AMCA MHTC cheat sheet

10% neutral buffered formalin (NBF) is approximately 4% formaldehyde w/v; phosphate buffer holds pH near 7.0-7.4 and limits formalin pigment.

Recommended fixation volume is at least 10:1 fixative-to-tissue ratio; thin sections (2-3 mm) fix faster than thick blocks.

Formalin fixation rate is roughly 1 mm per hour at room temperature; dense tissues such as uterus and bone fix more slowly.

Standard fixation time for routine small biopsies is 6-24 hours; overfixation can mask antigens and harden tissue.

Isotonic saline is the preferred transport medium for fresh tissue sent for flow cytometry or cytogenetics; formalin cross-links DNA and protein.

Bouin solution contains picric acid, formalin, and acetic acid; picric acid is explosive when dry and requires moist storage.

Decalcification uses acids (e.g., formic, hydrochloric) or EDTA chelation; EDTA is slower but preserves antigenicity and morphology better.

Specimens must be labeled with patient identifiers and orientation; small biopsies are wrapped in lens paper or biopsy bags to prevent loss.

Processing sequence: dehydration in graded alcohols, clearing in xylene or substitute, infiltration with molten paraffin.

Graded alcohol series ascends (e.g., 70%, 80%, 95%, 100%) to prevent tissue distortion from osmotic shock.

Xylene is the standard clearing agent because it is miscible with both alcohol and paraffin; it is toxic and requires ventilation.

Paraffin infiltration typically occurs at 56-60 degrees C; higher temperatures increase tissue hardening and antigen loss.

Vacuum and heat accelerate processing; pressure processing reduces time but risks tissue overheating and shrinkage.

Embedding orients tissue so the cut surface faces the block face; multiple pieces are aligned in one plane for serial sections.

Cassette thickness and block trimming determine section depth; overtrimming can exhaust small biopsies.

Incomplete processing shows as soft, wet, or opaque areas; re-processing may be needed before sectioning.

Routine paraffin sections are cut at 3-5 micrometers; thin sections (2-3 micrometers) suit small biopsies and immunohistochemistry.

A clearance angle of 5-10 degrees between knife and block prevents rubbing; too low causes chatter, too high causes compression.

Section floating bath is set near 40-45 degrees C; too hot causes tissue expansion and artifacts, too cold causes wrinkles.

Chatter (vibration marks) results from loose knife, loose block, hard tissue, or excessive cutting speed.

Compression (thick-thin alternation) is reduced by slower cutting, sharp blade, and adequate block cooling.

Frozen sections are cut at 4-6 micrometers at approximately -20 degrees C; colder tissue shatters, warmer tissue smears.

Serial sections are mounted on charged or coated slides (e.g., poly-L-lysine, silane) for special stains and IHC.

Section thickness is verified by micrometer setting and visual assessment; uniform ribbon indicates consistent cutting.

Hematoxylin stains nuclei blue-purple (basophilic); eosin stains cytoplasm and collagen pink (eosinophilic).

Progressive hematoxylin uses controlled stain time; regressive hematoxylin overstains then differentiates in acid alcohol.

Bluing of hematoxylin occurs in alkaline solution (e.g., ammonia water or lithium carbonate) after differentiation.

PAS stains glycogen and mucins magenta; diastase digestion distinguishes glycogen (removed) from mucin (retained).

Masson trichrome stains collagen blue or green, muscle red; useful for fibrosis and muscle architecture.

Gordon and Sweets or Gomori silver stains reticulin fibers black; nuclei are counterstained.

Ziehl-Neelsen (acid-fast) stain detects mycobacteria as red bacilli against a blue or green background.

Prussian blue stains hemosiderin (iron) blue; Perls reaction depends on acid release of ferric iron.

Formalin is a known carcinogen and irritant; use in a ventilated area with gloves, goggles, and formalin-safe waste disposal.

Xylene is flammable and neurotoxic; store in approved cabinets and use local exhaust ventilation.

Universal precautions treat all human tissue and body fluids as potentially infectious; wear gloves and lab coat.

Sharps (blades, needles, glass) go in rigid sharps containers; never recap or bend blades by hand.

Chemical spills are managed with spill kits and SDS guidance; know location of eyewash and safety shower.

Biological waste is segregated and autoclaved or incinerated per local regulations; formalin waste is collected separately.

Needlestick or splash exposures require immediate washing and reporting to occupational health.

Daily QC includes checking stain quality with control slides and documenting reagent lot numbers and expiration dates.

Poor H&E staining (pale nuclei) suggests exhausted hematoxylin, inadequate bluing, or over-differentiation.

Floaters in sections come from contaminated water bath, dirty knife, or carryover; change bath water and clean instruments.

Tissue folding or wrinkles indicate poor floating-bath temperature or rushed mounting; refloat or re-cut.

Formalin pigment (brown-black deposit) forms in acidic, unbuffered formalin; remove with saturated alcoholic picric acid.

Bubbles or cracks in blocks indicate incomplete infiltration or rapid cooling; re-embed if necessary.

Troubleshooting follows a systematic check: fixation, processing, sectioning, staining, and reagent integrity in sequence.

Microtomes require daily cleaning of blade and stage; lubricate moving parts per manufacturer schedule.

Rotary microtome knife angles and blade holders must be aligned to prevent chatter and uneven sections.

Tissue processors need regular cleaning, wax changes, and verification of reagent concentrations and temperatures.

Embedding stations maintain molten paraffin at 56-60 degrees C; temperature monitoring prevents block defects.

Reagents are labeled with preparation date, lot, and expiration; discard when QC fails or expiration passes.

Staining instruments and automated platforms require daily function checks and periodic calibration.

Water baths and floatation tables are cleaned daily to prevent cross-contamination and contamination artifacts.