue sections as the "gold standard" for immunohistochemical analysis, we evaluated frozen sections with various conditions of fixation and antigen retrieval (AR). Fresh human tissues were frozen in OCT. An adjacent tissue block was fixed in 10% neutral buffered formalin (NBF) and paraffin embedded (FFPE). Frozen sections were fixed by 6 protocols: acetone, ethanol, NBF (2 durations), and NBF + calcium chloride (2 durations). AR was used for all NBF-fixed sections. More than half of the antibodies (16/26 [62%]) showed immunohistochemical results indistinguishable between acetone- and NBF-fixed sections; 8 (31%) showed better immunohistochemical signals following NBF and AR; 2 gave better immunohistochemical results for acetone-fixed sections. Most cytoplasmic proteins (10/13) showed comparable immunohistochemical signals between acetone- and NBF-fixed sections. For nuclear proteins, NBF-fixed sections gave better immunohistochemical signals than did acetone-fixed sections. In most cases, NBF yielded stronger signals with less background and better morphology. The data do not support the use of acetone-fixed frozen tissue sections as the gold standard for immunohistochemical analysis. In evaluating new antibodies, a combination of acetone- and NBF-fixed frozen sections should be used, although in practice, FFPE tissue sections may serve as the standard for most antigens for immunohistochemical analysis.

Evaluation of the post-rotational nystagmus test (PRN) in determining alcohol intoxication.

This study evaluated the accuracy of the post-rotational nystagmus test (PRN) on the basis of the results of 1006 PRN tests performed at the Institute for Forensic Medicine in Ljubljana between 1998 and 2002 during standardized medical examinations in cases of suspected drunk driving. The evaluation of PRN test results with blood alcohol concentration (BAC) as a reference was based on classification into the following categories and characteristics: true positives (TP), true negatives (TN), false positives (FP), false negatives (FN), sensitivity, specificity, positive predictive values (PPV), negative predictive values (NPV), and accuracy. An optimal cut-off value of 10 s for post-rotational nystagmus time was chosen with the help of a receiver operating characteristic (ROC) curve for the BAC limit of 0.5 g/kg. The results of the decision analyses were: TP = 584, FP = 43, FN = 229, TN = 150, sensitivity = 0.718, specificity = 0.777, PPV= 0.931, NPV= 0.396, and accuracy = 0.730. The area under the ROC curve (AUC) was 0.813. Based on the AUC, the post-rotational nystagmus test is a good test for predicting alcohol intoxication over 0.5 g/kg. As a part of the physician's examination, it contributes significantly to the description of the clinical state.

Evaluation of chromatin integrity in human sperm using acridine orange staining with different fixatives and after cryopreservation.

Staining of cells with acridine orange (AO) has been widely accepted as a predictor of DNA damage in many cell types. Because of variability of protocols used in previous studies, the AO staining technique has not been widely accepted as a screening test to predict DNA damage in human sperm. In order to further validate the use of AO staining, sperm were evaluated using numerous variations in the staining protocol. This study also elucidated the effects of cryopreservation on sperm DNA. Sperm fixation in Carnoy's solution showed significantly (P < 0.05) more DNA damage (29.9 +/- 4.5%) than 2% glutaraldehyde (14.4 +/- 2.1%), 4% paraformaldehyde (5.5 +/- 1.7%), no fixation (15.8 +/- 4.3%) but did not differ from Diff Quik solution (19.2 +/- 5.8%). No difference was observed for sperm DNA damage assessment using a 0.2 m (15.5 +/- 3.2%) or 0.3 m (14.9 +/- 3.3%) concentration of Na(2)HPO(4).7H(2)O in the AO staining solution. Frozen-thawed semen samples showed increased damage to sperm DNA under both Carnoy's (fresh: 10.9 +/- 1.3%; frozen: 30.8 +/- 2.9%; P < 0.05) and Diff Quik fixation (fresh: 6.2 +/- 0.8; frozen: 17.1 +/- 2.5%P < 0.05). Present data also showed that spermatozoa from some individuals are more prone to DNA damage after freezing and thawing procedures than others. In conclusion, Carnoy's fixative provides a better predictive value for DNA damage to sperm using AO staining. Additionally, cryopreservation increased damage to the sperm DNA.