Appunti Anatomia patologica (Anatomical Pathology, Biomolecular Basis of Anatomical Pathology)

Anatomical Pathology 1 (08/03/2023)

Pathological anatomy

Pathological anatomy: binding bridge between basic sciences and clinical medicine belonging to lab medicine➔ represents the fundamental step in the clinical diagnostic approach.

Surgical pathology

Surgical pathology: surgical samples from the operating room (often constituted by small biopsies, e.g. liver, renal, etc.)

Intraoperative histological diagnosis

Pathologist able to study samples while patient is still on the surgical table in order to indicate the surgeon about pathology features, e.g. benign/malignant neoplastic lesions. This operating way helps the surgeon to avoid unnecessary tissue resection.

Autopsy

Autopsy: important role in the past in anatomic-clinical correlation. Remains the last due of the pathologist to reach a diagnosis in very difficult and complex cases and maintains an important role in student learning➔

• Autopsy requirement: dead bodies without medical assistance
• Dead patients in public or private hospitals if diagnostic confirmation is needed and/or for scientific purposes
• Forensic autopsy: usually done by the coroner when data must be collected as legal evidence in judicial processes

Pathological anatomy subspecialties

Many branches of pathological anatomy such as hematopathology, dermatopathology, neuropathology due to the big progress of knowledge needing different and innovative methods and techniques to perform the final diagnosis.

Cytopathology

Cytopathology➔ studies nuclear and cytoplasmic alteration in single cells which must be related to pathological processes of the tissues from which cells are derived. It is a powerful technique: easy, cheap, and quick technique mainly used in tumor screening, particularly in uterine cervix tumor (PAP Test).

Sampling methods

• Natural exfoliation: cells from saliva and cervical-vaginal secretion
• Brushing: cells from bronchial, nasal, esophageal, gastrointestinal, cervical brushing or brocho-alveolar lavage (BAL) samples richer in cells
• Cytocentrifugation/gradient centrifugation: cells from physiological liquid are examined such as cerebrospinal fluid, urine, pleural, pericardial, or peritoneal exudate
• FNAB/fine needle biopsy: cells from superficial lesions are fine needle biopsied (breast, salivary glands, thyroid, lymph node) or, sound guided, from deep lesions (lung, kidney, liver, pancreas, etc.)

Methods

PAP test: samples smeared on the slide and fast fixed (within 10 min) in ethanol 95% or other commercial fixation solution➢ nuclei stained in blue with hematoxylin and counterstained with Orange G (cytoplasm orange if it has a lot of cytokeratin) and with an eosin and light green mixture (cells with an acidophilic cytoplasm are stained with eosin, cells with basophilic cytoplasm are stained in light blue)➢ adequacy of the sample (cells in the samples are detectable)➢ cell population (cell number, cell debris, necrotic material, inflammatory cells)➢ nuclear and cytoplasmic characteristics.

Main cytological features

• Nucleus morphology: distinguish benign from malignant cells
• Cytoplasm morphology: define cell differentiation (atypia is considered a pre-neoplastic condition)
• Normal, reactive, degenerating, dysplastic (cell in transition between the normal to neoplastic morphology➔ which can be graded in low and high grade) definition of low/high grade based on involvement of different layers in the epithelium (dysplasia mainly related to modifications in epithelial cells)
• Low-grade dysplasia = only cells in the basal layer are involved
• Moderate = 2/3 layer
• Severe/high grade = all the thickness of the epithelium is involved by the presence of dysplastic cells

Neoplastic cells are often aggregated in overlapping layers with a severe pleomorphism, nuclear atypia, mitoses (if we see mitosis in the smears we know that there is a malignant tumor: proliferation rate in benign tumor is not so efficient so it is not visible in the samples), prominent and angulated, fragmented cytoplasm with anomalous differentiation features nucleoli.

Neoplastic

Malignant tumors can be divided into differentiated and undifferentiated malignant tumors➢ if we have an anaplastic tumor we have a complete loss of differentiation (= complete anaplasia) and cells are only able to proliferate.

Important: only one single parameter is not enough to define a cell as a neoplastic one.

Normal pattern

Group of normal epithelial cells within a cytological smear➢ the epithelial cell is already a well-differentiated cell➢ low ratio between nucleus/cytoplasm➢ nucleus is not so big, because cytoplasm has a lot of functions➢ in this case the nucleus is normal in size.

Reactive pattern – herpes infection

Reactive pattern in which there are inflammatory mediators/infiltrates➢ it is possible to see leukocytes polymorphonucleates (probably neutrophils)➢ cells are altered by presence of the virus➢ bigger nucleus in which there is prominent chromatin more staining➢ morphology of cells and nucleus are quite normal = mild dysplasia➢ possible to see the shape of the nucleus polylobate nucleus.

Dysplasia

Different transformation from a normal cell to a severe dysplasia➔ when describing an epithelium, which is a dysplastic one, we can also define the status of the dysplasia.

Dysplastic classification

• Mild: if the layer involved in this dysplastic mechanism is 1/3 of the total epithelium
• Moderate: if the layer involved in this dysplastic mechanism is 2/3 of the total
• Severe: when all the epithelium is dysplastic (also known as in situ neoplasia)

From a cytological point of view we can see differences between normal and dysplastic cells➔ change in nucleus size is one of the first alterations in the cell➔ low nucleus/cytoplasm ratio normal cells➔ high nucleus/cytoplasm ratio dysplasia (either mild/moderate/severe)➔ small cytoplasm indicates a loss of normal function.

In mild dysplasia we have an increase of nuclear size + modification of the shape of cells➢ mild dysplastic cells are characterized by big nuclei with a cell that has an elongated shape➢ severe dysplastic cells are smaller and characterized by a high ratio between nucleus and cytoplasm➔ the nucleus has a prominent nucleolus and it means that there is a neoplastic cell (normally it is difficult to see the nucleolus)➢ in neoplastic cells there is an increase of the metabolic activity.

Neoplastic

We can have proliferation of abnormal cells with respect to the shape and size of epithelial cells; here it is possible to see an active inducing proliferation of multilayer structures➢ undifferentiated anaplastic tumors present different types of cells, e.g. small-cells undifferentiated tumors might seem similar to stem cells➢ when we have a very undifferentiated tumor cells can be big cells giant-cell tumor or small cells small-cell tumor, or completely undifferentiated.

FNAP – fine needle aspiration biopsy

Fine needle biopsy in which we have cells which can be organized as a normal tissue (like an epithelial layer)➢ cells are completely normal➢ morphology of the cell is normal➢ possible to see that the epithelial cells seem to constitute a sort of gland lumen➢ they can be recognized in a quite normal structure➢ in this case there is not a tumor, even if these cells are neoplastic ones. It is a differentiated benign neoplasia (such as adenoma, which is a benign neoplasia derived from epithelial cells of gland luminal) even if they are proliferating cells they are differentiated and maintain their function with limited proliferation activity and quite normal structures.

Necropsy assessment

In each death case the following steps are needed➢ ascertainment of death➢ necropsy assessment (hypostatic spots, rigor mortis) used to define if patient is really dead. The pathologist must go and observe if there are changes in the body of the patient➢ observe the external aspect of the body to see if there are specific lesions➢ observation period (24/48 hrs for sudden death)➢ assessment of brain death.

Transplant law

The problem for organ transplant is the assessment of brain death➔ cardiac death: clinical examination evidences silence of heart tones, absence of arterial pulses, impossibility of measuring blood pressure and immobility of cardiac shadow to the scopia + stopping breathing (chest and diaphragm immobility)➔ brain death: stop of cerebral activity and vegetative functions survival by resuscitation maneuvers➔ there is a difference between the two = we could have brain death even if there is still cardiac activity. It is difficult to say, when the patient is related to respiratory devices, if they are dead or not➢ in brain death the heart continues its activity: the patient is still alive however cerebral activity is not present. Necessity to have a medical committee in order to establish if we have a “real” brain death + evaluate organ transplantation.

Brain death due to massive brain necrosis caused by primary lesions such as tumors, trauma, and cerebral bleeding➔ after cerebral tissue death many events follow, such as stop of spontaneous breathing and stop of cerebral electric activity➔ only cardiac activity spontaneously survives. This kind of dissociate death is only possible by resuscitation techniques which allow a patient in a coma dépassé to maintain for an indefinite period of time vegetative functions (skin colorful, body temperature, metabolism, digestion, and normal renal function). If the tumor is so big, the lesion goes and compresses the cerebral hemispheres: because the cerebral hemispheres are delimited by bones, it is impossible for them to increase their area. These hemispheres are pushed to the vertebral canal in this case, it is possible to have herniation, which causes the compression of medulla oblongata and the stop of the breathing activity.

Anatomical Pathology 2 (10/03/2023)

Histopathology

Histopathology➔ main histological activities: intraoperative histological diagnosis, surgical pathology, cytopathology, autopsy➔ biological sample procedures: fixation, dehydration, embedding, sectioning, staining➔ special techniques➔ histochemical stains for different types of staining, depending on what we are looking for, e.g. PAS➔ immunohistochemistry to define the immunophenotype of the tumor (using different antibodies)➔ flux cytometry➔ electron microscopy used to see the structure of the inner part of the cells (all of the organelles) + see if cells are differentiated or not.

Molecular biology

Biological samples are usually associated with a request containing: personal details, clinical history, clinical suspicion, site of origin, and referring doctor and hospital ward; code number for each biological sample; quality assurance procedures need that each step is recognizable (technician, pathologist, nurse, secretary).

Gross examination

Pathologist and resident pathologist aided by technician describe the specimen and select significant area putting them into small boxes contained in a fixative solution (10% formalin buffer).

Tissue fixation

The principal aim of the fixation step is to stop immediately all the metabolic vital mechanisms and maintain➔ the tissue structure that is during the complete processing mechanism need to block everything because, after fixation, there are other steps that use chemical substances (no interactions)➔ fixation is the most critical step in biological material processing to avoid artifacts which could be limiting in the interpretation➔ inappropriate fixation is able to impede structure reading.

Formaldehyde is a colorless gas water-soluble➔ product available as a water solution 40% called formalin➔ slow fixative➔ in microscopic applications, formalin is used as a buffered solution➔ policard formalin is a 10% solution + NaCl 0.9%.

After fixation tissue specimens must be washed vigorously to eliminate excess formalin which may cause➔ staining artifacts following steps are dehydration, clarification, and embedding. Dehydration is carried out by many steps in ascending graded ethanol from 25% to 100% (15 min each, depending on sample size)➔ clearing to cut sections, the tissue has to be embedded in paraffin wax, but wax is not soluble in water or alcohol; however, it is soluble in a paraffin solvent called xylene. The water in the tissue needs to be replaced by xylene: finally, once the water has been replaced by 100% alcohol, the alcohol is replaced with xylene which is miscible with alcohol➔ final step is called clearing➔ inclusion/embedding the tissue is placed in warm paraffin wax and the melted wax fills the spaces that used to have water in them; after cooling, the tissue hardens and can be used to cut slices (sectioning)➢ the tissue is trimmed, and mounted on a cutting device called a microtome➢ thin sections are cut, 4-6 microns, which can be stained and mounted on a microscope slide

Staining and mounting

Unfortunately, most staining solutions are aqueous, so to stain the sections, the wax has to be dissolved and replaced with water (rehydration)➢ this is essentially a step 2 in reverse. Sections are passed through xylene, and then decreasing strengths of alcohol (100% to 0%) and finally water➢ once stained, the section is then dehydrated once again, and placed in xylene➢ it is then mounted on the microscope slide in mounting medium dissolved in xylene with a coverslip placed on top to protect the sample➢ evaporation of xylene around the edges of the coverslip, dries the mounting medium and bonds the coverslips firmly to the slide.

What kind of histological stains are there?

Most cells are colorless and transparent, and therefore histological sections have to be stained in some way to make the cells visible➢ the techniques used can either be non-specific, staining most of the cells in much the same way, or specific, selectively staining particular chemical groupings or molecules within cells or tissues➢ staining usually works by using a dye that stains some of the cells components a bright color, together with a counterstain that stains the rest of the cell a different color.

Basophilic and acidophilic staining

Acidic dyes react with cationic or basic components in cells: proteins and other components in the cytoplasm are basic, and will bind to acidic dyes. Another way of saying this is that cytoplasmic proteins are acidophilic. Basic dyes react with anionic or acidic components in cells. Nucleic acids are acidic, and therefore bind to basic dyes.

H&E staining

The most commonly used staining system is called H&E (hematoxylin and eosin). Eosin is an acidic dye: it is negatively charged (general formula for acidic dyes is: Na dye)➢ it stains basic (or acidophilic) structures red or pink, this is also sometimes termed eosinophilic, thus, the cytoplasm is stained pink in the picture below, by H&E staining.

Hematoxylin can be considered as a basic dye. Hematoxylin is actually a dye called hematein (obtained from the log-wood tree) used in combination with aluminum ions (Al³⁺)➢ it is used to stain acidic (or basophilic) structures a purplish blue➢ thus the nucleus is stained in purple. This means that the nucleus, and parts of the cytoplasm that contain RNA stain up in one color (purple), and the rest of the cytoplasm stains up a different color (pink).

What structures are stained in purple (basophilic)?

DNA (heterochromatin and the nucleolus) in the nucleus, and RNA in ribosomes and in the rough endoplasmic reticulum are both acidic, and so hematoxylin binds to them and stains them purple; some extracellular materials (i.e. carbohydrates in cartilage) are also basophilic.

What structures are stained in pink (eosinophilic/acidophilic)?

Most proteins in the cytoplasm are basic, and so eosin binds to these proteins and stains them pink; this includes cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibers. Hematoxylin and eosin stain is useful for quite all diagnoses, e.g. Ewing sarcoma: small round cell tumor, bone tumor, high proliferative tumor with cells stained in blue. Cells have a very high nucleus/cytoplasmic ratio, so we have the prevalence of hematoxylin which stains in blue.

Questions in oncology

• What is tumor origin (histogenetic diagnosis)?
• What is tumor behavior (benign or malignant tumor)?
• What is tumor extension (tumor staging)?

Special techniques

Useful for diagnosis, etiology, histogenesis, pathogenesis➢ histoenzymology➢ cell culture from primary tumor or cell culture used in genetic disorders to study features➢ morphometry➢ electron microscopy.

Special stains

• PAS
• Bacteria, fungi, parasites
• Argentaffin and argyrophilic stains to evidence in tissue the presence of neuroendocrine cells
• Amyloid deposition
• Reticulin
• Tri-chrome staining to see the cells, the nucleus, cytoplasm, and also staining of stroma, ECM
• PTAH
• Hemosiderin (Perls)
• Melanin (Masson-Fontana)
• Calcium (von Kossa) presence of calcium due to dystrophic mechanisms, also can appear in breast cancer (marker for malignancy of the tumor)

Masson's trichrome

Often used to stain connective tissue➔ tri-chrome = the technique produces three colors. Nuclei and other basophilic (basic-liking) structures are stained blue. Cytoplasm, muscle, erythrocytes, and keratin are stained in bright-red. Collage is stained green or blue, depending on which variant of the technique is used.

Giemsa

Usually used for staining blood and bone-marrow smears➢