Diagnostic Flow Cytometry in Cytology: Pranab Dey

Diagnostic Flow
Cytometry in
Cytology
Pranab Dey
123
Diagnostic Flow Cytometry in Cytology
Pranab Dey
Diagnostic Flow
Cytometry in Cytology
Pranab Dey
Professor, Department of Cytology and Gynec Pathology
Post Graduate Institute of Medical Education
and Research (PGIMER)
Chandigarh, Chandigarh
India
ISBN 978-981-16-2654-8 ISBN 978-981-16-2655-5 (eBook)

https://doi.org/10.1007/978-981-16-2655-5
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2021
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Dedicated to
Shree Shree Satyananda Giri, Shree Shree
Paramahansa Yogananda,
Rini, and Madhumanti
Preface
This book highlights the practical aspect of flow cytometry and its application in
diagnostic cytology. The book has two parts: The fundamental and technical details
(Part I) and the diagnostic application part (Part II). Part I highlights the basic prin-
ciple and techniques of flow cytometry, sample preparation, data acquisition, differ-
ent fluorochrome dyes and quality control. Part II contains the diagnostic applications
of flow cytometry in cytology. The book contains numerous figures, graphs of flow
cytometry, boxes and tables. I hope the present book will be beneficial to understand
the subject and apply it in daily work.
Chandigarh, India Pranab Dey

June 2021
vii
Acknowledgements
I am thankful to Dr. Naren Aggarwal and Ms. Jagjeet Kaur Saini of Springer Nature,
who encouraged me to write this book. I wish to thank Saanthi Shankhararaman of
Springer Nature for her continuous support at the time of preparation of the manu-
script of the book. My colleagues in the department also deserve my thanks for their
support in day-to-day work.
My heartiest thanks to my wife Rini and my daughter Madhumanti for ongoing
support and encouragement. They were constantly with me in every stage of
the book.
Finally, I wish to express my gratitude to Almighty God for His immense
blessings.
ix
Contents
Part I Practical Aspects of Flow Cytometry

1 Introduction and History of Flow Cytometry �� 3
1.1 Introduction�� 3
1.2 History of Flow Cytometry �� 3
1.2.1 Early Motivation �� 3
1.2.2 Counting the Flowing Cells�� 4
1.2.3 Haematology Sample�� 4
1.2.4 Differential Count in the Blood Sample �� 4
1.2.5 Flow Sorter�� 5
1.3 Fluorescence Stain in the Flow Cytometer �� 5
1.4 Further Improvements in Flow Cytometry �� 6
References�� 7
2 Basic Principles and Instrumentation of Flow Cytometry�� 9
2.2 Principles of Flow Cytometry �� 9
2.2.1 The Fluidics System�� 11
2.3 Optical System�� 13
2.4 Fluorescence Emission�� 15
2.4.1 Collection of Light�� 15
2.4.2 Optical Filters�� 16
2.4.3 Electronics System�� 17
2.4.4 Computer System�� 19
2.4.5 Flow Cytometric Cell Sorting�� 19
References�� 21
3 Sample Preparation and Data Acquisition in Flow Cytometry �� 23
3.2 Basic Requirements�� 23
3.3 Cytology Samples for Flow Cytometry�� 24
3.4 Sample Collection�� 25
3.5 Single-Cell Preparation�� 25
3.5.1 Limitations of the Enzymatic Method�� 26
3.6 Fixation �� 26
xi
xii Contents
3.7 Permeabilization �� 27

3.8 RBC Lysing Solution�� 27
3.9 Staining �� 28
3.9.1 DNA Flow Cytometry�� 28
3.9.2 Control�� 30
3.10 Data Acquisitions�� 31
References�� 34
4 Display and Interpretation of Data in Flow Cytometry �� 35
4.1 Distribution of Fluorescence Intensity�� 38
4.2 Gating�� 42
4.2.1 The Crucial Gating in Flow Cytometry�� 43
4.3 Backgating�� 46
References�� 46
5 Quality Control in Flow Cytometry �� 47
5.2 Internal Quality Control�� 47
5.3 Instrument Quality Control �� 48
5.4 The Critical Factors to Have Good Quality FCM Data�� 49
5.4.1 Sensitivity �� 49
5.5 PMT Voltage Setting �� 51
5.5.1 Compensation �� 53
5.6 Daily Cytometer Set up�� 54
5.7 External Quality Assessment (EQA)�� 55
References�� 55
6 Fluorescent Probes and Different Useful Markers
for Flow Cytometry�� 57
6.1 Staining by the Fluorochrome Dye �� 59
6.1.1 Applications of FRET �� 62
6.1.2 The Desirable Characteristic of a Fluorochrome Dye�� 64
6.1.3 Fluorochrome Dyes Used in a Flow Cytometer �� 64
6.1.4 Multicoloured Flow Cytometry�� 69
6.1.5 Basic Principles of Panel Design�� 70
References�� 73
7 Nuclei Acid Dye and DNA Content Measurement in Flow Cytometry 75
7.1 Types of DNA Dye�� 75
7.2 Description of Different DNA Dyes �� 77
7.2.1 Intercalator Dyes�� 77
7.2.2 Minor Groove Binding Dye�� 77
7.2.3 Bis-Intercalator Dyes�� 78
7.3 DNA Content and Ploidy Analysis �� 78
7.4 Standard Nomenclature�� 79
7.4.1 Control Diploid Population�� 80
7.4.2 Staining for DNA FCM�� 81
Contents xiii
7.5 Data Acquisition�� 81

7.6 Interpretation�� 82
References�� 82
Part II Diagnostic Applications of Flow Cytometry in Cytology

8 Classification of Lymphoma, Different Markers and Approach �� 85
8.1 Non-Hodgkin Lymphoma �� 87
8.2 Markers of Lymphoid Cell Lineage�� 89
8.2.1 NK Cell �� 90
8.3 Hodgkin Lymphomas (HL)�� 90
8.4 Limitations�� 90
8.5 Approach to Flow Cytometry of Lymph Node �� 91
8.6 Cytology Smear and Panel of Antibody�� 92
References�� 95
9 Markers for Immunophenotyping in Flow Cytometry�� 97
9.2 CD Markers�� 97
9.2.1 CD2 �� 97
9.2.2 CD3 �� 98
9.2.3 CD4 �� 98
9.2.4 CD8 �� 98
9.2.5 CD5 �� 99
9.2.6 CD7 �� 100
9.2.7 CD10 �� 100
9.2.8 CD11B�� 100
9.2.9 CD14 �� 100
9.2.10 CD15 �� 101
9.2.11 CD19 �� 101
9.2.12 CD20 �� 101
9.2.13 CD23 �� 102
9.2.14 CD25 �� 102
9.2.15 CD30 �� 102
9.2.16 CD38 �� 103
9.2.17 CD43 �� 103
9.2.18 CD45 �� 104
9.2.19 CD56 �� 104
9.2.20 CD79a �� 104
9.2.21 CD103 �� 105
9.2.22 CD117 �� 105
9.2.23 CD138 �� 105
9.3 Other Markers Used in Lymphoma�� 106
9.3.1 Terminal Deoxynucleotidyl Transferase (TdT)�� 106
9.3.2 HLA-DR �� 106
9.3.3 PAX5�� 106
References�� 107
xiv Contents
10 Detection of Lymphoma: Clonality Demonstration

by Flow Cytometry�� 109
10.2 Clonal Proliferation of B Cells �� 109
10.2.1 Light Chain Restriction�� 109
10.2.2 Aberrant Expression of Certain Antigen�� 109
10.3 Immature B Cells�� 110
10.3.1 Reactive Lymph Node�� 111
10.3.2 B Cell Lymphoma with no Light Chain Expression�� 112
10.3.3 Clonal Proliferation of T Cells�� 113
10.3.4 Aberrant Expression or Loss of T Cell Antigen�� 113
10.3.5 Abnormality of CD4 and CD8 Expression �� 114
10.3.6 Increased Forward Scatter�� 115
10.3.7 The Expression of Other Markers�� 115
10.3.8 Presence of Markers of Blasts�� 115
11 Flow Cytometry of B-Non Hodgkin Lymphoma�� 117
11.2 Diagnosis of Individual NHL�� 117
11.2.1 Small Lymphocytic Lymphoma (SLL) �� 117
11.2.2 Mantle Cell Lymphoma (MCL)�� 122
11.2.3 Follicular Lymphoma (FL) �� 123
11.2.4 Marginal Zone Lymphoma (MZL) �� 126
11.2.5 Lymphoplasmacytic Lymphoma (LPL)�� 127
11.3 Lymphomas of Large-Sized Cells�� 130
11.3.1 Diffuse Large B-Cell Lymphoma (DLBCL)�� 130
11.3.2 Burkitt Lymphoma (BL) �� 133
11.3.3 Hairy Cell Leukaemia (HCL) �� 136
11.4 Immature B Cell�� 137
11.4.1 B-Lymphoblastic Lymphoma �� 137
11.5 Plasma Cell Neoplasm�� 138
11.5.1 Differential Diagnosis �� 140
11.6 CD5 Positive B-Cell Lymphomas�� 140
11.6.1 CD10 Positive Lymphomas�� 141
11.7 CD5 and CD10 Negative Lymphoma �� 141
12 Flow Cytometry of Mature and Immature T-Cell Lymphoma�� 143
12.2 Mature T-Cell Lymphomas �� 144
12.3 Mycosis Fungoides and Sezary Syndrome �� 144
12.4 Peripheral T-Cell Lymphoma (PTCL)�� 147
12.5 Angio-Immunoblastic T-Cell Lymphoma (AITL)�� 148
Contents xv
12.6 Hepatosplenic T-Cell Lymphoma (HSTL)�� 149

12.7 Extranodal Natural Killer/T-Cell Lymphoma �� 149
12.8 Immature T-Cell Lymphoma�� 150
12.8.1 T-Cell Lymphoblastic Leukaemia (T-LBL) or Lymphoma
(T-ALL)�� 150
13 Flow Cytometry of Body Cavity Fluid �� 153
13.2 Detection of Malignancy in Fluid �� 153
13.3 DNA Flow Cytometry�� 154
13.3.1 Immunophenotyping to Detect Malignancy �� 155
13.3.2 Precautions to Take for the Best Result�� 159
13.3.3 Possible Pitfalls�� 159
13.4 Detection of Lymphoma in Fluid�� 160
13.4.1 Panel of Markers in Leukaemia/Lymphoma�� 160
13.4.2 Diagnostic Features�� 160
13.5 Primary Effusion Lymphoma (PEL) �� 164
13.6 Urine Flow Cytometry�� 164
14 Flow Cytometry of Solid Tumours �� 169
14.2 Advantages of Flow Cytometry in the Detection of Carcinoma�� 172
14.3 Diagnosis of the Small Round Cell Tumours �� 173
14.4 DNA Content Analysis and Synthetic Phase Assessment�� 174
14.5 Limitation of DNA Analysis by FCM�� 176
14.6 The Response of Cancer Chemotherapeutic Drugs�� 176
14.7 Expression of Oncogene Markers and Receptor Expression�� 177
15 Self-Assessment Test in Flow Cytometry �� 179
15.1 Answer Key of Chap. 15�� 193
About the Author
Pranab Dey is Professor in the Department of Cytology and Gynaecologic

Pathology at Postgraduate Institute of Medical Education and Research (PGIMER),
Chandigarh. Professor Dey completed his M.D. (pathology) from PGIMER,
Chandigarh, and FRCPath (cytopathology) from Royal College of Pathologist,
London. He has been a Consultant of cytology and gynaecologic pathology in
PGIMER for the last 29 years and Professor for the last 11 years. Professor Dey has
keen interest in flow cytometry and artificial intelligence and conducted many
research projects and has pioneered works on DNA flow cytometry, image mor-
phometry, mono-layered cytology and cytomorphologic findings of various lesions
on cytology smears. He is a well published author, has published several books in
the field of cytology and gynaecologic pathology, 300 publications in international
journals, and 150 original research works and 30 review articles. Professor Dey is a
member of various societies.
xvii
Abbreviations
ADC Analogue to digital conversion

AITCL Angioimmunoblastic T cell lymphoma
ALCL Anaplastic large cell lymphoma
ALL Acute lymphoblastic leukaemia
AML Acute myeloid leukaemia
AO Acridine orange
APC Allophycocyanin
APD Avalanche photodiode
ATLL Adult T-cell leukaemia/lymphoma
BL Burkitt lymphoma
BP Bandpass
CLL Chronic lymphocytic leukaemia
CSF Cerebrospinal fluid
CV Coefficient of variation
DAPI 4´,6-Diamidino-2-phenylindole
DI DNA index
DLBCL Diffuse large B cell lymphomas
EATL Enteropathy-associated T-cell lymphoma
EpCAM Epithelial cell adhesion molecule
EQA External quality assessment
ER Estrogen receptor
FCM Flow cytometry
FITC Fluorescence isothiocyanate
FL Follicular lymphoma
FNAC Fine needle aspiration cytology
FRET Fluorescence resonance energy transfer
FSC Forward scatter
GFP Green fluorescent proteins
HCL Hairy cell leukaemia
HSTL Hepatosplenic T-cell lymphoma
IQC Internal quality control
LGL Large granular lymphocytic leukaemia
LPL Lymphoplasmacytic lymphoma
MCL Mantle cell lymphoma
xix
xx Abbreviations
MF Mycosis fungoides
MRD Minimal residual disease
MZL Marginal zone lymphoma
NB Neuroblastoma
NHL Non-Hodgkin lymphoma
PBS Phosphate buffer solution
PD Photodiodes
PE Phycoerythrin
PerCP Peridinin chlorophyll
PI Propidium iodide
PLL Prolymphocytic leukaemia
PMT Photomultiplier tubes
PNET Primitive neuroectodermal tumour
PR Progesterone receptors
PTCL Peripheral T cell lymphoma
QC Quality control
QD Quantum dots
RMS Rhabdomyosarcoma
SI Stain index
SiPM Silicon photomultiplier
SLL Small lymphocytic lymphoma
SRCT Small round cell tumours
SS Sezary syndrome
SSC Side scatter
TdT Terminal nucleotidase transferase
WHO World Health Organization
Part I
Practical Aspects of Flow Cytometry
Introduction and History of Flow
Cytometry 1
1.1 Introduction
Flow cytometry is the measurement of the various parameters of the cell/object in

the flowing fluid suspension. The cell/objects should be present singly in the fluid
suspension. The cells/objects are tagged with fluorescence dye. The beam of laser
light hits the cells. The scattered light and emitted fluorescence are measured and
recorded in the flow cytometer instrument. The various characters of the cells, such
as cell size, granularity, antigen expression, and nuclear DNA content, are measured
from the recorded data. The light scattered is directly related to the morphological
characteristics of the cell, and the intensity of the emitted fluorescence from the
cells is related with the quantity of the fluorescent probes attached with the cell. The
flow cytometry (FCM) can give valuable information on antigen bound with mem-
brane, cytoplasm or nuclei, DNA content of the cell, cellular organelles, RNA, and
information on the protein. Over time, FCM is changed remarkably, and the follow-
ing paragraphs describe a brief history of the FCM.
1.2 History of Flow Cytometry
1.2.1 Early Motivation
The early motivation of measuring the cell counts came at the Second World War by
the USA’s army. They tried to detect the quick and sensitive method to measure the
aerosol concentration to develop the biowarfare instrument. Gucker et al. injected
the sample air in a sheath of filtered flowing air [1]. The aerosol particle in the
sample air scattered light when passing through the tube. The lens detected the scat-
tered light and then collected it as an electronic impulse by the photodetector system.
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 3
P. Dey, Diagnostic Flow Cytometry in Cytology,
https://doi.org/10.1007/978-981-16-2655-5_1
4 1 Introduction and History of Flow Cytometry
1.2.2 Counting the Flowing Cells
Moldvan, in 1934, the first time, tried to measure the cells in suspension. They
applied the photoelectric method to identify the cells suspended in water [2].
The red blood cells in the suspension were forced to pass through a capillary tube
under the microscope. The photoelectric device was attached with the eyepiece of
the microscope to record each passing cell. The clumping of the RBCs in the tube
was a significant problem. The need for a sensitive photoelectric device to detect the
cells was also felt.
Cornwell and Davidson developed the upgraded version of the instrument in
1950 by using trypan blue-stained cells [3]. However, the cells were frequently
aggregated and clogged the capillary tube. Crosland-Taylor overcame clogging by
slowly injecting the jet of the sample in the centre of the faster stream of fluid pass-
ing in the same direction through a wide bore tube [4]. If there is no turbulence, then
the wide column of sample fluid is accelerated and form a narrow column. All mod-
ern flow cytometer has adopted this basic principle of laminar flow.
1.2.3 Haematology Sample
In 1953, Walter Coulter, designed the first successful instrument that counted blood
cells flowing in a liquid suspension one at a time [5]. He noted that due to the lipid
membrane covering, the cells are a poor conductor of electricity compared to the
saline solution. The saline solution containing the cells flowed through a narrow
orifice between the two electrodes that maintained a constant voltage difference.
The cells are relatively nonconductive, so they cause a voltage drop as they pass
through the constricted aperture and generates an electrical signal. The resulting pulse
is amplified, and the pulses that exceed the threshold are recorded. The number and
volume of the cells were measured successfully by this machine. The WBCs was
counted by lysing the RBCs, and the RBCs were counted by diluting the blood sample.
In the successive years, the machine was modified significantly, particularly the count-
ing chamber and the machine’s electronic. The Coulter counter was used successfully
in the haematology laboratory for cell counting and measurement of cell size [6].
1.2.4 Differential Count in the Blood Sample
In 1964, Hallermann L et al. [7] used Acridine orange, a fluorescence dye, to count
RBCs and WBCs. Acridine orange dye stains the WBCs relatively more brightly
than the RBCs. Therefore from the fluorescence signals, they counted WBCs, and
from the scattered signal, the RBCs were counted.
Ornstein et al. [8] in 1974 used cytochemical stains to recognize the various
types of WBCs. They used peroxidase stain to identify the neutrophils and eosino-
phils, esterase for monocytes and Alcian blue stain for basophils. The light scatter-
ing and absorption of the chromogens in different cells were measured and recorded
1.3 Fluorescence Stain in the Flow Cytometer 5
in different channels. Ornstein et al. used Technicon Hemalog D instrument with

Tungstein Halogen lamp as the light source.
1.2.5 Flow Sorter
Mack Fulwyler, in the year 1965, applied an electrostatic deflection ink-jet record-
ing technique to isolate the charged droplets and made the successful cell-sorting
machine [9]. He prepared the charged droplets of the cell in a liquid medium that
flow in an electrostatic field and are deflected into a container with the help of the
ink writing oscillography technique described by Sweet et al. [10].
Fulwyler [9] used piezoelectric crystal that produces high vibration (at a fre-
quency of 72,000 cy/s). In this, higher vibration the sheath stream in the flow cytom-
eter was broken into tiny droplets. According to the operator’s set criteria, whenever
a cell droplet satisfies the parameters, the system applies an electrical charge to the
droplet. The charged droplet then deflected by the electrostatic field and collected in
a separate container.
Herzenberg et al. [11, 12] realized the importance of fluorescence FCM and tried
to build a cell-sorting system based on fluorescence.
They initially took the help of the machine built by Louis A. Kamentsky that can
sort out the fluorescein stained cells. Herzenberg et al. [11] made a series of changes
to the machine and developed an improved version of fluorescence-activated cell
sorter. When the fluorescent labelled cells generated an optimum signal, a voltage
pulse was applied to the droplets. The charged droplets were deflected by an electric
field between a pair of deflection plates and collected. As the cells were sorted based
on fluorescence measurement, the device was labelled as “Fluorescence-activated
cell sorter” (FACS). Becton Dickinson Company, USA, commercially introduced
the machine in 1974.
1.3 Fluorescence Stain in the Flow Cytometer
In late 1960, several workers tried to introduce fluorescence dyes and laser beam as
light source [13, 14, 15]. The using of the fluorescence dye in the flow cytometer
had several advantages. The fluorescein staining gives greater sensitivity, helps
quantitation of antigen, and assess the presence of multiple antigens in the same cell
by using multiple fluorescein dyes.
The laser as a light source for excitation provides a high-intensity light beam
having a single wavelength. The laser beam can be aligned to focus on the cell more
precisely. The most commonly argon laser was used in the flow cytometer with a
wavelength of 488 nm. The argon laser was suitable for the widely used fluores-
cence dyes such as fluorescein, propidium iodide and Acridine orange. The other
lasers used in the flow cytometer are ultraviolet laser, krypton lasers, helium–neon
lasers and helium–cadmium lasers. Currently, the flow cytometer uses lasers of 350-
to 800-nanometre wavelengths.
In the year 1965, Kamentsky et al. developed a microscope-based flow cytome-

ter using the micro spectrophotometry [16, 17]. It was a two-parameter flow cytom-
eter, and it could measure the cell size and nucleic acid content of the cell.
Subsequently, he developed an instrument that was well connected with a computer
and could measure four parameters. At the same time, Dittrich and Gohde [18] in
Germany built a fluorescence dye-based flow cytometer to measure the cells’ DNA
content. The instrument helped to measure the cell kinetics and the DNA content of
the tumour cells. They used a mercury arc light source in their flow cytometer.
In 1969, Van Dilla et al. [19] in Los Alamos quantitatively measured nuclear
DNA content with the help of fluorescent Feulgen stain by using an argon laser light
source of the 488-nanometre wavelength of the excitation beam. They used the flow
cytometer’s orthogonal body plan where the excitation beam of laser light beam and
the light collection was perpendicular to the flow cells. It was anticipated that the
technique would help use multiple fluorochrome dyes and multiparametric assess-
ment by collecting the forward-angle light scattering.
1.4 Further Improvements in Flow Cytometry
Curbelo et al. [20] at Block Engineering, Cambridge, Massachusetts, designed a

flow cytometer with multiple wavelength laser excitation beam of light. This
machine was able to record eight optical measurements of a single cell. Subsequently,
the group developed a multiple laser source multiparameter flow cytometer [21].
Table 1.1 Milestones in the history of flow cytometry

Researcher Work
Moldvan [2] The cells flowing through a capillary tube counted by the
photoelectric method
Gucker [1] of American The detection of bacteria in the flowing air stream to make an
Army (1940) instrument that can be used for the biowarfare at the second world
war
Walter Coulter [5] Counted blood cells flowing in a liquid suspension by measuring
the drop of voltage when each cell passes through a static
electrostatic filed
Kamentsky [13] They designed a two-parameter flow cytometer to measure the cell
size and nucleic acid content of the cell by recording the emitted
fluorescence
Dittrich [14] in Germany A fluorescence dye-based flow cytometer to measure DNA content
and Van Dillain [15] Los of the cells
Alamos in (1969)
Fulwyler [9] Electrostatic deflection ink-jet recording technique was used to
isolate the charged droplet containing the desired cell and made
the successful cell-sorting machine
Herzenberg [11] The cells were sorted based on fluorescence measurement. The
device was labelled as “fluorescence-activated cell sorter” (FACS).
Becton Dickinson company, USA, commercially introduced the
machine in 1974
References 7
They used three separate laser beams of different wavelengths that measured at least
five measurements of a single cell at a speed of 1000 cells/second.
The development of monoclonal antibodies had a significant impact on flow
cytometry. The monoclonal antibody identifies the particular antigen in the cell. The
antibody tagged with fluorochrome dye emits a specific colour of light when the
laser beam hits it. The different antibody may be labelled with different fluoro-
chrome dyes, and the emitted colours are recorded. Therefore, the use of different
fluorochrome tagged antibody may help to categorize the different subset of cells.
The modern flow cytometers have the facility of multiple fluorochromes colours
(more than ten colours) to identify with multiple laser beams of different wave-
lengths (Table 1.1).
References
1. Gucker FT Jr, Pickard HB, O'Konski CT. A photoelectric instrument for comparing the con-
centrations of very dilute aerosols, and measuring low light intensities. J Am Chem Soc.
1947;69(2):429–38.
2. Moldavan A. Photo-electric technique for the counting of microscopical cells. Science.
1934;80(2069):188–9.
3. Cornwall JB, Davison RM. Rapid counter for small particles in suspension. J Sci Instrum.
1950;37:414–7.
4. Crosland-taylor PJ. A device for counting small particles suspended in a fluid through a tube.
Nature. 1953;171(4340):37–8.
5. Coulter WH. High speed automatic blood cell counter and cell size analyzer. Proc Natl Electron
Conf. 1956 (Vol. 12). Chicago: National Electronics Conference, Inc.; 1957; pp 1034–1040.
6. Mattern CF, Brackett FS, Olson BJ. Determination of number and size of particles by
electrical gating: blood cells. J Appl Physiol. 1957;10(1):56–70. https://doi.org/10.1152/
jappl.1957.10.1.56.
7. Hallermann L, Thom R, Gerhartz H. Elektronische differentialzaehlung von granulocyten
and lymphocyten nach intravitaler fluochromierung mit acridinorange [Electronic differential
counting of granulocytes and lymphocytes after intravital fluorochrome staining with acridine
orange]. Verh Dtsch Ges Inn Med. 1964;70:217–9.
8. Ornstein L, Ansley HR. Spectral matching of classical cytochemistry to automated cytology. J
Histochem Cytochem. 1974;22(7):453–69. https://doi.org/10.1177/22.7.453.
9. Fulwyler MJ. Electronic separation of biological cells by volume. Science.
1965;150(3698):910–1.
10. Sweet RG. Stanford University Technical Report 1722–1 (Report SU-SEL-64-004, Defense
Document Center), Washington, DC, 1964.
11. Herzenberg LA, Sweet RG, Herzenberg LA. Fluorescence-activated cell sorting. Sci Am.
1976;234(3):108–17.
12. Bonner WA, Hulett HR, Sweet RG, Herzenberg LA. Fluorescence activated cell sorting. Rev
Sci Instrum. 1972;43(3):404–9.
13. Kamentsky LA, Melamed MR. Rapid multiple mass constituent analysis of biological cells.
Ann N Y Sci. 1969;157:310–23.
14. Dittrich W, Göhde W. Impulsfluorometrie bei Einzelezellen in Suspensionen [Impulse fluo-
rometry of single cells in suspension]. Z Naturforsch B. 1969;24(3):360–1.
15. Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR. Cell microfluorometry: a method for
rapid fluorescence measurement. Science. 1969;163(3872):1213–4.
16. Kamentsky LA, Melamed MR. Rapid multiple mass constituent analysis of biological cells.
Ann N Y Sci. 1969;157:310–23.
17. Kamentsky LA, Melamed MR, Derman H. Spectrophotometer: new instrument for ultrarapid
cell analysis. Science. 1965;150(3696):630–1.
18. Dittrich W, Göhde W. Impulsfluorometrie bei Einzelezellen in Suspensionen [Impulse fluo-
rometry of single cells in suspension]. Z Naturforsch B. 1969;24(3):360–1.
19. Van Dilla MA, Trujillo TT, Mullaney PF, Coulter JR. Cell microfluorometry: a method for
rapid fluorescence measurement. Science. 1969;163(3872):1213–4.
20. Curbelo R, Schildkraut ER, Hirschfeld T, Webb RH, Block MJ, Shapiro HM. A gen-

eralized machine for automated flow cytology system design. J Histochem Cytochem.
1976;24(1):388–95.
21. Shapiro HM, Schildkraut ER, Curbelo R, Turner RB, Webb RH, Brown DC, Block

MJ. Cytomat-R: a computer-controlled multiple laser source multiparameter flow cytopho-
tometer system. J Histochem Cytochem. 1977;25(7):836–44.
Basic Principles and Instrumentation
of Flow Cytometry 2
2.1 Introduction
Over the years, there are massive developments in flow cytometry regarding fluid-
ics, optical system, data collection, etc. However, the basic principle of flow cytom-
etry (FCM) is almost the same [1]. It measures the optical characteristic and the
emitted fluorescence of the object/cell and procures valuable information. The tech-
nique enables us to quantitate the multiple characteristics of the cell one at a time in
a short period. This higher rate of measurement helps us collect the data from a large
number of cells, which promotes good sensitivity and accuracy. For the proper uti-
lization of FCM, it is essential to know the basic principles of the technique and the
fundamental working principles of the flow cytometer’s various components.
2.2 Principles of Flow Cytometry
• The basic principles of flow cytometry are the following (Fig. 2.1, Box 2.1):
• The single dissociated cells in a liquid medium.
• The cells are stained with one or multiple fluorochromes tagged marker/s.
• A laser beam of light strikes the individual cells.
• The cells tagged with the fluorochrome dye absorb photons of light and emits the
fluorescence.
• The forward light scatters, and the emitted fluorescence from the individual cells
are detected by the multiple photomultiplier tubes.
• The electronic impulse is converted to electrical current and then converted to
digital data (analogue to digital) and is recorded by the computer.
• The computer interprets the recorded data.
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10 2 Basic Principles and Instrumentation of Flow Cytometry
Fig. 2.1 Basic principles of flow cytometry is explained in this schematic diagram
Box 2.1 Components and Basic Principles of Flow Cytometer

Components
1. Fluidics.
2. Optical system:
• The excitation of the fluorochrome dye tagged with the cell the light
collection.
3. Electronics system.
4. Computer.
Basic Principle
• The single dissociated cells.
• The cells stained with one or multiple fluorochromes tagged marker/s.
• A laser beam strikes the cells.
• Fluorescence emitted.
• The fluorescence signals recorded.
• The light signal of photon is converted into electrical signal followed by
digital conversion.
• Digital data is recorded and analyzed in the computer.
The primary component of the flow cytometer instruments include:

1. Fluidics.
2. Optical system: It essentially has two parts: a) The optical system which is
responsible for the excitation of the fluorochrome dye tagged with the cell, b) the
light collection.
2.2 Principles of Flow Cytometry 11
3. Electronics system.
4. Computer.
2.2.1 The Fluidics System
The name “flow cytometer” is derived from the “flow” of the cells. The fluidics
system’s primary aim is to maintain a stable flow of cells one at a time without
forming any turbulence to the laser hit point (Box 2.2). The coaxial nature of the
stream of cells is used to maintain the steady flow of cells at the laser interrogation
point. The “coaxial stream” means two streams of fluid, one outer and the other
inner stream [1, 2] (Fig. 2.2).
The sheath fluid serves as the outer stream of fluid surrounding the inner sample
fluid containing the cells. Thus, the outer sheath fluid reduces any turbulence that
Fig. 2.2 Coaxial stream of flow where the outer stream is formed by sheath fluid and inner stream
by the sample
b c
Fig. 2.3 (a) Sample fluid is sent by high air pressure. (b) Lower air pressure causes a low flow rate
of cells and a narrow coaxial stream. (c) Higher air pressure creates a high flow rate of the cells and
a wider coaxial stream
could occur due to the resistance of the flow by the tube wall. The pressure in the
sample fluid is always kept much higher than the sheath fluid. It is done by injecting
the sample fluid by the high air pressure in the flow tube (Fig. 2.3). So the high pres-
sure of the sample fluid, differences of density, and speed of the sample fluid are
responsible for preventing the mixing of the two streams. The coaxial flow helps in
the uniform illumination of cells by a laser beam. It is also known as hydrodynamic
focusing. The rate of flow of cells can be manipulated by altering the air pressure of
the sample in the flow chamber. If the sample pressure is less, then the flow rate of
the cells in the flow chamber decreases. Lowered sample pressure makes the beam
narrow, and that may cause the single cell to pass at the lesser hit point at a single
point of time [3] (Fig. 2.3b).
2.3 Optical System 13
The higher flow rate of the cells makes the stream wide. So more than one cell
may pass through the beam in a single point of time (Fig. 2.3c). The slow rate of
cells is needed for DNA measurement. In contrast, a higher rate may be required to
assess the cell population in flow cytometric immunophenotyping, where a large
number of cells have to be studied. It is essential to note that the fluidics system
should always be free from any air bubble or debris.
Box 2.2 Fluidics of the Flow Cytometer

Aim: To maintain a stable flow of cells.
The “coaxial stream”: Inner sample fluid and outer sheath fluid.
Functions of the sheath fluid:

• To prevent turbulence of the sample fluid.
• Uniform illumination of cells by a laser beam.
Pressure:
• Pressure of the sample fluid is always higher than sheath fluid.
• Increasing sample fluid pressure increases the rate of flow and wider the
coaxial stream.
• Lowering the sample fluid pressure decreases rate of flow and narrower the
coaxial stream.
2.3 Optical System
Light source: The beam of light should hit each single cells. For this, there is a need
to focus the laser light on the cell by the lens.
Laser: Laser is the acronym, and the complete form of laser is “Light amplifica-
tion by stimulated emission of radiation”. Unlike the other sources of light, the laser
emits light with a specific wavelength. Box 2.3 highlights the essential characteris-
tics of the laser beam.
Box 2.3 The Characteristics of Laser Light

• Monochromatic light of single wave length.
• Highly intense.
• Coherent: All the photons have same phase and same polarization.
• Unidirectional.
• Simulated emission.
• Very sharply focussed.
Table 2.1 Laser sources and Laser source Wavelength (nm)

wavelengths of light Argon 351–528,488
Krypton 350–799 nm
Helium–neon 543, 594, 612, 633
Helium– 325–442
cadmium
UV 300–400
The laser beam can emit fluorescence of the particular fluorochrome dye/s that
match the excitation wavelength of that dye/s. So the selection of fluorochrome
dyes should be appropriate with the wavelength of the laser of FCM. The com-
monly used laser source of the flow cytometer is the argon laser source that gener-
ates lights of the wavelength of 488 nm. The flow cytometers may have other laser
sources such as krypton laser, helium–neon lasers, helium–cadmium lasers and
UV laser [3, 4].
Table 2.1 shows the wavelength of various laser sources.
Advantages of argon laser: The advantages of argon laser includes:

• It can generate multiple wavelengths of light.
• It produces a high power output.
• Argon laser is a higher gain system.
• It has a relatively less divergence.
Disadvantages of argon laser: It includes:

• Difficult to construct.
• Huge power requirement.
• The overall efficiency of argon laser is less (0.1%).
Detection of light: When the laser hits the cell/object, the light is scattered, and
in the case of fluorochrome dye, the fluorescence is emitted. The scattered light and
emitted fluorescence are detected with the help of appropriate filters.
Light scattering: The light is composed of photons. When the photons hit the
cells/object, two types of light scattering may occur (Fig. 2.4): (FSC) and side scat-
ter (SSC). In the case of FSC, the light is diffracted along the same axis of the laser
beam. The total amount of FSC is directly proportional to the size and surface area
of the cell/object. The forward scatter (FSC) of light is collected by the forward
scatter detector located on the same axis as the laser beam.
2.4 Fluorescence Emission 15
Fig. 2.4 Schematic diagram showing forward and side scattering of light.
Some light enters the cell and is then reflected and refracted by the various cyto-
plasmic organelles and nucleus. This light is collected at a 90° angle to the laser
beam and is known as SSC. The amount of SSC light is directly proportional to the
granularity and internal complexity of the cell.
2.4 Fluorescence Emission
Both the FSC and SSC lights have the same wavelength and colour as the laser
light. Therefore, no fluorochrome probe is needed to detect the signals of FSC
and SSC. The fluorochrome dye is used to characterize the various properties
of the cells. The fluorochrome dye is commonly tagged with the antibody.
However, the dye can be tagged with hormones, lectins, or different other
proteins.
The fluorochrome dye has the unique property to absorb light of a specific wave-
length and emits fluorescence of a higher wavelength of light. The emitted fluores-
cence is recorded in the detectors of the flow cytometer. The details of the
fluorescence and fluorochrome have been described in the subsequent chapter
(Chap. 6).
2.4.1 Collection of Light
The light collection is done by a set of special filters and optical mirrors (Box 2.4).
Modern flow cytometers are using multiple lenses. The multiple lasers beams may
hit the cells sequentially or simultaneously, all at a time. In sequential lasers, the
same cells are hit by different laser beam sequentially, and accordingly, the lenses
collect the light. However, in the simultaneous use of lasers, special filters and mir-
rors are required to separate the mixed light.
Box 2.4 Optical System of Flow Cytometer

Light source: LASER is the source of light.
Types of LASER source: Argon, krypton, helium–neon, helium–cadmium.
Multiple LASERs: Used sequentially or simultaneously.
Light scattering:
• Forward scatter (FSC): Light is diffracted along the same axis of the
laser beam.
–– FSC quantity represents the size and surface area of the cell.
• Side scatter (SSC): Light is collected at a 90° angle to the laser beam.
–– SSC represents the cytoplasmic granularity of the cell.
Collection of light: Light is collected by a set of special filters and optical

mirrors.
2.4.2 Optical Filters [2]
The dichroic mirrors are put at the right angle to the laser beam of light. This type
of mirror helps to eliminate the unwanted light and allows to pass light of higher
wavelength. The combination of different types of mirror and filters help to pass the
light of a specific wavelength.
Long-pass optical filter: This type of filter allows to pass light of a specific wave-
length and longer than the specified.
Short-pass optical filter: This type of filter allows transmission of light with an
equal or shorter wavelength.
Bandpass optical filter: This type of filter allows passing light within a narrow
range of wavelengths only.
Dichroic mirror: This type of dichroic mirror is also known as a beam splitter.
They may be of two types; dichroic long-pass and dichroic short-pass filter.
The dichroic long-pass filter reflects the light below the specific wavelength and
transmits light above the cut-off wavelength.
The dichroic short-pass filter reflects light above the specific cut-off wavelength
and transmits light only below the cut-off wavelength.
Figure 2.5 shows different types of optical filters, and the arrangement of these
filter are shown in Fig. 2.6.
Fig. 2.5 Different types of optical filters are demonstrated
2.4.3 Electronics System
The electronic system converts the light signal into the voltage, followed by digital
data (Box 2.5). The photons of light hit the photodetectors, which convert the pho-
ton into electrons that produce an electric current. So basically, the photodetectors
are “a light-driven current source”. The electric current is amplified in the photo-
multiplier, and a voltage pulse is produced. This voltage pulse is directly propor-
tional to the number of photons detected in the sensor system. From the pulse height,
width and area, the digital data is generated, known as analogue to digital conver-
sion (ADC). This digital data is recorded on the computer (Fig. 2.7). There are four
types of photodetectors: photodiodes (PD), photomultiplier tubes (PMT), avalanche
photodiode (APD and silicon photomultiplier (SiPM). The PD has lesser sensitivity
and is used to detect the stronger signal of FSC, whereas the PMT has a higher sen-
sitivity and is used to collect the weaker signals of SSC and fluorescence. There are
two types of amplification of the electrical current: linear and logarithmic. Linear
amplification is used when a limited dynamic range of data is needed, such as DNA
measurement, whereas logarithmic amplification is needed when a wide dynamic
range of data is required, such as measurement of fluorescence. Figure 2.8 illus-
trates the mechanism of a photomultiplier tube.
Fig. 2.6 Optical arrangements of the flow cytometer
Box 2.5 Electronics System of Flow Cytometer

• Photon of the light is detected by the photodetector.
• Photodetector converts the photon signal into electronic signal that pro-
duces electric current.
• Electric current is amplified by the photomultiplier and a voltage pulse is
generated.
• The voltage pulse is converted into digital data (analogue to digital).
• Data is recorded in the computer.
Figure 2.9 explains the generation of the signal for each event. When the cell
enters the laser beam, the voltage pulse is generated. This pulse attains its peak
when the cell completely reaches the centre of the laser beam. Subsequently, the
pulse reaches the baseline as the cell comes out from the laser beam [5].
Fig. 2.7 Events in the electronic system is summarized
Fig. 2.8 Schematic diagram of the electronics system of the photodetector
2.4.4 Computer System
Every individual event in the flow cytometer is digitized by analogue to digital con-
version, as mentioned before. Each event is given a channel number, and the numer-
ical value is generated for the pulse height, width, etc. The channel number is
transferred to the computer and recorded for analysis.
2.4.5 Flow Cytometric Cell Sorting
The flow cytometry-based cell sorter helps to capture and separation of the cells of inter-
est (Box 2.6). The separated cells may be analyzed further for various studies such as
cytomorphological analysis, functional analysis, etc [6] The basic principle of cell sort-
ing is the electrostatic deflection of the charged droplet containing the cell of interest [7].
In the cell sorter, the cells are rapidly injected through a narrowed orifice to
stream cells into droplets. The high-frequency vibration is applied with a piezoelec-
tric crystal when the cells are passed through the orifice. The vibration makes the
droplet containing cells more stable. The stream now simulates like a wavelength,
and each droplet is separated one wavelength apart.
When the droplets pass through the laser interrogation point, the fluorescence data
is analyzed. According to the set criteria, the individual droplets are charged
immediately by the charging electrode plate. The charged droplets are then deflected
with the help of a deflecting plate. The positively charged droplet goes towards the
negatively charged plate, and the negatively charged droplets are deflected towards
positively charged plates. Thus the cells of interest are separated from the mainstream
of flow.
Figure 2.10 shows the schematic diagram of cell sorting.
Box 2.6 Flow Cytometric Cell Sorting

The cell sorter capture and separation of the cells of interest.
Basic principle:
• Cells are made as small droplets.
• The droplets pass through the laser interrogation point and the fluores-
cence data is analyzed.
• According to the set criteria, the individual droplets are charged immedi-
ately by the charging electrode plate.
• The charged droplets are then deflected with the help of a deflecting plate
and collected separately.
Fig. 2.9 Height of the pulse indicates the maximum amount of electric current that is passed. The
area of the pulse indicates the integral pulse and the width represents the interval between
two pulses
References 21
Fig. 2.10 Basic principle of cell sorting
References
1. Wilkerson MJ. Principles and applications of flow cytometry and cell sorting in companion
animal medicine. Vet Clin North Am Small Anim Pract. 2012;42(1):53–71.
2. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applica-
tions. Crit Rev Biotechnol. 2017;37(2):163–76.
3. Shapiro HM. Lasers for flow cytometry. Curr Protoc Cytom. 2004;Chapter 1:Unit 1.9.
4. Shapiro HM, Telford WG. Lasers for flow cytometry: current and future trends. Curr Protoc
Cytom. 2018;83:1.9.1–1.9.21.
5. Snow C. Flow cytometer electronics. Cytometry A. 2004;57(2):63–9.
6. McKinnon KM. Flow cytometry: an overview. Curr Protoc Immunol. 2018;120:5.1.1–5.1.11.
7. Ibrahim SF, van den Engh G. Flow cytometry and cell sorting. Adv Biochem Eng Biotechnol.
2007;106:19–39.
Sample Preparation and Data
Acquisition in Flow Cytometry 3
3.1 Introduction
Proper preparation of the sample for flow cytometry is a crucial part of the proce-
dure. The optimum result from the flow cytometry is obtained only in an adequately
prepared sample. In this chapter, I will focus only on the preparation of the cytology
samples.
3.2 Basic Requirements
The essential goals of cell preparation are the following (Box 3.1):
1. To make single-cell preparation.
2. To suspend the cells in the proper medium, which is usually isotonic, having a
pH of 7.3.
3. To have adequate cells in the preparation, usually at a concentration of 105
cells per ml.
4. To label the cells of interest by the appropriate fluorochrome dye or to stain the
nuclei with fluorochrome dye for DNA flow cytometry.
Box 3.1 Basic Requirements for Flow Cytometry

• Single-cell preparation.
• Suspension of the cells in the isotonic medium.
• Optimum cell concentration (105 cells per ml).
• Appropriate fluorochrome to stain the cells.
https://doi.org/10.1007/978-981-16-2655-5_3
24 3 Sample Preparation and Data Acquisition in Flow Cytometry
3.3 Cytology Samples for Flow Cytometry
The commonly used cytology samples for flow cytometry procedures are:
1. Fine needle aspiration cytology (FNAC): The material is obtained usually from
the lymph node. However, the FNAC materials are also collected from the breast,
lung, and other solid organs.
2. The body cavity fluids: Effusion, urine, bronchoalveolar lavage, CSF, etc.
3. Washing and brushing sample.
Advantages of the cytology sample: There are certain advantages of the cytol-
ogy samples for flow cytometry. These include:
• Easy to obtain the sample.
• Samples can be obtained from the multiple sites.
• Easy to have single-cell preparation because the cells can be dissociated easily.
• If necessary, one can do the various functional studies as the cells are mostly viable.
Factors influencing sample preparation: The following factors influence the

sample preparation (Box 3.2):
A. The methods of sample procurement:
Collecting fluid: The methods of sample procurement are critical. If the sample
is not collected in the appropriate buffer solution, there is a high chance of damage
to the cells.
Freezing: If the sample is frozen at a very low temperature, the cells may be dam-
aged considerably.
Anticoagulant: The fluid sample may be needed to collect along with an
anticoagulant.
B. Storage:
Temperature: The cells may be damaged at an extreme low or high temperature.
Duration of storage: The sample should not be stored a long time before pro-
cessing. It should be processed within a couple of hours.
C. Single-cell preparation: The vigorous mechanical dispersion of the cells
may damage the cells.
Box 3.2 Factors Influencing Sample Preparation

1. The methods of sample procurement:
• Collecting fluid: Buffered solution.
• Freezing: Cells are damaged in very low temperature.
• Anticoagulant.
2. Storage.
• Temperature.
• Duration of storage.
3. Single-cell preparation: Excessive mechanical force damages the cells.
3.5 Single-Cell Preparation 25
3.4 Sample Collection
The proper choice of buffered fluid is essential. If the cells are processed immedi-
ately for FCM, then simple phosphate-buffered saline (PBS) or citrate buffer solu-
tions are sufficient for the sample procurement from the FNAC material. The
collected sample should be kept at 4 °C until further processing.
Phosphate-buffered saline:
• 8 g NaCl
• 0.2 g KCl
• 1.15 g Na2HPO4
• 0.2 g KH2PO4
• Add the material in 900 mL distilled water.
• Adjust the pH to 7.4 with HCl.
• Make the final volume to one litre by adding distilled water.
Citrate buffer solution

• Sucrose: 85.3 g.
• Trisodium citrate (Sigma): 11.8 g.
• 50 ml of Dimethyl Sulfoxide in 100 ml of water
• pH at 7.6.
The body cavity effusion sample can be collected in suitable anticoagulant (such
as ammonium oxalate solution: fluid should be 1:9).
Bronchoalveolar lavage can be collected in a normal saline solution; however, it
needs immediate processing for FCM.
3.5 Single-Cell Preparation
Single-cell preparation is one of the most critical steps of flow cytometry. Unless the
cells are adequately dissociated, it is impossible to get the data from the individual
cells. The cells are attached by the cell to cell junctions such as tight junction and
gap junction. The desmosomes and hemidesmosomes help to connect the cells with
the extracellular matrix. The cell to cell attachment can be broken by the enzymatic
digestion such as papain, trypsin.
The mechanical procedure usually does the dissociation of the cytology sample
[1]. The enzymatic digestion is better to avoid for the cell surface marker’s immuno-
phenotyping as the enzymes may affect the cell surface antigen.
Mechanical dissociation: The cells suspension is repeatedly rinsed through a
thin bore needle. The repeated rinsing usually dissociates the cells. Finally, the cells
should be filtered through the nylon mesh kept between the needle and syringe.
Enzymatic digestion: Enzymatic digestion can also be used to make the single-
cell preparation. The enzyme breaks the cell to cell junction and also the attachment
of the cells with collagenase material. The enzymes are sensitive to the temperature
and may not work at a lower temperature. The commonly used enzymes for cell
dissociation are trypsin and papain. Usually, 1- to 2-h incubation of the enzyme is
enough to make a single-cell preparation. After a certain period, one should block
the enzyme’s action by using a serum with a balanced salt solution.
3.5.1 Limitations of the Enzymatic Method
• Close supervision is needed to prevent over enzymatic reaction as this may dam-
age the cells completely.
• The enzymatic method is unsuitable for immunophenotyping.
• The enzyme is temperature sensitive.
Currently, many commercially available cocktails of enzymes are available in the

market. Accutase is one such enzymatic cocktail that contains proteolytic, colla-
genolytic, and DNAse. TrypLE is another such cocktail of enzymes with the unique
property of preserving the cell surface antigen.
Table 3.1 shows the comparison of mechanical and enzymatic methods of single-
cell preparation.
3.6 Fixation
Cellular fixation is not needed if the sample is processed immediately. Before the
fixation, the cells should be dissociated first.
The cellular fixation is needed for the following reasons:

• To study intracellular antigen.
• The sample can be further processed whenever time and opportunity allow.
• It may be easy and convenient for the person to do FCM.
Table 3.1 Comparison of mechanical and enzymatic methods of single-cell preparation

Features Mechanical Enzymatic
Methods Repeated rinsing the cell suspension Enzymatic digestion
through the syringe hub and nylon mesh
Procedure cost Cheap Cost of the enzyme is
relatively high
Supervision Not much supervision needed Continuous
supervision needed
Suitability for Suitable Not suitable
immunophenotyping
3.8 RBC Lysing Solution 27
Formaldehyde: The procedure of formaldehyde fixation

• The sample is centrifuged.
• The supernatant fluid is discarded.
• The cells are fixed by 0.4% formaldehyde for 10–15 min at 37 °C.
• The sample is centrifuged.
• The cells are washed thoroughly by centrifuging in PBS.
The commercially available solutions are also available that contain fixative
along with RBC lysing agents.
Alcohol: 95% of ethanol is a good fixative for the cells. It is suitable for DNA
analysis because the coagulation of protein in the cytoplasm helps the dye have bet-
ter access to nuclear DNA.
3.7 Permeabilization
The permeabilization of the cells may be required in the case of DNA content analy-
sis and demonstration of intracellular enzymatic analysis such as terminal nucleo-
tidase transferase (Tdt). The permeability reagents should increase the plasma
membrane’s permeability and should retain the cellular antigen for the immunos-
taining. The commonly used permeability reagents are saponin and other non-ionic
detergents such as Tween 20, Triton X, and NP40. Table 3.2 shows the list of the
various commercially available permeabilizing reagents.
3.8 RBC Lysing Solution
The cytology sample is often admixed with blood, and RBCs may contaminate the
cells of interest. The immunophenotyping of the lymphoid cells is usually unaf-
fected by the RBCs. However, it is wise to get rid of the RBCs. These RBC lyse
solutions contain ammonium chloride. The RBC lysing solutions often include
fixative to fix the leucocytes. Table 3.3 lists some commercially available lys-
ing agents.
Table 3.2 Commercially available permeabilizing agents

Company Product Permeabilizing agents Time
Beckman ItraPrep Permeabilizing Saponin and formaldehyde 45 min
coulter reagent
BD bioscience BD perm Saponin 1 h
Invirogen Fix and perm Not disclosed 40 min
Dako IntraStain Reagent A: Formaldehyde for 40 min
fixation
Reagent B: Permeabilization
Table 3.3 Commonly used lysing solutions in flow cytometry

Time to
Company Product name Lysing agent Fixation keep
BD FACS ™ Diethylene glycol, formaldehyde Yes 30 min
bioscience lysing solution
Beckman Immuno- Formaldehyde, formic acid Yes 2 min
coulter prep® Three parts: Reagent A: It lyses the red
blood cells. Reagent B: Stops lysing
reagent C cell fixation.
Dako Uti-lyse ™ Formaldehyde Yes 20 min
Sigma – Red blood cell Ammonium chloride, tris-HCL No
Aldrich lysing buffer
DAKO EasyLyse ™ Ammonium chloride No
3.9 Staining
3.9.1 DNA Flow Cytometry [2]
Reagents
Stock solution of propidium iodide (PI).
• Propidium iodide (PI) 5 mg

• Double distilled water 10 ml
Kindly store the stock solution in the dark at 4 °C.
Solution A
1. RNAse A 2 mg

2. Triton X-100 10 ml: 0. 1% (v/v)
3. Propidium iodide 0.40 ml of stock solution (500 μg/ml)
The freshly prepared solution is needed.
Steps
• The single-cell suspension in PBS buffer with the cell concentration in the buffer
is kept as at least 2 × 106 cells per ml.
• Add 500 μl of solution A.
• Keep the mixture in the dark for 30 min at room temperature.
• Run the sample for FCM.
Flow cytometric immunophenotyping (FCI).

3.9 Staining 29
Direct stain:
• Centrifuge the sample in PBS at 1500 round per minute for 3–5 min.
• Discard the supernatant fluid.
• Prepare the single-cell suspension by repeated syringing of the sample through
nylon mesh.
• Maintain the cell concentration in the buffer as at least 2 × 106 cells per ml.
• Centrifuge the sample at 1500 round per minute (RPM) for 10 min.
• Discard the supernatant fluid, and add 5 ml of lysing solution (commercially
available).
• Keep the solution for 5–10 min to lyse the red blood cells.
• Centrifuge the sample at 1500 RPM for 5 min, and discard the supernatant.
• Dissolve the cell pellet in PBS solution.
• Take 100 μl in PBS (pH 7.4).
• Add 10 μl of fluorochrome labelled primary antibody and keep it for 30 min in
the dark place at room temperature. In this step, multiple antibodies labelled with
different fluorochromes may be added for multicoloured flow cytometry.
• Wash the cells three times in PBS solution by 1500 round per minute for 3–5 min.
• Discard the supernatant fluid. Now add 50μl of fluorescent conjugated secondary
antibody and keep it at room temperature for 30 minutes in dark. Wash in PBS.
• Resuspend the cells in 250 μl PBS solution.
• Run in FCM.
Indirect staining procedure [3, 4]:

• Centrifuge the sample in PBS at 1500 round per minute for 3–5 min.
• Resuspend the cells in PBS.
• Prepare the single-cell suspension by repeated syringing of the sample through
nylon mesh.
• Maintain the cell concentration in the buffer as at least 2 × 106 cells per ml.
• Centrifuge the sample at 1500 round per minute (RPM) for 10 min.
• Discard the supernatant fluid, and add 5 ml of lysing solution (commercially
available).
• Keep the solution for 5–10 min to lyse the red blood cells.
• Centrifuge the sample at 1500 RPM for 5 min, and discard the supernatant.
• Dissolve the cell pellet in PBS solution.
• Take 100 μl solution of cells, and incubate with 50 μl of the primary nonconju-
gated antibody for 30 min at room temperature in the dark.
• Wash the cells three times in PBS solution by 1500 round per minute for 3–5 min.
• Resuspend the cells in 250 μl PBS solution.
• Run in FCM.
a
100 150 200 250 b
50 100 150 200 250

SSC-A (x 1,000)
SSC-A (x 1,000)
50
1
010
1
102 103 104 105 010 102 103 104 105
PE-Cy7-A CD45 APC-Cy7-A
Fig. 3.1 (a) Negative control in flow cytometry. No antibody is added. (b). Test sample that con-
tains the antibody
3.9.2 Control
It is mandatory to use an unstained control tube in each case.

Unstained control (Fig. 3.1): In the case of negative control, no fluorochrome
labelled primary antibody is applied. The unstained sample is used to know about
the background autofluorescence without addition by any fluorochrome tagged anti-
body. The knowledge of unstained autofluorescence helps to adjust the voltage and
also to have appropriate negative gating.
Isotype control: The use of isotype control is controversial, and it should not be
used in the routine FCM procedure. Isotype control means that antibodies are used
for the surface antigen absent in the cells of the sample. Isotype control helps to
know that the staining result in the FCM is due to the specific antibody and elimi-
nates the possibility of the artefact. An isotype control should not be used for posi-
tive control.
Fc block control: Fc receptor is present in the monocytes and macrophages.
These receptors can bind with the antibodies bypassing the Fab domain. So multiple
antibodies may bind with Fc receptors and produce false fluorescence signals. The
false-positive signal can be avoided by blocking the antibody with the help of
Human Fc seroblock or mouse seroblock FcR addition in the staining protocol.
The following points to remember in the FCM procedure

• The fresh sample processing always gives better results than a frozen sample.
• The volume and concentration of the primary antibody should always be titrated
beforehand.
• The RBC lysing solution followed by washing should be done before the addi-
tion of the labelled antibody.
• All high-grade lymphomas should be processed in a fresh sample.
• The proper selection of fluorochrome dye labelled antibody is essential in the
case of multicoloured FCM.
3.10 Data Acquisitions 31
Fig. 3.2 Threshold of the

signal was set to eliminate
the background noise
Threshold level
3.10 Data Acquisitions
The following aspects are critical for the acquisitions of data in flow cytometry
(Box 3.3).
Cells in suspension: The target cells should be in suspension evenly at the time
of data acquisition. So the tube containing cells can be gently vortexed, or gently
pipetting can be done.
The threshold of fluorescence (Fig. 3.2): The background noise signal may be
produced due to fragmented cells, small particles in the buffer, or the instrument
itself. It is crucial to eliminate background noise. The threshold value is defined as
the minimum fluorescence signal intensity which is recognized by the flow cytom-
eter as the event to record. The setup of the threshold in flow cytometry eliminates
unnecessary data to record. At the time, a combination of parameters can be used to
include the target cells. In this condition, the event is only included if both the
parameters are fulfilled. Forward scatter (FSC) can be used for setting the threshold.
The height of the pulse in each event indicates the brightness and width indicates
duration. Therefore height of the pulse can be kept as one of the threshold criteria.
Live gating: Gating means selecting the scatter plot area generated at the time of
flow cytometry. At the time of acquisition of the events, the live gating of the target
of interest can be done to select only the events of interest. For the implementation
of the successful gating strategy, one should know the following information:
1 . The approximate size of the cells.

2. The antibody marker that is commonly expressed by the cells.
3. The probable size variation of the target cells.
4. Any positive control of the cells.
The gating can be done based on forward scatter (FSC) and side scatters (SSC).
The debris or fragmented cells have low FSC and high SSC. In contrast, normal
cells should have high FSC and relatively low SSC (Fig. 3.3).
When the data of two parameters are collected, then one can make a bivariate
histogram (Fig. 3.4). In that condition, there will be four quadrants that represent
1. (b) and (d) one marker positive and other one negative,
Fig. 3.3 Gating of the
250
(x 1,000)
lymphoid cells were done
based on forward, and side
scatter
200150
SSC-A
100
50
50 100 150 200 250

FSC-A (x 1,000)
Fig. 3.4 Bivariate
histogram was made to
have a distinct population
of positive and negative
fluorescence markers
Fluorescent 2
b c
a d
Fluorescent 1
2 . (c) both the markers positive,

3. (a) both the markers negative.
Therefore, based on the target of interest (such as b in Fig. 3.4) where one par-
ticular marker is positive and the other one is negative), one can gate that population
and collect those events only.
Number of events to acquire: The “event” in the flow cytometry means detect-
ing a single object by the flow cytometer.
3.10 Data Acquisitions 33
Earlier FCM was used for mainly DNA content analysis. In that case, only
10,000 events acquisition was probably enough. However, fluorescence immuno-
phenotyping needs more than 10,000 cells in the list mode. It is due to the need for
selective gating for the cells of interest. As we do the gating of the target population,
the number of target cells become much less. It is particularly true for the acquisi-
tion of rare cells. Therefore at least 100,000 cells or events should be acquired. The
number of events required for analysis in the FCM, therefore, depends on:
1 . The signal versus noise ratio.

2. The amount of debris in the sample.
3. The possible frequency of the cells in the sample.
The formula of the number of events collection is [5].

R (number of cells to collect) = (100/CV)2.
CV: coefficient of variation of a known positive control.
The live display of the data profile, voltage adjustments, and data acquisition
termination is now available in the software attached with the machine.
Rate of data acquisition: The usual flow cytometer can acquire data at the rate
of 10,000 events or more per second. However, the best practice is to run the
machine at low differential pressure so that the event rate is near about 4 to 5000 per
second. A higher rate of events collection needs more differential pressure between
the sheath fluid and sample fluid. It may cause the widening of the central core fluid
sample, and so more number of cells may pass in front of the laser beam (Fig. 3.5).
Fig. 3.5 Impact of the high flow rate of the cells

It may have two impacts: (a) the laser may consider the two cells at doublet, (b) the
edge of the cells may be poorly illuminated, and therefore the data will have a high
coefficient of variation (CV) with low resolution. It may lead to the partial overlap
of the fluorescence data of the two different markers.
Box 3.3 Data Acquisitions in Flow Cytometry

Cells in suspension: Evenly presence of the target cells.
Threshold of fluorescence: The minimum fluorescence signal intensity
which is recognized by the flow cytometer as the event to record. Optimum
threshold is needed to eliminate the debris.
Live gating: It is done to select only the events of interest at the time of
data acquisitions. For live gating one should know:
• Size of the target cells.

• Size variation.
• Possible antigenic expression.
Number of cells/events to acquire: At least 10,000 cells.

Number of cells/events to acquire: Optimum speed is needed (approxi-
mately 4000/s).
References
1. Reichard A, Asosingh K. Best practices for preparing a single cell suspension from solid tissues
for flow Cytometry. Cytometry A. 2019;95(2):219–26. https://doi.org/10.1002/cyto.a.23690.
Epub 2018 Dec 6
2. Saikia UN, Dey P, Vohra H, Gupta SK. DNA flow cytometry of non Hodgkin's Lymphomas:
correlation with cytologic grade and clinical relapse. Diagn Cytopathol. 2000;22:153–6.
3. Kentrou NA, Tsagarakis NJ, Tzanetou K, Damala M, Papadimitriou KA, Skoumi D, Stratigaki
A, Anagnostopoulos NI, Malamou-Lada E, Athanassiadou P, Paterakis G. An improved flow
cytometric assay for detection and discrimination between malignant cells and atypical meso-
thelial cells, in serous cavity effusions. Cytometry B Clin Cytom. 2011;80(5):324–34.
4. Dey P, Amir T, Al Jassar A, et al. Combined applications of fine needle aspiration cytology
and flow cytometric immunphenotyping for diagnosis and classification of non Hodgkin lym-
phoma. Cytojournal. 2006;3:24.
5. Hedley BD, Keeney M. Technical issues: flow cytometry and rare event analysis. Int J Lab
Hematol. 2013;35(3):344–50.
Display and Interpretation of Data
in Flow Cytometry 4
Once the data is acquired, the next important task is an interpretation of the data.
The steps to interpret data are: (1) data threshold set, (2) data acquisition, (3) gating
of data, (4) data display, and (5) the extraction of information.
Data Display: All FCM data at first recorded in the “list mode” file. It is so
named because the data is recorded as a list of various parameters that contains both
FSC, SSC and fluorescent values. “List mode” file is also known as flow cytometry
standard file [1]. The flow cytometry standard file is now modified to updated ver-
sion FCS 3.1, which most FCM vendors now uses [2]. After recording the essential
information in the list mode file, the data is displayed further by the various soft-
ware packages provided by the vendors (Box 4.1).
Univariate data analysis or univariate histogram: This is the simplest form of
data display. The univariate histogram displays a single parameter. The histogram
may be displayed in the total data or the gated population. The univariate histogram
helps to assess the percentage of cells in the total population. The data is displayed
in two dimensions: X-axis and Y-axis. The magnitude of the variable or parameter
is plotted in the X-axis, and the frequency of the events is displayed in the Y-axis
(Fig. 4.1). In the case of fluorescence labelled marker, the signal intensity is repre-
sented digitally in the X-axis.
DNA content analysis is usually displayed by the univariate histogram (Fig. 4.2).
The distribution of the cells in the X-axis (or DNA content) is best demonstrated by
the coefficient of variation (CV). CV is calculated as standard deviation divided by
mean. As the CV is a dimensionless quantity, it is also the best way to compare cells
or DNA content in two graphs. The broader CV indicates that the staining of the
cells and alignment of the instrument is faulty. So it is preferable to maintain a low
CV in the case of DNA histogram.
Bivariate histogram: The bivariate histograms are used to display two different
parameters. One parameter is represented on X-axis, and the other parameter is
displayed on the Y-axis. The bivariate histogram may be in the following
format:Scatter plot /Dot plot: In the scatter plot (dot plot) histogram, each cell or
https://doi.org/10.1007/978-981-16-2655-5_4
36 4 Display and Interpretation of Data in Flow Cytometry
Fig. 4.1 Histogram Histogram

showing the distribution of
500 750 1,000 1,250

fluorescence intensity of
the CD45 positive cells
Count
250
0
102 103 104 105

CD45 APC-Cy7-A
Fig. 4.2 Schematic Count

diagram of DNA content 500
400
300
200
100
0
100 200 300 400
DNA
event is represented by a small dot (Fig. 4.3). This scatter plot may be displayed at
the time of acquisition or offline. In this graph, each axis represents a specific
parameter. Figure 4.3 shows the scatter plot diagram, where X-axis represents FSC,
and Y-axis indicated SSC. The acquisition of a large number of cells may slowly
obscure the finer details.
Contour plot: It is also a two parameter histogram where the individual events
are placed according to each parameter’s intensity (Fig. 4.4).
Density plot: This type of graphical representation of data is similar to that of
the dot plot. However, in the density plot graph, the dots’ colour represents the
events with the same intensity (Fig. 4.5).
4 Display and Interpretation of Data in Flow Cytometry 37
Fig. 4.3 Scatter plot
250
(x 1,000)
histogram
200
150
SSC-A
100
50
50 100 150 200 250

FSC-A (x 1,000)
Fig. 4.4 Contour plot

105
diagram
104
EPCam APC-A
−103 0 103−5,260
−474 −102 0 102 103 104 105

CD45 FITC-A
Fig. 4.5 Density plot

diagram
105
104
CD10 APC-A
103
−11,402
−798 0 103 104 105

PerCP-Cy5-5-A
Box 4.1 Commonly Used Graphs in Flow Cytometry

• Univariate histogram: The magnitude of the variable is plotted in the
X-axis, and the frequency of the events is displayed in the Y-axis.
• Bivariate histogram: Two parameters. One parameter is represented on
X-axis, and the other parameter is displayed on the Y-axis.
–– Scatter plot: Each cell or event is represented by a small dot.
–– Contour plot: The individual events are placed according to each
parameter’s intensity.
–– Density plot: The dots’ colour represents the events with the same
intensity.
Fluorescence intensity: The intensity of any fluorochrome stained marker is

directly proportional to the number of the attached fluorescent dyes with the
marker.
The cell population that is away from the origin will be more intensely positive
for fluorescence. Therefore the cells nearer to the origin of the graph are dim posi-
tive followed by moderately positive cell population and the intensely positive cells
(Fig. 4.6).
4.1 Distribution of Fluorescence Intensity
Linear scale: Here, the fluorescence intensity is distributed in the linear scale with
gradually increasing intensity (Fig. 4.7). It is usually shown when the fluorescence
intensity is not widely distributed, and it can be accommodated in the graph. Usually,
the DNA content distribution of the cell population is demonstrated on a linear scale.
4.1 Distribution of Fluorescence Intensity 39
Fig. 4.6 Schematic
diagram of fluorescence
intensity
Count
500
400 A645-19-Tube_002
50 100 150 200 250
SSC-A (x 1,000)
300
200 P2
100
0 50 100 150 200 250

100 200 300 400 FSC-A (x 1,000)
Fluorescence intensity
Fig. 4.7 The graph displayed in linear scale
Logarithmic scale: Here, the fluorescence intensity is distributed in the logarith-

mic scale. When the signal intensity varies widely in the population, then the data is
presented in the logarithmic scale to include the entire population in the graph.
In one log parameter graph, only one parameter is displayed as a logarithmic
graph (Fig. 4.8). In two log parameters, both the parameters are expressed in loga-
rithm. (Fig. 4.9).
Count
500
A645-19-Tube_002
400
100 150 200 250

SSC-A (x 1,000)
300
200
P3
100
50
0 102 103 104 105
101 102 103 104
CD45 PerCP-Cy5-5-A
Fluorescence
Fig. 4.8 only one parameter is displayed as a logarithmic graph
Count
105
Lymph
104
105
103
103 104
CD23 PE-A
102
−205 0102
101
−100 0 102 103 104 105

0 CD5 FITC-A
101 102 103 104
Fig. 4.9 Biexponential two log parameters
Logicle: Many investigators prefer the “logicle” to display the data. It is a biex-
ponential data display that includes minimal or even near-zero intensity (Fig. 4.10).
Logicle displays the cells that are piled up in the ‘logarithmic’ display.
Two parameter interpretation: The distribution of specific fluorescent stained
cells can be better demonstrated in a four-quadrant display (Fig. 4.11).
4.1 Distribution of Fluorescence Intensity 41
A-1395/2020-Tube_003
105
104
Lombdo PE-A
103 102
0
−275 −102
−085 −102 0 102 100 102

Koppo FITC-A
101
Fig. 4.10 Logicle graph where the exponential data displays minimal or even near-zero intensity
In this graph, the X-axis may display one type of fluorescence labelled marker
(such as FITC), and Y-axis may show the other type of fluorescence labelled marker
(such as PE). The population of cells closer to the origin are interpreted as negative
for fluorescence, and therefore, the cells left to the vertical dotted line are negative
for fluorescence tagged cells (Fig. 4.12, here FITC labelled cells). As we move away
from the origin and more right to the vertical line, the value of the fluorochromes are
more positive. Similarly, the cell population below the horizontal dotted lines are
negative for the fluorescence tagged cell (Fig. 4.12 here PE).
Fig. 4.11 Two parameter

histogram displaying the
positivity of the markers
according to the
fluorescence intensity
Fig. 4.12 The graph A5531/19-Tube_002

displays the positivity of
the two markers in
105
different quadrant
CD23+ CD5+ and CD23+
104
CD23 PE-A
103
Both − CD5+
0
−278
−162 0 102 103 104 105

CD5 FITC-A
4.2 Gating
Gating is defined as the identification of a group of cells from the collected events
with the help of marking the specific regions of the plot (Box 4.2). The plot may be
linear, logarithmic or biexponential scales. The gating may be live gating at the time
of acquisition of the data, or it can be done at the time of data display.
Live gating or real-time gating has been discussed in the previous chapter of data
acquisition.
The gating on the acquired data can be done by Boolean logic (using AND/ OR/
NOT) or by sequential order to have the population hierarchy.
4.2 Gating 43
Manual gating: Here, the regions are drawn around the population of interest
manually by drawing polygon, rectangle or quadrant. The drawing of the boundary
may be changed manually. The essential points in this type of gating:
• Gating done on the log of the population remains as same type of scale.
• It is essential to include all the events in the selected gating, so the boundary of
the gate should be extended below the axis to include all the events.
Automatic gating: Here, the autopolygon is created around the cells of interest.
The cells of interest with a particular type are displayed within the gated boundary.
One should always verify that the desired events are included or not in the gating.
Sequential gating is the most popular gating system. Here the cells and subsets
of the cell population are defined by sequential gating.
4.2.1 The Crucial Gating in Flow Cytometry
Single-cell gating: This gating is very important to remove the clusters of cells. The
single-cell gating is done based on pulse geometry. In doublet, the pulse’s height
remains the same, but the integral area of the pulse increases (Fig. 4.13). Therefore,
a b
100 150 200 250
(x 1,000)
Height
Voltage
FSC-H
Area Area: Total integral pulse
Width: Time interval

50
between two pulses
Time 50 100 150 200 250

FSC-A (x 1,000)
c Singlet d
Single cell gating
50 100 150 200 250
FSC-H (x 1,000)
Voltage
50 100 150 200 250

Time
FSC-A (x 1,000)
Doublet
Fig. 4.13 (a) The single cells will have increased pulse area along with the height. (b). Single-cell
gating by adjusting the FSC area versus height. (c) The doublets have the same height but increased
pulse area. (d) Selective gating has excluded the doublets
in the graph, one should gate the cells with increasing area and height (as shown in
the graph), and outside the rectangular gate, the cells should be considered doublet.
Debris elimination by forward and side scatter gating: This gating done on
forward and side scatter (FSC versus SSC) to eliminate the debris and non-cellular
elements. The events with very low FSC or low FSC but high SSC are eliminated by
the rectangular gating (Fig. 4.14).
Sequential gating to identify subset: The subset gating depends on the markers
used to analyse the cell population. It is usually sequential gating. In the following
example (Fig. 4.15), we have done sequential gating. At first CD45 population fol-
lowed by CD19 population was gated, and in that population, CD5 and CD23 popu-
lation of cells were gated for analysis.
Fig. 4.14 The debris is Scatter plot

eliminated by gating in
250
(x 1,000)
FSC-A and SSC-A. The

debris will have low
FSC-A and high SSC-A
200
150
SSC-A
100
50
50 100 150 200 250

FSC-A (x 1,000)
a b c
Fig. 4.15 Sequential gating pictures are shown. (a) CD45 population of cells are gated, (b) CD19
population among those CD45 population are identified by gating. (c) CD5 and CD23 population
are further gated
4.2 Gating 45
a b
100 150 200 250
SSC-A (x 1,000)
105
CD30 PE-A
0 103 104
50
−1,929
−1,342 0 103 104 105 −8,833 0 104 105
PE-Cy7-A CD1a APC-A
c d
100 150 200 250

SSC-A (x 1,000)
105
CD30 PE-A
103 104
0
50
−1,929
−1,342 0 103 104 105

−8,833 0 104 105
PE-Cy7-A
CD1a APC-A
Fig. 4.16 Backgating: (a). CD3 population, (b). CD30 population, (c). Gated CD30 is high-
lighted, (d). CD30 population in CD3 after backgating
From the graph, one can analyse the following value:
• Number of events: Total number of events and number of events in the particular
population.
• Percentage of cell population: Percentage of cells in the defined gated population.
• Mean: Average linear value of the cell population.
• Geometric mean: Logarithmic average of the events in the gated population.
• Standard deviation (SD): SD represents the measure of the spread of the events
around the mean events.
• The median fluorescence intensity value of the defined population.
4.3 Backgating
The backgating helps to detect the population of cells that may be missed during the
initial gating (Fig. 4.16). So it helps to minimise the missing of the desired cells.
Backgating helps to confirm the present gating pattern particularly in those situa-
tions where we are not sure of the present gating strategy.
Box 4.2 Gating

Definition: It is the identification of a group of cells from the collected events
with the help of marking the specific regions of the plot.
Type of gating:
• Live gating: At the time of data acquisition.

• Gating on the acquired data: Gating on the recorded data.
• Manual gating: The regions are drawn around the population of interest
manually.
• Automatic gating: The autopolygon is created around the cells of interest.
• Single-cell gating: Single cells are gated with increasing area and height.
• Debris elimination: This gating done on forward and side scatter to elimi-
nate the debris and non-cellular elements.
• Sequential gating: Sequential cell population is selected to assess the sub-
set of cell population.
• Backgating: To detect the population of cells that may be missed during
the initial gating.
References
1. Dean PN, Bagwell CB, Lindmo T, et al. Data file standard for flow cytometry. Cytometry 1990;
11:323–332.
2. Spidlen J, Moore W, Parks D, Goldberg M, Bray C, Bierre P, Gorombey P, Hyun B, Hubbard M,
Lange S, Lefebvre R, Leif R, Novo D, Ostruszka L, Treister A, Wood J, Murphy RF, Roederer
M, Sudar D, Zigon R, Brinkman RR. Data file standard for flow cytometry, version FCS 3.1.
Cytometry A. 2010;77(1):97–100.
Quality Control in Flow Cytometry
5
5.1 Introduction
The flow cytometer is now widely used in the clinical area to diagnose and sub-
classify lymphoproliferative lesions, detect malignancy in body cavity fluids, and
assess the tumour’s prognosis. So it is essential to have proper quality control (QC)
in flow cytometry.
The QC in the flow cytometry should cover two aspects: Internal quality control
(IQC) and external quality assessment (EQA). Herein I will discuss both IQC and
EQA and focus on the various problems in this area.
5.2 Internal Quality Control
The internal quality control (IQC) includes the sample receiving, processing, fluo-
rochrome selection, instrumental control data display, and interpretation (Box 5.1).
Box 5.1: Internal Quality Control

• Specimen integrity
• Specimen processing
• Antibody
• Reagent
• Instrument control
Integrity and processing: The specimen integrity represents the proper fresh
specimen free of the clot, properly labelled sample, no gross haemolysis and an
optimum number of cells for the analysis. The fresh specimen is always better for
immunophenotyping. Frozen sample analysis never provides a good result because
https://doi.org/10.1007/978-981-16-2655-5_5
48 5 Quality Control in Flow Cytometry
there may be a substantial loss of different subsets of lymphoid cells. Similarly, the
time interval between the sample’s collection and processing may also have adverse
effects on the FCM result [1, 2].
The RBC lysis procedure should be optimum as the over lysis RBC may be
responsible for the change of FSC and SSC pattern and cell loss [3]. Similarly,
under lysis of RBC may seriously impair the detection of different subsets of lym-
phoid cells. Brando et al. have shown that excessive vortexing may also be respon-
sible for considerable cells loss [4]. The excessive vortexing causes fragmentation
of the cells and generation of excess debris. It is also needed to mention that the
under-vortexing of the sample may be responsible for many doublets and cell
aggregates.
Cell count in the sample should be optimum in number for the analysis. Usually,
5–20 × 109 cells/litre is needed for analysis.
Antibody: The proper selection of antibody for flow cytometry is essential. The
choice of the antibody depends on the intention of the identification of the popula-
tion. The polyclonal antibody may bind with other than the target antigen contain-
ing population. Therefore, the monoclonal antibody is always preferable. It is
necessary to know whether the epitope is on the surface of the cell or intracellular
because the detection of the intracellular epitope needs permeabilization of the
cells. The selection of the particular clone and the mention of the clone are also
required. It has been shown that the same antibody from a different clone may give
a variable result [5, 6].
Fluorescence Conjugate: In present days, multiple fluorochromes tagged anti-
bodies are used. The correct choice of fluorochrome is an essential pre-requisite for
this purpose. It has been noted that some fluorochrome conjugates such as FITC or
PE tagged with the antibodies show dim positivity. Therefore, the use of such fluo-
rochrome tagged antibody may have low sensitivity. The weaker antigen such as
CD19, CD13, etc., should be labelled with well sensitive fluorochrome whereas, the
strongly expressed antigen such as CD45 should be tagged with a lesser bright dye
FITC or PE.
Data Acquisition: The acquisition of the number of events takes a vital role to
produce a valid result. Overall in each experiment tube, 10,000 to 20,000 viable
cells are needed for immunophenotyping. In the case of lymphoid neoplasms, at
least 5000 lymphocytes events should be recorded. The diagnosis of minimal resid-
ual disease (MRD) is based on the detection of an abnormal population of cells that
are usually absent or rarely present in the normal population. The total number of
acquired events for the detection of MRD should be at least 100,000 cells.
5.3 Instrument Quality Control
Nowadays, the flow cytometer is a routinely used machine and is no more handled
by specially trained technologists. The FCM is a sensitive machine, and it needs
proper standardization before its use. The device needs calibration of the optical
alignment, electronic setup, the laser and photomultiplier tube and compensation
5.4 The Critical Factors to Have Good Quality FCM Data 49
setup (Box 5.2). The skilled and specially trained engineers carry out all these works
at six-monthly intervals.
Optical alignment of the instrument, sensitivity and linearity are the necessary
tasks and should be carried out by the operator at least once a week. Regular daily
calibration of the device is needed to have vigilance on instrument performance
monitoring [7].
Box 5.2: Instrument Control

Periodical control:
• Laser alignment
• Laser time delay
• Sensitivity
Everyday control:
• Setting the voltage of the photomultiplier tube
• Spectral overlap and compensation
• Gating control in multicolour flow cytometry
• FMO control
• Biological control
5.4 The Critical Factors to Have Good Quality FCM Data
Efficient FCM should have the following performances:

1 . High sensitivity: It should have the ability to identify (resolution) the various
subpopulations, the particularly dim population of cells.
2. Relative measured values of fluorescence: The relative measured values of fluo-
rescence depend on the instrument’s linearity accuracy.
3. Regular assessment of the reproducibility of the result and the performance of
the flow cytometer.
5.4.1 Sensitivity
The sensitivity of the FCM is measured in two ways (Box 5.3):

Resolution: It represents the separation of the dim population of cells from the
unstained population (Fig. 5.1).
Threshold: It is the capability to distinguish the dim population of cells from the
particle-free background.
The measurement index of sensitivity.
Fluorescence detection efficiency: The fluorescence detection efficiency indi-
cates how bright the reagent in the sample when detected in a particular detector.
The fluorescence detection efficiency depends on the following factors: laser power,
the sensitivity of the photomultiplier tube, efficiency of the optics, and performance
of the filter.
Negative Positive
population population
Low
resolution
Count
Count
High
resolution
FITC FITC
Fig. 5.1 Schematic diagram showing how the good resolution can separate two populations
of cells
Count
Count
Low optical High optical

background background
FITC FITC
Negative Dim
Fig. 5.2 Schematic diagram showing the effect of the optical background
Optical background: The optical background is related to detecting the dim

signal from the unstained cells by the detector. It is influenced by the intact optical
component and dirty flow cells (Fig. 5.2).
Electronic noise: Electronic noise indicates the background signal due to elec-
tronics. The increased electronic noise harms the resolution sensitivity of the
5.5 PMT Voltage Setting 51
instrument. It increases due to ineffective photomultiplier tube connections and

digital error.
Stain index: The stain index (SI) indicates the performance of the reagent in the
flow cytometer. It is calculated as the difference between the mean fluorescence
intensity of the positive and negative population divided by 2 × standard deviation
of the negative population. The width of the negative population depends on several
factors of the instrument, such as fluorescence detection efficiency, optical back-
ground and electronic noise (Fig. 5.3). The higher the stain index better the resolu-
tion. The dye with the high SI is used for the antigen with low expression, and the
dye with low SI is used for the antigen with high expression.

MFI of the positive population　 MFI of the negative population
SI
2 Standard deviation of the negative population
Box 5.3: Sensitivity of the Instrument

Fluorescence detection efficiency
• Laser power
• Sensitivity of the photomultiplier tube
• Efficiency of the optics
• Performance of the filter
Optical background
• Intact optical component
• Dirty flow cells
Electronic noise
• Ineffective photomultiplier tube connections
• Digital error
5.5 PMT Voltage Setting
The photomultiplier tube (PMT) voltage setting is a crucial step in getting the opti-
mal resolution and discriminating the different population of cells in the experi-
ment. The PMT setting is done by adjusting the voltage in the unstained population.
The fluorescence intensity comes at the first quarter in a four-decade logarithmic
scale in each fluorochrome dye (Fig. 5.4).
As the PMT voltage increases, the CV of the cytometry set up beads also
decrease, and at a certain point of PMT voltage, there is no improvement of CV. The
green arrow points out the optimal PMT voltage, and from this point, the increment
of PMT voltage does not improve the CV [7] (Fig. 5.5).
Count
SD
MFI of negative MFI of positive

MFI of positive population - MFI of the

Stain negative population
index 2 x SD of negative population
MFI means mean fluorescence intensity, SD means standard deviation
Fig. 5.3 Schematic diagram explaining stain index
104 Unstained 104

cells
PMO voltage
103 adjustment 103
102 102 Unstained

cells
101 101
100 101 102 103 104 100 101 102 103 104
Fluorescence intensity Fluorescence intensity
Fig. 5.4 The adjustment of the voltage of the photomultiplier tube

5.5 PMT Voltage Setting 53
Fig. 5.5 The setting of

104
PMT voltage and CV
103
Optimum
CV 102 voltage
101
100 200 300 400 500

PMT voltage
Fig. 5.6 Schematic 525/550 BP 585/640 BP

diagram showing the
overlapping emission
1.0 FITC PE
spectra of Spectral
flurochrome dyes overlap
0.8
Emission
0.6
0.4
0.2
400 500 600 700

Wavelength (nm)
5.5.1 Compensation
The fluorochrome in the flow cytometer emits the photons when it is hit by an exci-
tation beam of laser light. The energy of the photons is of variable range. So the
emission spectrum of each fluorochrome dye covers a wide range of the wavelength
of light. The wavelength of the emitted light by fluorescence is always of the higher
wavelength of light. In the case of multicolour flow cytometry, the fluorescence
light’s emission may spill over to the detection range of the other fluorochrome dye
(Fig. 5.6). Such as the fluorescence of FITC dye is detected by the bandpass (BP)
filter of 525–550 nm wavelength. However, some fluorescence light is also detected
in the 585–640 nm BP filter used for PE fluorescence. We should always subtract
the spillover fluorescence cells from the wrong channel to identify the correct popu-
lation of cells. The correction of the fluorescence spillover in the multi-coloured
flow cytometry is known as compensation (Box 5.4). For the compensation to cal-
culate, we need a series of beads or samples of cells that are stained with single fluo-
rescent dyes. The compensation should be calculated after the PMT voltage set up.
For compensation to calculate, the background fluorescence of positive and nega-
tive control should be the same, and the compensation control should be brighter
than any sample whose compensation is measured [8].
Box 5.4: Compensation

Definition: In the case of multicolour flow cytometry, the fluorescence light’s
emission may spill over to the detection range of the other fluorochrome dye.
The correction of the spill over fluorescence intensity is known as
compensation.
How to do compensation:
• A series of beads or samples of cells that are stained with single fluorescent
dyes are used.
• The compensation should be calculated after the PMT voltage set up.
• Both positive and negative control of the cell/beads are used.
• The background of the positive and negative control should be same.
• Software is used from the data of positive and negative control beads.
5.6 Daily Cytometer Set up
The daily cytometer set up is mandatory for the optimum performance of the instru-
ment. Most of the company provides the ready-made commercially available beads
and the necessary software for the device set up. The daily cytometer set up helps to
get consistent and high-quality data and designing the multicolour flow cytometry
test (Box 5.5). It also helps to identify the early dysfunction of the instrument.
The company uses uniform beads that are excited by the laser beams supplied by
the company in the machine. The beads emit fluorescence after hit by the laser
beam, and the fluorescence is detected by the machine’s respective detectors. The
daily use of running the beads supports the configuration of the laser beam, voltage
control of the photomultiplier tube, compensation setting and the overall setting of
the instrument for the application.
Box 5.5: Importance of the Cytometer Set up

• Assessment of the baseline performance.
• Generation of the consistent high-quality data.
• Allows the comparison between the performance of different instruments.
• Designing the multicolour flow cytometry experiments.
• The detection of the early dysfunction of the flow cytometer.
References 55
5.7 External Quality Assessment (EQA)
External Quality Assessment of flow cytometry procedure provides a snapshot of

the performance. The specimens are sent at a few monthly intervals to the laborato-
ries. The final data generated in the laboratory help to compare the performance of
different laboratories. The separation and counting of different population and CV
of the controlled population of different laboratories can be compared.
References
1. Borowitz MJ, Bray R, Gascoyne R, Melnick S, Parker JW, Picker L, Stetler-Stevenson M. U.S.-
Canadian consensus recommendations on the immunophenotypic analysis of hematologic neo-
plasia by flow cytometry: data analysis and interpretation. Cytometry. 1997;30(5):236–44.
2. Ekong T, Kupek E, Hill A, Clark C, Davies A, Pinching A. Technical influences on immu-
nophenotyping by flow cytometry. The effect of time and temperature of storage on the
viability of lymphocyte subsets. J Immunol Methods. 1993;164(2):263–73. doi: https://doi.
org/10.1016/0022-1759(93)90319-3. Erratum in: J Immunol Methods 1993 Dec 3;166(2):301.
3. Romeu MA, Mestre M, González L, Valls A, Verdaguer J, Corominas M, Bas J, Massip E,
Buendia E. Lymphocyte immunophenotyping by flow cytometry in normal adults. Comparison
of fresh whole blood lysis technique, Ficoll-Paque separation and cryopreservation. J Immunol
Methods. 1992;154(1):7–10.
4. Brando B, Göhde W Jr, Scarpati B. D'Avanzo G; European working group on clinical cell
analysis. The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting
in phosphate-buffered saline-diluted leukapheresis samples. Cytometry. 2001;43(2):154–60.
5. Molica S, Dattilo A, Alberti A. Myelomonocytic associated antigens in B-chronic lymphocytic
leukemia: analysis of clinical significance. Leuk Lymphoma. 1991;5(2–3):139–44.
6. Morabito F, Prasthofer EF, Dunlap NE, Grossi CE, Tilden AB. Expression of myelomonocytic
antigens on chronic lymphocytic leukemia B cells correlates with their ability to produce inter-
leukin 1. Blood. 1987;70(6):1750–7.
7. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of
positivity. Cytometry A. 2006;69(9):1037–42.
8. (Szalóki G, Goda K. Compensation in multicolor flow cytometry. Cytometry A
2015;87(11):982–985. doi: https://doi.org/10.1002/cyto.a.22736. Epub 2015 Sep 8..
Fluorescent Probes and Different Useful
Markers for Flow Cytometry 6
For the successful application of flow cytometry, it is essential to know the fluores-
cence mechanism and the working principle of fluorochrome dyes. The properties
of the various fluorescence dyes also help to understand their appropriate uses. It is
particularly true at the time of simultaneous application of the multiple fluoro-
chromes tagged markers. In this chapter, the basic principles of fluorescence and the
properties of various fluorochromes have been described.
Light as quantum mechanics: According to quantum mechanics, the light is
both in wave and particle form. When the light interacts with the atom, then it is in
the particle form known as photons. The photon is the elementary particle with no
mass or charge. When the photon hits a molecule, the energy is absorbed. The
energy of the molecule is raised from the ground state to the excited state. When the
molecule releases the energy, the photon is emitted, and the molecule comes to the
ground state.
What is fluorescence: Fluorescence is a phenomenon where some molecules
absorb light of a specific wavelength (high energy and lower wavelength) and then
release energy by emitting light (low energy, higher wavelength) (Box 6.1). The
emitted light is of a different colour than that of light of absorption. The molecules
that can emit fluorescence are known as fluorochrome, such as fluorescent isothio-
cyanate (FITC), DAPI, etc.
Events in fluorescence: The Jablonski diagram explains the events of fluores-
cence (Fig. 6.1). Here. I describe the critical events in the fluorescence [1].
Ground state: It is the stable state of the fluorescence molecules (S0). In the
ground state, the molecule is in the relatively low-energy level and do not emit any
fluorescence.
Excited state: When the light of a particular wavelength hits the dye molecule,
the molecule absorbs the photon. It is known as excitation (S2). The excited fluoro-
chrome molecule attains a higher energy state depending on the light’s energy level
and wavelength. The excitation of the molecule by the photon is an extremely tem-
porary phenomenon and takes only in femtoseconds. The fluorochrome molecule
https://doi.org/10.1007/978-981-16-2655-5_6
58 6 Fluorescent Probes and Different Useful Markers for Flow Cytometry
Fig. 6.1 Jablonski diagram explaining the fluorescence
Fig. 6.2 Summary of fluorescence phenomena
eventually releases energy by the vibration and reaches a lowest-energy excited

state (S1). It is a semi-stable condition and comparatively slower process that occurs
in picoseconds.
Emission state: In this state, the fluorochrome molecule releases energy and
returns to the stable ground state. The released energy is lower than the initial
absorbed energy. Therefore the wavelength of the light is higher at the time of emis-
sion. It indicates that the colour of the emitted life is different from the colour of the
absorbed light. Figure 6.2 shows a summary of the fluorescence.
6.1 Staining by the Fluorochrome Dye 59
Fig. 6.3 Difference of

wavelength of the
absorption peak of
excitation and emission
light is the “Strokes shift”
The difference in the peak wavelength of the absorption and emission spectra is
known as “Stroke’s shift” (Fig. 6.3). The more the “Stroke’s shift”, the more is the
separation of the excitation and emission light.
The absorption of light by the fluorochrome dye is a very fast process as it takes
10−15 s only. However, the emission of fluorescence is a much slower process. It is
essential to have a strong intensity of fluorescent light for better detection.
Box 6.1: Fluorescence

What it is: Fluorescence is a phenomenon where some molecules absorb
light of a specific wavelength (and then release energy by emitting light).
Events in fluorescence
• Ground state: In this state the molecule is in the relatively low-energy

level and do not emit any fluorescence.
• Excited state: Here the molecule absorbs the photon and attains a higher
energy state.
• Emission state: In this state, the fluorochrome molecule releases energy
and returns to the stable ground state.
Stroke’s shift: It is the difference in the peak wavelength of the absorption

and emission spectra.
6.1 Staining by the Fluorochrome Dye
The fluorochrome can do staining in the following ways:
(a) The fluorochrome dye is linked with the primary antibody in case of direct
staining and secondary antibody in indirect staining. The dye is covalently con-
jugated with the antibodies.
(b) Some fluorochrome dyes have an affinity for specific substances and accumu-
late around them in higher concentrations, such as lipophilic dye binds with the
lipid-rich membrane.
(c) Fluorochroming/hyperchroming: Certain DNA binding dyes such as propidium
iodide (PI) and Ethidium bromide (Et Br) intercalate with nuclei acid. The
quantum yield of these dye increases, and they fluorescence strongly. It is
known as fluorochroming/hyperchroming.
Properties of fluorochrome dye; the various properties of the fluorochrome

dyes are described here (Box 6.2):
1. Molar extinction coefficient: The fluorescence intensity of the fluorochrome is
directly proportional to molar extinction coefficient. The molar extinction coef-
ficient represents how intensely a chemical absorbs light of a specific wave-
length. It is an intrinsic property of the chemical substance that depends on the
compound’s chemical composition and structure.
2. Quantum yield: The fluorescence quantum yield represents the efficiency of the
compound to emit photons to lose energy. It is measured as the ratio of the num-
ber of emitted photons divided by the absorbed photons by the substance. The
fluorescence quantum yield value may range from 0 to 1, and the higher the
value, the greater the fluorescence intensity. The quantum yield of a fluoro-
chrome dye is always less than one because of internal conversion and quenching.
Number of emitted photons

Fluorescence quantum yield =
Number of absorbed photons

The energy is lost due to internal conversion by collision with various non-
fluorescent molecules in the solution. In quenching, the fluorescence is lost due
to the transfer of energy to the neighbouring non-fluorescent molecules.
3. Absorption and emission spectra and spectral overlap: Each fluorochrome
dye has a specific absorption spectrum. The peak wavelength of the maximum
excitation of the dye is known as absorption maximum. The dye emits fluores-
cence of a range of wavelength of light, and the peak wavelength of emission is
known as emission maximum. As the emission of the fluorochrome dye may be
in a certain range, so there is a good chance to overlap the emission of the differ-
ent dyes. It is known as spectral overlap. The appropriate filter is needed to elimi-
nate the light from the unwanted fluorochrome dye. Due to the spectral overlap,
each fluorochrome may give rise to a signal to the multiple detectors. It is, there-
fore mandatory to correct the spectral overlap. Moreover, in the case of multico-
loured flow cytometry, the selected dye should be used to reduce spectral
overlapping.
4. Fluorescence resonance energy transfer (FRET): At times, the two fluoro-
chrome dye may be closely placed, and the outer electronic orbit may overlap.
One of the fluorochrome dyes (donor) is excited by the shorter wavelength, and
Fig. 6.4 Schematic diagram describing FRET
Fig. 6.5 The emission spectrum of the donor overlaps with the absorption spectrum of the
acceptor dye
the energy is transferred to the other dye (acceptor). This phenomenon of

energy transfer is known as FRET (Fig. 6.4) (Box 6.3). The donor fluoro-
chrome dye reaches the ground state and is quenched. In contrast, the acceptor
fluorochrome emits fluorescence (Fig. 6.5). The requirements for FRET is
following:
(a) To have the overlapping range of the emission spectrum of the donor with
the absorption spectrum of the acceptor dye. It means than the energy loss of
the donor dye should exactly match to excite the acceptor dye (resonance).
(b) The donor dye should have a good molar extinction coefficient and quantum
yield to release the energy for the excitation of the receptor dye.
(c) The distance between the donor and acceptor fluorochrome dye should be
2–10 nm. The intensity of the FRET is inversely proportional to the sixth
power of the distance between the donor and acceptor dye.
Fig. 6.6 Acceptor dye receives the energy and emits fluorescence
6.1.1 Applications of FRET
The FRET phenomenon is applied in the following areas:

(a) Multicoloured flow cytometry: The wise combination of two fluorescent dyes may
help to make multiple newer dyes. The combined dye can be tagged with the anti-
bodies at the time of multicoloured flow cytometry. These dyes are known as a
tandem dye. PE-Cy™ 5, PerCP-Cyanin™ 5.5 are examples of tandem dye. Here
the one fluorochrome dye (donor) is coupled with the second dye (acceptor). The
donor dye is excited by light, and the energy is transferred to the acceptor dye for
excitation. The acceptor dye gives fluorescence at a longer wavelength (Fig. 6.6).
(b) FRET may help to co-localize the two closely spaced different antigens in the
cell. One antibody is labelled with a donor fluorescent dye, and the other anti-
body is labelled with the acceptor fluorescent dye. If the antigens are co-
localized, then the acceptor fluorescent dye will show increased fluorescence
due to FRET compared to separately labelled fluorescent conjugated antibodies.
(c) FRET can help to make substrate analogues. In this situation, the donor and accep-
tor dyes are attached to either side of the cleavage site. Suppose the enzyme breaks
the protein, then there will be no FRET (Fig. 6.7). However, the intact protein will
produce FRET effect, and there will be fluorescence from the acceptor dye.
Box 6.2: Properties of the Fluorochrome Dye

Properties
(a) Molar extinction coefficient: It represents how intensely a chemical
absorbs light of a specific wavelength.
(b) Quantum yield: It represents the efficiency of the compound to emit
photons to lose energy.
–– It is the ratio of the number of emitted photons divided by the absorbed
photons by the substance.
–– The higher the value, the greater the fluorescence intensity.
(c) Absorption and emission spectra:
–– Absorption maximum: The peak wavelength of the maximum excita-
tion of the dye is known as absorption maximum.
–– Emission maximum: The peak wavelength of emission.
(d) Fluorescence resonance energy transfer (FRET): The two fluoro-

chrome dye is closely located and the donor dye is excited at lower wave-
length and subsequently transfer the energy to the acceptor dye to excite it.
Box 6.3: Fluorescence Resonance Energy Transfer (FRET)

What it is: The two closely located fluorochrome dye are closely placed, and
the donor fluorochrome dyes is excited by the shorter wavelength, and trans-
fer the energy to the acceptor dye so that it emits fluorescence.
Essential requirements for FRET:
1 . The overlapping range of the emission spectrum of the donor with the
absorption spectrum of the acceptor dye.
2. Good molar extinction coefficient and quantum of the donor dye.
3. The donor and acceptor fluorochrome dye should be closely located

(2–10 nm).
Applications:
• To make multiple newer dyes in multicoloured flow cytometry such as
PE-Cy™5, PerCP-Cyanin™ 5.5.
• Co-localization the two closely spaced different antigens in the cell.
• To make substrate analogues in the enzymatic reaction.
Fig. 6.7 Schematic
diagram showing the
breakage of protein and no
FRET effect
6.1.2 The Desirable Characteristic of a Fluorochrome Dye
The successful use in the multicoloured flow cytometry studies, the fluorochrome
dye should have specific desirable characteristic
(a) Brightness: The dye should have good brightness represented by a high molar
extinction coefficient.
(b) Quantum yield: The quantum yield of dye should be high that means the emit-
ted photons should be good in number in relation to absorbed photons.
(c) Spectral overlapping: The multiple dyes should not have overlapping emission
spectra. However, the spectral overlap is more or less an inevitable consequence
in using multiple fluorochrome dyes. Selective use of the bandpass filters in
each detector and the correction of the spectral overlap mathematically are
helpful remedies.
(d) Biological inertness: The dye should be biologically inert. The fluorochrome
dye should not take part in any chemical reaction, and it should not affect the
cell. It is also essential that the dye should not stain the background material.
(e) Antibody binding: The dye could be easily bound with the antibody.
The brightness of fluorescence stain: The intensity of fluorescence depends on

the following factors:
(a) Molar extinction coefficient: The higher the molar extinction, the more is the
brightness.
(b) Quantum yield: Higher the quantum yield, the more intense is the brightness.
(c) Multiple conjugations with the marker: If multiple fluorochrome dye mole-
cules combine with a single antibody, then the chances of brightness increase
significantly.
(d) Sensitive detector: The highly sensitive fluorescence detector shows more

brightness of the staining.
Overall the brightness is represented by:

Brightness Molar extinction coefficient Quantum yield

6.1.3 Fluorochrome Dyes Used in a Flow Cytometer
A large number of fluorochrome dyes are used in the flow cytometer [2].
We can categorize them as:

1 . Single fluorochrome dye conjugated with antibodies, protein and other ligands.
2. Tandem dye.
3. Quantum dots.
4. Reporter molecules.
5. Green fluorescent proteins (GFP).
Fluorochrome dye conjugated with antibodies, protein and other ligands:
6.1.3.1 Single Fluorochrome Dye

FITC (Fluorescent isothiocyanate): FITC is the most popular fluorescent dye in
flow cytometry (Box 6.4). The dye readily conjugates with the protein with moder-
ate stability. The quantum yield of FITC is 0.5 and is considered relatively high. The
maximum excitation spectrum of the dye is 495 nm which is close to the 488 nm
wavelength of argon laser wavelength. The argon laser is used in almost all the flow
cytometer instrument in the market. The maximum emission spectrum of FITC is
519 nm. The disadvantages of FITC dye include:
(a) the long trail of emission so high chance of spectral overlapping,

(b) high pH sensitivity and,
(c) quick photobleaching effect.
Therefore FITC Is not a suitable dye in multicoloured flow cytometry.

Phycoerythrin: The commonly used phycoerythrins are R-PE and B-PE. R-PE
is a 196 kDa protein with maximum excitation spectra are 480, 546, 565 nm. The
maximum emission spectrum of R-PE is 578 nm.
The B-PE is 241 kDa water-soluble protein with maximum excitation and emis-
sion spectra are 545 and 565, respectively. The PE labelled antibodies are also popu-
lar in flow cytometry.
Allophycocyanin (APC): APC is a 104 kDa protein. The maximum absorption
and emission spectra of APC are 650 and 660, respectively. So the dye is generally
excited by helium–neon and diode lasers. The disadvantages of APC are its large
size that can cause steric hindrance during binding with the protein. The other limi-
tation is the possibility of background staining.
Phycocyanin(PC): C-PC is a phycobiliprotein with a molecular weight of
70,000 to 11,000 daltons. It has maximum excitation and emission spectra of 620
and 650 nm, respectively. The C-PC has a large stroke shift and a very high quan-
tum yield.
Table 6.1 shows the maximum excitation and emission spectrum of the various
fluorochrome dyes.
Table 6.1 The maximum excitation and emission spectrum of the various fluorochrome dyes
Fluorochrome Excitation maximum (nm) Emission maximum (nm)
Fluorescein Isothiocyanate (FITC) 495 519
Phycoerythrin (PE) 496 576
Allophycocyanin (APC) 650 660
Rhodamine red-X 570 590
Texas red® 595 613
Peridinin chlorophyll (PerCP) 477 678
Box 6.4: Fluorescent Isothiocyanate

Maximum excitation spectrum: 495 nm.
Maximum emission spectrum: 519 nm.
Advantages
• FITC have stable conjugation with protein and antibodies.
• Excitation spectrum is close to 488 nm wavelength and fit for the com-
monly used argon laser in flow cytometry.
• High quantum yield (0.5).
Disadvantages
• The long trail of emission and high chance of spectral overlapping.
• Highly sensitive to change in pH.
• Quick rate of photobleaching.
• Unsuitable dye in multicoloured flow cytometry.
6.1.3.2 Tandem Dyes

The tandem dyes are the conjugates of two dyes that follow the principle of FRET
(Box 6.5). One of the dyes behave as donor that transfer the energy to the acceptor
dye that emits fluorescence.
PE attached with an acceptor: PE-Cy5.5, PE-Cyanine7, PE-Texas Red and
PE-Fire 700.
APC attached with an acceptor: APC-Cy 5.5, APC-H7, APC-Fire 750.
PerCP attached with an acceptor: PerCP-Cyanine 5.5.
Advantages of tandem dye: The excitation and emission spectrum of tandem
dyes are different from the commonly used single fluorochrome dye. So the combi-
nation of single fluorochrome dyes along with different tandem dyes can be used in
the multicoloured flow cytometry. The tandem dyes may have the same excitation
wavelength of the donor but different emission spectra of the acceptor. Therefore,
the donor dye can be used along with its tandem dye, and the different emission
spectra may be detected by the different detectors (Table 6.2).
Table 6.2 The maximum excitation and emission spectrum of the tandem fluorochrome dyes
Fluorochrome Excitation maximum (nm) Emission maximum (nm)
PE-Cy5.5 496, 565 695
PE-Cyanine7 565 785
PE-Texas red 565 615
PE-Alexa Fluor 700 496, 546 723
PE-Alexa Fluor 750 496, 546 779
APC-cy 5.5 650 695
APC-H7 650 780
APC-fire 750 650 787
PerCP-cyanine 5.5 482 695
Precautions of Using Tandem Dye

The following precautions should be taken:
1. Photobleaching: The tandem dyes have a very high rate of photobleaching. So,
they should be kept away from sunlight.
2. Freezing: The tandem dye-antibody conjugate should never be frozen as freezing
may denature the donor fluorochrome.
3. Isotype control: Isotype control is necessary to use the tandem dyes in multico-
loured flow cytometry.
Box 6.5: Tandem Dyes

What are tandem dyes: Conjugates of two dyes that follow the principle
of FRET.
Commonly used tandem dyes:

• PE attached with an acceptor: PE-Cy5.5, PE-Cyanine7, PE-Texas Red and
PE-Fire 700.
• APC attached with an acceptor: APC-Cy 5.5, APC-H7, APC-Fire 750.
• PerCP attached with an acceptor: PerCP-Cyanine.
Advantages of tandem dye: These dyes can be used in multicoloured flow

cytometry as the emission spectra of the acceptor dye is different than that of
donor dyes.
Precautions of using tandem dye:

1 . Photobleaching: Due to very high rate of photobleaching the dyes should
be kept away from sunlight.
2. Freezing: freezing may denature the donor fluorochrome.
3. Isotype control: Isotype control is necessary for using these dyes.
6.1.3.3 Quantum Dots(QD)

The quantum dots are fluorescent semiconductor nanocrystals (Fig. 6.8) (Box 6.6).
Properties: These dots have specific unique properties: (a) QD absorbs light of
all wavelengths for their excitation, (b) the emission spectra of the different types of
quantum dots do not overlap, (c) the emission spectra of the different QD are sym-
metrical, and the tail of emission spectra are remarkably short, (d) the quantum
yield of the QDs are very high, and more than 90% of the absorbed energy is
released as fluorescence.
Structure: QDs are made of nanocrystals of Cadmium, Selenium and Tellurium
by high temperature. The size of the QD is modified depending on the emission of
the wavelength. There are three layers of QDs: (a) innermost semiconductor core,
Fig. 6.8 (a) Schematic diagram of the quantum dot, (b): Size of the quantum dot determines the
maximum emission spectrum
(b) covering shell, (c) outermost coating. The size of the nanocrystals is finely tuned
to adjust the emission spectra of each QDs.
Advantages of QD
The applications of QD in flow cytometry have the following advantages [3]:
1 . Brighter stain due to high quantum yield.

2. There is no overlapping of emission spectra of different QD.
3. Only a single laser beam can excite all types of QD at once.
4. No photobleaching effect.
Box 6.6: Quantum Dots (QD)

The quantum dots are fluorescent semiconductor nanocrystals.
Properties of QD:
• Absorbs light of all wavelengths for their excitation.
• The emission spectra do not overlap.
• The emission spectra of the different QD are symmetrical, and.
• High quantum yield.
Structure: The size of the nanocrystals is finely tuned to adjust the emission
spectra of each QDs.
Made of three layers:

• Innermost semiconductor core.
• Covering shell.
• Outermost coating.
Advantages:
• Brighter stain.
• No overlapping of emission spectra.
• Only a single laser beam can excite all types of QD at once.
• No photobleaching effect.
6.1.3.4 Reporter Molecules

The green fluorescent protein (GFP) and GFP-like proteins such as enhanced cyan
fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the
enhanced yellow fluorescent protein (EYFP), and Discosoma coral red fluorescent
protein (DsRed) have been used as fluorescein reporter molecules in flow cytome-
try. These reporter molecules have overlapping excitation spectra, and so they can
be excited simultaneously by a single excitation wavelength [4].
6.1.4 Multicoloured Flow Cytometry
In multicoloured FCM, more than one fluorochrome dye labelled markers are used
simultaneously (Box 6.7). In advanced flow cytometry, 10 to 17 fluorochrome
tagged dye can be used.
There are several advantages of multicoloured FCM:

1 . It saves time and reagents.
2. A small amount of sample can be used for multicoloured FCM.
3. Several subsets of cell population can be detected.
4. Identification of a rare subset of the cell population.
5. A large amount of information from the small samples.
The disadvantages of multicoloured FCM:

1 . Carefully chosen fluorochrome dye is needed.
2. Often compensation is needed.
3. Many markers may not have the appropriate conjugation of fluorochrome dye.
6.1.5 Basic Principles of Panel Design
The basic principles of panel design in multicoloured flow cytometry include:

• The assessment of the capability of the instrument.
• Proper selection of the dye with appropriate non-overlapping excitation and
emission spectra.
• No trailing of the emission spectra.
• Relative brightness of the fluorochrome dye.
• Stable fluorochrome-conjugated antibody.
6.1.5.1 Machine Understanding

The following information about the machine are needed:
Laser: The knowledge of the number of lasers source and the wavelength of the
beam of light are needed. In the case of four lasers, the available wavelength
of the lasers are:
1. Violet: The wavelength of the violet laser is 405 nm. Krypton-ion lasers can
generate a violet laser beam of 405, 413 and 425 nm [5]. Many low molecular
weight fluorochrome dyes such as Cascade Blue and Pacific Blue can be excited
by the violet laser. Several QD fluorochromes can also be excited by a vio-
let laser.
2. Cyan laser: The wavelength of the blue laser is 488 nm. It is the most commonly
used laser source in commercial flow cytometry. The argon ion laser beam gener-
ates 488 nm wavelength laser beam and can excite FITC, PE, many tandem dyes
and GFP.
3. Red: Krypton-ion lasers can generate a red laser beam of the wavelength of 641
and 647 nm. They excite the dye Cy7, APC, and APC tandem dye APC-Cy 5.5
and APC-Cy 5.7.
4. Yellow-green laser: The wavelength of the Yellow-green laser is 561 nm. Yellow-
Green laser is just an additional fourth laser and has limited uses. Phycoerythrin
(PE) and its tandem dyes can be excited adequately by this laser.
Detector channels for each laser: It is also essential to know the number of
detector channels in each laser. The knowledge of the detector channels may help to
pick up the emission spectra of the fluorochrome dye.
Fluorochrome measured by the detector: The number of the available fluoro-
chrome dye detected by the detector channel of each laser helps to select the appro-
priate dye for the multicoloured FCM.
Table 6.3 shows the laser line, wavelength and available fluorochrome dyes for
multicoloured FCM.
Antigen density: Antigen density plays a critical role in selecting the fluoro-
chrome dye. Some antigens are expressed in low number, and therefore brighter
fluorochrome dye is needed to demonstrate them. Whereas some antigens are
expressed in higher concentration, and a relatively less brighter stain can demon-
strate these antigens.
Table 6.3 Laser line, wavelength and fluorochrome dye used

Excitation
Laser line Wavelength (nm) Fluorochrome dyes
Violet 405, 413 and 425 Brilliant violet 605, brilliant violet 421, Pacific blue,
Alexa-flour 430, QD545, QD 655
Blue 488 FITC, PerCP, PE-Cy7, PErCP-Cy5.5, PI, GFP, YFP
Red 641 APC, Cy5, APC-Cy7, APC-H7, Alexa Fluor 647
Yellow- 561 PE,PE-Texas red, PE-Cy5, PE-Cy7
green
Box 6.7: Multi-Coloured Flow Cytometry

Here, more than one fluorochrome dye labelled markers are used
simultaneously.
Advantages:
1 . Saves time and reagents.
2. Needs small amount of sample.
3. Several subsets of cell population can be detected.
4. Identification of a rare subset population.
5. Huge information from the small samples.
The disadvantages:
• Needs careful selection of the fluorochrome dye.
• Compensation is needed.
• Absence of the appropriate conjugation of fluorochrome dye for many
markers.
Basic principles of panel design

• Depends on capability of the flow cytometer: Number of the lasers, wave-
length and the number of detectors.
• Proper selection of the dye with appropriate non-overlapping excitation
and emission spectra.
• No trailing of the emission spectra.
• Relative brightness of the fluorochrome dye.
• Stable fluorochrome-conjugated antibody.
Designing a staining panel:

• Select the bright dye.
• Avoid the fluorescent spillover.
• Avoid degradation of the tandem dye.
• Choose the brightest dye for the antigen with low expression.
Table 6.4 Stain index of dif- Brightness Fluorochrome Stain index

ferent fluorochrome dye High
APC 193
PE 232
PE-CY5 216
Alexa-flour 91
488
Medium
FITC 51
PE-Texas red 40
Q dot 605 35
Low
Q dot 655 20
Q dot 705 18
APC-Cy7 24
PERCP 25
Designing a staining panel

1. Select the bright dye: The brightness of the dye is measured by stain index.
Stain index. The higher stain index means brighter fluorochrome dye. The dye
with the high SI is used for the antigen with low expression, and the dye with low
SI is used for the antigen with high expression. The Table 6.4 shows SI of differ-
ent fluorochrome dyes.
2. Avoid the fluorescent spillover (long tail of emission spectra): Whenever
more than one fluorochrome is used, there will be the probability of overlapping
emission spectra. This spillover of the emission fluorescence is overcome by
compensation. However, it may not be possible to remove completely the spill
over. Therefore it may be wise to include those dyes that have minimal spillover.
The spillover between the two brightest fluorochrome dye may be challenging as
it may affect the correction of spillover.
3. Avoid degradation of the tandem dye: The tandem dyes have the tendency to
degrade in the sun light, fixation or in the elevated temperature. In that situation
the fluorescence will be emitted in the main dye (parent dye) detector and may
give false positive result. Such as in APC-Cy7 tandem dye degradation the detec-
tor will detect positive events in APC (parent dye).
The rules of selection if fluorochrome dyes in multi coloured FCM are the
following:
Rule 1: Assess the capability of the instrument: Number of the lasers, wavelength
and the number of detectors.
Rule 2: Choose the dye with a high stain index (brightness).
Rule 3: Choose the dye with the high SI (brightest dye) for the antigen with low
expression, and the dye with low SI (less bright dye) is used for the antigen with
high expression.
References 73
Rule 4: Select the combination of dyes with minimum spectral overlapping.

Rule 5: Never select the brightest dye with overlapping emission spectra.
Rule 6: Be careful with the tandem dye as they may degrade easily in the pres-
ence of sunlight.
References
1. Houston JP, Yang Z, Sambrano J, Li W, Nichani K, Vacca G. Overview of fluorescence lifetime
measurements in flow Cytometry. Methods Mol Biol. 1678;2018:421–46.
2. McKinnon KM. Flow Cytometry: an overview. Curr Protoc Immunol. 2018;120:5.1.1–5.1.11.
3. Ibáñez-Peral R, Bergquist PL, Walter MR, Gibbs M, Goldys EM, Ferrari B. Potential use of
quantum dots in flow cytometry. Int J Mol Sci. 2008;9(12):2622–38. https://doi.org/10.3390/
ijms9122622. Epub 2008 Dec 17
4. Beavis AJ, Kalejta RF. Simultaneous analysis of the cyan, yellow and green fluorescent pro-
teins by flow cytometry using single-laser excitation at 458 nm. Cytometry. 1999;37(1):68–73.
5. Telford WG. Lasers in flow cytometry. In: Darzykiewicz Z, et al., editors. Methods in cell biol-
ogy, Academic Press, vol. 102. NY: New York; 2011. p. 375–409.
Nuclei Acid Dye and DNA Content
Measurement in Flow Cytometry 7
Fluorescent DNA dyes are used to measure the cell’s DNA content and analyse the
cell cycle. The DNA dye used in flow cytometer should have certain essential char-
acteristics, as mentioned in the Box 7.1. The most important requisite of the DNA
dye is the DNA specificity.
Box 7.1: Basic Requirements of a Good DNA Dye

• Dye should be specific to DNA.
• Dye should bind stoichiometric to the DNA content.
• Dye should show strong enhancement of fluorescence after binding with
the target.
7.1 Types of DNA Dye
The DNA dye may be primarily of two types (Box 7.2):

1. The dye that binds in the minor groove of DNA: DAPI, Hoechst, Mithramycin
The minor grooves are the narrow bend of the DNA helix molecule. The minor
groove binding dyes are less flexible and have less affinity. The dye binds by
hydrogen bonding to the base pair by non-covalent means.
2. The dye that is intercalated within the base pair: Propidium iodide (PI), Ethidium
Bromide, Acridine orange.
The intercalated DNA-binding dye binds between two sets of base pair by non-
covalent bonding. The dye molecule distorts the DNA. Intercalated dye only
binds with the double-stranded DNA and can identify the nuclear fragments
from the debris.
https://doi.org/10.1007/978-981-16-2655-5_7
76 7 Nuclei Acid Dye and DNA Content Measurement in Flow Cytometry
3. Bis-intercalator: Some DNA dyes are bis-intercalator such as TOTO, YOYO. Bis-

intercalator dyes have two intercalating parts which are further connected by a
link. These dyes have more affinity than single intercalator dyes.
Box 7.2: DNA Dye

Type:
1 . Dye that binds with minor groove of DNA: DAPI, Hoechst, Mithramycin.
2. The dye that is intercalated within the base pair: Propidium iodide,

Ethidium bromide, Acridine orange.
3. Bis-intercalator: TOTO, YOYO.
Cell permeability:
• Permeable to the cell membrane: DAPI, Hoechst.
–– Non permeable to the cell membrane: Propidium iodide, Ethidium
bromide.
Staining:
• Dye that stains both DNA and RNA: Propidium iodide, Ethidium bromide,
Acridine orange.
• Dye that stains only DNA: DAPI, Hoechst, Mithramycin, TOTO.
Cell permeability of the dye: Some DNA dyes are permeable to the cell mem-
brane and can enter the living cells. DAPI and Hoechst 33342 are permeable and
can be used as a supravital cell cycle. PI is not cell permeable, and the cell needs to
be fixed before staining with PI.
DNA stain of the fixed cells: The optimum DNA stain of the fixed cells depends
on the following factors:
1. Time of incubation: The period of incubation is the crucial factor of DNA stain
by the dye.
2. Dye concentration: The concentration of the dye is essential, particularly in cell
cycle measurement. The DNA should be saturated with the dye; otherwise CV of
the graph will be broad.
3. Fixation of the cells: The type of fixative may have an impact on DNA staining.
Ethanol gives better fixation for DNA and cell cycle analysis than formalin
fixation.
4. Temperature: The staining temperature is also a factor of the optimum staining.
The cells are better stained at 37 °C than room temperature.
7.2 Description of Different DNA Dyes 77
7.2 Description of Different DNA Dyes
7.2.1 Intercalator Dyes
Propidium iodide (PI) and Ethidium bromide (EB): Both these two dyes PI and
EB, are similar in chemical structure. PI enters the cells much faster and has higher
water solubility than EB. Both the dyes stain both DNA and RNA, and they interca-
late in between the base pairs. These dyes do not have any selective preference on
the nucleotide sequence of DNA. The excitation/ emission spectra of PI are
488,532/617 nano micron (nm). In contrast, the EB had different excitation/emis-
sion spectra (518/605 nm). The PI and EB both stain DNA and also RNA (Table 7.1).
Therefore, RNAse enzyme should be used to digest RNA in the solution before the
quantitation of DNA. The cells should be fixed before staining with PI. It is prefer-
able to use ethyl alcohol than formalin fixation of the cell as ethanol provides a
better CV. In general, PI gives better CV in DNA histogram than EB. The entry of
EB within the cell is slow, and the cells with intact cell membrane rapidly pump out
this dye. Therefore, the double-charged PI with higher binding affinity is preferable
for DNA ploidy study.
Acridine orange (AO): AO is another intercalated DNA dye. It stains both DNA
and RNA. The sensitivity of AO is variable due to a mild variation of operational
parameters. Therefore, its use in FCM is restricted. The excitation and emission
spectra of AO are 460/650 and 502/536 nm, respectively.
7.2.2 Minor Groove Binding Dye
Hoechst 33342 and Hoechst 33258: The Hoechst dyes bind in the minor outer
grooves of DNA double helix. These dyes have a preference in A-T base pair regions
of DNA. The Hoechst dye is permeable to the cell membrane and can stain the live
cells faster than the fixed cells. However, the increased concentration of the Hoechst
dyes is required to stain the live cells than the fixed cells. The excitation and emis-
sion spectra of dye are 350/461 nm.
Table 7.1 The comparison Features PI EB

of PI and EB Charge Double charge Single charge
Dye binding Intercalated Intercalated
RNA stain Yes Yes
Cell permeability Fast Slow
Excitation 488 and 518 nm
spectrum 532 nm
Emission spectrum 617 nm 605 nm
Cell fixation Ethanol fixation Ethanol fixation
Table 7.2 shows the basic information of different commonly used fluorescent DNA dye
Mode of DNA/RNA Cell Sequence Excitation Emission
Dye binding stain permeability specificity (nm) (nm)
PI Intercalated Both No No 488,532 617
EB Intercalated Both No No 518 605
AO Intercalated Both No No 460/650 502/536
DAPI Minor groove DNA Little A-T 358 452
Hoechst Minor groove DNA Yes A-T 350 461
33342
Mithramycin Minor groove DNA No G-C 441 575
TOTO Bis- Both No No 514 533
intercalator
DAPI (4’,6-Diamidino-2-phenylindole): DAPI is a minor groove binding dye.

It also has a high affinity to A-T base pair. The dye is highly specific for DNA and
does not stain RNA. It is less permeable to the cell membrane. The excitation/ emis-
sion spectra of DAPI are 358/452 nm, respectively.
Mithramycin: Mithramycin is a chemotherapeutic agent which binds in the
minor grooves of DNA helix. It is a DNA-specific dye and does not stain
RNA. Mithramycin dye has a special preference to G-C base pair of DNA. The dye
is not permeable to the cell membrane and the cells should be fixed in ethyl alcohol
for staining with mithramycin. The excitation/ emission spectra of this dye are
441/575 nm respectively.
7.2.3 Bis-Intercalator Dyes
TOTO: It is a bis-intercalator dye with very strong affinity to DNA. The dye has
very low quantum yield (less than 0.01), however, it increases thousand fold when
binds with DNA. The dye is impermeable to cell membrane. TOTO binds with both
DNA and RNA. The excitation/ emission spectra of this dye are 514/533 nm,
respectively (Table 7.2).
7.3 DNA Content and Ploidy Analysis
The gametes contain haploid chromosome (n). Human somatic cells have a diploid
(2n) chromosome. The normal cell divides into the following phases:
(a) G1 (Gap 1): The G1 cells prepare to replicate DNA to undergo the synthetic
phase (S-phase). These cells contain diploid (2n) DNA.
(b) S-phase: DNA replication occurs in the S-phase, and the nuclei contain a vari-
able amount of DNA from 2n to 4n. The S-phase fraction of the cells is also
known as cell proliferative fraction.
7.4 Standard Nomenclature 79
a b
Fig. 7.1 (a) Schematic diagram of the cell cycle, (b) Schematic diagram of DNA histogram in
flow cytometry
(c) G2 (Gap 2): The cells in this phase prepare for final mitosis, and they con-
tain 4n DNA.
(d) M-phase: In the mitotic phase, the cell divides into two daughter cells contain-
ing 4n DNA.
The resting and non-proliferative cells are in G0 phase with 2n DNA. These cells
may undergo in G1 phase or may remain lifelong in the G0 phase. The details of the
cell cycle are highlighted in Fig. 7.1a. On a morphological basis, it is impossible to
distinguish the cells in G0, G1, S and G2 phase. Only mitotic cells can be identified.
The DNA-binding dye binds with DNA in a stoichiometric manner. The cells with
4n DNA content cells will show double the fluorescence intensity than the 2n DNA
content cells. It means the channel number of 4nDNA cells will be double the
2nDNA containing cells. As most of the cells are in the G0-G1 phase of the cell
cycle, we get a diploid peak and a small tetraploid peak for G2-M cell population in
the double the channel number (Fig. 7.1b). The S-phase cells remain in between the
diploid and tetraploid peak.
7.4 Standard Nomenclature
According to the consensus report of the task force on standardisation of DNA flow
cytometry, the following nomenclature is applied [1, 2]:
DNA index (DI): DI represents the mean channel number of the G1 peak of the
tumour divided by the mean channel number of G1 peak of the normal cells. The
diploid tumour shows DI as 1.
Aneuploidy: The diploid tumour makes a peak in the G0/ G1 region of the DNA
histogram. The DNA tetraploid peak forms in the DNA index region 1.9 to 2.1. Any
peak other than the diploid and tetraploid peak is considered aneuploidy peak.
Coefficient of variation (CV): CV in DNA histogram is measured as:
CV: (standard deviation / mean channel number) X 100.
A good DNA histogram should have less than 8% CV. However, tumour DNA
peak in the histogram may be broader due to the coexistence of multiple subpopula-
tion of the neoplastic cells.
Source of sample in cytology: The common sources of DNA FCM are:
• Fine needle aspiration cytology (FNAC).

• Body fluids: Effusion sample, bladder wash, CSF, etc.
Sampling: The quality of the DNA FCM depends on the correct handling of the
sample. A fresh sample always gives a better result. If not possible to do the fresh
sample then the specimen can be stored by freezing. FNAC material can be col-
lected in the phosphate buffer solution. The body fluid should be collected along
with an anticoagulant to prevent coagulation. Heparin, EDTA, or ammonium oxa-
late can be used as anticoagulant. The cytology specimen should always be checked
by light microscopic examination after a rapid Giemsa stained smear.
Fixation: In the case of cytology material, the cells should be fixed by ice-cold
ethanol. Alternatively, buffered formalin can be used for cell fixation. It is prefera-
ble to avoid over fixation or fixation of the cells at higher temperature.
Single cell preparation: It is essential to have single cells in the flow cytometry
examination. The single cell can be prepared by:
1. Mechanically: The sample can be passed repeatedly through a nylon mesh with
a 50 micron pore size. The nylon mesh is kept in between the needle and syringe
hub, and the specimen should be passed repeatedly through the nylon mesh.
Advantage: Easy procedure and usually gives good result.
Disadvantage: The cells may be damaged.
2. Enzymatic: The trypsin enzyme can be used for disaggregation of the cells.
However, the cytology material does not require enzymatic disaggregation [3].
Minimum cell requirements for DNA histogram: The overall cell concentra-
tion should be at least 106 per ml. If the cell concentration is low, then the CV of the
graph may be substandard as the flow rate of the cells has to be increased at the time
of the acquisition. Too much-concentrated cells may affect the DNA saturation by
the dye as the dye may be needed much.
Microscopic examination: It is always advisable to check the quality of the
final sample. If possible, this can be done under a fluorescent microscope. The fol-
lowing factors may affect the staining:
(a) The presence of excessive debris,

( b) A large number of the clumped cells,
(c) The concentration of the cells.
7.4.1 Control Diploid Population
There is a definite need of a reference DNA sample to know the diploid peak. In
neoplastic sample, there may not be any normal diploid peak and therefore it may
7.5 Data Acquisition 81
be difficult to ascertain the diploid or aneuploidy peak. Normal healthy lympho-

cytes of blood can be used as controlled diploid peak in the cytology sample.
7.4.2 Staining for DNA FCM [4]
7.4.2.1 Materials
Chemicals
• 70% Ethyl alcohol
• Phosphate buffered solution (PBS).
• Propidium Iodide.
• Triton X-100 (Sigma).
• RNase.
Centrifuged tubes: 12 × 75 mm.

Flow cytometer having 488-nm argon laser.
DNA content measuring software.
PI-Triton X–RNAse solution:

• 10 ml of 0.1% (v/v) Triton X in PBS
• 2 mg RNase (free from DNase)
• 200 μl of 1 mg/ml Propidium Iodide.
Staining steps:
• Fill the centrifuged tube with 4.5 ml of 70% ethyl alcohol and keep it in ice to
make ice-cold ethanol.
• At first wash 106 cells in 5 ml of PBS by centrifuging at 200 x g.
• The supernatant fluid is discarded, and the cells are resuspended in 0.5 ml PBS.
• The cells are mechanically dispersed by repeatedly syringing through nylon mesh.
• Now transfer the cells into ice-cold ethanol and keep them for 2 h for fixation.
• Centrifuge the ethanol fixed cells at 200 × g for 5 min.
• Resuspend the cells in 5 ml PBS.
• Centrifuge at 200 × g for 5 min.
• Resuspend the cells in I ml of PI-Triton-X mixture.
• Keep for 15 min at 37 C.
• Measure DNA content of the cells in a 488 nm argon laser by flow cytometer.
7.5 Data Acquisition
• At first the alignment of the instrument should be checked as routine procedure.

• The control diploid DNA sample should be run.
• In the case of PI stained DNA 488 nm argon laser beam should be used. The
excitation beam of the other laser beam should be set according to the dye used.
• The voltage of the photomultiplier tube should be adjusted in such a way that
both diploid and tetraploid peak are seen in the histogram.
• One should collect all the signals, both debris and cells.
• At least 10,000 events should be acquired, excluding the debris.
• The fluorescent intensity in the X-axis should be linear than a logarithmic scale.
• It is important to run the sample in a moderate speed (200 to 300 events/second)
to get the good quality DNA histogram.
7.6 Interpretation
All the DNA histogram is not adequate for the interpretation. The causes of
inadequate DNA histogram for interpretation include:
• High CV (more than 8%).
• Too much debris.
• Too many aggregates and less number of singly dispersed cells.
Ploidy and S-phase: A single peak in the channel number of the control’s dip-
loid peak indicates the diploid peak of the tumour cells. The diploid peak of the
tumour should be present within ±5% of the channel number of the normal dip-
loid peak.
A tetraploid peak (4n) remains with 1.9 to 2.1 DNA index range. If another peak
appears in double the channel number of the tetraploid peak (8n) with an S-phase
fraction, then the tetraploid peak should be considered as aneuploidy population.
There are various softwares available in the market to estimate the S-phase frac-
tion. These sophisticated mathematical based softwares can eliminate the debris and
can accurately measure the S-phase fraction. It is preferable not to count the G2M
phase cells at the time of assessing the proliferative rate of the tumour cell.
Doublet discrimination: The inclusion of the doublets may erroneously increase
the G2M phase cells. Therefore, the doublets in the specimen should be excluded
from the DNA estimation. Simple gating of pulse width versus height may eliminate
the doublets from the graph.
References
1. Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, Mayall BH,
Wheeless L, Cox C. Guidelines for implementation of clinical DNA cytometry. International
Society for Analytical Cytology Cytometry. 1993;14(5):472–7.
2. Ormerod MG, Tribukait B, Giaretti W. Consensus report of the task force on standardisation
of DNA flow cytometry in clinical pathology. DNA flow cytometry task force of the European
Society for Analytical Cellular Pathology. Anal Cell Pathol. 1998;17(2):103–10.
3. Vindeløv LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of
nuclei for flow cytometric DNA analysis. Cytometry. 1983 Mar;3(5):323–7.
4. Darzynkiewicz Z, Huang X, Zhao H. Analysis of cellular DNA content by flow cytometry. Curr
Protoc Immunol. 2017 Nov 1;119:5.7.1–5.7.20.
Part II
Diagnostic Applications of Flow
Cytometry in Cytology
Classification of Lymphoma, Different
Markers and Approach 8
Abbreviations

BL Burkitt lymphoma
DLBCL Diffuse large B-cell lymphoma
FL Follicular lymphoma
LPL Lymphoplasmacytic lymphoma
MCL Mantle cell lymphoma
MZL Marginal zone lymphoma
SLL Small lymphocytic lymphoma
Fine needle aspiration cytology (FNAC) along with flow cytometry (FCM) is useful
both in the diagnosis and also sub classifying non-Hodgkin lymphoma (NHL).
World Health Organization Classification (WHO) classified NHL [1] based pre-
dominantly on:
1 . Individual cell morphology.

2. Immunophenotype.
3. Molecular cytogenetics.
4. Besides, the clinical features should also be considered for the successful clas-
sification of lymphoid neoplasms.
WHO approaches to classify the lymphomas based on the lineage of the cells: B
cell and T/NK cell. NHL was classified into two main types:
(a) B-NHL: Precursor and mature.

(b) T-NHL: Precursor and mature.
https://doi.org/10.1007/978-981-16-2655-5_8
86 8 Classification of Lymphoma, Different Markers and Approach
The lineage of the precursor neoplasms and certain mature neoplasms may not
be very rigid. Lymphoma is a clonal disorder and usually corresponds to the various
stages of differentiation.
Figures 8.1 and 8.2 highlight the brief outline of the WHO classification of non-
Hodgkin and Hodgkin lymphomas.
Fig. 8.1 Classification of Non-Hodgkin lymphoma according to WHO
Fig. 8.2 Classification of Hodgkin lymphoma according to WHO

8.1 Non-Hodgkin Lymphoma 87
8.1 Non-Hodgkin Lymphoma
The B-cell NHL greatly mimics the normal stages of B-cell differentiation.
So the classification and nomenclature of B-NHL are mainly based on the stages
of B-cell maturation. Figure 8.3 shows the B-cell differentiation and development
of lymphoma.
B cells experience a series of development in the bone marrow. The mature
naïve B cells circulate in the blood and subsequently shift to the primary follicles
of the lymph node. After the antigenic stimulation, the mature B cell undergoes
a series of changes such as germinal centre B cells, memory B cells, and plasma
cells. Each B cell is destined to develop only one type of specific immunoglobu-
lin by heavy chain gene rearrangement. This gene rearrangement occurs in the
pre-B cell.
The mature B-cell lymphomas are more frequently present among all the cases
of NHL. In general, low-grade B-cell NHL includes small lymphocytic lymphoma
(SLL), mantle cell lymphoma (MCL), low-grade follicular lymphoma (FL), lym-
phoplasmacytic lymphoma (LPL), marginal zone lymphoma (MZL) (Box 8.1).
Among all these low-grade lymphoma cases, MCL has the worst prognosis as its
behaviour is indolent. The “high grade” NHL included diffuse large B-cell lym-
phoma (DLBCL), Burkitt lymphoma (BL), grade III follicular lymphoma, plasma-
blastic lymphoma.
Fig. 8.3 Ontogenesis of B cell

Box 8.1: Grade of mature B-NHL

Low grade
• SLL
• MCL
• Low grade FL
• LPL
• MZL
High grade
• DLBCL
• BL
• Follicular lymphoma (Grade III)
• Plasmablastic lymphoma
T lymphocyte develops from the bone marrow from its precursor cells, and then
it migrates to the thymus. In the thymic cortex, the T cell undergoes maturation. The
immature T cells in the cortex of the thymus gland express TdT, CD1a, CD3, CD5
and CD7. These T cells are dual negative for CD4 and CD8. Later on, T cells show
either CD4 or CD8 antigen (Fig. 8.4).
Medullary thymocytes contain two types of T cells based on the surface recep-
tors: alpha-beta and gamma-delta T cells. Unlike T cells, the CD3 surface expres-
sion is absent in NK cells. NK cells show CD2, CD7 and CD8 expression and also
CD56 and CD16 markers.
T cell migrates from the thymus to blood, and a population of T cells finally
settle in the lymph node.
Fig. 8.4 Ontogenesis of T cell

8.2 Markers of Lymphoid Cell Lineage 89
In comparison to B-cell NHL, T-cell NHL is a more heterogeneous group of

lymphomas. T-lymphoblastic lymphoma develops from cortical and medullary thy-
mocytes. In contrast, mature T-lymphoma develops from the follicular T-helper
cells. T-NHL comprises only 15% of all NHL. The clinical presentation, immuno-
phenotype, histopathology, and prognosis of T-NHL varies widely. Certain T-NHL
types are mainly nodal such as peripheral T-cell lymphoma, angioimmunoblastic
T-cell lymphoma, anaplastic large cell lymphomas, and follicular T-cell lympho-
mas. The predominantly extranodal T-NHL includes mycosis fungoides, extranodal
natural killer T-cell lymphoma, hepatosplenic T-cell lymphoma, and enteropathy
associated T-cell lymphoma. Adult T-cell leukaemia / lymphoma may show both
leukemic and nodal presentation.
8.2 Markers of Lymphoid Cell Lineage
Both B-lymphoid and T-lymphoid cells are originated from the lymphoid-progenitor
cells (Fig. 8.3). The immature cells show CD34 and TdT expression. The early
B-lymphoid cells show relatively poor expression of CD45. The CD45 antigenic
expression increases from the immature to more mature cells.
B-cells markers: The expression of B-cell markers is highlighted in Fig. 8.3.
CD19, CD22 and cytoplasmic CD79a expression are noted in the earliest
B-lymphoid cells.
• The B cells exhibit bright CD10 expression in pre-B stage. Then subsequently,
CD10 becomes dim in immature B cells and absent in mature naïve B cells.
• The heavy chain μ is at first expressed within the cytoplasm of the pre-B cells.
Subsequently, surface IgM is expressed in the immature B cells, followed by the
mature B cells.
• The mature B lymphocytes express polyclonal light chain, and in contrast,
B-NHL expresses only a single type of light chain, either kappa or lambda chain.
• Upon antigenic stimulation, B lymphocytes in the germinal centre express CD10.
• Normal non-neoplastic plasma cells are the end stage of B-cell differentiation
and express CD38 and CD138. These cells are negative for CD20.
T-cell markers: The differentiation of T-lymphoid cells and the expression of

the T-cell markers have been highlighted in Fig. 8.4.
• The pro-thymocytes express the immaturity markers such as CD34, TdT and
HLA-DR. Besides, the cells also show intracytoplasmic CD3 and surface expres-
sion of CD2 and CD7.
• The pro-thymocyte reaches the thymus and matures further. T-cell receptor gene
rearrangement occurs in the cortical thymocytes.
• Dual positive CD4 and CD8 occurs in the medullary thymocytes.
8.2.1 NK Cell
• NK cells develop and mature in the bone marrow from the precursor cells.
However, the exact developmental stages of NK cells are still unknown.
• NK cells show surface expression of CD2, CD7, CD56 and CD16.
8.3 Hodgkin Lymphomas (HL)
HL is classified into nodular lymphocyte-predominant HL and classical HL. The

classical HL contains classical Reed-Sternberg (R-S) cells. Based on the relative
abundance of R-S cells, eosinophils, inflammatory cells, and fibrosis, the classical
HL is further subdivided into lymphocyte rich, mixed cellularity, lymphocyte deple-
tion type and nodular sclerosis variety.
Fine needle aspiration cytology (FNAC) in diagnosing and subclassification
of lymphomas.
FNAC plays an essential role in the initial diagnosis and further subclassification
of non-Hodgkin lymphomas (NHL) [2–6]. FNAC is a simple technique and can be
done in the routine outpatient department. The patient can tolerate this technique
better. The sample can be taken from multiple lymph nodes. It is advisable to have
a rapid on-site evaluation (ROSE) of the cytology smear for other ancillary tests.
The advantages of FNAC are highlighted in Box 8.2.
Box 8.2: Advantages of FNAC in Flow Cytometry of Lymph Node

• Easy technique
• Rapid procedure
• Cheaper than biopsy
• Well tolerated by the patient
• Multiple sampling can be done
• Better cytomorphology of the smear
8.4 Limitations
There are certain limitations of FNAC in the subclassification of NHL

(Box 8.3). There is a loss of architectural information in cytology smear. Grade 3
follicular lymphoma is prognostically worse [1], and it should be identified from
Grade 1 and grade 2. On FNAC smear, it is impossible to diagnose different grades
of follicular lymphomas. Moreover, the other newly described entities such as in
situ follicular lymphoma and in situ mantle cell lymphomas cannot be diagnosed by
FNAC. The other difficulty in FNAC is the archival preservation of the sample for
further molecular tests in the future.
8.5 Approach to Flow Cytometry of Lymph No 91
Box 8.3: Limitations of FNAC

• Loss of architecture.
• Inability to grade follicular lymphomas.
• Inability to diagnose in situ follicular and mantle cell lymphomas.
• Archival preservation may not be possible.
False-negative in Flow cytometry (Box 8.4): The causes of failure to diagnose

and subclassify lymphomas are mainly technical reasons. The predominantly aspi-
ration of the necrotic tissue, or FNAC of a scarred lymph nodes, and especially
blood mixed material may be a significant hindrance to get a reliable graph in flow
cytometry. Besides, scattered atypical cells, particularly in Hodgkin lymphoma,
may be missed in flow cytometry.
Box 8.4: False-Negative in Flow Cytometry

• Predominantly necrotic tissue.
• Scarred or fibrosed lymph node.
• Blood mixed diluted material.
• Scattered atypical cells: Hodgkin lymphoma.
8.5 Approach to Flow Cytometry of Lymph Node
The steps of flow cytometry in the lymph nodes are mentioned below (Fig. 8.5):
• At first, take a good clinical history and examine the swelling. It is advisable to
take a complete physical examination of the patient, including the abdominal
examination.
• Do FNAC of the lymph node and have a quick examination of the smear.
• Do multiple FNAC and take the sample for FCM in phosphate-buffered solu-
tion (PBS).
• Take one part of the sample for karyotyping.
• Take the other part of the sample for cell block to do immunocytochemistry and
fluorescent in situ hybridisation.
• Interpret the FCM graph along with FNAC smear.
Fig. 8.5 Flow chart showing the approach to do flow cytometry of lymph node
8.6 Cytology Smear and Panel of Antibody
Most of the NHL cases show a monomorphic population of cells on FNAC smears.
The polymorphic population is noted only a few types of lymphomas such as T-cell
rich B-cell lymphoma, follicular lymphoma, and T-cell lymphomas. The lympho-
mas with a monomorphic population can be divided into three groups based on the
size of the cell: small, medium and large-sized cells (Fig. 8.6). In all cases of lym-
phoma at first single cell, gating is done based on FSC-A versus FSC-H (Fig. 8.7).
With the help of forward scatter versus side scatter, the lymphoid cells are identified
(Fig. 8.8). The lymphoid cells are further gated based on CD45 positive population
(Fig. 8.9). Subsequently, for the identification of B cells, pan B-cell markers such as
CD19, CD20 and CD22 are used. For T cells, T-cell markers such as CD3, CD2,
CD5, CD7 are used. Besides, several additional markers are also used for further
sub classifying NHL cases.
8.6 Cytology Smear and Panel of Antibody 93
Fig. 8.6 Cytology smear and probable types of lymphoma
Fig. 8.7 Single-cell
250
(x 1,000)
population is gated by
using FSC-A versus Single cells
200
FSC-H are gated

150
FSC-H
100
50
50 100 150 200 250

FSC-A (x 1,000)
Fig. 8.8 Scatter diagram
250
(x 1,000)
of FSC-A versus SSC-A to
get the location of
200
lymphoid cells
150
SSC-A
100
50
Lymphoid
cells
50 100 150 200 250

FSC-A (x 1,000)
Fig. 8.9 Scatter diagram

250
(x 1,000)
of CD45 versus SSC-A,

showing the distinct
population of CD45
200
positive lymphoid cells for

analysis
150
SSC-A
100
CD45
positive
50
cells
102 103 104 105

CD45 APC-Cy7-A
Table 8.1 shows the fluorochrome combinations that are used in the Post Graduate
Institute of Medical Education and Research Chandigarh, India.
References 95
Table 8.1 Flow cytometry panel for lymphoma subtyping

Tube number FITC PE APC Per Cpcy5.5 PECy 7 APC H7
B1 CD5 CD 23 CD 43 CD 19 CD 45
B2 Kappa Lambda CD 10 CD 19 CD 45
B3 HLA-DR CD 20 CD 38 CD 34 CD 19 CD 45
B4 FMC 7 CD 4 CD 8 CD 3 CD 19 CD 45
B5 IgM IgD CD 19 CD 45
B6 CD 38 CD 200 CD 10 CD 34 CD 19 CD 45
T1 CD 8 CD 4 CD 7 CD 5 CD 3 CD 45
T2 CD 2 CD 1a CD 5 CD 3 CD 45
T3 TCαβ TCʏð CD 45
Additional tubes
A1 CD 13 CD 33 CD 117 CD 45
A2 CD 16 CD 56 CD 3 CD 45
A3 CD 64 CD 14 CD 11c CD 45
A4 cTdT Ccd3 C79a CD 45
A5 Ccd22 CD 45
References
1. Cazzola M. Introduction to a review series: the 2016 revision of the WHO classification of
tumors of hematopoietic and lymphoid tissues. Blood 2016; 127(20):2361–4.
2. Barroca H, Marques C. A basic approach to lymph node and flow cytometry fine-needle cytol-
ogy. Acta Cytol. 2016;60(4):284–301. https://doi.org/10.1159/000448679. Epub 2016 Sep 17
3. Cozzolino I, Rocco M, Villani G, Picardi M. Lymph node fine-needle cytology of non-Hodgkin
lymphoma: diagnosis and classification by flow cytometry. Acta Cytol. 2016;60(4):302–14.
https://doi.org/10.1159/000448389. Epub 2016 Aug 24
4. Ensani F, Mehravaran S, Irvanlou G, Aghaipoor M, Vaeli S, Hajati E, Khorgami Z, Nasiri
S. Fine-needle aspiration cytology and flow cytometric immunophenotyping in diagnosis and
classification of non-Hodgkin lymphoma in comparison to histopathology. Diagn Cytopathol.
2012;40(4):305–10. https://doi.org/10.1002/dc.21561. Epub 2010 Nov 12
5. Zeppa P, Marino G, Troncone G, Fulciniti F, De Renzo A, Picardi M, Benincasa G, Rotoli B,
Vetrani A, Palombini L. Fine-needle cytology and flow cytometry immunophenotyping and
subclassification of non-Hodgkin lymphoma: a critical review of 307 cases with technical sug-
gestions. Cancer. 2004;102(1):55–65. https://doi.org/10.1002/cncr.11903.
6. Dey P, Amir T, Al Jassar A, Al Shemmari S, Jogai S, Bhat M G, Al Quallaf A, Al Shammari
Z. Combined applications of fine needle aspiration cytology and flow cytometric immunphe-
notyping for diagnosis and classification of non Hodgkin lymphoma. Cytojournal. 2006; 3: 24.
doi: https://doi.org/10.1186/1742-6413-3-24.
Markers for Immunophenotyping
in Flow Cytometry 9
Abbreviations
AITL Angioimmunoblastic T cell lymphoma

AML Acute myeloid leukaemia
ATLL Adult T-cell leukaemia/ lymphoma
EATL Enteropathy associated T-cell lymphoma
LGL Large granular lymphocytic leukaemia
MF Mycosis fungoides
PLL Prolymphocytic leukaemia
PTCL Peripheral T cell lymphoma
SS Sezary syndrome
9.1 Introduction
The various markers are used in flow cytometry for immunophenotyping of lym-
phomas. These markers are often used judicially in combination with other markers
to identify the subpopulation of cells.
9.2 CD Markers
9.2.1 CD2
CD2 is a T-cell marker and is present in precursor T cell and mature T/NK cells.
https://doi.org/10.1007/978-981-16-2655-5_9
98 9 Markers for Immunophenotyping in Flow Cytometry
9.2.1.1 Diagnostic Applications

• CD2 is expressed in mature T and NK cell lymphomas.
• T-lymphoblastic lymphomas.
• Tumours of the mast cell.
9.2.2 CD3
CD3 is present in the cytoplasm of immature T cells and membranous expression in

mature T cells. It is also positive in NK cells. CD3 is a pan T cell marker and a spe-
cific marker of T cells.

• T-lymphoblastic lymphomas.
• All cases of mature T and NK cell lymphomas.
• Occasionally positive in primary effusion lymphoma, which is a B-lineage
lymphoma.
9.2.3 CD4
CD4 is expressed in the helper T cells, monocytes, histiocytes and dendritic cells. It
represents the major population of T cells. CD4 may not be a very reliable T cell
marker as it may not be expressed in T-NHL.

• In most cases of PTCL
• AITL
• ATLL
• ALCL
• Mycosis fungoides / Sezary syndrome
• T-PLL
• AML
• Chronic CMML
• Histiocytic tumours
9.2.4 CD8
CD8 is present in cytotoxic T cells and a subset of NK cell.

• PTCL
• T-PLL
• ALCL
9.2 CD Markers 99
• A subset of T- LGL
• Some cases of EATL
The expression of CD4 and CD8 in combination is highlighted in Box 9.1.
Box 9.1: Expression of CD4/CD8

CD4 positive lymphomas
• Sezary syndrome.
• T prolymphocytic leukaemia.
• Acute T-cell leukaemia/lymphoma.
• Anaplastic large cell lymphoma.
• Angioimmunoblastic T cell lymphoma.
CD8 positive lymphomas

• T-cell large granular lymphocytic leukaemia.
• Chronic proliferation of NK cells.
• Hepatosplenic T cell lymphoma.
Both CD4 and CD8 positive

• T-prolymphocytic leukaemia.
• Acute T-cell leukaemia/ lymphoma.
Both CD4 and CD8 negative T cell lymphoma

• Aggressive NK/T-cell neoplasm.
• Enteropathy-associated T-cell lymphoma.
9.2.5 CD5
CD5 is a T-cell antigen related to the signalling of T- cell receptor and antigen-
presenting cells. Mature and a subset of immature T cells is positive for CD5. In
addition, benign B cells in the peripheral blood and lymphocytes are often positive
for CD5. Loss of CD5 expression in the lymph node may be suggestive of T-cell
lymphoma [1].

• T-ALL/ T-lymphoblastic lymphoma.
• PTCL.
• Mantle cell lymphoma.
• Small lymphocytic lymphoma (SLL): SLL shows both CD5 and CD23
co-expression.
• Rare cases of DLBCL and mantle zone lymphomas.
9.2.6 CD7
CD7 is a pan T-cell marker related to cell proliferation, cell adhesion and signal
transmission. It is also positive in NK cell, foetal marrow B cell and some myeloid
precursor cell. Loss of CD7 may be seen in T-cell lymphomas.

• T-NHL (Mature).
• NK cell lymphomas.
• T-ALL.
• AML.
9.2.7 CD10
CD10 is metalloproteinase located on the cell membrane. It is expressed in imma-

ture B and T cells and B cells present in germinal centres and polymorphs.

• Follicular lymphoma.
• DLBCL subset.
• Burkitt lymphoma.
• ALL (B and T).
• Mixed phenotype leukaemia.
• Nodular T-cell lymphoma.
• Subset of HCL.
9.2.8 CD11B
CD11b is seen in macrophages, polymorphs, dendritic cells, natural killer cells and
activated CD8 T cells.

• T-LGL.
• Some cases of AML.
9.2.9 CD14
CD14 is mainly seen in monocytes.

• Chronic myelomonocytic leukaemia.
• Acute myelomonocytic leukaemia.
9.2 CD Markers 101
9.2.10 CD15
CD15 is expressed in neutrophils, promyelocytes and monocytes.

• Reed-Sternberg cells and Hodgkin cells of classical Hodgkin lymphoma.
• CML: Myeloid cells.
• AML: subset.
9.2.11 CD19
CD19 is a pan B cell marker and is seen in both mature and immature B cells. It
appears in the Pro-B cell and remains throughout the differentiation of B cells.
CD19 disappears in plasma cells. It is an excellent B-cell marker. The follicular
dendritic cells also express CD19.

• B-NHL (Mature and immature).
• Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL).
• AML: Certain types (AML-M0).
• Blast phase of CML.
9.2.12 CD20
CD20 is also a reliable B cell marker and appears in Pre-B cells. It remains through-
out the B cell differentiation and disappears in plasma cells.

• All B-cell lymphoma.
• B-ALL.
• A subset of classical Hodgkin lymphoma.
• NLPHL.
Notes
1. CD20 negative NHL: CD 20 may be poorly expressed or negative in certain

B cell lymphomas that include:
• DLBCL: Rarely.
• Primary effusion lymphoma.
• B -ALL.
• Classical Hodgkin lymphoma.
2. Combination: The combined use of CD20/CD19 or CD20/PAX5 is more reliable

B cell marker.
3. Rituximab: The monoclonal antibody, Rituximab, therapy against CD20 in the
treatment of B-NHL may cause loss of CD20 positive B cells, and the interpreta-
tion of CD20 positive cells may be difficult.
9.2.13 CD23
CD23 is a membrane glycoprotein and is present in mature B lymphocytes, acti-

vated histiocytes and follicular dendritic cells. It is an IgE receptor that controls IgE
response.

• SLL/CLL is positive for both CD5 and CD23, whereas MCL is negative for
CD23. So CD23 expression helps to differentiate SLL/CLL from MCL.
• Follicular dendritic cell tumour.
• A subset of follicular lymphoma.
• Mediastinal large B-cell lymphoma.
• Hairy cell leukaemia (HCL).
• Lymphoplasmacytic lymphoma.
9.2.14 CD25
CD25 is an interleukin-2 receptor.

• HCL.
• ALCL.
• ATLL.
• A subset of PTCL.
• A subset of AML.
9.2.15 CD30
CD30 is a membrane-bound phosphorylated glycoprotein. It is one of the type of

tumour necrosis factor receptors. CD30 is seen in activated normal T and B
lymphocytes.
Macrophages and granulocytes also show CD30 expression.

• ALCL.
• Hodgkin lymphoma: R-S cells are positive for CD30.
9.2 CD Markers 103
• A subset of DLBCL.
• Primary Mediastinal large B cell lymphoma.
• ATCL.
• NK cell neoplasms.
• Non-lymphomatous tumours: Embryonal carcinoma, nasopharyngeal carci-
noma, melanoma, angiosarcoma, mesothelioma, adenocarcinoma of the
pancreas.
9.2.16 CD38
CD38 is present in pluripotent stem cells, precursors of B and T lymphocytes,

myeloid cells and plasma cells.

• Plasma cell tumours.
• Plasmablastic lymphoma.
• Pre-T ALL.
• Certain subsets of CLL, DLBCL and FL.
Notes Higher CD38 expression in CLL (>30%) is related to advanced stage, poor
chemotherapy sensitivity, and shorter survival [2].
9.2.17 CD43
CD43 is expressed in T cells, NK cells, activated B cells and plasma cells.

• PTCL and NK cell lymphomas.
• B-ALL.
• Burkitt lymphoma.
• MCL.
• MZL.
• CLL.
• Multiple myeloma.
• Langerhans cell histiocytosis.
• Adenoid cystic carcinoma.
Notes
• Normal lymphocytes do not express CD43. So, CD43 positive B lymphocytes
should always be considered neoplastic.
• Among the non-hematopoietic tumours, CD43 is positive in adenoid cystic
carcinoma.
9.2.18 CD45
CD45 is also called as leukocyte common antigen (LCA). It is expressed in all

hematopoietic cells and their precursors. However, RBCs and megakaryocytes do
not show CD45 expression. CD45 is weakly expressed in immature cells and
strongly expressed in mature cells.

• Majority of lymphomas and leukaemias.
• Granulocytic sarcoma.
• Histiocytic sarcoma.
• Giant cell tumour of tendon sheath.
Haemato-lymphoid tumours that lack CD45 expression:

• Multiple myeloma.
• Hodgkin lymphoma (R-S cells).
• Erythroid leukaemia.
• B-ALL/lymphoma.
• Plasmablastic lymphoma.
• Some rare cases of ALCL.
9.2.19 CD56
CD56 is a neural cell adhesion molecule and is related to neural cell maturation. It
is mainly expressed in NK cells and activated T cells.

• NK cell lymphomas.
• Peripheral T cell lymphoma.
• ALCL.
• Plasma cell myeloma.
• Acute monoblastic leukaemia.
Notes
• CD56 is expressed in non-lymphoid tumours such as small cell carcinoma, pheo-
chromocytoma, synovial sarcoma.
9.2.20 CD79a
CD79a is an immunoglobulin that is expressed in B cells. It first appears in Pre-B

lymphocytes and persists till the development of plasma cells. A subset of CD3
positive T cells also expresses CD79a.
9.2 CD Markers 105

• B-cell leukaemia and lymphoma.
• AML-M3.
Notes: CD79a is not much reliable marker for B cell lymphomas. CD19 and
CD20 are more preferable markers for B-cell NHL than CD79a.
9.2.21 CD103
CD103 is expressed in hairy cell leukaemia. It is also positive in EATL.
9.2.22 CD117
CD117 is a c-kit gene product.

• AML.
• Erythroid leukaemia.
• Plasma cell tumours.
• Non-haematolymphoid tumours: Gastrointestinal stromal tumour (GIST) and
poorly differentiated carcinomas.
9.2.23 CD138
CD138 is expressed in precursor B cells, plasma cells, squamous epithelial cells and
hepatocytes.

• Plasma cell neoplasms.
• Plasmablastoma.
• Lymphoplasmacytic lymphoma.
• Primary effusion lymphomas.
• Squamous cell carcinomas.
• Malignant melanoma neuroendocrine tumours.
Notes
• Many non-haematolymphoid tumours such as squamous cell carcinomas and
adenocarcinomas express CD138. Therefore, at times to diagnose plasma cell
tumours, one should also consider the pattern of light chain expression (light
chain restriction).
9.3 Other Markers Used in Lymphoma
9.3.1 Terminal Deoxynucleotidyl Transferase (TdT)
TdT is an intracellular DNA nuclear polymerase enzyme. This enzyme is mainly

present in precursor B and T cells and cortical thymocytes.

• B- and T-lymphoblastic leukaemia and lymphoma.
• AML.
• Merkel cell carcinoma.
• Cortical thymoma.
9.3.2 HLA-DR
HLA-DR is seen in the B cell during its differentiation. It is absent in the plasma cells.

• Precursors and mature B-cell lymphomas.
• Most of the cases of AML.
• APL.
9.3.3 PAX5
PAX5 is a transcription factor and is responsible for tissue and organ differentiation.
It is expressed in the Pre-B cell to mature B cell.

• B-cell lymphoma.
• R-S cells of classical Hodgkin lymphoma.
Note:
• PAX5 may also be positive in Merkel cell carcinoma and small cell carcinoma of
the lung.
• Rarely PAX5 is positive in breast and endometrial carcinoma.
The markers of B and T/NK cells are highlighted in Box 9.2.

References 107
Box 9.2: Markers of B and T cells

B cells
• CD19
• CD20
• CD79a
• PAX5
• CD10
• CD23
T cells
• CD2
• CD3
• CD4
• CD5
• CD7
• CD8
• CD30
NK cells
• CD2
• CD3
• CD56
Plasma cells
• CD38
• CD138
• CD56
References
1. Kawano H, Minagawa K, Wakahashi K, Kawano Y, Sada A, Matsui T, Katayama Y. Diminished
expression of CD5 and/or CD7 surface antigens as the first clue of diagnosis for monoclonal T
lymphocytosis. Rinsho Ketsueki. 2012;53(8):785–7.
2. D'Arena G, Musto P, Cascavilla N, Dell'Olio M, Di Renzo N, Perla G, Savino L, Carotenuto
M. CD38 expression correlates with adverse biological features and predicts poor clinical out-
come in B-cell chronic lymphocytic leukemia. Leuk Lymphoma. 2001;42(1–2):109–14.
Detection of Lymphoma: Clonality
Demonstration by Flow Cytometry 10
10.1 Introduction
One of the main applications of flow cytometry in diagnostic cytology is the dif-
ferentiation of reactive lymphoid hyperplasia and lymphoma. Lymphoma is a clonal
disorder, and therefore, the demonstration of clonality is a critical feature of its
diagnosis.
10.2 Clonal Proliferation of B Cells
10.2.1 Light Chain Restriction
The B lymphoid cells contain immunoglobulin that is made of two heavy chains and
two light chains. The light chains are made of either κ or λ. The average ratio of κ
or λ chains in polyclonal B cells in the reactive lymphoid tissue varies from 1 to
2.7:1. In monoclonal B cell proliferation, the ratio of κ or λ chain is significantly
altered. The predominant expression of κ or λ chains in FCM is known as light chain
restriction (Fig. 10.1). In a practical situation, the κ or λ ratio is more than 4:1 or 1:2
may be taken as the firm evidence of monoclonality. In some instances of B lym-
phomas, there may not be demonstrable surface immunoglobulin in FCM. So, light
chain restriction cannot be demonstrated in these cases. The non-demonstrable light
chain restriction in FCM is seen in some instances of follicular lymphoma (FL) fol-
lowed by diffuse large B cell lymphomas (DLBCL).
10.2.2 Aberrant Expression of Certain Antigen
The following features suggest
https://doi.org/10.1007/978-981-16-2655-5_10
110 10 Detection of Lymphoma: Clonality Demonstration by Flow Cytometry
Fig. 10.1 Predominantly
Lambda chain expression
105
is present indicating light
chain restriction and
monoclonal B cell
104
population.
Lambda PE-A
103 −342 −102 0 102
−235 0 102 103 104 105

Kappa FITC-A
Fig. 10.2 CD38 positive

105
cell with no CD20

expression in plasma cell
tumour
104
CD200 PE-A
103
0
−915
−304 −102 0 102 103 104 105

CD38 FITC-A
1. The co-expression of certain antigens such as CD5 and CD10 may indicate a B
cell NHL.
2. Positive CD38 and CD138, high FSC along with no expression of CD19, CD20
and CD45 in plasma cell tumour (Fig. 10.2). The clonal plasma cells usually
show bright fluorescence of CD138 and mildly dimmer expression of CD38.
10.3 Immature B Cells
The immature B cells may show (Figs. 10.3, 10.4, and 10.5):

• Positive CD34 / TdT markers
• No demonstrable surface light chain
10.3 Immature B Cells 111
Fig. 10.3 CD34 positive

cells indicate immature
105
cells
104
HLA DR FITC-A
103
102
0
−313
−837 0 103 104 105

CD34 PerCP-Cy5-5-A
Fig. 10.4 TdT positive

5
cells indicate immature

10
cells
4
10
FITC-A
3
10
0
−250
−278 0 103 104 105

TDT APC-A
• No CD20 expression
• CD10 expression
• Dim CD45
10.3.1 Reactive Lymph Node
Reactive lymphoid cells show both κ and λ light chain expression (Fig. 10.6). The
ratio of κ and λ chain expression in FCM is not altered. However, rarely monoclonal
B cell population has been described in reactive lymphoid hyperplasia [1]. In all
these three cases, FNAC of the lymph node (in one case) and excisional biopsy of
Fig. 10.5 CD10
expression in B cell
105
lymphoma is seen in
immature cells of Burkitt
lymphoma
104
CD10 APC-A
103
−3,210 −103 0
−807 0 103 104 105

PerCP-Cy5-5-A
Fig. 10.6 Reactive
105
lymphoid hyperplasia:
Both kappa and lambda
chain expression in
reactive lymphoid
104
LAMBDA PE-A
hyperplasia and the ratio is

not altered
3
10
102
0−236
−205 0 102 103 104 105

KAPPA FITC-A
the lymph node showed light chain restriction in FCM. None of the cases showed
any evidence of lymphoma on biopsy or follow up.
10.3.2 B Cell Lymphoma with no Light Chain Expression
Certain B cell lymphomas such as mediastinal large B cell lymphomas, certain sub-
set of follicular lymphoma and DLBCL [2] (Fig. 10.7). In many such cases, a small
subset of clonal proliferation of B cells may be submerged in large polytypic B
cells. The forward scatter (FSC) of the light chain negative B cells were high, indi-
cating the large size of these cells. The lack of Surface Ig was defined by deMartini
10.3 Immature B Cells 113
Fig. 10.7 No expression

of light chain in a mature
105
B-NHL
104
Lambda PE-A
103 0
−474
−238 0 102 103 104 105

Kappa FITC-A
et al. as the less than 15% kappa and less than 10% lambda light chain in B-NHL
[3]. However Li et al. [2] re-defined it as the complete absence of kappa and lambda
light chain Ig in the B-NHL. In this strict criteria, only 2.25% of all peripheral
B-NHL demonstrates a lack of light chain expression.
10.3.3 Clonal Proliferation of T Cells
In the comparison of B cells, it is much difficult to identify T cell NHL. There are

only indirect evidence of T-cell clonality.
10.3.4 Aberrant Expression or Loss of T Cell Antigen
Loss or aberrant expression of various T cell antigens such as CD2, CD3, CD5 and
CD7 usually are abnormal and indicate T cell NHL. The antigenic expression may
be dim or partial or completely absent. CD7 antigen is the most commonly deranged
T cell antigen (40%) in mature T-NHL (Fig. 10.8). The other T-cell antigens such as
CD3, CD5 and CD2 are also affected variably. Most of the time (52%), at least one
antigen is affected by mature T-NHL. Occasionally two antigen (20%), three anti-
gens (7%) and rarely all four T-cell antigens are affected (<1%).
Aberrant expression of T-cell antigen may not always indicate a T-NHL. The
reduced or loss of T-cell antigen expression or increased expression may be noted in
reactive lymphoid hyperplasia or Hodgkin’s lymphoma. It is therefore always rec-
ommended to correlate the FCM findings with cytomorphology of the smear.
Fig. 10.8 High expression

of CD7 in a T-NHL
105
104
PE-A
103
0
−734
−213 0 102 103 104 105

CD7 FITC-A
Fig. 10.9 Both CD4 and

105
CD8 coexpressed in
T-NHL
104
CD4 PE-A
103 0
−1,081
−213 −102 0 102 103 104 105

CD8 FITC-A
10.3.5 Abnormality of CD4 and CD8 Expression
Dual positive: The dual positive CD4 and CD8 should raise the suspicion of
T-NHL. The dual positive CD4 and CD8 population are uncommon in peripheral
T-NHL (Fig. 10.9). However, approximately one third (32%) of T-ALL or T lym-
phoblastic lymphoma cases show dual positive CD4 and CD8 cells.
Dual negative: Near about half of the cases (52%) of T-ALL or T lymphoblastic
lymphomas show dual negative CD4 and CD8 cells. The dual negative phenotype is
uncommon in mature T-NHL.
Deranged ratio: The CD4:CD8 ratio in the reactive lymph node aspirate varies
from 0.2 to 14 with an average of 4. Gross abnormality of CD4:CD8 ratio also
References 115
increases the possibility of T-neoplasms. There may be either marked increase of

CD4 or CD8 population of cells in such cases.
10.3.6 Increased Forward Scatter
The normal T cells have low FSC. However, the neoplastic T cells usually show
high FSC.
10.3.7 The Expression of Other Markers
The expression of the additional markers may indicate a T-NHL.

CD30: CD30 is usually expressed in ALCL and also peripheral T cell lymphomas.
CD10: CD10 expression is commonly seen in AITL (60%) and occasionally
in PTCL.
CD103: CD103 is a marker of HCL.
10.3.8 Presence of Markers of Blasts
The certain markers are expressed in blasts such as CD34, TdT and CD1a. T cells
showing these markers suggest the possibility of immature (blast) T cells.
Loss or aberrant expression of CD45
The completely negative or dim positive CD45 markers are highly suggestive of
T-NHL. The abnormality of CD45 expression is noted mainly in T-PLL, PTCL and
ALCL (4%). However, the majority of T-NHL express strong CD45 expression.
References
1. Kussick SJ, Kalnoski M, Braziel RM, Wood BL. Prominent clonal B-cell populations identi-
fied by flow cytometry in histologically reactive lymphoid proliferations. Am J Clin Pathol.
2004 Apr;121(4):464–72.
2. Li S, Eshleman JR, Borowitz MJ. Lack of surface immunoglobulin light chain expression by
flow cytometric immunophenotyping can help diagnose peripheral B-cell lymphoma. Am J
Clin Pathol. 2002 Aug;118(2):229–34.
3. De Martini RM, Turner RR, Boone DC, et al. Lymphocyte immunophenotyping of B-cell lym-
phomas: a flow cytometric analysis of neoplastic and nonneoplastic cells in 271 cases. Clin
Immunol Immunopathol. 1988;49:365–79.
Flow Cytometry of B-Non Hodgkin
Lymphoma 11
11.1 Introduction
The application of Flow cytometry (FCM) to subclassify the non-Hodgkin lym-

phoma (NHL) is well recognized [1, 2]. Based on cell size, the cytology smear of B
cell NHL may be categorized into two groups:
1. Small and medium-sized cells: This group includes

• Small lymphocytic lymphoma (SLL)/chronic lymphocytic leukaemia (CLL)
• Mantle cell lymphoma (MCL)
• Marginal zone lymphoma (MZL)
• Follicular lymphoma grade I and II (FL) and
• Lymphoplasmacytic lymphoma (LPL).
2. Large-sized cells: This group includes
• Diffuse large B cell lymphoma (DLBCL)
• Burkitt lymphoma (BL) and
• Follicular grade III lymphoma
11.2 Diagnosis of Individual NHL
11.2.1 Small Lymphocytic Lymphoma (SLL)
SLL, the neoplasm of mature B cells, is the commonest NHL in adult population.
The patients usually are asymptomatic and detected by routine blood examination.
Occasionally the patients may have lymphadenopathy and splenomegaly.
Cytology (Fig. 11.1): The cytology smear shows:
• Dissociated lymphoid cells

• Small cell with no nuclear pleomorphism
https://doi.org/10.1007/978-981-16-2655-5_11
118 11 Flow Cytometry of B-Non Hodgkin Lymphoma
a b
c d
Fig. 11.1 Cytological features of small lymphocytic lymphoma. (a) Discrete small round cells,
(b) The lymphoid cells are small and round with mild nuclear pleomorphism, (c) The cells show
homogenous nuclear chromatin with inconspicuous nucleoli, (d) Cellblock section showing abun-
dant small lymphoid cells
• Regular nuclear margin

• Clumped chromatin
• Absent nucleoli
Flow cytometry (Figs. 11.2, 11.3, and 11.4): SLL cases show the following
FCM immunophenotyping:
• Light chain restriction: Altered light chain ratio and either kappa or lambda chain
is present.
• SLL/CLL in the lymph node aspirates shows different B-cell markers that include
CD19, CD20, CD79a.
• The tumour cells show characteristic co-expression of CD5 and CD23.
• Often CD43 is positive.
• Absence of FMC7 and CD10 expression.
• A subset of SLL may show CD38 expression that carries a bad prognosis.
• Positive for CD200.
11.2 Diagnosis of Individual NHL 119
a 250 b
(x 1,000)
105
200
Lambda PE-A
104
150
SSC-A
103
100
50
0
−342
−480 0 103 104 105 −1,899 103 104 105
0
CD19 PE-Cy7-A CD19 PE-Cy7-A
c d
105
105
Kappa FITC-A
104
Lambda PE-A
104
103
103
−342 1020 102
−235 0
−1,899 0 103 104 105

−235 0 103 103 104 105
CD19 PE-Cy7-A
Kappa FITC-A
Fig. 11.2 Flow cytometry of small lymphocytic lymphoma: (a) Predominantly CD19 cell popula-
tion, (b) CD19 positive lymphoid cells are also positive for lambda light chains, (c) CD19 positive
lymphoid cells are negative for Kappa, (d) The lymphoid cells showing only lambda positivity
indicating light chain restriction
Scoring to diagnose CLL/SLL: A scoring system was proposed to distinguish

SLL from other lymphoproliferative diseases. The expression of 5 markers was con-
sidered for this proposal (Fig. 11.4b). A cut off score of 4, or higher is highly sug-
gestive of SLL/CLL [3].
Genetic markers:
• Trisomy 12
• The tumour often shows deletion of 13q14 (50% cases)
• Deletion of 11q22-23
• Deletion of 6q21
Differential diagnosis
• MCL: MCL shows CD5+ and CD23- phenotype. Besides, MCL cases are posi-
tive for FMC7. The cells show a bright expression of CD45 and surface light
a 105 b
105
104
104
CD5 FITC-A
CD23 PE-A
103
103
102
0
−63 0
−447
−1,480 0 103 104 105 −1,480 0 103 104 105
c d
105
105
CD19 PE-Cy7-A
104
104
CD23 PE-A
103
103
0
0
−1,899
−447
−63 0 102 103 104 105 −111 −103 0 103 104 105
CD5 FITC-A CD10 APC-A
Fig. 11.3 Flow cytometry of small lymphocytic lymphoma: (a) The CD19 positive lymphoid
cells show CD5 positivity, (b) The cells also show CD23 positivity. (c) Dual expression of CD5
and CD23, (d) The lymphoid cells are negative for CD10
chain (Table 11.1). Certain cases of MCL may show CD23 expression, and SLL
cases may be negative for CD23. These cases are difficult to diagnose on
FCM. The demonstration of characteristic translocation of t (11;14) in MCL may
be needed in such cases.
• Follicular lymphoma (FL): FL shows CD10 expression and bright CD20 positiv-
ity (Table 11.2).
• Lymphoplasmacytic lymphoma (LPL): CD5- and CD23- cells.
• Marginal zone lymphoma (MZL): CD5- and CD23- cells.
• Adult T-cell leukaemia/lymphoma (ATLL): No light chain expression and pre-
dominantly CD3 positive cell population.
• Hairy cell leukaemia (HCL): Positive for 11c and CD103. The cells show a dual
expression of CD20 and CD103.
a
105
104
CD5 FITC-A
103
102
−63 0
0 101 102 103 104 105

CD43 APC-A
b
Score
CD5 Positive 1
CD23 Positive 1
FMC7 Negative 1
Surface lg Dim 1
CD79b Negative 1
Total score 4 or high indicates strong suggestion of SLL/CLL
Fig. 11.4 (a) Flow cytometry of small lymphocytic lymphoma: The cells show both CD5 and
CD43 expression. (b) Marker expression and scoring of CLL/SLL
Table 11.1 Differentiating Markers expression SLL MCL

features of small lymphocytic
CD5 + +
lymphoma (SLL) and mantle
cell lymphoma (MCL) CD23 + -
FMC7 - +
CD45 Dim Bright
Surface light chain Dim Bright
Positive = +, Negative = −.
Table 11.2 Differentiating Markers expression SLL FL

features of small lymphocytic CD5 + Usually negative
lymphoma (SLL) and
CD23 + -
Follicular lymphoma (FL)
CD10 - +
CD45 Dim Bright
Surface light chain Dim Moderate
Positive = +, Negative = −.
11.2.2 Mantle Cell Lymphoma (MCL)
MCL consists of 10% of NHL. The median age of the patient is approximately
60 year. The typical presentation of MCL cases is generalized lymphadenopathy,
weakness and splenomegaly. MCL has an indolent course, and the patients are usu-
ally not cured.
Cytology smear (Fig. 11.5):

• The cells are small.
• Monomorphic nuclei.
• Irregular nuclear outline.
• Fine chromatin.
• No nucleoli.
Flow cytometry (Fig. 11.6 and 11.7):

• Moderately intense light chain expression. Light chain restriction is present.
• B-cell indicators: CD19, CD20, PAX5, CD79a positive.
• The cells are characteristically CD5+ and CD23-. Occasionally, the cells may
not show CD5 expression.
• The cells show CD43, BCL2 and strong surface expression of IgD and IgM.
• Negative to dim CD71.
• CD38 and occasionally CD11c expression are present.
• Negative for CD10.
Genetic markers:
• The tumour cells show characteristic translocation of t (11;14), resulting in the
fusion of CYCLIN D1 and immunoglobulin heavy chain (IgH).
a b
c d
Fig. 11.5 Cytology of Mantle cell lymphoma. (a) Abundant small lymphoid cells, (b) The cells
are small, round and monomorphic, (c) Nuclei have regular margin, (d) Homogenous chromatin
with absent of nucleoli
Differential diagnosis:
• SLL: Discussed before.
• FL: The cells are typically CD5-, and CD23- phenotype and positive for CD10.
• Peripheral T-cell lymphoma: Positive for CD2, CD3, CD7.
• DLBCL: The cells are relatively large, and negative for CD 23 and CD5.
11.2.3 Follicular Lymphoma (FL)
FL represents nearly 20% of all non-Hodgkin lymphomas. The median age of the
patients is 58 years. They commonly have enlarged lymph node and
hepatosplenomegaly.
Cytological features (Fig. 11.8):

• Smears show an admixture of small and large cells.
• Small cells: Cleaved nucleus, inconspicuous nucleoli, coarser chromatin.
• Large cells: Nuclei cleaved and non-cleaved, opened chromatin and conspicuous
nucleoli.
a 250 b
(x 1,000)
105
200
104
Lambda PE-A
SSC-A
150
103
100
50
0
−575
−8,902 −103 0 103 104 105
−8,084 −103 0 103 104 105
CD19 PE-Cy7-A
CD19 PE-Cy7-A
c d
105
105
104
104
Kappa FITC-A
Lambda PE-A
103
103
0 102
0
−238
−575
−8,084 −103 0 103 104 105 −238 0 102 103 104 105
CD19 PE-Cy7-A Kappa FITC-A
Fig. 11.6 Flow cytometry of Mantle cell lymphoma.) (a) Predominantly CD19 cell population
indication a B-cell lymphoma, (b) B lymphoid cells are also negative for lambda light chains, (c)
CD19 positive lymphoid cells are also positive for Kappa, (d) The light chain restriction
Flow cytometry (Fig. 11.9)

• Light chain restriction is present. However, occasionally there may be no demon-
strable surface light chain.
• B-cell markers: Positive: CD19, CD20, PAX5 and CD79a.
• Positive for CD10, BCL2 and BCL6.
• Negative: CD 5, CD 43, CD23.
• A subset of FL may show absence of CD10 or BCL2 expression.
• Rarely FL may show expression of CD30, CD43 and CD5.
Genetic markers: The translocation of t(14:18) (q32:q21) is the most frequent

(90%). This translocation involves the juxtaposition of the BCL2 gene with IgH and
leads to overexpression of BCL2. Near about 5 to 10% of FL may not show t(14:18),
and they may show other chromosomal abnormalities involving BCL6 or breaks in
chromosome 1,2,4,5 and 17.
a 105 b
105
104
104
CD5 FITC-A
CD23 PE-A
103
103
−65 0 102
0
−657
−8,902 −103 0 103 104 105 −8,902 −103 0 103 104 105
c
105
104
CD23 PE-A
103
0
−657
−65 0 102 103 104 105

CD5 FITC-A
Fig. 11.7 Flow cytometry of Mantle cell lymphoma. (a) CD19 positive B cell are also positive for
CD5, (b) The lymphoid cells do not express CD23, (c) Only CD5 expression
Transformation of follicular lymphoma: FL may transform in FL grade III,

DLBCL, BL and plasmablastic lymphoma and B-acute lymphoblastic leukaemia
(B-ALL). The transformed cells of FL have a high Ki67 index. The transformed
lymphoblastic lymphoma shows all the markers as mentioned above, along
with TdT.
Differential diagnosis:
• MCL: CD5 +/ CD23 - /CD10- immunophenotype.
• Reactive lymphoid hyperplasia: No light chain restriction, CD10 positive and
BCL2 negative cells.
• SLL/CLL: CD5+ and CD23+ phenotype with negative CD10.
• HCL: Co-expression of CD20 and CD103.
a b
c d
Fig. 11.8 Cytological features of Follicular lymphoma. (a) The small and large cells, (b) The
large cells are moderately pleomorphic and admixed with small lymphoid cells, (c) Haematoxylin
and Eosin stained smear showing small and relatively larger cells, (d) The higher magnification
showing the cells with fine chromatin and occasional prominent nucleoli
11.2.4 Marginal Zone Lymphoma (MZL)
MZL represents 8% of all B-NHL. The median age of MZL patient is the seventh
decade. The patients are usually asymptomatic. Near about 30 to 40% of patients
may show extranodal involvement.
Types of MZL: Splenic MZL, extranodal MZL and nodal MZL.
Cytological features (Figs. 11.10, 11.11, 11.12, 11.13, and 11.14):

• A mixed population of cells.
• Small cell with an irregular nuclear margin.
• Coarse chromatin and absence of nucleoli.
• Many plasma cells/plasmacytoid cells.
• Monocytoid B cells: Pale cytoplasm, cleaved nuclei, absence of nucleoli.
Flow cytometry:
• Light chain restriction present: Bright expression of kappa or lambda chain.
• B-cell markers: The cells express CD19, CD20, CD22, PAX5 and CD79a.
• Negative: CD5, CD10 and CD23.
• Dim expression of CD11c and CD43.
a 250 b
(x 1,000)
105
200
Kappa FITC-A
104
150
SSC-A
103
100
50
102
−8,098 0 103 104 105 −8,204 0 103 104 105
c d
250
(x 1,000)
105
200
Lambda PE-A
104
150
SSC-A
103
100
50
102
−8,204 0 103 104 105 −8,639 0 103 104 105

CD19 PE-Cy7-A CD10 APC-A
Fig. 11.9 Flow cytometry of Follicular lymphoma. (a) Majority of the cells are positive for CD19,
(b) CD19 positive lymphoid cells express kappa. (c) The cells are negative for lambda, (d) The
cells are positive for CD10
Genetic markers: The tumour cells show trisomy 3 (60% cases), translocation
of t (11; 18) (50% cases), and deletion of 7q, 6q and 8p.
11.2.5 Lymphoplasmacytic Lymphoma (LPL)
LPL is a tumour of mature B cell, plasma cell and plasmacytoid cell. It represents
1.5% of all lymphomas. The median age of the patient is seventh decade. The patient
usually presents with weakness, malaise and lymphadenopathy.
Cytological features (Fig. 11.15)

• Lymphocytes: The small cells having monomorphic nuclei and homogenous
chromatin.
a b
c d
Fig. 11.10 Extra nodal lymphoma in parotid gland

(a) Discrete lymphoid cells along with lymphoid cells. (b) The lymphoid cells show mixed popula-
tion of large and small cells. (c) Haematoxylin and Eosin stained smear showing mixed small and
large cells. (d) The higher magnification of the same showing large cells with moderate nuclear
pleomorphism
a b c
200 250
(x 1,000)
105
105
PerCP-Cy5-5-A
Lambda PE-A
104
104
150
SSC-A
103
103
100
50
0
−696
−620
−3,358 −103 0 103 104 105 −610 0 103 104 105 −3,023 −103 0 103 104 105
CD19 PE-Cy7-A Kappa FITC-A CD10 APC-A
d e f
105
105
105
CD34 PerCP-Cy5-5-A
PerCP-Cy5-5-A
104
104
104
CD20 PE-A
103
103
103
0
0
−659
−626
−626
−1,262 0 103 104 105 −1,919 0 103 104 105 −3,099−103 0 103 104 105
CD34 PerCP-Cy5-5-A CD43 APC-A CD38 APC-A
Fig. 11.11 Extra nodal lymphoma in parotid gland

(a) Predominant CD19 population. (b) Only kappa chain expression indicating light chain restric-
tion. (c) Absence of CD10 expression. (d) Predominant CD20 expression. (e) Cells show CD43
expression. (f) Cells show CD38 expression
a b
c d
Fig. 11.12 (a) Thyroid follicular cells along with discrete lymphoid cells. (b) The thyroid folli-
cles are infiltrated with lymphoid cells. (c) The thyroid follicles are infiltrated with lymphoid cells.
(d) Higher magnification showing cells with monomorphic nuclei having condensed chromatin
• Plasma cells.
• Plasmacytoid cells.
• Occasionally immunoblasts.
• Epithelioid cells.
• Mast cells.
Flow cytometry (Fig. 11.16):

• B-cell markers: Positive for CD19, CD20, CD22, and CD79a.
• Negative: CD5 and CD10.
• The subset of cells may show expression of CD23, CD11c, CD43 and CD38. The
CD38 expression is usually dimmer. The plasma cells show a bright expression
of CD38 and CD138.
• The lymphoid cells may show light chain restriction.
Genetic markers: There is no specific cytogenetic hallmark in LPL. Chromosomal

translocation of t (9:14) may be present in 50% of PLP cases.
Table 11.3 shows the antigenic expression of different small- and medium-sized
lymphomas.
a 250 b
(x 1,000)
105
200
104
Lambda PE-A
150
SSC-A
103
100
0
50
−539
−166 0 102 103 104 105
−50 0 103 104 105 Kappa FITC-A
CD19 PE-Cy7-A
c d
105
105
PerCP-Cy5-5-A
104
104
CD23 PE-A
103
103
0
0
−456
−477
−7,886 −103 0 103 104 105

−185 0 102 103 104 105
CD10 APC-A
CD5 FITC-A
Fig. 11.13 Flow cytometry of lymphoma of thyroid. (a) Predominant CD19 positive cells. (b)
The cells express only Lambda chain indicating light chain restriction, (c) The cells are positive for
both CD5 and CD23, (d) Negative for CD10
11.3 Lymphomas of Large-Sized Cells
11.3.1 Diffuse Large B-Cell Lymphoma (DLBCL)
DLBCL represents 35% of all NHL. The median age of DLBCL patient is in sev-
enth decade. The patient may have nodal or extranodal involvement of the tumour.
Molecular profile highlights that there are two distinct subtypes of DLBCL [4]:
1. Germinal centre B-cell-like (GCB): Possible origin is from the germinal cen-
tre cells.
2. Activated B-cell-like: The lymphoma originates from the post-germinal cen-
tre cells.
11.3 Lymphomas of Large-Sized Cells 131
a 105 b
105
CD34 PerCP-Cy5-5-A
104
104
CD43 APC-A
103
103
0
0
−640
−,198
−1,689 0 103 104 105

−100 −102 0 102 103 104 105 CD38 APC-A
PerCP-Cy5-5-A
c d
105
105
HLA DR FITC-A
104
104
CD4 PE-A
103
103
0
0
−552
−320
−640 0 103 104 105 −1,968 0 103 104 105

CD34 PerCP-Cy5-5-A CD8 APC-A
Fig. 11.14 Flow cytometry of lymphoma of thyroid (a) CD43 positive cell population. (b) The
cells are positive for CD38, (c) The lymphoid cells are positive for HLA DR. (d) Both CD4 and
CD8 expression
a b
Fig. 11.15 Cytological features of lymphoplasmacytic lymphoma. (a) Discrete plasma cells and
small lymphoid cells. (b) The cells with enlarged nuclei and condensed chromatin. Occasional
cells show cartwheel chromatin
a 250 b
(x 1,000)
105
200
Lambda PE-A
104
SSC-A
150
0 103
100
50
−4,929
−6,474 −103 0 103 104 105 −2,846 0 103 104 105
c d
105
105
CD38 APC-A
CD23 PE-A
104
104
0 103
0
−13,280
−3,037
−2,919 0 103 104 105 −3,162 0 103 104 105

CD34 PerCP-Cy5-5-A CD5 FITC-A
Fig. 11.16 Flow cytometry of lymphoplasmacytic lymphoma. A. Predominantly CD19 positive

cells. (b) Light chain restriction indicated by only kappa chain expression. (c) Cells show CD38
expression, (d) The lymphoid cells show CD23 expression and absence of CD5 positivity
Table 11.3 FCM markers of small and medium-sized lymphoma

Antigen SLL/CLL MCL FL MZL LPL
CD19 + + + + +
CD20 Dim + + + + +
CD5 + + - - -
CD23 + - - - -
SIg Dim + + + + ±
FMC7 - + + + +
CD38 + + (occasional) - +(occasional) +
Positive = +, Negative = -
a b
Fig. 11.17 Cytological features of diffuse large B cell lymphoma. (a) Abundant discrete large
cells, (b) The cell with enlarged nuclei having fine nuclear chromatin
Cytological features (Fig. 11.17): The cytomorphology varies depending on the

cytological type. However, overall, DLBCL shows
• Large cells.
• Scanty cytoplasm.
• Large nucleus.
• Fine chromatin.
• Multiple nucleoli.

• B-cell markers: Positive for CD19, CD20, CD22 and CD79a.
• Light chain restriction may not be demonstrable.
• Occasional DLBCL may also express CD30 (15% case).
• A subset of DLBCL may be positive for CD10 and needs to be distinguished
from BL.
Genetic markers: The GCB subtype shows mutation of EZH2 and GNA13.
ABC subtype show mutation of CARD11, CD79b and MYD88.
Distinguishing GCB from ABC of DLBCL cases:
CD10, BCL6 and MUM1 immunocytochemistry may help to distinguish GCB
from ABC type of DLBCL (Fig. 11.19).
11.3.2 Burkitt Lymphoma (BL)
BL represents 3% of all NHL cases. It is a major bulk of childhood lymphoma and

consists of approximately 40% of childhood lymphomas. BL in the endemic areas
may show involvement of jaw bone and orbit. In sporadic cases, it occurs mainly in
intra-abdominal region.
a 250 b
(x 1,000)
105
200
104
CD20 PE-A
150
SSC-A
103
100
50
−405 0
−503 0 103 104 105
−999 0 103 104 105
CD34 PerCP-Cy5-5-A
CD19 PE-Cy7-A
c d
105
105
PerCP-Cy5-5-A
104
104
Lambda PE-A
103
103
−296 0
−495 0
−255 0 102 103 104 105 −533 0 103 104 105

Kappa FITC-A CD10 APC-A
Fig. 11.18 Flow cytometry of diffuse large B cell lymphoma. (a) Predominant CD19 positive cell
population, (b) The cells are also positive for CD20 and negative for CD34. (c) Light chain restric-
tion indicated by only lambda chain expression. (d) The cells are positive for CD10
GCB
GCB Negative
Positive
Positive MUM1
CD10
DLBCL
Positive
Negative BCL6 Non-GCB
Negative
Non-GCB
Fig. 11.19 Immunocytochemistry to distinguish germinal centre versus non-germinal centre type
of diffuse large B cell lymphoma
a b
Fig. 11.20 Cytology features of Burkitt lymphoma. (a) Discrete lymphoid cells in a vacuolated
foamy background. (b) The individual cells have moderate amount of vacuolated cytoplasm and
enlarged pleomorphic nuclei having prominent nucleoli
Cytology smear (Fig. 11.20):

• Cellular smear with starry sky type of look in lower magnification.
• Intermediate-sized cells.
• Bluish cytoplasm.
• Enlarged nucleus.
• Moderately pleomorphic nucleus.
• Coarse nuclear chromatin having multiple prominent nucleoli.
• Increase mitotic activity and apoptotic cells.
• Many scattered tangible body macrophages.
• Background lymphoglandular bodies.

• B-cell markers: Positive for CD19, CD20, CD22 and CD79a.
• The cells also show CD10 positivity indicating the germinal centre origin of
the cells.
• The tumour is negative for TdT and CD34, the markers of blast cells.
• In addition, BL may be positive for CD38 and CD43.
• The cell proliferation marker Ki67 is nearly 100%.
Genetic markers: The translocation of t(8:14) (q24;q32) is the hallmark of

BL. It causes juxtaposition of MYC gene against the immunoglobulin heavy chain
gene. Less commonly, there may be translocation of t(2:8) (p11:q24), or t(8:22)
(q24: q11.2), resulting in a rearrangement of MYC with light chain κ orλ.
Differential diagnosis
• DLBCL: No starry sky pattern in DLBCL, absence of co-expression of CD10

and CD43, relatively low ki67 index and low S-phase fraction.
• B-ALL: B-ALL is positive for TdT and CD34. The tumour cells show dim posi-
tive CD45.
a 250 b c
105
(x 1,000)
105
200
104
Lambda PE-A
104
CD20 PE-A
150
SSC-A
103
103
100
50
0
0
−566
−999
−530 0 103 104 105 −610 0 103 104 105 −89 0 103 104 105
CD19 PE-Cy7-A Kappa FITC-A HLA DR FITC-A
d e
f
105
105
105
CD10 APC-A
104
104
CD38 APC-A
104
CD23 PE-A
103
103
−210 −1030 103
0
−871
−506
−807 0 103 104 105 −1000 0 103 104 105 −69 0 103 104 105
PerCP-Cy5-5-A CD34 PerCP-Cy5-5-A CD43 APC-A
Fig. 11.21 Cytology features of Burkitt lymphoma. Other B-cell lymphoma. (a) Predominantly
CD19 positive cells. (b) Light chain restriction. (c) The cells express both CD20 and HLA DR. (d)
The cells are positive for CD10. (e) The cells also express CD38. (f) CD43 positive cell population
11.3.3 Hairy Cell Leukaemia (HCL)
FNAC of HCL is uncommon as a lymph node is rarely involved in this lymphoma.
Cytology smears:
• Abundant discrete monomorphic cells.
• Many plasmacytoid cells with eccentric nuclei having condensed chromatin.
• Large mononuclear atypical cells:
–– Oval to kidney shaped nuclei.
–– Homogenous chromatin.
–– Conspicuous nucleoli.
–– Moderate to abundant cytoplasm with hair like processes.
• Many atypical mitotic figures.
Flow cytometry:
• Pan B cell: Positive for CD19 and CD20.
• Positive for CD123, CD11c.
• Co-expression of CD25 and CD103.
• Occasional HCL may have aberrant CD10 and CD2 expression.
11.4 Immature B Cell 137
11.4 Immature B Cell
11.4.1 B-Lymphoblastic Lymphoma
B-lymphoblastic lymphoma is common in children. The patient presents with

lymphadenopathy and hepatosplenomegaly.

• The monomorphic population of cells.
• Monomorphic nucleus.
• Medium cell size.
• Scanty bluish cytoplasm.
• Fine chromatin.
• Small to absent nucleoli.

• B-cell markers: Positive for CD19 and CD79a. CD20 is dim positive.
• B lymphoblasts often express CD38 and HLA DR.
• The blasts are positive for immaturity marker TdT and CD34.
The different types of ALL show the following pattern of immunophenotyping:

Pro B-ALL: Positive for CD19 and TdT; Negative CD10.
Common ALL: Positive for CD19, CD34, TdT, CD10.
Pre B-ALL: CD10±, TdT±, CD34±.
Mature B-ALL: Positive for CD10, Mostly negative: TdT, CD34.
a b
Fig. 11.22 Cytology of B-lymphoblastic lymphoma. (a) Abundant dissociated medium to large
lymphoid cells. (b) Cells with scanty cytoplasm having finely dispersed chromatin and inconspicu-
ous nucleoli
a 250 b c
(x 1,000)
105
105
200
PerCP-Cy5-5-A
104
HLA DR FITC-A
104
150
SSC-A
103
103
100
0102
50
−13
−57
−692 −103 0 103 104 105 −236 0 103 104 105 −19 0 103 104 105
CD19 PE-Cy7-A CD10 APC-A CD38 APC-A
d e f
105
105
105
CD5 PerCP-Cy5-5-A
104
104
104
CD5 FITC-A
CD4 PE-A
103
103
103
0102
0
−86
−13
−25
0 103 104 105 −47 0 103 104 105 −4 0 103 104 105
CD43 APC-A CD8 FITC-A CD7 APC-A
Fig. 11.23 Flow cytometry of B-lymphoblastic lymphoma. (a) Predominantly CD19 positive
cells. (b) The cells are positive for CD10. (c) CD38 positive cells, (d) The cells also express CD43,
(e, f) The cells are negative for CD4, CD8, CD5 and CD7
11.5 Plasma Cell Neoplasm
Plasma cell neoplasm is a clonal proliferation of plasma cells. The tumour com-
monly occurs in an elderly patient. Occasionally the may be a collection of neoplas-
tic plasma cells forming as nodule without any evidence of systemic plasma cell
neoplasm. These nodular lesions are known as plasmacytoma.

• Abundant discrete plasma cells.
• Many bi and multinucleated cells.
• Occasional loose aggregates of the plasma cells.
• Plasma blasts may be present.
Flow cytometry (Fig. 11.25)

• The tumour cells show CD38 and CD138 expression.
• The cells show light chain restriction.
• The myeloma cells lose the expression of CD45, CD19, CD27 and gain of
CD117, CD56, CD28 and CD33.
• The neoplastic plasma cells show the characteristical absence of CD19, dim
positive CD45 along with CD38 and CD138 expression. Whereas, the normal
plasma cells show CD45+, CD19+, CD38+, CD138+, CD56+ phenotype
(Table 11.4).
11.5 Plasma Cell Neoplasm 139
a b
Fig. 11.24 Cytology of plasma cell tumour. (a) Abundant plasma cells. (b) Many cells show
eccentric nuclei and bi-nucleation
a b
105
105
104
LAmbda PE-A
104
CD20 PE-A
103
103
−213 0 102
102
102 103 104 105

−153 0 102 103 104 105
CD19 PerCp-Cy5-5-A
FITC-A
c d
105
105
LAmbda PE-A
104
104
CD138 PE-A
103
103
−467 −102 0102
102
102 103 104 105

Kappa FITC-A −566−102 0 102 103 104 105
CD38 FITC-A
Fig. 11.25 Flow cytometry of plasma cell tumour. (a) Predominant CD20 population. (b) CD19
population of cells showing lambda expression. (c) Light chain restriction as no kappa chain is
expressed. (d) The cells are positive for CD38
Table 11.4 Distinguishing neoplastic plasma cells from normal plasma cells
Marker Normal plasma cells Neoplastic plasma cells
CD45 Positive Absent or dim positive
CD19 Positive Negative
CD38 Positive Positive
CD138 Positive Positive
CD56 Negative Positive
Table 11.5 Immunophenotyping to distinguishing lymphoplasmacytoid lymphoma versus

plasma cell neoplasms
Markers Lymphoplasmacytoid lymphoma Plasma cell neoplasms
CD45 Positive Dim or negative
CD19 Positive Negative
CD38 Negative/positive Positive
Surface immunoglobulin Positive Negative
11.5.1 Differential Diagnosis
• Lymphoplasmacytoid lymphoma: It is often difficult to distinguish lympho-

plasmacytoid lymphoma from the plasma cell neoplasm on the basis of morphol-
ogy. However, the lymphoma cases are positive for CD45, CD19 and negative
for CD56. Whereas, the plasma cell neoplasms show CD45 dim positive, CD
negative, CD56 positive, CD39 and CD138 positive cell population (Table 11.5).
11.6 CD5 Positive B-Cell Lymphomas
CD5 is a marker of T cell. However, the expression of CD5 marker in certain B-cell
lymphomas helps to diagnose these lesions (Box 11.1). When a B-cell lymphoma
expresses CD5 antigen in FCM, the diagnostic possibilities become limited. The
CLL/SLL, MCL and a small number of cases of MZL and LPL are positive for the
CD5 marker. Besides, the dual positivity of CD5 and CD23 indicates the possibility
of CLL/SLL.
Box 11.1: CD5 Positive B Cell NHL

• SLL
• MCL
• Certain type of marginal zone lymphoma.
• Rarely FL
• Denovo DLBCL
11.7 CD5 and CD10 Negative Lymphoma 141
11.6.1 CD10 Positive Lymphomas
CD10 is a marker of germinal centre cells, and it is often expressed in a large variety
of B cell lymphomas. This marker is commonly positive in FL, BL and
B-lymphoblastic lymphoma (Box 11.2). CD 10 expression is also noted in non-
neoplastic germinal centre cells of reactive lymphoid cells.
Box 11.2: CD10 Positive Lymphomas

• FL
• BL
• B- lymphoblastic lymphoma
• Subset of MCL
• Subset of DLBCL
• Subset of HCL
11.7 CD5 and CD10 Negative Lymphoma
A large group of mature B cell lymphomas are negative for CD5 and CD10 markers
such as marginal zone lymphoma, lymphoplasmacytic lymphoma and diffuse large
B cell lymphomas (Box 11.3).
Box 11.3: CD5 and CD10 Negative Lymphoma

• MZL
• Lymphoplasmacytic lymphoma
• DLBCL
• Plasmablastic lymphoma
• HCL
• Rarely FL
• Rarely MCL
Table 11.6 highlights the immunophenotyping pattern of commonly encoun-

tered B-NHL.
Table 11.6 immunophenotyping pattern of the common B-non Hodgkin lymphomas

Type of lymphoma Immunophenotype
Small lymphocytic leukemialeukaemia/ • CD45+,CD19+,CD20+,
lymphoma • Dual expression of CD5, and CD22
• Light chain restriction
• CD200+
• CD10 negative
• FMC negative
• CD38 varablyvariably positive
Mantle cell lymphoma • CD45+,CD19+,CD20+
• CD5+
• CD23 negative
• CD200 negative
• FMC7 positive
Lymphoplasmacytic lymphoma • CD45+,CD19+,CD20+
• Positive for CD38
• Negative for CD5, 23, 10
Follicular lymphoma • CD45+,CD19+,CD20+
• Positive for bcl2
• Negative for CD5
Marginal zone lymphoma • CD45+,CD19+,CD20+
• Negative for CD5, 23, 10
Burkitt’s lymphoma • CD45+,CD19+,CD20+
• Negative for CD5, 23
Diffuse large B-cell lymphoma • CD45+,CD19+,CD20+
• Light chain restriction+/-
• CD10 +/-
• Negative for CD23, 5
References
1. Kroft SH, Harrington AM. Flow Cytometry of B-cell neoplasms. Clin Lab Med.
2017;37(4):697–723. https://doi.org/10.1016/j.cll.2017.07.001. Epub 2017 Aug 31
2. Dey P, Amir T, Al Jassar A, Al Shemmari S, Jogai S, Bhat MG, Al Quallaf A, Al SZ. Combined
applications of fine needle aspiration cytology and flow cytometric immunphenotyping for
diagnosis and classification of non Hodgkin lymphoma. Cytojournal. 2006;3:24.
3. Moreau EJ, Matutes E, A'Hern RP, Morilla AM, Morilla RM, Owusu-Ankomah KA, Seon
BK, Catovsky D. Improvement of the chronic lymphocytic leukemia scoring system with the
monoclonal antibody SN8 (CD79b). Am J Clin Pathol. 1997;108(4):378–82.
4. Lenz G, Wright GW, Emre NC, et al. Molecular subtypes of diffuse large B-cell lymphoma
arise by distinct genetic pathways. Proc Natl Acad Sci U S A. 2008;105(36):13520–5.
Flow Cytometry of Mature and Immature
T-Cell Lymphoma 12
12.1 Introduction
T-non Hodgkin lymphoma (NHL) consists of only 10% of all lymphomas [1].
Unlike B-cell NHL, the T-NHL cases are challenging to diagnose by FCM as the
clonal origin of these tumours are difficult to establish with certainty. Moreover,
T-NHLs are a mixed group of tumours with widely variable clinical, histological,
and molecular genetics features.
Identification of monoclonal proliferation of T-NHL in FCM is a challenging
area. As mentioned before, there is only indirect evidence of T-NHL on FCM. The
evidences are:
• Dual expression of CD4 and CD8.

• Loss of either CD4 or CD8 marker.
• CD4 and CD8 ratio if more than 10:1 or less than 1:10.
• Loss of CD5 and CD7.
• Aberrant positivity of T-cell antigen.
• Expression of CD16, CD56, CD57 and CD25.
Therefore to confirm or eliminate the possibility of T-NHL, we need the panel of

T-cell markers as mentioned in the Fig. 12.1. It is important to note that some T-cell
NHL may also express B cell markers such as CD19/CD20. In such conditions, one
should always see the expression of the other T-cell markers such as CD3, CD5,
CD7, CD4 and CD8. In addition, the expression of other B-cell antigen (such as
CD79a) should also be verified in such cases.
At times, the viral lymphadenitis may be responsible for T-cell proliferation and
aberrant expression of T-cell antigens. Such cases should be carefully evaluated
with the cytology findings.
https://doi.org/10.1007/978-981-16-2655-5_12
144 12 Flow Cytometry of Mature and Immature T-Cell Lymphoma
Fig. 12.1 Essential markers of T-cell lymphoma
The differentiation of precursor T cells from the mature T cells is also necessary
(Box 12.1). TdT and CD34 are the reliable precursor T-cell markers. Besides, the
other features such as dim CD45 expression, loss of CD3, bright, positive expres-
sion of CD7, CD10 and CD117 positivity.
Box 12.1: Markers of Immature T Cells

• Positive CD34
• TdT expression
• Positive CD117
• Dim CD45
• Bright CD7
• Loss of surface CD3
• Dual positivity or dual negativity CD4 and CD8
12.2 Mature T-Cell Lymphomas
Table 12.1 highlights the positivity of the different antigens in mature T-cell
lymphomas.
12.3 Mycosis Fungoides and Sezary Syndrome
Mycosis Fungoides and Sezary Syndrome are uncommon T-cell lymphomas

(<0.5%).
Cytology features [2]:
Occasionally the cases may infiltrate the lymph node:
12.3 Mycosis Fungoides and Sezary Syndrome 145
Table 12.1 Antigenic markers of T-cell markers

Mycosis Extranodal Natural
Markers Fungoides ALCL PTCL AITL HSTL killer/T-cell lymphoma
CD2 + + + + + +
CD3 + ± + + + + (cytoplasmic)
CD5 + ± + + - or −
dim +
CD7 − ± + − + ±
CD25 + (rare) − +(occasional) − − −
CD30 − + Dim + − − + (occasional)
CD10 − − +(nodal) + − −
CD56 − − − − + +
+ = positive, − = negative
ALCL Anaplastic large cell lymphoma, PTCL Peripheral T-cell lymphoma, AITL Angio-
immunoblastic T-cell lymphoma, HSTL Hepatosplenic T-cell lymphoma
• Atypical cell with enlarged nucleus having high nucleo-cytoplasmic ratio.

• Nuclei have irregular contour with deep infolding giving rise to a “cerebriform”
appearance.
• Many histiocytes.
Flow cytometry:
• Positive for CD3, CD4 and CD5.
• Loss of CD7 and CD26.
Anaplastic large cell lymphoma (ALCL).

ALCL represents 30% NHL in children and 3% in adults.
Cytology features (Fig. 12.2) [3]:

• Large cells.
• Moderately nuclear pleomorphism.
• Nuclei are multilobated.
• Many “hallmark” cells are present that show “horse shoe” shaped convoluted
nuclei with prominent nucleoli.
• Doughnut shaped cells showing peripherally arranged nuclei like a garland.
• Many plasmacytoid cells.

• T-cell markers: Positive for CD3, CD2, CD5, and CD4.
• Loss of CD7 marker.
• The expression of CD30 of the tumour cells is the characteristic of ALCL.
• Many cases show additional positivity of the myeloid markers CD13, CD15
and CD33.
• Most of the ALCL cases show derangement of at least one T-cell marker.
a b
c d
Fig. 12.2 Anaplastic large cell lymphoma: (a) Abundant discrete atypical cells admixed with
reactive lymphoid cell population. (b) Doughnut shaped cells present. (c) Atypical cell with
horseshoe-shaped nucleus. (d). Haematoxylin and eosin-stained smear showing large mono and
multinucleated cells
a b c
(x 1,000)
250
105
105
200
CD5 PerCP-Cy5-5-A
104
104
CD4 PE-A
150
SSC-A
103
103
100
0
50
0
−64
−342
−954 0 103 104 105 −954 0 103 104 105 −2,828 −1030103 104 105
CD3 PE-Cy7-A CD3 PE-Cy7-A CD8 APC-A
d e f
105
105
105
104
104
104
CD2 FITC-A
CD30 PE-A
CD20 PE-A
103
103
0 103
0102
0
−929
−929
−92
−2,833 −103 0103 104 105 −2,833 −103 0103 104 105 −2,751 0 103 104 105
CD1a APC-A CD1a APC-A CD34 PerCP-Cy5-5-A
Fig. 12.3 Anaplastic large cell lymphoma: (a) Predominantly CD3 population cells. (b). CD5 and
CD3 positive cells. (c). Cells are positive for CD4 and CD8. (d). CD2 positive cell population. (e).
The cells are characteristically positive for CD30. (f). Both CD20 and CD34 negative population
12.4 Peripheral T-Cell Lymphoma (PTCL) 147
Molecular genetics
ALCL shows characteristic chromosomal translocation of t(2;5) (q23’q25),
resulting in the fusion of ALK gene in chromosome 2 with nucleophosmin gene in
chromosome 5.
12.4 Peripheral T-Cell Lymphoma (PTCL)
PTCL is an aggressive disease that may occur both in nodal and extranodal sites.
Cytology features (Fig. 12.4):

• Dissociated cells.
• Pleomorphic nuclei.
• Prominent nucleoli.
• Nuclear cleaving.
• Coarsely clumped chromatin.
Flow cytometry:
• The tumour may show dual negativity of CD4/CD8.
• Aberrant expression of various T-markers (CD2, CD3, CD5 and CD7).
• The tumours may have expression of CD11c, CD38 and CD56.
a b c
105
105
104
104
CD5 FITC-A
CD4 PE-A
103
103
0
0
−770
−850
−778 0 103 104 105 −9,142 0 104 105

PerCP-Cy5-5-A CD8 APC-A
d e f
105
105
105
CD34 PerCP-Cy5-5-A
104
104
104
CD5 FITC-A
CD4 PE-A
103
103
103
0
0
−770
−850
−790
−9,922 −103 0 103 104 105 −9,008 0 103 104 105

−9,410 0 102 103 104 105
CD38 APC-A CD43 APC-A
CD7 FITC-A
Fig. 12.4 Cytology and flow cytometry of Peripheral T-cell lymphoma (a) Dissociated lymphoid
cells with scanty cytoplasm and round nuclei. (b) The cells show CD5 positivity. (c) CD4 and CD8
expression. (d) The cells show CD7 expression. (e) CD38 positive cell population, (f) Both CD5
and CD43 expression
12.5 Angio-Immunoblastic T-Cell Lymphoma (AITL)
AITL is a disease of elderly patient. The patient presents with fever, weight loss,
lymphadenopathy and skin rash.
Cytology features [4]:

• Intermediate and small cells.
• Small lymphocytes, immunoblasts, and plasma cells.
• Many atypical lymphoid cells resembling Reed-Sternberg cells.
• Large dendritic cells with pale cytoplasm having a regular nuclear margin.
• Many thin-walled capillaries.

• The tumour cells show CD4 expression.
• Positive for T-cell markers CD2, CD3 and CD5.
• The lymphoma may have loss of CD7. AITL cases are positive for CD10.
a b
250
250
(x 1,000)
(x 1,000)
200
200
150
SSC-A
SSC-A
150
100
100
50
50
101 102 103 104 105 2,124 0 103 104 105

CD45 APC-Cy7-A CD3 PE-Cy7-A
c d
105
105
104
104
CD23 PE-A
CD4 PE-A
103
103
0
102
−726
−113 0 102 103 104 105 −375 −102 0102 103 104 105
CD5 FITC-A CD8 FITC-A
Fig. 12.5 Flow cytometry of Angio-immunoblastic T-cell lymphoma (a) The cells are positive for
CD45. (b) Predominantly, the cells are CD3 positive. (c) CD5 positive cells. (d) CD4 and CD8
expression
12.7 Extranodal Natural Killer/T-Cell Lymphoma 149
12.6 Hepatosplenic T-Cell Lymphoma (HSTL) [5]
HSTL is an aggressive disease that usually affects young male patients. The tumour
cells show positive gamma delta receptors.
Cytological features:
• Discrete large cells.
• Enlarged pleomorphic nucleus.
• Irregular nuclear contour.
• Prominent nucleolus.
Flow cytometry:
• Positive for CD3, CD2, CD7.
• Absence of CD5 expression.
• Negative for both CD4 and CD8.
• About 25% of cases show CD56 expression.
Molecular genetics: HSTL shows isochromosome 7q. Besides, trisomy 8 is

also seen.
12.7 Extranodal Natural Killer/T-Cell Lymphoma
It is an aggressive lymphoma and was previously known as midline lethal granuloma.
Cytomorphology:
• Discrete large cells.
• Large convoluted nucleus.
• Multiple prominent nucleoli.
• Cytoplasmic granularity present.
• Necrotic tissue.
Flow cytometry:
• The tumour cells show expression of CD2, cytoplasmic CD3, and CD56.
• Absence of CD5, CD7, CD4 and CD8 expression.
Molecular cytogenetics
• The most common cytogenetic abnormality is 6 del (6) (q21q25).
• Loss of 17p del or 11q.
12.8 Immature T-Cell Lymphoma
12.8.1 T-Cell Lymphoblastic Leukaemia (T-LBL) or Lymphoma

(T-ALL) [6]
T-LBL and T-ALL develop from the precursor T cell. T-LBL contains more than
25% blasts, whereas T-ALL has less than 25% blasts in the bone marrow.
T-LBL comprises the main bulk (90%) of lymphoblastic lymphoma. LBL is
commonly seen in children, and the patient presents with mediastinal lymphade-
nopathy or cervical lymph nodal enlargement.
Cytological features (Fig. 12.6):

• Discrete medium to large cells.
• Vacuolated cytoplasm.
• Enlarge nuclei with a regular margin.
• Condensed chromatin.
• Inconspicuous nucleoli.
Flow cytometry (Fig. 12.7) (Box 12.2):

• Immaturity markers: Positive for CD34 and TdT. TdT is positive in the majority
(>90%) cases in comparison to CD34 expression (40%). The tumours also
express CD1a and lack of surface CD3 marker.
• Dim positive CD45.
• Either partial or loss of T-cell markers (CD2/CD3/CD5/CD7).
• Dual positive or dual negative CD4 /CD8 expression.
• CD7 antigenic expression is the most common finding (<90%).
a b
Fig. 12.6 Cytology smear of T-cell lymphoblastic lymphoma. (a) Abundant large lymphoid cells.
(b) The cells show nuclear convolution with fine chromatin and prominent nucleoli
12.8 Immature T-Cell Lymphoma 151
a 105 b c
105
105
FMC-7 FITC-A
104
104
104
Kappa FITC-A
Lambda PE-A
103
103
103
−65 0 102
0
0
−323
−425
0102 103 104 105 −103 0 103 104 105 −102 0102 103 104 105
Kappa FITC-A CD10 APC-A CD3 PerCP-Cy5-5-A
d e f
105
105
105
104
104
104
CD5 FITC-A
CD4 PE-A
FITC-A
103
103
103
−151 0 102
0
0
−250
−377
0 103 104 105 −278 0 103 104 105

0 103 104 105
CD43 APC-A TDT APC-A
CD8 APC-A
Fig. 12.7 Flow cytometry of T-cell lymphoblastic lymphoma. (a) No light chain restriction. (b)
CD10 positive cell population. (c) Cells are positive for CD3. (d) Both CD4 and CD8 positive
cells. (e) CD5 and CD43 positive cell population, (f) The cells are positive for TdT
Near about 30% of cases show CD10 expression.

Certain groups of T-LBL may express myeloid antigen CD13 or CD33.
Box 12.2: FCM Findings of T-LBL

• Positive TdT (90%)
• Positive CD34 (30%)
• Positive CD1a (30%)
• Positive CD10(35%)
• Negative HLA-DR
• Dual Positive CD4/CD8 (33%)
• Dual negative CD4/CD8 (50%)
Table 12.2 summarizes the immunophenotyping markers of commonly noted T/

NK cell lymphomas.
Table 12.2 Flow cytometric immunophenotyping of commonly encountered T- and NK-cell

lymphomas
Lymphoma Immunophenotyping
Peripheral T- cell lymphoma • CD3+, loss of CD5, and CD7.
• Dual negative CD4 and CD8
Mycosis fungoides/ Sezary syndrome • CD3+, CD4+ and CD5+
• Loss of CD7 and CD26
Angio-immunoblastic T-cell lymphoma • CD2+, CD3+, CD5+, CD4+, CD10+
• Loss of CD7
Anaplastic large cell lymphoma • CD30+
• CD2+, CD3+, CD5+, CD4+
• Loss of CD7 marker
Extranodal NK cell lymphomas • CD2+, cytoplasmic CD3+, and CD56+
• Loss of CD5, CD7, CD4 and CD8
T-cell lymphoblastic leukaemia • CD34+ and TdT+
• Lack of surface CD3 marker
• Variable expression of CD2/CD3/CD5/CD7
• CD10+ (30% cases)
References
1. Dey P. Fine Needle Aspiration Cytology: Interpretation and diagnostic difficulties. Jaypee
Medical Publisher, New Delhi, India. Second edition. Chapter 9: Lymph node.
2. Pai RK, Mullins FM, Kim YH, Kong CS. Cytologic evaluation of lymphadenopathy associated
with mycosis fungoides and Sezary syndrome: role of immunophenotypic and molecular ancil-
lary studies. Cancer. 2008;114(5):323–32.
3. Kolonic SO, Prasek-Kudrna K, Roso V, et al. Value of fine-needle aspiration cytology in diag-
nosis of Hodgkin's lymphoma and anaplastic large cell lymphoma: one Centre experience. Coll
Antropol. 2010;34(1):75–9.
4. Dey P, Radhika S, Das A. Fine-needle aspiration biopsy of angio-immunoblastic lymphade-
nopathy. Diagn Cytopathol. 1996;15(5):412–4.
5. Yabe M, Miranda RN, Medeiros LJ. Hepatosplenic T-cell lymphoma: a review of clinicopatho-
logic features, pathogenesis, and prognostic factors. Hum Pathol. 2018 Apr;74:5–16.
6. Cortelazzo S, Ferreri A, Hoelzer D, Ponzoni M. Lymphoblastic lymphoma. Crit Rev Oncol
Hematol. 2017 May;113:304–17.
Flow Cytometry of Body Cavity Fluid
13
13.1 Introduction
Flow cytometry is helpful in the detection of metastatic carcinoma in the body cav-
ity fluid. It may also be beneficial in the identification and sub-classification of
lymphoma in effusion sample and CSF. The indications of FCM in the fluid are
shown in Box 13.1.
Box 13.1: Indications of FCM in the Fluid Sample

Effusion
• Detection of carcinoma.
• Diagnosis of lymphoma.
• Subclassification of lymphoma.
Urine
• Diagnosis of urothelial cell carcinoma of bladder.
CSF
• Diagnosis of lymphoma.
13.2 Detection of Malignancy in Fluid
The detection of metastatic carcinoma is often a challenge to the cytologist. Flow

cytometry plays an important role in this aspect (Box 13.2).
https://doi.org/10.1007/978-981-16-2655-5_13
154 13 Flow Cytometry of Body Cavity Fluid
The following features can help to indicate malignancy in the fluid sample:
• Demonstration of aneuploidy in fluid: The presence of DNA aneuploidy in the

fluid indicates malignancy. The benign cells are seldom aneuploid.
• High S-phase fraction of cells: Higher synthetic phase cells may be indirect evi-
dence of malignancy.
• Presence of epithelial cells: The epithelial cells are usually absent in the effusion
sample. Epithelial cell adhesion molecule (EpCAM) EpCAM (CD326/) is a
transmembrane glycoprotein related to intercellular cell-adhesion [1]. Since
EpCAM is expressed exclusively in epithelial and epithelial-derived neoplasms,
therefore EpCAM can be used as a diagnostic marker in this study to diagnose
malignancy in effusion samples [2]. Ber-EP4 is an antibody against EpCAM, and
it also identifies the epithelial cells [3]. Therefore, we can use either BER-EP4 or
EpCAM to recognize the epithelial cells.
13.3 DNA Flow Cytometry
Aneuploidy in the effusion fluid is defined as the presence of a distinct peak other
than the G0/G1 peak or a G2M phase that consists of more than 20% cells (Figs. 13.1
and 13.2). The DNA index of the aneuploidy peak should be either less than 0.9%
or more than 1.1% [4].
The sensitivity and specificity of DNA FCM vary from 32.1 to 96.67% and 82%
to 100% (Table 13.1). The sensitivity range of DNA FCM is wide. The causes of
variable sensitivity and specificity may be due to the following factors:
• The selection of the type of cases.

• The criteria of aneuploidy.
• Variable processing methodology.
Fig. 13.1 Cytology smear

of a case of metastatic
adenocarcinoma
13.3 DNA Flow Cytometry 155
Fig. 13.2 DNA flow

cytometry showing
hyperdiploid aneuploidy
Table 13.1 Sensitivity and specificity of DNA FCM

Authors Year Total number of cases Sensitivity (%) Specificity (%)
Puri et al. [5] 2020 90 96.67 100
Kundu et al. [4] 2011 100 86.2 97.1
Krishan et al. [6] 2006 130 38 82
Motherby et al. [7] 2002 200 32 100
Both et al. [8] 2001 67 57 93
Saha et al. [9] 2000 100 59 98.6
Causes of false-negative DNA FCM:

1. The tumour may be diploid.
2. A small percentage of aneuploid cells may be diluted with a large number of
mesothelial cells.
3. The chromosomal changes may be minor, and a small peak may be missed.
13.3.1 Immunophenotyping to Detect Malignancy
Epithelial cells are not commonly present in the effusion sample. So, the demonstra-
tion of epithelial cells by a suitable epithelial marker such as BER-EP4 or EpCAM
helps to detect metastatic carcinoma. In immunophenotyping of fluid,
multiple antibodies are tagged with different fluorochromes. The mesothelial cells
(CD14), lymphoid cells (CD 45) and epithelial cells (EpCAM/BER-EP4) are identi-
fied. By proper gating, one can eliminate the reactive mesothelial cells and lympho-
cytes. The percentage positivity of the epithelial cells can be calculated to assess
malignant epithelial cells in the remaining population (Figs 13.3, 13.4, and 13.5).
a b
Fig. 13.3 Cytology smear of metastatic adenocarcinoma in peritoneal effusion. (a) Multiple clus-
ters of malignant cells. (b) The cells with a moderate amount of cytoplasm having hyperchromatic
pleomorphic nuclei
a b
(x 1,000)
(x 1,000)
250
250
Scatter
200
200
Singlet
150
150
FSC-H
SSC-A
100
100
50
50
50 100 150 200 250 50 100 150 200 250

(x 1,000) (x 1,000)
FSC-A FSC-A
c d
5
5
10
10
CD45-/CD: 4-/EPCAM+
4
4
10
10
EPCam APC-A
CD14 PE-A
3
10
3
10
2
299 -10 0 10
0
2
1,144
2 2 3 4 5 2 3 4 5
008 -10 0 10 10 10 10 299 0 10 10 10 10
APC-Cy7-A CD14 PE-A
Fig. 13.4 Flow cytometry of the metastatic adenocarcinoma in peritoneal effusion. (a) Single-cell
gating. (b) All the other cells are acquired. (c) CD45 and CD14 negative population gated. (d)
EpCAM percentage in the CD45 and CD14 negative population is calculated (6%)
5
10
4
10
CD45 FITC-A
3
10
CD45 -CD14- Cells
0
-508
a 2
-361 -10 0 10
2
10
3
10
4
10
5
CD14 PE-A
c d
5
5
10
10
4
4
10
10
EPCam APC-A
EPCam APC-A
3
3
10
10
0
0
1,098
1,098
1 2 3 4 5 2 2 3 4 5
0 10 10 10 10 10 -508 -10 0 10 10 10 10
CD45 FITC-H CD45 FITC-A
Fig. 13.5 Metastatic adenocarcinoma in a peritoneal effusion. (a) Multiple ball-like cohesive
clusters of malignant cells. (b) CD45 and CD14 negative population is gated. (c) EpCAM percent-
age in the CD45 and CD14 negative population is calculated (80.5%). (d) Contour diagram show-
ing EpCAM positive cells
Table 13.2 Sensitivity and specificity of EpCAM

Author Year Total number of cases Sensitivity (%) Specificity (%)
Sahu et al. [10] 2020 75 87 100
Pillai et al. [11] 2013 195 88.15 97.64
Kentrou et al. [12] 2011 125 80.6 90.7
Sayed et al. [13] 2009 119 73 95.5
Davidson et al. [14] 2002 92 93 93
Risberg et al. [15] 2000 49 95 81
The sensitivity of EpCAM/BER-EP4 in FCM varies from 77 to 95%. In contrast,

the specificity of FCM varies from 81 to 100% (Table 13.2).
The positive cut off value of EpCAM percentage in the effusion fluid for detect-
ing metastatic carcinoma varies in the individual laboratory. Pillai et al. [11] consid-
ered 1.5% as the cut off value of EpCAM percentage in the CD45 and CD14
negative population. In contrast, Sahu et al. noted that 2% EpCAM value maybe the
suitable cut off value in the effusion fluid for detecting metastatic carcinoma cases
a b
c d EpCAM 33.8%
5
5
10
10
4
4
10
10
CD45 FITC-A
EPCam APC-A
3
10
3
0 10
0
CD45-CD14-EPCam-
8,275 -10
-762
3 4 5 1 2 3 4 5
-979 0 10 10 10 0 10 10 10 10 10
CD14 PE-A CD45 FITC-H
Fig. 13.6 A 56 year female with pleural effusion. The case was initially diagnosed on cytology as
atypia suspicious for malignancy. However, flow cytometry shows a high EpCAM percentage
(33.8%). (a) Occasional clusters of mildly pleomorphic cells along with abundant discrete meso-
thelial cells. (b) Discrete round to oval cells. (c) CD45 and CD14 negative population was gated.
(d) The percentage of EpCAM positive cells in the CD45 and CD14 negative population was
calculated
[10]. The EpCAM positive cells are beneficial to detect malignancy in atypical cells
(Fig. 13.6).
The application of the EpCAM/ BER-EP4 positive cells in FCM for metastatic
carcinoma has certain advantages over the immunostaining of BER-EP4 or MOC31.
Advantages of FCM.
The advantage of FCM include:
1 . A quick procedure.
2. A large population of cells can be assessed within a short period (few minutes).
3. FCM gives a quantitative result.
4. Multiple antibodies can be used in a small volume of fluid.
5. Malignant cell can be sorted out, and further experiment can be done.
13.3.2 Precautions to Take for the Best Result
• The sample should be in proper anticoagulant material.

• It is better to process the sample within a few hours.
• The cells should be dislodged to have single cells. The sample should be passed
repeatedly through the nylon mesh.
• There should be a pre-planned and consistent gating strategy.
13.3.3 Possible Pitfalls
The possible pitfalls of FCM include:

BER-EP4/EpCAM can be positive for both benign and malignant epithelial cells.
Many times poorly differentiated carcinoma may not express epithelial marker.
Therefore, it is preferable to use multiple epithelial markers.
Rarely mesothelial cells may also express EpCAM.
In abdominal instrumentation, such as laparoscopy or recent surgery, epithelial cells
may shed out in the effusion fluid.
Peritoneal washing may be a source of false positivity as the increased number of
epithelial cells may be present in the fluid. So, peritoneal washing should not be
considered for the examination [15].
Box 13.2: Metastatic Carcinoma in Fluid

Indicators of carcinoma.
DNA flow cytometry (FCM)

• Presence of aneuploidy population.
• High S-phase fraction of cells.
Multicoloured FCM
• Presence of Epithelial cells: Demonstrated by EpCAM or BER-EP4
markers.
Causes of false-negative DNA FCM

• A small fraction of aneuploid cells.
• Minor chromosomal changes and no demonstrable aneuploidy.
• Diploid tumours.
Advantages of FCM
• Rapid.
• A large population of cells can be assessed.
• A quantitative result,
–– Multiple antibodies can be used in a small volume of fluid.
• Malignant cell can be sorted out, and further experiment can be done.
Possible pitfalls of FCM

• Epithelial markers do not distinguish benign and malignant epithelial cells.
• Poorly differentiated carcinoma may not express epithelial marker.
• In abdominal instrumentation, may shed out epithelial cells in the effu-
sion fluid.
• Peritoneal washing may show increased number of epithelial cells in the
effusion fluid.
13.4 Detection of Lymphoma in Fluid
Effusion due to lymphoreticular neoplasm is uncommon. The causes of the effusion

may be due to the secondary involvement of lymphoma, the primary involvement of
the disease, or secondary infection. Out of the various lymphoreticular neoplasms,
NHL is the most frequent cause of the effusion and it comprises 15% of the total
cases [16]. Infiltration of lymphoreticular neoplasms in the effusion fluid has an
overall poor prognostic outcome [17].
FCM plays an essential role in diagnosing lymphoma because the diagnosis of
lymphoma may not be possible on cytology alone [18]. FCM, along with cytology,
has 100% sensitivity and 94% specificity [19]. A panel of antibody is needed: The
selection of the panel of antibody in effusion fluid depends on the provisional
diagnosis.
13.4.1 Panel of Markers in Leukaemia/Lymphoma
Acute leukaemia: CD45/CD34/CD7/CD13, CD45/CD34/CD2.CD7, CD45/CD33/

CD56/CD19.
Lymphoma: CD45/CD3/CD4/CD8 and CD45/CD19/ CD3/CD56, CD45/
CD20/CD10.
In suspected T-NHL: CD3/ CD2/CD5/CD7 should be done (Figs. 13.7 and 13.8).
In suspected B-NHL: CD19 and CD5/CD23/CD10/CD38/CD138/FMC7/sur-
face Kappa and lambda light chain (Fig. 13.9).
In lymphocyte rich effusion in the absence of lymphoma, the flow cytometry
may not show any light chain restriction or any evidence of T-NHL (Figs. 13.10
and 13.11).
13.4.2 Diagnostic Features
It is relatively easy to detect a B-cell lymphoma in fluid in the presence of light

chain restriction. However, many cases of non-Hodgkin lymphoma (NHL) may not
show light chain restriction such as lymphoblastic lymphoma, diffuse large B-cell
13.4 Detection of Lymphoma in Fluid 161
a b
Fig. 13.7 Effusion cytology in a case of T-non-Hodgkin lymphoma: (a) Abundant discrete cells
along with mesothelial cells. (b) The cells show mildly pleomorphic nuclei with irregular
nuclear margin
a b c
(x 1,000)
200 250
5
10
10
PerCP-Cy5-5-A
4
10
4
10
cd7 APC-A
150
SSC-A
3
10
3
100
10
50
2
0
10
-534
3 4 5 2 3 4 5 2 2 3 4 5
064 0 10 10 10 152 0 10 10 10 10 827 -10 0 10 10 10 10
cd3 PE-Cy7-A CD5 FITC-A cd5 PerCP-Cy5-5-A
d e f
5
5
5
10
10
10
HLA DR FITC-A
4
4
10
4
10
CD23 PE-A
10
cd4 PE-A
3
3
10
10
3
10
2
-95 0 10
2
0
10
-514
2 3 4 5 1 2 3 4 5 3 4 5
231 0 10 10 10 10 0 10 10 10 10 10 2,411 0 10 10 10
cd8 FITC-A CD43 APC-A CD38 APC-A
Fig. 13.8 Flow cytometry of the T-non-Hodgkin lymphoma (NHL): (a) CD3 population is gated.
(b) The cells are positive for CD5. (c) The cells show both CD7 and CD5 positivity. (d) Dual posi-
tive CD4 and CD8 suggests T-NHL. (e) CD43 positive cell population. (f) CD38 positive cells
lymphoma or Burkitt lymphoma at times. Similarly T-NHL is difficult to identify in

effusion sample.
The following immunophenotyping expression may help in diagnosing lympho-
mas in effusion (Table 13.3):
• CD1 expression: It is a common thymocyte marker and is abnormal outside the

thymus. Therefore, CD1 expression in effusion fluid indicates lymphoblastic
lymphoma.
• CD10 expression: CD10 positive cells are normally noted in the germinal centre
of the lymph node and in the bone marrow. The significant number of CD10
a b
c F-6446-Tube_010 d F-6446-Tube_010
5
5
10
10
4
LAMBDA PE-A
4
10
10
CD79a PE-A
Q1 Q1 Q2
Q2
3
3
10
10
2
2
10
10
Q3 Q3 Q4
Q4
2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10
CD19 FITC-A KAPPA FITC-A
Fig. 13.9 B-non-Hodgkin lymphoma (NHL). (a) Discrete immature lymphoid cells. (b) The cells
have enlarged nuclei with prominent nucleoli. (c) Flow cytometry show predominantly CD19 posi-
tive cells. (d) Light chain restriction is evident by predominant Lambda chain positive cell
population
a b
Fig. 13.10 Lymphocyte rich effusion: (a) Abundant mature lymphocytes. (b) The round cells
with condensed chromatin
13.4 Detection of Lymphoma in Fluid 163
a 200 250
(x 1,000)
b c
(x 1,000)
200 250
5
10
4
CD20 PE-A
10
150
150
SSC-A
SSC-A
3
100
100
10
50
50
2
10
2 3 4 5 2 3 4 5 2 3 4 5
099 0 10 10 10 10 016 0 10 10 10 10 377 0 10 10 10 10
CD45 PerCP-Cy5-5-A FITC-A CD19 PerCP-Cy5-5-A
d e f
5
5
10
10
10
4
4
4
LAMBDA PE-A
10
10
10
CD3 PE-A
CD3 PE-A
3
3
10
10
10
2
2
0 10
2
0 10
0 10
-235
-210
-456
2 3 4 5 2 3 4 5 2 2 3 4 5
182 0 10 10 10 10 225 0 10 10 10 10 428 -10 0 10 10 10 10
KAPPA FITC-A CD2 FITC-A CD8 FITC-A
Fig. 13.11 Flow cytometry of the above case of lymphocyte rich effusion: (a) Predominant CD45
positive cells. (b) and (c)The cells are positive for CD20 and CD19, (d) No light chain restriction,
(e) CD3 and CD2 positive cell population. (f) The cells are positive for CD4 and CD8
Table 13.3 Indicator of lymphoma in effusion fluid

Marker Commonly present Combination Suggestion
CD1 Thymocyte markerA CD1 along with mature Lymphoblastic
T marker (CD3) lymphoma
CD5 T-cell marker and CD5+/CD20+ Small lymphocytic
also present in small lymphoma
population of B cells
CD10 Germinal Centre cells CD10+/CD10+ • Follicular lymphoma
• Burkitt lymphoma
• Lymphoblastic
lymphoma
Either kappa or B cell express surface κ or λ ratio is more than B-non-Hodgkin
lambda light chain 4:1 or 1:2 lymphoma
expression
CD3, CD4, CD3 is a panT cell • CD3 positive cells T-non-Hodgkin
CD8 marker showing dual lymphoma
CD4: Helper cell • CD4/CD8 positivity
marker CD3 positivity with
CD8: Suppressor cell dual negative CD4/CD8
marker
positive cells in the effusion fluid is abnormal and indicates the possibility of
lymphoblastic lymphoma or follicular lymphoma.
• CD5: CD5 is noted in T cells and minor fraction of B cells. Significant popula-
tion of CD5+/CD20+ cells indicate the possibility of B-NHL particularly infiltra-
tion of small lymphocytic lymphoma/ chronic lymphocytic lymphoma in the
effusion fluid.
• Predominant CD3 positive population expressing dual positive CD4/CD8 or dual
negative CD4/CD8 indicates the possibility of a T-NHL.
13.5 Primary Effusion Lymphoma (PEL)
PEL is related with Kaposi’s sarcoma associate human herpes virus 8. It usually
affects the body cavity. However, PEL may occur in the skin, lung and intestine.
Cytomorphology
• Discrete large pleomorphic cells.
• Moderate to marked nuclear pleomorphism.
• Prominent nucleoli.
Flow cytometry
• CD45 positive cells.
• Positive for CD38, CD138, CD43.
• Positive for CD30.
• Negative for B-cell markers: CD19, CD20.
• No light chain restriction.
• Absent or aberrant expression of T-markers: CD2, CD3, CD5, CD7.
13.6 Urine Flow Cytometry
DNA FCM is helpful to identify the malignant cells in urine. The diagnostic fea-
tures of malignancy are:
1. DNA aneuploidy.
2. High synthetic (S) phase.
The sensitivity of DNA FCM in urine cytology is 55 to 78% [20, 21]

Specimen collection.
The specimen of urine is collected as bladder washing. The bladder is irrigated
with normal saline with the help of a catheter. Alternatively, the bladder wash is col-
lected at the time of cystoscopy. One part of the sample is processed for cytological
preparation, and the other part is processed for flow cytometry,
13.6 Urine Flow Cytometry 165
Sample processing
• The bladder wash is centrifuged at 1500 round per minute for 10 min.
• The supernatant fluid is discarded, and the cell pellet is resuspended in phosphate
buffer solution.
• The cell is dissociated by vortexing the suspension and passing the sample
through a 54 micron nylon mesh.
• The concentration of the cell is measured by a haemocytometer and microscope.
• The concentration of the cell is maintained as 2 × 106 per ml.
• One ml of the above sample is added with solution containing propidium iodide
(see Chap. 7).
• The sample is kept in the dark for 1 h and then examined in a flow cytometer, and
at least 5000 cells are examined.
Causes of false negativity in DNA FCM

• Diploid tumour.
• Small unrecognizable aneuploidy peak due to less number of cells.
• Scanty tumour cells.
• The diluted malignant cells by other inflammatory cells.
Multicolour FCM
Cytokeratin antibody has been used to identify the epithelial cells, and the gated
population of cells are studied for DNA flow cytometry. The use of the epithelial
markers in DNA FCM increases the sensitivity of the detection of aneuploidy in
bladder wash sample [22].
Cerebrospinal fluid (CSF)
Detection of metastatic carcinoma: DNA FCM of CSF has limited value as the
CSF as CSF usually is scanty in volume for analysis. However, Cibas et al. have
shown 69% sensitivity and 95% specificity in detecting leptomeningeal infiltration
of malignancy based on DNA ploidy and high S-phase [23].
Detection of lymphoma/leukaemia: Immunophenotyping helps diagnose and
subclassify lymphoma in CSF [24]. FCM immunophenotyping has significantly
higher sensitivity than cytomorphology in the detection of lymphoma in CSF sam-
ple [25, 26].
The systemic review of 27 studies on CSF flow cytometric immunophenotyping
showed that 24/27 studies demonstrated an increased number of positive cases
using FCM of CSF [26]. The combined use of FCM and cytology increases the
detection rate of lymphoma in the CSF sample.
Advantages of FCM
• Highly specific, particularly in B-cell NHL.
• FCM is a sensitive test.
• FCM helps to differentiate the reactive lymphocytes versus neoplastic lesion
(Figs. 13.12 and 13.13). The demonstration of light chain restriction is reliable
for the diagnosis in a specific clinical setting.
a b
c d
Fig. 13.12 A 35-year-old male is a known treated case of diffuse large B-cell lymphoma
(DLBCL). Now the patient presented with disorientation. CSF was examined along with flow
cytometry. (a) Abundant discrete lymphoid cells. (b) The lymphoid cells are admixed with neutro-
phils. (c) Individual lymphoid cells have enlarged nuclei with fine chromatin. (d) Papanicolaou’s
stained smear is showing the cytomorphology of the cells
a b c
150 200 250
(x 1,000)
5
10
10
Lambda PE-A
4
4
10
CD23 PE-A
10
SSC-A
3
10
3
100
10
50
2
10
-514
3 4 5 3 2 3 4 5 2 3 4 5
1,526 0 10 10 10 26 0 10 10 10 10 -79 0 10 10 10 10
CD19 PE-Cy7-A FITC-A CD5 FITC-A
d e f
5
5
10
10
10
CD4 PE-A
4
4
10
10
10
CD5 FITC-A
CD8 APC-A
3
10
3
10
3
4,019 -10 0 10
2
0 10
3
0
682
-79
3 4 5 3 3 4 5 3 4 5
1,698 0 10 10 10 1,019 -10 0 10 10 10 -542 0 10 10 10
CD43 APC-A CD8 APC-A CD3 PerCP-Cy5-5-A
Fig. 13.13 Flow cytometry findings of the above case indicating reactive population. (a)
Predominant CD19 population. (b) No light chain restriction. (c) CD5 positive and CD23 negative
population. (d) CD5 and CD43 positive cell population. (e) Both CD4 and CD8 positive cells.
(f) CD3 positive cells
References 167
• A large panel of antibodies can be used in a small CSF sample by using multiple
fluorochrome-tagged antibodies.
Limitations and challenges of CSF- FCM

• A small amount of fluid and less cellularity in CSF.
• Traumatic tap may be the source of contamination of blood cells in CSF.
• Rapid degradation of cells in CSF is a significant problem. The CSF sample
should be processed immediately.
• It is difficult to diagnose a T-NHL by flow cytometry.
References
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Flow Cytometry of Solid Tumours
14
14.1 Introduction
Flow cytometry (FCM) is widely used to diagnose and classify haemato-lymphoid

neoplasms and body cavity fluids. Till now, the application of FCM in solid tumours
is limited. However, many non-haematopoietic solid tumours can be accessed by
Fine needle aspiration cytology (FNAC), and a vast amount of information may be
available with the help of FCM. The major areas of applications of FCM in solid
tumours are highlighted in Table 14.1.
Detection of metastatic and primary malignancies (Box 14.1): The epithelial
cells are generally absent in the lymph node. So, the metastatic carcinoma in the
lymph node can be detected by the demonstration of the cells having epithelial cell
markers positive in the FCM. The commonly used epithelial markers are cytokera-
tin (CK), epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA),
cancer antigen 125 (CA-125) and epithelial cell adhesion molecule (EpCAM,
CD326). Most of these antigens are unavailable for use in FCM. EpCAM (CD326)
or anti CD326 (known as Ber-EP4) are commonly used epithelial markers in FCM
and can successfully pick up the epithelial cells in the lymph node [1, 2] (Figs. 14.1
and 14.2). A combination of antibodies such as CD45, CD14 and EpCAM (CD 326)
will be able to detect epithelial cells. EpCAM percentage of cells can be estimated
in the CD45 and CD14 negatively gated population. Dorwal et al. [2] applied a
combined panel of Ber-EP4, CD45 and CD19/CD56 to detect metastatic
Table 14.1 Applications of 1. Detection of metastatic malignancies in the

flow cytometry in lymph node
solid tumours 2. Diagnosis of primary malignancy
3. Diagnosis of the small blue round cell tumours
4. DNA content analysis and synthetic phase
assessment
5. Response of cancer chemotherapeutic drugs
https://doi.org/10.1007/978-981-16-2655-5_14
170 14 Flow Cytometry of Solid Tumours
5
10
CD45-/CD14-
CD14 PE-A
4
10
3
10
2
-10 0 10
2
439
2 2 3 4 5
a 522 -10 0 10 10 10
CD45 APC-Cy7-A
10
c d
5
5
10
CD45-/CD14-/EPCAM+ 10
4
10
4
EPCam APC-A
10
EPCam APC-A
3
3
10
10
0
0
399
-399
2 2 3 4 5 2 2 3 4 5
239 -10 0 10 10 10 10 229 -10 0 10 10 10 10
CD14 PE-A CD14 PE-A
Fig. 14.1 (a). Fine needle aspiration cytology smear of a cervical lymph node showing discrete
and small clusters of malignant cells. (b). CD45 and CD14 negative population of cells are gated.
(c). High (7%) epithelial cell adhesion molecule (EpCAM) population indicating metastatic carci-
noma in the lymph node. (d). Contour diagram showing the EpCAM positive cells
malignancies in the lymph node biopsy tissue or FNAC material. They detected
BER-EP4 positive population of cells in 21 out of 41 cases (50%) of the lymph node
sample, and all these cases showed metastasis in the lymph node on subsequent
histopathology. Chang et al. [1] applied a panel of antibody consisting of EpCAM,
myogenin, CD56 and CD99 on formalin-fixed tissues of proven cases of non-hae-
matopoietic neoplasms. They noted that EpCAM was able to detect 11 out of 12
carcinoma cases. The antigen positivity in various tumours is highlighted in
Table 14.2:
Micrometastasis in sentinel lymph node: Breast carcinoma at first metastasize
in the sentinel lymph node (SLN) followed by other local and distant metastasis. It
is assumed that if the SLN is free of metastasis then it is unlikely to have metastasis
in the other lymph nodes. It is important to detect the status of SLN for the manage-
ment of the patients. The dissection of the SLN followed by histopathology and
immunocytochemistry of cytokeratin (CK) can detect the micrometastasis in
14.1 Introduction 171
(x 1,000)
200 250
150
FSC-H
100
50
50 100 150 200 250
a (x 1,000)
FSC-A
c d A-984/2021-Tube_002
250
(x 1,000)
105
200
CD45-/CD14-/EPCAM+
EPCam APC-A
104
150
SSC-A
100
103
-103 0
50
-87
50 100 150 200 250

(x 1,000) 877 0 103 104 105
FSC-A CD14 PE-A
Fig. 14.2 (a) Fine needle aspiration cytology smear of a cervical lymph node showing reactive
lymphoid cells. (b). Single cells gating was done. (c). All the single cells are acquired. (d). Very
low (0.01) percentage of EpCAM positive cells indicating no evidence of metastatic carcinoma
Table 14.2 Flow cytometric markers positivity

Markers in flow cytometry
Tumour CD45 CD14 EpCAM CD56 MYOD1 CD81
Majority of carcinomas N N P N N N
Neuroblastoma N N N P N P
Small cell carcinoma N N P P N P
Nasopharyngeal carcinoma N N P N N P
Germ cell tumour N N N P N P
P = Positive, N = Negative.
SLN. However, the false-positive diagnosis can occur due to the inclusion of the
cytokeratin positive cells that are not epithelial in origin [3]. Multiparameter flow
cytometry was done to detect the micrometastasis in SLN in breast carcinoma. It
was claimed that FCM was more efficient to detect micrometastasis than histopa-
thology and immunocytochemistry [4].
FCM was also used to identify and quantitate the melanoma cells in the sentinel
lymph nodes by the various workers. They noted that FCM is a promising tool in
such cases [5]. FCM was also performed in SLN of gastric carcinoma cases.
Combined markers of anti CD326, CD45 and anti CEACAM5 were used to gate the
desired cell population. It was seen that FCM is a rapid, sensitive, cost-effective and
highly specific method to detect micrometastasis [6].
14.2 A
dvantages of Flow Cytometry in the Detection
of Carcinoma
The advantages of flow cytometry in the detection of metastatic or primary

carcinoma include:
• FCM is a very rapid technique in comparison to immunocytochemistry or histo-
pathology. The whole procedure takes only a few hours, whereas immunocyto-
chemistry on histopathology takes at least two days.
• FCM gives an overall idea of the diagnosis, and then further subsequent details
immunocytochemistry can be done.
• A large number of cells can be studied with the help of flow cytometry. In a
paucicellular sample, immunocytochemistry may not be possible. However,
FCM may be helpful in that case.
• FCM immunophenotyping is the more objective and exact percentage of the
positive cell population can be assessed. Moreover, the intensity of the antigen-
positive cells can also be assessed by FCM.
• In FCM, there is no need for antigenic retrieval, and also, there is no chance of
the loss of antigen. Therefore, the reliability of the antigenic expression is much
more in FCM.
• Lastly, excluding the machine’s initial cost, the cost of multiple antigen use in
FCM is relatively less.
Limitation of FCM
• The major limitation of FCM is the loss of the histopathological architectural
pattern of the tissue. So, the morphological examination is mandatory in FCM.
• The technique of multicoloured FCM needs proper standardization.
Box 14.1: Detection of Carcinoma by Flow Cytometry (FCM)

Role of FCM
• Detection of metastatic carcinoma particularly micrometastasis.
• Typing of carcinoma.
Micro metastasis detection:

• A combined panel of Ber-EP4, CD45 and CD19.
• Ber-EP4 positive cells in the CD45 and CD19 negative population.
14.3 Diagnosis of the Small Round Cell Tumours 173
Advantages
• FCM is a very rapid technique.
• A large number of cells can be studied in a small volume of sample.
• Objective assessment.
Limitation of FCM
• Loss of the histopathological architectural pattern and cell morphol-
ogy in FCM.
• The technique needs proper standardization.
Primary carcinoma detection: A panel of antibodies needed.
14.3 Diagnosis of the Small Round Cell Tumours
Small round cell tumours (SRCT) are neoplasms that show almost similar morphol-
ogy but different origin. SRCT usually occurs in the paediatric age group and the
group includes neuroblastoma (NB), Ewing’s tumour/primitive neuroectodermal
tumour (EW/PNET), rhabdomyosarcoma (RMS), non-Hodgkin lymphoma (NHL).
As these tumours have different clinical management and prognosis, so it is
essential to recognize the individual tumours. FNAC and subsequent immunocyto-
chemistry can distinguish the individual SRCT [7]. However, immunophenotyping
of the SRCT can also be done by flow cytometry [8, 9, 10]. A panel of antibodies
consisting of CD45, CD56, CD99, MYOD1 may be helpful to distinguish the
majority of the SRCT.
CD56 is present on the surface of the cell and is expressed in the cells of neuro-
ectodermal derivatives, NK cells and also small cell carcinomas. Therefore, the use
of CD56 can identify EW/PNET and NB. CD99 is another cell surface glycoprotein
that is present in EW/PNET and precursor T/B cell leukaemia. CD45 negative pop-
ulation showing CD99 positivity indicates the possible diagnosis of EW/PNET
(Table 14.3). CD45 is the marker of lymphoid cells. The presence of predominant
CD45 cells indicates the use of more markers to determine the B/T lineage of the
lymphoid cells and also monoclonality.
Table 14.3 Flow cytometric markers positivity

Immunophenotypic marker
Tumour CD45 CD99 CD56 MYOD1
Neuroblastoma N N P N
EW/PNET N P P N
Rhabdomyosarcoma N N P P
Non-Hodgkin lymphoma P N N N
Wilms’ tumour N N P N
P = Positive, N = Negative
14.4 DNA Content Analysis and Synthetic Phase Assessment
DNA ploidy analysis and synthetic phase estimation can be done relatively quickly
in fine needle aspiration cytology (FNAC) material of the solid tumours (Figs. 14.3
and 14.4). Many studies on the correlation of DNA ploidy and S-phase fraction
(proliferative activity) with the clinical outcome are available. Overall, it has been
shown that tumour with diploid DNA pattern has a better prognosis, whereas, aneu-
ploidy tumour is related to poor differentiation and poor prognosis [11–13].
On the other hand, a good number of articles show that DNA ploidy has no prog-
nostic importance in solid tumours [14, 15, 16].
DNA ploidy in diagnosis: Many malignant tumours may be DNA diploid or
there may be minor chromosomal changes in the tumour that may not be reflected
in the flow cytometry. So DNA ploidy estimation has no diagnostic value.
200
150
Number
Debris
100
Aggregates
Dip G1
Dip G2
Dip S
50
An1 G1
An1 G2
An1 S
0
0 50 100 150 200

Channels (PI-A)
Fig. 14.3 DNA flow cytometry in breast carcinoma. The first peak is diploid, and the second peak
is aneuploid. (Courtesy by Professor Alka Bhatia, Department of Experimental Medicine and
Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India)
14.4 DNA Content Analysis and Synthetic Phase Assessment 175
120
90
Number
60
Debris
Aggregates
Dip G1
30
Dip G2
Dip S
An1 G1
0
0 50 100 150 200 250

Channels (PI-A)
Fig. 14.4 DNA flow cytometry showing a diploid and a hyperdiploid aneuploidy peak in breast
carcinoma. (Courtesy by Professor Alka Bhatia, Department of Experimental Medicine and
Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh, India)
Prognostic assessment: As mentioned above, it is assumed that aneuploidy

tumour has bad prognosis in certain solid tumours (Box 14.2).
Box 14.2: Aneuploid Solid Tumour that Have Bad Prognosis

• Carcinoma of ovary
• Carcinoma of breast
• Colonic carcinoma
• Carcinoma of prostate
• Non-small cell carcinoma of lung
• Carcinoma of endometrium
• Advanced cases of neuroblastoma
In case of advanced neuroblastoma, aneuploid tumour is better cured by surgical

chemotherapy than diploid tumours [17]. In case of prostatic carcinoma, the
advanced diploid carcinoma responds better than aneuploidy carcinoma [18].
Studies have shown that DNA aneuploidy in carcinoma of breast is related with
short disease free survival rate and higher chances of recurrence [19, 20].
Table 14.4 DNA ploidy and prognosis of solid tumours

DNA
Type of tumour histogra Prognosis References
Neuroblastoma Aneuploid Better cured by chemotherapy [17]
Prostate Aneuploid Worse survival [18]
Carcinoma of breast Aneuploid Short disease free survival rate and higher [19, 20]
chances of recurrence
Non-small carcinoma Aneuploid Worse survival [21]
of lung
Ovarian carcinoma Aneuploid Lower survival [22]
Malignant melanoma Aneuploid Worse survival [23]
Non-small carcinoma of lung showed 87% DNA aneuploidy. The correlation of

aneuploidy and survival of the patient was noted only in advanced stages of carci-
nomas [21]. DNA aneuploidy is seen in 20–40% of ovarian carcinoma cases. Volm
et al. have shown lower survival in these aneuploidy ovarian carcinoma cases [22].
Aneuploidy is noted in malignant melanoma in higher Clark’s level. The aneu-
ploid malignant melanoma showed worse prognosis. However the aneuploidy as an
independent prognostic factor was not established [23]. Table 14.4 highlight the
prognosis and DNA ploidy in different solid tumours.
14.5 Limitation of DNA Analysis by FCM
The technical factors that may modify the prognostic significance of DNA ploidy
are the following:
1 . Lack of the standardization of the DNA content measurement in different studies.

2. Tumour heterogeneity may be a significant problem.
3. Many studies used formalin-fixed paraffin-embedded tissue, which may not be a
suitable DNA content measurement method compared to FNAC.
4. Admixture of stromal tissue and other non-epithelial cells may interfere in the
analysis. There is a need to have a multiparameter-based DNA content measure-
ment such as cytokeratin, or EpCAM positive gated population can be studied
for DNA content measurement.
14.6 The Response of Cancer Chemotherapeutic Drugs
With the simultaneous use of multiple markers, one can see the DNA content and
cyclin A2 of the tumour cells in the presence of chemotherapeutic drugs. The level
of cyclin A2 may indicate the response of the chemotherapeutic drugs in the man-
agement of cancer [24].
References 177
14.7 E
xpression of Oncogene Markers
and Receptor Expression
The demonstration of various receptors and oncogene is essential for assessing

molecular classification, management and prognosis of tumour. Several markers are
necessary to evaluate breast carcinomas, such as estrogen receptor (ER) and proges-
terone receptors (PR) and Her2/neu oncogene expression. These markers are usu-
ally demonstrated by immunohistochemistry. Flow cytometry is successful in
demonstrating ER/PR and Her2/neu in breast carcinoma [25]. Multiparameter FCM
of breast carcinoma cases showed a significant correlation between FCM findings
and immunohistochemistry data [26].
The Her2/neu positive group of cells can be correlated with ER receptor-positive
population and DNA content. Therefore, the distinct subpopulation of cells can be
assessed by multiparameter FCM.
References
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2. Dorwal P, Moore H, Stewart P, Harrison B, Monaghan J. CD326 (EpCAM) testing by flow
cytometric BerEP4 antibody is a useful and rapid adjunct to histopathology. Cytometry B Clin
Cytom. 2018;94(3):536–41.
3. Bostick PJ, Chatterjee S, Chi DD, Huynh KT, Giuliano AE, Cote R, Hoon DS. Limitations of
specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases
in the lymph nodes and blood of breast cancer patients. J Clin Oncol. 1998;16(8):2632–40.
4. Leers MP, Schoffelen RH, Hoop JG, Theunissen PH, Oosterhuis JW. Vd Bijl H, Rahmy a,
tan W, Nap M. multiparameter flow cytometry as a tool for the detection of micrometastatic
tumour cells in the sentinel lymph node procedure of patients with breast cancer. J Clin Pathol.
2002;55(5):359–66.
5. Hellmich L, Witte KE, Ebinger M, Ulmer A. Flow Cytometry for detection and quantification
of micrometastases in sentinel lymph nodes from patients with primary melanoma. J Surg Res.
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6. Jagric T, Mis K, Gorenjak M, Goropevsek A, Kavalar R, Mars T. Can flow cytometry reinvent
the sentinel lymph node concept in gastric cancer patients? J Surg Res. 2018;223:46–57.
7. Brahmi U, Rajwanshi A, Joshi K, Ganguly NK, Vohra H, Gupta SK, Dey P. Role of immuno-
cytochemistry and DNA flow cytometry in the fine-needle aspiration diagnosis of malignant
small round-cell tumors. Diagn Cytopathol. 2001;24(4):233–9.
8. Ferreira-Facio CS, Milito C, Botafogo V, Fontana M, Thiago LS, Oliveira E, da Rocha-Filho
AS, Werneck F, Forny DN, Dekermacher S, de Azambuja AP, Ferman SE, de Faria PA, Land
MG, Orfao A, Costa ES. Contribution of multiparameter flow cytometry immunophenotyping
to the diagnostic screening and classification of pediatric cancer. PLoS One. 2013;8(3):e55534.
9. Leon ME, Hou JS, Galindo LM, Garcia FU. Fine-needle aspiration of adult small-round-cell
tumors studied with flow cytometry. Diagn Cytopathol. 2004;31(3):147–54.
10. Brahmi U, Rajwanshi A, Joshi K, Dey P, Vohra H, Ganguly NK, Gupta SK. Flow cytometric
immunophenotyping and comparison with immunocytochemistry in small round cell tumors.
Anal Quant Cytol Histol. 2001;23(6):405–12.
11. Beerman H, Kluin PM, Hermans J, van de Velde CJ, Cornelisse CJ. Prognostic significance of
DNA-ploidy in a series of 690 primary breast cancer patients. Int J Cancer. 1990;45(1):34–9.
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12. Lanza G, Gafà R, Santini A, Maestri I, Dubini A, Gilli G, Cavazzini L. Prognostic significance
of DNA ploidy in patients with stage II and stage III colon carcinoma: a prospective flow cyto-
metric study. Cancer. 1998;82(1):49–59.
13. Porschen R, Remy U, Bevers G, Schauseil S, Hengels KJ, Borchard F. Prognostic signifi-
cance of DNA ploidy in adenocarcinoma of the pancreas. A flow cytometric study of paraffin-
embedded specimens. Cancer. 1993;71(12):3846–50.
14. Dreinhöfer KE, Baldetorp B, Akerman M, Fernö M, Rydholm A, Gustafson P. DNA ploidy
in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93
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15. Lee JH, Noh SH, Lee KY, Choi SH, Min JS. DNA ploidy patterns in advanced gastric carcinoma;
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16. Ørbo A, Rydningen M, Straume B, Lysne S. Significance of morphometric, DNA cytometric
features, and other prognostic markers on survival of endometrial cancer patients in northern
Norway. Int J Gynecol Cancer. 2002;12(1):49–56.
17. Look AT, Hayes FA, Nitschke R, McWilliams NB, Green AA. Cellular DNA content as a pre-
dictor of response to chemotherapy in infants with unresectable neuroblastoma. N Engl J Med.
1984;311(4):231–5. https://doi.org/10.1056/NEJM198407263110405.
18. Stephenson RA, James BC, Gay H, Fair WR, Whitmore WF Jr, Melamed MR. Flow cytometry
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20. Ewers SB, Långström E, Baldetorp B, Killander D. Flow-cytometric DNA analysis in primary
breast carcinomas and clinicopathological correlations. Cytometry. 1984;5(4):408–19.
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23. Søndergaard K, Larsen JK, Møller U, Christensen IJ, Hou-Jensen K. DNA ploidy-characteristics
of human malignant melanoma analysed by flow cytometry and compared with histology and
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org/10.1007/BF02890369.
24. Chang Q, Hedley D. Emerging applications of flow cytometry in solid tumor biology. Methods.
2012;57(3):359–67.
25. Lostumbo A, Mehta D, Setty S, Nunez R. Flow cytometry: a new approach for the molecular
profiling of breast cancer. Exp Mol Pathol. 2006;80(1):46–53.
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carcinomas: comparison of immunohistochemistry and multiparameter DNA flow cytometry.
Anticancer Res. 2003;23(2A):999–1006.
Self-Assessment Test in Flow Cytometry
15
Q1. In 1953, Walter Coulter first time did:

(A) Nucleic acid content of the normal and abnormal cells.
(B) Counting blood cells flowing in a liquid suspension.
(C) Cell sorting.
(D) Counting RBCs and WBCs by using Acridine orange.
Q2. Mack Fulwyler in the year 1965 did:

(A) Multiple laser beam to measure simultaneous multiple parameters.
(B) Fluorescence dye based flow cytometer to measure DNA content of
the cells.
(C) Differential count in flow cytometer.
(D) Electrostatic deflection ink-jet recording technique for cell sorting.
Q3. The function of the sheath fluid is:

(A) Reduction of cellular aggregates.
(B) Give resistance to the flow of cells.
(C) Reduction of any turbulence of the inner sample containing cells.
(D) Fixation of the cell.
Q4. Which is true in flow cytometer.

(A) The pressure in the sample fluid is always kept much higher than the
sheath fluid.
(B) The pressure in the sample fluid is always kept much lower than the
sheath fluid.
(C) The pressure in the sample fluid is always kept equal to the sheath fluid.
(D) The lowered sample pressure makes the beam narrow.
https://doi.org/10.1007/978-981-16-2655-5_15
180 15 Self-Assessment Test in Flow Cytometry
Q5. Which one is a false statement regarding the forward scatter (FSC) of light:
(A) FSC of light is collected by the detector located on the same axis as the
laser beam.
(B) The total amount of FSC is directly proportional to the granularity and
internal complexity of the cell.
(C) The FSC light has the same wavelength and colour as that of the
laser light.
(D) No fluorochrome probe is needed to detect the signals of FSC.
Q6. Which statement is false for argon laser beam.

(A) It can generate multiple wavelengths of light.
(B) It produces a high power output.
(C) Argon laser is a higher gain system.
(D) Low power requirement.
Q7. This filter reflects the light below the specific wavelength and transmits light
above the cut-off wavelength:
(A) Long pass filter.
(B) Short pass optical filter.
(C) Dichroic short pass filter.
(D) Dichroic long pass filter.
Q8. The maximum electric current generated by the laser hit cells is repre-
sented by:
(A) Width of the pulse.
(B) Area of the pulse.
(C) Height of the pulse.
(D) None of the above.
Q9. Which one is not a permeabilizing agent to study intracellular antigen:

(A) Tween 20.
(B) Phosphate buffer solution.
(C) Triton X.
(D) NP40.
Q10. The cell permeabilization in flow cytometry is needed to study:

(A) CD4.
(B) EpCAM.
(C) CD45.
(D) TdT.
Q11. The graph (Fig. 15.1) is best known as:

(A) Density plot.
(B) Contour plot.
(C) Bivariate histogram.
(D) Scatter plot.
15 Self-Assessment Test in Flow Cytometry 181
Fig. 15.1 The
fluorescence intensity of
105
CD43 and CD5 is shown in
this graph
104
CD5 FITC-A
103102
0
-79
-1,698 0 103 104 105

CD43 APC-A
Fig. 15.2 The distribution

105
of EpCAM and CD14

104
EPCam APC-A
103 0
-2,350 -103
-1,406 0 103 104 105

CD14 PE-A
Q12. The graph (Fig. 15.2) is best known as:

(A) Density plot.
(B) Contour plot.
(C) Bivariate histogram.
(D) Scatter plot.
Q13. The gating in the graph (Fig. 15.3) is to:

(A) Include only single cells.
(B) Exclude the debris.
(C) Include lymphocytes.
(D) None of the above.
Fig. 15.3 Graph showing
(x 1,000)
250
FSC-A versus FSC-H
200 150
FSC-H
100
50
50 100 150 200 250

(x 1,000)
FSC-A
Fig. 15.4 Graph showing

CD19 versus Kappa
105 104
Kappa FITC-A
103 0 102
-225
-1,472 0 103 104 105

CD19 PE-Cy7-A
Q14. Flow cytometry of a lymph node aspirate. The graph in Fig. 15.4 shows a
population of:
( A) Both CD19 and Kappa chain.

(B) Only CD19.
(C) Only Kappa chain.
(D) Negative for both Kappa chain and CD19.
Q15. Everyday quality control includes all except.

(A) Setting the voltage of the photomultiplier tube.
(B) Spectral overlap and compensation.
(C) Gating control in multicolour flow cytometry.
(D) Laser alignment.
Q16. The separation of the dim population of cells from the unstained population
is recognized by:
(A) Resolution.
(B) Threshold.
(C) Sensitivity of the photomultiplier tube.
(D) Performance of the optical filter.
Q17. The correction of the fluorescence spillover in the multi-coloured flow cytom-
etry is known as:
(A) Stain index.
(B) Compensation.
(C) Performance of the filter.
(D) Sensitivity of the photomultiplier tube.
Q18. The difference of wavelength of the absorption peak of excitation and emis-
sion light in fluorescence is known as:
(A) Stroke shift.
(B) Excitation spectrum.
(C) Emission spectrum.
(D) Excited state.
Q19. The efficiency of the fluorescence dye to emit photons to lose energy is
known as:
(A) Quantum yield.
(B) Stain index.
(C) Molar extinction coefficient.
(D) Fluorescence resonance energy transfer.
Q20. The intensity of a chemical to absorb light of a specific wavelength is mea-

sured by.
a. Quantum yield.
b. Stain index.
c. Molar extinction coefficient.
d. Fluorescence resonance energy transfer.
Q21. The dye that binds in the minor groove of DNA:

(a) Mithramycin.
(b) Propidium Iodide.
(c) Acridine orange.
(d) Ethidium Bromide.
Q22. Which fluorescent dye stains only DNA:

(A) Propidium Iodide.
(B) Ethidium Bromide.
(C) Acridine orange.
(D) DAPI.
Q23. A tumour with DNA index 1.2 is:

(A) Diploid.
(B) Aneuploid.
(C) Tetraploid.
(D) Hypodiploid.
Q24. The factor that affects optimum DNA stain by propidium iodide:
(A) Incubating the final solution in the sunlight for 30 minutes.
(B) Use of RNAse.
(C) Using Tween 20 as surfactant.
(D) Ethanol fixation of the cells.
Q25. The causes of inadequate DNA-histogram for interpretation include:

(A) High CV (more than 8%).
(B) Too much debris.
(C) Too many aggregates and less number of singly dispersed cells.
(D) All of the above.
Q26. Mantle cell lymphoma will be in which group:

(A) Precursor B cell.
(B) Mature B cell.
(C) Precursor T cell.
(D) Mature T cell.
Q27. The lymphoma that develops from the germinal centre cells:
(A) Lymphoblastic lymphoma.
(B) Small lymphocytic lymphoma.
(C) Follicular lymphoma.
(D) Mantle cell lymphoma.
Q28. Which marker is positive for plasma cell tumour:

(A) CD10.
(B) CD5.
(C) CD23.
(D) CD138.
Q29. Which marker is present in the B-lymphoblastic lymphoma:

(A) TdT.
(B) CD34.
(C) CD19.
Q30. The characteristic marker/s of mantle cell lymphoma:

(A) Positive for both CD5 and CD23.
(B) Positive for CD5 and negative for CD23.
(C) Negative for CD5 and positive for CD23.
(D) Negative for both CD5 and CD23.
Q31. CD7 marker is positive in all except.

(A) Follicular lymphoma.
(B) NK cell lymphomas.
(C) T-ALL.
(D) AML.
Q32. This type of graph in Fig. 15.5 is seen in all except:

(A) Follicular lymphoma.
(C) Burkitt lymphoma.
(D) B-ALL.
Q33. CD30 is positive in:

(A) ALCL.
(B) Hodgkin lymphoma.
(C) Primary effusion lymphoma.
Q34. This type of expression in Fig. 15.6 is noted in all except:

(A) Mantle cell lymphoma.
(B) Lymphoplasmacytic lymphoma.
(C) Peripheral T cell lymphoma.
(D) Diffuse large cell lymphoma.
Fig. 15.5 The X-axis

represents the CD10
105
positivity
104
PerCP-Cy5-5-A
103 0
-495
-1,533 0 103 104 105

CD10 APC-A
Fig. 15.6 The distribution

of Kappa and Lambda
105
expression
104
Lambda PE-A
1030 102
–296
–255 0 102 103 104 105

Kappa FITC-A
Fig. 15.7 CD8 and CD4

expression
105
104
cd4 PE-A
103 102
–231 0 102 103 104 105

cd8 FITC-A
Q35. This type of expression in Fig. 15.7 indicates the possibility of:

(A) Burkitt lymphoma.
(B) T cell lymphoma.
(C) Reactive lymphoid hyperplasia.
(D) Plasma cell tumour.
Q36. This type of expression (Fig. 15.8) may be noted in:

(B) Burkitt lymphoma.
(C) Small lymphocytic lymphoma.
(D) Diffuse large B cell lymphoma.
Fig. 15.8 The expression

of CD5 and CD23
105
104
CD23 PE-A
103 0
–525
–294 –102 0 102 103 104 105

CD5 FITC-A
Fig. 15.9 The expression

of CD19 and Lambda
105
104
Lambda PE-A
103
102
–1,204 0 103 104 105

CD19 PE-Cy7-A
Q37. The graph in Fig. 15.9 indicates:

(A) CD19 cell populations are positive for Lambda.
(B) CD19 cells are not expressing Lambda.
(C) The cells are negative for both CD19 and Lambda.
(D) The cells expressing Lambda chains are not positive for CD19.
Q38. One 55-year-old female having multiple enlarged upper right cervical lymph
nodes for 5 months. Fine needle aspiration cytology was done from the lymph
node (Fig. 15.10 a) along with flow cytometry (Fig. 15.10 b to f).
The most likely diagnosis is:
(B) Reactive lymphoid hyperplasia.
(D) Small lymphocytic lymphoma.
Q39. A 51-year-old male presented with multiple left cervical lymph nodes 3–4 cm
diameter for 2 months. Fine needle aspiration cytology was done (Fig. 15.11a,
b) along with flow cytometry (Fig. 15.11c-f).
The most likely diagnosis is:
Q40. A 38-year-old male presented with generalized lymphadenopathy with occa-

sional history of fever for 20 days. Aspiration cytology of the lymph node
(Fig. 15.12 a, b) and flow cytometry were performed (Fig. 15.12 c–f). The
most likely diagnosis is:
(B) Lymphoplasmacytic lymphoma.
b c
(x 1,000)
50 100 150 200 250
105
104
Lambda PE-A
SSC-A
0 102 103
29
103 104 105 0 0 102 103 104 105

a 335 0
d e f
105
5
105
10
104
PerCP-Cy5-5-A
4
104
PerCP-Cy5-5-A
10
CD23 PE-A
103
3
103
10
2
102
10
238
0 101 102 103 104 105 203 -1030103 104 105 0,288 0 103 104 105
CD5 FITC-A CD10 APC-A CD43 APC-A
Fig. 15.10 (a) Cytology smear, (b) CD19 expression, (c) Kappa and Lambda expression, (d)
CD5 and CD23 expression, (e) CD10 expression, (f) CD43 expression
104 105
Lambda PE-A
103
534 0
-168 0 102
3
10 104 105
a b Kappa FITC-A
d e f
105
104 105
5
10
CD34 PerCP-Cy5-5-A
0 102 103 104

CD5 FITC-A
4
CD20 PE-A
10
3
375 0 102 10
3
10
2
10
293
2 2
-828 0 103 104 105 0,548 -103 0 103 104 105 844-10 0 10 103 104 105
CD19 PE-Cy7-A CD10 APC-A HLA DR FITC-A
Fig. 15.11 (a, b) Cytology smear, (c) Kappa and Lambda expression, (d) CD5 and CD19 expres-
sion, (e) CD10 and CD34 expression, (f) HLA DR and CD20 expression
c (x 1,000)
50 100 150 200 250
SSC-A
0,275 0 103 104 5

a b CD19 PE-Cy7-A
10
d e f
105
105
105
4
CD19 PE-Cy7-A
Lambda PE-A
CD23 PE-A
10
104
104
103
103
103
0
102
102
282
2
233 0 102 103 104 105 0,639 0 103 104 105 286 -10 0 102 103 104 105
Kappa FITC-A CD10 APC-A CD5 FITC-A
Fig. 15.12 (a, b) Cytology smear, (c) CD 19 expression, (d) Kappa and Lambda expression, (e)
CD10 and CD19 expression, (f) CD5 and CD23 expression
Q41. A 55-year-old male had a 3 cm diameter firm upper cervical lymph node for
3 months. Fine needle aspiration cytology was done (Fig. 15.13a, and b)
along with flow cytometry (Fig. 15.13c–i). The possible diagnosis is:
50 100 150 200 250

(x 1,000)
SSC-A
a b 303 0 10
3
104 105
CD19 PE-Cy7-A
d e f
105
105
5
103 104 10
103 104
Lambda PE-A
4
CD5 FITC-A
CD23 PE-A
10
3
10
102
2
10
0
0 102 103 104 5

0 102 103 104 105 10 103 104 105
92
2,102 0
90 Kappa FITC-A 246 CD5 FITC-A CD43 APC-A
g h i
105
105
105
CD19 PE-Cy7-A
CD3 PE-Cy7-A
104
103 104
4
CD4 PE-A
10
0 103
0 103
0
081
335
806
029 -103 0 103 104 105 277 0 102 103 104 105 009 -103 0 103 104 105
CD10 APC-A CD8 FITC-A CD7 APC-A
Fig. 15.13 (a, b) Cytology smear, (c) CD 19 expression, (d) Kappa and Lambda expression, (e)
CD5 and CD23 expression, (f) CD 43 and CD5 expression, (g) CD10 and CD19 expression, (h)
CD8 and CD4 expression, (i) CD7 and CD3 expression
Q42. A 60-year-old male presented with right-sided pleural effusion. Cytology

smear (Fig. 15.14 a) and flow cytometry figures (Fig. 15.14 b to f) are pre-
sented here. The most likely diagnosis is:
(A) Small lymphocytic lymphoma infiltration.
(B) Reactive lymphocytes.
(C) Adenocarcinoma metastasis.
(D) Follicular lymphoma.
Q43. A 57-year-old female presented with abdominal distension. The ascetic fluid
was examined for cytological examination (Fig. 15.15 a and b). Flow cytom-
etry was done using a cocktail of CD45, CD14 and EpCAM (Fig. 15.16 a to
d). The most likely diagnosis is:
(A) Metastatic adenocarcinoma.
(B) Reactive effusion (no malignancy).
(C) Mesothelioma.
(D) Non-Hodgkin lymphoma in fluid.
b c
50 100 150 200 250

(x 1,000)
62 0 102 103 104 105

Lambda PE-A
SSC-A
0 103 104 10
5
199 0 102 103 104 105
a CD19 PE-Cy7-A Kappa FITC-A
d e f
105
105
5
10
4
4
104
103 10
CD10 APC-A
10
CD23 PE-A
CD4 PE-A
3
10
0 103
0
0
638
388
322
0 101 102 103 104 105 394-102 0 102 103 104 105 -245 0 102 103 104 105
CD5 FITC-A CD19 PE-Cy7-A CD8 FITC-A
Fig. 15.14 (a) Cytology smear, (b) CD19 expression, (c) Kappa and Lambda expression, (d)
CD5 and CD23 expression, (e) CD19 and CD10 expression, (f) CD8 and CD4 expression
a b
Fig. 15.15 (a) May Grunwald Giemsa stained smear of fluid, (b) Papanicolaou’s stained smear
Q44. The lytic lesion over the frontal bone in a 65-year-old male. FNAC was done
(Fig. 15.17 a) along with flow cytometry. The possible diagnosis is:
(A) Plasmacytoma.
(B) Inflammatory lesion.
(C) Metastatic carcinoma.
(D) Chondrosarcoma.
Q45. A 61-year-old female presented with 3 cm diameter swelling in the right lobe
of the thyroid for 2 years. Fine needle aspiration cytology (Fig. 15.18 a to c)
and flow cytometry (Fig. 15.18 d to i) were done. The most likely diagnosis is:
(A) Lymphocytic thyroiditis.
(C) Mantle cell lymphoma.
(D) Diffuse large B cell lymphoma.
a b
(x 1,000)
250
(x 1,000)
250
200
200
150
150
SSC-A
FSC-H
Singlet
100
100
50
50
50 100 150 200 250 50 100 150 200 250

(x 1,000) (x 1,000)
FSC-A FSC-A
c d 5
105
10
CD45+CD14+
104
EpCAM:44%
104
EPCam APC-A
CD14 PE-A
103
103 0
102
2,350
102 103 104 105 1,406 0 103 104 105

CD45 APC-Cy7-A CD14 PE-A
Fig. 15.16 (a) Singlet cell gating, (b) All the scattered cells acquired, (c) CD45 and CD14 posi-
tive population, (d) EpCAM positive cells (44%) in CD45 and CD14 negative population
b
5
10
CD34 PerCP-Cy5-5-A
4
10
3
0 10 –1,150
–3,130 –103 0 103 104 105

a CD38 APC-A
Fig. 15.17 (a) Fine needle aspiration cytology smear, (b) CD38 and CD34 expression
15.1 Answer Key of Chap. 15 193
a b c
d e f
(x 1,000)
50 100 150 200 250
5
5
10
10
CD23 PE-A
4
Lambda PE-A
-10 0102 103 104
10
SSC-A
3
10 0
2
014
064
269 -103 0 103 104 105 2
003 -10 010
2
103 104 105 -102 0 102 103 104 10
5
246
CD19 PE-Cy7-A Kappa FITC-A CD5 FITC-A
g h i
105
5
5
10
10
CD10 APC-A
104
-102 0 102 103 104
4
CD5 FITC-A
10
CD4 PE-A
103
3
84 -10 0 10
3
0
96
599
3
-10 0 10
3
10
4 5
10 71 0 103 104 105 213 -103 0 103 104 105
CD43 APC-A CD19 PE-Cy7-A CD8 APC-A
Fig. 15.18 (a–c) Aspiration cytology smear, (d) CD19 expression, (e) Kappa and Lambda
expression, (f) CD5 and CD23 expression, (g) CD43 and CD5 expression, (h) CD19 and CD10
expression, (i) CD8 and CD4 expression.
15.1 Answer Key of Chap. 15
Answer
Q1. B. Counting blood cells flowing in a liquid suspension.
Q2. D. Electrostatic deflection ink-jet recording technique for cell sorting.
Q3. C. Reduction of any turbulence of the inner sample containing cells.
Q4. A. The pressure in the sample fluid is always kept much higher than the
sheath fluid.
Q5. B. The total amount of FSC is directly proportional to the granularity and inter-
nal complexity of the cell.
Q6: D. Low power requirement.
Q7. D. Dichroic long pass filter.
Q8. C. Height of the pulse.
Q9. B. Phosphate buffer solution.
Q10. D. TdT.
Q11. C. Bivariate histogram.
Q12 B. Contour plot.

Q13. A. Include only single cells.
Q14. A. Both CD19 and Kappa chain.
Q15 D. Laser alignment.
Q16. A. Resolution.
Q17. B. Compensation.
Q18. A. Stroke shift.
Q19. A. Quantum yield.
Q2O. C. Molar extinction coefficient.
Q21. A. Mithramycin.
Q22. D. DAPI.
Q23. B. Aneuploid.
Q24 A. Incubating the final solution in the sunlight for 30 minutes.
Q25. D. All of the above.
Q26. B. Mature B cell.
Q27. C. Follicular lymphoma.
Q28. D. CD138.
Q30. B. Positive for CD5 and negative for CD23.
Q31. A. Follicular lymphoma.
Q32. B. Small lymphocytic lymphoma.
Q34. C. Peripheral T cell lymphoma.
Q35. B. T cell lymphoma.
Q36. A. Mantle cell lymphoma.
Q37. B. CD19 cells are not expressing Lambda.
Q38. D. Small lymphocytic lymphoma.
Q39. A. Mantle cell lymphoma.
Q40. C. Follicular lymphoma.
Q41. B. Reactive lymphoid hyperplasia.
Q42. A. Small lymphocytic lymphoma infiltration.
Q43. A. Metastatic adenocarcinoma.
Q44. A. Plasmacytoma.
Q45. A. Lymphocytic thyroiditis.

Diagnostic Flow Cytometry in Cytology: Pranab Dey

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Diagnostic Flow

ISBN 978-981-16-2654-8 ISBN 978-981-16-2655-5 (eBook)

Chandigarh, India Pranab Dey

Part I Practical Aspects of Flow Cytometry

3.7 Permeabilization �������������������������������������������������������������������������������� 27

7.5 Data Acquisition���������������������������������������������������������������������������������� 81

Part II Diagnostic Applications of Flow Cytometry in Cytology

10 Detection of Lymphoma: Clonality Demonstration

12.6 Hepatosplenic T-Cell Lymphoma (HSTL)���������������������������������������� 149

Pranab Dey is Professor in the Department of Cytology and Gynaecologic

ADC Analogue to digital conversion

Flow cytometry is the measurement of the various parameters of the cell/object in

1.2 History of Flow Cytometry

1.2.1 Early Motivation

1.2.2 Counting the Flowing Cells

1.2.3 Haematology Sample

1.2.4 Differential Count in the Blood Sample

in different channels. Ornstein et al. used Technicon Hemalog D instrument with

1.2.5 Flow Sorter

1.3 Fluorescence Stain in the Flow Cytometer

In the year 1965, Kamentsky et al. developed a microscope-based flow cytome-

1.4 Further Improvements in Flow Cytometry

Curbelo et al. [20] at Block Engineering, Cambridge, Massachusetts, designed a

Table 1.1 Milestones in the history of flow cytometry

2.2 Principles of Flow Cytometry

Box 2.1 Components and Basic Principles of Flow Cytometer

The primary component of the flow cytometer instruments include:

2.2.1 The Fluidics System

Box 2.2 Fluidics of the Flow Cytometer

Functions of the sheath fluid:

2.3 Optical System

Box 2.3 The Characteristics of Laser Light

Table 2.1 Laser sources and Laser source Wavelength (nm)

Advantages of argon laser: The advantages of argon laser includes:

Disadvantages of argon laser: It includes:

2.4 Fluorescence Emission

2.4.1 Collection of Light

Box 2.4 Optical System of Flow Cytometer

Collection of light: Light is collected by a set of special filters and optical

2.4.2 Optical Filters [2]

Fig. 2.5 Different types of optical filters are demonstrated

2.4.3 Electronics System

Fig. 2.6 Optical arrangements of the flow cytometer

Box 2.5 Electronics System of Flow Cytometer

Fig. 2.7 Events in the electronic system is summarized

Fig. 2.8 Schematic diagram of the electronics system of the photodetector

2.4.4 Computer System

2.4.5 Flow Cytometric Cell Sorting

Box 2.6 Flow Cytometric Cell Sorting

Fig. 2.10 Basic principle of cell sorting

3.2 Basic Requirements

Box 3.1 Basic Requirements for Flow Cytometry

3.3 Cytology Samples for Flow Cytometry

Factors influencing sample preparation: The following factors influence the

Box 3.2 Factors Influencing Sample Preparation

3.4 Sample Collection

Citrate buffer solution

3.5 Single-Cell Preparation

3.5.1 Limitations of the Enzymatic Method

Currently, many commercially available cocktails of enzymes are available in the

The cellular fixation is needed for the following reasons:

Table 3.1 Comparison of mechanical and enzymatic methods of single-cell preparation

Formaldehyde: The procedure of formaldehyde fixation

Chandigarh, India Pranab Dey

Part I Practical Aspects of Flow Cytometry

3.7 Permeabilization �� 27

7.5 Data Acquisition�� 81

Part II Diagnostic Applications of Flow Cytometry in Cytology

10 Detection of Lymphoma: Clonality Demonstration

12.6 Hepatosplenic T-Cell Lymphoma (HSTL)�� 149

1.2 History of Flow Cytometry

1.2.1 Early Motivation

1.2.2 Counting the Flowing Cells

1.2.3 Haematology Sample

1.2.4 Differential Count in the Blood Sample

1.2.5 Flow Sorter

1.3 Fluorescence Stain in the Flow Cytometer

1.4 Further Improvements in Flow Cytometry

2.2 Principles of Flow Cytometry

2.2.1 The Fluidics System

2.3 Optical System

2.4 Fluorescence Emission

2.4.1 Collection of Light

2.4.2 Optical Filters [2]

2.4.3 Electronics System

2.4.4 Computer System

2.4.5 Flow Cytometric Cell Sorting

3.2 Basic Requirements

3.3 Cytology Samples for Flow Cytometry

3.4 Sample Collection

3.5 Single-Cell Preparation

3.5.1 Limitations of the Enzymatic Method

3.8 RBC Lysing Solution

3.9.1 DNA Flow Cytometry [2]

• Propidium iodide (PI) 5 mg

1. RNAse A 2 mg

3.10 Data Acquisitions

4.1 Distribution of Fluorescence Intensity

4.2.1 The Crucial Gating in Flow Cytometry

5.2 Internal Quality Control

5.3 Instrument Quality Control

5.4 The Critical Factors to Have Good Quality FCM Data