Immunohistochemical Stains to Differentiate Hodgkin from Non-Hodgkin Lymphoma
A core IHC panel including CD30, CD15, CD20, CD3, CD45, and PAX5 is essential to distinguish Hodgkin lymphoma from non-Hodgkin lymphoma, with CD30 and CD15 positivity in Reed-Sternberg cells being the hallmark of classical Hodgkin lymphoma. 1, 2, 3
Primary Diagnostic Panel
The following markers form the essential first-line panel for differentiating these entities:
For Classical Hodgkin Lymphoma (CHL) Identification:
- CD30: Positive in 100% of CHL cases; Reed-Sternberg cells show consistent strong expression 1, 2, 3
- CD15: Positive in approximately 80% of CHL cases; shows paranuclear deposit with cell surface and cytoplasmic staining 1, 4
- CD20: Positive in <40% of CHL cases (weak/variable expression) 1, 2
- CD45: Negative in CHL (positive in most NHL) 1, 2, 3
- PAX5: Weak positive in CHL (strong positive in B-cell NHL) 2, 3
- CD3: Negative in CHL (positive in T-cell NHL) 2, 3, 5
For Non-Hodgkin Lymphoma Identification:
- CD20: Strong diffuse positivity indicates B-cell NHL 6, 5
- CD79a: Alternative B-cell marker when CD20 is negative or after rituximab therapy 6, 5
- CD3: Pan T-cell marker for T-cell NHL 7, 5
- CD45: Positive in most NHL (negative in CHL) 1, 3
Algorithmic Approach to Staining Interpretation
Step 1: Initial Assessment
Begin with CD30, CD15, CD20, CD3, CD45, and PAX5 1, 2, 3:
- If CD30+/CD15+/CD45-: Strongly suggests classical Hodgkin lymphoma 1, 2
- If CD20 strong+/CD45+: Indicates B-cell non-Hodgkin lymphoma 6, 5
- If CD3+/CD45+: Indicates T-cell non-Hodgkin lymphoma 7, 5
Step 2: Expanded Panel for Equivocal Cases
When B-cell lineage overexpression occurs in suspected CHL or differential includes large B-cell lymphoma, add 3:
- OCT2: Negative in CHL, positive in DLBCL
- BOB1: Negative in CHL, positive in DLBCL
- MUM1/IRF4: Variable in both
- BCL6: May be positive in both 6, 8
Step 3: Subtype-Specific Markers
For nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) vs. CHL 1, 3:
- NLPHL: CD20+, CD45+, CD15-, CD30-
- CHL: CD30+, CD15+ (majority), CD45-, CD20+ (<40%)
For distinguishing specific NHL subtypes 6, 8, 9:
- Cyclin D1: Positive in mantle cell lymphoma
- CD10: Positive in follicular lymphoma and some DLBCL
- BCL2: Positive in follicular lymphoma
- CD5: Positive in mantle cell lymphoma and some DLBCL
- Ki-67: Proliferation index (>90% suggests Burkitt lymphoma) 8, 5
Critical Diagnostic Pitfalls
CD20 expression in CHL: Up to 40% of classical Hodgkin cases show CD20 positivity, which can mislead toward NHL diagnosis 1, 2. The key distinguishing feature is that CD20 in CHL is weak/variable, whereas NHL shows strong diffuse positivity 3.
CD30 expression in NHL: Some peripheral T-cell lymphomas and anaplastic large cell lymphoma express CD30 7, 3. Differentiate by checking ALK status, pan T-cell antigens (CD2, CD5, CD7), and cytotoxic markers (granzyme, perforin, TIA1) 7, 3.
GATA3 utility: GATA3 is positive in 80% of CHL cases and negative in 100% of NLPHL cases, providing 100% negative predictive value for excluding NLPHL 10. However, 20% of CHL can be GATA3-negative 10.
Technical Considerations
Specimen requirements: Excisional or core needle biopsy is mandatory; fine-needle aspiration alone is insufficient for initial diagnosis 6, 1, 11, 2. Adequate tissue must be available for both morphological assessment and comprehensive immunophenotyping 6, 1.
Antigen retrieval: Microwave citrate antigen retrieval method significantly improves immunoreactivity and detection of Reed-Sternberg cells in formalin-fixed paraffin-embedded tissues 4.
Panel approach: A panel of markers is always necessary rather than relying on single markers, as no individual antibody is entirely specific 3, 12, 4.