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

2. 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

3. 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

4. Mantle Zone
polarized toward antigen entry
express bcl-2 protein

5. 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

6. Medullary areas B cells predominate especially plasma cells
histiocytes

7. 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

8. Frozen Section Diagnostic frozen section should be discouraged
Use frozen to assess adequacy or triage tissue

9. 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

10. Fixation Node sliced in 2-3 mm intervals
One metal based fixative (B5, Zenkers, zinc sulfate)
One neutral buffered formaldehyde (formalin)

11. Processing
Single most important factor for optimal histology is section thickness
Sections should be one cell layer thick

12. 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

13. 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)

14. 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

15. Antibodies Employed in Paraffin Tissue Sections

16. Antibodies Employed in Paraffin Tissue Sections

17. Antibodies Employed in Paraffin Tissue Sections

18. Antibodies Employed in Paraffin Tissue Sections

19. Antibodies Employed in Paraffin Tissue Sections

20. Antibodies Employed in Paraffin Tissue Sections

21. Antibodies Employed in Paraffin Tissue Sections

22. Antibodies Employed in Paraffin Tissue Sections

23. Antibodies Employed in Fresh or Frozen Tissue

24. Antibodies Employed in Fresh or Frozen Tissue

25. Antibodies Employed in Fresh or Frozen Tissue

26. Antibodies Employed in Fresh or Frozen Tissue

27. Antibodies Employed in Fresh or Frozen Tissue

28. Antibodies Employed in Fresh or Frozen Tissue

29. Antibodies Employed in Fresh or Frozen Tissue

30. Antibodies Employed in Fresh or Frozen Tissue

31. Antibodies Employed in Fresh or Frozen Tissue