Basic Histology Part 4

‬ ‭Fixatives for Exfoliative Cytology‬ ‭Common preparation methods of fluid specimens‬ ‭Smears‬ ‭Cell block‬ ‭2 smears are usually made‬ ‭___________________________‬ ‭BEST‬‭yet highly flammable‬ ‭ ixatives:‬ F ‭1.‬‭Neutral buffered formalin‬‭:‬‭universal‬ ‭2. Formalin/95% alcohol rinse‬ ‭3. Nathan alcohol formalin substitute: less‬ ‭toxic‬ ‭4. Methanol: for automated cell block‬ ‭______________________‬ ‭Most commonly used‬ ‭Carnoy’s fluid‬ ‭ ade of equal parts of tertiary butyl‬ M ‭alcohol and 95% EtOh‬ ‭Saccomano’s fixative‬ ‭Made of 50% EtOh and 2% carbowax‬ ‭Schaudinn’s fluid‬ ‭Made of mercuric chloride & absolute EtOh‬ ‭Spray fixative‬ ‭Secure‬‭_____‬‭distance away for safe use‬ ‭ ethods of preparation after‬ M ‭centrifugation and obtaining sediment‬ ‭ irect‬ D ‭filtration‬ ‭ dd‬‭_______________‬ A ‭Cons: easily washed out‬ ‭ lasma‬ P ‭thrombin‬ ‭ ost popular‬ M ‭Add‬‭_______________‬ ‭★‬ ‭Forms a gel-like substance‬ ‭★‬ ‭Place in filter paper‬ ‭★‬ ‭Add 10% formalin‬ ‭Pros: minimized wash-outs‬ ‭Carbowax‬ A ‭ dd‬‭carbowax‬‭;‬‭uses paraffin block‬ ‭Pros: no dehydration step‬ ‭Cytospin‬ ‭ est for body fluids‬ B ‭Centrifuge‬‭___________________‬ ‭Clot and Scrape‬ ‭1. Aspirate contents onto glass slide‬ ‭2. After drying, scrape & wrap in tissue paper‬ ‭3. Process as routine tissue‬ ‭ ther methods include BBC cell block fixative, Collodion Bag cell‬ O ‭block, Shandon cytoblock, Cellient Automated Cell Block System,‬ ‭and Histogel.‬ ‭ ‭ xfoliated cells decompose rapidly, destroying cellular and nuclear‬ E ‭details, which results to inadequate results for diagnosis‬ ‭Therefore, Fixation is needed to preserve them.‬ ‭Fixation time:‬‭______________________‬ ‭Papanicolau’s Smear and Stain‬ ‭ redited to‬‭George Nicolas Papanicolau‬‭(1940)‬ C ‭Primary usage‬‭:‬‭___________________________‬ ‭Smears are prepared by‬‭rotary motion‬‭, and air dried‬‭2 hours after fixation‬ ‭Uses‬‭glycerin‬‭as adhesive‬ ‭➔‬ ‭Egg albumin is‬‭NOT RECOMMENDED‬ ‭Methods for Pap smear‬ ‭ . Conventional‬ 1 ‭2. Liquid Based (SurePath and ThinPrep): placed in vial with preservative‬ ‭Pros of Pap Stain‬ ‭❖‬ O ‭ bservable transparent blue‬ ‭staining of cytoplasm‬ ‭❖‬ ‭Excellent nuclear staining‬ ‭❖‬ ‭Color range is predictable‬ ‭Cons of Pap Stain‬ ‭ ‬L ❖ ‭ engthy and complicated‬ ‭❖‬ ‭Does not give accurate acidophilic‬ ‭index‬ ‭Stains in Pap Stain‬ ‭Harris Hematoxylin‬ ‭Nuclear Stain‬ ‭_______________‬ 1 ‭ st counterstain;‬‭for cytoplasm‬ ‭Strong affinity for‬‭______________‬ ‭ omposition:‬ C ‭1. Orange Green 6 (0.5% in 95% ROH)‬ ‭2. Phosphotungstic acid‬ ‭_______________‬ 2 ‭ nd counterstain;‬‭for cytoplasm‬ ‭Strong affinity for‬‭______________‬ ‭ emember: EA-50 is similar as EA-36‬ R ‭Whereas, EA-65 differs from EA 50/36 in the‬ ‭concentration of the‬‭light green stock solution‬ ‭➔‬ ‭EA-50/36 is 2 times greener‬ ‭ omposition:‬ C ‭1. Light green SF (0.1% in 95% ROH)‬ ‭2. Bismarck brown (0.5% in 95% ROH)‬ ‭3. Eosin Y (0.5% in 95% ROH)‬ ‭4. Phosphotungstic acid‬ ‭5. Lithium carbonate (saturated aqueous soln.)‬ ‭____________‬‭is the stain that stains‬‭nothing‬‭; since‬‭it was included in the‬ ‭Color of cellular components using Paps under microscope‬ ‭Nucleus‬ ‭Vesicular = Blue; Pyknotic = Dark blue to black‬ ‭Cytoplasm‬ O ‭ G-6: Orange with a hint of green‬ ‭EA-36/50: Oliver green with a hint of brown/red‬ ‭Bacteria‬ ‭Dark blue‬ ‭Mycelia‬ ‭Violet‬ ‭__________‬ ‭Pale greenish blue blob of cytoplasm‬ ‭Hormonal Cytology‬ ‭Based on vaginal epithelium response to hormones‬ ‭standard Pap stain, it was removed, changing it to “Modified Pap stain”‬ ‭Produced by‬ ‭Acts upon‬ ‭_____________‬ ‭_____________‬ ‭Granulose theca cells‬ ‭Corpus luteum after ovulation‬ ‭S‬‭uperficial Cells‬ ‭I‬‭ntermediate Cells‬ ‭ pecimen Collection‬ S ‭❖‬ ‭For hormonal changes, the‬‭best site‬‭is the‬‭upper third‬‭of vagina‬ ‭❖‬ ‭For more access and less contamination, the‬‭lateral‬‭walls‬‭can do‬ ‭Normal cells in cervico-vaginal smears‬ ‭ uperficial‬ S ‭cells‬ ‭ arge, polyhedral, and flat (30-60 um)‬ L ‭Small, dark, pyknotic nuclei present‬‭(<6um)‬ ‭Under estrogen influence,‬‭true acidophilia‬‭is observed‬ ‭ ntermediate‬ P I ‭ olyhedral or elongated‬ ‭cells‬ ‭With vesicular nuclei & basophilic cytoplasm w/ vacuoles‬ ‭Types of intermediate cells‬ ‭ ‭__________‬ ‭Boat-shaped cells‬ ‭ trong tendency to fold/curl edges‬ S ‭From “estrogen-progesterone effect”‬ ‭Pregnancy cells‬ ‭Round or oval to oval shape‬ ‭With‬‭double cell wall‬‭appearance‬ ‭_______‬‭pushes the nucleus aside‬ ‭ arabasal‬ P ‭cells‬ ‭ maller than intermediate cells (15-25 um)‬ S ‭With‬‭sunny side up-like‬‭appearance‬ ‭Found as early of 2 weeks of age‬ ‭Abnormal cellular components in cervico-vaginal smears‬ ‭ andida‬ C ‭albicans‬ ‭ udding yeast‬ B ‭Seen among diabetics, taking oral contraceptives,‬ ‭immunocompromised patients, prolonged steroid therapy‬ ‭ richomonas‬ T ‭vaginalis‬ ‭ ear-shaped parasite‬ P ‭Causes __________________‬ ‭ ardnerella‬ G ‭vaginalis‬ ‭ occobacilli that clings to epithelial cytoplasm‬ C ‭Presence of‬‭_______________________‬ ‭Koilocytes‬ ‭ Low Grade Squamous Intraepithelial Lesion”‬ “ ‭Squamous cells that show CPE of‬‭HPV‬ ‭➔‬ ‭Nucleus appears like‬‭________________‬‭that is‬ ‭surrounded by perinuclear halo‬ ‭HSV II‬ ‭ PE characterized by‬‭macrosomia, multinucleation,‬ C ‭nuclear molding,‬‭and‬‭ground glass chromatin‬ ‭pattern‬ ‭__________‬ ‭Slightly oval‬ ‭ ucleus is occupying at least half of the cell size‬ N ‭Found only before pregnancy and after menopause‬ ‭ ndometrial‬ E ‭cells‬ ‭ ndocervical‬ E ‭cells‬ ‭ ound in groups of‬‭three‬‭during menstruation‬ F ‭Found in singles 1-4 days after cessation of period‬ ‭Endometrial stromal cells:‬‭tight clusters of oval‬‭cells‬ ‭Endometrial glandular cells:‬‭slightly larger‬ ‭If found post-menopause, may indicate carcinoma‬ ‭ olumnar epithelium; slightly cylindrical,‬‭honeycomb-like‬ C ‭Mucin-filled basophilic cytoplasm‬ ‭Denotes‬‭satisfactory for collection‬ ‭ actobacillus acidophilus‬‭:‬‭Gram-positive slender rods‬ ‭ _________‬ L _ ‭__________‬ ‭Predominant normal flora organism; denotes low pH‬ ‭ nergy is from glycogen fermentation of dead cells‬ E ‭Numerous in the‬‭luteal phase‬‭and‬‭pregnancy‬ ‭Neutrophils‬ ‭Increased just before, during, and shortly after period‬ ‭ ed blood‬ R ‭cells‬ ‭ een during traumatic collection and period‬ S ‭To avoid, collect 10 days after Last Menstrual Period‬ ‭ eptothrix‬ L ‭spp.‬ ‭ ong, thin, filamentous bacilli‬ L ‭Increased in high pH; may indirectly indicate infection‬ ‭Talcum‬ ‭Ovoid bodies that are usual contaminants‬ ‭Other cells‬‭such as‬‭cancerous lesions (carcinomas)‬‭and‬‭dysplastic cells‬ ‭Ferning (Arborization)‬ ‭ ‬B ★ ‭ asis for diagnosis of‬‭________________‬ ‭★‬ ‭Mucus on drying exhibits a‬‭fern/palm leaf pattern‬ ‭★‬ ‭Due to salt crystal formation‬‭(high NaCl concentration)‬‭u‬‭nder the‬ ‭influence of‬‭estrogen‬ ‭(+) Ferning: High persistent estrogen effect in the‬‭______________________‬ ‭ ‭Criteria for Cytologic Diagnosis of Normal Pregnancy‬ ‭ . Marked Progesterone Effect‬ 1 ‭2. At least‬‭50%‬‭of‬‭____________________________‬ ‭3. Presence of‬‭3-7‬‭typical pregnancy cells‬ ‭4.‬‭<30%‬‭superficial cells‬ ‭5. Presence of‬‭Doderlein bacilli‬ ‭ or evaluating hormonal status based on‬‭cell distrubution:‬ F ‭Distribution of‬‭______________________________________‬ ‭➔‬ ‭Results should be correlated with age and LMP of patient‬ ‭CHMI: Cytohormonal Maturation Index (% per 100 cells)‬ ‭Expressed in the form of:‬‭Parabasal/Intermediate/Superficial‬‭(P/I/S)‬ ‭ xample 1:‬‭100/0/0‬ E ‭100% of the cells found are parabasals; no intermediates or superficials.‬ ‭1. Parabasal cells dominating indicate an atrophic effect (no estrogen)‬ ‭2. It is‬‭shift to the left‬‭since parabasal cells are‬‭at the leftmost part.‬ ‭It may indicate a‬‭premenopausal/postmenopausal condition‬‭.‬ ‭ xample 2:‬‭0/70/30‬ E ‭There are 0% parabasals, 70% intermediates, and 30% superficials.‬ ‭1. If intermediate cells dominate,‬‭progesterone‬‭is‬‭in effect.‬ ‭2. Therefore, it is‬‭shift to midzone‬‭since intermediate‬‭is in the middle‬ ‭ xample 3:‬‭0/10/90‬ E ‭There are 0% parabasals, 10% intermediates, and 90% superficials.‬ ‭1. Since‬‭superficials dominate with 90%‬‭, estrogen‬‭is taken into account.‬ ‭2. Since superficial is at the rightmost portion, it means‬‭shift to the right.‬ ‭Examples of CHMIs and their probable conditions associated‬ ‭Pregnancy‬ ‭MI: 0/90/10 (Increased progesterone)‬ ‭Newborn (8 weeks)‬ ‭MI: 0/90/10 (Progesterone from mother)‬ ‭Infancy to puberty‬ ‭MI: 0/90/10‬ ‭Late menopausal‬ ‭MI: 0/90/10‬ ‭ 5 y/o woman‬ 7 ‭w/ estrogen therapy‬ ‭MI: 0/90/10‬ ‬ ‭Manner of Reporting Paps Smears‬ ‭Systems:‬‭Class‬‭(No longer used), and‬‭Bethesda‬ ‭Class System‬ ‭Based on presence of malignant cells‬ ‭Class‬ ‭Interpretation‬ ‭I‬ ‭Negative for malignant cells‬ ‭II‬ ‭Atypical cells present; Negative for malignancy‬ ‭III‬ ‭Suspicious for malignant cells‬ ‭IV‬ ‭Strongly suggestive for malignant cells‬ ‭V‬ ‭Conclusive for malignant cells‬ , ‭Cytohormonal Smear‬ ‭Bethesda System‬ ‭Based on specimen adequacy, general categorization, and descriptive diagnosis‬ ‭Acronym‬ ‭NILM‬ ‭Interpretation‬ ‭Negative for intraepithelial lesion or malignancy‬ ‭ASCUS‬ ‭Atypical squamous cells of unknown significance‬ ‭L-SIL‬ ‭Low grade squamous intraepithelial lesion‬ ‭H-SIL‬ ‭High grade squamous intraepithelial lesion‬ ‭SCC‬ ‭Squamous cell carcinoma‬ ‭Class‬ ‭Bethesda‬ ‭Normal‬ ‭I‬ ‭NILM‬ ‭Atypical‬ ‭II‬ ‭ASCUS‬ ‭HPV‬ ‭II‬ ‭L-SIL‬ ‭Vaginal Cytology Quantitation‬ ‭Mild dysplasia‬ ‭II‬ ‭L-SIL‬ ‭Acidophilic Index (AI)‬ P ‭ ercentage of cells that stain (1) pink-orange to red‬ ‭with Pap’s,and (2) red in Shorr method‬ ‭Moderate dysplasia‬ ‭III‬ ‭H-SIL‬ ‭Severe dysplasia‬ ‭III‬ ‭H-SIL‬ ‭Carcinoma in situ‬ ‭IV‬ ‭H-SIL‬ ‭Invasive carcinoma‬ ‭V‬ ‭SCC‬ ‭ ‭Basophilic Index (BI)‬ “ ‭ Karyopyknotic index”‬ ‭Percentage of cells with shrunken and dark nuclei‬ ‭Maturation Index (MI)‬ ‭Percentage proportion of cells from the‬‭three‬ ‭layers of the vaginal epithelium‬ ‭Description‬ ‭Fresh Tissue Examination‬ ‭It also allows examination of protoplasmic activities‬ ‭ et, tissues are not permanent‬ Y ‭Microtome‬ ‭Fresh Tissue Examination Methods‬ ‭ easing/‬ T ‭Dissociation‬ ‭ issue‬‭immersed in a‬‭watch glass with NSS‬ T ‭Tissue is then disected and stained with‬‭methylene‬‭blue‬ ‭Squash/Crushing‬ ‭ mall‬‭(1mm)‬‭tissues‬‭placed in slide‬ S ‭Tissue‬‭compressed‬‭with‬‭another slide‬‭or coverslip‬ ‭Pull-Apart‬ ‭ hick secretions‬‭placed between‬‭2 slides‬ T ‭The slides are being pulled in opposite direction‬ ‭Spreading‬ ‭ ample is‬‭teased‬‭with an applicator stick‬ S ‭Advantage: maintains intracellular relationships‬ ‭Smear Preparation‬ S ‭ ample is‬‭spread lightly over a slide‬ ‭Uses an‬‭applicator stick‬‭in a‬‭zigzag‬‭fashion‬ ‭ ain purpose:‬‭rapid diagnosis‬ M ‭It allows demonstration of lipids, nervous tissues,‬‭and enzymes‬ ‭BEST TISSUE: unfixed tissue‬ ‭If fixed, it can be used to localize‬‭hydrolytic‬‭enzymes and other antigens‬ ‭Facts, Materials, and Equipment for Frozen Sections‬ ‭Formol calcium (4‬‭°C for 18 hours)‬ ‭Freeze-drying‬ D ‭ oes not use any chemical fixative‬ ‭1. Quenching: rapid freezing (-160°C)‬ ‭2. Sublimation: removing water in the form‬ ‭of ice using vacuum (-40°C)‬ ‭ ‭Dehydration‬ ‭Freeze-‬ ‭substitution‬ ‭ amel hair brush: for‬‭removal of ribbons that stick‬‭to the knife‬ C ‭Dew line: point in which sections may be cut at 10 um‬ ‭Cold Knife‬ ‭Old‬ ‭Freezing microtome‬ ‭Stains‬ ‭Mountant‬ ‭Clearing‬ ‭Cold Microtome (Cryostat)‬ ‭Currently used‬ ‭Rotary microtome inside‬ ‭ ‬‭olychrome methylene blue‬ P ‭A‬‭lcoholic pinacyanol‬ ‭T‬‭hionine‬ ‭H‬‭ematoxylin and Eosin‬‭(Progressive type)‬ ‭OCT (Optimal Cutting Temperatures)‬ ‭Glycerin and Gum Syrup (to‬‭increase Refractive index)‬ ‭Immunohistochemistry‬ ‭Frozen Sections‬ ‭Fixative‬ ‭ ryostat:‬‭-18 to -‬‭20°C‬ C ‭Knife temperature:-40 to -60‬‭°C‬ ‭Tissue temperature: 5 to -10‬‭°C‬ ‭Environment temperature: 0 to -10‬‭°C‬ , ‭Touch/Impression‬ T ‭ issue‬‭brought into contact‬‭onto the slide‬ ‭Slide should be sterile with polished edges‬ ‭ ‬‭reon 2.2‬ F ‭I‬‭sopentane - liquid at RT‬ ‭C‬‭arbon Dioxide gas‬ ‭A‬‭erosol sprays - made of fluorinated hydrocarbons‬ ‭L‬‭iquid Nitrogen‬‭(most rapid)‬ ‬ ‭Freezing‬ ‭Agents‬ ‭Miscellaneous Topics‬ ‭ ses‬‭Rossman’s solution/1% acetone‬ U ‭Then dehydrated using absolute alcohol‬ ‭Main purpose:‬‭identify specific antigens or phenotypic‬‭markers on tissues‬ I‭ t is also used for detection of organisms in‬‭cytologic specimens‬ ‭Makes use of antigen-antibody reactions‬ ‭Antigens‬ ‭The component that is being detected; found in cells‬ ‭Antibodies‬ T ‭ he reagents used to detect antigens in cells‬ ‭IgG: most commonly used Ab class‬ ‭Storage: -80‬‭°C in aliquots‬ ‭Types of antibodies used‬ ‭Polyclonal‬ P ‭ roduced by different clones of cells‬ ‭Main source:‬‭Rabbits‬ ‭Other sources: Goat, Pig, Sheep, Horse, Guinea pig‬ ‭Monoclonal‬ ‭Produced by individual clones of cells‬ ‭Source:‬‭mice‬ ‭Enzymes‬ ‬ ‭Types of Labels Used in Immunohistochemistry‬ ‭ ost widely used‬ M ‭Incubation to link Ab with peroxidases: ______________‬ ‭Suitable for viewing under light microscopy‬ ‭Examples‬ ‭ . Horseradish peroxidase‬‭(most common)‬ 1 ‭2. Alkaline phosphatase‬ ‭Chromogens‬ 1 ‭ . Diaminobenzidine (DAB) -‬‭Brown‬‭(HRP)‬ ‭2. Aminoethyl Carbazole (AEC) - Red (HRP)‬ ‭3. Bromochloroindolylphosphate/Nitroblue‬ ‭tetrazolium (BCIP/NBT) - Blue-purple (ALP)‬ ‭4. Fast Red - Red (ALP)‬ ‭Fluorochromes‬ ‭Lectins‬ ‭Fixatives for Immunohistochemistry‬ ‭ ses‬‭fluorescein isothiocyanate‬ U ‭Needs the use of‬‭fluorescent microscope‬ ‭10% Neutral Buffered Formalin‬ ‭Most commonly used‬ ‭Plant/animal proteins that bind to tissue carbohydrates‬ ‭Zamboni‬ ‭Others:‬‭Colloidal metals (gold), and radiolabels‬ ‭Labelling Methods for Immunohistochemistry‬ ‭Traditional Direct‬ ‭Primary Ab is labelled‬ ‭ xtended Polymer‬ E ‭One-Step Staining‬ ‭ nder direct labelling‬ U ‭Primary Ab is conjugated with dextran polymer‬ ‭2-step Indirect‬ ‭ ore sensitive than direct;‬‭↑‬‭signal amplification‬ M ‭Uses labelled secondary antibody‬ ‭Polymer 2-step Indirect‬ ‭ mmunogold Silver‬ I ‭Staining‬ ‭ econdary Ab is conjugated to‬‭enzyme labelled‬ S ‭polymer chain‬ ‭Silver is added to enhance reaction‬ ‭Others:‬‭Unlabeled antibody enzyme complex, Streptavidin‬‭Biotin‬ ‭Specimens allowed for IHC‬ ‭ ‭ ‬ ‭Frozen sections‬‭fixed‬‭with‬‭Absolute methanol or acetone‬ ❖ ‭ ‬ ‭Formalin-fixed‬‭or‬‭paraffin-embedded‬‭tissues‬ ❖ ‭Natatandaan mo pa ba si “No FaBeBe” ng FITC? Halos similar principles nila!‬ ‭If electron microscopy is used‬ ‭Others:‬‭4% formaldehyde, Bouin’s‬ ‭ ntigen retrieval,‬‭especially for‬‭paraffin-embedded‬‭tissues, is needed.‬ A ‭➔‬ ‭Tissues are de-waxed, rinsed in alcohol, and washed with water‬ ‭Methods of Antigen Retrieval‬ ‭Proteolytic Enzyme Retrieval‬ M ‭ ost commonly used‬ ‭Ex:‬‭Trypsin, protease‬‭, pepsin, bromelain‬ ‭Microwave Antigen Retrieval‬ A ‭ .k.a. Heat Induced Epitope Removal‬ ‭Microwave, then boil using citric acid‬ ‭ thers:‬‭Pressure Cooking, Autoclave Heating, Water‬‭Bath Heating, Steamer‬ O ‭Heating, Decloaking, and combination of Microwave and Enzyme digestion‬ ‭ rior to staining,‬‭inactivators‬‭or‬‭blocking agents‬‭are used‬ P ‭➔‬ ‭This is to prevent non-specific binding (false positive results)‬ ‭Inactivators‬ I‭ nhibits endogenous enzymes‬ ‭For HRP: 3% hydrogen peroxide‬ ‭For ALP: Levamisole‬ ‭Blocking agents‬ B ‭ locks residual sites to prevent 2’ antibody binding‬ ‭Incubation: 10-30 minutes at RT‬ ‭Example:‬‭Animal serum‬ ‭Stains and Counterstains in Enzyme Immunohistochemistry‬ ‭Enzyme‬ ‭Chromogen‬ ‭Counterstain‬ ‬ ‭Tumor Markers for Immunohistochemistry‬ ‭ . Keratin‬‭: Epithelial Cell Marker‬ ‭Epithelial Tumor‬ 1 ‭Cytokeratins (CK): CK7 and CK20‬ ‭HRP‬ ‭DAB‬ ‭Hematoxylin, Methyl Green, Methyl Blue‬ ‭(+) CK20‬ ‭Stomach, Colon‬ ‭HRP‬ ‭AEC‬ ‭Hematoxylin, Methyl Blue‬ ‭(+) CK7‬ ‭Lung, Uterus, Breast, Ovary‬ ‭ALP‬ ‭BCIP/NBT‬ ‭Nuclear Fast Red‬‭, Brilliant Green‬ ‭ALP‬ ‭Fast Red‬ ‭Hematoxylin, Methyl Green, Brilliant Green‬ ‭(+) CK20 & CK7‬ ‭Bladder, Mucinous ovarian tumor‬ ‭(-) CK20 & CK7‬ R ‭ enal, liver, prostate, thyroid,‬ ‭squamous cell‬ ‭ . Epithelial Membrane Antigen (EMA)‬ 2 ‭Helpful for determining tumor site‬ ‭(+) Breast adenocarcinoma‬ ‭Immunohistochemistry Adhesives and Mountants‬ ‭Adhesives‬ ‭APES diluted in acetone‬ ‭Mountant‬ ‭Neutral pH resins with 50% glycerol‬ ‭ . Carcinoembryonic Antigen (CEA)‬ 3 ‭(+) Adenocarcinoma,‬‭(-) Mesothelioma‬ ‭ . Thyroid Transcription Factor 1 (TTF-1)‬ 4 ‭Present in thyroid gland neoplasms‬ ‭(+) Lung adenocarcinoma‬ ‭ RP w/ DAB or ALP w/ BCIP/NBT: Neutral pH mountants‬ H ‭HRP w/ AEC or ALP w/ Fast Red: Water-soluble mountants‬ ‭ . Prostate Specific Antigen (PSA)‬ 5 ‭(+) Prostate‬‭, pancreatic, and salivary tumors‬ ‭Controls in Immunohistochemistry‬ ‭Positive‬ ‭Antigen is proven present in a known section‬ ‭Negative‬ ‭ mits the primary antibody from staining‬ O ‭Then it is replaced by an antibody to unrelated antigen‬ ‭Internal‬ “‭ Built-in control”‬ ‭Contains the target antigen‬‭but‬‭not the tissue sample‬‭itself‬ ‭ . Estrogen/Progesterone Receptor (ER/PR)‬ 6 ‭Prognosis and treatment of breast cancer‬ ‭ ntermediate‬ I ‭Filament‬ ‭ . Actin‬ 1 ‭Sensitive marker for muscle differentiation‬ ‭Tumors from smooth, skeletal, and cardiac muscle‬ ‭ . Vimentin‬ 2 ‭Melanomas and schwannomas always stain positive‬ ‭Present in mesenchymal cells and neoplastic cells‬ ‭ . Desmin‬ 3 ‭Highly specific for myogenic tumors‬ ‭ ‭ . Glial Fibrillary Acidic Protein (GFAP)‬ 4 ‭For diagnosis of‬‭astrocytoma‬‭(Astrocytes in CNS)‬ ‭ . Neurofilament (NF)‬ 5 ‭From cells of neural origin such as neuroblastomas‬ ‭ . S100 - Calcium Binding Protein‬ 6 ‭Expressed in various cell types (muscles, CNS, etc)‬ ‭ . Neuron-specific Enolase‬ ‭Neuroendocrine‬ 1 ‭For neural/neuroendocrine tumors‬ ‭ erm Cell‬ G ‭Tumor‬ ‭Laboratory Safety and Troubleshooting‬ ‭ . Chromogranin‬ 2 ‭Found in neural secretory granules (endocrine)‬ ‭Keratin & Chromogranin (+) =‬‭neuroendocrine CA‬ ‭ emove Stains on‬ R ‭Skin‬ ‭0.5% acid alcohol, then flush with tap water‬ ‭ . Synaptophysin‬ 3 ‭Found in presynaptic vesicles of neurons‬ ‭ estaining of Old‬ R ‭Sections‬ ‭. 1 ‭2. ‭3. ‭3. ‭3. ‭4. ‭5. ‭6. ‭7. ‭ roken Slides;‬ B ‭Transferring the‬ ‭Section to a New‬ ‭Slide‬ ‭ . Removal of coverslip: use Xylene‬ 1 ‭2. Removal of mountant: incubate at 37‬‭°C‬ ‭3. Place in 6:1 butyl acetate:durofix (30 mins)‬ ‭4. Incubate at 37‬‭°C‬ ‭5. Cut the film around the section‬ ‭6. Place in cold water to float off the film‬ ‭7. Mount the section on another clean slide‬ ‭8. Incubate at 37‬‭°C‬ ‭9. Wash gently‬‭with butyl acetate‬ ‭10. Use xylene, then mount again‬ ‭Explosives‬ ‭ ‬ ★ ‭★‬ ‭★‬ ‭★‬ ‭★‬ ‭★‬ ‭Carcinogenic Dyes‬ ‭ yes derived from benzene such as:‬ D ‭★‬ ‭Auramine‬ ‭★‬ ‭Basic fuchsin‬ ‭★‬ ‭Congo red‬ ‭★‬ ‭Diaminobenzidine‬ ‭ auses Death upon‬ C ‭Inhalation,‬ ‭Ingestion, or Skin‬ ‭Contact‬ ‭ ‬ ★ ‭★‬ ‭★‬ ‭★‬ ‭ . Human Chorionic Gonadotropin (HCG)‬ 1 ‭Synthesized by placental‬‭syncytiotrophoblasts‬‭early‬ ‭(+) Choriocarcinoma‬ ‭ . Alpha Fetoprotein (AFP)‬ 2 ‭(+) Hepatocellular carcinoma‬ ‭ . Placental-like Alkaline Antigen (PLAP)‬ 3 ‭Synthesized by placental‬‭syncytiotrophoblasts‬‭late‬ ‭(+) Choriocarcinoma, endodermal sinus tumors‬ ‭ esenchymal‬ M ‭Tumor‬ ‭ yogenic‬‭: skeletal muscle origin‬ M ‭Actin, Desmin, Myo D-1, Myoglobin, Myogenin‬ ‭ ibrohistiocytic:‬‭for Malignant Fibrohistiosarcoma‬ F ‭CD68, FAM56, A-1 Antitrypsin, A-1 Antichymotrypsin‬ ‭ ascular:‬‭endothelial tumors (Angiosarcoma)‬ V ‭Factor-VII related antigen, CD31,‬‭Ulex europaeus‬ ‭ elanoma‬ M ‭From melanocytes: (+) S100‬ ‭From melanosomes: (+) HMB45‬ ‭Lymphoma:‬‭Leukocyte Common Antigen (LCA) / CD45‬ ‭ ‬ ‭BEST SCREENING MARKER FOR LYMPHOMA‬ ★ ‭ -cells: CD3, CD4, CD8‬ T ‭B-cells: CD19, CD20, CD23‬ ‭Hodgkins: CD15, CD30, L and H chain Ig‬ ‭Genes‬ ‭ 53:‬‭expression absent in normal cells‬ P ‭HER2/neu:‬‭genes associated with breast cancer‬ ‭ nfectious‬ I ‭Agents‬ ‭ AV, HBsAg, HCV, HPV, CMV,‬‭Toxoplasma, P. jirovecii,‬‭H.‬ H ‭pylori, C. neoformans, Histoplasma, Entamoeba,‬‭TB‬ ‭ ‭ ell‬ C ‭Proliferation‬ ‭ . KI-67 (MIB-1)‬ 1 ‭2. Proliferating Cell Nuclear Antigen (PCNA)‬ ‭Both are to assess proliferation of tumors‬ ‭(+) greater possibility and aggressiveness of malignancy‬ Remove coverslip: Xylene (24 hours) or heat‬ Lift the section with dissecting needle‬ Put again in xylene for 30 minutes‬ Rinse with water‬ Put in 0.5% Potassium permanganate (5-10 mins)‬ Rinse with water‬ Put in 5% Oxalic Acid (5 mins)‬ Rinse with water‬ Restain‬ ‭ icric acid‬ P ‭Sodium iodate (Oxidizer)‬ ‭Mercuric oxide (Oxidizer)‬ ‭Chromic acid (Oxidizer)‬ ‭Sodium azide (When thrown in sink)‬ ‭Low Viscosity Nitrocellulose‬ ‭ ethanol‬ M ‭Chromic acid‬ ‭Osmium tetroxide‬ ‭Uranyl nitrate‬ ‭Autotechnicon‬ A ‭ n automatic tissue processor‬ ‭Able to do‬‭fixation, dehydration, clearing, infiltration‬ ‭Types of Autotechnicon‬ ‭ issue Transfer‬ T ‭(“Dip and Dunk”)‬ ‭ pecimens are transferred from‬ S ‭container to container‬ ‭ luid Transfer‬ F ‭(“Enclosed)‬ ‭ pecimens are held in a chamber‬ S ‭and fluids are pumped in and out‬ ‬ ‭Reports, Results, and Retentions‬ ‭Kinds of Reports‬ ‭(1) Surgical Pathology, (2) Autopsy, and (3) Cytology‬ ‭ of Report‬ # ‭Copies‬ ‭ hree copies‬ T ‭1. Patient (Original copy)‬ ‭2. Doctor‬ ‭3. File to histopathology section‬ ‭Signatories‬ ‭ equest Form: Attending physician‬ R ‭Pathology Result Form: Pathologist‬ , ‭Automation and Other Essential Equipment‬ ‭Result Turn-Over‬ F ‭ rozen Sections: 5-15 minutes‬ ‭Surgical Pathology and Cytology: 2 days‬ ‭Autopsy Report: 7 days‬ ‭ etentions of‬ R ‭Specimen,‬ ‭Reports, and‬ ‭Others‬ ‭Microscope‬ ‭ issue Tek‬ T ‭Embedding‬ ‭System‬ ‭ model of plastic embedding rings‬ A ‭Has a paraffin reservoir, tissue tank, warm & cold plates‬ ‭Pros:‬‭easy to use, less paraffin needed, faster embedding,‬ ‭firmer tissues and holder, and permanent identification‬ ‭ ith temperature of 10‬‭°C below MP of wax‬ W ‭Black coating is to see folds and creases in sections‬ ‭Water filled within ½ to 1 cm from the top‬ ‭ ‭ loatation‬ F ‭Water Bath‬ ‭ bjective magnifications: 4x, 10x, 40x, 100x‬ O ‭Eyepiece magnifications: 10x‬ ‭Normal tube length: 160 mm‬ ‭Working tube length: 180 mm‬ ‭Drying Oven‬ ‭With temperature of 5‬‭°C above MP of wax‬ ‭Records‬ ‭Requisition Forms‬ ‭2 years‬ ‭Instrument Maintenance‬ ‭2 years‬ ‭QC (General lab)‬ ‭2 years‬ ‭QC (Blood bank)‬ ‭5 years‬ ‭BB Employee Signatures‬ ‭10 years‬ ‭BB Donor/Recipient Records‬ ‭Indefinitely‬ ‭Reports‬ ‭Clinical Pathology Lab Reports‬ ‭2 years‬ ‭Surgical Pathology and BM Reports‬ ‭10 years‬ ‭Cytogenetics Lab Reports‬ ‭20 years‬ ‭Autopsy Forensic Reports‬ ‭Indefinitely‬ ‭Specimens‬ ‭Serum/Other Body Fluids‬ ‭48 hours (2 days)‬ ‭Routine Blood Smears‬ ‭7 days‬ ‭Microbiology Smears‬ ‭7 days‬ ‭ lood Bank Donor/Recipient‬ B ‭Specimens‬ ‭ days‬ 7 ‭post-transfusion‬ ‭Pathology Blocks‬ ‭10 years‬ ‭Cytogenetics Slides‬ ‭3 years‬ ‭Cytogenetics Diagnostic Images‬ ‭Indefinitely‬ ‭Forensic Cases‬ ‭Body Fluids‬ ‭1 year‬ ‭Toxicology Tissues‬ ‭1 year‬ ‭Wet Tissues‬ ‭3 years‬ ‭Paraffin Blocks‬ ‭Indefinitely‬ ‭Slides‬ ‭Indefinitely‬ ‭Reports‬ ‭Indefinitely‬ ‭Gross Photographs/Negatives‬ ‭Indefinitely‬ ‭Dried Blood Films‬ ‭Indefinitely‬ ‭Frozen Tissue for DNA‬ ‭Indefinitely‬ ‭ atapos mo ba ang handout kaya nababasa mo ito?‬ N ‭Kung oo, congrats! Ulit-ulitin mo hanggang maisapuso mo!‬ ‭Kung hindi, bumalik ka uli at simulan, or pumunta sa kung saan ka‬ ‭tumigil.‬ ‭Additional tips for Review:‬ ‭ ‭ . Medyo alanganin magsaulo ng recalls dito kasi madalang sila nauulit,‬ 1 ‭pero may iba na gasgas rin, Kaya this is a reminder to study everything.‬ ‭2. Correlate to other subjects; kung titignan mabuti, connected parin‬ ‭siya sa ibang subjects. Very helpful!‬ ‭3. Remember SEPSIS!‬ ‭Study, Eat, Pray, Stretching (Exercise), Inom (Water), Sleep‬ ‭Then repeat. para prepared and disciplined for the board exam!‬ ‭Further Readings for You ‘pag may time:‬ ‬ ‭10 years‬ ‭ . Atlas of Histology 1st Ed. (Cui, et al.), 2011‬ 1 ‭-‬‭For Basic Histology. Gawing Cross Referencing lang‬ ‭ . Robbins Basic Pathology 9th Ed. (Kumar, et al.), 2013‬ 2 ‭-For General Pathology. Gawing Cross Referencing lang‬ ‭ . Bancroft’s Theory and Practice of Histological Techniques 9th‬ 3 ‭Ed. (Suvarna, et al.), 2019‬ ‭-For further readings sa Histopathologic techniques; Secondary‬ , ‭Pathology/BM Slides‬ ‭ . Histopathologic Techniques 3rd Ed. (Gregorios), 2018‬ 4 ‭-The Primary Book for Histopath‬ ‭Galingan mo, Board Taker! Do your best sa lahat ng subjects!‬