Technical aspects: how do we best prepare bone samples for proper histological analysis?

Introduction

Histological analysis of bone is a critical step for the diagnosis of malignancies. It allows direct identification of malignant cells inside marrow spaces in case of bone metastases or hematological disorders. Bone biopsy is superior to marrow aspiration because the microarchitecture of the bone marrow is preserved, a parameter that is especially important in hematological disorders. Because marrow cells are in direct contact with bone cells (lining cells, osteoblasts, osteoclasts, and their precursors), an abnormal bone remodeling rate has been described in a variety of malignant cell proliferations when developing and expanding inside marrow spaces. Bone cells elaborate and synthesize a variety of cytokines acting on hematological precursors (e.g., M-CSF) [ ] and malignant cells release other cytokines active on bone remodeling [ ]: it is likely that bone changes are almost always associated with bone marrow alterations and vice versa. Histomorphometric analysis is a powerful tool in the evaluation of bone remodeling in metabolic bone diseases and was also successfully applied to hematological disorders and metastases from solid tumors [ , ]. Bone histomorphometry is a powerful method in the early diagnosis of B-cell malignancies, and smoldering myeloma or lymphomas can be characterized in patients with a monoclonal gammopathy of undetermined significance (MGUS) several years before the tumor has shown clinical expression. Bone histomorphometry is also useful in animal models of cancer bone lesions, since it permits a precise evaluation of the bone remodeling changes induced by tumor cells [ ]. However, bone histomorphometry must be done on undecalcified bone sections which allow a perfect identification of osteoid tissue (the unmineralized bone matrix recently synthesized by osteoblasts), a precise identification of osteoclasts (by using histoenzymatic detection) and histodynamic analyses (after a double tetracycline labeling in humans or using a variety of other fluorochromes in the animal). These methods cannot be used on decalcified and paraffin embedded bone, since decalcification abolishes the osteoid/bone matrix differential staining and removes the fluorochrome labels, and hot paraffin embedding destroys enzyme activities. However, decalcification and paraffin remain useful for immunohistochemistry, which is difficult and hazardous on plastic sections. The main disadvantage of polymer embedding was formerly the prolonged time for preparing bone specimens (several months when polyester resins were used). With the development of histological techniques, it is now possible to have polymer embedding methods that are as fast as conventional paraffin methods. The following techniques have been developed and improved in our laboratory during the last two decades and used on more than 3000 human bone biopsies and a large number of animal studies performed in a variety of animal species (for example, mouse, rat, chicken, dog, goat, sheep, pig).

Bone biopsy in humans or large animals

As recommended since the 1970s, bone trephines with a large inner diameter must be used: a 7-mm trephine is necessary to preserve bone microarchitecture and to analyze a representative area of marrow spaces. We have proposed several modifications to the ergonomics of the Meunier's trephine, making it easier to handle and providing better preserved bone cores [ ]. The trephine developed in our laboratory is manufactured by Commeca (Commeca, Beaucouzé-Angers, France) ( www.commeca.com ). Bone biopsy in human patients is painless when performed with a double cortical anesthesia. The technique (including video) is described elsewhere ( https://www.gerom-angers.fr/bone_biopsy.htm ).

Bone fixation

Classically, the collagenous bone matrix is said to be better preserved in a 70-degree ethanol fixative. However, it induces marked cell shrinkage incompatible with cytological examination. Formalin fixation provides very good cell preservation, but induces poor staining of the collagen when using a trichrome method. The combination of both ethanol and formalin was proposed by Beebe and works perfectly well [ ]. The formula known as BB's fluid is: 95 degrees ethanol, 900 ml; 37%–40% formaldehyde, 100 ml; deionized water, 150 ml.

Bone biopsies are fixed over 24 h at 4 °C in a refrigerator. The fixative is then discarded and replaced by acetone. BB's fluid allows the preservation of bone cell enzymes and retains the staining properties of collagen. Fixation in the cold (4–8 °C in a refrigerator) improves the quality of the tissues. After 24 h, the fixative is discarded and replaced either by acetone or by the fast dehydrating fluid as above.

Micro–computed tomography

Micro–computed tomography (micro-CT) is a new microscopic technique developed over the past few decades [ , ]. It is a miniaturized version of computed tomographs commonly used by radiologists and the systems now have a resolution in the order of 2 μm. Micro-CT is based on a sealed microfocus X-ray source, a CDD camera, and a step-by-step platform that receives the bone samples. Bone biopsies or animal bones can be analyzed when still in the fixative ( Fig. 8.1 ). They are transferred into an Eppendorf test tube filled with polyester fibers impregnated with the fixative (which are radiolucent and immobilize the samples). Micro-CT scans are obtained within an hour, and reconstruction of images, 3D model building, and morphometry are done within 2 h in human or animal samples [ , ]. Examples of micro-CT images appear in Fig. 8.1A–D . We have shown that micro-CT could be a very useful tool to provide a prediagnosis within 4 h in a bone laboratory. In a large series of 247 patients who presented with bone metastasis, overt myeloma, lymphoma, or MGUS, micro-CT was compared with 2D histomorphometry and histopathology [ ]. On the 3D reconstructed models provided by micro-CT, signs of osteolysis/osteosclerosis were searched for, including excess of bone resorption, focal disorganization of microarchitecture, bone metaplasia, and osteosclerosis. Strong agreement was obtained between histological method and micro-CT results using Cohen's kappa test. Micro-CT identified excess bone resorption on trabecular surfaces when eroded surfaces were >10.5% by histomorphometry but failed to identify some patients with smoldering myeloma, some lymphomas with microresorption or minute micrometastasis. These micro-CT changes are, however, not specific and must be confirmed by histopathological analysis. The stacks of micro-CT images can be used to develop new methods based on image analysis. In mice with the 5T2 myeloma model, osteolysis is characterized by a massive destruction of trabecular bone associated with perforation of cortical bone. Because the femur of these animals has a cylindrical shape, an unwrapping technique of the images can be used to measure the size and amount of perforations [ ].

The use of vascular injection products can be used to image the vascular bed of bone metastases in animal models. However, because the radio-opaque product (Microfil®: Flow Tech, Carver, MA, USA) has the same density as the mineralized bone, a double scan procedure must be used: undecalcified bones (containing the radio-opaque vascular casts) are first analyzed by micro-CT, and a 3D model is reconstructed [ ]. Bones are then decalcified over 4 days in a mixture of formic acid (4%) and 10% formalin. This decalcifying fluid has been adopted because it does not soften the bone matrix and does not provoke collagen swelling or distort the sample. Decalcified femurs are rinsed in tap water to remove acid remnants, and kept in 10% formalin until reanalysis by micro-CT. A second model (comprising only the vessels) is obtained and overimposed on the former, thus providing clear visualization of vessel trajectories in the invaded tissues, and allowing quantitative evaluation of the vascular volume and vessel diameter ( Fig. 8.2 ). Recently, we have developed a simpler method for preparing a radiopaque injection medium that can be used in animals and formalized human cadavers. A solution composed of 5% of gelatin and 95% of a commercial barium sulfate solution used for digestive X-ray analysis (Micropaque®, Guerbet, Roissy, France) is prepared made by heating at 37 °C. Addition of sodium azide prevents moldiness. The medium is stored in the fridge at +4 °C until use. A suitable amount of medium was warmed at 37 °C just before use [ , ].

Dehydration and infiltration of bone samples

Rapid dehydration and defatting is done by combining acetone and xylene at the same time [ ]. Acetone is preferred to ethanol, since it does not inactivate bone cell activities. We found that using a rotative device (running at a low speed) considerably shortens the dehydration time ( Fig. 8.3A ). Bone biopsies are transferred in screw-capped test tubes that allow complete infiltration. Dehydration and defatting is accomplished over 3 h in three consecutive baths (1 h each). A final bath in pure xylene (1 h) will ensure complete clearing.