Pathology

01 Tissue Processing & Histology

Specimen Fixation

The first and most critical step in histologic processing is fixation, which preserves tissue morphology by cross-linking proteins and preventing autolysis. The universal standard fixative is 10% neutral buffered formalin (NBF), a 4% aqueous solution of formaldehyde buffered to pH 6.8-7.2 with monobasic and dibasic sodium phosphate. Formalin penetrates tissue at approximately 1 mm per hour; therefore, specimens should be sliced to no more than 4 mm thickness and fixed for a minimum of 6-8 hours, though 24-48 hours is standard for most surgical specimens. Breast cancer specimens for HER2 testing require fixation for 6-72 hours per ASCO/CAP guidelines to ensure reliable immunohistochemistry and FISH results. Underfixation leads to poor nuclear morphology and unreliable IHC staining; overfixation (beyond 72 hours) causes excessive cross-linking that masks epitopes and makes antigen retrieval difficult.

Tissue Processing

After fixation, tissues undergo automated processing through a series of graded reagents. Dehydration removes water through ascending concentrations of ethanol (70%, 80%, 95%, 100%). Clearing replaces ethanol with xylene or xylene substitutes, which are miscible with both ethanol and paraffin wax. Infiltration replaces xylene with molten paraffin wax at 56-60°C under vacuum. Modern automated tissue processors complete this cycle in 8-12 hours for routine specimens. Rapid processors can produce sections in 1-2 hours for urgent cases. The processed tissue is then embedded in a paraffin wax block with careful orientation to ensure the correct plane of section.

Microtomy & Section Cutting

A microtome cuts thin sections from the paraffin block. Standard histologic sections are 4-5 micrometers (microns) thick. Thinner sections (2-3 µm) are used for renal biopsies to better visualize glomerular basement membranes. Sections are floated on a warm water bath (42-45°C) to smooth wrinkles, then picked up on glass slides. Serial sections (multiple consecutive levels through the tissue) are critical when searching for small foci — e.g., sentinel lymph node examination for micrometastases or small nerve invasion in prostate biopsies.

H&E Staining

The hematoxylin and eosin (H&E) stain is the foundation of diagnostic histopathology. Hematoxylin is a basic (cationic) dye that binds negatively charged (basophilic) structures, staining them blue-purple: nuclei (DNA/RNA), ribosomes, and rough endoplasmic reticulum. The active staining moiety is hematein, formed by oxidation of hematoxylin and complexed with a metal mordant (aluminum salts). Eosin is an acidic (anionic) dye that binds positively charged (eosinophilic/acidophilic) structures, staining them pink-red: cytoplasmic proteins, collagen, and muscle fibers. H&E sections are dehydrated, cleared, and coverslipped with permanent mounting medium for long-term storage.

Decalcification

Bone and calcified tissues must be decalcified after fixation to enable sectioning without damaging the microtome blade. Methods include: strong acid decalcification (hydrochloric or nitric acid — fast, 1-3 days, but damages nuclear detail and impairs IHC), weak acid decalcification (formic acid — moderate speed, 2-5 days, preserves morphology and most IHC), and chelating agents (EDTA — slow, days to weeks, but best preservation of morphology, IHC, and molecular studies). The endpoint of decalcification is assessed by physical testing (needle penetration) or chemical testing (calcium oxalate precipitation from the decalcifying solution). Bone marrow trephine biopsies are typically decalcified with EDTA or formic acid to preserve antigens for immunohistochemistry.

Frozen Sections

The frozen section technique bypasses formal fixation and processing entirely. Fresh tissue is snap-frozen at -20 to -30°C in a cryostat (a microtome housed inside a freezing chamber) and cut at 5-8 µm. Sections are briefly fixed in cold acetone or formalin, stained with rapid H&E, and interpreted within 15-20 minutes. Frozen sections are used intraoperatively for: margin assessment, tumor identification, lymph node evaluation, and tissue adequacy for special studies. Accuracy is approximately 95-98% compared to permanent sections. Limitations include: freezing artifact (ice crystal formation that distorts nuclear detail), thicker sections with poorer morphology, and difficulty with adipose tissue and calcified specimens. False-negative rates are highest for lobular carcinoma in situ and low-grade lymphomas.

Tissue Microarray (TMA)

A tissue microarray is a paraffin block containing small cylindrical cores (0.6-2.0 mm diameter) from dozens to hundreds of different tissue samples arranged in a grid. TMAs enable simultaneous IHC or FISH testing of many specimens on a single slide, conserving tissue and reagents. Applications include: biomarker validation studies, tumor banks, quality control (control TMA with known positive/negative tissues for each IHC antibody), and research cohort analysis. Limitations: small core size may not represent tumor heterogeneity; loss of cores during sectioning.

Electron Microscopy

Transmission electron microscopy (TEM) provides ultrastructural detail at 1,000-100,000x magnification. Specimens must be fixed in glutaraldehyde (not formalin) and processed with osmium tetroxide, embedded in epoxy resin, and cut at 60-90 nm with an ultramicrotome. Diagnostic applications include: renal biopsy (glomerular basement membrane abnormalities — thin basement membrane disease, Alport syndrome, dense deposit disease, immune complex deposition pattern), mesothelioma vs. adenocarcinoma (long microvilli vs. short microvilli), neuroendocrine tumors (dense-core granules), rhabdomyosarcoma (Z-band material), and poorly differentiated tumors of uncertain lineage. Though IHC has replaced TEM for most diagnostic purposes, it remains essential in renal pathology and selected tumor diagnostics.

02 Special Stains & Their Targets

Special stains exploit the chemical properties of tissue components to highlight structures not visible on routine H&E. Each stain has specific targets, characteristic colors, and defined clinical applications.

Immunofluorescence (IF)

Direct immunofluorescence (DIF) uses fluorescent-labeled antibodies applied directly to fresh-frozen tissue (or Michel transport medium for delayed processing). Patterns are diagnostic in autoimmune blistering diseases and renal pathology: pemphigus vulgaris — IgG and C3 in intercellular (chicken-wire) pattern in epidermis; bullous pemphigoid — linear IgG and C3 at the basement membrane zone (BMZ); dermatitis herpetiformis — granular IgA at the dermal papillae tips; linear IgA bullous dermatosis — linear IgA at the BMZ; lupus band test — granular IgG, IgM, IgA, and C3 at the BMZ (positive in involved and uninvolved skin in SLE). In renal pathology, IF identifies: IgA nephropathy (mesangial IgA dominant), membranous nephropathy (granular IgG and C3 along capillary walls), lupus nephritis ("full house" pattern with IgG, IgA, IgM, C3, C1q), and anti-GBM disease (linear IgG along GBM). Indirect IF (IIF) detects circulating autoantibodies in patient serum using substrate tissue; examples: ANA (HEp-2 cells), anti-dsDNA, anti-smooth muscle, anti-mitochondrial.

Staining Mechanism Principles

Silver stains (GMS, reticulin, Warthin-Starry) exploit the reduction of silver nitrate to metallic silver by tissue components, depositing black silver on target structures — termed argyrophilic (require an external reducing agent) or argentaffin (contain their own reducing agent). Trichrome stains (Masson, Gomori) use differential dye penetration: small dye molecules penetrate dense collagen while large molecules stain only permeable structures like cytoplasm. The PAS reaction involves periodic acid oxidizing vicinal diols in carbohydrates to create aldehyde groups, which then react with Schiff reagent to produce the characteristic magenta color.

03 Immunohistochemistry — Principles & Key Markers

IHC Technique

Immunohistochemistry (IHC) uses antibodies to detect specific protein antigens in tissue sections. The basic steps are: (1) antigen retrieval — heat-induced epitope retrieval (HIER) using citrate buffer (pH 6) or EDTA buffer (pH 9), or enzymatic retrieval with proteinase K or pepsin, to reverse formalin cross-links and expose epitopes; (2) blocking of endogenous peroxidase and nonspecific protein binding; (3) primary antibody application (monoclonal or polyclonal, directed against the target antigen); (4) secondary antibody conjugated to an enzyme (horseradish peroxidase or alkaline phosphatase); (5) chromogen application — most commonly DAB (3,3'-diaminobenzidine) producing a brown reaction product, or AEC (red), or Fast Red. The section is counterstained with hematoxylin to visualize nuclei. Positive and negative controls must be run with every IHC batch.

Key IHC Markers by Category

Epithelial Markers

Cytokeratins (CK) are intermediate filaments that define epithelial differentiation. The CK7/CK20 profile is one of the most useful tools for determining carcinoma origin: CK7+/CK20- suggests lung, breast, ovary, endometrium, thyroid, or upper GI; CK7-/CK20+ suggests colorectal; CK7+/CK20+ suggests urothelial (bladder), pancreatic, or mucinous ovarian; CK7-/CK20- suggests hepatocellular, renal, prostate, or squamous cell carcinoma. Pan-cytokeratin (AE1/AE3, CAM 5.2) stains virtually all carcinomas. CK5/6 marks squamous differentiation and basal cells. CK19 is positive in cholangiocarcinoma but negative in hepatocellular carcinoma.

Organ-Specific Markers

TTF-1 (thyroid transcription factor-1) — positive in lung adenocarcinoma (~75%) and thyroid carcinoma (both papillary and follicular); negative in squamous cell carcinoma of lung. Napsin A — positive in lung adenocarcinoma, increases specificity when combined with TTF-1. PAX8 — positive in renal, ovarian, thyroid, and endometrial carcinomas; key for distinguishing ovarian from colon primary in the pelvis. GATA3 — positive in breast and urothelial carcinoma; one of the most sensitive markers for metastatic breast cancer. CDX2 — nuclear transcription factor positive in colorectal adenocarcinoma (>95%); also marks other GI primaries. Hepatocyte Paraffin 1 (HepPar1) and arginase-1 — positive in hepatocellular carcinoma. PSA and NKX3.1 — prostate carcinoma. WT1 — ovarian serous carcinoma, mesothelioma, Wilms tumor.

Hormone Receptors & HER2

Estrogen receptor (ER) and progesterone receptor (PR) are nuclear stains scored by the Allred system (proportion score 0-5 + intensity score 0-3 = total score 0-8) or by percentage of positive cells. ASCO/CAP considers ER/PR positive at ≥1% nuclear staining, though tumors with 1-10% staining are now reported as "ER low positive" with a note about uncertain benefit from endocrine therapy. HER2 (ERBB2) IHC is scored: 0 (no staining or faint incomplete membrane staining in ≤10% of cells — negative), 1+ (faint incomplete membrane staining in >10% — negative), 2+ (weak-to-moderate complete membrane staining in >10% — equivocal, reflex to FISH), 3+ (strong complete membrane staining in >10% — positive). HER2-low (1+ or 2+/FISH non-amplified) is now therapeutically relevant with trastuzumab deruxtecan.

Proliferation & Tumor Suppressor Markers

Ki-67 (MIB-1) is a nuclear antigen expressed in all phases of the cell cycle except G0. It serves as a proliferation index: low (<15%) suggests indolent behavior; high (>30%) suggests aggressive biology. In breast cancer, Ki-67 helps distinguish Luminal A (<20%) from Luminal B (≥20%). In neuroendocrine tumors, Ki-67 defines WHO grade: G1 <3%, G2 3-20%, G3 >20%. p53 staining patterns: wild-type shows weak, scattered nuclear staining; missense mutation shows strong, diffuse nuclear overexpression; nonsense/frameshift mutation shows complete absence of staining (null pattern); cytoplasmic staining is an artifact. Aberrant p53 is characteristic of high-grade serous ovarian carcinoma and Li-Fraumeni syndrome.

Lymphoid Markers

CD20 — pan-B cell marker (target of rituximab). CD3 — pan-T cell marker. CD15/CD30 — classic Hodgkin lymphoma Reed-Sternberg cells (CD15+/CD30+/CD20-/CD45-). CD10 — follicular lymphoma, Burkitt lymphoma, precursor B-ALL. CD5 — CLL/SLL and mantle cell lymphoma (normal T cells also CD5+). Cyclin D1 — mantle cell lymphoma (due to t(11;14) CCND1-IGH translocation). BCL-2 — follicular lymphoma (t(14;18) overexpression); germinal center B cells are normally BCL-2 negative, so BCL-2 positivity in follicles is abnormal. BCL-6 — germinal center marker, positive in follicular lymphoma and GCB-type DLBCL. MUM1 — post-germinal center marker, positive in non-GCB DLBCL. CD138 — plasma cell marker (multiple myeloma).

Immune Checkpoint & Predictive Markers

PD-L1 (CD274) — membranous staining on tumor cells and/or immune cells; interpretation varies by scoring system (TPS, CPS, IC%) and antibody clone (22C3, 28-8, SP142, SP263) — see section 19 for details. MSI/MMR — loss of any MMR protein (MLH1, MSH2, MSH6, PMS2) by IHC indicates dMMR and eligibility for immunotherapy — see section 19. TMB — assessed by NGS from FFPE tissue; ≥10 mut/Mb defines TMB-high, a tissue-agnostic predictive marker for pembrolizumab. LAG-3 — an emerging immune checkpoint target; relatlimab + nivolumab (Opdualag) is FDA-approved for unresectable/metastatic melanoma; LAG-3 IHC is not yet routinely required for treatment selection.

Melanocytic & Soft Tissue Markers

S100 — most sensitive melanocytic marker (also marks nerve sheath tumors, Langerhans cells, chondrocytes, adipocytes). HMB-45 — more specific for melanoma but less sensitive; stains junctional component preferentially. Melan-A/MART-1 — melanocyte marker, also positive in adrenal cortex and steroid-producing tumors. SOX10 — nuclear marker for melanocytes and nerve sheath tumors; increasingly used as first-line melanocytic marker due to clean nuclear staining. Desmin — muscle marker (smooth and skeletal). SMA (smooth muscle actin) — smooth muscle, myofibroblasts, pericytes. MyoD1/Myogenin — skeletal muscle differentiation (rhabdomyosarcoma). CD34 — vascular endothelium, dermatofibrosarcoma protuberans, GIST, solitary fibrous tumor. DOG1/CD117 (c-KIT) — GIST (gastrointestinal stromal tumor); CD117 also marks mast cells and seminoma.

04 Key Terminology & Abbreviations

Fundamental Pathology Terms

Hyperplasia — increase in cell number (reversible). Hypertrophy — increase in cell size. Atrophy — decrease in cell size or number. Metaplasia — reversible replacement of one differentiated cell type by another (e.g., squamous metaplasia in bronchi, intestinal metaplasia in stomach/Barrett). Dysplasia — disordered, premalignant cellular growth with architectural and cytologic abnormalities (loss of polarity, increased mitoses, nuclear pleomorphism) that does not breach the basement membrane. Carcinoma in situ (CIS) — full-thickness dysplasia confined by the basement membrane; high-grade intraepithelial neoplasia. Invasion — tumor cells penetrate the basement membrane into the stroma — this is the defining event that distinguishes carcinoma from CIS. Anaplasia — loss of differentiation; features include marked pleomorphism, abnormal mitoses (tripolar, ring), high N:C ratio, and tumor giant cells.

Neoplasia Terminology

Benign tumors are named by cell of origin + suffix: -oma (adenoma, leiomyoma, lipoma, chondroma). Malignant epithelial tumors are carcinomas (adenocarcinoma, squamous cell carcinoma). Malignant mesenchymal tumors are sarcomas (leiomyosarcoma, liposarcoma, osteosarcoma). Exceptions to naming rules: melanoma (malignant but ends in -oma), lymphoma (malignant), hepatoma (obsolete term for hepatocellular carcinoma), seminoma/teratoma (malignant germ cell tumors). Differentiation — how closely the tumor resembles its tissue of origin: well-differentiated (low grade) vs. poorly differentiated (high grade). Desmoplasia — dense fibrous stroma induced by invasive carcinoma (especially pancreatic and breast ductal carcinoma).

Margin Terminology

Positive margin — tumor cells at the inked edge of the specimen (tumor at the resection margin). Close margin — tumor within 1-2 mm of the inked edge (definition varies by organ). Negative margin — no tumor at the inked edge. For breast lumpectomy, ASCO/SSO/ASTRO consensus defines adequate negative margin as "no ink on tumor" for invasive carcinoma and ≥2 mm for DCIS. For colorectal resection, a circumferential resection margin (CRM) ≤1 mm is considered positive and predicts local recurrence.

Cell Injury & Death

Necrosis — pathologic, unregulated cell death. Patterns: coagulative necrosis (cell outlines preserved, "ghost cells" — most common; ischemic infarcts of solid organs except brain), liquefactive necrosis (enzymatic digestion destroying tissue — brain infarcts, abscesses), caseous necrosis (amorphous, cheese-like, eosinophilic debris — tuberculosis, fungal infections), fat necrosis (lipase action on adipose — acute pancreatitis, breast trauma; saponification with calcium = dystrophic calcification), fibrinoid necrosis (bright pink, smudgy material in vessel walls — malignant hypertension, vasculitis, rheumatic fever), gangrenous necrosis (dry = coagulative in extremities; wet = secondary bacterial infection with liquefaction). Apoptosis — programmed, energy-dependent cell death: cell shrinkage, chromatin condensation, cytoplasmic budding into apoptotic bodies, no inflammatory response (unlike necrosis). Apoptotic bodies are phagocytosed by neighboring cells or macrophages. Councilman bodies (viral hepatitis), Civatte bodies (lichen planus), and tingible body macrophages (germinal centers) all represent apoptosis.

Inflammation Basics

Acute inflammation — neutrophil-predominant; vascular changes (vasodilation, increased permeability) and cellular events (margination, rolling, adhesion, transmigration via selectins and integrins). Cardinal signs: rubor (redness), calor (heat), tumor (swelling), dolor (pain), functio laesa (loss of function). Outcomes: resolution, abscess formation, fibrosis, or progression to chronic inflammation. Chronic inflammation — mononuclear cell infiltrate (lymphocytes, macrophages, plasma cells); granulation tissue formation; fibrosis. Granulomatous inflammation — collections of activated macrophages (epithelioid histiocytes) ± multinucleated giant cells (Langhans type with peripheral horseshoe-shaped nuclei = TB; foreign body type with scattered nuclei = foreign material). Causes: TB (caseating), sarcoidosis (non-caseating, "naked" granulomas), fungal infections, foreign body, Crohn disease, berylliosis, cat scratch disease.

05 Specimen Handling & Grossing

Specimen Orientation & Inking

Proper orientation of surgical specimens is essential for accurate margin assessment. Surgeons mark specimens with sutures, clips, or designated inks: a common convention is short suture = superior, long suture = lateral. The pathology assistant or pathologist applies colored inks to the specimen surfaces before cutting to identify specific margins: a typical scheme uses black (deep/posterior), blue (superior), red (inferior), green (lateral), orange (medial), though protocols vary by institution. Ink must be fixed (with Bouin solution, acetone, or brief formalin exposure) before sectioning to prevent ink run.

Grossing Techniques

Bread-loafing — the specimen is sliced at 2-3 mm intervals perpendicular to a defined axis (usually the longest axis or the margin of interest) to create serial cross-sections. This is the standard method for breast lumpectomies, colectomy segments, and uterine specimens. Perpendicular sections through the margin are preferred over en face (shave) margins when assessing the closest distance between tumor and margin. Shave margins sample more surface area but only indicate presence/absence of tumor at the margin without measuring distance. All grossly abnormal areas, representative normal tissue, and all margins must be submitted for microscopic examination.

Lymph Node Dissection

Lymph node retrieval is critical for staging. A minimum of 12 lymph nodes is the standard for adequate colorectal cancer staging (AJCC/CAP). Lymph nodes are identified by palpation and visual inspection of the mesenteric fat; fat-clearing solutions (Carnoy fixative, acetone, or xylene) can increase yield when palpation is insufficient. Each lymph node ≤3 mm is submitted entirely; larger nodes are bisected or serially sectioned at 2 mm intervals. The sentinel lymph node (SLN) protocol for breast and melanoma involves serial sectioning at 2 mm intervals with H&E and cytokeratin IHC (AE1/AE3 or CAM5.2) at each level. Isolated tumor cells (ITCs, ≤0.2 mm) are classified as pN0(i+); micrometastases (0.2-2.0 mm) as pN1mi; macrometastases (>2.0 mm) as pN1.

Fresh Tissue Triage

Certain studies require fresh (unfixed) tissue: flow cytometry (lymphoma subtyping — requires viable single-cell suspension), cytogenetics/karyotyping (requires viable dividing cells in culture medium like RPMI), molecular studies (snap-frozen tissue for RNA extraction), electron microscopy (fixed in glutaraldehyde, not formalin), and biobanking (snap-frozen at -80°C or liquid nitrogen for future research). A critical role of the pathologist is to triage fresh tissue appropriately — allocating sufficient material for diagnosis, biomarkers, and ancillary studies while ensuring that the primary diagnostic specimen is not compromised.

Specimen Types Requiring Special Handling

Frozen Section — Indications & Limitations

Indications for intraoperative frozen section include: (1) determining whether a lesion is neoplastic vs. non-neoplastic to guide extent of surgery, (2) assessing surgical margins (e.g., Mohs surgery, pancreatic transection margin), (3) evaluating sentinel lymph nodes, (4) confirming tissue adequacy for diagnosis or culture, and (5) identifying unexpected findings. Accuracy ranges from 95-98% with a false-negative rate of 1-3% and false-positive rate <1%. Entities with high deferral rates include: follicular thyroid lesions (cannot distinguish adenoma from carcinoma on frozen section — capsular and vascular invasion require permanent sections), low-grade lymphoproliferative disorders, and spindle cell lesions. The pathologist should communicate uncertainty via a "defer to permanent" response rather than risk an incorrect intraoperative diagnosis.

06 Breast Pathology

Ductal Carcinoma In Situ (DCIS)

DCIS is a neoplastic proliferation of malignant epithelial cells confined to the ductal system without invasion through the basement membrane. Architectural patterns include: comedo (central necrosis with calcification), cribriform (punched-out spaces), papillary, micropapillary, and solid. The Van Nuys Prognostic Index (VNPI) stratifies DCIS recurrence risk using four parameters, each scored 1-3: tumor size (≤15 mm = 1, 16-40 mm = 2, >40 mm = 3), margin width (≥10 mm = 1, 1-9 mm = 2, <1 mm = 3), nuclear grade (low = 1, intermediate = 2, high = 3), and age (>60 = 1, 40-60 = 2, <40 = 3). Total score 4-6: excision alone; 7-9: excision plus radiation; 10-12: consider mastectomy.

Invasive Breast Carcinoma — Nottingham Grading

The Nottingham histologic grade (modified Scarff-Bloom-Richardson) is the standard grading system for invasive breast carcinoma. Three components are scored 1-3 and summed:

ER/PR Scoring — Allred System

The Allred scoring system for ER and PR quantifies both the proportion and intensity of nuclear staining in tumor cells. The proportion score (PS): 0 = no cells staining, 1 = <1%, 2 = 1-10%, 3 = 11-33%, 4 = 34-66%, 5 = 67-100%. The intensity score (IS): 0 = none, 1 = weak, 2 = intermediate, 3 = strong. The total Allred score = PS + IS (range 0-8). A score of 0 is negative; scores 2-8 are considered positive (though scores of 2 are rare and may warrant retesting). In current ASCO/CAP guidelines, ER and PR are reported as positive if ≥1% of tumor cells show nuclear staining, but tumors with 1-10% staining are now categorized as "ER low positive" because the benefit of endocrine therapy in this range is uncertain. True ER-negative breast cancers (<1% staining) account for approximately 25-30% of invasive breast carcinomas and include most HER2-enriched and triple-negative subtypes.

Invasive Carcinoma — Histologic Types

Invasive ductal carcinoma, no special type (NST) — 70-80% of all breast cancers. Forms irregular nests, cords, and sheets with desmoplastic stroma. Invasive lobular carcinoma (ILC) — 10-15%; characterized by discohesive single-file infiltration (Indian-file pattern), targetoid pattern around ducts, and frequent multifocality/bilaterality. Classic ILC is typically ER+/PR+/HER2-, E-cadherin negative (due to CDH1 mutation/loss). Absence of E-cadherin staining by IHC is a key diagnostic feature distinguishing lobular from ductal carcinoma. Special types with favorable prognosis: tubular (>90% well-formed tubules), mucinous/colloid (abundant extracellular mucin), cribriform, adenoid cystic. Special types with poor prognosis: micropapillary (frequent lymphovascular invasion), metaplastic (chemo-resistant, often triple-negative).

Molecular Subtypes

Breast cancer molecular classification is determined by ER, PR, HER2, and Ki-67 status:

07 GI Pathology

Gynecologic Pathology — Endometrial & Ovarian

Endometrial carcinoma is classified by the dualistic model: Type I (endometrioid) — 80% of cases, grade 1-3 by FIGO (grade 1: ≤5% solid, grade 2: 6-50% solid, grade 3: >50% solid); ER/PR positive, associated with estrogen excess, obesity, PCOS, tamoxifen; molecular: POLE ultramutated (excellent prognosis), MSI-H/dMMR, copy number-low (p53 wild-type), and copy number-high (p53 aberrant — worst prognosis even in endometrioid histology). Type II (non-endometrioid) — serous (p53 aberrant in ~90%, aggressive, TP53 mutation), clear cell, undifferentiated, carcinosarcoma (malignant mixed Mullerian tumor); estrogen-independent, arise in atrophic endometrium, poor prognosis. The TCGA molecular classification (ProMisE/TransPORTEC) now guides adjuvant therapy decisions: POLE-mutated (no adjuvant), dMMR (consider immunotherapy), p53-wildtype/NSMP (intermediate risk), p53-abnormal (aggressive therapy). All endometrial carcinomas should undergo MMR/MSI testing to screen for Lynch syndrome.

Ovarian carcinoma — high-grade serous carcinoma (HGSC) is the most common and lethal subtype, accounting for ~70% of ovarian cancers. Characterized by: solid/papillary architecture, high-grade nuclear atypia, slit-like spaces, psammoma bodies, ubiquitous TP53 mutations (~96%), BRCA1/2 mutations (germline ~15-20%, somatic ~6%), and HRD (~50%). IHC: PAX8+, WT1+, p53 aberrant (overexpression or null pattern), ER+ (variable), CK7+. Other epithelial ovarian tumors: low-grade serous (BRAF/KRAS mutated, p53 wild-type, indolent), endometrioid (associated with endometriosis, ARID1A/PIK3CA mutated), clear cell (associated with endometriosis, HNF1-beta+, ARID1A mutated, resistant to platinum), mucinous (CK20+/CDX2+ — must exclude GI metastasis).

Genitourinary Pathology — Prostate

Prostate cancer is diagnosed on needle biopsy (typically 12-core systematic biopsy ± MRI-targeted biopsy). Key elements in the pathology report: Gleason score and ISUP grade group (see section 25), percentage of each core involved by tumor, number of positive cores out of total, perineural invasion (associated with extraprostatic extension but not an independent adverse prognostic factor), lymphovascular invasion (rare on biopsy but adverse when present), extraprostatic extension (pT3a), seminal vesicle invasion (pT3b), and surgical margin status on radical prostatectomy. Intraductal carcinoma of the prostate (IDC-P) — a distinct pattern with solid/dense cribriform growth filling pre-existing ducts, often associated with high-grade and locally advanced disease; must be distinguished from high-grade PIN (less architecturally complex, smaller gland involvement). IHC for basal cell markers (p63, HMWCK/CK903, CK5/6) is used to confirm invasion: presence of basal cells = benign glands or PIN; absence = invasive carcinoma. AMACR (P504S, racemase) is positive in ~80% of prostate carcinomas but has limited specificity.

Genitourinary Pathology — Bladder

Urothelial carcinoma is the most common malignancy of the urinary bladder. The critical pathologic distinction is between non-muscle-invasive (NMIBC) and muscle-invasive (MIBC) disease, as this determines treatment (TURBT + intravesical therapy vs. radical cystectomy). Staging: Ta (papillary, non-invasive), Tis (CIS — flat, high-grade), T1 (lamina propria invasion), T2 (muscularis propria invasion — T2a inner half, T2b outer half), T3 (perivesical tissue), T4 (adjacent organs). Grading (WHO 2022): papillary urothelial neoplasm of low malignant potential (PUNLMP), low-grade urothelial carcinoma, high-grade urothelial carcinoma. Histologic variants with prognostic/therapeutic implications: micropapillary (aggressive, often upstaged), plasmacytoid (aggressive, diffuse invasion), nested (deceptively bland), sarcomatoid (very poor prognosis), squamous differentiation, and glandular differentiation. Muscularis propria (detrusor muscle) must be present in the specimen to adequately stage a T1 tumor — if absent, re-TURBT is indicated.

Soft Tissue Tumor Pathology

Soft tissue sarcomas are classified by line of differentiation and assigned a histologic grade using the FNCLCC (French Federation) grading system, the most widely used system: three parameters are scored: tumor differentiation (1-3), mitotic count (1-3: 0-9 mitoses per 10 HPF = 1, 10-19 = 2, ≥20 = 3), and tumor necrosis (0 = 0 points, <50% = 1, ≥50% = 2). Total 2-3 = Grade 1 (low), 4-5 = Grade 2 (intermediate), 6-8 = Grade 3 (high). Common sarcomas and their defining features: liposarcoma — well-differentiated (atypical lipomatous tumor, MDM2/CDK4 amplified by FISH or IHC), dedifferentiated (abrupt transition to non-lipogenic sarcoma, also MDM2-amplified), myxoid (t(12;16) FUS-DDIT3, characteristic chicken-wire vasculature, round cell component worsens prognosis), pleomorphic (high-grade, diagnosis of exclusion). Undifferentiated pleomorphic sarcoma (UPS) — diagnosis of exclusion after IHC and molecular testing exclude specific lineage. Solitary fibrous tumor — CD34+, STAT6+ (due to NAB2-STAT6 fusion, the defining molecular alteration); STAT6 nuclear IHC is highly sensitive and specific. Dermatofibrosarcoma protuberans (DFSP) — CD34+, storiform pattern, t(17;22) COL1A1-PDGFB translocation; locally aggressive but rarely metastasizes; imatinib is effective for unresectable/recurrent.

Bone Tumors — Key Entities

Osteosarcoma — the most common primary malignant bone tumor in children/adolescents; located in the metaphysis of long bones (distal femur, proximal tibia, proximal humerus); produces osteoid matrix. Subtypes: conventional (osteoblastic, chondroblastic, fibroblastic), telangiectatic, small cell, surface (parosteal — low grade; periosteal — intermediate grade; high-grade surface). Ewing sarcoma — second most common primary bone tumor in children; diaphyseal; small round blue cell tumor with membranous CD99+; characteristic translocations: t(11;22) EWSR1-FLI1 (~85%), t(21;22) EWSR1-ERG (~10%). Chondrosarcoma — most common primary malignant bone tumor in adults; produces hyaline cartilage matrix; graded 1-3; IDH1/2 mutations in ~50% of conventional type. Giant cell tumor of bone (GCTB) — epiphysis of long bones in young adults; multinucleated osteoclast-like giant cells uniformly distributed among mononuclear stromal cells; H3.3 G34W mutation (IHC stain available); denosumab (RANKL inhibitor) is effective.

Barrett Esophagus

Barrett esophagus is defined as intestinal metaplasia of the distal esophageal mucosa, characterized by the presence of goblet cells (confirmed by Alcian blue/PAS staining at pH 2.5). Dysplasia grading follows a two-tier system: low-grade dysplasia (LGD) — nuclear enlargement, hyperchromasia, and pseudostratification confined to the basal half of the epithelium with preserved surface maturation; high-grade dysplasia (HGD) — marked cytologic atypia extending to the luminal surface with loss of surface maturation, architectural complexity (cribriform, villiform), and atypical mitoses. HGD carries a 30-40% risk of concurrent or progression to adenocarcinoma and is an indication for endoscopic eradication therapy (radiofrequency ablation or endoscopic mucosal resection).

Gastric Carcinoma

The Lauren classification divides gastric adenocarcinoma into two main types: intestinal type — well-formed glands, cohesive cells, expanding growth pattern, associated with H. pylori, intestinal metaplasia, and environmental risk factors (diet, smoking); and diffuse type — discohesive cells with signet-ring morphology (intracellular mucin displacing the nucleus), infiltrative growth pattern (linitis plastica when transmural), associated with E-cadherin (CDH1) germline mutations in hereditary diffuse gastric cancer, and carries a worse prognosis. A third type, indeterminate/mixed, contains features of both.

Colon Polyp Classification

Colorectal Carcinoma — MSI/MMR Testing

All colorectal carcinomas should undergo mismatch repair (MMR) testing by IHC for MLH1, MSH2, MSH6, and PMS2, or microsatellite instability (MSI) testing by PCR. Loss of any MMR protein = mismatch repair deficient (dMMR) = MSI-high. MLH1 loss with concurrent PMS2 loss suggests either Lynch syndrome (germline MLH1 mutation) or sporadic hypermethylation of the MLH1 promoter — distinguish by BRAF V600E testing or MLH1 promoter methylation testing (if BRAF mutated or methylated, sporadic; if wild-type and unmethylated, refer for Lynch syndrome germline testing). Loss of MSH2 (with MSH6) or isolated MSH6 or PMS2 loss strongly suggests Lynch syndrome.

Colorectal Carcinoma — Key Pathologic Features

Colorectal adenocarcinoma is staged using the AJCC TNM system (8th edition). T-staging: Tis (carcinoma in situ / intramucosal), T1 (submucosa), T2 (muscularis propria), T3 (through muscularis into pericolorectal tissues — most common at presentation), T4a (penetrates visceral peritoneum), T4b (directly invades other organs). Key pathologic features: tumor type (adenocarcinoma NOS, mucinous ≥50% mucin, signet ring cell ≥50% signet ring cells — poor prognosis, medullary — MSI-H associated, good prognosis despite poor differentiation), tumor grade (low = well/moderately differentiated vs. high = poorly differentiated/undifferentiated), lymphovascular invasion, perineural invasion, tumor budding (single cells or clusters <5 cells at invasive front — Bd1 0-4 buds, Bd2 5-9, Bd3 ≥10 per 0.785 mm² hotspot — high budding predicts nodal metastasis and poor prognosis), and tumor deposits (discrete tumor foci in pericolorectal fat without identifiable lymph node tissue or vessel wall).

Crohn Disease vs. Ulcerative Colitis — Histologic Features

08 Pulmonary Pathology

Lung Carcinoma — WHO Classification

Adenocarcinoma (40% of lung cancers) — the most common histologic type, typically peripheral. Classified by predominant pattern: lepidic (growth along alveolar walls without invasion — formerly BAC), acinar (gland-forming), papillary, micropapillary (tufts without fibrovascular cores — aggressive), and solid (sheets without glands, confirmed by mucin stains). Grading: lepidic-predominant = well-differentiated; acinar/papillary = moderately differentiated; micropapillary/solid = poorly differentiated. IHC: TTF-1+, Napsin A+, CK7+, CK20-. Biomarker testing mandatory: EGFR, ALK, ROS1, BRAF, KRAS, MET, RET, NTRK, PD-L1.

Squamous cell carcinoma (25-30%) — typically central/hilar, arises from bronchial epithelium. Features: keratinization, intercellular bridges (desmosomes), keratin pearls. IHC: p40+ (most specific), CK5/6+, p63+, TTF-1 negative. Often associated with smoking. Cavitation common radiographically. Hypercalcemia via PTHrP secretion (paraneoplastic).

Small cell lung carcinoma (SCLC) (15%) — neuroendocrine, highly aggressive, almost exclusively in smokers. Small cells with scant cytoplasm, finely granular "salt-and-pepper" chromatin, nuclear molding, crush artifact, high mitotic rate (>10 mitoses/2 mm²), and extensive necrosis. IHC: synaptophysin+, chromogranin+, CD56+, TTF-1+ (~90%), Ki-67 typically >80%. Paraneoplastic syndromes: SIADH, Cushing syndrome (ectopic ACTH), Lambert-Eaton myasthenic syndrome. Treatment: chemo-immunotherapy (not surgery), limited stage may add radiation.

Large cell carcinoma — diagnosis of exclusion: undifferentiated NSCLC lacking features of adenocarcinoma, squamous, or neuroendocrine differentiation. Large cells with vesicular nuclei, prominent nucleoli, moderate cytoplasm. Large cell neuroendocrine carcinoma (LCNEC) has neuroendocrine morphology (rosettes, palisading, organoid nests) with positive neuroendocrine markers — behaves similarly to SCLC.

Mesothelioma

Malignant pleural mesothelioma is associated with asbestos exposure (latency 20-40 years). Three histologic types: epithelioid (best prognosis, 60%), sarcomatoid (worst prognosis, 20%), and biphasic (mixed, 20%). IHC for epithelioid mesothelioma: calretinin+ (nuclear and cytoplasmic), WT1+ (nuclear), D2-40 (podoplanin)+, CK5/6+; negative for carcinoma markers: CEA-, MOC31-, BerEP4-, TTF-1-, Napsin A-. BAP1 loss by IHC (nuclear loss) supports mesothelioma diagnosis and indicates BAP1 mutation. MTAP loss (due to 9p21 CDKN2A deletion) by IHC or CDKN2A deletion by FISH supports the diagnosis, especially in sarcomatoid type where IHC markers are less reliable.

Interstitial Lung Disease Patterns

Lung Cancer Biomarker Testing — Reflex Panel

NCCN and CAP/IASLC/AMP guidelines mandate reflex biomarker testing on all newly diagnosed advanced NSCLC (adenocarcinoma and non-squamous histology, and consider for squamous in never-smokers). The minimum required panel includes: EGFR mutations (exon 19 deletion, L858R, exon 20 insertion — prevalence ~15% in Western, ~50% in Asian populations), ALK rearrangement (~5%), ROS1 rearrangement (~1-2%), BRAF V600E (~2%), KRAS G12C (~13%), MET exon 14 skipping (~3%), RET rearrangement (~1-2%), NTRK fusion (~0.2%), HER2 (ERBB2) mutations (~2%), and PD-L1 expression by IHC (all NSCLC). Broad NGS panels that test all of these simultaneously are preferred over sequential single-gene testing because they conserve tissue, reduce turnaround time, and may identify rare actionable mutations. If tissue is insufficient, liquid biopsy (ctDNA) should be performed. PD-L1 testing must still be performed on tissue (not ctDNA) using a validated antibody/platform combination.

09 Dermatopathology

Melanoma

Breslow thickness is the single most important prognostic factor for cutaneous melanoma, measured in millimeters from the granular layer of the epidermis (or ulcer base if ulcerated) to the deepest point of tumor invasion. AJCC 8th edition T-staging: T1 ≤1.0 mm (T1a <0.8 mm without ulceration; T1b <0.8 mm with ulceration or 0.8-1.0 mm with or without ulceration); T2 >1.0-2.0 mm (a=no ulceration, b=ulceration); T3 >2.0-4.0 mm; T4 >4.0 mm.

The Clark levels describe anatomic depth of invasion: Level I — in situ (confined to epidermis); Level II — invasion into papillary dermis; Level III — fills papillary dermis to the papillary-reticular junction; Level IV — invasion into reticular dermis; Level V — invasion into subcutaneous fat. Clark level has been largely supplanted by Breslow thickness for staging but remains relevant for thin melanomas where it may influence sentinel node biopsy decisions.

Additional prognostic factors reported in the melanoma pathology report: ulceration (upstages T category; associated with worse prognosis), mitotic rate (number per mm²; previously part of T1 staging in AJCC 7th edition, remains prognostically important), lymphovascular invasion, perineural invasion, microsatellites (discrete tumor nests >0.05 mm separated from the main tumor by normal tissue — classified as N2c in AJCC 8th edition), regression (fibrosis replacing melanoma — controversial significance), and tumor-infiltrating lymphocytes (TILs) (brisk = better prognosis).

Basal Cell Carcinoma (BCC)

Most common skin cancer. Histology: basaloid cells with peripheral palisading of nuclei, retraction artifact (clefting between tumor and stroma), myxoid stroma. Subtypes: nodular (most common; large nests with central necrosis), superficial (buds from epidermis, multifocal), infiltrative/morpheiform (thin cords and strands in sclerotic stroma — aggressive, higher recurrence), micronodular (small nests, aggressive). BCC virtually never metastasizes (<0.1%) but is locally destructive. Hedgehog pathway mutations (PTCH1) are central; vismodegib/sonidegib target this pathway in advanced BCC.

Squamous Cell Carcinoma (SCC)

Cutaneous SCC arises from keratinocytes, often in sun-damaged skin with actinic keratoses (AK) as precursors. Histology: irregular nests of squamous cells with keratinization, intercellular bridges, and variable differentiation. Well-differentiated: abundant keratinization and keratin pearl formation. Poorly differentiated: minimal keratinization, high mitotic rate, may require IHC (p40+, CK5/6+) for confirmation. High-risk features: thickness >6 mm, invasion beyond subcutaneous fat, perineural invasion (nerve diameter ≥0.1 mm), poor differentiation, and immunosuppression (transplant recipients have 65-250x increased SCC risk).

Melanoma — Immunohistochemistry & Molecular

Melanocytic lesions are evaluated with a panel of IHC markers. SOX10 — most sensitive nuclear marker for melanocytes (~98% sensitivity), also marks nerve sheath tumors. S100 — highly sensitive but less specific (also positive in nerves, cartilage, Langerhans cells, adipocytes). HMB-45 — more specific for melanoma; characteristically shows a maturation pattern in benign nevi (positive superficially, negative deep — loss of this pattern favors melanoma). Melan-A/MART-1 — can highlight single melanocytes along the dermal-epidermal junction but overstains in sun-damaged skin, potentially overcalling pagetoid spread. PRAME (Preferentially Expressed Antigen in Melanoma) — a newer marker with diffuse nuclear positivity in melanoma (~75-88% of melanomas) and negativity in most benign nevi; increasingly used to distinguish ambiguous melanocytic lesions. p16 loss (CDKN2A deletion) — seen in melanoma but retained in benign nevi; useful in atypical Spitz tumors and ambiguous lesions. Molecular testing for melanoma: BRAF V600E/K (40-50%), NRAS (15-20%), KIT (acral/mucosal, 10-15%), and NF1 mutations. Gene expression profiling (e.g., DecisionDx-Melanoma, myPath Melanoma) provides additional prognostic or diagnostic information in challenging cases.

Inflammatory Dermatoses — Pattern-Based Approach

10 Cervical Cytology — Pap Smear (Bethesda System)

The Bethesda System for Reporting Cervical Cytology (TBS, 3rd edition 2014) standardizes cervical Pap smear reporting into the following categories:

Cervical Histology — CIN Terminology

The histologic correlate of Pap smear findings is graded by cervical intraepithelial neoplasia (CIN): CIN 1 (mild dysplasia, limited to lower 1/3 of epithelium; corresponds to LSIL on cytology; most regress spontaneously, especially in young women — HPV cytopathic effect), CIN 2 (moderate dysplasia, extending into middle 1/3; heterogeneous — some regress, some progress; p16 IHC is used to determine biologic significance: p16+ CIN 2 is treated as precancer, p16- CIN 2 may be observed in women <25), CIN 3 / carcinoma in situ (severe dysplasia, full-thickness atypia; high risk of progression to invasive carcinoma if untreated; treated with excision — LEEP or cold knife cone). p16 IHC (block-type positive = continuous strong nuclear and cytoplasmic staining) serves as a surrogate marker for HPV E7 oncoprotein activity and is recommended by the LAST Project to distinguish CIN 2+ from mimics (immature squamous metaplasia, atrophy, repair).

Specimen Adequacy

Satisfactory for evaluation — minimum 5,000 well-preserved, well-visualized squamous cells on a liquid-based preparation (or 8,000-12,000 on conventional smear); presence of endocervical/transformation zone (EC/TZ) component is noted but does not determine adequacy. Unsatisfactory for evaluation — obscuring blood, inflammation, or insufficient cellularity rendering >75% of cells uninterpretable; specimen must be repeated.

Bethesda Categories — Full Enumeration

HPV Types

High-risk HPV types (oncogenic): 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68. HPV 16 and 18 together account for approximately 70% of cervical cancers and are individually genotyped in most testing platforms. HPV 16 has the highest oncogenic potential and is associated with squamous cell carcinoma. HPV 18 is disproportionately associated with adenocarcinoma. Low-risk HPV types (non-oncogenic): 6, 11, 42, 43, 44 — cause condylomata acuminata (genital warts) and low-grade changes but do not progress to cancer. The 9-valent HPV vaccine (Gardasil 9) covers types 6, 11, 16, 18, 31, 33, 45, 52, and 58.

11 Fine Needle Aspiration — Thyroid (Bethesda System)

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), 3rd edition (2023), classifies thyroid FNA results into six diagnostic categories, each with an implied risk of malignancy (ROM) and recommended management:

Thyroid Histology — Carcinoma Subtypes

Papillary thyroid carcinoma (PTC) — most common thyroid malignancy (~85%); characteristic nuclear features: "Orphan Annie" cleared nuclei, nuclear grooves, intranuclear pseudoinclusions, papillary architecture, psammoma bodies; BRAF V600E (~45%) and RAS mutations; variants include classic, follicular (NIFTP — noninvasive follicular thyroid neoplasm with papillary-like nuclear features is now reclassified as non-malignant). Follicular thyroid carcinoma — cannot be diagnosed on FNA (requires capsular/vascular invasion on excision); RAS mutations, PAX8-PPARG fusion; minimally invasive (capsular only) has excellent prognosis; widely invasive has poor prognosis. Medullary thyroid carcinoma (MTC) — C-cell origin, produces calcitonin (serum calcitonin is the tumor marker); amyloid deposition (Congo red+) from calcitonin polymerization; IHC: calcitonin+, chromogranin+, synaptophysin+, CEA+, TTF-1+; 25% are hereditary (MEN2A, MEN2B, familial MTC) — all patients should be tested for RET mutation. Anaplastic (undifferentiated) thyroid carcinoma — most aggressive thyroid cancer with median survival ~5 months; rapidly growing neck mass; often arises from preexisting differentiated carcinoma; TP53 and BRAF mutations common; BRAF V600E+ tumors may respond to dabrafenib + trametinib.

FNA Technique & Adequacy

Fine needle aspiration uses a 22-27 gauge needle (with or without suction via syringe) to obtain cellular material from palpable or image-guided lesions. Rapid on-site evaluation (ROSE) by a cytopathologist or cytotechnologist at the bedside involves immediate Diff-Quik staining of air-dried smears to assess specimen adequacy and provide a preliminary diagnosis, reducing the non-diagnostic rate from ~15-20% to <5% and enabling appropriate triage of material for ancillary studies (cell block, flow cytometry, molecular). Cell block preparation (formalin-fixed clot of aspirated material) is essential — it enables IHC and molecular testing identical to tissue biopsies. FNA advantages: minimally invasive, rapid, low cost, high specificity. Limitations: small sample (may not represent tumor heterogeneity), architecture not preserved (cannot assess invasion), and certain diagnoses require excision (follicular thyroid neoplasm, well-differentiated lymphoma vs. reactive).

Molecular Testing in Thyroid FNA

Afirma Genomic Sequencing Classifier (GSC) — a "rule-out" test with high negative predictive value (~96%); a benign GSC result in Bethesda III/IV allows observation instead of surgery. ThyroSeq v3 — a "rule-in" and "rule-out" test that sequences 112 genes for mutations (BRAF V600E, RAS, RET/PTC, PAX8/PPARG, TERT promoter) and gene fusions; a negative result has ~97% NPV; specific mutations (BRAF V600E, TERT promoter) have near-100% PPV for malignancy. These molecular tests are most useful for Bethesda III and IV nodules where the ROM is intermediate and clinical decision-making is uncertain.

12 Non-Gynecologic Cytology

Effusion Cytology

Pleural, peritoneal (ascitic), and pericardial fluids are evaluated for malignant cells, infection, and other abnormalities. Key processing: cell block preparation (centrifuged pellet processed like tissue — allows IHC and molecular studies) and cytospin/ThinPrep (for morphologic evaluation). Reactive mesothelial cells (activated by inflammation, heart failure, or infection) can mimic malignancy — they show "windows" (intercellular spaces), two-tone cytoplasm (dense ectoplasm and pale endoplasm), and bland nuclei. Malignant effusions are most commonly caused by: lung adenocarcinoma (pleural), ovarian carcinoma (peritoneal), and breast carcinoma (pleural). IHC on cell block is critical: BerEP4+/calretinin- favors adenocarcinoma; calretinin+/WT1+/BerEP4- favors mesothelioma; TTF-1/Napsin A for lung primary; PAX8/WT1 for ovarian/Mullerian primary.

Urine Cytology — The Paris System (TPS)

The Paris System for Reporting Urinary Cytology (TPS) (2nd edition, 2022) standardizes urine cytology reporting into categories with implied risk of high-grade urothelial carcinoma (HGUC). The key diagnostic criterion is the nuclear-to-cytoplasmic (N:C) ratio, with >0.7 being the threshold for HGUC. Important: voided specimens have higher background degeneration than instrumented specimens (catheterized, barbotage, upper tract washings); interpretation must account for collection method:

CSF & Bronchoalveolar Lavage (BAL) Cytology

CSF cytology — indicated for suspected leptomeningeal carcinomatosis, CNS lymphoma, and leukemic meningitis. Sensitivity improves with: larger volume (≥10 mL), immediate processing (cells degrade rapidly in CSF), and multiple samples (sensitivity ~50% first tap, ~80% after 3 taps). Malignant cells appear as clusters (carcinoma) or single cells (lymphoma, melanoma) with high N:C ratio and hyperchromatic nuclei. Flow cytometry on CSF is more sensitive than cytology for lymphoma/leukemia. BAL cytology — bronchoalveolar lavage evaluates: differential cell count (normal: >80% macrophages, <3% neutrophils, <1% eosinophils, 10-15% lymphocytes); elevated lymphocytes suggest sarcoidosis (CD4:CD8 >3.5:1), hypersensitivity pneumonitis (CD4:CD8 <1), drug reaction; elevated eosinophils (>25%) suggest eosinophilic pneumonia; elevated neutrophils suggest infection or ARDS; Pneumocystis jirovecii (GMS stain on BAL has ~95% sensitivity in HIV patients); pulmonary alveolar proteinosis (milky BAL, PAS-positive granular material).

Pancreaticobiliary FNA — Papanicolaou Society

The Papanicolaou Society of Cytopathology (PSC) guidelines for pancreatobiliary cytology standardize reporting using a six-tier system: Nondiagnostic, Negative (benign), Atypical, Neoplastic (benign — e.g., serous cystadenoma), Neoplastic (other — e.g., mucinous cystic neoplasm, IPMN, solid pseudopapillary neoplasm, NET), Suspicious for Malignancy, and Positive (Malignant — adenocarcinoma). For pancreatic cyst fluid, key ancillary tests include CEA level (>192 ng/mL suggests mucinous cyst), amylase (elevated in pseudocyst and IPMN), cytology (mucin-producing columnar cells), and molecular analysis (KRAS/GNAS mutations support mucinous neoplasm; VHL mutation supports serous cystadenoma).

13 Peripheral Blood Smear & CBC

RBC Morphology & Disease Associations

WBC Differential

Normal differential: neutrophils 40-70% (2,000-7,500/µL), lymphocytes 20-40% (1,500-4,000/µL), monocytes 2-8% (200-800/µL), eosinophils 1-4% (100-500/µL), basophils 0.5-1% (25-100/µL). Left shift = increased band forms (>6%) and/or immature granulocytes (metamyelocytes, myelocytes) — indicates acute infection, inflammation, or stress. Leukoerythroblastic picture (nucleated RBCs + immature WBCs in peripheral blood) suggests bone marrow infiltration (myelofibrosis, metastatic carcinoma, granulomatous disease). Auer rods — pink rod-shaped crystalline inclusions in myeloblasts; pathognomonic for AML (especially APL with t(15;17) — "faggot cells" contain bundles of Auer rods).

Platelet Estimate

Each platelet observed per oil-immersion field (100x) in the platelet-rich monolayer zone represents approximately 10,000-15,000/µL. Average 7-21 platelets per field suggests a normal count of ~150,000-300,000/µL. Large/giant platelets suggest accelerated thrombopoiesis (ITP, MPN) or inherited macrothrombocytopenias (Bernard-Soulier, MYH9-related). Platelet clumping (pseudothrombocytopenia) is an EDTA-dependent artifact — repeat with citrate or heparin anticoagulant.

RBC Indices & Anemia Classification

Anemias are classified by MCV: microcytic (MCV <80 fL) — iron deficiency (low ferritin, high TIBC), thalassemia (normal/high iron, target cells, Mentzer index MCV/RBC <13 favors thalassemia), anemia of chronic disease (low iron, low TIBC, normal/high ferritin), sideroblastic anemia (ring sideroblasts on marrow iron stain), lead poisoning (basophilic stippling). Normocytic (MCV 80-100 fL) — acute blood loss, hemolytic anemias (elevated LDH, low haptoglobin, elevated indirect bilirubin, reticulocytosis), anemia of chronic disease, aplastic anemia, bone marrow infiltration, early iron/B12 deficiency. Macrocytic (MCV >100 fL) — megaloblastic (B12 or folate deficiency: hypersegmented neutrophils ≥5 lobes, macro-ovalocytes) vs. non-megaloblastic (alcoholism, liver disease, hypothyroidism, MDS, reticulocytosis, medications — methotrexate, hydroxyurea, azathioprine).

14 Bone Marrow Evaluation

Aspiration vs. Biopsy

The bone marrow aspirate provides material for: morphologic assessment on smear (cell counts, blast percentage, dysplasia), flow cytometry (immunophenotyping), cytogenetics (karyotype), molecular studies (PCR, NGS), and iron stain. The bone marrow trephine biopsy (core biopsy, typically 1.5-2 cm from the posterior superior iliac spine) provides: overall cellularity assessment, architecture (fibrosis, granulomas, metastatic deposits), megakaryocyte morphology, and reticulin/trichrome staining for fibrosis grading. Both aspirate and biopsy should be performed together for a complete evaluation. A "dry tap" (failure to aspirate) suggests fibrosis (myelofibrosis) or packed marrow (acute leukemia, metastatic carcinoma) — the biopsy becomes essential.

Cellularity Assessment

Marrow cellularity is reported as the percentage of hematopoietic tissue relative to fat. The rough guideline is that normal cellularity = 100 minus the patient's age (±10%): a 30-year-old should have ~70% cellularity, a 70-year-old ~30%. Hypercellularity suggests myeloproliferative neoplasm, leukemia, or regenerating marrow. Hypocellularity suggests aplastic anemia, post-chemotherapy, or age-related change.

M:E Ratio & Blast Percentage

The myeloid-to-erythroid (M:E) ratio is normally 2:1 to 4:1. Increased M:E suggests myeloid hyperplasia (CML, infection, G-CSF effect). Decreased M:E suggests erythroid hyperplasia (hemolytic anemia, blood loss, megaloblastic anemia) or myeloid hypoplasia. The blast percentage is determined from a 500-cell differential on the aspirate smear: normal <5%; AML is defined as ≥20% blasts (WHO 5th edition, 2022), except for AML with defining genetic abnormalities (e.g., t(8;21), inv(16), t(15;17)) where the 20% threshold is not required. MDS-related blast thresholds: MDS with increased blasts-1 (MDS-IB1) = 5-9%; MDS-IB2 = 10-19%.

Iron Stores

Iron staining (Prussian blue) on the aspirate reveals: storage iron (hemosiderin in macrophages — graded 0-6 on the Gale scale; absent = iron deficiency, increased = iron overload/sideroblastic anemia) and ring sideroblasts (erythroblasts with ≥5 iron granules encircling ≥1/3 of the nucleus — indicates mitochondrial iron loading). Ring sideroblasts are the hallmark of: MDS with ring sideroblasts (MDS-RS, SF3B1 mutation in ~90%), sideroblastic anemia, and are also seen with lead poisoning, isoniazid, alcohol, and copper deficiency.

Flow Cytometry

Flow cytometry analyzes cell surface and intracellular markers on single-cell suspensions using fluorochrome-conjugated antibodies and laser excitation. In hematopathology, flow cytometry is essential for: immunophenotyping of leukemia/lymphoma (blast vs. mature, B-cell vs. T-cell vs. myeloid), detecting aberrant antigen expression (e.g., CD5 on B cells = CLL or MCL; CD10 on mature B cells = follicular lymphoma), MRD detection (sensitivity 10−3 to 10−4), PNH screening (loss of GPI-anchored proteins CD55/CD59 on RBCs and granulocytes using FLAER), and DNA ploidy analysis. Standard panels include: a screening tube (CD45 vs. side scatter to gate populations), B-cell panel (CD19, CD20, CD10, CD5, CD23, kappa/lambda light chains, CD38, CD200), T-cell panel (CD3, CD4, CD8, CD7, CD2, CD5, CD56), and myeloid panel (CD13, CD33, CD117, HLA-DR, CD34, CD64, CD14, CD11b). Light chain restriction (kappa:lambda ratio >4:1 or <0.5:1) is the hallmark of B-cell monoclonality.

Reticulin Fibrosis Grading

15 Lymphoma Classification — WHO

Hodgkin Lymphoma (HL)

Characterized by Reed-Sternberg (RS) cells — large, binucleated or multinucleated cells with prominent eosinophilic nucleoli ("owl-eye" appearance) in a background of reactive inflammatory cells (lymphocytes, eosinophils, histiocytes, plasma cells). IHC of classic HL RS cells: CD15+, CD30+, CD20- (or weak), CD45/LCA-, PAX5 weak. EBV-associated in ~40% (EBER in situ hybridization positive).

Classic Hodgkin Lymphoma (cHL) Subtypes

Nodular Lymphocyte-Predominant HL (NLPHL)

A distinct entity separate from cHL. Features "LP cells" (lymphocytic and histiocytic cells, "popcorn cells") — large cells with folded, lobulated nuclei. IHC: CD20+, CD45+, BCL-6+, OCT2+, BOB.1+, CD15-, CD30- (opposite of cHL RS cells). Indolent course, excellent prognosis; may transform to DLBCL (T-cell/histiocyte-rich variant) in ~5-10% of cases.

Non-Hodgkin Lymphoma (NHL) — Major Entities

B-Cell Lymphomas

Diffuse large B-cell lymphoma (DLBCL) — most common NHL (30-40%). Large cells with vesicular nuclei, prominent nucleoli, diffuse growth pattern. Cell of origin by IHC (Hans algorithm): GCB type (CD10+ or BCL6+/MUM1-) vs. non-GCB/ABC type (MUM1+). GCB has better prognosis. R-CHOP is standard therapy.

Follicular lymphoma (FL) — second most common NHL. Neoplastic follicles with loss of normal mantle zone, composed of centrocytes (small cleaved) and centroblasts (large). Grading by centroblast count per HPF: Grade 1 (0-5 centroblasts/HPF), Grade 2 (6-15/HPF), Grade 3A (>15/HPF but centrocytes still present), Grade 3B (sheets of centroblasts without centrocytes — treated as DLBCL). IHC: CD20+, CD10+, BCL-2+ (abnormal in follicles — due to t(14;18) IGH-BCL2), BCL-6+, CD5-, cyclin D1-. Indolent but incurable with standard therapy; watch-and-wait for low-burden disease.

Mantle cell lymphoma (MCL) — small-to-medium cells with irregular nuclear contours. IHC: CD20+, CD5+, cyclin D1+ (due to t(11;14) CCND1-IGH), SOX11+, CD23-, CD10-. Aggressive despite small cell morphology. Blastoid and pleomorphic variants are highly aggressive.

Marginal zone lymphoma (MZL) — three types: extranodal MZL of mucosa-associated lymphoid tissue (MALT lymphoma) — stomach (H. pylori-associated, may regress with H. pylori eradication), lung, thyroid, orbit, salivary gland; splenic MZL; nodal MZL. IHC: CD20+, CD5-, CD10-, CD23-, cyclin D1-. Indolent.

Burkitt lymphoma — highly aggressive with the fastest doubling time of any human tumor (~24 hours). Medium-sized cells with "starry sky" pattern (tingible body macrophages clearing apoptotic debris). IHC: CD20+, CD10+, BCL-6+, BCL-2-, Ki-67 ~100%. Translocation: t(8;14) MYC-IGH (most common), t(2;8), t(8;22). Three clinical forms: endemic (African jaw tumors, EBV+), sporadic (abdominal, children), and immunodeficiency-associated (HIV). Treatment requires intensive chemotherapy regimens (not R-CHOP).

CLL/SLL — chronic lymphocytic leukemia (peripheral blood) and small lymphocytic lymphoma (tissue) are the same disease. Small mature lymphocytes with smudge cells on blood smear. IHC: CD20+ (dim), CD5+, CD23+, CD200+, cyclin D1-, LEF1+. Proliferation centers (pseudofollicles) in tissue. Prognostic markers: IGHV mutation status, TP53 deletion/mutation, del(17p), del(11q), trisomy 12, del(13q).

T-Cell Lymphomas

Peripheral T-cell lymphoma, NOS (PTCL-NOS) — most common T-cell NHL; heterogeneous group with poor prognosis. Angioimmunoblastic T-cell lymphoma (AITL) — CD4+ T follicular helper cell origin; expanded follicular dendritic cell meshworks, high endothelial venules, EBV+ B cells in background. Anaplastic large cell lymphoma (ALCL) — large pleomorphic cells ("hallmark cells" with kidney-shaped nuclei); ALK+ type (t(2;5) NPM1-ALK; younger patients, better prognosis) vs. ALK- type (older patients, worse prognosis). IHC: CD30+, EMA+, ALK+ or ALK-, CD20-, PAX5-. Adult T-cell leukemia/lymphoma (ATLL) — caused by HTLV-1; "flower cells" on smear; acute form has poor prognosis. Mycosis fungoides/Sézary syndrome — cutaneous T-cell lymphoma; epidermotropic atypical lymphocytes with cerebriform nuclei, Pautrier microabscesses in epidermis.

Myeloma & Plasma Cell Neoplasms

Multiple myeloma — clonal plasma cell neoplasm with ≥10% bone marrow plasma cells or biopsy-proven plasmacytoma plus one or more myeloma-defining events: CRAB criteria (Calcium >11 mg/dL, Renal insufficiency Cr >2, Anemia Hgb <10, Bone lesions) or biomarkers of malignancy (bone marrow plasma cells ≥60%, serum free light chain ratio ≥100, >1 focal MRI lesion). IHC: CD138+, CD38+, with aberrant expression of CD56 (absent on normal plasma cells), CD117, or CD20. Light chain restriction confirmed by ISH for kappa/lambda mRNA or IHC. Revised International Staging System (R-ISS): Stage I (serum beta-2 microglobulin <3.5, albumin ≥3.5, standard risk cytogenetics, normal LDH), Stage II (not I or III), Stage III (beta-2 microglobulin ≥5.5 + high-risk cytogenetics or elevated LDH). High-risk cytogenetics: t(4;14), t(14;16), t(14;20), del(17p), gain(1q). MGUS (monoclonal gammopathy of undetermined significance) — M protein <3 g/dL, bone marrow plasma cells <10%, no CRAB; 1% per year risk of progression to myeloma.

Ann Arbor Staging (Modified, Lugano Classification)

International Prognostic Index (IPI) for DLBCL — 5 adverse factors: age >60, stage III/IV, elevated LDH, ECOG PS ≥2, >1 extranodal site. Score: 0-1 = low risk, 2 = low-intermediate, 3 = high-intermediate, 4-5 = high risk. The Follicular Lymphoma IPI (FLIPI) uses: age >60, stage III/IV, Hgb <12, >4 nodal areas, elevated LDH.

Suffix A = no B symptoms; B = B symptoms present (fever >38°C, drenching night sweats, unintentional weight loss >10% body weight in 6 months). "Bulky disease" is defined as a single nodal mass ≥10 cm or >1/3 of the transthoracic diameter on chest X-ray (specific to Hodgkin lymphoma).

16 Leukemia Classification

Acute Myeloid Leukemia (AML) — WHO 5th Edition

AML is defined by ≥20% myeloid blasts in blood or marrow, except for AML with defining genetic abnormalities (which can be diagnosed regardless of blast count). Major categories:

Acute Promyelocytic Leukemia (APL) — Medical Emergency

APL (t(15;17) PML-RARA) is a medical emergency due to life-threatening DIC with hyperfibrinolysis. The diagnosis must be suspected based on morphology (hypergranular promyelocytes) and confirmed with FISH or RT-PCR. ATRA (all-trans retinoic acid) must be started immediately on clinical suspicion — do not wait for confirmatory testing. ATRA + arsenic trioxide (ATO) has transformed APL from the most fatal to the most curable AML subtype (>90% cure rate). The microgranular/hypogranular variant has bilobed nuclei and few visible granules but still carries the t(15;17) — flow cytometry is key (CD13+, CD33+, CD117+, HLA-DR-).

Acute Lymphoblastic Leukemia (ALL)

B-cell ALL (B-ALL) — 85% of childhood ALL. Blasts are TdT+, CD19+, CD10+ (CALLA), CD22+, PAX5+. Favorable cytogenetics: hyperdiploidy (>50 chromosomes), t(12;21) ETV6-RUNX1. Adverse: Philadelphia chromosome (Ph+) t(9;22) BCR-ABL1 (25% of adult ALL, 3% of pediatric), Philadelphia-like (Ph-like) ALL, KMT2A (MLL) rearrangement, hypodiploidy (<44 chromosomes). T-cell ALL (T-ALL) — 15% of childhood ALL. Blasts are TdT+, CD3 (cytoplasmic)+, CD7+, CD2+, CD5+, CD4/CD8 may be double positive. Often presents with mediastinal mass (thymic). NOTCH1/FBXW7 mutations common (favorable).

Chronic Myeloid Leukemia (CML)

CML is defined by the Philadelphia chromosome t(9;22)(q34.1;q11.2) producing the BCR-ABL1 fusion protein (p210), a constitutively active tyrosine kinase. Three phases: chronic phase (leukocytosis with full myeloid maturation, basophilia, <10% blasts), accelerated phase (10-19% blasts, ≥20% basophils, persistent thrombocytopenia, additional cytogenetic abnormalities), blast crisis (≥20% blasts — myeloid in 70%, lymphoid in 30%). Treatment: tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, bosutinib, ponatinib). Molecular monitoring by RT-qPCR for BCR-ABL1 transcript levels (IS scale); major molecular response = BCR-ABL1 ≤0.1% IS.

Chronic Lymphocytic Leukemia (CLL) — Staging

Myeloproliferative Neoplasms (MPN)

MPNs are clonal stem cell neoplasms with effective (non-dysplastic) hematopoiesis and increased peripheral blood counts. The classic BCR-ABL1-negative MPNs share driver mutations in JAK2 (V617F in ~95% of PV, ~55% of ET and PMF; exon 12 in ~3% of PV), CALR (exon 9 frameshift in ~25% of ET and PMF), or MPL (W515L/K in ~5% of ET and PMF). Polycythemia vera (PV) — panmyelosis with erythroid predominance; diagnostic: Hgb >16.5 g/dL (male) or >16 g/dL (female), bone marrow hypercellularity with trilineage proliferation, JAK2 mutation, low EPO. Essential thrombocythemia (ET) — megakaryocytic proliferation with large, hyperlobulated megakaryocytes; platelets ≥450,000/µL; must exclude CML, PV, PMF, MDS. Primary myelofibrosis (PMF) — megakaryocytic atypia (cloud-like, hypolobulated nuclei in clusters) with reticulin/collagen fibrosis; prefibrotic PMF (MF-0 to MF-1) vs. overt fibrotic PMF (MF-2 to MF-3); leukoerythroblastic picture, dacrocytes, splenomegaly. The DIPSS-plus score stratifies PMF prognosis.

Myelodysplastic Syndromes (MDS)

MDS are clonal hematopoietic stem cell neoplasms characterized by ineffective hematopoiesis, cytopenias, and morphologic dysplasia in ≥1 lineage (≥10% dysplastic cells). WHO 5th edition classifies MDS by: morphology, blast percentage, ring sideroblasts, cytogenetics, and specific mutations. The IPSS-R (Revised International Prognostic Scoring System) stratifies MDS into 5 risk categories (very low, low, intermediate, high, very high) based on: cytogenetics (5 categories — very good through very poor), marrow blast percentage, hemoglobin, platelet count, and absolute neutrophil count. IPSS-R very-low risk: median survival ~8.8 years; very-high risk: ~0.8 years. Treatment ranges from observation (low risk) to hypomethylating agents (azacitidine, decitabine) and allogeneic stem cell transplant (high risk, if eligible).

17 PCR & Sequencing

Polymerase Chain Reaction (PCR)

Conventional PCR amplifies a specific DNA target using primers flanking the region of interest, Taq polymerase, and thermal cycling (denaturation 95°C, annealing 50-65°C, extension 72°C). Product is visualized by gel electrophoresis. Used for: pathogen detection, T-cell and B-cell clonality studies (TCR/IgH gene rearrangement), translocation detection. Reverse transcription PCR (RT-PCR) first converts RNA to cDNA using reverse transcriptase, then amplifies — used to detect fusion transcripts (BCR-ABL1) and viral RNA (HIV, HCV). Real-time/quantitative PCR (qPCR) monitors amplification in real-time using fluorescent probes (TaqMan) or dyes (SYBR Green); results expressed as Ct (cycle threshold) value — lower Ct = more target. Applications: viral load monitoring (HIV, HCV, CMV, EBV, BK virus), BCR-ABL1 molecular monitoring in CML, minimal/measurable residual disease (MRD) detection.

Digital PCR (dPCR)

Digital PCR partitions the sample into thousands to millions of individual reactions (droplet digital PCR uses water-in-oil emulsion droplets). Each partition contains zero or one target molecule, enabling absolute quantification without a standard curve. Sensitivity reaches 0.01-0.1% variant allele frequency — superior to qPCR. Clinical applications: detecting low-frequency resistance mutations (EGFR T790M in plasma), MRD monitoring (BCR-ABL1 at very low levels), cfDNA quantification, and copy number variation analysis. dPCR is being adopted for liquid biopsy applications where detecting rare mutations in a high background of wild-type DNA is critical.

Sanger Sequencing

Sanger sequencing (dideoxy chain termination) remains the gold standard for confirming point mutations identified by other methods. It reads one strand at a time with a limit of ~800-1000 base pairs per read. Sensitivity is limited to detecting mutations present in ≥15-20% of alleles — inadequate for low-frequency variants or heterogeneous tumor samples. It is still used for: BRAF V600E confirmation, single-gene testing (IDH1/2, KRAS), and verification of NGS findings.

RNA Sequencing & Gene Expression Profiling

RNA sequencing (RNA-seq) quantifies gene expression levels and detects fusion transcripts from total RNA extracted from tumor tissue. In clinical practice, RNA-based fusion panels (e.g., Archer FusionPlex, TruSight RNA) are increasingly used alongside DNA-based NGS because some gene fusions (ALK, ROS1, RET, NTRK) are more reliably detected at the RNA level — DNA-based detection can miss fusions with intronic breakpoints in large genes. Gene expression profiling platforms include: Oncotype DX (21-gene recurrence score for breast cancer — see section 6), Decipher (22-gene classifier for prostate cancer — predicts metastasis after prostatectomy), MammaPrint (70-gene signature for breast cancer risk stratification), and NanoString Prosigna (PAM50) (50-gene intrinsic subtype classifier for breast cancer — Luminal A/B, HER2-enriched, basal-like). RNA-based assays require adequate RNA quality (RNA integrity number RIN ≥4-5) and are more susceptible to degradation from prolonged fixation or decalcification than DNA-based tests.

Next-Generation Sequencing (NGS)

NGS enables massively parallel sequencing of millions of DNA fragments simultaneously. Three major approaches: targeted panels (50-500 genes; most common in clinical oncology — e.g., MSK-IMPACT, FoundationOne CDx, Tempus xT; sufficient for actionable mutation detection with ~500x coverage), whole exome sequencing (WES) (~20,000 genes, all coding regions; used in research and some inherited disease diagnostics), and whole genome sequencing (WGS) (entire genome including non-coding regions; highest resolution for structural variants and regulatory mutations). Clinical NGS requires ≥20% tumor cellularity for reliable somatic variant detection, though some assays perform at lower cellularity with increased sequencing depth.

Methylation Profiling & Epigenetics

DNA methylation profiling analyzes genome-wide methylation patterns (typically using Illumina EPIC array with ~850,000 CpG sites) to classify tumors based on their epigenetic signature. This is particularly powerful in CNS tumors where the WHO 5th edition recognizes methylation-defined entities and methylation profiling can reclassify up to 10-15% of histologically diagnosed brain tumors. Applications: distinguishing medulloblastoma subgroups (WNT, SHH, Group 3, Group 4), classifying ependymoma subtypes, resolving diagnostic uncertainty in glial tumors, and detecting MGMT promoter methylation status. MGMT promoter methylation — detected by methylation-specific PCR or pyrosequencing; methylated tumors have silenced MGMT (DNA repair enzyme) and respond better to temozolomide in glioblastoma. Methylation profiling is also emerging in sarcoma classification and carcinoma of unknown primary identification.

Circulating Tumor DNA (ctDNA) & Liquid Biopsy

Liquid biopsy analyzes cell-free DNA (cfDNA) in plasma, of which a fraction is circulating tumor DNA (ctDNA) shed from tumor cells. Applications: detection of resistance mutations (EGFR T790M in lung cancer without re-biopsy), MRD monitoring post-treatment, tumor genotyping when tissue is insufficient, and treatment response monitoring. Tumor mutational burden (TMB) is the number of somatic mutations per megabase (mut/Mb) of coding genome — TMB-high is defined as ≥10 mut/Mb (FDA-approved tissue-based threshold for pembrolizumab across solid tumors via FoundationOne CDx). Higher TMB generally correlates with greater neoantigen burden and better response to immune checkpoint inhibitors.

18 FISH & Cytogenetics

Fluorescence In Situ Hybridization (FISH)

FISH uses fluorescently labeled DNA probes that hybridize to complementary sequences on chromosomes or tissue sections. Probe types: break-apart probes (two differently colored probes flank a gene; separation indicates rearrangement — used for ALK, ROS1, RET, MYC), dual-fusion probes (probes on two different chromosomes; fusion signal indicates translocation — used for BCR-ABL1, IGH-BCL2, CCND1-IGH), and enumeration/centromere probes (count chromosome copies — detect aneuploidy, polysomy). FISH can be performed on tissue sections (interphase FISH) or metaphase spreads, and on paraffin-embedded tissue — a major advantage over conventional cytogenetics.

HER2 FISH Interpretation (ASCO/CAP 2018)

HER2 FISH evaluates gene amplification by counting HER2 signals and chromosome 17 centromere (CEP17) signals in ≥20 tumor cells. Results are reported as HER2/CEP17 ratio and average HER2 copy number per cell:

Key Translocations Detected by FISH

RNA-Based FISH & ISH

In situ hybridization (ISH) detects specific DNA or RNA sequences in tissue sections. Beyond FISH, other ISH methods include: chromogenic ISH (CISH/SISH) — uses chromogenic (DAB) or silver-based detection instead of fluorescence, allowing visualization on standard light microscope; used for HER2 (Dual ISH = DISH) as an alternative to FISH. EBER ISH (EBV-encoded small RNA) — the gold standard for detecting EBV in tissue; positive in EBV-associated malignancies: nasopharyngeal carcinoma, post-transplant lymphoproliferative disorder (PTLD), some Hodgkin lymphoma, Burkitt lymphoma, and NK/T-cell lymphoma (nasal type). RNA ISH (RNAscope) — a newer technology that detects mRNA transcripts in FFPE tissue with single-molecule sensitivity; applications include HPV genotyping (detects E6/E7 transcripts, more specific than p16 IHC for HPV-driven oropharyngeal carcinoma), PD-L1 mRNA, and pathogen detection.

Conventional Karyotyping & Microarray

Conventional karyotyping (G-banding) requires viable dividing cells cultured in vitro; analyzes all 46 chromosomes for numerical and structural abnormalities. Resolution ~5-10 Mb — cannot detect small mutations or microdeletions. Essential for: AML/MDS cytogenetic risk stratification, CLL prognostic markers, constitutional karyotyping. Chromosomal microarray (CMA) — either comparative genomic hybridization (aCGH) or SNP array — detects copy number variations (gains, losses) and regions of loss of heterozygosity (LOH) at ~50-100 kb resolution. Does not detect balanced translocations (no copy number change). Used for: constitutional anomalies (intellectual disability, developmental delay), tumor profiling, and uniparental disomy detection (SNP array).

19 Companion Diagnostics & Precision Medicine

PD-L1 Scoring

PD-L1 (programmed death-ligand 1) expression by IHC guides immune checkpoint inhibitor therapy. Two major scoring methods: Tumor Proportion Score (TPS) — percentage of viable tumor cells with partial or complete membrane PD-L1 staining (used in NSCLC with the 22C3 antibody clone for pembrolizumab: TPS ≥50% = first-line monotherapy, TPS 1-49% = pembrolizumab + chemotherapy); Combined Positive Score (CPS) — number of PD-L1+ cells (tumor cells + lymphocytes + macrophages) divided by total viable tumor cells × 100 (used in gastric/GEJ, HNSCC, cervical, urothelial, TNBC, endometrial, biliary cancers). Different antibody clones are validated for different drugs: 22C3 (pembrolizumab), 28-8 (nivolumab), SP142 (atezolizumab — also scores immune cells), SP263 (durvalumab). These clones are not fully interchangeable.

MSI/MMR Testing

Microsatellite instability (MSI) testing is required for all colorectal and endometrial carcinomas and recommended for many other solid tumors. Methods: (1) IHC for MMR proteins (MLH1, MSH2, MSH6, PMS2) — loss of nuclear staining = dMMR; (2) PCR-based MSI (Bethesda panel: BAT-25, BAT-26, D5S346, D2S123, D17S250; MSI-H = instability in ≥2 of 5 markers); (3) NGS-based MSI (computational analysis of microsatellite loci from targeted panel data). MSI-H/dMMR tumors are eligible for pembrolizumab regardless of tumor type (first tissue-agnostic FDA approval). dMMR testing also identifies Lynch syndrome candidates (germline mutations in MLH1, MSH2, MSH6, PMS2, or EPCAM).

Key Companion Diagnostic Biomarkers

Homologous Recombination Deficiency (HRD)

HRD testing evaluates the ability of tumor cells to repair DNA double-strand breaks via homologous recombination. HRD-positive tumors are sensitive to PARP inhibitors and platinum chemotherapy. Testing methods: BRCA1/2 mutation testing (germline and somatic — detected by NGS), genomic instability score (GIS) (Myriad myChoice CDx — composite score of LOH, telomeric allelic imbalance, and large-scale state transitions; score ≥42 = HRD-positive), and genomic scar assays (Foundation Medicine LOH score). HRD is relevant in high-grade serous ovarian carcinoma (~50% have HRD), breast cancer (BRCA-mutated), pancreatic cancer, and castration-resistant prostate cancer. Beyond BRCA1/2, other HRR pathway genes (ATM, PALB2, RAD51C/D, BRIP1, CHEK2) may confer PARP inhibitor sensitivity.

20 Clinical Chemistry

Comprehensive Metabolic Panel (CMP) Interpretation

Sodium (Na+) — 136-145 mEq/L; hyponatremia (<135): SIADH, heart failure, cirrhosis, thiazide diuretics, adrenal insufficiency; hypernatremia (>145): diabetes insipidus, dehydration, osmotic diuresis. Potassium (K+) — 3.5-5.0 mEq/L; hypokalemia: diuretics, vomiting, diarrhea, RTA type I/II, hyperaldosteronism; hyperkalemia: renal failure, ACE inhibitors, spironolactone, rhabdomyolysis, tumor lysis syndrome, hemolyzed specimen (pseudohyperkalemia). Chloride (Cl-) — 98-106 mEq/L; tracks with sodium; isolated low Cl: metabolic alkalosis (vomiting); high Cl: non-anion gap metabolic acidosis. CO2 (bicarbonate, HCO3-) — 22-29 mEq/L; low: metabolic acidosis; high: metabolic alkalosis or respiratory compensation. BUN — 7-20 mg/dL; Creatinine — 0.7-1.3 mg/dL (male); BUN/Cr ratio >20:1 suggests pre-renal azotemia (dehydration, heart failure, GI bleed). Glucose (fasting) — 70-99 mg/dL; 100-125 = prediabetes; ≥126 on two occasions = diabetes.

Hepatic Panel — AST/ALT Patterns

ALT (alanine aminotransferase) — more specific for liver injury (primarily hepatocytes). AST (aspartate aminotransferase) — found in liver, heart, muscle, RBCs — less specific. The AST/ALT ratio (De Ritis ratio) provides diagnostic clues: ratio >2:1 strongly suggests alcoholic hepatitis (both usually <300 U/L; rarely >500); ratio ~1:1 with high values (>1000 U/L) suggests viral hepatitis or ischemic hepatitis; ratio <1 suggests NAFLD/NASH. Alkaline phosphatase (ALP) — elevated in cholestasis (biliary obstruction, infiltrative disease), bone disease (Paget, bone metastases), and pregnancy (placental isoenzyme). GGT — confirms hepatic origin of elevated ALP (vs. bone); markedly elevated in alcohol use. Bilirubin — total normal <1.2 mg/dL; direct (conjugated) elevated in obstructive jaundice, hepatocellular disease; indirect (unconjugated) elevated in hemolysis, Gilbert syndrome (mild, benign, ~5% population), Crigler-Najjar.

Urinalysis — Laboratory Interpretation

Urinalysis combines dipstick chemistry and microscopic sediment examination. Key findings: Protein — dipstick detects albumin (≥30 mg/dL); trace to 1+ may be normal after exercise; persistent proteinuria requires quantification (spot urine protein:creatinine ratio or 24-hour collection; nephrotic range ≥3.5 g/day). Blood — positive dipstick with no RBCs on microscopy = myoglobinuria (rhabdomyolysis) or hemoglobinuria (intravascular hemolysis). Leukocyte esterase — indicates pyuria; nitrites — indicate bacteriuria with nitrate-reducing organisms (Enterobacteriaceae, not Enterococcus or Pseudomonas). Glucose — positive when serum glucose exceeds renal threshold (~180 mg/dL); also positive in Fanconi syndrome (proximal tubular dysfunction). Microscopic sediment: RBC casts = glomerulonephritis (nephritic syndrome); WBC casts = pyelonephritis, interstitial nephritis; granular ("muddy brown") casts = acute tubular necrosis (ATN); waxy casts = advanced CKD; fatty casts / oval fat bodies = nephrotic syndrome (Maltese cross under polarized light from cholesterol); hyaline casts = nonspecific (concentrated urine, exercise). Dysmorphic RBCs (acanthocytic, with irregular membranes) and RBC casts together are highly specific for glomerular origin of hematuria.

Anion Gap & Acid-Base

Anion gap (AG) = Na - (Cl + HCO3); normal 8-12 mEq/L (or 10-14 if albumin-corrected: add 2.5 for each 1 g/dL albumin below 4). Elevated AG metabolic acidosis (MUDPILES mnemonic): Methanol, Uremia, DKA, Propylene glycol/Paraldehyde, Isoniazid/Iron, Lactic acidosis, Ethylene glycol, Salicylates. Non-anion gap (hyperchloremic) metabolic acidosis: diarrhea (GI HCO3 loss), renal tubular acidosis (Type 1 distal — unable to secrete H+, urine pH >5.5, hypokalemia; Type 2 proximal — HCO3 wasting, hypokalemia; Type 4 — hypoaldosteronism, hyperkalemia), normal saline infusion (dilutional). Osmolal gap = measured osmolality - calculated osmolality (2Na + glucose/18 + BUN/2.8); gap >10 mOsm/kg suggests unmeasured osmoles: ethanol, methanol, ethylene glycol, isopropyl alcohol, mannitol.

Cardiac Biomarkers

Troponin — the gold standard for myocardial injury/infarction. Cardiac troponin I (cTnI) and T (cTnT) are structural proteins of the contractile apparatus specific to cardiac myocytes. High-sensitivity troponin (hs-cTn) assays detect troponin at concentrations 10-100x lower than conventional assays, enabling earlier detection (within 1-3 hours of symptom onset vs. 4-6 hours). The 99th percentile upper reference limit (URL) defines the decision threshold — values above this indicate myocardial injury. The delta (rise/fall) pattern distinguishes acute MI from chronic elevation: a ≥20% change (using 0/1h or 0/3h algorithms) with at least one value above the 99th percentile supports acute MI. Chronically elevated troponin without delta: heart failure, CKD, myocarditis, pulmonary embolism, sepsis.

Tumor Markers

21 Coagulation & Transfusion Medicine

Coagulation Cascade — Laboratory Correlates

PT (prothrombin time) / INR — measures the extrinsic pathway (factor VII) and the common pathway (factors X, V, II/prothrombin, fibrinogen). Normal PT 11-13.5 seconds; INR 0.8-1.1. Prolonged by: warfarin (targets vitamin K-dependent factors II, VII, IX, X), liver disease, vitamin K deficiency, DIC. aPTT (activated partial thromboplastin time) — measures the intrinsic pathway (factors XII, XI, IX, VIII) and the common pathway. Normal 25-35 seconds. Prolonged by: heparin, hemophilia A (factor VIII deficiency) or B (factor IX deficiency), von Willebrand disease (secondary FVIII decrease), lupus anticoagulant (paradoxically thrombotic), factor XII deficiency (prolonged aPTT but no bleeding). Thrombin time (TT) — measures fibrinogen to fibrin conversion; prolonged by heparin, fibrin degradation products, dysfibrinogenemia, and direct thrombin inhibitors (dabigatran).

Mixing Studies

When PT or aPTT is prolonged, a mixing study (1:1 mix with normal plasma) distinguishes: factor deficiency (corrects with mixing — the normal plasma supplies the missing factor) from inhibitor (does not correct — an antibody in the patient's plasma neutralizes the factor in normal plasma). Time-dependent inhibitors (e.g., factor VIII inhibitor in acquired hemophilia) may correct immediately but prolong after 1-2 hour incubation at 37°C. Lupus anticoagulant is a phospholipid-dependent inhibitor — mixing study does not correct, but confirmatory tests include dilute Russell viper venom time (dRVVT) and hexagonal phase phospholipid neutralization.

DIC — ISTH Scoring System

Thrombophilia Workup

The thrombophilia evaluation includes: Factor V Leiden (most common inherited thrombophilia in Caucasians, ~5% prevalence; activated protein C resistance screening, confirmed by PCR for the R506Q mutation), Prothrombin gene mutation G20210A (~2% prevalence; elevated prothrombin levels), Antithrombin deficiency (functional assay; most thrombogenic inherited deficiency; heparin resistance), Protein C deficiency (functional assay; warfarin-induced skin necrosis risk — must bridge with heparin), Protein S deficiency (free protein S antigen; decreased in pregnancy, OCP use, liver disease, acute thrombosis), Antiphospholipid syndrome (lupus anticoagulant by dRVVT, anticardiolipin IgG/IgM, anti-beta-2 glycoprotein I IgG/IgM — must be positive on 2 occasions ≥12 weeks apart). Testing pitfalls: acute thrombosis and anticoagulation affect most results — protein C/S decreased by warfarin, antithrombin decreased by heparin, lupus anticoagulant affected by all anticoagulants. Ideally test ≥2 weeks after completing anticoagulation or use PCR-based tests (Factor V Leiden, prothrombin mutation) that are unaffected by clinical status.

Blood Bank — ABO/Rh & Transfusion

ABO typing involves forward typing (patient RBCs tested with anti-A and anti-B reagents) and reverse typing (patient serum tested with A1 and B reagent cells). Discrepancies require investigation. Rh typing — D antigen is the most immunogenic; Rh(D)-negative patients must receive Rh(D)-negative blood to prevent alloimmunization. Type and screen (T&S) — ABO/Rh typing + antibody screen (indirect antiglobulin test with screening cells); if negative, blood can be issued via electronic/computer crossmatch. Crossmatch — final compatibility test: immediate spin (ABO), followed by antiglobulin phase if antibody screen is positive.

Transfusion Reactions

22 Microbiology & Point-of-Care

Gram Stain Interpretation

The Gram stain is the most important rapid diagnostic tool in microbiology. Gram-positive organisms (thick peptidoglycan wall retains crystal violet) appear purple/blue: cocci in clusters (Staphylococcus), cocci in chains (Streptococcus), cocci in pairs/lancet-shaped (Pneumococcus), large rods (Clostridium, Bacillus), branching filaments (Actinomyces, Nocardia). Gram-negative organisms (thin peptidoglycan, outer membrane, decolorized, counterstained) appear pink/red: diplococci (Neisseria), coccobacilli (Haemophilus), rods (Enterobacteriaceae, Pseudomonas), curved rods (Vibrio, Campylobacter). Gram stain also provides semi-quantitative assessment of the inflammatory response (WBCs) and specimen adequacy — sputum with >25 PMNs and <10 squamous epithelial cells per low-power field is considered adequate.

Culture & Sensitivity

Blood cultures — 2-3 sets (aerobic + anaerobic bottles each) from separate venipuncture sites before antibiotics; volume is the single most important variable (20-30 mL total). Time to positivity: most significant pathogens detected within 48 hours; 5-day incubation standard (extended for endocarditis, HACEK organisms). Urine culture — ≥10&sup5; CFU/mL from clean-catch midstream is diagnostic of UTI; ≥10³ CFU/mL from catheterized specimen; any growth from suprapubic aspiration is significant. CSF culture — aerobic and anaerobic; hold for 5 days; concurrent CSF Gram stain (positive in ~60-90% of bacterial meningitis), cell count, protein, glucose, and latex agglutination or PCR panel (FilmArray meningitis/encephalitis panel detects 14 pathogens in ~1 hour).

Sensitivity Testing

Minimum inhibitory concentration (MIC) — the lowest concentration of an antibiotic that inhibits visible growth; reported as µg/mL; interpreted by CLSI (US) or EUCAST (Europe) breakpoints as susceptible (S), intermediate (I), or resistant (R). Kirby-Bauer disk diffusion — antibiotic-impregnated disks placed on agar inoculated with bacteria; zone of inhibition diameter correlates with MIC. ESBL detection: phenotypic (clavulanate synergy with cephalosporins) or molecular (CTX-M, SHV, TEM gene detection). MRSA: cefoxitin disk or mecA gene PCR. Carbapenem resistance: modified carbapenem inactivation method (mCIM) or molecular (KPC, NDM, OXA-48, VIM, IMP gene detection).

Antimicrobial Resistance Mechanisms

Key resistance patterns with laboratory significance: MRSA — methicillin-resistant Staphylococcus aureus carries the mecA gene encoding PBP2a (altered penicillin-binding protein); resistant to all beta-lactams except ceftaroline and ceftobiprole; detected by cefoxitin disk test, PBP2a latex agglutination, or mecA PCR. ESBL (Extended-Spectrum Beta-Lactamase) — enzymes (CTX-M, SHV, TEM types) that hydrolyze 3rd-generation cephalosporins; detected by phenotypic clavulanate synergy test or molecular methods; treatment: carbapenems (drug of choice), piperacillin-tazobactam (debate on efficacy for serious infections). CRE (Carbapenem-Resistant Enterobacterales) — mechanisms include KPC (most common in US), NDM, OXA-48, VIM, IMP; detected by mCIM/eCIM (distinguishes serine from metallo-carbapenemases) or molecular; treatment: ceftazidime-avibactam (KPC), cefiderocol, meropenem-vaborbactam. VRE — vancomycin-resistant Enterococcus; vanA (inducible, high-level MIC ≥64) vs. vanB (inducible, moderate, MIC 16-32); treatment: linezolid, daptomycin. The lab must quickly communicate MDR organisms to infection control for isolation precautions.

Molecular Microbiology & POCT

GeneXpert (Cepheid) — rapid PCR platform for: MTB/RIF (Mycobacterium tuberculosis + rifampin resistance in ~2 hours), MRSA, C. difficile, Group B Streptococcus, SARS-CoV-2, influenza/RSV. FilmArray (BioFire) — multiplex PCR panels: blood culture identification (BCID, 43 targets), respiratory panel (RP, 20 targets), GI panel (22 targets), meningitis/encephalitis panel (14 targets). Turnaround ~1 hour. Point-of-care testing (POCT) requires: CLIA waiver or certificate, trained operators, daily/periodic QC, proficiency testing, competency assessment, and correlation with laboratory reference methods. Common POCT: glucose meters, i-STAT (blood gases, electrolytes, lactate, creatinine, troponin), rapid strep, rapid influenza/COVID, urine pregnancy (hCG), urinalysis dipstick.

23 Autopsy Pathology

Renal Pathology — Overview

Renal biopsies require a coordinated approach: tissue is divided for light microscopy (H&E, PAS, trichrome, silver stain — Jones methenamine silver highlights basement membranes), immunofluorescence (IF) (fresh/frozen tissue stained with fluorescent antibodies to IgG, IgA, IgM, C3, C1q, kappa, lambda, fibrinogen — pattern and location of deposition are diagnostic), and electron microscopy (glutaraldehyde-fixed for ultrastructural evaluation of basement membranes and electron-dense deposits). Glomerular deposit patterns: mesangial (IgA nephropathy), subepithelial (membranous nephropathy — "spike and dome" on silver stain), subendothelial (lupus nephritis class IV — "wire loop" lesions), intramembranous (dense deposit disease/C3GN). Adequate renal biopsy for glomerular disease requires a minimum of 10 glomeruli on light microscopy and 2-3 glomeruli on EM.

Head & Neck Pathology

HPV-associated oropharyngeal squamous cell carcinoma (OPSCC) — a distinct entity from HPV-negative OPSCC with markedly better prognosis. Typically nonkeratinizing, basaloid morphology arising from the palatine tonsils or base of tongue. Surrogate marker: p16 overexpression by IHC (strong, diffuse nuclear and cytoplasmic staining in ≥70% of tumor cells). p16 positivity is used for TNM staging (AJCC 8th edition separates p16+ and p16- OPSCC with distinct staging systems). For non-oropharyngeal sites, p16 alone is insufficient — confirmatory HPV testing (ISH for high-risk HPV DNA or RNA) is recommended. Salivary gland tumors — the most common benign tumor is pleomorphic adenoma (mixed tumor; risk of malignant transformation to carcinoma ex pleomorphic adenoma if present >10 years). The most common malignant tumor is mucoepidermoid carcinoma (graded low/intermediate/high; MAML2 rearrangement is diagnostic and associated with favorable prognosis). Adenoid cystic carcinoma has a characteristic cribriform ("Swiss cheese") pattern, perineural invasion is hallmark, MYB-NFIB fusion is defining; prolonged clinical course but late distant metastases (lung) are common.

Neuropathology Basics

Brain tumor grading follows the WHO CNS Classification (5th edition, 2021), which integrates molecular markers with histology for the first time. Adult-type diffuse gliomas are classified into three types: astrocytoma (IDH-mutant, graded 2-4), oligodendroglioma (IDH-mutant and 1p/19q-codeleted, graded 2-3), and glioblastoma (IDH-wildtype, grade 4). Key molecular markers: IDH1/2 mutation (favorable prognosis; IDH1 R132H detected by IHC in ~90%), 1p/19q codeletion (obligatory for oligodendroglioma diagnosis; detected by FISH or NGS), ATRX loss (astrocytoma), MGMT promoter methylation (predicts temozolomide response in glioblastoma), TERT promoter mutation, EGFR amplification, +7/-10 (chromosome 7 gain/10 loss — diagnostic of glioblastoma even without necrosis or microvascular proliferation if IDH-wildtype).

Types of Autopsy

Hospital (clinical/academic) autopsy — performed at the request of the attending physician and with consent of the next of kin to determine the cause of death, evaluate the accuracy of clinical diagnosis, assess treatment effects, and identify unsuspected disease. Autopsy rates have declined from ~50% in the 1960s to <5% today, despite studies showing significant diagnostic discrepancies in 10-30% of cases. Forensic (medicolegal) autopsy — ordered by the medical examiner or coroner for deaths that are suspicious, unexplained, sudden, due to injury, or of public health concern; no consent required. A limited/restricted autopsy examines only specific body cavities or organs as directed by the consent.

Autopsy Procedure

External examination — identification, body habitus, scars, tattoos, medical devices, evidence of trauma, postmortem changes (livor mortis, rigor mortis, decomposition). Evisceration methods: Virchow technique — organs removed one at a time in situ, allowing examination of relationships; Rokitansky technique — in situ dissection with minimal organ removal; Letulle technique — en bloc removal of all thoracic and abdominal organs as a single mass, then dissected on the cutting table (most common method in US academic centers). The brain is removed after reflecting the scalp, using an oscillating saw for the calvarium; it is typically fixed in formalin for 2-3 weeks before sectioning due to its soft consistency.

Normal Organ Weights

Standard Microscopic Sections

Standard blocks for a complete autopsy typically include: heart (left ventricle, right ventricle, septum, left anterior descending artery cross-section, valves), lungs (all 5 lobes, hilar sections), liver (right and left lobes, gallbladder), kidneys (bilateral cortex and medulla), spleen, pancreas (head and body), adrenals (bilateral), thyroid, brain (frontal, temporal, parietal, and occipital cortex, hippocampus, basal ganglia, thalamus, cerebellum, brainstem, pituitary), GI tract (esophagus, stomach, small bowel, colon), aorta, and any lesion-specific sections. For forensic cases, additional sections include skin from wound/injury sites, vitreous humor (for postmortem chemistry — glucose, electrolytes, BUN), and toxicology specimens (blood from femoral vein, urine, gastric contents, bile, liver tissue, hair).

Cause & Manner of Death

Cause of death — the disease or injury that initiated the chain of events leading to death; stated on the death certificate in a sequential manner (immediate cause, due to, due to, due to underlying cause). Example: (a) pulmonary thromboembolism, due to (b) deep vein thrombosis, due to (c) immobilization following hip fracture, due to (d) osteoporosis. Manner of death — the circumstances: natural (disease process), accident, suicide, homicide, or undetermined. In forensic autopsies, the pathologist determines cause of death; the manner of death is determined by the medical examiner/coroner considering all circumstances (scene investigation, history, toxicology, autopsy findings).

24 Quality Assurance & Laboratory Accreditation

CAP Accreditation & CLIA

The College of American Pathologists (CAP) laboratory accreditation program is the gold standard, accepted by CMS as equivalent to CLIA inspection. CLIA (Clinical Laboratory Improvement Amendments, 1988) regulates all clinical laboratory testing performed on human specimens in the US. CLIA classifies tests by complexity: waived (simple tests with low error risk — home pregnancy, glucose meters, rapid strep/flu; require certificate of waiver), moderate complexity (automated analyzers, routine chemistry, hematology), and high complexity (manual IHC, flow cytometry, cytogenetics, molecular testing). High-complexity labs require a laboratory director (pathologist, PhD), technical supervisor, and general supervisor. Inspections occur every 2 years.

Digital Pathology & Artificial Intelligence

Whole slide imaging (WSI) — glass slides are scanned at 20x or 40x magnification to create high-resolution digital images (whole slide images, typically 1-3 GB per slide). FDA-cleared WSI systems (Philips IntelliSite, Leica Aperio GT450 DX, Hamamatsu NanoZoomer) are approved for primary diagnostic use in surgical pathology. Benefits: remote diagnosis/teleconsultation, digital archiving, integration with AI algorithms, standardized teaching, and reduced glass slide storage. Limitations: large file sizes, scanning time (1-3 minutes per slide), color calibration challenges, and the need for pathologist validation. AI in pathology — FDA-cleared algorithms include: Paige Prostate (detects areas suspicious for cancer on prostate biopsies), Proscia for IVD (image analysis), and various research-stage tools for mitosis counting, Ki-67 quantification, PD-L1 scoring, HER2 scoring, and metastatic cancer detection in lymph nodes. AI assists but does not replace the pathologist — current algorithms are designed as "second reader" or "pre-screening" tools to improve efficiency and reduce errors, not for autonomous diagnosis.

Quality Control & Proficiency Testing

Quality control (QC) — run known-value control materials with every batch and at defined intervals; results plotted on Levey-Jennings charts with Westgard rules to detect systematic and random errors (e.g., 1:2s warning, 1:3s rejection, 2:2s shift, R:4s range). Proficiency testing (PT) — external QA program (CAP surveys) where unknown samples are sent to laboratories and results compared to peer group; unsatisfactory performance on 2 consecutive or 2 of 3 events triggers investigation and potential sanctions. All laboratories performing regulated testing must participate in PT for each analyte tested.

Turnaround Time (TAT) Benchmarks

Pre-Analytic, Analytic, & Post-Analytic Errors

Laboratory errors are categorized by phase: pre-analytic (60-70% of errors) — patient misidentification, incorrect specimen labeling, hemolyzed/clotted/insufficient specimen, improper transport/storage (e.g., failure to keep specimen on ice for ammonia, arterial blood gas), wrong tube type; analytic (10-15%) — instrument malfunction, reagent deterioration, interference (lipemia, hemolysis, icterus, biotin), calibration drift, pipetting errors; post-analytic (20-25%) — transcription error, result reported on wrong patient, failure to notify critical values, delayed reporting, misinterpretation. Critical values (aka panic values) — results requiring immediate clinician notification: examples include potassium >6.5 or <2.5 mEq/L, glucose <40 or >500 mg/dL, platelet count <20,000/µL, INR >5.0, positive blood culture, malignant cells in CSF. CAP requires documentation of critical value notification within 30 minutes of result verification.

Amended Reports & Root Cause Analysis

An amended report is issued when an error in a previously signed-out report is identified. The amendment must clearly state: the original diagnosis, the amended diagnosis, the reason for amendment, and the date/time. The CAP requires documentation of all amended reports and tracking of amendment rates as a quality metric. Root cause analysis (RCA) is performed for significant errors, near-misses, and sentinel events using structured investigation (e.g., fishbone/Ishikawa diagram, 5 Whys) to identify system failures rather than individual blame. Lean methodology and Six Sigma principles are increasingly applied in the laboratory to reduce waste (lean) and reduce defects to <3.4 per million opportunities (six sigma). Value stream mapping, 5S (Sort, Set in order, Shine, Standardize, Sustain), and DMAIC (Define, Measure, Analyze, Improve, Control) are common frameworks.

25 Grading & Staging Systems

Nottingham Histologic Grade — Breast

See section 6 for full enumeration. Summary: 3 components (tubule formation, nuclear pleomorphism, mitotic count) scored 1-3 each. Total 3-5 = Grade 1; 6-7 = Grade 2; 8-9 = Grade 3.

Gleason Grading — Prostate

The Gleason grading system assigns a primary pattern (most common) + secondary pattern (second most common), each scored 1-5. The Gleason score is the sum (range 2-10; in practice, scores <6 are no longer reported). Patterns: Pattern 3 — well-formed, discrete glands of varying size; Pattern 4 — fused glands, poorly formed glands, cribriform glands, glomeruloid structures; Pattern 5 — sheets, cords, single cells, comedonecrosis. The ISUP Grade Groups simplify Gleason into 5 prognostic categories:

Fuhrman/ISUP Grading — Renal Cell Carcinoma

The older Fuhrman nuclear grade (I-IV) has been replaced by the WHO/ISUP nucleolar grading system for clear cell and papillary RCC:

Bethesda Systems

Cervical Cytology (Bethesda) — see section 10 for full enumeration: NILM, ASC-US, ASC-H, LSIL, HSIL, SCC, AGC, AIS. Thyroid Cytopathology (Bethesda) — see section 11: categories I through VI with implied risk of malignancy.

Hereditary Cancer Syndromes Requiring Pathology-Driven Screening

TNM Staging — General Principles

The AJCC/UICC TNM staging system (8th edition, effective January 2018) stages solid tumors based on: T (primary tumor size/extent: Tis = in situ, T1-T4 = increasing size/invasion), N (regional lymph node involvement: N0 = none, N1-N3 = increasing nodal burden), M (distant metastasis: M0 = none, M1 = present). Prefix modifiers: c = clinical staging; p = pathologic staging; y = post-neoadjuvant therapy; r = recurrence. The TNM combination determines the overall stage group (I-IV). R classification: R0 = no residual tumor (negative margins); R1 = microscopic residual tumor (positive margin on microscopy); R2 = macroscopic residual tumor (visible residual disease left behind).

The ypTNM (post-neoadjuvant pathologic staging) is increasingly important as neoadjuvant therapy expands. Pathologic complete response (ypT0N0) — no viable invasive tumor in the primary site or lymph nodes after neoadjuvant treatment — is a favorable prognostic indicator in breast cancer (especially triple-negative and HER2+), esophageal cancer, and rectal cancer. Tumor regression grading (TRG) systems quantify the degree of treatment response: the AJCC TRG for rectal cancer scores 0-3 (0 = complete response/no viable cancer cells, 1 = near-complete/single cells or small groups, 2 = partial response/residual cancer with fibrosis, 3 = poor or no response). The Mandard TRG (1-5) is used for esophageal carcinoma. Accurate TRG requires thorough gross examination and sampling of the entire tumor bed, including areas of fibrosis where residual tumor may be microscopically present.

Liver Fibrosis Staging

Liver fibrosis is assessed on trichrome-stained needle biopsies using standardized scoring systems:

Adequate liver biopsy requires a minimum of 1.5-2.0 cm length with ≥11 complete portal tracts for reliable staging. Activity (necroinflammation) is graded separately using the Histologic Activity Index (HAI / Knodell score, modified by Ishak) or the Metavir Activity grade (A0-A3). Steatosis is reported as a percentage of hepatocytes containing fat vacuoles: <5% = none, 5-33% = mild (S1), 34-66% = moderate (S2), >66% = severe (S3). The NAS (NAFLD Activity Score) combines steatosis (0-3) + lobular inflammation (0-3) + hepatocyte ballooning (0-2) for a total of 0-8; NAS ≥5 correlates with NASH, <3 correlates with non-NASH.

WHO Neuroendocrine Tumor Grading

26 IHC Marker Reference Table

Pediatric Small Round Blue Cell Tumors

A critical differential diagnosis in pediatric pathology involves small round blue cell tumors — all have similar morphology (small cells with scant cytoplasm, hyperchromatic nuclei) but distinct biology and treatment. Differentiation requires IHC and molecular testing:

27 Normal Laboratory Values

Complete Blood Count (CBC)

Comprehensive Metabolic Panel (CMP)

Coagulation Panel

Cardiac Biomarkers

Iron Studies & Hemoglobin Electrophoresis

Hemoglobin electrophoresis / HPLC separates hemoglobin variants: Normal adult: HbA (~96-97%), HbA2 (2-3%), HbF (<1%). Beta-thalassemia trait: elevated HbA2 (>3.5%), mild microcytic anemia, normal iron studies. Sickle cell disease (HbSS): HbS ~80-90%, HbF variable (higher HbF = milder disease), HbA absent. Sickle trait (HbAS): HbA ~55-60%, HbS ~35-40%. HbC disease: HbC ~90%, target cells prominent. HbH disease (3-gene alpha-thal deletion): HbH inclusions on supravital stain, fast-migrating band on electrophoresis.

Endocrine & Other

28 Abbreviations Master List