Non-Hodgkin's Lymphoma Diagnostics

Last updated: 16 July 2025
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Laboratory Tests and Ancillaries

Biopsy  

Lymph Node Biopsy  

Lymph node biopsy is the cornerstone of diagnosis. This is indicated for nodes >1.5 cm in diameter, firm, irregular, clustered, and fixed palpable nodes. This is also used for suspected small lymphocytic lymphoma (SLL) patients with lymphadenopathy and/or splenomegaly, and abnormal peripheral blood count. Excisional or incisional biopsies are preferred.  

Core Needle Biopsy  

Core needle biopsy is suggested when lymph nodes are not accessible. This is recommended before initiation of radioimmunotherapy (RIT).  

Bone Marrow Biopsy  

Bone marrow aspiration and trephine biopsy are recommended for staging, especially for those with no access to positron emission tomography (PET)-computed tomography (CT) scans and for non-fluorodeoxyglucose (FDG)-avid NHL. This is indicated for specimens ≥1.6 cm and clinical stage I-II FL, marginal zone lymphoma (MZL), MCL and BL. This is indicated for primary cutaneous DLBCL leg type. Bone marrow aspiration is utilized in cases where lymph nodes are inaccessible for biopsy.  

Skin Biopsy  

Skin biopsy is indicated for MF/SS and cutaneous B-cell lymphoma. Incisional/excisional/punch biopsy is preferred over shave biopsy. This may also be performed for diagnosis of acute T-cell lymphoma/leukemia.



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Molecular and Genetic Analysis  

Molecular and genetic analysis effectively differentiates NHL subtypes by identifying the specific cell lineage using antibodies. Lymph node and spleen tissue samples are preferred over bone marrow tissue samples for cytogenic analysis. This detects immunoglobulin heavy chain variable (IGHV) mutational status. This provides prognostic information for most NHL subtypes.  

Flow Cytometry  

Flow cytometry is a fast and reliable method of identifying single-cell populations of surface antigens. This uses antibodies/markers to identify the presence and proportion of surface antigens by using antibodies/markers. This is used in FL, T-cell prolymphocytic lymphoma, MF/SS (expanded CD4 and cells with increased CD4/CD8 ratio), BL (BCL2 via cerebrospinal fluid analysis), lymphoblastic lymphoma (LL), and nodal and splenic MZL. This is suggested for confirmation of clonality of B cells in chronic lymphocytic leukemia (CLL)/SLL.



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Fluorescence in situ Hybridization (FISH)  

Fluorescence in situ hybridization detects chromosomal abnormalities (11q, 13q, 17p deletion, trisomy 12) and is highly sensitive for known detectable translocation during initial diagnosis. This is used in FL (t[14;18], BCL2 and BCL6 rearrangements), MCL (t[11;14] translocation), CLL (t[11;14] translocation with specific CCND1/IGH probes or CCND1 break-apart probe); DLBCL (MYC gene arrangements). This is recommended for diagnosis of BL (t[8;14]).  

Immunohistochemistry (IHC) Panel  

An immunohistochemistry panel aids in identification and characterization of the immunophenotype of most lymphomas.  

The immunohistochemistry panel used for identification of specific lymphomas:

  • FL: CD20, CD3, CD5, CD10, BCL2, BCL6, CD21, CD23
  • Gastric mucosa-associated lymphatic tissue (MALT) lymphoma: CD20, CD3, CD5, CD10, BCL2, kappa/ lambda, CD21 or CD23, cyclin D1, BCL6
  • Nongastric MALT lymphoma (noncutaneous), nodal MZL: CD20, CD3, CD5, CD10, BCL2, kappa/lambda, CD21 or CD23, cyclin D1
  • Splenic MZL: CD20, CD3, CD5, CD10, BCL2, kappa/lambda, CD21 or CD23, cyclin D1, IgD, CD43, annexin A1
  • MCL: CD20, CD3, CD5, cyclin D1, CD10, CD21, CD23, BCL2, BCL6, TP53, Ki-67, SOX11
  • DLBCL: CD20, CD3, CD5, CD10, CD21, CD45, BCL2, BCL6, Ki-67, IRF4/MUM1, MYC
  • BL: CD45, CD20, CD3, CD5, CD10, Ki-67, BCL2, BCL6, TdT
  • Peripheral T-cell lymphomas: CD20, CD3, CD10, BCL6, Ki-67, CD5, CD30, CD2, CD4, CD8, CD7, CD56, CD21, CD23, EBER-ISH, TCRβ, TCRδ, PD1/CD279, ALK, TP63
  • Primary cutaneous B-cell lymphomas: CD20, CD3, CD10, BCL2, BCL6, IRF4/MUM1
  • MF/SS: CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD30
  • Subcutaneous panniculitis-like T-cell lymphoma (SPTCL): TCRβ, TCRδ, CD2, CD3, CD20, CD4, CD5, CD8, CD30, CD56
  • HCL: CD19, CD20, CD5, CD10, CD11c, CD22, CD103, CD123, cyclin D1, CD200




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Polymerase Chain Reaction (PCR)  

Polymerase chain reaction detects specific abnormal DNA translocations and immunoglobulin gene rearrangements that may be the origin of leukemic cells, such as:

  • bcl gene rearrangements: B-cell lymphoma, FL, DLBCL
  • T-cell receptor gene rearrangements: MF/SS, MCL (CCND2 gene - 55%), SPTCL
  • MYC gene rearrangements: DLBCL (2-11%)

Laboratory Examinations

  • Complete blood count, with differential and platelet count
    • Peripheral monoclonal B lymphocyte count ≥5x109/L – CLL
    • B lymphocytes ≤5x109/L with lymphadenopathy and/or splenomegaly may indicate SLL
  • Metabolic panel, including LDH levels and serum beta-2-microglobulin
  • Serum uric acid levels
  • Hepatitis B screening is recommended for patients with FL, MALT, MZL, HCL, MCL, DLBCL, BL, AIDS-related BCL, LL, post-transplant lymphoproliferative disorders (PTLD)
  • Testing for hepatitis C is also suggested
  • Testing for Helicobacter pylori for gastric MALT
  • Viral etiology of NHL should also be examined (eg human T-cell lymphoma virus [HTCLV], Epstein Barr virus [EBV], HIV)
    • ENKL patients with ≥6.1x107 copies/mL may suggest inferior disease-free survival rates
    • HIV testing is suggested for patients with DLBCL, AIDS-related BCL and BL




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Other Procedures  

Lumbar Puncture  

Lumbar puncture is used for patients with possible CNS involvement with overt symptoms. This is indicated for DLBCL patients with paranasal sinus, testicular, epidural, HIV-associated, bone marrow with large cells, >2 extranodal sites and elevated LDH levels, for LL and ATLL.

Imaging

Positron Emission Tomography (PET)  

Positron emission tomography is recommended for localized diseases and to identify occult sites of the disease or histologic transformation. This aids in evaluating patients' response to treatment. This is indicated in FL, DLBCL, nongastric MALT lymphoma, MZL, BL, MCL, AIDS-related BCL, LL, PTLD, ATLL, PCBCL, ENKL and MF/SS. 



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Computed Tomography (CT) Scan  

Computed tomography scan of the chest, abdomen and pelvic area are recommended for patients with suspected/diagnosed CLL, FL, MALT, MZL, HCL, MF/SS, MCL, BL, AIDS-related BCL, LL, PTLD, peripheral T-cell lymphoma (PTCL), ATLL, PCBCL, ENKL, MF/SS. CT scan of the neck is suggested in FL, primary T-cell lymphoma, DLBCL, MF/SS, MCL, BL, AIDS-related BCL and ATLL. Head CT may be performed for patients with possible CNS involvement, especially in DLBCL, PTCL, BL and ATLL. The use of contrast medium is recommended for patients with FL, MALT, MZL, HCL, MCL, BL, AIDS-related BCL, LL, PTCL and ENKL. A CT scan is more sensitive and specific when combined with PET.   

Magnetic Resonance Imaging (MRI)  

Magnetic resonance imaging is indicated for ATLL. MRI of the brain is suggested for nongastric MALT lymphoma, BL, AIDS-related BCL, LL and ATLL. An MRI of the nasal cavity, hard palate, anterior fossa and nasopharynx is recommended for ENKL.  

Endoscopy/Endoscopic Ultrasound (EU)  

Endoscopy/endoscopic ultrasound may be performed as endoscopy alone or via ultrasound-guided endoscopy. This is suggested for patients with suspected gastric MALT, nongastric MALT lymphoma, MCL, AIDS-related BCL and ATLL (upper GI endoscopy).  

Echocardiogram and/or Multi-Gated Acquisition (MUGA) Scan  

Echocardiogram and/or Multi-Gated Acquisition (MUGA) scan is performed when Anthracycline/Anthracenedione treatment is anticipated. This is indicated for FL, gastric and nongastric MALT lymphoma, nodal MZL, MCL, DLBCL, PTCL, BL and ENKL.