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
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.. 2006 Bethesda International Consensus Recommendations on the Flow Cytometric Immunophenotypic Analysis of Hematolymphoid Neoplasia: Medical Indications, Clinical Cytometry. 72B:S5-S13, 2007
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
Chronic Lymphocytic Leukemia (CLL) CLL Incidence: by country
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;111:5446 5456)
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
Flow Cytometric Diagnosis of CLL: Not a Chemistry Test But a Clinical Diagnosis SF13 228 pb_01_s-1.fcs SF13 211 pb_01_s-1.fcs 406 314 305 236 Count 203 54% B-cells Count 157 54% B-cells 102 0 10 0 10 1 CD19 PE HCL 79 0 10 0 10 1 CD19 PE CLL SF12 904 pb_01_s-1.fcs SF13 198 pb_01_s-1.fcs 422 153 Count 317 211 106 48% B-cells Polyclonal Count 115 77 38 46% B-cells Polyclonal 0 10 0 10 1 CD19 PE 0 10 0 10 1 CD19 PE Need more than a B-cell lymphocytosis for Diagnosis of CLL
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.
Flow Cytometric Diagnosis of CLL: Not a Chemistry Test But a Clinical Diagnosis SF13 211 pb_08_b-5 new.fcs Can we just count CD5+ B-cells? SF12 324 pb_08_b-6+ K-p APC.fcs SF13 228 pb_08_b-5 new.fcs SF12 904 pb_08_b-6+ K-p.fcs CD5 PerCP Cy55 CLL CD5 PerCP Cy55 MCL CD5 PerCP Cy55 HCL CD5 PerCP Cy55 Polyclonal 103 104 105 103 104 105 103 104 105 103 104 105 SF13 211 pb_08_b-5 new.fcs SF12 904 pb_08_b-6+ K-p.fcs SF12 904 pb_08_b-6+ K-p.fcs Lambda-p FITC CD5 PerCP Cy55 Gate 222 Lambda-p FITC Kappa-p APC 103 104 105 103 104 105 Kappa-p APC 103 104 105
Flow Cytometric Diagnosis of CLL: Similar to Looking at H&E Slide CLL with vaguely nodular growth centers Follicular Lymphoma Reactive
Flow Cytometric Diagnosis of CLL : Appropriate Immunophenotype 105 105 105 CD19+CD5+ CD5 v450 104 103 CD23 APC 104 103 CD81 FITC 104 103 CD23+ Dim to negative CD81 Dim CD20, dim CD22 102 102 103 104 105 102 102 103 104 105 102 102 103 104 105 Dim to negative CD79b Monoclonal 105 105 105 CD22 PE 104 103 CD79b PE 104 103 Lambda-m PE 104 103 CLL 102 102 102 CD20 AH7 102 103 104 105 102 103 104 105 Kappa-m FITC 102 103 104 105
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
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)
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
Differentiation of reactive from clonal 422 SF12 904 pb_01_s-1.fcs Count 317 211 48% B-cells 106 0 10 0 10 1 CD19 PE SF12 904 pb_08_b-6+ K-p.fcs SF12 904 pb_08_b-6+ K-p.fcs SF12 904 pb_08_b-6+ K-p.fcs CD5 PerCP Cy55 CD5 PerCP Cy55 Gate 222 Lambda-p FITC 103 104 105 103 104 105 Kappa-p APC 103 104 105
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
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
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
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
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