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

Cortex Primary B-Cell Follicles Secondary 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 Dark 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 -70°C ideal but -20°C 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

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Paraffin Tissue Sections

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue

Antibodies Employed in Fresh or Frozen Tissue