Manual of Molecular and Clinical Laboratory Immunology (2 Volume Set)

VOLUME 1

Editorial Board 

Contributors 

Preface 

Author and Editor Conflicts of Interest 

Section A

Molecular Methods 1

Volume Editor: John L. Schmitz

Section Editor: Eric T. Weimer

• 1 Introduction 3
  Eric T. Weimer
• 2 Molecular Methods for Genetic Diagnosis of Inborn Errors of Immunity 5
  Sarah L. Anzick and Amy P. Hsu
• 3 Immunoglobulin and T- Cell Receptor Gene Assessment 23
  Paul A. Wadsworth, Chandler C. Ho, and Bing M. Zhang

Section B

Immunoglobulin Methods 39

Volume Editor: Barbara Detrick

Section Editor: David F. Keren

• 4 Introduction 41
  David F. Keren
• 5 Clinical Indications and Applications of Serum and Urine Protein Electrophoresis 43
  David M. Manthei and David F. Keren
• 6 Immunochemical Characterization of Immunoglobulins in Serum, Urine, and Cerebrospinal Fluid 62
  Caitlin O. Schein and William G. Finn
• 7 Cryoglobulins and Cryofibrinogenemia 79
  Peter D. Gorevic, Dennis Galanakis, and Jeffrey S. Warren
• 8 Strategy for Detecting and Following Monoclonal Gammopathies 90
  David F. Keren and Lee Schroeder

Section C

Complement 107

Volume Editor: Barbara Detrick

Section Editor: Ashley Frazer-Abel

• 9 Introduction 109
  Ashley Frazer- Abel
• 10 Methods of Complement Analysis 111
  Diana Pauly and Michael Kirschfink
• 11 Assessing Complement Dysregulation in the Thrombotic Microangiopathies and Ultrarare Complement- Mediated Kidney Disease 119
  Amanda K. Slagle, Jill J. Hauer, and Richard J. H. Smith
• 12 Current and Future of Complement Therapeutics and their Impact on the Clinical Immunology Laboratory 129
  Bradley Dixon, Shrey Purohit, and Ashley Frazer- Abel

Section D

Flow Cytometry 139

Volume Editor: Maurice R. G. O’gorman

Section Editor: Paul K. Wallace

• 13 Introduction: Clinical Flow Cytometry Applications and Quality Control 141
  Paul K. Wallace
• 14 Polychromatic Flow Cytometry 144
  Ankit Saxena, Angelique Biancotto, Pradeep Dagur, And John Philip Mccoy, Jr
• 15 High- Sensitivity Detection of Paroxysmal Nocturnal Hemoglobinuria by Multiparameter Flow Cytometry 166
  Ahmad Al- Attar, Stephen J. Richards, Iuri Marinov, Andrea Illingworth, And D. Robert Sutherland
• 16 Standardized Flow Cytometry Assays for Enumerating CD34 + Hematopoietic Stem Cells 189
  Ahmad Al- Attar and D. Robert Sutherland
• 17 The Detection of Fetal Red Cells in Fetomaternal Hemorrhage by Flow Cytometry 213
  Bruce Greig
• 18 Functional Flow Cytometry- Based Assays of Myeloid and Lymphoid Functions for the Diagnostic Screening of Inborn Errors of Immunity 221
  Caleb Cornaby and Maurice R. G. O’gorman
• 19 Quality Assurance and Validation in Clinical Flow Cytometry 231
  Teri Oldaker and Antony Bakke
• 20 Future Cytometric Technologies and Applications 249
  Holden T. Maecker

Section E

Functional Cellular Assays 253

Volume Editor: Maurice R. G. O’gorman

Section Editors: Miranda B. Carper And Thomas N. Denny

• 21 Introduction 255
  Miranda B. Carper and Thomas N. Denny
• 22 Cryopreservation of Blood Mononuclear Cells 257
  Cassandra Porth, Ambrosia Garcia, Raul Louzao, Krystle Garth, Emilie Jalbert, and Adriana Weinberg
• 23 Functional Assays for B Cells and Antibodies 266
  David C. Lafon and Moon H. Nahm
• 24 Methods for Detection of Antigen- Specific T Cells by Enzyme- Linked Immunospot Assay 277
  Mark Berrong, Danielle Nettere, and Guido Ferrari
• 25 Regulatory T Cell (Treg) Characterization: Phenotype, Function, and Clinical Importance of Human Treg 284
  Alma- Martina Cepika and Rosa Bacchetta
• 26 Measurement of Natural Killer Cell Phenotype and Activity in Humans 292
  Heinrich Schlums, Tessa M. Campbell, and Yenan T. Bryceson
• 27 Functional Assays for the Diagnosis of Chronic Granulomatous Disease 304
  Debra Long Priel and Douglas B. Kuhns
• 28 Interferon Gamma Release Assays for Tuberculosis Diagnosis 318
  Sofia Zavala and Jason E. Stout
• 29 Methods for Evaluating Antigen- Specific Cytotoxic T Lymphocyte Frequency and Activity 324
  Danielle Nettere and Guido Ferrari

Section F

Cytokines 333

Volume Editor: Barbara Detrick

Section Editors: John J. Hooks And Timothy B. Niewold

• 30 Introduction 335
  John J. Hooks and Timothy B. Niewold
• 31 Overview of the Cytokine Assay Multiverse: Systems and Applications 337
  Julio C. Valencia, Ngoc Thuy T. Nguyen, And Howard A. Young
• 32 Monitoring Interferons in Autoimmune Diseases and Interferonopathies 344
  Andrra Nimoni and Timothy B. Niewold
• 33 Detection of Anti-Cytokine Autoantibodies and Clinical Applications 352
  Serena J. Lee, Eszter Toth, Lindsey B. Rosen, Sarah K. Browne, and Steven M. Holland

Section G

Laboratory Management 363

Volume Editor: John L. Schmitz

Section Editor: Thomas S. Alexander

• 34 Licensure and Accreditation 365
  Thomas S. Alexander
• 35 Validation and Quality Control: General Principles and Application to the Clinical Immunology Laboratory 369
  Terri Lebo, Patricia A. Merkel, and Vijaya Knight
• 36 Quality Assurance and Quality Management of the Medical Laboratory 384
  Paul R. Johnson

Section H

Infectious Diseases Caused By Bacteria, Mycoplasmas, Chlamydiae, and Rickettsiae 393

Volume Editor: John L. Schmitz

Section Editor: Christine M. Litwin

• 37 Introduction 395
  Christine M. Litwin
• 38 Diagnostic Methods for Group A Streptococcal Infections 396
  Christine M. Litwin, Sheldon E. Litwin, And Harry R. Hill
• 39 Laboratory Diagnosis of Syphilis 404
  Tyson Chiaro and John L. Schmitz
• 40 Lyme Disease, Relapsing Fever, and Leptospirosis 412
  Guiqing Wang and Maria E. Aguero- Rosenfeld
• 41 Immunological Tests in Tuberculosis 424
  Christine M. Litwin
• 42 Mycoplasma: Serological Methods 433
  Ken B. Waites, Mary B. Brown, and Jerry W. Simecka
• 43 Chlamydia Infections 438
  Rosemary C. She and Valentina Zanfagnin
• 44 The Rickettsiaceae, Anaplasmataceae, and Coxiellaceae 447
  Lucas S. Blanton and David H. Walker
• 45 The Bartonellaceae, Brucellaceae, and Francisellaceae 458
  Christine M. Litwin, Burt Anderson, Renee Tsolis, and Amy Rasley

Section I

Mycotic and Parasitic Diseases 467

Volume Editor: John L. Schmitz

Section Editor: Elitza S. Theel

• 46 Introduction 469
  Elitza S. Theel
• 47 Immunological and Molecular Approaches for the Diagnosis of Parasitic Infections 470
  Marc R. Couturier and Elitza S. Theel
• 48 Serological Diagnosis of Fungal Infections 489
  Mark D. Lindsley

Section J

Viral Diseases 521

Volume Editor: John L. Schmitz

Section Editor: Jennifer Dien Bard

• 49 Introduction 523
  Jennifer Dien Bard
• 50 Herpes Simplex Virus and Varicella- Zoster Virus 524
  Susan Realegeno
• 51 Epstein- Barr Virus and Cytomegalovirus 535
  Henry H. Balfour, Jr., Kristin A. Hogquist, And Priya S. Verghese
• 52 Human Herpesviruses 6A, 6B, 7, and 8 550
  Shangxin (shaun) Yang
• 53 Parvovirus B19 563
  Rebecca Yee and Jose A. Lucar
• 54 Severe Acute Respiratory Syndrome Coronavirus- 2 572
  Patricia Slev
• 55 Enteroviruses 583
  Bijal A. Parikh and Allison R. Eberly
• 56 Measles, Mumps, and Rubella Viruses 592
  Ryan F. Relich and Elitza S. Theel
• 57 Viral Hepatitis 600
  Kileen L. Shier
• 58 Arboviruses 616
  Mimi R. Precit and Rachael M. Liesman
• 59 Hantavirus Infections 634
  William Marciel De Souza and Luiz Tadeu Moraes Figueiredo
• 60 Rabies Virus 640
  D. Craig Hooper
• 61 Human T- Cell Lymphotropic Virus Types 1 and 2 649
  Laura Gillim and Ajay Grover
• 62 Human Immunodeficiency Virus 658
  S. Michele Owen
• 63 Bandavirus 668
  Amanda E. Calvert and Holly R. Hughes

VOLUME 2

Section K

Errors Of Immunity 679

Volume Editor: Maurice R. G. O’gorman

Section Editor: Roshini S. Abraham

• 64 Introduction to Inborn Errors of Immunity 681
  Roshini S. Abraham
• 65 Newborn Screening for Severe Combined Immunodeficiency 683
  Lisa Kobrynski and Mirjam Van Der Burg
• 66 Combined Immunodeficiencies 692
  Tamar S. Rubin, Kelli W. Williams, and Monica G. Lawrence
• 67 Predominantly Antibody Deficiencies 717
  Sara Barmettler, Daniel Digiacomo, and Jocelyn R. Farmer
• 68 Primary Immune Regulatory Disorders 732
  Shanmuganathan Chandrakasan, Gulbu Uzel, Carrie L. Lucas, And Andrew L. Snow
• 69 Primary Innate Immune Defects (Including Phagocytic Disorders, TLR Defects, and NK Cell Deficiencies) 752
  Jenna R. E. Bergerson, Alexandra F. Freeman, Emily M. Mace, And Keith A. Sacco
• 70 Systemic Autoinflammatory Diseases 768
  Xiao Peng, Oskar Schnappauf, Adriana A. De Jesus, and Ivona Aksentijevich
• 71 Complement Defects 796
  Ann M. Moyer, Meera Sridharan, and Maria Alice Vieira Willrich
• 72 Inherited Bone Marrow Failure Syndromes: Focus on Immunodeficiency and Diagnostic Evaluation 813
  Abhishek A. Mangaonkar, Alejandro Ferrer, Asma Ali, Mira Kohorst, and Mrinal M. Patnaik
• 73 Phenocopies of Inborn Errors of Immunity 825
  Elena W. Y. Hsieh, Cullen M. Dutmer, and Vijaya Knight

Section L

llergic Diseases 845

Volume Editor: Barbara Detrick

Section Editor: Robert G. Hamilton

• 74 Introduction to Allergic Diseases 847
  Robert G. Hamilton
• 75 Quantitation and Standardization of Allergens 849
  Michael Brad Strader, Robert Zagorski, Ronald L. Rabin, and Jay E. Slater
• 76 Immunological Methods in the Diagnostic Allergy Laboratory 860
  Robert G. Hamilton
• 77 In Vitro Assay Methods for Measurement of Mediators and Markers of Allergic Inflammation 866
  John T. Schroeder

Section M

Systemic Autoimmune Diseases 877

Volume Editor: Barbara Detrick

Section Editor: Westley H. Reeves

• 78 Introduction 879
  Westley H. Reeves
• 79 Standardization of Fluorescent Antinuclear Antibody Testing 881
  Angela Ceribelli, Antonio Tonutti, Luis E. C. Andrade, and Edward K. L. Chan
• 80 Immunodiagnosis and Laboratory Assessment of Systemic Lupus Erythematosus 891
  Westley H. Reeves, Shuhong Han, and Haoyang Zhuang
• 81 Immunodiagnosis of Autoimmune Myopathies 900
  Minoru Satoh, Angela Ceribelli, Tomoko Hasegawa, Shin Tanaka, Michito Hirakata, and Edward K. L. Chan
• 82 Immunodiagnosis of Scleroderma 913
  Yasuhito Hamaguchi and Masataka Kuwana
• 83 Immunodiagnosis of Rheumatoid Arthritis 925
  Johan Rönnelid, Alf Kastbom, and Carl Turesson
• 84 Antiphospholipid Antibody Syndrome: Clinical Manifestations and Laboratory Diagnosis 935
  Pier Luigi Meroni, Caterina Bodio, Claudia Grossi, and Maria Orietta Borghi
• 85 Immunodiagnosis of Systemic Vasculitis 945
  Jan Damoiseaux
• 86 Immunodiagnosis of Sjögren’s Disease 952
  R. Hal Scofield and Martha Tsaliki
• 87 Diagnostic Testing for Deficiency of Adenosine Deaminase 2 962
  Kailey E. Brodeur, Liang Chen, And Pui Y. Lee
• 88 Future Direction of Laboratory Testing for Autoimmune Diseases 968
  Gerson Dierley Keppeke, Renan De Almeida Agustinelli, Diana Maria Landoni, Wilson Melo Cruvinel, Edward K. L. Chan, And Luis Eduardo Coelho Andrade

Section N

Organ-Localized Autoimmune Diseases 983

Volume Editor: Barbara Detrick

Section Editor: Stanley J. Naides

• 89 Introduction 985
  Stanley J. Naides
• 90 Endocrinopathies: Hashimoto Thyroiditis, Graves Disease, Hypophysitis, Addison Disease, Premature Ovarian Failure, Male Infertility, and Diabetes 987
  Amna Kahn, Giuseppe Barbesino, Jonathan Perez, Ziyan Xu, Xiaofan Jia, Fatima Zaman, Liping Yu, and Patrizio Caturegli
• 91 Myasthenia Gravis 1014
  Hans Frykman
• 92 Autoantibodies to Brain and Spinal Cord 1024
  Raquel Ruiz- García, Laura Naranjo, and Francesc Graus
• 93 Autoantibodies to Glycolipids in Peripheral Neuropathy 1037
  John A. Goodfellow and Hugh J. Willison
• 94 Detection of Antimitochondrial Autoantibodies in Primary Biliary Cholangitis and Liver Kidney Microsomal Antibodies in Autoimmune Hepatitis 1044
  Patrick S. C. Leung, Michael P. Manns, Ross L. Coppel, and M. Eric Gershwin
• 95 Cardiovascular Diseases 1054
  Jin li
• 96 Use of Laboratory Tests for Patients with Inflammatory Bowel Diseases 1071
  Louma B. Rustam, David E. Elliott, and M. Nedim Ince
• 97 Celiac Disease 1085
  Tracey M. Dafonte, Katherine L. Olshan, And Alessio Fasano
• 98 Primary Membranous Nephropathy 1090
  Ajay Grover, Andre Valcour, and Stanley J. Naides
• 99 Autoantibodies Directed against Erythrocytes in Autoimmune Hemolytic Anemia and Other Anemias 1098
  Laura Cooling
• 100 Immune Thrombocytopenia 1105
  Qizhao Li, Johan Rebetz, Drew Provan, Leendert L. Porcelijn, Rick Kapur, Suzanne Hofstede- Van Egmond, Elisabeth Semple, Li Guo, Shuqian Xu, and John W. Semple
• 101 Monitoring Autoimmune Reactivity within the Retina 1113
  John J. Hooks, Renee Liu, Lucia Sobrin, And Barbara Detrick
• 102 Diagnosis of Autoimmune Blistering Diseases 1120
  Mareike Becker and Enno Schmidt

Section O

Cancer 1133

Volume Editor: Maurice R. G. O’gorman

Section Editor: Brent L. Wood

• 103 Introduction 1135
  Brent L. Wood
• 104 Immunoassay- Based Tumor Marker Measurement: Assays, Applications, Algorithms, and Additional Approaches 1136
  Chinwe Madubata and Alex J. Rai
• 105 Immune Monitoring during Cancer Immunotherapy 1144
  Hesham M. Shehata, Pranay Dogra, and Shomyseh Sanjabi
• 106 Immunophenotyping by Flow Cytometry of Acute Lymphoblastic Leukemia Lymphoma 1168
  Karin P. Miller and Alexandra E. Kovach
• 107 Acute Myeloid Leukemia: Diagnosis and Measurable Residual Disease Detection by Flow Cytometry 1180
  Xueyan Chen and Brent L. Wood
• 108 Chronic Lymphocytic Leukemia: Diagnosis and Minimal Residual Disease Detection 1193
  Min Shi, Dragan Jevremovic, Curtis A. Hanson, and Horatiu Olteanu
• 109 Evaluation of Plasma Cell Neoplasms 1206
  Mikhail Roshal, Qi Gao, Shelby Hutcherson, Katie Thoren, Menglei Zhu, And Kazunory Murata

Section P

Transplantation Immunology 1225

Volume Editor: John L. Schmitz

Section Editor: Gerald P. Morris

• 110 Introduction: Immunogenetics, Histocompatibility Testing, and Transplantation 1227
  Gerald P. Morris
• 111 Human Leukocyte Antigen Biology and Genetics 1229
  Bing Melody Zhang and Marcelo Fernandez- Vina
• 112 Human Leukocyte Antigen Genotyping for Allogeneic Transplantations 1240
  Brian F. Duffy, Mei San Tang, and Chang Liu
• 113 Human Leukocyte Antigen Antibodies 1262
  Olga A. Timofeeva and Sandra Rosen- Bronson
• 114 Non- HLA Antibodies 1280
  Carrie L. Butler, Michelle J. Hickey, Rebecca A. Sosa, and Elaine F. Reed
• 115 ABO Blood Groups and Transplantation 1291
  Anne Halpin, Bruce Motyka, and Lori J. West
• 116 Cellular Immune Responses in Transplantation 1301
  Clarkson Crane and Gerald P. Morris
• 117 Molecular Characterization of Injury and Rejection in Solid Organ Transplant 1312
  Meagan Barner and Zahra Kashi
• 118 Killer Cell Immunoglobulin- Like Receptors (KIR) in Clinical Transplantation 1323
  Raja Rajalingam
• 119 Chimerism Monitoring 1335
  Caroline R. Alquist

Index 1341

THE authoritative guide for clinical laboratory immunology

For nearly 50 years, the Manual of Molecular and Clinical Laboratory Immunology has been the premier resource for laboratories, students, and professionals involved in the clinical and technical details of diagnostic immunology testing. The 9th Edition continues its tradition of providing comprehensive clinical and technical information on the latest technologies used in medical and diagnostic immunology.

Led by a world-renowned group of authors and editors, this new edition reflects substantial changes aimed at improving and updating the Manual’s utility while reflecting the significant transformations that have occurred since the last edition, including the revolution of gene editing and the widespread adoption of molecularly engineered cellular therapies. Topical highlights include:

• Laboratory Management: three new chapters cover essential aspects of quality assurance, quality improvement, and quality management, aligning with the increasingly stringent and demanding regulatory environment.
• Inborn Errors of Immunity: the primary immunodeficiency section has been completely updated to align with the latest International Union of Immunological Societies’ classifications of inborn errors of immunity.
• Functional Cellular Assays: expanded content includes detailed discussions on various functional assays critical for modern immunologic testing.
• Autoimmune Diseases: expanded chapters on systemic and organ-specific autoimmune disorders, including new chapters on Sjögren’s syndrome and deficiency of ADA2, as well as significant updates on organ-specific autoimmune diseases.
• Transplantation Immunology: updated chapters detail the assessment of immune reconstitution and ABO testing, reflecting latest practices.

The 9th Edition of the Manual of Molecular and Clinical Laboratory Immunology serves as an invaluable resource for laboratory directors, clinicians, laboratory managers, technologists, and students. It provides critical insights into the selection, application, and interpretation of immunologic tests, offering practical guidance on troubleshooting, clinical application, and an understanding of test limitations. This comprehensive and up-to-date manual remains an essential tool for anyone involved in the diagnosis, evaluation, and management of immune-mediated and immune system-related disorders.

John L. Schmitz, University of North Carolina, Chapel Hill, NC

Barbara Detrick, The Johns Hopkins University, Baltimore, MD

Maurice R. G. O’Gorman, University of Southern California, Los Angeles, CA