There are different embedding techniques utilized by histotechnology professionals, but no matter which method your lab uses, there are key factors that can affect the quality of embedding.  These include: sample thickness, tissue orientation, quality of paraffin wax, mold and cassette types, and availability of light.  Let's take a closer look at these factors:

Sample Thickness:

During grossing the tissue may be thicker or larger than the capability of the mold being used for the embedding of the tissue.  This can occur if the pathologist or pathology assistant is not aware of what mold sizes are being used in the histology laboratory.  To fix this problem, the tissue may be divided and embedded into more blocks, but be aware great care and understanding of the types of tissue is crucial and this should only be done by an experienced histology laboratory professional.  If you are unsure, consult a senior tech.  It is also important to communicate this issue to your pathologist or PA to avoid the problem in the future.

Tissue Orientation:

Improper orientation will lead to disorderly arranged histological features on the slide.  For example, when orienting skin, the specimen must be positioned so that the epithelial edges, the subcutaneous tissue and deeper layers, are aligned at the bottom so that the strata will be seen in the finished slide. When embedding more than one specimen, all the pieces of tissue should be embedded firmly to the bottom of the container so that the cut section will present a valid presentation of the tissue submitted.

Artifacts due to improper orientation are frequently encountered during embedding procedures which can lead to damage to microtome, tearing of the section thereby making microscopic study of tissue difficult.  The result is the need to re-embed cause lengthy turnaround time (TAT).

Paraffin Wax Quality/Composition:

Paraffin wax is as one of the most important elements of tissue embedding and its quality is fundamental for the production of a block.  Paraffin wax formulations should be developed with consistent quality and dependability to minimize tissue distortion and provide exceptional compression resistance and ribbon continuity. Different paraffins feature a variety of set-point temperatures, additives, and characteristics, there is a paraffin to meet your processing, embedding, and sectioning needs – so consider your requirements carefully and do your research.

For example, Paraplast Regular is recommended for general tissue embedding. It is composed of a refined mixture of highly purified paraffin containing plastic polymers of regulated molecular weights. This supports improved tissue infiltration and superior quality sections with minimal compression.

A histotech may also encounter impurities. Impurities such as white floccus should not appear. The presence of white floccus indicate excessive clearing agent in paraffin. To avoid this, increase the frequency in which you replace the paraffin.  Besides; It is highly recommended to use a bolt like structure material to make the tissue as flat as possible to equally to be sectioned by the blade while microtomy

Mold Size and Cassette Type:

Although not as common an issue, the knowledge of availability of different sizes of molds by a pathologist or pathologist assistant (PA) is important to understand in advance to plan the size of tissues selected for analysis (see sample thickness), and the same is true for the selection of cassettes for tissues to be processed. Some cassettes have very small holes that make difficulty to pass the embedding medium to pass through to build tissue blocks. Others have very big holes, as a result small tissues or fragments can be lost during tissue processing.

Availability of Light:

Tiny tissues may be lost of left un-embedded because of the ability for the histology laboratory professional to see when embedding. That is why modern embedding centers are available with removable/non-removable magnifying glass.  Well lighted work area is also vital and these embedding centers and led lights are also available.

References:

• L Carson, C. Cappellano, Histotechnology, A self-Instructional text, America Society for Clinical Pathology press, 4th ed. Page 48, 2016

• Bancrofti, M. Gamble, Theory and Practice of Histological Techniques, sixth edition, Churchil Livingstone Elsevier, 2008

• Izak B. Dimenstein, Grossing Technology Today and Tomorrow, ASCP, Lab Medicine 2020; 51;337-344

• Taqi SA, Sami SA, Sami LB, Zaki SA, A review of artifacts in histopathology, J Oral Maxillofac Pathol 2018;22:279

• Victor O, G Eze, Common artifacts and remedies in histopathology (a review), African Journal of Cellular Pathology, ajcp; 2015;(4):6-12

• Geoffrey R, Microtomy and Paraffin Section Preparation, Knowledge Pathway, Leica BioSystems, 2019

• Material Safety Data Sheet, Columbus Chemical Industries, ScholAR Chemistry, 2009, Giorgis Yeabyo, ASCP, HTL

• Tissue Tek^R Tec^TM 6 Embedding Console System; Specification, Sakura, available at https://www.sakuraus.com/Products/ Embedding/Tissue-Tek%c2%ae- TEC%e2%84%a2-6.html; [Accessed date April 11, 2022]

• Leica Biosystems, (https://shop.leicabiosystems.com/) available; at https://shop.leicabiosystems.com/ [ Accessed date April 3, 2022]

• Paraffin wax blended with synthetic polymers, Solidification point: 56°C, Surgipath® Paraplast Regular; https://us.vwr.com/store/product/202 69832/paraffin-wax-blended-with- synthetic-polymers-solidification- point-56c-surgipath-paraplast- regular), Leica Bio-system, avantor, [ Accessed date April 3, 2022]

• Ouyang Z, Xiaoying Y, Hui G, Xiangning L, Paraffin-embedding for large volume bio-tissue, Scientific Reports, nature research , 2020; 1-8
  
  
  
  
  Written By Giorgis Yeabyo, HTL(ASCP)