Section Cutting

section cutting - types, principles & trouble shooting

1. Section Cutting Dr Tamil Nila

2. Microtomy It is the means by which tissue is cut into thin sections & attached to a surface of a slide Performed on Tissue blocks embedded in paraffin wax & frozen sections Instrument : Microtome

3. Microtomes Mechanical device designed for accurate cutting of thin, uniform tissue sections for microscopic examination An advancing mechanism moves the block towards the cutting tool for a predetermined distance The tissue moves vertically towards the cutting tool Thickness is graduated in microns A ribbon of sections is produced Ideal thickness : 4 – 6 microns

4. Parts of a microtome Knife holder with base Anchors the knife holder to the stage Knife holder Blade clamp Knife tilt Face plate Coarse handwheel – for vertical motion (up and down) Micron adjustment Safety lock Advancement hand wheel – forward and backward movement

5. Types of Microtome Rocking microtome Rotary microtome Sledge microtome Sliding Microtome Freezing microtome

6. Rocking Microtome Oldest Knife is fixed Block moves through an arc Advantages Cheap, reliable, low maintenance Very small blocks can be cut Disadvantages Size of block that can be cut is limited Sections are cut in a curved plane It is to be fitted to avoid movement while cutting

7. Rotary Microtome Basic Mechanism : rotation of the hand wheel by 360 degrees Most commonly used Knife is fixed and the tissue moves vertically Mechanism to advance the block Retracting Non retracting Types Manual Semi Automated Fully automated

8. Rotary Microtomes Advantages Cuts thin sections 2 -3 microns Large number of sections Cuts all type of tissues Heavier & stable Large blocks can be cut Cutting angle can be adjusted Perfectly flat sections Disadvantages Repetitive motion disorder in histotechnicians Greater chance to cut finger

9. Sledge Microtome Specimen is held stationary Knife slides across the top of the specimen Advantages Very large & hard blocks of tissue Very stable & not subject to vibration 24 cm length knife requires less honing Angle of tilt can be changed Freezing stage is available Disadvantages Slower Thin sections are difficult to produce

10. Sliding Microtome Knife moves horizontally against a fixed tissue block Advantage Cuts both celloidin embedded & paraffin embedded sections Best results for cutting frozen sections Advantages Diagnosis in minutes Enzyme studies can be done Freezing Microtome

11. Microtome knives Types Heiffor knife Larger knives Classification Metal knives Steel Razor blades Non metallic knives Glass Diamond

12. Standard Steel knives Plano concave Used in sledge & rotary microtomes Very concave is used for Celloidin work Plane –Wedge Most common Easy to sharpen Biconcave Used in rocking & sledge microtome Less rigid & prone to vibration Wedge Originally designed for cutting frozen sections Tool Edge for very hard tissues Tungsten carbide at edge

13. Parts of metal knives Heel of the knife Angle between cutting edge & end of the knife nearest to handle Toe of the knife Angle between cutting edge & end of the knife farthest from handle Honing guide/ knife back sheath Gives angle while honing & stropping Handle

14. Microtome knife angles Wedge angle Angle between the sides of the knife Facet angle Angle between the actual cutting edges Greater than the wedge angle Clearance angle or tilt angle Angle between the block face and lower cutting facet of knife Only knife angle that can be adjusted Cutting angle Angle between the block face & upper cutting facet of the knife Rake angle 90 degree – angle of upper cutting facet of knife Larger rake angle easier cutting

15. Trimming the block At least 2mm of wax is left surrounding the tissue on all 4 sides Exposes the embedded tissue Edges of the block should be parallel

16. Fixing the block on the microtome Heat a flat spatula ( 1 – 2 inches) in a Bunsen flame Melt the wax by placing block on spatula Press the block on the holder & wait for it to harden

17. Technique Place the microtome on a sturdy table Insert knife & screw tight Mount the block within the clamp of the holder Adjust tilt of the knife = 2 – 6 degree Adjust the block holder – wax block almost touching the level of the knife Tighten all the adjusting screws

18. Technique Bevel of the knife face is parallel to the cutting motion Section thickness = 10 – 15 microns Operate until complete sections are cut Reset section thickness to 4 – 5 microns Ice can be applied to the surface of block -> wiped -> cut Right hand operates the microtome Left hand holds sections away from the knife Gentle breathing on the block removes creases

19. Technique Ribbon formation Heat formation between the block & knife Sections adhere to each other Sections are held with Blunt forceps Camel hair brush Fingers Sections are floated on a warm water bath Collected with slides

20. Fixing the sections to slides Water bath method 25 – 30 cm in dm 8 – 10 cm in depth Black colored water baths To see the creases in the sections easily Thermostatically controlled Temperature is 5 – 10 degree Celsius below the melting point of paraffin wax

21. Water bath method Sections lifted from the microtome & transferred to the surface of water One end followed by other end of the tissue section is gently lowered onto the water bath Use dissecting needles to iron out the creases Albuminised slide is dipped obliquely While withdrawing one end of the ribbon is attached to the slide Reposition the section on the slide if necessary Slide is marked immediately

22. Water bath method Slides in upright position to drain excess water Mounted slides are placed in an incubator at 37 degree celcius for 1 hr For albuminised slides Place in 60 degree inside oven x 2 hrs To dry & coagulate the albumin Disadvantages Bacterial contamination Floaters

23. Adhesives Mayer’s glycerol albumin 1 volume or glycerol 1 volume of egg white Pinch of thymol Starch paste Chrome gelatin Poly – L – lysine 3 – Aminopropyltriethoxysilane Araldite Charged or plus slides

24. Hot stage/ Plate method Metal top heated to 45 – 50 degrees Slide placed on the stage Flooded with distilled water Sections are floated When sections are completely flat, remove the plate Drain excess water

25. Warmed slide method Same as hot stage After the creases are removed Slide is held over a Bunsen burner for a seond Place in oven for drying

26. Why creases appear? Block wasn’t cold when cut Compression of wax by a blunt knife Poor bevel on the knife Incorrectly tilted knife

27. Why sections disappear? Exposure to strong alkaline solution during staining Tissue containing blood & mucous Decalcified tissues CNS tissues Sections exposed to extreme temperatures

28. Problems faced Problems Reason Remedy Scored sections Knife edge is damaged with small nicks Hard particles in the tissue or paraffin Use different part of the blade or replace it Decalcify if needed Remove the hard particle with scalpel Curled sections Dirty Knife Blunt knife Too much Tilt in the knife Clean Sharpen Reduce the tilt Thick & thin zones in the same section Blade / block is loose in holders Too much tilt Hard tissue / Paraffin Dull blade Tighten by adjusting screws Reduce the tilt angle Use Softening agent Replace the blade / remove excess paraffin

29. Problems faced Problem Reason Remedy Crumbling of sections on cutting Blunt knife Soft wax Crystallised wax Sharpen or replace Apply ice to cool the cutting surface Rapidly cool the wax Curving of ribbon/ consecutive sections Block edges are not parallel to each other & to the knife edge Dull blade Excessive paraffin Tissue varying in consistency Trim block edges until parallel to each other Correct block edge parallel to knife Sharpen/ Replace the blade Trim the excess Re – orient the block Thick & Thin Sections Paraffin too soft for tissue Insufficient clearance angle Faulty microtome mechanisms Blade or block loose in holders Cool block with ice / re-embed in wax with higher melting point Increase clearance angle Lubricate microtome Tighten holders

30. Problems faced Problems reasons Remedies Sections not forming ribbons Paraffin too hard for sectioning Debris on knife Incorrect clearance angle Re-embed in lower melting point paraffin Clean with xylene Keep optimal clearance angle Sections attach to block on return stroke Insufficient clearance angle Edge of the blade/Block with debris Static electricity on the ribbon Increase clearance angle Clean knife with xylene Trim edges of the block Place static guards or dryer sheets Incomplete section Improper impregnation of tissue with paraffin Incorrect embedding Superficially cut sections Reprocess the block Re embed the tissue Reface the block or cut deeper

31. Problems faced Problems Reason Remedy Excessive compression Dull blade Soft paraffin Sharpen/ Replace Cold block face Sections expand or disintegrate on water bath Improper impregnation of tissue High temperature of water Re process Reduce the temperature Sections roll into coil Dull blade Rake angle too small Thick sections Sharpen or replace Reduce blade tilt Reduce thickness of section

32. Microtomy requirements for good histological sections High quality sharp blades Optimize knife tilt angle for each microtome & blade type Trim blocks carefully Avoid freezing damage to tissue block Use cold blocks Cut sections slowly/ gently with a uniform, slow rotation

33. References Bancroft’s theory and Practice of histological techniques Histopathology techniques & its management by Ramadas Nayak Basic & Advanced laboratory techniques in Histopathology & cytology by Pranab Dey