Diagnostic Flow Cytometry in Cytology 9811626545, 9789811626548

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Diagnostic Flow Cytometry in Cytology Pranab Dey

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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 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

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 principle and techniques of flow cytometry, sample preparation, data acquisition, different 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 June 2021

Pranab Dey

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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 manuscript 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.

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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 2.1 Introduction����������������������������������������������������������������������������������������   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 3.1 Introduction����������������������������������������������������������������������������������������  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

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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 5.1 Introduction����������������������������������������������������������������������������������������  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

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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 9.1 Introduction����������������������������������������������������������������������������������������  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

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10 Detection of Lymphoma: Clonality Demonstration by Flow Cytometry������������������������������������������������������������������������������������ 109 10.1 Introduction�������������������������������������������������������������������������������������� 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 References���������������������������������������������������������������������������������������������������� 115 11 Flow Cytometry of B-Non Hodgkin Lymphoma������������������������������������ 117 11.1 Introduction�������������������������������������������������������������������������������������� 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 References���������������������������������������������������������������������������������������������������� 142 12 Flow Cytometry of Mature and Immature T-Cell Lymphoma�������������� 143 12.1 Introduction�������������������������������������������������������������������������������������� 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

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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 References���������������������������������������������������������������������������������������������������� 152 13 Flow Cytometry of Body Cavity Fluid ���������������������������������������������������� 153 13.1 Introduction�������������������������������������������������������������������������������������� 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 References���������������������������������������������������������������������������������������������������� 167 14 Flow Cytometry of Solid Tumours ���������������������������������������������������������� 169 14.1 Introduction�������������������������������������������������������������������������������������� 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 References���������������������������������������������������������������������������������������������������� 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 morphometry, 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

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

Abbreviations

Part I Practical Aspects of Flow Cytometry

1

Introduction and History of Flow Cytometry

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 membrane, cytoplasm or nuclei, DNA content of the cell, cellular organelles, RNA, and information on the protein. Over time, FCM is changed remarkably, and the following 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 scattered 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 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_1

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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 passing 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 modern 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 counting 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 eosinophils, esterase for monocytes and Alcian blue stain for basophils. The light scattering 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 recording 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 frequency of 72,000 cy/s). In this, higher vibration the sheath stream in the flow cytometer 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 fluorescence 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 350to 800-nanometre wavelengths.

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1  Introduction and History of Flow Cytometry

In the year 1965, Kamentsky et al. developed a microscope-based flow cytometer using the micro spectrophotometry [16, 17]. It was a two-parameter flow cytometer, 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 assessment 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 Moldvan [2] Gucker [1] of American Army (1940) Walter Coulter [5]

Kamentsky [13]

Dittrich [14] in Germany and Van Dillain [15] Los Alamos in (1969) Fulwyler [9]

Herzenberg [11]

Work The cells flowing through a capillary tube counted by the photoelectric method The detection of bacteria in the flowing air stream to make an instrument that can be used for the biowarfare at the second world war Counted blood cells flowing in a liquid suspension by measuring the drop of voltage when each cell passes through a static electrostatic filed They designed a two-parameter flow cytometer to measure the cell size and nucleic acid content of the cell by recording the emitted fluorescence A fluorescence dye-based flow cytometer to measure DNA content of the cells Electrostatic deflection ink-jet recording technique was used to isolate the charged droplet containing the desired cell and made the successful cell-sorting machine 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

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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 fluorochrome 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 wavelengths (Table 1.1).

References 1. Gucker FT Jr, Pickard HB, O'Konski CT. A photoelectric instrument for comparing the concentrations 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 fluorometry 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.

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1  Introduction and History of Flow Cytometry

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 fluorometry 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 cytophotometer system. J Histochem Cytochem. 1977;25(7):836–44.

2

Basic Principles and Instrumentation of Flow Cytometry

2.1

Introduction

Over the years, there are massive developments in flow cytometry regarding fluidics, optical system, data collection, etc. However, the basic principle of flow cytometry (FCM) is almost the same [1]. It measures the optical characteristic and the emitted fluorescence of the object/cell and procures valuable information. The technique 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 utilization 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.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_2

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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

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2  Basic Principles and Instrumentation of Flow Cytometry

a

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 pressure 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 amplification 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 characteristics 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.

14 Table 2.1  Laser sources and wavelengths of light

2  Basic Principles and Instrumentation of Flow Cytometry Laser source Argon Krypton Helium–neon Helium– cadmium UV

Wavelength (nm) 351–528,488 350–799 nm 543, 594, 612, 633 325–442 300–400

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 commonly used laser source of the flow cytometer is the argon laser source that generates 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 scatter (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 cytoplasmic 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 wavelength and emits fluorescence of a higher wavelength of light. The emitted fluorescence 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

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2  Basic Principles and Instrumentation of Flow Cytometry

collect the light. However, in the simultaneous use of lasers, special filters and mirrors 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 wavelength 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.

2.4 Fluorescence Emission

17

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 photon into electrons that produce an electric current. So basically, the photodetectors are “a light-driven current source”. The electric current is amplified in the photomultiplier, and a voltage pulse is produced. This voltage pulse is directly proportional 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 conversion (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 sensitivity 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 illustrates the mechanism of a photomultiplier tube.

18

2  Basic Principles and Instrumentation of Flow Cytometry

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 produces 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].

2.4 Fluorescence Emission

19

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 conversion, as mentioned before. Each event is given a channel number, and the numerical 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 interest (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 sorting 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 piezoelectric 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

20

2  Basic Principles and Instrumentation of Flow Cytometry

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 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 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 applications. 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.

3

Sample Preparation and Data Acquisition in Flow Cytometry

3.1

Introduction

Proper preparation of the sample for flow cytometry is a crucial part of the procedure. 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.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_3

23

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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 cytology 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 damaged 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 processing. 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

3.4

25

Sample Collection

The proper choice of buffered fluid is essential. If the cells are processed immediately for FCM, then simple phosphate-buffered saline (PBS) or citrate buffer solutions 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 immunophenotyping 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

26

3  Sample Preparation and Data Acquisition in Flow Cytometry

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 damage 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, collagenolytic, 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

Procedure cost

Mechanical Repeated rinsing the cell suspension through the syringe hub and nylon mesh Cheap

Supervision

Not much supervision needed

Suitability for immunophenotyping

Suitable

Features Methods

Enzymatic Enzymatic digestion Cost of the enzyme is relatively high Continuous supervision needed Not suitable

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 better access to nuclear DNA.

3.7

Permeabilization

The permeabilization of the cells may be required in the case of DNA content analysis and demonstration of intracellular enzymatic analysis such as terminal nucleotidase transferase (Tdt). The permeability reagents should increase the plasma membrane’s permeability and should retain the cellular antigen for the immunostaining. 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 unaffected 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 lysing agents. Table 3.2  Commercially available permeabilizing agents Company Beckman coulter BD bioscience Invirogen Dako

Product ItraPrep Permeabilizing reagent BD perm Fix and perm IntraStain

Permeabilizing agents Saponin and formaldehyde

Time 45 min

Saponin Not disclosed Reagent A: Formaldehyde for fixation Reagent B: Permeabilization

1 h 40 min 40 min

28

3  Sample Preparation and Data Acquisition in Flow Cytometry

Table 3.3  Commonly used lysing solutions in flow cytometry Company BD bioscience Beckman coulter

Product name FACS ™ lysing solution Immuno-­ prep®

Dako Sigma – Aldrich DAKO

Uti-lyse ™ Red blood cell lysing buffer EasyLyse ™

3.9

Lysing agent Diethylene glycol, formaldehyde

Fixation Yes

Time to keep 30 min

Formaldehyde, formic acid Three parts: Reagent A: It lyses the red blood cells. Reagent B: Stops lysing reagent C cell fixation. Formaldehyde Ammonium chloride, tris-HCL

Yes

2 min

Yes No

20 min

Ammonium chloride

No

Staining

3.9.1 DNA Flow Cytometry [2] Reagents Stock solution of propidium iodide (PI).  •   • 

Propidium iodide (PI) Double distilled water

5 mg 10 ml

Kindly store the stock solution in the dark at 4 °C. Solution A  1.   2.   3. 

RNAse A Triton X-100 Propidium iodide

2 mg 10 ml: 0. 1% (v/v) 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. • Discard the supernatant fluid. • 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 nonconjugated 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. • Discard the supernatant fluid. • Resuspend the cells in 250 μl PBS solution. • Run in FCM.

30

3  Sample Preparation and Data Acquisition in Flow Cytometry

b

50

SSC-A (x 1,000) 100 150 200 250

SSC-A (x 1,000) 50 100 150 200 250

a

1 010

102

103 104 PE-Cy7-A

105

1 010

104 102 103 CD45 APC-Cy7-A

105

Fig. 3.1 (a) Negative control in flow cytometry. No antibody is added. (b). Test sample that contains 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 antibody. 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 eliminates the possibility of the artefact. An isotype control should not be used for positive 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 addition 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 cytometer 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,

50

100

SSC-A 150

Fig. 3.3  Gating of the lymphoid cells were done based on forward, and side scatter

3  Sample Preparation and Data Acquisition in Flow Cytometry (x 1,000) 200 250

32

50

100

150 FSC-A

200

250 (x 1,000)

Fluorescent 2

Fig. 3.4 Bivariate histogram was made to have a distinct population of positive and negative fluorescence markers

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 particular 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 detecting 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 immunophenotyping 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 acquisition 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

34

3  Sample Preparation and Data Acquisition in Flow Cytometry

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 (approximately 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 mesothelial 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 lymphoma. 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.

4

Display and Interpretation of Data in Flow Cytometry

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 version 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 software 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 represented 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 © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_4

35

36

Count 500 750 1,000 1,250

Histogram

0

250

Fig. 4.1 Histogram showing the distribution of fluorescence intensity of the CD45 positive cells

4  Display and Interpretation of Data in Flow Cytometry

102

Fig. 4.2 Schematic diagram of DNA content

103 104 CD45 APC-Cy7-A

105

Count 500 400 300 200 100 0

100

200

300 DNA

400

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).

50

100

SSC-A 150

Fig. 4.3  Scatter plot histogram

37

(x 1,000) 200 250

4  Display and Interpretation of Data in Flow Cytometry

150 FSC-A

200 250 (x 1,000)

EPCam APC-A −103 0 103 104 −5,260

Fig. 4.4  Contour plot diagram

100

105

50

−474 −102 0 102

104 103 CD45 FITC-A

105

38

4  Display and Interpretation of Data in Flow Cytometry

104 −11,402

103

CD10 APC-A

105

Fig. 4.5  Density plot diagram

−798

103

0

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 positive 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

SSC-A (x 1,000) 50 100 150 200 250

400 300

200

100 0

A645-19-Tube_002

P2

50 100

200 300 Fluorescence intensity

400

100 150 200 250 FSC-A (x 1,000)

Fig. 4.7  The graph displayed in linear scale

Logarithmic scale: Here, the fluorescence intensity is distributed in the logarithmic 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 logarithm. (Fig. 4.9).

40

4  Display and Interpretation of Data in Flow Cytometry

Count 500 A645-19-Tube_002 SSC-A (x 1,000) 100 150 200 250

400 300

200

0

P3

50

100

101

102 103 Fluorescence

102 103 104 105 CD45 PerCP-Cy5-5-A

104

Fig. 4.8  only one parameter is displayed as a logarithmic graph Count 105 Lymph 105

104

CD23 PE-A 103 104

103

−205 0102

102 101

−100 0 102

0

101

102 103 104 Fluorescence intensity

103 104 CD5 FITC-A

105

Fig. 4.9  Biexponential two log parameters

Logicle: Many investigators prefer the “logicle” to display the data. It is a biexponential 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

−275 −102

0

102

Lombdo PE-A 103

104

105

A-1395/2020-Tube_003

−085 101

−102

0

102

100 Koppo FITC-A

102

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).

42

4  Display and Interpretation of Data in Flow Cytometry

Fig. 4.11  Two parameter histogram displaying the positivity of the markers according to the fluorescence intensity

105

A5531/19-Tube_002

CD23+

CD5+ and CD23+

CD23 PE-A 104 103

Fig. 4.12  The graph displays the positivity of the two markers in different quadrant

CD5+

−278

0

Both −

−162

4.2

0

102

103 104 CD5 FITC-A

105

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,

Area

Area: Total integral pulse

FSC-H

Voltage

Height

(x 1,000)

b

50

Width: Time interval between two pulses

100 150 200 250

a

Time

c

Singlet

50

100 150 FSC-A

200 250 (x 1,000)

d FSC-H (x 1,000)

Voltage

Time Doublet

50 100 150 200 250

Single cell gating

50

100

150

FSC-A

200

250

(x 1,000)

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

44

4  Display and Interpretation of Data in Flow Cytometry

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 followed by CD19 population was gated, and in that population, CD5 and CD23 population of cells were gated for analysis.

(x 1,000) 200 250

Scatter plot

50

100

SSC-A 150

Fig. 4.14  The debris is eliminated by gating in FSC-A and SSC-A. The debris will have low FSC-A and high SSC-A

50

100

150 FSC-A

a

b

200

250 (x 1,000)

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

b

103

−1,342 0

104

105

−1,929

50

CD30 PE-A 104 0 103

105

SSC-A (x 1,000) 100 150 200 250

a

−8,833

PE-Cy7-A

104

0

105

CD1a APC-A

d

−1,929

−8,833

50

0

CD30 PE-A 104 103

105

SSC-A (x 1,000) 100 150 200 250

c

0

104

105

−1,342 0

CD1a APC-A

103

104

105

PE-Cy7-A

Fig. 4.16  Backgating: (a). CD3 population, (b). CD30 population, (c). Gated CD30 is highlighted, (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.

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4  Display and Interpretation of Data in Flow Cytometry

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 situations 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 eliminate the debris and non-cellular elements. • Sequential gating: Sequential cell population is selected to assess the subset 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.

5

Quality Control in Flow Cytometry

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, fluorochrome 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 © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_5

47

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 lymphoid cells. Brando et al. have shown that excessive vortexing may also be responsible 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 population. The polyclonal antibody may bind with other than the target antigen containing 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 antibodies 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 fluorochrome 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 residual 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 fluorescence 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 indicates 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.

50

5  Quality Control in Flow Cytometry

Negative population

Positive population

High resolution

Count

Count

Low resolution

FITC

FITC

Count

Count

Fig. 5.1  Schematic diagram showing how the good resolution can separate two populations of cells

Low optical background

FITC

High optical background

FITC Negative Dim population population

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 electronics. 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 background and electronic noise (Fig. 5.3). The higher the stain index better the resolution. 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 optimal resolution and discriminating the different population of cells in the experiment. 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).

5  Quality Control in Flow Cytometry

Count

52

SD

Fluorescence intensity MFI of positive population

MFI of negative population MFI of positive population - MFI of the negative population

Stain index

2 x SD of negative population

MFI means mean fluorescence intensity, SD means standard deviation

Fig. 5.3  Schematic diagram explaining stain index

Unstained cells

104

104 PMO voltage adjustment

103

103

102

102

101

101

100

101

102

103

104

100

Fluorescence intensity

Fig. 5.4  The adjustment of the voltage of the photomultiplier tube

Unstained cells

101

102

103

Fluorescence intensity

104

5.5 PMT Voltage Setting

53

Fig. 5.5  The setting of PMT voltage and CV

104

103 Optimum voltage

CV 102

101

100

200

300

400

500

PMT voltage

525/550 BP FITC

1.0 Emission

Fig. 5.6 Schematic diagram showing the overlapping emission spectra of flurochrome dyes

585/640 BP PE

0.8

Spectral overlap

0.6 0.4 0.2 400

500 600 Wavelength (nm)

700

5.5.1 Compensation The fluorochrome in the flow cytometer emits the photons when it is hit by an excitation 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 population of cells. The correction of the fluorescence spillover in the multi-coloured

54

5  Quality Control in Flow Cytometry

flow cytometry is known as compensation (Box 5.4). For the compensation to calculate, we need a series of beads or samples of cells that are stained with single fluorescent dyes. The compensation should be calculated after the PMT voltage set up. For compensation to calculate, the background fluorescence of positive and negative 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 instrument. 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

5.7

55

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 laboratories. 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 neoplasia 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 immunophenotyping 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 interleukin 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..

6

Fluorescent Probes and Different Useful Markers for Flow Cytometry

For the successful application of flow cytometry, it is essential to know the fluorescence 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 fluorochromes 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 isothiocyanate (FITC), DAPI, etc. Events in fluorescence: The Jablonski diagram explains the events of fluorescence (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 fluorochrome 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 temporary phenomenon and takes only in femtoseconds. The fluorochrome molecule © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_6

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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 emission. 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 conjugated with the antibodies.

60

6  Fluorescent Probes and Different Useful Markers for Flow Cytometry

(b) Some fluorochrome dyes have an affinity for specific substances and accumulate 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 coefficient represents how intensely a chemical absorbs light of a specific wavelength. 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 number 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 fluorochrome dye is always less than one because of internal conversion and quenching. Fluorescence quantum yield =

Number of emitted photons 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 fluorescence 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 different dyes. It is known as spectral overlap. The appropriate filter is needed to eliminate 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, therefore mandatory to correct the spectral overlap. Moreover, in the case of multicoloured flow cytometry, the selected dye should be used to reduce spectral overlapping. 4. Fluorescence resonance energy transfer (FRET): At times, the two fluorochrome 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

6.1  Staining by the Fluorochrome Dye

61

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 fluorochrome 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.

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6  Fluorescent Probes and Different Useful Markers for Flow Cytometry

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 antibodies 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 antibody 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 acceptor 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 excitation of the dye is known as absorption maximum. –– Emission maximum: The peak wavelength of emission.

6.1  Staining by the Fluorochrome Dye

(d) Fluorescence resonance energy transfer (FRET): The two fluorochrome dye is closely located and the donor dye is excited at lower wavelength 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 transfer 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

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6  Fluorescent Probes and Different Useful Markers for Flow Cytometry

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 emitted 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 molecules 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).

6.1  Staining by the Fluorochrome Dye

65

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 moderate 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 emission spectra are 545 and 565, respectively. The PE labelled antibodies are also popular 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 limitation 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 quantum 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 Fluorescein Isothiocyanate (FITC) Phycoerythrin (PE) Allophycocyanin (APC) Rhodamine red-X Texas red® Peridinin chlorophyll (PerCP)

Excitation maximum (nm) 495 496 650 570 595 477

Emission maximum (nm) 519 576 660 590 613 678

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6  Fluorescent Probes and Different Useful Markers for Flow Cytometry

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 commonly 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 combination 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 PE-Cy5.5 PE-Cyanine7 PE-Texas red PE-Alexa Fluor 700 PE-Alexa Fluor 750 APC-cy 5.5 APC-H7 APC-fire 750 PerCP-cyanine 5.5

Excitation maximum (nm) 496, 565 565 565 496, 546 496, 546 650 650 650 482

Emission maximum (nm) 695 785 615 723 779 695 780 787 695

6.1  Staining by the Fluorochrome Dye

67

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 multicoloured 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 symmetrical, 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,

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6  Fluorescent Probes and Different Useful Markers for Flow Cytometry

a

b

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.

6.1  Staining by the Fluorochrome Dye

69

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 cytometry. 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.

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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 violet 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 generates 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 fluorochrome dye detected by the detector channel of each laser helps to select the appropriate 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 fluorochrome 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 demonstrate these antigens.

6.1  Staining by the Fluorochrome Dye Table 6.3  Laser line, wavelength and fluorochrome dye used Laser line Violet Blue Red Yellow-­ green

Excitation Wavelength (nm) 405, 413 and 425 488 641 561

Fluorochrome dyes Brilliant violet 605, brilliant violet 421, Pacific blue, Alexa-flour 430, QD545, QD 655 FITC, PerCP, PE-Cy7, PErCP-Cy5.5, PI, GFP, YFP APC, Cy5, APC-Cy7, APC-H7, Alexa Fluor 647 PE,PE-Texas red, PE-Cy5, PE-Cy7

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, wavelength 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.

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Table 6.4  Stain index of different fluorochrome dye

Brightness Fluorochrome High APC PE PE-CY5 Alexa-flour 488 Medium FITC PE-Texas red Q dot 605 Low Q dot 655 Q dot 705 APC-Cy7 PERCP

Stain index 193 232 216 91

51 40 35 20 18 24 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 different 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 detector 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

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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 presence 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 proteins 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 biology, Academic Press, vol. 102. NY: New York; 2011. p. 375–409.

7

Nuclei Acid Dye and DNA Content Measurement in Flow Cytometry

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 characteristics, 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 noncovalent 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.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_7

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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 membrane 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

7.2

77

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 intercalate 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/emission 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 preferable 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 emission spectra of dye are 350/461 nm. Table 7.1 The comparison of PI and EB

Features Charge Dye binding RNA stain Cell permeability Excitation spectrum Emission spectrum Cell fixation

PI Double charge Intercalated Yes Fast 488 and 532 nm 617 nm Ethanol fixation

EB Single charge Intercalated Yes Slow 518 nm 605 nm Ethanol fixation

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7  Nuclei Acid Dye and DNA Content Measurement in Flow Cytometry

Table 7.2  shows the basic information of different commonly used fluorescent DNA dye Dye PI EB AO DAPI Hoechst 33342 Mithramycin TOTO

Mode of binding Intercalated Intercalated Intercalated Minor groove Minor groove

DNA/RNA stain Both Both Both DNA DNA

Cell permeability No No No Little Yes

Sequence specificity No No No A-T A-T

Excitation (nm) 488,532 518 460/650 358 350

Emission (nm) 617 605 502/536 452 461

Minor groove Bis-­ intercalator

DNA Both

No No

G-C No

441 514

575 533

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/ emission 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 variable 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

a

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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 contain 4n DNA. (d) M-phase: In the mitotic phase, the cell divides into two daughter cells containing 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.

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7  Nuclei Acid Dye and DNA Content Measurement in Flow Cytometry

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 subpopulation 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 collected in the phosphate buffer solution. The body fluid should be collected along with an anticoagulant to prevent coagulation. Heparin, EDTA, or ammonium oxalate 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 preferable 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 concentration 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 following 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

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be difficult to ascertain the diploid or aneuploidy peak. Normal healthy lymphocytes 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. • Discard the supernatant fluid. • 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.

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• 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 diploid 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 diploid 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 fraction. 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

8

Classification of Lymphoma, Different Markers and Approach

Abbreviations ALCL BL DLBCL FL LPL MCL MZL SLL

Anaplastic large cell lymphoma Burkitt lymphoma Diffuse large B-cell lymphoma Follicular lymphoma Lymphoplasmacytic lymphoma Mantle cell lymphoma Marginal zone lymphoma 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 predominantly on: 1 . Individual cell morphology. 2. Immunophenotype. 3. Molecular cytogenetics. 4. Besides, the clinical features should also be considered for the successful classification 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. © The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_8

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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

8.1

87

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 immunoglobulin 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), lymphoplasmacytic 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 lymphoma (DLBCL), Burkitt lymphoma (BL), grade III follicular lymphoma, plasmablastic lymphoma.

Fig. 8.3  Ontogenesis of B cell

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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 receptors: alpha-beta and gamma-delta T cells. Unlike T cells, the CD3 surface expression 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

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In comparison to B-cell NHL, T-cell NHL is a more heterogeneous group of lymphomas. T-lymphoblastic lymphoma develops from cortical and medullary thymocytes. In contrast, mature T-lymphoma develops from the follicular T-helper cells. T-NHL comprises only 15% of all NHL. The clinical presentation, immunophenotype, 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 lymphomas. 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 expression 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.

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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 depletion 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

• • • • • •

8.4

Easy technique Rapid procedure Cheaper than biopsy Well tolerated by the patient Multiple sampling can be done Better cytomorphology of the smear

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

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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 aspiration 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

• • • •

8.5

Predominantly necrotic tissue. Scarred or fibrosed lymph node. Blood mixed diluted material. Scattered atypical cells: Hodgkin lymphoma.

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 solution (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.

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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 lymphomas 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 lymphoma 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

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250 200 150 100

FSC-H

Single cells are gated

50

Fig. 8.7 Single-cell population is gated by using FSC-A versus FSC-H

(x 1,000)

Fig. 8.6  Cytology smear and probable types of lymphoma

50

100

150 FSC-A

200

250 (x 1,000)

250 200 150 50

100

SSC-A

Fig. 8.8  Scatter diagram of FSC-A versus SSC-A to get the location of lymphoid cells

8  Classification of Lymphoma, Different Markers and Approach (x 1,000)

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Lymphoid cells 50

100

150

200

250

150

200

250

(x 1,000)

100

SSC-A

Fig. 8.9  Scatter diagram of CD45 versus SSC-A, showing the distinct population of CD45 positive lymphoid cells for analysis

(x 1,000)

FSC-A

50

CD45 positive 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

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Table 8.1  Flow cytometry panel for lymphoma subtyping Tube number B1 B2 B3 B4 B5 B6 T1 T2 T3

FITC CD5 Kappa HLA-DR FMC 7 IgM CD 38 CD 8 CD 2 TCαβ

Additional tubes A1 A2 CD 16 A3 CD 64 A4 cTdT A5

PE CD 23 Lambda CD 20 CD 4 IgD CD 200 CD 4

APC CD 43 CD 10 CD 38 CD 8

Per Cpcy5.5

CD 10 CD 7 CD 1a

CD 34 CD 5 CD 5

CD 34 CD 3

PECy 7 CD 19 CD 19 CD 19 CD 19 CD 19 CD 19 CD 3 CD 3

APC H7 CD 45 CD 45 CD 45 CD 45 CD 45 CD 45 CD 45 CD 45 CD 45

CD 117 CD 3

CD 45 CD 45 CD 45 CD 45 CD 45

TCʏð CD 13 CD 14 Ccd3

CD 33 CD 56 CD 11c C79a Ccd22

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 cytology. 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 suggestions. 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 immunphenotyping for diagnosis and classification of non Hodgkin lymphoma. Cytojournal. 2006; 3: 24. doi: https://doi.org/10.1186/1742-­6413-­3-­24.

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Markers for Immunophenotyping in Flow Cytometry

Abbreviations AITL ALCL AML ATLL EATL LGL MF PLL PTCL SS

Angioimmunoblastic T cell lymphoma Anaplastic large cell lymphoma Acute myeloid leukaemia Adult T-cell leukaemia/ lymphoma Enteropathy associated T-cell lymphoma Large granular lymphocytic leukaemia Mycosis fungoides Prolymphocytic leukaemia Peripheral T cell lymphoma Sezary syndrome

9.1

Introduction

The various markers are used in flow cytometry for immunophenotyping of lymphomas. 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.

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_9

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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 specific marker of T cells.

9.2.2.1 Diagnostic Applications • 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.

9.2.3.1 Diagnostic Applications • 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.

9.2.4.1 Diagnostic Applications • PTCL • T-PLL • ALCL

9.2  CD Markers

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• 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].

9.2.5.1 Diagnostic Applications • 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.

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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.

9.2.6.1 Diagnostic Applications • 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 immature B and T cells and B cells present in germinal centres and polymorphs.

9.2.7.1 Diagnostic Applications • 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.

9.2.8.1 Diagnostic Applications • T-LGL. • Some cases of AML.

9.2.9 CD14 CD14 is mainly seen in monocytes.

9.2.9.1 Diagnostic Applications • Chronic myelomonocytic leukaemia. • Acute myelomonocytic leukaemia.

9.2  CD Markers

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9.2.10 CD15 CD15 is expressed in neutrophils, promyelocytes and monocytes.

9.2.10.1 Diagnostic Applications • 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.

9.2.11.1 Diagnostic Applications • 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 throughout the B cell differentiation and disappears in plasma cells.

9.2.12.1 Diagnostic Applications • 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.

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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 interpretation of CD20 positive cells may be difficult.

9.2.13 CD23 CD23 is a membrane glycoprotein and is present in mature B lymphocytes, activated histiocytes and follicular dendritic cells. It is an IgE receptor that controls IgE response.

9.2.13.1 Diagnostic Applications • 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.

9.2.14.1 Diagnostic Applications • 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.

9.2.15.1 Diagnostic Applications • ALCL. • Hodgkin lymphoma: R-S cells are positive for CD30.

9.2  CD Markers

• • • • • •

103

A subset of DLBCL. Primary effusion lymphoma. Primary Mediastinal large B cell lymphoma. ATCL. NK cell neoplasms. Non-lymphomatous tumours: Embryonal carcinoma, nasopharyngeal carcinoma, 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.

9.2.16.1 Diagnostic Applications • Plasma cell tumours. • Plasmablastic lymphoma. • Pre-T ALL. • Primary effusion lymphoma. • 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.

9.2.17.1 Diagnostic Applications • 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.

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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.

9.2.18.1 Diagnostic Applications • 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.

9.2.19.1 Diagnostic Applications • 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, pheochromocytoma, 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

9.2.20.1 Diagnostic Applications • 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.

9.2.22.1 Diagnostic Applications • 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.

9.2.23.1 Diagnostic Applications • 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).

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9.3

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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.

9.3.1.1 Diagnostic Applications • 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.

9.3.2.1 Diagnostic Applications • 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.

9.3.3.1 Diagnostic Applications • 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

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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 outcome in B-cell chronic lymphocytic leukemia. Leuk Lymphoma. 2001;42(1–2):109–14.

Detection of Lymphoma: Clonality Demonstration by Flow Cytometry

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10.1 Introduction One of the main applications of flow cytometry in diagnostic cytology is the differentiation 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 lymphomas, 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) followed by diffuse large B cell lymphomas (DLBCL).

10.2.2 Aberrant Expression of Certain Antigen The following features suggest

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 P. Dey, Diagnostic Flow Cytometry in Cytology, https://doi.org/10.1007/978-981-16-2655-5_10

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Fig. 10.1 Predominantly Lambda chain expression is present indicating light chain restriction and monoclonal B cell population.

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10  Detection of Lymphoma: Clonality Demonstration by Flow Cytometry

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Fig. 10.2  CD38 positive cell with no CD20 expression in plasma cell tumour

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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

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Fig. 10.3  CD34 positive cells indicate immature cells

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• 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

10  Detection of Lymphoma: Clonality Demonstration by Flow Cytometry

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Fig. 10.5 CD10 expression in B cell lymphoma is seen in immature cells of Burkitt lymphoma

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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 subset 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, indicating the large size of these cells. The lack of Surface Ig was defined by deMartini

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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 antigens (7%) and rarely all four T-cell antigens are affected (