Flow Cytometry in Hematopathology

Although instrumentation and laboratory techniques for flow cytometry (FCM) immunophenotyping of hematologic malignancies are well documented, there is relatively little information on how best to perform data analysis, a critical step in FCM testing. In Flow Cytometry in Hematopathology: A Visual Approach to Data Analysis and Interpretation, three physicians highly experienced in laboratory hematopathology and FCM offer a unique systematic approach to FCM data analysis and interpretation based on the visual inspection of dual parameter FCM graphics. This step-by-step approach to optimal FCM data analysis is demonstrated by means of numerous FCM graphics derived from actual well-documented clinical cases. The authors include notes on "tricks of the trade" and pitfalls to avoid. The discussion, covering leukemias, lymphomas, and other conditions, moves from simple to complex specimens, with an emphasis on visual pattern analysis. The companion CD-ROM with 80 detailed case studies provides additional opportunities to gain a deeper understanding of FCM data analysis.
Richly illustrated and highly instructive, Flow Cytometry in Hematopathology: A Visual Approach to Data Analysis and Interpretation offers clinical pathologists, hematopathologists, and specialists in laboratory medicine a much-needed guide with a practical and logical approach for sharpening of their FCM data analysis skills on a wide spectrum of hematologic disorders.

Klappentext

Flow cytometry immunophenotyping of hematopoietic disorders is a complex and demanding exercise that requires a good understanding of cell lineages, developmental pathways, and physiological changes, as well as broad experience in hematopathology. The process includes several interrelated stages, from the initial medical decision regarding which hematologic c- dition is appropriate for FCM assay, to the final step of diagnosis whereby the FCM data is correlated with other relevant clinical and laboratory information. The actual FCM testing involves three major steps: pre-analytical (specimen processing, antibody staining), analytical (acquiring data on the flow cytometer) and post-analytical (data analysis and interpretation). The literature, including the latest FCM textbooks, provides ample information on the te- nical principles of FCM such as instrumentation, reagents and laboratory methods, as well as quality control and quality assurance. Similarly, correlations of morphologic findings and p- notypic profiles have been well covered in many publications. In contrast, much less attention has been given to the other equally important aspects of FCM immunophenotyping, especially data analysis. The latter is a crucial step by which a phenotypic profile is established. To bridge this gap in the literature, the focus of this book is more on FCM data analysis than laboratory methods and technical details. For the reader to become familiar with our data analysis strategy, an overview of our approach to the pre-analytical and analytical steps is also presented, with an emphasis on the pre-analytical aspects, which have been rarely touched upon in the literature.

Inhalt

Chapter 1. Approach to Flow Cytometry: General Considerations

1.1 Reasons for the necessity of proper data analysis
The pitfalls of the FCM data format of "percent positive" per antibody tested.
1.2 General aspects of FCM data analysis and interpretation
1.3 Other applications of FCM in hematopathology
1.4 Overview of lineage-associated markers

Chapter 2. FCM Immunophenotyping and DNA Analysis: Practical Aspects That Can Affect Data Analysis and Interpretation
2.1 Sample selection
Liquid specimens
Solid tissue specimens
2.2 Preparing nucleated cell suspensions
2.3 Cell yield and viability
2.4 Sample staining
Surface antigens
Intracellular antigens
DNA content
2.5 Data acquisition
Calibration
Color compensation
List mode data collection
Exclusion of nonviable cells
2.6 Antibody panel design
Antibody selection
Anti-light chain antibodies
Fluorochrome conjugation
2.7 Comprehensive antibody panels
Disease-oriented antibody panels
Antibody panels oriented by specimen type
2.8 Tailored panels and add-on testing
Minimal residual disease
2.9 FCM immunophenotyping data representation
Analysis panels
Color display
2.10 Approach to DNA data analysis
DNA ploidy
S-phase

Chapter 3. FCM Data Analysis on Nearly Homogeneous Samples

3.1 FCM parameters
Forward scatter
Side scatter
Fluorescence
Heterogeneous fluorescence intensity (bimodal, variable)
3.2 Fluorescence dynamic range
3.3 Strategy to the visual review of FCM immunophenotyping data
3.4 Common SSC/CD45 patterns
Assessment of the blast population
Immature neoplastic cells with downregulated CD45
SSC/CD45 in mature lymphoid disorders
3.5 Other dot plot patterns useful in acute leukemia diagnosis
Useful antigenic features in AML
Myeloid phenotypic abnormalities and MRD detection
Precursor B-ALL vs bone marrow precursor B-cells (hematogones)
3.6 Evaluation of mature lymphoid malignancies
Assessment of surface light chain expression
Assessment of pan B-cell antigens
Useful antigenic features in mature B-cell malignancies
CD10 expression: Follicular center cell lymphomas
Pattern of CD20 and CD11c coexpression
CD5 expression
Aberrant B-cell profile
Identification of abnormal T-cells
Useful antigenic features in mature T-cell malignancies
3.7 Dot plot patterns in histiocytic proliferations and nonhematopoietic malignancies

Chapter 4. FCM data analysis on heterogeneous specimens

4.1 Identifying normal FCM samples
Benign/reactive solid lymphoid tissue (e.g., lymph nodes, tonsils)
Pattern of CD10/CD20 coexpression. Distinction between FRFH and FCC lymphoma
Normal peripheral blood and normal bone marrow
Blast region
Bone marrow B-cell precursors (hematogones)
Lymphocytes
Monocytes
Plasma cells
Erythroid precursors
Maturing myeloid cells
4.2 Abnormal samples with a detectable immature neoplastic population
Blasts of lymphoid lineage
Blasts of myeloid lineage
AML
High-grade MDS and MPD with increased blasts
4.3 Minimal residual disease
4.4 Abnormal samples with detectable mature neoplastic populations
Abnormal mature B-cells. Evaluation of CD5 and CD23
FCM features suggestive of anti-CD20 therapy
Abnormal mature T-cells. Abnormal plasma cells present
4.5 Abnormal blood or bone marrow samples with no detectable neoplastic cells
Altered cellular composition and abnormal SSC
Abnormal antigenic maturation in myeloid or erythroid precursors
4.6 Coexisting malignancies

Chapter 5 FCM interpretation and reporting

5.1 Immature hematopoietic malignancies
ALL/lymphoblastic lymphoma
Myeloid malignancies
AML-M3
AML with minimal maturation
AML with maturation
AML with monocytic differentiation
AML with erythroid hyperplasia
AML with megakaryocytic differentiation
MPD and MDS
Acute leukemias with a multilineage antigenic profile
5.2 Mature lymphoid malignancies
B-cell LPD/NHL. CD10 expression
Coexpression of CD11c, CD25, and CD103
Coexpression of CD5 and CD23. CD5+CD23¯ B-cell neoplasms
CD45 and/or pan B-cell antigens markedly downregulated
Nondescript B-cell phenotype and high FSC
Nondescript B-cell phenotype and low FSC
Monoclonal B-cells of undetermined significance
Plasma cell dyscrasias
T-cell LPD/NHL
CD4+ T-cell LPD/NHL. CD8+ disorders
CD30+ lymphoma
5.3 FCM reporting
Suggested Reading
Appendix
Index