ASCP HTL cheat sheet

10% neutral buffered formalin contains about 4% w/v formaldehyde in phosphate buffer at pH 7.0-7.4.

Formalin penetration is approximately 1 mm per hour; tissue blocks should generally be no thicker than 3-4 mm.

Recommended fixative-to-tissue volume ratio is at least 10:1, commonly 15:1 to 20:1.

Routine formalin fixation is usually 6-24 hours; prolonged fixation can mask antigens and produce formalin pigment.

Bouin solution contains picric acid and causes yellow staining; remove with lithium carbonate or saturated alcohol.

Zenker and B5 fixatives contain mercury; remove mercury pigment with iodine followed by sodium thiosulfate.

Alcohol-based fixatives coagulate proteins and can preserve nucleic acids better than formalin in some settings but cause shrinkage.

Glutaraldehyde is used for electron microscopy because it cross-links proteins and preserves fine structure.

Hypertonic fixatives shrink cells; hypotonic fixatives swell cells.

Routine processing sequence is dehydration in graded ethanol, clearing in xylene or substitute, and infiltration with molten paraffin.

Paraffin infiltration typically uses paraffin with a melting point of 56-58°C; higher melting point paraffin gives harder blocks.

Routine paraffin sections are cut at 3-5 µm; sections for immunohistochemistry are often 3-4 µm.

Microtome clearance angle is usually 5-10°; too small causes chatter, and too large causes scraping.

Floating bath water temperature is typically 40-45°C, about 5-10°C below the paraffin melting point.

EDTA decalcification is slow but preserves nucleic acids and antigens; acid decalcifiers are faster but can damage DNA and antigenicity.

Cassette overfilling prevents proper infiltration and causes retained processing reagent and block voids.

Section folds appear as dark overlapping bands and can be reduced by slower cutting and proper blade angle.

Embedding should orient the tissue so the diagnostic surface is parallel to the block face and fully exposed.

Hematoxylin is a basic dye that stains nucleic acids blue-purple; eosin is an acidic dye that stains proteins pink.

Progressive hematoxylin is stained to final intensity and does not require differentiation; regressive hematoxylin is overstained then differentiated in acid alcohol.

Bluing after hematoxylin is done in weakly alkaline water such as Scott's tap water or ammonia water, converting hematoxylin to blue.

Eosin Y is commonly used as a 0.5-1% alcoholic or aqueous solution at pH 4-5.

Harris hematoxylin is a regressive hematoxylin that may contain mercuric oxide as an oxidizing agent.

Overdifferentiation removes too much hematoxylin and causes pale nuclei; underdifferentiation leaves background blue.

H&E quality is assessed by crisp blue nuclei, distinct pink cytoplasm, and minimal precipitate.

Acid decalcification and prolonged acid exposure can cause hematoxylin staining to appear pale or red.

A too-thin section appears pale; a too-thick section appears intensely stained and may show overlapping nuclei.

PAS stains glycogen, mucins, basement membranes, and fungi magenta; diastase digestion distinguishes glycogen from other PAS-positive material.

Masson trichrome stains collagen blue or green and muscle, cytoplasm, and keratin red.

Reticulin stain uses silver impregnation to demonstrate reticulin fibers black.

Prussian blue demonstrates hemosiderin iron as blue granules.

Congo red stains amyloid and shows apple-green birefringence under polarized light.

Ziehl-Neelsen acid-fast stain demonstrates mycobacteria as red bacilli against a blue background.

Grocott methenamine silver (GMS) stains fungal cell walls black with a green counterstain.

Mucicarmine stains epithelial mucin rose to red and is used for adenocarcinoma and cryptococcal capsules.

Masson-Fontana silver stain demonstrates melanin as black pigment.

Heat-induced antigen retrieval commonly uses citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0.

Endogenous peroxidase is blocked with hydrogen peroxide, usually 3% H2O2, before peroxidase-based detection.

Endogenous biotin can cause false-positive staining with avidin-biotin detection; polymer-based detection avoids this.

Every IHC run should include positive and negative controls; a negative reagent control replaces primary antibody with buffer.

DAB produces a brown, alcohol-insoluble reaction product; AEC produces a red product that is alcohol-soluble.

Antibody titration determines the optimal dilution that gives strong specific staining with minimal background.

Enzyme histochemistry often requires unfixed or lightly fixed frozen sections to preserve enzyme activity.

Alkaline phosphatase, acid phosphatase, and nonspecific esterase are demonstrated with enzyme histochemical methods.

Prolonged formalin fixation can alter epitopes; antigen retrieval may restore some but not all immunoreactivity.

Reagent containers must be labeled with identity, concentration, preparation date, and expiration date.

Molarity = moles of solute per liter of solution; % w/v = grams of solute per 100 mL of solution.

pH meters are calibrated with fresh standard buffers at pH 4.0, 7.0, and 10.0 before use.

Beer-Lambert law: absorbance is proportional to concentration and path length at a given wavelength.

Water quality for histology should be monitored for resistivity, microbial contamination, and organic content.

Microtomes require regular cleaning, lubrication, and blade-angle checks; debris causes section chatter and tears.

Reagent lot numbers and expiration dates must be recorded to allow traceability and recall.

Centrifuges should be balanced, and tubes checked for cracks before use.

Temperature-sensitive reagents and paraffin are stored within validated temperature ranges and monitored daily.

OSHA permissible exposure limit for formaldehyde is 0.75 ppm as an 8-hour time-weighted average and 2 ppm as a 15-minute short-term exposure limit.

Formaldehyde is a known human carcinogen; work with it in a chemical fume hood and use appropriate gloves.

Xylene is flammable and toxic; store away from ignition sources and use local exhaust ventilation.

The Chemical Hygiene Plan and Safety Data Sheets must be accessible to all laboratory personnel.

Quality control records should include daily temperature checks, instrument maintenance, and reagent lot verification.

Levey-Jennings charts plot QC values over time; Westgard rules detect systematic and random error.

Proficiency testing is required under CLIA and CAP to monitor laboratory performance.

Sharps must be discarded in puncture-resistant containers; broken glass is not placed in biohazard bags.

Chemical, biohazard, and sharps waste must be segregated according to facility and regulatory requirements.

Frozen sections are typically cut at 4-6 µm in a cryostat at about -20 to -25°C and are used for intraoperative diagnosis and margin assessment.

Cytology specimens are commonly fixed in 95% ethanol or processed as liquid-based preparations; Pap stain is used for cervical cytology.

Cell blocks allow cytology specimens to be processed like paraffin tissue sections for ancillary testing.

Molecular testing such as PCR, FISH, and sequencing requires specimens with adequate cellularity and preserved nucleic acids.

Frozen section artifacts include ice crystals, chatter, and folds; rapid freezing and proper cryostat temperature reduce them.

FISH uses fluorescent probes to detect gene amplifications, deletions, and translocations on interphase nuclei or metaphase spreads.

HPV testing detects high-risk viral DNA or RNA and is used in cervical cancer screening and triage.

Competency assessment for laboratory personnel includes direct observation, review of records, and testing of sample problems.

Formalin fixation can degrade nucleic acids; molecular testing may require ethanol-fixed or fresh-frozen tissue.