Immunophenotyping for Haematologists. Barbara J. Bain

to special, incidental, consequential, or other damages.

       Library of Congress Cataloging‐in‐Publication Data

      Names: Bain, Barbara J., author. | Leach, Mike, (Haematologist), author.

      Title: Immunophenotyping for haematologists : principles and practice / Barbara J. Bain, Mike Leach.

      Other titles: Immunophenotyping for hematologists

      Description: Hoboken, NJ : Wiley‐Blackwell, 2020. | Includes bibliographical references and index.

      Identifiers: LCCN 2020021078 (print) | LCCN 2020021079 (ebook) | ISBN 9781119606116 (hardback) | ISBN 9781119606147 (adobe pdf) | ISBN 9781119606154 (epub)

      Subjects: MESH: Immunophenotyping | Hematologic Tests

      Classification: LCC QR187.I486 (print) | LCC QR187.I486 (ebook) | NLM QW 525.5.I36 | DDC 616.07/582–dc23

      LC record available at https://lccn.loc.gov/2020021078 LC ebook record available at https://lccn.loc.gov/2020021079

      Cover Design: Wiley

      Cover Images: (background) © KTSDESIGN/SCIENCE PHOTO LIBRARY/ Getty Images, (inset) courtesy of Mike Leach

Preface

      The increasing centralisation of specialised tests and the divorce of clinical from laboratory haematology in many countries means that many haematologists now have no direct contact with an immunophenotyping laboratory. Despite this, the results from the laboratory are often crucial in the management of their patients. This book is intended to help haematologists and trainees understand and interpret immunophenotyping results. It is not directed at those working in an immunophenotyping laboratory and technical details are therefore outlined only briefly. Such laboratories may, however, find it a useful source of information. For further reading on the subject, see the bibliography of each chapter.

      Barbara J. Bain and Mike Leach

Acknowledgement

      We should like to thank Allyson Doig, Senior Biomedical Scientist, Gartnavel Hospital, for assistance with the flow cytometry plots in Part 4.

Abbreviations Used in the Book

κkappa (light chain)λlambda (light chain)ALCLanaplastic large cell lymphomaALLacute lymphoblastic leukaemiaAMLacute myeloid leukaemiaAMoLacute monoblastic/monocytic leukaemiaAPCallophycocyanineAPLacute promyelocytic leukaemiaASTaspartate transaminaseATLLadult T‐cell leukaemia lymphomaccytoplasmicCAR T cellschimaeric antigen receptor T cellsCDcluster of differentiationCLLchronic lymphocytic leukaemiaCMLchronic myeloid leukaemiaCMMLchronic myelomonocytic leukaemiaCSFcerebrospinal fluidCTcomputed tomographyDLBCLdiffuse large B‐cell lymphomaDNAdeoxyribonucleic acidEBNAEpstein–Barr virus nuclear antigenEBVEpstein–Barr virusEMAepithelial membrane antigenETP‐ALLearly T‐cell precursor acute lymphoblastic leukaemiaFBCfull blood countFISHfluorescence in situ hybridisationFITCfluorescein isothiocyanateFLAERfluorescent aerolysinFSCforward scatter (of light)G‐CSFgranulocyte colony‐stimulating factorGPIglycosylphosphatidylinositolHbhaemoglobin concentrationHHVhuman herpesvirusHIVhuman immunodeficiency virusHLAhuman leucocyte antigenHLHhaemophagocytic lymphohistiocytosisHTLV‐1human lymphotropic virus 1IgimmunoglobulinILinterleukinLDHlactate dehydrogenaseLGLLlarge granular lymphocytic leukaemiaLMPlatent membrane proteinLLlymphoblastic lymphomaMALTmucosa‐associated lymphoid tissueMDSmyelodysplastic syndromeMDS/MPNmyelodysplastic/myeloproliferative neoplasmMoAbmonoclonal antibodyMPALmixed phenotype acute leukaemiaMPNmyeloproliferative neoplasmMPOmyeloperoxidaseMRDminimal residual diseaseNHLnon‐Hodgkin lymphomaNKnatural killerNRnormal rangePEphycoerythrinPerCPperidinin chlorophyllPLLprolymphocytic leukaemiaPNHparoxysmal nocturnal haemoglobinuriaRNAribonucleic acidSmsurface membraneSSCside or sideways scatter (of light)TCRT‐cell receptorTdTterminal deoxynucleotidyl transferaseULNupper limit of normalWBCwhite blood cell countWHOWorld Health Organization

Part 1 Purpose and Principles of Immunophenotyping

      CONTENTS

        Flow Cytometric Immunophenotyping

        Immunohistochemistry

        Interpretation and Limitations of Flow Cytometric Immunophenotyping

        Problems and Pitfalls

        References

        Bibliography

      Immunophenotyping is the process by which the pattern of expression of antigens by a population of cells is determined. The presence of a specific antigen is recognised by its binding to a labelled antibody. Antibodies can be present in a polyclonal antiserum that is raised in an animal but more often they are well characterised monoclonal antibodies produced by hybridoma technology; a hybridoma is a clone of cells created by the fusion of an antibody‐producing cell with a mouse myeloma cell. Monoclonal antibodies can be labelled with an enzyme or with a chemical, known as a fluorochrome, that under certain circumstances will fluoresce. Immunophenotyping is carried out primarily by flow cytometry or immunohistochemistry. Flow cytometric immunophenotyping is applicable to cells in peripheral blood, bone marrow, body fluids (pleural, pericardial, ascitic and cerebrospinal fluids) and fine needle aspirates. Immunohistochemistry of relevance to haematological disease is applied particularly to trephine biopsy and lymph node biopsy specimens, but also to biopsy specimens from any other tissues where infiltration by haemopoietic or lymphoid cells is suspected.

Flow Cytometric Immunophenotyping

This technique determines cell size, structure (to some extent) and antigen expression. Cells in suspension are first exposed to a combination of fluorochrome‐labelled monoclonal antibodies (or other lectins or ligands) and then pass in a focused stream through a beam of light generated by a laser. Laser‐generated light is coherent (waves of light are parallel) and monochromatic (single wave length/colour). Large multichannel instruments with multiple lasers are used to identify, count, size and otherwise characterise cells that are hydrodynamically focused and pass in a single file through a narrow orifice in a flow cell. The passing of the cell through a light beam leads to both the scattering of light and the excitation of fluorochromes so that they emit a fluorescence signal. Forward scatter (FSC) of light at a narrow angle is detected and measured and is proportional to cell size. Sideways or side scatter (SSC) of light is detected and measured and is proportional to cell granularity and complexity. Antigens expressed on the surface membrane of cells or, with modified techniques, within cells are detected. After ‘permeabilisation’, both cytoplasmic and nuclear antigens can be detected.

Figure 1.1 Diagrammatic representation of the principles of flow cytometric immunophenotyping.

For each fluorochrome, a selected laser emits light of a specified wavelength that will be absorbed by the fluorochrome. This leads to excitation of the fluorochrome with subsequent emission of light of lower energy and a longer wavelength as the fluorochrome returns