Exogenic proteins in the human exhaled breath condensate

The analysis of the protein composition of exhaled breath to diagnose diseases of the respiratory system raises a problem of differentiation proteins of expressed in the tissues of the lungs and respiratory tract (endogenous) and got in the respiratory system from the ambient air in the process of respiration (exogenous). In this work an attempt was made to estimate a set of exhaled exogenic proteins by mass spectrometry coupled with nanoflow HPLC. Six-month isolation of healthy donors indoors with air cleaned of dust leads to removal from the spectrum of exhaled proteins of some keratins that are considered therefore to be exogenic. Non-keratin proteins may also circulate between the ambient air and human respiratory ways, but their concentration appears to be significantly lower the keratin concentrations (especially epidermis keratin). Among non-keratins dermcidin seems to be the most significant exogenic protein of exhaled air. The conclusion of the diagnostic value of exhaled proteins can be done only after careful comparison of the results of quantitative and qualitative analysis of their composition in norm and pathology for a statistically significant sample of donors.     

A new strategy based on real-time secondary electrospray ionization and high-resolution mass spectrometry to discriminate endogenous and exogenous compounds in exhaled breath

Breath is considered to be an easily accessible matrix, whose chemical composition relates to compounds present in blood. Therefore many metabolites are expected in exhaled breath, which may be used in the future for the development of diagnostic methods. In this article, a new strategy to discriminate between exhaled endogenous metabolites and exhaled exogenous contaminants by direct high-resolution mass spectrometry is introduced. The analysis of breath in real-time by secondary electrospray ionization mass spectrometry allows to interpret the origin of exhaled compounds. Exhaled metabolites that originate in the respiratory system show reproducible and significant patterns if plotted in real-time (>1 data point per second). An exhaled metabolite shows a signal that tends to rise at the end of a complete (forced) exhalation. In contrast, exogenous compounds, which may be present in room air, are gradually diluted by the air from the deeper lung and therefore show a trend of falling intensity. Signals found in breath by using this pattern recognition are linked to potential metabolites by comparison with online databases. In addition to this real-time approach, it is also shown how to combine this method with classical analytical methods in order to potentially identify unknown metabolites. Finally exhaled compounds following smoking a cigarette, chewing gum, or drinking coffee were investigated to underline the usefulness of this new approach.     

Proteomic analysis of exhaled breath condensate for diagnostics of respiratory system diseases

Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).     

Desaturation of exhaled air in camels

We have found that camels can reduce the water loss due to evaporation from the respiratory tract in two ways: (1) by decreasing the temperature of the exhaled air and (2) by removal of water vapour from this air, resulting in the exhalation of air at less than 100% relative humidity (r.h.). Camels were kept under desert conditions and deprived of drinking water. In the daytime the exhaled air was at or near body core temperature, while in the cooler night exhaled air wat at or near ambient air temperature. In the daytime the exhaled air was fully saturated, but at night its humidity might fall to approximately 75% r.h. The combination of cooling and desaturation can provide a saving of water of 60% relative to exhalation of saturated air at body temperature. The mechanism responsible for cooling of the exhaled air is a simple heat exchange between the respiratory air and the surfaces of the nasal passageways. On inhalation these surfaces are cooled by the air passing over them, and on exhalation heat from the exhaled air is given off to these cooler surfaces. The mechanism responsible for desaturation of the air appears to depend on the hygroscopic properties of the nasal surfaces when the camel is dehydrated. The surfaces give off water vapour during inhalation and take up water from the respiratory air during exhalation. We have used a simple mechanical model to demonstrate the effectiveness of this mechanism.     

A simple method to sample exhaled NO not contaminated by ambient NO from children and adults in epidemiological studies.

We previously showed that contamination of exhaled air by ambient NO could be avoided by 1 min of breathing and final inhalation of clean air (clean air procedure) prior to exhaled air sampling in balloons. This approach is, however, unsuitable for sampling large groups in epidemiological studies, because it is time consuming and laborious. We therefore discarded the initial part of exhaled air, which may contain ambient NO, in prebags of 250, 540, 775, 1000, and 2000 ml. The subsequent part of exhaled air was sampled in balloons and the NO content was measured. Inflation of a prebag of 500 ml to prevent ambient NO contamination proved to be effective only at low ambient NO levels (<20 ppb). Larger sizes of the prebag (1000 ml for adults and 775 ml for children) are, however, required so that contamination of the air sample at higher levels of ambient NO (up to 115 ppb) is excluded. Using different prebags of gradually increasing size, it was shown that the initial part of exhaled air (<500 ml) contained relatively high amounts of NO that gradually decreased, but attained a constant level in the subsequent air volumes. Using rather large prebags of 2000 and 1000 ml, respectively, in adults and children yielded exhaled NO levels even below those obtained the clean air procedure was applied in combination with a prebag of 540 ml. As this reduction also occurs at ambient NO levels of nearly zero, we suggest that this reduction was due to interference by the water vapor arising from the lowest part of the lungs. In conclusion, the use of a prebag to discard the initial volume of exhaled air ensures accurate measurement of exhaled endogenous NO in large-scale epidemiological studies not biased by ambient NO.     

Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus.

We examined the human cytotoxic T-cell repertoire of nine adults to 9 of the 10 proteins of respiratory syncytial (RS) virus. Peripheral blood mononuclear cells from normal adults were stimulated with RS virus in vitro. The resulting polyclonal cultures were tested for lysis of B-lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing each of nine individual RS virus proteins. The use of peripheral blood dendritic cells to present antigen gave more easily reproducible results over a shorter culture period than conventional methods. The six RS virus proteins most strongly recognized were the nucleoprotein N (nine of nine donors with greater than 10% above background lysis; P = 0.0004), the surface proteins SH (six of nine donors; P = 0.002) and F (five of nine donors; P = 0.008), the matrix proteins M (five of nine donors; P = 0.004) and 22K (three of nine donors; P = 0.01) and the nonstructural protein 1b (six of nine donors; P = 0.004). There was no significant recognition of the major surface glycoprotein G (two of nine donors), the internal phosphoprotein P (one of nine donors), or the nonstructural protein 1c (one of nine donors). Recognition was major histocompatibility complex class I restricted, but no association between major histocompatibility complex phenotype and protein specificity of T cells was seen. Recognition of F and 22K appeared to be associated with recent infection indicated by increased levels of anti-RS virus immunoglobulin G antibody in serum measured by enzyme-linked immunosorbent assay. Since cytotoxic T-cell recognition of RS virus proteins has been demonstrated to be important in the clearance of virus from infected hosts, the N, M, SH, 1b, F, and 22K proteins should be considered potential vaccine components.     

Increased heme catabolism in critically ill patients: correlation among exhaled carbon monoxide, arterial carboxyhemoglobin, and serum bilirubin IXalpha concentrations

It has been reported that exhaled carbon monoxide (CO) concentrations and arterial carboxyhemoglobin (CO-Hb) concentration in blood may be increased in critically ill patients. However, there was no study that examined correlation among amount of CO in exhaled air, CO-Hb concentrations in erythrocytes, and bilirubin IXalpha (BR) in serum, i.e., the three major indexes of heme catabolism, within the same subject. Here, we examined CO concentrations in exhaled air, CO-Hb concentrations in arterial blood, and BR levels in serum in 29 critically ill patients. Measurements of exhaled CO, arterial CO-Hb, and serum total BR have been done in the intensive care unit. As control, exhaled CO concentration was also measured in eight healthy volunteers. A median exhaled CO concentration was significantly higher in critically ill patients compared with control. There was significant correlation between CO and CO-Hb and CO and total BR level. We also found CO concentrations correlated with indirect BR but not direct BR. Multivariate linear regression analysis for amount of exhaled CO concentrations also showed significant correlation with CO-Hb and total BR, despite the fact that respiratory variables of study subjects were markedly heterogeneous. We found no correlation among exhaled CO, patients' severity, and degree of inflammation, but we found a strong trend of a higher exhaled CO concentration in survivors than in nonsurvivors. These findings suggest there is an increased heme breakdown in critically ill patients and that exhaled CO concentration, arterial CO-Hb, and serum total BR concentrations may be useful markers in critically ill conditions.     

Nasal respiratory turbinate function in birds

Nasal respiratory turbinates are complex, epithelially lined structures in nearly all birds and mammals that act as intermittent countercurrent heat exchangers during routine lung ventilation. This study examined avian respiratory turbinate function in five large bird species (115-1,900 g) inhabiting mesic temperate climates. Evaporative water loss and oxygen consumption rates of birds breathing normally (nasopharyngeal breathing) and with nasal turbinates experimentally bypassed (oropharyngeal breathing) were measured. Water and heat loss rates were calculated from lung tidal volumes and nasal and oropharyngeal exhaled air temperatures (T(ex)). Resulting data indicate that respiratory turbinates are equally adaptive across a range of avian orders, regardless of environment, by conserving significant fractions of the daily water and heat budget. Nasal T(ex) of birds was compared to that of lizards, which lack respiratory turbinates. The comparatively high nasal T(ex) of the lizards in similar ambient conditions suggests that their relatively low metabolic rates and correspondingly reduced lung ventilation rates may have constrained selection on similar respiratory adaptations.     

呼出气温度的研究现状

呼出气温度是一种新的呼吸系统疾病监测手段。从被发现开始,呼出气温度就引起了众多学者的兴趣。人们发现在哮喘等呼吸系统疾病患者身上测出的呼出气温度较正常健康人的呼出气温度要高。大量的研究结果表明呼出气温度的变化与患者肺部的炎症改变有关,学者们研究推断肺部的炎症改变使得肺部病变部位局部血管增生及血流量增加因而导致了肺部气道热量交换和损失的改变,最终导致患者呼出气温度的改变。这一发现在呼吸系统疾病中有着重要意义,科学家们认为呼出气温度可以作为一种非侵入性的新监测手段。     

The proteins of the keratin component of bird's beaks.

Birds' beaks have an outer shell of hard keratin which consists almost entirely of proteins which are very rich in glycine [about 30 residues per 100 residues (residues %)], contain moderate levels of tyrosine and serine (each about 8 residues %), and which have relatively low contents of cystine (about 2-5 residues %), lysine, histidine, isoleucine and methionine. Major protein fractions in the S-carboxymethyl form isolated from the beaks of six different orders of birds have similar amino acid compositions, isoelectric points (pH 4-2-4-9) and molecular weights (13,000-14,500). Detailed chromatographic electrophoretic and compositional studies of the proteins of kookaburra beak reveal them to be a family of closely related proteins with only limited heterogeneity, in contrast to mammalian keratin systems. The major kookaburra beak fraction is similar in overall composition and molecular weight to fowl epidermal scale, kookaburra claw and turtle scute proteins and shows some resemblance to reptile claw protein. Beaks also contain small amounts of protein which are distinctly different from the major fraction but which resemble feather keratin proteins in composition and size.     

Respiratory water loss during rest and flight in European Starlings (Sturnus vulgaris)

Respiratory water loss in Starlings (Sturnus vulgaris) at rest and during flight at ambient temperatures (T(amb)) between 6 and 25 degrees C was calculated from respiratory airflow and exhaled air temperature. At rest, breathing frequency f (1.4+/-0.3 Hz) and tidal volume Vt (1.9+/-0.4 ml) were independent of T(amb), but negatively correlated with each other. Mean ventilation at rest was 156+/-28 ml min(-1) at all T(amb). Exhaled air temperature (T(exh)) at rest increased with T(amb) (T(exh) = 0.92.T(amb)+12.45). Respiratory water loss at rest averaged 0.18+/-0.09 ml h(-1) irrespective of T(amb). In flying Starlings f was 4.0+/-0.4 Hz and independent of T(amb). Vt during flight averaged 3.6+/-0.4 ml and increased with T(amb) (Vt = 0.06.T(amb)+2.83) as, correspondingly, did ventilation. T(exh) during flight increased with T(amb) (T(exh) = 0.85.T(amb)+17.29). Respiratory water loss during flight (average REWL(f) = 0.74+/-0.22 ml h(-1)) was significantly higher than at rest and increased with T(amb). Our measurements suggest that respiratory evaporation accounts for most water loss in flying Starlings and increases more than cutaneous evaporation with rising ambient temperature.     

Unravelling the proteome of wool: towards markers of wool quality traits

With ongoing efforts to make wool more competitive alongside other fibres, notably synthetics, there is a need to obtain a better understanding of the relationship between protein composition and characteristic wool properties to assist sheep breeding programmes. Before this can be achieved, the wool proteome needs to be mapped, by gel and non-gel techniques, and methods developed to reliably quantitate protein expression. Nevertheless, in setting out to achieve this, there are numerous challenges to be faced in the application of proteomics to wool, including the relative lack of wool protein sequence information in the publically accessible databases, the wide variety of proteins in the wool fibre, the high homology within the Type I and Type II keratins, the high degree of homology and polymorphism within individual keratin associated protein families, the dominance of the keratin proteins over others in wool and the peculiar chemistries found in keratins and their associated proteins. This review will discuss the various strategies that have been developed to both identify these proteins in the wool protein map and quantify them with the view to their application to the identification of markers for wool quality traits.     

Keratin biosynthesis in normal mouse epithelia and in squamous cell carcinomas. mRNA-dependent alterations of the primary structure of distinct keratin subunits in tumors

The keratin polypeptide patterns of two murine transplantable squamous cell carcinomas--originally induced by chemical means in the back skin and in the forestomach epithelium--are deficient in high molecular weight keratin subunits (greater than 60 kDa) invariably present in the corresponding normal tissues. In addition, the keratin polypeptide composition within the low molecular weight range showed further alterations with regard to the corresponding keratin subset of normal tissues in that both tumors expressed a 40-kDa protein, and a 56-kDa protein was selectively found in the forestomach tumor. A comparison of the charge properties of normal and tumor keratin polypeptides revealed that the two uppermost tumor proteins at 60 and 58 kDa were basic in nature whereas their normal molecular weight counterparts belonged to the acidic subset of the pattern. These tumor proteins also showed mutually identical peptide maps which, however, were considerably different from those of the normal proteins. The remaining tumor keratin subunits at 52, 50, 48, and 45 kDa, common also to the normal tissues, had retained their normal charge properties. In vitro translation of mRNA, isolated from both normal and tumor tissue, revealed that every tumor keratin polypeptide is encoded by its own mRNA. In contrast to normal keratinizing tissues, there is therefore no indication of post-translational protein processing in tumors. The in vitro translation products of tumor RNAs had all properties in common with the in vivo tumor proteins, thus indicating that every deviation of the tumor keratin spectrum from the normal state is determined at the mRNA level.     

Hydrogen peroxide in breath condensate during a common cold

BACKGROUND: Hydrogen peroxide (H2O2) in exhaled air condensate is elevated in inflammatory disorders of the lower respiratory tract. It is unknown whether viral colds contribute to exhaled H2O2. AIM: To assess exhaled H2O2 during and after a common cold. METHODS: We examined H2O2 in the breath condensate of 20 normal subjects with acute symptoms of a common cold and after recovery 2 weeks later and, similarly, in 10 subjects without infection. H2O2 was measured with a fluorimetric assay. RESULTS: At the time of infection exhaled H2O2 (median, ranges) was 0.20 microM (0.03-1.2 microM), and this decreased to 0.09 microM (< 0.01-0.40 microM) after recovery (p = 0.006). There was no significant difference in lung function (forced vital capacity and forced expiratory volume in 1 sec) during and after colds. In the controls, exhaled H2O2 did not change over a 2-week period. CONCLUSIONS: H2O2 in exhaled air condensate is elevated during a common cold, and returns to normal within 2 weeks of recovery in healthy subjects. Hence, symptomatic upper respiratory tract infection may act as a confounder in studies of H2O2 as a marker of chronic lower airway inflammation.     

Evaluation of H2O2 and pH in exhaled breath condensate samples: methodical and physiological aspects

This veterinary study is aimed at further standardization of H₂O₂ and pH measurements in exhaled breath condensate (EBC). Data obtained in the study provide valuable information for many mammalian species including humans, and may help to avoid general pitfalls in interpretation of EBC data. EBC was sampled via the 'ECoScreen' in healthy calves (body weight 63-98 kg). Serum samples and condensates of ambient (indoor) air were collected in parallel. In the study on H₂O₂, concentrations of H₂O₂ in EBC, blood and ambient air were determined with the biosensor system 'ECoCheck'. In EBC, the concentration of H₂O₂ was found to be dependent on food intake and increased significantly in the course of the day. Physiologically, lowest H₂O₂ concentrations at 06:00 varied within the range 138-624 nmol l^-1 EBC or 0.10-0.94 nmol per 100 l exhaled breath and individual concentrations were significantly different indicating a remarkable intersubject variability. Highly reproducible results were seen within each subject (three different days within 4 weeks). No correlation existed between H₂O₂ concentrations in EBC and blood, and EBC-H₂O₂ was not influenced by variables of spontaneous breathing. Further results confirmed that standardization of H₂O₂ measurements in EBC requires (1) the re-calculation of the concentration exhaled per 100 l exhaled breath (because the analyzed concentration in the liquid condensate underlies multiple methodological sources of variability given by the collection process), and (2) subtracting the concentration of inspired indoor H₂O₂. In the study on pH use of the ISFET electrode (Sentron, the Netherlands) and a blood gas analyzer ABL 550 (Radiometer, Denmark) led to comparable results for EBC-pH (r=0.89, R²=79.3%, p≤0.001). Physiological pH data in non-degassed EBC samples varied between 5.3 and 6.5, and were not significantly different between subjects, but were significantly higher in the evening compared with the morning. EBC-pH was not dependent on variables of spontaneous breathing pattern or ambient conditions, and no significant correlation was found between serum and EBC for pH.     

Determination of proteomic and metabolic composition of exhaled breath condensate of newborns

Here, the possibility of proteomic and metabolomic analysis of the composition of exhaled breath condensate of neonates with respiratory support. The developed method allows non-invasive collecting sufficient amount of the material for identification of disease-specific biomarkers. Samples were collected by using a condensing device that was incorporated into the ventilation system. The collected condensate was analyzed by liquid chromatography coupled with high resolution mass spectrometry and tandem mass spectrometry. The isolated substances were identified with a use of databases for proteins and metabolites. As a result, a number of compounds that compose the exhaled breath condensate was determined and can be considered as possible biomarkers of newborn diseases or stage of development.     

Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues

We have constructed cDNA libraries with poly(A)+ RNA from normal mouse footpad epidermis and from a squamous cell carcinoma of mouse back skin. Both libraries were screened for type I keratin clones. We present sequence data of three keratin cDNA clones which selected mRNAs coding for two 52-kDa proteins (clones pke 52 and pkSCC 52) as well as for a 50-kDa protein (clone pkSCC50). According to their carboxyl-terminal sequences, the two 52-kDa keratin proteins belong to a group of keratins with serine-rich subdomains adjacent to the alpha-helix, whereas the short carboxyl-terminus of the 50-kDa protein lacks a distinct substructure. Sequentially the two 52-kDa keratins are more closely related to each other than to any other mouse type I keratin. However, in situ hybridization with specific subclones reveals a distinctly different pattern of expression in mouse epithelia. Clone pkSCC 52 contains sequence information for a 52-kDa keratin present in basal cells of epidermis and other stratified epithelia, whereas the pke 52 cDNA encodes a keratin which is predominantly expressed in suprabasal cells of nonepidermal tissues. In terms of nucleotide sequence identities, it cannot precisely be decided which of the two mouse 52-kDa proteins is the equivalent of the human epidermal 50-kDa keratin protein (Hanukoglu, I., and Fuchs, E. (1982) Cell 31, 243-252). In the case of the bovine keratin VII, however (Jorcano, J.L., Rieger, M., Franz, J.K., Schiller, D.L., Moll, R., and Franke, W.W. (1984) J. Mol. Biol. 179, 257-281) the sequence identity values speak for an equivalence with the mouse ke 52 keratin. Obviously, in situ hybridization experiments would best be suited to unravel the precise interspecies relationship between the four highly similar keratins. The discriminatory efficacy of this technique is further emphasized by the demonstration that the mRNA for a 50-kDa keratin is present not only in hyperproliferative epithelia, but also in normal cells of hair follicles.     

Concentration of light hydrocarbons in exhaled air depending on metabolic syndrome risk factors

The effect of metabolic syndrome risk factors on the content of light hydrocarbons (C₂-C₃) in exhaled air has been studied with the participation of students of Novosibirsk State Medical University. Gas chromatography was performed in an EKHO-EW-FID chromatograph with a short multichannel column. The study revealed sex-related differences between the concentrations of light hydrocarbons in exhaled air. In addition, some factors, such as smoking and type 2 diabetes mellitus in relatives, affect the content of C₂-C₃ compounds in exhaled air of women only. However, overweight is correlated with the content of acetone (C₃) in exhaled air only in men. Thus, the metabolic changes caused by the presence of age-specific metabolic syndrome risk factors lead to the changes in the gas composition of exhaled air and can be detected and used for early diagnosis.     

Alternatives for lung research: stuck between a rat and a hard place

The respiratory system acts as a portal into the human body for airborne materials, which may gain access via the administration of medicines or inadvertently during inhalation of ambient air (e.g. air pollution). The burden of lung disease has been continuously increasing, to the point where it now represents a major cause of human morbidity and mortality worldwide. In the UK, more people die from respiratory disease than from coronary heart disease or non-respiratory cancer. For this reason alone, gaining an understanding of mechanisms of human lung biology, especially in injury and repair events, is now a principal focus within the field of respiratory medicine. Animal models are routinely used to investigate such events in the lung, but they do not truly reproduce the responses that occur in humans. Scientists committed to the more robust Three Rs principles of animal experimentation (Reduction, Refinement and Replacement) have been developing viable alternatives, derived from human medical waste tissues from patient donors, to generate in vitro models that resemble the in vivo human lung environment. In the specific case of inhalation toxicology, human-oriented models are especially warranted, given the new REACH regulations for the handling of chemicals, the rising air pollution problems and the availability of pharmaceutically valuable drugs. Advances in tissue-engineering have made it feasible and cost-effective to construct human tissue equivalents of the respiratory epithelia. The conducting airways of the lower respiratory system are a critical zone to recapitulate for use in inhalation toxicology. Three-dimensional (3-D) tissue designs which make use of primary cells, provide more in vivo-like responses, based on the targeted interactions of multiple cell types supported on artificial scaffolds. These scaffolds emulate the native extracellular matrix, in which cells differentiate into a functional pulmonary tissue. When 3-D cell cultures are employed for testing aerosolised chemicals, drugs and xenobiotics, responses are captured that mirror the events in the in situ human lung and provide human endpoint data.     

Nitric oxide in the breath of bottlenose dolphins: Effects of breath hold duration,feeding, and lung disease

Breath analysis, including measurement of nitric oxide (NO), is a noninvasive diagnostic tool that may help evaluate cetacean health. This is the first report on the effects of breath hold duration, feeding, and lung disease on NO in dolphin exhaled breath. Three healthy dolphins were trained to hold their breath for 30, 60, 90, and 120 s and then exhale into an underwater funnel. Exhaled NO values from 157 breath samples were compared among three healthy dolphins by breath hold time and after fasting and feeding. Exhaled NO values were also measured in two dolphins with pulmonary disease. NO in dolphin breath was higher compared to ambient air; healthy dolphins had higher NO concentrations in their breath after feeding compared to after overnight fasting; and there were no significant differences in exhaled NO levels by breath hold duration. A dolphin with Mycoplasma‐associated pneumonia and chronic gastrointestinal disease had higher postprandial exhaled NO levels compared to healthy controls. This study demonstrates, contrary to previous publications, that dolphins exhale NO. Given the high standard deviations present in exhaled breath NO values, future studies are needed to further standardize collection methods or identify more reliable samples (e.g., blood).