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

2. 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

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

4. 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

5. 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

6. Chemical Components Involved
Paraffin Wax
Polymers (Plasticizers)
Microcrystalline & Natural Waxes
Other Additives
DMSO
Oils
Color

7. 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

8. Paraffin Wax
Majority Derived From Petroleum – Mixture of Solid Hydrocarbons
Simple Straight Chain Structure – Alkane
CnH2n+2 (20-40 carbon units)
Melting Point of Paraffin Is Vast
- Ranges From High 40’s To About 70oC
- Depends On How It Is Refined & How
Much Stearic Acid Is Added

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

10. 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

11. Paraffin Shrinkage To 15% When Cooled
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

12. 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

13. Quiz Time What is Paraffin miscible (able to mix) with?
Absolute Alcohol?
Xylene?
Water?
Formalin?
Xylene Substitutes?
Stains?

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

15. 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

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

17. 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

18. 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

19. 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

20. 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??

21. Paraffin Matrix
Low Polymer Content
High Polymer Content

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

23. Paraffin With Color?

24. Chemical Characteristics of Paraffin
Refractive Index
Viscosity
Melting Point
Colorimetry

25. 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

26. 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

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

28. What Type of Paraffin Is This?

29. Tissue Infiltration & Embedding

30. 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

31. 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

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

33. 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 uploaderThe source code of this SVG is valid.This vector image was created with Inkscape., Public Domain,

34. 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

35. Complete Infiltration is a necessity
Incomplete Infiltration

36. General Guide Following this guide MP Temp Amount /size of polymers
Cutting-microns
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

37. Specimen Types Harder vs. Softer Type of Tissues Soft or Hard
Temperature
Comments
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 may have coagulant effect on fat which results in poor cutting
Brain, Eyes, G.I., Liver, spleen
Soft
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
Low fat content, tissue can become dry and brittle, long infiltration adds to the effect

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

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

40. 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

41. Embedding

42. 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

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

44. 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

45. What is the issue?

46. 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

47. What is the Issue?

48. 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.

49. What is the Issue?

50. What is the Issue?

51. 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

52. ??

53. 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.

54. What is the Issue?

55. 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.

56. 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

57. 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

58. 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

59. 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

60. Questions