sgall17aWhat is a tissue processor.
Living tissue is naturally soft, non-uniform and almost transparent. Tissue must be "processed" before it is suitable for microscopic examination. Processing hardens the tissue and replaces the water with wax. This allows it to be embedded into a block before it is cut into thin slices about 4um thick.
Wax and water do not mix so we have to replace water in tissue with wax with a series of solvents. The standard method replaces water with alcohol (ethanol), then replaces the alcohol with xylene . Finally the xylene is replaced with wax.
Tissue is soaked in a series of chemicals. One way is to have a series of beakers (usually about 1-2 litres) and then to move tissue from one beaker to the next. This is often done by a machine called a "rotary processor". The alternative approach is to leave the tissue in one container (the "retort") and pump the chemicals in and out of the retort. This project takes the second approach.
The older rotary processing machines had about 10 beakers and mechanically moved a basket of tissue around. These were large (about 1.2metres in maximal dimension) and constituted "heavy engineering" compared to most laboratory machines. Sequencing was done by clockwork. Refurbished machines are available secondhand for a few thousand dollars, up to to about $12,000 for a new one.
Having a fixed retort with pumped chemical makes it easier to change conditions in the retort such as vacuum and heat. Most modern machines use this model. They are complex and expensive (about $42,000+). Secondhand machines, are still quite expensive and have a reputation of being unreliable.
Brief description of this project.
This project takes advantage of microcomputers, commonly available components such as plastic bottles, servo motors and a few 3d printed parts to duplicate most of the functions of pumped type tissue processor at much lower cost. Even using new components from China, material costs should be below $100 or so.
The processor is build up from a number of chemical modules. A module comprises the retort (shared between modules) and for each chemical a plastic bottle with silicone tuning connecting the retort to the bottle. A 3d printed pinch valve cuts off flow in silicone tube by external pressure. The retort fills with chemical by suction and is emptied by positive pressure blowing the chemicals back into the bottle. A microprocessor coordinates the pinch valve, and the two micro air pumps.
The retort can operate at room temperature and normal atmospheric pressure (like the old rotary processing machines ) for most stages. A magnetic flea stirs the liquids. Suction is used to fill the retort and this suction could also supply some vacuum for the retort.
Why are Pinch valves and air pumps are used as the pumping mechanism? Air pumps are cheap and reliable compared to fluid pumps for noxious chemicals and are never exposed directly to the chemicals. Using a pinch valve means that there are no valves or other mechanisms exposed to chemical to clog or corrode. It is easy to replace the silicone tubing if required.
Formalin, alcohol and xylene are liquid at room temperature but wax needs to be kept at 60C to remain liquid. The easiest approach would be to make the final wax handling a manual process ie put the basket of fixed tissue into wax by hand. Automatically wax handling would require a heating mechanism, possibly pumped domestic hot water. There is a good case for automating the wax step because on a typical overnight cycle tissue is ready to be placed in wax in the early hours of the morning. Since wax is the final step, a good compromise would be to automate the filling of the retort with wax in the night but to remove the tissue blocks and remaining wax manually the next morning.
Details of the processing cycle.
1. Extra fixation. Fixation or hardening of tissue is basically a process of protein denaturation and can be done by heat or chemicals. Denaturation by heat is basically cooking. Results are better with chemicals, like formalin.
Freezing is an alternate method to harden tissue. This is much faster than normal processing and is used for rapid diagnosis during an operation. Unfortunately visual quality in the resultant slides is terrible. On the other hand there is less denaturation of tissue and this may be an advantage for some (rare ) tests.
Fixation in formalin normally just occurs while transporting tissue to the laboratory. However, inadequately fixed tissue causes problems throughout the rest of the process so usually some extra fixation is provided at the beginning of tissue processing (say 30-60 minutes).
2. Alcohol. Alcohol (ethanol) removes water from tissue and this is probably the most important single step in processing . One alcohol bath will work, but changes of alcohol are better. Processing in alcohol takes about 1-2 hours, ideally with 3 changes. Other alcohols can work but ethanol is cheap. Apparently methylated spirits can substitute for pure ethanol.
3l. Xylene. Xylene is miscible (soluble) in both alcohol and wax and enables final wax impregnation. The xylene stage is about 30-60 minutes. Other short aromatic or chain hydrocarbons or even citrus derived chemicals can be substituted. Substitution is usually done because of the potential toxicity and carcinogenesis of xylene.
4. Wax. Molten wax will impregnate tissue in which the water has been replaced by xylene. This usually takes a couple of hours and must be done at 60C.
Timing of a complete processing cycle.
A typical cycle takes about 8-10 hours. Cycles can be shorter ("fast cycles") for small tissue fragments. Longer and more specialised cycles can be used for fatty tissue and brain tissue and some other tissue like bone.
Overview Tissue processing as part of microscopic diagnosis.
1. Take the tissue (operation or biopsy).
2. Transport tissue to the laboratory in formalin (say 3-4 hours).
3. Process a basket of tissue (say 30-100 pieces or blocks in a small laboratory) (typically overnight).
4. Embed and orient the tissue in a wax block. (Few minutes per block)
5. Cut thin slices and place on glass slides ( Few minutes per block)
6. Stain the usually (usually with pink and blue vegetable dyes) (Say half an hour per basket).
7. Examine under microscope and report. This can take the pathologist take a few minutes for a simple case to days or more for more difficult cases. Typically 80+% of cases can be reported in one day.
Citizen science
"Citizen science" is a movement to make scientific process cheaper and more accessible outside large institutions. Tissue diagnosis from biopsies, surgical specimens and autopsies is an important part of medicine especially when it evolves beyond basic emergency medicine . There are citizen science projects developing cheap or open source microscopes which are one of the steps required. Another critical process that would be required before tissue process is widely available is the process of cutting very thin slices or "microtomy". As far as I know there is not much progress in this area.
What is histopathology?
Histology is the examination of thin slices (or "sections") of tissue under the microscope. Usually the tissue is embedded in wax block from which very thin sections about 4um are cut (typically about 4um thick). In their natural state sections are transparent and need to be stained. The most common stains are cheap vegetable dyes.
Most diagnoses of tumours, infections and inflammatory conditions can be made from these thin wax sections. The basic diagnosis, although requiring skilled interpretation, is low tech. However even advanced molecular and protein studies are done from this simple material.
Tissue diagnosis is not necessary for emergency medicine. However when we progress beyond basic medicine, more accurate diagnosis is essential for treatment and assessing the level and type of disease in the community.
What is the point of a tissue processor if other stages remain expensive or inaccessible?
Tissue processing turns a hunk of soft, bulky fragile degradable tissue in a bottle of noxious formalin into a small robust block of wax which is the basis for an enormous range of subsequent tests. This block can be mailed by standard post to larger centers. The processing stage could be done locally with relatively low tech, enabling a much higher level of medical care to remote communities.
There are, I believe, further advantages which I will expand on another article.
Cost.
Plastic drink bottles are free.
Wax temperatures are similar to those achieved by domestic hot water temperatures.
Silicone tube costs tens of dollars.
Valves are 3d printed and use standard servo motors ( Say $5-10 each). About 12 valves would be needed.
Arduino Mega - say $15 from China.
ESP32 - say $10.
Retort - something like a casserole dish with a custom made lid that can be pressurised and passes the silicone tubing.