20 Immunohistochemistry quality control
Factors affecting stain quality
Tissue factors
Fixation
Tissue fixation has a significant influence on immunohistochemistry as most antigens are altered during this process (Williams et al. 1997). The purpose of fixation is to preserve tissue and prevent further degradation by the action of tissue enzymes or microorganisms. As discussed in an earlier chapter, good fixation requires tissue to have adequate time in the fixative to allow the solution to penetrate, retaining uniform cellular detail throughout the tissue. However, in the routine laboratory this ideal may be compromised as it is difficult to define a standard tissue size, fixation time, and fixative for each specimen type. Tissues need to be adequately, but not over-fixed, so that antigenicity is preserved without excessive alteration. Prolonged fixation can result in the irretrievable loss of many antigens, particularly membrane-associated antigens such as CD20 and immunoglobulin (Ig) light chains (Miller et al. 1995; Ashton-Key et al. 1996). Lack of adequate fixation, or delay in fixation, may also be equally detrimental to labile antigens (Donhuijsen et al. 1990; von Wasielewski et al. 1998; GEFPICS-FNCLCC 1999).
Any fixative used must be compatible with immunohistochemical staining methods and formalin is still the most universal of fixatives. Formulations differ between laboratories and include 10% neutral buffered formalin (NBF), 10% formalin in tap water, 10% formal saline, and 10% NBF with saline (Angel et al. 1989; Williams 1993; Williams et al. 1997). Even though it may be the pathologist’s choice, it can create a challenge for demonstrating certain antigens. Dabbs (2006) characterizes formalin as:
Processing
Regarding paraffin processing of tissues for immunohistochemistry, as with fixation, there is no standard protocol for the optimal demonstration of all antigens, as concluded by Williams et al. (1997). In a study of laboratories in the UK, Williams (1993) found nearly as many different schedules as the number of laboratories participating in the survey. Of the nine tissue-processing factors investigated, only two had any significant effect on immunoreactivity. Increasing the temperature of processing from ambient to 45°C, as well as longer processing times for dehydration and wax infiltration, were both found to improve immunostaining. Other factors including type of processor, type and quality of reagents, time in clearing agent, use of vacuum, most of which had been suggested as possible causes of poor processing (Horikawa et al. 1976; Trevisan et al. 1982; Anderson 1988; Slater 1988), were found to have no effect on subsequent immunohistochemistry.
Reversal of fixation/epitope retrieval
The quality and reproducibility of immunohistochemical staining relies upon the reversal of fixation, which results in the targeted epitope being exposed, allowing for the antigen binding site to be available. The revolution of reversing the hydrogen cross-bonds formed by formalin was introduced by Shi et al. (1991). There are now numerous methods for epitope retrieval including protein digestion techniques or, more commonly, heating the slides in a buffered solution. Cattoretti et al. (1993) introduced the solution most commonly used in standardized retrieval methods. They used a citrate buffer at pH 6.0, which is inexpensive, stores easily, and is readily available commercially or easily prepared in the laboratory. Other buffers used include EDTA-based solutions at a higher pH range, which produce more intense staining of some antibodies. These methods have allowed for the successful demonstration of a much greater range of antigens in tumors, including proliferation markers and oncogene expression. The use of automated immunostainers has brought greater standardization of retrieval methods, as these use standard retrieval solutions with defined reproducible protocols. Non-automated laboratories may have a number of variables that require internal standardization in the antigen retrieval technique including the choice of heating method (e.g. pressure cooker, microwave, etc.), retrieval solution, pH, temperature, volume of the fluid, and the temperature and exposure time while heating and cooling slides.
Procedural factors
Block and slide storage conditions
Pre-cutting control slides is more time efficient and serial sections result in minimal tissue loss. The correct storage of pre-cut slides is important and frequently overlooked as a potential source of error in staining. Some studies have found deterioration of antigens in stored sections (Raymond & Leong 1990; Bromley et al. 1994; Prioleau & Schnitt 1995). Others have found no deterioration of some markers investigated, including estrogen receptor (ER), CD3, CD20, CD45RO, vimentin, and Ig light chains, in sections stored for up to four months at room temperature (Williams et al. 1997; Eisen & Goldstein 1999).