MIDTERM-SPC-MLS-2B-HISTOPATH-LEC.pdf

This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 1: PRE-ANALYTICAL FACTORS AND GROSS DESCRIPTION MIDTERM | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD...

This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 1: PRE-ANALYTICAL FACTORS AND GROSS DESCRIPTION MIDTERM | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE ○ requisition form and specimen container I. Pre-Analytical Factors label must match A. Pre-Analytic Fixation Unlabeled, mislabeled, and inappropriately B. Specimen Reception identified specimens (last resort: DNA identification) C. Specimen Accessioning Leaking specimen containers II. Gross Examination Absent clinical data or history, and other necessary A. Materials for Gross Examination info B. Specimen Categories 1. Specimen for Gross Description ○ one of the basis of the pathologist in only reading or diagnosing the patient 2. Specimen Excluded from Mandatory Submission C. SPECIMEN ACCESSIONING III. Describing Specimens and Gross Description IV. Sectioning SPECIMEN ACCESSIONING V. Other Specimen Considerations 1st and most important step in histopath outside the tissue processing procedures I. PRE-ANALYTICAL FACTORS ○ the rest of the process will be affected, if there is mislabeling PRE-ANALYTICAL FACTORS Specimens are given a unique identification Factors being identified in order to know or have a number that will identify each specimen for each better result in performing or for the overall patient processing of the tissue ○ for easier retrieval of specimens of the slides, as well as the tissue blocks A. PRE-ANALYTIC FIXATION ○ It will depend on the protocol of the hospital PRE-ANALYTIC FIXATION Indicating codes may be used for the following: All parts to be examined must be initially fixed Surgical, Autopsy, Cytology before the actual gross examination Sample Format of Accession Number: Indicating ○ Gross examination must not be performed Code-Year-ID Number of Specimen to a fresh sample; the sample must be ○ E.g. #S94-12345 prefixed (preserved) Earlier fixation → better tissue preservation Improper fixation → impede tissue processing Observe proper tissue-to-fixative ratio (1:20) ○ Most common fixative in the laboratory is 10% formaldehyde 3-5mm thick tissues: fixed for 6-48 hrs ○ The longer the fixation time, the better 5mm thick tissues and large tissues (such as Avoid serial accessioning of similar specimen types limbs): sectioned prior to fixation, or else, fixation to reduce mix-up of specimens, and will not be complete and may occur only at the cross-contamination periphery of the tissue ○ E.g. gastric biopsies are interspersed with ○ To allow proper fixation of the sample other tissue types B. SPECIMEN RECEPTION SPECIMEN RECEPTION Specimens must be put in a container labeled with patient’s name and specimen source/site, time and date of collection, name of the surgeon, and with pathology requisition form ○ Ideal setup → placed in between of the same/similar specimen type to avoid CRITERIA FOR REJECTION OF SPECIMEN confusion Discrepancies between requisition form and specimen labels DEOMAMPO | SPC MLS 2B | 1 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com II. GROSS EXAMINATION B. SPECIMEN CATEGORIES GROSS EXAMINATION SPECIMEN CATEGORIES Consists of describing the specimen and placing Specimens only requiring transfer all or parts of it into a plastic cassette, in from container to tissue cassette preparation for tissue processing One of the basis of pathologists’ diagnosis Where the pathologist will choose a representation of the tissues, most especially if the tissue is large Ideally, it must be wrapped in a filter in size A paper to ensure that the sample is Involves selection of elements that appear to be of clinical significance for histologic examination still present until the last processing (infiltration) Examples A. MATERIALS FOR GROSS EXAMINATION ○ Endometrium ○ Breast core biopsies MATERIALS FOR GROSS EXAMINATION ○ Colonic series 1. Cutting Tools ○ Scissors Specimens requiring transfer but ○ Forceps with standard sampling, counting, ○ Blade Holders weighing or slicing ○ Blade B Examples ○ Small lipoma (made of fatty tissues) ○ Small skin biopsy ○ Cervical LLETZ Simple dissection required with sampling needing a low level of diagnostic assessment and/or The ideal chopping board is white — to clearly see the C preparation tissues Example ○ Prepuce 2. Gross Table or Gross Workstations ○ Gallbladder ○ Sink ○ Haemorrhoids ○ Tabletop ○ Appendix ○ Water supply Dissection and sampling required needing a moderate level of ○ Irrigation system assessment ○ Fume extraction/ventilation system Example ○ Water disposal unit D ○ Pigmented skin lesions ○ Large intestine (Crobin’s) ○ Skin with markers ○ Salivary gland tumour Specimens requiring complex dissection and sampling methods E Example ○ Thyroid (medullary Ca) ○ Breast cancer ○ Testis (seminoma) ○ Uterus (endomet, Ca) DEOMAMPO | SPC MLS 2B | 2 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com Exempted for routine microscopic review: ○ Accessory digits Excess finger/toe will be removed and will be submitted to the histopath lab for description only ○ Bunions (aka hallux valgus) & hammer toes Bony bump that forms at the joints of the base of the big toe ○ Extraocular muscle from corrective surgery ○ Inguinal hernia sacs in adult ○ Nasal bone & cartilage from rhinoplasty ○ Prosthetic breast implant 1. SPECIMENS FOR GROSS DESCRIPTION ONLY SPECIMEN FOR GROSS DESCRIPTION ONLY There are specimens received in the histopath lab but are not subjected for further processing; for gross description only Are removed in the body not because of an abnormality, but because of some instances where the tissue/part must be removed in the body Sent in the lab for disposal Disease is not histologic level DEOMAMPO | SPC MLS 2B | 3 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ○ Prosthetic heart valves without Foreskin (from circumcision) attached tissue IUDs (Intrauterine Device) ○ no soft tissue attached ○ form of contraceptive for female Medical devices ○ catheters, gastrostomy tubes, stents, and sutures ○ have not contributed to the patient’s illness, injury, or death Middle ear ossicles Orthopedic hardware and other radiopaque medical devices ○ Tonsils and adenoids from children ○ provided that there is an alternative policy not benign or malignant type for documentation for these types of samples during surgical removal Placentas ○ do not meet with the criteria of the hospital or laboratory for examination Rib segments or other tissues ○ removed only for purposes of gaining surgical access but is not suggestive of ○ Umbilical hernia sacs in children any form of malignancy Saphenous vein ○ harvested from a coronary artery bypass Skin or other normal tissue removed ○ such as cosmetic surgery, reconstructive procedures, as long as there is no lesion or any tumor or malignancies Therapeutic radioactive sources Normal toenails and fingernails ○ Varicose veins III. DESCRIBING SPECIMENS AND GROSS DESCRIPTION GROSS DESCRIPTION 1. Identify the specimen. Note and verify all anatomical structures. ○ Type of organ/ tissue ○ Left or right 2. SPECIMENS EXCLUDED FROM 2. Identify orientation markers used by surgeons, if MANDATORY SUBMISSION available ○ Inks - used to identify and orient the SPECIMEN EXCLUDED FROM MANDATORY specimen’s components, distinguish SUBMISSION samples, for embedding instructions Bone donated ○ Nicks - indicates laterality ○ usually submitted in the Bone Bank ○ Sutures - represented by LL: long lateral; Bone segments or SS - short superior ○ usually submitted in the Bone Bank Cataracts I. INKING ○ cloudy vision Purpose: to accurately and faithfully Dental appliances and teeth transmit information to allow accurate and ○ with no attached tissue reliable microscopic assessment of this Fat margin ○ removed by liposuction ○ Resection margins Foreign bodies ○ Embedding instructions ○ bullet or other medico-legal evidence; will ○ Orientation be given directly to the law enforcement ○ Distinguish between samples personnel ○ Identify the cut surface DEOMAMPO | SPC MLS 2B | 4 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ○ Acetic acid ○ Weight - of intact organs are rounded to Will set as an instruction for the embedding the nearest 0.1g process Only required for sample that are ○ Ink dots instruct the embedder to hyperplastic such as the uterus, embed the tissues a certain way endocrine, breast, kidneys India ink is used IV. SECTIONING SECTIONING Taking a representative sample of the tissue Indicate number of sections and blocks on the gross description ○ Indicate also the number of cassettes used Specimen must fit easily into the standard cassette, which measures 3 x 2.5 x 0.4cm Thickness: not more than 0.3cm to allow for closing of cassette and fixative penetration Different color scheme used to identify When possible, edges of tissue should be squared its orientation: ○ Blue (Superior) A. SECTIONING ○ Green (Inferior) Cut serially about 2mm thick to look ○ Black (Posterior or Deep) for small lesions. Lesions are then ○ Red (Medial) sampled for histologic exam. Filter ○ Yellow (Anterior) SMALL paper may be used in wrapping SPECIMENS small sample ○ Orange (Lateral) ○ If 2 or 3 colors are needed, the preferred color to be used is black, blue, and orange Acetic Acid is used to remove the ink Cut an interval of 1cm thickness (termed as breadloafing) to ensure II. SUTURING that pathologic areas or tumoral The pathologist will identify the location of areas are identified the suture LARGE SPECIMENS “12 o’clock” marker Cystoprostatectomy - an en bloc excision of all cancer bearing tissues in the pelvis “3 o’clock” marker including the bladder, the prostate 3. Describe all notable characteristics: LABELING ○ Type of specimen Paper tags are embedded in the cassette ○ Shape Labeled with accession number using (lead) ○ Color pencil. Markers and pens will dissolve upon ○ Texture/Consistency processing ○ Odor - noted only if the smell is obvious If printed, dot matrix must be used ○ Dimensions - (length, width, depth) are Original containers with specimen are saved until rounded to nearest 1.0cm. For multiple case is signed out (backup evidence in cases of pieces, indicate size of the largest piece discrepancies) DEOMAMPO | SPC MLS 2B | 5 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com 3. Dermatologic Specimen ○ Vertical orientation is always maintained (using markers) ○ Punch biopsies are submitted whole ○ Tissues greater than 4mm are dissected ○ Skin ellipses: serially cut along the short axis at 2 to 3 mm interval. The two most distal sections or tips are submitted in two separate cassettes. Remainder is submitted in one or more cassettes 4. Eyes ○ Inject fixative first then gross 5. Hard Tissues ○ Wash in running water then immerse in TSE softeners Example of result ○ Must undergo the process of decalcification 6. Hollow Structures ○ Must be cut open longitudinally and fixed with cottons inside (cottons soaked in fixative) ○ Ex. stomach Sample Gross Description 7. Lymph Nodes ○ Most important component of tumor V. OTHER SPECIMEN CONSIDERATIONS resections because they are essential for prognosis and planning therapeutic options OTHER SPECIMEN CONSIDERATIONS ○ Should be received fresh and not 1. Brain immersed in formalin ○ Brain is fixed first before grossed ○ Node is bivalved, and entirely submitted ○ Tied at the Circle of Willis and suspended ○ Sentinel lymph nodes: usually the first Circle of Willis - circulation where lymph node to be involved during blood flows or the pathway of the metastasis. Entirely submitted. However, blood to supply oxygen to the large specimens may be bisected, and brain as well as the surrounding submitted in one or two cassettes structures 8. Mastectomy ○ Must not touch side of container to avoid ○ Note for weight, size of breast and axillary deformity dissection, skin ellipse, nipple scar, basal ○ Recommended: In 10% NBF (Neutral margins Buffered Formalin) for 2-3 weeks 9. Pediatric Specimen 2. Colon Cancers ○ Additional processes such as IHC, flow ○ Polyps: base (the area where cautery cytometry, cytogenetics and molecular arteries are located) is always inked genetics is often done. These may require Small polyps: bisected and fresh, frozen, or specially processed places in one cassette tissues Large polyps: sides are trimmed 10. Specimen with Tumor away from the stalk, and stalk is ○ Identify: placed in a separate cassette Site & size of tumor different sides = placed Location & structure invaded by on same cassette; middle tumor parts = in different Vascular invasion cassettes Presence of lymph node Distance from resection margin Large polyps DEOMAMPO | SPC MLS 2B | 6 of 6 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 2: FIXATION MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE C. FACTORS AFFECTING FIXATION I. Fixation A. Effects of Fixatives Fixative of Choice B. Characteristics of a Good Fixative ○ 10% Neutral Buffered Formalin C. Factors Affecting Fixation Morphologic criteria for dx have D. Types of Fixative been established based on E. Aldehydes Fixative Formalin-Fixed Paraffin F. Metallic Fixatives Embedded Specimen (FFPES) G. Picric Acid Fixatives H. Alcohol Fixatives Time I. Osmic Acid Fixatives ○ Must be performed as soon as possible; J. Heat Fixation 20-30 mins after blood supply is cut off II. Secondary Fixation Tissue-to-Fixative Ratio III. Washing Out ○ 1:10 or 1:20 (tissue to fixative ratio) IV. Difficulties in Improper Fixation Penetration Rate ○ Formalin: 1mm/hr (but slows down as it I. FIXATION goes deeper into the tissue) Thickness of Specimen What is fixation? ○ Larger → Longer fixation time, more ○ Defined as the killing, penetration, and fixative hardening of tissues ○ Light Microscopy: 2cm2 x 0.4cm ○ First and most critical step in tissue ○ Electron Microscopy: 1-2mm2 processing Tissue Components ○ Primary purpose: Preserve the ○ Longer Fixation time: morphologic and chemical integrity of the Fibrous Tissue cell in a life-like manner as possible Mucus → wash with NSS Fat → cut into thin slices → fixed A. EFFECTS OF FIXATIVES longer Hardens soft tissues in preparation for further Blood → flushed out with saline tissue processing ○ Shorter Fixation time: Render cells resistant to damage caused by Small of loosely textured tissues chemicals used in further processing pH Inhibit decomposition caused by bacteria and fungi ○ Optimal pH: 6 to 8 Minimize the risk of occupational infection ○ Use buffers Act as mordant for certain stains, thus promoting or ○ For Electron Microscopy: pH should hastening staining, or inhibit certain dyes match physiologic pH Reduce the risk of infections during handling and Temperature actual processing of tissues ○ Higher temp → faster fixation rate and autolysis B. CHARACTERISTICS OF A GOOD FIXATIVE Room temp to 45ºC Optimal Temp Cheap (routine) Stable 40ºC Tissue processors Safe Quick Up to 65ºC Microwave Inhibits bacterial decomposition processing Produce minimum shrinkage 0 - 4ºC Electron microscopy Rapid and even penetration 100ºC Tuberculosis Hardens the tissue Makes cellular contents resistant to further 60ºC Rapid biopsy processing Permit staining Osmolality ○ Hypertonicity → cell shrinkage Note: No single fixative has all the mentioned ○ Isotonicity and Hypotonicity → cell swelling characteristics. Each of them has its own advantages and ○ Thus, maintain tissues at slightly disadvantages. hypertonic solution (400-450 mOsm) AQUE | SPC MLS 2B | 1 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com Agitation, Vacuum ○ Concentrations: ○ Hastens fixation 100% - gas form 37% to 40% - stock concentration D. TYPES OF FIXATIVE (causes overhardening of the external surfaces of tissues) Based on Composition 10% - working solution; most ○ Simple commonly used Made of only one component ○ Usually buffered to pH 7 with phosphate ○ Compound buffer Consists of two or more components of fixatives ADVANTAGES DISADVANTAGES Based on Action ○ Microanatomical Cheap, readily available, Irritating to the nose and easy to prepare eyes (allergic rhinitis); General study of tissues w/o may cause Dermatitis structure alteration ○ Cytological Compatible with many If unbuffered, may reduce stains both basophilic and Nuclear eosinophilic staining pH ≤ 4-6 Glacial acetic acid has Penetrates tissue well Prolonged fixation may cause bleaching of the affinity to nuclear specimen chromatin Cytoplasmic Allows natural tissue color to be restored pH ＞ 4-6 HAc destroys mitochondria and Golgi EFFECT CAUSE REMEDY bodies White Prolonged - Filter ○ Histochemical paraformald storage - Add 10% methanol Preserves chemical constituents if ehyde (but dentures proteins cells and tissues precipitate thus unsuitable for EM) Formation of Unbuffered - Buffer or Methanol Microanatomical - 10% NBF Formic acid - 10% formol saline + - 10% Formol-Saline Mg++ / Ca++ carbonate in - Heidenhain’s Susa jar with marbles - Zenker’s Formalin Action of - Saturated alcoholic - Zenker-formol pigments formic acid picric acid (Helly’s) brown / with excess - 1% KOH in 80& ROH - Bouin’s black blood - Kardasewitch’s Method - Brail’s precipitate (70% ETOH & 28% Nuclear - Flemming’s with ammonia H2O2 70% glacial acetic acid ETOH & 28% ammonia - Carnoy’s water - Bouin’s - Newcomer’s 2. 10% Formol-Saline - Heidenhain’s ○ Saturated formaldehyde + 10% NaCl Cytoplasmic - Helly’s ○ Recommended for fixation of CNS tissues - Orith’s and general post mortem tissues for - Regaud’s / Molter’s - Formalin with histochemical examination Post-charming ○ Advantage: Ideal for Silver impregnation - Fleming’s w/o glacial staining technique acetic acid ○ Disadvantage: Slower; tissue shrinks Histochemical - 10% Formol Saline during alcohol dehydration - Absolute ethanol Remedy: Secondary Fixation - Newcomer’s 3. 10% Neutral Buffered Formalin (NBF) - Acetone ○ Formaldehyde + Na Dihydrogen Phosphate + Disodium Hydrogen E. ALDEHYDE FIXATIVES Phosphate ○ pH 7 1. Formaldehyde (Formalin) ○ Best general tissue fixative ○ Gas produced by oxidation of methanol AQUE | SPC MLS 2B | 2 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ○ Best for iron-containing pigments and F. METALLIC FIXATIVES elastic fibers which do not stain well after Susa, Zenker, or Chromate fixation Mercuric Chloride Zenker ○ Disadvantage: Longer to prepare, inert to Zenker-Formol (Helly’s) phospholipids and neutral fats Carnoy-Lebron ○ Fixation Time: 4 to 24hrs Heidenhain’s Susa B5 4. Formol-Corrosive (Formol Mercuric Chloride) Schaudinn’s ○ Saturated aq. Mercuric chloride + 40% Formaldehyde Chromates Chromic acid Regaurd’s/Muller’s ○ Recommended for routine post mortem Orth’s tissues and Silver Reticulum staining Potassium dichromate methods ○ Advantage: does not need washing, fixes Lead lipids ○ Disadvantage: Forms mercuric chloride MERCURIC CHLORIDE deposits Most common metallic fixative; 5-7% 5. Gendre’s (Alcoholic Formalin) Routine fixative of choice for preservation of cell ○ Has 95% ETOH, Picric acid, and GHAc detail in tissue photography ○ Advantage: good for microincineration Advantage: penetrates and hardens tissue rapidly techniques; Fixes sputum Disadvantage: Banned worldwide due to extreme 6. Hollande’s toxicity; marked cell shrinkage ○ For gastrointestinal (GI) tissues, prostate ○ Remedy: add acid biopsies, and bone marrow (BM) Precautions: 7. Glutaraldehyde ○ May form black precipitates of mercury ○ Made up of 2 formaldehyde residues except in Heidenhain’s Susa linked by 3 carbon chains; Container must ○ Remedy: Dezenkerization be refrigerated 0.5% iodine + 70% ETOH → H2O ○ For enzyme histochemistry and electron → 5% Na thiosulfate → H2O microscopy 1. Zenker’s Fluid ○ Advantage: more pleasant and less ○ HgCl2 + Potassium dichromate + glacial irritation compared to formalin acetic acid ○ Disadvantage: less stable and more ○ Good general fixative for adequate expensive than formalin preservation of all kinds of tissues ○ Concentrations: 2. Zenker-Formol (Helly’s Solution) ○ HgCl2 + potassium dichromate + strong 0.25% For immune electron formalin (40%) microscopy ○ For pituitary gland, bone marrow, & 2.5% For small TSE fragments blood-containing organs ○ Brown pigments are removed with 3% Most common saturated alcoholic picric acid or NaOH 3. Heidenhain’s Susa solution 4% For large TSE fragments ○ Su = sublimate; Sa = saure (acid) 8. Paraformaldehyde ○ HgCl2 + NaCl + TCA + glacial acetic acid + ○ Polymer of Formalin formalin ○ Powder in form, used in 4% ○ Skin tumor biopsy ○ Plastic embedding ○ Advantage: minimum cell shrinkage and ○ For ultrathin and electron microscopy tissue hardening due to counter-ba;ance 9. Karnovsky’s Paraformaldehyde / effect of acids and mercury Glutaraldehyde Acids: swelling ○ Acrolein in glutaraldehyde or formalin Mercury: shrinkage ○ For Electron Histochemistry & Electron ○ Does not produce black pigments Immunocytochemistry ○ Disadvantage: Weigert’s staining of 10. 40% Aqueous Glyoxal elastic fibers not possible ○ Advantage: no smudging of nuclei and 4. B-5 Fixative distortion of staining compared with ○ Recommended for hematopoietic and formalin lymphoid tissues ○ Disadvantage: reduced staining capacity ○ Fixation Time: 4-8hrs Remedy: increase staining time ○ Bone marrow biopsies AQUE | SPC MLS 2B | 3 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com 5. Shaudinn’s Fluid ○ Incorporated in compound fixatives ○ Recommended for making smears of loose ○ Solidifies at 17ºC cells on slides ○ Important for nuclear fixatives (precipitates nucleoproteins, chromatins) CHROMATE FIXATIVES ○ Destroys mitochondria and Golgi elements, 1. Chromic Acid 1-2% thus not for cytoplasmic fixation ○ Preserves carbohydrates; precipitates all protein H. ALCOHOL FIXATIVES 2. Regaud’s / Muller’s Fluid ○ For chromatin, mitochondria, mitotic Advantage: good for glycogen figures, golgi bodies, RBC and colloid Causes polarization of glycogen (granules will containing tissues move towards the poles or ends of the cells) ○ Disadvantage: prolonged fixation may Disadvantage: dissolves Fats and Lipoproteins lead to blackening of tissue pigment Effect: rapidly denatures and precips CHONs, Remedy: Wash in running tap preserves nuclear stains H2O before dehydration 1. Carnoy’s Fluid 3. Orths’ Fluid ○ Most rapid tissue fixative (1-3 hrs) ○ Study of early degenerative processes and ○ Fixing brain tissues for rabies diagnosis necrosis; Rickettsiae and other bacteria ○ Fixes Nissl granules and cytoplasmic ○ Preserves myelin granules 4. Potassium Dichromate 2. Ethanol (70 - 100%) ○ Preserves lipids & mitochondria at pH 4.5 ○ Enzyme studies; does not fix but preserves to 5.2; cytoplasm, chromatin and glycogen chromosome are fixed 3. Methanol / Wood Alcohol (100%) ○ Corrosive, thus avoid skin contact ○ Dry and wet smears, Bone Marrow smears, bacterial smears LEAD FIXATIVES 4. Isopropanol Preserves acid mucopolysaccharide ○ Touch prep smears to be Wright-stained Disadvantage: Prolonged standing → formation of 5. Newcomer’s Fluid insoluble lead carbonate ○ For mucopolysaccharide (12-18 hrs) ○ Remedy: add drops of acetic acid to 6. Gendre’s (Alcoholic Formalin) dissolve residue ○ For sputum (4 - 18hrs) G. PICRIC ACID FIXATIVES I. OSMIC ACID FIXATIVES Normally used in strong aqueous solution (1%) Pale yellow powder in water (6% in 20ºC) Glycogen demonstration Ultrathin section in Electron Microscopy Highly explosive when dry Disadvantage: very expensive, very volatile, inhibits ○ Remedy: add distilled H2O or 0.5% - 1% hematoxylin saturated alcohol Tissue-to-fixative ratio: 1:5 Yellowing effect 1. Flemming’s Solution w/ GAC ○ Remedy: immerse in Li2CO3 with ○ Nuclear fixative (24 - 48hrs) 70%ROH → water → 70% ethanol → 5% ○ Most common osmic acid fixative Na thiosulfate → water ○ Effect: permanently fixes fat 1. Bouin’s ○ Advantage: needs less amount of fixative ○ For embryo and pituitary biopsies, and 2. Flemming’s Solution w/o GAC tissues to be stained with Masson’s ○ Cytoplasmic Fixative (24 - 48hrs) Trichrome 3. Trichloroacetic Acid ○ Advantage: minimum cell shrinkage and ○ Incorporated into compound fixatives tissue hardening due to counter=balance ○ Marked swelling effect on tissues effect of glacial HAc (swelling) and picric ○ Poor penetration thus for small pieces of acid (shrinking) tissues or bones ○ Disadvantage: poorly penetrates large ○ Weak decalcifying agent, this has tissue, thus limited to small fragments of softening effect on dense fibrous tissues tissues 4. Acetone 2. Brasil’s Alcoholic Picroformol ○ Use at ice cold temperature (-5 to 4ºC) ○ Better and less messy than Bouin’s ○ For water-diffusible enzymes 3. Glacial Acetic Acid (Phosphatase, Lipase) AQUE | SPC MLS 2B | 4 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ○ Fixes brain tissue (for rabies) ○ Disadvantage: Dissolves fat, evaporates rapidly J. HEAT FIXATION Involves thermal coagulation of tissue proteins for rapid diagnosis Frozen tissue sections & bacteriologic smears 10 - 15mins thickness of tissue Microwave Technique ○ Increases movement of molecules to accelerate fixation, staining, and decalcification Advantage: Can preserve neurochemical substances of tissues Disadvantage: Cannot penetrate tissues that are 10 - 15mm in thickness Optimum temperature: 45º - 55ºC II. SECONDARY FIXATION To improve the demonstration of particular substances To make special staining possible (secondary fixatives as mordants) To ensure further and complete hardening and preservation of tissues Post-Chromatization ○ Technique whereby a primary fixed tissue is placed in aq. Solution of 2.5% - 3% potassium dichromate for 24hrs III. WASHING OUT Process of removing excess fixatives Tap water ○ Excess chromates, formalin, osmic acid 50% - 70% Alcohol ○ Picric acid Alcoholic Iodine ○ Mercuric fixatives IV. DIFFICULTIES IN IMPROPER FIXATION Failure to arrest autolysis Tissues are brittle and too hard ○ Too long fixation time Shrinkage and swelling of cells ○ No balance Tissue soft consistency Presence of artifact pigments Removal of soluble substances Incomplete result in other procedures AQUE | SPC MLS 2B | 5 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 3: DECALCIFICATION MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE B. UNIQUE CHARACTERISTICS I. Decalcification A. Characteristics of a Good Decalcifying Stable Agent Easily available B. Unique Characteristics Inexpensive C. Factors Affecting Rate of Decalcification Easy to prepare II. Four Types of Decalcifying Agents A. Acid B. Chelating Agents C. FACTORS AFFECTING C. Ion Exchange Resin RATE OF DECALCIFICATION D. Electrophoresis Concentration I. DECALCIFICATION Less concentrated = slow acting Tissue-to-volume Ratio Purpose: 1:20 Removal of calcium and lime salts ○ 1 parts tissue and 20 parts decalcifying Done after fixation and before dehydration and agent impregnation Temperature Calcium might interfere with accurate evaluation Must be well regulated and examination Mechanical Agitation ○ Calcium should be removed in the tissue It is the key to a rapid acting or rapid decalcifying of samples because it can potentially the sample interfere with the accurate evaluation and Size and Consistency of Tissue Sample examination of the metabolic bone Large, thick, rigid = longer decalcification process disease, differentiate mineralized bone from osteoid, and morphometric III. FOUR TYPES OF DECALCIFYING AGENTS measurement of the tissue sample Significance: A. ACID Facilitate normal cutting of tissue in sectioning Prevent obscuring microanatomical detail of tissue Organs that require decalcification: Bones ○ Calcium is present in the bones Tuberculous Lungs ○ Calcified lungs due to TB bacteria Arteriosclerotic Vessels ○ Accumulated lipids, calcium, cellular debris in the lumen of the blood vessels Nitric Acid Teeth 5-10% Routine or most common decalcifying agent In order to facilitate good cutting and evaluation of your Fastest decalcifying agent in the market now tissue samples, they should undergo removal of calcium Imparts yellow coloration to the tissue sample and lime salts first. through nitrous acid formation ○ Remedy: A. CHARACTERISTICS OF A GOOD DECALCIFYING Add urea or Sodium AGENT Thiosulfate/sulfate Do not cause cell destruction 70% ROH (DHD) Rapid, cheap, inexpensive ROH is an alkyl alcohol ○ Should also render best and accurate Variation/s: result ○ 10% Aqueous Nitric Acid Solution Safe ○ Formol-Nitric Acid Readily available ○ Perenyi’s Fluid Acts as tissue softener Nitric acid + chromic acid + ROH FRONDA | SPC MLS 2B | 1 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ○ Phloroglucinol-Nitric Acid Common brand: Fastest agent (simple or Cal-Ex and Versene compound) ○ Contains Na2EDTA Simple solution = 1 ingredient/component only C. ION EXCHANGE RESIN Compound solution = 2 or more ingredients/ components Hydrochloric Acid Provides good nuclear staining at 1% Slower and causes more distortion compared to nitric acid Variation/s: ○ Von Ebner’s Fluid Principle: For teeth and small bones Increase solubility HCL + 36% NaCl + distilled water ○ Uses formic acid with TSE : Formic acid of Formic Acid 1:20-30 Good for routine decalcification of post-mortem Remove calcium ions from formic acid containing research tissues, small pieces of bone and teeth, decalcifying solutions ISH staining Not recommended for hydrochloric acid and nitric If the formic acid contains a large amount of nitric acid fluid containing mineral acids acid, it produces better nuclear staining and less Decalcification Extent: tissue distortion Can be measured using physical method by simply Variation/s: bending or poking the tissue sample and/or X-ray ○ Formic Acid method ○ Formic Acid-Sodium Citrate Solution Trichloroacetic Acid D. ELECTROPHORESIS Best for small bone spicules Good nuclear staining Slow, weak Chromic Acid (Flemming’s Fluid) Best for minute bone spicules Sulfurous Acid Best for minute pieces of bone Weak Citric Acid Citrate Buffered Solution pH: 4.5 Principle: Attracting calcium ions going to the cathode part of B. CHELATING AGENTS the agarose gel Advantage: Time is shortened due to heat and electrolytic reaction produced in the process ○ Shorter time is needed to remove calcium ions or calcium salts ○ It utilizes 88% formic acid Principle: Procedure: Use of other salts to form complexes with Calcium Used for small bone fragments salts for ease of removal Decalcification Extent: Procedure: Best Measured Using: Utilized in Immunohistochemistry and enzyme ○ Physical or Mechanical Test staining with the help of electron microscope ○ X-ray or Radiologic Method Duration: ○ Chemical Method 1-3 weeks for small tissue samples 6-8 weeks for longer and dense bones pH: 7-7.4 ○ Should be maintained at this pH in order to work properly FRONDA | SPC MLS 2B | 2 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 4: DEHYDRATION MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE ○ Kasi pag ang dehydrating solution is I. Dehydration capable of removing stains, we will not be II. Characteristics of Ideal Dehydrating Solution able to appreciate the cellular details of the III. Commonly Used Dehydrating Agents tissue samples A. Alcohol Non-toxic, and not a fire hazard 1. Ethanol ○ As much as possible 2. Methanol 3. Butyl Alcohol B. Acetone III. COMMONLY USED DEHYDRATING AGENTS C. Dioxane D. Cellosolve A. ALCOHOL E. Triethyl phosphate F. Tetrahydrofuran ALCOHOL IV. Additives to Dehydrating Agent Done in ascending grades to avoid distortion of tissue: 70% ROH 90% ROH 100% ROH I. DEHYDRATION (absolute alcohol) For delicate tissues, start at 30% DEHYDRATION ○ Must start with weak alcohol. Why? Removal of fixative and water from the tissue If strong and initial concentration siya nag start, it will tend to shrink or tend to brittle TAKE NOTE!!! Prolonged dehydration in less From fixation, it will undergo decalcification - if the than 70% alcohol, tissue sample contains a high amount of calcium salts or migh tend to macerate lime salts. Then after decalcification, dehydration is Macerate - ma almost mag the next procedure pino pino siya, ma turn into pieces. Same consistency sa Dehydrating time must be brief as much as pastil possible and at a tissue-to-fixative ratio of 1:10 Initial alcohol concentration depends on the size ○ 1 part tissue and 10 parts dehydrating and nature of the tissue and the fixative used solutions. 37°C will hasten dehydration rate (for urgent ○ Since most fixatives are aqueous exams) solutions, placing the fixed tissue in a ○ You can speed up the process by simply melted paraffin wax will not achieve exposing your sample with alcohol at 37°C impregnation because paraffin wax and To ensure complete dehydration: water do not mix, hence dehydration is ○ Add at least ¼ deep layer of anhydrous required and must be done. Cu2SO4 at bottom of container, and cover with filter paper II. CHARACTERISTICS OF IDEAL DEHYDRATING ○ Bluing of copper sulfate crystals indicates SOLUTION full saturation of dehydrating fluids with water. Thus alcohol must be changed with Rapid Action, with minimal tissue shrinkage a fresh solution. and distortion Should not evaporate fast ETHANOL ○ Kasi pag mag evaporate fast siya, ang Best dehydrant under alcohol because it is tendency ma retain lang din ang water. fast-acting, mixes with water (it is miscible with Hindi magsama ang water during the water) and many organic solvents, and penetrates evaporation process tissues easily Able to dehydrate even fatty tissues Not poisonous and not very expensive ○ Kasi sa fatty tissues, nandoon ang medyo Good thing is it is clear, colorless, flammable mahirap ipa evaporate na liquid particles Should not harden tissues excessively METHANOL Should not remove stains Toxic Ideal for blood and smear preparation fixation TRIPOLE | SPC MLS 2B | 1 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com BUTYL ALCOHOL However, its disdavantage is it produces minimum Slow-acting shrinkage Made up of plant and animal microtechnique Not commonly available in the market ○ Mahirapan ka bumili ng triethyl phosphate NOTE: ○ And good thing if istore mo siya for a Tissue is passed through a series of progressive longer period, wala siyang side effects sa changes in alcohol concentration. In an ascending tissue. It can be stored for months without manner of concentration. causing tissue distortion kumbaga. B. ACETONE F. TETRAHYDROFURAN (THF) ACETONE TETRAHYDROFURAN Fastest dehydrant; will only require 30 minutes to 2 Dual in action: can act as a dehydrant and clearing hours to complete the dehydration process agent Highly flammable, evaporates fast Advantages: Not recommended for routine dehydration ○ Staining procedures are improved purposes ○ Miscible in water and paraffin wax ○ Kasi sa sobrang bilis niya, di na Disadvantages: makasama ang water sa pag evaporate ○ Dissolve fats ○ Aside from that one, urgent biopsies lipids ○ Eye and skin irritant (conjunctival irritation) are removed if we will be using acetone. ○ The vapors, or the fumes can cause The limited for small tissues only because nausea, dizziness, headache, and they are volatile. anesthesia effect to the histotechnicians C. DIOXANE IV. ADDITIVES TO DEHYDRATING AGENTS DIOXANE ADDITIVES TO DEHYDRATING AGENTS Also known as Diethylene Dioxide 4% Phenol Has dual purpose: dehydrating and clearing agent Glycerol/alcohol mixture Graupner pure dioxane capable of interacting paraffin REFERENCES: Weiseberger’s Method ○ Wrapping tissue in gauze and suspension to a bottle containing dioxane with anhydrous calcium oxide to quicklime Expensive and extremely dangerous Miscible with water, melted paraffin wax, alcohol, and xylene, an example is your Graupner pure dioxane Vapor produces highly cumulative toxic action in man, so that’s why it is not recommended to utilize and should not be recycled, and it is also high risk for creating explosive peroxides D. CELLOSOLVE CELLOSOLVE Example would be Glycol Monoethyl Ether Rapid, prolonged storage does not cause distortion to tissues Toxic inhalation, skin irritant and combustible at 110-120F Alternative; propylene based glycol ethers E. TRIETHYL PHOSPHATE TRIETHYL PHOSPHATE Soluble in alcohol, water, ether benzene, chloroform, acetone, and xylene TRIPOLE | SPC MLS 2B | 2 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 5: CLEARING MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE IV. TYPES OF CLEARING AGENT I. Introduction to Clearing II. Purpose of Clearing A. ADVANTAGE AND DISADVANTAGES OF ALCOHOL III. Characteristics of Good Clearing Agent AGENTS IV. Types of Clearing Agents A. Advantages and Disadvantages of Alcohol ALCOHOLS Clearing Agents AGENT ADVANTAGES DISADVANTAGES B. Common Clearing Agents C. Others Clearing Agents XYLENE rapid, suitable Highly 30mins to for urgent flammable 1 hr biopsies > 3 hours, CLEARING Makes the tse makes tse hard transparent Not suitable for I. INTRODUCTION Miscible both in nervous tissues alcohol and and lymph FIXATION ➨ DECALIFICATION ➨ DEHYDRATION ➨ paraffin nodes CLEARING (DE-ALCOHOLIZATION) Cheap TOLUENE Miscible both in Slower than 1hr to 2hrs alcohol and xylene “In the process of Clearing, we can also term this one as paraffin More de-alcholization because we are removing alcohol, which Does not make expensive is the commonly used dehydrating agent during the process tse hard and of dehydration.” brittle Not II. PURPOSE OF CLEARING carcinogenic BENZENE For URGENT Carcinogenic 15mins to biopsies It may lead to 1. Removing dehydrant from tissue, and replacing 1hr Fastest aplastic anemia it with a fluid miscible to both dehydrant and clearing agent embedding agent. 2. Makes tissues “translucent” or transparent, B. COMMON CLEARING AGENTS hence the term clearing. 3. Most are flammable fluids and have low boiling CHLOROFORM (6-24 hours) points Tough tissues and large specimens 4. Excessive clearing may cause brittleness ○ Skin, fibroid and decalcified tissues (requires longer processing time) The main purpose of performing clearing is to Toxic to liver (prolong inhalation) create a bridge between the process of dehydration and infiltration. CEDARWOOD OIL (2-3 days) It is because our paraffin wax is not miscible or For smooth muscle, CNS compatible with our alcohol, so we need to (requires deeper penetration) remove the alcohol thru the process of clearing, Milky on prolonged storage and preparing our tissue for the next process, which is the infiltration. ANILINE OIL Prolonged exposure to clearing agents may result For insects, embryos, and delicate tissues to some respiratory problems. CLOVE OIL III. CHARACTERISTICS OF GOOD CLEARING AGENT Minimum shrinkage of tissues Slow, expensive and difficult to use It should be miscible with alcohol. (not usually used in the laboratory) It should be miscible with paraffin wax. It should not produce tissue shrinkage. CARBON TETRACHLORIDE It should make tissue transparent. Tough tissues and large specimens It should not dissolve aniline dyes. (same tse of choice to chloroform) It should not evaporate quickly in water bath ○ Skin, fibroid and decalcified tissues Similar to chloroform but cheaper Dangerous to inhale on prolonged exposure CALATRAVA | SPC MLS 2B | 1 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com TETRAHYDRFURAN Nontoxic but with offensive odor and should be used in a well-ventilated room This considered superior among other agents since it can process both DEHYDRATION AND CLEARING thus shorter processing time It has an offensive, ethereal odor so the room must be well-ventilated DIOXANE Causes greater shrinkage than xylene Dangerous, toxic to liver C. OTHER CLEARING AGENTS OTHERS GUM SYRUP & Used when clearing directly from GLYCERIN water ( as in a frozen section) OIL OF For skin and smooth muscle BERGAMOT OIL OF For skin ORIGANUM OIL OF Artificial oil, for delicate tissues WINTERGREEN CARBON For smooth muscle, has foul DISULFIDE odor CARBOL For friable tissues XYLENE TERPINEOL For delicate tissues like eyes PHENOL For smooth muscles HIGH TEST Excellent clearing agent AVIATION LEAD-FREE GASOLINE CALATRAVA | SPC MLS 2B | 2 of 2 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 6: IMPREGNATION & EMBEDDING MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE PARAFFIN WAX I. Impregnation ADVANTAGES DISADVANTAGES A. 3 Types of General Tissue Impregnation Rapid (24 hours) Prolonged 1. Paraffin Wax Impregnation Maybe cut with ease impregnation will 2. Celloidin Impregnation without undue cause excessive 3. Gelatin Impregnation distortion shrinkage II. Embedding Many staining Not recommended to A. Orientations procedures are fatty tissues B. Types of Molds permitted (ex. breast) C. Other Embedding Methods Overheated paraffin makes the specimen IMPREGNATION & EMBEDDING brittle I. IMPREGNATION IMPREGNATION (a.k.a. Infiltration) Process that removes the clearing agent Fills the tissue cavities Permeates tissue with a support medium Incomplete impregnation ➨ tissue airholes In this process, we are still using different changes to remove the clearing agent then eventually fill the Figures 1 & 2. Paraffin Wax in Solid and Liquid form tissue cavities. We need to fill the holes in order for us to create a support and it could be easier during sectioning. A. 3 TYPES OF GENERAL TISSUE IMPREGNATION 1. PARAFFIN WAX IMPREGNATION Paraffin wax is solid in form upon procurement thus we need to melt it first in order for us to have it in liquid form. Figure 3. Paraffin Oven Requires 2 or more changes of melted paraffin wax Melting point: 45°C, 52°C, 56°C, 58°C @RT (20- 3 Ways of Paraffin Wax Impregnation: 24°C) ➔ By manual processing ○ Upon performing, we need to use a hot ◆ This is what we are performing in the plate in order to maintain the liquid state of laboratory. In this illustration, there are 3 the paraffin wax since it quickly solidifies at changes of paraffin; 1 hour per room temperature. beaker/change.The changes will depend Never overheat; >60°C causes brittleness, on the protocol of the laboratory. shrinkage, hardening; destruction of lymph tissue Paraffin oven must be maintained 2-5°C above the melting point of wax Used pure ○ Wax must be filtered first using coarse filter paper such as Green’s No. 904 in oven at 2°C higher than the melting point of wax. ○ Reusable only once, but heat it first @ 100-105°C (remove water) ○ In the actual practice, it can be actually used a multiple of times until a cloudiness can be seen. CALATRAVA | SPC MLS 2B | 1 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ➔ By automatic processing ○ Harder than paraffin thus used with ◆ Our automatic processor is usually sliding/sledge-type microtome designed with 12 stations. The infiltration ○ Water insoluble but soluble in 95% will take place up at stations 11 & 12. ethanol, thus prior clearing is not needed ◆ The infiltration time will be faster because ○ But Cellosolve, and xylene may be used if there is constant agitation in the machine. indicated Hence, a shorter processing time. Water Soluble Waxes (Polyethylene glycol) ○ Melting Point: 38-42°C or 45-56°C ○ Ex. Carbowax - most common No need for dehydration and clearing thus sections are difficult to float out during the fishing method and mount Remedy: add soap to water or 10% PEG 900 in water Neutral fats and lipids can be Figure 4 & 5. Autotechnicon & Elliott bench-type demonstrated For enzyme histochemistry ➔ By vacuum embedding Used for URGENT ◆ Among these 3 processes, this type is the fastest. However, it is expensive because 2. CELLOIDIN IMPREGNATION we need to acquire first the machine itself, ◆ it can only perform the embedding/ A.k.a Colloidin infiltration process. Unlike with our Purified form of nitrocellulose/gun cotton automatic processing, from filtration down Specimen with large and hollow cavities which tend to infiltration. to collapse; hard and dense tissues; neurologic ◆ This is faster because it will remove the air tissues directly on our tissue thus allowing the Concentration: in 2%, 4%, 8% dissolved in equal infiltration to happen. parts of ether and alcohol - increasing ◆ 25-75% reduced time than the usual processing time. CELLOIDIN ADVANTAGES DISADVANTAGES Does not require heat; Very slow (days or less shrinkage week) Cutting of thicker Thin sections are tissues difficult to cut Recommended for Very volatile neurological tissues METHODS OF CELLOIDIN INFILTRATION Fixation & Dehydration ⬇ Figure 6. VEU (Vacuum Embedding Unit) Place tissue in ether-alcohol ⬇ Thin celloidin (2%) SUBSTITUTE FOR PARAFFIN WAX ⬇ Paraplast Medium celloidin (4%) ○ Melting Point: 56-57°C ⬇ ○ Mixture of pure paraffin and synthetic Thick celloidin (8%) plastic polymer (Dimethyl sulfoxide); more ⬇ elastic and resilient Remove specimen and put in a fresh thick celloidin Embeddol ⬇ ○ Melting Point: 56-57°C Keep in jar or desiccator until ether-alcohol evaporates ○ Less brittle, and less compressible ⬇ Bioloid WET: Store tissue block in ○ semisynthetic; for embedding of dyes 70%-80% alcohol - Wet Ester Wax Gilson’s Mixture (chloroform and cedarwood oil) - Dry ○ Melting Point: 46-48°C CALATRAVA | SPC MLS 2B | 2 of 5 Flexcil - The Smart Study Toolkit & PDF, Annotate, Note  This Document has been modified with Flexcil app (Android) https://www.fexcil.com ➔ Wet - for bones, brain, teeth II. EMBEDDING ◆ Store tissue block in 70%-80% alcohol ◆ The purpose of storing it in this a.k.a. Casting, Blocking, Molding concentration is to avoid dehydration and Placing the impregnated tissue into a mold with shrinkage of the tissue. embedding media, and then allowing the media to solidify ➔ Dry - for whole eye sections Orientation: Arrangement of the tissue in a precise ◆ Store tissue block in Gilson’s Mixture position in the mold during embedding (chloroform and cedarwood oil) Surface to be cut should be parallel to bottom of the mold Molds should bear the accession number ➔ Nitrocellulose/Low Viscosity Nitrocellulose ◆ Has lower viscosity, thus can be used in higher concentration, and rapid tissue penetration ◆ Advantages: Harder tissues blocks, thus thinner sections are possible ◆ Disadvantages: Explosive when dry due to nitrates That is why this is usually suspended with alcohol to maintain its liquid state Figure 7. Embedded Tissues ◆ It consists Plasticizer E.g. oleum ricini or castor oil A. ORIENTATIONS Needed to prevent tissue cracking Arrangement of the tissue in a precise position in in chrome mordanted tissues the mold during embedding This promotes plasticity as well as We need to follow a specific type of orientation for a flexibility in order to reduce specific type of tissue sample: brittleness. 3. GELATIN IMPREGNATION ➔ Tubular tissue: ◆ All layers in transverse sections Rarely used ◆ If not: we will only see the sides of the For histochemical, enzyme studies, and frozen sec. sample, not the layers Advantage: Water soluble (no dehydration and ◆ Ex. Fallopian tube, Appendix clearing needed) Disadvantage: may decay Tissue must be