Applied Technologies in Histology

Histology, simply defined, is the study of tissues. The term comes from the Greek words ‘‘histos,’’ meaning web (or tissue), and ‘‘logos,’’ meaning study; and the word histology first appeared in 1819. Without the microscope, there would be no modern field of histology; however Marie Francois Bichat, an anatomist and surgeon in Montpellier, France, defined 21 types of tissues without this technology. Other technologies used in histology pertain to preparation, preservation, and visualization of tissue samples.

The basic techniques of sectioning and staining used in the early part of the twentieth century depended on the ability of a tissue or cell to retain enough of its morphological integrity to be useful after being processed. One problem with preparing specimens was obtaining cuts of tissue thin enough for visualization without destruction. The microtome, a device for cutting specimens, developed synchronously in France and Germany.

A handheld model by Nachet in 1890 was followed by a rotary microtome by Bausch and Lomb in 1901, and Leitz manufactured a sledge chain-driven machine in 1905. The basic principle involves the operation of a hand wheel that activates the advancement of a block of tissue embedded in wax toward a fixed knife blade that slices it into very thin pieces. Microtome knives were hand sharpened by histotechnologists until the 1960s when machines with disposable blades were introduced.

One challenge to early scientists was the death of cells after exposure to air and light: tissue scrapings placed on slides soon lost their shape and size. When tissues are removed from the body, they lose circulating nutrients and will deform unless treated with appropriate chemicals. Tissue had to be fixed, dehydrated, cleared, and infiltrated.

During the first half of the century, each tissue was taken through a series of baths of formalin, alcohols, dioxane, and then paraffin. Formalin denatures the proteins so that they do not get damaged during the subsequent chemical baths. After rinsing, they were placed in 70 percent alcohol and successive baths of more concentrated alcohol until the application of xylene, a solvent for paraffin. Dioxane (diethyl dioxide alcohol) was used as the final dehydrant until the 1970s when its toxicity and pathogenicity were recognized.

After the tissue was cleared (alcohol removed) and made transparent, a material to support it was necessary. Liquid paraffin was embedded to infiltrate, support and enclose the specimens. In 1949, another embedding media, butyl metacrylate was introduced for the ultrathin sections used in electron microscopy.

Celloidin, a nitrocellulose compound, was used in Europe instead of paraffin because it was considered superior with regard to support of tissues that were hard to infiltrate, such as bone or eyes. Carbowax, a water-soluble wax, was first used in the early 1960s. It took less time, but since it was hygroscopic, it required more care with regard to environmental moisture.

Until the 1970s, the technologist had to fabricate paper ‘‘boats’’ into which paraffin was poured to embed the prepared tissue. Some laboratories used plastic trays but until the introduction of an automated technology known as an embedding center (Tissue-Tek), the work was tedious and time consuming. After the paraffin cooled and solidified, the paraffin-containing specimen was cut on the microtome and mounted on a slide.

An alternative to the paraffin method of preparation is the cryostatic method, introduced in 1932 by Schultz-Brauns. This technology consisted of a refrigerated knife and microtome that could prepare the tissue at a temperature between -10 and —20°C. Its advantage was that so-called frozen specimens could be examined while the patient was still in surgery. If the tissue was pathological (usually cancerous), it could then be removed in the same surgical procedure.

By the 1990s, the technique used was historadiography. Quickly- frozen tissues were dried, then prepared and photographed. The relative mass could be determined because there is a relationship between the film contrast and the various parts of the specimen.

Since cells are made from proteins, each subcellular organelle reacts to a dye by either staining to a color or not. The two most common stains are eosin, which stains the cytoplasm pink, and hematoxylin, a blue color used for nuclear material. The process of staining formerly required labor-intensive work in which a technologist applied stain to one slide at a time.

The process took 12 hours from start to availability for examination in order to make a diagnosis. With the introduction of an automated system (Dako Autostainer) in the 1990s, 48 slides could be processed in a period of two hours and with more than one stain. This equipment applies the stain, advances the slides, and dries them in a uniform process.

Preservation of live material is only temporary, but in certain cases one can observe both structure and function. The phase contrast microscope allowed for optimal visualization of difficult materials, but a challenge to progress in histology has been the inverse relationship between magnification and light. The power of a microscope depends on the wavelength of the light and the lightgathering capacity (numerical aperture) of the objective.

Most histological work is performed with lenses of numerical apertures of 1.0 or less. If the wavelength of light is reduced, it is possible to increase the resolving power of a microscope to 0.1 micrometers with ultraviolet light. Smaller particles can be seen in dark field, but shape and dimension are not accurate.

The challenge to increase both the light and the resolving power of the microscope resulted in a phase contrast microscope developed by Zeiss in the 1930s that was able to film a cell division. Such microscopes were not widespread until the 1950s. In 1955 improvements were made in the prism design, known as differential interference contrast (DIC), and this allowed visualization of living cells without staining.

In the 1960s a technology for diagnosing cervical cancer was developed by George Papanicolaou. Known as the PAP smear, the technique consisted of lightly scraping the cervical mucosa, spreading the sample on the slide, fixing it, all in the doctor’s office, and then sending it to a laboratory for analysis. In 1996, a ThinPrep (Cytyc) test was approved by the U.S. Food and Drug Administration that collected the cells, rinsed them into a vial of preservative solution, and after filtration, applied them to a microscope slice uniformly. This technique was extended to nongynecologic specimens such as fine needle aspirations and endoscopic brushings.

The halogen lamp, powered by a circuit board that prolonged its life, solved the light versus resolution relationship. Microscopes with multiple eyepieces allowed more than one person to view a specimen. By adding a tube to the viewing chamber and attaching another set of eyepieces, it was possible for two pathologists to view the same slide simultaneously (Olympus split microscope). Variations on this technology allowed for a class arranged in a circle to observe the same material. By the 1990s, technology had arrived at infinity- corrected optics; optical microscopy allowed investigation at the micron and submicron level.

Perhaps the most revolutionary development in twentieth century medicine has been the emergence of biotechnology. Monoclonal antibodies, immu- nohistochemistry, tumor markers, and flow cytometry depend on the ability to work with DNA and fluorescent labeling of organelles such as microtubules and endoplasmic reticulum. New diseases such as AIDS required new technologies to detect antibodies. By the end of the twentieth century, automation technology had affected and improved every division of histology departments in hospitals, from cytology and microbiology to pathology.

One microscope could be accessorized with DIC, fluorescence, polarized light, phase contrast, and photomicrography using several film formats and digital image capture (Olympus Provis AX-70). The entire paradigm of a lengthy fixation time and preservation was modified because immunohistochemistry, electron microscopy, and molecular biology techniques required change. An autostainer can produce 2000 slides per hour, 100 at a time.

NASA technology led to the development of an automated cellular information system (ACIS) using automated microscopy and computerized image analysis. The slide software captures hundreds of fields and projects them on a screen, and the observer selects those of interest and adjusts the magnification to a higher order.

A quantitative score is then computed with regard to staining intensity and other parameters. The data is converted to a format suitable for export to a spreadsheet or database program. At the close of the century, fiber optics, digital cameras, and camcorders as well as computer software used in microscopy were used in programs for both students and continuing professional education.