5.1 Residue in LPG is a contaminant that can lead to operational problems in some end use applications. Engines, micro-turbines, fuel cells and other equipment may be sensitive to residue levels as low as 10 mg/kg.5.2 Contamination of LPG can occur during production, transport, delivery, storage and use. A qualitative indication of the contaminants can help track down the source of the contamination from manufacture, through the distribution system, and to the end user.5.3 This test method is designed to provide a lower detection limit, wider dynamic range, and better accuracy than gravimetric methods like Test Method D2158.5.4 This test method can be performed with little or no discharge of LPG vapors, compared to Test Method D2158 which requires evaporation of 100 mL of sample per test.5.5 Sampling for residue in LPG using sorbent tubes can be performed in the field, and the sorbent tubes sent to a laboratory for analysis. This saves significant costs in shipping (weight of tube is approximately 10 grams), and is much safer and easier than transporting LPG cylinders.5.6 This test method determines total residues from C6 to C40, compared to a thermal gravimetric residue method such as Test Method D2158 which heat the residue to 38°C, resulting in a lower recovery due to loss of lighter residue components.5.7 If there is a need to decrease the detection limit of residue or individual compounds of interest below 10 µg/g, the procedures in this test method can be modified to achieve 50 times enhanced detection limit, or 0.2 µg/g.1.1 This test method covers the determination of residue in LPG by automated thermal desorption/gas chromatography (ATD/GC) using flame ionization detection (FID).1.2 The quantitation of residue covers a component boiling point range from 69°C to 522°C, equivalent to the boiling points of C6 through C40 n-paraffins.1.2.1 The boiling range covers possible LPG contaminants such as gasoline, diesel fuel, phthalates and compressor oil. Qualitative information on the nature of the residue can be obtained from this test method.1.2.2 Materials insoluble in LPG and components which do not elute from the gas chromatograph or which have no response in a flame ionization detector are not determined.1.2.3 The reporting limit (or limit of quantitation) for total residue is 6.7 µg/g.1.2.4 The dynamic range of residue quantitation is 6.7 to 3300 µg/g.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

定价： 0元 / 折扣价： 0 元

Cyanides are known to be toxic to man, but more so to fish and other aquatic life. The complexity of the chemistry of cyanides has led to the coexistence of several cyanide species in the environment. The presence of cyanides in industrial, domestic, and surface water is cause for concern. Several regulations and standards require continuous monitoring of cyanides in different types of water and wastes. The automated test methods presented offer useful tools for such monitoring. (See also Practice D 4193.)1.1 These test methods cover the determination of different species of cyanides and thiocyanate in water and waste water, namely weak acid dissociable cyanide, total cyanide, and thiocyanate ().1.1.1 Total Cyanide This test method determines all the weak acid dissociable cyanides and the strong metal-cyano-complexes, such as ferrocyanide [Fe(CN)6] 4, ferricyanide [Fe(CN)6]3, hexacyanocolbaltate [Co(CN) 6]3, and those of gold and platinum.1.1.2 Weak Acid Dissociable CyanideThis test method basically determines free cyanides, as CN and HCN, and weak metal-cyano-complexes such as [Cd(CN) 4] 2 and [Mn(CN)6] 3. Iron complexes are not included.1.1.3 Strong cyanide complexes, like those of iron, cobalt, etc., can be determined by difference, that is, cyanide complexes = total cyanides weak acid dissociable cyanides.1.1.4 Thiocyanate This test method determines the thiocyanate as the difference between another measurement that includes total cyanide plus thiocyanate and the value of total cyanide, that is, thiocyanate = total cyanide plus thiocyanate total cyanide.1.2 Cyanates and cyanogen halides are not detected. Cyanogen chloride hydrolyzes to cyanate at the pH of sample preservation (12).1.3 Most of the organo-cyano-complexes are not measured, with the exception of the weak cyanohydrins.1.4 These test methods apply to different types of water, waste water (raw sewage, sludge, and effluent), sludge, some industrial waste, and sediments. Sample matrixes should be evaluated by the user. The reported precision and bias (see Section ) may not apply to all samples.1.5 The values stated in SI units are to be regarded as the standard.This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific precautionary statements, see Section 9.

定价： 0元 / 折扣价： 0 元

5.1 This test method allows for the measurement of the torque retention properties of container/continuous thread closure systems of various designs, materials, and manufacture, and is suitable for packaging development and engineering evaluation.5.2 This test method can be used for the evaluation of container/continuous thread closure systems under controlled conditions (where the application torque is known and the applied downward force to the closure is zero).5.3 This test method measures torque retention properties of container/continuous thread closure systems with the use of a non-automated, spring torque-meter (with either a dial indicator or a digital readout) or a torque wrench.1.1 These test methods evaluate the torque retention of continuous thread closures on containers, with matching finishes, for predetermined environmental conditions over time.1.2 The values stated in either SI units or inch-pound units are to be regarded separately as standard. The values stated in each system may not be exact equivalents; therefore, each system shall be used independently of the other. Combining values from the two systems may result in non-conformance with the standard.NOTE 1: The SI unit system is the recommended system.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 515元 / 折扣价： 438 元 加购物车

5.1 Many petroleum products and some non-petroleum products are used as lubricants in the equipment, and the correct operation of the equipment depends upon the appropriate viscosity of the lubricant being used. Additionally, the viscosity of many petroleum fuels is important for the estimation of optimum storage, handling, and operational conditions. Thus, the accurate determination of viscosity is essential to many product specifications.5.2 The viscosity of used oils is a commonly determined parameter in the oil industry to assess the effect of engine wear on the lube oils used, as well as the degradation of the engine parts during operation.5.3 The Houillon viscometer tube method offers automated determination of kinematic viscosity. Typically a sample volume of less than 1 mL is required for the analysis.1.1 This test method covers the measurement of the kinematic viscosity of transparent and opaque liquids; such as base oils, formulated oils, diesel oil, biodiesel, biodiesel blends, residual fuel oils, marine fuels, and used lubricating oils using a Houillon viscometer in automated mode.1.2 The range of kinematic viscosity capable of being measured by this test method is from 2 mm2/s to 2500 mm2/s (see Fig. 1). The range is dependent on the tube constant utilized. The temperature range that the apparatus is capable of achieving is between 20 °C and 150 °C, inclusive. However, the precision has only been determined for the viscosity range; 2 mm2/s to 478 mm2/s at 40 °C for base oils, formulated oils, diesel oil, biodiesel, and biodiesel blends; 3 mm2/s to 106 mm2/s at 100 °C for base oils and formulated oils; 25 mm2/s to 150 mm2/s at 40 °C and 5 mm2/s to 16 mm2/s at 100 °C for used lubricating oils; 25 mm2/s to 2500 mm2/s at 50 °C and 6 mm2/s to 110 mm2/s at 100 °C for residual fuels. As indicated for the materials listed in the precision section.FIG. 1 Houillon Viscometer Typical Viscosity Range of Tube ConstantsNOTE 1: Viscosity range of a Houillon tube is based on most practical flow time of 30 s to 200 s.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For specific warning statements, see Section 7.1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

This specification establishes the performance-based and prescriptive-based methods of evaluating various classes of automated gate constructions that are used for vehicular traffic. The gate types addressed in this specification include horizontal slide gates, horizontal swing gates, vertical lift gates, vertical pivot gates, and overhead pivot gates. Conversely, the four classes of gates covered here are as follows: Class I, a gate for the garage or parking area intended for use in a home of a one-to-four single family dwelling; Class II, a gate intended for use in a commercial location or building such as a multi-family housing unit (five or more single family units), hotel, garages, retail store, or other building servicing the general public; Class III, a gate intended for use in an industrial location or building such as a factory, loading dock area, or other locations not intended to service the general public; and Class IV, a gate intended for use in a guarded industrial location or building such as an airport security area, or other restricted access locations not servicing the general public, in which unauthorized access is prevented by means of supervision by security personnel.1.1 This specification defines performance-based and prescriptive-based methods of evaluating various classes of gates that are used for vehicular traffic and are to be automated.1.2 Gate types addressed in this specification include horizontal slide gates, horizontal swing gates, vertical lift gates, vertical pivot gates, and overhead pivot gates.1.3 Gate types not listed in this specification will be subject to any applicable provisions contained in this specification.1.4 Automated vehicular gate systems shall comply with this specification and shall be compliant with UL 325.1.5 Units—The values stated in inch-pound units are to be regarded as standard. The values given in parentheses are mathematical conversions to SI units that are provided for information only and are not considered standard.1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 515元 / 折扣价： 438 元 加购物车

5.1 Shot peening is a process for cold working surfaces by bombarding the product with shot of a solid and spherical nature propelled at a relatively high velocity. In general, shot peening will increase the fatigue life of a product that is subject to bending or torsional stress. It will improve resistance to stress corrosion cracking. It can be used to form parts or correct their shapes. See Appendix X1 for additional information.5.2 It is essential that the shot peening process parameters be controlled rigidly to ensure repeatability from part to part and lot to lot.5.3 This specification covers techniques and methods necessary for proper control of the shot peening process.AbstractThis specification covers the requirements for automated, controlled shot peening of metallic articles prior to electrolytic or autocatalytic deposition of nickel or chromium, or as a final finish, using shot made of cast steel, conditioned cut wire, or ceramic media. The process is applicable to those materials on which test work has shown it to be beneficial within given intensity ranges. It is not suitable for brittle materials. Hand peening and rotary flap peening are excluded specifically. Shot peening induces residual compressive stresses in the surface and near-surface layers of metallic articles, controlling or limiting the reduction in fatigue properties that occurs from nickel or chromium plating of the article, or the fatigue properties of unplated articles. It is a process for cold working surfaces by bombarding the product with shot of a solid and spherical nature propelled at a relatively high velocity. Cast steel, cut wire, and ceramic shot shall all be spherical in shape and shall all be free of sharp edges, corners, and broken pieces. Prior to shot peening, the following operations shall be done first: heat treatment, machining, grinding, flaw test, crack test, corrosion detection, cleaning, and masking. Peened surfaces shall be uniform in appearance and completely dented so that the original surface is obliterated entirely. After shot peening, the following methods shall be done: residual shot removal, surface finishing, chemical cleaning of nonferrous metals and their alloys, thermal and heat treatments, and corrosion protection.1.1 This specification covers the requirements for automated, controlled shot peening of metallic articles prior to electrolytic or autocatalytic deposition of nickel or chromium, or as a final finish, using shot made of cast steel, conditioned cut wire, or ceramic media. The process is applicable to those materials on which test work has shown it to be beneficial within given intensity ranges. It is not suitable for brittle materials. Hand peening and rotary flap peening are excluded specifically.1.2 Shot peening induces residual compressive stresses in the surface and near-surface layers of metallic articles, controlling or limiting the reduction in fatigue properties that occurs from nickel or chromium plating of the article, or the fatigue properties of unplated articles.1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

4.1 For hydrocarbon resins and rosin based resins, softening does not take place at a definite temperature. As the temperature rises, these materials gradually change from brittle solids or very viscous liquids to less viscous liquids. For this reason, determination of the softening point must be made by a fixed, arbitrary, and closely defined method if the results obtained are to be comparable.1.1 These test methods are intended for determining the softening point of hydrocarbon resins, rosin based resins and similar materials by means of an automated ring-and-ball apparatus. Portions are similar in technical content to the automated-apparatus versions of Test Methods D36, E28, and ISO 4625.1.1.1 The ring-and-ball softening point of a hydrocarbon resin and rosin based resins may also be determined with lower precision using the manual ring-and-ball softening point procedure in Test Methods E28.1.2 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

5.1 This test method allows for the measurement of the torque retention properties of container/continuous thread closure systems of various designs, materials, and manufacture, and is suitable for package development and engineering evaluation.5.2 Each test method can be used for the evaluation of non child resistant container/continuous thread closure systems under controlled conditions such as when the application torque is known and the applied downward force to the closure is zero or for Type I, style “A” push down and turn child resistant container/continuous thread closure systems under controlled conditions such as when the application torque and the applied downward force to the closure is known.5.3 This test method measures torque retention properties of container/continuous thread closure systems with the use of an automated transducer based torque meter operating at a known rotational velocity (rpm) or known torque ramp.5.4 This test method is intended for measurement of dry torque only.1.1 These test methods evaluate the torque retention of continuous thread closures on containers with matching finishes, for predetermined environmental conditions over time. Methods are defined for both Type I, style “A” push down and turn Type II2 child resistant and non child resistant type closures.1.2 The values stated in either SI units or inch-pound units are to be regarded separately as standard. The values stated in each system may not be exact equivalents; therefore, each system shall be used independently of the other. Combining values from the two systems may result in non-conformance with the standard.NOTE 1: The SI unit system is the recommended system.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

定价： 1820元 / 折扣价： 1547 元

4.1 Individual pellet hardness is related to several carbon black characteristics. Among these are mass strength and attrition. The subsequent level of dispersion obtained in some mixed compounds containing the carbon black may be affected by pellet hardness. Acceptable pellet hardness must be agreed to by the user and the producer.1.1 This test method covers a procedure for measuring individual pellet hardness of carbon black by the automated pellet hardness tester.1.2 The values stated in SI units are to be regarded as the standard. The values in parentheses are for information only.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

定价： 455元 / 折扣价： 387 元

5.1 This test method provides a means of automatically separating and collecting atmospheric particulate and acidic gaseous fluoride samples.5.2 Since the samples are collected on dry tapes, the samples are in a form which allows elution of the fluoride content with a small volume of eluent. Consequently, the method allows analyses of air samples taken for a time period as short as several minutes.1.1 This test method describes the automatic separation and collection on chemically treated paper tapes of particulate and gaseous forms of acidic fluorides in the atmosphere by means of a double paper tape sampler. The sampler may be programmed to collect and store individual air samples obtained over time periods from several minutes to 3 h. A 30.5-m (100-ft) tape will allow unattended operation for the automatic collection of up to 600 samples.1.2 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are included for information only and are not considered standard.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

5.1 This guide is intended to provide machinery maintenance and monitoring personnel with a guideline for performing filter debris analysis as a means to determine machine condition. Correlating the filter contaminants to ‘normal’ and ‘abnormal’ lube system operation provides early indication of a contaminant or component wear related lube system problem. Analysis of the contaminant collected within the lube filter element provides a tool to identify the failure mode, its rate of progression, and the source of the contamination.5.2 FDA differs from traditional oil analysis in that the filter is sampled instead of the fluid. Debris from the filter is removed for analysis. FDA is an effective means of monitoring equipment wear because the wear history is efficiently captured in the filter matrix. Typically, more than 95 % of all released metal particles larger than the filter pore size are captured in the filter (1).5 In addition, other types of particulate contamination, including seal wear material and environmental contaminations are captured, which can also provide diagnostic information.1.1 This guide pertains to removal and analysis techniques to extract debris captured by in-service lubricant and hydraulic filters and to analyze the debris removed.1.2 This guide suggests techniques to remove, collect and analyze debris from filters in support of machinery health condition monitoring.1.3 Debris removal techniques range from manual to automated.1.4 Analysis techniques vary from visual, particle counting, microscopic, x-ray fluorescence (XRF), atomic emission spectroscopy (AES), and scanning electron microscopy energy dispersive x-rays (SEMEDX).1.5 This guide is suitable for use with the following filter types: screw on, metal mesh, and removable diagnostic layer filters.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

5.1 This test method is of particular use as a quality control tool for a molding or synthesis operation. Acetaldehyde is a volatile degradation product generated during melt processing of PET. Thus, it becomes trapped in the sidewalls of a molded article and desorbs slowly into the contents packaged therein. In some foods and beverages AA can impart an off-taste that is undesirable, thus, it is important to know its concentration in PET articles that are to be used in food contact applications.5.2 The desorption conditions of 150 °C for 60 min are such that no measurable AA is generated by the sample during the desorption process.1.1 This test method covers a gas chromatographic procedure for the determination of the ppm residual acetaldehyde (AA) present in poly(ethylene terephthalate) (PET) homo-polymers and co-polymers which are used in the manufacture of beverage bottles. This includes sample types of both amorphous and solid-stated pellet and preform samples, as opposed to the bottle test, Test Method D4509, an acetaldehyde test requiring 24 h of desorption time at 23 °C into the bottle headspace and then the concentration of the headspace quantified by a similar GC method.1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

定价： 590元 / 折扣价： 502 元 加购物车

4.1 The Manual Observer-Dependent Assay—The manual quantification of cell and CFU cultures based on observer-dependent criteria or judgment is an extremely tedious and time-consuming task and is significantly impacted by user bias. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell- and colony-based assays often require that a single observer count cells and colonies in hundreds, and potentially thousands of cultures. Due to observer fatigue, both accuracy and reproducibility of quantification suffer severely (5). When multiple observers are employed, observer fatigue is reduced, but the accuracy and reproducibility of cell and colony enumeration is still significantly compromised due to observer bias and significant intra- and inter-observer variability (2, 4) . Use of quantitative automated image analysis provides data for both the number of colonies as well as the number of cells in each colony. These data can also be used to calculate mean cells per colony. Traditional methods for quantification of colonies by hand-counting coupled with an assay for cell number (for example, DNA or mitochondrial) remains a viable method that can be used to calculate the mean number of cells per colony. These traditional methods have the advantage that they are currently less labor intensive and less technically demanding (8, 9). However, the traditional assays do not, provide colony level information (for example, variation and skew), nor do they provide a means for excluding cells that are not part of a colony from the calculation of mean colony size. As a result, the measurement of the mean number of cells per colony that is obtained from these alternative methods may differ when substantial numbers of cells in a sample are not associated with colony formation. By employing state-of-the-art image acquisition, processing and analysis hardware and software, an accurate, precise, robust and automated analysis system is realized.4.1.1 Areas of Application—Cell and colony enumeration (CFU assay) is becoming particularly important in the manufacture, quality assurance/control (QA/QC), and development of product safety and potency release criteria for cell-based regenerative medicine and cellular therapy. The U.S. Food and Drug Administration (FDA) has a guidance document that indicates that the CFU assay may be appropriate for testing stability of placental and umbilical cord blood-derived stem cells (7). Since cell source validation and QA/QC comprise approximately 50 % of the manufacturing cost of cellular therapies (10), developing a precise, robust, and cost-effective means for enumerating cells and colonies is vital to sustainability and growth in this industry. The broad areas of use for automated analysis of colony forming unit assays include:4.1.1.1 Characterization of a cell source by correlating biological potential and functional potency with CFU formation.4.1.1.2 Characterization of the effect of processing steps or biological or physical manipulation (for example, stimuli) on cells or colony formation.4.1.1.3 Cell and colony characterization using specific fluorescent and non-fluorescent (differentiation) markers.4.1.1.4 Extrapolation of the biological potency (for example, differentiation, proliferative, and so forth) of a larger sample from application of colony forming assay to sub-samples.4.1.1.5 Provision of criteria for sub-colony selection of preferred colonies (specific tissue type, proliferation rate, and so forth) for use and/or further expansion.4.2 The Technology (image acquisition, processing, and analysis)—Current standards utilize user input for defining the presence and location of colonies based on visualization of an entire culture surface at low magnification through the eyepieces of a microscope. In this case, the sample may be viewed in transmission light mode (unstained or with a histochemical marker) or fluorescently with a dye or antibody. For this practice, the colony count is the only measurable output parameter. Utilizing a microscope-based imaging system to stitch together high resolution image tiles into a single mosaic image of the entire culture surface and subsequently “clustering” segmented cells using image processing algorithms to delineate colonies, provides a fully automated, accurate, and precise method for characterizing the biological potential and functional potency of the cultured cells. Furthermore, extracted parameters in addition to colony number provide means of further characterization and sub-classification of colony level statistics. These parameters include, but are not limited to, cell/nuclear count, cell/nuclear density, colony morphology (shape and size parameters), secondary marker coverage, effective proliferation rates, and so forth (Fig. A1.2). In addition to human connective tissue progenitors (CTPs) derived from bone, bone marrow, cartilage, adipose tissue, muscle, periosteum, and synovium, this practice and technology has been implemented in the cell and colony identification and characterization of several cell and tissue types including: umbilical cord blood hematopoietic stem cells (Fig. X1.2); adipose-derived stem cells (Fig. X1.3); and human epidermal (Fig. X1.4) and dermal (Fig. X1.5) stem cells.4.3 Benefits of Automated Analysis of CFU Assays—Automated analysis is expected to provide more rapid, reproducible, and precise results in comparison to the manual enumeration of cells and colonies utilizing a microscope and hemocytometer. In addition to being time consuming, labor intensive, and subjective, manual enumeration has been shown to have a significant degree of intra- and inter-observer variability, with coefficients of variation (CV) ranging from 8.1 % to 40.0 % and 22.7 % to 80 %, respectively. Standard CVs for cell viability assessment and progenitor (colony) type enumeration have been shown to range from 19.4 % to 42.9 % and 46.6 % to 100 %, respectively (4, 11, 12). In contrast, studies focusing on bacteria, bone marrow-derived stem cells and osteogenic progenitor cells have collectively concluded that automated enumeration provides significantly greater accuracy, precision, and/or speed for counting and sizing cells and colonies, relative to conventional manual methodologies (4-6). Automated methods for enumerating cells and colonies are less biased, less time consuming, less laborious, and provide greater qualitative and quantitative data for intrinsic characteristics of cell and colony type and morphology.4.4 Selection of Cell Culture Surface Area and Optimal Cell Seeding Density—When performing a CFU assay, optimizing the cell culture surface area and cell seeding density is critical to developing methods for generating reliable and reproducible colony- and cell-level data. If seeding density is too low, then the frequency of observed colonies is decreased. This can result in a sampling size that is inadequate to characterize the population of CFUs in the sample. If seeding density is too high, the colonies that are formed may be too closely spaced. Overlapping colony footprints compromise colony counting and characterization. Because the intrinsic range of CFU prevalence in a given cell source may vary widely, in many cases, a trial and error approach to optimizing cell seeding density (or range of densities) that are needed for a given cell source will be necessary. It is important to note that the more heterogeneous the cell source (for example, bone marrow), the more colonies that are needed to accurately represent the stem and progenitor cell constituents. Further, the cell type, effective proliferation rate (EPR) and specific cell culture conditions (for example, media, serum, factors, oxygen tension, and so forth) can impact colony formation. For example, the automated CFU assay depicted in Fig. A1.2 employs a six-day culture period, two media changes, 20 % oxygen tension, alpha-MEM media (with 25 % fetal bovine serum, ascorbate, dexamethasone and streptomycin), an optimized cell seeding density of 250 000 nucleated cells per cm2 (250 000 cells per 1 mL of cell culture medium) and a cell culture surface area of 22 mm by 22 mm (dual-chamber Lab-Tek culture slides) (12, 13).4.5 Useful Documents—A number of useful documents are available that address best practices for conducting quantitative measurements of cells using imaging approaches: Guide F2998, Guide F3294, ISO 20391-1, ISO 20391-2, and “FDA Guidance on Technical Performance Assessment of Digital Pathology Whole Slide Imaging Devices,” (14).1.1 This practice, provided its limitations are understood, describes a procedure for quantitative measurement of the number and biological characteristics of colonies derived from a stem cell or progenitor population using image analysis.1.2 This practice is applied in an in vitro laboratory setting.1.3 This practice utilizes: (a) standardized protocols for image capture of cells and colonies derived from in vitro processing of a defined population of starting cells in a defined field of view (FOV), and (b) standardized protocols for image processing and analysis.1.4 The relevant FOV may be two-dimensional or three-dimensional, depending on the CFU assay system being interrogated.1.5 The primary unit to be used in the outcome of analysis is the number of colonies present in the FOV. In addition, the characteristics and sub-classification of individual colonies and cells within the FOV may also be evaluated, based on extant morphological features, distributional properties, or properties elicited using secondary markers (for example, staining or labeling methods).1.6 Imaging methods require that images of the relevant FOV be captured at sufficient resolution to enable detection and characterization of individual cells and over a FOV that is sufficient to detect, discriminate between, and characterize colonies as complete objects for assessment.1.7 Image processing procedures applicable to two- and three-dimensional data sets are used to identify cells or colonies as discreet objects within the FOV. Imaging methods may be optimized for multiple cell types and cell features using analytical tools for segmentation and clustering to define groups of cells related to each other by proximity or morphology in a manner that is indicative of a shared lineage relationship (that is, clonal expansion of a single founding stem cell or progenitor).1.8 The characteristics of individual colony objects (cells per colony, cell density, cell size, cell distribution, cell heterogeneity, cell genotype or phenotype, and the pattern, distribution and intensity of expression of secondary markers) are informative of differences in underlying biological properties of the clonal progeny.1.9 Under appropriately controlled experimental conditions, differences between colonies can be informative of the biological properties and underlying heterogeneity of colony founding cells (CFUs) within a starting population.1.10 Cell and colony area/volume, number, and so forth may be expressed as a function of cell culture area (square millimeters), or initial cell suspension volume (milliliters).1.11 Sequential imaging of the FOV using two or more optical methods may be valuable in accumulating quantitative information regarding individual cells or colony objects in the sample. In addition, repeated imaging of the same sample will be necessary in the setting of process tracking and validation. Therefore, this practice requires a means of reproducible identification of the location of cells and colonies (centroids) within the FOV area/volume using a defined coordinate system.1.12 To achieve a sufficiently large field-of-view (FOV), images of sufficient resolution may be captured as multiple image fields/tiles at high magnification and then combined together to form a mosaic representing the entire cell culture area.1.13 Cells and tissues commonly used in tissue engineering, regenerative medicine, and cellular therapy are routinely assayed and analyzed to define the number, prevalence, biological features, and biological potential of the original stem cell and progenitor population(s).1.13.1 Common applicable cell types and cell sources include, but are not limited to: mammalian stem and progenitor cells; adult-derived cells (for example, blood, bone marrow, skin, fat, muscle, mucosa) cells, fetal-derived cells (for example, cord blood, placental/cord, amniotic fluid); embryonic stem cells (ESC) (that is, derived from inner cell mass of blastocysts); induced pluripotent cells (iPC) (for example, reprogrammed adult cells); culture expanded cells; and terminally differentiated cells of a specific type of tissue.1.13.2 Common applicable examples of mature differentiated phenotypes which are relevant to detection of differentiation within and among clonal colonies include: hematopoietic phenotypes (erythrocytes, lymphocytes, neutrophiles, eosinophiles, basophiles, monocytes, macrophages, and so forth), adult tissue-specific progenitor cell phenotypes (oteoblasts, chondrocytes, adipocytes, and so forth), and other tissues (hepatocytes, neurons, endothelial cells, keratinocyte, pancreatic islets, and so forth).1.14 The number of stem cells and progenitor cells in various tissues can be assayed in vitro by liberating the cells from the tissues using methods that preserve the viability and biological potential of the underlying stem cell and/or progenitor population, and placing the tissue-derived cells in an in vitro environment that results in efficient activation and proliferation of stem and progenitor cells as clonal colonies. The true number of stem cells and progenitors (true colony forming units (tCFU)) can thereby be estimated on the basis of the number of colony-forming units observed (observed colony forming units (oCFU)) to have formed (1-3)2 (Fig. A1.1). The prevalence of stem cells and/or progenitors can be estimated on the basis of the number of observed colony-forming units (oCFU) detected, divided by the number of total cells assayed.1.15 The automated image acquisition and analysis approach (described herein) to cell and colony enumeration has been validated and found to provide superior accuracy and precision when compared to the current “gold standard” of manual observer defined visual cell and colony counting under a brightfield or fluorescent microscope with or without a hemocytometer (4), reducing both intra- and inter-observer variation. Several groups have attempted to automate this and/or similar processes in the past (5, 6) . Recent reports further demonstrate the capability of extracting qualitative and quantitative data for colonies of various cell types at the cellular and even nuclear level (4, 7).1.16 Advances in software and hardware now broadly enable systematic automated analytical approaches. This evolving technology creates the need for general agreement on units of measurement, nomenclature, process definitions, and analytical interpretation as presented in this practice.1.17 Standardized methods for automated CFU analysis open opportunities to enhance the value and utility of CFU assays in several scientific and commercial domains:1.17.1 Standardized methods for automated CFU analysis open opportunities to advance the specificity of CFU analysis methods though optimization of generalizable protocols and quantitative metrics for specific cell types and CFU assay systems which can be applied uniformly between disparate laboratories.1.17.2 Standardized methods for automated CFU analysis open opportunities to reduce the cost of colony analysis in all aspects of biological sciences by increasing throughput and reducing work flow demands.1.17.3 Standardized methods for automated CFU analysis open opportunities to improve the sensitivity and specificity of experimental systems seeking to detect the effects of in vitro conditions, biological stimuli, biomaterials and in vitro processing steps on the attachment, migration, proliferation, differentiation, and survival of stem cells and progenitors.1.18 Limitations are described as follows:1.18.1 Colony Identification—Cell Source/Colony Type/Marker Variability—Stem cells and progenitors from various tissue sources and in different in vitro environments will manifest different biological features. Therefore, the specific means to detect cells or nuclei and secondary markers utilized and the implementation of their respective staining protocols will differ depending on the CFU assay system, cell type(s) and markers being interrogated. Optimized protocols for image capture and image analysis to detect cells and colonies, to define colony objects and to characterize colony objects will vary depending on the cell source being utilized and CFU system being used. These protocols will require independent optimization, characterization and validation in each application. However, once defined, these can be generalized between labs and across clinical and research domains.1.18.2 Instrumentation-Induced Variability in Image Capture—Choice of image acquisition components described above may adversely affect segmentation of cells and subsequent colony identification if not properly addressed. For example, use of a mercury bulb rather than a fiber-optic fluorescent light source or the general misalignment of optics could produce uneven illumination or vignetting of tiled images comprising the primary large FOV image. This may be corrected by applying background subtraction routines to each tile in a large FOV image prior to tile stitching.1.18.3 CFU Assay System Associated Variation in Imaging Artifacts—In addition to the presentation of colony objects with unique features that must be utilized to define colony identification, each image from each CFU system may present non-cell and non-colony artifacts (for example, cell debris, lint, glass aberrations, reflections, autofluorescence, and so forth) that may confound the detection of cells and colonies if not identified and managed.1.18.4 Image Capture Methods and Quality Control Variation—Variation in image quality will significantly affect the precision and reproducibility of image analysis methods. Variation in focus, illumination, tile registration, exposure time, quenching, and emission spectral bleeding, are all important potential limitations or threats to image quality and reproducibility.1.19 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.20 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.1.21 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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