REVIEW OF PRANALYTICAL FACTORS TO IMPROVE THE QUALITY OF IHC

REVIEW OF PRANALYTICAL FACTORS TO IMPROVE THE QUALITY OF IHC

Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue (compare to immunocytochemistry). The procedure was conceptualized and first implemented by Dr. Albert Coons in 1941.  Immunohistochemistry (IHC) is a method used to determine the expression of biomarkers in tissue.

Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. The histological process, which begins with the acquisition of tissue samples and continues through to the interpretation of IHC or ISH results, can be broadly broken down into three main stages:

v  pre-analytical,

v  analytical and

v  post-analytical.

In this review we are going to discuss about the pre analytical procedures that should be taken into account while preparing section for IHC.

IHC: What can go wrong?

v  PREANALYTIC

Ø Fixation

Ø Selection of markers

v  ANALYTIC

Ø Choice of antibody, retrieval

Ø Use of controls

v  POST-ANALYTIC

Ø Interpretation of immunostaining (+/-)

Ø Interpretation of immmunoprofile

Pre-analytical pitfall: Fixation

Ø  Tissue does NOT fix at 1mm/hr

Ø  mm/hour is permeation time

Ø  Cross-linking of proteins takes much longer

Ø  Optimal fixation: 8 – 24 hours

Fixation

Ø  Small biopsies need as long fixation time as large tissue blocks

Ø  Under-fixation causes more problems than over-fixation

Pre-analytical pitfall:Choice of immunomarker

Ø  Critical

Ø  Should always be based on the morphological differential diagnosis

Fixation modifies the physicochemical state, including redox and membrane potentials, of the tissue, and thereby it changes the reactivity of cellular components with the stain. Consequently, the results of various histological and histochemical staining methods are modified depending on the prefixation used. In addition, many other parameters influence the quality and reliability of immunoreactivities, such as the thickness of histological sections, the dilution range of the antisera used as first layers, and the type or composition of the buffers used for dilution of antisera and of the chromogens (e.g., DAB or FITC), or as the rinsing solution. However, a critical comparison of different fixation media is still missing, although bad fixation quality generally strongly impairs the exact localisation of reaction products in the tissues and organs investigated. An ideal fixation should preserve the original structure of the tissue as good as possible and should be able to provide an equivalent close to the natural state. This demand can be accomplished, for example, by fast penetration of the fixation fluid into the tissue, thus avoiding autolysis and guaranteeing rapid conservation

The pre-analytical stage begins as soon as a piece of tissue is removed from its nutritional source (blood supply) and the time to fixation is critical. Degeneration is caused primarily by autolysis, which is a process of self-digestion by enzymes contained within cells; and this begins immediately. This process is accelerated by increased temperatures. Fixatives are used to stop degeneration, while preserving the structure and integrity of the tissue elements as much as possible.

Fixation:

Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability. The most common fixatives used for immunohistochemistry are the followings:

v  4% paraformaldehyde in 0.1M phosphate buffer

v  2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer

v  PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer

v  4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry

However, fixation itself introduces artifacts and the ideal fixative would also maintain the structure of all of the epitopes in the tissues. This is not achievable, as the alteration in chemical structure caused by fixation necessarily modifies at least some epitopes. For IHC and ISH procedures it is critical that the tissue does not dry out during any stage of the tissue handling and slide preparation. Drying may cause morphological changes, such as poorly defined chromatin; and subsequently alter the structure of the target particularly along the edge of the tissue. This could inhibit ligand binding and is particularly applicable to small specimens such as endoscopic biopsies. Additionally, dry tissue is more adsorbent, which increases the risk of non-specific or unwanted adsorption of reagents during staining procedures, thereby interfering with interpretation of results.

Influence of the temperature and the duration of fixation

An often forgotten but critical aspect is the temperature during fixation. Whereas fixation in Ca-formol and Bouin`s solution are fairly independent of temperature influences and can be mainly conducted at room temperature. In general, low temperatures retard autolysis, but they also decrease the diffusion rate and thus prolong penetration. In our experience, it can be concluded that it is particularly important to have a temperature of not more than 4°C during the incubation. Even only slightly higher temperatures during this fixation process resulted in a reduced quality of tissue preservation.

Furthermore, the duration of fixation influences fixation quality. Fixing tissue in formalin-based solutions for a time less than 24 hrs generally results in a mixture of formalin and ethanol fixation. The latter aspect happens during postfixative rinsing of the samples in 70% ethanol and during embedding. This means that an interruption of the formalin fixation before it is completed will lead to cross-linking only at the tissue periphery promoting a crust formation. In other words, near the centre coagulation occurs, caused by the ascending ethanol solutions during dehydration, or the centre of the tissue sample remains unfixed including tissue hardening.

Moreover, Ca-formol solutions are very susceptible for over fixation problems. Even a controlled prolonged storing in formaldehyde media may lead to excessive cross-linking and cause irreversible damage of epitopes, which diminishes the immunoreactivity during IHC experiments. This advantage is more relevant regarding Bouin`s fixative that is better suitable for a longer fixation, because it generally produces no over fixation effects. Such quality supports the view of Pol André Bouin, who recommended his solution particularly for embryonic tissues. Other restrictions of fixation quality may occur realizing that many organs are composed of different tissues types, which may include varying structural densities with the consequences of varying penetration times of the fixatives within the organ, producing an only more or less acceptable tissue structure. For example, the esophagus epithelium of mammals contains great amounts of keratins, in contrast to most of the other organs, and it is surrounded by a rather voluminous tunica muscularis. Thus, reduced tissue preservation could be a result of such particular cell or organ stabilizing characteristics. Especially the diminished structural quality of Ca-formol fixed tissue might origin from a slow penetration rate of the fluid in such structures, in comparison to Bouin`s solution. As already emphasised earlier, a mixture of different fixatives is still the best way to achieve relatively high quality tissue preservation by a rather steady progress of solution penetration.

Cold Ischemic Time

Recently, there has been more of a focus on “cold ischemic time” and the impact this may have on IHC and ISH results. The duration of cold ischemia is calculated from when the tissue is removed from the body to when the tissue is placed into fixative. This time should be as short as possible, with published guidelines of one hour or less . The deleterious effects of delayed fixation are increased, decreased or de-localized immunoreactivity. It should be noted that deterioration of an epitope due to ischemia cannot be recovered using antigen retrieval techniques. Relatively little has been published on the ischemic effects for specific antigens or molecular targets which are Class I. Perhaps, a broader understanding of the interrelationship between ischemic time and different targets will be easier, once the recording of ischemic times becomes a part of required documentation for all specimens.

Accessioning and Documentation

When the specimen is received in the laboratory it is ‘accessioned’ and given a unique, traceable number. The documentation (requisition) which accompanies each surgical specimen should include: patient and physician information, date of procurement, clinical information, specimen site and type, collection time, cold ischemic time, type of fixative and duration of fixation. If it is necessary to decalcify a specimen, then that information must also be recorded, including: time in fixative before decalcification, the type of decalcification used, the length of decalcification and any post-decalcification treatment. Part of the sample verification process during accessioning is to confirm that the information on the requisition matches that on the specimen container. The specimen container should have a minimum of two identifiers such as patient name and date of birth.

Grossing

Once a specimen is deemed acceptable, it is examined macroscopically. This is referred to as grossing and it is a critical pre-analytical step which requires proper training. Larger specimens should be ‘bread loafed’ (sliced) into approximately 5 mm sections and placed in 10% NBF. Gauze or paper towel may be placed between the slices to facilitate exposure to the fixative. Care must be taken to handle each type of tissue in a standardized manner and not to physically damage the tissue. Usually, it is necessary to select areas of interest from a larger specimen. These pieces of tissue, or blocks, should be trimmed such that the size does not exceed 20 mm in length and width, or 4-5 mm in depth. Trimmed tissue is then placed into a processing cassette and submerged immediately into the desired fixative (usually 10% NBF). The volume of fixative should be approximately 10 to 20 times that of the specimen . Formalin enters the tissue relatively quickly, but the chemical processes which actually fix the tissue are more time consuming, taking at least 24 hours. When calculating total time in fixative, the time the specimen sits in 10% NBF in the grossing area and on the automated tissue processor must be included.

Tissue Processing

During tissue processing, fixation reagents containing water are replaced by wax (polymer, non-polymer and microcrystalline formulas exist) which is done through a series of passages through increasing concentrations of alcohol, up to 100% (absolute) alcohol. This process is followed by clearing the alcohol from the tissue (for example by using xylene) and replacing it with molten wax. Low melting temperature (45 °C) as opposed to higher melting temperature (65 °C) waxes have been reported to produce better staining results for IHC, particularly in T-lymphocyte staining. Next, the paraffin infiltrated pieces of tissue are embedded to form blocks, which are easily handled, cut and subsequently stored.

Rapid Tissue Processing

There is increasing pressure to shorten turnaround times (TATs) in tissue pathology, so that patients do not have to wait days to receive their pathological diagnoses. However, the laboratory staff still need to ensure that samples are properly fixed (>24 hours in NBF, even for needle biopsies), to make sure that validated IHC and ISH methods are used. As noted above, tests used after alternative fixation and processing must be fully re-validated. This requirement is particularly applicable to the modern rapid tissue processors which employ alternative fixatives and microwave enhanced processing (as well as small specimen size). This combination allows an H&E diagnosis on paraffin sections the same day. Nonetheless, the morphology will differ from routine FFPE processed samples; and IHC and ISH methods will require complete re-validation, as some of these will not need pre-staining antigen retrieval, whether this is of the heating or proteolytic type (personal observation). Section Preparation Generally, unless otherwise specified by a protocol of choice, sections for IHC or ISH are cut at 3 μm, 4 μm or 5 μm. Thicker sections may cause difficulty during staining, and also problems in interpretation due to the multi-layering of cells. After sections are cut they are usually floated on water and picked up onto glass slides that are coated with some adherent material. Sections must lay flat against the glass to prevent lifting during staining or bubble formation, which may trap staining reagents. The more points of adhesion the more likely the tissue will remain fixed to the slide, supporting the need for thinner sections. Some commercially available slides come with a positive charge that attracts the negative charges of tissue proteins. These charged slides are especially effective following formalin fixation of tissues, since formalin blocks amino acids in tissues, rendering the tissue more acidic and therefore more negatively charged. Different manufactures of staining platforms may recommend the use of particular slides to achieve optimal results. As with every other pre-analytical step, cutting and mounting sections onto glass slides, and all steps prior to staining must be standardized. For example, if the slides are to left at room temperature for 15 minutes, in an upright position to allow draining of excess water and then heated in staining rack at 60 °C for 30 minutes prior to staining, this step must be repeated every time sections for IHC or ISH are prepared. Finally, the changes resulting from block and section storage prior to IHC and ISH staining may also affect staining results. For example, it is recommended that sections cut for HER2 testing should not be used if they are more than 6 weeks old.

Dewaxing and Hydration

Wax must be removed completely from the tissue sections, so that aqueous antibodies, molecular probes and detection reagents can penetrate and adhere to the tissue. Traditionally, dewaxing was done by immersing the sections into a dewaxing solution (such as xylene), with or without prior brief heating. This step was followed by absolute and graded hydrating solutions (generally alcohols), until the final solution: water. If xylene is used to dewax sections, approximately 50 slides per 50 mL of xylene is the limit, before it is no longer effective and residual wax begins to cause artifacts in the stained tissue. Today, there are many commercially available staining platforms which include onboard removal of wax and rehydration of the tissue sections. The accumulation of residual wax may be a problem with these instruments, if rinsing is insufficient or if solutions are not replenished regularly.

Conclusion

Patient safety based on accurate interpretation of results depends heavily on this standardization of all pre-analytical variables. Prognostic tests using IHC and ISH are being developed and they will independently forecast clinical outcomes for patients. HER2, ER and PgR are considered predictive markers that influence the selection of patients who will respond more favorably to therapies, emphasizing further the need for standardization. Even if it is not possible to perfectly optimize every pre-analytical step, it is possible to perform each step in the same manner each time it is done. Rigorous adherence to this approach will yield more meaningful results and will, if necessary, facilitate problem solving