Immunophenotyping and stains in hematologic malignancies
Hematologic IHC (Reference)
How to Read These Tables
- Notation: + positive, ++ strong/uniform, +/- variable (often positive), -/+ variable (often negative), - negative.
- Lineage-defining pearls: cytoplasmic CD3 is a T-lineage criterion in acute-leukemia phenotyping, but CD3-epsilon is also expressed by NK cells (surface CD3-negative), e.g. extranodal NK/T-cell lymphoma, so it is not strictly T-specific (also TdT+ and frequently CD7+). B-lineage in mixed-phenotype acute leukemia (WHO/ICC): strong CD19 plus >=1 of CD10, CD22, or CD79a strongly expressed, or weak CD19 plus >=2 of these; this is not a general rule for all B cells.; mature B cells are surface Ig+ and usually CD20+. Many precursor B-ALLs are CD10+, HLA-DR+, TdT+; both B- and T-lineage blasts frequently express CD38.
Clinically Useful CD Markers and Lineage Association
Clinically useful CD markers and lineage association
| Marker | Lineage association |
|---|---|
| Progenitor cells | |
| CD34 | Progenitor cells, endothelium |
| CD38 | Myeloid progenitors, T cells, B cells, NK cells, plasma cells, monocytes, CLL subset |
| B-cell markers | |
| CD10 | Pre-B lymphocytes, germinal center cells, neutrophils |
| CD19 | B cells; normal/reactive plasma cells usually CD19+ (dim), most myeloma plasma cells CD19- |
| CD20 | B cells (not plasma cells) |
| CD21 | Mature B cells, follicular dendritic cells, subset of thymocytes |
| CD22 | Mature B cells including germinal center cells (GC-derived lymphomas eg FL, Burkitt are CD22+) |
| CD23 | B cells, CLL |
| CD79b | B cells (not typical CLL) |
| CD103 | Intraepithelial lymphocytes, hairy cell leukemia, T cells in enteropathic T-cell lymphoma |
| FMC7 | B cells (not typical CLL), hairy cell leukemia |
| T-cell markers | |
| CD1a | Cortical thymocytes and certain T-cell leukemias; also positive in epidermal Langerhans and dendritic cells |
| CD2 | Pro- and pre-T cells, T cells, thymocytes, NK cells, some lymphocytes in CLL and B-ALL |
| CD3 | Thymocytes, mature T cells, cytoplasm of immature T cells |
| CD5 | Thymocytes, T cells, B cells in CLL, B cells in mantle cell lymphoma |
| CD4 | Helper T cells, monocytes, dendritic cells, activated eosinophils, thymocytes |
| CD7 | Pro- and pre-T cells, T cells, thymocytes, NK cells, some myeloblasts |
| CD8 | Suppressor T cells, NK cells, thymocytes |
| CD25 | Activated T and B cells, adult T-cell leukemia/lymphoma |
| NK / cytotoxic T-cell markers | |
| CD16 | NK cells, monocytes, macrophages, neutrophils |
| CD56 | NK cells, myeloma cells |
| CD57 | NK cells, T-cell subset |
| Myeloid and monocytic markers | |
| CD13 | Monocytes, neutrophils, eosinophils, basophils |
| CD14 | Monocytes, macrophages, subset of granulocytes |
| CD33 | Myeloid lineage cells and monocytes |
| CD117 | Immature myeloid cells, AML, mast cells |
| Monocytes | |
| CD11c | Monocytes, macrophages, granulocytes, activated B and T cells, NK cells, hairy cell leukemia |
| CD15 | Myeloid lineage cells and monocytes |
| CD64 | Monocytes, immature myeloid cells, activated neutrophils |
| Megakaryocytic markers | |
| CD41 | Platelets and megakaryocytes (GPIIb) |
| CD42 | Platelets and megakaryocytes (CD42a GPIX; CD42b GPIb) |
| CD61 | Platelets, megakaryocytes, endothelial cells (GPIIIa) |
| Erythroid markers | |
| CD71 | Transferrin receptor, upregulated on cell activation |
| CD235a | Glycophorin A |
| Hemoglobin | Stains hemoglobin in mature red cells and precursors |
| E-cadherin | Stains immature erythroid precursors |
Common Stains (Blood, Marrow, Tissue)
Common stains for hematologic disorders
| Stain | Description |
|---|---|
| Myeloperoxidase | Primary granules of neutrophils and secondary granules of eosinophils. Monocytic lysosomal granules stain faintly. Mature lymphocytes do not stain. By cytochemistry, IHC, or flow. Sudan black B shows a similar pattern. |
| Specific esterase | Naphthol AS-D chloroacetate esterase (Leder stain). Neutrophils and mast cells stain; lymphocytes and monocytes do not. |
| Nonspecific esterase | α-Naphthyl butyrate or α-naphthyl acetate. Stains monocytes, macrophages, histiocytes. Does not stain neutrophils or eosinophils. Dot-like pattern in mature T lymphocytes. Megakaryocytes stain with α-naphthyl acetate but not α-naphthyl butyrate. |
| TdT | Intranuclear enzyme. Stains thymocytes and lymphoblasts but not mature lymphocytes. Some myeloblasts stain. By cytochemistry, IHC (fixed tissue), or flow. |
| TRAP | Acid phosphatase isoenzyme. Positive in hairy cell leukemia, Gaucher cells, activated T lymphocytes. By cytochemistry or IHC (fixed tissue). |
| PAS | Periodic acid-Schiff. Detects intracellular glycogen and neutral mucosubstances. Positive in ALL, AML, erythroleukemia, and Gaucher cells. |
| Toluidine blue | Detects acid mucopolysaccharides. Positive in mast cells and basophils. More specific mast cell stains exist by IHC. |
| Iron | Perls / Prussian blue. Identifies hemosiderin in NRBCs (sideroblastic iron) and histiocytes (reticuloendothelial iron). Ring sideroblasts are abnormal NRBCs with ≥5 blue iron granules (in perinuclear mitochondria) around ≥1/3 of the nucleus. Marrow core biopsy can underestimate stores due to iron loss during decalcification. |
| GMS | Grocott methenamine silver. Identifies fungal organisms; carbohydrates in fungal cell walls stain brown to black. |
| Acid-fast | Ziehl-Neelsen or Kinyoun. Acid-fast organisms have mycolic-acid-rich walls; carbol-fuchsin (lipid-soluble) penetrates the wall, highlighting mycobacteria pink/red. |
AML Phenotyping (FAB Classification, by Morphology)
AML phenotyping by FAB subtypeFAB is no longer used; swipe sideways on phone
| FAB | Subtype | HLA-DR | CD34 | CD33 | CD13 | CD11c | CD14 | CD41 | CD235a | CD61 |
|---|---|---|---|---|---|---|---|---|---|---|
| M0 | AML with minimal differentiation | + | + | + | +/- | +/- | - | - | - | |
| M1 | Minimal maturation | + | + | + | + | +/- | +/- | - | - | |
| M2 | With maturation | +/- | +/- | + | + | +/- | +/- | - | - | |
| M3 | Acute promyelocytic (APL) | - | - | + | + | +/- | - | - | - | |
| M4 | Acute myelomonocytic (plus or minus eos) | + | +/- | + | + | + | + | - | - | |
| M5 | Acute monocytic | + | - | + | + | + | + | - | - | |
| M6 | Acute erythroid | +/- | - | - | - | +/- | - | - | + | |
| M7 | Acute megakaryoblastic | +/- | +/- | +/- | - | - | - | + | - | + |
B-lineage ALL Phenotyping
B-lineage ALL phenotypingBy stage of maturation
| Stage | TdT | CD19 | CD10 | CD20 | Cyto-mu | Surface Ig |
|---|---|---|---|---|---|---|
| Pro-B | + | + | - | - | - | - |
| Pre-pre-B (common ALL) | + | + | + | - | - | - |
| Pre-B | + | + | + | +/- | + | - |
| Mature B (Burkitt) | - | + | + | + | - | + |
Cyto-mu, cytoplasmic mu; Ig, immunoglobulin.
T-lineage ALL Phenotyping
T-lineage ALL phenotypingBy stage of maturation
| Stage | TdT | CD7 | CD2 | CD5 | CD1a | sCD3 | cCD3 | CD4/CD8 |
|---|---|---|---|---|---|---|---|---|
| Prothymocyte | + | + | + | - | - | - | + | -/- |
| Immature thymocyte | + | + | + | + | - | - | + | -/- |
| Common thymocyte | + | + | + | + | + | +/- | + | +/+ |
| Mature thymocyte | - | + | + | + | - | + | + | CD4 or CD8+ |
| Mature T cell | - | + | + | + | - | + | + | CD4 or CD8+ |
cCD3, cytoplasmic CD3; sCD3, surface CD3.
Common B-cell Neoplasms
Common B-cell neoplasms: immunophenotypeTable 12-10; swipe sideways on phone
| Entity | CD20 | CD5 | CD10 | CD23 | CD43 | cIg | sIg | Cyclin D1 | Other |
|---|---|---|---|---|---|---|---|---|---|
| CLL/SLL | + | ++ | - | ++ | ++ | 5%+ | + | - | CD200+, CD79b dim |
| LPL | ++ | - | - | - | +/- | + | + | - | |
| PLL | ++ | +/- | - | ++ | - | ||||
| HCL | ++ | - | - | - | - | - | + | +/- | CD11c+, CD25+, CD103+ |
| MCL | ++ | ++ | - | - | ++ | - | ++ | ++ | CD200- |
| MZL | ++ | - | - | - | +/- | +/- | ++ | - | |
| FL | ++ | - | 60%+ | -/+ | - | - | ++ | - | BCL2+, BCL6+ |
| LCL | ++ | 10%+ | 25% to 50%+ | - | +/- | +/- | +/- | - | BCL2+ in 30% to 40% |
| BL | ++ | - | + | - | - | + | + | - | BCL2- |
| Myeloma | -/+ | - | Occ + | - | + | ++ | - | ~25%+ | CD56+, CD38+, CD138+, MUM1+ |
BL, Burkitt lymphoma; cIg, cytoplasmic Ig; FL, follicular lymphoma; HCL, hairy cell leukemia; LCL, large-cell lymphoma; LPL, lymphoplasmacytic lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; Occ, occasionally; PLL, B-cell prolymphocytic leukemia; sIg, surface Ig; SLL, small lymphocytic lymphoma.
Common Mature T-cell and NK-cell Neoplasms
Common mature T-cell and NK-cell neoplasms: immunophenotypeTable 12-11; swipe sideways on phone
| Entity | sCD3 | cCD3 | CD5 | CD7 | CD4 | CD8 | CD30 | CD16 | CD56 | EBV | Other |
|---|---|---|---|---|---|---|---|---|---|---|---|
| T-PLL | +dim | + | + | + | +/- | -/+ | - | - | - | - | TCL1+, CD52+ |
| T-LGL | + | + | + | + | - | + | - | + | - | - | Granzyme+, TIA1+ |
| NK leukemia | - | + | - | +/- | - | +/- | - | +/- | + | + | Granzyme+, TIA1+ |
| EN-NK/T | - | + | - | +/- | - | - | - | - | + | + | Granzyme+, TIA1+ |
| HSTL | + | + | - | + | - | - | - | + | +/- | - | Gamma delta TCR+, alpha beta TCR- |
| Ent-T lym | + | + | - | + | - | +/- | +/- | - | - | - | Granzyme+, TIA1+, perforin+, CD103+ |
| SCPTL | + | + | + | + | - | + | +/- | - | - | - | Granzyme+, TIA1+, perforin+, alpha beta TCR+, CD123- |
| PTCL-NOS | + | + | +/- | +/- | +/- | +/- | +/- | - | -/+ | +/- | |
| AILT | + | + | + | + | +/- | - | - | - | - | +/- | CD10+, CXCL13+, BCL6+, PD1+; EBV +/- reflects background B immunoblasts, not the neoplastic TFH cells (EBV in the malignant T/NK cells suggests EBV-positive nodal T/NK-cell lymphoma) |
| ALCL | -/+ | - | +/- | +/- | +/- | +/- | ++ | - | - | - | sys ALCL ALK+ or ALK-; pc-ALCL, BIA-ALCL ALK- |
Hodgkin Lymphoma IHC
Immunohistochemical diagnosis of Hodgkin lymphomaNeoplastic cell phenotype
| Entity | CD45 | CD30 | CD15 | CD20 | CD3 | PAX5 |
|---|---|---|---|---|---|---|
| CHL (RS cells) | - | + | + | - | - | dim+ |
| NLPHL (LP cells) | + | - | - | + | - | + |
| B-cell lymphoma | + | +/- | - | + | - | + |
| T-cell lymphoma | + | +/- | +/- | - | + | - |
CHL, classical Hodgkin lymphoma; RS, Reed-Sternberg; NLPHL, nodular lymphocyte-predominant Hodgkin lymphoma; LP, lymphocyte-predominant.
- cHL subtypes: nodular sclerosis (~60%), mixed cellularity (15% to 25%, EBV+), lymphocyte-rich (~5%), lymphocyte-depleted (~1%, worst prognosis). CD30 is the target of brentuximab vedotin.
- NLPHL (WHO 2022) / NLPBL (ICC 2022): popcorn LP cells CD20+, CD45+, BCL6+, OCT-2+, BOB.1+, CD15-, CD30-; managed stage- and risk-adapted: involved-site RT alone for early favorable (eg stage IA), observation for selected excised cases, rituximab-based systemic therapy for advanced/symptomatic disease.
NHL Phenotype & Translocations
Common chromosomal translocations in selected NHL subtypesStandard reference
| Subtype | Translocation | Genes involved |
|---|---|---|
| Follicular lymphoma | t(14;18)(q32;q21) | IGH::BCL2 |
| Mantle cell lymphoma | t(11;14)(q13;q32) | CCND1 (cyclin D1)::IGH |
| Burkitt lymphoma | t(8;14)(q24;q32); variants t(2;8), t(8;22) | MYC::IGH (variants MYC with IGK or IGL) |
| Extranodal MZL (MALT) | t(11;18)(q21;q21) | BIRC3 (API2)::MALT1 |
| DLBCL | 3q27 rearrangement; double-hit (DLBCL/HGBL with MYC and BCL2 rearrangements, WHO 2022) = MYC plus BCL2; MYC plus BCL6 alone is excluded (WHO) or provisional (ICC) | BCL6; MYC, BCL2, BCL6 |
| ALCL (ALK+) | t(2;5)(p23;q35) | NPM1::ALK |
| LPL / Waldenstrom | Point mutation (not a translocation) | MYD88 L265P |
Quick Lineage Marker Summary
- Blasts: CD34+, CD117+, CD123+, HLA-DR+.
- Myeloid (AML): CD13+, CD33+, MPO+, CD11b+.
- B-cell: CD19+, CD20+, CD22+, CD79a+, PAX5+.
- T-cell: CD2+, CD3+, CD5+, CD7+, CD30+ (in some). Cytoplasmic CD3 is the T-lineage criterion in acute leukemia, but also expressed by NK cells (not strictly T-specific).
- B-ALL: almost always CD19+, cCD79a+, cCD22+ (CD19 and CD22 targetable); most also CD10+, surface CD22, PAX5+, TdT+; CD20/CD34 variable (CD20 targetable).
- T-ALL (20% to 30% of adult ALL): TdT+, cCD3+, CD7+; often CD1a+, CD4, CD8. Variable CD1a, CD2, CD3, CD4, CD5, CD7, CD8.
- ETP-ALL (5% to 10%): CD7+, CD8-, CD1a-, CD5 absent/weak (under 75% of blasts), plus at least one myeloid/stem-cell marker (CD34, CD117, HLA-DR, CD13, CD33).
- Burkitt: strong surface IgM with light-chain restriction, strong CD20, plus CD19, CD22, CD79a, PAX5; often CD10, BCL6.
- Hairy cell leukemia: CD11c+, CD103+ (also CD25+, annexin A1+, TRAP+).
Practical Distinctions & Targets
- CLL vs MCL: CLL = CD5+ CD23+ CD20 dim CD79b dim CD200+; MCL = CD5+ CD23- cyclin D1+ t(11;14), FMC7+, CD200-. SOX11 marks MCL (including cyclin D1-negative); leukemic non-nodal MCL is SOX11-.
- Hans algorithm (DLBCL): CD10+ → GCB; CD10- BCL6- → non-GCB; CD10- BCL6+ → MUM1+ non-GCB, MUM1- GCB. Hans assigns GCB versus non-GCB (not molecular ABC): CD10+ is GCB regardless of BCL6/MUM1; CD10-/BCL6+/MUM1- is also GCB; remaining patterns are non-GCB.
- Burkitt vs DLBCL-GCB: Ki-67 near 100%, BCL2-, monomorphic medium-sized cells with deeply basophilic, vacuolated cytoplasm favor Burkitt.
- Plasma cell neoplasms: CD138+ (syndecan-1), CD38+, CD56+, MUM1+, kappa/lambda-restricted; CD19- (rarely +), CD20- in most, but CD20 expressed in about 15 to 20% (can be strong), enriched in t(11;14), CD45 dim/-. Targets: CD38 (daratumumab, isatuximab), BCMA (teclistamab, elranatamab; ide-cel, cilta-cel), SLAMF7 (elotuzumab). Cyclin D1+ in ~25% by IHC; t(11;14) in ~15 to 20% (enriched in AL amyloidosis and plasma cell leukemia; predicts venetoclax sensitivity).
- Mast cell & histiocytic: systemic mastocytosis = tryptase+, CD117+, aberrant CD25+ (often CD2+), KIT D816V. LCH = CD1a+, S100+, langerin (CD207)+, Birbeck granules. Rosai-Dorfman = S100+, CD68+, CD1a-, emperipolesis. FDC sarcoma = CD21+, CD23+, CD35+. BPDCN = CD4+, CD56+, CD123+, TCL1+, CD303+.
- Solid-tumor IHC pearls: BAP1 loss (mesothelioma, RCC, uveal melanoma); SOX10 (melanoma, salivary, nerve sheath); TTF-1 (lung adenocarcinoma, thyroid); CDX2 (GI origin); GATA3 (urothelial, breast); p63/p40 (squamous, urothelial, salivary).
- Therapeutic target panel: CD20 (rituximab, obinutuzumab, ofatumumab; bispecifics mosunetuzumab/epcoritamab/glofitamab); CD19 (blinatumomab, tafasitamab; CAR-T axi-cel/tisa-cel/liso-cel/brexu-cel); CD22 (inotuzumab ozogamicin; moxetumomab withdrawn from US market 2023); CD30 (brentuximab vedotin); CD33 (gemtuzumab ozogamicin); CD38 (daratumumab, isatuximab); BCMA (ide-cel, cilta-cel; teclistamab, elranatamab, linvoseltamab); GPRC5D (talquetamab); SLAMF7 (elotuzumab).
Veli Bakalov MD, Board Review Notes 2026