Flow Cytometric Diagnosis of Low Grade B-cell Leukemia/Lymphoma

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1 Flow Cytometric Diagnosis of Low Grade B-cell Leukemia/Lymphoma Maryalice Stetler-Stevenson, M.D., Ph.D. Flow Cytometry Unit, Laboratory of Pathology, DCS, NCI,NIH DEPARTMENT OF HEALTH & HUMAN SERVICES

2 Flow Cytometric Analysis of Hematolymphoid Neoplasia Standard of care-not research Guidelines available for medical indications Plays a vital role in diagnosis and subclassification Davis et al Bethesda International Consensus Recommendations on the Flow Cytometric Immunophenotypic Analysis of Hematolymphoid Neoplasia: Medical Indications, Clinical Cytometry. 72B:S5-S13, 2007

3 Flow Cytometric Diagnosis of Chronic Lymphocytic Leukemia (CLL) CLL is the most common leukemia in the western world Diagnosis is routinely made by a combination of flow cytometry, WBC and review of clinical history Flow cytometric diagnosis is accepted medical practice

4 Chronic Lymphocytic Leukemia (CLL) CLL Incidence: by country

5 Chronic Lymphocytic Leukemia (CLL) Guidelines for the Diagnosis and Treatment of Chronic Lymphocytic Leukemia: A Report from the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) updating the National Cancer Institute Working Group (NCI WG) 1996 guidelines: The diagnosis of CLL requires the presence of more than or equal to 5x 10 9 /L B lymphocytes (5000/µL) in the peripheral blood for the duration of at least 3 months. Clinical history is relevant Blood. 2008;111: )

6 Flow Cytometric Diagnosis of CLL : If detect by flow cytometry the presence of monoclonal B-cells with appropriate immunophenotype in the peripheral blood and at least 5x 10 9 /L B lymphocytes (5000/µL)*- Diagnosis is CLL If less than 5x 10 9 /L B lymphocytes (5000/µL)- Diagnosis is Monoclonal B-Cell Lymphocytosis Absolute lymphocyte count elevated: Monoclonal lymphocytosis with clinical lymphocytosis: Absolute lymphocyte count not elevated: Low count monoclonal lymphocytosis * duration of at least 3 months Clinical history and WBC results relevant

7 Flow Cytometric Diagnosis of CLL: Not a Chemistry Test But a Clinical Diagnosis SF pb_01_s-1.fcs SF pb_01_s-1.fcs Count % B-cells Count % B-cells CD19 PE HCL CD19 PE CLL SF pb_01_s-1.fcs SF pb_01_s-1.fcs Count % B-cells Polyclonal Count % B-cells Polyclonal CD19 PE CD19 PE Need more than a B-cell lymphocytosis for Diagnosis of CLL

8 Differential Diagnosis in CLL: Mantel Cell lymphoma Splenic Lymphoma with Villous Lymphocytes (SLVL) Lymphoplasmacytic lymphoma Follicular Lymphoma PLL HCL And if not determining lymphocytosis is B-cellother non-hodgkin s lymphomas, including T- cell.

9 Flow Cytometric Diagnosis of CLL: Not a Chemistry Test But a Clinical Diagnosis SF pb_08_b-5 new.fcs Can we just count CD5+ B-cells? SF pb_08_b-6+ K-p APC.fcs SF pb_08_b-5 new.fcs SF pb_08_b-6+ K-p.fcs CD5 PerCP Cy55 CLL CD5 PerCP Cy55 MCL CD5 PerCP Cy55 HCL CD5 PerCP Cy55 Polyclonal SF pb_08_b-5 new.fcs SF pb_08_b-6+ K-p.fcs SF pb_08_b-6+ K-p.fcs Lambda-p FITC CD5 PerCP Cy55 Gate 222 Lambda-p FITC Kappa-p APC Kappa-p APC

10 Flow Cytometric Diagnosis of CLL: Similar to Looking at H&E Slide CLL with vaguely nodular growth centers Follicular Lymphoma Reactive

11 Flow Cytometric Diagnosis of CLL : Appropriate Immunophenotype CD19+CD5+ CD5 v CD23 APC CD81 FITC CD23+ Dim to negative CD81 Dim CD20, dim CD Dim to negative CD79b Monoclonal CD22 PE CD79b PE Lambda-m PE CLL CD20 AH Kappa-m FITC

12 Flow Cytometric Diagnosis of CLL : Appropriate Immunophenotype CD5 APC CD23 APC CD81 FITC CD19+CD5+ Dim Partial CD23+ Moderate CD81 Moderate CD20&CD22 Moderate CD79b Monoclonal CD22 PE CD79b APC Lambda-m PE MCL CD20 AH7 Kappa-m FITC

13 CLL Diagnosis is Routinely Performed by Flow Cytometry: 5x 10 9 /L B-cells (5000/µL) At least 3 months duration Monoclonal Appropriate immunophenotype (CD19+, CD5+, CD23+, dim CD20+, dim CD22+, dim to negative CD79b, dim to negative CD81, CD43+, dim CD45+, dim surface immunoglobulin Diagnosis made by medical professional based upon identification of abnormal pattern and correlating this with history and other test results (WBC)

14 Flow Cytometry Used in Diagnosis and Sub-Classification of B- LPD Differentiation of reactive from clonal Sub-classification of CD5+ B-cell neoplasms: CLL, MCL, PLL, LCL, HCL, FL, Sub classification of CD10+ B-cell neoplasms: FL, Burkitt s. LCL, MCL, HCL Sub-classification of B-cell neoplasms with hairy/villous cytoplasmic borders HCL, HCLv, SLVL

15 Differentiation of reactive from clonal 422 SF pb_01_s-1.fcs Count % B-cells CD19 PE SF pb_08_b-6+ K-p.fcs SF pb_08_b-6+ K-p.fcs SF pb_08_b-6+ K-p.fcs CD5 PerCP Cy55 CD5 PerCP Cy55 Gate 222 Lambda-p FITC Kappa-p APC

16 Subclassification of B- Cell Neoplasms: CD5+ MCL: +CD5, +CD19, - CD23 (may be dim+), moderate CD20, CD22, sig, CD79b, CD81, CD45, CLL: +CD5, +CD19, +CD23, dim CD20, CD22, sig, CD79b, CD81, CD45 PLL and LCL CD5 CD5 CD19 CD19

17 Subclassification of B- Cell Neoplasms: CD10 Positivity FL: + CD10, dim CD19, +CD20, +CD22, +sig, +/-CD23, -CD5, -CD25, -CD11c, -CD103 Burkitt s: +CD10, moderate CD19, +CD20, +CD22, +sig, -CD23, -CD5 ALL:Immature +CD10, dim CD19, +CD22, +/-CD20, +TdT, -sig, +/-CD34 LCL and MCL CD10 CD10 CD19 CD19

18 Subclassification of B- Cell Neoplasms: Hairy/Villous Cytoplasmic Borders HCL + CD19, Bright CD20, Bright CD22, Bright CD25, Bright CD11c, CD103, CD123 HCLv + CD19, Bright CD20, Bright CD22, ++CD11c(can be moderate or dim), +CD103, - CD25, - or dimcd123 SLVL + CD19, Moderate CD20, Moderate CD22, +/- CD11c (dim when +), CD25-, CD103-, CD123- CD11c CD11c CD20 CD20

19 HCL CD19 PerCP Cy55 CD22 1:1 PE CD11c APC CD25 PE CD123 PE CD3 APC CD20 PerCP CD103 FITC CD20 PerCP CD20 PerCP HCLv CD19 PerCP Cy55 SLVL CD3 APC CD22 1:1 PE CD20 PerCP CD11c APC CD103 FITC CD25 PE CD20 PerCP CD123 PE CD20 PerCP CD19 PerCP Cy55 CD3 APC CD22 1:1 PE CD20 PerCP CD11c APC CD103 FITC CD25 PE CD20 PerCP CD123 PE CD19 PerCP Cy 55

20 Summary Flow cytometry is routinely used for diagnosis and sub-classification of B-cell neoplasia The diagnosis is made by a medical professional with intimate knowledge of normal immunophenotypic patterns Diagnosis is based upon recognition of an abnormal pattern, not a single value, similar to evaluation of morphology. Flow cytometric results are correlated with clinical history and the results of other tests for the diagnosis

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