LN review

Review of normal Lymph node architecture and cellularity

	features							staining
Capsule	·         thin fibrous connective tissue covering of lymph node ·         may be thicker at hilus ·         connected to fibrous trabeculae which penetrate the node ·         capsule may contain smooth muscle cells
cortex	subcapsular portion of node with largest number of follicles
Germinal center	·         Centroblasts/ Centrocytes ·         Macrophage/ Follicular dendritic cells ·         Surrounded by mantle zone small B lymphocytes						Bcl6, CD10 (strong/dense) Negative: Bcl2 CD10 BCL-6
Follicles (10/20)	Secondary follicle: GC develops due to antigenic stimulation of B cells and production of AB. Contains pale staining GC: polarized towards site of antigen entry ·          B cells ·          Follicular dendritic cells (CD21+, CD35+) ·          Tingible body macrophage   Primary Follicle: round aggregates of small dark-staining lymphocytes – usually near capsule – within a network of follicular dendritic cell processes **no GC**
Mantle zone	small B cells surrounding pale staining germinal centers				CD79a: B cell marker with higher affinity for mantle B cells ·         staining within GC = plasma cell
Marginal zone	·      light zone on outer rim of mantle zone ·      contains post-follicular memory B cells derived after stimulation from T cell dependent antigen
medulla	·            portion closest to hilum               contains vessels/sinuses/mast cells               minimal follicles
Paracortex	Tissue between cortical follicle and medulla – mostly dark staining ·         Contains post-capillary venules – lined by high endothelial cells ·         Dendtitic cells + T cells present ·         Expands during cell mediated immunologic reactions ·         Coarse network reticulin fibers			Stromal marker transglutaminase highlights perfollicular sinus nearly encircling GC with a small collar connecting the follicles to the paracortex
Medullary cord
sinuses	Carry lymph from afferent à efferent lymphatics Subcapsular: below capsule and partially lined by endothelium à medullary as it approaches the hilum and is lined by macrophages
Vessels:	Afferent lymph vessels: penetrate capsule à enter marginal sinus, communicate with intranodal sinuses àEfferent lymph vessels: lack endothelial lining Intradnodal vessels: littoral cells or histiocytes (phagocytes)
Immune cells:
Centrocytes centroblasts
Lymphoid stem cells:	monoclonal proliferation: assume neoplastic								Tdt+, CD34+, HLA-DR+ Then develop along B/T pathway
T cells T cells develop in BM = prothymocytes à migrate to thymus to mature ·         Precursor cell: TdT+, CD34+, HLA-DR+ ·         Drops HLA-DR, adds CD2, CD5, CD7 = early thrombocytes ·         Gamma/beta chain rearrangement + CD1 + loss CD34 = common thrombocyte ·         Beta/alpha rearrangement à CD4+/CD8+ ·         Splits into helper (CD4) or cytotoxic (CD8) o    Loss: TdT, CD1, CD5, CD7 o    CD2 + 3  remain o    Note: NK and T cells arise from progenitor that express CD3 (and cannot become B cell) T cells are mainly present in paracortex of LN ·         Little cytoplasm ·         Small round/irregular nuclei with dark and condensed chromatin ·         May have blastic form with moderate cytoplasm and irregular nuclei and marginal nucleoli
B cells ·         B cells express surface Ig: 2 heavy (H) and 2 light (L) chains (κ, λ) ·         Antigen receptor loci have 4 types of rearrangement: o    Rearrangement of variable, diversity, joining regions (VDJ) o    Somatic hypermutation of V o    Ig heavy chain class switching o    Receptor editing Early B precursor: contain Ig-related components but no immunoglobins ·         CD179a/b = precursor to light chains in pre-B-receptor à disappears when replaced by light chains ·         TdT+, CD34+, HLA-DR+ H chain rearrangement ·         +CD19 à +CD10 à IgM – Heavy chain Light chain rearrangement ·         IgM & IgD CD20 + = pre-B cell ·         Ig surface, CD21, CD22 Drops TdT = B cell ·         B-cells express surface immunoglobin which is associated with CD79a/79b complex to form a B cell antigen receptor complex
Plasma cells	·         Makes antibodies ·         Abundant basophilic cytoplasm -- ↑ RER ·         Paranuclear hof (Giemsa stain: due to Golgi) ·         Eccentric nucleus with spoke wheel (clock face) chromatin – small clumps of chromatin on nuclear membrane ·         Round/clear nucleus ·         +/- Russell bodies: intracytoplsamic PAS+ globules
NK cells	·         Lymphoid origin ·        innate immunity granules: perforin + granzyme 5-20% peripheral WBC kill cells lacking MHC1 look like large granular lymphocyte					·         CD56 (>90%, adhesion molecule) ·         CD57 ·         CD16 (low affinity IgG FcR/FCRIII: responsible for AB-dep cellular cytotixicity in NK, PMN, monocyte) ·         CD3, CD2 , CD7, CD8,  CD11b, CD11c, perforin, granzyme B, TIA-1 Negative (CD3 - surface, Ig)
Mast cells	Present in T cell areas ·         granular cytoplasm ·         some cells are elongated and resemble fibroblasts					Giemsa & Toludidine blue (purple granules), Leder stain, microphthalmia TF, tryptase, CD2, CD25, CD45, CD68, CD117
Accessory immune system ·         APC: dendritic / Langerhan ·         Processing: monocytes, macrophages ·         Histiocytes: macrophage/monocyte & dendritic/Langerhan
Macrophages  APC		·         Abundant cytoplasm ·         Medium/large nuclei ·         Vesicular chromatin Tingible body: clear cytoplasm + cellular debris (starry sky)								CD68 Lysozyme
Langerhan cells:		APC Related to interdigitating denderic cells and dermal dendrocytes Contain large birbeck granules (EM) Found in all layers of epidermis (most prominent in stratum spinosum) Langerhan cell histiocytosis (seen in LN)								CD1a S100 Vimentin Langerin Variable CD68 (negative: CD21, CD35, CD86)
Indeterminate cells		·         Resemble langerhan – NO Birbeck granules ·         Derived from langerhan cells/interstitial dendritic cells reside in skin/mucosa								CD1a, S100, fascin
Interdigitating dendritic cells		Associated with interfollicular (T-cell) zone in lymphoid tissue may cause mottled appearance to paracortex abundant clear and plae cytoplasm , ill defined borders large bizarre nucleus - deep clefts/folds EM: no desmosomes/birbeck granules have long complex processes -- interdigitate								S100, vimentin, fascin, CD45RB, CD68 (focal), variable CD1a / CD13
Follicular dendritic cells		·         Associated with germinal centers ·         Have complex cell processes (desmosomes) ·         no Birbeck’s granules ·         Control B cell maturation and isotype switching ·         FDC like cells can be produced from monocytes ·         Upregulated by Tamoxifin treatment for breast cancer mesh-work of follicular dendritic cells: CD21								CD21, CD35, fascin, ER, epidermal GFr, CD11b, CD14, CD19, CD20, variable (CD68, s100, EBV)
Fibroblastic reticulum cells		·         Stromal support cells ·         Spindled cells ·         Parafolicular/deep cortex ·         EM: filaments with focal condensations								Vimentin, SM actin, desmin, alk phosphatase, CD68(focal), +/- keratin (negative: CD21, CD35, S100, EBV)

Not all enlarged LN are neoplastic....

Reactive follicular hyperplasia
·          Most common pattern ·          Primarily seen young adults/kids ·          Usually localized ·          Cervical and axillary	·          Autoimmune ·          Kimura ·          Syphilis ·          Toxoplasmosis ·          Castleman disease ·          HIV – related benign LAD ·          Progressive transformation of germinal centers
Reactive follicles Follicular lymphoma Low density of follicles High density of follicles Follicles usually limited to subcortical region Follicles distributed throughout node Follicles rarely extend beyond capsule Follicles extend beyond capsule Follicles of uneven size/shape Similar size/shaped follicles Mixture of cell types in germinal center Monomorphic or polymorphic population Tangible body macrophages present Tangible body macrophages absent Moderate à high mitotic rate Low à moderate mitotic rate Distinct mantle zone Indistinct or absent mantle zone Cell polarization +/- Cell polarization absent Large interfollicular areas evident Compressed interfollicular areas Nodal effacement +/- nodal effacement BCL-2 negative in B cells of GC BCL-2 + (90%) GC No light chain restriction on immunos Light chain restriction No light chain restriction on flow cytometry Light chain restriction/absence Ig rearrangement absent Ig rearrangement detected (80%) T(14;18) absent T(14;18) present
Reactive paracortical hyperplasia
·          Expansion of paracortical region à mottled appearance with mixed infiltrate ·          Small & large lymphoid cells without cytologic atypia ·          HEV, ↑ vasculature	·          Acute infectious mononucleosis ·          CMV lymphadenitis ·          Post-vaccinial reactive paracortical hyperplasia ·          Drug-induced reactive paracortical hyperplasia ·          Dermatopathic lymphadenitis
Reactive paracortical hyperplasia T cell lymphoma Cortical hyperplasia ***Obliteration or marked diminution of cortical region*** No atypia in mixed infiltrate Cytologic atypia Large B cell population Diminution B cell components (cortical/subcapsular) Typical phenotypic markers seen Loss of T-cell phenotypic markers (loss bcl-2, or gain CD10) No clonal TCR gene rearrangements Clonal T cell receptor gene rearrangements
Benign LAD w/ extensive necrosis
·          Complete necrosis: Liquefactive: o    abundant karyorrhectric debris, ↑ PMN, often abscess o     fulminant (no rim fibrosis/granulation tissue) o    Usually associated with massive infection ·          Complete necrosis: infarct o    Architecture preserved (coagulative) o    Inflammatory background o    Subacute – fibrosis/granulation tissue (often) o    NHL: most important cause of infarctive necrosis ·          Kikuchi histiocytic necrotizing lymphadenitis ·          SLE ·          Kawasaki
Kikuchi-Fujimoto disease; Kikuchi Lymphadenitis: histiocytic necrotizing lymphadenitis, Kikuchi’s lymphadenitis, subacute necrotizing lymphadenitis Cause: ·          Unknown; likely related to infection ·          Some patients have SLE on follow-up – suggesting possible autoimmune process? Epidemiology: ·          More prevalent among young adults (mean = 30s), Asians Presentation/clinical progression: ·          Unilateral, fq painful, cervical LAD – less fq seen in other LN o    Uncommon: Extranodal sites (skin) ·          Grossly: Moderate LN enlargement ·          Clinical presentation: o    50% present with fever +/- flulike symptoms o    Anemia, neutropenia o    Atypical lymphocytosis, 25% cases o    <5% patients have leukocytosis o    +/- rash o    Self-limited disease + most patients recover w/o therapy (1-4 months) o    5%  develop recurrent LAD Pathology: ·          Paracortical hyperplasia with one or eosinophilic areas (discrete or confluent): o    histiocytes/lymphocytes/immunoblasts/plasmacytoid monocytes/karyorrhectic & eosinophilic debris o    Necrotic debris in the center and immunoblasts found in the periphery ·          Aggregates of plasmacytoid monocytes-commonly non-necrotizing areas o    medium sized cells, round nuclei, dispersed chromatin, moderate faintly amphophilic cytoplasm ·          Plasma cells, granulocytes (PMN), granumolas absent ·          The stages occur on a continuum (over time: CD4 à CD8 predominance) Proliferative type Necrotizing type Xanthomatous type Mixture of cells with apoptosis but w/o coagulative necrosis ·          patchy involvement – lymphoid cells, histiocytes, plasmacytoid monocytes ·          Histiocytes: bland chromatin +/- irregular nuclear outline, tend to cluster at edge of lesion Large areas of necrosis + characteristic changes seen in proliferative type ·          Focal à extensive ·          Eosinophilic appearance + karyorrhectic debris ·          Lymphoid cells/histiocytes at margins of necrosis Predominance of foamy histiocytes +/- necrosis ·          Scattered CD20 positive B immunoblasts IHC: ·          lymphoid cells: pan-T antigens o    CD8>>CD4 o    CD56+ NK rare ·          Histiocytes: lysozyme+, CD68+, coexpress MPO (this is an unusual feature that is noted in SLE) ·          Plasmacytoid monocytes: CD2, CD4, CD43 – lack T-cell-specific antigens (CD3) and express monocyte-associated antigens (CD68) ·          B cells are found if reactive follicles are present Differential: ·          NHL: Kikuchi proliferative stage looks similar to diffuse large-cell lymphoma o    Predominance of medium-large lymphoid cells, histiocytes, plasmacytoid monocytes/dendritic cells seen in both o    Abundance of karyorrhectic debris suggestive Kikuchi o    ↓B-cells ↑ CD8 à suggestive Kikuchi ·          HL: focal necrosis o    No RS cells o    Not supported by IHC ·          Infection: high PMNs à less likely Kikuchi o    Bartonella (cat scratch) à cervical/axillary LN §   Granulomatous §   Epithelioid histiocytes à stellate microabscesses (opposed to patchy necrosis) ·          SLE: histological features may be identical to Kikuchi o    Clinical SLE: malar/discoid rash, photosensitivity, oral ulcers, arthritis, serositis, renal disorder, neurologic disorder, hematologic disorder, immunologic disorder, ANA o    Hematoxylin (basophilic) bodies / deposition hematoxiphilic substance on necrotic foci (PAS+, Feulgen +) o    Abundant plasma cells o    Prominent reactive follicular hyperplasia, arteritis, capsular inflammation, deposition nuclear material in BV (azzopardi phenomenon)à more likely SLE ·          Kawasaki: distinguish based on clinical picture and greater extent vascular change (fibrin thrombi) o    Clinical setting (asian children <5) §   High fever, conjunctivitis, rash, oral mucosal inflammation, desquamating rash §   Vasculitis coronary arteries o    Vascular/nodal changes: §   Abundant fibrin deposition, PMN, karyorrhectic debris §   Inflammation capsule §   Fibrinoid necrosis §   Numerous fibrin thrombi (also seen in TTP, Rickettsial infection)
Sinus hyperplasia
·          Nonspecific sinus histiocytosis ·          Monocytoid B-cell hyperplasia ·          Whipple LAD ·          LAD due to deposition of exogenous material ·          LAD due to endogenous lipid material ·          Rosai-Dorfman (sinus histiocytosis + massive LAD) ·          Hemophagocytic lymphohistiocytosis
Monocytopid B-cell hyperplasia Nodal Marginal Zone B cell lymphoma Usually comprises <50% node Usually >50% node Lacks atypia Nuclear atypia Scattered PMNs Cells with plasmacytic differentiation BCL-2+ BCL-2- Polytypic light chains Often monotypic light chains CD43- Maybe CD43+
Reactive lymphadenopathies with primary granulomatous pattern
·          Cluster macrophages (àepithelioid histiocytes) o    larger cytoplasm o    ↑ secretory/killing capacity o    ↓ phagocytic capability ·          Giant cells: fusion epitheliod histiocytes o    Nuclei randomly distributed: foreign-body type o    Nuclei align towards periphery: Langhan type		Non-infectious ·          Granulomatous inflammation in malignant neoplasm ·          Berrylliosis ·          Crohns ·          Sarcoidosis Infectious: non-suppurative ·          TB ·          Atypical mycobacterial infection ·          Lepromatous lymphadenitis ·          Fungal lymphadenitis Infectious: suppurative ·          Cat scratch lymphadenitis ·          Lymphogranuloma venereum ·          Yersinial lymphadenitis
LAD due to alteration of CT framework
·          Inflammatory pseudotumor of LN ·          Vascular transformation of LN sinuses ·          Bacillary angiomatosis (bartonella hensalae infection: cause of cat scratch) form seen in immunosuppressed /AIDS
Deposition of interstitial substance
·          Proteinaceous LAD ·          Ig deposition LAD ·          Pneumocystis lymphadenitis