Bone Marrow IHC 0 Front Matter [Torlakovic].indd i 0 Front Matter [Torlakovic].indd i 8/18/ :30:45 AM 8/18/ :30:45 AM

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1 Bone Marrow IHC 0 Front Matter [Torlakovic].indd i 8/18/ :30:45 AM

2 Dedication I dedicate this book to my husband Goran and to my children Lino, Jakov, Marija Milena, and Marta Danica Emina Emilia Torlakovic, MD, PhD 0 Front Matter [Torlakovic].indd ii 8/18/ :31:23 AM

3 Affiliations Emina Emilia Torlakovic, MD, PhD Associate Professor Department of Pathology and Laboratory Medicine Royal University Hospital University of Saskatchewan Saskatoon, SK Canada Professor Kikkeri Naresh, MBBS, CCP, MD, FRCPath Consultant Haematopathologist Department of Histopathology Hammersmith Hospital London UK Richard D Brunning, MD Professor Emeritus Department of Laboratory Medicine and Pathology University of Minnesota Minneapolis, MN USA iii 0 Front Matter [Torlakovic].indd iii 8/18/ :31:23 AM

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5 Bone Marrow IHC Emina Emilia Torlakovic, MD, PhD Kikkeri N Naresh, MBBS, CCP, MD, FRCPath Richard D Brunning, MD Chicago 0 Front Matter [Torlakovic].indd v 8/18/ :31:24 AM

6 Publishing Team Erik Tanck (design and production) Joshua Weikersheimer (publishing direction) Notice Trade 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 therefore 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 Printed in Hong Kong 0 Front Matter [Torlakovic].indd vi 8/18/ :31:27 AM

7 Abbreviations Ab antibody Ag antigen ALL acute lymphoblastic leukemia ALPS autoimmune lymphoproliferative syndrome AMF acute myelofibrosis AMKL acute megakaryoblastic leukemia AML acute myeloid leukemia APMF acute panmyelosis with myelofibrosis B-ALL B-cell acute lymphoblastic leukemia BM bone marrow chl classical Hodgkin lymphoma CLL chronic lymphocytic leukemia CLL/SLL chronic lymphocytic leukemia/small lymphocytic lymphoma CML chronic myeloid leukemia CMML chronic myelomonocytic leukemia DLBCL diffuse large B-cell lymphoma DS Down syndrome EBV Epstein-Bar virus EGIL European Group for the Immunologic Classification of Leukemia ET essential thrombocythemia FISH fluorescent in situ hybridization FL follicular lymphoma HCL hairy cell leukemia HIER heat-induced epitope retrieval IHC immunohistochemistry ISH in situ hybridization LF lactoferrin LPL lymphoplasmacytic lymphoma LZ lysozyme MCD mast cell disease MCL mantle cell lymphoma MCT mast cell tryptase MDS myelodysplastic syndrome MGUS monoclonal gammopathy of undetermined significance MPD myeloproliferative disease MPO myeloperoxidase MZL marginal zone lymphoma OMIM Online Mendelian Inheritance in Man PROW Protein Reviews on the Web ( PTCL peripheral T-cell lymphoma RA refractory anemia RAEB refractory anemia with excess blasts RARS refractory anemia with ringed sideroblasts RCMD refractory anemia with multilineage dysplasia T-ALL T-cell acute lymphoblastic leukemia vii 0 Front Matter [Torlakovic].indd vii 8/18/ :31:27 AM

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9 TABLE OF CONTENTS Preface xiii Acknowledgements xiv Chapter 1 Introduction References Chapter 2 The Role of Tissue Processing In Bone Marrow Immunohistochemistry BM Biopsy Fixation Decalcification Embedding Tissue Sectioning Antigen Retrieval Turnaround Time Protocols Protocol for Bone Marrow Processing and Staining Used at Hammersmith Hospital Protocol for Bone Marrow Processing and Staining Suitable for Rapid Turnaround Time References ix 0 Front Matter [Torlakovic].indd ix 8/18/ :31:27 AM

10 Table of Contents Chapter 3 Cluster of Differentiation Markers CD1a CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD CD31 (PECAM) CD CD CD CD45 (CD45RA) CD45 (CD45RO) CD CD CD61 and CD42b CD CD79a CD x 0 Front Matter [Torlakovic].indd x 8/18/ :31:28 AM

11 Table of Contents 3.31 CD CD CD CD235a (Glycophorin A) References Chapter 4 Other Common Markers Myeloperoxidase Hemoglobin A (HgbA) Mast Cell Tryptase Terminal Deoxynucleotidyl transferase (TdT) Factor 8-Related Antigen (von Willebrand Factor) Pax PU Fli Cyclin D Lysozyme Bcl Immunoglobulins Ki Cytotoxic Molecules Stromal Markers MUM TRAP Bcl References xi 0 Front Matter [Torlakovic].indd xi 8/18/ :31:28 AM

12 Table of Contents Chapter 5 Special Diagnostic Considerations Acute Leukemia Metastatic Tumors Recommended Panels References Index xii 0 Front Matter [Torlakovic].indd xii 8/18/ :31:28 AM

13 PREFACE Bone Marrow IHC is intended to provide a collection of illustrations of various immunohistochemical tests that are routinely used in diagnostic practice. The choice of tests and the frequency of use vary greatly depending not only on the type of practice 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 published 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 important in the future. The intrinsic value of the in situ tests will be difficult to replace by any other type of evaluation. This work does not include descriptions and possible uses of all currently available markers, 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 associated 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 suboptimal results are also illustrated to depict certain common problems. Most BM biopsies that are illustrated here were fixed in 10% buffered formalin, aceto-zinc-formalin fixative, or B5, and most were decalcified by rapid decalcifier or Gooding and Stewart s decalcification 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 ( The names PROW, Entrez Gene, and OMIM are used throughout the text to designate the following Web sites as a source of information: and respectively. A number of internet sites serve those who study bone marrow protein expression by immunohistochemistry. Search at offers IHC images of relatively high quality showing various proteins that can be evaluated in TMA samples. Hematopoietic tissue markers are searchable at It includes many good images, but not all markers are illustrated, and most do not show bone marrow expression patterns. xiii 0 Front Matter [Torlakovic].indd xiii 8/18/ :31:28 AM

14 ACKNOWLEDGEMENTS We are indebted to many colleagues and collaborators 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 applications. 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 laboratory 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 immunohistochemistry 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, particularly 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 xiv 0 Front Matter [Torlakovic].indd xiv 8/18/ :31:28 AM