Spleen Flow Cytometry: Normal vs Reactive Profiles

Flow cytometry is a powerful tool for analyzing cellular composition and immunophenotypic changes in splenic samples. It distinguishes between normal immune cell distributions and reactive or pathological alterations, providing insights into various hematologic and immunologic conditions.

Understanding how to differentiate normal from reactive spleen profiles requires careful analysis of specific cell populations and their expression patterns.

Indications For Flow Cytometry In Splenic Samples

Flow cytometry is used to evaluate splenic samples in hematologic disorders, lymphoid proliferations, and unexplained splenomegaly. The spleen’s complex cellular composition makes it a key site for diagnosing malignancies, immune dysregulation, and infections. When histopathology and cytology yield inconclusive results, flow cytometry offers a detailed immunophenotypic profile, enabling precise classification of abnormal cells.

A primary indication is the assessment of lymphoproliferative disorders. Conditions such as splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), and diffuse large B-cell lymphoma (DLBCL) present distinct immunophenotypic markers detectable through multiparametric flow cytometry. SMZL typically exhibits a CD19⁺CD20⁺CD5⁻CD10⁻CD23⁻ profile with strong surface immunoglobulin expression, while HCL is characterized by CD11c, CD25, and CD103 positivity. Identifying these markers differentiates B-cell neoplasms with overlapping histologic features but distinct therapeutic approaches.

Flow cytometry also evaluates non-neoplastic splenic conditions. Reactive lymphoid hyperplasia, often seen in chronic infections or autoimmune diseases, can mimic malignancies on histology. Flow cytometry distinguishes reactive expansions from clonal proliferations by assessing light chain restriction and aberrant antigen expression. In systemic infections like Epstein-Barr virus (EBV) or cytomegalovirus (CMV), it detects atypical lymphocyte populations with activated phenotypes, aiding diagnosis.

Another key application is investigating unexplained cytopenias, particularly in suspected hypersplenism. The spleen’s role in sequestration and destruction of blood cells makes flow cytometry useful in determining whether cytopenias stem from increased phagocytic activity, immune-mediated destruction, or an underlying malignancy. For example, in Evans syndrome, it detects autoantibody-coated red blood cells and platelets, supporting an immune-mediated cytopenia diagnosis. In hemophagocytic lymphohistiocytosis (HLH), an aggressive hyperinflammatory syndrome, it identifies activated T cells and macrophages expressing CD25 and HLA-DR, aiding early diagnosis and management.

Cell Subsets Commonly Analyzed

Flow cytometry of splenic samples examines immune cell populations, each with distinct phenotypic markers. The primary subsets analyzed include T lymphocytes, B lymphocytes, and myeloid cells, which play different roles in normal function and pathology.

T Lymphocytes

T cells make up a significant portion of the splenic lymphoid population and are divided into CD4⁺ helper T cells and CD8⁺ cytotoxic T cells. Normal splenic T cells express CD3, with CD4⁺ cells generally outnumbering CD8⁺ cells in a 2:1 ratio. Markers like CD45RA and CD45RO distinguish naïve from memory T cells, while CD25 and HLA-DR indicate activation.

In reactive conditions, such as viral infections or autoimmune disorders, activated T cells expressing CD38 and HLA-DR increase. In T-cell malignancies like hepatosplenic T-cell lymphoma, aberrant immunophenotypes with loss of CD5 or CD7 and γδ T-cell receptor expression are detected. Flow cytometry also identifies regulatory T cells (CD4⁺CD25⁺FOXP3⁺), which may be expanded in immunoregulatory disorders.

B Lymphocytes

B cells in the spleen, primarily in the white pulp, exhibit various maturation stages. Normal splenic B cells express CD19, CD20, and surface immunoglobulins, with subsets including naïve (IgD⁺CD27⁻), memory (IgD⁻CD27⁺), and marginal zone B cells (CD19⁺CD20⁺CD21⁺). Polyclonal immunoglobulin light chain expression indicates a non-clonal B-cell population.

Reactive conditions like chronic infections or autoimmune diseases show polyclonal B-cell expansion with increased CD38 and CD21 expression. In contrast, B-cell lymphoproliferative disorders exhibit restricted light chain expression and aberrant markers. SMZL typically lacks CD5 and CD10 but expresses CD11c and strong surface IgM, while hairy cell leukemia is marked by CD103, CD25, and CD11c co-expression.

Myeloid Cells

Myeloid cells in the spleen include monocytes, macrophages, and granulocytes, which contribute to immune surveillance and hematopoiesis. Normal splenic myeloid cells express CD14 and CD16 (monocytes), CD11b and CD68 (macrophages), and CD15 and CD66b (granulocytes). Dendritic cells are identified by CD11c and HLA-DR expression.

Reactive states, such as infections or inflammation, increase activated monocytes and macrophages expressing CD163 and CD206. In hemophagocytic lymphohistiocytosis, macrophage hyperactivation leads to increased CD25 and HLA-DR expression, along with phagocytosed hematopoietic cells. Myeloid malignancies like myelodysplastic syndromes or chronic myelomonocytic leukemia may show aberrant CD34 and CD117 expression, indicating immature myeloid precursors.

Interpreting Normal Vs Reactive Profiles

Distinguishing normal from reactive splenic profiles requires an understanding of cellular distributions, antigen expression patterns, and population dynamics. A normal spleen maintains balanced lymphoid and myeloid cell populations, with consistent lineage-specific marker expression. B cells exhibit polyclonal immunoglobulin light chains, reflecting a diverse immune repertoire. The absence of aberrant antigen expression is a hallmark of physiologic stability.

Reactive conditions, arising from infections, autoimmune activity, or systemic inflammation, alter these baseline characteristics. A key feature is the expansion of activated lymphocytes with increased HLA-DR, CD38, and CD25 expression. In viral infections, CD8⁺ T cells often show heightened CD57 expression, reflecting antigen-driven expansion. Reactive B-cell populations may display increased CD21 expression but retain polytypic light chain expression, distinguishing them from neoplastic clones. Myeloid cells in reactive states upregulate CD163 and CD206, markers of macrophage activation, particularly in inflammatory or hemophagocytic conditions.

While reactive patterns can mimic malignancies, distinctions lie in clonality and antigen aberrancy. Flow cytometry identifies these differences when histopathology is inconclusive. Reactive lymphoid hyperplasia maintains a heterogeneous B-cell immunophenotype, whereas lymphoproliferative disorders show a dominant monoclonal population with restricted κ or λ light chain expression. Reactive T-cell expansions preserve a polyclonal T-cell receptor (TCR) repertoire, while malignant counterparts may exhibit skewed or monoclonal TCR rearrangements. Aberrant antigen combinations, such as CD5 expression on B cells in chronic lymphocytic leukemia or CD7 loss in T-cell neoplasms, further aid differentiation.

Sample Preparation And Processing

Accurate spleen flow cytometry results depend on proper sample preparation and processing. Fresh splenic tissue or aspirates should be processed promptly to preserve cellular viability. If immediate analysis is not feasible, cold storage in buffered media like RPMI-1640 is recommended. Delays beyond 24 hours can cause apoptosis and altered antigen expression.

Mechanical dissociation is preferred for single-cell suspensions, as enzymatic digestion can cleave surface proteins and interfere with antigen detection. Filtration through a 70-μm mesh removes debris, ensuring a uniform suspension. Red blood cell lysis with ammonium chloride-based buffers enhances leukocyte resolution but must be carefully timed to prevent damage.

Fluorochrome-conjugated antibodies targeting lineage-specific and activation markers are selected based on diagnostic needs. Compensation controls using single-stained beads or cells ensure accurate signal interpretation, particularly in multicolor assays. Viability dyes like 7-AAD or propidium iodide exclude necrotic and apoptotic cells, preventing misinterpretation.

Advanced Multiparametric Approaches

Advances in flow cytometry have refined splenic sample analysis. Multiparametric approaches using expanded fluorochrome-conjugated antibody panels improve the ability to distinguish overlapping cell populations and detect subtle immunophenotypic shifts. High-color configurations allow simultaneous evaluation of lineage markers, activation states, and functional properties, enhancing diagnostic precision.

Computational tools such as t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) aid in visualizing complex cellular relationships. These methods are particularly useful in detecting rare populations, such as minimal residual disease (MRD) in lymphoproliferative disorders. Spectral flow cytometry, capturing full fluorochrome emission spectra, further enhances resolution between closely related cell subsets.