12.5 Scrolls, RNase-Free Technique & Laser Capture Microdissection
Key Takeaways
- Scrolls are thick 5 to 10 micrometer curls cut directly into a nuclease-free tube, bracketed by hematoxylin and eosin bookend sections that confirm the target lesion is still present in the block.
- RNase-free microtomy requires a dedicated decontaminated area, a fresh blade for every block, nuclease-free water, and gloves changed between specimens, because ribonucleases are ubiquitous and heat-stable.
- Macrodissection scrapes a marked region from an unstained slide using a matching hematoxylin and eosin roadmap, and is the routine way to reach a 20 to 30 percent neoplastic cellularity threshold.
- Laser capture microdissection requires special membrane-coated slides, minimal or rapid staining, complete dehydration, and no coverslip, because a coverslip and aqueous mountant make capture impossible.
- Infrared systems melt a thermoplastic film onto the target and lift it off, while ultraviolet systems cut around the target and catapult or drop it into a collection cap.
1. Where the Histotechnologist Sits in the Molecular Workflow
When a molecular laboratory reports that a specimen "failed extraction" or that "tumor content was insufficient," the cause is almost always something that happened at the microtome. The ASCP BOC content outline places preparation for molecular testing under Embedding and Microtomy and marks it HTL only, listing three specific examples: RNase-free areas, scrolls, and laser capture microdissection.
2. Scrolls (Curls) for Nucleic Acid Extraction
A scroll, also called a curl, is a thick paraffin section cut directly into a labeled nuclease-free microcentrifuge tube rather than floated onto a slide.
Standard scroll protocol
- Face the block and discard the facing ribbon.
- Cut a leading hematoxylin and eosin bookend at routine thickness onto a slide.
- Cut the requested scrolls, typically 5 to 10 micrometers each, and drop them straight into the tube using clean forceps or a fresh applicator stick. The number of scrolls is set by the tissue surface area on the block face: a large resection may need two or three curls, a small core biopsy may need eight or ten.
- Cut a trailing hematoxylin and eosin bookend.
- Review both bookends. If the lesion is present on the leading slide but exhausted on the trailing slide, part of the collected material contained no tumor, and the cellularity estimate must be revised.
[!IMPORTANT] The bookend sections are not a formality. They are the only evidence that the material in the tube actually contains the lesion the molecular assay is being asked about, and they document how much target tissue remains in the block for repeat or reflex testing.
Why thicker sections
Scrolls are cut thicker than diagnostic sections because the goal is nucleic acid yield per section, not optical resolution. There is no water bath, no slide, and no staining, so the section thickness is limited only by what the microtome can cut cleanly.
3. RNase-Free and Contamination-Free Microtomy
Ribonucleases are present on skin, in dust, and on laboratory surfaces, and they are heat stable and refold after autoclaving. DNA carryover between blocks is equally damaging because modern sequencing panels amplify from very small inputs.
| Control | Practice |
|---|---|
| Dedicated area | A designated microtome and bench used for molecular cutting, physically separated from routine sectioning |
| Surface decontamination | Wipe microtome, forceps, and bench with a commercial RNase-inactivating reagent or 10 percent bleach followed by nuclease-free water or alcohol |
| Fresh blade | A new blade or a clean, previously unused blade facet for every block, never a shared facet |
| Water | Nuclease-free or molecular-grade water; ideally no flotation bath at all, using dry pickup or a disposable bath |
| Gloves | Changed between every block; hands never touch the block face or the section |
| Consumables | Nuclease-free tubes, filter tips, and disposable forceps or applicator sticks |
| Order of work | Cut low-tumor-burden and precious specimens before high-tumor-burden blocks to limit carryover risk |
4. Macrodissection: The Everyday Enrichment Method
Most tumor enrichment in a hospital laboratory is done by hand.
- The pathologist circles the target region on a hematoxylin and eosin slide and estimates neoplastic nuclei as a percentage of total nuclei.
- The technologist aligns unstained serial sections against the marked slide, transferring the outline to the back of the unstained slide.
- The region is scraped off with a sterile scalpel blade directly into a nuclease-free tube, using a fresh blade for each case.
Macrodissection routinely lifts a specimen from below an assay threshold to above it. Common minimum neoplastic cellularity requirements sit near 20 percent, with some panels requiring 30 percent or more.
5. Laser Capture Microdissection
When the target is a structure rather than a region, such as individual glomeruli, a few hundred tumor cells, or an amyloid deposit, hand scraping is not precise enough. Laser capture microdissection (LCM) isolates a microscopic population under direct visual control.
5.1 Slide preparation is the technologist's job
| Requirement | Reason |
|---|---|
| Membrane slides (polyethylene naphthalate or similar) or plain uncharged glass, per platform | The membrane supports the excised fragment; charged slides can hold tissue too tightly for capture |
| Minimal or rapid staining — rapid hematoxylin and eosin, methyl green, or cresyl violet | Long aqueous staining leaches nucleic acid and degrades RNA |
| Complete dehydration and air drying before capture | Residual moisture prevents film adhesion and causes incomplete cuts |
| No coverslip and no mountant | The laser and capture cap must reach the tissue surface directly |
| Sections about 5 to 8 micrometers | Thick enough for yield, thin enough for a clean laser cut |
5.2 The two platform families
| Platform | Mechanism | Notes |
|---|---|---|
| Infrared capture | A near-infrared laser melts a thermoplastic transfer film on a cap placed over the target; the melted polymer bonds the cells, and lifting the cap removes them | Gentle, no cutting beam through the tissue; well suited to small clusters |
| Ultraviolet cutting | A focused ultraviolet laser ablates a path around the target, and the isolated piece is either catapulted upward into a cap or dropped by gravity into a tube | Precise contour cutting; suited to larger or irregular structures |
5.3 Clinical applications
- Pure tumor populations for sequencing when tumor is admixed with abundant stroma or inflammatory cells
- Individual glomeruli for renal research and for proteomic analysis
- Amyloid subtyping, where deposits are captured and identified by liquid chromatography with tandem mass spectrometry
- Single-population transcriptomics, where contaminating cell types would obscure the signal
5.4 Verification
After capture, the operator inspects both the cap (target present) and the residual slide (target absent) to document that the intended cells were actually removed. That before-and-after check is the quality control record for the dissection.
A molecular laboratory reports that a colectomy specimen sent as ten scrolls contained almost no tumor DNA. Review of the case shows a leading hematoxylin and eosin bookend with abundant carcinoma and a trailing bookend showing only normal colonic wall. What does this indicate?
A technologist prepares sections for laser capture microdissection using the routine workflow: charged slides, a full hematoxylin and eosin stain, dehydration, and coverslipping with resinous mountant. Why will capture fail?
Which practice is essential when cutting scrolls for RNA-based fusion testing but is not required for routine diagnostic microtomy?