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淋巴瘤 - Normal LN & immunophenotype - importance of quality


更新时间:2008-06-27 14:45:34 作者: 陈国璋



NORMAL LYMPH NODE AND IMMUNOPHENOTYPE; Importance of technical quality
John K.C. Chan
Queen Elizabeth Hospital
Hong Kong
 

QUALITY OF HISTOLOGIC SECTION
Good-quality histologic section is essential for assessment of lymphoid proliferations; even top expert cannot help for poorly fixed or poorly processed tissues
The most important modalities of “investigation” in hematopathology are: morphology … morphology … and morphology


One cannot practise good hematopathology
without access to high-quality
immunohistochemistry service


Most useful antibodies for identifying normal cell types and corresponding tumors
B lineage            CD20
                     (Reserve: CD79a, PAX-5)
T lineage            CD3
                     (Reserve: CD45RO, CD2)
NK lineage          CD56 [Note: Not CD57]
Myeloid                Myeloperoxidase
Follicle center cell CD10, bcl-6

Most useful antibodies for identifying normal cell types and corresponding tumors
Mantle zone cell                    IgD
Immature precursor cell             TdT
Plasma cell                         CD20-, CD138+, MUM1+
                                    (Reserve: Oct-2, Bob.1)
Histiocyte                          CD68, CD163
Follicular dendritic cell           CD21, CD35
Langerhans cell                     S100, CD1a, Langerin

LYMPH NODE ARCHITECTURE
Cortex
Paracortex
Medulla
Capsule and fibrous trabeculae
Sinuses


The cortex: B-zone of lymph node
Primary lymphoid follicle
Virgin B cells and mature B cells
Follicular dendritic cells


The cortex: B-zone of lymph node
Secondary lymphoid follicle
Centroblasts, centrocytes (follicle center B cells; CD10+, Bcl6+)
Follicular dendritic cells
T cells (CD10 strongly +; usually CD4+ and CXCL13+; subset CD57+)
Tingible body macrophages
Mantle zone B cells (IgM+D)


The cortex: B zone of lymph node
Marginal zone B cells
In most lymph nodes, it is very difficult to find marginal zone B cells around the reactive follicles
Marginal zone cells (slightly larger than mantle zone cells, with a greater amount of pale to clear cytoplasm) are easiest to find in intraabdominal lymph nodes


Reactive changes involving the cortex
Reactive follicular hyperplasia
Secondary follicles are formed as a reaction to T cell dependent antigens
Germinal center typically shows dark and light zones, i.e. exhibiting polarity. Polarity of germinal centers is easiest to appreciate in the tonsils
May be accompanied by an increase of plasma cells


The paracortex: T-zone of lymph node
Predominated by T cells (CD4>CD8): small lymphocytes; occasional large T cells (some large cells scattered in paracortex are activated B cells)
High endothelial venules (permitting lymphocytes to traffic through them)
S100+ antigen presenting cells (interdigitating dendritic cells)
Cytokeratin+ dendritic cells (fibroblastic dendritic cells?)


Reactive changes involving paracortex
Commonest causes: viral infection, hypersensitivity, tumor in drainage area
Paracortex expanded, sometimes forming nodules
High endothelial venules increased; mixture of small lymphocytes and immunoblasts


Medullary cords
Rich in plasma cells
May be inconspicuous or prominent
Expanded in B cell reaction, e.g. reactive follicular hyperplasia, plasma cell type Castleman disease


Sinuses
Afferent lymphatics Subcapsular sinus Intermediate sinus Medullary sinus Efferent lymphatics
Sinuses are lined by sinus lining cells (with desmosomes). They contain some histiocytes and lymphoid cells
Sinuses are often dilated and prominent in intraabdominal lymph nodes


Connective tissue framework
Fibrous capsule, with fibrous trabeculae projecting into the node (sinuses are also found adjacent to the trabeculae)
Normally inconspicuous
Prominent in any reactive condition with a prominent component of perilymphadenitis


Special cell type: Monocytoid B cells
Known as “immature sinus histiocytes” in the past
Occur as non-circumferential bands in a perifollicular location or in sinuses, adjacent to hyperplastic follicles
Medium-sized cells with slightly notched nuclei, fine chromatin, and pale cytoplasm; often sprinkled with neutrophils; central necrosis can occur
Relationship with marginal zone B cells unknown. Previously thought to be post-germinal center B cells, but recent studies indicate naïve B cells


Monocytoid B cell reaction
Not pathognomonic of any condition
Prominent in:
Toxoplasmosis
HIV-associated lymphadenopathy
Cat scratch disease
Lymphogranuloma venereum
CMV lymphadenitis

Special cell type: Plasmacytoid dendritic cells
Known as “plasmacytoid monocytes” or “plasmacytoid T cells” in the past
Occur in 10-17% of non-specific reactive lymphadenopathies, but up to 87% if immunostains (LN2 or CD68) are used to detect them
Particularly common in:
Kikuchi lymphadenitis
Hyaline-vascular Castleman disease


Special cell type: Plasmacytoid dendritic cells
Nature of plasmacytoid dendritic cells
Believed to represent dendritic cell precursors (can differentiate into dendritic cells in vitro)
The natural interferon-producing cells

Plasmacytoid dendritic cells: phenotype
CD2- CD3- CD5- CD7-
CD4+ CD8-
CD123+ (IL-3 receptor)
Interferon-α+
CD36+ CD43+ CD45RA+ CD68+ CD74+
B markers negative


Special cell type: Polykaryocyte
“Warthin-Finkeldey giant cell”
Multiple, closely packed nuclei; scanty cytoplasm
Found in germinal centers (may represent fused follicular dendritic cells) or paracortex (may represent fused endothelial cells)
Particularly common in:
HIV-associated lymphadenopathy
Kimura disease lymphadenopathy
Measles infection


Immunohistochemistry


Pan-hematolymphoid marker
CD45 (CD45RB):
Leukocyte common antigen
Positive in all normal lymphoid cells (B, T or NK lineage) except plasma cells
Reed-Sternberg cells (Hodgkin lymphoma) are CD45-
But usually not necessary to stain for LCA in a definite lymphoma: can proceed directly to lineage markers (e.g. CD20 and CD3)


B lineage markers
CD20 (e.g. L26):
Very sensitive (>95% B cell lymphomas positive), although B-CLL may not stain well and B-cell lymphoblastic lymphoma may be negative
Negative in plasma cells, plasmacytomas and plasmablastic lymphomas
Nucleolar staining alone should not be considered positive. With heat-induced antigen retrieval, any cell can show this phenomenon
Most important B-cell marker for B-lymphomas since availability of anti-CD20 (rituximab) therapy

B lineage markers
CD79a:
B cell receptor complex: sensitive and specific B cell marker (normal plasma cells are positive; germinal center cells are stained weakly)
~50% plasmacytomas are positive
But can be positive in T-lymphoblastic lymphoma/leukemia
More expensive than CD20
Immunoglobulin

B-lineage markers
PAX5:
B-cell-specific transcription factor
Expressed from early stage of B cell development to mature B cells, except plasma cells
B cell neoplasms are PAX-5 positive, except plasmacytoma. Reed-Sternberg cells: weak/moderate positive
Main applications:
Confirm B-lineage of lymphoblastic neoplasms
Classical Hodgkin lymphoma (PAX5+) versus anaplastic large cell lymphoma (PAX5-)

B-lineage markers
Oct-2, Bob.1:
In B cells, Ig gene expression requires interaction between an octamer and its transactivating factors, e.g. Oct-2, Bob.1
B cells (including plasma cells) are Oct-2+, Bob.1+
Main applications:
Classical Hodgkin lymphoma (both negative or only 1 positive) vs large B cell lymphoma (both positive)
Suspected plasmacytoma, especially when other markers are negative
Can be positive in some T-cell lymphomas, esp. Bob.1

T-lineage markers
Cytoplasmic CD3 (polyclonal against CD3e, monoclonal also available, e.g. PS1):
Highly sensitive and specific for T lineage
Staining stronger in perinuclear space, often accentuating the nuclear foldings
Stains >90% of T and NK cell lymphomas
May add other T markers (e.g. CD45RO, CD2) if CD3 is negative and yet T-cell lymphoma is suspected

T-lineage markers
Surface CD3 (e.g. Leu4, T3, OKT3):
Needs fresh or frozen tissue for demonstration
Most but not all peripheral T cell lymphomas are positive for surface CD3
NK cell lymphoma typically shows the following distinctive phenotype: surface CD3-, cytoplasmic CD3+. The discrepancy is explained by the presence of the incomplete CD3 molecule in the cytoplasm of NK cells; surface CD3 antibody reacts with the complete CD3 molecule

T-lineage markers
CD45RO (e.g. UCHL1, A6):
Highly sensitive for T lineage; some T cell lymphomas are CD3- CD45RO+
But also stains histiocytes/myeloid cells and their tumors
CD43 (e.g. MT1, DFT1, Leu22):
Highly sensitive for T lineage, but not very specific
Also stains histiocytes/myeloid cells and their tumors
Expression in B cells can be upregulated in EBV infection
More useful for diagnosis of small B-cell lymphomas (looking for aberrant coexpression)

T lineage markers
CD4, CD8:
Helper versus cytotoxic phenotype
CD2:
Sensitive and specific pan T cell marker
Expensive: use as back-up
CD7:
Pan T cell marker expressed early in T cell development
Particularly helpful for demonstrating T lineage in lymphoblastic lymphoma (which can be CD3-)

NK cell markers
CD56 (e.g. 123C3):
Best marker for NK cell lymphomas
But CD56 is not entirely specific for NK lymphomas; some T cell lymphomas can also be positive, especially those expressing gd T-cell receptor
CD56 also expressed in non-hematolymphoid cells (especially neural cells and neuroendocrine cells) and their tumors: diagnosis of NK cell lymphoma must be supported by expression of other lymphoid markers
CD57 (Leu7): No use for diagnosis of NK neoplasms

Follicular center cell markers
CD10
CD10 is the common acute lymphoblastic leukemia antigen, normally expressed by follicle center B cells and follicle center T cells
Some spindly stromal cells are also CD10+ (serving as internal positive control)
Value in lymphoma typing:
Follicular center cell differentiation (follicular lymphoma, Burkitt lymphoma, some large B-cell lymphomas)
Angioimmunoblastic T-cell lymphoma

Follicular center cell markers
Bcl-6
Normally expressed (nuclear staining) in follicular center B cells, CD30+ perifollicular cells, and rare subpopulation of T cells
Positive in the following lymphoma types:
follicular lymphoma
some large B cell lymphomas
Burkitt lymphomas
some anaplastic large cell lymphomas
Angioimmunoblastic T-cell lymphoma

Other markers
Follicular dendritic cell markers (such as CD21, CD35, clusterin) can aid in the diagnosis of:
Follicular lymphoma
Mantle cell lymphoma (loose or dispersed meshworks)
Angioimmunoblastic T cell lymphoma (extrafollicular meshworks)
Diagnosis of anaplastic large cell lymphoma
BerH2/CD30, ALK1, clusterin

Other markers
CD30:
A marker for activated lymphoid cells; some CD30+ cells are found in perifollicular region
CD30+ cells are increased in lymphoid hyperplasia
Staining should be in the cell membrane +/- Golgi zone; pure diffuse cytoplasmic staining should not be considered positive
CD30 is positive in classical Hodgkin lymphoma, anaplastic large cell lymphoma and some large cell lymphomas

Other markers
Proliferation marker Ki67: can aid in diagnosis of Burkitt lymphoma (~100% cells positive)
Cytotoxic markers, e.g. TIA1, granzyme B, perforin: may add in the diagnosis of NK/T cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma
Most useful marker for diagnosis of lymphoblastic lymphoma/leukemia: TdT

Important markers for classification of lymphomas
Lymphoblastic lymphoma: TdT (Reserve: CD99)
B-CLL: CD5, CD23
Mantle cell lymphoma: Cyclin D1
Follicular lymphoma: CD10 (or bcl-6)
Burkitt lymphoma: Ki67 (~100% proliferation index)
Angioimmunoblastic T cell lymphoma: CD10, follicular dendritic cell markers (extrafollicular meshworks)
Anaplastic large cell lymphoma: CD30, ALK

 

Flow cytometry Immunohistochemistry
Rapid (hours) Less rapid (1-2 days)
Quantitative Subjective evaluation
Can wash cell surface Strong background for Ig
Can assess coexpression of several antigens At most double staining
Limited morphologic correlation (cell size only) Good architectural and cytologic correlation
Difficult to refer/send out Easy to refer/send out
Possible loss of cell viability No such problem


 

 

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