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Lymph Node Normal Morphology
Cortex
Primary Follicle
Secondary Follicle
Mantle Zone
Paracortex
Medulla
Sinuses
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Cortex
Primary B-Cell Follicles
Nodules of small lymphocytes
Lack germinal centers
Secondary B-Cell Follicles
Result of stimulation
Germinal Centers
Mantle zone
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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
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Mantle Zone
polarized toward antigen entry
express bcl-2 protein
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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
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Medullary areas
B cells predominate especially plasma cells
histiocytes
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Handling the Fresh Specimen
Surgeon should excise the largest and most
abnormal node
Tissue for histology
Touch imprints
Fresh / frozen tissue for immunologicstudies
Sterile portion for cytogenetics
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Frozen Section
Diagnostic frozen section should be
discouraged
Use frozen to assess adequacy or triage
tissue
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Freezing for Immunologic
Studies
Liquid nitrogen or isopentane / dry ice mix
is best
Thin sections (
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Fixation
Node sliced in 2-3 mm intervals
One metal based fixative (B5, Zenkers, zinc
sulfate)
One neutral buffered formaldehyde
(formalin)
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Processing
Single most important factor for optimal
histology is section thickness
Sections should be one cell layer thick
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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
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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)
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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
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
LCA
CD45RB
Lymphocytes,
myeloid cells andmacrophages
Plasma cells and RS
cells negative
Immuno-
globulin
B cells and plasma
cells
Paraffin c to lasmic
Frozen surface
L26
CD20
B cells occasional
macrophages
Plasma cells negative
Poor reactivity in
decalcified tissue
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
CD79 B cells Plasma cells negative
LN1
CDw75
Germinal center B
cells
Better in B5
LN2CD74
B cells,interdigitating
dendritic cells,
macrophages
Better in B5
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
MB1
CD45RA
B cells and some T
cells
Plasma cells negative
CD3 T cells May require
pretreatment
Leu-22CD43 T cells tissue CD5useful in small
lymphocytic
neoplasms
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
UCHL1
CD45RO
T cells May see nonspecific
cytoplasmic staining
Leu-7
CD57
GC T cells and some
NK cells
Better in frozen tissue
TCR betachain
T cells Requires proteolyticdigestion; better in
frozen tissue
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
LeuM1
CD15
Granulocytes,
macrophages
Carcinomas and
CMV infected cellsmay be reactive
BerH2
CD30
Activated B and T
cells, plasma cells
Less reactive in B5,
embryonal ca. and
few other non-hematolymphoid
neoplasms positive
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
KP1
CD68
Macrophages
myeloid cells
Reactive in
melanoma, MFH,granular cell tumors
and renal cell ca.
Lysozyme Macrophages
myeloid cells
Reactive with many
non-hematolymphoidneoplasms
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
S100 Langerhans cells,
interdigitating andsome follicular
dendritic cells
Reactive with many
non-hematolymphoidneoplasms
Mac-387 Macrophages
myeloid cells
Reactive with some
squamous cell ca.
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Antibodies Employed in Paraffin
Tissue Sections
Antibody /
CD Anti en
Normal Reactivity Comments
bcl-2 Non-germinal center
B cells, most T cellsand plasma cells
Best in B5;
differentiate benignfrom malignant
follicular lesions
Myeloper-
oxidase
Myeloid cells Most sensitive and
specific myeloid
marker
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD45 Hematolymphoid
cells
Plasma cells and
ALCL may benegative
Immuno-
globulin
B cells Monoclonal Ig less
sensitive than
polyclonal Ig
Precursor Blymphomas and
leukemias may be
negative
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD19 B cells Earliest expressed B
cell antigen
CD20 B cells Most plasma cells
unreactive
CD79 B cells May be negative inplasma cells
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD10
CALLA
Precursor and GC
B cells
Follicular vs. other
low grade MLEwings and MFH
positive
CD21 Mantle and marginal
zone B cells
Most mantle and
marginal zone MLCD23 CLL / SLL reactive
Mantle cell ML
unreactive
Upregulated by EBV
infection
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD32 Mantle zone B cells Reactive with MCL
and some follicularML, myeloid
leukemias
CD35 Mantle and marginal
zone B cells
Similar to CD21
CD38 Lymphoid progenitor
cells, NK cells and
plasma cells
One of few markers
expressed by plasma
cell neoplasms
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD2, CD3,
CD5 andCD7
T cells Pan T cell antigens
CD7 earliestexpressed and most
commonly deleted
CD1a Cortical thymocytes Good for precursor T
cell neoplasms;thymomas rich in
CD1A cells
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD4 Helper/Inducer T
cells macrophagesand dendritic cells
May be expressed in
some plasma cellneoplasms
CD8 Cytotoxic/Suppressor
T cells and NK cells
Minority T subset;
may be increased in
early phase viralinfections or late HIV
infection
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD11c Myelomonocytic
cells
Hairy cell leukemia
and monocytoid Bcell ML
CD13 Myelomonocytic
cells
Most AML M1-M5
CD14 Monocytes andmacrophages
Many M4-M5 AML
CD33 Myeloid cells and
monocytes
Most myeloid
leukemias
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD16 NK cells and
granulocytes
CD56 NK cells and a few T
cells
Reactive with neural
and neuroendocrine
cells and neoplastic
plasma cellsCD57 Some NK cells and T
cells
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Antibodies Employed in Fresh or
Frozen Tissue
Antibody /
CD Anti en
Normal Reactivity Comments
CD25 Activated T cells, B
cells and monocytes
ATLL, HCL, ALCL
and some other T andB cell ML
CD34 Progenitor cells and
endothelial cells
Stem cell marker
Good for vascular
neoplasmsTdT Precursor cells in
marrow and cortical
thymocytes
Lymphoblastic
leukemia / l m homa
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