Bone specimens received in the laboratory can generate comments that range from:
“This block is impossible to cut!”… to …“This block cuts like butter!”
How can one tissue behave like Dr. Jekyll and the other like Mr. Hyde? As with many things in histology, the answer is in the details. Similar to dermatopathology, the histologist must understand the histology of bone tissue in order to produce optimal microscope slides.
Everyone knows that bone is basically hard and brittle. This characteristic is due to the cell biology of bone growth and development. Bone collagen is laid down in bands that are parallel to one another. These bands, or lamellae, become mineralized with a polysaccharide containing calcium and phosphate which provides strength necessary in cortical bone to provide support to the skeleton.
Trabecular bone is mineralized in a similar fashion, however, many spaces remain where bone marrow is located. The bone marrow stem cells divide and grow into the specialized blood cell components (Figure 1).
Bone is a dynamic, living tissue. New bone is made by osteoblasts located on the surface of newly formed bone. The most recent material is not mineralized and is referred to as the osteoid seam. This material is mineralized later to form mature bone. Osteoclasts are also located on the bone surface. These are large, multinucleated cells responsible for “eating up” mature bone to release calcium into the blood stream. If the balance between these two bone cell types is disturbed, disease may result. Osteoporosis is a disease where the osteoclast activity outpaces the osteoblast activity; weak, porotic bones prone to breakage, can result (Figure 2).
The million dollar question in the histology laboratory is: How can we prepare bone specimens to be cut from a paraffin block on a rotary microtome, and keep the sections on the slide for optimal staining?
As with all specimens, the answer begins with proper fixation. The “20 to 1” rule applies in that the bone specimen should be placed into unbuffered formalin fixative, in a volume that is 20 times that of the size of the specimen. Then, since bone is very dense, the fixation time should be measured in days – not hours; with an average of 3-5 days of fixation for larger specimens.
Once the specimen is fixed, it must be decalcified. Methods must be used that will remove the calcium from the bone tissue, to render it soft and amenable to subsequent processing and cutting. There are methods for processing mineralized bone into plastic, however these methods are not within the scope of this discussion.
Before we discuss what can be used as decalcifying agents, we must understand that all act in the same way. That is, the solutions are used to leach out and remove calcium ions from the tissue. To that end, specimens should be placed into a decalcifying solution that is 100 times the volume of the specimen, thereby provided plenty of solution. Since the solution will now become saturated with calcium during the process, it should be changed every 24 hours.
Large pieces of bone should be cut with a scalpel into smaller pieces as they become softer, yielding pieces of tissue not exceeding 2 mm in thickness. Finally, the solution should be agitated using a rotary shaker, or stirring mechanism as shown in Figure 3.
So then, what decalcifying solution should be used? Below are a few that are in use today:
Rapid decalcifer: These solutions usually contain hydrochloric and/or nitric acid, and are used on bone marrow biopsies. The advantage of rapid decalcifying over some hours is offset by decreased histology quality, and possibility of antigenicity damage.
Bouin’s solution: The picric acid contained in Bouin’s solution can decalcify bone in small bone marrow biopsies, as described above. The disadvantage is that picric acid is a dangerous chemical to use and dispose of.
10% formic acid in 10% unbuffered formaldehyde: This method yields the best histology. The disadvantage of this solution, is the time it takes to decalcify is measured in days.
Chelating agents: These are mostly used in research applications, as histology and antigenicity are maximally preserved. However, the time frame is measured in weeks.
The endpoint of decalcification can be determined subjectively, via manual manipulation of the tissue to determine softness. Conversely, the chemical method detailed in the procedure “Determining the Endpoint of Decalcification, found later in this blog, can be used (Luna, 1968). After decalcification is complete, the tissue should be rinsed thoroughly in running tap water. Processing should be done using a procedure for large, dense tissues.
During microtomy, if there are times that the decalcification seems to be incomplete, the histologist may soak the block face in decalcification solution for 30 minutes to an hour. Then, after rinsing, the first few sections should cut easier and be floated out on the water bath for section pick up. Finally, microscope slides coated with 0.5% gelatin should be used to pick up sections. This will help the sections to adhere to the microscope slide during staining. Some eosin background staining on the slide may result, but it does not affect the final H and E stain.
By following the above guidelines, all of your bone specimens should now;
“Cut like butter!”
- Theory and Practice of Histological Techniques. Chapter 10. JD Bancroft, A Stevens ed. Churchill Livingstone, NY. Fourth edition. 1996
- Theory and Practice of Histotechnology. Chapter 9. DC Sheehan, BB Hrapchak. CV Mosby Company, St. Louis. First edition. 1980.
- Chapman CM: Histology Hardball: Solutions for Hard Tissue. Advance for Medical Laboratory Professionals: Vol. 24 No2, February 20, 2012.
- Luna L. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed., McGraw-Hill, New York, 1968, page 10.