When minerals are present within the tissues, such as bone and dentin, or there is pathological calcification in soft tissues, it is difficult to get good sections either from paraffin embedded material or from frozen samples. In these cases, it is necessary to remove the minerals from the tissue by a process known as decalcification, because they are mostly calcium minerals. However, there are cases where this is not possible, or the tissue must be as unmodified as possible, and sections are obtained from the original mineralized tissue (see below).
Hydroxyapatite is the hard mineral found in the animal tissues. It is a calcium phosphate mineral [Ca10(PO4)6(OH)2] found among the collagen fibers in bone tissue. The mineral is in equilibrium with the interstitial saturated solution of the tissue, according to the reaction:
Ca10(PO4)6(OH)2 = 10Ca2+ + 6PO43- + 2OH-
If some component is removed from the right, the mineral (on the left) is dissolved to compensate the equation. This is how decalcificant substances work. The selection of decalcificant substances and incubation time depends on what type of bone we are interested in: compact or spongy bone. For example, minerals are slowly solved in compact bones.
Decalcification is usually performed after fixation, although it is sometimes done after paraffin embedding, and then the minerals are removed from just a few superficial micrometers. A good fixation is important because the sample may be incubate for long time in relatively strong acid solutions. Furthermore, it is a good practice to completely remove the fixative with thorough washes to prevent possible reactions between the fixative and decalcification agents. The fixation time of bone samples are usually much longer than other non-mineralized tissues. The best fixation method is perfusion. If it is not possible, it is a good procedure to trim very small pieces of bone with a small saw with very small saw teeth, and remove the surrounding non-bone tissue before the immersion in the fixative. The best fixatives are Bouin, formaldehyde-zinc mixes, and FAA (formaldehyde, acetic acid and alcohol).
Dhere are two major decalcifacting agents: acids and calcium chelating substances. The following are the more common.
Acids
Acids substances provide hydrogens and then hydroxyl ions are removed. Therefore, the mineral is dissolved according to the reaction described above.
H+ + 2OH- = H2O
There are two groups of acid substances, strong (inorganic) and weak (organic) acids. The strong acids remove the mineral very fast but very aggressively, so that the incubation time has to be short. Otherwise, the tissue is damaged. The weak acids remove the mineral slowly, but they preserve the tissue. The most common strong acids are chlorhydric acid and nitric acid, whereas formic acid is the most used weak acid. During decalcification, molecules such as antigens and enzymes are more deteriorated as stronger is the acid. The ARN is also damaged with stronger acid solutions, so that in situ hybridization may be also compromised.
Chloridric acid and nitric acid are diluted at 5 to 10 % in distilled water. They remove the minerals very fast, and the incubation of samples should not last longer than 24 to 48 hours. This treatment is not recommended for immunostaining or enzymatic essays. The strong acid treatment is intended for small samples and when a fast diagnostic is needed.
The formic acid is the most common weak acid used for decalfication. It can be used diluted at 5 to 10% in distilled water, buffered with citric acid, or combined with formaldehyde. In this last case, decalcification and fixation happen at the same time. The decalcification time may last from 1 to 10 days, depending on the size of the sample and the type of bone tissue. The decalcification solution is replaced for fresh solution every 24 to 48 hours.
Other weak acids, such as acetic acid o ascorbic acid, can be used when the tissue is intended for immunohistochemistry. In these cases, the decalfication is performed at pH above 2.5.
Calcium chelants
Chelants are organic substances that chemically bind metallic ions, resulting in a soluble compound referred as metal chelate. EDTA (Ethylenediaminetetraacetic Acid) joins calcium ions from the surface of the bone minerals, so that the mineral is progressively dissolved. EDTA is commonly used at 10 to 14 % in aqueous solutions, that can be buffered. Although it is a slow process, decalcificaton may last for weeks, it preserves the tissular features. Thus, if the time is not a problem, calcium chelation is the recommended method for decalcification. The working solution is replaced every 3 to 5 days with fresh solution. The process can be speed up by increasing the pH (above 7), but it can affect the tissues.
The decalcification time, in both acid and chelant methods, can decrease by changing some parameters, such as a higher temperature or gentle agitation of the samples. There are more aggressive ones like sonication or microwaves treatments, but tissular damages have to be tested.
It is important to know how decalcification is progressing with time, because it determines the total time of decalcification. It was mentioned above that longer times may deteriorate the tissue quality. However, the tissue processing is difficult when mineral remains after a short decalcification time. During decalcification, the calcium removed from the mineral is dissolved in the decalcification solution. The calcium concentration can be measured during the periodical replacement of the used decalcification solution for a new one. When acids are the decalcification agents, the calcium concentration can be obtained by chemical reactions: ammonium oxalate is added after sodium hydroxide neutralization, and then calcium oxalate precipitates are formed. The dedcalcificaction process should continue as long as calcium oxalate precipitates are observed.
However, it is sometimes necessary to maintain the mineral content of the sample and, therefore, decalcification is not carried out. Thus, sections by non-conventional methods have to be obtained. For example, the sample can be sawed in thin slides, which are latter filed to get a very thin section suitable for light microscopy observation. Thin sections from mineralized samples can also be obtained by resin embedding, and semithin sections are then cut with an ultramicrotome and diamond knifes. Another way is to get sections from frozen samples.
Immunohistochemistry may be affected by decalcification treatments. In general, the impact of decalcificaton depends on both the antigen to be studied and the type of treatment. Anyway, the strong acids should be avoided.