Cutting - tips

                   Soaking blocks with water 

When tissue is exposed on the surface of a paraffin block by rough trimming it has the capacity to absorb water. It will penetrate a small distance into the tissue softening and swelling it. With poorly processed tissue (both under- and over-processed) this effect may helps to cut sections.

With difficult blocks the procedure entails very careful trimming (rough cut) to expose the tissue while avoiding too much damage to the specimen surface, then placing it face up on the top of the ice tray. Add few drops of water just to cover the tissue on the block (PICTURE A).  Keep it  for at least 2-3 minutes.  The block should be re-chilled in the ice tray before attempting to obtain sections.  It is vital that alignment of the block face in the microtome is preserved so that no tissue is wasted in obtaining a full-face section. Generally, after this procedure, the best quality sections are achieved by cutting very slowly. 

This technique helps to cut any difficult blocks such as skin, bone, nail etc. Especially tissues containing blood (e.g. endometrial samples) can be easily cut by using this method and can avoid creased and flapped sections.  

For heavily keratinised skin or nail specimens treat block face with NairTM or VeetTM (hair removal products). But  soaking the block in water is the easiest way to cut sections without any damage in the tissue architecture. 

 PICTURE A (Skin punch Biopsy)

                                                                                        

Cutting Levels

This is a simple and easy way to cut levels. When you cut levels, leave sections in the water bath and after finish cutting all levels you can  pick sections on the slide. This allows saving time and it helps to get rid of the creases. Make sure that the sections are attached to the side of the water bath to avoid confusion (PICTURE B). 

PICTURE B (3levels of a skin punch)

https://www.youtube.com/watch?v=PafHxS5bq9A

Perls’ Prussian Blue

Perls’ Prussian Blue – IRON

Perls’ Prussian blue stains are used to detect and identify ferric (Fe3+) iron in tissue preparations, blood smears, or bone marrow smears. Minute amounts of ferric acid are commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate haemochromatosis and hemosiderosis. Lack of iron identification can indicate deficient anemia.

Haemochromatosis is caused by excessive iron uptake. In the liver, the deposits may lead to cirrhosis following hepatocyte death.

What is Prussian Blue?

Prussian blue (PB) was actually the first synthetic color to be discovered during the Industrial Revolution. It was developed accidentally in 1704 by a chemist who was trying to produce another color. It wasn’t used as a histochemical stain until 1867 when German pathologist, Max Perls – hence described its original formula why is often known as ‘Perls’ Prussian blue’.

What does it Stain?

PB is used to detect the presence of IRON in tissues. It is an extremely sensitive test, and can even detect single granules of iron in cells.

Although iron is essential for life, it is also toxic due to its ability to form free radicals that can damage cells. So the body must protect itself from this element, and it does so by using iron-storage proteins. Haemosiderin is one type of iron-storage complex that is found inside cells. It is mostly found within phagocytic macrophages, and is especially prominent in these cells following haemorrhage, when haemoglobin breaks down.

Microscopically, the presence of iron is therefore identified by demonstrating iron-containing haemosiderin, which shows up as a granular brown pigment in cells when examined with haematoxylin and eosin (H&E) stain. Although visible with H&E, other pigments can stain a similar color, so haemosiderin needs to be differentiated from other brown pigments using the PB histochemical staining technique.

General Principles of the Stain

Various staining protocols have emerged since Perls’ original description, but all serve to achieve the same result – demonstration of iron in tissues.

The procedure involves 3 basic steps:

1.            Tissue sections are treated with hydrochloric acid to denature the binding proteins of the haemosiderin molecule, and thereby release ferric (3+) ions.

2.            Potassium ferrocyanide is then introduced. The ferric ions combine with this solution, resulting in the formation of ferric ferrocyanide, an insoluble bright blue pigment – otherwise known as Prussian blue. Although the hydrochloric acid and potassium ferrocyanide can be introduced as separate solutions, most formulations now typically use them in combination.

3.            If required, the PB reaction can then be followed by a red counterstain, such as neutral red.

Consequently, the end product of the reaction is such that iron in tissue sections is seen as blue or purple deposits, while other tissue components, such as nuclei and cytoplasm, are highlighted red by the counterstain (1% neutral red).

Who Uses Prussian Blue Stain?

This stain is used widely for both diagnostic and research purposes. In diagnostic labs, PB is widely used by pathologists to detect the presence of iron in biopsy specimens, especially in tissues such as bone marrow and spleen.

This procedure is particularly helpful when they need to evaluate patients with pathological conditions that involve haemosiderin deposits. In addition to haemorrhage, this can occur in patients with haemolytic anaemia, as well in conditions such as haemochromatosis (where excessive amounts of iron may form in organs due to iron overload), some liver diseases, and in the lungs of patients with congestive heart failure.

RESULTS:

Ferric iron (haemosiderin) – Blue

Nuclei – Red

Background (cytoplasm) – Pink

Microtome

The rotary microtome is the most common instrument found in a histology laboratory. Although most microtomes are manual, some are automatic or semi-automatic. In order for tissue to be viewed under the microscope it must be thin enough to allow light to pass through and be mounted on a glass slide. A microtome is a mechanical device which helps to make thin sections of paraffin wax embedded tissues using a very sharp blade. care must be taken when handling blades or cutting section. 

1. Microtome base plate or stage: A platform which has rails that secure the knife holder base.
2. Knife holder base: A part that anchors the knife holder to the microtome stage. The knife holder base can be moved toward or away from the block, but MUST be stationary and locked during microtomy.
3. Knife holder: This part is comprised of several components including the blade clamp that holds the blade, the knife tilt for adjusting the knife angle, and the face plate that guides that ribbons away from the blade and towards the operator.
4. Cassette clamp or block holder: Holds the paraffin block in place. Typically, the block moves up and down with each revolution while the blade is stationary. The block holder may have knobs that allow the user to manipulate the block face in various directions to bring the tissue in alignment with the blade.
5. Coarse handwheel: Moves the block holder either toward the knife or away from the knife.
6. Advancement handwheel: Turns in one direction and advances the block toward the knife at the specified microns. Most handwheels are equipped with a safety lock to prevent the wheel from releasing and having the block holder come down towards the blade while a block is inserted or removed. The safety lock should be used anytime the microtomist is not actively sectioning paraffin blocks.
7. Micron adjustment: Micron settings for section thickness can range from 1 to 60 microns on most microtomes.

What is Histopathology?

Histopathology is the microscopical study of a diseased tissue.
                                                             OR

Histopathology is the macroscopic and microscopic examination of tissue samples to diagnose and evaluate disease.
                                                            OR

Histopathology is the  microscopical examination of biological tissues to observe the appearance of diseased cells and tissues in very fine details.
                                               
                                                  Histos-pathos-logos 

The word 'Histopathology'  is derived from a combination of 3 Greek words

Histos- Tissue

pathos- disease 

logos- study