ASCP H cheat sheet

10% neutral buffered formalin (NBF) contains 4% formaldehyde w/v buffered to pH 7.2–7.4; the recommended fixative-to-tissue volume ratio is 10:1.

Formalin penetrates tissue at roughly 1 mm per hour from the surface, so specimens are sliced 3–4 mm thick to allow complete fixation.

Additive fixatives (formalin, glutaraldehyde) cross-link proteins; coagulant/non-additive fixatives (Bouin, Zenker, B-5, Carnoy) precipitate proteins.

Carnoy fluid (60% ethanol, 30% chloroform, 10% acetic acid) fixes and dehydrates rapidly and is used for glycogen and lymphocyte-rich tissues.

Fixation beyond 48–72 hours in NBF over-cross-links proteins and masks epitopes; heating above 45 °C also degrades antigens.

Processing order: graded ethanol dehydration, xylene clearing (miscible with both ethanol and paraffin), then paraffin infiltration at 56–60 °C.

Incomplete dehydration leaves xylene cloudy and blocks soft and wet; incomplete clearing leaves milky white areas in the paraffin block.

Formalin pigment (acid hematin) is a brown artifact formed in acidic, blood-rich tissue and is removed with saturated alcoholic picric acid.

Routine paraffin sections for H&E are cut at 3–5 µm; small biopsies and immunohistochemistry sections are often cut at 2–3 µm.

The clearance angle between the blade facet and the block face is normally 3–5 degrees; too little causes compression, too much causes chatter.

Chatter appears as fine parallel lines or vibration and results from hard tissue, a loose blade, a dull blade, or cutting too rapidly.

Alternating thick and thin sections ('venetian blind' artifact) indicate a loose blade or block, a soft warm block, or an incorrect clearance angle.

Sections are floated on a water bath at about 40–45 °C, roughly 5–10 °C below the paraffin melting point, to flatten without melting the wax.

Cryostat chambers are usually held near −20 °C; warmer temperatures cause smearing and folds, colder temperatures cause shattering and cracking.

Charged slides (poly-L-lysine or silane) are used for immunohistochemistry and for small, fatty, or calcified sections prone to detachment.

Embedding orientation: skin and mucosa are placed on edge perpendicular to the blade, and multiple fragments are arranged in a honeycomb pattern at one level.

Hematoxylin is a basic (cationic) dye complexed with an aluminum mordant that binds nucleic acids and stains nuclei blue-purple; such structures are basophilic.

Eosin Y is an acidic (anionic) dye applied at slightly acidic pH that stains cytoplasm, collagen, and red blood cells pink-red; such structures are eosinophilic.

Progressive staining stops when the desired intensity is reached; regressive staining deliberately overstains and then differentiates in 1% acid alcohol (HCl in 70% ethanol).

Bluing in running tap water or Scott's tap water (pH about 8) converts the soluble red aluminum-hematin complex into an insoluble blue form.

Harris hematoxylin is ripened with mercuric oxide and contains acetic acid; Mayer's and Gill's hematoxylins are chemically ripened with sodium iodate and contain no mercury.

Over-differentiated nuclei appear pale gray-blue; overly blued or thick sections appear densely blue with obscured chromatin detail.

Eosin is water-soluble and is decolorized by prolonged aqueous steps, so dehydration after eosin must be prompt; lithium carbonate or ammonia water removes excess eosin.

Most H&E staining defects are preanalytical in origin: delayed fixation, thick sections, or incomplete processing rather than the dye itself.

PAS: periodic acid oxidizes 1,2-glycol groups to aldehydes that react with Schiff reagent to give magenta glycogen, mucin, and basement membranes; diastase digestion (PAS-D) removes glycogen.

Masson trichrome: collagen stains blue (aniline blue) or green (light green), muscle and cytoplasm red with Biebrich scarlet–acid fuchsin, and nuclei black with iron hematoxylin.

Gomori reticulin: silver impregnation renders reticulin fibers black; it requires acid-cleaned glassware, ammoniacal silver, a formalin developer, and gold chloride toning.

Prussian blue: hydrochloric acid liberates iron from hemosiderin, which reacts with potassium ferrocyanide to form an insoluble blue pigment; nuclear fast red is the usual counterstain.

Congo red stains amyloid pink-red in ordinary light and produces apple-green birefringence under polarized light.

Ziehl–Neelsen AFB: hot carbol fuchsin, decolorization in 1% acid alcohol, methylene blue counterstain, giving red bacilli on a blue background.

Alcian blue at pH 2.5 stains both carboxylated and sulfated acid mucins; at pH 1.0 only the strongly acidic sulfated mucins stain.

Von Kossa demonstrates calcium indirectly: silver replaces phosphate in calcium salts and is reduced to black metallic silver by light.

Silver methods are finished by toning (gold chloride) and removal of unreduced silver with sodium thiosulfate (hypo) to prevent background darkening.

NBF is the preferred routine fixative for IHC; overfixation masks epitopes, and antigen retrieval restores many but not all of them.

Heat-induced epitope retrieval uses citrate buffer pH 6.0 or Tris-EDTA pH 9.0 at 95–100 °C for 20–40 minutes; high-pH EDTA is favored for many nuclear antigens.

Protease-induced epitope retrieval (trypsin, proteinase K, pepsin) is used for antigens that are destroyed by heat.

Endogenous peroxidase is blocked with 3% hydrogen peroxide; endogenous biotin in liver and kidney is blocked with free avidin followed by biotin.

Polymer (two-step) detection avoids the endogenous biotin and avidin-binding problems of avidin–biotin complex methods and is generally more sensitive.

DAB produces a brown, alcohol-insoluble precipitate that allows permanent resinous mounting; AEC produces a red, alcohol-soluble product that requires aqueous mounting.

Controls include a positive tissue control, a negative reagent control using an irrelevant antibody of the same isotype and concentration, and an absorption control.

Enzyme histochemistry such as alkaline phosphatase with naphthol AS-BI phosphate and Fast Red usually requires unfixed frozen sections to preserve enzyme activity.

A very high antibody concentration can produce the prozone (hook) effect, in which a dilution series paradoxically shows weaker staining at higher concentration.

A new lot of stain or antibody is tested in parallel with the in-use lot on known positive and negative control material before being released for patient use.

Specimens that are unlabeled, mislabeled, leaked, or in the wrong fixative require documented resolution with the submitting source before accessioning.

FIFO stock rotation consumes the oldest in-date reagents first, with expired material segregated and removed from the working area.

Daily temperature logs are maintained for tissue processors, cryostats, water baths, refrigerators, incubators, and paraffin dispensers.

Reagent-grade or deionized water should have resistivity of at least 1 MΩ·cm; organic or microbial contamination degrades hematoxylin and silver stains.

Sectioning quality falls when humidity is very low (static and curled sections) or very high (soft blocks); the usual working range is about 18–24 °C with moderate humidity.

Formalin and xylene waste streams are segregated as hazardous waste and are never discharged to sinks or drains.

Batch staining trades turnaround time for efficiency, while continuous or single-slide staining is used for urgent and stat cases.

Quality control is the testing of a product or process against specifications; quality assurance is the broader system of policies and monitors that ensures the result.

Levey–Jennings charts plot control values over time with lines at the mean and ±1s, ±2s, and ±3s, making systematic shifts and random scatter visible.

Westgard rules: 1-3s and R-4s signal random error; 2-2s, 4-1s, and 10x signal systematic error and require run rejection.

The majority of laboratory errors are preanalytical (identification, fixation, transport), which is why specimen labeling is a leading quality indicator.

Proficiency testing and external quality assessment compare performance with peer laboratories and are required for accreditation.

Corrective action repairs a specific identified problem; preventive action modifies the system before an error recurs (CAPA).

Root cause analysis tools include the five whys, the fishbone (Ishikawa) diagram, and process mapping.

Tracked quality indicators include amended report rate, mislabeled specimen rate, turnaround time, and control outlier rate.

The OSHA permissible exposure limit for formaldehyde is 0.75 ppm as an 8-hour time-weighted average, with a 2 ppm short-term exposure limit over 15 minutes; formaldehyde is an IARC Group 1 carcinogen.

The OSHA permissible exposure limit for xylene is 100 ppm; xylene is flammable with a flash point near 27 °C, so containers are grounded and stored in flammable-liquid cabinets.

Class B extinguishers are used for flammable liquids such as xylene and alcohols, Class A for paper and wood, and Class C or carbon dioxide for electrical fires.

Formalin spills are contained with absorbent material and neutralized with ammonium carbonate or a commercial neutralizer rather than diluted with water.

A cryostat holding unfixed tissue is a biohazard; decontamination uses 70% ethanol or a tuberculocidal disinfectant, with UV cycles when the unit is idle.

The bloodborne pathogens standard requires offering hepatitis B vaccination, providing PPE, and using engineering controls such as sharps containers and safety devices.

GHS-compliant secondary container labels require the product identifier, hazard pictograms, signal word, hazard statements, and precautionary statements; an SDS contains 16 sections.

Fume hood face velocity is typically maintained at about 100–120 feet per minute, and the sash is kept low while working.

Waste is segregated by stream: halogenated versus non-halogenated solvents, formalin, heavy metals such as silver and osmium, and sharps.