Table 2.

Initial diagnostic steps for differentiating non-neoplastic causes of generalized lymphadenopathy

Diagnostic example workup for differential of generalized lymphadenopathy
All • CBC
• Chest radiograph
• HIV-1/2 testing (antigen/antibody combination immunoassay)
• SPEP
• CRP/ESR 
• Assessments of end-organ function (eg, creatinine, urinalysis, LFTs, coagulation studies, albumin)
• Variable: US of LN, CT with IV contrast, 18F-FDG PET, LN biopsy (excisional biopsy is the gold standard for CD) 
Infectious disease differential example workup (if indicated) • Syphilis: RPR testing (nontreponemal) followed by confirmation with a treponemal test (eg, FTA-ABS)
• HBV/HCV: HBV DNA PCR (or HBsAg with anti-HBc IgM antibody confirmation), HCV RNA PCR
• EBV: heterophile antibody test (or VCA IgM/IgG [ELISA])
• Other pathogen-specific serology and PCR testing (depending on clinical indication):
  PCR: CMV (plasma, DNA), HHV8 (plasma, DNA), adenovirus (swab, DNA), HSV (swab/CSF/etc, DNA), HHV6
  (plasma/tissue, DNA)
  ELISA (IgM/IgG): toxoplasmosis (or DNA PCR of tissue/plasma in immunocompromised)
• Urine/serum antigen test (Histoplasma, Coccidioides, Cryptococcus
• Atypical pathogen testing (if clinically indicated): leishmaniasis (DNA PCR), measles (RNA PCR), mumps (RNA PCR), rubella (RNA PCR), dengue (RNA PCR or NS1 antigen testing), Lyme disease (ELISA + Western blot for confirmation), leptospirosis (DNA PCR), brucellosis (DNA PCR), bartonellosis (DNA PCR), tularemia (DNA PCR), diphtheria (toxin PCR), plague (DNA PCR), ehrlichiosis (DNA PCR)
• Sputum samples (if clinically indicated)
• Blood cultures (with stain request including pathogens of interest)
• LN histopathology specific tests (eg, LANA1 for HHV8) 
Autoimmune
differential example workup
(if indicated) 
• ANA (only if clinically indicated, due to false positives)
• Disease-specific antibody testing (eg, anti-dsDNA, anti-Ro/SSA, anti-La/SS-B, rheumatoid factor, anti-CCP, anti-MPO, anti-PR3 if clinically indicated)
• Serum complement levels (eg, C3, C4)
• Serum immunoglobulin levels (eg, IgG1-4)
• Genetic testing (eg, FAS/FASL/CASP10/NRAS/CASP8 mutations if ALPS is suspected) 
• Flow cytometry for immune cell populations (eg, CD4/CD8 T cells in ALPS, if suspected)
• Organ-specific imaging evaluation to assess organ involvement (disease specific)
• Biopsy of other involved tissue (eg, skin) 
Inflammatory disease differential example workup (if indicated) • HLH: ferritin, triglycerides, sCD25, fibrinogen (additional specialized tests and tests to identify the trigger)
• Disease-specific inflammatory biomarkers (eg, sIL-2RA, IL-18, CXCL9 for HLH)
• Bone marrow examination (disease-specific studies; eg, evidence of hemophagocytosis for HLH) 
• LN pathology–specific tests (S100, CD68, CD163, etc)
• Serum eosinophils and IgE
• Other genetic testing (eg, UBA1 gene sequencing for VEXAS) 
Diagnostic example workup for differential of generalized lymphadenopathy
All • CBC
• Chest radiograph
• HIV-1/2 testing (antigen/antibody combination immunoassay)
• SPEP
• CRP/ESR 
• Assessments of end-organ function (eg, creatinine, urinalysis, LFTs, coagulation studies, albumin)
• Variable: US of LN, CT with IV contrast, 18F-FDG PET, LN biopsy (excisional biopsy is the gold standard for CD) 
Infectious disease differential example workup (if indicated) • Syphilis: RPR testing (nontreponemal) followed by confirmation with a treponemal test (eg, FTA-ABS)
• HBV/HCV: HBV DNA PCR (or HBsAg with anti-HBc IgM antibody confirmation), HCV RNA PCR
• EBV: heterophile antibody test (or VCA IgM/IgG [ELISA])
• Other pathogen-specific serology and PCR testing (depending on clinical indication):
  PCR: CMV (plasma, DNA), HHV8 (plasma, DNA), adenovirus (swab, DNA), HSV (swab/CSF/etc, DNA), HHV6
  (plasma/tissue, DNA)
  ELISA (IgM/IgG): toxoplasmosis (or DNA PCR of tissue/plasma in immunocompromised)
• Urine/serum antigen test (Histoplasma, Coccidioides, Cryptococcus
• Atypical pathogen testing (if clinically indicated): leishmaniasis (DNA PCR), measles (RNA PCR), mumps (RNA PCR), rubella (RNA PCR), dengue (RNA PCR or NS1 antigen testing), Lyme disease (ELISA + Western blot for confirmation), leptospirosis (DNA PCR), brucellosis (DNA PCR), bartonellosis (DNA PCR), tularemia (DNA PCR), diphtheria (toxin PCR), plague (DNA PCR), ehrlichiosis (DNA PCR)
• Sputum samples (if clinically indicated)
• Blood cultures (with stain request including pathogens of interest)
• LN histopathology specific tests (eg, LANA1 for HHV8) 
Autoimmune
differential example workup
(if indicated) 
• ANA (only if clinically indicated, due to false positives)
• Disease-specific antibody testing (eg, anti-dsDNA, anti-Ro/SSA, anti-La/SS-B, rheumatoid factor, anti-CCP, anti-MPO, anti-PR3 if clinically indicated)
• Serum complement levels (eg, C3, C4)
• Serum immunoglobulin levels (eg, IgG1-4)
• Genetic testing (eg, FAS/FASL/CASP10/NRAS/CASP8 mutations if ALPS is suspected) 
• Flow cytometry for immune cell populations (eg, CD4/CD8 T cells in ALPS, if suspected)
• Organ-specific imaging evaluation to assess organ involvement (disease specific)
• Biopsy of other involved tissue (eg, skin) 
Inflammatory disease differential example workup (if indicated) • HLH: ferritin, triglycerides, sCD25, fibrinogen (additional specialized tests and tests to identify the trigger)
• Disease-specific inflammatory biomarkers (eg, sIL-2RA, IL-18, CXCL9 for HLH)
• Bone marrow examination (disease-specific studies; eg, evidence of hemophagocytosis for HLH) 
• LN pathology–specific tests (S100, CD68, CD163, etc)
• Serum eosinophils and IgE
• Other genetic testing (eg, UBA1 gene sequencing for VEXAS) 

ALPS, autoimmune lymphoproliferative syndrome; CCP, cyclic citrullinated peptide; CSF, cerebrospinal fluid; FASL, FAS ligand; HBV, hepatitis B; HCV, hepatitis C; HLH, hemophagocytic lymphohistiocytosis; MPO, myeloperoxidase; US, ultrasound; VCA, viral capsid antigen.

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