Histology and immunohistochemistry (IHC) are commonly used to visualize the cellular and biochemical constituents of tissues. Whether the attributes to be probed are detected based on chemical reactivities (histochemistry) or are detected using antibodies (IHC) or lectins with known specificities, the preparation of the specimen for staining is a critical phase of the analysis. Preparation typically involves (1) tissue fixation, (2) mounting the tissue in an embedding medium to permit sectioning that is sufficiently thin for the tissue to be viewed microscopically, and then (3) removal of any embedding medium before histochemical or IHC evaluation.

The purpose of this chapter is to identify factors that should be controlled to optimize the consistency of tissue staining results. The following sections address tissue fixation used to prevent degradation and to prepare the tissue for sectioning and staining. Since these methods are often used to characterize tissue-based therapeutic products and the adequacy of their process methods, USP sometimes includes visual tools to support those product monographs. USP Authentic Visual References (AVRs) are often histology images that have been prepared as described in this chapter and are used as reference standards associated with product monographs. The purpose of histologic analysis is to generate visual images that can be used either for illustration or as a visual reference. When used as AVRs, the image set should include representative images of both failed and passing samples at different magnifications. AVRs can be used to better clarify specifications and acceptance criteria related to, for example, cell content, collagen structure, or integrity.

The sample tissue must be properly and adequately treated or fixed to limit changes to the extracellular matrix elements or to specific constituents such as cells or proteins. Tissue fixation typically employs a chemical that can rapidly permeate the tissue to effectively cross-link proteins and limit degradation caused by either chemical or enzymatic action. Standard fixation methods typically use 10% neutral buffered histological-grade formalin and are adequate for most, if not all, histochemical analyses. Formalin fixation provides material suitable for IHC analysis provided that proper attention is paid to postfixation treatments (collectively known as antigen retrieval). Once tissue is properly fixed, it can be embedded.

The tissue is embedded in a medium that is sufficiently stiff to maintain sample geometry and the desired orientation for sectioning but is soft enough to be easily and rapidly cut through without distortion. The most common medium used is paraffin, although it is not suitable for nondecalcified hard tissues and other stiff materials (e.g., hard polymers or metals). Because paraffin is not water soluble, tissue generally is passed through a graded series of increasing concentrations of ethanol and finally into xylene (or a suitable substitute) in which the paraffin wax is soluble. Sufficient time is allowed for the paraffin to permeate the tissue, and then the block is sectioned to the desired thickness. Hard tissues commonly are embedded in hard plastics such as poly(methyl methacrylate).

After sectioning, the samples are floated on warm water with or without gelatin so they can be transferred to microscope slides. If the sections are to be used for IHC, it is recommended that gelatin not be used in the water because it can contribute significantly to background staining. Positively charged slides also are recommended to aid in sample retention throughout the IHC processing regime. The sections are allowed to dry in air, in a slide warmer, or in an oven at no more than 50. Because most histochemical and IHC procedures are carried out under aqueous conditions, the paraffin within the mounted tissue sections must be removed. This is accomplished by reversing the xylene and alcohol rinses until the samples are rinsed in water immediately before staining.

The mounted sections are now ready for histochemical staining. If an immunological staining method is to be used, in most cases an antigen retrieval step will be necessary to break the protein cross-links formed by formalin fixation. Many procedures are available to accomplish this, and although heat treatment in acid vapor appears to be effective in recovering the reactivity of most targets in tissues, the ultimate choice may depend on the characteristics of the target to be evaluated.

Histological methods sometimes are used to demonstrate that cells within a tissue sample are still viable (e.g., when evaluating a cryopreservation method's performance). In other circumstances, the method is used to evaluate the absence of viable cells following a decellularization process. Each case presents specific validation challenges.

H&E staining provides a useful tool for determining whether significant quantities of cells are still viable after preservation. During method validation, immunohistological stains can be used to ensure that specific cell types are still present in the preserved samples. Testing samples for all expected cell types during the validation phase provides support for the less specific H&E staining (that will stain all cells and not just specific cell types) during routine monitoring.

When validating any histological method, analysts should demonstrate that different observers can detect the same quality (or quantity) of an object, tissue type, cell body, nucleus, etc., and that the characterization by different observers can be replicated in several specimens following the same process. Samples for evaluation may consist of multiple types of cells, extracellular matrix proteins, and glycosaminoglycans, or other components. Some of these components may be resistant to decellularization processes or may be difficult to stain. During assessment of method reproducibility, analysts should evaluate multiple samples throughout the sample to ensure that they are representative of the types of materials expected to be found in the matrix. In addition, because inadequate decellularization procedures may not remove all cells throughout the material, it is important to take samples from multiple locations in the tissue so that remaining cells are not underestimated. Intra-operator reproducibility also should be a part of the assessment because the relatively weaker signal from fewer positively stained cells may make visualization more challenging for some technicians. The method validation should use samples that underwent worst-case decellularization processing conditions as determined by statistical modeling to ensure that residual cells can still be identified and quantified in these marginal samples. Finally, the validation of the method should include control samples to confirm the suitability of the staining system (see Hematoxylin and Eosin Staining of Sectioned Tissue for Microscopic Examination 1285.1).

The histological assessment of decellularized tissue-based matrix products is one tool to characterize the matrix but cannot be the sole indicator of product quality. On the one hand, processing may generate decellularized matrix products that have no detectable cells but may not possess other critical quality attributes. On the other hand, tissue matrix products undergoing other decellularization processes may result in products with greatly reduced cellular content (although some cells may remain) that retain the necessary performance characteristics for that therapeutic purpose.

This chapter outlines common steps to prepare tissue or organ specimens for light microscopic analysis following either staining or treatment with structure-specific probes. Each of these steps can produce specimen artifacts that either interfere with the observation or actually introduce structures that were not present in the original sample (e.g., certain well-known chemical artifacts). Therefore, proper attention and consistent execution of each step are critical factors in generating sections that reflect the character of the tissue and that stain in a reproducible manner.