General steps of Histopatholgy and Cytopatholgy
The figure below shows the general steps of histopathology and cytopathology. First step is the sample fixation. This step is crucial to get good microscopic slides. Second steps, is only for histopathology which is sample processing. The next step is sample staining. This steps basically to distinguish which part of cell are nucleus or cytoplasm. Last but not least is microscopic examination.
Histopathology
The histology samples from patients have several steps in order to diagnose their cells. It is involved several steps which; 1- sample fixation with formalin, 2- sample processing, 3- sample staining. and 4- Microscopic examination.
1: Sample Fixation
Sample fixation is a crucial step in histology studies. Fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. Fixation of tissue is done for several reasons. One reason is to kill the tissue so that postmortem decay (autolysis and putrefaction) is prevented. Fixation preserves a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this, several conditions usually must be met:
First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes—which otherwise digests or damages the sample.Second, a fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.
Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis. In this cases, the sample from patient is immersed in 10% formalin.
2: Sample processing
To provide slides for microscopic examination, the sample must be process first. This step involved another several steps. First is sample grossing- Is the process referred to as grossing a specimen, where tissue specimens taken from routine surgical cases, autopsies, or other scientific investigations are examined, described and trimmed to proper size. The grossing method is vary from hospital to hospital. From this lab, the sample that fixed by 10% formalin is cut. The part of sample that being cut is depend on pathologist. Pathologist may choose the tumour part or healthy part as control.
Next step is sample dehydration by tissue processor which is automated or can be done manually. The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Biological tissue must be supported in a hard matrix to allow sufficiently thin sections to be cut. For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. This is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene.The sample processing firstly immersed in formalin for 30 minutes twice, after that in Alcohol solution for 1 hour six times, next in xylene for 1 hour 3 times and in wax also 1hour 3 times.
Next is the embedding step. After the tissues have been dehydrated, cleared, and infiltrated with the embedding material, they are ready for external embedding. During this process the tissue samples are placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned.
Because Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, FFPE tissues are an important resource for historical studies in medicine. The last step for sample processing is the sample sectioning by microtome. This step can be divide by 2 which are trimming and sectioning. This step provide thin slide for microscopic examination.
3: Sample staining
Before we observed the morphology of cell/sample under microscope, first we must stained the sample. In histopathology studies, the routine stain is Haematoxylin and Eosin stain (H &E). The purpose of H & E staining is to distinguish the nucleus and cytoplasm. Haematoxylin which stains blue or purple on nuclei. Haematoxylin is oxidative agent that positively charged and bind to negatively charged phosphate on nuclear-DNA. Eosin which stains pink to red blood cell or connective tissue in cytoplasm. Eosin is acidic colourant that mainly bind to protein.Besides routine H & E staining, it is also has another type of staining which histochemistry staining and Immunohistochemistry staining. The need to do the staining is depend on the patient cases and diseases.
4: Microscopic examination
Under the light microscopes, the pathologist can observed the morphology of the cell to determine the type of tumour whether benign or malignant, the level of certain cancer and whether the tumour are metastatic or not.
Cytopathology
Cytology laboratory test can be divide by two which “Gynecological Cytology” and “non-gynecological cytology”. This is the test that everyone known by as “Pap Smear”. It name comes from the Papanicolau test for cancer cells, developed in the 1940’s. Smears are made from cells collected from the cervix of the uterus, stained, and then examined microscopically for evidence of any abnormal cells. Non-gynecological cytology can be body fluid such as sputum,urine and spinal fluid and also fine needle aspiration.
Cytology samples are fixed in 95% alcohol at least for 30 minutes. After that, the samples are stained by Papanicalou stain. After staining, observed the morphology under light microscope.
Principle of Papanicolou staining
It is involves five dyes in three solutions, A nuclear stain, haematoxylin, is used to stain cell nuclei. The unmordanted haematein may be responsible for the yellow color imparted to glycogen.First OG-6 counterstain (-6 denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8). Orange G is used. It stains keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
Second EA (Eosin Azure) counterstain, comprising three dyes; the number denotes the proportion of the dyes, eg. EA-36, EA-50, EA-65. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells. Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, and however it produces visually different results and is not considered satisfactory by some. Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.
How to distinguish the benign tumour and malignant tumour in the cases of breast cancer
From this lab, what interesting that I can learn is how to distinguish benign tumour and malignant tumour specific to breast cancer. During practical here, I try to compare between various slides of breast cancer patients and observed the doctor comment on the patients. By H & E staining, we know that Haematoxyline stain purple on nuclei, so that under microscopic observation I try to observed nuclei morphology on benign tumour and malignant tumour.
From microscopic observation, the nuclei in benign tumour cell are big, and clump together in one place but in malignant tumour, nuclei very small and disperse in all cytoplasm or tissues. The presence of nuclei in all tissues indicates that the tumour is invasive (malignant tumour).
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| Benign Breast Tumour |
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| Malignant Breast Tumour |
It has always interested me to hear about how others view these things. I have been teaching using histology dyes for awhile now and the reactions are always different
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