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Chapter 23 : Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis

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Abstract:

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a neoplasm of mature B lymphocytes involving peripheral blood (PB), bone marrow (BM), spleen, and lymph nodes (LN). CLL is the most common lymphoproliferative disorder in western countries and is primarily a disease of adults, often occurring during or after middle age. The diagnosis is established by blood counts, blood smears, and immunophenotyping by flow cytometry (FC) of circulating B lymphocytes (1). Although usually an indolent disease, some patients have a more rapid disease progression and require treatment earlier. Survival in patients varies from 1 year to 20 years with an 82% 5-year survival rate (2). FC demonstration of the typical CLL immunophenotype is vital for diagnosis. The differential diagnosis of CLL/SLL would primarily include monoclonal B-cell lymphocytosis (MBL) and mantle cell lymphoma (MCL), although B-prolymphocytic leukemia, marginal zone lymphoma, diffuse large B-cell lymphoma, and lymphoplasmacytic lymphoma can on occasion have some features of CLL/SLL (Table 1) (3).

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
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Figures

Image of FIGURE 1
FIGURE 1

Peripheral blood specimen with CLL cells (red), patient's residual nonneoplastic B cells (blue), and T cells (green). (A) The red CLL cells are CD19 and CD5 positive. The blue nonneoplastic B cells are CD19 positive but CD5 negative. The normal T cells are CD5 positive but CD19 negative. (B) The nonneoplastic B cells (blue) show normal levels of CD20 and CD79b expression. The CLL cells (red) have dim CD20 and dim CD79b expression. (C) The nonneoplastic B cells have normal levels of CD20 and CD22, while the CLL cells are dim for both. (D) The nonneoplastic B cells have normal CD81 intensity and are dim to negative for CD43. The CLL cells are negative for CD81 and have bright CD43 expression. The normal T cells (green) provide an internal CD81 and CD43 positive control. (E, F) CD20 on the axis helps to separate the B cells from non-B-cell elements and the CLL cells from the nonneoplastic B cells. The nonneoplastic B cells are polyclonal for kappa and lambda, while the CLL cells have dim kappa expression but are negative for lambda. PE, phycoerythrin; PerCP, peridinin chlorophyll protein; FITC, fluorescein isothiocyanate; APC, allophycocyanin v450, violet 450; PC7, phycoerythrin cyanin 7; AH7, allophycocyanin H7 (analog dye to APC-cyanin 7 dye).

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
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Image of FIGURE 2
FIGURE 2

Flow cytometric analysis of ZAP-70 expression. The upper row is an example of a ZAP-70-negative case, while the lower row is an example of a ZAP-70-positive CLL case. Different methods of ZAP-70 reporting are shown. Panel A shows the percentile method, where the marker was placed so that the gated T cells (CD3, CD19) and natural killer (NK) cells (CD16/56, CD19) with a high level of ZAP-70 expression would appear in the upper right quadrant. Gated CLL cells (CD19, CD20 dim, CD5) were plotted using the same quadrant, and the percentage of CLL cells that showed ZAP-70 positivity (the lower right quadrant) was calculated using 20% as a cutoff value for positivity. Panels B and C show histograms demonstrating the overlay of normal donor (ND) B cells (green), CLL clonal cells (blue), ND T cells (red), and ND NK cells (orange) using ZAP-70 1E7.2 AF488 (B) and SBZAP PE (C). Note the high signal-to-noise ratio seen with the SBZAP PE clone compared to ZAP-70 1E7.2 AF488.

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
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Image of FIGURE 3
FIGURE 3

ERIC method of flow cytometric CLL MRD analysis. (A) All cells shown. A quadrant type of gate (gate 1) is set including the lymphocytes and monocytes in quadrant Q1 and quadrant Q3 (gate 1: CD43 and CD43, low SSC). (B) Cells from gate 1 shown. Gate 2 is restricted to the cells in gate 1 and the CD19-positive and CD3-negative cells in quadrant Q1-1 (gate 2: CD19, CD3, CD43, and CD43, low SSC). (C) Cells from gate 2 shown. Gate 3 is restricted to the cells in gate 2 and P1 (gate 3: CD19, CD3, CD43, and CD43, low SSC, FSC consistent with lymphocytes). (D) Cells in gate 3 shown. Gate P2 targets abnormal CD43-bright and CD81-dim-to-negative cells in gate 3 (gate 3: CD19, CD3, CD43, and CD43, low SSC, FSC consistent with lymphocytes). (E) Cells in gate 3 shown. Gate P3 targets abnormal CD79b-dim-to-negative and CD5-positive cells in gate 3 (gate 3: CD19, CD3, CD43, and CD43, low SSC, FSC consistent with lymphocytes). (F) Cells in gate 3 shown. Gate P4 targets abnormal CD20-dim and CD22-dim CD19-positive cells in gate 3 (gate 3: CD19, CD3, CD43, and CD43, low SSC, FSC consistent with lymphocytes). The final combined analysis gate for CLL MRD includes only cells in gates 1, 2, 3, P2, P3, and P4 which are CD19 positive, CD3 negative, CD43 bright positive, CD81 dim to negative, CD79b dim to negative, CD5 positive, CD20 dim positive, CD22 positive, with low SSC and FSC consistent with lymphocytes.

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
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Image of FIGURE 4
FIGURE 4

(A) CD19-positive lymphocytes (upper panel) and CD19- and CD5-positive cells (lower panel) are shown. In the upper panel, the CD19-positive and CD5-positive B cells are gated (in red), and in the lower panel, the lack of light chain restriction is demonstrated. No CLL MRD was detected. (B) CD19-positive lymphocytes (upper panel) and CD20-dim and CD5-positive cells (lower panel) are shown. In the upper panel, CD20-dim and CD5-positive B cells are gated (light purple), and in the lower panel, CLL MRD is demonstrated by light chain restriction. (C) CD19-positive lymphocytes (upper panel) and CD20-dim, CD22-dim, and CD5-positive cells (lower panel) are shown. In the upper panel, CD22-dim, CD5-positive, CD20-dim, and CD22-dim cells are gated (brown). In the lower panel, CLL MRD is demonstrated by light chain restriction and a cleaner analysis gate is achieved.

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
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Tables

Generic image for table
TABLE 1

Immunophenotype of B-cell chronic lymphoproliferative disorders

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23
Generic image for table
TABLE 2

Immunophenotypes of T-cell chronic lymphoproliferative disorders

Citation: Degheidy H, Salem D, Yuan C, Stetler-Stevenson M. 2016. Chronic Lymphocytic Leukemia, the Prototypic Chronic Leukemia for Flow Cytometric Analysis, p 226-234. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch23

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