ProcessControl for Standardization of Multiple Step Staining Methods
Hadi Yaziji MD; Vitro Molecular LAboratories, LLC; Alton D. Floyd PhD; ImagePath Systems, Inc.;
Content:
Context: Procedures to stain biological objects, such as tissue specimens for diagnostic purposes, often contain a number of sequentially applied steps. To insure reproducible results from one staining run to another, each sequential step must be rigidly controlled. In the case of immunostains, this is particularly difficult as the most common staining protocols involve two or more amplification steps, which may vary from one stain run to another, even when the same staining protocol is used. A further complication is that many modern immunostain detection systems consist of mixtures (polyvalent) of secondary antibodies to recognize different species of primary antibody. The user has great difficulty in determining whether the various components of such polyvalent mixtures perform and age at identical rates.
Technology:
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Design:
Design: A new patented technology has been developed to monitor staining processes. This new process consists of a control device (microscope slide) containing precise dilution series of reactants which monitor each step of a sequential stain procedure. In the case of an immunostain, the device contains dilutions for each species of secondary antibody used, dilutions for each of the coupling chemistries used, as well as the enzyme(s) used to generate the visible staining result.
Results:
Results: By utilizing this control device for each staining run, a visual record of changes in any sequential step of a process is evidenced by either a complete failure of a particular analyte to react, or a change in sensitivity level. The device is particularly well suited to monitor mixtures of secondary antibodies such polyvalent detection systems, and identifies aging differences in individual components of secondary antibody cocktails. Image analysis may be used on the stained device, permitting run to run variations in staining to be normalized, as there is a standard to calibrate variation in amplification in any given amplification step.
Conclusion:
Conclusions: The use of a process standard is recommended to eliminate much of the subjectivity associated with run to run variations in sequential staining methods such as immunostains.
