Histopathologic Techniques - 03 Infiltration and Embedding

HISTOPATHOLOGIC AND CYTOGENETIC TECHNIQUES • Methods of Paraffin Wax Infiltration o Manual ▪ Carried out using paraffin oven (55-60C) • Oven keeps wax melted to efficiently fill up the tissue spaces; 2-5C higher than wax MP • Heat accelerates the process ▪ Requires 4 changes of wax at 15 minutes interval • Completes the entire process in one hour ▪ Place small tissues in a container (dissecting pan) and add paraffin wax. Place entire preparation in the oven o Automatic ▪ Requires autotechnicon (automatic tissue processor) ▪ Use of autotechnicon is rapid than manual • There is heat generated and constant agitation which accelerate processing time ▪ 2-3 changes of wax, more rapid o Vacuum ▪ Under negative atmospheric pressure inside the oven ▪ The most rapid method among the three ▪ Recommended for specimens with mucha ir ▪ Recommended for urgent biopsies, for specimens like lungs, brain, eyes, spleen and CNS ▪ Melting point for routine work: 56C • Substitutes for Paraffin Wax o Paraplast ▪ Not a pure paraffin ▪ mixture of highly purified paraffin and synthetic plastic polymers ▪ Melting point 56-57C; used for boensa nd brain specimens o Embeddol – similar to paraplast ▪ melting point of 56-58C o Bioloid - used for infiltrating eye specimens o Ester wax ▪ Wax with a much lower melting point (46-48C) ▪ Harder than paraffin wax and requires heavy duty type of microtome (sliding microtome) ▪ Soluble in alcohol which does not require clearing; only dehydration is required o Tissue mat – product of paraffin with rubber o Carbowax ▪ a water-soluble wax for enzyme histochemistry • advantage because it will eliminate dehydration and clearing process • disadvantage because tissues will be difficult to float out when cut; remedy: add soap to water or 10% polyethylene glycol 900 to promote floating out of sections ▪ no longer require dehydration and clearing INFILTRATION & EMBEDDING OUTLINE • • • Introduction Infiltration o Definition and Purpose o Infiltration Methods Embedding o Embedding Molds o What is Double Embedding? • INTRODUCTION • • • • Impregnation (Infiltration) is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the cavities, thereby giving a firm consistency to the specimen, and allowing easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components Embedding (Casting or Blocking) is the process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify. The medium used to infiltrate the tissue is usually the same medium utilized for impregnation, and for general purposes is known as Embedding Medium. Infiltration → Embedding → Sectioning INFILTRATION / IMPREGNATION DEFINITION & PURPOSE • • • • Infiltration - step in tissue processing involves removal of clearing agents from tissues to be replaced by medium hat will fill all cavities and tissue spaces Two reasons o To remove clearing agents from tissues o To fill up cavities and tissue spaces Tissue spaces/cavities must be filled up to give the tissue a firm consistency which is important to facilitate easy cutting Three general infiltration methods o Paraffin wax o Celloidin o gelatin INFILTRATION METHODS PARAFFIN WAX INFILTRATION • • • • • • • • Paraffin wax - medium used to fill up cavities/tissue spaces o Most commonly used infiltrating agent o Usually carried out in routine due to rapid processing Allows cutting of serial sections with ease Compatible with any staining procedures Must be carried out correctly and adequately Prolonged process may cause excessive shrinkage and tissue hardening Inadequate process may cause retention of clearing agent Not suited for some specimens (fatty tissues) o Requires heat which can dissolve fats Melting point for routine work: 56C CELLOIDIN INFILTRATION TRIMMING • • • • • • • • • • a.k.a. collodion uses celloidin as infiltrating agent A purified form of nitrocellulose soluble in alcohol & ether Do not require heat because highly flammable For tissues with large & hollow cavities that tend to collapse Very Slow, thin sections, sections for photomicrography are all difficult to obtain Procedure: Thin Celloidin → Medium Celloidin → Thick Celloidin 2-4% 4-6% 8-12% 5-7 days 5-7 days 3-5 days Methods of Celloidin Infiltration: o Wet Celloidin - Uses 70% alcohol for storage ▪ For infiltrating bones, brain, teeth, whole organs o Dry celloidin - Uses Gilson’s Mixture for storage ▪ For infiltrating eye specimens ▪ Gilson’s Mixture - chloroform + cedarwood oil Nitrocellulose Method (Low Viscosity Nitrocellulose) o Has lower viscosity compared to usual celloidin ▪ Can be used in higher concentrations o Another form of celloidin soluble in equal concentrations of ether and alcohol; highly explosive o Produces harder tissue blocks and allows cutting of thinner sections o Do not use heat o Tissues with LVN has tendency to crack ▪ Remedy: Add Plasticizers (Oleum ricini & castor oil) to prevent cracking of tissues cutting off excess paraffin wax to form a 4-sided prism EMBEDDING MOLDS • • • • • • 2 general types of embedding molds o Disposable o Non-disposable ▪ leukharts ▪ compound ▪ plastic ▪ tissue tek Leuckhart’s (L-mold) – consist of 2 L- shaped strips of heavy brass or metal o Requires assembling prior to use ▪ Inefficient for busy laboratories o Can produce blocks of different sizes Compound embedding - used when there is a need to embed more than 1 specimen Plastic embedding rings and base molds Tissue Tek Disposable molds (peel away, plastic ice trays, paper boat) o Paper boat - economical, easy to prepare o Plastic ice tray - requires smearing of inner mold with glycerin (to facilitate easy removal) o Peel away - can produce perfect blocks without trimming GELATIN INFILTRATION • • • • • • • Rarely used; required volume Recommended for frozen section and histochemical and enzyme studies Water soluble No longer require dehydration and clearing Tissue for processing should not be more than 2-3 mm thick Procedure o Place specimen in a container with 10% gelatin with 1% phenol for 24 hours o After 24 hours, transfer tissue in 20% gelatin with 1% phenol in the next 12 hours o After 12 hours, transfer tissue to fresh 20% gelatin solution with 1% phenol that is allowed to cool in fridge until infiltration and embedding (including immersion in 10% formalin for 12-24 hrs) are complete Add phenol to prevent growth of molds EMBEDDING/ CASTING/ BLOCKING • • • • • Placing infiltrated tissue in a mold containing the embedding medium which is allowed to solidify Requirements: embedding mold, medium, & infiltrated tissue o Paraffin wax - commonly used embedding medium o Plastic resins - for light microscopy Temperature of melted paraffin used for embedding: 5-10C above melting point Tissue block - product of embedding To allow solidification of block, there are two methods o Place preparation in refrigerator at -5C o Immerse it in cold water WHAT IS DOUBLE EMBEDDING? • • Infiltrating using celloidin then embedding using paraffin wax Different medium for infiltration and embedding o Usually, infiltration and embedding medium are same