Flow cytometric immunophenotyping for hematologic neoplasms by Fiona E. Craig, and Kenneth A. Foon Blood Volume 111(8):3941-3967 April 15, 2008 ©2008 by American Society of Hematology

Mantle cell lymphoma. Mantle cell lymphoma. (A) Histologic section from a submandibular gland biopsy specimen demonstrating an abnormal diffuse infiltrate of small to intermediate-size lymphoid cells. Several mitotic figures are present. Hematoxylin & eosin stain, magnification ×40. (B) Representative flow cytometric dot plots with population of interest highlighted in green: CD19 versus CD5 demonstrates CD5+ B-cell population with weak intensity staining for CD19; FMC-7 versus CD5 demonstrates positivity for FMC-7; CD20 versus kappa and CD20 versus lambda demonstrate moderate intensity staining for CD20 and kappa immunoglobulin light chain restriction. In addition, B cells were CD10− and CD23− (data not shown). The flow cytometric data was acquired using a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA) and the dot plots were created using BD FACSDiva software v5.0.2 (BD Biosciences). (C) Cyclin-D1 paraffin section immunohistochemical stain, demonstrating many positive cells with characteristic nuclear staining; magnification ×40. (D) FISH studies demonstrating the IGH/CCND1 [t(11,14)(q13;q32)] rearrangement. Hybridization with the LSI IGH/CCND1-XT dual color, dual fusion DNA probe demonstrates one green signal from the unrearranged chromosome 14q32, one red signal from the unrearranged 11q13, and 3 fusion signals: one from the derivative chromosome 11, one from the derivative chromosome 14, and an extra signal suggesting the presence of an additional copy of all or part of one of the derivative chromosomes involved in the IGH/CCND1rearrangement. Courtesy of the Pittsburgh Cytogenetics Laboratory, Magee-Womens Hospital, Pittsburgh, PA. The images were taken through an Olympus BX40 microscope (Olympus, Tokyo, Japan) and acquired with a SPOT Insight 2 megapixel 3-shot color camera and SPOT Advanced imaging software (Diagnostic Instruments, Sterling Heights, MI). Fiona E. Craig, and Kenneth A. Foon Blood 2008;111:3941-3967 ©2008 by American Society of Hematology

Sézary Syndrome. Sézary Syndrome. (A) Peripheral blood smear demonstrating an abnormal lymphoid cell with an irregular folded nucleus. Wright Giemsa stain, magnification ×100. Images were acquired as in Figure 1. (B) Representative flow cytometric dot plots with population of interest highlighted in green: CD3 versus CD7, demonstrating many CD7− CD3+ T cells; CD3 versus CD16 and/or CD57, demonstrating lack of NK-associated antigen expression; CD26 versus CD4, demonstrating many CD4+ cells, including more than 30% CD26− cells; and CD3 versus CD25, demonstrating only partial weak intensity staining of T cells for CD25. Fiona E. Craig, and Kenneth A. Foon Blood 2008;111:3941-3967 ©2008 by American Society of Hematology

Acute myeloid leukemia with MLL rearrangement and monocytic differentiation. Acute myeloid leukemia with MLL rearrangement and monocytic differentiation. Although there is minimal staining with CD14, flow cytometric studies demonstrate other features associated with monocytic differentiation and a butyrate esterase cytochemical stain is positive. (A) Bone marrow aspirate smear demonstrating abnormal cells with moderately abundant cytoplasm, a few cytoplasmic granules, and some irregularity in nuclear outlines. Wright Giemsa stain, magnification ×100. (B) Representative flow cytometric dot plots: CD45 versus side scatter demonstrates a small population of lymphoid cells indicated in red, and a population of interest highlighted in green with weak intensity CD45 and variable side (orthogonal) light scatter; CD14 versus CD13+33 demonstrates staining for the myeloid antigens CD13 and/or CD33 and minimal staining for CD14; CD13+33 versus CD34 demonstrates absence of staining for CD34; CD15 versus CD33 demonstrates relatively bright staining for CD33 and variable intensity staining for CD15; CD36 versus CD64 demonstrates staining for both CD36 and CD64; and CD13+33 versus CD56 demonstrates partial aberrant expression of CD56. (C) Butyrate esterase cytochemical stain demonstrating many positive cells; magnification ×100. Staining was inhibited with fluoride incubation (not shown). (D) Classical cytogenetic studies demonstrating 47,XX,+8,t(11,19)(q23;p13.3). Courtesy of the Pittsburgh Cytogenetics Laboratory, Magee-Womens Hospital, Pittsburgh, PA. (E) FISH studies demonstrating an MLL gene rearrangement. Hybridization with the LSI MLL dual color DNA probe demonstrates one cell (lower left) with one fusion signal (corresponding to the unrearranged chromosome 11 at band 11q23) and separate green and red signals corresponding to the split MLL gene, and one normal cell (top right) with 2 fusion signals. Courtesy of the Pittsburgh Cytogenetics Laboratory, Magee-Womens Hospital, Pittsburgh, PA. Images were acquired as in Figure 1. Fiona E. Craig, and Kenneth A. Foon Blood 2008;111:3941-3967 ©2008 by American Society of Hematology

Flow cytometric detection of minimal residual CLL Detection using the protocol published by Rawstron and colleagues.10 Top row of plots demonstrate preliminary gating on CD19+ B cells with low orthogonal (side) light scatter and refinement of the gate using... Flow cytometric detection of minimal residual CLL Detection using the protocol published by Rawstron and colleagues.10 Top row of plots demonstrate preliminary gating on CD19+ B cells with low orthogonal (side) light scatter and refinement of the gate using a plot of forward versus low orthogonal (side) light scatter. Bottom 3 rows of plots demonstrate representative dot-plots from 3 separate tubes evaluating CD5, CD20, and CD38, (second row); CD5, CD22, and CD81 (third row); and CD5, CD43, and CD79b (bottom row). Only the CD19+ gated events are displayed. Regions are placed around events with phenotypic features characteristic of CLL: R5 indicates dim CD20 and CD5+; R6, dim CD20 and dim to moderate intensity CD38 staining; R7, CD5+ and CD22+; R8, CD22+ and dim CD81; R9, CD5+; and CD79bdim, R10, CD5+ and CD43+. Residual CLL is identified by more than 50 events meeting the criteria for 2 or more of the following: R5 and R6 (displayed in second row right dot plot), R7 and R8 (displayed in third row right dot plot), R9 and R10 (displayed in bottom row right dot plot). The flow cytometric data were acquired using a BD FACS Calibur flow cytometer (BD Biosciences) and the dot plots were created using BD CellQuest software v3.3 (BD Biosciences). Fiona E. Craig, and Kenneth A. Foon Blood 2008;111:3941-3967 ©2008 by American Society of Hematology