Which immunohistochemical markers should be used to identify B‑cell lineage in tissue sections?

B-Cell Immunohistochemical Markers

For B-cell lineage identification in tissue sections, use CD20 and/or CD79a as first-line markers, with PAX5 serving as the most reliable backup marker when conventional markers are negative or equivocal. 1

Core B-Cell Lineage Markers

Primary Panel

• CD20: Most sensitive marker for mature B-cell lymphomas in routine practice, expressed in the vast majority of B-cell neoplasms 1, 2
• CD79a: Most specific marker alongside CD20; use together with CD20 for optimal sensitivity and specificity in mature lymphoid neoplasms 1, 2
• PAX5: Essential backup marker that remains positive even when CD20 and CD79a are negative or weak; expressed in nearly all B-cell neoplasms including those lacking conventional markers 3, 4, 5, 6

When to Add Secondary Markers

• Surface and cytoplasmic immunoglobulins (sIg/cIg): Confirm B-cell lineage and detect monotypic expression on frozen and paraffin sections respectively 1
• Oct2 and Bob1: Critical for CD20-negative cases, particularly plasmablastic lymphomas, primary effusion lymphomas, and post-rituximab recurrent lymphomas where conventional markers fail 4, 7
• CD19: More sensitive than CD79a but less specific (can be positive in rare T-cell lymphomas); useful in acute leukemias and precursor B-cell neoplasms 2

Context-Specific Marker Selection

For Cutaneous B-Cell Lymphomas

• CD3: Simultaneously assess to quantify admixed reactive T cells 1
• Bcl-2, Bcl-6, CD10: Distinguish primary cutaneous from secondary systemic lymphoma; strong expression of all three in follicular structures indicates systemic follicular lymphoma with skin involvement 1, 3
• CD5 and cyclin D1: Differentiate primary cutaneous marginal zone lymphoma (both negative) from mantle cell lymphoma (both positive) and chronic lymphocytic leukemia (CD5+, cyclin D1−) 1, 8

For CD20-Negative B-Cell Neoplasms

This scenario occurs in three main contexts:

• Precursor B-lymphoblastic leukemia/lymphoma: PAX5 (100% positive), BOB.1 (93%), CD79a (79%), OCT.2 (22%) 4
• Post-rituximab recurrent lymphomas: PAX5 (88%), CD79a (84%), OCT.2 (81%), BOB.1 (73%) 4
• Plasmablastic/primary effusion lymphomas: OCT.2 and BOB.1 are most useful when CD20, CD79a, and PAX5 are all negative; combined Oct2/Bob1 positivity reaches 94% 4, 7

For Hodgkin Lymphoma

• PAX5: Expressed in 88% of Hodgkin lymphoma cases, often the only detectable B-cell marker in Reed-Sternberg cells 6
• Hierarchy of expression in Reed-Sternberg cells: PAX5 > CD20 > CD79a > CD19 2

Ancillary Markers for Subtyping

Once B-cell lineage is confirmed, add these markers for specific diagnoses:

• Ki-67: Proliferative fraction; distinguishes neoplastic from reactive follicles 1
• CD21 or CD35: Visualize follicular dendritic networks and reactive follicle remnants 1
• MUM-1 and FOXP1: Aid in distinguishing CBCL subtypes and secondary involvement 1

Critical Diagnostic Pitfalls

• Never rely on a single marker: CD20 alone misses 12% of Hodgkin lymphomas and essentially all plasmablastic lymphomas 6, 7
• PAX5 specificity: While highly specific for B-cells, <1% of non-hematolymphoid tumors may show aberrant expression 5
• Post-rituximab cases: CD20 may be completely absent; always include PAX5 in the initial panel for any suspected recurrent lymphoma 4
• Adequate tissue: Obtain excisional biopsy when possible; punch biopsies should be ≥4mm diameter to preserve architecture 1, 3

Recommended Algorithmic Approach

1. Initial panel: CD20 + CD79a + CD3 (to exclude T-cell) 1, 2
2. If CD20/CD79a negative or equivocal: Add PAX5 immediately 4, 5, 6
3. If all three negative: Add Oct2 + Bob1 before concluding non-B-cell lineage 7
4. Once B-cell confirmed: Add subtyping markers (CD5, CD10, Bcl-2, Bcl-6, cyclin D1) based on morphology and clinical context 1, 3, 8