Quantifiable Internal Reference standardization

Indeed, there are potentially several different levels to approach standardization of IHC For the greatest scientific rigor, it is required to perform serial experiments based upon the hypothesis mentioned above to investigate and validate, if possible, standardization of IHC based on the AR technique (Chapter 5 in detail). Another approach, that may be combined with AR, is the systematic development of Quantifiable Internal Reference Standards (QIRS) that will allow assessment of the degree of protein degradation... 

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. 

Taylor CR. Quantifiable internal reference standards for immunohistochemistry the measurement of quantity by weight. Appl. Immunohistochem. Mol. Morphol. 2006 14 253-259. 

In order to quantify compound X (with mass Mx) in a mixture, a quantity p mg of reference compound R (with mass MR) is added to P mg of the sample containing compound X. The internal reference standard R is chosen so that the signal it generates does not interfere with the signal used to quantify compound X (Fig. 9.25). 

Figure 9.25—Spectrum of a sample into which an internal standard R has been added. Peak X belongs to the compound to be quantified and peak R to the internal reference compound.

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

The colloidal state inevitably brings about difficulties for the experimentalist when separation of the disperse phase from the dispersion medium is needed. This is the case when the speciation and concentration of only the free soluble species have to be determined. Separation of the ionic solution from the small colloidal particles for conventional chemical analysis is nontrivial, although separation techniques such as ultracentrifugation, dialysis, and field-flow fractionation have been successfully used. If the soluble species of interest have an active nuclear spin, the liquid NMR technique wiU constitute an alternative and simpler way to characterize and quantify those species without being affected by the disperse phase. An exception is the case where the colloidal species gives a signal that fully overlaps the sharp resonance of the solution entity. As NMR is quantitative, the absolute concentration of the species can be estimated based on an internal reference of known concentration but different chemical shift relative to the sample signals. Alternatively, a calibration curve can be established from a set of external standard solutions (preferably the same substance found in the sample) measured under the same experimental NMR conditions as those applied to the sample. 

Note The analytical problems of inorganic MS often require only certain selected isotopes or narrow m/z ranges to be measured. Multicollector systems, for example, are adjusted to simultaneously detect a few isotopes for the purpose of accurate isotope ratio determinations or to quantify a low-abundant isotope together with an isotopic standard for internal reference. Thus, the data is more often presented in tabular form or in plots of concentration versus variables such as depth of invasion, age of samples, or location on a surface. Mass spectra covering a wider range are only acquired for survey multi-element detection. 

For trace analysis, it is preferable to use a method that relies on the relative response factor for a compound against a reference compound. The areas of the compounds to be quantified are compared to the area of a reference compound, called an internal standard, present at a given concentration in each one of the samples (see Fig. 4.13). This approach can compensate for imprecision due to the injected volume and instrument instability between successive injections. It is superior to the preceding method where all of these factors influence the quantification. 

GC-MS can be used to analyze organochlorine pesticides, for example, a-, [3-, y-HCH, HCB, and polychlorinated biphenyls (PCB). The components are quantified by using an internal standard. Furthermore, a calibration is performed with a standard mixture containing known concentrations of the components to be measured and one or more components not contained in the sample (internal standards). The calibration is followed by injection of the sample containing known amounts of internal standards. Quantification is relative to the internal standard. In this way, the sample extract volume will not be included in the calculations, and it is not necessary to accurately determine the final sample volume after evaporation of the injection volume. The GC-MS instrument should be calibrated every day. The sensitivity of the mass spectrometer can, for instance, be controlled daily by determining the signal-to-noise ratio for a given amount of a chosen component (PCB-101 could be one such component). For further details of the method, the reader is referred to different manuals and papers on the subject.417... 

This method is based on a comparison of the intensities of the signal corresponding to the product that has to be quantified with the one of a reference compound called the internal standard. This method allows the elimination of various error sources other than the minimal intrinsic error due to statistical reasons. In fact, if we choose as an internal standard a molecule with chemical and physical properties as close as possible to the properties of the molecule to be measured, the latter and the internal standard undergo the same loss in the extraction steps and in the derivative or the same errors in the introduction of the sample into the mass spectrometer, when the source conditions are varied. As both... 

Obtaining appropriate internal standards and standard reference materials has also proven difficult [94]. As a result, researchers have employed a variety of chemicals for internal standards [93]. For example, Hansen et al. [105] used the 6 2 FTS/THPFOS to quantify PFHxS, PFOS, PFOA and PFOSA in biological matrices. This internal standard was subsequently measured in groundwater samples from military bases [98] and illustrates the importance for researchers to scan samples for any analyte being used as an internal standard. A suite of and O-mass labelled PFCs have become commercially available and it is recommended they be employed as internal standards [93,97,128]. Prior to use, it is suggested that the purity of these labelled standards be confirmed to ensure the absence of native (unlabelled) PFCs. 

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