Laboratory Workup for Atypical Lymphocytes
When atypical lymphocytes are identified on peripheral blood smear, the initial laboratory workup should include a complete blood count with manual differential to quantify the atypical lymphocytes, peripheral blood smear examination to confirm morphology, and flow cytometry to assess for clonality and differentiate reactive from neoplastic causes. 1
Initial Core Laboratory Tests
The foundational workup requires:
- Complete blood count with manual differential to quantify absolute lymphocyte count and percentage of atypical lymphocytes 1
- Peripheral blood smear examination with manual review to confirm atypical lymphocyte morphology and characterize cell types (Downey type I, II, or III) 1, 2
- Comprehensive metabolic panel including lactate dehydrogenase (LDH), which may be elevated in lymphoproliferative disorders 3
- Liver function tests to assess for hepatic involvement 3
Flow Cytometry Analysis
Flow cytometry is essential to differentiate reactive from neoplastic causes by assessing clonality and immunophenotype 1. The panel should evaluate:
- T-cell markers: CD2, CD3, CD4, CD5, CD7, CD8 to assess for antigen loss patterns 3
- CD30 expression to evaluate for anaplastic large cell lymphoma or CD30+ lymphoproliferative disorders 3
- B-cell markers: CD19, CD20, CD23 with light chain restriction (kappa/lambda) if chronic lymphocytic leukemia is suspected 1
- NK cell markers: CD16, CD56 to quantify natural killer cell populations 4
- Activation markers: CD25, CD69, CD95, CD57, CD28, CD45RO to characterize activated T-cell subsets 4, 5
Research demonstrates that atypical lymphocytosis is characterized by marked increases in activated CD8+ T cells (particularly CD8+/CD57-, CD8+/CD95+, CD8+/CD48- subsets), moderate increases in NK cells, and no increase in CD4+ T cells or B cells 4.
Infectious Disease Serologies
The viral workup should be guided by clinical context, as 70% of heterophile-negative patients with atypical lymphocytosis have identifiable viral infections 2:
- Epstein-Barr virus (EBV) serology: VCA-IgM, VCA-IgG, EBNA, and early antigen to distinguish acute from past infection 1, 2
- Cytomegalovirus (CMV) serology: CMV IgM and IgG, as CMV accounts for approximately 39% of heterophile-negative atypical lymphocytosis cases 1, 2
- Human herpesvirus 6 (HHV-6) testing if EBV and CMV are negative, present in 25% of cases 1, 2
- HTLV-1/2 serology in endemic areas or when adult T-cell leukemia/lymphoma is suspected (atypical lymphocytes may appear as "flower cells") 3, 1
- HIV testing in appropriate clinical contexts 2
A critical caveat: Downey type II atypical lymphocytes are significantly associated with EBV positivity, Downey type III with HHV-6, and Downey type I show a trend toward CMV positivity 2. This morphologic correlation can guide targeted testing.
Additional Testing for Suspected Malignancy
When hematologic malignancy is suspected based on persistent atypical lymphocytosis, cytopenias, or abnormal flow cytometry:
- Cytogenetic studies (FISH) for recurrent chromosomal abnormalities including t(9;22) BCR-ABL1, t(12;21) TEL-AML1, MLL rearrangements, and hyperdiploidy/hypodiploidy 3, 1
- T-cell receptor (TCR) gene rearrangement to demonstrate clonality in suspected T-cell lymphoproliferative disorders 3
- Bone marrow biopsy is indicated for persistent unexplained atypical lymphocytosis, associated cytopenias, or when flow cytometry suggests clonal disease 3, 1
Clinical Context-Specific Considerations
Immunocompromised patients (HIV, transplant recipients, chronic immunosuppression) may have atypical lymphocytes due to opportunistic infections and require expanded infectious workup including blood cultures and tissue sampling if systemically ill 1.
Persistent atypical lymphocytosis warrants monitoring with serial CBCs 1. Research shows that isolated atypical lymphocytosis without the classic triad of splenomegaly, pharyngitis, and adenopathy does not reliably indicate infectious mononucleosis 6.
Predictive thresholds: Peripheral blood lymphocyte count ≥2.375 × 10⁹/L or lymphocyte proportion >35.9% has moderate accuracy (AUC 0.80-0.87) for predicting the presence of atypical lymphocytes 7.
Pitfalls to Avoid
- Do not assume all atypical lymphocytes represent infectious mononucleosis—multiple viral and neoplastic causes exist 6, 2
- Multiple positive viral serologies occur frequently (up to 70% of cases), complicating definitive diagnosis; morphologic correlation with Downey types helps guide interpretation 2
- Atypical lymphocytes resembling leukemic blasts are not uncommon at peak CAR-T expansion in patients receiving cellular therapy—clinical context is essential 3
- CD30+ cells on flow cytometry may represent reactive T cells rather than lymphoma—small CD30+ cells with preserved T-cell antigens (CD2, CD3, CD5, CD7) indicate reactive populations 3