Reference materials preparative amounts

Use of Complex Standards for Cell Proteins and Two-Dimensional Electrophoresis. One of the more widely-used cell lines chosen as a reference standard is the lymphoblastoid cell line GM607, derived from a normal individual and available from the Human Genetic Mutant Cell Depository, Camden, NJ 08103. This cell line may be grown in defined media, labeled with a radioactive tracer, and reproducibly separated in a 2-DE system. Heat shock proteins may readily be isolated and visualized from this cell line, as shown by Anderson et al. (42). For serum, a reference preparation for serum proteins is available as a certified reference material prepared and assayed by the College of American Pathologists (CAP) and by the U. S. Centers for Disease Control. A widely available human serum standard is that provided by the National Bureau of Standards as SRM 909. If sufficient interest from the user community is evident, a full electrophoretic characterization of this material can be included in the documentation. If the amount of selected standard proteins loaded on a gel is known, "relative" quantification of similar proteins could be obtained. In addition, the National Bureau of Standards could serve as an impartial evaluator of potential national standards (e.g. molecular weight standards, "tie-point" proteins, and Isoelectric point standards) to assess suitability and stability. 

In those cases where there are any doubts about the feasibility of producing a sufficiently homogeneous and stable reference material, a feasibility study might be needed. For this study, an extra amount of material is needed. Questions regarding the best way of preparing the sample, the stability of the material, or the fitness for purpose might justify the inclusion of a feasibility study in the project. In the BCR projects, it is common practice to have a feasibility study, which usually has as the sole purpose of assessing the performance of the laboratories in the collaborative study in relation to the certification of the reference material. The feasibility study allows the participants to fine-tune their equipment, their methods, and their procedures in view of the characterization measurements. In each of these cases, a considerable extra number of samples is needed. 

In the preparation of many solid state reference materials, reduction of the grain size plays an important role. Usually this reduction is required because of the measurement methods to be used both in the projects and later by the users of the reference material, as well as to come to an acceptable minimum sample intake. The minimum sample intake can be defined as the minimum amount of material needed, so that the heterogeneity of the material does not affect the repeatability of the measurement method. The reduction of the grain size is usually implemented by crushing and/or grinding techniques. The techniques employed and the equipment used must be suitable for the purpose of processing the material. Potential problems of contamination, loss of volatile components, and/or other physical and... 

Two different and possibly complementary approaches have been explored. One utilizes a panel of quantifiable internal reference standards (QIRS), which are common proteins present widely in tissues in relatively consistent amounts.11,22 In this instance because the reference proteins are intrinsic to the tissue they are necessarily subjected to identical fixation and processing, and incur no additional handling or cost, other than synchronous performance of a second IHC assay (stain), such that the intensity of reaction for the QIRS and the test analyte can be compared by IA, allowing calculation of the amount of test analyte (protein) present on a formulaic standard curve basis. The other approach seeks to identify external reference materials and to introduce these into each step of tissue preparation for cases where IHC studies are anticipated in this instance the logistical issues of production, distribution, and inclusion of the reference standard into all phases of tissue processing also must be considered, along with attendant costs. 

The starting material (batch) after appropriate homogenization should be stored in appropriate containers. Later these containers would be distributed to the interested scientists in order to support their measurements. Of course, first, the appropriate containers, in terms of size (large, small), shape (bottle, vial), properties (hard, soft, coloured), material (glass, plastic) have to be selected. Some other important items at this stage are the preparation of the units imder the appropriate conditions (e.g. freeze-dried material tmder low humidity), each unit should contain an appropriate amount of material (depending on the amoimt needed for each measurement and the availability of the material) and the appropriate number of units has to be decided (taking into account the needs for this specific certified reference material). 

An example of a primary procedure to establish the mass fraction of a pure reference material is the use of coulometry to prepare an amount of an... 

Extraction to gas phase, liquid phase and solid phase can be used in the preparation of SVOC/POM samples. It is essential to estimate the recovery of each extraction step. One method is spiking the sample with known amounts of internal standards similar to the analytes. The problem of this method is that spiked standards may not bind to the matrix in the same way as the analytes. Another method is to find the method with the highest recovery of a number of methods. Probably a combination of the two methods will give the most reliable results. Finally, use of certified reference materials, if available, will be the best way to determine the total recovery. 

A third and very important example is one where the unknown amount is not measured by a PMM directly in SI- or other units, but against commonly accepted references, usually values carried by reference materials (in whatever units). This is especially important in cases where the substance cannot (yet) be unequivocally identified as a unique substance (e.g. protein in beef or fibre in com flakes, both very important in trade). Since an amount-of-substance measurement is not (yet) possible because the substance to be measured is not (yet) uniquely defined, we go back to the traditional definition of calibration as illustrated in Fig. 5 [2], Several reference samples of different contents are prepared (or agreed) and their contents ex-... 

The reference materials have the form of pellets, globules, shot, wires or bars. They are intended for use as amount of substance standard (traceability to SI) and are applied for preparation of calibration solutions. They are available only for producer of calibration standards or for national metrology institutes. 

For both consistency over each day, and to check day-to-day reproducibility, it is very useful to analyse at regular intervals either synthetic standard solutions, prepared in an appropriate matrix, or, better still, standard or certified reference materials, which contain precisely known amounts of the elements or species of interest. Routine long-term time-plots of the results of such regularly repeated analyses are very useful in showing up errors arising as a consequence of hitherto unrealized procedural changes. 

Sometimes, during the course of determining the capacity of the stationary phase and the adsorption isotherms, it turns out that significant preparative amounts of reference material can easily be obtained even with analytical... 

Control samples should have a high degree of similarity to the actual samples analyzed otherwise, one cannot draw reliable conclusions on the measurement system s performance. Control samples must be so homogeneous and stable that individual increments measured at various times will have less variability than the measurement process itself. Quality Control samples are prepared by adding known amounts of analytes to blank specimens. They can be purchased as certified reference material (CRM) or may be prepared in-house. In the latter case, sufficient quantities should be prepared to allow the same samples to be used over a longer period of time. Their stability over time should be proven and their accuracy verified, preferably through interlaboratory tests or by other analysis methods. 

Secondary reference materials are solutions whose concentrations cannot be prepared by weighing the solute and dissolving a loiown amount into a volume of solution. The concentration of secondary reference materials is usually determined by analysis of an aliquot of the solution by an acceptable reference method, using a primary reference material to calibrate the method. 

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