Full download test bank at ebook textbookfull.

com

Flow Cytometry in Neoplastic

Hematology Morphologic-
immunophenotypic Correlation
Includes Digital Download 3rd
CLICK LINK TO DOWLOAD

https://textbookfull.com/product/flow-
cytometry-in-neoplastic-hematology-
morphologic-immunophenotypic-correlation-
includes-digital-download-3rd-edition-
wojciech-gorczyca/

textbookfull
More products digital (pdf, epub, mobi) instant
download maybe you interests ...

Complications in Uveitis Includes Digital Download

Francesco Pichi (Editor)

https://textbookfull.com/product/complications-in-uveitis-
includes-digital-download-francesco-pichi-editor/

Pediatric Neurology: includes digital download 2nd

Edition James Bale

https://textbookfull.com/product/pediatric-neurology-includes-
digital-download-2nd-edition-james-bale/

Operative Thoracic Surgery Includes Digital Download

6th Edition Larry R. Kaiser

https://textbookfull.com/product/operative-thoracic-surgery-
includes-digital-download-6th-edition-larry-r-kaiser/

Plasma Cell Neoplasms: A Morphologic, Cytogenetic and

Immunophenotypic Approach 1st Edition Michael A. Linden

https://textbookfull.com/product/plasma-cell-neoplasms-a-
morphologic-cytogenetic-and-immunophenotypic-approach-1st-
edition-michael-a-linden/
Flow Cytometry Basics for the Non Expert Christine
Goetz

https://textbookfull.com/product/flow-cytometry-basics-for-the-
non-expert-christine-goetz/

Harrison’s Hematology and Oncology 3rd Edition Dan L.

Longo

https://textbookfull.com/product/harrisons-hematology-and-
oncology-3rd-edition-dan-l-longo/

Creating flow with OmniFocus 3rd Edition Koroush Dini

https://textbookfull.com/product/creating-flow-with-
omnifocus-3rd-edition-koroush-dini/

International Digital Imaging Correlation Society

Proceedings of the First Annual Conference 2016 1st
Edition Michael Sutton

https://textbookfull.com/product/international-digital-imaging-
correlation-society-proceedings-of-the-first-annual-
conference-2016-1st-edition-michael-sutton/

Molecular hematology Fourth Edition Gribben

https://textbookfull.com/product/molecular-hematology-fourth-
edition-gribben/
Flow Cytometry in Neoplastic Hematology
Morphologic–Immunophenotypic Correlation
Third Edition
Flow Cytometry in Neoplastic Hematology
Morphologic–Immunophenotypic Correlation
Third Edition

Wojciech Gorczyca, MD, PhD

BioReference Laboratories
Elmwood Park, New Jersey, USA
CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742

© 2017 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-4987-7502-1 (Pack- Hardback)

This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information,
neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that
any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the
publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement
to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best
practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified.
The reader is strongly urged to consult the relevant national drug formulary and the drug companies’ and device or material manufacturers’ printed instructions, and
their websites, before administering or utilizing any of the drugs, devices or materials mentioned in this book. This book does not indicate whether a particular treatment
is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so
as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication
and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let
us know so we may rectify in any future reprint.

Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or
other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written
permission from the publishers.

For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright
Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a
variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged.

Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to
infringe.

Library of Congress Cataloging-in-Publication Data

Names: Gorczyca, Wojciech, 1959- author.

Title: Flow cytometry in neoplastic hematology : morphologic-immunophenotypic correlation / Wojciech Gorczyca.
Description: Third edition. | Boca Raton, FL : CRC Press/Taylor & Francis Group, 2017. | Includes bibliographical references and index.
Identifiers: LCCN 2016045629| ISBN 9781498775021 (pack-book and ebook : alk. paper) | ISBN 9781498775038 (ebook) | ISBN 9781498775045 (ebook)
Subjects: | MESH: Hematologic Neoplasms—diagnosis | Flow Cytometry | Immunophenotyping | Leukemia, Lymphoid—diagnosis | Leukemia,
Myeloid, Acute—diagnosis
Classification: LCC RC280.H47 | NLM WH 525 | DDC 616.99/418—dc23
LC record available at https://lccn.loc.gov/2016045629

Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com

and the CRC Press Web site at

http://www.crcpress.com
Inhalt

Preface to the Third Edition vi

Abbreviations vii

1. Introduction 1
2. Gating Strategies 15
3. Classification of Hematopoietic Tumors 25
4. Identification of Clonal B-Cell Populations 29
5. Identification of Abnormal T-Cell Populations 44
6. Identification of Myeloblasts 60
7. Identification of B-Lymphoblasts 84
8. Identification of T-Lymphoblasts 94
9. Antigen Expression during Myeloid and Lymphoid Maturation 99
10. Identification of Myelodysplastic Syndrome by Flow Cytometry 105
11. Phenotypic Markers 115
12. Morphologic–Flow Cytometric Correlation 126
13. Molecular-Flow Cytometric Correlation 145
14. Flow Cytometry Limitations 150
15. Phenotypic Classification of B-Cell Lymphoproliferations 156
16. Mature B-Cell Lymphoproliferations 163
17. Plasma Cell Neoplasms 227
18. Mature T/NK-Cell Lymphoproliferations 240
19. Hodgkin Lymphoma 302
20. B-Cell Lymphoblastic Leukemia/Lymphoma 309
21. T-Cell Lymphoblastic Leukemia/Lymphoma 322
22. Myelodysplastic Syndromes 331
23. Chronic Myelomonocytic Leukemia and Other Myelodysplastic/Myeloproliferative Neoplasms 341
24. Myeloproliferative Neoplasms 353
25. Acute Myeloid Leukemia: Introduction 378
26. Acute Myeloid Leukemia with Recurrent Genetic Changes 392
27. Acute Myeloid Leukemia, Not Otherwise Specified 412
28. Other Tumors 434

Index 451

v
Preface to the Third Edition

This third edition is the result of the author’s 25 years of experi- MPN, chronic myelomonocytic leukemia (CMML), and other
ence with flow cytometry (1991–2016). It incorporates the revised chronic myeloid neoplasms is significantly expanded. This
2016 World Health Organization (WHO) classification of hema- third edition provides expanded criteria for identifying blasts,
topoietic tumors. The number of illustrations has increased from with emphasis on differential diagnosis between various types
372 in the second edition to 489 in this edition, the majority being of acute leukemias and between blasts and maturing elements
composites in color. The text is significantly expanded and bet- with or without dysmaturation, and detailed discussion of leu-
ter organized to help early as well as experienced flow cytometry kemia-associated phenotype (LAP). Acute myeloid leukemias
users dealing with both standard and difficult cases. Chapters (AMLs) and their differential diagnoses are now discussed in
1–14 present flow cytometry features helpful in identification of greater detail, utilizing the revised WHO classification, includ-
abnormal population(s), and Chapters 15–28 provide systematic ing distinct immunophenotypic patterns of acute promyelocytic
descriptions of hematopoietic neoplasms. Each chapter has its leukemia (APL) and AML with NPM1 mutations, pure erythroid
own up-to-date references. leukemia versus high-grade MDS, differential diagnosis between
Although the book covers the wide spectrum of hematopoi- hypogranular APL, acute monoblastic leukemia, and AML with
etic tumors, the focus remains on the most important clinical NPM1 and/or FLT3 mutations, and acute monoblastic leukemia
diagnoses, such as acute promyelocytic leukemia, identification versus blastic plasmacytoid dendritic cell neoplasm (BPDCN).
of blasts, identification of clonal B-cell population, differentiat- The statistical analysis of immunophenotypic features of T-cell
ing mature vs. immature T-cell proliferations, differential diag- disorders, CMML, and acute leukemias incorporates hundreds
nosis between hematogones and B-lymphoblastic leukemia/ of new cases. Additional detailed criteria are provided for iden-
lymphoma (B-ALL), or distinction between chronic and acute tification of abnormal B- and T- populations, with significantly
monocytic proliferations. expanded differential diagnosis. New tables and expanded tables
The number of chapters has increased from 13 to 28. New from the previous edition summarize phenotypic features, clas-
chapters include topics such as the correlation of immunophe- sification, prognosis, and differential diagnosis on most of the
notypic features with specific morphologic or laboratory find- entities discussed.
ings (e.g., cytopenia, hyperleukocytosis, circulating plasma cells,
etc.) and the correlation between flow cytometry findings and Wojciech Gorczyca MD, PhD
molecular (genetic) changes. Chapter 13 of the previous edition Chief for Hematopathology Services
(Myelodysplastic syndromes and chronic myeloproliferative Medical Director of Flow Cytometry
neoplasms) is now expanded to three chapters (myelodysplas- BioReference Laboratories, an OPKO Health Company
tic syndrome [MDS], myeloproliferative neoplasm [MPN], Elmwood Park, New Jersey
and mixed myeloproliferative/myelodysplastic neoplasms). The September 2016
description of flow cytometric features associated with MDS,

vi
Abbreviations

ABL1 Abelson leukemia homolog 1 gene ENKTL extranodal NK/T-cell lymphoma, nasal type
aCML atypical chronic myeloid leukemia (BCR-ABL1-) ET essential thrombocythemia
AITL angioimmunoblastic T-cell lymphoma ETP-ALLearly T-cell precursor ALL
ALC absolute lymphocyte count FAB French-American-British [classification of acute
ALCL anaplastic large cell lymphoma leukemia]
ALIP abnormal localization of immature precursors FC flow cytometry
ALK anaplastic lymphoma kinase FGFR1 fibroblast growth factor receptor 1 gene
ALL acute lymphoblastic leukemia FISH fluorescence in situ hybridization
AML acute myeloid leukemia FITC fluorescein isothiocyanate
AMML acute myelomonocytic leukemia FL follicular lymphoma
ANC absolute neutrophil count FLT3 Fms-like tyrosine kinase 3 gene
AP accelerated phase FSC forward scatter
APL acute promyelocytic leukemia GCB germinal center B-cell
ATLL adult T-cell lymphoma/leukemia G-CSF granulocyte-colony stimulating factor
ATRA all-trans retinoic acid GPI glycosylphosphatidylinositol
B-ALL B-lymphoblastic leukemia/lymphoma HGBL high-grade B-cell lymphoma
BCL1 B-cell lymphoma 1 (cyclin D1) protein encoded by H&E hematoxylin and eosin
CCND1 gene HCL hairy cell leukemia
BCL2 B-cell lymphoma 2 apoptosis regulating protein HCL-v hairy cell leukemia variant
encoded by BCL2 gene HES hypereosinophilic syndrome
BCR breakpoint cluster region gene HHV-8 human herpesvirus-8
BL Burkitt lymphoma HL Hodgkin lymphoma
BM bone marrow HLA-DR human leukocyte antigen D-related
BP blast phase HPF high-power field
BPDCN blastic plasmacytoid dendritic cell neoplasm HSTL hepatosplenic T-cell lymphoma
B-PLL B-cell prolymphocytic leukemia ICUS idiopathic cytopenia of undetermined significance
BRAF v-raf murine sarcoma viral oncogene homolog B1 IDUS idiopathic dysplasia of undetermined significance
CALLA common acute lymphoblastic leukemia antigen IGVH immunoglobulin heavy-chain variable gene
CBF core-binding factor ISFN in situ follicular neoplasia
CD cluster designation ISMCN in situ mantle cell neoplasia
CEL chronic eosinophilic leukemia ITD internal tandem duplication
CGH comparative genomic hybridization IVLBCL intravascular large B-cell lymphoma
CHIP clonal hematopoiesis of indeterminate potential JMML juvenile myelomonocytic leukemia
CLL chronic lymphocytic leukemia LAP leukemia-associated phenotype
CML chronic myeloid leukemia (BCR-ABL1+) LBL lymphoblastic lymphoma
CMML chronic myelomonocytic leukemia LCH Langerhans cell histiocytosis
CMR complete molecular response LESA lymphoepithelial sialadenitis
CNL chronic neutrophilic leukemia LGL large granular lymphocyte
CP chronic phase L&H lymphocyte and histiocyte cell [popcorn cell;
CR complete response neoplastic cells in NLPHL]
CSF cerebrospinal fluid LP lymphocyte predominant (cell) [neoplastic cells in
DH double hit NLPHL]
DIC disseminated intravascular coagulopathy LPL lymphoplasmacytic lymphoma
DLBCL diffuse large B-cell lymphoma LYG lymphomatoid granulomatosis
EATL enteropathy-associated T-cell lymphoma LyP lymphomatoid papulosis
EBER Epstein–Barr virus early RNA MALT mucosa associated lymphoid tissue
EBV Epstein–Barr virus positivity by in situ hybridization MBL monoclonal B-cell lymphocytosis
EMA epithelial membrane antigen MBR major breakpoint region
EMT extramedullary myeloid tumor (granulocytic MCL mantle cell lymphoma
sarcoma) mcr minor cluster region

vii
 abbreviations

MDS myelodysplastic syndrome PLL prolymphocytic leukemia

MDS-EB MDS with excess blasts PMBL primary mediastinal large B-cell lymphoma
MDS-f MDS with fibrosis PMF primary myelofibrosis
MDS-MLD MDS with multilineage dysplasia PML promyelocytic leukemia gene
MDS-RS MDS with ring sideroblasts PNH paroxysmal nocturnal hemoglobinuria
MDS-SLD MDS with single lineage dysplasia PV polycythemia vera
MDS/MPN myelodysplastic/myeloproliferative PTCL peripheral T-cell lymphoma, unspecified
neoplasms PTLD posttransplant lymphoproliferative disorder
MDS/MPN-RS-T MDS/MPN with ring sideroblasts and QR quantitative real-time
thrombocytosis RARA retinoic acid receptor α gene
MEITL monomorphic epitheliotropic intestinal T-cell R-CHOP Rituximab+Cyclophosphamide+Hydroxydauno
lymphoma rubicin+Oncovin+Prednisone
MF mycosis fungoides ROC receiver operating characteristics
MGUS monoclonal gammopathy of undetermined RQ-PCR real-time quantitative PCR
significance RT-PCR reverse transcriptase PCR
MPAL mixed phenotype acute leukemia R-S Reed–Sternberg [cell]
MPN myeloproliferative neoplasm SBP solitary bone plasmacytoma
MPO myeloperoxidase SLE systemic lupus erythematous
MRD minimal residual disease SLL small lymphocytic lymphoma
MRI magnetic resonance imaging SM systemic mastocytosis
MZL marginal zone B-cell lymphoma SMZL splenic marginal zone lymphoma
NHL non-Hodgkin lymphoma SPTL subcutaneous panniculitis-like T-cell lymphoma
NK natural killer SS Sézary syndrome
NLPHL nodular lymphocyte predominant Hodgkin SSB Sudan Black B
lymphoma SSC side scatter
NOS not otherwise specified t-AML therapy-related acute myeloid leukemia
NPM nucleophosmin gene TAM transient abnormal myelopoiesis
NSE nonspecific esterase T-ALL T-cell lymphoblastic leukemia/lymphoma
PAS periodic acid-Schiff stain TFH follicular helper T-cell
PBL plasmablastic lymphoma TCR T-cell receptor
PCM plasma cell myeloma THRLBCL T-cell/histiocyte-rich large B-cell lymphoma
PCR polymerase chain reaction TdT terminal deoxynucleotidyl transferase
PDGFRA alpha-type platelet-derived growth factor T-LGL T-cell large granular lymphocyte [leukemia]
receptor gene t-MDS therapy-related myelodysplastic syndrome
PDGFRB beta-type platelet-derived growth factor t-MDS/MPN therapy-related myelodysplastic/
receptor gene myeloproliferative neoplasms
PE phycoerythrin T-PLL T-cell prolymphocytic leukemia
PEL primary effusion lymphoma TTF1 thyroid transcription factor 1
PerCP peridinium chlorophyll protein complex WBC white blood cell [count]
PFL pediatric follicular lymphoma WHO World Health Organization
Ph Philadelphia chromosome [result of t(9;22) WM Waldenström macroglobulinemia
translocation]

viii
1 Introduction

Flow cytometry (FC) plays a very important role in diagnosis, of thiazole orange (TOT-1) [38]. Data are displayed in single-
subclassification, and post-treatment monitoring of hematologic parameter histogram and 2-parameer dot plots [8,13,39,40].
neoplasms [1–26]. FC results allow one to choose in a timely Most of the antigens detected by FC are located on the cell sur-
manner the most appropriate further testing (such as FISH or face. In order to stain intracellular components, cells have to
PCR) to establish the definite diagnosis and to further character- be permeabilized (so that the antibody can enter cells through
ize the malignant process. Multiparameter FC measures simul- “holes” in their membrane). The optimal number of reagents
taneously several surface and/or intracytoplasmic markers on a required to evaluate hematolymphoid neoplasms depends on the
single cell, allowing for accurate phenotypic characterization of clinical diagnosis, relevant laboratory data, and differential diag-
analyzed population(s). While no single marker permits a defi- nosis based on cytomorphology [26,41]. Flow cytometry is much
nite lineage assignment, analysis with panels of antibodies allows faster than immunohistochemistry and can analyze thousands
for separation of hematologic tumors into very precise subtypes of cells within seconds. Another advantage of FC immunophe-
with different prognosis and treatment requirements, as defined notyping is that it allows correlation of several markers on a sin-
by current World Health Organization (WHO) classification of gle cell, and detects intensity of staining and aberrant expression
hematopoietic and lymphoid tumors [27]. FC analysis can pre- of antigens. FC has high sensitivity for B-cell lymphoprolifera-
cisely differentiate between B- and T-cell malignancies, between tive disorders and acute leukemia and high specificity for several
mature (peripheral) and precursor tumors, and among the latter, categories of those neoplasms. All these properties are used in
determine the myeloid or lymphoid lineage. In acute leukemias, diagnostic hematopathology for subclassification of neoplasms.
the role of FC is not limited to identification of blasts, but expands The major disadvantage of FC is a need for liquid cell suspension
to determine the lineage and specific phenotype, which often and therefore lack of correlation with histomorphologic features
prompts additional testing for final subclassification of leukemia. (tissue architecture). FC requires viable fresh (unfixed) mate-
For example, blasts (or blast equivalents) with high side scatter rial. In a subset of neoplasms, especially high-grade lymphomas,
(SSC), positive CD117, CD13, and CD33 and negative HLA-DR decreased viability often precludes accurate FC analysis. Flow
raise the possibility of acute promyelocytic leukemia (APL), cytometry analysis requires at least 10,000–20,000 cells (events)
which can be confirmed by testing for PML-RARA. Myeloblasts acquired by tube, which often limits its use in specimens from
with aberrant co-expression of CD19 and CD56 suggest acute CSF, fine needle aspirates, and paucicellular (or fibrotic) lesions.
myeloid leukemia (AML) with t(8;21) and B-lymphoblasts with Dropout of neoplastic cells due to low viability or sample bias
aberrant expression of CD13 or CD33 suggest either BCR-ABL1 due to focal (partial) tissue involvement may lead to false nega-
or ETV6-RUNX1 rearrangements. Availability of new markers tive flow results (see also Chapter 14).
(antibodies), new fluorochromes, and improvement in instru-
mentation increases accuracy of FC studies and allows for identi-
fication of a minute abnormal population among the majority of
the major role of flow cytometry analysis
Identification of Acute Promyelocytic Leukemia
benign cells, evaluates minimal residual disease (MRD), and also
Acute promyelocytic leukemia (APL) is characterized by high
expands FC applications into such disorders as myelodysplastic
SSC, positive CD117, negative CD11c, CD34, and HLA-DR, and
syndromes, paroxysmal nocturnal hemoglobinuria (PNH), and
positive myeloid markers (myeloperoxidase, CD13, and CD33)
myeloproliferative neoplasms [9,18,26,28–34].
(Figure 1.1). The less common, hypogranular variant of APL
FC analysis requires fresh (unfixed) material. Types of speci-
shows a similar phenotype to classic APL, except for low SSC
mens suitable for FC include blood, bone marrow (BM) aspirate,
and often positive expression of CD2 and CD34. For details see
fresh tissue samples, fine needle aspirates, effusions (pleural,
Chapters 6, 25, and 26.
peritoneal), and other body fluids (e.g., cerebrospinal fluid; CSF).
In FC protocol, the sample is incubated with antibodies, followed
by red blood cell lysis, washing, fixation in paraformaldehyde, Identification of Blasts, Their Lineage and Phenotype
and FC analysis. Whole blood lysis represents the most com- Myeloblasts are usually characterized by expression of CD117
monly used technique for sample preparation [35–37]. Routinely and/or CD34, HLA-DR, CD133, CD13, CD33, and CD38. The
5,000 to 10,000 cells are collected (in MRD protocols 500,000 expression of CD45 is moderate and SSC is low (Figure 1.2).
events). Monoclonal antibodies used in FC are conjugated with Subsets of myeloblasts may be positive for TdT, CD123, CD71,
fluorochromes, which are excited or stimulated by laser(s) in flow CD7, and CD11c. Occasional cases may be HLA-DR-negative.
cytometer. The most common fluorochromes excited at 488 nm AML is diagnosed with ≥20% blasts (or blasts equivalents) in
(argon laser) include fluorescein isothiocyanate (FITC), phyco- the BM, except for AML with t(15;17), inv(16), or t(8;21), which
erythrin (PE), propidium iodide (PI), 7-amino-actinomcyin D do not require a 20% threshold for the diagnosis. For details see
(7AAD), peridin-chloryophyl-A-protein (PerCP), and dimmers Chapters 6 and 25 through 27.

1
 flow cytometry in neoplastic hematology

(b)

High SSC
Side scatter
CD45
(a)

(c) (d) (e)

Negative Positive Negative
Forward scatter

CD34 CD117 HLA-DR

(f ) (g) (h)
Dim Bright Dim

CD13 CD33 CD64

Figure 1.1 APL: typical flow cytometry pattern. Neoplastic promyelocytes are characterized by hypergranular cytoplasm with Auer rods (a). Flow cytometry fea-
tures includes high side scatter (b; arrows), lack of CD34 (c) and HLA-DR (e), and positive CD117 (d), CD13 (f), CD33 (g), and CD64 (h; dim).

Monoblasts are characterized by positive CD64 (moderate range from dim to moderate expression. SSC is low. For details
or bright), bright CD45 (usually stronger than in myeloblasts), see Chapter 28.
positive HLA-DR, positive CD11b, CD11c, CD4, CD36, and B-lymphoblasts from B lymphoblastic leukemia/lymphoma
often positive CD2, CD56, CD71, and/or CD123 (Figure 1.3). The (B-ALL) are typically positive or CD34, TdT, HLA-DR, CD19,
expression of CD14 varies from positive (bright), heterogeneous CD22, CD38, and CD10 with negative to dim CD45 and negative
(variable), dim, or partial to completely negative, depending CD20 (Figure 1.5). A subset of B-ALL cases is negative for CD10
on the degree of maturation (immature monoblasts are usually and subsets of cases may be CD71+, CD123+, or CD20+. Blasts may
CD14–). Expression of CD11b, although often positive, may be express myeloid antigens (usually CD13 or CD33, rarely CD15).
partial, dim, or variable (“smeary”). Rare cases may be posi- For details see Chapters 8 and 20.
tive for blastic markers (CD34 and/or CD117). For details see T-lymphoblasts from T lymphoblastic leukemia/lymphoma
Chapters 6 and 25 through 27. (T-ALL) are positive for CD34 and/or TdT, do not express surface
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is CD3 (cytoplasmic CD3 is positive), and are positive for one or
positive for CD4, CD36, CD38, CD43, CD45, CD56 (bright), more of the T-cell markers (CD2, CD5 and/or CD7) (Figure 1.6).
CD71, HLA-DR, CD303 (blood dendritic cell antigen-2; BDCA- They are either dual CD4/CD8+ or dual CD4/CD8–. The subset of
2), CD123, and HLA-DR (Figure 1.4). CD45 is positive and may T-ALL is positive for CD1a and/or CD10. Early T-cell precursor

2
 introduction

(a)
Granulocytes

Side scatter
Myeloblasts

R1

CD45
(b) (c)
HLA-DR

CD33

CD117 CD34
Figure 1.2 Identification of myeloblasts (AML with maturation). Myeloblasts (green dots) show moderate CD45 and low side scatter placing them in “blastic” gate
on CD45 versus SSC display (a). They are usually positive for HLA-DR (b), CD117 (b), CD33 (c), and CD34 (c). Granulocytes/maturing myeloid precursors are rep-
resented by gray dots.

(a) (b) (c) (d)

Side scatter

CD45 CD34 CD117 HLA-DR

(e) (f) (g) (h)

CD64--FITC-IgG1
Side scatter

CD11b CD13 CD33 CD14

Figure 1.3 Acute monoblastic leukemia. Monoblasts (green dots, arrow) show bright expression of CD45 and slightly increased side scatter (a). CD34 and CD117 are
not expressed (b–c). HLA-DR is positive (d) and both CD33 and CD64 are brightly expressed (g–h). There is aberrant expression of CD11b (e; variable with subset
negative and dimly positive), CD13 (f; mostly negative, only minor population is positive), and CD14 (h; mostly negative with only minute population showing
variable expression).

3
 flow cytometry in neoplastic hematology

Forward scatter
Side scatter

(a) (b) (c) (d)

CD45 CD4 CD56 CD123

Forward scatter

(e) (f ) (g) (h)

HLA-DR CD33 CD34 CD117

Figure 1.4 BPDCN shows low side scatter (a), negative to dim CD45 (a), positive CD4 (b), CD56 (c), CD123 (d), and HLA-DR (f), and does not express CD33 (f),
CD34 (g), and CD117 (h).
Forward scatter
Side scatter

(a) (b) (c) (d)

CD45 CD34 CD19 CD10

Figure 1.5 B-lymphoblasts (green dots, arrow) show negative to dim CD45 (a), low side scatter (a), and positive expression of CD34 (b), CD19 (c), and CD10 (d).
Forward scatter
Side scatter

(a) (b) (c) (d) (e)

CD45 TdT CD7 CD4 CD8

Figure 1.6 T-lymphoblasts (green dots; arrow) are CD45+ (a), have low side scatter (a), express TdT (b), CD7 (c), and are dual CD4/CD8- (d–e).

T-ALL (ETP-ALL) often shows aberrant expression of CD13 and and/or partial expression of CD10 or CD123 may be seen. For
CD117, and lack of CD5. For details see Chapters 9 and 21. details see Chapter 28.
Acute undifferentiated leukemia is positive for some of the Mixed phenotype acute leukemia (MPAL) co-expresses
blastic markers (TdT, CD34), CD38, and HLA-DR, and does not myeloid (or monocytic) markers with B- or T-cell lineage-
express markers specific for myeloid or lymphoid lineages. Dim specific marker (rarely B- and T-cell markers). MPALs are often

4
 introduction

positive for CD34, TdT, and HLA-DR. Lineage assignment crite- are positive. A subset of B-cell lymphoproliferations is positive
ria for myeloid lineage include (1) myeloperoxidase (MPO) or (2) for CD5 or CD10. For details see Chapters 4, 15, and 16.
monocytic differentiation (at least two of the following: CD11c, Plasma cells are positive for CD38 and CD138. Benign plasma
CD14, CD64, nonspecific esterase, and lysozyme) [42]. T-lineage cells express CD19, CD27, CD45, and a polytypic pattern of expres-
is assigned with (1) strong cytoplasmic CD3 or (2) surface CD3 sion of cytoplasmic light chain immunoglobulins, and are negative
expression. B-lineage is assigned when there is (1) strong CD19 for CD28, CD56, and CD117. Malignant plasma cells have increased
expression with at least one of the following strongly expressed: forward scatter (FSC) and are CD19–, CD45–/+, CD20–, CD27–,
CD79a, cytoplasmic CD22, or CD10, or (2) weak CD19 with at CD28+, CD56+/–, CD117+/– and show restricted expression of either
least two of the following strongly expressed: CD79a, cytoplas- cytoplasmic κ or cytoplasmic λ. Clonal plasma cells accompanying
mic CD22, or CD10. For details see Chapter 28. low grade B-cell lymphomas with plasmacytic differentiation show
low to medium FSC, may be CD19+ and/or CD45+, and are usually
Identification of Clonal B-Cells and Clonal Plasma Cells CD56– and CD117–. For details see Chapter 17.
B-cell lymphomas show prominent and cohesive population
of light chain-restricted B-cells (Figure 1.7). Normal (mature)
B-cells consist of two populations, one expressing kappa (κ) and Identification of Atypical T-Cells
the other lambda (λ) light chain immunoglobulins. A subset of T-cell lymphomas can be suspected by FC based on loss or aber-
mature B-cell proliferation may be surface light chain immuno- rant (dim or variable) expression of the T antigens (CD2, CD3,
globulin negative. B-cell markers (CD19, CD20, CD22, CD79a) CD5, and/or CD7), aberrant expression of CD4 or CD8 (subset
restriction, dual-positive CD4/CD8 expression, or lack of both
markers), increased FSC, lack of CD26, or presence of additional
(a) (b) markers such as CD10, CD25, CD30, CD56, CD57, or CD103
(Figure 1.8). For details see Chapters 5 and 18.
κ+
Lambda
Kappa

Identification of MRD; e.g., Acute Leukemias,

CLL, Plasma Cell Myeloma
λ- Evaluation of PNH

CD20 pattern of antigen expression

Figure 1.7 Identification of clonal B-cells: B-cells (red dots, arrow) show CD20 FC immunophenotyping includes evaluation of the expression of
expression and κ restriction (a). There is no λ expression (b). specific antigens (markers) along with light scatter properties of

(a) (b) (c) (d)

Tumor cells Tumor cells
Tumor cells Tumor cells
Forward scatter

Benign (residual) B-cells

Benign (residual) T-cells Benign (residual) T-cells Benign (residual) T-cells Benign (residual) T-cells

CD2 CD3 CD5 CD7

(e) (f ) (g)
Tumor cells
Forward scatter

Benign (residual) CD4+ T-cells Benign (residual) CD8+ T-cells

Benign (residual) B-cells

CD4 CD8 CD20

Figure 1.8 Identification of abnormal T-cells: increased forward scatter. PTCL with positive expression of all pan T-cell antigens (a–d). The neoplastic cells have
increased forward scatter (compare with residual B-cells [g] and benign T-cells) and show dim CD3 (b). The expression of CD5 is slightly dimmer when compared
to benign T-cells, and the expression of both CD2 and CD7 is slightly brighter (a and d). Neoplastic T-cells are CD4+ (e–f).

5
 flow cytometry in neoplastic hematology

the cells. The analyzed parameters are compared to that expected in evaluation of blood or BM samples for features of dyspoiesis
in normal (benign) counterpart. Presence of any difference in the (dysmaturation).
pattern of expression, e.g., lack of antigen expression, decreased Once the abnormal population is identified, evaluation of
or increased intensity of staining, variable (heterogeneous) additional markers may be helpful to further characterize the
expression, or presence of a marker which is not typically seen process and often allows disease monitoring. Based on the
in normal counterpart (e.g., lymphoid markers in myeloid cells immunophenotypic features, possible entities or specific diag-
or pan-myeloid markers in lymphoid cells), allows for identifica- nosis (whenever possible) is suggested along with recommenda-
tion of abnormal population. In many cases, and especially in tion for additional testing (when applicable), such as morphology
tumors with predominance of neoplastic population (without (e.g., blast enumeration; nodular or diffuse pattern of lymph
benign cells which could be used as controls) correlation with node involvement), cytogenetics, FISH (e.g., PML-RARA, IGH-
staining of negative controls maybe invaluable in excluding non- BCL2, MYC), PCR (e.g., BCR-ABL1, JAK2 mutation, T-cell clon-
specific (“background”) staining and thus deciding which results ality), radiologic imaging studies (e.g., bone lesions in patients
are truly positive. Although numeric evaluation of flow results with plasma cell neoplasm) and laboratory data (e.g., serum M
(percent of cells positive for specific antigen) is crucial in render- protein, serum erythropoietin level, HTLV-1, etc.).
ing flow diagnosis (e.g., in analysis of number of CD34+ blasts,
percent of CD34+ hematogones among all CD34+ precursors,
CD4:CD8 ratio, or number of CD10 – granulocytes, etc.), analysis intensity of staining
of pattern of antigen expression is equally important, as often The results of the staining are determined by the comparison
only an aberrant pattern of expression helps in establishing the between negative controls and the intensity of staining with
correct diagnosis. Figure 1.9 shows a case of B-cell lymphoma each antibody (Figures 1.10 through 1.12). Negative staining
with biclonal κ and λ expression. This case may be misdiagnosed can be defined by the fluorescence intensity similar to that of
as benign (polytypic) if analysis is based solely on numbers and negative controls (Figure 1.10). The staining is positive when
not pattern of antigen expression. Careful correlation of flow the expression (fluorescence intensity) of any given marker
results with normal pattern seen in benign cases is important (antibody) is greater than that of a negative (isotypic) control.

(a) (a’)
κ+ = 40% λ+ = 35%
Lambda
Kappa

CD19

(b) (b’)
Lambda
Kappa

CD20
Figure 1.9 Analysis of percent of κ+ and λ+ B-cells in this CLL may suggest polytypic process (a, aʹ), but careful evaluation of pattern of antigen expression (CD20
versus κ and λ) indicate bi-clonal B-cell lymphoproliferative process: κ+ B-cells show variable CD20 expression (b; red arrows) whereas λ+ B-cells show more cohe-
sive cluster of cells (bʹ; grey arrow).

6
 introduction

Negative CD14

Forward scatter

Forward scatter
(a)
(b)

Control-lgG2b-FITC CD14-lgG2b-FITC

Figure 1.10 Assessing the staining results: the staining intensity for each antibody is compared with the negative control. Myelomonocytic population (green and
blue dots) in panel b appears to express CD14, when compared to overtly negative lymphocytes (red dots). However, the intensity of the expression of CD14 is similar
to nonspecific staining with isotypic control (panel a), and therefore the results for CD14 have to be interpreted as negative. In this sample, only the cells located
within the dotted circle (close to and beyond 103 on the x-axis) would be considered positive. This panel also shows that the “built-in” negative controls (in this
case lymphocytes, which do not express CD14) cannot be used reliably as a negative control, since the population of interest may be characterized by a very high
nonspecific “background” staining (a).

Blasts
Blasts
Granulocytes Granulocytes

(a) (a’)
IgG2/FITC HLA-DR/FITC

Blasts Blasts
Granulocytes

(b) (b’)
Forward scatter

IgG2a/PE CD33/PE

Blasts Blasts

(c) (c’)
IgG2a/PE-Cy7 CD2/PE-Cy7

Lymphocytes (d) Lymphocytes (d’)

IgG2a/PerCP-Cy5-5 CD3/PerCP-Cy5-5
Figure 1.11 High nonspecific (background) staining. Flow cytometry shows three distinct populations: blasts (green dots), granulocytes (gray dots), and lympho-
cytes (red dots). Based on control staining (left column; a–d) blasts are positive for CD33 (bʹ) and CD2 (cʹ), granulocytes are positive for CD33 (bʹ), and lymphocytes
are positive for CD2 (cʹ) and CD3 (dʹ). All three populations do not express HLA-DR (aʹ).

7
 flow cytometry in neoplastic hematology

Negative Dim

Dim to moderate
Forward scatter

(a) (b) Moderate (c)

CD3 CD3 CD2

Bright
Moderate Bright Dim

(d) (e) (f )

CD3 CD5 CD10

Figure 1.12 Intensity of antigen expression; see text for details.

Even in heterogeneous samples (e.g., BM specimen) the use of lymphocytes are negative for CD3. Dim staining is defined by
“built-in” negative controls is limited and may lead to misinter- the fluorescence intensity, which is slightly increased when
pretation of the results. Different cell populations often display compared to negative control. The cells in Figure 1.12b have
variable intensity of “background” (nonspecific) staining. Some dim to moderate expression of CD3. Moderate staining is
cells (e.g., monocytic cells, atypical promyelocytes, and large defined by at least one log decade brighter than negative control
lymphomatous cells with decreased viability) often display (Figure 1.12c,d). Figure 1.12c shows two populations of cells:
high nonspecific staining (Figures 1.10 and 1.11). Therefore, one with dim and other with moderate expression of CD2. The
the threshold between positive and negative expression should abnormal population of lymphocytes (dim CD2) has increased
be established for each cell population based on the control FSC, suggesting larger cell size when compared to benign
(negative) sample and not by comparison with other popula- lymphocytes (moderate CD2). Bright staining is at least two
tion known to be negative for a specific marker. As illustrated log decades brighter than negative control. T-PLL cells (red;
in Figure 1.10, if only staining with CD14 was performed, each Figure 1.12e) and B-lymphoblasts (Figure 1.12f) display bright
population with intensity greater than observed in lymphocytes expression of CD5 and CD10, respectively. Based on the 2006
(red dots) which do not express CD14 would be considered pos- Bethesda International Consensus flow cytometry meeting,
itive. However, as it is evident from the staining with negative the recommended descriptions of antibody distribution are
(isotypic) control antibody (Figure 1.10a), the abnormal cells “negative,” “positive,” or “partially expressed” (relative to an
(green and blue dots) have very high nonspecific staining which appropriate negative control population) and the recommended
is similar to that observed with CD14 (Figure 1.10b). Figure 1.11 descriptions of antibody fluorescence intensity are “dim,”
illustrates the different levels of background (nonspecific) stain- “bright,” and “heterogeneous,” with the intensity relative to the
ing among different populations (myeloblasts, granulocytes, closest normal hematolymphoid population [26].
and benign T-lymphocytes) in the sample from BM involved by
AML. Only careful comparison of antigen expression for each
population with that of nonspecific staining (control) allows for the co-expression of antigens
the characterization of identified cells. The diagnosis and subclassification of hematopoietic tumors
The intensity of expression of any antigen can be categorized rely not only on the distinction between positive and negative
into dim, moderate, bright, and variable (Figure 1.12). The expression of analyzed marker or the fluorescence intensity, but
intensity of staining of any population is compared to that also on the proper identification of the co-expression of two or
of a benign counterpart and reported as either normal or more markers by the same population of cells. The possibility
abnormal. Each cell type has a different pattern of antigen to analyze the co-expression of several antigens on a single cell
expression; for example, bright CD38 expression and negative is one of the biggest advantages of multicolor (multiparameter)
CD45 expression are normal for benign plasma cells, whereas FC analysis. Among B-cell lymphoproliferations, co-expression
bright CD45 expression and moderate CD20 expression are of CD5 and CD23 is seen typically in CLL/SLL, CD25/CD103 in
normal for benign B-cells. In Figure 1.12a, red dots representing hairy cell leukemia (HCL), CD10/BCL2 in follicular lymphoma

8
 introduction

CD3+/CD7+ CD25+/CD103+

CD23+/CD5+

CD23

CD25
CD3

CD3–/CD7+ (a) CD23–/CD5+ (b) (c)

CD7 104
CD5 CD103
CD2+/TdT+ CD33+/CD34+
CD4+/CD8– CD4+/CD8+

CD33
CD4

CD2
CD2+/TdT–

CD4–/CD8+

(d) (e) (f )

CD8 TdT CD34

Figure 1.13 Co-expression of antigens. (a) Two populations of T-cells are identified by comparing the expression of CD3 and CD7. Normal (benign) T-cells (upper
right quadrant) co-express CD3 and CD7, whereas neoplastic cells (lower right quadrant) lack CD3. (b) B-CLL/SLL is characterized by co-expression of CD5 and
CD23 (upper right quadrant) in contrast to benign T-cells, which have brighter expression of CD5 and do not express CD23 (lower right quadrant). (c) HCL is
characterized by co-expression of CD25 and CD103. (d) Immature T-cell population (thymocytes from thymoma) display co-expression of CD4 and CD8 (upper
right quadrant). Mature T-cells are either CD4+ (upper left quadrant) or CD8+ (lower right quadrant). (e) Immature T-cell population co-expressing CD2 and TdT
(upper right quadrant); benign T-cells express CD2 but are TdT- (upper left quadrant). (f) Myeloblasts co-expressing CD34 and CD33 (arrow).

(FL), CD5 in mantle cell lymphoma (MCL), and CD10/CD43 in by monoblasts, or pan-T-cell antigens by B-cells), absence of an
Burkitt lymphoma (BL). T-cell lymphomas may show co-expres- antigen which is normally positive (e.g., lack of HLA-DR or CD14
sion of T-cell antigens with CD30 (e.g., anaplastic large cell in monocytes, surface CD3 in T-ALL, CD20 in B-cells, and CD45
lymphoma), CD10 (nodal T-cell lymphomas with T-follicular in B-ALL), or unusually dim or bright expression of marker (e.g.,
helper (TFH) phenotype, including angioimmunoblastic T-cell bright CD10 in B-ALL, dim CD13 or CD33 in AML, and dim
lymphoma and follicular-cell lymphoma), CD103 (enteropathy- pan-T-cell markers in T-cell lymphomas) helps to identify neo-
associated T-cell lymphoma; EATL), and CD16/CD56/CD57 plastic process and allows for identification of MRD or early
(e.g., T-LGL leukemia, T/NK-cell lymphoma). Figure 1.13 illus- relapse in follow-up studies [1,2,7,24,43–58].
trates co-expression of different antigens by hematopoietic Aberrant expression of T-cell–associated antigens (other than
tumors. CD5 and CD43) on B-cell NHL is a known but uncommonly
observed phenomenon [59–61]. Neoplastic plasma cells often
express CD56 and CD117 [62], and T-cells in angioimmunoblas-
aberrant expression of antigens tic T-cell lymphoma are often positive for CD10 and BCL6 [7,63].
Benign B-cells are positive for B-cell markers (e.g., CD19, Maturing myeloid cells in myelodysplastic syndrome or chronic
CD20, CD22, and CD79a) and negative for T-cell markers, and myeloproliferative disorders often display abnormal expression
conversely, normal T-cells are positive for T-antigens and are of CD56 (N-CAM) [46]. Inaba et al. reported aberrant expres-
negative for B-cell markers. Other cell lineages also have char- sion of T-cell antigens in 24.2% of B-cell lymphomas [61] and
acteristic immunophenotypic profiles; e.g., benign monocytes Quintanilla-Martinez et al. reported CD20+ T-cell lymphoma
display bright expression of CD11b, CD11c, CD14, and CD64 [64]. The co-expression of CD5 and CD23 is typical for CLL/SLL,
and are positive for HLA-DR, granulocytes are positive for pan- but a subset of cases may show aberrant lack of CD23. Similarly,
myeloid antigens (CD13, CD33), CD10, and CD16, and plasma co-expression of CD5 is typical for MCL but ~10% of cases show
cells are positive for CD38, CD138, cytoplasmic light and heavy lack of CD5.
chain immunoglobulins, and are negative for CD45. Figure 1.14 presents several examples of aberrant antigen
Identification of aberrant antigen expression, such as posi- expression: diffuse large B-cell lymphoma with aberrant expression
tive expression of marker(s) not associated with the specific cell of CD56 (Figure 1.14a); HCL with aberrant expression of CD2
type (e.g., CD20 by T-cell lymphoma, CD33 by B-ALL, CD56 (T-cell marker) on a subset of leukemic cells (Figure 1.14b; arrow);

9
 flow cytometry in neoplastic hematology

Diffuse large B-cell lymphoma with aberrant CD56 Hairy cell leukemia with aberrant CD2

Forward scatter
CD56

CD19 Cytoplasmic CD3

(a) (b)

B-chronic lymphocytic leukemia with aberrant CD8 Peripheral T-cell lymphoma, NOS with aberrant CD20

CD20
CD8

CD19 Cytoplasmic CD3

(c) (d)

Marginal zone lymphoma with aberrant CD13

CD19

CD19

CD13

(e) Kappa Lambda CD20

Figure 1.14 Aberrant antigen expression; see text for details.

CLL/SLL with co-expression of CD8 (Figure 1.14c; arrow), loss of CD20 (Figure 1.15c), and peripheral T-cell lymphoma
and peripheral T-cell lymphoma with aberrant expression of with aberrant loss of surface CD3 expression (Figure 1.15d).
CD20 (arrow) on a subset of T-cells (Figure 1.14d). Similar to Table 1.1 presents the frequency of aberrant antigen expression
aberrant expression of an antigen, lack of a marker helps to in B-cell lymphomas, Table 1.2 presents aberrant expression of
identify and define abnormal cell population (Figure 1.15): acute T antigens in non-T-cell disorders, and Table 1.3 shows aberrant
monoblastic leukemia with aberrant loss of HLA-DR expression expression of B-cell markers in non-B-cell neoplasms. Aberrant
(Figure 1.15a), B-lymphoblastic leukemia with loss of CD45 antigen expression in B- and T-cell neoplasms is discussed in
expression (Figure 1.15b), diffuse large B-cell lymphoma with detail in Chapters 4, 5, and 15.

10
 introduction

Acute monoblastic leukemia with aberrant B-lymphoblastic leukemia with aberrant

lack of HLA-DR lack of CD45

Forward scatter

Side scatter
HLA-DR CD45
(a) (b)

Diffuse large B-cell lymphoma with aberrant Peripheral T-cell lymphoma with aberrant
lack of CD20 lack of surface CD3

Lymphomatous cells

Forward scatter
Kappa

Benign T-cells

(c) CD20 (d) CD3

Figure 1.15 Aberrant lack of antigen expression; see text for details.

Table 1.1 Frequency of Aberrant Antigen Expression in B-Cell Table 1.2 Aberrant Expression of T-Cell Antigens in Non-T-Cell
Lymphomas Disorders
CD5− mantle cell lymphoma 11% CD2 APL, BPDCN, mast cell disease, rare cases of CLL/SLL
CD23− CLL/SLL 4% CD3 PEL
CD10+ hairy cell leukemia 12% CD5 SLL/CLL, MCL, some MZL, rare cases of FL, de novo
CD19+ plasma cell myeloma 2% DLBCL, thymoma/thymic carcinoma
CD10− follicular lymphoma 6% CD7 AML, APL, BPDCN, monocytic leukemia
CD5+ follicular lymphoma 1% CD8 CLL (rare cases)
CD5+ marginal zone lymphoma 10%–15%
CD56+ DLBCL <1%
CD2+ CLL/SLL <1% Table 1.3 Aberrant Expression of Pan-B Antigens in Non-B-Cell
Disorders
CD19 Acute myeloid leukemia with t(8;21)
forward and side (orthogonal) light scatter CD20 Peripheral T-cell lymphoma, NOS (rare cases)
In flow cytometry analysis, cells are tagged with fluorochrome- CD79a Megakaryocytes (non-specific staining)
conjugated monoclonal antibodies directed toward specific
surface, cytoplasmic, or nuclear antigens. Intrinsic physical
properties of the cells, especially their size and cytoplasmic neutrophils, eosinophils, hypergranular promyelocytes) have
granularity, are measured simultaneously with fluorescence high SSC. Forward angle light scatter (FSC) corresponds to cell
emission as the fluorochrome-tagged cells pass through laser size (Figures 1.17 and 1.18). Large cells have higher FSC when
light. The side scatter (SSC; right angle/orthogonal light scatter) compared to smaller cells: note the difference between myelo-
corresponds to the granularity of the cytoplasm (Figure 1.16; blasts and monoblasts in Figure 1.17 and between small lym-
y-axis). The cells with agranular cytoplasm (i.e., lymphocytes) phocytes (red dots), large lymphocytes (blue dots), and cancer
have low SSC, whereas cells with granular cytoplasm (i.e., cells (orange dots) in Figure 1.18.

11
 flow cytometry in neoplastic hematology

CLL T-LGL
Granulocytes

Side scatter
Lymphocytes

Side scatter
T-LGL cells
(a) (b)

CD45

AML HCL

Side scatter
Side scatter

HCL cells
Granulocytes

Blasts
(c) (d)

NY04052134GT09.001 CD45 0269695 AE03.001

Carcinoma APL

Side scatter
Extrinsic cells
Side scatter

Leukemic
promyelocytes

(e) (f )

CD45
Figure 1.16 Side scatter (SSC; orthogonal, right angle scatter) on y-axis corresponds to granularity of the cytoplasm (x-axis presents CD45 expression). Lymphocytes
(a–f; red dots) have low SSC, whereas neutrophils (b–c), cancer cells (e), and atypical promyelocytes (f) have high SSC. Monocytes (b; blue dots), blasts (c; green dots),
and HCL cells (d; blue dots) have higher SSC than lymphocytes (compare with red dots).

Blasts Blasts
Forward scatter

(a) (b)

CD45 CD33

Monocytic Monocytic
cells cells

(c) (d)

CD45 CD33
Figure 1.17 Forward scatter (FSC). Comparison of FSC (y-axis) between myeloblasts (a–b; green dots) and monoblasts (c–d; blue dots). Myeloblasts are smaller and
therefore have lower FSC when compared to monocytic cells (compare with the location of dotted line in upper and lower panels or in relation to small lymphocytes
marked with red dots).

12
 introduction

10. Borowitz, M.J., Flow cytometry defended. Am J Clin Pathol, 2000.

113(4): p. 596–8.
CD20+ DLBCL 11. Borowitz, M.J., et al., U.S.-Canadian Consensus recommenda-
tions on the immunophenotypic analysis of hematologic neopla-
Forward scatter

sia by flow cytometry: data analysis and interpretation. Cytometry,

1997. 30(5): p. 236–44.
12. Borowitz, M.J., et al., Guidelines for the diagnosis and monitor-
ing of paroxysmal nocturnal hemoglobinuria and related dis-
Benign lymphocytes orders by flow cytometry. Cytometry B Clin Cytom, 2010. 78(4):
p. 211–30.
(a) 13. Braylan, R.C., Flow cytometry is becoming an indispensable tool
CD20
in leukemia diagnosis and classification. Cancer Invest, 1997.
15(4): p. 382–3.
14. Braylan, R.C., Impact of flow cytometry on the diagnosis and
characterization of lymphomas, chronic lymphoproliferative
CD45– cancer cells
disorders and plasma cell neoplasias. Cytometry A, 2004. 58(1):
Forward scatter

p. 57–61.
15. Braylan, R.C., et al., U.S.-Canadian Consensus recommenda-
tions on the immunophenotypic analysis of hematologic neo-
plasia by flow cytometry: data reporting. Cytometry, 1997. 30(5):
p. 245–8.
Benign lymphocytes 16. Braylan, R.C., N.A. Benson, and J. Iturraspe, Analysis of lympho-
mas by flow cytometry. Current and emerging strategies. Ann N
(b)
Y Acad Sci, 1993. 677: p. 364–78.
CD45 17. Cannizzo, E., et al., Multiparameter immunophenotyping by flow
Figure 1.18 Forward scatter (FSC; y-axis): comparison between benign small cytometry in multiple myeloma: The diagnostic utility of defining
lymphocytes (red dots; a), large lymphocytes of diffuse large B-cell lymphoma ranges of normal antigenic expression in comparison to histol-
(blue dots; a) and metastatic cancer cells (orange dots; b). ogy. Cytometry B Clin Cytom, 2010.
18. Craig, F.E. and K.A. Foon, Flow cytometric immunophenotyping
for hematologic neoplasms. Blood, 2008. 111(8): p. 3941–67.
19. D’Archangelo, M., Flow cytometry: new guidelines to support
references its clinical application. Cytometry B Clin Cytom, 2007. 72(3):
1. Gorczyca, W., Flow cytometry immunophenotypic character- p. 209–10.
istics of monocytic population in acute monocytic leukemia 20. Davis, B.H., et al., U.S.-Canadian Consensus recommendations
(AML-M5), acute myelomonocytic leukemia (AML-M4), and on the immunophenotypic analysis of hematologic neoplasia
chronic myelomonocytic leukemia (CMML). Methods Cell Biol, by flow cytometry: medical indications. Cytometry, 1997. 30(5):
2004. 75: p. 665–77. p. 249–63.
2. Gorczyca, W., Differential diagnosis of T-cell lymphoprolifera- 21. Jennings, C.D. and K.A. Foon, Flow cytometry: recent advances
tive disorders by flow cytometry multicolor immunophenotyp- in diagnosis and monitoring of leukemia. Cancer Invest, 1997.
ing. correlation with morphology. Methods Cell Biol, 2004. 75: 15(4): p. 384–99.
p. 595–621. 22. Ogata, K., Diagnostic flow cytometry for low-grade myelodys-
3. Gorczyca, W., ed., Flow Cytometry in Neoplastic Hematology. 2 plastic syndromes. Hematol Oncol, 2008. 26(4): p. 193–8.
ed. 2010, Informa Healthcare: London. 23. Porwit, A., Role of flow cytometry in diagnostics of myelodys-
4. Gorczyca, W., Acute promyelocytic leukemia: four distinct pat- plastic syndromes—beyond the WHO 2008 classification. Semin
terns by flow cytometry immunophenotyping. Pol J Pathol, 2012. Diagn Pathol, 2011. 28(4): p. 273–82.
63(1): p. 8–17. 24. Weir, E.G. and M.J. Borowitz, Flow cytometry in the diagnosis of
5. Gorczyca, W., et al., Immunophenotypic pattern of myeloid pop- acute leukemia. Semin Hematol, 2001. 38(2): p. 124–38.
ulations by flow cytometry analysis. Methods Cell Biol, 2011. 103: 25. Weisberger, J., W. Gorczyca, and M.C. Kinney, CD56-positive
p. 221–66. large B-cell lymphoma. Appl Immunohistochem Mol Morphol,
6. Gorczyca, W., et al., Flow cytometry in the diagnosis of medi- 2006. 14(4): p. 369–74.
astinal tumors with emphasis on differentiating thymocytes 26. Wood, B.L., et al., 2006 Bethesda International Consensus rec-
from precursor T-lymphoblastic lymphoma/leukemia. Leuk ommendations on the immunophenotypic analysis of hema-
Lymphoma, 2004. 45(3): p. 529–38. tolymphoid neoplasia by flow cytometry: optimal reagents and
7. Gorczyca, W., et al., An approach to diagnosis of T-cell lymphop- reporting for the flow cytometric diagnosis of hematopoietic neo-
roliferative disorders by flow cytometry. Cytometry, 2002. 50(3): plasia. Cytometry B Clin Cytom, 2007. 72 Suppl 1: p. S14–22.
p. 177–90. 27. Swerdlow, S.H., Campo, E., Harris, N. L., Jaffe, E. S., Pileri, S. A.,
8. Baumgarth, N. and M. Roederer, A practical approach to Stein, H., Thiele, J., Vardiman, J.W., ed. WHO Classification of
multicolor flow cytometry for immunophenotyping. J Immunol Tumors of Haematopoietic and Lymphoid Tissues. 2008, IARC: Lyon.
Methods, 2000. 243(1–2): p. 77–97. 28. Boveri, E., et al., Bone marrow histology in marginal zone B-cell
9. Borowitz, M.J., Flow cytometry testing in PNH. How much is lymphomas: correlation with clinical parameters and flow cytom-
enough? Cytometry, 2000. 42(4): p. 221–2. etry in 120 patients. Ann Oncol, 2009. 20(1): p. 129–36.

13
 flow cytometry in neoplastic hematology

29. van de Loosdrecht, A.A., et al., Standardization of flow cytometry 46. Lanza, F., B.S. Castoldi, J.M. Goldman, Abnormal expression
in myelodysplastic syndromes: report from the first European of N-CAM (CD56) adhesion molecule on myeloid and pro-
LeukemiaNet working conference on flow cytometry in myelo- genitor cells from chronc myeloid leukemia. Leukemia, 1993. 7:
dysplastic syndromes. Haematologica, 2009. 94(8): p. 1124–34. p. 1570–1575.
30. Benesch, M., et al., Flow cytometry for diagnosis and assess- 47. Tabernero, M.D., et al., Adult precursor B-ALL with BCR/ABL
ment of prognosis in patients with myelodysplastic syndromes. gene rearrangements displays a unique immunophenotype based
Hematology, 2004. 9(3): p. 171–7. on the pattern of CD10, CD34, CD13 and CD38 expresssion.
31. Greig, B., et al., 2006 Bethesda International Consensus recom- Leukemia, 2001. 15(3): p. 406–14.
mendations on the immunophenotypic analysis of hematolym- 48. Porwit, A., et al., B-cell chronic lymphocytic leukaemia with
phoid neoplasia by flow cytometry: recommendations for training aberrant expression of CD8 antigen. Eur J Haematol, 1987. 39(4):
and education to perform clinical flow cytometry. Cytometry B p. 311–7.
Clin Cytom, 2007. 72 Suppl 1: p. S23–33. 49. Schmidt, C.J., et al., Aberrant antigen expression detected by
32. Malcovati, L., et al., Flow cytometry evaluation of erythroid and multiparameter three color flow cytometry in intermediate and
myeloid dysplasia in patients with myelodysplastic syndrome. high grade B-cell lymphomas. Leuk Lymphoma, 1999. 34(5–6):
Leukemia, 2005. 19(5): p. 776–83. p. 539–44.
33. Richards, S.J. and D. Barnett, The role of flow cytometry in the 50. Dong, H.Y., et al., B-cell lymphomas with coexpression of CD5
diagnosis of paroxysmal nocturnal hemoglobinuria in the clini- and CD10. Am J Clin Pathol, 2003. 119(2): p. 218–30.
cal laboratory. Clin Lab Med, 2007. 27(3): p. 577–90, vii. 51. Jasionowski, T.M., et al., Analysis of CD10+ hairy cell leukemia.
34. Fromm, J.R., A. Thomas, and B.L. Wood, Flow cytometry can Am J Clin Pathol, 2003. 120(2): p. 228–35.
diagnose classical hodgkin lymphoma in lymph nodes with 52. Ludwig, W.D., et al., Ambiguous phenotypes and genotypes in 16
high sensitivity and specificity. Am J Clin Pathol, 2009. 131(3): children with acute leukemia as characterized by multiparameter
p. 322–32. analysis. Blood, 1988. 71(6): p. 1518–28.
35. Borowitz, M.J., et al., Immunophenotyping of acute leukemia by 53. Macedo, A., et al., Phenotypic analysis of CD34 subpopulations in
flow cytometric analysis. Use of CD45 and right-angle light scat- normal human bone marrow and its application for the detection
ter to gate on leukemic blasts in three-color analysis. Am J Clin of minimal residual disease. Leukemia, 1995. 9(11): p. 1896–901.
Pathol, 1993. 100(5): p. 534–40. 54. Macedo, A., et al., Immunological detection of blast cell sub-
36. Carter, P.H., et al., Flow cytometric analysis of whole blood lysis, populations in acute myeloblastic leukemia at diagnosis: implica-
three anticoagulants, and five cell preparations. Cytometry, 1992. tions for minimal residual disease studies. Leukemia, 1995. 9(6):
13(1): p. 68–74. p. 993–8.
37. Fleisher, T.A., C. Hagengruber, and G.E. Marti, 55. Macedo, A., et al., Characterization of aberrant phenotypes in
Immunophenotyping of normal lymphocytes. Pathol acute myeloblastic leukemia. Ann Hematol, 1995. 70(4): p. 189–94.
Immunopathol Res, 1988. 7(5): p. 305–18. 56. Oelschlagel, U., et al., Shift of aberrant antigen expression at
38. Baumgarth, N. and M. Bigos, Optimization of emission optics for relapse or at treatment failure in acute leukemia. Cytometry, 2000.
multicolor flow cytometry. Methods Cell Biol, 2004. 75: p. 3–22. 42(4): p. 247–53.
39. Marti, G.E., et al., Introduction to flow cytometry. Semin Hematol, 57. Ross, C.W., et al., Immunophenotypic aberrancy in adult acute
2001. 38(2): p. 93–9. lymphoblastic leukemia. Am J Clin Pathol, 1990. 94(5): p. 590–9.
40. Roederer, M., Z. Darzynkiewicz, and D.R. Parks, Guidelines for 58. Terstappen, L.W., et al., Flow cytometric characterization of acute
the presentation of flow cytometric data. Methods Cell Biol, 2004. myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis.
75: p. 241–56. Leukemia, 1992. 6(1): p. 70–80.
41. Braylan, R.C., et al., Optimal number of reagents required to 59. Kennedy, G.A., et al., Identification of tumours with the CD43
evaluate hematolymphoid neoplasias: results of an international only phenotype during the investigation of suspected lymphoma:
consensus meeting. Cytometry, 2001. 46(1): p. 23–7. a heterogeneous group not necessarily of T cell origin. Pathology,
42. Arber, D.A., et al., The 2016 revision to the World Health 2002. 34(1): p. 46–50.
Organization classification of myeloid neoplasms and acute leu- 60. Kaleem, Z., G. White, M.M. Zutter, Aberrant expression of T-cell-
kemia. Blood, 2016. 127(20): p. 2391–405. associated antigens on B-cell non-Hodgkin lymphomas. Am J
43. Juco, J., J.T. Holden et al, Immunophenotypic analysis of anaplas- Clin Pathol, 2001. 115: p. 396–403.
tic large cell lymphoma by flow cytometry. Am J Clin Pathol, 2003. 61. Inaba, T., et al., T-cell associated antigen-positive B-cell lym-
119: p. 205–212. phoma. Leuk Lymphoma, 2001. 42(6): p. 1161–71.
44. Kampalath, B., M.P. Barcos, and C. Stewart, Phenotypic hetero- 62. Lin, P., et al., Flow cytometric immunophenotypic analysis of 306
geneity of B cells in patients with chronic lymphocytic leukemia/ cases of multiple myeloma. Am J Clin Pathol, 2004. 121(4): p. 482–8.
small lymphocytic lymphoma. Am J Clin Pathol, 2003. 119(6): 63. Attygalle, A., et al., Neoplastic T cells in angioimmunoblastic
p. 824–32. T-cell lymphoma express CD10. Blood, 2002. 99(2): p. 627–33.
45. Bahia, D.M., et al., Aberrant phenotypes in acute myeloid leuke- 64. Quintanilla-Martinez, L., et al., CD20+ T-cell lymphoma.
mia: a high frequency and its clinical significance. Haematologica, Neoplastic transformation of a normal T-cell subset. Am J Clin
2001. 86(8): p. 801–6. Pathol, 1994. 102(4): p. 483–9.

14
2 Gating Strategies

flow cytometry of blood and bone Hematogones Gate

marrow-gating strategy Normal B-cell precursors (Figure 2.4) have very low SSC and
The analysis of CD45 expression combined with side scatter dimmer expression of CD45 than in normal lymphocytes.
(SSC; orthogonal light scatter) provides a simple and reproduc-
ible method for distinguishing major cell lineages in bone mar-
Blast Gate
row [1–10]. Figure 2.1 illustrates characteristic patterns seen in
Blasts usually have moderate CD45 expression and slightly
using this method and gating strategy applied in the flow cyto-
increased SSC (Figure 2.5). B- and T-lymphoblastic leukemias,
metric (FC) analysis of blood and bone marrow (BM). Based on
blastic plasmacytoid dendritic cell neoplasm (BPDCN), and
the intensity of CD45 staining (x-axis) and SSC (y-axis), one
occasional B- and T-cell lymphomas (most often large cell type)
can distinguish several major cell populations in normal BM
display similar location of neoplastic cells in the “blast” gate.
or blood: lymphocytes (bright CD45 and low SCC; red dots),
monocytes (bright CD45 and increased SSC; blue dots), and
granulocytes (moderate CD45 and high SSC; grey dots). In Plasmacytic Gate
the normal BM samples additional populations include blasts Neoplastic plasma cells usually are CD45– and have high forward
(moderate CD45 and low SSC), hematogones (moderate CD45, scatter (FSC) and low to slightly increased SSC (Figure 2.6).
very low SSC), and plasma cells (negative CD45 and low or Similar properties are typical for nucleated red cell precursors.
intermediate SSC). The majority of red blood cells and their Many cases of B lymphoblastic leukemias (B-ALL) and occa-
precursors are eliminated from FC analysis by lysis; the nucle- sional cases of acute myeloid leukemia (AML), blastic plasma-
ated red cell precursors are characterized by low SCC, nega- cytoid dendritic cell neoplasm (BPDCN), chronic lymphocytic
tive CD45, and bright CD71. Figures 2.2 through 2.8 present leukemia (CLL) or T-cell prolymphocytic leukemia (T-PLL) may
distributions of some of the neoplastic populations in regard to be located in this gate.
the gates defined by CD45 vs. SSC properties. Since malignant
populations do not always display typical antigen expression Metastatic Gate
or light scatter properties and do not fall into a specific gate, Extrinsic elements (e.g., small cell carcinoma) are CD45– and
FC analysis should also include evaluation of antigen expres- usually display high SSC (Figure 2.7). Erythroid precursors and
sion in open (ungated) mode (cluster analysis). For example, occasional plasma cell tumors may be located in this gate.
some myeloblasts display high SSC and dim or negative CD45,
and might be underestimated if CD34 and/or CD117 analysis
includes only cells with low SSC and moderate CD45. Based on Granulocytic Gate
antigen expression of ungated cells, the predefined gates can be Granulocytes/maturing myeloid cells are characterized by
modified (back-gating). moderate expression of CD45 and high SSC (Figure 2.8). Similar
SSC properties are represented by classic (hypergranular) acute
promyelocytic leukemia, mast cells, and occasional acute myeloid
Lymphocytic Gate leukemias.
Benign lymphocytes and the majority of mature (peripheral) After the data are acquired, each population is analyzed for
lymphoproliferative disorders including CLL/SLL, T-LGL specific markers (e.g., lymphocytes are analyzed for B-cell
leukemia, and follicular lymphoma (FL) have bright CD45 and clonality and T-cell antigen expression, cells with dimmer CD45
low SSC (Figure 2.2). expression and low SSC are analyzed for leukemic blasts, etc.).
Flow data analysis should always include ungated display as
well, so that the abnormal population with different than typical
Monocytic Gate CD45 versus SSC properties (see below) is not omitted.
Benign and neoplastic monocytes (including chronic myelo-
monocytic leukemia and acute monoblastic leukemia) have
bright CD45 expression, but SSC is higher than in lympho- flow cytometry of solid tissues and
cytes (Figure 2.3). A similar distribution is typical for hairy cell effusions-exclusion of nonviable cells
leukemia (HCL). Occasional cases of myelodysplastic syndrome Figure 2.9 illustrates gating strategy applied in the FC analysis
(MDS) with prominent dyspoiesis (dysgranulation) of granulo- of lymph node and other solid organs or effusions (the spleen
cytic cells display markedly decreased SSC with granulocytes is most often analyzed using BM/blood strategy). The nonvi-
located in “monocytic” gate. able cells are excluded from analysis by gating on “viability”

15
 flow cytometry in neoplastic hematology

Red cells are lysed and do not appear on

flow cytometry dot plots

Bone marrow aspirate

Granulocytes (grey dots) have dimmer

expression of CD45 (x-axis), than
lymphocytes and monocytes, but display
much higher side scatter. The latter
corresponds to cytoplasmic granularity

Monocytes (blue dots) have bright CD45

expression (x-axis) and mildly increased
side scatter (y-axis), when compared to
lymphocytes. Side scatter corresponds to
minimal cytoplasmic granularity of
monocytes.
Side scatter

Lymphocytes (red dots) are characterized

by bright CD45 expression (x-axis) and
agranular cytoplasm. Therefore they
display low side scatter (y-axis).

CD45

Plasms cells are Blasts have dim to

CD45 negative and moderate expression of
have low side CD45 (x-axis) and low to
scatter minimally increased side
scatter (y-axis)

Figure 2.1 Gating strategy. Part of the sample from the bone marrow aspirate or blood (tube) is smeared on the microscope glass slide for morphologic correlation,
while the rest is incubated with antibodies, lysed, fixed, and submitted for flow cytometry analysis; see text for details.

16
 gating strategies

“Lymphocytic” gate
Side scatter

Benign lymphocytes

B-cell lymphomas

T-cell lymphomas

T-large granular lymphocytes leukemia

CD45

Chronic lymphocytic leukemia Sezary’s syndrome

Side scatter

Side scatter

CD45 CD45

T-LGL leukemia Lymphoplasmacytic lymphoma

Side scatter

Side scatter

CD45 CD45
Figure 2.2 “Lymphocytic” gate. Benign lymphocytes and the majority of mature lymphoproliferative disorders are characterized by low side scatter (SSC) and bright
expression of CD45 (red dots; arrow).

17
 flow cytometry in neoplastic hematology

“Monocytic” gate

Side scatter Monocytes, monoblasts

Hairy cell leukemia
Occasional T/NK-LGL leukemias
Mast cell leukemia
Rare plasma cell neoplasms
Granulocytes in MDS
CD45

Acute monoblastic leukemia Myelodysplastic syndrome

Side scatter

Side scatter

CD45 CD45

Peripheral T-cell lymphoma, nos Chronic myelomonocytic leukemia

Side scatter

Side scatter

CD45 CD45

Hairy cell leukemia Acute myelomonocytic leukemia

Side scatter

Side scatter

CD45 CD45
Figure 2.3 “Monocytic” gate. Benign monocytes and majority of neoplastic monocytic proliferations are characterized by bright CD45 expression and slightly
increased side scatter (SSC). They appear above lymphocytic gate on CD45 versus SSC display (arrow). Apart from monocytic cells, similar SSC and CD45 properties
are often seen in hairy cell leukemia and occasional lymphoproliferations, mast cell leukemia (unusual and rare form of mast cell disease), and markedly dysplastic
granulocytes.

18
 gating strategies

(a’)

CD10
(a)
Side scatter

CD20

(a’’)

CD10
CD45

Hematogones gate
CD34
Hematogones
B-lymphoblastic leukemia
T-lymphoblastic leukemia
Occasional AML (non-M3)
Occasional B- and T-cell lymphoproliferative disorders

NK-LGL leukemia Chronic lymphocytic leukemia

(b) (c)
Side scatter

Side scatter

CD45 CD45

B-lymphoblastic leukemia T-lymphoblastic leukemia

(d) (e)
Side scatter

Side scatter

CD45 CD45
Figure 2.4 “Hematogones” gate (a). Hematogones are characterized by very low side scatter (SSC) and dimmer expression of CD45 when compared to lymphocytes
(red dots); they have variable (“smeared”) expression of CD20 (aʹ; broken arrows), are positive for CD10 and partially for CD34 (aʹʹ). Occasional lymphoproliferative
disorders and blasts may have similar to hematogones SSC and CD45 properties (b–e).

19
 flow cytometry in neoplastic hematology

“Blast” gate

Acute myeloid leukemia

Side scatter

Acute promyelocytic leukemia,

microgranular variant
Precursor B-cell leukemia/lymphoma
Plasmacytoid dendritic cell lymphoma/leukemia
Occasional B-cell lymphomas
Occasional plasma cell neoplasia
CD45

Acute myeloid leukemia Follicular lymphoma

Side scatter

Side scatter

CD45 CD45

Diffuse large B-cell lymphoma CML, Myeloid blast crisis

Side scatter

Side scatter

CD45 CD45

Plasma cell “leukemia” (blood) Microgranular APL (APLv)

Side scatter

Side scatter

CD45 CD45
Figure 2.5 “Blast” gate. Myeloid precursors (blasts) have moderate CD45 and low side scatter (SSC). Occasional lymphoproliferative disorders, plasma cell tumors,
as well as granulocytes with decreased granularity and microgranular APL have similar CD45 versus SSC characteristics.

20
Another random document with
no related content on Scribd:
"How do you come on, my dear?" she asked. "I'll hear you
now."

"Ma, my head aches so dreadfully."

She looked directly in his eyes.

"It does truly, ma; I began right earnest, Aunt Sarah will tell
you, but—"

There was something in her face which prevented him from

going further.

"Where does your last lesson commence, my dear?"

He reluctantly pointed out the page.

"Let me hear how well you can play it."

"O Frankie!" she exclaimed, after he had touched a few

chords. "What will your teacher say?"

"I couldn't play with such a bad headache, ma."

"Well, you may go to your room now, and lie down, while I
carry the yarn to Nancy."

Frank sprang from the stool with an eagerness which

surprised them, exclaiming,—

"Can't I go nutting, then?"

"Of course not. Take off your clothes and go to bed till I
return."
 CHAPTER IV.
FRANKIE'S NEW LESSON.

IN the afternoon, Mrs. Colvin went with her sewing to the

nursery, where Frankie was still in bed. Nurse had carried
some bread and milk to him there.

Tony was on the bed, too, and the boy was amusing himself
with hiding his pocket-handkerchief under the sheets and
telling her to find it.

"Is your head better?" asked his mother, seating herself

near him.

"It felt better right off, as soon as I stopped playing," he

said, earnestly.

"I was very sorry that you could not enjoy your favorite
pudding, my dear. Cook said she made it on purpose for
Master Frank."

"Did she have that nice, foaming sauce, ma?"

"Yes, it was very nice."

Frankie's face flushed, and then with a start, he covered his

head with the pillows to hide his tears. At last he said,—

"I think you might have given me some, ma."

She made no answer; and presently he recovered himself

and began his play with Tony again.
He hid the handkerchief here and there, now under the
pillows, and then beneath the tick, when he would say
authoritatively,—

"Go find it, Tony!" and she instantly obeyed.

"She is a very good dog," at last the lady remarked.

"Yes, ma, she minds me real quick. Sam Lambert has a

dog; and he tried to make the boys believe that Fox—that's
his name—was smarter than Tony."

"I saw him once. Sam had a stick and he threw it away and
told Fox to get it; but the lazy thing never stirred. He just
whisked his tail and stood still. I wouldn't have such a dog.
Now see, ma, how quick Tony minds me."

He hid the handkerchief again, for the twentieth time, and

then said, "Find it, Tony;" and she instantly darted here and
there, pulling the pillow up with her teeth.

"I see, Frankie; and I think I know a little boy who could
learn a good lesson from his dog."

"Learn from a dog, ma?"

"Yes, indeed! Don't you remember how Solomon, the wisest

man, told us to go to the ant, and learn industry and
perseverance? We can learn many lessons from animals, if
we notice their particular traits."

Frank looked very thoughtful for a few moments, and then

said, frankly,—

"I'm sorry, ma, I didn't obey you better. I acted just like
Sam Lambert's Fox. I didn't think of that before."
Mrs. Colvin leaned over the bed, and kissed her boy.

What do you suppose she was thinking of? She was

thanking God, who had put it into Frankie's heart to confess
his sin.

"Why, ma," the boy went on, "if Tony had acted that way,
when I told her to do something,—I mean if she had fooled
away her time,—I should have got a stick and whipped her.
Why didn't you whip me, ma?"

The lady smiled, as she said, "I preferred to punish you by

sending you here, and giving you time to reflect; and I
rejoice that you have learned so good a lesson from your
pet. Obedience, prompt, cheerful obedience, is the best
foundation for a good character. You see what a nuisance
Tony would become if she did not obey you; and there is no
sight more dreadful to good people, than an unruly,
disobedient child. Such a one is sure to grow up
disregardful of the laws of God and man, and very often
ends his days in a prison, or on the gallows."

"But now I want to talk with you about your music. You can
remember how very anxious you were to learn, and how
many promises you made to practise regularly. Your father
is very fond of music, and cheerfully paid the twenty-five
dollars, which Mr. Lenox asks for teaching you."

"But, ma," urged the boy, "I didn't know how it would make
my fingers ache. If I could play nice marches like Etty
Bowles, I'd like it first-rate."

"Etty began when you did, Frankie; and I dare say her
fingers ached till they became accustomed to the motion. If
you persevere, you will soon be able to play marches. Every
lesson you learn thoroughly is a good step in advance. You
know how easy your first lessons have become."
"Oh, yes, ma! I can diddle 'em off tip-top." He laughed
merrily, as he began to practise with his finger on Tony's
back. Presently, he said,—

"Ma, if you'll let me get up, I'll play steady at it. I'll try to
obey you just as nice as Tony does me. Wont I, doggy?"

"I'd like to see you doing that," replied ma.

Frankie lost no time in tumbling out of bed, followed by

Tony, who seemed, by her joyful bark, to think that his
young master had got into the right track again.

I wish you could have seen the boy as he splashed the

water over his face and head. There was a look of resolution
on his countenance which his mother was very much
pleased to see.

"Come, ma! Come, Tony! Now you'll hear some playing!"

And down he ran, happier than he had been for many a day.

When he was in the right humor, it only took one minute to

find his place, arrange the stool, and seat the dog in a chair
near him.

"It looks awful hard, ma," he said; "but I'll do it." So he

began, slowly at first, picking out the chords of one line and
then repeating the strains until it became familiar. Then he
went on to the next, until he was himself surprised at his
success.

"Bravo! Bravo! My dear child," exclaimed his loving father.

"I doubt whether I could play better myself."

Frankie jumped. "Why, pa, I didn't know you were here.

Stop a minute, please, and hear how nicely I have learned
my whole lesson."
"How long do you think you have been practising?" asked
his mother, kissing him.

"I don't know, ma; I never once thought of the time."

"One hour and twenty minutes. You have done well, my

dear child."

"And my fingers don't ache at all. They only feel stiff a little.
Now, ma, I feel as if I could dance, I'm so very glad. But
wont Mr. Lenox be surprised, though? He'll say, 'Frankie
Colvin, take your place on the stool;' and he'll frown and be
all ready to scold when he finds I haven't practised the last
lesson. Oh, it will be fun to see him!"

"Why, Frank," said his father; "you dance and hop about for
all the world like Tony."

"I'm so happy, pa. This dear little doggy has given me one
good lesson, and I love her better than ever," he added,
hugging the faithful creature.

"It is a lesson," said his mother, gravely, "which I hope you

will remember as long as you may live. Prompt, cheerful
obedience, in the performance of duty, makes old and
young happy. Now, my darling, you shall take a walk with
me; and then it will be time for tea."

CHAPTER V.
THE STOLEN DOG.
ONE day, Frankie was going an errand for his mother; and
Tony, of course, was following closely at his heels, when he
heard the sound of a hand-organ down a lane, and he ran
to find it.

The organ-grinder had a box with dolls dressed in lace,

dancing and bowing in the funniest manner. There was a
crowd of boys around him, who were laughing merrily at
the novel sight. Frankie laughed, too. Indeed, I don't think
one of the boys enjoyed it more than he did.

By and by, he thought of his errand, and was frightened

when he found he had lost the package of thread for which
his mother had sent him to the store. He looked on the
ground near the place where he had been standing; but it
was nowhere to be seen, neither could it be found.

Then he looked behind him for Tony; but she, too, was
missing. He called, loudly, "Tony! Tony!" But there was no
answer back.

"I saw a dog running off that way," said a boy, pointing in
the direction of Frankie's home. "I guess that was your
dog."

Frankie looked in the boy's face, and, child though he was,

he knew it was not an honest one; then he started off for
home, resolved to tell his mother the whole story. The fright
about Tony had made him very warm; and he put his hand
in his pocket to find his handkerchief to wipe the
perspiration from his forehead. But to his surprise, his
handkerchief had followed Tony and the package.

He ran now as fast as he could go, looking in every direction

for the lost dog, and calling, "Tony! Tony! Good doggy!"
When he reached home, his mother was standing at the
door, and she wondered to see him so flushed, and that the
dog was not with him.

"O ma!" he began. "Have you seen Tony, and my

handkerchief? and your thread is lost, too!"

Half crying, he told his mother what had occurred, at the

same time running from room to room in search, for the
dog.

Mrs. Colvin was soon convinced that Tony had been stolen.
She told Frankie to sit down and cool himself; and then she
sent Edward to a neighbor, who was a constable, to ask him
what they should do.

Unfortunately the man was not at home; and they were

obliged to wait until night, when Captain Colvin returned
from the city.

Poor Frank cried until his head ached, and could not eat a
mouthful of dinner. He kept saying, "Oh, I wish I hadn't
gone down the lane! I'm afraid they'll kill Tony, or starve
him to death."

Captain Colvin accompanied the constable to the lane,

where there were a number of shabby tenement houses;
but nobody had seen the dog, or knew anything about her.
At a late hour, he returned home, and found Frankie had
cried himself to sleep.

The next morning, advertisements were put up at all the

principal stores, at the post-office, and the depot; but that
day and the next passed without any news of poor Tony.

"I know she's dead," said poor Frankie, who now looked
really ill. "I know I shall never see her again."
A fresh burst of grief prevented him from saying more. He
laid his arm on the table and cried as if his heart would
break.

His mother tried to soothe him, when suddenly she heard a

feeble whine at the gate.

Frankie heard it, too, and ran quickly across the room to
open time door. There stood poor Tony, with the lost
handkerchief between her teeth. She had grown so thin that
she could hardly stand, but tried to crawl forward to her
young master's feet.

With a scream of joy, Frankie lay down on the floor by her

side and, patting her, covered her with kisses.

Mrs. Colvin rang the bell, and sent nurse for some food for
the poor, starved creature.

"We must feed her very cautiously at first," she said, as her
boy began to cut the meat from the bones. "Let her eat but
a little now, and give her more at the end of an hour."

"I wish Tony could tell where she has been. I'd get the
constable to put the people in prison, for treating her so.
Look, ma! Here is the rope they tied her with. I do believe
the good creature gnawed it off, and ran away!"

Tony seemed to understand what they were saying, for she

tried to bark; but she had not strength, and, instead, made
such a mournful sound, that Frankie's tears began to flow
again. He brought a soft cushion for her to lie upon, and
soon had the pleasure of seeing her enjoying a refreshing
nap!

Toward the close of the day, cook came into the parlor and
told her mistress that there were two boys at the end of the
garden, and she had overheard them talking about the dog.

Mrs. Colvin at once sent the hired man to bring them to the
house. But the boys saw him coming and ran away,
laughing, and shouting loudly, "Catch me if you can."

The man said he knew the boys, and that they were none
too good to steal a dog.

When Mr. Colvin came home, he thought it was best to let

the constable know that he had found out the names of the
thieves. When he reached the gentleman's house, he found
out that the two boys had already been detected of crime,
having only an hour before been arrested for stealing goods
from a store, and were now on their way to the county jail.

When Frankie heard this, he lavished more caresses on his

pet, and told her she was safe now.

CHAPTER VI.
TONY'S LOVE FOR HER MASTER.

I AM sure the little boys and girls who read this, will be glad
to know that good care, nourishing food, and Frankie's
caresses, at last restored Tony; though it was a long time
before she could run and jump as she had before. It was
quite affecting to see her try to spring after Frankie's ball
when he bid her bring it to him. She would seem to forget
for a moment how feeble she was, and then, quite
exhausted, she would lie down puffing and panting for
breath, but keeping her eyes on her master as if to say, "I
would obey you, if I could."

Then Frank would blame himself for telling her to do it; and
he would ask her pardon, and kiss her over and over again.
Oh, they were very good friends, indeed!

Frankie's school was about a quarter of a mile from the

house. Before Tony was stolen, she used to go with him
until they were in sight of the school-house, and then
scamper back as fast as she could go; but now Tony was
not allowed beyond the gates.

The faithful creature seemed to know when it was time for

Frank to come home. The first day she could crawl down the
yard, she stationed herself where she could see far down
the road, and at the first glimpse of his figure, began to
bark out her welcome.

One day, for whispering, he was kept after school was

dismissed. Tony watched and watched, and, after Eddy
came home, showed great impatience. She even stole
through the gate, and walked cautiously toward the school-
house.

At last Frankie came in sight, not running and jumping as

usual; but walking slowly, with his handkerchief at his eyes.

Tony was sure, now, that something was the matter. She
sprang up, licked his hand, and tried, in every way that a
dog could try, to show her sympathy.

Frankie was a proud boy; that is, he would not like anybody
to know that he had been punished, and was crying for it;
but he didn't mind telling his troubles to Tony.
"It was real mean of the master," he began, "to ferule me
for just saying one word. Sam Lambert asked me to lend
him my new ball; and I said it was at home. Master
punished me; but Sam got nothing but praise."

Tony had the poor, swollen hand now, and was kissing it
with all her might.

"Well," Frankie went on, "after all, I had rather have a good
whipping every day, than to lie as Sam does. The master
will find him out some time; and if he don't, God sees him.
I'd be afraid ever to go to sleep, if I was such a liar."

They were now nearly home, and Frankie concluded to wet

his handkerchief at the pump and wipe his eyes before he
presented himself in the parlor.

Tony stood by, looking very wishful; and then they went in
together. During all the intermission, Tony did not leave her
master a minute; but watched him closely, every now and
then standing on her hind feet to lick his hand.

When the boy started to go to school, she held the skirt of

his coat in her mouth as if she were afraid he would be hurt
again.

"Frankie Colvin, you may walk up to the desk," called out

the master, when all the boys had taken their seats.

Poor Frankie began to tremble, but instantly obeyed.

"Samuel Lambert, take your place by his side."

"Now," continued the teacher, "I wish you, Samuel, to tell

me exactly the words Frank Colvin whispered to you this
morning."
"I can't remember exactly, sir." Sam's face was as red as
fire.

"You may tell, Frank."

"He said, 'Will you let me take your new ball at recess?' and
I told him that I had left it at home."

"Yes, those were the words. I can believe you, Frank, for I
have never known you to tell a lie. You may take your seat."

"Now, Samuel, let me tell you, that I heard you boasting to

the boys that you got off from a whipping by a nice little
white lie as you called it. There are no white lies. Every lie
is, in God's sight, black; and he has told us how he will
punish liars who do not repent and forsake their sins. You
broke the rule of the school by whispering to your
companion, and thus tempting him to answer; and you
have done what was far worse, and broken God's holy law.
You denied that you had whispered, and bore false witness
against your neighbor. How mean for you to sit by and see
him punished for an offence which you had led him to
commit! I am distressed that I have such a scholar, and
shall be compelled to punish you as you deserve."

He then took a ruler from his desk, and punished Samuel on

both hands,—the boy yelling with all his might, while poor
Frankie laid his head on the desk before him and cried.

When Sam returned to his seat, he raised the cover of his

desk to screen his burning cheeks. He was very, very angry;
and, if he had dared, would have been glad to knock
Frankie down for daring to tell the truth against him.

It was very hard for the good boy to study his lesson, while
his seat-mate was muttering so angrily behind the cover;
but, remembering his victory over himself with his music
lesson, he bent his whole mind to the task, and soon
learned all the map questions in his geography lesson.

The class was called to recite directly after recess; and

Frankie was standing in front of the teacher, when he heard
a familiar bark, and suddenly Tony bounded into the room
and rushed up to him.

Then there was a knock at the door, and presently Colonel

Jameson and Mrs. Colvin walked in and took seats on the
platform.

You can easily imagine how glad Frankie was that he had
learned his lesson so perfectly. He stood very erect, his eyes
sparkling, his cheeks rosy-red, and was ready with every
answer, the moment the question was asked.

"Your class appears uncommonly well," remarked the

visitor. "I am glad to see that a little friend of mine is so
studious."

"Bow! Wow! Wow!" barked Tony. "So am I, — so am I."

Mrs. Colvin smiled and was very happy. Nothing makes

mothers so happy as to have their children do well.

CHAPTER VII.
CONCLUSION.
WHEN the classes had all recited, the visitors took their
leave, Mrs. Colvin asking permission for Frankie to
accompany them home, as their friend would remain only
one night.

So they all left together, Frankie turning, as he went out, to

make a bow to his master. Tony thinking, I suppose, it was
the rule of the school, turned round also, and made such a
funny little motion with her head as set the whole school
laughing.

I need not tell you that our little friend felt very happy.
Instead of walking along steadily with his mother and the
colonel, he and Tony had a chase, here and there, every
minute or two returning to the side of their friends.

Before they were half-way home, he determined to tell his

mother all that had occurred at school, ending with, "It's
bad enough to be whipped for whispering, ma; but if I had
been talked to before the school for lying, I should have felt
so mean, I never could have shown my face there again."

"And that is not the worst of it, my dear son," said the lady;
"the exposure before the school was mortifying, to be sure;
but that is nothing compared to the displeasure of our
heavenly Father. There is no sin which appears to good
people more mean and despicable than lying, and there is
none which God abhors more."

"Well, mother," said Frankie, "I'm resolved I'll never, never

tell another lie. You know I haven't told one for a long
time."

"May God help you to keep so good a resolution!" said

Colonel Jameson, very solemnly, laying his hand on the
boy's head. "Be truthful and obedient, and you will not only
have the friendship of all good people; but God, your
heavenly Father, will reward you with his favor."

Tony, at this, sprang into Frankie's arms, and laid her head
on his breast.

Colonel Jameson smiled as he saw the little boy lay his

cheek against Tony's nose. He loved Frankie for the kind
care he took of the dog. After a while he asked,—

"Frankie, can you guess what brought me out here to day?"

"No, sir."

"I came to get permission for you and Tony to go with me to

the sea-shore. There is nice bathing there."

"Oh! Oh!" screamed the boy. "I'm so glad. May I go, ma?"

"Yes, dear. I have consented, on condition that you never go

near the water, unless in company with Colonel Jameson."

"I never will, ma. I'll do everything he says. Oh, wont I

have a good time! Say, Tony;" and he hugged the dog
tightly in his arms. "But when are we going, Colonel
Jameson?"

"On Monday morning. I shall come from the city in an early

train; and you will meet me at the depot. Your mother has
promised to fit you up in a bathing-suit, and I will provide
fish-poles."

Frankie sprang up the floor, clapping his shouting with

delight,—

"Thank you, ma, for letting me go. I mean to be a real good

boy after this."
 Col. Jameson, Frankie, and Tony going to fish.

It was now Friday; but immediately after the Colonel left,

the young boy insisted that it would be safer to pack his
clothes and have everything in readiness for a start. He ran
to the attic to bring a small valise, and asked his father's
permission to take it.

An hour later, his mother entered his chamber and found

the floor covered with caps, boots, shoes, books, balls, etc.,
etc.

"Why, what is this, Frankie?" she asked.

"I can't get all my things in, ma," said the boy, in a
desponding tone.

"You will only want one change, my dear, besides your

bathing clothes, and that, with some clean collars, a comb
and brush, and plenty of dry socks, will be all that you will
need."

"Would you carry Tony's new suit, ma? I wish I could; she
does look so funny in it."

"I think there'll be room in the valise."

"How kind you are, ma!" Frankie jumped up and gave his
mother a warm kiss.

Monday morning came at last; and the little fellow,

determined to be in season, had privately engaged cook to
wake him as soon as it was light. To his mother's great
surprise, he walked into her chamber, just as the clock was
striking five, all dressed for his journey.

It was still two hours before the train would leave the city;
and Mrs. Colvin tried to persuade him to return to bed; but
his father laughed and said,—

"Let him stay up if he wishes."

At eight o'clock, he met Colonel Jameson and started off in

high glee. At the end of a week, he walked into the sitting-
room with Tony at his heels. His face was as brown as a
nut; but he exclaimed, as he kissed his mother,—"I've been
a good boy, ma; Colonel Jameson told me to tell you so."
 *** END OF THE PROJECT GUTENBERG EBOOK FRANKIE'S
DOG TONY ***

Updated editions will replace the previous one—the old editions will
be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States copyright in
these works, so the Foundation (and you!) can copy and distribute it
in the United States without permission and without paying copyright
royalties. Special rules, set forth in the General Terms of Use part of
this license, apply to copying and distributing Project Gutenberg™
electronic works to protect the PROJECT GUTENBERG™ concept
and trademark. Project Gutenberg is a registered trademark, and
may not be used if you charge for an eBook, except by following the
terms of the trademark license, including paying royalties for use of
the Project Gutenberg trademark. If you do not charge anything for
copies of this eBook, complying with the trademark license is very
easy. You may use this eBook for nearly any purpose such as
creation of derivative works, reports, performances and research.
Project Gutenberg eBooks may be modified and printed and given
away—you may do practically ANYTHING in the United States with
eBooks not protected by U.S. copyright law. Redistribution is subject
to the trademark license, especially commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE