ASCP HT cheat sheet

10% neutral buffered formalin (NBF) contains 4% formaldehyde buffered to pH 7.0-7.4; it cross-links proteins and penetrates tissue at roughly 1 mm per hour at room temperature.

Minimum fixative-to-tissue volume ratio is 10:1; blocks are sliced 2-3 mm thick so fixative reaches the center within a practical time frame.

Routine NBF fixation is 6-24 hours for small biopsies and 24-48 hours for large specimens; fixation time is documented when IHC or molecular testing is planned.

Cold ischemia time runs from devascularization in the patient to immersion in fixative; prolonged warm ischemia degrades DNA, RNA, and phosphorylated epitopes.

Alcohol fixatives such as 95% ethanol are used for cytology smears and preserve glycogen, but they coagulate proteins and cause more shrinkage than formalin.

EDTA decalcification at neutral pH chelates calcium slowly and preserves antigenicity and nucleic acids better than strong acids such as nitric or hydrochloric acid.

Acid hematin (formalin pigment) forms in acidic or unbuffered formalin, especially in blood-rich tissue, and is removed with saturated alcoholic picric acid.

Mercury-based fixatives (B5, Zenker) require post-fixation in alcohol and pigment removal with iodine followed by sodium thiosulfate.

10% NBF is markedly hypertonic relative to plasma; for electron microscopy use 2-3% glutaraldehyde in buffer at approximately 300-400 mOsm/kg.

Standard processing sequence: dehydration in graded ethanol (70-100%), clearing in xylene, then infiltration with paraffin at 56-60 degrees C.

Incomplete dehydration leaves the clearing agent milky and causes poor paraffin infiltration, section crumbling, and holes in the tissue.

Routine H&E sections are cut at 3-5 micrometres; small biopsies and kidney at 2-3 micrometres; IHC and FISH sections at 4-5 micrometres.

The flotation bath is set 5-10 degrees C below the paraffin melting point (about 40-45 degrees C); overheated water over-expands and distorts sections.

Clearance angle between the knife bevel and block face is 3-8 degrees; too little produces chatter, too much causes uneven or skipped sections.

Cryostat chamber temperature for routine frozen sections is approximately minus 20 to minus 25 degrees C, with sections cut at 4-6 micrometres.

Decalcification endpoint is confirmed by the ammonium oxalate chemical test or by radiography before the specimen is processed.

Blades are changed frequently; tungsten carbide blades are used for hard tissue and glass or diamond knives for resin and electron microscopy sections.

Section adhesives such as Mayer's egg albumin, poly-L-lysine, or APES are required for IHC, bone, and other sections prone to detachment.

Hematoxylin is a basic (cationic) dye oxidized to hematein and complexed with aluminum; the complex stains nucleic acids blue-purple.

Eosin is an acidic (anionic) dye applied at slightly acidic pH, staining cytoplasm, collagen, and red cells pink after hematoxylin bluing.

Differentiation with 1% acid alcohol removes excess hematoxylin; bluing in Scott's tap water or lithium carbonate converts red alum-hematin to blue.

Progressive hematoxylin (Gill, Mayer) stains to endpoint, while regressive hematoxylin (Harris) deliberately overstains and is then differentiated.

PAS relies on periodic acid oxidizing 1,2-glycol groups to aldehydes that react with Schiff reagent, giving magenta staining of glycogen, mucin, and basement membrane.

Every special stain requires a positive control slide; PAS also uses a diastase-digested section to separate glycogen from other PAS-positive substances.

Prussian blue (hydrochloric acid plus potassium ferrocyanide) demonstrates hemosiderin; Masson trichrome stains collagen blue or green and muscle red.

Silver stains require oxidation and a reduction step: GMS uses chromic acid oxidation and stains fungal walls black against a green counterstain.

Gram stain sequence is crystal violet, iodine mordant, alcohol decolorizer, and safranin counterstain, leaving gram-negative organisms pink.

Fixation in 10% NBF for 6-24 hours is preferred; both under-fixation and over-fixation reduce antigen detection and alter staining intensity.

HIER uses citrate pH 6.0 or Tris-EDTA pH 8.0-9.0 with a pressure cooker, steamer, or microwave; PIER uses enzymatic digestion with trypsin or pepsin at 37 degrees C.

Endogenous peroxidase is blocked with 3% hydrogen peroxide before primary antibody; endogenous biotin is blocked when avidin-biotin detection systems are used.

Each run includes a positive tissue control, a negative reagent (isotype) control, and an internal control element where one exists in the section.

DAB produces an insoluble brown precipitate resistant to alcohol, whereas AEC and Fast Red are alcohol soluble and require aqueous mounting media.

ASCO/CAP HER2 interpretation: IHC 3+ is positive; FISH is positive when HER2/CEP17 is 2.0 or greater or HER2 copies are 6.0 or greater per nucleus.

Estrogen receptor is reported positive when 1% or more of invasive tumor nuclei stain; the same threshold applies to progesterone receptor.

Molecular testing from FFPE depends on controlled cold ischemia and fixation time; 10 micrometre curls are cut with clean technique and an H&E slide guides tumor enrichment.

OSHA formaldehyde permissible exposure limit is 0.75 ppm as an 8-hour time-weighted average, with a 2 ppm short-term exposure limit.

Xylene is flammable and neurotoxic; it is dispensed in a fume hood with nitrile gloves, eye protection, and no ignition sources nearby.

Spilled formalin is absorbed with pads and neutralized with ammonium hydroxide or sodium bisulfite before disposal as hazardous waste.

A 16-section safety data sheet must be accessible for every hazardous chemical present in the laboratory.

Daily quality control includes documented temperature logs for processors, embedding centers, flotation baths, refrigerators (2-8 degrees C), and freezers.

Each slide is labeled with two patient identifiers plus specimen source and collection date, and is verified against the requisition before staining.

CAP/CLIA proficiency testing is required for predictive marker immunohistochemistry such as ER, PR, and HER2, with documented review of results.

Equipment maintenance records, reagent lot verification, and documented corrective action for failed QC are core components of the quality management system.