30 Histochemistry Bone and Cartilage
Histological and histochemical analysis of bone are useful techniques for examining growth and development as well as diseases associated with bone. To obtain high-quality data, every step from the choice of fixative through mounting of slides can affect the final outcome of the stain quality. Unfortunately, all specimen-handling procedures may be suitable for one of the analyses but not for others. For example, the fixation and embedding step affects the histological appearance of morphological details in bone specimens, whereas it is the most problematic step for the detection of bone specimens in regard to immunohistochemistry. In this chapter, we will discuss the technical difficulties associated with different histological and histochemical stainings.
30.1 Tissue Fixation
After tissue collection, the first step of the procedure is tissue preservation for future investigation. The purpose of fixation is to stabilize the proteins in the tissues and preserve their microarchitecture for investigation. Fixatives also inhibit the growth of bacteria and molds that give rise to putrefactive changes. Currently, 10% buffered formalin, 2.5% gluteraldehyde, and alcohol fixatives are the most widely used fixatives for routine light microscopy and ultrastructural studies. However, every fixative has different capabilities for staining certain organelles and maintaining tissue integrity, and the choice of fixative should be based on the tissue being stained.
30.1.1 Fixatives
Formaldehyde (4% buffered formaldehyde, 10% buffered formalin)
Widely used universal fixative for paraffin embedded sections
Requires a relatively short fixation time but can also be used for long-term storage
Produces no deleterious effects on tissue morphology with nuclear and cytoplasmic detail
Slow penetration
Requires fixation at temperatures of 1 to 4°C
Fixed tissue specimen can be stored in buffer solution for many months
Alcoholic fixatives
Include Clarke′s fluid (ethanol and acetic acid, 3:1 by volume), Carnoy′s fluid (ethanol, chloroform, and acetic acid, 60:30:10 by volume), and 70% ethanol diluted in acetic acid
Better histological preservation and retain reactivity for labile lymphocyte membrane antigens
30.1.2 Factors that Affect Fixation
Volume ratio: Ratio of the fixative volume to the tissue volume needs to be more than 10 times.
Temperature: For standard histology, fixation can be carried out at room temperature; for electron microscopy and histochemical procedures, fixation should be done at 4°C.
Size: Tissue blocks should either be small or thin. Recommended thickness is ≤ 3 mm.
Time: 2 mm thick tissue blocks = 4 to 8 hours; large specimens = overnight.
30.2 Decalcification
Decalcification is usually carried out between the fixation and processing steps. The removal of calcium deposits in bone or other tissues that contain calcified areas is essential to improve sectioning and staining quality.
30.2.1 Decalcifying Agents
Mineral acids: dilute nitric or hydrochloric acid
Fast decalcification, suitable for dense cortical bone
Damage cellular morphology
Not recommended for delicate tissues such as bone marrow
Organic acids: acetic acid, formic acid
Not as harsh as mineral acid
Damage cellular morphology if expose too long
Suitable for bone marrow or other soft tissues
Require longer decalcification time
Chelating agents: ethylenediaminetetraacetic acid
Slow penetration and slow decalcification
Take weeks for decalcification
Better for histochemical methods
30.3 Histology and Histochemical Staining Methods
30.3.1 Hematoxylin and Eosin Stain
The hematoxylin and eosin (H&E) technique is widely used in the histopathology laboratory and is essential to demonstrate the general tissue structures, enabling recognition of malignant and nonmalignant cells as well as several intracellular and extracellular substances necessary for medical diagnosis. This staining has been used for a long time because it works well with a variety of fixatives and displays a broad range of cytoplasm, nuclear, and extracellular matrix features.
Hematoxylin is a natural dye, obtained from the heartwood of the logwood tree. It has no staining properties until it is oxidized to hematein, a compound that forms strongly colored complexes with metal ions such as iron. In H&E staining, hematoxylin stains the nuclei blue, enabling recognition of the nuclear features of different types of cells, such as malignant and nonmalignant cells. Hematoxylin can be used as a nuclear counterstain.
Eosin is a fluorescent red dye resulting from the action of bromine on fluorescein. Eosin Y is most often used as a counterstain to hematoxylin in H&E staining. It is typically used in concentration of 1 to 5% by weight volume dissolved in 1% acetic acid.
Before using a new batch of H&E solutions, a simple test should be performed for quality assurance. From that, we should be able to (1) determine the performance of each new container of stain, (2) decide suitable staining times, (3) find out how many slides can be stained satisfactorily by a given volume of each stain, (4) how many rinses should be changed, and (5) troubleshoot the cause of an observed staining problem. After staining times and stain and rinse change schedules have been determined, the use of control sections is not necessary for the remainder of the life of the particular stain that has been validated.
Key Concepts: The Final Staining Pattern of Hematoxylin and Eosin
Cytoplasm: Pink to orange
Nuclei: Blue
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