lymph node normal morphology cortex –primary follicle –secondary follicle –mantle zone...
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Lymph Node Normal Morphology
• Cortex– Primary Follicle– Secondary Follicle– Mantle Zone
• Paracortex
• Medulla– Sinuses
Cortex
• Primary B-Cell Follicles– Nodules of small lymphocytes– Lack germinal centers
• Secondary B-Cell Follicles– Result of stimulation– Germinal Centers– Mantle zone
Germinal Centers
• Pale zone– toward antigen entry– small cleaved cells / centrocytes– follicular dendritic cells
• Dark zone– toward paracortex– large lymphoid cells / centroblasts– tingible body macrophages
Mantle Zone
• polarized toward antigen entry
• express bcl-2 protein
Paracortex
• rich in T cells• CD4:CD8 ratio variable• interdigitating dendritic cell
– S-100 positive– irregular vesicular nuclei
• high endothelial venules– postcapillary vessel– cuboidal epithelium
Medullary areas
• B cells predominate especially plasma cells
• histiocytes
Handling the Fresh Specimen
• Surgeon should excise the largest and most abnormal node
• Tissue for histology
• Touch imprints
• Fresh / frozen tissue for immunologic studies
• Sterile portion for cytogenetics
Frozen Section
• Diagnostic frozen section should be discouraged
• Use frozen to assess adequacy or triage tissue
Freezing for Immunologic Studies
• Liquid nitrogen or isopentane / dry ice mix is best
• Thin sections (<2 mm )may be frozen in OCT
• OCT must be wrapped in foil / plastic to avoid desiccation
• Store at -70C ideal but -20C suitable for many antigens
Fixation
• Node sliced in 2-3 mm intervals
• One metal based fixative (B5, Zenkers, zinc sulfate)
• One neutral buffered formaldehyde (formalin)
Processing
• Single most important factor for optimal histology is section thickness
• Sections should be one cell layer thick
Routine Stains
• H&E
• Giemsa - highlight nuclear features, cytoplasmic granules and plasmacytoid features
• PAS - highlights mucin and glycogen, immunoglobulin inclusions and blood vessels
• Methyl-green pyronin - highlights plasmacytoid features
Common Errors in Fixation and Processing
• Drying of specimen - dark edge artifact; autolysis if prolonged
• Section >3 mm thick - soft unfixed core; center cells show ballooning and are pale
• Overfixation in B5 - brittle tissue; decreased nuclear staining
• Inadequate dehydration - numerous cracks (dry earth look)
Common Errors in Fixation and Processing
• Paraffin too hot - muddy staining with poor detail
• Improper sectioning - Venetian-blind effect; poor cytologic detail
• Section drying too hot - bubbled nuclei and antigen loss
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
LCACD45RB
Lymphocytes,myeloid cells andmacrophages
Plasma cells and RScells negative
Immuno-globulin
B cells and plasmacells
Paraffin – cytoplasmicFrozen – surface
L26CD20
B cells occasionalmacrophages
Plasma cells negativePoor reactivity indecalcified tissue
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
CD79 B cells Plasma cells negative
LN1CDw75
Germinal center Bcells
Better in B5
LN2CD74
B cells,interdigitatingdendritic cells,macrophages
Better in B5
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
MB1CD45RA
B cells and some Tcells
Plasma cells negative
CD3 T cells May requirepretreatment
Leu-22CD43
T cells “tissue CD5”useful in smalllymphocyticneoplasms
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
UCHL1CD45RO
T cells May see nonspecificcytoplasmic staining
Leu-7CD57
GC T cells and someNK cells
Better in frozen tissue
TCR betachain
T cells Requires proteolyticdigestion; better infrozen tissue
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
LeuM1CD15
Granulocytes,macrophages
Carcinomas andCMV infected cellsmay be reactive
BerH2CD30
Activated B and Tcells, plasma cells
Less reactive in B5,embryonal ca. andfew other non-hematolymphoidneoplasms positive
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
KP1CD68
Macrophagesmyeloid cells
Reactive inmelanoma, MFH,granular cell tumorsand renal cell ca.
Lysozyme Macrophagesmyeloid cells
Reactive with manynon-hematolymphoidneoplasms
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
S100 Langerhans cells,interdigitating andsome folliculardendritic cells
Reactive with manynon-hematolymphoidneoplasms
Mac-387 Macrophagesmyeloid cells
Reactive with somesquamous cell ca.
Antibodies Employed in Paraffin Tissue Sections
Antibody /CD Antigen
Normal Reactivity Comments
bcl-2 Non-germinal centerB cells, most T cellsand plasma cells
Best in B5;differentiate benignfrom malignantfollicular lesions
Myeloper-oxidase
Myeloid cells Most sensitive andspecific myeloidmarker
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD45 Hematolymphoidcells
Plasma cells andALCL may benegative
Immuno-globulin
B cells Monoclonal Ig lesssensitive thanpolyclonal IgPrecursor Blymphomas andleukemias may benegative
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD19 B cells Earliest expressed Bcell antigen
CD20 B cells Most plasma cellsunreactive
CD79 B cells May be negative inplasma cells
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD10CALLA
Precursor and GCB cells
Follicular vs. otherlow grade MLEwing’s and MFHpositive
CD21 Mantle and marginalzone B cells
Most mantle andmarginal zone ML
CD23 CLL / SLL reactiveMantle cell MLunreactive
Upregulated by EBVinfection
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD32 Mantle zone B cells Reactive with MCLand some follicularML, myeloidleukemias
CD35 Mantle and marginalzone B cells
Similar to CD21
CD38 Lymphoid progenitorcells, NK cells andplasma cells
One of few markersexpressed by plasmacell neoplasms
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD2, CD3,CD5 andCD7
T cells Pan T cell antigensCD7 earliestexpressed and mostcommonly deleted
CD1a Cortical thymocytes Good for precursor Tcell neoplasms;thymomas rich inCD1A cells
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD4 Helper/Inducer Tcells macrophagesand dendritic cells
May be expressed insome plasma cellneoplasms
CD8 Cytotoxic/SuppressorT cells and NK cells
Minority T subset;may be increased inearly phase viralinfections or late HIVinfection
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD11c Myelomonocyticcells
Hairy cell leukemiaand monocytoid Bcell ML
CD13 Myelomonocyticcells
Most AML M1-M5
CD14 Monocytes andmacrophages
Many M4-M5 AML
CD33 Myeloid cells andmonocytes
Most myeloidleukemias
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD16 NK cells andgranulocytes
CD56 NK cells and a few Tcells
Reactive with neuraland neuroendocrinecells and neoplasticplasma cells
CD57 Some NK cells and Tcells
Antibodies Employed in Fresh or Frozen Tissue
Antibody /CD Antigen
Normal Reactivity Comments
CD25 Activated T cells, Bcells and monocytes
ATLL, HCL, ALCLand some other T andB cell ML
CD34 Progenitor cells andendothelial cells
Stem cell markerGood for vascularneoplasms
TdT Precursor cells inmarrow and corticalthymocytes
Lymphoblasticleukemia / lymphoma