Lymph Node Normal Morphology Cortex –Primary Follicle –Secondary Follicle –Mantle Zone Paracortex Medulla –Sinuses

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




CD79 B cells Plasma cells negative

LN1CDw75

Germinal center Bcells

Better in B5

LN2CD74

B cells,interdigitatingdendritic cells,macrophages

Better in B5




MB1CD45RA

B cells and some Tcells

Plasma cells negative

CD3 T cells May requirepretreatment

Leu-22CD43

T cells “tissue CD5”useful in smalllymphocyticneoplasms




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




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




KP1CD68

Macrophagesmyeloid cells

Reactive inmelanoma, MFH,granular cell tumorsand renal cell ca.

Lysozyme Macrophagesmyeloid cells

Reactive with manynon-hematolymphoidneoplasms




S100 Langerhans cells,interdigitating andsome folliculardendritic cells

Reactive with manynon-hematolymphoidneoplasms

Mac-387 Macrophagesmyeloid cells

Reactive with somesquamous cell ca.




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



CD45 Hematolymphoidcells

Plasma cells andALCL may benegative

Immuno-globulin

B cells Monoclonal Ig lesssensitive thanpolyclonal IgPrecursor Blymphomas andleukemias may benegative




CD19 B cells Earliest expressed Bcell antigen

CD20 B cells Most plasma cellsunreactive

CD79 B cells May be negative inplasma cells




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




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




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




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




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




CD16 NK cells andgranulocytes

CD56 NK cells and a few Tcells

Reactive with neuraland neuroendocrinecells and neoplasticplasma cells

CD57 Some NK cells and Tcells




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

Documents

Lymph Node Normal Morphology Cortex –Primary Follicle –Secondary Follicle –Mantle Zone Paracortex Medulla –Sinuses