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HISTOPATHOLOGY, FRESH TISSUE PREPARATION (FDCIETS SMoL), CYTOLOGY, HTP…

- - - - According to ORIGIN
        
        Synthetic
        coal tar & benzene derivatives
        
        Natural
        plant, insects, lichen (from fungi & algae)
        
        Saffron (Crocus sativus)
        
        Holy dye
        
        Yellow - yellow-red
        
        Orcein (from lichen)
        
        Blue-violet
        
        litchen-derived
        
        Carmine (Coccus cacti)
        
        Female cochineal bug
        
        Dark red
        
        Alizarin (Rubia tinctorium)
        
        Roots of Madder plant
        
        Red
        
        Hematoxylin (Haematoxylum campechianum)
        
        Logwood dye
        
        Blue
        
        Litmus (from lichen)
        
        pH indicator
        
        CH SOAL
      - According to ELECTRICAL CHARGE
        
        Basic
        
        Basic fuschin
        
        Crystal violet
        
        Methyl violet
        
        Methyl green
        
        Malachite green
        
        Saffranin
        
        Methylene blue
        
        Acid
        
        Acid fuschin
        
        Congo red
        
        Aniline blue
        
        Picric acid
        
        Eosin B
        
        Eosin Y
        
        Saffron
        
        Methyl blue
        
        🤡 Ano nga dye ang neat and clean?
        EOSIN kay permi siya gapang himos (HAHA as a funny person)
        
        Amphoteric
        
        Hematein/ Hematoxylin
        
        🤡 Ampho-10 HAHA LORD
        
        Neutral
        
        Giemsa stain
        
        Wright’s stain
        
        May-grunwald stain
        
        🤡 May Wright si Giemsa mangin Zero (neutral)
      - According to
        CHEMICAL CONFIGURATION AT CHROMOPHORE
        
        Nitro
        Chromophore: Nitrogen dioxide (NO2)
        
        Non-aromatic/Alipathic compounds
        
        Aromatic compounds
        
        Azo chromophore
        Chromophore: Azo group
        
        Monoazo
        
        Orange
        Ponceau 2R
        🤡 (Orange nga Ponkan)
        
        Diazo
        
        Sudan III
        Sudan IV
        Oil Red O
        🤡 (Basta Sudan? Fats!)
        
        Triazo & Polyazo
        
        Blue ink dye
        Sirius light blue
        🤡 (Sirius Blue)
        
        Aniline
        
        Quininoid Chromophore
        Chromophore: Quininoid ring
        
        Antra-quininioid Group
        
        Alizarin red S
        
        Xanthene Group
        
        Triarylamine Group
        
        Basic Dyes
        (BC MMA)
        Basic fuchsin
        Crystal violet
        Methyl green
        Malachite green
        Aniline Blue
        
        Acid Dyes
        (ALaM)
        Acid fuschin
        Light green SF
        Methyl blue
        
        Hematein
        
        Quinonimine
        
        basta may -ine sa punta 😩
    - - IMMUNOHISTOCHEMICAL (IHC)
        Antigens (epitopes/ markers) in the tissue will be demonstrated
        
        Antibody is used to 'deliver' dye to the tissue
        
        Combination of immunologic and histochemical techniques
      - HISTOLOGICAL
        dye binds with/adsorbed on tissue
        
        Microanatomical stains
        
        Bacterial stains
        
        Gomori Trichrome Blue stain
      - HISTOCHEMICAL
        chemical reacts with the tissue component to produce color
        
        Prussian blue stain: Hgb
        
        Periodic Acid Schiff: CHO
    - - DIRECT
        (Absorption)
        WITHOUT mordant/ accentuator
        
        Giving color to the sections using aqueous/ alcoholic dye solutions
        
        Dye is electrically attracted & penetrates the cell
      - INDIRECT
        WITH mordant/ accentuator
        
        Mordant binds with the dye to form a “lake"
        
        Ginause sa regressive staining
        
        Tissue-Mordant-Dye Complex
        
        MORDANT: serves as link/ bridge between the tissue and the dye to make staining reaction possible
        
        ACCENTUATOR: accelerates or hastens the speed of staining power and selectivity; not essential to the union of tissue and dye
    - - METACHROMATIC
        staining
        
        tissue stained is different from the color of the stain itself
        
        Mast cells: magenta red in Toluidine blue
        
        Babes granules (Diphtheria): red in MB
        
        Amyloid: brilliant red in Congo red
      - COUNTERSTAIN
        
        application with a different color to
        provide contrast and background
        
        Eosin: counterstains Hematoxylin
        
        NUCLEAR STAINS
        
        BLUE
        
        Methylene blue
        Toluidine blue
        Celestine blue
        
        RED
        
        Neutral Red
        Safranin O
        Carmine
        Hematoxylin
        
        CYTOPLASMIC STAINS
        
        YELLOW
        
        Picric acid
        Orange G
        Rose bengal
        
        GREEN
        
        Light Green SF
        Lissamine Green
        
        RED
        
        Phloxine B
        Eosin B
        Eosin Y
      - VITAL
        Staining
        
        INTRAvital
        
        application of stain via injecting:
        
        IV
        
        Intraperitoneal
        
        Subcutaneous
        
        SUPRAvital
        
        application of stain to cells removed from body
        
        N methylene blue
        
        Brilliant Cresyl Blue
        
        Reticulocytes
        
        Heinz bodies
        
        staining of living cells
      - PROGRESSIVE
        staining
        
        stained in sequence/ chronological order
        once dye is taken up, tissue is not moved
        
        Masson’s Trichrome staining
        
        No decolorization or washing
      - REGRESSIVE
        staining
        
        primary stain applied in excess/ overstained to obliterate cellular details
        overstained → de-stained (differentiated) - decolorized using alcohols (e.g. acid alcohol)
        
        H&E, Gram's PAP's
        
        DIFFERENTIATION/ DECOLORIZATION: selective removal of excess stain from tissue during regressive staining
      - METALLIC IMPREGNATION
        
        usage of silver/ gold esp. CNS, argyrophilic
        consists of hypnotic drugs (e.g. barbital)
        
        2. Argentaffin reaction
        
        melanin, enterochromaffin, APUD cells
        
        3. Induced Argentaffin reaction
        
        systemic fungi, basement membranes
        
        1. Argyrophyl reaction
        
        for reticulin fibers, nerve fibers, calcium salts, spirochetes
      - FLUORESCENT STAINING
        
        tissue is labelled with fluorescent dye
        Fluorescence is the property of a substance that absorbs light of a shorter wavelength (UV light) and then emitting light of a longer wavelength (Visible light)
        
        FISH
        Fluorescent in-situ hybridization
        
        Probe w/ dye + unknown ssDNA
      - ORTHOCHROMATIC
        staining
        
        tissue stained is similar to the dye itself
    - - PHYSICAL
        
        Selective solubility
        Selective solubility of dyes in oils
        
        Sudan III
        orange color
        CNS tissue
        
        Orange III? Orange Tree.
        
        Sudan IV
        dark red color
        neutral lipids (TAGs)
        Scarlet red, Scharlach B
        
        B-naphtols
        
        Sudan Black
        black 😪
        most sensitive
        neutral lipids, phospholipids
        
        Sundan black?
        
        Porosity/ Permeability
        permeability of dye thru protein meshwork on the surface of the tissue
        
        Fixation precipitates proteins
        
        Meshwork is formed on the surface of the tissue, pore size differs in each protein.
        
        Used in Trichrome staining of the connective tissue
        
        Adsorption
        Metallic Impregnation Technique
        
        Ag+ become electrically bound to the negative electric cloud that surrounds the tissue
        💥 Strong negative charge is provided by the use of a very acidic fixative solution (i.e., Ramon y Cajal, Conn & Darrow, Davenport’s)
        
        “Dyes” is easily differentiated in water or alcohol.
      - CHEMICAL
        
        Chemical reagent (not a dye) is used
        
        Involves:
        
        Dye or dye precursor binds to the tissue by slat-linkages (ionic bonds)
        
        Covalent bonding
        
        Formation of chelates
        
        Production of insoluble pigment (Prussian blue)
        
        Prussian Blue Method (for hemosiderin/ Fe+++)
        Fe+++ (Ferric) + Potassium ferrocyanide → Prussian blue color
        
        Turnbull blue method (for Fe++)
        Fe++ (Ferrous) + Potassium ferrocyanide → Turnblue blue color
        
        PAS (for polysaccharides)
        Carbohydarte + Periodic acid + Schiff’s rgt → Oxidized Schiff’s rgt (magenta red)
    - - Component of Fixative
        
        Broad spectrum
        
        Zenker's
        
        10% buffered neutral formalin
        
        Helly's
        
        Bouin’s Susa
        
        Carnoy's
        
        Hg++
        
        Enchaces nuclear staining and cellular detail
        
        Best used in Trichrome staining
        
        Interferes with hematoxylin
        
        Formalin is not the best fixative for aniline dyes (Trichrome staining)
        = Prolonged fixation inhibits eosin action
      - Electric Charge of stain
      - pH of Fixative
      - Mordant
        reacts with stain creates insoluble colored precipetate on tissue
      - Presence of Oxidizers and Reducers
        
        Hematoxylin is only a potential dye; ineffective unless oxidized to hematein
        
        In Metallic Impregnation technique the metallic salt is reduced into a metallic precipitate (silver precipitate) which settles on the surface of the tissue when acted upon by reducing substances present on the tissue surface or in the staining solution.
- - - - SLIDE PREPARATION/SMEAR
        
        Solid lesion
        
        most diagnostic material for cytological evaluation
        
        usually few drops from the tip of the needle
        
        if Bloody specimen
        
        diagnostic cells are diluted...hard to find on direct smear
        
        Ideal aspirate 💘
        
        creamy consistency with numerous cells
        
        no blood
        
        Transbrochial FNA
        
        1-2 drops sa slide
        
        smeared by 2 slide pull method
        
        Lymph node aspiration
        
        cell suspension prepared in addtion to direct smears
      - FIXATION
        Smears obatined by FNA are fixed based sa requirements sang stain na gamiton
        
        Colloid, mucins and smears - stained with hematological stains
        
        May-Grunwald-Giemsa
        should be air dried quickly (if not quickly = artifacts)
        
        Rapid stains = Diff Quick (2-3min)
        useful in prelimenary assessment of adequacy of sample before px is released
        
        Smears to be stained with PAPANICOLAOU or H&E
        should be rapidly fixed with alcohol (wet fixation)
        
        shows nuclear details
        
        better identification of malignant cells
        
        ⚠️ if smears not quickly fixed and dried
        
        cytoplasm will be eosinophilic
        
        nuclear details = Fuzzy
        
        drying artifact occurs
  - - - Direct
        
        Exudate
        
        Transudate
      - Cytocentrifuge/ Cytospin
  - - - Sampling & Preparation
        
        Smear
        
        Liquid-Based Cytology
      - Cells in the Cervico-Vagina
        (Superficial - Deep)
        
        Parabasal
        
        round to oval cells w/ small dense basophilic cytoplasm (15-30um)
        
        young cells
        
        2 weeks of age, after childbirth, with abortions, after menopause
        
        Endometrial cells
        
        small cells, cylindrical w/ less basophilic cyotplasm
        
        tightly packed groups of 3 or more
        
        1-10 days after menstruation; shed in response to ovarian hormones
        
        FERNING - indicator of fertility, cervical mucous crystal
        
        Intermediate Cells
        
        polyhedral/elongated cells with basophilic vacuolated cytoplasm)
        
        present in all stages
        
        Navicular cells
        
        boat shape
        
        strong tendency to fold/curl
        
        presence suggest combined estrogen-progesteron effect
        found on all stages except pregancy
        
        Pregnancy cells
        
        round, oval, boatshape
        
        translucent basophilic cytoplasm, due to; glycogen accumulation pushing nucleus at the side
        
        deeper blue stain at the periphery
        
        ''double boundary'' (cell may ara 2 hues of blue)
        
        Endocervical Glandular Cells
        
        honeycomb appearance
        
        small sheets or large groups
        
        cytoplasm stained pale blue or gray, finely vacuolated
        
        Mature Superficial
        
        Polygonal squamous cell (45-50um, pale pink cytoplasm, dark pyknotic nuclei >6um)
        
        uppermost layer (superficial vaginal cells - under influence by estrogen) = true acidophilia
        
        The Bethesda System
        (TBS)
        
        Group III: moderate to severe dysplasia (doubtful result)
        
        Group IV: carcinoma in situ
        
        Group II: normal variant but not suspicious for malignancy (negative)
        
        Group V: invasive carcinoma
        
        Group I: exclusively normal cells (negative)
        
        Cervical Intraepithelial Lesion
        (CIN)
        
        CIN 2: for moderate changes
        
        CIN 3: for more severe changes (high-grade)
        
        CIN 1: for mild changes (low-grade)
        
        CIN IV
        
        CIN IVa: carcinoma in situ
        
        CIN IVb: invasive carcinoma
        
        CIN 0: negative
        
        PAPs Grading
        
        Class III: presence of dysplasia; cytologic picture suggestive but not conclusive of malignancy
        
        Class VIa: carcinoma in situ; cancer has not yet metastasized; capsule of cancer cells are intact
        
        Class II: presence of atypical cytologic picture but no evidence of malignancy
        
        Class IVb: invasive carcinoma
        
        Class I: absence if atypical cells (normal)
    - - SMEAR PREPARATIONS
        
        preferably on frosted end slide
        
        smears fixed in 95% ethanol
        
        IMPRESSION SMEAR
        
        SPUTUM SMEAR
        
        collect 3 consecutive days
        
        deep cough
        
        cytological fixative - saccomanno fluid ( 50% alcohol & 2% carbowax)
        
        if inde kaubo, inhalation of aerosol soln' for 20 mins.
        
        BRONCHOSCOPY SPECIMENS
        
        Methods
        
        Bronchial washing
        
        1 more item...
        
        Bronchial aspirates
        
        1 more item...
        
        Bronchial brushing
        
        1 more item...
        
        SMEARS FOR GASTRIC SECRETIONS AND ASPIRATES
        
        need 8 hours fasting
        
        if not fixed within 30 min. gastric juice will digest cells fir evaluation
        
        usually collected by:
        
        simple irrigaiton
        
        aspiration technique
        
        bronchial lavage
        
        gastric brush
        
        FNA (fine needle aspiration) : submucosal
        
        SMEARS FOR BREAST SECRETION
        
        1 more item...
        
        recommended 2-4 slides e prepare
        1 is air dried - giemsa stain
        other 2 - for papanicolaou
        
        Alveolar Macrophages = sputum
        
        no Alveolar macrophages = saliva
        
        used for ulcerated surface lesion, gna use for assessment of lesion
        
        for cases of tumors especially lymph nodes
        
        quickly air dried then stained
        
        cytological detection - air drying not allowed, changes appearance of cells
        blood/BM smear - allowed air drying
- - - - N0: lymph nodes are not involved
      - N1: mobile lymph nodes are involved
      - N2: fixed lymph nodes are involved
    - - Mx: suspected metastases
      - M1: evidence of metastases
      - M0: no distant metastases
      - M2: blood-borne metastases
    - - T1: Tumor 1 cm in diameter
      - T2: Tumor less than 2cm in diameter
      - T0: in-situ tumor
      - T3: Tumor less than 3cm in diameter
  - - - 75% - 100%
        
        Grade I
        
        50% - 75%
        
        25% - 50%
        
        0% - 25%
        
        Grade IV
        
        Grade III
        
        Grade II
    - - 0% - 25%
        
        25% - 50%
        
        50% - 75%
        
        75% - 100%
- - - - Repeated mutation
        
        Malignant tumor
        
        Loss of control and regulation
        
        Rapid cell proliferation
        
        Elevated level of normla protein
        
        De-differention
        
        Carcino-embryonic antigen
        (Ectopic markers)