Paraffin What Histologists Need To Know and Understand

Paraffin: What Histologists Need To Know and Understand For Success Debbie Siena & Gary Wiederhold Stat. Lab Medical Products

Outline • • Overview of Paraffin Wax Chemical Basis of Paraffin Performance Parameters in the Histology Lab Common Problems Associated With Paraffin Related Products & Techniques Polymers & Wax Quality Control Equipment Maintenance Troubleshooting – Q and A

Key Milestones • 1830 – paraffin discovered in beechwood tars • 1836 – produced from crude oil • 1869 – Klebs – introduced infiltration & embedding • 1882 – 1 st ribbon from paraffin block from first rotary microtome • 1890 – 1 st floating section on warm water bath • 1905 – 1 st “thick” quality section produced

Requirements For Successful Paraffin Use in Histology • Rapidly Converted From Solid to Liquid Form Upon Heating • Permeates Tissue in a Liquid State • Solidifies Rather Quickly on Cooling • Provides Support For Tissue Cellular Structure • Ability to Cut Thin or Thicker Sections • Cost Effective

Why Use Paraffin In Histology? • • Easy To Use History Relatively Ease to Obtain Sections of Tissue Very Soluble & Easy to Remove With Common Laboratory Reagents • Labor Effective • Cost Effective Compared To Other Methods

Chemical Components Involved • • Paraffin Wax Polymers (Plasticizers) Microcrystalline & Natural Waxes Other Additives 1. DMSO 2. Oils 3. Color

Where Does Paraffin Come From? • Oil – Petroleum • Petroleum Companies “harvest” crude oil & process and separate into different components 1. Gasoline 2. Motor Oil 3. Kerosene/Lubrication Oil • Refinery will clean the wax & produce clear liquid or solid block • Plants – product a layer of wax for protection • Bees - Beeswax

Paraffin Wax • Majority Derived From Petroleum – Mixture of Solid Hydrocarbons • Simple Straight Chain Structure – Alkane Cn. H 2 n+2 (20 -40 carbon units) • Melting Point of Paraffin Is Vast - Ranges From High 40’s To About 70 o. C - Depends On How It Is Refined & How Much Stearic Acid Is Added

Paraffin Wax Structure • • • Simple Straight Chain Bonds Are Not Strong Chain Length Varied Odorless Petroleum Contains Waxy Substances and These Are Removed and Refined

Paraffin Structure – Crystal Formation • Pure Paraffin Is A Blend – Some Variation of Molecular Chains, Weak Covalent Bonding, and Vast Melting Point Range • Resulting In A Wide Range Of Crystals • Causes Paraffin to Harden Unevenly With Large & Small Crystals

Paraffin Shrinkage • Paraffin Can Shrink In Volume Up To 15% When Cooled • Tissue Is Compressed Similarly In The Paraffin Block • A Number Of Difficulties Arose During Sectioning And On The Heated Water Bath 1. Decompression 2. Expansion

Paraffin Remedies • Goal For Manufacturers Is To Provide A Homogeneous Molecular Composition • Limit The Number Of Blends And Other Factors • Upgrades In Refinement & Purity • Vendor Selection Is Significant

Quiz Time • What is Paraffin miscible (able to mix) with? 1. Absolute Alcohol? 2. Xylene? 3. Water? 4. Formalin? 5. Xylene Substitutes? 6. Stains?

Paraffin Wax Additives • Hydrocarbon Polymers (Resins) • Rubber Polymers • Microcrystalline & Other Natural Waxes • DMSO (Dimethyl Sulfoxide) • Anti-Oxidant

Polymers/Plasticizers • A Polymer is a Large Molecule Composed of Many Monomer Units (50 – 10, 000 repeating units) • Want a moderate molecular weight polymer to properly infiltrate & produce optimized crystals • These Resins Allow Improved Support Of The Cell & Cutting Thinner Sections 1. Increase Hardness Of Paraffin 2. Reduce Shrinkage 3. Also Have Affect On Melting Point of Paraffin & Prevent Their Fluctuation

Polymer - Resin • Thermoplastic Properties 1. Stable When Heated 2. Resistant to Discoloration 3. Soluble in Xylene/Xylene Substitutes 4. Must Have Softening Point That Allows It To Blend With Paraffin

Rubber Polymers • Increase elasticity • Increase stickiness so that sections will stick together to form a ribbon (desirable trait to a certain extent, if overdone, then customers may complain that paraffin is too sticky) • Can impede infiltration • Can oxidize (yellow) – extended times above paraffin melting point

Microcrystalline Wax • Modifies the crystalline properties of paraffin wax • Useful when some desired functional changes are needed such as flexibility, higher or lower melting point and increased opacity

DMSO • Analgesic treatment “Ben-Gay” for horses among other uses speeds up infiltration • Penetrates skin & tissue very readily • Not a regulated or listed carcinogen but you will hear that it is carcinogenic from some suppliers/customers • Smells like garlic & is hygroscopic

Multiple Polymers • It is Very Common Manufacturers Will Use Multiple Polymers • Multiple Resins; Resin & Rubber Polymer • There Are a Few Paraffin Products That Contain No Polymer Or Other Additives • Why? ?

Paraffin Matrix Low Polymer Content High Polymer Content

Block Color • Clear – pure paraffin and paraffin with little amount of polymer • Opaque – paraffin with a varying degree of polymer

Paraffin With Color?

Chemical Characteristics of Paraffin • • Refractive Index Viscosity Melting Point Colorimetry

Definitions • Refractive Index - determines how much light is bent, or refracted, when entering a material. • Viscosity - a measure of its resistance to gradual deformation by tensile stress. For liquids, it corresponds to the informal concept of "thickness". • Melting Point - is the temperature at which it changes state from solid to liquid at atmospheric pressure. • Colorimetry - technology used to quantify and describe physically the human color perception. - APHA, Saybolt tests

Viscosity • Increased molecular weight of paraffin increases the overall viscosity • Increase viscosity means an increase infiltration and cooling time • Examples being 10 – 20% polymer content • Especially difficult with fatty or dense tissues

Basic Paraffin Mixture Formula Chemicals Percentage Paraffin Wax Balance Polymer(s) 1 -20 Antioxidant 1 -2 Microcrystalline Wax (optional) 0 -5 Other Additives (DMSO, Color, etc. ) <1 (optional) Blend and Filter

What Type of Paraffin Is This?

Tissue Infiltration & Embedding

Tissue Processing-Infiltration • Infiltration-Latin word to saturate or fill • Histologically speaking-permeation and subsequent saturation of tissue by specific media resulting in tying together of tissue constituents, forming a matrix which prevents tissue structure disruption during microtomy

Paraffin infiltration- What happens in the tissue processor – Top-Air (arrows) being pulled from the tissue interstices by the action of vacuum – Middle-Long chain paraffin wax hydrocarbons (small dashes) entering and filling voids (interstices) left by the effects of vacuum – Bottom-Compacted paraffin wax hydrocarbon molecule (dark areas) due to vacuum followed by pressure

Paraffin… What is it? • Paraffin is a blend– simple hydrocarbon chains will penetrate first – long polymer chain needs time to fully infiltrate

Infiltration Time • Dependent on the size or molecular weight of each component • Paraffin with no additives will penetrate fast due to simple carbon chains • Adding branched carbon chain waxes or polymers increases the time of infiltration proportional to its size • Larger the molecule the higher the viscosity and thus slower rate of penetration By Ilmari Karonen - Own work by uploader. The source code of this SVG is valid. This vector image was created with Inkscape. , Public Domain, https: //commons. wikimedia. org/w/index. php? curid=6681265

Infiltration times • Problem-Turn around time needs to be reduced • Solution-cut down on processing time • Outcome-wrinkles on harder tissues due to long polymer chains not infiltrating

Complete Infiltration is a necessity Incomplete Infiltration Complete Infiltration

General Guide • Following this guide MP Temp Amount /size of polymers Cuttingmicrons Infiltration time Hardness 52˚-55˚C Least/smallest 4 -5 1 -2 hours Soft 56˚-58˚C ↓ ↓ 2 -4 hours Medium 60˚-62˚C Most 2 Min 4 hours Hardest

Specimen Types • Harder vs. Softer Type of Tissues Soft or Hard Bones, Uterus Hard 60 -62 Bones infiltrate better at higher temperatures. Helps prevent the tissue from popping out of a too soft paraffin block Skin and Breast Medium 56 -58 Fat content in the dermis has the most tendency to collapse during cutting, harder paraffin can give more support but heat over 60 -62 may have coagulant effect on fat which results in poor cutting Brain, Eyes, Soft G. I. , Liver, spleen 52 -55 Small and bloody tissues have the most tendency to literally cook in the infiltration step of processing. Artifact can be seen as dry parchment powder, H&E stains darker on outside than the inside Animal Tissues 52 -55 Low fat content, tissue can become dry and brittle, long infiltration adds to the effect Soft Temperature Comments

Paraffin Selection • Most labs have adopted a paraffin that is in the mid-range, not too hard and not too soft Infiltrating paraffin-Soft Mid. Range 55˚-58˚ Embedding Paraffin- Hard

INFILTRATING and EMBEDDING PARAFFIN INFILTRATING PARAFFIN • Used in Tissue Processors • Pro: – Infiltrates quickly • Con: – Too soft for cutting thin sections – 2 or more paraffins to store and inventory EMBEDDING PARAFFIN • Used for Embedding • Pro – May be able to cut thinner • Con: – May not be recommended for use in tissue processors – Additional Inventory

How to Evaluate Quality of Paraffin Impregnation Moisture Exposure Quality of Impregnation Processing Remedial Step 5 seconds Extremely poor (all specimens turn white) Requires drastic processing schedule changes in every step. (change solutions. Increase time in all steps by 30 minutes each 10 seconds Very poor (90% of the specimens turn white) Requires drastic processing schedule changes in most steps (change solutions. Increase time in all steps by 30 minutes each) 20 seconds Poor (50% of the specimens turn white) Requires moderate processing changes in most steps (change solutions. Increase time in all steps by 15 minutes) 45 seconds Good (20% of the specimens turn white) Requires minor changes (usually an increase of 15 minutes in the last dehydrant, clearant and paraffin 2 minutes Excellent (5% of the specimens turn white) Requires no changes

Embedding

Specimen Embedding • Embed-operation which results in the “fixation” or setting of a tissue specimen in a firm medium • Keeps the tissue intact during microtomy – Very important in determining the quality of microscope slides

IMPORTANCE OF RAPID CHILLING Room Temperature Zero Degrees C Zero degrees C Smaller Crystals support tissues better and cut better than larger crystals

Embedding • Tissue should be centered in the block so that a margin of paraffin surrounds it. – Makes it easier to separate sections on water bath – Makes it easier to center section on slide – Makes it easier for ribbons to form when cutting

What is the issue?

What is the Issue? • Fatty Tissue, mushy • Processing has failed to remove lipid from areas of the specimen and these areas have not been properly infiltrated with wax and are unsupported for cutting. • This is commonly with large breast specimens that have been processed using too short a cycle • Reprocessing, using a schedule of sufficient length, should produce blocks that can be sectioned

What is the Issue?

What is the Issue? • The solvent has not been displaced by wax • Re-infiltration of the specimen with wax may produce blocks that can be sectioned – because specimens like this often contain traces of water, complete reprocessing may be the best option.

What is the Issue?

What is the Issue?

What is the Issue? • Paraffin infiltration was not complete, May be undissolved lipids or incomplete infiltration with paraffin • Central area where cells are swollen and demonstrate typical “blue hue” • Retained solvent is one of the causes of this problem where tissue has had insufficient dehydration and clearing and the clearing solvent has not been replaced with paraffin • Reprocessing may help but morphology may be permanently affected

What is the Issue? • The tissue is not completely dehydrated and/or cleared and is therefore not properly infiltrated • (the wax could not penetrate due to the presence of water). Reprocessing should help • Another possibility is that the specimen has been properly dehydrated and cleared but has had insufficient exposure to wax to produce complete infiltration – Melt down the block and apply additional infiltration.

What is the Issue?

What is the Issue? • Wax on the specimen surface has been allowed to solidify before positioning the specimen in the embedding mould. – Careful re-embedding would overcome this problem. • Extreme under-processing can also produce this effect as the specimen shrinks when solvent evaporates from the blockface, causing separation of the specimen surface from the wax – Thorough reprocessing can overcome this problem.

Block Chilling • Chilling hardens the wax to better match the hardness of the infiltrated tissue • -15˚C freezer may be used but “dry” cold can causes cracks at the tissue/wax interface – May be difficult to get cohesive sections, particularly from difficult blocks • A refrigerated plate can be used and if its surface is wet it is most effective (0 - 4˚C) • Placing the block face in contact with the surface of melting ice is an effective means of chilling for difficult blocks • Pressurized cold spray can be used directly on the block face however this must be done carefully because can cause cracking

Paraffin Rotation Schedule • Xylene and Paraffin: • Xylene and Paraffin-rotated and/or discarded every 3 days (2 x week) if cycled with half of more of its volume of cassettes • • Xylene and Paraffin-rotated and/or discarded every four days if cycled with less than half of more of its volume of cassettes

QA/Safety • If paraffin melts during transportation-the wax itself is not affected and will perform in usual manner • Avoid temperatures over 60ºC in your tissue processor as that may affect tissue morphology • Rotate solutions according to your laboratory SOPs • Record tissue processor solution changes • Train all users of the tissue processor in maintenance and operation procedures

QA/Safety • Handle hot wax carefully • Paraffin shavings considered contaminated by CLIA inspector – Red bagged – Self closing lids • Dispose of wax according to local, state, territorial, providencial and national regulations– Contains xylene • Paraffin is a petroleum based product

Questions