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Flow Cytometry of Hematological Malignancies
To my wife, Angela, and to my sons Stefano, Paola, and Marzia
Flow Cytometry
of Hematological
Malignancies
Second Edition

CLAUDIO ORTOLANI md
Adjunct Professor of Laboratory of Diagnostic Cytometry
Urbino University
Urbino, Italy;

Consultant Clinical Pathologist (retired)

Ospedale dell’Angelo
Venice, Italy
This edition first published 2021
© 2021 John Wiley & Sons Ltd
Edition History
Blackwell Publishing Ltd (1e, 2011)

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Library of Congress Cataloging‐in‐Publication Data

Names: Ortolani, C. (Claudio), author.
Title: Flow cytometry of hematological malignancies / Claudio Ortolani.
Description: Second edition. | Hoboken, NJ : Wiley-Blackwell, 2021. |
Includes bibliographical references and index. | Description based on
print version record and CIP data provided by publisher; resource not
viewed.
Identifiers: LCCN 2020028333 (print) | LCCN 2020028334 (ebook) | ISBN
9781119611301 (epub) | ISBN 9781119611271 (Adobe PDF) | ISBN 9781119611257
(cloth)
Subjects: | MESH: Hematologic Neoplasms–diagnosis | Flow Cytometry–methods
Classification: LCC RC280.H47 (ebook) | LCC RC280.H47 (print) | NLM WH 525 |
DDC 616.99/418–dc23
LC record available at https://lccn.loc.gov/2020028333
LC record available at https://lccn.loc.gov/2020028334

Cover Design: Wiley

Cover Image: courtesy of Claudio Ortolani

Set in 9.5/12pt Minion by SPi Global, Pondicherry, India

10 9 8 7 6 5 4 3 2 1
 Contents

Foreword to the Second Edition

by Michael J. Borowitz xi
Foreword to the First Edition
by Maryalice Stetler-Stevenson xii
Foreword to the First Edition
by Bruno Brando xiii
Preface to the Second Edition xv
Preface to the First Edition xvi
Abbreviationsxvii

1 Antigens 1

Clustered (CD) Antigens

CD13
CD25
CD38
CD417
CD521
CD724
CD826
CD1030
CD11b35
CD11c38
CD1340
CD1444
CD1546
CD1649
CD1952
CD2055
CD2259
CD2361
CD2464
CD2566
CD2667

v
Contents

CD2769
CD2870
CD3071
CD3373
CD3477
CD3879
CD4381
CD4582
CD45 Isoforms 87
CD4990
CD5693
CD5796
CD6197
CD62L98
CD6499
CD65101
CD66c102
CD71103
CD79104
CD81107
CD103108
CD117110
CD123112
CD138113
CD200114
CD305116
CD307 (IRTA) Antigen Family 117
CD371118
Non clustered (or primarily known with other names) antigens
Bcl‐2 Protein 119
Chemokines and Chemokine Receptors 121
CRLF2128
Cytotoxic Proteins 129
HLA‐DR130
Immunoglobulins132
KIR, CD158 isoforms 136
Myeloperoxidase (MPO) 139
NG2140
PCA‐1141
ROR1141
SLAM Molecules and SLAM‐associated Protein (SAP) 142
SOX11144
T‐cell Receptor (TCR) 145
Terminal Deoxy‐nucleotidyl‐transferase (TdT) 148
Toll‐like Receptors (TLR) 150
VS38151
ZAP‐70152

2 Diseases 155

Myeloproliferative neoplasms 157

Chronic myeloid leukemia (CML) 157

vi
 Contents

Myeloproliferative neoplasms other than CML 160

Chronic neutrophilic leukemia (CNL) 160
Polycythemia vera (PV) 160
Primary myelofibrosis (PMF) 160
Essential thrombocythemia (ET) 160
Chronic eosinophilic leukemia (CEL) 161
Mastocytosis162
Acute mast‐cell leukemia (AMCL) 162
Chronic mast‐cell leukemia (CMCL) 163
Myelomastocytic leukemia (MML) 163
Myelodysplastic/myeloproliferative neoplasms 164
Chronic myelomonocytic leukemia (CMML) 164
Other myelodysplastic/myeloproliferative neoplasms and related conditions 167
Juvenile myelomonocytic leukemia (JMML) 167
Atypical CML bcr/abl negative (ACML) 167
RAS‐associated autoimmune leukoproliferative disorder (RALD) 167
Myelodysplastic syndromes 168
Myeloid neoplasms with germline predisposition 171
Acute myeloid leukemias 172
AMLs with recurrent genetic anomalies 173
AMLs with chromosomal anomalies 173
AMLs with gene mutations 180
Relationships between genotype and phenotype in cases of AML not recognized as separate entities in WHO 2017 181
AMLs with myelodysplasia‐related changes (AML‐MRC) 182
AMLs not otherwise specified 182
AML with minimal differentiation 182
AML without maturation 183
AML with maturation 183
Acute myelomonocytic leukemia (AMMoL) 183
Acute monoblastic and monocytic leukemia (AMoL) 184
Pure erythroid leukemia (PEL) 185
Acute megakaryoblastic leukemia (AMKL) 186
Acute basophilic leukemia (ABL) 188
Myeloid proliferations associated with Down syndrome 188
Transient abnormal myelopoiesis (TAM) 189
AMLs in patients with Down syndrome 189
Blastic plasmacytoid dendritic cell neoplasm (BPDCN/PDCL) 189
Acute leukemias with ambiguous lineage attribution (ALAL) 192
Acute undifferentiated leukemias (AUL) 192
Mixed phenotype acute leukemias (MPAL) 192
Neoplastic diseases of B and T lymphatic precursors 194
B lymphoblastic leukemia/lymphoma, not otherwise specified (B‐ALL/LBLnos) 195
B lymphoblastic leukemia/lymphoma with recurrent genetic anomalies 197
Relationships between genotype and phenotype in cases of B‐ALL not recognized as separate entities in WHO 2017 201
T lymphoblastic leukemia/lymphoma (T‐ALL/LBL) 202
Early T‐cell precursor lymphoblastic leukemia (ETP‐ALL) 205
NK lymphoblastic leukemia/lymphoma (NK‐ALL/LBL) 205
Neoplastic diseases of mature B cells 206
Chronic lymphocytic leukemia/small
lymphocytic lymphoma (B‐CLL/SLL) 206
Familial B‐CLL 215
Richter syndrome 215
Monoclonal B‐cell lymphocytosis (MBL) 216

vii
Contents

CLL‐like monoclonal B lymphocytosis 216

Non‐CLL‐like monoclonal B lymphocytosis 216
B‐cell prolymphocytic leukemia (B‐PLL) 216
Lymphoplasmacytic lymphoma (LPL) 218
Heavy chain disease (HCD) 221
γ heavy chain disease 222
μ heavy chain disease 222
α heavy chain disease 222
Hairy cell leukemia (HCL) 222
Hairy cell leukemia, variant (HCL‐v) 226
Hairy cell leukemia, Japanese variant (HCL‐J) 227
Splenic diffuse red pulp lymphoma (SDRPL) 227
Marginal zone lymphomas (MZL) 228
Nodal marginal zone lymphoma (NMZL) 229
Splenic marginal zone lymphoma (SMZL) 230
Extranodal marginal zone lymphoma (EMZL/MALToma) 232
Clonal B‐cell lymphocytosis with MZL‐like phenotype (CBL‐MZ) 233
Follicular lymphoma (FCL) 234
Testicular follicular lymphoma 237
Duodenal type follicular lymphoma 237
Pediatric type follicular lymphoma 237
Primitive cutaneous follicular lymphoma (PCFL) 237
Large B‐cell lymphoma with IRF4 rearrangement 237
Mantle‐cell lymphoma (MCL) 237
Blastic mantle‐cell lymphoma (BMCL) 240
Leukemic non nodal mantle‐cell lymphoma 240
DLBCL not otherwise specified (DLBCLnos) 240
CD5(+) diffuse large cell lymphoma (CD5(+) DLBCL) 243
T‐cell/histiocyte‐rich B‐cell lymphoma (THRLBCL) 243
Primary DLBCL of the CNS (PCNSL) 244
Primary cutaneous DLBCL, “leg type” 244
EBV(+) DLBCLnos 244
DLBCL associated with chronic inflammation (PAL) 245
Fibrin associated DLBCL 245
Lymphomatoid granulomatosis (LYG) 245
Primary mediastinal B‐cell lymphoma (PMBCL) 245
Intravascular large B‐cell lymphoma (IVBCL) 246
ALK‐positive large cell lymphoma (ALK(+) LBCL) 246
Plasmablastic lymphoma (PBL) 247
Primary effusion lymphoma (PEL) 247
HHV8‐associated lymphoproliferative disorders 247
HHV8‐positive DLBCL 248
HHV8‐positive germinotropic lymphoproliferative disorder 248
Burkitt lymphoma (BL) 248
Burkitt leukemia with immature phenotype 250
Burkitt‐like lymphoma with 11q aberrations 251
High‐grade B‐cell lymphoma (HGBL) 251
Plasma cell neoplasms 251
Monoclonal gammopathies of undetermined significance (MGUS) 253
Multiple myeloma (MM) 253
Plasma cell leukemia (PCL) 257

viii
 Contents

Neoplastic diseases of mature T and NK cells 258

T‐cell prolymphocytic leukemia (T‐PLL) 258
T‐cell large granular lymphocytic leukemia (T‐LGL) 261
Chronic lymphoproliferative disorders of NK cells (CLPD‐NK/CNKL) 263
Aggressive NK‐cell leukemia (ANKL) 266
Adult T‐cell leukemia/lymphoma (ATLL) 266
Extranodal NK/T-cell lymphoma, “nasal type” (ENKTL) 269
Intestinal T‐cell lymphomas (ITCL) 270
Enteropathy‐associated T‐cell lymphoma (EATCL) 270
Monomorphic epitheliotropic intestinal T‐cell lymphoma (MEITL) 272
Indolent gastro‐intestinal T lymphoproliferative disorder (indolent GI T‐LPD) 273
Hepatosplenic T‐cell lymphoma (HSTCL) 273
Subcutaneous panniculitis‐like T‐cell lymphoma (SPTCL) 275
Mycosis fungoides (MF) 275
Sézary syndrome (SS) 277
Primary cutaneous CD30(+) lymphoproliferative disorders 279
Lymphomatoid papulosis (LyP) 279
Primary cutaneous anaplastic T‐cell lymphoma (pcALCL) 279
Primary cutaneous peripheral T‐cell lymphoma (PTCL) 280
Primary cutaneous TCRγδ(+) T‐cell lymphoma (PCGD‐TCL) 280
Primary cutaneous CD8(+) aggressive epidermotropic cytotoxic T‐cell lymphoma (PCAETL) 280
Primary cutaneous acral CD8(+) T‐cell lymphoma (PCATCL) 280
Primary cutaneous lymphoma of the medium/small CD4(+) T cells (PCSM‐TCL) 281
Peripheral T‐cell lymphoma, not otherwise specified (PTCLnos) 281
Nodal lymphomas of follicular T‐helper derivation 283
Angioimmunoblastic T‐cell lymphoma (AITL) 283
Follicular T‐cell lymphoma (FTCL) 285
Nodal PTCL with follicular T‐helper phenotype 285
Anaplastic large cell lymphoma ALK(+) (ALCL ALK(+)) 285
Anaplastic large cell lymphoma ALK(‐) (ALCL ALK(‐)) 288
Breast implant–associated anaplastic large cell lymphoma (biaALCL) 288
Hodgkin lymphomas 289
Classic Hodgkin lymphoma (CHL) 289
Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) 290
Neoplastic diseases of histiocytic and dendritic cells 291
Histiocytic sarcoma (HS) 292
Langerhans cell histiocytosis (LCH) 292
Indeterminate dendritic cell tumor (IDCT) 292
Interdigitating dendritic cell sarcoma (IDCS) 292
Follicular dendritic cell sarcoma (FDCS) 292
Erdheim–Chester disease (EDC) 292

3 Appendix 293

Acute leukemias not recognized by the 2017 WHO ­classification 294

Acute leukemia of myeloid/NK precursors (M/NK‐AL) 294
Acute leukemia of myeloid dendritic cells (MDCL) 294
Acute leukemia of Langerhans cells 294
Composite lymphomas 294
Hypereosinophilic syndrome (HES), lymphocyte variant 295

ix
Contents

Indolent T lymphoblastic proliferations (iT‐LBP) 295

Polyclonal lymphocytoses of B lymphocytes 298
Persistent polyclonal B‐cell lymphocytosis (PPBL) 298
Persistent polyclonal CD5(+) B‐cell lymphocytosis 298
Persistent polyclonal B‐cell lymphocytosis, Japanese (hairy) variant 298
Polyclonal plasmacytoses 299
Small round (blue) cell tumors (SR(B)CT) 300
References301
Index429

x
 Foreword to the Second Edition
It has been about a decade since Dr. Claudio Ortolani gave us the
first edition of Flow Cytometry in Hematologic Malignancies, and
in that time flow cytometry has solidified its place as a routine
diagnostic test in the diagnosis and classification of leukemias
and lymphomas. Over the decade, dozens of new markers have
been discovered, many of which are now a standard part of the
flow cytometry arsenal. It is thus timely that Dr. Ortolani has cho-
sen to update his work with this new second edition. As before,
this work is extensively researched and prodigiously referenced,
with more than 1300 new references not included in the first edi-
tion, bringing the total to more than 4500 relevant citations.
These references not only cover the new markers, but also include
new information about common markers in use for years, and
descriptions of the markers covered by the first edition have been
expanded to discuss these new advances.
In compiling this book, Dr. Ortolani has maintained the
unique structure of the first edition, with the first part of the
book oriented around individual antigens, and the second
around diseases, supplementing the comprehensive text with
characteristic images to illustrate the key points raised. The first
section includes detailed information about normal tissue dis-
tribution of antigens, and also includes a discussion of some key
pitfalls in the interpretation of cytometric findings of many
antigens, before discussing pathologic conditions in which the
antigens may be found. The disease section has been extensively
revised and now follows the 2016 version of the WHO classifi-
cation, while also including some newer entities not mentioned
in the WHO monograph. In keeping with the changes in classi-
fication, this book now includes a much more extensive discus-
sion of phenotypic properties of the different types of T‐cell
lymphomas and histiocytic neoplasms than was available
previously.
This book will be a valuable resource for anyone interested in
flow cytometry and hematologic malignancies. Trainees can eas-
ily find detailed phenotypic information about any disease they
are learning about, while even an expert, struggling with an unu-
sual case with a phenotype that appears contradictory to an
expected diagnosis, can quickly learn whether others have found
similar patterns in that diagnosis; moreover, because the cita-
tions are so extensive, it is possible simply by considering the
numbers of references to a phenotype to assess the weight of evi-
dence for a particular interpretation. Those of us in the field are
grateful that Dr. Ortolani took the time to update his work and
produce a reference that will be useful for many years to come.
Michael J. Borowitz, MD, PhD
Professor of Pathology and Oncology
Director of Hematopathology and Flow Cytometry
Johns Hopkins Medical Institutions
Baltimore, MD, USA
xi
 Foreword to the First Edition
Flow cytometry is a crucial tool in the diagnosis of hematolym-
phoid neoplasms, determining prognosis and monitoring
response to therapy. Clinical flow cytometric immunophenotyp-
ing, however, is a complex field requiring extensive expertise in
normal and abnormal patterns before clinical tests can be appro-
priately interpreted. Those new to the field are left with the
conundrum of how best to achieve this expertise. At particular
disadvantage is the resident or clinical fellow seeking to interpret
flow cytometric data on a specific patient. The typical flow cytom-
etry reference text is written in an encyclopedic format with
extensive narrative that is not conducive to looking up the mean-
ing of unusual test results. Furthermore, the general flow cytom-
etry textbook, although a useful reference, cannot completely
cover all the aspects needed to interpret clinical flow cytometry
data. Therefore, Flow Cytometry of Hematological Malignancies
fills a much needed role in hematopathology and hematology/
oncology. The presentation is oriented toward the diagnostic
laboratory in the academic center as well as in the general
­ plasms, with a particular interest in lymphoproliferative dis-
hospital.
Flow Cytometry of Hematological Malignancies is organized
in a novel manner that makes it especially useful for the medical
student and residents/fellows still in training, while still provid-
ing a valuable resource for hematopathologists, hematologists/­
oncologists and experts in the field of clinical flow cytometry.
It lists antigens typically studied in clinical flow cytometry
­laboratories, from CD1 to CD138, followed by a discussion of
general as well as flow cytometric features and hematolymphoid
neoplasms expressing each antigen. Thus, when interpreting a
clinical flow cytometry report, one can easily research an unu-
sual antigen expressed by a leukemia or lymphoma. This pattern
xii
of organization makes more sense than only presenting lists of
neoplastic processes and the expected flow cytometric findings.
One has to first know the diagnosis on a particular patient before
such a reference can be useful. Flow Cytometry of Hematological
Malignancies also provides the usual description of typical flow
cytometric immunophenotypical findings in the various hema-
tolymphoid neoplasms. This is useful as a reference for panel
design as well as diagnosis.
Flow Cytometry of Hematological Malignancies is being pub-
lished at a time when the field is expanding rapidly and flow
cytometry is assuming an even greater role in management of
patients with hematolymphoid neoplasia. Dr Ortolani, an out-
standing flow cytometrist, possesses extensive expertise in the
clinical arena. For over 30 years he was employed in the Clinical
Pathology Department of the Venice General Hospital, running
one of the first diagnostic flow cytometry units in Italy. His
main clinical activity was the diagnosis of hematological neo-
eases. Dr Ortolani has also been very active in teaching flow
cytometry in many national and international courses. He has
written what I believe to be an outstanding textbook covering
the essential aspects of clinical flow cytometry. Dr Ortolani is
to be commended for this brilliant contribution that is sure to
become a well-used textbook in clinical centers around the
world.
Maryalice Stetler-Stevenson, PhD, MD
Chief, Flow Cytometry Laboratory
National Cancer Institute, National Institutes of Health
Bethesda, MD, USA
 Foreword to the First Edition
The European Society for Clinical Cell Analysis (ESCCA) is
proud to present this volume by Dr Claudio Ortolani. Flow
Cytometry of Hematological Malignancies is a benchtop
­companion for all who are involved in the complex process of
characterization and diagnosis of leukemias and lymphomas by
immunophenotypical techniques and flow cytometry.
This volume is a useful quick reference text for the matching
of CD antigens with malignant hematological diseases, as defined
by the WHO 2008 classification, taking into account antibody
clones, features and behavior, with particular emphasis on variant
forms and unexpected presentations.
After several decades of clinical and laboratory practice in
this field, Dr Claudio Ortolani has meticulously prepared this
book under the auspices of the ESCCA. It represents a major
achievement for the dissemination of knowledge in one of the
most important specialties within clinical cell analysis, as the
book aims to improve and standardize the diagnostic process of
malignant blood diseases. As a result, communication between
clinicians and laboratory operators should benefit!
Bruno Brando
ESCCA President
Director, Hematology Laboratory and Transfusion Center,
Legnano Hospital, Milan, Italy
xiii
 Preface to the Second Edition

Ten years have passed since the first edition of this book, 10 fruit-
ful years full of new acquisitions in the field of Hematology. Many
doubts have been clarified, new questions have been raised,
and – above all – new therapeutic targets have been reached.
Although molecular biology keeps assuming ever greater impor-
tance, especially in the field of acute leukemias and myeloprolif-
erative neoplasms; nevertheless, flow cytometry still remains one
of the fundamental pillars of hematopathology and becomes even
more and more important in particular areas such as the evalua-
tion of the minimal residual disease and the study of the pharma-
codynamic characteristics of experimental therapies with
monoclonal antibodies.
In these 10 years, technology has also made substantial pro-
gress, and today we have much more efficient tools than before,
but also much more complex ones, and consequently the
pre‐analytical and analytical components have become consid-
erably complicated. Even if an attempt to account for these
problems has been made, in‐depth treatment of most of them is
beyond the scope of this book; they will be dealt with –
hopefully – in the next edition, together with the many other
topics neglected this time.
Claudio Ortolani MD
Adjunct Professor of Laboratory of Diagnostic Cytometry
Urbino University
Urbino, Italy
Consultant Clinical Pathologist (retired)
Ospedale dell’Angelo
Venice, Italy

xv
 Preface to the First Edition
The cytometric analysis of hematological malignancies is one of
the most difficult applications of flow cytometry, requiring both a
good knowledge of hematopathology and good control of the
technique. Moreover, the effort of operators is made harder by the
continuous evolution of the technology and by the continuous
progress in the comprehension of the nature of the diseases.
This book was compiled from a series of notes originally
intended for people practically involved in the field of diagnos-
tic flow cytometry, and it is an example of what the author would
have liked to consult at the beginning of his own career. The
goal of this book is to offer the reader a quick and updated
source of information on the phenotype of the hematological
malignancies recognized by the last WHO classification, with
the major exception of Hodgkin lymphoma which because of its
peculiar nature is still beyond the limits of flow cytometry, even
if things promise to change in the next few years.
xvi
The author may have unwittingly sown a number of mis-
takes and imprecisions, and he will be grateful to all colleagues
who report these to him. He also realizes that this book could
not have been written without the help of many friends and
colleagues. Being unable to cite all of them, the author wants to
particularly thank his friend Bruno Brando, current President
of the European Society for Clinical Cell Analysis, for the
­continuous moral and practical support he has given over
the years.
Claudio Ortolani MD
Former Director of the Flow Cytometry Unit
Clinical Pathology Department
Venice General Hospital
 Abbreviations

3‐FAL 3‐fucosyl‐N‐acetyllactosamine BALF bronchoalveolar lavage fluid

ABC antibody‐binding capacity B‐ALL B‐cell acute lymphoblastic leukemia
ABC age/autoimmune‐associated B cell BCL B‐cell lymphoma (gene)
ABC‐like activated B‐cell like B‐CLL B‐cell chronic lymphocytic leukemia
a‐B‐CLL Atypical B‐cell chronic lymphatic leukemia B‐CLL/LPL B‐CLL in lymphoplasmacytoid transformation
ABL acute basophilic leukemia B‐CLL/PL B‐CLL in prolymphocytoid transformation
acPGP N‐acetyl Proline–Glycine–Proline B-CLPD B-cell chronic lymphoproliferative disease
aCML atypical chronic myeloid leukemia BCR B‐cell receptor
ADA adenosine deaminase BFU‐E burst‐forming units/erythroid
ADP adenosine diphosphate biaALCL breast implant–associated anaplastic large cell
AITL angioimmunoblastic T‐cell lymphoma lymphoma
aka also known as BIM bcl‐2‐interacting mediator of cell death
ALAL acute leukemia of ambiguous lineage BL Burkitt lymphoma
ALALnos acute leukemia of ambiguous lineage not B‐LBL B-cell lymphoblastic lymphoma
otherwise specified BM bone marrow
ALCL anaplastic large cell lymphoma B‐NHL B‐cell non‐Hodgkin lymphoma
ALDH aldehyde dehydrogenase BPDCN blastic plasmacytoid dendritic cell neoplasm
ALK anaplastic lymphoma kinase B‐PLL B‐cell prolymphocytic leukemia
ALL acute lymphoblastic leukemia B‐SLL B‐cell small lymphocytic lymphoma
ALPS autoimmune lymphoproliferative syndrome BVMCL blastic variant mantle‐cell lymphoma
AMCL acute mast cell leukemia C9RP complement 9 related protein
AMKL acute megakaryoblastic leukemia CA I carbonic anhydrase isoenzyme I
AML acute myeloid leukemia CBCL cutaneous B‐cell lymphoma
AMLL acute mixed lineage leukemia CBL‐MZ clonal B‐cell lymphocytosis of marginal zone
AML‐MRC AML with myelodysplasia‐related changes origin
AMM agnogenic myeloid metaplasia CCND1 cyclin D1
AMoL acute monoblastic and monocytic leukemia CEA carcinoembryonic antigen
ANKL aggressive NK‐cell leukemia CEL chronic eosinophilic leukemia
ANXA‐1 Annexin‐1 CEL nos chronic eosinophilic leukemia not otherwise
APC antigen‐presenting cell specified
APL acute promyelocytic leukemia CFU colony‐forming unit
ASM aggressive systemic mastocytosis CFU‐EO colony‐forming unit/eosinophil
ATLL adult T‐cell leukemia/lymphoma CFU‐G colony‐forming unit/granulocyte
atMSC adipose‐tissue‐derived mesenchymal stromal CFU‐GEMM colony‐forming unit/granulocyte, erythrocyte,
cells monocyte, megakaryocyte
ATRA all‐trans retinoic acid CFU‐GM colony‐forming unit granulocyte‐macrophage
AUL acute undifferentiated leukemia CFU‐M colony‐forming unit/macrophage
AZT azidothymidine (zidovudine) CGH comparative genomic hybridization

xvii
Abbreviations

CHL classic Hodgkin lymphoma FCL follicular lymphoma

CINCA chronic infantile neurological cutaneous FCM flow cytometry
articular syndrome FcRH Fc receptor homologues
CLA cutaneous lymphocyte antigen FCRL Fc receptor–like proteins
CLPD chronic lymphoproliferative disease FDC follicular dendritic cell
CLPD‐NK chronic lymphoproliferative disorders of NK FDCS follicular dendritic cell sarcoma
cells FISH fluorescence in situ hybridization
CM cutaneous mastocytosis FITC fluorescein isothiocyanate
CMCL chronic mast cell leukemia FL follicular lymphoma
CML chronic myeloid leukemia FLT3 fms‐related tyrosine kinase 3 (CD135)
CML‐BC CML in blastic crisis FLT3‐ID FLT3 internal tandem duplication
CMML chronic myelomonocytic leukemia FOXP FOX (forkhead box) protein
CMPD chronic myeloproliferative disease FSC forward scatter
CMPN chronic myeloproliferative neoplasm FTCL follicular T‐cell lymphoma
CMV cytomegalovirus FTH follicular helper T cells
CNKL chronic NK cell lymphocytosis FUT fucosyl‐transferase
CNL chronic neutrophilic leukemia GCB‐like germinal center B‐cell like
CNS central nervous system GCC germinal center cell
CPLD chronic lymphoproliferative disease G‐CSF granulocyte‐colony stimulating factor
CRLF2 cytokine receptor‐like factor 2 GEP gene expression profile
CRS chemokine receptor status GLPD germinotropic lymphoproliferative disorder
CSF cerebrospinal fluid GM‐CSF granulocyte‐macrophage colony‐stimulating
CSR class switch recombination factor
CTCL cutaneous T‐cell lymphoma HABP4 hyaluronan binding protein 4
CY5.5 cyanine 5.5 HAL hybrid acute leukemia
CY7 cyanine 7 HBME‐1 human bone marrow endothelium marker‐1
cyμ cytoplasmic μ heavy chain HBLD hairy B‐cell lymphoproliferative disorder
DFS disease‐free survival HCD heavy chain disease
DH‐JH diversity and joining segments of the Ig heavy HCL hairy cell leukemia
chain gene HCL‐J hairy cell leukemia, Japanese variant
DHL double‐hit B‐cell lymphoma HCL‐v hairy cell leukemia, variant
DLBCL diffuse large B‐cell lymphoma HCV hepatitis C virus
DLBCLnos diffuse large B‐cell lymphoma not otherwise HE hematoxylin–eosin
specified HES hypereosinophilic syndrome
DLBCL‐SS sanctuary site DLBCL HEV high endothelial venules
DPP IV dipeptidyl peptidase‐IV HGAL protein human germinal center–associated lymphoma
DRESS DRug‐induced Eosinophilia with Systemic protein
Symptoms HGBL high‐grade B‐cell lymphoma
EATCL enteropathy‐associated T‐cell lymphoma HGL high‐grade lymphoma
EBER Epstein–Barr virus‐encoded RNA HLH hemophagocytic lymphohistiocytosis
EBV Epstein–Barr virus HN hematodermic neoplasm
ECD Erdheim–Chester disease HPC hemopoietic precursor cell
EFS event‐free survival HRS Hodgkin and Reed–Sternberg
EGIL European Group for the Immunological HS histiocytic sarcoma
Characterization of Leukemias HSTCL hepatosplenic T‐cell lymphoma
EMA epithelial membrane antigen HUMARA human androgen receptor
EML extramedullary myeloid leukemia ICOS inducible T‐cell COStimulator (CD278)
EMZL extranodal marginal zone lymphoma IDCS interdigitating dendritic cell sarcoma
ENKTL extranodal NK/T‐cell lymphoma IDCT indeterminate dendritic cell tumor
EPC endothelial progenitor cells IEL intraepithelial intestinal lymphocyte
ET essential thrombocythemia Ig immunoglobulin
ETP early T‐cell precursor i‐GIT‐T‐LPD indolent T‐cell lymphoproliferative disorder of
FAB French–American–British the gastrointestinal tract
FCCL follicular cell cutaneous lymphoma IHC immunohistochemistry

xviii
 Abbreviations

IL interleukin MAIT mucosal‐associated invariant T cell

iNK immature NK cells MALD1 monoclonal asymptomatic lymphocytosis, cyclin
IPSID immune proliferative small intestinal disease D1–positive
IPTCLB intralymphatic proliferation of T‐cell lymphoid MALT mucosa‐associated lymphoid tissue
blasts MALToma lymphoma of the mucosa‐associated lymphoid
IRF4 interferon regulatory factor 4 protein, tissue
aka mum1 MAMP microbe‐associated molecular patterns
IRTA immune receptor translocation‐associated MATK megakaryocyte‐associated tyrosine kinase
proteins MBC myeloid blastic crisis
ISM indolent systemic mastocytosis MBCL monocytoid B‐cell lymphoma
ITAM immunoreceptor tyrosine‐based activation MBL monoclonal B‐cell lymphocytosis
motif MCC Merkel cell carcinoma
ITCL intestinal T‐cell lymphoma MCCHL mixed cellularity classic Hodgkin lymphoma
ITIM immunoreceptor tyrosine‐based inhibition MCD multicentric Castleman disease
motif MCL mantle‐cell lymphoma
iT‐LBP indolent T‐lymphoblastic proliferation MCL‐BV mantle‐cell lymphoma – blastic variant
IT‐LPD indolent T‐cell lymphoproliferative disorder MDC myeloid dendritic cell
ITSM immunoreceptor tyrosine‐based switch motif MDCL myeloid dendritic cell leukemia
IVL intravascular lymphoma MDS myelodysplastic syndrome
IVLBCL intravascular large B‐cell lymphoma MDSC myeloid‐derived suppressor cells
IWCLL International Workshop on Chronic MDS‐U myelodysplastic syndrome, unclassified
Lymphocytic Leukemia MEITL monomorphic epitheliotropic intestinal T‐cell
JAM junctional adhesion molecule lymphoma
JMML juvenile myelomonocytic leukemia MEP megakaryocyte/erythroid progenitor
KIR killer cell immunoglobulin‐like receptor MESF molecules of equivalent soluble fluorochrome
LAIP leukemia‐associated immune phenotype MF mycosis fungoides
LANA latency‐associated nuclear antigen MFI mean fluorescence intensity
LBC lymphoid blastic crisis MGUS monoclonal gammopathy of undefined
LBCL large B‐cell lymphoma significance
LBP lipopolysaccharide‐binding protein MHC major histocompatibility complex
LCA leukocyte common antigen MLBCL mediastinal large B‐cell lymphoma
LCH Langerhans cell histiocytosis MLC mixed leukocyte culture
LDBCL large diffuse B‐cell lymphoma MLP multiple lymphomatous polyposis
LDCHL lymphocyte‐depleted classic Hodgkin MM multiple myeloma
lymphoma MML myelomastocytic leukemia
L/DCS Langerhans/dendritic cell sarcoma MMoL myelomonocytic leukemia
LDH lactate dehydrogenase MNDA myeloid cell nuclear differentiation antigen
LEF1 lymphoid enhancer‐binding factor 1 M/NK‐AL acute leukemia of myeloid/NK precursors
L&H cells lymphocytic & histiocytic Reed–Sternberg MoAb monoclonal antibody
variant cells MoAg monocytic antigen
LHL lymphoepithelioid lymphoma MPAL mixed phenotype acute leukemia
LinAg lineage antigen MPDMN mature PDC proliferations associated with
LIR leukocyte Ig‐like receptors myeloid neoplasms
LMO2 LIM domain only 2 (rhombotin‐like 1) MPN myeloproliferative neoplasm
LP cells lymphocyte predominant cells MPO myeloperoxidase
LPL lymphoplasmacytic lymphoma MRD minimal residual disease
LPS lipopolysaccharide MS myeloid sarcoma
LRCHL lymphocyte‐rich classic Hodgkin lymphoma mTOR mammalian target of rapamycin
LRP lung resistance protein MUM1 multiple myeloma 1 protein, aka IRF4
LSC leukemic stem cell MVL microvillous lymphoma
LyAg lymphoid antigen MyAg myeloid antigen
LYG lymphomatoid granulomatosis MZL marginal zone lymphoma
LyGa lymphomatoid gastropathy NBS Nijmegen breakage syndrome
LyP lymphomatoid papulosis NCA non‐specific cross‐reacting antigen

xix
Abbreviations

NCAM neural cell adhesion molecule PPBL persistent polyclonal B‐cell lymphocytosis
NEC nucleated erythroid cells pPNET peripheral primitive neuroectodermic tumors
NGC next‐generation cytometry PPO platelet peroxidase
NK natural killer PRCA pure red cell aplasia
NKCE NK-cell enteropathy PTCL peripheral T‐cell lymphoma
NKP NK‐cell precursors PTCLnos peripheral T lymphoma not otherwise specified
NKR NK‐cell receptors PTFL pediatric‐type follicular lymphoma
NLPHL nodular lymphocyte predominant Hodgkin PTLD post‐transplant lymphoproliferative disease
lymphoma PV polycythemia vera
NMZL nodal marginal zone lymphoma RA refractory anemia
NPM nucleophosmin RAEB refractory anemia with excess of blasts
NRBC nucleated red blood cell RALD RAS‐associated autoimmune leukoproliferative
NSCHL nodular sclerosis classic Hodgkin lymphoma disorder
OS overall survival RARS refractory anemia with ring sideroblasts
PAL pyothorax‐associated lymphoma RCMD refractory cytopenia with multilineage dysplasia
PB peripheral blood RCUD refractory cytopenia with unilineage dysplasia
PBL plasmablastic lymphoma RER rough endoplasmic reticulum
PCATCL primary cutaneous acral T‐cell lymphoma RN refractory neutropenia
PCAETL primary cutaneous aggressive epidermotropic ROR1 receptor‐tyrosine‐kinase‐like orphan receptor 1
cytotoxic T‐cell lymphoma RRV rhesus rhadinovirus
pcALCL primary cutaneous anaplastic large cell RT refractory thrombocytopenia
lymphoma RTE recent thymic emigrants
PCBCL‐LT primary cutaneous diffuse large B‐cell lym- sALCL systemic anaplastic large cell lymphoma
phoma “leg type” SAP SLAM‐associated protein
PC‐DLBCL primary cutaneous diffuse large B‐cell SCF stem cell factor
lymphoma SCLC small cell lung cancer
PC‐FCL primary cutaneous follicle center lymphoma SDRPL splenic diffuse red pulp lymphoma
PC‐ENKTL primary cutaneous extranodal NK/T‐cell SFTS severe fever with thrombocytopenia syndrome
lymphoma SLAM signaling lymphocytic activation molecules
PCGD‐TCL primary cutaneous TCRγδ(+) T‐cell lymphoma SLF steel factor
PCH pseudo Chediak–Higashi SLL small lymphocytic lymphoma
PCL plasma cell leukemia SLVL splenic lymphoma with villous lymphocytes
PCMZL primary cutaneous marginal zone lymphoma (obsolete)
PCNSL primary CNS lymphoma SM systemic mastocytosis
PcP peridinin‐chlorophyll‐protein SM‐AHNMD systemic mastocytosis with an associated clonal
PCSM‐TCL primary cutaneous lymphoma of medium/small hematological non‐mast cell disorder
CD4(+) T lymphocytes SMZL splenic marginal zone lymphoma
PD‐1 programmed death‐1 SPTCL subcutaneous panniculitis‐like T‐cell lymphoma
PDC plasmacytoid dendritic cell SR(B)CT small‐round (blue) cell tumor
PDCL plasmacytoid dendritic cell leukemia SRCT small‐round‐cell tumor
PE phycoerythrin SS Sézary syndrome
PEL primary effusion lymphoma SSC side scatter
PEL pure erythroid leukemia SSEA stage‐specific embryonic antigen
PFS progression‐free survival T‐ALL T‐cell acute lymphoblastic leukemia
PHA phytohemagglutinin TAM transient abnormal myelopoiesis
PID primary immunodeficiency disease TCL1 T‐cell leukemia/lymphoma 1
PLEVA pityriasis lichenoides et varioliformis acuta T‐CLL T‐cell chronic lymphocytic leukemia
PMA phorbol myristate acetate T-CLPD T-cell chronic lymphoproliferative disease
PMBC peripheral mononuclear blood cells TCM T central memory
PMBCL primary mediastinal (thymic) large B‐cell TCR T‐cell receptor
lymphoma TCRAV T‐cell receptor A variable (region)
PMF primary myelofibrosis TCRBCL T‐cell‐rich large B‐cell lymphoma
PNET primitive neuroectodermic tumors TCRBV T‐cell receptor B variable (region)
PNH paroxysmal nocturnal hemoglobinuria TdT terminal deoxy‐nucleotidyl transferase

xx
 Abbreviations

TEM T effector memory TRAF TNF receptor‐associated factor

TFR therapy‐free remission TSCM T stem cell memory
THL true histiocytic lymphoma (obsolete) TSLP stromal thymic lymphopoietin
THRLBCL T‐cell/histiocyte‐rich B‐cell lymphoma TTE T terminal effector
TKI tyrosine kinase inhibitor TTM T transitional memory
TKR tyrosine kinase receptor TTT time to treatment
T‐LBL T‐cell lymphoblastic lymphoma TXR Texas Red
T‐LGL T‐cell large granular lymphocytic leukemia TZL T zone lymphoma
TLR Toll‐like receptor VDJ variable, diversity, joining segments of the Ig
TMD transient myeloproliferative disorder heavy chain gene
tMF mycosis fungoides in transformation VH variable segment of the Ig heavy chain gene
TMPD transient myeloproliferative disorder VLA very late activation
TN T naïve vWF von Willebrand factor
TNF tumor necrosis factor WBC white blood cells
TNFAIP2 TNF alpha induced protein 2 WD‐EMT well‐differentiated extramedullary myeloid
T‐NHL T‐cell non‐Hodgkin lymphoma tumor
T‐PLL T‐cell prolymphocytic leukemia WHO World Health Organization
TPO thrombopoietin ZAP‐70 zeta‐chain‐associated protein‐70
TPO‐R thrombopoietin receptor

xxi
 1 Antigens

Clustered (CD) Antigens CD48, see SLAM molecules

CD1, 3 CD49, 90
CD2, 5 CD56, 93
CD3, 8 CD57, 96
CD4, 17 CD61, 97
CD5, 21 CD62L, 98
CD7, 24 CD64, 99
CD8, 26 CD65, 101
CD10, 30 CD66c, 102
CD11b, 35 CD71, 103
CD11c, 38 CD79, 104
CD13, 40 CD81, 107
CD14, 44 CD84, see SLAM molecules
CD15, 46 CD103, 108
CD16, 49 CD117, 110
CD19, 52 CD123, 112
CD20, 55 CD138, 113
CD22, 59 CD150, see SLAM molecules,
CD23, 61 CD158 isoforms, see KIRs
CD24, 64 CD181–186, CD191–199, see Chemokines and
CD25, 66 Chemokine Receptors
CD26, 67 CD200, 114
CD27, 69 CD229, see SLAM molecules
CD28, 70 CD244, see SLAM molecules
CD30, 71 CD280–290, see Toll‐like Receptors
CD33, 73 CD305, 116
CD34, 77 CD307 (IRTA) Antigen Family, 117
CD38, 79 CD319, see SLAM molecules
CD43, 81 CD352–353, see SLAM molecules
CD45, 82 CD371, 118
CD45 Isoforms, 87

Flow Cytometry of Hematological Malignancies, Second Edition. Claudio Ortolani.

© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.

1
Antigens

Non clustered (or primarily known with other names) NG2, 140
antigens PCA‐1, 141
Bcl‐2 Protein, 119 ROR‐1, 141
Chemokines and Chemokine Receptors, 121 SLAM Molecules and SLAM‐associated Protein (SAP), 142
CRLF2, 128 SOX11, 144
Cytotoxic Proteins, 129 T‐cell Receptor (TCR), 145
HLA‐DR, 130 Terminal Deoxy‐nucleotidyl‐transferase (TdT), 148
Immunoglobulins, 132 Toll‐like Receptors (TLR), 150
KIR, CD158 isoforms, 136 VS38, 151
Myeloperoxidase (MPO), 139 ZAP‐70, 152

2
 CD1 Antigens

newborns [4], and in the peripheral blood of subjects submitted

General features
to autologous or allogeneic bone marrow transplantation during
the first year following transplant [8]
CD1 antigens are a group of five different glycoproteins that
• CD1d has been demonstrated on the membrane of bone mar-
weigh 43–49 kD and are encoded by a group of genes situated on
row B precursors [10], on B lymphocytes in peripheral
the long arm of chromosome 1 [1].
blood [11,12], and on a subset of B lymphocytes in the mantle of
CD1 antigens play a role in the presentation of lipidic and
the germinal center [11] and in the spleen [13].
glycolipidic antigens to T and NKT cells [1,2], and can be
As for the APC, CD1a, CD1b, and CD1c antigens have been
divided in three groups, namely:
commonly reported on dendritic cells [6]. Moreover:
• Group I, encompassing CD1a, CD1b, and CD1c
• CD1a has been demonstrated on Langerhans cells [14], where it
• Group II, encompassing CD1d
is expressed at an intensity of 1600 molecules per cell [15], on
• Group III, encompassing CD1e, which is an intracytoplasmic
some CD11b(+) CD14(+) mononuclear cells reported in the
protein.
peripheral blood of burnt subjects and interpreted as Langerhans
CD1 antigens are mainly expressed on cells belonging to T
cell precursors migrating from bone marrow to epidermis [16],
and B lineages and on antigen‐presenting cells (APC).
on monocytes activated with granulocyte‐macrophage colony‐
As for the T lineage, CD1a, CD1b, and CD1c antigens have
stimulating factor (GM‐CSF) in vitro [17], and on in vitro mono-
been demonstrated on the membrane of the cortical or “com-
cyte‐derived dendritic cells [18]
mon” thymocytes [3] and on the membrane of some T‐lympho-
• CD1b has been demonstrated on monocytes activated with
cyte subsets in cord and neonatal peripheral blood [4]; according
GM‐CSF in vitro [17] and on a subset of Langerhans cells [19]
to some authors, a low expression of CD1 antigens can be dem-
• CD1c has been demonstrated on monocytes activated with
onstrated in the cytoplasm of T lymphocytes activated in vitro
GM‐CSF in vitro, on Langerhans cells [19], and on a minor subset
by phytohemagglutinin (PHA) [5]. CD1d has been reported at a
of myeloid dendritic cells (MDC) characterized by CD11c(++)
high density on common thymocytes, and at a lower density on
CD123(±) phenotype [20]
medullary thymocytes [6].
• CD1d has been demonstrated on “resting” monocytes [11], on
As for the B lineage, both CD1c and CD1d have been demon-
dendritic cells of the dermis [21], on dendritic cells in peripheral
strated on the precursors and on some subsets of mature B lym-
blood [6], and on in vitro monocyte‐derived dendritic cells [21].
phocytes. More precisely:
CD1d has also been demonstrated in keratinocytes of psoriatic
• CD1c has been demonstrated on some subsets of B lympho-
skin [22], in the cells of human scalp hair follicles [22], in the epithe-
cytes in the peripheral blood [7,8], in the spleen [7,8], and in the
lial cells of the gut and other organs [23], and in adipocytes [24].
mantle of the germinal center [7]
CD1a, CD1b, CD1c, and CD1d have been demonstrated in
• CD1c(+) B lymphocytes account for the majority of B cells in
the “foam cells” of the atherosclerotic plaque [25].
tonsils [9], in cord blood [4], in the peripheral blood of

Flow Cytometry of Hematological Malignancies, Second Edition. Claudio Ortolani.

© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.

3
Antigens

The expression of CD1d has been reported on the cells of the

Cytometric features
juvenile myelomonocytic leukemia (JMML) [32].
The cytometric demonstration of molecules belonging to the
CD1 antigens in neoplastic diseases of B‐cell precursors
CD1 family should be performed taking the following points into
In a group of 80 patients affected by childhood B‐cell acute
account:
lymphoblastic leukemia (B‐ALL), the expression of CD1d has
• cytometric studies have demonstrated that activated T lympho-
been demonstrated in 15% of the cases [10]. CD1d expression is
cytes express CD1c on the membrane only when kept at room
significantly associated with pre‐B phenotype, rearrangement of
temperature, and fail to mount the molecule on the surface when
the gene KMT2A (formerly known as MLL), and shorter global
kept at +4 or to +37°C [26]
survival [10].
• the expression of CD1a on the surface of the leukemic blasts can
fluctuate spontaneously after a short period of incubation in vitro [27].
CD1 antigens in neoplastic diseases of T‐cell precursors
The antibodies specific for CD1 antigens do not behave in the
In T‐cell acute lymphoblastic leukemia (T‐ALL) the expression
same way.
of CD1a antigen has been detected in a percentage of cases rang-
It should be kept in mind that CD1a features four different
ing from 40% to 50% of cases [27,4565]. CD1a antigen is gener-
epitopes, the first of which is recognized by clones D47, Na1/34,
ally expressed on the cells of T lymphoblastic leukemia/
and L119, the second by clone L404, and the third by clone
lymphoma (T‐ALL/LBL) related to the stage of cortical or “com-
L504 [28]; it should be noted that CD1a on the cells of B‐cell
mon” thymocyte [3,27] (Fig. 1.1). According to the European
chronic lymphocytic leukemia (B‐CLL) can be demonstrated
Group for the Immunological Characterization of Leukemias
only with clones other than OKT6 or Na1/34 [29].
(EGIL) classification of T‐ALL, CD1a antigen is typically present
The clones 7C4 and IOT6b recognize different epitopes of
in the T III form but missing in the T I, T II, and T IV forms [34].
CD1b [30].
If CD13 is negative, the expression of CD1a is related to good
survival [35], while the expression of CD1a together with CD10
is associated with the presence of the t(5;14)(q35;q32)
Diagnostic features
translocation [33,4594,4595].
CD1 in myelodysplastic and chronic myeloproliferative
CD1 antigens in acute myeloid leukemias
diseases
The expression of CD1a and of CD1d has been repeatedly
CD1a can be detected by immunohistochemistry (IHC) in the
reported on the surface of the blasts of the acute myeloid leuke-
neoplastic cells of the cutaneous localization of chronic myelo-
mias (AML) [27,32,33]. According to some authors, the expres-
monocytic leukemia (CMML); a case is known displaying
sion of CD1a and CD1d is restricted to French–American–British
S‐100(−), CD1a(+), CD4(+), CD56(+), CD123(−), and Langerin/
(FAB) subtypes characterized by a monocytic component [32]. In
CD207(−) phenotype, mimicking the phenotype of the indeter-
a percentage of cases, the expression of CD1a can be accompanied
minate dendritic cell tumor (IDCT) [31].
by the presence of other histiocytic markers (S‐100, CD163,
CD1a, CD1b, and CD1c are expressed on the membrane of
Langerin/CD207) and is interpreted as a sign of histiocytic
the blast cells in the 20% of cases of chronic myeloid leukemia in
differentiation [36].
blastic crisis (CML‐BC) [27].

SSC (A) (B) CD8 (C)

CD1a

CD45 CD3 CD4

Figure 1.1 Peripheral blood from a subject affected by T‐ALL. The blasts (red) express phenotype CD45(dim+) (A), CD1a(+) (B), CD3(+) (heterogeneous) (B), CD4(+) (C),
CD8(+) (partial) (D).

4

Table 1.1 Differential diagnosis of CD5(−) CD10(−) B‐CLPDs
B‐CLPD CD1d CD200
HCL Positive Positive
LPL Dimly positive Positive
MZL Positive Negative
B‐CLPD: B chronic lymphoproliferative disease; HCL: hairy cell leukemia;
LPL: lymphoplasmacytic lymphoma; MZL: marginal zone lymphoma.
CD1 antigens in neoplastic diseases of mature B cells
It has been reported that B‐cell prolymphocytic leukemia (B‐PLL)
cells express CD1c [9,37], that Burkitt lymphoma (BL) cells do
not express CD1c [7], and that hairy cell leukemia (HCL) cells
express CD1a [38] and CD1c [9]. The cells of B‐CLL have been
reported to express CD1a [9,29], CD1c [7], and CD1d; it is note-
worthy that CD1a on B‐CLL cells could be demonstrated only
with clones other than OKT6 or Na1/34 [29].
CD1d is usually expressed by the cells of the B‐CLPDs, but at
an intensity depending on the type of disease. Its intensity in
B‐CLL is typically lower than in normal B cells, but it is pro-
gressively higher on MCL, HCL‐variant (HCL‐v), lymphoplas-
macytic lymphoma (LPL), splenic marginal zone lymphoma
(SMZL), and HCL cells [12]. The combined exploration of
CD1d and CD200 seems very promising in the differential
diagnosis of CD5(−) CD10(−) B‐CLPDs as well, inasmuch as a
recent study has shown that HCL is CD1d(+) CD200(+), LPL is
CD1d(−/±) CD200(+), and MZL is CD1d(+) CD200(−) [39]
(Table 1.1).
CD1d antigen seems of particular interest in B‐CLL workup,
because its intensity of expression and the percentage of
CD19(+) CD1d(+) cells are bad prognostic predictors [40,41];
according to some authors [42,43], but not to others [40], CD1d
intensity seems to correlate with the absence of somatic
hypermutations [43].
Multiple myeloma (MM) cells express CD1d in the early
stages but tend to reduce its expression with disease
progression [44].
CD2 Antigen
General features
CD2 is a 45–58 kD glycoprotein belonging to the superfamily of
the immunoglobulins, which is encoded by a gene situated on the
short arm of chromosome 1. CD2 is an adhesion molecule,
CD2 Antigen
CD1 antigens in neoplastic diseases of mature T
and natural killer (NK) cells
The expression of CD1a antigens has been reported in rare cases
of peripheral T‐cell lymphoma (PTCL) [45] and in an isolated
case of adult T‐cell leukemia/lymphoma (ATLL) [46].
Overexpression of CD1d mRNA has been detected in Sezary’s
cells by RT‐PCR [47], but this report has not yet been confirmed
by phenotypic studies.
CD1 antigens in neoplasms of histiocytes and dendritic cells
As for the neoplasms of histiocytic and dendritic cells, CD1a,
CD1b, and CD1c have been demonstrated with immunohisto-
chemical techniques in the Langerhans cell histiocytosis
(LCH) [19,48,49]. One of the most typical features of the pulmo-
nary involvement in LCH is the occurrence of more than 5% of
CD1(+) cells in the bronchoalveolar lavage fluid (BALF) [50].
Cells with CD1a(+), CD207(+), CD11b(±), and CD11c(++) phe-
notype have been detected in the peripheral blood of patients with
active LCH [51]. CD1a expression has been reported in an anecdo-
tal case interpreted as acute leukemia of Langerhans cell precur-
sors based on the presence of Birbeck granules and of the ability of
blasts to develop dendritic processes when cultured in vitro [52].
CD1a has been demonstrated with immunohistochemical
techniques in the IDCT [53], but neither in the follicular den-
dritic cell sarcoma (FDCS) nor in the interdigitating dendritic
cell sarcoma (IDCS) [49].
Partial expression of CD1a and expression of CD1c have been
reported on the cells of an exceptional case of leukemia classified
as Langerhans cell/dendritic cell leukemia, which occurred in a
patient suffering from myelodysplastic syndrome (MDS). This
case displayed CD11c(+), CD123(−), CD141(+), CD303(−),
CD304(±/−), and CD207/Langerin(+/−) phenotype [54].
CD1 antigens in other pathological conditions
CD1a has been reported on the cells of the so called “indolent T‐
lymphoblastic proliferation” (iT‐LBP) [55], an entity not recognized
by the 2017 WHO classification (see also the dedicated paragraph).
constitutes the ligand of the CD58 molecule [56] and interacts
with CD48 and CD59 molecules as well [57].
CD2 is normally expressed on thymocytes, on whose mem-
brane it begins to appear at the prothymocyte level [58], and on
mature T lymphocytes [59].
5
Antigens
Not all mature T lymphocytes co‐express CD2; in the periph-
eral blood small subsets of T lymphocytes exist that show
CD3(+) CD2(−) phenotype and are characterized by the expres-
sion of T‐cell receptor (TCR) either with αβ [60] or γδ
chains [61].
The expression of CD2 is not restricted to the T lineage.
Indeed, it is well known that CD2 is expressed:
• on 70–90% of the NK cells negative for CD3 [62,63], where it is
upregulated by activation [64]
• on a minority of follicular dendritic cells (FDC) [65]
• on a subset of mononuclear peripheral cells interpreted as pre-
cursors of MDCs [66]
• on a subset of peripheral monocytes characterized by the co‐
expression of Fcε receptor (FcεRI) [67]
• on a subset of plasmacytoid dendritic cells (PDCs) [68]
• on a small subset of B cells in fetal liver [69], in fetal bone mar-
row [69], in thymus [70], in peripheral blood [71], and in the
bone marrow of normal subjects [71].
Cytometric features
The staining of peripheral normal lymphocytes with an anti‐CD2
monoclonal antibody (MoAb) generates a positive histogram
with a narrow gaussian‐like peak, clearly separated from the nega-
tive component, with a channel peak representing the presence of
24 ± 7 E03 ABC (antibody‐binding capacity) [72].
Bimodal histograms can often be seen, especially when
immune system activation is ongoing, because a higher number
of CD2 molecules is expressed on activated cells [73] (Fig. 1.2).
In these cases, the CD2(bright+) population tends to show
higher values of forward and side scatter than the CD2(dim+)
population.
SSC
(A)
CD2
Figure 1.2 The histogram produced by the cytometric analysis of CD2 is bimodal (A), because the activated CD25(+) lymphocytes (red) express more CD2 molecules than
CD25(−) lymphocytes (blue) (B).
6
CD2(bright+) cells nearly exclusively express CD45R0, while the
CD2(dim+) population display either CD45R0 or CD45RA [74];
the staining of CD2(+) CD45RA(+) cells with an anti‐CD2 MoAb
generates a histogram with a channel peak representing the pres-
ence of 21 ± 4 E03 ABC while the staining of CD2(+) CD45R0(+)
lymphocytes with the same MoAb generates a histogram with a
channel peak representing the presence of 55 ± 9 E03 ABC [72].
In accordance with the state of chronic activation caused by
HIV infection, the lymphocytes of HIV‐infected subjects seem
to express a higher amount of CD2 molecules [75], whereas a
reduced expression has been documented on the lymphocytes
of elderly subjects [76]. According to some authors, the expres-
sion of CD2 on NK cells is inhomogeneous, being more intense
in the CD16 dim CD56 bright subset than in the CD16 bright
CD56 dim subset [77].
Not all the anti‐CD2 MoAbs behave in the same way; some
clones are able to inhibit the E‐rosette formation [78], while
others are able to activate T lymphocytes in vitro [79].
Diagnostic features
CD2 in myelodysplastic and chronic myeloproliferative
diseases
CD2 has been reported in a third of cases of CMML [80].
CD2 in neoplastic diseases of mast cells
CD2 has been reported together with CD22 and CD25 on the
neoplastic mast cells in systemic mastocytosis (SM) and acute
mast cell leukemia (AMCL) [81–83]. However, an aberrant CD2
(and CD25) expression can be found on mast cells in the bone
marrow of subjects without evidence of neoplasms of mast or
myeloid stem cell [84].
CD25
(B)
CD2

CD2 in neoplastic diseases of B‐cell precursors
CD2 has been reported in 1–4% of the globally considered neo-
plastic diseases of B‐cell precursors [85–87], where it is associated
with a worse prognosis [88].
Nonetheless, the expression of CD2 has been detected in just
less than 25% of the cases of B‐ALL with KMT2A‐MLLT3
translocation [4565].
CD2 in neoplastic diseases of T‐cell precursors
According to the EGIL classification of T‐lymphoblastic leuke-
mias, CD2 is typically present in the T II, T III, and T IV forms,
but is missing in the most immature form, T I [34,89]. In a pedi-
atric group of more than 100 cases of T‐ALL, CD2 has been
detected in 80% of the patients [4565].
CD2 is generally expressed by the cases with TCRαβ, but only
by some cases with TCRγδ [90,91]; its presence in childhood
cases is correlated with an increased probability of maintaining
complete remission [92].
CD2 in AML and BPDCN
Depending on the survey, the expression of CD2 has been
reported in 3–34% of the observed cases [93–100]. CD2 has been
reported:
• on the blasts of pediatric AML‐M2 negative for translocation
t(8;21) [101]
• on the promyelocytes of AML‐M3, with predilection for the
microgranular variant (AML‐M3v) [99,102,103], and for the
presence of the “short” type of the PML‐RARA fusion
gene [100,103]
• on both the monocytic and non‐monocytic neoplastic cells of
AML‐M4 [80,99,104]
• on the blasts of AML‐M5 [80]
• on the blasts of de novo AML with inv3(q21q26.2) and mono-
somy 7 [105–107].
The presence of CD2 (and also of CD4, CD7, and CD56)
on the blasts of AML is correlated with an increased risk of
extramedullary disease (granulocytic sarcoma, and cutane-
ous, gingival, and meningeal involvement) [108], and with a
lower incidence of complete remission [109]. The CD2
expression has been reported in cases of AML with morpho-
logical anomalies mimicking the picture of Chediak–Higashi
disease (pseudo Chediak–Higashi, PCH) [110], and in some
cases of BPDCN, also known as plasmacytoid dendritic cell
leukemia (PDCL) [111]. The presence of CD2 on AML‐M3
promyelocytes correlates with the occurrence of thrombotic
events [112].
In AML‐M4 with inv(16)/t(16;16), the expression of CD2 is
variable, and has been reported as weaker in cases with fusion
transcript CBFβ‐MYH11 other than type A [113].
CD2 in neoplastic diseases of mature B cells
Sporadic reports exist signaling the presence of CD2 in isolated
cases of B lineage non‐Hodgkin lymphoma [114,115]. Since CD2
CD2 Antigen
has been demonstrated on the surface of normal B lympho-
cytes [71], it is theoretically possible that these cases constitute a
clonal expansion of very infrequent normal B cells rather than an
expansion of B‐cell with an aberrant phenotype.
The expression of CD2 has occasionally been demonstrated
in the sporadic B‐CLL [71], but it seems particularly frequent in
familial B‐CLL, where it appears in 13% of the cases [116]; the
demonstration of CD2 on the cells of a patient affected by B‐
CLL suggests that clinical investigations should be extended to
the relatives as well [116].
Furthermore, CD2 has been demonstrated in some cases of
follicular lymphoma (FCL) [71], in some cases of diffuse large
B‐cell lymphoma (DLBCL) [71,117–120], in some cases of
DLBCL associated with pyothorax (PAL) [121], in some cases of
HCL [71], and in a case of MM [122].
CD2 in neoplastic diseases of mature T and NK cells
CD2 is generally expressed on the cells of the neoplastic diseases
of mature T and NK cells, but it may also be missing or expressed
in an aberrant way. In the peripheral T lymphoma not otherwise
specified (PTCLnos), about a third of the cases has been reported
to show an aberrant antigen expression [123,124]. An aberrant
CD2 expression has been reported with immunohistochemical
methods in atypical cutaneous T‐cell infiltrates of subjects
affected by mycosis fungoides (MF) [125], and with flow cytomet-
ric (FCM) methods on neoplastic lymphocytes of subjects affected
by Sézary syndrome (SS) [126], by T‐cell chronic lymphocytic
leukemia (T‐CLL) and by ATLL [127].
The CD2 expression is more constant in the cases of angioim-
munoblastic T‐cell lymphoma (AITL) [124], while in T‐cell
large granular lymphocytic leukemia (T‐LGL) it has been
reported either as constant [128] or as variable [129]. The cases
of CD8(+) cutaneous T‐cell lymphoma (CD8(+) CTCL) with
CD2(+) CD7(−) phenotype show a better prognosis than those
with phenotype CD2(−) CD7(+) [130].
In the neoplastic diseases of mature NK cells, CD2 may be
missing [131] but it has been reported in most cases of chronic
NK cell lymphocytosis (CLPD‐NK/CNKL) [131–133], of
aggressive NK cell leukemia (ANKL) [134,135], and of NK
lymphoma [136].
CD2 in Hodgkin lymphomas
CD2 has been demonstrated by IHC in the Hodgkin and Reed–
Sternberg (HRS) cells of isolated cases of classic Hodgkin lym-
phoma (CHL), but in most instances, its expression has been
judged as aberrant [4626,4627].
Increased expression of CD5 has been documented by FCM
on non‐neoplastic T cells in the background of pathological tis-
sues from CHL [4636,4637].
CD2 in neoplasms of histiocytic and dendritic cells
CD2 has been demonstrated by IHC on the membrane of the cells
of LCH [137].
7
Antigens

CD3 Antigen

lymphocytes with an anti‐CD3ε MoAb generates a positive histo-

General features
gram with a narrow gaussian‐like peak, clearly separated from the
negative component, with a channel peak representing the pres-
CD3 is made up of five different chains, i.e. γ, δ, ε, ζ, and η. Chains
ence of 57 ± 7 E03 ABC [72].
γ, δ, ε, and η are encoded by a gene on the long arm of chromo-
Evidence does exist that the number of CD3ε chains is not the
some 11 [138], while chain ζ, separately clustered as CD247 [139],
same for every T lymphocyte but is particularly high on γδ T
is encoded by another gene on the long arm of chromosome
cells [166], which express about 116 ± 15 E3 ABC per cell [167].
1 [140]. In T cells, CD3 transmits the activation signal produced
Among peripheral T lymphocytes, CD3 expression tends to vary
by the engagement of TCR [141,142].
depending on the T lymphocyte subset. Evidence does exist that, in
There is evidence that the CD3/TCR complex forms a multi-
comparison to CD8(bright+) T lymphocytes, the CD3 mean fluo-
meric array together with the tyrosine‐phosphatase CD45, with
rescence intensity (MFI) of positive cells is almost twice as intense
a tyrosine‐kinase, and with the CD7, which takes part in signal
in CD4(+) T cells, while T CD8(dim+) lymphocytes behave simi-
transmission [143].
larly to CD4(+) lymphocytes. This behavior does not depend on
The expression of the δ and ε chains is restricted to T lym-
cellular dimensions, inasmuch as in CD4(+) lymphocytes the scat-
phocytes, with two important exceptions:
ter values are even lower than in CD8(bright+) ones [75].
• the fetal and adult activated NK cells, which can contain δ and
A reduced expression of CD3 has been reported in other
ε chains in the cytoplasm [144,145]
cases:
• the PDCs, in the cytoplasm of which ε chains have been
• in alveolar T cells, with a greater negative modulation in
demonstrated [146].
CD4(+) cells [168]
As a rule, γ and ζ chains are present in NK cells as well [147],
• in activated T cells that infiltrate nasal polyps [169]
as either homodimers or heterodimers [148]. In NK cells, both
• in intrathyroidal T lymphocyte subsets in autoimmune thyroid
chains are not covalently linked with the transmembrane tail of
disease [170]
the CD16, and transmit the signal produced by the linkage
• in intestinal intraepithelial T lymphocytes [171]
between CD16 and the IgG crystallizable fragment [149–151].
• in T cells of patients given OKT3 rescue treatment for trans-
Signal transduction by ζ chain is carried out by the intracyto-
plant rejection [172]
plasmic protein ZAP‐70 [152,153].
• in T cells of patients with HIV infection [75,173]
During normal T‐cell maturation, the CD3 appears in the
• in T cells of aged subjects [76]
cytoplasm at the prothymocyte level but is only expressed on the
• in a minor subset of peripheral T lymphocytes characterized by
membrane from the common thymocyte stage on [3,154–159].
low CD4 expression, and positivity for CD25 and
During normal T‐cell maturation, the TCR and the CD3
HLA‐DR [174].
complex are assembled together before they are expressed on
This CD3 downmodulation might be due to the activation
the surface [160]; consequently, the TCR is not normally
state common to the great majority of the reported cases; it is
expressed on the membrane in the absence of CD3, and vice
important to bear in mind that a CD3 downmodulation can be
versa [161,162].
caused by apoptosis as well [175].
CD3 has been demonstrated by IHC in the cytoplasm of a
In some cases, the positive histogram can appear with a
subset of Warthin–Finkeldey polykaryocytes, which can be seen
bimodal shape, mostly due to the presence of a consistent sub-
in tonsils during the measles prodromal period and are proba-
set of γδ T cells, which actually bear more TCR/CD3ε com-
bly derived from T lymphocytes [163].
plexes than αβ T cells on the membrane [167] (Fig. 1.3). In our
experience, this behavior does not occur with the γδ T lympho-
cytes mounting Vδ1/Jδ1 sequences stained by δTCS1 MoAb
Cytometric features (Fig. 1.4).
Sometimes it is possible that the bimodality of the CD3(+)
Almost all anti‐CD3 monoclonal antibodies are specific for an ε peak is due to the presence of a clonal T‐cell population, homoge-
chain epitope [164,165]. The staining of peripheral normal neously expressing the molecule at an intensity that differs from

8
 CD3 Antigen

(A) (B)

CD3 CD3

(C) TCR γδ (D)

CD3 CD3

Figure 1.3 Pattern of expression of T‐specific CD3 antigen on peripheral T lymphocytes. The positive peak can show a bimodal appearance (A–C), because γδ(+) T
lymphocytes (red) express more CD3 molecules than αβ(+) T lymphocytes (B, D).

TCR γδ (A) Vδ1/Jδ1 (B) (C)

CD3 CD3 CD3

Figure 1.4 Different patterns of expression of T‐specific CD3 antigen on peripheral γδ(+) T lymphocytes. Vδ1/Jδ1(+) γδ(+) T lymphocytes (red) express CD3 less brightly
than the Vδ1/Jδ1(−) γδ(+) T lymphocytes (blue) (A–C). Vδ1/Jδ1(+) γδ(+) T lymphocytes were recognized by MoAb δ‐TCS‐1, and γδ(+) T lymphocytes were recognized by
MoAb 11F2.

9
Antigens
other normal residual T cells. This behavior is frequently reported
in patients affected by mature T‐cell malignancies [123,176].
In comparison to mature T cells, thymocytes express CD3
with a different intensity; as a rule, most common or cortical
CD1(+) CD4(+) CD8(+) thymocytes express low amounts of
CD3, while mature or medullar CD1(−) and CD4(+) or CD8(+)
thymocytes express the molecule in the same way as mature T
lymphocytes [155,177], with a differential higher expression on
CD4(+) CD8(−) T cells [75].
A CD4(+) CD8(+) thymocyte subset has been reported
expressing high levels of CD3; it is hypothesized that this subset
is a late differentiation stage between cortical and medullar
thymocytes [178].
As mentioned previously, the CD3 can be looked for both
on the membrane and in the cytoplasm of the cell. The demon-
stration of the intracytoplasmic molecule requires the use of
permeabilization techniques which allow intracellular entry of
the antibody. Although they could be improved by some opti-
mization procedures [179], such techniques can rely on the
use of standardized commercial permeabilizing solutions
[180–183].
Great care should be spent in the evaluation of membrane CD3
expression in T‐CLPDs; it is indeed possible that in some cases the
neoplastic cells dismiss the antigen after prolonged staining proce-
dures. This possibility is also suggested by the cytograms pub-
lished in a recent report which demonstrated a bimodal CD3(−)/
CD3(dim+) population in directly stained samples but only a
monomodal CD3(−) population in permeabilized ones [184].
MoAb OKT3, SK7/Leu4 and UCHT‐1
The three monoclonal antibodies OKT‐3, SK7/Leu4 and UCHT‐1
recognize CD3ε chains in cells transfected with genes coding for ε
and δ chains or for ε and γ chains, but do not recognize CD3ε
chains in cells transfected with genes coding for ε chains
only [185]. This behavior suggests that the three antibodies recog-
nize a conformational ε chain epitope, depending on the associa-
tion of ε chain with δ or γ chain, and are not able to detect isolated
intracytoplasmic ε chains [185].
Consequently, a negativity for intracytoplasmic ε chains
accomplished with one of the aforementioned antibodies is not
sufficient proof of ε chain absence, and should be validated
using an antibody specific for isolated ε chains, such as SP34 and
APA 1/1, or a polyclonal rabbit antiserum raised against a syn-
thetic polypeptide mimicking a sequence on the intracytoplas-
mic tail of the ε chain [186]. This point is of some practical
importance. Given that in thymocyte cytoplasm δ and ε chains
are simultaneously expressed from the prothymocyte level
onwards [187], these three antibodies are perfectly suitable for
demonstrating the intracytoplasmic CD3 antigen in T‐cell
malignancies but could miss it in some cases of NK neoplasms.
It has been also reported that MoAb UCHT‐1 is able to stain
the cerebellar Purkinje cells [188].
10
MoAb WT31
In the same way as OKT‐3, SK7/Leu4 and UCHT‐1, the MoAb
WT31 recognizes CD3ε chains in cells transfected with genes
coding for ε and δ chains or for ε and γ chains, but do not recog-
nize CD3ε chains in cells transfected with genes coding for ε
chains only [185]. This behavior confirms that, contrary to the
original hypothesis [189] and in keeping with successive
remarks [164], MoAb WT31 is not specific for a TCRαβ determi-
nant, but binds a conformational epitope on CD3ε chains, and
should be considered a bona fide anti‐CD3 antibody.
Nevertheless, it should be stressed that the epitope recog-
nized by MoAb WT31 is particularly accessible to this MoAb in
the case of TCRαβ co‐expression; this condition makes MoAb
WT31 fit for the presumptive identification of TCRαβ T cells,
especially if used in combination with a second antibody spe-
cific for the same chain. In this case, the sterical hindrance
between the two antibodies blocks the binding between WT31
and the ε chain of T cells bearing TCRγδ, and WT31 behaves
like a MoAb specific for TCRαβ only.
In these conditions, the staining of peripheral normal T lym-
phocytes with the WT31 MoAb generates a histogram with a
negative peak encompassing T cells bearing TCRγδ (Fig. 1.5).
The removal of the sterical hindrance allows the WT31 MoAb
to bind the ε chain of T cells with TCRγδ, although in a weaker
way than TCRαβ T cells. Indeed, if we stain a sample containing
a high number of γδ T cells using both the WT31 MoAb and a
second MoAb specific for TCRγδ, the WT31 MoAb will generate
a histogram with a first positive peak which encompasses γδ
negative T cells, and a second positive but intermediate peak
which encompasses γδ positive ones (Fig. 1.6).
From a practical point of view, the possibility of sterical hin-
drance between the WT31 MoAb and another anti‐CD3ε MoAb
suggests that a sequential staining procedure should be per-
formed, in which the sample is incubated first with WT31 alone
and then with the other anti‐CD3ε antibody.
MoAb T3
The FITC‐conjugated form of T3 displays unexpected behav-
ior [190]. In a multicolor analysis which combines a MoAb spe-
cific for TCRγδ (clone 11F2) and a second anti‐CD3ε MoAb
(clone SK7), the FITC‐conjugated form of T3 does not recognize
γδ T cells (Fig. 1.7).
It is interesting to notice that in this model, T3‐FITC behaves
very similarly to WT31, which is shown for comparison
(Fig. 1.8).
The anomalous behavior of T3‐FITC is difficult to explain.
The small molecular volume of FITC rules out a sterical hin-
drance effect, and the independence of the phenomenon from
the length of incubation does not suggest affinity variations
induced by the conjugation procedures.
It has been observed that, owing to a different glycosylation
pattern, CD3δ chains in γδ T cells display a more acidic isoionic
 CD3 Antigen

CD3 (A) TCR γδ (B)

(SK7) (11F2)

WT31 WT31

TCR γδ (C) (D)

(11F2)

CD3
(SK7) WT31

Figure 1.5 If lymphocytes are stained with an anti CD3ε antibody (MoAb SK7 in the reported example), MoAb WT31 does not recognize γδ(+) T lymphocytes (red) (A,B,D)
and behaves like a “bona‐fide” anti TCR αβ MoAb.

TCR γδ (B)
(A)
(11F2)

WT31 WT31

Figure 1.6 Without the steric hindrance caused by the anti CD3ε antibody, Moab WT31 recognizes γδ(+) T lymphocytes (red) as well, although weaker than αβ(+)
ones (A, B).

11
Antigens
CD3 SK7 (A) TCR γδ
(11F2)
CD3 T3
(D)
TCR γδ
(11F2)
Figure 1.7 When conjugated with FITC, MoAb T3 (A, B, F) does not recognize γδ(+) T lymphocytes (red) (B, C, D). γδ(+) T lymphocytes express more CD3 molecules than
γδ(−) T lymphocytes (E).
point than CD3δ chains in αβ T cells [191]. It could be hypoth-
esized perhaps that FITC increases the total negative charge of
the FITC‐conjugated antibody, allowing its binding with CD3δ
chains in αβ T cells, but preventing its binding with the more
glycosylated CD3δ chains in γδ T cells.
Other antibodies
Some antibodies do exist that are able to recognize isolated CD3ε
chains. These include the MoAb SP‐34, APA 1/1 [185] and
F7.2.38 [192], as well as a polyclonal rabbit antiserum which dis-
plays a high cross‐specificity and is even able to react with
Australian koala’s T lymphocytes [193].
MoAb SP‐34 can recognize isolated ε chain either on the
membrane or in the cytoplasm [185] and is able to identify T
cells from all but two non‐human primate species tested and
from the Siberian tiger as well [194].
Clones F7.2.38 and APA 1/1 have been raised against an
intracellular epitope and can consequently recognize only intra-
cellular chains [192]. Either the rabbit polyclonal antibody or
the MoAb F7.2.38 reacts with isolated CD3ε chains in formalin‐
fixed paraffin‐embedded tissue samples. This is a very impor-
tant point, because their use in IHC allows the detection of
12
(B) TCR γδ (C)
(11F2)
CD3 T3 CD3 SK7
(E) (F)
CD3 SK7 CD3 T3
intracellular isolated CD3ε chains in NK lymphoma
cells [195–197].
Other interesting clones are clone F101.01, which displays a
behavior similar to clone WT31 [198], and clone 446, which
cross‐reacts with a determinant in the cytoplasm of basal
keratinocytes [199].
Furthermore, several antibodies anti‐CD3ζ chains are com-
mercially available, among which clone TIA‐2 should be
cited [200]. This antibody reacts with an intracytoplasmic
epitope and requires the permeabilization of the sample [201].
Diagnostic features
It is important to bear in mind that in acute leukemia characteriza-
tion as well as in every doubtful case, the CD3 antigen must be
looked for both on the surface and in the cytoplasm [202] (Fig. 1.9).
Nevertheless, it should be stressed that the presence of intra-
cytoplasmic CD3ε chains is not necessarily to be interpreted as
a proof of T lineage attribution, given that CD3ε chains can be
demonstrated in NK cell lines [144,203], in NK malignan-
cies [197,203,204], in some cases of the so‐called myeloid/NK
 CD3 Antigen

CD3 SK7 (A) TCR γδ (B)

(11F2)

CD3 T3 CD3 T3

CD3 SK7 (C) TCR γδ (D)

(11F2)

WT31 WT31

Figure 1.8 Comparison between WT31 and T3‐FITC monoclonal antibodies. Neither WT31 (C, D) nor T3‐FITC (A, B) recognize γδ(+) T lymphocytes (red).

mCD3

cyCD3

Figure 1.9 Combined membrane and cytoplasmic CD3 staining in a case of T‐ALL. The technique allows the distinction between mCD3(+)/cyCD3(+) residual lymphocytes
(blue) and mCD3(−)/cyCD3(+) leukemic blasts (red).

13
Antigens
cell precursor acute leukemia (M/NK-AL) [204,205], in
PDCs [146], and in some cases of BPDCN [206,207].
CD3 in myelodysplastic and chronic myeloproliferative
diseases
The bone marrow of patients affected by refractory anemia with
blast excess can host a consistent percentage of cells characterized
by the expression of lymphoid antigens CD3 and CD7 along with
the expression of myeloid antigens CD13 and CD33. This feature
has been explained by the neoplastic transformation of a pluripo-
tent precursor able to retain the phenotypic features of both
lineages [208].
CD3 in neoplastic diseases of B‐cell precursors
As a rule, CD3 is not detected in neoplastic diseases of B‐cell pre-
cursors [86,210,211]. Nevertheless, cytoplasmic CD3 has been
detected in the blasts of two isolated cases of B‐ALL, one of which
featured the combined expression of CD2 and CD19 [212]. Owing
to the high likelihood of technical artifacts in intracellular antigen
detection, the greatest care must be taken in the interpretation of
these data.
CD3 in neoplastic diseases of T‐cell precursors
CD3 is usually detectable in the cytoplasm of every case of neo-
plastic disease arising from T‐cell precursors [4565]. According to
the EGIL classification of T‐lymphoblastic leukemias, CD3 is
detectable on the membrane of the cases pertaining to the most
mature form, T IV, but is lacking on the membrane of the cases
pertaining to the more immature forms T I, T II, and T III [34,213].
On the other hand, it is possible to find cells co‐expressing CD1
and CD3 on the membrane at least in one subset of cases.
This classification is perhaps too limited and could be
amended by introducing the “early T‐cell precursor lympho-
blastic leukemia” (ETP-ALL), recognized by the 2017 WHO
classification as a separate entity [214], in which the mCD3
expression is mandatory missing.
The absence of the CD3 surface antigen correlates with the
expression of CD13, CD33, CD34, and CD56 [215].
The CD3 surface antigen can be demonstrated in a propor-
tion of cases varying from 30% to 50% of total
cases [215,216,4565]; in childhood leukemias, the presence of
the CD3 surface antigen seems to confer an increased risk of
treatment failure [217].
However, the CD3 surface antigen is expressed by T‐ALL
blasts more faintly than by mature residual T cells [74], and this
difference can be used in the cytometric determination of the
minimal residual disease (MRD).
Some cases of lymphoblastic lymphoma have been reported,
categorized as NK precursor lymphomas because of the CD16
expression, in which the CD3 has been demonstrated in the
cytoplasm but not on the membrane [218–220]. One case of
acute lymphoblastic leukemia has been reported, categorized as
T/NK precursor leukemia, whose cells displayed a mCD3(−)/
14
cyCD3(+) phenotype and expressed the following antigens:
CD1a, CD2, CD4, CD13, CD19, CD30, CD33, and CD56 [221].
CD3 in AML and BPDCN
It should be observed that many cases which in the past have been
defined as AML with T‐related antigens could probably be relo-
cated into particular subgroups of T‐ALL not yet identified at that
time (in this regard see Part 2, section “Early T‐Precursor Acute
Lymphoblastic Leukemia (ETP‐ALL)”).
This could be probably true for a group of mCD3(−)/
cyCD3(+) cases, sometimes defined as “acute myeloid leukemia
with T‐lymphoid features,” which displayed special features
such as high blast count, presence of lymphadenopathies, poor
response to induction protocols specific to myeloblastic leuke-
mias, and good response to induction protocols specific for
lymphoblastic leukemias [222–225]. Anyway, insufficient phe-
notypic data often prevent the reclassification of these cases.
CD3 has been sporadically detected either on membrane or
in cytoplasm of AML cells [99,226–231]. CD3 intracytoplasmic
antigen has been detected in 2 out of 13 cases of AML‐M3v
[232]. CD3 has also been detected on the blasts of transient
abnormal myelopoiesis (TAM), also known as transient myelo-
proliferative disorder (TMD or TMPD), occurring in newborns
affected by Down syndrome [209].
The phenotype mCD3(−)/cyCD3(+) has been detected in the
so‐called “acute leukemia of myeloid/NK precursors” [233,234],
a clinical entity not recognized by the 2017 WHO classifica-
tion [235] (Fig. 1.10) whose cases can probably be re‐classified
as AML‐M0 or “true” NK‐ALL.
This phenotype has also been detected in the cells of some
cases of BPDCN (aka PDCL) [206,207].
Few cases of cyCD3(+) AML‐M7 have been recently
reported [236,237]; some of them co‐expressed also CD19
[237].
CD3 in neoplastic diseases of mature B cells
As a rule, CD3 expression is not be detected in neoplastic diseases
of mature B cells [119], and in a cohort of 501 cases of mature B‐
cell lymphoma no CD3(+) cases were reported [120].
Nevertheless, some anecdotal cases positive by FCM and/or
IHC have been reported, i.e.:
• one B‐CLL case characterized by the presence of translocation
t(18;22), whose cells expressed CD3 and CD8 beside the normally
expected B‐cell markers [238]
• four cases of otherwise typical HCL whose cells reacted with
UCHT‐1 but not with OKT‐3 MoAb [239]
• a little group of patients made up of one B‐cell non‐Hodgkin
lymphoma (B‐NHL) and six B‐CLL cases, whose cells co‐
expressed CD2 and CD3, and displayed IgH but not TCR
rearrangement [114]
• isolated cases of MM [122,240,241]
• one case of the so‐called EBV(+) DLBCL of the elderly [242]
• isolated cases of plasmablastic lymphoma (PBL) [118,243,244]

SSC (A) SSC
FSC
CD7 (D)
CD56
Figure 1.10 A putative case of M/NK‐AL whose blasts (red) display SSC values typical of small/medium‐size mononuclear cells (A), express CD45 at an intensity lower than
normal lymphocytes (B), and display phenotype CD7(+), CD56(+), CD34(+) (C, D) and weak positivity for cyCD3 (E, F). M/NK‐AL is not recognized by the 2017 WHO
Classification of Tumours of Hematopoietic and Lymphoid Tissues.
• one case of primary mediastinal (thymic) large B‐cell lym-
phoma (PMBCL) [245]
• one case of pyothorax‐associated lymphoma (PAL) [246]
• rare cases of DLBCL [117–120, 247–249]
• two cases of BL [118]
• one case of FCL [118]
• one case of CD30(+) anaplastic DLBCL [250].
Moreover, contrary to these assumptions and according to
studies performed on selected patients, some groups of B‐
CLPDs seem to exist in which an aberrant expression of CD3 is
more frequently detected. This groups encompass:
• the primary effusion lymphomas (PELs) and HHV8(+) DLBCL,
in which about a third of cases demonstrated an aberrant CD3
expression [121,251]
• the B‐cell neoplasms with plasmablastic differentiation (PBL
and plasmablastic myelomas), in which most cases expressed the
antigen with a strong cytoplasmic pattern [252]
• the HHV8(+) germinotropic lymphoproliferative disorder
(GLPD), in which CD3 has been detected by IHC in just under a
third of cases [4579–4581].
CD3 Antigen
(B) CD34 (C)
CD45 CD117
(E) (F)
CTRL cyCD3
CD3 in neoplastic diseases of mature T and NK cells
The cells of the neoplastic diseases of mature T cells are usually
but not always positive for CD3 (Fig. 1.11). An immunohisto-
chemical study detected the presence of CD3 in no more than
71% of the cases of PTCLnos, in no more than 60% of the cases of
AITL, and in no more than 26% of the cases of systemic anaplastic
large cell lymphoma (sALCL), with a significantly more frequent
expression in the ALK1(+) cases [253]. Another independent
study has confirmed the particularly low frequency of CD3 posi-
tivity in anaplastic large cell lymphoma (ALCL) and other
CD30(+) T chronic lymphoproliferative diseases [254].
Sometimes the CD3 can be expressed in an aberrant way, i.e.
with less or more intensity than in normal mature T cells, and
sometimes it may be detected in the cytoplasm, but not on the
cell surface [123].
In PTCLnos, CD3 is aberrantly expressed in a proportion of
cases varying from 6% to 66% of the total [45,123,255–263]. In
the more recent studies, the higher percentage of aberrations is
probably due to the increasing sensitivity of modern cytometric
techniques.
15
Antigens
CD4
(A)
CD3
CD4
(C)
CD3
Figure 1.11 Aberrant CD3 expression on neoplastic CD4(+) cells (red) in four cases of neoplastic disease of mature T cells. (A) AITL, (B) ALCL, (C) T‐PLL, (D) PTCLnos.
In ATLL, CD3 can be missing or dimly expressed [264–270],
and it has been reported that a bivariate analysis carried out for
CD3 and CD7 can allow the staging of the disease, distinguish-
ing asymptomatic HTLV‐I carriers and patients with smolder-
ing, chronic and acute disease on the basis of the quantitation of
three subsets of cells, i.e. CD7(−) CD3(±), CD7(±) CD3(±), and
CD7(+) CD3(+) [271].
Missing or aberrant (decreased) expression of CD3 has been
reported in T‐cell prolymphocytic leukemia (T‐PLL) [268,272,273],
AITL [274,275], T‐LGL [268,276,277], SS, and MF [176,278–280],
enteropathy‐associated T‐cell lymphoma (EATCL) [281], mono-
morphic epitheliotropic intestinal T‐cell lymphoma (MEITL) [282],
and PTCLnos [45,124]. According to a recent report, the γδ T cells
of hepatosplenic T‐cell lymphoma (HSTCL) typically express CD3/
TCR complex at a lesser intensity than normal γδ T cells [283];
moreover, the neoplastic cells, either TCRγδ(+) or TCRαβ(+), can
lose the expression of CD3 [284–286].
Neoplastic populations of T cells with mCD3(−)/cyCD3(+)
phenotype have been detected in the peripheral blood in
patients affected by AITL [287] and in a subset of patients
16
CD4
(B)
CD3
CD4
(D)
CD3
affected by hypereosinophilic syndrome. In this last case, hyper-
eosinophilia represents a paraneoplastic response to the over-
production of IL‐5 by the pathological T clone [288].
The cells of the neoplastic diseases of mature NK cells do not
express the CD3/TCR complex on the membrane but can con-
tain free CD3ε and CD3δ chains in the cytoplasm. Accordingly,
the phenotype mCD3(−)/cyCD3(+) is a typical although not
exclusive feature of this type of disease, and it has been docu-
mented in extranodal NK/T-cell lymphoma “nasal type” (ENKTL)
[195] [197] [203] [204] [289] [291] [293] [294] [297–299], and in
ANKL [134,294,300].
CD3 in Hodgkin lymphomas
CD3 has been demonstrated by IHC in the HRS cells of isolated
cases of CHL, but in most instances, its expression has been
judged as aberrant [4625–4627].
CD3 in neoplasms of histiocytic and dendritic cells
An aberrant focal cytoplasmic expression of CD3ε chain has been
detected by IHC in one case of Langerhans cell sarcoma [301].
 CD4 Antigen

CD3 in other pathological conditions In some cases of so‐called “myeloid and lymphoid neoplasm
CyCD3 has also been reported in the cells of the so‐called iT‐ with FGFR1 abnormalities,” also known as “8p11 stem cell syn-
LBP [55], an entity not recognized by the 2017 WHO drome,” the expression of CD3 has been demonstrated by IHC
classification. not only in T lymphoblasts but in myeloid blasts as well [302].

CD4 Antigen

• Th9 lymphocytes, mainly responsible for pro‐inflammatory

General features
effector functions and protection from helminth infections
• Th17 lymphocytes, mainly responsible for pro‐inflammatory
CD4 is a 55 kD glycoprotein belonging to the superfamily of the
effector functions
immunoglobulins and encoded by a gene situated on chromo-
• Th21 lymphocytes, mainly responsible for the regulation of
some 12 [303]. CD4 is a transmembrane molecule constituted by
innate immunity and active in the differentiation and repair of
four extracellular domains similar to those of immunoglobu-
epithelial cells
lins [304]. Amino‐terminal domain 1 contains an epitope acting
• Treg lymphocytes, mainly responsible for immunosuppressive
as a receptor for the gp120 of HIV‐1 virus [305], while domains 1
functions
and 2 act together as a receptor for the HLA‐DR antigen [306],
• Follicular T helper (FTH) lymphocytes, mainly responsible for
binding its non‐polymorphic domain β2 [307]. CD4 acts as a
the formation of follicular centers in lymph nodes and for the
receptor for IL‐16 as well [308].
cooperation in the production of high‐affinity antibodies.
CD4 is mainly expressed by a heterogeneous population of T
During T‐cell maturation CD4 begins to appear at the thymo-
lymphocytes, which encompasses the following functional sub-
cyte level. In particular, CD4 appears first on immature thymo-
sets (Table 1.2) [309,310]:
cytes lacking CD8. These “single‐positive” immature thymocytes
• Th1 lymphocytes, mainly responsible for the regulation of cell‐
are characterized by the CD1a(+) CD4(+) CD8(−) phenotype. In
mediated immunity against intracellular pathogens
the course of their maturation, these elements begin to co‐
• Th2 lymphocytes, mainly responsible for the regulation of
express CD8, giving origin to the CD1a(+) CD4(+) CD8(+)
humoral immunity against extracellular pathogens
“double‐positive” thymocytes, and then finally segregate into
two populations of “single‐positive” mature thymocytes, which

Table 1.2 Functional subsets of CD4(+) lymphocytes

Th1 Th2 Th9 Th17 Th22 Treg FTh

Membrane CD366 CD365 CD124 CCR4 CCR4 CD25(++) CXCR4

antigens CCR1 CCR3 IL17RB CCR6 CCR6 CD127(±) CXCR5
CCR5 CCR4 TGFβRII TCFβRII CCR10 CD357 CD10
CXCR3 CCR8 CD121a FR4 CD278
CXCR4 CD161 CD279
Transcription Tbet GATA3 GATA3 RORγt
factors STAT1 STAT5 STAT6 RORα AHR FoxP3 BCL6
STAT4 STAT6 PU.I STAT3
IRF4
Cytokine expression IFNγ IL4 IL9 IL17A IL22 IL10 IL4
LT IL5 IL10 IL17F TGFβ IL21
IFNα IL6 IL21 IL21 IFNγ
IL2 IL10 IL22 CXCL13
IL13

17
Antigens
express the mutually exclusive phenotypes CD4(+) CD8(−) and
CD4(−) CD8(+) [155,311].
Unlike T lymphocytes with TCRαβ, T lymphocytes with
TCRγδ do not express CD4. Nevertheless, it has been reported
that at least in some subjects, γδ T lymphocytes express CD4 at
a frequency ranging between 1% and 4% [312]; this frequency
can be increased in vitro by infection with HHV‐6 virus [313].
CD4 is also expressed by immature NKT cells [2], by a subset of
mature NKT cells [2], and by APC, including monocytes [314],
macrophages [314], MDCs [315], PDCs [315], Langerhans
cells [314], activated microglial cells [316], dendritic cells of
cord blood [317], and FDCs [65], which seem selectively nega-
tive for the epitope recognized by the clone OKT4D [65].
CD4 is expressed on hemopoietic precursors [318], erythroid
precursors [319] and megakaryocytes [4735], and it has been
reported on a subset of neutrophils in some subjects either healthy
or affected by HIV infection [320]. CD4 can be induced on baso-
phils [321] and it is constitutively although weakly expressed on
eosinophils [322], but not on mast cells [81]. Finally, CD4 has
been documented on tonsillar activated B cells [323], on a
­minority of NK cells [324], and on activated CD8(bright+) T
lymphocytes in the course of HIV infection [325].
Cytometric features
The monoclonal antibodies OKT4, OKT4A, OKT4B, OKT4C, and
OKT4D recognize different epitopes [326], and it is known that some
clones, such as Leu3a, OKT4A, and F101–69, recognize epitopes
related to the binding site for the gp120 protein of HIV‐1 virus [327].
In the human, the distribution of CD4 epitopes depends on a
genetically determined polymorphism, consisting of the substi-
tution of a molecule of tryptophan for a molecule of arginine in
position 240 [328]. Subjects carrying this polymorphism do not
CD4 (A)
CD3
Figure 1.12 Differential CD4 expression on T lymphocytes in a subject affected by HIV infection. CD3(+) CD4(dim+) lymphocytes (A, red) tend to display CD45RA(−),
CD62L(+) phenotype (B).
18
express the epitope recognized by the MoAb OKT4, but nor-
mally express the epitopes recognized by OKT4A and Leu3a
MoAbs. In these subjects, OKT4 MoAb does not recognize
CD4, or produces positive histograms with a peak channel con-
sistent with an intensity of expression about half that of normal
controls [329]. This anomaly is exceptionally reported in
Caucasians [330], is rare (<1%) in the Japanese population [329],
but is relatively frequent in populations of African origin [331].
It must finally be remembered that the phenotype OKT4(−)/
Leu3a(+) is not the only possible anomalous phenotype; an
isolated report exists about an apparently normal subject selec-
tively lacking the epitope recognized by Leu3a MoAb [332].
The staining of peripheral normal lymphocytes with an anti‐
CD4 MoAb generates a positive histogram with a narrow gauss-
ian‐like peak, clearly separated from the negative component,
with a channel peak representing the presence of 50 ± 10 E03
ABC [72], equivalent to the presence of around 100 E03 mole-
cules of CD4 per cell [333].
Besides this population, operationally defined CD4(bright+),
there is sometimes a second little population of CD4(+) lym-
phocytes characterized by a reduced expression of the antigen
(about 50 E03 molecules per cell). These CD4(dim+) lympho-
cytes account for 5–10% of all the peripheral lymphocytes, are
characterized by a reduced expression of CD3, co‐express CD25
and HLA‐DR, are increased in old age, and have been inter-
preted as chronically activated and apoptosis‐resistant lympho-
cytes [76,174] (Fig. 1.12).
A reduced expression of CD4 on T lymphocytes has been
documented in subjects affected by B‐CLL [334] and by
Nijmegen breakage syndrome (NBS) [335].
Finally, it should be borne in mind that thymocytes and
monocytes express the antigen at a lesser extent than mature T
lymphocytes [336]; in particular, CD4 is expressed on periph-
eral monocytes with an intensity of 17 ± 5 E0 ABC [72]
CD62L
CD62L
(B)
CD45RA

SSC (A)
CD4
Figure 1.13 CD4 antigen is expressed by monocytes (red) at a lower intensity than by T lymphocytes (blue) (A, B).
(Fig. 1.13). Such intensity is further reduced on the monocytes
of newborns [337] and traumatized patients [338].
Diagnostic features
CD4 in myelodysplastic and chronic myeloproliferative
diseases
CD4 has been demonstrated with FCM techniques on the surface
of the granulocytes of a patient affected by myelodysplasia charac-
terized by the presence of translocation t(5;12); in this case, it was
hypothesized that the breakage of chromosome 12 was able to
upregulate the expression of the antigen, encoded by a gene in
12p12 [339].
CD4 in neoplastic diseases of mast cells
In isolated cases of AMCL, CD4 has been reported on the
­neoplastic cells either by FCM [340] or by IHC [2444].
CD4 in neoplastic diseases of B‐cell precursors
As a rule, the expression of CD4 is missing in the neoplastic dis-
eases of B‐cell precursors, but it has been reported in some isolated
cases [86,87], where it seems related to a worse prognosis [88].
CD4 in neoplastic diseases of T‐cell precursors
On the blasts of the neoplastic diseases of T‐cell precursors, CD4
can be expressed alone or together with CD8 on the forms derived
from the common thymocytes.
The isolated expression of CD4 suggests a disease stemming
from an immature single‐positive precursor characterized by a
CD1a(+) CD4(+) CD8(−) phenotype and situated immediately
before the stage of double‐positive (DP) CD4(+) CD8(+) com-
mon thymocyte.
In a pediatric group of more than 100 cases of T‐ALL, CD4
has been detected in 53% of the patients [4565].
CD4 Antigen
(B)
CD4
In a group of cases made up of 18 patients affected by T‐ALL,
the “single‐positive” CD4(+) CD8(−) phenotype has been
reported in 39% of cases, while the “double‐positive” CD4(+)
CD8(+) phenotype has been reported in 22% [341]. Contrary to
what happens in the neoplastic diseases of mature T cells, in the
neoplastic diseases of T‐cell precursors the presence of the
TCRγδ does not rule out CD4 expression [342].
CD4 in AML
Depending on the survey, the expression of CD4 has been
reported in 36–74% of the observed cases [94,96–98,100,343].
According to some authors, CD4 expression is strongly indic-
ative of a myeloid lineage [344] regardless of a monocytic com-
mitment [345], but for others CD4 expression is frequent in the
AML‐M4 and AML‐M5 forms [100,226], with a preference for
the most mature cases [96]. In a survey made up of 495 adult
patients, CD4 expression has been documented in 45.9% of the
cases, and in 37.4% of AML‐M1, 33.7% of AML‐M2, 35.4% of
AML‐M3, 65% of AML‐M4, 78.3% of AML‐M5, and 55.6% of
AML‐M6 [343]. According to some authors, the absence of CD4
characterizes the pediatric AML‐M2 with translocation
t(8;21) [94], while other authors report that in a survey of 59
pediatric cases, CD4 was regularly co‐expressed in all the six
observed cases [100]. It is interesting to note that in the study of
Abdelhaleem and co‐workers, CD4 was co‐expressed in all the
AML‐M7 cases [100]; the expression of CD4 in this FAB sub-
type has been reported in another case [343]. In an isolated case
of monoblastic leukemia, CD4 was the only antigen found posi-
tive in the absence of myeloid and monocytic lineage‐specific
markers [346].
The presence of CD4 on the blasts of AML correlates to an
increased risk of extramedullary disease (granulocytic sarcoma,
and cutaneous, gingival, and meningeal involvement) [108], to
anomalies of chromosome 11 [343], and to the pericentric
inversion of chromosome 16 [343].
19
Antigens
CD4 has been demonstrated by IHC in an isolated case
of myeloid sarcoma (MS) [347], and in some cases of
M/NK-AL [348], a clinical entity not recognized by the 2017
WHO classification [235].
CD4 in the BPDCN
CD4 expression, together with the expression of CD56 and
CD123 and the absence of other lineage‐specific markers, consti-
tutes the characteristic phenotype of the BPDCN (aka
PDCL) [111,349–353]. Nonetheless, a CD4(−) case has been
reported characterized by solitary skin involvement [354].
CD4 in neoplastic diseases of mature B cells
CD4 expression is usually missing in neoplastic diseases of mature
B cells, but it has been reported with immunohistochemical
­techniques in most of the cases belonging to a rare variety of
DLBCL, named ALK(+) large B‐cell lymphoma (ALK(+)
LBCL) [355–357].
CD4 expression has also been reported in sporadic cases of
DLBCLnos [118,119,358], DLBCL with primary splenic
onset [359], DLBCL associated with pyothorax (PAL) [121],
HCL [360], MM [122,241,361], PBL [117,361], B‐CLL [362],
and BL with plasmacytoid differentiation arising in a subject
with HIV infection [363].
CD4 has been reported in two very similar cases of trans-
formed FCL, which lost B‐cell antigens and expressed aber-
rantly CD30 as well [364].
CD4 in neoplastic diseases of mature T and NK cells
In the neoplastic diseases of mature T cells CD4 is generally
expressed:
• in most cases of T‐PLL [581]
• in most cases of ATLL [365,366,4167]
• in most cases of SS [126,367–369] and MF [370]
• in virtually all cases of lymphomatoid papulosis (LyP), variants
A and B, and in most cases of LyP variant C [371]
• in most cases of primary cutaneous anaplastic large cell lym-
phoma (pcALCL) [372]
• in virtually all cases of primary cutaneous lymphoma of T
medium/small CD4(+) T cells (PCSM‐TCL)
• in most cases of breast implant‐associated anaplastic large cell
lymphoma (biaALCL) [373]
• in most cases of ALCL [374,375], with predilection for the
ALK(+) cases [376]
• in most cases of AITL [124,377–379] and other follicular T‐
cell‐derived lymphomas [380]
• in most cases of (mostly nodal) PTCLnos [45,123,124, 381,
382], with preference for the GATA3 subtype [383]
20
• in most cases of the so‐called “indolent T‐cell lymphoprolifera-
tive disorder of the gastrointestinal tract (indolent GI
T‐LPD)” [384,385]
• in a subgroup of cases of T‐LGL [386–389].
The so‐called Lennert lymphoma, described by Lennert and
Mestdagh in 1968 [390] and also known as lymphoepithelioid
lymphoma (LHL), is considered by the 2017 WHO classifica-
tion as the only remaining morphological variant of
PTCLnos [391]. Lennert lymphoma is reported by some authors
as a CD4(+)neoplasm [392], while according to others its cells
would express CD8 [393,394]. In comparison to CD8(+) cases,
LHL CD4(+) cases seem fare better from a prognostic point of
view [382].
CD4 is seldom expressed on the cells of extranodal lympho-
mas [395], and as a rule is always missing on the cells of lympho-
mas with TCRγδ, with only two exceptions reported so
far [396,397].
In accordance with the fact that neoplastic diseases of mature
T cells very often display an abnormal T‐related antigen expres-
sion, CD4 may be missing or display an abnormally low or high
expression [398]; this behavior is frequently found in
SS [123,126], but it has also been reported in AITL [378] and in
PTCLnos [123].
As for the neoplastic diseases of mature NK cells, CD4 has
been reported in isolated cases of extranodal NK/T lymphoma
(ENKTL) “nasal‐type” [399], and in a case of ANKL [400].
PTCLnos can be subdivided in two groups depending on the
expression of either GATA3 or TBX21; different prognosis but
no difference in CD4 or CD8 expression was found between the
groups [401].
CD4 in Hodgkin lymphomas
CD4 has been demonstrated by IHC in the HRS cells of isolated
cases of CHL, but in most instances, its expression has been
judged as aberrant [4625–4627].
CD4 in neoplasms of histiocytes and dendritic cells
CD4 has been demonstrated with immunohistochemical tech-
niques on the cells of LCH [19], HS, often referred in the past as
true histiocytic lymphoma (THL) [402,403], and in isolated cases
of tumor of the indeterminate dendritic cells (IDCT), a solid neo-
plasm of the dendritic cells [53]. CD4 has been demonstrated
with immunohistochemical techniques in an isolated case of
FDCS [404].
CD4 in other pathological conditions
Together with CD8, CD4 has also been reported in the cells of the
so called iT‐LBP [55], an entity not recognized by the 2017 WHO
classification.

CD5 Antigen
General features
CD5 is a 67 kD glycoprotein which is encoded by a gene situated
on the long arm of chromosome 11 [405]. It is a T‐related antigen
that appears during T lymphocyte maturation, and it is normally
expressed after the prothymocyte level [58,155]. CD5 is expressed
on most mature T lymphocytes, but not on all of them, as either a
subset of T lymphocytes with TCR γδ [406] or a little subset of T
lymphocytes with TCRαβ [407] does not express the antigen.
Expanded populations of CD3(+) CD5(−) T lymphocytes
have been reported in the peripheral blood of subjects who have
undergone allogeneic bone marrow transplantation [408].
CD5 can be considered a T‐associated antigen, but not a T‐
specific molecule, inasmuch as it is expressed on the membrane
of a subset of peripheral blood B lymphocytes, ranging between
17% and 25% of all the B lymphocytes [409–411]. Furthermore,
CD5 is expressed on mature hematogones [412] and on B lym-
phocytes in fetal spleen [410], in the mantle of the germinal
center [413], and in thymus [414]; it is expressed on most B lym-
phocytes in the cord blood or in the peripheral blood of the
newborn [410], and on most peripheral B lymphocytes after
autologous or allogeneic bone marrow transplantation [8].
CD5 is usually not expected to be expressed on NK lympho-
cytes, but some isolated reports exist documenting the antigen
on a NK cell subset in normal subjects [415], in subjects affected
by pulmonary tuberculosis [415], in subjects affected by MM
TCR γδ (A)
(11F2)
CD5
Figure 1.14 γδ(+) T lymphocytes (red) tend to express CD5 at a lower intensity than αβ(+) T lymphocytes (A, B).
CD5 Antigen
and plasmacytoma [416], and in two subjects with NK lympho-
cytosis induced by Epstein–Barr virus (EBV) reactivation [417].
CD5 has also been reported on a dendritic cell subset [146].
Cytometric features
The staining of peripheral normal lymphocytes with an anti‐CD5
MoAb generates a positive histogram with a gaussian‐like peak,
clearly separated from the negative component, but rather broad
because of a certain variability in antigen expression.
The histogram produced by the staining of TCRαβ(+) T lym-
phocytes generates a histogram with a channel peak represent-
ing the presence of 57 ± 7 E03 ABC [72], but the CD5 intensity
of expression is progressively decreasing in γδ T lympho-
cytes [312,406] (Fig. 1.14), in the CD5(+) CD19(+) neoplastic B
lymphocytes of B‐CLL [418], and in the CD5(+) CD19(+) nor-
mal B lymphocytes, which express the antigen at a level lower
than 2 E03 ABC [72].
Among peripheral T lymphocytes, CD5 expression tends to vary
according to the subset; evidence exists that CD5 expression on
CD4(+) cells is 1.5 times more intense than that on CD8(bright+)
cells, and twice as intense as on CD8(dim+) cells [74].
CD5 is also expressed dimly on CD3(+) CD6(−) T lympho-
cytes [419], on TCRαβ(+) intraepithelial T lymphocytes in the
gut [420], and on most thymocytes [155].
(B)
CD5
21
Antigens
A decreased expression of CD5 on CD8(+) lymphocytes has
been also reported in EBV‐associated [494–496] and in familial
hemophagocytic lymphohistiocytosis (FHLH) with perforin
gene mutations [497].
According to some authors [421], but not to others [422],
cryopreservation decreases the antigen expression.
Diagnostic features
CD5 in myelodysplastic and chronic myeloproliferative
diseases
CD5 is frequently expressed on the blasts of MDS [423].
CD5 in neoplastic diseases of B‐cell precursors
As a rule, CD5 is not expressed in the globally considered
­neoplastic diseases of B‐cell precursors [86], even if it has been
reported sporadically in isolated cases [88,424–429], where it is
associated with a particularly aggressive behavior [430].
The expression of CD5 has been detected in just under half of
the cases with mutated MEF2D, and is associated with a poor
prognosis; CD5 has been detected in half cases of CD10(+) with
KMT2A‐MLLT3 translocation, but not in cases with the same
genetic anomaly without CD10 expression [4565].
CD5 in neoplastic diseases of T‐cell precursors
CD5 is generally expressed in neoplastic diseases of T‐cell precur-
sors [341], but it can be missing in the most immature forms, also
known as “early T,” “pro/pre‐T,” or “T‐stem cell leukemias” [89].
This is in agreement with the EGIL classification of T lymphoblas-
tic leukemias, according to which CD5 is typically present in the T
II, T III and T IV forms, but missing in the most immature, T I [34].
In a pediatric group of more than 100 cases of T‐ALL, CD5
has been detected in 91% of the patients [4565].
In ETP‐ALL the CD5 antigen should be missing; if present, it
should be expressed only in a subset of cells, and/or with an MFI
at least 1 log dimmer than normal mature T cells [431,432].
Nevertheless, it should not be forgotten that a negative or very
dim expression of CD5 can be documented also in non‐ETP T‐
ALL with TCRγδ expression [431].
CD5 in AML
In the AML CD5 has been reported in less than 10% of cases [226];
CD5 expression seems related to the AML‐M5a [96] and AML‐
M0 subtypes [433]; in the AML‐M0 subtype, CD5 seems to cor-
relate with hypertriploid chromosome number [433].
The expression of CD5 has been reported in a case of acute
basophilic leukemia (ABL) arisen in a subject affected by
MDS [434].
CD5 in neoplastic diseases of mature B cells
The presence of CD5 divides the neoplastic diseases of mature B
cells into two groups, the first made up of CD5(+) diseases
22
including B‐CLL and mantle cell lymphoma (MCL), the second
comprising CD5(−) diseases and including virtually all the
remaining forms. Of course, this distinction only has didactic
value and there are many important exceptions. Moreover, it is
possible that new CD5(+) clinical entities exist, not yet recog-
nized as such by current classifications, as perhaps in the case of
CD5(+) DLBCL with primary splenic onset.
From an operative point of view, a percentage of CD5(+) B
cells greater than 35% has been considered indicative for B‐cell
lymphoma in the analysis of a lymph node biopsy without
demonstrable light chain restriction [435].
Traditionally CD5(+) diseases
As mentioned above, CD5 expression is a typical trait of B‐CLL
and MCL [436–439]. In comparison with B‐CLL, the neoplastic
lymphocytes of MCL express CD5 at a higher intensity; this point
has been confirmed by either FCM [440] or IHC [441].
Nevertheless, it must not be forgotten that rare cases of CD5(−)
MCL have been reported [439,442,443]; moreover, a case of
CD5(+) MCL is known which relapsed as CD5(−) [444], and a
specular case also which was CD5(−) at the onset but relapsed as
CD5(+) [445].
CD5 is expressed either by “typical” or “atypical” B‐CLL [446];
in B‐CLL its increased expression is correlated with deletion of
the long arm of chromosome 13 [447]. CD5 is also expressed on
the elements of B‐CLL in plasmacytoid transformation [448]; in
these cases, CD5 expression can constitute a useful element in
the differential diagnosis with LPL [449].
In some surveys B‐PLL expresses CD5 in 50–70% of the
cases [450–452], but in others it is consistently negative for the
antigen [440]; this discrepancy can probably be explained either
by the fact that in some surveys the cases derived from a pre‐
existing B‐CLL are merged together with cases arising “de novo”
or by the fact that leukemized MCL can sometimes present pro-
lymphocytoid morphology, consequently being confused with
B‐PLL [453].
Traditionally CD5(−) diseases
Marginal zone lymphoma (MZL), HCL, LPL, splenic diffuse red
pulp lymphoma (SDRPL), FCL, and plasma cell neoplasms (mon-
oclonal gammopathy of undefined significance (MGUS), MM,
plasma cell leukemia (PCL)) are traditionally considered negative
for the expression of CD5.
Nevertheless, it must not be forgotten that CD5 has been
reported in 25% of cases of SMZL [455], in 8% of cases of nodal
marginal zone lymphoma (NMZL) [456], in isolated cases of
either mucosa‐associated lymphoid tissue (MALT) or non‐
MALT extranodal marginal zone lymphoma (EMZL) [457–
462], in rare cases of either primary cutaneous [463] or
nodal [464–467] FCL and in rare cases of HCL [360,468–471].
CD5 has been demonstrated on lymphocytes [472–475] and
plasma cells [472] in rare cases of LPL, and on neoplastic plasma
cells in a case of MCL in plasmacytic transformation [476].

When present in usually negative lymphomas, CD5 carries
an unfavorable prognostic significance [115], especially in
MALT type lymphomas, where it is correlated with leukemiza-
tion and dissemination to bone marrow and other sites [457,459].
The demonstration of CD5 on HCL cells is of some practical
importance because the antigen expression is related to resist-
ance to α‐interferon [470], but to sensitivity to cladribine (2‐
chloro‐2′‐deoxyadenosine, 2‐CdA) [471]. It is noteworthy that
in a case reported by Usha and collaborators, CD5 was expressed
by hairy cells in bone marrow, but not by hairy cells in periph-
eral blood [471].
Sporadically CD5(+) diseases
Apart from Richter syndrome, in which it is expected [477], CD5
has been reported either by FCM or IHC in 10% of cases of
DLBCL [478,479].
CD5(+) DLBCL probably constitutes a separate entity not yet
recognized as such and is characterized by poor prognosis and
frequent extranodal presentation [3857,3855].
CD5 expression has been reported in a case of primary cuta-
neous DLBCL “leg type” [480], and with high frequency in
CD5 (A) CD5
CD3
CD5 (D) CD5
CD3
Figure 1.15 Aberrantly low CD5 expression on neoplastic CD3(+) cells (red) in 6 cases of T‐LGL (A–F).
CD5 Antigen
intravascular large B‐cell lymphoma (IVBCL) [481], where it
seems devoid of prognostic significance [482]; CD5 expression
has been reported in a case of T‐cell‐rich large B‐cell lymphoma
(TCRBCL) [483], in some cases of BL in leukemic phase [484],
and in a subset of cases of large B‐cell lymphoma with IRF4
rearrangement [485].
CD5 in neoplastic diseases of mature T and NK cells
CD5 is generally expressed on the cells of the neoplastic diseases
of mature T cells, but it can also be missing or expressed in an
aberrant way [398].
An irregular expression of CD5 has been sporadically
reported in ATLL [486], AITL [124], and PTCLnos [45,123,124],
and it is frequently found in T‐LGL with either
TCRαβ [128,129,487] or TCRγδ [488] (Fig. 1.15).
CD5 is missing in most TCRγδ(+) T‐cell lymphomas, such as
primary cutaneous TCRγδ(+) T‐cell lymphoma (PCGD‐
TCL) [283,488,489] and other mucocutaneous [488,490] or
nodal [491] lymphomas.
Although reported in a subset of normal NK cells, CD5 is not
expressed in the neoplastic diseases of mature NK cells [203].
(B) CD5 (C)
CD3 CD3
(E) CD5 (F)
CD3 CD3
23
Antigens
CD5 in Hodgkin lymphomas
CD5 has been demonstrated by IHC in the HRS cells of isolated
cases of CHL, but in most instances, its expression has been
judged as aberrant [4626,4627].
CD5 has been detected by FCM on HRS cells of CHL and on
LP cells of nodular lymphocyte predominant Hodgkin lym­
phoma (NLPHL), but it has been attributed to the non‐neoplas-
tic T lymphocytes rosetting the neoplastic cells [4603].
Increased expression of CD5 has been documented by FCM
on non‐neoplastic T cells in the background of pathological tis-
sues from CHL [4636,4637].
CD7 Antigen
General features
CD7 is a 40 kD glycoprotein encoded by a gene situated on chro-
mosome 17 [4552]. In T and NK cells CD7 plays an important
role in the activation and regulation of cytokine production [498];
among other activities, CD7 binds Galectin and is necessary for
Galectin‐mediated apoptosis [499].
CD7 is a T linage‐associated antigen but is devoid of a true T
lineage specificity [500]. It is the first T‐associated antigen to
appear during the maturation of T lymphocytes [58,155] but is
physiologically missing in important subsets of T lymphocytes
in peripheral blood [501] and in epidermis [502].
The absent or reduced expression of CD7 seems to corre-
late with activation; CD3(+) CD7(−) cells increase with
age [503], and can be increased in the peripheral blood of
patients with conditions characterized by acute and chronic
immune system activation, such as primary EBV infec-
tion [504], rheumatoid arthritis [505], allogeneic transplanta-
tion [506], and HIV infection [501,507]. Likewise, expanded
populations of CD3(+) CD7(−) cells can be found in the skin
of patients affected by HIV infection [508], eosinophilic cel-
lulitis (Wells syndrome) [509], and other inflammatory skin
diseases [510].
CD7 is expressed by 80–90% of CD3(−) NK cells [63], and it
has been demonstrated in a subset of bone marrow myeloid pre-
cursors [510,511], in the PDCs [146], and in a subset of CD19(+)
lymphoid precursors in fetal [512] and pediatric bone
marrow [513].
24
CD5 in neoplasms of histiocytic and dendritic cells
CD5 has been reported in a case of leukemized Langerhans cell
sarcoma [492].
CD5 in other pathological conditions
CD5 has also been reported in the cells of the so called iT‐
LBP [55], an entity not recognized by the 2017 WHO
classification.
CD5 has been anecdotally reported in some cases of persis-
tent polyclonal B lymphocytosis (PPBL) [493].
Cytometric features
The staining of peripheral normal lymphocytes with an anti‐CD7
MoAb generates a positive histogram characterized by a rather
heterogeneous distribution, with a channel peak representing the
presence of 28 ± 7 E03 ABC [72].
On mature T lymphocytes, CD7 intensity of expression seems
to be inversely proportional to their degree of immunological
competence, inasmuch as it has been demonstrated that CD7(+)
CD45RA(+) T lymphocytes expressed CD7 with an intensity of
27 ± 5 E03 ABC, while CD7(+) CD45R0(+) T lymphocytes
express CD7 with an intensity of 14 ± 3 E03 ABC [72].
CD3(−) NK lymphocytes expressed CD7 more intensely than
any other T subset [74] and can downregulate it after
activation [64].
Diagnostic features
CD7 in myelodysplastic and chronic myeloproliferative
diseases
Together with CD117, CD7 is preferentially expressed on the
blasts of high‐risk MDS, while the blasts of low‐risk cases tend to
express CD10 and CD15, which are related to more mature stages
of myeloid differentiation [514].
The bone marrow blasts of patients affected by CML in
chronic phase can express CD7, whose expression on more than
20% of CD34(+) blasts is related to progression of the

disease [515]. Experiments of gene expression corroborate this
point, inasmuch as a low expression of the gene coding for CD7,
together with a high expression of the gene coding for protein-
ase 3, can predict a longer global survival [516].
Likewise, the CD34(+) blasts of CML‐BC can co‐express
CD7 in an elevated percentage of cases [517].
Moreover, CD7 has been reported in about 10% of cases of
CMML [80], and on the blasts of TAM, also known as TMPD,
occurring in newborns affected by Down syndrome [209].
Adding CD7 to the antigens detected by the so‐called “Ogata
score” improves the sensitivity of the procedure [518].
CD7 in neoplastic diseases of B‐cell precursors
As a rule, CD7 is missing in the neoplastic diseases of B‐cell pre-
cursors, even if it has been reported in isolated cases [86,87,519],
where it is associated to a worse prognosis [88].
Nonetheless, the expression of CD7 has been detected in just
more half of the cases of B‐ALL with KMT2A‐MLLT3
translocation [4565].
CD7 in neoplastic diseases of T‐cell precursors
As a rule, CD7 is always positive on the blasts of neoplastic dis-
eases of T‐cell precursors [34,341,4565], and it is expressed at an
elevated intensity [519]; in this regard, CD7 expression can be
exploited to define an immunological gate able to restrict the
cytometric analysis to pathological cells only [520].
CD7 in AML
Depending on the survey, the expression of CD7 has been
reported in 12–42% of the observed cases, with particular predi-
lection for the M0, M1, and M2 subtypes [93–95, 97, 100, 101,
226, 521, 522], but it has also been reported in sporadic cases of
AML‐M4 and M5 [80,523], with a particular predilection for the
most immature monoblastic cases [93].
CD7 expression is rarely reported in the AML‐M3 of the
adult [524] and is virtually missing in the AML‐M3 of child-
hood [94,100]. In a group of 59 cases of pediatric AML, CD7
was co‐expressed with CD4 in all cases of AML‐M7 [100]. In
CD7(+) AML, CD7 is expressed at an intensity lower than T‐
ALL and is brighter in AML‐M0 than in other FAB
subtypes [519].
According to some authors, the co‐expression of CD7 and
CD56 defines a subgroup of AML‐M0, characterized by more
frequent extramedullary involvement, fewer circulating leuke-
mic blasts, less anemia, and higher platelet counts than usually
expected in AML‐M0 [525]. This group of cases shows features
very similar to the so‐called M/NK‐AL [348], a clinical entity
not recognized by the 2017 WHO classification [235], which has
also been reported to express CD7 [205].
According to some authors [108,521] but not others [526],
the presence of CD7 on AML blasts correlates with an increased
risk of extramedullary disease (granulocytic sarcoma, and cuta-
neous, gingival, and meningeal involvement) [108], and with a
CD7 Antigen
lower incidence of complete remission [109], especially in cases
characterized by the co‐expression of CD14 [93].
CD7 on AML blasts correlates with alterations of chromosome
5 [527], and with the expression of the thrombopoietin (TPO)
receptor [528]. In some cases of AML, molecular studies have
demonstrated a correlation between CD7 expression and the
silencing of CEBPA and NOTCH1 genes [529], or the presence of
Fms‐like tyrosine kinase‐3 internal tandem duplication (FLT3/
ITD) [530]. It is interesting to observe that in AML‐M2, CD7
seems mutually exclusive with translocation t(8;21) [100,101].
CD7 has been reported in some cases of BPDCN, where it corre-
lates positively with the expression of BDCA‐2 and negatively
with the presence of TdT, defining a subset of cases thought to
stem from a more mature precursor [531].
The co‐existence of CD7 and myeloid antigens correlated
both to the early and advanced stages of myeloid maturation is
frequently detected in AML with biallelic mutations of the
CEBPA gene [532,533].
CD7 in neoplastic diseases of mature B cells
As a rule, CD7 is missing in the neoplastic diseases of mature B
cells, but it has sporadically been reported in a case of B‐CLL
which was also CD4(+) [362], in a case of diffuse blastoid B‐cell
lymphoma, a histological variant of t(14;18)‐negative FCL [534],
in some cases of DLBCL [117,119,120], in a case of PBL [535], in
a case of DLBCL associated with chronic inflammation (also
known as PAL) [536], and in two cases of PEL [537,538].
CD7 in neoplastic diseases of mature T and NK cells
In neoplastic diseases of mature T cells, CD7 can be expressed in
different ways. In some diseases CD7 is missing, in others it is
constantly and strongly expressed, in others it is generally
expressed but sometimes is missing or expressed in an aberrant
way.
CD7 is missing in MF [366,370], in SS [367,369], in
AITL [124,275,378, 379], and in mucocutaneous γδ T‐cell lym-
phomas [488], renamed as PCGD‐TCL by the 2017 WHO
classification [539].
In ATLL, CD7 can be missing or dimly expressed [366,540],
and it has been reported that a bivariate analysis carried out for
CD3 and CD7 can allow the staging of the disease, distinguish-
ing asymptomatic HTLV‐I carriers and patients with smolder-
ing, chronic and acute disease on the basis of the quantitation of
three subsets of cells, i.e. CD7(−) CD3(±), CD7(±) CD3(±), and
CD7(+) CD3(+) [271].
CD7 is usually expressed in T‐PLL [541], but it can also be
absent [272], particularly in the so‐called small cell vari-
ant [542]. In PTCLnos, an aberrant expression of the antigen
has been reported in 45–80% of the observed cases, depending
on the survey [45,123,124].
Moreover, an aberrant expression of the antigen has been
reported in some cases of T‐LGL [128,388,487], in a case of
hepatosplenic αβ T‐cell lymphoma (HSTCL) [489], and in
25
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21, three days prior to discontinuance, there were only 34,271
female applicants for out-of-work donations.[271] Yet on the whole,
even though there was for a few months an alarming amount of
unemployment among women workers, officials held that British
industry adjusted itself to peace more quickly than it had to war. A
long list of factories which had changed from war to peace products,
for instance from airplanes to furniture and from fuses to electric
equipment, was given as early as February. Government control of
raw materials was used to aid the transition, and priority was given to
certain essentials in using the productive capacity set free from war
work.
The independence among women workers which had developed
during the war was reflected in their attitude during the period of
great unemployment. In the similar crisis at the beginning of the war
they had been inarticulate. But on February 15, 1919, their
organizations arranged a meeting in Albert Hall, London, attended by
women representing nearly every trade, at which women speakers
dwelt on the folly of unemployment while the country was in need of
all kinds of manufactured articles. Resolutions were passed giving
the three points of the “Women’s Charter”—“the right to work, the
right to live and the right to leisure.” It was held that all workers by
hand or brain should unite, and that work should be provided for the
unemployed. An adequate living wage, an eight hour day and a forty
hour week were advocated as standards for working conditions. A
deputation was organized to take the resolutions to the Prime
Minister, but apparently he did not reply to them.[272]
The measures actually adopted by the government show many
traces of the Civil War Workers Committee recommendations,
though, hastily put in force as they were, they were much less
complete, and in some cases widely different. The arrangements
made but little distinction between men and women workers. The
whole process of “demobilizing” war workers was put in charge of a
“controller general” responsible to the Ministry of Labor, who
controlled the employment exchanges, a new “Appointments
Branch” for “men of office rank” and the labor departments of the
Ministry of Munitions, the Admiralty and the War Office. The
employment exchanges were made the center for the transfer of war
workers. By the day after the armistice the recall of employment
exchange officials from the army had been arranged. Staff and
premises were enlarged and additional local advisory committees
formed. Various efforts were made to provide raw materials and to
hasten the change to peace time work by munition manufacturers.
Instructions to manufacturers asked them to avoid an immediate
general discharge of workers, to abolish all overtime and piece work
at once, and to retain as many workers as possible on short time. If
wages under this plan fell below certain levels, which were for
women 25s. ($6.00) a week, the government agreed to make up the
difference. In case of actual discharge, a week’s notice or a week’s
pay was to be given, and free railway passes home or to new work
places were provided. “The loyal and cordial cooperation of all
employers” in carrying out the directions was invited, but nothing is
at hand to show to what extent they were observed or how far they
lessened unemployment. It will be noted that men and women
workers were treated practically alike under this scheme. The
“Waacs” and other women auxiliaries of the army and navy were
demobilized under the same conditions as all members of the
military forces, receiving, besides certain gratuities, a civilian outfit,
four weeks’ pay and a railway pass.
Special provision for unemployed women through training
courses was outlined in a pamphlet issued by the government in the
spring of 1919.[273] It was stated that a large number of typical
women’s trades, such as clothing, textiles, food manufacture and
laundry work, would be covered by short training courses of from
one to six months’ duration, usually three months. In addition a
special course in housekeeping would be offered. The courses might
be given in any suitable place, such as a factory, as well as in trade
schools and the government instructional factories formerly used for
training munition workers. Approved students were to receive 15s. to
25s. ($3.50-$6.00) a week while taking the course, with traveling
fares if necessary, and an additional 10s. ($2.40) weekly if obliged to
live away from home.
 When the government adopted for immediate action the plans for
relieving unemployment previously outlined it also put forward
certain other schemes for decreasing unemployment during the later
reconstruction period, which included the stimulation of orders and
contracts, public and private, an increase in public works and
improvements and the extension of contributory unemployment
insurance to practically all workers.
The chief reliance of the government in dealing with
unemployment after the armistice was not a contributory insurance
plan, but a system of unemployment “donations.” Before the war
contributory unemployment insurance, paying 7s. ($1.68) a week to
unemployed workers for fifteen weeks a year from a fund created
through small weekly contributions for employers, employes and the
government, covered 2,200,000 workers in six trades, almost all of
whom were males. In 1916 the law was extended for a period of
from three to five years after the end of the war to include most of
the chief war industries with an additional 1,500,000 employes,
including many women. But by an emergency order made within a
few weeks after the armistice, the contributory insurance law was
temporarily superseded by a scheme of “donations” applying also to
all war workers not previously covered and all ex-soldiers and
sailors. Free policies were issued, at first good in the case of civilians
for six months beginning November 25, 1918, and in the case of
soldiers, for twelve months from the date of demobilization. The
policies provided their holders with donations while unemployed for
thirteen weeks if civilians and twenty-six weeks if soldiers. The
original scale was 20s. ($4.80) weekly for women workers, which
was raised after a few weeks to 25s. ($6.00). Additional payments
were made for dependent children, amounting to 6s. ($1.44) weekly
for the first and 3s. (72 cents) for each succeeding child. A later
amendment permitted payments to civilians for an additional thirteen
weeks at a reduced rate, which was, for women, 15s. ($3.60) weekly.
Later, in May, 1919, when according to the terms of the original order
all donation policies held by civilians would have expired, they were
renewed for an additional six months. Except for ex-service men and
women, the system was finally discontinued on November 25, 1919.
At this date 137,000 civilians were receiving donations, of whom
29,000 were females. All donations were paid through the
employment exchanges and could be stopped if the recipients
refused “to accept suitable employment.”
Undoubtedly the system of unemployment donations prevented
much suffering among thousands of wage earners to whom the
country was indebted for their war work. But as a whole its operation
can not be said to have been satisfactory, particularly among women
employes. An entire session of the House of Commons was devoted
mainly to criticisms of the system and its defence by the Minister of
Labor. Complaints of “slackers” who were taking a vacation at the
taxpayers’ expense were met by charges that women were being
forced to take places at sweated wages by refusals to pay the
unemployment donations. In the five months ending April 25, 1919,
claims for donations numbering 141,770 were disallowed, in 100,442
of which cases appeals to the referees were made. Only 27,536 of
the appealed claims were finally allowed, 81 per cent of the women’s
claims being denied, about half of them on the ground of “refusal to
accept suitable employment.”[274]
The Ministry of Labor, which administered the unemployment
donations, admitted that an unsatisfied demand for women workers
existed in domestic service, laundries, the needle work trades and in
some districts in the textile industry at the same time that half a
million women were out of work. But the places open were either
very highly skilled or grossly underpaid and unattractive. For one firm
which needed 5,000 workers, the employment exchanges could find
only fifty women who seemed qualified, of whom the firm hired only
fifteen.
The association of laundrymen even appealed to the government
to bring pressure to bear on the women to accept work, but
apparently no action was taken in answer to the demand. The
women workers themselves said that when the government had
raised the rate of unemployment donations from 20s. to 25s. weekly
on the ground that a single woman could not live on less, they could
not be expected to enter laundries at 18s. ($4.32) a week.
 Other less prominent difficulties of adjustment were the
reluctance of soldiers’ wives to enter new kinds of work when they
would retire from industry in a few months, and the unwillingness of
women in general to go from the comparatively high wages of
munitions to the low wages of learners and to factories lacking the
conveniences of the new munitions plants.
Criticism of the system was so widespread that an official
investigating committee was formed which issued two reports.[275]
The committee concluded that there had been no widespread fraud,
though under the plan as first put in operation it was possible legally
for persons who were not genuinely seeking work to abuse the
scheme. The committee felt, however, that the emergency had been
great and that if the later safeguards had been introduced in the
beginning the whole system might have broken down. They
recommended, among other points, swifter prosecution of fraud, a
contributory rather than a noncontributory plan, and discontinuance
of allowances based on the number of dependents. They felt that
applicants must not expect exactly the same sort of work or wage
rates that they had had during the war, and that donations should be
stopped if similar work was refused.

The Domestic Service Problem

Some of the main difficulties and the keenest discussion centered
on the question of domestic service. That the Ministry of
Reconstruction found it advisable to appoint a “Women’s Advisory
Committee on the Domestic Service Problem,” which made a formal
report, indicates the extent of agitation on the subject. It will be
recalled that during the war the number of household servants
decreased by 400,000. Householders seemingly expected that as
soon as the war was over this shortage would be made up from the
ranks of ex-munition workers. But this failed to occur. Some
dissatisfaction with the wages offered, most frequently 10s. to 13s.
($2.40 to $3.12 a week, with board) was expressed, but the chief
complaint was that of long hours and unsatisfactory personal
treatment.
 Various schemes for attracting workers by improving conditions
were put forward, some of which involved radical changes from the
usual customs. The majority of the official Women’s Advisory
Committee, however, placed its chief emphasis in solving the
problem merely on the provision of improved methods of training,
notably a two year course to be entered by girls of fourteen. Other
groups, such as the Fabian Women’s Group and the Women’s
Industrial Council, advocated plans which in essence abolished all
“living in,” and provided for hostels giving training which would send
qualified workers into the homes for a fixed number of hours. By May
the Young Women’s Christian Association was ready to open a
hostel in London from which workers were to be sent out on an eight
hour basis. Employers were to pay 10d. (20 cents) an hour to the
hostel, and the workers were to receive 30s. ($7.20) for a forty-eight
hour week, and to pay the hostel £1 ($4.80) weekly for board, for a
guarantee against unemployment, for use of uniform and club
privileges. If the hostel was successful, others were to be started.
[276]

Meanwhile an active movement for union organization among

domestic servants was begun, and forty branches having 4,000
members were formed in the four or five months after the armistice.
The chief aim of the union was said to be the raising of the status of
domestic service so that the workers would be proud of it. Its
standards seemed to be comparatively modest—a minimum weekly
wage of 12s. 6d. ($2.40) for general servants and 15s. ($3.60) for
cooks, a ten hour work day during a fourteen hour period, part of
Sunday and another half day off weekly and abolition of uniforms.
This last demand perhaps represented the sharpest departure from
prevailing customs. In Glasgow a “Mistresses’ League” was formed
to cooperate with the union, and it was the general opinion of
persons interested that both sides needed organizing.
Still “a house is not a factory,” and there were not wanting friends
of the women worker to point out that domestic service must
necessarily remain to some extent individual and unstandardized.
I am profoundly sceptical as to the various
“industrialised” suggestions put forward—the
 introduction of shifts, etc. How could a household
worker strictly on a shift system deal with the
irregular incursion of visitors, children home for the
holidays, measles, influenza, spring cleaning and
other ills to which mortal flesh is heir?...
From the maid’s point of view, I take it the main
disadvantages of domestic service are twofold; the
question of free evenings and the uncertainty as to
the type of household. Time off in the afternoon is
naturally of less value than time off at night.
Similarly a maid may find herself on taking a new
situation in a comfortable home or very much the
reverse.
In a house organized on proper lines, domestic
service has compensations as well as drawbacks. A
just mistress will arrange for adequate time off, even
if the home can not be laid down each week with
mathematical exactness. She will see that her
maids are properly housed, that their food is
adequate and properly cooked, that their work is
organized on sensible lines and gives as much
scope as possible for individual responsibility. In a
household which lives literally as a family and is
inspired with mutual consideration and good will
“that servant problem” simply does not exist. When
mutual consideration and good will are lacking
neither corps, caps, correspondence nor
conferences will create the cement by which a
contented household is held together.[277]
It is difficult to tell how far these new schemes will change the
conditions of housekeeping and lessen unemployment by attracting
women to domestic service. But the fact that they were put forward is
an interesting sign of the extent of the movement for reconstructing
the national life on better lines.
 Dilution and Equal Pay
The other two chief problems of the women workers in the
reconstruction period, that of the “dilutees,” who had taken up men’s
work during the war, and that of “equal pay for equal work” and an
adequate standard of wages for women workers generally, were
closely related to each other. Much of the opposition of the men
workers to the entrance of women into new occupations was based
on the fact that women’s wage standards were lower than those of
men. In most cases, it will be remembered, dilution had taken place
under promises that it would last only during the war. Parliament, by
the Munitions Act, had given the government’s pledge that
departures from prewar practices should be merely temporary in the
establishments covered.[278] Similar clauses, often even more
explicit, were found in practically all the substitution agreements
made by private employers with labor organizations.[279] Meanwhile
the fixing of women’s wages by law had been widely extended, and,
in the opinion of close students of labor problems, “a removal of the
statutory regulations might well be followed by a serious and
immediate fall in wages.”[280]
The government in several instances took action on matters
connected with women’s wages and occupations after the war, but it
is not too harsh to say that a disposition to tide over difficulties
temporarily rather than to define any very clear line of policy was
evident. Two laws were passed affecting the after war wages of
women. The Trade Boards (minimum wage) Act was extended in
1918, before the close of the war, as a measure of preparedness for
peace. “There is reason to fear that the after war dislocation of
industry will make the problem of adequate wages for unskilled and
unorganized workers, especially women, very acute,” said an official
explanation of the changes in the act.[281] “Eight years’ satisfactory
results of Trade Boards pointed to these as the best way of meeting
the situation.” The new law provided that boards might be formed
wherever wages were unduly low, instead of exceptionally low as in
the original law. The general wage level for women workers was so
low before the war that it had often been difficult to prove an
“exceptional” condition. Provisions were also made to have minimum
wage awards come into force more quickly. By the spring of 1919
new Trade Boards had been formed in eight industries.[282] They
apparently fixed wages for women on the basis of the necessary
cost of living for a single woman—28s. ($6.72) for a forty-eight hour
week in laundries, for example.
But the Trade Boards covered only a fraction of the industries of
the country, and further measures were considered necessary to
prevent a dislocation of wages. Following the advice of a committee
appointed by the Ministry of Reconstruction, the Wages (Temporary
Regulation) Bill was passed November 21, 1918. This act required
employers to pay the “prescribed” or “substituted” rate which
prevailed at the time of the armistice for a period of six months. In
May, 1919, the provisions of the act were extended for another six
months. Under this law an Interim Court of arbitration was set up
which handled the arbitration of disputed wage cases. During the
year of its existence it made 932 awards and advised on several
others. On November 20, 1919, this Interim Court was displaced by
the Industrial Courts Act, which in addition to its function of voluntary
arbitration, extended certain parts of the Wages Temporary
Regulation Act until September 30, 1920.[283] At the close of the war
the greatest number of women were substituting for men on semi-
skilled and repetition processes, and it was therefore semi-skilled
men who were menaced most immediately by the danger of
undercutting by the women. But in the rapid extension of specialized
work during the war lay an evident threat to the position of the skilled
worker. A right solution of the two questions, in which the interests of
all the groups concerned would be safeguarded, would apparently
involve a modification of prewar conditions, rather than a return to
them.
Three points of view were evident in English opinion about
women’s work and wages after the armistice. The first point of view
was, briefly, that women workers would and should return to their
prewar occupations. But little attention was given to the question of
their wage level. Whether such a return was possible or just to the
women themselves, or whether they might not be excluded for a time
but remain potential competitors with low wage standards, thus
bringing about the very danger they were trying to avoid—all this
was seemingly not considered. Though relatively seldom expressed
in print it was a viewpoint held widely and tenaciously. Government
officials, visiting America in November, 1917, for instance, said that
marriage, the return of married women to their homes and the revival
of the luxury trades and domestic service, would relieve the situation.
Many old line trade unionists also believed that women should not be
allowed to remain in most of their new lines of work, and demanded
the literal fulfilment of all pledges to that effect. The general
secretary of the Postal and Telegraph Clerks’ Association, at a
conference of “Working Class Associations” said as to the basis of
suitable occupations:
My own view, for what it is worth, is that this
problem could be solved with very little trouble. I
think a careful study of the census returns for the
last thirty years would help to solve the problem of
the basis of suitability. We could safely conclude
that the occupations which, according to the census,
show a steady and persistent increase in the
number of women employed are suitable
occupations for the extension of women’s labour. I
think we must face it ... that, as far as we can see at
present, the prewar standard for fixing wages as
between men and women is likely to remain.
A second point of view, which might be termed the “moderate”
one, compromising between prewar and war conditions, advocated
the retention of women in all “suitable” occupations, together with an
extension of protective labor legislation, protection of the wage level
by minimum wage fixing, and “equal pay for equal work” where men
and women remained in the same occupations. This opinion was
evident in the two chief official reports on women’s work which have
been issued since the armistice, that of the Home Office on
“Substitution of Women in Nonmunition Factories during the War”
and that of the “War Cabinet Committee on Women in Industry.” The
former described a fairly large range of new employments as
“suitable” for women, including positions in scientific laboratory work,
supervision and management, as well as factory processes. Even
with all unsuitable occupations set aside, there remained “a body of
industries and operations offering a hopeful field of fresh
employment to women, where their war experience can be turned to
account, and should prove a national asset of great value.” Among
the approved trades were light leather tanning, fancy leather
manufacture, box and packing case making, furniture, scientific
instrument making, flint glass cutting and engraving, and cutlery,
except scissors manufacture. The factors causing an occupation to
be disapproved were the heaviness of the work, the use of
dangerous machinery or poisonous substances, the presence of
exceptional heat, wet or dirt and the necessity for night work or
solitary employment.[284] Basing its conclusions on considerations of
“efficiency” and relative output, the War Cabinet Committee decided
that women would probably not remain in heavy manual labor and
out door work. There had not been time during the war to judge of
their effectiveness in skilled work, but in routine and repetition
processes, into which the war had hastened their “normal”
movement, they had been successful and were likely to stay
permanently. Repetition work in the metal trades, light work in
chemical plants, certain processes in printing, woodworking and
manufacture, agriculture, commerce and government positions, and
many of the new administrative and professional openings for
educated women, were mentioned by the War Cabinet Committee as
providing possibilities for the continued work of women.[285] But both
reports recognized that many other factors besides suitability,
notably the attitude of the trade unions, would play an important part
in determining the position of the woman worker.
The chief purpose of the investigations of the War Cabinet
Committee was to decide on the proper relation between the wages
of men and of women. The majority of the committee concluded that
when men and women did radically different work, it was “not
possible to lay down a relation between their wages.” However, for
the protection of women workers they urged a universal minimum
wage for adult women, sufficient to cover the necessary cost of living
for a single woman. The extension and strengthening of protective
labor laws was also endorsed, and the possibility of such regulation
through international action was welcomed. But when the two sexes
had entered the same occupations, the committee subscribed to the
principle of equal pay for equal work, “in the sense that pay should
be in proportion to efficient output.” The committee believed that
piece rates should be equal and time rates should be fixed by trade
union negotiation. In the frequent case in which a woman was doing
part of a man’s job, the total rate should be unchanged, and the
different workers should be paid in proportion to the value of their
contribution. Where processes were simplified on the introduction of
women, the women should be paid the unskilled men’s rate, unless it
could be proved that their work was of less value.
The third position regarding women’s wages and women on
men’s jobs was clear cut and uncompromising and was perhaps
typified in a minority report to the War Cabinet Committee by Mrs.
Sidney Webb. In this report Mrs. Webb expressed the belief that
existing relations between men’s and women’s employment were
harmful to individuals and to the nation. All occupations should be
opened to qualified persons regardless of sex, at the same standard
rates and under the same working conditions. “Equal pay for equal
work” was an ambiguous and easily evaded phrase. A national legal
minimum wage should also be fixed, in which “there should be no
sex inequality.” As a corollary to the proposals Mrs. Webb believed
that some form of public provision for the needs of maternity and
childhood should be established. “There seems no alternative—
assuming that the nation wants children—to some form of state
provision, entirely apart from wages.”[286]
Eighteen months after the signing of the armistice it was still
hardly possible to know definitely what the after war wages and
occupations of the woman worker would be. After war industrial
conditions in themselves naturally stimulated some return of women
to their former occupations. Many of the women substitutes were
found in munition making which was immediately curtailed, while the
luxury, the needle and other “women’s” trades, depressed during the
war may be expected to revive in time. The reluctance of women to
enter these trades under the prevailing wage standards was very
pronounced, however. Another important factor in forcing women
back to prewar lines of work was the carrying out of certain war time
substitution agreements. For example, the newly formed industrial
council of the wool textile industry, representing employers and
employes, adopted on February 3, 1919, the substitution agreement
made between employers and work people of the West Riding of
Yorkshire three years before. By the terms of this agreement, the
returning soldiers were to get their places back when fit for
employment. Women were not to be employed on men’s work if men
were available and were to be the first discharged if there was a
shortage of work. As long as women substitutes remained in the
industry they were to be paid on a basis equivalent to that of men
workers.
But in other cases, even though similar agreements exist, it
appears probable that they will be modified to allow women to keep
at least some of their new jobs. Although the Amalgamated Society
of Engineers had the legal sanction of the Munitions Acts for
excluding women from engineering at the end of the war, at a
conference between employers and the union for drafting an after
war trade agreement their president expressed his willingness to
allow women to remain in semi-skilled repetition work. According to
this official much of this kind of work would be carried on in munition
plants converted into factories for the manufacture of articles
formerly imported. Officials expect the so-called “Whitley” industrial
councils of employers and employes to make many similar
adjustments, but it has been noted that the council in the woolen
industry merely reverted to prewar conditions and arranged to shut
out the women. Moreover, in many new occupations, notably clerical
and commercial work, which women entered without conditions, and
where their efficiency has been demonstrated, it seems almost
certain that they will remain. The awakened spirit among women
workers and the growth of labor organizations among them, which
will give voice to their demands, must also not be forgotten in judging
whether women will not continue to occupy at least a part of their
new field of work. The radical point of view, that there should be no
barriers against their continuing all their new occupations has
attracted much attention from its logical presentation and the new
note that it strikes.
The position of the government on “dilution” is not wholly clear.
During the Parliamentary campaign of December, 1918, Lloyd
George, in answer to questions from Lady Rhondda of the Women’s
Industrial League, stated that he intended to carry out the terms of
the Treasury Agreement of 1915, which promised to restore prewar
practices. But “the government had never agreed that new industries
come under the Treasury Agreement.” Women could find
employment in these, which were already extensive, and in their
prewar occupations. The Prime Minister also stated that he was “a
supporter of the principle of equal pay for equal output. To permit
women to be the catspaw for reducing the level of wages is
unthinkable.” In his stand at this time, Lloyd George appeared to
approach the middle-of-the-road compromising position of the
majority of the War Cabinet Committee on Women in Industry.
A somewhat similar stand was taken in the “Restoration of
Prewar Practices Act” of August 15, 1919, which arranged for the
fulfilment of pledges made in the Treasury Agreement. It required the
owners of the establishments covered—mainly those engaged in
munitions work—to restore or permit the restoration of prewar trade
rules and customs, and to allow such prewar practices to be
continued for a year.
Rules laid down by the Ministry of Labour are quoted, however,
which would turn out all the “dilutees,” both male and female, and
give back to the skilled men their former monopoly. The rules state
that wherever a part of the force must be discharged, the “dilutees”
must go first and that if a skilled man applies for work, a “dilutee”
must be discharged if necessary.[287] It is probable that these rules
apply only to establishments covered by the Munition Acts, but, as
far as they go, they leave the women nothing of their war time gains.
On the other hand, in assenting to the recommendations of the
national Industrial Conference, the government agreed with those
who argued for the same protective legislation for both sexes along
with state maternity provisions. This national industrial conference,
representing employers and employes was called in the spring of
1919 during great labor unrest. It urged legislation for a forty-eight
hour week and a universal minimum wage for both sexes, and such
bills were pending in Parliament in September, 1919.
The conference also proposed that public provision for maternity
care be extended and centralized under the Ministry of Health to
whose creation the government was pledged. Maternity protection
will undoubtedly hold a prominent place in legislation during the next
few years. The successful strike of the women bus workers for equal
pay, supported as they were by their male coworkers and by the
public, gave hope for the coming of industrial equality between men
and women. Such equality immediately raises the question of pay for
the services which married women render to the state. The rearing of
healthy children is of vital national importance and the endowment of
motherhood, provision of milk and proper food for pregnant and
nursing mothers and the extension of maternity centers and
hospitals with medical and nursing care, are already under
consideration by the newly created Ministry of Health.

Child Workers After the War

On the needs of children there was much more general
agreement. The most pressing problem was prevention of
unemployment during the readjustment from war to peace time
production. The larger issue lay in greater public control over the first
years of working life, to the end that the young workers might grow
into better citizens. Both problems were undoubtedly made more
difficult by the harm done to boys and girls in body and character by
the war. But at the same time the war had roused a greater
appreciation of the value of these future citizens and a greater
determination to improve their chances.
Alarming forecasts were made as to the probable extent of
unemployment among boys and girls at the end of the war by a
committee of enquiry appointed by the Ministry of Labour at the
suggestion of the Ministry of Reconstruction.[288] A number of
munition firms which were canvassed said that they intended to
discharge nearly half their boys and three quarters of their girls when
peace was declared. It was estimated that 60,000 out of the 200,000
working boys and girls in London would be thrown out of a job. Acute
unemployment was predicted in occupations that had engaged more
than three-tenths of all working girls—the metal, woodworking and
chemical trades, government establishments, transport and perhaps
commerce.
It was likewise anticipated that it would be particularly difficult for
boys and girls dismissed at the end of the war to find new places.
Not only would openings be few and the numbers of adults seeking
work be large, but the high wages children had received for
repetition work on munitions would make them unwilling to learn
trades or to accept lower pay. When a number of boys were
discharged from munition plants in 1916-1917, although labor at that
time was very scarce, great difficulty was found in getting them new
places because of their unwillingness to accept ordinary wages. To
meet the crisis the Ministry of Reconstruction committee suggested a
comprehensive program for unemployment prevention. The
discharge of war workers should be regulated and placement
centered in the employment exchanges, whose juvenile employment
committees were to be strengthened. Government establishments
should hold back dismissals until notified that places were open. A
canvass for possible openings and for probable dismissals should be
made in advance of the end of the war.
The second point in the committee’s plan was keeping
newcomers out of industry. The exemptions allowing children under
fourteen to leave school should be abolished, scholarships provided
for many capable children at secondary schools, and the working
weeks for all under eighteen reduced to forty-eight hours. For those
still uncared for, training during unemployment should be provided.
Training centers should be opened in all towns of over 20,000
population and allowances made to parents whose children
attended. For the boys most demoralized by war work it might even
be necessary to open residential training camps where they could
remain at least six or eight weeks.
The third main point in the program was the improvement of
working conditions, including for all occupations a week of forty-eight
hours for work and continuation school together, the abolition of night
work, and a searching physical examination before entering industry.
A novel recommendation was that it should be made a legal offence
to employ young persons under conditions “impeding their training.”
But as was the case with the women workers, the comprehensive
plans worked out under the Ministry of Reconstruction had not been
adopted when the armistice was signed, and juvenile workers were
helped through the unemployment crisis only by the incomplete
makeshifts hastily adopted in the first few days after November 11.
Chief among these was the provision of unemployment donations,
the payment of which was conditional on attendance at a training
center wherever one was available. The donations were payable for
the same period as those for adults, that is, for thirteen weeks during
the first six months of peace, later extended for a second six months,
but were less in amount, being 14s. 6d. ($5.48) weekly for boys and
12s. 6d. ($3.00) for girls. During the first few months of 1919, about
50,000 young persons received the donations.
The number receiving donations steadily declined until on
November 21, 1919, when civilian donations ceased, there were
8,000 boys and 2,287 girls on the Labor Exchange donation lists.
[289] By February of that year 116 training centers had been opened,
providing nearly sufficient in London, and a smaller number
elsewhere. More were opening daily, but it was hard to find teachers
and rooms. The centers were managed by the Board of Education,
in close cooperation with the employment exchanges. About 13,500
boys and girls were in attendance daily.[290]
The Fisher Education Law is, to date, the chief constructive
measure looking toward a permanent improvement in the condition
of juvenile workers. This measure was the result of proposals made
by 1917 by an official committee on “Juvenile Education in Relation
to Employment after the War,” which were strikingly like those put
forward by a number of workers’ organizations. All exceptions
allowing children to leave school before the age of fourteen were
abolished. Any gainful employment by children under twelve was
forbidden, and children between twelve and fourteen might work only
on Saturdays and for a few hours after school. Attendance at
continuation schools by all young workers was required, and the age
limit will be eighteen years when the law goes into full effect. Eight
hours a week and two hundred and eighty hours a year must be
given to continuation school, the time for attendance being taken out
of working hours. Unfortunately, those who in some ways most need
the protection of the law, namely, the boys and girls who left school
for work prematurely during the war, do not come under its
provisions. Two special sections exempted those who had already
left school from returning, and those fourteen years old or more
when the law was passed, from attendance at continuation classes.
Nevertheless by the enactment of this law the final effect of the war
on English child labor standards should be to lift them to a higher
level than ever before.
Even at this time of writing it is difficult to measure the final
effects of the war upon the economic conditions of the women and
children. Too many unfinished plans and unfulfilled pledges still
remain for action by the government. Far reaching changes are,
however, in prospect and some of them actually under way.
Foremost among these is the aroused spirit among the workers, who
are demanding and peacefully securing a real share in the
management of industry. In this awakening the woman worker has
fully participated. The disadvantages of war work, in long hours,
overstrain, the disruption of home life, may pass as industrial
conditions return to normal. The gains in the way of better working
conditions, higher wages and a wider range of occupations seem
likely to be more permanent. Most important of all is the fact that
because of her broader and more confident outlook on life, the
woman worker is able consciously to hold to the improved economic
position to which the fortunes of war have brought her.
 APPENDICES
Appendix A
The following table, from a “Report to the Board of Trade on the State of Employment in the United
Kingdom,” of February, 1915, compares the number of males and females on full time, on overtime, on
short time, and unemployed, between September, 1914, and February, 1915.

STATE OF EMPLOYMENT IN SEPTEMBER, OCTOBER

AND DECEMBER, 1914, AND FEBRUARY, 1915
(Numbers Employed in July = 100 per cent.)
September, 1914 October, 1914 December, 1914 February, 1915
M F M F M F M F
60.2 53.5 66.8 61.9 65.8 66.6 68.4 75.0
Full time
3,913,000 1,337,500 4,342,000 1,547,500 4,277,000 1,665,000 4,446,000 1,875,000
3.6 2.1 5.2 5.9 12.8 10.8 13.8 10.9
Overtime
234,000 52,500 338,000 147,500 832,000 270,000 897,000 272,500
26.0 36.0 17.3 26.0 10.5 19.4 6.6 12.6
Short time
1,690,000 900,000 1,124,500 650,000 682,500 485,000 390,000 15,000
Contraction in 10.2 8.4 10.7 6.2 10.9 3.2 11.8 1.5
Nos. employed 663,000 210,000 695,000 155,000 708,500 80,000 767,000 37,500
8.8 ... 10.6 ... 13.3 ... 15.4 ...
Enlisted
572,000 ... 689,000 ... 864,500 ... 1,010,000 ...
Net displacement (-) -1.4 -8.4 -0.1 -6.2 +2.4 -3.2 +3.6 -1.5
or replacement (+) -91,000 -210,000 -6,500 -155,000 +156,000 -80,000 +243,000 37,500

Appendix B
The following table indicates some of the processes formerly reserved for men on which the factory
inspectors found women employed by the end of 1915:
INDUSTRY PROCESSES
Linoleum Attending cork grinding and embossing machines,
machine printing, attending stove, trimming
and packing.
Woodworking—
Brush making Fibre dressers, brush makers and on boring
machinery.
Furniture Light upholstery, cramping, dowelling,
glueing, fret-work, carving by hand or
machine, staining and polishing.
Saw mills On planing, moulding, sand-papering, boring,
mortising, dovetailing, tenoning, turning and
nailing machines. Taking off from circular
saws; box making, printing and painting.
Cooperage Barrel making machines.
Paper mills In rag grinding and attending to beating and
breaking machines, and to coating machines,
calenders and in certain preparations and
finishing and warehouse processes.
Printing Machine feeding (on platen machines and
 INDUSTRY PROCESSES
on guillotines) and as linotype operators.
Wire rope On stranding and spinning machines.
Chemical works Attending at crystallising tanks and for
yard work.
Soap As soap millers and in general work.
Paint At roller mills, filling tins and kegs,
labeling and packing.
Oil and cake mills Trucking, feeding and drawing off from chutes,
attending to presses.
Flour mills Trucking.
Bread and biscuits Attending to dough-breaks, biscuit machines,
and at the ovens assisting bakers.
Tobacco Leaf cutting, cigarette making, soldering,
trucking and warehouse work.
Rubber At washing machines, grinding mills, dough
rolls, solutioning, motor tube making.
Malting Spreading and general work.
Breweries Cask washing, tun-room work, beer bottling
and bottle washing.
Distilleries In the mill and yeast houses.
Cement Attending weighing machines, trucking.
Foundries Core making, moulding.
Tanning and currying At the pits, in finishing and drying, and in
oiling, setting up, buffing and staining.
Woolen mills Beaming and overlooking, attending drying
machines, carding, pattern weaving.
Jute mills On softening machines, dressing yarn,
calendering.
Cotton mills In blowing room on spinning mules, beaming,
twisting and drawing, and in warehouse.
Hosiery Folding and warehouse work.
Lace Threading.
Print, bleach and Beetling, assisting printers at machines,
dye works warehouse processes.

Appendix C
The following tables from the second report of the British Association for the
Advancement of Science bring out in detail, first, the gradual disappearance of
unemployment and short time and the increase of women’s numbers in
industry from September, 1914, to April, 1916; second, the changes in
numbers of women in the various occupations, both industrial and nonindustrial
in December, 1915, and April, 1916, compared with July, 1914, and, third,
similar details as to the number of women who were undertaking “men’s work.”

STATE OF EMPLOYMENT OF WOMEN AT VARIOUS DATES

SINCE THE OUTBREAK OF WAR, COMPARED WITH STATE
OF EMPLOYMENT IN JULY, 1914
(“Industrial” employment only.
Numbers employed July, 1914 = 100 per cent.)