Bone Marrow IHC

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DedicationI dedicate this book to my husband Goran and to my children Lino, Jakov, Marija Milena, and Marta Danica—Emina Emilia Torlakovic, MD, PhD

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AffiliationsEmina Emilia Torlakovic, MD, PhDAssociate ProfessorDepartment of Pathology and Laboratory Medicine Royal University Hospital University of SaskatchewanSaskatoon, SK Canada

Professor Kikkeri Naresh, MBBS, CCP, MD, FRCPathConsultant HaematopathologistDepartment of HistopathologyHammersmith HospitalLondonUK

Richard D Brunning, MDProfessor EmeritusDepartment of Laboratory Medicine and Pathology University of Minnesota Minneapolis, MN USA

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Bone Marrow IHC

Chicago

Emina Emilia Torlakovic, MD, PhD

Kikkeri N Naresh, MBBS, CCP, MD, FRCPath

Richard D Brunning, MD

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Publishing TeamErik Tanck (design and production)Joshua Weikersheimer (publishing direction)

NoticeTrade names for equipment and supplies described herein are included as suggestions only. In no way does their inclusion constitute an endorsement or preference by the American Society for Clinical Pathology. The ASCP did not test the equipment, supplies, or procedures and there-fore urges all readers to read and follow all manufacturers’ instructions and package insert warnings concerning the proper and safe use of products.

Copyright © 2009 by the American Society for Clinical Pathology.All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the publisher.

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Printed in Hong Kong

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Abbreviations

Ab – antibodyAg – antigenALL – acute lymphoblastic leukemiaALPS – autoimmune lymphoproliferative syndromeAMF – acute myelofibrosis AMKL – acute megakaryoblastic leukemiaAML – acute myeloid leukemiaAPMF – acute panmyelosis with myelofibrosisB-ALL – B-cell acute lymphoblastic leukemiaBM – bone marrowcHL – classical Hodgkin lymphomaCLL – chronic lymphocytic leukemiaCLL/SLL – chronic lymphocytic leukemia/small lymphocytic lymphomaCML – chronic myeloid leukemiaCMML – chronic myelomonocytic leukemiaDLBCL – diffuse large B-cell lymphomaDS – Down syndromeEBV – Epstein-Bar virusEGIL – European Group for the Immunologic Classification of Leukemia ET – essential thrombocythemia FISH – fluorescent in situ hybridizationFL – follicular lymphomaHCL – hairy cell leukemiaHIER – heat-induced epitope retrievalIHC – immunohistochemistryISH – in situ hybridizationLF – lactoferrinLPL – lymphoplasmacytic lymphomaLZ – lysozymeMCD – mast cell diseaseMCL – mantle cell lymphomaMCT – mast cell tryptaseMDS – myelodysplastic syndromeMGUS – monoclonal gammopathy of undetermined significanceMPD – myeloproliferative diseaseMPO – myeloperoxidaseMZL – marginal zone lymphoma OMIM – Online Mendelian Inheritance in ManPROW – Protein Reviews on the Web (http://mpr.nci.nih.gov/PROW/)PTCL – peripheral T-cell lymphomaRA – refractory anemiaRAEB – refractory anemia with excess blastsRARS – refractory anemia with ringed sideroblastsRCMD – refractory anemia with multilineage dysplasiaT-ALL – T-cell acute lymphoblastic leukemia

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TABLE OF CONTENTS

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiv

Chapter 1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2

Chapter 2

The Role of Tissue Processing In Bone Marrow Immunohistochemistry . . .3

2.1 BM Biopsy Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2 Decalcification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.3 Embedding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.4 Tissue Sectioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.5 Antigen Retrieval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.6 Turnaround Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.7 Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Protocol for Bone Marrow Processing and Staining Used at Hammersmith Hospital . . . . . . . . . . . . 11

Protocol for Bone Marrow Processing and Staining Suitable for Rapid Turnaround Time . . . . . . . . 11

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

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Chapter 3

Cluster of Differentiation Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.1 CD1a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3.2 CD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

3.3 CD3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.4 CD4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3.5 CD5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

3.6 CD7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.7 CD8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.8 CD9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

3.9 CD10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3.10 CD14 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

3.11 CD15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.12 CD16 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.13 CD20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3.14 CD21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

3.15 CD22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

3.16 CD23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

3.17 CD25 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

3.18 CD30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

3.19 CD31 (PECAM). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

3.20 CD33 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

3.21 CD34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

3.22 CD35 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

3.23 CD45 (CD45RA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.24 CD45 (CD45RO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

3.25 CD56 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

3.26 CD57 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

3.27 CD61 and CD42b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

3.28 CD68 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

3.29 CD79a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

3.30 CD99 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

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3.31 CD117 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

3.32 CD123 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

3.33 CD138 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

3.34 CD235a (Glycophorin A) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Chapter 4

Other Common Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

4.1 Myeloperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

4.2 Hemoglobin A (HgbA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

4.3 Mast Cell Tryptase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

4.4 Terminal Deoxynucleotidyl transferase (TdT). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

4.5 Factor 8-Related Antigen (von Willebrand Factor) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

4.6 Pax-5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

4.7 PU.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

4.8 Fli-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

4.9 Cyclin D1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

4.10 Lysozyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

4.11 Bcl-6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

4.12 Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

4.13 Ki-67 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

4.14 Cytotoxic Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

4.15 Stromal Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

4.16 MUM1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

4.17 TRAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

4.18 Bcl-2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

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Chapter 5

Special Diagnostic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

5.1 Acute Leukemia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

5.2 Metastatic Tumors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

5.3 Recommended Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

Table of Contents

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Bone Marrow IHC is intended to provide a collection of illus-trations of various immunohistochemical tests that are rou-tinely used in diagnostic practice. The choice of tests and the

frequency of use vary greatly depending not only on the type of prac-tice and personal experience, but also on availability of the tests. The menus of primary antibodies that can be applied to bone marrrow (BM) biopsy are logarithmically growing, and the number of pub-lished articles describing newly available tests is following the trend. Despite the advance of flow cytometry and molecular methods, immunohistochemistry remains an integral part of the diagnostic evaluation of most BM biopsies and is not likely to become less impor-tant in the future. The intrinsic value of the in situ tests will be dif-ficult to replace by any other type of evaluation. This work does not include descriptions and possible uses of all currently available mark-ers, but results of the most common markers are depicted in detail. Some not so commonly used markers are also portrayed to illustrate results that can be expected and help in their interpretation, which may be difficult if the range of expected outcomes is unfamiliar. The number of rare diseases and unusual findings may be overrepresented because of the greater need for immunohistochemistry studies in such cases. Also, some representative cases were used to illustrate several different markers solely for practical reasons. The comments associ-ated with the findings and their interpretation are, however, based on the literature and wider experience and were not deduced from such individual samples except where it is explicitly stated to be the case.

Bone Marrow IHC does not promote or recommend use of any particular test, testing method, protocol, primary antibody, or other material and methods, but rather focuses on the results with optimized protocols. It cannot be overemphasised that the path to

optimization and standardization of any test starts with agreed on expected/optimal results. The great majority of images in this book are, in our opinion, examples of what results may be expected with optimal methods, with the assumption that any method and/or reagent that can produce such results is acceptable. Some subopti-mal results are also illustrated to depict certain common problems. Most BM biopsies that are illustrated here were fixed in 10% buff-ered formalin, aceto-zinc-formalin fixative, or B5, and most were decalcified by rapid decalcifier or Gooding and Stewart’s decalci-fication fluid (10% formic acid and 5% formaldehyde). All but a few tests used the EnVision+ detection system simply because this is what is currently used, but with the clear acknowledgment that many other detection systems could produce the same if not better results. When any other method is used for detection, it is stated in the figure legend. The great majority of antibody clones or polyclonal antibodies that are illustrated here are listed and were evaluated by NordiQC (www.nordiqc.org). The names “PROW,” “Entrez Gene,” and “OMIM” are used throughout the text to designate the fol-lowing Web sites as a source of information: http://mpr.nci.nih.gov/PROW, http://www.ncbi.nlm.nih.gov/sites/entrez, and http://www.ncbi.nlm.nih.gov/omim, respectively.

A number of internet sites serve those who study bone marrow protein expression by immunohistochemistry. Search at http://www.proteinatlas.org offers IHC images of relatively high quality showing various proteins that can be evaluated in TMA samples. Hematopoietic tissue markers are searchable at http://www.tissuemarkers.org.uk/htm/search.php. It includes many good images, but not all markers are illustrated, and most do not show bone marrow expression patterns.

PREFACE

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We are indebted to many colleagues and collabora-tors who made this book possible. Professor Dr. Jan Nesland, Head of the Department of Pathology, The

National Hospital–The Norwegian Radium Hospital, University of Oslo, Norway, was generous in his support and professional mentorship that created a platform for learning and experience in diagnostic immunohistochemistry as well as reasearch appli-cations. About one third of the images included in the Atlas are from the cases E. Torlakovic evaluated while working at the Norwegian Radium Hospital. We are aslo indebted to labora-tory technologists Grette Mykkelbost, Eva Gustavson, and Don Trihn, whose dedication to patient care, technical expertise, hard work, and perfectionism have facilitated the application of various new techniques and antibodies to bone marrow biopsies at the Norwegian Radium Hospital. In particular, gratitude is due to Grette Mykklebost, a collaborator and a friend, who was eager to quickly adopt not-so-routine methods like double and

triple immunohistochemistry applications and any combination of immunohistochemistry and in situ hybridization and whose commitment to excellence in all things is unprecedented. E. Torlakovic currently works with Shannon Klassen and her team whose working motto could be “nothing is impossible in immu-nohistochemistry” and who is able to accomodate 3 months of developmental testing into a couple of weeks.

The authors thank the biomedical scientists in the department of histopathology, Hammersmith Hospital, London, UK, particu-larly Donna Horncastle and Kay Elderfield for maintaining high standards of bone marrow trephine biopsy processing, sectioning, immunohistochemistry, and in situ hybridization.

On a more personal level, we thank our families for their loving patience and support.

—Emina Emilia Torlakovic—Kikkeri N Naresh—Richard D Brunning

ACKNOWLEDGEMENTS

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