定价： 481元 / 折扣价： 409 元

5.1 This test method is used to measure the apparent viscosity of thermoplastic pavement marking at elevated temperatures. Elevated temperature viscosities of thermoplastic pavement marking may be related to the properties of coatings, adhesives, and composite thermoplastics. This method is helpful in determining the flow properties which can be used in determining processability when applied to the road surface.5.2 Thermoplastic pavement markings may be applied to the road surface in several different ways. Typical methods of application are screed extrude, ribbon extrude, thin film spray, and standard spray. Proper application depends on the viscosity of the thermoplastic material at application temperatures for the method being used. Thin-line applied thermoplastic pavement marking, for example, requires a relatively lower viscosity. Screed extrude applied thermoplastic requires a higher viscosity.5.3 Materials of the type described in this procedure may be non-Newtonian, and as such, the apparent viscosity will be a function of shear rate under the conditions of test. Although the viscometer described in this test method operates under conditions of relatively low shear rate, differences in shear effect can exist depending upon the spindle and rotational speed conditions selected for the test program. Comparisons between non-Newtonian viscosity values should be made only for measurements made with similar viscometers under conditions of equivalent shear. For this method, “torpedo” spindles are recommended. Spindles considered torpedo spindles are ~1-in. long and come in many diameters with a 45° conical bottom. A diameter that is half the diameter of the thimbles used is recommended. If large glass beads are used in the pavement marking formulation, a smaller diameter spindle may be needed so the beads do not cause an impedance of the spindle due to a jamming between the inside wall of the thimble and the spindle.1.1 This test method covers the sample preparation and testing procedure needed to determine the apparent viscosity of a thermoplastic pavement marking formulation at elevated temperatures to the specimen.1.2 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are customary units and are provided as a courtesy to the user.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 is intended either as an index test or as a performance test used to determine and compare the flow rate per unit width of one or several candidate geosynthetics under specific conditions.5.2 This test method may be used as an index test for acceptance testing of commercial shipments of geosynthetics, but caution is advised since information on between-laboratory precision of this test method is incomplete. Comparative tests as directed in 5.2.1 may be advisable.5.2.1 In case of a dispute arising from differences in reported test results when using this test method for acceptance testing of commercial shipments, the purchaser and the supplier should first confirm that the tests were conducted using comparable test parameters including specimen conditioning, normal stress, seating period, hydraulic gradient, test water temperature, etc., then conduct comparative tests to determine if there is a statistical bias between their laboratories. Competent statistical assistance is recommended for the investigation of bias. As a minimum, the two parties should take a group of test specimens that are as homogenous as possible and that are formed from a lot of the material of the type in question. The test specimens should then be randomly assigned in equal numbers to each laboratory for testing. The average results from the two laboratories should be compared using the Student's t-test for unpaired data and an acceptable probability level chosen by the two parties before the testing is begun. If bias is found, either its cause must be found and corrected or the purchaser and supplier must agree to interpret future test results in light of the known bias.1.1 This test method covers the procedure for determining the flow rate per unit width within the manufactured plane of geosynthetics under varying normal compressive stresses and a constant head. The test is intended primarily as an index test but can be used also as a performance test when the hydraulic gradients and specimen contact surfaces are selected by the user to model anticipated field conditions.1.2 This test method is limited to geosynthetics that allow continuous in-plane flow paths to occur parallel to the intended direction of flow.1.3 The values stated in either SI units or inch-pound units are to be regarded separately as standard. The values stated in each system are not necessarily exact equivalents; therefore, to ensure conformance with the standard, each system shall be used independently of the other, and values from the two systems shall not be combined.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： 646元 / 折扣价： 550 元 加购物车

4.1 Compaction tests on soils performed in accordance with Test Methods D698, D1557, D4253, and D7382 place limitations on the maximum size of particles that may be used in the test. If a soil contains cobbles or gravel, or both, test options may be selected which result in particles retained on a specific sieve being discarded (for example the 4.75-mm [No. 4], the 19-mm [3/4-in.] or other appropriate size) and the test performed on the finer fraction. The unit weight-water content relations determined by the tests reflect the characteristics of the actual material tested, and not the characteristics of the total soil material from which the test specimen was obtained.4.2 It is common engineering practice to use laboratory compaction tests for the design, specification, and construction control of soils used in earth construction. If a soil used in construction contains large particles, and only the finer fraction is used for laboratory tests, some method of correcting the laboratory test results to reflect the characteristics of the total soil is needed. This practice provides a mathematical equation for correcting the unit weight and water content of the finer fraction of a soil, tested to determine the unit weight and water content of the total soil.4.3 Similarly, as utilized in Test Methods D1556/D1556M, D2167, D6938, D7698, and D7830/D7830M, this practice provides a means for correcting the unit weight and water content of field compacted samples of the total soil, so that values can be compared with those for a laboratory compacted finer fraction.NOTE 2: The quality of the result produced by this standard is dependent on the competence of the personnel performing it, and the suitability of the equipment and facilities used. Agencies that meet the criteria of Practice D3740 are generally considered capable of competent and objective testing/sampling/inspection/etc. Users of this standard are cautioned that compliance with Practice D3740 does not in itself ensure reliable results. Reliable results depend on many factors; Practice D3740 provides a means of evaluating some of those factors.NOTE 3: When this practice is used for construction control, the using agency should specify whether the maximum unit weight value used for reference is the unit weight including oversize fraction or the unit weight of the finer fraction. Calculated values of percent compaction based on this correction practice will vary depending on which unit weight value is used for reference.1.1 This practice presents a procedure for calculating the unit weights and water contents of soils containing oversize particles when the data are known for the soil fraction with the oversize particles removed.1.2 This practice also can be used to calculate the unit weights and water contents of soil fractions when the data are known for the total soil sample containing oversize particles.1.3 This practice is based on tests performed on soils and soil-rock mixtures in which the portion considered oversize is that fraction of the material retained on the 4.75-mm [No. 4] sieve. Based on these tests, this practice is applicable to soils and soil-rock mixtures in which up to 40 % of the material is retained on the 4.75-mm [No. 4] sieve. The practice also is considered valid when the oversize fraction is that portion retained on some other sieve, but the limiting percentage of oversize particles for which the correction is valid may be lower. However, the practice is considered valid for materials having up to 30 % oversize particles when the oversize fraction is that portion retained on the 19-mm [3/4-in.] sieve.1.4 The factor controlling the maximum permissible percentage of oversize particles is whether interference between the oversize particles affects the unit weight of the finer fraction. For some gradations, this interference may begin to occur at lower percentages of oversize particles, so the limiting percentage must be lower for these materials to avoid inaccuracies in the computed correction. The person or agency using this practice shall determine whether a lower percentage is to be used.1.5 This practice may be applied to soils with any percentage of oversize particles subject to the limitations given in 1.3 and 1.4. However, the correction may not be of practical significance for soils with only small percentages of oversize particles. The person or agency specifying this practice shall specify a minimum percentage of oversize particles below which the practice need not be applied. If a minimum percentage is not specified, 5 % shall be used.1.6 This practice may not be applicable to soil-rock mixtures which degrade under field compaction.1.7 Units—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.1.7.1 It is common practice in the engineering profession to concurrently use pounds to represent both a unit of mass (lbm) and a force (lbf). This implicitly combines two separate systems of units; that is, the absolute system and the gravitational system. It is scientifically undesirable to combine the use of two separate sets of inch-pound units within a single standard. This standard has been written using the gravitational system of units when dealing with the inch-pound system. In this system, the pound (lbf) represents a unit of force (weight). However, the use of balances or scales recording pounds of mass (lbm) or the recording of density in lbm/ft3 shall not be regarded as a non conformance with this standard.NOTE 1: Sieve size is identified by its standard designation in Specification E11. The alternative designation given in brackets is for information only and does not represent a different standard sieve size.1.8 All observed and calculated values shall conform to the guidelines for significant digits and rounding established in Practice D6026.1.8.1 The procedures used to specify how data are collected/recorded and calculated in this standard are regarded as the industry standard. In addition, they are representative of the significant digits that generally should be retained. The procedures used do not consider material variation, purpose for obtaining data, special purpose studies, or any considerations for the user‘s objectives; and it is common practice to increase or reduce significant digits of reported data to commensurate with these considerations. It is beyond the scope of these test methods to consider significant digits used in analysis methods for engineering design.1.9 This practice offers a set of instructions for performing one or more specific operations. This document cannot replace education or experience and should be used in conjunction with professional judgment. Not all aspects of this practice may be applicable in all circumstances. This ASTM standard is not intended to represent or replace the standard of care by which the adequacy of a given professional service must be judged, nor should this document be applied without consideration of a project's many unique aspects. The word “Standard” in the title of this document means only that the document has been approved through the ASTM consensus process.1.10 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 This test method provides a cooling time versus temperature curve (profile) that can be related to physical properties, such as the hardness obtainable upon quenching of a metal. The results obtained by this test method may be used as a guide in quenchant selection or as a comparison of quench severities of different quenchants, new or used.1.1 This test method covers the equipment and the procedure for evaluation of quenching characteristics of a quenching fluid by cooling rate determination.1.2 This test method is designed to evaluate quenching fluids with agitation, using the Drayton Agitation Unit.1.3 The values stated in SI units are to be regarded as standard. The values given in parentheses are for information only.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

5.1 This test method can be used to determine the in-place density and unit weight of natural inorganic soil deposits, soil-aggregate mixtures, or other similar firm materials. It is often used as a basis of acceptance for earthen material compacted to a specified density or percentage of a maximum density determined by a test method, such as Test Methods D698, D1557 or D4253.5.1.1 Test Methods D698 and D1557 require that mass measurements of laboratory compacted test specimens be determined to the nearest 1 g so that computed water contents and densities can be reported to three and four significant digits, respectively. This standard is a field procedure requiring mass measurements to the nearest 5 g. As such, water content calculations should only be reported to two significant digits and density to three significant digits.5.2 This test method may be used to determine the density and unit weight of compacted soils used in construction of earth embankments, road fill, and structural backfill. This test method often is used as a basis of acceptance for soils compacted to a specified density or a percentage of maximum density or unit weight, as determined by a standard test method.5.3 The use of this test method is generally limited to soil in an unsaturated condition and is not recommended for soils that are soft or that deform easily. Such soils may undergo a volume change during the application of pressure during testing. This test method may not be suitable for soils containing crushed rock fragments or sharp edge materials, which may puncture the rubber membrane.NOTE 1: The quality of the result produced by this standard is dependent on the competence of the personnel performing it and the suitability of the equipment and the facilities used. Agencies that meet the criteria of Practice D3740 are generally considered capable of competent and objective testing. Users of this standard are cautioned that compliance with Practice D3740 does not in itself ensure reliable results. Reliable results depend on many factors; Practice D3740 provides a means of evaluating some of those factors.1.1 This test method covers the determination of the in-place density and unit weight of compacted or firmly bonded soil using a rubber balloon apparatus.1.2 This test method is suitable for use as a means of acceptance for compacted fill or embankments constructed of fine-grained soils or granular soils without appreciable amounts of rock or coarse material.1.3 This test method also may be used for the determination of the in-place density and unit weight of undisturbed or in situ soils, provided the soil will not deform under the pressures imposed during the test.1.4 This test method is not suitable for use in organic, saturated, or highly plastic soils that would deform under the pressures applied during this test. This test method may require special care for use on (1) soils consisting of unbonded granular materials that will not maintain stable sides in a small hole, (2) soils containing appreciable amounts of coarse material in excess of 37.5 mm (11/2 in.), (3) granular soils having high void ratios, or (4) fill materials containing particles with sharp edges. For soils containing appreciable amounts of particles in excess of 37.5 mm (11/2 in.), Test Methods D4914 or D5030 should be used.1.5 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.5.1 In the engineering profession it is customary to use units representing both mass and force interchangeably, unless dynamic calculations are involved. This implicitly combines two separate systems of units; that is, the absolute system and the gravitational system. It is scientifically undesirable to combine the use of two separate sets of inch-pound units within a single standard. This standard has been written using the gravitational system of units when dealing with the inch-pound system. In this system the pound (lbf) represents a unit of force (weight). However, conversions are given in the SI system. The use of balances or scales recording pounds of mass lbm/ft3 should not be regarded as nonconforming with this test method.1.6 All observed and calculated values shall conform to the guidelines for significant digits and rounding established in Practice D6026 unless superseded by this standard.1.6.1 The procedures used to specify how data are collected, recorded or calculated in this standard are regarded as the industry standard. In addition they are representative of the significant digits that generally should be retained. The procedures used do not consider material variation, purpose for obtaining the data, special purpose studies, or any considerations for the user’s objectives; it is common practice to increase or reduce significant digits of reported data to be commensurate with these considerations. It is beyond the scope of this standard to consider significant digits used in analytical methods for engineering design.1.7 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

4.1 This test method is often used to determine bulk density values that are necessary for use for many methods of selecting proportions for concrete mixtures.4.2 The bulk density also may be used for determining mass/volume relationships for conversions in purchase agreements. However, the relationship between degree of compaction of aggregates in a hauling unit or stockpile and that achieved in this test method is unknown. Further, aggregates in hauling units and stockpiles usually contain absorbed and surface moisture (the latter affecting bulking), while this test method determines the bulk density on a dry basis.4.3 A procedure is included for computing the percentage of voids between the aggregate particles based on the bulk density determined by this test method.1.1 This test method covers the determination of bulk density (“unit weight”) of aggregate in a compacted or loose condition, and calculated voids between particles in fine, coarse, or mixed aggregates based on the same determination. This test method is applicable to aggregates not exceeding 125 mm [5 in.] in nominal maximum size.NOTE 1: Unit weight is the traditional terminology used to describe the property determined by this test method, which is weight per unit volume (more correctly, mass per unit volume or density).1.2 The values stated in either SI units or inch-pound units are to be regarded separately as standard, as appropriate for a specification with which this test method is used. An exception is with regard to sieve sizes and nominal size of aggregate, in which the SI values are the standard as stated in Specification E11. Within the text, inch-pound units are shown in brackets. 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.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

1.1 This test method covers procedures for the use of oxygen analyzers to measure the percentage of oxygen in an insulating glass unit where normal atmospheric air has been replaced with other gases such as argon, krypton, xenon, or sulfur hexafluoride (SF6). The procedure shows how to convert the measured percentage of oxygen in an insulating glass unit to the percentage of air in the unit, and subtracts the air percentage from 100 % to calculate the percentage of fill gas in the unit.1.2 This test method does not determine the type of fill gas. It only measures the percentage of oxygen in the gas in the space between the lites of an insulating glass unit.1.3 This test method is not applicable to insulating glass units containing open capillary/breather tubes.1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.5 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

定价： 546元 / 折扣价： 465 元

1.1 This terminology covers terms, definitions of terms, descriptions of terms, nomenclature, and explanations of abbreviations, acronyms, and symbols specifically associated with standards under the jurisdiction of ASTM Committee C12 on Mortars and Grouts for Unit Masonry.1.2 The definitions and descriptions of terms in this terminology pertain to Test Methods C780, C1019, C1148, C1324, and C1403; Specifications C144, C270, C404, C476, C887, and C1384; Practice C946; and Guide C1586.1.3 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

定价： 546元 / 折扣价： 465 元

定价： 5415元 / 折扣价： 4603 元

5.1 Stress is applied as pressure over the area of the flatjack. In the case of multi-wythe masonry, stress is estimated only in the wythe in which the flatjack is inserted. Stress in other wythes may be different.1.1 This test method covers the determination of the average compressive stress in existing unreinforced solid-unit masonry (see Note 1). This test method concerns the measurement of in-situ compressive stress in existing masonry by use of thin, bladder-like flatjack devices that are installed in cut mortar joints in the masonry wall. This test method provides a relatively non-destructive means of determining masonry properties in place.NOTE 1: Solid-unit masonry is that built with stone, concrete, or clay units whose net area is equal to or greater than 75 % of the gross area.1.2 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.1.3 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 Turbidity is monitored to help control processes, monitor the health and biology of aquatic environments and to determine the impact of environmental events such as storms, floods, runoff, etc. Turbidity is undesirable in drinking water, plant-effluent waters, water for food and beverage production, and for a large number of other water-dependent manufacturing processes. Turbidity is often reduced by coagulation, sedimentation and water filtration. The measurement of turbidity may indicate the presence of particle-bound contaminants and is vital for monitoring the completion of a particle-waste settling process. Significant uses of turbidity measurements include:5.1.1 Compliance with permits, water-quality guidelines, and regulations;5.1.2 Determination of transport and fate of particles and associated contaminants in aquatic systems;5.1.3 Conservation, protection and restoration of surface waters;5.1.4 Measure performance of water and land-use management;5.1.5 Monitor waterside construction, mining, and dredging operations;5.1.6 Characterization of wastewater and energy-production effluents;5.1.7 Tracking water-well completion including development and use; and5.1.8 As a surrogate for other constituents in water including sediment and sediment-associated constituents.5.2 The calibration range of a turbidimeter shall exceed the expected range of TU values for an application but shall not exceed the measurement range specified by the manufacturer.5.3 Designs described in this standard detect and respond to a combination of relative absorption, intensity of light scattering, and transmittance. However, they do not measure these absolute physical units as defined in 3.2.15 and 3.2.19.5.4 Several different turbidimeter designs may be used for this test method and one design may be better suited for a specific type of sample or monitoring application than another. The selection flowchart in Annex A1 provides guidance for the selection of an appropriate turbidimeter design for a specific application.5.5 Report turbidity in units that reflect the design of the turbidimeter used as recommended in 4.3. See Table 1 and Section 7 for a discussion of the design criteria and derivation of reporting units.5.6 Table 1 and Section 7 lists the turbidimeter designs currently used for in-situ measurements. Future revisions of the method may include additional designs.1.1 This test method covers the in-situ field measurements of turbidity in surface water. The measurement range is greater than 1 TU and the lesser of 10 000 TU or the maximum measurable TU value specified by the turbidimeter manufacturer.1.1.1 Precision data was conducted on both real world and surrogate turbidity samples up to about 1000 TU. Many of the technologies listed in this test method are capable of measuring above that provided in the precision section (see Section 16).1.2 “In-situ measurement” refers in this test method to applications where the turbidimeter sensor is placed directly in the surface water in the field and does not require transport of a sample to or from the sensor. Surface water refers to springs, lakes, reservoirs, settling ponds, streams and rivers, estuaries, and the ocean.1.3 Many of the turbidity units and instrument designs covered in this test method are numerically equivalent in calibration when a common calibration standard is applied across those designs listed in Table 1. Measurement of a common calibration standard of a defined value will also produce equivalent results across these technologies. This test method prescribes the assignment of a determined turbidity values to the technology used to determine those values. Numerical equivalence to turbidity standards is observed between different technologies but is not expected across a common sample. Improved traceability beyond the scope of this test method may be practiced and would include the listing of the make and model number of the instrument used to determine the turbidity values.1.4 In this test method, calibration standards are often defined in NTU values, but the other assigned turbidity units, such as those in Table 1 are equivalent. For example, a 1 NTU formazin standard is also a 1 FNU, a 1 FAU, a 1 BU, and so forth.1.5 This test method was tested on different natural waters and with standards that served as surrogates for samples. It is recommended to validate the method response for waters of untested matrices.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： 646元 / 折扣价： 550 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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