UDDD2124 ESSENTIAL PATHOLOGY

JAN 2024

LABORATORY REPORT 1 & 2

Tissue Processing and
H & E Staining
Name : Chew Yu Xin
Student ID : 2202373
Year/Sem : Y2/S3
Group : P2
Lecturer : Ms. Kokila a/p Thiagarajah
1.0 INTRODUCTION

A major facet of clinical analytical work is histology, the microscopic study of cells and
tissues through sectioning, staining, and examining them under a microscope. Histology sees
widespread use for diagnosis, study of diseased tissues or malignant cancers, investigation on
the effectiveness of treatment, and autopsy (Gurina and Simms, 2023).

The preparation of a tissue sample for microscopic analysis can be broken down into five
central steps: fixation, processing, embedding, sectioning, and staining (Alturkistani,
Tashkandi, and Mohammedsaleh, 2016). Technological advancements in the medical field
have allowed these processes to be automated, although this experiment will demonstrate
manual processing of tissue samples and staining with hematoxylin and eosin (H and E)
staining.

2.0 MATERIALS & METHODS

 Materials: Paraffin wax, microscope slides, cassettes
 Reagents: xylene, graded alcohol series (100%, 95%, 80%), hematoxylin, eosin, DPX
 Biological sample: Chicken liver
3.0 SAMPLE PREPARATION
3.1 SAMPLE COLLECTION
The sample to be prepared was chicken liver.
3.2 FIXATION
 The sample was fixed with formalin before the experiment.
3.3 PROCESSING
 A piece of the sample was loaded into a cassette.
 Melted paraffin was poured into the cassette from the tissue processor,
enough to cover the sample without overflowing.
 The cassette was moved to a cooled steel surface for the paraffin to
solidify.
3.4 SECTIONING
 After the paraffin block had solidified, the cassette was loaded onto the
microtome, with the blade locked and knife guard lifted to cover the blade.
 The mechanism locks were released, and the hand crank was turned to let
the blade slice into the block to produce a thin ribbon of paraffin.

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  Once a desirable length had been achieved, the microtome was locked
again, and the ribbon was transferred to a warm water bath.
 The ribbon was allowed to float for approximately 3 minutes, before being
picked up with a clean microscope slide.
 The slide was placed on a hot plate to allow the paraffin sheet to dry.
3.5 STAINING
 The next week, the slides were loaded into slide holders and put through a
regressive staining protocol, consisting of xylene, graded ethanol series,
and the two dyes, hematoxylin and eosin.
 A drop of DPX was dropped onto the slide, near the tissue sample.
 A cover slip was gently placed onto the slide, allowing the DPX to spread
underneath it and cover the tissue.
4.0 VISUALIZATION

A total of two sheets of chicken liver tissue managed to be fixed onto the slide and stained.

5.0 ARTIFACTS
Several artifacts from the tissue preparation could be seen in the tissue samples upon
microscopic examination.

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 Tears and cracks

Chicken liver, 40x

The first is a series of prominent cracks in the tissue. This commonly happens when the
paraffin ribbon was floating in the water bath, during which bubbles of air may be trapped
underneath the tissue. When the tissue is picked up with the slide, areas of the tissue with air
bubbles entrapped beneath remain dry and cannot adhere to the glass properly (Jimson et al.,
2016). This can be remedied by using freshly boiled water for the floatation bath (Rastogi et
al., 2013).

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 Tissue wrinkles

Chicken liver, 100x

The other artifact visible are wrinkle marks in the tissue, which appear as darker coloured
lines. Wrinkles and folds are common when thin sections of tissue are forced to stretch
around other structures with a different consistency. They can be corrected by gentle teasing
with forceps (Krishnanand et al., 2010).

6.0 DISCUSSION

Histopathology techniques have been a part of the clinical laboratory for a long time, and the
manual tissue preparation and staining method conducted in this experiment is considered the
gold standard for histological tissue viewing. As technology and medical research and

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understanding grows, other methods of tissue viewing have emerged as easier to use, more
reliable alternatives to the manual method.

A study by Freudiger et al. (2012) pointed out that most of the time, histopathological
techniques cannot be conducted in vivo, as the tissue of interest must be frozen or fixed, and
then thinly sliced and stained for it to be fit for viewing under the microscope, which is not
ideal in instances where a biopsy may not be possible. To address this, the study explored an
alternative dye-free imaging technique known as coherent Raman imaging (CRI), which
allows chemical imaging based on the intrinsic vibrational properties of the tissue molecules
without the need of any staining or labelling. Their study proved that CRI was able to
produce stain-free histopathological images that are similar to those obtained with
conventional techniques, all without requiring tissue fixation, sectioning, or staining.

A study by Zhang et al. (2020) mentioned another downside of tissue processing, namely that
the process is destructive in nature and may exhaust the tissue sample, requiring repeat
biopsies. It is also laborious, delicate, and time-consuming, especially if special stains are
required. One alternative that can address these issues is digital staining of samples, by
applying a contrast map to transform images of unstained tissue sections into images that
simulate the same tissue after a conventional staining. Zhang et al.’s study demonstrated the
use of machine learning and deep neural networks to generate virtually stained images from
label-free tissue images, with the digital stains matching microscopic images of the same
tissues stained with H and E, as well as Jone’s stain and Masson’s trichome stain. Their
network was also able to produce an image virtually stained by a combination of those stains,
which would be impossible to do in conventional staining.

There were other attempts to bring histopathology to the digital sphere, including the use of
automated slide scanners that can scan entire histology slides and save them as digital
images, known as whole slide images (WSIs), or virtual slides. These images can be viewed
through a computer interface that has applications to simulate a light microscope (Webster
and Dunstan, 2013). Virtual slide technology has potential to greatly improve clinical
workflow, as the digital format allows remote consultation with other hospitals, side-by-side
comparison, which would not be possible with glass slides, and easy storage and retrieval,
without the risk of physical damage to the samples. However, slide scanners are still an
expensive investment as of today, and equally expensive is the IT infrastructure required to

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manage the WSIs and their metadata, which can range up to several gigabytes per image
without compression (Treanor, 2009).

7.0 REFERENCES
1. Alturkistani, H. A., Tashkandi, F. M., and Mohammedsaleh, Z. M., 2016. Histological
Stains: A Literature Review and Case Study. Global Journal of Health Science, [e-
journal] 8(3), pp. 72-79. https://doi.org/10.5539%2Fgjhs.v8n3p72
2. Freudiger, C. W., Pfanni, R., Orringer, D. A., Saar, B. G., Ji, M., Zeng, Q., Ottoboni,
L., Ying, W., Waeber, C., Sims, J. R., De Jager, P. L., Sagher, O., Philbert, M. A., Xu,
X., Kesari, S., Xie, X. S., and Young, G. S., 2012. Multicolored stain-free
histopathology with coherent Raman imaging. Laboratory Investigation, [e-journal]
92(10), pp. 1492-1502. https://doi.org/10.1038/labinvest.2012.109
3. Gurina, T. S. and Simms, L., 2023. Histology, Staining. [e-book] Treasure Island:
StatPearls. Available at: National Library of Medicine.
<https://www.ncbi.nlm.nih.gov/books/NBK557663/> [Accessed 10 March 2024]
4. Jimson, S., Malathi, L., Kumar, G. M. K., and Balachander, N., 2016. Artifact in
Histological Section. Biomedical and Pharmacology Journal, [e-journal] 9(2), pp.
843-845. https://dx.doi.org/10.13005/bpj/1014
5. Krishnanand, P. S., Kamath, V. V., Nagaraja, A., and Badni, M., 2010. Artefacts in
Oral Mucosal Biopsies – A Review. Journal of Orofacial Sciences, [e-journal] 2(1),
pp. 57-62.
6. Rastogi, V., Puri, N., Arora, S., Kaur, G., Yadav, L., and Sharma, R., 2013. Artefacts:
A Diagnostic Dilemma – A Review. Journal of Clinical and Diagnostic Research, [e-
journal] 7(10), pp. 2408-2413. https://dx.doi.org/10.7860/JCDR/2013/6170.3541
7. Taqi, S. A., Sami, S. A., Sami, L. B., and Zaki, S. A., 2018. A review of artifacts in
histopathology. Journal of Oral and Maxillofacial Pathology, [e-journal] 22(2), p.
279. https://doi.org/10.4103%2Fjomfp.JOMFP_125_15
8. Treanor, D., 2009. Virtual slides: an introduction. Diagnostic Histopathology, [e-
journal] 15(2), pp. 99-103. https://doi.org/10.1016/j.mpdhp.2009.01.006
9. Webster, J. D. and Dunstan, R. W., 2013. Whole-Slide Imaging and Automated Image
Analysis: Considerations and Opportunities in the Practice of Pathology. American
College of Veterinary Pathologists, [e-journal] 51(1), pp. 211-223.
https://doi.org/10.1177/0300985813503570

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10. Zhang, Y., de Haan, K., Rivenson, Y., Li, J., Delis, A., and Ozcan, A., 2020. Digital
synthesis of histological stains using micro-structured and multiplexed virtual staining
of label-free tissue. Light: Science and Applications, [e-journal] 9(78).
https://doi.org/10.1038/s41377-020-0315-y

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