Flow Cytometry in Hematological Disorders

• Flow cytometry is relevant to hematological disorders.
• Flow cytometry indications include:
• Suspicion of hematological neoplasia, such as cytopenia or organomegaly
• Pathological indications like lineage determination, lymphoma classification, or prognostic factors in acute leukemia cases
• Clinical integration, like with T-cell lymphoma or myelodysplastic syndrome

Advantages of Immunophenotyping by Flow Cytometry

• Rapid results are possible.
• It surpasses IHC in detecting light chain restriction.
• Flow Cytometry offers an extensive range of complementary markers to IHC for immunophenotyping, for example CD103, CD52, FMC7, and CD200.
• It's effective with cytological aspiration for lymphoma diagnosis and classification with minimally invasive methods.
• It simultaneously analyzes multiple surface antigens on individual cells.
• High sensitivity enables detection of minimal residual disease (MRD) at 1 in 10^4 cells.
• Standardization facilitates higher quality control.

Disadvantages of Immunophenotyping by Flow Cytometry

• Lack of tissue architecture evaluation
• Correlation of cytological and immunophenotypic findings is impacted
• Viable, fresh cells are necessary.

Specimen Collection and Quality

• Various specimens can be used, but fixation is not needed.
• Common specimens include bone marrow aspirates and lymph node biopsies.
• It's ideal in situations where a biopsy is not feasible (FNA).
• Deliver specimens immediately, ideally within 48 hours in RPMI medium to the lab.
• Suboptimal samples involve insufficient medium or exposure to fixatives.
• 7-AAD can be used to assess cell viability.

Instrumentation and Theory

• The major components of instrumentation include:
• A fluid sample suspension with cells and fluorochrome-conjugated antibodies
• A light source, such as a laser and lens
• Detectors for measuring fluorescence and electronic systems which convert signals to digital data
• Software to acquire, save and interpret data

Gating Strategies

• Traditional selection uses forward scatter/side scatter or CD45/side scatter.
• Effective when the target population is distinct and large
• Less effective with small or polymorphous cell populations, or subtle immunophenotypic alterations
• Limitations include not all populations fitting into a gate, and gates comprising multiple cell types

Cluster Analysis

• It serves as an alternative to gating
• It's not based on the assumption that a cell population maintains a certain position in flow
• It characterizes cell populations by their position in flow and relative to other populations
• The benefits of this type of analysis include allowing comparison of all populations and detection of slight aberrations.

Common Markers

• Common markers for cell lineage identification include:
• Lymphocytes B: CD19, CD20, CD22, CD79a, and kappa/lambda.
• Lymphocytes T: CD2, CD3, CD4, CD5, CD7, CD8.
• Granulocytes: Myeloperoxidase, CD33, CD13, CD15, CD16.
• Blasts: CD34, TdT, CD117.

Techincal and Biological Considerations

• Technical and biological considerations include:
• Fluorescence non-specificity
• Inadequate color compensation
• Carryover artefacts
• Doublets/aggregates
• Atypical immunophenotype
• Flawed selection strategy

Causes for False Negatives

• Causes for false negatives include:
• Sampling errors in biopsy specimens
• Loss of aberrant cells
• Rarity of neoplastic cells in lymphomas such as LH or LBDGC
• Surface light chain expression absence

Mature B-cell Neoplasms

• Mature B cell lymphomas identification is based on phenotypic anomalies involving:
• Light chain restriction
• Aberrant antigen expression.

Role of Flow Cytometry in Classifying B Cell Lymphomas

• Four major groups, classified by CD5 and CD10 expression:
• CD5+/CD10-
• CD5-/CD10+
• CD5+/CD10+
• CD5-/CD10-
• Subsequent markers associated with morphology are used to refine diagnoses and guide additional tests.

CD5+/CD10- Subgroup

• Mantle cell lymphoma and small lymphocytic lymphoma (SLL) are the most common in this group.
• Characteristic SLL immunophenotype: weak CD20, CD23-positivity, dim surface light chains, and FMC7 negativity.
• The mantle lymphomas in this group are FMC7-positive, confirmination with BCL1 in IHC or t(11;14) is required.
• LBDGC CD5+ displays large cells morphologically.
• Normal B lymphocyte sub-population, exhibits CD5+

CD5-/CD10+ Subgroup

• Follicular lymphoma and LBDGC are common in this group.
• Morphology is essential for refining the diagnosis.
• In a clinically appropriate context and with suggestive morphology, more studies are needed to rule out Burkitt lymphoma.

CD5-/CD10- Subgroup

• Marginal zone lymphomas and LBDGC are most frequent.
• Follicular lymphoma with aberrant CD5-/CD10- is rare.
• Differential diagnosis should include hairy cell leukemia (CD11c, CD25 and CD103).

CD5+/CD10+ Subgroup

• Immunophenotype is relatively rare.
• Most are LBDGC.

Role of Flow Cytometry in T Lymphoid Neoplasms

• The Role of Flow Cytometry in T Lymphoid Neoplasms:
• There is no clonality marker, unlike B-cell lymphomas.
• T-cell lymphomas express aberrant antigens, losing CD3, CD5, or CD7.
• T-cell populations suggest a lymphoproliferative process such as with CD4/CD8 ratio.
• Clinical, histological, and molecular studies required for diagnosis.

Key Takeaways

• Flow cytometry is a powerful method for detecting antigen presence or absence in individual cells.
• Understanding of normal and abnormal immunophenotypes is vital for differential diagnosis.
• It's generally superior to immunohistochemistry because of greater sensitivity, less subjectivity, and faster results.
• Flow cytometry correlated with clinical and morphological findings is a significant asset, providing accurate diagnostics to lymphoproliferative processes.