Germ layers Categories of tissues
Covering Epithelia Simple squamous Simple cuboidal Simple columnar Stratified squamous Stratified columnar Pseudostratified columnar Glandular Epithelia Exocrine glands
Endocrine glands Pancreas Merocrine Apocrine Holocrine Connective Tissues Connective tissue Collagen
MUST TO KNOW IN HISTOPATHOLOGIC TECHNIQUES 1. Ectoderm 2. Mesoderm 3. Endoderm 1. Epithelial = 3 germ layers 2. Nervous = endoderm 3. Muscular = mesoderm 4. Connective = mesoderm Bowman’s, endothelium, loop of Henle, alveoli Ducts of glands, walls of thyroid follicles Gallbladder (nonciliated) Uterine tube (ciliated) Skin (keratinized) Vagina, esophagus, cervix (nonkeratinized) Male urethra Female reproductive tract (nonciliated) Trachea (ciliated), Epididymis w/ ducts Tubular = stomach, uterus Acinar/alveolar = pancreas, salivary glands Tubuloacinar = prostate Ductless Exocrine = enzymes Endocrine = hormones No loss of cytoplasm Goblet cells, sweat glands w/ cytoplasmic loss Distal portion is pinched off Mammary glands Disintegrating cell and its constituents released Complete breakdown of cell Sebaceous gland Support Framework Cells are widely separated Major ingredient of connective tissues Stains: “VgMMAK” Van Gieson Mallory’s aniline blue Masson’s trichrome Alcian blue Krajian’s aniline blue
General Connective Tissues Loose CT Wharton’s jelly (acid MPS) BM (reticular) Lymph node (reticular) Embryo (mesenchyme) Hypodermis lec.mt 04 |Page | 255
Dense CT
Dermis Capsules Tendons Stroma of cornea
Special Connective Tissues Cartilage Hyaline = trachea Fibrous = intervertebral discs Elastic = ear, epiglottis Bone Cancellous/spongy/trabecular = epiphysis/ends of long bones Compact/cortical = diaphysis/shaft Others Blood Lymph Hematopoietic tissues Acid mucopolysaccharides Fixative: Lead fixatives Stain: Alcian blue Osteogenesis imperfect Brittle bone disease Defective production of collagen Deposits found in Connective Tissues (Eosinophilic) Fibrin Early: yellow Old: blue Stains: Mallory’s PTAH Lendrum’s MSB Fibrinoid Necrotizing vasculitis Staining reactions identical to fibrin Mixture of exudates & altered cytoplasmic constituents Hyaline Degenerated collagen Hypertension, atheroma, diabetic kidney Stain: PAS Amyloid TB, leprosy, osteomyelitis Stains: “CoMT” Congo Red Metachromatic stain Thioflavine Muscle Tissues Smooth Involuntary Intestines, blood vessels Skeletal Striated, voluntary Skeletal muscles Cardiac Striated, involuntary Heart Nervous Tissues CNS Brain, spinal cord PNS Peripheral nerves Special receptors Ear, eye, nose Inflammation Inflammation Latin word: Inflammare (to set afire) 5 Cardinal Signs of Inflammation 1. Rubor Redness Blood flow Injury 2. Calor Heat lec.mt 04 |Page | 256
3. Tumor
Swelling
4. Dolor
Pain
Fluid extravasation Sensory nerves Loss of function Destruction of functional units Acute inflammation Vascular & exudative ---(Tissue)---> Microphages Subchronic inflammation Intergrade between acute & chronic Chronic inflammation Vascular & fibroblastic ---(Tissue)---> Macrophages Inflammation according to Characterisics of Exudate Serous inflammation Serum/secretions from serosal mesothelial cells (3P’s) Pulmonary TB Fibrinous inflammation Diphtheria, rheumatoid pericarditis Early stage of pneumonia Catarrhal inflammation Hypersecretion of mucosa Hemorrhagic inflammation Blood + exudates Bacterial infections & other infections Suppurative/purulent inflammation 5. Functio laesa
Retrogressive Changes = Organ/Tissue smaller than normal Developmental defects: AAHA Aplasia Incomplete/defective development of a tissue/organ Ex. amastia (breast aplasia) Atresia Failure to form an opening Hypoplasia Failure of an organ to reach its matured size Agenesia Complete non-appearance of an organ Atrophy Physiologic atrophy Natural Thymus, brain, sex organs Pathologic atrophy Vascular atrophy Pressure atrophy Atrophy of disuse Exhaustion atrophy Endocrine atrophy Brown atrophy Lipofuscin Progressive Changes = Organ/Tissue larger than normal Hypertrophy Increased tissue size due to increased cell size Physiologic: ásize of uterus Pathologic: Systemic hypertension Hyperplasia Increased tissue size due to increased cell number Physiologic: Glandular proliferation of the female breast, ásize of uterus (preg.) Pathologic: Skin warts due to HPV Compensatory hyperplasia Ex. Enlargement of one kidney Pathologic hyperplasia Ex. Endometrial hyperplasia Congenital hypertrophy Phenytoin-induced Degenerative Changes = Tissues have abnormalities Metaplasia Reversible One adult cell type ↔ Another adult cell type lec.mt 04 |Page | 257
Dysplasia Anaplasia/ Dedifferentiation Neoplasia/tumor Oncology Parts of a tumor Types of tumor
Benign Malignant Leukemia Lymphoma Squamous cell papilloma Squamous cell carcinoma Hepatoma/ hepatocarcinoma Melanoma/ melanocarcinoma Ectopic pregnancy Grading
Grade I II III IV Staging UICC AJCS TNM system T
Reversible One type of adult cell ↔ Changes in structural components Irreversible Criterion toward malignancy Adult cell More primitive cells (release tumor markers) Continuous abnormal proliferation of cells w/o control (no purpose/function) Ex. Leukemia Study of neoplasm Tumors 1. Parenchyma = active elements (tumor cells) 2. Stroma = CT framework 1. Capacity to produce death: - Benign (Ex. mole) - Malignant 2. Histologic characteristics: - Medullary = cells (parenchyma) > supporting tissues (stroma) - Scirrhous = supporting tissues (stroma) > cells (parenchyma) “-oma” “SaMe CarE” “-sarcoma” = mesenchymal/CT “-carcinoma” = epithelial tissues Malignant Benign Malignant Malignant Malignant Fallopian tube pregnancy Grading Aggressiveness/level of malignancy Differentiated cells = resemble normal cells Undifferentiated cells = younger cells Broder’s Classification (Grading) Differentiated Cells Undifferentiated Cells 75-100% 0-25% 50-75% 25-50% 25-50% 50-75% 0-25% 75-100% Staging Size, extent of spread to lymph nodes, +/- metastases TNM classification Grading + staging TNM System Applicable to all forms of neoplasia 1’ tumor #: denotes the size of tumor and its local extent Tis = carcinoma in situ Ta = non-invasive Tx = cannot be evaluated T0 = free of tumor
Treatment Surgery ↓ ↓ Radiation
lec.mt 04 |Page | 258
N
M
Teratomas
Apoptosis Necrobiosis Necrosis Types of Necrosis Coagulation necrosis
Liquefaction/colliquative necrosis Caseous/caseation necrosis Gangrenous necrosis Fat necrosis Fatty degeneration 1’ changes 2’ changes Algor mortis (1st)
T1 = lesion <2 cm (T1a = <0.5 cm | T1b = <1 cm | T1c = <2 cm) T2 = lesion 2-5 cm (invasion in muscle) T3 = skin and/or chest wall involved by invasion (T3a = deep muscle | T3b = through organ) T4 = tumor invasion/fixation (T4a = adjacent organ | T4b = fixation to bladder or colonic wall, in breast, edema) Regional lymph node involvement High # denotes increasing extent of involvement Nx = not evaluable N0 = no axillary nodes involved N1 = 1 mobile regional (axillary) node involved N2 = multiple, mobile regional nodes involved N3 = fixed regional lymph node involved N4 = beyond regional lymph node involvement Metastasis M0 = no evidence of metastases M1 = distant metastases are present Mx = distant metastases not evaluable Compound tumors Greek: Monstrous tumors May contain hair, teeth, bones w/ heartbeat Cellular Death Programmed cell death (cellular suicide) Physiologic cell death Ex. normal sloughing off of skin cells Pathologic cell death Most common Tombstone formation “MyLKS” Myocardium Lungs Kidneys Spleen Pus formation Brain & spinal cord Yellow, cheesy, crumbly material TB, syphilis, tularemia, lymphogranuloma inguinale Sulfide gas production a. Dry gangrene = arterial occlusion b. Wet gangrene = venous occlusion Chalky white precipitates Pancreatic degeneration Liver Somatic death During somatic death “CRC”: circulatory, respiratory, CNS failure After somatic death “ARLP DPA”: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting, Dessication, Putrefaction, Autolysis Postmortem cooling lec.mt 04 |Page | 259
Cooling: 7’F/hr Rigor mortis Stiffening 1st: neck & head (2-3 hrs) Persists for 3-4 days Livor mortis Lividity/suggillations Purplish discoloration After 10-12 hrs, it does not blanch on pressure or shift when the body is moved Postmortem Lividity vs. Ecchymosis Postmortem Lividity Ecchymosis Disappears on pressure (reappears when pressure is Opposite of postmortem lividity released) Oozing of blood (incision) No oozing of blood (incision) Postmortem Clot vs. Antemortem Clot Postmortem Clot Antemortem Clot Settling of RBCs from plasma Not readily detachable from the blood vessels Chicken fat No chicken fat Currant jelly No currant jelly Assumes the shape of the vessel Seldom assumes the shape of blood vessels Rubbery consistency Granular & friable Dessication Drying & wrinkling of the anterior chamber of the eye Putrefaction Invasion of intestinal microorganisms Autolysis Self digestion of cells Lysosome: suicide sac of the cell, releases lysozyme Organ Weights Liver 1,100 – 1,600g Brain 1,150 – 1,450g Right lung 300-400g Left lung 250-350g Heart 250-300g Spleen 60-300g Thyroid 10-50g Adrenals 4g or so each Exfoliative Cytology Exfoliative cytology Desquamated cells Pap smear stain method Method of choice Barr body PAP smear 3 anatomical sites 1. Upper lateral third of the vagina 2. Ectocervix (Stratified Squamous Epithelium) --------------------------------T zone: detect cervical cancer-------------------------------(Simple Squamous Epithelium) 3. Endocervix ♫ 50% alcohol = All types Fixation 50% alcohol = pleural & peritoneal 70% alcohol = sputum 95% alcohol = urine, bronchial & gastric ♫ Saccomanno’s fixative = 50% ETOH + 2% carbowax Smear preparation Fix immediately 2-3 slides/patient a. streaking b. spreading lec.mt 04 |Page | 260 (2nd)
Fixing smear Sputum
BAL Jelly-like clots GI specimen Urine Pap smear
Adhesive agents 3 primary materials used for obtaining specimen for Pap smear Strawberry cervix
Shift to the left Shift to the right Shift to the midzone Superficial cells
Intermediate cells
c. touch preparation/impression d. pull-apart Never touch the bottom of the fixative container Equal parts of ethanol & ether = BEST (but highly flammable) 95% ethanol = commonly used Spray fixatives = 1 ft away Saccomanno’s fixative 3 specimen (+) alveolar macrophage = sputum (-) alveolar macrophage = saliva P. carinii/P. jiroveci Prevent by adding 300U heparin/100mL aspirate If >½ hr delay of fixation digestion of cells Fasting: 8 hrs 50mL = cytology 10-15mL = UA 2nd urine = preferred Dr. George Papanicolau (1940) Smears: prepared by rotary motion 1. Mailing of specimens - air drying after 2 hrs fixation - glycerin technique 2. Egg albumin = not recommended as adhesive agent (intensifies stain by Light Green) Pooled human serum/plasma Celloidin ether alcohol Leuconostoc culture 1. Speculum 2. Ayer’s spatula = rotate 3600 3. Cytobrush = Os T. vaginalis Cells (Cervicovaginal Smears) Parabasal | Intermediate | Superficial ↓ ----------Estrogen----------↑
45-50μm Pyknotic nucleus True acidophilia Folds/curls on edges a. Navicular cells = boat-shaped
Parabasal cells
15-30μm Fried eggs w/ sunny side up
Endometrial cells
Groups of 3 or more 1-10 days after menst Endocervical glandular cells Honeycomb appearance Similar appearance to parabasal cells lec.mt 04 |Page | 261
Doderlein bacillus
L. acidophilus Pap’s stain: blue to lavender Diabetic patients Sish kebab appearance Pear-shaped, blue-gray to blue-green Pigs on a scruff appearance Indicates T. vaginalis infection Clue cells Wrinkled prune appearance w/ perinuclear halo HPV (LSIL) Formation of salt crystals
C. albicans T. vaginalis Leptothrix G. vaginalis Koilocytes Ferning
Early pregnancy Quantitative Evaluation: Cytohormonal Maturation Index (CHMI) MI = P/I/S
CHMI Pregnancy Newborn (8 weeks) Infancy (8 weeks-puberty) Late menopausal 75 y.o. woman w/ estrogen therapy
MI = 100/0/0 (no estrogen) MI = 0/20/80 Quality Assurance
3 copies/report
1. Doctor 2. Patient = original copy 3. File Reports Surgical pathology report Cytopathology report Autopsy report Signatories Request forms = patient’s doctor Result forms = pathologist Turnover of results Surgical pathology & cytology = 24 hrs Frozen section = 5-15 mins Autopsy report = 1 week (Autopsy procedure: 24 hrs) Storage Specimen (tissue) = 1 month to 1 year Tissue blocks (paraffin) = 3 to 10 years Slides = indefinite Suggested Guidelines for Record and Specimen Retention (Henry, 21st Ed.) Records Requisitions 2 years QC 2 years Instrument maintenance 2 years BB QC 5 years BB employee signatures 10 years BB donor/recipient records Indefinitely Reports Clinical pathology lab 2 years reports Surgical pathology (and 10 years BM) reports Cytogenetics reports 20 years Autopsy forensic reports Indefinitely lec.mt 04 |Page | 262
Specimens Serum/other body fluids Blood smears (routine) Microbiology smears BB donor/recipient specimens Pathology/BM slides Pathology blocks Cytogenetic slides Cytogenetics diagnostic images Forensic Cases Body fluids Tissue for toxicology Wet tissues Paraffin blocks Slides Reports Gross photographs/ negatives Dried blood films Frozen tissue for DNA Autopsy
Types of autopsy
Preliminaries for PME
48 hours 7 days 7 days 7 days post-transfusion 10 years 10 years 3 years 20 years 1 year 1 year 3 years Indefinitely Indefinitely Indefinitely Indefinitely Indefinitely indefinitely Autopsy (Postmortem Examination) Gold standard for confirmation of a medical disease Wherever scientific medicine of high quality is practiced, postmortem exams are performed Whenever a conscientious physician knows why he lost his patient, a postmortem exam has been performed Whenever criminal law is enforced Whenever a death certificate shows accurately the causes of death & confirmed medical diagnosis for the assembling of vital statistics, a postmortem has been performed Whenever there is medical research on the causes & nature of diseases such as cancer, heart diseases & stroke, the investigative method is the postmortem exam An informed society requires a postmortem exam in human death for the good of medical science, for the public’s health & for the future care of the living patient 1. Complete autopsy - Requires consent - Complete examination of all organs, including the brain 2. Partial autopsy - Part of the anatomy 3. Selective autopsy - Restricted to at least a single organ (Ex. MI – heart) 1. Written consent from the next kin-abide by the extent or restrictions allowed - Relative: oriented by the attending physician, not the pathologist 2. Death certificate (Old: Blue form | New: Blue border/frame) - Signed by: a. Physician b. Pathologist (back): will sign when PME has been performed 3. Medical abstract or clinical data lec.mt 04 |Page | 263
4. Medico-legal clearance - Suspicious evidence of foul play - Ex. physical injury Other Uses of Death Certificate
PME is permitted w/o consent in the following circumstances
If pathologist is not available The coroner has authority in the following cases
Somatic death Criteria for the pronouncement of death
Burial & cremation purposes Transport of body from hospital funeral cemetery Medical insurance claiming - If suicide: (-) insurance - Acts of God (lightning, flood): (-) insurance - Civil war: (-) insurance 1. When it is ordered by the police or coroner (NBI) 2. When it is necessary to complete the death certificate 3. When the deceased himself has given consent before he died (advanced directive) - Stipulate that in the event you will die, you will be giving out a consent for autopsy - Donate your organs for medical purposes or for transplantations 4. Deceased military personnel who dies in active services/training duty or military services The medico-legal examiner or the coroner has jurisdiction in medico-legal cases & may authorize the pathologist to proceed w/ an autopsy 1. All natural deaths occurring in the hospital w/in 24 hrs of admission, unless the case was attended by a private physician w/in 36 hrs of death 2. Newborns in the 1st 24 hrs of life 3. All injury cases, old or recent 4. All deaths due to unknown cases 5. All deaths due to suspicious cases 6. All abortion, whether self-induced or otherwise 7. All violent deaths 8. All accidental deaths 9. All sudden deaths 10. All cases w/o medical attendance w/in 36 hrs prior to the hour of death 11. All deaths due to drowning, hanging or strangulation (asphyxia) 12. All deaths due to shooting, stab wounds, burns, electricity, lightning, tetanus, etc. 13. Homicides 14. All suicides 15. All poisoning 16. Stillborn = omission 17. Premature death Death of an organism Cessation of circulation & respiration (1960’s) 1. Advanced resuscitation techniques that are capable of reviving effectively cases of clinical death *Clinical death: cessation of heartbeat & respiration but the brain is still alive but injured 2. Advance life-sustaining equipment capable of maintaining cardiovascular & respiratory functions despite severe brain injury 3. Redefinition from cessation to irreversible cessation of cardiorespiratory functions after resuscitation attempts 4. Brain death: cannot be revived anymore [National institute of neurological diseases & stroke in the US (1977)] - Clinically dead & dead are the same lec.mt 04 |Page | 264
Criteria for brain death
American bar association & national conference of commission of uniform state laws legislative definition of death (1980) American academy of neurology
Postmortem changes
Technique of Virchow
Brain death: perpetual state of deep sleed a. Coma (patient will not respond) & cerebral unresponsiveness b. Apnea c. Absent cephalic (brainstem) reflexes d. Electrocerebral silence criteria should be present for 30 mins at least 6 hrs after onset of coma & apnea 1. irreversible cessation of circulation & respiratory functions 2. Irreversible cessation of all functions of the entire brain, including the brainstem is dead Death: 1. Coma 2. Absence of the following: - Motor response - Pupillary response to light & pupils at mid-position - Corneal reflexes - Caloric responses - Gag reflexes - Coughing in response to tracheal suctioning - Sucking & rooting reflexes 1. Algor mortis - 1st demonstrable change after death is cooling of the body - At room temp: 2’-2.5’F/hr (1st hr) - 1.5-2’F/hr (next 12 hrs) - 1’F/hr (next 12-18 hrs) - As a rule, the body cools at 1.5’F/hr (50% of cases) - Not a reliable indicator as to the time of death 2. Rigor mortis - Rigidity of the body due to hardening of the skeletal muscles caused by a series of physiochemical events after death - ( formation of locking-chemical bodies between actin & myosin - This interlocking is fixed & produces rigor mortis w/o shortening of the muscles - Sets w/in 2 hrs after death (head & neck) - Complete w/ 12 hrs - Persists about 3-4 days 3. Livor mortis (postmortem lividity/hypostasis) - Blood supply gravitates to the skin vessels w/c becomes toneless & dilate after circulation ceases - Becomes evident as early as 20 mins after death - Fully evident w/in 4-8 hrs - Tardien spots: petechiae 4. Postmortem clotting of blood 5. Discoloration of tissue - Abdomen: green - Formation of sulfur gases (bacteria) 6. Putrefaction 7. Dessication (mummification) Techniques of Autopsy Organs removed & dissected individually in the body Most widely used metohd lec.mt 04 |Page | 265
Technique of Rokitansky
Technique of Ghon Technique of Letulle
Autopsy: Larynx Rectum
Teasing/Dissociation Crushing/squash preparation Smear preparation Frozen section Freezing of unfixed tissue Freezing of fixed tissue Formal (formol) calcium Commonly used methods of freezing Staining methods (frozen sections)
H&E
Freeze-drying Freeze-substitution Cold knife procedure
In-situ dissection in part combined w/ en bloc technique ♫ En bloc: - By cavity - Interrelated to each other - Systemic dissection - Ex. thoracic cavity (lungs, heart, diaphragm), respiratory system En bloc technique En masse technique ♫ En masse: - All organs of thoracic, abdominal, & pelvic are removed at the same time - Sweeping of all organs Very popular, easy to do, convenient Part of consent: organs should be retained completely or partially Organs set aside later Body undertaker of the body Fresh Tissue Examination Tissue specimen Watchglass (isotonic solution) BF/PC microscope Tissue (<1mm) Sandwich bet. 2 slides/coverslip Vital stain Spread lightly over a slide (wireloop/applicator) Frozen Section (-) Fixative Best frozen section To localize hydrolytic enzymes & other antigens Derivative of formaldehyde Fix at 4’C for 18 hrs Liquid nitrogen = most rapid Isopentane cooled by liquid nitrogen CO2 gas Aerosol sprays “PATH” Polychrome methylene blue Alcoholic pinacyanol Thionine H & E = progressive, no decolorizer a. Progressive - w/o decolorizer - for frozen sections b. Regressive - w/ decolorizer (acid-alcohol) - for routine histology staining w/o use of any chemical fixative ♫ Quenching: rapid freezing (-160’C) ♫ Sublimation: removal of H2O in the form of ice (-40’C) – vacuum Similar to freeze drying but: Frozen tissue Rossman’s formula/1% acetone Dehydrated in absolute alcohol Any microtome Uses CO2 Knife: -40 to -60’C Tissue: 5 to -10’C Environment: 0 to -10’C lec.mt 04 |Page | 266
Cryostat procedure (Cold microtome) O.C.T. (Optimal Cutting Temperature)
Steps
Fixation
pH Temperature Microanatomical fixatives
Cytological Fixatives
Nuclear fixatives
Cytoplasmic fixatives
Temperature: -18 to -20’C Cryostat: refrigerated cabinet w/ rotary microtome Best mounting media for cryostat sections
Tissue Processing “FDCIETS SMoL” 1. Fixation (Decalcification) 2. Dehydration 3. Clearing/Dealcoholization 4. Impregnation/Infiltration 5. Embedding/Casting/Blocking 6. Trimming 7. Sectioning/Microtomy 8. Staining 9. Mounting 10. Labeling (slides) Fixation st 1 and most critical step 1’ aim: preserve cell (life-like) 2’ aim: harden & protect tissues Most important: stabilization of proteins 6.0-8.0 Room temp = Surgical specimen 0 to 4’C = EM and Histochem. General microscopic study of tissues a. 10% Formol saline b. 10% NBF c. Heidenhain’s SuSa d. Formol sublimate (formol corrosive) e. Zenker’s solution f. Zenker-formol (Helly’s) g. Bouin’s solution h. Brasil’s solution Specific parts of the cell a. Nuclear fixatives: w/ glacial acetic acid – destroys mitochondria & golgi bodies (pH ≤4.6) b. Cytoplasmic fixatives: w/o glacial acetic acid c. Histochemical fixatives: preserves chemical constituents “BFNCH” Bouin’s Flemming’s w/ acetic acid Newcomer’s Carnoy’s Heidenhain’s SuSa “HORFF” Helly’s Orth’s Regaud’s lec.mt 04 |Page | 267
Histochemical fixatives
Aldehyde Fixatives Formaldehyde
10% Formol saline 10% NBF
Formol-Corrosive (formol sublimate) Glutaraldehyde Karnovsky’s paraformaldehydeglutaraldehyde Acrolein Formol-calcium Mercuric Chloride
Heidenhain’s SuSa HgCl2 G.HAc De-zenkerization Chromate fixatives
Flemming’s w/o acetic acid Formalin w/ post chroming “FANA” 10% Formol saline Absolute alcohol Newcomer’s fluid Acetone Concentrated solutions should not be neutralized (explosion) Stock solution: 37-40% Working solution: 10% (no buffer: unstable) Formalin pigments: a. Paraformaldehyde - White crystalline precipitates - Due to prolonged standing - Removed by: 10% METOH/filtration b. Acid formaldehyde hematin - Brown/black granular deposits that may obscure microscopic details CNS Best general tissue fixative Best fixative for tissue containing iron granules w/ double phosphate buffer 1 mm/hr = rate of tissue penetration w/ HgCl2 EM EM: electron histochemistry & electron immunocytochemistry Mixture w/ formaldehyde/formaldehyde Lipids (frozen section) Fixatives Tissue photography For Trichrome stain (excellent) Produce black granular deposits except SuSa “BOSCHZZ” a. B5 = for BM biopsies b. Ohlmacher’s c. Schaudinn’s d. Carnoy-Lebrun e. Heidenhain’s SuSa = (-) black pigments f. Zenker’s = recommended for trichrome staining g. Zenker-formol (Helly’s) = pituitary gland, BM, & blood containing organs Su = sublimat (HgCl2) Sa = saure (acid) Shrinks tissues Swells tissues, counteracts HgCl2 Removal of mercuric deposits H2O I2 H2O Sodium thiosulfate H2O “ROCK” a. Regaud’s (Moller’s) = chromatin, mitochondria, mitotic figures… b. Orth’s = for Rickettsia, tissue necrosis lec.mt 04 |Page | 268
Chromate pigments Lead fixatives Picric acid fixatives
Glacial acetic acid Alcoholic fixatives
Osmium tetroxide (Osmic acid)
Trichloroacetic acid Acetone Heat fixation
2’ fixation Post-chromatization Washing out
EM fixatives
Stains (EM)
c. Chromic acid = preserves CHO d. K2CrO4 = mitochondria (if acidified, fixes chromatin bodies & chromosomes but destroys mitochondria) Fine, yellow brown Used in 4% aqueous solution of basic lead acetate For acid MPS and mucin Highly explosive when dry Excessive yellow staining of tissues Picrates Protein Ppt. (H2O soluble) Add 70% ETOH Insoluble Never wash in H2O before dehydration For glycogen (excellent) a. Bouin’s = for embryos, Masson’s trichrome stain, glycogen b. Brasil’s alcoholic picroformol = less messy than Bouin’s, glycogen (excellent) Solidifies at 17’C Fixes & precipitates nucleoproteins, chromosomes, & chromatin material Most commonly combined w/ other fixatives Disadvantage: polarization (glycogen granules poles/ends of the cells) “MEICAN” a. Methanol = BM & blood smears b. Ethanol = preserves but does not fix glycogen (Disadv: polarization) c. Isopropanol = for touch preparations d. Carnoy’s = most rapid (1-3 hrs) | for chromosomes | Dx: rabies (acetone) e. Alcoholic formalin (Gendre’s) = sputum f. Newcomer’s = for MPS | nuclear & histochemical fixative Inhibits hematoxylin Produce black precipitate crystals (osmium oxide) For lipids a. Flemming’s = permanently fixes fat, for nuclear structures (excellent) - Fixative & decalcifying agent (chromic acid) b. Flemming’s w/o acetic acid = for mitochondria Precipitates proteins Swelling effect counteract shrinkage by other fixatives Weak decalcifying agent (softening effect) Recommended for H2O-diffusible enzymes (phosphatases, lipases) Rabies Bacteriologic smears Microwave: 45-55’C Underheating: poor sectioning Overheating (>65’C): vacuolation, overstained cytoplasm Placing an already fixed tissue in a 2nd fixative Primarily fixed tissue 2.5-3% K2CrO4 (mordant) Removing excess fixative a. Tap H2O = remove excess chromates, formalin, osmic acid (NOT Bouin’s) b. 50-70% alcohol = wash out excess picric acid (Bouin’s) c. Alcoholic I2 = remove excess mercuric fixatives Glutaraldehyde PtCl3 PtCl3 – formalin (Zamboni’s) AuCl Osmium tetroxide 10% NBF = acceptable but not recommended “PUL” lec.mt 04 |Page | 269
1. PTA = 1st general stain 2. Uranyl acetate = Best 3. Lead Factors that Affect Fixation of Tissues Retarded by: Size & thickness (+) Mucus
Prevents complete penetration of fixative Wash w/ NSS Fatty tissues: cut in thin sections, fixed longer Flush out w/ NSS fix Inactivates enzymes
(+) Fat (+) Blood Cold temperature Enhanced by: Size & thickness Agitation Automatic/mechanical tissue processing Moderate heat 37-56’C Principles and Precautions in Handling and Fixation of Specimens in General Autopsy materials Fixed ASAP If not possible mortuary refrigerator (4’C) or arterial embalming Surgical specimens Fixed ASAP If not possible refrigerate If placed in NSS during Autolysis may occur before fixation operation If tissues are refrigerated Avoid slow freezing (ice crystal formation) Repeated freezing & thawing destroy organelles, release enzymes… Not more than 5mm thick Size of tissues Except lung edema: 1-2 cm thick 20:1 Ratio of fixative to tissue Except osmium tetroxide (expensive) = ratio is 5-10:1 50-100:1 Ratio of fixative to tissue in prolonged fixation (ex. museum preparation) Avoid drying of small tissue To prevent: place in a petri dish w/ moistened filter paper biopsies Hollow organs Stomach, intestines Packed w/ cotton soaked fixative or completely opened before being immersed in adequate fixing solution Air-filled lungs Float on fixative To prevent: cover w/ several layers of gauze to maintain it under surface Human brains Suspended by a cord tied under the Circle of Willis to prevent flattening Avoid Ringer’s lactate for washing out of blood intravascular perfusion Fixation time: 2 weeks Eyes Not dissected before fixation tissue collapse & wrinkling (escape of vitreous humor) Inject formol-alcohol before immersing the organ in the fixative Glycogen-containing tissues Do not use water Glycogen is water-soluble Hard tissues Cervix, uterus, fibroids, hyperkeratotic skin, fingernails Wash in running water overnight immerse in 4% aqueous phenol for 1-3 days (Lendrum’s method) Difficulties Encountered because of Improper Fixation Problem Cause Failure to arrest early cell autolysis Failure to fix immediately (tissue was allowed to dry before fixing) Insufficient fixative lec.mt 04 |Page | 270
Removal of substances soluble in fixing agent Wrong choice of fixative Presence of artifact pigments on tissue sections Incomplete washing of fixative Tissues are soft & feather-like in consistency Incomplete fixation Loss/inactivation of enzymes needed for study Wrong choice of fixative Shrinkage & swelling of cells & tissue structure Overfixation Tissue blocks are brittle & hard Prolonged fixation ♫ An incompletely fixed tissue may lead to improper & incomplete clearing & impregnation, and may later prove to be a hindrance to normal sectioning & staining of specimen Pigment Acid formaldehyde hematin
Mercuric chloride pigment Chromate pigment Osmium tetroxide pigment Crush artifact 20:1 37’C 55’C RT (18-30’C) 24-48 hrs Decalcifying agents
HNO3
5% Formic acid HCl (Von Ebner’s) EDTA Ion exchange resins Electrophoresis Measuring extent of decalcification Post-Decalcification Tissue softeners
Color Brown/black granules
Removed by: “SAKaL” a. Saturated picric acid b. Alcoholic KOH c. Kardasewitsch method d. Lillie’s method Black granules Alcoholic iodine Fine, yellow brown Acid-alcohol Black precipitate crystals Cold H2O Intense eosinophilic staining at the center of the tissue (H & E) Due to partial coagulation of partially fixed protein Decalcification Ratio of decalcifying agent to tissue Impaired nuclear stain by Van Gieson’s stain Tissue Digestion (24-48 hrs) Optimum temperature Time Acids Chelating agents (EDTA/versene) Ion exchange resins Elec. ionization (electrophoresis) Most common a. Perenyi’s = tissue softener & decalcifying agent b. Phloroglucin-HNO3 = most rapid - Disadvantage: Yellow color on tissue (neutralize w/ sodium thiosulfate) Both fixative & decalcifying agent Best general decalcifying agent For small pcs of bones & teeth For small pcs of bones & teeth For surface decalcification (HCl) For EM, IHC, & enzyme staining Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions Ca2+ are attracted to negative electrode (cathode) Physical method Chemical method = CaOx test (routine) | Turbidity = (+) Ca2+ X-ray = most ideal, most sensitive, most reliable but very expensive - X-ray paper = Kodak X-omat or Faxitron Removal/neutralization of acid from the tissues after decalcification Lithium carbonate or sodium bicarbonate solution 4% phenol Molliflex = tissues appear swollen & soapy 2% HCl lec.mt 04 |Page | 271
Dehydration 10:1 Ethanol Methanol Butanol Denatured alcohol Acetone Dioxane (Diethylene dioxide) Tetrahydrofuran (THF) Graupner’s method Weiseberger’s method Cellosolve Triethyl phosphate Additives to dehydrating agents Methods of determining incomplete dehydration Xylene (Xylol) Toluene
Chloroform Benzene Methyl salicylate Methyl benzoate Cedarwood oil Clove oil CCl4 Aniline oil Glycerin Gum syrup Others
1% HCl in 70% alcohol Dehydration Aim: To remove fixative & H2O Ascending grades of alcohol (Start: 65%) Embryonic & animal tissues: 30% ETOH Ratio of dehydrating agent to tissue Best dehydrating agent Blood & tissue films Plants & animals Ethanol + methanol Both fixative & dehydrating agent Both dehydrating & clearing agents Dehydration w/ dioxane Ethylene glycol monoethyl ether Combustible and toxic -a. 4% phenol + 95% ETOH = softener b. Anhydrous CuSO4 (Last ETOH bath) - both dehydrating agent & indicator of H2O content of 100% ETOH - (+) H2O = White Blue 1. Anhydrous CuSO4 method 2. Xylene Milky Clearing Most commonly used Clearing time: ½ to 1 hr Block size: <5mm Substitute for xylene/benzene Clearing time: 1-2 hrs Not carcinogenic Toxic fumes Toxic to liver For clearing tough tissues Does not make tissue translucent but removes alcohol For urgent biopsies Minimum shrinkage Aplastic anemia For double embedding techniques For CNS, smooth muscles, skin Minimum shrinkage Has tendency to become adulterated Similar to chloroform but is cheaper Disadvantage: similar to chloroform For delicate tissues, embryos and insects No dealcoholization but make the tissues clearer Citrus fruits oil Trichloroethane & petrol Impregnation lec.mt 04 |Page | 272
25:1 Medium
Paraffin
Paraffin filters Paraffin oven
Ratio of infiltrating medium to tissue Paraffin wax Celloidin (collodion) Gelatin = H2O soluble, not a wax Plastic = EM Introduced by Bütschlii Not recommended for fatty tissues Low MP = paraffin is soft High MP = paraffin is hard Manual: At least 4 changes of wax at 15mins interval Filtration at 2’C above the MP of wax Ex. Green’s no. 904 (coarse filter paper) 2-5’C above the melting point of wax (55-60’C) >60’C = shrinkage & hardening of tissues
Substitutes for Paraffin Wax Paraplast 56-57’C MP More elastic & resilient Bones & brain Embeddol 56-58’C MP Bioloid Eyes Tissue mat Contains rubber Ester wax 46-48’C MP Soluble in 95% ETOH & clearing agent Impregnation w/o prior clearing H2O soluble wax 38-42’C/45-56’C MP Mostly PEG ♫ Carbowax: most commonly used - Does not require dehydration and clearing - For enzyme histochemistry Celloidin/Collodion Purified form of nitrocellulose Wet celloidin Equal parts of ether & alcohol Bones, brain, teeth sections Dry celloidin Gilson’s mixture: equal parts of chloroform & cedarwood oil Whole eye sections LVN Another form of celloidin Soluble in equal concentrations of ether & alcohol Plastic/Resins Epoxy (EPON™) Polyester Acrylic Waterbath 6-10’C below the MP of wax (45-50’C) Autotechnicon Fixes, dehydrates, clears & infiltrates tissues Constant tissue agitation Elliott Bench-Type processor Wax bath: 3’C above the MP of wax Vacuum embedding Wax impregnation under negative pressure (hasten removal of air bubbles) Time is reduced from 25-75% of the normal time required for tissue processing 2-4’C above the MP of wax (+) Odor in the clearing Indicates that the paraffin wax should be changed agent Embedding Orientation Arranging in precise positions in the mold, microtome & slide Oven 5-10’C above the MP of wax (Impregnation: 2-5’C above) lec.mt 04 |Page | 273
Blocking-out Molds Leuckhart’s embedding mold Compound embedding unit Plastic embedding rings & basse mold Tissue Tek Disposable embedding molds Celloidin/Nitrocellulose method Double embedding method
Cold knife procedure Cryostat (Cold microtome) Fixation Freeze-drying Algor mortis Formol calcium fixation Impregnation Embedding Flotation water bath Trimming Routine histopath. (Rotary) Freezing EM (Ultrathin) Rocking/Cambridge microtome Rotary/Minot microtome Sliding microtome
2 L-shaped strips Adjustable Several compartments Special stainless steel base mold fitted w/ a plastic embedding ring (block holder) Warm plate Cold plate (-5’C) 1. Peel-away: perfect even block w/o trimming 2. Plastic ice trays: ordinary refrigerators 3. Paper boats: cheap & easy to make Bones, teeth, eyes Bell jars: control evaporation 1st: celloidin 2nd: paraffin Brain Recall Temperatures Knife = -40 to -60’C Tissue = 5 to -10’C Environment = 0 to -10’C -18 to -20’C Surgical specimen: room temp HC & EM: 0-4’C Quenching: -160’C Sublimation: -40’C 7’F/hr (3.89’C/hr) 4’C Manual = 2-5’C above MP of wax (55-60’C) Automated = 3’C above MP of wax Vacuum = 2-4’C above MP of wax 5-10’C above MP of wax 6-10’C below MP of wax (45-50’C) Trimming Removing excess wax after embedding Ideal: four-sided prism/truncated pyramid Microtomy 4-6μm 10-15μm 0.5μm Trefall Simplest Minot Most commonly used for paraffin embedded tissues Adams Most dangerous (movable knife) a. Base-sledge - For all forms of media - Block: moving - Knife: stationary b. Standard sliding - Block: stationary - Knife: moving lec.mt 04 |Page | 274
Vibrotome Ultrathin microtome Freezing microtome Clearance angle Bevel angle Honing Types of hones Stropping
Natural dyes
Synthetic dyes Chromophores Auxochrome Chromogen Dye Chromophores
Auxochromes Dye modifiers
Van der Waals forces Sudanophilia Hematoxylin
For unfixed, unfrozen tissues For enzyme demonstration EM Diamond knives or broken plate glass Queckett Knife to tissue block: 0-150 angle 27-320 angle Removal of gross nicks Heel to toe (Edge 1st) a. Belgium yellow: Best b. Arkansas c. Fine carborundum: for badly nicked knives Removal of gross burrs Toe to heel (Edge last) Paddle strop (horseleather) - Mineral oil = not recommended - Vegetable oil (castor oil) = applied into the back of the strop, not the surface Staining From plants & animals a. Hematoxylin b. Cochineal dyes: female Coccus cacti c. Orcein: from lichens d. Saffron A.k.a. coal tar dyes Derived from benzene & collectively known as aniline dyes Greek: “color-bearer” Coloring property Greek: “increasers” Dyeing property Benzene + Chromophore Imparts color temporarily Chromogen + auxochrome Imparts color to tissue almost permanently a. Quinoid ring: Basic fuchsin b. Azo groups: Congo red c. Xanthene: Eosin d. Quinone-imine group - Oxazin: cresyl fast violet - Thiazins: toluidine blue Cationic auxochromes: amino group (NH3+) Anionic auxochromes: hydroxyl (OH-) and carboxyl (COO-) groups Attached on benzene ring a. Ethyl group b. Methyl group c. Sulphonic acid Alum hematoxylin Tissue stained w/ fat or oil-soluble dyes H & E Staining Hematoxylin capechianum/ Hematoxylon campechianum Nuclear/basic/1’ stain Waldeyer: 1st to use hematoxylin lec.mt 04 |Page | 275
Lake Oxidizing agents
Alum hematoxylin
Iron hematoxylin
Tungsten hematoxylin Copper hematoxylin Eosin (Eosin Y)
Coplin jar Slotted staining dishes Metal/glass staining racks/ carriers H & E staining steps
Hematoxylin ---(Ripening)---> Hematein (active coloring substance) Tissue-Mordant-Dye complex H 2O 2 HgO2 = Harris’ K2MnO4 Na perborate Na iodate = Mayer’s, Ehrlich’s, Gill’s Routine H & E = Red Mordant: K Alum “MEGDH” Mayer’s = Na iodate (ripening agent) Ehrlich’s = Na iodate (ripening agent) Gill’s Delafield’s Harris’ = HgO2 (ripening agent) Mordant = oxidizing/ripening agent = Iron a. Weigert’s - Mordant: FeCl3 - Weigert’s + Van Gieson’s = CT & E. histolytica b. Heidenhain’s - Mordant: Ferric ammonium sulfate a. Mallory’s PTAH - Mordant = sunlight/K+ - Stain fibrin Spermatogenesis Cytoplasmic/acidic/2’ stain Counterstain a. Eosin Y (Yellowish) = most commonly used b. Eosin B (Bluish) = deep red c. Ethyl eosin/Eosin S/Eosin alcohol soluble Holds 5-9 slides Holds 5-19 slides Holds 10-30 slides 1. Xylol (2) = deparaffinization 2. Descending grade of alcohol = rehydration 3. H2O 4. Remove fixative artifact pigments after rehydration & before staining 5. Stain: Nucleus = light blue 6. H2O 7. Acid alcohol (differentiator): Nucleus = light blue 8. Ammonia water (blueing agent): Nucleus = blue - NH4OH - LiCO3 - Scott’s tap H2O 9. Wash 10. Stain: Eosin Y 11. Ascending grade of alcohol = dehydration 12. Xylene = dealcoholization/clearing 13. Mount & label Nuclei: blue to blue black Cytoplasm: pale pink lec.mt 04 |Page | 276
Pap Smear staining
Benzidine Acridine orange Gentian violet Congo red Iodine Malachite green Janus green Night blue Victoria blue Lysochromes
Adhesives
Mounting media
Stains on skin Restaining of old sections
Broken slides
Hematoxylin = nuclear stain OG-6 = cytoplasmic stain (mature superficial cells) EA (Eosin Azure) = cytoplasmic stain (immature cells: parabasal/intermediate) EA 65 = for body fluids EA 36/50 = for gynecologic smears Other Stains Hgb RNA (red fluorescence) DNA (green fluorescence) Crystal violet + methyl violet + dextrin Elastic tissue, amyloid, myelin Oldest stain Ascaris eggs Mitochondria (intravital stain) Substitute for carbolfuchsin Neuroglia Oil soluble dyes a. SBB = Black (most sensitive) b. Sudan III = orange c. Sudan IV (Scharlach R) = red a. Mayer’s egg albumin = add thymol crystals (inhibit mold growth) b. Dried albumin c. Gelatin d. Gelatin-formaldehyde e. Starch paste f. Plasma g. Poly-L-lysine = IHC h. 3-APES: 3-aminopropyltriethoxysilane = Best (cytology) ♫ 1.518 = refractive index of glass 1. Resinous media = contains xylene - a. DPX = 1.532 - b. XAM = 1.52 - c. Canada balsam (Abus balsamea) = 1.524 - d. Clarite = 1.544 2. Aqueous media = for lipids (no xylene) - Water = temporary mounting, low refractive index - Glycerin jelly = 1.47 | standard mounting medium (fat stains) - Gum Arabic (Farrant’s) = 1.43 - Apathy’s medium = 1.52 - Brun’s fluid = for frozen sections Others: - Permount - HSR - Clearmount Remove by using 0.5% acid alcohol tap water Slide Xylene (24hrs) or gently heat until mounting medium begins to bubble Remove coverslip Section: Xylene (30mins) H2O 0.5% K2MnO4 (5mins) H2O 5% Oxalic acid (5 mins or ‘til decolorized) H2O Restain Shortcut: “X-XhKhOhR” SlideXyleneRemove coverslipXyleneK2MnO4Oxalic acid Restain 1. Mount the broken slide to another clean xylene-moist slide w/ drop of lec.mt 04 |Page | 277
mounting media 2. If replacement not possible, the section (if intact) may be transferred to another slide: Broken slide Xylene (rem. coverslip) incubate (rem. mountant) 6 parts butyl acetate + 1 part durofix incubate (mixture film) Cut the film around the section Cold H2O until the film & section float off Film w/ section mount on a clean slide incubate butyl acetate xylene mount
Ringing
Enzyme histochemistry IgG Polyclonal Monoclonal Epithelial Tumor Markers (+) CK 7 (-) CK 20
Shortcut: “Xi6B1DiCuCoFSMiBXM” Broken slide Xylene Incubate 6 Butyl acetate + 1 Durofix Incubate Cut film Cold H2O to float film & section Film w/ section mount incubate butyl acetate xylene mount Sealing the margins of the coverslip Prevent escape/evaporation of fluid Immobilize the coverslip Prevent sticking of slides a. Kronig cement = 2 parts paraffin + 4-9 parts colophonium resin b. Durofix (cellulose adhesives) Immunohistochemistry Trypsin & protease = most commonly used Most commonly used antibody Rabbits (1’) > Goat (2’) > Pig (3’) > Sheep (4’) > Horse (5’) > Guinea pig (6’) Mice
“LUBO” = paired Lung Uterus Breast Ovary (+) CK 20 Stomach (-) CK 7 Colon (+) CK 7 Transitional cell carcinoma of the bladder (+) CK 20 Mucinous ovarian tumor (-) CK 7 HCC (-) CK 20 RCC SCC Thyroid carcinoma Prostatic adenocarcinoma EMA (Epithelial membrane (+) carcinoma “BuLK” = paired antigen) Breast Lungs Kidney CEA Oncofetal antigen GI carcinoma Differentiates adenocarcinoma (+) & mesothelioma (-) TTF-1 (Thyroid Differentiates lung adenocarcinoma & mesothelioma Transcription Factor) (+): Thyroid, lung, neuroendocrine tumors PSA Prostate cancer Intermediate Filament Markers Actin Smooth muscle Skeletal muscle lec.mt 04 |Page | 278
Vimentin Desmin GFAP (Glial Fibrillary Acidic Protein) NF (Neurofilament)
S100 protein Neuroendocrine Markers NSE (Neuron-specific enolase) Others
Cardiac muscle Melanomas Schwannomas Leiomyoma (smooth muscle) Rhabdomyosarcoma (skeletal muscle) Astrocytoma Neuroblastoma Ganglioneuromas Neuroma Chemodectoma Pheochromocytoma Low MW Ca2+-binding protein CNS glial cells, Schwann cells Strong evidence of neural/neuroendocrine differentiation Chromogranin Synaptophysin
Germ Cell tumor markers HCG
Synthesized by syncytiotrophoblasts Choriocarcinoma AFP Endodermal sinus tumors showing yolk sac differentiation PLAP (Placenta-like ALP) Germinomas Mesenchymal Tumor Markers Myogenic tumors Myo-D1 Myoglobin Myogenin Fibrohistiocytic tumors -Vascular tumors Factor VII-related antigen CD31 UEA: Ulex europaeus I Melanomas -Lymphomas LCA: Leukocyte common antigen (CD45) Cell Proliferation Markers Ki67 MIB-1: reference monoclonal antibody for Ki67 demonstration PCNA Proliferating cell nuclear antigen Controls Positive control Known Contains antigen in question Negative control Done using a parallel section from the tissue Internal tissue control A.k.a. “built-in control” Contains the target antigen Other Topics Faults During Tissue Processing Brittle/hard tissue
Clearing agent Milky
Incomplete dehydration lec.mt 04 |Page | 279
On trimming, tissue smells of clearing agent Tissue is opaque Tissue shrinks away from wax Tissue is soft when block is trimmed Air holes on tissue Wax appears crystalline Paraffin block is moist & crumbles Sections fail to form ribbons
Sections roll up on cutting… adhere & get broken against the knife edge Ribbon is curved, crooked, or uneven Sections are compressed, wrinkled or jammed
Sections are squashed Hole in section Sections of unequal thickness Sections adhere to knife or other parts of the microtome
Insufficient impregnation Insufficient clearing Insufficient dehydration Incomplete clearing & impregnation Incomplete fixation Incomplete impregnation Contaminated wax Block not cooled rapidly enough Insufficient paraffin impregnation Surface & edges of block not parallel Wax too hard Knife tilted too much Thick sections Dull knife Blunt knife Dirty knife edge Irregular knife edge Edges of block are not parallel Knife not parallel to the block Impure paraffin Blunt/dull knife Block is warm & soft Knife edge coated w/ paraffin Thin sections Microtome screw is loose Tilt: vertical Bevel of knife is lost Incorrect sharpening Bubble/dirt Hard spot in the tissue (Ca2+) Screw/holder is loose Large & hard blocks Static electricity Dirty knife edge Dull knife edge
Ribbon is split
Nicks/damage on knife Dirty embedding Dirty knife
Chatters are seen
Knife vibrates (hard tissue)
Section: sometimes thin & thick
Blunt knife
Frozen tissue crumbles & comes off when the block
Knife/block holder is loose Inadequate freezing lec.mt 04 |Page | 280
holder when cut Frozen tissue chips into fragments
Tissue is frozen too hard
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PAS w/ diastase ctrl
Stains Substance Stained (+) Color/Result CHO, Glycogen, Mucins, PAS (+):Magenta red Bacteria & Fungi, basement membrane Glycogen Red
Best Carmine
Glycogen
Bright red
Langhan’s iodine method
Glycogen
Mahogany brown
Alcian blue
Acid mucins
Blue
Alcian Blue-PAS
Any mucins (acid/neutral) Acid MPS Sulfated mucins Carboxylated mucins Cryptococcus neoformans Mucins Acid mucins
Acid mucin: blue Neutral mucin: magenta Sulfated mucins: purple Carboxylated mucins: blue Mucin: red
Acid mucins/MPS Fungi
Stain PAS
Gomori’s aldehyde fuchsin stain Mucicarmine stain Colloidal (Dialyzed) iron technique Acridine orange Sudan black Sudan IV (Scharlach R)
Lipids Lipids (TAG)
Oil red O Osmium tetroxide Nile blue sulfate method
Lipids Lipids Neutral fat Cholesterin esters Cholesterin fatty acids Fatty acids & soap Cerebrosides
Brilliant red Black = Pinkish red = Light red = Light red = Deep blue to violet = Light blue
Toluidine blue-acetone mtd
Sulfatide
Borohydride-PeriodicSchiff method Alkaline fast-green method
Gangliosides
Metachromatic redbrown or yellow Red
Peracetic acid-Alcian blue Sakaguchi’s test Gomori calcium method Gomori lead method Lead method Metal precipitation Calcium cobalt method α-naphthyl acetate method
Avoid Ehrlich’s hematoxylin
Dark blue Acid MPS: black Fungi: greenish red fluorescence Blue black red
Histones Protamines Cystine Cysteine Arginine Alkaline phosphatase Acid phosphatase 5’-nucleotidase ATPase ATPase Nonspecific esterase
Comments Basic fuchsin: essential component of Schiff reagent Method of choice for glycogen staining Selective & highly specific for glycogen Obsolete Not specific for glycogen Avoid celloidinization of slides Avoid Ehrlich’s hematoxylin
Green
Lasts for only 2 hrs
Most commonly used stain Nile blue: preliminary indicator of the type of lipid present -Red oxazone (dissol. neutral lipids) -Blue oxazone (reacts w/ PL and FFA)
Fast green stains basic groups in tissues
Blue-green Orange-red Brownish-black Black Blackish brown deposits Dark brownish-black ppt Cobalt phosphate ppt Reddish brown
Uses Milton reagent Substrate: β-glyceroPO4 For skel. muscle biopsies For skel. muscle biopsies lec.mt 04 |Page | 282
Stain Indoxyl acetate method Tetrazolium method Feulgen technique
Substance Stained Nonspecific esterase Monoamine oxidase DNA
(+) Color/Result Blue Bluish black Red-purple
Methyl green-pyronin
RNA DNA Reticulin fibers
RNA (nucleoli): red DNA (chromatin): green black
Collagen Muscle, cytoplasm, RBC, fibrin Collagen & mucus Muscle, RBC & keratin Collagen fibers, cytoplasm, fibroglia fibrils, axon cylinders, neuroglia Elastic fibers RBCs, myelin sheets CT Glomerular basement membrane Amyloid & mucous colloid Elastic fibers Elastic fibers Elastic fibers Elastic fibers Fibrin & CT RBC RBCs Muscle Collagen Fibrin Fibrin, muscle striations, neuroglia, amoeba RBCs Myelin Collagen, osteoid, cartilage, elastic fibers Amyloid Amyloid
= Pink/deep red = Yellow
Amyloid
Yellow fluorescence
Muscle fibers Collagen Muscles, RBC Collagen
= Red = Green = Red = Yellow
Gomori’s silver impregnation stain Van Gieson’s stain Masson’s trichrome stain Mallory’s aniline blue
Azocarmine
Weigert’s Verhoeff’s Taenzer-Unna-Orcein mtd Krajian’s technique Martius-Scarlet-Blue
Mallory’s PTAH
Congo red Methyl violet-crystal violet method Thioflavin-T fluorescent staining Modified Gomori’s Trichrome stain Lissamine fast red
= Blue = Red = Red
Comments Most reliable & specific histochemical staining technique for DNA Contains Schiff’s reagent Reticulin = Argyrophilic (silver stain) Contains acid fuchsin & picric acid
(-) Fuchsin: Excellent & colorful method of demonstrating CT fibers
= Pale pink/yellow = Yellow
= Deep blue Dark-blue/blue-black Black Dark-brown = Bright red = Dark blue = Orange-yellow = Yellow = Red = Blue = Red = Dark blue
Heidenhain’s modification of Mallory’s aniline blue stain
Rapid method Early fibrin = yellow Old fibrin = blue
= Blue = Lighter blue = Deep brownish-red Red Purplish red
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Stain Schmorl’s Picro-Thionin method
Substance Stained Lacunae & canaliculi Bone matrix
Bielschowsky’s technique
Neurofibril, axons & dendrites Neuroglia & collagen Bodian’s stain Nerve fibers & nerve endings Sevier-Munger technique Peripheral neuritis Axons Myelin sheath Neuritic plaques & tangles Argentaffin granules Toluidine blue Nissl granules & nucleoli Polychrome methylene blue Nissl granules & nucleoli Thionine Nissl granuls & nucleoli Cresyl fast violet Nissl substance Neurons Weigert-Pal technique Myelin sheath Luxol fast blue Myelin Weil’s method Myelin Cajal’s gold sublimate Astrocytes method Perl’s Prussian blue Hemosiderin Gomori’s Prussian blue Iron pigments Turnbull’s blue Ferrous iron (Hemosiderin) Benzidine-nitroprusside Hemoglobin & oxidase stain granules Mod. Fouchet’s technique Bile pigments Gmelin technique Bile & hematoidin Stein’s iodine test Bile pigments Schmorl’s ferric ferricyanide method Gomori’s aldehyde fuchsin Mallory’s fuchsin stain Masson Fontana technique
Von Kossa’s silver nitrate method Lindquist modified rhodanine technique
(+) Color/Result = Dark brown-black = Yellow/brownishyellow = Black on a grayish BG
Comments
= Lightly stained Diagnosis of Alzheimer’s disease = Black = Black = Light brown = Black = Black Deep blue Deep blue Purple = Purple-dark blue = Pale purple blue Blue black Blue-green Black Black on a light brownish BG Deep blue Bright blue Blue
Nissl granules: a.k.a. Tigroid substances
Dark blue Emerald to blue green Blue-purple then green Depend on the oxidation of the pigment to green biliverdin by iodine
Bile, lipofuscins, melanin, argentaffin cells, chromaffin, thyroid colloid Lipofuscin Hemofuscin Melanin Argentaffin cell granules
Dark blue
Calcium
Black
Copper
Red to orange-red
Purple Red = Black = Black
Argentaffin reaction: melanin reduces ammoniacal silver solutions w/o use of a reducer
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Stain Gram-Twort stain
Brown & Brenn method Wade-Fite technique Toluidine blue
Substance Stained Gram (+) organisms Gram (-) organisms RBCs Elastic fibers Gram (+) bacteria Gram (-) bacteria M. leprae H. pylori
Cresyl violet acetate mtd Dieterle method
H. pylori L. pneumophila & spirochetes Levaditi’s method Spirochetes Modified Steiner & Steiner Spirochetes, Donovan technique bodies, fungi, bacteria Warthin-Starry method Spirochetes Grocott Methenamine Silver Fungi
Lendrum’s phloxinetartrazine method Orcein method Giemsa stain
In situ hybridization PCR Chondrocalcinosis Kardasewitsch method 0.1% urea + 5% NaSO4 Metastasis Degree of localization Dunn-Thompson K2MnO4 H2 O 2 Helly’s Formalin ammonium bromide Alcohol as 1’ fixative Glutaraldehyde Carnoy’s Orth’s Zenker’s Formaldehyde Ethanol
Mucin & glycogen Mycelia & hyphae RBCs Viral inclusions
(+) Color/Result = Blue-black = Pink-red = Green = Black = Blue = Red Golden yellow Dark blue against blue BG Blue-violet Dark brown to black
Comments
Black on a yellowish BG Black Black = Sharply outlined I black = Gray-black = Old rose = Yellow Bright red
HBsAg Brown-black Bacteria = Blue Recommended for blood Mast cell granules = Deep blue and BM parasites, Eosinophilic granules = Red inclusion conjunctivitis, Nuclei = Blue Toxoplasma, spirochetes Cytoplasm = Pink & other bacteria Most sensitive technique for identifying DNA DNA amplification Pseudogout Pigment removal 70% ETOH + 28% NH3 water Remove yellow color of HNO3 Most definitive of malignancy Most reliable indicator of prognosis of malignant tumors Hgb = emerald green Removes excess melanin Contains formalin, K2CrO4 and HAc Fixative for CNS (gold/silver stain) Increased tissue shrinkage Not satisfactory for PAS Nonaqueous fixative Pheochromocytoma PTAH for cross-striations Wash tissue in water after fixation in Zenker’s Combines w/ amino group Nonadditive fixative lec.mt 04 |Page | 285
pH >6.0 (formalin) Universal solvents Soft paraffin Weigert’s hematoxylin Natural resins (mounting) Formalin-alcohol Churukian-Schenk technique Masson-Fontana Muscle biopsies Paraffin sections Zamboni’s PAF Glutaraldehyde 10% NBF Paraformaldehyde Warthin-Starry stain Iris diaphragm Substage condenser Pathology Cornelius Celsus Littoral cells Hoffbauer cells Cancer Biohazards Mercuric chloride According to the presence of a mordant According to the presence of differentiator According to the resultant color Vital staining
Neurons
Prevent brown microcrystalline deposits in H & E stain Both dehydrates & clears For thick sections Not easily decolorized w/ acidic solutions Inherently acidic Microincineration Substance that can bind silver but need a chemical reducer Substance that can both bind & reduce silver Isopentane at -150’C If isopentane is low, dust muscle w/ talc then freeze in liquid nitrogen Naphthol AS-D chloroacetate esterase Specimens may remain indefinitely Specimens may be removed after 2-4 hrs pH 7.2-7.4 Pure polymer of formaldehyde Calibrate pH meter to 7.0 Increase amount of light Adjust to focus the image of the substage diaphragm Greek: Pathos = suffering 1st to describe the 4 signs of inflammation Splenic macrophages Placental macrophages Latin: Cancrum = crab Infectious agents Contaminated solutions Contaminated specimens Corrode all metals except for the nickel alloy Monel Methods of Staining a. Direct staining = w/o mordant b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue & the dye a. Progressive = w/o differentiator/decolorizer b. Regressive = w/ differentiator/decolorizer *1’ stain = acidic (decolorizer: basic) *2’ stain = basic (decolorizer: acidic) a. Orthochromatic = “ortho”: correct/same | same color = dye & tissue b. Metachromatic = “meta”: after/change | different color = dye & tissue Selective staining of living cells a. Intravital stain = injection of dye animal body - Ex. Lithium, Carmine, India ink b. Supravital stain = staining of cells immediately after removal from the animal body. Examples are: - Neutral red = Best vital dye - Janus green = mitochondria - Trypan blue - Nile blue - Thionine - Toluidine blue Functional cells of the CNS Nerve fibers: a. Dendrites (Greek: “Tree”) = conduct impulses to the cell body lec.mt 04 |Page | 286
b. Axons = conduct impulses away from the cell body Criteria for the diagnosis of Marked progesterone effect normal pregnancy At least 50% of intermediate cells in clusters At least some typical pregnancy cells present <30% of matured superficial cells Doderlein-filled dirty BG Staining solutions used in a. Hematoxylin = dark purple to black Pap’s staining method b. OG-6 = orange w/ a hint of green (Macroscopic) c. EA 36/50 = olive green w/ a hint of brown & red EA 36/50 Components: a. Eosin Y b. Bismarck brown c. Light green SF # in EA designates the proportion of SF ♫ Affinity: OG-6 Matured superficial cells Keratinizing malignant cells ♫ Cytoplasmic: Bright orange to yellow orange ♫ Affinity: EA 36/50 Immature vaginal cells (parabasal, intermediate) ♫ Cytoplasm color: Transparent blue to gray to brown hue Presently, the Bethesda system divides squamous cell abnormalities into 4 categories: ASCUS Atypical cells of undetermined significance L-SIL Low-grade squamous intraepithelial lesion H-SIL High-grade squamous intraepithelial lesion SCC Squamous cell carcinoma Description Bethesda 2001 Papanicolau Normal (-) for intraepithelial lesion/malignancy Class I Atypical ASCUS Class II HPV L-SIL Class II Mild dysplasia L-SIL Class II Moderate dysplasia H-SIL Class III Severe dysplasia H-SIL Class III Carcinoma in situ H-SIL Class IV Invasive carcinoma Invasive carcinoma Class V Microtome Usual Description Tissues embedding in Microtome Knives Length Plane concave 25 mm One side flat, other Less concave: celloidin-impregnated tissues Sliding is concave More concave: paraffin embedded tissues
Biconcave
120 mm
Plane wedge
100 mm
Carmine Best Carmine Mucicarmine
Both sides are Paraffin concave Both sides are Frozen sections straight Hard, tough tissue specimen (paraffin) Chromatin stain Carmine + Aluminum chloride = For glycogen Carmine + Aluminum hydroxide = For C. neoformans and mucin
Base-sledge Rotary Rocking Rotary Sliding Base-sledge
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Picrocarmine Carmine + Picric acid = for neuropathological studies Duke’s staging for neoplasia One of the most frequently applied for staging individual tumors of the rectum Biopsy Biopsy Excision and exam (living subject) Preferred: perform the biopsy at the periphery of the tumor (advancing tumor margin) Types of Biopsy Exfoliative cytology Desquamated cells Sex hormonal status in females Sex chromatin phenotype Excisional biopsy Complete removal of a lesion Most reliable Incisional biopsy Removal of part of a lesion/small piece of tumor directly incising the tumor capsule Preferred for large tumors that can’t be excised completely Needle biopsy Aspiration of fluid Bite biopsy Small pcs of tumor are removed w/ special forceps Cutaneous biopsy Skin fragments Punch biopsy For specimens >2mm embed in a single paraffin block Shave biopsy Curettage specimens Wedge biopsy Specimen is subdivided w/ a razor blade Marginal excision Shell-out end
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